阅读说明：本技术 抗体-药物偶联物及其用途 (Antibody-drug conjugates and uses thereof ) 是由 郑镇沅 金柱嬉 朴泳燉 宋大海 严在铉 廉东勋 李宝罗 洪荣恩 于 2020-03-06 设计创作，主要内容包括：本发明涉及一种包含半乳糖触发部分和环丙基苯并吲哚(CBI)的新型化合物,以及使用其制备的抗体-药物偶联物。(The present invention relates to a novel compound comprising a galactose trigger moiety and Cyclopropylbenzindole (CBI), and an antibody-drug conjugate prepared using the same.)

1. A compound represented by the following formula (I) or a pharmaceutically acceptable salt or solvate thereof:

wherein R is¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br; and is

R³Is a single bond, -O-or-NH-.

2. A compound represented by the following formula (II):

wherein R is¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer; and is

L¹Is a joint.

3. The compound of claim 2, wherein W is-R⁴-A-R⁵-、-R⁴-A-、-(CH₂CH₂R⁶)_x-、-(CH₂)_r(R⁷(CH₂)_p)_q-、-((CH₂)_pR⁷)_q-、-(CH₂)_r(R⁷(CH₂)_p)_qR⁸-、-((CH₂)_pR⁷)_q(CH₂)_r-、-R⁸((CH₂)_pR⁷)_q-or- (CH)₂)_r(R⁷(CH₂)_p)_qR⁸CH₂-，

Wherein R is⁴And R⁵Each independently is- (CH)₂)_r(V(CH₂)_x)_p(CH₂)_qWherein A is a direct bond or a peptide bond; and V is a single bond, O or S; and is

R⁶is-O-, C₁-C₈Alkylene, -NR⁹-or-C (O) NR¹³-; and is

R⁷And R⁸Each independently a single bond, -O-, -NR¹⁰-、-C(O)NR¹¹-、-NR¹²C (O) -or C₃-C₂₀(ii) a heteroaryl group, wherein,

wherein R is⁹To R¹³Each independently is hydrogen, C₁-C₆Alkyl, (C)₁-C₆Alkyl) C₆-C₂₀Aryl or (C)₁-C₆Alkyl) C₃-C₂₀A heteroaryl group;

x is an integer of 1 to 5;

r is an integer from 0 to 10;

p is an integer of 0 to 10; and is

q is an integer of 0 to 20,

wherein 1 to 10 hydrogen atoms in W are optionally substituted by hydroxyl, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl substitution.

4. The compound of claim 2, wherein L¹Is hydroxy, aldehyde, ONH₂、NH₂Or from 1 to 3 selected fromN, O and S, or a structure represented by the following formula (I-a) or (I-b), wherein the heteroaryl group can be substituted with 1 to 5 substituents independently selected from hydroxyl, aldehyde, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂And substituent substitution of carbonyl:

wherein Q is¹Is cyclooctynyl or heterocyclooctynyl, wherein the cyclooctynyl or heterocyclooctynyl is optionally and independently selected from C₃-C₁₂Cycloalkyl radical, C₃-C₁₂Aryl and C₃-C₁₂1 or 2 rings of heteroaryl being fused and optionally substituted by hydroxy, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl substitution;

R¹³is selected from C₁-C₂₄Alkyl radical, C₃-C₂₄Cycloalkyl radical, C₃-C₂₄Aryl radical, C₃-C₂₄Heteroaryl group, C₃-C₂₄Alkylaryl group, C₃-C₂₄Alkyl heteroaryl, C₃-C₂₄Arylalkyl and C₃-C₂₄Heteroarylalkyl, wherein the heteroaryl contains a group selected from O, S and NR¹⁴Wherein R is¹⁴Is hydrogen or C₁-C₄An alkyl group;

Sp¹、Sp²、Sp³and Sp⁴Are spacer moieties and are each independently selected from: a single bond, or C being straight or branched₁-C₂₀₀Alkylene radical, C₂-C₂₀₀Alkenylene radical, C₂-C₂₀₀Alkynylene, C₃-C₂₀₀Cycloalkylene radical, C₅-C₂₀₀Cycloalkenylene group, C₈-C₂₀₀Cycloalkynylene, C₇-C₂₀₀Alkylarylene, C₇-C₂₀₀Arylalkylene radical, C₈-C₂₀₀ArylaryleneAlkenyl and C₉-C₂₀₀Arylalkynylene, wherein said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene are optionally selected from O, S and NR¹⁴Is substituted or contains heteroatoms selected from O, S and NR¹⁴A heteroatom of (a);

Z¹and Z²Each independently selected from O, C (O) and N (R)¹³)；

a is each independently 0 or 1;

each b is independently 0 or 1;

c is 0 or 1;

d is 0 or 1;

e is 0 or 1;

f is an integer of 0 to 150;

g is 0 or 1; and is

i is 0 or 1.

5. The compound of claim 2, comprising a compound represented by the formula:

6. the compound of claim 2, comprising a compound represented by the formula:

7. an antibody-drug conjugate comprising a compound represented by the following formula (III):

wherein

R¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer;

L²is a joint; and is

Ab is an antibody or antigen-binding fragment thereof.

8. The antibody-drug conjugate of claim 7, wherein W is-R⁴-A-R⁵-、-R⁴-A-、-(CH₂CH₂R⁶)_x-、-(CH₂)_r(R⁷(CH₂)_p)_q-、-((CH₂)_pR⁷)_q-、-(CH₂)_r(R⁷(CH₂)_p)_qR⁸-、-((CH₂)_pR⁷)_q(CH₂)_r-、-R⁸((CH₂)_pR⁷)_q-or- (CH)₂)_r(R⁷(CH₂)_p)_qR⁸CH₂-，

Wherein R is⁴And R⁵Each independently is- (CH)₂)_r(V(CH₂)_x)_p(CH₂)_qWhich isWherein A is a direct bond or a peptide bond; and V is a single bond, O or S; and is

R⁶is-O-, C₁-C₈Alkylene, -NR⁹-or-C (O) NR₂-; and is

R⁷And R⁸Each independently a single bond, -O-, -NR¹⁰-、-C(O)NR¹¹-、-NR¹²C (O) -or C₃-C₂₀(ii) a heteroaryl group, wherein,

wherein R is⁹To R¹³Each independently is hydrogen, C₁-C₆Alkyl, (C)₁-C₆Alkyl) C₆-C₂₀Aryl or (C)₁-C₆Alkyl) C₃-C₂₀A heteroaryl group;

x is an integer of 1 to 5;

r is an integer from 0 to 10;

p is an integer of 0 to 10; and is

q is an integer of 0 to 20,

wherein 1 to 10 hydrogen atoms in W are optionally substituted by hydroxyl, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl substitution.

9. The antibody-drug conjugate of claim 7, wherein L²is-CH₂NH-、-ON＝C(CH₃) -, -ON ═, -CH ═ N-, or a4 to 7-membered heterocyclic ring containing 1 to 3 heteroatoms selected from N, O and S, or has a structure represented by the following formula (II-a) or (II-b), wherein the heterocyclic ring may be substituted with 1 to 5 substituents independently selected from hydroxyl, aldehyde, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂And substituent substitution of carbonyl:

wherein Q is²Is a cyclooctenyl group fused with triazole or a heterocyclooctyenyl group fused with triazole, wherein the cyclooctenyl or hetero-octenyl groupCyclooctenyl is optionally further selected from C independently of each other₃-C₁₂Cycloalkyl radical, C₃-C₁₂Aryl and C₃-C₁₂1 or 2 rings of heteroaryl fused, optionally with hydroxy, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl substitution, wherein Q²Is attached to Ab through the nitrogen atom contained in the triazole;

Sp¹、Sp²、Sp³and Sp⁴Are spacer moieties and are each independently selected from: a single bond, or C being straight or branched₁-C₂₀₀Alkylene radical, C₂-C₂₀₀Alkenylene radical, C₂-C₂₀₀Alkynylene, C₃-C₂₀₀Cycloalkylene radical, C₅-C₂₀₀Cycloalkenylene group, C₈-C₂₀₀Cycloalkynylene, C₇-C₂₀₀Alkylarylene, C₇-C₂₀₀Arylalkylene radical, C₈-C₂₀₀Arylalkenylene and C₉-C₂₀₀Arylalkynylene, wherein said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene are optionally selected from O, S and NR¹⁴Is substituted or contains heteroatoms selected from O, S and NR¹⁴A heteroatom of (a);

Z¹and Z²Each independently selected from O, C (O) and N (R)¹³)；

a is each independently 0 or 1;

each b is independently 0 or 1;

c is 0 or 1;

d is 0 or 1;

e is 0 or 1;

f is an integer of 0 to 150;

g is 0 or 1; and is

i is 0 or 1.

10. The antibody-drug conjugate of claim 7, comprising a compound represented by the formula:

11. the antibody-drug conjugate of claim 7, wherein the linker is attached to the antibody by introduction of a cysteine, lysine, azido, or keto group of the antibody or by the N-terminus of an antibody protein.

12. The antibody-drug conjugate of claim 7, wherein the antibody is selected from the group consisting of an anti-BCMA antibody, an anti-ROR 1 antibody, an anti-Her 2 antibody, an anti-NaPi 2b antibody, and an anti-CLL 1 antibody.

13. A pharmaceutical composition for preventing or treating a proliferative disease, comprising the antibody-drug conjugate according to any one of claims 7 to 12.

Technical Field

The present invention relates to a novel compound comprising a galactose trigger moiety and Cyclopropylbenzindole (CBI), and an antibody-drug conjugate prepared using the same.

Background

In recent years, methods for diagnosing or treating various diseases using antibodies have been studied. In particular, various therapeutic methods using antibodies have been developed due to the targeting specificity of antibodies, and various types of drugs including antibodies, such as antibody-drug conjugates (ADCs), are being developed. Therefore, methods of increasing the in vivo stability and maximizing the therapeutic effect of antibodies or antibody-drug conjugates are being continuously studied.

Among them, ADCs generally have lower in vivo stability than natural antibodies, but they have been developed in order to improve the disadvantage that the conventional therapeutic effect of natural antibodies is low by combining with drugs. Drugs having specific drug effects, such as cytotoxins conjugated with target-specific antibodies, have been developed in various ways, and antibody-drug conjugates capable of inducing cancer cell death by binding drugs to cancer cell-specific antibodies have been commercialized.

Clinical trials of ADCs containing DNA minor groove alkylating agents as cargo in drugs are ongoingIn the row. Examples of such drugs include pyrrolobenzodiazepines(PBD) dimers and Cycloprophenylindole (CBI) based duocarmycin derivatives. In particular, CBI is known to be cytotoxic to various types of cancer, and CBI dimers have also been reported to be highly cytotoxic (titze et al, angel w. chem. int. ed. engl.2010,49, 7336-.

The linker may be accidentally cleaved from the ADC before it is delivered to the target cancer cell, from where the drug may be released prematurely, which poses a risk of systemic toxicity. The risk may be greater if the drug is highly cytotoxic. To prevent this phenomenon, when the cleaved derivative in the lysosome is used as a drug, the derivative acts as a trigger (prodrug functional group), and therefore, before releasing the active cytotoxic drug, the trigger should be cleaved in addition to the linker, so that the risk can be reduced.

It has been thought that either carbamate or phosphate groups may be attached to the CBI as a trigger. When a carbamate group is used as a trigger, it can be cleaved by carboxylesterases in lysosomes and/or cytoplasm. When phosphate groups are used as triggers, the phosphate groups may be cleaved by phosphatases in lysosomes and/or cytoplasm. However, these conventional triggers still have safety issues.

In the background of the art, as a result of extensive efforts to develop a trigger moiety capable of effectively exhibiting pharmaceutical activity and improved safety due to higher target-specific toxicity than conventional triggers, the present inventors have found that a prodrug containing galactose as a trigger can be used for ADC, and completed the present invention on the basis thereof.

Disclosure of Invention

An object of the present invention is to provide a compound represented by the following formula (I) or (II), which is a CBI dimer compound bound to a trigger moiety, or a pharmaceutically acceptable salt or solvate thereof.

[ formula I ]

[ formula II ]

It is another object of the present invention to provide an antibody-drug conjugate represented by formula (III), wherein the antibody binds to a compound represented by formula (II), which is a CBI dimer compound bound to a trigger moiety.

[ formula III ]

It is another object of the present invention to provide a composition for preventing or treating a proliferative disease, comprising the antibody-drug conjugate.

In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a compound represented by the following formula (I):

[ formula I ]

Wherein R is¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br; and is

R³Is a single bond, -O-or-NH-.

According to another aspect of the present invention, there is provided a compound represented by the following formula (II):

[ formula II ]

Wherein R is¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer; and is

L¹Is a joint.

According to another aspect of the present invention, there is provided an antibody-drug conjugate comprising a compound represented by the following formula (III):

[ formula III ]

Wherein

R¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer;

L²is a joint; and is

Ab is an antibody or antigen-binding fragment thereof.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a proliferative disease, comprising the conjugate.

Drawings

FIG. 1 is a schematic diagram illustrating the process by which a prodrug CBI dimer is converted to the activated form.

FIG. 2 shows the results of detection of β -galactosidase expression in various tumor cell lines.

Figure 3 shows the results of testing the efficacy of prodrug CBI dimers according to the present invention on free drug.

Fig. 4 shows the results of confirming the purity of the antibody-drug conjugate according to the present invention using SE-HPLC.

Fig. 5 shows the results of confirming the purity of the antibody-drug conjugate according to the present invention using SE-HPLC.

Figure 6 shows the results of analysis of DAR using LC/MS for antibody-drug conjugates according to the invention.

Figure 7 shows the results of analysis of DAR using LC/MS for antibody-drug conjugates according to the invention.

Fig. 8 shows the results of confirming the in vitro cytotoxicity of the antibody-drug conjugate according to the present invention using the H929 cell line.

Fig. 9 shows the results of confirming the in vitro cytotoxicity of the antibody-drug conjugate according to the present invention using each of the Mino and Jeko-1 cell lines.

FIG. 10 shows the results of confirming the in vitro cytotoxicity of the antibody-drug conjugate according to the present invention using each of NCI-N87 and HCC1954 cell lines.

Figure 11 shows the results of confirming the in vitro cytotoxicity of antibody-drug conjugates according to the invention using the OVCAR-3 cell line.

Fig. 12 shows the results of confirming the in vivo cytotoxicity of the antibody-drug conjugate according to the present invention using Jeko-1 model.

Fig. 13 shows the results of confirming the in vivo cytotoxicity of the antibody-drug conjugate according to the present invention using multiple myeloma model xenograft.

Fig. 14 shows the results of confirming the in vivo cytotoxicity of the antibody-drug conjugate according to the present invention using AML model xenograft.

Figure 15 shows the change in body weight of animals administered ADC.

Figure 16 shows the change in blood leukocyte levels of animals administered ADC.

Fig. 17 shows the results of determining the toxicity of the ADCs detected by analysis of blood biochemical parameters such as ALT (alanine aminotransferase), AST (aspartate aminotransferase) and Blood Urea Nitrogen (BUN).

Figure 18 shows the change in body weight in single dose rat toxicity tests.

Fig. 19 shows changes in the level of white blood cells detected by a hematological examination.

Figure 20 shows the presence of toxicity expression and recovery determined based on the changes in body weight and hematological biochemical changes observed after administration of a single dose to SD rats.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, nomenclature used herein is well known in the art and commonly employed.

In one aspect, the present invention relates to a compound represented by the following formula (I):

wherein

R¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br; and is

R³Is a single bond, -O-or-NH-.

In another aspect, the present invention relates to a compound represented by the following formula (II):

wherein R is¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer; and is

L¹Is a joint.

According to another aspect of the present invention, there is provided an antibody-drug conjugate comprising a compound represented by the following formula (III):

wherein

R¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose;

R²is Cl or Br;

R³is a single bond, -O-or-NH-;

w is a spacer;

L²is a joint; and is

Ab is an antibody or antigen-binding fragment thereof.

The compounds of formula (I), (II) and (III) are novel compounds previously unknown, which can be used as drug-tether combinations (drug-tethercombination) for the preparation of antibody-drug complexes and linker-drug complexes; the compounds of formula (II) may be used as linker-drug complexes for the preparation of antibody-drug conjugates; while the compound of formula (III) may be used as an antibody-drug conjugate or a prodrug thereof, the present invention may not be limited thereto.

According to formula (I), (II) or (III), R¹Represents a trigger of a prodrug and, as shown in fig. 1, is removed by an enzyme in the cell to activate CBI dimer, which is a cytotoxic drug. Trigger R¹Is D-galactose beta-pyranose or D-galactose alpha-pyranose.

As used herein, the term "prodrug" refers to a compound that: by enzymatic oxidation, reduction and/or hydrolysis under specific in vivo physiological conditions, by a trigger R¹Inactive CBI dimers may be converted to active CBI dimers, which exhibit activity, such as cytotoxicity.

The present inventors found that when a phosphate group is included as a trigger, cytotoxicity efficacy is excellent, but severe nephrotoxicity may be induced, which may be accompanied by irreversible renal damage. In addition, ADC containing a phosphate group as a trigger is considered to be unstable in serum, but is not limited to such a theory.

However, the present inventors have found that R¹Comprising as a trigger a D-galactose beta-pyranose or a D-galactose alpha-pyranose, which are isomers of galactose, thus enabling the maintenance of the desired targeting-specific cytotoxicity and enabling the solution of the problem of undesired in vivo toxicity.

When galactose is used as a trigger, an enzyme capable of activating galactose may be highly expressed in target cells such as cells having a proliferative disease, particularly cancer cells.

In one embodiment, R¹Is a D-galactose β -pyranose or a D-galactose α -pyranose, and in this case the enzyme may be a galactosidase, in particular a β -galactosidase. Beta-galactosidase is a lysosomal enzyme and is expressed at high levels in most cancer cells, indicating that beta-galactosidase is considered a tumor-selective activating enzyme (fig. 2).

In one embodiment, the following moieties in formula (II) or (III) are capable of linking CBI dimers to each other to enhance pharmacological effects, and represent useful for linking the spacer W and linker L in formula (II)¹Or a spacer W and a linker L in the formula (III)²A tether stably coupled to the drug.

In the tether, R³Is a single bond, O-or-NH-.

Specifically, the conjugate according to the present invention may include a compound represented by the following formula:

in formulas (II) and (III), linker L is bound to spacer W, and the spacer serves to maintain a sufficient distance between the antibody and the drug when the antibody is bound to deliver the drug to the target. Optionally, hydrophilic moieties may be provided to enhance solubility. In some embodiments, the term "linker" may also be referred to as comprising a spacer.

In one embodiment, the spacer W is-R⁴-A-R⁵-、-R⁴-A-、-(CH₂CH₂R⁶)_x-、-(CH₂)_r(R⁷(CH₂)_p)_q-、-((CH₂)_pR⁷)_q-、-(CH₂)_r(R⁷(CH₂)_p)_qR⁸-、-((CH₂)_pR⁷)_q(CH₂)_r-、-R⁸((CH₂)_pR⁷)_q-or- (CH)₂)_r(R⁷(CH₂)_p)_qR⁸CH₂-，

Wherein R is⁴And R⁵Each independently is- (CH)₂)_r(V(CH₂)_x)_p(CH₂)_qA is a direct bond or a peptide bond, and V is a single bond, O or S,

R⁶is-O-, C₁-C₈Alkylene, -NR⁹-or-C (O) NR₂-，

R⁷And R⁸Each independently a single bond, -O-, -NR¹⁰-、-C(O)NR¹¹-、-NR¹²C (O) -or C₃-C₂₀(ii) a heteroaryl group, wherein,

R⁹to R¹²Each independently is hydrogen, C₁-C₆Alkyl, (C)₁-C₆Alkyl) C₆-C₂₀Aryl or (C)₁-C₆Alkyl) C₃-C₂₀(ii) a heteroaryl group, wherein,

x is an integer from 1 to 5, r is an integer from 0 to 10, p is an integer from 0 to 10, and q is an integer from 0 to 20, and

1 to 10 hydrogen atoms in W may optionally be replaced by hydroxyl, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl (oxo) substitution.

