Antibody-drug conjugate having linking system

Abstract

Provided are an antibody-drug conjugate having a fixed-point linking system, a preparation method therefor, and a use thereof. The linking system has high linking stability and enhanced solubility, allows efficient coupling of hydrophobic drugs, and effectively implements drug activity and selectivity.

Description

Antibody-drug conjugates with linker systems

[0001] This application requests the following:

[0002] Priority is claimed for the earlier application filed with the China National Intellectual Property Administration on October 30, 2024, with patent application number 202411531454.1 and title "Antibody-Drug Conjugate with Linkage System".

[0003] Priority is claimed for the earlier application filed with the China National Intellectual Property Administration on November 27, 2024, with patent application number 202411717146.8 and title "Antibody-Drug Conjugate with Linkage System";

[0004] The full text of the prior application is incorporated herein by reference. Technical Field

[0005] This invention belongs to the field of pharmaceuticals, specifically relating to a class of antibody-drug conjugates with a linker system. Background Technology

[0006] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active drugs via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of both normal and tumor cells, as well as the high efficacy of drugs, while avoiding the drawbacks of lower efficacy with antibodies and excessive toxicity with drugs. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0007] Microtubules are powerful, filamentous cytoskeletal proteins involved in a wide range of cellular functions, including intracellular migration and transport, cell signaling, and maintaining cell shape. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, necessary for chromosomes to divide into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization kinetics, which occur through the reversible, non-covalent addition of α- and β-tubulin dimers to the ends of the microtubules. This kinetic behavior and the resulting control over microtubule length are essential for the proper functioning of the mitotic spindle. Even minor alterations in microtubule kinetics can involve axon checkpoints, inhibiting cell cycle progression during mitosis and subsequently leading to cell death. Because cancer cells divide rapidly, they are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Tubulin inhibitors, such as MMAE and eribulin, hold promise as potential drugs for cancer treatment.

[0008] Eribulin is a spongin-like microtubule dynamics inhibitor with unique binding properties. Besides its mechanism of action of inhibiting microtubule dynamic growth, non-clinical studies have shown that eribulin has unique effects on the tumor microenvironment, such as increasing vascular perfusion and permeability of the tumor core, promoting epithelial status, and reducing the migration ability of breast cancer cells. Currently, eribulin has been approved in more than 70 countries and regions, including Europe, the Americas, and Asia, for the treatment of breast cancer. The most common grade 3-4 adverse reaction in eribulin mesylate treatment is neutropenia (64%). Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, combining the tumor-targeting activity of antibodies with the activity of bioactive molecules, becoming a kind of biological missile with very promising efficacy and safety advantages. Antibodies guide ADCs to bind to target cells, which are then internalized. The small molecule drug is then released intracellularly through enzymatic decomposition under the action of specific enzymes, treating the disease. Therefore, it is expected that the ADC composed of eribulin and tumor-targeting antibodies can maintain the therapeutic effect of eribulin while eliminating or reducing the toxic side effects caused by eribulin acting on non-disease tissues, thereby improving the therapeutic effect. Summary of the Invention

[0009] The present invention also provides an antibody-drug conjugate of formula (I) or a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound thereof: Ab-[Link-(D)2] β (I)

[0010] Where Ab (i.e., the target ligand) is the antibody or its antigen-binding fragment, D is the structural fragment of the bioactive molecule, Link is the linker part connecting the target ligand Ab and the bioactive molecule D; β is selected from integers or decimals between 1 and 10;

[0011] Wherein, Link is B-(MZ-Tr-L)2;

[0012] Furthermore, the Link structure is illustrated below:

[0013] Where B represents the portion of Link that is connected to the target ligand Ab, # represents the connection site between Link and the target Ab, and * represents the connection site between Link and the bioactive molecule D.

[0014] B is selected from the structure shown below:

[0015] # represents the connection site between B and the target part Ab. B is formed by the reactive group B' and the target group Ab (specifically, it is formed by the reactive group B' B1' and the target group).

[0016] **Represents the site where B and M are connected; B2 is formed by reactive groups B2' and M'; B3 is formed by reactive groups B3' and M'; M is formed by reactive group M' and intermediate B”.

[0017] Wherein, B0 is selected from unsubstituted or optionally substituted by one, two or more Rs. B The following groups are substituted: amino acid residues (e.g., lysine residues) C 6-14 Aryl, 3-14 heterocyclic; each R B They are the same or different, and are independently selected from -C(O)CH2-(OCH2CH2) b -OCH3; b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0018] B1 is selected from unsubstituted or arbitrarily assigned to one, two or more R... B1 The following groups are substituted: -C(O)CH2-(OCH2CH2) b11 -NH-#、-NHC(O)CH2-(OCH2CH2) b11 -NH-#、 Each R B1 Same or different, selected independently from C 1-10 Alkyl group; b11 and b12 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5 or 6;

[0019] B1' is selected from unsubstituted or optionally by one, two or more R's. B1 The following groups are substituted: -C(O)CH2-(OCH2CH2) b11 -NH2、-NHC(O)CH2-(OCH2CH2) b11 -NH2、 Preferably, the amino group in B1' is optionally protected by an amino protecting group commonly used in the art, such as Fmoc.

[0020] B2 and B3 may be the same or different, and are independently selected from each other without substitution or arbitrarily selected by one, two or more Rs. B2 The following groups are substituted: Each R B2 Same or different, selected independently from C 1-10 Alkyl group; b21, b22 and b23 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5 or 6;

[0021] B2' and B3' may be the same or different, and are independently selected from each other without substitution or arbitrarily selected by one, two or more R's. B2The following groups are substituted:

[0022] M is selected from Each R 21 R 22 R 23 Whether the two are the same or different, they are selected independently from H and C. 1-6 Alkyl, C 1- 10 Alkyl group; t is selected from 1, 2, 3, 4, 5 or 6; m1 is selected from 0, 1, 2, 3 or 4; m2 is selected from 0, 1, 2, 3 or 4; m3 is selected from 0, 1 or 2; reactive group M' is selected from...

[0023] Z is selected from single bond, -N(R) z )-(CH2)n1-C(=O)-, -N(R1)-(CH2CH2O)z1-(CH2)n2-C(=O)-, Among them, ring A is selected from cyclohexane ring or six-membered heterocycle;

[0024] R1, R z They are either the same or different, and are independently selected from H, -(CH2CH2O)z3-CH3,

[0025] z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers from 1 to 36;

[0026] n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5, and 6;

[0027] Tr is a divalent triggering group selected from glycine-glycine-phenylalanine-glycine (GGFG, SEQ ID NO.1), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG, SEQ ID NO.2), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG, SEQ ID NO.3), and lysine-glycine-glycine-phenylalanine-glycine (KGGFG, SEQ ID NO.4).

[0028] Tr is formed by Tr' and reactive group M'; Tr' is a monovalent triggering group, the N-terminus of which is NH2 and the C-terminus is connected to L, and is selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), lysine-glycine-glycine-phenylalanine-glycine (KGGFG);

[0029] L is the linker between Tr and the bioactive molecular structural fragment D, formed by the reaction of the reactive group L' with the bioactive molecule D. L is selected from single bonds, -NH-C... 1-10 Alkyl-, -N(CH3)-C 1-10 Alkylene-

[0030] The L' is selected from single bonds, -NH-C 1-10 Alkyl-X, -N(CH3)-C 1-10 Alkylene-X, X is selected from leaving groups, such as halogens.

[0031] According to an embodiment of the present invention, β can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or from 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9; β can be selected from an integer or a decimal of 1-3.

[0032] According to embodiments of the present invention, Ab is an antibody or an antigen-binding fragment, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb; and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody.

[0033] According to an embodiment of the present invention, Ab is an anti-HER2 antibody or its antigen-binding fragment.

[0034] According to an embodiment of the present invention, the Ab is trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0035] According to an embodiment of the present invention, the heavy chain constant region of the Ab is deglycosylated or mutated to remove N-glycosylation.

[0036] According to an embodiment of the present invention, the Ab is modified by mutating amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region to A (alanine, Alanine, Ala) to remove N-glycosylation.

[0037] According to an embodiment of the present invention, the Ab is a pertuzumab with a mutation of amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region to A (alanine, Alanine, Ala), for example, the amino acid sequence of its heavy chain constant region is shown in SEQ ID NO.7; preferably, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO.6, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.7, the sequence of the light chain variable region is shown in SEQ ID NO.8, and the sequence of the light chain constant region is shown in SEQ ID NO.9.

[0038] According to an embodiment of the present invention, D is selected from structural fragments of an antitumor compound.

[0039] According to an embodiment of the present invention, D is selected from the structural fragment of the dehydrogenated antitumor compound.

[0040] According to an embodiment of the present invention, the antitumor compound is selected from eribulin.

[0041] According to an embodiment of the present invention, D is selected from...

[0042] According to an embodiment of the present invention, the Link is selected from the following structures:

[0043] The left amino linker is connected to the antibody portion Ab, and the right carbonyl linker is connected to the drug portion D.

[0044] According to embodiments of the present invention, B0 is selected from unsubstituted or optionally replaced by one, two or more R. B The following groups are substituted: C 3-8 Alkyl, C 6-10 Aryl, 6-12 heterocyclic;

[0045] According to an embodiment of the present invention, B0 is selected from...

