Antibody-conjugated drugs of N-haloalkyl-substituted camptothecin derivatives

Abstract

The present invention relates to an antibody-drug conjugate represented by formula (I) having a haloalkyl-substituted camptothecin derivative as a drug toxic moiety, wherein the definitions of each group in the formula are as described in the specification, and the antibody-drug conjugate has a significant antitumor effect. [Formula 1] JPEG2026501617000115.jpg57170

Description

[Technical Field]

[0001] The present invention relates to the medical field. Specifically, the present invention provides an antibody-conjugated drug of an N-fluoroalkyl-substituted camptothecin derivative, its preparation method and its application in the antitumor field. [Background technology]

[0002] Camptothecin and its derivatives are broad-spectrum antitumor drugs, and many camptothecin derivatives have been approved for use in the treatment of various types of cancer in many countries.

[0003] Antibody-drug conjugates (ADCs) are monoclonal antibodies (mAbs) that are chemically linked to biologically active small molecule drugs, which then serve as carriers for delivering the small molecule drugs to target cells. In recent years, several camptothecin derivatives have been used as small molecule drugs in antibody-drug conjugates, demonstrating significant antitumor effects. For example, Chinese Patent Application No. 201380053256.2 discloses an antibody-drug conjugate using exatecan as its small molecule drug payload, which has demonstrated significant inhibitory effects against melanoma and non-small cell lung cancer. However, the number of commercially available ADC drugs is limited, and further research is needed to determine the structure and appropriate linker structure of camptothecin derivatives used as ADC payloads. Therefore, there is a need to develop antibody-drug conjugates with antitumor activity. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide antibody-drug conjugates having haloalkyl-substituted camptothecin derivatives as toxic moieties, particularly fluoroalkyl-substituted camptothecin derivatives as toxic moieties, which have remarkable antitumor effects. [Means for solving the problem]

[0005] In one embodiment of the present invention, there is provided an antibody-drug conjugate according to formula (I): [ka] (In the formula, R 1 is selected from halogen-substituted methyl groups; R a is hydrogen and -L 1 -NH-L p -Z-Ab, and R b is hydrogen, -L 2 -NH-L p -Z-Ab and -L 3 -NH-Z-Ab, with the proviso that R a and R b is not hydrogen at the same time, where: L 1 is -C(=O)-L 11 -L 12 -L 13 - and -CH2-NR 2 -L 14 -CHR 3 - selected from L 11 , L 12 , and L 13 are each independently selected from —O—, a C1-C3 alkylene group, and a phenyl group; R 2 is selected from hydrogen, deuterium, a C1-C6 alkyl group, a C1-C6 alkoxy group, an acyl group, and a sulfonyl group; L 14 is selected from —C(═O)— and a C1-C3 alkylene group; R 3 is selected from hydrogen, deuterium, a C1-C6 alkyl group, a C1-C6 alkoxy group, and a phenyl-substituted C1-C6 alkyl group; L 2 is -C(=O)-NH-L 21 -CHR 4 - selected from L 21is selected from -C(=O)- and -NH-C(=O)-; R 4 is selected from hydrogen, deuterium, a C1-C6 alkyl group, and a phenyl-substituted C1-C6 alkyl group; L 3 is -C(=O)-NR 5 -NR 6 -L 31 -L 32 - selected from R 5 and R 6 are each independently selected from hydrogen, deuterium, and a C1-C6 alkyl group; L 31 is selected from -C(=O), -(C1-C3 alkylene)-C(=O), -5- to 8-membered aromatic ring group -C(=O)-, -5- to 8-membered nitrogen heteroaromatic ring group -C(=O)- and -O-(C1-C3 alkylene)-C(=O)-; L 32 is selected from -NH-(C1-C3 alkylene), -N(CH3)2-(C1-C3 alkylene) and -NH-(C1-C3 alkylene)-NH-; L P is a peptide residue consisting of 2 to 7 amino acids, Z is -L z -L j - selected from, where L z is a -C(=O)-C1-C8 alkylene group, -C(=O)-CHO-(CHCHO) 2~5 -CH2CH2NH-, or -C(=O)-(CH2CH2O) 2~6 -CH2CH2NH-, L j is a linker capable of being attached to an antibody, n1 is selected from integers of 1 to 3; Ab is an antibody.

[0006] In the antibody-drug conjugate of formula (I), the drug-antibody ratio (DAR) is selected from the range of 2 to 8, and may further be 6 to 8 or 6.2 to 7.6, for example, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.5, or 7.6.

[0007] One aspect of the present invention provides an antibody-drug conjugate shown in formula (IA), (IB) or (IC): [ka] (each group in the formula is defined as above). [ka] (each group in the formula is defined as above). [ka] (n' is selected from 2 to 8, and may further be 6 to 8 or 6.2 to 7.6, for example 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.5 or 7.6; Each group in the formula is defined as above.

[0008] In some specific embodiments, R 1 is selected from fluorine, chlorine or bromine substituted methyl groups.

[0009] In some specific embodiments, R 1 is selected from fluorine-substituted methyl groups.

[0010] In some specific embodiments, R 1 is selected from a fluoromethyl group, a difluoromethyl group, and a trifluoromethyl group.

[0011] In some specific embodiments, R 1 is selected from a difluoromethyl group and a trifluoromethyl group.

[0012] In some specific embodiments, n1 is 1.

[0013] In some specific embodiments, L 11 , L12 , and L 13 are each independently selected from —O—, a methylene group, and a phenyl group.

[0014] In some specific embodiments, L 11 -L 12 -L 13 is selected from -O-CH2-phenyl- and -CH2-O-CH2-.

[0015] In some specific embodiments, R 2 is selected from hydrogen, deuterium, a C1-C6 alkyl group, a C1-C6 alkoxy group, a -C(=O)C1-C6 alkyl group, and a -S(=O)2C1-C6 alkyl group.

[0016] In some specific embodiments, R 2 is selected from hydrogen, deuterium, a C1-C3 alkyl group, a C1-C3 alkoxy group, a -C(=O)C1-C3 alkyl group, and a -S(=O)2C1-C3 alkyl group.

[0017] In some specific embodiments, R 2 is selected from hydrogen, deuterium, methyl, ethyl, methoxy, ethoxy, formyl, acetyl, methanesulfonyl, and ethanesulfonyl.

[0018] In some specific embodiments, R 2 is selected from hydrogen, methyl, methoxy, formyl, and methanesulfonyl.

[0019] In some specific embodiments, R 2 is hydrogen.

[0020] In some specific embodiments, L 14 is selected from -C(=O)- and a C1 to C3 alkylene group.

[0021] In some specific embodiments, R14 is selected from -C(=O)- and a methylene group.

[0022] In some specific embodiments, R 3 is selected from hydrogen, deuterium, a C1-C6 alkyl group, a C1-C6 alkoxy group, and a phenyl-substituted C1-C6 alkyl group.

[0023] In some specific embodiments, R 3 is selected from hydrogen, a C1-C6 alkyl group, and a phenyl-substituted C1-C6 alkyl group.

[0024] In some specific embodiments, R 3 is selected from hydrogen, C1-C4 alkyl groups, and phenyl-substituted C1-C3 alkyl groups.

[0025] In some specific embodiments, R 3 is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenylmethyl, and phenylethyl.

[0026] In some specific embodiments, R 3 is selected from hydrogen, methyl, isopropyl, isobutyl, and phenylmethyl.

[0027] In some specific embodiments, R 3 is hydrogen.

[0028] In some specific embodiments, L 1 is selected from -C(=O)-O-CH2-phenyl-, -C(=O)-CH2-O-CH2-, -CH2-NH-C(=O)-CH2- and -CH2-NH-CH2-CH2-.

[0029] In some specific embodiments, L 1is selected from -C(=O)-O-CH2-phenyl-, -C(=O)-CH2-O-CH2- and -CH2-NH-C(=O)-CH2-.

[0030] In some specific embodiments, L 1 is selected from -C(=O)-O-CH2-phenyl- and -C(=O)-CH2-O-CH2-.

[0031] In some specific embodiments, R 4 is selected from hydrogen, deuterium, a C1-C6 alkyl group, and a phenyl-substituted C1-C6 alkyl group.

[0032] In some specific embodiments, R 4 is selected from hydrogen, deuterium, a C1-C3 alkyl group, and a phenyl-substituted C1-C3 alkyl group.

[0033] In some specific embodiments, R 4 is selected from hydrogen, deuterium, methyl, isopropyl, isobutyl, and phenylmethyl.

[0034] In some specific embodiments, R 4 is selected from hydrogen.

[0035] In some specific embodiments, L 2 is selected from -C(=O)-NH-C(=O)-CH2- and -CH2-NH-NH-C(=O)-CH2-.

[0036] In some specific embodiments, L 2 is -CH2-NH-NH-C(=O)-CH2-.

[0037] In some specific embodiments, R 5 and R 6 are each independently selected from hydrogen, deuterium, and a C1-C3 alkyl group.

[0038] In some specific embodiments, R 5 and R 6 are each independently selected from hydrogen, deuterium, and a methyl group.

[0039] In some specific embodiments, R 5 is hydrogen, R 6 is hydrogen.

