Method for preparing antibody-drug conjugates and their use

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 tautomeric, meso-, racemic, enantiomeric, diastereomeric 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] This application relates to the field of biopharmaceuticals, and particularly to a method for preparing an antibody-drug conjugate and its use.

Background Art

[0002] An antibody-drug conjugate (ADC) as a novel targeted therapeutic drug delivers a drug to an affected area through an appropriate carrier. However, due to the technical limitations of antibodies and highly active cytotoxic drugs, only a limited number of antibody-drug conjugates have been approved for marketing, and rapid development of the application of antibody-drug conjugates in the field of tumor treatment is needed.

[0003] Cytotoxic substances are essential for ADC drugs to exert their effects, and camptothecin drugs have great potential for application. Trodelvy and Enhertu, which have launched ADC drugs, use the camptothecin drugs SN38 and DX-8951f, respectively, as warhead molecules. However, the ADCs ultimately prepared through camptothecin drugs significantly alter the properties of the monoclonal antibody, and their stability and half-life are drastically 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 expression breast cancer was 37.0%, and the efficacy rate of the treatment was significantly lower than that for high- or moderate-expression breast cancer. Furthermore, new symptoms in terms of toxic side effects have appeared with ADC drugs, such as pneumonia and interstitial pneumonia with the drug Enhertu. Patent Document 1 discloses a camptothecin compound containing a valine-citrulline (Val-Cit)-PAB linker, but it is not possible to obtain a quality-compliant ADC molecule by conjugating this molecule with an antibody (the aggregate content in a satisfactory ADC product is less than 5%). Therefore, in this field, it is an urgent task to provide a more suitable antibody-drug conjugate based on camptothecin drugs (exatecan, berotecan, etc.), achieve efficient, simple, and practical chemical preparation and conjugation, and improve the pharmaceutical properties, metabolic properties, efficacy, and safety (improvement of molecular stability of ADCs, expansion of therapeutic range, etc.) of conventional antibody-drug conjugates. [Prior art documents] [Patent Documents]

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

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

[0006] This application provides antibody-drug conjugates, intermediates thereof, methods for preparation, and uses. The antibody-drug conjugates of this application enable a wide range of applications of cytotoxic drugs in the ADC field for the treatment of tumor diseases. The main technical effect of this application is that the novel linker provided can conjugate highly hydrophobic antitumor drugs (such as exatecan, berotecan, and other topoisomerase inhibitors) to antibodies by a specific chemical method, and the resulting conjugates have high hydrophilicity and stability. Compared to conventional ADC linkers, the linker provided in this application is less likely to form aggregates in antibody conjugates at high drug-antibody ratios. Compared to similar ADC drugs, the toxin molecules released by this application are the original molecules of the topoisomerase inhibitors exatecan and berotecan, and tests have shown that these drug molecules have better bioactivity, safety, and other drug-related properties than drug derivatives released by similar ADC drugs. Therefore, this application aims to improve the antitumor activity of drugs and / or broaden the overall therapeutic range by extending the in vivo half-life of drugs and increasing drug concentrations in tumor tissue.

[0007] In one embodiment, the present application provides a compound, or a tautomer thereof, a meso compound, a racemic compound, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure represented by formula (C-HER2).

[0008] [ka] [In the formula, Q1 includes the linker, L1 is -L 1a -C(=O)- is included, Here, L 1aThis is selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkylylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted aliphatic heterocyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups. L2 may contain substituted polypeptide residues. L3 may include a substituted spacer group, Here, L2 and / or L3 may include substituted polysarcosine residues, T includes drug units, Ab is a ligand that can bind to HER2, and m is a number from 1 to 8.

[0009] In another embodiment, the present application provides compounds, or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof. Here, the compound includes a structure selected from the group consisting of the following:

[0010] [ka] [ka] [Ab is a ligand that can bind to HER2, and m is a number from 1 to 8.]

[0011] In another embodiment, the present application provides a pharmaceutical composition comprising a compound described in any one of the present applications, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and optionally a selected pharmaceutically acceptable carrier.

[0012] In another embodiment, the present application provides the use of any compound described in any one of the present applications, or in the form of its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, and / or pharmaceutical compositions described in the present application, in the preparation of drugs for treating and / or preventing tumors.

[0013] In another embodiment, the present invention provides an antibody-drug conjugate formed by conjugating the present compound with an antibody.

[0014] In one embodiment, the complex of the present invention has one or more pharmaceutical components covalently bonded together.

[0015] In one embodiment, the antibody and drug of the present application are conjugated by a covalent bond (for example, they may be covalently bonded to a linker, respectively).

[0016] In another embodiment, the present invention provides a method for preparing an antibody-drug conjugate, which involves generating a pair of cysteine ​​residues through the reduction of the disulfide bond in the hinge region of an antibody fragment, and conjugating the compound to the cysteine ​​mercapto group of the cysteine ​​of the antibody or antibody fragment via a substitution reaction between the mercapto group of the cysteine ​​residue and a linking group such as the maleimide group of the compound in the present invention, thereby obtaining an antibody-drug conjugate, the drug-antibody ratio DAR (such as m in the present invention) is controlled depending on the reaction conditions, for example, typically between 2 and 8.

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

[0018] In another embodiment, the present invention provides a method for preparing a compound, comprising the steps of: contacting an amino acid active ester having an amino protecting group N1 with an amino acid to obtain intermediate M1; contacting intermediate M1 with a substituted / unsubstituted p-aminobenzyl alcohol in the presence of a bonding agent such as EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) to obtain intermediate M2; removing N1 from 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 fluorenyl methoxycarbonyl.

[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 brought into contact with trifluoroacetic acid and then into contact with acetylated polysarcosine.

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

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

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

[0024] In one embodiment, the ligand is brought into contact with the compound of the present invention at a temperature of approximately 0 to approximately 37°C.

[0025] In one embodiment, the ligand is reacted with a reducing agent in a buffer solution to obtain a reduced ligand before contacting the compound of the present invention.

[0026] In one embodiment, after obtaining a reduced ligand, the step includes removing the reducing agent before contacting the ligand with the compound of the present invention.

[0027] In one embodiment, the step of removing the reducing agent includes 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 solution 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 dimethyl sulfoxide (DMSO).

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

[0032] In this application, we have found that, compared to conventional antibody-drug conjugates, the hydrophobicity of drugs such as exatecan and berotecan is too high. Therefore, by introducing polysarcosine, the hydrophilicity of the conjugate is significantly increased, resulting in a more stable and less aggregated antibody-drug conjugate overall. Furthermore, this application provides a carbamate that can release the drug through rapid 1,6-elimination after enzymatic digestion, exhibiting better in vitro and in vivo stability and biological activity. Based on these findings, we have completed the present invention.

[0033] Those skilled in the art will readily recognize other aspects and advantages of the Application from the following detailed description. Only exemplary embodiments of the Application are shown and described in the following detailed description. Modifications to specific embodiments disclosed can be made based on the content of the Application, and such modifications will be understood to those skilled in the art to be within the spirit and scope of the Invention. The accompanying drawings and descriptions in the Specification are illustrative and not intended to limit the Application.

[0034] The specific features of the invention described herein are described in the attached claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments in the following detailed description and the attached drawings. A brief description of the attached drawings is as follows: [Brief explanation of the drawing]

[0035] [Figure 1] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 1 of the present invention. [Figure 2] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 2 of the present invention. [Figure 3] This is a chromatogram of the hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) of antibody complex 2 of the present invention. [Figure 4] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 3 of the present invention. [Figure 5] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 4 of the present invention. [Figure 6] This is a chromatogram of the hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) of antibody complex 4 of the present invention. [Figure 7] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 5 of the present invention. [Figure 8]This is a chromatogram of the hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) of antibody complex 5 of the present invention. [Figure 9] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 6 of the present invention. [Figure 10] This is a chromatogram of the hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) of antibody complex 6 of the present invention. [Figure 11] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 7 of the present invention. [Figure 12] This is a chromatogram of the hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) of antibody complex 7 of the present invention. [Figure 13] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody conjugate 8 of the present invention. [Figure 14] This is a chromatogram of the antibody complex 8 of the present invention obtained by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). [Figure 15] This is a chromatogram obtained by size exclusion chromatography (SEC-HPLC) of antibody complex 9 of the present invention. [Figure 16] This is a chromatogram of the antibody complex 9 of the present invention obtained by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). [Figure 17] This is a chromatogram of the antibody conjugate 10 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 18] This is a chromatogram of the antibody complex 10 of the present invention obtained by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). [Figure 19] This is a chromatogram of the antibody conjugate 11 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 20] This is a chromatogram of the antibody complex 11 of the present invention obtained by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). [Figure 21]This is a chromatogram of the antibody conjugate 12 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 22] This is a chromatogram of the antibody complex 12 of the present invention obtained by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). [Figure 23] This is a graph showing the concentration change during accelerated stability testing of several antibody conjugates of the present invention. [Figure 24] This graph shows the change in the increase in aggregates during accelerated stability tests of several antibody conjugates of the present invention. [Figure 25] The results of in vitro NCI-N87 cell (human gastric cancer cell) proliferation inhibitory activity tests of several antibody conjugates of this invention are shown. [Figure 26] The results of in vitro OV-CAR3 cell (human ovarian adenocarcinoma cell) proliferation inhibitory activity tests of several antibody conjugates of this invention are shown. [Figure 27] The results of in vitro NCI-N87 cell (human gastric cancer cell) proliferation inhibitory activity tests of several antibody conjugates of this invention are shown. [Figure 28] The results of in vitro SK-BR-3 cell (human breast cancer cell) proliferation inhibitory activity tests of several antibody conjugates of this invention are shown. [Figure 29] The results of in vivo efficacy of several anti-HER2 antibody conjugates in the COLO205 human colon cancer are shown. [Figure 30] The results of in vivo efficacy of several anti-HER2 antibody conjugates of this invention in HCC1954 human breast cancer are shown. [Figure 31] This is a chromatogram of the antibody conjugate 13 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 32] This is a chromatogram of the antibody conjugate 14 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 33] This is a chromatogram of the antibody conjugate 15 of the present invention obtained by size exclusion chromatography (SEC-HPLC). [Figure 34] This is the mass spectrogram of the naked trastuzumab antibody after reduction. [Figure 35] This is a mass spectral gram corresponding to the light and heavy chains of antibody complex 5 of the present invention after reduction. [Figure 36] This is a mass spectral gram corresponding to the light and heavy chains of antibody complex 6 of the present invention after reduction. [Modes for carrying out the invention]

[0036] The embodiments of the present invention will be described below with specific examples, but those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed herein.

[0037] Term definition In this application, the term "ligand" generally refers to a macromolecule that can recognize and bind to an antigen or receptor associated with a target cell. The role of a ligand may be to deliver a drug to a target cell population bound to the ligand. These ligands include, but are 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, a ligand may be represented as Ab, and the ligand antigen may form a linker bond with a linking unit via a heteroatom on the ligand and may be an antibody or its antigen-binding fragment. The antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or mouse antibodies. The antibody may also be a monoclonal antibody. For example, the antibody may be an antibody or its antigen-binding fragment that targets a target selected from the group consisting of HER2.

[0038] In this application, the term "alkyl" generally refers to a residue obtained by removing a hydrogen atom from an alkane. Alkyls may be substituted or unsubstituted. The term "alkyl" generally refers to a saturated linear or branched aliphatic hydrocarbon group having a residue obtained by removing a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, which may be a linear or branched group containing 1 to 20 carbon atoms, for example, an alkyl group containing 1 to 12 carbon atoms, or an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkyls include, but are not limited to, methyl, ethyl, propyl, propyl, and butyl. Alkyls may be substituted or unsubstituted. For example, if alkyl is substituted, this substituent may be substituted at any available linkage point, and this substituent may optionally be substituted independently by one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo, for example, 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, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 It can be an aliphatic group.

[0039] In this application, the term "alkylene" generally refers to a saturated linear or branched aliphatic hydrocarbon group having residues obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which may be a linear or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" may refer to a residue obtained by removing two hydrogen atoms from a single carbon atom group. Methylene may be substituted or unsubstituted, and may be an alkylene containing, for example, 1 to 12 carbon atoms, or 1 to 6 carbon atoms. Non-limiting examples of alkylenes include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). Alkylene may be substituted or unsubstituted, and for example, if alkylene is substituted, substituents may be substituted at any available linkage point, and substituents may optionally be independently substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo, for example substituents 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, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 It may be an aliphatic group. The methylene or alkylene may be substituted or unsubstituted.

[0040] In this application, the term "alkenyl" generally refers to a linear or branched hydrocarbon group containing one or more double bonds. Specific examples of alkenyls include allyl, homoallyl, vinyl, clotyl, butenyl, pentenyl, and hexenyl. 2-6 Specific examples of alkenyl groups include butadienyl, pentadienyl, hexadienyl, hexatrienyl, and their branched forms. The unsaturated bond (double bond) can be located at any position within the carbon chain. The alkenyl may be substituted or unsubstituted.