In one embodiment, W in formula (II) or formula (III) may include the following:

(1)-R⁴-A-R⁵-or-R⁴-A-, wherein R⁴And R⁵Each independently is- (CH)₂)_r(V(CH₂)_x)_p(CH₂)_qA is a direct bond or a peptide bond, and V is a single bond, O or S,

x is an integer from 1 to 5, in particular 1,2, 3, 4 or 5,

r is an integer from 0 to 10, in particular 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10,

p is an integer from 0 to 10, specifically 1,2, 3, 4, 5, 6, 7, 8, 9 or 10, or specifically 0 to 7, or 0 to 5,

q is an integer from 0 to 20, specifically 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or specifically an integer from 0 to 10, an integer from 0 to 7, or an integer from 0 to 5; and

(2) from- (CH)₂CH₂R⁶)_xA compound represented by- ((CH)₂)_pR⁷)_q(CH₂)_rA compound represented by- (CH) or a salt thereof₂)_r(R⁷(CH₂)_p)_qR⁸CH₂-a compound of (a).

In this case, R⁶is-O-, C₁-C₈Alkylene, -NR⁹-or-C (O) NR¹³-；

R⁷And R⁸Each independently a single bond, -O-, -NR¹⁰-、-C(O)NR¹¹-、-NR¹²C (O) -or C₃-C₂₀Heteroaryl, in particular C₃-C₁₅Heteroaryl, more particularly C₃-C₁₂(ii) a heteroaryl group, wherein,

R⁹to R¹³Each independently is hydrogen, C₁-C₆Alkyl, (C)₁-C₆Alkyl) C₆-C₂₀Aryl, in particular (C)₁-C₆Alkyl) C₃-C₁₅Aryl, more specifically (C)₁-C₆Alkyl) C₃-C₁₂Aryl, or (C)₁-C₆Alkyl) C₃-C₂₀Heteroaryl, in particular (C)₁-C₆Alkyl) C₃-C₁₅Heteroaryl, more specifically (C)₁-C₆Alkyl) C₃-C₁₂A heteroaryl group.

x is an integer from 1 to 5, in particular 1,2, 3, 4 or 5,

r is an integer from 0 to 10, specifically 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, or more specifically 0 to 7, or 0 to 5,

p is an integer from 0 to 10, specifically 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, or more specifically 0 to 7, or 0 to 5,

q is an integer from 0 to 20, specifically 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more specifically 0 to 10,0 to 7, or 0 to 5.

Linker L in formula (II)¹Or the linker L in the formula (III)²The antibody is stably bound to the drug, and when the antibody-drug conjugate reaches a target cell after circulating in vivo, it allows the antibody-drug conjugate to enter the cell, allowing the drug to be easily released therefrom by dissociation of the antibody from the drug, and providing a drug effect on the target cancer cell.

Linker L in formula (II)¹Is hydroxy, aldehyde, ONH₂、NH₂Or a 4-to 7-or 5-to 7-membered heteroaryl group containing 1 to 3 heteroatoms selected from N, O and S, wherein the heteroaryl group may be substituted with 1 to 5 heteroatoms independently selected from hydroxyl, aldehyde, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂And carbonyl substituents.

L in the formula (III)²is-CH₂NH-、-ON＝C(CH₃) -, -ON ═ or a 4-to 7-or 5-to 7-membered heterocycle containing 1 to 3 heteroatoms selected from N, O and S, wherein,the heterocyclic ring can be substituted by 1 to 5 groups independently selected from hydroxyl, aldehyde, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂And carbonyl substituents.

In particular, the joint L¹Or L²May include NH₂、-OH、-ONH₂(hydroxylamine), -NH₂Aldehyde (formyl), -CO₂H. -SH, 2-formylpyridine, sulfonamide, (hetero) cyclooctyne, azide (-N)₃) Or a maleimide.

In some cases, linker L in formula (II)¹Or the linker L in the formula (III)²A compound represented by the following formula (IV) may be included:

wherein, a is 0 or1,

R¹³is selected from C₁-C₂₄Alkyl radical, C₃-C₂₄Cycloalkyl radical, C₃-C₂₄Aryl radical, C₃-C₂₄Heteroaryl group, C₃-C₂₄Alkylaryl group, C₃-C₂₄Alkyl heteroaryl, C₃-C₂₄Arylalkyl and C₃-C₂₄Heteroarylalkyl, wherein heteroaryl contains a residue selected from O, S and NR¹⁴Wherein R is¹⁴Is hydrogen or C₁-C₄An alkyl group.

In particular, the linker L in formula (II)¹May have a structure represented by the following formula (I-a) or (I-b):

wherein Q is¹Is cyclooctynyl or heterocyclooctynyl, wherein cyclooctynyl or heterocyclooctynyl is optionally each independently and independently selected from C₃-C₁₂Cycloalkyl radical, C₃-C₁₂Aryl and C₃-C₁₂1 or 2 rings of the heteroaryl group are fused,and optionally substituted by hydroxy, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or a substitution of a carbonyl group,

R¹³is selected from C₁-C₂₄Alkyl radical, C₃-C₂₄Cycloalkyl radical, C₃-C₂₄Aryl radical, C₃-C₂₄Heteroaryl group, C₃-C₂₄Alkylaryl group, C₃-C₂₄Alkyl heteroaryl, C₃-C₂₄Arylalkyl and C₃-C₂₄Heteroarylalkyl, wherein heteroaryl contains a residue selected from O, S and NR¹⁴Wherein R is¹⁴Is hydrogen or C₁-C₄An alkyl group, a carboxyl group,

Sp¹、Sp²、Sp³and Sp⁴Are spacer moieties and are each independently selected from: a single bond, or C being straight or branched₁-C₂₀₀Alkylene radical, C₂-C₂₀₀Alkenylene radical, C₂-C₂₀₀Alkynylene, C₃-C₂₀₀Cycloalkylene radical, C₅-C₂₀₀Cycloalkenylene group, C₈-C₂₀₀Cycloalkynylene, C₇-C₂₀₀Alkylarylene, C₇-C₂₀₀Arylalkylene radical, C₈-C₂₀₀Arylalkenylene and C₉-C₂₀₀Arylalkynylene, wherein alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are optionally selected from O, S and NR¹⁴Is substituted or contains heteroatoms selected from O, S and NR¹⁴The heteroatom(s) of (a),

Z¹and Z²Each independently selected from O, C (O) and N (R)¹³)，

a is each independently 0 or1,

b is each independently 0 or1,

c is a number of 0 or1,

d is a number of 0 or1,

e is a number of 0 or1,

f is an integer of 0 to 150,

g is 0 or1, and

i is 0 or 1. For example, Q¹May be selected from the following formulae:

wherein U is O or NR¹⁵Wherein R is¹⁵Is hydrogen, straight-chain or branched C₁-C₁₂Alkyl radical, C₄-C₁₂Aryl or C₄-C₁₂Heteroaryl, and more specifically, R¹⁵Is hydrogen or C₁-C₄An alkyl group.

In particular, the linker L in formula (III)²May have a structure represented by the following formula (II-a) or (II-b):

wherein Q is²Is a cyclooctenyl radical fused with triazole or a heterocyclooctyenyl radical fused with triazole, wherein the cyclooctenyl or heterocyclooctyl radical is optionally further selected from C independently of one another₃-C₁₂Cycloalkyl radical, C₃-C₁₂Aryl and C₃-C₁₂1 or 2 rings of heteroaryl fused, and optionally substituted by hydroxy, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, amino, ONH₂Or carbonyl substitution, wherein Q²Attached to Ab through the nitrogen atom contained in the triazole;

Sp¹、Sp²、Sp³and Sp⁴Are spacer moieties and are each independently selected from: a single bond, or C being straight or branched₁-C₂₀₀Alkylene radical, C₂-C₂₀₀Alkenylene radical, C₂-C₂₀₀Alkynylene, C₃-C₂₀₀Cycloalkylene radical, C₅-C₂₀₀Cycloalkenylene group, C₈-C₂₀₀Cycloalkynylene, C₇-C₂₀₀Alkylarylene, C₇-C₂₀₀Arylalkylene radical, C₈-C₂₀₀Arylalkenylene and C₉-C₂₀₀Arylalkynylene, wherein alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are optionally selected from O, S and NR¹⁴Is substituted or contains heteroatoms selected from O, S and NR¹⁴A heteroatom of (a);

Z¹and Z²Each independently selected from O, C (O) and N (R)¹³)；

a is each independently 0 or 1;

each b is independently 0 or 1;

c is 0 or 1;

d is 0 or 1;

e is 0 or 1;

f is an integer of 0 to 150;

g is 0 or 1; and is

i is 0 or 1.

For example, Q²Is cyclooctenyl fused with triazole, wherein the cyclooctenyl is further fused with C₃-C₆Cycloalkyl and/or C₃-C₆The aryl groups are fused.

For example Sp as a spacer moiety¹、Sp²、Sp³And Sp⁴Each independently selected from: a single bond, or C being straight or branched₁-C₂₀Alkylene radical, C₂-C₂₀Alkenylene radical, C₂-C₂₀Alkynylene, C₃-C₂₀Cycloalkylene radical, C₅-C₂₀Cycloalkenylene group, C₈-C₂₀Cycloalkynylene, C₇-C₂₀Alkylarylene, C₇-C₂₀Arylalkylene radical, C₈-C₂₀Arylalkenylene and C₉-C₂₀Arylalkynylene, wherein alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene andarylalkynyl is optionally substituted with a substituent selected from the group consisting of O, S and NR¹⁴Is substituted by 1 to 5 heteroatoms or contains groups selected from O, S and NR¹⁴1 to 5 heteroatoms of (a).

As used herein, the term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound, and includes, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and the like, saturated straight-chain alkyl includes, for example, methyl, ethyl, n-propyl, n-butyl, n-pentyl (pentyl), n-hexyl, n-heptyl, and the like, and saturated branched-chain alkyl includes, for example, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and the like.

As used herein, the term "alkenylene" refers to a straight or branched chain monovalent hydrocarbon radical of any length and having at least one site of unsaturation of a carbon atom, i.e., a carbon-carbon double bond, and an alkenyl group may be optionally independently substituted with one or more of the following substituents and may include groups having "cis" and "trans" orientations.

As used herein, the term "cycloalkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of an alicyclic ring of a cyclic hydrocarbon compound. Examples of cycloalkyl groups include: saturated monocyclic hydrocarbon compounds such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane and methylcyclohexane; and

unsaturated monocyclic hydrocarbon compounds, such as cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene and methylcyclohexene.

As used herein, the term "heterocyclyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.

Prefix (e.g., C) as used herein_1-12、C_3-8Etc.) means the number of ring atoms or ring atomsThe range of sub-numbers regardless of whether the prefix is preceded by a carbon atom or a heteroatom. For example, the term "C" as used herein_3-6Heterocyclyl "means a heterocyclyl having 3 to 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N₁: aziridine, azetidine, pyrrolidine, pyrroline, 2H-or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine and azepine；

N₂: imidazolidine, pyrazolidine, imidazoline, pyrazoline, and piperazine;

O₁: oxirane, oxetane, oxolane, furan (oxol), oxacyclohexane, dihydropyran, pyran, and oxepine;

O₂: dioxolane, dioxane and dioxepane;

O₃: trioxane;

N₁O₁: tetrahydrooxazole, dihydrooxazole, tetrahydroisooxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, and oxazine;

S₁: thiirane, tetrahydrothiophene, thiacyclohexane and thiepane;

N₁S₁: dihydrothiazoles, thiazolidines and thiomorpholines;

N₂O₁: oxadiazines;

O₁S₁: oxathiol and oxathiacyclohexane; and

N₁O₁S₁: oxathiazines.

As used herein, the term "aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom. "C₆-C₂₀Aryl "refers to a moiety having from 6 to 20 ring atoms, formed byObtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and prefixed (C)₆-C₂₀) Is meant to refer to a range of ring or ring atoms, regardless of whether the formula pertains to a carbon atom or a heteroatom, and indicates that the formula may include one carbon atom or at least one heteroatom.

As used herein, the term "heteroaryl" refers to an aryl group containing one or more heteroatoms, and may, for example, include: pyridine, pyrimidine, benzothiophene, furyl, dioxolanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl or pyrimidinyl, in particular C having two condensed rings derived from benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindolinone, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, benzodioxazole, benzofuran, benzotriazole, benzothiofuran, benzothiazole or benzothiadiazole₉C with two condensed rings derived from chromene, isochromene, chroman, isochroman, benzodioxan, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine or pteridine₁₀Having a structure derived from benzodiazepineC of two condensed rings of₁₁C13 having three fused rings derived from carbazole, dibenzofuran, dibenzothiophene, carboline, perimidine or pyridoindole, and C13 having three fused rings derived from acridine, xanthene, thianthrene, dibenzo-p-dioxin (oxanthrene), phenoxathiin, phenazine, phenoxazine, phenothiazine, thianthrene, phenanthridine, phenanthroline or phenazine₁₄。

As used herein, the term "alkoxy" refers to-OR [ wherein R is alkyl ], and examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "alkylene" refers to a hydrocarbon compound containing a double bond, and may represent an alkylene group having 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, or1 to 4 carbon atoms. The alkylene group may be a linear, branched or cyclic alkylene group, and may be optionally substituted with one or more substituents.

As used herein, the term "pharmaceutically acceptable salt" can be an acid addition salt formed from a pharmaceutically acceptable free acid, and the free acid can be an organic acid or an inorganic acid.

Organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid. In addition, inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid.

For example, when a compound is anionic or has a functional group that can become anionic (e.g., -COOH can be converted to-COO-), it can form a salt with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (e.g., Na)⁺And K⁺) Alkaline earth metal cations (e.g. Ca)²⁺And Mg²⁺) And other cations (e.g., Al)³⁺). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)⁴⁺) And substituted ammonium ions (e.g., NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺)。

Some examples of suitable substituted ammonium ions include those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids such as lysine and arginine. A typical example of a quaternary ammonium ion is N (CH)₃)⁴⁺。

When the compound is cationic or has a functional group which can become cationic (e.g., -NH)₂Can be converted into-NH 3⁺) When used, it may form a salt with a suitable anion. Examples of suitable inorganic anionsIncluding but not limited to those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, adipic acid, oleic acid, oxalic acid, palmitic acid, sulfamic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, valeric acid, and the like. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose, and the like.

As used herein, the term "solvate" refers to a molecular complex between a compound according to the present invention and a solvent molecule, and for example, solvates include, but are not limited to, compounds according to the present invention in combination with water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethyl acetate, acetic acid, ethanolamine, or mixed solvents thereof.

Optionally, amino acids or peptides, such as dipeptides, may be further included. Specifically, dipeptides such as Val-Cit, Phe-Lys, Val-Glu, Val-Asp, Val-Ser, or Val-Gly may be included. Preferably a Val-Cit dipeptide linker. When single amino acids are included, for example, Cit, Glu, Lys, or Ser may be included. Linkers comprising such amino acids or peptides may be cleaved by cathepsin B.

Specifically, the compound of formula (II) may be represented by the following formula:

the compound of formula (II) according to the present invention may be a linker-drug complex having a linker moiety bound to an antibody to form an antibody-drug conjugate. In one embodiment, the linker may include a spacer.

The linker of the compound of formula (II) according to the present invention may have maleimide, aldehyde, aminoxy, 2-PCA or cyclooctynyl for binding a drug, but is not limited thereto.

For example, when the linker of the compound of formula (II) according to the present invention includes a maleimide group, the linker may be linked to the antibody by introducing a cysteine amino acid of the antibody to form an antibody-drug conjugate according to the present invention. For example, cysteine amino acids can be engineered and used for drug conjugation, but can be through Thiomab^TMAntibody binding, Thiomab^TMAn antibody is an antibody substituted with a cysteine at a position that does not prevent antibody folding and does not alter antigen binding or effector function. It can be conjugated to cytotoxic drugs via engineered cysteine thiol groups, enabling the production of THIOMAB with uniform stoichiometry (e.g., in antibodies with a single engineered cysteine, each antibody contains up to 2 drugs)^TMAntibody-drug conjugates (TDCs).

Except for Thiomab^TMIn addition to antibodies, any antibody containing a cysteine amino acid can be bound to a compound of formula (II) having a maleimide linker by the following Michael reaction to produce an antibody-drug conjugate.

For example, an engineered cysteine may be present at a different position for drug attachment, such as a specific amino acid position within the light chain-Fab, heavy chain-Fab, or heavy chain-Fc of an antibody.

Cysteine substitutions in the heavy chain are for example selected from the group consisting of Y33C, G162C, V184C, I195C, S420C, Y432C, Q434C, R19C, E46C, T57C, Y59C, a60C, M100cC, W103C, G162C, I195C, S420C, H425C and N430C, according to the numbering of Kabat. The cysteine substitutions in the light chain are for example selected from the group consisting of Y55C, G64C, T85C, T180C, N430C, T31C, S52C, G64C, R66C, a193C and N430C, according to the Kabat numbering, or may optionally be selected from the group consisting of LC-I106C, LC-R108C, LC-R142C and LC-K149C.

For example, when the linker of the compound of formula (II) according to the present invention includes an aldehyde group, the linker may be through the N-terminus of the following antibody protein or NH of lysine amino acid₂Reductive alkylation with the aldehyde in the linker binds to the antibody to produce an antibody-drug conjugate according to the invention.

For example, when the linker of the compound of formula (II) according to the present invention includes an aminooxy group, the linker may be bound to the antibody through an oxime linkage between a ketone group in an amino acid of the following antibody and the aminooxy group in the linker to generate an antibody-drug conjugate according to the present invention.

For example, when the linker of the compound of formula (II) according to the invention comprises 2-pyridinecarboxaldehyde (2-PCA), the linker may be through NH in the following antibody amino acids₂The N-terminal imidazolidinone formation between the 2-PCA of the linker binds to the antibody to produce an antibody-drug conjugate according to the present invention.

For example, when the linker of the compound of formula (II) according to the present invention contains cyclooctynyl group, the linker is modified to have azido group (-N)₃) To produce antibody-drug conjugates according to the invention. The linker can be bound to the antibody by a click reaction between the azido group in the following antibody and the cyclooctyne group in the linker.

The types of linkers and the specific reactions for forming antibody-drug conjugates by conjugating each linker to an antibody are known in the art and can be readily performed by those skilled in the art without undue effort. In addition to the above linkers, various linkers known in the art and available to those skilled in the art may be used to generate the antibody-drug conjugates of the present invention, and those skilled in the art will also readily recognize the structure of the antibody-drug conjugates that bind to such linkers.

The antibodies used herein recognize antigens that are naturally expressed or overexpressed by target cells (e.g., cancer cells), and are capable of delivering drug moieties to cancer cells with a high degree of specificity as targeting agents. When the antibody binds to the antigen, the antigen-conjugate forms a complex, is internalized, and eventually enters the lysosome, and the linker between the drug moiety and the antibody is cleaved to release the drug moiety, thereby providing a cytotoxic effect.

