Camptothecin derivatives

文档序号：1942440 发布日期：2021-12-07 浏览：7次中文

阅读说明：本技术 喜树碱衍生物 (Camptothecin derivatives ) 是由 R·查里 W·C·威迪森李为 D·P·普莱内特于 2020-04-10 设计创作，主要内容包括：本文公开了新型细胞毒性化合物,以及包含这些细胞毒性化合物和细胞结合剂的细胞毒性缀合物。更具体来说,本公开涉及其新型喜树碱衍生物、其中间体、其缀合物和其药学上可接受的盐,其可用作药剂,特别是可用作抗增殖剂(抗癌剂)。(Disclosed herein are novel cytotoxic compounds, as well as cytotoxic conjugates comprising these cytotoxic compounds and a cell-binding agent. More particularly, the present disclosure relates to novel camptothecin derivatives thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as pharmaceutical agents, particularly as antiproliferative agents (anticancer agents).)

1. A compound of formula I or a pharmaceutically acceptable salt thereof,

Z—L¹d (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R²is-H, -F, -OR³、-SR³、-S(O)R⁴、-S(O)₂R⁴、C₁-C₆Alkyl or C₁-C₆A fluoroalkyl group; or R¹And R²Taken together with the carbon atom to which it is attached to form a methylenedioxy or difluoromethylenedioxy ring;

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R ⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

L¹and L²Each independently optionally substituted by 1-4 substituents selected from halogen, -CN, -OR⁷、-SR⁷、-N(R⁷)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, or heteroaryl; and is

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

with the proviso that if R¹Is F, then L¹Is- (C)₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z; and Z is-X²(ii) a And is

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂。

2. A compound of formula I or a pharmaceutically acceptable salt thereof,

Z—L¹d (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C ₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂(ii) a And is

With the proviso that if R¹Is F and R²is-Me, then-L ¹-Z cannot be-CH₂OH。

3. A compound as claimed in claim 1 or claim 2 wherein R is¹is-H or-F.

4. The compound of any one of claims 1 to 3, wherein R¹is-F.

5. A compound according to any one of claims 1 to 4, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl.

6. The compound of any one of claims 1 to 5, wherein R²is-F.

7. The compound of any one of claims 1 to 5, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

8. The compound of any one of claims 1 to 5, wherein R²Is methyl.

9. A compound as claimed in claim 1 or claim 2 wherein R is¹is-F and R²is-F.

10. A compound as claimed in claim 1 or claim 2 wherein R is¹Is methyl and R²is-F.

11. A compound as claimed in claim 1 or claim 2 wherein R is¹is-F and R²Is a-methyl group.

12. The compound of any one of claims 1-11, wherein-L¹-Z is-H.

13. The compound of any one of claims 1-11, wherein-L¹-Z is- (C)₁-C₆Alkylene) -H or- (C)₁-C₆Alkylene) -X²。

14. The compound of claim 13, wherein-L¹-Z is methyl, ethyl, propyl or butyl.

15. The compound of any one of claims 1-11, wherein-L¹-Z is- (C)₁-C₄Alkylene) -OR⁶、-(C₁-C₄Alkylene) -SR⁶Or- (C)₁-C₄Alkylene) -N (R)⁶)₂。

16. The compound of claim 15, wherein-L¹-Z is-CH₂OH、-(CH₂)₂OH、-(CH₂)₃OH、-(CH₂)₄OH、-CH₂OMe、-(CH₂)₂OMe、-(CH₂)₃OMe、-(CH₂)₄OMe、-CH₂SH、-(CH₂)₂SH、-(CH₂)₃SH、-(CH₂)₄SH、-CH₂SMe、-(CH₂)₂SMe、-(CH₂)₃SMe、-(CH₂)₄SMe、-CH₂NH₂、-(CH₂)₂NH₂、-(CH₂)₃NH₂、-(CH₂)₄NH₂。

17. The compound of any one of claims 1-11, wherein-L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SR⁶Or- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶。

18. The compound of claim 17, wherein-L¹-Z is-CH₂NHC(＝O)CH₂OH、-CH₂NHC(＝O)(CH₂)₂OH、-CH₂NHC(＝O)(CH₂)₃OH、-CH₂NHC(＝O)(CH₂)₄OH、-CH₂NHC(＝O)(CH₂)₅OH、-CH₂NHC(＝O)CH₂OMe、-CH₂NHC(＝O)(CH₂)₂OMe、-CH₂NHC(＝O)(CH₂)₃OMe、-CH₂NHC(＝O)(CH₂)₄OMe、-CH₂NHC(＝O)(CH₂)₅OMe、-CH₂NHC(＝O)CH₂SH、-CH₂NHC(＝O)(CH₂)₂SH、-CH₂NHC(＝O)(CH₂)₃SH、-CH₂NHC(＝O)(CH₂)₄SH、-CH₂NHC(＝O)(CH₂)₅SH、-CH₂NHC(＝O)CH₂SMe、-CH₂NHC(＝O)(CH₂)₂SMe、-CH₂NHC(＝O)(CH₂)₃SMe、-CH₂NHC(＝O)(CH₂)₄SMe、-CH₂NHC(＝O)(CH₂)₅SMe、-CH₂SCH₂OH、-CH₂S(CH₂)₂OH、-CH₂S(CH₂)₃OH、-CH₂S(CH₂)₄OH、-CH₂S(CH₂)₅OH、-CH₂SCH₂OMe、-CH₂S(CH₂)₂OMe、-CH₂S(CH₂)₃OMe、-CH₂S(CH₂)₄OMe、-CH₂S(CH₂)₅OMe、-CH₂SCH₂SH、-CH₂S(CH₂)₂SH、-CH₂S(CH₂)₃SH、-CH₂S(CH₂)₄SH、-CH₂S(CH₂)₅SH、-CH₂SCH₂SMe、-CH₂S(CH₂)₂SMe、-CH₂S(CH₂)₃SMe、-CH₂S(CH₂)₄SMe or-CH₂S(CH₂)₅SMe。

19. The compound of claim 17 or claim 18, wherein each R is⁵independently-H, methyl or benzyl.

20. The compound of any one of claims 15 to 18, wherein each R⁶independently-H, methyl or benzyl.

21. The compound of any one of claims 1-11, wherein-L¹-Z is-X^1'-(C₁-C₄Alkylene) -X²。

22. The compound of claim 21, wherein-L¹-Z is-OCH₂OH、-O(CH₂)₂OH、-O(CH₂)₃OH、-O(CH₂)₄OH、-SCH₂OH、-S(CH₂)₂OH、-S(CH₂)₃OH、-S(CH₂)₄OH、-S(O)CH₂OH、-S(O)(CH₂)₂OH、-S(O)(CH₂)₃OH、-S(O)(CH₂)₄OH、-S(O)₂CH₂OH、-S(O)₂(CH₂)₂OH、-S(O)₂(CH₂)₃OH、-S(O)₂(CH₂)₄OH、-OCH₂SMe、-O(CH₂)₂SMe、-O(CH₂)₃SMe、-O(CH₂)₄SMe、-SCH₂SMe、-S(CH₂)₂SMe、-S(CH₂)₃SMe、-S(CH₂)₄SMe、-S(O)CH₂SMe、-S(O)(CH₂)₂SMe、-S(O)(CH₂)₃SMe、-S(O)(CH₂)₄SMe、-S(O)₂CH₂SMe、-S(O)₂(CH₂)₂SMe、-S(O)₂(CH₂)₃SMe or-S (O)₂(CH₂)₄SMe。

23. The compound of any one of claims 1-11, wherein-L¹-Z is- (C)₁-C₆Alkylene) -X¹-L²-X²。

24. The compound of claim 23, wherein-L¹-Z is

25. The compound of claim 1, wherein the compound is any one of the compounds selected from the group consisting of:

26. The compound of claim 1, wherein the compound is any one selected from the compounds of table 1B.

27. A compound of formula II or a pharmaceutically acceptable salt thereof,

E—A—Z'—L¹d (formula II)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

L¹and L²Each independently optionally substituted by 1-4 substituents selected from halogen, -CN, -OR⁷、-SR⁷、-N(R⁷)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C ₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, or heteroaryl; and is

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols; and is

E is-C (═ O) -L³-X³；

L³Is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X³The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

wherein L is³Optionally substituted by 0-4 substituents selected from halogen, -CN, -OR¹¹、-SR¹¹、-N(R¹¹)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, heteroarylAnd substituent substitution of polyols;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X³ -C(＝O)-CR^bbR^cc-W'、-NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰；

Each W' is independently-H, -N (R)^gg)₂、C₁-C₁₀Alkyl radical, C₁-C₁₀Alkenyl radical, C₁-C₁₀Alkynyl, C₃-C₆Cycloalkyl, aryl, heteroaryl or- (CH)₂CH₂O)_q-R^ff；

q is 1 to 24;

each R^aa、R^bb、R^cc、R^eeAnd R ^ffIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R^ggeach independently is-H or C₁-C₆An alkyl group; and is

R⁹And R¹⁰Each independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

28. The compound of claim 27, wherein R¹Is-H or-F.

29. The compound of claim 27 or claim 28, wherein R¹is-F.

30. The compound of any one of claims 27-29, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl.

31. The compound of any one of claims 27-30, wherein R²is-F.

32. The compound of any one of claims 27-30, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

33. The compound of any one of claims 27-30, wherein R²Is methyl.

34. The compound of claim 27, wherein R¹is-F and R²is-F.

35. The compound of claim 27, wherein R¹Is methyl and R²is-F.

36. The compound of claim 27, wherein R¹is-F and R²Is a-methyl group.

37. The compound of any one of claims 27-36, wherein-L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH ₂-NR⁸-*、-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (C)₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

38. The compound of claim 37, wherein-L¹-Z' -is-CH₂O-CH₂NH-*、-(CH₂)₂O-CH₂NH-*、-(CH₂)₃O-CH₂NH-*、-(CH₂)₄O-CH₂NH-*、-CH₂S-CH₂NH-*、-(CH₂)₂S-CH₂NH-*、-(CH₂)₃S-CH₂NH-*、-(CH₂)₄S-CH₂NH-*、-CH₂NH-*、-(CH₂)₂NH-*、-(CH₂)₃NH- [ or- (CH)₂)₄NH-。

39. The compound of any one of claims 27-36, wherein-L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-, wherein is a site covalently linked to a.

40. The compound of claim 39, wherein-L¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-, wherein is a site covalently linked to a.

41. The compound of claim 39 or claim 40, wherein each R⁵independently-H, methyl or benzyl.

42. The compound of any one of claims 37-41, wherein each R⁸independently-H, methyl or benzyl.

43. The compound of any one of claims 27-36, wherein-L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

44. The compound of claim 43, wherein-L¹-Z' -is-OCH₂O-CH₂-NH-*、-O(CH₂)₂O-CH₂-NH-*、-O(CH₂)₃O-CH₂-NH-*、-O(CH₂)₄O-CH₂-NH-*、-SCH₂O-CH₂-NH-*、-S(CH₂)₂O-CH₂-NH-*、-S(CH₂)₃O-CH₂-NH-*、-S(CH₂)₄O-CH₂-NH-*、-S(O)CH₂O-CH₂-NH-*、-S(O)(CH₂)₂O-CH₂-NH-*、-S(O)(CH₂)₃O-CH₂-NH-*、-S(O)(CH₂)₄O-CH₂-NH-*、-S(O)₂CH₂O-CH₂-NH-*、-S(O)₂(CH₂)₂O-CH₂-NH-*、-S(O)₂(CH₂)₃O-CH₂-NH-*、-S(O)₂(CH₂)₄O-CH₂-NH-*、-OCH₂S-CH₂-NH-*、-O(CH₂)₂S-CH₂-NH-*、-O(CH₂)₃S-CH₂-NH-*、-O(CH₂)₄S-CH₂-NH-*、-SCH₂S-CH₂-NH-*、-S(CH₂)₂S-CH₂-NH-*、-S(CH₂)₃S-CH₂-NH-*、-S(CH₂)₄S-CH₂-NH-*、-S(O)CH₂S-CH₂-NH-*、-S(O)(CH₂)₂S-CH₂-NH-*、-S(O)(CH₂)₃S-CH₂-NH-*、-S(O)(CH₂)₄S-CH₂-NH-*、-S(O)₂CH₂S-CH₂-NH-*、-S(O)₂(CH₂)₂S-CH₂-NH-*、-S(O)₂(CH₂)₃S-CH₂-NH-*、-S(O)₂(CH₂)₄S-CH₂-NH-*、-OCH₂-NH-*、-O(CH₂)₂-NH-*、-O(CH₂)₃-NH-*、-O(CH₂)₄S-NH-*、-SCH₂-NH-*、-S(CH₂)₂-NH-*、-S(CH₂)₃-NH-*、-S(CH₂)₄-NH-*、-S(O)CH₂-NH-*、-S(O)(CH₂)₂-NH-*、-S(O)(CH₂)₃-NH-*、-S(O)(CH₂)₄-NH-*、-S(O)₂CH₂-NH-*、-S(O)₂(CH₂)₂-NH-*、-S(O)₂(CH₂)₃-NH-or-S (O)₂(CH₂)₄-NH-*。

45. The compound of any one of claims 27-36, wherein-L ¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -, wherein is a site covalently linked to a.

46. The compound of claim 45, wherein-L¹-Z' -is

Wherein is a site covalently linked to a.

47. The compound of any one of claims 27-46, wherein A is a peptide comprising 2 to 8 amino acids.

48. The compound of any one of claims 27-47, wherein A is a peptide comprising 2 to 4 amino acids.

49. The compound of any one of claims 27-48, wherein at least one amino acid in the peptide is an L amino acid.

50. The compound of any one of claims 27-49, wherein each amino acid in the peptide is an L amino acid.

51. The compound of any one of claims 27-48, wherein at least one amino acid in the peptide is a D amino acid.

52. The compound of any one of claims 27-46, wherein A is- (AA)¹)-(AA²)_a1-, wherein is a position covalently linked to EPoint; AA¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

53. The compound of claim 52, wherein-AA 1- (AA2) a 1-is-Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Lys-, -Lys-Cit-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Phe-Ala-, -, -Ala-Phe-, -Phe-N9-tosyl-Arg-, -N9-tosyl-Arg-Phe-, -Phe-N9-nitro-Arg-, -N9-nitro-Arg-Phe-, -Phe-Lys-, -Lys-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Leu-, -Val-Ala-Val-, -Ala-Leu-, -, -Leu-Ala-, - β -Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -Val-Arg-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, - -Met-, -Met-Thr-, -Leu-Ala-, - -Leu-Cit, -Cit-Val-, -Gln-Val-, -Leu-, -, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr-, and-Tyr-Met-).

54. The compound of claim 52, wherein-AA¹-(AA²)_a1-Val-D-Lys-, -Val-D-Arg-, -L-Val-Cit-, -L-Val-Lys-, -L-Val-Arg-, -L-Val-D-Cit-, -L-Phe-Phe-Lys-, -L-Val-D-Arg-, -L-Arg-D-Arg-, -L-Ala-Ala-Ala-, -L-Ala-D-Ala-, -Val-D-Cit-, -, -L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val- ".

55. The compound of claim 52, wherein-AA¹-(AA²)_a1-:

-Ala-Ala-*、

-Ala-Val-*、

-Val-Ala-*

-Gln-Leu-*、

-Leu-Gln-*

-Ala-Ala-Ala-*、

-Ala-Ala-Ala-Ala-*、

-Gly-Ala-Gly-Gly-*、

-Gly-Gly-Ala-Gly-*、

-Gly-Val-Gly-Gly-*、

-Gly-Gly-Val-Gly-*、

-Gly-Phe-Gly-Gly-or

-Gly-Gly-Phe-Gly-*。

56. The compound of claim 52, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-*、

-L-Ala-L-Val-*、

-L-Ala-D-Val-*、

-L-Val-L-Ala-*、

-L-Val-D-Ala-*

-L-Gln-L-Leu-*、

-L-Gln-D-Leu-*、

-L-Leu-L-Gln-*、

-L-Leu-D-Gln-*、

-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-*、

-L-Ala-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-D-Ala-*、

-Gly-L-Ala-Gly-Gly-*、

-Gly-Gly-L-Ala-Gly-*、

-Gly-D-Ala-Gly-Gly-*、

-Gly-Gly-D-Ala-Gly-*、

-Gly-L-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-D-Val-Gly-Gly-*、

-Gly-Gly-D-Val-Gly-*、

-Gly-L-Phe-Gly-Gly-or

-Gly-Gly-L-Phe-Gly-*。

57. The compound of claim 52, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-LAla-*、

-L-Ala-L-Ala-L-Ala-or

-L-Ala-L-Ala-L-Ala-L-Ala-*。

58. The compound of any one of claims 27-57, wherein A is substituted with one or more polyols.

59. The compound of any one of claims 27-58, wherein E is substituted with one or more polyols.

60. The compound of any one of claims 27-59, wherein polyol is- (C)₁-C₆Alkylene) -X⁵-Y³；

Wherein:

X⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；

Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and is

R¹²is-H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

61. The compound of claim 60, wherein the polyol isWherein R is¹²Is H or methyl.

62. The compound of any one of claims 27-61, wherein E is-C (═ O) - (C)₁-C₁₀Alkylene) -X³。

63. The compound of claim 62, wherein E is

64. The compound of any one of claims 27-61, wherein E is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X³；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X³Is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

65. The compound of any one of claims 27-61, wherein E is-C (C: (I)) (＝O)-Y¹-(CH₂)₂-X⁴-(CH₂)₂-X³；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X³is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

66. The compound of claim 27, wherein the compound is any one selected from the compounds of table 2.

67. A compound of formula III or a pharmaceutically acceptable salt thereof,

CBA—E'—A—Z'—L¹d (formula III)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴Is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols;

e' is-C (═ O) -L³-X⁶-; wherein is a site covalently attached to the CBA;

L³is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X⁶The site of (a);

Y¹Is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA;

each R^aa、R^bb、R^ccAnd R^eeIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R⁹independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

CBA is a cell binding agent.

68. The compound of claim 67, wherein R¹is-H or-F.

69. The compound of claim 67 or claim 68, wherein R¹is-F.

70. The compound of any one of claims 67-69, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl。

71. The compound of any one of claims 67-70, wherein R ²is-F.

72. The compound of any one of claims 67-70, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

73. The compound of any one of claims 67-70, wherein R²Is methyl.

74. The compound of claim 67, wherein R¹is-F and R²is-F.

75. The compound of claim 67, wherein R¹Is methyl and R²is-F.

76. The compound of claim 67, wherein R¹is-F and R²Is a-methyl group.

77. The compound of any one of claims 67-76, wherein-L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (C)₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

78. The compound of claim 77, wherein-L¹-Z' -is-CH₂O-CH₂NH-*、-(CH₂)₂O-CH₂NH-*、-(CH₂)₃O-CH₂NH-*、-(CH₂)₄O-CH₂NH-*、-CH₂S-CH₂NH-*、-(CH₂)₂S-CH₂NH-*、-(CH₂)₃S-CH₂NH-*、-(CH₂)₄S-CH₂NH-*、-CH₂NH-*、-(CH₂)₂NH-*、-(CH₂)₃NH- [ or- (CH)₂)₄NH-。

79. The compound of any one of claims 67-76, wherein-L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-, wherein is a site covalently linked to a.

80. The compound of claim 79, wherein-L¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-*。

81. The compound of claim 79 or claim 80, wherein each R ⁵independently-H, methyl or benzyl.

82. The compound of any one of claims 77-81, wherein each R⁸independently-H, methyl or benzyl.

83. The compound of any one of claims 67-76, wherein-L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

84. The compound of claim 83, wherein-L¹-Z' -is-OCH₂O-CH₂-NH-*、-O(CH₂)₂O-CH₂-NH-*、-O(CH₂)₃O-CH₂-NH-*、-O(CH₂)₄O-CH₂-NH-*、-SCH₂O-CH₂-NH-*、-S(CH₂)₂O-CH₂-NH-*、-S(CH₂)₃O-CH₂-NH-*、-S(CH₂)₄O-CH₂-NH-*、-S(O)CH₂O-CH₂-NH-*、-S(O)(CH₂)₂O-CH₂-NH-*、-S(O)(CH₂)₃O-CH₂-NH-*、-S(O)(CH₂)₄O-CH₂-NH-*、-S(O)₂CH₂O-CH₂-NH-*、-S(O)₂(CH₂)₂O-CH₂-NH-*、-S(O)₂(CH₂)₃O-CH₂-NH-*、-S(O)₂(CH₂)₄O-CH₂-NH-*、-OCH₂S-CH₂-NH-*、-O(CH₂)₂S-CH₂-NH-*、-O(CH₂)₃S-CH₂-NH-*、-O(CH₂)₄S-CH₂-NH-*、-SCH₂S-CH₂-NH-*、-S(CH₂)₂S-CH₂-NH-*、-S(CH₂)₃S-CH₂-NH-*、-S(CH₂)₄S-CH₂-NH-*、-S(O)CH₂S-CH₂-NH-*、-S(O)(CH₂)₂S-CH₂-NH-*、-S(O)(CH₂)₃S-CH₂-NH-*、-S(O)(CH₂)₄S-CH₂-NH-*、-S(O)₂CH₂S-CH₂-NH-*、-S(O)₂(CH₂)₂S-CH₂-NH-*、-S(O)₂(CH₂)₃S-CH₂-NH-*、-S(O)₂(CH₂)₄S-CH₂-NH-*、-OCH₂-NH-*、-O(CH₂)₂-NH-*、-O(CH₂)₃-NH-*、-O(CH₂)₄S-NH-*、-SCH₂-NH-*、-S(CH₂)₂-NH-*、-S(CH₂)₃-NH-*、-S(CH₂)₄-NH-*、-S(O)CH₂-NH-*、-S(O)(CH₂)₂-NH-*、-S(O)(CH₂)₃-NH-*、-S(O)(CH₂)₄-NH-*、-S(O)₂CH₂-NH-*、-S(O)₂(CH₂)₂-NH-*、-S(O)₂(CH₂)₃-NH-or-S (O)₂(CH₂)₄-NH-*。

85. The compound of any one of claims 67-76, wherein-L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -, wherein is a site covalently linked to a.

86. The compound of claim 85, wherein-L¹-Z' -is

Wherein is a site covalently linked to a.

87. The compound of any one of claims 67-86, wherein a is a peptide comprising 2 to 8 amino acids.

88. The compound of any one of claims 67-87, wherein a is a peptide comprising 2 to 4 amino acids.

89. The compound of any one of claims 67-88, wherein at least one amino acid in the peptide is an L amino acid.

90. The compound of any one of claims 67-89, wherein each amino acid in the peptide is an L amino acid.

91. The compound of any one of claims 67-88, wherein at least one amino acid in the peptide is a D amino acid.

92. The compound of any one of claims 67-86, wherein A is- (AA)¹)-(AA²)_a1-¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

93. The compound of claim 92, -AA1- (AA2) a 1-is-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -, -Phe-N9-tosyl-Arg-, -N9-tosyl-Arg-Phe-, -Phe-N9-nitro-Arg-, -N9-nitro-Arg-Phe-, -Phe-Lys-, -Lys-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Leu-, -Ala-Leu-, -Leu-Ala-Leu-Ala-Leu-, -Leu-Ala-Leu-, -, - β -Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -Val-Arg-, -Arg-Val-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, - -Met-, - -Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, -Val-Gln-, -Ser-Val-, -Leu-, -Ser-Val-, -, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr- & Tyr-Met- & ltr-Gln- & ltr- & gt.

94. The compound of claim 92, wherein-AA¹-(AA²)_a1-Val-D-Lys-, -Val-D-Arg-, -L-Val-Cit-, -L-Val-Lys-, -L-Val-Arg-, -L-Val-D-Cit-, -L-Phe-Phe-Lys-, -L-Val-D-Arg-, -L-Arg-D-Arg-, -L-Ala-Ala-Ala-, -L-Ala-D-Ala-, -Val-D-Cit-, -, -L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val- ".

95. The compound of claim 92, wherein-AA¹-(AA²)_a1-:

-Ala-Ala-*、

-Ala-Val-*、

-Val-Ala-*

-Gln-Leu-*、

-Leu-Gln-*

-Ala-Ala-Ala-*、

-Ala-Ala-Ala-Ala-*、

-Gly-Ala-Gly-Gly-*、

-Gly-Gly-Ala-Gly-*、

-Gly-Val-Gly-Gly-*、

-Gly-Gly-Val-Gly-*、

-Gly-Phe-Gly-Gly-or

-Gly-Gly-Phe-Gly-*。

96. The compound of claim 92, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-*、

-L-Ala-L-Val-*、

-L-Ala-D-Val-*、

-L-Val-L-Ala-*、

-L-Val-D-Ala-*

-L-Gln-L-Leu-*、

-L-Gln-D-Leu-*、

-L-Leu-L-Gln-*、

-L-Leu-D-Gln-*、

-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-*、

-L-Ala-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-D-Ala-*、

-Gly-L-Ala-Gly-Gly-*、

-Gly-Gly-L-Ala-Gly-*、

-Gly-D-Ala-Gly-Gly-*、

-Gly-Gly-D-Ala-Gly-*、

-Gly-L-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-D-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-L-Phe-Gly-Gly-or

-Gly-Gly-L-Phe-Gly-*。

97. The compound of claim 92, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-or

-L-Ala-L-Ala-L-Ala-L-Ala-*。

98. The compound of any one of claims 67-97, wherein a is substituted with one or more polyols.

99. The compound of any one of claims 67-98, wherein E' is substituted with one or more polyols.

100. The compound of any one of claims 67-99, wherein polyol is- (C)₁-C₆Alkylene) -X⁵-Y³；

Wherein:

X⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；

Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and is

R¹²is-H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

101. The compound of claim 100, wherein the polyol isWherein R is¹²Is H or methyl.

102. The compound of claims 63-93, wherein E' is-C (═ O) - (C)₁-C₁₀Alkylene) -X⁶-*。

103. The compound of claim 102, wherein E' is

-C(＝O)CH₂CH₂-C(＝O)-CR^bbR^cc- (O) CH₂CH₂-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

104. The compound of any one of claims 63-93, wherein E' is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X⁶-*；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X⁶Is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

105. The compound of any one of claims 63-93, wherein E' is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X⁶-*；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

106. The compound of any one of claims 63-105, wherein the CBA comprises an-SH group covalently attached to E' to provide for -C(＝O)-CR^bbR^cc-S-CBA or-NR^ee-C(＝O)-CR^bbR^cc-S-CBA。

107. A compound according to any one of claims 67 to 106 wherein CBA is an antibody and-E '-a-Z' -L¹-D is a drug-linker moiety, the average number of drug-linker moieties conjugated per antibody being in the range of 2 to 10.

108. The compound of claim 107, wherein the average number of drug-linker moieties conjugated per antibody is in the range of 2 to 10.

109. The compound of claim 107, wherein the average number of drug-linker moieties conjugated per antibody is in the range of 6 to 8.

110. The compound of claim 107, wherein the average number of drug-linker moieties conjugated per antibody is 8.

111. The compound of any one of claims 67-110, wherein the CBA is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a hexabody, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient-transporting molecule.

112. The compound of any one of claims 67-111, wherein the CBA binds to a target cell selected from the group consisting of: tumor cells, virally infected cells, microbially infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; expression of 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B2-H2, BCMA, C4.4a, CA 2, CanAg, CD123, CD138, CD142, CD166, CD184, CD2, CD205, CD2, CD 36248, CD2, CD 36352, CD2, CD40 2, CD44v 2, CD79 2, CDH 2, CEACAM 2, cKIT, cKIN 18.2, N2, CLL-1, EGFC-MET, Cripto-1, CSP-2, GCCLDLLR-1-DLL 72, EPTC-1-GCDLP-2, EPTC-2, EPDCP-2, EPTC-GCDLP-2, EPTC-3, EPTC-GCD-3, EPTC-2, EPTC-3, EPTC-CTC-2, EPC-2, EPDCHA-2, EPTC-3, EPTC-2, EPTC-3, EPC-2, EPDCHA-3, EPDCHA-2, EPDCP-3, EPDCHA-3, EPTC-3, EPDCHA-3, EPDCP-2, EPDCP-3, EPTC-3, EPDCP-2, EPDCP, EPTC-3, EPDCHA-3, EPTC-3, EPDCP, EPTC-3, EPDCHA-3, EPTC-3, EPDCHA-3, EPTC-3, EPDCHA-3, EPDCP-3, EPDCHA-3, FGFR-3, EPDCHA-3, EPTC-3, FGFR-3, EPTC-3, EPDCHA-3, EPTC-3, FGFR-3, EPDCHA-3, EPTC-3, EPDCHA-3, EPDCE 3, EPDCHA-3, EPTC-3, EPDCHA-3, FGFR-3, LGALS3BP, LGR5, LH/hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2B, Nectin-4, NOTCH3, OAcGD2, L, p-cadherin, PD-L1, Phosphatidylserine (PS), Polymorphic Epithelial Mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLC AMF7, SLC 3644A 7, SLITK 7, STER 7, STEAP-1, STING, MUR 1, TRA 1, MUR 7, TIM-72, TIM-3, TIM-72, TAG 7, TAG 3, TAG, TA.

113. The compound of any one of claims 67-110, wherein the cell binding agent is an anti-folate receptor antibody or antibody fragment thereof, an anti-EGFR antibody or antibody fragment thereof, an anti-CD 33 antibody or antibody fragment thereof, an anti-CD 19 antibody or antibody fragment thereof, an anti-Muc 1 antibody or antibody fragment thereof, an anti-CD 37 antibody or antibody fragment thereof, or an anti-EpCAM antibody or antibody fragment thereof.

114. A pharmaceutical composition comprising a compound of any one of claims 1-113, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

115. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 114.

116. The method of claim 115, wherein the cancer is lymphoma or leukemia.

117. The method of claim 116, wherein the cancer is Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), Acute Lymphoblastic Leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), Chronic Lymphocytic Leukemia (CLL), Hairy Cell Leukemia (HCL), Acute Promyelocytic Leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), non-hodgkin's lymphoma (NHL), Mantle Cell Leukemia (MCL), Small Lymphocytic Lymphoma (SLL), hodgkin's lymphoma, systemic mastocytosis, and burkitt's lymphoma.

118. The method of claim 115, wherein the cancer is endometrial, lung, colorectal, bladder, gastric, pancreatic, renal cell, prostate, esophageal, breast, head and neck, uterine, ovarian, liver, cervical, thyroid, testicular, bone marrow, melanoma, and lymphatic cancer.

119. The method of claim 115, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

Technical Field

Disclosed herein are novel compounds, and conjugates thereof. More particularly, the present disclosure relates to novel camptothecin derivatives, intermediates, metabolites and conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as pharmaceutical agents, particularly as antiproliferative agents (anticancer agents).

Background

Cell-binding agent-drug conjugates, including antibody-drug conjugates (ADCs), emerge as a class of potent agents with efficacy in a variety of abnormal cell growth or proliferative diseases, such as cancer. Cell-binding agent-drug conjugates (e.g., ADCs) are typically composed of three distinct elements: cell binding agents (e.g., antibodies); a joint; and a cytotoxic moiety.

Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and trunk of Camptotheca acuminata (Camptotheca acuminata/Camptotheca/Happy tree) which is a tree native to china. Camptothecin inhibits topoisomerase I, which causes cell death. Because of the cytotoxic mechanisms and broad-spectrum antitumor activity of camptothecin, much effort has been directed towards developing clinical analogs of camptothecin. However, poor solubility and inactivity under physiological conditions have limited the clinical development of suitable camptothecin analogs. Camptothecin and most of its derivatives are insoluble in aqueous buffers. In addition, camptothecin in the form of an active lactone and in the form of an inactive hydrolyzed carboxylate is in equilibrium, thereby limiting its therapeutic efficacy.

There is a need for therapeutically effective camptothecin derivatives with increased solubility, potency, lactone stability and bioavailability.

Disclosure of Invention

In one aspect, the present invention provides a compound of formula I:

Z-L¹-D (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; whereinIs a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂。

In some embodiments, the compounds of formula I have the further proviso that if R is¹Is F and R²is-Me, then-L₁-Z cannot be-CH₂OH。

In some embodiments, R¹is-H or-F. In some embodiments, R¹is-F. In some embodiments, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl. In some embodiments, R²is-F. In some embodiments, R²is-OMe, -SMe, -S (O) Me or methyl. In some embodiments, R²Is methyl. In some embodiments, R¹is-F and R²is-F. In some embodiments, R¹Is methyl and R²is-F. In some embodiments, R¹is-F and R²Is a-methyl group.

In some embodiments, -L¹-Z is-H. In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -H or- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is- (C) ₁-C₆Alkylene) -H. In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is methyl, ethyl, propyl or butyl.

In some embodiments, -L¹-Z is- (C)₁-C₄Alkylene) -OR⁶、-(C₁-C₄Alkylene) -SR⁶Or- (C)₁-C₄Alkylene) -N (R)⁶)₂. In some embodiments, -L¹-Z is- (C)₁-C₄Alkylene) -OR⁶. In some embodiments, -L¹-Z is- (C)₁-C₄Alkylene) -SR⁶. In some embodiments, -L¹-Z is- (C)₁-C₄Alkylene) -N (R)⁶)₂。

In some embodiments, -L¹-Z is-CH₂OH、-(CH₂)₂OH、-(CH₂)₃OH、-(CH₂)₄OH、-CH₂OMe、-(CH₂)₂OMe、-(CH₂)₃OMe、-(CH₂)₄OMe、-CH₂SH、-(CH₂)₂SH、-(CH₂)₃SH、-(CH₂)₄SH、-CH₂SMe、-(CH₂)₂SMe、-(CH₂)₃SMe、-(CH₂)₄SMe、-CH₂NH₂、-(CH₂)₂NH₂、-(CH₂)₃NH₂Or- (CH)₂)₄NH₂。

In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SR⁶Or- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) compoundsSR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶。

In some embodiments, -L¹-Z is-CH₂NHC(＝O)CH₂OH、-CH₂NHC(＝O)(CH₂)₂OH、-CH₂NHC(＝O)(CH₂)₃OH、-CH₂NHC(＝O)(CH₂)₄OH、-CH₂NHC(＝O)(CH₂)₅OH、-CH₂NHC(＝O)CH₂OMe、-CH₂NHC(＝O)(CH₂)₂OMe、-CH₂NHC(＝O)(CH₂)₃OMe、-CH₂NHC(＝O)(CH₂)₄OMe、-CH₂NHC(＝O)(CH₂)₅OMe、-CH₂NHC(＝O)CH₂SH、-CH₂NHC(＝O)(CH₂)₂SH、-CH₂NHC(＝O)(CH₂)₃SH、-CH₂NHC(＝O)(CH₂)₄SH、-CH₂NHC(＝O)(CH₂)₅SH、-CH₂NHC(＝O)CH₂SMe、-CH₂NHC(＝O)(CH₂)₂SMe、-CH₂NHC(＝O)(CH₂)₃SMe、-CH₂NHC(＝O)(CH₂)₄SMe、-CH₂NHC(＝O)(CH₂)₅SMe、-CH₂SCH₂OH、-CH₂S(CH₂)₂OH、-CH₂S(CH₂)₃OH、-CH₂S(CH₂)₄OH、-CH₂S(CH₂)₅OH、-CH₂SCH₂OMe、-CH₂S(CH₂)₂OMe、-CH₂S(CH₂)₃OMe、-CH₂S(CH₂)₄OMe、-CH₂S(CH₂)₅OMe、-CH₂SCH₂SH、-CH₂S(CH₂)₂SH、-CH₂S(CH₂)₃SH、-CH₂S(CH₂)₄SH、-CH₂S(CH₂)₅SH、-CH₂SCH₂SMe、-CH₂S(CH₂)₂SMe、-CH₂S(CH₂)₃SMe、-CH₂S(CH₂)₄SMe or-CH₂S(CH₂)₅SMe。

In some embodiments, each R is⁶independently-H, methyl or benzyl. In some embodiments, each R is⁶Independently is-H. In some embodiments, each R is⁶Is methyl. In some embodiments, each R is⁶Is benzyl.

