Novel cyclic dinucleotide derivatives and antibody drug conjugates thereof

文档序号：664428 发布日期：2021-04-27 浏览：39次中文

阅读说明：本技术 新型环状二核苷酸衍生物及其抗体药物偶联物 (Novel cyclic dinucleotide derivatives and antibody drug conjugates thereof ) 是由津田敏史田渊俊树渡边秀昭小林宏行石崎雅之原香生子和田悌司荒井雅巳于 2019-09-06 设计创作，主要内容包括：期望开发具有STING激动剂活性的新型CDN衍生物以及使用该新型CDN衍生物的STING激动剂活性相关的疾病的治疗剂和/或治疗方法。还期望开发能够将该新型CDN衍生物特异性地送达至目标细胞或脏器的STING激动剂活性相关的疾病的治疗剂和/或治疗方法。本发明提供一种具有强的STING激动剂活性的新型CDN衍生物、包含新型CDN衍生物的抗体CDN衍生物偶联物。(It is desirable to develop novel CDN derivatives having STING agonist activity and therapeutic agents and/or therapeutic methods for diseases associated with STING agonist activity using the novel CDN derivatives. It is also desired to develop a therapeutic agent and/or a therapeutic method for a disease associated with STING agonist activity, which can deliver the novel CDN derivative specifically to a target cell or organ. The present invention provides a novel CDN derivative having a strong STING agonist activity, an antibody CDN derivative conjugate comprising the novel CDN derivative.)

1. An antibody drug conjugate represented by the following formula (II):

in the formula, m¹In the range of 1 to 10, in the case of a liquid crystal,

ab represents an antibody, the sugar chain of which may optionally be reconstituted, or a functional fragment of the antibody,

l represents a linker linking Ab to D,

ab may optionally be bound to L directly from its amino acid residue, or bound to L from the sugar chain or reconstituted sugar chain of Ab,

d represents a compound represented by the following formula (I):

wherein L and L¹Or L²any-NH contained therein₂Or the combination of hydroxyl groups,

L¹represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino]1 to 3 groups of ethyl substituted at arbitrary positions and selected fromA group of the group consisting of:

and

in the formula, R⁶And R^6’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH ₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl,

R⁷and R^7’Each independently represents a hydrogen atom or a C1-C6 alkyl group, which C1-C6 alkyl group may be optionally substituted by 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group,

R⁸and R^8’Each independently represents a hydrogen atom or a halogen atom,

Z⁴represents-CH₂-, -NH-or an oxygen atom,

Z⁵represents a nitrogen atom or-CH ═ CH,

L²represents a group selected from the following (i) or (ii):

(i)L²in combination with L, L²represents-NHR ', hydroxyl C1-C6 alkyl or amino C1-C6 alkyl, wherein R' represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl can be optionally substituted by 1-6 halogen atoms; or

(ii)L²When not bound to L, L²Represents a hydrogen atom or a halogen atom,

Q¹and Q^1’Each independently represents a hydroxyl group, a thiol group or a boryl group BH₃－，

Q²And Q^2’Each independently represents an oxygen atomOr a sulfur atom,

X¹and X²Each independently represents an oxygen atom, a sulfur atom or-CH₂－，

Y¹And Y²Represents an oxygen atom or-CH₂－，

X³And X⁴Represents a group selected from the following (iii) or (iv):

(iii)Y¹when it is an oxygen atom, X³－X⁴represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH ₂－CF₂-; or

(iv)Y¹is-CH₂When is, X³－X⁴represents-O-CH₂－，

X⁵And X⁶Represents a group selected from the following (v) or (vi):

(v)Y²when it is an oxygen atom, X⁵－X⁶represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or

(vi)Y²is-CH₂When is, X⁵－X⁶represents-O-CH₂－，

R¹、R²And R³Each independently represents a hydrogen atom, a halogen atom, -OR ', -OC (═ O) R', -N₃-NHR ', -NR' R "or-NHC (═ O) R ', wherein R' is as defined hereinbefore, R" represents C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl,

W¹represents a nitrogen atom, an oxygen atom, a sulfur atom or-CH-,

W²represents a nitrogen atom or-CH ═ CH,

R⁴represents a hydrogen atom, a halogen atom or-NH₂，

R⁵Represents a group selected from the following (vii) to (x):

(vii)W¹when it is a nitrogen atom, R⁵Represents a hydrogen atom, C1-C6 alkyl, hydroxy C1-C6 alkyl or amino C1-C6 alkyl;

(viii)W¹when it is an oxygen atom, R⁵Is absent;

(ix)W¹when it is a sulfur atom, R⁵Is absent; or

(x)W¹When is-CH-, R⁵Represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂Or C1-C6 alkyl,

Z¹－Z²－Z³taken together represent a group selected from: -CH₂－CH₂－CH₂－、－CH₂－CH₂－R”’－、－CH＝CH－CH₂－、－CH＝CX－CH₂－、－CX＝CH－CH₂－、－CX＝CX－CH₂－、－C(＝O)－CH₂－CH₂－、－CH₂－CH₂－C(＝O)－、－CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃) -or a group of any of the formulae, wherein R' "represents-O-or-CH₂－CH₂-, X represents a halogen atom,

Here, the asterisk indicates¹In combination, the wavy line indicates bonding to the carbon atom of ═ C-.

2. The antibody drug conjugate of claim 1, wherein,

W¹is a nitrogen atom.

3. The antibody drug conjugate of claim 2,

W¹is a nitrogen atom, R⁵Is a hydrogen atom.

4. The antibody drug conjugate of claim 1, wherein,

W¹is an oxygen atom.

5. The antibody drug conjugate of claim 1, wherein,

W¹is a sulfur atom.

6. The antibody drug conjugate of claim 1, wherein,

W¹is-CH-.

7. The antibody drug conjugate of claim 6, wherein,

W¹is-CH-, R⁵Is a hydrogen atom.

8. The antibody drug conjugate according to any one of claims 1 to 7,

Z¹、Z²and Z³Taken together as-CH₂－CH₂－CH₂-or-CH ═ CH-CH₂－。

9. The antibody drug conjugate according to any one of claims 1 to 7,

Z¹、Z²and Z³Taken together as-CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃)－。

10. The antibody drug conjugate according to any one of claims 1 to 7,

Z¹、Z²and Z³Taken together as-CH₂－CH₂-R '"-, wherein R'" represents-O-or-CH₂－CH₂－。

11. The antibody drug conjugate according to any one of claims 1 to 10, wherein,

W²is-CH ═ CH.

12. The antibody drug conjugate according to any one of claims 1 to 10, wherein,

W²is a nitrogen atom.

13. The antibody drug conjugate according to any one of claims 1 to 12,

R⁴represents a hydrogen atom.

14. The antibody drug conjugate according to any one of claims 1 to 12,

R⁴represents a fluorine atom.

15. The antibody drug conjugate according to any one of claims 1 to 14,

L¹r in (1)⁸And R^8’Each independently a hydrogen atom.

16. The antibody drug conjugate according to any one of claims 1 to 15, wherein,

L¹is a group selected from the group consisting of:

and

herein, R is⁹And R^9’Represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，

R¹⁰Represents a hydroxyl group, -NH₂、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH、－CH₂CH₂NHC(＝O)CH₂OH, hydroxy C1-C3 alkyl or amino C1-C3 alkyl,

R¹¹and R^11’Each independently represents a hydrogen atom, a fluorine atom or a methyl group, or R¹¹And R^11’Taken together to represent a cyclopropane ring,

Z⁴represents-CH₂-, -NH-or an oxygen atom.

17. The antibody drug conjugate according to any one of claims 1 to 15, wherein,

L¹is a group selected from the group consisting of:

and

herein, R is¹³And R^13’Each independently represents a hydrogen atom, a hydroxyl group or-NH ₂，

R¹²Represents a hydroxyl group, -NH₂、－CH₂OH、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH or-CH₂CH₂NHC(＝O)CH₂OH，

Z⁴As defined above.

18. The antibody drug conjugate according to any one of claims 1 to 15, wherein,

L¹is a group selected from the group consisting of:

and

herein, R is¹⁴Represents a hydrogen atom or-NH₂，

R¹⁵Represents a hydrogen atom or-C (═ O) CH₂OH，

R¹⁶Represents a hydroxyl group, -NH₂、－CH₂OH、－CH₂CH₂OH、－CH₂NH₂or-CH₂CH₂NH₂。

19. The antibody drug conjugate of any one of claims 1 to 18,

L²combined with L to represent-NH₂、－CH₂NH₂or-CH₂OH。

20. The antibody drug conjugate of any one of claims 1 to 18,

L²is not bonded to L and represents a hydrogen atom or a fluorine atom.

21. The antibody drug conjugate according to any one of claims 1 to 20, wherein,

Q¹and Q^1’Each independently represents a hydroxyl group or a thiol group.

22. The antibody drug conjugate of any one of claims 1 to 21,

X¹and X²Represents an oxygen atom.

23. The antibody drug conjugate according to any one of claims 1 to 22,

Y¹and Y²Represents an oxygen atom.

24. The antibody drug conjugate of any one of claims 1 to 23,

X³and X⁴represents-CH₂－O－。

25. The antibody drug conjugate of any one of claims 1 to 24,

X⁵And X⁶represents-CH₂－O－。

26. The antibody drug conjugate of any one of claims 1 to 25,

R¹、R²and R³Each independently a hydrogen atom, a hydroxyl group or a fluorine atom.

27. The antibody drug conjugate of any one of claims 1 to 26,

d is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, the above-mentioned definition,

R¹⁷、R^17’、R¹⁸and R^18’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，

W³represents-NH-, an oxygen atom, a sulfur atom or-CH₂－，

W⁴represents-CH ═ or a nitrogen atom.

28. The antibody drug conjugate of claim 27, wherein,

d is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²、Q^2’、R¹⁷、R^17’、R¹⁸And R^18’As defined above.

29. The antibody drug conjugate of claim 27 or 28, wherein D is represented by any one of the 8 structural formulae below:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, the above-mentioned definition,

R¹⁹、R^19’、R²⁰and R^20’Each independently represents a hydrogen atom or a fluorine atom.

30. The antibody drug conjugate of any one of claims 27 to 29, wherein,

d is represented by any one of the following 4 structural formulae:

here, L¹As defined above.

31. The antibody drug conjugate of any one of claims 27 to 30,

d is represented by any one of the following 4 structural formulae:

here, L¹As defined above.

32. The antibody drug conjugate of any one of claims 27 to 30,

d is represented by any one of the following 4 structural formulae:

here, L¹As defined above.

33. The antibody drug conjugate of any one of claims 1 to 26,

d is represented by the formula:

here, L¹As defined above, the above-mentioned definition,

Q³and Q^3＇Each independently represents a hydroxyl group or a thiol group,

R²¹and R²²Each independently represents a hydroxyl group or a fluorine atom,

W⁵represents an-NH-or sulfur atom.

34. The antibody drug conjugate of claim 33, wherein,

d is represented by any one of the following 2 structural formulae:

here, L¹、Q³And Q^3＇、W⁵As defined above.

35. The antibody drug conjugate of any one of claims 1 to 34,

L¹represented by any one of the following 4 structural formulae:

36. the antibody drug conjugate of any one of claims 1 to 34,

L¹represented by any one of the following 4 structural formulae:

In the formula, an asterisk indicates the binding to L.

37. The antibody drug conjugate of any one of claims 33, 34 or 36,

d is represented by any one of the following 4 structural formulae:

here, the asterisk indicates the bond to L, Q³、Q^3＇And W⁵As defined above.

38. The antibody drug conjugate of any one of claims 33, 34, 36 or 37,

d is represented by any one of the following 4 structural formulae:

here, an asterisk indicates binding to L.

39. The antibody drug conjugate of any one of claims 33, 34, 36 or 37,

d is represented by any one of the following 3 structural formulae:

here, an asterisk indicates binding to L.

40. The antibody drug conjugate of any one of claims 33, 34, 36 or 37,

d is represented by any one of the following 4 structural formulae:

here, an asterisk indicates binding to L.

41. The antibody drug conjugate according to any one of claims 1 to 40,

the linker L is represented by-Lb-La-Lp-Lc-,

wherein the asterisk indicates that the drug D is bound,

lp represents a linker formed by an amino acid sequence that can be cleaved in a target cell or is absent,

La represents any one selected from the group consisting of:

－C(＝O)－(CH₂CH₂)n²－C(＝O)－、

－C(＝O)－(CH₂CH₂)n²－CH₂－C(＝O)－、

－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－C(＝O)-、

－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－CH₂－C(＝O)－、

－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂O)n³－CH₂－C(＝O)－、

－(CH₂)n⁴-O-C (═ O) -, and,

－(CH₂)n⁹－C(＝O)－，

Here, n is²Represents an integer of 1 to 3, n³Represents an integer of 1 to 5, n⁴Represents an integer of 0 to 2, n⁹Represents an integer of 2 to 7,

lb represents a spacer binding La to the sugar chain or the reconstituted sugar chain of Ab or a spacer binding La to the cysteine residue of Ab, and,

lc represents-NH-CH₂-, -NH-phenyl-CH₂-O (C ═ O) -or-NH-heteroaryl-CH₂-O (C ═ O) -, or is absent.

42. The antibody drug conjugate of claim 41, wherein,

lc is absent.

43. The antibody drug conjugate of claim 41, wherein,

lc is-NH-CH₂－。

44. The antibody drug conjugate of any one of claims 41 to 43,

lp represents any one selected from the group consisting of:

-GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-, and-ICit-.

45. The antibody drug conjugate of claim 44, wherein,

lp is any one of-GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-or-FCit-.

46. The antibody drug conjugate of any one of claims 41 to 43,

lp is any one of-GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-or-GGPP-.

47. The antibody drug conjugate of claim 46, wherein,

lp is-GGFG-or-GGPI-.

48. The antibody drug conjugate of any one of claims 41 to 47, wherein,

la represents any one selected from the group consisting of:

－C(＝O)－CH₂CH₂－C(＝O)－、

－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₃－CH₂－C(＝O)-、

－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₄－CH₂-C (═ O) -, and

－(CH₂)₅－C(＝O)－。

49. the antibody drug conjugate of any one of claims 41 to 48, wherein,

lb is represented by any one of the following structural formulae:

in the structural formula of Lb described above, asterisks indicate the binding to La, wavy lines indicate the binding to sugar chains or restructured sugar chains of Ab.

50. The antibody drug conjugate of any one of claims 41 to 48, wherein,

lb is- (succinimidin-3-yl-N) -,

herein, - (succinimidin-3-yl-N) -represents the following structural formula:

here, the asterisk indicates that La is bonded, and the wavy line indicates that the side chain of the cysteine residue of the antibody is bonded via thioether formation.

51. The antibody drug conjugate of any one of claims 41, 46 to 49, wherein,

the linker L is represented by-Lb-La-Lp-Lc-,

wherein the asterisk indicates that the drug D is bound,

lp is-GGFG-, or-GGPI-,

la represents-C (═ O) -CH₂CH₂－C(＝O)－，

Lb represents the following formula:

in the structural formula of Lb shown above, the asterisk indicates the binding to La, the wavy line indicates the binding to the sugar chain or the reconstituted sugar chain of Ab,

lc represents-NH-CH₂－。

52. The antibody drug conjugate of any one of claims 1 to 51, wherein,

the average drug binding number of each molecule of antibody in the antibody drug conjugate is in the range of 1-10.

53. The antibody drug conjugate of claim 52,

the average drug binding number of each molecule of antibody in the antibody drug conjugate is in the range of 1-5.

54. The antibody drug conjugate of any one of claims 1 to 53,

the antibody binds to L from a sugar chain bound to Asn297 of the antibody, which is an N297 sugar chain.

55. The antibody drug conjugate of claim 54, wherein,

the N297 sugar chain is a reconstituted sugar chain.

56. The antibody drug conjugate of claim 54 or 55,

The N297 sugar chain is N297- (Fuc) MSG1 or N297- (Fuc) SG.

57. The antibody drug conjugate of any one of claims 1 to 56,

the antibody is an anti-HER 2 antibody, an anti-HER 3 antibody, an anti-DLL 3 antibody, an anti-FAP antibody, an anti-CDH 11 antibody, an anti-CDH 6 antibody, an anti-a 33 antibody, an anti-CanAg antibody, an anti-CD 19 antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 98 antibody, an anti-TROP 2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUC 1 antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, an anti-Mesothelin antibody, an anti-ENPP 3 antibody, an anti-CD 47 antibody, an anti-EGFR antibody, an anti-GPR 58.

58. The antibody drug conjugate of claim 57, wherein,

the antibody is an anti-HER 2 antibody.

59. The antibody drug conjugate of claim 58, wherein,

the antibody is an antibody comprising a light chain formed by the amino acid sequence of sequence number 1 and a heavy chain formed by the amino acid sequence of sequence number 2, or an antibody comprising a light chain formed by the amino acid sequence of sequence number 1 and a heavy chain formed by the amino acid sequence of sequence number 3.

60. The antibody drug conjugate of claim 58, wherein,

the antibody is an antibody comprising a light chain formed by the amino acid sequence of sequence number 28 and a heavy chain formed by the amino acid sequence of sequence number 29, or an antibody comprising a light chain formed by the amino acid sequence of sequence number 28 and a heavy chain formed by the amino acid sequence of sequence number 30.

61. The antibody drug conjugate of claim 57, wherein,

the antibody is an antibody comprising a light chain formed by the amino acid sequence of sequence number 31 and a heavy chain formed by the amino acid sequence of sequence number 32, an antibody comprising a light chain formed by the amino acid sequence of sequence number 33 and a heavy chain formed by the amino acid sequence of sequence number 34, or an antibody comprising a light chain formed by the amino acid sequence of sequence number 35 and a heavy chain formed by the amino acid sequence of sequence number 36.

62. A compound represented by the following formula (Ia) or a pharmacologically acceptable salt thereof:

here, L¹Represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino ]1 to 3 groups of the group consisting of ethyl groups are substituted at arbitrary positions, and are selected from the group consisting of the following formulae:

and

herein, R is⁶And R^6’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl,

R⁸and R^8’Each independently represents a hydrogen atom or a halogen atom,

Z⁴represents-CH₂-, -NH-or an oxygen atom,

Z⁵represents a nitrogen atom or-CH ═ CH,

L³represents a hydrogen atom, a halogen atom, -NH₂A hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group,

Q¹and Q^1’Each independently represents a hydroxyl group, a thiol group or a boryl group BH₃－，

Q²And Q^2’Each independently represents an oxygen atom or a sulfur atom,

X¹and X²Each independently represents an oxygen atom, a sulfur atom or-CH₂－，

Y¹And Y²Represents an oxygen atom or-CH₂－，

X³And X⁴Represents a group selected from the following (iii) or (iv):

(iii)Y¹when it is an oxygen atom, X³－X⁴represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or

(iv)Y¹is-CH₂When is, X³－X⁴represents-O-CH₂－，

X⁵And X⁶Represents a group selected from the following (v) or (vi):

(v)Y²When it is an oxygen atom, X⁵－X⁶represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or

(vi)Y²is-CH₂When is, X⁵－X⁶represents-O-CH₂－，

R¹、R²And R³Each independently represents a hydrogen atom, a halogen atom, -OR ', -OC (═ O) R', -N₃NHR ', -NR' R 'or-NHC (═ O) R', wherein R 'represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, said C1-6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl being optionally substituted by 1 to 6 halogen atoms, R' represents C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-C6 cycloalkyl,

W¹represents a nitrogen atom, an oxygen atom, a sulfur atom or-CH-,

W²to representA nitrogen atom or-CH ═ c,

R⁴represents a hydrogen atom, a halogen atom or-NH₂，

R⁵Represents a group selected from the following (vii) to (x):

(vii)W¹when it is a nitrogen atom, R⁵Represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group;

(viii)W¹when it is an oxygen atom, R⁵Is absent;

(ix)W¹when it is a sulfur atom, R⁵Is absent; or

(x)W¹When is-CH-, R⁵Represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂Or C1-C6 alkyl,

here, the asterisk indicates¹In combination, the wavy line indicates bonding to the carbon atom of ═ C-.

63. The compound according to claim 62, or a pharmacologically acceptable salt thereof,

W¹is a nitrogen atom.

64. The compound according to claim 63, or a pharmacologically acceptable salt thereof,

W¹is a nitrogen atom, R⁵Is a hydrogen atom.

65. The compound according to claim 62, or a pharmacologically acceptable salt thereof,

W¹is an oxygen atom.

66. The compound according to claim 62, or a pharmacologically acceptable salt thereof,

W¹is a sulfur atom.

67. The compound according to claim 62, or a pharmacologically acceptable salt thereof,

W¹is-CH-.

68. The compound according to claim 67, or a pharmacologically acceptable salt thereof,

W¹is-CH-, R⁵Is a hydrogen atom.

69. The compound according to any one of claims 62 to 68, or a pharmacologically acceptable salt thereof,

Z¹、Z²and Z³Taken together as-CH₂－CH₂－CH₂-or-CH ═ CH-CH₂－。

70. The compound according to any one of claims 62 to 68, or a pharmacologically acceptable salt thereof,

Z¹、Z²and Z³Taken together as-CH₂－CH(CH₃)－CH₂-or-CH ₂－CH₂－CH(CH₃)－。

71. The compound according to any one of claims 62 to 68, or a pharmacologically acceptable salt thereof,

Z¹、Z²and Z³Taken together as-CH₂－CH₂-R '"-, wherein R'" represents-O-or-CH₂－CH₂－。

72. The compound according to any one of claims 62 to 71, or a pharmacologically acceptable salt thereof,

W²is-CH ═ CH.

73. The compound according to any one of claims 62 to 71, or a pharmacologically acceptable salt thereof,

W²is a nitrogen atom.

74. The compound according to any one of claims 62 to 73, or a pharmacologically acceptable salt thereof,

R⁴represents a hydrogen atom.

75. The compound according to any one of claims 62 to 73, or a pharmacologically acceptable salt thereof,

R⁴represents a fluorine atom.

76. The compound according to any one of claims 62 to 75, or a pharmacologically acceptable salt thereof,

L¹r in (1)⁸And R^8’Each independently a hydrogen atom.

77. The compound according to any one of claims 62 to 76, or a pharmacologically acceptable salt thereof,

L¹is a group selected from the group consisting of:

and

herein, R is⁹And R^9’Represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，

R¹⁰Represents a hydroxyl group, -NH ₂、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH、－CH₂CH₂NHC(＝O)CH₂OH, hydroxy C1-C3 alkyl or amino C1-C3 alkyl,

R¹¹and R^11’Each independently represents a hydrogen atom, a fluorine atom or a methyl group, or R¹¹And R^11’Taken together to represent a cyclopropane ring,

Z⁴represents-CH₂-, -NH-or an oxygen atom.

78. The compound according to any one of claims 62 to 76, or a pharmacologically acceptable salt thereof,

L¹is a group selected from the group consisting of:

and

herein, R is¹³And R^13’Each independently represents a hydrogen atom, a hydroxyl group or-NH₂，

R¹²Represents a hydroxyl group, -NH₂、－CH₂OH、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH or-CH₂CH₂NHC(＝O)CH₂OH，

Z⁴As defined above.

79. The compound according to any one of claims 62 to 76, or a pharmacologically acceptable salt thereof,

L¹is a group selected from the group consisting of:

and

herein, R is¹⁴Represents a hydrogen atom or-NH₂，

R¹⁵Represents a hydrogen atom or-C (═ O) CH₂OH，

R¹⁶Represents a hydroxyl group, -NH₂、－CH₂OH、－CH₂CH₂OH、－CH₂NH₂or-CH₂CH₂NH₂。

80. The compound according to any one of claims 62 to 79, or a pharmacologically acceptable salt thereof,

L³represents a hydrogen atom, a fluorine atom, -NH₂、－CH₂OH or-CH₂NH₂。

81. The compound according to any one of claims 62 to 80, or a pharmacologically acceptable salt thereof,

Q¹and Q^1’Each independently represents a hydroxyl group or a thiol group。

82. The compound according to any one of claims 62 to 81, or a pharmacologically acceptable salt thereof,

X¹And X²Represents an oxygen atom.

83. The compound according to any one of claims 62 to 82, or a pharmacologically acceptable salt thereof,

Y¹and Y²Represents an oxygen atom.

84. The compound according to any one of claims 62 to 83, or a pharmacologically acceptable salt thereof,

X³and X⁴represents-CH₂－O－。

85. The compound according to any one of claims 62 to 84, or a pharmacologically acceptable salt thereof,

X⁵and X⁶represents-CH₂－O－。

86. The compound according to any one of claims 62 to 85, or a pharmacologically acceptable salt thereof,

R¹、R²and R³Each independently a hydrogen atom, a hydroxyl group or a fluorine atom.

87. The compound according to any one of claims 62 to 86, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, the above-mentioned definition,

R¹⁷、R^17’、R¹⁸and R^18’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，

W³represents-NH-, an oxygen atom, a sulfur atom or-CH₂－，

W⁴represents-CH ═ or a nitrogen atom.

88. The compound according to claim 87, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of the following 2 structural formulae:

Here, L¹、Q¹、Q^1’、Q²、Q^2’、R¹⁷、R^17’、R¹⁸And R^18’As defined above.

89. The compound or pharmacologically acceptable salt thereof according to claim 87 or 88, wherein,

the compound is represented by any one of the following 8 structural formulae:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, the above-mentioned definition,

R¹⁹、R^19’、R²⁰and R^20’Each independently represents a hydrogen atom or a fluorine atom.

90. The compound according to any one of claims 87 to 89, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of 4 structural formulas:

here, L¹As defined above.

91. The compound according to any one of claims 87 to 90, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of 4 structural formulas:

here, L¹As defined above.

92. The compound according to any one of claims 87 to 90, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of 4 structural formulas:

here, L¹As defined above.

93. The compound according to any one of claims 62 to 86, or a pharmacologically acceptable salt thereof,

the compound is represented by the formula:

Here, L¹As defined above, the above-mentioned definition,

Q³and Q^3＇Each independently represents a hydroxyl group or a thiol group,

R²¹and R²²Each independently represents a hydroxyl group or a fluorine atom,

W⁵represents an-NH-or sulfur atom.

94. The compound according to claim 93, or a pharmacologically acceptable salt thereof,

the compound is represented by any one of the following 2 structural formulae:

here, L¹、Q³And Q^3＇、W⁵As defined above.

95. The compound according to any one of claims 62 to 94, or a pharmacologically acceptable salt thereof,

L¹represented by any one of the following structural formulae:

96. the compound according to any one of claims 62 to 94, or a pharmacologically acceptable salt thereof,

L¹represented by any one of the following 4 structural formulae:

97. the compound according to any one of claims 93, 94 or 96, or a pharmacologically acceptable salt thereof,

d is represented by any one of the following 4 structural formulae:

here, Q³、Q^3＇And W⁵As defined above.

98. The compound of any one of claims 93, 94, 96 or 97, or a pharmaceutically acceptable salt thereof,

d is represented by any one of the following 4 structural formulae:

99. the compound of any one of claims 93, 94, 96 or 97, or a pharmaceutically acceptable salt thereof,

D is represented by any one of the following 3 structural formulae:

100. the compound of any one of claims 93, 94, 96 or 97, or a pharmaceutically acceptable salt thereof,

d is represented by any one of the following 4 structural formulae:

101. a STING agonist comprising any one selected from the group consisting of an antibody drug conjugate according to claims 1 to 61 and a compound according to claims 62 to 100 or a pharmacologically acceptable salt thereof.

102. A pharmaceutical composition comprising any one selected from the group consisting of the antibody drug conjugate according to claim 1 to 61 and the compound according to claim 62 to 100 or a pharmacologically acceptable salt thereof.

103. An antitumor agent comprising any one selected from the group consisting of the antibody drug conjugate according to claim 1 to 61 and the compound according to claim 62 to 100 or a pharmacologically acceptable salt thereof.

104. The anti-neoplastic agent of claim 103, wherein,

the tumor is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, uterine corpus cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor GIST, gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

105. A method of treating cancer, comprising: administering any one selected from the group consisting of an antibody drug conjugate according to claim 1 to 61, a compound according to claim 62 to 100 or a pharmacologically acceptable salt thereof, a STING agonist according to claim 101, a pharmaceutical composition according to claim 102, and an antitumor agent according to claim 103 or 104.

106. The method of claim 105, wherein,

the cancer is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, uterine corpus cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor GIST, gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

Technical Field

The present invention relates to a cyclic dinucleotide derivative having a novel structure and having STING agonist activity, an antibody drug conjugate obtained by binding the novel cyclic dinucleotide derivative to an antibody against a target cell via a linker, a pharmaceutical composition containing the antibody drug conjugate, and the like.

Background

STING (Stimulator of Interferon Genes: Interferon gene stimulating proteins) is a transmembrane-type adaptor protein (adaptor protein) located in the endoplasmic reticulum (non-patent document 1). STING functions as a central molecule of natural immune activation in mammals and is the first line of entry to protect against pathogens such as bacteria, viruses, and the like. It is known that STING is activated by signals when exogenous and endogenous DNAs are sensed by a plurality of cytoplasmic DNA sensors. cGAS (Cyclic GMP-AMP synthsase: Cyclic GMP-AMP Synthase) is considered as an important DNA sensor among cytoplasmic DNA sensors. When cGAS senses DNA, a cyclic dinucleotide (2 ', 3' -cGAMP) is generated, and the 2 ', 3' -cGAMP directly binds to STING to activate STING (non-patent document 2). The activated STING migrates to the Golgi apparatus where it promotes autophosphorylation of TBK1 (Tank-binding kinase 1: Tank-binding kinase 1). TBK-1 activated by autophosphorylation activates both IRF3(Interferon Regulatory Factor 3) transcription pathway (non-patent document 3) and NF-. kappa.B transcription pathway (non-patent document 4), increasing the production of inflammatory proteins called interferons or cytokines (type I IFN (Interferon: Interferon), IL-6 (Interleukin-6: Interleukin-6), TNF-alpha (Tumor Necrosis Factor-alpha)). These proteins initiate an adaptive immune system comprising T cells that destroy pathogens or cancer cells through a complex cascade (cascade).

According to recent studies it has been shown that: STING not only promotes host defense against microorganisms, but also promotes anti-tumor immunity. For example, when an immunogenic tumor is transplanted into a STING-deficient mouse, the tumor is proliferated rapidly as compared with a wild-type mouse or a TRIF (Toll/Interleukin-1 (IL-1) receptor domain connecting adaptor-inducing interferon- β -receptor domain-containing) deficient mouse. In addition, the mice deficient in STING have spontaneous CD8 for tumors, unlike TLR (Toll-like receptor), MyD88(Myeloid differentiation primary response 88), and MAVS (mitochondrion antiviral signal protein) deficient mice⁺Sensitization of T cells (priming) also disappeared. This suggests that the STING pathway initiated by cytoplasmic DNA sensing is involved in the control of tumor proliferation (non-patent document 5). Other studies have also shown that: STING in radiation therapy (non-patent literature)Document 6) or anti-CD 47 antibody therapy (non-patent document 7). After the DNA from the dead tumor cells treated with the radiation or the anti-CD 47 antibody moves to the cytoplasm of dendritic cells to activate cGAS-STING pathway, IFN production is induced as a mediator of innate immunity and acquired immunity. This study showed that: dendritic cell mediated cross-priming following activation via the STING pathway is important for eliciting adaptive immunity to tumors.

A flavonoid-based low-molecular compound DMXAA, which is widely known as a vascular disrupting agent, has been shown to have strong antitumor activity in a mouse tumor model because it induces macrophage type I IFN (non-patent document 8). DMXAA is expected as an immunotherapeutic agent for non-small cell lung cancer because of its excellent antitumor effect in the preclinical field, but has failed in human clinical trials (non-patent document 9). It is clear from recent studies that DMXAA is a specific agonist for STING in mice, and STING in humans cannot bind because of no species cross-linkage (non-patent document 10). In terms of results, DMXAA was not effective in humans, but studies based on mouse models showed that low molecular drugs intervened in STING to effectively sensitize CD8⁺T cells, can enhance anti-tumor immunity.

As other low molecular compounds, Cyclic Dinucleotides (CDN) show: when administered to tumor-bearing mice, the antitumor immune response is enhanced by STING intervention, and tumor proliferation is significantly inhibited, and the survival rate of the mice can be improved (non-patent document 11). CDNs are classified into CDNs (cyclic-di-GMPs, cyclic-di-AMPs, 3 ', 3' -cGAMP) having 2 standard 3 '-5' phosphate bonds derived from bacteria and mixed-binding CDNs (2 ', 3' -cGAMP) having non-standard 2 '-5' phosphate bonds produced by mammalian cGAS. Recent studies have shown that a hybrid CDN can activate multiple STING more generally than a standard CDN (non-patent document 12).

Natural CDN is not administered in the original form because it is rapidly decomposed by a nucleolytic enzyme in blood like many nucleic acid molecules. Therefore, synthetic low-molecular compounds having STING agonist activity in vivo have been developed (for example, patent documents 1 to 26).

At present, STING agonists MIW-815 (sometimes referred to as ADU-S100, ML RR-S2 CDA or ML-RR-CDA.2Na) as clinical trials for the advancement of antitumor agents⁺) Administered directly into the tumor. The method of directly administering STING agonists into tumors has a problem that only a limited range of the drug can be administered into tumors, and it is difficult to directly administer all of a plurality of distant metastatic tumors, so that treatable tumors are limited. Non-patent document 13 describes that antitumor effect is exhibited by ML RR-S2 CDA administration, but only intratumoral administration, and not antitumor effect by systemic administration (e.g., intravenous administration). Non-patent document 14 describes that the STING agonist SB11285 exhibits an antitumor effect when administered intravenously to a mouse tumor model, but it is not clear what structure SB11285 has specifically been. Patent document 14 describes a conjugate containing an immunostimulatory compound, an antibody structure, and a linker, but does not describe a specific example of a conjugate using a STING agonist as the immunostimulatory compound. Patent document 26 describes a conjugate in which a CDN having a specific structure and an antibody are bound via a linker, but does not describe an example in which the conjugate is administered in vivo (in vivo), and does not confirm the antitumor effect of the conjugate.

Documents of the prior art

Patent document

Patent document 1: WO2014/099824

Patent document 2: WO2014/179335

Patent document 3: WO2014/189805

Patent document 4: WO2014/189806

Patent document 5: WO2015/074145

Patent document 6: WO2015/185565

Patent document 7: WO2016/096714

Patent document 8: WO2016/012305

Patent document 9: WO2016/145102

Patent document 10: WO2017/027646

Patent document 11: WO2017/027645

Patent document 12: WO2017/075477

Patent document 13: WO2017/093933

Patent document 14: WO2017/100305

Patent document 15: WO2017/123669

Patent document 16: WO2017/161349

Patent document 17: WO2017/175147

Patent document 18: WO2017/175156

Patent document 19: WO2018/009466

Patent document 20: WO2018/045204

Patent document 21: WO2018/060323

Patent document 22: WO2018/067423

Patent document 23: WO2018/065360

Patent document 24: WO2014/093936

Patent document 25: WO2018/009648

Patent document 26: WO2018/100558

Non-patent document

Non-patent document 1: nature 2008,455,674-678

Non-patent document 2: cell,2013,51,226-235

Non-patent document 3: science 2015a,347, aaa2630

Non-patent document 4: J.Virol.2014,88, 5328-one 5341

Non-patent document 5: immunity 2014,41,830-842

Non-patent document 6: immunity 2014,41, 843-852-

Non-patent document 7: nat. Med.2015,21,1209-

Non-patent document 8: J.Immunol.1994,153,4684-4693

Non-patent document 9: J.Clin.Oncol.2011,29,2965-

Non-patent document 10: J.Immunol.2013,190,5216-5225

Non-patent document 11: sci. Rep.2016,6,19049

Non-patent document 12: cell,2015,59,891-903

Non-patent document 13: cell Rep.2015,11,1018-

Non-patent document 14: AACR Tumor Immunology and Immunotherapy,2017, Poster # A25

Disclosure of Invention

Problems to be solved by the invention

It is desired to develop CDN derivatives having a novel backbone that have STING agonist activity, activate immune cells by increasing the production of inflammatory proteins such as interferon or cytokine (cytokine), and therapeutic agents and/or therapeutic methods for diseases associated with STING agonist activity using the novel CDN derivatives, such as diseases that can be treated based on immune activation (e.g., cancer). In addition, it is desired to develop an antibody drug conjugate in which the novel CDN derivative and an antibody against a target cell are bound via a linker, which can be systemically administered and which can specifically deliver a STING agonist to the target cell or organ (e.g., tumor site), and a therapeutic agent and/or a therapeutic method for a disease associated with the STING agonist activity using the antibody drug conjugate, for example, a disease (e.g., cancer) that can be treated by immune activation.

Means for solving the problems

The present inventors have found that, in order to solve the above-mentioned technical problems, a novel CDN derivative characterized by having a fused tricyclic substituent group and found that the novel CDN derivative has a strong STING agonist activity and exhibits a strong antitumor activity. The present inventors have also found an antibody drug conjugate obtained by binding the novel CDN derivative of the present invention to an antibody via a linker, and have found that when the antibody drug conjugate is administered systemically, an antitumor effect is exhibited in a tumor expressing an antigen, thereby completing the present invention.

Namely, the present invention is as follows.

[1] An antibody drug conjugate represented by the following formula (II):

in the formula, m¹1 to 10, Ab represents an antibody or a functional fragment of the antibody, the sugar chain of the antibody is optionally modified (remodelling), L represents a linker linking Ab to D, Ab is optionally directly bound to L from the amino acid residue thereof, or bound to L from the sugar chain of Ab or the modified sugar chain, and D represents a compound represented by the following formula (I):

here, L and L¹Or L²any-NH contained therein₂Or a hydroxyl group is bonded, L¹Represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino ]1 to 3 groups of the group consisting of ethyl groups are substituted at arbitrary positions, and are selected from the group consisting of the following formulae:

and

herein, R is⁶And R^6’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, R⁷And R^7’Each independently represents a hydrogen atom or a C1-C6 alkyl group, which C1-C6 alkyl group may be optionally substituted by 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group, R⁸And R^8’Each independently represents a hydrogen atom or a halogen atom, Z⁴represents-CH₂-, -NH-or an oxygen atom, Z⁵Represents a nitrogen atom or-CH ═ L²Represents a group selected from the following (i) or (ii): (i) l is²In combination with L, L²represents-NHR ', hydroxyl C1-C6 alkyl or amino C1-C6 alkyl, wherein R' represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl can be optionally substituted by 1-6 halogen atoms; or (ii) L²When not bound to L, L²Represents a hydrogen atom or a halogen atom, Q¹And Q^1’Each independently represents a hydroxyl group, a thiol group or a boryl group (BH)₃－)，Q²And Q^2’Each independently represents an oxygen atom or a sulfur atom, X ¹And X²Each independently represents an oxygen atom, a sulfur atom or-CH₂－，Y¹And Y²Represents an oxygen atom or-CH₂－，X³And X⁴Represents a group selected from the following (iii) or (iv): (iii) y is¹When it is an oxygen atom, X³－X⁴represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (iv) Y¹is-CH₂When is, X³－X⁴represents-O-CH₂－，X⁵And X⁶Represents a group selected from the following (v) or (vi): (v) y is²When it is an oxygen atom, X⁵－X⁶represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (vi) Y²is-CH₂When is, X⁵－X⁶represents-O-CH₂－，R¹、R²And R³Each independently represents a hydrogen atom, a halogen atom, -OR ', -OC (═ O) R', -N₃-NHR ', -NR' R "or-NHC (═ O) R ', wherein R' is as defined hereinbefore, R" represents C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, W is¹Represents a nitrogen atom, an oxygen atom, a sulfur atom or-CH-, W²Represents a nitrogen atom or-CH ═ R⁴Represents a hydrogen atom, a halogen atom or-NH₂，R⁵Represents a group selected from the following (vii) to (x):(vii)W¹when it is a nitrogen atom, R⁵Represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group; (viii) w¹When it is an oxygen atom, R⁵Is absent; (ix) w¹When it is a sulfur atom, R⁵Is absent; or (x) W¹When is-CH-, R⁵Represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH ₂Or C1-C6 alkyl, Z¹－Z²－Z³Taken together represent a group selected from: -CH₂－CH₂－CH₂－、－CH₂－CH₂－R”’－、－CH＝CH－CH₂－、－CH＝CX－CH₂－、－CX＝CH－CH₂－、－CX＝CX－CH₂－、－C(＝O)－CH₂－CH₂－、－CH₂－CH₂－C(＝O)－、－CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃) -or a group of any of the formulae, wherein R' "represents-O-or-CH₂－CH₂-, X represents a halogen atom.

Here, the asterisk indicates¹In combination, the wavy line indicates bonding to the carbon atom of ═ C-.

[2]According to [1]The antibody drug conjugate of (A), wherein W¹Is a nitrogen atom.

[3]According to [2 ]]The antibody drug conjugate of (A), wherein W¹Is a nitrogen atom, R⁵Is a hydrogen atom.

[4]According to [1]The antibody drug conjugate of (A), wherein W¹Is an oxygen atom.

[5]According to [1]The antibody drug conjugate of (A), wherein W¹Is a sulfur atom.

[6]According to [1]The antibody drug conjugate of (A), wherein W¹is-CH-.

[7]According to [6]The antibodyA bulk drug conjugate wherein W¹is-CH-, R⁵Is a hydrogen atom.

[8]According to [1]～[7]The antibody drug conjugate according to any one of the above, wherein Z¹、Z²And Z³Taken together as-CH₂－CH₂－CH₂-or-CH ═ CH-CH₂－。

[9]According to [1]～[7]The antibody drug conjugate according to any one of the above, wherein Z¹、Z²And Z³Taken together as-CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃)－。

[10]According to [1]～[7]The antibody drug conjugate according to any one of the above, wherein Z¹、Z²And Z³Taken together as-CH ₂－CH₂-R '"- (wherein R'" represents-O-or-CH)₂－CH₂－)。

[11]According to [1]～[10]The antibody drug conjugate according to any one of the above items, wherein W is²is-CH ═ CH.

[12]According to [1]～[10]The antibody drug conjugate according to any one of the above items, wherein W is²Is a nitrogen atom.

[13]According to [1]～[12]The antibody drug conjugate according to any one of the above items, wherein R is⁴Represents a hydrogen atom.

[14]According to [1]～[12]The antibody drug conjugate according to any one of the above items, wherein R is⁴Represents a fluorine atom.

[15]According to [1]～[14]The antibody drug conjugate according to any one of the above items, wherein L¹R in (1)⁸And R^8’Each independently a hydrogen atom.

[16]According to [1]～[15]The antibody drug conjugate according to any one of the above items, wherein L¹Is a group selected from the group consisting of:

and

herein, R is⁹And R^9’Represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，R¹⁰Represents a hydroxyl group, -NH₂、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH、－CH₂CH₂NHC(＝O)CH₂OH, hydroxy C1-C3 alkyl or amino C1-C3 alkyl, R¹¹And R^11’Each independently represents a hydrogen atom, a fluorine atom or a methyl group, or R¹¹And R^11’Combined to represent cyclopropane, Z⁴represents-CH₂-, -NH-or an oxygen atom.

[17]According to [1]～[15]The antibody drug conjugate according to any one of the above items, wherein L¹Is a group selected from the group consisting of:

and

herein, R is ¹³And R^13’Each independently represents a hydrogen atom, a hydroxyl group or-NH₂，R¹²Represents a hydroxyl group, -NH₂、－CH₂OH、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH or-CH₂CH₂NHC(＝O)CH₂OH，Z⁴As defined above.

[18]According to [1]～[15]The antibody drug conjugate according to any one of the above items, wherein L¹Is a group selected from the group consisting of:

and

herein, R is¹⁴Represents a hydrogen atom or-NH₂，R¹⁵Represents a hydrogen atom or-C (═ O) CH₂OH，R¹⁶Represents a hydroxyl group, -NH₂、－CH₂OH、－CH₂CH₂OH、－CH₂NH₂or-CH₂CH₂NH₂。

[19]According to [1]～[18]The antibody drug conjugate according to any one of the above items, wherein L²Combined with L to represent-NH₂、－CH₂NH₂or-CH₂OH。

[20]According to [1]～[18]The antibody drug conjugate according to any one of the above items, wherein L²Is not bonded to L and represents a hydrogen atom or a fluorine atom.

[21]According to [1]～[20]The antibody drug conjugate according to any one of the above, wherein Q¹And Q¹' independently represent a hydroxyl group or a thiol group, respectively.

[22]According to [1]～[21]The antibody drug conjugate according to any one of the above items, wherein X is¹And X²Represents an oxygen atom.

[23]According to [1]～[22]The antibody drug conjugate according to any one of the above, wherein Y is¹And Y²Represents an oxygen atom.

[24]According to [1]～[23]The antibody drug conjugate according to any one of the above items, wherein X is³And X⁴represents-CH₂－O－。

[25]According to [1]～[24]The antibody drug conjugate according to any one of the above items, wherein X is⁵And X⁶represents-CH₂－O－。

[26]According to [1]～[25]The antibody drug conjugate according to any one of the above items, wherein R is ¹、R²And R³Each independently a hydrogen atom, a hydroxyl group or a fluorine atom.

[27] The antibody drug conjugate according to any one of [1] to [26], wherein D is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, R¹⁷、R^17’、R¹⁸And R^18’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，W³represents-NH-, an oxygen atom, a sulfur atom or-CH₂－，W⁴represents-CH ═ or a nitrogen atom.

[28] The antibody drug conjugate according to [27], wherein D is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²、Q^2’、R¹⁷、R^17’、R¹⁸And R^18’As defined above.

[29] The antibody drug conjugate according to [27] or [28], wherein D is represented by any one of the following 8 structural formulae:

here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, R¹⁹、R^19’、R²⁰And R^20’Each independently represents a hydrogen atom or a fluorine atom.

[30] The antibody drug conjugate according to any one of [27] to [29], wherein D is represented by any one of 4 structural formulae below:

here, L¹As defined above.

[31] The antibody drug conjugate according to any one of [27] to [30], wherein D is represented by any one of 4 structural formulae below:

Here, L¹As defined above.

[32] The antibody drug conjugate according to any one of [27] to [30], wherein D is represented by any one of 4 structural formulae below:

here, L¹As defined above.

[33] The antibody drug conjugate according to any one of [1] to [26], wherein D is represented by the following formula:

here, L¹As defined above, Q³And Q^3＇Each independently represents a hydroxyl group or a thiol group, R²¹And R²²Each independently represents a hydroxyl group or a fluorine atom, W⁵Represents an-NH-or sulfur atom.

[34] The antibody drug conjugate according to [33], wherein D is represented by any one of the following 2 structural formulae:

here, L¹、Q³And Q^3＇、W⁵As defined above.

[35]According to [1]～[34]The antibody drug conjugate according to any one of the above items, wherein L¹Represented by any one of the following 4 structural formulae:

[36]according to [1]～[34]The antibody drug conjugate according to any one of the above items, wherein L¹Represented by any one of the following 4 structural formulae:

in the formula, an asterisk indicates the binding to L.

[37] The antibody drug conjugate according to any one of [33], [34] or [36], wherein D is represented by any one of 4 structural formulae below:

here, the asterisk indicates the bond to L, Q ³、Q^3＇And W⁵As defined above.

[38] The antibody drug conjugate according to any one of [33], [34], [36] or [37], wherein D is represented by any one of 4 structural formulae below:

here, an asterisk indicates binding to L.

[39] The antibody drug conjugate according to any one of [33], [34], [36] or [37], wherein D is represented by any one of 3 structural formulae below:

here, an asterisk indicates binding to L.

[40] The antibody drug conjugate according to any one of [33], [34], [36] or [37], wherein D is represented by any one of 4 structural formulae below:

here, an asterisk indicates binding to L.

[41]According to [1]～[40]The antibody drug conjugate according to any one of the above items, wherein the linker L is represented by-Lb-La-Lp-Lc-, wherein an asterisk indicates that the drug D is bound thereto, Lp indicates that the linker formed by an amino acid sequence that can be cleaved in a target cell is present or absent, and La indicates any one selected from the group consisting of: -C (═ O) - (CH)₂CH₂)n²－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－CH₂－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－CH₂－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂O)n³－CH₂－C(＝O)－、－(CH₂)n⁴-O-C (═ O) -, and- (CH)₂)n⁹－C(O) -, where n²Represents an integer of 1 to 3, n³Represents an integer of 1 to 5, n⁴Represents an integer of 0 to 2, n⁹Represents an integer of 2 to 7, Lb represents a spacer for bonding La to the sugar chain or the reconstructed sugar chain of Ab or a spacer for bonding La to the cysteine residue of Ab, and Lc represents-NH-CH ₂-, -NH-phenyl-CH₂-O (C ═ O) -or-NH-heteroaryl-CH₂-O (C ═ O) -, or is absent.

[42] The antibody drug conjugate according to [41], wherein Lc is absent.

[43]According to [ 41)]The antibody drug conjugate is characterized in that Lc is-NH-CH₂－。

[44] The antibody-drug conjugate according to any one of [41] to [43], wherein Lp represents any one selected from the group consisting of: -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-, and-ICit-.

[45] The antibody-drug conjugate according to [44], wherein Lp is any one of-GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-or-FCit-.

[46] The antibody-drug conjugate according to any one of [41] to [43], wherein Lp is any one of-GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-or-GGPP-.

[47] The antibody-drug conjugate according to [46], wherein Lp is-GGFG-or-GGPI-.

[48]According to [ 41)]～[47]The antibody drug conjugate according to any one of the preceding claims, wherein La represents any one selected from the group consisting of: -C (═ O) -CH₂CH₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₃－CH₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₄－CH₂-C (═ O) -, and- (CH)₂)₅－C(＝O)－。

[49] The antibody drug conjugate according to any one of [41] to [48], wherein Lb is represented by any one of the following structural formulae:

or the like, or, alternatively,

in the structural formula of Lb described above, asterisks indicate the binding to La, wavy lines indicate the binding to sugar chains or restructured sugar chains of Ab.

[50] The antibody drug conjugate according to any one of [41] to [48], wherein Lb is- (succinimid-3-yl-N) -, wherein- (succinimid-3-yl-N) -represents the following structural formula:

here, the asterisk indicates that La is bonded, and the wavy line indicates that the side chain of the cysteine residue of the antibody is bonded via thioether formation.

[51]According to [ 41)]、[46]～[49]The antibody-drug conjugate according to any one of the above items, wherein the linker L is represented by-Lb-La-Lp-Lc-, wherein the asterisk indicates that the linker is bonded to the drug D, and Lp is-GGFG-, or-GGPI-, and La represents-C (═ O) -CH₂CH₂-C (═ O) -, Lb represents the formula:

in the structural formula of Lb shown above, the asterisk indicates the binding to La, and the wavy line indicates the binding to Ab Is bonded to the sugar chain or the reconstructed sugar chain of (2), Lc represents-NH-CH₂－。

[52] The antibody-drug conjugate according to any one of [1] to [51], wherein the average number of drug-binding molecules per antibody in the antibody-drug conjugate is in the range of 1 to 10.

[53] The antibody drug conjugate according to [52], wherein the average number of drug-binding molecules per antibody in the antibody drug conjugate is in the range of 1 to 5.

[54] The antibody drug conjugate according to any one of [1] to [53], wherein the antibody binds to L from a sugar chain (N297 sugar chain) bound to Asn297 of the antibody.

[55] The antibody drug conjugate according to [54], wherein the N297 sugar chain is a reconstituted sugar chain.

[56] The antibody drug conjugate according to [54] or [55], wherein the N297 sugar chain is N297- (Fuc) MSG1 or N297- (Fuc) SG.

[57] The antibody drug conjugate according to any one of [1] to [56], wherein the antibody is an anti-HER 2 antibody, an anti-HER 3 antibody, an anti-DLL 3 antibody, an anti-FAP antibody, an anti-CDH 11 antibody, an anti-CDH 6 antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-CD 19 antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 98 antibody, an anti-TROP 2 antibody, an anti-CEA antibody, an anti-Criptto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUC 1 antibody, an anti-GPNMB antibody, an anti-Integrin (Integrin) antibody, an anti-PSMA antibody, an anti-Tenascin-C (Tenascin-C) antibody, an anti-HER 44A4 antibody, an anti-Mesothelin (CD Mesothelin) antibody, an anti-SLC antibody, an anti-GPR 20 antibody, or an anti-EGFR antibody.

[58] The antibody drug conjugate according to [57], wherein the antibody is an anti-HER 2 antibody.

[59] The antibody drug conjugate according to [58], wherein the antibody comprises a light chain formed from the amino acid sequence of SEQ ID NO. 1 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 2, or comprises a light chain formed from the amino acid sequence of SEQ ID NO. 1 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 3.

[60] The antibody drug conjugate according to [58], wherein the antibody comprises a light chain formed from the amino acid sequence of SEQ ID NO. 28 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 29, or comprises a light chain formed from the amino acid sequence of SEQ ID NO. 28 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 30.

[61] The antibody drug conjugate according to [57], wherein the antibody is an antibody comprising a light chain formed from the amino acid sequence of SEQ ID NO. 31 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 32, an antibody comprising a light chain formed from the amino acid sequence of SEQ ID NO. 33 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 34, or an antibody comprising a light chain formed from the amino acid sequence of SEQ ID NO. 35 and a heavy chain formed from the amino acid sequence of SEQ ID NO. 36.

[62] A compound represented by the following formula (Ia):

here, L¹Represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino]1 to 3 groups of the group consisting of ethyl groups are substituted at arbitrary positions, and are selected from the group consisting of the following formulae:

and

herein, R is⁶And R^6’Are respectively provided withIndependently represent a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, R⁷And R^7’Each independently represents a hydrogen atom or a C1-C6 alkyl group, which C1-C6 alkyl group may be optionally substituted by 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group, R⁸And R^8’Each independently represents a hydrogen atom or a halogen atom, Z⁴represents-CH₂-, -NH-or an oxygen atom, Z⁵Represents a nitrogen atom or-CH ═ L³Represents a hydrogen atom, a halogen atom, -NH₂Hydroxy C1-C3 alkyl or amino C1-C3 alkyl, Q¹And Q^1’Each independently represents a hydroxyl group, a thiol group or a boryl group (BH)₃－)，Q²And Q^2’Each independently represents an oxygen atom or a sulfur atom, X¹And X²Each independently represents an oxygen atom, a sulfur atom or-CH₂－，Y¹And Y ²Represents an oxygen atom or-CH₂－，X³And X⁴Represents a group selected from the following (iii) or (iv): (iii) y is¹When it is an oxygen atom, X³－X⁴represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (iv) Y¹is-CH₂When is, X³－X⁴represents-O-CH₂－，X⁵And X⁶Represents a group selected from the following (v) or (vi): (v) y is²When it is an oxygen atom, X⁵－X⁶represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (vi) Y²is-CH₂When is, X⁵－X⁶represents-O-CH₂－，R¹、R²And R³Each independently represents a hydrogen atom, a halogen atom, -OR ', -OC (═ O) R', -N₃-NHR ', -NR ' R ' or-NHC (═ O) R ' (R ' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group, the C1-6 alkaneThe group, C2-C6 alkenyl or C2-C6 alkynyl may be optionally substituted by 1 to 6 halogen atoms, R 'represents C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-C6 cycloalkyl, W' represents a C3-C6 cycloalkyl group¹Represents a nitrogen atom, an oxygen atom, a sulfur atom or-CH-, W²Represents a nitrogen atom or-CH ═ R⁴Represents a hydrogen atom, a halogen atom or-NH₂，R⁵Represents a group selected from the following (vii) to (x): (vii) w¹When it is a nitrogen atom, R⁵Represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group; (viii) w¹When it is an oxygen atom, R ⁵Is absent; (ix) w¹When it is a sulfur atom, R⁵Is absent; or (x) W¹When is-CH-, R⁵Represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂Or C1-C6 alkyl, Z¹－Z²－Z³Taken together represent a group selected from: -CH₂－CH₂－CH₂－、－CH₂－CH₂－R”’－、－CH＝CH－CH₂－、－CH＝CX－CH₂－、－CX＝CH－CH₂－、－CX＝CX－CH₂－、－C(＝O)－CH₂－CH₂－、－CH₂－CH₂－C(＝O)－、－CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃) -or a group of any of the formulae, wherein R' "represents-O-or-CH₂－CH₂-, X represents a halogen atom.

Here, the asterisk indicates¹In combination, the wavy line indicates bonding to the carbon atom of ═ C-.

[63]According to [ 62)]The compound or a pharmacologically acceptable salt thereof, wherein W¹Is a nitrogen atom.

[64]According to [ 63)]The compound or a pharmacologically acceptable salt thereof, wherein W¹Is a nitrogen atom, R⁵Is a hydrogen atom.

[65]According to [ 62)]The compound or a pharmacologically acceptable salt thereof, wherein W¹Is an oxygen atom.

[66]According to [ 62)]The compound or a pharmacologically acceptable salt thereof, wherein W¹Is a sulfur atom.

[67]According to [ 62)]The compound or a pharmacologically acceptable salt thereof, wherein W¹is-CH-.

[68]According to [67 ]]The compound or a pharmacologically acceptable salt thereof, wherein W¹is-CH-, R⁵Is a hydrogen atom.

[69]According to [ 62)]～[68]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein Z ¹、Z²And Z³Taken together as-CH₂－CH₂－CH₂-or-CH ═ CH-CH₂－。

[70]According to [ 62)]～[68]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein Z¹、Z²And Z³Taken together as-CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃)－。

[71]According to [ 62)]～[68]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein Z¹、Z²And Z³Taken together as-CH₂－CH₂-R '- (R' represents-O-or-CH)₂－CH₂－)。

[72]According to [ 62)]～[71]The compound of any one of the above, or a pharmacologically acceptable salt thereof, wherein W²is-CH ═ CH.

[73]According to [ 62)]～[71]The compound of any one of the above, or a pharmacologically acceptable salt thereof, wherein W²Is a nitrogen atom.

[74]According to [ 62)]～[73]The compound of any one of above or a pharmacologically acceptable salt thereof, wherein R⁴Represents a hydrogen atom.

[75]According to [ 62)]～[73]The compound of any one of or the compound of any one ofA pharmacologically acceptable salt thereof, wherein R⁴Represents a fluorine atom.

[76]According to [ 62)]～[75]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L¹R in (1)⁸And R^8’Each independently a hydrogen atom.

[77]According to [ 62)]～[76]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L¹Is a group selected from the group consisting of:

and

herein, R is⁹And R^9’Represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH ₂，R¹⁰Represents a hydroxyl group, -NH₂、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH、－CH₂CH₂NHC(＝O)CH₂OH, hydroxy C1-C3 alkyl or amino C1-C3 alkyl, R¹¹And R^11’Each independently represents a hydrogen atom, a fluorine atom or a methyl group, or R¹¹And R^11’Combined to represent cyclopropane, Z⁴represents-CH₂-, -NH-or an oxygen atom.

[78]According to [ 62)]～[76]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L¹Is a group selected from the group consisting of:

and

herein, R is¹³And R^13’Each independently represents a hydrogen atom, a hydroxyl group or-NH₂，R¹²Represents a hydroxyl group, -NH₂、－CH₂OH、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH or-CH₂CH₂NHC(＝O)CH₂OH，Z⁴As defined above.

[79]According to [ 62)]～[76]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L¹Is a group selected from the group consisting of:

and

[80]According to [ 62)]～[79]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L³Represents a hydrogen atom, a fluorine atom, -NH₂、－CH₂OH or-CH₂NH₂。

[81]According to [ 62)]～[80]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein Q¹And Q^1’Each independently represents a hydroxyl group or a thiol group.

[82]According to [ 62)]～[81]Any one ofA compound or a pharmacologically acceptable salt thereof, wherein X ¹And X²Represents an oxygen atom.

[83]According to [ 62)]～[82]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein Y¹And Y²Represents an oxygen atom.

[84]According to [ 62)]～[83]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein X³And X⁴represents-CH₂－O－。

[85]According to [ 62)]～[84]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein X⁵And X⁶represents-CH₂－O－。

[86]According to [ 62)]～[85]The compound of any one of above or a pharmacologically acceptable salt thereof, wherein R¹、R²And R³Each independently a hydrogen atom, a hydroxyl group or a fluorine atom.

[87] The compound according to any one of [62] to [86], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of the following 2 structural formulae:

[88] The compound according to [87], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of the following 2 structural formulae:

here, L¹、Q¹、Q^1’、Q²、Q^2’、R¹⁷、R^17’、R¹⁸And R^18’As defined above.

[89] The compound according to [87] or [88], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of the following 8 structural formulae:

Here, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, R¹⁹、R^19’、R²⁰And R^20’Each independently represents a hydrogen atom or a fluorine atom.

[90] The compound according to any one of [87] to [89], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of 4 structural formulae below:

here, L¹As defined above.

[91] The compound according to any one of [87] to [90], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of 4 structural formulae below:

here, L¹As defined above.

[92] The compound according to any one of [87] to [90], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of 4 structural formulae below:

here, L¹As defined above.

[93] The compound according to any one of [62] to [86], or a pharmacologically acceptable salt thereof, wherein the compound is represented by the following formula:

[94] The compound according to [93], or a pharmacologically acceptable salt thereof, wherein the compound is represented by any one of the following 2 structural formulae:

Here, L¹、Q³And Q^3＇、W⁵As defined above.

[95]According to [ 62)]～[94]The compound of any one of them or a pharmacologically acceptable salt thereof, wherein L¹Represented by any one of the following structural formulae:

[96]according to [ 62)]～[94]A compound of any one of them, or a pharmacologically acceptable salt thereof, L¹Represented by any one of the following 4 structural formulae:

[97] the compound according to any one of [93], [94] or [96], or a pharmacologically acceptable salt thereof, wherein D is represented by any one of 4 structural formulae below:

here, Q³、Q^3＇And W⁵As defined above.

[98] The compound according to any one of [93], [94], [96] or [97], or a pharmacologically acceptable salt thereof, wherein D is represented by any one of the following 4 structural formulae:

[99] the compound according to any one of [93], [94], [96] or [97], or a pharmacologically acceptable salt thereof, wherein D is represented by any one of 3 structural formulae below:

[100] the compound according to any one of [93], [94], [96] or [97], or a pharmacologically acceptable salt thereof, wherein D is represented by any one of the following 4 structural formulae:

[101] a STING agonist comprising any one selected from the group consisting of the antibody drug conjugates according to [1] to [61] and the compounds according to [62] to [100] or pharmacologically acceptable salts thereof.

[102] A pharmaceutical composition comprising any one selected from the group consisting of the antibody drug conjugate according to any one of [1] to [61] and the compound according to any one of [62] to [100] or a pharmacologically acceptable salt thereof.

[103] An antitumor agent comprising any one selected from the group consisting of the antibody drug conjugates according to [1] to [61] and the compounds according to [62] to [100] or pharmaceutically acceptable salts thereof.

[104] The antitumor agent according to [103], wherein the tumor is lung cancer, kidney cancer, urothelial cancer, large intestine cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, uterine corpus cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma;

[105] a method of treating cancer, comprising: administering any one selected from the group consisting of the antibody drug conjugates of [1] to [61], the compounds of [62] to [100] or pharmaceutically acceptable salts thereof, the STING agonists of [101], the pharmaceutical compositions of [102], and the antitumor agents of [103] or [104 ].

[106] The method of [105], wherein the cancer is lung cancer, kidney cancer, urothelial cancer, large intestine cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, uterine corpus cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

Effects of the invention

The invention provides a novel CDN derivative. The novel CDN derivative of the present invention has a strong STING agonist activity and shows a high antitumor activity. In addition, the present invention also provides a novel antibody CDN derivative conjugate which can be administered systemically and shows an anti-tumor effect in a tumor expressing an antigen.

Drawings

Fig. 1 schematically shows a drug conjugate (molecule (II) in fig. 1) obtained from an SG-type sugar chain reshaped antibody and a drug conjugate (molecule (II) in fig. 1B) obtained from an MSG-type sugar chain reshaped antibody, which are drug conjugates (molecule (II) in fig. 1) of the present invention. (a) The drug D (b) represents a linker L (c) represents a PEG linker (L (PEG)), (D) represents an N297 sugar chain (here, white circles represent NeuAc (sia), white hexagons represent Man, black hexagons represent GlcNAc, white diamonds represent Gal, and white inverted triangles represent Fuc). The white pentagons represent the triazole rings formed by the reaction of the alkyne from linker L with the azide from the PEG linker. Antibody Ab is shown in Y-letter form. The PEG linker is linked to the carboxyl group at position 2 of the sialic acid at the non-reducing terminus via an amide bond. Unless otherwise specified, this expression method is applied to all the embodiments in the present specification.

Fig. 2 is a schematic diagram showing the structures of a (Fuc α 1, 6) GlcNAc-antibody (molecule of (III) in fig. 2 a), an SG-type sugar chain reconstituted antibody (molecule of (IV) in fig. 2B), and an MSG-type sugar chain reconstituted antibody (molecule of (IV) in fig. 2C), which are intermediates for producing the drug conjugate of the present invention. In all figures, the letter "Y" indicates the antibody Ab as in FIG. 1. In fig. 2 a, (e) represents an N297 sugar chain composed of a disaccharide having an α -glycoside bonded to the 1-position of Fuc and the 6-position of GlcNAc. In fig. 2B and C, (d) represents an N297 sugar chain similar to that in fig. 1, and (f) represents a PEG linker having an azide group and represents an azide group provided at the end to bond to the linker L. The PEG linker with azido groups is attached in the same manner as the PEG linker of FIG. 1.

FIG. 3 is a schematic diagram of a process for producing an SG type sugar chain reconstituted antibody and an MSG type sugar chain reconstituted antibody from an antibody produced in an animal cell. The molecules (III) and (IV) in the figure represent (Fuc. alpha.1, 6) GlcNAc-antibody and SG-type sugar chain-modified antibody or MSG-type sugar chain-modified antibody, respectively, as in FIG. 2. The molecule (V) is an antibody produced in an animal cell, and is a mixture of molecules having heterogeneous N297 sugar chains. A in FIG. 3 shows a step of preparing a uniform (Fuc. alpha.1, 6) GlcNAc-antibody (III) by treating the non-uniform N297 sugar chains of (V) with a hydrolase such as Endos. FIG. 3B shows a step of preparing an SG-type sugar chain-reconstituted antibody (IV) by sugar chain transfer of an SG-type sugar chain donor molecule to GlcNAc of N297 sugar chain of antibody (III) using a glycosyltransferase such as Endos D233Q/Q303L variant. In FIG. 3, C is a step of preparing the MSG-type sugar chain reconstructed antibody of (IV) by subjecting the MSG-type sugar chain donor molecule to sugar chain transfer with respect to the antibody (III), similarly to B in FIG. 3. The SG-type sugar chain donor molecule and the MSG-type sugar chain donor molecule used herein were each modified by a PEG linker having an azide group for sialic acid at a non-reducing end, and the SG-type N297 sugar chain recombinant antibody and the MSG-type N297 sugar chain recombinant antibody prepared were similarly modified for sialic acid at a non-reducing end as shown in B and C in fig. 2.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 1) of the light chain and the amino acid sequence (SEQ ID NO: 2) of the heavy chain of Trastuzumab (Trastuzumab).

Fig. 5 shows the amino acid sequence of the light chain (seq id No. 1) and the amino acid sequence of the heavy chain (seq id No. 3) of the engineered anti-HER 2 antibody.

FIG. 6 shows (a) the amino acid sequence of human STING wild-type, (b) the amino acid sequence of human STING REF variant (R232H), and (c) the amino acid sequence of human STING HAQ variants (R71H, G230A, R293Q).

Figure 7 shows the antitumor effect of the CDN derivative based intratumoral administration. In the figure, black square lines indicate vehicle (vehicle) groups, white square lines indicate compound number 6a administration groups, white inverted triangular lines indicate compound number 8b administration groups, and white circular lines indicate compound number 9b administration groups. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 8 shows the antitumor effect based on intravenous administration of anti-HER 2 antibody-CDN conjugate (1) and anti-LPS antibody-CDN conjugate (1). In the figure, the black square line represents the vehicle group, and the white triangle line represents the modified anti-HER 2 antibody prepared in reference example 1 andthe group to which the anti-HER 2 antibody-CDN conjugate (1) obtained by coupling the compound of example 8b was administered is indicated by a black triangle line, and the group to which the anti-LPS antibody-CDN conjugate (1) obtained by coupling the modified anti-LPS antibody prepared in reference example 2 and the compound of example 8b was administered is indicated by a black triangle line. The vertical axis represents the tumor volume (mm) ³) The horizontal axis represents the number of days after tumor implantation.

Figure 9 shows the antitumor effect based on intravenous administration of anti-HER 2 antibody-CDN conjugates (2) and (3). In the figure, black square lines indicate the vehicle group, white square lines indicate the group to which the anti-HER 2 antibody-CDN conjugate (2) was administered, and white triangular lines indicate the group to which the anti-HER 2 antibody-CDN conjugate (3) was administered. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 10 shows the antitumor effect based on intravenous administration of anti-HER 2 antibody-CDN conjugate (19). The black square line in the figure indicates the vehicle group, and the white triangle line indicates the group to which the anti-HER 2 antibody-CDN conjugate (19) was administered. anti-HER 2 antibody-CDN conjugate (19) is a drug linker conjugated to an antibody with cysteine conjugation. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Fig. 11 shows the antitumor effect of intravenous administration based on anti-HER 2 antibody-CDN conjugates (1) and (9) to (12). In the figure, black squares indicate vehicle groups, white triangles indicate anti-HER 2 antibody-CDN conjugate (9) administration groups, white inverted triangles indicate anti-HER 2 antibody-CDN conjugate (10) administration groups, white diamonds indicate anti-HER 2 antibody-CDN conjugate (11) administration groups, white circles indicate anti-HER 2 antibody-CDN conjugate (12) administration groups, and white squares indicate anti-HER 2 antibody-CDN conjugate (1) administration groups. anti-HER 2 antibody-CDN conjugates (9), (10), (11), (12), (1) were conjugated with different linkers to the compound of example 8b, respectively. The vertical axis represents the tumor volume (mm) ³) The horizontal axis represents the number of days after tumor implantation.

Figure 12 shows the anti-tumor effect based on intravenous administration of anti-HER 2 antibody 2-CDN conjugate (1), anti-HER 2 antibody 2 and compound No. 8 b. The black square line in the figure represents the vehicle group and the white triangle line represents the 60 μ g administration of the anti-HER 2 antibody 2-CDN conjugate (1)In the drug combination, the black inverted triangle line indicates the 59. mu.g administration group of anti-HER 2 antibody 2, and the black circle line indicates the 1.2. mu.g administration group of Compound No. 8 b. The amounts of anti-HER 2 antibody 2 and compound No. 8b administered were equivalent to each component constituting anti-HER 2 antibody 2-CDN conjugate (1). The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 13(a) shows the anti-tumor effect of intravenous administration based on anti-HER 2 antibody 2-CDN conjugates (2) and (3). Fig. 13(b) shows the antitumor effect of intravenous administration of 2-CDN conjugates (4), (5), (7) and (8) based on anti-HER 2 antibody. Figure 13(c) shows the anti-tumor effect based on intravenous administration of anti-HER 2 antibody 2-CDN conjugate (6). The black square line in the figure indicates the vehicle group, and the white symbol lines indicate the groups to which the anti-HER 2 antibody 2-CDN conjugates (2) to (8) were administered. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 14 shows the antitumor effect of intravenous administration based on anti-HER 2 antibody 2-CDN conjugates (9) and (10). In the figure, black square lines indicate the vehicle group, white triangular lines indicate the group administered with the anti-HER 2 antibody 2-CDN conjugate (9), and white circular lines indicate the group administered with the anti-HER 2 antibody 2-CDN conjugate (10). The anti-HER 2 antibody 2-CDN conjugates (9) and (10) were antibody-CDN conjugates using MSG-type sugar chain recombinant antibodies with an average number of drug conjugates of about 2. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 15 shows the anti-tumor effect based on intravenous administration of anti-EphA 2 antibody and anti-EphA 2 antibody-CDN conjugate (1). In the figure, black square lines indicate the vehicle group, white circular lines indicate the anti-EphA 2 antibody administration group, and white triangular lines indicate the anti-EphA 2 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation.

Figure 16 shows the anti-tumor effect based on intravenous administration of anti-CD 33 antibody and anti-CD 33 antibody-CDN conjugate (1). In the figure, black square lines indicate vehicle groups, white circular lines indicate anti-CD 33 antibody administration groups, and white triangular lines indicate anti-CD 33 antibody-CDN conjugate (1) administration groups. The vertical axis represents the tumor volume (mm) ³) Transverse directionThe axis represents the number of days after tumor implantation.

FIG. 17 shows the amino acid sequence (SEQ ID NO: 28) of the light chain and the amino acid sequence (SEQ ID NO: 29) of Pertuzumab (Pertuzumab).

Fig. 18 shows the amino acid sequence of the light chain (seq id No. 28) and the amino acid sequence of the heavy chain (seq id No. 30) of the engineered anti-HER 2 antibody 2.

FIG. 19 shows the amino acid sequence of the light chain (SEQ ID NO: 31) and the amino acid sequence of the heavy chain (SEQ ID NO: 32) of the anti-CD 33 antibody.

FIG. 20 shows the amino acid sequence of the light chain (SEQ ID NO: 33) and the amino acid sequence of the heavy chain (SEQ ID NO: 34) of the anti-EphA 2 antibody.

FIG. 21 shows the amino acid sequence of the light chain (SEQ ID NO: 35) and the amino acid sequence of the heavy chain (SEQ ID NO: 36) of the anti-CDH 6 antibody.

Figure 22 shows the antitumor effect of intravenous administration of 2-CDN conjugates (11) and (12) based on anti-HER 2 antibody. In the figure, black square lines indicate vehicle groups, white triangular lines indicate groups administered with anti-HER 2 antibody 2-CDN conjugate (11), and white circular lines indicate groups administered with anti-HER 2 antibody 2-CDN conjugate (12).

Detailed Description

The present invention relates to novel CDN derivatives having STING agonist activity, antibody drug conjugates thereof, and uses thereof. The novel CDN derivative has the STING agonist activity, activates immune cells and induces the production of interferon and cytokine. In addition, the novel CDN derivative of the present invention exerts an antitumor effect by activation of the immune cell. The novel CDN derivative may be directly administered to a target tissue to activate an immune function, or may be systemically administered via an arbitrary linker in conjunction with an antibody capable of recognizing and binding to a target cell (e.g., a tumor cell or an immune cell).

Sting (stimulant of interference genes) refers to transmembrane-type adaptor proteins located in the endoplasmic reticulum. It is known that there are congenital polytypes (PLoS One, 2013Oct, 21, 8(10), e77846) at STING high frequencies. For example, a variation of R232H in which the 232 th amino acid is changed from arginine (R) to histidine (H), a variation of HAQ in which the 71 th arginine (R) is changed to histidine (H), the 230 th glycine (G) is changed to alanine (A), and the 293 th arginine (R) is changed to glutamine (Q), is known as a STING variant. It is known that such a polymorphism of STING has a difference in response intensity such as the amount of cytokine production induced by stimulation with a STING agonist (Genes and Immunity, 2011, 12, 263-269). Therefore, in order for STING agonists to act stably in humans, it is preferable to have activity on each type of STING.

In the present specification, "cancer" and "tumor" have the same meaning.

In the present invention, the term "immunoactivating activity" refers to any type of activation of immune cells involved in antitumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, and neutrophils, and is a structural or functional change of all immune cells, for example, the activation of immune cells involved in antitumor immunity, such as the production of cytokines and chemokines, the increase in the expression of immunoactivating markers, the decrease in the expression of immunosuppressive markers, the change in intracellular signal transduction systems, such as phosphorylation, and the change in gene expression. The term "inducing a change in antitumor immunity induced by tumor cells" includes, for example, activation of immune cells, induction of production of free cytokines and chemokines, induction of increased sensitivity to immune cells, and the like.

In the present invention, the "antitumor effect" means that tumor cells are affected directly or indirectly by a drug, thereby inducing reduction or regression of tumors. For example, the reduction and damage of the number of tumor cells or the regression of tumors are called an antitumor effect, because the antitumor immune activation is caused by directly damaging the tumor cells with a drug, the tumor cells are stimulated with a drug, and the antitumor immune activation is caused around the tumor cells by, for example, releasing the drug delivered to the tumor cells to the outside of the cells.

In the present invention, "cytotoxicity" refers to any pathological change that is caused to a cell in any form, and refers to not only direct trauma but also structural and functional damages of all cells that cause DNA cleavage, formation of a base dimer, chromosome cleavage, damage to cell division apparatus, and reduction in various enzyme activities.

In the present invention, "cell" also includes cells in an animal individual, and cultured cells.

In the present specification, "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In the present specification, "C1-C6 alkyl" refers to a C1-6 linear or branched alkyl group. "C1-C6 alkyl" may have cyclopropane in the alkyl group if the total number of carbon atoms is not more than 6. Examples of the "C1-C6 alkyl group" include, but are not limited to, the following structures.

The wavy line indicates the substitution position.

As used herein, the term "C2-C6 alkenyl group" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms.

As used herein, the "C2-C6 alkynyl group" means a straight-chain or branched alkynyl group having 2 to 6 carbon atoms.

As used herein, the term "C3-C6 cycloalkyl group" refers to a saturated cyclic hydrocarbon group having 3 to 6 carbon atoms. "C3-C6 cycloalkyl" may be substituted with more than one alkyl group if the total number of carbon atoms is not more than 6. Examples of the "C3-C6 cycloalkyl group" include, but are not limited to, the following structures.

The wavy line indicates the substitution position.

In the present specification, "hydroxyC 1-C6 alkyl" refers to an alkyl group substituted with 1 or 2 hydroxyl groups at any position of a linear or branched alkyl group having 1 to 6 carbon atoms. "hydroxy C1-C6 alkyl" may also have cyclopropane on the alkyl group if the total number of carbon atoms is not more than 6. Examples of the "hydroxy C1-C6 alkyl group" include, but are not limited to, the following structures.

The wavy line indicates the substitution position.

In the present invention, the "amino C1-C6 alkyl group" refers to an alkyl group substituted with 1 or 2 amino groups at any position of a linear or branched alkyl group having 1 to 6 carbon atoms. "amino C1-C6 alkyl" may also have cyclopropane on the alkyl group if the total number of carbon atoms is not more than 6. Examples of the "amino C1-C6 alkyl group" include, but are not limited to, the following structures.

The wavy line indicates the substitution position.

<1. novel CDN derivative >

The novel CDN derivative of the present invention has a structure represented by the following formula (Ia):

L¹represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino]1 to 3 groups of the group consisting of ethyl groups are substituted at arbitrary positions, and are selected from the group consisting of the following formulae:

and

(in the formula, R⁶And R^6’Respectively independent earth surfaceRepresents a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, R⁷And R^7’Each independently represents a hydrogen atom or a C1-C6 alkyl group, which C1-C6 alkyl group may be optionally substituted by 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group, R⁸And R^8’Each independently represents a hydrogen atom or a halogen atom, Z⁴represents-CH₂-, -NH-or an oxygen atom, Z⁵Represents a nitrogen atom or-CH ═ c).

In addition, L¹Represents optionally selected from the group consisting of hydroxy, -NH₂2-hydroxyacetamidomethyl and 2- [ (2-hydroxyacetyl) amino]1 to 3 groups of the group consisting of ethyl groups are substituted at arbitrary positions, and are selected from the group consisting of the following formulae:

(in the formula, R⁶And R^6’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, R⁷And R^7’Each independently represents a hydrogen atom or a C1-C6 alkyl group, which C1-C6 alkyl group may be optionally substituted by 1 or 2 substituents selected from the group consisting of a halogen atom and an oxo group, R⁸And R^8’Each independently represents a hydrogen atom or a halogen atom, Z⁴represents-CH₂-, -NH-or an oxygen atom, Z⁵Represents a nitrogen atom or-CH ═ c).

L¹Preferably a group selected from the group consisting of:

and

(in the formula, R⁹And R^9’Represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，R¹⁰Represents a hydroxyl group, -NH₂、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH、－CH₂CH₂NHC(＝O)CH₂OH, hydroxy C1-C3 alkyl or amino C1-C3 alkyl, R¹¹And R^11’Each independently represents a hydrogen atom, a fluorine atom or a methyl group, or R¹¹And R^11’Combined to represent cyclopropane, Z⁴represents-CH₂-, -NH-or an oxygen atom).

In addition, L¹Preferably a group selected from the group consisting of:

(in the formula, R¹³And R^13’Each independently represents a hydrogen atom, a hydroxyl group or-NH₂，R¹²Represents a hydroxyl group, -NH₂、－CH₂OH、－NHC(＝O)CH₂OH、－CH₂NHC(＝O)CH₂OH or-CH₂CH₂NHC(＝O)CH₂OH，Z⁴As previously described. ).

Furthermore, L¹Preferably a group selected from the group consisting of:

And

(in the formula, R¹⁴Represents a hydrogen atom or-NH₂，R¹⁵Represents a hydrogen atom or-C (═ O) CH₂OH，R¹⁶Represents a hydroxyl group, -NH₂、－CH₂OH、－CH₂CH₂OH、－CH₂NH₂or-CH₂CH₂NH₂)。

L¹More preferably a group selected from the group consisting of the following formulae:

L³selected from hydrogen atoms, halogen atoms, -NH₂Hydroxyl C1-C3 alkyl or amino C1-C3 alkyl.

Q¹And Q^1’Each independently represents a hydroxyl group, a thiol group or a boryl group (BH)₃－)。Q¹Preferably a hydroxyl or thiol group. Q^1’Preferably a hydroxyl or thiol group. More preferably Q¹And Q^1’In a combination of (A) and (B)¹And Q^1’Is a thiol group, or Q¹And Q^1’Is a hydroxyl group.

Q²And Q^2’Each independently represents an oxygen atom or a sulfur atom. Preferably Q²And Q^2’All are oxygen atoms or all are sulfur atoms.

Q¹And Q²Preferably Q¹Is a thiol group and Q²Is an oxygen atom, or Q¹Is a thiol group and Q²Is a sulfur atom.

Q^1’And Q^2’Preferably Q^1’Is a thiol group and Q^2’Is an oxygen atom, or Q^1’Is hydroxy and Q^2’Is an oxygen atom, or Q^1’Is a thiol group and Q^2’Is a sulfur atom.

X¹And X²Each independently represents an oxygen atom, a sulfur atom or-CH₂－。X¹Preferably an oxygen atom. X²Preferably an oxygen atom. More preferably X¹And X²Is an oxygen atom.

Y¹And Y²Represents an oxygen atom or-CH₂－。Y¹Oxygen atoms are preferred. Y is²Oxygen atoms are preferred. More preferably Y¹And Y ²Is an oxygen atom.

X³And X⁴Represents a group selected from the following (iii) or (iv): (iii) y is¹When it is an oxygen atom, X³－X⁴represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (iv) Y¹is-CH₂When is, X³－X⁴represents-O-CH₂－。X³And X⁴Preferred is-CH of the above-mentioned (iii)₂－O－。

X⁵And X⁶Represents a group selected from the following (v) or (vi): (v) y is²When it is an oxygen atom, X⁵－X⁶represents-CH₂－O－、－CH₂－S－、－CH₂－CH₂-or-CH₂－CF₂-; or (vi) Y²is-CH₂When is, X⁵－X⁶represents-O-CH₂－。X⁵And X⁶preferably-CH of the above-mentioned (v)₂－O－。

R¹、R²And R³Each independently represents a hydrogen atom, a halogen atom, -OR ', -OC (═ O) R', -N₃NHR ', -NR' R 'or-NHC (═ O) R' (wherein R 'represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl may be substituted with 1 to 6 halogen atoms, and R' represents C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl).

R¹Preferably a hydrogen atom, a hydroxyl group or a fluorine atom.

R²Preferably a hydrogen atom, a hydroxyl group or a fluorine atom.

R³Preferably a hydrogen atom, a hydroxyl group or a fluorine atom.

W¹Represents a nitrogen atom, an oxygen atom, sulfurAn atom or-CH-.

R⁵Represents a group selected from the following (vii) to (x): (vii) w¹When it is a nitrogen atom, R ⁵Represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group or an amino C1-C6 alkyl group; (viii) w¹When it is an oxygen atom, R⁵Is absent; (ix) w¹When it is a sulfur atom, R⁵Is absent; or (x) W¹When is-CH-, R⁵Represents a hydrogen atom, a halogen atom, a hydroxyl group, -NH₂Or C1-C6 alkyl. W¹When it is a nitrogen atom, R⁵Preferably a hydrogen atom. W¹When is-CH-, R⁵Preferably a hydrogen atom.

W²Represents a nitrogen atom or-CH ═ CH. W²Preferably, -CH ═ CH.

R⁴Represents a hydrogen atom, a halogen atom or-NH₂。R⁴Preferably a hydrogen atom.

Z¹－Z²－Z³Taken together represent a group selected from: -CH₂－CH₂－CH₂－、－CH₂－CH₂－R”’－、－CH＝CH－CH₂－、－CH＝CX－CH₂－、－CX＝CH－CH₂－、－CX＝CX－CH₂－、－C(＝O)－CH₂－CH₂－、－CH₂－CH₂－C(＝O)－、－CH₂－CH(CH₃)－CH₂-or-CH₂－CH₂－CH(CH₃) - (in the formula, R' "represents-O-or-CH)₂－CH₂-, X represents a halogen atom) or a group represented by any one of the following formulae:

(in the formula, asterisks indicate the relation with W¹Binding, wavy line indicates binding to the carbon atom of ═ C "). Z¹、Z²And Z³Preferably taken together represent-CH₂－CH₂－CH₂－、－CH＝CH－CH₂－、－CH₂－CH(CH₃)－CH₂－、－CH₂－CH₂－CH(CH₃) -, or-CH₂－CH₂-R '- (wherein R' represents-O-or-CH)₂－CH₂－)。

In addition, the novel CDN derivatives of the present invention preferably have a structure represented by the following formula:

L¹as defined above.

Q³And Q^3＇Each independently represents a hydroxyl group or a thiol group. Preferably Q³And Q^3＇Are all thiol groups.

R²¹And R²²Each independently represents a hydroxyl group or a fluorine atom. R ²¹Hydroxyl groups are preferred. R²²Fluorine atoms are preferred.

W⁵Represents an-NH-or sulfur atom.

The method for producing the novel CDN derivative of the present invention is described in <3 > production method >.

<2 > antibody drug conjugate >

The novel CDN derivative of the present invention may be administered directly to a target tissue (e.g., intratumoral administration), or may be administered as an antibody drug conjugate in which the novel CDN derivative of the present invention and an antibody capable of recognizing and binding to a target cell (e.g., a tumor cell or an immune cell) are linked via an arbitrary linker.

The antibody drug conjugate of the present invention is represented by the following formula (II):

m¹represents the number of drug-binding molecules per molecule of antibody in the antibody-drug conjugate, Ab represents an antibody or a functional fragment of the antibody, L represents a linker linking Ab to D,d represents the above-mentioned novel CDN derivative (in the present specification, in the case where the novel CDN derivative is used as part of an antibody drug conjugate, it is also simply referred to as "drug").

Drug D is a compound having an activity of activating immune cells, specifically, having a STING agonist activity. When a part or all of the linker is cleaved in a target cell (e.g., a tumor cell or an immune cell), the drug D is released as an original structure and exerts an immune activation effect. The target function is exerted by the increased sensitivity of the target cell to the immune cell or by the activation of the target cell by the immune cell. The target function is not particularly limited as long as it is a function associated with STING agonist activity, and antitumor activity is preferred. That is, the drug D linked to an antibody targeting a tumor (e.g., an anti-HER 2 antibody) via an arbitrary linker is delivered to a target cell or tissue, and a part or all of the linker is cleaved, whereby the anti-tumor effect is exerted by the increased sensitivity of the target cell to an immune cell or the activation of the immune cell via the target cell (e.g., the production of interferon or cytokine).