For example, an antibody may bind to the following antigens, but is not limited thereto:

(1) BMPR1B (bone morphogenetic protein receptor-type IB, GenBank accession No. NM _ 001203);

(2) e16(LAT1, SLC7A5, GenBank accession NM-003486);

(3) STEAP1 (six transmembrane epithelial antigen of prostate 1, GenBank accession No. NM _ 012449);

(4)0772P (CA125, MUC16, GenBank accession AF 361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin, GenBank accession No. NM _ 005823);

(6) napi3B (Napi-3B, NPTIIb, SLC34a2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3B, GenBank accession No. NM — 006424);

(7) sema5B (FLJ10372, KIAA1445, mm.42015, Sema5B, SEMAG, Semaphorin (Semaphorin)5B Hlog, Sema domain, 7 thrombospondin repeats (type 1 and pseudotype 1), transmembrane domain (TM) and a short cytoplasmic domain, (Semaphorin)5B, GenBank accession No. AB 040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, GenBank accession No. AY 358628);

(9) ETBR (endothelin type B receptor, GenBank accession No. AY 275463);

(10) MSG783(RNF124, hypothetical protein FLJ20315, GenBank accession No. NM _ 017763);

(11) STEAP2(HGNC _8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate 6-transmembrane epithelial antigen 2, 6-transmembrane prostate protein, GenBank accession No. AF 455138);

(12) TrpM4(BR22450, FLJ20041, TrpM4, TrpM4B, transient receptor potential cation channel, subfamily M member 4, GenBank accession No. NM _ 017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, GenBank accession No. NP _003203 or NM _ 003212);

(14) CD21(CR2 (complement receptor 2), C3DR (C3d/Epstein Barr virus receptor) or Hs.73792GenBank accession number M26004);

(15) CD79B (CD79B, CD79 β, IGb (immunoglobulin-related protein β), B29, GenBank accession No. NM — 000626);

(16) FcRH2(IFGP4, IRTA4, spa 1A (SH 2 domain containing phosphatase dockerin 1a), spa 1B, spa 1C, GenBank accession No. NM — 030764);

(17) HER2(GenBank accession number M11730);

(18) an ErbB receptor selected from EGFR, HER3, and HER 4;

(19) NCA (GenBank accession number M18728);

(20) MDP (GenBank accession number BC 017023);

(21) IL20R α (GenBank accession No. AF 184971);

(22) short proteoglycans (GenBank accession No. AF 229053);

(23) EphB2R (GenBank accession No. NM _ 004442);

(24) ASLG659(GenBank accession No. AX 092328);

(25) PSCA (GenBank accession No. AJ 297436);

(26) GEDA (GenBank accession No. AY 260763);

(27) BAFF-R (B cell activator receptor, BLyS receptor 3, BR3, NP _ 443177.1);

(28) CD22(B cell receptor CD22-B isoform, NP-001762.1);

(29) CD79a (CD79A, CD79 α, immunoglobulin-related protein α, which are B-cell specific proteins that covalently interact with Ig β (CD79B), form complexes with IgM on the surface and transmit signals involved in B-cell differentiation, GenBank accession No. NP _ 001774.1);

(30) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokine, believed to act on lymphocyte migration and humoral defense, involved in HIV-2 infection, and associated with the onset of AIDS, lymphoma, myeloma, and leukemia, GenBank accession No. NP _ 001707.1);

(31) HLA-DOB (β subunit of MHC class II molecule (Ia antigen), which is bound to a peptide and delivered to CD4+ T lymphocytes, GenBank accession No. NP _ 002111.1);

(32) P2X5 (purinoceptor P2X ligand-gated ion channel 5, which is an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, the deficiency of which may lead to pathophysiology of idiopathic detrusor instability GenBank accession No. NP _ 002552.2);

(33) CD72(B cell differentiation antigens CD72, Lyb-2, GenBank accession No. NP _ 001773.1);

(34) LY64 (lymphocyte antigen 64(RP105), a type I membrane protein of the Leucine Rich Repeat (LRR) family, regulates B cell activation and apoptosis, and its loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, GenBank accession No. NP _ 005573.1);

(35) FcRH1 (Fc receptor homolog 1, a putative receptor for the immunoglobulin Fc domain comprising C2-like Ig and ITAM domains, which may be involved in B lymphocyte differentiation, GenBank accession No. NP _ 443170.1);

(36) IRTA2 (immunoglobulin superfamily receptor translocation related protein 2, a putative immunoreceptor that can act on B-cell and lymphoma genesis, and gene deregulation by translocation occurs in several B-cell malignancies, GenBank accession No. NP _ 112571.1); and

(37) TENB2 (putative transmembrane proteoglycan associated with EGF/regulin family of growth factors and follistatin, GenBank accession No. AF 179274);

(38) MAGE-C1/CT7 (protein overexpressed in testicular cancer);

(39) androgen receptor, PTEN, human kallikrein-related peptidase 3 (protein overexpressed in prostate cancer);

(40)CD20；

(41)CD30；

(42)CD33；

(43)CD52；

(44)EpCam；

(45)CEA；

(46)gpA33；

(47) mucin;

(48)TAG-72；

(49) carbonic anhydrase IX;

(50)PSMA；

(51) folate receptors (which are a family of proteins expressed by the FOLR gene, have high affinity for folate, and transport 5-methyltetrahydrofolate into the cell);

(52) gangliosides (GD2, GD3, GM 2);

(53) a sugar hydrate Lewis-Y;

(54)VEGF；

(55)VEGFR；

(56)aVb3；

(57)a5b1；

(58)ERB3；

(59)c-MET；

(60)EphA3；

(61) TRAIL-R1 and TRAIL-R2;

(62)RANKL；

(63)FAP；

(64) tenascin;

(65)ROR1；

(66) BCMA; or

(67)CLL1。

For example, the antibody may be selected from the group consisting of an anti-BCMA antibody, an anti-ROR 1 antibody, an anti-Her 2 antibody, an anti-NaPi 2b antibody, and an anti-CLL 1 antibody, but is not limited thereto. In a specific example, the following antibodies were used as anti-BCMA antibodies and anti-ROR 1 antibodies for ADC construction.

[ Table 1]

Known sequences of trastuzumab are used for anti-Her 2 antibody, known sequences of 10H1 antibody (10H1VL: SEQ ID NO:17, 10H1VH: SEQ ID NO:18) are used for anti-NaPi 2b antibody, and known sequences of 6E7(N54A) antibody (6E7(N54A) VL: SEQ ID NO:18, 6E7(N54A) VH: SEQ ID NO:19) are used for anti-CLL 1 antibody.

As used herein, the term "antibody" refers to a polypeptide or protein that specifically binds to a particular antigen. Antibodies include not only complete antibodies that specifically bind to an antigen, but also antigen-binding fragments of antibodies.

The term "full antibody" refers to a structure having two full-length light chains and two full-length heavy chains, wherein each light chain is linked to a corresponding heavy chain by a disulfide bond. The heavy chain constant region has the gamma (γ), spurious (μ), alpha (α), delta (δ), and eppirone (ε) types, and is subdivided into gamma 1(γ 1), gamma 2(γ 2), gamma 3(γ 3), gamma 4(γ 4), alpha 1(α 1), and alpha 2(α 2). The light chain constant region has kappa (. kappa.) and lanrda (. lamda.) types.

An antigen-binding fragment of an antibody or an antibody fragment is a fragment having an antigen-binding ability, and includes Fab, F (ab')2, Fv, and the like. In antibody fragments, Fab refers to a structure comprising the variable region of each of the heavy and light chains, the constant region of the light chain, and the first constant domain of the heavy chain (CH1), each of which has an antigen binding site. Fab' differs from Fab in that it further comprises a hinge region comprising at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F (ab ')2 is produced by disulfide bonding between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment with only heavy and light chain variable regions. A two-chain Fv is a fragment in which the variable region of the heavy chain and the variable region of the light chain are joined by a non-covalent bond; whereas single-chain Fv (scFv) are fragments in which the variable region of the heavy chain and the variable region of the light chain are linked, usually by a covalent bond via a peptide linker therebetween, or directly at the C-terminus, to form a dimeric structure, such as a two-chain Fv. Such antibody fragments can be obtained using proteases (e.g., Fab can be obtained by restriction cleavage of the complete antibody with papain, while F (ab')2 fragments can be obtained by cleavage of the complete antibody with pepsin), or can be prepared using genetic recombination techniques.

The heavy chain constant region may be selected from the gamma (γ), spurious (μ), alpha (α), delta (δ) and epplon (ε) isoforms. For example, the constant region may be γ 1(IgG1), γ 3(IgG3), or γ 4(IgG 4). The light chain constant region may be kappa or lambda.

As used herein, the term "heavy chain" includes full-length heavy chains, including variable domains (VH) comprising amino acid sequences having variable region sequences sufficient to confer specificity to an antigen, three constant domains (CH1, CH2, and CH3), and fragments thereof. As used herein, the term "light chain" includes full-length light chains, including variable domains (VL), constant domains (CL) comprising amino acid sequences having variable region sequences sufficient to confer specificity to an antigen, and fragments thereof.

The antibody of the present invention includes, but is not limited to, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single chain fv (scfv), a single chain antibody, a Fab fragment, a F (ab') fragment, a disulfide bond fv (sdfv), an epitope-binding fragment of such an antibody, and the like.

As used herein, the term "monoclonal antibody" refers to the same antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprising the population, excluding possible naturally occurring mutations that may be present in negligible amounts. Monoclonal antibodies are highly specific and are therefore induced against a single antigenic site. Unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The term "epitope" refers to a protein determinant to which an antibody is capable of specifically binding. Epitopes usually comprise a group of chemically active surface molecules, such as amino acids or sugar side chains, and usually have not only specific three-dimensional structural characteristics, but also specific charge characteristics. The three-dimensional epitope is distinguished from a non-three-dimensional epitope in that binding to the former is disrupted, while binding to the latter is not disrupted, in the presence of a denaturing solvent.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.

As used herein, the term "human antibody" refers to a molecule derived from a human immunoglobulin in which all amino acid sequences constituting the antibody, including complementarity determining regions and structural regions, are composed of a human immunoglobulin.

A portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains comprise "chimeric" antibodies (immunoglobulins) that are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

As used herein, the term "antibody variable domain" refers to the amino acid sequences of the light and heavy chain regions of an antibody molecule, including the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the Framework Regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

The term "complementarity determining regions" (CDRs, i.e., CDR1, CDR2 and CDR3) refer to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR 3.

The term "framework region" (FR) refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3 and FR 4.

The antibodies or antigen-binding fragments of the invention may include the sequences of the antibodies mentioned herein as well as their biological equivalents. For example, additional changes may be made to the amino acid sequence of an antibody to further improve the binding affinity and/or other biological properties of the antibody. For example, such alterations include deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody. Such amino acid changes are based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size. As can be seen by analyzing the size, shape and type of amino acid side-chain substituents, arginine, lysine and histidine are all positively charged residues, and alanine, glycine and serine are of similar size, and phenylalanine, tryptophan and tyrosine are of similar shape. Thus, based on these considerations, arginine, lysine and histidine are considered to be biologically functional equivalents, alanine, glycine and serine are considered to be biologically functional equivalents, and phenylalanine, tryptophan and tyrosine are considered to be biologically functional equivalents.

The antibody or the nucleotide molecule encoding the same according to the present invention is construed to include a sequence having substantial identity to the sequence shown as the sequence number in consideration of variations having a bioequivalent activity. The term "substantial identity" means that when a sequence of the invention is aligned with any other sequence to correspond as closely as possible thereto and the aligned sequences are analyzed using algorithms commonly used in the art, the sequences have at least 90% homology, most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible through NCBI and can be used in conjunction with sequence analysis programs such as BLASTP, BLASTM, BLASTX, TBLASTN and TBLASTX on the Internet. BLAST is available at www.ncbi.nlm.nih.gov/BLAST/N. Methods for comparing sequence homology using this program can be found on www.ncbi.nlm.nih.gov/BLAST/BLAST _ help.

Based on this, the antibody or antigen binding fragment thereof according to the invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequences disclosed herein or all thereof. Homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, the percent sequence homology of a nucleic acid or protein according to the present invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

In some cases, an antibody or antigen-binding fragment thereof can be produced recombinantly by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof according to the invention. The nucleic acid is isolated and inserted into a replicable vector and then further cloned (amplification of the DNA) or further expressed. Based on this, in another aspect, the invention relates to a vector comprising the nucleic acid.

The term "nucleic acid" is intended to include DNA (gDNA and cDNA) and RNA molecules, as well as nucleotides (which are the basic building blocks of nucleic acids) including naturally derived nucleotides and analogs thereof, wherein the sugar or base moiety is modified. The sequences of the nucleic acids encoding the heavy chain variable region and the light chain variable region of the present invention may be changed. Such changes include additions, deletions or non-conservative or conservative substitutions of nucleotides.

The DNA encoding the antibody can be readily isolated or synthesized using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to DNA encoding the heavy and light chains of the antibody). A variety of vectors are available. Carrier components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.

As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, and viral vectors, such as phage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

The term "operably linked" refers to a functional linkage between one nucleic acid expression control sequence (e.g., a series of promoters, signal sequences, or binding sites for transcriptional regulators) and another nucleic acid sequence, and such that the control sequence is capable of regulating the transcription and/or translation of the other nucleic acid sequence.

When a prokaryotic cell is used as a host, it generally includes a promoter effective for transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL lambda promoter, pR lambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter), a ribosome binding site for translation initiation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, it includes a promoter derived from the genome of a mammalian cell (for example, metallothionein promoter, β -actin promoter, human hemoglobin promoter, or human muscle creatine promoter), or a promoter derived from a mammalian virus (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, Cytomegalovirus (CMV) promoter, HSV tk promoter, Mouse Mammary Tumor Virus (MMTV) promoter, HIV LTR promoter, moloney virus promoter, epstein-barr virus (EBV) promoter, or Rous Sarcoma Virus (RSV) promoter), and generally has a polyadenylation sequence as a transcription termination sequence.

Optionally, the vector may be fused to another sequence to facilitate purification of the antibody expressed thereby. For example, sequences to which fusion is made may include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6 XHis (hexahistidine; Qiagen, USA), and the like.

The vector includes antibiotic resistance genes commonly used in the art as a selectable marker, and examples thereof include genes that confer resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

In another aspect, the present invention relates to a cell transformed with the above-described vector. The cells used to produce the antibodies of the invention may be prokaryotic, yeast, or higher eukaryotic cells, but are not limited thereto.

Prokaryotic host cells, such as E.coli (Escherichia coli), strains of Bacillus (Bacillus), such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces spp, Pseudomonas spp, such as Pseudomonas putida, Proteus mirabilis and Staphylococcus spp, such as Staphylococcus carnosus (Staphylococcus carnosus), may be used.

Examples of host cell lines of greatest interest and utility for animal cells include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER. C6, SP2/0, NS-0, U20S and HT 1080.

Cells can be cultured in various media. Any commercially available medium can be used as the medium without limitation. All other necessary supplements known to those skilled in the art may be included in suitable concentrations. Culture conditions, such as temperature and pH, are those typically used to select host cells for expression, as will be apparent to those skilled in the art.

For example, recovery of the antibody or antigen-binding fragment thereof can be performed by centrifugation or ultrafiltration to remove impurities from the resulting product and purify the resulting product using, for example, affinity chromatography. Other additional purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography.

In another aspect, the present invention relates to a composition for preventing or treating a proliferative disease (e.g., tumor or cancer), or to a pharmaceutical composition containing the antibody-drug conjugate as an active ingredient.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a proliferative disease (e.g., tumor or cancer), comprising: (a) a pharmaceutically effective amount of an antibody-drug conjugate and (b) a pharmaceutically acceptable carrier. In another aspect, the invention relates to a method of preventing or treating a tumor comprising administering an antibody-drug conjugate according to the invention to a patient suffering from a proliferative disease (e.g. a tumor or cancer). In another aspect, the invention relates to the use of the antibody-drug conjugate for the prevention or treatment of a proliferative disease (e.g., a tumor or cancer).

With respect to such tumors or cancers, non-limiting examples of tumors or cancers that can be treated include, but are not limited to, renal cancer, pancreatic cancer, ovarian cancer, lymphoma, colon cancer, mesothelioma, gastric cancer, lung cancer, prostate cancer, adenocarcinoma, liver cancer, breast cancer, and the like. The tumor or cancer may comprise refractory or recurrent cancer.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may include those commonly used for preparing medicines, for example, one or more selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, without being limited thereto. The pharmaceutical composition may further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives.

The pharmaceutical composition can be administered orally or parenterally. Parenteral administration may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, rectal administration, and the like. Upon oral administration, the protein or peptide is digested and therefore the oral composition should be coated or formulated with the active agent to prevent its degradation in the stomach. Furthermore, the pharmaceutical composition may be administered using any device capable of delivering an active substance to a target cell.

The effective dose of the pharmaceutical composition according to the present invention may vary depending on factors such as formulation method, administration method, and age, body weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate and reactivity of the patient. For example, the daily dose of the pharmaceutical composition may be in the range of 0.001mg/kg to 1,000mg/kg, 0.01mg/kg to 100mg/kg, 0.1mg/kg to 50mg/kg, or 0.1mg/kg to 20mg/kg, but is not limited thereto. The formulations may be prepared in unit dosage form containing a daily dose of the pharmaceutical composition, or the daily dose of the formulation may be divided into multiple doses or may be incorporated in a multi-dose container.

The pharmaceutical composition may be formulated in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be formulated in the form of an extract, powder, suppository, granule, tablet or capsule. The composition may further contain a dispersant or stabilizer for formulation.

Examples

Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be apparent to those skilled in the art that these examples are provided only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Preparation examples: pharmaceutical synthesis

In the following preparation examples, all were recorded at 400MHz using Bruker Avance¹H NMR spectrum.

The LCMS was measured using a Shimadzu LCMS 2011 quadrupole mass spectrometer (column: Shim-pack XR-ODS (3.0 x 30mm, 2.2m)) operated in ESI (+) ionization mode. Flow rate: 0.8mL/min, collection time: 3min or 1.5min, wavelength: UV220, furnace temperature: at 50 ℃.

Preparative HPLC was performed under the following conditions: column: fuji C18(300 × 25), YMC (250 × 20); wavelength: 220 nm; mobile phase: CH (CH)₃CN(0.05％NH₃H₂O or 0.225% FA); b Water (0.1% NH)₃·H₂O or 0.225% FA); flow rate: 30 mL/min; sample introduction amount: 3 mL; operating time: 20 minutes; balancing: 3 minutes

Preparation example 1: synthesis of PD001

1) Preparation of Compound 1

A mixture of Compound 1A (700mg, 1.65mmol) and 10% dry Pd/C (140mg) in MeOH (7mL) and EtOAc (7mL) was degassed in vacuo and H₂And (5) purifying for multiple times. Mixing the mixture in H₂(balloon, 15psi) at 20 ℃ for 4 hours. TLC showed the formation of a new spot with greater polarity. The mixture was filtered through celite, and the filtrate was concentrated and dried to give compound 1(549mg, yield 99.6%) as a white solid.

¹H NMR(400MHz，CDCl₃)δ1.56(9H，s)，3.43(1H，t，J＝10.4Hz)，3.85-4.01(2H，m)，4.07-4.16(1H，m)，4.19-4.30(1H，m)，6.24(1H，brs)，7.34(1H，t，J＝7.6Hz)，7.50(1H，t，J＝7.6Hz)，7.59-7.80(2H，m)，8.17(1H，d，J＝8.0Hz)。

2) Preparation of Compound 5-2

4A MS (2g) was added to a stirred solution of compound 1(300mg, 0.899mmol) in anhydrous DCM (10mL) at 20 ℃. Subsequently, the mixture was stirred at 20 ℃ for 30 minutes. After addition of Compound 5-1(576mg, 1.17mmol), the resulting mixture was cooled to-10 ℃ and BF in DCM (5mL) was added dropwise thereto₃·Et₂O (64mg, 0.45 mmol). The mixture was then stirred at-10 ℃ for 1 hour. The mixture was then heated at 0 ℃ for 2 hours. TLC showed that a trace of starting material remained and a new main spot with large polarity was formed. The mixture was filtered off and the filtrate was taken up with NaHCO₃Quenched with saturated aqueous solution (20 mL). The mixed organic layers were separated. The remaining aqueous phase was extracted with DCM (15mL × 2). The combined organic layers were washed with Na₂SO₄Dried, concentrated and dried. The residue was mixed with batch 2 and PE: EtOAc/5: 1 as eluent in a silica gel column.

4A MS (1.5g) was further added to a stirred solution of compound 1(249mg, 0.746mmol) in DCM (10mL) at 20 ℃. Subsequently, the mixture was stirred at 20 ℃ for 30 minutes. After addition of Compound 5-1(478mg, 0.970mmol), the mixture was cooled to-10 ℃ and BF in DCM (5mL) was added dropwise thereto₃·Et₂O (53mg, 0.373 mmol). The mixture was then stirred at-10 ℃ for 1 hour. The mixture was then heated at 0 ℃ for 2 hours. TLC showed that a trace of starting material remained and a new main spot with large polarity was formed. The mixture was filtered off and the filtrate was taken up with NaHCO₃Quenched with saturated aqueous solution (20 mL). The organic layer was separated. The remaining aqueous phase was washed with DCM (15 m)L × 2) extraction. The combined organic layers were washed with Na₂SO₄Dried, concentrated and dried. The residue was mixed with batch 1 and PE: EtOAc/5: 1 as eluent on silica gel column to give compound 5-2(1.19g, crude) as a white solid. 35mg of the starting material of Compound 1 was recovered as a white solid.

¹H NMR(400MHz，CDCl₃) δ 1.61(9H, s, overlapping water signal), 2.02(3H, s), 2.06(3H, s, overlapping EtOAc signal), 2.11(3H, s), 2.24(3H, s), 3.45(1H, t, J ═ 10.8Hz), 3.91-4.05(2H, m), 4.10-4.19(1H, m, overlapping signal), 4.20-4.43(4H, m), 5.20(1H, dd, J ═ 10.4, 3.6Hz), 5.37(1H, d, J ═ 8.0Hz), 5.53(1H, d, J ═ 3.2Hz), 5.72(1H, dd, J ═ 10.4, 7.6Hz), 7.34-7.40(1H, m), 7.49-7.57(1H, 7.57), 7.68(1H, 8Hz), 7.8H, 8H, 1.6 Hz).