In some embodiments, -L¹-Z is-X^1'-(C₁-C₄Alkylene) -X². In some embodiments, -L¹-Z is-OCH₂OH、-O(CH₂)₂OH、-O(CH₂)₃OH、-O(CH₂)₄OH、-SCH₂OH、-S(CH₂)₂OH、-S(CH₂)₃OH、-S(CH₂)₄OH、-S(O)CH₂OH、-S(O)(CH₂)₂OH、-S(O)(CH₂)₃OH、-S(O)(CH₂)₄OH、-S(O)₂CH₂OH、-S(O)₂(CH₂)₂OH、-S(O)₂(CH₂)₃OH、-S(O)₂(CH₂)₄OH、-OCH₂SMe、-O(CH₂)₂SMe、-O(CH₂)₃SMe、-O(CH₂)₄SMe、-SCH₂SMe、-S(CH₂)₂SMe、-S(CH₂)₃SMe、-S(CH₂)₄SMe、-S(O)CH₂SMe、-S(O)(CH₂)₂SMe、-S(O)(CH₂)₃SMe、-S(O)(CH₂)₄SMe、-S(O)₂CH₂SMe、-S(O)₂(CH₂)₂SMe、-S(O)₂(CH₂)₃SMe or-S (O)₂(CH₂)₄SMe。

In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -X¹-L²-X². In some embodiments, -L¹-Z isIn some embodiments, -L¹-Z isIn some embodiments, -L¹-Z is

In some embodiments, the compound is any one of the compounds selected from:

in some embodiments, the compound is any one of the compounds selected from:

in some embodiments, the compound is any one selected from the compounds of table 1B.

In another aspect, the present invention provides a compound of formula II:

E-A-Z^'-L¹-D (formula II)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is covalently linked to Z^'The site of (a);

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Z^'is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to A；

Each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

A is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols; and is

E is-C (═ O) -L³-X³；

L³Is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X³The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- - (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X³is that -C(＝O)-CR^bbR^cc-W'、-NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰；

Each W' is independently-H, -N (R)^gg)₂、C₁-C₁₀Alkyl radical, C₁-C₁₀Alkenyl radical, C₁-C₁₀Alkynyl, C₃-C₆Cycloalkyl, aryl, heteroaryl or- (CH)₂CH₂O)_q-R^ff；

q is 1 to 24;

each R^aa、R^bb、R^cc、R^eeAnd R^ffIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R^ggeach independently is-H or C₁-C₆An alkyl group; and is

R⁹And R¹⁰Each independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C ₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

In some embodiments, -L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (C)₁-C₄Alkylene) -NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₄Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₄Alkylene) -NR⁸-*。

In some embodiments, -L¹-Z' -is-CH₂O-CH₂NH-*、-(CH₂)₂O-CH₂NH-*、-(CH₂)₃O-CH₂NH-*、-(CH₂)₄O-CH₂NH-*、-CH₂S-CH₂NH-*、-(CH₂)₂S-CH₂NH-*、-(CH₂)₃S-CH₂NH-*、-(CH₂)₄S-CH₂NH-*、-CH₂NH-*、-(CH₂)₂NH-*、-(CH₂)₃NH- [ or- (CH)₂)₄NH-*。

In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸- *. In some embodiments, -L ¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-*。

In some embodiments, -L¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-*。

In some embodiments, each R is⁵independently-H, methyl or benzyl. In some embodiments, each R is⁵Independently is-H. In some embodiments, each R is⁵Is methyl. In some embodiments, each R is⁵Is benzyl. In some embodiments, each R is⁸independently-H, methyl or benzyl. In some embodiments, each R is⁸Independently is-H. In some embodiments, each R is⁸Is methyl. In some embodiments, each R is⁸Is benzyl.

In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸- *. In some embodiments, -L¹-Z^'is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L ¹-Z' -is-X^1'-(C₁-C₄Alkylene) -NR⁸-*。

In some embodiments, -L¹-Z' -is-OCH₂O-CH₂-NH-*、-O(CH₂)₂O-CH₂-NH-*、-O(CH₂)₃O-CH₂-NH-*、-O(CH₂)₄O-CH₂-NH-*、-SCH₂O-CH₂-NH-*、-S(CH₂)₂O-CH₂-NH-*、-S(CH₂)₃O-CH₂-NH-*、-S(CH₂)₄O-CH₂-NH-*、-S(O)CH₂O-CH₂-NH-*、-S(O)(CH₂)₂O-CH₂-NH-*、-S(O)(CH₂)₃O-CH₂-NH-*、-S(O)(CH₂)₄O-CH₂-NH-*、-S(O)₂CH₂O-CH₂-NH-*、-S(O)₂(CH₂)₂O-CH₂-NH-*、-S(O)₂(CH₂)₃O-CH₂-NH-*、-S(O)₂(CH₂)₄O-CH₂-NH-*、-OCH₂S-CH₂-NH-*、-O(CH₂)₂S-CH₂-NH-*、-O(CH₂)₃S-CH₂-NH-*、-O(CH₂)₄S-CH₂-NH-*、-SCH₂S-CH₂-NH-*、-S(CH₂)₂S-CH₂-NH-*、-S(CH₂)₃S-CH₂-NH-*、-S(CH₂)₄S-CH₂-NH-*、-S(O)CH₂S-CH₂-NH-*、-S(O)(CH₂)₂S-CH₂-NH-*、-S(O)(CH₂)₃S-CH₂-NH-*、-S(O)(CH₂)₄S-CH₂-NH-*、-S(O)₂CH₂S-CH₂-NH-*、-S(O)₂(CH₂)₂S-CH₂-NH-*、-S(O)₂(CH₂)₃S-CH₂-NH-*、-S(O)₂(CH₂)₄S-CH₂-NH-*、-OCH₂-NH-*、-O(CH₂)₂-NH-*、-O(CH₂)₃-NH-*、-O(CH₂)₄S-NH-*、-SCH₂-NH-*、-S(CH₂)₂-NH-*、-S(CH₂)₃-NH-*、-S(CH₂)₄-NH-*、-S(O)CH₂-NH-*、-S(O)(CH₂)₂-NH-*、-S(O)(CH₂)₃-NH-*、-S(O)(CH₂)₄-NH-*、-S(O)₂CH₂-NH-*、-S(O)₂(CH₂)₂-NH-*、-S(O)₂(CH₂)₃-NH-or-S (O)₂(CH₂)₄-NH-*。

In some embodiments, -L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -. In some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -is

Disclosed herein as-L¹In various embodiments of-Z' -, is a site covalently linked to a.

In some embodiments, a is a peptide comprising 2 to 8 amino acids. In some embodiments, a is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid.

In some casesIn embodiments, A is- (AA)¹)-(AA²)_a1-, wherein is a site covalently linked to E; AA¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

In some embodiments, -AA¹-(AA²)_a1-is-Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-N ⁹-tosyl-Arg-, -N⁹-tosyl-Arg-Phe-, -Phe-N⁹-nitro-Arg-, -N⁹-nitro-Arg-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu-, -Leu Ala-Leu-Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -, -Val-Arg-, -Arg-Val-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, - -Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -y-Gln-, -al Ser-Gln-, -Ser-, -y-Gln-, -al, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr- & lty-Met- & ltx.

In some embodiments, -AA¹-(AA²)_a1-Val-D-Lys-, -Val-D-Arg-, -L-Val-Cit-, -L-Val-Lys-, -L-Val-Arg-, -L-Val-D-Cit-, -L-Phe-Phe-Lys-, -L-Val-D-Arg-, -L-Arg-D-Arg-, -L-Ala-Ala-Ala-, -L-Ala-D-Ala-, -Val-D-Cit-, -, -L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val- ".

In some embodiments, -AA¹-(AA²)_a1-: -Ala-, -Ala-Val-, -Val-Ala-, -Gln-Leu-, -Leu-Gln-, -Ala-, -Gly-Ala-Gly-, -Gly-Ala-Gly-, -al-Leu-, -andGly-Val-Gly-Gly-, -Gly-Gly-Val-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Phe-Gly-,.

In some embodiments, -AA¹-(AA²)_a1-: -L-Ala-, -L-Ala-D-Ala-, -L-Ala-L-Val-, -L-Ala-D-Val-, -L-Val-L-Ala-, -L-Val-D-Ala-, -L-Gln-L-Leu-, -L-Gln-D-Leu-, -L-Leu-L-Gln-, -L-Leu-D-Gln-, -L-Ala-, -L-Ala-D-Ala-L-Ala-, -L-D-Ala-L-Ala-, -al-L-D-Ala-L-Ala-, -, -L-Ala-L-Ala-D-Ala-, -L-Ala-L-Ala-L-Ala-L-Ala-, -L-Ala-D-Ala-L-Ala-L-Ala-, -L-Ala-L-Ala-D-Ala-L-Ala-, -L-Ala-L-Ala-L-Ala-D-Ala-, -Gly-L-Ala-Gly-Gly-, -Gly-Gly-L-Ala-Gly-, -Gly-D-Ala-Gly-, -Ala-Gly-, -Ala-L-Ala-Gly-, -Ala-Gly-, -Ala-Gly-, -, -Gly-L-Val-Gly-, Gly-L-Val-Gly-, -Gly-D-Val-Gly-, -Gly-D-Val-Gly-, -Gly-L-Phe-Gly-, or-Gly-L-Phe-Gly-,.

In some embodiments, -AA¹-(AA²)_a1-: -L-Ala-, -L-Ala-D-Ala-LAla-, -L-Ala-, -L-Ala-, or-L-Ala- >.

Disclosed herein is-AA ¹-(AA²)_a1In various embodiments, is a site covalently attached to E.

In some embodiments, a is substituted with one or more polyols. In some embodiments, E is substituted with one or more polyols. In some embodiments, the polyol is — (C)₁-C₆Alkylene) -X⁵-Y³(ii) a Wherein: x⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and R is¹²is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

In some embodiments, wherein the polyol isWherein R is¹²Is H or methyl.

In some embodiments, E is-C (═ O) - (C)₁-C₁₀Alkylene) -X³. In some embodiments, E is

In some embodiments, E is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X³；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X³Is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

In some embodiments, E is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X³；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X³is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

In some embodiments, the compound is any one selected from the compounds of table 2.

In another aspect, the present invention provides a compound of formula III, or a pharmaceutically acceptable salt thereof:

CBA-E'-A-Z'-L¹-D (formula III)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols;

E' is-C (═ O) -L³-X⁶-; wherein is a site covalently attached to the CBA;

L³is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X⁶The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA;

each R^aa、R^bb、R^ccAnd R^eeIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R⁹independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

CBA is a cell binding agent.

In some embodiments, R¹is-H or-F. In some embodiments, R¹is-F. In some embodiments, R ²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl. In some embodiments, R²is-F. In some embodiments, R²is-OMe, -SMe, -S (O) Me or methyl. In some embodiments, R²Is methyl. In some embodiments, R¹is-F and R²is-F. In some embodiments, R¹Is methyl and R²is-F. In some embodiments, R¹is-F and R²Is a-methyl group.

In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L ¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-*。

In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene radical)-O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -NR⁸-*。

In some embodiments, -L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -. In some embodiments, -L ¹-Z' -isIn some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -is

Disclosed herein as-L¹In various embodiments of-Z' -, is a site covalently linked to a.

In some embodiments, a is- (AA)¹)-(AA²)_a1-¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

In some embodiments, -AA¹-(AA²)_a1-is-Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-N⁹-tosyl-Arg-, -N⁹-tosyl-Arg-Phe-, -Phe-N⁹-nitro-Arg-, -N ⁹-nitro-Arg-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu-, -Leu Ala-Leu-Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -, -Val-Arg-, -Arg-Val-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, - -Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -y-Gln-, -al Ser-Gln-, -Ser-, -y-Gln-, -al, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr- & lty-Met- & ltx.

In some embodiments, -AA¹-(AA²)_a1-: -Ala-, -Ala-Val-, -Val-Ala-, -Gln-Leu-, -Leu-Gln-, -Ala-, -Gly-Ala-Gly-, -Gly-Ala-Gly-, -Gly-Val-Gly-, -Gly-Val-Gly-, -Gly-Phe-Gly-, or-Gly-Phe-Gly-,.

In some embodiments, -AA¹-(AA²)_a1-: -L-Ala-, -L-Ala-D-Ala-L-Ala-, -L-Ala-, or-L-Ala- >.

Disclosed herein is-AA ¹-(AA²)_a1In various embodiments, is a site covalently linked to E'.

In some embodiments, a is substituted with one or more polyols. In some embodiments, E' is substituted with one or more polyols. In some embodiments, the polyol is — (C)₁-C₆Alkylene) -X⁵-Y³(ii) a Wherein: x⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and R is¹²is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

In some embodiments, the polyol isWherein R is¹²Is H or methyl.

In some embodiments, E' is-C (═ O) - (C)₁-C₁₀Alkylene) -X⁶- *. In some embodiments, E' is

-C(＝O)CH₂CH₂-C(＝O)-CR^bbR^cc- (O) CH₂CH₂-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

In some embodiments, E' is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X⁶-*；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X⁶Is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

In some embodiments, E' is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X⁶-*；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

In some embodiments, the CBA comprises an-SH group covalently attached to E' to provide -C(＝O)-CR^bbR^cc-S-CBA or-NR^ee-C(＝O)-CR^bbR^cc-S-CBA。

In some embodiments, the CBA is an antibody and-E '-A-Z' -L¹-D is a drug-linker moiety, the average number of drug-linker moieties conjugated per antibody being in the range of 2 to 10.

In some embodiments, the average number of drug-linker moieties conjugated per antibody is in the range of 2 to 10. In some embodiments, the average number of drug-linker moieties conjugated per antibody is in the range of 6 to 8. In some embodiments, the average number of drug-linker moieties conjugated per antibody is 8.

In some embodiments, the CBA is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a hexabody, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient-transporting molecule.

In some embodiments, the CBA binds to a target cell selected from the group consisting of: tumor cells, virally infected cells, microbially infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; expression of 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B2-H2, BCMA, C4.4a, CA 2, CanAg, CD123, CD138, CD142, CD166, CD184, CD2, CD205, CD2, CD 36248, CD2, CD352, CD2, CD40 2, CD44v 2, CD79 2, CDH 2, CEACAM 2, cKIT, cKIN 18.2, GCN 72, CLDL-1, EGFC-MET, Criptto, CSP-2, GCCLDLLR-1-DLL 72, EPDCP-2, EPDCP-GCDLP-2, EPDCP-GCD-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-2, EPDCP-3, EPDCP-3, EPDCP-2, EPDCP-3, lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC class I chain-associated proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2B, Nectin-4, NOTCH3, OAcGD2, 001 OX L, p-cadherin, PD-L1, Phosphatidylserine (PS), Polymorphic Epithelial Mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, TRAMF 7, TRT44A 7, STK 7, STEAP-1, STAP-72, SLC-1, SLC-72, SLC 7, SLC-associated glycoprotein, SLC 7, SLC-3-72, SLC 7, SLC-3-72, SLC 7, or any epitope specific for a tumor associated with a tumor.

In some embodiments, the cell binding agent is an anti-folate receptor antibody or antibody fragment thereof, an anti-EGFR antibody or antibody fragment thereof, an anti-CD 33 antibody or antibody fragment thereof, an anti-EpCAM antibody or antibody fragment thereof, an anti-CD 19 antibody or antibody fragment thereof, an anti-Muc 1 antibody or antibody fragment thereof, or an anti-CD 37 antibody or antibody fragment thereof.

The invention also includes a composition (e.g., a pharmaceutical composition) comprising a cytotoxic compound or conjugate of the invention described herein and a carrier (pharmaceutically acceptable carrier). The compounds, conjugates or compositions of the invention are useful for inhibiting abnormal cell growth or treating a proliferative disorder (e.g., cancer), an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis or a kidney disease in a mammal (e.g., a human).

The compounds, conjugates or compositions of the invention are useful for treating cancer in a subject in need thereof. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), Acute Lymphoblastic Leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), Chronic Lymphocytic Leukemia (CLL), Hairy Cell Leukemia (HCL), Acute Promyelocytic Leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), non-Hodgkin lymphoma (non-Hodgkin lymphoma, NHL), Mantle Cell Leukemia (MCL), Small Lymphocytic Lymphoma (SLL), Hodgkin lymphoma (Hodgkin's lymphoma), Systemic mastocytosis and Burkitt's lymphoma. In some embodiments, the cancer is endometrial, lung, colorectal, bladder, gastric, pancreatic, renal cell, prostate, esophageal, breast, head and neck, uterine, ovarian, liver, cervical, thyroid, testicular, bone marrow, melanoma, and lymphatic cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

The invention also includes the use of a cytotoxic compound, conjugate or composition of the invention for the manufacture of a medicament for inhibiting abnormal cell growth or treating a proliferative disorder (e.g., cancer), an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis or a kidney disease in a mammal (e.g., a human).

The compounds, conjugates or compositions of the invention are useful in the manufacture of a medicament for treating cancer in a subject in need thereof. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), Acute Lymphoblastic Leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), Chronic Lymphocytic Leukemia (CLL), Hairy Cell Leukemia (HCL), Acute Promyelocytic Leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), non-hodgkin's lymphoma (NHL), Mantle Cell Leukemia (MCL), Small Lymphocytic Lymphoma (SLL), hodgkin's lymphoma, systemic mastocytosis, and burkitt's lymphoma. In some embodiments, the cancer is endometrial, lung, colorectal, bladder, gastric, pancreatic, renal cell, prostate, esophageal, breast, head and neck, uterine, ovarian, liver, cervical, thyroid, testicular, bone marrow, melanoma, and lymphatic cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

Drawings

Figure 1 depicts the first part of the synthesis of camptothecin building blocks.

Figure 2 depicts the second part of the synthesis of camptothecin building blocks.

Figure 3 depicts the first part of the synthesis of the side chain.

Figure 4 depicts the second part of the synthesis of the side chain.

Figure 5 depicts the coupling of camptothecin building blocks to the first part of the side chain.

Figure 6 depicts the coupling of camptothecin building blocks to the second part of the side chain.

Figure 7 depicts the coupling of camptothecin building blocks to the third part of the side chain.

Figure 8 depicts the synthesis of additional camptothecin metabolites.

Figure 9 depicts the coupling of camptothecin building blocks to side chains.

Figure 10 depicts the synthesis of additional camptothecin compounds.

Figure 11 depicts compounds for comparison including a generic Ab-999 structure of an ADC with an ADC moiety linked via a reducing interchain disulfide of an antibody.

Figure 12 depicts the cytotoxicity of sulfide bearing compound 8c and its sulfoxides 34a and sulfones 34 b.

FIG. 13 depicts the pharmacokinetics of ML66-999 in mice. The top panel depicts a plot of concentration of mAb component (mean) and payload component (μ g/mL) versus time at time points 2min, 1 day, and 3 days post-administration in mice. The bottom panel depicts the mAb component (mean) and concentration (μ g/mL) at which biological activity is maintained (pooled samples) versus time at time points 2min, 1 day, and 3 days post-administration in mice.

FIG. 14 depicts the pharmacokinetics of ML66-22a in mice. The top panel depicts a plot of concentration of mAb component (mean) and payload component (μ g/mL) versus time at time points 2min, 1 day, and 3 days post-administration in mice. The bottom panel depicts the mAb component (mean) and concentration (μ g/mL) at which biological activity is maintained (pooled samples) versus time at time points 2min, 1 day, and 3 days post-administration in mice.

FIG. 15 depicts the in vitro cytotoxicity of ADCs against Ag + and Ag-cells. Formulations (standards) containing ADC taken 2min, 1 day, or 3 days after administration of ML66-999 in mice or ADC standards in serum (pooled).

FIG. 16 depicts in vitro cytotoxicity of ADCs against Ag + and Ag-cells. Formulations (standards) containing ADC taken 2min, 1 day, or 3 days after administration of ML66-22a in mice or ADC standards in serum (pooled).

FIG. 17 depicts the efficacy of ADC in HSC-2 xenograft models. Dosing was based on payload (75. mu.g/kg and 250. mu.g/kg based on antibody about 3mg/kg and about 10 mg/kg).

Fig. 18 depicts the efficacy of ADC in the FaDu xenograft model. Dosing was based on payload (75. mu.g/kg and 250. mu.g/kg based on antibody about 3mg/kg and about 10 mg/kg).

FIG. 19 depicts tolerance of mice to ML66-999, ML66-22a, and ML66-28a ADCs.

Figure 20 depicts the anti-tumor activity of ADCs in the H1703 mouse xenograft model. Dosing was based on payload (75. mu.g/kg and 250. mu.g/kg based on antibody about 3mg/kg and about 10 mg/kg).

Figure 21 depicts mouse tolerance of non-cross-reactive ADCs at a 5000 μ g/kg payload dose (about 200mg/kg based on Ab component). Ab_FHumanized anti-folate receptor antibodies.

Detailed Description

In order that the invention described herein may be fully understood, the following detailed description is set forth. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize a variety of methods and materials similar or equivalent to those described herein, which may be used in the practice of the present invention.

The term "herein" refers to the entire application.

Unless defined otherwise herein, scientific and technical terms used herein shall have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used in connection with the compounds, compositions, and methods described herein are those well known and commonly employed in the art.

It is to be understood that any embodiment described herein, including those described under different aspects of the invention and different portions of this specification (including embodiments described only in the examples), may be combined with one or more other embodiments of the invention, unless expressly disclaimed or otherwise inappropriate. Combinations of embodiments are not limited to those specific combinations claimed via the various dependent claims.

Chemical nomenclature used herein is used according to conventional usage in The art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", eds, Parker S.A., McGraw-Hill, San Francisco, C.A. (1985).

All of the above and any other publications, patents and published patent applications mentioned in this application are specifically incorporated herein by reference. Any information in any material that has been incorporated by reference herein is incorporated by reference only to the extent that no conflict exists between that information and other statements and drawings set forth herein. Any such conflicting information in such incorporated by reference material is not specifically incorporated by reference herein, and will control this specification, including its specific definitions, if such conflict exists.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer (or component) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

Throughout this specification, where a composition is described as having, including, or comprising (or variations thereof) a particular component, it is contemplated that the composition can also consist essentially of, or consist of, the recited component. Similarly, where a method or process is described as having, including, or comprising specific process steps, the process may also consist essentially of, or consist of, the recited process steps. Further, it should be understood that the order of steps or order for performing certain actions is not important so long as the compositions and methods described herein remain operable. Further, two or more steps or actions may be performed simultaneously.

The term "including" is used to mean "including but not limited to". "include" and "include but are not limited to" are used interchangeably.

As used herein, "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing elements (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "or" is understood to mean "and/or" unless clearly indicated otherwise herein.

Unless otherwise indicated herein, ranges of values set forth herein are intended merely to serve as shorthand methods of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

Definition of

As used herein, the term "alkyl" or "straight or branched chain alkyl" refers to a saturated straight or branched chain monovalent hydrocarbon group. In a preferred embodiment, straight orBranched alkyl groups having thirty or fewer carbon atoms (e.g., C for straight chain alkyl groups)₁-C₃₀And C for branched alkyl₃-C₃₀) And more preferably twenty or less carbon atoms. Even more preferably, the straight or branched alkyl group has ten or fewer carbon atoms (i.e., C for straight chain alkyl groups)₁-C₁₀And C for branched alkyl₃-C₁₀). In other embodiments, the straight or branched alkyl group has six or fewer carbon atoms (i.e., C for straight chain alkyl groups)₁-C_6-And C for branched alkyl₃-C₆). Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH₂CH(CH₃)₂2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, 1-heptyl, 1-octyl and the like. Furthermore, the term "alkyl" as used throughout the specification, examples and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", wherein the latter refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. As used herein, (C) _x-C_xx) Alkyl or C_x-xxAlkyl refers to straight or branched chain alkyl groups having a number of carbon atoms from x to xx.

As used herein, the term "alkylene" refers to a saturated straight or branched chain divalent hydrocarbon radical. In a preferred embodiment, the linear or branched alkylene group has thirty or less carbon atoms (e.g., C for linear alkylene groups)₁-C₃₀And C for branched alkylene₃-C₃₀) And more preferably twenty or less carbon atoms. Even more preferably, the linear or branched alkylene group has ten or less carbon atoms (i.e., C for linear alkylene groups)₁-C₁₀And as to the branched alkylene group areC₃-C₁₀). In other embodiments, the linear or branched alkylene has six or fewer carbon atoms (i.e., C for linear alkylene)₁-C₆And C for branched alkylene₃-C₆). As used herein, (C)_x-C_xx) Alkylene or C_x-xxAlkylene means a straight or branched chain alkylene group having a number of carbon atoms from x to xx.

The term "alkenyl" or "straight or branched chain alkenyl" refers to a straight or branched chain monovalent hydrocarbon group having two to twenty carbon atoms and having at least one site of unsaturation (i.e., a carbon-carbon double bond), wherein alkenyl includes groups having "cis" and "trans" orientations, or alternatively "E" and "Z" orientations. Examples include, but are not limited to, ethenyl (-CH ═ CH) ₂) Allyl (-CH)₂CH＝CH₂) And the like. Preferably, the alkenyl group has two to ten carbon atoms. More preferably, the alkyl group has two to four carbon atoms.

The term "alkynyl" or "straight or branched chain alkynyl" refers to a straight or branched chain monovalent hydrocarbon group having two to twenty carbon atoms and having at least one site of unsaturation (i.e., a carbon-carbon triple bond). Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably, the alkynyl group has two to ten carbon atoms. More preferably, the alkynyl group has two to four carbon atoms.

The terms "cyclic alkyl" and "cycloalkyl" are used interchangeably. As used herein, the term refers to a saturated carbocyclic group. In a preferred embodiment, the cycloalkyl group has 3 to 10 carbon atoms in its ring structure, and more preferably 5 to 7 carbon atoms in the ring structure. In some embodiments, the two rings may have two or more atoms in common, e.g., the rings are "fused rings. Suitable cycloalkyl groups include, but are not limited to, cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. In some embodiments, the cycloalkyl group is a monocyclic group. In some embodiments, the cycloalkyl group is a bicyclic group. In some embodiments, the cycloalkyl group is a tricyclic group.

The term "cycloalkylalkyl" refers to an alkyl group as described above substituted with a cycloalkyl group.

The term "cyclic alkenyl" refers to a carbocyclic group having at least one double bond in the ring structure.

The term "cyclic alkynyl" refers to a carbocyclic group having at least one triple bond in the ring structure.

As used herein, the term "aryl" or "aromatic ring" includes a substituted or unsubstituted monocyclic aromatic group, wherein each atom of the ring is carbon. Preferably, the ring is a 5 to 7 membered ring, more preferably a 6 membered ring. Aryl groups include, but are not limited to, phenyl, phenol, aniline, and the like. The term "aryl" also includes "polycyclyl," "polycyclyl," and "polycyclic" ring systems having two or more rings in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings," in which at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, or aromatic rings. In some preferred embodiments, the polycyclic ring has 2 to 3 rings. In certain preferred embodiments, the polycyclic ring system has two rings, wherein both rings are aromatic. Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains from 3 to 10 carbon atoms in the ring, preferably from 5 to 7. For example, aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7, 8-tetrahydronaphthyl, and the like. In some embodiments, aryl is a monocyclic aromatic group. In some embodiments, aryl is a bicyclic aromatic group. In some embodiments, aryl is a tricyclic aromatic group.

The term "heteroalkyl" refers to an alkyl group wherein one or more of the backbone atoms of the alkyl group is selected from an atom other than carbon, such as O, S, N (e.g., -NH, -N (alkyl) -), or combinations thereof. The heteroalkyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkyl group. In one aspect, heteroalkyl is C₁-C₃₀A heteroalkyl group. C₁-C₃₀Heteroalkyl means an alkane having from 1 to 30 carbon atoms and from 1 to 15 heteroatomsAnd (4) a base. C₁-C₃₀Examples of heteroalkyl groups include, but are not limited to, ethers (e.g., -CH)₂-O-CH₃、-(CH₂)₂-O-CH₃、-(CH₂)₃-O-(CH₂)₂-O-CH3、-(CH₂)₂-O-(CH₂)₃CH₃、CH₂-O-CH₂-O-CH₃、-CH₂-O-(CH₂)₃-O-CH₃) Polyethylene glycol (PEG) derivatives (e.g., [ (CH)₂)₂O]₁₀CH₂CH₃) Thioethers (e.g. -CH)₂-S-CH₃、-(CH₂)₂-S-CH₃、-(CH₂)₃-S-(CH₂)₂CH₃、-((CH₂)₂S)₁₀CH₂CH₃)、-CH₂-S-S-CH₂、-(CH₂)₂-S-(CH₂)₃CH₃、CH₂-S-CH₂-S-CH₃、-CH₂-S-(CH₂)₃-S-CH₃) Amines (e.g. -CH)₂-NH-CH₃、-(CH₂)₂-N(alkyl)-CH₃、-(CH₂)₃-NH-(CH₂)₂CH₃、-(CH₂)₂-N(alkyl)-(CH₂)₃CH₃、CH₂-NH-CH₂-NH-CH₃、-CH₂-NH-(CH₂)₃-NH-CH₃) Or a combination thereof. The disclosure also encompasses C₁-C₃₀Heteroalkyl groups in which one of 1 to 15 heteroatoms occupies a terminal position of the alkyl group, for example to produce an alcohol (i.e., OH), thiol (i.e., SH) or amine (e.g., -NH) in the terminal position of the moiety₂)。

The term "heteroalkenyl" refers to an alkenyl group, as defined herein, in which one or more carbon atoms have been replaced by a heteroatom, such as O, S, N (e.g., -NH, -N (alkyl) -). A heteroalkenyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkenyl group. In one aspect, heteroalkenyl is C₁-C₃₀A heteroalkenyl group. C₁-C₃₀Heteroalkenyl means having from 1 to 30 carbonsAn alkenyl group of atoms and 1 to 15 heteroatoms (e.g., 1 to 10 heteroatoms or 1 to 5 heteroatoms). The disclosure also encompasses C ₁-C₃₀Heteroalkenyl where one of 1 to 15 heteroatoms occupies a terminal position of the alkenyl group, e.g., produces an alcohol (i.e., OH), a thiol (i.e., SH), an amine (e.g., -NH) in the terminal position of the moiety₂) Or an imine (-C ═ N).

The term "heteroalkynyl" refers to an alkenyl group, as defined herein, in which one or more carbon atoms have been replaced by a heteroatom, such as O, S, N (e.g., -NH, -N (alkyl) -). Heteroalkynyl is attached to the remainder of the molecule at a carbon atom of the heteroalkynyl. In one aspect, heteroalkynyl is C₁-C₃₀A heteroalkynyl group. C₁-C₃₀Heteroalkynyl refers to an alkynyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms (e.g., 1 to 10 heteroatoms or 1 to 5 heteroatoms). The disclosure also encompasses C₁-C₃₀Heteroalkynyl wherein one of 1 to 15 heteroatoms occupies a terminal position of the alkynyl, e.g., produces an alcohol (i.e., OH), thiol (i.e., SH), amine (e.g., -NH) in the terminal position of the moiety₂) Or a nitrile (-C.ident.N).

As used herein, the terms "heterocycle", "heterocyclyl" and "heterocyclic ring" refer to a substituted or unsubstituted non-aromatic ring structure of a 3-to 18-membered ring, preferably a 3-to 10-membered ring, more preferably a 3-to 7-membered ring, which ring structure includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. In certain embodiments, the ring structure may have two rings. In some embodiments, the two rings may have two or more atoms in common, e.g., the rings are "fused rings. Heterocyclic groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. Heterocycles are described in Paquette, Leo a.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York,1968), in particular chapters 1,3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of monograms" (John Wiley & Sons, New York, 1950) especially volumes 13, 14, 16, 19 and 28; and J.am.chem.Soc. (1960)82: 5566. Examples of heterocycles include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, tetrahydropyran, dihydropyran, tetrahydrothiopyran, thiomorpholine, thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine, piperidine, piperazine, pyrrolidine, morpholine, oxepane, thietane, oxazepine, diazepine, thiazepine, 2-pyrroline, 3-pyrroline, indoline, 2H-pyran, 4H-pyran, dioxane, 1, 3-dioxolane, pyrazoline, dithiane, dithiolane, dihydropyran, dihydrothiophene, dihydrofuran, pyrazolylimidazoline, imidazolidine, 3-azabicyclo [3.1.0] hexane, 3-azabicyclo [4.1.0] heptane and azabicyclo [2.2.2] hexane. The spiro moiety is also included within the scope of this definition. Examples of heterocyclic groups in which the ring atom is partially substituted by oxo (═ O) are pyrimidinones and 1, 1-dioxo-thiomorpholine.

As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a substituted or unsubstituted aromatic monocyclic structure, preferably a 6-to 18-membered ring, preferably a 5-to 7-membered ring, more preferably a 5-to 6-membered ring, the ring structure of which comprises at least one heteroatom (e.g. O, N or S), preferably one to four or one to three heteroatoms, more preferably one or two heteroatoms. When two or more heteroatoms are present in the heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "polycyclyl", and "polycyclic" ring systems having two or more rings in which two or more ring atoms are common to two adjoining rings, e.g., the rings are "fused rings", in which at least one of the rings is heteroaromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaromatics, and/or heterocyclyls. In some preferred embodiments, the polycyclic heteroaryl has 2 to 3 rings. In certain embodiments, preferred polycyclic heteroaryls have two rings, wherein both rings are aromatic. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms in the ring, preferably 5 to 7 atoms in the ring. For example, heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole, benzofuran, benzothiophene, cinnoline, phthalazine, quinazoline, carbazole, phenoxazine, quinoline, purine, and the like. In some embodiments, heteroaryl is a monocyclic aromatic group. In some embodiments, the heteroaryl group is a bicyclic aromatic group. In some embodiments, the heteroaryl group is a tricyclic aromatic group.