[0041] In this application, the term "alkenylene" generally refers to a residue obtained by removing two hydrogen atoms from an alkene carbon atom. Examples include allylene, vinylene, butenylene, pentenylene, and hexenylene. Alkenylene may be substituted or unsubstituted.

[0042] In this application, the term "alkynyl" generally refers to unsaturated linear or branched alkynyls such as ethynyl, 1-propynyl, propargyl, and butynyl. The alkynyl may be substituted or unsubstituted.

[0043] In this application, the term "alkylylene group" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atom of an alkyne. For example, it may be ethynylene, propynylene, propargylene, butynylene, etc. The alkylylene may be substituted or unsubstituted.

[0044] In this application, the term "aryl" generally refers to a residue obtained by removing one hydrogen atom from an aromatic ring. The term "aromatic ring" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring having a conjugated π-electron system (i.e., a ring sharing adjacent pairs of carbon atoms), which can be 6- to 10 members, such as benzene and naphthalene. The aromatic ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, with the ring bonded to the parent structure being an aryl ring. The aryl may be substituted or unsubstituted, and if the aryl is substituted, the substituent 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. The aryl may be substituted or unsubstituted.

[0045] In this application, the term "arirene" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atom of an aromatic ring. For example, it may be phenylene, naphthylene, etc. Arirene may be substituted or unsubstituted.

[0046] In this application, the term "heteroaryl" generally refers to a residue obtained by removing one hydrogen atom from the carbon atoms of a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms can be selected from the group consisting of oxygen, sulfur, and nitrogen. Heteroaryls may have 5 to 10 members, or 5 or 6 members, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, and the ring bonded to the parent structure is a heteroaryl ring. The heteroaryl may be substituted or unsubstituted, and if the heteroaryl is substituted, the substituent 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. The heteroaryl may be substituted or unsubstituted.

[0047] In this application, the term "heteroarylene" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atom of a heteroaromatic ring. For example, it may be furanylene, thienylene, pyridylene, pyrrolylene, pyrimidinylene, pyradinylene, imidazoylene, tetrazoylene, etc. Heteroarylene may be substituted or unsubstituted.

[0048] In this application, the term "aliphatic cyclyl" generally refers to a residue obtained by removing a hydrogen atom from the same carbon atom or several different carbon atoms of an aliphatic ring. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon whose carbocyclic ring contains 3 to 20 carbon atoms, which 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, cycloheptatrielinyl, and cyclooctanyl. Polycyclic carbocyclic rings may include spirocyclic, fused, and bridging carbocyclic rings. Aliphatic cyclyls may be substituted or unsubstituted. In this application, the term "carbocykrill" generally refers to a residue obtained by removing one hydrogen atom from a carbon atom of a carbocyclic ring. The term "carbocyclic" generally refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbons, which contain 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 carbocyclics include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, and cyclooctane. Polycyclic carbocyclics may include spirocyclics, fused rings, and bridging rings. The carbocyclyl group may be substituted or unsubstituted. In some cases, alicyclic rings and carbocyclics can be used interchangeably.

[0049] In this application, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between ring molecules. The term "partially unsaturated" encompasses cyclic structures having multiple unsaturated sites, but is not intended to include aromatic rings or heteroaromatic rings as defined in this application. The term "unsaturated" means that the part has one or more degrees of unsaturation.

[0050] In this application, the term "aliphatic cyclylene" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atoms of an alicyclic ring. Examples include cyclopropanylene, cyclobutanylene, cyclopentanylene, cyclopentenylene, cyclohexanylene, cyclohexenylene, cyclohexadienylene, cycloheptanylene, cycloheptatrieylene, and cyclooctanylene. Polycyclic carbocyclic rings may include spiro rings, fused rings, and bridging rings. Aliphatic cyclylenes may be substituted or unsubstituted.

[0051] In this application, the term "aliphatic heterocyclyl" generally refers to a stable, non-aromatic 3-7 member monocyclic carbocyclic structure, a condensed 7-10 member bicyclic heterocyclic structure, or a bridged 6-10 member bicyclic heterocyclic structure. These cyclic structures may be saturated or partially saturated, and in addition to carbon atoms, they may also contain one or more heteroatoms, which may be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these cyclic structures may contain 1-4 heteroatoms as defined above. When used to represent atoms on an aliphatic heterocyclic cyclic structure, the term "nitrogen" may include substituted nitrogen that has undergone a substitution reaction. For example, aliphatic heterocyclyls can include "heterocycloalkyls," which can refer to stable non-aromatic 3-7 membered monocyclic alkane structures, condensed 7-10 membered bicyclic heterocyclic structures, or bridged 6-10 membered bicyclic heterocyclic structures. In addition to carbon atoms, these cyclic structures also include one or more heteroatoms, which can be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, these cyclic structures contain 1-4 heteroatoms as defined above. Heterocycloalkyls may be substituted or unsubstituted. Aliphatic heterocyclyls may be substituted or unsubstituted.

[0052] In this application, the term "aliphatic heterocyclylene" generally refers to a residue obtained by removing two hydrogen atoms from the carbon atoms of an aliphatic heterocycle. Aliphatic heterocyclylenes may be substituted or unsubstituted.

[0053] In this application, the terms "depending on the circumstances" or "depending on the circumstances" generally mean that the event or situation described thereafter may or may not occur, and therefore the description includes both cases in which the event or situation occurs and cases in which it does not occur. For example, "a heterocyclic group that may be substituted with an alkyl group" means that an alkyl group may or may not be present, and the description may include both cases in which the heterocyclic group is substituted with an alkyl group and cases in which it is not.

[0054] In this application, the term "substituted" generally means that one or more (e.g., up to five) hydrogen atoms in a group, for example, one to three hydrogen atoms, are independently substituted by a corresponding number of substituents. Substituents exist only in possible chemical positions, and those skilled in the art can verify possible or impossible substitutions (by experiment or theory) without excessive effort. For example, an amino group or hydroxyl group with free hydrogen may become unstable when bonded to a carbon atom by an unsaturated (e.g., olefinic) bond.

[0055] In this application, as is known to those skilled in the art, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by symbols indicating the number of atoms present in the group in specific cases, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., where the subscript following "C" represents the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having 3 carbon atoms (e.g., n-propyl, isopropyl), C 1-10 Therefore, the members of the group can have any number of carbon atoms in the range of 1 to 10.

[0056] One or more hydrogen atoms (e.g., up to five) in the group, for example, one to three hydrogen atoms, are independently substituted by a corresponding number of substituents. Substituents exist only in possible chemical positions, and those skilled in the art can verify possible or impossible substitutions (experimentally or theoretically) without excessive effort. For example, an amino group or hydroxyl group with free hydrogen can become unstable when bonded to a carbon atom by an unsaturated (e.g., ethylenically) bond.

[0057] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound in this application may be an organic compound. For example, the compound in this application may be a compound with a molecular weight of 500 or less, a compound with a molecular weight of 1000 or less, a compound with a molecular weight of 1000 or more, or a compound with a molecular weight of 10000 or more or 100000 or more. In this application, a compound may also refer to a compound linked by chemical bonds, which may be a compound in which one or more molecules with a molecular weight of 1000 or less are linked to a biomacromolecule by chemical bonds. Biomacromolecules may be polysaccharides, proteins, nucleic acids, polypeptides, etc. For example, the compound in this application may be a compound in which a protein is linked to one or more molecules with a molecular weight of 1000 or less, a compound in which a protein is linked to one or more molecules with a molecular weight of 10000 or less, or a compound in which a protein is linked to one or more molecules with a molecular weight of 100000 or less.

[0058] In this application, the term "comprise" generally refers to including a particularly specified feature, but does not exclude other elements. The terms "more than" and "less than" generally mean including that number.

[0059] In this application, the term "approximately" generally refers to a range of 0.5% to 10% above or below the specified value, for example, within the range of 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.

[0060] In the present application, the compounds of the present application include their tautomers, meso forms, racemates, enantiomers, and / or diastereomers. In the present application, the term "diastereomer" generally refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers may have different physical properties such as melting point, boiling point, spectral characteristics, and reactivity. In the present application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that can be interconverted via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via the movement of protons such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. In the present application, the term "meso form" generally refers to a molecule that contains chiral atoms but has a plane of symmetry within the molecule, resulting in a zero optical rotation within the entire molecule. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomers.

[0061] In the present application, specific atoms of the compounds of the present application may exist in one or more isotopes. For example, hydrogen may exist as protium ( 1 H), deuterium ( 2 H), and tritium ( 3 H), and carbon may exist naturally as three different isotopes ( 12 C, 13 C, and 14 C). Examples of isotopes that can be incorporated into the compounds of the present application include 15 N, 18 O, 17 O, 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125Examples include, but are not limited to, isotopes I or similar isotopes. Therefore, compared to their natural abundances, the compounds of this invention can be enriched with one or more of these isotopes. Such isotope-enriched compounds can be used in a variety of applications, as is known to those skilled in the art. For example, heavier isotopes, such as deuterium ( 2 Substitution with H) may yield certain therapeutic benefits due to greater metabolic stability. For example, deuterium ( 2 The natural abundance of H) is approximately 0.015%. Therefore, in nature, there is one deuterium atom for every approximately 6500 hydrogen atoms. For this reason, the deuterium-containing compounds of this application have a deuterium abundance greater than 0.015% at one or more positions (in some cases). Unless otherwise explicitly indicated, 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 in which a hydrogen atom is substituted with deuterium or tritium, or a carbon atom is substituted with carbon-13 or carbon-14, except that the rest of the compound matches the structure of this application, fall within the scope of this application.

[0062] In this application, the term "pharmaceutical composition" generally refers to a mixture comprising one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, and other chemical components and other components (e.g., physiologically / pharmaceutically acceptable carriers and excipients). Pharmaceutical compositions can facilitate administration to a living organism and facilitate the absorption of the active ingredient to exert biological activity. The preparation of conventional pharmaceutical compositions can refer to commonly used techniques in the art.

[0063] In this application, the term “pharmaceutically acceptable salt” generally refers to a salt of the compound or ligand-drug conjugate described herein, or a salt of the compound described herein. Such salts may be safe and / or effective and may have the desired biological activity when used in the body of a mammal. The antibody-antibody-drug conjugate compounds of this application may form salts with acids, and non-limiting examples of pharmaceutically 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.

[0064] In this application, the term "conjugate" generally refers to a compound prepared by the compound of this application through one or more chemical reactions, or a compound prepared by linking the compound of this application via one or more linking structures such as bridges, spacers, or connecting portions.

[0065] In this application, the term “pharmaceutically acceptable carrier” generally refers to a carrier used for administering a therapeutic agent (e.g., antibodies or polypeptides, genes, and other therapeutic agents). This term refers to a drug carrier that does not induce the production of antibodies harmful to the individual to which the composition is administered and does not exhibit excessive toxicity after use. For example, a pharmaceutically acceptable carrier can be distinguished from a nucleic acid vector used in genetic engineering to contain a target gene. Suitable carriers may be large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid polymers, and inactivated virus particles. Such carriers are well known to those skilled in the art. The pharmaceutically acceptable carrier in a therapeutic composition may include liquids such as water, saline, glycerin, and ethanol. Auxiliary substances such as wetting agents or emulsifiers and pH buffers may also be present in these carriers.

[0066] In this application, the terms "Trop2" and "TROP2" generally refer to a single-pass transmembrane type 1 cell membrane protein. In this application, the term "Trop2" may also include homologs, variants, and isoforms (including splicing isoforms) of Trop2. The term "Trop" includes proteins having one or more sequences in Trop2 homologs, variants, and isoforms, as well as fragments of such sequences, insofar as variant proteins (including isoforms) exist. Trop2 may be human Trop2. For example, Uniprot accession number P09758 contains a description of Trop2 and its sequence.

[0067] 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. The term encompasses "full-length" and unprocessed HER2, as well as any form of HER2 resulting from processing in cells (e.g., mature protein). The term also encompasses naturally occurring variants and isoforms of HER2, such as splicing variants or allele variants. For example, Uniprot accession number P04626 contains a description of HER2 and its sequence.

[0068] 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 of 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 splicing variants or allele variants. For example, Uniprot accession number Q96NY8 contains a description of nectin-4 and its sequence.

[0069] 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, thereby reducing the immune response induced by the mouse antibody. To produce a chimeric antibody, a hybridoma that secretes a specific monoclonal antibody derived from a mouse is established, the variable region gene is cloned from the mouse hybridoma cells, and if necessary, the constant region gene of a human antibody is cloned. The mouse variable region gene and the human constant region gene are then linked to a chimeric gene, and this is inserted into an expression vector to express the chimeric antibody molecule in a eukaryotic or prokaryotic system.

[0070] In this application, the term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody produced by transplanting a mouse CDR sequence into a variable region framework of a human antibody, that is, a framework sequence of a different type of human germline antibody. Because it retains a large amount of mouse protein components, it can overcome heterologous reactions caused by chimeric antibodies. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database.

[0071] In this application, the terms "fully human antibody," "fully human-type antibody," or "fully human-derived antibody" are also known as "fully human monoclonal antibody," and both the variable and constant regions of the antibody are of human origin, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has progressed through four stages: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibodies or ligands described in this application may be fully human monoclonal antibodies. 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.

