Antibody-drug conjugate and preparation method and use thereof

Abstract

Provided are an antibody-drug conjugate and a preparation method and a use thereof, which has a structure shown in the formula Ab-[M-L-E-D] x, the drug is selected from DNA topoisomerase inhibitors, the prepared antibody-drug conjugate has a relatively good drug-antibody conjugation ratio and has excellent targeted killing effects on solid tumors such as gastric cancer, breast cancer, lung cancer, and urothelium carcinoma.

Description

Antibody drug conjugates and preparation methods and uses thereof

[0001] This application is based on the application with CN application number 202310538792.7 and application date of May 12, 2023, the application with CN application number 202311258765.0 and application date of September 27, 2023, and the application with CN application number 202410173874.0 and application date of February 6, 2024, and claims the priority of the aforementioned applications. All contents of the said CN applications are hereby introduced into this application as a whole. Technical Field

[0002] The present application relates to the field of targeted therapy, and specifically to an antibody-drug conjugate. Background Art

[0003] Antibody drug conjugates (ADCs) for tumor treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a tumor-killing cytotoxin), and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells, where it is internalized. The bioactive molecule is then released inside the cancer cells and kills them by inhibiting their microtubules or damaging their DNA, minimizing damage to normal tissue cells.

[0004] The ErbB family of receptor tyrosine kinases is an important mediator of cell growth, differentiation, and survival. This family includes four members: epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4). The anti-ErbB2 antibody trastuzumab (trade name Herceptin) is commonly used clinically to treat breast cancer with high ErbB2 expression, but the clinical response rate is low. In order to improve the therapeutic effect, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansines (such as DM1) and auristatins (such as MMAE) or DNA topoisomerase I inhibitors (such as Dxd) (Trastuzumab emtansine, Disitamab vedotin, Trastuzumab deruxtecan) have been used in clinical treatment in recent years.

[0005] With the widespread use of the above-mentioned ADC drugs in the clinical treatment of Her2-expressing tumors, safety issues or drug resistance issues including neurotoxicity, hematotoxicity, hepatotoxicity, and interstitial pneumonia have gradually emerged (Pharmacology & Therapeutics 2019, 200, 110-125; Breast Cancer Research and Treatment 2020, 183, 23-39; JAMA Oncol. 2021, 7, 1873-1881; Drug Deliv. 2022, 29, 1335-1344; Cancers 2023, 15, 1130; Cancers 2023, 15, 1278.).

[0006] Specifically, the linker portion of both Disitamab vedotin and Trastuzumab deruxtecan uses a maleimide linker (MC). Literature reports that MC linkers are prone to reverse Michael reactions and sulfhydryl exchange under physiological conditions, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). In terms of the hydrophilicity of the linker, Disitamab vedotin and Trastuzumab deruxtecan use the more hydrophobic valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively. Literature reports that the hydrophilicity of the linker significantly affects the hydrophilicity of the ADC, thereby affecting the aggregation, pharmacokinetic properties, and toxicity of the ADC (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3.). Regarding the biologically active molecules that kill cancer cells, Trastuzumab emtansine uses the microtubule inhibitor DM1 as a cytotoxin, which results in a weak bystander effect when paired with a non-cleavable linker; Disitamab vedotin uses the auristatin-like toxin MMAE as a biological effector molecule, which is prone to problems such as neurotoxic accumulation after continuous repeated use; and both of the above ADC drugs are non-site randomly coupled, with a drug loading ratio (DAR) of approximately 4 and poor uniformity.

[0007] Summary of the Invention

[0008] The present invention aims to improve the above-mentioned problems in existing pharmaceutical technologies, specifically to provide a class of anti-Her2 antibody-drug conjugates that can be used to treat Her2-expressing tumors. The antibody-drug conjugates have good stability, homogeneity, hydrophilicity, efficacy, and safety.

[0009] The present application relates to an antibody drug conjugate, and exemplarily discloses an antibody drug conjugate having the general formula Ab-[MLED] with trastuzumab as the targeting moiety. x The antibody-drug conjugate of the structure shown. The results showed that the conjugate had a relatively good drug-antibody coupling ratio (e.g., 6.5-8.5), and the conjugate had excellent binding activity and proliferation inhibition on Her2-positive cells, and had a good targeted killing effect on Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, the present application provides an antibody-drug conjugate for treating Her2-expressing cancers, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating Her2-expressing cancers.

[0010] Antibody Drug Conjugates

[0011] In one aspect, the present application provides an antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0012] Ab is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases:

[0013] M is a linker site with an antibody or antigen-binding fragment thereof;

[0014] L is the structural fragment connecting the linkers M and E;

[0015] E is a structural fragment connecting L and D;

[0016] D is the cytotoxic drug fragment;

[0017] X is 1 to 10.

[0018] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via the "MLE" fragment shown in this application.

[0019] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0020] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0021] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0022] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0023] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0024] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0025] In some embodiments, M is selected from the following substituted or unsubstituted structural fragments:

[0026] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclic group (for example, substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO: 46), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO: 47), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Va l-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-G ly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG,SEQ ID NO:41), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:42), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:43), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:44), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:45)), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -C 1-6 Alkylene-heterocycle, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 alkyl)2-C1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), -C 1-6 Alkylene-N(C 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C 1-6 alkyl)-NOTA, wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0027] In some embodiments, "-NOTA" refers to

[0028] In some embodiments, "-DOTA" refers to

[0029] In some embodiments, "-DOTAGA" refers to

[0030] In some embodiments, Lys(R') refers to the structure

[0031] In some embodiments, Glu(R') refers to the structure

[0032] In some embodiments, r is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0033] In some embodiments, s is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0034] In some embodiments, s is represented by n.

[0035] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more (eg, 1, 2, 3, 4, 5, 6, or 7) of the following: 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Va l-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gl u-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), wherein R' is composed of one or more (eg, 1, 2, 3, 4, 5, 6, or 7) of the following groups, including but not limited to hydrogen, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkyl)2, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), -NHC 1-6 Alkylene-SO3H, -C 1-6 Alkylene-N(C 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C 1-6 alkyl)-NOTA, wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0036] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more (eg, 1, 2, 3, 4, 5, 6, or 7) of the following: 1-6Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Ly s, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), wherein R' is composed of one or more (eg, 1, 2, 3, 4, 5, 6, or 7) of the following groups, including but not limited to hydrogen, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkyl)2, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0037] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen-containing heterocyclic group, -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-V al-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1- 6 alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC 1-6 Alkylene -SO3H, -CH2N(C1-6 Alkyl)C 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2- 6-alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O)r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0038] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 9-12 membered nitrogen-containing heterocyclic group, -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val- Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val -Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 alkyl)2-C 1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r-C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0039] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Ci t, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1- 6 alkyl, C 1-6Alkylene, amino, hydroxyl, carboxyl, acyl, -O-, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0040] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Va l-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gl u-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, or polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), wherein r is selected from an integer of 1-20; s is selected from an integer of 1-20.

[0041] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6Alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclic group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gl y-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys- Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1-20.

[0042] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, A la-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly , Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, wherein s is selected from an integer of 1-20.

[0043] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0044] In some embodiments, the structure of L contains the following fragment:

[0045] In some embodiments, the structure of L contains the following fragment:

[0046] In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following Group I and one or more substituted or unsubstituted structural fragments selected from the following Group II:

[0047] Group I:

[0048] wherein s is an integer selected from 1 to 20;

[0049] Group II:

[0050] In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from the following Group I and one or more substituted or unsubstituted structural fragments selected from the following Group II:

[0051] Group I:

[0052] wherein s is an integer selected from 1 to 20;

[0053] Group II:

[0054] In some embodiments, L is formed by linking one or more substituted or unsubstituted structural fragments selected from Group I and one or more substituted or unsubstituted structural fragments selected from Group II:

[0055] Group I:

[0056] Group II:

[0057]

[0058] s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0059] In some embodiments, Group 1 consists of composition.

[0060] In some embodiments, Group 1 consists of composition.

[0061] In some embodiments, Group II consists of

[0062] s is selected from an integer of 1-20, for example, an integer of 1-15, 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0063] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following:

[0064] In some embodiments, L is selected from the following substituted or unsubstituted structural fragments:

[0065] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0066] s is an integer selected from 1-20.

[0067] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0068] s is an integer selected from 1-20.

[0069] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0070] s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0071] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0072] s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0073] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0074] s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0075] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more of the following groups:

[0076] s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0077] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more (e.g., 1, 2, 3, 4, or 5) of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0078] In some embodiments, L is selected from a substituted or unsubstituted structural fragment consisting of one or more (e.g., 1, 2, 3, 4, or 5) of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 8, 10.