Where possible, the heterocycle or heteroaryl may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked. For example, and without limitation, a carbon-bonded heterocycle or heteroaryl is bonded at the 2, 3, 4, 5, or 6 position of a pyridine, the 3, 4, 5, or 6 position of a pyridazine, the 2, 4, 5, or 6 position of a pyrimidine, the 2, 3, 5, or 6 position of a pyrazine, the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2, 4, or 5 position of an oxazole, imidazole, or thiazole, the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole, the 2 or 3 position of an aziridine, the 2, 3, or 4 position of an azetidine, the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline, or the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline.

By way of example and not limitation, the nitrogen-bonded heterocycle or heteroaryl is bonded to aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine and the 9-position of carbazole or O-carboline.

Heteroatoms present in heteroaryl or heterocyclyl groups include oxidized forms, e.g. NO, SO and SO ₂。

In some embodiments, the heteroaryl ring is a 5 to 18 membered ring.

The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). In some embodiments, the halogen is fluorine. In some embodiments, the halogen is chlorine. In some embodiments, the halogen is bromine. In some embodiments, the halogen is iodine. The term "haloalkyl" as used herein refers to an alkyl group, as defined herein, substituted with one or more halo groups, as defined herein. The haloalkyl group may be a monohaloalkyl group, a dihaloalkyl group, or a polyhaloalkyl group. The monohaloalkyl group may have one fluorine, chlorine, bromine or iodine substituent. The dihaloalkyl or polyhaloalkyl groups may be substituted with two or more of the same halogen atoms or a combination of different halogen groups. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl (difluoroopropyl), dichloroethyl, and dichloropropyl.

The term "alkoxy" as used herein refers to alkyl-O-, wherein alkyl is defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, t-butoxy, pentoxy, hexoxy, and the like.

The alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl groups described above may be optionally substituted with one or more (e.g. 2, 3, 4, 5, 6 or more) substituents.

Unless explicitly stated as "unsubstituted," references herein to chemical moieties are to be understood as also including substituted variants. For example, reference to "alkyl" or a moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on a chemical moiety include, but are not limited to, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aryl or heteroaryl moiety.

"optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the application includes instances where said circumstance occurs and instances where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the application includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

The term "substituted" refers to a moiety having a substituent that replaces a hydrogen on one or more carbon, nitrogen, oxygen, or sulfur atoms. It is understood that "substitution" or "substituted" includes the implicit limitation that the substitution is consistent with the allowed valences of the substituted atom or group and that the substitution results in a stable compound, e.g., the compound does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Permissible substituents can be one or more and the same or different for appropriate organic compounds. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein which satisfy the valency of the heteroatom. Substituents may include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. To illustrate, a monofluoroalkyl group is an alkyl group substituted with one fluorine substituent, and a difluoroalkyl group is an alkyl group substituted with two fluorine substituents. It will be appreciated that if more than one substitution is present on a substituent, each non-hydrogen substituent may be the same or different (unless otherwise specified).

If a carbon of a substituent is described as optionally substituted with one or more of a list of substituents, then one or more hydrogens on that carbon (to the extent that some are present) may be replaced independently and/or together with an independently selected optional substituent. If a substituent nitrogen is described as optionally substituted with one or more of a list of substituents, then one or more hydrogens on the nitrogen (to the extent that some is present) may each be replaced with an independently selected optional substituent. One exemplary substituent may be depicted as-NR 'R ", where R' and R", together with the nitrogen atom to which they are attached, may form a heterocyclic ring. The heterocyclic ring formed by R' and R "together with the nitrogen atom to which they are attached may be partially or fully saturated. In some embodiments, the heterocycle consists of 3 to 7 atoms. In other embodiments, the heterocycle is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl, and thiazolyl.

The terms "substituent", "radical" and "group" are used interchangeably herein.

If groups of substituents are collectively described as optionally substituted with one or more of the list of substituents, the groups may include: (1) unsubstituted substituents, (2) substitutable substituents which are unsubstituted by optional substituents, and/or (3) substitutable substituents which are substituted by one or more of the optional substituents.

If a substituent is described as being optionally substituted with up to the specified number of non-hydrogen substituents, then that substituent may be (1) unsubstituted; or (2) by the specified number of non-hydrogen substituents or by up to the maximum number (whichever is smaller) of substitutable positions on the substituent. Thus, for example, if a substituent is described as a heteroaryl group optionally substituted with up to 3 non-hydrogen substituents, any heteroaryl group having fewer than 3 substitutable positions will be optionally substituted with up to as many non-hydrogen substituents as the heteroaryl group has substitutable positions. In non-limiting examples, such substituents may be selected from linear, branched or cyclic alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [ -NH (C ═ NH) NH₂]、-OR¹⁰⁰、NR¹⁰¹R¹⁰²、-NO₂、-NR¹⁰¹COR¹⁰²、-SR¹⁰⁰of-SOR¹⁰¹Sulfoxide represented by the formula-SO₂R¹⁰¹Sulfone, sulfonate group-SO of₃M, sulfate-OSO₃M, is of-SO₂NR¹⁰¹R¹⁰²Sulfonamide, cyano, azido, -COR¹⁰¹、-OCOR¹⁰¹、-OCONR¹⁰¹R¹⁰²And polyethylene glycol unit (-OCH)₂CH₂)_nR¹⁰¹Wherein M is H or a cation (e.g. Na)⁺Or K⁺)；R¹⁰¹、R¹⁰²And R¹⁰³Each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, polyethylene glycol units (-OCH) ₂CH₂)_n-R¹⁰⁴(wherein n is an integer of 1 to 24), an aryl group having 6 to 10 carbon atoms, a heterocyclic ring having 3 to 10 carbon atoms and a heteroaryl group having 5 to 10 carbon atoms; and R is¹⁰⁴Is H or a linear or branched alkyl group having 1 to 4 carbon atoms, wherein the group is represented by R¹⁰⁰、R¹⁰¹、R¹⁰²、R¹⁰³And R¹⁰⁴The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl groups represented are optionally independently selected from halogen, -OH, -CN, -NO₂And one or more (e.g., 2, 3, 4, 5, 6, or more) substituents of an unsubstituted, straight or branched chain alkyl group having 1 to 4 carbon atoms. Preferably, substituents for the optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl groups described above include halogen, -CN, -NR¹⁰²R¹⁰³、-CF₃、-OR¹⁰¹Aryl, heteroaryl, heterocyclyl, -SR¹⁰¹、-SOR¹⁰¹、-SO₂R¹⁰¹and-SO₃M。

With respect to-SOR as indicated in the preceding paragraph¹⁰¹The sulfoxides, as represented, encompass both optical isomers (R and S configurations at the sulfur atom of the sulfoxide group).

The number of carbon atoms in a group may be referred to herein by the prefix "C_x-xx"or" C_x-C_xx"specify, where x and xx are integers. For example, "C_1-4Alkyl "or" C1-C4 alkyl "is an alkyl group having 1 to 4 carbon atoms.

The terms "compound" or "cytotoxic compound", "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include compounds whose structures or formulae or any derivatives have been disclosed in the present invention or whose structures or formulae or any derivatives have been incorporated by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts), and prodrugs, as well as prodrug salts, of the compounds of all formulae disclosed herein. The term also includes any solvates, hydrates and polymorphs of any of the foregoing. Specific recitation of "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salts", "prodrugs", "prodrug salts", "conjugates", "conjugate salts", "solvates", "hydrates" or "polymorphs" of certain aspects of the invention described in this application should not be interpreted as an intent to omit such forms of other aspects of the invention where the term "compound" is used without recitation of such other forms.

The term "conjugate" as used herein refers to a compound described herein or a derivative thereof linked to a cell binding agent.

The term "chiral" refers to a molecule having the non-superimposable properties of mirror partners, while the term "achiral" refers to a molecule that is superimposable on its mirror partner.

The term "stereoisomer" refers to a compound that has the same chemical composition and connectivity, but whose atoms are not interconvertible in different orientations in space by rotation about a single bond.

The term "diastereomer" refers to a stereoisomer that has two or more chiral centers and the molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated according to high resolution analytical procedures such as crystallization, electrophoresis, and chromatography.

The term "enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

The stereochemical definitions and conventions used herein generally follow the codes of S.P. Parker, McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention are contemplated to form part of the present invention, including but not limited to diastereomers, enantiomers, and hindered isomers, as well as mixtures thereof, e.g., racemic mixtures. Many organic compounds exist in optically active form, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to indicate the sign of the compound rotating plane-polarized light, where (-) or 1 indicates that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. With respect to a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Particular stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may exist without stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, which lack optical activity.

The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can be converted to each other via a low energy barrier. For example, proton tautomers (also referred to as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions through recombination of some of the bonded electrons.

As used herein, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate ", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1' -methylene-bis- (2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) and ammonium salts. Pharmaceutically acceptable salts may be directed to include another molecule, such as an acetate ion, succinate ion, or other counterion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. The case where the plurality of charged atoms are part of a pharmaceutically acceptable salt can have a plurality of counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

If the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example by treatment of the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranonic acid (e.g. glucuronic acid or galacturonic acid), an alpha hydroxy acid (e.g. citric acid or tartaric acid), an amino acid (e.g. aspartic acid or glutamic acid), an aromatic acid (e.g. benzoic acid or cinnamic acid), a sulfonic acid (e.g. p-toluenesulfonic acid or ethanesulfonic acid) and the like.

If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example by treating the free acid with an inorganic or organic base, for example an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, and the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine, and piperazine, as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

As used herein, the term "solvate" refers to a compound that also includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces, such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine, dichloromethane, 2-propanol, and the like. Solvates or hydrates of the compounds are readily prepared by addition of at least one molar equivalent of a hydroxy solvent (e.g., methanol, ethanol, 1-propanol, 2-propanol, or water) to the compound to produce solvation or hydration of the imine moiety.

The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients comprising the formulation and/or the mammal being treated therewith.

The term "leaving group" refers to a group of charged or uncharged moieties that leave during substitution or displacement. Such leaving groups are well known in the art and include, but are not limited to, halogens, esters, alkoxy, hydroxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, thioether, and diazo compounds.

The term "reactive ester" refers to an ester having an easily displaceable leaving group that can be easily reacted with an amine group to form an amide bond. Examples of reactive esters include, but are not limited to, N-hydroxysuccinimide ester, N-hydroxysulfosuccinimide ester, nitrophenyl (e.g., 2 or 4-nitrophenyl) ester, dinitrophenyl (e.g., 2, 4-dinitrophenyl) ester, sulfo-tetrafluorophenyl (e.g., 4 sulfo-2, 3,5, 6-tetrafluorophenyl) ester, or pentafluorophenyl ester.

The term "reactive group" refers to a group that can react with a moiety located on another molecule (e.g., a cell-binding agent or cytotoxic compound) to form a covalent bond. Reactive groups include, but are not limited to, amine reactive groups and thiol reactive groups.

The term "amine-reactive group" refers to a group that can react with an amine group to form a covalent bond. Exemplary amine-reactive groups include, but are not limited to, a reactive ester group, an acid halide, a sulfonyl halide, an imide ester, or a reactive thioester group. In certain embodiments, the amine-reactive group is a reactive ester group. In one embodiment, the amine reactive group is an N-hydroxysuccinimide ester or an N-hydroxysulfo-succinimide ester.

The term "thiol-reactive group" refers to a group that can react with a thiol (-SH) group to form a covalent bond. Exemplary thiol-reactive groups include, but are not limited to, maleimide, haloacetyl, haloacetamide, vinylsulfone, vinylsulfonamide, or vinylpyridine. In one embodiment, the thiol-reactive group is maleimide.

The term "bifunctional crosslinker", "bifunctional linker" or "crosslinker" refers to a modifying agent having two reactive groups; one of the reactive groups is capable of reacting with a cell-binding agent, while the other reacts with a cytotoxic compound to link the two moieties together. Such bifunctional crosslinking agents are well known in the art (see, e.g., Isalm and Dent Bioconjugation, Chapter 5, pp. 218-363, Groves diagnostics Inc. New York, 1999). For example, bifunctional crosslinkers that enable linkage via a thioether bond include N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group, or N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB) to introduce an iodoacetyl group. Introduction of maleic anhydride on cell binding agent Other bifunctional crosslinking agents of imide groups or haloacetyl groups are well known in the art (see U.S. patent applications 2008/0050310, 20050169933, available from Pierce Biotechnology inc. p.o.box117, Rockland, IL 61105, USA) and include, but are not limited to, bismaleimide-based polyethylene glycol (BMPEO), bm (peo)₂、BM(PEO)₃N- (beta-maleimidopropoxy) succinimidyl ester (BMPS), N-succinimidyl gamma-maleimidobutanoate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), NHS 5-maleimidopentanoate, HBVS, N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate which is a "long chain" analogue of SMCC (LC-SMCC)), M-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), 4- (4-N-maleimidophenyl) -butanoic acid hydrazide or HCl salt (MPBH), N-succinimidyl 3- (bromoacetamido) propionate (SBAP), N-Succinimidyl Iodoacetate (SIA), N-succinimidyl undecanoate kappa-maleimidyl undecanoate (KMUA), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), succinimidyl-6- (β -maleimidopropionamido) hexanoate (SMPH), succinimidyl- (4-vinylsulfonyl) benzoate (SVSB), Dithiobismaleimidoethane (DTME), 1, 4-bismaleimidobutane (BMB), 1, 4-bismaleimido-2, 3-dihydroxybutane (BMDB), Bismaleimidohexane (BMH), Bismaleimido ethane (BMOE), sulfosuccinimido 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimido (4-iodo-acetyl) aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimido ester (sulfo-MBS), n- (γ -maleimidobutyryloxy) sulfosuccinimidyl ester (sulfo-GMBS), N- (e-maleimidocaproyloxy) sulfosuccinimidyl ester (sulfo-EMCS), N- (κ -maleimidoundecenyloxy) sulfosuccinimidyl ester (sulfo-KMUS) and sulfosuccinimidyl 4- (p-maleimidoyl). Phenyl) butyrate (sulfo-SMPB).

Heterobifunctional crosslinkers are bifunctional crosslinkers having two different reactive groups. Heterobifunctional crosslinkers containing both amine-reactive N-hydroxysuccinimide groups (NHS groups) and carbonyl-reactive hydrazine groups can also be used to link the cytotoxic compounds described herein with cell-binding agents (e.g., antibodies). Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamideacetonhydrazone (SANH), succinimidyl 4-hydrazinoterephthalic acid ester hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate ester hydrochloride (SHNH). Conjugates with acid labile bonds can also be prepared using benzodiazepine derivatives with hydrazines of the present invention. Examples of bifunctional crosslinking agents that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

Bifunctional cross-linkers that enable the attachment of cell-binding agents to cytotoxic compounds via disulfide bonds are known in the art and include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl-4- (2-pyridyldithio) valerate (SPP), N-succinimidyl-4- (2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4- (2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) to introduce dithiopyridyl groups. Other bifunctional crosslinkers that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Pat. nos. 6,913,748, 6,716,821 and U.S. patent publications 20090274713 and 20100129314, all of which are incorporated herein by reference. Alternatively, crosslinkers which introduce thiol groups, such as 2-iminothiolane, homocysteine thiolactone or S-acetylsuccinic anhydride, may also be used.

As defined herein, the term "linker," "linker moiety," or "linking group" refers to a moiety that links two groups, e.g., a cell-binding agent and a cytotoxic compound, together. Typically, the linker is substantially inert under the conditions of the attachment of the two groups to which it is attached. The bifunctional crosslinking agent may comprise two reactive groups, one group at each end of the linker moiety, such that one reactive group may first react with the cytotoxic compound to provide a compound bearing the linker moiety and a second reactive group, which may then react with the cell binding agent. Alternatively, one end of the bifunctional crosslinking reagent may first be reacted with a cell-binding agent to provide a cell-binding agent with a linker moiety and a second reactive group, which may then be reacted with a cytotoxic compound. The linking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds (see, e.g., U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Disulfide, thioether and peptidase labile bonds are preferred. Other linkers useful in the present invention include non-cleavable linkers, such as those described in detail in U.S. publication No. 20050169933, or charged or hydrophilic linkers described in US 2009/0274713, US 2010/01293140, and WO 2009/134976, each of which is expressly incorporated herein by reference.

The term "self-immolative linker" refers to a linker that will allow the release of a cytotoxic compound when the remote site is activated. In certain embodiments, the linker comprises a p-aminophenyl methyl unit. In some such embodiments, the p-aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and a carbamate, methyl carbamate, or carbonate is produced between the benzyl alcohol and the drug (Hamann et al (2005) Expert opin. ther. patents (2005)15: 1087-. In some embodiments, the linker comprises a p-aminobenzyloxycarbonyl group (PAB). Other examples of self-immolative linkers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al (1999) bioorg.Med.chem.Lett.9:2237) and anthranyl acetals or p-aminophenyl methyl acetals. In some embodiments, spacers which undergo cyclization upon hydrolysis of the amide bond may be used, for example substituted and unsubstituted 4-aminobutanoic acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo [2.2.1] and bicyclo [2.2.2] ring systems (Storm et al (1972) J.Amer. chem.Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al (1990) J.org.chem.55: 5867). The attachment of a drug to the alpha-carbon of a glycine residue is another example of a self-immolative linker that may be suitable for use in an ADC (Kingsbury et al (1984) J. Med. chem.27: 1447).

The term "amino acid" refers to a naturally occurring amino acid or a non-naturally occurring amino acid. In some embodiments, the amino acid consists of NH₂-C(R^aa'R^aa) -C (═ O) OH, wherein R is^aaAnd R^aa'Each independently is H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl, or R^aaAnd the N-terminal nitrogen atom may be taken together to form a heterocyclic ring (e.g., as in proline). The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and/or carboxyl terminus of an amino acid, e.g., -NH-C (R)^aa'R^aa)-C(＝O)-。

The term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the peptide contains 2 to 20 amino acid residues. In other embodiments, the peptide contains 2 to 10 or 2 to 8 amino acid residues. In other embodiments, the peptide contains 2 to 5 amino acid residues. As used herein, when a peptide is part of a cytotoxic agent or linker described herein represented by a particular sequence of amino acids, the peptide may be attached to the remaining part of the cytotoxic agent or linker in both directions.

The term "cation" refers to an ion having a positive charge. The cation may be monovalent (e.g., Na)⁺、K⁺Etc.), divalent (e.g., Ca)²⁺、Mg²⁺Etc.) or polyvalent (e.g., Al)³⁺Etc.). Preferably, the cation is monovalent.

The term "antibody" refers to an immunoglobulinA molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" as used herein encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab ', F (ab')₂And Fv fragments), single chain Fv (scfv) mutants, multispecific antibodies (e.g., bispecific antibodies, biparatopic antibodies, etc.), multivalent antibody (e.g., trivalent, tetravalent, etc. antibodies with three, four, or more antigen binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five main classes of immunoglobulins based on the identity of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively): IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibodies may be naked or conjugated to other molecules, such as toxins, radioisotopes, and the like. As used herein, "antibody" also includes activatable antibodies (e.g., probodies). Activatable refers to an activatable antibody exhibiting a first level of binding to a target when the activatable antibody is in an inhibited, masked, intact or uncleaved state (i.e., a first conformation) and a second level of binding to the target in an uninhibited, unmasked and/or cleaved state (i.e., a second conformation), wherein the second level of target binding is greater than the first level of binding.

In some embodiments, the antibody is a non-naturally occurring antibody. In some embodiments, the antibody is purified from a native component. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma.

The term "antibody fragment" refers to a portion of an intact antibody and refers to an epitope of an intact antibodyA constant variable region. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')₂And F_vFragments, linear antibodies, single chain antibodies, and multispecific antibodies (e.g., bispecific, biparatopic) formed from antibody fragments. The term "antigen-binding fragment" of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (but are not limited to): (i) fab fragments, i.e. consisting of V_L、V_H、C_LAnd C_H1Monovalent fragments of domain composition (e.g., antibodies digested by papain produce three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind antigen); (ii) single chain Fab (scFab), i.e. consisting of V _L、V_H、C_LAnd C_H1A fragment consisting of a domain wherein C_LAnd V_HThe domains are linked via a linker peptide; (iii) f (ab')₂Fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge of the hinge region (e.g., antibody digested by pepsin yields two fragments: bivalent antigen binding F (ab')₂Fragments and pFc 'fragments that do not bind antigen) and their associated F (ab') monovalent units; (iv) f_dFragment of V_HAnd C_H1Domain composition (i.e., the portion of the heavy chain included in the Fab); (v) v with one arm consisting of antibody_LAnd V_HDomain composed of F_vFragments, and related disulfide-linked F_v(ii) a (vi) dAb (domain antibody) or sdAb (single domain antibody) fragments (Ward et al, Nature 341:544-546,1989) consisting of V_HDomain composition; (vii) an isolated Complementarity Determining Region (CDR); (viii) single chain variable fragments (scFv), i.e. composed of V linked via a linker peptide_HAnd V_LA fragment consisting of a domain; and (ix) tetravalent antibodies, which may include multiple forms (structures), whereby the antibody comprises 4 antigen binding sites.

The term "monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab ', F (ab') ₂、F_v) Single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in a variety of ways, including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

The term "humanized antibody" refers to a form of a non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin or fragment thereof that contains minimal non-human (e.g., murine) sequences. In general, humanized antibodies are human immunoglobulins in which residues from the Complementarity Determining Regions (CDRs) are replaced by residues from CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al, Nature 321:522-525, 1986; Riechmann et al, Nature 332:323-327, 1988; Verhoeyen et al, Science 239:1534-1536, 1988).

In some cases, F of human immunoglobulin_vFramework Region (FR) residues are replaced with corresponding residues in antibodies from non-human species having the desired specificity, affinity, and capacity. The humanized antibody can be produced by F _vSubstitutions of additional residues in the framework regions and/or within the replaced non-human residues are further modified to refine and optimize antibody specificity, affinity, and/or capacity. In general, the humanized antibody will comprise substantially all of at least one and typically two or three variable domains which comprise all or substantially all of the CDR regions corresponding to a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibodies may also comprise immunoglobulin constant regions or domains (F)_c) (typically, a constant region or domain of a human immunoglobulin). Example description of a method for generating humanized antibodiesIn U.S. Pat. Nos. 5,225,539 and 5,639,641, Roguska et al, Proc. Natl. Acad. Sci. USA 91(3): 969-; and Roguska et al, Protein Eng.9(10):895-904,1996, all of which are incorporated herein by reference. In some embodiments, a "humanized antibody" is a resurfaced antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

The term "variable region" of an antibody refers to either the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains each consist of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held tightly together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on sequence variability of cross species (i.e., Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, 1991, National Institutes of Health, Bethesda Md.); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et Al, J.Molec.biol.273:927-948, 1997). Furthermore, a combination of these two methods is sometimes used in the art to determine CDRs.

When referring to residues in the variable domain (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is commonly used (e.g., Kabat et al, Sequences of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Amino acid position numbering as in Kabat refers to the numbering system used in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (incorporated herein by reference) for the assembled heavy or light chain variable domain of an antibody. Using such numbering systems, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening of, or insertion into, the FRs or CDRs of the variable domains. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and residues inserted (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment with a "standard" Kabat numbered sequence at the region of homology of the sequence of the antibody. Chothia instead mentions the position of the structural loops (Chothia and Lesk, J.mol.biol.196: 901. 917, 1987). The ends of the Chothia CDR-H1 loops when numbered using the Kabat numbering convention vary between H32 and H34, depending on the length of the loops. This is because the Kabat numbering scheme places the insertions at H35A and H35B-if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if 35A and 35B are both present, the loop ends at 34. The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering system of human IgG1 EU antibody based on Edelman et al, 1969, Proc Natl Acad Sci USA 63:78-85 (incorporated herein by reference).

The term "human antibody" refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. In certain embodiments, the human antibody does not have a non-human sequence. This definition of human antibody includes whole or full-length antibodies, or antigen-binding fragments thereof.

The term "chimeric antibody" refers to an antibody in which the amino acid sequences of immunoglobulin molecules are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody derived from another species (typically human) to avoid or reduce the probability of eliciting an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody may comprise an antibody or antigen-binding fragment thereof comprising at least one human heavy and/or light chain polypeptide, e.g., an antibody comprising a murine light chain and a human heavy chain polypeptide.

The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that is capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope may be formed by contiguous amino acids and non-contiguous amino acids juxtaposed by triple folding of the protein. Epitopes formed by contiguous amino acids are typically retained when proteins are denatured, while epitopes formed by triple folding are typically lost when proteins are denatured. Epitopes typically comprise at least 3 and more typically at least 5 or 8-10 amino acids in a unique spatial configuration.

"binding affinity" generally refers to the aggregate strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen), unless otherwise indicated. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_d) Or half maximal Effective Concentration (EC)₅₀) And (4) showing. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate quickly, while high affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. Certain illustrative embodiments are described herein.

By "specifically binds" is generally meant that the antibody binds to an epitope via its antigen binding domain, and that the binding is such that some complementarity between the antigen binding domain and the epitope is required. According to this definition, an antibody is said to "specifically bind" to a random, unrelated epitope when it binds to that epitope more rapidly via its antigen binding domain than it would. The term "specificity" is used herein to define the relative affinity of a particular antibody when bound to a particular epitope. For example, antibody "a" can be considered to have a higher specificity for a given epitope than antibody "B", or antibody "a" can be said to bind to epitope "C" with a higher specificity than the specificity of antibody "a" for the relevant epitope "D".

As used herein, the term "immunoconjugate", "conjugate" or "ADC" refers to a compound or derivative thereof that is linked to a cell-binding agent (e.g., an antibody or antigen-binding fragment thereof).

The term "cysteine-engineered antibody" includes antibodies having at least one Cys that is not normally present at a given residue of an antibody light chain or heavy chain. The Cys, which may also be referred to as an "engineered Cys," may be engineered using any conventional molecular biology or recombinant DNA techniques (e.g., by replacing the coding sequence for a non-Cys residue at a target residue with the coding sequence for a Cys). For example, if the original residue is Ser with a coding sequence of 5'-UCU-3', the coding sequence can be mutated (e.g., induced by site-directed mutagenesis) to 5'-UGU-3', which encodes Cys. In certain embodiments, a Cys engineered antibody of the invention has an engineered Cys in the heavy chain. In certain embodiments, the engineered Cys is in or near the CH3 domain of the heavy chain. In certain embodiments, the engineered Cys is at residue 442 of the heavy chain (EU/OU numbering). The C442 residue can be conjugated to the cytotoxic drug/agent via the free thiol group of the C442 residue, e.g., via reaction with a thiol-reactive agent (e.g., maleimide group) of the cytotoxic drug.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. "tumor" and "neoplasm" refer to one or more cells that are benign (non-cancerous) or malignant (cancerous), including precancerous lesions, resulting from excessive cell growth or proliferation.

Examples of cancer include endometrial cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, and lymphatic cancer. In certain embodiments, the cancer is non-small cell lung cancer, colorectal cancer, gastric cancer, or pancreatic cancer. In certain embodiments, the cancer is non-small cell lung cancer (squamous cell, non-squamous cell, adenocarcinoma or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, or squamous cell carcinoma), or breast cancer (e.g., Triple Negative Breast Cancer (TNBC)). In certain embodiments, the cancer is lymphoma and leukemia. In certain embodiments, examples of cancer include AML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndrome, blastic plasmacytoid DC lymphoma (BPDCN) leukemia, B-cell lymphoma (including non-hodgkin's lymphoma (NHL)), precursor B-cell lymphoblastic leukemia/lymphoma, and mature B-cell neoplasm (e.g., B-cell chronic lymphocytic leukemia (B-CLL)/Small Lymphocytic Lymphoma (SLL)), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL) (including low, intermediate and high grade FL), cutaneous follicular central lymphoma, marginal zone B-cell lymphoma (MALT type, lymph node and spleen type), Hairy Cell Leukemia (HCL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, Anaplastic Large Cell Lymphoma (ALCL), and Hodgkin's Leukemia (HL). In certain embodiments, the cancer is BPDCN leukemia. In certain embodiments, the cancer is ALL. In other embodiments, the cancer is AML.

The term "subject" refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, and the like. In general, the terms "subject", "patient" and "individual" are used interchangeably herein with respect to a human subject.

The term "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of an active ingredient to be effective and that is free of additional components having unacceptable toxicity to the subject to which the composition will be administered. Such compositions may be sterile.

As used herein, a "therapeutically effective amount" is an amount of a compound or composition sufficient for a particular stated purpose. Administration of one dose may not necessarily result in complete therapeutic effect, but may only occur after administration of a series of doses. The particular "therapeutically effective amount" will depend, for example, on the age, weight, and medical condition of the subject, as well as the method of administration and the therapeutic agent or combination of therapeutic agents selected for administration. The "therapeutically effective amount" can be determined empirically and in a routine manner for the stated purpose.

As used herein, the terms "treating", "treating" or "treatment" include reversing, reducing or preventing symptoms, clinical signs or underlying pathology of a condition in a subject in a manner that improves or stabilizes the condition. As used herein, and as is well understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired results include, but are not limited to, prevention, alleviation, amelioration or slowing of one or more symptoms or conditions associated with the condition, diminishment of extent of disease, stabilization of the disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an increase in survival compared to expected survival in the absence of treatment.

Compound (I)

In one aspect, the present invention provides a compound of formula I:

Z-L¹-D (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C ₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂。

In some embodiments, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof:

Z-L¹-D (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R²is-H, -F, -OR³、-SR³、-S(O)R⁴、-S(O)₂R⁴、C₁-C₆Alkyl or C₁-C₆A fluoroalkyl group; or R¹And R²Together with the carbon atom to which it is attached to formA methylenedioxy or difluoromethylenedioxy ring;

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

with the proviso that if R¹Is F, then L¹Is- (C)₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z; and Z is-X²；

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂(ii) a And is

With the proviso that if R¹Is F and R²is-Me, then-L₁-Z cannot be-CH₂OH。

In some embodiments, R¹Is F and-L¹-Z is- (C)₁-C₆Alkylene) -X²、-(C₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -X²、-X^1'-(C₁-C₆Alkylene) -X²Or- (C)₁-C₆Alkylene) -X¹-L²-X². In some embodiments, R¹Is F and-L¹-Z is- (C)₁-C₆Alkylene) -OR⁶、-(C₁-C₆Alkylene) -SR⁶、-(C₁-C₆Alkylene) -S (O) R⁶、-(C₁-C₆Alkylene) -S (O)₂R⁶、-(C₁-C₆Alkylene) -SSR⁶Or- (C)₁-C₆Alkylene) -N (R)⁶)₂. In some embodiments, R¹Is F and-L¹-Z is- (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -OR⁶、-(C₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -SR⁶、-(C₁-C₆Alkylene) -X ¹-(C₁-C₆Alkylene) -S (O) R⁶、-(C₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -S (O)₂R⁶、-(C₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -SSR⁶Or- (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -N (R)⁶)₂. In some embodiments, R¹Is F and-L¹-Z is-X^1'-(C₁-C₆Alkylene) -OR⁶、-X^1'-(C₁-C₆Alkylene) -SR⁶、-X^1'-(C₁-C₆Alkylene) -S (O) R⁶、-X^1'-(C₁-C₆Alkylene) -S (O)₂R⁶、-X^1'-(C₁-C₆Alkylene) -SSR⁶or-X^1'-(C₁-C₆Alkylene) -N (R)⁶)₂. In some embodiments, R¹Is F and-L¹-Z is- (C)₁-C₆Alkylene) -X¹-L²-OR⁶、-(C₁-C₆Alkylene) -X¹-L²-SR⁶、-(C₁-C₆Alkylene) -X¹-L²-S(O)R⁶、-(C₁-C₆Alkylene) -X¹-L²-S(O)₂R⁶、-(C₁-C₆Alkylene) -X¹-L²-SSR⁶Or- (C)₁-C₆Alkylene) -X¹-L²-N(R⁶)₂。

In some embodiments, -L¹-Z is-H. In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -H or- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -H. In some embodiments, -L ¹-Z is- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -X². In some embodiments, -L¹-Z is methyl, ethyl, propyl or butyl.

In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SR⁶Or- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SR⁶. In some embodiments, -L¹-Z is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶。

In some embodiments, -L¹-Z is- (C)₁-C₆Alkylene) -X¹-L²-X². In some embodiments, -L¹-Z isIn some embodiments, -L¹-Z isIn some embodiments, -L¹-Z is

In another aspect, the present invention provides a compound of formula II:

E-A-Z'-L¹-D (formula II)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X ¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols; and is

E is-C (═ O) -L³-X³；

L³Is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X³The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

Each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X³is that -C(＝O)-CR^bbR^cc-W'、-NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰；

Each W' is independently-H, -N (R)^gg)₂、C₁-C₁₀Alkyl radical, C₁-C₁₀Alkenyl radical, C₁-C₁₀Alkynyl, C₃-C₆Cycloalkyl, aryl, heteroaryl or- (CH)₂CH₂O)_q-R^ff；

q is 1 to 24;

each R^aa、R^bb、R^cc、R^eeAnd R^ffIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R^ggeach independently is-H or C₁-C₆An alkyl group; and is

R⁹And R¹⁰Each independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

In some embodiments, R¹is-H or-F. In some embodiments, R¹is-F. In some embodiments, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl. In some embodiments, R²is-F. In some embodiments, R²is-OMe, -SMe, -S (O) Me or methyl. In some embodiments, R ²Is methyl. In some embodiments, R¹is-F and R²is-F. In some embodiments, R¹Is methyl and R²is-F. In some embodiments, R¹is-F and R²Is a-methyl group.

In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-*。

In some embodiments, -L ¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-*。

In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -NR⁸-*。

In some embodiments, -L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -. In some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -is

Disclosed herein as-L¹In various embodiments of-Z' -, is a site covalently linked to a.

In some embodiments, a is- (AA)¹)-(AA²)_a1-, wherein is a site covalently linked to E; AA¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

In some embodiments, -AA¹-(AA²)_a1-is-Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-N⁹-tosyl-Arg-, -N⁹-tosyl-Arg-Phe-, -Phe-N⁹-nitro-Arg-, -N⁹-nitro-Arg-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu-, -Leu Ala-Leu-Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -, -Val-Arg-, -Arg-Val-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, - -Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -y-Gln-, -al Ser-Gln-, -Ser-, -y-Gln-, -al, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr- & lty-Met- & ltx.