3) Preparation of Compound 5-3

BF is brought to 0 DEG C₃·Et₂O (890mg, 6.27mmol) was added dropwise to a stirred solution of compound 5-2(1.19g, crude) in anhydrous DCM (15 mL). The mixture was then allowed to warm to 20 ℃ and stirred for 2 hours. TLC showed the reaction was complete. The mixture is washed with NaHCO₃Quenched with saturated aqueous solution (15 mL). The organic layer was separated. The remaining aqueous phase was extracted with DCM (15mL × 2). The combined organic layers were washed with Na₂SO₄Dried, concentrated and further dried. The use of PE: EtOAc/2: 1 to 1: 1 as eluent the residue was purified on silica gel column to give the de-Boc intermediate (782mg, 84% over two steps yield) as a white solid. 28mg of Compound 5-2 was recovered.

¹H NMR(400MHz，CDCl₃) δ 2.06(3H, s), 2.07(3H, s, overlapping EtOAc signal), 2.11(3H, s), 2.24(3H, s), 3.52(1H, t, J ═ 10.8Hz), 3.77-3.87(2H, m), 3.88-3.94(1H, m), 3.96-4.05(1H, m), 4.12-4.25(3H, m, overlapping EtOAc signal), 4.31(1H,dd, J-10.8, 6.8Hz), 5.11-5.20(2H, m), 5.52(1H, dd, J-3.2, 0.8Hz), 5.70(1H, dd, J-10.4, 8.0Hz), 6.66(1H, s), 7.20-7.20(1H, m, overlapping CDCl)₃Signal), 7.45-7.52(1H, m), 7.60(1H, dd, J ═ 8.4, 1.2Hz), 8.00(1H, d, J ═ 8.0 Hz).

EDCI (219mg, 1.14mmol) was added to a mixture of the de-Boc intermediate (643mg, 1.14mmol) and compounds 1-7(190mg, 0.380mmol) in dry DMF (6mL) at 20 ℃. Then, the mixture was stirred at 20 ℃ for 16 hours. TLC showed starting material still present and the desired product was observed. The solvent was removed under reduced pressure. The mixture was quenched with water (15mL) and extracted with EtOAc (15mL x 3). The combined organic layers were washed with water (20mL) and Na₂SO₄Concentrating and drying. The use of PE: EtOAc/2: 1 to 1: 2 as an eluent, the residue was purified on a silica gel column to give compound 5-3(448mg, yield: 74%) as a yellow solid. In addition, 163mg of the de-Boc intermediate was recovered (LCMS: purity 75%).

¹H NMR(400MHz，CDCl₃) Δ 2.01-2.20(18H, m, overlap EtOAc signal), 2.23(6H, s), 3.43-3.57(2H, m), 3.69-3.82(2H, m), 3.95-4.05(2H, m), 4.06-4.10(1H, m, overlap EtOAc signal), 4.19-4.65(15H, m), 5.12-5.27(2H, m), 5.32-5.44(2H, m, overlap CH signal)₂Cl₂Signal), 5.50-5.62(2H, m), 5.70-5.82(2H, m), 6.92-7.13(2H, m), 7.19-7.39(7H, m, overlapping CDCl)₃Signal), 7.40-7.50(2H, m), 7.51-7.64(4H, m), 7.65-7.76(5H, m), 7.84(1H, d, J ═ 15.2Hz), 8.13-8.27(3H, m), 8.47(2H, d, J ═ 18.4 Hz).

4) Preparation of Compounds 5-4

A solution of compound 5-3(250mg, 0.157mmol) and piperidine (134mg, 1.57mmol) in dry DCM (4mL) was stirred at 20 deg.C for 16 h. TLC showed the reaction had proceeded to completion. The solvent was removed under reduced pressure. Using DCM: MeOH/50: 1 to 25: 1 as an eluent, the impurity was purified on a silica gel column to give compound 5-4(176mg, yield: 82%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 2.01-2.24(24H, m), 3.35-3.59(4H, m), 3.99-4.62(16H, m), 5.20-5.30(2H, m), 5.37-5.47(2H, m), 5.56(2H, d, J ═ 1.2Hz), 5.75(2H, dd, J ═ 8.0, 2.0Hz), 6.94(1H, d, J ═ 15.6Hz), 7.10-7.20(2H, m), 7.26-7.34(1H, m, overlapping CDCl)₃Signal), 7.36-7.74(7H, m), 7.80(1H, d, J ═ 15.2Hz), 8.05-8.20(3H, m), 8.44(2H, d, J ═ 7.6 Hz).

5) Preparation of Compounds 5-5

A solution of NaOMe (8.0mg, 0.15mmol) in MeOH (0.5mL) was added dropwise to a solution of compound 5-4(100mg, 0.073mmol) in MeOH (2mL) and DCM (2mL) at 0 deg.C. Then, the mixture was stirred at 0 ℃ for 2 hours. Crude LCMS showed desired product MS observed. The mixture was quenched with AcOH (9.0mg, 0.15 mmol). Subsequently, the mixture was concentrated and dried at 25 ℃ to give crude compound 5-5(79mg) as a yellow solid. Next, 79mg of crude compound 5-5 was used directly in the next step.

6) Preparation of PD001

A mixture of compounds 5-5(79mg, crude), compounds 1-3(24mg, 0.076mmol) and DIEA (20mg, 0.15mmol) in DMF (2mL) was stirred at 20 ℃ for 16 h. Crude LCMS showed desired product MS observed. By preparative HPLC (0.05% NH)₃·H₂O) purifying the solvent. Most of the MeCN was removed under reduced pressure. The remaining aqueous phase was lyophilized to give pure PD001(20mg, two-step yield: 22%) as a yellow solid.

¹H NMR(400MHz，DMSO-d₆)δ1.15-1.27(2H，m)，1.42-1.60(4H，m)，2.13(2H，t，J＝7.6Hz)，3.48-3.74(10H，m)，3.75-3.88(4H，m)，3.89-4.08(4H，m)，4.24-4.40(4H，m)，4.48-4.74(8H，m)，4.92-5.05(4H，m)，5.38(2H，d，J＝5.6Hz)，6.96(2H，s)，7.29-7.45(4H，m)，7.48(1H，d，J＝8.0Hz)，7.52-7.63(2H，m)，7.66(1H，s)，7.90(1H，d，J＝15.6Hz)，7.86-8.01(4H，m)，8.20-8.25(1H，m)，8.33(2H，dd，J＝8.4，4.8Hz)，8.38-8.48(2H，m)。

7) Preparation of Compounds 1-2

Furan-2, 5-dione (374mg, 3.81mmol) was added to a solution of 6-aminocaproic acid (500mg,3.81mmol) in AcOH (5 mL). The mixture was heated at 25 ℃ under N₂Stirred for 2 hours. Subsequently, the mixture was heated at 110 ℃ under N₂Stirred for 16 hours. TLC showed another spot formed. The reaction mixture was concentrated in vacuo. Water (10mL) was added to the residue, the resulting solution was extracted with EtOAc (20mL x 3), and the combined organic layers were extracted with anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. The use of petroleum ether: ethyl acetate/1: 1 to 1: 2 the residue was purified by silica gel chromatography to give compound 1-2(525mg, yield: 65%) as a white solid.

¹H NMR(400MHz，DMSO-d₆) δ 1.11-1.29(2H, m), 1.40-1.57(4H, m), 2.18(2H, t, J ═ 7.2Hz), 3.36-3.41(2H, m, overlap with water signal), 6.93-7.07(2H, s), 12.00(1H, brs).

8) Preparation of Compounds 1-3

A solution of compound 1-2(525mg, 2.49mmol), 1-hydroxy-2, 5-pyrrolidinedione (300mg, 2.61mmol) and DIC (336mg, 2.66mmol) in DCM (5mL) at 25 ℃ in N₂Stirred for 16 hours. TLC showed another spot formed. To the reaction mixtureWater (10mL) was added and the resulting solution was extracted with EtOAc (15mL 4). The combined organic phases were washed with anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. The use of petroleum ether: ethyl acetate/2: 1 to 1: 1 to 1: 2 as eluent the residue was purified by silica gel chromatography to give impure compounds 1-3(643mg) as white solids.

¹H NMR(400MHz，CDCl₃)δ1.32-1.45(2H，m)，1.61-1.65(2H，m)，1.73-1.81(2H，m)，2.59(2H，t，J＝7.2Hz)，2.82(4H，s)，3.52(2H，t，J＝7.2Hz)，6.68(2H，m)。

9) Preparation of Compounds 1-5

5-bromo-2-iodo-phenol (1.0g, 3.35mmol), tert-butyl acrylate (1.50g, 11.7mmol), Pd (OAc)₂(15mg, 0.67mmol), tris (o-methylphenyl) phosphine compound (79mg, 0.26mmol) and Et₃A solution of N (1.02g, 10.0mmol) in DMF (10mL) at 110 ℃ in N₂Stirred for 16 hours. TLC showed the desired product formed. The reaction mixture was quenched in 60mL water and the resulting solution was extracted with EtOAc (40mL 4). The combined organic layers were washed with water (100mL) and anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. The use of petroleum ether: ethyl acetate/24: 1 to 20:1 to 6: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compound 1-5(963mg, yield: 83%) as a yellow solid.

¹H NMR(400MHz，DMSO-d₆)δ1.48(18H，s)，6.41(1H，d，J＝15.6Hz)，6.56(1H，d，J＝16.0Hz)，7.07(1H，s)，7.19(1H，d，J＝8.0Hz)，7.45(1H，d，J＝15.6Hz)，7.63(1H，d，J＝8.0Hz)，7.75(1H，d，J＝16.4Hz)，10.42(1H，brs)。

10) Preparation of Compounds 1-6

A solution of compounds 1-5(2.00g, 5.77mmol) and 1-5A (2.45g, 8.66mmol) in THF (20mL) at 0 deg.C in N₂Stirred for 30 minutes. When the clear solution turned into a white emulsion, Ph3P (2.57g, 9.81mmol) was added to the mixture at 0 deg.C and N₂Stirred for 10 minutes. Subsequently, DIAD (1.75g, 8.66mmol) was added dropwise to the mixture at 0 deg.C (white emulsion turned into orange clear solution). Then, the resulting solution was allowed to stand in N₂The temperature was raised to 25 ℃ for 1 hour. TLC showed the desired product formed. 1N aqueous HCl (60mL) was added to the reaction mixture and the resulting solution was extracted with EtOAc (50mL × 3). The combined organic layers were washed with brine (80mL) and anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. Using petroleum ether, then using petroleum ether: ethyl acetate/7: 1 the residue was purified on a CombiFlash apparatus as eluent to yield impure compounds 1-6(3.67g) as colorless oils.

11) Preparation of Compounds 1-7

TFA (19.3g, 169mmol) was added to a solution of compounds 1-6(3.67g, impure) in DCM (80 mL). The mixture was stirred at 25 ℃ for 16 hours without monitoring. The reaction mixture was concentrated in vacuo. The residue was triturated with methyl tert-butyl ether (60mL) and then filtered. The filter cake was washed with methyl tert-butyl ether (20mL x 3). The filter cake was dried in vacuo to give compounds 1-7(2.12g, 2-step yield: 74%) as a white solid.

¹H NMR(400MHz，DMSO-d₆)δ3.43-3.46(2H，m)，4.13-4.27(3H，m)，4.31-4.43(2H，m)，6.59(1H，d，J＝16.2Hz)，6.69(1H，d，J＝16.2Hz)，7.29-7.35(3H，m)，7.36-7.48(3H，m)，7.55-7.65(2H，m)，7.66-7.77(3H，m)，7.80-7.92(3H，m)，12.40(2H，brs)。

Preparation example 2: synthesis of PD005

1) Preparation of Compounds 5-5

A solution of NaOMe (4.0mg, 0.073mmol) in MeOH (0.1mL) was added dropwise to a solution of compound 5-4(50mg, 0.036mmol) in MeOH (1mL) and DCM (1mL) at 0 deg.C. Subsequently, the mixture was stirred at 0 ℃ for 3.5 hours. Crude LC-MS was shown to be at retention time 0.828(MS calculation: 1031; MS: 1034[ M + 3H)]⁺) The purity of the product was 93%. The mixture was quenched with AcOH (4.6 mg). The mixture was then concentrated to dryness at 25 ℃ to give crude compound 5-5(41mg) as a yellow solid. 41mg of crude compound 5-5 was used directly in the next step.

2) Preparation of PD005

A mixture of compounds 5-5(41mg, crude), 5-6(11mg, crude) and DIPEA (10mg, 0.079mmol) in DMF (1mL) was stirred at 15 ℃ for 16 h. Crude LC-MS showed a retention time of 0.845(MS calculation: 1143; MS found: 1149[ M +6H ]]⁺) The purity of the product was 89%. The reaction mixture was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure. The remaining aqueous phase was lyophilized to give crude PD005(11mg) as a yellow solid. Furthermore, HNMR showed that traces of aldehyde signal protons were observed.

3) Preparation of Compound 5-5A

HOBt (9.5mg, 0.070mmol), EDCI (13mg, 0.070mmol) and DIEA (9.1mg, 0.070mmol) were added to a solution of compound 5-6b (8.4mg, 0.064mmol) in DCM (2 mL). Subsequently, the mixture was stirred at 15 ℃ for 10 minutes. Compound 5-4(80mg, 0.058mmol) was added to the reaction mixture. The prepared mixture was stirred at 15 ℃ for 2 hours. TLC showed complete consumption of starting material. A new spot is formed. The mixture was quenched with water (10mL) and extracted with DCM (6mL × 3). The combined organic layers were washed with Na₂SO₄Dried and concentrated to dryness. The residue was purified on a silica gel column using EtOAc as an eluent to give compound 5-5A (81mg, yield: 94%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) Δ 1.58-1.75(4H, m), 1.95-2.05(12H, m), 2.06-2.16(12H, m), 2.24-2.31(2H, m), 2.40-2.49(2H, m), 3.42-3.55(3H, m), 3.69-3.79(2H, m), 3.92-4.05(2H, m), 4.10-4.38(9H, m, overlap EtOAc signal), 4.42-4.64(4H, m), 5.12-5.26(2H, m), 5.30-5.42(2H, m), 5.50-5.60(2H, m), 5.67-5.77(2H, m), 6.91-7.06(2H, m), 7.13-7.22(2H, m, overlap Cl)₃Signal), 7.35-7.48(3H, m), 7.50-7.60(2H, m), 7.65-7.76(3H, m), 7.77-7.84(1H, m), 8.09-8.19(2H, m), 8.20-8.30(1H, m), 8.37-8.50(2H, m), 9.71(1H, s).

4) Preparation of PD005

A solution of NaOMe (1.8mg, 0.034mmol) in MeOH (0.1mL) was added dropwise to a stirred solution of compound 5-5A (25mg, 0.017mmol) in MeOH (0.5mL) and DCM (0.5mL) at 0 deg.C. Then, the mixture was stirred at 0 ℃ for 2 hours. Crude LC-MS showed a retention time of 0.834(MS calculation: 1143; MS found: 1144[ M + H ]]⁺) The purity of the product was 98%. The mixture was then quenched with water (5mL) and the solvent removed under reduced pressure at 15 ℃. The resulting yellow precipitate was collected by filtration. Will be provided withThe filter cake was diluted with water (10mL) and lyophilized to give crude PD005(11mg) as a yellow solid. About 6mg of crude was used for HNMR analysis and 5mg of crude was provided.

¹H NMR (400MHz, DMSO). delta.1.19-1.60 (4H, m), 2.05-2.22(2H, m), 2.35-2.45(2H, m, overlapping DMSO signals), 3.45-4.76(30H, m), 4.90-5.05(4H, m), 5.35-5.53(2H, m), 7.18-8.05(13H, m), 8.15-8.50(4H, m), 9.61(0.5H, m).

A solution of NaOMe (4.1mg, 0.076mmol) in MeOH (0.2mL) was added dropwise to a stirred solution of compound 5-5A (56mg, 0.038mmol) in MeOH (1mL) and DCM (1mL) at 0 deg.C. Subsequently, the mixture was stirred at 0 ℃ for 2 hours. Crude LCMS showed the formation of the desired product M. Crude LC-MS showed a retention time of 0.857(MS calculation: 1143; MS found: 1169[ M + Na;)]⁺) The purity of the product was 98%.

The mixture was then quenched with water (10mL) and the solvent removed under reduced pressure at 15 ℃. The resulting yellow precipitate was collected by filtration. The filter cake was diluted with water (10mL) and lyophilized to give crude PD005(27mg) as a yellow solid.

¹H NMR (400MHz, DMSO). delta.1.19-1.60 (4H, m), 2.05-2.21(2H, m), 2.35-2.45(2H, m, overlapping DMSO signals), 3.45-4.76(30H, m), 4.90-5.05(4H, m), 5.35-5.51(2H, m), 7.21-8.01(13H, m), 8.15-8.50(4H, m), 9.61(0.4H, m).

5) Preparation of Compounds 5-6b

Ethyl 6-oxohexanoate (546mg, 3.45mmol), TsOH (15mg, 0.08mmol) and H₂O (1.2mL) solution at 90 ℃ in N₂Stirred for 16 hours. TLC showed another spot formed. The reaction mixture was quenched in water (10mL) and the resulting solution was extracted with EtOAc (15mL × 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. The use of petroleum ether: ethyl acetate/1: 1 to 2: 3 as eluent, the residue is subjected to a silica gel columnPurification gave compound 5-6b (383mg, yield: 85%) as colorless oil.

¹H NMR(400MHz，DMSO-d₆)δ1.49-1.53(4H，m)，2.22(2H，t，J＝6.8Hz)，2.44(2H，t，J＝7.2Hz)，9.66(1H，s)，12.01(1H，brs)。

6) Preparation of Compounds 5-6

In N₂Next, DIC (397mg, 3.15mmol) was added to a solution of compound 5-6b (383mg, 2.94mmol) and N-hydroxysuccinimide (356mg, 3.09mmol) in DCM (4 mL). The mixture was stirred at 25 ℃ for 3 hours. TLC showed the formation of another less polar spot. The reaction mixture was filtered, the filtrate quenched with water (10mL), and the organic layer separated. The aqueous phase was then extracted with DCM (10mL × 3). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of petroleum ether: ethyl acetate/3: 2 to 2: 3 the residue was purified on a silica gel column as eluent to give impure compound 5-6(204mg) as a white solid.

¹H NMR(400MHz，CDCl₃)δ1.76-1.78(4H，m)，2.49-2.51(2H，m)，2.64(2H，t，J＝6.8Hz)，2.84(4H，s)，9.77(1H，s)。

Preparation example 3: synthesis of PD006

1) Preparation of Compound 6-4

The mixture of Compound 6-3(349mg, 0.635mmol), Compound 6-2(220mg, 0.635mmol) and K₂CO₃A mixture of (105mg, 0.762mmol) in DMF (3mL) was heated to 60 deg.CAnd stirred for 16 hours. Crude LC-MS showed a retention time of 1.088(MS calculation: 723.4; MS found: 746.3[ M + Na;)]⁺) The purity of the product was 59%. Most of the solvent was removed under reduced pressure. The mixture was quenched with water (20mL) and extracted with EtOAc (15mL × 3). The combined organic layers were washed with water (20mL x 2) and Na₂SO₄Drying, concentration and further drying gave compound 6-4(0.45g, yield 98%) as a brown oil.

¹H NMR(400MHz，CDCl₃)δ1.51-1.56(18H，m)，3.39(2H，t，J＝4.8Hz)，3.61-3.68(24H，m)，3.69-3.72(2H，m)，3.92(2H，t，J＝4.8Hz)，4.21(2H，t，J＝4.8Hz)，6.37(1H，d，J＝16.0Hz)，6.50(1H，d，J＝16.0Hz)，7.02(1H，s)，7.10(1H，d，J＝8.0Hz)，7.47-7.55(2H，m)，7.86(1H，d，J＝16.0Hz)。

The synthesis of compound 6-2 was the same as in the above example of PD 001.

2) Preparation of Compounds 6-5

PPh at 10 ℃₃(196mg, 0.746mmol) was added to a solution of Compound 6-4(450mg, 0.622mmol) in THF (4mL), and the mixture was stirred at1 deg.C for 1 hour. Then, H was added to the mixture₂O (1mL), and the resulting mixture was stirred at 10 ℃ for 16 hours. Crude LC-MS showed retention time of 0.967(MS calculation: 697.4; MS found: 698.4[ M + H ]]⁺) The purity of the product was 21%. The solvent was removed under reduced pressure. Using EtOAc, DCM: MeOH/5: 1, then MeOH: NH (NH)₃·H₂O/100: 1 as eluent, the residue was purified to give compound 6-5(285mg, yield 66%) as a brown oil.