[0046] According to an embodiment of the present invention, R BIt is -C(O)CH2-(OCH2CH2)6-OCH3.

[0047] According to an embodiment of the present invention, B1 is selected from -C(O)CH2-(OCH2CH2)3-NH-#, -NHC(O)CH2-(OCH2CH2)3-NH-#,

[0048] According to an embodiment of the present invention, B1' is selected from -C(O)CH2-(OCH2CH2)3-NH2, -NHC(O)CH2-(OCH2CH2)3-NH2,

[0049] According to an embodiment of the present invention, B2 and B3 may be the same or different, and are independently selected from each other.

[0050] According to an embodiment of the present invention, B2' and B3' may be the same or different, and are independently selected from each other.

[0051] According to an embodiment of the present invention, B is selected from:

[0052] According to an embodiment of the present invention, R 21 R 22 R 23 Selected from H.

[0053] According to an embodiment of the present invention, t is selected from 2, 3 or 4.

[0054] According to an embodiment of the present invention, M is selected from...

[0055] According to an embodiment of the present invention, M' is selected from...

[0056] According to an embodiment of the present invention, ring A is selected from...

[0057] According to an embodiment of the present invention, R z Selected from the following groups: -(CH2CH2O)z3-CH3,

[0058] According to an embodiment of the present invention, R z Selected from the following groups: -(CH2CH2O)9-CH3, -(CH2CH2O) 13 -CH3、

[0059] According to an embodiment of the present invention, z1, z2, z3, z4, and z5 may be the same or different, and are independently selected from integers of 6-15.

[0060] According to an embodiment of the present invention, z1, z2, z3, z4, and z5 may be the same or different, and are independently selected from integers of 8-14.

[0061] According to an embodiment of the present invention, n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, and 3.

[0062] According to an embodiment of the present invention, Z is selected from single bonds, -N(R) z )-(CH2)n1-C(=O)-, -NH-(CH2CH2O)z1-(CH2)n2-C(=O)-, Wherein, X1 and X2 may be the same or different, and are independently selected from CH or N; preferably, at least one of X1 and X2 is an N atom; R z z1, z2, n1, n2, n3, and n4 have the definitions described herein.

[0063] According to an embodiment of the present invention, when X2 is CH, n4 is selected from 0.

[0064] According to an embodiment of the present invention, Z is selected from single bonds, -NH-(CH2CH2O)8-CH2CH2-C(=O)-, and -NH-(CH2CH2O). 10 -CH2CH2-C(=O)-、-N(R z -CH2-C(=O)-、-N(R) z )-(CH2)2-C(=O)-、-N(R z )-(CH2)3-C(=O)-、-N(R z )-(CH2)4-C(=O)-、 Among them, R z It has the definition described in this article.

[0065] According to an embodiment of the present invention, Z is selected from single bonds,

[0066] According to an embodiment of the present invention, Tr is selected from glycine-glycine-phenylalanine-glycine (GGFG), i.e.

[0067] According to an embodiment of the present invention, Tr is selected from valine-alanine (VA), that is:

[0068] According to an embodiment of the present invention, Tr' is selected from

[0069] According to an embodiment of the present invention, L is selected from a single bond.

[0070] According to an embodiment of the present invention, L is selected from

[0071] According to an embodiment of the present invention, the antibody-drug conjugate is selected from:

[0072] Among them, Ab and β have the definitions above;

[0073] According to an embodiment of the present invention, the antibody-drug conjugate is selected from:

[0074] Wherein, β has the definition as above.

[0075] According to an embodiment of the present invention, the antibody-drug conjugate is selected from the compounds in the examples: Pertuzumab(A)-L001-001, Pertuzumab(A)-L001-002, Pertuzumab(A)-L002-001, Pertuzumab(A)-L002-002, Pertuzumab(A)-L002-011, Pertuzumab(A)-L002-012, Pertuzumab(A)-L002-013, Pertuzumab(A)-L002-014, Pertuzumab(A)-L002-015 and / or Pertuzumab(A)-L002-016.

[0076] The present invention also provides an intermediate B”, the structure of which is as follows: Among them, B1', B0, B2', and B3' are as defined above.

[0077] According to an embodiment of the present invention, the intermediate B” is selected from:

[0078] The present invention also provides a connector “Link”, the structure of which is: B'-(MZ-Tr-L')2;

[0079] Where B' is

[0080] Wherein, B1', B0, B2, B3, M, Z, Tr, and L' have the definitions described herein independently of each other;

[0081] According to an embodiment of the present invention, B' is selected from:

[0082] According to an embodiment of the present invention, the "Link" is selected from the following structures:

[0083] The present invention also provides a linker-drug or its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound represented by formula (II), having the structure: B'-(MZ-Tr-LD)2; wherein B', M, Z, Tr, L and D have each independently the definitions described above;

[0084] According to an embodiment of the present invention, the linker-drug structure is as follows:

[0085] According to an embodiment of the present invention, the linker-drug shown in formula (II) is selected from the following structures:

[0086] The present invention also provides a linker-drug fragment of formula (II-1) with the structure: B-(MZ-Tr-LD)2; wherein B, M, Z, Tr, L and D have the definitions described above independently;

[0087] According to an embodiment of the present invention, the structure of the linker-drug fragment is as follows:

[0088] According to an embodiment of the present invention, the linker-drug fragment shown in formula (II-1) is selected from the following structures:

[0089] The present invention also provides a linker-drug represented by formula (III): M'-Z-Tr-LD (III)

[0090] Among them, Z, Tr, L, and D are independent of each other and have the definitions described herein; M' has the definitions described herein;

[0091] According to an embodiment of the present invention, the linker-drug represented by formula (III) is selected from the following structures:

[0092] Among them, Z and R z X1, X2, Tr, L, D, z1, z3, n1, n3, n4 have the definitions described herein.

[0093] According to an embodiment of the present invention, the linker-drug shown in formula (III) is selected from:

[0094] The present invention also provides an intermediate having a structure of Tr'-LD;

[0095] Wherein, Tr' is the structure before Tr is connected to Z; Tr', Tr, L and D have the definitions described in this paper independently;

[0096] According to embodiments of the present invention, the amino group in Tr' is optionally protected by an amino protecting group commonly used in the art, such as Fmoc.

[0097] According to an embodiment of the present invention, the intermediate is selected from:

[0098] The present invention also provides an intermediate having the following structure:

[0099] Among them, Ab, B0, B1, B2', B3', and β have the definitions described herein independently.

[0100] According to an embodiment of the present invention, the intermediate is selected from:

[0101] Ab and β are independently defined as described in this paper.

[0102] According to an embodiment of the present invention, the intermediate is selected from:

[0103] Where β has the definition described in this article.

[0104] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate of formula (I) or a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound thereof.

[0105] According to embodiments of the present invention, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0106] According to embodiments of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0107] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of at least one of the antibody-drug conjugate of formula (I), its racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound thereof.

[0108] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of the above-described pharmaceutical composition.

[0109] The tumor diseases mentioned are selected from breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia.

[0110] In some implementations, the patient includes mammals, preferably humans.

[0111] The present invention also provides at least one of the following: an antibody-drug conjugate of formula (I) for treating tumor diseases, a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or a prodrug compound thereof, or a pharmaceutical composition thereof.

[0112] The present invention also provides the use of at least one of the antibody-drug conjugate of formula (I), its racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, or the above-described pharmaceutical compositions in the preparation of a medicament for the prevention or treatment of a disease or condition.

[0113] In some implementations, the disease or condition is a tumor, including breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia. Beneficial effects

[0114] The present invention also provides antibody-drug conjugates of formula (I), their racemic, stereoisomers, tautomers, solvates, polymorphs, pharmaceutically acceptable salts or prodrug compounds, which have good inhibitory activity and selectivity against cancer cells. Attached Figure Description

[0115] Figure 1 is a schematic diagram of the efficacy evaluation of the test drug on NCI-N87 tumor-bearing mice.

[0116] Figure 2 is a schematic diagram of the efficacy evaluation of the test drug on JIMT-1 tumor-bearing mice.

[0117] Terminology Definitions and Explanations

[0118] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0119] The "antibody-drug conjugate" (ADC) described in this invention refers to a target portion that is linked to a biologically active drug via a stable linker unit.

[0120] Unless otherwise stated, the numerical ranges described in this specification and claims are equivalent to describing at least each specific integer value within them. For example, the numerical range "1-15" is equivalent to describing each integer value within the numerical range "1-15", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. The numerical range "1-10" is equivalent to describing each integer value within the numerical range "1-15", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Furthermore, when certain numerical ranges are defined as "numbers", it should be understood that they describe the two endpoints of the range, each integer within the range, and each decimal within the range. For example, the numerical range "1-10" further includes 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.

[0121] The term "integers from 0 to 10" refers to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0122] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0123] “C 1-10 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. 1-8 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. 1-6"Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0124] Wavy lines intersecting chemical bonds Used to indicate the connection position of a group with other atoms or groups in a general formula.

[0125] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkyloxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, or heterocycloalkyloxy.