[0072] In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., Chothia et al., and MacCallum et al. When used in this application, Kabat's definition of CDR applies only to the heavy chain variable domains CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) in addition to the light chain variable domains CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3).

[0073] In this application, the term "group capable of binding to mercapto" means that compound A has a group capable of binding to mercapto, compound B has a group capable of binding to mercapto, and compound B reacts with the mercapto of compound A via the group capable of binding to mercapto, thereby achieving linkage between compound A and compound B.

[0074] In this application, the term “linker” generally refers to a chemical structural fragment or bond that is linked to one group at one end and to another group at the other end, and which may also be linked to other linkers before linking to a drug and / or ligand. Direct or indirect linking to a ligand may mean that the group is directly linked to the ligand via a covalent bond, or it may be linked to the ligand via a linker. For example, the linker may be the structure shown in Q1 described in this application. For example, chemical fragments or bonds including acid-unstable linker structures (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker structures, photo-unstable linker structures, dimethyl linker structures, or disulfide-containing linker structures can be used as linkers.

[0075] In this application, the term "linking group" generally refers to a group that has the ability to link 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.

[0076] In this application, the term "drug unit" generally refers to a chemical portion that directly or indirectly binds to an antibody or antigen-binding fragment to form an immune complex. For example, "drug units" include, but are not limited to, the compounds having antitumor activity described herein. For example, drug units include topoisomerase inhibitors.

[0077] In this application, the term "compound having 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 during the treatment period, stabilization or reduction of tumor size, or an extension of survival time with treatment compared to an untreated control. Antitumor activity can be evaluated using an approved in vitro or in vivo tumor model (e.g., a xenograft model).

[0078] In some embodiments of the present invention, the bioactive 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 Alternatively, radioactive isotopes of Lu, metal complexes such as metal-platinum complexes (such as oxaliplatin) or metal-gold complexes, glycopeptide antibiotics such as bleomycin or pinyanmycin, topoisomerase inhibitors such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, and other drugs that interfere with DNA synthesis, drugs that act on structural proteins such as microtubule system inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, meitansinoids, auristatin, 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, and cyclin-dependent kinase inhibitors. In this application, the term "topoisomerase inhibitor" generally refers to compounds or derivatives of topoisomerase I inhibitors and topoisomerase II inhibitors. Examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogues, and topoisomerase II inhibitors (e.g., actinomycin D, adriamycin, doxorubicin, amikacin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). Topoisomerase may refer to an enzyme that modifies the number of DNA strands by cleaving phosphodiester bonds in one or both strands of DNA, and then unwinding and sealing them.

[0079] In this application, the term "camptothecin analog" generally refers to compounds that are structurally similar to or derived from camptothecin. For example, the structure of camptothecin is described in CAS number 7689-03-4. For example, a camptothecin analog may refer to exatecan (CAS number 171335-80-1) or belotecan (CAS number 256411-32-2). The term "non-camptothecin-type topoisomerase I inhibitor" generally refers to heterocyclic molecules of indolocarbazoles, indenoisoquinolinones, benzophenanthidines, and dibenzonaphthyridinones that have topoisomerase I inhibitory activity, and mainly refers to Genz-644282 (CAS number 529488-28-6).

[0080] In this application, the term “disease associated with the expression of a certain target” generally means that the onset and / or progression of the disease is associated with the level of expression of the target. For example, the level of expression of a certain target is increased, i.e., high, in cells derived from a diseased area (e.g., a specific tissue or organ of the patient) compared to the level of expression of a normal cell derived from a tissue or organ. Or, for example, the level of expression of a certain target is decreased, i.e., low, in cells derived from a diseased area (e.g., a specific tissue or organ of the patient) compared to the level of expression of a normal cell derived from a tissue or organ. Or, for example, cells derived from a diseased area (e.g., a specific tissue or organ of the patient) express a certain target, i.e., positive. Or, for example, cells derived from a diseased area (e.g., a specific tissue or organ of the patient) do not express a certain target, i.e., negative. Target expression can be characterized by standard assays known in the art.

[0081] In this application, the term "effective dose" generally refers to the amount of therapeutic agent that treats, alleviates, or prevents a target disease or symptom, or that exhibits a detectable therapeutic or preventive effect. The exact effective dose for a subject will depend on the subject's body type and health status, the nature and severity of the disease, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For this reason, it is not useful to pre-specify the exact effective dose. However, for a given situation, a clinician can determine the effective dose through routine experimentation.

[0082] Unless otherwise specified, all compounds described herein include all possible optical isomers, such as single chiral compounds or mixtures of various chiral compounds (i.e., racemates). In all compounds described herein, each chiral carbon atom may optionally be in an R configuration, an S configuration, or a mixture of R and S configurations.

[0083] As used herein, the term “the compound of the application” refers to the compound of the application. This term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compound of the application.

[0084] In this specification, when a trade name is used, it is intended to include the formulation of the trade name product, its corresponding generic drug, and the active pharmaceutical ingredient of the trade name product.

[0085] As used herein, the term “antibody” is used in its broadest sense and includes, in particular, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity. Antibodies may be derived from mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens typically have numerous binding sites, also called epitopes, that are recognized by various antibody CDRs. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen may have one or more corresponding antibodies. Antibodies are molecules containing a full-length immunoglobulin molecule or the immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen or a portion thereof that specifically binds to a target, such targets 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 molecules. Immunoglobulins can originate from any species. However, in one embodiment, immunoglobulins are derived from humans, mice, or rabbits. "Antibody fragment" may include a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, minibodies, Fab expression library preparation fragments, anti-idiotype (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 this application may maintain their antigen-binding ability in their original wild state. Therefore, the antibody in this application can specifically bind to the antigen.Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules related to tissue growth and differentiation (such as known or predicted functional molecules), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (for example, antigens to which known antibodies bind may be one or a subset of the above classification, while other subsets include other molecules / antigens with specific properties (compared to target antigens)). 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 produced 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 more than on the surface of non-cancer cells.

[0086] 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 containing 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 containing the heavy chain variable region and the light chain variable region of enfortumab.

[0087] 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 containing the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 of pertuzumab. In this application, pertuzumab may refer to an antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of pertuzumab.

[0088] 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 containing the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 of trastuzumab. In this application, trastuzumab may refer to an antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of trastuzumab.

[0089] 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 containing the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 of sacituzumab. In this application, sacituzumab may refer to an antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of sacituzumab.

[0090] In this application, the term "patritumab" generally refers to an antibody that targets HER3-related antibodies. For example, patritumab is described in Patent Document 6. In this application, patritumab may refer to any antibody or antigen-binding fragment containing the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 of patritumab. In this application, patritumab may refer to an antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of patritumab.

[0091] In this application, the term "antibody H01L02" generally refers to an antibody that targets CDH6. For example, the monoclonal antibody H01L02 is described in Patent Documents 7 and 8. For example, the H01L02 monoclonal antibody may be the monoclonal antibody used in the drug DS6000. In this application, the H01L02 monoclonal antibody may refer to any antibody or antigen-binding fragment containing the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of the H01L02 monoclonal antibody. In this application, the H01L02 monoclonal antibody may refer to an antibody or antigen-binding fragment containing the heavy chain variable region and the light chain variable region of the H01L02 monoclonal antibody.

[0092] In this application, the term "polypeptide residue" generally refers to a residue containing one or more linked amino acid residues. For example, one or more amino acids within a polypeptide residue may be substituted as appropriate. For example, polypeptide residues in this application can 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).

[0093] In this application, the term "polyethylene glycol" generally refers to a residue containing one or more ethylene glycol residues bonded together. For example, polyethylene glycol is -(CH2CH2O) p - may be included, where p is at least one. For example, polyethylene glycol in this application may be substituted in some cases.

[0094] In this application, the term "glycol" generally refers to polyethylene glycol. For example, glycol in this application may be substituted as it may be. For example, the number preceding glycol may represent the number of ethylene glycol units in the glycol, and for example, a diethylene glycol group may refer to two polymerization residues of ethylene glycol.

[0095] In this application, the term "polysarcosine residue" generally refers to a residue containing one or more sarcosine residues linked together. For example, a polysarcosine residue is -(COCH2N(CH3)) q - may be included, where q is at least one. For example, the polysarcosine residue in this application may be substituted in some cases. For example, a structure containing a polysarcosine residue is, [ka] This is possible, where n² is a number between 4 and 18.

[0096] In this application, the term "sodium dodecyl sulfate-polyacrylamide gel electrophoresis" generally refers to a material analysis and characterization technique. For example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can detect the molecular weight of a substance.

[0097] In this application, the term "hydrophobic chromatography" generally refers to an analytical technique based on differences in the hydrophobicity of substances.

[0098] In this application, the term "liquid chromatography-mass spectrometry" generally refers to an analytical method for identifying the components of a substance. For example, liquid chromatography-mass spectrometry can analyze the molecular weight of a test substance using liquid chromatography and mass spectrometry.

[0099] In this application, the term "tumor" generally refers to a mass of tissue formed by the autonomous and excessive growth of tissue. For this application, angiogenesis is part of the characteristics of a tumor. Tumors can be benign or malignant. The term "tumor" is generally used to refer to benign or malignant tumors, while the term "cancer" is generally used to refer to metastatic or non-metastatic malignant tumors. Tumors that can be diagnosed by the method of this application are selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic 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 tumors, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma, and mesothelioma. When used in research, these tissues can be isolated from readily available sources in a manner well known to those skilled in the art.

[0100] (Detailed description of the invention) In one embodiment, the present application provides a compound, or a tautomer thereof, a meso compound, a racemic compound, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound comprises a structure represented by formula (C-HER2).

[0101] [ka] [In the formula, Q can be a linking group, L1 is -L 1a -C(=O)- may be included, Here, L 1a This may be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkylylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted aliphatic heterocyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups. L2 may contain substituted polypeptide residues, L3 may optionally include a substituted spacer group, for example the spacer group of the present application may have self-decomposing ability. [ka] , or replaced in some cases [ka] May include, Here, L2 and / or L3 may include optionally substituted polysarcosine residues, T may include drug units. Ab is a ligand that can bind to HER2, and m can be a number between 1 and 8.

[0102] In another embodiment, the present application provides compounds, or their tautomers, meso compounds, racemic compounds, enantiomers, and diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, wherein the compounds may include a structure represented by formula (CM-HER2).

[0103] [ka] [In the formula, Q1 may include a linker, L1 is -L 1a -C(=O)- may be included, Here, L 1aThis may be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkylylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted aliphatic heterocyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups. L2 may contain substituted polypeptide residues, L3 may optionally include a substituted spacer group, for example the spacer group of the present application may have self-decomposing ability. [ka] Or, depending on the case, replaced [ka] May include, Here, L2 and / or L3 may include the substituted structural unit-X, T may include drug units. Ab is a ligand that can bind to HER2, and m can be an integer from 1 to 8.

[0104] For example, here, L3 is replaced depending on the case. [ka] and, if applicable, replaced [ka] It is selected from the group consisting of the following.

[0105] For example, the benzene ring of L3 here may be substituted with a substituted structural unit -X. For example, the structural unit -X may be substituted [ka] X1 may be 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S; X2 may be 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S; and X3 may be hydrogen, The group consists 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 the heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and the 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 independently optionally substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0106] For example, here the benzene ring of L3 may be substituted with optionally substituted structural unit -X. For example, the structural unit -X may be substituted [ka] The heteroalkyl group may include, where X1 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl groups containing 1 to 8 atoms, and linear-cyclic heteroalkyl groups containing 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 atoms selected from N, O, or S, and the C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl groups containing 1 to 8 atoms, and linear-cyclic heteroalkyl groups containing 1 to 8 atoms are each optionally substituted independently with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0107] For example, here, structural unit-X is substituted depending on the case. [ka] That is the case.

[0108] For example, the present application provides compounds, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, wherein the compounds may include a structure represented by formula (C-M2).

[0109] [ka] [In the formula, L may optionally comprise a substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 may optionally comprise a substituted isopropyl or an optionally substituted benzyl; and R2 may optionally comprise a substituted methyl or an optionally substituted [ka] Or, depending on the case, replaced [ka] It could be R3 or R5, and R5 may be substituted. [ka] This could be the case, and T may include Exatecan (CAS number 171335-80-1) and / or Belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0110] For example, here, Q1 may include a linker coupled with a mercapto.

[0111] For example, here, Q1 is replaced depending on the case. [ka] , replaced in some cases [ka] , replaced in some cases [ka] , and, if applicable, replaced [ka] A selection can be made from the group consisting of the following:

[0112] For example, here, L 1aThis can be selected from the group consisting of optionally substituted C1-C7 alkylene groups, optionally substituted diethylene glycol to octaethylene glycol, optionally substituted C3-C6 aliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups.

[0113] For example, here, L 1a This can 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.

[0114] For example, here, L 1a This can be selected from the group consisting of optionally substituted methylene groups, optionally substituted ethylene groups, optionally substituted propylene groups, optionally substituted butylene groups, and optionally substituted pentylene groups.