In some embodiments, -AA¹-(AA²)_a1-: -Ala-, -Ala-Val-, -Val-Ala-, -Gln-Leu-, -Leu-Gln-, -Ala-, -Gly-Ala-Gly-, -Gly-Ala-Gly-, -Gly-Val-Gly-, -Gly-Val-Gly-, -Gly-Phe-Gly-, or-Gly-Phe-Gly-,.

In some embodiments, -AA¹-(AA²)_a1-: -L-Ala-, -L-Ala-D-Ala-LAla-, -L-Ala-, -L-Ala-, or-L-Ala- >.

Disclosed herein is-AA¹-(AA²)_a1In various embodiments, is a site covalently attached to E.

In some embodiments, wherein the polyol isWherein R is¹²Is H or methyl.

In some embodiments, E is-C (═ O) - (C)₁-C₁₀Alkylene) -X³. In some embodiments, E is

In some embodiments, E is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X³；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X³Is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

In some embodiments, E is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X³；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X³Is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

In another aspect, the present invention provides a compound of formula III, or a pharmaceutically acceptable salt thereof:

CBA-E'-A-Z'-L¹-D (formula III)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols;

e' is-C (═ O) -L³-X⁶-; wherein is a site covalently attached to the CBA;

L³is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X⁶The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA;

each R^aa、R^bb、R^ccAnd R^eeIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R⁹independently is-H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

CBA is a cell binding agent.

In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C) ₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene radical)-S-(C₁-C₅Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-*。

In some embodiments, each R is⁵independently-H, methyl or benzyl. In some embodiments, each R is⁵Independently is-H. In some embodiments, each R is⁵Is methyl. In some embodiments, each R is⁵Is benzyl. In some embodiments, eachR⁸independently-H, methyl or benzyl. In some embodiments, each R is⁸Independently is-H. In some embodiments, each R is⁸Is methyl. In some embodiments, each R is⁸Is benzyl.

In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸- *. In some embodiments, -L ¹-Z' -is-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- *. In some embodiments, -L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -NR⁸-*。

In some embodiments, -L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -. In some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -isIn some embodiments, -L¹-Z' -is

Disclosed herein as-L¹In various embodiments of-Z' -, is a site covalently linked to a.

In some embodiments, a is- (AA)¹)-(AA²)_a1-¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

In some embodiments of the present invention, the substrate is,-AA¹-(AA²)_a1-Val-D-Lys-, -Val-D-Arg-, -L-Val-Cit-, -L-Val-Lys-, -L-Val-Arg-, -L-Val-D-Cit-, -L-Phe-Phe-Lys-, -L-Val-D-Arg-, -L-Arg-D-Arg-, -L-Ala-Ala-Ala-, -L-Ala-D-Ala-, -Val-D-Cit-, -, -L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val- ".

In some embodiments, -AA¹-(AA²)_a1-: -Ala-, -Ala-Val-, -Val-Ala-, -Gln-Leu-, -Leu-Gln-, -Ala-, -Gly-Ala-Gly-, -Gly-Ala-Gly-, -Gly-Val-Gly-, -Gly-Val-Gly-, -Gly-Phe-Gly-, or-Gly-Phe-Gly-,.

In some embodiments, -AA¹-(AA²)_a1-: -L-Ala-, -L-Ala-D-Ala-L-Ala-, -L-Ala-, or-L-Ala- >.

Disclosed herein is-AA ¹-(AA²)_a1In various embodiments, is a site covalently linked to E'.

In some embodiments, a is substituted with one or more polyols. In some embodiments, E' is substituted with one or more polyols. In some embodiments, the polyol is — (C)₁-C₆Alkylene) -X⁵-Y³(ii) a Wherein:X⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and R is¹²is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

In some embodiments, the polyol isWherein R is¹²Is H or methyl.

In some embodiments, wherein the polyol isWherein R is¹²Is H or methyl.

In some embodiments, E' is-C (═ O) - (C)₁-C₁₀Alkylene) -X⁶- *. In some embodiments, E' is

-C(＝O)CH₂CH₂-C(＝O)-CR^bbR^cc- (O) CH₂CH₂-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA. In some embodiments, R^YY、R^XX、R^aa、R^bbEach independently is-H or C₁-C₆An alkyl group.

In some embodiments, E' is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X⁶-*；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X⁶Is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

In some embodiments, E' is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X⁶-*；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

In some embodiments, the CBA comprises an-SH group covalently attached to E' to provide -C(＝O)-CR^bbR^cc-S-CBA or-NR^ee-C(＝O)-CR^bbR^cc-S-CBA。

In another aspect, the present invention provides a compound of formula IV:

E-A-Z'-L¹-D (formula IV)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

L¹And L²Each independently optionally substituted by 1-4 substituents selected from halogen, -CN, -OR⁷、-SR⁷、-N(R⁷)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, or heteroaryl;

each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols;

e is-C (═ O) O-L³-X³；

L³Is- (C)₁-C₁₀Alkylene) -;

wherein L is³Optionally substituted by 0-4 substituents selected from halogen, -CN, -OR⁹、-SR⁹、-N(R⁹)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, heteroaryl, and polyol;

each R⁹Independently isH、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

X³Is that

In some embodiments, E is-C (═ O) O-L³-X³And L is³Is- (C)₁Alkylene) -. In some embodiments, L is³is-CH₂-。

In another aspect, the present invention provides a compound of formula V or a pharmaceutically acceptable salt thereof:

A-Z'-L¹-D (formula V)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

A is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols.

In some embodiments, the compound is a compound of formula I and is any one of the following compounds from the following table:

Table 1A.

In some embodiments, the compound is a compound of formula I and is a compound from any one of the following table:

TABLE 1B

In some embodiments, the compound is a compound of formula II and is a compound from any one of the following tables:

TABLE 2

In some embodiments, wherein the compound comprises a sulfoxide and the sulfoxide is in the R or S configuration. In some embodiments, the compound is any one of:

in some embodiments, the compound is any one of:

synthesis of Compounds

The compounds described herein are prepared by the general synthetic routes described in the schemes below.

In some embodiments, the compounds described herein are prepared as outlined in scheme 1.

Scheme 1 for the preparation of RNH₂General Synthesis scheme of Compound (A-6)

In some embodiments, the compounds described herein are prepared as outlined in scheme 2.

Scheme 2 general synthetic scheme for the preparation of ROH Compound (B-2)

In some embodiments, the compounds described herein are prepared as outlined in scheme 3.

Scheme 3 for preparing RNHR_C1General Synthesis scheme for Compound (C-4)

In some embodiments, the compounds described herein are prepared as outlined in scheme 4.

Scheme 4 for preparation of RS (CH)₂)_nGeneral Synthesis scheme for OH Compound (D-2)

In some embodiments, the compounds described herein are prepared as outlined in scheme 5.

Scheme 5 general procedure for the preparation of thiol-bearing camptothecin derivatives (E-4)

In some embodiments, v is 1 to 4. In some embodiments, R is alkyl, aryl, or heteroaryl. In some embodiments, R is methyl, ethyl, phenyl or pyridyl.

In some embodiments, the compounds described herein are prepared as outlined in scheme 6.

Scheme 6 general procedure for the preparation of thioketal-peptide bonds that can be further derivatized to incorporate thiols or maleimides

In some embodiments, v is 1 to 4. In some embodiments, v2 is 1 to 4. In some embodiments, R is alkyl, aryl, or heteroaryl. In some embodiments, R is methyl, ethyl, phenyl or pyridyl.

In some embodiments, the compounds described herein are prepared as outlined in scheme 7.

Scheme 7.

In some embodiments, n is 1 to 10. In some embodiments, n is 1 to 5. In some embodiments, v is 1 to 10. In some embodiments, v is 1 to 5.

In some embodiments, the compounds described herein are prepared as outlined in scheme 8.

Scheme 8.

In some embodiments, v is 1 to 10. In some embodiments, v is 1 to 5.

Cell binding agents

The cell-binding agents in the immunoconjugates of the invention can be of any species that are currently known, or that become known, including peptides and non-peptides that bind to cells or cellular components (e.g., receptors, proteins, DNA, RNA, etc.). In general, these cell binding agents may be antibodies (e.g. polyclonal and monoclonal antibodies, especially monoclonal antibodies) or fragments thereof, lymphokines, hormones, growth factors, vitamins (e.g. folate etc., which can bind to its cell surface receptor, e.g. folate receptor), nutrient transport molecules (e.g. transferrin), probodies, nanobodies or any other cell binding molecule or substance.

In certain embodiments, the cell-binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, a chimeric antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, a domain antibody (e.g., sdAb), or a domain antibody fragment that specifically binds to a target cell.

In certain embodiments, the cell-binding agent is a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment (or "antigen-binding portion" or "antigen-binding fragment").

In certain embodiments, the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment (or "antigen-binding portion" or "antigen-binding fragment").

In certain embodiments, the cell-binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives), and the CBA can bind to a ligand on the target cell, such as a cell-surface ligand, including a cell-surface receptor.

In certain embodiments, the cell-binding agent (CBA) binds to a target cell selected from a tumor cell, a virally-infected cell, a microbially-infected cell, a parasitically-infected cell, an autoimmune cell, an activated cell, a myeloid cell, an activated T cell, a B cell, or a melanocyte. In some embodiments, the CBA binds to cells expressing 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B2-H2, BCMA, C4.4a, CA 2, CanAg, CD123, CD138, CD142, CD166, CD184, CD2, CD205, CD2, CD248, CD2, CD352, CD2, CD40 2, CD44v 2, CD79 2, CDH 2, ACAM 2, CEM 2, ACACACACACACACAKIT, cKIT, N18.2, EGFN 2, CLL-1, CLL-METC-CSP, GCCLDLLR 1-CTP, EPR-2, EPTC-2, EPR-2, EPTC-3, EPTC-2, EPTC-3, EPTC-hAD 2, EPTC-CTP, EPTC-2, EPTC-3, EPTC-2, EPDChAD 2, EPTC-3, EPDChAD 2, EPDCHA-2, EPC 3, EPDChAD 2, EPTC-2, EPDChAD 2, EPTC-CTH-2, EPTC-3, EPTC-CTP, EPTC-2, EPTC-CTP, EPTC-3, EPTC-CTD 2, EPR-3, EPTC-CTH-3, EPR-2, EPTC-3, EPTC-2, EPTC-3, EPR-3, EPTC-3, EPR-CTD 2, EPR-3, EPTC 2, EPTC-3, EPTC-CTP, EPTC-3, EPTC 2, EPTC-3, EPR-3, EPTC 2, EPTC-3, EPTC-CTC 3, EPTC 2, EPDCHA-3, EPDChAD 2, EPTC-3, EPTC-CTC 3, EPR-3, EPTC 2, EPDChAD 2, EPTC-3, EPR-CTC 3, EPTC-3, EPTC 2, KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC class I chain-associated proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2B, Nectin-4, CH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), Polymorphic Epithelial Mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF- α, TRA, TROP-2, tumor-associated glycoprotein 72(TAG-72), a tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigen.

In certain embodiments, the cell binding agent is a cysteine engineered antibody or antigen binding fragment thereof that specifically binds to epitope 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD 36352, CD25, CD40, CD44v 25, CD 3679 25, CDCEM 25, ACEPECEM 25, ACACACACACACACACACACECK, KIN 18. CLCLCLCLCLCLCLCLCLDLN, CLCLCLCLCLCLCLCLCLCLD 25, EPH-25, EPR-FLhAD 25, EPH-25, EPR-25, EPTC-25, EPDR-25, EPTC-25, EPH-EPTC-25, EPTC-EPC-25, EPH-25, EPTC-EPR-25, EPTC-25, EPC-EPDChAD 25, EPTC-CTP-25, EPDChA-25, EPC-CTP-EPTC-EPC-25, EPR-EPTC-EPH-25, EPC-EPTC-EPC-25, EPTC-25, EPC-EPTC-EPC-EPH-3, EPH-25, EPC-EPH-EPC-25, EPTC-EPH-EPTC-3, EPTC-EPH-25, EPTC-25, EPTC-EPTC 25, EPTC-3, EPTC-EPTC 25, EPTC-3, EPTC-3, EPTC-, IL-13R, IL RAP, IL7R, interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2B, Nectin-4, NOTCH3, OAcGDA 2, OX L, p-cadherin, PD-L1, Phosphatidylserine (PS), Polymorphic Epithelial Mucin (PEM), prolactin receptor (PRLRR), STEMA, PTK7, RNF43, ROR 43, SAR 72, SART 72, SLC 72, SLS-L72, SLC 43, SLS-related proteins (SLSC 43, SLS-SSTA-72, SLS-associated tumor epitope (43), SLS-72, SLS-1-72, SLC 43, SLS-1, SLR-72, SLS-1, SLRP-11, SLS-1, SLS-4, SLS 3, Cells of any one or more of CD5, TIM-3, UPK2, or UPK1b antigens.

In certain embodiments, the CBA is an antibody selected from the group consisting of: anti-CD 37 antibodies (e.g., as disclosed in USPN 8,765,917, the contents of which are incorporated herein by reference in their entirety), anti-CD 19 antibodies (e.g., huB4 antibodies as disclosed in USPN 9,555,126, the contents of which are incorporated herein by reference in their entirety), and anti-EGFR antibodies (e.g., huML66 antibodies as disclosed in USPN 9,238,6908,790,649 and 9,125,896, the contents of which are incorporated herein by reference in their entirety).

In certain embodiments, the CBA is an anti-CD 123 antibody or antigen-binding fragment thereof, which may comprise: a) at least one light chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_L1、CDR_L2 and CDR_L3 wherein CDR_L1 has the amino acid sequence RASQDINSYLS (SEQ ID NO:1), CDR_L2 has the amino acid sequence RVNRLVD (SEQ ID NO:2) and CDR_L3 has the amino acid sequence LQYDAFPYT (SEQ ID NO: 3); and b) at least one heavy chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_H1、CDR_H2 and CDR_H3 wherein CDR_H1 has the amino acid sequence SSIMH (SEQ ID NO:4), CDR_H2 has an amino acid sequence YIKPYNDGTKYNEKFKG (SEQ ID NO:5) and CDR_H3 has the amino acid sequence EGGNDYYDTMDY (SEQ ID NO: 6).

In certain embodiments, the anti-CD 123 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V)_H) Having an amino acid sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and light chain variable region (V)_L) Having an amino acid sequence

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-CD 123 antibody has the full-length heavy chain sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and the full-length sequence of the light chain

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-CD 123 antibody or antigen-binding fragment thereof is provided as an activatable antibody or an activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-CD 123 activatable antibody or activatable CD123 antibody binding antibody fragment may be conjugated to a compound of formula I.

In certain embodiments, the CBA is an anti-CD 123 antibody or antigen-binding fragment thereof as described in U.S. patent nos. 7,342,110 and 7,557,189, which are incorporated herein by reference.

In certain embodiments, the anti-CD 33 antibody or antigen-binding fragment thereof may comprise: a) at least one light chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively _L1、CDR_L2 and CDR_L3 wherein CDR_L1 has the amino acid sequence KSSQSVFFSSSQKNYLA (SEQ ID NO:11), CDR_L2 has the amino acid sequence WASTRES (SEQ ID NO:12), and a CDR_L3 has an amino acid sequence HQYLSSRT (SEQ ID NO: 13); and b) at least one heavy chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_H1、CDR_H2 and CDR_H3 wherein CDR_H1 has the amino acid sequence SYYIH (SEQ ID NO:14), CDR_H2 has an amino acid sequence VIYPGNDDISYNQKFQG (SEQ ID NO:15) and a CDR_H3 has the amino acid sequence EVRLRYFDV (SEQ ID NO: 16).

In certain embodiments, the anti-CD 33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V)_H) Having an amino sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and light chain variable region (V)_L) Having an amino acid sequence

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-CD 33 antibody has the full-length heavy chain sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and the full-length sequence of the light chain

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-CD 33 antibody is a huMy9-6 antibody.

In certain embodiments, the anti-CD 33 antibody or antigen-binding fragment thereof is provided as an activatable antibody or an activatable antibody-binding antibody fragment as described further below. In certain other embodiments, the anti-CD 33 activatable antibody or activatable CD33 antibody binding antibody fragment may be conjugated to a compound of formula I.

In certain embodiments, the CBA is an anti-ADAM 9 antibody or antigen-binding fragment thereof as described in WO2018/119196 and U.S. provisional applications nos. 62/690052 and 62/691342 (each of which is incorporated herein by reference).

In certain embodiments, the anti-ADAM 9 antibody or antigen-binding fragment thereof is a humanized anti-ADAM 9 antibody or antigen-binding fragment thereof that specifically binds to human ADAM9 and cynomolgus monkey ADAM 9.

In certain embodiments, the humanized anti-ADAM 9 antibody or ADAM9 binding fragment thereof is optimized to have at least 100-fold greater binding affinity for cynomolgus monkey ADAM9 and retains high affinity binding to human ADAM9 compared to the chimeric or murine parent antibody.

In certain embodiments, an anti-ADAM 9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAM 9 antibody or antigen-binding fragment thereof) comprises: a) at least one light chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_L1、CDR_L2 and CDR_L3 wherein CDR_L1 has the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO:21), CDR_L2 has the amino acid sequence AASDLES (SEQ ID NO:22), and a CDR_L3 has the amino acid sequence QQSHEDPFT (SEQ ID NO: 23); and b) at least one heavy chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively _H1、CDR_H2 and CDR_H3 wherein CDR_H1 has the amino acid sequence SYWMH (SEQ ID NO:24), CDR_H2 has an amino acid sequence EIIPIFGHTNYNEKFKS (SEQ ID NO:25) and a CDR_H3 has the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO: 26).

In certain embodiments, the anti-ADAM 9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAM 9 antibody or antigen-binding fragment thereof) comprises a heavy chain variable region (V)_H) Having an amino sequence

Wherein the CDR_H1、CDR_H2 and CDR_H；3 double underlined;

and light chain variable region (V)_L) Having an amino acid sequence

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-ADAM 9 antibody has a heavy chain full-length sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and the full-length sequence of the light chain

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-ADAM 9 antibody or antigen-binding fragment thereof is provided as an activatable antibody or an activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-ADAM 9 activatable antibody or activatable ADAM9 antibody binding antibody fragment can be conjugated to a compound of formula I.

In certain embodiments, the CBA is an anti-folate receptor antibody (i.e., FOLR1 or FR α) (e.g., as described in U.S. patent 8,709,432, U.S. patent No. 8,557,966, and WO2011106528, all of which are incorporated herein by reference).

In certain embodiments, an anti-FR α antibody or antigen-binding fragment thereof can comprise: a) at least one light chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_L1、CDR_L2 and CDR_L3 wherein CDR_L1 has the amino acid sequence KASQSVSFAGTSLMH (SEQ ID NO:31), CDR_L2 has the amino acid sequence RASNLEA (SEQ ID NO:32) and a CDR_L3 has the amino acid sequence QQSREYPYT (SEQ ID NO: 33); and b) at least one heavy chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_H1、CDR_H2 and CDR_H3 wherein CDR_H1 has an amino acid sequence of GYFMN (SEQ ID NO:34) or GYTFTGYFMN (SEQ ID NO:37), CDR_H2 has an amino acid sequence of RIHPYDGDTFYNQKFQG (SEQ ID NO:35) or RIHPYDGDTF (SEQ ID NO:38) and a CDR_H3 has the amino acid sequence YDGSRAMDY (SEQ ID NO: 36). In certain embodiments, the anti-FR α antibody or antigen-binding fragment thereof comprises a) a light chain variable region comprising a CDR having the amino sequence set forth in SEQ ID NO:31_L1. CDR having the amino sequence shown in SEQ ID NO. 32_L2 andCDR having the amino sequence shown in SEQ ID NO. 33_L3; and b) a heavy chain variable region comprising a CDR having the amino sequence set forth in SEQ ID NO 34 _H1. CDR having amino sequence shown by SEQ ID NO. 35_H2 and a CDR having the amino sequence shown in SEQ ID NO:36_H3. In certain embodiments, the anti-FR α antibody or antigen-binding fragment thereof comprises a) a light chain variable region comprising a CDR having the amino sequence set forth in SEQ ID NO:31_L1. CDR having the amino sequence shown in SEQ ID NO. 32_L2 and a CDR having the amino sequence shown in SEQ ID NO:33_L3; and b) a heavy chain variable region comprising a CDR having the amino sequence set forth in SEQ ID NO:37_H1. CDR having the amino sequence shown in SEQ ID NO. 38_H2 and a CDR having the amino sequence shown in SEQ ID NO:36_H3。

In certain embodiments, the anti-FR α antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V)_H) Having an amino sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and light chain variable region (V)_L) Having an amino acid sequence

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-fra antibody has the full-length heavy chain sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and the full-length sequence of the light chain

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-FR α antibody is huMov19 or M9346A antibody.

In certain embodiments, the anti-FR α antibody or antigen-binding fragment thereof is provided as an activatable antibody or an activatable antibody-binding antibody fragment as described further below. In certain other embodiments, the anti-fra activatable antibody or activatable fra antibody binding antibody fragment may be conjugated to a compound of formula I.

In certain embodiments, the CBA is an anti-EpCAM antibody or antigen-binding fragment thereof. In certain embodiments, an anti-EpCAM antibody or antigen-binding fragment thereof can comprise: a) at least one light chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_L1、CDR_L2 and CDR_L3 wherein CDR_L1 has the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:45), CDR_L2 has an amino acid sequence QTSNLAS (SEQ ID NO:46) and a CDR_L3 has the amino acid sequence AQNLELPNT (SEQ ID NO: 47); and b) at least one heavy chain variable region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_H1、CDR_H2 and CDR_H3 wherein CDR_H1 has the amino acid sequence NYIH (SEQ ID NO:48), CDR_H2 has an amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO:49), and a CDR_H3 has the amino acid sequence DGPWFAY (SEQ ID NO: 50).

In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V) _H) Having an amino acid sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and light chain variable region (V)_L) Having an amino acid sequence

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the anti-EpCAM antibody has a full-length heavy chain sequence

Wherein the CDR_H1、CDR_H2 and CDR_H3 double underlined;

and the full-length sequence of the light chain

Wherein the CDR_L1、CDR_L2 and CDR_L3 is double underlined.

In certain embodiments, the CBA is an anti-EpCAM antibody or antigen-binding fragment thereof, which can comprise: a) at least one light chain Variable (VL) region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs, respectively_L1、CDR_L2 and CDR_L3 wherein CDR_L1 has an amino acid sequence selected from the group consisting of SEQ ID NOs 78, 45, 79, 80, and 82; CDR_L2 has the amino acid sequence SEQ ID NO 46; and CDR_L3 has an amino acid sequence selected from the group consisting of SEQ ID NOs 47, 81, 83, 84, 85, 86 and 87, and b) at least one heavy chain Variable (VH) region or fragment thereof comprising three consecutive Complementarity Determining Region (CDR) CDRs_H1、CDR_H2 and CDR_H3 wherein CDR_H1 has an amino group selected from SEQ ID NOs 48, 57, 58, 60, 61, 62, 68, 69, 70, 77 and 88A sequence; CDR_H2 has an amino acid sequence selected from the group consisting of SEQ ID NOs 49, 56, 59, 63, and 64; and CDR _H3 has an amino acid sequence selected from the group consisting of SEQ ID NOs 55, 65, 66, 67, 71, 72, 73, 74, 75 and 76.

In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is provided as an activatable antibody or an activatable antibody-binding antibody fragment as described further below. In certain other embodiments, the anti-EpCAM activatable antibody or activatable EpCAM antibody-binding antibody fragment may be conjugated to a compound of formula I.

The variants in the CDRs of the anti-EpCAM antibody or antigen-binding fragment are summarized in tables 3-6 below.

TABLE 3 HuEpCAM-234.2 (CDR and variable Domain)

TABLE 4 humanized variants of huEpCAM-23 (full-Length sequence)

TABLE 5 EpCAM23 variant heavy chain CDR sequences

TABLE 6 EpCAM23 variant light chain CDR sequences

In certain embodiments, the antibodies described herein are murine, non-human mammalian, chimeric, humanized, or human antibodies. For example, the humanized antibody can be a CDR-grafted antibody or a resurfaced antibody. In certain embodiments, the antibody is a full length antibody. In certain embodiments, the antigen binding fragment thereof is Fab, Fab ', F (ab')₂、F_dSingle chain Fab (scFab), single chain Fv or scFv, disulfide linked F_vV-NAR domain, IgNar, endosome, IgG Δ CH ₂Minibody, F (ab')₃Tetrafunctional antibody, trifunctional antibody, bifunctional antibody, diabody, single domain antibody, DVD-Ig, Fcab, mAb₂、(scFv)₂Or scFv-Fc.

In certain embodiments, the cell-binding agent is an alternative protein scaffold, such as a Peptide targeting a somatostatin receptor (see Barbieri F, Bajetto A, Pattarozzi A, Gatti M, Wurth R, Thellung S ET al, Peptide receptor targeting in cancer: the ligand binding Peptide. int J Peptide 2013; 2013:926295), inhibitor cystine knot (also known as ICK or Knottin; see knotting-bound thermal and diagnostic peptides. drug delivery technology 2012; 9(1) ore 1-e 70; Mochran JR. engineering coding Peptide. N. E. G. J. C. P. J. P. A. B. A. B. A. B. A. B. Berrskens FJ, Verploegen S, Strumane K, van Kampen MD, Voorhorst M et al, A Novel Platform for the location of Therapeutic Antibodies Based on anti-gene-Dependent format of IgG Hexamers at the Cell surface. PLoS Biol 2016; 14(1) e 1002344); single-chain Fab (scFab) fragments (see Koerber JT, Hornsby MJ, Wells JA. an improved Single-chain Fab platform for expression and recombinant expression. J Mol Biol; 427(2): 576-86; Hust M, Jostock T, Menzel C, Voedisch B, Mohr A, Brenneis M et al, Single chain Fab (scFab) fragment. BMC Biotechnology 2007; 7:14), targets identified by Drug Affinity Reactive Target Stability (DARTS) (see Pai MY, Lonicek B, Hwang H, Schiestl R, McJade W, Loo Jay et al, Drug Affinity Reactive Target Stability (DARTS), protein joining of consensus protein 3598, consensus protein 3, and consensus protein joined by the consensus protein 3, and consensus protein 3 (see Pat M et al, J. Ser. No. 7:14, target C, marker C, and D, Ankyrin repeat proteins (e.g., designed ankyrin repeat proteins, referred to as DARPins; see U.S. patent publication Nos. 2004/0132028, 2009/0082274, 2011/0118146 and 2011/0224100, incorporated herein by reference, and also C.Zahnd et al, Cancer Res. (2010)70:1595-, incorporated herein by reference), Knottin (a small disulfide-rich protein characterized by disulfide bonding via a disulfide junction), a bicyclic peptide (also referred to as bicyclic; see Heinis C, Rutherford T, Freund S, Winter g. phase-encoded combinatorial chemical basic groups on bicyclic peptides. nat Chem Biol 2009; 502-7 parts of (5), (7); teufel DP, Bennett G, Harrison H, van Rietschoten K, Pavan S, Stace C et al, Stable and Long-Lasting, Novel bicycylic Peptide plasmid Kallikrein Inhibitors for the Treatment of diabetes mac Edema.J Med Chem 2018; 2823-36), Avibody (including bifunctional, trifunctional, and tetrafunctional antibodies; see U.S. publication nos. 2008/0152586 and 2012/0171115), dual receptor retargeting (DART) molecules (p.a. moore et al, Blood, 2011; 117(17) 4542-4551; veri MC et al, Arthritis Rheum, 30 months 3 2010; 1933-43 in 62 (7); johnson S et al, J Mol Biol, 9/4/2010; 399(3):436-49) and cell penetrating supercharged proteins (Methods in enzymol.502,293-319 (2012)).

Activatable CBA

In additional embodiments, the provided CBA is an activatable antibody or an activatable antigen-binding antibody fragment (collectively referred to as AA). In some embodiments, the activatable antibody or activatable antigen-binding antibody fragment comprises an antibody or antigen-binding antibody fragment (e.g., an antibody or antigen-binding antibody fragment described herein) that specifically binds to a ligand on a target cell (or "target") coupled to a Masking Moiety (MM) such that coupling of the MM reduces the ability of the antibody or antigen-binding antibody fragment to bind to the target. In some embodiments, the MM is coupled via a sequence that includes a substrate for a protease, e.g., a protease active in diseased tissue and/or a protease that is co-localized with the target at a treatment site in a subject. The activatable antibody is preferably stable in circulation, activated at the intended site of therapy and/or diagnosis but not in normal (e.g., healthy) tissue or other tissue not targeted for therapy and/or diagnosis, and when activated, exhibits binding to the target that is at least comparable to the corresponding, unmodified antibody. In some embodiments, the AA are those described in WO 2009/025846, WO 2010/081173, WO 2015/048329, WO 2015/116933, and WO 2016/118629, each of which is incorporated by reference in its entirety.

In some embodiments, the activatable antibody or antibody fragment comprises:

(a) a Cleavable Moiety (CM) coupled to the antibody or antibody fragment (collectively "AB"), wherein the CM is a polypeptide that serves as a protease substrate; and

(b) a Masking Moiety (MM) coupled to the antibody or antibody fragment, wherein the MM inhibits binding of the antibody or antibody fragment to a ligand when the activatable antibody is in an uncleaved state,

wherein the activatable antibody in an uncleaved state has the following structural arrangement from N-terminus to C-terminus: (MM) - (CM) - (AB) or (AB) - (CM) - (MM).

In some embodiments, the masking moiety (or "mask") is an amino acid sequence that is coupled or otherwise linked to the antibody and is positioned within an activatable antibody construct such that the masking moiety reduces the ability of the antibody to specifically bind the target. Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in WO 2009/025846, the contents of which are incorporated herein by reference in their entirety.

K targeting with MM-modified AB_dCan be directed against the K of the target compared to AB or parent AB not modified with MM _dAt least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more times as large as or larger than 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-10,000, 1,000-10,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000, 10,000-100-000-100-000-one-100,000, 10,000-one-100,000-one-100-000-one-100,000-one-100-000-one-100,000, 10,000-one-000, 10,000-one-100-000, 10,000-one-100-000, 10,000, or more-one-100-one-100-one-100-one, 10,000-100-one-100-one, 10,000, or more-one, or more-one, or more times as one-one. In contrast, the binding affinity of an AB modified with MM to a target can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more times, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000, 100,000-1,000,000 or 100,000-10,000,000 times.

Dissociation constant (K) of MM for AB_d) K against target generally greater than AB_d. K of MM for AB_dK against target comparable to AB_dAt least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater. In contrast, the binding affinity of MM for AB is generally lower than the binding affinity of AB for the target. The binding affinity of the MM to the AB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 fold lower than the binding affinity of the AB to the target.

When the AB is modified with MM and in the presence of the target, specific binding of the AB to its target can be reduced or inhibited, as compared to specific binding of the AB to the target without the modification with MM or specific binding of the parent AB to the target. The ability of an AB to bind a target when modified with MM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more when compared to the binding of an AB to a target or the binding of a parent AB to a target when not modified with MM, as measured in vivo or in a target displacement in vitro immunoadsorbent assay as described in WO 2010/081173.

MM may inhibit binding of AB to the target. MM can bind to the antigen binding domain of AB and inhibit binding of AB to its target. MM can sterically inhibit AB binding to the target. MM can inhibit AB binding to its target. In these embodiments, when the AB is modified or coupled to MM with MM and in the presence of the target, the AB does not bind or does not substantially bind to the target, or there is only.001%,. 01%,. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, when measured in vivo or in a target displacement in vitro immunoadsorbent assay as described in WO 2010/081173, 10. 11, 12 months or longer.

When AB is coupled to or modified by MM, the MM may 'mask' or reduce or inhibit specific binding of AB to its target. When an AB is coupled to or modified by MM, the coupling or modification may effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target.

An AB coupled to or modified with MM can be represented by the following formulae (in order from the amino (N) terminal region to the carboxy (C) terminal region):

(MM)-(AB)

(AB)-(MM)

(MM)-L-(AB)

(AB)-L-(MM)

wherein MM is a masking moiety, Ab is an antibody or target-binding antigen fragment, and L is a linker. In various embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the composition in order to provide flexibility.

In certain embodiments, the MM is not a natural binding partner of the Ab. In some embodiments, the MM is free or substantially free of homology to any natural binding partner of the Ab. In some embodiments, the MM is only 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the Ab. In some embodiments, the MM is only 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 25% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 50% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 20% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 10% identical to any natural binding partner of the Ab.

Activatable antibodies provided herein can further comprise a Cleavable Moiety (CM). Such AA exhibit activatable/switchable binding to the target of AB. An AA generally includes an antibody or antibody fragment (AB) modified or conjugated to a Masking Moiety (MM) and a modifiable or Cleavable Moiety (CM) by the Masking Moiety (MM) and the modifiable or Cleavable Moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for a protease of interest. In other embodiments, the CM provides a cysteine-cysteine disulfide bond that is cleavable by reduction. In other embodiments, the CM provides a photolytic substrate that can be activated by photolysis.

In some embodiments, the cleavable moiety (or "substrate") comprises an amino acid sequence that is a substrate for a protease, typically an extracellular protease. Suitable substrates are identified using any of a variety of known techniques. For example, the use of peptide substrates is described in U.S. patent nos. 7,666,817 and 8,563,269; and the method of WO 2014/026136, the contents of each of which are incorporated herein by reference in their entirety. (see also Boulware et al, Biotechnol Bioeng.106(3):339-346 (2010)). Optionally, the CM comprises a cysteine-cysteine pair capable of forming a disulfide bond, which disulfide bond can be cleaved by the action of a reducing agent. The CM is positioned such that when the CM is cleaved by a cleavage agent in the presence of the target (e.g., a protease substrate of the CM is cleaved by a protease and/or a cysteine-cysteine disulfide bond is disrupted via reduction by exposure to a reducing agent), resulting in a cleaved state, the Ab binds to the target, and in an uncleaved state, in the presence of the target, binding of the Ab to the target is inhibited by the MM. It is noted that the amino acid sequence of CM may overlap or be included within MM such that all or a portion of CM facilitates "masking" of Ab when the activatable antibody is in an uncleaved conformation.

In some embodiments, CM may be selected based on proteases that co-localize with the desired target of the activatable antibody in the tissue. A number of different conditions are known in which a target of interest is co-localized with a protease whose substrate is known in the art. For example, the target tissue may be cancerous tissue, in particular cancerous tissue of a solid tumor. Proteases with known substrates have been reported in the literature many times at increased levels in various cancers (e.g. solid tumors). See, e.g., La Rocca et al, (2004) British J.of Cancer 90(7) 1414-.