[0126] Those skilled in the art will understand that the antibody-drug conjugates in the embodiments of the present invention, through transglutaminase action, catalytically transfer the NH2 of the Link” containing the amine donor to the glutamine residue of the acceptor, forming -CONH-, and the COOH at the other end of the Link” condenses with the NH2 on D to form -NHCO-. Therefore, the antibody-drug conjugate structure shown in the exemplary Pertuzumab(A)-L002-001 below is... and They can represent the same structure, both illustrating that through the action of transglutaminase, the NH in Link is catalytically transferred to the glutamine residue of the Pertuzumab(A) receptor to form -CONH-, that is, the NH in Link is linked to the CO on the Pertuzumab(A) fragment, and the CO in Link is linked to the NH on the eribulin fragment.

[0127] The term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When the term "antibody" is used, unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). The antibody light chain can be classified as κ (kappa) and λ (lambda) light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites. The allocation of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3.The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this invention, the CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the Kabat numbering system. As used herein, the term “frame region” or “FR” residue refers to those amino acid residues in the variable region of the antibody other than the CDR residues as defined above. The term “antibody” is not limited to any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; this fragment is also referred to as the “antigen-binding moiety.” See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical fragmentation of the intact antibody.Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, single-domain antibody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136. As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of the VH and CH1 domains). As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site. As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has a variety of functions but does not participate in antigen binding. "Effective functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. The Fc domain can include both native and variant Fc regions. Native Fc regions contain amino acid sequences consistent with those found in naturally occurring Fc regions, such as the native human IgG1 Fc region; the native human IgG2 Fc region; the native human IgG3 Fc region; and the native human IgG4 Fc region, as well as their naturally occurring variants. Variant Fc regions contain amino acid sequences that differ from the amino acid sequence of native Fc regions due to at least one amino acid modification. In some implementations, the variant Fc region may have altered effector functions compared to the native Fc region (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence (SEQ ID NO. 5) or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL of the scFv. In some embodiments of the invention, scFv may form di-scFv, which refers to two or more individual scFvs linked in tandem to form an antibody. In some embodiments of the invention, scFv may form (scFv)2, which refers to two or more individual scFvs linked in parallel to form an antibody. As used herein, the term "biantibody" means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)). As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind the same antigen bound by the full-length antibody. As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity to at least two (e.g., three or four) different antigens (or epitopes). Bispecific or multispecific antibodies contain multiple antigen-binding domains with binding specificity to different antigens (or epitopes), thereby enabling them to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain in a bispecific or multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or its antigen-binding fragments (e.g., Fv fragments, Fab fragments, F(ab')2 fragments, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers. Each of these antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific antigen binding.Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody fragments provided in this invention) can be obtained from a given antibody (e.g., the antibody fragments described above) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as for intact antibodies. As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Generally, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or the constant region) is derived from a human immunoglobulin (receptor antibody). In some embodiments, the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or the constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In this application, the donor antibody may be a murine antibody with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human frame sequence using methods known in the art. In some cases, the human frame sequence may contain amino acid mutations substituted by corresponding non-human residues. Furthermore, the humanized antibody may also contain residues not found in either the variable region (e.g., the light chain variable region or the heavy chain variable region) of the initial donor antibody or in the human frame sequence to further improve or optimize the performance of the humanized antibody. As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains binding activity to the target antigen in any case. In some embodiments, the term "chimeric antibody" may include an antibody in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, while the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0128] In this invention, the compounds involved also include isotopically labeled compounds, which are the same as those shown in Formula I, but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of this invention include isotopes of H, C, N, O, S, F, and Cl, respectively such as 2 H, 3 H, 13C 11 C 14 C 15 N、 18 O、 17 O、 32 P, 35 S, 18 F and 36 Cl. Compounds of the present invention, their prodrugs, or pharmaceutically acceptable salts of said compounds or prodrugs containing the aforementioned isotopes and / or other isotopes are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those doped with radioactive isotopes (e.g.,... 3 H and 14 Compounds in (C) can be used for drug and / or substrate tissue distribution assays. Tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Furthermore, heavier isotopes (such as deuterium, i.e., 2 Hydrogen (H or D) substitution can provide certain therapeutic advantages derived from greater metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain circumstances. The compounds of the invention claimed in the claims are particularly defined as being substituted with deuterium or tritium. Furthermore, the presence of hydrogen in the substituents without a separate mention of the terms deuterium or tritium does not exclude deuterium or tritium, but may also include deuterium or tritium.

[0129] Those skilled in the art will understand that the compounds shown in formula (I) can exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they can form acid addition salts; if these compounds have an acidic center, they can form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they can also form internal salts.

[0130] The compounds of the present invention may exist as solvates (such as hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0131] Depending on their molecular structure, the compounds of the present invention can be chiral, and therefore may exist in various enantiomeric forms. Thus, these compounds can exist in racemic or optically active forms. The compounds of the present invention encompass isomers of each chiral carbon in the R or S configuration, or mixtures thereof, and racemates. The compounds of the present invention or their intermediates can be isolated as enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are obtained from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as tartaric acid in both R and S forms, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g., N-benzoylproline or N-benzenesulfonylproline), or various optically active camphorsulfonic acids. Chromatographic enantiomeric separation can also be advantageously performed using optically active resolving agents (e.g., dinitrobenzoylphenylglycine immobilized on silica gel, cellulose triacetate or other carbohydrate derivatives, or chiral derivatized isobutylene ester polymers). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, such as hexane / isopropanol / acetonitrile.

[0132] The corresponding stable isomers can be separated using known methods, such as extraction, filtration, or column chromatography.

[0133] The term "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, with humans being the most preferred.

[0134] The term “therapeutic effective amount” refers to the amount of an active compound or drug that researchers, veterinarians, physicians, or other clinicians are searching for in tissues, systems, animals, individuals, or humans to elicit a biological or medical response. It includes one or more of the following: (1) prevention of disease: e.g., prevention of disease, disorder, or condition in individuals susceptible to disease, disorder, or symptom but not yet experiencing or exhibiting the pathology or symptoms of the disease; (2) suppression of disease: e.g., suppression of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., prevention of further development of the pathology and / or symptoms); (3) relief of disease: e.g., relief of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., reversal of the pathology and / or symptoms).

[0135] In this document, the term "DAR" refers to the "drug-to-antibody ratio." Those skilled in the art will understand that, as in the general formula Ab-[Link-(D)2] β In this context, Ab represents the antibody and D represents the drug, then DAR = 2β. Detailed Implementation

[0136] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0137] Unless otherwise stated, the structure of the compounds was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0138] MS determination was performed using an Agilent 1260 / 1290 Infinity II liquid chromatography system (manufacturer: Agilent, MS model: 1260 / 1290 Infinity II) and a Shimadzu Prominence UFLC+LCMS-2020 system (manufacturer: Shimadzu, MS model: Prominence UFLC+LCMS-2020).

[0139] High performance liquid chromatography (HPLC) analysis was performed using a Thermo UltiMate 3000 (manufacturer: Thermo, MS model: UltiMate 3000).

[0140] Chiral HPLC analysis was performed using a YMC K-PrepLAB100G high-performance liquid chromatograph.

[0141] High-performance liquid chromatography (HPLC) was performed using a Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, GX-281 Liquid Handler without pump, 4020 Syringe pump, 333-H3 Pump, 334-H3 Pump, and 1741 UV Detector preparative chromatograph.

[0142] The rapid chromatograph used is the Biotage Isolera One rapid chromatograph.

[0143] The silica gel plates used for thin-layer chromatography are Yantai Xinnuo GF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15mm to 0.2mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4mm to 0.5mm.

[0144] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0145] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0146] The known starting materials of this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0147] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0148] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0149] A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

[0150] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0151] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0152] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0153] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0154] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0155] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0156] Example A: Preparation of the A-payload (connector)

[0157] Synthesis of intermediate Int 1

[0158] Synthesis of the first-step compound (Int 1b)

[0159] Eribulin mesylate Int 1a (700 mg, 0.85 mmol), N-[fluorenylmethoxycarbonyl]-L-valine-L-alanine (369 mg, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL), and HATU (486 mg, 1.28 mmol) and DIPEA (348 mg, 2.7 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, water was added, and the system was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography to obtain compound Int 1b (900 mg, yield: 94%).

[0160] MS m / z(ESI): 1122.5(M+1) + .

[0161] The second step involves the synthesis of compound (Int 1).

[0162] Compound Int 1b (700 mg, 0.62 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (1 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was evaporated to obtain compound Int 1 (505 mg, yield: 90%).

[0163] MS m / z(ESI): 900.5(M+1) + .

[0164] Example A-1 Synthesis of Compound 001

[0165] Synthesis of compound 001b in step one

[0166] Compound 001a (320 mg, 0.73 mmol), 2,5-dioxo-1-pyrrolyl 11,12-didehydro-γ-oxodibenzo[b,f]azaaroctyl-5(6H)-butyrate (350 mg, 0.870 mmol), and DIEA (187 mg, 1.45 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography system A to give compound 001b (400 mg, yield: 71%).

[0167] MS m / z(ESI): 729.3(M+1) +

[0168] The second step involves the synthesis of compound 001.

[0169] Compound 001b (100 mg, 0.14 mmol), compound Int 1 (123 mg, 0.14 mmol), and HATU (63 mg, 0.17 mmol) were dissolved in DMF (5 mL), and DIEA (27 mg, 0.21 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C182 1.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% NH3); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 40%-70%, flow rate: 25 mL / min) to obtain compound 001 (3.9 mg, yield: 2%).