[0115] For example, here, L 1a This can be selected from the group consisting of optionally substituted diethylene glycol groups, optionally substituted triethylene glycol groups, optionally substituted tetraethylene glycol groups, optionally substituted pentaethylene glycol groups, optionally substituted hexaethylene glycol groups, optionally substituted heptaethylene glycol groups, and optionally substituted octaethylene glycol groups.

[0116] For example, here, L 1a This can be selected from the group consisting of optionally substituted cyclopropylene groups, optionally substituted cyclobutylene groups, and optionally substituted cyclohexylene groups.

[0117] For example, here, L2 may include optionally substituted polypeptide residues composed 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.

[0118] For example, here, L2 may include optionally substituted polypeptide residues composed of amino acids selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine, and lysine.

[0119] For example, here, L2 may include optionally substituted polypeptide residues composed of amino acids selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamate-valine-alanine (Glu-Val-Ala), glutamate-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).

[0120] For example, here, L2 may include optionally substituted polypeptide residues selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), and valine-lysine (Val-Lys).

[0121] For example, if L2 contains a lysine residue, the lysine residue may be substituted with structure R1, which contains a polysarcosine residue.

[0122] For example, any H contained in L2 may be substituted with R1.

[0123] For example, here, R1 may be replaced [ka] This is possible, 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.

[0124] For example, here n1 could be 4-18, 8-18, 4-12, or 8-12. For example, here n1 could be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0125] For example, here, L3 is replaced depending on the case. [ka] and, if applicable, replaced [ka] It is selected from the group consisting of the following.

[0126] For example, the benzene ring of L3 may be substituted with structure R2, which contains a polysarcosine residue.

[0127] For example, any H in the benzene ring of L3 may be substituted with R2.

[0128] For example, here the benzene ring of L3 is linked to a optionally substituted polysarcosine residue via structural unit -X-, and structural unit -X- is optionally substituted [ka] 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, wherein the heteroalkyl contains 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S; and X3 is covalently bonded or The group consists 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and the 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 independently optionally substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0129] For example, the benzene ring of L3 is linked to a optionally substituted polysarcosine residue via structural unit -X-, and structural unit -X- is Replaced in some cases [ka] X1 is selected from, but is not limited to, 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 the heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and each of the 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 is optionally substituted with one or more substituents independently selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxyl, alkoxy, or cycloalkyl.

[0130] For example, here, the structural unit -X- is substituted depending on the case. [ka] The polysarcosine residue substituted in the above case is [ka] This includes, 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.

[0131] For example, here, the structural unit -X- is substituted depending on the case. [ka] 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, wherein the heteroalkyl contains 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S; and X3 is covalently bonded or hydrogen, C1-C8 Selected from the group consisting of alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and each of the 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 is 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 [ka] This includes, 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.

[0132] For example, here, the structural unit -X- is substituted depending on the case. [ka] The polysarcosine residue substituted in the above case is [ka] This includes, 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, here, the structural unit -X- is substituted depending on the case. [ka] 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, wherein the heteroalkyl contains 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, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S; and X3 is covalently bonded or hydrogen, C1-C8 Selected from the group consisting of alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl containing 1 to 8 atoms, and linear-cyclic heteroalkyl containing 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 atoms selected from N, O, or S, and each of the 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 is 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 [ka] This includes, 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, here n2 could be 4-18, 8-18, 4-12, or 8-12. For example, here n2 could be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0135] For example, here, T may include a compound having antitumor activity.

[0136] For example, here, T may include a topoisomerase inhibitor.

[0137] For example, here, T may include topoisomerase I inhibitors of camptothecines and non-camptothecines.

[0138] For example, here T may include exatecan (CAS number 171335-80-1) and / or belothecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0139] For example, here T is [ka] The structure of the group can be selected from the following:

[0140] For example, the present application provides a compound, or a tautomer thereof, a meso compound, a racemic compound, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may include a structure represented by formula (C-HER2).

[0141] [ka] [In the formula, Q1 may include a linker coupled with a mercapto, L1 is -L 1a -C(=O)- may be included, where L 1a This may be selected from the group consisting of optionally substituted C1-C7 alkylene groups, optionally substituted diethylene glycol to octaethylene glycol, optionally substituted C3-C6 aliphatic cyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups. L2 may contain optionally substituted polypeptide residues composed of amino acids selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine, and lysine. L3 is replaced in some cases. [ka] May include, Here, L2 may contain a polysarcosine residue. T may include a topoisomerase inhibitor.

[0142] For example, the present application provides a compound, or a tautomer thereof, a meso compound, a racemic compound, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may include a structure represented by formula (C-HER2).

[0143] [ka] [In the formula, Q1 may include a linker coupled with a mercapto, L1 is -L 1a -C(=O)- may be included, where L 1amay be a C1-C7 alkylene group which may be substituted, diethylene glycol to octaethylene glycol which may be substituted, a C3-C6 aliphatic cyclylene group which may be substituted, an arylene group which may be substituted, or a heteroarylene group which may be substituted, and may be selected from the group consisting of L2 may contain a polypeptide residue which may be substituted and is composed of an amino acid selected from the group consisting of glycine, phenylalanine, valine-alanine, arginine, citrulline, aspartic acid, asparagine, and lysine. L3 may be

Chemical formula

[0144] For example, the present application provides a compound, or a tautomer, meso form, racemic form, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may contain a structure represented by formula (C-HER2).

[0145]

Chemical formula

Chemical formula

[0146] For example, the present application provides a compound, or a tautomer thereof, a meso compound, a racemic compound, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, wherein the compound may include a structure represented by formula (C-HER2).

[0147] [ka] [In the formula, Q1 is substituted depending on the case.] [ka] , replaced in some cases [ka] , replaced in some cases [Chemistry] and optionally substituted [Chemistry] may be selected from the group consisting of L1 may contain -L 1a -C(=O)-, where L 1a may contain an optionally substituted C1-C7 alkylene group L2 may contain 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 may be optionally substituted [Chemistry] and may contain where L3 may be optionally substituted [Chemistry] and may be replaced by, where n2 is a number from 4 to 18 T may contain 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 a tautomer, meso form, racemic form, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, where the compound may contain a structure represented by formula (C2-HER2).

[0149] [Chemistry] [In the formula, L may optionally comprise a substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 may optionally comprise a substituted isopropyl or an optionally substituted benzyl; and R2 may optionally comprise a substituted methyl or an optionally substituted [ka] Or, depending on the case, replaced [ka] R3 may be hydrogen or optionally substituted methyl, where R2 may be optionally substituted [ka] This can be substituted with, where n1 is a number between 4 and 18, and T may include exatecan (CAS number 171335-80-1) and / or belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0150] For example, the present application provides compounds, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, wherein the compounds may include a structure represented by the formula (C2-HER2).

[0151] [ka] [In the formula, L may optionally comprise a substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 may optionally comprise a substituted isopropyl or an optionally substituted benzyl; and R2 may optionally comprise a substituted methyl or an optionally substituted [ka] Or, depending on the case, replaced [ka] R3 may be hydrogen or optionally substituted methyl, where R2 may be optionally substituted [ka] This can be substituted with n², where n² is a number between 4 and 18, and T may include exatecan (CAS number 171335-80-1) and / or belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0152] For example, the present application provides compounds, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, wherein the compounds may include a structure represented by the formula (C2-HER2).

[0153] [ka] [wherein L may optionally comprise a substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 may optionally comprise isopropyl or optionally comprise benzyl; and R2 may optionally comprise [ka] R3 may be hydrogen or optionally substituted methyl, where R4 may be optionally substituted [ka] This is possible, where n1 is a number between 4 and 18, and T may include Exatecan (CAS number 171335-80-1) and / or Belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0154] For example, the present application provides compounds, their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, wherein the compounds may include a structure represented by the formula (C2-HER2).

[0155] [ka] [In the formula, L may optionally comprise a substituted alkylene group, an optionally substituted polyethylene glycol group, and an optionally substituted aliphatic cyclylene group; R1 may optionally comprise a substituted isopropyl or an optionally substituted benzyl; and R2 may optionally comprise a substituted methyl or an optionally substituted [ka] Or, depending on the case, replaced [ka] It could be R3 or R5, and R5 may be substituted. [ka] This is possible, where n² is a number between 4 and 18, and T can be Exatecan (CAS number 171335-80-1) and / or Belotecan (CAS number 256411-32-2) and / or Genz-644282 (CAS number 529488-28-6).

[0156] For example, Ab may include an anti-HER2 antibody or its antigen-binding fragment.

[0157] For example, the antibody may be selected from the group consisting of mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0158] For example, the antibody may include a monoclonal antibody.

[0159] For example, the antibody may include a bispecific antibody.

[0160] 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.

[0161] For example, the heavy chains HCDR1, HCDR2, and HCDR3 and light chains LCDR1, LCDR2, and LCDR3 of Ab each include the heavy chains HCDR1, HCDR2, and HCDR3 and light chains LCDR1, LCDR2, and LCDR3 of the anti-HER2 antibody, respectively.

[0162] For example, the heavy chain variable region VH and light chain variable region VL of Ab each include the heavy chain variable region VH and light chain variable region VL of the anti-HER2 antibody, respectively.

[0163] For example, the heavy chain and light chain of Ab each include the heavy chain and light chain of an anti-HER2 antibody.

[0164] For example, Ab may include trastuzumab or pertuzumab.

[0165] For example, here, 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 the antibody-drug conjugate obtained after a single monoclonal antibody molecule is conjugated with a cytotoxic drug, and m can be an integer or decimal between 1 and 8, for example m can 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 can be about 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.

[0166] In another embodiment, the present application also provides compounds, or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof. Here, the compound may include a structure selected from the group consisting of the following:

[0167] [ka] [ka]

[0168] 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 of this application may be anti-Her2 antibodies or their antigen-binding fragments.

[0169] In this application, the ligand comprises at least one CDR within the variable region (VL) of the antibody light chain. The CDR described herein may be defined according to Kabat, or according to Chothia, and CDR sequences defined in various ways are included within the scope of protection of this application.

[0170] For example, the antigen-binding protein of the present invention may include CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region, where CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region may be CDR1-3 of trastuzumab and CDR1-3 of pertuzumab, respectively. For example, the antigen-binding protein of the present invention may have the ability to bind to HER2.

[0171] For example, the antigen-binding protein of the present invention may include 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 trastuzumab and pertuzumab, respectively. For example, the antigen-binding protein of the present invention may have the ability to bind to HER2.

[0172] For example, the antigen-binding protein of the present invention may include a heavy chain and a light chain, where the heavy chain and light chain may be the heavy chain and light chain of trastuzumab and pertuzumab, respectively.

[0173] For example, the heavy chain amino acid sequence of trastuzumab may be shown in SEQ ID NO:3, and the light chain amino acid sequence of trastuzumab may be shown in SEQ ID NO:4.

[0174] To verify the outstanding advantages of the compound of the present invention, the ligand of the present invention may be, for example, as follows: For example, the heavy chain amino acid sequence of sacituzumab may be shown in SEQ ID NO:1, and the light chain amino acid sequence of sacituzumab may be shown in SEQ ID NO:2. For example, the heavy chain amino acid sequence of trastuzumab may be shown in SEQ ID NO:3, and the light chain amino acid sequence of trastuzumab may be shown in SEQ ID NO:4. For example, the heavy chain amino acid sequence of pertuzumab may be shown in SEQ ID NO:5, and the light chain amino acid sequence of pertuzumab may be shown in SEQ ID NO:6. For example, the heavy chain amino acid sequence of enfortumab may be shown in SEQ ID NO:7, and the light chain amino acid sequence of enfortumab may be shown in SEQ ID NO:8. For example, the heavy chain amino acid sequence of patritumab may be shown in SEQ ID NO:9, and the light chain amino acid sequence of patritumab may be shown in SEQ ID NO:10. For example, the heavy chain amino acid sequence of antibody H01L02 may be shown in SEQ ID NO:11, and the light chain amino acid sequence of antibody H01L02 may be shown in SEQ ID NO:12.

[0175] The antibodies of this application can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthesis techniques, or combinations thereof, or other techniques known in the art. Variants may refer to amino acid sequence variants of antibodies and covalent derivatives of natural polypeptides, as long as they retain biological activity comparable to that of the natural polypeptide. The difference between an amino acid sequence variant and a natural amino acid sequence generally lies in the substitution of one or more amino acids in the natural amino acid sequence, or the deletion and / or insertion of one or more amino acids in the polypeptide sequence. Deletion variants include fragments of natural polypeptides and N-terminal and / or C-terminal cleavage variants. Typically, amino acid sequence variants have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more homology to the natural sequence.

[0176] The antibody-drug conjugate provided in this application can target specific cell populations and bind to specific proteins (antigens) on the cell surface, thereby releasing the drug into the cell in an active form through endocytosis or drug infiltration of the conjugate. Therefore, the antibody-drug conjugate of this application can be used to treat targeted diseases and can be administered to subjects (such as humans) in therapeutically effective doses via appropriate routes. Subjects requiring treatment may be patients at risk of, or suspected of having, a disease associated with the activity or expression of a specific antigen. Such patients can be identified through conventional health examinations.