The drug D bound to the antibody drug conjugate of the present invention is represented by the following formula (I):

wherein L and L¹Or L²Any of-NH contained in₂Or a hydroxyl group is bonded, L¹As described above<1. Novel CDN derivative>Is defined in (1), L²Represents a group selected from the following (i) or (ii): (i) l is²In combination with L, L²represents-NHR ', hydroxyl C1-C6 alkyl or amino C1-C6 alkyl, wherein R' represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl can be optionally substituted by 1-6 halogen atoms; or (ii) L²When not bound to L, L²Represents a hydrogen atom or a halogen atom. Q¹、Q^1’、Q²、Q^2’、X¹、X²、X³、X⁴、X⁵、X⁶、Y¹、Y²、R¹、R²、R³、R⁴、R⁵、W¹、W²、Z¹、Z²And Z³As described above<1. Novel CDN derivative>The method as defined in (1).

L²In combination with L, preferably-NH₂、－CH₂NH₂or-CH₂OH。L²When L is not bonded, a hydrogen atom or a fluorine atom is preferable.

The novel CDN derivative of the present invention or the drug D used in the antibody drug conjugate of the present invention is preferably represented by any one of the following 2 structural formulae:

(in the formula, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, R¹⁷、R^17’、R¹⁸And R^18’Each independently represents a hydrogen atom, a halogen atom, a hydroxyl group or-NH₂，W³represents-NH-, an oxygen atom, a sulfur atom or-CH ₂－，W⁴represents-CH ═ or a nitrogen atom).

In addition, the drug D used in the novel CDN derivative of the present invention or the antibody drug conjugate of the present invention is preferably represented by any one of the following 2 structural formulae:

(in the formula, L¹、Q¹、Q^1’、Q²、Q^2’、R¹⁷、R^17’、R¹⁸And R^18’As previously described. ).

(in the formula, L¹、Q¹、Q^1’、Q²And Q^2’As defined above, R¹⁹、R^19’、R²⁰And R^20’Each independently represents a hydrogen atom or a fluorine atom).

(in the formula, L¹As previously described. ).

(in the formula, L¹As previously described. )

In addition, the novel CDN derivative of the present invention or the drug D used in the antibody drug conjugate of the present invention is preferably represented by the following formula:

(in the formula, L¹、Q³、Q^3＇、R²¹、R²²And W⁵As described above<1. Novel CDN derivative>As specified in (1).

In addition, in the novel CDN derivative of the present invention or the drug D used in the antibody drug conjugate of the present invention, L¹Preferably represented by any one of the following structural formulae:

in addition, in the novel CDN derivative of the present invention or the drug D used in the antibody drug conjugate of the present invention, L¹Preferably represented by any one of the following 4 structural formulae:

(in the formula, an asterisk indicates the binding to L).

(wherein an asterisk indicates the bond to L, Q³、Q^3＇And W⁵As described above<1. Novel CDN derivative>As specified in (1).

<2.1. linker Structure >

The structure of a linker for binding a drug to an antibody in the antibody drug conjugate of the present invention will be described. The linker used in the antibody drug conjugate of the present invention is not particularly limited as long as it is a linker that links an antibody and a drug, and is understood by those skilled in the art. Examples of the linker used in the antibody drug conjugate of the present invention include Protein Cell, 2018, 9 (1): 33-46, Pharm Res, 2015, 32: 3526-. The linker may be a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but is preferably a linker that is cleaved in vivo.

Examples of the linker used in the antibody drug conjugate of the present invention include, but are not limited to, a linker (sometimes referred to as "sugar chain conjugation" in the present specification) for binding a drug to a sugar chain or a reconstituted sugar chain on the Fc portion of an antibody, and a linker (for example, described in WO2018/003983) for binding a drug to an arbitrary amino acid residue (for example, a cysteine residue or a lysine residue) of an antibody, and a linker (for example, described in WO 2014/057687). Preferred linkers for binding a drug to an arbitrary amino acid residue of an antibody include those that form a thioether bond with a thiol group (SH group) of cysteine of Ab (sometimes referred to as "cysteine coupling" in the present specification), and those that form an amino group (NH) of lysine of Ab₂Group) to form an amide (which may be referred to as "lysine coupling" in the present specification), cysteine coupling is preferable.

Preferred linkers L of the present invention are represented by the following formula.

－Lb－La－Lp－Lc－＊

(in the formula, an asterisk indicates the L-form of the drug D¹Or L²Any of the amino groups or hydroxyl groups contained in (1) is bonded. ).

First, Lp will be explained.

Lp represents a linker formed of an amino acid sequence (hereinafter, also referred to as a peptide linker in the present specification) which can be cleaved in vivo or in a target cell, or is absent.

Lp is cleaved by an enzyme such as peptidase or esterase. Lp is a peptide consisting of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond with a carbonyl group at the right end of La described later at the N-terminal end thereof, and forms an amide bond with an amino group (-NH-) of Lc at the C-terminal end thereof. The amide bond at the C-terminal side of Lp is cleaved by an enzyme such as the peptidase.

The amino acid constituting Lp is not particularly limited, but is, for example, an L-or D-amino acid, preferably an L-amino acid. In addition, the amino acid may be an unnatural amino acid such as an N-methylated amino acid, in addition to an α -amino acid, e.g., an amino acid having a structure of β -alanine, e-aminocaproic acid, or γ -aminobutyric acid. The amino acid sequence of Lp is not particularly limited, and examples of the constituent amino acids include glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), methionine (Met; M), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among them, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe: F), citrulline (Cit) are preferable. These amino acids may be repeated, having an amino acid sequence comprising arbitrarily selected amino acids. In addition, the pattern of drug release can be controlled by the type of amino acid.

Specific examples of Lp include-GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-and ICit-. The linker Lp is preferably-GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, -FCit-. The linker Lp is more preferably-GGVA-, -GGFG-, -GGVCit-. In addition, the linker Lp is preferably-GGFG-or-GGPI-.

La will be described below.

La represents any one selected from the group consisting of: -C (═ O) - (CH)₂CH₂)n²－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－CH₂－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂)n³－CH₂－C(＝O)－、－C(＝O)－(CH₂CH₂)n²－C(＝O)－NH－(CH₂CH₂O)n³－CH₂－C(＝O)－、－(CH₂)n⁴-O-C (═ O) -, and- (CH)₂)n⁹－C(＝O)－。

(in the formula, n²Represents an integer of 1 to 3 (preferably 1 or 2), n³Represents an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably 3 or 4), n⁴Represents an integer of 0 to 2 (preferably 0 or 1), n⁹Represents an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably 2, 3 or 5)

La preferably represents any one selected from the group consisting of: -C (═ O) -CH₂CH₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₃－CH₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₄－CH₂－C(＝O)－、－C(＝O)－(CH₂CH₂)₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂)₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂)₂－CH₂－C(＝O)－、－CH₂-OC (═ O) -, and- (CH)₂)₅－C(＝O)－。

La is more preferably-C (═ O) -CH₂CH₂－C(＝O)－、－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₃－CH₂-C (═ O) -, or- (CH)₂)₅－C(＝O)－。

La is more preferably-C (═ O) -CH ₂CH₂－C(＝O)－。

Next, Lb will be described.

Lb represents a spacer used for sugar chain-coupled linkers (also referred to as "spacer of sugar chain-coupled linker" in the present specification), or a spacer used for cysteine coupling (also referred to as "spacer of cysteine-coupled linker" in the present specification).

< Lb is a "spacer for linker having sugar chain coupling" >

When Lb is a "spacer of a linker to which a sugar chain is coupled", Lb is not particularly limited, and examples thereof include spacers represented by the following formula.

In each of the structural formulae shown above, the asterisk (@) indicates- (C ═ O) -or-CH with respect to the left end of La₂Binding, wavy line means binding to sugar chain of Ab or restructured sugar chain.

When Lb is any of Lb-1, Lb-2 or Lb-3, the triazole ring site has a geometric isomeric structure, and 1 Lb contains any of 2 structures, or a mixture containing these. The antibody drug conjugate of the present invention can bind a plurality of drugs to 1 molecule antibody. When a plurality of drugs are bound to the 1-molecule antibody, a plurality of Lb may exist (for example, see the following description)<3. Manufacturing method>Scheme (1E)) of the antibody drug conjugate shown in method E. Lb is selected from any of Lb-1, Lb-2 and Lb-3, when a plurality of Lbs are present in 1-molecule antibody (for example, m described later) ²1 or 2), the triazole ring site has a geometric isomeric structure in each Lb, containing any of 2 structures in one Lb, or a mixture comprising these.

< Lb is a "spacer for cysteine-coupled linker" >

When Lb is "spacer of linker for cysteine coupling", Lb is not particularly limited, and examples thereof include- (succinimide-3-yl-N) -. In the present invention, "- (succinimid-3-yl-N) -" has a structure represented by the following formula:

in the structural formula shown above, the asterisk indicates that La is bonded, and the wavy line forms a thioether bond with the side chain of the cysteine residue of the antibody.

Next, Lc will be explained.

Lc represents-NH-CH₂-, -NH-phenyl-CH₂-O (C ═ O) -or-NH-heteroaryl-CH₂-O (C ═ O) -, or is absent. In the formula, as the phenyl group, a 1, 4-phenyl group is preferable, and as the heteroaryl group, a 2, 5-pyridyl group, a 3, 6-pyridyl group, a 2, 5-pyrimidyl group or a 2, 5-thienyl group is preferable. Lc is preferably-NH-CH₂-or is absent.

As for the linker L more preferred in the present invention, when the mode of binding the drug to the antibody is "sugar chain coupling", the linker L is-Z ^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFG－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGVA－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGVCit－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFCit－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGICit－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFM－、－Z¹－C(＝O)－CH₂CH₂－C(＝O)－GGPI－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGLM－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－FG－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－VA－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFG－NH－CH₂－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGVA－NH－CH₂－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGVCit－NH－CH₂－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFCit－NH－CH₂－、－Z^L1－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₃－CH₂-C (═ O) -, or-Z^L1－C(＝O)－CH₂CH₂－C(＝O)－NH－(CH₂CH₂O)₄－CH₂-C (═ O) -, where Z^L1The following structural formula representing the above Lb:

alternatively, where the mode of drug binding to the antibody is "cysteine coupled", linker L is-Z^L2－(CH₂)₅－C(＝O)－GGFG－、－Z^L2－(CH₂)₅－C(＝O)－GGVA－、－Z^L2－(CH₂)₅－C(＝O)－GGVCit－、－Z^L2－(CH₂)₅－C(＝O)－GGFCit－、－Z^L2－(CH₂)₅－C(＝O)－GGICit－、－Z^L2－(CH₂)₅－C(＝O)－GGFM－、－Z^L2－(CH₂)₅－C(＝O)－GGPI－、－Z^L2－(CH₂)₅－C(＝O)－GGLM－、－Z^L2－(CH₂)₅－C(＝O)－FG－、－Z^L2－(CH₂)₅－C(＝O)－VA－、－Z^L2－(CH₂)₅－C(＝O)－GGFG－NH－CH₂－、－Z^L2－(CH₂)₅－C(＝O)－GGVA－NH－CH₂－、－Z^L2－(CH₂)₅－C(＝O)－GGVCit－NH－CH₂－、－Z^L2－(CH₂)₅－C(＝O)－GGFCit－NH－CH₂－、－Z^L2－(CH₂)₅－C(＝O)－NH－(CH₂CH₂O)₃－CH₂-C (═ O) -, or-Z^L2－(CH₂)₅－C(＝O)－NH－(CH₂CH₂O)₄－CH₂-C (═ O) -, where Z^L2(succinimide-3-yl-N) -, which is represented by the following structural formula, and which represents the Lb.

A more preferred mode of binding of the drug to the antibody as the linker L in the present invention is "sugar chain coupling" and is-Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGFG－NH－CH₂-, or-Z^L1－C(＝O)－CH₂CH₂－C(＝O)－GGPI－NH－CH₂-, in the formula, Z^L1The following structural formula representing the above Lb:

the right end of the aforementioned "preferred linker L" and "more preferred linker L" is linked to L of the formula (I)¹Or L²optionally-NH contained therein₂Or a combination of hydroxyl groups.

<2.2 > antibodies and sugar chain modifications thereof >

<2.2.1 antibodies >

In the present specification, "gene" refers to a nucleotide or a nucleotide sequence contained in a nucleotide sequence encoding an amino acid of a protein or a complementary strand thereof, and for example, a nucleotide sequence contained in a nucleotide sequence encoding an amino acid of a protein or a polynucleotide, an oligonucleotide, DNA, mRNA, cDNA, RNA, or the like as a complementary strand thereof is also included in the meaning of "gene".

In the present specification, "nucleotide", "polynucleotide" or "nucleotide sequence" has the same meaning as "nucleic acid", and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of "nucleotide" or "nucleotide sequence".

In the present specification, "polypeptide", "peptide" and "protein" are used indiscriminately.

In the present specification, the term "functional fragment of an antibody" is also referred to as "antigen-binding fragment of an antibody", and refers to a partial fragment of an antibody having an activity of binding to an antigen, and includes Fab, F (ab') 2, Fv, scFv, diabody, linear antibodies, multispecific antibodies formed from antibody fragments, and the like. In addition, a monovalent fragment Fab 'of the variable region of an antibody, which is obtained by treating F (ab') 2 under reducing conditions, is also included in the antigen-binding fragment of the antibody. However, the molecule is not limited to these molecules as long as it has an ability to bind to an antigen. In addition, these antigen-binding fragments include not only molecules obtained by treating the full-length molecules of antibody proteins with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

The functional fragment of the present invention contains asparagine (Asn297) modified by an N-linked sugar chain well preserved in the Fc region of an IgG heavy chain and amino acids in the vicinity thereof, and has a binding ability to an antigen.

The antibody used in the antibody drug conjugate of the present invention is an immunoglobulin, and is a molecule containing an antigen binding site that immunospecifically binds to an antigen. The antibody of the present invention may be any of IgG, IgE, IgM, IgD, IgA and IgY, and preferably IgG. The subclass (subclass) may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and preferably IgG1, IgG2, or IgG4 (an antibody having a mutation affecting ADCC and ADCP activity in the Fc region of an IgG heavy chain).

When IgG1 is used as the isotype (isotype) of the antibody of the present invention, a part of the amino acid residues in the constant region may be substituted to adjust the effector function (effector) (see WO88/07089, WO94/28027, and WO 94/29351). Examples of IgG1 variants include IgG1 LALA variants (IgG 1-L234A, L235A). L234A and L235A indicate that the leucine at positions 234 and 235 specified by EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol.63, No.1(May 15, 1969), pp.78-85) is substituted by alanine.

The antibody may be derived from any species, and preferably, human, rat, mouse and rabbit may be exemplified. When the antibody is derived from a species other than human, it is preferably chimerized or humanized by using a known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. The monoclonal antibody includes a monoclonal antibody derived from a non-human animal such as a rat antibody, a mouse antibody, and a rabbit antibody, a chimeric antibody, a humanized antibody, a human antibody, a functional fragment thereof, or a modified form thereof.

The antibody is preferably an antibody targeting a tumor cell or an immune cell, but is not limited thereto. The antibody is more preferably an antibody targeting a tumor cell.

When an antibody that uses a tumor cell as a target cell is used, the antibody preferably has at least one of a property of recognizing the tumor cell, a property of binding to the tumor cell, a property of being engulfed into the tumor cell and internalized, and a property of damaging the tumor cell. Drugs conjugated to the antibodies of the invention via a linker have STING agonist activity. The drug of the present invention induces interferon by activating the signal of interferon control factor 3(IRF 3). Therefore, when an antibody targeting a tumor cell is used as the antibody drug conjugate of the present invention, the antibody drug conjugate is administered in vivo, delivered to the tumor site, engulfed into the tumor cell, and then the linker moiety is cleaved by a peptidase or the like to release the drug moiety. It is thought that the free drug moiety activates anti-tumor immunity by STING agonist activity to exert an anti-tumor effect.

The binding of the antibody to the tumor cells can be confirmed using a flow cytometer. The engulfment of the antibody into the tumor cells can be confirmed using the following method: (1) an assay for visualizing antibodies engulfed into cells by fluorescence microscopy using a secondary antibody (fluorescent label) bound to a therapeutic antibody (Cell Death and Differentiation (2008)15, 751-. As the immunotoxin, a recombinant complex protein of the catalytic region of diphtheria toxin and protein G may be used.

In the present invention, the term "high internalization ability" refers to the survival rate (expressed as a relative rate of cell survival rate without addition of an antibody of 100%) of a target antigen-expressing cell (for example, a cell expressing HER2 when an anti-HER 2 antibody is used) to which the antibody and a saporin-labeled anti-mouse or rat IgG antibody are added, preferably 70% or less, and more preferably 60% or less.

When the antibody drug conjugate of the present invention is an antibody targeting a tumor cell, the antibody itself preferably has an anti-tumor effect, but it is not essential. The antibody used in the antibody drug conjugate of the present invention preferably has internalization properties into tumor cells.

The antitumor activity of the drug and the antibody-drug conjugate refers to cytotoxicity to tumor cells, an anti-cell effect, and atrophy of tumor volume. The antitumor activity can be confirmed using a known in vitro (in vitro) or in vivo (in vivo) evaluation system.

The immunoactivating activity of the drug and the antibody-drug conjugate refers to the increase in the sensitivity of tumor cells to immune cells or the activation of immune cells by tumor cells. The immunoactivating activity can be confirmed using a known in vitro (in vitro) or in vivo (in vivo) evaluation system.

Examples of the antibody used in the present invention include, but are not limited to, an anti-HER 2 antibody, an anti-HER 3 antibody, an anti-DLL 3 antibody, an anti-FAP antibody, an anti-CDH 11 antibody, an anti-CDH 6 antibody, an anti-a 33 antibody, an anti-CanAg antibody, an anti-CD 19 antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 98 antibody, an anti-TROP 2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUC 1 antibody, an anti-gpgb antibody, an anti-integran antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, an anti-Mesothelin antibody, an anti-ENPP 3 antibody, an anti-CD 5 antibody, an anti-EGFR antibody, an anti-20 antibody. As the antibody of the present invention, an anti-HER 2 antibody (e.g., trastuzumab or pertuzumab), an anti-CDH 6 antibody, an anti-CD 33 antibody, or an anti-EphA 2 antibody is preferable, and an anti-HER 2 antibody is more preferable.

The antibody of the present invention can be obtained by immunizing an animal with a polypeptide as an antigen using a method generally practiced in the art, and collecting and refining the antibody produced in vivo. The source of the antigen is not limited to humans, and an animal may be immunized with an antigen derived from an animal other than humans, such as a mouse or a rat. In this case, antibodies that can be suitably used for human diseases can be selected by testing the crossability between the antibody that binds to the obtained foreign antigen and the human antigen.

Alternatively, a Monoclonal antibody can be obtained by fusing an antibody-producing cell that produces an antibody to an antigen with a myeloma cell to establish a hybridoma according to a known method (for example, Kohler and Milstein, Nature (1975)256, p.495-497, Kennett, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)).

It is stated that an antigen can be obtained by producing a gene encoding an antigenic protein in a host cell by genetic manipulation.

The humanized antibody of the present invention can be obtained by a known method (for example, Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984), Nature (1986)321, p.522-525, WO 90/07861).

For example, anti-HER 2 antibodies (US5821337, WO2004/008099, etc.), anti-CD 33 antibodies (WO2014/057687, etc.), anti-CD 70 antibodies (WO2004/073656, etc.), anti-EphA 2 antibodies (WO2009/028639, etc.), anti-CDH 6 antibodies (WO2018/212136, etc.) can be obtained using a known method.

The anti-HER 2 antibody of the present invention is not particularly limited, but is preferably an antibody having the following properties, for example.

(1) An anti-HER 2 antibody, having the following properties: (a) specifically binds to HER 2. (b) Has activity of internalizing in HER2 expressing cells by binding to HER 2.

(2) The antibody of (1) above, which binds to the extracellular domain of HER 2.

(3) The antibody according to the above (1) or (2), which is a monoclonal antibody.

(4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).

(5) The antibody according to any one of the above (1) to (4), which is a mouse monoclonal antibody, a chimeric monoclonal antibody or a humanized monoclonal antibody.

(6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and comprises a mutation that causes ADCC and ADCP activity to decrease.

(7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO. 2 and a light chain having the amino acid sequence of SEQ ID NO. 1.

(8) The antibody according to (5) above, wherein the heavy chain constant region is human IgG1, wherein the leucine amino acids at positions 234 and 235 are replaced by alanine according to EU INDEX.

(9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain formed from the amino acid sequence of SEQ ID NO. 3 and a light chain formed from the amino acid sequence of SEQ ID NO. 1.

(10) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO. 29 and a light chain having the amino acid sequence of SEQ ID NO. 28.

(11) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO. 30 and a light chain having the amino acid sequence of SEQ ID NO. 28.

(12) The antibody according to any one of (1) to (11) above, wherein 1 or 2 amino acids are deleted from the carboxyl terminal of the heavy chain.

(13) An antibody obtained by a method for producing the antibody, comprising the steps of: culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above; and collecting the target antibody from the culture obtained in the step.

<2.2.2 sugar chain reconstitution of antibody >

In recent years, a method of reconstructing uneven sugar chains of antibodies by an enzyme reaction and uniformly introducing sugar chains having functional groups has been reported (ACS chem.biol.2012, 7, 110-. Attempts have been made to synthesize uniform ADCs by site-specifically introducing drugs using this sugar chain reconstruction technique (Bioconjugate chem.2015, 26, 2233. sup. -) 2242, Angew. chem. int. Ed.2016, 55, 2361. sup. -) 2367, US 2016361436).

Reconstitution of sugar chains first, heterogeneous sugar chains attached to a protein (antibody or the like) are cleaved by a hydrolase to leave only GlcNAc at the terminal, and a homogeneous protein fraction (hereinafter referred to as "acceptor") to which GlcNAc has been added is prepared. Subsequently, an arbitrary sugar chain (hereinafter referred to as "donor") prepared separately is prepared, and the acceptor and the donor are linked by glycosyltransferase. Thereby, a uniform glycoprotein having an arbitrary sugar chain structure can be synthesized.

In the present invention, the "sugar chain" refers to a structural unit in which two or more monosaccharides are bonded via glycosidic bonds. Specific monosaccharides or sugar chains may sometimes be represented in abbreviated forms, such as "GlcNAc-", "SG-". When these abbreviations are used in the structural formulae for description, unless otherwise specified, an oxygen atom or a nitrogen atom at the reducing end ascribed to the glycosidic bond with other structural units is not included in the abbreviations representing the sugar chains.

In the present invention, unless otherwise specified, for convenience, in the description of a monosaccharide as a basic unit of a sugar chain, a carbon atom bonded to an oxygen atom constituting a ring in a ring structure and directly bonded to a hydroxyl group (or an oxygen atom ascribed to a glycosidic bond) is labeled as the 1-position (the 2-position in sialic acid only). The names of the example compounds are labeled as a whole with the chemical structure, and this rule does not necessarily apply.

In the present invention, when a sugar chain is described by a symbol (for example, SG, MSG, GlcNAc, etc.), unless otherwise specified, the carbon at the reducing end is included in the symbol, but N or O that is ascribed to an N-or O-glycosidic bond is not included in the symbol.

The antibody drug conjugates of the present invention are represented by the following formula:

the antibody Ab or functional fragment thereof is bound to L directly from the side chain of its amino acid residue (e.g., cysteine, lysine, etc.), or bound to L from the sugar chain or reconstituted sugar chain of Ab.

The sugar chain of Ab of the present invention is an N-linked sugar chain or an O-linked sugar chain, and preferably an N-linked sugar chain.

The N-linked sugar chain is bonded to the amino acid side chain of the antibody via an N-glycosidic bond, and the O-linked sugar chain is bonded to the amino acid side chain of the antibody via an O-glycosidic bond.

Ab of the present invention is IgG, preferably IgG1, IgG2 or IgG 4.

IgG is known to have a highly conserved N-linked sugar chain in the asparagine residue at position 297 (hereinafter referred to as "Asn 297 or N297") in the Fc region of its heavy chain, contributing to the activity or dynamics of the antibody molecule, etc. (Eon-Duval, a.et al, biotechnol. prog.2012, 28, 608-.

The amino acid sequence in the constant region of IgG is highly conserved, and in the report by Edelman et al (proc.natl.acad.sci.u.s.a.,63,78-85, (1969)), each amino acid is determined by EU numbering (EU INDEX). For example, Asn297 with an N-linked sugar chain added to the Fc region corresponds to position 297 in the EU numbering, and even if the actual amino acid position changes due to fragmentation of the molecule or region defect, the amino acid can be uniquely identified by expression in the EU numbering.

The following figure shows the case where the antibody drug conjugate of the present invention is bound to L from the N297 sugar chain of an antibody or a functional fragment thereof.

The antibody having the reconstituted sugar chain is referred to as a sugar chain reconstituted antibody.

SGP (. alpha.2, 6-SGP) is a short name for sialoglycopeptide, and is a representative of N-linked glycopeptides. SGP can be isolated and purified from the yolk of eggs according to, for example, the method described in WO 2011/0278681. In addition, the refined product of SGP is sold by Tokyo chemical industry and pharmaceutical industry. In the present specification, the sugar chain portion of SGP is referred to as SG, and sugar chains lacking GlcNAc at the reducing end of one SG are referred to as SG (10). SG (10) can be prepared by enzymatic hydrolysis of SGP, for example, in accordance with a report of Meichuan et al (Biochim. Biophys. acta 2010,1800, 1203-1209). SG (10) is also commercially available from Tokyo chemical industry and pharmaceutical industry.

In this specification, a sugar chain structure in which sialic acid at a non-reducing terminal is deleted only in any one of β -Man branches of SG (10) is referred to as MSG (9), a sugar chain having sialic acid only in branched 1-3 sugar chains is referred to as MSG1, and a sugar chain having sialic acid only in branched 1-6 sugar chains is referred to as MSG 2.

The sugar chain to be reconstituted in the present invention is N297- (Fuc) SG, N297- (Fuc) MSG1, N297- (Fuc) MSG2, or a mixture of N297- (Fuc) MSG1 and N297- (Fuc) MSG2, preferably N297- (Fuc) SG, N297- (Fuc) MSG1 or N297- (Fuc) MSG2, more preferably N297- (Fuc) SG or N297- (Fuc) MSG 1.

N297- (Fuc) SG is represented by the following structural formula or sequence formula.

In the above formula, the wavy line indicates Asn297 binding to the antibody, and L (PEG) indicates- (CH)₂－CH₂－O)n⁵－CH₂－CH₂-NH-represents a bond in which the amino group at the right end forms an amide with the carboxyl group at the 2-position of sialic acid at the non-reducing terminal of both the 1-3 chain side and the 1-6 chain side of the beta-Man branch of the N297 sugar chain, and the asterisk represents a bond with the nitrogen atom at the 1-position or 3-position of the linker L, particularly the 1, 2, 3-triazole ring of Lb of the linker L, wherein N is⁵Is an integer of 2 to 10, preferably an integer of 2 to 5.

N297- (Fuc) MSG1 is shown in the following structural formula or sequence formula.

In the above formula, the wavy line indicates Asn297 binding to the antibody, and L (PEG) indicates- (CH)₂－CH₂－O)n⁵－CH₂－CH₂-NH-represents a bond with the amino group at the right end forming an amide with the carboxyl group at the 2-position of sialic acid at the 1-3 chain side of the beta-Man branch of the N297 sugar chain, and the asterisk represents a bond with the nitrogen atom at the 1-or 3-position of the linker L, particularly the 1, 2, 3-triazole ring of Lb of the linker L, wherein N is⁵Is an integer of 2 to 10, preferably an integer of 2 to 5.

N297- (Fuc) MSG2 is shown in the following structural formula or sequence formula.

In the above formula, the wavy line indicates Asn297 binding to the antibody, and L (PEG) indicates- (CH) ₂－CH₂－O)n⁵－CH₂－CH₂-NH-represents a structure in which the amino group at the right end is bonded to the carboxyl group at the 2-position of sialic acid at the 1-6 chain side of the beta-Man branch of the N297 sugar chain through amide bond, and the asterisk represents a structure in which the bond is bonded to the nitrogen atom at the 1-position or 3-position of the 1, 2, 3-triazole ring of the linker L, particularly Lb in the linker LIn which n is⁵Is an integer of 2 to 10, preferably an integer of 2 to 5.

When the N297 sugar chain of the antibody drug conjugate of the present invention is N297- (Fuc) SG, the antibody is a dimer, and therefore the antibody drug conjugate is a molecule in which 4 linkers L and 4 drugs D are bonded (m above)²＝2)。

When the N297 sugar chain of the antibody drug conjugate of the present invention is N297- (Fuc) MSG1, N297- (Fuc) MSG2, or a mixture thereof, the antibody is a dimer, and therefore the antibody drug conjugate is a molecule in which 2 linkers L and 2 drugs D are bonded (m above)²1) (see fig. 1).

The N297 sugar chain is preferably N297- (Fuc) SG or N297- (Fuc) MSG1 or N297- (Fuc) MSG2, more preferably N297- (Fuc) SG.

When the N297 sugar chain of the antibody in the antibody drug conjugate of the present invention is N297- (Fuc) SG, N297- (Fuc) MSG1, or N297- (Fuc) MSG2, ADC having high uniformity can be obtained.

<3 > production method

A representative production method of the novel CDN derivative of the present invention, the antibody drug conjugate thereof, or a production intermediate thereof will be described. In the following, the numbers of the compounds shown in the respective reaction formulae are used to represent the compounds. Namely, the compound is referred to as "the compound of formula (1)", "the compound (1)", etc. The compounds having other numbers are also described in the same manner.

In the following methods A to E, the substituent R¹～R⁵、L¹、L²、W¹、W²、Z¹～Z³The same meanings as above are given. Substituent R^a、R^c、R^eAnd R^gRepresents the side chain of a natural alpha-amino acid. Examples thereof include methyl, isopropyl, sec-butyl, isobutyl and benzyl. PRO¹Represents a protecting group of a primary alcohol. Preferred are 4, 4' -dimethoxytrityl group, 4-methoxytrityl group and the like. PRO²、PRO³、PRO⁷、PRO⁸Represents a protecting group for a secondary alcohol. Preferred are tert-butyldimethylsilyl group and triisopropyl groupSilyloxymethyl, benzoyl, 2-nitrobenzyl, 4-methoxytetrahydropyran-4-yl and the like. PRO⁶Represents a protecting group of a carboxylic acid. Preferred are t-butyl, benzyl, etc. PRO⁵、PRO⁹Represents a protecting group of an amine. PRO⁵Preferred are t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, benzyloxycarbonyl and the like, PRO ⁹Preference is given to 9-fluorenylmethyloxycarbonyl or 2- (trimethylsilyl) ethoxycarbonyl. PRO⁴Represents a protective group for an alcohol or an amine. The alcohol is preferably tert-butyldimethylsilyl, benzoyl or the like, and the amine is preferably 2- (trimethylsilyl) ethoxycarbonyl, allyloxycarbonyl, tert-butyloxycarbonyl or the like. Q^aRepresents an oxygen atom or a sulfur atom, Q^bRepresents a hydroxyl group or a thiol group. Q^a’And Q^b’Each independently representing a negatively charged oxygen atom (O)^－) Or a sulfur atom (S)^－)。R^xAnd R^yEach independently represents a halogen atom or-O-PRO². n represents an integer of 1 to 3.

Method A

The CDN derivative represented by (1) of the present invention can be produced according to method a described below.

The present production method is a method for producing a compound represented by the general formula (1). The steps A-1 to A-5 of the present production method can be carried out by one-pot synthesis (one pot synthesis) with reference to the report of Gaffney et al (org. Lett.2010, 12, 3269-3271).

(A-1 Process)

This step is a step of producing a compound of formula (2a) by continuously performing hydrolysis reaction and removal of cyanoethyl group on a compound of formula (1a) by a known organic chemistry method.

The compound (1a) is hydrolyzed by treating with water and an acid (such as pyridine trifluoroacetate, 4, 5-dicyanoimidazole or 1H-tetrazole) in a solvent (acetonitrile, tetrahydrofuran, N-dimethylformamide or a mixed solvent thereof) at a temperature of from-10 ℃ to the boiling point of the solvent used in the reaction, preferably at a temperature of from 15 ℃ to 35 ℃. The amount of water is 2 to 10 mol, preferably 2 to 10 mol, and the amount of acid is 1 to 5 mol, based on 1 mol of the compound (1 a). The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Subsequently, a base (e.g., tert-butylamine) is added to the reaction solution to remove the cyanoethyl group. The base is used in an excess molar amount, preferably 30 to 50 molar amount, based on 1 molar amount of the compound (1 a). The reaction time is 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound (2 a). The crude product of the compound (2a) may be subjected to the next step without purification.

(A-2 Process)

This step is a step of removing a protecting group of a hydroxyl group from the compound of formula (2a) by a known organic chemical method to produce a compound of formula (3 a). Before starting the reaction in this step, the crude product of formula (2a) is dried by azeotropic distillation with acetonitrile 1 to 3 times as necessary.

PRO¹In the case of 4, 4 '-dimethoxytrityl group, the compound (2a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid or the like) in a solvent (dichloromethane, chloroform, dichloroethane or the like) at a temperature of from-10 ℃ to the boiling point of the solvent used in the reaction, preferably at a temperature of from 15 ℃ to 35 ℃ to remove the 4, 4' -dimethoxytrityl group. The acid is diluted to 1 to 50% (v/v), preferably 5 to 10% (v/v), with the solvent used in the reaction, and the diluted solution is used in an excess molar amount, preferably 5 to 15 mol. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Adding pyridine into the reaction solutionReaction stopping treatment is carried out. Pyridine is used in an amount sufficient to neutralize the acid used, and preferably 2 to 10 moles based on 1 mole of the acid. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound (3 a). The crude product of compound (3a) was azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile remained at the time of the final azeotropic distillation, and a 0.01M to 1M acetonitrile solution of the compound (3a) was obtained. The obtained acetonitrile solution is directly fed into the next working procedure.

(step A-3)

This step is a step of producing a compound of formula (5a) by continuously performing a coupling reaction with a compound of formula (4a) and a sulfurization reaction of the obtained coupled body, using a known organic chemical method, on a compound of formula (3 a).

Before starting the reaction in this step, compound (4a) was azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile remained at the time of the final azeotropic distillation, and a 0.01M to 1M acetonitrile solution of the compound (4a) was prepared. A drying agent (molecular sieve 3A or molecular sieve 4A in the form of powder or cake) was added to the solution, and the solution was stored under a nitrogen or argon atmosphere before use.

The coupling reaction is carried out by adding an acetonitrile solution of the compound (4a) which has been azeotropically dried to an acetonitrile solution of the compound (3a) at a temperature of 5 to 35 ℃. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Then, a sulfurizing agent (e.g., N-dimethyl-N' - (3-sulfenyl-3H-1, 2, 4-dithiazol-5-yl) formamidine, 3H-1, 2-benzodithiol-3-one) is added to the reaction solution to conduct a sulfurization reaction. The sulfidizing agent is used in an amount of 1 to 5 moles, preferably 1 to 2 moles, based on 1 mole of the compound (3 a). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound (5 a). The obtained crude product of the compound (5a) was directly subjected to the next step.

(A-4 Process)

This step is a step of removing a protecting group of a hydroxyl group from the compound of formula (5a) by a known organic chemical method to produce a compound of formula (6 a).

PRO¹Is 4, 4' -diIn the case of methoxytrityl, the compound of the compound (5a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at a temperature of from-10 ℃ to the boiling point of the solvent used in the reaction, preferably at a temperature of from 15 ℃ to 35 ℃, to remove 4, 4' -dimethoxytrityl. The acid is diluted to 1 to 50% (v/v), preferably 5 to 10% (v/v), with the solvent used in the reaction, and the diluted solution is used in an excess molar amount, preferably 5 to 15 mol, based on 1 mol of the compound (5 a). The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Pyridine was added to the reaction solution to stop the reaction. Pyridine is used in an amount sufficient to neutralize the acid used, and is preferably used in an amount of 10 to 200 moles based on 1 mole of the acid. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound (6 a). The obtained crude product of the compound (6a) was directly subjected to the next step.

(A-5 Process)

This step is a step of producing a compound of formula (7a) by continuously performing a cyclization reaction and a sulfurization reaction on a compound of formula (6a) by a known organic chemical method.

After dissolving the compound (6a) in pyridine, the solution was concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. Adding a dehydration condensation agent (2-chloro-5, 5-dimethyl-1, 3, 2 lambda) to the pyridine solution at a temperature of 5 to 35 DEG C⁵Dioxyphosphocyclohexane (phosphinane) -2-one, etc.) to effect cyclization. The dehydration condensation agent is used in an amount of 1 mol to an excess mol, preferably 3 mol to 5 mol, based on 1 mol of the compound (6 a). The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Then, water and a vulcanizing agent (e.g., 3H-1, 2-benzodithiol-3-one, N-dimethyl-N' - (3-sulfydryl-3H-1, 2, 4-dithiazol-5-yl) formamidine) are added to the reaction solution to carry out a vulcanization reaction. An excess mole, preferably 30 to 50 moles, of water is used, and 1 to 5 moles, preferably 1 to 2 moles, of a sulfurizing agent is used, based on 1 mole of the compound (6 a). Reaction time of 5 minutes-12 hours, preferably 30 minutes to 3 hours. The reaction solution is added to an aqueous sodium bicarbonate solution (0.1M to 1M), and then stirred for 15 minutes to 24 hours to stop the reaction. The reaction solution was extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixed solvent thereof), and the extracts were combined and dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was filtered off and the filtrate was concentrated under reduced pressure. The obtained residue is subjected to silica gel column chromatography [ dichloromethane/methanol, ethyl acetate/methanol, hexane/ethyl acetate, etc. ] ]C18 silica gel column chromatography [ buffer/acetonitrile]Or a combination thereof, and purifying the mixture to obtain the compound (7a) as a mixture of 2 or more diastereomers or as 2 or more pure diastereomers. In this step, 2 kinds of diastereomers are obtained in many cases, but depending on the starting materials (1a) and (4a), 1 kind or 2 kinds of diastereomers may be further obtained in some cases. Even if the compound (7a) obtained is a mixture of a plurality of diastereomers, the next step may be carried out without purification.

(A-6 Process)

This step is a step of simultaneously removing cyanoethyl groups and all the acyl protecting groups from the compound of formula (7a) by a known organic chemical method to produce a compound of formula (8 a). This step is carried out in an autoclave or a sealed tube as required.

PRO⁴In the case of a benzoyl group, the cyanoethyl group and the benzoyl group are removed by treating the compound of compound (7a) with 28% (v/v) aqueous ammonia in a solvent (methanol, ethanol, tetrahydrofuran or a mixed solvent thereof) at a temperature of from 5 ℃ to the boiling point of the solvent used in the reaction. Ammonia is used in an excess molar amount, preferably 300 to 3000 molar amount, based on 1 molar amount of the compound (7 a). The reaction time is 30 minutes to 96 hours, preferably 2 hours to 48 hours. The reaction mixture is concentrated as necessary, and the residue is subjected to preparative HPLC [ buffer/acetonitrile, buffer/methanol, etc. ] ]C18 silica gel column chromatography [ buffer/acetonitrile, buffer/methanol, etc. ]]Or a combination thereof, to give compound (8 a). Even if the obtained compound (8a) is a diastereomer mixture, it may be subjected to the next step without purification. In this step, the reaction mixture may be subjected to the next step without purification.

(step A-7)

This step is a step of producing a compound of formula (9a) by simultaneously removing all silyl protecting groups from a compound of formula (8a) by a known organic chemical method.

PRO²And PRO³In the case of t-butyldimethylsilyl group, the compound (8a) is treated directly with triethylamine trihydrofluoride at a temperature of 5 to 100 ℃ preferably 35 to 60 ℃ to remove the t-butyldimethylsilyl group. The triethylamine trihydrofluoride salt is used in an excess molar amount, preferably 100 to 200 moles, based on 1 mole of the compound (8 a). The reaction time is 30 minutes to 24 hours, preferably 2 hours to 12 hours. After the reaction solution was cooled to room temperature, ice-cooled 1M triethylammonium bicarbonate aqueous solution and 3: 1-10: 1(v/v) of the mixed solution was subjected to a reaction stop treatment. The reaction mixture may be poured into an ice-cooled mixed solution of 1M aqueous triethylammonium bicarbonate and triethylamine, if necessary. In this case, the reaction vessel was washed with acetonitrile and water. The amount of triethylamine used is sufficient to make the liquid of the reaction solution weakly basic, and preferably about 2 moles of triethylamine is used per 1 mole of triethylamine trihydrofluoride salt. The organic solvent component of the reaction solution was distilled off under reduced pressure, and the remaining aqueous solution was subjected to preparative HPLC [ buffer/acetonitrile, buffer/methanol, etc. ] ]C18 silica gel column chromatography [ buffer/acetonitrile, buffer/methanol, etc. ]]Or a combination of these may be purified to obtain the compound (9a) as a single diastereomer.

(A-8 Process)

This step is a step of producing the compound of formula (1) by ion-exchanging the compound of formula (9a) by a known organic chemical method.

A cation exchange resin (BT AG (registered trademark) 50W-X2 resin, 100-mesh 200-mesh, hydrogen form) was suspended in pure water and packed in an empty column tube (column cartridge). The cation exchange resin is used in an amount of 10 to 50 times by weight the amount of the compound (9 a). After allowing excessive pure water to flow down naturally, 1M sodium hydroxide aqueous solution was allowed to flow down naturally for 3 column volumes, and then 6 column volumes of pure water were allowed to flow down naturally. Compound (9a) was dissolved in about 3 column volumes of pure water and packed in the column. When the compound is difficult to dissolve in pure water, a mixture with a small amount of an organic solvent (acetonitrile, methanol, or the like) may be used. After the solution flowing down naturally is separated and extracted, the extracted fraction (fraction) is further eluted with 6 column volumes of pure water or the like. The fractions containing the desired product were combined and lyophilized to give compound (1) as a single diastereomer.

A' method

The CDN derivative represented by (1 ') of the present invention can be produced by the method a' described below.

The present production method is a method for producing a compound represented by the general formula (1') by modifying a part of the method A. Specifically, the compound of the general formula (1 ') can be produced by changing the process of A method A-5 to the process of A' -5 shown below. In addition, the substituent R^xAnd R^yWhen all are halogen atoms, the step A-7 may be omitted.

(A' -5 Process)

This step is a step of producing a compound of formula (7a ') by continuously performing a cyclization reaction and an oxidation reaction on a compound of formula (6 a') by a known organic chemical method.

After dissolving the compound (6 a') in pyridine, the mixture was concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. Adding a dehydration condensation agent (2-chloro-5, 5-dimethyl-1, 3, 2 lambda) to the pyridine solution at a temperature of 5 to 35 DEG C⁵Dioxaphosphorinan-2-one, etc.) to carry out a cyclization reaction. The dehydration-condensation agent is used in an amount of 1 to excessive mol based on 1 mol of the compound (6 a')Preferably, 3 to 5 moles are used. The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Subsequently, water and an oxidizing agent (iodine or the like) are added to the reaction solution to perform an oxidation reaction. The amount of water is 0 to excess mole, preferably 30 to 50 mole, and the amount of the oxidizing agent is 2 to 10 mole, preferably 3 to 5 mole, based on 1 mole of the compound (6 a'). The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction solution is added into sodium bicarbonate aqueous solution (0.1M-1M), and stirred for 15 minutes-24 hours to stop the reaction. The reaction solution is extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixed solvent thereof), and the combined extracts are dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The obtained residue is subjected to silica gel column chromatography [ dichloromethane/methanol, ethyl acetate/methanol, hexane/ethyl acetate, etc. ] ]C18 silica gel column chromatography [ buffer/acetonitrile]Or a combination thereof to obtain compound (7 a').

A' method

The CDN derivative represented by (1 ") of the present invention can be produced according to the method a" described below.

The present production method is a method for producing a compound represented by the general formula (1 ") by modifying a part of the method a. Specifically, the compound of the general formula (1 ") can be produced by changing the step a-3 of method a to the step a" -3 shown below. In addition, the substituent R^xAnd R^yWhen both are halogen atoms, the A-7 step can be omitted.

(A' -3 Process)

This step is a step of producing a compound of formula (5a ") by continuously performing a coupling reaction with a compound of formula (4 a") and an oxidation reaction of the obtained coupled body, using a known organic chemical method, on the compound of formula (3a ").

Before starting the reaction in this step, compound (4a ") was azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile remained at the time of the final azeotropic distillation, and a 0.01M to 1M acetonitrile solution of the compound (4 a') was prepared. A drying agent (molecular sieve 3A or molecular sieve 4A in the form of powder or cake) was added to the solution, and the solution was stored under a nitrogen or argon atmosphere before use.

The coupling reaction is carried out by adding an acetonitrile solution of the compound (4a ") azeotropically dried to an acetonitrile solution of the compound (3 a") at a temperature of 5 to 35 ℃. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, an oxidizing agent (e.g., t-butyl hydroperoxide) is added to the reaction solution to perform an oxidation reaction. The oxidizing agent is used in an amount of 1 to 5 moles, preferably 2 to 3 moles, based on 1 mole of the compound (3a "). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. Adding saturated sodium thiosulfate aqueous solution into the reaction solution, and stirring for 10 minutes to 12 hours to stop the reaction. The reaction solution is extracted 1 to 5 times with an organic solvent (e.g., a mixed solvent of dichloromethane and methanol), and the combined extracts are dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give the crude product of compound (5a "). The crude product of the compound (5a ") obtained was directly subjected to the next step.

A' ″ method

The CDN derivative represented by (1 "') of the present invention can be produced by the method a"' described below.

The present production method is a method for producing a compound represented by the general formula (1') by modifying a part of method A. Specifically, the compound of the general formula (1 ') can be produced by changing the step A-3 of method A to the step A ' -3 and the step A-5 to the step A ' -5. In addition, get Substituent R^xAnd R^yWhen both are halogen atoms, the A-7 step can be omitted.

Method B: coupling precursor (sugar chain coupling)

The coupling precursor represented by (2) of the present invention can be produced by the method B described below.

The manufacturing method is used for manufacturing L¹Substituted at any position by-NH₂The method of coupling the precursor (2).

(step B-1)

This step is a step of producing a compound of formula (2b) by removing a protecting group from a compound of formula (1b) by a known organic chemical method.

PRO⁵In the case of t-butyloxycarbonyl, the protecting group is removed by treating compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixed solvent thereof) at a temperature of from-10 ℃ to the boiling point of the solvent used in the reaction, preferably at a temperature of from 15 ℃ to 35 ℃. The trifluoroacetic acid is used in an excess amount of mol, preferably 20 to 50 mol, based on 1 mol of the compound (1 b). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction mixture was concentrated under reduced pressure, suspended in toluene, and concentrated under reduced pressure. This operation was repeated 2 to 5 times. A solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixed solvent thereof) was added to prepare a slurry, and the solid was filtered to obtain a crude product of the compound (2 b). Compound (A) to (B) 2b) The crude product of (2) is subjected to the next step without purification.

(step B-2)

This step is a step of amidating the compound of formula (2b) with the compound of formula (3b) using a known organic chemical method to produce the compound of formula (4 b).

Amidation is carried out by reacting compound (2b) with a base (triethylamine, N-diisopropylethylamine, etc.) and compound (3b) in a solvent (N, N-dimethylformamide, N-methylpyrrolidone, N-dimethylacetamide, acetonitrile, etc.) at a temperature of 5 to 35 ℃. The amount of the base is 1 to 5 mol and the amount of the compound (3b) is 0.5 to 1.5 mol based on 1 mol of the compound (2 b). The reaction time is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution is poured into two layers of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol or a mixed solvent thereof) and water or an acidic aqueous solution (0.1 to 1M hydrochloric acid, citric acid aqueous solution, or the like), and extracted with the organic solvent for 1 to 5 times. The combined extracts were washed with saturated brine and dried with anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was filtered off and the filtrate was concentrated under reduced pressure. The above-mentioned separation operation can be omitted, and the reaction solution can be concentrated under reduced pressure as it is and subjected to the next purification step using a silica gel column. The obtained residue was purified by silica gel column chromatography [ dichloromethane/methanol, ethyl acetate/methanol, etc. ], whereby compound (4b) was obtained. The obtained compound (4b) is dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol, or a mixed solvent thereof) as needed, and then a poor solvent (diethyl ether, diisopropyl ether, hexane, or the like) is added thereto to reprecipitate, and the solid is collected by filtration to improve the purity.

(step B-3)

This step is a step of producing a compound of formula (5b) by esterifying the compound of formula (4b) by a known organic chemical method.

The compound (4b) is esterified by reacting it with N-hydroxysuccinimide and a condensing agent (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride or the like) in a solvent (N, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, acetonitrile or the like) at a temperature of 5 to 35 ℃. The N-hydroxysuccinimide and the condensing agent are used in an amount of 1 to 3 moles, respectively, based on 1 mole of the compound (4 b). The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. The reaction solution was diluted with an organic solvent (dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof) and washed with ice water 3 to 5 times. The organic layer was dried with anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering off the drying agent, the filtrate was concentrated under reduced pressure to give the crude product of compound (5 b). The obtained compound (5b) can be purified by C18 silica gel column chromatography [ acetonitrile only ] as required. The obtained compound (5b) can be dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane, or a mixed solvent thereof), and then reprecipitated using a poor solvent (diethyl ether, diisopropyl ether, hexane, or the like) to extract a solid by filtration to improve the purity.

(step B-4)

This step is a step of producing a compound of formula (2) by subjecting a compound of formula (5b) to a condensation reaction with a compound of formula (6b) by a known organic chemical method.

The condensation reaction is carried out by reacting the compound (6b) with a base (triethylamine, N-diisopropylethylamine, etc.) and the compound (5b) in a solvent (N, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at a temperature of-10 to 100 ℃ and preferably at a temperature of 15 to 35 ℃. The amount of the base is 2 to 5 moles and 1 to 2 moles of the compound (5b) to 1 mole of the compound (6 b). The reaction time is 5 minutes to 24 hours, preferably 1 hour to 6 hours. Benzylamine was added to the reaction solution to stop the reaction. Benzylamine is used in an amount of 4 to 10 moles based on 1 mole of compound (6 b). The reaction mixture was partially concentrated under reduced pressure as required, and the remaining solution was purified by preparative HPLC [ buffer/acetonitrile, buffer/methanol, etc. ], C18 silica gel column chromatography [ buffer/acetonitrile, buffer/methanol, etc. ], or a combination thereof, to obtain compound (2).

B' method: coupling precursor (cysteine coupling)

B' method

The coupling precursor represented by (2 ') of the present invention can be produced by the method B' described below.

The manufacturing method is used for manufacturing L¹Substituted at any position by-NH₂The method of coupling the precursor (2').

(step B-5)

This step is a step of producing a compound of formula (8b) by amidating a compound of formula (7b) with a compound of formula (2 b') using a known organic chemical method. Compound (8B) was obtained according to the method described in the step B-2 of method B except that no base was used.

(step B-6)

This step is a step of producing a compound of formula (9b) by removing a protecting group from a compound of formula (8b) by a known organic chemical method. PRO⁶For tert-butyl, except that silica gel column chromatography [ dichloromethane/methanol ] is used in the purification operation]In addition, compound (9B) was obtained according to the method described in the step B-1 of Process B.

(step B-7)

This step is a step of producing a compound of formula (10b) by esterifying the compound of formula (9b) by a known organic chemical method. Compound (10B) was obtained according to the method described in Process B-3 of Process B.

(step B-8)

This step is a step of producing a compound of formula (2') by subjecting a compound of formula (6b) to a condensation reaction with a compound of formula (10b) by a known organic chemical method. Compound (2') was obtained according to the method described in Process B-4 of Process B.

C method

The coupling precursor represented by (3) of the present invention can be produced by the method C described below.

The manufacturing method is used for manufacturing L¹A method for coupling the precursor (3) when the hydroxyl group is substituted at an arbitrary position.

(C-1 Process)

This step is a step of producing a compound of formula (3c) by amidating a compound of formula (2c) with a compound of formula (1c) using a known organic chemical method. Compound (3c) was obtained according to the method described in Process B-2 of Process B.

(C-2 Process)

This step is a step of producing a compound of formula (4c) by esterifying the compound of formula (3c) by a known organic chemical method. Compound (4c) was obtained according to the method described in Process B-3.

(step C-3)

This step is a step of producing a compound of formula (7c) by successively carrying out a coupling reaction (aminomethylation) with a compound of formula (6c) and deprotection of the obtained coupled body on the compound of formula (5c) by a known organic chemical method.

PRO⁹In the case of 9-fluorenylmethyloxycarbonyl, aminomethylation is carried out by reacting compound (5c) with compound (6c) and an acid (p-toluenesulfonic acid, etc.) in tetrahydrofuran at a temperature of 5 to 35 ℃. The amount of the compound (6c) is 1 to 20 mol, preferably 2 to 10 mol, and the amount of the acid is 0.05 to an excess mol, preferably 0.1 to 3 mol, based on 1 mol of the compound (5 c). The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. Subsequently, a base (1, 8-diazabicyclo [5.4.0 ] was added to the reaction mixture ]-7-undecene, etc.) to carry out deprotection. When the reaction solution is suspended, a solvent (e.g., N-dimethylformamide) may be added and dissolved as necessary, followed by reaction. The base is used in an excess molar amount, preferably 5 to 20 moles, based on 1 mole of the compound (5 c). The reaction time is 10 minutes to 24 hours, preferably 2 hours to 12 hours. Adding water to the reaction solution, and directly subjecting to C18 silica gel column chromatography [ buffer/acetonitrile, etc. ]]Purification was performed to obtain compound (7 c).

(C-4 Process)

This step is a step of producing a compound of formula (8c) by removing a protecting group from a compound of formula (7c) by a known organic chemical method. PRO⁷And PRO⁸In the case of a tert-butyldimethylsilyl group, the compound (8c) can be obtained according to the method described in the step A-7.

(C-5 Process)

This step is a step of producing a compound of formula (3) by subjecting a compound of formula (8c) to a condensation reaction with a compound of formula (4c) by a known organic chemical method. Compound (3) is obtained according to the method described in Process B-4 of Process B.

C' method

The coupling precursor represented by (3 ') of the present invention can be produced by the C' method described below.

The manufacturing method is used for manufacturing L¹A method of coupling the precursor (3') when the hydroxyl group is substituted at an arbitrary position of (3).

(C' -1 step)

This step is a step of producing a compound of formula (2c ') by continuously performing hydrolysis reaction and removal of a cyanoethyl group on the compound of formula (1 c') by a known organic chemical method. Compound (2 c') was obtained according to the method described in Process A-1.

(C' -2 Process)

This step is a step of producing a compound of formula (3c ') by removing a protecting group for a hydroxyl group from a compound of formula (2 c') by a known organic chemical method. Compound (3 c') was obtained according to the method described in Process A-2.

(C' -3 Process)

This step is a step of producing a compound of formula (5c ') by continuously performing a coupling reaction with a compound of formula (4c ') and a sulfurization reaction or an oxidation reaction of the obtained coupled body on the compound of formula (3c ') by a known organic chemical method. Compound (5c ') was obtained according to the method described in Process A-3 or Process A' -3.

(C' -4 Process)

This step is a step of producing a compound of formula (6c ') by removing a protecting group for a hydroxyl group from a compound of formula (5 c') by a known organic chemical method. Compound (6 c') was obtained according to the method described in Process A-4.

(C' -5 Process)

This step is a step of producing a compound of formula (7c ') by continuously performing a cyclization reaction and a sulfurization reaction or an oxidation reaction on a compound of formula (6 c') by a known organic chemical method. Compound (7c ') was obtained according to the method described in Process A-5 or Process A ' to Process A ' -5.

(C' -6 Process)

This step is a step of producing a compound of formula (8c ') by simultaneously removing the cyanoethyl group and all the acyl protecting groups from the compound of formula (7 c') by a known organic chemical method. Compound (8 c') was obtained according to the method described in Process A-6.

(C' -7 Process)

This step is a step of producing a compound of formula (9c ') by simultaneously removing all silyl-based protecting groups from a compound of formula (8 c') by a known organic chemical method. PRO⁹In the case of 2- (trimethylsilyl) ethoxycarbonyl, the compound (8 c') is reacted with tetra-ethyl-phenyl-ethyl at a temperature of 5 ℃ to 100 ℃, preferably at a temperature of 35 ℃ to 60 ℃A solution of butylammonium fluoride in tetrahydrofuran was treated to thereby remove the 2- (trimethylsilyl) ethoxycarbonyl group. The tetrabutylammonium fluoride is used in an excess amount, preferably 10 to 30 moles, based on 1 mole of the compound (8 c'). The reaction time is 1 to 48 hours, preferably 4 to 24 hours. After the reaction mixture was diluted with a buffer, the organic solvent component was distilled off under reduced pressure as needed. The residue was subjected to preparative HPLC [ buffer/acetonitrile, buffer/methanol, etc. ] ]C18 silica gel column chromatography [ buffer/acetonitrile, buffer/methanol, etc. ]]Or a combination thereof, to obtain compound (9 c').

(C' -8 Process)

This step is a step of producing a compound of formula (3 ') by subjecting a compound of formula (9 c') to a condensation reaction with a compound of formula (4c) by a known organic chemical method. Compound (3') was obtained according to the method described in Process B-4 of Process B.

Method D: production of sugar chain-reconstituted antibody

The sugar chain-restructured antibody can be produced by the method shown in the following formula according to, for example, the method described in WO2018/003983 or the like.

(step D-1)

This step is a step of producing a sugar chain-cleaved antibody by cleaving a glycoside bond between GlcNAc β 1-4 GlcNAc of a reduced terminal chitobiose structure of an N-linked sugar chain (N297-linked sugar chain) bound to asparagine at position 297 of the amino acid sequence of the antibody by hydrolysis using a known enzyme reaction on a target antibody.

The target antibody (1d) (10mg/mL) was subjected to hydrolysis reaction of the glycosidic bond between GlcNAc β 1 and 4GlcNAc of the chitobiose structure at the reduction end in a buffer (such as a phosphate buffer) at a temperature of 0 ℃ to 40 ℃ using a hydrolase such as a wild-type Endos enzyme. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type Endos enzyme is used in an amount of 0.1 to 10mg, preferably 0.1 to 3mg, per 100mg of the antibody (1 d). After completion of the reaction, the antibody (Fuc. alpha.1, 6) GlcNAc was obtained by purification using affinity chromatography (HiTrap protein A FF (5ml) (manufactured by GE HEALTH CARE)) and/or a hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5ml) (manufactured by BIO-RAD)).

(step D-2)

This step is a step of producing a sugar chain-reshaped antibody (3D) by binding SG-type or MSG (MSG1, MSG2) -type sugar chain oxazoline bodies (hereinafter referred to as "azido sugar chain oxazoline bodies") having an azido-containing PEG linker to the (Fuc. alpha.1, 6) GlcNAc antibody (2D) obtained in the D-1 step by using a known enzyme reaction.

The antibody (2D) is reacted with an azido sugar chain oxazoline body in a buffer solution (such as a phosphate buffer solution) at a temperature of 0 ℃ to 40 ℃ in the presence of a glycosyltransferase such as Endos (D233Q/Q303L) to carry out a sugar chain transfer reaction. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. The EndoS enzyme (D233Q/Q303L) is used in an amount of 1mg to 10mg, preferably 1mg to 3mg, and the azido sugar chain oxazoline compound is used in an amount of 2 equivalents to an excess amount, preferably 4 equivalents to 20 equivalents, based on 100mg of the antibody. After completion of the reaction, the antibody was purified by affinity chromatography (HiTrap rProtein A FF (5ml, manufactured by GEHEALTH CARE)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5ml, manufactured by BIO-RAD)) to obtain a sugar chain-reconstituted antibody (3 d).

In the above-described production of the sugar chain-reconstituted antibody, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to the common procedures A to C described later.

Incidentally, SG type azido sugar chain oxazoline was synthesized by the method described in WO 2018/003983. As an example, [ N ]₃－PEG(3)]₂The synthesis method of-SG (10) -Ox (compounds 1-10 described in WO 2018/003983) is shown as the following formula.

An MSG type azido sugar chain oxazoline was also synthesized according to the method described in WO 2018/003983. As an example, [ N ]₃－PEG(3)]The synthesis method of-MSG 1(9) -Ox (compounds 1-11 described in WO 2018/003983) is shown as the following formula.

E method: conjugation of antibody to drug (sugar chain conjugation 1)

(wherein 2 asterisks (, C) on the left side of the antibody-drug conjugate (1e) represent the drug linker moiety indicated by the asterisk on the right side.)

The present production method is a method for producing an antibody drug conjugate (1e) by reacting and binding a sugar chain-reconstituted antibody (3D) obtained in the D-method step (2) and a conjugate precursor (2) obtained in the B-method step (4) with SPAAC (Strain-promoted azidoyne cycloaddition: J.Am.chem.Soc.2004, 126, 15046-15047).

(E-1 Process)

The SPAAC reaction is carried out by mixing a buffer solution (phosphate buffer, acetate buffer, borate buffer, etc.) of the sugar chain-restructured antibody (3d) with a solution obtained by dissolving the coupling precursor (2) in an appropriate solvent (dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixed solvent of these). The coupling precursor (2) is used in an amount of 2 to excessive moles, preferably 4 to 30 moles, based on 1 mole of the sugar chain reshaped antibody (3d), and the ratio of the organic solvent to the buffer solution of the antibody is preferably 1 to 200% (v/v). The reaction temperature is 0 ℃ to 37 ℃, preferably 15 ℃ to 25 ℃, and the reaction time is 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution is preferably 5 to 9. The reaction solution was purified by the method described in the common procedure D to obtain an antibody-drug conjugate (1 e).

E' method: conjugation of antibodies to drugs (cysteine conjugation)

The antibody drug conjugate of the present invention having cysteine conjugation can be produced using the target antibody prepared according to reference example 1 or the like and the conjugation precursor (2 ') having a maleimide group obtained in the step of B' method B-8, according to the method described in WO2014/057687 or the like.

E' method: conjugation of antibody to drug (sugar chain conjugation 2)

In the E method, the conjugate precursor (2) was changed to the conjugate precursor (3 ') obtained in the C ' method C ' -8 step, whereby an antibody drug conjugate (1E ") represented by the following formula was obtained.

(in the formula, the antibody drug conjugate (1 e') is shown with 2 asterisks on the left side¹) Representing the drug linker portion shown by the asterisk on the right. )

The antibody drug conjugate can be identified by performing buffer exchange, purification, measurement of the antibody concentration, and measurement of the average number of drug bound per molecule of antibody by the common operations D to G described later.

Common operation a: concentration of aqueous antibody solution

An antibody or antibody-drug conjugate solution was added to an Amicon (registered trademark) Ultra centrifugal filter device (50000NMWL, Merck Millipore Ltd.), and the antibody and antibody-drug conjugate solution were concentrated by a centrifugation operation (centrifugation at 2000G to 4000G for 5 minutes to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

Common operation B: determination of antibody concentration

Antibody concentrations were determined according to the manufacturer's specified method using a UV-meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.). In this case, different 280nm absorbances (1.3 mLmg) were used for each antibody^－1cm^－1～1.8mLmg^－1cm^－1)。

And C, common operation: buffer exchange of antibodies

To the antibody aqueous solution, a buffer (phosphate buffered saline (pH6.0), phosphate buffer (pH6.0), or the like) was added, and the mixture was concentrated according to the method described in general procedure A. After this operation was carried out several times, the antibody concentration was measured according to the method described in the common operation B. To the antibody buffer solution, an appropriate buffer (phosphate buffered saline (ph6.0), phosphate buffer (ph6.0), or the like) is added to prepare an antibody buffer solution at a target concentration (e.g., about 10 mg/mL).

Common operation D: refining of antibody drug conjugate (gel filtration chromatography)

The NAP column (NAP-5, NAP-10, NAP-25 (manufactured by GEHEALTH CARE)) was equilibrated with an Acetate Buffer (10mM Acetate Buffer, 5% Sorbitol, pH 5.5; referred to as ABS in the present specification) or other appropriate Buffer. The NAP column was filled with the antibody drug conjugate reaction solution, and the antibody fraction was collected by allowing a manufacturer-specified amount of the buffer to flow down naturally. This fraction was again loaded into a NAP column, and the antibody fraction was separated by allowing the manufacturer-specified amount of buffer to flow down naturally. This operation was repeated 2 to 3 times in total to obtain an antibody drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol were removed. The concentration of the antibody drug conjugate solution was adjusted by common procedures a and C as needed.

Common operation E: determination of antibody concentration of antibody drug conjugate and average number of drug bound per molecule of antibody (UV method)

The bound-drug concentration of the antibody-drug conjugate was calculated by measuring the absorbance at 2 wavelengths, 280nm and 260nm (wavelengths other than 260nm may be used) of an aqueous solution of the antibody-drug conjugate using an absorptiometer (UV/VIS Spectrometer Lambda 25, PerkinElmer, Inc.). Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all the absorbing chemical species present in the system (additive absorbance), the antibody concentration and the drug concentration of the antibody-drug conjugate are shown in the following relational expressions, assuming that the molar absorption coefficients of the antibody and the drug are unchanged before and after the antibody-drug conjugate.

A₂₈₀＝A_D，₂₈₀+A_A，₂₈₀＝ε_D，₂₈₀C_D+ε_A，₂₈₀C_AFormula (I)

A₂₆₀＝A_D，₂₆₀+A_A，₂₆₀＝ε_D，₂₆₀C_D+ε_A，₂₆₀C_AFormula (II)

In the formula, A₂₈₀The absorbance of an aqueous solution of the antibody drug conjugate at 280nm, A₂₆₀Represents the absorbance at 260nm of an aqueous solution of an antibody drug conjugate, A_A，₂₈₀Represents the absorbance of the antibody at 280nm, A_A，₂₆₀Represents the absorbance of the antibody at 260nm, A_D，₂₈₀Represents the absorbance, A, of the conjugate precursor at 280nm_D，₂₆₀Represents the absorbance,. epsilon., of the conjugate precursor at 260nm _A，₂₈₀Represents the molar absorptivity, ε, of an antibody at 280nm_A，₂₆₀Represents the molar absorptivity, ε, of an antibody at 260nm_D280 denotes the molar absorptivity,. epsilon.of the conjugate precursor at 280nm_D，₂₆₀Denotes the molar absorption coefficient, C, of the conjugate precursor at 260nm_AIndicates the antibody concentration, C, in the antibody drug conjugate_DIndicates the concentration of the drug in the antibody drug conjugate. In the formula, epsilon_A，₂₈₀、ε_A，₂₆₀、ε_D，₂₈₀、ε_D，₂₆₀A value prepared in advance (calculation estimated value or actual measurement value) may be used. E.g. epsilon_A，₂₈₀Can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol.4, 2411-2423). Epsilon_A，₂₆₀Using the measured values obtained by UV measurement of the antibody and from ε_A，₂₈₀The estimated value of (2). In the examples, the molar absorptivity of the engineered anti-HER 2 antibody was determined using epsilon_A，₂₈₀215380 and ε_A，₂₆₀110117. Engineering molar absorptivity of anti-LPS antibodies Using ε_A，₂₈₀227300 andε_A，₂₆₀＝110710。ε_D，₂₈₀and epsilon_D，₂₆₀The absorbance of a solution obtained by dissolving the conjugate precursor used in a certain molar concentration can be measured according to Lambert-beer (absorbance ═ molar concentration × molar absorption coefficient × channel optical path length) law. The molar absorption coefficients of the conjugate precursors in the examples were each determined by UV. Determination of aqueous solution of antibody drug conjugate A ₂₈₀And A₂₆₀These values are substituted into the equations (I) and (II) to solve the simultaneous equations to obtain C_AAnd C_D. Further by mixing C_DIs divided by C_AThe average number of drug binding per molecule of antibody can be determined.

Common operation F: determination of antibody concentration in antibody drug conjugates and average number of drug binding per molecule of antibody (reversed phase high performance liquid chromatography: RP-HPLC)

The antibody concentration of the antibody-drug conjugate and the average number of drug bound per molecule of the antibody can be determined by high performance liquid chromatography analysis using the following method in addition to the above-described common procedure E.

[ F-1. preparation of sample for HPLC analysis (reduction of antibody-drug conjugate) ]

The antibody drug conjugate solution (about 1mg/mL, 60. mu.L) was mixed with Dithiothreitol (DTT) in water (100mM, 15. mu.L). The mixture was incubated at 37 ℃ for 30 minutes (incubate) to cleave the disulfide bond between the L chain and H chain of the antibody drug conjugate. The reaction solution was used directly for HPLC analysis.

[ F-2. HPLC analysis ]

Representative assay conditions are as follows.

HPLC system: agilent 1290HPLC System (Agilent Technologies)

A detector: ultraviolet absorption photometer (measuring wavelength: 280nm)

Column: acquisty BEH Phenyl (2.1X 50mm, 1.7 μm, made by Waters)

Column temperature: 75 deg.C

Flow rate: 0.8mL/min

Sample injection amount: 10 μ L

Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% aqueous isopropanol

Mobile phase B: 0.075% TFA, 15% Isopropanol acetonitrile solution

Gradient program (mobile phase B): 14% -36% (0 min-15 min), 36% -80% (15-17 min), 80% -14% (17 min-17.1 min), 14% -14% (17.1 min-23 min)

[ F-3. data analysis ]

[ F-3-1 ] the H chain to which the drug was bound (H chain to which one drug was bound: H1, H chain to which 2 drugs were bound: H2) was increased in hydrophobicity in proportion to the number of bound drugs as compared with the L chain (L0) and H chain (H0) of the antibody to which no drug was bound, and the retention time was prolonged, and therefore, elution was performed in the order of L0, H0, H1, and H2 in principle. By comparing the retention times with L0 and H0, the detected peak can be assigned to any one of L0, H0, H1, H2.

[ F-3-2 ] since the drug linker has UV absorption, the peak area was corrected according to the following formula using the molar absorption coefficients of the H chain and the drug linker depending on the number of the drug linker to be bonded.

In the formula, the molar absorption coefficients (280nm) of the L chain and H chain of each antibody were estimated by a known calculation method described in common operation E. In the case of the engineered anti-HER 2 antibody, 26213 was used for the molar absorption coefficient of the L chain and 81478 was used for the molar absorption coefficient of the H chain. Similarly, in the case of the modification of anti-LPS antibody, 27703 was used as the molar absorption coefficient of L chain and 85948 was used as the molar absorption coefficient of H chain. The molar absorption coefficient (280nm) of the drug linker was determined using the observed values of the coupling precursors in the case of coupling under the SPAAC reaction, and using the observed values of the compounds in which each coupling precursor was reacted with mercaptoethanol or N-acetylcysteine to convert the maleimide group to succinimide sulfide in the case of cysteine coupling.

[ F-3-3 ] the peak area ratio (%) of each chain to the sum of the peak area correction values was calculated according to the following formula.

[ F-3-4 ] the average number of drug-bound (DAR) per molecule of antibody of the antibody-drug conjugate was calculated according to the following formula.

[ F-3-5 ] the antibody concentration of the antibody-drug conjugate was calculated according to the following formula.

Here, the absorbance (280nm) of the antibody-drug conjugate was measured using an aqueous solution of the antibody-drug conjugate. The dilution factor means that the antibody-drug conjugate aqueous solution is diluted several times, usually 4 times, when the absorbance is measured. The molar absorption coefficient (280nm) of the antibody was estimated by a known calculation method described in the common operation E. The average number of drug combinations was determined by using the value obtained in [ F-3-4 ]. The molar absorption coefficient (280nm) of the drug linker was measured for the coupling under SPAAC reaction using the coupling precursor, and for the cysteine coupling using the compound obtained by reacting each drug linker with mercaptoethanol or N-acetylcysteine to convert the maleimide group into succinimide thioether.

Common operation G: determination of antibody concentration of antibody drug conjugate and average number of drug bound per molecule of antibody (hydrophobic interaction-high Performance liquid chromatography: HI-HPLC)

The antibody concentration of the antibody-drug conjugate and the average number of drug bound per molecule of antibody can be determined by high performance liquid chromatography analysis using the following method in addition to the above-described common procedures E and F.

[ G-1. preparation of sample for HPLC analysis ]

The antibody drug conjugate solution (about 1mg/mL, 60. mu.L) was used directly for HPLC analysis.

[ G-2. HPLC analysis ]

Representative analysis conditions are as follows.

HPLC system: SHIMADZU CBM-20A (Shimadzu corporation)

A detector: ultraviolet absorption photometer (measuring wavelength: 280nm)

Column: TSK-gel Butyl-NPR (4.6X 100mm, 2.5 μm, manufactured by TOSOH)

Column temperature: fixed temperature around 25 DEG C

Mobile phase A: 25mM phosphate buffer (pH 7.0) containing 1.5M ammonium sulfate

Mobile phase B: 25mM phosphate buffer (pH 7.0)/Isopropanol mixture (3: 1)

Flow rate: 0.8mL/min

Sample injection amount: 15 μ L

Gradient program (mobile phase B): 10% -15% (0 min-5 min), 15% -65% (5 min-20 min)

Or, HPLC system: SHIMADZU CBM-20A (Shimadzu corporation)

A detector: ultraviolet absorption photometer (measuring wavelength: 280nm)

Column: polypypyl a (4.6 x 100mm, 3 μm,PolyLC system

Column temperature: fixed temperature around 40 deg.C

Mobile phase A: 20mM phosphate buffer (pH 7.4) containing 1.5M ammonium sulfate

Mobile phase B: 20mM phosphate buffer (pH 7.4)

Flow rate: 0.8mL/min

Sample injection amount: 15 μ L

Gradient program (mobile phase B): 40% -80% (0 min-20 min)

[ G-3. data analysis ]

Since the hydrophobicity increases in proportion to the number of drugs bound to the antibody and the retention time increases, DAR-0, DAR-2 and DAR-4 are eluted in that order in principle in the case of coupling in the SPAAC reaction. By comparing the retention time to DAR ═ 0, the detected peak can be assigned to either DAR ═ 2 or DAR ═ 4. The detection of DAR ═ 1 and DAR ═ 3 peaks sometimes depends on the type of antibody or drug linker. The DAR that detected a peak was also estimated by measuring the mass spectrum after separating the peak by HI-HPLC.

[ G-3-2 ] since the drug linker has UV absorption, the peak area value was corrected according to the following formula using the molar absorption coefficients of the antibody and the drug linker according to the number of bound drug linkers.

In the formula, the molar absorption coefficient (280nm) of the antibody was estimated by a known calculation method described in common operation E. Molar absorptivity of drug linker (280nm) the observed value of the coupled precursor was used.

[ G-3-3 ] the total antibody peak area ratio (%) was calculated from the corrected peak area value according to the following formula.

[ G-3-4 ] the average number of drug-bound antibody molecules of the antibody-drug conjugate was calculated according to the following formula.

The antibody concentration of [ G-3-5 ] antibody-drug conjugate was calculated according to the formula described in [ F-3-5 ]. In this case, the average number of drug bound was the value obtained in [ G-3-4 ].

The novel CDN derivative, the antibody drug conjugate, or the intermediate for producing the same according to the present invention may include stereoisomers, optical isomers such as optical isomers, geometric isomers, tautomers, or d-, l-, atropisomers derived from asymmetric carbon atoms, and these isomers, optical isomers, and mixtures thereof are included in the present invention.

In the antibody drug conjugate, the binding number of the drug and one molecule of antibody is an important factor influencing the effectiveness and safety of the antibody drug conjugate. The production of antibody drug conjugates is carried out under reaction conditions such as the amounts of raw materials and reagents used in the reaction are determined so that the number of drug conjugates is fixed, but in general, a mixture in which different numbers of drugs are bound is obtained, unlike the chemical reaction of low-molecular compounds. The number of drug binding to a molecule of antibody can be specified as the average, i.e., the average number of drug binding (DAR). The number of cyclic dinucleotide derivatives bound to antibody molecules can be controlled, and the average number of drug binding per molecule of antibody can be in the range of 1 to 10, preferably 1 to 8, and more preferably 1 to 5 cyclic dinucleotide derivatives bound thereto.

In the antibody drug conjugate of the present invention, when the antibody Ab is bound to L from the reconstituted sugar chain of the antibody Ab, the number of drug binding m per molecule of the antibody in the antibody drug conjugate²Is an integer of 1 or 2. When the sugar chain is N297 sugar chain and the sugar chain is N297- (Fuc) SG, m is²2 and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297- (Fuc) MSG1, N297- (Fuc) MSG2 or a mixture of N297- (Fuc) MSG1 and N297- (Fuc) MSG2, m is²1 and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

Further, those skilled in the art can design a reaction of binding an antibody to a desired number of drugs according to the description of examples of the present application, and can obtain an antibody in which the number of binding of cyclic dinucleotide derivatives is controlled.

The CDN derivative, the antibody drug conjugate, and the intermediate for producing these of the present invention may be left in the air or recrystallized to absorb water, and may be adsorbed by water or form a hydrate, and compounds and salts containing water are also included in the present invention.

When the CDN derivative, the antibody drug conjugate, or the intermediate for producing these compounds of the present invention has a basic group such as an amino group, a pharmaceutically acceptable salt can be produced as expected. Examples of such salts include hydrohalic salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonic acid salts such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formic acid, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate and aspartate.

The CDN derivative or the antibody drug conjugate of the present invention may form a base addition salt in general because it contains a phosphate group and/or a phosphorothioate group in its structure. When these production intermediates have an acidic group such as a carboxyl group, a base addition salt can be generally formed. Examples of the pharmaceutically acceptable salt include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N' -dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N- (2-phenylethoxy) amine salt, piperazine salt, tetramethylammonium salt, and tris (hydroxymethyl) aminomethane salt.

The CDN derivative, the antibody drug conjugate, and the production intermediate thereof of the present invention may exist in the form of a hydrate by absorbing moisture in the air, for example. The solvate of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and specifically, a hydrate, an ethanolate, a 2-propanol complex, and the like are preferable. In addition, when a nitrogen atom is present in the CDN derivative, the antibody drug conjugate, or the production intermediate thereof of the present invention, an N-oxide may be formed, and these solvates and N-oxides are also included in the scope of the present invention. In addition, the CDN derivative, the antibody drug conjugate, and the intermediate for producing the same of the present invention may form a sulfoxide in the presence of a sulfur atom, and such solvates and sulfoxides are also included in the scope of the present invention.

In addition, the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. One or more of the atoms constituting the CDN derivative, the antibody drug conjugate, and the production intermediate thereof of the present invention may contain an unnatural proportion of an atomic isotope. Examples of the atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), and carbon-14 (14C). Alternatively, the compounds of the invention may be radiolabeled with a radioisotope, such as tritium (3H), iodine-125 (125I) or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents (e.g., analytical reagents), and diagnostic agents (e.g., in vivo diagnostic imaging agents). All isotopic variations of the antibody drug conjugates of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

<4. medicine >

The CDN derivative or the antibody drug conjugate of the present invention exhibits antitumor immune activity or cytotoxicity against cancer cells, and thus can be used as a medicine, particularly, a therapeutic and/or prophylactic agent against cancer, or an antitumor agent.

Examples of the cancer type to which the CDN derivative or the antibody drug conjugate of the present invention is suitably applied include lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), kidney cancer, urothelial cancer, large intestine cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (e.g., epithelial superficial tumors, interstitial tumors, germ cell tumors), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, uterine corpus cancer, testicular cancer (e.g., seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain Tumor, head and neck cancer, thyroid cancer, mesothelioma, Gastrointestinal Stromal Tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, and the like The cancer cell expressing a protein that can be recognized is not limited to this.

The CDN derivative or the antibody drug conjugate of the present invention can be administered to mammals, more preferably to humans.

The substance used in the pharmaceutical composition containing the CDN derivative or the antibody drug conjugate of the present invention can be appropriately selected and used from pharmaceutical additives and the like generally used in the art according to the dose and administration concentration.

The CDN derivative or the antibody drug conjugate of the present invention can be administered in the form of a pharmaceutical composition comprising one or more pharmaceutically compatible ingredients. For example, typically, such pharmaceutical compositions comprise 1 or more pharmaceutical carriers (carriers) (e.g., sterile liquids (e.g., comprising water and oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case where the above-mentioned pharmaceutical composition is administered intravenously, water is a more representative carrier. Saline solution, as well as aqueous dextrose (dextrose) and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are well known in the art. The above composition may further contain a trace amount of a wetting agent or an emulsifying agent, or a pH buffering agent, as required. Examples of suitable Pharmaceutical carriers are described in e.w. martin, "Remington's Pharmaceutical Sciences". The prescription corresponds to the mode of administration.

Various delivery systems are known and may be used for the administration of the CDN derivatives or antibody drug conjugates of the invention. Examples of the introduction method include intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes, but are not limited thereto. Administration may be by infusion or bolus injection (bolus injection), for example. In a particularly preferred embodiment, the administration of the CDN derivative or the antibody drug conjugate described above is by infusion. Parenteral administration is the preferred route of administration.

In representative embodiments, the pharmaceutical composition comprising the antibody drug conjugate described above is formulated according to conventional procedures for pharmaceutical compositions suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the above-mentioned medicine may further contain a solubilizing agent and a local anesthetic (e.g., lidocaine) for alleviating pain at the injection site. Generally, the above ingredients are supplied separately, for example, in the form of a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container such as an ampoule or a Sachet (Sachet) showing the amount of the active agent, or mixed together in a unit dosage form. In the case where the above-described pharmaceutical composition is intended to be administered by infusion, it may be administered, for example, by means of an infusion bottle containing sterile pharmaceutical grade water or saline. When the above-mentioned medicines are administered by injection, an ampoule of sterile water for injection or saline may be provided, for example, in such a manner that the above-mentioned components are mixed before administration. The above pharmaceutical compositions are sometimes provided in the form of solutions.

The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the CDN derivative or the antibody drug conjugate of the present invention, or may be a pharmaceutical composition containing the CDN derivative or the antibody drug conjugate and at least one cancer therapeutic agent other than the CDN derivative or the antibody drug conjugate. The CDN derivative or the antibody drug conjugate of the present invention may be administered together with other cancer therapeutic agents, whereby the anticancer effect may be enhanced. The other anticancer agents used for this purpose may be administered to the subject simultaneously, separately or sequentially with the CDN derivative or the antibody drug conjugate, or may be administered with varying intervals between the respective administrations. Examples of such a cancer therapeutic agent include albumin-bound paclitaxel (abraxane), carboplatin (carboplatin), cisplatin (cissplatin), gemcitabine (gemcitabine), irinotecan (CPT-11) (irinotecan), paclitaxel (paclitaxel), pemetrexed (pemetrexed), sorafenib (sorafenib), vinblastine (vinblastine), or a drug and LH-RH analog (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonist (tamoxifen, raloxifene, etc.), aromatase inhibitor (anastrozole, letrozole, exemestane, etc.), immune checkpoint inhibitor (nivolumab), ipilimumab (ipilimumab), and the like. The drug is not limited as long as it has an antitumor activity.

Such pharmaceutical compositions may be formulated as lyophilized preparations or as liquid preparations having the selected composition and the desired purity. When the formulation is a lyophilized formulation, it may be a formulation comprising appropriate formulation additives used in the art. In addition, the liquid preparation may be formulated as a liquid preparation containing various formulation additives used in the art as well.

The composition and concentration of the pharmaceutical composition also vary depending on the administration method, and the antibody drug conjugate contained in the pharmaceutical composition of the present invention can exert a drug effect even in a small amount of administration, as the affinity of the antibody drug conjugate for an antigen, that is, the dissociation constant (Kd value) for the antigen is higher (Kd value is lower). Therefore, when determining the amount of the antibody drug conjugate to be administered, the amount to be administered can be set according to the affinity between the antibody drug conjugate and the antigen. When the CDN derivative or the antibody drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100mg/kg may be administered once, or may be administered multiple times at intervals of once every 1 to 180 days.

The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Examples

In the following examples, room temperature is 15 ℃ to 35 ℃. Dehydrated acetonitrile was used as acetonitrile (dehydrated) -Super-sold by kanto chemical or as acetonitrile (Super-dehydrated) sold by Wako pure chemical industries. Pyridine the pyridine (dehydrated) -Super-sold by kanto chemical was used. Silica gel chromatography was performed using Biotage SNAP Ultra (manufactured by Biotage), chromatex Q-Pack SI (manufactured by Silysia, Fuji) or Purif-Pack-Ex SI (manufactured by Showa Science). DIOL silica gel column chromatography was performed using Chromatorex Q-pack DIOL (manufactured by Silysia, Fuji). The C18 silica gel column chromatography was performed using Biotage SNAP Ultra C18 (Biotage). Amino silica gel column chromatography Using BiotageSNAP Isolute NH₂(Biotage Co., Ltd.). Preparative HPLC was performed using a SHIMADZU SPD-M10A HPLC system (Shimadzu corporation) or the like. The column was prepared using a kinetic column of Kinetex (5 μm, C18,250X 30.0mm, manufactured by Phenomenex) or Kinetex (5 μm, C18,250X 21.2mm, manufactured by Phenomenex).

The following instruments were used to measure various spectral data.¹H-NMR spectra were measured using JEOL ECS-400 (400MHz), Varian 400-MR (400MHz) or Varian Unity Inova 500(500 MHz).³¹P-NMR spectra were determined using JEOL ECS-400 (160 MHz). Mass spectra were determined using an Agilent 6130Quadrupole LC/MS system (Agilent Technologies). The LC/MS assay was performed under the following conditions [ column: develosil Combi-RP, 5 μm, 50X 2.0mm (manufactured by Nomura chemical Co., Ltd.), mobile phase: 0.1% formic acid acetonitrile solution/0.1% formic acid aqueous solution, 0.1% formic acid acetonitrile solution: 2% -100% (0 min-5 min or 0 min-10 min) ]。

Example 1: synthesis of CDN1

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5-iodo-7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

In a solution of 5-iodotubercidin (1.0g) in N, N-dimethylformamide (10mL) known in the literature (Tetrahedron 2007, 63, 9850-. After imidazole (868mg) was added at 0 ℃, the temperature was raised to room temperature and the mixture was stirred for 30 minutes. T-butyldimethylsilyl chloride was added at room temperature and stirred at the same temperature overnight. Saturated aqueous sodium bicarbonate was added to the reaction solution, and after stopping the reaction, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (910 mg).

MS(ESI)m/z：647(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.25(1H，s)，7.03(1H，s)，6.10(1H，s)，5.63(2H，brs)，4.49－4.44(2H，m)，4.26(1H，dd，J＝9.7，4.8Hz)，4.17(1H，m)，4.00(1H，t，J＝9.7Hz)，1.09(9H，s)，1.04(9H，s)，0.91(9H，s)，0.13(3H，s)，0.11(3H，s).

(step 2)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (3, 3-diethoxyprop-1-yn-1-yl) -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

To a mixed solution of the compound (910mg) obtained in the above step 1 in N, N-dimethylformamide (3.0mL) -tetrahydrofuran (9.0mL) were added propargylaldehyde dimethylacetal (1.01mL), triethylamine (0.392mL), tetrakis (triphenylphosphine) palladium (0) (163mg), and copper (I) iodide (53.6mg) in this order, and the mixture was stirred at 40 ℃ for 18 hours. Saturated aqueous sodium bicarbonate and ethyl acetate were added to the reaction mixture, and extraction was performed with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (878 mg).

MS(ESI)m/z：647(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.27(1H，s)，7.17(1H，s)，6.09(1H，s)，5.56(2H，brs)，5.50(1H，s)，4.48(1H，dd，J＝9.1，4.9Hz)，4.42(1H，d，J＝4.9Hz)，4.25(1H，dd，J＝9.4，4.6Hz)，4.17(1H，m)，4.00(1H，t，J＝9.7Hz)，3.85－3.77(2H，m)，3.66(2H，m)，1.28(6H，t，J＝7.3Hz)，1.08(9H，s)，1.04(9H，s)，0.91(9H，s)，0.13(3H，s)，0.11(3H，s).