¹H NMR(400MHz，CDCl₃) δ 1.52-1.64(18H, m, superposed water signal), 2.88(2H, t, J ═ 5.2Hz), 3.63(2H, t, J ═ 5.2Hz), 3.62-3.75(22H, m), 3.76-3.80(2H, m), 3.94(2H, t, J ═ 5.2Hz), 4.24(2H, t, J ═ 5.2Hz), 6.39(1H, d, J ═ 1H, t, J ═ 5.2Hz)＝16.0Hz)，6.52(1H，d，J＝16.4Hz)，7.04(1H，s)，7.12(1H，d，J＝8.4Hz)，7.49-7.57(2H，m)，7.89(1H，d，J＝16.4Hz)。

No two active protons were observed.

3) Preparation of Compounds 6-11

Fmoc-Cl (96mg, 0.37mmol) and NaHCO were combined at 10 deg.C₃(31mg, 0.37mmol) was added to compound 6-5(235mg, 0.337mmol) in THF (1mL) and H₂O (1 mL). Then, the mixture was stirred at 10 ℃ for 2 hours. The crude LC-MS showed a retention time of 1.155(MS calculation: 919.5; found MS: 942.5[ M + Na)]⁺) The purity of the product was 84%. The mixture was quenched with water (5mL) and extracted with EtOAc (15mL × 3). Mixing the organic layer with Na₂SO₄Drying, concentration and further drying gave crude compound 6-11(325mg) as a colourless oil containing FmocCl.

4) Preparation of Compounds 6-12

A mixture of compound 6-11(325mg, crude) and DCM (3mL) in TFA (2mL) was stirred at 10 ℃ for 16 h. Crude LC-MS showed a retention time of 0.890(MS calculation: 807.4; MS found: 830.3[ M + Na;)]⁺) The purity of the product was 87%. The solvent was removed under reduced pressure. The residue was triturated with EtOAc (5mL) to give compound 6-12(191mg, two-step yield: 70%) as a white solid.

¹H NMR (400MHz, DMSO) δ 3.10-3.17(2H, m), 3.30-3.43(2H, m, overlapping water signal), 3.45-3.58(24H, m), 3.59-3.64(2H, m), 3.78-3.84(2H, m), 4.18-4.32(5H, m), 4.42(2H, d, J ═ 7.2Hz), 5.46(1H, brs), 6.65(2H, dd, J ═ 16.0, 14.4Hz), 7.27-7.36(4H, m), 7.38-7.46(3H, m), 7.57(1H, d, J ═ 16.0Hz), 7.66-7.74(3H,m)，7.81(1H，d，J＝16.4Hz)，7.89(2H，d，J＝7.6Hz)，12.4(2H，brs)。

5) preparation of Compounds 6-13

A mixture of compound 6-12(90mg, 0.11mmol), compound 6-9(188mg, 0.334mmol) and EDCI (64mg, 0.33mmol) in DMF (2mL) was stirred at 10 ℃ for 16 h. TLC showed the desired product formed. The solvent is removed under greatly reduced pressure. The use of PE: EtOAc/2: 1, then using EtOAc: MeOH/50: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compound 13(96mg, yield 45%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 2.04-2.09(12H, m, overlapping EtOAc signals), 2.16(6H, d, J ═ 10.4Hz), 2.24(6H, s), 3.35-3.43(2H, m), 3.47-3.84(30H, m), 3.98-4.09(4H, m), 4.11-4.62(15H, m, overlapping EtOAc signals), 5.17-5.27(2H, m), 5.34-5.43(2H, m), 5.46-5.62(3H, m), 5.69-5.80(2H, m), 6.97(1H, d, J ═ 14.8Hz), 7.17(1H, s), 7.25-7.36(4H, m, overlapping CDCl), 2.16 (1H, m, overlapping CDCl)₃Signals), 7.37-7.50(4H, m), 7.53-7.66(5H, m), 7.69-7.80(4H, m), 7.83(1H, d, J ═ 15.2Hz), 8.00(1H, d, J ═ 15.2Hz, overlaid DMF signals), 8.17(2H, dd, J ═ 8.4, 2.0Hz), 8.46(2H, d, J ═ 12.0 Hz).

The preparation of compounds 6-9 was the same as in the above example for PD 001.

6) Preparation of Compounds 6-14

A solution of piperidine (86mg, 1.0mmol, 0.1mL) and compound 6-13(96mg, 0.051mmol) in DCM (1mL) was stirred at 10 deg.C for 3 h. TLC showed complete consumption of the starting material and formed a new main spot. The solvent was removed under reduced pressure. Using DCM: MeOH/100: 1 to 9: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compounds 6-14(75mg, yield: 78%) as yellow solids.

¹H NMR(400MHz，CDCl₃) δ 2.03-2.09(12H, m, overlap EtOAc signal), 2.17(6H, d, J ═ 10.4Hz), 2.24(6H, s), 3.11-3.18(2H, m), 3.48-3.87(30H, m), 3.97-4.70(18H, m, overlap EtOAc signal), 5.16-5.26(2H, m), 5.36-5.46(2H, m), 5.51-5.63(2H, m), 5.68-5.79(2H, m), 7.13(1H, d, J ═ 15.2Hz), 7.24-7.37(3H, m, overlap CDCl), 2.17(6H, d, J ═ 10.4Hz), 2.24-5.70 (2H, m, overlap EtOAc signal), 5.16-5.26(2H, m), 5.36-5.46(2H, m), 5.51-5.63(2H, m), 5.79(2H, m), 7.13(1H, J ═ 15.2Hz), 7.37(3H, m, overlap CDCl)₃Signals), 7.40-7.49(2H, m), 7.53-7.65(3H, m), 7.73(2H, d, J ═ 8.0Hz), 7.83(1H, d, J ═ 15.2Hz), 8.02(1H, d, J ═ 15.2Hz), 8.16(2H, d, J ═ 8.4Hz), 8.46(2H, d, J ═ 8.4 Hz).

7) Preparation of Compounds 6-15

Adding K at 10 ℃₂CO₃(14mg, 0.10mmol) was added to a solution of compounds 6-14(87mg, 0.052mmol) in MeOH (2mL) and DCM (1 mL). Then, the mixture was stirred at 10 ℃ for 1 hour. Crude LC-MS showed retention time of 0.836(MS calculation: 1339.5; MS found: 1340.9[ M + H ]]⁺) The purity of the product was 90%. TLC showed complete consumption of the starting material. The mixture was quenched with AcOH (10 mg). The mixture was concentrated to dryness to give crude compound 6-15(74mg) as a yellow solid which was used directly in the next step.

8) Preparation of PD006

A mixture of compound 6-15(74mg, crude), compound 6-6(16mg, 0.061mmol) and DIEA (8.6mg, 0.066mmol) in DMF (1mL) was stirred at 10 ℃ for 5 hours. Crude LC-MS showed a retention time of 0.842(MS calculation: 1490.5; found MS: 1493.5[ M + 2H)]⁺) The purity of the product was 94%. The solvent was removed under reduced pressure. By preparative HPLC (0.225% FA) purification residue. The remaining aqueous solution was lyophilized to give PD006(8mg) and 7mg of impurity. Impure PD006 was further purified by preparative HPLC (0.225% FA). The remaining aqueous solution was lyophilized to give PD006(4mg) as a yellow solid. A total of 12mg PD006 was obtained, with a yield of 16% in two steps.

¹H NMR (400MHz, DMSO) δ 2.24-2.37(2H, m, overlapping DMSO signals), 3.08-3.21(2H, m), 3.24-4.10(46H, m, overlapping water signals), 4.26-4.77(12H, m), 4.88-5.02(4H, m), 5.34-5.47(2H, m), 6.99(1.1H, s), 7.30-7.64(8H, m), 7.74(1H, d, J ═ 15.2Hz), 7.83-7.94(4H, m), 7.98-8.04(1H, m), 8.29-8.46(4H, m).

9) Preparation of Compound 6-3B

TsCl (14.7g, 77.2mmol) was added to a mixture of tetraethyleneglycol (30.0g, 154mmol), EtN (11.7g, 116mmol) and DMAP (944mg, 7.72mmol) in DCM (300mL) at 0 ℃. The reaction mixture was stirred at 10 ℃ for 16 hours. TLC showed two new spots and showed complete consumption of the starting material. The reaction mixture was quenched with water (100mL) and the organic layer was separated. The remaining mixture was then extracted with DCM (100mL 4). The combined organic layers were washed with brine (400 mL). Na for organic layer₂SO₄Dried, filtered and concentrated in vacuo. Using EtOAc, then DCM: MeOH/20: 1 as an eluent, the residue was purified on a silica gel column to give 6-3B (9.33g) and 6-3B (7.16g) as colorless oily compounds. Then, using DCM: MeOH/50: 1 to 20:1 As eluent 7.16g of impure oil was purified on a Combiflash apparatus to give compound 6-3B (4.89g) as a colourless oil. Thus, a total of 14.2g of compound 6-3B was obtained, and the yield was 26%.

¹H NMR(400MHz，CDCl₃)δ2.45(3H，s)，2.48-2.62(1H，brs)，3.58-3.71(14H，m)，4.17(2H，t，J＝4.8Hz)，7.34(2H，d，J＝8.0Hz)，7.80(2H，dd，J＝6.4Hz，1.6Hz)。

Case 2:

TsCl (9.82g, 51.5mmol), KI (855mg, 5.15mmol) and Ag were mixed at 15 deg.C₂A mixture of O (14.3g, 61.8mmol) was added to a solution of tetraethylene glycol (10.0g, 51.5mmol) in DCM (300 mL). The reaction mixture was stirred at 15 ℃ for 16 hours. TLC showed another spot with greater polarity and showed complete consumption of the starting material. The reaction mixture was filtered through celite, and the filtrate was concentrated in vacuo. The use of petroleum ether: ethyl acetate/1: 1 with MeOH as an eluent, the residue was purified on a Combiflash apparatus to give compound 6-3B (11.8g, yield: 66%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ2.32-2.44(1H，brs)，2.45(3H，s)，3.60-3.71(14H，m)，4.17(2H，t，J＝4.4Hz)，7.33(2H，d，J＝8.0Hz)，7.80(2H，d，J＝8.0Hz)。

10) Preparation of Compound 6-3C

Adding NaN₃(465mg, 7.15mmol) was added to a solution of Compound 6-3B (1.66g, 4.76mmol) in DMF (20 mL). The reaction mixture was stirred at 60 ℃ for 16 hours. TLC showed another less polar spot and showed complete consumption of the starting material. The reaction mixture was quenched with water (50mL) and the resulting solution was extracted with EtOAc (50mL × 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. Using DCM: MeOH/40: 1 to 30: 1 the residue was purified on a silica gel column as eluent to give compound 6-3C as an impure pale yellow oil (800 mg).

¹H NMR(400MHz，CDCl₃)δ2.46-2.72(1H，brs)，3.34-3.45(2H，m)，3.59-3.64(2H，m)，3.66-3.71(10H，m)，3.71-3.75(2H，m)。

11) Preparation of Compound 6-3D

EtN (517mg, 5.11mmol) and TsCl (974mg, 5.11mmol) were added to a solution of compound 6-3C (800mg, impure) in DCM (16mL) at 0 deg.C, then allowed to warm to 15 deg.C for 16 h. TLC showed another spot of low polarity. The mixture was quenched with water (20mL) and the organic layer was separated. The remaining mixture was then extracted with DCM (15mL × 3). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of petroleum ether: ethyl acetate/2: 1 to 1: 1 as an eluent, the residue was purified on a silica gel column to give compound 6-3D (1.01g, two-step yield: 57%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ2.47(3H，s)，3.35-3.46(2H，m)，3.62-3.72(12H，m)，4.18(2H，t，J＝4.8Hz)，7.36(2H，d，J＝8.0Hz)，7.82(2H，d，J＝8.4Hz)。

12) Preparation of Compound 6-3E

A solution of compound 6-3A (3.41g, 17.6mmol) in THF (3mL) at 0 deg.C under N₂Add dropwise to a suspension of NaH (780mg, 60% pure in 19.5mmol mineral oil) in THF (33 mL). The reaction was stirred at 10 ℃ for 1.5 hours. Then, a solution of compound 6-3D (3.28g impure) in THF (3mL) was added dropwise to the refluxing sodium alkoxide solution. The mixture was then refluxed (70 ℃ C.) for 16 hours. TLC showed the formation of a new spot. After the reaction mixture was cooled to room temperature, THF was removed in vacuo. The residue was quenched with water (30mL) and the resulting solution was extracted with mixed solvents (DCM: MeOH/10: 1) (50mL × 5). Mixing the organic layer with anhydrous Na₂SO₄Drying, filtration, and concentration in vacuo afforded crude compound 6-3E (3.47g, crude) as receivedUsed in the next step.

13) Preparation of Compound 6-3

Adding Et₃N (1.12g, 11.1mmol) was added to a solution of compound 6-3E (3.03g, crude) in DCM (30mL) and TsCl (2.12g, 11.1mmol) was added to the mixture at 0 deg.C. The reaction mixture was warmed to 10 ℃ and stirred for 16 hours. TLC showed another new spot formed. The reaction mixture was quenched with water (40mL) and the organic layer was separated. The remaining aqueous phase was then extracted with DCM (30mL × 5). The combined organic layers were then washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of petroleum ether: ethyl acetate (EtOAc)/3: 1 as eluent the residue was purified on a silica gel column to give impure compound 6-3(1.82 g). The use of petroleum ether: ethyl acetate/2: 1, the impure solid was then purified using a CombiFlash apparatus using EtOAc as eluent to give compound 6-3(1.26g, two steps yield: 26%) as a yellow oil.

¹H NMR(400MHz，CDCl₃)δ2.47(3H，s)，3.40-3.53(2H，m)，3.54-3.83(28H，m)，4.14-4.24(2H，m)，7.36(2H，d，J＝8.4Hz)，7.81(2H，d，J＝8.0Hz)。

14) Preparation of Compounds 6-6

EDCI (1.21g, 6.33mmol) was added to a solution of 3-maleimidopropionic acid (1.00g, 5.91mmol) and N-hydroxysuccinimide (714mg, 6.21mmol) in DCM (10mL) and the resulting mixture was stirred at 15 ℃ in N₂Stirring in the presence for 16 hours. TLC showed the formation of another spot with lower polarity. The reaction was quenched with water (15mL) and the organic layer was separated. The remaining aqueous phase was then extracted with DCM (20mL × 3). The combined organic layers were washed with anhydrous Na₂SO₄Drying, filtering, and vacuum dryingAnd (4) concentrating in air. The use of petroleum ether: ethyl acetate/1: 2 as an eluent, the residue was purified on a silica gel column to give compound 6-6(1.18g, yield: 75%) as a white solid.

¹H NMR(400MHz，CDCl₃)δ2.84(4H，s)，3.04(2H，t，J＝14.0Hz)，3.95(2H，t，J＝7.2Hz)，6.76(2H，s)。

Preparation example 4: synthesis of PD008

1) Preparation of Compound 8-1

A mixture of (2, 5-dioxopyrrolidin-1-yl) 6-oxohexanoate (16mg, 0.072mmol), DIPEA (15mg, 0.12mmol) and compound 6-14(100mg, 0.0596mmol) in DCM (1mL) was stirred at 10 ℃ for 16 h. TLC showed the formation of a new spot with lower polarity. The mixture was quenched with water (8mL) and extracted with DCM (8mL × 3). The combined organic layers were washed with anhydrous Na₂SO₄Dried and concentrated to dryness. The reaction was performed using EtOAc: MeOH/10: 1 as an eluent, the residue was purified by preparative TLC to give compound 8-1(59mg, yield: 55%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 1.55-1.76(4H, m, overlap water signal), 2.03-2.10(12H, m), 2.13-2.27(14H, m), 2.45-2.50(2H, m), 3.42-3.48(2H, m), 3.49-3.85(30H, m), 3.99-4.63(16H, m), 5.22(2H, dd, J ═ 10.4, 3.2Hz), 5.40(2H, dd, J ═ 7.2, 2.0Hz), 5.57(2H, d, J ═ 2.8Hz), 5.70-5.79(2H, m), 6.41(1H, brs), 6.98(1H, d, J ═ 15.2Hz), 7.18(1H, s), 7.26-7.36H, cdm (2H, cdh, cdm, overlap water signal), 2.03-2H, m, 3.42H, d, m, c₃Signals), 7.41-7.48(2H, m), 7.54-7.65(3H, m), 7.70-7.78(2H, m), 7.84(1H, d, J ═ 15.2Hz), 8.01(1H, d, J ═ 15.6Hz), 8.14-8.20(2H, m), 8.46(2H, d, J ═ 11.6Hz), 9.77(1H，t，J＝1.6Hz)。

The synthesis of (2, 5-dioxopyrrolidin-1-yl) 6-oxohexanoate ester and compound 6-14 was the same as in the preparation example of PD 006.

2) Preparation of compound PD008

The reaction mixture of Compound 8-1(59mg, 0.033mmol) and K₂CO₃A mixture of (9.1mg, 0.065mmol) in MeOH (1mL) and DCM (0.5mL) was stirred at 15 deg.C for 1 h. Crude LC-MS showed a retention time of 0.796(MS calculation: 1451.5; MS found: 1455.1[ M +3H ]]⁺) The purity of the product was 99.9%. The mixture was quenched with AcOH (4 mg). The resulting mixture was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure. The remaining mixture was lyophilized to give PD008(16mg, yield: 33%) as a yellow solid.

¹H NMR (400MHz, DMSO). delta.1.36-1.56 (4H, m), 1.95-2.12(2H, m), 2.33-2.44(2H, m, overlapping DMSO-d)₆Signal), 3.08-3.22(2H, m, overlap water signal), 3.24-4.11(44H, m, overlap water signal), 4.24-4.74(12H, m), 4.86-5.04(4H, m), 5.24-5.57(2H, m), 7.27-7.64(8H, m), 7.68-7.94(6H, m), 8.28-8.49(4H, m), 9.64(0.6H, s).

Preparation example 5: synthesis of PD009

1) Preparation of Compound 2

In N₂Next, tris (o-tolyl) -phosphine (433mg, 1.42mmol) and palladium acetate (80mg, 0.36mmol) were added to a solution of 2, 5-dibromobenzene (5.00g, 17.8mmol) and tert-butyl acrylate (6.84g, 53.4mmol)In solution. The mixture was stirred at 100 ℃ for 16 hours. TLC showed that compound 1 was not completely consumed. Subsequently, tert-butyl acrylate (4.00g), tri (o-tolyl) -phosphine (224mg) and palladium acetate (80mg) were added to the reaction mixture. Then, the mixture was stirred at 100 ℃ for 20 hours. TLC showed the formation of another spot with greater polarity. The reaction mixture was concentrated in vacuo, EtOAc (50mL) was added to the residue, the mixture was filtered, and the filtrate was concentrated in vacuo. The use of PE: EtOAc/50: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compound 2(5.72g, yield: 85%) as a yellow solid.

¹H NMR(400MHz，CDCl₃)δ1.54(18H，s)，6.34(1H，d，J＝15.6Hz)，6.48(1H，d，J＝16.0Hz)，7.56(1H，d，J＝16.0Hz)，7.65(1H，d，J＝8.0Hz)，7.72(2H，dd，J＝8.0Hz，1.6Hz)，7.98(1H，d，J＝16.0Hz)，8.12(1H，d，J＝1.6Hz)。

2) Preparation of Compound 3

Zn (9.73g, 149mmol) was added to a solution of Compound 2(6.98g, 18.6mmol) in acetone (70mL) cooled in an ice bath, and NH was then added thereto₄Cl (3.98g, 74.4mmol) in H₂O (35mL) solution. The mixture was stirred at 5 ℃ for 4 hours. TLC showed another spot with greater polarity formed and the starting material was completely consumed. EtOAc (50mL 4) was added to the reaction mixture and the clear solution present as the uppermost layer was collected. The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of PE: EtOAc/16: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compound 3(5.22g, yield: 81%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 1.54(18H, s, superposed water signal), 3.99(2H, brs), 6.26-6.38(2H, m), 6.81(1H, s), 6.93(1H, d, J ═ 9.6Hz), 7.37(1H, d, J ═ 8.4Hz), 7.46(1H, d,J＝16.0Hz)，7.68(1H，d，J＝16.0Hz)。

3) preparation of Compound 6

A mixture of compound 3(300mg, 0.484mmol), TsOH (8mg, 0.05mmol), compound 9-5(251mg, 0.726mmol) and EDCI (464mg, 2.42mmol) in DMA (6mL) was stirred at 10 ℃ for 16 h. TLC showed the formation of another spot with greater polarity. The DMA was removed in vacuo. The residual solution was quenched in water (10mL), and the resulting solution was quenched with EtOAc: MeOH ═ 3: 1 extraction (30mL x 6). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. EtOAc was used, then EtOAc: MeOH/10: 1 as an eluent, the residue was purified on a silica gel column to give compound 6(319mg, yield: 69%) as a yellow oil.