[0177] When treated with the antibody-drug conjugate of this invention, it can be delivered by conventional methods in the art. For example, it 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 using an infusion pump. Other methods may include the use of various delivery and carrier systems using the conjugate and biodegradable polymers.

[0178] In one embodiment, the present application provides a pharmaceutical composition that may comprise a compound described in any one of the present applications, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and optionally a pharmaceutically acceptable carrier.

[0179] The pharmaceutical composition described in this 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, and excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0180] The pharmaceutical composition containing the active ingredient may be in dosage forms suitable for oral use, such as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups. The oral composition may be prepared by methods for preparing pharmaceutical compositions known in the art, and the composition may include binders, fillers, lubricants, disintegrants, or pharmaceutically acceptable wetting agents. The composition may also contain one or more components selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives.

[0181] The aqueous suspension may contain excipients suitable for mixing with the active substance and for preparing the aqueous suspension. The aqueous suspension may contain one or more preservatives, such as one or more colorants, one or more flavoring agents, and one or more sweeteners. The oily suspension can be prepared by suspending the active ingredient in vegetable oil. The oily suspension may contain thickeners. The aforementioned sweeteners and flavoring agents may also be added.

[0182] The pharmaceutical composition may also contain one or more dispersants, wetting agents, suspending agents, or preservatives as active ingredients, which are obtained by mixing dispersible powders and granules with water to prepare an aqueous suspension. Other excipients, such as sweeteners, flavoring agents, and colorants, may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid. The pharmaceutical composition of this application may also be in the form of an oil-in-water emulsion.

[0183] The pharmaceutical composition may be in the form of a sterile, injectable aqueous solution. Acceptable vehicles or solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile oil-in-water microemulsion for injection in which the active ingredient is dissolved in the oil phase. For example, the active ingredient can be dissolved in a mixture of soybean oil and lecithin, and then the oil solution can be added to a mixture of water and glycerol and treated to form a microemulsion. The injectable solution or microemulsion can be injected into the patient's bloodstream by local bolus injection. Alternatively, the solution and microemulsion can be administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous delivery device can be used. For example, the device may be an intravenous infusion pump.

[0184] The pharmaceutical composition may be in the form of a sterile aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension may be prepared using the appropriate dispersants or wetting agents and suspending agents described above in accordance with known techniques. The sterile injection formulation may be a sterile injection solution or suspension prepared in a non-toxic, parenterally acceptable diluent or solvent. Alternatively, a sterile fixative oil may be conveniently used as the solvent or suspension medium. Sterile fixative oil is commonly used as the solvent or suspension medium.

[0185] The compounds of this 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 dissolves in the rectum to release the drug. Such substances include, for example, cocoa butter, glycerin-gelatinized, hydrogenated vegetable oil, polyethylene glycol of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

[0186] 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 specific compound used, the patient's age and weight, the patient's health condition, the patient's behavior, the patient's diet, the time of administration, the method of administration, the rate of excretion, the combination of drugs, etc. Also, the optimal treatment method (e.g., treatment regimen), the form of the compound described in this application, or its tautomers, meso, racemic, enantiomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts thereof, the daily dose of the compound, or its tautomers, meso, racemic, enantiomers, diastereomers or mixtures thereof, or the type of pharmaceutically acceptable salt thereof can be determined according to conventional treatment plans.

[0187] The pharmaceutical composition of this application may contain a safe and effective amount of the antibody-drug conjugate of this application and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Typically, pharmaceutical formulations need to be adapted to a method of administration, and the pharmaceutical composition of this application can be manufactured in solution form, for example, by conventional methods using an aqueous solution containing physiological saline, glucose, or other adjuvants. The pharmaceutical composition can be manufactured under sterile conditions. The dose of the active ingredient may be a therapeutically effective dose.

[0188] The effective dose of the antibody-drug conjugate described herein may vary depending on the mode of administration, the severity of the disease awaiting treatment, etc. The selection of the effective dose can be determined by a person skilled in the art based on various factors (e.g., through clinical trials). These factors may include, but are not limited to, the pharmacokinetic parameters of the dual-function antibody conjugate (bioavailability, metabolism, half-life, etc.), the severity of the disease being treated in the patient, the patient's weight, the patient's immune status, and the route of administration. Typically, a satisfactory effect can be obtained by administering the antibody-drug conjugate of this invention in an appropriate daily dose. For example, depending on the urgency of the treatment situation, it may be administered in several divided doses daily, or the dose may be reduced proportionally.

[0189] The compound of this application can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. When used in a pharmaceutical composition, a safe and effective amount of the compound of this application can be applied to mammals (such as humans) in need of treatment, and the dose administered may be a medically and pharmaceutically approved dose, with the specific dose taking into consideration factors such as the route of administration and the patient's health condition.

[0190] This application provides the use of the compounds of this application, or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, and / or pharmaceutical compositions thereof, in the preparation of drugs that can be used for the treatment and / or prevention of tumors. For example, the tumors may be selected from tumors associated with the expression of the target, which is HER2. For example, tumors associated with the expression of the target include tumors with high expression of the target and / or target-positive tumors. For example, the tumors include solid tumors and / or hematological malignancies. For example, the tumors may be selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic 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 tumors, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, glioma, and mesothelioma.

[0191] This application provides the use of the compounds of this application, or their tautomers, meso-molecules, racemics, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, and / or pharmaceutical compositions of this application in drugs that can be used for the treatment and / or prevention of tumors. For example, the tumors may be selected from tumors with expression of the target, which is HER2. For example, tumors with expression of this target include tumors with high expression of the target and / or tumors that are positive for the target. For example, the tumors include solid tumors and / or hematological malignancies. For example, the tumors may be selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic 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 tumors, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, gliomas, and mesotheliomas.

[0192] The present application provides a method for the prevention and / or treatment of a tumor, which may include administering to a subject the compound of the present application, or its tautomers, meso-molecules, racemics, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and / or pharmaceutical compositions of the present application. For example, the tumor may be selected from tumors with expression of the target HER2. For example, tumors with expression of the target include tumors with high expression of the target and / or tumors that are positive for the target. For example, the tumors include solid tumors and / or hematological malignancies. For example, the tumors may be selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic 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 tumors, pancreatic cancer, prostate cancer, colon cancer, gastric cancer, gliomas, and mesotheliomas.

[0193] While not intended to be limited by any theory, the following examples are solely for illustrative purposes of illustrating the compounds, preparation methods, and uses of the present invention and are not intended to limit the scope of the invention. [Examples]

[0194] (Example 1: Synthesis and preparation of the compound) The raw materials described herein are commercially available or prepared according to methods known in the art or methods described herein. Here, Fmoc is a 9-fluorenylmethyloxycarbonyl protecting group, Boc is a tert-butyldimethylsilyl protecting group, and TBDMS / TBS is a tert-butyldimethylsilyl protecting group.

[0195] Synthesis of compound (A-1)

[0196] [ka] Synthesis pathway:

[0197] [ka]

[0198] 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 500 ml of aqueous solution, respectively. The reaction mixture was stirred at room temperature for 48 hours, and the completion of the reaction was detected. The reaction mixture was adjusted to pH 6 with 1N dilute hydrochloric acid, extracted with 500 ml of ethyl acetate, separated the organic phase, washed once with water and saturated brine, 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, yield 82%) as a white solid. LC-MS (ESI, m / z) calculated value: 567.29, measured value: 568.26 (M+H).

[0199] Step 2: Synthesis of intermediates 1-2 Intermediate 1-1 (60 g, 106 mmol) was dissolved in a mixed solvent 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 mixture was stirred at room temperature for 24 hours, evaporated to dryness under reduced pressure, diethyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and dried to obtain intermediate 1-2 (51 g, 72% yield). LC-MS (ESI, m / z) theoretical value: 672.35, experimental value: 673.37 (M+H).

[0200] Step 3: Synthesis of intermediates 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 mixture was stirred at room temperature for 2 hours, evaporated to dryness under reduced pressure, ethyl acetate and diethyl ether were added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and dried to obtain intermediate 1-3 (31.5 g, 94% yield). LC-MS (ESI, m / z) theoretical value: 450.28, experimental value: 451.32 (M+H).

[0201] Step 4: Synthesis of intermediates 1-4 Intermediate 1-3 (5g, 11.10 mmol) is dissolved in 100 ml of DMF, and N-succinimidyl maleimide acetate (2.80 g, 11.10 mmol) is added at room temperature. The reaction mixture is stirred overnight at room temperature, evaporated to dryness under reduced pressure, methyl tert-butyl ether is added, and the mixture is stirred until a solid precipitates. The mixture is filtered, the solid is rinsed several times with diethyl ether, and the solid is dried to obtain intermediate 1-4, which is then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 587.30, experimental value: 588.31 (M+H).

[0202] Step 5: Synthesis of intermediates 1-5 Intermediate 1-4 (2g, 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. The mixture was stirred overnight at room temperature under an argon atmosphere, methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 1-5, which was then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 752.30, experimental value: 753.31 (M+H).

[0203] Step 6: Synthesis of intermediates 1-6 Intermediate 1-5 (142 mg, 0.188 mmol) was dissolved in 400 μL of anhydrous DMF, 100 μL of anhydrous pyridine was added, followed by 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 an argon atmosphere, the reaction mixture was purified by reverse-phase HPLC to obtain intermediate 1-6 (80 mg, 40% yield). LC-MS (ESI, m / z) theoretical value: 1048.43, experimental value: 1045.45 (M+H).

[0204] Step 7: Synthesis of compound (A-1) Intermediate 1-6 (100 mg, 0.095 mmol) was dissolved in 1 mL of anhydrous dichloromethane, 500 μL of trifluoroacetic acid was added under ice bath, the mixture was allowed to return to room temperature and stirred for 30 minutes, and the solvent was removed under reduced pressure to obtain the final product. The final compound (A-1) (70 mg, yield 77%) was then purified by reverse-phase HPLC. LC-MS (ESI, m / z) theoretical value: 1048.43, experimental value: 1045.45 (M+H). LC-MS (ESI, m / z) theoretical value: 948.38, experimental value: 949.37 (M+H).

[0205] Synthesis of compound (A-2)

[0206] [ka]

[0207] The synthesis of compound (A-2) was the same as that of compound (A-1), except that the starting material N-succinimidyl maleimide acetate in step 4 was replaced with N-hydroxysuccinimidide 6-maleimidohexanoate. After several reaction steps, product (A-2) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1004.44, measured value: 1005.45 (M+H).

[0208] Synthesis of compound (A-3)

[0209] [ka]

[0210] Compound (A-1) (100 mg, 0.105 mmol) was dissolved in 1 ml of anhydrous DMF. 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain the final compound (A-3) (87 mg, yield 49%). LC-MS (ESI, m / z) theoretical value: 1700.76, experimental value: 1701.78 (M+H).

[0211] Synthesis of compound (A-4)

[0212] [ka]

[0213] 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain the final compound (A-4) (95 mg, 54% yield). LC-MS (ESI, m / z) theoretical value: 1756.83, experimental value: 1757.85 (M+H).

[0214] Synthesis of compound (A-5)

[0215] [ka]

[0216] The synthesis of compound (A-5) followed the same procedure as that for compound (A-4), except that the final step involved replacing acetylated-10 polysarcosine (Ac-Sar10-COOH) with acetylated-4 polysarcosine (Ac-Sar4-COOH). After several reaction steps, product (A-5) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1330.60, experimental value: 1331.61 (M+H).

[0217] Synthesis of compound (A-6)

[0218] [ka]

[0219] The synthesis of compound (A-6) was the same as that of compound (A-4), except that the exatecan mesylate in step 6 was replaced with berotecan hydrochloride (purchased from Shanghai Haoyuan Co., Ltd.). After several reaction steps, product (A-6) was obtained as a white amorphous powder. LC-MS (ESI, m / z) theoretical value: 1754.87, measured value: 1755.88 (M+H).

[0220] Synthesis of compound (A-7)

[0221] [ka]

[0222] The synthesis of compound (A-7) followed the same procedure as for compound (A-4), except that Fmoc-Val-OSu in step 1 was replaced with Fmoc-Phe-OSu. After several reaction steps, product (A-7) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1804.83, measured value: 1805.85 (M+H).

[0223] Synthesis of compound (A-8)

[0224] [ka]

[0225] The synthesis of compound (A-8) followed the same procedure as that for compound (A-7), except that the exatecan mesylate in step 6 was replaced with berotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-8) was obtained as a white amorphous powder. LC-MS (ESI, m / z) theoretical value: 1802.87, measured value: 1803.86 (M+H).

[0226] Synthesis of compound (A-9)

[0227] [ka] Synthesis pathway:

[0228] [ka]

[0229] Step 1: Synthesis of intermediate 9-1 Intermediates 9-1A (1.3g, 2.69 mmol) and 9-1B (1.35g, 2.69 mmol) were dissolved in a mixed solvent of dichloromethane and methanol (v:v=2:1, 90 ml). p-EEDQ (800 mg, 3.23 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 24 hours, then evaporated to dryness under reduced pressure, and 9-1 (1.8g, 69% yield) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 966.49, experimental value: 967.50 (M+H).