[0040] In some specific embodiments, L 31 is selected from -C(=O), -methylene-C(=O), -phenyl-C(=O)-, -pyridyl-C(=O)- and -O-CH2-C(=O)-.

[0041] In some specific embodiments, L 32 is selected from -NH-CH2-CH2-, -N(CH3)2-CH2-CH2- and -NH-CH2-NH-.

[0042] In some specific embodiments, L 3 is -C(=O)-NH-NH-phenyl-C(=O)-NH-CH2-CH2-.

[0043] In some specific embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn).

[0044] In some specific embodiments, L p is selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn).

[0045] In some specific embodiments, L pis selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), and lysine (Lys).

[0046] In some specific embodiments, L p is selected from peptide residues consisting of 2, 3 or 4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu) and lysine (Lys).

[0047] In some specific embodiments, L p is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe-, and -Gly-Gly-Phe-Gly-.

[0048] In some specific embodiments, L p is selected from the group represented by the formula: [ka] The carbonyl end is linked to -NH- and the other end is linked to Z.

[0049] In some specific embodiments, L p is selected from the group represented by the formula: [ka] The carbonyl end is linked to -NH- and the other end is linked to Z.

[0050] In some specific embodiments, L j is selected from the group represented by the formula: [ka] [ka] The position indicated by indicates that it is linked to an antibody, [ka] The position indicated by L Z represents that the group is linked to the

[0051] In some specific embodiments, L z is a -C(=O)-C1-C8 alkylene group, -C(=O)-CHO-(CHCHO) 2~5 -CH2CH2NH- and -C(=O)-(CH2CH2O) 2~6 -CH2CH2NH-.

[0052] In some specific embodiments, L z is a -C(=O)-C1-C6 alkylene group, -C(=O)-CHO-(CHCHO) 2~3 -CH2CH2NH- and -C(=O)-(CH2CH2O) 2~4 -CH2CH2NH-.

[0053] In some specific embodiments, L z is selected from —C(═O)—(CH2CH2O)2—CH2CH2NH—, —C(═O)—CH2O—CH2CH2O—CH2CH2NH—, and —C(═O)—CH2O—(CH2CH2O)3—CH2CH2NH.

[0054] In some specific embodiments, Z is selected from the group represented by the formula: [ka] [ka] The position indicated by indicates that it is linked to an antibody, [ka] The position indicated by L p Group or L 3 represents that the group is linked to the

[0055] In some specific embodiments, the antibody is a tumor-associated antigen antibody.

[0056] In some specific embodiments, the tumor-associated antigen antibody is selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody.

[0057] In some specific embodiments, the antibody-drug conjugate of formula (I) has the structure shown in formula (I-1), formula (I-1′), formula (I-2′), or formula (I-2). [ka] JPEG2026501617000015.jpg47170 (in the formula, R 1 , n1, L p , Z, and Ab are each defined as in the compound of formula (I), and n' is defined as above.

[0058] In some specific embodiments, the antibody-drug conjugate of Formula (I) has the structure shown in Formula (I-3), Formula (I-3'), Formula (I-4'), or Formula (I-4). [ka] JPEG2026501617000017.jpg47170 (in the formula, R 1 , R 2 , n1, L p, Z, and Ab are each defined as in the compound of formula (I), and n' is defined as above.

[0059] In some specific embodiments, the antibody-drug conjugate of formula (I) has the structure shown in formula (I-5) or formula (I-5'). [ka] (In the formula, R 1 , R 5 , R 6 , n1, and Ab are each defined as in the compounds of formula (I), and n' is defined as above. n2 is selected from 1 or 2. Z is -L z -L j’ where L j is selected from the group represented by the formula: [ka] [ka] The position indicated by indicates that it is linked to an antibody, [ka] The position indicated by L Z represents that the group is connected to the

[0060] In some specific embodiments, L z is -C(=O)-CH2O-(CH2CH2O) 1~5 -CH2CH2NH-.

[0061] In some specific embodiments, L z is -C(=O)-CH2O-(CH2CH2O) 3~5 -CH2CH2NH-.

[0062] In some specific embodiments, L zis selected from —C(═O)—CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—.

[0063] In some specific embodiments, Z is selected from the group represented by the formula: [ka] [ka] The position indicated by indicates that it is linked to an antibody, [ka] The position indicated by L p Group or L 3 represents that the group is linked to the

[0064] In some specific embodiments, the antibody-drug conjugate of formula (I) has the structure shown in formula (I-6) or formula (I-6'). [ka] (In the formula, R 1 , R 5 , R 6 , n1, L p , Z, and Ab are each defined as in the compound of formula (I), and n' is defined as above.

[0065] The present invention provides the following antibody-drug conjugates: [ka] [ka] (wherein Ab is defined as in the compound of formula (I)).

[0066] In some specific embodiments, the Ab is selected from a tumor-associated antigen antibody, and further selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody.

[0067] Preferably, the Ab is an HS627 antibody or an IP140B antibody, the heavy chain amino acid sequence of HS627 being shown in SEQ ID NO:1 and the light chain amino acid sequence being shown in SEQ ID NO:2, and the heavy chain amino acid sequence of IP140B antibody being shown in SEQ ID NO:3 and the light chain amino acid sequence being shown in SEQ ID NO:4.

[0068] In some specific embodiments, n is selected from 6 to 8, and may further be 6.2 to 7.6, such as 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.5, or 7.6.

[0069] The present invention provides the following antibody-drug conjugates: [ka] (n is selected from 6.8 to 7.0.) [ka] (n is selected from 7.4 to 7.5.) [ka] (n is selected from 7.5 to 7.6.) [ka] (n is selected from 7.1 to 7.2.) [ka] (n is selected from 7.2 to 7.3.) [ka] (n is selected from 7.5 to 7.6.) [ka] (n is selected from 7.0 to 7.1.) [ka] (n is selected from 6.2 to 6.5.) [ka] (n is selected from 6.3 to 6.6.) [ka] (n is selected from 7.3 to 7.5.) [ka] (n is selected from 7.0 to 7.1.) [ka] (n is selected from 7.3 to 7.5.) [ka] (n is selected from 6.3 to 6.6.)

[0070] wherein Ab is defined as in the compound of formula (I); Preferably, the Ab is the HS627 antibody or the IP140B antibody, the heavy chain amino acid sequence of HS627 being shown in SEQ ID NO:1 and the light chain amino acid sequence being shown in SEQ ID NO:2, and the heavy chain amino acid sequence of the IP140B antibody being shown in SEQ ID NO:3 and the light chain amino acid sequence being shown in SEQ ID NO:4.

[0071] In another aspect of the present invention, there is provided a method for producing the above antibody-drug conjugate, the method being selected from the following synthetic routes:

[0072] Synthetic Route 1: The compound of formula (a) is z -L j ' to obtain a compound of formula (b), and linking the compound of formula (b) to an antibody to obtain a compound of formula (I), wherein R a -L 1 -NH-L p -Z-Ab and R b is hydrogen. [ka]

[0073] Synthetic Route 2: The compound of formula (c) is z -L j ' to obtain a compound of formula (d), and linking the compound of formula (d) to an antibody to obtain a compound of formula (I), wherein R a is hydrogen and R b -L 2 -NH-L p -Z-Ab. [ka]

[0074] Synthetic Route 3: The compound of formula (e) is z -L j ' to obtain a compound of formula (f), and linking the compound of formula (f) to an antibody to obtain a compound of formula (I), wherein R a is hydrogen and R b -L 3 -NH-Z-Ab. [ka] In the above synthesis route, R 1 , n1, L 1 , L 2 , L 3 , L p , L z and Ab are each defined as in the compound of formula (I), L j is selected from the groups represented by the following formulas: [ka]

[0075] In another aspect of the present invention, there is provided a pharmaceutical composition comprising the antibody-drug conjugate or the antibody-drug conjugate produced by the above-described method and a pharmaceutically acceptable carrier.

[0076] In another aspect of the present invention, there is provided use of the above antibody-drug conjugate, the antibody-drug conjugate produced by the above method, or the above pharmaceutical composition in the manufacture of an anti-tumor drug.

[0077] In another aspect of the present invention, there is provided a method for suppressing or treating a tumor disease in a patient in need thereof, the method comprising administering to a patient in need thereof the above-described antibody-drug conjugate or pharmaceutical composition.

[0078] In some specific embodiments, the amount of the antibody-drug conjugate or pharmaceutical composition administered is a therapeutically effective amount.

[0079] In some specific embodiments, the tumor is selected from a solid tumor.

[0080] In some specific embodiments, the tumor is selected from esophageal cancer, lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, ovarian cancer, lung cancer, or breast cancer.