(step 3)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (878mg) obtained in the above step 2 in ethanol (8.8mL), 10% palladium on carbon (M) wet (500mg) was added, and the mixture was stirred at room temperature for 9 hours under a hydrogen atmosphere. After removing the catalyst by filtration, the mixture was washed with methylene chloride, and the filtrate was concentrated under reduced pressure. To a solution of the residue in acetic acid (8.8mL) was added 10% palladium on carbon (M) wet (500mg), and the mixture was stirred under hydrogen atmosphere at 40 ℃ for 2 days. After removing the catalyst by filtration, the mixture was washed with methylene chloride, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (603 mg).

MS(ESI)m/z：561(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.47(1H，brs)，8.07(1H，s)，6.70(1H，s)，6.14(1H，s)，4.47－4.43(2H，m)，4.29(1H，dd，J＝9.1，4.8Hz)，4.15(1H，m)，3.99(1H，t，J＝9.7Hz)，3.55(2H，m)，2.89(2H，t，J＝5.4Hz)，2.04(2H，m)，1.09(9H，s)，1.04(9H，s)，0.90(9H，s)，0.10(3H，s)，0.10(3H，s).

(step 4)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Pyridine (1.56mL), N-dimethylaminopyridine (94.5mg) and benzoyl chloride (0.898mL) were added in this order to a solution of the compound (2.17g) obtained in the above step 3 in dichloromethane (21.7mL) at room temperature, and the mixture was stirred at 50 ℃ for 15 hours. To the reaction mixture was added a saturated aqueous sodium bicarbonate solution to stop the reaction. After extraction with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (1.91 g).

MS(ESI)m/z：665(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.08(1H，s)，7.37－7.33(3H，m)，7.23(2H，t，J＝7.6Hz)，6.97(1H，s)，6.21(1H，s)，4.50－4.46(2H，m)，4.37－4.30(2H，m)，4.28－4.09(2H，m)，4.02(1H，t，J＝10.0Hz)，3.03(2H，t，J＝6.3Hz)，2.29－2.17(2H，m)，1.10(9H，s)，1.05(9H，s)，0.90(9H，s)，0.10(6H，s).

(step 5)

6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (1.91g) obtained in the above step 4 in dichloromethane (15mL) was added a mixture of hydrogen fluoride-pyridine (0.30mL) and pyridine (1.88mL) prepared at 0 ℃ and the mixture was stirred at 0 ℃ for 2 hours. The reaction mixture was added with a saturated aqueous sodium bicarbonate solution to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (15mL), and 4, 4' -dimethoxytrityl chloride (1.17g) was added thereto, followed by stirring at 0 ℃ for 12 hours. After adding methanol and stirring for 30 minutes, a saturated aqueous sodium bicarbonate solution was added to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (1.98 g).

MS(ESI)m/z：827(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.07(1H，s)，7.47(2H，m)，7.37－7.19(13H，m)，6.84(4H，m)，6.37(1H，d，J＝5.5Hz)，4.75(1H，t，J＝5.2Hz)，4.38－4.20(4H，m)，3.80(6H，s)，3.53(1H，dd，J＝10.7，2.8Hz)，3.40(1H，dd，J＝11.0，3.1Hz)，2.83(1H，d，J＝3.7Hz)，2.78(2H，t，J＝6.4Hz)，2.17(2H，m)，0.81(9H，s)，－0.03(3H，s)，－0.21(3H，s).

(step 6)

6-benzoyl-2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino (phosphinoyl) } -beta-D-ribofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (1.98g) obtained in the above step 5 in dichloromethane (23.9mL) were added N, N-diisopropylethylamine (1.02mL) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (1.07mL), and the mixture was stirred at room temperature for 15 hours. The reaction mixture was added with a saturated aqueous sodium bicarbonate solution to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to obtain the title compound (2.06g) in the form of a diastereomer mixture (diastereomer ratio ═ 7: 3) on the phosphorus atom.

MS(ESI)m/z：1027(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.06(0.3H，s)，8.04(0.7H，s)，7.50－7.16(15H，m)，6.85－6.79(4H，m)，6.35(0.7H，d，J＝6.7Hz)，6.31(0.3H，d，J＝6.1Hz)，4.84(0.7H，dd，J＝7.0，4.6Hz)，4.78(0.3H，t，J＝5.8Hz)，4.43－4.17(4H，m)，4.04－3.85(1.3H，m)，3.80－3.76(6H，m)，3.69－3.43(3H，m)，3.50(0.7H，dd，J＝10.6，3.3Hz)，3.33－3.26(1H，m)，2.87－2.76(2H，m)，2.74－2.60(1.4H，m)，2.31(0.6H，t，J＝6.7Hz)，2.23－2.11(2H，m)，1.21－1.13(7.8H，m)，1.04(4.2H，d，J＝6.7Hz)，0.73(2.7H，s)，0.72(6.3H，s)，－0.03(0.9H，s)，－0.06(2.1H，s)，－0.24(3H，s).

(step 7)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl]-3-O- [ hydroxy (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulene derivatives

To an acetonitrile (6.67mL) solution of the compound (1.37g) obtained in the above step 6 were added water (48. mu.L) and pyridinium trifluoroacetate (335mg), and the mixture was stirred at room temperature for 15 minutes. Tert-butylamine (6.67mL) was added to the reaction solution, and the mixture was stirred at room temperature for 15 minutes. After the reaction solution was concentrated under reduced pressure, the residue was azeotroped 2 times in acetonitrile (5 mL). After water (0.240mL) was added to a dichloromethane (16.7mL) solution of the residue, a dichloromethane (16.7mL) solution of dichloroacetic acid (0.953mL) was added, and the mixture was stirred at room temperature for 15 minutes. Pyridine (1.82mL) was added to stop the reaction, and then the reaction mixture was concentrated under reduced pressure. The residue was azeotroped 3 times with dehydrated acetonitrile (10mL), and about 5mL of acetonitrile remained in the last time. The obtained acetonitrile solution of the title compound was used directly in the next reaction.

(step 8)

Commercially available (Chemgenes) N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (1.31g) was azeotroped 3 times with dehydrated acetonitrile (10mL) to leave about 5mL of acetonitrile in the last step, and molecular sieves 3A, 1/16 (5 pieces in a lump) were added. The acetonitrile solution was added to the solution synthesized in the step 7, and the mixture was stirred at room temperature for 20 minutes under a nitrogen atmosphere. N, N-dimethyl-N' - (3-sulfydryl-3H-1, 2, 4-dithiazol-5-yl) formamidine (300mg) was added to the reaction solution, and after stirring at room temperature for 30 minutes, the reaction solution was concentrated under reduced pressure. After water (0.240mL) was added to a solution of the residue in dichloromethane (19.0mL), a solution of dichloroacetic acid (1.20mL) in dichloromethane (19.0mL) was added, and the mixture was stirred at room temperature for 15 minutes. Pyridine (13.2mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. The crude product obtained was used directly in the next reaction.

(step 9)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } benzamide

The crude pyridine (39.6mL) solution obtained in step 8 was concentrated to about 25mL, and 2-chloro-5, 5-dimethyl-1, 3, 2. lambda. was added⁵Dioxophosphohexane-2-one (908mg), stirred at room temperature for 30 min. Water (0.84mL) and 3H-1, 2-benzodithiol-3-one (336mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was poured into an aqueous solution (180mL) of sodium hydrogencarbonate (5.25g), stirred at room temperature for 30 minutes, and then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [ hexane/ethyl acetate/methanol ] ]Purification was carried out to give the title compound (507mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1219(M+H)⁺.

(Process 10)

Bis (N, N-diethyl)Ethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a solution of the compound (507mg) obtained in the above step 9 in methanol (5mL), 28% aqueous ammonia (5mL) was added, and the mixture was stirred at room temperature for 14 hours. The reaction mixture was concentrated, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give the title compound (301mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：958(M+H)⁺.

(step 11)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Triethylamine trihydrofluoride (3.84mL) was added to the compound (301mg) obtained in the above step 10, and the mixture was stirred at 45 ℃ for 3 hours. To the reaction mixture was added an ice-cooled mixture of 1M aqueous triethylammonium bicarbonate (20mL) and triethylamine (4mL) at room temperature. The reaction mixture was concentrated under reduced pressure, and subjected to C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 0% to 25% (0 part to 40 parts) to separate diastereoisomers on the phosphorus atom. The obtained compound (triethylamine salt) was converted into a sodium salt by the following method.

[ conversion to sodium salt ]

BT AG (registered trademark) 50W-X2 Resin (biological grade, 100-. After allowing an excessive amount of pure water to flow down naturally, a 1M aqueous sodium hydroxide solution (5mL) and pure water (10mL) were flowed down naturally in this order. The compound obtained above was dissolved in pure water (5mL) and packed in a column. The solution flowing down naturally was separated and extracted, and then dissolved and precipitated with pure water (10 mL). Fractions containing the objective product were combined and lyophilized to give diastereomer 1(83.4mg), diastereomer 2(44.8mg), and diastereomer 3(13.1mg) of the title compound (retention time on HPLC: diastereomer 1>2, 3).

Diastereomer 1

MS(ESI)m/z：730(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.74(1H，s)，8.17(1H，s)，8.02(1H，s)，7.10(1H，s)，6.34(1H，d，J＝8.5Hz)，6.30(1H，d，J＝4.8Hz)，5.41－5.34(1H，m)，5.19－5.13(1H，m)，4.85(1H，d，J＝3.6Hz)，4.79(1H，t，J＝4.5Hz)，4.52－4.41(2H，m)，4.40－4.31(2H，m)，4.07－3.97(2H，m)，3.52－3.47(2H，m)，2.90－2.76(2H，m)，2.05－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，54.5(s).

Diastereomer 2

MS(ESI)m/z：730(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.82(1H，s)，8.17(1H，s)，8.02(1H，s)，7.13(1H，s)，6.35(1H，d，J＝2.4Hz)，6.33(1H，s)，5.50－5.43(2H，m)，4.80(1H，dd，J＝6.7，4.2Hz)，4.52－4.28(5H，m)，4.02(1H，d，J＝12.1Hz)，3.93－3.86(1H，m)，3.54－3.47(2H，m)，2.95－2.88(2H，m)，2.05－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：63.0(s)，60.2(s).

Diastereomer 3

MS(ESI)m/z：730(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.16(1H，s)，8.17(1H，s)，8.02(1H，s)，7.12(1H，s)，6.35(1H，d，J＝8.5Hz)，6.29(1H，d，J＝6.7Hz)，5.63－5.56(1H，m)，5.54－5.46(1H，m)，4.79(1H，dd，J＝6.7，4.8Hz)，4.53－4.43(2H，m)，4.36－4.28(2H，m)，4.26－4.19(1H，m)，4.16－4.09(1H，m)，3.93－3.86(1H，m)，3.52－3.47(2H，m)，2.92－2.87(2H，m)，2.04－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：62.8(s)，58.7(s).

Example 2: synthesis of CDN2

(diastereomer 4 of Compound 1 described in example 1)

[ synthetic route ]

(step 1)

N-benzoyl-3' -O- [ tert-butyl (dimethyl) silyl]-2' -O- [ hydroxy (oxo) -lambda⁵-phosphino]Adenosine (I)

Using commercially available (Chemgenes) N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (962mg), a reaction was carried out in the same manner as in step 7 of example 1 to give an acetonitrile solution of the title compound. This acetonitrile solution was used as it was in the next reaction.

(step 2)

Using the compound obtained in the above step 1 and the compound (1.00g) obtained in the step 6 of example 1, a reaction was carried out in the same manner as in the step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 3)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2- (2-cyanoethoxy) -10-oxo-10-mercapto-2-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } benzamide

Using the crude product obtained in the above step 2, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (367mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1219(M+H)⁺.

(step 4)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (367mg) obtained in the above step 3, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(115 mg: containing impurities) and diastereomer 4(101 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：958(M+H)⁺.

Diastereomer 4 (high polarity)

MS(ESI)m/z：958(M+H)⁺.

(step 5)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 4 of Compound 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 4) (101 mg: containing impurities) obtained in the above step 4 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -25% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 5% -100% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (28.5 mg).

MS(ESI)m/z：730(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.11(1H，s)，8.19(1H，s)，8.02(1H，s)，7.08(1H，s)，6.35(1H，d，J＝8.5Hz)，6.27(1H，d，J＝4.8Hz)，5.43－5.36(1H，m)，5.29－5.21(1H，m)，4.95－4.88(1H，m)，4.80(1H，dd，J＝4.5，2.3Hz)，4.50－4.43(1H，m)，4.42－4.33(2H，m)，4.30－4.22(1H，m)，4.20－4.03(2H，m)，3.52－3.46(2H，m)，2.85－2.66(2H，m)，2.05－1.90(2H，m).

³¹P－NMR(CD₃OD)δ：58.1(s)，54.1(s).

Example 3: synthesis of CDN3

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -2, 10, 15, 16-tetrahydroxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-bis (alkoxide)

To a solution of the compound (diastereomer 1) obtained in step 11 of example 1 (30.0mg) in acetone (0.5mL) -water (0.2mL) were added triethylamine (0.27mL) and iodomethane (60. mu.L), and the mixture was stirred for 1 day. After the reaction solution was concentrated under reduced pressure, the residue was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 0 to 20 percent (0 to 40 portions) for refining. The obtained compound was subjected to salt conversion in the same manner as in [ conversion to sodium salt ] in step 11 of example 1 to obtain the title compound (21.2 mg).

MS(ESI)m/z：698(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.55(1H，s)，8.18(1H，s)，8.01(1H，s)，7.32(1H，s)，6.26(1H，s)，6.13(1H，s)，5.00－4.85(2H，m)，4.68－4.64(1H，m)，4.48－4.23(5H，m)，4.15－4.04(2H，m)，3.49－3.39(2H，m)，2.90－2.66(2H，m)，1.98－1.83(2H，m).

³¹P－NMR(CD₃OD)δ：－0.22(s).

Example 4: synthesis of CDN4

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

The reaction of step 7 in example 1 was carried out on the following scale (starting material: 1.01 g). Using the obtained acetonitrile solution of the compound and commercially available (Wuhu Nuowei Chemistry) 5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -N- (2-methylpropanoyl) guanosine (954mg), a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 2)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-2-yl } -2-methylpropanamide

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (357mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1201(M+H)⁺.

(step 3)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] amide]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (357mg) obtained in the above step 3, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (241mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：974(M+H)⁺.

(step 4)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraaza-neBenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (241mg) obtained in the above step 3, a reaction was carried out in the same manner as in the step 11 of example 1, and then diastereoisomers on a phosphorus atom were separated under the following [ purification conditions ], to obtain 2 kinds of diastereoisomers of the title compound as triethylamine salt, respectively.

[ refining conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -20% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ conversion to sodium salt ] in step 11 of example 1 to give diastereomer 1(56.7mg) and diastereomer 2(25.9mg) of the title compound (retention time of HPLC: diastereomer 1> 2).

Diastereomer 1 (low polarity)

MS(ESI)m/z：746(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.03(1H，s)，8.00(1H，s)，7.11(1H，s)，6.27(1H，d，J＝3.0Hz)，5.99(1H，d，J＝8.5Hz)，5.67－5.61(1H，m)，5.27－5.21(1H，m)，4.85(1H，d，J＝3.6Hz)，4.73(1H，dd，J＝3.9，2.0Hz)，4.48－4.39(2H，m)，4.38－4.30(2H，m)，4.18－4.08(2H，m)，3.51－3.45(2H，m)，2.80－2.71(1H，m)，2.63－2.53(1H，m)，2.02－1.84(2H，m).

³¹P－NMR(CD₃OD)δ：57.6(s)，53.5(s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：746(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.20(1H，s)，8.01(1H，s)，7.19(1H，s)，6.32(1H，d，J＝6.0Hz)，6.05(1H，d，J＝8.5Hz)，5.67－5.53(1H，m)，5.47－5.40(1H，m)，4.77－4.71(1H，m)，4.51－4.46(1H，m)，4.45－4.30(3H，m)，4.28－4.25(1H，m)，4.19－4.08(1H，m)，3.96－3.89(1H，m)，3.53－3.46(2H，m)，2.92－2.79(2H，m)，2.05－1.93(2H，m).

³¹P－NMR(CD₃OD)δ：61.7(s)，59.5(s).

Example 5: synthesis of CDN5

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2 ', 3 ', 5 ' -tri-O-acetyl-1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] inosine

After 2- (trimethylsilyl) ethyl (2-hydroxyethyl) carbamate (3.12g) and triphenylphosphine (3.99g) were added to a suspension of 2 ', 3 ', 5 ' -tri-O-acetylinosine (5.00g) in tetrahydrofuran (90mL), a solution of diprop-2-yl (E) -diazene-1, 2-dicarboxylate (3.05mL) in tetrahydrofuran (10mL) was added and the mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol ] to give the title compound (3.01 g).

MS(ESI)m/z：582(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.96(1H，s)，7.93(1H，s)，6.10(1H，d，J＝4.8Hz)，5.86(1H，t，J＝5.4Hz)，5.58(1H，t，J＝5.1Hz)，4.96(1H，t，J＝7.3Hz)，4.47－4.40(2H，m)，4.36(1H，dd，J＝13.0，5.1Hz)，4.24(2H，t，J＝5.4Hz)，4.15(2H，t，J＝8.8Hz)，3.55(2H，q，J＝6.0Hz)，2.15(3H，s)，2.13(3H，s)，2.10(3H，s)，0.97(2H，t，J＝8.8Hz)，0.03(9H，s).

(step 2)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] inosine

To a solution of the compound (3.01g) obtained in the above step 1 in tetrahydrofuran (15mL) -methanol (15mL) was added potassium carbonate (100mg), and the mixture was stirred at room temperature for 2 hours. Acetic acid (83. mu.L) was added to the reaction mixture, and the mixture was concentrated under reduced pressure, and the residue was azeotroped with pyridine. After dissolving the resulting mixture in pyridine (30mL) again, 4' -dimethoxytrityl chloride (2.10g) was added thereto at 0 ℃ and the mixture was stirred for 30 minutes and then stored in a refrigerator overnight. Methanol (1mL) was added to the reaction mixture, and after stirring for 30 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (3.61 g).

MS(ESI)m/z：758(M+H)⁺.

¹H－NMR(CDCl₃) δ: 7.92(1H, s), 7.76(1H, s), 7.37(2H, d, J ═ 7.3Hz), 7.29-7.14 (7H, m), 6.78(4H, d, J ═ 8.5Hz), 5.94(1H, d, J ═ 5.4Hz), 5.63(1H, brs), 4.81-4.74 (1H, m), 4.46-4.41 (1H, m), 4.36-4.31 (1H, m), 4.19-4.05 (4H, m), 3.76(6H, s), 3.52-3.44 (2H, m), 3.44-3.31 (2H, m), 0.99-0.91 (2H, m), 0.02(9H, s) · observable peaks only are described

(step 3)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] inosine to a dichloromethane (18mL) solution of the compound (3.61g) obtained in the above step 2, imidazole (811mg) and tert-butyl (chloro) dimethylsilane (861mg) were added, and the mixture was stirred at room temperature for 17 hours. Saturated aqueous sodium bicarbonate was added to the reaction solution, and extraction was performed with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (1.61g) and 5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] inosine (1.31g), a positional isomer of the title compound, respectively.

MS(ESI)m/z：872(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.98(1H，s)，7.85(1H，s)，7.39(2H，d，J＝7.9Hz)，7.32－7.15(7H，m)，6.78(4H，d，J＝9.1Hz)，5.93(1H，d，J＝4.8Hz)，5.22－5.11(1H，m)，4.60(1H，q，J＝5.6Hz)，4.47(1H，t，J＝4.2Hz)，4.28－4.08(5H，m)，3.77(6H，s)，3.59－3.49(2H，m)，3.45(1H，dd，J＝10.3，3.0Hz)，3.26(1H，dd，J＝10.3，3.9Hz)，3.15－3.08(1H，m)，0.95(2H，t，J＝8.5Hz)，0.88(9H，s)，0.07(3H，s)，0.02(9H，s)，0.00(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：872(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(1H，s)，7.82(1H，s)，7.46－7.41(2H，m)，7.35－7.19(7H，m)，6.84－6.78(4H，m)，5.98(1H，d，J＝5.4Hz)，5.06－4.96(1H，m)，4.84(1H，t，J＝5.4Hz)，4.34－4.08(6H，m)，3.78(6H，s)，3.54(2H，q，J＝5.8Hz)，3.48(1H，dd，J＝10.6，2.7Hz)，3.39(1H，dd，J＝10.6，3.9Hz)，2.71(1H，d，J＝4.2Hz)，0.99－0.91(2H，m)，0.85(9H，s)，0.03(9H，s)，0.02(3H，s)，－0.12(3H，s).

(step 4)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] inosine

To a solution of the compound (1.61g) obtained in the above step 3 in dichloromethane (18.5mL) were added 4, 5-dicyanoimidazole (240mg) and 2-cyanoethyl N, N, N ', N' -tetraisopropylphosphorodiamidite (0.703mL), and the mixture was stirred at room temperature for 15 hours. The reaction mixture was added with a saturated aqueous sodium bicarbonate solution to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.95g) as a diastereomer mixture (diastereomer ratio 61: 39) on the phosphorus atom.

MS(ESI)m/z：1072(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.04(0.39H，s)，7.99(0.61H，s)，7.83(0.39H，s)，7.82(0.61H，s)，7.42(2H，d，J＝7.3Hz)，7.35－7.15(7H，m)，6.85－6.77(4H，m)，6.15(0.61H，d，J＝6.0Hz)，6.09(0.39H，d，J＝4.8Hz)，5.34－5.24(0.61H，m)，5.12－5.03(0.39H，m)，4.86－4.76(0.39H，m)，4.72－4.62(0.61H，m)，4.47－4.42(0.39H，m)，4.42－4.36(0.69H，m)，4.31－4.05(6H，m)，3.78(6H，s)，3.78－3.65(1H，m)，3.61－3.39(7H，m)，3.35(0.61H，dd，J＝10.6，3.9Hz)，3.28(0.39H，dd，J＝10.9，4.2Hz)，2.49(0.78H，t，J＝6.0Hz)，2.29(1.22H，t，J＝5.7Hz)，1.30－0.94(12H，m)，0.85(5.49H，s)，0.84(3.51H，s)，0.09(1.17H，s)，0.08(1.83H，s)，0.03(9H，s)，0.02(1.83H，s)，0.00(1.17H，s).

(step 5)

The same reaction as in step 7 of example 1 (starting material: 910mg) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (950mg) obtained in the above step 4, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 6)

2- (trimethylsilyl) ethyl (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) carbamate

Using the crude product obtained in the above step 5, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (602mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1303(M+H)⁺.

(step 7)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-7- { 6-oxo-1- [ 2- ({ [ 2- (trimethylsilyl) ethoxy group)]Carbonyl } amino) ethyl]-1, 6-dihydro-9H-purin-9-yl } -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (602mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(205 mg: containing impurities) and diastereomer 2(244 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1146(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1146(M+H)⁺.

(step 8-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Triethylamine trihydrofluoride (1.31mL) was added to the compound (diastereomer 1) (145 mg: containing impurities) obtained in the above step 7, and the mixture was stirred at 45 ℃ for 3 hours. To the reaction mixture was added an ice-cooled mixed solution of 1M triethylammonium bicarbonate solution (10mL) and triethylamine (2mL) at room temperature. The reaction mixture was concentrated under reduced pressure and purified by column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ]. To a tetrahydrofuran (4mL) solution of the obtained compound was added a tetrabutylammonium fluoride solution in tetrahydrofuran (about 1M, 2mL), and the mixture was stirred at room temperature for 39 hours. To the reaction mixture was added 10mM triethylammonium acetate aqueous solution (4mL) and the mixture was concentrated under reduced pressure. The residue was subjected to C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5 to 50 percent (0 to 40 portions) of the raw materials are refined. In the case of salt conversion of the obtained compound (triethylamine salt), salt conversion was performed in the same manner as in [ sodium salt conversion ] described in step 11 of example 1 except that a mixed solution of acetonitrile-methanol-pure water (1: 1: 1) was used as a solvent for dissolving the compound and as a part of the eluent, to obtain the title compound (72.5 mg).

MS(ESI)m/z：774(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.60(1H，s)，8.15(1H，s)，8.02(1H，s)，7.11(1H，s)，6.26(1H，d，J＝4.8Hz)，6.24(1H，t，J＝5.1Hz)，5.47(1H，dt，J＝8.2，4.2Hz)，5.23－5.17(1H，m)，4.77－4.73(2H，m)，4.52－4.44(2H，m)，4.36－4.19(3H，m)，4.14－4.02(3H，m)，3.48(2H，t，J＝4.8Hz)，3.31－3.26(2H，m)，2.90－2.74(2H，m)，2.01－1.93(2H，m).

³¹P－NMR(CD₃OD)δ：57.7(s)，54.7(s).

(step 8-2)

(diastereomer 2)

Using the compound (diastereomer 2) (133 mg: containing impurities) obtained in the above step 7, a reaction and salt conversion were carried out in the same manner as in the above step 8-1 to obtain the title compound (55.4 mg).

MS(ESI)m/z：774(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，s)，8.25(1H，s)，8.02(1H，s)，7.11(1H，s)，6.31(1H，d，J＝6.7Hz)，6.28(1H，d，J＝8.5Hz)，5.47－5.38(2H，m)，4.77(1H，dd，J＝6.7，4.2Hz)，4.48(1H，d，J＝4.2Hz)，4.46－4.37(2H，m)，4.37－4.29(3H，m)，4.27－4.18(1H，m)，4.08－4.02(1H，m)，3.92－3.85(1H，m)，3.53－3.46(2H，m)，3.28－3.23(2H，m)，2.93－2.86(2H，m)，2.04－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：62.6(s)，60.0(s).

Example 6: synthesis of CDN6

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis(mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2 ', 3 ', 5 ' -tri-O-acetyl-1- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) inosine

After adding 2- { [ tert-butyl (dimethyl) silyl ] oxy } ethan-1-ol (5.37g) and triphenylphosphine (7.69g) to a commercial (Ark Pharm) suspension of 2 ', 3 ', 5 ' -tri-O-acetylinosine (10.0g) in tetrahydrofuran (100mL), dipropan-2-yl (E) -diazene-1, 2-dicarboxylate (6.10mL) was further added, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate/dichloromethane ] to give the title compound as a mixture (10.6g) with triphenylphosphine oxide.

MS(ESI)m/z：553(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.05(1H，s)，7.92(1H，s)，6.12(1H，d，J＝5.4Hz)，5.86(1H，t，J＝5.4Hz)，5.59(1H，dd，J＝5.4，4.2Hz)，4.47－4.41(2H，m)，4.38－4.31(1H，m)，4.22－4.17(2H，m)，3.89(2H，t，J＝4.8Hz)，2.15(3H，s)，2.14(3H，s)，2.08(3H，s)，0.83(9H，s)，－0.06(3H，s)，－0.06(3H，s).

(step 2)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -1- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) inosine

Using the compound (10.6g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 5 to obtain the title compound in the form of a mixture (7.21g) with triphenylphosphine oxide.

MS(ESI)m/z：729(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.01(1H，s)，7.97(1H，s)，7.35－7.30(2H，m)，7.25－7.17(7H，m)，6.81－6.76(4H，m)，5.95(1H，d，J＝5.4Hz)，5.13(1H，brs)，4.68－4.61(1H，m)，4.43－4.36(2H，m)，4.31－4.23(1H，m)，4.15－4.08(1H，m)，3.89(2H，t，J＝4.5Hz)，3.77(6H，s)，3.42(1H，dd，J＝10.3，3.6Hz)，3.34(1H，dd，J＝10.3，3.6Hz)，3.10(1H，brs)，0.83(9H，s)，－0.06(3H，s)，－0.07(3H，s).

(step 3)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) inosine

Using the compound (7.21g) obtained in the above step 2, a reaction was carried out in the same manner as in step 3 of example 5 to give the title compound (2.17g) and 5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) inosine (2.55g), which is a positional isomer of the title compound.

MS(ESI)m/z：843(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(1H，s)，7.97(1H，s)，7.43－7.39(2H，m)，7.33－7.19(7H，m)，6.83－6.77(4H，m)，5.96(1H，d，J＝4.2Hz)，4.56－4.50(2H，m)，4.33－4.25(1H，m)，4.19－4.02(2H，m)，3.89(2H，t，J＝4.8Hz)，3.78(6H，s)，3.45(1H，dd，J＝10.9，4.2Hz)，3.27(1H，dd，J＝10.9，4.2Hz)，3.03(1H，d，J＝6.0Hz)，0.88(9H，s)，0.82(9H，s)，0.07(3H，s)，－0.01(3H，s)，－0.07(3H，s)，－0.07(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：843(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.98(1H，s)，7.94(1H，s)，7.46－7.42(2H，m)，7.35－7.20(7H，m)，6.85－6.79(4H，m)，5.99(1H，d，J＝5.4Hz)，4.83(1H，t，J＝5.1Hz)，4.33－4.29(1H，m)，4.27－4.24(1H，m)，4.24－4.12(2H，m)，3.90(2H，t，J＝4.5Hz)，3.79(3H，s)，3.78(3H，s)，3.48(1H，dd，J＝10.3，3.0Hz)，3.40(1H，dd，J＝10.3，3.0Hz)，2.71(1H，d，J＝3.6Hz)，0.86(9H，s)，0.83(9H，s)，0.01(3H，s)，－0.07(3H，s)，－0.07(3H，s)，－0.11(3H，s).

(step 4)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } inosine

Using the compound (2.17g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (2.65g) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1043(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.03(0.53H，s)，8.01(0.47H，s)，7.97(0.53H，s)，7.93(0.47H，s)，7.45－7.41(2H，m)，7.35－7.19(7H，m)，6.83－6.78(4H，m)，6.17(0.53H，d，J＝4.2Hz)，6.05(0.47H，d，J＝4.2Hz)，4.87－4.80(0.47H，m)，4.64－4.58(0.53H，m)，4.46－4.40(1H，m)，4.30－4.05(3H，m)，3.92－3.87(2H，m)，3.78(6H，s)，3.86－3.40(5H，m)，3.33－3.24(1H，m)，2.54(0.94H，t，J＝6.0Hz)，2.43(1.06H，t，J＝6.7Hz)，1.16－1.09(9H，m)，1.01－0.97(3H，m)，0.83(4.23H，s)，0.83(4.77H，s)，0.82(9H，s)，0.07(1.41H，s)，0.04(1.59H，s)，－0.02(3H，s)，－0.07(1.41H，s)，－0.08(1.59H，s)，－0.08(3H，s).

(step 5)

The same reaction as in step 7 of example 1 (raw material: 935mg) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (950mg) obtained in the above step 4, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 6)

3- ({ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 1- (2- { [ tert-butyl (dimethyl) silyl)]Oxy } ethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyn-10-yl } oxy) propionitrile

Using the crude product obtained in the above-mentioned step 5, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (494mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1274(M+H)⁺.

(step 7)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 1- (2- { [ tert-butyl (dimethyl) silyl)]Oxy } ethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (494mg) obtained in the above-mentioned step 6, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(88.5 mg: containing impurities) and diastereomer 2(70.7 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1003(M－C₆H₁₅Si+2H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1003(M－C₆H₁₅Si+2H)⁺.

(step 8-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (88.5 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (25.7 mg).

MS(ESI)m/z：775(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.63(1H，s)，8.22(1H，s)，8.02(1H，s)，7.11(1H，s)，6.30－6.24(2H，m)，5.46－5.37(1H，m)，5.23－5.15(1H，m)，4.83－4.79(1H，m)，4.78－4.74(1H，m)，4.53－4.42(2H，m)，4.35－4.16(3H，m)，4.16－3.97(3H，m)，3.83－3.78(2H，m)，3.52－3.47(2H，m)，2.88－2.81(2H，m)，2.03－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s)，54.4(s).

(step 8-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (70.7 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (17.8 mg).

MS(ESI)m/z：775(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，s)，8.23(1H，s)，8.02(1H，s)，7.11(1H，s)，6.30(2H，dd，J＝13.6，7.6Hz)，5.48－5.39(2H，m)，4.78(1H，dd，J＝6.7，4.2Hz)，4.51－4.28(5H，m)，4.26－4.13(2H，m)，4.06－4.00(1H，m)，3.93－3.86(1H，m)，3.85－3.80(2H，m)，3.52－3.47(2H，m)，2.94－2.88(2H，m)，2.05－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：62.9(s)，60.0(s).

Example 7: synthesis of CDN7

N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) -2-hydroxyacetamide

[ synthetic route ]

(step 1-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- { 1- [ 2- (2-hydroxyacetamide) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Triethylamine (8. mu.L) and 1- [ (hydroxyacetyl) oxy ] pyrrolidine-2, 5-dione (5.3mg) were added to a solution of the compound (10.0mg) obtained in step 8-1 of example 5 in N, N-dimethylformamide (0.5mL), and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with 10mM aqueous triethylammonium acetate, and subjected to C18 silica gel column chromatography [10mM aqueous triethylammonium acetate/acetonitrile ] and preparative HPLC [10mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 0 to 30 percent (0 to 40 portions) for refining. The obtained compound (triethylamine salt) was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (10.5 mg).

MS(ESI)m/z：832(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.57(1H，s)，8.04(1H，s)，8.03(1H，s)，7.13(1H，s)，6.26(1H，d，J＝4.2Hz)，6.24－6.19(1H，m)，5.57－5.49(1H，m)，5.26－5.18(1H，m)，4.80(1H，d，J＝3.6Hz)，4.76(1H，t，J＝4.5Hz)，4.51－4.41(2H，m)，4.35－4.17(3H，m)，4.11－3.95(3H，m)，3.91(2H，s)，3.62－3.55(2H，m)，3.52－3.45(2H，m)，2.89－2.65(2H，m)，2.02－1.91(2H，m).

³¹P－NMR(CD₃OD)δ：57.6(s)，54.3(s).

(Process 1-2)

(diastereomer 2)

Using the compound (30.0mg) obtained in step 8-2 of example 5, a reaction was carried out in the same manner as in step 1-1, and then purification was carried out according to the following purification conditions, whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (23.6 mg).

MS(ESI)m/z：832(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.69(1H，s)，8.14(1H，s)，8.02(1H，s)，7.11(1H，s)，6.31(1H，d，J＝6.7Hz)，6.26(1H，d，J＝7.9Hz)，5.49－5.40(2H，m)，4.77(1H，dd，J＝6.7，4.8Hz)，4.48(1H，d，J＝4.2Hz)，4.46－4.28(4H，m)，4.22(2H，t，J＝5.4Hz)，4.06－4.00(1H，m)，3.94(2H，s)，3.92－3.86(1H，m)，3.70－3.55(2H，m)，3.52－3.47(2H，m)，2.92－2.86(2H，m)，2.04－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：62.7(s)，59.9(s).

Example 8: synthesis of CDN8

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2 ', 3 ' -bis-O- [ tert-butyl (dimethyl) silyl ] -2-chloroadenosine

Imidazole (16.4g) and tert-butyldimethylchlorosilane (18.2g) were added to a solution of 5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-chloroadenosine (29.1g) in N, N-dimethylformamide (145mL) known in the literature (J.Med.chem.1989, 32, 1135-Asa 1140), and the mixture was stirred at room temperature for 18 hours. After the reaction was stopped by adding water to the reaction solution, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (34.9 g).

MS(ESI)m/z：832(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.02(1H，s)，7.47－7.42(2H，m)，7.36－7.32(4H，m)，7.31－7.18(3H，m)，6.84－6.79(4H，m)，5.90(1H，d，J＝4.8Hz)，5.72(2H，brs)，4.74(1H，dd，J＝4.5，2.3Hz)，4.25(1H，dd，J＝4.2，2.3Hz)，4.21(1H，q，J＝4.2Hz)，3.78(6H，s)，3.58(1H，dd，J＝10.9，4.2Hz)，3.33(1H，dd，J＝10.9，4.2Hz)，0.84(9H，s)，0.82(9H，s)，0.04(3H，s)，－0.01(3H，s)，－0.02(3H，s)，－0.17(3H，s).

(step 2)

N-acetyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2 ', 3 ' -bis-O- [ tert-butyl (dimethyl) silyl ] -2-chloroadenosine

Acetic anhydride (140mL) and 4-dimethylaminopyridine (515mg) were added to a pyridine (210mL) solution of the compound (34.9g) obtained in the above step 1, and the mixture was stirred at room temperature for 21 hours under a nitrogen atmosphere. The reaction mixture was diluted with dichloromethane (100mL), and a saturated aqueous sodium bicarbonate solution was added thereto, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Methylene chloride (210mL) and morpholine (7.30mL) were added to the residue, and the mixture was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride solution was added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (45.4 g: containing impurities). The obtained compound was used in the next reaction without purification.

¹H－NMR(CDCl₃)δ：8.95(1H，brs)，8.22(1H，s)，7.45－7.42(2H，m)，7.34－7.20(7H，m)，6.82(4H，dq，J＝9.4，2.7Hz)，5.96(1H，d，J＝4.8Hz)，4.72－4.69(1H，m)，4.23(2H，brs)，3.79(6H，s)，3.59(1H，dd，J＝10.9，3.6Hz)，3.35(1H，dd，J＝10.3，3.6Hz)，2.73(3H，s)，0.83(9H，s)，0.82(9H，s)，0.04(3H，s)，0.00(3H，s)，－0.03(3H，s)，－0.18(3H，s).

(step 3)

N-acetyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-chloroadenosine

A tetrahydrofuran solution (about 1.0M, 100mL) of tetrabutylammonium fluoride was added to a tetrahydrofuran (200mL) solution of the compound (45.4 g: containing impurities) obtained in the above step 2, and the mixture was stirred at room temperature for 3 hours under a nitrogen atmosphere. Saturated aqueous ammonium chloride solution was added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/acetone/0.1% triethylamine ] to obtain the title compound (23.0 g).

¹H－NMR(CDCl₃)δ：8.71(1H，brs)，8.16(1H，s)，7.28－7.16(9H，m)，6.78－6.73(4H，m)，5.99(1H，d，J＝5.4Hz)，4.86(1H，t，J＝5.1Hz)，4.49(1H，dd，J＝5.1，2.7Hz)，4.39(1H，q，J＝3.2Hz)，3.78(3H，s)，3.77(3H，s)，3.42(1H，dd，J＝10.9，3.6Hz)，3.35(1H，dd，J＝10.6，3.3Hz)，2.66(3H，s).

(step 4)

N-acetyl-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -2-chloroadenosine

Imidazole (5.94g) and tert-butyldimethylsilyl chloride (6.44g) were added to a solution of the compound (23.0g) obtained in the above step 3 in N, N-dimethylformamide (178mL), and the mixture was stirred at room temperature for 18 hours under a nitrogen atmosphere. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (9.01 g).

¹H－NMR(CDCl₃)δ：8.42(1H，brs)，8.14(1H，s)，7.38－7.35(2H，m)，7.29－7.18(7H，m)，6.80－6.76(4H，m)，5.99(1H，d，J＝4.2Hz)，4.71－4.66(2H，m)，4.17－4.14(1H，m)，3.78(6H，s)，3.49(1H，dd，J＝10.6，3.3Hz)，3.29(1H，dd，J＝10.9，4.2Hz)，3.02(1H，d、J＝5.4Hz)，2.67(3H，s)，0.89(9H，s)，0.11(3H，s)，0.02(3H，s).

(step 5)

N-acetyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2-chloro-2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine

Using the compound (9.01g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 6 of example 1 to obtain the title compound (10.6g) in the form of a diastereomer mixture (diastereomer ratio 65: 35) on the phosphorus atom.

¹H－NMR(CDCl₃)δ：8.40(1H，brs)，8.31(0.35H，s)，8.25(0.65H，s)，7.38(2H，d，J＝7.3Hz)，7.29－7.19(7H，m)，6.80(4H，dd，J＝9.1，2.4Hz)，6.27(0.35H，d，J＝3.0Hz)，6.13(0.65H，d，J＝3.6Hz)，4.88－4.83(0.65H，m)，4.69－4.65(0.35H，m)，4.56(1H，t，J＝4.8Hz)，4.23－4.17(1H，m)，3.93－3.78(1H，m)，3.78(6H，s)，3.64－3.54(4H，m)，3.33－3.28(1H，m)，2.67(3H，s)，2.56(1.3H，t，J＝6.3Hz)，2.52(0.7H，t，J＝6.3Hz)，1.16(2.1H，d，J＝7.3Hz)，1.14(3.9H，d，J＝6.0Hz)，1.12(3.9H，d，J＝6.0Hz)，1.02(2.1H，d，J＝6.7Hz)，0.83(5.9H，s)，0.82(3.1H，s)，0.10(1.9H，s)，0.07(1.1H，s)，0.01H(3H，s).

(step 6)

N, N-diethylethanaminium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-acetamido-2-chloro-9H-purin-9-yl) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-thiolate

The same reaction as in step 7 of example 1 (starting material: 2.06g) was carried out on the following scale. Using the obtained acetonitrile solution of the compound and the compound (1.98g) obtained in the above step 5, reactions were carried out in the same manner as in the step 8 of example 1 and the step 9 of example 1 to obtain diastereomer 1(180mg) and diastereomer 2(167 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1191(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.10(1H，s)，8.00(1H，s)，7.40－7.36(2H，m)，7.30－7.23(4H，m)，6.38(1H，d，J＝8.5Hz)，6.35(1H，d，J＝3.0Hz)，5.67－5.60(1H，m)，5.09－5.04(1H，m)，4.72(1H，d，J＝3.6Hz)，4.50－4.29(8H，m)，4.07(1H，dd，J＝12.4，4.5Hz)，3.91－3.83(1H，m)，3.54－3.45(1H，m)，3.17(6H，q，J＝7.3Hz)，3.08(2H，t，J＝6.0Hz)，2.45－2.42(2H，m)，2.42(3H，s)，2.28－2.23(2H，m)，1.29(9H，t，J＝7.3Hz)，1.01(9H，s)，0.90(9H，s)，0.29(3H，s)，0.28(3H，s)，0.25(3H，s)，0.10(3H，s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：1191(M+H)⁺.

(step 7-1)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl ] silaneAlkyl radical]Oxy } -2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-dione was prepared by adding ethylenediamine (256. mu.L) to a methanol (1.28mL) solution of the compound (diastereomer 1) (49.6mg) obtained in the above step 6, stirring at 60 ℃ for 2 hours, and reacting at 120 ℃ for 2 hours using a microwave reaction apparatus. The reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 40% -70% (0 min-30 min)]Purification was performed to give the title compound (37.2 mg).

MS(ESI)m/z：1016(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.27(1H，s)，7.99(1H，s)，7.16(1H，s)，6.25(1H，d，J＝4.2Hz)，6.09(1H，d，J＝8.5Hz)，5.41－5.35(1H，m)，5.12－5.08(1H，m)，4.86－4.80(2H，m)，4.69(1H，t，J＝4.5Hz)，4.51－4.45(1H，m)，4.29－4.23(2H，m)，4.11－4.03(2H，m)，3.51－3.44(4H，m)，3.13－3.04(2H，m)，2.80－2.76(2H，m)，2.02－1.92(2H，m)，0.99(9H，s)，0.84(9H，s)，0.32(3H，s)，0.29(3H，s)，0.24(3H，s)，0.07(3H，s).

(step 7-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl) ]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Ethylenediamine (25.8. mu.L) was added to a methanol (1.29mL) solution of the compound (diastereomer 2) (50.0 mg: containing impurities) obtained in the above step 6, and the mixture was stirred at 60 ℃ for 2 hours, followed by reaction at 120 ℃ for 2 hours using a microwave reaction apparatus. The reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 30% to 50% (0 part to 30 parts) was purified to give the title compound (23.7 mg).

MS(ESI)m/z：1016(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.16(1H，s)，8.00(1H，s)，7.08(1H，s)，6.33(1H，d，J＝7.3Hz)，6.13(1H，d，J＝8.5Hz)，5.51－5.48(1H，m)，5.30(1H，t，J＝4.8Hz)，5.13－5.06(1H，m)，4.95(1H，d，J＝4.2Hz)，4.66－4.55(2H，m)，4.24(1H，s)，4.08(1H，dd，J＝12.4，4.5Hz)，3.89－3.83(1H，m)，3.69－3.61(1H，m)，3.50－3.33(4H，m)，3.12－3.01(14H，m)，2.89(2H，t，J＝5.4Hz)，2.03－1.96(2H，m)，1.25(18H，t，J＝7.3Hz)，0.99(9H，s)，0.74(9H，s)，0.27(6H，s)，0.18(3H，s)，－0.08(3H，s).

(step 8-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (37.2mg) obtained in the above step 7-1, a reaction was carried out in the same manner as in step 11 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (16.5 mg).

MS(ESI)m/z：788(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.21(1H，brs)，8.01(1H，s)，7.07(1H，s)，6.27(1H，d，J＝3.6Hz)，6.10(1H，d，J＝8.5Hz)，5.51－5.41(1H，m)，5.16－5.11(1H，m)，4.83(1H，d，J＝3.6Hz)，4.73(1H，t，J＝4.2Hz)，4.50－4.45(2H，m)，4.35－4.29(2H，m)，4.16－4.04(2H，m)，3.50－3.42(4H，m)，3.17－3.05(2H，m)，2.82－2.66(2H，m)，2.04－1.92(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，54.2(s).

(step 8-2)

(diastereomer 2)

Using the compound (23.7mg) obtained in the above step 7-2, a reaction was carried out in the same manner as in step 11 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (14.9 mg).

MS(ESI)m/z：788(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.27(1H，brs)，8.02(1H，s)，7.15(1H，s)，6.31(1H，d，J＝6.0Hz)，6.12(1H，d，J＝8.5Hz)，5.45－5.33(2H，m)，4.75(1H，dd，J＝5.7，4.5Hz)，4.50(1H，d，J＝4.2Hz)，4.47－4.30(4H，m)，4.17－4.13(1H，brm)，3.94－3.89(1H，m)，3.69－3.59(1H，brm)，3.51－3.44(3H，m)，3.21－3.07(2H，m)，2.88－2.85(2H，m)，2.03－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：62.2(s)，59.8(s).

Example 9: synthesis of CDN9

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-hydroxyethyl) amino group ]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1-1)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-hydroxyethyl) amino]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-dione was prepared by adding 2-aminoethanol (258. mu.L) to a methanol (1.29mL) solution of the compound (diastereomer 1) (50.1mg) obtained in step 6 of example 8, stirring at 60 ℃ for 2 hours, and reacting at 120 ℃ for 2 hours using a microwave reaction apparatus. Preparative HPLC [10mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% -60% (0 min-30 min)]Purification was performed to obtain the title compound (39.4mg) as a mixture containing the ethanolamine-derived compound.

MS(ESI)m/z：1017(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.32(1H，s)，8.01(1H，s)，7.16(1H，s)，6.28(1H，d，J＝5.4Hz)，6.13(1H，d，J＝9.1Hz)，5.44－5.38(1H，m)，5.19－5.14(1H，m)，4.98－4.83(2H，m)，4.78－4.75(1H，m)，4.45－4.39(1H，m)，4.28－4.22(1H，m)，4.18(1H，s)，4.13－4.07(1H，m)，4.04－3.99(1H，m)，3.67(2H，t，J＝5.4Hz)，3.51－3.42(4H，m)，2.86(2H，t，J＝5.4Hz)，2.04－1.98(2H，m)，0.98(9H，s)，0.82(9H，s)，0.31(3H，s)，0.27(3H，s)，0.22(3H，s)，0.05(3H，s).

(Process 1-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-hydroxyethyl) amino]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

2-aminoethanol (254. mu.L) was added to a methanol (1.27mL) solution of the compound (diastereomer 2) (49.3 mg: containing impurities) obtained in step 6 of example 8, and the mixture was stirred at 60 ℃ for 2 hours and then reacted at 120 ℃ for 3 hours using a microwave reaction apparatus. Preparative HPLC [10mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% to 60% (0 part to 30 parts) was purified to give the title compound (26.1 mg).

MS(ESI)m/z：1017(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.25(1H，s)，8.01(1H，s)，7.10(1H，s)，6.36(1H，d，J＝7.3Hz)，6.17(1H，d，J＝7.9Hz)，5.59－5.53(1H，m)，5.41(1H，t，J＝4.5Hz)，5.21－5.14(1H，m)，5.02－4.95(2H，m)，4.70－4.61(2H，m)，4.18(1H，s)，4.03(1H，dd，J＝12.1，4.8Hz)，3.91－3.86(1H，m)，3.75－3.69(2H，m)，3.52－3.43(4H，m)，3.14(12H，q，J＝7.3Hz)，2.93－2.91(2H，m)，2.04－1.99(2H，m)，1.28(18H，t，J＝7.6Hz)，0.99(9H，s)，0.75(9H，s)，0.27(6H，s)，0.21(3H，s)，－0.05(3H，s).

(step 2-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-hydroxyethyl) amino group]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The reaction was carried out in the same manner as in step 11 of example 1 using the mixture (39.4mg) obtained in step 1-1 above, and then purification was carried out according to the following purification conditions, thereby obtaining the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (16.8 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.25(1H，s)，8.02(1H，s)，7.10(1H，s)，6.30(1H，d，J＝3.6Hz)，6.15(1H，d，J＝8.5Hz)，5.49－5.42(1H，m)，5.21－5.16(1H，m)，4.87－4.85(1H，m)，4.77(1H，t，J＝4.2Hz)，4.50－4.35(3H，m)，4.31(1H，s)，4.12－4.10(2H，m)，3.64(2H，t，J＝5.4Hz)，3.51－3.38(4H，m)，2.85－2.70(2H，m)，2.02－1.94(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s)，53.9(s).

(step 2-2)

The structure of disodium (5R, 7R,8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-hydroxyethyl) amino]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The compound (26.1mg) obtained in the above step 1-2 was reacted in the same manner as in step 11 of example 1, and then purified under the following purification conditions to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (18.6 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.35(1H，s)，8.03(1H，s)，7.17(1H，s)，6.33(1H，d，J＝6.0Hz)，6.16(1H，d，J＝8.5Hz)，5.49－5.41(2H，m)，4.80(1H，t，J＝5.4Hz)，4.51－4.26(5H，m)，4.07(1H，d，J＝12.7Hz)，3.94－3.89(1H，m)，3.67(2H，5，J＝5.7Hz)，3.53－3.39(4H，m)，2.89(2H，t，J＝5.4Hz)，2.03－1.99(2H，m).

³¹P－NMR(CD₃OD)δ：62.8(s)，60.3(s).

Example 10: synthesis of CDN10

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-amino-2-methylpropyl) amino]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-amino-2-methylpropyl) amino ]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

1, 2-diamino-2-methylpropane (210. mu.L) was added to a methanol (1.10mL) solution of the compound (diastereomer 1) (41.0mg) obtained in step 6 of example 8, and the mixture was stirred at 60 ℃ for 2 hours, followed by reaction at 120 ℃ for 6 hours using a microwave reaction apparatus. Preparative HPLC [10mM triethylammonium acetate solution/acetonitrile, acetonitrile: 20% -60% (0 min-30 min) was purified by a simple procedure to give the title compound (16.3 mg: containing impurities). The obtained compound was used in the next reaction without purification.

MS(ESI)m/z：1044(M+H)⁺.

(step 2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-amino-2-methylpropyl) amino]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzene And [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The compound (16.3 mg: containing impurities) obtained in the above step 1 was reacted in the same manner as in the step 11 of example 1, and then purified according to the following purification conditions to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -25% (0 min-30 min) ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (6.4 mg).

MS(ESI)m/z：816(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.28(1H，brs)，8.01(1H，s)，7.05(1H，s)，6.27(1H，d，J＝4.2Hz)，6.15(1H，d，J＝7.9Hz)，5.41－5.27(1H，m)，5.13－5.08(1H，m)，4.84(1H，d，J＝3.6Hz)，4.73(1H，t，J＝4.5Hz)，4.50－4.44(2H，m)，4.36－4.31(2H，m)，4.16－4.00(2H，m)，3.49(2H，dd，J＝6.3，3.3Hz)，3.31－3.25(2H，m)，2.84－2.70(2H，m)，2.03－1.91(2H，m)，1.34(3H，s)，1.30(3H，s).

³¹P－NMR(CD₃OD)δ：57.9(s)，54.5(s).

Example 11: synthesis of CDN11

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- (aminomethyl) -9H-purin-9-yl radical]-15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-cyanoadenosine

To a pyridine (8.00mL) solution of 2-cyanoadenosine (440mg) known in the literature (J.Am.chem.Soc.1989, 111, 8502-8504) was added 4, 4' -dimethoxytrityl chloride (642mg), and the mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. Methanol (10mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (528 mg).

¹H－NMR(CDCl₃)δ：8.21(1H，s)，7.31－7.17(9H，m)，6.79－6.70(4H，m)，5.99(1H，d，J＝5.4Hz)，5.86(2H，brs)，4.86(1H，q，J＝4.6Hz)，4.65(1H，t，J＝3.6Hz)，4.48－4.45(1H，m)，4.41(1H，q，J＝3.0Hz)，3.79(6H，s)，3.46(1H，dd，J＝10.9，3.6Hz)，3.34(1H，dd，J＝10.6，3.3Hz)，2.93(1H，d，J＝2.4Hz).

(step 2)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2- [ ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) methyl ] adenosine

To a tetrahydrofuran (500mL) solution of the compound (14.3g) obtained in the above step 1 was added a tetrahydrofuran solution of lithium aluminum hydride (about 2.5M, 29.0mL), and the mixture was stirred at 40 ℃ for 2 hours under a nitrogen atmosphere. The reaction mixture was cooled with ice, and a saturated aqueous sodium bicarbonate solution (450mL) was added thereto, followed by stirring for 10 minutes, followed by addition of 1- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } oxy) pyrrolidine-2, 5-dione (25.0g), and reaction at room temperature for 18 hours. Saturated aqueous Rochelle's salt (Rochelle salt) was added, stirred for 2.5 hours, and then extracted with a mixture of dichloromethane/methanol. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (10.8 g).

¹H－NMR(CDCl₃)δ：8.01(1H，s)，7.26－7.15(9H，m)，6.75－6.71(4H，m)，6.37(1H，brs)，5.93(1H，d，J＝6.0Hz)，5.67(2H，brs)，5.59(1H，brs)，4.77－4.74(1H，m)，4.46－4.37(4H，m)，4.21(2H，t，J＝8.5Hz)，3.76(3H，s)，3.76(3H，s)，3.42(1H，dd，J＝10.6，3.3Hz)，3.25(1H，dd，J＝10.6，3.3Hz)，3.16(1H，brs)，1.03(2H，t，J＝8.5Hz)，0.05(9H，s).

(step 3)

N-benzoyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2- [ ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) methyl ] adenosine

Chlorotrimethylsilane (15.0mL) was added to a pyridine (70.0mL) solution of the compound (10.8g) obtained in the above step 2, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. Benzoyl chloride (8.44mL) was added to the reaction solution and the mixture was stirred for an additional 2 hours. The reaction mixture was cooled to 0 ℃ and water (21.0mL) was added thereto and the mixture was stirred for 10 minutes, followed by addition of 28% aqueous ammonia (31.4mL) and further stirring at the same temperature for 20 minutes. After further stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure. The residue was suspended in ethyl acetate and filtered to remove solids. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (9.47 g).

MS(ESI)m/z：847(M+H)⁺.

¹H－NMR(CDCl₃)δ：9.54(1H，brs)，8.17(2H，d，J＝6.7Hz)，7.91(1H，brs)，7.66－7.52(3H，m)，7.35－7.10(9H，m)，6.75(4H，d，J＝8.5Hz)，6.45(1H，brs)，6.23(1H，brs)，6.03(1H，d，J＝6.7Hz)，4.70－4.65(2H，m)，4.45－4.19(5H，m)，3.73(6H，s)，3.38－3.32(2H，m)，2.65(1H，brs)，1.05(2H，t，J＝8.8Hz)，0.00(9H，s).

(step 4)

N-benzoyl-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -2- [ ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) methyl ] adenosine

The title compound (3.13g) was obtained in the same manner as in step 4 of example 8 using the compound (9.47g) obtained in step 3.

MS(ESI)m/z：961(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.87(1H，brs)，8.24(1H，brs)，8.02(2H，d，J＝7.3Hz)，7.64－7.51(3H，m)，7.40－7.18(9H，m)，6.81－6.77(4H，m)，6.08(1H，d，J＝4.8Hz)，5.85(1H，brs)，4.70－4.52(4H，m)，4.23－4.17(3H，m)，3.77(6H，s)，3.50(1H，dd，J＝10.9，3.0Hz)，3.29(1H，dd、J＝10.9，4.2Hz)，3.21(1H，d，J＝6.0Hz)，1.06－1.02(2H，m)，0.89(9H，s)，0.09(3H，s)，0.05(9H，s)，0.01(3H，s).

(step 5)

N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -2- [ ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) methyl ] adenosine

To a solution of the compound (1.49g) obtained in the above step 4 in dichloromethane (15.5mL) were added N, N-diisopropylethylamine (1.58mL) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (1.04mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] and C18 silica gel column chromatography [ acetonitrile: 100% ] purification gave the title compound (1.39g) as a mixture of diastereomers on the phosphorus atom (diastereomer ratio ═ 6: 4).

¹H－NMR(CDCl₃)δ8.84(1H，s)，8.33(0.6H，s)，8.28(0.4H，s)，8.02－7.99(2H，m)，7.64－7.59(1H，m)，7.55－7.51(2H，m)，7.42－7.20(9H，m)，6.82－6.79(4H，m)，6.30(0.4H，d，J＝4.2Hz)，6.25(0.6H，d，J＝4.2Hz)，5.95－5.88(1H，m)，4.89－4.77(1H，m)，4.60－4.58(2H，m)，4.51－4.45(1H，m)，4.25－4.18(3H，m)，3.86－3.46(5H，m)，3.78(6H，s)，3.35－3.29(1H，m)，2.53(1.2H，t，J＝6.3Hz)，2.38(0.8H，t，J＝6.3Hz)，1.16－0.98(14H，m)，0.85(3.6H，s)，0.84(5.4H，s)，0.10(1.8H，s)，0.08(1.2H，s)，0.05(9H，s)，0.01(1.2H，s).－0.01(1.8H，s).

(step 6)

N, N-diethylethanaminium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-benzamide-2- [ ({ [ 2- (trimethylsilyl) ethoxy ] amide]Carbonyl } amino) methyl]-9H-purin-9-yl } -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-thiolate

The same reaction as in step 7 of example 1 (raw material: 1.94g) was carried out on the following scale. Using the obtained acetonitrile solution of the compound and the compound (2.19g) obtained in the above step 5, reactions were carried out in the same manner as in the step 8 of example 1 and the step 9 of example 1 to obtain diastereomer 1(138mg) and diastereomer 2(82.8mg) of the title compound.

Diastereomer 1 (low polarity)

¹H－NMR(CD₃OD)δ：9.08(1H，s)，8.11(2H，d，J＝7.3Hz)，7.98(1H，s)，7.67(1H，t，J＝7.6Hz)，7.57(2H，t，J＝7.9Hz)，7.37(2H，d，J＝7.9Hz)，7.28－7.22(4H，m)，6.54(1H，d，J＝8.5Hz)，6.36(1H，d，J＝1.8Hz)，5.66－5.59(1H，m)，5.07－5.02(1H，m)，4.85－4.83(1H，m)，4.72(1H，d，J＝3.6Hz)，4.58－4.08(12H，m)，3.88－3.78(1H，m)，3.49－3.38(1H，m)，3.21(6H，q，J＝7.3Hz)，3.05－3.00(2H，m)，2.49－2.40(1H，m)，2.34－2.26(1H，m)，2.09－2.03(2H，m)，1.31(9H，t，J＝7.6Hz)，1.21－1.09(2H，m)，1.02(9H，s)，0.91(9H，s)，0.30(3H，s)，0.29(3H，s)，0.26(3H，s)，0.12(3H，s)，0.05(9H，s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：1392(M+H)⁺.

¹H－NMR(CD₃OD) δ: 8.91(1H, s), 8.10-8.07 (2H, m), 7.94(1H, s), 7.67-7.63 (1H, m), 7.59-7.54 (2H, m), 7.41-7.20 (6H, m), 6.54(1H, d, J ═ 8.5Hz), 6.22(1H, d, J ═ 5.4Hz), 5.36-5.30 (1H, m), 3.20(6H, q, J ═ 7.3Hz), 3.04-3.00 (2H, m), 2.85-2.75 (2H, m), 2.23-2.13 (2H, m), 1.30(9H, t, J ═ 7.3Hz), 1.03(9H, s), 0.79(9H, s), 0.05(9H, s) (only the peaks observed)

(step 7-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) methyl]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 1) obtained in the above step 6 (42.3mg), a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (25.9 mg).

MS(ESI)m/z：1131(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，s)，8.00(1H，s)，7.26(1H，s)，6.35(1H，d，J＝9.1Hz)，6.26(1H，d，J＝4.8Hz)，5.42－5.36(1H，m)，5.20－5.15(1H，m)，4.91－4.87(2H，m)，4.80－4.78(1H，m)，4.43(1H，t，J＝11.2Hz)，4.36－4.28(3H，m)，4.20－4.15(3H，m)，4.09－3.99(2H，m)，3.51(2H，d，J＝6.7Hz)，3.13(12H，q，J＝7.3Hz)，2.85(2H，brs)，2.01－1.97(2H，m)，1.25(18H，t，J＝7.3Hz)，1.07－1.00(2H，m)，1.00(9H，s)，0.82(9H，s)，0.32(3H，s)，0.28(3H，s)，0.25(3H，s)，0.07(12H，s).

(step 7-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) methyl]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 2) (82.8mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (45.2 mg).

¹H－NMR(CD₃OD)δ：8.61(1H，s)，8.01(1H，s)，7.08(1H，s)，6.34(2H，t，J＝7.9Hz)，5.49(1H，dd，J＝10.6，4.5Hz)，5.42(1H，t，J＝5.1Hz)，5.24－5.17(1H，m)，5.00－4.95(2H，m)，4.69－4.57(2H，m)，4.36(2H，t，J＝17.8Hz)，4.22－4.15(3H，m)，4.05(1H，dd，J＝12.4，5.1Hz)，3.90－3.85(1H，m)，3.51(2H，d，J＝9.1Hz)，3.17(12H，q，J＝7.3Hz)，2.92(2H，t，J＝5.4Hz)，2.04－1.99(2H，m)，1.29(18H，t，J＝7.3Hz)，1.07－0.98(2H，m)，1.00(9H，s)，0.74(9H，s)，0.28(3H，s)，0.28(3H，s)，0.21(3H，s)，0.07(9H，s)，－0.06(3H，s).

(step 8-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) methyl]-9H-purin-9-yl } -15, 16-diHydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Triethylamine trihydrofluoride (700. mu.L) was added to the compound (25.9mg) obtained in the above step 7-1, and the mixture was stirred at 55 ℃ for 2 hours. To the reaction mixture was added an ice-cold mixture of 1M aqueous triethylammonium carbonate (3.5mL) and triethylamine (1.10mL) at room temperature, followed by preparative HPLC [10mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 10% to 40% (0 part to 30 parts) was purified to give the title compound (19.1 mg).

MS(ESI)m/z：903(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.71(1H，s)，8.03(1H，s)，7.10(1H，s)，6.37(1H，d，J＝7.9Hz)，6.28(1H，d，J＝4.2Hz)，5.38－5.33(1H，m)，5.18－5.13(1H，m)，4.84－4.80(2H，m)，4.50－4.40(2H，m)，4.35－4.40(4H，m)，4.21－4.16(2H，m)，4.07－4.00(2H，m)，3.51－3.49(2H，m)，3.07(12H，q，J＝7.3Hz)，2.85(2H，t，J＝5.4Hz)，2.02－1.97(2H，m)，1.23(18H，t，J＝7.3Hz)，1.04(2H，t，J＝8.2Hz)，0.07(9H，s).

(step 8-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) methyl]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (45.2mg) obtained in the above step 7-2, a reaction was carried out in the same manner as in the above step 8-1 to obtain the title compound (37.1 mg).

MS(ESI)m/z：903(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.75(1H，s)，8.02(1H，s)，7.13(1H，s)，6.36(1H，d，J＝9.1Hz)，6.33(1H，d，J＝6.7Hz)，5.51－5.42(2H，m)，4.81(1H，dd，J＝6.7，4.8Hz)，4.51－4.28(7H，m)，4.18(2H，dt，J＝8.3，2.6Hz)，4.02(1H，d，J＝12.7Hz)，3.92－3.87(1H，m)，3.51－3.47(2H，m)，3.13(12H，q，J＝7.3Hz)，2.93－2.90(2H，m)，2.04－1.98(2H，m)，1.27(18H，t，J＝7.3Hz)，1.06－0.99(2H，m)，0.06(9H，s).

(step 9-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- (aminomethyl) -9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

To a tetrahydrofuran (576. mu.L) solution of the compound (19.1mg) obtained in the above step 8-1, a tetrabutylammonium fluoride tetrahydrofuran solution (about 1M, 288. mu.L) was added, and after stirring overnight at room temperature under a nitrogen atmosphere, purification was performed according to the following [ purification conditions ] to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 0% to 30% (0 min to 40 min) and Sep-Pak (registered trade Mark) C18[ water/acetonitrile ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (8.4 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.48(1H，s)，8.03(1H，s)，7.04(1H，s)，6.27(1H，d，J＝3.6Hz)，6.24(1H，d，J＝8.5Hz)，5.98－5.93(1H，m)，5.04－4.99(1H，m)，4.81－4.79(2H，m)，4.45－4.39(2H，m)，4.31－4.27(2H，m)，4.12－3.99(4H，m)，3.54－3.44(2H，m)，2.88－2.85(2H，m)，2.02－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：57.6，55.5.

(step 9-2)

(diastereomer 2)

Using the compound (37.1mg) obtained in the above step 8-2, a reaction was carried out in the same manner as in the above step 9-1 to obtain the title compound (12.8 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.61(1H，s)，8.02(1H，s)，7.13(1H，s)，6.31(1H，d，J＝6.0Hz)，6.28(1H，d，J＝8.5Hz)，5.61－5.55(1H，m)，5.38－5.35(1H，m)，4.80(1H，t，J＝5.1Hz)，4.54(1H，d，J＝4.2Hz)，4.48－4.28(4H，m)，4.13(2H，s)，4.08－4.04(1H，m)，3.94－3.90(1H，m)，3.52－3.49(2H，m)，2.90－2.88(2H，m)，2.03－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：62.2(s)，60.0(s).

Example 12: synthesis of CDN12

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- (hydroxymethyl) -9H-purin-9-yl]-15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H,10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

6-chloro-2-iodo-9- {2, 3, 5-tri-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -9H-purine

To a commercial (Amadis Chemical) solution of 6-chloro-2-iodo-9-. beta. -D-ribofuranosyl-9H-purine (9.65g) in ethylene glycol dimethyl ether (120mL) was added N, N-diisopropylethylamine (40.7mL) and tert-butyldimethylsilyl triflate (26.9mL) at 0 ℃ under a nitrogen atmosphere, warmed to room temperature, and stirred for 19 hours. The reaction mixture was cooled to 0 ℃, and a saturated aqueous sodium bicarbonate solution was added to the reaction mixture to stop the reaction, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (13.7 g).

¹H－NMR(CDCl₃)δ：8.48(1H，s)，6.02(1H，d，J＝4.2Hz)，4.54(1H，t，J＝4.5Hz)，4.29(1H，t，J＝4.5Hz)，4.18－4.15(1H，m)，4.04(1H，dd，J＝11.5，4.2Hz)，3.80(1H，dd，J＝11.5，2.4Hz)，0.96(9H，s)，0.93(9H，s)，0.84(9H，s)，0.17(3H，s)，0.16(3H，s)，0.10(3H，s)，0.09(3H，s)，0.01(3H，s)，－0.16(3H，s).

(step 2)

2- [ (benzoyloxy) methyl ] -6-chloro-9- {2, 3, 5-tri-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -9H-purine

To a solution of the compound (13.7g) obtained in the above step 1 in tetrahydrofuran (121mL) were added tetrakis (triphenylphosphine) palladium (0) (2.10g) and benzyloxymethyl zinc iodide (ca. 0.9M, 30.2mL) prepared according to the following method under a nitrogen atmosphere, and the mixture was stirred at room temperature for 20 hours. After the reaction solution was added with a saturated aqueous ammonium chloride solution to stop the reaction, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (7.29 g).

[ preparation of Benzyloxymethyl Zinc iodide ]

A suspension of zinc powder (5.99g) in tetrahydrofuran (17.1mL) was treated with ultrasonic waves under a nitrogen atmosphere, and then a solution of iodomethyl benzoate (12.0g) in tetrahydrofuran (21.3mL) was added at 10 to 15 ℃ and stirred at the same temperature for 1.5 hours to obtain a tetrahydrofuran solution of benzyloxymethyl zinc iodide (ca. 0.9M, 38.4 mL).

MS(ESI)m/z：763(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.58(1H，s)，8.13(1H，dd，J＝8.5，1.2Hz)，7.60－7.56(1H，m)，7.47－7.43(2H，m)，6.09(1H，d，J＝4.8Hz)，5.60(1H，d，J＝13.9Hz，)，5.56(1H，d，J＝13.9Hz)，4.48(1H，t，J＝4.5Hz)，4.27(1H，t，J＝4.2Hz)，4.15－4.11(1H，m)，4.03(1H，dd，J＝11.5，3.0Hz)，3.80(1H，dd，J＝11.5，2.4Hz)0.96(9H，s)，0.90(9H，s)，0.76(9H，s)，0.16(3H，s)，0.15(3H，s)，0.08(3H，s)，0.06(3H，s)，－0.07(3H，s)，－0.27(3H，s).

(step 3)

2- [ (benzoyloxy) methyl ] -6-chloro-9-beta-D-ribofuranosyl-9H-purine

A tetrahydrofuran solution (about 1M, 38mL) of tetrabutylammonium fluoride was added to a tetrahydrofuran (47.7mL) solution of the compound (7.29g) obtained in the above step 2 at 0 ℃ under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 2.5 hours. After the reaction mixture was quenched by addition of a saturated aqueous ammonium chloride solution, the reaction mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (3.69 g).

¹H－NMR(CDCl₃)δ：8.23(1H，s)，8.15(2H，dd，J＝8.5，1.2Hz)，7.63－7.59(1H，m)，7.48(2H，t，J＝7.9Hz)，5.89(1H，d，J＝6.0Hz)，5.61(1H，d，J＝13.9Hz)，5.56(1H，d，J＝14.5Hz)，4.90(1H，q，J＝5.6Hz)，4.46－4.43(1H，m)，4.28(1H，q，J＝2.2Hz)，4.02(1H，dd，J＝10.0，2.7Hz)，3.84－3.79(1H，m)，3.71－3.65(1H，m)，3.56－3.53(1H，m)，2.70(1H，d，J＝2.4Hz).

(step 4)

2- [ (benzoyloxy) methyl ] -9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-ribofuranosyl } -6-chloro-9H-purine

To a pyridine (56mL) solution of the compound (2.36g) obtained in the above step 3 was added 4, 4' -dimethoxytrityl chloride (2.30g), and the mixture was stirred at room temperature for 17 hours under a nitrogen atmosphere. Ethanol (20mL) was added to the reaction mixture, and the mixture was stirred for about 10 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.41 g).

MS(ESI)m/z：745(M+Na)⁺.

¹H－NMR(CDCl₃)δ：8.32(1H，s)，8.14－8.11(2H，m)，7.64－7.59(1H，m)，7.49－7.44(2H，m)，7.23－7.12(9H，m)，6.72(4H，d，J＝7.9Hz)，5.94(1H，d，J＝5.4Hz)，5.64(1H，d，J＝15.1Hz)，5.59(1H，d，J＝14.5Hz)，4.83－4.77(2H，m)，4.37－4.33(2H，m)，3.77(6H，s)，3.35(1H，dd，J＝10.6，3.3Hz)，3.28(1H，dd，J＝10.9，3.6Hz)，2.64(1H，s).

(step 5)

2- [ (benzoyloxy) methyl ] -9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -6-chloro-9H-purine

To a solution of the compound (2.61g) obtained in the above step 4 in ethylene glycol dimethyl ether (72.0mL) were added N, N-diisopropylethylamine (1.89mL) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.24mL), and the mixture was stirred at room temperature for 1.5 hours under a nitrogen atmosphere. After the reaction solution was quenched by addition of a saturated aqueous sodium bicarbonate solution, the reaction solution was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (1.10 g).

¹H－NMR(CDCl₃)δ：8.35(1H，s)，8.12－8.10(2H，m)，7.60－7.56(1H，m)，7.46－7.42(2H，m)，7.37－7.35(2H，m)，7.28－7.20(7H，m)，6.81－6.75(4H，m)，6.00(1H，d，J＝4.8Hz)，5.50(2H，s)，4.69(1H，q，J＝5.6Hz)，4.39(1H，dd，J＝5.1，3.9Hz)，4.16(1H，q，J＝3.8Hz)，3.77(6H，s)，3.45(1H，dd，J＝10.6，3.3Hz)，3.31(1H，dd，J＝10.9，4.2Hz)，3.06(1H，d，J＝6.7Hz)，0.86(9H，s)，0.04(3H，s)，－0.02(3H，s).

(step 6)

2- [ (benzoyloxy) methyl ] -9- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -2-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -6-chloro-9H-purine

Using the compound (511mg) obtained in the above step 5, a reaction was carried out in the same manner as in the step 6 of example 1 to obtain the title compound (569mg) as a mixture of diastereomers relating to phosphorus atoms (diastereomer ratio: 6: 4).

¹H－NMR(CDCl₃)δ：8.43(0.6H，s)，8.39(0.4H，s)，8.10(2H，d，J＝7.9Hz)，7.58(1H，t，J＝7.6Hz)，7.47－7.38(4H，m)，7.31－7.18(7H，m)，6.82－6.78(4H，m)，6.25(0.4H，d，J＝4.8Hz)，6.21(0.6H，d，J＝4.8Hz)，5.49(1H，d，J＝13.9Hz)，5.45(1H，d，13.9Hz)，4.98－4.93(0.6H，m)，4.83－4.78(0.4H，m)，4.45(0.6H，t，J＝4.2Hz)，4.36(0.4H，t，J＝4.2Hz)，4.21－4.17(1H，m)，3.78(6H，s)，3.76－3.32(6H，m)，2.46(1.2H，t，J＝6.3Hz)，2.30(0.8H，t，J＝6.3Hz)，1.28－0.86(12H，m)，0.825(5.4H，s)，0.815(3.6H，s)，0.08(1.8H，s)，0.04(1.2H，s)，－0.01(1.8H，s)，－0.02(1.2H，s).

(step 7)

N, N-diethylethanaminium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 2- [ (benzoyloxy) methyl]-6-chloro-9H-purin-9-yl } -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-thiolate

The same reaction as in step 7 of example 1 (starting material: 2.72g) was carried out on the following scale. Using the obtained acetonitrile solution of the compound and the compound (3.03g) obtained in the above step 6, reactions were carried out in the same manner as in the step 8 of example 1 and the step 9 of example 1 to obtain diastereomer 1(351mg) and diastereomer 2(351mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1268(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.30(1H，s)，8.14(2H，d，J＝7.3Hz)，7.99(1H，s)，7.64(1H，t，J＝7.3Hz)，7.52(2H，t，J＝7.9Hz)，7.40－7.36(2H，m)，7.30－7.23(4H，m)，6.43(1H，d，J＝8.5Hz)，6.37(1H，d，J＝3.0Hz)，5.62－5.56(1H，m)，5.62(2H，s)，5.06－5.01(1H，m)，4.83(1H，dd，J＝4.5，2.7Hz)，4.69(1H，d，J＝4.2Hz)，4.49－4.28(7H，m)，4.08(1H，dd，J＝12.1，4.8Hz)，3.81－3.70(1H，m)，3.46－3.38(1H，m)，3.17－3.10(8H，m)，2.29－2.23(4H，m)，1.28(9H，t，J＝7.3Hz)，0.91(9H，s)，0.90(9H，s)，0.29(3H，s)，0.20(3H，s)，0.16(3H，s)，0.11(3H，s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：1268(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.97(1H，s)，8.14(2H，d，J＝8.5Hz)，7.94(1H，s)，7.68－7.63(1H，m)，7.55－7.50(2H，m)，7.39－7.36(2H，m)，7.26－7.21(4H，m)，6.41(1H，d，J＝7.9Hz)，6.21(1H，d，J＝5.4Hz)，5.64(1H，d，J＝15.1Hz)，5.57(1H，d，J＝15.1Hz)，5.25－5.18(2H，m)，5.13－5.10(1H，m)，5.04－5.01(1H，m)，4.94－4.78(3H，m)，4.51(1H，t，J＝10.9Hz)，4.33－4.06(6H，m)，3.15(6H，q，J＝7.3Hz)，3.08－2.96(2H，m)，2.84－2.71(2H，m)，2.25－2.19(2H，m)，1.28(9H，t，J＝7.3Hz)，0.91(9H，s)，0.79(9H，s)，0.19(6H，s)，0.14(3H，s)－0.07(3H，s).

(step 8-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 6-amino-2- (hydroxymethyl) -9H-purin-9-yl]-15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a solution of the compound (diastereomer 1) obtained in the above step 7 (37.3mg) in methanol (0.500mL) was added a 28% aqueous ammonia solution (0.500mL), and the mixture was stirred in a sealed tube at 60 ℃ for 3 hours. The reaction was directly purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 35% -55% (0 min-30 min) to give the title compound (22.0 mg: containing impurities).

MS(ESI)m/z：988(M+H)⁺.

(step 8-2)

Bis (N, N-diethylethanaminium) (5R, 7)R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 6-amino-2- (hydroxymethyl) -9H-purin-9-yl]-15, 16-bis { [ tert-butyl (dimethyl) silyl ]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a tetrahydrofuran (0.500mL) solution of the compound (diastereomer 2) (37.7mg) obtained in the above step 7 was added a 28% aqueous ammonia solution (0.500mL), and the mixture was stirred in a sealed tube at 60 ℃ for 3 hours. A28% aqueous ammonia solution (0.500mL) was added thereto, and the mixture was stirred for further 3 hours. A28% aqueous ammonia solution (0.500mL) was added thereto and the mixture was stirred overnight. The reaction was directly purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 20% to 50% (0 part to 30 parts) was purified to give the title compound (19.3 mg).

MS(ESI)m/z：988(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.64(1H，s)，8.02(1H，s)，7.09(1H，s)，6.35(2H，d，J＝7.9Hz)，5.48－5.41(2H，m)，5.26－5.19(1H，m)，5.00－4.95(2H，m)，4.70－4.53(4H，m)，4.22(1H，s)，4.06(1H，dd，J＝12.1，4.8Hz)，3.91－3.86(1H，m)，3.53－3.48(2H，m)，3.18(12H，q，J＝7.3Hz)，2.92(2H，t，J＝5.4Hz)，2.04－1.99(2H，m)，1.29(18H，t，J＝7.3Hz)，1.00(9H，s)，0.74(9H，s)，0.28(6H，s)，0.21(3H，s)，－0.06(3H，s).

(step 9-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- (hydroxymethyl) -9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (22.0 mg: containing impurities) obtained in the above step 8-1, a reaction was carried out in the same manner as in the step 11 of example 1, and then purification was carried out according to the following purification conditions, thereby obtaining the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (8.0 mg).

MS(ESI)m/z：760(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.70(1H，s)，8.03(1H，s)，7.14(1H，s)，6.39(1H，d，J＝8.5Hz)，6.29(1H，d，J＝3.6Hz)，5.40－5.35(1H，m)，5.15(1H，dt，J＝9.1，3.8Hz)，4.84(1H，d，J＝3.6Hz)，4.80(1H，t，J＝4.5Hz)，4.57(2H，s)，4.51－4.43(2H，m)，4.38－4.31(2H，m)，4.09－4.00(2H，m)，3.52－3.48(2H，m)，2.89－2.76(2H，m)，2.01－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：58.1(s)，54.4(s).

(step 9-2)

The compound (19.3mg) obtained in the above step 8-2 was reacted in the same manner as in step 11 of example 1, and then purified under the following purification conditions to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (6.5 mg).

MS(ESI)m/z：760(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.76(1H，s)，8.05(1H，s)，7.18(1H，s)，6.38(1H，d，J＝8.5Hz)，6.33(1H，d，J＝6.7Hz)，5.49－5.43(2H，m)，4.81(1H，dd，J＝6.3，4.5Hz)，4.58(2H，s)，4.51－4.29(5H，m)，4.04(1H，d，J＝12.1Hz)，3.93－3.88(1H，m)，3.54－3.52(2H，m)，2.92(2H，t，J＝5.4Hz)，2.05－2.00(2H，m).

³¹P－NMR(CD₃OD)δ：63.0(s)，60.3(s).

Example 13: synthesis of CDN13

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6- [ (2-aminoethyl) amino]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -6-chloro-9H-purine

Using 9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] - β -D-ribofuranosyl } -6-chloro-9H-purine (15.3g), which is known in the literature (J.org.chem.2000, 65, 5104-5113), a reaction was carried out in the same manner as in step 3 of example 5 to give the title compound (8.44g) and the positional isomer of the title compound, 9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] - β -D-ribofuranosyl } -6-chloro-9H-purine (5.77 g).

MS(ESI)m/z：703(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.71(1H，s)，8.37(1H，s)，7.40－7.37(2H，m)，7.31－7.19(7H，m)，6.82－6.78(4H，m)，6.06(1H，d，J＝4.9Hz)，4.79－4.74(1H，m)，4.59(1H，dd，J＝4.9，3.9Hz)，4.20(1H，dd，J＝3.9，1.9Hz)，3.79(3H，s)，3.78(3H，s)，3.52(1H，dd，J＝10.7，3.4Hz)，3.29(1H，dd，J＝10.7，3.9Hz)，3.08(1H，d，J＝6.8Hz)，0.90(9H，s)，0.10(3H，s)，0.03(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：703(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.67(1H，s)，8.36(1H，s)，7.46－7.42(2H，m)，7.36－7.20(7H，m)，6.84－6.80(4H，m)，6.11(1H，d，J＝5.4Hz)，5.00－4.97(1H，m)，4.40－4.35(1H，m)，4.31－4.28(1H，m)，3.79(3H，s)，3.79(3H，s)，3.52(1H，dd，J＝10.7，2.9Hz)，3.42(1H，dd，J＝10.7，3.9Hz)，2.68(1H，d，J＝3.9Hz)，0.84(9H，s)，0.00(3H，s)，－0.16(3H，s).

(step 2)

9- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -2-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -6-chloro-9H-purine

Using the compound (5.39g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (5.78g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

MS(ESI)m/z：903(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.69(0.5H，s)，8.67(0.5H，s)，8.43(0.5H，s)，8.41(0.5H，s)，7.43－7.37(2H，m)，7.32－7.19(7H，m)，6.83－6.78(4H，m)，6.30(0.5H，d，J＝4.4Hz)，6.21(0.5H，d，J＝4.9Hz)，5.06－5.00(0.5H，m)，4.86－4.80(0.5H，m)，4.56－4.50(1H，m)，4.27－4.20(1H，m)，3.79(6H，s)，3.75－3.62(1H，m)，3.57－3.46(4H，m)，3.30(1H，dt，J＝10.7，3.9Hz)，2.50(1H，t，J＝6.3Hz)，2.37(1H，t，J＝6.6Hz)，1.13－1.06(9H，m)，0.90(1.5H，s)，0.89(1.5H，s)，0.86(4.5H，s)，0.85(4.5H，s)，0.12(1.5H，s)，0.08(1.5H，s)，0.02(1.5H，s)，0.02(1.5H，s).

(step 3)

The same reaction as in step 7 of example 1 (raw material: 1.84g) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.62g) obtained in the above step 2, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 4)

3- { [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- (6-chloro-9H-purin-9-yl) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-10-yl]Oxy-propionitrile

Using the crude product obtained in the above step 3, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (626mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1134(M+H)⁺.

(step 5)

Bis (N, N-Diethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6- [ (2-aminoethyl) amino]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Ethylenediamine (0.352mL) and triethylamine (0.735mL) were added to a solution of the compound (299 mg: diastereomer mixture) obtained in the above step 4 in ethanol (10mL), and the mixture was stirred at 60 ℃ for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(122 mg: containing impurities) and diastereomer 2(111 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1001(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1001(M+H)⁺.

(step 6-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6- [ (2-aminoethyl) amino]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (122 mg: containing impurities) obtained in the above step 5 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (29.6 mg).

MS(ESI)m/z：773(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.77(1H，s)，8.27(1H，s)，8.03(1H，s)，7.10(1H，s)，6.35(1H，d，J＝8.5Hz)，6.27(1H，d，J＝4.8Hz)，5.41(1H，ddd，J＝7.9，4.2，2.1Hz)，5.21－5.14(1H，m)，4.84－4.77(2H，m)，4.49－4.38(2H，m)，4.35－4.26(2H，m)，4.09－3.99(2H，m)，3.92－3.80(2H，m)，3.51－3.45(2H，m)，3.22(2H，t，J＝6.0Hz)，2.89－2.81(2H，m)，2.02－1.94(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s)，55.0(s).

(step 6-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (119 mg: containing impurities) obtained in the above step 5 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (15.6 mg).

MS(ESI)m/z：773(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.83(1H，s)，8.27(1H，s)，8.02(1H，s)，7.10(1H，s)，6.35(1H，d，J＝7.9Hz)，6.32(1H，d，J＝6.7Hz)，5.55－5.43(2H，m)，4.81(1H，dd，J＝7.0，4.5Hz)，4.52－4.29(5H，m)，4.06－4.00(1H，m)，3.93－3.80(3H，m)，3.52－3.47(2H，m)，3.21(2H，t，J＝5.7Hz)，2.94－2.88(2H，m)，2.05－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.1(s).

Example 14: synthesis of CDN14

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- { 6- [ (2-hydroxyethyl) amino group ]-9H-purin-9-yl } -2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- { 6- [ (2-hydroxyethyl) amino]-9H-purin-9-yl } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2-yl)H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a solution of the compound (313mg) obtained in step 4 of example 13 in ethanol (10mL) were added 2-aminoethanol (0.330mL) and triethylamine (0.769mL), and the mixture was stirred at 60 ℃ for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(111 mg: containing impurities) and diastereomer 2(102 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1002(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1002(M+H)⁺.

(step 2-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- { 6- [ (2-hydroxyethyl) amino]-9H-purin-9-yl } -2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (111 mg: containing impurities) obtained in the above step 1 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (47.1 mg).

MS(ESI)m/z：774(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.70(1H，s)，8.22(1H，s)，8.03(1H，s)，7.10(1H，s)，6.34(1H，d，J＝8.5Hz)，6.29(1H，d，J＝4.8Hz)，5.41－5.34(1H，m)，5.19－5.13(1H，m)，4.84(1H，d，J＝4.2Hz)，4.79(1H，dd，J＝4.8，2.4Hz)，4.52－4.41(2H，m)，4.39－4.31(2H，m)，4.07－3.96(2H，m)，3.81－3.66(4H，m)，3.52－3.47(2H，m)，2.90－2.77(2H，m)，2.03－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，54.4(s).

(step 2-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (102 mg: containing impurities) obtained in the above step 1 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (27.1 mg).

MS(ESI)m/z：774(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.78(1H，s)，8.22(1H，s)，8.02(1H，s)，7.12(1H，s)，6.34(1H，d，J＝1.8Hz)，6.32(1H，s)，5.52－5.42(2H，m)，4.80(1H，dd，J＝6.7，4.8Hz)，4.50－4.28(5H，m)，4.05－3.98(1H，m)，3.93－3.86(1H，m)，3.81－3.68(4H，m)，3.53－3.47(2H，m)，2.95－2.88(2H，m)，2.05－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：63.0(s)，60.2(s).

Example 15: synthesis of CDN15

N- [ 2- ({ 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } amino) ethyl]-2-hydroxyacetamide

[ synthetic route ]

(step 1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- (6- { [ 2- (2-hydroxyacetamide) ethyl]Amino } -9H-purin-9-yl) -2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound obtained in the step 6-2 of example 13 (10.0mg), a reaction was carried out in the same manner as in the step 1-1 of example 7, and then purification was carried out according to the following purification conditions, thereby obtaining the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (6.6 mg).