[0230] Step 2: Synthesis of intermediate 9-2 Intermediate 9-1 (900 mg, 0.93 mmol) was dissolved in 20 ml of anhydrous tetrahydrofuran. Under an argon atmosphere, pyridine hydrogen fluoride complex (1.8 g, 18.61 mmol) was added 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, dried over anhydrous sodium sulfate, and column chromatography yielded intermediate 9-2 (610 mg, 77% yield). LC-MS (ESI, m / z) theoretical value: 852.41, experimental value: 853.43 (M+H).

[0231] 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. The mixture was stirred overnight at room temperature under an argon atmosphere, the solvent was removed by distillation under reduced pressure, then methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 9-3, which was then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 10¹⁷.41, experimental value: 10¹⁸.38 (M+H).

[0232] 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. Then, exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 157 mg, 0.295 mmol) and HOBt (40 mg, 0.295 mmol) were added. After stirring overnight at room temperature under an argon atmosphere, the reaction mixture was purified by reverse-phase HPLC to obtain intermediate 9-4 (160 mg, yield 41%). LC-MS (ESI, m / z) theoretical value: 1313.54, experimental value: 1314.51 (M+H).

[0233] 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 mixture was stirred at room temperature for 2 hours, then evaporated to dryness under reduced pressure to obtain intermediate 9-5, which was used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 1091.48, measured value: 1092.51 (M+H).

[0234] Step 6: Synthesis of intermediates 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain intermediate compound 9-6 (105 mg, 52% yield). LC-MS (ESI, m / z) theoretical value: 1843.86, experimental value: 1844.84 (M+H).

[0235] 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. The mixture was then returned to room temperature and stirred for 30 minutes. 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, yield 60%). LC-MS (ESI, m / z) theoretical value: 1048.43, experimental value: 1045.45 (M+H). LC-MS (ESI, m / z) theoretical value: 1743.81, experimental value: 1744.85 (M+H).

[0236] Synthesis of compound (A-10)

[0237] [ka]

[0238] The synthesis of compound (A-10) followed the same procedure as that for compound (A-9), except that the exatecan mesylate in step 4 was replaced with berotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-10) was obtained as a white amorphous powder. LC-MS (ESI, m / z) theoretical value: 1741.85, measured value: 1742.83 (M+H).

[0239] Synthesis of compound (A-11)

[0240] [ka]

[0241] Synthesis pathway:

[0242] [ka]

[0243] Step 1: Synthesis of intermediate 11-1 Intermediates 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 mixed solvent 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 mixture was stirred at room temperature for 24 hours, then evaporated to dryness under reduced pressure, and intermediate 11-1 (3.9 g, yield 71%) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 867.46, experimental value: 868.49 (M+H).

[0244] Step 2: Synthesis of intermediate 11-2 Intermediate 11-1 (2 g, 2.3 mmol) was dissolved in 50 ml of anhydrous tetrahydrofuran. Under an argon atmosphere, pyridine hydrogen fluoride complex (4.6 g, 46 mmol) was added 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, dried over anhydrous sodium sulfate, and column chromatography yielded intermediate 11-2 (1.1 g, 76% yield). LC-MS (ESI, m / z) theoretical value: 629.34, experimental value: 630.31 (M+H).

[0245] 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. The mixture was stirred overnight at room temperature under an argon atmosphere, the solvent was removed by distillation under reduced pressure, then methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 11-3, which was then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 794.35, experimental value: 795.41 (M+H).

[0246] 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. Then, exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 234 mg, 0.44 mmol) and HOBt (60 mg, 0.44 mmol) were added. After stirring overnight at room temperature under an argon atmosphere, the reaction mixture was purified by reverse-phase HPLC to obtain intermediate 11-4 (230 mg, yield 48%). LC-MS (ESI, m / z) theoretical value: 1090.48, experimental value: 1091.53 (M+H).

[0247] 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. The mixture was then allowed to return to room temperature and stirred for 30 minutes. The solvent was removed under reduced pressure to obtain the trifluoroacetate salt of the final intermediate 11-5, which was used directly in the next step of the reaction without further purification. LC-MS (ESI, m / z) theoretical value: 990.43, measured value: 991.47 (M+H).

[0248] Step 6: Synthesis of compound (A-11) Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 ml of anhydrous DMF. 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain compound (A-11) (74 mg, yield 38%). LC-MS (ESI, m / z) theoretical value: 1742.81, experimental value: 1743.85 (M+H).

[0249] Synthesis of compound (A-12)

[0250] [ka]

[0251] The synthesis of compound (A-12) followed the same procedure as that for compound (A-11), except that the starting compound Ac-Sar10-COOH in step 6 was replaced with Ac-Sar4-COOH. After several reaction steps, product (A-12) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1316.59, measured value: 1317.62 (M+H).

[0252] Synthesis of compound (A-13)

[0253] [ka]

[0254] The synthesis of compound (A-13) was the same as that of compound (A-11), except that the starting material compound 11-1A in step 1 was replaced with Mc-Val-Cit-OH (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-13) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1828.86, measured value: 1829.88 (M+H).

[0255] Synthesis of compound (A-14)

[0256] [ka]

[0257] The synthesis of compound (A-14) followed the same procedure as for compound (A-11), except that the starting material compound 11-1A (Mc-VA-OH) in step 1 was replaced with Mc-GGFG-OH (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-13) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1890.84, measured value: 1891.90 (M+H).

[0258] Synthesis of compound (A-15)

[0259] [ka]

[0260] Synthesis pathway:

[0261] [ka]

[0262] Step 1: Synthesis of intermediate 15-2 Intermediates 15-1A (2.3 g, 8.57 mmol) and 15-1B (3.74 g, 8.57 mmol) were dissolved in a mixed solvent 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 mixture at room temperature for 24 hours, the reaction mixture was evaporated to dryness under reduced pressure, and intermediate 15-2 (3.23 g, 4.70 mmol, yield 54.9%) was obtained by column chromatography. LC-MS (ESI, m / z) theoretical value: 686.27, experimental value: 687.21 (M+H).

[0263] 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, respectively. The mixture was stirred overnight at room temperature under an argon atmosphere, the solvent was removed by distillation under reduced pressure, then methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 15-3 (2.1 g, 2.465 mmol, yield 73.6%), which was then used directly in the next step of the reaction.

[0264] 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). The mixture was stirred overnight at room temperature under an argon atmosphere, and after evaporating the reaction mixture to dryness, it was purified by column chromatography (DCM:MeOH = 30:1) to obtain intermediate 15-4 (244 mg, 0.213 mmol, yield 45.3%). LC-MS (ESI, m / z) theoretical value: 1147.41, experimental value: 1148.48 (M+H).

[0265] 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. The mixture was then allowed to return to room temperature and stirred for 30 minutes. The solvent was removed under reduced pressure to obtain the trifluoroacetate of the final product, intermediate 15-5, which was used directly in the next step of the reaction without further purification. LC-MS (ESI, m / z) theoretical value: 1047.36, experimental value: 1048.40 (M+H).

[0266] 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The final product was then purified by reverse-phase HPLC to obtain compound A-15 (107 mg, 0.059 mmol, yield 45.4%). LC-MS (ESI, m / z) theoretical value: 1799.74, experimental value: 1800.77 (M+H).

[0267] Synthesis of compound (A-16)

[0268] [ka]

[0269] The synthesis of compound (A-16) followed the same procedure as that for compound (A-15), except that the exatecan mesylate in step 3 was replaced with berotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-16) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1797.78, experimental value: 1798.81 (M+H).

[0270] Synthesis of compound (A-17)

[0271] [ka]

[0272] The synthesis of compound (A-17) followed the same procedure as that for compound (A-11), except that the exatecan mesylate in step 4 was replaced with Genz-644282 (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-17) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1714.80, measured value: 1715.83 (M+H).

[0273] Synthesis of compound (A-18)

[0274] [ka]

[0275] Synthesis pathway:

[0276] [ka]

[0277] Step 1: Synthesis of intermediate 18-2 Intermediate 18-1A (purchased from Shanghai Bi De Pharmaceutical, 4.94 g, 14.82 mmol) and 15-1B (synthesized according to the method in Patent Document 9, 6.04 g, 14.82 mmol) were dissolved in a mixed solvent 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 mixture was stirred at room temperature for 24 hours, then evaporated to dryness under reduced pressure. The product was stirred, filtered through diethyl ether, and then vacuum-dried to obtain intermediate 18-2 (6.3 g, yield 59%). LC-MS (ESI, m / z) theoretical value: 726.40, experimental value: 727.41 (M+H).

[0278] Step 2: Synthesis of intermediate 18-3 Intermediate 18-2 (3.0 g, 4.13 mmol) was dissolved in 50 ml of THF, 10% Pd-C (800 mg) was added, and hydrogenation was carried out at atmospheric pressure for 8 hours. The completion of the reaction was detected by TLC, palladium carbon was filtered and removed, and the reaction mixture was evaporated to dryness and used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 636.36, measured value: 637.34 (M+H).

[0279] 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. 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. This product was then purified by column chromatography to obtain compound 18-4 (2.2 g, 71% yield). LC-MS (ESI, m / z) theoretical value: 758.40, experimental value: 759.42 (M+H).

[0280] Step 4: Synthesis of intermediate 18-5 Intermediate 18-4 (1.0 g, 1.32 mmol) was dissolved in 20 ml of anhydrous tetrahydrofuran. Under an argon atmosphere, pyridine hydrogen fluoride complex (2.6 g, 26.4 mmol) was added 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, dried over anhydrous sodium sulfate, and column chromatography yielded intermediate 18-5 (570 mg, yield 67%). LC-MS (ESI, m / z) theoretical value: 644.32, experimental value: 645.35 (M+H).

[0281] 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, respectively. The mixture was stirred overnight at room temperature under an argon atmosphere, the solvent was removed by distillation under reduced pressure, then methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 18-6 (530 mg, yield 84%), which was then used directly in the next step of the reaction.

[0282] 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. Then, exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd., 263 mg, 0.494 mmol) and HOBt (66.7 mg, 0.494 mmol) were added. After stirring overnight at room temperature under an argon atmosphere, the reaction mixture was purified by reverse-phase HPLC to obtain intermediate 18-7 (320 mg, yield 58%). LC-MS (ESI, m / z) theoretical value: 1105.46, experimental value: 1106.48 (M+H).

[0283] Step 7: Synthesis of intermediate 18-8 Intermediate 15-4 (300 mg, 0.27 mmol) is dissolved in 3 mL of anhydrous dichloromethane, 1 mL of trifluoroacetic acid is added under ice bath, the mixture is returned to room temperature and stirred for 30 minutes, and the solvent is removed under reduced pressure to obtain the trifluoroacetate salt of the final intermediate 18-8, which is used directly in the next step of the reaction without further purification. LC-MS (ESI, m / z) theoretical value: 1005.40, measured value: 1006.41 (M+H).

[0284] 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain compound A-18 (170 mg, yield 36%). LC-MS (ESI, m / z) theoretical value: 1757.78, experimental value: 1758.82 (M+H).

[0285] Synthesis of compound (A-19)

[0286] [ka]

[0287] The synthesis of compound (A-19) followed the same procedure as that for compound (A-18), except that N-(2-aminoethyl)maleimide hydrochloride in step 3 was replaced with 2-(methylsulfonyl)benzo[d]thiazol-6-amine. Product (A-19) was obtained through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1845.73, experimental value: 1846.75 (M+H).

[0288] Synthesis of compound (A-20)

[0289] [ka]

[0290] The synthesis of compound (A-20) followed the same procedure as that for compound (A-18), except that N-(2-aminoethyl)maleimide hydrochloride in step 3 was replaced with 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline. Product (A-20) was obtained through several reaction steps. LC-MS (ESI, m / z) theoretical value: 1856.76, experimental value: 1857.80 (M+H).

[0291] Synthesis of compound (A-21)

[0292] [ka]

[0293] The synthesis of compound (A-21) is the same as the synthesis process for compound (A-11), and the intermediate in step 1 [ka] of [ka] By simply substituting this, the product (A-21) was obtained after several reaction steps. LC-MS (ESI, m / z) theoretical value: 1742.81, experimental value: 1743.85 (M+H).

[0294] Synthesis of compound (A-22)

[0295] [ka]

[0296] The synthesis of compound (A-22) followed the same procedure as that for compound (A-21), except that the starting compound Mc-Val-Ala-OH in step 1 was replaced with Mc-Val-Cit-OH. After several reaction steps, product (A-22) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1828.86, experimental value: 1829.87 (M+H).

[0297] Synthesis of compound (A-23)

[0298] [ka]

[0299] The synthesis of compound (A-23) followed the same procedure as that for compound (A-21), except that the starting compound Mc-Val-Ala-OH in step 1 was replaced with Mc-GGFG-OH. After several reaction steps, product (A-23) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1890.84, experimental value: 1891.86 (M+H).

[0300] Synthesis of compound (A-28)

[0301] [ka]

[0302] Synthesis pathway:

[0303] [ka]

[0304] 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, then stirred overnight. Methyl tert-butyl ether (MTBE) was added, the mixture was stirred, filtered, and the solid was washed several times with MTBE. After vacuum drying, intermediate 28-1 (13.6 g, 72% yield) was obtained. LC-MS (ESI, m / z) theoretical value: 339.14, experimental value: 340.17 (M+H).