[0081] In some specific embodiments, the lung cancer is non-small cell lung cancer. [Brief explanation of the drawings]

[0082] [Figure 1] 1 is a graph showing the tumor growth curve of the Calu-6 human non-small cell lung cancer CDX model in Test Example 2. [Figure 2]1 shows photographs of tumors after dissection of a Calu-6 human non-small cell lung cancer CDX model in Test Example 2. [Figure 3] 1 is a graph showing the tumor growth curve of the KYSE-150 human esophageal squamous cell carcinoma CDX model in Test Example 2. [Figure 4] 1 shows photographs of tumors after dissection of a KYSE-150 human esophageal squamous cell carcinoma CDX model in Test Example 2. [Figure 5] 1 is a graph showing the tumor growth curve of the ES-2 human ovarian cancer human CDX model in Test Example 2. [Figure 6] 1 shows photographs of tumors after dissection of the ES-2 human ovarian cancer CDX model in Test Example 2. [Figure 7] 1 is a graph showing the tumor growth curve of the NCI-H1975 lung cancer CDX model in Test Example 2. [Figure 8] 1 shows photographs of tumors after dissection of an NCI-H1975 lung cancer CDX model in Test Example 2. [Figure 9] 1 is a graph showing the tumor growth curve of the MDA-MB-231 triple-negative breast cancer CDX model in Test Example 2. [Figure 10] 1 shows photographs of tumors after dissection of an MDA-MB-231 triple-negative breast cancer CDX model in Test Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0083] I. Definition: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the relevant terms and experimental procedures used herein are terms and routine steps that are widely used in the corresponding fields.In addition, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0084] As used herein, unless otherwise specified, the terms "comprise," "include," "have," and "contain," including their grammatical equivalents, are generally to be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps.

[0085] The compounds of the present disclosure may be asymmetric, e.g., may have one or more stereoisomers. Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are included. The stereoisomers include geometric isomers (e.g., cis- and trans-configurations) and optical isomers (e.g., enantiomers), and are therapeutic agents consisting of individual compounds, racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all within the scope of the present invention.

[0086] Numerical ranges herein refer to individual integers within the specified range, for example, "C1-C6" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. When any variable (e.g., Rn) occurs more than one time in any composition or structure of a compound, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1 to 5 R, then that group is optionally substituted with up to 5 R, and each occurrence of R is an independent option. Further, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0087] The term "C1-C6 alkyl group" refers to a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched-chain isomers thereof.

[0088] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. An alkoxy group can be optionally substituted or unsubstituted, and if substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydrogen, nitro, chloro, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxylic acid, or carboxylic acid ester groups.

[0089] The term "acyl group" refers to -C(=O)R, where R is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heteroalkyl group.

[0090] The term "sulfonyl" refers to -S(=O)R, where R is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted heteroalkyl group.

[0091] [ka] indicates the point of attachment of a chemical bond. z -L j -) represents a linking of the corresponding groups in left to right order unless otherwise specified. For example, -L 12 is C1-C2 alkylene-O-, this means that the left side of C1-C2 alkylene-O- is -L 11 -L on the right 13 - indicates that it will be linked to.

[0092] As will be understood by those skilled in the art, in the antibody-drug conjugates of the present application, for example, in formulae (I), (I-1), (I-2), (I-3), (I-4), (I-5), and (I-6), the "-" between the drug-linker fragment and the antibody is intended to represent the linkage between the antibody and the fragment, and is not intended to limit the linkage of one antibody to only one drug-linker fragment. As is well known in the art, one antibody can have multiple intermolecular disulfide bonds, and therefore one antibody can be linked to one or more drugs.

[0093] Drug or pharmaceutical composition The drugs or pharmaceutical compositions of the present invention can be administered orally, topically, parenterally, or mucosally (e.g., sublingually, by inhalation, or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. Oral administration is generally preferred. The active agent can be administered orally in the form of capsules, tablets, or the like.

[0094] The term "treatment" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0095] The use of the term "inhibition" refers to a control. Those skilled in the art can easily determine the appropriate control for each experiment. For example, the reduction in the response in subjects or cells treated with a compound is compared with the response in subjects or cells not treated with a compound.

[0096] The term "patient" refers to an animal, preferably a mammal, and more preferably a human.

[0097] The term "pharmaceutical composition" means a composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the group consisting of, but not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersing agents, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, and suspension aids, depending on the nature of the administration method and dosage form.

[0098] The term "effective amount" or "therapeutically effective amount" refers to a nontoxic but sufficient amount of a drug or agent to achieve a desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug to be administered will depend on many factors, such as the specific administration protocol, the type and severity of the disease or condition, and the specific characteristics (e.g., body weight) of the subject or host requiring treatment. However, dosages can be routinely determined by methods known in the art depending on the specific circumstances, including, for example, the specific drug employed, the route of administration, the condition being treated, and the subject or host being treated. Generally, dosages used in adult treatment typically range from 0.02 to 5000 mg / day, e.g., about 1 to 1500 mg / day. This required dose can conveniently be expressed as a single dose or as divided doses administered simultaneously (or shortly thereafter) or at appropriate intervals, e.g., two, three, four, or more divided doses per day. Although the above dosage ranges are given, it will be understood by those skilled in the art that the specific effective amount may be appropriately adjusted depending on the condition of the patient and in conjunction with the diagnosis of a physician.

[0099] The term "antibody-conjugated drug" as used herein has the same meaning as antibody-drug conjugate (ADC).

[0100] The term "drug-to-antibody ratio (DAR)" refers to the average number of effective bearing molecules (anti-tumor compounds or drugs) that bind to a single monoclonal antibody.

[0101] Abbreviation: Fmoc: 9-fluorenylmethoxycarbonyl group The amino acid constituting the peptide residue LP is Val: valine, and its structural formula is [ka] and Ala: Alanine, its structural formula is [ka] and Gly: Glycine, whose structural formula is [ka] and Phe: Phenylalanine, its structural formula is [ka] and L P can be obtained by amino acid condensation methods known in the art. PABC: [ka] ha: [ka] m: [ka] In the present invention, a linker L for linking antibodies j can be obtained by linking by methods known in the art. For example, L j The structure of [ka] (or [ka] ), then it is [ka] (or [ka] ) with a reactive group such as thiol on the antibody, and the present invention is not particularly limited thereto.

[0102] II. Working Examples The antibodies used to produce the ADC in this example are not limited to pertuzumab and IP140B antibodies. The heavy chain amino acid sequence of pertuzumab (HS627, PERTUZUMAB) is as follows (SEQ ID NO: 1): [ka]

[0103] The light chain amino acid sequence is as follows (SEQ ID NO:2): [ka]

[0104] The heavy chain amino acid sequence of the IIP140B antibody is as follows (SEQ ID NO:3): [ka]

[0105] The light chain amino acid sequence is as follows (SEQ ID NO:4): [ka]

[0106] Unless otherwise specified, the materials and equipment used in specific embodiments of the present invention are known products and can be obtained commercially.

[0107] Intermediate HX-16a: N-(2',2'-difluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin [ka] Difluoroethylamine (1.0 g, 12 mmol), hydrochloric acid (1.4 mL, 16 mmol), 7-methyl-10,11-methylenedioxycamptothecin (1.0 g, 2.46 mmol), and DMSO (5 mL) were added to a reaction flask, and the mixture was heated to 120°C with stirring and reacted for 1 hour. The mixture was cooled, and isopropanol was added. The mixture was suction filtered and purified by silica gel column chromatography to obtain the title compound (393 mg, yield 32%, HPLC 95%). 1 H NMR(500MHz,DMSO-d6)δ7.68(s,1H),7.52(s,1H),7.28(s,1H),6.58(s,1H),6.33(s,2H),6.02(t,J=5 6.6Hz,1H),5.47(s,2H),5.26(s,2H),3.26(s,3H),2.94(s,4H),1.92(s,2H),0.93(s,3H);LC-MS(M+H) + 500.13 (theoretical value 499.16).

[0108] Intermediate HX-22e: N-(2',2',2'-trifluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin [ka] Trifluoroethylamine hydrochloride (100 mg, 0.74 mmol) and 7-methyl-10,11-methylenedioxycamptothecin (100 mg, 0.25 mmol) were dissolved in DMSO (3 mL), heated to 120°C with stirring, and reacted for 1 hour. After cooling, methyl tert-butyl ether was added, filtered, and purified by silica gel column chromatography to obtain the title compound (49 mg, yield 38%, HPLC 99%). 1 H NMR(500MHz,DMSO-d6)δ7.71(s,1H),7.54(s,1H),7.29(s,1H),6.55(s,1H),6.34(d,J=2.0Hz,2H),5.48(d,J=3.0Hz,2H) ),5.30(s,2H),3.42(s,2H),3.42(s,2H),3.29(s,2H),1.92(dd,J=14.4,7.3Hz,2H),0.94(t,J=7.3Hz,4H);LC-MS(M+H)+ 518.34 (theoretical value 17.15).