MS(ESI)m/z：831(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.78(1H，s)，8.24(1H，s)，8.02(1H，s)，7.12(1H，s)，6.33(2H，d，J＝6.7Hz)，5.52－5.42(2H，m)，4.80(1H，dd，J＝6.7，4.2Hz)，4.50－4.27(5H，m)，4.04－3.98(1H，m)，3.95(2H，s)，3.93－3.86(1H，m)，3.83－3.73(2H，m)，3.58－3.46(4H，m)，2.95－2.88(2H，m)，2.05－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.4(s).

Example 16: synthesis of CDN16

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 2-amino-6- [ (2-hydroxyethyl) amino group]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2-acetamide-2 ', 3 ', 5 ' -tri-O-acetyl-N- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) adenosine

To a solution of commercially available (Tokyo chemical industry) 6-chloro-9-. beta. -D-ribofuranosyl-9H-purin-2-amine (5.00g) in ethanol (30mL) were added 2- { [ tert-butyl (dimethyl) silyl ] oxy } ethane-1-amine (3.49g) and N, N-diisopropylethylamine (4.33mL), and the mixture was stirred at 80 ℃ for 65 hours. After the reaction mixture was concentrated under reduced pressure, pyridine (15mL) and acetic anhydride (15mL) were added to the residue, and the mixture was stirred at 70 ℃ for 4 hours. The reaction mixture was concentrated under reduced pressure, and then saturated aqueous sodium bicarbonate solution was added thereto, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (8.91 g).

MS(ESI)m/z：609(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.84(1H，s)，7.77(1H，s)，6.14(1H，brs)，6.03(1H，d，J＝4.8Hz)，5.92(1H，t，J＝5.1Hz)，5.72(1H，t，J＝5.1Hz)，4.50－4.40(2H，m)，4.35(1H，dd，J＝11.8，4.5Hz)，3.83(2H，t，J＝5.1Hz)，3.76－3.63(2H，m)，2.54(3H，s)，2.14(3H，s)，2.10(6H，s)，0.91(9H，s)，0.07(6H，s).

(step 2)

2-acetamide-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -N- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) adenosine

To a solution of the compound (4.00g) obtained in the above step 1 in dichloromethane (40mL) was added a methanol solution of sodium methoxide (1.0M, 6.64mL), and the mixture was stirred at 0 ℃ for 1 hour. After acetic acid (0.413mL) and pyridine (0.5mL) were added to the reaction mixture to stop the reaction, the reaction mixture was concentrated under reduced pressure. After pyridine was added to the residue, a portion was concentrated under reduced pressure to prepare a pyridine (about 20mL) solution. To the solution was added 4, 4' -dimethoxytrityl chloride (4.68g) at 0 ℃ and, after stirring at the same temperature for 30 minutes, the mixture was stored at 4 ℃ overnight. Methanol (2mL) was added to the reaction mixture, and after stirring for 30 minutes, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (4.41 g).

MS(ESI)m/z：785(M+H)⁺.

¹H－NMR(CDCl₃) δ: 7.97(1H, s), 7.90(1H, s), 7.21-7.08 (9H, m), 6.73-6.67 (4H, m), 6.14(1H, s), 5.88(1H, d, J ═ 6.7Hz), 4.91-4.85 (1H, m), 4.46(1H, t, J ═ 3.0Hz), 4.32(1H, d, J ═ 5.4Hz), 3.86-3.80 (2H, m), 3.76(3H, s), 3.70-3.66 (1H, m), 3.42-3.34 (2H, m), 3.13(1H, dd, J ═ 10.6, 2.7Hz), 2.24(3H, s), 0.90(9H, s), 0.07 (H, s), and only the peaks described in the text that can be observed

(step 3)

2-acetamide-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -N- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) adenosine

Using the compound (4.41g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (1.75g) and the positional isomer of the title compound, 2-acetamide-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -N- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) adenosine (1.31 g).

MS(ESI)m/z：899(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.89(1H，s)，7.69(1H，brs)，7.39－7.34(2H，m)，7.29－7.18(7H，m)，6.80－6.75(4H，m)，6.11(1H，brs)，5.89(1H，d，J＝5.4Hz)，4.65(1H，dd，J＝5.4，2.7Hz)，4.43(1H，dd，J＝5.1，3.3Hz)，4.19－4.15(1H，m)，3.83(2H，dd，J＝5.4，2.7Hz)，3.77(6H，s)，3.74－3.63(2H，m)，3.40(1H，dd，J＝10.9，3.6Hz)，3.22(1H，dd，J＝10.9，3.9Hz)，2.43(3H, s), 0.90(9H, s), 0.88(9H, s), 0.10(3H, s), 0.06(6H, s), 0.03(3H, s) ("only the peaks observed in memory")

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：899(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.84(1H，s)，7.52(1H，brs)，7.48－7.44(2H，m)，7.37－7.32(4H，m)，7.29－7.18(3H，m)，6.83－6.78(4H，m)，6.12(1H，brs)，5.89(1H，d，J＝6.0Hz)，4.96－4.89(1H，m)，4.32－4.27(1H，m)，4.24(1H，dd，J＝3.2，1.6Hz)，3.84(2H，dd，J＝5.4，2.7Hz)，3.78(6H，s)，3.75－3.65(2H，m)，3.48(1H，dd，J＝10.6，2.7Hz)，3.35(1H，dd，J＝10.6，3.6Hz)，2.72(1H，d，J＝3.0Hz)，2.37(3H，s)，0.91(9H，s)，0.84(9H，s)，0.07(6H，s)，－0.01(3H，s)，－0.17(3H，s).

(step 4)

2-acetamide-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -N- (2- { [ tert-butyl (dimethyl) silyl ] oxy } ethyl) -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine

Using the compound (1.75g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (2.08g) in the form of a diastereomer mixture (diastereomer ratio: 6: 4) on the phosphorus atom.

MS(ESI)m/z：1099(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.90(0.6H，s)，7.88(0.4H，s)，7.61(1H，d，J＝7.3Hz)，7.45－7.18(9H，m)，6.81(4H，m)，6.10(0.6H，d，J＝5.4Hz)，6.09(1H，brs)，6.06(0.4H，d，J＝4.8Hz)，4.95－4.85(0.6H，m)，4.76－4.69(0.4H，s)，4.45－4.41(0.6H，m)，4.40－4.36(0.4H，m)，4.19－4.13(1H，m)，3.86－3.80(2H，m)，3.78(6H，s)，3.75－3.41(8H，m)，3.32－3.22(1H，m)，2.55－2.45(3H，m)，2.36－2.30(1H，m)，1.30－1.10(9H，m)，0.92(1.2H，d，J＝6.7Hz)，0.90(9H，s)，0.85(9H，s)，0.76(1.8H，d，J＝6.7Hz)，0.10(1.8H，s)，0.07(1.2H，s)，0.06(6H，s)，0.00(3H，s).

(step 5)

The same reaction (raw material: 981mg) as in step 7 of example 1 was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.05g) obtained in the above step 4, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 6)

Using the crude product obtained in the above step 5, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (413mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1330(M+H)⁺.

(step 7)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 2-amino-6- [ (2- { [ tert-butyl (dimethyl) silyl) ]Oxy } ethyl) amino]-9H-purin-9-yl } -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The compound (413mg) obtained in the above step 6 was dissolved in methanol (5mL) and 28% aqueous ammonia (5mL), and stirred at room temperature for 63 hours. After the reaction mixture was concentrated under reduced pressure, the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], to separate diastereomer 1 having low polarity from diastereomer 2 having high polarity. The individual diastereoisomers were dissolved again in methanol (5mL) and 28% aqueous ammonia (5mL) and stirred at 100 ℃ for 2 days. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(70.4 mg: containing impurities) and diastereomer 2(65.1 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1131(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1131(M+H)⁺.

(step 8-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 2-amino-6- [ (2-hydroxyethyl) amino group]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (70.4 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (20.8 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.24(1H，s)，8.02(1H，s)，7.09(1H，s)，6.29(1H，d，J＝4.2Hz)，6.12(1H，d，J＝8.5Hz)，5.46－5.39(1H，m)，5.22－5.15(1H，m)，4.84(1H，d，J＝3.6Hz)，4.80(1H，t，J＝4.5Hz)，4.50－4.32(3H，m)，4.32－4.28(1H，m)，4.12－3.98(2H，m)，3.74(2H，t，J＝5.4Hz)，3.68－3.60(2H，m)，3.48(2H，t，J＝5.4Hz)，2.87－2.70(2H，m)，2.01－1.93(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s)，54.1(s).

(step 8-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 2-amino-6- [ (2-hydroxyethyl) amino group ]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (65.1 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -30% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 0% -60% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (7.9 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.39(1H，s)，8.02(1H，s)，7.13(1H，s)，6.33(1H，d，J＝6.7Hz)，6.13(1H，d，J＝8.5Hz)，5.51－5.40(2H，m)，4.83－4.78(1H，m)，4.51－4.29(4H，m)，4.28－4.23(1H，m)，4.07－4.00(1H，m)，3.94－3.87(1H，m)，3.75(2H，t，J＝5.7Hz)，3.69－3.62(2H，m)，3.53－3.47(2H，m)，2.91(2H，t，J＝5.7Hz)，2.05－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：62.9(s)，60.3(s).

Example 17: synthesis of CDN17

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 6- (hydroxymethyl) -9H-purin-9-yl ]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

6- [ (benzoyloxy) methyl ] -9- {2, 3, 5-tri-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -9H-purine

Using 6-chloro-9- {2, 3, 5-tri-O- [ tert-butyl (dimethyl) silyl ] - β -D-ribofuranosyl } -9H-purine (10.7g) which is known in the literature (J.org.chem.1997, 62, 6833-6841), a reaction was carried out in the same manner as in step 2 of example 12 to give the title compound (10.4 g).

MS(ESI)m/z：729(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.94(1H，s)，8.50(1H，s)，8.18－8.13(2H，m)，7.60－7.54(1H，m)，7.48－7.41(2H，m)，6.13(1H，d，J＝4.2Hz)，5.88(2H，s)，4.63(1H，dd，J＝4.5，2.1Hz)，4.34(1H，dd，J＝4.2，2.1Hz)，4.17－4.14(1H，m)，4.04(1H，dd，J＝11.5，3.6Hz)，3.80(1H，dd，J＝11.5，2.4Hz)，0.94(9H，s)，0.93(9H，s)，0.80(9H，s)，0.14(3H，s)，0.13(3H，s)，0.11(3H，s)，0.10(3H，s)，－0.02(3H，s)，－0.20(3H，s).

(step 2)

6- [ (benzoyloxy) methyl ] -9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-ribofuranosyl } -9H-purine

A tetrahydrofuran solution (about 1M, 49.4mL) of tetrabutylammonium fluoride was added to a tetrahydrofuran (50mL) solution of the compound (10.3g) obtained in the above step 1, and the mixture was stirred at 0 ℃ for 4 hours. After adding acetic acid (2.83mL) to the reaction mixture, the mixture was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [ dichloromethane/methanol ] to conduct crude purification. To a solution of the crude product in pyridine (50mL), 4' -dimethoxytrityl chloride (10.1g) was added at 0 ℃ and the mixture was stirred at 4 ℃ overnight. Methanol (2mL) was added to the reaction mixture, and the mixture was stirred for 1 hour. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (4.62 g).

MS(ESI)m/z：689(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.91(1H，s)，8.33(1H，s)，8.15－8.11(2H，m)，7.60－7.54(1H，m)，7.46－7.40(2H，m)，7.27－7.12(9H，m)，6.75－6.70(4H，m)，6.04(1H，d，J＝6.0Hz)，5.88(2H，s)，5.43(1H，brs)，4.90－4.84(1H，m)，4.48－4.41(2H，m)，3.76(6H，s)，3.44(1H，dd，J＝10.6，3.3Hz)，3.30(1H，dd，J＝10.6，3.3Hz)，3.09(1H，brs).

(step 3)

6- [ (benzoyloxy) methyl ] -9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -9H-purine

Using the compound (4.53g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (2.09g) and the positional isomer of the title compound, 6- [ (benzoyloxy) methyl ] -9- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] - β -D-ribofuranosyl } -9H-purine (1.81 g).

MS(ESI)m/z：803(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.92(1H，s)，8.32(1H，s)，8.16－8.12(2H，m)，7.60－7.54(1H，m)，7.47－7.36(4H，m)，7.31－7.17(7H，m)，6.81－6.76(4H，m)，6.07(1H，d，J＝4.8Hz)，5.87(2H，s)，4.81－4.74(1H，m)，4.62－4.57(1H，m)，4.22－4.17(1H，m)，3.77(6H，s)，3.52(1H，dd，J＝10.9，3.6Hz)，3.26(1H，dd，J＝10.9，3.6Hz)，3.12(1H，d，J＝6.7Hz)，0.89(9H，s)，0.09(3H，s)，0.02(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：803(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.87(1H，s)，8.31(1H，s)，8.17－8.13(2H，m)，7.60－7.55(1H，m)，7.48－7.42(4H，m)，7.35－7.31(4H，m)，7.28－7.17(3H，m)，6.83－6.78(4H，m)，6.12(1H，d，J＝5.4Hz)，5.88(2H，s)，4.99(1H，dd，J＝5.1，2.6Hz)，4.40－4.34(1H，m)，4.28(1H，dd，J＝3.6，2.0Hz)，3.77(6H，s)，3.53(1H，dd，J＝10.9，3.0Hz)，3.40(1H，dd，J＝10.9，3.9Hz)，2.68(1H，d，J＝4.8Hz)，0.84(9H，s)，0.00(3H，s)，－0.14(3H，s).

(step 4)

6- [ (benzoyloxy) methyl ] -9- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -2-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -9H-purine

Using the compound (2.04g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (2.37g) in the form of a diastereomer mixture (diastereomer ratio: 56: 44) on the phosphorus atom.

MS(ESI)m/z：1003(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.88(0.44H，s)，8.87(0.56H，s)，8.37(0.44H，s)，8.35(0.56H，s)，8.16－8.11(2H，m)，7.60－7.54(1H，m)，7.47－7.38(4H，m)，7.33－7.16(7H，m)，6.83－6.76(4H，m)，6.31(0.44H，d，J＝4.8Hz)，6.21(0.56H，d，J＝4.8Hz)，5.87(0.88H，s)，5.86(1.12H，s)，5.08－5.01(0.56H，m)，4.86－4.80(0.44H，m)，4.56－4.50(1H，m)，4.27－4.20(1H，m)，3.85－3.54(2H，m)，3.78(6H，s)，3.54－3.44(3H，m)，3.34－3.25(1H，m)，2.51(1.12H，t，J＝6.3Hz)，2.34(0.88H，t，J＝6.3Hz)，1.14－1.05(9H，m)，0.90(3H，d，J＝6.7Hz)，0.85(5.04H，s)，0.85(3.96H，s)，0.11(1.68H，s)，0.08(1.32H，s)，0.01(3H，s).

(step 5)

The same reaction as in step 7 of example 1 (raw material: 783mg) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (765mg) obtained in the above step 4, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 6)

{ 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } methylbenzoate

Using the crude product obtained in the above step 5, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (345mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1234(M+H)⁺.

(step 7)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl ]Oxy } -7- [ 6- (hydroxymethyl) -9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (345mg) obtained in the above-mentioned step 6, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(101 mg: containing impurities) and diastereomer 2(89.9 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：973(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：973(M+H)⁺.

(step 8-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 6- (hydroxymethyl) -9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Oxygen fiveHeterodiphosphocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (101 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (59.2 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.07(1H，s)，8.88(1H，s)，8.03(1H，s)，7.10(1H，s)，6.48(1H，d，J＝8.5Hz)，6.29(1H，d，J＝4.8Hz)，5.46(1H，dt，J＝7.9，4.2Hz)，5.21－5.15(1H，m)，5.10(2H，s)，4.84－4.80(1H，m)，4.79(1H，t，J＝4.5Hz)，4.51－4.42(2H，m)，4.37－4.34(1H，m)，4.33－4.24(1H，m)，4.09－3.97(2H，m)，3.52－3.47(2H，m)，2.89－2.82(2H，m)，2.03－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，54.8(s).

(step 8-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 6- (hydroxymethyl) -9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (88.5 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -30% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 10% -70% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (27.7 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.16(1H，s)，8.87(1H，s)，8.03(1H，s)，7.11(1H，s)，6.51(1H，d，J＝8.5Hz)，6.33(1H，d，J＝6.7Hz)，5.58－5.45(2H，m)，5.12－5.09(2H，m)，4.81－4.76(1H，m)，4.53－4.49(1H，m)，4.48－4.36(2H，m)，4.34(2H，s)，4.09－4.02(1H，m)，3.92－3.85(1H，m)，3.53－3.47(2H，m)，2.94－2.87(2H，m)，2.05－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.1(s).

Example 18: synthesis of CDN18

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 4-amino-5- (3-aminopropyl) -7H-pyrrolo [2, 3-d ]]Pyrimidin-7-yl]-15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

4-azido-5-iodo-7- (2, 3, 5-tri-O-benzoyl-beta-D-ribofuranosyl) -7H-pyrrolo [2, 3-D ] pyrimidine

Tetrabutylammonium azide (10.1g) was added to a solution of 4-chloro-5-iodo-7- (2, 3, 5-tri-O-benzoyl-. beta. -D-ribofuranosyl) -7H-pyrrolo [2, 3-D ] pyrimidine (17.96g) in N, N-dimethylformamide (90mL) known in the literature (Synth. Commun.2012, 42, 358-374), and the mixture was stirred at 80 ℃ for 30 minutes. The reaction mixture was returned to room temperature, and a saturated aqueous sodium bicarbonate solution and ethyl acetate were added to the reaction mixture to conduct extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (17.66 g).

¹H－NMR(CDCl₃)δ：9.18(1H，s)，8.13－8.09(2H，m)，8.03－7.99(2H，m)，7.95－7.90(2H，m)，7.64－7.34(10H，m)，6.71(1H，d，J＝5.1Hz)，6.20(1H，t，J＝5.5Hz)，6.11(1H，t，J＝5.3Hz)，4.96(1H，dd，J＝12.2，3.2Hz)，4.88－4.84(1H，m)，4.70(1H，dd，J＝12.2，3.2Hz).

(step 2)

4-azido-7- (2, 3, 5-tri-O-benzoyl- β -D-ribofuranosyl) -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) prop-1-yn-1-yl ] -7H-pyrrolo [2, 3-D ] pyrimidine

To a mixed solution of the compound (9.64g) obtained in the above step 1 in N, N-dimethylformamide (30mL) -tetrahydrofuran (100mL) were added 2- (trimethylsilyl) ethylprop-2-yn-1-ylcarbamate (6.58g), triethylamine (4.57mL), tetrakis (triphenylphosphine) palladium (0) (763mg), and copper (I) iodide (251mg) in this order, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (7.20 g).

¹H－NMR(CDCl₃)δ：9.16(1H，s)，8.14－8.08(2H，m)，8.03－7.99(2H，m)，7.94－7.90(2H，m)，7.66(1H，s)，7.65－7.35(9H，m)，6.66(1H，d，J＝5.1Hz)，6.22(1H，t，J＝5.5Hz)，6.10(1H，t，J＝5.3Hz)，5.07(1H，brs)，4.95(1H，dd，J＝12.3，2.9Hz)，4.88－4.84(1H，m)，4.70(1H，dd，J＝12.3，3.7Hz)，4.29(2H，d，J＝5.5Hz)，4.21(2H，t，J＝8.6Hz)，1.02(2H，t，J＝8.6Hz)，0.05(9H，s).

(step 3)

7- (2, 3, 5-tri-O-benzoyl-beta-D-ribofuranosyl) -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

To a mixed solution of the compound (7.20g) obtained in the above step 2 in methanol (70mL) -tetrahydrofuran (70mL) was added 20% palladium hydroxide on carbon (2g), and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. After the catalyst was removed by filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (5.45 g).

¹H－NMR(CDCl₃)δ：8.26(1H，s)，8.19－8.15(2H，m)，8.02－7.97(2H，m)，7.96－7.92(2H，m)，7.65－7.48(5H，m)，7.43－7.32(4H，m)，6.82(1H，s)，6.73(1H，d，J＝6.3Hz)，6.17(1H，t，J＝5.9Hz)，6.11(1H，dd，J＝5.9，3.9Hz)，5.23(2H，s)，4.90(1H，dd，J＝12.2，3.4Hz)，4.77－4.70(2H，m)，4.63(1H，dd，J＝12.2，3.4Hz)，4.15(2H，t，J＝8.6Hz)，3.20－3.10(2H，m)，2.62(2H，t，J＝7.6Hz)，1.72－1.62(2H，m)，1.01－0.94(2H，m)，0.04(9H，s).

(step 4)

N, N-dibenzoyl-7- (2, 3, 5-tri-O-benzoyl-beta-D-ribofuranosyl) -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

Pyridine (1.69mL) and benzoyl chloride (2.01mL) were added to a solution of the compound (5.45g) obtained in step 3 in methylene chloride (60mL) in this order at 0 ℃ and stirred at 0 ℃ for 5 minutes, then triethylamine (2.91mL) was added and stirred at room temperature for 2 hours. Saturated aqueous sodium bicarbonate and dichloromethane were added to the reaction solution, and extraction was performed with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ], followed by powderization with ethyl acetate and hexane. The resulting solid was filtered to give the title compound (6.16 g).

¹H－NMR(CDCl₃)δ：8.54(1H，s)，8.20－8.16(2H，m)，8.02－7.98(2H，m)，7.96－7.92(2H，m)，7.82－7.76(4H，m)，7.65－7.32(15H，m)，7.18(1H，brs)，6.82(1H，d，J＝5.9Hz)，6.17(1H，t，J＝6.1Hz)，6.13(1H，dd，J＝5.9，3.5Hz)，4.94(1H，dd，J＝12.2，3.2Hz)，4.79－4.75(1H，m)，4.65(1H，dd，J＝12.2，3.2Hz)，4.41(1H，brs)，4.08(2H，t，J＝8.6Hz)，2.95－2.87(2H，m)，2.58－2.50(2H，m)，1.54－1.52(2H，m)，0.97－0.91(2H，m)，0.02(9H，s).

(step 5)

N-benzoyl-7-beta-D-ribofuranosyl-5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

To a tetrahydrofuran (250mL) solution of the compound (6.16g) obtained in the above step 4 was added dropwise a methanol solution (1.0M, 12.0mL) of sodium methoxide at 0 ℃ and the mixture was stirred at room temperature for 1 hour. Acetic acid (1.07mL) was added to the reaction mixture at 0 ℃ and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ ethyl acetate/methanol ], and further powdered with ethyl acetate and hexane. The resulting solid was filtered to give the title compound (3.35 g).

¹H－NMR(CD₃OD)δ：8.62(1H，s)，8.04(2H，d，J＝7.0Hz)，7.69－7.62(1H，m)，7.60－7.54(3H，m)，6.26(1H，d，J＝6.3Hz)，4.59(1H，t，J＝5.7Hz)，4.31(1H，dd，J＝5.1，3.5Hz)，4.12－4.01(3H，m)，3.86(1H，dd，J＝12.3，2.9Hz)，3.76(1H，dd，J＝12.1，3.5Hz)，3.01(2H，t，J＝6.7Hz)，2.76(2H，t，J＝7.4Hz)，1.86－1.76(2H，m)，0.93(2H，t，J＝8.2Hz)，0.03(9H，s).

(step 6)

N-benzoyl-7- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-ribofuranosyl } -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

The compound (3.35g) obtained in the above step 5 was azeotroped with pyridine, and 4, 4' -dimethoxytrityl chloride (2.38g) was added to a solution of the residue in anhydrous pyridine (50mL) at 0 ℃ and stirred at room temperature overnight. Ethanol (5mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (4.34 g).

¹H－NMR(CDCl₃)δ：8.51(1H，brs)，8.30(1H，brs)，8.05－7.90(1H，m)，7.56－7.42(3H，m)，7.39－7.31(2H，m)，7.29－7.16(10H，m)，6.81－6.75(4H，m)，6.22(1H，d，J＝5.9Hz)，4.91(1H，brs)，4.76－4.67(2H，m)，4.45－4.41(1H，m)，4.38－4.29(1H，m)，4.05－3.95(2H，m)，3.77(6H，s)，3.50(1H，dd，J＝10.6，3.1Hz)，3.37－3.27(1H，m)，3.19－2.94(4H，m)，2.66(1H，brs)，1.82(1H，brs)，0.93－0.76(2H，m)，－0.01(9H，s).

(step 7)

N-benzoyl-7- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine (3' -O-TBS body)

Using the compound (4.34g) obtained in the above-mentioned step 6, a reaction was carried out in the same manner as in the step 4 of example 8 to give the title compound (1.51g), and the positional isomer of the title compound, N-benzoyl-7- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] - β -D-ribofuranosyl } -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine (2' -O-TBS) (2.01g), respectively.

(3' -OTBS body) (high polarity)

¹H－NMR(CDCl₃)δ：8.38－8.26(2H，m)，8.10－8.03(1H，m)，7.58－7.40(5H，m)，7.36－7.20(10H，m)，6.86－6.77(4H，m)，6.30－6.22(1H，m)，5.02－4.89(1H，m)，4.63－4.45(2H，m)，4.17－4.10(1H，m)，4.04－3.92(2H，m)，3.79(3H，s)，3.79(3H，s)，3.59－3.52(1H，m)，3.32－3.23(1H，m)，3.17－2.80(4H，m)，1.86－1.64(2H，m)，0.89(9H，s)，0.86－0.72(2H，m)，0.09(3H，s)，0.01(3H，s)，－0.01(9H，s).

(2' -OTBS body) (Low polarity)

¹H－NMR(CDCl₃)δ：8.39－8.25(2H，m)，8.04(1H，s)，7.57－7.42(5H，m)，7.37－7.18(10H，m)，6.87－6.80(4H，m)，6.33(1H，d，J＝5.1Hz)，5.01(1H，brs)，4.78(1H，t，J＝5.7Hz)，4.40－4.33(1H，m)，4.29－4.25(1H，m)，4.05－3.92(2H，m)，3.80(3H，s)，3.79(3H，s)，3.59－3.51(1H，m)，3.44－3.37(1H，m)，3.16－3.03(1H，m)，2.99－2.81(3H，m)，1.74－1.61(2H，m)，0.83(9H，s)，0.80－0.70(2H，m)，0.00－－0.03(12H，m)，－0.20(3H，s).

(step 8)

N-benzoyl-7- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- [ tert-butyl (dimethyl) silyl ] -2-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -5- [ 3- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) propyl ] -7H-pyrrolo [2, 3-D ] pyrimidin-4-amine

Using the compound (3' -OTBS form) (1.51g) obtained in the above step 7, a reaction was carried out in the same manner as in the step 6 of example 1 to obtain diastereomer 1(0.77g) and diastereomer 2(0.48g) of the title compound, which are diastereomers on a phosphorus atom, respectively.

Diastereomer 1 (low polarity)

¹H－NMR(CDCl₃)δ：8.36－8.27(2H，m)，8.02(1H，brs)，7.57－7.40(5H，m)，7.37－7.21(10H，m)，6.87－6.77(4H，m)，6.39(1H，d，J＝5.1Hz)，4.98(1H，brs)，4.85－4.73(1H，m)，4.56－4.47(1H，m)，4.20－4.15(1H，m)，4.05－3.92(2H，m)，3.88－3.67(8H，m)，3.63－3.47(3H，m)，3.33－3.23(1H，m)，3.15－2.82(3H，m)，2.60－2.45(2H，m)，1.83－1.64(2H，m)，1.10(6H，d，J＝6.7Hz)，0.92(6H，d，J＝6.7Hz)，0.87(9H，s)，0.81－0.71(2H，m)，0.12(3H，s)，0.03(3H，s)，－0.01(9H，s).

Diastereomer 2 (high polarity)

¹H－NMR(CDCl₃)δ：8.36－8.28(2H，m)，8.05(1H，brs)，7.57－7.38(5H，m)，7.36－7.19(10H，m)，6.84－6.79(4H，m)，6.48－6.40(1H，m)，5.02(1H，brs)，4.65－4.42(2H，m)，4.19－4.15(1H，m)，4.06－3.90(2H，m)，3.80－3.48(11H，m)，3.32－3.24(1H，m)，3.17－3.03(2H，m)，2.99－2.86(1H，m)，2.51－2.40(2H，m)，1.86－1.69(2H，m)，1.18－1.02(12H，m)，0.85(9H，s)，0.81－0.71(2H，m)，0.08(3H，s)，0.02(3H，s)，－0.01(9H，s).

(step 9)

2- (trimethylsilyl) ethyl (3- { 4-benzamide-7- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano- 2λ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-7H-pyrrolo [2, 3-d]Pyrimidin-5-yl } propyl) carbamates

The same reaction as in step 7 of example 1 (raw material: 1.08g) was carried out on the following scale. Using the obtained acetonitrile solution of the compound and the compound (1.25 g: diastereomer mixture) obtained in the above step 8, reactions were carried out in the same manner as in the step 8 of example 1 and the step 9 of example 1 to obtain the title compound in the form of a diastereomer mixture on a phosphorus atom. The reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 10% -50% (0 min. to 35 min.) diastereoisomers on the phosphorus atom were separated to give diastereoisomer 1(0.19g) and diastereoisomer 2(0.043g) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1419(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1419(M+H)⁺.

(step 10-1)

2- (trimethylsilyl) ethyl (3- { 4-amino-7- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl group]Oxy } -2, 10-di-oxo-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-7H-pyrrolo [2, 3-d]Pyrimidin-5-yl } propyl) carbamates

Using the compound (diastereomer 1) obtained in the above step 9 (0.19g), a reaction was carried out in the same manner as in the step 8-1 of example 12. Preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 40% to 70% (0 part to 35 parts) was purified to give the title compound (58 mg).

MS(ESI)m/z：1158(M+H)⁺.

(step 10-2)

2- (trimethylsilyl) ethyl (3- { 4-amino-7- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl group]Oxy } -2, 10-di-oxo-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-7H-pyrrolo [2, 3-d]Pyrimidin-5-yl } propyl) carbamates

Using the compound (diastereomer 2) obtained in the above step 9 (43mg), a reaction was carried out in the same manner as in the step 8-1 of example 12. Preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 30% to 60% (0 part to 35 parts) was purified to obtain the title compound (15 mg).

MS(ESI)m/z：1158(M+H)⁺.

(step 11-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 4-amino-5- (3-aminopropyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-7-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

A tetrahydrofuran solution (about 1M, 5mL) of tetrabutylammonium fluoride was added to a tetrahydrofuran (1mL) solution of the compound (58mg) obtained in the above step 10-1, and the mixture was stirred at room temperature overnight. Since the reaction had not yet ended, stirring was continued at 40 ℃ for 3 hours. After the reaction solution was concentrated under reduced pressure, the residue was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: refining 10-40% (0-35 min). And then mixed with Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile, acetonitrile: 0% -30% ] to give the title compound (25 mg).

MS(ESI)m/z：786(M+H)⁺.

(step 11-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 4-amino-5- (3-aminopropyl) -7H-pyrrolo [2, 3-d) ]Pyrimidin-7-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (15mg) obtained in the above step 10-2, a reaction was carried out in the same manner as in the above step 11-1 to obtain the title compound (7.2 mg).

MS(ESI)m/z：786(M+H)⁺.

(step 12-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 4-amino-5- (3-aminopropyl) -7H-pyrrolo [2, 3-d ]]Pyrimidin-7-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (25mg) obtained in the above step 11-1, salt conversion was carried out in the same manner as in [ conversion to sodium salt ] in the step 11 of example 1 to obtain the title compound (17 mg).

MS(ESI)m/z：786(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.028(1H，s)，8.025(1H，s)，7.71(1H，s)，7.09(1H，s)，6.51(1H，d，J＝8.2Hz)，6.26(1H，d，J＝5.1Hz)，5.38－5.29(1H，m)，5.17－5.10(1H，m)，4.85－4.80(1H，m)，4.75(1H，d，J＝3.9Hz)，4.44－4.37(1H，m)，4.33－4.23(3H，m)，4.09－3.97(2H，m)，3.52－3.46(2H，m)，3.00－2.72(6H，m)，2.13－2.02(2H，m)，2.02－1.93(2H，m).

³¹P－NMR(CD₃OD)δ：56.65(s)，55.31(s).

(step 12-2)

(diastereomer 2)

Using the compound (7.2mg) obtained in the above step 11-2, salt conversion was carried out in the same manner as in [ conversion to sodium salt ] in the step 11 of example 1 to obtain the title compound (6.0 mg).

MS(ESI)m/z：786(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.02(1H，s)，8.01(1H，s)，7.75(1H，s)，7.07(1H，s)，6.52(1H，d，J＝8.6Hz)，6.29(1H，d，J＝7.4Hz)，5.61－5.55(1H，m)，5.47－5.37(1H，m)，4.86－4.83(1H，m)，4.56－4.43(2H，m)，4.31－4.21(3H，m)，4.06－3.99(1H，m)，3.91－3.84(1H，m)，3.54－3.46(3H，m)，3.00－2.87(5H，m)，2.29－2.07(2H，m)，2.06－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：63.08(s)，58.68(s).

Example 19: synthesis of CDN19

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-penta-azaHetero-benzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

3-bromo-N- (prop-2-en-1-yl) -1H-pyrazolo [3, 4-d ] pyrimidin-4-amine

Commercially available (Bepharm) 3-bromo-4-chloro-1H-pyrazolo [3, 4-d ] pyrimidine (8.50g) was suspended in dioxane (140mL), and N, N, -diisopropylethylamine (12.5mL) and allylamine (11mL) were added thereto at room temperature, followed by stirring at the same temperature for 70 hours. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with dichloromethane and ethyl acetate and filtered to obtain a solid (solid 1). After concentrating the filtrate under reduced pressure, the same operation was repeated 2 times to obtain solid 2 and solid 3, respectively. The final filtrate was purified by silica gel column [ hexane/ethyl acetate ] to obtain solid 4. Further, the solid 1 was purified by a silica gel column [ hexane/ethyl acetate ] to obtain a solid 5. Combine solid 4 and solid 5 to give the title compound (4.48 g). Combine solid 2 and solid 3 to give the title compound (3.78g) containing impurities.

MS(ESI)m/z：254(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：13.82(1H，s)，8.26(1H，s)，7.23(1H，t，J＝5.9Hz)，5.96(1H，m)，5.18(1H，dd，J＝17.1，1.5Hz)，5.10(1H，dd，J＝10.3，1.5Hz)，4.19(2H，m).

(step 2)

3-bromo-N- (prop-2-en-1-yl) -1- (2, 3, 5-tri-O-benzoyl- β -D-ribofuranosyl) -1H-pyrazolo [3, 4-D ] pyrimidin-4-amine

The compound (1.00g) obtained in step 1 above and commercially available (Ark Pharm) 1-O-acetyl-2, 3, 5-tri-O-benzoyl-. beta. -D-ribofuranose (2.58g) were suspended in nitromethane (50mL) and then dissolved by heating. Under reflux with heating, boron trifluoride diethyl ether complex (0.63mL) was added and stirred at the same temperature for 1 hour. 1-O-acetyl-2, 3, 5-tri-O-benzoyl-. beta. -D-ribofuranose (0.40g) and boron trifluoride diethyl ether complex (0.10mL) were added thereto, and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.80 g).

MS(ESI)m/z：698(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.40(1H，s)，8.13(2H，m)，7.96(4H，m)，7.59－7.32(9H，m)，6.79(1H，d，J＝3.4Hz)，6.39(1H，dd，J＝5.1，3.7Hz)，6.25(1H，t，J＝5.6Hz)，6.17(1H，t，J＝5.6Hz)，6.00(1H，m)，5.31(1H，dd，J＝17.1，1.0Hz)，5.24(1H，dd，J＝10.5，1.2Hz)，4.81(1H，m)，4.75(1H，dd，J＝12.0，3.7Hz)，4.63(1H，dd，J＝12.2，4.4Hz)，4.30(2H，m).

(step 3)

3-vinyl-N- (prop-2-en-1-yl) -1- (2, 3, 5-tri-O-benzoyl-beta-D-ribofuranosyl) -1H-pyrazolo [3, 4-D ] pyrimidin-4-amine

The solution was degassed by sonication under reduced pressure in toluene (120mL) of the compound (11.65g) obtained in step 2. Tributylvinyltin (12.8mL) and tetrakis (triphenylphosphine) palladium (0) (2.73g) were added to the reaction mixture under a nitrogen atmosphere, and the mixture was refluxed for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/ethyl acetate ] to give the title compound (10.07 g).

MS(ESI)m/z：646(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.42(1H，s)，8.09(2H，m)，7.99(2H，m)，7.95(2H，m)，7.59－7.50(3H，m)，7.42－7.33(6H，m)，6.90－6.84(2H，m)，6.43(1H，dd，J＝5.4，3.4Hz)，6.32(1H，t，J＝5.9Hz)，6.00(1H，m)，5.94(1H，dd，J＝17.6，1.0Hz)，5.67(1H，dd，J＝11.2，1.0Hz)，5.56(1H，t，J＝5.6Hz)，5.27(1H，dd，J＝17.1，1.0Hz)，5.22(1H，dd，J＝10.3，1.0Hz)，4.82(1H，m)，4.77(1H，dd，J＝12.2，3.9Hz)，4.62(1H，dd，J＝11.7，4.9Hz)，4.29(2H，m).

(step 4)

2- (2, 3, 5-tri-O-benzoyl- β -D-ribofuranosyl) -6, 7-dihydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

The compound (9.0g) obtained in the above step 3 was azeotroped with benzene 2 times. To a solution of the residue in methylene chloride (480mL) were added (+) -10-camphorsulfonic acid (4.2g) and benzylidene [1, 3-bis (2, 4, 6-trimethylphenyl) imidazolidin-2-ylidene]Dichloride (tricyclohexyl-lambda)⁵Phosphino) ruthenium (second generation Grubbs catalyst) (360mg), heated at reflux for 3 hours. A second generation Grubbs catalyst (360mg) was added and the mixture was heated under reflux for an additional 1 hour. The reaction mixture was cooled to room temperature, washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [ hexane/ethyl acetate ] ]Purification was carried out to give the title compound (6.39 g).

MS(ESI)m/z：618(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.39(1H，s)，8.11(2H，m)，7.99(2H，m)，7.96(2H，m)，7.57－7.52(3H，m)，7.42－7.35(6H，m)，6.83(1H，d，J＝2.9Hz)，6.76(1H，d，J＝10.7Hz)，6.43(1H，dd，J＝5.1，3.2Hz)，6.33(1H，t，J＝5.9Hz)，6.12(1H，m)，5.61(1H，brs)，4.83(1H，m)，4.78(1H，dd，J＝12.2，3.9Hz)，4.63(1H，dd，J＝12.0，4.6Hz)，4.16(2H，m).

(step 5)

2-beta-D-ribofuranosyl-6, 7-dihydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

To a mixed solution of the compound (620mg) obtained in the above step 4 in methanol (10mL) and tetrahydrofuran (5.0mL), a methanol solution of sodium methoxide (1.0M, 0.10mL) was added at room temperature, and the mixture was stirred at the same temperature for 18 hours. The reaction mixture was neutralized with 1N hydrochloric acid, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (279 mg).

MS(ESI)m/z：306(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.23(1H，s)，6.79(1H，m)，6.22－6.17(2H，m)，4.74(1H，t，J＝5.1Hz)，4.42(1H，t，J＝4.6Hz)，4.14(2H，dd，J＝5.9，1.5Hz)，4.11(1H，q，J＝4.1Hz)，3.81(1H，dd，J＝12.4，3.2Hz)，3.68(1H，dd，J＝12.4，4.6Hz).

(step 6)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -6, 7-dihydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

Using the compound (2.81g) obtained in the above step 5, a reaction was carried out in the same manner as in step 1 of example 1 to obtain the title compound (4.72 g).

¹H－NMR(CDCl₃)δ：8.38(1H，s)，6.85(1H，dt，J＝11.0，1.4Hz)，6.28(1H，s)，6.16－6.08(1H，m)，5.78(1H，brs)，4.71－4.63(2H，m)，4.39(1H，dd，J＝9.0，5.1Hz)，4.22－4.10(3H，m)，3.96(1H，dd，J＝10.6，9.0Hz)，1.11(9H，s)，1.05(9H，s)，0.90(9H，s)，0.11(3H，s)，0.09(3H，s).

(step 7)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

To a tetrahydrofuran (20mL) solution of the compound (2.12g) obtained in the above step 6, acetic acid (3 drops in a Pasteur pipette) and 10% palladium on carbon (AD) wet (0.82g) were added, and the mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The catalyst was filtered off, washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.09 g).

¹H－NMR(CDCl₃)δ：8.29(1H，s)，6.33(1H，brs)，6.27(1H，s)，4.70(1H，d，J＝4.7Hz)，4.62(1H，dd，J＝9.6，4.9Hz)，4.39(1H，dd，J＝9.0，5.1Hz)，4.21－4.08(1H，m)，3.95(1H，dd，J＝10.4，9.2Hz)，3.65－3.58(2H，m)，3.15－2.99(2H，m)，2.22－2.10(2H，m)，1.11(9H，s)，1.05(9H，s)，0.90(9H，s)，0.10(3H，s)，0.09(3H，s).

(step 8)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

Using the compound (2.09g) obtained in the above step 7, a reaction was carried out in the same manner as in the step 4 of example 1 to obtain the title compound (2.23 g).

¹H－NMR(CDCl₃)δ：8.19(1H，s)，7.47－7.40(3H，m)，7.33－7.28(2H，m)，6.34(1H，s)，4.72(1H，d，J＝4.7Hz)，4.66(1H，dd，J＝9.4，4.7Hz)，4.49(1H，dd，J＝14.7，8.0Hz)，4.40(1H，dd，J＝9.0，5.1Hz)，4.24－4.09(2H，m)，3.96(1H，dd，J＝10.6，9.0Hz)，3.26－3.11(2H，m)，2.45－2.23(2H，m)，1.12(9H，s)，1.05(9H，s)，0.90(9H，s)，0.12(3H，s)，0.10(3H，s).

(step 9)

6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

Using the compound (2.23g) obtained in the above step 8, a reaction was carried out in the same manner as in step 5 of example 1 to obtain the title compound (2.69 g).

¹H－NMR(CDCl₃)δ：8.22(1H，s)，7.55－7.51(2H，m)，7.46－7.36(7H，m)，7.32－7.26(2H，m)，7.26－7.16(3H，m)，6.80－6.73(4H，m)，6.43(1H，d，J＝5.5Hz)，5.32(1H，t，J＝5.5Hz)，4.43(1H，dd，J＝14.3，8.0Hz)，4.34－4.29(1H，m)，4.25－4.13(2H，m)，3.78(3H，s)，3.77(3H，s)，3.46(1H，dd，J＝10.4，3.3Hz)，3.17－3.05(3H，m)，2.79(1H，d，J＝3.5Hz)，2.40－2.19(2H，m)，0.82(9H，s)，0.03(3H，s)，－0.13(3H，s).

(Process 10)

6-benzoyl-2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene

Using the compound (2.69g) obtained in the above-mentioned step 9, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (2.93g) in the form of a diastereomer mixture (diastereomer ratio: 6: 4) on the phosphorus atom.

¹H－NMR(CDCl₃)δ：8.22(0.4H，s)，8.21(0.6H，s)，7.56－7.50(2H，m)，7.44－7.37(7H，m)，7.28－7.18(5H，m)，6.81－6.73(4H，m)，6.44(0.6H，d，J＝6.3Hz)，6.40(0.4H，d，J＝6.3Hz)，5.34－5.28(1H，m)，4.47－4.35(2.4H，m)，4.32－4.26(0.6H，m)，4.25－4.16(1H，m)，4.03－3.84(1H，m)，3.80－3.73(6H，m)，3.69－3.44(4H，m)，3.18－3.00(3H，m)，2.73－2.59(1H，m)，2.40－2.19(3H，m)，1.22－1.14(8.4H，m)，1.01(3.6H，d，J＝6.7Hz)，0.76(3.6H，s)，0.74(5.4H，s)，0.02(1.2H，s)，0.01(1.8H，s)，－0.12(1.8H，s)，－0.15(1.2H，s).

(step 11)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrakis)Hydrogen-2H-1, 2, 3, 5, 6-pentazabenzo [ cd]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

The reaction was carried out in the same manner as in example 1, step 7 using the compound (1.04g) obtained in the above step 10 to give a solution of 6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3-O- (dihydroxyphosphino) - β -D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] azulene in acetonitrile. Using this acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (1.20g), a reaction was carried out in the same manner as in step 8 of example 1, step 9 of example 1, and step 10 of example 1 to obtain the title compound as a diastereomer mixture on the phosphorus atom. The reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: diastereoisomers on the phosphorus atom were separated at 25% to 50% (0 min to 35 min) to give diastereoisomer 1(15mg) and diastereoisomer 2(55mg) of the title compound (retention time of HPLC: diastereoisomer 1> 2).

Diastereomer 1 (low polarity)

MS(ESI)m/z：959(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：959(M+H)⁺.

(step 12-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] pentazab]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine ringsTetradecyne-2, 10-bis (thiolate)

Triethylamine trihydrofluoride (1mL) was added to the compound (diastereomer 1) obtained in the above step 11 (20mg), and the mixture was stirred at 45 ℃ for 2 hours. To the reaction mixture was added an ice-cooled mixed solution of 1M aqueous triethylammonium bicarbonate (3mL) and triethylamine (1mL) to stop the reaction. With Sep-Pak (registered trademark) C18[ 0.1% triethylamine water/acetonitrile, acetonitrile: 0% -17% ] to give the title compound (15 mg).

MS(ESI)m/z：731(M+H)⁺.

(step 12-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] pentazab ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 2) obtained in the above step 11 (43mg), a reaction was carried out in the same manner as in the above step 12-1 to obtain the title compound (34 mg).

MS(ESI)m/z：731(M+H)⁺.

(step 13-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] pentazab]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (15mg) obtained in the above step 12-1, salt conversion was carried out in the same manner as in [ conversion to sodium salt ] in the step 11 of example 1 to obtain a diastereomer mixture (12mg) of the title compound.

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.79(1H，s)，8.18(1H，s)，8.14(1H，s)，6.37－6.34(2H，m)，5.50(1H，dd，J＝10.8，5.3Hz)，5.40－5.33(1H，m)，5.06(1H，dd，J＝4.5，2.9Hz)，4.94(1H，d，J＝3.5Hz)，4.67－4.30(4H，m)，4.03(1H，ddd，J＝12.5，6.1，2.2Hz)，3.93(1H，dt，J＝18.1，6.1Hz)，3.58(2H，d，J＝7.4Hz)，3.02(2H，t，J＝5.9Hz)，2.18－2.04(2H，m).

³¹P－NMR(CD₃OD)δ：56.5(s)，54.2(s).

(step 13-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-1, 2, 3, 5, 6-pentazabenzo [ cd ] pentazab ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound obtained in the above step 12-2 (34mg), salt conversion was performed in the same manner as in [ conversion to sodium salt ] described in the step 11 of example 1 to obtain a diastereomer mixture of the title compound (28 mg: diastereomer ratio: 4: 1).

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.16(0.2H，s)，8.85(0.8H，s)，8.19(0.2H，s)，8.17(0.8H，s)，8.14(0.8H，s)，8.13(0.2H，s)，6.38－6.30(2H，m)，5.67－5.61(0.8H，m)，5.57－5.36(1.2H，m)，5.22(0.8H，dd，J＝5.9，4.3Hz)，5.10(0.2H，t，J＝4.5Hz)，4.64－4.26(4.8H，m)，4.16－4.10(0.2H，m)，4.05－3.98(1H，m)，3.86－3.79(1H，m)，3.62－3.54(2H，m)，3.07(1.6H，t，J＝5.7Hz)，2.98(0.4H，t，J＝5.9Hz)，2.17－2.04(2H，m).

³¹P－NMR(CD₃OD)δ：63.5(s)，63.4(s)，60.0(s)，59.9(s).

Example 20: synthesis of CDN20

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-oxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -4-chloro-5-iodo-7H-pyrrolo [2, 3-D ] pyrimidine

Using 4-chloro-5-iodo-7-. beta. -D-ribofuranosyl-7H-pyrrolo [2, 3-D ] pyrimidine (3.15g) known in the literature (J.Med.chem.2008, 51, 3934-one 3945), a reaction was carried out in the same manner as in step 1 of example 1 to give the title compound (2.97 g).

¹H－NMR(CDCl₃)δ：8.62(1H，s)，7.38(1H，s)，6.18(1H，s)，4.53－4.47(1H，m)，4.44(1H，d，J＝3.9Hz)，4.24－4.18(2H，m)，4.06－3.98(1H，m)，1.09(9H，s)，1.05(9H，s)，0.92(9H，s)，0.13(3H，s)，0.12(3H，s).

(step 2)

4- (benzyloxy) -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5-iodo-7H-pyrrolo [2, 3-D ] pyrimidine

To a solution of the compound (4.97g) obtained in the above step 1 and benzyl alcohol (1.0mL) in tetrahydrofuran (50mL) was added sodium hydride (containing 37% mineral oil) (426mg) under ice-cooling, and the mixture was stirred overnight at room temperature. The reaction was stopped by adding a saturated aqueous ammonium chloride solution to the reaction mixture under ice-cooling. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with water and saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (3.38 g).

¹H－NMR(CDCl₃)δ：8.43(1H，s)，7.62－7.57(2H，m)，7.43－7.37(2H，m)，7.36－7.30(1H，m)，7.13(1H，s)，6.15(1H，s)，5.65(1H，d，J＝13.7Hz)，5.62(1H，d，J＝13.7Hz)，4.50－4.43(2H，m)，4.27(1H，dd，J＝9.2，4.9Hz)，4.21－4.12(1H，m)，4.01(1H，dd，J＝10.4，9.2Hz)，1.09(9H，s)，1.04(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 3)

4- (benzyloxy) -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (3-hydroxypropan-1-yn-1-yl) -7H-pyrrolo [2, 3-D ] pyrimidine

Using the compound (3.38g) obtained in the above step 2 and 2-propyn-1-ol (1.35mL), a reaction was carried out at room temperature in the same manner as in the step 2 of example 1 to obtain the title compound (2.22 g).

¹H－NMR(CDCl₃)δ：8.46(1H，s)，7.59－7.55(2H，m)，7.43－7.33(3H，m)，7.22(1H，s)，6.16(1H，s)，5.60(1H，d，J＝12.7Hz)，5.57(1H，d，J＝12.7Hz)，4.51－4.41(4H，m)，4.27(1H，dd，J＝9.5，5.0Hz)，4.19(1H，dt，J＝9.9，5.0Hz)，4.01(1H，dd，J＝9.9，9.5Hz)，1.52(1H，t，J＝6.3Hz)，1.08(9H，s)，1.04(9H，s)，0.91(9H，s)，0.13(3H，s)，0.11(3H，s).

(step 4)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (3-hydroxypropyl) -3, 7-dihydro-4H-pyrrolo [2, 3-D ] pyrimidin-4-one

Using the compound (3.38g) obtained in the above step 3, a reaction was carried out in the same manner as in step 7 of example 19 using a mixed solution of methanol (20mL) and tetrahydrofuran (20mL) as a reaction solvent to obtain the title compound (0.92 g).

¹H－NMR(CDCl₃)δ：11.36(1H，brs)，7.86(1H，s)，6.69(1H，s)，6.10(1H，s)，4.48(1H，dd，J＝9.2，4.9Hz)，4.37(1H，d，J＝5.1Hz)，4.23(1H，dd，J＝9.3，4.8Hz)，4.16(1H，dt，J＝9.8，4.8Hz)，4.02(1H，dd，J＝9.8，9.3Hz)，3.84(1H，t，J＝6.3Hz)，3.58(2H，dd，J＝11.9，6.1Hz)，3.03－2.90(2H，m)，1.86(2H，s)，1.09(9H，s)，1.04(9H，s)，0.90(9H，s)，0.10(6H，s).

(step 5)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) - β -D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triazabenzo [ cd ] azulene

Triphenylphosphine (0.62g) and diisopropyl azodicarboxylate (0.47mL) were added to a tetrahydrofuran (32mL) solution of the compound (0.92g) obtained in step 4 above under ice cooling, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (0.77 g).

¹H－NMR(CDCl₃)δ：8.44(1H，s)，6.85(1H，s)，6.19(1H，s)，4.56－4.51(2H，m)，4.50－4.44(2H，m)，4.35(1H，dd，J＝9.5，5.0Hz)，4.17(1H，dt，J＝10.0，5.0Hz)，4.00(1H，dd，J＝10.0，9.5Hz)，2.98－2.92(2H，m)，2.28－2.20(2H，m)，1.10(9H，s)，1.05(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 6)

2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (0.95g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 5 of example 1 to obtain the title compound (1.13 g).

¹H－NMR(CDCl₃)δ：8.42(1H，s)，7.48－7.43(2H，m)，7.37－7.21(7H，m)，7.19(1H，s)，6.85－6.79(4H，m)，6.35(1H，d，J＝5.4Hz)，4.71(1H，t，J＝5.4Hz)，4.55－4.48(2H，m)，4.35(1H，dd，J＝8.2，4.3Hz)，4.22(1H，q，J＝3.0Hz)，3.79(3H，s)，3.79(3H，s)，3.53(1H，dd，J＝10.6，3.1Hz)，3.37(1H，dd，J＝10.6，3.1Hz)，2.80(1H，d，J＝4.3Hz)，2.71(2H，t，J＝5.5Hz)，2.23－2.15(2H，m)，0.83(9H，s)，－0.04(3H，s)，－0.16(3H，s).

(step 7)

2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triaza-benzo [ cd ] azulene

A reaction was carried out in the same manner as in step 4 of example 5 except for using the compound (1.13g) obtained in step 6 above and purifying the compound by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ], so as to obtain the title compound (1.00g) in the form of a diastereomer mixture on a phosphorus atom (diastereomer ratio ═ 6: 4).

¹H－NMR(CDCl₃)δ：8.41(0.4H，s)，8.39(0.6H，s)，7.51－7.43(2H，m)，7.39－7.17(8H，m)，6.85－6.79(4H，m)，6.34(0.6H，d，J＝6.7Hz)，6.30(0.4H，d，J＝5.9Hz)，4.82(0.6H，dd，J＝6.7，4.7Hz)，4.76(0.4H，dd，J＝5.9，4.7Hz)，4.55－4.47(2H，m)，4.43－4.35(1.2H，m)，4.30－4.25(0.8H，m)，4.05－3.85(1H，m)，3.81－3.76(6H，m)，3.70－3.47(4H，m)，3.32－3.24(1H，m)，2.79－2.64(3.2H，m)，2.31(0.8H，t，J＝6.5Hz)，2.23－2.14(2H，m)，1.20－1.15(8.4H，m)，1.03(3.6H，d，J＝7.0Hz)，0.75(3.6H，s)，0.73(5.4H，s)，－0.03(1.2H，s)，－0.08(1.8H，s)，－0.20(1.2H，s)，－0.22(1.8H，s).

(step 8)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] silyl]Oxy } -10- (2-cyanoethoxy) -14- (8, 9-dihydro-6-oxo-2, 3, 5-triaza-eno [ cd)]Azulene-2 (7H) -yl) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } benzamide

Using the compound (1.00g) obtained in the above-mentioned step 7, a reaction was carried out in the same manner as in the step 7 of example 1 to give a solution of 2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3-O- (dihydroxyphosphino) - β -D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triaza-benzo [ cd ] azulene in acetonitrile. Using this acetonitrile solution and commercially available (Cool Pharm) N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (1.28g), reactions were carried out in the same manner as in step 8 of example 1 and step 9 of example 1 to obtain a mixture containing diastereomer 1 and a mixture containing diastereomer 2 of the title compound, respectively.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1116(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1116(M+H)⁺.

(step 9-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl) ]Oxy } -14- (8, 9-dihydro-6-oxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The reaction was carried out in the same manner as in step 10 of example 1 using the total amount of the compound obtained in step 8 (mixture containing diastereomer 1), and then purification was carried out according to the following [ purification conditions ] to obtain the title compound (73mg) as a triethylamine salt.

[ purification conditions ] HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 30-60% (0-35 min).

MS(ESI)m/z：959(M+H)⁺.

(step 9-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl)]Oxy } -14- (8, 9-dihydro-6-oxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The reaction was carried out in the same manner as in step 10 of example 1 using the total amount of the compound obtained in step 8 (diastereomer 2-containing mixture), and then purification was carried out according to the following [ purification conditions ] to obtain the title compound (58mg) as a triethylamine salt.

[ purification conditions ] HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -50% (0 min-35 min).

MS(ESI)m/z：959(M+H)⁺.

(step 10-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-oxo-2, 3, 5-triaza-benzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (68mg) obtained in the above step 9-1, a reaction was carried out in the same manner as in the step 12-1 of example 19, and then salt conversion was carried out in the same manner as in the step 11 of example 1 [ to sodium salt ] to obtain the title compound (42 mg).

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，s)，8.31(1H，s)，8.17(1H，s)，7.36(1H，s)，6.38(1H，d，J＝4.7Hz)，6.34(1H，d，J＝8.2Hz)，5.42－5.34(1H，m)，5.24－5.16(1H，m)，4.86－4.81(2H，m)，4.64－4.29(6H，m)，4.12－4.01(2H，m)，2.98－2.81(2H，m)，2.28－2.13(2H，m).

³¹P－NMR(CD₃OD)δ：58.2(s)，54.4(s).

(step 10-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-oxo-2, 3, 5-triaza-benzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l ][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (58mg) obtained in the above step 9-2, a reaction was carried out in the same manner as in the step 12-1 of example 19, and then salt conversion was carried out in the same manner as in the step 11 of example 1 [ conversion to sodium salt ] to obtain the title compound (35 mg).

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.81(1H，s)，8.31(1H，s)，8.17(1H，s)，7.38(1H，s)，6.41(1H，d，J＝6.7Hz)，6.34(1H，d，J＝8.6Hz)，5.56－5.41(2H，m)，4.87(1H，m)，4.64－4.27(7H，m)，4.06－3.99(1H，m)，3.92－3.88(1H，m)，2.99(2H，t，J＝5.5Hz)，2.28－2.19(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.5(s).

Example 21: synthesis of drug linker 1

[ synthetic route ]

(step 1)

N- (ammonium (azaniumyl) acetyl) glycyl-L-phenylalanyl glycine trifluoroacetate salt

To a solution of commercial (BACHEM) N- (tert-butoxycarbonyl) glycylglycyl-L-phenylalanylglycine (3.00g) in methylene chloride (30mL) was added trifluoroacetic acid (15mL) at room temperature, and the mixture was stirred at the same temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, suspended in toluene, and concentrated again under reduced pressure. This concentration was repeated 2 further times. The residue was slurried with diethyl ether (100mL) and filtered to give the title compound (3.27g) as a crude product.

MS(ESI)m/z：337(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.60(1H，brs)，8.48(1H，t，J＝5.6Hz)，8.44(1H，t，J＝5.9Hz)，8.31(1H，d，J＝8.8Hz)，7.97(3H，brs)，7.28－7.16(5H，m)，4.58(1H，m)，3.87(1H，dd，J＝16.8，5.6Hz)，3.78(2H，d，J＝5.9Hz)，3.67(1H，dd，J＝17.1，5.4Hz)，3.56(2H，brd，J＝4.4Hz)，3.05(1H，dd，J＝13.7，3.9Hz)，2.74(1H，dd，J＝13.7，10.3Hz).

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanylglycine

Triethylamine (0.804mL) and 1- { [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] oxy } pyrrolidine-2, 5-dione (1.87g) were added to a solution of the compound (2.09g) obtained in the above step 1 in N, N-dimethylformamide (46.4mL), and the mixture was stirred at room temperature for 21 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/methanol ]. Diethyl ether was added to a dichloromethane solution of the obtained compound to prepare a slurry, which was then filtered to obtain the title compound (2.10 g).

MS(ESI)m/z：624(M+H)⁺.

¹H－NMR(DMSO－d₆) δ: 8.20 to 7.91(4H, m), 7.68 to 7.13(13H, m), 4.98(1H, dd, J ═ 13.9, 3.2Hz), 4.51 to 4.46(1H, m), 3.73 to 3.47(7H, m), 3.00(1H, dd, J ═ 13.9, 4.1Hz), 2.73(1H, t, J ═ 11.7Hz), 2.67 to 2.57(1H, m), 2.29 to 2.22(1H, m), 2.06 to 2.01(1H, m), 1.80 to 1.73(1H, m). (only the peaks observed are described)

(step 3)

2, 5-Bisoryloxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanyl glycinate

To a solution of the compound (2.10g) obtained in the above step 2 in N, N-dimethylformamide (33.7mL) were added N-hydroxysuccinimide (426mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (710mg), and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. The reaction solution was diluted with dichloromethane, washed with ice water 3 times, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. To the oily residue was added ethyl acetate to precipitate a solid. The solvent was distilled off under reduced pressure, and diethyl ether was added to the resulting solid to prepare a slurry, which was then filtered to give the title compound (2.18 g).

¹H－NMR(DMSO－d₆)δ：8.74－8.69(1H，m)，8.16－8.08(2H，m)，8.00－7.93(1H，m)，7.71－7.15(13H，m)，5.00(1H，dd，J＝13.9，3.0Hz)，4.55－4.49(1H，m)，4.27(2H，t，J＝6.0Hz)，3.77－3.68(1H，m)，3.64－3.50(4H，m)，3.02(1H，dd，J＝13.9，4.2Hz)，2.82－2.73(5H，m)，2.69－2.58(1H，m)，2.33－2.24(1H，m)，2.10－2.02(1H，m)，1.83－1.75(1H，m).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] d]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) glycinamide

(drug linker 1)

Triethylamine (8. mu.L) and the compound (17.6mg) obtained in the above step 3 were added to a solution of the compound (10.0mg) obtained in the step 8-2 of example 5 in N, N-dimethylformamide (1mL), and the mixture was stirred at room temperature for 2 hours. Benzylamine (3. mu.L) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. To the reaction solution were added 10mM triethylammonium acetate aqueous solution and methanol, and column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% to 50% (0 part to 40 parts) was purified to give the title compound (10.9 mg).

MS(ESI)m/z：1379(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，d，J＝10.0Hz)，8.15(1H，d，J＝10.0Hz)，8.02(1H，s)，7.63－7.50(2H，m)，7.42－7.37(3H，m)，7.32－7.13(8H，m)，7.12(1H，s)，6.31(1H，d，J＝6.7Hz)，6.25(1H，d，J＝8.5Hz)，5.51－5.40(2H，m)，5.09－4.99(1H，m)，4.85－4.77(1H，m)，4.53－4.42(2H，m)，4.42－4.15(5H，m)，4.04－3.96(1H，m)，3.92－3.46(12H，m)，3.18(12H，q，J＝7.3Hz)，

3.16－2.73(5H，m)，2.40－2.23(2H，m)，2.06－1.94(4H，m)，1.29(18H，t，J＝7.3Hz).

Example 22: synthesis of drug linker 2

[ synthetic route ]

(step 1)

1- [ 2- (benzoyloxy) ethyl ] -5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine

To a solution of inosine (10.0g) in pyridine (50mL) and N, N-dimethylacetamide (50mL) was added 4, 4' -dimethoxytrityl chloride (15.2g) at 0 ℃ and the mixture was stirred at 4 ℃ for 64 hours. Methanol (2mL) was added to the reaction mixture, and after stirring for 10 minutes, the mixture was concentrated to about 50 mL. To the residue were added 2-bromoethyl benzoate (7.02mL) and 2, 3, 4, 6, 7, 8, 9, 10-octahydropyrimido [1, 2-a ] ]Aza derivatives(azepine) (13.9mL) was stirred at room temperature for 1 day. To the reaction mixture were added saturated aqueous sodium bicarbonate and water, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine]Purification was carried out to give the title compound (15.2 g).

MS(ESI)m/z：719(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.00(1H，s)，7.98(1H，s)，7.98－7.94(2H，m)，7.62－7.15(12H，m)，6.80－6.75(4H，m)，5.95(1H，d，J＝5.4Hz)，4.82－4.79(1H，m)，4.72－4.64(3H，m)，4.55－4.34(5H，m)，3.77(6H，s)，3.43(1H，dd，J＝10.6，3.9Hz)，3.34(1H，dd，J＝10.6，3.6Hz).

(step 2)

1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] inosine

The title compound (1.20g) and 1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] inosine (1.22g), a positional isomer of the title compound, were synthesized in the same manner as in step 3 of example 5 using the compound (3.01g) obtained in step 1.

MS(ESI)m/z：833(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.03(1H，s)，7.98－7.96(1H，m)，7.96(1H，s)，7.96－7.94(1H，m)，7.59－7.52(1H，m)，7.44－7.38(4H，m)，7.32－7.15(7H，m)，6.83－6.77(4H，m)，5.94(1H，d，J＝4.8Hz)，4.69－4.63(2H，m)，4.59－4.35(4H，m)，4.16(1H，dd，J＝3.8，1.9Hz)，3.77(6H，d，J＝1.8Hz)，3.47(1H，dd，J＝10.9，3.0Hz)，3.27(1H，dd，J＝10.9，4.2Hz)，3.00(1H，d，J＝6.7Hz)，0.87(9H，s)，0.06(3H，s)，－0.01(3H，s).

(2' -O-TBS body)

MS(ESI)m/z：833(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.01(1H，s)，7.97－7.93(2H，m)，7.91(1H，s)，7.59－7.53(1H，m)，7.45－7.38(4H，m)，7.35－7.17(7H，m)，6.83－6.77(4H，m)，5.97(1H，d，J＝6.0Hz)，4.84(1H，t，J＝5.4Hz)，4.71－4.60(2H，m)，4.52－4.37(2H，m)，4.33－4.28(1H，m)，4.28－4.24(1H，m)，3.78(3H，s)，3.77(3H，s)，3.47(1H，dd，J＝10.9，3.0Hz)，3.38(1H，dd，J＝10.9，3.6Hz)，2.71(1H，d，J＝3.0Hz)，0.80(9H，s)，－0.03(3H，s)，－0.19(3H，s).

(step 3)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } inosine

The compound (1.20g) obtained in the above step 2 was synthesized in the same manner as in the step 4 of example 5 to obtain the title compound (1.41g) as a diastereomer mixture (diastereomer ratio: 0.55: 0.45) on the phosphorus atom.

MS(ESI)m/z：1033(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.05(0.45H，s)，8.04(0.55H，s)，7.99－7.95(2H，m)，7.95(0.55H，s)，7.92(0.45H，s)，7.59－7.53(1H，m)，7.45－7.39(4H，m)，7.35－7.10(7H，m)，6.83－6.78(4H，m)，6.15(0.55H，d，J＝5.4Hz)，6.08(0.45H，d，J＝6.0Hz)，4.86－4.49(3H，m)，4.49－4.35(3H，m)，4.25－4.10(1H，m)，3.78(6H，s)，3.72－3.41(5H，m)，3.35－3.25(1H，m)，2.47(1H，t，J＝6.7Hz)，2.32(1H，t，J＝6.3Hz)，1.33－1.24(6H，m)，1.13－1.03(6H，m)，0.84(4.05H，s)，0.84(4.95H，s)，0.08(1.35H，s)，0.05(1.65H，s)，0.00(1.35H，s)，－0.01(1.65H，s).

(step 4)

The same reaction (raw material: 1.40g) as in step 7 of example 1 was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.41g) obtained in the above step 3, a reaction was carried out in the same manner as in step 8 of example 1. The crude product obtained was used directly in the next reaction.

(step 5)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2)-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 4, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (778mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1264(M+H)⁺.

(step 6)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (778mg) obtained in the above step 5, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(255mg) and diastereomer 2 (containing impurities) of the title compound. Diastereomer 2 was purified by preparative HPLC [ water/0.2% acetonitrile containing triethylamine, 0.2% acetonitrile containing triethylamine: from 5% to 50% (0 min to 40 min) was again purified to give diastereomer 2 of the title compound (94.6 mg).

Diastereomer 1 (low polarity)

MS(ESI)m/z：1003(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.66(1H，s)，8.21(1H，s)，8.04(1H，s)，7.33(1H，s)，6.27(1H，d，J＝5.1Hz)，6.25(1H，d，J＝3.6Hz)，5.39－5.29(1H，m)，5.18－5.11(1H，m)，4.85－4.81(1H，m)，4.79－4.74(1H，m)，4.71－4.66(1H，m)，4.50－4.42(1H，m)，4.36－4.21(2H，m)，4.09－3.98(2H，m)，3.85－3.78(2H，m)，3.78－3.69(2H，m)，3.55－3.46(2H，m)，3.17(12H，q，J＝7.3Hz)，2.98－2.75(2H，m)，2.05－1.88(2H，m)，1.28(18H，t，J＝7.3Hz)，0.98(9H，s)，0.85(9H，s)，0.31(3H，s)，0.27(3H，s)，0.25(3H，s)，0.09(3H，s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：1003(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.50(1H，s)，8.22(1H，s)，8.07(1H，s)，7.20(1H，s)，6.33(1H，d，J＝7.3Hz)，6.26(1H，d，J＝9.1Hz)，5.59－5.44(1H，m)，5.38－5.32(1H，m)，5.21－5.11(1H，m)，4.99－4.89(2H，m)，4.68－4.54(2H，m)，4.25－4.12(3H，m)，4.09－4.03(1H，m)，3.90－3.80(3H，m)，3.59－3.51(2H，m)，3.20(12H，q，J＝7.3Hz)，2.96－2.89(2H，m)，2.07－1.98(2H，m)，1.30(18H，t，J＝7.3Hz)，0.99(9H，s)，0.74(9H，s)，0.27(3H，s)，0.27(3H，s)，0.20(3H，s)，－0.05(3H，s).

(step 7-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl ]Oxy } -7- (1- { 2- [ (glycylamino) methoxy)]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

To a solution of the compound (diastereomer 1) obtained in the above-mentioned step 6 (30mg) in tetrahydrofuran (0.5mL) were added [ (N- { [ (9H-fluoren-9-yl) methoxy ] carbonyl } glycyl) amino ] methyl acetate (91.7mg) and p-toluenesulfonic acid 1 hydrate (11.8mg), and the mixture was stirred at room temperature for 6 hours. N, N-dimethylformamide (0.5mL) and 1, 8-diazabicyclo [5.4.0] -7-undecene (56. mu.L) were added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours. To the reaction mixture was added 10mM triethylammonium acetate aqueous solution, and purification was performed by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to obtain the title compound (25.6mg) containing the starting material as an impurity.

MS(ESI)m/z：1089(M+H)⁺.

(step 7-2)

(diastereomer 2)

Using the compound (diastereomer 2) (84.6mg) obtained in the above step 6, a reaction was carried out in the same manner as in the above step 7-1 to obtain the title compound (70.9mg) containing the starting material as an impurity.

MS(ESI)m/z：1089(M+H)⁺.

(step 8-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] s]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Diastereomer 1

Triethylamine trihydrofluoride (2mL) was added to the compound (25.6mg) obtained in the above step 7-1, and the mixture was stirred at 45 ℃ for 3 hours. To the reaction mixture was added an ice-cooled mixture of 1M triethylammonium bicarbonate solution (10mL) and triethylamine (2mL) at room temperature. The reaction mixture was concentrated under reduced pressure and purified by C18 silica gel column chromatography (10mM triethylammonium acetate aqueous solution/acetonitrile) to give the title compound (16.6 mg: containing the impurities derived from the starting material in step 7-1).

MS(ESI)m/z：861(M+H)⁺.

(step 8-2)

(diastereomer 2)

Using the compound (70.9mg) obtained in the above step 7-2, a reaction was carried out in the same manner as in the above step 8-1 to obtain the title compound (51.7 mg: containing impurities derived from the starting materials in the step 7-2).

MS(ESI)m/z：861(M+H)⁺.

(step 9-1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]PentaoxaDiphosphocyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 2 a: diastereomer 1)

Triethylamine (6. mu.L) and the compound (15.5mg) obtained in step 11 described below were added to a solution of the compound (16.6mg) obtained in step 8-1 in N, N-dimethylformamide (0.5mL), and the mixture was stirred at room temperature for 3 hours. Benzylamine (3. mu.L) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. 10mM triethylammonium acetate aqueous solution and methanol were added, and column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% to 45% (0 part to 30 parts) was purified to give the title compound (5.1 mg).

MS(ESI)m/z：1409(M+H)⁺.

¹H－NMR(CD₃OD) δ: 8.66-8.60 (1H, m), 8.17(1H, s), 8.02(1H, s), 7.65-7.48 (2H, m), 7.43-7.36 (3H, m), 7.31-7.13 (8H, m), 7.11(1H, s), 6.30-6.21 (2H, m), 5.46-5.37 (1H, m), 5.23-5.16 (1H, m), 5.08-4.99 (1H, m), 4.86-4.81 (1H, m), 4.80-4.75 (1H, m), 4.70-4.40 (7H, m), 4.40-4.20 (3H, m), 4.10-3.97 (3H, m), 3.86-3.58 (8H, m), 3.51-3.43 (3H, 3 m), 3.18H, 3H

(step 9-2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 2 b: diastereomer 2)

Using the compound (51.7mg) obtained in the above step 8-2, a reaction was carried out in the same manner as in the above step 9-1, and then purification was carried out under the following [ purification conditions ] to obtain the title compound (33.7mg)

MS(ESI)m/z：1409(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，d，J＝6.7Hz)，8.19(1H，d，J＝3.0Hz)，8.02(1H，s)，7.66－7.50(2H，m)，7.43－7.37(3H，m)，7.33－7.13(8H，m)，7.11(1H，s)，6.33－6.23(2H，m)，5.51－5.38(2H，m)，5.04(1H，t，J＝13.6Hz)，4.83－4.77(1H，m)，4.64－4.55(2H，m)，4.52－4.26(6H，m)，4.25－3.97(2H，m)，3.93－3.45(13H，m)，3.19(12H，q，J＝7.3Hz)，3.17－3.11(1H，m)，3.02－2.92(1H，m)，2.91－2.73(3H，m)，2.40－2.24(2H，m)，2.07－1.95(3H，m)，1.30(18H，t，J＝7.3Hz).

(Process 10)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanine

Triethylamine (2.56mL) and 1- { [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] oxy } pyrrolidine-2, 5-dione (3.69g) which was commercially available (Click Chemistry Tools) were added to a solution of commercially available (BACHEM) in (2S) -2- [ [ 2- [ (2-aminoacetyl) amino ] acetyl ] amino ] -3-phenylpropionic acid (2.86g) in N, N-dimethylformamide (51.2mL), and stirred at room temperature for 24 hours. A solution of citric acid monohydrate (24.0g) in water (500mL) was added to the reaction mixture, which was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in an ethyl acetate/acetonitrile mixed solution, and then precipitated with diisopropyl ether, followed by filtration to give the title compound (4.30 g).

¹H－NMR(DMSO－d₆)δ：12.8(1H，brs)，8.15－7.95(3H，m)，7.68－7.17(13H，m)，5.01(1H，d，J＝14.2Hz)，4.41－4.37(1H，m)，3.74－3.57(5H，m)，3.05－3.01(1H，m)，2.87(1H，dd，J＝14.2，9.3Hz)，2.68－2.59(1H，m)，2.32－2.25(1H，m)，2.09－2.03(1H，m)，1.82－1.76(1H，m).

(step 11)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanine ester

To a solution of the compound (2.10g) obtained in the above step 10 in N, N-dimethylformamide (75.9mL) were added N-hydroxysuccinimide (961mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.60g), and the mixture was stirred at room temperature under a nitrogen atmosphere for 21 hours. The reaction solution was diluted with dichloromethane, washed with ice water 3 times, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Toluene was added to the residue, and concentration was performed again under reduced pressure. The residue was dissolved in acetonitrile and subjected to C18 silica gel column chromatography [ acetonitrile: 100% ] refining. After the fraction containing the target substance was concentrated under reduced pressure, diisopropyl ether was added to the residue to prepare a slurry. The resulting solid was filtered to give the title compound (2.59 g).

¹H－NMR(DMSO－d₆)δ：8.58－8.51(1H，m)，8.17－8.00(2H，m)，7.66－7.20(13H，m)，5.02－4.98(1H，m)，4.90－4.85(1H，m)，3.78－3.57(5H，m)，3.24－3.19(1H，m)，3.06－3.00(1H，m)，2.82(4H，brs)，2.67－2.58(1H，m)，2.32－2.23(1H，m)，2.09－2.02(1H，m)，1.82－1.75(1H，m).

Example 23: synthesis of drug linker 3

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ] ]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Glycine amides

(drug linker 3)

Triethylamine (1.9. mu.L) and the compound (5.2mg) obtained in the step 3 of example 21 were added to a solution of the compound (4.8mg) obtained in the step 8-2 of example 8 in N, N-dimethylformamide (0.29mL), and the mixture was stirred at room temperature for 1.5 hours under a nitrogen atmosphere. After the reaction was stopped by addition of benzylamine (3.2 μ L), the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) to give the title compound (8.0 mg).

MS(ESI)m/z：1393(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.35(1H，brs)，8.02(1H，s)，7.61－7.15(14H，m)，6.33(1H，dd，J＝6.0，3.0Hz)，6.15－6.09(1H，m)，5.49－5.37(2H，m)，5.05(1H，dd，J＝13.9，12.1Hz)，4.85－4.79(1H，m)，4.53－4.21(6H，m)，4.06－3.61(8H，m)，3.51－3.13(6H，m)，3.16(12H，q，J＝7.3Hz)，3.06－2.65(7H，m)，2.35－1.94(4H，m)，1.28(18H，t，J＝7.6Hz).

Example 24: synthesis of drug linker 4

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 6-amino-2- ({ 2- [ (glycylamino) methoxy ]Ethyl } amino) -9H-purin-9-yl]-15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (17.6mg) obtained in the step 1-2 of example 9, a reaction was carried out in the same manner as in the step 7-1 of example 22 to obtain the title compound (8.3 mg).

MS(ESI)m/z：1103(M+H)⁺.

(step 2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- ({ 2- [ (glycylamino) methoxy]Ethyl } amino) -9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (10.7mg) obtained in the above step 1, a reaction was carried out in the same manner as in the step 8-1 of example 22 to obtain the title compound (7.6 mg).

MS(ESI)m/z：875(M+H)⁺.

(step 3)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b,f]azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- { [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethoxy]Methyl glycinamide

(drug linker 4)

Using the compound (7.6mg) obtained in the above step 2, a reaction was carried out in the same manner as in the step 9-1 of example 22. Purification was carried out according to the following [ purification conditions ] to give the title compound (7.6mg) as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) ].

MS(ESI)m/z：1423(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.37(1H，brs)，8.01(1H，d，J＝2.4Hz)，7.63－7.11(14H，m)，6.33(1H，d，J＝6.7Hz)，6.17(1H，d，J＝7.3Hz)，5.51－5.36(2H，m)，5.09－5.03(1H，m)，4.84－4.80(1H，m)，4.63－4.25(8H，m)，4.07－3.58(9H，m)，3.50－3.41(4H，m)，3.28－2.72(8H，m)，3.18(12H，q，J＝7.3Hz)，2.45－1.96(4H，m)，1.29(18H，t，J＝7.3Hz).

Example 25: synthesis of sugar chain-reconstituted antibody 1

Engineered anti-HER 2 antibodies- [ SG- (N)₃)₂]₂Synthesis of (2)

[ synthetic route ]

(step 1)

Preparation of (Fuc α 1, 6) GlcNAc-engineered anti-HER 2 antibodies

To a phosphate-buffered saline solution (20mL, 12.6mg/mL, pH6.0) of the anti-HER 2 antibody engineered according to reference example 1 was added a phosphate-buffered saline solution of wild-type EndoS (0.147mL, 7.70mg/mL, pH6.0), and the mixture was shaken at 37 ℃ for 2 hours and 15 minutes. The progress of the reaction was confirmed by an Experion electrophoresis workstation (manufactured by BIO-RAD). After the completion of the reaction, purification by affinity chromatography and purification by hydroxyapatite column chromatography were carried out in accordance with the following methods.

(1) Affinity chromatography based purification

A refining device: AKTA avant 25 (manufactured by GE HEALTH CARE)

Column: HiTrap rProtein A FF (5mL) (manufactured by GE HEALTH CARE)

Flow rate: 5mL/min (1.25 mL/min when filling)

The reaction solution obtained above was purified in two steps. When bound to the column, the reaction solution was added to the column, and a binding buffer [20mM phosphate buffer (pH6.0) ] was flowed in at 1.25mL/min and 2CV and further at 5mL/min and 5 CV. For the intermediate washing, a washing solution [20mM phosphate buffer (pH7.0), 0.5M sodium chloride solution ] was poured in at 15 CV. At the time of Elution, an Elution buffer (ImmunoPure IgG Elution buffer, manufactured by PIERCE) was flowed in at 6 CV. The eluate was immediately neutralized with 1M Tris buffer (pH 9.0). The fractions containing the target were subjected to buffer exchange with 5mM phosphate buffer, 50mM 2-morpholinoethanesulfonic acid (MES) solution (pH6.8) according to the method described in the common operation C. The antibody concentration of the resulting buffer solution was measured by the method described in general procedure B to obtain a crude and purified title antibody solution (26.57mg/mL, 9.0 mL).

(2) Refining based on hydroxyapatite chromatography

A refining device: AKTA avant 25 (manufactured by GE HEALTH CARE)

Column: Bio-Scale Mini CHT Type I tube (5mL) (manufactured by BIO-RAD)

Flow rate: 5mL/min (1.25 mL/min when filling)

The solution obtained in (1) above was added to the column, and solution A [5mM phosphate buffer, 50mM MES solution (pH 6.8) ] was allowed to flow in at 1.25mL/min and 2CV, and further at 5mL/min and 3 CV. Then, elution was carried out using solution A and solution B [5mM phosphate buffer, 50mM MES solution (pH 6.8), 2M sodium chloride solution ]. The elution conditions were solution a: and B, 100: 0 to 0: 100(5 CV). In addition, a washing solution [500mM phosphate buffer (pH 6.5) ] was flowed in at 5 CV. The fractions containing the target were subjected to buffer exchange with 20mM phosphate buffer (pH6.0) according to the method described in common procedure C. The antibody concentration of the obtained buffer solution was measured according to the method described in the common operation B to obtain the title antibody solution (17.29mg/mL, about 13 mL).

(step 2)

Engineered anti-HER 2 antibodies- [ SG- (N)₃)₂]₂Preparation of

[ N ] was added to a 20mM phosphate buffer solution (17.29mg/mL, 13mL, pH6.0) of the antibody obtained in step 1₃－PEG(3)]₂SG (10) Ox (compounds 1 to 10 of WO 2018/003983) (52mg) in 20mM phosphate buffer (pH6.0) (3.0mL + Wash 1.0mL) and Endos (D233Q/Q303L) in phosphate buffered saline solution (0.698mL, 5.8mg/mL, pH6.0) with shaking at 30 ℃ for 4 hours. The reaction mixture was kept at-80 ℃ for 15 hours, then thawed at 30 ℃ and [ N ] was added ₃－PEG(3)]₂Phosphate-buffered saline solution of-SG (10) Ox (7.4mg) and Endos (D233Q/Q303L) (0.155mL, 5.8mg/mL, pH6.0) was shaken at 30 ℃ for 2 hours. The progress of the reaction was confirmed by an Experion electrophoresis workstation (manufactured by BIO-RAD). After completion of the reaction, purification by affinity chromatography and purification by hydroxyapatite chromatography were carried out in the same manner as in step 1. The fractions containing the target substance (total 7 parts) were divided into the first 4 parts and the second 3 parts, and buffer exchange with phosphate buffered saline (pH6.0) was performed according to the method described in common procedure C. The antibody concentration of the resulting buffer solution was measured in accordance with the method described in general procedure B to give the title antibody solution (first 4 parts: 14.99mg/mL, 10mL) and the title antibody solution (second 3 parts: 10.97mg/mL, 6.2mL), respectively.

Example 26: synthesis of sugar chain-reconstituted antibody 2

Engineering anti-LPS antibodies－[SG－(N₃)₂]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc α 1, 6) GlcNAc-engineered anti-LPS antibodies

The same procedures as in step 1 of example 25 were carried out using a phosphate buffered saline solution (8.5mL, 10.96mg/mL, pH6.0) modified with the anti-LPS antibody prepared in reference example 2 to give a 20mM phosphate buffered solution (11.70mg/mL, 7.5mL, pH6.0) of the title antibody.

(step 2)

Engineering anti-LPS antibodies- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (11.70mg/mL, 7.5mL, pH6.0) of the antibody obtained in the above step 1 and [ N ]₃－PEG(3)]2-SG (10) Ox (20.3mg) was subjected to the same procedure as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (10.55mg/mL, 7.5mL, pH 6.0).

Example 27: synthesis of antibody drug conjugate 1 (Synthesis of anti-HER 2 antibody-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (10.97mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.0907mL, 24 equivalents per 1 molecule of antibody) of drug linker 3 and propylene glycol (0.159mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified according to the method described in general procedure D, to obtain an ABS (10mM Acetate Buffer, 5% Sorbitol, pH5.5) solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.97mg/mL

Antibody yield: 3.41mg (62%)

Mean drug binding number: 3.6

Example 28: synthesis of antibody drug conjugate 2 (Synthesis of anti-HER 2 antibody-CDN conjugate 2)

A phosphate-buffered saline (pH6.0) solution (10.97mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.0907mL, 24 equivalents per 1 molecule of antibody) of drug linker 4 and propylene glycol (0.159mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.08mg/mL

Antibody yield: 3.78mg (69%)

Mean drug binding number: 3.2

Example 29: synthesis of antibody drug conjugate 3 (Synthesis of anti-HER 2 antibody-CDN conjugate 3)

A phosphate-buffered saline (pH6.0) solution (10.97mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.0907mL, 24 equivalents per 1 molecule of antibody) of drug linker 2a and propylene glycol (0.159mL), and the mixture was reacted at room temperature for 47 hours using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.91mg/mL

Antibody yield: 3.17mg (58%)

Mean drug binding number: 3.6

Example 30: synthesis of antibody drug conjugate 4 (Synthesis of anti-LPS antibody-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (10.55mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 2 was diluted with propylene glycol (0.500 mL). To this solution, a mixture of a dimethylsulfoxide solution (10mM, 0.174mL, 24 equivalents per 1 molecule of antibody) of drug linker 3 and propylene glycol (0.326mL) was added, and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (6.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.11mg/mL

Antibody yield: 7.23mg (69%)

Mean drug binding number: 3.9

Example 31: synthesis of CDN21

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd ]Inden-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

4- (benzyloxy) -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (4-hydroxybut-1-yn-1-yl) -7H-pyrrolo [2, 3-D ] pyrimidine

Using the compound (3.37g) obtained in step 2 of example 20 and 3-butyn-1-ol (1.72mL), a reaction was carried out in the same manner as in step 2 of example 1 with the reaction temperature set to room temperature to obtain the title compound (2.74 g).

¹H－NMR(CDCl₃)δ：8.44(1H，s)，7.58－7.53(2H，m)，7.42－7.31(3H，m)，7.16(1H，s)，6.16(1H，s)，5.61(2H，dd，J＝14.9，12.5Hz)，4.47(1H，dd，J＝9.2，4.9Hz)，4.42(1H，d，J＝4.7Hz)，4.26(1H，dd，J＝9.4，4.7Hz)，4.17(1H，td，J＝9.9，4.8Hz)，4.01(1H，t，J＝9.6Hz)，3.71－3.64(2H，m)，2.65(2H，t，J＝6.1Hz)，1.87(1H，t，J＝6.5Hz)，1.08(9H，s)，1.04(9H，s)，0.91(9H，s)，0.11(3H，s)，0.10(3H，s).

(step 2)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (4-hydroxybutyl) -3, 7-dihydro-4H-pyrrolo [2, 3-D ] pyrimidin-4-one

Using the compound (2.74g) obtained in the above step 1, a reaction was carried out in the same manner as in step 7 of example 19 using a mixed solvent of methanol (30mL) and tetrahydrofuran (30mL) as a reaction solvent, so as to obtain the title compound (2.21 g).

