Methods for preparing and using antibody drug conjugates

Abstract

The objective of the present invention is to provide a method for preparing and using an antibody-drug conjugate. [Solution] The present application relates to methods for the preparation and use of antibody-drug conjugates, in particular to compounds or their tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or pharma-ceutically acceptable salts, prodrugs or solvates thereof, as well as methods for the preparation of said compounds and related antibody-drug conjugates and their use in the preparation of drugs for the treatment of cancer.

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to methods for preparing and using antibody-drug conjugates. [Background technology]

[0002] Antibody drug conjugates (ADCs) as novel targeted therapeutic agents deliver drugs to the affected area via suitable carriers. However, due to technical limitations of antibodies and highly active cytotoxic drugs, only a limited number of ADCs have been approved for market launch, and rapid development is needed for the application of ADCs in the field of tumor therapy.

[0003] ADC drugs require cytotoxic substances to exert their effects, and camptothecin drugs have great prospects for application. Trodelvy and Enhertu, which have launched ADC drugs, use camptothecin drugs SN38 and DX-8951f as warhead molecules, respectively. However, the ADC finally prepared through camptothecin drugs has significantly changed the properties of monoclonal antibodies, and the stability and half-life have been greatly shortened. In the DESTINY-Breast03 clinical trial, the objective response rate (ORR) confirmed by Enhertu reached 79.7%, but according to the DS8201-A-J101 clinical trial, the objective response rate (ORR) confirmed by Enhertu for HER2 low-expressing breast cancer was 37.0%, and the treatment efficacy rate was significantly lower than that of high-expressing or medium-expressing breast cancer. In addition, ADC drugs have new symptoms in terms of toxic side effects, such as side effects such as pneumonia and interstitial pneumonia caused by the drug Enhertu. Patent Document 1 discloses a camptothecin compound containing a valine-citrulline (Val-Cit)-PAB linker, but the molecule cannot be conjugated with an antibody to obtain a quality-qualified ADC molecule (the aggregate content in a qualified ADC product is less than 5%). Therefore, it is an urgent task in this field to provide a more suitable antibody-drug conjugate based on camptothecin drugs (such as exatecan, belotecan, etc.), achieve efficient, simple and practical chemical preparation and conjugation, and improve the pharmaceutical properties, metabolic properties, efficacy and safety of conventional antibody-drug conjugates (such as improving the molecular stability of ADC, widening the therapeutic window, etc.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Application Publication No. WO2020233174A1 [Patent Document 2] International Application Publication No. WO2017042210A1 [Patent Document 3] International Application Publication No. WO2014172371A2 [Patent Document 4] U.S. Patent Publication No. US20060275305A1 [Patent Document 5] International Application Publication No. WO2003074566 [Patent Document 6] Chinese Patent No. CN102174105B Publication [Patent Document 7] International Application Publication No. WOWO2018212136 [Patent Document 8] U.S. Patent Publication No. US20200171163A1 [Patent Document 9] International Application Publication No. WO2016038383 [Patent Document 10] U.S. Patent Publication No. US11173213B2 [Non-patent literature]

[0005] [Non-Patent Document 1] Protein Science,1995,vol.4,2411-2423 Summary of the Invention [Means for solving the problem]

[0006] The present application provides antibody-drug conjugates, their intermediates, preparation methods and uses. The antibody-drug conjugates of the present application can realize the wide application of cytotoxic drugs in the ADC field to treat tumor diseases. The main technical effect of the present application is that the novel linker provided can bind highly hydrophobic antitumor drugs (such as topoisomerase inhibitors exatecan and belotecan) to antibodies through a specific chemical method, and the resulting conjugates have high hydrophilicity and stability; compared with conventional ADC linkers, the linkers provided in the present application make the antibody conjugates less likely to form aggregates at high drug-antibody ratios; compared with the same ADC drug, the toxin molecules released by the present application are the original molecules of topoisomerase inhibitors exatecan and belotecan, and tests have shown that the drug molecules have better biological activity, safety and other drug-related properties than the drug derivatives released by the same ADC drug. Therefore, the present application is capable of extending the in vivo half-life of the drug and increasing the drug concentration in tumor tissue, thereby improving the antitumor activity of the drug and / or widening the therapeutic window of the entire drug.

[0007] In one aspect, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure represented by formula (C-Trop-2):

[0008] [ka] [In the formula, Q1 comprises a linker, L1 is -L 1a Contains -C(=O)-, Here, L 1ais selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted heteroaliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L2 comprises an optionally substituted polypeptide residue; L3 comprises an optionally substituted spacer group; wherein L2 and / or L3 comprise optionally substituted polysarcosine residues; T comprises a Drug unit; Ab is a ligand capable of binding to Trop-2, and m is a number from 1 to 8.

[0009] In another aspect, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure selected from the group consisting of:

[0010] [ka] [ka] [ka]

[0011] In another aspect, the present application provides a pharmaceutical composition comprising a compound according to any one of the claims herein, or a tautomer, meso, racemate, enantiomeric, and diastereomeric form thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, and optionally a pharma- ceutically acceptable carrier.

[0012] In another aspect, the present application provides the use of a compound described in any one of the present application, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, and / or a pharmaceutical composition described in the present application, in the preparation of a medicament for treating and / or preventing a tumor.

[0013] In another aspect, the present application provides an antibody-drug conjugate formed by conjugating a compound of the present application with an antibody.

[0014] In one embodiment, there is one or more pharmaceutical moieties covalently attached to the conjugate of the present application.

[0015] In one embodiment, the antibody and drug of the present application are linked by a covalent bond (eg, each may be covalently attached to a linker).

[0016] In another aspect, the present application provides a method for preparing an antibody-drug conjugate, comprising generating a pair of cysteine ​​residues through reduction of disulfide bonds in a hinge region of an antibody or a fragment thereof, and binding a compound of the present application to the mercapto group of cysteine ​​of the antibody or antibody fragment through a substitution reaction between the mercapto group of the cysteine ​​residue and a linking group such as a maleimide group of the compound of the present application, thereby obtaining an antibody-drug conjugate, in which the drug-antibody ratio DAR (e.g., m in the present application) is controlled depending on the reaction conditions and is usually, for example, 2 to 8.

[0017] In another embodiment, m in the present application represents the molar ratio of cytotoxic drug molecules to Ab (DAR, also known as drug-antibody ratio), which may be an integer or a decimal number, and is understood to be the average molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after conjugation of a single monoclonal antibody molecule with a cytotoxic drug, and can generally be measured by methods such as hydrophobic-interaction chromatography (HIC), reverse phase HPLC (RP-HPLC), SDS polyacrylamide gel electrophoresis (SDS PAGE, elecTrophoresis), liquid chromatography-mass spectrometer (LC-MS), ultraviolet / visible spectroscopy (UV / Vis), etc.

[0018] In another aspect, the present application provides a method for preparing a compound of the present application, comprising the steps of contacting an amino acid activated ester having an amino protecting group N1 with an amino acid to obtain intermediate M1, contacting the intermediate M1 with a substituted / unsubstituted p-aminobenzyl alcohol in the presence of a condensing agent such as EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) to obtain intermediate M2, removing the N1 of the intermediate M2 to obtain intermediate M3, contacting intermediate M3 with a compound containing a maleimide group to obtain intermediate M4, contacting intermediate M4 with bis(4-nitrophenyl)carbonate to obtain intermediate M5, and contacting intermediate M5 with a Drug unit.

[0019] In one embodiment, N1 comprises fluorenylmethoxycarbonyl.

[0020] In one embodiment, when preparing a compound in which L2 is substituted with a structure containing a polysarcosine residue, an intermediate containing an amino protecting group N2 is contacted with trifluoroacetic acid and then contacted with an acetylated polysarcosine.

[0021] In one embodiment, said N2 comprises tert-butoxycarbonyl.

[0022] In another aspect, the present application provides a method for preparing a compound of the present application, comprising contacting a ligand with a compound of the present application under conditions suitable for forming a bond between the ligand and the compound.

[0023] In one embodiment, the ligand is contacted with the compound in a mixture of a buffer and an organic solvent.

[0024] In one embodiment, the ligand is contacted with the compound at about 0 to 37°C.

[0025] In one embodiment, the method includes reacting the ligand with a reducing agent in a buffer to obtain a reduced ligand prior to contacting the ligand with the compound of the present application.

[0026] In one embodiment, after obtaining the reduced ligand, the method includes removing the reducing agent prior to contacting the ligand with a compound of the present application.

[0027] In one embodiment, the step of removing the reducing agent comprises passing the reaction product through a desalting column and / or ultrafiltration.

[0028] In one embodiment, the reducing agent is selected from the group consisting of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), β-mercaptoethanol, β-mercaptoethylamine hydrochloride and dithiothreitol (DTT).

[0029] In one embodiment, the buffer is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA) buffer, disodium hydrogen phosphate-citric acid / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA), boric acid-borax / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA), histidine-sodium hydroxide / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA) and PBS / diethyltriaminepentaacetic acid (DTPA).

[0030] In one embodiment, the organic solvent is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA) and dimethylsulfoxide (DMSO).

[0031] In one embodiment, the organic solvent does not exceed 30% by volume in the mixture of buffer and organic solvent.

[0032] The present application has found that, compared with conventional antibody-drug conjugates, the hydrophobicity of the conjugates of the present application is significantly increased by the introduction of polysarcosine, since the drugs such as exatecan and belotecan are too hydrophobic, and the resulting antibody-drug conjugates are more stable overall and less prone to aggregation. The present application provides carbamates that can undergo rapid 1,6-elimination to release the drug after enzymatic cleavage, and have better in vitro and in vivo stability and bioactivity. Based on these findings, the present invention has been completed.

[0033] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. Based on the contents of the present application, it will be understood by those skilled in the art that modifications can be made to the specific embodiments disclosed, and such modifications are within the spirit and scope of the invention of the present application.

[0034] It should be noted that the accompanying drawings and the description in the present application are merely illustrative and are not intended to be limiting.

[0035] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments in the following detailed description and the accompanying drawings.

[0036] A brief description of the accompanying drawings follows: [Brief description of the drawings]

[0037] [Figure 1] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 1 of the present application. [Diagram 2] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 2 of the present application. [Diagram 3] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 2 of the present application. [Figure 4] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 3 of the present application. [Diagram 5] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 4 of the present application. [Figure 6] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 4 of the present application. [Figure 7] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 5 of the present application. [Figure 8] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 5 of the present application. [Figure 9]FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 6 of the present application. [Figure 10] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 6 of the present application. [Figure 11] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 7 of the present application. [Figure 12] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 7 of the present application. [Figure 13] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 8 of the present application. [Figure 14] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 8 of the present application. [Figure 15] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 9 of the present application. [Figure 16] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 9 of the present application. [Figure 17] 1 is a chromatogram of size-exclusion chromatography (SEC-HPLC) of the antibody conjugate 10 of the present application. [Figure 18] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 10 of the present application. [Figure 19] FIG. 2 shows a chromatogram of size exclusion chromatography (SEC-HPLC) of antibody conjugate 11 of the present application. [Figure 20] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 11 of the present application. [Figure 21] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 12 of the present application. [Figure 22] FIG. 2 shows a chromatogram of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) of antibody conjugate 12 of the present application. [Diagram 23] FIG. 1 shows concentration evolution graphs of accelerated stability studies of several antibody conjugates of the present application. [Figure 24] FIG. 1 shows a graph of aggregate increase in accelerated stability studies of several antibody conjugates of the present application. [Diagram 25] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 13 of the present application. [Figure 26] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 14 of the present application. [Figure 27] FIG. 2 shows a chromatogram of size-exclusion chromatography (SEC-HPLC) of antibody conjugate 15 of the present application. [Figure 28] FIG. 1 shows a comparative graph of in vivo efficacy results of several antibody conjugates of the present application in human lung squamous cell carcinoma NCI-H2170 cells. [Figure 29] FIG. 13 is a graph showing changes in mouse body weight in an in vivo efficacy experiment of several antibody conjugates of the present application against human lung squamous cell carcinoma NCI-H2170 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Hereinafter, embodiments of the present invention will be described with reference to specific examples. Those skilled in the art will easily understand the advantages and effects of the present invention and other aspects from the contents disclosed in this specification.

[0039] Definition of Terms In this application, the term "ligand" generally refers to a polymeric compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand may be to deliver a drug to a target cell population that is bound to the ligand, which may include, but is not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigen molecules. In this application, the ligand may be represented as an Ab, where the ligand antigen forms a linker bond with the linking unit via a heteroatom on the ligand. The ligand may be an antibody or an antigen-binding fragment thereof, which may be selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody. The antibody may be a monoclonal antibody. For example, the antibody may be an antibody or an antigen-binding fragment thereof that targets a target selected from the group consisting of HER2.

[0040] In this application, the term "alkyl" generally refers to a residue obtained by removing a hydrogen atom from an alkane. The alkyl may be substituted or unsubstituted. The term "alkyl" generally refers to a saturated straight or branched chain aliphatic hydrocarbon group having a residue obtained by removing a hydrogen atom from the same carbon atom or from two different carbon atoms of a parent alkane, which may be a straight or branched chain group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 12 carbon atoms, such as an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl may be substituted or unsubstituted. For example, when an alkyl is substituted, the substituent may be substituted at any available point of attachment and the substituent may be optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, e.g., the substituent may be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SON2NH2, -OC(O)H, -N(H)SO2H, or C(O)NH2. 1-6 It may be an aliphatic group.

[0041] As used herein, the term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues obtained by removing two hydrogen atoms, typically from the same or two different carbon atoms of a parent alkane, and may be a straight or branched chain group containing 1 to 20 carbon atoms, e.g., the term "methylene" may refer to a residue obtained by removing two hydrogen atoms from a group of one carbon atom. The methylene may be substituted or unsubstituted, e.g., alkylene containing 1 to 12 carbon atoms, e.g., alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH-), 1,1-ethylene (-CH(CH)-), 1,2-ethylene (-CHCH)-, 1,1-propylene (-CH(CHCH)-), 1,2-propylene (-CHCH(CH)-), 1,3-propylene (-CHCHCHCH-), 1,4-butylene (-CHCHCHCHCH-), and 1,5-butylene (-CHCHCHCHCHCH-). An alkylene group can be substituted or unsubstituted, for example, when an alkylene is substituted, the substituents can be substituted at any available point of attachment, and the substituents can be optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo. For example, the substituents can be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SON2NH2, -OC(O)H, -N(H)SO2H, or C(O)NH2. 1-6 It may be an aliphatic group. The methylene or alkylene group may be substituted or unsubstituted.

[0042] In this application, the term "alkenyl" generally refers to a straight or branched chain hydrocarbon group containing one or more double bonds. Specific examples of alkenyl include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, hexenyl, etc. 2-6 Specific examples of alkenyl groups include butadienyl, pentadienyl, hexadienyl, hexatrienyl, and branched forms thereof. The position of the unsaturated bond (double bond) may be anywhere in the carbon chain. The alkenyl group may be substituted or unsubstituted.