[0109] Example 1: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (1) [ka] A reaction flask was charged with HX-11b (0.1 g, 0.16 mmol), 3 mL of DMF, HATU (74.6 mg, 0.2 mmol), DIPEA (25.3 mg, 0.2 mmol), and HX-16a (82 mg, 0.16 mmol). The mixture was reacted at room temperature for 1.5 hours, and purified by silica gel column chromatography to give a yellow solid, HX-16b (153 mg, 85% yield). LCMS: (M+1) + 1093.11 (theoretical value: 1093.11)

[0110] HX-16b (153 mg, 0.14 mmol) and piperidine (140 mg, 1.6 mmol) were added sequentially to a 10 mL reaction flask and stirred at room temperature for 1 hour. 30 mL of methyl tert-butyl ether was added to the reaction mixture, which was then centrifuged (5 minutes, 10,000 rpm). The supernatant was removed, 1 mL of methanol was added again, and the mixture was clarified by ultrasound. 30 mL of methyl tert-butyl ether was added again, the mixture was centrifuged (5 minutes, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure to give HX-16c (103.5 mg, 72.7% yield). LCMS: (M+1) + 871.21 (theoretical value: 870.34), which was used directly in the next reaction without purification.

[0111] To a 50 mL single-neck flask, DCM (20 mL), 3-[2-(2-aminoethoxy)ethoxy]propionic acid (2 g, 11.3 mmol), and bromoacetyl bromide (2.27 g, 11.3 mmol) were added sequentially and stirred for 30 minutes. The reaction mixture was extracted with 10 mL of saturated sodium chloride solution, and the DCM phase was dried over anhydrous magnesium sulfate to give intermediate HX-3b (3.1 g, 89% yield). LCMS: (M+1) + 298.00 (theoretical value: 297.02).

[0112] To a 10 mL single-neck flask, DCM (3 mL), HX-16c (60 mg, 0.069 mmol), HX-3b (25 mg, 0.083 mmol), and DIC (10.5 mg, 0.083 mmol) were added sequentially and stirred at room temperature for 30 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% HCOOH in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give yellow solid product 6 (24.5 mg, 30.9% yield). LCMS: (M+1) + 1150.35 (theoretical value: 1149.35).

[0113] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0114] Compound HX-16 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20, using a NAP-5 gel column (Cytiva), to obtain the antibody conjugate ADC 1 (3.0 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.8.

[0115] Example 2: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (2) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0116] Compound HX-16 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 2 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.0.

[0117] Example 3: 7-[N-(HS627-Ac-PEG2-Gly-Val-Ala-Gly-ha-Ac),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (3) [ka] HX-9b (3 g, 6.32 mmol) was dissolved in 100 mL of nitrile, and DBU (0.48 g, 3.16 mmol) was added. The mixture was allowed to react at room temperature for 2 hours. PPTS (0.79 g, 3.16 mmol), HOBT (0.85 g, 6.32 mmol), Fmoc-Gly-Val-Ala-OH (2.61 g, 5.36 mmol), and EDCI (1.21 g, 6.32 mmol) were added sequentially and the mixture was allowed to react at room temperature for 15 hours. The mixture was then dried under reduced pressure on a rotary evaporator and purified by silica gel column chromatography to give HX-17a (2.75 g, 62% yield) as a white powdery solid. LCMS: (M+1) + 702.15 (theoretical value: 701.31).

[0118] In a 500 mL single-neck flask, HX-17a (2.75 g, 3.9 mmol) was dissolved in 50 mL of methanol and 25 mL of DCM mixed solvent, 1 g of 10% palladium carbon was added, and the mixture was purged with hydrogen gas. The mixture was reacted at room temperature and atmospheric pressure for 2 hours. After the reaction was completed, the mixture was filtered and concentrated to give product HX-17b (2.2 g, 92% yield). LCMS: (M+1) + 612.18 (theoretical value: 611.26).

[0119] HX-16a (50 mg, 0.1 mmol) was added with 2 mL of DMF, HATU (38 mg, 0.1 mmol), DIPEA (26 mg, 0.2 mmol), and HX-17b (61 mg, 0.1 mmol). The mixture was allowed to react at room temperature for 15 hours, diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give a yellow solid, HX-17c (85 mg, 78% yield). LCMS: (M+1) + 1093.11 (theoretical value: 1092.40).

[0120] HX-17c (78 mg, 0.07 mmol) was dissolved in 0.2 mL of DMF, piperidine (55 mg, 0.7 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 30 mL of methyl tert-butyl ether was added to the reaction mixture, which was then centrifuged (5 min, 10,000 rpm). The supernatant was removed, the solvent was removed under reduced pressure, and the mixture was dried to give solid HX-17d (56 mg, 73.4% yield). LCMS: (M+1) + 871.32 (theoretical: 870.34). The intermediate was used directly in the next reaction without further purification.

[0121] To a 10 mL single-neck flask, DCM (3 mL), HX-17d (56 mg, 0.064 mmol), HX-3b (18.6 mg, 0.064 mmol), and DIC (8.1 mg, 0.064 mmol) were added sequentially and stirred at room temperature for 90 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give a yellow solid, HX-17 (23 mg, 31% yield). LCMS: (M+1) + 1150.33 (theoretical value: 1149.35).

[0122] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0123] Compound HX-17 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 3 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.4.

[0124] Example 4: 7-[N-(IP140B-Ac-PEG2-Gly-Val-Ala-Gly-ha-Ac),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (4) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0125] Compound HX-17 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain the antibody conjugate drug ADC 4 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0126] Example 5: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Phe-Gly-m),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (5) [ka] In a 100 mL three-neck flask, HX-9a (1 g, 2.7 mmol) was dissolved in 10 mL of DCM, 8 mL of TMSCl was added, and the reaction mixture was stirred at room temperature under argon gas for 2 hours. The mixture was then concentrated under reduced pressure to give a white solid, HX-18a, which was used directly in the next step. LCMS: (M+1) + 344.80 (theoretical value: 344.09).

[0127] HX-16a (100 mg, 0.2 mmol) was dissolved in 5 mL of anhydrous DCM, HX-18a (0.93 g, 2.7 mmol) was added, and the reaction mixture was allowed to react at room temperature for 2 hours. Water (10 mL) was added, and the mixture was extracted three times with DCM (20 mL x 3). The organic phase was directly concentrated and purified by silica gel column chromatography to give compound HX-18b (121 mg, 75% yield). LCMS: (M+1) + 808.13 (theoretical value: 807.28).

[0128] HX-18b (121 mg, 0.149 mg) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-18c (68 mg, 78% yield). LCMS: (M+1) + 586.21 (theoretical value: 585.20).

[0129] HX-18c (68 mg, 0.11 mmol) was dissolved in 2 mL of DMF, and DIPEA (15 mg, 0.11 mmol) was added by stirring to completely dissolve the compound. 0.138 g of Fmoc-Gly-Gly-Phe-OH (56 mg, 0.11 mmol) and HATU (42 mg, 0.11 mmol) were added sequentially, and the resulting mixture was stirred at room temperature. The resulting mixture was purified by column chromatography to give product HX-18d (107 mg, 86% yield). LCMS: (M+1) + 1069.09 (theoretical value: 1068.38).

[0130] HX-18d (107 mg, 0.1 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-18e (56 mg, 66% yield). LCMS: (M+1) + 847.23 (theoretical value: 846.31).

[0131] To a 10 mL single-neck flask, DCM (3 mL), HX-18e (56 mg, 0.066 mmol), HX-3b (19.2 mg, 0.066 mmol), and DIC (8.4 mg, 0.066 mmol) were added sequentially, and the mixture was stirred at room temperature for 90 minutes. The mixture was concentrated and purified by silica gel column chromatography to give the yellow solid product HX-18 (12 mg, 16% yield). LCMS: (M+1) + 1126.24 (theoretical value: 1125.33).

[0132] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0133] Compound HX-18 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 5 (3.4 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0134] Example 6: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Phe-Gly-m),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (6) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0135] Compound HX-18 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 6 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 6 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.6.

[0136] Example 7: 7-[N-(HS627-Ac-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (7) [ka] HX-16a (100 mg, 0.2 mmol), 1 mL of NMP, DIPEA (129 mg, 1 mmol), Fmoc-GK-PAB-PNP (187 mg, 0.2 mmol), and HOBt (27 mg, 0.2 mmol) were added to a 10 mL single-neck flask in this order, and the mixture was stirred at room temperature for 4 hours. The resulting HX-19a was then directly added to the next reaction. LCMS: (M+1) + 1298.41 (theoretical value: 1297.50).

[0137] To the HX-19a reaction mixture, 0.1 mL of piperidine (V:V = 10%) was added and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added to the reaction mixture, and the mixture was centrifuged (5 minutes, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure to give HX-19b (155 mg, 72% yield). LCMS: (M+1) + 1076.39 (theoretical: 1075.43), the intermediate was used directly in the next reaction without the need for further purification.

[0138] To a 10 mL single-neck flask, DCM (2 mL), HX-19b (50 mg, 0.046 mmol), HX-3b (13.4 mg, 0.046 mmol), and DIC (5.85 mg, 0.046 mmol) were added sequentially and stirred at room temperature for 90 minutes. 0.1 mL of TFA was added and the mixture was allowed to react for an additional 30 minutes. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product HX-19 (21 mg, 40% yield). LCMS: (M+1) +1 113.24 (theoretical value: 1112.33).

[0139] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0140] Compound HX-19 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 7 (3.0 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.1.

[0141] Example 8: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (8) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0142] Compound HX-19 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate ADC 8 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.2.