[0170] MS m / z(ESI): 828.0(M+Na / 2) +

[0171] 1H NMR(400MHz,CD3OD)δ7.65(d,1H),7.60(dd,1H),7.50–7.41(m,3H),7.39–7.29(m,2H),7.25(dd,1H),5.25–5.07(m,3H),5.02(s,2H),4.70 (t,1H),4.62–4.58(m,2H),4.47(d,1H),4.39–4.24(m,4H),4.19–4.1 5(m,2H),4.13–4.06(m,2H),4.00–3.95(m,1H),3.86–3.81(m,4H),3.7 9–3.67(m,6H),3.65–3.57(m,31H),3.57–3.53(m,3H),3.48(s,1H),3 .46–3.38(m,4H),3.25–3.22(m,4H),2.93–2.84(m,2H),2.77–2.63(m, 3H),2.58–2.50(m,2H),2.49–2.27(m,4H),2.26–1.91(m,8H),1.90–1 .65(m,4H),1.59–1.44(m,4H),1.35(d,4H),1.10(d,4H),0.96(t,6H).

[0172] Example A-2 Synthesis of Compound 002

[0173] Synthesis of compound 002a in step one

[0174] Compound 001b (200 mg, 0.27 mmol), (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutyramide (89 mg, 0.30 mmol), and HATU (157 mg, 0.41 mmol) were dissolved in DMF (10 mL), and DIEA (71 mg, 0.55 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography system A to give compound 002a (85 mg, yield: 27%).

[0175] MS m / z(ESI): 1004.4(M+1) + .

[0176] The second step involves the synthesis of compound 002b.

[0177] Compound 002a (200 mg, 0.20 mmol) and bis(4-nitrophenyl) carbonate (182 mg, 0.60 mmol) were dissolved in DMF (10 mL), and DIEA (154 mg, 1.20 mmol) was added. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography system A to give compound 002b (160 mg, yield: 61%).

[0178] MS m / z(ESI): 1169.4(M+H) +

[0179] The third step involves the synthesis of compound 002.

[0180] Compound 002b (50 mg, 0.04 mmol) and eribulin mesylate (31 mg, 0.043 mmol) were dissolved in DMF (3 mL), and DIEA (11 mg, 0.09 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% NH4HCO3); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 52%-62%, flow rate: 15 mL / min) to obtain compound 002 (38.8 mg, yield: 51%).

[0181] MS m / z (ESI): 889.4 (M+Na / 2) + .

[0182] 1H NMR (400MHz, CDCl3) δ7.77–7.62(m,3H),7.51(dd,1H),7.43–7.27(m,7H),5. 27(d,1H),5.15(d,1H),5.10–5.06(m,1H),5.03(s,2H),4.94(s,1H),4.90–4. 77(m,2H),4.73–4.64(m,2H),4.61(t,1H),4.39–4.25(m,3H),4.24–4.17(m, 2H),4.16–4.08(m,1H),4.05–4.02(m,1H),3.97–3.88(m,3H),3.84–3.79(m,2 H),3.67–3.56(m,31H),3.49–3.44(m,2H),3.42(s,3H),3.39–3.30(m,3H),3 .27(d,1H),3.19–3.15(m,1H),2.92–2.79(m,3H),2.71(dd,2H),2.61–2.39(m ,6H),2.35–2.06(m,12H),2.03–1.88(m,7H),1.74–1.70(m,4H),1.62–1.57( m,3H),1.44(t,4H),1.38–1.32(m,1H),1.12–1.07(m,3H),1.03–0.95(m,6H).

[0183] Synthesis of Compound 014 in Example A-3

[0184] Synthesis of compound 014b (Step 1)

[0185] Compound 014a (200 mg, 0.40 mmol) was dissolved in DMF (3 mL), and 2,5-dioxo-1-pyrrolidinyl-11,12-disehydro-γ-oxodibenzo[b,f]azaaroctyl-5(6H)-butyrate (161 mg, 0.40 mmol) and triethylamine (81 mg, 0.80 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was purified by silica gel column chromatography system A to give compound 014b (220 mg, yield: 70%).

[0186] MS m / z(ESI): 787.4(M+1) + .

[0187] The second step involves the synthesis of compound 014c.

[0188] Compound 014b (220 mg, 0.28 mmol) was dissolved in DMF (3 mL), and (2S)-2-amino-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]-3-methylbutyramide (82 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (56 mg, 0.56 mmol) were added. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was purified by silica gel column chromatography system A to give compound 014c (200 mg, yield: 67%).

[0189] MS m / z(ESI): 1084.5(M+23) + .

[0190] The third step involves the synthesis of compound 014d.

[0191] Compound 014c (200 mg, 0.18 mmol) was dissolved in DMF (2 mL), and bis(4-nitrophenyl) carbonate (169 mg, 0.56 mmol) and triethylamine (114 mg, 1.13 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was purified by silica gel column chromatography system A to give compound 014d (120 mg, yield: 52%).

[0192] MS m / z(ESI): 1227.5(M+1) + .

[0193] Step 4: Synthesis of compound 014

[0194] Compound 014d (100 mg, 0.08 mmol) was dissolved in DMF (2 mL), and eribulin mesylate Int 1a (59 mg, 0.08 mmol) and DIPEA (16 mg, 0.16 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% NH4HCO3); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 70%-80%, flow rate: 30 mL / min) to obtain compound 014 (40 mg, yield: 27%).

[0195] 1H NMR (400MHz, CDCl3) δ7.71–7.59(m,3H),7.56–7.51(m,1H),7.48–7.40(m, 3H),7.39–7.34(m,1H),7.33–7.27(m,4H),7.25–7.20(m,2H),5.09–5.07(m ,1H),5.06–4.98(m,3H),4.95–4.90(m,1H),4.88(t,2H),4.79(d,2H),4.6 9(t,1H),4.61(t,1H),4.38–4.27(m,2H),4.26–4.06(m,3H),4.06–4.00(m, 2H),3.99–3.86(m,3H),3.85–3.74(m,3H),3.66–3.58(m,30H),3.57–3.50 (m,8H),3.43–3.41(m,4H),3.37(s,4H),3.20–3.08(m,3H),2.92–2.83(m,3 H),2.77–2.64(m,3H),2.60–2.44(m,5H),2.22(s,7H),2.12–2.02(m,4H),1 .64–1.57(m,4H),1.50–1.40(m,3H),1.14–1.08(m,3H),1.06–0.96(m,6H).

[0196] Synthesis of Compound 011 in Example A-4

[0197] Synthesis of compound 011b in step one

[0198] 4-Bromobutyrate tert-butyl ester 011a (2 g, 9.0 mmol), 4-aminobutyrate benzyl ester (2.61 g, 13.5 mmol), potassium carbonate (2.48 g, 18.0 mmol), and potassium iodide (1.48 g, 9.0 mmol) were dissolved in acetonitrile (40 mL) and stirred at 80 °C for 16 hours. After the reaction was complete, the reaction solution was concentrated by rotary evaporation to obtain compound 011b (3.0 g). The product was used directly in the next step without purification.

[0199] MS m / z(ESI): 336.1(M+1) +

[0200] The second step involves the synthesis of compound 011c.

[0201] Compound 011b (3 g, crude product) and potassium carbonate (2.5 g, 18.1 mmol) were dissolved in acetonitrile (50 mL), and benzyl chloroformate (2.3 g, 13.5 mmol) was slowly added. The reaction was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography system A to give product 011c (0.95 g, yield: 22%).

[0202] MS m / z (ESI): 492.2 (M+Na) +

[0203] The third step involves the synthesis of compound 011d.

[0204] Compound 011c (450 mg, 0.96 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was evaporated to dryness to obtain product 011d (390 mg, yield: 98%).

[0205] MS m / z(ESI): 414.1(M+H) +

[0206] The fourth step is the synthesis of compound 011f.

[0207] Compound 011d (396 mg, 0.96 mmol), N-(2-(dimethylamino)-2-oxoethyl)-2-(N,5,8,11,14,17-hexamethyl-4,7,10,13,16-pentoxo-2,5,8,11,14,17-hexaazanonadecan-19-amide)-N-methylacetamide 011e (588 mg, 0.96 mmol), and HATU (546 mg, 1.44 mmol) were dissolved in DMF (10 mL), and DIEA (248 mg, 1.92 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography system A to give compound 011f (269 mg, yield: 28%).

[0208] MS m / z(ESI): 1009.4(M+H) +

[0209] Step 5: Synthesis of compound 011g

[0210] Compound 011f (240 mg, 0.238 mmol) and palladium on carbon (20 mg) were dissolved in methanol (30 mL), and the reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was collected and evaporated to dryness to give compound 011 g (190 mg, 100%).

[0211] MS m / z(ESI): 785.3(M+H) +

[0212] Step 6: Synthesis of compound 011h

[0213] Compound 011g (85 mg, 0.11 mmol) and 2,5-dioxo-1-pyrrolyl 11,12-disehydro-γ-oxodibenzo[b,f]azaaroctyl-5(6H)-butyrate (65 mg, 0.16 mmol) were dissolved in DMF (3 mL), and triethylamine (22 mg, 0.22 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated, and the crude product was purified by silica gel column chromatography system A to give product 011h (86 mg, yield: 73%).