[0043] As used herein, the term "alkenylene" generally refers to a residue obtained by removing two hydrogen atoms from an alkene carbon atom. Examples include arylene, vinylene, butenylene, pentenylene, hexenylene, etc. Alkenylene groups may be substituted or unsubstituted.

[0044] In this application, the term "alkynyl" generally refers to unsaturated straight or branched chain alkynyl, for example, ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl may be substituted or unsubstituted.

[0045] In this application, the term "alkynylene" generally refers to a residue obtained by removing two hydrogen atoms from a carbon atom of an alkyne. Examples include ethynylene, propynylene, propargylene, butynylene, etc. The alkynylene group may be substituted or unsubstituted.

[0046] As used herein, the term "aryl" generally refers to the residue obtained by removing one hydrogen atom from an aromatic ring. The term "aromatic ring" refers to a 6-14 membered all-carbon monocyclic or fused polycyclic ring (i.e., rings sharing adjacent pairs of carbon atoms) having a conjugated pi-electron system, which may be 6-10 membered, such as benzene and naphthalene. The aromatic ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, with the ring attached to the parent structure being the aryl ring. The aryl group may be substituted or unsubstituted, and when the aryl is substituted, the substituents may be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups. The aryl group may be substituted or unsubstituted.

[0047] In this application, the term "arylene" generally refers to a residue obtained by removing two hydrogen atoms from a carbon atom of an aromatic ring. Examples include phenylene, naphthylene, etc. Arylene may be substituted or unsubstituted.

[0048] As used herein, the term "heteroaryl" generally refers to the residue obtained by removing one hydrogen atom from a carbon atom of a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1-4 heteroatoms and 5-14 ring atoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur and nitrogen. Heteroaryl may be 5-10 membered, 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. Heteroaryl rings may be fused to aryl, heterocyclyl or cycloalkyl rings, where the ring attached to the parent structure is a heteroaryl ring. Heteroaryl groups can be optionally substituted or unsubstituted, and when heteroaryl is substituted, the substituents can be one or more groups independently selected from the group consisting of alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen groups, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups. Heteroaryl groups can be substituted or unsubstituted.

[0049] In this application, the term "heteroarylene" generally refers to a residue obtained by removing two hydrogen atoms from a carbon atom of a heteroaromatic ring. Examples include furanine, thienylene, pyridylene, pyrrolylene, pyrimidinylene, pyrazinylene, imidazolylene, tetrazolylene, etc. Heteroarylene may be substituted or unsubstituted.

[0050] As used herein, the term "aliphatic cyclyl" generally refers to a residue obtained by removing a hydrogen atom from the same or different carbon atoms of an aliphatic ring. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocyclic ring containing 3 to 20 carbon atoms, and may contain 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of aliphatic cyclyls include cyclopropyl, cyclobutanyl, cyclopentanyl, cyclopentenyl, cyclohexanyl, cyclohexenyl, cyclohexadienyl, cycloheptanyl, cycloheptatrienyl, cyclooctanyl, and the like. Polycyclic carbocycles may include spiro, fused, and bridged carbocycles. Aliphatic cyclyls may be substituted or unsubstituted. As used herein, the term "carbocyclyl" generally refers to a residue obtained by removing a hydrogen atom from a carbon atom having a carbocyclic ring. The term "carbocycle" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocycle containing 3 to 20 carbon atoms, and may contain 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, and the like. Polycyclic carbocycles may include spiro, fused, and bridged carbocycles. Carbocyclyl groups may be substituted or unsubstituted. In some cases, alicyclic and carbocycles may be used interchangeably.

[0051] As used herein, the term "partially unsaturated" generally refers to cyclic structures that contain at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures that have multiple sites of unsaturation, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" means that the moiety has one or more degrees of unsaturation.

[0052] In this application, the term "aliphatic cyclylene" generally refers to a residue obtained by removing two hydrogen atoms from an aliphatic carbon atom. Examples include cyclopropanylene, cyclobutanylene, cyclopentanylene, cyclopentenylene, cyclohexanylene, cyclohexenylene, cyclohexadienylene, cycloheptanylene, cycloheptatrienylene, cyclooctanylene, etc. Polycyclic carbocycles may include spiro, fused, and bridged carbocycles. Aliphatic cyclylenes may be substituted or unsubstituted.

[0053] As used herein, the term "aliphatic heterocyclyl" generally refers to a stable non-aromatic 3- to 7-membered monocyclic carbocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. These ring structures may be saturated or partially saturated, and in addition to carbon atoms, these ring structures also contain one or more heteroatoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these ring structures contain 1 to 4 heteroatoms as defined above. When used to represent an atom on an aliphatic heterocyclic ring structure, the term "nitrogen" may include a substituted nitrogen that has undergone a substitution reaction. For example, aliphatic heterocyclic groups can include "heterocycloalkyl", which can refer to a stable non-aromatic 3- to 7-membered monocycloalkane structure, a fused 7- to 10-membered bicyclic heterocyclic structure, or a bridged 6- to 10-membered bicyclic heterocyclic structure, where in addition to carbon atoms, these ring structures also contain one or more heteroatoms, which can be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these ring structures contain 1 to 4 heteroatoms as defined above. Heterocycloalkyl groups can be substituted or unsubstituted. Aliphatic heterocyclyl can be substituted or unsubstituted.

[0054] As used herein, the term "heteroaliphatic heterocyclylene" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atoms of an aliphatic heterocycle. The heteroaliphatic heterocyclylene may be substituted or unsubstituted.

[0055] In this application, the term "optionally" or "optionally" generally means that the subsequently described event or circumstance may or may not occur, and thus the description includes the event or circumstance in which it occurs and in which it does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may or may not be present, and may describe the heterocyclic group in which it is substituted with an alkyl group and in which it is not substituted.

[0056] In this application, the term "substituted" generally refers to one or more (e.g., up to 5) hydrogen atoms in a group, e.g., 1 to 3 hydrogen atoms, being independently replaced by a corresponding number of substituents. Substituents are present only at possible chemical positions, and one of ordinary skill in the art can identify (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when attached to carbon atoms with unsaturated (e.g., olefinic) bonds.

[0057] As used herein, as will be appreciated by those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl" and the like may in certain cases be preceded by a symbol indicating the number of atoms present in the group, e.g., C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., where the subscript following the "C" represents the number of carbon atoms present in the group. For example, a C3 alkyl group refers to an alkyl group having 3 carbon atoms (e.g., n-propyl, isopropyl), ... 1-10 In the formula, the group members can have any number of carbon atoms ranging from 1 to 10.

[0058] It refers to the fact that one or more (e.g., up to 5) hydrogen atoms in a group, e.g., 1 to 3 hydrogen atoms, are independently replaced by a corresponding number of substituents. Substituents are present only at possible chemical positions, and one of ordinary skill in the art can identify (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when attached to carbon atoms with unsaturated (e.g., ethylenic) bonds.

[0059] In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, a compound having a molecular weight of 500 or less, a compound having a molecular weight of 1000 or less, or a compound having a molecular weight of 1000 or more, or a compound having a molecular weight of 10,000 or more, or a compound having a molecular weight of 100,000 or more. In the present application, the compound may also refer to a compound linked by a chemical bond, for example, a compound in which one or more molecules having a molecular weight of 1000 or less are linked to a biopolymer by a chemical bond. The biopolymer may be a polysaccharide chain, a protein, a nucleic acid, a polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is linked to one or more molecules having a molecular weight of 1000 or less, may include a compound in which a protein is linked to one or more molecules having a molecular weight of 10,000 or less, or may include a compound in which a protein is linked to one or more molecules having a molecular weight of 100,000 or less.

[0060] In this application, the term "comprising" generally means the inclusion of the specifically specified feature but without excluding other elements. The terms "more than" and "less than" generally mean an inclusive number.

[0061] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above and below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0062] In the present application, the present compounds include their tautomers, meso-isomers, racemates, enantiomers, and / or diastereomers. In the present application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers may have different physical properties such as melting points, boiling points, spectral properties, and reactivity. In the present application, the terms "tautomers" or "tautomeric forms" are used interchangeably and generally refer to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions by migration of a proton, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by rearrangement of some bond electrons. As used herein, the term "meso isomer" generally refers to a molecule that contains an asymmetric atom, but has a plane of symmetry within the molecule, resulting in zero intramolecular optical rotation. The term "racemate" or "racemic mixture" refers to a composition consisting of equimolar amounts of two enantiomers.

[0063] In this application, certain atoms of the compounds of the present application may exist in one or more isotopes. For example, hydrogen is represented by protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), and carbon can exist as three different isotopes ( 12 C. 13 C and 14 Examples of isotopes that can be incorporated into the compounds of the present invention include: 15 N, 18 O. 17 O. 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125I, or similar isotopes. Thus, the compounds of the present invention can be enriched in one or more of these isotopes compared to their natural abundance. Such isotopically enriched compounds can be used in a variety of applications, as known to those skilled in the art. For example, heavy isotopes (e.g., deuterium ( 2 Substitution with deuterium (H) may confer certain therapeutic advantages due to greater metabolic stability. 2 The natural abundance of deuterium (H) is about 0.015%. Thus, in nature, there is about one deuterium atom for every 6500 hydrogen atoms. Thus, the deuterium-containing compounds of the present application have a deuterium abundance of greater than 0.015% at one or more positions (in some cases). Unless expressly indicated otherwise, the structures described herein may include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are identical to the structures of the present application except that a hydrogen atom is replaced by deuterium or tritium, or a carbon atom is replaced by carbon-13 or carbon-14, are within the scope of the present application.

[0064] In the present application, the term "pharmaceutical composition" generally refers to a mixture of one or more compounds described in the present application, or their physiologically / pharmaceutical acceptable salts or prodrugs, with other chemical components, and other components (e.g., physiologically / pharmaceutical acceptable salt carriers and excipients). Pharmaceutical compositions can facilitate administration to a living body and facilitate the absorption of active ingredients to exert biological activity. Conventional pharmaceutical compositions can be prepared by referring to techniques commonly used in the art.

[0065] In the present application, the term "pharmaceutical acceptable salt" generally refers to a salt of the present compound or ligand-drug conjugate, or a salt of a compound described in the present application. Such salts may be safe and / or effective when used in a mammalian body and have the desired biological activity. The antibody-antibody-drug conjugate compound of the present application may form a salt with an acid, and non-limiting examples of pharmaceutical acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, esylate, benzenesulfonate, or p-toluenesulfonate.

[0066] As used herein, the term "conjugate" generally refers to a compound prepared by one or more chemical reactions of the compounds of the present application, or by linking the compounds of the present application to one another via one or more linking structures, such as a bridge, spacer, or linking moiety.

[0067] In this application, the term "pharmaceutical acceptable carrier" refers to a carrier generally used for administration of therapeutic agents (e.g., antibodies or polypeptides, genes, and other therapeutic agents). This term refers to a pharmaceutical carrier that does not induce the production of antibodies harmful to the individual to whom the composition itself is administered, and does not have excessive toxicity after use. For example, a pharmaceutical acceptable carrier can be distinguished from a nucleic acid vector used to contain a target gene in genetic engineering. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid polymers, and inactivated virus particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerin, and ethanol. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, etc. may also be present in these carriers.

[0068] In the present application, the term "Trop2" and "TROP2" generally refer to a type 1 cell membrane protein with a single transmembrane. In the present application, the term "Trop2" may also include Trop2 homologs, variants and isoforms (including splicing isoforms). The term "Trop" also includes proteins having one or more sequences in Trop2 homologs, variants and isoforms, and fragments of said sequences, so long as variant proteins (including isoforms) exist. Trop2 may be human Trop2. For example, Uniprot accession number P09758 describes Trop2 and the sequence.

[0069] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2). For example, the term "HER2" refers to any native HER2 from any human source. This term encompasses "full-length" and unprocessed HER2, as well as any form of HER2 resulting from processing in cells (e.g., mature protein). This term also encompasses naturally occurring variants and isoforms of HER2, such as splice variants or allelic variants. For example, Uniprot accession number P04626 provides a description of HER2 and sequence.

[0070] In this application, the term "nectin-4" generally refers to adhesion molecule 4. For example, the term "nectin-4" refers to any native nectin-4 from any human source. This term encompasses "full-length" and unprocessed nectin-4, as well as any form of nectin-4 resulting from processing in cells (e.g., mature protein). This term also encompasses naturally occurring variants and isoforms of nectin-4, such as splice variants or allelic variants. For example, Uniprot accession number Q96NY8 provides a description of nectin-4 and the sequence.

[0071] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and which can reduce the immune response induced by the mouse antibody. To prepare a chimeric antibody, a hybridoma secreting a specific monoclonal antibody derived from a mouse is established, the variable region genes are cloned from the mouse hybridoma cells, and if necessary, the constant region gene of a human antibody is cloned, and the mouse variable region genes and the human constant region genes are linked to the chimeric gene, which is then inserted into an expression vector to express the chimeric antibody molecule in a eukaryotic or prokaryotic system.

[0072] In this application, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to an antibody produced by grafting mouse CDR sequences onto the variable region framework of a human antibody, i.e., the framework sequences of a different type of human germline antibody. By retaining a large amount of mouse protein components, it is possible to overcome the xenogeneic reactions caused by chimeric antibodies. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.

[0073] In this application, the term "fully human antibody", "fully human type antibody", or "fully human-derived antibody" is also known as "fully human monoclonal antibody", in which the variable and constant regions of the antibody are both of human origin, which can eliminate immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibody or ligand described in this application may be a fully human monoclonal antibody. Technologies for producing fully human antibodies include human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody production technology, and single B cell antibody production technology.

[0074] In this application, the term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that generally contributes mainly to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., Chothia et al., MacCallum et al. As used in this application, the Kabat CDR definition applies only to the CDR1, CDR2 and CDR3 (CDR H1, CDR H2, CDR H3 or H1, H2, H3) of the heavy chain variable domain in addition to the CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain.

[0075] In the present application, the term "a group capable of bonding to a mercapto" means that compound A has a group capable of bonding to a mercapto, compound B has a group capable of bonding to a mercapto, and compound B reacts with the mercapto of compound A via the group capable of bonding to a mercapto, thereby realizing the linkage between compound A and compound B.

[0076] In this application, the term "linker" generally refers to a chemical structure fragment or bond that is linked to one group at one end and another group at the other end, and can be linked to another linker and then linked to a drug and / or a ligand. Direct or indirect linkage to a ligand can mean that the group is directly linked to the ligand via a covalent bond, or it can be linked to the ligand via a linker. For example, the linker can be a structure shown in Q1 described in this application. For example, chemical fragments or bonds including acid-labile linker structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linker structures, photolabile linker structures, dimethyl linker structures, or disulfide-containing linker structures can be used as linkers.

[0077] In this application, the term "linking group" generally refers to a group that has the ability to be linked to another group.For example, in the case of a compound having a linking group, the compound can be linked to another group through a coupling reaction between the linking group and another group.For example, a maleimide group can be used as a linking group.

[0078] As used herein, the term "drug unit" generally refers to a chemical moiety to which an antibody or antigen-binding fragment binds, directly or indirectly, to form an immunoconjugate. For example, a "drug unit" includes, but is not limited to, a compound having antitumor activity as described herein. For example, a drug unit includes a topoisomerase inhibitor.