[0079] In some embodiments, E is a single bond, a substituted or unsubstituted -NH-CH2-, or a substituted or unsubstituted structural fragment selected from the following:

[0080] In some embodiments, E is a single bond, a substituted or unsubstituted -NH-CH2-,

[0081] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0082] In some embodiments, E is a single bond, -NH-CH2- or

[0083] In some embodiments, E is substituted or unsubstituted -NH-CH2- or

[0084] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0085] n is selected from integers of 1-20.

[0086] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0087] n is selected from integers of 1-20.

[0088] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0089] n is selected from integers of 1-20.

[0090] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0091] n is selected from integers of 1-20.

[0092] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0093] wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 8 or 10.

[0094] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0095] wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 8 or 10.

[0096] In some embodiments, Selected from the following substituted or unsubstituted structural fragments:

[0097] wherein n is selected from an integer of 1-20, preferably n is selected from an integer of 1-15, such as an integer of 1-12, 3-12, 5-15, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 8 or 10.

[0098] In some embodiments, the cytotoxic drug is selected from an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, an RNA polymerase inhibitor, and a gene transcription inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound, a maytansine compound, or an eribulin compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, trabectedin, or rubectedin. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belotecan, rubitecan, diflomotecan, lurtotecan, karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogs, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin. In some embodiments, the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs.

[0099] In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor (e.g., Camptothecin, Hydroxycamptothecin, 9-aminocamptothecin, SN-38, Irinotecan, Isotecan, Topotecan, Belotecan, Rubitecan, Diflomotecan, Lurtotecan, Karenitecin, Gimatecan, Namitecan, Simmitecan, Chimmitecan, Silatecan, or Elomotecan).

[0100] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR1H), tertiary amine (-NR2R3), wherein R1, R2, and R3 here represent only non-hydrogen substituents on N, or sulfhydryl (-SH), and these functional groups can react with appropriate functional groups in the rest of the conjugate to achieve linkage.

[0101] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate via a -OH, primary amino, secondary amine, or tertiary amine group, or -SH group. In some embodiments, D is a monovalent structure obtained by losing one H from a -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0102] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0103] Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0104] In some embodiments, the cytotoxic drug is selected from the following compounds or isotopically labeled compounds thereof:

[0105] Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0106] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0107] Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0108] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0109] In some embodiments, the cytotoxic drug is selected from the following formula III and formula IV

[0110] Wherein, R5, R6 are each independently selected from H, OH, -NH2, -NH(C 1-6 Alkyl), C 1-6 Alkyl and halogen; the C 1-6The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 Alternatively, R5 and R6 together with adjacent carbon atoms (ring-forming carbon atoms connecting R5 and R6, respectively) form a five-membered oxygen-containing heterocyclic ring

[0111] R7 is selected from H, -OH, -NH2, -NH(C 1-6 alkyl), and -NH-CO-(C 1-6 Alkylene)-OH; the C 1-6 Alkyl and C 1-6 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 substituted by a cycloalkyl substituent;

[0112] q is 1 or 2;

[0113] R8 and R9 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; the 5-6 membered oxygen-containing heterocyclic ring is optionally substituted with one or more selected from deuterium (D), halogen, hydroxyl, C 1-6 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0114] R 10 Selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0115] R 11 Selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;and

[0116] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, -SO2-C 1-6 Alkyl, -(C=O)-OC 1-6 Alkyl and -CO-C 1-6 Alkyl; the C 1-6 Alkyl and C 1-4 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0117] In some embodiments, R5, R6 are each independently selected from OH, -NH2, C 1-4 Alkyl and halogen; the C 1-4 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 In some embodiments, R5 and R6 together with adjacent carbon atoms (ring-forming carbon atoms connecting R5 and R6, respectively) form a five-membered oxygen-containing heterocyclic ring.

[0118] In some embodiments, R7 is selected from -H, -NH2, -NH(C 1-4 alkyl), and -NH-CO-(C 1-4 Alkylene)-OH; the C 1-4 Alkyl and C 1-4 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 The cycloalkyl group is substituted with a substituent.

[0119] In some embodiments, R a 、R b Each occurrence is independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, -SO2-C 1-4 Alkyl and -CO-C 1-4 Alkyl; the C 1-4 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3- 6-cycloalkyl substituents.

[0120] In some embodiments, R5, R6 are each independently selected from H, OH, -NH2, C 1-6 Alkyl and halogen;

[0121] R7 is selected from -H, -NH2, -NH(C 1-6 alkyl), and -NH-CO-(C 1-6 Alkylene)-OH; the C 1-6 The alkylene group is optionally replaced by C 3-6 cycloalkyl substituted; and

[0122] q is 2.

[0123] In some embodiments, R5, R6 are each independently selected from OH, -NH2, C 1-4In some embodiments, R5, R6 are each independently selected from OH, -NH2, methyl, ethyl, n-propyl, isopropyl and halogen. In some embodiments, R5, R6 are each independently selected from OH, -NH2, methyl, fluorine and chlorine.

[0124] In some embodiments, R7 is selected from -H, -NH2, -NH(C 1-4 alkyl), and -NH-CO-(C 1-4 In some embodiments, R7 is selected from -H, -NH2, -NHCH3, -NHCH2CH3, -NHCH(CH3)2, -NH-CO-CH2-OH, -NH-CO-CH2CH2-OH, -NH-CO-CH2(CH3)-OH, and -NH-CO-CH2CH(CH3)-OH. In some embodiments, R7 is selected from -H, -NH2, -NH-CO-CH2-OH, and -NH-CO-CH2CH(CH3)-OH.

[0125] In some embodiments, R8 and R9 are each independently selected from H, halogen, or hydroxyl, and R 10 is selected from hydrogen; and

[0126] R 11 Selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0127] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, -SO2-C 1-6 Alkyl, -(C=O)-OC 1-6 Alkyl and -CO-C 1-6 Alkyl; the C 1-6 Alkyl and C 1-4 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0128] In some embodiments, R 11 Selected from C 1-4 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0129] where R a 、Rb Each occurrence is independently selected from H, C 1-4 Alkyl, C 3-6 Cycloalkyl, -SO2-C 1-4 Alkyl, -(C=O)-OC 1-4 Alkyl and -CO-C 1-4 Alkyl; the C 1-4 Alkyl and C 1-4 The alkylene group is optionally further substituted with one or more selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0130] In some embodiments, R 11 Selected from ethyl, -CH2NH-CO-CH(OH)-cyclopropyl, -CH2NH-CO-CH2CH(OH)-CH3 and -CH2CH2-N(CH(CH3)2)(SO2-CH3).

[0131] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0132] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0133] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0134] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0135] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0136] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0137] In some embodiments, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0138] In some embodiments, the structure of the cytotoxic drug fragment D is shown in Formula III-A, Formula III-B or Formula III-C below:

[0139] Among them, R 5A Selected from -O- or -N-; R 7A Selected from chemical bonds, -O-, -NH-, -N(C 1-6 alkyl)-, and -NH-CO-(C 1-6 Alkylene)-O-; the C 1-6 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 3-6 substituted by a cycloalkyl substituent;

[0140] R5 to R7 and q are as described above.

[0141] In some embodiments, R 7A Selected from chemical bonds, -O-, -NH-, -N(C 1-4 alkyl)-, and -NH-CO-(C 1-4 Alkylene)-O-; the C 1-4 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-4 Halogenated alkyl, C 3-6 In some embodiments, R 7A is selected from the group consisting of a chemical bond, -O-, -NH-, -N(CH3)-, -N(CH2CH3)-, -N(CH(CH3)2)-, -NH-CO-CH2-O-, -NH-CO-CH2CH2-O-, -NH-CO-CH2(CH3)-O-, -NH-CO-CH2CH(CH3)-O-. In some embodiments, R 7A Selected from -NH-, -NH-CO-CH2-O-, -NH-CO-CH2CH(CH3)-O-.

[0142] In some embodiments, the cytotoxic drug fragment D is selected from the following formula IV-A or IV-B

[0143] Among them, R8~R 11 As described in any of the above; R' 11 -C 1-4 Alkylene-NH(C 1-6 Alkylene)-O- or -C 1-4 Alkylene-N(C 1- 6 alkyl)(C 1-6 Alkylene)-O-; the C 1-4Alkylene, C 1-6 Alkylene or C 1-6 The alkyl group is optionally further substituted with one or more radicals selected from halogen, hydroxy, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0144] In some embodiments, D is selected from:

[0145] Those skilled in the art will appreciate that the antibody-drug conjugates described herein can be prepared modularly. For example, a free-form "drug-linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking to the antibody or its antigen-binding fragment) is first obtained, and then covalently linked to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described herein. Accordingly, M' in the free-form "drug-linker" is linked to one or more sulfhydryl (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment by a substitution reaction (e.g., removal of structures such as -SO2Me or pentafluorophenol thereon) or by an addition reaction.