[0143] Example 9: 7-[N-(HS627-isonicotinoyl-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (9) [ka] HX-16a (100 mg, 0.2 mmol), 1 mL of NMP, DIPEA (129 mg, 1 mmol), Fmoc-GK-PAB-PNP (187 mg, 0.2 mmol), and HOBt (27 mg, 0.2 mmol) were added to a 10 mL single-neck flask, and the mixture was stirred at room temperature for 4 hours to give HX-20a. LCMS: (M+1) + 1298.41 (theoretical value: 1297.50). Used directly in the next reaction.

[0144] To the HX-20a reaction mixture, 0.1 mL of piperidine (V:V = 10%) was added and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added to the reaction mixture, and the mixture was centrifuged (5 minutes, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure to give HX-20b (155 mg, 72% yield). LCMS: (M+1) + 1076.42 (theoretical value: 1075.43). Used directly in the next reaction.

[0145] HX-20b (155 mg, 0.14 mmol) was dissolved in 2 mL of DMF, and DIPEA (18.6 mg, 0.14 mmol) was added by stirring to completely dissolve the compound. 0.138 g of Fmoc-NH-PEG-CHCH-COOH (57 mg, 0.14 mmol) and HATU (55 mg, 0.14 mmol) were added sequentially, and the resulting mixture was stirred at room temperature. The resulting mixture was purified by column chromatography to give the solid product HX-20c (168 mg, 81% yield). LCMS: (M+1) + 1443.57 (theoretical value: 1442.57).

[0146] HX-20c (168 mg, 0.11 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to obtain the solid product HX-20d (126 mg, 88% yield). LCMS: (M+1) + 1221.31 (theoretical value: 1220.50).

[0147] To a 10 mL single-neck flask, DCM (2 mL), HX-20d (126 mg, 0.10 mmol), 2-(bromomethyl)isonicotinic acid (22 mg, 0.10 mmol), and DIC (13 mg, 0.10 mmol) were added sequentially and stirred at room temperature for 90 minutes. 0.1 mL of TFA was added and the mixture was allowed to react for an additional 30 minutes. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product HX-20 (32 mg, 25% yield). LCMS: (M+1) + 1176.28 (theoretical value: 1175.34).

[0148] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0149] Compound HX-20 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 9 (3.1 mg / mL, 2 mL). Average value calculated by UV-HPLC: n=7.3

[0150] Example 10: 7-[N-(IP140B-isonicotinoyl-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (10) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0151] Compound HX-20 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 10 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 10 (3.4 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.2.

[0152] Example 11: 7-[N-(HS627-Ac-PEG2-Val-Ala-PABC), N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (11) [ka] To a 10 mL single-neck flask, HX-16a (100 mg, 0.20 mmol), 0.5 mL of NMP, DIPEA (130 mg, 1 mmol), Fmoc-VA-PAB-PNP (138 mg, 0.20 mmol), and HOBt (27.4 mg, 0.20 mmol) were sequentially added and the mixture was stirred at room temperature for 4 hours to give HX-21a, which was directly used in the next step without further purification. LCMS: (M+1) + 1041.07 (theoretical value: 1040.38).

[0153] To the HX-21a reaction mixture, 0.5 mL of piperidine (V:V = 10%) was added and the mixture was allowed to react at room temperature for 30 minutes. 20 mL of methyl tert-butyl ether was added to the reaction mixture, and the mixture was centrifuged (5 minutes, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure to obtain the solid product HX-21b (123 mg, 75% yield). LCMS: (M+1) + 819.22 (theoretical value: 818.31).

[0154] To a 10 mL single-neck flask, DCM (1 mL), HX-17b (123 mg, 0.15 mmol), HX-3b (43.45 mg, 0.15 mmol), and DIC (18.9 mg, 0.15 mmol) were added sequentially and stirred at room temperature for 90 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-21 (44 mg, 26% yield). LCMS: (M+1) + 1098.21 (theoretical value: 1097.32).

[0155] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0156] Compound HX-21 (0.88 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 11 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0157] Example 12: 7-[N-(IP140B-Ac-PEG2-Val-Ala-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (12) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0158] Compound HX-21 (0.88 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 12 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.6.

[0159] Example 13: 7-[N-(HS627-Ac-PEG2-Gly-Leu-Gly-m),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycampto (13) [ka] To a 1000 mL single-neck flask, 500 mL of dichloromethane was added. With stirring, Fmoc-NH-PEG-2-CH2CH2-COOH (30 g, 75 mmol), HOBT (12.15 g, 90 mmol), EDCI (17.25 g, 90 mmol), glycine tert-butyl ester (10.32 g, 78.75 mmol), and DIPEA (10.16 g, 90 mmol) were sequentially added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with saturated aqueous sodium chloride (300 mL x 3), extracted sequentially with saturated sodium bicarbonate (300 mL x 3) and 0.5 M hydrochloric acid (300 mL x 3). The phases were separated, and the DCM phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to give product HX-22a (36.2 g, 94% yield). LCMS: (M+1) + 513.14 (theoretical value: 512.25).

[0160] In a 1000 mL single-neck flask, 400 mL of dichloromethane was added, and HX-22a (36.2 g, 70 mmol) was added. After stirring uniformly, 100 mL of trifluoroacetic acid was added dropwise, and the mixture was stirred at room temperature overnight. The mixture was then concentrated to give product HX-22b (32.2 g, 100% yield). LCMS: (M+1) + 457.08 (theoretical value: 456.19).

[0161] To a 1000 mL single-neck flask, 300 mL of dichloromethane was added. With stirring, HX-22b (32.2 g, 70 mmol), HOBT (11.34 g, 84 mmol), EDCI (16.10 g, 84 mmol), glycine tert-butyl ester (9.62 g, 73.5 mmol), and DIPEA (10.84 g, 84 mmol) were added sequentially. The mixture was stirred at room temperature overnight. The reaction mixture was washed with saturated aqueous sodium chloride (150 mL x 3), and then extracted sequentially with saturated sodium bicarbonate (150 mL x 3) and 0.5 M hydrochloric acid (150 mL x 3). The phases were separated, and the DCM phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to give product HX-22c (42 g, 95% yield). LCMS: (M+1) + 626.18 (theoretical value: 625.34).

[0162] A 1000 mL single-neck flask was charged with 400 mL of dichloromethane, HX-22c (42 g, 67.2 mmol), and stirred until uniform. 100 mL of trifluoroacetic acid was added dropwise, and the mixture was stirred at room temperature overnight. The reaction was then concentrated to give product HX-22d (38.23 g, 100% yield). LCMS: (M+1) + 570.14 (theoretical value: 569.27).

[0163] HX-22e (100 mg, 0.19 mmol) was dissolved in 5 mL of anhydrous DCM, HX-18a (665.3 mg, 1.9 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours, concentrated, and purified by silica gel column chromatography to give compound HX-22f (47 mg, 30% yield). LCMS: (M+1) + 826.19 (theoretical value: 825.26).

[0164] HX-22f (47 mg, 0.056 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to obtain HX-22g (21 mg, 60% yield). LCMS: (M+1) + 604.03 (theoretical value: 603.19).

[0165] HX-22g (21 mg, 0.034 mmol) was dissolved in 2 mL of DMF, DIPEA (4.49 mg, 0.034 mmol) was added, and the mixture was stirred uniformly. HX-22d (19.8 mg, 0.034 mmol) and HATU (13.23 mg, 0.034 mmol) were added sequentially, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated and purified by silica gel column chromatography to give compound HX-22h (35 mg, 87.5% yield). LCMS: (M+1) + 11555 (theoretical value: 1154.46).

[0166] HX-22h (35 mg, 0.03 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. 100 μL of piperidine was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-22i (22 mg, 78.9% yield). LCMS: (M+1) + 933.65 (theoretical value: 932.95).

[0167] DCM (3 mL), HX-22i (22 mg, 0.023 mmol), HX-3b (7 mg, 0.023 mmol), and DIC (2.97 mg, 0.023 mmol) were added sequentially to a 10 mL single-neck flask, and the mixture was stirred at room temperature for 90 minutes. The mixture was concentrated and purified by silica gel column chromatography to give the yellow solid product HX-22 (19 mg, 77% yield). LCMS: (M+1) + 1053.27 (theoretical value: 1052.31).

[0168] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0169] Compound HX-22 (0.84 mg, 0.8 mmol) was dissolved in 0.08 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 13 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.1.

[0170] Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Leu-Gly-m),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (14) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0171] Compound HX-22 (0.84 mg, 0.8 mmol) was dissolved in 0.08 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 14 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 14 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.0.

[0172] Example 15: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Phe-Gly-ha-Ac),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (15) [ka] To HX-22e (70 mg, 0.11 mmol), DMF (2 mL), HATU (41.8 mg, 0.11 mmol), DIPEA (14.19 mg, 0.11 mmol), and Fmoc-Gly-Gly-Phe-OH (55.11 mg, 0.11 mmol) were added, and the mixture was stirred at room temperature for 6 hours. The mixture was diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give yellow solid product HX-23a (108 mg, 85% yield). LCMS: (M+1) + 1087.25 (theoretical value: 1086.37).