[0214] MS m / z(ESI): 1072.3(M+H) +

[0215] Step 7: Synthesis of compound 011

[0216] Compound 011h (30 mg, 0.028 mmol), compound 011i (29 mg, 0.03 mmol), and HATU (16 mg, 0.04 mmol) were dissolved in DMF (3 mL), and triethylamine (6 mg, 0.056 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% NH4HCO3); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 35%-65%, flow rate: 15 mL / min) to obtain compound 011 (7.9 mg, yield: 13%).

[0217] MS m / z (ESI): 1062.9 (M+Na / 2) +

[0218] 1H NMR (400MHz, CDCl3) δ7.86–7.81(m,1H),7.78–7.76(m,1H),7.67–7.59(m,2H),7.44 –7.31(m,6H),7.22–7.16(m,1H),7.13–7.07(m,1H),5.38–5.32(m,2H),5.10–5.06(m ,2H),5.01(s,2H),4.95(s,2H),4.88(s,2H),4.80(s,2H),4.71–4.68(m,2H),4.62– 4.60(m,2H),4.40–4.34(m,4H),4.28–4.22(m,6H),4.21–4.16(m,7H),4.13–4.07(m, 6H),4.05–4.02(m,3H),3.97(m,3H),3.92–3.84(m,4H),3.82–3.76(m,2H),3.69–3. 63(m,5H),3.42(s,5H),3.27–3.25(m,1H),3.05–3.03(m,8H),2.99–2.97(m,8H),2.9 4–2.92(m,10H),2.53–2.45(m,4H),2.35–2.29(m,4H),2.24–2.15(m,8H),2.02–2.00 (m,3H),1.45–1.43(m,6H),1.26–1.25(m,6H),1.10–1.09(m,6H),0.89–0.85(m,6H).

[0219] Following a similar method disclosed above, the following compounds were synthesized, and their structural characterization data are shown in Table 1 below:

[0220] Table 1

[0221] Example B: Preparation of Linker B

[0222] Example B-1 Synthesis of compound L-001

[0223] Synthesis of compound L-001b in step one

[0224] 1,4,7-triazacyclononane L-001a (500 mg, 3.87 mmol) was dissolved in DMF (5 mL), and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid (2.7 g, 11.6 mmol), HATU (2.2 g, 5.8 mmol), and DIEA (1.5 g, 11.6 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5 μm C18 150*19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 22%-32%, flow rate: 20 mL / min) to obtain compound L-001b (400 mg, yield: 18%).

[0225] MS m / z(ESI): 560.3(M+1) + .

[0226] The second step involves the synthesis of compound L-001c.

[0227] Compound L-001b (200 mg, 0.36 mmol) was dissolved in DMF (4 mL), and 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid (132 mg, 0.43 mmol), HATU (204 mg, 0.54 mmol), and DIEA (92 mg, 0.71 mmol) were added. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5 μm C18 150*19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 40%-50%, flow rate: 20 mL / min) to obtain compound L-001c (200 mg, yield: 66%).

[0228] MS m / z(ESI): 749.4(M+1) + .

[0229] The third step involves the synthesis of compound L-001.

[0230] Compound L-001c (200 mg, 0.24 mmol) was dissolved in DCM (2 mL), and 4 M HCl dioxane solution (2 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5 μm C18 150*19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 16%-26%, flow rate: 20 mL / min) to obtain compound L-001 (50.0 mg, yield: 28%).

[0231] MS m / z(ESI): 749.4(M+1) + .

[0232] 1 H NMR (400MHz, CDCl3) δ5.00–4.90(m,2H),4.28–4.23(m,1H),4.22–4.18(m,6H),3.84–3.80(m,2 H),3.74–3.71(m,2H),3.70–3.64(m,33H),3.54–3.47(m,4H),3.42–3.39(m,4H),3.16(s,2H).

[0233] Example B-2 Synthesis of compound L-002

[0234] Synthesis of compound L-002b in step one

[0235] 5-Amino-isophthalic acid L-002a (700 mg, 3.86 mmol) was dissolved in DMF (10 mL), and 2-(-2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl-1-amine (1.85 g, 8.49 mmol), EDCI (1.78 g, 9.26 mmol), and HOBt (1.36 g, 10.04 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was purified by reverse-phase column chromatography to give compound L-002b (600 mg, yield: 27%).

[0236] MS m / z(ESI): 582.3(M+1) + .

[0237] The second step involves the synthesis of compound L-002c.

[0238] Compound L-002b (300 mg, 0.52 mmol) was dissolved in DMF (3 mL), and 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecan-15-acid (268 mg, 6.2 mmol), HATU (296 mg, 0.78 mmol), and DIPEA (134 mg, 1.04 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250mm 10μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 52%-62%, flow rate: 20mL / min) to obtain compound L-002c (300mg, yield: 58%).

[0239] MS m / z(ESI): 993.3(M+1) + .

[0240] The third step involves the synthesis of compound L-002.

[0241] Compound L-002c (100 mg, 0.10 mmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 22%-32%, flow rate: 25 mL / min) to obtain compound L-002 (50 mg, yield: 65%).

[0242] MS m / z(ESI): 771.3(M+1) + .

[0243] 1H NMR(400MHz, CDCl3)δ9.81(s,1H),8.32–8.15(m,3H),8.05(s,1H),7.83(s,3H),5.43–5.24(m,2H),4.14(s,3H),3.89–3.78(m,2H),3 .78–3.49(m,22H),3.42–3.33(m,3H),3.29–3.16(m,3H),2.32–2.15(m,2H),2.07–1.98(m,2H),1.74–1.56(m,2H),1.45–1.28(m,5H).

[0244] Example C: Preparation of the antibody portion

[0245] In some embodiments, the antibody in the antibody-drug conjugate (ADC) is a known antibody, including but not limited to pertuzumab or its antigen-binding fragment.

[0246] Example C-1 Preparation of anti-human HER2 monoclonal antibody Pertuzumab (A)

[0247] In this disclosed embodiment, the antibody-drug conjugate (ADC) containing the anti-HER2 antibody pertuzumab includes a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. The anti-HER2 antibody pertuzumab, when the amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region sequence is mutated to A (alanine, Alanine, Ala), becomes the anti-human HER2 monoclonal antibody Pertuzumab (A).

[0248] The heavy chain variable region sequence of the anti-HER2 antibody pertuzumab, namely the heavy chain variable region sequence of Pertuzumab(A) (SEQ ID NO.6), is as follows:

[0249] The sequence following the mutation of amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region of the anti-HER2 antibody pertuzumab to A (alanine, Alanine, Ala), i.e., the heavy chain constant region sequence of Pertuzumab (A) (SEQ ID NO.7), is as follows:

[0250] The light chain variable region sequence of the anti-HER2 antibody pertuzumab, namely the light chain variable region sequence of Pertuzumab(A) (SEQ ID NO.8), is as follows:

[0251] The light chain constant region sequence of the anti-HER2 antibody pertuzumab, namely the light chain constant region sequence of Pertuzumab(A) (SEQ ID NO.9), is as follows:

[0252] The antibody gene was synthesized using a plasmid and expressed in Expi-CHO-S cells. The antibody was purified using the standard AmMag™ Protein A Magnetic Beads method and stored in PBS solution at pH 7.2. The antibody batch number was C8559028G0-3 / P36JI001. The antibody concentration was quantified using the A280 method to be 9.76 mg / ml. The purity was 95% by non-reducing SDS-PAGE, 98% by SEC-HPLC, and the endotoxin level was less than 0.1 EU / mg.

[0253] Example D: Preparation of Antibody-Drug Conjugates

[0254] Example D-1a Preparation of Pertuzumab(A)-L002

[0255] Transfer 60 mg of Pertuzumab(A) antibody to a new 50 mL centrifuge tube. Under pre-cooling conditions, add compound L-002 (prepared with DMSO) at a final drug-to-antibody molar ratio of 50:1. Add 8.5 times the molar amount of 80 mg / mL transglutaminase (prepared with water for injection), and then add 50 mM PBS. Adjust the pH to 7.4 until the antibody concentration reaches 5 mg / mL. Incubate overnight at 25°C, mixing continuously. Purify the co-incubated conjugate using a HiLoad 26 / 600 Superdex 200 pg column. After purification, concentrate the sample and change the buffer to storage buffer (20 mM His, 150 mM NaCl, pH 5.5) to obtain the antibody linker B conjugate Pertuzumab(A)-L002.

[0256] Determination of β ratio (LC-MS):

[0257] An Agilent 1260 series high-performance liquid chromatography system coupled with TOF mass spectrometry was used, with PLRP-S column model. (8 μm, 50*2.1 mm), 80℃. Ultrapure water containing 0.025% TFA (trifluoroacetic acid) and 0.1% FA (formic acid) and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Mobile phase B was used for gradient elution from 34% to 45%. The sample loading amount was 3–10 μg. β = 1.87 was calculated based on the abundance of the corresponding peaks of the relevant molecular weights after deconvolution.