Exemplary substrates may include, but are not limited to, substrates cleavable by one or more of the following enzymes: MMP-I, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAMLO, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14 and TACE.

In some embodiments, the CM is a polypeptide up to 15 amino acids long.

In some embodiments, the CM is a polypeptide comprising a first cleavable moiety (CM1) that is a substrate for at least one Matrix Metalloproteinase (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one Serine Protease (SP). In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide of up to 15 amino acids in length.

In some embodiments, the CM is a substrate of legumain, plasmin, TMPRSS-3/4, MMP-9, MTl-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA.

K against target with MM and CM modified Ab_dCan be directed against the K of the target than an Ab or parent Ab not modified with MM and CM_dAt least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more times as large as or larger than 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-10,000, 1,000-10,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000, 10,000-100-000-100-000-one-100,000, 10,000-one-100,000-one-100-000-one-100,000-one-100-000-one-100,000, 10,000-one-000, 10,000-one-100-000, 10,000-one-100-000, 10,000, or more-one-100-one-100-one-100-one, 10,000-100-one-100-one, 10,000, or more-one, or more-one, or more times as one-one. In contrast, the binding affinity of an Ab modified with MM and CM for a target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000-fold or more lower than the binding affinity of an Ab or parent Ab not modified with MM and CM for a target, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000, 100-containing 1,000, 100-containing 10,000, 100-containing 100,000, 100-containing 1,000,000, 100-containing 10,000, 1,000-containing 10,000, 10,000-containing 100-containing 10,000, 10,000-containing 100-containing 100,000, 10,000-containing 10,000, 10,000-containing 100-containing 10,000, 10,000-containing-100,000, 10,000-containing-100-containing-100,000, 10,000-containing 10,000, 10-containing 10,000, 10,000-containing-10,000, 10-containing-10,000, 10-containing-10,000, 10,000-containing-10,000, 10-containing-10,000, 10,000-containing-10,000, 10-containing-10,000, 10-containing-.

When an Ab is modified with MM and CM and in the presence of the target but not in the presence of a modifying agent (e.g., an enzyme, protease, reducing agent, light), specific binding of the Ab to its target can be reduced or inhibited, as compared to specific binding of the Ab, or a parent Ab, to the target that is not modified with MM and CM. The ability of an Ab to bind a target when modified with MM and CM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer when compared to the binding of the parent Ab to its target or the Ab that is not modified with MM and CM, when measured in vivo or in a target displacement in vitro immunoadsorbent assay as described in WO201008117 (incorporated herein by reference in its entirety).

As used herein, the term cleavage state refers to the state of AA following protease modification of CM and/or reduction and/or photoactivation of the cysteine-cysteine disulfide bond of CM. As used herein, the term uncleaved state refers to the state of AA in the absence of protease cleavage of CM and/or in the absence of reduction of the cysteine-cysteine disulfide bond of CM and/or in the absence of light. As discussed above, the term AA is used herein to refer to AA in its uncleaved (native) state as well as in its cleaved state. It should be apparent to the ordinarily skilled artisan that in some embodiments, the cleaved AA may be deficient in MM due to protease cleavage of CM resulting in release of at least MM (e.g., where MM is not attached to the AA by covalent bonds such as disulfide bonds between cysteine residues).

Activatable or convertible refers to an AA exhibiting a first level of binding to a target when in an inhibited, masked, or uncleaved state (i.e., a first conformation) and a second level of binding to the target in an uninhibited, unmasked, and/or cleaved state (i.e., a second conformation), wherein the second level of target binding is greater than the first level of binding. In general, the proximity of the Ab of the target to AA is greater in the presence of a cleavage agent capable of cleaving CM than in the absence of such a cleavage agent. Thus, when AA is in an uncleaved state, the Ab is inhibited from target binding and may be masked from target binding (i.e., the first conformation renders the Ab unable to bind to the target), and in a cleaved state, the Ab is uninhibited or unmasked with respect to target binding.

The CM and AB of the AA can be selected such that AB represents a binding moiety for the target of interest and CM represents a substrate for a protease that is co-localized with the target at a treatment site in the subject. Alternatively or additionally, the CM is a cysteine-cysteine disulfide bond, which may be cleaved as a result of reduction of this disulfide bond. The AA contains at least one of a protease cleavable CM or a cysteine-cysteine disulfide bond, and in some embodiments includes two species of CM. The AA may alternatively or further comprise a photolabile substrate which can be activated by a light source. The AA disclosed herein finds particular use where proteases capable of cleaving sites in the CM are present at relatively higher levels in target-containing tissue (e.g., diseased tissue; e.g., for therapeutic or diagnostic treatment) at a treatment site, e.g., as compared to tissue at a non-treatment site (e.g., in healthy tissue). The AA disclosed herein also find particular use where a reducing agent capable of reducing a site in the CM is present at relatively higher levels in target-containing tissue at a treatment or diagnostic site, e.g., as compared to tissue at a non-treatment non-diagnostic site. The AA disclosed herein also finds particular use where, for example, a light source capable of photolyzing a site in the CM is introduced (e.g., with the aid of a laser) to target-containing tissue at a treatment or diagnostic site.

In some embodiments, the AA can provide reduced toxicity and/or adverse side effects that may otherwise result from Ab binding at the non-treatment site if the Ab is not masked or otherwise inhibited from binding to its target. Where an AA contains CM that can be cleaved by a reducing agent that promotes disulfide bond reduction, the AB of such AA can be selected to study AB activation in the presence of the target of interest at the desired treatment site, characterized by elevated reducing agent levels, such that the environment has a higher reducing potential than, for example, the environment of the non-treatment site.

In general, AA can be designed by selecting the Ab of interest and constructing the remainder of the AA such that, when conformationally constrained, MM provides masking of the Ab or reduction of the Ab's binding to its target. Structural design criteria should be considered to provide such functional characteristics.

AA of a switchable phenotype exhibiting a desired dynamic range for target binding in the inhibited conformation, as opposed to the uninhibited conformation. Dynamic range generally refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection value of the parameter under a second set of conditions. For example, in the case of AA, dynamic range refers to the ratio of (a) the maximum detection level of a target protein that binds to AA in the presence of a protease capable of cleaving CM of AA to (b) the minimum detection level of a target protein that binds to AA in the absence of the protease. The dynamic range of AA can be calculated as the ratio of the equilibrium dissociation constant of an AA cleaving agent (e.g., enzyme) treatment to the equilibrium dissociation constant of the AA cleaving agent treatment. The greater the dynamic range of AA, the better the convertible phenotype of AA. An AA having a relatively high dynamic range value (e.g., greater than 1) exhibits a more desirable switching phenotype such that target protein binding of the AA occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving CM of the AA than in the absence of the cleavage agent.

AA can be provided in a variety of structural configurations. Exemplary formulas for AA are provided below. It is specifically contemplated that the N-terminal to C-terminal order of AB, MM, and CM may be reversed within AA. It is also specifically contemplated that the amino acid sequences of CM and MM may overlap, e.g., such that the CM is contained within the MM.

For example, AA can be represented by the following formula (in order from the amino (N) -terminal region to the carboxyl (C) -terminal region):

(MM)-(CM)-(AB)

(AB) - (CM) - (MM), wherein MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. It should be noted that although MM and CM are indicated as distinct components in the above formula, in all exemplary embodiments (including the various formulae) disclosed herein, it is contemplated that the amino acid sequences of MM and CM may overlap, e.g., such that CM is completely or partially contained within MM. In addition, the above formulae provide additional amino acid sequences that can be positioned N-terminal or C-terminal to the AA element.

In certain embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the AA construct in order to provide flexibility at one or more of the MM-CM junction, CM-AB junction, or both. For example, the AB, MM, and/or CM may not contain a sufficient number of residues (e.g., GIy, Ser, Asp, Asn, particularly GIy and Ser, particularly GIy) to provide the desired flexibility. Thus, the switchable phenotype of such AA constructs may benefit from the introduction of one or more amino acids in order to provide a flexible linker. In addition, as described below, where AA is provided as a conformationally constrained construct, a flexible linker is operably inserted to facilitate the formation and maintenance of a cyclic structure in the uncleaved AA.

For example, in certain embodiments, AA comprises one of the following formulae (wherein the following formula represents an amino acid sequence in the N-terminal to C-terminal direction or C-terminal to N-terminal direction):

(MM)-L₁-(CM)-(AB)

(MM)-(CM)-L₁-(AB)

(MM)-L₁-(CM)-L₂-(AB)

ring [ L ]₁-(MM)-L₂-(CM)-L₃-(AB)]

Wherein MM, CM and AB are as defined above; wherein L is₁、L₂And L₃Each independently and optionally present or absent, is the same or different flexible linker comprising at least 1 flexible amino acid (e.g., Gly); and wherein in the presence of a loop, AA is in the form of a cyclic structure due to the presence of a disulfide bond between a pair of cysteines in AA. In addition, the above formulae provide additional amino acid sequences that can be positioned N-terminal or C-terminal to the AA element. It is understood that in the formula ring [ L ]₁-(MM)-L₂-(CM)-L₃-(AB)]The cysteines responsible for the disulfide bonds may be positioned in the AA to allow for one or two tails, thereby creating a lasso or ω structure when the AA is in a disulfide-bonded structure (and thus conformationally constrained state). The amino acid sequence of the tail may provide additional AA features, such as binding to a target receptor to facilitate the localization of AA, thereby increasing the serum half-life of AA, and the like. A target moiety (e.g., a ligand for a receptor for a cell present in the target tissue) and a serum half-life extending moiety (e.g., a polypeptide that binds a serum protein, such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., Human Serum Albumin (HSA))).

Linkers suitable for use with the AA described herein are generally linkers that provide the flexibility of the modified AB or AA to facilitate inhibition of binding of the AB to a target. Such joints are commonly referred to as flexible joints. Suitable linkers can be readily selected and can be of any suitable different length, for example 1 amino acid (e.g., GIy) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (including, for example, (GS)_n、(GSGGS)_nAnd (GGGS)_nWhere n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral tethers between components. Glycine approaches even significantly more phi-psi space than alanine and is less restricted than residues with longer side chains (see Scheraga, rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly-Gly, GIy-Ser-Gly-Ser-Gly, Gly-Ser-Gly-Gly-Gly, Gly-Gly-Gly-Ser-Gly, Gly-Ser-Ser-Gly, and the like. One of ordinary skill will recognize that the design of an AA may include joints that are flexible in whole or in part, such that the joints may include flexible joints and one or more portions that impart less flexible structure to provide the desired AA structure.

In addition to the elements described above, the modified AB and AA can contain additional elements, such as the N-terminus or C-terminus of the amino acid sequence of AA. For example, an AA may include a targeting moiety to facilitate delivery to a target cell or tissue. In addition, AA may be provided in the case of a scaffold protein to facilitate display of AA on the cell surface.

In some embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is bound to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly conjugated to the MM of the activatable antibody. In some embodiments, the spacer is directly conjugated to the MM of the activatable antibody in a structural arrangement having a spacer-MM-CM-AB from the N-terminus to the C-terminus.

Suitable spacers and spacer techniques are known in the art and can be routinely used to incorporate spacers in some embodiments of the provided activatable antibodies. See, for example, WO 2016/179285 (e.g., on pages 52-53), the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the activatable antibody is exposed to and cleaved by a protease such that, in an activated or cleaved state, the activatable antibody comprises a light chain sequence comprising at least a portion of LP2 and/or CM sequence after the protease has cleaved the CM.

The CM is composed of at least one protease in the range of about 0.001-1500X 10⁴M^-1S^-1Or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10⁴M^-1S^-1Is specifically cleaved. In some embodiments, the CM is at about 100,000M^-1S^-1Is specifically cleaved. In some embodiments, the CM is at about 1 x 10²To about 1X 10⁶M^-1S^-1(i.e., about 1X 10)²To about 1X 10⁶M^-1S^-1) Is specifically cleaved. With respect to specific cleavage by an enzyme, contact is achieved between the enzyme and the CM. An activatable antibody comprising an Ab (e.g., an antibody or EpCAM-binding antibody fragment) coupled to MM and CM can be cleaved when the antibody is in the presence of the target and sufficient enzymatic activity. Sufficient enzymatic activity may refer to the ability of the enzyme to contact the CM and effect cleavage. It is readily envisioned that an enzyme may be in the vicinity of the CM, but not cleaved due to other cytokine or protein modifications of the enzyme.

Production of cell-binding agent-drug conjugates

To link any of the cytotoxic compounds described herein, or derivatives thereof, to a cell-binding agent, the cytotoxic compound may comprise a linking moiety having a reactive group bonded thereto. These compounds may be linked directly to the cell binding agent. Representative processes for linking a cytotoxic compound having a reactive group bonded thereto with a cell-binding agent to produce a cell-binding agent-cytotoxic agent conjugate are described in the examples.

In some embodiments, the bifunctional crosslinking agent may first be reacted with the cytotoxic compound to provide a compound with a linking moiety having one reactive group bonded thereto (i.e., a drug-linker compound), which may then be reacted with the cell-binding agent. Alternatively, one end of the bifunctional crosslinking reagent may first be reacted with a cell-binding agent to provide a cell-binding agent with a linking moiety having one reactive group bonded thereto, which may then be reacted with a cytotoxic compound. The linking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds (see, e.g., U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Disulfide, thioether and peptidase labile bonds are preferred. Other linkers useful in the present invention include non-cleavable linkers, such as those described in detail in U.S. publication No. 2005/0169933, or charged or hydrophilic linkers described in US 2009/0274713, US 2010/01293140, and WO 2009/134976, each of which is expressly incorporated herein by reference.

In some embodiments, a solution of a cell-binding agent (e.g., an antibody) in an aqueous buffer can be incubated with a molar excess of a bifunctional crosslinking agent such as N-succinimidyl-4- (2-pyridyldithio) valerate (SPP), N-succinimidyl-4- (2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4- (2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) to introduce dithiopyridyl groups. The modified cell-binding agent (e.g., modified antibody) is then reacted with a thiol-containing cytotoxic compound described herein to produce the disulfide-linked cell-binding agent-cytotoxic agent conjugates of the invention.

In another embodiment, the thiol-containing cytotoxic compounds described herein can be reacted with a bifunctional crosslinking agent such as N-succinimidyl-4- (2-pyridyldithio) valerate (SPP), N-succinimidyl-4- (2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4- (2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) to form a cytotoxic agent-linker compound, which can then be reacted with a cell-binding agent to produce the disulfide-linked cell-binding agent-cytotoxic agent conjugates of the present invention. The cytotoxic agent-linker compound can be prepared in situ without purification, followed by reaction with a cell-binding agent. Alternatively, the cytotoxic agent-linker compound may be purified prior to reaction with the cell-binding agent.

The cell-binding agent-cytotoxic agent conjugate can be purified using any purification method known in the art, such as those described in U.S. patent No. 7,811,572 and U.S. publication No. 2006/0182750, both incorporated herein by reference. For example, the cell-binding agent-cytotoxic agent conjugate can be purified using tangential flow filtration, adsorption chromatography, adsorptive filtration, selective precipitation, non-absorptive filtration, or a combination thereof. Preferably, tangential flow filtration (TFF, also known as cross-flow filtration, ultrafiltration and diafiltration) and/or adsorption chromatography resins are used for the purification of the conjugate.

The number of cytotoxic molecules bound per antibody molecule can be determined spectrophotometrically by measuring the ratio of absorbance at 280nm and 330 nm. In some embodiments, an average of 1-10 cytotoxic compounds per antibody molecule may be attached by the methods described above. In some embodiments, the average number of cytotoxic compounds attached per antibody molecule (DAR) is 2-12. In some embodiments, the DAR value is 2-10. In some embodiments, the DAR value is 2-8. In some embodiments, the DAR value is 2.5-4.0. In some embodiments, the DAR value is 4-8. In some embodiments, the DAR value is 5-8. In some embodiments, the DAR value is 6-8. In some embodiments, the DAR value is 6.5 to 8. In some embodiments, the DAR value is 7-8. In some embodiments, the DAR value is 7.1 to 8. In some embodiments, the DAR value is 7.2-8. In some embodiments, the DAR value is 7.3-8. In some embodiments, the DAR value is 7.4-8. In some embodiments, the DAR value is 7.5-8. In some embodiments, the DAR value is 7.6-8. In some embodiments, the DAR value is 7.7-8. In some embodiments, the DAR value is 7.8-8. In some embodiments, the DAR value is 7.9-8. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a conjugate of the invention has a DAR value of between 2 and 12, between 2 and 10, more specifically between 6 and 8. In some embodiments, the average number of cytotoxic compounds attached per antibody molecule (DAR) is 7-8, and more specifically 8.

In some embodiments, when the antibody is linked to a cytotoxic agent via a cysteine thiol group, the conjugate has 1 cytotoxic compound per antibody molecule. In some embodiments, the conjugate has 1 or 2 cytotoxic compounds per antibody molecule. In some embodiments, the conjugate has 2 cytotoxic compounds per antibody molecule. In some embodiments, the average number of cytotoxic compounds attached per antibody molecule (DAR) is 1.5-2.5, more specifically 1.8-2.2. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a conjugate of the invention has a DAR value of between 1.0 and 2.5, between 1.5 and 2.5, more specifically between 1.8 and 2.2, or between 1.9 and 2.1.

Representative processes for preparing the cell-binding agent-drug conjugates of the invention are described in 8,765,740 and U.S. application publication No. 2012/0238731. The full teachings of these references are incorporated herein by reference.

Composition comprising a metal oxide and a metal oxide

The present invention includes compositions (e.g., pharmaceutical compositions) comprising any of the compounds described herein, derivatives thereof, or conjugates thereof (and/or solvates, hydrates, and/or salts thereof) and a carrier (pharmaceutically acceptable carrier).

The pharmaceutical compositions described herein may be administered in a variety of ways for local or systemic treatment. Administration can be to surfaces (e.g., to mucous membranes, including vaginal and rectal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; lungs (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); orally taking; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some particular embodiments, the administering is intravenous. The pharmaceutical compositions described herein may also be used in vitro or ex vivo.

Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and will be determined by the skilled person in the clinical setting. Examples of suitable carriers, diluents and/or excipients include: (1) dulbecco phosphate buffered saline, about pH 7.4, with or without about 1mg/mL to 25mg/mL human serum albumin, (2) 0.9% physiological saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

Application method

The compositions of the present invention are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human).

The present invention includes a method of inhibiting abnormal cell growth or treating a proliferative disorder, an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis or a kidney disease in a mammal (e.g., a human) comprising administering to the mammal a therapeutically effective amount of a cytotoxic compound, derivative thereof, or conjugate thereof (and/or solvates and salts thereof), or a composition thereof, as described herein.

In certain embodiments, the proliferative disorder of a mammal is cancer, including a hematological cancer, leukemia, or lymphoma. In certain embodiments, the proliferative disorder is a cancer of the lymphoid organ, or a hematologic malignancy. In some embodiments, the cancer is lymphoma or leukemia.

For example, the cancer may be selected from the group consisting of: acute myeloid leukemia (AML, including CD33 low AML, P-glycoprotein positive AML, relapsed AML or refractory AML), Chronic Myeloid Leukemia (CML) (including blast crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation)), myelodysplastic syndrome (MDS), Acute Lymphoblastic Leukemia (ALL) (including but not limited to acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL)), Chronic Lymphocytic Leukemia (CLL) (including Richter syndrome or Richter CLL transformation), Hairy Cell Leukemia (HCL), Acute Promyelocytic Leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia (preferably with marker CD11c expression), diffuse large B-cell lymphoma (DLBCL), Blast Plasmacytoid Dendritic Cell Neoplasm (BPDCN), non-hodgkin's lymphoma (NHL) (including Mantle Cell Leukemia (MCL) and Small Lymphocytic Lymphoma (SLL), hodgkin's lymphoma, systemic mastocytosis, and burkitt's lymphoma).

In some embodiments, the cancer is endometrial, lung, colorectal, bladder, gastric, pancreatic, renal cell, prostate, esophageal, breast (e.g., Triple Negative Breast (TNBC)), head and neck, uterine, ovarian, liver, cervical, thyroid, testicular, bone marrow, melanoma, and lymphatic cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. In other embodiments, the compounds of the invention may be useful for the treatment of non-small cell lung cancer (squamous cell, non-squamous cell, adenocarcinoma or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma or squamous cell carcinoma), or breast cancer (e.g., Triple Negative Breast Cancer (TNBC)).

The invention thus generally described will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

Examples

The following abbreviations are used for the following terms:

DAR drug to antibody ratio;

DIPEA diisopropylethylamine;

DMF dimethyl formamide;

DMSO dimethyl sulfoxide;

DMTMM chloride 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholine;

EDTA ethylene diamine tetraacetic acid;

EPPS 3- [4- (2-hydroxyethyl) -1-piperazinyl ] propanesulfonic acid;

fmoc 9-fluorenylmethoxycarbonyl;

HMPA hexamethylphosphoramide;

i.v. intravenously;

NMM N-methylmorpholine;

PPTS pyridine p-toluenesulfonate;

SEC size exclusion chromatography;

standard error of SEM mean;

TCEP 3,3',3 "-phosphinotripropionate;

TEA triethylamine; and

TFA trifluoroacetic acid.

Figures 1 and 2 depict the synthesis of camptothecin building blocks. Fig. 3, 4 and 5 depict the synthesis of side chains. Fig. 6 and 7 depict the coupling of camptothecin building blocks to side chains. Figure 8 depicts the synthesis of additional camptothecin metabolites. Figure 9 depicts the coupling of camptothecin building blocks to side chains. Figure 10 depicts the synthesis of additional camptothecin compounds.

Figure 11 depicts compounds for comparison including a generic Ab-999 structure of an ADC with an ADC moiety linked via a reducing interchain disulfide of an antibody. Compounds 998 and 999 were prepared as described in us patent 2016/0297890a1 and references therein.

EXAMPLE 1 Synthesis of Compounds 2a-2e

2 a: to a flask containing anhydrous 1, 2-dichloroethane (80mL) was added dichloromethane (16mL, 16mmol) containing 1M boron trichloride, which was then cooled to 0 ℃ with an ice water bath. 1a (2.5g, 20mmol) was added in portions, followed by stirring at 0 ℃ for 10min, followed by chloroacetonitrile (2.7mL, 23.5mmol), followed by aluminum chloride (3.5g, 26 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10min, the reaction mixture was heated at reflux for 39 h. The reaction solution was cooled to room temperature and cold water (40mL) was slowly added, followed by addition of 5% aqueous HCl. After 30min, it was diluted with dichloromethane (80 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was passed through reverse phase HPLC (200g C18 column, 80mL/min, CH)₃CN/H₂O，25％CH₃CN lasted for 5min and then reached 95% CH within 22min₃CN, then 95% CH₃CN for 5min) to give compound 2a as an off-white solid (1.52g, 38% yield). Ms (esi): (M + H)⁺Calculated 202.0 and experimental 202.2.

2 b: mixing AlCl₃(1.0g, 7.7mmol) and toluene (3.4mL) were added to a 100mL flask equipped with magnetic stirring. A solution of 1b (1.0g, 6.4mmol) in toluene (3.85mL) was added to the flask under a nitrogen atmosphere, followed by the addition of 1M BCl ₃Toluene (3.85mL, 7.6 mmol). 2-Chloroacetonitrile (1.7mL, 26.6mmol) was added dropwise over approximately 2min, then the flask was equipped with a reflux condenser and heated in an oil bath at 114 ℃ for 3 h. Remove the heating bath and place the thermometer 4min laterIn the flask. After cooling to 50 ℃, the reaction mixture was poured into deionized water (50mL) and then extracted with ethyl acetate (2 × 60 mL). Separating the organic layer over anhydrous Na₂SO₄Dried and then the solvent removed by rotary evaporation under vacuum. The residue was dissolved in a minimum volume of ethyl acetate and then purified on an 80g silica column eluting with 92:8 hexanes: ethyl acetate. Fractions containing pure desired product were combined and the solvent was removed by rotary evaporation under vacuum to give 0.69g of desired product 2b as a yellow solid (46% yield). MS (M + H) + 233.8;¹h NMR (400MHz, chloroform-d) δ 7.79(d, J ═ 7.9Hz,1H),6.40(d, J ═ 11.0Hz,1H),4.62(s,3H),2.39(s,3H),1.54(s, 4H).

2 e: to a solution of compound 2a (155mg, 0.77mmol) in anhydrous dichloromethane (1.5mL) was added benzylamine (0.68mL, 6.2mmol) and the reaction mixture was stirred at room temperature for 6 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (12g silica column, CH) ₂Cl₂MeOH, 0-10% MeOH over 16 min) to give 156mg 2e (74% yield). MS (ESI) M/z 273.4(M + H)⁺。

2 c: to a flask containing anhydrous 1, 2-dichloroethane (50mL) was added dichloromethane (9.95mL, 9.95mmol) containing 1M boron trichloride, followed by cooling to 0 ℃ with an ice water bath. 1a (1.56g, 12.4mmol) was added in portions, followed by stirring at 0 ℃ for 10 minutes, and 5-bromovaleronitrile (1.72mL, 14.9mmol) was added, followed by aluminum chloride (2.16g, 16.2 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was heated at reflux for 39.5 hours. The reaction solution was cooled to room temperature and cold water (25mL) was added slowly, followed by 5% HAqueous Cl solution. After 30 minutes, it was diluted with dichloromethane (50 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was passed through reverse phase HPLC (100g C18 column, CH)₃CN/H₂O，25％CH₃CN lasted for 5 minutes, then reached 95% CH in 15 minutes₃CN, then 95% CH₃CN for 5 min) to give compound 2c as an off-white solid (1.42g, 40% yield). MS (ESI) M/z 289.2(M + H)⁺。

2 d: to a dry flask containing anhydrous 1, 2-dichloroethane (40mL) was added boron trichloride (4.73mL, 4.73mmol, 1M in CH ₂Cl₂Medium) and cooled to 0 ℃ with an ice-water bath. After compound 1b (0.93g, 5.92mmol) was added in portions and stirred at 0 ℃ for 10 minutes, 5-bromovaleronitrile (0.819mL, 7.1mmol) was added, followed by aluminum chloride (1.025g, 7.69 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was heated at reflux for 45 hours. The reaction solution was cooled to room temperature and cold water (25mL) was added slowly, followed by 5mL of 5% aqueous HCl. After 30 minutes, it was diluted with dichloromethane (50 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was passed through reverse phase HPLC (100g C18 column, CH)₃CN/H₂O，25％CH₃CN lasted for 3 minutes, then reached 95% CH in 15 minutes₃CN, then 95% CH₃CN for 5 min) to afford compound 2d as an off-white solid (0.776g, 41% yield). MS (ESI) M/z 321.0(M + H)⁺。

Example 2 general procedure for Compounds 4a-4d

4 b: compound 3(777mg, 2.95mmol), 2b (690mg, 2.95mmol) and PPTS (37mg, 0.15mmol) were suspended in a 50mL flask equipped with a reflux condenser containing dry toluene (10 mL). The reaction was heated to reflux under an argon atmosphere with magnetic stirring overnight and then allowed to cool to room temperature. The mixture was filtered under vacuum and the solid washed with toluene (5mL) to give 1.08g of the desired product 4b as a brown solid (79% yield). MS (M + H) +461, MS (M-H) 459.

4 a: prepared by the general procedure, a solution of compound 2a (3.15g, 15.64mmol) and compound 3(3.92g, 14.89mmol) in toluene (200mL) containing PPTS (187mg, 0.75mmol) was refluxed for 40 hours to give 4a (4.74g), 74% yield. MS (ESI) M/z 429.2.431.0(M + H)⁺。¹H NMR(400MHz,DMSO-d6)δ8.37(d,J＝8.3,1.2Hz,1H),8.10-7.90(m,1H),7.33(d,J＝2.1Hz,1H),6.55(d,J＝4.8Hz,1H),5.85-5.20(m,6H),2.61-2.45(m,3H),1.98-1.76(m,2H),0.89(t,J＝7.3Hz,3H)。HRMS(M+H)⁺Calculated 429.1017, experimental 429.1023.

4 c: following the general procedure, a solution of compound 2c (1.695g, 5.88mmol) and compound 3(1.548g, 5.88mmol) in toluene (50mL) containing PPTS (0.074g, 0.294mmol) was refluxed for 21.5 hours. The solvent was evaporated and the residue was dissolved in a minimum volume of CH containing 10% methanol₂Cl₂Then chromatographed on silica gel (40g silica column, CH)₂Cl₂MeOH, 20min 0-10% MeOH) and chromatography on silica gel (40g silica column, CH)₂Cl₂MeOH, 0-10% MeOH over 20 min) to give 4c (2.56g), 84% yield. MS (ESI) M/z 515.2 and 517.1(M + H)⁺513.1 and 515.1(M-H)^-。¹H NMR(400MHz,DMSO-d6)δ8.39-8.17(m,1H),7.89(d,J＝10.9Hz,1H),7.31(s,1H),6.52(d,J＝6.2Hz,1H),5.44(s,2H),5.31(s,2H),3.65(t,J＝6.7Hz,2H),3.28-3.09(m,2H),2.51(t,J＝1.8Hz,3H),2.15-1.95(m,2H),1.95-1.71(m,4H),0.88(t,J＝7.4Hz,3H)。HRMS(M+H)⁺Calculated 515.0981, experimental 515.0992.

4 d: following the general procedure, a solution of compound 2d (776mg, 2.42mmol) and compound 3(670mg, 2.54mmol) in toluene (20mL) containing PPTS (30mg, 0.12mmol) was refluxed for 24 hours and chromatographed on silica gel (40g silica column, CH ₂Cl₂MeOH, 0-10% MeOH over 23 min) to give 4d (1.02g), 77% yield. MS (ESI) M/z 547.0 and 548.9(M + H)⁺544.8 and 546.8(M-H)^-。

4 e: to a solution of 6a (10mg, 0.024mmol) in DMF (0.5mL) was added NMM (3 μ L, 0.027mmol) and 6-bromohexanoic acid (7.2mg, 0.037mmol) and the solution was cooled to 0 ℃ with an ice bath and deionized water containing DMTMM (13.5mg, 0.049mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g column, CH)₂Cl₂MeOH, 0-10% MeOH over 15 min) to yield 15mg of 4e (98% yield). MS (ESI) M/z 586.1(M + H)⁺,588.0(M+H)⁺。

EXAMPLE 3 Synthesis of Compounds 8a-8e and 8p

8 a: a solution of compound 4c (860mg, 1,67mmol) in HMPA (5mL) and deionized water (0.9mL) was heated at 101 ℃ for 18 hours. The reaction mixture was cooled to room temperature and the solution was cooledThe solution was loaded onto a C18 cartridge and subjected to reverse phase HPLC (30g C18 column, CH)₃CN/H₂O，25％CH₃CN lasted for 3 minutes, then reached 95% CH in 15 minutes₃CN, then 95% CH₃CN for 5 minutes) to afford 8a as a solid, contaminated with impurities. It is further chromatographed on silica gel (CH)₂Cl₂MeOH, 0-20% MeOH) to give 392mg of 8a as an off-white solid (51% yield). MS (ESI) M/z 453.2(M + H) ⁺,451.1(M-H)^-。¹H NMR (400MHz, methanol-d)₄)δ8.00(dd,J＝8.2,1.1Hz,1H),7.52-7.33(m,2H),5.53(d,J＝16.1Hz,1H),5.33(d,J＝16.1Hz,1H),5.18-4.96(m,2H),3.66(q,J＝7.2,6.7Hz,2H),3.24-3.06(m,2H),2.45(dd,J＝20.1,2.7Hz,3H),1.94(tdd,J＝7.1,6.0,2.0Hz,2H),1.88-1.66(m,4H),1.00(td,J＝7.4,2.5Hz,3H)。¹³C NMR (101MHz, methanol-d)₄)δ173.32,163.68,161.19,157.56,151.44,151.09,148.68,148.56,146.06,144.46,128.10,127.89,127.22,125.66,125.60,124.26,118.92,111.91,111.69,97.96,72.74,65.28,60.97,49.40,32.15,30.80,29.00,25.96,14.09,14.06,6.75；HRMS(M+H)⁺Calculated 453.1826, experimental 453.1832.

8 b: prepared from 4d using the 8a procedure. Yield (48% yield). MS (ESI) M/z (M + H) + 485.7.

8 c: compound 4a (100mg, 0.23mmol) was dissolved in anhydrous DMF (2mL), to which was added 2-mercaptoethanol (0.2mL, 2.8mmol), followed by TEA (0.13mL, 0.94mmol) and magnetic stirring continued for 20 min. The reaction mixture was poured onto a 250g medium pressure C18 column, which was pre-equilibrated with 95:5 deionized water containing 0.2% formic acid, acetonitrile. The column was eluted with 95:5 deionized water containing 0.2% formic acid acetonitrile at 50mL/min for 5min, then 5% acetonitrile at 5min to 95% acetonitrile at 35minElution with a linear gradient. Fractions containing the desired product were combined, frozen and lyophilized to give 85mg 8c (78% yield). MS (ESI) M/z 471.4(M + H)⁺。¹H NMR (400MHz, DMSO-d6) δ 8.25(d, J ═ 8.2Hz,1H),7.78(d, J ═ 10.8Hz,1H),7.27(s,1H),6.52(s,1H),5.42(s,2H),5.22(s,2H),4.87(t, J ═ 5.4Hz,1H),4.47-4.28(m,2H),3.56(q, J ═ 6.3Hz,2H),3.33(s,2H),2.62(t, J ═ 6.6Hz,2H),2.48-2.44(m,3H),1.87 (heptad, J ═ 7.1, Hz,2H),0.88(t, J ═ 7.3Hz, 3H).¹³C NMR(101MHz,DMSO-d6)δ172.44,163.07,160.58,156.69,152.16,149.97,148.66,148.53,145.48,140.06,128.18,128.16,127.28,127.07,126.61,126.54,123.59,119.09,112.56,112.35,96.77,72.36,65.26,60.83,49.69,34.38,30.31,28.78,15.24,15.20,7.75。HRMS(M+H)⁺Calculated 471.1390, experimental 471.1393.

8 d: prepared by reaction of 1, 3-propanedithiol with 4a in analogy to 8c (60% yield). MS (ESI) M/z 501.7(M + H)⁺

8 e: prepared by reaction of 1, 2-ethanedithiol with 4a (47% yield) in analogy to 8 c. MS (ESI) (M + H)⁺Calculated 487.1, experimental 487.3.