¹H－NMR(CDCl₃)δ：11.55(1H，brs)，7.90(1H，s)，6.63(1H，s)，6.09(1H，s)，4.47(1H，dd，J＝9.0，5.1Hz)，4.38(1H，d，J＝4.7Hz)，4.24(1H，dd，J＝9.4，4.7Hz)，4.19－4.11(1H，m)，4.01(1H，t，J＝9.8Hz)，3.82－3.74(2H，m)，2.88(1H，brs)，2.83－2.74(2H，m)，1.85－1.75(2H，m)，1.71－1.62(2H，m)，1.09(9H，s)，1.04(9H，s)，0.90(9H，s)，0.102(3H，s)，0.099(3H，s).

(step 3)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd ] indene ] octane

Using the compound (1.89g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 5 of example 20 to obtain the title compound (1.40 g).

¹H－NMR(CDCl₃)δ：8.41(1H，s)，6.84(1H，s)，6.21(1H，s)，4.54－4.43(4H，m)，4.31(1H，dd，J＝9.6，4.9Hz)，4.17(1H，td，J＝10.0，5.1Hz)，4.00(1H，dd，J＝10.4，9.2Hz)，2.87－2.73(2H，m)，2.05－1.87(4H，m)，1.10(9H，s)，1.05(9H，s)，0.91(9H，s)，0.12(3H，s)，0.10(3H，s).

(step 4)

2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd ] indene ] octane

Using the compound (1.61g) obtained in the above step 3, a reaction was carried out in the same manner as in step 5 of example 1 to obtain the title compound (1.95 g).

¹H－NMR(CDCl₃)δ：8.40(1H，s)，7.49－7.43(2H，m)，7.38－7.20(8H，m)，6.86－6.78(4H，m)，6.38(1H，d，J＝5.5Hz)，4.70(1H，t，J＝5.3Hz)，4.54－4.45(2H，m)，4.38－4.31(1H，m)，4.26－4.18(1H，m)，3.79(3H，s)，3.79(3H，s)，3.53(1H，dd，J＝10.6，2.3Hz)，3.37(1H，dd，J＝10.6，3.1Hz)，2.81(1H，d，J＝3.9Hz)，2.56－2.45(2H，m)，2.00－1.91(2H，m)，1.86－1.76(2H，m)，0.82(9H，s)，－0.04(3H，s)，－0.17(3H，s).

(step 5)

2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd ] indene

The same procedures as in step 4 of example 5 were carried out using the compound (1.95g) obtained in step 4 above to give the title compound (2.20g) as a mixture of diastereomers on the phosphorus atom (diastereomer ratio: 6: 4).

¹H－NMR(CDCl₃)δ：8.39(0.4H，s)，8.37(0.6H，s)，7.51－7.45(2H，m)，7.40－7.20(8H，m)，6.86－6.78(4H，m)，6.37(0.6H，d，J＝6.7Hz)，6.32(0.4H，d，J＝6.3Hz)，4.83－4.78(0.6H，m)，4.77－4.70(0.4H，m)，4.56－4.44(2H，m)，4.42－4.33(1.4H，m)，4.29－4.24(0.6H，m)，4.07－3.86(1H，m)，3.82－3.75(6H，m)，3.70－3.46(4H，m)，3.32－3.24(1H，m)，2.76－2.65(1H，m)，2.63－2.49(2H，m)，2.32(1H，t，J＝6.7Hz)，2.01－1.90(2H，m)，1.87－1.74(2H，m)，1.23－1.12(8.4H，m)，1.03(3.6H，d，J＝6.7Hz)，0.74(3.6H，s)，0.72(5.4H，s)，－0.04(1.2H，s)，－0.08(1.8H，s)，－0.21(1.2H，s)，－0.23(1.8H，s).

(step 6)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl ]Oxy } -2, 10-bis (mercapto) -14- (7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd]Inden-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

Using the compound (1.18g) obtained in the above step 5, a reaction was carried out in the same manner as in step 7 of example 1 to obtain 2- { 2-O- [ tert-butyl (dimethyl) silyl group]-3-O- [ hydroxy (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd]Indene in acetonitrile. This acetonitrile solution was used together with commercially available (Cool Pharm) N-benzoyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl group]-3' -O- [ tert-butyl (dimethyl) silyl]-2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino]Phosphino } adenosine (1.49g),the reaction was carried out in the same manner as in steps 8, 9 and 10 of example 1 to obtain the title compound as a mixture of diastereomers at the phosphorus atom. The mixture was purified using HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 25% -60% (0 min-35 min) ]Purification was performed to give diastereomer 1(50mg) and diastereomer 2(34mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：973(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：973(M+H)⁺.

(step 7-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd]Inden-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (diastereomer 1) obtained in the above step 6 (50mg), a reaction was performed in the same manner as in the step 11 of example 1, and then the reaction mixture was reacted with Sep-Pak (registered trademark) C18[ 0.1% triethylammonium aqueous solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (33 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，s)，8.30(1H，s)，8.17(1H，s)，7.40(1H，s)，6.39(1H，d，J＝4.3Hz)，6.34(1H，d，J＝8.6Hz)，5.40－5.36(1H，m)，5.22－5.17(1H，m)，4.87－4.84(1H，m)，4.80(1H，t，J＝4.5Hz)，4.64－4.31(6H，m)，4.11－4.03(2H，m)，2.82(1H，dd，J＝16.4，8.6Hz)，2.68(1H，dd，J＝16.2，8.8Hz)，2.06－1.71(4H，m).

³¹P－NMR(CD₃OD)δ：58.1(s)，54.2(s).

(step 7-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (7, 8, 9, 10-tetrahydro-2H-6-oxo-2, 3, 5-triazacyclooctane [1, 2, 3-cd ]Inden-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (diastereomer 2) (34mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 11 of example 1, and then the reaction mixture was reacted with Sep-Pak (registered trademark) C18[ 0.1% triethylammonium aqueous solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.81(1H，s)，8.30(1H，s)，8.17(1H，s)，7.40(1H，s)，6.43(1H，d，J＝6.7Hz)，6.34(1H，d，J＝8.6Hz)，5.55－5.42(2H，m)，4.87－4.84(1H，m)，4.59－4.28(7H，m)，4.06－3.99(1H，m)，3.94－3.86(1H，m)，2.96－2.81(2H，m)，2.07－1.94(2H，m)，1.93－1.80(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.5(s).

Example 32: synthesis of CDN22

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-14- [ (7S) -7-methyl-8, 9-dihydro-6-oxo-2, 3, 5-triazabenzo [ cd]Azulene-2 (7H) -yl]-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

4- (benzyloxy) -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- [ (3R) -3-hydroxybut-1-yn-1-yl ] -7H-pyrrolo [2, 3-D ] pyrimidine

Using the compound (2.54g) obtained in the step 2 of example 20 and (R) - (+) -3-butyn-2-ol (1.36mL), a reaction was carried out in the same manner as in the step 2 of example 1 with the reaction temperature set at room temperature to obtain the title compound (1.85 g).

¹H－NMR(CDCl₃)δ：8.46(1H，s)，7.57(2H，d，J＝7.0Hz)，7.44－7.32(3H，m)，7.20(1H，s)，6.16(1H，s)，5.58(1H，d，J＝12.9Hz)，5.55(1H，d，J＝13.7Hz)，4.71－4.63(1H，m)，4.48(1H，dd，J＝9.2，4.9Hz)，4.42(1H，d，J＝4.7Hz)，4.26(1H，dd，J＝9.4，4.7Hz)，4.23－4.14(1H，m)，4.01(1H，t，J＝9.6Hz)，1.70(1H，d，J＝5.5Hz)，1.42(3H，d，J＝6.7Hz)，1.09(9H，s)，1.04(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 2)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- [ (3R) -3-hydroxybutyl ] -3, 7-dihydro-4H-pyrrolo [2, 3-D ] pyrimidin-4-one

Using the compound (1.85g) obtained in the above step 1, a reaction was carried out in the same manner as in step 7 of example 19 using a mixed solvent of methanol (15mL) and tetrahydrofuran (15mL) as a reaction solvent, so as to obtain the title compound (1.34 g).

¹H－NMR(CDCl₃)δ：11.81(1H，s)，7.89(1H，s)，6.68(1H，s)，6.12(1H，s)，4.49(1H，dd，J＝9.2，4.5Hz)，4.34(1H，d，J＝4.3Hz)，4.27－4.14(3H，m)，4.03(1H，t，J＝9.6Hz)，3.77－3.65(1H，m)，3.17－3.06(1H，m)，2.85－2.74(1H，m)，1.84－1.73(1H，m)，1.71－1.61(1H，m)，1.15(3H，d，J＝6.3Hz)，1.09(9H，s)，1.04(9H，s)，0.90(9H，s)，0.10(6H，s).

(step 3)

(7S) -2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -7-methyl-2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (1.34g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 5 of example 20 to obtain the title compound (0.70 g).

¹H－NMR(CDCl₃)δ：8.43(1H，s)，6.82(1H，s)，6.19(1H，s)，4.58－4.43(3H，m)，4.33(1H，dd，J＝9.6，5.0Hz)，4.17(1H，td，J＝10.0，5.0Hz)，4.00(1H，dd，J＝10.4，9.2Hz)，3.06－2.97(1H，m)，2.89－2.79(1H，m)，2.23－2.08(2H，m)，1.60(3H，d，J＝6.3Hz)，1.10(9H，s)，1.05(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 4)

(7S) -2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -7-methyl-2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (0.70g) obtained in the above step 3, a reaction was carried out in the same manner as in step 5 of example 1 to obtain the title compound (0.82 g).

¹H－NMR(CDCl₃)δ：8.41(1H，s)，7.49－7.43(2H，m)，7.37－7.20(7H，m)，7.16(1H，s)，6.85－6.78(4H，m)，6.35(1H，d，J＝5.5Hz)，4.72(1H，t，J＝5.3Hz)，4.57－4.47(1H，m)，4.36(1H，dd，J＝9.0，3.9Hz)，4.22(1H，q，J＝3.1Hz)，3.79(3H，s)，3.79(3H，s)，3.52(1H，dd，J＝10.6，2.7Hz)，3.37(1H，dd，J＝10.6，3.1Hz)，2.81(1H，d，J＝3.9Hz)，2.78－2.58(2H，m)，2.16－2.07(2H，m)，1.59(3H，d，J＝6.7Hz)，0.83(9H，s)，－0.03(3H，s)，－0.15(3H，s).

(step 5)

(7S) -2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -beta-D-ribofuranosyl) -7-methyl-2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triaza-benzo [ cd ] azulene

The same procedures as in step 4 of example 5 were carried out using the compound (0.82g) obtained in step 4 above to give the title compound (0.85g) in the form of a diastereomer mixture on the phosphorus atom (diastereomer ratio: 6: 4).

¹H－NMR(CDCl₃)δ：8.40(0.4H，s)，8.38(0.6H，s)，7.50－7.43(2H，m)，7.39－7.16(8H，m)，6.86－6.78(4H，m)，6.34(0.6H，d，J＝6.7Hz)，6.30(0.4H，d，J＝5.9Hz)，4.86－4.80(0.6H，m)，4.79－4.74(0.4H，m)，4.55－4.46(1H，m)，4.44－4.35(1.4H，m)，4.29－4.24(0.6H，m)，4.05－3.85(1H，m)，3.82－3.75(6H，m)，3.69－3.47(4H，m)，3.31－3.24(1H，m)，2.82－2.61(3H，m)，2.31(1H，t，J＝6.7Hz)，2.17－2.07(2H，m)，1.58－1.55(3H，m)，1.22－1.13(8.4H，m)，1.03(3.6H，d，J＝6.7Hz)，0.75(3.6H，s)，0.74(5.4H，s)，－0.03(1.2H，s)，－0.07(1.8H，s)，－0.19(1.2H，s)，－0.21(1.8H，s).

(step 6)

(5R，7R，8R，12aR，14R，15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -14- [ (7S) -7-methyl-8, 9-dihydro-6-oxo-2, 3, 5-triaza-benzo [ cd)]Azulene-2 (7H) -yl]-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

Using the compound (0.85g) obtained in the above step 5, a reaction was carried out in the same manner as in step 7 of example 1 to obtain (7S) -2- { 2-O- [ tert-butyl (dimethyl) silyl group ]-3-O- [ hydroxy (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -7-methyl-2, 7, 8, 9-tetrahydro-6-oxo-2, 3, 5-triazabenzo [ cd]Acetonitrile solution of azulene. This acetonitrile solution was used together with commercially available (Cool Pharm) N-benzoyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl group]-3' -O- [ tert-butyl (dimethyl) silyl]-2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino]Phosphino } adenosine (1.12g), was reacted in the same manner as in steps 8, 9 and 10 of example 1 to give the title compound as a mixture of diastereomers on the phosphorus atom. The mixture was purified by HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 25% -60% (0 min-35 min)]Purification was carried out to give diastereomer 1(95mg) and diastereomer 2(44mg) of the title compound (retention time of HPLC: diastereomer 1)>2)。

Diastereomer 1 (low polarity)

MS(ESI)m/z：973(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：973(M+H)⁺.

(step 7-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-14- [ (7S) -7-methyl-8, 9-dihydro-6-oxo-2, 3, 5-triaza-pyrido [ cd ] S ]Azulene-2 (7H) -yl]－2，10-di-oxo-octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (diastereomer 1) (95mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 11 of example 1, and then the reaction mixture was purified by Sep-Pak (registered trademark) C18[ 0.1% triethylammonium aqueous solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (57 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，s)，8.30(1H，s)，8.17(1H，s)，7.35(1H，s)，6.37(1H，d，J＝4.3Hz)，6.34(1H，d，J＝8.2Hz)，5.40－5.35(1H，m)，5.22－5.17(1H，m)，4.85－4.81(2H，m)，4.66－4.58(1H，m)，4.53－4.40(2H，m)，4.39－4.30(2H，m)，4.12－4.01(2H，m)，3.02－2.93(1H，m)，2.80－2.68(1H，m)，2.23－2.14(1H，m)，2.12－2.00(1H，m)，1.57(3H，d，J＝6.3Hz).

³¹P－NMR(CD₃OD)δ：58.1(s)，54.3(s).

(step 7-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-14- [ (7S) -7-methyl-8, 9-dihydro-6-oxo-2, 3, 5-triaza-pyrido [ cd ] S]Azulene-2 (7H) -yl]-2, 10-dialkoxyoctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (diastereomer 2) (44mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 11 of example 1, and then the reaction mixture was reacted with Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (30 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.81(1H，s)，8.30(1H，s)，8.17(1H，s)，7.36(1H，s)，6.41(1H，d，J＝6.7Hz)，6.34(1H，d，J＝8.6Hz)，5.55－5.42(2H，m)，4.87－4.84(1H，m)，4.65－4.57(1H，m)，4.55－4.28(5H，m)，4.06－3.99(1H，m)，3.93－3.86(1H，m)，3.11－3.01(1H，m)，2.95－2.83(1H，m)，2.29－2.19(1H，m)，2.16－2.03(1H，m)，1.57(3H，d，J＝6.3Hz).

³¹P－NMR(CD₃OD)δ：63.7(s)，61.2(s).

Example 33: synthesis of CDN23

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

4- (benzyloxy) -5- { 3- [ bis (4-methoxyphenyl) (phenyl) methoxy ] prop-1-yn-1-yl } -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -7H-pyrrolo [2, 3-D ] pyrimidine

Using the compound obtained in step 3 of example 20 (4.17g), a reaction was carried out in the same manner as in step 1 of example 11 using a mixed solvent of dichloromethane (40mL) -pyridine (40mL) as a reaction solvent to obtain the title compound (5.70 g).

¹H－NMR(CDCl₃)δ：8.44(1H，s)，7.53－7.47(4H，m)，7.41－7.35(4H，m)，7.33－7.11(7H，m)，6.86－6.79(4H，m)，6.17(1H，s)，5.61(1H，d，J＝13.7Hz)，5.58(1H，d，J＝13.7Hz)，4.49(1H，dd，J＝9.0，5.0Hz)，4.44(1H，d，J＝4.8Hz)，4.29(1H，dd，J＝9.6，5.0Hz)，4.23－4.15(1H，m)，4.04(1H，t，J＝9.8Hz)，3.98(2H，s)，3.78(6H，s)，1.10(9H，s)，1.05(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 2)

5- { 3- [ bis (4-methoxyphenyl) (phenyl) methoxy ] propyl } -7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -3, 7-dihydro-4H-pyrrolo [2, 3-D ] pyrimidin-4-one

Ammonium formate (3.71g) and 10% palladium on carbon (AD) wet (2g) were added to a methanol (100mL) -tetrahydrofuran (50mL) mixed solution of the compound (5.70g) obtained in the above step 1, and the mixture was stirred at room temperature for 3 hours. The catalyst was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (4.56 g).

¹H－NMR(CDCl₃)δ：11.61(1H，brs)，7.75(1H，s)，7.48－7.44(2H，m)，7.37－7.14(7H，m)，6.84－6.79(4H，m)，6.57(1H，s)，6.05(1H，s)，4.45(1H，dd，J＝9.2，4.9Hz)，4.35(1H，d，J＝5.1Hz)，4.23(1H，dd，J＝9.6，4.9Hz)，4.16－4.10(1H，m)，3.97(1H，t，J＝9.8Hz)，3.78(6H，s)，3.19－3.08(2H，m)，2.90(2H，t，J＝7.8Hz)，2.07－1.98(2H，m)，1.09(9H，s)，1.04(9H，s)，0.89(9H，s)，0.09(3H，s)，0.08(3H，s).

(step 3)

Pyridine (0.461mL) was added to a dichloromethane (10mL) solution of the compound (1.01g) obtained in the above step 2, and trifluoromethanesulfonic anhydride (0.385mL) was added dropwise under ice cooling, followed by stirring for 30 minutes. A suspension of sodium hydrogensulfite (2.54g) in N, N-dimethylformamide (25mL) was added to the reaction mixture at the same temperature, and the mixture was stirred at room temperature for 2 hours. To the reaction mixture were added a saturated aqueous sodium bicarbonate solution and ethyl acetate, and the mixture was filtered through celite and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (0.51 g).

¹H－NMR(CDCl₃)δ：10.83(1H，s)，7.83(1H，s)，7.50－7.44(2H，m)，7.39－7.14(7H，m)，6.87－6.79(4H，m)，6.72(1H，s)，6.07(1H，s)，4.48－4.42(1H，m)，4.31(1H，d，J＝4.3Hz)，4.21－4.09(2H，m)，3.99－3.92(1H，m)，3.79(6H，s)，3.19－3.09(4H，m)，2.09－1.98(2H，m)，1.08(9H，s)，1.04(9H，s)，0.90(9H，s)，0.09(3H，s)，0.09(3H，s).

(step 4)

7- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) -beta-D-ribofuranosyl } -5- (3-hydroxypropyl) -3, 7-dihydro-4H-pyrrolo [2, 3-D ] pyrimidine-4-thione

Distilled water (4mL) was added to a dichloromethane (80mL) solution of the compound (2.79g) obtained in the above step 3, and dichloroacetic acid (1.28mL) was added dropwise under ice cooling, followed by stirring for 30 minutes. Pyridine (2.50mL) was added to the reaction mixture at the same temperature, and then the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (0.96 g).

¹H－NMR(CDCl₃)δ：11.16(1H，s)，7.89(1H，s)，6.83(1H，s)，6.11(1H，s)，4.51－4.45(1H，m)，4.36－4.29(1H，m)，4.22－4.13(2H，m)，4.06－3.97(1H，m)，3.68(2H，t，J＝6.1Hz)，3.27－3.09(2H，m)，2.25－2.13(1H，brm)，2.00－1.93(2H，m)，1.08(9H，s)，1.04(9H，s)，0.90(9H，s)，0.10(6H，s).

(step 5)

2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-O- (di-tert-butylsilylidene) - β -D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (0.35g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 5 of example 20 to obtain the title compound (0.25 g).

¹H－NMR(CDCl₃)δ：8.53(1H，s)，6.92(1H，s)，6.21(1H，s)，4.50－4.44(2H，m)，4.30(1H，dd，J＝9.6，4.9Hz)，4.17(1H，td，J＝10.0，5.0Hz)，4.00(1H，dd，J＝10.4，9.2Hz)，3.18－3.12(2H，m)，3.06－3.00(2H，m)，2.39－2.30(2H，m)，1.09(9H，s)，1.05(9H，s)，0.91(9H，s)，0.12(3H，s)，0.11(3H，s).

(step 6)

2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (0.41g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 5 of example 1 to obtain the title compound (0.46 g).

¹H－NMR(CDCl₃)δ：8.52(1H，s)，7.48－7.42(2H，m)，7.37－7.20(8H，m)，6.85－6.78(4H，m)，6.36(1H，d，J＝5.1Hz)，4.70(1H，t，J＝5.1Hz)，4.37(1H，dd，J＝8.8，4.1Hz)，4.23－4.19(1H，m)，3.79(3H，s)，3.79(3H，s)，3.53(1H，dd，J＝10.6，2.7Hz)，3.38(1H，dd，J＝10.6，3.1Hz)，3.16－3.09(2H，m)，2.78(1H，d，J＝4.1Hz)，2.76－2.70(2H，m)，2.30－2.22(2H，m)，0.83(9H，s)，－0.03(3H，s)，－0.14(3H，s).

(step 7)

Using the compound (0.46g) obtained in the above step 6, the title compound (0.48g) was obtained in the form of a diastereomer mixture (diastereomer ratio 6: 4) on the phosphorus atom in the same manner as in the step 4 of example 5.

¹H－NMR(CDCl₃)δ：8.51(0.4H，s)，8.49(0.6H，s)，7.50－7.43(2H，m)，7.38－7.23(8H，m)，6.86－6.78(4H，m)，6.36(0.6H，d，J＝6.7Hz)，6.32(0.4H，d，J＝5.5Hz)，4.83－4.78(0.6H，m)，4.76－4.71(0.4H，m)，4.45－4.35(1.4H，m)，4.29－4.24(0.6H，m)，4.04－3.84(1H，m)，3.82－3.75(6H，m)，3.70－3.48(4H，m)，3.32－3.25(1H，m)，3.16－3.09(2H，m)，2.84－2.73(2H，m)，2.73－2.61(1H，m)，2.36－2.20(3H，m)，1.22－1.13(8.4H，m)，1.03(3.6H，d，J＝7.0Hz)，0.76(3.6H，s)，0.75(5.4H，s)，－0.03(1.2H，s)，－0.07(1.8H，s)，－0.18(1.2H，s)，－0.20(1.8H，s).

(step 8)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] silyl]Oxy } -10- (2-cyanoethoxy) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triaza-eno [ cd)]Azulene-2 (7H) -yl) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Oxygen fiveHetero diphosphacyclotetradecyn-7-yl]-9H-purin-6-yl } benzamide

Using the compound (0.48g) obtained in the above step 7, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 2- { 2-O- [ tert-butyl (dimethyl) silyl group]-3-O- [ hydroxy (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Acetonitrile solution of azulene. This acetonitrile solution was used together with commercially available (Cool Pharm) N-benzoyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl group]-3' -O- [ tert-butyl (dimethyl) silyl]-2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino]Phosphino adenosine (0.61g) was reacted in the same manner as in steps 8 and 9 of example 1 to obtain the title compound as a mixture of diastereomers on the phosphorus atom. The mixture was purified by HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 40% -90% (0 min-35 min)]Purification was carried out to obtain diastereomer 1(40mg) and diastereomer 2(18mg) of the title compound, respectively.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1132(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1132(M+H)⁺.

(step 9-1)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl ]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

Using the compound (diastereomer 1) (40mg) obtained in the above step 8, a reaction was carried out in the same manner as in step 10 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 30% to 60% (0 part to 35 parts) was purified to obtain the title compound (35 mg).

MS(ESI)m/z：975(M+H)⁺.

(step 9-2)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

Using the compound (diastereomer 2) (18mg) obtained in the above step 8, a reaction was carried out in the same manner as in step 10 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -50% (0 min-35 min) to give the title compound (15 mg).

MS(ESI)m/z：975(M+H)⁺.

(step 10-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (35mg) obtained in the above step 9-1, a reaction was carried out in the same manner as in the step 11 of example 1, and then the reaction mixture was purified by filtration using Sep-Pak (registered trademark) C18[ 0.1% triethylammonium aqueous solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (18 mg).

MS(ESI)m/z：747(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，s)，8.41(1H，s)，8.17(1H，s)，7.48(1H，s)，6.40(1H，d，J＝4.3Hz)，6.34(1H，d，J＝8.6Hz)，5.40－5.35(1H，m)，5.23－5.17(1H，m)，4.86－4.79(2H，m)，4.54－4.41(2H，m)，4.39－4.31(2H，m)，4.12－4.01(2H，m)，3.23－3.16(2H，m)，3.05－2.86(2H，m)，2.36－2.20(2H，m).

³¹P－NMR(CD₃OD)δ：58.1(s)，54.2(s).

(step 10-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15, 16-dihydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (15mg) obtained in the above step 9-2, a reaction was carried out in the same manner as in step 11 of example 1, and then the reaction mixture was purified by filtration using Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (8.1 mg).

MS(ESI)m/z：747(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.81(1H，s)，8.41(1H，s)，8.17(1H，s)，7.49(1H，s)，6.43(1H，d，J＝6.7Hz)，6.34(1H，d，J＝8.2Hz)，5.55－5.41(2H，m)，4.87－4.82(1H，m)，4.57－4.27(5H，m)，4.07－3.99(1H，m)，3.94－3.86(1H，m)，3.24－3.16(2H，m)，3.12－3.03(2H，m)，2.39－2.25(2H，m).

³¹P－NMR(CD₃OD)δ：63.0(s)，60.5(s).

Example 34: synthesis of CDN24

1- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]Pyrimidine-2, 4(1H, 3H) -diones

[ synthetic route ]

(step 1)

1- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] silyl]Oxy } -2, 10-di-oxo-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]Pyrimidine-2, 4(1H, 3H) -diones

The reaction of step 7 in example 1 (raw material: 1.01g) was carried out on the following scale. Using the acetonitrile solution of the obtained compound and commercially Available (ANGENE) 5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } uridine (1.03g), a reaction was carried out in the same manner as in steps 8, 9 and 10 of example 1 to obtain the title compound as a diastereomer mixture on the phosphorus atom. The mixture was purified by HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: purification from 25% to 60% (0 min to 35 min) gave diastereomer 1(50mg) and diastereomer 2(23mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：935(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：935(M+H)⁺.

(step 2-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (2, 4-Dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -15, 16-dihydroxy-2, 10-Dioxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-Tetraazabenzo [ cd ] s ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (diastereomer 1) (50mg) obtained in the above step 1, a reaction was performed in the same manner as in the step 11 of example 1, and then the reaction mixture was purified by Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (30 mg).

MS(ESI)m/z：707(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.09(1H，d，J＝8.2Hz)，8.03(1H，s)，7.16(1H，s)，6.32(1H，d，J＝8.6Hz)，6.28(1H，d，J＝4.3Hz)，5.82(1H，d，J＝8.2Hz)，5.08－5.01(1H，m)，4.93－4.84(1H，m)，4.73(1H，t，J＝4.5Hz)，4.68(1H，d，J＝3.9Hz)，4.48－4.38(2H，m)，4.33－4.24(1H，m)，4.23(1H，d，J＝2.3Hz)，4.09－3.99(2H，m)，3.56－3.46(2H，m)，2.96－2.83(2H，m)，2.07－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：58.3(s)，54.6(s).

(step 2-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (2, 4-Dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -15, 16-dihydroxy-2, 10-Dioxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-Tetraazabenzo [ cd ] s]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (diastereomer 2) (23mg) obtained in the above step 1, a reaction was carried out in the same manner as in the step 11 of example 1, and then the reaction mixture was reacted with Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile 5: 1] to obtain the triethylamine salt of the title compound.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (11 mg).

MS(ESI)m/z：707(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.08(1H，d，J＝7.8Hz)，8.01(1H，s)，7.16(1H，s)，6.35(1H，d，J＝8.6Hz)，6.31(1H，d，J＝6.7Hz)，5.85(1H，d，J＝7.8Hz)，5.38－5.33(1H，m)，5.04－4.96(1H，m)，4.75(1H，dd，J＝6.5，4.5Hz)，4.50－4.34(3H，m)，4.33－4.26(1H，m)，4.22－4.17(1H，m)，4.04－3.97(1H，m)，3.91－3.84(1H，m)，3.53－3.46(2H，m)，2.97－2.87(2H，m)，2.05－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：63.2(s)，60.2(s).

Example 35: synthesis of CDN25

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- (6-oxo-1, 6-dihydro-9H-purin-9-yl) -2, 10-bis (mercapto) -14-) (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-7- (6-oxo-1, 6-dihydro-9H-purin-9-yl) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a tetrahydrofuran (5.0mL) solution of the compound (313mg) obtained in step 4 of example 13, N- [ (E) - (pyridin-2-yl) methylidene ] hydroxylamine (337mg) and N, N, N ', N' -tetramethylguanidine (0.346mL) were added, and the mixture was stirred at room temperature for 1 day. Methanol (5.0mL) and 28% aqueous ammonia (5.0mL) were added to the reaction mixture, and the mixture was stirred at 50 ℃ for 5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(106 mg: containing impurities) and diastereomer 2(105 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：959(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：959(M+H)⁺.

(step 2-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-7- (6-oxo-1, 6-dihydro-9H-purin-9-yl) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (106 mg: containing impurities) obtained in the above step 1 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 7% -50% (0 min-40 min) ]

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (43.4 mg).

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.66(1H，s)，8.02(2H，s)，7.09(1H，s)，6.30(1H，d，J＝6.8Hz)，6.28(1H，d，J＝4.8Hz)，5.45－5.38(1H，m)，5.20－5.13(1H，m)，4.82(1H，d，J＝4.2Hz)，4.77(1H，t，J＝4.5Hz)，4.52－4.41(2H，m)，4.36－4.27(2H，m)，4.08－3.97(2H，m)，3.53－3.46(2H，m)，2.88－2.80(2H，m)，2.04－1.95(2H，m).

³¹P－NMR(CD₃OD)δ：57.7(s)，54.6(s).

(step 2-2)

Disodium salt (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-Dihydroxy-2, 10-di-oxo-7- (6-oxo-1, 6-dihydro-9H-purin-9-yl) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (105 mg: containing impurities) obtained in the above step 1 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21.5 mg).

MS(ESI)m/z：731(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.72(1H，s)，8.03(1H，s)，8.02(1H，s)，7.11(1H，s)，6.32(1H，d，J＝6.0Hz)，6.30(1H，d，J＝8.0Hz)，5.49－5.40(2H，m)，4.77(1H，dd，J＝6.7，4.2Hz)，4.49(1H，d，J＝4.5Hz)，4.47－4.29(4H，m)，4.07－4.01(1H，m)，3.93－3.86(1H，m)，3.52－3.47(2H，m)，2.90(2H，t，J＝5.4Hz)，2.05－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：62.9(s)，60.0(s).

Example 36: synthesis of CDN26

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 1- (3-hydroxypropyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetranitrogenHetero-benzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

1- [ 3- (benzoyloxy) propyl ] -5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine

Triethylamine (1.83mL) and benzoyl chloride (1.43mL) were added to a solution of 3-bromopropan-1-ol (1.14mL) in tetrahydrofuran (25mL), and the mixture was stirred at room temperature for 6 hours. The reaction solution was filtered, washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure. To a dehydrated N, N-dimethylacetamide (25mL) solution of the residue were added commercially available (Aamdis Chemical) 5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine (5.0g) and 1, 8-diazabicyclo [5.4.0] -7-undecene (2.75mL), and the mixture was stirred at room temperature for 3 days. Water was added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol ] to give the title compound (4.41 g).

MS(ESI)m/z：733(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.00－7.98(1H，m)，7.98－7.96(1H，m)，7.97(1H，s)，7.96(1H，s)，7.58－7.52(1H，m)，7.46－7.39(2H，m)，7.35－7.30(2H，m)，7.26－7.16(7H，m)，6.81－6.75(4H，m)，5.87(1H，d，J＝6.0Hz)，4.85(1H，d，J＝3.6Hz)，4.67－4.62(1H，m)，4.43－4.34(3H，m)，4.30－4.16(2H，m)，3.78－3.75(1H，m)，3.77(6H，s)，3.42(1H，dd，J＝10.3，3.6Hz)，3.33(1H，dd，J＝10.3，3.6Hz)，3.02(1H，d，J＝2.4Hz)，2.32(2H，dd，J＝11.8，5.7Hz).

(step 2)

1- [ 3- (benzoyloxy) propyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] inosine

Using the compound (4.41g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (1.60g) and 1- [ 3- (benzoyloxy) propyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] inosine (1.70g), which is a positional isomer of the title compound.

MS(ESI)m/z：847(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.01(1H，d，J＝1.2Hz)，8.00(1H，s)，7.99(1H，d，J＝1.2Hz)，7.93(1H，s)，7.60－7.52(1H，m)，7.46－7.38(4H，m)，7.33－7.16(7H，m)，6.83－6.77(4H，m)，5.90(1H，d，J＝4.8Hz)，4.58－4.52(1H，m)，4.50－4.46(1H，m)，4.39(2H，t，J＝6.0Hz)，4.28－4.12(3H，m)，3.78(3H，s)，3.77(3H，s)，3.46(1H，dd，J＝10.6，3.9Hz)，3.26(1H，dd，J＝10.6，3.9Hz)，2.99(1H，d，J＝6.7Hz)，2.30(2H，dd，J＝13.3，6.0Hz)，0.88(9H，s)，0.07(3H，s)，0.00(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：847(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.01(1H，d，J＝1.8Hz)，7.99(1H，d，J＝1.8Hz)，7.98(1H，s)，7.87(1H，s)，7.59－7.54(1H，m)，7.47－7.40(4H，m)，7.36－7.17(7H，m)，6.84－6.78(4H，m)，5.94(1H，d，J＝5.4Hz)，4.84(1H，t，J＝5.4Hz)，4.41－4.35(2H，m)，4.33－4.28(1H，m)，4.28－4.19(3H，m)，3.78(3H，s)，3.78(3H，s)，3.48(1H，dd，J＝10.9，3.0Hz)，3.37(1H，dd，J＝10.9，3.0Hz)，2.68(1H，d，J＝3.6Hz)，2.34－2.26(2H，m)，0.84(9H，s)，0.01(3H，s)，－0.13(3H，s).

(step 3)

1- [ 3- (benzoyloxy) propyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } inosine

Using the compound (1.60g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (1.91g) in the form of a diastereomer mixture (diastereomer ratio: 67: 33) on the phosphorus atom.

MS(ESI)m/z：1047(M+H)⁺.

¹H－NMR(CDCl3)δ：8.04－8.00(0.33H，m)，8.03(1H，s)，8.02(1H，s)，7.91(0.67H，d，J＝14.5Hz)，7.60－7.53(1H，m)，7.49－7.40(4H，m)，7.35－7.17(7H，m)，6.84－6.78(4H，m)，6.14(0.67H，d，J＝5.1Hz)，6.06(0.33H，d，J＝6.0Hz)，4.86－4.78(0.33H，m)，4.68－4.61(0.67H，m)，4.44－4.35(2H，m)，4.29－4.09(4H，m)，3.78(6H，s)，3.65－3.42(6.33H，m)，3.34－3.24(0.67H，m)，2.76(1.34H，t，J＝6.6Hz)，2.50(0.66H，t，J＝6.6Hz)，2.38(1.34H，t，J＝6.6Hz)，2.30(0.66H，t，J＝6.6Hz)，1.30－1.24(6H，m)，1.15－1.07(4.02H，m)，0.95(1.98H，d，J＝6.6Hz)，0.84(9H，s)，0.09(0.99H，s)，0.05(2.01H，s)，0.00(3H，s).

(step 4)

The same reaction (raw material: 981mg) as in step 7 of example 1 was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.03g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 5)

3- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) methylSilane radical]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } propyl benzoate

Using the crude product obtained in the above step 4, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (774mg) as a mixture of diastereomers at the phosphorus atom.

MS(ESI)m/z：1278(M+H)⁺.

(step 6)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 1- (3-hydroxypropyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (774mg) obtained in the above-mentioned step 5, a reaction was carried out in the same manner as in the step 10 of example 1, and purification was carried out by C18 silica gel column chromatography [ 0.2% aqueous triethylamine solution/acetonitrile ] to obtain diastereomer 1(101 mg: containing impurities) and diastereomer 2(90.8 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1017(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1017(M+H)⁺.

(step 7-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 1- (3-hydroxypropyl) -6-oxo-1, 6-dihydro-9H-purine-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound (diastereomer 1) (101 mg: containing impurities) obtained in the above step 6, a reaction was carried out in the same manner as in the step 11 of example 1, and then purification was carried out under the following purification conditions to obtain the title compound as a triethylamine salt.

[ refining conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (48.8 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.65(1H，s)，8.28(1H，s)，8.03(1H，s)，7.09(1H，s)，6.28(1H，s)，6.27(1H，d，J＝4.8Hz)，5.46－5.38(1H，m)，5.21－5.13(1H，m)，4.83－4.80(1H，m)，4.79－4.75(1H，m)，4.52－4.39(2H，m)，4.36－4.28(2H，m)，4.26－4.17(1H，m)，4.17－4.08(1H，m)，4.08－3.97(2H，m)，3.59(2H，t，J＝5.7Hz)，3.49(2H，t，J＝4.8Hz)，2.91－2.74(2H，m)，2.02－1.92(4H，m).

³¹P－NMR(CD₃OD)δ：57.6(s)，54.6(s).

(step 7-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 1- (3-hydroxypropyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (90.8 mg: containing impurities) obtained in the above step 6 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (22.3 mg).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.71(1H，s)，8.29(1H，s)，8.02(1H，s)，7.11(1H，s)，6.32(1H，d，J＝6.7Hz)，6.28(1H，d，J＝8.5Hz)，5.48－5.38(2H，m)，4.80－4.74(1H，m)，4.51－4.47(1H，m)，4.47－4.28(4H，m)，4.27－4.14(2H，m)，4.07－4.01(1H，m)，3.92－3.86(1H，m)，3.60(2H，t，J＝6.0Hz)，3.53－3.47(2H，m)，2.93－2.87(2H，m)，2.06－1.94(4H，m).

³¹P－NMR(CD₃OD)δ：62.8(s)，59.9(s).

Example 37: synthesis of CDN27

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 2-amino-1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

1- [ 2- (benzoyloxy) ethyl ] -5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -N- [ (dimethylamino) methenyl ] guanosine

To a mixed solution of N- [ (dimethylamino) methine ] guanosine (10.0g) in N, N-dimethylacetamide (50mL) -pyridine (50mL) known in the literature (Journal of Organic Chemistry, 1994, 59, 7243-7248), 4' -dimethoxytrityl chloride (10.5g) was added at 0 ℃ and stirred at 4 ℃ for 16 hours. 2-bromoethyl benzoate (6.54mL) and 1, 8-diazabicyclo [5.4.0] -7-undecene (11.0mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 2 days. To the reaction mixture were added saturated aqueous sodium bicarbonate and water, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol ] to give the title compound as a mixture with triphenylphosphine oxide (16.7 g).

MS(ESI)m/z：789(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.17(1H，s)，7.93－7.89(2H，m)，7.55(1H，s)，7.54－7.48(1H，m)，7.42－7.36(4H，m)，7.31－7.26(4H，m)，7.25－7.19(2H，m)，7.16－7.11(1H，m)，6.82－6.76(4H，m)，5.96(1H，d，J＝6.7Hz)，4.79－4.70(1H，m)，4.70－4.61(2H，m)，4.60－4.52(1H，m)，4.52－4.45(1H，m)，4.41－4.38(1H，m)，4.34－4.30(1H，m)，3.75(3H，s)，3.75(3H，s)，3.39－3.36(2H，m)，2.89(3H，s)，2.80(3H，s).

(step 2)

1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -N- [ (dimethylamino) methine ] guanosine

Using the compound (15.7g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (4.82g) and 1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -N- [ (dimethylamino) methine ] guanosine (6.01g), which is a positional isomer of the title compound.

MS(ESI)m/z：903(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.24(1H，s)，7.99－7.95(2H，m)，7.85(1H，s)，7.56－7.50(1H，m)，7.44－7.38(4H，m)，7.34－7.27(6H，m)，7.24－7.18(1H，m)，6.84－6.79(4H，m)，5.98(1H，d，J＝4.2Hz)，4.87－4.77(2H，m)，4.72－4.61(2H，m)，4.41－4.36(2H，m)，4.16－4.09(1H，m)，3.78(3H，s)，3.78(3H，s)，3.45(1H，dd，J＝10.6，3.9Hz)，3.25(1H，dd，J＝10.6，3.9Hz)，3.05(1H，d，J＝5.4Hz)，2.91(3H，s)，2.78(3H，s)，0.86(9H，s)，0.05(3H，s)，－0.04(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：903(M+H)⁺.

1H－NMR(CDCl3)δ：8.23(1H，s)，7.97－7.91(2H，m)，7.82(1H，s)，7.56－7.50(1H，m)，7.45－7.36(4H，m)，7.35－7.26(6H，m)，7.25－7.19(1H，m)，6.85－6.79(4H，m)，5.98(1H，d，J＝5.4Hz)，4.88－4.77(2H，m)，4.73－4.62(3H，m)，4.32－4.27(1H，m)，4.23－4.19(1H，m)，3.79(3H，s)，3.79(3H，s)，3.47(1H，dd，J＝10.9，3.6Hz)，3.37(1H，dd，J＝10.9，3.6Hz)，2.90(3H，s)，2.76(1H，d，J＝2.5Hz)，2.75(3H，s)，0.84(9H，s)，0.02(3H，s)，－0.15(3H，s).

(step 3)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -N- [ (dimethylamino) methenyl ] guanosine

Using the compound (4.81g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (5.41g) in the form of a diastereomer mixture (diastereomer ratio: 7: 3) on the phosphorus atom.

MS(ESI)m/z：1103(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.33(0.7H，s)，8.31(0.3H，s)，7.98－7.94(2H，m)，7.87(0.3H，s)，7.80(0.7H，s)，7.56－7.50(1H，m)，7.46－7.37(4H，m)，7.35－7.27(6H，m)，7.24－7.18(1H，m)，6.85－6.80(4H，m)，6.14(0.7H，d，J＝6.0Hz)，6.13(0.3H，d，J＝5.4Hz)，4.92－4.59(4H，m)，4.55－4.48(0.7H，m)，4.33－4.29(0.3H，m)，4.25－4.21(0.6H，m)，4.17－4.13(1.4H，m)，3.79(3H，s)，3.79(3H，s)，3.60－3.38(5H，m)，3.33－3.23(1H，m)，2.93(2.1H，s)，2.92(0.9H，s)，2.82(2.1H，s)，2.81(0.9H，s)，2.47－2.42(0.6H，m)，2.34－2.27(1.4H，m)，1.09(4.2H，d，J＝6.7Hz)，1.07(1.8H，d，J＝7.3Hz)，1.05(4.2H，d，J＝6.7Hz)，0.91(1.8H，d，J＝7.3Hz)，0.84(9H，s)，0.07(0.9H，s)，0.04(2.1H，s)，－0.02(3H，s).

(step 4)

The same reaction as in step 7 of example 1 (starting material: 1.68g) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.81g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 5)

2- (9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2λ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-2- { (E) - [ (dimethylamino) methine]Amino } -6-oxo-6, 9-dihydro-1H-purin-1-yl) ethyl benzoate

Using the crude product obtained in the above step 4, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (1.15g) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1334(M+H)⁺.

(step 6)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 2-amino-1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl ]-15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (1.15g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(134 mg: containing impurities) and diastereomer 2(127 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1018(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1018(M+H)⁺.

(step 7-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 2-amino-1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (134 mg: containing impurities) obtained in the above step 6 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (36.0 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.01(1H，s)，7.99(1H，s)，7.17(1H，s)，6.23(1H，d，J＝2.4Hz)，5.96(1H，d，J＝8.5Hz)，5.67－5.58(1H，m)，5.29－5.22(1H，m)，4.95－4.85(1H，m)，4.83－4.79(1H，m)，4.48－4.40(2H，m)，4.39－4.31(2H，m)，4.22－4.09(3H，m)，3.73－3.66(2H，m)，3.56－3.50(1H，m)，3.50－3.44(2H，m)，2.80－2.68(1H，m)，2.48－2.35(1H，m)，2.00－1.88(1H，m)，1.87－1.77(1H，m).

³¹P－NMR(CD₃OD)δ：57.6(s)，53.1(s).

(step 7-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 2-amino-1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Oxygen fiveHeterodiphosphocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

Using the compound (diastereomer 2) (127 mg: containing impurities) obtained in the above step 6, a reaction was carried out in the same manner as in the step 11 of example 1, and then purification was carried out under the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (20.1 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.20(1H，s)，8.02(1H，s)，7.19(1H，s)，6.31(1H，d，J＝6.0Hz)，6.05(1H，d，J＝8.5Hz)，5.62－5.52(1H，m)，5.47－5.40(1H，m)，4.80－4.75(1H，m)，4.51－4.47(1H，m)，4.47－4.21(5H，m)，4.16－4.09(1H，m)，3.99－3.89(2H，m)，3.84－3.78(2H，m)，3.52－3.46(2H，m)，2.93－2.83(1H，m)，2.82－2.72(1H，m)，2.03－1.92(2H，m).

³¹P－NMR(CD₃OD)δ：61.6(s)，59.6(s).

Example 38: synthesis of CDN28

N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dioxaneoxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-2-hydroxyacetamide

[ synthetic route ]

(step 1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-2- { [ 2- (2-hydroxyacetamide) ethyl]Amino } -9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (6.6mg) obtained in the step 8-2 of example 8, a reaction was carried out in the same manner as in the step 1-1 of example 7, and then purification was carried out under the following purification conditions, so as to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (2.9 mg).

MS(ESI)m/z：846(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.33(1H，s)，8.02(1H，s)，7.16(1H，s)，6.33(1H，d，J＝6.0Hz)，6.18(1H，d，J＝8.5Hz)，5.53－5.45(2H，m)，4.79(1H，t，J＝5.1Hz)，4.50－4.25(5H，m)，4.10(1H，d，J＝11.5Hz)，3.97(2H，s)，3.94－3.89(1H，m)，3.53－3.39(6H，m)，2.88(2H，t，J＝5.7Hz)，2.04－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：62.6，60.1.

Example 39: synthesis of CDN29

N- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } methyl) -2-hydroxyacetamide

[ synthetic route ]

(step 1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-hydroxyacetamide) methyl]-9H-purin-9-yl } -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l ][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound obtained in step 9-2 of example 11 (4.6mg), a reaction was carried out in the same manner as in step 1-1 of example 7, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min) to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (4.0 mg).

MS(ESI)m/z：817(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.75(1H，s)，8.02(1H，s)，7.12(1H，s)，6.36(1H，d，J＝8.5Hz)，6.33(1H，d，J＝6.7Hz)，5.49－5.41(2H，m)，4.80(1H，dd，J＝6.7，4.8Hz)，4.50－4.29(7H，m)，4.07(2H，s)，4.05－4.01(1H，m)，3.92－3.87(1H，m)，3.51－3.49(2H，m)，2.93－2.90(2H，m)，2.03－1.99(2H，m).

³¹P－NMR(CD₃OD)δ：63.2，60.3.

Example 40: synthesis of CDN30

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-2- { [ (1-aminocyclopropyl) methyl]Amino } -9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2-chloro-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] amide ]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 1) (590mg) obtained in step 6 of example 8, a reaction was carried out in the same manner as in step 10 of example 1 to obtain the title compound (420 mg).

MS(ESI)m/z：992(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.76(1H，s)，7.97(1H，s)，7.30(1H，s)，6.23(1H，d，J＝4.8Hz)，6.22(1H，d，J＝7.9Hz)，5.43－5.37(1H，m)，5.19－5.15(1H，m)，4.85－4.77(3H，m)，4.44－4.31(2H，m)，4.22(1H，brs)，4.08－4.00(2H，m)，3.52－3.48(2H，m)，3.14(12H，q，J＝7.3Hz)，2.84－2.81(2H，m)，2.02－1.92(2H，m)，1.26(18H，t，J＝7.3Hz)，1.00(9H，s)，0.85(9H，s)，0.33(3H，s)，0.28(3H，s)，0.27(3H，s)，0.10(3H，s).

(Process 1-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2-chloro-9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl ] amide]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 2) obtained in the step 6 of example 8 (710mg), a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (452 mg).

MS(ESI)m/z：992(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.55(1H，s)，8.01(1H，s)，7.07(1H，s)，6.34(1H，d，J＝7.3Hz)，6.20(1H，d，J＝8.5Hz)，5.52－5.44(1H，m)，5.38－5.36(1H，m)，5.17－5.10(1H，m)，4.98－4.95(2H，m)，4.67－4.57(2H，m)，4.25(1H，brs)，4.11－4.07(1H，m)，3.89－3.84(1H，m)，3.52－3.49(2H，m)，3.18(12H，q，7.3Hz)，2.93－2.91(2H，m)，2.03－1.99(2H，m)，1.30(18H，t，J＝7.3Hz)，1.00(9H，s)，0.74(9H，s)，0.27(6H，s)，0.20(3H，s)，－0.28(3H，s).

(step 2-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ (1-aminocyclopropyl) methyl ]Amino } -9H-purin-9-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a solution of 1- (aminomethyl) cyclopropane-1-amine-2 HCl (309mg) in methanol (40mL) was added MP-Carbonate resin (5.45g), and the mixture was stirred at room temperature for 2 hours. The resin was removed by filtration, and the filtrate was concentrated under reduced pressure. A methanol (0.837mL) solution of the residue was added to the compound (30.0mg) obtained in the above step 1-1, and the mixture was reacted at 120 ℃ for 4 hours using a microwave reaction apparatus. After the reaction solution was concentrated under reduced pressure, the residue was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: from 30% to 60% (0 part to 30 parts) to give a mixture containing the title compound. The obtained mixture was used as it is in the next step.

MS(ESI)m/z：1042(M+H)⁺.

(step 2-2)

Using the compound (58.6mg) obtained in the above step 1-2, a mixture containing the title compound was obtained in the same manner as in the above step 2-1. The resulting mixture was used directly in the next reaction.

MS(ESI)m/z：1042(M+H)⁺.

(step 3-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-2- { [ (1-aminocyclopropyl) methyl]Amino } -9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the mixture obtained in the above step 2-1, a reaction was carried out in the same manner as in step 11 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (2.0 mg).

MS(ESI)m/z：812(M－2Na+1H)^－.

¹H－NMR(CD₃OD)δ：8.25(1H，s)，8.01(1H，s)，7.04(1H，s)，6.28(1H，d，J＝4.2Hz)，6.12(1H，d，J＝7.9Hz)，5.43－5.35(1H，m)，5.15－5.11(1H，m)，4.75(1H，t，J＝4.2Hz)，4.65－4.56(1H，m)，4.49－4.43(2H，m)，4.37－4.31(2H，m)，4.15－4.01(2H，m)，3.73－3.59(1H，m)，3.50－3.47(2H，m)，3.22－3.15(1H，m)，2.85－2.68(2H，m)，2.00－1.93(2H，m)，0.87－0.80(4H，m).

³¹P－NMR(CD₃OD)δ：57.7(s)，54.3(s).

(step 3-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS,16R) -7- (6-amino-2- { [ (1-aminocyclopropyl) methyl]Amino } -9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the mixture obtained in the above step 2-2, a mixture containing the title compound was obtained in the same manner as in step 11 of example 1. The resulting mixture was used directly in the next reaction.

(step 4)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- [ 6-amino-2- ({ [ 1- ({ [ 2- (trimethylsilyl) ethoxy ] ethoxy]Carbonyl } amino) cyclopropyl]Methyl } amino) -9H-purin-9-yl]-15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Triethylamine (40.8. mu.L) and 2, 5-dimonoxypyrrolidin-1-yl (2- (trimethylsilyl) ethyl) carbonate (39.5mg) were added to a solution of the mixture obtained in the above step 3-2 in N, N-dimethylformamide (1.0mL), and the mixture was stirred at room temperature for 1 hour. After quenching the reaction mixture by adding water thereto, the reaction mixture was quenched by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% to 50% (0 part to 30 parts) was purified to obtain the title compound (11.0 mg).

MS(ESI)m/z：958(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.40(1H，s)，8.03(1H，s)，7.12(1H，s)，6.32(1H，d，J＝6.7Hz)，6.18(1H，d，J＝8.5Hz)，5.53－5.49(1H，m)，5.46－5.39(1H，m)，4.83(1H，dd，J＝6.3，4.5Hz)，4.53－4.30(4H，m)，4.26－4.24(1H，m)，4.13－4.08(2H，m)，4.04－4.00(1H，m)，3.93－3.88(1H，m)，3.60(1H，d，J＝13.9Hz)，3.52－3.49(2H，m)，3.40(1H，d，J＝13.9Hz)，3.04(12H，q，7.3Hz)，2.93－2.90(2H，m)，2.04－1.99(2H，m)，1.23(18H，t，J＝7.3Hz)，0.96－0.92(2H，m)，0.84－0.80(2H，m)，0.76－0.73(2H，m)，0.02(9H，s).

(step 5)

(diastereomer 2)

A tetrahydrofuran solution (about 1M, 237. mu.L) of tetrabutylammonium fluoride was added to a tetrahydrofuran (474. mu.L) solution of the compound (11.0mg) obtained in the above step 4, and the mixture was stirred at 40 ℃ for 3 hours under a nitrogen atmosphere. After the reaction mixture was quenched by adding 10mM aqueous triethylammonium acetate, the reaction mixture was quenched by preparative HPLC [10mM aqueous triethylammonium acetate/acetonitrile, acetonitrile: 2% to 30% (0 min to 30 min) and Sep-Pak (registered trademark) C18[ water/acetonitrile/0.1% triethylamine ] to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (4.1 mg).

MS(ESI)m/z：812(M－2Na+1H)^－.

¹H－NMR(CD₃OD)δ：8.34(1H，s)，8.01(1H，s)，7.14(1H，s)，6.32(1H，d，J＝6.0Hz)，6.16(1H，d，J＝8.5Hz)，5.43－5.37(2H，m)，4.78(1H，t，J＝5.4Hz)，4.50－4.28(5H，m)，4.12－4.08(1H，m)，3.95－3.89(1H，m)，3.69－3.64(1H，m)，3.51－3.48(2H，m)，3.26(1H，d，J＝14.5)，2.89－2.86(2H，m)，2.03－1.98(2H，m)，0.83－0.79(4H，m).

³¹P－NMR(CD₃OD)δ：62.3(s)，60.0(s).

Example 41: synthesis of CDN31

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 2- (hydroxymethyl) -6- (methylamino) -9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 2- (hydroxymethyl) -6- (methylamino) -9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-dione was added to a methanol (1.00mL) solution of diastereomer 2 (high polarity) (56.5mg) obtained in step 7 of example 12 in 40% aqueous methylamine (1.00mL), and the mixture was stirred in a sealed tube at 60 ℃ for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a mixture containing the title compound. The resulting mixture was used directly in the next reaction.

MS(ESI)m/z：1002(M+H)⁺.

(step 2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-7- [ 2- (hydroxymethyl) -6- (methylamino) -9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the mixture obtained in the above step 1, a reaction was carried out in the same manner as in step 11 of example 1, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -20% (0 min-30 min) to obtain the title compound in the form of triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (16.0 mg).

MS(ESI)m/z：774(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.70(1H，s)，8.02(1H，s)，7.13(1H，s)，6.38(1H，d，J＝9.1Hz)，6.33(1H，d，J＝7.3Hz)，5.49－5.42(2H，m)，4.80(1H，dd，J＝6.7，4.2Hz)，4.60(2H，s)，4.50－4.28(5H，m)，4.05－4.00(1H，m)，3.92－3.87(1H，m)，3.52－3.49(2H，m)，3.14(3H，brs)，2.91(2H，t，J＝5.4Hz)，2.04－1.99(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，60.3(s).

Example 42: synthesis of CDN32

(5R, 7R, 8R, 12aR, 14R, 15aS, 16R) -16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

5- (3, 3-diethoxyprop-1-yn-1-yl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-amine

To a solution of commercially available (PharmaBlock) 5-iodo-7H-pyrrolo [2, 3-d ] pyrimidin-4-amine (22g) in N, N-dimethylformamide (70mL) were added copper iodide (1.61g), bis (triphenylphosphine) palladium dichloride (5.94g), and triethylamine (35 mL). Propargyl propionaldehyde diethyl acetal (22mL) was added over 2 hours and stirred at room temperature overnight. Chloroform (350mL) was added to the reaction solution, and the mixture was washed with water 2 times. The organic layer was dried over magnesium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate (350mL) was added to the residue and stirred overnight. The precipitated solid was filtered to give the title compound (9.60 g).

MS(ESI)m/z：261(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.04(1H，brs)，8.11(1H，brs)，7.57(1H，s)，6.56(2H，brs)，5.59(1H，s)，3.68(2H，m)，3.57(2H，m)，1.17(6H，t，J＝7.3Hz).

(step 2)

5- (3, 3-diethoxypropyl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-amine

10% Palladium on carbon (M) wet (25.0g) was added to a tetrahydrofuran (160mL) -ethanol (80mL) mixed solution of the compound (17.9g) obtained in the above step 1, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The catalyst was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to give the title compound (17.0g) as a crude product.

MS(ESI)m/z：265(M+H)⁺.

(step 3)

6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

The compound (50.21g) obtained in step 2 was dissolved in 90% aqueous acetic acid (344mL) and stirred at 50 ℃ overnight. After confirming the disappearance of the starting material, palladium on carbon (M) wet (60g) was added to the reaction mixture, and the mixture was stirred overnight at 40 ℃ under a hydrogen atmosphere. The catalyst was removed by filtration through celite, and the filtrate was concentrated under reduced pressure. To the residue was added a saturated aqueous sodium bicarbonate solution (350mL), and the mixture was extracted 7 times with chloroform/methanol (9: 1). The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ chloroform/methanol ] to give the title compound (18.47 g).

MS(ESI)m/z：175(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：11.26(1H，brs)，7.97(1H，s)，7.37(1H，brs)，6.86(1H，brs)，3.35(2H，m)，2.80(2H，t，J＝5.4Hz)，1.88(2H，m).

(step 4)

Phenyl (2, 7, 8, 9-tetrahydro-6H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-6-yl) methanone

Dehydrated pyridine (39.2mL), N-dimethylaminopyridine (2.38g) and benzoyl chloride (22.6mL) were added in this order to a suspension of the compound (8.47g) obtained in step 3 in dichloromethane (120mL), and the mixture was stirred at room temperature for 1 hour. After the reaction mixture was concentrated under reduced pressure, chloroform (150mL), methanol (60mL) and triethylamine (50mL) were added to the residue, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was poured into two layers of chloroform and water, and extracted with chloroform. The organic layer was washed 2 times with 25 w/v% aqueous potassium hydrogensulfate solution and dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. To the residue were added ethyl acetate (50mL) and hexane (125mL) in this order to prepare a slurry, and the mixture was stirred for 1 hour. The precipitated solid was filtered to obtain the title compound (9.89 g).

MS(ESI)m/z：279(M+H)⁺.

¹H－NMR(CDCl₃)δ：10.07(1H，brs)，8.11(1H，s)，7.39－7.21(5H，m)，7.12(1H，s)，4.32(2H，m)，3.06(2H，m)，2.26(2H，m).

(step 5)

6-benzoyl-2- [ 2-deoxy-3, 5-bis-O- (4-methylbenzoyl) - β -D-erythro-pentofuranosyl ] -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a suspension of the compound (7.10g) obtained in the above step 4 in acetonitrile (80mL) was added potassium hydroxide (2.9g) and tris [ 2- (2-methoxyethoxy) ethyl ] amine (0.41mL) in the form of powder under a nitrogen atmosphere, and the mixture was stirred at room temperature for 15 minutes. Under ice-cooling, 2-deoxy-3, 5-bis-O- (4-methylbenzoyl) - α -D-erythro-pentofuranosyl chloride (10.12g) known in the literature (Synlett 2004 (2): 335-337) and acetonitrile (60mL) were added, and the mixture was warmed to room temperature and stirred overnight. The reaction solution was added with a saturated aqueous ammonium chloride solution to stop the reaction. The precipitated solid was filtered, and washed with water to give the title compound (9.70 g).

MS(ESI)m/z：631(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.10(1H，s)，8.00－7.96(4H，m)，7.37－7.22(9H，m)，7.10(1H，s)，6.88(1H，dd，J＝8.5，5.4Hz)，5.76(1H，m)，4.77(1H，dd，J＝11.8，3.9Hz)，4.65－4.57(2H，m)，4.32(1H，m)，4.20(1H，m)，2.90－2.78(3H，m)，2.73(1H，ddd，J＝2.1，5.7，8.5Hz)，2.44(6H，s)，2.19(2H，m).

(step 6)

6-benzoyl-2- (2-deoxy-beta-D-erythro-pentofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a mixed solution of the compound (8.69g) obtained in the above step 5 in methanol (45mL) and tetrahydrofuran (135mL) was added a 2N aqueous solution of sodium hydroxide (27.6mL) at-10 ℃ for 20 minutes. After stirring at the same temperature for 2 hours, 1N hydrochloric acid (58mL) was added to stop the reaction. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ chloroform/methanol ] to give the title compound (4.09 g).

MS(ESI)m/z：395(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.04(1H，s)，7.39－7.23(5H，m)，7.05(1H，s)，6.27(1H，dd，J＝9.7，5.4Hz)，6.00(1H，d，J＝10.9Hz)，4.77(1H，d，J＝4.8Hz)，4.41(1H，dd，J＝14.5，7.9Hz)，4.19(1H，s)，4.15(1H，dd，J＝14.5，7.9Hz)，3.94(1H，d，J＝12.7Hz)，3.74(1H，m)，3.16－2.95(3H，m)，2.30－2.14(3H，m)，1.96(1H，s).

(step 7)

6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-deoxy- β -D-erythro-pentofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound (4.55g) obtained in the above step 6, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (6.38 g).

MS(ESI)m/z：697(M+H)⁺.

¹H－NMR(CDCl₃) δ: 8.08(1H, s), 7.43(2H, d, J ═ 7.9Hz), 7.36-7.19 (13H, m), 6.84-6.76 (5H, m), 4.66(1H, brs), 4.31(1H, m), 4.21(1H, m), 4.07(1H, m), 3.79(6H, s), 3.44(1H, dd, J ═ 10.0, 3.9Hz), 3.38(1H, dd, J ═ 10.3, 4.8Hz), 2.85(2H, t, J ═ 6.3Hz), 2.66(1H, m), 2.47(1H, ddd, J ═ 4.1, 6.5, 13.9Hz), 2.18(2H, m) (only the peaks observed)

(step 8)

6-benzoyl-2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -2-deoxy- β -D-erythro-pentofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

The reaction was carried out in the same manner as in step 6 of example 1 using the compound obtained in step 7 (6.37g) to obtain the title compound (6.05g) as a diastereomer mixture (diastereomer ratio: 1).

¹H－NMR(CDCl₃)δ：8.09(0.5H，s)，8.08(0.5H，s)，7.46－7.41(2H，m)，7.35－7.19(13H，m)，6.84－6.75(5H，m)，4.82－4.76(1H，m)，4.35－4.19(3H，m)，3.89－3.54(4H，m)，3.79(1.5H，s)，3.79(1.5H，s)，3.78(1.5H，s)，3.78(1.5H，s)，3.42(1H，td，J＝9.8，3.8Hz)，3.38－3.30(1H，m)，2.81(2H，t，J＝6.3Hz)，2.74－2.66(1H，m)，2.63－2.49(1H，m)，2.62(1H，t，J＝6.0Hz)，2.45(1H，t，J＝6.3Hz)，2.21－2.14(2H，m)，1.20－1.17(9H，m)，1.11(3H，d，J＝6.7Hz).

(step 9)

Using the compound (868mg) obtained in the above step 8, a reaction was carried out in the same manner as in step 7 of example 1 to obtain 6-benzoyl-2- { 2-deoxy-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]- β -D-erythro-pentofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Acetonitrile solution of azulene. Using this acetonitrile solution and the compound (1.00g) obtained in step 3 of example 22, a reaction was carried out in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(Process 10)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15aS, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 9, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (702mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1134(M+H)⁺.

(step 11)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15aS, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (702mg) obtained in the above step 10, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(119 mg: containing impurities) and diastereomer 2(113 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：873(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：873(M+H)⁺.

(step 12-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15aS, 16R) -16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (119 mg: containing impurities) obtained in the above step 11 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (51.8 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.69(1H，s)，8.24(1H，s)，8.02(1H，s)，7.09(1H，s)，6.68(1H，t，J＝7.0Hz)，6.29(1H，d，J＝8.5Hz)，5.36－5.27(2H，m)，4.75－4.71(1H，m)，4.41－4.30(3H，m)，4.28－4.12(3H，m)，4.06－4.00(1H，m)，3.94－3.84(1H，m)，3.82(2H，t，J＝4.8Hz)，3.52－3.47(2H，m)，2.91－2.69(4H，m)，2.04－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：57.3(s)，54.9(s).

(step 12-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15aS, 16R) -16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The compound (diastereomer 2) (113 mg: containing impurities) obtained in the above step 11 was reacted by the same method as in the step 11 of example 1, and then purified under the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (65.4 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.78(1H，s)，8.24(1H，s)，8.02(1H，s)，7.08(1H，s)，6.73(1H，dd，J＝9.1，5.4Hz)，6.29(1H，d，J＝8.5Hz)，5.59－5.52(1H，m)，5.45－5.37(1H，m)，4.45(1H，d，J＝4.2Hz)，4.40－4.16(6H，m)，4.01(1H，d，J＝12.7Hz)，3.87－3.75(3H，m)，3.53－3.46(2H，m)，2.93－2.88(2H，m)，2.87－2.65(2H，m)，2.06－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，57.3(s).

Example 43: synthesis of CDN33

(5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

1- (2, 7, 8, 9-tetrahydro-6H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-6-yl) ethane-1-one

The compound (6.88g) obtained in step 3 of example 42 was dissolved in acetic anhydride (48mL) and stirred at 90 ℃. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in a mixture of chloroform (100mL) -methanol (50mL) -triethylamine (30 mL). After stirring at room temperature for 4 hours, the reaction mixture was poured into two layers of chloroform and water, and extracted with chloroform. The organic layer was dried over magnesium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. After hexane/ethyl acetate (1: 2) was added to the residue to make a slurry, the solid was filtered to give the title compound (7.18 g).

MS(ESI)m/z：217(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.47(1H，s)，7.21(1H，s)，4.11(2H，d，J＝8.5Hz)，2.97(2H，t，J＝6.3Hz)，2.46(3H，s)，2.06(2H，m).

(step 2)

6-acetyl-2- (3, 5-di-O-benzoyl-2-deoxy-2-fluoro- β -D-arabinofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound obtained in the above step 1 (6.00g) and commercially available [ (2R, 3R, 4S, 5R) -3-benzoyloxy-5-bromo-4-fluoro-tetrahydro-furan-2-yl ] methylbenzoate (14.1g) (CARBOSYTH), a reaction was carried out in the same manner as in the step 5 of example 42 to obtain the title compound (11.45 g).

MS(ESI)m/z：559(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.58(1H，s)，8.12(4H，t，J＝7.3Hz)，7.69－7.44(6H，m)，7.28(1H，d，J＝2.4Hz)，6.92(1H，dd，J＝23.3，2.7Hz)，5.77(1H，dd，J＝17.5，3.0Hz)，5.34(1H，dd，J＝50.2，3.0Hz)，4.83(2H，dd，J＝11.8，4.5Hz)，4.76(2H，dd，J＝11.8，5.1Hz)，4.56(1H，m)，4.14(2H，m)，2.90(2H，t，J＝6.7Hz)，2.07(3H，s).

(step 3)

6-acetyl-2- (2-deoxy-2-fluoro- β -D-arabinofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a mixed solution of the compound (8.70g) obtained in the above step 2 in methanol (58mL) -tetrahydrofuran (117mL) was added dropwise a 2N aqueous sodium hydroxide solution (32mL) at-20 ℃ for 12 minutes. After stirring at the same temperature for 3 hours, the reaction was stopped by adding 1N hydrochloric acid (66 mL). The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ chloroform/methanol ] to give the title compound (3.27 g).

MS(ESI)m/z：351(M+H)⁺.

¹H－NMR(CDCl₃) δ: 8.54(1H, s), 7.20(1H, d, J ═ 1.8Hz), 6.68(1H, dd, J ═ 17.5, 4.2Hz), 5.16(1H, ddd, J ═ 52.0, 2.4, 1.2Hz), 4.75(1H, ddd, J ═ 19.2, 2.6, 1.3Hz), 4.16(1H, m), 4.08(2H, m), 3.99(1H, dd, J ═ 12.1, 3.6Hz), 3.92(1H, dd, J ═ 12.1, 4.2Hz), 2.93(2H, m), 2.52(3H, s), 2.07(2H, m). (only the peaks that can be observed are described)

(step 4)

6-acetyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-deoxy-2-fluoro- β -D-arabinofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound (3.95g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (5.66 g).

MS(ESI)m/z：653(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.56(1H，s)，7.50－7.20(10H，m)，6.85－6.78(5H，m)，5.07(1H，dt，J＝51.8，3.2Hz)，4.59(1H，brd，J＝18.1Hz)，4.21－4.02(3H，m)，3.80(3H，s)，3.79(3H，s)，3.50(1H，dd，J＝10.3，5.4Hz)，3.44(1H，dd，J＝10.0，5.1Hz)，2.88(2H，m)，2.54(3H，s)，2.42(1H，brs)，2.07(2H，m).

(step 5)

6-acetyl-2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -2-deoxy-2-fluoro- β -D-arabinofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

The reaction was carried out in the same manner as in step 6 of example 1 using the compound obtained in step 4 (5.68g) to obtain the title compound (5.08g) in the form of a diastereomer mixture (diastereomer ratio: 1) on the phosphorus atom.

MS(ESI)m/z：853(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.58(0.5H，s)，8.57(0.5H，s)，7.51－7.20(10H，m)，6.85－6.76(5H，m)，5.24－5.02(1H，m)，4.76－4.60(1H，m)，4.21－4.05(3H，m)，3.91－3.74(1H，m)，3.80(1.5H，s)，3.79(1.5H，s)，3.79(1.5H，s)，3.79(1.5H，s)，3.69－3.55(3H，m)，3.49－3.37(2H，m)，2.95－2.80(2H，m)，2.61(1H，t，J＝6.3Hz)，2.54(3H，s)，2.43(1H，t，J＝6.7Hz)，2.11－2.03(2H，m)，1.21－1.17(9H，m)，1.11(3H，d，J＝7.3Hz).

(step 6)

Using the compound (1.00g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 6-acetyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]- β -D-arabinofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The reaction was carried out in the same manner as in step 8 of example 1 using the acetonitrile solution and the compound (1.21g) obtained in step 3 of example 22, and the obtained crude product was used as it was in the next reaction.

(step 7)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -14- (6-acetyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] b ]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 6, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (757mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1090(M+H)⁺.

(step 8)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (757mg) obtained in the above step 7, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(113 mg: containing impurities) and diastereomer 2(108 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：891(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：891(M+H)⁺.

(step 9-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (113 mg: containing impurities) obtained in the above step 8 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (28.8 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.67(1H，s)，8.24(1H，s)，8.04(1H，s)，7.05(1H，s)，6.73(1H，dd，J＝23.9，2.7Hz)，6.28(1H，d，J＝8.5Hz)，5.43－5.24(3H，m)，4.77－4.72(1H，m)，4.51－4.32(4H，m)，4.26－4.12(2H，m)，4.06－3.91(2H，m)，3.82(2H，t，J＝5.1Hz)，3.50(2H，t，J＝5.1Hz)，2.92－2.85(2H，m)，2.06－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s)，54.7(s).

(step 9-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (108 mg: containing impurities) obtained in the above step 8 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (3.2 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.76(1H，s)，8.23(1H，s)，8.04(1H，s)，7.06(1H，s)，6.73(1H，dd，J＝24.5，2.1Hz)，6.28(1H，d，J＝8.5Hz)，5.53－5.45(1H，m)，5.43－5.27(2H，m)，4.62－4.49(1H，m)，4.45－4.41(1H，m)，4.40－4.27(2H，m)，4.27－4.15(3H，m)，4.03－3.92(2H，m)，3.87－3.78(2H，m)，3.51(2H，t，J＝5.6Hz)，2.90(2H，t，J＝5.6Hz)，2.08－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：63.0(s)，57.8(s).

Example 44: synthesis of CDN34

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl ]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a dichloromethane (322mL) -pyridine (35mL) mixed solution of the compound (35.80g) obtained in step 4 of example 1, a hydrogen fluoride-pyridine (6.33g) solution in dichloromethane (36mL) was added under ice-cooling for 5 minutes, and the mixture was stirred at the same temperature for 3 hours. To the reaction mixture were added a saturated aqueous sodium bicarbonate solution (268mL) and a saturated brine (143mL) in this order to stop the reaction, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Hexane/ethyl acetate (1: 1) (108mL) was added to the residue to prepare a slurry, which was then stirred at 50 ℃ for 30 minutes, followed by addition of hexane (161mL) and further stirring for 2 hours. The precipitated solid was filtered and washed with hexane/ethyl acetate (4: 1) (143mL) to give the title compound (26.81 g).

MS(ESI)m/z：525(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：7.98(1H，s)，7.65(1H，s)，7.39(1H，m)，7.26－7.20(4H，m)，6.19(1H，d，J＝6.5Hz)，5.15(1H，t，J＝5.6Hz)，5.00(1H，d，J＝4.8Hz)4.48(1H，t，J＝5.6Hz)，4.27(1H，m)，4.11－4.02(2H，m)，3.97(1H，m)，3.67－3.57(2H，m)，2.99(2H，m)，2.23－2.07(2H，m)，0.68(9H，s)，－0.11(3H，s)，－0.26(3H，s).

(step 2)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3, 5-bis-O- (tetrahydropyran (Oxane) -2-yl) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (19.93g) obtained in the above step 1 and 3, 4-dihydro-2H-pyran (35mL) in N, N-dimethylformamide (200mL) was added p-toluenesulfonic acid monohydrate (7.25g) under ice cooling, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate under ice-cooling, and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (24.73 g).

¹H－NMR(CDCl₃)δ：8.10－8.07(1H，m)，7.59－7.35(1H，m)，7.35－7.27(3H，m)，7.25－7.17(2H，m)，6.44－6.36(1H，m)，4.90－3.36(13H，m)，3.06－2.96(2H，m)，2.31－2.15(2H，m)，2.01－1.43(12H，m)，0.84－0.73(9H，m)，0.04－(－0.35)(6H，m).

(step 3)

6-benzoyl-2- [3, 5-bis-O- (tetrahydropyran-2-yl) - β -D-ribofuranosyl ] -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (24.73g) obtained in the above step 2 and acetic acid (3.1mL) in tetrahydrofuran (250mL) was added a tetrabutylammonium fluoride solution in tetrahydrofuran (about 1M, 55mL) under ice-cooling, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue, followed by washing with water and saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (18.74 g).

¹H－NMR(CDCl₃)δ：8.12－8.09(1H，m)，7.49－7.30(4H，m)，7.28－7.20(2H，m)，6.41－6.30(1H，m)，4.83－4.18(7H，m)，4.12－3.50(7H，m)，3.06－2.97(2H，m)，2.31－2.17(2H，m)，1.96－1.47(12H，m).

(step 4)

6-benzoyl-2- [3, 5-bis-O- (tetrahydropyran-2-yl) - β -D-arabinofuranosyl ] -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (18.74g) obtained in the above step 3 and pyridine (13.1mL) in dichloromethane (300mL) was added dropwise trifluoromethanesulfonic anhydride (11mL) under ice-cooling, and the mixture was stirred for 10 minutes. The reaction mixture was quenched by addition of saturated brine, extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (300mL), and a solution of tetrabutylammonium nitrite (28.34g) in tetrahydrofuran (150mL) was added dropwise under ice-cooling, followed by stirring at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue, followed by washing with water and saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (10.46 g).

¹H－NMR(CDCl₃)δ：8.13－8.06(1H，m)，7.63－7.30(4H，m)，7.29－7.18(2H，m)，6.79－6.55(1H，m)，4.93－3.45(14H，m)，3.11－2.95(2H，m)，2.32－2.14(2H，m)，1.98－1.44(12H，m).

(step 5)

6-benzoyl-2- [ 2-deoxy-2-fluoro-3, 5-bis-O- (tetrahydropyran-2-yl) - β -D-ribofuranosyl ] -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (10.46g) obtained in the above step 4 and pyridine (7.3mL) in dichloromethane (200mL) was added dropwise trifluoromethanesulfonic anhydride (6.1mL) under ice-cooling, and the mixture was stirred for 10 minutes. The reaction mixture was quenched by addition of saturated brine, extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (200mL), and a tetrabutylammonium fluoride solution in tetrahydrofuran (about 1M, 150mL) was added under ice-cooling, followed by stirring at the same temperature for 3 hours. Saturated aqueous ammonium chloride was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered off the drying agent, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (7.65 g).

¹H－NMR(CDCl₃)δ：8.13－8.08(1H，m)，7.53－7.31(4H，m)，7.26－7.22(2H，m)，6.68－6.53(1H，m)，5.42－5.08(1H，m)，4.93－4.18(6H，m)，4.10－3.76(3H，m)，3.71－3.47(3H，m)，3.06－2.96(2H，m)，2.29－2.18(2H，m)，1.96－1.47(12H，m).

(step 6)

6-benzoyl-2- (2-deoxy-2-fluoro- β -D-ribofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

To a solution of the compound (7.65g) obtained in the above step 5 in ethanol (150mL) was added pyridinium p-toluenesulfonate (6.62g), and the mixture was stirred at 50 ℃ for 3 hours. The reaction mixture was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (3.55 g).

¹H－NMR(CDCl₃)δ：8.05(1H，s)，7.41－7.35(3H，m)，7.30－7.24(2H，m)，7.06(1H，s)，6.07－6.00(2H，m)，5.85(1H，ddd，J＝52.8，6.7，4.7Hz)，4.66(1H，d，J＝3.9Hz)，4.42－4.31(2H，m)，4.20(1H，m)，3.93(1H，dd，J＝12.9，1.6Hz)，3.74(1H，td，J＝12.3，1.6Hz)，3.12－2.96(2H，m)，2.51(1H，s)，2.33－2.15(2H，m).

(step 7)

6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-deoxy-2-fluoro- β -D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound (3.55g) obtained in the above step 6, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (5.77 g).

¹H－NMR(CDCl₃)δ：8.09(1H，s)，7.45－7.41(2H，m)，7.36－7.17(13H，m)，6.85－6.79(4H，m)，6.53(1H，dd，J＝17.2，2.3Hz)，5.40(1H，ddd，J＝53.2，4.8，2.3Hz)，4.83－4.72(1H，m)，4.32－4.21(2H，m)，4.19－4.14(1H，m)，3.79(3H，s)，3.79(3H，s)，3.59(1H，dd，J＝11.0，2.7Hz)，3.45(1H，dd，J＝11.0，3.5Hz)，2.79(2H，t，J＝6.3Hz)，2.45(1H，s)，2.24－2.11(2H，m).

(step 8)

6-benzoyl-2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -2-deoxy-2-fluoro-. beta. -D-ribofuranosyl) -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

The reaction was carried out in the same manner as in step 6 of example 1 using the compound obtained in step 7 (5.77g) above to obtain the title compound (5.95g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

¹H－NMR(CDCl₃)δ：8.10(0.5H，s)，8.09(0.5H，s)，7.45－7.12(15H，m)，6.84－6.75(4H，m)，6.57－6.46(1H，m)，5.61－5.33(1H，m)，5.07－4.83(1H，m)，4.34－4.18(3H，m)，3.93－3.72(7H，m)，3.69－3.49(4H，m)，3.38－3.27(1H，m)，2.87－2.68(2H，m)，2.61(1H，td，J＝6.3，1.6Hz)，2.40(1H，td，J＝6.4，2.1Hz)，2.21－2.12(2H，m)，1.21－1.13(9H，m)，1.03(3H，d，J＝6.7Hz).

(step 9)

Using the compound (1.02g) obtained in the above step 8, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ] ⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (1.15g) obtained in step 3 of example 22 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(Process 10)

2－{9－[(5R，7R，8R，12aR，14R，15R，15aR，16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 9, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (818 mg: containing impurities) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1152(M+H)⁺.

(step 11)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl ]Oxy } -15-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (818mg) obtained in the above step 10, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(107 mg: containing impurities) and diastereomer 2(101 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：891(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：891(M+H)⁺.

(step 12-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (107 mg: containing impurities) obtained in the above step 11 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (29.1 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，m)，8.11(1H，m)，8.03(1H，s)，7.11(1H，s)，6.47(1H，d，J＝17.5Hz)，6.26(1H，d，J＝8.5Hz)，5.53－5.36(2H，m)，5.29－5.17(1H，m)，4.77(1H，d，J＝4.2Hz)，4.54－4.46(1H，m)，4.44－4.38(1H，m)，4.35－4.32(1H，m)，4.30－4.25(2H，m)，4.25－4.16(1H，m)，4.06－3.99(1H，m)，3.96－3.85(1H，m)，3.82－3.71(2H，m)，3.54－3.42(2H，m)，2.77－2.68(1H，m)，2.66－2.55(1H，m)，2.02－1.81(2H，m).

³¹P－NMR(CD₃OD)δ：57.5(s)，53.0(s).

(step 12-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (101 mg: containing impurities) obtained in the above step 11 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (11.2 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.61(1H，m)，8.16(1H，m)，8.02(1H，m)，7.36(1H，s)，6.49(1H，dd，J＝16.0，2.1Hz)，6.28(1H，d，J＝8.5Hz)，5.56－5.33(3H，m)，4.58－4.49(2H，m)，4.45－4.37(2H，m)，4.31－4.27(1H，m)，4.25－4.16(1H，m)，4.10－3.98(3H，m)，3.80(2H，t，J＝5.1Hz)，3.48(2H，dd，J＝6.7，3.6Hz)，2.90－2.72(2H，m)，2.00－1.90(2H，m).

³¹P－NMR(CD₃OD)δ：59.5(s)，57.7(s).

Example 45: synthesis of CDN35

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15-fluoro-16-hydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd [)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2λ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] inosine

To a suspension of commercially available (Aamdis Chemical) 5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine (13.0g) in N, N-dimethylacetamide (60mL) were added N- (2-bromoethyl) phthalimide (7.02g) and 1, 8-diazabicyclo [5.4.0] -7-undecene (4.1mL), and the mixture was stirred at room temperature overnight. N- (2-bromoethyl) phthalimide (1.75g) and 1, 8-diazabicyclo [5.4.0] -7-undecene (1.1mL) were added thereto, and the mixture was stirred for additional 1 day. Water was added to the reaction solution to stop the reaction, followed by extraction with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ ethyl acetate/methanol ] to give the title compound (12.4 g).

¹H－NMR(CDCl₃)δ：7.83(1H，s)，7.76－7.67(4H，m)，7.64(1H，s)，7.35－7.33(2H，m)，7.25－7.11(7H，m)，6.74－6.70(4H，m)，5.93(1H，d，J＝5.1Hz)，5.68(1H，d，J＝3.9Hz)，4.71(1H，q，J＝4.8Hz)，4.43(1H，m)，4.37－4.18(3H，m)，4.10－4.06(2H，m)，3.730(3H，s)，3.728(3H，s)，3.51(1H，m)，3.36(1H，dd，J＝10.6，3.9Hz)，3.32(1H，dd，J＝11.0，5.5Hz).

(step 2)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -1- [ 2- (1, 3-di-oxo-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] inosine

Using the compound (12.4g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (4.18g) and 5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] inosine (6.31g), which is a positional isomer of the title compound.

¹H－NMR(CDCl₃)δ：8.00(1H，s)，7.82－7.77(2H，m)，7.74(1H，s)，7.72－7.67(2H，m)，7.41－7.39(2H，m)，7.32－7.19(7H，m)，6.83－6.78(4H，m)，5.90(1H，d，J＝5.1Hz)，4.53－4.41(3H，m)，4.32－4.25(1H，m)，4.19－4.11(3H，m)，3.79(3H，s)，3.78(3H，s)，3.46(1H，dd，J＝10.6，3.1Hz)，3.24(1H，dd，J＝10.8，4.1Hz)，2.98(1H，d，J＝6.7Hz)，0.85(9H，s)，0.04(3H，s)，－0.03(3H，s).

Positional isomers (2' -O-TBS body)

¹H－NMR(CDCl₃)δ：7.97(1H，s)，7.82－7.78(2H，m)，7.73－7.69(2H，m)，7.66(1H，s)，7.44－7.41(2H，m)，7.33－7.18(7H，m)，6.81(4H，d，J＝7.8Hz)，5.91(1H，d，J＝5.9Hz)，4.82(1H，t，J＝5.5Hz)，4.43(1H，m)，4.34－4.23(3H，m)，4.18－4.08(2H，m)，3.79(6H，s)，3.46(1H，dd，J＝10.6，2.7Hz)，3.36(1H，dd，J＝10.6，3.5Hz)，2.70(1H，d，J＝3.1Hz)，0.83(9H，s)，－0.04(3H，s)，－0.19(3H，s).

(step 3)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -1- [ 2- (1, 3-di-oxo-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] inosine

The reaction was carried out in the same manner as in step 6 of example 1 using the compound obtained in step 2 (8.89g) to obtain the title compound (9.45g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

¹H－NMR(CDCl₃)δ：8.01(0.5H，s)，8.00(0.5H，s)，7.82－7.77(2H，m)，7.74(0.5H，s)，7.72－7.67(2.5H，m)，7.42(2H，d，J＝7.8Hz)，7.33－7.18(7H，m)，6.81(4H，d，J＝8.6Hz)，6.10(0.5H，d，J＝5.5Hz)，6.04(0.5H，d，J＝5.1Hz)，4.75(0.5H，m)，4.60(0.5H，m)，4.49－4.41(1H，m)，4.38－4.23(2H，m)，4.22－4.05(3H，m)，3.79(6H，s)，3.78－3.65(1H，m)，3.62－3.39(4H，m)，3.33－3.23(1H，m)，2.49(1H，t，J＝6.3Hz)，2.34(1H，t，J＝6.7Hz)，1.12－1.08(9H，m)，0.91(3H，d，J＝7.0Hz)，0.82(9H，s)，0.06(1.5H，s)，0.03(1.5H，s)，－0.03(3H，s).

(step 4)

Using the compound (1.30g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (1.50g) obtained in the above step 3 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 5)

3- { [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -7- { 1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-10-yl ]Oxy-propionitrile

Using the crude product obtained in the above step 4, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (828mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1177(M+H)⁺.

(step 6)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Hydrazine monohydrate (0.342mL) was added to a mixed solution of the compound (828mg) obtained in the above step 5 in ethanol (5.0mL) -tetrahydrofuran (5.0mL), and the mixture was stirred at 50 ℃ for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(90.9 mg: containing impurities) and diastereomer 2(91.1 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：890(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：890(M+H)⁺.

(step 7-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15-fluoro-16-hydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (90.9 mg: containing impurities) obtained in the above step 6 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (20.2 mg).

MS(ESI)m/z：776(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.54(1H，s)，8.03(1H，s)，7.99(1H，s)，7.13(1H，s)，6.44(1H，d，J＝18.1Hz)，6.22(1H，d，J＝7.9Hz)，5.56－5.38(2H，m)，5.33－5.21(1H，m)，4.72(1H，d，J＝4.2Hz)，4.58－4.49(1H，m)，4.41－4.24(4H，m)，4.24－4.17(1H，m)，4.05－3.98(1H，m)，3.86－3.76(1H，m)，3.50－3.42(2H，m)，3.27－3.16(2H，m)，2.78－2.68(1H，m)，2.59－2.49(1H，m)，1.98－1.80(2H，m).

³¹P－NMR(CD₃OD)δ：57.5(s)，53.1(s).

(step 7-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (91.1 mg: containing impurities) obtained in the above step 6 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (26.3 mg).