¹H NMR(400MHz，CDCl₃) δ 1.52(18H, s), 2.71(2H, t, J ═ 5.6Hz), 3.44-3.67(26H, m), 3.71-3.75(2H, m), 3.82-3.89(2H, m), 4.16-4.24(1H, m), 4.38-4.40(2H, m), 5.45(1H, brs), 6.33-6.45(2H, m), 7.26-7.32(3H, m, overlapping CDCl), and the like₃Signal), 7.39(2H, t, J ═ 7.6Hz), 7.51-7.61(4H, m), 7.69-7.76(3H, m), 8.00(1H, s), 8.76(1H, brs).

4) Preparation of Compound 7

TFA (1.09g, 9.52mmol) was added to a solution of compound 6(319mg, 0.337mmol) in DCM (3 mL). The mixture was stirred at 10 ℃ for 16 hours. Crude LC-MS was shown to be at retention time 0.801(MS calculation: 834.3; MS found: 835.0[ M + H ]]⁺) The purity of the product was 95%. The reaction mixture was concentrated in vacuo. The residue was triturated with EtOAc (10mL) and filtered. The filter cake was washed with EtOAc (5mL) to give compound 7(244mg, yield: 86%) as a white solid.

¹H NMR(400MHz，DMSO-d₆) Delta.2.51-2.68 (2H, m, overlapping DMSO-d)₆Signal), 3.09-3.17(2H, m), 3.44-3.58(26H, m, overlap water signal), 3.62-3.79(2H, m), 4.16-4.21(1H, m), 4.22-4.42(2H, m), 6.48-6.61(2H, m), 7.28-7.34(3H, m), 7.36-7.49(2H, m), 7.54-7.58(2H, m), 7.66-7.72(4H, m), 7.84-7.90(3H, m), 9.88(1H, brs), 12.48(2H, brs).

5) Preparation of Compound 8

EDCI (168mg, 0.877mmol) was added to a solution of compound 7(244mg, 0.292mmol) and compound 6-9(494mg, 0.877mmol) in DMF (4mL) at 10 ℃. Subsequently, the mixture was stirred at 10 ℃ for 16 hours. TLC showed the formation of another spot with greater polarity. The DMF was removed in vacuo and the residue was quenched with water (10 mL). The resulting solution was purified with EtOAc: MeOH (20: 1) extraction (15mL × 6). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of PE: EtOAc/1: 1 to 0:1, then using EtOAc: MeOH/20: 1 as an eluent, the residue was purified on a silica gel column to give compound 8(469mg, yield: 83%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 1.97-2.04(12H, m, overlap water signal), 2.11-2.14(6H, m), 2.15-2.23(6H, m), 2.71-2.82(2H, m), 3.39-3.62(26H, m), 3.67-3.73(2H, m), 3.75-3.83(2H, m), 3.92-4.06(4H, m), 4.11-4.57(15H, m) (overlap signal), 5.22(2H, d, J ═ 7.6Hz), 5.35-5.40(2H, m), 5.50-5.56 (EtOAc 3H, m), 5.74(2H, t, J ═ 8.0Hz), 6.95-7.03(2H, m), 7.27-7.36(2H, m, overlap cl) (2H, m, overlap cl, cdh, m)₃Signals), 7.37-7.46(5H, m), 7.54-7.61(4H, m), 7.71-7.76(5H, m), 7.84(1H, d, J ═ 15.6Hz), 7.98-8.04(1H, m), 8.17(3H, d, J ═ 8.0Hz), 8.43(2H, d, J ═ 8.0Hz), 8.95(1H, brs).

The synthesis of compounds 6-9 was the same as in the preparation example of PD 005.

6) Preparation of Compound 9

Piperidine (0.3mL) was added to a solution of compound 8(197mg, 0.102mmol) in DCM (3mL) at 10 ℃. The mixture was stirred at 10 ℃ for 1.5 hours. TLC showed the formation of another spot with greater polarity. The reaction mixture was concentrated in vacuo, and the residue was quenched with water (10 mL). The resulting solution was extracted with DCM (15mL 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. Using EtOAc, then DCM: MeOH/50: 1 to 10: 1 as an eluent, the residue was purified on a silica gel column to give compound 9(96mg, yield: 55%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 2.03-2.10(12H, m, superposed water signal), 2.11-2.18(6H, m), 2.19-2.28(6H, m), 2.95-3.06(2H, m), 3.16-3.16(2H, m), 3.48-3.76(28H, m), 3.76-3.82(2H, m), 3.84-3.90(2H, m), 3.91-4.06(4H, m), 4.13-4.39(8H, m), 4.46-4.63(4H, m), 5.23(2H, d, J ═ 10.8Hz), 5.40(2H, d, J ═ 8.0Hz), 5.52-5.61(2H, m), 5.68-5.80(2H, m), 6.98(2H, t, 6J ═ 8, 7.7, 7H, 7.7H, 7, 7.7, 7.3H, 7, 7.7, 7H, 7 Hz, m), 8.12-8.20(2H, m), 8.34-8.51(2H, m), 10.04(1H, brs).

7) Preparation of Compound 10

Will K₂CO₃(19mg, 0.14mmol) was added to a solution of compound 9(120mg, 0.0704mmol) in DCM (1mL) and MeOH (2 mL). The mixture was stirred at 10 ℃ for 30 minutes. The crude LC-MS showed a new peak and showed that compound 9 was completely consumed (MS calculation: 1702.5) (MS of compound 10 exceeded 1500,and therefore not detectable). Will CH₃COOH (19mg) was added to the reaction mixture. The mixture was stirred at 10 ℃ for 5 minutes and concentrated in vacuo to give compound 10(92mg, crude) as a yellow solid.

8) Preparation of PD009

Compound 6-6(28mg, 0.11mmol) was added to a solution of compound 10(92mg, crude) and DIEA (14mg, 0.11mmol) in DMF (1mL) at 10 ℃. The mixture was stirred at 10 ℃ for 16 hours. Crude LC-MS showed a new peak and showed complete consumption of compound 10 (MS calculation: 1366.5) (MS of PD009 exceeded 1500 and could not be detected). The resulting mixture was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure. The remaining mixture was lyophilized to give PD009(40mg, two-step yield: 37%) as a yellow solid.

¹H NMR(400MHz，DMSO-d₆) δ 2.32(2H, t, J ═ 7.2Hz), 2.64-2.74(2H, m)3.09-3.18(2H, m), 3.46-3.71(33H, m, superposed water signal), 3.73-4.12(12H, m), 4.23-4.70(11H, m)4.91-5.00(4H, m), 5.34-5.44(2H, m), 6.99(1.4H, s), 7.27(2H, d, J ═ 15.2Hz), 7.43(2H, t, J ═ 8.0Hz), 7.57(2H, t, J ═ 7.6Hz), 7.66-7.81(2H, m), 7.84-7.91(4H, m), 7.98-8.10(2H, m), 8.32-7.91 (4H, m), 10.00 (4H, m), 1.00 (1H, m).

The synthesis of compound 6-6 was the same as in the preparation example of PD 006.

9) Preparation of Compound 9-2

Batch 1

At 0 ℃ and in N₂Next, a solution of triethylene glycol (2.41g, 16.1mmol) in THF (5mL) is added dropwise to a suspension of NaH (536mg, 60% in 13.4mmol mineral oil) in THF (20 mL). The reaction was stirred at 5 ℃ for 1.5 hours.Next, a solution of Compound 6-3D (2.00g,5.36mmol) in THF (5mL) was added dropwise to the refluxing sodium alkoxide solution. Then, the mixture is added to N₂Reflux (70 ℃ C.) for 16 hours. TLC showed another new position and showed complete consumption of compound 6-3D. The reaction mixture was mixed with batch 2.

Batch 2

At 0 ℃ and in N₂Next, a solution of triethylene glycol (2.41g, 16.1mmol) in THF (5mL) is added dropwise to a suspension of NaH (536mg, 60% in 13.4mmol mineral oil) in THF (20 mL). The reaction was stirred at 5 ℃ for 1.5 hours. Next, a solution of Compound 6-3D (2.00g,5.36mmol) in THF (5mL) was added dropwise to the refluxing sodium alkoxide solution. Then, the mixture is added to N₂Reflux (70 ℃ C.) for 16 hours. TLC showed another new position and showed complete consumption of compound 6-3D. The reaction mixture was quenched with batch 1 with water (50 mL). The organic layer was separated and then washed with DCM: MeOH (10: 1) extracted the remaining mixture (50mL 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. Using DCM: MeOH/100: 1 to 50: 1 as an eluent, the residue was purified on a silica gel column to give compound 9-2' (3.03g, two-batch yield: 80%) as a pink oil.

¹H NMR(400MHz，CDCl₃)δ3.41(2H，t，J＝5.2Hz)，3.62-3.76(26H,m)。

The synthesis of compound 6-3D was the same as in the preparative example for PD 006.

10) Preparation of Compound 9-3

NaH (4.0mg, 0.093mmol, 60% in mineral oil) was added to a solution of compound 9-2' (328mg, 0.933mmol) in THF (5mL) at 0 deg.C and the mixture was stirred at 0 deg.C for 1 h. Next, tert-butyl acrylate (239mg, 1.87mmol) was added to the mixture. The reaction mixture was stirred at 5 ℃ for 16 hours. TLC showed another spot with low polarity. A trace amount of Compound 9-2 'was remained'. The reaction was quenched with water (10mL) and the resulting solution was quenched with DCM: MeOH (10: 1) extraction (15mL 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of PE: EtOAc/1: 1 to 1: 4 the residue was purified on a silica gel column as an eluent to give compound 9-3(322mg, yield: 72%) as colorless oil.

¹H NMR(400MHz，CDCl₃)δ1.45(9H，s)，2.50(2H，t，J＝6.8Hz)，3.39(2H，t，J＝5.2Hz)，3.62-3.72(28H，m)。

11) Preparation of Compound 9-3

In N₂The following reaction₃(896mg, 3.42mmol) was added to a solution of compound 9-3(1.49g, 3.11mmol) in THF (10 mL). The mixture was stirred at 10 ℃ for 1 hour, and then H was added to the mixture₂O (5 mL). Subsequently, the mixture was heated at 10 ℃ under N₂Stirred for 16 hours. Crude LC-MS showed a retention time of 0.742(MS calculation: 453.2; MS found: 454.2[ M + H)]⁺) The purity of the product was 3%. THF was removed in vacuo. The remaining solution was quenched with water (10mL), and the resulting solution was quenched with EtOAc: PE (3: 1) extraction (30mL × 2). The remaining aqueous layer was concentrated in vacuo to afford compound 9-3' (1.52g, crude) (containing water) as a colorless oil.

12) Preparation of Compound 9-4

Fmoc-Cl (897mg, 3.47mmol) and NaHCO₃(291mg, 3.47mmol) was added to compound 9-3' (1.52g) (crude, water) in THF (10mL) and H₂O (10 mL). After stirring the mixture at 10 ℃ for 16h, TLC showed another less polar spot. THF was removed in vacuo and the remaining solution was quenched with water (20 mL). The resulting solution was extracted with EtOAc (40mL × 5). The combined organic layers were washed with anhydrous Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of PE: EtOAc/1: 1 to 0:1 as an eluent, the residue was purified on a silica gel column to give compound 9-4' (1.25g, two-step yield: 59%) as colorless oil.

¹H NMR(400MHz，CDCl₃)δ1.46(9H，s)，2.51(2H，t，J＝6.8Hz)，3.39-3.43(2H，m)，3.62-3.73(28H，m)，4.22-4.26(1H，m)，4.42(2H，d，J＝7.2Hz)，5.45(1H，brs)，7.31-7.35(2H，m)，7.38-7.47(2H，m)，7.62(2H，d，J＝7.2Hz)，7.78(2H，d，J＝7.2Hz)。

13) Preparation of Compounds 9-5

TFA (5.95g, 52.2mmol) was added to a solution of compound 9-4' (1.25g, 1.85mmol) in DCM (12 mL). The mixture was stirred at 10 ℃ for 16 hours. Crude LC-MS showed a retention time of 0.816(MS calculation: 619.3; MS found: 642.0[ M + Na;)]⁺) The purity of the product was 95%. The reaction mixture was concentrated in vacuo to give compound 9-5(1.17g, yield: 100%) as colorless oil.

¹H NMR(400MHz，DMSO-d₆) δ 2.43(2H, t, J ═ 6.4Hz), 3.13(2H, dd, J ═ 11.6Hz, 5.6Hz), 3.40(2H, t, J ═ 6.0Hz), 3.48-3.49(24H, m), 3.59(2H, t, J ═ 6.4Hz), 4.19-4.23(1H, m), 4.29(2H, d, J ═ 6.4Hz), 7.29-7.39(3H, m), 7.42(2H, t, J ═ 7.6Hz), 7.69(2H, d, J ═ 7.2Hz), 7.89(2H, d, J ═ 7.2 Hz). No active protons were observed.

Preparation example 6: synthesis of PD010

1) Preparation of Compound 10

A mixture of compound 9(120mg, 0.0704mmol), (2, 5-dioxopyrrolidin-1-yl) 6-oxohexanoate (24mg, 0.11mmol) and DIEA (18mg, 0.14mmol) in DCM (2mL) was stirred at 10 ℃ for 16 h. TLC showed one main spot with lower polarity. The reaction mixture was purified by preparative TLC (20: 1 EtOAc: MeOH as eluent) to give compound 10(72mg, yield: 56%) as a yellow solid.

¹H NMR(400MHz，CDCl₃)δ1.57-1.76(4H，m)，2.01-2.10(12H，m)，2.11-2.30(14H，m)，2.41-2.50(2H，m)，2.77-2.88(2H，m)，3.36-3.76(28H，m)，3.78-3.86(2H，m)，3.90-4.07(4H，m)，4.12-4.39(8H，m)，4.45-4.63(4H，m)，5.23(2H，d，J＝10.0Hz)，5.35-5.45(2H，m)，5.56(2H，s)，5.74(2H，d，J＝9.2Hz)，6.42(1H，brs)，6.93-7.07(2H，m)，7.40-7.50(3H，m)，7.52-7.63(2H，m)，7.69-7.78(3H，m)，7.85(1H，d，J＝15.6Hz)，8.02(1H，d，J＝14.8Hz)，8.13-8.21(3H，m)，8.43(2H，d，J＝7.2Hz)，9.10(1H，brs)，9.75(1H，s)。

The synthesis of (2, 5-dioxopyrrolidin-1-yl) 6-oxohexanoate was the same as in the preparation example of PD 006. The synthesis of compound 9 was the same as in the preparation example of PD 009.

2) Preparation of PD010

The reaction mixture of Compound 10(72mg, 0.040mmol) and K₂CO₃A mixture of (11mg, 0.079mmol) in DCM (1mL) and MeOH (2mL) was stirred at 10 ℃ for 1 h. Crude LC-MS was shown to be at retention time 0.831(MS calculation: 1478.5; MS found: 1481.1[ M + 3H)]⁺) The purity of the product was 91%. The reaction mixture was quenched with AcOH (12 mg). The resulting mixture was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure. The remaining mixture was lyophilized to give PD010(24mg, yield: 41%) as a yellow solid. The batch (24 mg)) Mixed with batch es8455-193-p1(2 mg). Then, a total of 26mg of PD010 was obtained.

¹H NMR (400MHz, DMSO). delta.1.30-1.54 (4H, m), 1.95-2.10(2H, m), 2.63-2.78(2H, m, overlapping DMSO signals), 3.08-3.24(2H, m, overlapping water signals), 3.25-4.11(46H, m), 4.26-4.42(2H, m), 4.45-4.77(8H, m), 4.81-5.10(4H, m), 5.29-5.54(2H, brs), 7.18-7.33(2H, m), 7.36-7.49(2H, m), 7.51-7.62(2H, m), 7.64-7.96(7H, m), 8.04-8.14(1H, m), 8.26-8.47(4H, m), 9.64(1H, m), 1H (10H, m).

Preparation example 7: synthesis of PD011

1) Preparation of Compound 11-2

Mixing compound 11-1(1.30g, 1.94mmol), compound 3(672mg, 1.94mmol) and K₂CO₃A mixture of (322mg, 2.33mmol) in DMF (6mL) was stirred at 60 ℃ for 16 h. TLC showed that the starting material of Compound 11-1 was still present. A new spot was observed. The mixture was quenched with water (50mL) and extracted with DCM (20mL × 3). The combined organic layers were washed with water (50mL) and Na₂SO₄Dried and concentrated to dryness. The residue was purified with EtOAc: PE/1: 1 with EtOAc to give crude compound 11-2(710mg, containing DMF) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ1.55(18H，d，J＝4.0Hz)，3.56-3.73(22H，m)，3.75-3.80(2H，m)，3.86-3.90(2H，m)，3.92-3.96(2H，m)，4.21-4.26(2H，m)，4.37-4.42(2H，m)，6.30(1H，d，J＝16.0Hz)，6.52(1H，d，J＝16.0Hz)，7.04(1H，s)，7.12(1H，d，J＝8.4Hz)，7.48-7.57(2H，m)，7.75-7.79(2H，m)，7.84-7.91(3H，m)。

The synthesis of compound 11-1 was the same as in the example of PD 001.

2) Preparation of Compound 11-3

Reacting NH₃And a solution of compound 11-2(738mg, crude) in MeOH (7M, 10mL) was stirred at 15 deg.C for 16 h. A white precipitate formed and was then filtered. The filtrate was concentrated and dried to give compound 11-3(582mg) as a colorless oil.

¹H NMR(400MHz，DMSO)δ1.48(18H，d，J＝4.4Hz)，3.46-3.54(22H，m)，3.55-3.66(6H，m)，3.79-3.85(2H，m)，4.24-4.29(2H，m)，4.37-4.42(2H，m)，5.98(2H，brs)，6.60-6.71(2H，m)，7.28(1H，d，J＝7.6Hz)，7.45(1H，s)，7.53(1H，d，J＝16.0Hz)，7.73(1H，t，J＝8.0Hz)，7.81(1H，d，J＝18.4Hz)。

3) Preparation of Compound 11-4

Compound 11-3(580mg, 0.812mmol), Fmoc-Cl (231mg, 0.894mmol) and NaHCO₃(75mg, 0.89mmol) in H₂A mixture of O (5mL) and THF (5mL) was stirred at 15 ℃ for 2 h. Crude LC-MS showed a retention time of 1.003(MS calculation: 935.5; MS found: 958.5[ M + Na;)]⁺) The purity of the product was 43%. The mixture was extracted with EtOAc (6mL 4). The combined organic layers were washed with anhydrous Na₂SO₄Dried, concentrated, and further dried. The use of PE: EtOAc/1: 1, EtOAc, then using EtOAc: MeOH/10: 1 as an eluent, the residue was purified on a silica gel column to give compound 11-4(558mg, yield: 73%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ1.55(18H，d，J＝4.8Hz)，3.61-3.79(26H，m)，3.90-3.96(2H，m)，4.01-4.06(2H，m)，4.19-4.30(3H，m)，4.50(2H，d，J＝6.8Hz)，6.38(1H，d，J＝16.0Hz)，6.52(1H，d，J＝16.0Hz)，7.03(1H，s)，7.12(1H，d，J＝7.6Hz)，7.31-7.36(2H，m)，7.42(1H，t，J＝7.6Hz)，7.48-7.57(2H，m)，7.62(1H，d，J＝7.6Hz)，7.78(1H，d，J＝7.2Hz)，7.88(1H，d，J＝16.0Hz)，8.27(1H，brs)。

4) Preparation of Compound 11-5

A solution of compound 11-4(558mg, 0.596mmol) and TFA (3.08g, 27.0mmol, 2mL) in DCM (5mL) was stirred at 15 deg.C for 16 h. Crude LC-MS showed a retention time of 0.827(MS calculation: 823.3; MS found: 846.3[ M + Na;)]⁺) The purity of the product was 85%. The solvent was removed under reduced pressure. The residue was purified by PE: EtOAc/1: 1(10mL) to give compound 11-5(433mg, yield: 88%) as a white solid.

¹H NMR (400MHz, DMSO) δ 3.41-3.87(30H, m, overlapping water signal), 4.21-4.32(3H, m), 4.40(2H, d, J ═ 6.8Hz), 6.59-6.72(2H, m), 7.26-7.37(3H, m), 7.38-7.47(3H, m), 7.58(1H, d, J ═ 16.0Hz), 7.65-7.75(3H, m), 7.80(1H, d, J ═ 16.4Hz), 7.89(2H, d, J ═ 7.6Hz), 10.46(1H, brs), 12.43(2H, brs).