[0146] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof having a structure shown in formula DEL-M', wherein:

[0147] M' is -M-Lg, wherein Lg is a leaving group for nucleophilic substitution reaction, and M is a structural fragment that binds to the antibody or antigen-binding fragment thereof;

[0148] L is the structural fragment connecting M and E;

[0149] E is a structural fragment connecting L and D;

[0150] D is a cytotoxic drug fragment.

[0151] In some embodiments, Lg is selected from halogen (eg, F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH2), nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, the halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl group-containing structural fragments are optionally substituted with one or more suitable substituents.

[0152] In some embodiments, Lg is selected from halogen (eg, F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkylsulfoxide, halogenated phenoxy, hydroxyl (-OH), thiol (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and structural fragments containing alkynyl.

[0153] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6 Alkylsulfonyl (C 1-6 alkyl-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2).

[0154] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0155] In some embodiments, Lg is selected from C 1-6 Alkylsulfonyl and halophenoxy.

[0156] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0157] In some embodiments, the free form of the "drug-linker" is selected from A-1 to A-204 and B-1 to B-24 shown below:

[0158] wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, 5-15, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0159] In some embodiments, the free form of the "drug-linker" is selected from the above-mentioned A-1 to A-201 and B-1 to B-17.

[0160] In some embodiments, the free form of the "drug-linker" is selected from the above-mentioned A-1 to A-150, B-1 to B-3, and B-5 to B-11.

[0161] In some embodiments, the free form of the "drug-linker" is selected from A-1 to A-86 described above.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0163] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0164] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0165] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0166] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0167] or,

[0168] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0169] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0170] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0171] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0172] or,

[0173] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0174] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0175] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0176] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0177] or,

[0178] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0179] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0180] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0181] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises

[0183] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0184] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0185] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0186] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0187] or,

[0188] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0189] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0190] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0191] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0192] or,

[0193] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0194] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0195] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0196] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0197] or,

[0198] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0199] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0200] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0201] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0203] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0204] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0205] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0206] or,

[0207] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0208] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0209] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0210] or,

[0211] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0212] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0213] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0214] or,

[0215] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0216] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0217] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0219] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0220] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0221] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0222] or,

[0223] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0224] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0225] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0226] or,

[0227] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0228] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0229] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0230] or,

[0231] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0232] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0233] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0234] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0235] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0236] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0237] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0239] (a) VH shown in SEQ ID NO: 1, and / or VL shown in SEQ ID NO: 2; or

[0240] (b) VH represented by SEQ ID NO: 3, and / or VL represented by SEQ ID NO: 4.

[0241] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0242] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0243] (b) VH shown in SEQ ID NO: 3 or a variant thereof, and VL shown in SEQ ID NO: 4 or a variant thereof.

[0244] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0245] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0246] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0247] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0248] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and

[0249] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

[0250] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0251] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0252] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof having up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 36.

[0253] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:35 and a light chain constant region (CL) as shown in SEQ ID NO:36.

[0254] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0255] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0256] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0257] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0258] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 37, and a light chain comprising the sequence shown in SEQ ID NO: 38;

[0259] (2) A heavy chain comprising the sequence shown in SEQ ID NO: 39, and a light chain comprising the sequence shown in SEQ ID NO: 40.

[0260] In some embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab or Pertuzumab, the amino acid sequence of Trastuzumab has an accession number in the IMGT database (IMGT / mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has an accession number in the IMGT database (IMGT / mAb-DB ID): 80.

[0261] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0262] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0263] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0264] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0265] In certain embodiments, the N-terminal glutamine of the VH or variant thereof shown in SEQ ID NO: 1 or 3 or the heavy chain or variant thereof shown in SEQ ID NO: 37 or 39 undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0266] In certain embodiments, the heavy chain constant region (CH) of SEQ ID NO: 35 or a variant thereof, or the heavy chain of SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0267] In some embodiments, M in the antibody drug conjugate is linked to a sulfhydryl (-SH) group on Ab.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the antibodies or antigen-binding fragments thereof described in this application; -MLED is selected from the structural compounds shown in this application; x is 1 to 10; preferably, x is 1 to 9, and more preferably x is 5 to 8.

[0269] In some embodiments, in the presence of Ab-[MLED] x In the antibody drug conjugate of the structure shown, x is 1 to 10, MLED is formed by the compounds shown by A-1 to A-204 or B-1 to B-24, preferably by removing -SO2Me from the compounds, and Ab is as defined above.

[0270] In some embodiments, the antibody drug conjugate is selected from ADC A-1 to ADC A-204 and ADC B-1 to ADC B-24 shown below:

[0271] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases; n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, 5-15, or 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0272] in, Or it represents the specific connection method between the sulfhydryl group in the antibody or its antigen-binding fragment and the M fragment.

[0273] In some embodiments, the HA in each antibody drug conjugate comprises:

[0274] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0275] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0276] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0277] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0278] or,

[0279] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0280] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0281] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0282] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0283] or,

[0284] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0285] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0286] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0287] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0288] or,

[0289] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0290] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0291] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0292] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0293] In some embodiments, the HA in each antibody drug conjugate comprises:

[0294] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0295] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0296] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0297] wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0298] or,

[0299] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0300] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0301] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0302] wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0303] or,

[0304] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0305] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0306] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0307] wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0308] or,

[0309] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0310] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0311] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0312] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0313] In some embodiments, the HA in each antibody drug conjugate comprises:

[0314] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0315] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0316] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0317] or,

[0318] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0319] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0320] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0321] or,

[0322] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0323] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0324] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0325] or,

[0326] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0327] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0328] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0329] In some embodiments, the HA in each antibody drug conjugate comprises:

[0330] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0331] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0332] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0333] or,

[0334] (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0335] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0336] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0337] or,

[0338] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0339] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or

[0340] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0341] or,

[0342] (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0343] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or

[0344] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0345] In some embodiments, the HA in each antibody drug conjugate comprises:

[0346] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0347] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0348] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0349] In some embodiments, the HA in each antibody drug conjugate comprises:

[0350] (a) VH shown in SEQ ID NO: 1, and / or VL shown in SEQ ID NO: 2; or

[0351] (b) VH represented by SEQ ID NO: 3, and / or VL represented by SEQ ID NO: 4.

[0352] In some embodiments, the HA in each antibody drug conjugate comprises:

[0353] (a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

[0354] (b) VH shown in SEQ ID NO: 3 or a variant thereof, and VL shown in SEQ ID NO: 4 or a variant thereof.

[0355] In some embodiments, the HA in each antibody drug conjugate comprises:

[0356] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0357] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

[0358] In some embodiments, the HA in each antibody drug conjugate further comprises:

[0359] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and

[0360] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

[0361] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0362] In some embodiments, the HA in each antibody drug conjugate comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof having up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions).

[0363] In some embodiments, the HA in each antibody drug conjugate comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:35.

[0364] In some embodiments, the HA in each antibody drug conjugate comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof having up to 20 conservative amino acid substitutions compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions).

[0365] In some embodiments, the HA in each antibody drug conjugate comprises a light chain constant region (CL) as set forth in SEQ ID NO:36.

[0366] In some embodiments, the HA in each antibody drug conjugate comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0367] In some embodiments, the HA in each antibody drug conjugate comprises:

[0368] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0369] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0370] In some embodiments, the HA in each antibody drug conjugate is selected from Trastuzumab or Pertuzumab, the amino acid sequence of Trastuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 97, and the amino acid sequence of Pertuzumab has an IMGT database accession number (IMGT / mAb-DB ID): 80.

[0371] In certain embodiments, the HA in each antibody drug conjugate can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0372] In certain embodiments, the HA in each antibody drug conjugate may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in the heavy chain, conversion of an N-terminal glutamine or glutamic acid in the heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., CHO cells) or during purification / storage.

[0373] In certain embodiments, the N-terminal glutamine of the VH or variant thereof shown in SEQ ID NO: 1 or 3 or the heavy chain or variant thereof shown in SEQ ID NO: 37 or 39 undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0374] In certain embodiments, the heavy chain constant region (CH) of SEQ ID NO: 35 or a variant thereof, or the heavy chain of SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine.