[0079] In this application, the term "compound with antitumor activity" generally refers to a compound that has the ability to reduce the proliferation rate, survival rate, or metastatic activity of tumor cells. For example, antitumor activity may be demonstrated by a decrease in the proliferation rate of abnormal cells or a stabilization or reduction in tumor size during treatment, or an increase in survival time due to treatment compared to a control without treatment. Antitumor activity can be evaluated using recognized in vitro or in vivo tumor models (e.g., xenograft models).

[0080] In some embodiments of the present invention, the biologically active molecule in the complex is a compound having antitumor activity, specifically, for example, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 or radioisotopes of Lu, metal complexes such as metal-platinum complexes (such as oxaliplatin) or metal-gold complexes, glycopeptide antibiotics such as bleomycin or pingyangmycin, topoisomerase inhibitors, e.g. drugs that disrupt DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, agents acting on structural proteins such as microtubule inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansinoids, auristatins, Tubulysin B or eribulin), tumor signaling pathway inhibitors (such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors), proteasome inhibitors, epigenetic related target inhibitors, tumor angiogenesis inhibitors, cyclin dependent kinase inhibitors, In this application, the term "topoisomerase inhibitor" generally refers to compounds or derivatives thereof, including topoisomerase I inhibitors and topoisomerase II inhibitors. Examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs, topoisomerase II inhibitors (such as actinomycin D, adriamycin, doxorubicin, amikacin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide). Topoisomerase may refer to an enzyme that breaks the phosphodiester bond of one or both strands of DNA, and then rewinds and seals it to correct the number of DNA strands.

[0081] In this application, the term "camptothecin analog" generally refers to a compound that is structurally similar to or derived from camptothecin. For example, the structure of camptothecin is set forth in CAS No. 7689-03-4. For example, camptothecin analogs may refer to Exatecan (CAS No. 171335-80-1) or Belotecan (CAS No. 256411-32-2). The term "non-camptothecin type topoisomerase I inhibitors" generally refers to heterocyclic molecules with topoisomerase I inhibitory activity of indolocarbazoles, indenoisoquinolinones, benzophenanthridines, and dibenzonaphthyridinones, mainly Genz-644282 (CAS No. 529488-28-6).

[0082] In the present application, the term "disease associated with expression of a target" generally means that the occurrence and / or progression of the disease is associated with the expression level of the target. For example, the expression level of a target is increased, i.e., high expression, in cells derived from a diseased area (e.g., a specific tissue or organ of a patient) compared to the expression level of normal cells derived from the tissue or organ. Or, the expression level of a target is decreased, i.e., low expression, in cells derived from a diseased area (e.g., a specific tissue or organ of a patient) compared to the expression level of normal cells derived from the tissue or organ. Or, for example, cells derived from a diseased area (e.g., a specific tissue or organ of a patient) express a target, i.e., are positive. Or, for example, cells derived from a diseased area (e.g., a specific tissue or organ of a patient) do not express a target, i.e., are negative. Target expression can be characterized by standard assays known in the art.

[0083] In this application, the term "effective amount" generally refers to the amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or that exhibits a detectable therapeutic or preventive effect. The exact effective amount for a subject will depend on the subject's physical shape and health condition, the nature and extent of the disease, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For this reason, it is not useful to specify the exact effective amount in advance. However, for a given situation, a clinician can determine the effective amount by routine experimentation.

[0084] Unless otherwise indicated, all compounds presented herein are intended to include all possible optical isomers, including single chiral compounds or mixtures of various chiral compounds (i.e., racemates). In all compounds herein, each asymmetric carbon atom may optionally be in the R or S configuration, or a mixture of the R and S configurations.

[0085] As used herein, the term "compound of the present application" refers to a compound of the present application. This term also includes various crystal forms, pharma- ceutically acceptable salts, hydrates, or solvates of the compound of the present application.

[0086] When trade names are used herein, the trade names are intended to include formulations of the trade name products, their corresponding generic drugs, and the active pharmaceutical ingredients of the trade name products.

[0087] As used herein, the term "antibody" is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. A target antigen usually has multiple binding sites, also called epitopes, that are recognized by the CDRs of various antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have one or more corresponding antibodies. An antibody is a molecule that contains a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen or a portion thereof that specifically binds to a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins described herein may have any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins may be derived from any species. However, in one embodiment, the immunoglobulins are derived from human, mouse, or rabbit. An "antibody fragment" may include a portion of a full-length antibody, typically the antigen-binding region or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, minibodies, Fab expression library prepared fragments, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), any of the above epitope-binding fragments that bind to cancer cell antigens, viral antigens, or microbial antigens in an immunospecific manner, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. The antibodies constituting the antibody-drug conjugates in the present application may maintain their antigen-binding ability in their original wild state. Therefore, the antibody in this application is capable of specifically binding to the antigen.The antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (such as known or predicted functional molecules), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, molecules involved in angiogenesis (e.g., antigens bound by known antibodies may be one or a subset of the above classifications, while other subsets include other molecules / antigens with special properties (compared to the target antigen)). Antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be made based on antibody production methods and information well known in the art. These targets can be specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides can be overexpressed on the surface of cancer cells compared to the surface of non-cancer cells.

[0088] In this application, the term "enfortumab" refers to an antibody that targets nectin-4. For example, enfortumab is described in Patent Document 2. In this application, enfortumab may refer to any antibody or antigen-binding fragment that contains the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of enfortumab. In this application, enfortumab may refer to any antibody or antigen-binding fragment that contains the heavy chain variable region and the light chain variable region of enfortumab.

[0089] In this application, the term "pertuzumab" generally refers to an antibody that targets HER2. For example, pertuzumab is described in Patent Document 3. In this application, pertuzumab may refer to any antibody or antigen-binding fragment that includes the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of pertuzumab. In this application, pertuzumab may refer to an antibody or antigen-binding fragment that includes the heavy chain variable region and the light chain variable region of pertuzumab.

[0090] In this application, the term "trastuzumab" generally refers to an antibody that targets HER2. For example, trastuzumab is described in Patent Document 4. In this application, trastuzumab may refer to any antibody or antigen-binding fragment that contains the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of trastuzumab. In this application, trastuzumab may refer to an antibody or antigen-binding fragment that contains the heavy chain variable region and the light chain variable region of trastuzumab.

[0091] In this application, the term "sacituzumab" generally refers to an antibody that targets Trop2. For example, sacituzumab (hRS7) is described in Patent Document 5. In this application, sacituzumab may refer to any antibody or antigen-binding fragment that includes the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of sacituzumab. In this application, sacituzumab may refer to an antibody or antigen-binding fragment that includes the heavy chain variable region and the light chain variable region of sacituzumab.

[0092] In this application, the term "patritumab" generally refers to an antibody that targets HER3. For example, patritumab is described in Patent Document 6. In this application, patritumab may refer to any antibody or antigen-binding fragment that contains the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of patritumab. In this application, patritumab may refer to an antibody or antigen-binding fragment that contains the heavy chain variable region and the light chain variable region of patritumab.

[0093] In this application, the term "antibody H01L02" generally refers to an antibody that targets CDH6. For example, monoclonal antibody H01L02 is described in Patent Document 7 and Patent Document 8. For example, H01L02 monoclonal antibody may be a monoclonal antibody employed in pharmaceutical DS6000. In this application, H01L02 monoclonal antibody may refer to any antibody or antigen-binding fragment that includes the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of H01L02 monoclonal antibody. In this application, H01L02 monoclonal antibody may refer to an antibody or antigen-binding fragment that includes the heavy chain variable region and the light chain variable region of H01L02 monoclonal antibody.

[0094] In the present application, the term "polypeptide residue" generally refers to a residue that includes one or more amino acid residues linked together. For example, one or more amino acids in a polypeptide residue may be optionally substituted. For example, the polypeptide residue of the present application may be selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn), and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0095] As used herein, the term "polyethylene glycol" generally refers to a residue that contains one or more ethylene glycol residues linked together. For example, polyethylene glycol is -(CH2CH2O) p -, where p is a number of at least 1. For example, the polyethylene glycols herein may be optionally substituted.

[0096] In this application, the term "glycol" generally refers to polyethylene glycol. For example, the glycol in this application may be optionally substituted. For example, the number in front of glycol may represent the number of ethylene glycol units in the glycol, for example, a diethylene glycol group may refer to the polymerized residue of two ethylene glycols.

[0097] As used herein, the term "polysarcosine residue" generally refers to a residue that includes one or more sarcosine residues linked together. For example, a polysarcosine residue is -(COCHN(CH)). q -, where q is a number of at least 1. For example, the polysarcosine residues herein may be optionally substituted. For example, a structure comprising a polysarcosine residue may be [ka] where n2 is a number from 4 to 18.

[0098] As used herein, the term "sodium dodecyl sulfate-polyacrylamide gel electrophoresis" refers generally to a technique for analyzing and characterizing substances, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which can detect the molecular weight of a substance.

[0099] As used herein, the term "hydrophobic chromatography" generally refers to analytical techniques based on differences in the hydrophobicity of substances.

[0100] As used herein, the term "liquid chromatography-mass spectrometry" generally refers to an analytical method for identifying components of a substance. For example, liquid chromatography-mass spectrometry can analyze the molecular weight of a test substance by using liquid chromatography and mass spectrometry.

[0101] In the present application, the term "tumor" generally refers to a mass of tissue that is formed by the autonomous excessive proliferation of tissue. For the present application, angiogenesis is one of the characteristics of tumors. Tumors can be benign or malignant. The term "tumor" is generally used to refer to benign or malignant tumors, and the term "cancer" is generally used to refer to metastatic or non-metastatic malignant tumors. Tumors that can be diagnosed by the method of the present application are selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal stromal tumor, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma, and mesothelioma. When used in research, these tissues can be isolated from readily available sources by methods well known to those skilled in the art.

[0102] Detailed Description of the Invention In one aspect, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure represented by formula (C-Trop-2):

[0103] [ka] [Wherein, Q1 may include a linker, L1 is -L 1a may contain -C(=O)-, Here, L 1amay be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted heteroaliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L2 can include optionally substituted polypeptide residues; L3 may include an optionally substituted spacer group, for example the spacer group of the present application may have autolysis capability. For example, the spacer group of the present application may be an optionally substituted [ka] , or possibly replaced [ka] may include wherein L2 and / or L3 can comprise optionally substituted polysarcosine residues; T can include a Drug unit; Ab may be a ligand capable of binding to Trop-2, and m may be a number from 1 to 8.

[0104] In another aspect, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2-M):

[0105] [ka] [Wherein, Q1 can include a linker; L1 is -L 1a may contain -C(=O)-, Here, L 1a may be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted heteroaliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L2 can include optionally substituted polypeptide residues; L3 may include an optionally substituted spacer group, for example the spacer group of the present application may have autolysis capability. For example, the spacer group of the present application may be an optionally substituted [ka] or possibly replaced [ka] may include wherein L2 and / or L3 can comprise an optionally substituted structural unit -X; T can include a Drug unit; Ab may be a ligand capable of binding to Trop-2, and m may be a number from 1 to 8.

[0106] For example, where L3 is optionally substituted [ka] and optionally substituted [ka] is selected from the group consisting of:

[0107] For example, the benzene ring of L3 may be substituted with an optionally substituted structural unit -X. For example, the structural unit -X may be an optionally substituted [ka] wherein X1 is selected from the group consisting of carbonyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X2 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X3 is hydrogen, and selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, wherein said heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and said C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0108] For example, the benzene ring of L3 may be substituted with an optionally substituted structural unit -X. For example, the structural unit -X may be an optionally substituted [ka] wherein X1 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S, and said C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0109] For example, where the structural unit -X is [ka] It is.

[0110] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2-M2):

[0111] [ka] wherein L can include an optionally substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 can be an optionally substituted isopropyl or an optionally substituted benzyl; R2 can be an optionally substituted methyl, an optionally substituted [ka] or possibly replaced [ka] R3 may be hydrogen or R5, R5 may be optionally substituted [ka] and T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0112] For example, here, Q1 can include a linker attached to a mercapto.

[0113] For example, where Q1 is optionally substituted [ka] , optionally substituted [ka] , optionally substituted [ka] , and optionally substituted [ka] may be selected from the group consisting of:

[0114] For example, here, L 1amay be selected from the group consisting of optionally substituted C1-C7 alkylene groups, optionally substituted diethylene glycol to octaethylene glycol groups, optionally substituted C3-C6 aliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups.

[0115] For example, here, L 1a may be selected from the group consisting of optionally substituted methylene groups, optionally substituted ethylene groups, optionally substituted propylene groups, optionally substituted butylene groups, optionally substituted pentylene groups, optionally substituted diethylene glycol groups, optionally substituted tetraethylene glycol groups, optionally substituted hexaethylene glycol groups, optionally substituted octaethylene glycol groups, and optionally substituted cyclohexylene groups.

[0116] For example, here, L 1a may be selected from the group consisting of optionally substituted methylene, optionally substituted ethylene groups, optionally substituted propylene groups, optionally substituted butylene groups, and optionally substituted pentylene groups.

[0117] For example, here, L 1a may be selected from the group consisting of an optionally substituted diethylene glycol group, an optionally substituted triethylene glycol group, an optionally substituted tetraethylene glycol group, an optionally substituted pentaethylene glycol group, an optionally substituted hexaethylene glycol group, an optionally substituted heptaethylene glycol group, and an optionally substituted octaethylene glycol group.

[0118] For example, here, L 1a may be selected from the group consisting of optionally substituted cyclopropylene groups, optionally substituted cyclobutylene groups, and optionally substituted cyclohexylene groups.

[0119] For example, herein, L2 can comprise optionally substituted polypeptide residues consisting of amino acids selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine.

[0120] For example, herein, L2 can comprise optionally substituted polypeptide residues consisting of amino acids selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine, and lysine.

[0121] For example, herein, L2 may comprise an optionally substituted polypeptide residue selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn), and glycine-glycine-phenylalanine (Gly-Gly-Phe-Gly).

[0122] For example, herein, L2 can comprise an optionally substituted polypeptide residue selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit) and valine-lysine (Val-Lys).

[0123] For example, where L2 comprises a lysine residue, said lysine residue may be replaced with a structure R1 comprising a polysarcosine residue.

[0124] For example, any H contained in L2 may be substituted with R1.

[0125] For example, wherein R1 is optionally substituted [ka] where n1 is a number from 4 to 18, and R is selected from the group consisting of C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy.

[0126] For example, n1 here may be 4 to 18, 8 to 18, 4 to 12, or 8 to 12. For example, n1 here may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0127] For example, where L3 is optionally substituted [ka] and optionally substituted [ka] is selected from the group consisting of:

[0128] For example, herein the benzene ring of L3 may be substituted with structure R2 comprising a polysarcosine residue.

[0129] For example, any H contained in the benzene ring of L3 may be substituted with R2.