MS(ESI)m/z：776(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.56(1H，s)，8.08(1H，s)，8.02(1H，s)，7.37(1H，s)，6.48(1H，d，J＝16.2Hz)，6.23(1H，d，J＝7.9Hz)，5.58－5.32(3H，m)，4.65－4.27(6H，m)，4.07－3.96(3H，m)，3.48－3.42(2H，m)，3.38－3.23(2H，m)，2.85－2.75(1H，m)，2.69－2.59(1H，m)，1.99－1.81(2H，m).

³¹P－NMR(CD₃OD)δ：59.0(s)，57.6(s).

Example 46: synthesis of CDN36

N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-di-oxo-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) -2-hydroxyacetamide

[ synthetic route ]

(step 1-1)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -16-fluoro-15-hydroxy-7- { 1- [ 2- (2-hydroxyacetamide) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

Using the compound obtained in the step 7-1 of example 45 (15.0mg), a reaction was carried out in the same manner as in the step 1-1 of example 7, and then purification was carried out according to the following purification conditions, thereby obtaining the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (10.3 mg).

MS(ESI)m/z：834(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.46(1H，brm)，8.04(1H，s)，7.82(1H，brm)，7.15(1H，brm)，6.43(1H，d，J＝16.9Hz)，6.17(1H，dd，J＝9.1，4.5Hz)，5.70－5.24(3H，m)，4.81－4.75(1H，m)，4.52－4.44(1H，m)，4.43－4.26(4H，m)，4.24－3.94(2H，m)，3.89－3.84(2H，m)，3.72－3.37(5H，m)，2.75－2.65(1H，m)，2.48－2.32(1H，m)，1.98－1.76(2H，m).

³¹P－NMR(CD₃OD)δ：57.0(s)，53.0(s).

(Process 1-2)

(diastereomer 2)

Using the compound obtained in the step 7-2 of example 45 (15.0mg), a reaction was carried out in the same manner as in the step 1-1 of example 7, and then purification was carried out under the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (10.6 mg).

MS(ESI)m/z：834(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.54(1H，brs)，8.03(1H，s)，7.97(1H，brs)，7.35(1H，brs)，6.48(1H，d，J＝15.7Hz)，6.23(1H，d，J＝8.5Hz)，5.65－5.39(3H，m)，4.57－4.47(2H，m)，4.46－4.36(2H，m)，4.31－4.19(2H，m)，4.07－3.96(2H，m)，3.95－3.78(3H，m)，3.67－3.43(4H，m)，2.85－2.75(1H，m)，2.71－2.59(1H，m)，2.00－1.84(2H，m).

³¹P－NMR(CD₃OD)δ：59.1(s)，57.5(s).

Example 47: synthesis of CDN37

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15-fluoro-16-hydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2- [ (2-ammonioethyl) amino ] adenosine dichloride

To a solution of 2- ({ 2- [ (tert-butoxycarbonyl) amino ] ethyl } amino) adenosine (28.7g) in methanol (240mL) as known in the literature (WO2012/159072), a dioxane solution of hydrogen chloride (about 4M, 240mL) was added and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to about 50mL, and the precipitated solid was suspended in diethyl ether and filtered to give the title compound (28.8 g).

¹H－NMR(CD₃OD)δ：8.32(1H，s)，5.98(1H，d，J＝6.0Hz)，4.51(1H，t，J＝5.4Hz)，4.30(1H，dd，J＝5.1，3.3Hz)，4.14－4.11(1H，m)，3.86－3.72(4H，m)，3.23(2H，t，J＝6.0Hz).

(step 2)

2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Triethylamine (37mL) and 1- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } oxy) pyrrolidine-2, 5-dione (18.4g) were added to a tetrahydrofuran (330mL) -water (70mL) mixture of the compound (28.8g) obtained in the above step 1, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and azeotroped with toluene. The residue was suspended in toluene and the solid was filtered. The obtained solid was dissolved in methylene chloride and methanol, and concentrated under reduced pressure. Tetrahydrofuran was added to the residue, and the precipitated solid was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in methylene chloride and methanol, and concentrated under reduced pressure. The precipitated solid was suspended in methylene chloride and filtered to give the title compound (22.5 g).

¹H－NMR(CD₃OD)δ：7.92(1H，s)，5.83(1H，d，J＝6.0Hz)，4.77(1H，t，J＝5.4Hz)，4.33(1H，dd，J＝4.8，3.0Hz)，4.14－4.10(3H，m)，3.87(1H，dd，J＝12.4，2.7Hz)，3.73(1H，dd，J＝12.1，3.0Hz)，3.50－3.42(2H，m)，3.32－3.29(2H，m)，0.98－0.86(2H，m)，0.03(9H，s).

(step 3)

N-benzoyl-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Using the compound (24.7g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 3 of example 11 to obtain the title compound (22.1 g).

¹H－NMR(CD₃OD)δ：8.19(1H，s)，8.05(2H，d，J＝7.9Hz)，7.63(1H，t，J＝7.6Hz)，7.54(2H，t，J＝7.6Hz)，5.95(1H，d，J＝5.4Hz)，4.75(1H，t，J＝5.4Hz)，4.36(1H，t，J＝4.5Hz)，4.11－4.07(3H，m)，3.86(1H，dd，J＝12.4，3.3Hz)，3.75(1H，dd，J＝12.1，3.6Hz)，3.59－3.47(2H，m)，3.36－3.33(2H，m)，0.92(2H，t，J＝8.2Hz)，0.00(9H，s).

(step 4)

N-benzoyl-5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Using the compound (22.1g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (29.5 g).

¹H－NMR(DMSO－d₆)δ：10.7(1H，s)，8.09(1H，s)，8.01－7.99(2H，m)，7.64－7.60(1H，m)，7.52(2H，t，J＝7.6Hz)，7.36－7.34(2H，m)，7.26－7.17(7H，m)，7.05－7.02(1H，m)，6.95－6.92(1H，m)，6.85－6.80(4H，m)，5.88(1H，d，J＝4.8Hz)，5.53(1H，d，J＝5.4Hz)，5.20(1H，d，J＝5.4Hz)，4.72－4.63(1H，m)，4.34－4.26(1H，m)，4.05－3.99(3H，m)，3.72－3.71(6H，m)，3.31－3.10(6H，m)，0.89(2H，t，J＝8.5Hz)，0.00(9H，s).

(step 5)

N-benzoyl-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Using the compound (29.5g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 3 of example 5 to obtain the title compound (6.85 g).

¹H－NMR(DMSO－d₆，90℃)δ：10.3(1H，brs)，8.02(1H，s)，7.99(2H，d，J＝7.3Hz)，7.60(1H，t，J＝7.3Hz)，7.51(2H，t，J＝7.6Hz)，7.37(2H，d，J＝7.3Hz)，7.28－7.20(7H，m)，6.85－6.82(4H，m)，6.69－6.53(2H，m)，5.86(1H，d，J＝5.4Hz)，5.07－5.04(1H，m)，4.80－4.74(1H，m)，4.36－4.33(1H，m)，4.08－3.99(3H，m)，3.74(6H，s)，3.37－3.15(6H，m)，0.89(2H，t，J＝7.9Hz)，0.86(9H，s)，0.09(3H，s)，0.04(3H，s)，0.01(9H，s).

(step 6)

N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

The reaction was carried out in the same manner as in step 6 of example 1 using the compound obtained in step 5 (6.22g) to obtain the title compound (6.18g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

¹H－NMR(CD₃Cl)δ：8.82(1H，s)，8.00－7.97(2H，m)，7.88(0.5H，s)，7.85(0.5H，s)，7.59(1H，t，J＝7.0Hz)，7.51(2H，t，J＝7.6Hz)，7.43(2H，d，J＝7.3Hz)，7.33－7.20(7H，m)，6.81(4H，d，J＝8.5Hz)，6.08－6.01(1H，m)，5.19－4.88(2H，m)，4.51－4.42(1H，m)，4.20－4.08(3H，m)，3.82－3.24(11H，m)，3.78(6H，s)，2.52(1H，5，J＝6.3Hz)，2.38－2.34(1H，m)，1.14－1.09(9H，m)，0.90－0.85(2H，m)，0.87(9H，s)，0.12(1.5H，s)，0.09(1.5H，s)，0.03(3H，s)，－0.01(9H，s).

(step 7)

Using the compound (1.03g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ] ⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (999mg) obtained in the above step 6 were reacted in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 8)

2- (trimethylsilyl) ethyl [ 2- ({ 6-benzamide-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Carbamates, their preparation and their use

Using the mixture obtained in the above step 7, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain diastereomer 1(370 mg: containing impurities) and diastereomer 2(201 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1307(M－H)^－.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1307(M－H)^－.

(step 9-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 1) (370 mg: containing impurities) obtained in the above step 8, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (134 mg: containing impurities).

MS(ESI)m/z：1048(M+H)⁺.

(step 9-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 2) (201 mg: containing impurities) obtained in the above step 8, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (49.1 mg: containing impurities).

MS(ESI)m/z：1048(M+H)⁺.

(step 10-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15-fluoro-16-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The reaction was carried out in the same manner as in step 5 of example 40 using the compound (134mg) obtained in step 9-1, and then the reaction product was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (22 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.04(1H，brs)，8.01(1H，s)，7.06(1H，s)，6.43(1H，d，J＝16.9Hz)，6.00(1H，d，J＝7.3Hz)，5.78－5.55(1H，m)，5.39(1H，dd，J＝51.7，3.9Hz)，5.31－5.18(1H，m)，4.80(1H，d，J＝3.6Hz)，4.50－4.44(1H，m)，4.40－4.32(4H，m)，4.14－4.10(1H，m)，3.52－3.40(2H，m)，3.35－3.23(2H，m)，3.06－2.90(2H，m)，2.63－2.53(1H，m)，2.40－2.20(1H，m)，1.99－1.76(2H，m).

³¹P－NMR(CD₃OD)δ：57.2(s)，52.6(s).

(step 10-2)

Disodium salt (5R, 7R, 8R, 12aR, 14R, 15R, 15aR16R) -7- { 6-amino-2- [ (2-aminoethyl) amino]-9H-purin-9-yl } -15-fluoro-16-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The reaction was carried out in the same manner as in step 5 of example 40 using the compound obtained in step 9-2 (49.1 mg: containing impurities), and then the reaction product was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.02(1H，brs)，8.01(1H，s)，7.40(1H，s)，6.48(1H，d，J＝16.3Hz)，6.01(1H，d，J＝7.9Hz)，5.80－5.63(1H，m)，5.45－5.28(2H，m)，4.54－4.48(2H，m)，4.41－4.36(2H，m)，4.28－4.20(2H，m)，4.08－4.03(1H，m)，3.54－3.41(2H，m)，3.40－2.52(6H，m)，2.03－1.84(2H，m).

³¹P－NMR(CD₃OD)δ：57.8(s).

Example 48: synthesis of CDN38

(5S, 7R, 8R, 12aR, 14R, 15R, 15aS) -15-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2 ' -O- [ tert-butyl (dimethyl) silyl ] -3 ' -O- (1H-imidazole-1-thiocarbonyl) inosine

To a solution of the compound (2' -O-TBS isomer) (1.45g) obtained in step 2 of example 22 in N, N-dimethylformamide (8.7mL) were added N, N-dimethyl-4-aminopyridine (213mg) and bis (1H-imidazol-1-yl) methylthio ketone (1.55g) at room temperature, and the mixture was stirred for 20 hours. The reaction mixture was diluted with ethyl acetate, and washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.58 g).

MS(ESI)m/z：943(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.38(1H，s)，8.05(1H，s)，8.02－7.94(3H，m)，7.93(1H，s)，7.66－7.65(1H，m)，7.60－7.52(1H，m)，7.46－7.18(10H，m)，7.08(1H，s)，6.83－6.80(4H，m)，6.07(1H，dd，J＝5.4，3.0Hz)，6.02(1H，d，J＝6.0Hz)，5.20(1H，dd，J＝6.7，5.4Hz)，4.72－4.63(2H，m)，4.55－4.38(3H，m)，3.78(3H，s)，3.77(3H，s)，3.57－3.56(2H，m)，0.65(9H，s)，－0.11(3H，s)，－0.26(3H，s).

(step 2)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2 ' -O- [ tert-butyl (dimethyl) silyl ] -3 ' -deoxyinosine

Tributyltin hydride (1.42mL) and 2, 2' -azobis (isobutyronitrile) (29.4mg) were added to a benzene (10mL) solution of the compound (1.69g) obtained in the above step 1 at room temperature, and the mixture was stirred at 80 ℃ for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.12 g).

MS(ESI)m/z：817(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99－7.96(4H，m)，7.60－7.55(1H，m)，7.46－7.19(11H，m)，6.83－6.81(4H，m)，5.94(1H，d，J＝1.2Hz)，4.68－4.60(4H，m)，4.52－4.39(2H，m)，3.79(6H，s)，3.42－3.34(2H，m)，2.15－2.08(1H，m)，1.94－1.89(1H，m)，0.86(9H，s)，0.06(3H，s)，0.04(3H，s).

(step 3)

1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -deoxyinosine

To a tetrahydrofuran (11mL) solution of the compound (1.75g) obtained in the above step 2 was added a tetrabutylammonium fluoride tetrahydrofuran solution (about 1M, 2.6mL) at 0 ℃ and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.29 g).

MS(ESI)m/z：703(M+H)⁺.

¹H－NMR(CDCl₃) δ: 7.99-7.95 (4H, m), 7.60-7.55 (1H, m), 7.45-7.37 (4H, m), 7.29-7.17 (7H, m), 6.81-6.78 (4H, m), 5.88(1H, d, J ═ 3.0Hz), 4.76-4.63 (4H, m), 4.52-4.40 (2H, m), 3.78(6H, s), 3.36(1H, dd, J ═ 10.3, 3.0Hz), 3.29(1H, dd, J ═ 10.3, 4.8Hz), 2.30-2.23 (1H, m), 2.18-2.12 (1H, m) (only the peaks observed are described)

(step 4)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -3 ' -deoxyinosine

Using the compound (1.28g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (1.47g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

MS(ESI)m/z：820(M－C₆H₁₄N+OH+H)⁺.

¹H－NMR(CDCl₃)δ：7.99－7.94(4H，m)，7.59－7.56(1H，m)，7.46－7.42(4H，m)，7.33－7.18(7H，m)，6.81(4H，d，J＝8.5Hz)，6.13(0.5H，brs)，6.07(0.5H，brs)，4.84－4.39(6H，m)，3.84－3.52(10H，m)，3.42－3.34(2H，m)，2.59(1H，t，J＝6.3Hz)，2.52(1H，t，J＝6.3Hz)，2.30－2.07(2H，m)，1.18－1.07(12H，m).

(step 5)

The same reaction as in step 7 of example 1 (raw material: 836mg) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (735mg) obtained in the above step 4, a reaction was carried out in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 6)

2- { 9- [ (5S, 7R, 8R, 12aR, 14R, 15R, 15aR) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 5, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain diastereomer 1(67.6mg) and diastereomer 2(91.6mg) of the title compound (retention time of HPLC: diastereomer 1> 2).

Diastereomer 1 (low polarity)

MS(ESI)m/z：1134(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1134(M+H)⁺.

(step 7-1)

(5S, 7R, 8R, 12aR, 14R, 15R, 15aR) -15- { [ tert-butyl (dimethyl) silyl group]Oxy } -7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

Using the compound (diastereomer 1) obtained in the above step 6 (67.6mg), a reaction was carried out in the same manner as in step 10 of example 1, and the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 0% to 50% (0 part to 30 parts) was purified to give the title compound (34.9 mg).

MS(ESI)m/z：873(M+H)⁺.

(step 7-2)

Using the compound (diastereomer 2) (91.6mg) obtained in the above step 6, a reaction was carried out in the same manner as in step 10 of example 1, and the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 0% -50% (0 min-30 min)]Purification was performed to give the title compound (44.2 mg). MS (ESI) m/z: 873(M + H)⁺.

(step 8-1)

Disodium (5S, 7R, 8R, 12aR, 14R, 15R, 15aS) -15-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The compound (34.9mg) obtained in the above step 7-1 was reacted in the same manner as in the step 11 of example 1, and then purified by Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21.9 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.34(1H，brs)，8.23(1H，s)，8.02(1H，s)，7.24(1H，brs)，6.28(1H，d，J＝3.6Hz)，6.15(1H，d，J＝3.0Hz)，5.37－5.32(1H，m)，4.99－4.93(1H，m)，4.74－4.58(3H，m)，4.36－4.29(2H，m)，4.24－4.13(3H，m)，4.00－3.91(1H，m)，3.82(2H，t，J＝4.8Hz)，3.51－3.49(2H，m)，3.00－2.90(3H，m)，2.57－2.51(1H，m)，2.02－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：59.7(s)，56.2(s).

(step 8-2)

Disodium (5S, 7R, 8R, 12aR, 14R, 15R, 15aS) -15-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furanAnd [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The compound (44.2mg) obtained in the above step 7-2 was reacted in the same manner as in step 11 of example 1, and then purified by Sep-Pak (registered trademark) C18[ 0.1% aqueous triethylamine solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (26.5 mg).

MS(ESI)m/z：759(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.48－8.46(1H，brm)，8.24(1H，s)，8.03(1H，s)，7.41－7.38(1H，brm)，6.32(1H，d，J＝4.8Hz)，6.15(1H，d，J＝4.2Hz)，5.46－5.41(1H，m)，5.21－5.17(1H，m)，4.68－4.65(1H，m)，4.59－4.54(2H，m)，4.50－4.44(1H，m)，4.35－4.32(1H，m)，4.21－4.18(2H，m)，4.02－3.92(2H，m)，3.84－3.81(2H，m)，3.52－3.50(2H，m)，2.96－2.83(3H，m)，2.54－2.48(1H，m)，2.03－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：60.3(s)，59.1(s).

Example 49: synthesis of CDN39

(5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -16-fluoro-15-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

1- [ 2- (benzoyloxy) ethyl ] -2 ', 5' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine

To a commercially available (Amadis Chemical) mixed solution of 5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] inosine (20.0g) in pyridine (50mL) -N, N-dimethylacetamide (70.1mL) were added 4, 4' -dimethoxytrityl chloride (12.5g), 2-bromoethyl benzoate (6.60mL), and 1, 8-diazabicyclo [5.4.0] -7-undecene (13.1mL), and the mixture was stirred at room temperature for 20 hours. To the reaction mixture were added a saturated aqueous sodium bicarbonate solution and water to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (24.1 g: containing impurities).

MS(ESI)m/z：1121(M+H)⁺.

(step 2)

1- [ 2- (benzoyloxy) ethyl ] -2 ', 5 ' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- (trifluoromethanesulfonyl) inosine

Pyridine (19.0mL) was added to a dichloromethane (120mL) solution of the compound (24.1g) obtained in the above step 1, and then trifluoromethanesulfonic anhydride (5.96mL) was added dropwise slowly at 0 ℃ and stirred at the same temperature for 1 hour. After the reaction was terminated by adding a saturated aqueous sodium bicarbonate solution and water to the reaction mixture, the mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (19.7 g: containing impurities).

MS(ESI)m/z：1153(M+H)⁺.

(step 3)

9- {2, 5-bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-xylofuranosyl } -1- (2-hydroxyethyl) -1, 9-dihydro-6H-purin-6-one

Cesium acetate (8.20g) was added to a solution of the compound (19.7g) obtained in the above step 2 in N, N-dimethylformamide (85.4mL), and the mixture was stirred at room temperature for 16 hours. Methanol (85.4mL) and potassium carbonate (4.72g) were added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. After the reaction was stopped by adding water to the reaction solution, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (12.2 g: containing impurities).

MS(ESI)m/z：917(M+H)⁺.

(step 4)

1- [ 2- (benzoyloxy) ethyl ] -9- {2, 5-bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-xylofuranosyl } -1, 9-dihydro-6H-purin-6-one

Pyridine (1.61mL) and benzoic anhydride (3.16g) were added to a dichloromethane (48.8mL) solution of the compound (12.2g) obtained in the above step 3, and the mixture was stirred at room temperature for 64 hours. To the reaction mixture were added a saturated aqueous sodium bicarbonate solution and water, and the reaction mixture was stopped, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (7.36 g: containing impurities).

MS(ESI)m/z：1021(M+H)⁺.

(step 5)

1- [ 2- (benzoyloxy) ethyl ] -2 ', 5' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 '-deoxy-3' -fluoroinosine

After 2, 6-lutidine (3.34mL) was added to a solution of the compound (7.36g) obtained in the above step 4 in methylene chloride (37.0mL), N-diethylaminosulfur trifluoride (1.42g) was added thereto at-78 ℃ and the temperature was gradually raised to room temperature under a nitrogen atmosphere, followed by stirring overnight. After the reaction was stopped by slowly adding a saturated aqueous sodium bicarbonate solution, water was added thereto, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate 0.1% triethylamine ] to give the title compound (6.89 g: containing impurities).

MS(ESI)m/z：1023(M+H)⁺.

(step 6)

1- [ 2- (benzoyloxy) ethyl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-3 ' -fluoroinosine

Water (0.303mL) and dichloroacetic acid (1.39mL) were added to a dichloromethane (25.0mL) solution of the compound (6.89g) obtained in step 5, and the mixture was stirred at room temperature for 63 hours. Pyridine (2.0mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was subjected to crude purification by silica gel column chromatography [ hexane/ethyl acetate/methanol ] and DIOL silica gel column chromatography [ hexane/ethyl acetate ]. To a pyridine (25.0mL) solution of the obtained compound was added 4, 4' -dimethoxytrityl chloride (1.83g) at 0 ℃ and the mixture was stirred at 4 ℃ for 21 hours. Methanol (1.0mL) was added to the reaction mixture, and the mixture was stirred at 4 ℃ for 1 hour and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (1.33 g).

MS(ESI)m/z：721(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(1H，s)，7.95(1H，d，J＝1.2Hz)，7.93(1H，s)，7.93(1H，d，J＝1.5Hz)，7.58－7.51(1H，m)，7.43－7.37(2H，m)，7.35－7.30(2H，m)，7.26－7.13(7H，m)，6.82－6.75(4H，m)，5.98(1H，d，J＝7.3Hz)，5.13(1H，dd，J＝54.7，4.1Hz)，5.05－4.92(1H，m)，4.63(2H，t，J＝5.1Hz)，4.56－4.25(4H，m)，3.76(3H，s)，3.76(3H，s)，3.45(1H，dd，J＝10.9，3.6Hz)，3.34(1H，dd，J＝10.9，3.6Hz).

(step 7)

1- [ 2- (benzoyloxy) ethyl ] -5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -3 '-deoxy-3' -fluoroinosine

The reaction was carried out in the same manner as in step 4 of example 5 using the compound (1.33g) obtained in step 6 above to obtain the title compound (1.49g) in the form of a diastereomer mixture (diastereomer ratio 1: 1) on the phosphorus atom.

MS(ESI)m/z：921(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(0.5H，s)，7.98－7.93(3H，m)，7.88(0.5H，s)，7.58－7.52(1H，m)，7.45－7.37(4H，m)，7.33－7.18(7H，m)，6.85－6.77(4H，m)，6.14(0.5H，d，J＝7.3Hz)，6.10(0.5H，d，J＝7.9Hz)，5.27－5.01(2H，m)，4.70－4.61(2H，m)，4.57－4.30(3H，m)，3.78(3H，s)，3.77(3H，s)，3.62－3.33(6H，m)，2.56(1H，t，J＝6.3Hz)，2.32(1H，t，J＝6.3Hz)，1.12(6H，d，J＝6.3Hz)，1.04(3H，d，J＝6.3Hz)，0.76(3H，d，J＝6.3Hz).

(step 8)

The same reaction as in step 7 of example 1 (raw material: 948mg) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (850mg) obtained in the above step 7, a reaction was carried out in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 9)

2- { 9- [ (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -16-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 8, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (709mg) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1152(M+H)⁺.

(Process 10)

Bis (N, N-diethylethylammonium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -15- { [ tert-butyl (dimethyl) silyl ]Oxy } -16-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (709mg) obtained in the above step 9, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(108 mg: containing impurities) and diastereomer 2(102 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：891(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：891(M+H)⁺.

(step 11-1)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -16-fluoro-15-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine ringsTetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (108 mg: containing impurities) obtained in the above step 10 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (40.1 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.66(1H，m)，8.26(1H，s)，8.02(1H，s)，7.09(1H，s)，6.29－6.24(2H，m)，5.68－5.45(2H，m)，5.26－5.18(1H，m)，4.78－4.73(1H，m)，4.62－4.42(3H，m)，4.26－3.98(5H，m)，3.85－3.78(2H，m)，3.53－3.47(2H，m)，2.91－2.84(2H，m)，2.04－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：58.0(s)，56.9(s).

(step 11-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (102 mg: containing impurities) obtained in the above step 10 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (26.7 mg).

MS(ESI)m/z：777(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，m)，8.25(1H，s)，8.02(1H，s)，7.10(1H，brs)，6.33(1H，d，J＝6.7Hz)，6.28(1H，d，J＝9.1Hz)，5.65－5.50(1H，m)，5.49－5.43(1H，m)，5.31(1H，dd，J＝54.4，3.6Hz)，4.79(1H，dd，J＝6.3，4.5Hz)，4.62－4.34(4H，m)，4.27－4.14(2H，m)，4.08－4.01(1H，m)，3.93－3.87(1H，m)，3.86－3.80(2H，m)，3.53－3.47(2H，m)，2.95－2.89(2H，m)，2.06－1.97(2H，m).

³¹P－NMR(CD₃OD)δ：63.1(s)，59.7(s).

Example 50: synthesis of CDN40

(5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-16-fluoro-15-hydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd [ ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

3 '-deoxy-3' -fluoroinosine

An aqueous solution (48mL) of sodium nitrite (6.10g) was added little by little to a commercial (Angene) solution of 3 '-deoxy-3' -fluoroadenosine (2.38g) in acetic acid (120mL), and the mixture was stirred at room temperature for 43 hours. The reaction solution was concentrated under reduced pressure and azeotroped with toluene 2 times. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (3.76 g).

MS(ESI)m/z：271(M+H)⁺.

1H－NMR(CD₃OD)δ：8.30(1H，s)，8.05(1H，s)，6.03(1H，d，J＝7.8Hz)，5.08(1H，dd，J＝54.4，4.1Hz)，4.89－4.85(1H，m)，4.38(1H，dt，J＝26.8，3.2Hz)，3.82－3.73(2H，m).

(step 2)

3 '-deoxy-1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] -3' -fluoroinosine

To a solution of the compound (3.76g) obtained in the above step 1 in N, N-dimethylacetamide (37.6mL) were added 2- (2-bromoethyl) -1H-isoindole-1, 3(2H) -dione (6.60mL) and 1, 8-diazabicyclo [5.4.0] -7-undecene (3.12mL), and the mixture was stirred at room temperature for 3 days. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol ] to give the title compound (3.51 g: containing impurities).

MS(ESI)m/z：444(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.87(1H，s)，7.84(1H，s)，7.81(2H，dd，J＝5.4，3.0Hz)，7.73(2H，dd，J＝5.4，3.0Hz)，5.83(1H，d，J＝7.9Hz)，5.28(1H，dd，J＝11.5，2.4Hz)，5.18(1H，dd，J＝55.0，4.2Hz)，5.01－4.87(1H，m)，4.49(1H，d，J＝28.4Hz)，4.43－4.28(2H，m)，4.19－4.08(2H，m)，3.95(1H，d，J＝8.2Hz)，3.91－3.84(1H，m)，3.76(1H，d，J＝13.3Hz).

(step 3)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] -3 ' -fluoroinosine

Using the compound (3.51g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (4.05 g).

MS(ESI)m/z：746(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.95(1H，s)，7.79(2H，dd，J＝5.4，3.0Hz)，7.73(1H，s)，7.70(2H，dd，J＝5.4，3.0Hz)，7.34－7.29(2H，m)，7.25－7.18(7H，m)，6.81－6.76(4H，m)，5.92(1H，d，J＝7.3Hz)，5.12(1H，dd，J＝54.4，4.2Hz)，4.97－4.85(1H，m)，4.55－4.40(2H，m)，4.30－4.22(1H，m)，4.21－4.01(2H，m)，3.77(6H，s)，3.73(1H，d，J＝10.3Hz)，3.43(1H，dd，J＝10.9，3.6Hz)，3.32(1H，dd，J＝10.9，3.6Hz).

(step 4)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -3 '-deoxy-1- [ 2- (1, 3-di-oxo-1, 3-dihydro-2H-isoindol-2-yl) ethyl ] -3' -fluoroinosine

The reaction was carried out in the same manner as in step 4 of example 5 using the compound obtained in step 3 (4.05g), so as to obtain the title compound (4.35g) in the form of a diastereomer mixture (diastereomer ratio: 1) on the phosphorus atom.

MS(ESI)m/z：946(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.95(0.5H，s)，7.94(0.5H，s)，7.84－7.78(2H，m)，7.73－7.68(2H，m)，7.65(1H，s)，7.43－7.41(2H，m)，7.32－7.15(7H，m)，6.84－6.77(4H，m)，6.09(0.5H，d，J＝7.9Hz)，6.05(0.5H，d，J＝7.9Hz)，5.31－5.16(1H，m)，5.15－4.98(1H，m)，4.51－4.21(3H，m)，4.20－4.05(2H，m)，3.793(1.5H，s)，3.789(3H，s)，3.784(1.5H，s)，3.66－3.55(2H，m)，3.50－3.30(4H，m)，2.56(1H，t，J＝6.3Hz)，2.41(1H，t，J＝6.3Hz)，1.16(3H，d，J＝7.3Hz)，1.14(3H，d，J＝7.3Hz)，1.10(3H，d，J＝7.3Hz)，0.83(3H，d，J＝7.3Hz).

(step 5)

The same reaction as in step 7 of example 1 (starting material: 1.47g) was carried out on the following scale. Using an acetonitrile solution of the obtained compound and the compound (1.35g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 6)

3- { [ (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -7- { 1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -16-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-10-yl ]Oxy-propionitrile

Using the crude product obtained in the above step 5, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (1.25g) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1177(M+H)⁺.

(step 7)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-15- { [ tert-butyl (dimethyl) silyl]Oxy } -16-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a mixed solution of the compound (1.25g) obtained in the above step 6 in ethanol (7.5mL) -tetrahydrofuran (7.5mL) was added hydrazine monohydrate (0.599mL), and the mixture was stirred at 50 ℃ for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(145 mg: containing impurities) and diastereomer 2(198 mg: containing impurities) (retention time of HPLC: diastereomer 1>2) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：890(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：890(M+H)⁺.

(step 8-1)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-16-fluoro-15-hydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (145 mg: containing impurities) obtained in the above step 7 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (70.7 mg).

MS(ESI)m/z：776(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.64(1H，s)，8.23(1H，s)，8.02(1H，s)，7.07(1H，s)，6.27(1H，d，J＝4.5Hz)，6.25(1H，d，J＝6.7Hz)，5.67－5.43(2H，m)，5.23－5.16(1H，m)，4.76(1H，t，J＝5.1Hz)，4.63－4.45(3H，m)，4.31－4.03(5H，m)，3.51－3.46(2H，m)，3.31－3.26(2H，m)，2.90－2.83(2H，m)，2.03－1.94(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，56.9(s).

(step 8-2)

(diastereomer 2)

The compound (diastereomer 2) (198 mg: containing impurities) obtained in the above step 7 was reacted by the same method as in the step 11 of example 1, and then purified according to the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (69.7 mg).

MS(ESI)m/z：776(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，s)，8.27(1H，s)，8.02(1H，s)，7.09(1H，s)，6.31(1H，d，J＝6.7Hz)，6.26(1H，d，J＝8.5Hz)，5.62－5.47(1H，m)，5.47－5.41(1H，m)，5.30(1H，dd，J＝53.8，3.6Hz)，4.78(1H，dd，J＝6.7，4.2Hz)，4.58(1H，d，J＝26.0Hz)，4.51－4.41(2H，m)，4.40－4.23(3H，m)，4.11－4.05(1H，m)，3.93－3.86(1H，m)，3.53－3.46(2H，m)，3.36－3.28(2H，m)，2.90(2H，t，J＝5.7Hz)，2.05－1.96(2H，m).

³¹P－NMR(CD₃OD)δ：62.8(s)，59.4(s).

Example 51: synthesis of CDN41

N- (2- { 9- [ (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -16-fluoro-15-hydroxy-2, 10-dioxido-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) -2-hydroxyacetamide

[ synthetic route ]

(step 1-1)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound obtained in the step 8-1 of example 50 (25.0mg) in the same manner as in the step 1-1 of example 7 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (18.6 mg).

MS(ESI)m/z：834(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.60(1H，m)，8.12(1H，m)，8.02(1H，s)，7.11(1H，brs)，6.27(1H，d，J＝4.9Hz)，6.23(1H，d，J＝9.1Hz)，5.75－5.58(1H，m)，5.54(1H，dd，J＝53.5，3.0Hz)，5.28－5.20(1H，m)，4.75(1H，t，J＝5.2Hz)，4.62－4.52(1H，m)，4.52－4.42(2H，m)，4.29－4.01(5H，m)，3.92(2H，s)，3.64－3.58(2H，m)，3.53－3.47(2H，m)，2.93－2.76(2H，m)，2.06－1.93(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，56.7(s).

(Process 1-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by carrying out a reaction using the compound (diastereomer 2) (15.0mg) obtained in step 8-2 of example 50 in the same manner as in step 1-1 of example 7 and then purifying the reaction product according to the following [ purification conditions ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (7.4 mg).

MS(ESI)m/z：834(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.69(1H，m)，8.16(1H，s)，8.02(1H，s)，7.11(1H，s)，6.32(1H，d，J＝6.7Hz)，6.26(1H，d，J＝8.6Hz)，5.67－5.51(1H，m)，5.48－5.43(1H，m)，5.29(1H，dd，J＝54.0，3.7Hz)，4.77(1H，dd，J＝6.4，4.6Hz)，4.62－4.33(2H，m)，4.25－4.17(2H，m)，4.08－4.01(1H，m)，3.94(2H，s)，3.93－3.85(1H，m)，3.70－3.56(2H，m)，3.52－3.46(2H，m)，2.93－2.86(2H，m)，2.04－1.97(4H，m).

³¹P－NMR(CD₃OD)δ：62.8(s)，59.5(s).

Example 52: synthesis of CDN42

(5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -16-fluoro-15-hydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2 ', 5' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-iodoadenosine

Commercially available (Amadis Chemical) 2-iodoadenosine (1.35g) was azeotroped 3 times with pyridine. To a solution of the residue in dehydrated pyridine (17.0mL) was added 4, 4' -dimethoxytrityl chloride (2.35g), and the mixture was stirred at room temperature overnight under a nitrogen atmosphere. Methanol (5.00mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (1.96 g: containing impurities).

MS(ESI)m/z：998(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.88(1H，s)，7.29－7.12(18H，m)，6.73－6.60(8H，m)，6.31(1H，d，J＝7.9Hz)，5.69(2H，brs)，5.13(1H，dd，J＝7.6，4.5Hz)，4.07(1H，t，J＝3.3Hz)，3.77(6H，s)，3.76(3H，s)，3.74(3H，s)，3.14(2H，d，J＝3.6Hz)，2.87(1H，d，J＝4.2Hz)，2.28(1H，s).

(step 2)

2 ', 5 ' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-iodo-3 ' -O- (trifluoromethanesulfonyl) adenosine

The compound (100mg) obtained in the above step 1 was azeotroped 3 times with toluene. To a solution of the residue in dehydrated dichloromethane (1.0mL) under nitrogen at 0 ℃ were added pyridine (0.20mL) and trifluoromethanesulfonic anhydride (27.0. mu.L), and the mixture was stirred at the same temperature for 80 minutes. Trifluoromethanesulfonic anhydride (27.0. mu.L) was added thereto, and the mixture was stirred for further 80 minutes. After the reaction solution was quenched by addition of a saturated aqueous sodium bicarbonate solution, the reaction solution was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (57.2 mg).

MS(ESI)m/z：1130(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.73(1H，s)，7.32－7.09(18H，m)，6.79－6.70(6H，m)，6.60－6.56(2H，m)，6.19(1H，dd，J＝8.2，3.9Hz)，6.01(1H，d，J＝7.9Hz)，5.59(2H，brs)，4.05(1H，d，J＝3.6Hz)，3.98(1H，t，J＝6.7Hz)，3.78(3H，s)，3.77(3H，s)，3.71(3H，s)，3.70(3H，s)，3.39(1H，dd，J＝10.9，6.0Hz)，3.17(1H，dd，J＝10.6，7.0Hz).

(step 3)

9- {2, 5-bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -beta-D-xylofuranosyl } -2-iodo-9H-purin-6-amine

Cesium acetate (1.20g) was added to a solution of the compound (2.76g) obtained in the above step 2 in N, N-dimethylformamide (24.4mL), and the mixture was stirred at room temperature for 3 hours. Methanol (24.4mL) and potassium carbonate (675mg) were added to the reaction solution, which was then stirred for 2 hours. After water was added to the reaction mixture to precipitate a solid, the methanol component was distilled off under reduced pressure. The resulting solid was filtered to give the title compound (2.38 g).

¹H－NMR(CDCl₃)δ：8.02(1H，s)，7.42－7.13(18H，m)，6.79－6.72(8H，m)，5.68(2H，brs)，5.55(1H，d，J＝1.2Hz)，5.49(1H，d，J＝9.1Hz)，4.48(1H，s)，4.41－4.37(1H，m)，4.06(1H，dd，J＝9.1，3.6Hz)，3.77(9H，s)，3.76(3H，s)，3.56－3.48(2H，m).

(step 4)

2 ', 5' -bis-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 '-deoxy-3' -fluoro-2-iodoadenosine

Pyridine (1.13mL) was added to a dichloromethane (17.0mL) solution of the compound (2.54g) obtained in the above step 3, and then N, N-diethylaminosulfur trifluoride (0.397mL) was added thereto under ice cooling, followed by stirring at room temperature for 40 minutes under a nitrogen atmosphere. After the reaction solution was quenched by addition of a saturated aqueous sodium bicarbonate solution, the reaction solution was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/0.1% triethylamine ] to give the title compound (2.02 g: containing impurities).

MS(ESI)m/z：1000(M+H)⁺.

(step 5)

3 '-deoxy-3' -fluoro-2-iodoadenosine

To a dichloromethane solution (20.2mL) of the mixture (2.02g) obtained in the above step 4 were added water (0.364mL) and dichloroacetic acid (0.996mL) under ice-cooling, and the mixture was stirred at room temperature for 15 minutes. Pyridine (1.95mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.34 g: containing impurities).

¹H－NMR(CD₃OD) δ: 5.94(1H, d, J ═ 7.9Hz), 5.09(1H, dd, J ═ 54.7, 4.3Hz), 4.95-4.89 (1H, m), 4.40(1H, d, J ═ 27.3Hz), 3.87-3.76 (2H, m) (only the peaks observed are noted)

(step 6)

2- [ (2-aminoethyl) amino ] -3 '-deoxy-3' -fluoroadenosine

Ethylenediamine (1.35mL) was added to the compound (1.34g) obtained in the above step 5, and the mixture was stirred at 110 ℃ for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give the title compound (543 mg: containing impurities).

¹H－NMR(CD₃OD)δ：7.94(1H，s)，5.88(1，d，J＝7.9Hz)，5.09(1H，dd，J＝54.7，4.5Hz)，4.95(1H，dq，J＝25.1，4.2Hz)，4.38(1H，dt，J＝27.6，2.7Hz)，3.84－3.76(2H，m)，3.56－3.53(2H，m)，3.01(2H，t，J＝5.7Hz).

(step 7)

3 '-deoxy-3' -fluoro-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

1- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } oxy) pyrrolidine-2, 5-dione (961mg), N-dimethylformamide (5.0mL) and methanol (5.0mL) were added to a tetrahydrofuran solution (10mL) of the compound (543mg) obtained in the above step 6, and the mixture was stirred at room temperature for 1.5 hours. 1- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } oxy) pyrrolidine-2, 5-dione (800mg) was added thereto, and the mixture was stirred for additional 40 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (483 mg).

¹H－NMR(CD₃OD)δ：7.87(1H，s)，5.83(1H，d，J＝7.9Hz)，5.15－4.96(2H，m)，4.35(1H，dt，J＝27.4，2.7Hz)，4.11－4.04(2H，m)，3.84－3.73(2H，m)，3.48－3.38(2H，m)，2.96－2.83(2H，m)，0.95－0.83(2H，m)，0.00(9H，s).

(step 8)

N-benzoyl-3 '-deoxy-3' -fluoro-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Using the compound (483mg) obtained in the above step 7, a reaction was carried out in the same manner as in the step 3 of example 11 to obtain the title compound (374 mg).

¹H－NMR(CDCl₃) δ: 9.16-9.03 (1H, m), 8.01(2H, d, J ═ 7.3Hz), 7.63-7.33 (4H, m), 5.85-5.60 (2H, m), 5.21-4.91 (2H, m), 4.45(1H, d, J ═ 27.8Hz), 4.19-4.06 (2H, m), 3.92(1H, d, J ═ 12.7Hz), 3.77-3.34 (6H, m), 1.05-0.87 (2H, m), -0.02 (9H, s) (only the peaks observed are described)

(step 9)

N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-3 ' -fluoro-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

Using the compound (374mg) obtained in the above step 8, a reaction was carried out in the same manner as in step 1 of example 11 to obtain the title compound (398 mg).

MS(ESI)m/z：878(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.94(1H，brs)，8.01－7.98(2H，m)，7.88－7.81(1H，m)，7.62－7.49(3H，m)，7.40－7.20(9H，m)，6.83－6.78(4H，m)，6.12－5.48(2H，m)，5.22－4.98(2H，m)，4.52(1H，d，J＝27.8Hz)，4.24－4.10(2H，m)，3.77(6H，s)，3.65－3.29(8H，m)，1.04－0.88(2H，m)，0.01(9H，brs).

(Process 10)

N-benzoyl-5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -3 '-deoxy-3' -fluoro-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] carbonyl } amino) ethyl ] amino } adenosine

The reaction was carried out in the same manner as in step 6 of example 1 using the compound (398mg) obtained in step 9 above to obtain the title compound (434mg) as a mixture of diastereomers on the phosphorus atom (diastereomer ratio: 1).

¹H－NMR(CDCl₃)δ：8.79－8.75(1H，m)，8.00－7.97(2H，m)，7.83(0.5H，s)，7.77(0.5H，s)，7.61－7.21(12H，m)，6.84－6.79(4H，m)，6.07－5.96(1H，m)，5.58－5.10(3H，m)，4.50－4.40(1H，m)，4.11－4.06(2H，m)，3.87－3.80(1H，m)，3.79(3H，s)，3.78(3H，s)，3.65－3.20(10H，m)，2.63－2.59(1H，m)，2.39(1H，t，J＝6.3Hz)，1.17(3H，d，J＝6.7Hz)，1.15(3H，d，J＝6.7Hz)，1.10(3H，d，J＝6.7Hz)，0.92－0.86(2H，m)，0.81(3H，d，J＝6.7Hz)，－0.01(9H，s).

(step 11)

A compound A:

n, N-diethylethanaminium (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- (6-benzamide-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] amide]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -16-fluoro-2-oxo-10-ylideneMercapto octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-thiolate

Compound B:

bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- (6-benzamide-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] amide]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -16-fluoro-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The same reaction (starting material: 502mg) as in step 7 of example 1 was carried out on the following scale. Using the obtained acetonitrile solution of the compound and the compound (434mg) obtained in the above step 10, reactions were carried out in the same manner as in the step 8 of example 1 and the step 9 of example 1 to obtain diastereomer 1(43.9mg) and diastereomer 2(27.0mg) of the title compound a and diastereomer 1(55.0mg) and diastereomer 2(121mg) (each containing impurities) of the title compound B.

Diastereomer 1 of Compound A (Low polarity)

MS(ESI)m/z：1309(M+H)⁺.

Diastereomer 2 of Compound A (high polarity)

MS(ESI)m/z：1309(M+H)⁺.

Diastereomer 1 of Compound B (Low polarity)

MS(ESI)m/z：1256(M+H)⁺.

Diastereomer 2 of Compound B (high polarity)

MS(ESI)m/z：1256(M+H)⁺.

(step 12-1)

Bis (N, N-diethylethylammonium) (5R, 7R, 8S, 12aR,14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] 2]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -15- { [ tert-butyl (dimethyl) silyl ]Oxy } -16-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The title compound was obtained by carrying out a reaction using compound a (diastereomer 1) (43.9mg) obtained in the above step 11 in the same manner as in the step 10 of example 1.

MS(ESI)m/z：1048(M+H)⁺.

(step 12' -1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -16-fluoro-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The title compound was obtained by carrying out a reaction using compound B (diastereomer 1) (55.0mg) obtained in the above step 11 in the same manner as in the step 10 of example 1.

MS(ESI)m/z：1048(M+H)⁺.

(step 12-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -16-fluoro-2, 10-di-oxo-14- (6, 7, 8, 9-)tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using compound A (diastereomer 2) (27.0 mg: containing impurities) obtained in the above step 11, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound.

MS(ESI)m/z：1048(M+H)⁺.

(step 13-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -16-fluoro-15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The compounds obtained in the above steps 12-1 and 12' -1 were combined and reacted by the same method as in step 11 of example 1 to obtain the title compound (36.4 mg).

MS(ESI)m/z：934(M+H)⁺.

(step 13-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -16-fluoro-15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound obtained in the above step 12-2, a reaction was carried out in the same manner as in step 11 of example 1 to obtain the title compound (12.4 mg).

MS(ESI)m/z：934(M+H)⁺.

(step 14-1)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -16-fluoro-15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The compound (36.4mg) obtained in the above step 13-1 was reacted by the same method as in step 5 of example 40, and then purified by the following purification conditions to obtain the title compound as a triethylamine salt.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-40 min) and Sep-Pak (registered trademark) C18[ water/acetonitrile/0.1% triethylamine ].

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21.0 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.26(1H，brs)，8.01(1H，s)，7.04(1H，s)，6.27(1H，d，J＝4.8Hz)，6.13(1H，d，J＝7.9Hz)，5.69－5.50(1H，m)，5.57(1H，dd，J＝53.5，2.7Hz)，5.14－5.10(1H，m)，4.73(1H，t，J＝4.8Hz)，4.62－4.53(1H，m)，4.50－4.44(2H，m)，4.24－4.01(3H，m)，3.67－3.58(1H，m)，3.50－3.44(3H，m)，3.20－3.04(2H，m)，2.83－2.81(2H，m)，2.04－1.94(2H，m).

³¹P－NMR(CD₃OD)δ：58.4(s)，56.5(s).

(step 14-2)

Bis (N, N, N-tributylbutan-1-aminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -16-fluoro-15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The compound (29.5mg) obtained in the above step 13-2 was reacted by the same method as in step 5 of example 40, and then purified under the following purification conditions to obtain the title compound (24.2 mg: containing impurities).

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min) ], Sep-Pak (registered trademark) C18[ water/acetonitrile/0.1% triethylamine ], and preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 10% -25% (0 min-30 min) ].

¹H－NMR(CD₃OD)δ：8.27(1H，s)，8.02(1H，s)，7.13(1H，s)，6.32(1H，d，J＝6.0Hz)，6.15(1H，d，J＝7.9Hz)，5.63－5.51(1H，m)，5.39－5.35(1H，m)，5.30(1H，dd，J＝54.4，3.6Hz)，4.77(1H，t，J＝5.1Hz)，4.60－4.34(4H，m)，4.19－4.13(1H，m)，3.92－3.88(1H，m)，3.70－3.62(1H，m)，3.51－3.45(3H，m)，3.26－3.07(18H，m)，2.98－2.87(2H，m)，2.03－1.99(2H，m)，1.70－1.62(16H，m)，1.47－1.37(16H，m)，1.03(24H，t，J＝7.3Hz).

(step 14-2')

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aS, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -16-fluoro-15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furanPyrano [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The compound (19.0 mg: containing impurities) obtained in the above step 14-2 was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: refining 10-25% (0-30 min).

The obtained compound was subjected to salt conversion in the same manner as in [ conversion to sodium salt ] in step 11 of example 1 to obtain the title compound (8.4 mg).

MS(ESI)m/z：790(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.27(1H，s)，8.01(1H，s)，7.12(1H，s)，6.31(1H，d，J＝6.0Hz)，6.15(1H，d，J＝8.5Hz)，5.65－5.49(1H，m)，5.38－5.34(1H，m)，5.30(1H，dd，J＝55.0，3.0Hz)，4.77(1H，dd，J＝5.7，4.5Hz)，4.60－4.34(4H，m)，4.19－4.13(1H，m)，3.92－3.88(1H，m)，3.68－3.61(1H，m)，3.51－3.45(3H，m)，3.22－3.07(2H，m)，2.89－2.87(2H，m)，2.03－1.98(2H，m).

³¹P－NMR(CD₃OD)δ：62.6(s)，59.5(s).

Example 53: synthesis of CDN43

(5S, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -16-amino-7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15-hydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

2 '-O-acetyl-3' -azido-5 '-O-benzoyl-3' -deoxy-N- (2-methylpropanoyl) guanosine

To a solution of 1, 2-di-O-acetyl-3-azido-5-O-benzoyl-3-deoxy-D-ribofuranose (4.0g) in acetonitrile (60mL) known in the literature (Recl. Trav. Chim. Pay-Bas 1986, 105, 85-91) were added N2-isobutyrylguanine (3.65g) and N, O-bis (trimethylsilyl) acetamide (8.08mL) at room temperature, and the mixture was stirred at 70 ℃ for 3 hours. Trimethylsilyl trifluoromethanesulfonate (2.98mL) was added at 70 ℃ and stirred at the same temperature for 1 day. After the reaction solution was cooled, a saturated aqueous sodium hydrogencarbonate solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (4.82 g).

¹H－NMR(CDCl₃)δ：12.2(1H，s)，9.42(1H，s)，8.02(2H，m)，7.71(1H，s)，7.63(1H，m)，7，48(2H，m)，5.97(1H，d，J＝3.9Hz)，5.87(1H，dd，J＝5.5，3.9Hz)，5.10(1H，dd，J＝11.7，5.5Hz)，4.93(1H，t，J＝5.9Hz)，4.66(1H，dd，J＝11.7，5.5Hz)，4.14(1H，q，J＝7.2Hz)，2.77(1H，m)，2.21(3H，s)，1.31(6H，m).

(step 2)

3 '-azido-3' -deoxy-N- (2-methylpropanoyl) guanosine

To a mixed solution of the compound (5.48g) obtained in the above step 1 in tetrahydrofuran (64mL) and methanol (32mL) was added 5M sodium hydroxide (17mL) at 0 ℃ and the mixture was stirred for 15 minutes. Acetic acid (5.08mL) was added to the reaction mixture to stop the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (3.8 g).

¹H－NMR(CD₃OD)δ：8.29(1H，s)，5.97(1H，d，J＝5.7Hz)，4.86(1H，t，J＝5.3Hz)，4.29(1H，t，J＝5.3Hz)，4.10(1H，dt，J＝5.9，2.4Hz)，3.87(1H，dd，J＝12.1，3.1Hz)，3.76(1H，dd，J＝12.3，3.3Hz)，2.74(1H，m)，1.24(6H，d，J＝6.7Hz).

(step 3)

3 ' -azido-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-N- (2-methylpropionyl) guanosine

Using the compound (3.8g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 1 of example 11 to obtain the title compound (5.78 g).

¹H－NMR(CDCl₃)δ：11.9(1H，s)，7.68(2H，d，J＝7.4Hz)，7.60(1H，s)，7.57(2H，d，J－8.6Hz)，7.51(2H，d，J＝8.6Hz)，7.39－7.18(3H，m)，7.02(1H，d，J＝4.7Hz)，6.95(2H，d，J＝9.0Hz)，6.89(2H，d，J＝9.0Hz)，5.84(1H，m)，5.63(1H，d，J＝7.8Hz)，4.58(1H，dd，J＝6.7，2.3Hz)，3.99(1H，m)，3.83(3H，s)，3.81(3H，s)，3.63(1H，dd，J＝11.0，1.6Hz)，2.92(1H，dd，J＝10.8，2.5Hz)，0.91(1H，m)，69(3H，d，J＝6.7Hz)，0.21(3H，d，J＝7.0Hz).

(step 4)

3 ' -amino-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-N- (2-methylpropionyl) guanosine

Triphenylphosphine (3.98g) was added to a methanol (60mL) solution of the compound (5.17g) obtained in the above step 3, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.22 g).

¹H－NMR(CDCl₃)δ：7.77(2H，d，J＝6.7Hz)，7.49－7.17(8H，m)，6.86－6.77(4H，m)，5.93(1H，d，J＝2.7Hz)，5.85(1H，d，J＝3.5Hz)，5.02(1H，dd，J＝6.7，2.7Hz)，4.74(1H，m)，4.32(1H，m)，4.06(1H，m)，3.79(3H，s)，3.78(3H，s)，3.72(1H，t，J＝5.9Hz)，3.49(1H，dd，J＝10.4，3.3Hz)，3.33(1H，dd，J＝10.4，3.7Hz)，2.26(1H，m)，1.21－0.95(6H，m).

(step 5)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -deoxy-N- (2-methylpropionyl) -3 ' - (2, 2, 2-trifluoroacetamide) guanosine

Ethyl trifluoroacetate (4.0mL) was added to a solution of the compound (2.22g) obtained in the above step 4 in N, N-dimethylformamide (20mL), and the mixture was stirred at 50 ℃ for 2 days. After the reaction solution was cooled, a saturated aqueous sodium hydrogencarbonate solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (2.06 g).

MS(ESI)m/z：751(M+H)⁺.

¹H－NMR(CDCl₃)δ：12.0(1H，brs)，7.81(1H，brs)，7.65(1H，s)，7.59(2H，d，J＝7.4Hz)，7.47(2H，d，J＝9.0Hz)，7.43(2H，d，J＝8.6Hz)，7.27－7.18(3H，m)，6.87(2H，d，J＝9.0Hz)，6.82(2H，d，J＝9.0Hz)，5.72(1H，d，J＝6.3Hz)，5.67(1H，m)，5.02(1H，m)，4.33(1H，m)，3.80(3H，s)，3.78(3H，s)，3.60(1H，dd，J＝10.6，1.6Hz)，3.29(1H，dd，J＝10.6，2.7Hz)，1.36(1H，m)，0.82(3H，d，J＝7.0Hz)，0.44(3H，d，J＝6.7Hz).

(step 6)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -3 '-deoxy-N- (2-methylpropanoyl) -3' - (2, 2, 2-trifluoroacetamide) guanosine

Using the compound (1.0g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 4 of example 5 to obtain the title compound (780mg) as a diastereomer mixture on the phosphorus atom.

(step 7)

2-methylpropane-2-ammonium 6-benzoyl2- { 2-O- [ tert-butyl (dimethyl) silyl radical]-3-O- [ oxy (oxide) (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulene derivatives

To a solution of the compound (5.0g) obtained in step 6 of example 1 in acetonitrile (30mL) were added water (0.18mL) and a pyridinium trifluoroacetate (1.2g), and the mixture was stirred at room temperature for 30 minutes. Tert-butylamine (30mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. After the reaction solution was concentrated under reduced pressure, the residue was azeotroped with acetonitrile 2 times. To a solution of the residue in dichloromethane (50mL) were added water (0.88mL) and a solution of dichloroacetic acid (3.2mL) in dichloromethane (50mL) in this order, and the mixture was stirred at room temperature for 1 hour. After the reaction was stopped by adding methanol (5mL) and pyridine (6.3mL), the reaction mixture was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [ hexane/ethyl acetate → methylene chloride/methanol ] to give the title compound (3.0 g).

MS(ESI)m/z：589(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.01(1H，s)，7.65(1H，s)，7.44(1H，m)，7.34－7.26(4H，m)，7.02(1H，d，J＝639.3Hz)，6.24(1H，s)，4.89(1H，m)，4.81(1H，ddd，J＝10.6，5.1，1.6Hz)，4.43－4.27(3H，m)，3.90(2H，m)，3.14(2H，m)，2.30(2H，m)，1.42(9H，s)，0.80(9H，s)，－0.27(6H，s).

(step 8)

Dinucleotide A

Molecular sieves 3A, 1/16(500mg) and 4, 5-dicyanoimidazole (126mg) were added to a dichloromethane (6.0mL) -acetonitrile (6.0mL) mixed solution of the compound (543mg) obtained in the above step 7, and the mixture was stirred at room temperature for 15 minutes. The compound (780mg) obtained in the above-mentioned step 6 was added to the reaction mixture, and the mixture was stirred for 5 hours, followed by addition of N, N-dimethyl-N' - (3-sulfydryl-3H-1, 2, 4-dithiazol-5-yl) formamidine (219mg) and further stirring for 2 hours. The reaction mixture was filtered to remove molecular sieve 3A, and a saturated aqueous sodium bicarbonate solution was added to the filtrate to conduct extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ]. To a solution of the obtained compound (740mg) in dichloromethane (6.0mL) were added water (0.086mL) and a solution of dichloroacetic acid (0.158mL) in dichloromethane (6.0mL) in this order, and the mixture was stirred at room temperature for 1.5 hours. Pyridine (0.31mL) was added to stop the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (520mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1168(M+H)⁺.

(step 9)

N- { 9- [ (5S, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-sulfydryl-16- (2, 2, 2-trifluoroacetamide) octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-2-yl } -2-methylpropanamide

Using the compound (270mg) obtained in the above step 8, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (110mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1182(M+H)⁺.

(Process 10)

Bis (N, N-diethylethanaminium) (5S, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16-amino-7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15- { [ tert-butyl (dimethyl) silyl]Oxy } -2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiol)Salt)

Using the compound (110mg) obtained in the above step 9, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(34.2mg) and diastereomer 2(19.3mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：859(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：859(M+H)⁺.

(step 11-1)

Disodium (5S, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -16-amino-7- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -15-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The reaction was carried out in the same manner as in step 11 of example 1 using the compound (diastereomer 1) (34.2mg) obtained in step 10, and then the reaction product was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (8.9 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.01(1H，s)，7.93(1H，s)，7.01(1H，s)，6.20(1H，d，J＝3.9Hz)，6.01(1H，d，J＝8.6Hz)，5.65(1H，m)，5.19(1H，dt，J＝9.5，4.0Hz)，4.68(1H，t，J＝4.3Hz)，4.40－4.28(2H，m)，4.23(1H，m)，4.17(1H，m)，4.11－4.01(3H，m)，3.41(2H，m)，2.75－2.53(2H，m)，1.98－1.78(2H，m).

³¹P－NMR(CD₃OD)δ：58.0(s)，54.3(s).

(step 11-2)

(diastereomer 2)

The reaction was carried out in the same manner as in step 11 of example 1 using the compound (diastereomer 2) (19.3mg) obtained in step 10, and then the reaction product was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (5.6 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：10.7(1H，s)，8.40(2H，brs)，8.09(1H，s)，8.03(1H，s)，7.74(1H，brs)，7.22(1H，s)，6.95(1H，brs)，6.53(2H，brs)，61.6(2H，t，J＝8.2Hz)，5.57(1H，q，J＝8.0Hz)，5.25(1H，dd，J＝7.8，4.3Hz)，4.61(1H，dd，J＝7.6，4.5Hz)，4.38(1H，s)，4.27－4.11(3H，m)，3.85－3.71(3H，m)，3.35(2H，m)，2.80(2H，m)，1.93(2H，m).

³¹P－NMR(DMSO－d₆)δ：60.3(s)，58.3(s).

Example 54: synthesis of CDN44

(5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -15, 16-difluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl ]-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-l-2H-2, 3, 5, 6-tetraazabenzo [ cd]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (636mg) obtained in the step 8 of example 44, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (640mg) obtained in step 7 of example 49 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- { 9- [ (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -10- (2-cyanoethoxy) -15, 16-difluoro-2-endoxy-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (228 mg: containing impurities) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1040(M+H)⁺.

(step 3)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -15, 16-difluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The compound (228mg) obtained in step 2 was dissolved in methanol (5mL) and a 28% aqueous ammonia solution (5mL), and the mixture was stirred at 60 ℃ for 12 hours. The reaction mixture was concentrated under reduced pressure, and then subjected to C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 5% -30% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): purification from 7% to 50% (0 min to 40 min) gave diastereomer 1 and diastereomer 2, respectively, of the title compound as triethylamine salt (retention time on HPLC: diastereomer 1> 2).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to give diastereomer 1(12.5mg) and diastereomer 2(15.8mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：779(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.63(1H，s)，8.16(1H，s)，8.03(1H，s)，7.08(1H，s)，6.47(1H，dd，J＝17.5，1.8Hz)，6.27(1H，d，J＝7.9Hz)，5.64－5.36(3H，m)，5.31－5.20(1H，m)，4.62－4.50(2H，m)，4.44－4.39(1H，m)，4.32－4.12(3H，m)，4.10－4.03(1H，m)，3.99－3.91(1H，m)，3.83－3.72(2H，m)，3.52－3.46(2H，m)，2.78－2.72(2H，m)，2.04－1.85(2H，m).

³¹P－NMR(CD₃OD)δ：57.5(s)，55.0(s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：779(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.69(1H，s)，8.21(1H，s)，8.03(1H，s)，7.25(1H，s)，6.49(1H，dd，J＝15.1，3.0Hz)，6.28(1H，d，J＝9.1Hz)，5.64－5.28(4H，m)，4.61－4.39(4H，m)，4.26－4.17(1H，m)，4.13－3.95(3H，m)，3.84－3.77(2H，m)，3.53－3.45(2H，m)，2.92－2.77(2H，m)，2.02－1.92(2H，m).

³¹P－NMR(CD₃OD)δ：60.7(s)，57.4(s).

Example 55: synthesis of CDN45

(5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino]-9H-purin-9-yl } -15, 16-difluoro-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (696mg) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to obtain 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (738mg) obtained in the step 10 of example 52 were reacted in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- (trimethylsilyl) ethyl [ 2- ({ 6-benzamide-9- [ (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -10- (2-cyanoethoxy) -15, 16-difluoro-2-endoxy-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Carbamates, their preparation and their use

Using the mixture obtained in the above step 1, a reaction was carried out in the same manner as in step 9 of example 1 to obtain a mixture (1.31g) containing the title compound. The resulting mixture was used directly in the next reaction.

MS(ESI)m/z：1195(M－H)^－.

(step 3)

Bis (N, N-diethylethanaminium) (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -15, 16-difluoro-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the mixture (1.31g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(249 mg: containing impurities) and diastereomer 2(344 mg: containing impurities), respectively, of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：936(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：936(M+H)⁺.

(step 4-1)

Disodium (5R, 7R, 8S, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -15, 16-difluoro-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (249 mg: containing impurities) obtained in the above step 3 in the same manner as in the step 5 of example 40 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% -40% (0 min-30 min) and preparative HPLC [10mM triethylammonium acetate in water/acetonitrile, acetonitrile: 0% -30% (0 min-30 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (9.2 mg).

MS(ESI)m/z：792(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.17(1H，brs)，8.01(1H，s)，7.01(1H，s)，6.45(1H，d，J＝17.5Hz)，6.07(1H，d，J＝8.5Hz)，5.84－5.64(1H，m)，5.61(1H，dd，J＝53.5，3.3Hz)，5.40(1H，dd，J＝52.0，4.2Hz)，5.30－5.18(1H，m)，4.59－4.17(6H，m)，3.54－3.42(2H，m)，3.39－3.31(2H，m)，3.05－2.98(2H，m)，2.69－2.51(2H，m)，2.02－1.83(2H，m).

³¹P－NMR(CD₃OD)δ：57.3(s)，54.8(s).

(step 4-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (344 mg: including impurities) obtained in the above step 3 in the same manner as in the step 5 of example 40 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (4.7 mg).

MS(ESI)m/z：792(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.05(1H，brs)，8.01(1H，s)，7.35(1H，s)，6.50(1H，d，J＝16.3Hz)，6.06(1H，d，J＝8.5Hz)，5.98－5.75(1H，m)，5.48－5.28(3H，m)，4.59－4.28(3H，m)，4.29－4.22(1H，m)，4.04－3.98(1H，m)，3.56－3.42(2H，m)，3.36－2.61(6H，m)，2.05－1.86(2H，m).

³¹P－NMR(CD₃OD)δ：58.4(brs)，57.6(s).

Example 56: synthesis of CDN46

(5R，7R，8R，12aR，14R，15S，15aR，16R)－7－(6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) decahydro-2H, 10H-5, 8-methano-2 lambda⁵，10λ⁵-cyclopenta [ l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

(1R, 2S, 3R, 5R) -3- (4-chloro-5-iodo-7H-pyrrolo [2, 3-d ] pyrimidin-7-yl) -5- (hydroxymethyl) cyclopentane-1, 2-diol

To 7- [ (3aS, 4R, 6R, 6aR) -6- ({ [ tert-butyl (dimethyl) silyl ] oxy } methyl) -2, 2-dimethyltetrahydro-2H, 3 aH-cyclopenta [ d ] [1, 3] dioxol-4-yl ] -4-chloro-5-iodo-7H-pyrrolo [2, 3-d ] pyrimidine (6.0g) known in the literature (WO2015/199136) were added trifluoroacetic acid (36mL) and water (12mL) in this order, and the mixture was stirred at room temperature for 3 hours. The reaction was concentrated under reduced pressure to give the title compound as a crude product. The crude product obtained was used directly in the next reaction.

(step 2)

(1R, 2S, 3R, 5R) -3- (4-amino-5-iodo-7H-pyrrolo [2, 3-d ] pyrimidin-7-yl) -5- (hydroxymethyl) cyclopentane-1, 2-diol

To a solution of the compound (4.4g) obtained in the above step 1 in 1, 4-dioxane (40mL) was added 28% aqueous ammonia (40mL), and the mixture was stirred at 90 ℃ for 72 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [ water/methanol ] to give the title compound (3.4 g).

¹H－NMR(CD₃OD)δ：8.07(1H，s)，7.56(1H，s)，6.57(1H，brs)，4.89(1H，dd，J＝19.2，8.6Hz)，4.79(1H，d，6.7Hz)，4.70(1H，t，J＝5.3Hz)，4.59(1H，d，J＝4.3Hz)，4.15(1H，m)，3.78(1H，m)，3.45(2H，m)，2.14(1H，m)，2.03(1H，m)，1.48(1H，m).

(step 3)

7- [ (4aR, 6R, 7S, 7aR) -2, 2-di-tert-butyl-7- { [ tert-butyl (dimethyl) silyl ] oxy } hexahydro-2H-cyclopenta [ d ] [1, 3, 2] dioxasilane (dioxasilin) -6-yl ] -5-iodo-7H-pyrrolo [2, 3-d ] pyrimidin-4-amine

Using the compound (3.3g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 1 of example 1 to obtain the title compound (4.6 g).

¹H－NMR(CDCl₃)δ：8.27(1H，s)，7.00(1H，s)，5.69(1H，brs)，4.92(1H，dt，J＝9.0，1.2Hz)，4.32(3H，m)，3.98(1H，t，J＝10.8Hz)，2.58(1H，m)，2.26(1H，m)，1.74(1H，brs)，1.49(1H，dt，J＝12.5，8.6Hz)，1.13(9H，s)，1.09(9H，s)，0.86(9H，s)，0.06(3H，s)，0.00(3H，s).

(step 4)

7- [ (4aR, 6R, 7S, 7aR) -2, 2-di-tert-butyl-7- { [ tert-butyl (dimethyl) silyl ] oxy } hexahydro-2H-cyclopenta [ d ] [1, 3, 2] dioxasilan-6-yl ] -5- (3, 3-diethoxyprop-1-yn-1-yl) -7H-pyrrolo [2, 3-d ] pyrimidin-4-amine

Using the compound (4.6g) obtained in the above step 3, a reaction was carried out in the same manner as in the step 2 of example 1 to obtain the title compound (3.6 g).

¹H－NMR(CDCl₃)δ：8.30(1H，s)，7.15(1H，s)，5.61(1H，brs)，5.55(1H，s)，4.90(1H，dt，J＝9.0，1.2Hz)，4.32(3H，m)，3.97(1H，t，J＝10.8Hz)，3.86(2H，m)，3.71(2H，m)，2.58(1H，m)，2.28(1H，m)，1.68(1H，s)，1.48(1H，m)，1.32(6H，t，J＝7.0Hz)，1.13(9H，s)，1.09(9H，s)，0.86(9H，s)，0.05(3H，s)，0.04(3H，s).

(step 5)

2- [ (4aR, 6R, 7S, 7aR) -2, 2-di-tert-butyl-7- { [ tert-butyl (dimethyl) silyl ] oxy } hexahydro-2H-cyclopenta [ d ] [1, 3, 2] dioxasilan-6-yl ] -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound (3.60g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 3 of example 1 to obtain the title compound (2.44 g).

MS(ESI)m/z：589(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.18(1H，s)，6.66(1H，s)，6.55(1H，brs)，4.94(1H，m)，4.36－4.25(3H，m)，3.97(1H，t，J＝10.8Hz)，3.57(2H，m)，2.93(2H，t，J＝5.5Hz)，2.58(1H，m)，2.27(1H，m)，2.09(1H，m)，1.46(1H，dt，J＝12.5，8.6Hz)，1.29(1H，m)，1.12(9H，s)，1.08(9H，s)，0.86(9H，s)，0.04(3H，s)，0.03(3H，s).

(step 6)

{ 2- [ (4aR, 6R, 7S, 7aR) -2, 2-di-tert-butyl-7- { [ tert-butyl (dimethyl) silyl ] oxy } hexahydro-2H-cyclopenta [ d ] [1, 3, 2] dioxasilan-6-yl ] -2, 7, 8, 9-tetrahydro-6H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-6-yl } (phenyl) methanone

Using the compound (2.44g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 4 of example 1 to obtain the title compound (2.01 g).

MS(ESI)m/z：663(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.12(1H，s)，7.42－7.25(5H，m)，6.97(1H，s)，5.04(1H，t，J＝9.0Hz)，4.44(1H，m)，4.38(1H，dd，J＝10.0，4.9Hz)，4.33－4.23(3H，m)，4.00(1H，t，J＝10.8Hz)，3.06(2H，m)，2.60(1H，m)，2.29(3H，m)，1.57(1H，m)，1.14(9H，s)，1.10(9H，s)，0.84(9H，s)，0.06(3H，s)，0.05(3H，s).

(step 7)

{ 2- [ (1R, 2S, 3R, 4R) -4- { [ bis (4-methoxyphenyl) (phenyl) methoxy ] methyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } -3-hydroxycyclopentyl ] -2, 7, 8, 9-tetrahydro-6H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-6-yl } (phenyl) methanone

Using the compound (2.01g) obtained in the above step 6, a reaction was carried out in the same manner as in the step 5 of example 1 to obtain the title compound (2.13 g).

¹H－NMR(CDCl₃)δ：8.03(1H，s)，7.49(2H，m)，7.38－7.19(13H，m)，7.02(1H，s)，6.83(4H，m)，5.07(1H，m)，4.60(1H，dd，J＝8.6，5.1Hz)，4.29(2H，m)，3.99(1H，m)，3.79(6H，s)，3.33(1H，dd，J＝9.4，3.9Hz)，3.22(1H，dd，J＝9.2，4.1Hz)，2.97(2H，t，J＝6.5Hz)，2.68(1H，d，J＝1.6Hz)，2.37－2.18(5H，m)，0.73(9H，s)，－0.18(3H，s)，－0.47(3H，s).

(step 8)

(1R, 2S, 3R, 5R) -3- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-2-yl) -5- { [ bis (4-methoxyphenyl) (phenyl) methoxy ] methyl } -2- { [ tert-butyl (dimethyl) silyl ] oxy } cyclopentyl 4-oxo pentanoate

N, N-dicyclohexylcarbodiimide (2.63g) was added to a solution of levulinic acid (2.96g) in tetrahydrofuran (20mL), and the mixture was stirred at room temperature for 12 hours. After the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure. The compound (2.10g) obtained in step 7 and 4-dimethylaminopyridine (155mg) were added to a dichloromethane (20mL) solution of the residue, and the mixture was stirred at room temperature for 1 hour. After the reaction was terminated by adding a saturated aqueous solution of sodium hydrogencarbonate, the mixture was extracted with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.35 g).

¹H－NMR(CDCl₃)δ：8.02(1H，s)，7.49(2H，m)，7.40－7.19(13H，m)，7.04(1H，s)，6.84(4H，m)，5.26(1H，dd，J＝5.1，2.0Hz)，5.06(1H，q，J＝9.0Hz)，4.61(1H，dd，J＝8.8，4.9Hz)，4.28(2H，m)，3.79(6H，s)，3.39(1H，dd，J＝9.2，3.7Hz)，3.19(1H，dd，J＝9.4，3.9Hz)，3.00－1.85(14H，m)，0.64(9H，s)，－0.13(3H，s)，－0.45(3H，s).

(step 9)

(1R, 2S, 3R, 5R) -3- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-2-yl) -2- { [ tert-butyl (dimethyl) silyl ] oxy } -5- (hydroxymethyl) cyclopentyl-4-oxo-pentanoate

To a dichloromethane (25mL) solution of the compound (2.35g) obtained in the above step 8, water (0.24mL) and a dichloromethane (25mL) solution of dichloroacetic acid (1.05mL) were sequentially added, and the mixture was stirred at room temperature for 1 hour. After the reaction was stopped by adding methanol (1.0mL) and a saturated aqueous solution of sodium hydrogencarbonate to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.15 g).

MS(ESI)m/z：621(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.05(1H，s)，7.38－7.17(5H，m)，7.04(1H，s)，5.19(1H，dd，J＝4.5，1.4Hz)，4.82－4.69(2H，m)，4.47(1H，dd，J＝14.5，7.4Hz)，4.31(1H，d，J＝7.8Hz)，4.15－4.08(1H，m)，3.83(1H，m)，3.75(1H，d，J＝10.8Hz)，3.11－2.95(2H，m)，2.90－2.51(4H，m)，2.47－2.32(2H，m)，2.32－2.11(3H.m)，2.22(3H，s)，0.68(9H，s)，－0.16(3H，s)，－0.50(3H，s).

(Process 10)

N-benzoyl-2 ' -O- [ ({ (1R, 2R, 3S, 4R) -4- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-2-yl) -3- { [ tert-butyl (dimethyl) silyl ] oxy } -2- [ (4-oxopentanoyl) oxy ] cyclopentyl } methoxy) (2-cyanoethoxy) thiophosphoryl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] adenosine

To a solution of the compound (550mg) obtained in the above step 9 in acetonitrile (12mL) were added molecular sieves 3A, 1/16(500mg) and N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (1.23g), and the mixture was stirred at room temperature for 15 minutes. 4, 5-dicyanoimidazole was added to the reaction mixture, and after stirring for 1 hour, 3H-1, 2-thiophenol-3-one 1, 1-dioxide (355mg) was added thereto, followed by further stirring for 1 hour. The reaction mixture was filtered to remove molecular sieve 3A, and a saturated aqueous sodium bicarbonate solution was added to the filtrate to conduct extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.17g) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1539(M+H)⁺.

(step 11)

N-benzoyl-2 ' -O- [ { [ (1R, 2R, 3S, 4R) -4- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulen-2-yl) -3- { [ tert-butyl (dimethyl) silyl ] oxy } -2-hydroxycyclopentyl ] methoxy } (2-cyanoethoxy) thiophosphoryl ] -5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] adenosine

To an acetonitrile (2.0mL) solution of the compound (1.22g) obtained in the above step 10 was added a mixed solution of hydrazine monohydrate (0.25mL) in acetic acid (5.0mL) -pyridine (7.5mL), and the mixture was stirred at room temperature for 30 minutes. After the reaction was stopped by adding water to the reaction mixture, extraction was performed with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (770mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1442(M+H)⁺.

(step 12)

N-benzoyl-2' -O- [ { [ (1R, 2R, 3S, 4R) -4- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -3- { [ tert-butyl (dimethyl) silyl]Oxy } -2- { [ hydroxy (oxo) -lambda⁵-phosphino]Oxy } cyclopentyl group]Methoxy } (2-cyanoethoxy) thiophosphoryl group]-3' -O- [ tert-butyl (dimethyl) silyl]Adenosine (I)

Diphenyl phosphite (0.61mL) was added to a pyridine (8.0mL) solution of the compound (770mg) obtained in the above step 11, and the mixture was stirred at room temperature for 2 hours. To the reaction mixture were added water (20mL), acetonitrile (8.0mL) and triethylammonium acetate aqueous solution (2M, 1.6mL), and the mixture was stirred for 1 hour, after which the reaction mixture was concentrated under reduced pressure. To a solution of the residue in dichloromethane (5.0mL) were added water (0.096mL) and a solution of dichloroacetic acid (0.22mL) in dichloromethane (5.0mL) in this order, and the mixture was stirred for 30 minutes. Pyridine (0.43mL) was added to stop the reaction, and then the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (400mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1203(M+H)⁺.

(step 13)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -2-oxo-2-mercapto-10-mercaptodecahydro-2H, 10H-5, 8-methano-2. lambda⁵，10λ⁵-cyclopenta [ l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } benzamide

Using the compound (400mg) obtained in the above step 12, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (300mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1217(M+H)⁺.

(step 14)

Disodium (5R, 7R, 8R, 12aR, 14R, 15S, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) decahydro-2H, 10H-5, 8-methano-2 lambda⁵，10λ⁵-cyclopenta [ l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a methanol (10mL) solution of the compound (300mg) obtained in the above step 13, 28% aqueous ammonia (10mL) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and triethylamine trihydrofluoride (3.0mL) was added to the residue, and the mixture was stirred at 45 ℃ for 2 hours. The reaction mixture was added to an ice-cooled mixed solution of 1M triethylammonium bicarbonate solution (18mL) and triethylamine (6.0mL) to stop the reaction. The reaction mixture was concentrated under reduced pressure, and then purified by column chromatography on C18 silica gel [10mM aqueous triethylammonium acetate/acetonitrile ], to give diastereomer 1 and diastereomer 2 of the title compound as triethylamine salts, respectively.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to give diastereomer 1(45.6mg) and diastereomer 2(12.6mg) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：728(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.69(1H，s)，8.10(1H，s)，7.93(1H，s)，7.10(1H，s)，6.29(1H，d，J＝8.2Hz)，5.38(1H，dq，J＝9.7.2.7Hz)，4.99(2H，m)，4.79(1H，d，J＝3.9Hz)，4.61(1H，dd，J＝7.2，4.9Hz)，4.36(1H，m)，4.29(1H，m)，4.19(1H，m)，3.99(1H，m)，3.89(1H，q，J＝9.9Hz)，3.47(2H，t，J＝4.9Hz)，2.80(2H.q，J＝5.7Hz)，2.72(1H，m)，2.37(1H，dt，J＝16.4，6.7Hz)，1.94(2H，m)，1.61(1H，dt，J＝16.4，6.7Hz).

³¹P－NMR(CD₃OD)δ：58.3(s)，54.0(s).

Diastereomer 2 (high polarity)

MS(ESI)m/z：728(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.74(1H，s)，8.10(1H，s)，7.95(1H，s)，7.09(1H，s)，6.26(1H，d，J＝8.6Hz)，5.44(1H，m)，5.24(1H，m)，5.06(1H，q，J＝9.3Hz)，4.59(1H，dd，J＝9.4，4.3Hz)，4.51(1H，d，J＝4.3Hz)，4.34－4.21(3H，m)，3.94(1H，m)，3.74(1H，m)，3.47(2H，m)，2.85(2H，t，J＝5.5Hz)，2.54(1H，m)，2.45(1H，dt，J＝17.1，6.7Hz)，1.95(2H，m)，1.38(1H，ddd，J＝14.6，8.3，5.0Hz).

³¹P－NMR(CD₃OD)δ：62.7(s)，60.1(s).

Example 57: synthesis of CDN47

(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2, 10-bis (mercapto) -10-sulfydryl-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyn-2-ones

[ synthetic route ]

(step 1)

6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-O- [ tert-butyl (dimethyl) silyl ] -3-O- (4-oxopentanoyl) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound obtained in the step 5 of example 1, a reaction was carried out in the same manner as in the step 8 of example 56 to obtain the title compound (2.8 g).

¹H－NMR(CDCl₃)δ：8.08(1H，s)，7.48－7.19(15H，m)，6.37(4H，d，J＝6.7Hz)，5.48(1H，dd，J＝5.1，2.3Hz)，4.83(1H，dd，J＝6.7，5.1Hz)，4.37－4.00(3H，m)，3.80(3H，s)，3.79(3H，s)，3.69(1H，m)，3.54(1H，dd，J＝10.6，2.7Hz)，3.39(1H，dd，J＝11.0，2.7Hz)，2.90－2.57(6H，m)，2.20(3H，s)，2.20－2.11(2H，m)，0.69(9H，s)，－0.03(3H，s)，－0.29(3H，s).

(step 2)

6-benzoyl-2- { 2-O- [ tert-butyl (dimethyl) silyl ] -3-O- (4-oxopentanoyl) -beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] azulene

Using the compound (2.8g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 9 of example 56 to obtain the title compound (1.74 g).

¹H－NMR(CDCl₃)δ：8.06(1H，s)，7.38－7.20(5H，m)，7.04(1H，s)，6.32(1H，dd，J＝12.1，1.6Hz)，5.64(1H，d，J＝7.8Hz)，5.47(1H，d，J＝5.1Hz)，5.16(1H，dd，J＝7.8，5.1Hz)，4.40(1H，m)，4.29(1H，s)，4.17(1H，m)，3.91(1H，m)，3.75(1H，m)，3.02(2H，m)，2.90－2.60(4H，m)，2.30－2.14(2H，m)，2，22(3H，s)，0.68(9H，s)，－0.15(3H，s)，－0.46(3H，s).

(step 3)

N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- [ (2-cyanoethoxy) { [ (2, 4-dichlorophenyl) methyl ] mercapto } thiophosphoryl ] adenosine

Molecular sieves 3A, 1/16(1.0g) and 2, 4-dichlorobenzyl mercaptan (1.8mL) were added to a commercially available (Chemgenes) solution of N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } adenosine (3.73g) in acetonitrile (30mL) and stirred at room temperature for 10 minutes. Imidazole perchlorate (3.18g) was added to the reaction mixture, and the mixture was stirred for 2.5 hours, followed by addition of sulfur (242mg) and further stirring for 1 hour. The reaction solution was freed from molecular sieve 3A, and a saturated aqueous sodium hydrogencarbonate solution was added to the filtrate to conduct extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.73g) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1111(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.89(1H，s)，8.74(1H，d，J＝1.8Hz)，8.32(0.5Hs)，8.26(0.5H，s)，8.02(2H，m)，7.64－7.61(15H，m)，6.82(4H，d，J＝9.0)，6.45(0.5H，d，J＝6.7Hz)，6.38(0.5H，d，J＝6.3Hz)，5.88(0.5H，ddd，J＝14.2，6.6，4.8Hz)，5.74(0.5H，ddd，J＝14.4，6.4，4.8Hz)，4.75(0.5H，dd，J＝4.7，2.7Hz)，4.64(0.5H，dd，J＝4.7，2.3Hz)，4.22－3.82(5H，m)，3.78(6H，s)，3.58－3.53(2H，m)，3.34－3.29(2H，m)，2，57(1H，t，J＝6.3Hz)，2.48(1H，t，J＝6.3Hz)，0.90(4.5H，s)，0.88(4.5H，s)，0.16(1.5H，s)，0.11(1.5H，s)，0.07(1.5H，s)，0.04(1.5H，s).

(step 4)

N, N-Diethylethylammonium N-benzoyl-5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- [ { [ (2, 4-dichlorophenyl) methyl ] mercapto } (thio) phosphoryl ] adenosine

Triethylamine (30mL) was added to an acetonitrile (30mL) solution of the compound (2.66g) obtained in the above step 3, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by DIOL silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.5g) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1058(M+H)⁺.

(step 5)

Dinucleotide B

Molecular sieves 4A and 1/16(1.0g), the compound obtained in the above step 2 (930mg), and 1-methylimidazole (1.18mL) were added in this order to a dichloromethane (10mL) solution of the compound obtained in the above step 4 (2.5g), and the mixture was stirred at room temperature for 20 minutes. 2, 4, 6-triisopropylbenzenesulfonyl chloride (905mg) was added to the reaction solution, followed by stirring for 4 hours. The molecular sieve 4A was filtered off from the reaction solution, and a saturated aqueous sodium bicarbonate solution was added to the filtrate, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.04g) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1662(M+H)⁺.

(step 6)

Dinucleotide C

Using the compound (1.04g) obtained in the above step 5, a reaction was carried out in the same manner as in the step 11 of example 56 to obtain the title compound (820mg) as a diastereomer mixture on the phosphorus atom.

MS(ESI)m/z：1564(M+H)⁺.

(step 7)

Dinucleotide D

Using the compound (820mg) obtained in the above step 6, a reaction was carried out in the same manner as in the step 12 of example 56 to obtain the title compound (440mg) as a diastereomer mixture on the phosphorus atom.

(step 8)

N- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -10- { [ (2, 4-dichlorophenyl) methyl]Mercapto } -2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } benzeneCarboxamides

Using the compound (440mg) obtained in the above step 7, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (360 mg).

MS(ESI)m/z：1340(M+H)⁺.

(step 9)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-benzamide-9H-purin-9-yl) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a solution of the compound (360mg) obtained in the above step 8 in dimethyl sulfoxide were added 1-dodecanethiol (434mg) and 1, 8-diazabicyclo [5.4.0] -7-undecene (0.40mL), and the mixture was stirred at room temperature for 6 hours. The reaction mixture was directly purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give the title compound (130 mg).

MS(ESI)m/z：1182(M+H)⁺.

(Process 10)

Disodium (2S, 5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15, 16-dihydroxy-2-oxo-10-sulfydryl-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The reaction was carried out in the same manner as in example 56, step 14 using the compound (130mg) obtained in the above step 9, and then the product was purified by C18 silica gel column chromatography [10mM aqueous triethylammonium acetate/acetonitrile ], whereby the title compound was obtained as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (62.8 mg).

MS(ESI)m/z：746(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.70(1H，s)，8.07(1H，s)，8.03(1H，s)，7.25(1H，s)，6.26(1H，d，J＝8.6Hz)，6.21(1H，d，J＝5.9Hz)，5.52(1H，dq，J＝14.1，5.2Hz)，5.36(1H，m)，4.70(1H，dd，J＝5.7，4.5Hz)，4.42(1H，d，J＝4.3Hz)，4.37－4.26(3H，m)，4.21(1H，m)，3.96(1H，m)，3.84(1H，m)，3.50(2H，m)，2.81(2H，m)，1.93(2H，m).

³¹P－NMR(CD₃OD)δ：119.8(s)，59.4(s).

Example 58: synthesis of CDN48

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

4-chloro-5-iodo-7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidine

To a commercially available (PharmaBlock) solution of 4-chloro-5-iodo-7H-pyrrolo [2, 3-d ] pyrimidine (73.8g) in N, N-dimethylformamide (10mL) was added sodium hydride (containing 45% mineral oil) (13.3g) under ice cooling, and the mixture was stirred for 40 minutes while warming to room temperature. After cooling with ice again, [ 2- (chloromethoxy) ethyl ] (trimethyl) silane (51.0mL) was added over 10 minutes, and the mixture was stirred at the same temperature for 30 minutes. Water (260mL) was added little by little to the reaction solution at which most of the solidified matter was solidified to stop the reaction. The solid was filtered, washed with water (1500mL) and hexane (600mL), and dried under reduced pressure at 40 ℃ to give the title compound (97.63 g).

MS(ESI)m/z：410(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.64(1H，s)，7.54(1H，s)，5.61(2H，s)，3.52(2H，t，J＝8.3Hz)，0.92(2H，t，J＝8.3Hz)，－0.04(9H，s).