[0375] In some embodiments, HA in each antibody drug conjugate represents trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0376] In some embodiments, HA in each antibody drug conjugate represents the following antibody or antigen-binding fragment:

[0377] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL sequence of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 36; or

[0378] (2) A heavy chain comprising the VH sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0379] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 1-10, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0380] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 5-8, such as 5-6, 5-7, 5-8, 6-7, 6-8 or 7-8.

[0381] In some embodiments, x in the antibody drug conjugate represented by Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0382] The antibody-drug conjugates of the present invention are optionally substituted with one or more suitable substituents.

[0383] The present application further provides an antibody-drug conjugate as shown in the formula Ab-[L'-D]x, wherein:

[0384] Ab is an antibody or an antigen-binding fragment thereof;

[0385] D is a cytotoxic drug fragment;

[0386] L' is a linker connecting Ab and D;

[0387] x is an integer from 1 to 10;

[0388] The antibody or antigen-binding fragment thereof forms the antibody-drug conjugate with compounds A-1 to A-204, or B-1 to B-24 described above.

[0389] In certain embodiments, the antibody comprises the heavy chain amino acid sequence of SEQ ID NO:37, and the light chain amino acid sequence of SEQ ID NO:38.

[0390] In certain embodiments, the antibody is Trastuzumab.

[0391] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0392] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0393] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0394] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0395] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0396] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0397] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0398] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0399] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0400] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0401] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0402] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0403] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0404] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0405] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0406] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0407] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0408] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0409] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0410] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0411] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0412] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0413] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0414] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0415] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0416] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0417] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0418] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0419] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0420] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0421] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0422] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0423] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0424] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0425] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0426] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0427] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0428] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0429] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0430] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0431] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0432] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0433] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0434] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0435] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0436] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0437] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0438] Where * is the attachment point of the cysteine ​​sulfhydryl group of the conjugated antibody.

[0439] In certain embodiments, the antibody in the antibody drug conjugate is conjugated to 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8; preferably 5-8) of the following structures:

[0440] intermediates

[0441] In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

[0442] in

[0443] PG1 is independently H or an amino protecting group, such as an alkoxycarbonyl amino protecting group, for example, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, for example, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group, for example, trityl (Trt), C 1-6 Alkyl-substituted trityl, p-methoxytrityl (MMT), dimethoxytrityl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0444] PG2 is each independently H or a carboxyl protecting group, such as C 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

[0445] PG3 is each independently H or a hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, trityl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl;

[0446] s1 is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0447] Lg is as defined above.

[0448] In some embodiments, the present application provides an intermediate compound having the structure shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof:

[0449] In another aspect, the present application provides use of the aforementioned intermediate compound or its salt, stereoisomer, tautomer or isotope-labeled compound in preparing the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0450] Composition

[0451] In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug-linker as described herein, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Thus, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reverse phase chromatography or HPLC-MS.

[0452] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.

[0453] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

[0454] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 8, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, about 7.5 to 8.0.

[0455] In some embodiments, the DAR of the ADC compositions described herein is between about 6.0 and 9.0, preferably between about 6.0 and 8.0, for example, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.40, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.50, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55 .34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6. 55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6.73, about 6.74, about 6.75, about 6.7 6, about 6.77, about 6.78, about 6.79, about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.9, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97 , about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, about 7.1, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7.16, about 7.17, about 7.18, about 7.19, about 7.2, about 7.21, about 7.22, about 7.23, about 7.24, about 7.25, about 7.26, about 7.27, about 7.28, about 7.29, about 7.3, about 7.31, about 7.32, about 7.33, about 7.34, about 7.35, about 7.36, about 7.37, about 7.38, about 7.39, about 7.4, about 7.41, about 7.42, about 7.43, about 7.44, about 7.45, about 7.46, about 7.47, about 7.48, about 7.49, about 7.5, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about 7.6, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.7, about 7.71, about 7.72, about 7.73, about 7.74, about 7.75, about 7.76, about 7.77, about 7.78, about 7.79, about 7.8, about 7.81, about 7.82, about 7.83, about 7.84, about 7.85, about 7.86, about 7.87, about 7.88, about 7.89, about 7.9, about 7.91, about 7.92, about 7.93, about 7.94, about 7.95, about 7.96, about 7.97, about 7.98, about 7.99, about 8.0, about 8.01, about 8.02, about 8.03, about 8.04, about 8.05, about 8.06, about 8.07, about 8.08, about 8.09, about 8.1, about 8.11, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.23, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33, about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, about 8.42, about 8.43, about 8.44, about 8.45, about 8.46, about 8.47, about 8.48, about 8.49, about 8.5, about 8.51, about 8.52, about 8.53, about 8.54, about 8.55, about 8.56, about 8.57, about 8.58, about 8.59, about 8.6, about 8.61, about 8.62, about 8.63, about 8.64, about 8.65, About 8.66, about 8.67, about 8.68, about 8.69, about 8.7, about 8.71, about 8.72, about 8.73, about 8.74, about 8.75, about 8.76, about 8.77, about 8.78, about 8.79, about 8.8, about 8.81, about 8.82, about 8.83, about 8.84, about 8.85, about 8.86, about 8.87, about 8.88, about 8.89, about 8.9, about 8.91, about 8.92, about 8.93, about 8.94, about 8.95, about 8.96, about 8.97, about 8.98, about 8.99, about 9.0.

[0456] In certain embodiments, the composition is composed of an antibody drug conjugate as shown in Ab-[L'-D]x. In certain embodiments, the DAR of the composition is 1-8, such as 5-8, such as about 8.0, 7.89, 7.69, 7.91, 7.92, 7.85, 7.96, 7.42, 7.48, 7.91, 7.79, 7.99, or 7.9. In certain embodiments, antibody drug conjugates in which x is 8 account for more than 60%, more than 70%, more than 80%, or more than 90%.

[0457] Compound

[0458] In one aspect, the present application provides the following compounds or their pharmaceutically acceptable salts, isotope-labeled compounds, solvates, hydrates, isomers, or any crystalline forms or racemates thereof:

[0459] Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof:

[0460] Pharmaceutical composition

[0461] In another aspect, the present application provides a pharmaceutical composition comprising any of the above-described antibody-drug conjugates, any of the above-described compounds, or any of the above-described drug-linkers, and one or more pharmaceutical excipients. Alternatively, the present application provides a pharmaceutical composition comprising any of the above-described antibody-drug conjugates, any of the above-described compositions, and one or more pharmaceutical excipients.

[0462] The antibody drug conjugates, compositions, compounds or drug-linkers described herein are typically formulated in a unit injectable form with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same can be administered by any route appropriate to the subject to be treated.

[0463] application

[0464] The antibody drug conjugates, compounds, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematological malignancies, such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0465] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates, compounds, compositions, or pharmaceutical compositions containing the same in the preparation of drugs for treating Her2-expressing cancers.

[0466] At the same time, the present application provides any of the above-mentioned antibody-drug conjugates, compounds, compositions, or pharmaceutical compositions containing the same, and their use in drugs for treating Her2-expressing cancers.

[0467] At the same time, the present application also provides a method for treating Her2-expressing cancer, which comprises the step of administering an effective amount of any of the above-mentioned antibody-drug conjugates, compounds, compositions, or pharmaceutical compositions containing the same to a subject in need thereof.

[0468] In some embodiments, the antibody drug conjugate, compound, drug-linker, or pharmaceutical composition containing the same is sufficient (e.g., in a subject):

[0469] (1) Inhibit the proliferation of cells (such as tumor cells);

[0470] (2) inhibit tumor growth;

[0471] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0472] (4) inhibiting HER2-mediated signal transduction;

[0473] (5) preventing and / or treating HER2-mediated diseases / disorders; or

[0474] (6) Any combination of (1) to (5) above.

[0475] In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma), and urothelial carcinoma.

[0476] All technical features disclosed in this specification, such as the definitions of various groups, except for mutually exclusive technical features, and all embodiments can be combined in any manner to obtain different general formula ranges or specific solutions. These ranges and solutions are all within the scope of the present invention.

[0477] definition

[0478] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0479] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0480] The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to a pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, or have the N-terminal amino acid cyclized to a pyroglutamate.

[0481] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0482] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0483] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these generally constant features do not appear; however, Cys residues are the most conserved feature.

[0484] V H The entire amino acid sequence of a V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or according to Kabat numbering. H and V L The amino acid positions in are defined according to sequential numbering.

[0485] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0486] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0487] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0488] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0489] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0490] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0491] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0492] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 48) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 49) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, a VH-VH-COOH domain comprising NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0493] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0494] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0495] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0496] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0497] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0498] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0499] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0500] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0501] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0502] The term "linker" refers to a structural fragment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, a fragment of the formula Ab-[MLED] x -MLE- structure fragment in.