[0130] For example, where the benzene ring of L3 is linked to an optionally substituted polysarcosine residue via the structural unit -X-, the structural unit -X- being an optionally substituted [ka] wherein X1 is selected from the group consisting of carbonyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X2 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; and X3 is a covalent bond. or hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, wherein said heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and said C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0131] For example, the benzene ring of L3 is linked to an optionally substituted polysarcosine residue via the structural unit -X-, which is [ka] X1 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, wherein said heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and said C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0132] For example, where the structural unit -X- is an optionally substituted [ka] and said optionally substituted polysarcosine residue is [ka] where n2 is a number from 4 to 18, and R is selected from the group consisting of C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy.

[0133] For example, where the structural unit -X- is an optionally substituted [ka] wherein X1 is selected from the group consisting of carbonyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S; X2 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S; and X3 is a covalent bond or hydrogen, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S. wherein the heteroalkyl comprises 1 to 3 atoms selected from N, O or S; and the C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1 to 8 atoms, and linear-cyclic heteroalkyl comprising 1 to 8 atoms are each independently substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy or cycloalkyl; and the optionally substituted polysarcosine residue is [ka] where n2 is a number from 4 to 18, and R is selected from the group consisting of C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy.

[0134] For example, where the structural unit -X- is an optionally substituted [ka] and said optionally substituted polysarcosine residue is [ka] where n2 is a number from 4 to 18, and R is selected from the group consisting of C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy.

[0135] For example, where the structural unit -X- is an optionally substituted [ka] wherein X1 is selected from the group consisting of carbonyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S; X2 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S; and X3 is a covalent bond or hydrogen, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1-8 atoms, and linear-cyclic heteroalkyl containing 1-8 atoms, said heteroalkyl containing 1-3 atoms selected from N, O, or S. wherein the heteroalkyl comprises 1 to 3 atoms selected from N, O or S; and the C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1 to 8 atoms, and linear-cyclic heteroalkyl comprising 1 to 8 atoms are each independently substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy or cycloalkyl; and the optionally substituted polysarcosine residue is [ka] where n2 is a number from 4 to 18, and R is selected from the group consisting of C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy.

[0136] For example, n2 here may be 4 to 18, 8 to 18, 4 to 12, or 8 to 12. For example, n2 here may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0137] For example, where T may comprise a compound with anti-tumor activity.

[0138] For example, where T can include a topoisomerase inhibitor.

[0139] For example, here T can include camptothecin and non-camptothecin topoisomerase I inhibitors.

[0140] For example, herein, T can include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0141] For example, where T is [ka] The structure may be selected from the group consisting of:

[0142] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2):

[0143] [ka] wherein Q1 can include a linker bonded to mercapto; L1 is -L 1a -C(=O)-, where L 1a may be selected from the group consisting of optionally substituted C1-C7 alkylene, optionally substituted diethylene glycol to octaethylene glycol, optionally substituted C3-C6 aliphatic cyclylene groups, optionally substituted arylene, and optionally substituted heteroarylene; L2 can comprise optionally substituted polypeptide residues consisting of amino acids selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine and lysine; L3 is optionally substituted [ka] may include wherein L2 can include a polysarcosine residue; T may include a topoisomerase inhibitor.

[0144] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2):

[0145] [ka] wherein Q1 can include a linker bonded to mercapto; L1 is -L 1a -C(=O)-, where L 1amay be selected from the group consisting of optionally substituted C1-C7 alkylene, optionally substituted diethylene glycol to octaethylene glycol, optionally substituted C3-C6 aliphatic cyclylene groups, optionally substituted arylene, and optionally substituted heteroarylene; L2 can comprise optionally substituted polypeptide residues consisting of amino acids selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine and lysine; L3 is optionally substituted [ka] may include wherein L3 can include a polysarcosine residue; T may include a topoisomerase inhibitor.

[0146] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2):

[0147] [ka] wherein Q1 is an optionally substituted [ka] may include L1 is -L 1a -C(=O)-, where L 1a can include an optionally substituted C1-C7 alkylene; L2 can comprise a structure selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit) and valine-lysine (Val-Lys); L3 is optionally substituted [ka] may include wherein L2 can include a lysine residue, said lysine residue being optionally substituted [ka] where n1 is a number from 4 to 18; T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0148] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C-Trop-2):

[0149] [ka] wherein Q1 is an optionally substituted [ka] , optionally substituted [ka] , optionally substituted [ka] , and optionally substituted [ka] may be selected from the group consisting of L1 is -L 1a -C(=O)-, where L 1a can include an optionally substituted C1-C7 alkylene; L2 can comprise a structure selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit) and valine-lysine (Val-Lys); L3 is optionally substituted [ka] may include where L3 is an optionally substituted [ka] where n2 is a number from 4 to 18; T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0150] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C2-Trop-2):

[0151] [ka] wherein L can include optionally substituted alkylene, optionally substituted polyethylene glycol, and optionally substituted aliphatic cyclylene groups; R1 can be optionally substituted isopropyl or optionally substituted benzyl; R2 can be optionally substituted methyl, optionally substituted [ka] or possibly replaced [ka] and R3 may be hydrogen or an optionally substituted methyl, where R2 is an optionally substituted [ka] where n1 is a number from 4 to 18, and T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0152] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C2-Trop-2):

[0153] [ka] wherein L can include an optionally substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 can be an optionally substituted isopropyl or an optionally substituted benzyl; R2 can be an optionally substituted methyl, an optionally substituted [ka] or possibly replaced [ka] and R3 may be hydrogen or an optionally substituted methyl, where R2 is an optionally substituted [ka] where n2 is a number from 4 to 18, and T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0154] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C2-Trop-2):

[0155] [ka] wherein L can include optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, and optionally substituted aliphatic cyclylene groups; R1 can be optionally substituted isopropyl or optionally substituted benzyl; R2 can be optionally substituted [ka] R3 may be hydrogen or an optionally substituted methyl, where R4 may be an optionally substituted [ka] where n1 is a number from 4 to 18, and T may include Exatecan (CAS No. 171335-80-1) and / or Belotecan (CAS No. 256411-32-2) and / or Genz-644282 (CAS No. 529488-28-6).

[0156] For example, the present application provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may comprise a structure represented by formula (C2-Trop-2):

[0157] [ka] wherein L can include an optionally substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 can be an optionally substituted isopropyl or an optionally substituted benzyl; R2 can be an optionally substituted methyl, an optionally substituted [ka] or possibly replaced [ka] R3 may be hydrogen or R5, R5 may be optionally substituted [ka] where n2 is a number from 4 to 18, and T may be exatecan (CAS number 171335-80-1) and / or belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0158] For example, the Ab may comprise an anti-Trop-2 antibody or an antigen-binding fragment thereof.

[0159] For example, the antibody may be selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0160] For example, the antibody may comprise a monoclonal antibody.

[0161] For example, the antibody may comprise a bispecific antibody.

[0162] For example, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0163] For example, the heavy chain HCDR1, HCDR2 and HCDR3 and the light chain LCDR1, LCDR2 and LCDR3 of said Ab comprise the heavy chain HCDR1, HCDR2 and HCDR3 and the light chain LCDR1, LCDR2 and LCDR3 of an anti-Trop-2 antibody, respectively.

[0164] For example, the heavy chain variable region VH and the light chain variable region VL of said Ab comprise the heavy chain variable region VH and the light chain variable region VL of an anti-Trop-2 antibody, respectively.

[0165] For example, the heavy and light chains of said Ab comprise the heavy and light chains, respectively, of an anti-HER2 antibody.

[0166] For example, the Ab can include Sacituzumab (hRS7).

[0167] For example, m can be measured by a method selected from the group consisting of hydrophobic chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and liquid chromatography mass spectrometry. For example, m is the average molar ratio of drug molecules to monoclonal antibody molecules in an antibody-drug conjugate obtained after binding a single monoclonal antibody molecule to a cytotoxic drug, and m may be an integer or decimal number of 1 to 8, for example, m may be about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, or about 1 to about 8, for example, m may be 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0168] In another aspect, the present application also provides a compound or its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure selected from the group consisting of:

[0169] [ka] [ka] [ka]

[0170] The ligands described herein may be protein hormones, lectins, growth factors, antibodies or other molecules capable of binding to cells, receptors and / or antigens. For example, the ligands described herein may be anti-Trop-2 antibodies or antigen-binding fragments thereof.

[0171] In the present application, the ligand comprises at least one CDR in the antibody light chain variable region VL. The CDRs described in the present application can be defined according to Kabat or according to Chothia, and CDR sequences defined in various ways are included in the scope of protection of the present application.

[0172] For example, the antigen-binding protein of the present application may comprise CDR1-3 of a heavy chain variable region and CDR1-3 of a light chain variable region, where the CDR1-3 of the heavy chain variable region and the CDR1-3 of the light chain variable region may be the CDR1-3 of the heavy chain variable region and the CDR1-3 of the light chain variable region of Sacituzumab, respectively. For example, the antigen-binding protein of the present application may have the ability to bind to Trop-2.

[0173] For example, the antigen-binding protein of the present application may comprise a heavy chain variable region and a light chain variable region, where the heavy chain variable region and the light chain variable region may be the heavy chain variable region and the light chain variable region of Sacituzumab, respectively. For example, the antigen-binding protein of the present application may have the ability to bind to TROP2.

[0174] For example, an antigen binding protein of the present application can include a heavy chain and a light chain, where the heavy chain and the light chain can be the heavy chain and the light chain of Sacituzumab, respectively.

[0175] For example, the heavy chain amino acid sequence of Sacituzumab can be shown in SEQ ID NO:1, and the light chain amino acid sequence of Sacituzumab can be shown in SEQ ID NO:2.

[0176] To verify the significant advantages of the compounds of the present application, for example, the ligand of the present application can be: For example, the heavy chain amino acid sequence of Sacituzumab can be shown in SEQ ID NO:1, and the light chain amino acid sequence of Sacituzumab can be shown in SEQ ID NO:2. For example, the heavy chain amino acid sequence of Trastuzumab can be shown in SEQ ID NO:3, and the light chain amino acid sequence of Trastuzumab can be shown in SEQ ID NO:4. For example, the heavy chain amino acid sequence of Pertuzumab can be shown in SEQ ID NO:5, and the light chain amino acid sequence of Pertuzumab can be shown in SEQ ID NO:6. For example, the heavy chain amino acid sequence of Enfortumab can be set forth in SEQ ID NO:7, and the light chain amino acid sequence of Enfortumab can be set forth in SEQ ID NO:8. For example, the heavy chain amino acid sequence of Patritumab can be set forth in SEQ ID NO:9, and the light chain amino acid sequence of Patritumab can be set forth in SEQ ID NO:10. For example, the heavy chain amino acid sequence of antibody H01L02 can be set forth in SEQ ID NO:11, and the light chain amino acid sequence of antibody H01L02 can be set forth in SEQ ID NO:12.

[0177] The antibodies of the present application can be produced using techniques well known in the art, such as hybridoma techniques, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art. Variants can refer to amino acid sequence variants of antibodies and covalent derivatives of native polypeptides, so long as they retain biological activity comparable to the native polypeptide. The difference between the amino acid sequence variant and the native amino acid sequence generally consists in the substitution of one or more amino acids in the native amino acid sequence, or the deletion and / or insertion of one or more amino acids in the polypeptide sequence. Deletion variants include fragments of the native polypeptide, N-terminal and / or C-terminal truncation variants. Typically, the amino acid sequence variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more homology with the native sequence.

[0178] The antibody-drug conjugates provided herein can target specific cell populations and bind to specific proteins (antigens) on the cell surface, thereby releasing the drug in an active form into the cells through endocytosis of the conjugate or drug infiltration, so that the antibody-drug conjugates of the present application can be used to treat a target disease, and the antibody-drug conjugates of the present application can be administered to a subject (such as a human) in a therapeutically effective amount via a suitable route. A subject in need of treatment may be a patient at risk for or suspected of having a disease associated with the activity or expression of a specific antigen. Such patients can be identified by conventional medical examination.

[0179] When treated with the antibody-drug conjugates of the present application, they can be delivered by conventional methods in the art. For example, they can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump. Other methods may include the use of various delivery and carrier systems using the conjugates and biodegradable polymers.

[0180] In one aspect, the present application provides a pharmaceutical composition that may include a compound described in any one of the claims herein, or in the form of a tautomer, meso-isomer, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, and optionally a pharma- ceutically acceptable carrier.

[0181] The pharmaceutical composition described in the present application may contain one or more adjuvants in addition to the active compound, and the adjuvants may be selected from the group consisting of fillers (diluents), binders, wetting agents, disintegrants, excipients, etc. Depending on the method of administration, the composition may contain 0.1-99% by weight of the active compound.

[0182] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, such as, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups. The oral composition may be prepared by methods known in the art for preparing pharmaceutical compositions, and may include binders, fillers, lubricants, disintegrants, or pharma- ceutically acceptable wetting agents. The composition may also contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives.

[0183] Aqueous suspensions may contain excipients suitable for preparing aqueous suspensions that are used to mix with active substances. Aqueous suspensions may also contain one or more preservatives, such as one or more coloring agents, one or more flavoring agents, and one or more sweetening agents. Oily suspensions may be prepared by suspending active ingredients in vegetable oil. Oily suspensions may contain thickening agents. The above-mentioned sweetening agents and flavoring agents may also be added.

[0184] The pharmaceutical compositions may also contain one or more dispersing agents, wetting agents, suspending agents or preservatives, which are mixed with water to prepare aqueous suspensions of dispersible powders and granules as the active ingredient. Other excipients, such as sweeteners, flavoring agents and coloring agents, may also be added. These compositions are preserved by the addition of an antioxidant, such as ascorbic acid. The pharmaceutical compositions of the present application may also be in the form of oil-in-water emulsions.

[0185] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin, and the oil solution may then be added to a mixture of water and glycerol and processed to form a microemulsion. The injectable solutions or microemulsions may be injected into a patient's bloodstream by local bolus injection. Alternatively, the solutions and microemulsions may be administered in such a way as to maintain a constant circulating concentration of the compound of the present application. In order to maintain this constant concentration, a continuous intravenous delivery device may be used. For example, the device may be an I intravenous infusion pump.

[0186] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension for intramuscular and subcutaneous administration. The suspension may be prepared using the appropriate dispersing or wetting agent and suspending agent described above in this application according to known techniques. The sterile injectable preparation may be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent. Alternatively, sterile fixed oils are conveniently used as a solvent or suspension medium. Sterile fixed oils are usually used as a solvent or suspension medium.

[0187] The compounds of the present application can be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and melts in the rectum to release the drug. Such materials are, for example, cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

[0188] As is well known to those skilled in the art, the dosage of a drug depends on many factors, including but not limited to the activity of the particular compound used, the age and weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the method of administration, the rate of excretion, the combination of drugs, etc., as well as the optimal treatment method (e.g., treatment regimen), the form of the compounds described herein or their tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or their pharma- ceutically acceptable salts, and / or the daily dose of the compounds or their tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or their pharma-ceutically acceptable salts, or the type of pharma-ceutically acceptable salts, can be ascertained according to conventional treatment regimens.