(step 2)

4- (benzyloxy) -5-iodo-7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidine

To a solution of benzyl alcohol (27mL) in N, N-dimethylformamide (170mL) was added sodium hydride (containing 45% mineral oil) (12g) under ice-cooling, and the mixture was stirred for 40 minutes while warming to room temperature. After ice-cooling again, a suspension of the compound (97.63g) obtained in the above step 1 in N, N-dimethylformamide (360mL) was added thereto over 40 minutes, and the mixture was stirred at the same temperature for 35 minutes. Crushed ice and a saturated aqueous ammonium chloride solution were added to the reaction mixture to stop the reaction. The reaction mixture was poured into two layers of saturated aqueous ammonium chloride solution and ethyl acetate, and the mixture was poured into a flask using ethyl acetate: toluene (9: 1) was extracted. The organic layer was washed with water and saturated brine for 2 times, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (107.7 g).

MS(ESI)m/z：482(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.47(1H，s)，7.61(2H，d，J＝7.3Hz)，7.41(2H，t，J＝7.6Hz)，7.36－7.30(1H，m)，7.30(1H，s)，5.65(2H，s)，5.57(2H，s)，3.52(2H，t，J＝8.3Hz)，0.91(2H，t，J＝8.3Hz)，－0.05(9H，s).

(step 3)

4- (benzyloxy) -5- (3, 3-diethoxyprop-1-yn-1-yl) -7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidine

Copper iodide (4.49g), tetrakistriphenylphosphine palladium (0) (8.17g), and 3, 3-diethoxyprop-1-yne (104mL) were added to a mixed solution of the compound (113.4g) obtained in the above step 2 in acetonitrile (1000mL) -triethylamine (98mL) under a nitrogen atmosphere at room temperature, and the mixture was stirred at the same temperature for 4.5 hours. After the reaction mixture was concentrated under reduced pressure, ethyl acetate and hexane were added to the residue, and the precipitated solid was removed by filtration. The solid was taken up in ethyl acetate: after washing with a mixture of hexane (1: 1), the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (145.5 g: containing impurities).

MS(ESI)m/z：482(M+H)⁺.

(step 4)

5- (3, 3-diethoxypropyl) -7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-4-ol

To a solution of the compound (145.5g) obtained in the above step 3 in ethanol (900mL) was added 10% palladium on carbon catalyst (M) wet (50.2g), and the mixture was stirred at room temperature for 5 hours under a hydrogen atmosphere. Methylene chloride (500mL) was added to the reaction mixture, the catalyst was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified 2 times by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (59.6 g).

MS(ESI)m/z：418(M+Na)⁺，394[M－H]^－.

¹H－NMR(CDCl₃)δ：11.23(1H，brs)，7.85(1H，s)，6.79(1H，s)，5.47(2H，s)，4.58(1H，t，J＝5.9Hz)，3.69(2H，m)，3.57－3.49(4H，m)，2.90(2H，t，J＝7.8Hz)，2.07(2H，m)，1.23(6H，t，J＝7.1Hz)，0.91(2H，t，J＝8.1Hz)，－0.04(9H，s).

(step 5)

5- (3, 3-diethoxypropyl) -7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidine-4-thiol

To a solution of the compound (59.6g) obtained in the above step 4 in dehydrated dichloromethane (300mL) was added 2, 6-lutidine (42mL) under a nitrogen atmosphere. Trifluoromethanesulfonic anhydride (31mL) was added dropwise thereto over 20 minutes at-20 ℃ and stirred at the same temperature for 20 minutes. N, N-dimethylformamide (500mL) and sodium hydrogensulfite N-hydrate (33.5g) were added under ice cooling, and the mixture was stirred for 2.5 hours after warming to room temperature. The reaction mixture was concentrated under reduced pressure, and the low boiling point component was distilled off. The residue was poured into two layers of ethyl acetate and ice-cold saturated aqueous ammonium chloride and the mixture was washed with ethyl acetate: a mixed solution of toluene (9: 1) was extracted. The organic layer was washed with a saturated aqueous solution of ammonium chloride 1 time, washed with a saturated brine 2 times, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to obtain a mixture of the title compound and 2, 6-lutidine. The resulting mixture was poured into two layers of ethyl acetate and 1N hydrochloric acid, and extracted 2 times with ethyl acetate. The organic layer was washed with saturated brine for 3 times, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure to give the title compound (57.6 g).

MS(ESI)m/z：410[M－H]^－.

¹H－NMR(CDCl₃)δ：11.69(1H，brs)，7.90(1H，s)，6.96(1H，s)，5.49(2H，s)，4.61(1H，t，J＝5.9Hz)，3.71(2H，m)，3.55(2H，m)，3.49(2H，t，J＝8.1Hz)，3.14(2H，t，J＝7.8Hz)，2.08(2H，m)，1.23(6H，t，J＝7.1Hz)，0.90(2H，t，J＝8.3Hz)，－0.04(9H，s).

(step 6)

3- (4-mercapto-7- { [ 2- (trimethylsilyl) ethoxy ] methyl } -7H-pyrrolo [2, 3-d ] pyrimidin-5-yl) propan-1-ol

The compound (31.62g) obtained in the above step 5 was dissolved in 80% aqueous acetic acid (300mL) and stirred at room temperature for 30 minutes. After confirming the disappearance of the starting material, the mixture was ice-cooled, sodium borohydride (1.45g) was added little by little with caution, and stirred at the same temperature for 30 minutes. Then, sodium triacetoxyborohydride (24.4g) was added over 15 minutes, and stirred at the same temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure to about one fifth. After the residue was carefully neutralized to some extent by addition of sodium bicarbonate (solid), it was extracted with ethyl acetate. The organic layer was washed with saturated sodium hydrogencarbonate and saturated brine in this order, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (17.93 g).

MS(ESI)m/z：340[M+H]⁺.

¹H－NMR(CDCl₃)δ：11.92(1H，brs)，7.95(1H，s)，7.01(1H，s)，5.51(2H，s)，3.70(2H，t，J＝5.9Hz)，3.50(2H，t，J＝8.1Hz)，3.23(2H，t，J＝7.3Hz)，2.33(1H，brs)，1.99(2H，m)，0.91(2H，t，J＝8.3Hz)，－0.04(9H，s).

(step 7)

2- { [ 2- (trimethylsilyl) ethoxy ] methyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triaza-benzo [ cd ] azulene

Triphenylphosphine (25.4g) and diisopropyl azodicarboxylate (21.8g) were added to a solution of the compound (31.31g) obtained in the above step 6 in dehydrated tetrahydrofuran (600mL) at 0 ℃ under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/ethyl acetate ] and silica gel column chromatography [ hexane/ethyl acetate ] in this order to obtain the title compound (35.93 g: containing impurities).

MS(ESI)m/z：322[M+H]⁺.

¹H－NMR(CDCl₃)δ：8.57(1H，s)，7.08(1H，s)，5.58(2H，s)，3.52(2H，t，J＝8.3Hz)，3.17(2H，m)，3.06(2H，t，J＝5.6Hz)，2.36(2H，m)，0.92(2H，t，J＝8.3Hz)，－0.05(9H，s).

(step 8)

(8, 9-dihydro-6-thio-2, 3, 5-triazabenzo [ cd ] azulen-2 (7H) -yl) methanol

Trifluoroacetic acid (150mL) was added to a dichloromethane (150mL) solution of the compound (35.93g) obtained in the above step 7 at room temperature, and the mixture was stirred at the same temperature for 1.5 hours. After the reaction solution was concentrated under reduced pressure and azeotroped with toluene 4 times, dichloromethane was added to the residue: the mixture of hexane (1: 2) was filtered to remove the precipitated solid (solid 1). After the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column chromatography [ hexane/ethyl acetate → ethyl acetate/methanol ] to obtain solid 2. Combine solid 1 and solid 2 to give the title compound (20.13 g).

MS(ESI)m/z：222[M+H]⁺.

¹H－NMR(CDCl₃) δ: 8.60(1H, s), 7.19(1H, s), 5.71(2H, s), 3.21(2H, m), 3.07(2H, m), 2.38(2H, m) ("only the peaks observed" are recorded)

(step 9)

2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulenes

To a methanol (250mL) suspension of the compound (20.13g) obtained in the above step 8, 28% aqueous ammonia (150mL) was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure to about half. The precipitated solid was filtered and washed with ethanol to give a solid 1. The filtrate was concentrated under reduced pressure to obtain solid 2 in the same manner. The filtrate was coated with silica gel and purified by silica gel column chromatography [ dichloromethane/methanol ]. The fraction containing the target substance was concentrated under reduced pressure, slurried and washed with ethanol, and the solid (solid 3) was filtered. Combine solid 1, solid 2, and solid 3 to give the title compound (12.36 g).

MS(ESI)m/z：192[M+H]⁺.

¹H－NMR(CDCl₃)δ：10.53(1H，brs)，8.57(1H，s)，7.10(1H，s)，3.18(2H，m)，3.08(2H，t，J＝5.6Hz)，2.37(2H，m).

(Process 10)

2- (2, 3, 5-tri-O-benzyl- β -D-arabinofuranosyl) -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

To a suspension of the compound (13.47g) obtained in the above step 9 in dehydrated acetonitrile (350mL) was added potassium hydroxide (10.3g) and tris [ 2- (2-methoxyethoxy) ethyl ] amine (1.13mL) in the form of powder under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1.5 hours. A solution of 2, 3, 5-tri-O-benzyl-. alpha. -D-arabinofuranosyl chloride (40.2g) in acetonitrile (100mL) known in the literature (J.Med.chem.1976, 19, 6, 814-816) was added little by little under ice-cooling, and then the mixture was warmed to room temperature and stirred for 4 hours. Insoluble material was removed by filtration and washed with acetonitrile. The filtrate was concentrated under reduced pressure, and the residue was purified 2 times by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (26.19 g).

MS(ESI)m/z：594[M+H]⁺.

¹H－NMR(CDCl₃)δ：8.51(1H，s)，7.37－7.17(14H，m)，6.86(2H，m)，6.82(1H，d，J＝4.9Hz)，4.68(1H，d，J＝11.7Hz)，4.59(1H，d，J＝11.7Hz)，4.54(1H，d，J＝13.2Hz)，4.52(1H，d，J＝11.7Hz)，4.36－4.33(2H，m)，4.22(1H，d，J＝11.7Hz)，4.14－4.08(2H，m)，3.77(1H，dd，J＝10.7，3.9Hz)，3.72(1H，dd，J＝10.5，4.1Hz)，3.13(2H，m)，2.81(2H，m)，2.27(2H，m).

(step 11)

2-beta-D-arabinofuranosyl-2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

A solution of the compound (26.19g) obtained in the above step 10 in dehydrated dichloromethane (300mL) was added to a solution of boron trichloride in dichloromethane (1M, 200mL) at-78 ℃ under a nitrogen atmosphere, stirred at the same temperature for 2 hours, then heated to 0 ℃ and stirred for 4 hours. The reaction mixture was cooled to-78 ℃ again, and a solution of methanol (80mL) in methylene chloride (160mL) was added, followed by stirring at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure and azeotroped with ethanol 2 times. Ethanol (200mL) and diethyl ether (100mL) were added to the residue to make a slurry, and the solid (solid 1) was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/methanol ]. The fraction containing the target compound was concentrated under reduced pressure, ethanol was added to the concentrated fraction to prepare a slurry, and the slurry was filtered to obtain a solid (solid 2). Combine solid 1 and solid 2 to give the title compound (13.2 g).

MS(ESI)m/z：324[M+H]⁺.

¹H－NMR(CD₃OD)δ：8.73(1H，s)，7.96(1H，s)，6.70(1H，d，J＝4.9Hz)，4.32(1H，t，J＝4.6Hz)，4.25(1H，t，J＝4.6Hz)，3.97(1H，m)，3.90(1H，dd，J＝12.0，3.2Hz)，3.85(1H，dd，J＝12.0，4.6Hz)，3.53(2H，m)，3.17(2H，m)，2.43(2H，m).

(step 12)

2- [3, 5-bis-O- (tetrahydropyran-2-yl) - β -D-arabinofuranosyl ] -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

To a dehydrated dimethylsulfoxide (160mL) solution of the compound (15.35g) obtained in the above step 11, 3, 4-dihydro-2H-pyran (17.2mL) and p-toluenesulfonic acid monohydrate (9.02g) were added at 0 ℃ and the mixture was stirred at room temperature for 3 hours. 3, 4-dihydro-2H-pyran (8.6mL) was added thereto, and after stirring for 45 minutes, triethylamine (13mL) was added immediately to stop the reaction. The reaction mixture was poured into two layers of ethyl acetate and saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was washed once with water, 2 times with saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (10.81g) as a mixture of 4 diastereomers.

MS(ESI)m/z：492[M+H]⁺.

¹H－NMR(CDCl₃)δ：8.548(0.2H，s)，8.546(0.3H，s)，8.54(0.3H，s)，8.53(0.2H，s)，7.54(0.2H，s)，7.53(0.3H，s)，7.51(0.2H，s)，7.44(0.3H，s)，6.75(0.2H，d，J＝5.4Hz)，6.71(0.2H，d，J＝5.9Hz)，6.57(0.3H，d，J＝5.9Hz)，6.56(0.3H，d，J＝5.9Hz)，4.87－4.69(2H，m)，4.55－3.54(10H，m)，3.18－3.12(2H，m)，3.10－2.96(2H，m)，2.40－2.30(2H，m)，1.92－1.51(12H，m).

(step 13)

2- [ 2-deoxo-2-fluoro-3, 5-bis-O- (tetrahydropyran-2-yl) - β -D-ribofuranosyl ] -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

Pyridine (5.3mL) and trifluoromethanesulfonic anhydride (5.6mL) were added to a solution of the compound (10.81g) obtained in the above step 12 in dehydrated dichloromethane (150mL) at 0 ℃ under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 1 hour. After the reaction was stopped by adding crushed ice to the reaction solution, the reaction solution was poured into two layers of ethyl acetate and a saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was washed with saturated brine for 2 times, and then dried over anhydrous sodium sulfate. The drying agent was filtered off and the filtrate was concentrated under reduced pressure to give the crude amorphous triflate. The obtained crude trifluoromethanesulfonate was dissolved in dehydrated tetrahydrofuran (150mL), and a tetrabutylammonium fluoride tetrahydrofuran solution (about 1M, 154mL) was added little by little under ice cooling, followed by stirring at the same temperature overnight. The reaction solution was added with a saturated aqueous ammonium chloride solution to stop the reaction. The reaction mixture was concentrated under reduced pressure to about half the volume. The residue was poured into two layers of ethyl acetate and saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The organic layer was washed once with a saturated aqueous ammonium chloride solution and 2 times with a saturated saline solution. The aqueous layer was extracted with ethyl acetate again, and the extract was washed with saturated brine. The organic layers were combined and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure to give the title compound (40.37g) as a crude product.

MS(ESI)m/z：494[M+H]⁺.

(step 14)

2- (2-deoxy-2-fluoro- β -D-ribofuranosyl) -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

To a solution of the compound (40.37g) obtained in the above step 13 in methanol (400mL) was added p-toluenesulfonic acid monohydrate (2.09g), and the mixture was stirred at 60 ℃ for 4 hours. Triethylamine (16mL) was added to the reaction mixture to stop the reaction. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ hexane/ethyl acetate → ethyl acetate/methanol ]. The fraction containing the target substance was concentrated under reduced pressure to a slurry state, and filtered to obtain a solid. The resulting solid was washed with hexane/ethyl acetate (1: 1) to give solid 1. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to obtain solid 2. Combine solid 1 and solid 2 to give the title compound (5.32 g).

MS(ESI)m/z：326[M+H]⁺.

¹H－NMR(CDCl₃)δ：8.51(1H，s)，7.01(1H，s)，6.00(1H，dd，J＝13.7，6.3Hz)，5.95(1H，dd，J＝11.7，2.0Hz)，5.87(1H，ddd，J＝52.7，6.3，4.9Hz)，4.69(1H，m)，4.32(1H，brs)，3.96(1H，d，J＝12.7Hz)，3.77(1H，m)，3.17(2H，m)，3.04(2H，m)，2.41－2.31(3H，m).

(step 15)

2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2-deoxy-2-fluoro- β -D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

Using the compound (5.32g) obtained in the above step 14, a reaction was carried out in the same manner as in step 1 of example 11 to obtain the title compound (10.1 g).

MS(ESI)m/z：628[M+H]⁺.

¹H－NMR(CDCl₃)δ：8.54(1H，s)，7.42(2H，d，J＝7.3Hz)，7.32－7.21(8H，m)，6.81(4H，m)，6.52(1H，dd，J＝17.3，2.2Hz)，5.37(1H，ddd，J＝53.3，4.4，2.4Hz)，4.76(1H，m)，4.16(1H，m)，3.789(3H，s)，3.786(3H，s)，3.59(1H，dd，J＝10.7，2.4Hz)，3.44(1H，dd，J＝10.7，3.4Hz)，3.12(2H，m)，2.76(2H，t，J＝5.6Hz)，2.27(2H，m)，2.18(1H，dd，J＝7.8，2.9Hz).

(step 16)

2- (5-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3-O- { (2-cyanoethoxy) [ bis (prop-2-yl) amino ] phosphino } -2-deoxy-2-fluoro- β -D-ribofuranosyl) -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd ] azulene

The reaction was carried out in the same manner as in step 6 of example 1 using the compound (10.1g) obtained in step 15 to obtain the title compound (12.6g) in the form of a diastereomer mixture (diastereomer ratio: 1) on the phosphorus atom.

¹H－NMR(CDCl₃)δ：8.53(1H，s)，7.40(2H，m)，7.34－7.17(8H，m)，6.84－6.74(4H，m)，6.53(0.5H，dd，J＝17.3，2.2Hz)，6.48(0.5H，dd，J＝17.6，1.5Hz)，5.50－5.31(1H，m)，4.99(0.5H，m)，4.85(0.5H，m)，4.31－4.26(1H，m)，3.93－3.76(1H，m)，3.792(1.5H，s)，3.789(1.5H，s)，3.779(1.5H，s)，3.776(1.5H，s)，3.67－3.51(4H，m)，3.34－3.30(1H，m)，3.13－3.10(2H，m)，2.76－2.69(2H，m)，2.61(1H，td，J＝6.3，2.4Hz)，2.39(1H，m)，2.28－2.21(2H，m)，1.19－1.15(9H，m)，1.03(3H，d，J＝6.8Hz).

(step 17)

Using the compound (740mg) obtained in the above-mentioned step 16, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (924mg) obtained in step 3 of example 22 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 18)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl ]Oxy } -10- (2-cyanoethoxy) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triaza-eno [ cd)]Azulene-2 (7H) -yl) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 17, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (502 mg: containing impurities) as a mixture of diastereomers on the phosphorus atom. MS (ESI) m/z: 1065(M + H)⁺.

(step 19)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulen-2 (7H) -yl) -15-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-dialkoxyoctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (502mg) obtained in the above step 18, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(67.8 mg: containing impurities) and diastereomer 2(69.5 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：908(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：908(M+H)⁺.

(step 20-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-dialkoxyoctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (67.8 mg: containing impurities) obtained in the above step 19 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 10% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 25% -75% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (21.2 mg).

MS(ESI)m/z：794(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.55(1H，brs)，8.41(1H，s)，8.09(1H，brs)，7.51(1H，s)，6.58(1H，d，J＝16.9Hz)，6.25(1H，d，J＝7.9Hz)，5.55－5.34(2H，m)，5.30－5.17(1H，m)，4.74(1H，d，J＝4.2Hz)，4.55－4.47(1H，m)，4.46－4.40(1H，m)，4.37－4.30(2H，m)，4.28－4.16(2H，m)，4.05－3.99(1H，m)，3.90－3.70(3H，m)，3.28－3.20(1H，m)，3.18－3.10(1H，m)，2.91－2.82(1H，m)，2.76－2.64(1H，m)，2.33－2.22(1H，m)，2.21－2.09(1H，m).

³¹P－NMR(CD₃OD)δ：57.6(s)，52.7(s).

(step 20-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (69.5 mg: containing impurities) obtained in the above step 19 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 10% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 25% -75% (0 min-40 min).

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (14.9 mg).

MS(ESI)m/z：794(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.60(1H，s)，8.41(1H，s)，8.16(1H，s)，7.72(1H，s)，6.60(1H，d，J＝15.7Hz)，6.28(1H，d，J＝7.9Hz)，5.61－5.33(3H，m)，4.59－4.49(2H，m)，4.48－4.39(2H，m)，4.34－4.27(1H，m)，4.25－4.16(1H，m)，4.11－3.99(3H，m)，3.86－3.75(2H，m)，3.25－3.11(2H，m)，3.05－2.90(2H，m)，2.35－2.17(2H，m).

³¹P－NMR(CD₃OD)δ：59.1(s)，57.9(s).

Example 59: synthesis of CDN49

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Azulene-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (1.80g) obtained in step 16 of example 58, a reaction was carried out in the same manner as in step 7 of example 1 to give 2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (2.30g) obtained in step 3 of example 45 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

3- { [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -7- { 1- [ 2- (1, 3-dioxido-1, 3-dihydro-2H-isoindol-2-yl) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-10-yl]Oxy-propionitrile

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (1.22g) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1090(M+H)⁺.

(step 3)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-16- { [ tert-butyl (dimethyl) silyl]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15-fluoro-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The reaction was carried out in the same manner as in step 6 of example 45 using the compound (1.22g) obtained in step 2, and then the reaction product was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], whereby diastereomer 1(108 mg: containing impurities) and diastereomer 2(111 mg: containing impurities) of the title compound were obtained.

Diastereomer 1 (low polarity)

MS(ESI)m/z：907(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：907(M+H)⁺.

(step 4-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- [ 1- (2-aminoethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Azulene-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (108 mg: containing impurities) obtained in the above step 3 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (44.4 mg).

MS(ESI)m/z：793(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.50(1H，s)，8.42(1H，s)，7.92(1H，s)，7.56(1H，s)，6.56(1H，d，J＝16.3Hz)，6.21(1H，d，J＝6.0Hz)，5.57－5.40(2H，m)，5.35－5.22(1H，m)，4.73－4.67(1H，m)，4.58－4.49(1H，m)，4.45－4.26(4H，m)，4.24－4.15(1H，m)，4.05－3.96(1H，m)，3.78－3.51(1H，m)，3.26－3.06(4H，m)，2.93－2.82(1H，m)，2.70－2.51(1H，m)，2.29－2.07(2H，m).

³¹P－NMR(CD₃OD)δ：57.5(s)，52.9(s).

(step 4-2)

(diastereomer 2)

The compound (diastereomer 2) (111 mg: containing impurities) obtained in the above step 3 was reacted by the same method as in the step 11 of example 1, and then purified by the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (40.6 mg).

MS(ESI)m/z：793(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.57(1H，s)，8.41(1H，s)，8.13(1H，s)，7.72(1H，s)，6.59(1H，dd，J＝15.7，1.8Hz)，6.26(1H，d，J＝8.5Hz)，5.61－5.34(3H，m)，4.57－4.48(2H，m)，4.48－4.38(2H，m)，4.38－4.28(2H，m)，4.08－3.98(3H，m)，3.29－3.21(2H，m)，3.20－3.12(2H，m)，3.02－2.92(1H，m)，2.92－2.81(1H，m)，2.29－2.15(2H，m).

³¹P－NMR(CD₃OD)δ：58.7(s)，57.8(s).

Example 60: synthesis of CDN50

N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dialkoxy-2, 10-bis (mercapto) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) -2-hydroxyacetamide

[ synthetic route ]

(step 1-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15-fluoro-16-hydroxy-7- { 1- [ 2- (2-hydroxyacetamide) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -2, 10-di-oxo-octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound obtained in the step 4-1 of example 59 (20.0mg) in the same manner as in the step 1-1 of example 7 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (15.6 mg).

MS(ESI)m/z：851(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.43(1H，s)，8.40(1H，brs)，7.66(1H，brs)，7.58(1H，s)，6.53(1H，d，J＝16.3Hz)，6.14(1H，d，J＝8.5Hz)，5.73－5.64(1H，m)，5.59－5.42(1H，m)，5.42－5.29(1H，m)，4.80－4.74(1H，m)，4.53－4.26(5H，m)，4.21－4.12(1H，m)，3.99－3.92(1H，m)，3.83(2H，s)，3.66－3.56(1H，m)，3.43－3.26(2H，m)，3.23－3.06(2H，m)，2.89－2.79(1H，m)，2.49－2.33(1H，m)，2.27－2.15(1H，m)，2.15－2.02(1H，m).

³¹P－NMR(CD₃OD)δ：57.0(s)，52.6(s).

(Process 1-2)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazaBenzo [ cd)]Azulene-2 (7H) -yl) -15-fluoro-16-hydroxy-7- { 1- [ 2- (2-hydroxyacetamide) ethyl]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -2, 10-di-oxo-octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound obtained in the step 4-2 of example 59 (10.0mg) in the same manner as in the step 1-1 of example 7 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (6.6 mg).

MS(ESI)m/z：851(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.46(1H，s)，8.42(1H，s)，7.84(1H，s)，7.78(1H，s)，6.59(1H，d，J＝15.1Hz)，6.20(1H，d，J＝7.9Hz)，5.69－5.38(3H，m)，4.60－4.50(2H，m)，4.48－4.38(2H，m)，4.31－4.20(2H，m)，4.10－3.93(2H，m)，3.87(2H，s)，3.73－3.57(2H，m)，3.52－3.41(1H，m)，3.25－3.10(2H，m)，3.01－2.90(1H，m)，2.83－2.71(1H，m)，2.30－2.11(2H，m).

³¹P－NMR(CD₃OD)δ：58.2(s)，57.6(s).

Example 61: synthesis of CDN51

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-bis (mercapto) octahydro-2H, 10H,12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (1.80g) obtained in step 16 of example 58, a reaction was carried out in the same manner as in step 7 of example 1 to give 2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -2, 7, 8, 9-tetrahydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (3.10g) obtained in step 6 of example 47 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- (trimethylsilyl) ethyl [ 2- ({ 6-benzamide-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triaza-eno [ cd)]Azulene-2 (7H) -yl) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Carbamates, their preparation and their use

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (1.83 g: containing impurities).

MS(ESI)m/z：1222(M+H)⁺.

(step 3)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-2- { [ 2- ({ [ 2- (trimethylsilyl) ethoxy ] ethyl]Carbonyl } amino) ethyl]Amino } -9H-purin-9-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd)]Azulene-2 (7H) -yl) -15-fluoro-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the mixture (1.83g) obtained in the above step 2, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(151 mg: containing impurities) and diastereomer 2(103 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1065(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1065(M+H)⁺.

(step 4-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 6-amino-2- [ (2-aminoethyl) amino group]-9H-purin-9-yl } -14- (8, 9-dihydro-6-thioxo-2, 3, 5-triazabenzo [ cd]Azulene-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (151 mg: containing impurities) obtained in the above step 3 in the same manner as in the step 5 of example 40 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (10.6 mg).

MS(ESI)m/z：807(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.39(1H，s)，7.97(1H，brs)，7.55(1H，s)，6.51(1H，d，J＝16.3Hz)，6.00－5.92(1H，m)，5.83－5.65(1H，m)，5.44(1H，dd，J＝52.0，3.6Hz)，5.34－5.20(1H，m)，4.77(1H，d，J＝3.6Hz)，4.49－4.31(5H，m)，4.11－4.07(1H，m)，3.28－2.72(8H，m)，2.29－1.99(2H，m).

³¹P－NMR(CD₃OD)δ：57.3(s)，52.3(s).

(step 4-2)

(diastereomer 2)

The compound (diastereomer 2) (103 mg: containing impurities) obtained in the above step 3 was reacted by the same method as in the step 5 of example 40, and then purified according to the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (12.1 mg).

MS(ESI)m/z：807(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.39(1H，s)，7.95(1H，brs)，7.82(1H，s)，6.58(1H，d，J＝15.1Hz)，6.00－5.95(1H，m)，5.90－5.71(1H，m)，5.40(1H，dd，J＝51.7，3.3Hz)，5.38－5.25(1H，m)，4.53－4.39(4H，m)，4.28－4.18(2H，m)，4.10－4.05(1H，m)，3.27－2.54(8H，m)，2.34－2.10(2H，m).

³¹P－NMR(CD₃OD)δ：57.9(s)，57.3(s).

Example 62: synthesis of CDN52

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-2, 16-dihydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-10-mercapto-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (1.00g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. Using the obtained acetonitrile solution andthe compound (1.13g) obtained in step 3 of example 22 was reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-hydroxy-2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

The crude pyridine (32.5mL) solution obtained in step 1 was concentrated to about 25mL, and 2-chloro-5, 5-dimethyl-1, 3, 2. lambda. was added⁵Dioxophosphadin-2-one (945mg), stirred at room temperature for 30 min. Iodine (1.11g) was added to the reaction solution, and the mixture was stirred for 1 hour. The reaction mixture was poured into an aqueous solution (150mL) of sodium hydrogencarbonate (4.30g), stirred for 30 minutes, and then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [ hexane/ethyl acetate/methanol ]]Purification was conducted to give the title compound (671 mg: containing impurities) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1136(M+H)⁺.

(step 3)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl ]Oxy } -15-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-ol salt

Using the compound (671mg) obtained in the above-mentioned step 2, a reaction was carried out in the same manner as in the step 10 of example 1 to give diastereomer 1(55.6 mg: containing impurities) and diastereomer 2(65.7 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：875(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：875(M+H)⁺.

(step 4-1)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-ol salt

(diastereomer 1)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 1) (55.6 mg: containing impurities) obtained in the above step 3 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (11.3 mg).

MS(ESI)m/z：761(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.55(1H，m)，8.15(1H，m)，8.03(1H，s)，7.14(1H，d，J＝4.8Hz)，6.46(1H，d，J＝18.1Hz)，6.28(1H，d，J＝7.9Hz)，5.50－5.29(2H，m)，5.16－5.04(1H，m)，4.74－4.69(1H，m)，4.40－4.18(6H，m)，4.13－4.07(1H，m)，4.02－3.91(1H，m)，3.84－3.74(2H，m)，3.53－3.43(2H，m)，2.81－2.63(2H，m)，2.02－1.85(2H，m).

³¹P－NMR(CD₃OD)δ：53.4(s)，－0.86(s).

(step 4-2)

(diastereomer 2)

The title compound was obtained as a triethylamine salt by reacting the compound (diastereomer 2) (65.7 mg: containing impurities) obtained in the above step 3 in the same manner as in the step 11 of example 1 and then purifying the reaction mixture under the following purification conditions.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (23.5 mg).

MS(ESI)m/z：761(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，d，J＝3.0Hz)，8.19(1H，d，J＝2.4Hz)，8.03(1H，s)，7.40(1H，s)，6.49(1H，dd，J＝16.3，1.8Hz)，6.29(1H，d，J＝8.5Hz)，5.51－5.22(3H，m)，4.59－4.55(1H，m)，4.43－4.17(5H，m)，4.14－4.01(3H，m)，3.85－3.78(2H，m)，3.51－3.44(2H，m)，2.90－2.75(2H，m)，1.99－1.90(2H，m).

³¹P－NMR(CD₃OD)δ：59.7(s)，－0.75(s).

Example 63: synthesis of CDN53

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-10, 16-dihydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2-mercapto-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (1.02g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Acetonitrile solution of azulene (acetonitrile solution a). The compound (1.23g) obtained in step 3 of example 22 was azeotroped 3 times with dehydrated acetonitrile (10 mL). About 7mL of acetonitrile remained in the last step, and molecular sieves 3A and 1/16 (5 particles in a lump form) (acetonitrile solution B) were added. The acetonitrile solution A and the acetonitrile solution B were mixed and stirred at room temperature for 15 minutes under a nitrogen atmosphere. A decane solution (5.5M, 0.50mL) of tert-butyl hydroperoxide was added to the reaction mixture, and after stirring for 40 minutes, the reaction mixture was ice-cooled, and an aqueous solution (1.1mL) of sodium thiosulfate 5 hydrate (826mg) was added and stirred for 10 minutes. Adding into the reaction solutionWater (20mL) was added, and extraction was performed with a dichloromethane-methanol mixture. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. To a solution of the residue in dichloromethane (15.9mL) were added water (0.200mL) and a solution of dichloroacetic acid (1.00mL) in dichloromethane (15.9mL) in this order, and the mixture was stirred at room temperature for 15 minutes. Pyridine (11.0mL) was added to the reaction mixture to stop the reaction, and then the reaction mixture was concentrated under reduced pressure. The crude product obtained was used directly in the next reaction.

(step 2)

N, N-diethylethanaminium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- { 1- [ 2- (benzoyloxy) ethyl ]-6-oxo-1, 6-dihydro-9H-purin-9-yl } -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2, 10-dioxidooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-thiolate

The crude product obtained in the above step 1 was reacted by the same method as in the step 9 of example 1 to obtain the title compound (122 mg: containing impurities).

MS(ESI)m/z：1136(M+H)⁺.

(step 3)

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-10-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2-mercapto-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

The reaction was carried out in the same manner as in step 10 of example 1 using the compound (122mg, containing impurities) obtained in step 2, and the obtained crude product was used as it was in the next reaction.

MS(ESI)m/z：875(M+H)⁺.

(step 4)

Disodium (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-10-ol salt

The reaction was carried out in the same manner as in step 11 of example 1 using the crude product obtained in step 3, and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 2% -30% (0 min-30 min) to obtain the title compound in the form of triethylamine salt. The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (30 mg).

MS(ESI)m/z：761(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，s)，8.12(1H，s)，8.02(1H，s)，7.13(1H，s)，6.47(1H，d，J＝18.1Hz)，6.27(1H，d，J＝8.5Hz)，5.41(1H，dd，J＝51.7，3.9Hz)，5.29－5.16(2H，m)，4.58－4.52(2H，m)，4.36－4.17(5H，m)，4.05－3.90(2H，m)，3.82－3.71(2H，m)，3.52－3.43(2H，m)，2.76－2.63(2H，m)，2.02－1.85(2H，m).

³¹P－NMR(CD₃OD)δ：58.0(s)，－0.97(s).

Example 64: synthesis of CDN54

(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-2, 10, 16-trihydroxy-7- [ 1- (2-hydroxyethyl) -6-oxo-1, 6-dihydro-9H-purin-9-yl]-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H,12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-diones

[ synthetic route ]

(step 1)

Using the compound (1.00g) obtained in the step 8 of example 44 and the compound (1.13g) obtained in the step 3 of example 22, a reaction was carried out in the same manner as in the step 1 of example 63, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-hydroxy-2, 10-di-oxo-octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethylbenzoate

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 62 to obtain the title compound (602 mg: containing impurities) as a mixture of diastereomers on the phosphorus atom.

MS(ESI)m/z：1120(M+H)⁺.

(step 3)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-7- [ 1- (2-hydroxyethyl) -6-oxoRadical-1, 6-dihydro-9H-purin-9-yl]-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-bis (alkoxide)

Using the compound (602mg) obtained in the above step 2, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain the title compound (90.8 mg: containing impurities).

MS(ESI)m/z：859(M+H)⁺.

(step 4)

The compound (90.8 mg: containing impurities) obtained in the above step 3 was reacted in the same manner as in the step 11 of example 1, and then purified according to the following purification conditions to obtain the title compound as a triethylamine salt.

The triethylamine salt thus obtained was subjected to salt conversion in the same manner as in [ to sodium salt ] in step 11 of example 1 to obtain the title compound (40.4 mg).

MS(ESI)m/z：745(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.56(1H，s)，8.19(1H，s)，8.03(1H，s)，7.10(1H，s)，6.46(1H，d，J＝18.7Hz)，6.31(1H，d，J＝8.5Hz)，5.51－5.32(1H，m)，5.24－5.01(2H，m)，4.61－4.56(1H，m)，4.41－4.21(5H，m)，4.21－3.97(3H，m)，3.87－3.75(2H，m)，3.54－3.41(2H，m)，2.83－2.67(2H，m)，2.02－1.85(2H，m).

³¹P－NMR(CD₃OD)δ：－0.59(s)，－0.81(s).

Example 65: synthesis of drug linker 5

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15, 16-bis { [ tert-butyl (dimethyl) silyl]Oxy } -7- (6- { [ (glycylamino) methoxy)]Methyl } -9H-purin-9-yl) -2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (diastereomer 2) (80.6mg) obtained in step 7 of example 17, a reaction was carried out in the same manner as in step 7-1 of example 22 to obtain the title compound (66.1 mg: containing impurities).

MS(ESI)m/z：1059(M+H)⁺.

(step 2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6- { [ (glycylamino) methoxy-amino) ]Methyl } -9H-purin-9-yl) -15, 16-dihydroxy-2, 10-dialkoxy-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

Using the compound (66.1mg) obtained in the above step 1, a reaction was carried out in the same manner as in the step 8-1 of example 22 to obtain the title compound (40.4 mg: containing impurities).

MS(ESI)m/z：831(M+H)⁺.

(step 3)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ ({ 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-6-yl } methoxy) methyl]Glycine amides

(drug linker 5)

The reaction was carried out in the same manner as in step 9-1 of example 22 using the compound obtained in step 2 (40.4mg), and then column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% to 45% (0 part to 30 parts) was purified to obtain the title compound (5.0 mg).

MS(ESI)m/z：1379(M+H)⁺.

¹H－NMR(CD₃OD)δ：9.17(1H，s)，8.83(1H，d，J＝4.8Hz)，8.01(1H，d，J＝3.6Hz)，7.63－7.45(2H，m)，7.40－7.34(3H，m)，7.29－7.12(8H，m)，7.09(1H，s)，6.47(1H，d，J＝8.5Hz)，6.31(1H，d，J＝6.7Hz)，5.56－5.47(2H，m)，5.05－4.91(4H，m)，4.88－4.73(3H，m)，4.57－4.30(5H，m)，4.09－4.01(1H，m)，4.00－3.55(9H，m)，3.52－3.45(2H，m)，3.18(12H，q，J＝7.3Hz)，3.02－2.94(1H，m)，2.90－2.71(3H，m)，2.35－2.20(2H，m)，2.04－1.92(3H，m)，1.27(18H，t，J＝7.3Hz).

Example 66: synthesis of drug linker 6

[ synthetic route ]

(step 1)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanyl-N- ({ 2- [ (2, 5-dioxinopyrrolidin-1-yl) oxy ] -2-oxoethoxy } methyl) glycinamide

To a solution of { [ (N- { [ (9H-fluoren-9-yl) methoxy ] carbonyl } glycyl) amino ] methoxy } acetic acid (955mg) in N, N-dimethylformamide (8.0mL) known in the literature (WO2014/057687) was added 1, 8-diazabicyclo [5.4.0] -7-undecene (0.74mL), and the mixture was stirred at room temperature for 1 hour (reaction solution A). To a solution of the compound (938mg) obtained in step 10 of example 22 in N, N-dimethylformamide (8.0mL) were added N-hydroxysuccinimide (229mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (380mg), and the mixture was stirred at room temperature for 50 minutes (reaction solution B). The reaction solution A was added to the reaction solution B, and stirred at room temperature for 1 hour. Methylene chloride (50mL) and a 10% aqueous solution of citric acid (10mL) were added to the reaction mixture, and the mixture was extracted with methylene chloride. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ chloroform/(chloroform/methanol/water: 7: 3: 1 lower layer) ]. To a solution of the obtained compound in N, N-dimethylformamide (8.0mL) were added N-hydroxysuccinimide (229mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (380mg), and the mixture was stirred at room temperature for 30 minutes. Methylene chloride (100mL) and water (25mL) were added to the reaction mixture, and the mixture was extracted with methylene chloride. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ chloroform/methanol ]. The fraction containing the target substance was concentrated under reduced pressure, and diethyl ether was added to the residue to prepare a slurry. The resulting solid was filtered to give the title compound (412 mg).

¹H－NMR(DMSO－d₆)δ：8.72(1H，m)，8.32(1H，m)，8.17－7.96(3H，m)，7.71－7.15(13H，m)，5.01(1H，d，J＝13.9Hz)，4.70－4.48(5H，m)，3.81－3.51(7H，m)，3.05(1H，dd，J＝14.2，3.9Hz)，2.83(4H，s)，2.80(1H，m)，2.64(1H，m)，2.28(1H，m)，2.07(1H，m)，1.79(1H，m).

(step 2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- ({ 2- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) amino]-2-oximinoethoxy } methyl) glycinamide

(drug linker 6)

Using the compound obtained in step 8-2 of example 5 (20.0mg) and the compound obtained in step 1 (23.7mg), a reaction was carried out in the same manner as in step 4 of example 21, and then subjected to C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 30% to 80% (0 part to 40 parts) was purified to obtain the title compound (14.1 mg).

MS(ESI)m/z：1466(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.74－8.69(1H，m)，8.17－8.12(1H，m)，8.04－7.96(1H，m)，7.65－7.13(13H，m)，7.13－7.04(1H，m)，6.33－6.23(2H，m)，5.50－5.31(2H，m)，5.07－4.94(2H，m)，4.85－4.76(1H，m)，4.71－4.26(10H，m)，4.18－3.53(13H，m)，3.53－3.33(3H，m)，3.19(12H，q，J＝7.3Hz)，3.05－2.92(1H，m)，2.91－2.70(3H，m)，2.40－2.20(2H，m)，2.07－1.87(2H，m)，1.29(18H，t，J＝7.3Hz).

Example 67: synthesis of drug linker 7

[ synthetic route ]

(step 1)

[ (N- { [ (9H-fluoren-9-yl) methoxy ] carbonyl } -L-isoleucyl) amino ] methyl acetate

To a commercial (IRIS) mixture of N- { [ (9H-fluoren-9-yl) methoxy ] carbonyl } -L-isoleucyl glycine (2.50g) in tetrahydrofuran (45mL) -toluene (15mL) was added pyridine (0.588mL) and lead tetraacetate (3.24g) at room temperature, and the mixture was stirred at 65 ℃ for 3 hours. The reaction mixture was diluted with ethyl acetate, and washed with water and saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (2.06 g).

MS(ESI)m/z：447(M+Na)⁺.

¹H－NMR(CDCl₃)δ：7.77(2H，d，J＝7.9Hz)，7.58(2H，d，J＝7.3Hz)，7.40(2H，t，J＝7.3Hz)，7.32(2H，td，J＝7.6，1.2Hz)，6.97－6.92(1H，brm)，5.32－5.20(3H，m)，4.47－4.37(2H，m)，4.21(1H，t，J＝6.7Hz)，4.05－4.01(1H，m)，2.04(3H，s)，1.92－1.84(1H，brm)，1.51－1.41(1H，brm)，1.17－1.07(1H，brm)，0.93－0.89(6H，m).

(step 2)

Benzyl { [ (N- { [ (9H-fluoren-9-yl) methoxy ] carbonyl } -L-isoleucyl) amino ] methoxy } acetate

To a suspension of the compound (2.06g) obtained in the above step 1 in tetrahydrofuran (48mL) were added benzyl glycolate (1.38mL) and p-toluenesulfonic acid monohydrate (92.3mg) at 0 ℃ and the mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate, washed with saturated sodium bicarbonate water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (1.72 g).

MS(ESI)m/z：553(M+Na)⁺.

¹H－NMR(CDCl₃)δ：7.76(2H，d，J＝7.9Hz)，7.58(2H，d，J＝7.3Hz)，7.42－7.29(9H，m)，6.74－6.69(1H，brm)，5.24－5.21(1H，brm)，5.17(2H，s)，4.86(2H，d，J＝6.7Hz)，4.48－4.39(2H，m)，4.23－4.19(3H，m)，4.04－4.00(1H，m)，1.95－1.87(1H，brm)，1.50－1.41(1H，brm)，1.15－1.07(1H，brm)，0.93－0.89(6H，m).

(step 3)

N- [ (benzyloxy) carbonyl ] glycylglycinyl-L-prolyl-N- { [ 2- (benzyloxy) -2-epioxyethoxy ] methyl } -L-isoleucylamine

To a suspension of the compound (1.72g) obtained in the above step 2 in acetonitrile (40mL) was added 1, 8-diazabicyclo [5.4.0] -7-undecene (0.290mL) at room temperature, and the mixture was stirred for 1 hour (reaction solution A). To a suspension of commercially available N- [ (benzyloxy) carbonyl ] glycylglycinyl-L-proline (1.41g) in acetonitrile (20mL) was added 3H- [1, 2, 3] triazolo [4, 5-b ] pyridin-3-ol (529mg), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (746mg), and N, N-diisopropylethylamine (0.678mL) at room temperature, and the mixture was stirred for 1 hour. This reaction solution was added to the above reaction solution A at room temperature, stirred for 6 hours, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ chloroform/methanol ] to give the title compound (1.56 g).

MS(ESI)m/z：676(M+Na)⁺.

¹H－NMR(CD₃OD)δ：7.37－7.27(10H，m)，5.17(2H，s)，5.09(2H，s)，4.82－4.70(2H，m)，4.56－4.44(1H，m)，4.17－4.14(3H，m)，4.07－3.96(2H，m)，3.83－3.82(2H，m)，3.64－3.48(2H，m)，2.34－1.78(5H，m)，1.61－1.51(1H，m)，1.25－1.13(1H，m)，0.95－0.87(6H，m).

(step 4)

glycylglycinyl-L-prolyl-N- [ (carboxymethoxy) methyl ] -L-isoleucylamine

To a mixture of the compound (1.56g) obtained in the above step 3 in methanol (8mL) -tetrahydrofuran (24mL) -dichloromethane (8mL) was added 10% palladium on carbon (M) wet (1.4g), and the mixture was stirred at room temperature under a hydrogen atmosphere for 23 hours. A mixture (50mL) of methanol/tetrahydrofuran (1: 1) was added to the reaction mixture, and the mixture was filtered through celite. The celite was washed with a methanol/tetrahydrofuran (1: 1) mixture. After the filtrate was concentrated under reduced pressure, methanol (20mL), tetrahydrofuran (20mL), and 10% palladium on carbon (M) wet (1.0g) were added to the residue, and the mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered through celite, and the celite was washed with methanol/tetrahydrofuran (1: 1). The filtrate was concentrated under reduced pressure to give the title compound as a crude product (1.02 g).

MS(ESI)m/z：430(M+H)⁺.

(step 5)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-N- [ (carboxymethoxy) methyl ] -L-isoleucylamine

1- { [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] oxy } pyrrolidine-2, 5-dione (956mg) and N, N-diisopropylethylamine (0.496mL) were added to a solution of the compound (1.02g) obtained in the above step 4 in N, N-dimethylformamide (24mL), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ chloroform/methanol ]. After the fraction containing the target compound was concentrated under reduced pressure, ethyl acetate was added to the residue to solidify it. The resulting solid was filtered to give the title compound (1.06 g).

MS(ESI)m/z：739(M+Na)⁺，715(M－H)^－.

¹H－NMR(CD₃OD)δ：7.64－7.59(2H，m)，7.48－7.44(3H，m)，7.38－7.23(3H，m)，5.15－5.12(1H，m)，4.74－4.70(2H，m)，4.64－4.48(1H，m)，4.25－4.18(1H，m)，4.11－3.96(3H，m)，3.91－3.85(1H，m)，3.78－3.57(5H，m)，2.84－2.75(1H，m)，2.39－2.16(3H，brm)，2.08－1.84(5H，brm)，1.61－1.52(1H，brm)，1.23－1.15(1H，brm)，0.96－0.87(6H，m).

(step 6)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-N- ({ 2- [ (2, 5-dioxinopyrrolidin-1-yl) oxy ] -2-oxoethoxy } methyl) -L-isoleucylamine

To a suspension of the compound (1.06g) obtained in the above step 5 in N, N-dimethylformamide (16mL) were added N-hydroxysuccinimide (187mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (312mg), and the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with chloroform and washed with water 3 times. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the residue, and the resulting solid was filtered. To the solid obtained by filtration was added diethyl ether to prepare a slurry, and the solid was filtered to obtain the title compound (767 mg).

MS(ESI)m/z：836(M+Na)⁺.

¹H－NMR(CD₃OD)δ：7.68－7.56(2H，m)，7.48－7.44(3H，m)，7.38－7.23(3H，m)，5.15－5.12(1H，m)，4.83－4.70(2H，m)，4.64－4.50(3H，m)，4.24－3.55(8H，m)，2.83－2.76(5H，m)，2.38－2.18(3H，m)，2.11－1.84(5H，brm)，1.60－1.52(1H，m)，1.25－1.13(1H，m)，0.98－0.87(6H，m).

(step 7)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-prolyl-N- ({ 2- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l ][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) amino]-2-oximinoethoxy } methyl) -L-isoleucylamine

(drug linker 7)

Using the compound obtained in step 8-2 of example 5 (20.0mg) and the compound obtained in step 6 (20.9mg), a reaction was carried out in the same manner as in step 4 of example 21, and then subjected to preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 30% to 80% (0 part to 40 parts) was purified to give the title compound (11.9 mg).

MS(ESI)m/z：1472(M+H)⁺

¹H－NMR(CD₃OD)δ：8.71(1H，brs)，8.19－8.13(1H，m)，8.02(1H，s)，7.64－7.56(2H，m)，7.48－7.41(3H，m)，7.37－7.08(4H，m)，6.34－6.23(2H，m)，5.48－5.36(2H，m)，5.16－5.08(1H，m)，4.98－4.55(6H，m)，4.52－4.15(7H，m)，4.07－3.81(6H，m)，3.80－3.45(8H，m)，3.19(12H，q，J＝7.3Hz)，2.93－2.85(2H，m)，2.85－2.71(1H，m)，2.45－2.30(1H，m)，2.30－2.11(2H，m)，2.08－1.83(6H，m)，1.66－1.45(1H，m)，1.29(18H，t，J＝7.3Hz)，1.28－1.10(1H，m)，1.01－0.85(7H，m).

Example 68: synthesis of drug linker 8

[ synthetic route ]

(step 1)

N- (tert-butoxycarbonyl) glycylglycyl-L-valyl-L-alanine

To a commercial (ChemFun) solution of N- (tert-butoxycarbonyl) glycylglycine (5.00g) and N-hydroxysuccinimide (2.97g) in N, N-dimethylformamide (50mL) was added 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (4.95g) at 0 ℃ under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1 hour. Triethylamine (3.6mL) and commercially available (domestic chemical) L-valinyl-L-alanine (4.05g) were added to the reaction mixture, and the mixture was stirred at the same temperature for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [ dichloromethane/methanol ]. The fraction containing the target compound was concentrated under reduced pressure, and diethyl ether was added to the concentrated fraction to prepare a slurry. The resulting solid was filtered to give the title compound (4.99 g).

MS(ESI)m/z：401(M－H)^－.

¹H－NMR(DMSO－d₆)δ：12.43(1H，br)，8.27(1H，d，J＝6.8Hz)，7.92(1H，t，J＝5.4Hz)，7.79(1H，d，J＝9.3Hz)，6.99(1H，t，J＝5.9Hz)，4.19(1H，t，J＝8.1Hz)，4.13(1H，m)，3.73(2H，d，J＝5.4Hz)，3.52(2H，d，J＝5.9Hz)，1.92(1H，dd，m)，1.35(9H，s)，1.24(3H，d，J＝7.3Hz)，0.85(3H，d，J＝6.3Hz)，0.79(3H，d，J＝6.8Hz).

(step 2)

N- (ammonioacetyl) glycyl-L-valyl-L-alanine trifluoroacetate

Using the compound (1.00g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 1 of example 21 to obtain the title compound (1.10g) as a crude product.

MS(ESI)m/z：301(M－H)^－.

¹H－NMR(DMSO－d₆)δ：12.49(1H，br)，8.54(1H，t，J＝5.6Hz)，8.34(1H，d，J＝6.8Hz)，8.03(1H，d，J＝9.3Hz)，8.00(3H，brs)，4.24(1H，dd，J＝8.8，6.8Hz)，4.17(1H，m)，3.93－3.84(2H，m)，3.62－3.58(2H，m)，1.96(1H，m)，1.27(3H，d，J＝7.3Hz)，0.89(3H，d，J＝6.8Hz)，0.84(3H，d，J＝6.8Hz).

(step 3)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-valyl-L-alanine

Using the compound (861mg) obtained in the above step 2, a reaction was carried out in the same manner as in step 2 of example 21 to obtain the title compound (737 mg).

MS(ESI)m/z：590(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.46(1H，s)，8.28(1H，t，J＝8.1Hz)，8.18(1H，t，J＝5.9Hz)，8.13(0.5H，t，J＝5.6Hz)，8.04(0.5H，t，J＝5.9Hz)，7.99(0.5H，t，J＝5.9Hz)，7.73(0.5H，d，J＝1.5Hz)，7.67(1H，m)，7.61(1H，m)，7.53－7.44(3H，m)，7.40－7.28(3H，m)，5.03(0.5H，d，J＝6.3Hz)，5.00(0.5H，d，J＝6.3Hz)，4.21(1H，dd，J＝9.0，7.1Hz)，4.18－4.08(1H，m)，3.78－3.67(2H，m)，3.65－3.55(3H，m)，2.72－2.59(1H，m)，2.28(1H，d，J＝7.8Hz)，2.09－2.03(1H，m)，1.99－1.91(1H，m)，1.79(1H，m)，1.25(1.5H，d，J＝5.4Hz)，1.24(1.5H，d，J＝5.4Hz)，0.87(3H，d，J＝6.8Hz)，0.82－0.79(3H，m).

(step 4)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-valyl-L-alanine ester

Using the compound (260mg) obtained in the above step 3, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (84 mg).

MS(ESI)m/z：687(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.73(1H，t，J＝7.0Hz)，8.18(0.5H，t，J＝5.7Hz)，8.12(0.5H，t，J＝5.7Hz)，8.04(0.5H，t，J＝5.7Hz)，7.98(0.5H，t，J＝5.7Hz)，7.84－7.60(3H，m)，7.52－7.44(3H，m)，7.40－7.28(3H，m)，5.03(0.5H，d，J＝6.0Hz)，5.00(0.5H，d，J＝6.0Hz)，4.69－4.58(1H，m)，4.22(1H，t，J＝7.6Hz)，3.80－3.54(5H，m)，2.80(4H，s)，2.73－2.59(1H，m)，2.34－2.24(1H，m)，2.11－2.02(1H，m)，1.99－1.91(1H，m)，1.79(1H，m)，1.45(1.5H，d，J＝3.6Hz)，1.43(1.5H，d，J＝3.6Hz)，0.85(3H，d，J＝6.7Hz)，0.83－0.79(3H，m).

(step 5)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl ]glycylglycinyl-L-valyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-L-alaninamide

(drug linker 8)

Using the compound obtained in step 8-2 of example 8 (5.3mg) and the compound obtained in step 4 (4.5mg), a reaction was carried out in the same manner as in step 1 of example 23, followed by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% to 35% (0 part to 30 parts) was purified to give the title compound (5.1 mg).

MS(ESI)m/z：1359(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.37(1H，brs)，8.02(1H，s)，7.66－7.13(9H，m)，6.33(1H，d，J＝6.7Hz)，6.13(1H，d，J＝9.1Hz)，5.49－5.42(2H，m)，5.13－5.04(1H，m)，4.83－4.80(1H，m)，4.51－3.63(14H，m)，3.51－3.37(4H，m)，3.14(12H，q，J＝7.3Hz)，2.90－2.72(5H，m)，2.36－1.96(6H，m)，1.34－1.25(21H，m)，0.98－0.86(6H，m).

Example 69: synthesis of drug linker 9

[ synthetic route ]

(step 1)

N- (ammonioacetyl) glycyl-L-prolyl-L-isoleucine trifluoroacetate

Using N- (tert-butoxycarbonyl) glycylglycyl-L-prolyl-L-isoleucine (1.00g) obtained from a commercial product (Hangzhou Peptide Biochem), the reaction was carried out in the same manner as in step 1 of example 21 to give the title compound (1.02g) as a crude product.

MS(ESI)m/z：343(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.61(1H，s)，8.51(0.7H，t，J＝5.1Hz)，8.48(0.3H，t，J＝4.9Hz)，8.35(0.3H，d，J＝8.8Hz)，8.05(0.7H，d，J＝8.3Hz)，7.99(3H，brs)，4.55(0.3H，dd，J＝8.3，2.4Hz)，4.46(0.7H，dd，J＝8.8，2.9Hz)，4.23(0.3H，dd，J＝8.5，5.6Hz)，4.15(0.7H，dd，J＝8.3，5.9Hz)，4.06(0.7H，dd，J＝17.6，5.4Hz)，3.99－3.95(1H，m)，3.63－3.36(4.3H，m)，2.28－2.20(0.3H，m)，2.07－2.00(0.7H，m)，1.96－1.74(4H，m)，1.46－1.36(1H，m)，1.26－1.13(1H，m)，0.89－0.83(6H，m).

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-L-isoleucine

Using the compound (1.02g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 21 to obtain the title compound (993 mg).

MS(ESI)m/z：630(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.57(1H，s)，8.30(0.3H，dd，J＝8.5，3.6Hz)，8.14－8.06(1H，m)，8.00(0.7H，d，J＝7.9Hz)，7.88－7.80(1H，m)，7.70－7.66(1H，m)，7.63－7.60(1H，m)，7.53－7.27(6H，m)，5.04(0.7H，d，J＝14.5Hz)，5.02(0.3H，d，J＝14.5Hz)，4.56(0.3H，dd，J＝8.5，2.4Hz)，4.47－4.43(0.7H，m)，4.20(0.3H，dd，J＝8.2，5.7Hz)，4.12(0.7H，m)，3.99－3.36(7H，m)，2.68－2.58(1H，m)，2.34－2.18(1.3H，m)，2.09－1.74(6.7H，m)，1.44－1.34(1H，m)，1.25－1.12(1H，m)，0.87－0.80(6H，m).

(step 3)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-L-isoleucine ester

Using the compound (300mg) obtained in the above step 2, a reaction was carried out in the same manner as in the step 3 of example 21 to obtain the title compound (287 mg).

MS(ESI)m/z：727(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.77－8.67(0.3H，m)，8.50(0.5H，d，J＝8.3Hz)，8.31(0.2H，m)，8.10－8.04(1H，m)，7.88－7.79(1H，m)，7.68－7.57(2H，m)，7.49－7.26(6H，m)，5.01(1H，d，J＝14.2Hz)，4.80－4.42(2H，m)，3.97－3.84(1H，m)，3.79－3.34(6H，m)，2.78(4H，s)，2.64－2.57(1H，m)，2.30－1.74(8H，m)，1.56－1.17(2H，m)，1.00－0.80(6H，m).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-prolyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-di-bridgeOxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-L-isoleucylamine

(drug linker 9)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (51.8mg) and the compound obtained in step 3 (36.9mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% to 35% (0 part to 30 parts) was purified to give the title compound (62.5 mg).

MS(ESI)m/z：1397(M－H)^－.

Example 70: synthesis of drug linker 10

[ synthetic route ]

(step 1)

N- (ammonioacetyl) glycyl-L-phenylalanyl-L-methionine trifluoroacetate

The reaction was carried out in the same manner as in step 1 of example 21 using commercially available N- (tert-butoxycarbonyl) glycylglycyl-L-phenylalanyl-L-methionine (1.00g) of Hangzhou Peptide Biochem to obtain the title compound (1.19g) as a crude product.

MS(ESI)m/z：411(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.48(1H，t，J＝5.4Hz)，8.41(1H，d，J＝7.8Hz)，8.27(1H，d，J＝8.3Hz)，7.97(3H，m)，7.28－7.18(5H，m)，4.58(1H，m)，4.32(1H，m)，3.85(1H，dd，J＝17.1，5.9Hz)，3.68(1H，dd，J＝16.6，5.4Hz)，3.56(2H，m)，3.04(1H，dd，J＝13.9，3.7Hz)，2.74(1H，dd，J＝13.7，10.3Hz)，2.47(1H，m)，2.05(3H, s), 2.00(1H, m), 1.88(1H, m), 1.48(1H, d, J ═ 10.3Hz) (only the peaks observed are described)

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-phenylalanyl-L-methionine

Using the compound (1.03g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 21 to obtain the title compound (652 mg).

MS(ESI)m/z：698(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.67(1H，brs)，8.28(0.7H，d，J＝7.9Hz)，8.25(0.3H，d，J＝7.9Hz)，8.17(0.7H，t，J＝5.7Hz)，8.10(0.3H，t，J＝5.7Hz)，8.04－7.93(2H，m)，7.73－7.15(13H，m)，5.00(1H，d，J＝13.9Hz)，4.56－4.49(1H，m)，4.35－4.28(1H，m)，3.74－3.49(5H，m)，3.03(1H，dd，J＝13.9，3.6Hz)，2.79－2.22(5H，m)，2.11－1.76(4H，m)，2.02(2.1H，s)，2.02(0.9H，s).

(step 3)

2, 5-Bisoroxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-Oxdobutyryl ] glycylglycinyl-L-phenylalaninyl-L-methionine ester

Using the compound (201mg) obtained in the above step 2, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (95 mg).

MS(ESI)m/z：795(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.84－8.59(1H，m)，8.17－7.96(3H，m)，7.72－7.15(13H，m)，5.01(0.55H，d，J＝14.5Hz)，5.00(0.45H，d，J＝13.9Hz)，4.86－4.74(1H，m)，4.56－4.48(1H，m)，3.75－3.50(5H，m)，3.05－2.52(6H，m)，2.82(4H，brs)，2.33－1.73(4H，m)，2.05(3H，s).

(step 4)

Bis (N, N-Diethylammonium ethyl) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl ]-L-methionine amide

(drug linker 10)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (6.0mg) and the compound obtained in step 3 (5.5mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) to give the title compound (2.0 mg).

MS(ESI)m/z：1467(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.23(1H，brs)，7.93(1H，s)，7.54－7.07(14H，m)，6.23(1H，d，J＝6.7Hz)，6.05－5.98(1H，m)，5.41－5.31(2H，m)，5.00－4.93(1H，m)，4.73－4.69(1H，m)，4.41－3.52(14H，m)，3.44－2.62(12H，m)，3.08(12H，q，J＝7.3Hz)，2.38－2.13(3H，m)，2.02－1.80(8H，m)，1.19(18H，t，J＝7.3Hz).

Example 71: synthesis of drug linker 11

[ synthetic route ]

(step 1)

N- (ammonioacetyl) glycyl-L-valyl-N⁵-carbamoyl-L-ornithine trifluoroacetate

Use marketN- (tert-Butoxycarbonyl) glycylglycyl-L-valyl-N- (tert-Butoxycarbonyl) of commercial (Hangzhou Peptide Biochem)⁵Reaction of (E) -carbamoyl-L-ornithine (1.00g) was carried out in the same manner as in step 1 of example 21 to give the title compound (1.02g) as a crude product.

MS(ESI)m/z：389(M+H)⁺.

¹H－NMR(DMSO－d₆) δ: 12.52(1H, brs), 8.55(1H, t, J ═ 5.4Hz), 8.29(1H, d, J ═ 7.3Hz), 8.03-7.96 (4H, m), 5.97(1H, brs), 5.40(1H, brs), 4.26(1H, t, J ═ 7.8Hz), 4.11(1H, m), 3.93-3.85 (2H, m), 3.60(2H, d, J ═ 5.9Hz), 2.95(2H, brs), 1.97(1H, m), 1.69(1H, m), 1.56(1H, m), 1.39(2H, m), 0.88(3H, d, J ═ 6.8Hz), 0.84(3H, d, J ═ 6.8Hz), and the peaks only observed (6.8 Hz)

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-valyl-N⁵-carbamoyl-L-ornithine

Using the compound (1.02g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 21 to obtain the title compound (795mg)

MS(ESI)m/z：676(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.49(1H，s)，8.28－8.22(1H，m)，8.18(0.5H，t，J＝6.0Hz)，8.13(0.5H，t，J＝5.7Hz)，8.06(0.5H，t，J＝5.7Hz)，8.01(0.5H，t，J＝6.0Hz)，7.75－7.71(1H，m)，7.69－7.65(1H，m)，7.62－7.59(1H，m)，7.52－7.43(3H，m)，7.40－7.28(3H，m)，5.93(1H，t，J＝5.7Hz)，5.38(2H，brs)，5.02(0.5H，d，J＝13.9Hz)，5.01(0.5H，d，J＝13.9Hz)，4.24(1H，dd，J＝8.8，7.0Hz)，4.10(1H，m)，3.79－3.54(5H，m)，2.93(2H，q，J＝6.4Hz)，2.73－2.60(1H，m)，2.29(1H，m)，2.09－1.91(2H，m)，1.79(1H，m)，1.72－1.63(1H，m)，1.60－1.50(1H，m)，1.43－1.33(2H，m)，0.86(3H，d，J＝6.7Hz)，0.81(1.5H，d，J＝6.7Hz)，0.80(1.5H，d，J＝6.7Hz).

(step 3)

2, 5-Bibridging oxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-valyl-N⁵-carbamoyl-L-ornithine ester

Using the compound (300mg) obtained in the above step 2, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (177 mg).

MS(ESI)m/z：773(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.75(1H，t，J＝6.1Hz)，8.20(0.5H，t，J＝6.1Hz)，8.15(0.5H，t，J＝5.6Hz)，8.06(0.5H，t，J＝5.9Hz)，8.02(0.5H，t，J＝5.9Hz)，7.83(0.5H，d，J＝8.8Hz)，7.77(0.5H，d，J＝8.8Hz)，7.72(0.5H，d，J＝7.8Hz)，7.67(0.5H，d，J＝6.3Hz)，7.62－7.59(1H，m)，7.52－7.44(3H，m)，7.39－7.29(3H，m)，6.03(1H，brs)，5.43(2H，brs)，5.03(0.5H，d，J＝13.7Hz)，5.01(0.5H，d，J＝14.2Hz)，4.59－4.54(1H，m)，4.25(1H，m)，3.79－3.68(5H，m)，2.97(2H，q，J＝6.5Hz)，2.80(4H，s)，2.71－2.60(1H，m)，2.28(1H，m)，2.10－2.03(1H，m)，2.00－1.93(1H，m)，1.88－1.72(3H，m)，1.54－1.49(2H，m)，0.85(3H，d，J＝5.4Hz)，0.81(1.5H，d，J＝4.9Hz)，0.80(1.5H，d，J＝6.8Hz).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-valyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxa bisPhosphino cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]－N⁵-carbamoyl-L-ornithine amides

(drug linker 11)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (5.0mg) and the compound obtained in step 3 (3.8mg), and then the reaction mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% to 35% (0 part to 30 parts) was purified to give the title compound (3.0 mg).

MS(ESI)m/z：1443(M－H)^－.

Example 72: synthesis of drug linker 12

[ synthetic route ]

(step 1)

N- (ammonioacetyl) glycyl-L-phenylalanyl-N⁵-carbamoyl-L-ornithine trifluoroacetate

The commercially available N- (tert-butoxycarbonyl) glycylglycinyl-L-phenylalanyl-N-alaninyl of (Hangzhou Peptide Biochem) was used⁵Reaction of (E) -carbamoyl-L-ornithine (1.00g) was carried out in the same manner as in step 1 of example 21 to give the title compound (1.05g) as a crude product.

MS(ESI)m/z：437(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.63(1H，brs)，8.49(1H，t，J＝5.4Hz)，8.40(1H，d，J＝7.8Hz)，8.26(1H，d，J＝8.3Hz)，7.97(3H，brs)，7.28－7.17(5H，m)，6.00(1H，brs)，5.41(2H，brs)，4.60(1H，m)，4.17(1H，m)，3.85(1H，dd，J＝16.6，5.4Hz)，3.67(1H，dd，J＝16.6，5.4Hz)，3.56(2H，d，J＝5.9Hz)，3.03(1H，dd，J＝13.9，3.7Hz)，2.96(2H，brs)，2.73(1H，dd，J＝13.9，10.5Hz)，1.73(1H，m)，1.58(1H，m)，1.40(2H，m).

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl ]glycylglycinyl-L-phenylalanyl-N⁵-carbamoyl-L-ornithine

Using the compound (1.05g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 21 to obtain the title compound (550 mg).

MS(ESI)m/z：724(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.60(1H，s)，8.29(1H，t，J＝9.1Hz)，8.17(0.5H，t，J＝5.7Hz)，8.10(0.5H，t，J＝5.7Hz)，8.04－7.94(2H，m)，7.72－7.14(13H，m)，5.94(1H，br)，5.39(2H，s)，5.01(0.5H，d，J＝13.9Hz)，5.00(0.5H，d，J＝14.5Hz)，4.56(1H，m)，4.15(1H，m)，3.74－3.49(5H，m)，3.04－2.93(3H，m)，2.77－2.58(2H，m)，2.28(1H，m)，2.05(1H，m)，1.83－1.70(2H，m)，1.62－1.52(1H，m)，1.46－1.32(2H，m).

(step 3)

2, 5-Bibridging oxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N⁵-carbamoyl-L-ornithine ester

Using the compound (225mg) obtained in the above step 2, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (226 mg).

MS(ESI)m/z：821(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.80(1H，t，J＝7.6Hz)，8.19－7.96(3H，m)，7.72－7.15(13H，m)，6.03－5.89(1H，br)，5.43(2H，brs)，5.01(0.5H，d，J＝14.0Hz)，4.99(0.5H，d，J＝14.0Hz)，4.65－4.53(2H，m)，3.74－3.50(5H，m)，3.03－2.95(3H，m)，2.81(4H，s)，2.78－2.58(2H，m)，2.28(1H，m)，2.07(1H，m)，1.91－1.73(3H，m)，1.56－1.49(2H，m).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]－N⁵-carbamoyl-L-ornithine amides

(drug linker 12)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (5.1mg) and the compound obtained in step 3 (4.0mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% to 35% (0 part to 30 parts) was purified to give the title compound (4.6 mg).

MS(ESI)m/z：1493(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.31(1H，brs)，8.02(1H，s)，7.62－7.14(14H，m)，6.32(1H，d，J＝6.0Hz)，6.11(1H，brd，J＝5.4Hz)，5.55－5.41(2H，m)，5.06(1H，dd，J＝13.9，11.5Hz)，4.86－2.73(28H，m)，3.19(12H，q，J＝7.5Hz)，2.39－2.16(2H，m)，2.04－1.94(3H，m)，1.84－1.74(1H，m)，1.73－1.62(1H，m)，1.51－1.37(2H，m)，1.29(18H，t，J＝7.3Hz).

Example 73: synthesis of drug linker 13

[ synthetic route ]

(step 1)

N- (ammonioacetyl) glycyl-L-isoleucylaminoacyl-N⁵-carbamoyl-L-ornithine trifluoroacetate

The commercially available N- (tert-butoxycarbonyl) glycylglycinyl-L-isoleucyl-N-glycyl-L-glycyl-N was used (Hangzhou Peptide Biochem)⁵Reaction of (E) -carbamoyl-L-ornithine (1.00g) was carried out in the same manner as in step 1 of example 21 to give the title compound (1.09g) as a crude product.

MS(ESI)m/z：403(M+H)⁺.

¹H－NMR(DMSO－d₆) δ: 8.54(1H, t, J ═ 5.4Hz), 8.30(1H, d, J ═ 7.3Hz), 8.03(1H, d, J ═ 9.3Hz), 7.99(3H, brs), 5.99(1H, brs), 4.27(1H, t, J ═ 8.1Hz), 4.11(1H, m), 3.92-3.83 (2H, m), 3.60(2H, m), 2.95(2H, m), 1.71(2H, m), 1.56(1H, m), 1.48-1.33 (3H, m), 1.08(1H, m), 0.86(3H, d, J ═ 6.8Hz), 0.81(3H, t, J ═ 7.3Hz) (only the peaks observed)

(step 2)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-isoleucyl-N⁵-carbamoyl-L-ornithine

Using the compound (1.08g) obtained in the above step 1, a reaction was carried out in the same manner as in step 2 of example 21 to obtain the title compound (524 mg).

MS(ESI)m/z：690(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.47(1H，brs)，8.25(1H，t，J＝6.0Hz)，8.17(0.5H，t，J＝5.7Hz)，8.12(0.5H，t，J＝6.0Hz)，8.04(0.5H，t，J＝6.0Hz)，7.99(0.5H，t，J＝6.0Hz)，7.76－7.66(2H，m)，7.62－7.59(1H，m)，7.52－7.29(6H，m)，5.92(1H，t，J＝5.4Hz)，5.38(2H，s)，5.02(0.5H，d，J＝14.5Hz)，5.01(0.5H，d，J＝13.9Hz)，4.25(1H，t，J＝8.2Hz)，4.09(1H，m)，3.78－3.54(5H，m)，2.93(2H，q，J＝6.4Hz)，2.73－2.59(1H，m)，2.33－2.24(1H，m)，2.10－2.02(1H，m)，1.79(1H，ddd，J＝16.6，7.3，5.7Hz)，1.74－1.63(2H，m)，1.60－1.50(1H，m)，1.45－1.33(3H，m)，1.09－1.00(1H，m)，0.84(3H，d，J＝6.7Hz)，0.79(1.5H，t，J＝6.7Hz)，0.77(1.5H，t，J＝7.0Hz).

(step 3)

2, 5-Bibridging oxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-isoleucyl-N⁵-carbamoyl-L-ornithine ester

Using the compound (250mg) obtained in the above step 2, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (224 mg).

MS(ESI)m/z：787(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.75(1H，t，J＝6.4Hz)，8.19(0.5H，t，J＝5.8Hz)，8.14(0.5H，t，J＝5.8Hz)，8.06(0.5H，t，J＝5.8Hz)，8.01(0.5H，t，J＝5.8Hz)，7.89－7.65(2H，m)，7.63－7.58(1H，m)，7.53－7.27(6H，m)，5.96(1H，m)，5.42(2H，brs)，5.02(0.5H，d，J＝14.0Hz)，5.01(0.5H，d，J＝14.0Hz)，4.64－4.51(1H，m)，4.26(1H，t，J＝8.2Hz)，3.79－3.55(5H，m)，3.01－2.93(2H，m)，2.80(4H，brs)，2.73－2.58(1H，m)，2.28(1H，m)，2.12－2.02(1H，m)，1.87－1.68(4H，m)，1.54－1.38(3H，m)，1.11－1.02(1H，m)，0.83(3H，d，J＝6.7Hz)，0.78(1.5H，t，J＝6.7Hz)，0.76(1.5H，t，J＝6.7Hz).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-isoleucyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]－N⁵-carbamoyl-L-ornithine amides

(drug linker 13)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (5.4mg) and the compound obtained in step 3 (4.0mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -35% (0 min-30 min) to give the title compound (4.7 mg).

MS(ESI)m/z：1457(M－H)^－.

Example 74: synthesis of drug linker 14

[ synthetic route ]

(step 1)

N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycine

Using commercially available (manufactured by Tokyo chemical industry), glycylglycine (0.61g) was reacted in the same manner as in step 2 of example 21 to obtain the title compound (1.08 g).

MS(ESI)m/z：420(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.57(1H，s)，8.12(1H，t，J＝6.0Hz)，8.07(1H，t，J＝5.7Hz)，7.69－7.67(1H，m)，7.62－7.60(1H，m)，7.52－7.29(6H，m)，5.03(1H，d，J＝13.9Hz)，3.73(2H，d，J＝6.0Hz)，3.70－3.55(3H，m)，2.64(1H，m)，2.28(1H，m)，2.06(1H，ddd，J＝15.3，7.7，5.6Hz)，1.79(1H，ddd，J＝16.5，7.4，5.6Hz).

(step 2)

2, 5-Bisoryloxypyrrolidin-1-yl N- [ 4- (11, 12-Didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycine ester

Using the compound (1.07g) obtained in the above step 1, a reaction was carried out in the same manner as in the step 3 of example 21 to obtain the title compound (942 mg).

MS(ESI)m/z：517(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.40(1H，t，J＝6.0Hz)，8.19(1H，t，J＝6.0Hz)，7.69－7.67(1H，m)，7.64－7.62(1H，m)，7.53－7.29(6H，m)，5.05(1H，d，J＝14.5Hz)，4.26(1H，dd，J＝18.1，6.0Hz)，4.19(1H，dd，J＝18.1，6.0Hz)，3.72－3.59(3H，m)，2.81(4H，brs)，2.68－2.59(1H，m)，2.33－2.24(1H，m)，2.07(1H，ddd，J＝15.4，7.6，5.7Hz)，1.80(1H，ddd，J＝16.3，7.3，5.4Hz).

(step 3)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-L-leucine ester

N, N-diisopropylethylamine (0.25mL) was added to a suspension of the compound obtained in the above step 2 (250mg) and commercially available (domestic chemical) L-prolyl-L-leucine (166mg) in N, N-dimethylformamide (5.0mL), and the mixture was stirred at room temperature for 2.5 hours. L-prolyl-L-leucine (833mg) was added thereto and stirred overnight. After the reaction mixture was concentrated under reduced pressure, chloroform (50mL) and a 10% aqueous solution of citric acid (10mL) were added to the residue, followed by extraction with chloroform. The organic layer was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography [ chloroform/(chloroform: methanol: water: 7: 3: 1) lower layer ] to be roughly purified. To a solution of the crude product in N, N-dimethylformamide (5.0mL) were added N-hydroxysuccinimide (67mg) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (111mg), and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure, and chloroform (40mL) and water (15mL) were added to the residue to conduct extraction with chloroform. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ chloroform/methanol ] and [ ethyl acetate/methanol ] in this order to obtain the title compound (127 mg).

MS(ESI)m/z：727(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.85(0.3H，m)，8.56(0.5H，d，J＝7.3Hz)，8.33(0.2H，m)，8.09(1H，m)，7.91－7.79(1H，m)，7.70－7.28(8H，m)，5.04(0.7H，d，J＝14.5)，5.03(0.3H，d，J＝13.9)，4.71－4.29(2H，m)，4.01－3.30(7H，m)，2.79(4H，brs)，2.63(1H，m)，2.33－1.64(10H，m)，0.93－0.83(6H，m).

(step 4)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-prolyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-L-leucine amide

(drug linker 14)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (6.9mg) and the compound obtained in step 3 (4.7mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) was purified to give the title compound (7.2 mg).

MS(ESI)m/z：1397(M－H)^－.

Example 75: synthesis of drug linker 15

[ synthetic route ]

(step 1)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacycloocta-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-alanyl-L-glutamate

Using commercially available (Wako pure chemical industries, Ltd.) L-alanyl-L-glutamine (263mg) and the compound obtained in the step 2 of example 74 (250mg), a reaction was carried out in the same manner as in the step 3 of example 74 to obtain the title compound (90 mg).

MS(ESI)m/z：716(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.59(0.8H，d，J＝7.3Hz)，8.48(0.2H，m)，8.20－7.92(3H，m)，7.70－7.25(9H，m)，6.85(1H，brs)，5.02(1H，d，J＝13.9Hz)，4.62(1H，m)，4.33(1H，m)，3.75－3.56(5H，m)，2.80(4H，brs)，2.64(1H，m)，2.33－2.21(3H，m)，2.15－2.03(2H，m)，1.99－1.87(1H，m)，1.83－1.76(1H，m)，1.24－1.17(3H，m).

(step 2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-alanyl-N¹- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-L-Glutamine (glutamamide)

(drug linker 15)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (6.9mg) and the compound obtained in step 1 (4.7mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) was purified to give the title compound (3.9 mg).

MS(ESI)m/z：1388(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.38－8.35(1H，m)，8.02(1H，s)，7.66－7.13(9H，m)，6.33(1H，d，J＝6.7Hz)，6.14－6.10(1H，m)，5.51－5.42(2H，m)，5.14－5.06(1H，m)，4.84－4.79(1H，m)，4.53－3.64(14H，m)，3.56－3.11(6H，m)，3.15(12H，q，J＝7.5Hz)，2.92－2.69(4H，m)，2.42－1.93(8H，m)，1.45－1.24(3H，m)，1.27(18H，t，J＝7.3Hz).

Example 76: synthesis of drug linker 16

[ synthetic route ]

(step 1)

2, 5-Diendoxypyrrolidin-1-yl N- [ 4- (11, 12-didehydrodibenzo [ b, f ] azacyclooctatetra-5 (6H) -yl) -4-oxobutanoyl ] glycylglycinyl-L-prolyl-L-prolinamate

Using commercially available (Cool Pharm) 1- (tert-butoxycarbonyl) -L-prolyl-L-proline (777mg), a reaction was carried out in the same manner as in step 1 of example 21 to give a crude product of 1- [ (2S) -pyrrolidin-1-ium-2-carbonyl ] -L-proline trifluoroacetate. Using the crude product and the compound (428mg) obtained in step 2 of example 74, a reaction was carried out in the same manner as in step 3 of example 74 to obtain the title compound (238 mg).

MS(ESI)m/z：711(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.12－8.08(1H，m)，7.90(0.2H，s)，7.82(0.8H，d，J＝4.8Hz)，7.70－7.61(2H，m)，7.52－7.29(6H，m)，5.07－5.01(1H，m)，4.88－4.86(0.2H，m)，4.77－4.74(0.2H，m)，4.69－4.62(0.8H，m)，4.60－4.56(0.8H，m)，4.02－3.38(9H，m)，2.79(4H，brs)，2.68－2.58(1H，m)，2.37－2.24(2H，m)，2.16－1.73(9H，m).

(step 2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-prolyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]-L-proline amide

(drug linker 16)

The reaction was carried out in the same manner as in step 1 of example 23 using the compound obtained in step 8-2 of example 8 (6.9mg) and the compound obtained in step 1 (4.6mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -40% (0 min-30 min) was purified to give the title compound (5.8 mg).

MS(ESI)m/z：1383(M+H)⁺.

Example 77: synthesis of drug linker 17

[ synthetic route ]

(step 1)

Acetic acid [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methyl ester

Using commercially available N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycylglycine (9.32g) of (SUNDIA), a reaction was carried out in the same manner as in step 1 of example 67 to give the title compound (8.24 g).

¹H－NMR(CDCl₃)δ：7.14(1H，brs)，5.27(2H，d，J＝7.3Hz)，5.20(1H，brs)，4.22－4.16(2H，m)，3.88(2H，d，J＝6.0Hz)，2.08(3H，s)，1.04－0.97(2H，m)，0.05(9H，s).

(step 2)

2 ', 3 ', 5 ' -tri-O- [ tert-butyl (dimethyl) silyl ] -1- (2-hydroxyethyl) inosine

To a mixed solution of 2 ', 3 ', 5 ' -tri-O- [ tert-butyl (dimethyl) silyl ] inosine (31.3g) in tetrahydrofuran (75mL) -N, N-dimethylacetamide (75mL) known in the literature (chem. pharm. Bull.1987, 35(1), 72-79) was added 2-bromoethanol (4.82mL) and 1, 8-diazabicyclo [5.4.0] -7-undecene (7.65mL), and the mixture was stirred at room temperature for 23 hours. Water and ethyl acetate were added to the reaction solution, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered off the drying agent, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (29.4 g).

MS(ESI)m/z：655(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.16(1H，s)，7.99(1H，d，J＝2.4Hz)，5.97(1H，d，J＝4.2Hz)，4.40－4.25(3H，m)，4.18－4.06(3H，m)，4.03－3.92(2H，m)，3.79(1H，dd，J＝11.5，2.4Hz)，3.08－2.83(1H，brm)，0.96(9H，s)，0.92(9H，s)，0.82(9H，s)，0.15(3H，s)，0.14(3H，s)，0.09(3H，s)，0.08(3H，s)，－0.02(3H，s)，－0.15(3H，s).

(step 3)

2 ', 3 ', 5 ' -tri-O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methoxy } ethyl) inosine

The compound (10.4g) obtained in the above step 1 and pyridine (9.63mL) were added to a toluene (46.8mL) solution of the compound (15.6g) obtained in the above step 2, and the mixture was stirred at 110 ℃ for 12 hours. The compound (3.46g) obtained in the above step 1 was added to the reaction mixture, and the mixture was stirred at 110 ℃ for 1 day. Saturated aqueous sodium bicarbonate and dichloromethane were added to the reaction solution, and extraction was performed with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate ] to give the title compound (20.6 g: containing impurities).

MS(ESI)m/z：885(M+H)⁺.

(step 4)

5' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methoxy } ethyl) inosine

Triethylamine trihydrofluoride salt (10mL) was added to a tetrahydrofuran (50mL) solution of the compound (20.6g) obtained in the above step 3, and the mixture was stirred at room temperature for 17 hours. A mixture of a 1M triethylammonium bicarbonate solution (50mL) and triethylamine (10mL) was slowly added to the reaction mixture under ice-cooling, and the reaction mixture was concentrated under reduced pressure. The residue was subjected to crude purification by C18 silica gel column chromatography [ water/acetonitrile ], followed by lyophilization. The resulting crude product was azeotroped with pyridine, and 4, 4' -dimethoxytrityl chloride (4.73g) was added to a solution of the residue in pyridine (50mL) at 0 ℃ and stirred at 4 ℃ for 17 hours. Methanol (2mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ hexane/ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (9.18 g: containing impurities).

MS(ESI)m/z：845(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.94(1H，s)，7.88(1H，s)，7.65(1H，brs)，7.41－7.36(2H，m)，7.32－7.15(7H，m)，6.83－6.76(4H，m)，5.96(1H，d，J＝6.1Hz)，5.73－5.65(2H，m)，4.87－4.80(1H，m)，4.76－4.61(2H，m)，4.44－4.39(1H，m)，4.35－4.30(1H，m)，4.22－4.05(4H，m)，3.83－3.73(2H，m)，3.77(6H，s)，3.72－3.67(2H，m)，3.48－3.32(3H，m)，0.99－0.91(2H，m)，0.02(9H，s).

(step 5)

5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3' -O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methoxy } ethyl) inosine

Using the compound (5.96g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 3 of example 5 to give the title compound (2.33g) and 5 '-O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -2' -O- [ tert-butyl (dimethyl) silyl ] -1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methoxy } ethyl) inosine (2.45g), which is a positional isomer of the title compound.

MS(ESI)m/z：959(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(1H，s)，7.93(1H，s)，7.45－7.39(2H，m)，7.35－7.18(7H，m)，7.05(1H，brs)，6.84－6.77(4H，m)，5.92(1H，d，J＝5.4Hz)，5.46(1H，brs)，4.71－4.61(3H，m)，4.54－4.51(1H，m)，4.22－4.10(5H，m)，3.81－3.76(2H，m)，3.78(3H，s)，3.78(3H，s)，3.74(2H，d，J＝6.0Hz)，3.48(1H，dd，J＝10.9，4.2Hz)，3.26(1H，dd，J＝10.9，4.2Hz)，3.16(1H，d，J＝6.7Hz)，1.00－0.93(2H，m)，0.89(9H，s)，0.09(3H，s)，0.02(9H，s)，0.02(3H，s).

Positional isomers (2' -O-TBS body)

MS(ESI)m/z：959(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.99(1H，s)，7.91(1H，s)，7.48－7.42(2H，m)，7.37－7.18(8H，m)，6.85－6.78(4H，m)，5.96(1H，d，J＝5.4Hz)，5.63(1H，brs)，4.88(1H，t，J＝5.1Hz)，4.66(2H，d，J＝6.7Hz)，4.36－4.32(1H，m)，4.27－4.19(2H，m)，4.18－4.10(3H，m)，3.81－3.74(4H，m)，3.78(3H，s)，3.78(3H，s)，3.50(1H，dd，J＝10.9，3.6Hz)，3.38(1H，dd，J＝10.9，3.6Hz)，2.73(1H，d，J＝4.2Hz)，0.97－0.90(2H，m)，0.86(9H，s)，0.02(3H，s)，0.01(9H，s)，－0.09(3H，s).

(step 6)

5 ' -O- [ bis (4-methoxyphenyl) (phenyl) methyl ] -3 ' -O- [ tert-butyl (dimethyl) silyl ] -2 ' -O- { (2-cyanoethoxy) [ di (prop-2-yl) amino ] phosphino } -1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] carbonyl } glycyl) amino ] methoxy } ethyl) inosine

The reaction was carried out in the same manner as in step 4 of example 5 using the compound (2.33g) obtained in step 5 above to obtain the title compound (2.72g) in the form of a diastereomer mixture on the phosphorus atom (diastereomer ratio: 6: 4).

MS(ESI)m/z：1159(M+H)⁺.