[0258] Pertuzumab(A)-L002 purity determination (SEC-HPLC)

[0259] The Waters ARC system was used, with an analytical column of Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm) (Waters, 186007640). The mobile phase was 150mM PBS + 200mM Arg (pH 6.80) + 10% IPA. The sample purity was determined to be 100% using a UV detector at 280nm.

[0260] Example D-1 Preparation of Pertuzumab(A)-L002-001

[0261] Transfer 10 mg of Pertuzumab(A)-L002 antibody conjugate to a new 50 mL centrifuge tube, add 20 mM His, 150 mM NaCl, and adjust pH to 5.5 until the antibody concentration is 5 mg / mL. Add 1 M Tris and adjust pH to 7.2-7.4 from pH 8.0. Under pre-cooling conditions, add compound 001 (prepared with DMA) at a final drug-to-antibody molar ratio of 8:1. Incubate at 22 °C for 3 h, mixing continuously. Desalt and purify the conjugate using a 10 mL (40 K MWCO) Zeba column. Concentrate using Amicon (30 kDa, 15 mL) and transfer to storage buffer (30 mM His / HAc pH 5.5) to obtain the antibody-drug conjugate Pertuzumab(A)-L002-001 (6.7 mg).

[0262] Drug / antibody ratio determination (RP-DAR):

[0263] Using a Waters ARC system with a BioResolve RP mAb Polyphenyl (4.6×100mm, 2.7μm) (Waters, 186008946) column, ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Mobile phase B was used for gradient elution from 30% to 45%. Detection was performed at 280 nm using a UV detector. The chromatogram was integrated, and the peak area percentage of each peak was calculated using the peak area normalization method. The calculation formula is as follows. The final DAR was 3.59.

[0264] DAR 2n=2ⅹ[Σ(Weighted Peak Area of ​​light chain)+Σ(Weighted Peak Area of ​​heavy chain)] / 100

[0265] ADC purity detection (SEC-HPLC)

[0266] Using a Waters ARC system, the supernatant was loaded onto an Xbridge BEH200 SEC (7.8 × 300 mm, 3.5 μm) column (Waters, 186007640). The mobile phase was 150 mM PBS + 200 mM Arg (pH 6.80) + 10% IPA. Isocratic elution was performed, and the sample purity was determined to be 99.68% at 280 nm using a UV detector.

[0267] Detection of free small molecule drug content (RP-HPLC)

[0268] Residual free drug levels were determined using reversed-phase high-performance liquid chromatography (RP-HPLC). After protein precipitation, the supernatant was loaded onto an XBridge Premier BEH C18 column. A 2.5 μm, 4.6 x 50 mm column (Waters, 186009847) was used. Ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Gradient elution was performed in mobile phase B from 10% to 80%. Detection was performed using a UV detector at 280 nm. Comparison with the standard curve showed that the residual free drug content was <47.06 ppm.

[0269] Following the method of Example D-1, compound 001 was replaced with compounds from other examples of this application to prepare the following antibody-drug conjugates. The ADC structures are shown in Table 2, and the analytical characterization results are shown in Table 3.

[0270] Example D-2a Preparation of Pertuzumab(A)-L001

[0271] Transfer 80 mg of Pertuzumab(A) antibody to a new 50 mL centrifuge tube. Under pre-cooling conditions, add L-001 (prepared with DMSO) at a final drug-to-antibody molar ratio of 50:1. Add 8.5 times the molar amount of 80 mg / mL transglutaminase (prepared with water for injection), and then add 50 mM PBS. Adjust the pH to 7.4 until the antibody concentration reaches 5 mg / mL. Incubate at 30°C overnight, mixing continuously. Purify the co-incubated conjugate using a HiLoad 26 / 600 Superdex 200 pg column. After purification, concentrate the sample and change the buffer to storage buffer (20 mM His, 150 mM NaCl, pH 5.5) to obtain the antibody linker conjugate Pertuzumab(A)-L001.

[0272] Determination of β (LC-MS):

[0273] An Agilent 1260 series high-performance liquid chromatography system coupled with TOF mass spectrometry was used, with PLRP-S column model. 8 μm (50*2.1 mm), 80℃. Ultrapure water containing 0.025% TFA (trifluoroacetic acid) and 0.1% FA (formic acid) and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Mobile phase B was used for gradient elution from 34% to 45%. The sample loading amount was 3–10 μg. β = 1.84 was calculated based on the abundance of the corresponding peaks of the relevant molecular weights after deconvolution.

[0274] ADC purity detection (SEC-HPLC)

[0275] The Waters ARC system was used, with an analytical column of Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm) (Waters, 186007640). The mobile phase was 150mM PB + 200mM Arg (pH 6.80) + 10% IPA. The sample purity was determined to be 100% using a UV detector at 280nm.

[0276] Example D-2 Preparation method of Pertuzumab(A)-L001-001

[0277] Transfer 30 mg of Pertuzumab(A)-L001 antibody conjugate to a new 50 mL centrifuge tube. Add 20 mM His, 150 mM NaCl, and adjust the pH to 5.5 until the antibody concentration is 5 mg / mL. Add 1 M Tris and adjust the pH to 7.2-7.4 (pH 8.0). Under pre-cooling conditions, add 001 (prepared with DMA) at a final drug-to-antibody molar ratio of 8:1. Incubate at 22 °C for 3 h, mixing continuously. Desalt and purify the conjugate using a 10 mL (40 K MWCO) Zeba column. Concentrate using Amicon (30 kDa, 15 mL) and transfer to storage buffer (30 mM His / HAc pH 5.5) to obtain the antibody-drug conjugate Pertuzumab(A)-L001-001.

[0278] Drug / antibody ratio determination (RP-DAR):

[0279] Using a Waters ARC system with a BioResolve RP mAb Polyphenyl (4.6 × 100 mm, 2.7 μm) column (Waters, 186008946), ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Mobile phase B was used for gradient elution from 30% to 45%. Detection was performed at 280 nm using a UV detector. The chromatogram was integrated, and the peak area percentage of each peak was calculated using the peak area normalization method. The final DAR was 3.60. DAR = 2 × [Σ(Weighted Peak Area of ​​light chain) + Σ(Weighted Peak Area of ​​heavy chain)] / 100

[0280] ADC purity detection (SEC-HPLC)

[0281] Using a Waters ARC system, the supernatant was loaded onto an Xbridge BEH200 SEC (7.8×300mm, 3.5μm) column (Waters, 186007640). The mobile phase was 150mM PB + 200mM Arg (pH 6.80) + 10% IPA. Isocratic elution was performed, and the sample purity was determined to be 99.30% under a UV detector at 280nm.

[0282] Detection of free small molecule drug content (RP-HPLC)

[0283] Residual free drug levels were determined using reversed-phase high-performance liquid chromatography (RP-HPLC). After protein precipitation, the supernatant was loaded onto an XBridge Premier BEH C18 column. A 2.5 μm, 4.6 x 50 mm column (Waters, 186009847) was used. Ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Gradient elution was performed in mobile phase B from 10% to 80%. Detection was performed using a UV detector at 280 nm. Comparison with the standard curve showed that the residual free drug content was <37.47 ppm.

[0284] Example D-3: Preparation method of Pertuzumab(A)-L001-002

[0285] Transfer 30 mg of Pertuzumab(A)-L001 antibody conjugate to a new 50 mL centrifuge tube, add 20 mM His, 150 mM NaCl, and adjust pH to 5.5 until the antibody concentration is 5 mg / mL. Add 1 M Tris and adjust pH to 7.2-7.4 (pH 8.0). Under pre-cooling conditions, add 002 (prepared with DMA) at a final drug-to-antibody molar ratio of 8:1. Incubate at 22 °C for 3 h, mixing continuously. Desalt and purify the conjugate using a 10 mL (40 K MWCO) Zeba column. Concentrate using Amicon (30 kDa, 15 mL) and transfer to storage buffer (30 mM His / HAc pH 5.5) to obtain the antibody-drug conjugate Pertuzumab(A)-L001-002.

[0286] Drug / antibody ratio determination (RP-DAR):

[0287] Using a Waters ARC system with a BioResolve RP mAb Polyphenyl (4.6 × 100 mm, 2.7 μm) column (Waters, 186008946), ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Mobile phase B was used for gradient elution from 30% to 45%. Detection was performed at 280 nm using a UV detector. The chromatogram was integrated, and the peak area percentage of each peak was calculated using the peak area normalization method. The final DAR was 3.60. DAR = 2 × [Σ(Weighted Peak Area of ​​light chain) + Σ(Weighted Peak Area of ​​heavy chain)] / 100

[0288] ADC purity detection (SEC-HPLC)

[0289] Using a Waters ARC system, the supernatant was loaded onto an Xbridge BEH200 SEC (7.8 × 300 mm, 3.5 μm) column (Waters, 186007640). The mobile phase was 150 mM PB + 200 mM Arg (pH 6.80) + 10% IPA. Isocratic elution was performed, and the sample purity was determined to be 98.46% at 280 nm using a UV detector.