[0173] HX-23a (108 mg, 0.099 mmol) was dissolved in 0.2 mL of DMF, followed by the addition of piperidine (77 mg, 0.99 mmol). The mixture was stirred at room temperature for 1 hour, and then 30 mL of methyl tert-butyl ether was added to the reaction mixture. The mixture was centrifuged (5 min, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure to give the solid product HX-23b (43 mg, 50% yield). This was used directly in the next reaction without further purification. LCMS: (M+1) + 865.29 (theoretical value: 864.31).

[0174] To a 10 mL single-neck flask, DCM (2 mL), HX-23b (43 mg, 0.049 mmol), HX-3b (15 mg, 0.049 mmol), and DIC (6.27 mg, 0.049 mmol) were added sequentially and stirred at room temperature for 90 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-23 (21 mg, 37.5% yield). LCMS: (M+1) + 1144.28 (theoretical value: 1143.32).

[0175] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0176] Compound HX-23 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 15 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.2.

[0177] Example 16: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Phe-Gly-ha-Ac),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (16) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0178] Compound HX-23 (0.92 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 16 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.5.

[0179] Example 17: 7-[N-(HS627-Ac-PEG2-Gly-Val-Ala-Gly-m),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (17) [ka] To HX-22e (70 mg, 0.11 mmol), DMF (2 mL), HATU (41.8 mg, 0.11 mmol), DIPEA (14.19 mg, 0.11 mmol), and Fmoc-Gly-Val-Ala-OH (51.37 mg, 0.11 mmol) were added, and the mixture was allowed to react at room temperature for 6 hours. The mixture was then diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give the yellow solid product HX-25a (100.1 mg, 87% yield). LCMS: (M+1) + 1053.06 (theoretical value: 1052.39).

[0180] HX-25a (100.1 mg, 0.095 mmol) was dissolved in 0.2 mL of DMF, piperidine (74 mg, 0.95 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 30 mL of methyl tert-butyl ether was added, and the mixture was centrifuged (5 min, 10,000 rpm). The supernatant was removed, and the solvent was removed under reduced pressure. The solid product HX-24b (49 mg, 62% yield) was dried and used directly in the next reaction. LCMS: (M+1) + 831.27 (theoretical value: 830.32).

[0181] To a 10 mL single-neck flask, DCM (2 mL), HX-24b (49 mg, 0.059 mmol), HX-3b (17.6 mg, 0.059 mmol), and DIC (7.43 mg, 0.059 mmol) were added sequentially and stirred at room temperature for 90 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-24 (15 mg, 22% yield). LCMS: (M+1) + 1110.28 (theoretical value: 1109.33).

[0182] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0183] Compound HX-24 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 17 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.3.

[0184] Example 18: 7-[N-(IP140B-Ac-PEG2-Gly-Val-Ala-Gly-m),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (18) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0185] Compound HX-24 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 18 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.6.

[0186] Example 19: 7-[N-(HS627-Ac-PEG2-Gly-Val-Ala-Gly-ha-Ac),N-(2',2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (19) [ka] HX-22e (50 mg, 0.096 mmol) was added to DMF (2 mL), HATU (36.7 mg, 0.096 mmol), DIPEA (25 mg, 0.193 mmol), and HX-17b (118 mg, 0.11 mmol). The mixture was allowed to react at room temperature for 15 hours, diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give a yellow solid, HX-25a (92 mg, 85% yield). LCMS: (M+1) + 1111.10 (theoretical value: 1110.39).

[0187] HX-25a (92 mg, 0.082 mmol) was dissolved in 0.2 mL of DMF, piperidine (64 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 30 mL of methyl tert-butyl ether was added, and the mixture was centrifuged (5 min, 10,000 r / min). The supernatant was removed, and the solvent was removed under reduced pressure. The solid product HX-25b (52 mg, 71% yield) was dried and used directly in the next reaction. LCMS: (M+1) + 889.72 (theoretical value: 888.86).

[0188] To a 10 mL single-neck flask, DCM (2 mL), HX-25b (52 mg, 0.058 mmol), HX-3b (17.74 mg, 0.058 mmol), and DIC (7.4 mg, 0.058 mmol) were added sequentially and stirred at room temperature for 90 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-25 (15 mg, 22% yield). LCMS: (M+1) + 1168.27 (theoretical value: 1167.34).

[0189] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0190] Compound HX-25 (0.93 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. After thorough mixing, the mixture was reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 19 (3.0 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.3.

[0191] Example 20: 7-[N-(IP140B-Ac-PEG2-Gly-Val-Ala-Gly-ha-Ac),N-(2',2',2'-trifluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (20) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0192] Compound HX-25 (0.93 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 20 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0193] Example 21: 20-O-(HS627-Ac-PEG2-Gly-Val-Ala-Gly-NHNHC)-7-N-(2',2',2'-trifluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin (21) [ka] To a 50 mL single-neck flask, HX-22e (200 mg, 0.386 mmol) and 3 mL of dichloromethane were added, followed by Boc anhydride (92.8 mg, 0.425 mmol) and TEA (86.1 mg, 0.851 mmol), and the mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give the pale yellow solid product HX-28a (213.2 mg, 89.3% yield). LCMS: (M+1) + 618.20 (theoretical value: 617.58).

[0194] To a 50 mL single-neck flask, HX-28a (100 mg, 0.162 mmol), DMAP (98.9 mg, 0.81 mmol), and 2 mL of dichloromethane were added sequentially and stirred at 0 °C for 5 minutes. 2 mL of dichloromethane and triphosgene (38.6 mg, 0.131 mmol) were added and the mixture was stirred at room temperature for 2 hours to obtain a clear yellow solution of HX-28b. HX-26c (82.3 mg, 0.324 mmol) was added and the mixture was stirred at room temperature for 3 hours. The mixture was purified by gel column chromatography to obtain a pale yellow solid product, HX-28d (123.5 mg, 85.3% yield). LCMS: (M+1) + 898.29 (theoretical value: 897.27).

[0195] HX-28d (100 mg, 0.11 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. Piperidine (94.8 mg, 1.1 mmol) was added and the mixture was allowed to react at room temperature for 0.5 hours. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-28e (65.5 mg, 87% yield). LCMS: (M+1) + 676.22 (theoretical value: 675.22).

[0196] HX-28e (50 mg, 0.074 mmol) was added with 2 mL of DMF, HATU (33.8 mg, 0.089 mmol), DIPEA (11.5 mg, 0.089 mmol), and Fmoc-Gly-Val-Ala-Gly-OH (38.8 mg, 0.074 mmol). The mixture was allowed to react at room temperature for 2 hours, diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give a yellow solid, HX-28f (51.3 mg, 58.7% yield). LCMS: (M+1) + 1181.19 (theoretical value: 1180.44).

[0197] HX-28f (40 mg, 0.034 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. Piperidine (28.7 mg, 0.34 mmol) was added and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added, the mixture was centrifuged, the supernatant was removed, and the mixture was dried to give HX-28g (25.8 mg, 79.5% yield). LCMS: (M+1) + 958.37 (theoretical value: 959.37).

[0198] To a 10 mL single-neck flask, DCM (1 mL), HX-28g (20.2 mg, 0.021 mmol), HX-3b (7.6 mg, 0.026 mmol), and DIC (3.2 mg, 0.026 mmol) were added sequentially and stirred at room temperature for 30 minutes to give a clear yellow solution of HX-28h. 100 μL of trifluoroacetic acid was added and stirred at room temperature for 30 minutes. The reaction solution was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The solution was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product HX-28 (4.1 mg, 17% yield). LCMS: (M+1) + 1138.95 (theoretical value: 1137.33).

[0199] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0200] Compound HX-28 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 21 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.1.

[0201] Example 22: 20-O-(IP140B-Ac-PEG2-Gly-Val-Ala-Gly-NHNHC)-7-N-(2',2',2'-trifluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin (22) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0202] Compound HX-28 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 22 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.0.

[0203] Example 23: 20-O-(HS627-Ac-PEG4-daP-NHNHC)-7-N-(2',2',2'-trifluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin (23) [ka] To the clear yellow solution of HX-28b, HX-27a (113.8 mg, 0.274 mmol) was added directly, and the mixture was stirred at room temperature for 3 hours. The resulting mixture was purified by gel column chromatography to give a pale yellow solid, HX-29a (125.6 mg, 86.7% yield). LCMS: (M+1) + 1060.05 (theoretical value: 1059.36).

[0204] HX-27b (125.6 mg, 0.118 mmol) was added to a 10 mL single-neck flask, dissolved in 1 mL of DMF, piperidine (100.9 mg, 1.18 mmol) was added, and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added, the mixture was centrifuged, the supernatant was removed, and the mixture was dried to give HX-29b (93.5 mg, 94.2% yield). LCMS: (M+1) + 838.30 (theoretical value: 837.29).

[0205] To a 10 mL single-neck flask, DCM (2 mL), HX-29b (70 mg, 0.084 mmol), HX-4a (37.3 mg, 0.1 mmol), and DIC (12.6 mg, 0.1 mmol) were added sequentially and stirred at room temperature for 30 minutes to give a clear yellow solution, HX-29c. 200 μL of trifluoroacetic acid was added and stirred at room temperature for 30 minutes. The reaction solution was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The solution was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product, HX-29 (20.2 mg, 22.1% yield). LCMS: (M+1) + 1092.29 (theoretical value: 1091.89).