[0305] 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. TBS-Cl (6.66 g, 44.2 mmol) was added while cooling in an ice bath, and the mixture was stirred overnight at room temperature. Water was added to the reaction mixture to quench the reaction, and the organic phase was separated. After washing once with water and once with saturated brine, the mixture was 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, experimental value: 454.33 (M+H).

[0306] 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 under an argon atmosphere, 1 g of 10% Pd-C was added, followed by ammonium formate (8.34 g, 132 mmol). The mixture was stirred overnight at room temperature, palladium carbon was filtered off, the filtrate was evaporated to dryness, dichloromethane and water were added, the organic phase was separated, washed once each with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain intermediate 28-3 (5.6 g, 100%). LC-MS (ESI, m / z) theoretical value: 423.26, experimental value: 424.31 (M+H).

[0307] 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 mixed solvent 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 mixture at room temperature for 24 hours, the reaction mixture was evaporated to dryness under reduced pressure, diethyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 28-4 (6.4 g, yield 60%). LC-MS (ESI, m / z) theoretical value: 815.43, experimental value: 816.48 (M+H).

[0308] 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. The reaction solution was stirred at room temperature for 2 hours, then evaporated to dryness under reduced pressure. Ethyl acetate and diethyl ether were added, and the mixture was stirred until a solid precipitated. The solution was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 28-5 (4.3 g, 98% yield). LC-MS (ESI, m / z) theoretical value: 593.36, experimental value: 594.40 (M+H).

[0309] Step 6: Synthesis of intermediate 28-6 Intermediate 28-5 (4.0 g, 6.74 mmol) is dissolved in 100 ml of DMF, and maleimide acetate N-succinimidyl (2.07 g, 6.74 mmol) is added at room temperature. The reaction mixture is stirred overnight at room temperature, then evaporated to dryness under reduced pressure. Methyl tert-butyl ether is added and the mixture is stirred until a solid precipitates. The mixture is filtered, the solid is rinsed several times with diethyl ether, and the solid is dried to obtain intermediate 28-6, which is then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 786.43, experimental value: 787.45 (M+H).

[0310] Step 7: Synthesis of intermediate 28-7 Intermediate 28-6 (3.5 g, 4.45 mmol) was dissolved in 100 ml of anhydrous tetrahydrofuran. Under an argon atmosphere, pyridine hydrogen fluoride complex (8.8 g, 89 mmol) was added 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, dried over anhydrous sodium sulfate, and column chromatography yielded intermediate 28-7 (1.6 g, 53% yield). LC-MS (ESI, m / z) theoretical value: 672.35, experimental value: 673.37 (M+H).

[0311] 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 an argon atmosphere, the solvent was removed by distillation under reduced pressure, then methyl tert-butyl ether was added, and the mixture was stirred until a solid precipitated. The mixture was filtered, the solid was rinsed several times with diethyl ether, and the solid was dried to obtain intermediate 28-8, which was then used directly in the next step of the reaction. LC-MS (ESI, m / z) theoretical value: 837.35, experimental value: 838.36 (M+H).

[0312] Step 9: Synthesis of intermediate 28-9 Intermediate 28-8 (300 mg, 0.358 mmol) was dissolved in 4 mL of anhydrous DMF, 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 an argon atmosphere, the reaction mixture was purified by reverse-phase HPLC to obtain intermediate 28-9 (234 mg, yield 58%). LC-MS (ESI, m / z) theoretical value: 1133.49, experimental value: 1134.52 (M+H).

[0313] Step 10: Synthesis of intermediates 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. The mixture was then allowed to return to room temperature and stirred for 30 minutes. 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 step of the reaction without further purification. LC-MS (ESI, m / z) theoretical value: 1033.43, measured value: 1034.45 (M+H).

[0314] 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. The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to obtain the final product. The product was then purified by reverse-phase HPLC to obtain compound (A-11) (135 mg, yield 43%). LC-MS (ESI, m / z) theoretical value: 1785.82, experimental value: 1786.85 (M+H).

[0315] Synthesis of compound (A-29)

[0316] [ka]

[0317] The synthesis of compound (A-29) followed the same procedure as that for compound (A-11), except that the exatecan mesylate in step 4 was replaced with berotecan hydrochloride (purchased from Shanghai Haoyuan Chemical Co., Ltd.). After several reaction steps, product (A-29) was obtained as a white amorphous powder. LC-MS (ESI, m / z) theoretical value: 1740.85, measured value: 1741.82 (M+H).

[0318] Synthesis of PEG derivative AP-1 corresponding to molecule A-11

[0319] [ka]

[0320] The synthesis of compound (AP-1) followed the same procedure as that for compound (A-11), except that the starting compound Ac-Sar10-COOH in step 1 was replaced with m-PEG8-acid (CAS number 1093647-41-6). After several reaction steps, product (AP-1) was obtained as a beige amorphous powder. LC-MS (ESI, m / z) theoretical value: 1384.64, measured value: 1385.66 (M+H).

[0321] Synthesis of control product MC-Val-Cit-PABC-DX8951

[0322] Referring to the method described in 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 nitro ester, which was then reacted with exatecan mesylate (purchased from Shanghai Haoyuan Co., Ltd.) to obtain the control compound MC-Val-Cit-PAB-DX8951.

[0323] (Example 2: General method for ADC preparation) 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, 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 mixture was stirred at 35°C for 2.5 hours.

[0324] The reaction solution described above is cooled to 8°C, an appropriate amount of dimethylacetamide (DMA) is added without purification, and then a molar excess of 6 to 15 times the amount of control drug molecules or drug linker complexes A1 to A29 (10 mg / ml dissolved in DMA beforehand) is added, ensuring that the volume ratio of DMA in the reaction system does not exceed 20%, and the mixture is stirred at 37°C for 3 hours to bind.

[0325] The conjugation reaction mixture was filtered and purified using a desalting column on a pH 6.0 histidine-acetic acid / sucrose gel, and peak samples were collected based on UV280 ultraviolet absorption values. The mixture was then sterilized by passing it through a 0.15 micron filter and stored at -60°C.

[0326] 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 complex 1. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 1. The proportion of aggregate 1 was approximately 26%, and the proportion of aggregate 2 was approximately 20%. The drug-antibody ratio (DAR) was approximately 4-5 by UV analysis.

[0327] Preparation of antibody conjugate 2 (control ADC2) Referring to the general method described above, a 12-fold excess of the control product Deruxtecan (purchased from Shanghai Haoyuan Chemical Co., Ltd.) was conjugated to the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 2. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 2. The proportion of aggregates was approximately 2%. Hydrophobic interaction chromatography (HIC-HPLC) analysis showed a drug-antibody ratio (DAR) of approximately 7-8, and the analytical spectrum is shown in Figure 3.

[0328] Preparation of antibody complex 3 Referring to the general method described above, an 8-fold excess of compound (A-2) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 3. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 4. The proportion of aggregate 1 was approximately 6.8%, and the proportion of aggregate 2 was approximately 7.7%. The drug-antibody ratio (DAR) was approximately 6-7 by UV analysis.

[0329] Preparation of antibody complex 4 Referring to the general method described above, a 12-fold excess of compound (A-4) was conjugated to the reduced trastuzumab monoclonal antibody to obtain antibody complex 4 with a drug-to-antibody ratio (DAR) of approximately 7-8. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 5. The proportion of aggregates was approximately 0.3%. Hydrophobic interaction chromatography (HIC-HPLC) analysis showed a drug-to-antibody ratio (DAR) of approximately 7-8, and the analytical spectrum is shown in Figure 6.

[0330] Preparation of antibody complex 5 Referring to the general method described above, a 12-fold excess of compound (A-11) was conjugated to the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 5. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 7. The monomer purity of antibody complex 5 was 99.41%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 8. The accurate drug-antibody ratio obtained by comparing the mass difference between the light and heavy chains of reduced Trastuzumab (Figure 34) and the light and heavy chains of antibody complex 1 (Figure 35) by mass spectrometry was calculated to be 7.8.

[0331] Preparation of antibody complex 6 Referring to the general method described above, a 12-fold excess of compound (A-14) was conjugated to the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 6. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 9. The monomer purity of antibody complex 6 was 99.60%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 10. The accurate drug-antibody ratio obtained by comparing the mass difference between the light and heavy chains of reduced Trastuzumab (Figure 34) and antibody complex 2 (Figure 36) by mass spectrometry was 8.0.

[0332] Preparation of antibody complex 7 Referring to the general method described above, a 15-fold excess amount of compound (A-24) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 7. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 11. The monomer purity of antibody complex 7 was 96.36%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 12.

[0333] Preparation of antibody conjugate 8 (control ADC3) Referring to the general method described above, a 12-fold excess amount of compound AP-1 was conjugated to the reduced trastuzumab monoclonal antibody to obtain the corresponding antibody complex 8. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 13. The monomer purity of antibody complex 8 was 97.92%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 14.

[0334] Preparation of antibody complex 9 Referring to the general method described above, a 12-fold excess amount of compound (A-11) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 9. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 15. The monomer purity of antibody complex 9 was 97.92%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 16.

[0335] Preparation of antibody complex 10 Referring to the general method described above, a 12-fold excess amount of compound (A-11) was conjugated with the reduced H01L02 monoclonal antibody to obtain the corresponding antibody complex 10. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 17. The monomer purity of antibody complex 10 was 99.10%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 18.

[0336] Preparation of antibody complex 11 Referring to the general method described above, a 12-fold excess amount of compound (A-28) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 11. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 19. The monomer purity of antibody complex 11 was 99.59%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 20.

[0337] Preparation of antibody conjugate 12 Referring to the general method described above, a 12-fold excess amount of compound (A-29) was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding antibody complex 12. The aggregate content was analyzed by size exclusion chromatography (SEC-HPLC), and the analytical spectrum is shown in Figure 21. The monomer purity of antibody complex 12 was 99.67%. The drug-antibody ratio (DAR) obtained by hydrophobic interaction chromatography (HIC-HPLC) analysis was approximately 7-8, and the analytical spectrum is shown in Figure 22.

[0338] Preparation of antibody complex 13 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed and replaced with 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 cooled in an ice bath for 10 minutes. 2.5 times the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction mixture was stirred overnight at 4°C. Dimethylacetamide (DMA) was continuously added in appropriate amounts to the unpurified reaction mixture described above, and then a 6.0-fold molar excess of the control drug molecule or drug linker complex A-11 (10 mM pre-dissolved in DMA) was added, ensuring 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 binding was complete, 4.0 times the molar ratio of small molecules of cysteine ​​were added to the reaction mixture to consume the excess drug linker complex A-11, and the reaction was stopped by stirring at 4°C for 30 minutes. The binding reaction mixture was filtered and purified using a desalting column with histidine-acetic acid / sodium chloride at pH 5.5, and the filtered sample was collected. One-tenth the volume of the sample was added to a suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL), and the mixture was stirred at room temperature for 2 hours to sufficiently absorb the free drug molecules. The sample was then sterilized by passing it 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 31. The purity of antibody complex 13 monomer was 98.95%.

[0339] The concentration of the bound drug in the antibody-drug conjugate can be calculated as follows by measuring the ultraviolet absorbance of the antibody-drug conjugate aqueous solution at wavelengths of 280 nm and 370 nm.

[0340] The total absorbance at a given wavelength is equal to the sum of the absorbances 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 can be expressed by the following equations.

[0341]

number

[0342] ε A,280 This can be estimated from the amino acid sequence of the antibody using a known calculation method (Non-Patent Literature 1), and ε A,370 It is generally zero. From the absorbance of a solution of a drug linker complex at a specific molar concentration, ε can be calculated based on Lambert-Beer's law. D,280 and ε D,370 Obtain the result using a microplate reader or ultraviolet spectrophotometer. 280 and A 370 The value of is measured, and the values ​​of the four molar extinction coefficients above are substituted into the simultaneous equations (1) and (2) to obtain C A and C D The value can be calculated. Average number of bound drug molecules in each antibody molecule DAR = C D / C A Therefore, the ultraviolet absorption coefficient of compound A-11 is ε D,280 =6480, ε D,370The value was 16483, which was used to calculate the drug-antibody ratio in this embodiment, and the DAR value of antibody complex 13 was approximately 4.0.

[0343] Preparation of antibody complex 14 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed and replaced with 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 cooled in an ice bath for 10 minutes. 2.5 times the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction mixture was stirred overnight at 4°C. Dimethylacetamide (DMA) was continuously added in appropriate amounts to the unpurified reaction mixture described above, and then a 6.0-fold molar excess of the control drug molecule or drug linker complex A-14 (10 mM pre-dissolved in DMA) was added, ensuring 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 binding was complete, 4.0 times the molar ratio of small molecules of cysteine ​​were added to the reaction mixture to consume the excess drug linker complex A-14, and the reaction was stopped by stirring at 4°C for 30 minutes. The binding reaction mixture was filtered and purified using a desalting column with histidine-acetic acid / sodium chloride at pH 5.5, and the filtered sample was collected. One-tenth the volume of the sample was added to a suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL), and the mixture was stirred at room temperature for 2 hours to sufficiently absorb the free drug molecules. The sample was then sterilized by passing it 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 32. The purity of antibody complex 14 monomer was 98.20%. The ultraviolet extinction coefficient of compound A-14 was ε D,280 = 5932, ε D,370 The value was 14997, which was used to calculate the drug-antibody ratio in this embodiment, and the DAR value of antibody complex 14 was approximately 4.2.