[0189] The pharmaceutical composition of the present application may comprise a safe and effective amount of the antibody-drug conjugate of the present application and a pharma- ceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical formulation should be compatible with the method of administration, and the pharmaceutical composition of the present application may be prepared in a solution form, for example, by using an aqueous solution containing saline, glucose, or other auxiliary agents, in a conventional manner. The pharmaceutical composition may be prepared under sterile conditions. The dosage of the active ingredient may be a therapeutically effective amount.

[0190] The effective amount of the antibody-drug conjugate described herein may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the effective amount can be determined by one skilled in the art (e.g., through clinical trials) based on various factors. Such factors may include, but are not limited to, the pharmacokinetic parameters (bioavailability, metabolism, half-life, etc.) of the dual-function antibody conjugate, the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, the antibody-drug conjugate of the present application can be administered daily in an appropriate dose to achieve satisfactory effects. For example, it may be administered daily in several divided doses depending on the urgency of the treatment situation, or the dose may be proportionally reduced.

[0191] The compound of the present application can be administered alone or in combination with other pharma- ceutical acceptable therapeutic agents. When using a pharmaceutical composition, a safe and effective amount of the compound of the present application can be administered to a mammal (such as a human) in need of treatment, and the dosage at the time of administration can be a dosage approved in medical and pharmacological sciences, and the specific dosage can be determined by taking into consideration factors such as the route of administration and the health condition of the patient.

[0192] The present application provides the use of the compounds of the present application, or their tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or their pharma- ceutically acceptable salts, prodrugs, or solvates, and / or pharmaceutical compositions of the present application, in the preparation of a medicament that can be used for the treatment and / or prevention of a tumor. For example, the tumor can be selected from tumors associated with a target expression that is HER2. For example, tumors associated with the expression of the target include tumors with high expression of the target and / or tumors that are target positive. For example, the tumor can include solid tumors and / or hematological tumors. For example, the tumor is selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal stromal tumor, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma and mesothelioma.

[0193] The present application provides the use of the compounds of the present application, or in the form of their tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or pharma- ceutically acceptable salts, prodrugs, or solvates, and / or pharmaceutical compositions of the present application, in a drug that can be used for the treatment and / or prevention of a tumor. For example, the tumor can be selected from tumors associated with expression of a target that is HER2. For example, tumors associated with expression of the target include tumors with high expression of the target and / or tumors that are target positive. For example, the tumor can include solid tumors and / or hematological tumors. For example, the tumor can be selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal stromal tumor, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma, and mesothelioma.

[0194] The present application provides a method for preventing and / or treating tumors, which may include administering to a subject a compound of the present application, or a tautomer, meso, racemate, enantiomer, and diastereomer thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, and / or a pharmaceutical composition of the present application. For example, the tumor may be selected from tumors associated with target expression, which is HER2. For example, tumors associated with target expression include tumors with high expression of the target and / or tumors that are target positive. For example, the tumor may include solid tumors and / or hematological tumors. For example, the tumor is selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal stromal tumor, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma and mesothelioma.

[0195] Without intending to be limited by any theory, the following examples are only illustrative of the compounds, methods of preparation and use of the present application and are not intended to limit the scope of the present invention. EXAMPLES

[0196] Example 1 Synthesis and Preparation of Compounds Starting materials described herein are either commercially available or prepared according to methods known in the art or described herein, where Fmoc is the 9-fluorenylmethyloxycarbonyl protecting group, Boc is the tert-butyldimethylsilyl protecting group, and TBDMS / TBS are the tert-butoxycarbonyl protecting group.

[0197] Synthesis of compound (A-1)

[0198] [ka] Synthesis Route:

[0199] [ka]

[0200] Step 1: Synthesis of intermediate 1-1 Fmoc-Val-OSu (100 g, 229 mmol) was dissolved in 500 ml of tetrahydrofuran, and Nε-(tert-butoxycarbonyl)-L-lysine (59.3 g, 241 mmol) and sodium bicarbonate (20.21 g, 241 mmol) were added to the 500 ml aqueous solution, respectively. The reaction solution was stirred at room temperature for 48 hours, and the completion of the reaction was detected. The reaction solution was adjusted to about pH 6 with 1N dilute hydrochloric acid, and extracted by adding 500 ml of ethyl acetate. The organic phase was separated, washed once with water and saturated saline, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was recrystallized from methyl tert-butyl ether to obtain product 1-1 (107 g, 82% yield) as a white solid. LC-MS (ESI, m / z) calculated value: 567.29, observed value: 568.26 (M+H).

[0201] Step 2: Synthesis of intermediate 1-2 Intermediate 1-1 (60 g, 106 mmol) was dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 900 ml). p-Aminobenzyl alcohol (19.52 g, 159 mmol) was added at room temperature, followed by EEDQ (39.2 g, 159 mmol). The reaction was stirred at room temperature for 24 h, evaporated to dryness under reduced pressure, added diethyl ether, stirred until solid precipitated, filtered, and the solid was rinsed with diethyl ether several times and dried to give intermediate 1-2 (51 g, 72% yield). LC-MS (ESI, m / z) theoretical: 672.35, found: 673.37 (M+H).

[0202] Step 3: Synthesis of intermediate 1-3 Intermediate 1-2 (50 g, 74.3 mmol) was dissolved in 370 mL of DMF. Diethylamine (78 ml, 743 mmol) was added at room temperature. The reaction was stirred at room temperature for 2 hours, evaporated to dryness under reduced pressure, acetic acid, diethyl ether were added, stirred until solid precipitated, filtered, the solid was rinsed several times with diethyl ether, and after drying intermediate 1-3 (31.5 g, 94% yield) was obtained. LC-MS (ESI, m / z) theoretical: 450.28, found: 451.32 (M+H).

[0203] Step 4: Synthesis of intermediate 1-4 Intermediate 1-3 (5 g, 11.10 mmol) was dissolved in 100 ml of DMF, and N-succinimidyl maleimidoacetate (2.80 g, 11.10 mmol) was added at room temperature, the reaction mixture was stirred at room temperature overnight, and evaporated to dryness under reduced pressure, methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated, filtered, and the solid was rinsed with diethyl ether several times. After drying the solid, intermediate 1-4 was obtained, which was used directly in the next reaction. LC-MS (ESI, m / z) theoretical value: 587.30, actual value: 588.31 (M+H).

[0204] Step 5: Synthesis of intermediate 1-5 Intermediate 1-4 (2 g, 3.40 mmol) was dissolved in 40 ml of DMF, and DIPEA (1.189 ml, 6.81 mmol) and bis(4-nitrophenyl)carbonate (1.553 g, 5.10 mmol) were added at room temperature. Stir overnight at room temperature under argon atmosphere, add methyl tert-butyl ether, stir until solid precipitates, filter, rinse the solid with diethyl ether several times, and dry the solid to obtain intermediate 1-5, which is used directly in the next step reaction. LC-MS (ESI, m / z) theoretical: 752.30, found: 753.31 (M+H).

[0205] Step 6: Synthesis of intermediate 1-6 Intermediate 1-5 (142 mg, 0.188 mmol) was dissolved in 400 μL of anhydrous DMF and 100 μL of anhydrous pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 100 mg, 0.188 mmol) and HOBt (25.4 mg, 0.188 mmol). After stirring overnight at room temperature under argon atmosphere, the reaction was purified by reverse phase HPLC to give intermediate 1-6 (80 mg, 40% yield). LC-MS (ESI, m / z) theoretical: 1048.43, found: 1045.45 (M+H).

[0206] Step 7: Synthesis of compound (A-1) Intermediate 1-6 (100 mg, 0.095 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 500 μL of trifluoroacetic acid was added under ice bath, then the mixture was stirred at room temperature for 30 min, and the solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse phase HPLC to obtain the final compound (A-1) (70 mg, 77% yield). LC-MS (ESI, m / z) theoretical: 1048.43, actual: 1045.45 (M+H). LC-MS (ESI, m / z) theoretical: 948.38, actual: 949.37 (M+H).

[0207] Synthesis of compound (A-2)

[0208] [ka]

[0209] The synthesis of compound (A-2) was the same as that of compound (A-1), except that the raw material N-succinimidyl maleimidoacetate in step 4 was replaced with N-hydroxysuccinimide 6-maleimidohexanoate, and the product (A-2) was obtained as a beige amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1004.44, actual value: 1005.45 (M+H).

[0210] Synthesis of compound (A-3)

[0211] [ka]

[0212] Compound (A-1) (100 mg, 0.105 mmol) was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH, 97 mg, 0.126 mmol), HATU (48 mg, 0.126 mmol) and DIPEA (37 μL, 0.211 mmol) were added, respectively, and then stirred at room temperature overnight, and the solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse-phase HPLC to obtain the final compound (A-3) (87 mg, 49% yield). LC-MS (ESI, m / z) theoretical: 1700.76, observed: 1701.78 (M+H).

[0213] Synthesis of compound (A-4)

[0214] [ka]

[0215] Compound (A-2) (100 mg, 0.099 mmol) was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH, 92 mg, 119 mmol), HATU (45 mg, 119 mmol) and DIPEA (35 μL, 0.199 mmol) were added, respectively, and then stirred at room temperature overnight. The solvent was removed under reduced pressure to obtain the final product, which was then purified by reversed-phase HPLC to obtain the final compound (A-4). (95 mg, 54% yield). LC-MS (ESI, m / z) theoretical: 1756.83, observed: 1757.85 (M+H).

[0216] Synthesis of compound (A-5)

[0217] [ka]

[0218] The synthesis of compound (A-5) is the same as that of compound (A-4), except that acetylated-10 polysarcosine (Ac-Sar10-COOH) is replaced with acetylated-4 polysarcosine (Ac-Sar4-COOH) in the final step. After several reaction steps, the product (A-5) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1330.60, actual value: 1331.61 (M+H).

[0219] Synthesis of compound (A-6)

[0220] [ka]

[0221] The synthesis of compound (A-6) was the same as that of compound (A-4), except that exatecan mesylate in step 6 was replaced with belotecan hydrochloride (purchased from Shanghai Haoyuan Co., Ltd.), and the product (A-6) was obtained as a white amorphous powder after several reaction steps. LC-MS (ESI, m / z) theoretical value: 1754.87, actual value: 1755.88 (M+H).

[0222] Synthesis of compound (A-7)

[0223] [ka]

[0224] The synthesis of compound (A-7) was the same as that of compound (A-4), except that Fmoc-Val-OSu in step 1 was replaced with Fmoc-Phe-OSu. After several reaction steps, the product (A-7) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1804.83, actual value: 1805.85 (M+H).

[0225] Synthesis of compound (A-8)

[0226] [ka]

[0227] The synthesis of compound (A-8) was the same as that of compound (A-7), except that exatecan mesylate in step 6 was replaced with belotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.), and the product (A-8) was obtained as a white amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1802.87, actual value: 1803.86 (M+H).

[0228] Synthesis of compound (A-9)

[0229] [ka] Synthesis Route:

[0230] [ka]

[0231] Step 1: Synthesis of intermediate 9-1 Intermediate 9-1A (1.3 g, 2.69 mmol) and 9-1B (1.35 g, 2.69 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 90 ml). p-EEDQ (800 mg, 3.23 mmol) was added at room temperature. The reaction was stirred at room temperature for 24 h, then evaporated to dryness under reduced pressure and purified by column chromatography to give 9-1 (1.8 g, 69% yield). LC-MS (ESI, m / z) theoretical: 966.49, found: 967.50 (M+H).

[0232] Step 2: Synthesis of intermediate 9-2 Intermediate 9-1 (900 mg, 0.93 mmol) was dissolved in 20 ml of anhydrous tetrahydrofuran, and hydrogen fluoride pyridine complex (1.8 g, 18.61 mmol) was added under argon atmosphere while cooling in an ice bath. The reaction solution was stirred at 0°C for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was separated and dried over anhydrous sodium sulfate, and intermediate 9-2 (610 mg, 77% yield) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 852.41, actual value: 853.43 (M+H).

[0233] Step 3: Synthesis of intermediate 9-3 Intermediate 9-2 (600 mg, 0.703 mmol) was dissolved in 4 ml of anhydrous DMF, and DIPEA (0.84 ml, 1.05 mmol) and bis(4-nitrophenyl)carbonate (321 mg, 1.05 mmol) were added at room temperature. Stir overnight at room temperature under argon atmosphere, remove the solvent under reduced pressure, then add methyl tert-butyl ether, stir until solid precipitates, filter, rinse the solid with diethyl ether several times, and dry the solid to obtain intermediate 9-3, which is used directly in the next reaction. LC-MS (ESI, m / z) theoretical value: 1017.41, found value: 1018.38 (M+H).

[0234] Step 4: Synthesis of intermediate 9-4 Intermediate 9-3 (300 mg, 0.295 mmol) was dissolved in 400 μL of anhydrous DMF and 100 μL of anhydrous pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 157 mg, 0.295 mmol) and HOBt (40 mg, 0.295 mmol). After stirring overnight at room temperature under argon atmosphere, the reaction was purified by reverse phase HPLC to give intermediate 9-4 (160 mg, 41% yield). LC-MS (ESI, m / z) theoretical: 1313.54, found: 1314.51 (M+H).

[0235] Step 5: Synthesis of intermediate 9-5 Intermediate 9-4 (150 mg, 0.114 mmol) was dissolved in 1 mL of DMF. Diethylamine (120 μL, 1.14 mmol) was added at room temperature. The reaction was stirred at room temperature for 2 h and then evaporated to dryness under reduced pressure to give intermediate 9-5, which was used directly in the next reaction. LC-MS (ESI, m / z) theoretical: 1091.48, found: 1092.51 (M+H).

[0236] Step 6: Synthesis of intermediate 9-6 Compound 9-5 (120 mg, 0.110 mmol) was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH, 102 mg, 0.132 mmol), HATU (50 mg, 0.132 mmol) and DIPEA (38 μL, 0.22 mmol) were added, respectively, and stirred at room temperature overnight. The solvent was removed under reduced pressure to give the final product, which was then purified by reverse-phase HPLC to give intermediate compound 9-6 (105 mg, 52% yield). LC-MS (ESI, m / z) theoretical: 1843.86, found: 1844.84 (M+H).

[0237] Step 7: Synthesis of compound (A-9) Intermediate 9-6 (100 mg, 0.054 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 500 μL of trifluoroacetic acid was added under ice bath, then the mixture was stirred at room temperature for 30 min, and the solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse phase HPLC to obtain the final compound (A-9) (57 mg, 60% yield). LC-MS (ESI, m / z) theoretical: 1048.43, found: 1045.45 (M+H). LC-MS (ESI, m / z) theoretical: 1743.81, found: 1744.85 (M+H).

[0238] Synthesis of compound (A-10)

[0239] [ka]

[0240] The synthesis of compound (A-10) was the same as that of compound (A-9), except that exatecan mesylate in step 4 was replaced with belotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.), and the product (A-10) was obtained as a white amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1741.85, actual value: 1742.83 (M+H).