[0206] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0207] Compound HX-29 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 23 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.3.

[0208] Example 24: 20-O-(IP140B-Ac-PEG4-daP-NHNHC)-7-N-(2',2',2'-trifluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin (24) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0209] Compound HX-29 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 24 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0210] Example 25: 7-[N-(HS627-Qx-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (25) [ka] N-(2',2'-difluoroethyl)aminoethyl-10,11-methylenedioxycamptothecin (0.05 g, 0.1 mmol), NMP (1 mL), DIPEA (25.9 mg, 0.2 mmol), Fmoc-GK-PAB-PNP (93.9 mg, 0.1 mmol), and HOBt (20.3 mg, 0.15 mmol) were added and reacted at room temperature for 4 hours with stirring. The resulting HX-36a reaction solution was directly added to the next step. LCMS: (M+1) + 1298.41 (theoretical value: 1297.50).

[0211] HX-36a (130 mg, 0.1 mmol) and piperidine (85.3 mg, 1 mmol) were added to a 10 mL reaction flask, and the mixture was stirred at room temperature for 1 hour. 30 mL of methyl tert-butyl ether was added, the mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure. The resulting solid product, HX-36b (78.2 mg, 72.6% yield), was used directly in the next reaction. LCMS: (M+1) + LCMS:(M+1) + 1076.39 (theoretical value: 1075.43).

[0212] To a 10 mL single-neck flask, DCM (2 mL), HX-36b (78.2 mg, 0.073 mmol), 2-bromomethyl-3-oxo-dihydroquinoline-6-formyl-3,6-oxaoctanoic acid (38.6 mg, 0.087 mmol), and DIC (11 mg, 0.087 mmol) were added sequentially and stirred at room temperature for 90 min. 0.1 mL of TFA was added and the reaction mixture was further stirred for 30 min. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile and then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-36 (46.3 mg, 50.5% yield). LCMS:(M+1)1258.10 + (Theoretical value: 1256.33).

[0213] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0214] Compound HX-36 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 25 (3.2 mg / mL, 2 mL).

[0215] Mean value calculated by UV-HPLC: n = 6.3. Example 26: 7-[N-(IP140B-Qx-PEG2-Gly-Lys-PABC),N-(2',2'-difluoroethyl)]aminoethyl-10,11-methylenedioxycamptothecin (26) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0216] Compound HX-36 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 26 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.6.

[0217] Test Example 1: In vitro tumor cell proliferation inhibitory activity of ADC Test method for in vitro inhibitory activity of ADC: Human esophageal cancer cells OE-33, human lung cancer cells NCI-H1975, and human breast cancer cells MDA-MB-231 used for activity detection were cultured in RPMI1640 (Cellmax), RPMI1640 (Cellmax), and DMEM (Cellmax) media containing 10% fetal bovine serum until exponential growth phase. After trypsin digestion, the supernatant was discarded by centrifugation, and 3 × 10 cells were cultured in the media. 4 cells / mL, 0.5×10 4 cells / mL, 1.5×10 4The ADCs were diluted to 1000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, and 0.026 nM in medium. 100 μL of the diluted ADCs were added to each well of a 96-well cell culture plate. Each well was replicated for each concentration. For negative and blank controls, 100 μL of culture medium was added per well. After incubation, the plates were placed in a 37°C, 5% CO2 incubator for 6 days. After incubation, the cell culture plate was removed, the medium in the plate was aspirated and removed with a pipette, and then 100 μL of medium containing 10% CCK-8 was added per well and incubated at 37°C for 3 hours. After incubation, the culture plate was removed, shielded from light, and placed on an enzyme plate. The absorbance was measured using a reference wavelength of 630 nm and a measurement wavelength of 450 nm. IC 50 was calculated from the absorbance values ​​using a four-parameter regression in GraphPad (Table 2). The ADC drug corresponding to Dxd was used as a positive control drug and has the following structure: [ka] (where Ab is IP140B antibody.) [Table 1] I C 50 For values, "++++" is IC 50 <50 nM, and "++++" indicates IC 50 indicates that the IC is between 10 and 100 nM, and "++" indicates that the IC 50 indicates that the IC is between 100 and 250 nM, and "+" indicates that the IC 50 Indicates that the value is >250nM.

[0218] The ADC compounds in the examples of the present invention have excellent inhibitory activity against NCI-H1975, MDA-MB-231, and OE-33 cells, and the IC50 All of the values ​​were below 250 nM, and some ADC compounds, such as ADC2, 14, 16, and 18, had IC values ​​of inhibitory activity against these cancer cells. 50 The value is even less than 50 nM.

[0219] Test Example 2: In vivo tumor growth inhibitory activity of ADC Test method for in vitro inhibitory activity of ADC: Human lung cancer cells NCI-H292, human non-small cell lung cancer cells Calu-6, human lung cancer cells NCI-H1975, human esophageal squamous cell carcinoma cells KYSE-150, human ovarian cancer cells ES-2, and human breast cancer cells MDA-MB-231 were cultured in vitro as monolayers. When the cell density reached 80-90%, the cells were digested with trypsin-EDTA, centrifuged, and the supernatant discarded. The cells were resuspended in PBS and adjusted to the appropriate cell concentration. NCI-H292, Calu-6, NCI-H1975, KYSE-150, and MDA-MB-231 cells (2-10 × 10) were cultured in vitro as monolayers. 6 BALB / c nude mice were subcutaneously inoculated with ES-2 cells (0.1 mL / cells), and NOD-SCID mice were subcutaneously inoculated with ES-2 cells. The animals and tumor growth were monitored regularly until the tumor volume reached 100–200 mm. 3 Once the tumors reached a certain size, they were randomly divided into two groups based on tumor volume and body weight: a vehicle control group (physiological saline) and an ADC-treated group (dissolved in physiological saline), with six animals in each group. The tumors were administered intravenously. The administration frequency was Q4D, with a total of two doses (the first dose was on Day 1, and the second on Day 5). The tumor's major axis (a) and minor axis (b) were measured twice weekly using calipers, along with the mouse's body weight. Tumor volume (V) was calculated using the following formula: V = 1 / 2 × a × b. 2 (mm 3), where a and b represent the length and width of the tumor, respectively. A growth curve was then created, and the tumors were finally excised and weighed. Statistical analysis was performed using GraphPad Prism software using the tumor volume and weight data of the tumor-bearing mice at the end of the experiment to obtain the results of the antitumor effect. In the figure, "" in the control group indicates that there was no result for that group or that the tumor status was not measured. In the experimental group, "no tumor" in the corresponding group, "tumor shrinkage to 0," and "tumor disappearance to 0" indicate that no tumor was found in the corresponding dissected animal. [Table 2-1] [Table 2-2]

[0220] The foregoing description of specific exemplary embodiments of the present invention has been presented for purposes of explanation and illustration. It is not intended that these descriptions be construed as limiting the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary examples is to explain certain principles of the present invention and their practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention, as well as various alternatives and modifications. It is intended that the scope of the present invention be limited by the claims and their equivalents.

Claims

1. An antibody-drug conjugate as shown in formula (I): 【Chemistry 1】 (In the formula, R １ is selected from halogen-substituted methyl groups; R ａ is hydrogen and -L １ -NH-L ｐ -Z-Ab, R ｂ is hydrogen, -L ２ -NH-L ｐ -Z-Ab and -L ３ -NH-Z-Ab, with the proviso that R ａ and R ｂ is not hydrogen at the same time, where: L １ is -C(=O)-L １１ -L １２ -L １３ - and -CH ２ -NR ２ -L １４ -CHR ３ - is selected from, L １ , L １２ , and L １３ are each independently —O—, C １ ~C ３ and a phenyl group, R ２ is hydrogen, deuterium, C １ ~C ６ Alkyl group, C １ ~C ６ selected from an alkoxy group, an acyl group, and a sulfonyl group; L １４ is -C(=O)- and C １ ~C ３ alkylene groups, R ３ is hydrogen, deuterium, C １ ~C ６ Alkyl group, C １ ~C ６ Alkoxy groups, and phenyl-substituted C １ ~C ６ alkyl groups, L ２ is -C(=O)-NH-L ２１ -CHR ４ - is selected from, L ２１ is selected from —C(═O)— and —NH—C(═O)—; R ４ is hydrogen, deuterium, C １ ~C ６ Alkyl groups, and phenyl-substituted C １ ~C ６ alkyl groups, L ３ is -C(=O)-NR ５ -NR ６ -L ３１ -L ３２ - is selected from, R ５ and R ６ are each independently hydrogen, deuterium, and C １ ~C ６ alkyl groups, L ３１ is -C(=O), -(C １ ~C ３ alkylene)-C(=O), -5- to 8-membered aromatic ring group -C(=O)-, -5- to 8-membered nitrogen heteroaromatic ring group -C(=O)-, and -O-(C １ ~C ３ alkylene)-C(═O)—; L ３２ is -NH-(C １ ~C ３ alkylene), -N(CH ３ ) ２ -(C １ ~C ３ alkylene) and —NH—(C １ ~C ３ alkylene)-NH—; L Ｐ is a peptide residue consisting of 2 to 7 amino acids, Z is -L ｚ -L ｊ -, where L ｚ is -C(=O)-C １ ~C ８ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～５ -CH ２ CH ２ NH—, or —C(═O)—(CH ２ CH ２ O) ２～６ -CH ２ CH ２ NH—, and L ｊ is a linker capable of being attached to an antibody, n １ is selected from integers from 1 to 3, Ab is an antibody.