[0290] Detection of free small molecule drug content (RP-HPLC)

[0291] Residual free drug levels were determined using reversed-phase high-performance liquid chromatography (RP-HPLC). After protein precipitation, the supernatant was loaded onto an XBridge Premier BEH C18 column. A 2.5 μm, 4.6 x 50 mm column (Waters, 186009847) was used. Ultrapure water containing 0.1% TFA and acetonitrile were used as mobile phases A and B, respectively, at a flow rate of 0.5 mL / min. Gradient elution was performed in mobile phase B from 10% to 80%. Detection was performed using a UV detector at 280 nm. Comparison with the standard curve showed that the residual free drug content was <36.14 ppm.

[0292] Table 3 ADC Characterization Results

[0293] Biological evaluation

[0294] Test Example 1: ADC Bioactivity Detection

[0295] 1.1 Test Objective

[0296] The purpose of this experiment was to detect the inhibitory activity of ADC compounds on the in vitro proliferation of HER2-expressing NCI-N87 cells, JIMT-1 cells, and HER2-negative MDA-MB-468 cells. Cells were treated with different concentrations of the compounds in vitro, and after 5 days of culture, CTG was used for cell proliferation analysis. The Luminescent Cell Viability Assay detects cell proliferation based on IC50. 50 The value was used to evaluate the in vitro activity of the compound.

[0297] 1.2 Test Methods

[0298] (1) On the first day, tumor cells were seeded on 96-well plates, with 5000 cells / 100μL of culture medium in each well, and 100μL of DPBS in each empty well at the edge. The plates were incubated overnight at 37°C.

[0299] (2) On the second day, first aspirate 50 μL of the old culture medium per well; add ADC at different concentration gradients, with the initial concentration of ADC being 200 nM, diluted 5 times, resulting in 9 concentrations. The volume of drug added is 50 μL per well.

[0300] (3) On the sixth day, thaw CellTiter-Glo Buffer and CellTiter-Glo Substrate reagent at 4°C. Before use, aspirate 10 ml of Buffer and add it to the substrate, mix well, and equilibrate to room temperature.

[0301] (4) On the seventh day, equilibrate the 96-well plate at room temperature for 30 minutes, and add 100 μL of Cell-Titer-Glo to each well. Shake at room temperature in the dark for 5 minutes, incubate for 10 minutes, transfer 100 μL of the liquid in the well to the white plate, and then use a microplate reader to detect chemiluminescence.

[0302] 1.3 Data Analysis

[0303] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5 to test the inhibitory activity of the ADC compound on the in vitro proliferation of NCI-N87 cells, JIMT-1 cells, and MDA-MB-468 cells. The results are shown in Table 4 below.

[0304] Table 4. Inhibitory activity of ADCs on in vitro proliferation

[0305] Conclusion: The antibody-drug conjugates targeting HER2 in this invention have significant inhibitory activity against the proliferation of HER2-positive cells JIMT-1 and NCI-N87; at the same time, they have weak inhibitory activity against the proliferation of HER2-negative cells MDA-MB-468; and they have good selectivity.

[0306] Test Example 2: Efficacy Evaluation of NCI-N87 Tumor-Bearing Mice

[0307] 2.1 Experimental Objective

[0308] Using Balb / c nude mice as test animals, this study investigated whether tumor growth was inhibited, delayed, or cured, and evaluated the efficacy of the ADC in this application.

[0309] 2.2 Experimental Procedure

[0310] 2.2.1 Test Drug

[0311] Blank control / or vehicle control: PBS

[0312] Pertuzumab(A)-L001-001:1.1mg / kg

[0313] Pertuzumab(A)-L001-002:1.1mg / kg

[0314] Pertuzumab(A)-L002-001:1.1mg / kg

[0315] Pertuzumab(A)-L002-002:1.1mg / kg

[0316] 2.2.2 Preparation method: All were prepared by diluting with PBS.

[0317] 2.2.3 Test Methods

[0318] NCI-N87 cells were subcutaneously injected into the right rib area of ​​mice. After 7 days of tumor growth, the animals were randomly divided into 5 groups (n=6 per group, 4 treatment groups + 1 control group). The drugs were administered via tail vein injection once daily. Tumor volume (diameter) and body weight were measured twice weekly for four weeks. Data were recorded. Statistical analysis was performed using Excel 2023: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for inter-group differences were calculated using TTEST.

[0319] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0320] The antitumor efficacy of the compound was evaluated using TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0321] TGI(%) = [1-(Ti-T0) / (Ci-C0)]×100%, where Ti is the average tumor volume of a certain treatment group on a certain day, T0 is the average tumor volume of this treatment group at the beginning of administration; Ci is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the solvent control group at the beginning of administration.

[0322] 2.3 Experimental Results and Conclusions

[0323] Figure 1 shows a schematic diagram of the efficacy evaluation of NCI-N87 tumor-bearing mice. Table 5 shows the in vivo tumor inhibition effect (TGI) of the test drugs in the NCI-N87 transplantation model in the treatment group. The results show that the ADC molecules of this application can significantly reduce tumor volume.

[0324] Table 5: In vivo tumor suppression effect of ADC on NCI-N87 transplantation model

[0325] Test Example 3: Evaluation of JIMT-1 Efficacy in Tumor-Bearing Mice

[0326] 3.1 Experimental Objective

[0327] Using SCID Beige mice as test animals, this study investigates whether tumor growth is inhibited, delayed, or cured, and evaluates the efficacy of the ADC in this application.

[0328] 3.2 Experimental Procedure

[0329] 3.2.1 Test Drug

[0330] Blank control / or vehicle control: PBS

[0331] Pertuzumab(A)-L001-001:1.1mg / kg

[0332] Pertuzumab(A)-L001-002:1.1mg / kg

[0333] Pertuzumab(A)-L002-001:1.1mg / kg

[0334] Pertuzumab(A)-L002-002:1.1mg / kg

[0335] 3.2.2 Preparation method: All were prepared by diluting with PBS.

[0336] 3.2.3 Test Methods

[0337] JIMT-1 cells were subcutaneously injected into the right rib area of ​​mice. After 8 days of tumor growth, the animals were randomly divided into 7 groups (6 treatment groups + 1 control group). The drugs were administered via tail vein injection once. Tumor volume (diameter) and body weight were measured twice weekly for four weeks. Data were recorded. Statistical analysis was performed using Excel 2023: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for inter-group differences were calculated using TTEST.

[0338] The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0339] The antitumor efficacy of the compound was evaluated using TGI (%), and the tumor growth inhibition rate was calculated using the following formula:

[0340] TGI(%) = [1-(Ti-T0) / (Ci-C0)]×100%, where Ti is the average tumor volume of a certain treatment group on a certain day, T0 is the average tumor volume of this treatment group at the beginning of administration; Ci is the average tumor volume of the solvent control group on a certain day (the same day as Ti), and C0 is the average tumor volume of the solvent control group at the beginning of administration.

[0341] 3.3 Experimental Results and Conclusions

[0342] The in vivo tumor-suppressive effects of the treatment group on the JIMT-1 transplantation model are shown in Table 6, and the schematic diagram of the efficacy evaluation of JIMT-1 in tumor-bearing mice is shown in Figure 2. The results indicate that the ADC molecules in this application can significantly reduce tumor volume.

[0343] Table 6: In vivo tumor-suppressive effect of ADC on JIMT-1 transplantation model

[0344] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. The antibody-drug conjugate or its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound of formula (I): Ab-[Link-(D)2] β (I) in, Ab (i.e., the target ligand) is the antibody or its antigen-binding fragment, D is the structural fragment of the bioactive molecule, Link is the linker part that connects the target ligand Ab and the bioactive molecule D; β is selected from integers or decimals between 1 and 10; Wherein, Link is B-(MZ-Tr-L)2; Furthermore, the Link structure is illustrated below: Where B represents the portion of Link that is connected to the target ligand Ab, # represents the connection site between Link and the target Ab portion, and * represents the connection site between Link and the bioactive molecule D. B is selected from the structure shown below: # represents the connection site between B and the target part Ab. B is formed by the reactive group B' and the target group Ab (specifically, it is formed by the reactive group B' B1' and the target group). **Represents the site where B and M are connected; B2 is formed by reactive group B2' and reactive group M'; B3 is formed by reactive group B3' and reactive group M'; M is formed by reactive group M' and intermediate B”; Wherein, B0 is selected from unsubstituted or optionally substituted by one, two or more Rs. B The following groups are substituted: amino acid residues (e.g., lysine residues) C 6-14 Aryl, 3-14 heterocyclic; each R B They are the same or different, and are independently selected from -C(O)CH2-(OCH2CH2) b -OCH3; b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; B1 is selected from unsubstituted or arbitrarily assigned to one, two or more R... B1 The following groups are substituted: -C(O)CH2-(OCH2CH2) b11 -NH-#、-NHC(O)CH2-(OCH2CH2) b11 -NH-#、 Each R B1 Same or different, selected independently from C 1-10 Alkyl group; b11 and b12 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5 or 6; B1' is selected from unsubstituted or optionally by one, two or more R's. B1 The following groups are substituted: -C(O)CH2-(OCH2CH2) b11 -NH2、-NHC(O)CH2-(OCH2CH2) b11 -NH2、 Preferably, the amino group in B1' is optionally protected by an amino protecting group commonly used in the art, such as Fmoc; B2 and B3 may be the same or different, and are independently selected from each other without substitution or arbitrarily selected by one, two or more Rs. B2 The following groups are substituted: Each R B2 Same or different, selected independently from C 1-10 Alkyl group; b21, b22 and b23 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5 or 6; B2' and B3' may be the same or different, and are independently selected from each other without substitution or arbitrarily selected by one, two or more R's. B2 The following groups are substituted: M is selected from Each R 21 R 22 R 23 Whether the two are the same or different, they are selected independently from H and C. 1-6 Alkyl, C 1-10 Alkyl group; t is selected from 1, 2, 3, 4, 5 or 6; m1 is selected from 0, 1, 2, 3 or 4; m2 is selected from 0, 1, 2, 3 or 4; m3 is selected from 0, 1 or 2; reactive group M' is selected from... Z is selected from single bond, -N(R) z )-(CH2)n1-C(=O)-, -N(R1)-(CH2CH2O)z1-(CH2)n2-C(=O)-, Among them, ring A is selected from cyclohexane ring or six-membered heterocycle; R1, R z They are either the same or different, and are independently selected from H, -(CH2CH2O)z3-CH3, z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers from 1 to 36; n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5, and 6; Tr is a divalent triggering group selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), and lysine-glycine-glycine-phenylalanine-glycine (KGGFG). Tr is formed by Tr' and reactive group M'; Tr' is a monovalent triggering group, the N-terminus of which is NH2 and the C-terminus is connected to L, and is selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), lysine-glycine-glycine-phenylalanine-glycine (KGGFG); L is the linker between Tr and the bioactive molecular structural fragment D, formed by the reaction of the reactive group L' with the bioactive molecule D. L is selected from single bonds, -NH-C... 1-10 Alkyl-, -N(CH3)-C 1-10 Alkylene- The L' is selected from single bonds, -NH-C 1-10 Alkyl-X, -N(CH3)-C 1-10 Alkylene-X, X is selected from leaving groups, such as halogens.