8 p: compound 8e (28mg, 0.057mmol) was dissolved in anhydrous THF (1mL) and mineral oil (5mg, 2.1mmol) containing a 60% NaH emulsion was added thereto under magnetic stirring. After 2min, iodoethane (20 μ L, 0.32mmol) was added. After 20min, the solvent was evaporated under vacuum and the residue was dissolved in anhydrous DMF (1mL) and then purified by medium pressure C18 chromatography. Column 100g C18, 50mL/min, deionized water containing 0.2% formic acid, 5% acetonitrile from 0-5min, then a linear gradient from 5-95% from 5min-28 min. Fractions containing the desired product were combined, frozen and lyophilized to give 15mg of 8p as a yellow solid (54% yield).¹H NMR(400MHz,DMSO-d6)δ8.40-8.27(m,1H),7.89(d,J＝10.8Hz,1H),7.31(s,1H),6.52(s,1H),5.43(s,2H),5.39(s,1H),5.32(s,1H),4.57-4.39(m,2H),2.84-2.69(m,3H),2.44(m,1H),2.07(m,1H),2.02(s,3H),1.87(m,2H),1.23(s,1H),0.87(t,J＝7.3Hz,3H)。HRMS(M+H)⁺Calculated value 501.1318; experimental value 501.1322.

EXAMPLE 4 Synthesis of Compound 5a

5 a: a solution of 4a (2g, 4.66mmol) and sodium azide (0.455g, 7.0mmol) in 25mL of anhydrous DMSO was stirred at room temperature for 6h, followed by dilution with deionized water (200 mL). The product precipitated and was collected by vacuum filtration and then dried under vacuum to give 2.03g of 5a (100% yield). MS (ESI) (M + H) ⁺Calculated value 435.1, experimental value 436.2, MS (ESI): M-H^-Calculated 433.1 and experimental 434.2.

EXAMPLE 5 Synthesis of Compound 6a

6 a: to a solution of 5a (2.03g, 4.66mmol) in dry benzene (60mL) was added triethyl phosphite (1.94g, 11.66mmol) and the solution was flushed with argon and then heated at reflux for 4 hours. The reaction was cooled to room temperature and 3M methanolic HCl (30mL) was added and heated at reflux (80 ℃ bath) for 38 h. The reaction was cooled to room temperature and then filtered in vacuo to give 6a (1.016g) as an off-white solid. The filtrate was concentrated and the residue was chromatographed on silica gel (12g silica column, CH)₂Cl₂MeOH, 0-20% MeOH over 16 min) to give additional 6a (0.079 g). 1.09g of combination 6a (57% yield) was obtained.¹H NMR (400MHz, DMSO-d6) δ 8.62(s,3H),8.43(d, J ═ 8.0Hz,1H),7.99(d, J ═ 10.7Hz,1H),7.35(s,1H),5.59(s,2H),5.45(s,2H),4.70(d, J ═ 6.0Hz,2H),2.55(s,3H),1.88 (heptad, J ═ 7.1Hz,2H),0.88(t, J ═ 7.3Hz, 3H). MS (ESI) calculated m/z 410.2, experimentValue 410.4(M + H)⁺；408.1(M-H)^-。

EXAMPLE 6 Synthesis of Compound 7a

7 a: to a solution of 6a (7.5mg, 0.018mmol) in DMF (0.5mL) was added triethylamine (2.6. mu.L, 0.018mmol) and glycolic acid (1.5mg, 0.020 mmol). The solution was cooled to 0 ℃ with an ice bath and deionized water (0.1mL) containing DMTMM (10.1mg, 0.037mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g silica column, CH) ₂Cl₂MeOH, 0-20% MeOH over 15 min) to give the desired product 7a (8mg, 93% yield). MS (ESI) M/z 468.2(M + H)⁺,466.1(M-H)^-。¹H NMR(400MHz,DMSO-d6)δ0.88(t,J＝7.3Hz,3H),1.79-1.98(m,3H),2.55(s,0H),3.84(d,J＝5.6Hz,3H),4.85(d,J＝6.0Hz,2H),5.44(s,3H),5.51(s,2H),5.58(t,J＝5.7Hz,1H),6.53(s,1H),7.32(s,1H),7.90(d,J＝10.8Hz,1H),8.48(d,J＝1.2,8.3Hz,1H),8.76(t,J＝6.0Hz,1H)。¹³C NMR(101MHz,DMSO-d6)δ172.95,172.89,157.23,152.85,150.44,149.07,145.85,140.12,129.27,127.97,124.28,119.63,97.23,72.83,65.74,61.91,54.06,50.54,49.58,44.06,37.34,30.72,15.74,8.22。HRMS(M+H)⁺Calculated 468.1571, experimental 468.1593.

EXAMPLE 7 Synthesis of Compound 6c

6 c: a stirred solution of compound 2e (129mg, 0.47mmol), compound 3(125mg, 0.47mmol) and PPTS (131mg, 0.52mmol) in dry toluene (10mL) was placed briefly under vacuum and then heated at reflux for 20 hours. The reaction solution was cooled to room temperature and stripped and the residue chromatographed on silica gel (12g column, CH)₂Cl₂MeOH, 0-20% MeOH over 16 min) to yield196mg of impure product are present as a black solid. It was further subjected to reverse phase HPLC (30g C18 column, CH)₃CN/H₂O, 20% -60% CH in 15 min₃CN, then 95% CH₃CN for 5 min) to yield 6c (31mg, 13% yield). MS (ESI) M/z 500.4(M + H)⁺,498.3(M-H)^-。

EXAMPLE 8 Synthesis of Compound 7c

7 c: to a solution of 6c (8.5mg, 0.017mmol) in DMF (0.5mL) was added triethylamine (2.4. mu.L, 0.017mmol) and glycolic acid (1.9mg, 0.026 mmol). The solution was cooled to 0 ℃ with an ice bath and deionized water (0.1mL) containing DMTMM (9.4mg, 0.034mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35 ℃ bath) and the residue was passed through semi-preparative HPLC (C18 column, CH) ₃CN/H₂O, 25% -65% CH in 23 min₃CN) to yield 2.5mg 7c (26% yield). MS (ESI) M/z 558.5(M + H)⁺,556.3(M-H)^-。

Example 9 Synthesis of side chains

11: N-methyl-D-glucamine 9(3.94g, 20.19mmol) and Z-L-Glu-OBn 10(5g, 13.46mmol) were weighed into a 200mL flask, to which DMF (50mL) was added and magnetically stirred. DIPEA (2.351mL, 13.46mmol) was then added, followed by a suspension of DMTMM (5.22g, 18.85mmol) in DMF (30mL) and deionized water (10 mL). After 2h, one third of the reaction was loaded onto a 450g C18 cartridge, which was pre-equilibrated with 95:5 deionized water acetonitrile. The column was eluted with 95:5 deionized water acetonitrile at 100mL/min for 5min, followed by a linear gradient of 5% acetonitrile to 95% acetonitrile over 38 min. The chromatography was repeated two more times and fractions containing pure desired product were combined. By rotating under vacuumThe solvent was removed by evaporation. Methanol (100mL) was added and evaporated 2 times to give 5g of the desired product 11 as a viscous colorless oil (68% yield). MS (ESI) MS (M + H)⁺Calculated value 549.2, Experimental value 549.3, MS (ESI): M-H)^-Calculated 547.2, experimental 547.2.

12: compound 11(5g, 9.1mmol) was suspended in 95:5 methanol in a 250mL PARR shaker flask deionized water (100mL), to which was added 10% Pd-C (0.13g, 1.222mmol) and at 30PSI H ₂The downhydrogenation was continued for 45min, with periodic addition of H₂To reestablish pressure. The solution was vacuum filtered through a celite filter aid. The clear colorless filtrate was concentrated by rotary evaporation under vacuum and then left under vacuum overnight to give 2.8g of the desired product 12 as a white solid (94% yield).¹H NMR(400MHz,DMSO-d6)δ4.09-3.98(m,1H),3.84(dt,J＝6.1,3.1Hz,1H),3.79(d,J＝6.0Hz,1H),3.75(ddd,J＝7.5,4.2,2.3Hz,1H),3.72-3.59(m,3H),3.56-3.51(m,1H),3.48(dd,J＝15.0,3.6Hz,1H),3.13(s,2H),2.97(s,1H),2.78-2.49(m,2H),2.22-2.06(m,2H)。MS(ESI):MS(M-H)^-Calculated 323.1, experimental 323.4.

14 b: compound 13b (1.4, 4.5mmol) was added to a magnetically stirred solution of 12(1.4g, 4.3mmol) in DMF (10mL) and DIPEA (1.1mL, 6.3 mmol). After stirring for 15min, the reaction was poured onto a medium pressure 350g C18 column, which was pre-equilibrated with 98:2 deionized water containing 0.1% formic acid, acetonitrile. The column was eluted with 2% acetonitrile at 100mL/min for 5min, followed by a linear gradient of 2% acetonitrile at 5min to 60% acetonitrile at 35min, detected at 214 and 306 nm. Fractions containing pure product were combined, frozen and lyophilized to give 1.3g of the desired product 14b as a white solid (58% yield). MS (M + H) +518.5,¹H NMR(400MHz,DMSO-d6)δ8.03(dd,J＝15.3,7.7Hz,1H),6.99(s,3H),4.79(d,J＝65.9Hz,1H),4.33(d,J＝30.0Hz,1H),4.22-4.07(m,1H),3.75(p,J＝4.2Hz,1H),3.57(ddt,J＝11.4,6.7,3.6Hz,1H),3.52-3.39(m,2H),3.39-3.30(m,3H),3.30-3.19(m,1H),2.97(s,2H),2.80(s,2H),2.45-2.21(m,1H),2.08(h,J＝3.4Hz,2H),2.01-1.85(m,1H),1.76(qd,J＝8.5,3.9Hz,1H),1.59-1.39(m,3H),1.30-1.08(m,2H)。

14 a: compound 13a (1.4, 5.3mmol) was added to a magnetically stirred solution of 12(1.8g, 5.5mmol) in DMF (10mL) and DIPEA (1.1mL, mmol). After stirring for 15min, the reaction was poured onto a medium pressure 350g C18 column, which was pre-equilibrated with 98:2 deionized water containing 0.1% formic acid, acetonitrile. The column was eluted with 2% acetonitrile at 100mL/min for 5min, followed by a linear gradient of 2% acetonitrile at 5min to 60% acetonitrile at 35min, detected at 214 and 306 nm. Fractions containing pure product were combined, frozen and lyophilized to give 1.4g of 14a as a white solid (55% yield). ¹H NMR(400MHz,DMSO-d6)δ8.33(dd,J＝9.2,7.7Hz,1H),7.08(d,J＝1.1Hz,2H),4.24(qd,J＝8.8,4.8Hz,1H),3.86(dt,J＝8.2,4.0Hz,1H),3.76-3.63(m,4H),3.62-3.43(m,4H),3.41-3.29(m,1H),3.08(s,2H),2.91(s,2H),2.61(q,J＝1.8Hz,1H),2.40(dd,J＝10.0,6.4Hz,1H),2.02(tdd,J＝12.6,10.3,9.1,5.8Hz,1H),1.93-1.76(m,1H)。MS(ESI):MS(M+H)⁺Calculated 476.2, experimental 476.4.

Fmoc-protected peptides 15a, 15b, 15c and 15d were prepared by solid phase synthesis using standard procedures.

EXAMPLE 10 conversion of Fmoc-peptide-Gly-OH to Fmoc-peptide-NHCH₂Methods of OAc (Compounds 16a-16d)

16 a: compound 15a (2.5g, 4.9mmol) was dissolved in anhydrous DMF (40mL) and magnetically stirred in a 100mL flask, acetic acid was addedCopper (II) (0.334g, 1.84mmol), acetic acid (0.64mL, 11.1mmol), and lead tetraacetate (2.5g, 5.6 mmol). The flask was heated in a 60 ℃ oil bath for 15 min. The oil bath was removed and the reaction was allowed to cool to room temperature. Approximately 1/2 of the mixture was purified on a 350g medium pressure C18 column equilibrated with 90:10 deionized water containing 0.3% formic acid acetonitrile. The column was eluted with 10% acetonitrile at 100mL/min for 5min and then a linear gradient from 10% acetonitrile at 5min to 95% acetonitrile at 38 min. This procedure was repeated for additional 1/2 of the reaction mixture and the fractions containing the desired product 16a were combined, frozen and lyophilized to give 1.2g of a white semi-solid (62% yield).¹H NMR(400MHz,DMSO-d6)δ8.86(t,J＝6.9Hz,1H),7.97(dd,J＝16.1,7.4Hz,2H),7.89(dt,J＝7.6,0.9Hz,2H),7.72(t,J＝7.1Hz,2H),7.53(d,J＝7.5Hz,1H),7.42(td,J＝7.5,1.2Hz,2H),7.33(td,J＝7.5,1.2Hz,2H),5.13-5.01(m,2H),4.30-4.17(m,4H),4.06(t,J＝7.3Hz,1H),3.32(s,1H),1.99(s,3H),1.27-1.13(m,9H)。MS(ESI):MS(M+Na)⁺Calculated 547.2, experimental 547.5.

16b was prepared from Fmoc-Ala-Ala-Gly-OH 15b in 55% yield. MS (M + Na) + 476.7.

16c was prepared from Fmoc-Leu-Gln-Gly-OH 15c in 46% yield. MS (M + Na) + 575.6.

16D was prepared from Fmoc-Ala-D-Ala-Ala-Gly-OH 15D in 52% yield. MS (M + Na) + 547.3.

EXAMPLE 11 use of benzyl-2-Hydroxyacetate with Fmoc-peptide-NHCH₂Methods of OAc reaction (Compounds 17a-17d)

17 a: compound 16a (142mg, 0.27mmol) and benzyl-2-glycolate (226mg, 1.36mmol) were suspended in a solution of 20% TFA in dichloromethane (7mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in the minimum volume of DMF and then purified on a 200g C18 medium pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid acetonitrile. The column was then washed with 10% acetonitrile at 60mL/minThe stripping was continued for 5min, followed by a linear gradient from 10% acetonitrile at 5min to 95% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 102mg of 17a as a white solid (59% yield). MS (ESI) MS (M + Na)⁺Calculated 653.3, experimental 653.5.

17b was prepared from 16b in 61% yield. MS (M + Na) + 582.7.

17c was prepared from 16c in 52% yield. MS (M + H) + 659.5.

17d was prepared from 16d, 56% yield. MS (M + Na) + 653.4.

Example 12 Fmoc-peptide-NHCH₂OCH₂Conversion of COOBn to H-peptide-NHCH ₂OCH₂COOBn (Compounds 18a-18d)

18 a: compound 17a (100mg, 0.16mmol) was dissolved in DMF (4mL), to which was added morpholine (0.6mL, 6.9mmol) and magnetically stirred. After 1h, the reaction mixture was purified on a 200g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid acetonitrile. The column was then eluted with 5% acetonitrile at 60mL/min for 5min, followed by a linear gradient of 5% acetonitrile at 5min to 70% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 50mg of 18a as a white solid (76% yield). MS (ESI) MS (M + H)⁺Calculated 409.2 and experimental 409.6.

18b was prepared from 17b in 80% yield. MS (M + H) + 338.3.

18c was prepared from 17c in 61% yield. MS (M + H) + 437.6.

18d was prepared from 17d in 61% yield. MS (M + H) + 409.6.

Example 13H-peptide-NHCH₂OCH₂Conversion of COOBn to H-peptide-NHCH₂OCH₂COOH (Compounds 19a-19d)

19 a: in a 100mL PARR shaker flask, H-Ala-Ala-Ala-NHCH₂OCH₂COOBn 18a (50mg, 0.12mmol) was dissolved in 5:95 deionized water methanol (50mL), to which 10% palladium on carbon (0.1g) was added and the reaction hydrogenated in a PARR shaker at 30PSI H2 for 1H. The solution was filtered under vacuum through celite filter aid and the solvent was removed from the filtrate by rotary evaporation under vacuum to give 35mg of the desired product 19a as a viscous oil (91% yield). MS (ESI) MS (M + H) ⁺Calculated 319.2, experimental 319.3.

19b was prepared from 18b in 92% yield. MS (M + H) + 248.3.

19c was prepared from 18c in 83% yield. MS (M + H) + 347.4.

19d was prepared from 18d in 81% yield. MS (M + H) + 319.5.

Example 14 preparation of Mal- (CH)₂)₅-CO-peptide NHCH₂OCH₂Method for preparing COOH Compound (Compounds 20a-20d)

20 a: mixing Mal- (CH)₂)₅-COONHS 13b (46mg, 0.15mmol) was dissolved in anhydrous DMF (2mL), to which DIPEA (0.1mL, 0.31mmol) and H-Ala-Ala-Ala-NHCH were added₂OCH₂COOH 19a (30mg, 0.093 mmol). The reaction was magnetically stirred for 15min and then purified on a 50g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid, acetonitrile. The column was then eluted with 5% acetonitrile at 40mL/min for 5min, followed by a linear gradient of 5% acetonitrile at 5min to 90% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 30mg of white solid 20a (63% yield). MS (ESI) MS (M-H)^-Calculated 510.2 and experimental 510.1.¹H NMR(400MHz,DMSO-d6)δ12.16(s,1H),8.64-8.48(m,1H),7.97(dd,J＝7.3,3.2Hz,2H),7.89(d,J＝7.5Hz,1H),7.00(s,2H),4.3-4.7(m,4H),4.21(dt,J＝10.3,7.2Hz,4H),3.55(t,J＝6.4Hz,2H),3.37(t,J＝7.1Hz,2H),2.41(t,J＝6.4Hz,2H),2.08(t,J＝7.4Hz,2H),1.47(p,J＝7.2Hz,4H),1.27-1.10(m,6H)。

20b was prepared from 19b in 83% yield. MS (M + H) + 441.4.

20c was prepared from 19c in 83% yield. MS (M-H) -655.4.

20 d: to a solution of compound 18d (99.7mg, 0.24mmol) in methanol (3mL) was added Pd (10%/carbon, 26mg, 0.024mmol) and the reaction flask was purged with hydrogen. It was hydrogenated with hydrogen balloon at room temperature for 3 hours and then filtered. The filtrate was stripped to give compound 19d as a white solid (78mg, 100% yield). 41.9mg (0.13mmol) was taken out and dissolved in anhydrous DMF (0.5mL) and 6-maleimidocaproic acid N-hydroxysuccinimide ester 13b (40.6mg, 0.13mmol) was added. The obtained colorless clear solution was stirred at room temperature for 24 hours. It was stripped under reduced pressure and the residue was passed through reverse phase HPLC (C18 column with CH ₃CN/H₂O elution, 10-50% CH in 15 min₃CN, then 95% CH₃CN for 5 min) to give compound 20d (30.8mg, 45% yield). MS (ESI) M/z 512.4(M + H)⁺,510.4(M-H)^-。

EXAMPLE 15 Synthesis of Compounds 21a and 21b

21 a: compound 16a (300mg, 0.57mmol) and 6-mercaptohexanoic acid (254mg, 1.7mmol) were suspended in a solution of 20% TFA in dichloromethane (10mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in the minimum volume of DMF and then purified on a 200g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid acetonitrile. The column was then eluted with 10% acetonitrile at 60mL/min for 5min, followed by a linear gradient of 5% acetonitrile at 5min to 95% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 202mg of 21a as a white solid (58% yield). MS (M + Na) + 613.9.

21b was prepared analogously to 21a from 16d and 6-mercaptohexanoic acid in 60% yield. MS (M + H) + 613.7.

EXAMPLE 16 Synthesis of Compound 22a

22 a: compound 6a (20mg, 0.049mmol) and DMTMM (18mg, 0.065mmol) in 85:15DMF deionized water (0.8mL) was magnetically stirred as 20a (35mg, 0.068mmol) and TEA (0.04mL, 0.28mmol) were added sequentially. After 1h, the reaction mixture was loaded on a 50g medium pressure silica column equilibrated with dichloromethane and run with dichloromethane at 30mL/min using a linear gradient from 0% to 100% dichloromethane containing 20% methanol over 40 min. Fractions containing pure product were combined and the solvent was removed by rotary evaporation under vacuum to give 16mg of brown solid 22a (36% yield). MS (M + Na) + 925.6. ¹H NMR(400MHz,DMSO-d6)δ0.88(t,J＝7.3Hz,3H),1.11-1.23(m,15H),1.46(p,J＝7.3Hz,5H),1.79-1.95(m,2H),2.07(t,J＝7.4Hz,2H),3.04-3.16(m,2H),3.88(s,2H),4.18(dd,J＝7.1,11.0Hz,2H),4.50-4.65(m,2H),4.86(d,J＝5.9Hz,2H),5.44(s,2H),5.49(s,2H),6.53(s,1H),7.00(s,2H),7.32(s,1H),7.91(d,J＝10.0Hz,1H),7.97(d,J＝7.0Hz,2H),8.43(d,J＝8.3Hz,1H),8.65(t,J＝6.6Hz,1H),8.73(t,J＝5.9Hz,1H)。¹³C NMR(101MHz,DMSO-d6)δ180.20,173.96,172.96,172.89,172.56,172.36,171.54,170.03,163.99,157.27,156.21,152.90,150.48,145.85,139.87,134.92,129.28,128.06,124.24,119.65,97.28,72.84,70.18,67.29,65.74,50.53,48.81,48.68,48.60,46.26,41.01,37.44,35.26,30.71,28.24,26.23,25.09,18.33,18.24,18.18,15.77,15.74,9.12,8.21。HRMS(M+H)⁺Calculated 903.3688, experimental 903.3676.

EXAMPLE 17 Synthesis of Compound 22c

22 c: to a solution of 6a (8mg, 0.02mmol) in DMF (0.5mL) was added NMM (2.2. mu.L, 0.02mmol) and compound 20d (10mg, 0.02 mmol). The solution was cooled to 0 ℃ with an ice bath and deionized water (0.1mL) containing DMTMM (10.8mg, 0.04mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35 ℃ bath) and the residue was passed through reverse phase HPLC (30g C18 column, CH)₃CN/H₂O，25％CH₃CN lasted for 3 minutes, then reached 95% CH in 12 minutes₃CN, then 95% CH₃CN for 5 minutes). The product fractions were combined and lyophilized to give a white solid. It was further chromatographed on silica gel (4g silica column, CH)₂Cl₂MeOH, 0-20% MeOH over 15 min) to give the desired product 22c (9.8mg, 55% yield). MS (ESI) M/z 903.9(M + H)⁺,901.9(M-H)^-,947.9(M+HCOOH-H)^-。

EXAMPLE 18 Synthesis of Compound 22b

22 b: to a solution of 6c (10.7mg, 0.022mmol) in DMF (0.5mL) was added NMM (2.4. mu.L, 0.022mmol) and compound 20a (11mg, 0.022 mmol). The solution was cooled to 0 ℃ with an ice bath and deionized water (0.1mL) containing DMTMM (11.9mg, 0.043mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35 ℃ bath) and the residue was passed through reverse phase HPLC (30g C18 column, CH) ₃CN/H₂O，25％CH₃CN lasted for 3 minutes, then reached 95% CH in 12 minutes₃CN, then 95% CH₃CN for 5 min) to give the desired product 22b (4mg, 18% yield). MS (ESI) M/z 1015.9(M + Na)⁺,991.9(M-H)^-,1037.9(M+HCOOH-H)^-。

EXAMPLE 19 Synthesis of Compound 22d

22 d: prepared by reaction of 6c with 20d similarly to 22c (22% yield). MS (ESI) M/z1015.8(M + Na)⁺

EXAMPLE 20 Synthesis of Compound 22e

22 e: to a solution of compound 18c (15.2mg, 0.035mmol) in methanol (2mL) was added Pd (10%/carbon, 3.7mg, 0.0035mmol) and the reaction flask was purged with hydrogen. It was hydrogenated with hydrogen balloon at room temperature for 3 hours and then filtered. The filtrate was stripped to give compound 19c as a colorless foam. This was dissolved in anhydrous DMF (0.3mL) and 13d (15.9mg, 0.03mmol) was added. The obtained colorless clear solution was stirred at room temperature for 15 hours. It was diluted with DMF (0.2mL) followed by the addition of 6a (15.3mg, 0.037mmol) and NMM (4.1. mu.L, 0.037 mmol). Deionized water (0.1mL) containing DMTMM (20.7mg, 0.075mmol) was then added and the reaction solution was stirred at room temperature for 1.5 hours. The reaction mixture was stripped under reduced pressure and the residue was chromatographed on silica gel (CH)₂Cl₂MeOH, 0-20% MeOH) to give the desired 22e (10mg, 26% yield). MS (ESI) M/z 1049.1(M + H) ⁺,1093.2(M+HCOOH-H)^-。

EXAMPLE 21 Synthesis of Compound 23a

23 a: to a solution of 6a (32mg, 0.078mmol) in DMF (0.8mL) was added NMM (8.6. mu.L, 0.078mmol) and Fmoc protected L-Ala-L-Ala-L-Ala tripeptide linker 21a (53mg, 0.078mmol), followed by deionized water (0.16mL) containing DMTMM (43mg, 0.156 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g column, CH)₂Cl₂MeOH, 0-20% MeOH over 15 min) to give the desired compound 23a (78mg, 99% yield). MS (ESI) M/z 1004.5(M + H)⁺,1026.6(M+Na)⁺,1048.4(M+HCOOH-H)^-。

EXAMPLE 22 Synthesis of Compound 24a

24 a: to a solution of compound 23a (78mg, 0.078mmol) in anhydrous DMF (1mL) was added morpholine (0.24mL, 2.7mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g silica column, using CH)₂Cl₂MeOH elution, 0-20% MeOH over 9 min, then 20% MeOH for 11 min) to afford the desired compound 24a (39.6mg, 65% yield). MS (ESI) M/z 782.4(M + H)⁺,780.0(M-H)^-。

EXAMPLE 23 Synthesis of Compound 25a

25 a: to a solution of compound 24a (20mg, 0.026mmol) in anhydrous DMF (0.3mL) was added 13b (9.6mg, 0.031mmol) and the reaction mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with DMSO and passed through reverse phase semi-preparative HPLC (C18 column, using CH) ₃CN/H₂Elution with 25% -55% CH in 23 min₃CN, then 95% CH₃CN for 7 minutes). Fractions containing 25a were combined and lyophilized to give 25a as a white solid. It was further chromatographed on silica gel (4g silica column, CH)₂Cl₂MeOH, 0-20% MeOH over 15 min) to give the desired 25a (7.7mg, 30% yield). MS (ESI) M/z 975.8(M + H)⁺,997.8(M+Na)⁺,773.7(M-H)^-,1019.7(M+HCOOH-H)^-。

EXAMPLE 24 Synthesis of Compound 23b

23 b: to a solution of 6a (31mg, 0.076mmol) in DMF (0.8mL) was added NMM (M8.3. mu.L, 0.076mmol) and 21b (46.4mg, 0.076 mmol). The solution was cooled to 0 ℃ with an ice bath and deionized water (0.16mL) containing DMTMM (48mg, 0.16mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g silica column, CH)₂Cl₂MeOH, 0-20% MeOH over 15 min) to give the desired product, compound 23b (31mg, 40% yield). MS (ESI) M/z 1004.6(M + H)⁺,1048.7(M+HCOOH-H)^-。

EXAMPLE 25 Synthesis of Compound 24b

24 b: to a solution of 23b (31mg, 0.031mmol) in anhydrous DMF (0.4mL) was added morpholine (95 μ L, 1.08mmol) and the reaction mixture was stirred at room temperature for 2.5 h. The reaction solution was stripped under reduced pressure and the residue was chromatographed on silica gel (4g silica column, using CH) ₂Cl₂MeOH elution, 0-20% MeOH over 15 min, then 20% MeOH for 5 min) to give the product, compound 24b (18mg, 75% yield). MS (ESI) M/z 782.5(M + H)⁺,780.2(M-H)^-,826.4(M+HCOOH-H)^-。

EXAMPLE 26 Synthesis of Compound 25b

25 b: to a solution of 24b (12.3mg, 0.016mmol) in anhydrous DMF (0.3mL) was added N-hydroxysuccinimide 5-maleimidocaproate 13b (7.3mg, 0.024mmol) and the reaction mixture was stirred at room temperature for 4 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4g column, CH2Cl2/MeOH, 0-20% MeOH over 15 min) to give 3.2mg 25b and another 6.5mg of impure product. The impure product was further subjected to semi-preparative reverse phase HPLC (C18 column using CH)₃CN/H₂Elution with O, 25% within 23 minutes55％CH₃CN, then 95% CH₃CN for 7 min) to yield 2.8mg of 25 b. A total of 6mg of 25b was isolated (39% yield). MS (ESI) M/z 975.6(M + H)⁺,997.6(M+Na)⁺,973.7(M-H)^-,1019.6(M+HCOOH-H)^-。

EXAMPLE 27 Synthesis of Compound 26a

26 a: to a stirred solution of 8a (58mg, 0.106mmol) and compound 16a (55.8mg, 0.106mmol) in anhydrous DMF (1.5mL) was added HCl etherate (2M HCl in ether, 64. mu.L, 0.128 mmol). After stirring at room temperature for 22 hours, the reaction solution was stripped under reduced pressure (heating bath at 35 ℃). The residue was purified by reverse phase HPLC (30g C18 column, CH) ₃CN/H₂O，25％CH₃CN lasted 3 minutes, then reached 90% CH in 12 minutes₃CN, then 90% CH₃CN for 3 min) to give compound 26a as a white solid (47mg, 48% yield). MS (ESI) calculated M/z 917.4, Experimental 917.6(M + H)⁺,961.5(M+HCOOH-H)^-. Unreacted 8a was also recovered (12 mg).

EXAMPLE 28 Synthesis of Compound 27a

27 a: to a stirred solution of compound 26a (57mg, 0.062mmol) in anhydrous DMF (0.8mL) was added morpholine (27. mu.L, 0.31 mmol). After stirring at room temperature for 6 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30g C18 column with CH)₃CN/H₂O elution, 20% CH₃CN lasted for 3 minutes, then reached 90% CH in 15 minutes₃CN, then 90% CH₃CN for 3 min) to give compound 27a as an off-white solid (35.9mg, 83% yield). MS (ESI) M/z 695.5(M + H)⁺,739.3(M+HCOOH-H)^-。

EXAMPLE 29 Synthesis of Compound 28a

28 a: to a solution of compound 27a (18mg, 0.026mmol) in anhydrous DMF (0.3mL) was added N-hydroxysuccinimide ester of 5-maleimidocaproic acid 13b (12mg, 0.039mmol) and NMM (3.1. mu.L, 0.028 mmol). After stirring at room temperature for 4 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30g C18 column using CH) ₃CN/H₂O elution, 18 min run, 20% CH₃CN lasted for 3 minutes, then 20% -90% CH in 12 minutes₃CN, then 90% CH₃CN for 3min) to yield product 28a as a white solid (11.9mg, 51% yield). MS (ESI) M/z 888.5(M + H)⁺,932.5(M+HCOOH-H)^-。¹H NMR(400MHz,DMSO-d₆)δ8.45(t,J＝6.7Hz,1H),8.11(d,J＝8.2Hz,1H),7.90(t,J＝7.5Hz,2H),7.78(dd,J＝15.2,9.1Hz,2H),7.22(s,1H),6.92(s,2H),6.45(s,1H),5.36(s,2H),5.17(s,2H),4.54-4.38(m,2H),4.15-4.09(m,2H),3.38-3.33(m,1H),3.32-3.21(m,1H),3.15-3.06(m,1H),2.50-2.42(m,3H),1.99(t,J＝7.4Hz,2H),1.79(h,J＝7.0Hz,2H),1.67-1.55(m,6H),1.38(p,J＝7.4Hz,5H),1.15-1.04(m,12H),0.81(t,J＝7.4Hz,3H)。¹³C NMR(101MHz,DMSO-d₆)δ174.54,173.86,171.34,171.26,170.84,170.79,170.47,170.15,161.42,158.95,155.71,155.16,150.43,148.38,146.97,146.80,144.22,142.23,129.03,126.25,125.69,125.48,124.98,124.50,122.77,122.48,117.29,111.01,110.78,95.09,70.75,70.67,67.55,65.17,63.64,48.03,46.55,33.20,28.68,27.46,26.96,25.04,24.55,24.05,23.06,22.84,16.39,16.14,13.54,6.13,5.98；HRMS(M+H)⁺Calculated 888.3943, experimental 888.3966.

EXAMPLE 30 Synthesis of Compound 26b

26 b: to the mixture of 8a (73mg, 0.14mmol) and compound16b (64mg, 0.14mmol) to a stirred solution in anhydrous DMF (1.2mL) was added HCl etherate (2M HCl in ether, 0.14mL, 0.28 mmol). After stirring at room temperature for 6h, the reaction solution was stripped under reduced pressure (heating bath at 35 ℃). The residue was purified by reverse phase HPLC (30g C18 column, CH)₃CN/H₂O，25％CH₃CN lasted for 3min and then reached 90% CH within 12min₃CN, then 90% CH₃CN for 3min) to give compound 26b as an off-white solid (60mg, 50% yield). MS (ESI) M/z 846.4(M + H)⁺,890.3(M+HCOOH-H)^-。

EXAMPLE 31 Synthesis of Compound 26c

26 c: to a stirred solution of 8a (36mg, 0.08mmol) and 16c (40.2mg, 0.084mmol) in anhydrous DMF (0.6mL) was added HCl etherate (2M HCl in ether, 72. mu.L, 0.43 mmol). After stirring at room temperature for 15h, the reaction solution was stripped under reduced pressure (heating bath at 35 ℃). The residue was purified by reverse phase HPLC (30g C18 column, CH) ₃CN/H₂O，25％CH₃CN lasted for 3min and then reached 95% CH within 12min₃CN, then 95% CH₃CN for 3min) to give compound 26c as a white solid (41.9mg, 60% yield). MS (ESI) M/z 874.4(M + H)⁺,918.5(M+HCOOH-H)^-. Unreacted 8a was also recovered (12 mg).