5) Preparation of Compound 11-6

EDCI (168mg, 0.874mmol) was added to a mixture of compound 11-5(240mg, 0.291mmol) and compound 6-9(493mg, crude) in DMF (3mL) at 15 ℃. Subsequently, the mixture was stirred at 15 ℃ for 16 hours. TLC showed one major yellow spot. Most of the DMF was removed under reduced pressure. EtOAc was used, then EtOAc: MeOH/25: 1 the residue was purified on a silica gel column as eluent to give the product (541mg) as a yellow solid. About 165mg of the crude product was used for the next step (Fmoc removal reaction), but no positive results were obtained. Then, the residue is removedThe material (375mg) was dissolved in EtOAc (40mL) and washed with water (40mL × 3). Separating the organic layer with Na₂SO₄Dried and concentrated to dryness to give pure compound 11-6(340 mg).

¹H NMR(400MHz，CDCl₃) δ 2.00-2.11(12H, m, overlap EtOAc signal), 2.17(6H, d, J ═ 11.2Hz), 2.24(6H, s), 3.45-3.84(28H, m), 3.97-4.40(19H, m, overlap EtOAc signal), 4.42-4.63(5H, m), 5.18-5.26(2H, m), 5.36-5.44(2H, m), 5.52-5.60(2H, m), 5.70-5.80(2H, m), 6.97(1H, d, J ═ 15.2Hz), 7.17(1H, s), 7.25-7.37(4H, m, overlap CDCl), 2.17(6H, d, J ═ 11H, m, overlap EtOAc signal), 2.17(6H, d, J ═ 11H, m, 5H, overlap CDCl)₃Signals), 7.38-7.49(4H, m), 7.53-7.64(5H, m), 7.69-7.79(4H, m), 7.83(1H, d, J ═ 15.2Hz), 8.00(1H, d, J ═ 15.6Hz), 8.14-8.20(2H, m), 8.34(1H, brs), 8.46(1H, d, J ═ 11.6 Hz).

6) Preparation of Compounds 11-7

A solution of compound 11-6(280mg, 0.146mmol) and piperidine (172mg, 2.03mmol, 0.2mL) in THF (2mL) was stirred at 15 deg.C for 1 h. TLC showed a new spot was observed. The starting material was completely consumed. The solvent was removed under reduced pressure. The reaction was performed using EtOAc: MeOH/10: 1 as an eluent, the residue was purified by preparative TLC to give compound 11-7(116mg, yield: 47%) as a yellow solid.

¹H NMR(400MHz，CDCl₃) δ 2.06(12H, d, J ═ 4.4Hz), 2.17(6H, d, J ═ 10.4Hz), 2.24(6H, s), 3.47-3.85(30H, m), 3.97-4.63(18H, m), 5.17-5.27(2H, m), 5.35-5.44(2H, m), 5.52-5.62(2H, m), 5.70-5.80(2H, m), 6.98(1H, d, J ═ 15.2Hz), 7.18(1H, s), 7.26-7.37(2H, m, overlapping CDCl)₃Signal), 7.40-7.49(2H, m), 7.52-7.65(3H, m), 7.73(2H, d, J ═ 7.6Hz), 7.84(1H, d, J ═ 15.2Hz), 8.00(1H, d, J ═ 15.2Hz), 8.13-8.21(2H, m), 8.45(2H, d, J ═ 11.6 Hz).

7) Preparation of PD011

Compounds 11-7(116mg, 0.068mmol) and K₂CO₃A mixture of (19mg, 0.146mmol) in MeOH (1mL) and THF (1mL) was stirred at 15 deg.C for 1 h. Crude LC-MS was shown to be at retention time 0.772(MS calculation: 1355.5; MS found: 1357.2[ M + 2]]⁺) The purity of the product was 86%. The mixture was quenched with AcOH (50 mg). The mixture was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure, and the remaining aqueous solution was lyophilized to give PD011(45mg, yield: 48%) as a yellow solid.

¹H NMR (400MHz, DMSO) δ 3.18-4.10(48H, m, overlapping water signals), 4.27-4.72(12H, m), 4.87-5.02(2H, m), 5.39(2H, brs), 7.30-7.64(8H, m), 7.74(1H, d, J ═ 15.2Hz), 7.81-7.97(4H, m), 8.28-8.49(4H, m).

8) Preparation of Compound 2

TosCl (2.57g, 13.5mmol) was added to octaethylene glycol (5.00g, 13.5mmol) and Et at 15 deg.C₃N (1.37g, 13.5mmol) in DCM (50 mL). Subsequently, the mixture was stirred at 15 ℃ for 16 hours. TLC showed the starting material was consumed and two new spots formed. The mixture was quenched with water (50mL) and the organic layer was separated. The remaining aqueous phase was extracted with DCM (30mL × 2). Mixing the organic layer with Na₂SO₄Dried, concentrated, and further dried. EtOAc was used, then EtOAc: MeOH/9: 1 as eluent, the residue was purified on a CombiFlash apparatus to give compound 2(3.59g, yield: 51%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ3.59-3.76(30H，m)，4.18(2H，t，J＝4.8Hz)，7.36(2H，d，J＝8.0Hz)，7.82(2H，d，J＝8.4Hz)。

9) Preparation of Compound 3

At 0 ℃ in N₂2-Hydroxyisoindoline-1, 3-dione (1.11g, 6.82mmol) and PPh in the Presence of₃(2.33g, 8.87mmol) was added to a solution of Compound 2(3.58g, 6.82mmol) in THF (30 mL). Subsequently, the mixture was stirred at 0 ℃ for 30 minutes. Then, DIAD (1.66g,8.19mmol) was added to the mixture at 0 ℃. The resulting mixture was heated at 15 ℃ under N₂Stirring in the presence for 16 hours. TLC showed a new spot. Then, the solvent was removed under reduced pressure. The use of PE: EtOAc/1: 1 and EtOAc as eluent the residue was purified on a CombiFlash apparatus to give compound 3(3.91g, yield: 86%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ2.47(3H，s)，3.56-3.73(26H，m)，3.86-3.91(2H，m)，4.16-4.20(2H，m)，4.37-4.42(2H，m)，7.36(2H，d，J＝7.6Hz)，7.75-7.88(6H，m)。

Preparation 8: synthesis of PD012

1) Preparation of Compound 12-1

DIEA (13mg, 0.10mmol) was added to a solution of compounds 6-14(107mg, 0.0638mmol) and compound 12-1a (25mg, crude oil) in DMF (2 mL). The mixture was stirred at 15 ℃ for 16 hours. TLC showed another spot with a lower polarity. DMF was removed in vacuo. The residue was quenched with water (10 mL). Subjecting the obtained solution toDCM extraction (15mL 4). The combined organic layers were washed with water (50mL) and the organic layer was washed with anhydrous Na₂SO₄Dried, filtered and then concentrated in vacuo. The reaction was performed using EtOAc: MeOH/10: 1 as eluent the residue was purified by preparative TLC to give compound 12-1(81mg, crude oil) as a yellow oil.

¹H NMR(400MHz，CDCl₃) δ 1.94-2.08(12H, m), 2.09-2.24(12H, m), 3.50-3.84(32H, m), 3.98-4.37(13H, m), 4.43-4.62(4H, m), 5.20(2H, d, J ═ 10.0Hz), 5.38(2H, d, J ═ 7.2Hz), 5.50-5.57(2H, m), 5.72(2H, t, J ═ 8.8Hz), 6.96(1H, d, J ═ 15.2Hz), 7.15(1H, s), 7.27-7.35(2H, m, overlapping CDCl), 7.96 (1H, m, or m, or c, or m, or m, c, or m, or c, or a metal salt, or a metal salt, or a metal₃Signals), 7.39-7.47(2H, m), 7.51-7.62(4H, m), 7.71(2H, d, J ═ 8.0Hz), 7.81(1H, d, J ═ 15.2Hz), 7.94-8.09(2H, m), 8.11-8.20(2H, m), 8.36-8.48(3H, m), 10.07(1H, s).

The synthesis of compounds 6-14 was the same as in the example for PD 006.

2) Preparation of PD012

Will K₂CO₃(12mg, 0.090mmol) was added to a solution of Compound 12-1(81mg, crude) in DCM (0.2mL) and MeOH (0.4 mL). The mixture was stirred at 15 ℃ for 1 hour. Crude LC-MS showed a retention time of 0.787(MS calculation: 1474.2; MS found: 1496.2[ M + Na;)]⁺) The purity of the product was 99%. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (0.225% FA). Most of the MeCN was removed under reduced pressure, and the remaining mixture was lyophilized to give PD012(31mg, two-step yield: 33%) as a yellow solid.

¹H NMR(400MHz，DMSO-d₆)δ3.44-3.68(28H，m)，3.71-4.16(15H，m)，4.26-4.82(15H，m)，4.88-5.07(4H，m)，5.39(2H，brs)，7.25-7.64(8H，m)，7.57(1H，d，J＝5.2Hz)，7.81-8.02(4H，m)，8.07(1H，d，J＝7.6Hz)，8.17-8.36(4H，m)，8.37-8.46(2H，m)，8.81(1H，brs)，10.02(1H，s)。

3) Preparation of Compound 1

2, 6-Pyridinedicarboxylic acid (2.00g, 12.0mmol) was added to SOCl at 0 deg.C₂(8.83g, 74.2mmol) in MeOH (25 mL). The mixture was stirred at 70 ℃ for 3 hours. Crude LC-MS showed a retention time of 0.752(MS calculation: 195.1; MS found: 195.9[ M + H ]]⁺) The purity of the product was 99%. The reaction mixture was washed with NaHCO₃Saturated aqueous solution (30mL) was quenched and the resulting solution was extracted with EtOAc (30mL x 6). The combined organic layers were washed with Na₂SO₄Drying, filtration and then concentration in vacuo gave compound 1(2.33g, yield: 99%) as a white solid.

¹H NMR(400MHz，CDCl₃)δ4.03(6H，s)，8.03(1H，t，J＝8.0Hz)，8.32(2H，d，J＝7.2Hz)。

4) Preparation of Compound 2

NaBH is reacted at 0 DEG C₄(1.71g, 45.1mmol) was added portionwise to a stirred solution of compound 1(2.33g, 11.9mmol) in MeOH (35mL) and the mixture was stirred at 0 deg.C for 1 h. Crude LC-MS showed a retention time of 0.297(MS calculation: 167.1; MS found: 167.7[ M + H)]⁺) The purity of the product was 97%. The reaction mixture was washed with NaHCO₃Saturated aqueous solution (200mL) was quenched and the resulting solution was extracted with DCM (100mL × 6). The combined organic layers were washed with Na₂SO₄Dried, filtered, and concentrated in vacuo. The use of PE: EtOAc/1: 1, and then the residue was purified on a silica gel column using EtOAc as an eluent to give compound 2(832mg, yield: 42%) as a white solid.

¹H NMR(400MHz，CDCl₃)δ3.45(1H，brs)，4.02(3H，s)，4.88(2H，d，J＝4.8Hz)，7.55(1H，d，J＝7.6Hz)，7.87(1H，t，J＝7.6Hz)，8.06(1H，d，J＝7.2Hz)。

5) Preparation of Compound 3

MnO at 15 ℃₂(4.33g, 50.0mmol) was added to a solution of Compound 2(832mg, 4.98mmol) in 1, 2-dichloroethane (25mL), and the mixture was stirred at 90 ℃ for 2 hours. The crude LC-MS showed a retention time of 0.386(MS calculation: 165.1; MS found: 165.7[ M + H;)]⁺) The purity of the product was 98%. The mixture was filtered through celite and the filtrate was evaporated to give compound 3(606mg, yield: 73%) as a white solid.

¹H NMR(400MHz，CDCl₃)δ4.10(3H，s)，8.08(1H，t，J＝7.6Hz)，8.18(1H，d，J＝6.8Hz)，8.38(1H，d，J＝7.6Hz)，10.22(1H，s)。

6) Preparation of Compound 4

LiOH (132mg, 5.50mmol) was added to compound 3(606mg, 3.67mmol) in THF (6mL) and H at 0 deg.C₂O (6mL) and the mixture was stirred at 0 ℃ for 1 hour. TLC showed another new spot with greater polarity and showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo and the remaining solution was quenched with 1N aqueous HCl. The resulting solution was purified with EtOAc: THF 10: 1 extraction (20mL x 8) gave an impure product (466mg) as a white solid. Impure product was purified by TBME: PE ═ 1: 1(10mL) and filtered, and the filter cake was dried in vacuo to give compound 4(444mg, crude oil) as a white solid.

¹H NMR(400MHz，DMSO-d₆)δ8.09(1H，dd，J＝7.6Hz，1.2Hz)，8.20(1H，t，J＝7.6Hz)，8.29(1H，dd，J＝7.6Hz，1.2Hz)，10.03(1H, s). (Note: no active protons were observed).

7) Preparation of Compound 12-1a

At 15 ℃ in N₂EDCI (136mg, 0.708mmol) was added to a solution of compound 4(100mg, crude) and N-hydroxysuccinimide (80mg, 0.695mmol) in DMF (1.5mL) in the presence of stirring at 15 ℃ for 4 h. Crude LC-MS showed a retention time of 1.289(MS calculation: 248.1; MS found: 248.9[ M + H)]⁺) The purity of the product was 96%. DMF was removed in vacuo, and PE: EtOAc/2: 3 the residue was purified on a silica gel column as eluent to give compound 12-1a (51mg, impure) as a white solid.

Preparation example 9: synthesis of PD013

The linker was prepared according to the method described in U.S. patent No.9,636,421 to Synaffix. The prepared linker was used to prepare a linker-drug complex in the same manner as in preparation example 8.

Example 1 overexpression of beta-galactosidase in tumor cell lines compared to Normal cells

The expression of beta-galactosidase in tumor cell lines was identified by western blotting. 15 cell lines (1X 10 per cell line) were used⁷) Washed with PBS, and then centrifuged to obtain a cell pellet. By usingMammalian protein extraction reagents (Thermo, 78501) lyse cells and extract proteins contained in the cells. Mixing 200. mu.l ofMammalian protein extraction reagent added toIn the cell pellet, the solution was pipetted and allowed to react for 10 minutes. The reaction solution was centrifuged and the supernatant was collected. The concentration of protein in the supernatant was measured using BCA protein assay kit (Pierce, 23225). 10ug of protein extract from each cell line was subjected to SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes using the iBlot transfer system (Invitrogen, IB 401002). The membrane was blocked with 4% bsa (pbs) for 2 hours at room temperature, treated with anti- β -galactosidase antibody (1/10000, abcam, ab128993), and allowed to react for 15 hours at 4 ℃. The reaction product was washed 3 times with 1 XPBST (1 XPBS + 0.05% Tween20), treated with anti-rabbit IgG HRP (1/3000, Pierce, 65-6120) and allowed to react at room temperature for 1 hour. The resulting product was washed 3 times with 1 XPBST and treated with ECL solution (GE Healthcare, RPN2232) and tested for protein. Membranes that have been used to identify beta-galactosidase expression were peeled off to identify beta-actin expression. The peeled membrane was blocked with 4% BSA (PBS) at room temperature for 2 hours, and then reacted with an anti-actin antibody (1/1000, Santa Cruz, SC-47778) at 4 ℃ for 15 hours. The membranes were washed 3 times with 1 XPBST (1 XPBS + 0.05% Tween20), treated with anti-mouse IgG HRP (1/10000, Pierce, 31432) and allowed to react for 1 hour at room temperature. The reaction product was washed 3 times with 1 XPBST and treated with ECL solution, and the protein was detected.

The results of western blotting showed that β -galactosidase was expressed in 15 tumor cell lines. In addition, analysis of TCGA (The Cancer Genome Atlas) data showed higher levels of mRNA expression of β -galactosidase in tumor tissues compared to normal tissues (fig. 2). Fig. 2 shows the mRNA expression ratio of β -galactosidase gene GLB1 in tumor tissue and normal tissue, and indicates that β -galactosidase is highly expressed in tumor tissue compared to normal tissue. Especially in the case of uterine cancer, GLB1 is expressed in tumor tissues 2-fold higher than in normal tissues, and also in breast cancer tissues over-express GLB1 by more than 68%. This indicates that beta-galactosidase highly expressed in tumor cells can be used as a tumor-selective activating enzyme.

Example 2 evaluation of the efficacy of linker-drug complexes

The prepared linker-drug complex containing β -galactosidase was evaluated for cytotoxicity per se. Specifically, each linker-drug complex dissolved in DMSO was serially diluted from 200nM 3 times to 30pM in a medium for cell culture to prepare a sample, and various cancer cell lines were treated with the sample, and finally the cells were exposed to 100nM to 15pM of the linker-drug complex. Cells were cultured for 6 days, cell viability was observed and cytotoxicity was evaluated using CCK-8 cell counting kit. The evaluation results are shown in table 2, and a representative reaction curve is shown in fig. 3. As shown in table 2 and fig. 3, PD003 was not cytotoxic to most cells, and therefore IC50 could not be determined. On the other hand, PD001 was cytotoxic to various cells, and IC50 is shown in table 2.

These experimental results indicate that sufficient β -galactosidase to degrade β -galactose is present in cancer cells, and thus PD001 can be activated by β -galactosidase in various cancer cells and can exhibit cytotoxicity. The fact that PD003 is inactive indirectly indicates that the β -glucose moiety cannot be used as a substrate for β -glucosidase. In addition, the examples given below show that PD001 prepared in ADC form is more cytotoxic than the single agent, supporting the idea that ADC form is more effective for drug delivery and activation.

[ Table 2]

-: non-response

Example 3 preparation of antibody-drug conjugates (ADC) for binding of antibodies to drugs

To prepare an antibody-drug conjugated ADC, lysine (K) at position 149 (according to Kabat numbering, this also applies hereinafter) of the existing antibody light chain is mutated to cysteine (C) and reacted with a reducing agent, such as Dithiothreitol (DTT), to generate a thiol group (K153C T) on antibody light chain K153C, and the antibody is coupled to a drug through a disulfide bond generated between the thiol group and the drug. Specifically, an antibody was prepared at a concentration of 5mg/ml or more by ultrafiltration/diafiltration (UF/DF), and 1M Tris (hydroxymethyl) aminomethane (Tris-HCl) and 500mM ethylenediaminetetraacetic acid (EDTA) at pH 8.8 were added thereto to adjust the final antibody concentration to 5mg/ml, and 75mM Tris-HCl and 2mM EDTA were obtained. Dithiothreitol (DTT) was added at 100mM to the prepared antibody so that the molar ratio of antibody to DTT was adjusted to 1: 20 and allowed to react at 25 ℃ for 16.5 hours to remove the free cysteine at cysteine 149 of the antibody light chain which is disulfide-linked. This process is called "decapping" and then cation exchange Chromatography (CEX) is performed as a purification method to isolate the uncapped antibody. The reaction product was eluted on ｃA HiTrap SPHP column (GE Healthcare) equilibrated with SPHP-A buffer [10mM succinate, pH 5.0] and SPHP-B buffer [50mM Tris (hydroxymethyl) aminomethane (Tris-HCl), pH7.5, 0.5M sodium chloride ]. Preparing an antibody to be oxidized in 75mM Tris-HCl (pH7.5) by using 1M Tris (hydroxymethyl) aminomethane (Tris-HCl, pH7.5) to recombine the uncapped antibody; dehydroascorbic acid (DHAA), an oxidized vitamin C, was added at a ratio of 1: 20 antibody to DHAA was added and re-oxidized in the dark at 25 ℃ for 2 hours. Then, to isolate the reoxidized antibody, the resultant was eluted with SPHP-C buffer [10mM succinate, pH 5.0, 0.5M sodium chloride ] using cation exchange Chromatography (CEX) purification method. The purified antibody was concentrated to 5mg/ml or more by ultrafiltration/diafiltration (UF/DF). Then, in order to produce an antibody-drug conjugate, the antibody to be reacted was prepared in 100mM Tris (hydroxymethyl) aminomethane (Tris-HCl, final concentration: 5mg/ml, pH 8.0), and 1M Tris (hydroxymethyl) aminomethane (Tris-HCl, pH 8.0) was added so that the molar ratio of the antibody to the drug was 1: 10 and then allowed to react at 25 ℃ for 16.5 hours. Then, for isolation of the antibody-drug conjugate, elution was performed using cation exchange Chromatography (CEX) purification method with SPHP-C buffer [10mM succinate, pH 5.0, 0.5M sodium chloride ]. Then, Hydrophobic Interaction Chromatography (HIC) was used to isolate DAR2 antibody-drug conjugates. For this, the HiTrap butyl HP column (GE Healthcare) was equilibrated with HIC-A buffer [50mM potassium phosphate, pH 7.0, 1.0M ammonium sulfate ] and eluted using HIC-B buffer [50mM potassium phosphate, pH 7.0, 30% isopropanol (2-propanol) ]. The antibody was then exchanged with HA buffer [20mM histidine, pH 5.5, 240mM sucrose ] to remove isopropanol (2-propanol) by ultrafiltration/diafiltration (UF/DF) to prepare the final antibody-drug conjugate.