[0503] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described herein prior to linkage to the antibody or antigen-binding fragment thereof. For example, a "drug-linker" refers to M'-LED, where M' represents the structure of M prior to covalent linkage to the antibody or antigen-binding fragment thereof. The covalent linkage of the drug-linker to the antibody or antigen-binding fragment thereof yields the antibody-drug conjugate described herein.

[0504] The "drug-linker" also includes all pharmaceutically acceptable isotope-labeled compounds thereof, which are identical to the "drug-linker" compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); and sulfur isotopes (e.g. 35 S).

[0505] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0506] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20 Alkyl", "C 1-10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0507] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-6 Alkylene", "C 1-5 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0508] The term "aryl" refers to an unsaturated carbon ring group having a conjugated π electron system, such as a "6-10 membered aryl group", and specific examples include but are not limited to phenyl and naphthyl.

[0509] The term "heteroaryl" refers to an unsaturated group having a conjugated π electron system, wherein at least one (e.g., 1, 2, 3, or 4) of the ring atoms is a heteroatom, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized. For example, "5-12 membered heteroaryl", "5-11 membered heteroaryl", "5-10 membered heteroaryl", "5-9 membered heteroaryl", "5-6 membered heteroaryl", and specific examples include, but are not limited to, phenyl, naphthylfuryl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indazolyl, indolyl, quinolinyl, and isoquinolinyl.

[0510] The term "heterocyclyl" refers to a saturated or partially unsaturated cyclic group consisting of ring atoms and at least one (e.g., 1, 2, 3, 4, or 5 heteroatoms) ring atom being a heteroatom, such as N, O, and S, wherein the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms are optionally oxidized, and the carbon atom is optionally oxoed. The heterocyclyl includes a monocyclic, bicyclic, or polycyclic ring, including a spirocyclic, a paracyclic, or a bridged ring. The term "nitrogen-containing heterocyclyl" refers to a group in which at least one heteroatom is N, such as a "5-12 membered nitrogen-containing heterocyclyl," a "5-9 membered nitrogen-containing heterocyclyl," or a "9-12 membered nitrogen-containing heterocyclyl." Specific examples include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and 1,2,3,4-tetrahydroquinolinyl.

[0511] The term "isotopically labeled compound" means a compound that is identical in structure to a compound of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); and sulfur isotopes (e.g. 35 S).

[0512] As used herein, the term "suitable substituent" refers to modifications that can be made to a compound by one skilled in the art according to the needs of the compound substituent. "Suitable substituents" include oxo (=O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g. -C 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 、R 9 Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -C1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0513] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. In some embodiments, the substituents are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-8 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene), C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 (alkenyl), C2-C6 (alkynyl), C3-C8 (cycloalkyl), 3-10 membered (heterocyclyl), C6-C 10 (Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc., wherein R 8 、R 9 and R' are as defined above. For example, the substituent may be a suitable substituent as described above.

[0514] If a functional group or structural moiety is described as "substituted or unsubstituted," the functional group or structural moiety may be (1) unsubstituted or (2) substituted.

[0515] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, where reasonable.

[0516] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0517] Whether explicitly stated or not, the numerical values ​​in this application are modified by the term “about.” The term “about” means within ±20%, ±10%, preferably ±5%, and more preferably ±2% of the numerical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0518] Figure 1 Anti-tumor effect of anti-human HER2 antibody-drug conjugate ADC on the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0519] Figure 2 Anti-tumor effect of anti-human HER2 antibody-drug conjugate ADC on the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0520] Figure 3. Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0521] Figure 4 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0522] Figure 5 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0523] Figure 6. Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0524] Figure 7 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0525] Figure 8. Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0526] Figure 9 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0527] Figure 10. Changes in body weight of mice in each group in the human breast cancer cell JIMT-1CDX model.

[0528] Figure 11 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the HCC827 cell subcutaneous tumor-bearing mouse model.

[0529] Figure 12 shows changes in body weight of mice in each group in the HCC827 human non-small cell lung cancer CDX model.

[0530] Figure 13 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0531] Figure 14 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0532] Figure 15 shows changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0533] Figure 16 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0534] Figure 17 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0535] Figure 18 shows changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model.

[0536] Figure 19 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0537] Figure 20 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0538] Figure 21 shows changes in body weight of mice in each group in the human breast cancer cell JIMT-1CDX model.

[0539] Figure 22 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0540] Figure 23 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0541] Figure 24 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0542] Figure 25 shows changes in body weight of mice in each group in the human gastric cancer cell NCI-N87 CDX model. DETAILED DESCRIPTION

[0543] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0544] The information of the sequences involved in the present invention is described in the following table:

[0545] The abbreviations used in this document have the following meanings:

[0546] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1H NMR) or mass spectrometry (MS).

[0547] Nuclear magnetic resonance (1H NMR) measurements were performed using a Bruker 400 MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0548] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0549] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0550] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0551] Preparation Example 1: Preparation of (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (1-36)

[0552] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 52.64 μmol), (R)-3-hydroxybutyric acid (10.96 mg, 105.28 μmol), HATU (30.02 mg, 78.96 μmol) and DIPEA (20.41 mg, 157.93 μmol) were dissolved in DMF (2 mL). The reaction system was stirred at 25°C for 2 h. The reaction solution was prepared by HPLC and freeze-dried to obtain the title compound (17 mg, 31.42 μmol).

[0553] Its separation and purification method is as follows:

[0554] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0555] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0556] Its structural characterization data are as follows:

[0557] ESI-MS (m / z): 542.1 [M+H] +

[0558] 1 H NMR (400MHz, DMSOd6) δ8.43 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 10.4 Hz, 1H), 7.33 (s, 1H), 6. 54(s,1H), 5.63-5.55(m,1H), 5.43(s,2H), 5.27(d,J=18.8Hz,1H), 5.21(d,J=18.8Hz ,1H), 4.66(d,J=4.8Hz,1H), 4.09-3.98(m,1H), 3.32-3.26(m,2H), 2.32-2.25(m,1H) , 2.24-2.12(m,3H), 1.93-1.78(m,2H), 1.08(d,J=6.0Hz,3H), 0.87(t,J=7.2Hz,3H).

[0559] [Article 26 of the Rules, 08.05.2025] Preparation Example 2: Preparation of (S)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxybutanamide (1-39)

[0560] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 0.053 mmol, trifluoroacetate) and (S)-3-hydroxybutyric acid (16.5 mg, 0.158 mmol) were dissolved in DMF (0.5 mL). HATU (40.0 mg, 0.105 mmol) and DIPEA (20.4 mg, 0.158 mmol) were then added and reacted at room temperature for 2 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to give the title compound (13.5 mg, 0.024 mmol).

[0561] Its structural characterization data are as follows:

[0562] MS m / z(ESI):542.1[M+H] +

[0563] 1 HNMR(400MHz,DMSOd6)δ8.44(d,J=8.8Hz,1H),8.09(d,J=10.4Hz,1H),7.34(s ,1H),6.56(m,1H),5.64-5.60(m,1H),5.44(s,2H),5.32-5.25(m,2H),4.63(d ,J=4.8Hz,1H),4.06-4.01(m,1H),3.31-3.26(m,2H),2.28-2.23(m,1H),2.20 -2.13(m,3H),1.90-1.82(m,2H),1.08(d,J=6.4Hz,3H),0.87(t,J=7.2Hz,3H).

[0564] Its separation and purification method is as follows:

[0565] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0566] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0567] Intermediate Preparation Example 1: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexatriacontane-35-ynoic acid (INT-1):

[0568] Step 1: Preparation of 1-(9H-fluoren-9-yl)-4,7,10,13,16,19,22,25,28,31-decamethyl-3,6,9,12,15,18,21,24,27,30-decaoxo-2-oxa-4,7,10,13,16,19,22,25,28,31-decaazatricarboxane-33-oic acid (INT-1-2)

[0569] Use standard solid phase synthesis methods:

[0570] 1) Resin Preparation: 2-CTC resin (3.00 mmol, 3.70 g, 0.81 mmol / g), Fmoc-sarcosyl-sarcosine (9.00 mol, 3.44 g, 3.00 equivalents), and DIPEA (4.00 equivalents) were added to dichloromethane (10.0 mL) and reacted under a nitrogen atmosphere for 2 hours. MeOH (10.0 mL) was then added to the resin under a nitrogen bubbling atmosphere over 30 minutes, and the resin was filtered to obtain the obtained resin.