EXAMPLE 32 Synthesis of Compound 27b

27 b: to a stirred solution of compound 26b (60mg, 0.071mmol) in anhydrous DMF (0.4mL) was added morpholine (31. mu.L, 0.36 mmol). After stirring at room temperature for 3.5 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (15.5g C18 column with CH₃CN/H₂O elution, 10% CH₃CN lasted for 3 minutes and then reached 90 in 9 minutes％CH₃CN, then 90% CH₃CN for 3min) to give compound 27b as an off-white solid (36.7mg, 83% yield). MS (ESI) M/z 624.5(M + H)⁺,668.3(M+HCOOH-H)^-。

EXAMPLE 33 Synthesis of Compound 27c

27 c: to a stirred solution of compound 26c (41.9mg, 0.048mmol) in anhydrous DMF (0.4mL) was added morpholine (21. mu.L, 0.24 mmol). After stirring at room temperature for 5h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30g C18 column with CH)₃CN/H₂O elution, 20% CH₃CN lasted 3min, then reached 90% CH within 12min₃CN, then 90% CH₃CN for 5min) to give compound 27c as a white solid (25.5mg, 82% yield). MS (ESI) M/z 652.5(M + H) ⁺,650.2(M-H)^-.696.2(M+HCOOH-H)^-。

EXAMPLE 34 Synthesis of Compound 28b

28 b: to a stirred solution of compounds 27a (18mg, 0.026mmol) and 14a (18.5mg, 0.039mmol) in anhydrous DMF (0.2mL) was added DMTMM (17mg, 0.052mmol) and NMM (2.9. mu.L, 0.026mmol) at room temperature. After stirring at room temperature for 19h, the reaction solution was stripped under reduced pressure (bath temperature 35 ℃) and the residue was passed through reverse phase HPLC (30g C18 column, CH)₃CN/H₂O，20％CH₃CN lasted for 3min and then reached 90% CH within 12min₃CN, then 90% CH₃CN for 3 min). Fractions containing the product were combined and lyophilized to give 28b as a white solid (2.3mg, 7% yield). MS (ESI) M/z 1152.5(M + H)⁺,1150.0(M-H)^-,1196.5(M+HCOOH-H)^-。

EXAMPLE 35 Synthesis of Compound 28c

28 c: to a stirred solution of 27b (36.7mg, 0.059mmol) and 14a (42mg, 0.088mmol) in anhydrous DMF (0.6mL) at room temperature were added DMTMM (39mg, 0.12mmol) and NMM (3.2. mu.L, 0.029 mmol). After stirring at room temperature for 5h, the reaction solution was loaded directly onto a C18 cartridge and subjected to reverse phase HPLC (30g C18 column, CH)₃CN/H₂O, 20% CH3CN for 3min, then 90% CH within 12min₃CN, then 90% CH₃CN for 3 min). Fractions containing the product were combined and lyophilized to give 28c as a white solid (9.9mg, 15% yield). MS (ESI) M/z 1081.5(M + H) ⁺,1079.2(M-H)^-,1025.5(M+HCOOH-H)^-。

EXAMPLE 36 Synthesis of Compound 28d

28 d: to a solution of 27c (10.5mg, 0.016mmol) in anhydrous DMF (0.2mL) was added N-hydroxysuccinimide 5-maleimidocaproate 13b (6mg, 0.019mmol) and NMM (2.1. mu.L, 0.019 mmol). After stirring at room temperature for 4h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30g C18 column using CH)₃CN/H₂O elution, 18 min run, 20% CH₃CN lasted for 3 minutes, then 20% -90% CH in 12 minutes₃CN, then 90% CH₃CN for 3 min) to give product 28d as a white solid (8.7mg, 63% yield). MS (ESI) M/z 845.4(M + H)⁺,989.4(M+HCOOH-H)^-。

EXAMPLE 37 Synthesis of Compound 29a

29 a: compound 16a (30mg, 0.057mmol) and 8d (50mg, 0.1mmol) were suspended in a solution of 20% TFA in dichloromethane (10mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in the minimum volume of DMF and then purified on a 100g c18 medium pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid acetonitrile. The column was then eluted with 10% acetonitrile at 30mL/min for 5min, followed by a linear gradient of 10% acetonitrile at 5min to 95% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 31mg of white solid 29a (56% yield). MS (M + Na) + 987.5.

EXAMPLE 38 Synthesis of Compound 30a

30 a: compound 29a (28mg, 0.029mmol) was dissolved in anhydrous DMF (0.8mL) and magnetically stirred while morpholine (0.2mL) was added. After 1h, the reaction mixture was loaded directly onto a 100gC18 cartridge, 25:75CH₃CN/H₂O, run at 50mL/min, 25% CH₃CN lasted for 3min, then reached 90% CH from 3-23min₃A linear gradient of CN. Fractions containing the desired product were combined, frozen and lyophilized to give 18mg (83% yield) of 30a as a yellow solid. MS (M + H) + 743.5.

EXAMPLE 39 Synthesis of Compound 32a

32 a: compound 30a (16mg, 0.02mmol) was dissolved in 84:16DMF deionized water (0.5mL), to which was rapidly added DMTMM (15mg, 0.054mmol), TEA (0.02mL, 0.14mmol) and 14a (20mg, 0.042mmol) and magnetically stirred. After 35min, the reaction mixture was loaded on a 100g silica cartridge pre-equilibrated with dichloromethane, and then run with a linear gradient of 0% -100% 40:60 methanol: dichloromethane over 30min at 35 mL/min. Fractions containing the desired product were combined and the solvent was evaporated in vacuo to yield 7mg (29% yield) 32a as a viscous oil. MS (M + Na) + 1223.0.

EXAMPLE 40 Synthesis of Compound 33a

33 a: to a solution of 4c (11mg, 0.021mmol) in anhydrous DMF (0.2mL) was added sodium thiomethoxide (3.6mg, 0.052mmol) and the reaction mixture was stirred at room temperature for 18 hours. The reaction solution was loaded directly onto a 30g C18 cartridge, 25:75CH ₃CN/H₂O, run at 20mL/min, 25% CH₃CN lasts for 3 minutes, then 90% CH is reached in 3-12min₃A linear gradient of CN. Fractions containing the desired product were combined and evaporated in vacuo to afford compound 33a as a white solid (3.1mg, 30% yield). MS (ESI) M/z 483.4(M + H)⁺,481.3(M-H)^-。

EXAMPLE 41 Synthesis of Compound 34a and Compound 34b

34a and 34 b: compound 8c (30mg, 0.064mmol) was magnetically stirred in DMF (1mL) to which was added vanadyl acetylacetonate (3mg, 0.008mmol) and a 5M solution of tert-butyl hydroperoxide in decane (0.05mL, 0.25 mmol). After 5min, the solution was loaded directly onto 30g C18 cartridges at 25:75CH₃CN/H₂O, run at 20mL/min, 25% CH₃CN lasts for 3 minutes, then 90% CH is reached in 3-12min₃A linear gradient of CN. Fractions containing pure 34a and 34b were combined separately and the two were frozen and lyophilized separately to give 6mg 34a (19% yield) and 15mg 34b (47% yield), both as yellow solids. 34a MS (M + H) +487.4,34b MS (M + H) + 503.6.

EXAMPLE 42 Synthesis of Compound 35a

35 a: to a solution of compound 4e (17mg, 0.025mmol) in anhydrous DMF (0.3mL) was added sodium thiomethoxide (7mg, 0.1mmol) and the reaction mixture was stirred at room temperature for 24 h. The reaction solution was diluted with DMSO and injected into semi-preparative HPLC (C18 column, CH) for purification ₃CN/H₂O, 25% -65% CH in 23 min₃CN) to yield the desired product 35a (2.7mg, 19% yield). MS (ESI) M/z 554.4(M + H)⁺,552.5(M-H)^-。

EXAMPLE 43 Synthesis of Compound 29b

29 b: compound 16a (30mg, 0.057mmol) and 8e (50mg, 0.1mmol) were suspended in a solution of 20% TFA in dichloromethane (10mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in the minimum volume of DMF and then purified on a 100g c18 medium pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid acetonitrile. The column was then eluted with 10% acetonitrile at 30mL/min for 5min, followed by a linear gradient of 10% acetonitrile at 5min to 95% acetonitrile at 38 min. Fractions containing the desired product were combined, frozen and lyophilized to give 31mg of 29b as a white solid (56% yield). MS (ESI) MS (M + Na)⁺Calculated 973.3, experimental 973.7.

EXAMPLE 44 Synthesis of Compound 30b

30 b: compound 29b (28mg, 0.029mmol) was dissolved in anhydrous DMF (0.8mL) and magnetically stirred while morpholine (0.2mL, 2.32mmol) was added. After 1h, the reaction mixture was loaded directly onto a 100g C18 cartridge, 25:75CH₃CN/H₂O, run at 50mL/min, 25% CH₃CN lasts for 3min Then using the mixture for reaching 90% CH from 3-23min₃A linear gradient of CN. Fractions containing the desired product were combined, frozen and lyophilized to give 18mg (83% yield) of 30b as a yellow solid.¹H NMR(400MHz,DMSO-d6)δ8.52(t,J＝6.3Hz,1H),8.31(d,J＝8.3Hz,1H),8.11(d,J＝7.4Hz,2H),7.87(t,J＝9.1Hz,2H),7.69(d,J＝7.5Hz,1H),7.37(t,J＝7.4Hz,1H),7.31(t,J＝3.7Hz,2H),5.43(s,2H),5.31(s,2H),4.47(d,J＝2.6Hz,2H),4.32-4.09(m,4H),3.69(t,J＝4.6Hz,2H),3.36(q,J＝6.9Hz,1H),2.84(s,4H),2.61-2.54(m,3H),2.46-2.42(m,4H),1.94-1.75(m,2H),1.17(dd,J＝7.2,3.4Hz,6H),1.12(d,J＝6.8Hz,3H),0.87(t,J＝7.3Hz,3H)。MS(ESI):MS(M+H)⁺Calculated 729.3, experimental 729.4.

EXAMPLE 45 Synthesis of Compound 32b

32 b: compound 30b (16mg, 0.02mmol) was dissolved in 84:16DMF deionized water (0.5mL), to which was rapidly added DMTMM (15mg, 0.054mmol), TEA (0.02mL, 0.14mmol) and 14a (20mg, 0.042mmol) and magnetically stirred. After 35min, the reaction mixture was loaded on a 100g silica cartridge pre-equilibrated with dichloromethane, and then run with a linear gradient of 0% -100% 40:60 methanol: dichloromethane over 30min at 35 mL/min. Fractions containing the desired product were combined and the solvent was evaporated in vacuo to give 7mg of 32b as a viscous oil (29% yield).¹H NMR (400MHz, DMSO-d6) δ 7.86-7.83(m,1H),7.71(s,1H),7.69(d, J ═ 1.1Hz,1H),7.66(d, J ═ 8.2Hz,1H),7.51-7.47(m,2H),7.28(t, J ═ 1.0Hz,1H),6.61(s,2H),5.59-5.53(m,1H),5.40(dd, J ═ 3.3,1.1Hz,2H),5.29-5.26(m,2H),4.72(s,2H),4.52-4.46(m,1H),4.38(s,1H),4.35(dd, J ═ 3.5,1.8, 2H), 4.28-4.28 (m, 4.22, 4.70 (m,1H), 4.9.6H, 3, 3.5 (dd, 3.5, 1H), 3.6H, 7.7.6 (d, 3, 3.7.7.7H), 7.6H, 7H, 8H) 3.20(dd, J ═ 4.7,3.7Hz,2H),2.91(s,3H),2.89(s,3H),2.56(td, J ═ 6.4,1.7Hz,2H),2.31(s,3),2.37-2.21(m,2H),2.07-1.82(m,3H),1.72(dq, J ═ 13.7,8.1Hz,1H),1.53(d, J ═ 5.5Hz,6H),1.45(d, J ═ 5.9Hz,3H),0.86(t, J ═ 5.5Hz,6H), and so on 8.0Hz,3H)。¹³C NMR(101MHz,DMSO-d6)δ174.75,174.58,172.85,172.75,172.45,171.62,171.37,169.30,163.44,160.52,157.18,149.96,148.73,147.39,147.32,142.26,132.98,131.74,127.54,127.38,127.24.126.88,124.90,124.84,124.34,122.88,118.32,116.83,116.67,97.97,74.87,73.80,72.41,72.34,69.44,65.10,62.94,53.02,50.02,49.92,49.62,48.46,45.53,42.38,40.85,35.32,35.13,34.00,32.31,32.24,30.20,26.63,20.49,17.81,17.71,17.61,7.68。HRMS(M+H)⁺Calculated 1186.4237, experimental 1186.4220.

Example 46 general procedure for the preparation of conjugates

Humanized IgG1 antibodies were generated, such as anti-epidermal growth factor (ML66), anti-folate receptor alpha (FR α), and chimeric antibodies that bind to Kunitz soybean trypsin inhibitor (KTI). Conjugation of the antibody to the payload with maleimide was performed as described for the preparation of ML66-22 a. The resulting conjugate will be designated herein as a target binder-payload, e.g., ML66 conjugated to 22a is ML66-22a

EXAMPLE 47 Synthesis of ML66-22a

ML66-22 a: 5mg/ML ML66 was treated with 7.0 equivalents of TCEP for 1-1.5h at 37 ℃ in 50mM EPPS pH7.4, 5mM EDTA. The mixture was then cooled to room temperature. The conjugation reaction between antibody and payload is performed at 2mg/ml by adding 15-20 equivalents of 22a dissolved in DMSO in a buffer containing 20% DMSO (50mM EPPS, ph7.4) and spinning on a tube rotator for 1.5-2.5h at room temperature. The reaction mixture was immediately purified using a NAP desalting column (Illustra Sephadex G-25, GE Healthcare) into a formulation buffer (10mM acetate, 9% sucrose, 0.01% Tween-20, pH 5.0). The drug to antibody ratio (DAR) of the resulting conjugate was 7.5 and 99% monomer was determined by size exclusion chromatography.

Biophysical evaluation for interchain high DAR conjugates included determination of conjugate concentration, yield, DAR (drug: antibody ratio),Free drug, percent monomer, and DAR distribution. The conjugate concentration was determined to have a final protein concentration of 4.5mg/ml (using the extinction coefficient ε via UV-Vis)₂₈₀＝205520M^-1cm^-1) DAR 7.5 (use of extinction coefficient ε for 22a via UV-Vis)₂₈₀＝10764M^-1cm^-1，ε₃₇₀＝20982M^-1cm^-1) 99% monomer (via a size exclusion UPLC protein BEH SEC column),<1% free drug (via HiSEP HPLC column) and mainly 8 homogeneous drugs attached per antibody (via Q-ToF mass spectrometry, and Butyl-NPR HIC chromatography). The yield of this particular interchain conjugate ML66-22a was 75%.

UPLC protein BEH SEC method: in a column equipped with Acquity UPLC protein BEH SEC ((R))1.7um, 4.6mm × 150mm, part #186005225) on a Waters Acquity UPLC class H system. The mobile phase is 400mM sodium perchlorate, 50mM sodium phosphate, 5% IPA, pH7.0, flow rate of 0.30mL/min, and running time of 20 min.

HiSep HPLC method: percentage free drug analysis was performed on an Agilent HPLC system equipped with a Supelco analytical HiSep column (25cm × 4.6mm, 5um, catalog # 58919). The mobile phase consisted of 0.1M ammonium, pH7.0 (solvent a) and acetonitrile (solvent B). The process was run with solvent a using a linear gradient from 25% solvent B to 40% solvent B starting at 0.70mL/min using 0-25 min.

Example 48 in vitro cytotoxicity assays

Cytotoxic efficacy was assessed in flat bottom 96-well plates (Costar) using a water-soluble bisazo salt (WST-8) based cell viability assay (Dojindo, Molecular Technologies, Inc.) as previously described (Kovtun YV et al, Antibody-maytansinoid conjugates designed to bypass multidrug resistance. cancer Res 2010; 70(6): 2528-37). Briefly, human tumor cells (1,000-5,000 cells/well, depending on the cell line) in appropriate medium were incubated at 37 ℃ with 6% CO₂The conjugate is then used in the presence or absence of an excess of the corresponding unconjugated antibody or in the presence of an excess of unconjugated antibodyThe metabolite incubation lasted 5 days. Cell viability was determined from the background-corrected absorbance of WST-8.

The results of this study are summarized in the tables below. Figure 12 depicts the cytotoxicity of sulfide bearing compound 8c and its sulfoxides 34a and sulfones 34 b.

TABLE 7A. in vitro cytotoxicity (IC) of unconjugated Compounds₅₀Value)

TABLE 7B in vitro cytotoxicity (IC) of unconjugated Compounds₅₀Value)

N-Luc indicates Namalwa cells stably transfected with luciferase Gene

Example 49 bystander cell killing assay

Cell Titer Glo and One Glo reagents were purchased from Promega. The ability of the ADC to induce bystander killing was determined by one of two assays. Both assays were performed in U-bottom 96-well plates (Costar) to keep mixed antigen negative (Ag-) and antigen positive (Ag +) cells in close proximity to each other.

Namalwa-luciferase (N-Luc) Ag-cells (1000 cells/well) in appropriate medium at 37 ℃ 6% CO₂The wells of the U-bottom 96-well plate were incubated with the indicated number of MDA-MB-468Ag + cells and ADC (1.1nM) for 5 days. The concentration of conjugate used in the assay is high enough to kill all Ag + cells, but not Ag-cells, unless they are present. On day 5, Cell viability was determined by the Cell Titer Glo assay according to the manufacturer's protocol; the luminescence signal was read using a Victor3 plate reader. The following table shows in vitro cytotoxicity and bystander killing of the compounds described herein as obtained in the previous example.

TABLE 8 in vitro cytotoxicity and bystander killing of ADCs

N-Luc indicates Namalwa cells stably transfected with luciferase Gene

Example 50 methods for determining in vivo efficacy in xenograft models

6-week-old female CB.17SCID mice were received from Charles River Laboratories. All in vivo procedures were performed strictly according to NIH laboratory animal care and use guidelines. The in vivo efficacy of ADCs was evaluated in a given tumor xenograft model. Female SCID mice were inoculated subcutaneously in the right flank with the desired cell type in serum-free medium at a 1:1 ratio. The animals were then randomly distributed in groups of 6 or 8 mice each. Control mice were treated with phosphate buffered saline (vehicle). The desired concentration of ADC was made by diluting the stock sample with vehicle. Xenografts grown to approximately 100mm ³Mice were then administered ADC or vehicle by tail vein intravenous (i.v.) injection (200 μ L/mouse). All administrations are based on the weight of the antibody component of the conjugate. Three-dimensional tumor size was measured twice weekly using caliper gauges, where tumor volume was in mm calculated using the formula V ═ 1/2 (length x width x height)³And (4) showing. Body weight was also measured twice weekly. Data from these studies were interpreted using standardized methods as previously described (Bissery MC et al, Experimental reactor activity of taxotere (RP 56976, NSC 628503), a taxol analogue. cancer Res 1991; 51(18): 4845-52).

Example 51 tolerance of mice to ML66-999, ML66-22a, and ML-28a ADCs

ADC tolerance was assessed in female CD-1 mice by performing daily weight measurements and clinical observations for 2 weeks after ADC injection. The naked ML66 antibody at the pre-10 mg/kg dose was administered intravenously into 32 female CD-1 mice of 7 weeks of age, as it was shown to reduce the acute infusion response caused by ML66 alone at doses greater than 20 mg/kg. After this injection of naked antibody, one mouse died. After two hours, three groups of seven to eight mice per group were dosed with an intravenous bolus of 1500 μ g/kg payload ML66-999(58mg/kg antibody), ML66-22a (59mg/kg antibody), or ML66-28a (69mg/kg antibody). The Maximum Tolerated Dose (MTD) was defined as the highest dose at which euthanasia was required for the animals without death or due to > 20% weight loss or painful signs or morbidity (back of arch, lack of activity, no food or water intake or signs of pain/distress). Based on these criteria, all three ADCs were well tolerated at the 1500 μ g/kg payload dose. FIG. 19 depicts tolerance of mice to ML66-999, ML66-22a, and ML66-28a ADCs.

Example 52 method for determining ADC Pharmacokinetic (PK) parameters in mice

Three groups of eleven 7-week-old female CD-1 mice were each administered with a single intravenous bolus of either 10mg/kg naked antibody or ADC. Terminal blood samples were collected from 3 mice at 2 minutes, 3 mice at 24 hours, and 5 mice at 72 hours after injection of each ADC. Blood was processed into serum and the ADCs were purified using affinity capture with anti-human Fc beads. The samples were analyzed by anti-human Fc ELISA to determine the concentration of the antibody component (regardless of payload loading). Samples were also analyzed by Size Exclusion Chromatography (SEC) and Mass Spectrometry (MS). The partial loss of linker-payload from the captured ADC at all time points was measured by the full MS method. The ADC concentration is calculated based on the antibody concentration and the drug-to-antibody ratio (DAR).

Fig. 13 and table 9 show the pharmacokinetics of ML66-999 in mice. Fig. 14 and table 10 show the pharmacokinetics of ML66-999 in mice. Table 11 summarizes the binding of ADCs or naked antibodies to cell lines expressing the corresponding antigens. FIGS. 15 and 16 show in vitro cytotoxicity of ML66-999 and ML66-22a against Ag + and Ag-cells. The partial retention of biological activity of ADC in plasma samples over time was determined in cytotoxicity assays using Ag + or Ag-cells. The ADC retains most of its activity against Ag + cells at each time point while still being over 200-fold less active compared to Ag-cells, indicating that cytotoxicity is due to intact ADC, with little contribution from any released payload.

Table 9.

Table 10.

TABLE 11 binding of ADC or naked antibody to cell lines expressing the corresponding antigen

Example 53 anti-EGFR antibody drug conjugates anti-tumor Activity in HSC-2 human head and neck squamous cell carcinoma xenograft bearing nude mice

Antitumor activity of 1, 3 and 10mg/kg ML66-999 and ML66-22a was evaluated in female nude mice bearing HSC-2 cells (human head and neck squamous cell carcinoma xenograft model).

Mice were inoculated with 0.1ml of 1X 10 in 50% Matrigel/50% serum-free medium by subcutaneous injection in the area on the right posterolateral flank⁷And (4) HSC-2 cells. Obtaining female athymic Foxn1^nuMice (6 weeks old). After receipt, the animals were observed for 9 days prior to study initiation. Animals showed no signs of disease or illness after arrival or prior to treatment.

Forty-eight mice were randomly grouped into 8 groups (6 mice per group) according to tumor volume. Tumor volumes from 68.48 to 118.26 (93.42. + -. 11.25, mean. + -. SD) mm³Within the range. Mice were measured, randomized, and dosed based on tumor volume on day 4 post-implantation (11/12/18). The body weight of the mice ranged from 19.46 to 25.77(22.98 ± 1.50, mean ± SD) grams. Mice in each group were identified by the puncture method. By using a 1 equipped with a 27 gauge, 1/2 inch needle. Administration of test agent and vehicle was performed intravenously in a 0ml syringe. The antibody drug conjugate test agent is administered at 1, 3 or 10mg/kg qdx1, where 75 μ g/kg or 250 μ g/kg on a payload basis correlates to about 3 or 10mg/kg on an antibody concentration basis. The group comprising: control group dosed with vehicle (PBS, 200. mu.L), control group dosed with non-targeted KTI-999 at 10mg/kg, ML66-999 dosed at 1, 3 and 10mg/kg based on antibody concentration, and ML66-22a dosed at 1, 3 and 10mg/kg based on antibody concentration.

Three-dimensional tumor size was measured twice weekly using caliper gauges. Tumor volume was measured in mm using the formula length x width x height x 1/2³And (4) showing. When tumor volume is reduced by 50% or more, mice are considered to have Partial Regression (PR) and when palpable tumors cannot be detected, mice are considered to have complete tumor regression (CR). Tumor volume was determined by StudyLog software.

Tumor growth inhibition (% T/C) is determined at a predetermined time (e.g., when the median TV of the control tumors reaches a maximum tumor volume of about 1000mm³Time at which mice were euthanized) ratio of median Tumor Volume (TV) of treatment group (T) to median TV of control group (C). The% T/C was calculated on day 22 post inoculation when the median TV of the control group reached 1038mm ³. According to the NCI criterion, T/C.ltoreq.42% is the minimum level of antitumor activity and T/C<10% was considered as high anti-tumor activity level.

Body Weight (BW) of all mice was measured twice weekly as a rough index of drug toxicity and determined by StudyLog software. Body weight of the mice is expressed as percent change in body weight compared to body weight before treatment as follows: BW Change [ (% after BW/before BW) -1]X 100, where post BW is the weight after treatment and pre BW is the starting weight before treatment. Percent Body Weight Loss (BWL) is expressed as the mean change in body weight after treatment. If the tumor volume is greater than 1000mm³Tumor necrosis, mice losing their initial body weight>20%, or mice moribund at any time during the study, the animals were euthanized.

FIG. 17 and Table 12 depict the efficacy of ADC in the HSC-2 xenograft model. ML66-999 and ML66-22a conjugates have similar antitumor activity. ML66-KTI administered at 10mg/kg had a T/C value of 70% (no activity) and no tumor regression. This demonstrates that the activity of the ML66 conjugate is EGFR targeted because the control ADC is inactive. ML66-999 at 1mg/kg had a T/C value of 49% (no activity) and no tumor regression. ML66-999 administered at 3mg/kg had a T/C value of 7% (highly active), with 4 of 6 mice having partially regressed tumors and 1 having completely regressed. ML66-999 administered at 10mg/kg had a T/C value of 3% (highly active), with 6 of 6 mice having partial regression of the tumor and 2 complete regression. ML66-22a administered at 1mg/kg had a T/C value of 39% (activity), with 1 of 6 mice having partial tumor regression and no complete regression. ML66-22a administered at 3mg/kg had a T/C value of 5% (highly active), with 3 of 6 mice having partial regression of the tumor and 1 complete regression. ML66-22a administered at 10mg/kg had a T/C value of 2% (highly active), with 6 of 6 mice having partial regression of the tumor and 5 complete regression. With respect to any of the conjugates, no significant weight loss was observed at any of the indicated doses, indicating that the conjugate was well tolerated. The results of this study indicate that both ML66-999 and ML66-22a conjugates demonstrate dose-dependent anti-tumor activity and are effective in HSC-2 head and neck squamous cell carcinoma tumor xenograft models.

Table 12.

Example 54 antitumor Activity of conjugates in nude mice bearing non-Small cell Lung cancer (NSCLC) xenografts

The conjugates were assayed for anti-tumor activity in nude mice bearing non-small cell lung cancer (NSCLC) xenografts in a manner similar to that described for HSC-2 cells as in example 53.

6 week old female athymic nude mice were received from Charles River Laboratories (Foxn 1)^nu). All in vivo procedures were performed strictly according to NIH laboratory animal care and use guidelines. The antitumor activity of the ADCs was evaluated in a non-small cell lung cancer (NSCLC) squamous H1703 tumor xenograft model. Female nude mice were subcutaneously inoculated in the right flank with the desired cell type (5X 10) in a 1:1 ratio serum-free medium to Matrigel⁶Individual cells/mouse). Three-dimensional Tumor Volume (TV) was measured twice weekly using caliper gauges, where tumor volume was in mm calculated using the formula TV 1/2 (length x width x height)³And (4) showing. Xenografts grown to about 100mm³And mice were based on their TV (with 116.0+/-18.5 mm) on day 16 post cell inoculation³[ mean value +/-SD]TV) were randomly distributed in groups of 6 mice each. Stock ADCs were diluted with conjugate dilution buffer and mice were dosed according to subject body weight. Mice received a single Intravenous (IV) bolus of either vehicle (phosphate buffered saline (PBS), 200. mu.L/mouse) or ADC at a dose volume of 5mL/kg based on a payload of 75. mu.g/kg or 250. mu.g/kg (based on antibody concentrations of approximately 3 or 10 mg/kg). Tumor growth inhibition (T/C) is at a predetermined time (e.g., when the median TV of the control tumors reaches a maximum tumor volume of about 1000mm ³Time at which the mice were euthanized) the ratio of median Tumor Volume (TV) of the treated group (T) to the median TV of the control group (C). According to the NCI criterion, T/C.ltoreq.42% is the minimum level of antitumor activity and T/C<10% was considered as high anti-tumor activity level. When the TV is reduced by 50% or more, the mice are considered to have Partial Regression (PR) and when no palpable tumor can be detected, the mice are considered to have Complete Regression (CR). The T/C, PR and CR for both efficacy studies are listed in the following table. Body weight was also measured twice weekly as a rough index of drug toxicity.

Fig. 20 and table 14 depict the efficacy of ADC in the H1703 xenograft model. The results indicate that both the ML66-999 and ML66-22a conjugates demonstrate dose-dependent anti-tumor activity and are effective in the H1703 tumor xenograft model.

Table 14. results of H1703 efficacy study

Example 55 anti-EGFR antibody drug conjugates anti-tumor Activity in nude mice bearing FaDu human head and neck squamous cell carcinoma xenografts

Antitumor activity of 1, 3 and 10mg/kg ML66-999 and ML66-22a was evaluated in female nude mice (human head and neck squamous cell carcinoma xenograft model) carrying FaDu cells.

Mice were inoculated with 0.1ml of 1X 10 in 50% Matrigel/50% serum-free medium by subcutaneous injection in the area on the right posterolateral flank⁷A FaDu cell. Obtaining female athymic Foxn1^nuMice (6 weeks old). After receipt, the animals were observed for 7 days prior to study initiation. Animals showed no signs of disease or illness after arrival or prior to treatment.

Sixty-four mice were randomly grouped into 8 groups (8 mice per group) according to tumor volume. Tumor volumes from 74.07 to 128.73 (104.66. + -. 15.70, mean. + -. SD) mm³Within the range. Mice were measured, randomized, and dosed based on tumor volume at day 6 post-implantation (11/19/18). The body weight of the mice ranged from 20.48 to 25.77(23.55 ± 1.25, mean ± SD) grams. Mice in each group were identified by the puncture method. Administration of the test agents and vehicle was performed intravenously by using a 1.0ml syringe equipped with a 27 gauge, 1/2 inch needle. Antibody drug conjugate test agents were administered at 1, 3 or 10mg/kg qdx 1. The group comprising: control group dosed with vehicle (PBS, 200. mu.L), control group dosed with non-targeted KTI-999 at 10mg/kg, ML66-999 at 1, 3 and 10mg/kg, and ML66-22a at 1, 3 and 10 mg/kg.

Tumor growth inhibition (% T/C) is determined at a predetermined time (e.g., when the median TV of the control tumors reaches a maximum tumor volume of about 1000mm³Time at which mice were euthanized) ratio of median Tumor Volume (TV) of treatment group (T) to median TV of control group (C). The% T/C was calculated on day 21 post inoculation when the median TV of the control group reached 749mm³. According to the NCI criterion, T/C.ltoreq.42% is the minimum level of antitumor activity and T/C<10% was considered as high anti-tumor activity level.

Body Weight (BW) of all mice was measured twice weekly as a rough index of drug toxicity and determined by StudyLog software. Body weight of the mice is expressed as percent change in body weight compared to body weight before treatment as follows: BW Change [ (% after BW/before BW) -1]X 100, where post BW is the weight after treatment and pre BW is the starting weight before treatment. Percent Body Weight Loss (BWL) is expressed as the mean change in body weight after treatment. If the tumor volume is greater than 1000mm ³Tumor necrosis, mice losing their initial body weight>20%, or mice moribund at any time during the study, the animals were euthanized.

Fig. 18 and table 13 depict the efficacy of ADCs in the FaDu xenograft model. ML66-999 and ML66-22a conjugates have similar antitumor activity. ML66-KTI administered at 10mg/kg had a T/C value of 20% (activity), with 2 of 8 mice having partial tumor regression and 2 complete regression. This demonstrates that some of the antitumor activity in this model is non-targeted. ML66-999 administered at 1mg/kg had a T/C value of 12% (activity), with 4 of 8 mice having partial tumor regression and 2 complete regression. ML66-999 administered at 3mg/kg had a T/C value of 2% (highly active), with 8 of 8 mice having partial regression of the tumor and 4 complete regression. ML66-999 administered at 10mg/kg had a T/C value of 0% (highly active), with 8 of 8 mice having partial tumor regression and 8 complete regression. ML66-22a administered at 1mg/kg had a T/C value of 13% (activity), with 4 of 8 mice having partial regression of the tumor and 3 complete regression. ML66-22a administered at 3mg/kg had a T/C value of 0% (highly active), with 8 of 8 mice having partial tumor regression and 8 complete regression. ML66-22a administered at 10mg/kg had a T/C value of 0% (highly active), with 8 of 8 mice having partial tumor regression and 8 complete regression. With respect to any of the conjugates, no significant weight loss was observed at any of the indicated doses, indicating that the conjugate was well tolerated. The results of this study indicate that both the ML66-999 and ML66-22a conjugates demonstrate dose-dependent anti-tumor activity and are effective in the FaDu head and neck squamous cell carcinoma tumor xenograft model.

Table 13.

Example 56.Ab_F-999 and Ab_FMouse tolerance of-22 a ADC

Tolerability of non-cross-reactive ADCs was assessed in female CD-1 mice by performing daily weight measurements and clinical observations for 2 weeks after ADC injection. Three groups of three mice per group based on payload Ab_F22a (184 mg/kg based on antibody) or Ab_F999(198mg/kg antibody) was given as an IV bolus at 5000. mu.g/kg. Maximum Tolerated Dose (MTD) is defined as the non-death or attributable to animals>20% weight loss or signs of distress or morbidity (arch back, lack of activity, no food or water intake or signs of pain/distress) requiring the highest dose at which euthanasia was administered. GraphPad was used for statistical analysis of body weight in each group (Ab was shown using Tukey's multiple comparison test two-way ANOVA_FGroup-999 significantly differs from vehicle and Ab_F22a group, p<0.05). Fig. 21 depicts these results.

While certain embodiments have been illustrated and described, it will be appreciated that changes and modifications may be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which are not specifically disclosed herein.

The present disclosure is not limited to the specific embodiments described in this application. It will be apparent to those skilled in the art that many modifications and variations can be made without departing from the spirit and scope thereof. Functionally equivalent methods and compositions within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds or compositions, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are described in relation to markush groups, those skilled in the art will recognize that the disclosure is also thereby described in relation to any individual member or subgroup of members of the markush group.

As will be understood by one of skill in the art, with respect to any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily considered to fully describe the range and enable the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those of skill in the art, all languages such as "up to," "at least," "greater than," "less than," and the like include the recited number and refer to ranges that may subsequently be broken down into sub-ranges as discussed above. Finally, as will be understood by those of skill in the art, a range includes each individual member.

All publications, patent applications, issued patents, and other documents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the text incorporated by reference are excluded to the extent they contradict definitions in the present disclosure.

Embodiments of the present disclosure:

1. a compound of formula I or a pharmaceutically acceptable salt thereof,

Z-L¹-D (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O)-or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂。

2. A compound of formula I or a pharmaceutically acceptable salt thereof,

Z-L¹-D (formula I)

Wherein:

d is represented by the following structural formula:

R¹is-F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, -X^1'-(C₁-C₆Alkylene) - (or- (C) ₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z;

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z is-H or-X²；

X²is-OR⁶、-SR⁶、-S(O)R⁶、-S(O)₂R⁶、-SSR⁶or-N (R)⁶)₂；

Each R⁶Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

With the proviso that if R¹Is F and R²is-OMe, then-L¹-Z cannot be-NH₂(ii) a And is

With the proviso that if R¹Is F and R²is-Me, then-L¹-Z cannot be-CH₂OH。

3. The compound of embodiment 1 or embodiment 2, wherein R¹is-H or-F.

4. A compound according to any one of embodiments 1 to 3, wherein R¹is-F.

5. As in embodiments 1 to 4The compound of any one item, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl.

6. The compound of any one of embodiments 1-5, wherein R²is-F.

7. The compound of any one of embodiments 1-5, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

8. The compound of any one of embodiments 1-5, wherein R²Is methyl.

9. The compound of embodiment 1 or embodiment 2, wherein R¹is-F and R²is-F.

10. The compound of embodiment 1 or embodiment 2, wherein R¹Is methyl and R²is-F.

11. The compound of embodiment 1 or embodiment 2, wherein R¹is-F and R²Is a-methyl group.

12. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is-H.

13. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is- (C)₁-C₆Alkylene) -H or- (C)₁-C₆Alkylene) -X²。

14. The compound of embodiment 13, wherein-L¹-Z is methyl, ethyl, propyl or butyl.

15. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is- (C)₁-C₄Alkylene) -OR ⁶、-(C₁-C₄Alkylene) -SR⁶Or- (C)₁-C₄Alkylene) -N (R)⁶)₂。

16. The compound of embodiment 15, wherein-L¹-Z is-CH₂OH、-(CH₂)₂OH、-(CH₂)₃OH、-(CH₂)₄OH、-CH₂OMe、-(CH₂)₂OMe、-(CH₂)₃OMe、-(CH₂)₄OMe、-CH₂SH、-(CH₂)₂SH、-(CH₂)₃SH、-(CH₂)₄SH、-CH₂SMe、-(CH₂)₂SMe、-(CH₂)₃SMe、-(CH₂)₄SMe、-CH₂NH₂、-(CH₂)₂NH₂、-(CH₂)₃NH₂、-(CH₂)₄NH₂，

17. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -OR⁶、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -SR⁶、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SR⁶Or- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SSR⁶。

18. The compound of embodiment 17, wherein-L¹-Z is-CH₂NHC(＝O)CH₂OH、-CH₂NHC(＝O)(CH₂)₂OH、-CH₂NHC(＝O)(CH₂)₃OH、-CH₂NHC(＝O)(CH₂)₄OH、-CH₂NHC(＝O)(CH₂)₅OH、-CH₂NHC(＝O)CH₂OMe、-CH₂NHC(＝O)(CH₂)₂OMe、-CH₂NHC(＝O)(CH₂)₃OMe、-CH₂NHC(＝O)(CH₂)₄OMe、-CH₂NHC(＝O)(CH₂)₅OMe、-CH₂NHC(＝O)CH₂SH、-CH₂NHC(＝O)(CH₂)₂SH、-CH₂NHC(＝O)(CH₂)₃SH、-CH₂NHC(＝O)(CH₂)₄SH、-CH₂NHC(＝O)(CH₂)₅SH、-CH₂NHC(＝O)CH₂SMe、-CH₂NHC(＝O)(CH₂)₂SMe、-CH₂NHC(＝O)(CH₂)₃SMe、-CH₂NHC(＝O)(CH₂)₄SMe、-CH₂NHC(＝O)(CH₂)₅SMe、-CH₂SCH₂OH、-CH₂S(CH₂)₂OH、-CH₂S(CH₂)₃OH、-CH₂S(CH₂)₄OH、-CH₂S(CH₂)₅OH、-CH₂SCH₂OMe、-CH₂S(CH₂)₂OMe、-CH₂S(CH₂)₃OMe、-CH₂S(CH₂)₄OMe、-CH₂S(CH₂)₅OMe、-CH₂SCH₂SH、-CH₂S(CH₂)₂SH、-CH₂S(CH₂)₃SH、-CH₂S(CH₂)₄SH、-CH₂S(CH₂)₅SH、-CH₂SCH₂SMe、-CH₂S(CH₂)₂SMe、-CH₂S(CH₂)₃SMe、-CH₂S(CH₂)₄SMe or-CH₂S(CH₂)₅SMe。

19. The compound of embodiment 17 or embodiment 18, wherein each R is⁵independently-H, methyl or benzyl.

20. The compound of any one of embodiments 15-18, wherein each R⁶independently-H, methyl or benzyl.

21. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is-X^1'-(C₁-C₄Alkylene) -X²。

22. The compound of embodiment 21, wherein-L¹-Z is-OCH₂OH、-O(CH₂)₂OH、-O(CH₂)₃OH、-O(CH₂)₄OH、-SCH₂OH、-S(CH₂)₂OH、-S(CH₂)₃OH、-S(CH₂)₄OH、-S(O)CH₂OH、-S(O)(CH₂)₂OH、-S(O)(CH₂)₃OH、-S(O)(CH₂)₄OH、-S(O)₂CH₂OH、-S(O)₂(CH₂)₂OH、-S(O)₂(CH₂)₃OH、-S(O)₂(CH₂)₄OH、-OCH₂SMe、-O(CH₂)₂SMe、-O(CH₂)₃SMe、-O(CH₂)₄SMe、-SCH₂SMe、-S(CH₂)₂SMe、-S(CH₂)₃SMe、-S(CH₂)₄SMe、-S(O)CH₂SMe、-S(O)(CH₂)₂SMe、-S(O)(CH₂)₃SMe、-S(O)(CH₂)₄SMe、-S(O)₂CH₂SMe、-S(O)₂(CH₂)₂SMe、-S(O)₂(CH₂)₃SMe or-S (O)₂(CH₂)₄SMe。

23. A compound according to any one of embodiments 1 to 11, wherein-L¹-Z is- (C)₁-C₆Alkylene) -X¹-L²-X²。

24. The compound of embodiment 23, wherein-L¹-Z is

25. The compound of embodiment 1, wherein said compound is any one of the compounds selected from the group consisting of:

26. the compound of embodiment 1, wherein said compound is any one selected from the group of compounds of table 1B.

27. A compound of formula II or a pharmaceutically acceptable salt thereof,

E-A-Z'-L¹-D (formula II)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

Each R ⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols; and is

E is-C (═ O) -L³-X³；

L³Is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X³The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X³is that -C(＝O)-CR^bbR^cc-W'、-NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰；

Each W' is independently-H, -N (R)^gg)₂、C₁-C₁₀Alkyl radical, C₁-C₁₀Alkenyl radical, C₁-C₁₀Alkynyl, C₃-C₆Cycloalkyl, aryl, heteroaryl or- (CH)₂CH₂O)_q-R^ff；

q is 1 to 24;

each R^aa、R^bb、R^cc、R^eeAnd R^ffIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R^ggeach independently is-H or C ₁-C₆An alkyl group; and is

R⁹And R¹⁰Each independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

28. The compound of embodiment 27, wherein R¹is-H or-F.

29. A compound of embodiment 27 or embodiment 28, wherein R¹is-F.

30. The compound of any one of embodiments 27-29, R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl.

31. The compound of any one of embodiments 27-30, wherein R²is-F.

32. The compound of any one of embodiments 27-30, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

33. The compound of any one of embodiments 27-30, wherein R²Is methyl.

34. The compound of embodiment 27, wherein R¹is-F and R²is-F.

35. The compound of embodiment 27, wherein R¹Is methyl and R²is-F.

36. The compound of embodiment 27, wherein R¹is-F and R²Is a-methyl group.

37. The compound of any one of embodiments 27-36, wherein-L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (C)₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

38. The compound of embodiment 37, wherein-L ¹-Z' -is-CH₂O-CH₂NH-*、-(CH₂)₂O-CH₂NH-*、-(CH₂)₃O-CH₂NH-*、-(CH₂)₄O-CH₂NH-*、-CH₂S-CH₂NH-*、-(CH₂)₂S-CH₂NH-*、-(CH₂)₃S-CH₂NH-*、-(CH₂)₄S-CH₂NH-*、-CH₂NH-*、-(CH₂)₂NH-*、-(CH₂)₃NH- [ or- (CH)₂)₄NH-。

39. The compound of any one of embodiments 27-36, wherein-L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-, wherein is a site covalently linked to a.

40. The compound of embodiment 39, wherein-L¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-*。

41. The compound of embodiment 39 or embodiment 40, wherein each R is⁵independently-H, methyl or benzyl.

42. The compound of any one of embodiments 37-41, wherein each R⁸independently-H, methyl or benzyl.

43. The compound of any one of embodiments 27-36, wherein-L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

44. The compound of embodiment 43 wherein-L¹-Z' -is-OCH₂O-CH₂-NH-*、-O(CH₂)₂O-CH₂-NH-*、-O(CH₂)₃O-CH₂-NH-*、-O(CH₂)₄O-CH₂-NH-*、-SCH₂O-CH₂-NH-*、-S(CH₂)₂O-CH₂-NH-*、-S(CH₂)₃O-CH₂-NH-*、-S(CH₂)₄O-CH₂-NH-*、-S(O)CH₂O-CH₂-NH-*、-S(O)(CH₂)₂O-CH₂-NH-*、-S(O)(CH₂)₃O-CH₂-NH-*、-S(O)(CH₂)₄O-CH₂-NH-*、-S(O)₂CH₂O-CH₂-NH-*、-S(O)₂(CH₂)₂O-CH₂-NH-*、-S(O)₂(CH₂)₃O-CH₂-NH-*、-S(O)₂(CH₂)₄O-CH₂-NH-*、-OCH₂S-CH₂-NH-*、-O(CH₂)₂S-CH₂-NH-*、-O(CH₂)₃S-CH₂-NH-*、-O(CH₂)₄S-CH₂-NH-*、-SCH₂S-CH₂-NH-*、-S(CH₂)₂S-CH₂-NH-*、-S(CH₂)₃S-CH₂-NH-*、-S(CH₂)₄S-CH₂-NH-*、-S(O)CH₂S-CH₂-NH-*、-S(O)(CH₂)₂S-CH₂-NH-*、-S(O)(CH₂)₃S-CH₂-NH-*、-S(O)(CH₂)₄S-CH₂-NH-*、-S(O)₂CH₂S-CH₂-NH-*、-S(O)₂(CH₂)₂S-CH₂-NH-*、-S(O)₂(CH₂)₃S-CH₂-NH-*、-S(O)₂(CH₂)₄S-CH₂-NH-*、-OCH₂-NH-*、-O(CH₂)₂-NH-*、-O(CH₂)₃-NH-*、-O(CH₂)₄S-NH-*、-SCH₂-NH-*、-S(CH₂)₂-NH-*、-S(CH₂)₃-NH-*、-S(CH₂)₄-NH-*、-S(O)CH₂-NH-*、-S(O)(CH₂)₂-NH-*、-S(O)(CH₂)₃-NH-*、-S(O)(CH₂)₄-NH-*、-S(O)₂CH₂-NH-*、-S(O)₂(CH₂)₂-NH-*、-S(O)₂(CH₂)₃-NH-or-S (O)₂(CH₂)₄-NH-*。

45. The compound of any one of embodiments 27-36, wherein-L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z' -, wherein is a site covalently linked to a.

46. The compound of embodiment 45, wherein-L¹-Z' -is

Wherein is a site covalently linked to a.

47. The compound of any one of embodiments 27-46, wherein A is a peptide comprising 2 to 8 amino acids.

48. The compound of any one of embodiments 27-47, wherein A is a peptide comprising 2 to 4 amino acids.

49. The compound of any of embodiments 27-48, wherein at least one amino acid in said peptide is an L amino acid.

50. The compound of any one of embodiments 27-49, wherein each amino acid in the peptide is an L amino acid.

51. The compound of any one of embodiments 27-48, wherein at least one amino acid in said peptide is a D amino acid.

52. The compound of any one of embodiments 27-46, wherein A is- (AA)¹)-(AA²)_a1-, wherein is a site covalently linked to E; AA¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

53. The compound of embodiment 52, wherein-AA 1- (AA2) a 1-is-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-, -Ala-Lys-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Ala-, -Leu-Cit-, -Leu-Cit-, -Phe-Ala-, -, -Ala-Phe-, -Phe-N9-tosyl-Arg-, -N9-tosyl-Arg-Phe-, -Phe-N9-nitro-Arg-, -N9-nitro-Arg-Phe-, -Phe-Lys-, -Lys-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Val-, -Ala-Leu-Al a-Leu-, -Leu-), -Leu-Ala-, - β -Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -Val-Arg-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, - -Met-, -Met-Thr-, -Leu-Ala-, - -Leu-Cit, -Cit-Val-, -Gln-Val-, -Leu-, -, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr-, and-Tyr-Met-).

54. The compound of embodiment 52 wherein-AA¹-(AA²)_a1-Val-D-Lys-Val-D-Arg-L-Val-Cit-L-Val-Lys-L-Val-Arg-L-Val-D-Cit-L-Phe-Phe-Lys-L-Val-D-Lys-L-Val-D-Arg-L-Arg-D-Arg-L-Ala-Al a-L-Ala-D-Cit-L-Val-D-Cit-, L-Val, -L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val- ".

55. The compound of embodiment 52 wherein-AA¹-(AA²)_a1-:

-Ala-Ala-*、

-Ala-Val-*、

-Val-Ala-*

-Gln-Leu-*、

-Leu-Gln-*

-Ala-Ala-Ala-*、

-Ala-Ala-Ala-Ala-*、

-Gly-Ala-Gly-Gly-*、

-Gly-Gly-Ala-Gly-*、

-Gly-Val-Gly-Gly-*、

-Gly-Gly-Val-Gly-*、

-Gly-Phe-Gly-Gly-or

-Gly-Gly-Phe-Gly-*。

56. The compound of embodiment 52 wherein-AA¹-(AA²)_a1-: -L-Ala-L-Ala-),

-L-Ala-D-Ala-*、

-L-Ala-L-Val-*、

-L-Ala-D-Val-*、

-L-Val-L-Ala-*、

-L-Val-D-Ala-*

-L-Gln-L-Leu-*、

-L-Gln-D-Leu-*、

-L-Leu-L-Gln-*、

-L-Leu-D-Gln-*、

-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-*、

-L-Ala-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-D-Ala-*、

-Gly-L-Ala-Gly-Gly-*、

-Gly-Gly-L-Ala-Gly-*、

-Gly-D-Ala-Gly-Gly-*、

-Gly-Gly-D-Ala-Gly-*、

-Gly-L-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-D-Val-Gly-Gly-*、

-Gly-Gly-D-Val-Gly-*、

-Gly-L-Phe-Gly-Gly-or

-Gly-Gly-L-Phe-Gly-*。

57. The compound of embodiment 52 wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-LAla-*、

-L-Ala-L-Ala-L-Ala-or

-L-Ala-L-Ala-L-Ala-L-Ala-*。

58. The compound of any of embodiments 27-57 wherein A is substituted with one or more polyols.

59. The compound of any of embodiments 27-58 wherein E is substituted with one or more polyols.

60. The compound of any one of embodiments 27-59, whereinWherein the polyol is- (C)₁-C₆Alkylene) -X⁵-Y³；

Wherein:

X⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；

Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and is

R¹²is-H, C ₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

61. The compound of embodiment 60, wherein the polyol is

Wherein R is¹²Is H or methyl.

62. The compound of any one of embodiments 27-61, wherein E is-C (═ O) - (C)₁-C₁₀Alkylene) -X³。

63. The compound of embodiment 62, wherein E is

64. The compound of any one of embodiments 27-61 wherein E is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X³；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X³Is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

65. The compound of any one of embodiments 27-61 wherein E is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X³；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X³is that -C(＝O)-CR^bbR^cc-W'、NR^ee-C(＝O)-CR^bbR^cc-W' or-SR¹⁰。

66. The compound of embodiment 27, wherein said compound is any one selected from the compounds of table 2.

67. A compound of formula III or a pharmaceutically acceptable salt thereof,

CBA-E'-A-Z'-L¹-D (formula III)

Wherein:

d is represented by the following structural formula:

R¹is-H, -F, -CH₃or-CF₃；

R³Is H or C₁-C₆An alkyl group;

R⁴is C₁-C₆An alkyl group;

L¹is absent, is- (C₁-C₆Alkylene) -, - (C)₁-C₆Alkylene) -X¹-(C₁-C₆Alkylene) -, X^1'-(C₁-C₆Alkylene) - (or- (C)₁-C₆Alkylene) -X¹-L²-; wherein is a site covalently linked to Z';

X¹is-O-, -S (O)₂-、-C(＝O)-、-NR⁵-、-NR⁵C (═ O) -or-C (═ O) NR⁵-；

X^1'is-O-, -S (O) -or-S (O)₂-；

L²Is phenylene;

each R⁵Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

z' is-O-CH₂-NR⁸-*、-S-CH₂-NR⁸-*、-NR⁸-; wherein is a site covalently linked to a;

each R⁸Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

L¹and L²Each independentlyOptionally substituted by 1-4 substituents selected from halogen, -CN, -OR⁷、-SR⁷、-N(R⁷)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, or heteroaryl; and is

Each R⁷Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

a is a peptide comprising 2 to 10 amino acids; wherein a is optionally substituted with one or more polyols;

e' is-C (═ O) -L³-X⁶-; wherein is a site covalently attached to the CBA;

L³is- (C)₁-C₁₀Alkylene) -or-Y¹-(C₁-C₁₀Alkylene) -X⁴-Y²-(C₁-C₁₀Alkylene) -; wherein is covalently linked to X ⁶The site of (a);

Y¹is absent, is- (CR^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -or-C (═ O) NR⁹-；

Y²Is absent, is- (CR^cR^dO)_o-or- (CR)^cR^dCR^c'R^d'O)_p-；

n, m, o and p are each independently 1-10;

each R^a、R^b、R^a'、R^b'、R^c、R^d、R^c'And R^d'Independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

wherein L is³Optionally selected from 0-4From halogen, -CN, -OR¹¹、-SR¹¹、-N(R¹¹)₂、C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₁-C₆Heteroalkyl group, C₃-C₆Cycloalkyl radical, C₂-C₁₀Heterocycloalkyl, aryl, heteroaryl, and polyol;

each R¹¹Independently is H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA;

each R^aa、R^bb、R^ccAnd R^eeIndependently is-H or optionally substituted C₁-C₆An alkyl group;

each R^YYAnd R^XXIndependently is-H or C₁-C₆An alkyl group;

R⁹independently is-H, C₁-C₆Alkyl radical, C₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl; and is

CBA is a cell binding agent.

68. The compound of embodiment 67 wherein R¹is-H or-F.

69. The compound of embodiment 67 or embodiment 68, wherein R¹is-F.

70. The method of any one of embodiments 67 to 69Compound (I) R²is-H, -F, -OCF₃、-CF₃、-OMe、-OEt、-SMe、-S(O)Me、-S(O)₂Me、-SEt、-S(O)Et、-S(O₂) Et, methyl or ethyl.

71. The compound of any of embodiments 67-70, wherein R²is-F.

72. The compound of any of embodiments 67-70, wherein R²is-OMe, -SMe, -S (O) Me or methyl.

73. The compound of any of embodiments 67-70, wherein R²Is methyl.

74. The compound of embodiment 67 wherein R¹is-F and R²is-F.

75. The compound of embodiment 67 wherein R¹Is methyl and R²is-F.

76. The compound of embodiment 67 wherein R¹is-F and R²Is a-methyl group.

77. The compound of any of embodiments 67-76, wherein-L¹-Z' -is- (C)₁-C₄Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (C)₁-C₄Alkylene) -NR⁸-, wherein is a site covalently linked to a.

78. The compound of embodiment 77, wherein-L¹-Z' -is-CH₂O-CH₂NH-*、-(CH₂)₂O-CH₂NH-*、-(CH₂)₃O-CH₂NH-*、-(CH₂)₄O-CH₂NH-*、-CH₂S-CH₂NH-*、-(CH₂)₂S-CH₂NH-*、-(CH₂)₃S-CH₂NH-*、-(CH₂)₄S-CH₂NH-*、-CH₂NH-*、-(CH₂)₂NH-*、-(CH₂)₃NH- [ or- (CH)₂)₄NH-。

79. A compound of any of embodiments 67 to 76, whereinmiddle-L¹-Z' -is- (C)₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -O-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -NR⁵C(＝O)-(C₁-C₅Alkylene) -S-CH₂-NR⁸-*、-(C₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -S-CH₂-NR⁸- (C)₁-C₅Alkylene) -S- (C)₁-C₅Alkylene) -SS-CH₂-NR⁸-, wherein is a site covalently linked to a.

80. The compound of embodiment 79, wherein-L ¹-Z' -is-CH₂NHC(＝O)CH₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄O-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅O-CH₂-NH-*、-CH₂NHC(＝O)CH₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₂S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₃S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₄S-CH₂-NH-*、-CH₂NHC(＝O)(CH₂)₅S-CH₂-NH-*、-CH₂SCH₂O-CH₂-NH-*、-CH₂S(CH₂)₂O-CH₂-NH-*、-CH₂S(CH₂)₃O-CH₂-NH-*、-CH₂S(CH₂)₄O-CH₂-NH-*、-CH₂S(CH₂)₅O-CH₂-NH-*、-CH₂SCH₂S-CH₂-NH-*、-CH₂S(CH₂)₂S-CH₂-NH-*、-CH₂S(CH₂)₃S-CH₂-NH-*、-CH₂S(CH₂)₄S-CH₂-NH-or-CH₂S(CH₂)₅S-CH₂-NH-*。

81. The compound of embodiment 79 or embodiment 80, wherein each R⁵independently-H, methyl or benzyl.

82. The compound of any one of embodiments 77-81, wherein each R⁸independently-H, methyl or benzyl.

83. The compound of any of embodiments 67-76, wherein-L¹-Z' -is-X^1'-(C₁-C₄Alkylene) -O-CH₂-NR⁸-*、-X^1'-(C₁-C₄Alkylene) -S-CH₂-NR⁸- (O-X) - (Y-O) -or-X^1'-(C₁-C₄Alkylene) -NR⁸-*，

84. The compound of embodiment 83, wherein-L¹-Z' -is-OCH₂O-CH₂-NH-*、-O(CH₂)₂O-CH₂-NH-*、-O(CH₂)₃O-CH₂-NH-*、-O(CH₂)₄O-CH₂-NH-*、-SCH₂O-CH₂-NH-*、-S(CH₂)₂O-CH₂-NH-*、-S(CH₂)₃O-CH₂-NH-*、-S(CH₂)₄O-CH₂-NH-*、-S(O)CH₂O-CH₂-NH-*、-S(O)(CH₂)₂O-CH₂-NH-*、-S(O)(CH₂)₃O-CH₂-NH-*、-S(O)(CH₂)₄O-CH₂-NH-*、-S(O)₂CH₂O-CH₂-NH-*、-S(O)₂(CH₂)₂O-CH₂-NH-*、-S(O)₂(CH₂)₃O-CH₂-NH-*、-S(O)₂(CH₂)₄O-CH₂-NH-*、-OCH₂S-CH₂-NH-*、-O(CH₂)₂S-CH₂-NH-*、-O(CH₂)₃S-CH₂-NH-*、-O(CH₂)₄S-CH₂-NH-*、-SCH₂S-CH₂-NH-*、-S(CH₂)₂S-CH₂-NH-*、-S(CH₂)₃S-CH₂-NH-*、-S(CH₂)₄S-CH₂-NH-*、-S(O)CH₂S-CH₂-NH-*、-S(O)(CH₂)₂S-CH₂-NH-*、-S(O)(CH₂)₃S-CH₂-NH-*、-S(O)(CH₂)₄S-CH₂-NH-*、-S(O)₂CH₂S-CH₂-NH-*、-S(O)₂(CH₂)₂S-CH₂-NH-*、-S(O)₂(CH₂)₃S-CH₂-NH-*、-S(O)₂(CH₂)₄S-CH₂-NH-*、-OCH₂-NH-*、-O(CH₂)₂-NH-*、-O(CH₂)₃-NH-*、-O(CH₂)₄S-NH-*、-SCH₂-NH-*、-S(CH₂)₂-NH-*、-S(CH₂)₃-NH-*、-S(CH₂)₄-NH-*、-S(O)CH₂-NH-*、-S(O)(CH₂)₂-NH-*、-S(O)(CH₂)₃-NH-*、-S(O)(CH₂)₄-NH-*、-S(O)₂CH₂-NH-*、-S(O)₂(CH₂)₂-NH-*、-S(O)₂(CH₂)₃-NH-or-S (O)₂(CH₂)₄-NH-*。

85. The compound of any of embodiments 67-76, wherein-L¹-Z' -is- (C)₁-C₆Alkylene) -X¹-L²-Z'-*。

86. The compound of embodiment 85 wherein-L¹-Z' -is

87. The compound of any one of embodiments 67-86, wherein a is a peptide comprising 2 to 8 amino acids.

88. The compound of any of embodiments 67-87, wherein a is a peptide comprising 2 to 4 amino acids.

89. The compound of any of embodiments 67-88, wherein at least one amino acid in the peptide is an L amino acid.

90. The compound of any one of embodiments 67-89, wherein each amino acid in the peptide is an L amino acid.

91. The compound of any of embodiments 67-88, wherein at least one amino acid in the peptide is a D amino acid.

92. The compound of any one of embodiments 67-86, wherein a is- (AA)¹)-(AA²)_a1-¹And AA²Each independently is an amino acid residue; and a1 is an integer from 1 to 9.

93. The compound of embodiment 92, -AA1- (AA2) a 1-is-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe-, -Leu-Cit-, -Cit-Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Leu-Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -, -Phe-N9-tosyl-Arg-, -N9-tosyl-Arg-Phe-, -Phe-N9-nitro-Arg-, -N9-nitro-Arg-Phe-, -Phe-Lys-, -Lys-Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Leu-, -Ala-Leu-, -Leu-Ala-Leu-Ala-Leu-, -Leu-Ala-Leu-, -, - β -Ala-Leu-, -Gly-Phe-Leu-Gly-, -Gly-Leu-Phe-Gly-, -Val-Arg-, -Arg-Val-, -Arg-, -Ala-, -Ala-Met-, -Met-Ala-, -Thr-, - -Met-, - -Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, -Val-Gln-, -Ser-Val-, -Leu-, -Ser-Val-, -, -Val-Ser-, -Ser-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val-, -Met-Tyr- & Tyr-Met- & ltr-Gln- & ltr- & gt.

94. The compound of embodiment 92, wherein-AA¹-(AA²)_a1-Val-D-Lys-, -Val-D-Arg-, -L-Val-Cit-, -L-Val-Lys-, -L-Val-Arg-, -L-Val-D-Cit-, -L-Phe-Phe-Lys-, -L-Val-D-Arg-, -L-Arg-D-Arg-, -L-Ala-Ala-Ala-, -L-Ala-D-Ala-, -Val-D-Cit-, -, -L-Ala-L-Ala-, -L-Ala-L-Val-, -L-Gln-L-Leu-, -L-Ser-L-Val-,.

95. The compound of embodiment 92, wherein-AA¹-(AA²)_a1-:

-Ala-Ala-*、

-Ala-Val-*、

-Val-Ala-*

-Gln-Leu-*、

-Leu-Gln-*

-Ala-Ala-Ala-*、

-Ala-Ala-Ala-Ala-*、

-Gly-Ala-Gly-Gly-*、

-Gly-Gly-Ala-Gly-*、

-Gly-Val-Gly-Gly-*、

-Gly-Gly-Val-Gly-*、

-Gly-Phe-Gly-Gly-or

-Gly-Gly-Phe-Gly-*。

96. The compound of embodiment 92, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-*、

-L-Ala-L-Val-*、

-L-Ala-D-Val-*、

-L-Val-L-Ala-*、

-L-Val-D-Ala-*

-L-Gln-L-Leu-*、

-L-Gln-D-Leu-*、

-L-Leu-L-Gln-*、

-L-Leu-D-Gln-*、

-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-*、

-L-Ala-L-Ala-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-L-Ala-*、

-L-Ala-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-D-Ala-*、

-Gly-L-Ala-Gly-Gly-*、

-Gly-Gly-L-Ala-Gly-*、

-Gly-D-Ala-Gly-Gly-*、

-Gly-Gly-D-Ala-Gly-*、

-Gly-L-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-D-Val-Gly-Gly-*、

-Gly-Gly-L-Val-Gly-*、

-Gly-L-Phe-Gly-Gly-or

-Gly-Gly-L-Phe-Gly-*。

97. The compound of embodiment 92, wherein-AA¹-(AA²)_a1-:

-L-Ala-L-Ala-*、

-L-Ala-D-Ala-L-Ala-*、

-L-Ala-L-Ala-L-Ala-or

-L-Ala-L-Ala-L-Ala-L-Ala-*。

98. The compound of any of embodiments 67-97 wherein a is substituted with one or more polyols.

99. The compound of any of embodiments 67-98, wherein E' is substituted with one or more polyols.

100. The compound of any of embodiments 67-99 wherein the polyol is- (C)₁-C₆Alkylene) -X⁵-Y³；

Wherein:

X⁵is-NR¹²C (═ O) -or-C (═ O) NR¹²-；

Y³is-C₁-C₁₀Alkyl radical, wherein Y³Substituted with 0-10 OH groups; and is

R¹²is-H, C₁-C₆Alkyl radical, C ₁-C₆Fluoroalkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl or benzyl.

101. The compound of embodiment 100, wherein the polyol isWherein R is¹²Is H or methyl.

102. The compound of embodiments 63-93 wherein E' is-C (═ O) - (C)₁-C₁₀Alkylene) -X⁶-*。

103. The compound of embodiment 102 wherein E' is

-C(＝O)CH₂CH₂-C(＝O)-CR^bbR^cc- (O) CH₂CH₂-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

104. A compound according to any one of embodiments 63-93 wherein E' is-C (═ O) -Y¹-(C₁-C₁₀Alkylene) -X⁴-(C₁-C₁₀Alkylene) -X⁶-*；

Y¹Is- (CR)^aR^bO)_n-or- (CR)^aR^bCR^a'R^b'O)_m-；

X⁴is-NR⁹C (═ O) -; and is

X⁶Is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

105. The compound of any one of embodiments 63-93, wherein E' is-C (═ O) -Y¹-(CH₂)₂-X⁴-(CH₂)₂-X⁶-*；

Y¹Is- (CH)₂O)_n-or- (CH)₂CH₂O)_m-；

X⁴is-NHC (═ O) -;

n is 2; m is 2 to 6;

X⁶is that -C(＝O)-CR^bbR^cc- (O) -or-NR^ee-C(＝O)-CR^bbR^cc-; wherein is a site covalently attached to the CBA.

106. The compound of any of embodiments 63-105, wherein the CBA comprises an-SH group covalently attached to E' to provide -C(＝O)-CR^bbR^cc-S-CBA or-NR^ee-C(＝O)-CR^bbR^cc-S-CBA。

107. The compound of any one of embodiments 67-106, wherein CBA is an antibody and-E '-a-Z' -L¹-D is a drug-linker moiety, the average number of drug-linker moieties conjugated per antibody being in the range of 2 to 10.

108. The compound of embodiment 107, wherein the average number of drug-linker moieties conjugated per antibody is in the range of 2 to 10.

109. The compound of embodiment 107, wherein the average number of drug-linker moieties conjugated per antibody is in the range of 6 to 8.

110. The compound of embodiment 107, wherein the average number of drug-linker moieties conjugated per antibody is 8.

111. The compound of any one of embodiments 67-110, wherein the CBA is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a hexabody, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient-transporting molecule.

112. The compound of any one of embodiments 67-111, wherein the CBA binds to a target cell selected from the group consisting of: tumor cells, virally infected cells, microbially infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; expression of 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B2-H2, BCMA, C4.4a, CA 2, CanAg, CD123, CD138, CD142, CD166, CD184, CD2, CD205, CD2, CD 36248, CD2, CD 36352, CD2, CD40 2, CD44v 2, CD79 2, CDH 2, CEACAM 2, cKIT, cKIN 18.2, N2, CLL-1, EGFC-MET, Criptto, CSP-2, GCCLDLLR-1-DLL 72, EPCR 1-GCDLP-2, EPTC-GCDLP-2, EPTC-3-GCD-3, EPTC-2, EPTC-3, EPTC-CTC-2, EPC-2, EPTC-2, EPDCD-3, EPC-3, EPTC-3, EPDCHA-3, EPDCD-3, EPC-3, EPTC-3, EPDCHA-3, EPC-2, EPDCHA-3, EPDCD-3, EPTC-2, EPC-3, EPTC-3, EPDCD-3, EPC-3, EPTC-3, EPC-3, EPDCHA-3, EPTC-3, EPDCD-2, EPDCHA-3, EPTC-3, EPDCD-3, EPTC-3, EPDCHA-3, EPTC-3, EPDCHA-3, EPTC-3, EPDCHA-3, FGFR-3, EPTC-3, FGFR-3, EPTC-3, EPDCHA-3, EPTC-3, FGFR-3, EPTC-3, EPDCHA-3, EPTC-3, FGFR-3, lewis Y antigen, LGALS3BP, LGR5, LH/hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, Mesothelin (MSLN), MET, MHC class I chain-associated proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2B, Nectin-4, NOTCH3, OAcGD2, 001 OX L, p-cadherin, PD-L1, Phosphatidylserine (PS), Polymorphic Epithelial Mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, TRAMF 7, TRT44A 7, STK 7, STEAP-1, STAP-72, SLC-1, SLC-72, SLC 7, SLC-associated glycoprotein, SLC 7, SLC-3-72, SLC 7, SLC-3-72, SLC 7, or any epitope specific for a tumor associated with a tumor.

113. The compound of any one of embodiments 67-110, wherein the cell binding agent is an anti-folate receptor antibody or antibody fragment thereof, an anti-EGFR antibody or antibody fragment thereof, an anti-CD 33 antibody or antibody fragment thereof, an anti-CD 19 antibody or antibody fragment thereof, an anti-Muc 1 antibody or antibody fragment thereof, an anti-CD 37 antibody or antibody fragment thereof, or an anti-EpCAM antibody or antibody fragment thereof.

114. A pharmaceutical composition comprising a compound according to any one of embodiments 1-113, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

115. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 114.

116. The method of embodiment 115, wherein the cancer is lymphoma or leukemia.

117. The method of embodiment 116, wherein the cancer is Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), Acute Lymphoblastic Leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), Chronic Lymphocytic Leukemia (CLL), Hairy Cell Leukemia (HCL), Acute Promyelocytic Leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), non-hodgkin's lymphoma (NHL), Mantle Cell Leukemia (MCL), Small Lymphocytic Lymphoma (SLL), hodgkin's lymphoma, systemic mastocytosis, and burkitt's lymphoma.

118. The method of embodiment 115, wherein the cancer is endometrial, lung, colorectal, bladder, gastric, pancreatic, renal cell, prostate, esophageal, breast, head and neck, uterine, ovarian, liver, cervical, thyroid, testicular, bone marrow, melanoma, and lymphatic cancer.

119. The method of embodiment 115, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

Other embodiments are set forth in the following claims.

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