[0241] Synthesis of compound (A-11)

[0242] [ka]

[0243] Synthesis Route:

[0244] [ka]

[0245] Step 1: Synthesis of intermediate 11-1 Intermediate 11-1A (Mc-Val-Ala-OH, purchased from Shanghai Haoyuan Chemical Co., Ltd., 2.4 g, 6.29 mmol) and 11-1B (3.18 g, 6.29 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 90 ml). p-EEDQ (1.86 g, 7.55 mmol) was added at room temperature. The reaction solution was stirred at room temperature for 24 h, then evaporated to dryness under reduced pressure and purified by column chromatography to give intermediate 11-1 (3.9 g, 71% yield). LC-MS (ESI, m / z) theoretical: 867.46, found: 868.49 (M+H).

[0246] Step 2: Synthesis of intermediate 11-2 Intermediate 11-1 (2g, 2.3mmol) was dissolved in 50ml of anhydrous tetrahydrofuran, and hydrogen fluoride pyridine complex (4.6g, 46mmol) was added under argon atmosphere while cooling in an ice bath, the reaction solution was stirred at 0°C for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane, the organic layer was separated, dried over anhydrous sodium sulfate, and intermediate 11-2 (1.1g, 76% yield) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 629.34, actual value: 630.31 (M+H).

[0247] Step 3: Synthesis of intermediate 11-3 Intermediate 11-2 (700 mg, 1.11 mmol) was dissolved in 4 ml of anhydrous DMF, and DIPEA (0.39 ml, 2.23 mmol) and bis(4-nitrophenyl)carbonate (406 mg, 1.33 mmol) were added at room temperature. Stir overnight under argon atmosphere at room temperature, remove the solvent under reduced pressure, then add methyl tert-butyl ether, stir until solid precipitates, filter, rinse the solid with diethyl ether several times, and dry the solid to obtain intermediate 11-3, which is used directly in the next reaction. LC-MS (ESI, m / z) theoretical: 794.35, found: 795.41 (M+H).

[0248] Step 4: Synthesis of intermediate 11-4 Intermediate 11-3 (300 mg, 0.44 mmol) was dissolved in 400 μL of anhydrous DMF and 100 μL of anhydrous pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 234 mg, 0.44 mmol) and HOBt (60 mg, 0.44 mmol). After stirring overnight at room temperature under argon atmosphere, the reaction was purified by reverse phase HPLC to give intermediate 11-4 (230 mg, 48% yield). LC-MS (ESI, m / z) theoretical: 1090.48, found: 1091.53 (M+H).

[0249] Step 5: Synthesis of intermediate 11-5 Intermediate 11-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 300 μL of trifluoroacetic acid was added under ice bath, and the mixture was stirred at room temperature for 30 min. The solvent was removed under reduced pressure to give the trifluoroacetate salt of the final intermediate 11-5, which was used directly in the next reaction without further purification. LC-MS (ESI, m / z) theoretical value: 990.43, actual value: 991.47 (M+H).

[0250] Step 6: Synthesis of compound (A-11) Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH, 84 mg, 0.109 mmol), HATU (50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol) were added, respectively, and stirred at room temperature overnight. The solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse-phase HPLC to obtain compound (A-11) (74 mg, 38% yield). LC-MS (ESI, m / z) theoretical: 1742.81, observed: 1743.85 (M+H).

[0251] Synthesis of compound (A-12)

[0252] [ka]

[0253] The synthesis of compound (A-12) was the same as that of compound (A-11), except that the raw material compound Ac-Sar10-COOH in step 6 was replaced with Ac-Sar4-COOH. After several reaction steps, the product (A-12) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1316.59, actual value: 1317.62 (M+H).

[0254] Synthesis of compound (A-13)

[0255] [ka]

[0256] The synthesis of compound (A-13) was the same as that of compound (A-11), except that the raw material compound 11-1A in step 1 was replaced with Mc-Val-Cit-OH (purchased from Shanghai Haoyuan Chemical Co., Ltd.), and the product (A-13) was obtained as a beige amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1828.86, actual value: 1829.88 (M+H).

[0257] Synthesis of compound (A-14)

[0258] [ka]

[0259] The synthesis of compound (A-14) was the same as that of compound (A-11), except that the raw material compound 11-1A (Mc-VA-OH) in step 1 was replaced with Mc-GGFG-OH (purchased from Shanghai Haoyuan Chemical Co., Ltd.), and the product (A-13) was obtained as a beige amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1890.84, actual value: 1891.90 (M+H).

[0260] Synthesis of compound (A-15)

[0261] [ka]

[0262] Synthesis Route:

[0263] [ka]

[0264] Step 1: Synthesis of intermediate 15-2 Intermediate 15-1A (2.3 g, 8.57 mmol) and 15-1B (3.74 g, 8.57 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 90 ml). p-EEDQ (4.24 g, 17.15 mmol) was added at room temperature. After stirring the reaction at room temperature for 24 h, the reaction was evaporated to dryness under reduced pressure and purified by column chromatography to give intermediate 15-2 (3.23 g, 4.70 mmol, 54.9% yield). LC-MS (ESI, m / z) theoretical: 686.27, found: 687.21 (M+H).

[0265] Step 2: Synthesis of intermediate 15-3 Intermediate 15-2 (2.3 g, 3.35 mmol) was dissolved in 40 ml of anhydrous DMF, and DIPEA (1.170 ml, 6.70 mmol) and bis(4-nitrophenyl)carbonate (1.528 g, 5.02 mmol) were added. The mixture was stirred overnight at room temperature under argon atmosphere, the solvent was removed under reduced pressure, and then methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, rinsed with diethyl ether several times, and the solid was dried to obtain intermediate 15-3 (2.1 g, 2.465 mmol, 73.6% yield), which was used directly in the next reaction step.

[0266] Step 3: Synthesis of intermediate 15-4 Intermediate 15-3 (400 mg, 0.470 mmol) was dissolved in 4 mL of anhydrous DMF, 1 mL of anhydrous pyridine was added, followed by the addition of exatecan free base (purchased from Shanghai Haoyuan Chemical Co., Ltd.) (204 mg, 0.470 mmol) and HOBt (63.4 mg, 0.470 mmol). Stirring was carried out overnight at room temperature under argon atmosphere, and the reaction solution was evaporated to dryness and then purified by column chromatography (DCM:MeOH=30:1) to give intermediate 15-4 (244 mg, 0.213 mmol, 45.3% yield). LC-MS (ESI, m / z) theoretical: 1147.41, found: 1148.48 (M+H).

[0267] Step 4: Synthesis of intermediate 15-5 Intermediate 15-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 300 μL of trifluoroacetic acid was added under ice bath, and the mixture was stirred at room temperature for 30 min. The solvent was removed under reduced pressure to give the trifluoroacetate salt of the final intermediate 15-5, which was used directly in the next reaction without further purification. LC-MS (ESI, m / z) theoretical value: 1047.36, actual value: 1048.40 (M+H).

[0268] Step 5: Synthesis of Compound A-15 Intermediate compound 15-5 (150 mg, 0.131 mmol) was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH) (101 mg, 0.131 mmol), DIPEA (114 μl, 0.654 mmol) and HATU (74.6 mg, 0.196 mmol) were added, respectively, and stirred at room temperature overnight, and the solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse-phase HPLC to obtain compound A-15 (107 mg, 0.059 mmol, 45.4% yield). LC-MS (ESI, m / z) theoretical: 1799.74, observed: 1800.77 (M+H).

[0269] Synthesis of compound (A-16)

[0270] [ka]

[0271] The synthesis of compound (A-16) was the same as that of compound (A-15), except that exatecan mesylate in step 3 was replaced with belotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.) and the product (A-16) was obtained as a beige amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1797.78, actual value: 1798.81 (M+H).

[0272] Synthesis of compound (A-17)

[0273] [ka]

[0274] The synthesis of compound (A-17) was the same as that of compound (A-11), except that exatecan mesylate in step 4 was replaced with Genz-644282 (purchased from Shanghai Haoyuan Chemical Co., Ltd.) and the product (A-17) was obtained as a beige amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1714.80, actual value: 1715.83 (M+H).

[0275] Synthesis of compound (A-18)

[0276] [ka]

[0277] Synthesis Route:

[0278] [ka]

[0279] Step 1: Synthesis of intermediate 18-2 Intermediate 18-1A (purchased from Shanghai BiDe Pharmaceutical, 4.94 g, 14.82 mmol) and 15-1B (synthesized according to the method of Patent Document 9, 6.04 g, 14.82 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 120 ml). p-EEDQ (7.33 g, 29.6 mmol) was added at room temperature. The reaction solution was stirred at room temperature for 24 hours, then evaporated to dryness under reduced pressure, and the product was stirred, filtered with diethyl ether, and dried in vacuum to obtain intermediate 18-2 (6.3 g, 59% yield). LC-MS (ESI, m / z) theoretical value: 726.40, actual value: 727.41 (M+H).

[0280] Step 2: Synthesis of intermediate 18-3 Dissolve intermediate 18-2 (3.0g, 4.13mmol) in 50ml of THF, add 10% Pd-C (800mg), and hydrogenate at normal pressure for 8 hours. TLC indicates the reaction is complete. Filter and remove the palladium carbon. Evaporate the reaction solution to dryness, and use it directly in the next step. LC-MS (ESI, m / z) theoretical value: 636.36, actual value: 637.34 (M+H).

[0281] Step 3: Synthesis of intermediate 18-4 Intermediate compound 18-3 (2.6 g, 4.08 mmol) was dissolved in 100 ml of anhydrous DMF, and N-(2-aminoethyl)maleimide hydrochloride (721 mg, 4.08 mmol), DIPEA (1.42 mL, 8.17 mmol) and HATU (2.0 g, 5.31 mmol) were added, respectively, and then stirred at room temperature overnight. The solvent was removed under reduced pressure to give the final product, which was then purified by column chromatography to give compound 18-4 (2.2 g, 71% yield). LC-MS (ESI, m / z) theoretical: 758.40, found: 759.42 (M+H).

[0282] Step 4: Synthesis of intermediate 18-5 Intermediate 18-4 (1.0 g, 1.32 mmol) was dissolved in 20 ml of anhydrous tetrahydrofuran, and hydrogen fluoride pyridine complex (2.6 g, 26.4 mmol) was added under argon atmosphere while cooling in an ice bath. The reaction solution was stirred at 0°C for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was separated and dried over anhydrous sodium sulfate, and intermediate 18-5 (570 mg, 67% yield) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 644.32, actual value: 645.35 (M+H).

[0283] Step 5: Synthesis of intermediate 18-6 Intermediate 18-5 (500 mg, 0.776 mmol) was dissolved in 10 ml of anhydrous DMF, and DIPEA (271 μl, 1.55 mmol) and bis(4-nitrophenyl)carbonate (236 mg, 0.776 mmol) were added. The mixture was stirred overnight at room temperature under an argon atmosphere, the solvent was removed under reduced pressure, and then methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, rinsed with diethyl ether several times, and the solid was dried to obtain intermediate 18-6 (530 mg, 84% yield), which was used as it was in the next reaction step.

[0284] Step 6: Synthesis of intermediate 18-7 Intermediate 18-6 (400 mg, 0.494 mmol) was dissolved in 4 mL of anhydrous DMFF and 1 mL of anhydrous pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 263 mg, 0.494 mmol) and HOBt (66.7 mg, 0.494 mmol). After stirring overnight at room temperature under argon atmosphere, the reaction was purified by reverse phase HPLC to give intermediate 18-7 (320 mg, 58% yield). LC-MS (ESI, m / z) theoretical: 1105.46, found: 1106.48 (M+H).

[0285] Step 7: Synthesis of intermediate 18-8 Intermediate 15-4 (300 mg, 0.27 mmol) was dissolved in 3 mL of anhydrous dichloromethane, and 1 mL of trifluoroacetic acid was added under ice bath, then the mixture was allowed to warm to room temperature and stirred for 30 min. The solvent was removed under reduced pressure to give the trifluoroacetate salt of final intermediate 18-8, which was used directly in the next reaction without further purification. LC-MS (ESI, m / z) theoretical: 1005.40, found: 1006.41 (M+H).

[0286] Step 8: Synthesis of Compound A-18 Intermediate compound 15-5 (300 mg, 0.268 mmol) was dissolved in 5 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH) (206 mg, 0.268 mmol), DIPEA (94 μl, 0.536 mmol) and HATU (122 mg, 0.321 mmol) were added, respectively, and the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse phase HPLC to obtain compound A-18 (170 mg, 36% yield). LC-MS (ESI, m / z) theoretical value: 1757.78, actual value: 1758.82 (M+H).

[0287] Synthesis of compound (A-19)

[0288] [ka]

[0289] The synthesis of compound (A-19) was the same as that of compound (A-18), except that N-(2-aminoethyl)maleimide hydrochloride in step 3 was replaced with 2-(methylsulfonyl)benzo[d]thiazol-6-amine, and the product (A-19) was obtained through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1845.73, actual value: 1846.75 (M+H).

[0290] Synthesis of compound (A-20)

[0291] [ka]

[0292] The synthesis of compound (A-20) is the same as that of compound (A-18), except that N-(2-aminoethyl)maleimide hydrochloride in step 3 is replaced with 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline, and the product (A-20) is obtained through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1856.76, actual value: 1857.80 (M+H).

[0293] Synthesis of compound (A-21)

[0294] [ka]

[0295] The synthesis of compound (A-21) is the same as that of compound (A-11), and the intermediate of step 1 [ka] of [ka] By simply replacing with , the product (A-21) was obtained through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1742.81, observed value: 1743.85 (M+H).

[0296] Synthesis of compound (A-22)

[0297] [ka]

[0298] The synthesis of compound (A-22) was the same as that of compound (A-21), except that the raw material compound Mc-Val-Ala-OH in step 1 was replaced with Mc-Val-Cit-OH. After several reaction steps, the product (A-22) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1828.86, actual value: 1829.87 (M+H).

[0299] Synthesis of compound (A-23)

[0300] [ka]

[0301] The synthesis of compound (A-23) was the same as that of compound (A-21), except that the raw material compound Mc-Val-Ala-OH in step 1 was replaced with Mc-GGFG-OH. After several reaction steps, the product (A-23) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1890.84, actual value: 1891.86 (M+H).

[0302] Synthesis of compound (A-28)

[0303] [ka]

[0304] Synthesis Route:

[0305] [ka]

[0306] Step 1: Synthesis of intermediate 28-1 6-nitroisobenzofuran-1(3H)-one (10 g, 55.8 mmol) and tert-butyl (2-aminoethyl)carbamate (9.84 g, 61.4 mmol) were placed in a 100 mL round bottom flask and heated to 90 °C and stirred overnight. Methyl tert-butyl ether (MTBE) was added, stirred, filtered, and the solid was washed several times with MTBE and then dried in vacuum to give intermediate 28-1 (13.6 g, 72% yield). LC-MS (ESI, m / z) calculated: 339.14, found: 340.17 (M+H).

[0307] Step 2: Synthesis of intermediate 28-2 Intermediate 28-1 (10 g, 29.5 mmol) and imidazole (8.02 g, 118 mmol) were dissolved in 300 ml of dichloromethane, and TBS-Cl (6.66 g, 44.2 mmol) was added while cooling in an ice bath, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution to quench the reaction, and the organic phase was separated and washed once each with water and saturated saline, then dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography to obtain intermediate 28-2 (12.3 g, 92% yield). LC-MS (ESI, m / z) theoretical value: 453.23, actual value: 454.33 (M+H).