[0571] 2) Coupling: A solution of FMOC-sarcosyl-sarcosine (6.88 g, 6.00 equiv) and HATU (6.58 g, 5.70 equiv) in DMF (10.0 mL) was added to the resin under nitrogen bubbling. DIPEA (6.00 equiv) was added dropwise, and nitrogen was bubbled through at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

[0572] 3) Deprotection: A 20% piperidine DMF solution (30.0 mL) was added to the resin and bubbled with nitrogen at 20° C. for 30 minutes. The resin was then washed with DMF (30.0 mL) × 5.

[0573] 4) Repeat steps 2 and 3 using the amino acids in Table 1.

[0574] 5) The resin was then washed with DMF (30.0 mL x 5) and MeOH (30.0 mL x 5), and then dried under vacuum.

[0575] Table 1:

[0576] Peptide cleavage and purification:

[0577] 1) Add cleavage solution (TFA / DCM, 1 / 10, v / v, 200.0 mL) to the flask containing the side-chain protected peptide at room temperature and stir twice for 3 minutes.

[0578] 2) After filtration, the filtrates were combined and concentrated.

[0579] 3) The crude product was purified by HPLC and freeze-dried to give the title compound (2856 mg, TFA salt).

[0580] Its structural characterization data are as follows:

[0581] ESI-MS (m / z): 952.2 (M+H) + .

[0582] The purification method is as follows:

[0583] Step 2: Preparation of 5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-oic acid (INT-1-3)

[0584] To a solution of 1-(9H-fluoren-9-yl)-4,7,10,13,16,19,22,25,28,31-decamethyl-3,6,9,12,15,18,21,24,27,30-decaoxo-2-oxa-4,7,10,13,16,19,22,25,28,31-decaazatricarboxane-33-oic acid (200.0 mg, 0.21 mmol) in DMF (3 mL) was added diethylamine (46.1 mg, 0.63 mmol) and stirred at room temperature for 1 hour. After completion of the reaction, the solvent was removed by lyophilization and the mixture was recrystallized by adding 10 mL of ethyl acetate for 1 hour. The filter cake was filtered and the solvent was lyophilized to afford the title compound (145.4 mg, 0.20 mmol).

[0585] Its structural characterization data are as follows:

[0586] MS m / z(ESI):729.2[M+H] +

[0587] Step 3: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexatriacontane-35-ynoic acid (INT-1)

[0588] 5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decazatriacontane-31-oic acid (145.4 mg, 0.20 mmol) was dissolved in DMF (2 mL), DIPEA (77.5 mg, 0.6 mmol) was added, and A-14-1 (109.6 mg, 0.30 mmol) was added. After completion of the reaction, the product was purified by flash column chromatography (C18, water / acetonitrile = 0.4) and freeze-dried to give the title compound (146.2 mg, 0.15 mmol).

[0589] Its structural characterization data are as follows:

[0590] MS m / z(ESI):979.3[M+H]+

[0591] Intermediate Preparation Example 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0592] Step 1: Preparation of L-alanyl-L-alanine (INT-2-1)

[0593] Dissolve (tert-Butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) in dichloromethane (2 mL) and add trifluoroacetic acid (0.5 mL). The reaction is allowed to react at 20°C for 1 hour. After completion of the reaction, the reaction mixture is concentrated under reduced pressure to remove the solvent, yielding the light yellow title compound (160 mg, 370.93 μmol).

[0594] Its structural characterization data are as follows:

[0595] MS m / z(ESI):161.1[M+H] +

[0596] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0597] Dissolve L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (150.58 mg, 412.14 μmol) in N,N-dimethylacetamide (2 mL). Add N,N-diisopropylethylamine (266.33 mg, 2.06 mmol). The reaction is allowed to proceed at 20°C for 1 hour. After completion of the reaction, the product is purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (125 mg, 304.55 μmol).

[0598] Its structural characterization data are as follows:

[0599] MS m / z(ESI):428.1[M+H2O] +

[0600] Intermediate Preparation Example 3: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (INT-3)

[0601] Dissolve glycylglycine (1 g, 7.57 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.77 g, 7.57 mmol) in dimethyl sulfoxide (10 mL). Stir the reaction at room temperature for 2 hours. After completion, the product is purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to yield the title compound (2.52 g, 6.59 mmol).

[0602] Its structural characterization data are as follows:

[0603] MS m / z(ESI):383.2[M+H] +

[0604] Intermediate Preparation Example 4: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0605] Step 1: Preparation of glycylglycyl-L-phenylalanine (INT-4-2)

[0606] Dissolve ((Benzyloxy)carbonyl)glycylglycylglycyla-L-phenylalanine (200 mg, 483.77 μmol) in methanol (8 mL). After nitrogen replacement, add palladium on carbon (20 mg). Replace the atmosphere with hydrogen three times, and stir the reaction at room temperature for 3 hours. After completion of the reaction, remove the palladium on carbon by filtration. The filtrate is concentrated to remove the solvent to yield the title compound (135 mg, 483.37 μmol).

[0607] Its structural characterization data are as follows:

[0608] MS m / z(ESI):280.2[M+H] +

[0609] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0610] Dissolve glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (170.06 mg, 465.46 μmol) in N,N-dimethylformamide (8 mL), and add N,N-diisopropylethylamine (120.32 mg, 930.93 μmol). Stir the reaction at room temperature for 1 hour. After completion, the reaction is purified by flash column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to obtain the title compound (60 mg, 0.11 mmol).

[0611] Its structural characterization data are as follows:

[0612] MS m / z(ESI):530.3[M+H] +

[0613] Intermediate Preparation Example 5: Preparation of (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (INT-5)

[0614] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (INT-5-2)

[0615] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (10.0 g, 26.2 mmol) in dichloromethane (100 mL). Add DCC (8.09 g, 39.2 mmol, 7.93 mL) and 1-hydroxypyrrolidine-2,5-dione (4.51 g, 39.2 mmol). Stir at 25°C for 12 hours. Filter the reaction mixture directly to remove DCC, and rinse the filter cake three times with dichloromethane (10.0 mL x 3). Concentrate the filtrate, add it to tetrahydrofuran (100 mL), cool it to 0°C, and continue stirring for 1 hour. Filter again to remove DCC, and concentrate the filtrate to obtain the crude title compound (11.7 g), which is used directly in the next step without purification.

[0616] Its structural characterization data are as follows:

[0617] ESI-MS (m / z): 502.1 (M+Na) + .

[0618] Step 2: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine-glycine (INT-5-3)

[0619] 2,5-Dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (5.00 g, 10.4 mmol) and L-alanine glycine (1.52 g, 10.4 mmol) were added to a mixture of water (25.0 mL) and tetrahydrofuran (50.0 mL). Aqueous sodium bicarbonate (1.75 g, 20.8 mmol, 811 μL) was added, and the mixture was stirred at 25°C for 2 hours. The reaction mixture was adjusted to pH 2-3 with 1N dilute hydrochloric acid and extracted three times with dichloromethane (60.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (3.18 g, 6.23 mmol), which was used directly in the next step without purification.

[0620] Its structural characterization data are as follows:

[0621] ESI-MS (m / z): 511.1 (M+H) + .

[0622] Step 3: Preparation of (5S,8S,11S)-1-(9H-fluoro-9-yl)-5,8-11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradec-14-yl acetate (INT-5-4)

[0623] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-L-alanine-glycine (3.10 g, 6.07 mmol), copper acetate (882 mg, 4.86 mmol), and lead tetraacetate (6.46 g, 14.5 mmol) were dissolved in DMF (40.0 mL). Glacial acetic acid (1.60 g, 26.7 mmol, 1.53 mL) was added, and the mixture was heated to 60°C and stirred for 1 hour. The reaction mixture was concentrated to obtain a crude product, which was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the crude title compound (709 mg, 1.28 mmol), which was used in the next step without purification.

[0624] Its structural characterization data are as follows:

[0625] 1H NMR (400MHz, DMSO) δ8.85(m,1H),7.97(m,2H),7.89(d,J=7.6Hz,2H),7.72(m,2H),7.53(m,1H),7.39-7.44(m,2H ),7.30-7.35(m,2H),5.04-5.10(m,2H),4.24-4.28(m,3H),3.17(d,J=5.2Hz,3H),1.98(s,3H),1.18-1.22(m,9H)

[0626] Step 4: Preparation of (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (INT-5)

[0627] (5S,8S,11S)-1-(9H-fluoro-9-yl)-5,8-11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradec-14-yl acetate (300 mg, 571.90 μmol) and 2-hydroxyacetic acid (52.2 mg, 686 μmol, 41.7 μL) were dissolved in dichloromethane (3.00 mL). PPTS (144 mg, 572 μmol) was added, and the mixture was stirred at 25°C for 2 hours. Water (100 mL) was added to the reaction mixture, and the pH was adjusted to 4-5 with 1N dilute hydrochloric acid. The mixture was then extracted three times with dichloromethane (80.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by preparative HPLC and freeze-dried to give the title compound (176 mg, 325.10 μmol).