2. The antibody-drug conjugate or its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound according to claim 1, characterized in that, Ab is an antibody or antigen-binding fragment, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb; and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody; Preferably, Ab is an anti-HER2 antibody or its antigen-binding fragment; Preferably, the Ab is trastuzumab, pertuzumab, or an antigen-binding fragment thereof; Preferably, the heavy chain constant region of the Ab is deglycosylated or mutated to remove N-glycosylation; Preferably, the Ab is modified by mutating amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region to A (alanine, Alanine, Ala) to remove N-glycosylation; Preferably, the Ab is a pertuzumab with a mutation of amino acid N (asparagine, Asn) at position 297 of the heavy chain constant region to A (alanine, Alanine, Ala), for example, the amino acid sequence of its heavy chain constant region is shown in SEQ ID NO.7; preferably, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO.6, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.7, the sequence of the light chain variable region is shown in SEQ ID NO.8, and the sequence of the light chain constant region is shown in SEQ ID NO.9; Preferably, D is selected from structural fragments of antitumor compounds; Preferably, D is selected from the dehydrogenated structural fragment of an antitumor compound; Preferably, the antitumor compound is selected from eribulin; Preferably, D is selected from Preferably, β can be selected from an integer or a decimal number between 1 and 3; More preferably, β can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or can be selected from 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9; Preferably, the Link is selected from the following structures: Among them, the left amino linker is connected to the antibody part Ab, and the right carbonyl linker is connected to the drug part D; Preferably, B0 is selected from unsubstituted or optionally substituted by one, two or more R. B The following groups are substituted: C 3-8 Alkyl, C 6-10 Aryl, 6-12 heterocyclic; Preferably, B0 is selected from Preferably, R B It is -C(O)CH2-(OCH2CH2)6-OCH3; Preferably, B1 is selected from -C(O)CH2-(OCH2CH2)3-NH-#, -NHC(O)CH2-(OCH2CH2)3-NH-#, Preferably, B1' is selected from -C(O)CH2-(OCH2CH2)3-NH2, -NHC(O)CH2-(OCH2CH2)3-NH2, Preferably, B2 and B3 are the same or different, and are selected independently from each other. Preferably, B2' and B3' are the same or different, and are selected independently from each other. Preferably, B is selected from: Preferably, R 21 R 22 R 23 Selected from H; Preferably, t is selected from 2, 3, or 4; Preferably, M is selected from Preferably, M' is selected from Preferably, ring A is selected from Preferably, R z Selected from the following groups: -(CH2CH2O)z3-CH3, Preferably, R z Selected from the following groups: -(CH2CH2O)9-CH3, -(CH2CH2O) 13 -CH3、 Preferably, z1, z2, z3, z4, and z5 are the same or different, and are independently selected from integers between 6 and 15; Preferably, z1, z2, z3, z4, and z5 are the same or different, and are independently selected from integers from 8 to 14; Preferably, n1, n2, n3, n4, n5, n6, n7, and n8 are the same or different, and are independently selected from 0, 1, 2, and 3; Preferably, Z is selected from single bonds, -N(R) z )-(CH2)n1-C(=O)-, -NH-(CH2CH2O)z1-(CH2)n2-C(=O)-, Wherein, X1 and X2 may be the same or different, and are independently selected from CH or N; preferably, at least one of X1 and X2 is an N atom; R z z1, z2, n1, n2, n3, and n4 have the definitions as described in claim 1 or 2; Preferably, when X2 is CH, n4 is selected from 0; Preferably, Z is selected from single bonds, -NH-(CH2CH2O)8-CH2CH2-C(=O)-, and -NH-(CH2CH2O). 10 -CH2CH2-C(=O)-、-N(R z -CH2-C(=O)-、-N(R) z )-(CH2)2-C(=O)-、-N(R z )-(CH2)3-C(=O)-、-N(R z )-(CH2)4-C(=O)-、 Among them, R z It has the definition as described in claim 1 or 2; Preferably, Z is selected from single bonds, Preferably, Tr is selected from glycine-glycine-phenylalanine-glycine (GGFG), i.e. Preferably, Tr is selected from valine-alanine (VA), that is: Preferably, Tr' is selected from Preferably, L is selected from single bonds; Preferably, L is selected from 3. The antibody-drug conjugate or its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound according to claim 1 or 2, characterized in that, The antibody-drug conjugates mentioned are selected from: Wherein, Ab and β have the characteristics defined as in any one of claims 1-2; Preferably, the antibody-drug conjugate is selected from... Wherein, β has the characteristics defined as in any one of claims 1-2; More preferably, the antibody-drug conjugate is selected from the compounds in the examples: Pertuzumab(A)-L001-001, Pertuzumab(A)-L001-002, Pertuzumab(A)-L002-001, Pertuzumab(A)-L002-002, Pertuzumab(A)-L002-011, Pertuzumab(A)-L002-012, Pertuzumab(A)-L002-013, Pertuzumab(A)-L002-014, Pertuzumab(A)-L002-015 and / or Pertuzumab(A)-L002-016.

4. An intermediate B”, the structure of which is: in, B1', B0, B2', B3' are as defined in any one of claims 1-3; Preferably, the intermediate B” is selected from:

5. A connector “Link”, the structure of which is: B'-(MZ-Tr-L')2; in, B' is B1', B0, B2, B3, M, Z, Tr, and L' each have the definition of any one of claims 1-3 independently; Preferably, B' is selected from: Preferably, the "Link" is selected from the following structures:

6. A linker-drug of formula (II) or a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound thereof, having the structure: B'-(MZ-Tr-LD)2; wherein, B', M, Z, Tr, L, and D independently have the definitions described in any one of claims 1-3; Preferably, the linker-drug structure is as follows: Preferably, the linker-drug shown in formula (II) is selected from the following structures:

7. A linker-drug fragment of formula (II-1), having the structure: B-(MZ-Tr-LD)2; wherein, B, M, Z, Tr, L, and D independently have the definitions described in any one of claims 1-3; Preferably, the structure of the linker-drug fragment is as follows: Preferably, the linker-drug fragment shown in formula (II-1) is selected from the following structures:

8. A linker-drug represented by formula (III): M'-Z-Tr-LD (III) in, M', Z, Tr, L and D are independent of each other and have the definitions described in any one of claims 1-3; Preferably, the linker-drug represented by formula (III) is selected from the following structures: Among them, Z and R z X1, X2, Tr, L, D, z1, z3, n1, n3, n4 have the definitions as described in any one of claims 1-3; Preferably, the linker-drug shown in formula (III) is selected from:

9. An intermediate having the structure Tr'-LD; in, Tr' is the structure before Tr and Z are connected; Tr, L and D independently have the definitions described in any one of claims 1-3; Preferably, the amino group in Tr' is optionally protected by an amino protecting group commonly used in the art, such as Fmoc; Preferably, the intermediate is selected from:

10. An intermediate having the following structure: Wherein Ab, B0, B1, B2', B3', and β independently have the definitions described in any one of claims 1-3; Preferably, the intermediate is selected from: Wherein Ab and β independently have the definitions described in any one of claims 1-3; More preferably, the intermediate is selected from: Wherein β has the definition as described in any one of claims 1-3.

11. A pharmaceutical composition comprising a therapeutically effective amount of at least one of the antibody-drug conjugate of any one of claims 1-3 or a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound thereof.

12. Use of at least one of the antibody-drug conjugates according to any one of claims 1-3 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for the prevention or treatment of a disease or condition; Preferably, the disease or symptom is a tumor, including breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia.

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