[0308] Step 3: Synthesis of intermediate 28-3 Intermediate 28-2 (6.0 g, 13.23 mmol) was dissolved in THF:EtOH (1:1) (500 mL) and 1 g of 10% Pd-C was added under argon atmosphere, followed by ammonium formate (8.34 g, 132 mmol). Stirred overnight at room temperature, filtered off the palladium on carbon, evaporated the filtrate to dryness, added dichloromethane and water, separated the organic phase, washed once each with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to give intermediate 28-3 (5.6 g, 100% yield). LC-MS (ESI, m / z) calculated: 423.26, found: 424.31 (M+H).

[0309] Step 4: Synthesis of intermediate 28-4 Intermediate 28-3 (5.5 g, 12.98 mmol) and Fmoc-Val-Alal-OH (5.33 g, 12.98 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v=2:1, 300 ml). EEDQ (4.82 g, 19.47 mmol) was added at room temperature. After stirring the reaction solution at room temperature for 24 h, the reaction solution was evaporated to dryness under reduced pressure, diethyl ether was added and stirred until a solid precipitated, filtered, the solid was rinsed with diethyl ether several times, and intermediate 28-4 (6.4 g, 60% yield) was obtained after drying the solid. LC-MS (ESI, m / z) theoretical: 815.43, found: 816.48 (M+H).

[0310] Step 5: Synthesis of intermediate 28-5 Intermediate 28-4 (6.0 g, 7.35 mmol) was dissolved in 100 mL of DMF. Diethylamine (7.68 ml, 73.5 mmol) was added at room temperature. After stirring the reaction solution at room temperature for 2 hours, the reaction solution was evaporated to dryness under reduced pressure, ethyl acetate and diethyl ether were added, and the mixture was stirred until a solid precipitated, filtered, and the solid was rinsed with diethyl ether several times. After drying the solid, intermediate 28-5 (4.3 g, 98% yield) was obtained. LC-MS (ESI, m / z) theoretical value: 593.36, actual value: 594.40 (M+H).

[0311] Step 6: Synthesis of intermediate 28-6 Intermediate 28-5 (4.0 g, 6.74 mmol) was dissolved in 100 ml of DMF, and N-succinimidyl maleimidoacetate (2.07 g, 6.74 mmol) was added at room temperature, and the reaction solution was stirred at room temperature overnight, after which the reaction solution was evaporated to dryness under reduced pressure, methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated, filtered, and the solid was rinsed with diethyl ether several times. After drying the solid, intermediate 28-6 was obtained, which was used directly in the next reaction step. LC-MS (ESI, m / z) theoretical value: 786.43, actual value: 787.45 (M+H).

[0312] Step 7: Synthesis of intermediate 28-7 Intermediate 28-6 (3.5 g, 4.45 mmol) was dissolved in 100 ml of anhydrous tetrahydrofuran, and hydrogen fluoride pyridine complex (8.8 g, 89 mmol) was added under argon atmosphere while cooling in an ice bath. The reaction mixture was stirred at 0°C for 2 hours, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was separated and dried over anhydrous sodium sulfate, and intermediate 28-7 (1.6 g, 53% yield) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 672.35, actual value: 673.37 (M+H).

[0313] Step 8: Synthesis of intermediate 28-8 Intermediate 28-7 (1.0 g, 1.49 mmol) was dissolved in 20 ml of anhydrous DMF, and DIPEA (0.519 ml, 2.97 mmol) and bis(4-nitrophenyl)carbonate (678 mg, 2.23 mmol) were added at room temperature. The mixture was stirred overnight at room temperature under argon atmosphere, the solvent was removed under reduced pressure, and then methyl tert-butyl ether was added and stirred until a solid precipitated. The mixture was filtered, the solid was rinsed with diethyl ether several times, and the solid was dried to obtain intermediate 28-8, which was used directly in the next reaction. LC-MS (ESI, m / z) theoretical value: 837.35, actual value: 838.36 (M+H).

[0314] Step 9: Synthesis of intermediate 28-9 Intermediate 28-8 (300 mg, 0.358 mmol) was dissolved in 4 mL of anhydrous DMF and 1 mL of anhydrous pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 190 mg, 0.358 mmol) and HOBt (55 mg, 0.358 mmol). After stirring overnight at room temperature under argon atmosphere, the reaction was purified by reverse phase HPLC to give intermediate 28-9 (234 mg, 58% yield). LC-MS (ESI, m / z) theoretical: 1133.49, found: 1134.52 (M+H).

[0315] Step 10: Synthesis of intermediate 28-10 Intermediate 28-10 (200 mg, 0.176 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 300 μL of trifluoroacetic acid was added under ice bath, then the mixture was stirred at room temperature for 30 min, and the solvent was removed under reduced pressure to obtain the trifluoroacetate salt of the final intermediate 28-10, which was used directly in the next reaction without further purification. LC-MS (ESI, m / z) theoretical value: 1033.43, actual value: 1034.45 (M+H).

[0316] Step 11: Synthesis of compound A28 Compound 28-10 obtained in step 10 was dissolved in 1 ml of anhydrous DMF, and acetylated-10 polysarcosine (Ac-Sar10-COOH, 136 mg, 0.176 mmol), HATU (87 mg, 0.229 mmol) and DIPEA (154 μL, 0.88 mmol) were added, respectively, and then the mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure to obtain the final product, which was then purified by reversed-phase HPLC to obtain compound (A-11) (135 mg, 43% yield). LC-MS (ESI, m / z) theoretical value: 1785.82, actual value: 1786.85 (M+H).

[0317] Synthesis of compound (A-29)

[0318] [ka]

[0319] The synthesis of compound (A-29) was the same as that of compound (A-11), except that exatecan mesylate in step 4 was replaced with belotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.), and the product (A-29) was obtained as a white amorphous powder through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1740.85, actual value: 1741.82 (M+H).

[0320] Synthesis of AP-1, a PEG derivative corresponding to the A-11 molecule

[0321] [ka]

[0322] The synthesis of compound (AP-1) was the same as that of compound (A-11), except that the raw material compound Ac-Sar10-COOH in step 1 was replaced with m-PEG8-acid (CAS number 1093647-41-6). After several reaction steps, the product (AP-1) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1384.64, actual value: 1385.66 (M+H).

[0323] Synthesis of control product MC-Val-Cit-PABC-DX8951

[0324] Referring to the method of Patent Document 1, Mc-Val-Cit-OH (purchased from Shanghai Haoyuan Chemical Co., Ltd.) was reacted with bis(4-nitrophenyl)carbonate to obtain an active nitroester, which was then reacted with exatecan mesylate (purchased from Shanghai Haoyuan Chemical Co., Ltd.) to obtain the control compound MC-Val-Cit-PAB-DX8951.

[0325] Example 2 General method for preparing ADCs Antibodies or antigen-binding fragments, such as Trastuzumab stock solution targeting HER2 (heavy chain sequence shown in SEQ ID NO:3, light chain sequence shown in SEQ ID NO:4) and / or hRS7 monoclonal antibody (Sacituzumab) targeting Trop-2, for example, were diluted to 2 mg / mL in 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriaminepentaacetic acid (DTPA), pH 7 reaction buffer, a 6.0-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction was stirred at 35° C. for 2.5 hours.

[0326] The above reaction solution is cooled to 8°C, and an appropriate amount of dimethylacetamide (DMA) is added without purification, followed by 6-15 times molar excess of control drug molecules or drug-linker complexes A1-A29 (10 mg / ml pre-dissolved in DMA), ensuring that the volume ratio of DMA in the reaction system does not exceed 20%, and stirring at 37°C for 3 hours to couple.

[0327] The binding reaction mixture was filtered and purified through a pH 6.0 histidine acetate / sucrose gel using a desalting column, and peak samples were collected based on UV280 absorbance, sterilized by passing through a 0.15 micron filter, and stored at -60°C.

[0328] Preparation of antibody conjugate 1 (control ADC1) Referring to the general method described above, a 10-fold excess of the control product MC-Val-Cit-PAB-DX8951 was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 1, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC). The analysis spectrum is shown in Figure 1. The proportion of aggregate 1 was about 26%, and the proportion of aggregate 2 was about 20%. The drug-antibody ratio (DAR) was about 4-5 by UV analysis.

[0329] Preparation of antibody conjugate 2 (control ADC2) With reference to the above-mentioned general method, a 12-fold excess of the control product Deruxtecan (purchased from Shanghai Haoyuan Chemical Co., Ltd.) was combined with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 2, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analysis spectrum is shown in Figure 2. The aggregate percentage was about 2%. By hydrophobic interaction chromatography (HIC-HPLC) analysis, the drug-antibody ratio (DAR) was about 7-8, and the analysis spectrum is shown in Figure 3.

[0330] Preparation of antibody conjugate 3 Referring to the general method described above, an 8-fold excess of compound (A-2) was combined with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 3, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC). The analysis spectrum is shown in Figure 4. The proportion of aggregate 1 was about 6.8%, and the proportion of aggregate 2 was about 7.7%. The drug-antibody ratio (DAR) was about 6-7 by UV analysis.

[0331] Preparation of antibody conjugate 4 With reference to the above-mentioned general method, a 12-fold excess of compound (A-4) was combined with the Trastuzumab monoclonal antibody after reduction to obtain antibody conjugate 4 with a drug-antibody ratio (DAR) of about 7-8. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analysis spectrum is shown in Figure 5. The aggregate percentage was about 0.3%. By hydrophobic interaction chromatography (HIC-HPLC) analysis, the drug-antibody ratio (DAR) was about 7-8, and the analysis spectrum is shown in Figure 6.

[0332] Preparation of antibody conjugate 5 With reference to the above-mentioned general method, a 12-fold excess of compound (A-11) was conjugated with the Trastuzumab monoclonal antibody after reduction to obtain the corresponding antibody conjugate 5, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC). The analysis spectrum is shown in FIG. 7, and the purity of the monomer of antibody conjugate 5 was 99.41%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analysis spectrum is shown in FIG. 8.

[0333] Preparation of antibody conjugate 6 With reference to the above-mentioned general method, a 12-fold excess of compound (A-14) was conjugated with the Trastuzumab monoclonal antibody after reduction to obtain the corresponding antibody conjugate 6, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC). The analysis spectrum is shown in Figure 9, and the purity of the monomer of the antibody conjugate 6 was 99.60%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analysis spectrum is shown in Figure 10.

[0334] Preparation of antibody conjugate 7 With reference to the general method described above, a 15-fold excess of compound (A-24) was combined with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 7, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 11. The purity of the monomer of antibody conjugate 7 was 96.36%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 12.

[0335] Preparation of antibody conjugate 8 (control ADC3) With reference to the general method described above, a 12-fold excess of compound AP-1 was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 8, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 13. The purity of the monomer of antibody conjugate 8 was 97.92%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 14.

[0336] Preparation of antibody conjugate 9 With reference to the general method described above, a 12-fold excess of compound (A-11) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 9, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 15. The purity of the monomer of antibody conjugate 9 was 97.92%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 16.

[0337] Preparation of antibody conjugate 10 With reference to the general method described above, a 12-fold excess of compound (A-11) was combined with the reduced H01L02 monoclonal antibody to obtain the corresponding antibody conjugate 10, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 17. The purity of the monomer of the antibody conjugate 10 was 99.10%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 18.

[0338] Preparation of antibody conjugate 11 With reference to the general method described above, a 12-fold excess of compound (A-28) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 11, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 19. The purity of the monomer of antibody conjugate 11 was 99.59%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 20.

[0339] Preparation of antibody conjugate 12 With reference to the general method described above, a 12-fold excess of compound (A-29) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody conjugate 12, which was analyzed for aggregate content by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 21. The purity of the monomer of the antibody conjugate 12 was 99.67%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was about 7-8, and the analytical spectrum is shown in Figure 22.

[0340] Preparation of antibody conjugate 13 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed into 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4) / 50 mM sodium chloride (NaCl), pH 7.0 buffer. After measuring the monoclonal antibody concentration in the solution, the antibody was diluted to 5 mg / mL with the above buffer. The reaction tube was placed in an ice bath and cooled for 10 minutes. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added in a molar ratio of 2.5 times, and the reaction solution was stirred at 4°C overnight. After adding an appropriate amount of dimethylacetamide (DMA) to the above unpurified reaction solution, a 6.0-fold molar excess of the control drug molecule or drug linker conjugate A-11 (10 mM dissolved in DMA in advance) was added, so that the volume ratio of DMA in the reaction system did not exceed 10%, and the conjugation was carried out by stirring at 4°C for 2 hours. After the conjugation was completed, a 4.0-fold molar amount of cysteine ​​small molecule was added to the reaction solution to consume excess drug-linker conjugate A-11, and the reaction was stopped by stirring at 4°C for 30 minutes. The conjugation reaction mixture was filtered and purified with histidine-acetic acid / sodium chloride at pH 5.5 using a desalting column, and the filtered sample was collected. A suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL) was added to the sample in an amount of 1 / 10 of the volume, and stirred at room temperature for 2 hours to fully absorb the free drug small molecule. It was then sterilized by passing through a 0.22 micron filter and stored at -80°C. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 25. The purity of the antibody conjugate 13 monomer was 98.95%.

[0341] The bound drug concentration in the antibody-drug complex can be calculated as follows by measuring the ultraviolet absorbance of an aqueous solution of the antibody-drug complex at wavelengths of 280 nm and 370 nm.

[0342] The total absorbance at a given wavelength is equal to the sum of the absorbance of all light-absorbing chemicals in the system (additivity of absorbance). Therefore, assuming that the molar extinction coefficients of the antibody and drug do not change before and after binding, the antibody and drug concentrations in the antibody-drug complex are expressed by the following equations:

[0343]

number

[0344] ε A,280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Non-Patent Document 1), and ε A,370 is generally zero. From the absorbance of a solution of the drug-linker conjugate at a particular molar concentration, ε can be calculated based on the Beer-Lambert law: D,280 and ε D,370 Obtain the A using a microplate reader or UV spectrophotometer. 280 and A 370 The value of is measured, and by substituting the above four molar extinction coefficient values ​​into the simultaneous equations (1) and (2), C A and C D The average number of drug molecules bound to each antibody molecule can be calculated as DAR = C D / C A The ultraviolet absorption coefficient of compound A-11 is ε D,280 = 6480, ε D,370= 16483, which was used to calculate the drug-antibody ratio in this example, and the DAR value of antibody conjugate 13 was approximately 4.0.