[0628] Its structural characterization data are as follows:

[0629] ESI-MS (m / z): 558.2 [M+H] + .

[0630] The purification method is as follows:

[0631] Chromatographic column: Waters xbridge C18 (150mm*25mm*10μm)

[0632] Mobile phase A: acetonitrile; mobile phase B: water (0.05% ammonium methyl bicarbonate)

[0633] Intermediate Preparation Example 6: Preparation of 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (INT-6)

[0634] Step 1: Preparation of methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (INT-6-2)

[0635] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (300 g, 846 mmol) in 3 L of ethanol. Add lead tetraacetate (450 g, 1.02 mol), acetic acid (111 g, 1.86 mol, 106 mL), and copper acetate (61.5 g, 338 mmol). Heat to 60°C and stir for 1 hour. Add water (5 L) to the reaction mixture, and a large amount of solid precipitates. Filter and dry to obtain the crude product. The crude product is purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (225 g, 610 mmol).

[0636] Its structural characterization data are as follows:

[0637] ESI-MS (m / z): 390.9 (M+H) + .

[0638] Step 2: Preparation of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (INT-6-3)

[0639] Methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (225 g, 610 mmol) and benzyl 2-hydroxyacetate (202 g, 1.22 mol, 173 mL) were dissolved in dichloromethane (1.2 L). PPTS (23.0 g, 91.6 mmol) was added, and the mixture was heated to 50°C and stirred for 12 hours. Water (1 L) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (500 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (215 g, 453 mmol).

[0640] Its structural characterization data are as follows:

[0641] ESI-MS (m / z): 492.2 (M+H2O+H) + .

[0642] Step 3: Preparation of 2-((2-aminoacetamido)methoxy)benzyl acetate (INT-6-4)

[0643] Benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (210 g, 442 mmol) was dissolved in DMF (1.5 L). DBU (47.1 g, 309 mmol, 46.70 mL) was added and stirred at 25°C for 1 hour. The reaction mixture was used directly in the next step without further treatment.

[0644] Its structural characterization data are as follows:

[0645] ESI-MS (m / z): 252.5 (M+H) + .

[0646] Step 4: Preparation of 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-benzyl ester (INT-6-5)

[0647] To the reaction mixture from the previous step, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (161 g, 455 mmol), EDCI (96.1 g, 501 mmol), and HOBt (67.7 g, 501 mmol) were added, and the mixture was stirred at 25°C for 1 hour. Water (2 L) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (1000 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1), the product was concentrated again to obtain the title compound (150 g, 254 mmol).

[0648] Its structural characterization data are as follows:

[0649] ESI-MS (m / z): 558.2 [M+H] + .

[0650] Step 5: Preparation of 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (INT-6)

[0651] 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-benzyl ester (75 g, 127 mmol) and Pd / C (16 g, 10% purity) were added to a mixture of DMF (200 mL) and tetrahydrofuran (300 mL) and hydrogenated at 25°C for 2 hours. The reaction mixture was filtered directly, and the filter cake was rinsed with DMF three times (100 mL x 3). The filtrates were combined and concentrated to obtain the crude product. The title compound (30 g, 52.9 mmol) was obtained after recrystallization from methanol (300 mL).

[0652] Its structural characterization data are as follows:

[0653] ESI-MS (m / z): 516.1 [M+H2O+H] + .

[0654] Intermediate Preparation Example 7: Preparation of N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-N6-trityl-L-lysyl-L-alanyl-L-alanine (INT-7):

[0655] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-N6-trityl-L-lysine salt (INT-7-1)

[0656] Dissolve N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-N6-trityl-L-lysine (200 mg, 306.37 μmol) and 1-hydroxypyrrolidine-2,5-dione (38.79 mg, 337.01 μmol) in DMF (3 mL). Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (70.48 mg, 367.65 μmol). Stir at room temperature for 3 hours. After completion of the reaction, water was added to the stirred reaction mixture. A white solid precipitated. Stirring was continued for 1 hour, followed by filtration and drying of the filter cake to obtain the title compound (218 mg, 276.18 μmol).

[0657] Its structural characterization data are as follows:

[0658] MS m / z(ESI):750.4[M+H]+

[0659] Step 2: Preparation of N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-N6-trityl-L-lysyl-L-alanyl-L-alanine (INT-7)

[0660] Dissolve 2,5-dioxopyrrolidin-1-yl N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-N6-trityl-L-lysine salt (200 mg, 266.71 μmol) and L-alanyl-L-alanine (42.72 mg, 266.71 μmol) in DMF (3 mL). Stir at room temperature for 2 hours. After completion of the reaction, the reaction mixture was purified by reverse-phase column chromatography and lyophilized to obtain the title compound (174 mg, 218.88 μmol).

[0661] Its structural characterization data are as follows:

[0662] MS m / z(ESI):795.3[M+H] +

[0663] Example 1: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (A-2)

[0664] Step 1: Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (A-2-2)

[0665] Dissolve (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) in DMF (50 mL). Add DIPEA (1.18 g, 9.12 mmol, 1.59 mL) dropwise. Add acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) dropwise with ice-cooling and stirring. Continue stirring for 1 hour. Add the reaction solution to 0.1 M dilute hydrochloric acid to precipitate a solid, which is then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0666] Its structural characterization data are as follows:

[0667] ESI-MS (m / z): 552.2 [M+1] +

[0668] Step 2: Preparation of ethyl 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontazolylamino)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin-1-yl)amino)-2-oxoacetate (A-2-3)

[0669] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0°C under nitrogen protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise. The reaction was stirred and kept warm for 0.5 hour. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise and allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified on a silica gel column (MeOH / DCM = 0% to 5%) to obtain the title compound (498 mg, 0.304 mmol).

[0670] Its structural characterization data are as follows:

[0671] ESI-MS (m / z): 1352.8 [M+1] +

[0672] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (A-2-4)

[0673] 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,1 Ethyl 2-oxoacetate (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL). A solution of sodium carbonate (25.88 mg, 0.224 mmol) in water (1 mL) was added dropwise with stirring. Stirring was continued for 1 hour after the addition was complete. Dilute hydrochloric acid was added dropwise to the reaction mixture to neutralize the reaction. After concentration under reduced pressure, the mixture was directly transferred to the next step.

[0674] Its structural characterization data are as follows:

[0675] ESI-MS (m / z): 1596.7 [M+1] +

[0676] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (A-2-5)

[0677] 4-((S)-35-Azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and the organic phase was concentrated to obtain a crude product. The crude product was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0678] Its structural characterization data are as follows:

[0679] ESI-MS (m / z): 1323.6 [M+1] +

[0680] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (A-2)

[0681] 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ [3',4':6,7] indolizino[1,2-b]quinolin-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL). Cuprous bromide (9.42 mg, 0.066 mmol) was added and stirring continued for 2 hours. The reaction mixture was directly filtered and concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0682] Its structural characterization data are as follows:

[0683] ESI-MS (m / z): 1628.7 [M+1] +

[0684] The separation and purification method is as follows:

[0685] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0686] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0687] Example 2: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-14)

[0688] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (A-14-1, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetrahydro-3-oxo-5,8,11,14-tetraazahexadecanoic acid (A-14-2, 0.75 g, 1.77 mmol) were added to DMF (19 mL), and the temperature was raised to 35 ° C. After the reaction for 16 hours, (1S, 9S )-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-2, 1.00 g, 1.77 mmol) was cooled to 5-15°C with ice water, and DMTMM (0.98 g, 3.53 mmol) was added, followed by dropwise addition of DIPEA (1.14 g, 8.84 mmol). The reaction mixture was allowed to react at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated and washed with brine (100 mL). The organic phase was concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to obtain 0.98 g of compound A-14.

[0689] The A-14 separation and purification method is as follows:

[0690] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0691] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0692] The structural characterization data of A-14 are as follows:

[0693] MS m / z(ESI):1107.3[M+H] +

[0694] 1H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.31 (m, 1H), 8.21-8.19 (m, 1H), 8.17-8.09 (m, 2 H),8.08-8.04(m,1H),7.30(s,1H),7.26-7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H),4.6 4(d,J=6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41(s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2 .83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J=7.2Hz,3H).