[0344] Preparation of antibody complex 15 The stock solution of hRS7 monoclonal antibody targeting Trop-2 was dialyzed and replaced with 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 cooled in an ice bath for 10 minutes. 2.5 times the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction mixture was stirred overnight at 4°C. Dimethylacetamide (DMA) was continuously added in appropriate amounts to the unpurified reaction mixture described above, and then a 6.0-fold molar excess of the control drug molecule or drug linker complex A-24 (10 mM pre-dissolved in DMA) was added, ensuring 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 binding was complete, 4.0 times the molar ratio of small molecules of cysteine ​​were added to the reaction mixture to consume the excess drug linker complex A-24, and the reaction was stopped by stirring at 4°C for 30 minutes. The binding reaction mixture was filtered and purified using a desalting column with histidine-acetic acid / sodium chloride at pH 5.5, and the filtered sample was collected. One-tenth the volume of the sample was added to a suspension of activated carbon-histidine-acetic acid / sodium chloride (300 mg / mL), and the mixture was stirred at room temperature for 2 hours to sufficiently absorb the free drug molecules. The sample was then sterilized by passing it 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 33. The purity of antibody complex 15 monomer was 96.36%. The ultraviolet extinction coefficient of compound A-24 was ε D,280 =4723, ε D,370 The value was 12467, which was used to calculate the drug-antibody ratio in this embodiment, and the DAR value of antibody complex 15 was approximately 4.0.

[0345] The compound of this application and the ADC prepared therefrom exhibit higher monomer purity at higher drug-antibody ratios compared to the ADC prepared from the control molecule, suggesting that this type of ADC molecule has better stability. Furthermore, hydrophobic interaction chromatography (HIC-HPLC) analysis results showed that the ADC molecule of this application had a shorter retention time than the control molecule, indicating that it is closer to a naked antibody. This suggests that this type of ADC molecule has better hydrophilicity, a longer in vivo half-life, and may have stronger in vivo efficacy.

[0346] (Example 3: Stability test of antibody complex) ADC drugs based on camptothecin topoisomerase inhibitors, such as sacituzumab govitecan (Trodelvy) and trastuzumab deruxtecan (Enhertu), typically have a high drug-to-antibody ratio (DAR). Because camptothecin molecules possess a fused ring structure and strong hydrophobicity, the stability of this type of ADC molecule is bound to be affected to some extent. The ADCs prepared in this invention, by introducing polysarcosine peptide chains at specific positions, may have a better "barrier effect" and thus better stability. To test this hypothesis, we designed an accelerated stability test for the antibody.

[0347] The molecules to be 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

[0348] Dilute the test molecule sample to a concentration of 5 mg / ml in formulation buffer (20 mM Histidine-acetic acid, 150 mM NaCl, pH 5.5), take 50 μL and test directly or 55 oAfter incubating the samples in a water bath for 1, 2, 5, 24, 48, and 72 hours, respectively, tests (accelerated stability tests) were performed. Changes in sample concentration were detected by UV spectroscopy, and quantitative analysis of the content of normal structured ADC molecules (main peak) and polymerized molecules in the samples was performed using a TSK-GEL SWXL3000 molecular exclusion chromatography column on an Agilent 1260 Infinity II bioinert LC system.

[0349] 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.

[0350] Figure 23 shows the statistical results of the sample concentration changes. Trastuzumab and its corresponding antibody-drug conjugates 5 and 6 both exhibited good thermal stability, and the difference in concentration changes from 0 to 72 hours was small. However, the samples of antibody-drug conjugates 2, 7, and 8 showed a rapid decrease in ADC concentration after 24 hours, and the sample concentration decreased rapidly due to precipitation.

[0351] Further analysis of the changes in the rate of increase of polymer molecules in the accelerated stability test samples using molecular size exclusion chromatography yielded statistical results, shown in Figure 24. These results were consistent with the trend in sample concentration changes, and the order of sample stability from highest to lowest was trastuzumab ≈ antibody complex 5 > antibody complex 6 > antibody complex 7 > antibody complex 8 ≈ antibody complex 2.

[0352] The results described above confirm that the hydrophilic polysarcosine peptide chain at a specific position in the present invention has a "barrier effect" and provides an "unexpected" stability-enhancing effect to the antibody-drug conjugate molecule.

[0353] (Example 4: In vitro cytotoxicity test) NCI-N87 human gastric cancer cells were selected as the cell line for in vitro activity detection in this experiment, and the effective doses of antibody complex 2, antibody complex 5, antibody complex 6, trastuzumab monoclonal antibody, and IgG-deruxtecan for cell killing were observed. After adding the sample to the antibody-drug conjugate prepared in Example 2, the final concentration was set to 500 nM, and nine concentration levels (5-fold dilution) from 500 to 0.1 nM were designed from the initial concentration. Changes in killing (or suppression) were observed over 120 hours, chemiluminescent staining (Luminescent Cell Viability Assay) was performed, and fluorescence data was read before IC25. 50 I calculated it.

[0354] As shown in Figure 25, Her2-highly expressing NCI-N87 cells were processed with antibody complex 2, antibody complex 4, antibody complex 5, antibody complex 6, and Trastuzumab monoclonal antibody and IgG-deruxtecan. Antibody complexes 2, 4, 5, and 6 all significantly suppressed tumor cell proliferation, and were significantly more effective than Trastuzumab monoclonal antibody and IgG-deruxtecan.

[0355] As shown in Figure 26, Her2-low expressing OV-CAR3 human ovarian adenocarcinoma cells were processed with antibody complex 5, antibody complex 11, and DS8201a (antibody complex 2). All three antibody complexes significantly suppressed tumor cell proliferation, with antibody complex 5 and antibody complex 11 being superior to DS8201a (antibody complex 2).

[0356] As shown in Figures 27 and 28, when Her2-highly expressing NCI-N87 cells and SK-BR-3 cells were processed with antibody conjugate 12 and Belotecan small molecule, antibody conjugate 12 significantly suppressed tumor cell proliferation, and was significantly more effective than the supported small molecule drug Belotecan.

[0357] According to the activity test results described above, all ADCs prepared with the compounds of this invention exhibited good in vitro antitumor activity, and the advantages of the antibody conjugates of this invention are particularly evident against tumor cells with low expression of the relevant antigens.

[0358] (Example 5: Measurement of in vivo antitumor effect) The effects of the combination of the present invention could be measured in vivo, specifically by transplanting allografts or xenografts of cancer cells into rodents and processing the tumors with the combination. Test mice were processed with either the drug or a control and monitored for several weeks or more to measure the time of tumor doubling, logarithmic cell killing, and tumor suppression.

[0359] In vivo antitumor experiments (1) HER2-low-expressing COLO205 human colon cancer cells (ATCC) were suspended in physiological saline and 4 × 10⁶ cells were used. 7 Individual cells were transplanted subcutaneously into the right flank of female nude mice, and the mice were randomly divided into groups on day 6. Day 0 was designated as day 0, and on day 0, antibody complex 2 (DS8201), antibody complex 5, and 10 mg / kg of Kadcyla (T-DM1) were administered via the tail vein at doses of 3 mg / kg and 10 mg / kg, respectively. A control group administered PBS buffer was also provided.

[0360] The results are shown in Figure 29. In COLO205 tumors with low HER2 expression, administration of 10 mg / kg Kadcyla (T-DM1) did not show tumor growth inhibitory activity, while DS8201 at 3 mg / kg and 10 mg / kg, and antibody conjugate 5 all showed dose-dependent antitumor activity. Here, the efficacy of antibody conjugate 5 was significantly stronger than that of the control antibody conjugate 2.

[0361] Regarding HER2 expression in tumors, results obtained by immunohistochemical staining were used to classify tumors as high expression (score 3+), moderate expression (score 2+), and low expression (score 1+). It should be noted that even if a tumor scores 0 using this method, if other measurement methods, such as flow cytometry, yield positive results, it will still be classified as low expression.

[0362] In vivo antitumor experiments (2) HER2-expressing HCC1954 human breast cancer cells (ATCC) were suspended in physiological saline, and 4 × 10⁶ samples were taken. 7 Individual cells were transplanted subcutaneously into the right flank of female nude mice, and they were randomly divided into groups on day 6. Day 0 was designated as day 0, and on day 0, antibody complex 2 (DS8201) and antibody complex 5 were administered via the tail vein at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. As a control group, IgG was prepared and bound to the corresponding toxin linker, and these groups were administered the IgG-IgG-deruxtecan complex and the IgG-A11 complex, respectively.

[0363] The results are shown in Figure 30. In the case of HCC1954 tumors expressed in HER2, administration of antibody conjugate 2 (DS8201) at 1 mg / kg, 3 mg / kg, and 10 mg / kg, and antibody conjugate 5 all showed dose-dependent antitumor activity. Of these, antibody conjugate 5 showed significantly stronger efficacy than the control antibody conjugate 2.

[0364] According to the activity test results, all ADCs prepared with the compounds of this application showed a certain level of in vivo antitumor activity, and were able to exhibit significantly stronger antitumor activity compared to the control sample. The tumor mice were sufficiently resistant to the above drugs, and no symptoms such as weight loss occurred.

[0365] (Example 6: In vivo pharmacokinetic detection) In this example, the pharmacokinetic properties of antibody conjugate 5, antibody conjugate 6, and the control drug DS8201a were evaluated in rats. Specifically, in this example, 3 mg / kg of antibody conjugate 5, antibody conjugate 6, and DS8201 were administered to rats by tail injection, and the mean toxicity parameters of the bound antibodies in each dose group are shown in the table below. The order of the half-lives of the bound antibodies was antibody conjugate 5 ≈ antibody conjugate 6 > DS8201a.

[0366] [Table 1-1] [Table 1-2]

[0367] The PK results of the above antibody conjugate in rats indicate that the ADC obtained using the new technology scheme has better stability and a longer half-life in rats. Therefore, the ADC of this application can achieve superior safety and efficacy compared to DS-8201a, and thus significantly contributes to cancer patients with moderate to low HER2 expression.

[0368] The descriptions and examples provided in the section on the detailed description of the invention are intended solely to clarify the technical content of the present invention and do not limit the scope of the appended claims. The various variations of the embodiments described herein will be obvious to those skilled in the art and will fall within the scope of the appended claims and their equivalents.

Claims

1. The compound represented by formula (C-HER2), or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof. 【Chemistry 1】 [In the formula, Q １ It is a linker coupled with mercapto, L １ is, -L １ａ -C (=O)- is included, L １ａ It is selected from the group consisting of C1-C12 alkylene groups and arylene groups, L ２ It contains polypeptide residues 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). L ３ is replaced 【Chemistry 2】 , and replaced 【Transformation 3】 Selected from the group consisting of, the benzene ring of L3 is linked to a polysarcosine residue substituted via structural unit -X-, and the structural unit -X- is 【Chemistry 4】 ,or 【Transformation 5】 X1 is a C1-C8 alkyl group, and the substituted polysarcosine residue is 【Transformation 6】 n2 is 10, and R is selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, and C1-C6 alkoxy. T is a drug unit, Ab is a ligand that can bind to HER2, and m is a number from 1 to 8.

2. Q １ teeth, 【Transformation 7】 A compound according to claim 1, selected from the group consisting of the following, in the form of a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

3. L １ａ is an alkylene group of C １ -C ７ The compound according to claim 1, or a tautomer, meso form, racemic form, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, which is an alkylene group of

4. The aforementioned structural unit -X- is, in some cases, replaced 【Transformation 8】 The polysarcosine residue substituted in the above case is 【Chemistry 9】 It includes n2 is 10, and R is C １ -C ６ The compound according to claim 1, which is alkyl, or in the form of its tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

5. T is a compound according to claim 1, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, comprising a compound having antitumor activity.

6. T is, 【Chemistry 10】 A compound according to claim 1, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, selected from the group of structures comprising the above, or a pharmaceutically acceptable salt or solvate thereof.

7. The compound according to claim 1, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an anti-HER2 antibody or its antigen-binding fragment.

8. The antigen-binding fragments are Fab, Fab', Fv fragment, and F(ab'). ２ F(ab) ２ A compound according to claim 7, selected from the group consisting of scFv, di-scFv, VHH, and dAb, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

9. The aforementioned Ab is a compound according to claim 1, comprising trastuzumab or pertuzumab, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

10. A compound according to claim 1, selected from the group consisting of the following, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. 【Chemistry 11】

11. A compound selected from the group consisting of the following, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. 【Chemistry 12】

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, or a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

13. Use of a compound according to any one of claims 1 to 11, or in the form of a tautomer, meso, racemic, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a drug for treating or preventing tumors.

Images