[0345] Preparation of antibody conjugate 14 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed into 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4) / 50 mM sodium chloride (NaCl), pH 7.0 buffer. After measuring the monoclonal antibody concentration in the solution, the antibody was diluted to 5 mg / mL with the above buffer. The reaction tube was placed in an ice bath and cooled for 10 minutes. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added in a molar ratio of 2.5 times, and the reaction solution was stirred at 4°C overnight. After adding an appropriate amount of dimethylacetamide (DMA) to the above unpurified reaction solution, a 6.0-fold molar excess of the control drug molecule or drug linker conjugate A-14 (10 mM pre-dissolved in DMA) was added, so that the volume ratio of DMA in the reaction system did not exceed 10%, and the conjugation was carried out by stirring at 4°C for 2 hours. After the conjugation was completed, a 4.0-fold molar amount of cysteine ​​small molecule was added to the reaction solution to consume excess drug-linker conjugate A-14, and the reaction was stopped by stirring at 4°C for 30 minutes. The conjugation reaction mixture was filtered and purified with histidine-acetic acid / sodium chloride at pH 5.5 using a desalting column, and the filtered sample was collected. A suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL) was added to the sample in an amount of 1 / 10 of the volume, and stirred at room temperature for 2 hours to fully absorb the free drug small molecule. It was then sterilized by passing through a 0.22 micron filter and stored at -80°C. The content of aggregates was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 26. The purity of the antibody conjugate 14 monomer was 98.20%. The ultraviolet absorption coefficient of compound A-14 is ε D,280 =5932, ε D,370 = 14997, which was used to calculate the drug-antibody ratio in this example, giving a DAR value of about 4.2 for antibody conjugate 14.

[0346] Preparation of antibody conjugate 15 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed into 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4) / 50 mM sodium chloride (NaCl), pH 7.0 buffer. After measuring the monoclonal antibody concentration in the solution, the antibody was diluted to 5 mg / mL with the above buffer. The reaction tube was placed in an ice bath and cooled for 10 minutes. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added in a molar ratio of 2.5 times, and the reaction solution was stirred at 4°C overnight. After adding an appropriate amount of dimethylacetamide (DMA) to the above unpurified reaction solution, a 6.0-fold molar excess of the control drug molecule or drug linker conjugate A-24 (10 mM pre-dissolved in DMA) was added, so that the volume ratio of DMA in the reaction system did not exceed 10%, and the mixture was stirred at 4°C for 2 hours to allow binding. After the conjugation was completed, a 4.0-fold molar amount of cysteine ​​small molecule was added to the reaction solution to consume excess drug-linker conjugate A-24, and the reaction was stopped by stirring at 4°C for 30 minutes. The conjugation reaction mixture was filtered and purified with histidine-acetic acid / sodium chloride at pH 5.5 using a desalting column, and the filtered sample was collected. A suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL) was added to the sample in an amount of 1 / 10 of the volume, and stirred at room temperature for 2 hours to fully absorb the free drug small molecule. It was then sterilized by passing through a 0.22 micron filter and stored at -80°C. The content of aggregates was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 27. The purity of the antibody conjugate 15 monomer was 96.36%. The ultraviolet absorption coefficient of compound A-24 was ε D,280 = 4723, ε D,370 = 12467, which was used to calculate the drug-antibody ratio in this example, and the DAR value for antibody conjugate 15 was approximately 4.0.

[0347] The compounds of the present application and the ADCs prepared therefrom have higher monomer purity at higher drug-antibody ratios than the ADC molecules prepared with the control molecules, suggesting that this type of ADC molecule has better stability. Furthermore, the results of hydrophobic interaction chromatography (HIC-HPLC) analysis showed that the retention time of the ADC molecules of the present application is shorter than that of the control molecules and is closer to that of naked antibodies, suggesting that this type of ADC molecule has better hydrophilicity, a longer half-life in vivo, and may have stronger in vivo efficacy.

[0348] Example 3: Stability test of antibody complex Camptothecin topoisomerase inhibitor-based ADC drugs, such as Sacituzumab govitecan (Trodelvy) and Trastuzumab Deruxtecan (Enhertu), usually have a high drug-antibody ratio (DAR), and camptothecin molecules have aromatic condensed ring structures and strong hydrophobicity, so the stability of this type of ADC molecule should be affected to some extent. The ADC prepared in the present invention introduces polysarcosine peptide chains at specific positions, which may have better "barrier effect" and better stability, and to verify this hypothesis, we designed an accelerated stability test of the antibody.

[0349] The molecules compared are as follows: 1. Trastuzumab 2. Antibody complex 2 3. Antibody complex 5 4. Antibody complex 6 5. Antibody complex 7 5. Antibody complex 8

[0350] The test molecule sample was diluted to a concentration of 5 mg / ml with formulation buffer (20 mM histidine-acetic acid, 150 mM NaCl, pH 5.5), and 50 μL was taken for direct testing or 55 μL was taken for testing. oAfter incubation in a water bath at 37 °C for 1, 2, 5, 24, 48, and 72 hours, respectively, tests (accelerated stability tests) were performed. The concentration changes of the samples were detected by UV method, and the contents of normal structure ADC molecules (main peak) and polymerized molecules in the samples were quantitatively analyzed using a TSK-GEL SWXL3000 molecular exclusion chromatography column on an Agilent 1260 Infinity II bio-inert LC system.

[0351] The HPLC mobile phase was 200 mM phosphate buffer (pH 7.0) + 150 mM KCl + 15% IPA, the column temperature was 25°C, the injection volume was 7 μL, the flow rate was 0.75 ml / min, and the UV detection wavelengths were 280 nm and 370 nm.

[0352] The statistical results of the sample concentration change are shown in Figure 23. Trastuzumab and its corresponding antibody-drug conjugate 5 and antibody-drug conjugate 6 both have good thermal stability, and the difference in concentration change from 0 to 72 hours is small, but the samples of antibody-drug conjugate 2, antibody-drug conjugate 7, and antibody-drug conjugate 8 showed a sharp decrease in ADC concentration after 24 hours, and the precipitation phenomenon occurred, which caused a rapid decrease in the sample concentration.

[0353] The change results of the increase rate of polymerized molecules in the above accelerated stability test samples were further analyzed by molecular size exclusion chromatography. The statistical results are shown in Figure 24, which is consistent with the change trend of the sample concentration, and the ranking of the samples from high to low stability is Trastuzumab ≒ antibody conjugate 5> antibody conjugate 6> antibody conjugate 7> antibody conjugate 8 ≒ antibody conjugate 2.

[0354] The above results confirmed that the hydrophilic polysarcosine peptide chain at a specific position in the present invention has a "barrier effect" and brings about an "unexpected" stability-improving effect on the antibody-drug conjugate molecule.

[0355] Example 4: In vitro cytotoxic activity test Human lung squamous cell carcinoma cells NCI-H2170 and LK-2, and human breast cancer cells MDA-MB-231 were selected as cell lines for in vitro activity detection in this experiment, and the expression level of TROP2 antigen (represented by rMFI) on the surface of the above human tumor cells was detected by flow cytometry (FACS), while observing the effective amount status of antibody conjugate 13, DS-1062a (control drug, prepared with reference to the method described in Patent Document 10), hIgG-deruxtecan conjugate, and hIgG-A-11 conjugate for cell killing. After adding the sample to the antibody-drug conjugate, the final concentration was set to 500 nM, and 9 levels of concentration (5-fold dilution) from the initial concentration were designed from 500 to 0.1 nM. The killing (or inhibition) changes were observed for 120 hours, and chemiluminescent staining (Luminescent Cell Viability Assay) was performed, and the fluorescence data was read, followed by IC 50 was calculated.

[0356] As shown in Table 1, both antibody conjugate 13 and DS-1062a (control drug) can significantly inhibit tumor cell proliferation, and are significantly stronger than the negative control drugs IgG-deruxtecan conjugate and IgGA-1 conjugate. The antibody conjugate 13 of the present invention is significantly superior to the clinical drug DS-1062a.

[0357] [Table 1]

[0358] According to the above activity test results, all ADCs prepared with the compounds of the present application showed good in vitro antitumor activity, and the advantages of the antibody conjugates of the present application are even more prominent, especially against tumor cells with low expression of relevant antigens.

[0359] Example 5: Measurement of in vivo antitumor effect The effect of the combination of the present invention can be measured in vivo, i.e., rodents are implanted with allografts or xenografts of cancer cells and tumors are treated with the combination. Test mice are treated with drug or control and monitored over several weeks to measure tumor doubling time, log cell kill and tumor inhibition.

[0360] In vivo antitumor experiments Human lung squamous cell carcinoma NCI-H2170 (ATCC) cells were suspended in saline at 4 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice, and the mice were randomly divided into groups on day 6. The day of grouping was designated as day 0, and antibody conjugate 2 (DS8201) and antibody conjugate 13 were administered at a dose of 5 mg / kg into the tail vein on day 0. As control groups, DS-1062a (positive control drug) and hIgG-A-11 conjugate were used as negative controls.

[0361] The results are shown in Figure 28. In the case of human lung squamous cell carcinoma NCI-H2170, both DS-1062a and antibody conjugate 13 at 5 mg / kg showed antitumor activity, and at a single dose of 5 mg / kg, the antitumor activity of antibody conjugate 13 was significantly stronger than that of the control drug DS-1062a. The change in mouse body weight (Figure 29) suggests that antibody conjugate 13 has a good safety profile.

[0362] According to the results of the activity test, the ADCs prepared with the compounds of the present application all exhibited a certain in vivo antitumor activity, and showed significantly stronger antitumor activity than the control sample. The tumor-bearing mice tolerated the above drugs well, and did not suffer from symptoms such as weight loss.

[0363] The description and examples in the Detailed Description of the Invention section are merely intended to clarify the technical contents of the present invention, and are not intended to limit the scope of the appended claims. Various modifications of the embodiments set forth in this application are obvious to those skilled in the art and fall within the scope of the appended claims and their equivalents.

Claims

1. A compound comprising a structure represented by formula (C-Trop-2), or in the form of a tautomer, meso-isomer, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof. 【Chemistry 1】 [In the formula, Q １ comprises a linker, L １ Is, -L １ａ Contains -C(=O)-, L １ａ is selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted heteroaliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L ２ comprises optionally substituted polypeptide residues, L ３ comprises an optionally substituted spacer group, L ２ and / or L ３ comprises an optionally substituted polysarcosine residue, T comprises a Drug unit; Ab is a ligand capable of binding to Trop-2, and m is a number from 1 to 8.

2. Q １ or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

3. L １ａ is an optionally substituted C １ -C ７ an alkylene group of the formula: ３ -C ６ 2. The compound of claim 1, selected from the group consisting of an aliphatic cyclylene group, an optionally substituted arylene group and an optionally substituted heteroarylene group, or in the form of its tautomers, meso-isomers, racemates, enantiomers and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug or solvate thereof.

4. L ２ comprises an optionally substituted polypeptide residue consisting of an amino acid selected from the group consisting of glycine, phenylalanine, valine, alanine, arginine, citrulline, aspartic acid, asparagine and lysine, or in the form of a tautomer, meso form, racemate, enantiomeric, and diastereomeric form, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

5. L ２ comprises an optionally substituted polypeptide residue selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn) and glycine-glycine-phenylalanine (Gly-Gly-Phe-Gly), or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma-ceutically acceptable salt, prodrug, or solvate thereof.

6. L ２ contains a lysine residue, the lysine residue is represented by the structure R １ or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

7. The structure R １ is optionally replaced 【Chemistry 2】 where n1 is a number from 4 to 18, and R is C １ -C ６ Alkyl, C １ -C ６ Cycloalkyl, and C １ -C ６ 7. The compound of claim 6, selected from the group consisting of alkoxy, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

8. L ３ is optionally replaced 【Chemistry 3】 and optionally substituted 【Chemistry 4】 or in the form of a tautomer, meso-isomer, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

9. L ３ is linked to an optionally substituted polysarcosine residue via the structural unit -X-, which is an optionally substituted 【Chemistry 5】 X is selected from the group consisting of １ is carbonyl, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ２ is hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ３ is a covalent bond, or hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S, said C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ 2. The compound of claim 1, wherein cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma-ceutically acceptable salt, prodrug, or solvate thereof.

10. L ３ is linked to an optionally substituted polysarcosine residue via the structural unit -X-, which is Possibly replaced 【Chemistry 6】 X is selected from the group consisting of １ is C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S, said C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ 2. The compound of claim 1, wherein cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma-ceutically acceptable salt, prodrug, or solvate thereof.

11. The structural unit -X- is an optionally substituted 【Chemistry 7】 and said optionally substituted polysarcosine residue is 【Chemistry 8】 n2 is a number from 4 to 18, and R is C １ -C ６ Alkyl, C １ -C ６ Cycloalkyl, and C １ -C ６ 9. The compound of claim 8, selected from the group consisting of alkoxy, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

12. The structural unit -X- is an optionally substituted 【Chemistry 9】 X is selected from the group consisting of １ is carbonyl, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ２ is hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ３ is a covalent bond, or hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S, said C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ The cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl, and the optionally substituted polysarcosine residue is 【Chemistry 10】 n2 is a number from 4 to 18, and R is C １ -C ６ Alkyl, C １ -C ６ Cycloalkyl, and C １ -C ６ 10. The compound of claim 9, selected from the group consisting of alkoxy, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

13. The structural unit -X- is an optionally substituted 【Chemistry 11】 and said optionally substituted polysarcosine residue is 【Chemistry 12】 n2 is a number from 4 to 18, and R is C １ -C ６ Alkyl, C １ -C ６ Cycloalkyl, and C １ -C ６ 10. The compound of claim 9, selected from the group consisting of alkoxy, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

14. The structural unit -X- is an optionally substituted 【Chemistry 13】 X is selected from the group consisting of １ is carbonyl, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ２ is hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S; X ３ is a covalent bond, or hydrogen, C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ cycloalkyl, straight chain heteroalkyl containing 1 to 8 atoms, and straight chain-cyclic heteroalkyl containing 1 to 8 atoms, said heteroalkyl containing 1 to 3 atoms selected from N, O, or S, said C １ -C ８ Alkyl, C １ -C ８ Alkoxy, C １ -C ６ The cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms are each optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl, and the optionally substituted polysarcosine residue is 【Chemistry 14】 n2 is a number from 4 to 18, and R is C １ -C ６ Alkyl, C １ -C ６ Cycloalkyl, and C １ -C ６ 10. The compound of claim 9, selected from the group consisting of alkoxy, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

15. T comprises a compound having antitumor activity, the compound of claim 1, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

16. T is 【Chemistry 15】 or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

17. 2. The compound of claim 1, wherein the Ab comprises an anti-Trop-2 antibody or an antigen-binding fragment thereof, or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

18. The antigen-binding fragments include Fab, Fab', Fv fragments, F(ab') ２ , F(ab) ２ 18. The compound of claim 17, selected from the group consisting of: scFv, di-scFv, VHH and dAb, or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

19. The compound of claim 1, wherein the Ab comprises hRS7, or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof.

20. A compound comprising a structure selected from the group consisting of: or in the form of a tautomer, meso, racemate, enantiomer, and diastereomer, or mixture thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof. 【Chemistry 16-1】 【Chemistry 16-2】

21. A pharmaceutical composition comprising a compound according to claim 1, or in the form of its tautomers, meso-isomers, racemates, enantiomers, and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug, or solvate thereof, and optionally a pharma- ceutically acceptable carrier.

22. Use of a compound according to claim 1, or in the form of its tautomers, meso-isomers, racemates, enantiomers and diastereomers, or mixtures thereof, or a pharma- ceutically acceptable salt, prodrug or solvate thereof, and / or a pharmaceutical composition according to claim 21, in the preparation of a drug for treating and / or preventing tumors.

Images