2. R １ is selected from fluorine, chlorine or bromine substituted methyl groups; Preferably, R １ is selected from fluorine-substituted methyl groups; Preferably, R １ is selected from a fluoromethyl group, a difluoromethyl group, and a trifluoromethyl group; Preferably, R １ is selected from a difluoromethyl group and a trifluoromethyl group; Preferably, n １ is 1, The antibody-drug conjugate of claim 1.

3. L １１ , L １２ , and L １３ are each independently selected from —O—, a methylene group, and a phenyl group; Preferably, L １１ -L １２ -L １３ is -O-CH ２ -phenyl- and -CH ２ -O-CH ２ - is selected from, Preferably, R ２ is hydrogen, deuterium, C １ ~C ６ Alkyl group, C １ ~C ６ Alkoxy group, —C(═O)C １ ~C ６ Alkyl groups, and -S(=O) ２ C １ ~C ６ alkyl groups, Preferably, R ２ is hydrogen, deuterium, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, —C(═O)C １ ~C ３ Alkyl groups, and -S(=O) ２ C １ ~C ３ alkyl groups, Preferably, R ２ is selected from hydrogen, deuterium, methyl, ethyl, methoxy, ethoxy, formyl, acetyl, methanesulfonyl, and ethanesulfonyl; Preferably, R ２ is selected from hydrogen, methyl, methoxy, formyl, and methanesulfonyl; Preferably, R ２ is hydrogen, Preferably, L １４ is -C(=O)- and C １ ~C ３ alkylene groups, Preferably, L １４ is selected from —C(═O)— and a methylene group; Preferably, R ３ is hydrogen, deuterium, C １ ~C ６ Alkyl group, C １ ~C ６ Alkoxy groups, and phenyl-substituted C １ ~C ６ alkyl groups, Preferably, R ３ is hydrogen, C １ ~C ６ Alkyl groups, and phenyl-substituted C １ ~C ６ alkyl groups, Preferably, R ３ is C １ ~C ４ Alkyl groups, and phenyl-substituted C １ ~C ３ alkyl groups, Preferably, R ３ is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenylmethyl, and phenylethyl; Preferably, R ３ is selected from hydrogen, methyl, isopropyl, isobutyl, and phenylmethyl; Preferably, R ３ is hydrogen, Preferably, L １ is -C(=O)-O-CH ２ -phenyl-, -C(=O)-CH ２ -O-CH ２ -, -CH ２ -NH-C(=O)-CH ２ - and -CH ２ -NH-CH ２ -CH ２ - is selected from, Preferably, L １ is C(=O)-O-CH ２ -phenyl-, -C(=O)-CH ２ -O-CH ２ - and -CH ２ -NH-C(=O)-CH ２ - is selected from, Preferably, L １ is -C(=O)-O-CH ２ -phenyl- and -C(=O)-CH ２ -O-CH ２ - selected from The antibody-drug conjugate according to claim 1 or 2.

4. R ４ is hydrogen, deuterium, C １ ~C ６ Alkyl groups, and phenyl-substituted C １ ~C ６ alkyl groups, Preferably, R ４ is hydrogen, deuterium, C １ ~C ３ Alkyl groups, and phenyl-substituted C １ ~C ３ alkyl groups, Preferably, R ４ is selected from hydrogen, deuterium, methyl, isopropyl, isobutyl, and phenylmethyl; Preferably, R ４ is hydrogen, Preferably, L ２ is -C(=O)-NH-C(=O)-CH ２ - and -CH ２ -NH-NH-C(=O)-CH ２ - is selected from, Preferably, L ２ is -CH ２ -NH-NH-C(=O)-CH ２ - and Preferably, R ５ and R ６ are each independently hydrogen, deuterium, and C １ ~C ３ alkyl groups, Preferably, R ５ and R ６ are each independently selected from hydrogen, deuterium, and a methyl group; Preferably, R ５ is hydrogen, R ６ is hydrogen, Preferably, L ３１ is —C(═O), -methylene-C(═O), -phenyl-C(═O)—, -pyridyl-C(═O)— and —O—CH ２ -C(=O)-; Preferably, L ３２ is -NH-CH ２ -CH ２ -, -N(CH ３ ) ２ -CH ２ -CH ２ - and -NH-CH ２ -NH-; Preferably, L ３ is -C(=O)-NH-NH-phenyl-C(=O)-NH-CH ２ -CH ２ -is, The antibody-drug conjugate according to any one of claims 1 to 3.

5. the amino acids are selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn); Preferably, L ｐ is selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn); Preferably, L ｐ is selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu) and lysine (Lys); Preferably, L ｐ is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe- and -Gly-Gly-Phe-Gly-; Preferably, L ｐ is selected from the group represented by the formula: 【Chemistry 2】 The carbonyl group end is linked to —NH—, and the other end is linked to Z; Preferably, L ｊ is selected from the group represented by the formula: 【Transformation 3】 【Chemistry 4】 The position indicated by indicates that it is linked to an antibody, 【Transformation 5】 The position indicated by L Ｚ represents being linked to a group, Preferably, L ｚ is -C(=O)-C １ ~C ８ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～５ -CH ２ CH ２ NH— and —C(═O)—(CH ２ CH ２ O) ２～６ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-C １ ~C ６ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～３ -CH ２ CH ２ NH— and —C(═O)—(CH ２ CH ２ O) ２～４ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-(CH ２ CH ２ O) ２ -CH ２ CH ２ NH-, -C(=O)-CH ２ O-CH ２ CH ２ O-CH ２ CH ２ NH— and —C(═O)—CH ２ O-(CH ２ CH ２ O) ３ -CH ２ CH ２ NH; Preferably, Z is selected from the group represented by the formula: 【Transformation 6】 【Transformation 7】 The position indicated by indicates that it is linked to an antibody, 【Transformation 8】 The position indicated by L ｐ group or L ３ represents a bond to the group, The antibody-drug conjugate according to any one of claims 1 to 4.

6. the antibody is a tumor-associated antigen antibody, Preferably, the tumor-associated antigen antibody is selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody. The antibody-drug conjugate according to any one of claims 1 to 5.

7. The antibody-drug conjugate represented by formula (I) has a structure represented by formula (I-1), formula (I-2), formula (I-3), formula (I-4), formula (I-5) or formula (I-6), 【Chemistry 9】 【change】 In formula (I-1), formula (I-2), formula (I-3), and formula (I-4), R １ , R ２ , n １ , L ｐ , Z, and Ab, when present, are each defined as in compounds of formula (I); 【Chemistry 10】 In formula (I-5), R １ , R ５ , R ６ , n １ and Ab are each defined as in the compound of formula (I), n ２ is selected from 1 or 2, Z is -L ｚ -L ｊ’ wherein L is selected from ｊ is selected from the group represented by the formula: 【Chemistry 11】 【Chemistry 12】 The position indicated by indicates that it is linked to an antibody, 【Chemistry 13】 The position indicated by L Ｚ represents being linked to a group, Preferably, L ｚ is -C(=O)-CH ２ O-(CH ２ CH ２ O) １～５ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-CH ２ O-(CH ２ CH ２ O) ３～５ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-CH ２ O-(CH ２ CH ２ O) ３ -CH ２ CH ２ NH-; Preferably, Z is 【Chemistry 14】 is selected from 【Chemistry 15】 The position indicated by indicates that it is linked to an antibody, 【Chemistry 16】 The position indicated by L ｐ group or L ３ represents being linked to a group, 【Chemistry 17】 In formula (I-6), R １ , R ５ , R ６ , n １ , L ｐ , Z, and Ab are each defined as in the compound of formula (I), The antibody-drug conjugate according to any one of claims 1 to 6.

8. An antibody-drug conjugate comprising: [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 wherein Ab is defined as in the compound of formula (I), and preferably Ab is an HS627 antibody or an IP140B antibody, the heavy chain amino acid sequence of HS627 is represented by SEQ ID NO: 1, the light chain amino acid sequence of IP140B antibody is represented by SEQ ID NO: 2, and the heavy chain amino acid sequence of IP140B antibody is represented by SEQ ID NO: 3, and the light chain amino acid sequence of IP140B antibody is represented by SEQ ID NO:

4.

9. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of the antibody-drug conjugate of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 in the preparation of an antitumor drug, Preferably, the tumor is selected from solid tumors, more preferably selected from esophageal cancer, lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, ovarian cancer, esophageal adenocarcinoma, lung cancer or breast cancer.

11. A method for treating a tumor disease, comprising the step of administering to a patient in need thereof the antibody-drug conjugate of any one of claims 1 to 8 or the pharmaceutical composition of claim 9.

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