¹H－NMR(CDCl₃)δ：8.03(0.4H，s)，8.02(0.6H，s)，7.95(0.6H，s)，7.92(0.4H，s)，7.46－7.40(2H，m)，7.35－7.17(7H，m)，6.88(1H，brs)，6.84－6.78(4H，m)，6.15(0.6H，d，J＝4.2Hz)，6.10(0.4H，d，J＝4.8Hz)，5.34(1H，brs)，4.86－4.61(3H，m)，4.48－4.42(1H，m)，4.29－4.09(5H，m)，3.83－3.44(9H，m)，3.79(3H，s)，3.78(3H，s)，3.32－3.23(1H，m)，2.58－2.49(1H，m)，2.44－2.38(1H，m)，1.15(3.6H，d，J＝6.7Hz)，1.11(6H，d，J＝6.7Hz)，1.04－0.92(2H，m)，0.97(2.4H，d，J＝6.7Hz)，0.85(3.6H，s)，0.84(5.4H，s)，0.09(1.2H，s)，0.06(1.8H，s)，0.03(9H，s)，0.00(3H，s).

(step 7)

Using the compound (2.15g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (2.72g) obtained in the above step 6 were reacted in the same manner as in the step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 8)

2- (trimethylsilyl) ethyl (2- { [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) -16- { [ tert-butyl (dimethyl)Radical) silyl radical]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-oxo-2-mercapto-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl ]Amino } -2-oxoethyl) carbamate

Using the crude product obtained in the above step 7, a reaction was carried out in the same manner as in the step 9 of example 1 to obtain the title compound (1.47 g: containing impurities) in the form of a diastereomer mixture on a phosphorus atom.

MS(ESI)m/z：1278(M+H)⁺.

(step 9)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-2, 10-di-oxo-7- [ 6-oxo-1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] ethoxy]Carbonyl } glycyl) amino]Methoxy } ethyl) -1, 6-dihydro-9H-purin-9-yl]-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

To a mixed solution of the compound (1.47g) obtained in the above step 8 in methanol (10mL) -tetrahydrofuran (10mL) was added 28% aqueous ammonia (10mL), and the mixture was stirred at 50 ℃ for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] to give diastereomer 1(204 mg: containing impurities) and diastereomer 2(205 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1121(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1121(M+H)⁺.

(step 10-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

After a reaction was carried out in the same manner as in step 9-1 of example 11 using the compound obtained in step 9 (diastereomer 1) (204mg), column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 10% to 50% (0 part to 40 parts) was purified to obtain the title compound (40.7 mg: containing impurities).

MS(ESI)m/z：863(M+H)⁺.

(step 10-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy ]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphinocyclotetradecyne-2, 10-bis (thiolate)

The reaction was carried out in the same manner as in step 9-1 of example 11 using the compound (diastereomer 2) (205mg) obtained in step 9, and then subjected to column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 10% to 50% (0 part to 40 parts) was purified to obtain the title compound (50.8 mg: containing impurities).

MS(ESI)m/z：863(M+H)⁺.

(step 11-1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 17 a: diastereomer 1)

The reaction was carried out in the same manner as in step 9-1 of example 22 using the compound (40.7mg) obtained in step 10-1, and then column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 40% to 90% (0 part to 40 parts) was purified to give the title compound (25.1 mg).

MS(ESI)m/z：1411(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，s)，8.09(1H，s)，8.04(1H，s)，7.57－7.49(2H，m)，7.43－7.34(3H，m)，7.32－7.08(9H，m)，6.47(1H，d，J＝16.9Hz)，6.23(1H，d，J＝7.9Hz)，5.56－5.37(2H，m)，5.31－5.17(1H，m)，5.03(1H，d，J＝13.9Hz)，4.79(1H，d，J＝4.2Hz)，4.64－4.38(6H，m)，4.36－4.21(4H，m)，4.05－3.60(10H，m)，3.53－3.42(3H，m)，3.18(12H，q，J＝7.3Hz)，3.01－2.92(1H，m)，2.86－2.73(1H，m)，2.70－2.54(2H，m)，2.37－2.16(2H，m)，2.06－1.77(3H，m)，1.28(18H，t，J＝7.3Hz).

(step 11-2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 17 b: diastereomer 2)

The reaction was carried out in the same manner as in step 9-1 of example 22 using the compound (50.8mg) obtained in step 10-2, and then column chromatography on C18 silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 45% -90% (0 min-40 min) was purified to give the title compound (23.4 mg).

MS(ESI)m/z：1411(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.67(1H，s)，8.14(1H，s)，8.02(1H，s)，7.67－7.50(2H，m)，7.43－7.36(3H，m)，7.34－7.12(9H，m)，6.48(1H，d，J＝15.1Hz)，6.26(1H，t，J＝8.8Hz)，5.60－5.31(3H，m)，5.09－5.00(1H，m)，4.61－4.22(9H，m)，4.11－3.59(13H，m)，3.50－3.44(2H，m)，3.18(12H，q，J＝7.3Hz)，3.04－2.93(1H，m)，2.87－2.74(3H，m)，2.39－2.22(2H，m)，2.06－1.85(3H，m)，1.28(18H，t，J＝7.3Hz).

Example 78: synthesis of drug linker 18

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) glycinamide

(drug linker 18)

The reaction was carried out in the same manner as in step 4 of example 21 using the compound obtained in step 7-2 of example 45 (10.0mg) and the compound obtained in step 3 of example 21 (8.8mg), and then the mixture was subjected to preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -50% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 45% -90% (0 min-40 min) was purified to give the title compound (10.9 mg).

MS(ESI)m/z：1381(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.61(1H，s)，8.03(1H，s)，8.02(1H，s)，7.64－7.49(2H，m)，7.44－7.37(3H，m)，7.35－7.11(9H，m)，6.48(1H，dd，J＝15.1，1.8Hz)，6.24(1H，d，J＝8.5Hz)，5.62－5.39(3H，m)，5.03(1H，dd，J＝18.4，14.2Hz)，4.55－4.37(5H，m)，4.29－4.18(2H，m)，4.04－3.91(3H，m)，3.87－3.52(8H，m)，3.50－3.41(3H，m)，3.19(12H，q，J＝7.3Hz)，3.18－3.10(1H，m)，3.02－2.92(1H，m)，2.86－2.66(3H，m)，2.40－2.21(2H，m)，2.06－1.84(3H，m)，1.29(18H，t，J＝7.3Hz).

Example 79: synthesis of drug linker 19

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- ({ 2- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-dioxaneoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) amino]-2-oximinoethoxy } methyl) glycinamide

(drug linker 19)

The reaction was carried out in the same manner as in step 4 of example 21 using the compound obtained in step 7-2 of example 45 (20.0mg) and the compound obtained in step 1 of example 66 (19.8mg), and then the mixture was subjected to preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 20% -45% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 30% to 80% (0 part to 40 parts) was purified to give the title compound (22.1 mg).

MS(ESI)m/z：1468(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.66(1H，brs)，8.10(1H，brs)，8.04－7.96(1H，m)，7.66－7.35(5H，m)，7.33－7.12(9H，m)，6.52－6.41(1H，m)，6.28－6.22(1H，m)，5.64－5.31(3H，m)，5.07－4.99(1H，m)，4.70－4.20(9H，m)，4.11－3.51(14H，m)，3.50－3.30(3H，m)，3.27－3.10(1H，m)，3.19(12H，q，J＝7.3Hz)，3.07－2.93(1H，m)，2.89－2.65(3H，m)，2.41－2.18(2H，m)，2.05－1.82(2H，m)，1.29(18H，t，J＝7.3Hz).

Example 80: synthesis of drug linker 20

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thio-2, 3, 5-triazabenzo [ cd) ]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dialkoxy-2, 10-dithiolether octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) glycinamide

(drug linker 20)

After a reaction was carried out in the same manner as in step 4 of example 21 using the compound obtained in step 4-2 of example 59 (25.0mg) and the compound obtained in step 3 of example 21 (25.8mg), column chromatography was carried out using C18 silica gel column [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 30% -50% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 50% to 90% (0 part to 40 parts) was purified to give the title compound (33.1 mg).

MS(ESI)m/z：1398(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，brs)，8.39(1H，d，J＝8.5Hz)，8.00(1H，brs)，7.71(1H，brs)，7.64－7.49(2H，m)，7.44－7.37(3H，m)，7.32－7.10(8H，m)，6.59(1H，d，J＝15.1Hz)，6.23(1H，d，J＝8.5Hz)，5.65－5.36(3H，m)，5.03(1H，dd，J＝16.6，14.2Hz)，4.57－4.38(5H，m)，4.30－4.17(2H，m)，4.07－3.95(2H，m)，3.94－3.50(9H，m)，3.50－3.35(1H，m)，，3.19(12H，q，J＝7.3Hz)，3.18－3.07(3H，m)，3.04－2.73(4H，m)，2.40－2.11(4H，m)，2.05－1.92(1H，m)，1.29(18H，t，J＝7.3Hz).

Example 81: synthesis of drug linker 21

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- ({ 2- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thio-2, 3, 5-triazabenzo [ cd) ]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dialkoxy-2, 10-dithiolether octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethyl) amino]-2-oximinoethoxy } methyl) glycinamide

(drug linker 21)

After a reaction was carried out in the same manner as in step 4 of example 21 using the compound obtained in step 4-2 of example 59 (15.0mg) and the compound obtained in step 1 of example 66 (17.4mg), column chromatography was carried out using C18 silica gel column [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -50% (0 min-40 min), and preparative HPLC [10mM triethylammonium acetate in water/methanol, methanol: 40% to 90% (0 part to 40 parts) was purified to give the title compound (21.8 mg).

MS(ESI)m/z：1485(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.61(1H，brs)，8.41－8.34(1H，m)，8.10－8.05(1H，m)，7.72－7.37(6H，m)，7.32－7.12(8H，m)，6.63－6.50(1H，m)，6.27－6.22(1H，m)，5.65－5.31(3H，m)，5.07－4.94(1H，m)，4.68－4.20(10H，m)，4.11－3.53(14H，m)，3.26－3.08(2H，m)，3.19(12H，q，J＝7.3Hz)，3.06－2.67(4H，m)，2.40－2.11(4H，m)，2.05－1.88(1H，m)，1.29(18H，t，J＝7.3Hz).

Example 82: synthesis of drug linker 22

[ synthetic route ]

(step 1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (8, 9-dihydro-6-thio-2, 3, 5-triazabenzo [ cd) ]Azulen-2 (7H) -yl) -15-fluoro-16-hydroxy-2, 10-dialkoxy-2, 10-dithiolether octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Glycine amides

(drug linker 22)

The reaction was carried out in the same manner as in step 4 of example 21 using the compound obtained in step 4-2 of example 61 (4.4mg) and the compound obtained in step 3 of example 21 (3.5mg), and then the mixture was purified by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 25% -45% (0 min-30 min) was purified to give the title compound (6.4 mg).

MS(ESI)m/z：1412(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.37(1H，d，J＝1.2Hz)，8.10－7.98(1H，m)，7.75(1H，s)，7.62－7.13(13H，m)，6.59(1H，d，J＝16.3Hz)，6.01(1H，brs)，5.82－5.62(1H，m)，5.54－5.33(2H，m)，5.04(1H，d，J＝14.5Hz)，4.57－3.48(14H，m)，3.35－2.65(12H，m)，3.17(12H，q，J＝7.3Hz)，2.35－2.11(4H，m)，2.00－1.92(1H，m)，1.28(18H，t，J＝7.3Hz).

Example 83: synthesis of drug linker 23

[ synthetic route ]

(step 1)

Tert-butyl N- [ (benzyloxy) carbonyl ] glycylglycinyl-L-phenylalanyl glycinate

To a solution of commercially available N- [ (benzyloxy) carbonyl ] glycylglycinyl-L-phenylalanine (5.00g) and glycine tert-butyl ester hydrochloride (2.03g) in N, N-dimethylformamide (50mL) (BACHEM) were added N, N-diisopropylethylamine (4.11mL) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (3.01g) under ice-cooling, and the mixture was stirred overnight while warming to room temperature. The reaction mixture was poured into two layers of chloroform and a saturated aqueous sodium bicarbonate solution, and extracted with chloroform. The organic layer was washed with water and saturated brine, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure and azeotroped with toluene 2 times. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (4.51 g).

MS(ESI)m/z：527(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.40(1H，t，J＝5.7Hz)，8.15(1H，d，J＝9.1Hz)，8.00(1H，t，J＝5.7Hz)，7.51(1H，t，J＝6.0Hz)，7.38－7.16(10H，m)，5.03(2H，s)，4.52(1H，m)，3.80－3.54(6H，m)，3.05(1H，dd，J＝13.9，3.6Hz)，2.76(1H，dd，J＝14.2，10.6Hz)，1.41(9H，s).

(step 2)

Tert-butyl glycyl-L-phenylalanyl glycinate

To a mixture of the compound (4.51g) obtained in the above step 1 in methanol (20mL) and methylene chloride (80mL) was added 10% palladium on carbon (M) wet (750mg), and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. 10% Palladium on carbon (M) wet (1.5g) was added thereto, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (3.18 g).

MS(ESI)m/z：393(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.42(1H，t，J＝6.1Hz)，8.20(1H，d，J＝8.8Hz)，8.00(1H，brs)，7.28－7.16(5H，m)，4.53(1H，m)，3.78－3.73(3H，m)，3.61(1H，brd，J＝16.1Hz)，3.06(3H，dd，J＝15.4，5.6Hz)，2.76(1H，dd，J＝14.2，10.3Hz)，1.88(2H，br)，1.41(9H，s).

(step 3)

Tert-butyl N- [ 6- (2, 5-di-oxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl ] glycylglycinyl-L-phenylalanyl glycinate

The compound (2.65g) obtained in step 2 and a commercially available N-succinimidyl 6-maleimidocaproate (2.20g) were dissolved in N, N-dimethylformamide (20mL), and the mixture was stirred at room temperature for 7 hours. After the reaction solution was concentrated under reduced pressure, the residue was poured into two layers of methylene chloride and water, and extracted with methylene chloride. The organic layer was washed with water 3 times, saturated brine 1 time, and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (3.52 g).

MS(ESI)m/z：586(M+H)⁺.

¹H－NMR(CDCl₃)δ：7.28－7.17(5H，m)，7.09(1H，t，J＝5.1Hz)，6.96(1H，t，J＝5.1Hz)，6.91(1H，d，J＝8.3Hz)，6.68(2H，s)，6.52(1H，t，J＝4.9Hz)，4.86(1H，q，J＝7.3Hz)，4.02－3.88(5H，m)，3.82(1H，dd，J＝18.1，4.9Hz)，3.51(2H，t，J＝7.1Hz)，3.15(1H，dd，J＝14.2，6.3Hz)，3.04(1H，dd，J＝13.7，7.3Hz)，2.25(2H，t，J＝7.6Hz)，1.70－1.57(4H，m)，1.45(9H，s)，1.32(2H，s).

(step 4)

N- [ 6- (2, 5-Dicrossoxy-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl ] glycylglycinyl-L-phenylalanylglycine

Trifluoroacetic acid (4.5mL) was added to a solution of the compound (1.00g) obtained in the above step 3 in dichloromethane (9.0mL) at 0 ℃ and the mixture was stirred at room temperature for 2.5 hours. The reaction mixture was concentrated under reduced pressure, and azeotroped with toluene 2 times. The residue was purified by silica gel column chromatography [ dichloromethane/methanol ] to give the title compound (534 mg).

MS(ESI)m/z：530(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：12.59(1H，br)，8.34(1H，t，J＝5.9Hz)，8.10(1H，d，J＝8.8Hz)，8.07(1H，t，J＝5.9Hz)，7.99(1H，t，J＝5.6Hz)，7.27－7.16(5H，m)，7.00(2H，s)，4.52(1H，m)，3.76(2H，d，J＝5.9Hz)，3.74(1H，m)，3.66(2H，d，J＝5.4Hz)，3.57(1H，dd，J＝16.8，5.6Hz)，3.36(2H，t，J＝7.1Hz)，3.04(1H，dd，J＝13.9，4.1Hz)，2.77(1H，dd，J＝13.9，10.0Hz)，2.10(2H，t，J＝7.3Hz)，1.51－1.43(4H，m)，1.19(2H，m).

(step 5)

2, 5-Diconoxypyrrolidin-1-yl N- [ 6- (2, 5-Diconoxy-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl ] glycylglycinyl-L-phenylalanyl glycinate

Using the compound (462mg) obtained in the above step 4, a reaction was carried out in the same manner as in step 3 of example 21 to obtain the title compound (258 mg).

MS(ESI)m/z：627(M+H)⁺.

¹H－NMR(DMSO－d₆)δ：8.70(0.85H，t，J＝6.1Hz)，8.44(0.15H，t，J＝5.9Hz)，8.17(0.85H，d，J＝8.8Hz)，8.11(0.15H，d，J＝8.8Hz)，8.06(1H，t，J＝5.6Hz)，7.99－7.95(1H，m)，7.27－7.16(5H，m)，6.99(2H，s)，4.53(1H，m)，4.28(1.7H，d，J＝5.9Hz)，3.85(0.3H，d，J＝5.9Hz)，3.74(1H，dd，J＝17.1，5.9Hz)，3.66(2H，d，J＝5.9Hz)，3.58(1H，dd，J＝16.8，5.6Hz)，3.36(2H，t，J＝7.1Hz)，3.04(1H，dd，J＝13.9，4.1Hz)，2.81(4H，brs)，2.77(1H，dd，J＝10.0，3.7Hz)，2.10(2H，t，J＝7.3Hz)，1.51－1.44(4H，m)，1.20(2H，d，J＝7.3Hz).

(step 6)

Bis (N, N-diethylethanaminium) N- [ 6- (2, 5-dioxido-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl]glycylglycinyl-L-phenylalanyl-N- [ 2- ({ 6-amino-9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -15, 16-dihydroxy-2, 10-dialkoxy-2, 10-disulfide-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-9H-purin-2-yl } amino) ethyl]Glycine amides

(drug linker 23)

Using the compound obtained in step 8-2 of example 8 (9.5mg) and the compound obtained in step 5 (7.5mg), a reaction was carried out in the same manner as in step 1 of example 23, followed by preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 10% to 30% (0 part to 30 parts) was purified to give the title compound (7.8 mg).

MS(ESI)m/z：1299(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.35(1H，s)，8.02(1H，s)，7.29－7.15(6H，m)，6.76(2H，s)，6.32(1H，d，J＝6.7Hz)，6.11(1H，d，8.5Hz)，5.48－5.36(2H，m)，4.82－4.79(1H，m)，4.50－4.25(6H，m)，4.06－3.81(7H，m)，3.61－3.37(9H，m)，3.17－2.89(4H，m)，3.14(12H，q，J＝7.3Hz)，2.21－1.96(4H，m)，1.58－1.48(4H，m)，1.30－1.21(2H，m)，1.27(18H，t，J＝7.3Hz).

Example 84: synthesis of sugar chain-reconstituted antibody 3

anti-HER 2 antibody 2- [ SG- (N)₃)2]2 preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc α 1, 6) GlcNAc-anti-HER 2 antibody 2

A commercially available PERJETA (registered trademark) intravenous drip (420 mg/14 mL) (3.5mL) was subjected to buffer exchange according to the common procedure C to give a phosphate buffered saline solution (6.0mL, 15.39mg/mL, pH 6.0). The same procedures as in step 1 of example 25 were carried out to give a 20mM phosphate buffer solution (12.96mg/mL, 7.5mL, pH6.0) of the title antibody.

(step 2)

anti-HER 2 antibody 2- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (12.96mg/mL, 7.5mL, pH6.0) of the antibody obtained in the above step 1 and [ N ] ₃－PEG(3)]₂-SG (10) Ox (22.5mg) was subjected to the same procedures as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (10.21mg/mL, 9.0mL, pH 6.0).

Example 85: synthesis of sugar chain-reconstituted antibody 4

Engineered anti-HER 2 antibody 2- [ SG- (N)₃)₂]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc α 1, 6) GlcNAc-engineered anti-HER 2 antibody 2

The same procedures as in step 1 of example 25 were carried out using a phosphate buffered saline solution (24mL, 12.25mg/mL, pH6.0) of the anti-HER 2 antibody 2 prepared in reference example 5, to give a 20mM phosphate buffered solution (20.86mg/mL, 12.5mL, pH6.0) of the title antibody.

(step 2)

Engineered anti-HER 2 antibody 2- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (20.86mg/mL, 12.5mL, pH6.0) of the antibody obtained in the above step 1 and [ N ]₃－PEG(3)]₂The same procedures as in step 2 of example 25 were repeated except for using-SG (10) Ox (52mg) to obtain a phosphate-buffered saline solution (10.87mg/mL, 22mL, pH6.0) of the title antibody.

Example 86: synthesis of sugar chain-reconstituted antibody 5

anti-CD 33 antibody- [ SG- (N)₃)₂]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc. alpha.1, 6) GlcNAc-anti-CD 33 antibody

The same procedures as in step 1 of example 25 were carried out using a phosphate buffered saline solution (9.0mL, 11.56mg/mL, pH6.0) of the anti-CD 33 antibody prepared in reference example 6 to give a 20mM phosphate buffered solution (11.62mg/mL, 8mL, pH6.0) of the title antibody.

(step 2)

anti-CD 33 antibody- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (11.62mg/mL, 8mL, pH6.0) of the antibody obtained in the above step 1 and [ N₃－PEG(3)]₂－SG (10) Ox (21.5mg) was subjected to the same procedures as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (10.01mg/mL, 8mL, pH 6.0).

Example 87: synthesis of sugar chain-reconstituted antibody 6

anti-EphA 2 antibody- [ SG- (N)₃)₂]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc. alpha.1, 6) GlcNAc-anti-EphA 2 antibody

The same procedures as in step 1 of example 25 were carried out using a phosphate buffered saline solution (8.0mL, 12.83mg/mL, pH6.0) of the anti-EphA 2 antibody prepared in reference example 7 to give a 20mM phosphate buffered solution (13.51mg/mL, 7mL, pH6.0) of the title antibody.

(step 2)

anti-EphA 2 antibody- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (13.51mg/mL, 7mL, pH6.0) of the antibody obtained in the above step 1 and [ N₃－PEG(3)]₂-SG (10) Ox (21.7mg) was subjected to the same procedures as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (8.91mg/mL, 7.5mL, pH 6.0).

Example 88: synthesis of sugar chain-reconstituted antibody 7

anti-CDH 6 antibody- [ SG- (N) ₃)₂]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc. alpha.1, 6) GlcNAc-anti-CDH 6 antibody

The buffer (5.0mL, 20.0mg/mL) of the HBSor (25mM histidine/5% sorbitol, pH6.0) buffer (5.0mL, 20.0mg/mL) against CDH6 antibody prepared according to reference example 8 was buffer-exchanged with a phosphate buffered saline solution (pH 6.0) according to common procedure C, and then the same procedure as in step 1 of example 25 was performed to obtain a 20mM phosphate buffer solution (9.58mg/mL, 9.0mL, pH6.0) of the title antibody.

(step 2)

anti-CDH 6 antibody- [ SG- (N)₃)₂]₂Preparation of

Using 20mM phosphate buffer solution (9.58mg/mL, 9.0mL, pH6.0) of the antibody obtained in the above step 1 and [ N ]₃－PEG(3)]₂-SG (10) Ox (24.5mg) was subjected to the same procedures as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (10.69mg/mL, 7.5mL, pH 6.0).

Example 89: synthesis of sugar chain-reconstituted antibody 8

Engineered anti-HER 2 antibody 2- [ MSG 1- (N)₃)]₂Preparation of

[ synthetic route ]

(step 1)

Preparation of (Fuc α 1, 6) GlcNAc-engineered anti-HER 2 antibody 2

The same reaction as in step 1 of example 85 was carried out to obtain a 20mM phosphate buffer solution (14.84mg/mL, 7.5mL, pH6.0) of the title antibody from a buffer solution (10mL, 12.25mg/mL, pH6.0) of the starting antibody.

(step 2)

Engineered anti-HER 2 antibody 2- [ MSG 1- (N)₃)]₂Preparation of

Using 20mM phosphate buffer solution (14.84mg/mL, 7.5mL, pH6.0) of the antibody obtained in the above step 1 and [ N ]₃－PEG(3)]MSG1(9) -Ox (Compounds 1 to 11 in WO 2018/003983) (19mg) was subjected to the same procedures as in step 2 of example 25 to give the title antibody in the form of a phosphate-buffered saline solution (10.48mg/mL, 9.25mL, pH 6.0).

Example 90: synthesis of antibody drug conjugate 5 (Synthesis of anti-HER 2 antibody-CDN conjugate 4)

A phosphate-buffered saline (pH6.0) solution (10.97mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution, a mixture of a dimethylsulfoxide solution (10mM, 0.091mL, 24 equivalents per 1-molecule antibody) of drug linker 1 and propylene glycol (0.159mL) was added, and the mixture was reacted at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.49mg/mL

Antibody yield: 1.71mg (31%)

Mean drug binding number: 3.6

Example 91: synthesis of antibody drug conjugate 6 (Synthesis of anti-HER 2 antibody-CDN conjugate 5)

A phosphate-buffered saline (pH6.0) solution (10.97mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution, a mixture of a dimethylsulfoxide solution (10mM, 0.091mL, 24 equivalents per 1 molecule of antibody) of drug linker 2a and propylene glycol (0.159mL) was added, and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.91mg/mL

Antibody yield: 3.17mg (58%)

Mean drug binding number: 3.6

Example 92: synthesis of antibody drug conjugate 7 (Synthesis of anti-HER 2 antibody-CDN conjugate 6)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 4.00mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (2.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.707mL, 24 equivalents per 1 molecule of antibody) of drug linker 2b and propylene glycol (1.293mL), and the mixture was reacted at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (24.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.96mg/mL

Antibody yield: 23.57mg (59%)

Mean drug binding number: 3.7

Example 93: synthesis of antibody drug conjugate 8 (Synthesis of anti-HER 2 antibody-CDN conjugate 7)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 4.00mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (2.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.707mL, 24 equivalents per 1 molecule of antibody) of drug linker 5 and propylene glycol (1.293mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (24.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.10mg/mL

Antibody yield: 26.86mg (67%)

Mean drug binding number: 3.7

Example 94: synthesis of antibody drug conjugate 9 (Synthesis of anti-HER 2 antibody-CDN conjugate 8)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 4.00mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (2.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.707mL, 24 equivalents per 1 molecule of antibody) of drug linker 6 and propylene glycol (1.293mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (24.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.34mg/mL

Antibody yield: 32.92mg (82%)

Mean drug binding number: 3.7

Example 95: synthesis of antibody drug conjugate 10 (Synthesis of anti-LPS antibody-CDN conjugate 2)

A phosphate-buffered saline (pH6.0) solution (10.55mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 2 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.087mL, 24 equivalents per 1 molecule of antibody) of drug linker 2a and propylene glycol (0.163mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.88mg/mL

Antibody yield: 3.08mg (62%)

Mean drug binding number: 3.6

Example 96: synthesis of antibody drug conjugate 11 (Synthesis of anti-EphA 2 antibody-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (8.91mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 6 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.073mL, 24 equivalents per 1 molecule of antibody) of drug linker 2b and propylene glycol (0.177mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by analysis according to the methods described in common procedures E and F.

Antibody concentration: 0.60mg/mL

Antibody yield: 2.10mg (47%)

Mean drug binding number: 3.8

Example 97: synthesis of antibody drug conjugate 12 (Synthesis of anti-CD 33 antibody-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (10.01mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 5 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.083mL, 24 equivalents per 1 molecule of antibody) of the drug linker 2b and propylene glycol (0.167mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.73mg/mL

Antibody yield: 2.57mg (51%)

Mean drug binding number: 3.9

Example 98: synthesis of antibody drug conjugate 13 (Synthesis of anti-CDH 6 antibody-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (10.69mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 7 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 2b and propylene glycol (0.162mL), and the mixture was reacted at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.96mg/mL

Antibody yield: 3.37mg (63%)

Mean drug binding number: 3.8

Example 99: synthesis of antibody drug conjugate 14 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 1)

A phosphate-buffered saline (pH6.0) solution (10.21mg/mL, 1.50mL) of the sugar chain-reconstituted antibody 3 was diluted with propylene glycol (0.750 mL). To this solution, a mixture of a dimethylsulfoxide solution (10mM, 0.253mL, 24 equivalents per 1 molecule of antibody) of drug linker 3 and propylene glycol (0.497mL) was added, and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (9.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.02mg/mL

Antibody yield: 9.73mg (64%)

Mean drug binding number: 3.7

Example 100: synthesis of antibody drug conjugate 15 (Synthesis of anti-HER 2 antibody-CDN conjugate 9)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 8 and propylene glycol (0.162mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.93mg/mL

Antibody yield: 3.24mg (61%)

Mean drug binding number: 3.6

Example 101: synthesis of antibody drug conjugate 16 (Synthesis of anti-HER 2 antibody-CDN conjugate 10)

A phosphate-buffered saline (pH6.0) solution (10.35mg/mL, 13.50mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (6.750 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 2.310mL, 24 equivalents per 1 molecule of antibody) of drug linker 9 and propylene glycol (4.440mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified according to the method described in general procedure D to obtain an ABS solution (74.3mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.36mg/mL

Antibody yield: 100.8mg (72%)

Mean drug binding number: 3.7

Example 102: synthesis of antibody drug conjugate 17 (Synthesis of anti-HER 2 antibody-CDN conjugate 11)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 10 and propylene glycol (0.162mL), and the mixture was reacted at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.63mg/mL

Antibody yield: 2.22mg (42%)

Mean drug binding number: 3.5

Example 103: synthesis of antibody drug conjugate 18 (Synthesis of anti-HER 2 antibody-CDN conjugate 12)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 11 and propylene glycol (0.162mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.94mg/mL

Antibody yield: 3.29mg (62%)

Mean drug binding number: 3.6

Example 104: synthesis of antibody drug conjugate 19 (Synthesis of anti-HER 2 antibody-CDN conjugate 13)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 12 and propylene glycol (0.162mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.92mg/mL

Antibody yield: 3.20mg (60%)

Mean drug binding number: 3.5

Example 105: synthesis of antibody drug conjugate 20 (Synthesis of anti-HER 2 antibody-CDN conjugate 14)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 13 and propylene glycol (0.162mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.01mg/mL

Antibody yield: 3.52mg (66%)

Mean drug binding number: 3.5

Example 106: synthesis of antibody drug conjugate 21 (Synthesis of anti-HER 2 antibody-CDN conjugate 15)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution, a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 14 and propylene glycol (0.162mL) was added, and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.78mg/mL

Antibody yield: 2.74mg (51%)

Mean drug binding number: 3.6

Example 107: synthesis of antibody drug conjugate 22 (Synthesis of anti-HER 2 antibody-CDN conjugate 16)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 15 and propylene glycol (0.162mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.32mg/mL

Antibody yield: 4.61mg (86%)

Mean drug binding number: 3.6

Example 108: synthesis of antibody drug conjugate 23 (Synthesis of anti-HER 2 antibody-CDN conjugate 17)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.088mL, 24 equivalents per 1 molecule of antibody) of drug linker 16 and propylene glycol (0.162mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.93mg/mL

Antibody yield: 3.25mg (61%)

Mean drug binding number: 3.6

Example 109: synthesis of antibody drug conjugate 24 (Synthesis of anti-HER 2 antibody-CDN conjugate 18)

A phosphate-buffered saline (pH6.0) solution (10.70mg/mL, 4.00mL) of the sugar chain-reconstituted antibody 1 was diluted with propylene glycol (2.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.707mL, 24 equivalents per 1 molecule of antibody) of drug linker 7 and propylene glycol (1.293mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (24.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.33mg/mL

Antibody yield: 32.64mg (82%)

Mean drug binding number: 3.7

Example 110: synthesis of antibody drug conjugate 25 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 2)

A phosphate-buffered saline (pH6.0) solution (10.87mg/mL, 8.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (4.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 1.079mL, 18 equivalents per 1 molecule of antibody) of drug linker 17a and propylene glycol (2.921mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (44.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.16mg/mL

Antibody yield: 51.5mg (59%)

Mean drug binding number: 3.8

Example 111: synthesis of antibody drug conjugate 26 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 3)

A phosphate-buffered saline (pH6.0) solution (10.87mg/mL, 8.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (4.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 1.079mL, 18 equivalents per 1 molecule of antibody) of drug linker 17b and propylene glycol (2.921mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (44.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.24mg/mL

Antibody yield: 54.99mg (63%)

Mean drug binding number: 3.9

Example 112: synthesis of antibody drug conjugate 27 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 4)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.180mL, 24 equivalents per 1 molecule of antibody) of drug linker 19 and propylene glycol (0.320mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.79mg/mL

Antibody yield: 5.53mg (51%)

Mean drug binding number: 3.9

Example 113: synthesis of antibody drug conjugate 28 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 5)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.180mL, 24 equivalents per 1 molecule of antibody) of drug linker 21 and propylene glycol (0.320mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.84mg/mL

Antibody yield: 5.91mg (54%)

Mean drug binding number: 3.9

Example 114: synthesis of antibody drug conjugate 29 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 6)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.180mL, 24 equivalents per 1 molecule of antibody) of drug linker 22 and propylene glycol (0.320mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (5.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.92mg/mL

Antibody yield: 4.60mg (42%)

Mean drug binding number: 3.5

Example 115: synthesis of antibody drug conjugate 30 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 7)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.180mL, 24 equivalents per 1 molecule of antibody) of drug linker 18 and propylene glycol (0.320mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.08mg/mL

Antibody yield: 7.53mg (69%)

Mean drug binding number: 3.9

Example 116: synthesis of antibody drug conjugate 31 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 8)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.180mL, 24 equivalents per 1 molecule of antibody) of drug linker 20 and propylene glycol (0.320mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.09mg/mL

Antibody yield: 7.62mg (70%)

Mean drug binding number: 3.8

Example 117: synthesis of antibody drug conjugate 32 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 9)

A phosphate-buffered saline (pH6.0) solution (10.48mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 8 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.087mL, 12 equivalents per 1 molecule of antibody) of drug linker 17a and propylene glycol (0.413mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.77mg/mL

Antibody yield: 5.36mg (51%)

Mean drug binding number: 1.8

Example 118: synthesis of antibody drug conjugate 33 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 10)

A phosphate-buffered saline (pH6.0) solution (10.48mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 8 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.087mL, 12 equivalents per 1 molecule of antibody) of drug linker 17b and propylene glycol (0.413mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.91mg/mL

Antibody yield: 6.34mg (61%)

Mean drug binding number: 1.8

Example 119: synthesis of antibody drug conjugate 34 (Synthesis of anti-HER 2 antibody-CDN conjugate 19)

A phosphate buffer solution of the anti-HER 2 antibody prepared in accordance with reference example 1 was subjected to buffer exchange by the common procedure C to prepare a phosphate buffered saline/5 mM-EDTA antibody solution (7.69 mg/mL). To the antibody solution (0.65mL) were added an aqueous solution (10mM, 20.7. mu.L) of tris (2-carboxyethyl) phosphine hydrochloride and an aqueous solution (1M, 9.8. mu.L) of dipotassium hydrogen phosphate. After confirming that the pH of the reaction solution was within 7.4. + -. 1.0, the reaction solution was stirred at 37 ℃ for 2 hours. A DMSO solution (10mM, 0.0689mL) containing the drug linker 23 was added, and the reaction was carried out at room temperature for 1 hour using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction was stopped by adding an aqueous solution of N-acetyl-L-cysteine (100mM, 6.9. mu.L) to the reaction mixture. The reaction solution was subjected to buffer exchange by the method described in the common procedure C to obtain an ABS solution (1.6mL) of the target antibody drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.46mg/mL

Antibody yield: 2.34mg (47%)

Mean drug binding number: 6.5

Example 120: synthesis of drug linker 24

[ synthetic route ]

(step 1)

Using the compound (1.55g) obtained in step 8 of example 44, a reaction was carried out in the same manner as in step 7 of example 1 to give 6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda ]⁵-phosphino]-beta-D-furanRibosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Acetonitrile solution of azulene. The obtained acetonitrile solution and the compound (1.96g) obtained in step 6 of example 77 were reacted in the same manner as in step 8 of example 1, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- (trimethylsilyl) ethyl (2- { [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-hydroxy-2-oxo-10-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Amino } -2-oxoethyl) carbamate

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in the step 2 of example 62 to obtain the title compound (1.07 g: containing impurities) in the form of a diastereomer mixture on a phosphorus atom.

MS(ESI)m/z：1262(M+H)⁺.

(step 3)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 16R) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -15-fluoro-2, 10-di-oxo-7- [ 6-oxo-1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] ethoxy]Carbonyl } glycyl) amino]Methoxy } ethyl) -1, 6-dihydro-9H-purin-9-yl]-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-ol salt

After the reaction was carried out in the same manner as in step 10 of example 1 using (1.07g) obtained in step 2 above, column chromatography was carried out using C18 silica gel column [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol (1: 1), acetonitrile-methanol (1: 1): from 25% to 90% (from 0 min to 40 min) was purified to give diastereomer 1(67.8 mg: containing impurities) and diastereomer 2(56.6 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1105(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1105(M+H)⁺.

(step 4-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-ol salt

The reaction was carried out in the same manner as in step 9-1 of example 11 using the compound obtained in step 3 (diastereomer 1) (67.8mg), and then, column chromatography was carried out using C18 silica gel column [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol (1: 1), acetonitrile-methanol (1: 1): 10% -60% (0 min-30 min) was purified to give the title compound (33.7 mg: containing impurities).

MS(ESI)m/z：847(M+H)⁺.

(step 4-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy ]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-10-thioether-14- (67, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2-ol salt

The reaction was carried out in the same manner as in step 9-1 of example 11 using the compound obtained in step 3 (diastereomer 2) (56.6mg), and then subjected to C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol (1: 1), acetonitrile-methanol (1: 1): 10% -60% (0 min-30 min) was purified to give the title compound (32.6 mg: containing impurities).

MS(ESI)m/z：847(M+H)⁺.

(step 5-1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2-oxo-2, 10-di-oxo-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(diastereomer 1)

Using the compound (33.7mg) obtained in the above-mentioned step 4-1 and the compound (21.3mg) obtained in the step 11 of example 22, a reaction was carried out in the same manner as in the step 9-1 of example 22, and then purification was carried out according to the following [ purification conditions ] to obtain the title compound (11.2 mg).

MS(ESI)m/z：1395(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.57－8.54(1H，m)，8.16－8.10(1H，m)，8.04(1H，s)，7.64－7.49(2H，m)，7.44－7.34(3H，m)，7.33－7.11(9H，m)，6.46(1H，d，J＝18.1Hz)，6.26(1H，d，J＝8.5Hz)，5.54－5.30(2H，m)，5.18－5.00(2H，m)，4.75－4.71(1H，m)，4.65－4.20(10H，m)，4.13－3.93(2H，m)，3.88－3.60(8H，m)，3.55－3.40(2H，m)，3.26－3.15(1H，m)，3.18(12H，q，J＝7.3Hz)，3.02－2.91(1H，m)，2.87－2.61(3H，m)，2.38－2.18(2H，m)，2.06－1.80(3H，m)，1.28(18H，t，J＝7.3Hz).

(step 5-2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2-oxo-2, 10-di-oxo-10-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(diastereomer 2)

Using the compound (32.6mg) obtained in the above-mentioned step 4-2 and the compound (20.6mg) obtained in the step 11 of example 22, a reaction was carried out in the same manner as in the step 9-1 of example 22, and then purification was carried out under the following [ purification conditions ] to obtain the title compound (16.7 mg).

[ purification conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ], preparative HPLC [10mM triethylammonium acetate aqueous solution/methanol, methanol: 40% -90% (0 min-40 min), and preparative HPLC [100mM hexafluoro-2-propanol, 8mM aqueous triethylamine/acetonitrile, acetonitrile: 10% -45% (0 min-40 min).

MS(ESI)m/z：1395(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，s)，8.17－8.13(1H，m)，8.02(1H，s)，7.64－7.50(2H，m)，7.42－7.35(4H，m)，7.32－7.12(8H，m)，6.49(1H，d，J＝16.3Hz)，6.27(1H，dd，J＝8.2，6.3Hz)，5.50－5.21(3H，m)，5.08－5.00(1H，m)，4.66－4.23(9H，m)，4.14－3.98(3H，m)，3.91－3.52(9H，m)，3.48－3.41(2H，m)，3.23－3.12(1H，m)，3.18(12H，q，J＝7.3Hz)，3.03－2.94(1H，m)，2.85－2.75(3H，m)，2.40－2.23(2H，m)，2.06－1.87(3H，m)，1.28(18H，t，J＝7.3Hz).

Example 121: synthesis of drug linker 25

[ synthetic route ]

(step 1)

N, N-diethylethanaminium 6-benzoyl-2- { 5-O- [ bis (4-methoxyphenyl) (phenyl) methyl]-2-deoxy-2-fluoro-3-O- [ oxy (oxo) -lambda⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] ]Azulene derivatives

Diphenyl phosphite (599. mu.L) was added to a pyridine (10.4mL) solution of the compound (1.49g) obtained in step 7 of example 44 under ice cooling, and the mixture was stirred at room temperature for 30 minutes. Diphenyl phosphite (200. mu.L) was added thereto and stirred for 2 hours. Water (1.5mL) was added to the reaction mixture under ice-cooling, and the mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ ethyl acetate/methanol/0.1% triethylamine ] to give the title compound (1.41 g).

MS(ESI)m/z：779(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.01(1H，s)，7.54(1H，s)，7.42－7.39(2H，m)，7.32－7.19(10H，m)，7.14－7.09(2H，s)，6.84(1H，dd，J＝628.7，1.8Hz)，6.83－6.79(4H，m)，6.53(1H，dd，J＝17.5，1.8Hz)，5.54(1H，ddd，J＝52.1，4.0，2.0Hz)，5.37－5.27(1H，m)，4.31－4.21(3H，m)，3.55(1H，dd，J＝11.2，2.1Hz)，3.38(1H，dd，J＝10.9，3.0Hz)，3.34(6H，s)，3.19(6H，q，J＝7.5Hz)，2.84－2.69(2H，m)，2.18－2.12(2H，m)，1.29(9H，t，J＝7.3Hz).

(step 2)

6-benzoyl-2- { 2-deoxy-2-fluoro-3-O- [ hydroxy (oxo) -lambda [ ] -⁵-phosphino]-beta-D-ribofuranosyl } -6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulene derivatives

To a solution of the compound (1.41g) obtained in the above step 1 in dichloromethane (20.0mL) were added water (289. mu.L) and a solution of dichloroacetic acid (1.14mL) in dichloromethane (20.0mL), and the mixture was stirred at room temperature for 10 minutes. Pyridine (2.19mL) was added to the reaction mixture to stop the reaction, and then the mixture was concentrated under reduced pressure. To the residue was added a pyridinium trifluoroacetate (402mg), and the mixture was azeotroped 3 times with dehydrated acetonitrile (15mL) to leave about 10mL of acetonitrile in the last step. The obtained acetonitrile solution was used as it was in the next reaction.

MS(ESI)m/z：477(M+H)⁺.

(step 3)

Using an acetonitrile solution of the compound obtained in the step 6 of example 77 (2.15g) and the compound obtained in the step 2, a reaction was carried out in the same manner as in the step 1 of example 63, and the obtained crude product was used as it was in the next reaction.

(step 4)

2- (trimethylsilyl) ethyl (2- { [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2, 10-dioxido-2-mercaptooctahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Amino } -2-oxoethyl) carbamate

Using the crude product obtained in the above step 3, a reaction was carried out in the same manner as in step 9 of example 1 to obtain the title compound (1.00 g: containing impurities).

MS(ESI)m/z：1262(M+H)⁺.

(step 5)

Bis (N, N-diethylethylammonium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16- { [ tert-butyl (dimethyl) silyl ]Oxy } -15-fluoro-2, 10-di-oxo-7- [ 6-oxo-1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy ] ethoxy]Carbonyl } glycyl) amino]Methoxy } ethyl) -1, 6-dihydro-9H-purin-9-yl]-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-10-ol salt

Using the compound (1.00g) obtained in the above step 4, a reaction was carried out in the same manner as in the step 10 of example 1 to obtain diastereomer 1(191 mg: containing impurities) and diastereomer 2(369 mg: containing impurities) of the title compound.

Diastereomer 1 (low polarity)

MS(ESI)m/z：1105(M+H)⁺.

Diastereomer 2 (high polarity)

MS(ESI)m/z：1105(M+H)⁺.

(step 6-1)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd) ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo[3，2－l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-10-ol salt

Using the compound (diastereomer 1) (191mg) obtained in the above step 5, a reaction was carried out in the same manner as in the step 5 of example 40 to obtain the title compound (21.9 mg: containing impurities).

MS(ESI)m/z：847(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.39(1H，s)，8.03(1H，s)，7.71(1H，s)，7.24(1H，s)，6.45(1H，d，J＝17.5Hz)，6.15(1H，d，J＝8.5Hz)，5.79－5.65(2H，m)，5.46－5.36(1H，m)，4.63－4.23(9H，m)，4.01－3.95(1H，m)，3.65－3.57(2H，m)，3.52－3.37(5H，m)，3.15(12H，q，J＝7.3Hz)，2.76－2.68(1H，m)，2.39－2.30(1H，m)，1.92－1.82(2H，m)，1.28(18H，t，J＝7.6Hz).

(step 6-2)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd)]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-10-ol salt

Using the compound (diastereomer 2) (369mg) obtained in the above step 5, a reaction was carried out in the same manner as in the step 5 of example 40 to obtain the title compound (137 mg: containing impurities).

MS(ESI)m/z：847(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.96(1H，s)，8.14(1H，s)，8.02(1H，s)，7.04(1H，s)，6.46(1H，d，J＝19.3Hz)，6.28(1H，d，J＝8.5Hz)，5.61(1H，dd，J＝52.0，4.2Hz)，5.38－5.23(2H，m)，4.69－4.63(2H，m)，4.56(1H，d，J＝10.3Hz)，4.39－4.30(4H，m)，4.26－4.17(3H，m)，3.97－3.91(1H，m)，3.84－3.72(2H，m)，3.52－3.46(2H，m)，3.32－3.25(2H，m)，2.97(12H，q，J＝7.3Hz)，2.74－2.63(2H，m)，2.03－1.84(2H，m)，1.21(18H，t，J＝7.3Hz).

(step 7-1)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl ]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-10-oxo-2, 10-di-oxo-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 25 a: diastereomer 1)

The compound (21.9mg) obtained in the above step 6-1 was reacted by the same method as in the step 9-1 of example 22, and then purified by the following purification conditions to obtain the title compound (26.3 mg).

[ purification conditions ] preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile, acetonitrile: 30% -50% (0 min-30 min) and Sep-Pak (registered trademark) C18[ water/acetonitrile/0.1% triethylamine ].

MS(ESI)m/z：1395(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.58(1H，d，J＝4.2Hz)，8.08－8.02(2H，m)，7.64－7.13(14H，m)，6.46(1H，d，J＝18.1Hz)，6.26－6.21(1H，m)，5.45(1H，d，J＝53.2Hz)，5.35－5.18(2H，m)，5.07－5.01(1H，m)，4.62－4.13(10H，m)，4.09－3.90(2H，m)，3.87－3.43(11H，m)，3.21－3.09(1H，m)，3.18(12H，q，J＝7.3Hz)，3.00－2.89(1H，m)，2.83－2.52(3H，m)，2.37－2.21(2H，m)，2.03－1.79(3H，m)，1.28(18H，t，J＝7.3Hz).

(step 7-2)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]Azacyclooctan-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-10-oxo-2, 10-di-oxo-2-thioether-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] a ]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

(drug linker 25 b: diastereomer 2)

The compound (47.5mg) obtained in the above step 6-2 was reacted by the same method as in the step 9-1 of example 22, and then purified by the following purification conditions to obtain the title compound (15.1 mg).

MS(ESI)m/z：1395(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.99(1H，d，J＝3.6Hz)，8.14(1H，d，J＝1.8Hz)，8.02－8.00(1H，m)，7.64－7.13(13H，m)，7.01－6.98(1H，m)，6.47(1H，dd，J＝19.0，2.7Hz)，6.27(1H，dd，J＝8.5，4.2Hz)，5.67－5.52(1H，m)，5.37－5.16(2H，m)，5.08－5.01(1H，m)，4.67－4.14(11H，m)，4.10－3.61(10H，m)，3.47－3.42(2H，m)，3.17－3.10(1H，m)，3.15(12H，q，J＝7.5Hz)，3.00－2.92(1H，m)，2.84－2.75(1H，m)，2.63－2.54(2H，m)，2.32－2.20(2H，m)，2.03－1.79(3H，m)，1.28(18H，t，J＝7.3Hz).

Example 122: synthesis of drug linker 26

[ synthetic route ]

(step 1)

Using the compound (1.26g) obtained in the step 6 of example 77, a reaction was carried out in the same manner as in the step 7 of example 1 to obtain 3' -O- [ tert-butyl (dimethyl) silyl group]-2' -O- [ hydroxy (oxo) -lambda⁵-phosphino]-1- (2- { [ (N- { [ 2- (trimethylsilyl) ethoxy]Carbonyl } glycyl) amino]Methoxy } ethyl) inosine in acetonitrile. The obtained acetonitrile solution and the compound (1.00g) obtained in step 8 of example 44 were reacted in the same manner as in step 1 of example 63, and the obtained crude product was used as it was in the next reaction.

(step 2)

2- (trimethylsilyl) ethyl (2- { [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-cyanoethoxy) -15-fluoro-2-hydroxy-2, 10-di-oxo-octahydro-2H, 10H, 12H-5, 8-methano-2 lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Amino } -2-oxoethyl) carbamate

Using the crude product obtained in the above step 1, a reaction was carried out in the same manner as in step 2 of example 62 to obtain the title compound (678 mg: containing impurities).

MS(ESI)m/z：1246(M+H)⁺.

(step 3)

2- (trimethylsilyl) ethyl (2- { [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-benzoyl-6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ]]Azulen-2-yl) -16- { [ tert-butyl (dimethyl) silyl]Oxy } -10- (2-Cyanoethoxy) -15-fluoro-2-hydroxy-2, 10-dioxaneoxy octahydro-2H, 10H, 12H-5, 8-methano-2 lambda ⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Amino } -2-oxoethyl) carbamate

After the reaction was carried out in the same manner as in step 10 of example 1 using the compound (678mg) obtained in step 2, column chromatography was carried out using C18 silica gel column [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1): 25% to 90% (0 part to 30 parts) was refined to obtain the title compound (99.6 mg: containing impurities).

MS(ESI)m/z：1089(M+H)⁺.

(step 4)

Bis (N, N-diethylethanaminium) (5R, 7R, 8R, 12aR, 14R, 15R, 16R) -15-fluoro-7- (1- { 2- [ (glycylamino) methoxy]Ethyl } -6-oxo-1, 6-dihydro-9H-purin-9-yl) -16-hydroxy-2, 10-di-oxo-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] o]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyne-2, 10-bis (alkoxide)

The reaction was carried out in the same manner as in step 9-1 of example 11 using the compound obtained in step 3 (99.6mg), and then, column chromatography on silica gel [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [10mM triethylammonium acetate aqueous solution/acetonitrile-methanol solution (1: 1), acetonitrile-methanol solution (1: 1) ] using C18: 10% -60% (0 min-30 min) to give the title compound (60.8 mg: containing impurities).

MS(ESI)m/z：831(M+H)⁺.

(step 5)

Bis (N, N-diethylethanaminium) N- [ 4- (11, 12-didehydrodibenzo [ b, f ]]AzacyclooctanesTetralin-5 (6H) -yl) -4-oxobutanoyl]glycylglycinyl-L-phenylalanyl-N- [ (2- { 9- [ (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-16-hydroxy-2, 10-dioxy-2, 10-dioxido-14- (6, 7, 8, 9-tetrahydro-2H-2, 3, 5, 6-tetraazabenzo [ cd ] c]Azulen-2-yl) octahydro-2H, 10H, 12H-5, 8-methano-2. lambda⁵，10λ⁵-furo [3, 2-l][1，3，6，9，11，2，10]Pentaoxadiphosphine cyclotetradecyn-7-yl]-6-oxo-6, 9-dihydro-1H-purin-1-yl } ethoxy) methyl]Glycine amides

Using the compound obtained in the above-mentioned step 4 (60.8mg) and the compound obtained in the step 11 of example 22 (21.3mg), a reaction was carried out in the same manner as in the step 9-1 of example 22, and then purification was carried out under the following [ purification conditions ] to obtain the title compound (30.5 mg).

[ refining conditions ] C18 silica gel column chromatography [10mM triethylammonium acetate aqueous solution/acetonitrile ] and preparative HPLC [100mM hexafluoro-2-propanol, 8mM triethylamine aqueous solution/acetonitrile, acetonitrile: 10% -45% (0 min-40 min).

MS(ESI)m/z：1379(M+H)⁺.

¹H－NMR(CD₃OD)δ：8.56(1H，s)，8.16－8.10(1H，m)，8.05(1H，s)，7.63－7.49(2H，m)，7.45－7.35(3H，m)，7.33－7.12(9H，m)，6.45(1H，d，J＝18.3Hz)，6.27(1H，d，J＝9.8Hz)，5.55－4.99(4H，m)，4.65－4.42(5H，m)，4.38－4.01(8H，m)，3.89－3.60(9H，m)，3.52－3.42(2H，m)，3.18(12H，q，J＝7.3Hz)，3.00－2.91(1H，m)，2.87－2.60(3H，m)，2.38－2.19(2H，m)，2.04－1.81(3H，m)，1.28(18H，t，J＝7.3Hz).

Example 123: synthesis of antibody drug conjugate 35 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 11)

A phosphate-buffered saline (pH6.0) solution (10.87mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.135mL, 18 equivalents per 1 molecule of antibody) of the drug linker 24a and propylene glycol (0.365mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained according to the methods described in the common operations E and F.

Antibody concentration: 1.08mg/mL

Antibody yield: 7.54mg (69%)

Mean drug binding number: 3.8

Example 124: synthesis of antibody drug conjugate 36 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 12)

A phosphate-buffered saline (pH6.0) solution (10.87mg/mL, 1.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.500 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.135mL, 18 equivalents per 1 molecule of antibody) of the drug linker 24b and propylene glycol (0.365mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (7.0mL) of the target antibody-drug conjugate.

The following results were obtained by analysis according to the methods described in common procedures E and F.

Antibody concentration: 1.12mg/mL

Antibody yield: 7.85mg (72%)

Mean drug binding number: 3.8

Example 125: synthesis of antibody drug conjugate 37 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 13)

A phosphate-buffered saline (pH6.0) solution (10.87mg/mL, 2.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (1.00 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.270mL, 18 equivalents per 1 molecule of antibody) of drug linker 26 and propylene glycol (0.730mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (14.0mL) of the target antibody-drug conjugate.

The following results were obtained by analysis according to the methods described in the common procedures E and F.

Antibody concentration: 1.05mg/m

Antibody yield: 14.71mg (68%)

Mean drug binding number: 3.8

Example 126: synthesis of antibody drug conjugate 38 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 14)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 3.00mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (1.50 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.540mL, 24 equivalents per 1 molecule of antibody) of drug linker 25a and propylene glycol (0.960mL), and the mixture was stirred at room temperature for 3 days using a test tube rotator (MTR-103, AS ONE Co.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (19mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 1.38mg/mL

Antibody yield: 26.28mg (80%)

Mean drug binding number: 3.7

Example 127: synthesis of antibody drug conjugate 39 (Synthesis of anti-HER 2 antibody 2-CDN conjugate 15)

A phosphate-buffered saline (pH6.0) solution (10.89mg/mL, 0.500mL) of the sugar chain-reconstituted antibody 4 was diluted with propylene glycol (0.250 mL). To this solution was added a mixture of a dimethylsulfoxide solution (10mM, 0.090mL, 24 equivalents per 1 molecule of antibody) of drug linker 25b and propylene glycol (0.160mL), and the mixture was reacted at room temperature for 2 days using a test tube rotator (MTR-103, AS ONE Co., Ltd.). The reaction solution was purified as described in general procedure D to obtain an ABS solution (3.5mL) of the target antibody-drug conjugate.

The following results were obtained by conducting the analysis according to the methods described in common procedures E and G.

Antibody concentration: 0.93mg/mL

Antibody yield: 3.25mg (60%)

Mean drug binding number: 3.5

Reference example 1 preparation of anti-HER 2 antibody

In the present specification, "trastuzumab" is sometimes referred to as HERCEPTIN (registered trademark), huMAb4D 5-8, and rhuMAb4D 5-8, and is a humanized IgG1 antibody having a light chain formed of the amino acid sequence described in sequence No. 1 and a heavy chain formed of the amino acid sequence described in sequence No. 2. The amino acid sequence is referred to US 5821337. The light chain amino acid sequence (SEQ ID NO: 1) and the heavy chain amino acid sequence (SEQ ID NO: 2) of trastuzumab are shown in FIG. 4.

The anti-HER 2 antibody used in the present specification was produced by designing an IgG1 antibody (also referred to as an engineered anti-HER 2 antibody in the present specification) that modifies leucine (L) at positions 234 and 235 of EU INDEX of the heavy chain amino acid sequence of trastuzumab to alanine (a) (also referred to as a LALA modification in the present specification) to the constant region of trastuzumab. The light chain amino acid sequence (seq id No. 1) and the heavy chain amino acid sequence (seq id No. 3) of the engineered anti-HER 2 antibody are shown in figure 5.

Reference example 2 preparation of anti-LPS antibody

anti-LPS antibodies were made according to WO 2015/046505. The isotype of the anti-LPS antibody used in this example was IgG1, with a LALA variation (also referred to herein as engineered anti-LPS antibody). The amino acid sequences of the light chain and the heavy chain of the modified anti-LPS antibody used in this example are shown in seq id nos 26 and 27.

(reference example 3: ML-RR-CDA.2Na⁺Synthesis of (2)

ML-RR-CDA.2Na used as a reference compound in the present specification⁺Synthesized according to the method described in patent document 3(WO 2014/189805).

Reference example 4 Synthesis of 2 ', 3' -cGAMP

In the present specification, 2 ', 3' -cGAMP used as a reference compound is synthesized from ATP and GTP under enzymatic acceleration using cGAS. The preparation of cGAS and the enzymatic reaction were carried out by appropriately changing the methods described in the literature (Immunity, 2013, 39, 1019-1031, Cell Rep.2014, 6, 421-430). Refining WAs performed by column chromatography using a weakly basic anion exchange resin (DIAION WA10) and synthetic adsorbent (SEPABEADS SP207 SS).

Reference example 5 preparation of anti-HER 2 antibody 2

"pertuzumab" also referred to as PERJETA (registered trademark) is a humanized IgG1 antibody having a light chain formed from the amino acid sequence described by sequence No. 28 and a heavy chain formed from the amino acid sequence described by sequence No. 29. The amino acid sequence is referred to WO 2004/008099. Also referred to herein as anti-HER 2 antibody 2. The light chain amino acid sequence (SEQ ID NO: 28) and the heavy chain amino acid sequence (SEQ ID NO: 29) of pertuzumab are shown in FIG. 17.

In the present specification, an anti-HER 2 antibody 2 having a constant region of G1m3 allotype in which lysine (K) at position 214 of EU INDEX of the heavy chain amino acid sequence was mutated to arginine (R) in addition to the LALA mutation was designed and produced (also referred to as an engineered anti-HER 2 antibody 2 in the present specification). The light chain amino acid sequence (seq id No. 28) and the heavy chain amino acid sequence (seq id No. 30) of the engineered anti-HER 2 antibody 2 used in this example are shown in figure 18.

Reference example 6 preparation of anti-CD 33 antibody

anti-CD 33 antibodies were made with reference to WO 2014/057687. The isotype of the anti-CD 33 antibody used in this example was IgG1, with LALA variation. The light chain amino acid sequence (SEQ ID NO: 31) and the heavy chain amino acid sequence (SEQ ID NO: 32) of the anti-CD 33 antibody used in this example are shown in FIG. 19.

Reference example 7 preparation of anti-EphA 2 antibody

anti-EphA 2 antibodies were made with reference to WO 2009/028639. The isotype of the anti-EphA 2 antibody used in this example was IgG 1. The light chain amino acid sequence (SEQ ID NO: 33) and the heavy chain amino acid sequence (SEQ ID NO: 34) of the anti-EphA 2 antibody used in this example are shown in FIG. 20.

Reference example 8 preparation of anti-CDH 6 antibody

anti-CDH 6 antibodies were made with reference to WO 2018/212136. The isotype of the anti-CDH 6 antibody used in this example was IgG1, and had a variation in which proline (P) in EU INDEX No. 329 of the heavy chain amino acid sequence was changed to glycine (G) in addition to the LALA variation. The light chain amino acid sequence (SEQ ID NO: 35) and the heavy chain amino acid sequence (SEQ ID NO: 36) of the anti-CDH 6 antibody used in this example are shown in FIG. 21.

(test example 1) evaluation of STING agonist Activity Using reporter cells

< analysis of reporter Gene >

Human STING agonist activity using THP 1-Dual that confirms activation of the pathway of interferon control factor 3(IRF3) located downstream of the STING pathway^TMCells (HAQ variants) (InvivoGen, CA, US) were evaluated. Mouse STING agonist Activity RAW-Dual was used ^TMCells (InvivoGen) were evaluated.

The analysis is carried out in the following manner. First, the test compound diluted with PBS was dispensed into a transparent 96-well plate (Corning, NY, US) at 20. mu.L/well. Then, the mixture was heated at 180. mu.L/well (1X 10)⁵cells/well) reporter cells suspended in assay buffer (RPMI 1640 medium or DMEM medium containing 10% bovine serum albumin) were added to initiate stimulation. Recovering 5% CO at 37 deg.C₂The supernatant was centrifuged after 24 hours of incubation at ambient. mu.L of the recovered supernatant was added to a white 384-well plate, and 15. mu.L of QUANTI-Luc (InvivoGen) solution was added thereto. After thorough mixing, the luminescence was measured using a plate reader (PerkinElmer, MA, US). 1.37 to 100 mu M ML-RR-CDA.2Na is used⁺The maximum count value in (Compound 21in WO2014/189805) treated cells was set to 100%, the count in PBS treated cells was set to 0%, and the concentration required to give 50% of the counts of the test compounds was calculated as EC50(μ M) value using GraphPad Prism (GraphPad Software, CA, US). The results of the human STING agonist activity assay are shown in table 1.

[ Table 1]

This result indicates that the compounds of the present application have agonist activity to human STING. In addition, it was also confirmed that the agonist activity was equal to or higher than that of the existing CDN for mouse STING.

(test example 2) Protein thermal shift assay Using recombinant STING C-terminal binding Domain Protein

(i) Construction of various expression plasmids

< construction of human TMEM173 expression plasmid >

Plasmid for mammalian cell expression of human STING (also referred to herein as human TMEM173) human TMEM173 cDNA Clone (access NM _198282.3, H232(REF) variant STING expression plasmid) (GeneCopoeia, MD, US) was purchased with a histidine (H) variant (also referred to herein as H232 variant or REF variant) to arginine (R) at position 232. The amino acid sequence of human H232(REF) variant STING is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 5. Furthermore, site-specific mutagenesis was performed by the invader PCR method using the H232 mutant STING expression plasmid as a template to prepare expression plasmids for wild-type STING and mutant STING. Specifically, PCR was first carried out using 2 kinds of primers (Primer) (5'-CGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R (WT) fwd) (SEQ ID NO: 12) and 5'-GTCACCGGTCTGCTGGGGCAGTTTATC-3' (H232R (WT) rev)) (SEQ ID NO: 13) and KOD-Plus-Mutagenesis Kit (SMK-101) (Toyobo), and the construction of the target wild-type (R232) STING expression plasmid was confirmed by DNA sequencing. The amino acid sequence of human wild-type STING is shown in seq id No. 6, and the nucleotide sequence is shown in seq id No. 7.

Next, a mutant HAQ (R71H, G230A and R293Q) was prepared in the same manner as in the wild-type STING expression plasmid. Specifically, PCR was carried out using H232 mutant STING expression plasmid as a template, 2 kinds of primers (5'-GCTGACCGTGCTGGCATCAAGGATCGGGTTTAC-3' (H232R/G230A fwd) (SEQ ID NO: 14) and 5'-GGTCTGCTGGGGCAGTTTATCCAGG-3' (H232R/G230A rev) (SEQ ID NO: 15)) and Mutagenesis Kit. The mutation G230A was obtained by introducing mutation at position 2. Furthermore, PCR was carried out using 2 kinds of primers (5'-CACCACATCCACTCCAGGTACCGG-3' (R71H fwd) (SEQ ID NO: 16) and 5'-CAGCTCCTCAGCCAGGCTGCAGAC-3' (R71H rev) (SEQ ID NO: 17)) and the Mutagenesis Kit using the G230A variant STING expression plasmid as a template, to obtain the R71H/G230A variant STING expression plasmid.

Next, PCR was carried out using the STING expression plasmid of the R71H/G230A variant as a template, 2 kinds of primers (5'-CAGACACTTGAGGACATCCTGGCAG-3' (R293Q fwd) (SEQ ID NO: 18) and 5'-GCAGAAGAGTTTGGCCTGCTCAA-3' (R293Q rev) (SEQ ID NO: 19)) and the Mutagenesis Kit to obtain an expression plasmid of the HAQ (R71H/G230A/R293Q) variant. The amino acid sequence of human HAQ variant STING is shown in SEQ ID NO. 8, and the nucleotide sequence is shown in SEQ ID NO. 9.

The amino acid sequences of wild-type STING, REF-type STING, and HAQ-type STING in humans are shown in fig. 6.

< construction of plasmid for expressing recombinant STING C-terminal binding Domain protein >

Human STING C-terminal binding domain (aa 139-342) protein (unit entry Q86WV6) cDNA was prepared from full-length human TMEM173 cDNA clone expression plasmid (wild-type, H232 variant, and HAQ variant) by PCR using 2 primers (5'-ACCTGTATTTTCAGGGCCTGGCCCCAGCTGAGATCTCTG-3' (hST Fw _ V2) (seq id No. 20) and 5'-CAGAATTCGCAAGCTTTTAAGTAACCTCTTCCTTTTCCTCCTGC-3' (hST Rv _ V3) (seq id No. 21)). Using the In-Fusion HD Cloning Kit (Takara Bio), the PCR product was inserted into pET15b, which is a vector for expression of Escherichia coli, and expression plasmids of pET15 b-HisAviTEV-hSTING (139-342), the human wild type pET15 b-HisAviTEV-hSTING (139-342), the human REF variant pET15 b-HisAviTEV-hSTING (139-342), and the human AQ variant pET15 b-HisAviTEV-hSTING (139-342) were constructed by having a 6XHis tag consisting of histidine 6 bases, an Avidin (Avidin) tag, and a TEV protease cleavage site at the N-terminus.

For cDNA for expression of mouse STING C-terminal binding domain (aa 138-341) protein (Unit entry Q3TBT3), cDNA corresponding to cDNA sequence Nos. 138 to 341 of mouse TMEM173 was synthesized by eurofins Genomics. The amino acid sequence of mouse STING is shown in seq id No. 10, and the nucleotide sequence is shown in seq id No. 11. The synthesized cDNA was inserted into pET15b as a vector for Escherichia coli expression using the In-Fusion HD Cloning Kit, and the N-terminus was provided with a 6XHis tag consisting of histidine 6 base, an Avidin tag, and a TEV protease cleavage site, thereby constructing pET15 b-HisAviTEV-mSTING (138-.

An artificially synthesized Escherichia coli BirA (Unit entry P06709) cDNA was inserted into the pCDF _ Duet-1 vector to construct a pCDF _ Duet-1 BirA (1-321) expression plasmid.

(ii) Method for preparing protein of STING C-terminal binding domain

Each of the pET15 b-HisAviTEV-hSTING (139-) (STING C-terminal binding domain proteins of the human wild type, human REF variant and human AQ variant) expression plasmids and pET15 b-HisAviTEV-mSTING (138-) (341) (STING C-terminal binding domain protein of the mouse wild type) expression plasmids was converted into the plasmid pCDF _ Duet-1 BirA (1-321) expression plasmid, respectively, and then transformed into the plasmid E.coli Rosetta 2(DE3) (Merck Millipore, MA, US) to prepare each of the HisAviTEV-STING expression strains. These expression strains were added to TB medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL streptomycin and 30. mu.g/mL kanamycin, cultured at 37 ℃ and then induced to express with 100. mu.M IPTG, and further cultured at 16 ℃.

The culture medium was centrifuged, and the obtained cells were suspended in 50mM HEPES pH8.0, 500mM NaCl, 20mM imidazole, 1mM DTT, 5% (w/v) glycerol (glycerol), Complete EDTA free, and then frozen and dissolved. After addition of Lysozyme and DNase I, the protein was extracted by ultrasonication, and the supernatant was collected by centrifugation. The resulting supernatant was purified by using an AKTAexpress chromatography system (GE Healthcare, IL, US) on a HisTrap FF column (GE Healthcare), and eluted through a Superdex20016/60 column (GE Healthcare) with a buffer (20mM HEPES pH7.5, 120mM NaCl, 20% Glycerol, 0.8mM DTT). Fractions containing the protein of the target molecular weight were recovered by SEC as His-Avi-TEV-hSTING (139-) -human wild-type protein, HisAviTEV-hSTING (139-) -human REF variant protein, HisAviTEV-hSTING (139-) -human AQ variant protein and His-Avi-TEV-mSTING (138-) -341) mouse wild-type protein. Protein concentration was determined using Nanodrop2000(Thermo Fisher Scientific, MA, US) and stored frozen at-80 ℃ until use.

The amino acid sequence of the HisAviTEV-hSTING (139-342) human wild-type protein is shown as sequence No. 22, the amino acid sequence of the HisAviTEV-hSTING (139-342) human REF variant protein is shown as sequence No. 23, the amino acid sequence of the HisAviTEV-hSTING (139-342) human AQ variant protein is shown as sequence No. 24, and the amino acid sequence of the His-Avi-TEV-mSTING (138-341) mouse wild-type protein is shown as sequence No. 25.

(iii) STING binding assay

The binding of the compound to the Protein of the STING C-terminal binding domain is carried out by Protein thermal shift assay using the rise in the thermal denaturation temperature of the Protein as an indicator.

Specifically, 3. mu.L of test compound (final concentration 0.5mM), 3. mu.L of SYPRO Orange Protein Gel Stein (Thermo Fisher Scientific) (final concentration 20 Xconcentration), and 6. mu.L of STING Protein were mixed on a 384-well real-time PCR plate using assay buffer (20mM Tris-HCl pH 7.5, 120mM NaCl) and mixed with a plate shaker. The temperature of thermal denaturation of proteins was measured using a real-time PCR system (Thermo Fisher Scientific) by raising the temperature from 25 ℃ to 95 ℃ at a rate of 0.03 ℃ per second, using fluorescence emitted from SYPRO Orange as an indicator. The obtained measurement value was measured for Tm (the midpoint of the unfolding transition) (. degree. C.) as a temperature at which the rate of increase in fluorescence intensity shows a maximum value using analytical software Protein Thermal Shift software (Thermo Fisher Scientific). The shift in Tm of the test compound was calculated as Δ Tm (° c) by subtracting the Tm value of the wells to which no compound was added from the Tm value of each compound. Table 2 shows the results of the binding assay for STING proteins.

[ Table 2]

The results clearly show that the compounds of the present application have binding activity to wild type STING as well as variant STING in humans, and wild type STING in mice.

Test example 3 antitumor test (1)

Mice: female BALB/c mice (BALB/cAnNCrlCrlj) (Charles River, Japan) of 5 weeks of age were acclimatized under SPF conditions for 4 days or more before experimental use.

Measurement and calculation formula: in all studies, tumor volume (mm) was calculated by measuring the major and minor diameters of tumors 2-3 times per week using digital electronic calipers (CD 15-CX, Mitutoyo)³). The calculation formula is shown below.

Tumor volume (mm)³) 0.5 long diameter (mm) × [ short diameter (mm) ]]²

The test compound was used by diluting it with physiological saline (Otsuka pharmaceutical factory). 50 μ L was administered intratumorally.

Mouse colon cancer cell line CT26.WT (CRL2638) cells purchased from American Type Culture Collection were used. Suspending CT26.WT cells in physiological saline, and mixing 1.0X 10 cells⁶The cells were subcutaneously transplanted into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each test compound was administered in a total of three tumors of Day7, 9, and 11, respectively, in an amount of 10 μ g. As the vehicle group, a group to which physiological saline was administered was set. The number of mice in each group was either 5 or 6.

The results are shown in fig. 7a and 7 b. In the figure, black square lines indicate the vehicle group, white square lines indicate the compound number 6a administration group, white inverted triangular lines indicate the compound number 8b administration group, and white circular lines indicate the compound number 9b administration group. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. The vehicle group underwent tumor proliferation. In contrast, in the compound-administered group, tumor proliferation was significantly suppressed.

As described above, the antitumor effect of the cyclic dinucleotide derivative-based intratumoral administration was confirmed.

Test example 4 antitumor test (2)

CT26.WT-hHER 2 cells were prepared by introducing the human HER2 gene into a mouse large intestine cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection. Specifically, the cDNA was amplified using a plasmid containing cDNA of human HER2 (Clone ID IOH 82145; Thermo Fisher Scientific), which was inserted into pQCXIN retroviral vector (Takara Bio) using In-Fusion HD Cloning Kit (Clontech). Human HER2 insert pQCXIN retroviral vector was introduced into EcoPack 2-293 cell line (Takara Bio) using Lipofectamine 3000(Thermo Fisher Scientific), and the supernatant was recovered to infect CT26. WT. Cells were maintained in medium supplemented with 250. mu.g/mL Genetin (Thermo Fisher Scientific).

The antibody-CDN conjugate was diluted with Acetate Buffer (10mM Acetate Buffer, 5% Sorbitol, pH5.5) (Nacalai Tesque) for use. At the time of administration, 200. mu.L of the drug was administered into the tail vein.

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each antibody-CDN conjugate was administered in an amount of 30 μ g in tail vein at Day 7. As the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 8.

The results are shown in FIG. 8. In the figure, the black square line indicates the vehicle group, the white triangle line indicates the anti-HER 2 antibody-CDN conjugate (1) administration group in which the modified anti-HER 2 antibody prepared in reference example 1 was conjugated to the compound of example 8b, and the black triangle line indicates the anti-LPS antibody-CDN conjugate (1) administration group in which the modified anti-LPS antibody prepared in reference example 2 was similarly conjugated to the compound of example 8 b. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group and the anti-LPS antibody-CDN conjugate (1) administration group not binding to HER 2. In contrast, the anti-HER 2 antibody-CDN conjugate (1) administered group significantly inhibited tumor proliferation.

As described above, an antibody targeting-dependent anti-tumor effect based on intravenous administration of the anti-HER 2 antibody-CDN conjugate (1) was confirmed.

Test example 5 antitumor test (3)

CT26. WT-hHEThe R2 cells were suspended in physiological saline and 5.0X 10 cells were added⁶The cells were subcutaneously transplanted into the right axillary fossa of BALB/c mice (Day0), and randomized after 6 days. Each antibody-CDN conjugate was administered in tail vein one time at Day6 in an amount of 30 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 6 or 8.

The results are shown in fig. 9. In the figure, black square lines indicate the vehicle group, white square lines indicate the group administered with the anti-HER 2 antibody-CDN conjugate (2), and white triangular lines indicate the group administered with the anti-HER 2 antibody-CDN conjugate (3). The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. The vehicle group underwent tumor proliferation. In contrast, the anti-HER 2 antibody-CDN conjugates (2) and (3) significantly inhibited tumor proliferation in the group administered.

In conclusion, a strong anti-tumor effect of the anti-HER 2 antibody-CDN conjugate was confirmed.

Test example 6 antitumor test (4)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells ⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. antibody-CDN conjugate (19) was administered in tail vein at Day7 in an amount of 30 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 6.

The results are shown in fig. 10. The black square line in the figure indicates the vehicle group, and the white triangle line indicates the group to which the anti-HER 2 antibody-CDN conjugate (19) was administered. The drug linker of the anti-HER 2 antibody-CDN conjugate (19) was conjugated to the antibody via cysteine conjugation. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. The vehicle group underwent tumor proliferation. In contrast, the group administered with the anti-HER 2 antibody-CDN conjugate (19) significantly inhibited tumor proliferation.

In summary, strong antitumor effects of anti-HER 2 antibody-CDN conjugates coupling an antibody and a drug linker cysteine were confirmed.

Test example 7 antitumor test (5)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 6 days later. Each antibody-CDN conjugate was administered in tail vein one time at Day6 in an amount of 30 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 5.

The results are shown in fig. 11. In the figure, black squares indicate a vehicle group, white triangles indicate an anti-HER 2 antibody-CDN conjugate (9) administration group, white inverted triangles indicate an anti-HER 2 antibody-CDN conjugate (10) administration group, white diamonds indicate an anti-HER 2 antibody-CDN conjugate (11) administration group, white circles indicate an anti-HER 2 antibody-CDN conjugate (12) administration group, and white squares indicate an anti-HER 2 antibody-CDN conjugate (1) administration group. The anti-HER 2 antibody-CDN conjugates (9), (10), (11), (12) and (1) were each conjugated with a different linker to the compound of example 8 b. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. The vehicle group underwent tumor proliferation. On the other hand, in the groups administered with the anti-HER 2 antibody-CDN conjugates (9), (10), (11), and (12), tumor proliferation was significantly suppressed in the same manner as in the group administered with the anti-HER 2 antibody-CDN conjugate (1).

As described above, it was confirmed that the anti-HER 2 antibody-CDN conjugate showed an anti-tumor effect even though the linker was changed.

Test example 8 antitumor test (6)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each test subject was administered one tail vein at Day 7. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 5.

The results are shown in fig. 12. In the figure, the black square line indicates the vehicle group, the white triangle line indicates the 60 μ g administration group of the anti-HER 2 antibody 2-CDN conjugate (1), the black inverted triangle line indicates the 59 μ g administration group of the anti-HER 2 antibody 2, and the black circle line indicates the 1.2 μ g administration group of compound No. 8 b. anti-HER 2 antibody 2 and compoundsThe amount of substance No. 8b administered is the equivalent of each component constituting the anti-HER 2 antibody 2-CDN conjugate (1). The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. In contrast, in the group administered with the anti-HER 2 antibody 2-CDN conjugate (1), tumor proliferation was significantly inhibited. However, when the equivalent of anti-HER 2 antibody 2 and compound No. 8b was administered, tumor proliferation was not inhibited.

Taken together, anti-HER 2 antibody 2-CDN conjugate (1) showed anti-tumor effect, equivalent anti-HER 2 antibody 2 and compound No. 8b showed no anti-tumor effect in tail vein administration.

Test example 9 antitumor test (7)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each antibody-CDN conjugate was administered in tail vein one time at Day7 in an amount of 30 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was either 5 or 6.

The results are shown in FIGS. 13(a) to (c). In the figure, black square lines indicate vehicle groups, and white symbol lines indicate groups to which the anti-HER 2 antibody 2-CDN conjugates (2), (3), (4), (5), (6), (7), and (8) were administered, respectively, for evaluation. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. In contrast, the group administered with anti-HER 2 antibody 2-CDN conjugates (2), (3), (4), (5), (6), (7), and (8) significantly inhibited tumor proliferation.

Taken together, the strong anti-tumor effect of the anti-HER 2 antibody 2-CDN conjugate was confirmed.

Test example 10 antitumor test (8)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each antibody-CDN conjugate was administered in an amount of 60 μ g in tail vein at Day 7. In addition, as a vehicle group, an administration acetate buffer solution was setThe group (2). The number of mice in each group was 5.

The results are shown in fig. 14. In the figure, the black square line indicates the vehicle group, the white triangular line indicates the group administered with the anti-HER 2 antibody 2-CDN conjugate (9), and the white circular line indicates the group administered with the anti-HER 2 antibody 2-CDN conjugate (10). The anti-HER 2 antibody 2-CDN conjugates (9) and (10) are antibody-CDN conjugates using MSG type sugar chain recombinant antibodies with an average drug binding number of about 2. The vertical axis represents the tumor volume (mm) ³) And the horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. In contrast, the anti-HER 2 antibody 2-CDN conjugate (9) and (10) significantly inhibited tumor proliferation in the administered group.

In summary, a strong antitumor effect of the anti-HER 2 antibody 2-CDN conjugate with an average drug binding number of about 2 was confirmed.

Test example 11 antitumor test (9)

CT26. WT-hEPHA 2 cells were prepared by introducing the human EphA2 gene into the mouse large intestine cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection. In detail, the cDNA was amplified using pDONR221(Thermo Fisher Scientific) containing the cDNA of human EphA2, and inserted into pLNCX retroviral vector (Takara Bio) using a Gateway vector conversion system (Thermo Fisher Scientific). Human EphA2 was inserted into pLNCX retroviral vector and introduced into EcoPack 2-293 cell line (Takara Bio) using Lipofectamine 2000(Thermo Fisher Scientific), and the supernatant was recovered to infect CT26. WT. Cells were maintained in medium supplemented with 500. mu.g/mL Genetin (Thermo Fisher Scientific).

CT26. WT-hEPhEPHA 2 cells were suspended in phosphate buffer and 1.9X 10 cells were added ⁶The cells were transplanted subcutaneously into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. The anti-EphA 2 antibody and anti-EphA 2 antibody-CDN conjugate (1) were administered in tail vein at Day7 once in an amount of 60 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 8.

The results are shown in fig. 15. The black square lines in the figure represent the vehicle groups and the white circular lines represent the anti-EphThe a2 antibody administration group and the white triangle line indicate the anti-EphA 2 antibody-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. The anti-EphA 2 antibody-administered group did not inhibit tumor proliferation. In contrast, the anti-EphA 2 antibody-CDN conjugate (1) administered group significantly inhibited tumor proliferation.

As described above, using a model in which the anti-EphA 2 antibody did not exhibit an anti-tumor effect, a strong anti-tumor effect of the anti-EphA 2 antibody-CDN conjugate was confirmed.

Test example 12 antitumor test (10)

P388D 1-hCD 33 cells were prepared by introducing the human CD33 gene into the mouse lymphocyte-like cell line P388D1 (CCL-46) purchased from American Type Culture Collection. Specifically, a pLVSIN Lentivirus (Lentivirus) vector (Takara Bio) into which cDNA of human CD33 was inserted was prepared, introduced into a Lenti-X293T cell line (Takara Bio) using a Lentiviral High Titer Packaging Mix (Takara Bio), and the supernatant was collected and infected with P388D 1. Cells were maintained in medium supplemented with 2. mu.g/mL Puromycin (Thermo Fisher Scientific).

For the mice, female DBA/2 mice (DBA/2NCrl) (Charles River, Japan) of 4 weeks of age were purchased and acclimatized under SPF conditions for 5 days before experimental use.

The P388D 1-hCD 33 cells were suspended in phosphate buffer and 1.0X 10 cells were added⁶The cells were implanted subcutaneously in the right axillary region of DBA/2 mice (Day0), and randomized after 4 days. anti-CD 33 antibody and anti-CD 33 antibody-CDN conjugate (1) were administered in tail vein at Day4 once in an amount of 60 μ g. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 10.

The results are shown in fig. 16. In the figure, the black square line indicates the vehicle group, the white circular line indicates the group to which the anti-CD 33 antibody was administered, and the white triangular line indicates the group to which the anti-CD 33 antibody-CDN conjugate (1) was administered. The vertical axis represents the tumor volume (mm)³) And the horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. The group administered with the anti-CD 33 antibody did not inhibit tumor proliferation. And therewithIn contrast, the anti-CD 33 antibody-CDN conjugate (1) administered group significantly inhibited tumor proliferation.

As described above, a strong antitumor effect of the anti-CD 33 antibody-CDN conjugate was confirmed using a model in which the anti-CD 33 antibody did not show an antitumor effect.

Test example 13 antitumor test (11)

Suspending CT26. WT-hHER 2 cells in physiological saline, and mixing 5.0X 10 cells⁶The cells were subcutaneously transplanted into the right axillary fossa of BALB/c mice (Day0), and randomized 7 days later. Each antibody-CDN conjugate was administered in an amount of 60 μ g in tail vein at Day 7. In addition, as the vehicle group, a group to which an acetate buffer solution was administered was set. The number of mice in each group was 6.

The results are shown in fig. 22. In the figure, the black square line indicates the vehicle group, the white triangular line indicates the group administered with the anti-HER 2 antibody 2-CDN conjugate (11), and the white circular line indicates the group administered with the anti-HER 2 antibody 2-CDN conjugate (12). Compound numbers 52a and 52b conjugated to anti-HER 2 antibody 2-CDN conjugates (11) and (12) are CDNs having a phosphate group. The vertical axis represents the tumor volume (mm)³) The horizontal axis represents the number of days after tumor implantation. Tumor proliferation was performed in the vehicle group. In contrast, the anti-HER 2 antibody 2-CDN conjugate (11) and (12) significantly inhibited tumor proliferation in the administered group.

As described above, strong antitumor effects of the anti-HER 2 antibody 2-CDN conjugates (11) and (12) to which a CDN having a phosphate group was conjugated were confirmed.

Industrial applicability

The invention can provide a novel CDN derivative which has stronger STING agonist activity and shows strong anti-tumor effect. In addition, antibody drug conjugates comprising the novel CDN derivative can be provided based on the present invention. These are useful as therapeutic agents for diseases associated with STING agonist activity (e.g., cancer).

Sequence Listing free text

Sequence number 1: amino acid sequence of the light chain of trastuzumab

Sequence number 2: amino acid sequence of the heavy chain of trastuzumab

Sequence number 3: amino acid sequence of heavy chain of engineered anti-HER 2 antibody

Sequence number 4: amino acid sequence of human H232(REF) variant STING

Sequence number 5: nucleotide sequence of human H232(REF) variant STING

Sequence number 6: amino acid sequence of human wild type STING

Sequence number 7: nucleotide sequence of human wild type STING

Sequence number 8: amino acid sequence of human HAQ variant STING

Sequence number 9: nucleotide sequence of human HAQ variant STING

Sequence number 10: amino acid sequence of mouse STING

Sequence number 11: nucleotide sequence of mouse STING

Sequence number 12 ~ 21: sequence of the primer

Sequence number 22: amino acid sequence of HisAviTEV-hSTING (139-342) human wild-type protein

Sequence number 23: amino acid sequence of HisAviTEV-hSTING (139-342) human REF variant protein

Sequence number 24: amino acid sequence of HisAviTEV-hSTING (139-342) human AQ mutant protein

Sequence number 25: amino acid sequence of His-Avi-TEV-mSTING (138-

Sequence number 26: engineering the amino acid sequence of the light chain of an anti-LPS antibody

Sequence number 27: amino acid sequence engineering of heavy chain of anti-LPS antibody

Sequence number 28: amino acid sequence of pertuzumab light chain

Sequence number 29: amino acid sequence of pertuzumab heavy chain

Sequence number 30: amino acid sequence of heavy chain of engineered anti-HER 2 antibody 2

Sequence number 31: amino acid sequence of anti-CD 33 antibody light chain

Sequence number 32: amino acid sequence of heavy chain of anti-CD 33 antibody

Sequence number 33: amino acid sequence of anti-EphA 2 antibody light chain

Sequence number 34: amino acid sequence of anti-EphA 2 antibody heavy chain

Sequence number 35: amino acid sequence of anti-CDH 6 antibody light chain

Sequence number 36: amino acid sequence of the heavy chain of an anti-CDH 6 antibody.

Sequence listing

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Gly Lys

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

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Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

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Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

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Gly Lys

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Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr

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Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val

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Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys

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Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser

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Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly

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Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala

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Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln

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Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile

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Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala

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Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln

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Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly

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Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val

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Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys

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Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg Val Tyr

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Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr

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Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser

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Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala

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Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu

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Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp

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Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu

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Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro

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Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met

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Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser

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gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccggt gaccatgctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctccacgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttag 1140

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Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His

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Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr

20 25 30

Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val

35 40 45

Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys

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Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser

65 70 75 80

Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly

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Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala

100 105 110

Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln

115 120 125

Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile

130 135 140

Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala

145 150 155 160

Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln

165 170 175

Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly

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Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val

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Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys

210 215 220

Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr

225 230 235 240

Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr

245 250 255

Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser

260 265 270

Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala

275 280 285

Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu

290 295 300

Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp

305 310 315 320

Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu

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Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala Val Pro

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Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met

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Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser

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