Table 3 shows the names and specific configurations of ADCs according to the present invention, ADCs using D-glucose β -pyranose or glucuronide as triggers, and ADCs attached to different drugs. The VH and VL sequences of each antibody are shown in table 1.

[ Table 3]

Example 4 characterization of ADC

4-1. detection of purity of prepared ADC

The purity of the prepared ADCs was measured by size exclusion-high performance liquid chromatography (SE-HPLC). The elution position and area under the curve (AUC) of each sample were compared on an Agilent 1200 series HPLC instrument using a Tosoh TSKgel G3000SWxl column (Tosoh bioscience) to determine the purity of D20106 and D20109. It was confirmed that the purity of the major peak of ADC was 97% or more and the total process yield (based on antibody) was 20% or more. These results are shown in fig. 4 and 5, respectively.

4-2 detection of drug-antibody ratio of prepared ADC

The drug-antibody ratio (DAR) of the ADC was determined by a liquid chromatography-mass spectrometry (LC/MS) method. 1 unit of PNGaseF (NEB) was added per 100. mu.g of 1mg/ml ADC (in PBS) antibody, followed by incubation at 37 ℃ for 15 hours and removal of the N-glycans. An Acquity UPLC BEH200 SEC 1.7 μm (4.6 x 150mm) column was mounted on an LC/MS apparatus (including a Waters UPLC I-class device and Waters Synapt G2-S) and equilibrated with a mobile phase of 30% (v/v) acetonitrile, 0.1% (v/v) formic acid and 0.05% trifluoroacetic acid (TFA). A5. mu.g sample from which N-glycans were removed was loaded thereon and subjected to LC/MS. The DAR was determined by calculating a weighted average of the relative content of each chemical from the molecular weight distribution obtained as a result of LC/MS. These results are shown in fig. 6 and fig. 7, respectively. As shown in fig. 6 and 7, the DAR for D20106 and D20109 is about 2. These results indicate that PD006 and PD009 linked to the antibody with the expected DAR values.

4-3 detection of in vitro target-specific cytotoxicity of prepared ADCs

(1) In vitro cytotoxicity in multiple myeloma cell lines

In vitro cytotoxicity of ADCs containing anti-BCMA antibodies, D20103(B58PD001), D20106(B58PD006) and D20109(B58PD009), was tested using BCMA expressing multiple myeloma cell line MM NCI-H929. Specifically, 50. mu.l of H929 cell line (ATCC, CRL-9068) cultured in a medium containing RPMI (ATCC), 10% FBS (Gibco), 0.05 mM. beta. -mercaptoethanol, and antibiotic-antifungal agent (Gibco) was added^TM) The cells were seeded at a density of 20,000 cells/well in 96-well plates and 50 μ l of ADC diluted in the same medium was seeded per well. The concentration of ADC was changed from 200nM to 5pM by serial dilution. Then, the cells were incubated at 37 ℃ and 5% CO₂The incubation was continued for about 6 days. After incubation, 10. mu.l of WST-8(Dojindo) was added to each well, followed by further incubation. The absorbance was measured at a wavelength of 450nm using a SpectraMax microplate reader. Response curves between absorbance and ADC concentration were subjected to 4PL curve fitting to calculate IC₅₀Value (nM), IC₅₀Values refer to the concentration of 50% apoptosis. The results are shown in fig. 8. D20103, D20106 and D20109 all showed excellent potency, especially D20109 including PD009 had the best efficacy. The amount of PD009 obtained after coupling and refiltering was higher than in the case of PD006 and more ADCs with high DAR could be produced under the same conditions. In this regard, table 4 below shows the results of purification of the cation exchange resin after the reaction during the coupling. This process characteristic of PD009 is due to its relatively high solubility, so PD009 is considered more process-friendly.

[ Table 4]

(2) In vitro cytotoxicity of mantle cell lymphoma cell lines

D20204(C2E3-PD001), an ADC containing anti-ROR 1 antibody, was tested for cytotoxicity in vitro using the ROR1 expressing human mantle cell lymphoma cell lines Mino and Jeko-1. The control groups used here were: (i) C2E3-PD003, which comprises the same anti-ROR 1 antibody C2E3, but comprises PD003 as the drug, no D-glucose β -pyranose, and D-galactose β -pyranose as the trigger, and (ii) C2E3-CAAX-MMAE, which comprises the same anti-ROR 1 antibody C2E3, and comprises the conventional drug MMAE. Specifically, Jeko-1 cell line (ATCC, CRL-3006) cultured in a medium containing RPMI (ATCC), 20% FBS (Jeko-1) (Gibco) or 15% FBS (mini) and an antibiotic-antifungal agent (Gibco)^TM) And Mino cell line (ATCC, CRL-3000)^TM) 50 μ l each, was seeded at 20,000 cells/well in 96-well plates and then tested for cytotoxicity. The subsequent experimental procedure was the same as for anti-BCMA ADC.

The results are shown in table 5 and fig. 9. In the Mino cell line, D20204 was more cytotoxic than the control C2E3-CAAX-MMAE, and C2E3-PD003, which contained glucose as a trigger, was not cytotoxic. In Jeko-1, D20204 was slightly less cytotoxic than C2E3-CAAX-MMAE, while PD003 was not cytotoxic at all. This indicates that the ADC of the present invention containing galactose as a trigger exhibits significantly superior target-specific cytotoxicity compared to ADC containing glucose as a trigger.

[ Table 5]

(3) In vitro cytotoxicity in gastric and breast cancer cell lines

In vitro cytotoxicity of D20001(T-PD001), an ADC containing an anti-Her 2 antibody, was tested using Her2 expressing gastric cancer cell line (NCI-N87) and breast cancer cell line (HCC 1954). The control group used here was D20103(B58-PD001) comprising an anti-BCMA antibody linked to the same drug PD 001. Specifically, in the case of a composition containing RPMI (ATCC), 10% FBS (Gibco) and antibiotic-antimycoticNCI-N87 cell line (ATCC, CRL-5822) cultured in a medium of fungal drug (Gibco)^TM) And HCC1954 cell line (ATCC, CRL-2338)^TM) 50 μ l each, seeded at 10,000 cells/well or 5,000 cells/well in 96-well plates and then tested to determine target-specific cytotoxicity. The subsequent experimental procedure was the same as for anti-BCMA ADC.

The results are shown in table 6 and fig. 10. The NCI-N87 and HCC1954 cell lines expressing Her2 showed cytotoxicity of D20001, but did not show cytotoxicity of D20103 (an anti-BCMA ADC). This indicates that the ADCs according to the invention have target specific cytotoxicity, which depends on the type of antibody contained in the ADC.

[ Table 6]

IC50(nM)	D20001	D20103 (non-combination body)	Unconjugated body/conjugated body ratio
				NCl-N87	0.0365	16.7	457
HCC1954	0.263	Non-response	n.d.

(4) In vitro cytotoxicity of ovarian cancer cell lines

The in vitro cytotoxicity of D20002(10H1-PD001), an ADC containing anti-NaPi 2b antibody, was tested using an ovarian cancer cell line expressing NaPi2b (OVCAR-3). The control group used here was D20103(B58-PD001) in which an anti-BCMA antibody was linked to the same drug PD 001. Specifically, 50. mu.l of OVCAR-3 cell line (ATCC, HTB-161) cultured in a medium containing RPMI (ATCC), 20% FBS (Gibco) and antibiotic-antifungal (Gibco)^TM) Each well of a 96-well plate was seeded with 5,000 cells/well, and then tested to determine cytotoxicity in vitro. The subsequent experimental procedure was the same as for anti-BCMA ADC.

The results are shown in FIG. 11. As can be seen in FIG. 11, the ADC containing D20002 exhibited an IC of 1.53nM₅₀. The ADC containing D20002 had a non-binder/binder ratio of 100 or more, and thus showed the best reactivity in solid cancer cell lines. On the other hand, the ADC containing D20103 had no cytotoxicity of D20103 (an anti-BCMA ADC). This indicates that the ADCs according to the invention have target specific cytotoxicity, which depends on the type of antibody contained in the ADC.

(5) Conclusion

The in vitro cytotoxicity of ADCs of the invention prepared to include drugs containing D-galactose β -pyranose as a trigger was compared to that of various control ADCs. The results show that most ADCs according to the invention have an IC of 1nM or less in various cell lines₅₀And its potential as a potent drug was determined by in vitro experiments (table 7).

[ Table 7]

Sigmoidal response curves with incomplete response

ND: has been tested but not determined

NA: is not available

4-4. detection of in vivo target specific cytotoxicity

To ensureGiven the in vivo targeting specific cytotoxicity of ADC, tumor inhibition efficacy in xenografted mouse models of different cancer cell lines was tested. Specifically, 0.5 × 10⁷Or 1X 10⁷Individual cancer cells were mixed with Matrigel (Matrigel) and injected subcutaneously into 6-8 week old female Fox Chase SCID mice (CB 17/Icr-Prkdc)^scid/IcrIcoCrl) or in the flank of nude mice. When the average tumor size reaches 150mm³To 200mm³At the time, mice were sequentially classified into each experimental group. Drugs were administered through the tail vein of each mouse in the ADC group and the control group, and PBS (vehicle) was used as a negative control. The size of the tumor was determined by measuring the long and short axes of the tumor using a vernier caliper, and the tumor volume was calculated using the following equation:

tumor volume (mm)³) (0.5) × (major axis) × (minor axis)²

(1) Cytotoxicity in human mantle cell lymphoma cell xenograft mouse model

The cytotoxicity of D20204(C2E3-PD001) was tested in vivo in a xenografted mouse model using the ROR 1-expressing human mantle cell lymphoma cell line Jeko-1. C2E3-mc-MMAF including C2E3 as an antibody and MMAF as a drug was used as a control group and was administered 3 times per week at a single ADC dose of 4 mg/kg.

The results are shown in fig. 12. D20204 exhibits an IC of greater than 10nM in an in vitro assay₅₀(fig. 9), but showed significantly superior effect in vivo experiments compared to ADC coupled with MMAF.

(2) Cytotoxicity in human multiple myeloma cell xenograft mouse models

In vivo cytotoxicity of D20106(B58-PD006) and D20109(B58-PD009) was detected in a xenografted mouse model using BCMA-expressing multiple myeloma cell line NCI-H929. Specifically, 6-7 week old Fox Chase SCID mice were used, and 1 × 10 mice were used⁷Individual NCl-H929 cells were mixed with Matrigel (Matrigel) and implanted subcutaneously in the flank of each mouse. When the average tumor size reaches 180mm³To 200mm³The medication is administered thereto. The two substances D20106 and D20109 were administered in three doses (2.5mpk, 1.25mpk, 0.6)25mpk) and tumor size was measured twice weekly and observed for 3 weeks.

As shown in fig. 13, both D20106 and D20109 showed excellent cytotoxicity in multiple myeloma cell xenograft models. Specifically, D20106 and D20109 were effective in inhibiting the growth of NCI-H929 tumors, and tumors disappeared in the other treatment groups except the group to which D20106 was administered at a low dose (0.625 mpk).

(3) Cytotoxicity in mouse model of acute myelogenous leukemia cell xenograft

D20502(6E7-PD009) was tested for cytotoxicity in vivo in a xenografted mouse model using the Acute Myeloid Lymphoma (AML) cell lines EOL-1 and HL-60 expressing CLL-1. Specifically, 5X 10 mice were used, 6-7 weeks old Fox Chase SCID mice⁶The individual EOL-1 cells or HL-60 cells were mixed with Matrigel (Matrigel) and implanted subcutaneously in the flank of each mouse, and when the average tumor size was 180mm³To 200mm³The medication is administered thereto. D20502 was administered once in a single dose of 2mpk, and tumor size was measured twice weekly and observed for 3 weeks.

As can be seen from figure 14, at 2mpk dose, D20502 was effective in inhibiting tumor growth in both AML xenograft models, and all tumors disappeared 2 weeks after administration.

4-5 Single dose rodent toxicity test 1 for determining maximum tolerated dose

Single dose rodent toxicity assays were performed to assess toxicity of ADCs containing either PD001 or PD006 and to determine the maximum tolerated dose. Specifically, 7-8 week-old female SD rats were sequentially grouped so that the average body weight of each group was similar, as shown in table 8 below. ADCs were administered to their tail veins at doses ranging from 5mpk to 45mpk, and blood samples were collected on days 3, 18, and 35 for hematology and serum biochemical analysis. All other animals were sacrificed on day 34 and major organs were histopathologically analyzed. Group 1, the control group, was administered 20mM histidine and 7% trehalose at pH 6.0 as a vehicle.

[ Table 8]

(1) Weight change

All animals were weighed on day 1 (pre-application) and on days 2, 4, 8, 15, 19, 22, 25, 28, 31, 34 and 35 using a small animal scale. The weight of dead and moribund animals was measured and subsequently necropsied immediately after such dead or moribund animals were found.

The measurement results of the weight change are shown in fig. 15. As shown in fig. 15, no weight loss was observed in groups other than groups 4 and 7 administered at a dose of 45 mpk. In groups 4 and 7, which were administered at the maximum dose, all subjects died before the end of the experiment.

(2) Hematology analysis

The toxicity of the ADC was assessed by hematological testing. Following administration, intravenous blood collection was performed on day 3 (first blood collection), day 18 (second blood collection), and day 35 (third blood collection), and animals were fasted (but provided with water) overnight prior to blood collection. After injecting about 0.5mL of blood into a CBC vial containing EDTA-2K, an anticoagulant, changes in leukocyte levels were measured using an automatic hematology analyzer, and the results are shown in fig. 16. The analysis results showed that no significant toxicity was observed in groups other than groups 4 and 7 administered at high dose. Due to toxicity caused by the administered substance, the white blood cell count temporarily decreases but gradually recovers over time.

(3) Biochemical analysis of serum

The toxicity of ADCs was tested by analyzing blood biochemical parameters such as ALT (alanine aminotransferase), AST (aspartate aminotransferase) and Blood Urea Nitrogen (BUN). Following administration, venous blood collection was performed on day 3 (primary blood collection), day 18 (secondary blood collection), and day 35 (tertiary blood collection). About 1mL of blood was injected into a 5mL evacuated blood collection tube containing a coagulation activator, allowed to clot at room temperature for 15 to 20 minutes, and then centrifuged for 10 minutes. The resulting serum was examined using a blood biochemical analyzer. The results are shown in fig. 17. As a result of the analysis, no significant toxicity was observed in groups other than groups 4 and 7 administered at a dose of 45 mpk.

(4) Pathological analysis results

Table 9 shows the symptoms of each organ observed at necropsy. Atrophy of the thymus was observed in the group to which ADC was administered, i.e., G2, G3, G4 and G7, but this was considered to be due to leukopenia rather than a direct effect of the test substance. Other results were found in the spleen, stomach and lymph nodes of moribund animals, but were not accompanied by histopathological changes, or spontaneous changes, and were therefore not considered symptoms due to ADC toxicity.

Table 10 shows the histopathological results. Megakaryocyte formation was observed in the renal tubules and showed a dose-response correlation, which was considered to be due to the test substance. In particular, this phenomenon was observed only in D20103, but not in D20106, and thus it is presumed that the PEG group for the linker is involved in reducing renal toxicity.

[ Table 9]

[ Table 10]

4-6 Single dose rodent toxicity test 2 for determining maximum tolerated dose

Experimental groups were set up as shown in table 11 below and subjected to a single dose rodent toxicity test to assess toxicity of ADCs containing either PD006 or PD009 and determine the maximum tolerated dose.

[ Table 11]

Group of	Test substance	Dosage form
			G1	Carrier
G2	D20106	10mpk, once
			G3	D20106	20mpk, once
G4	D20106	30mpk, once
			G5	D20109	5mpk, once
G6	D20109	10mpk, once
			G7	D20109	20mpk, once

The application, feeding and weighing methods were the same as in 4-5 above. Blood was collected on days 3, 14 and 35 after administration and hematological examination was performed using an automatic hematology analyzer. After completion of the experiment, euthanasia, autopsy and pathological examination were performed. Group 1, the control group, was administered 20mM histidine and 7% trehalose at pH 6.0 as a vehicle.

The body weight changes of the test animals are shown in fig. 18. Acute toxic reactions involving weight loss were observed within 2 weeks after administration in all experimental groups, but recovered in groups other than G4 and G7, G4 and G7 being the groups administered with the highest dose. G4 and G7 recovered slowly within 3 weeks after administration. Weight loss in all groups on day 36 was the change caused by fasting prior to sacrifice.

In addition, fig. 19 shows the measurement results of the change in white blood cell count at the time of the hematological examination. A pattern similar to the body weight change was observed, and in groups other than the group administered at the highest dose, a decrease in leukocytotoxicity was observed at day 14 after administration, and recovery was observed at day 35 after administration (i.e., the end date of the experiment). However, in the groups receiving the highest dose (i.e., groups G4 and G7), there was no pattern of recovery of white blood cell counts.

Thus, based on the two indices of body weight and white blood cell count, the Maximum Tolerated Dose (MTD) of D20106 with PD006 was set to 20mpk, while the maximum tolerated dose of D20109 with PD009 was set to 10 mpk.

4.7. Single dose rodent toxicity test for comparison with similar substances

In order to demonstrate that the load containing β -galactose as a trigger is superior in toxicity to the load containing another trigger having a similar structure thereto, GT70 having the following structure and based on CBI and β -glucuronide was synthesized, on the basis of which ADC was prepared and toxicity was compared. An ADC was prepared in the same manner as in example 2, except that GT70 as a linker-drug was coupled to the B58 antibody and designated "D20111".

SD rats were administered 1 time each of D20109 and D20111, and changes in body weight and hematological biochemical changes were observed for 28 consecutive days, and toxicity expression and recovery were determined from these changes. Specific methods of single dose toxicity testing As described above in examples 4-5, the experimental groups were configured as shown in Table 12 below.

[ Table 12]

The test results are shown in fig. 20. All toxicological indices showed dose-dependent responses, and more toxic responses were observed in the high dose groups (groups 3 and 5) than in the low dose groups (groups 2 and 4). When weight changes were observed, at the same dose level, the group administered D20109 lost less weight than the group administered D20111 and recovered more weight at the end of the trial (fig. 20). Even if toxicity was compared according to the white blood cell count as a hematological index, the group to which D20109 was administered showed less leukopenia and showed a recovery pattern as compared to the group to which D20111 was administered (fig. 20). In particular, in the high dose group D20111, the red blood cell count of males did not recover until the end of the experiment (fig. 20). This supports the following results: beta-galactose has significantly superior stability when used as a trigger for ADCs compared to other materials with similar structures.

[ INDUSTRIAL APPLICABILITY ]

The ADC linked to a load containing a galactose trigger according to the present invention has a target-specific property, i.e. exhibits cytotoxicity only when galactose is removed by β -galactosidase highly expressed in cancer cells or tumor cells, thereby significantly reducing the risk of premature release of the drug and subsequent systemic toxicity resulting therefrom. Further, it was found that the ADC containing a galactose trigger according to the present invention exhibits superior drug efficacy and lower in vivo toxicity, compared to an ADC having another trigger of a similar structure thereto (e.g., an ADC having glucose or glucuronide as a trigger). That is, ADCs that are less toxic, highly stable in vivo, exhibit excellent efficacy, and have a wider therapeutic window than conventional prodrugs can be prepared using a cargo using a galactose trigger according to the present invention.

While specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this description is provided for purposes of illustration of preferred embodiments and should not be construed to limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and equivalents thereof.

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225 230 235 240

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

340 345 350

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 17

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 10H1 VL

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Glu Thr Leu Val His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Phe Asn Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Cys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 10H1 VH

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Gly Arg Val Ala Phe His Thr Tyr Tyr Pro Asp Ser Met

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Gly Phe Asp Val Gly His Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 19

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 6E7(N54A) VL

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Thr Ser

20 25 30

Ser Tyr Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp

85 90 95

Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Cys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 20

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 6E7(N54A) VH

<400> 20

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Ala Gly Ala Ala Phe Tyr Ser Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Glu Arg Gly Ala Asp Leu Glu Gly Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450