[0695] Example 3: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-26)

[0696] Step 1:

[0697] Compound INT-5 (657 mg, 1.22 mmol) and compound 1-2 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL). HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) were then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by HPLC and freeze-dried to obtain 700 mg of compound A-26-1.

[0698] The separation and purification method is as follows:

[0699] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0700] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0701] Step 2:

[0702] Compound A-26-1 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. After the reaction, the reaction solution was directly purified by HPLC and freeze-dried to obtain 307 mg of compound A-26-2.

[0703] The separation and purification method is as follows:

[0704] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0705] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0706] Step 3:

[0707] A-26-2 (170 mg, 0.226 mmol) and compound A-14-1 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by HPLC and freeze-dried to obtain 50.56 mg of compound A-26.

[0708] Its structural characterization data are as follows:

[0709] MS m / z(ESI):1002.4[M+H] +

[0710] The separation and purification method is as follows:

[0711] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0712] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0713] 1H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2H z,1H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H ),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.5 6(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J=7.2Hz,3H).

[0714] Example 4: N-((7S,10R,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-38)

[0715] Step 1: Preparation of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine glycine tert-butyl ester (A-38-2)

[0716] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (350 g, 1.12 mol) and glycine tert-butyl ester (147 g, 1.12 mol) in dichloromethane (2.5 L). EDCI (237 g, 1.24 mol), DIEA (159 g, 1.24 mol, 215 mL), and HOBt (167 g, 1.24 mol) were added sequentially, and the mixture was stirred at room temperature for 1 hour. Water (1 L) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (500 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified on a silica gel column (CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (220 g, 518 mmol).

[0717] Its structural characterization data are as follows:

[0718] MS m / z(ESI):442.2[M+H] +

[0719] Step 2: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine glycine (A-38-3)

[0720] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine glycine tert-butyl ester (120 g, 282 mmol) in dichloromethane (1 L). Add trifluoroacetic acid (32.2 g, 282 mmol, 20.9 mL) and stir at 25°C for 2 hours. Concentrate the reaction mixture to obtain the crude title compound (130 g), which is used in the next step without purification.

[0721] Its structural characterization data are as follows:

[0722] MS m / z(ESI):369.0[M+H2O+H] +

[0723] Step 3: Preparation of (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetic acid methyl ester (A-38-4)

[0724] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine glycine (128 g, 347 mmol) in DMF (1 L). Add copper acetate (25.2 g, 138 mmol), lead tetraacetate (184 g, 416.96 mmol), and AcOH (45.9 g, 764 mmol, 43.7 mL). Heat to 60°C and stir for 18 hours. Add water (1.5 L) to the reaction mixture, and extract with ethyl acetate three times (1 L x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify on a silica gel column (CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and reconcentrate to obtain the title compound (70 g, 183.05 mmol).

[0725] Its structural characterization data are as follows:

[0726] MS m / z(ESI):400.0[M+H2O+H] +

[0727] Step 4: Preparation of (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (A-38-5)

[0728] Methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate (2.7 g, 7.06 mmol) and glycolic acid (1.61 g, 21.18 mmol) were dissolved in THF (50 mL). p-Toluenesulfonic acid (36.78 mg, 706.05 μmol) was added and reacted at 20°C for 4 hours. The organic phase was diluted with water and adjusted to pH 6 with 1N dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (MeOH / DCM = 5-10%) and concentrated again to obtain the title compound (1.83 g, 4.14 mmol).

[0729] Its structural characterization data are as follows:

[0730] MS m / z(ESI):399.1[M+H] +

[0731] Step 5: Preparation of (9H-fluoren-9-yl)methyl((S)-1-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)carbamate (A-38-6)

[0732] (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (200 mg, 0.50 mmol) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (275 mg, 0.50 mmol) were dissolved in DMF (10 mL), followed by the addition of DMTMM (295.91 mg, 1.00 mmol) and DIPEA (259.52 mg, 2.01 mmol), and the mixture was stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 70%) and freeze-dried to obtain the title compound (220 mg, 264.33 μmol).

[0733] Its structural characterization data are as follows:

[0734] MS m / z(ESI):832.3[M+H] +

[0735] Step 6: Preparation of (S)-2-amino-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)propionamide (A-38-7)

[0736] (9H-fluoren-9-yl)methyl((S)-1-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino) Dissolve 2-(1-oxopropyl)-2-yl)carbamate (220 mg, 0.26 mmol) in N,N-dimethylformamide (2 mL), add diethylamine (38.66 mg, 0.53 mmol), and allow to react at room temperature for 1 hour. The reaction solution is directly purified by reverse-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (120 mg, 196.70 μmol).

[0737] Its structural characterization data are as follows:

[0738] MS m / z(ESI):610.4[M+H] +

[0739] Step 7: Preparation of (9H-fluoren-9-yl)methyl((7S,10R,13S)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)carbamate (A-38-8)

[0740] (S)-2-amino-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)propanamide (120 mg, 0.20 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-D-alanine (97.79 mg, 0.26 mmol) were dissolved in DMF (10 mL), followed by the addition of DMTMM (115.95 mg, 0.39 mmol) and DIPEA (101.69 mg, 0.79 mmol). The atmosphere was purged with nitrogen three times, and the mixture was stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 60%) and freeze-dried to obtain the title compound (18 mg, 18.47 μmol).

[0741] Its structural characterization data are as follows:

[0742] MS m / z(ESI):974.4[M+H] +

[0743] Step 8: Preparation of (S)-2-amino-N-((R)-1-(((S)-1-(2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino (A-38-9)

[0744] Dissolve (9H-fluoren-9-yl)methyl ((7S,10R,13S)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)carbamate (18 mg, 0.018 mmol) in DMF (10 mL). Add diethylamine (2.70 mg, 0.037 mmol), replace the atmosphere with nitrogen three times, and stir at room temperature for 1 hour. The reaction solution was directly drained of the solvent under reduced pressure to obtain 18 mg of a crude product of the title compound.

[0745] Its structural characterization data are as follows:

[0746] MS m / z(ESI):752.4[M+H] +

[0747] Step 9: Preparation of N-((7S,10R,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-38)

[0748] (S)-2-amino-N-((R)-1-(((S)-1-(2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino (18 mg, 0.024 mmol), ( 2,5-Dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl)hexyl-5-ynoate (8.74 mg, 0.024 mmol) was dissolved in DMF (5 mL), and DIPEA (9.28 mg, 0.072 mmol) was added to the reaction mixture. The atmosphere was purged with nitrogen three times, and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain the title compound (10.64 mg, 10.61 μmol).

[0749] Its structural characterization data are as follows:

[0750] MS m / z(ESI):1002.3[M+H] +

[0751] The separation and purification method is as follows:

[0752] Column: SHIMADZU Prep C18 OBD (5μm*19mm*150mm)

[0753] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0754] Example 5: N-(((S)-1-(((S)-1-(2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-40)

[0755] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (A-40-2)

[0756] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (2 g, 5.23 mmol) was dissolved in THF (20 mL), and HOSu (722.29 mg, 6.28 mmol) and DCC (1.19 g, 5.75 mmol) were added. The mixture was stirred at room temperature for 3 hours, and solids gradually precipitated. After the reaction was completed, the reaction solution was directly filtered, and the filter cake was washed with an appropriate amount of THF. The filtrate was then dried to obtain the title compound as a crude product (3.4 g), which was used directly in the next reaction without purification.

[0757] Its structural characterization data are as follows:

[0758] MS m / z(ESI):497.2[M+H+H2O] +

[0759] Step 2: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine glycine (A-40-3)

[0760] Dissolve 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1.8 g, 3.75 mmol) and glycine (563.61 mg, 7.51 mmol) in acetone (12 mL) and water (6 mL). Then add NaHCO₃ (630.73 mg, 7.51 mmol) and stir at room temperature for 2 hours. Add water (20 mL) to the reaction mixture, adjust the pH to 3-4 with concentrated hydrochloric acid, and extract with ethyl acetate three times (15 mL x 3). The combined organic phases are washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to yield 1.2 g of the crude title compound, which is used directly in the next step without purification.

[0761] Its structural characterization data are as follows:

[0762] MS m / z(ESI):440.2[M+H] +

[0763] Step 3: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundec-11-yl acetate (A-40-4)

[0764] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine-glycine (1.2 g, 2.73 mmol) was dissolved in THF (20 mL), and pyridine (647.97 mg, 8.19 mmol) and lead tetraacetate (3.63 g, 8.19 mmol) were added. The system turned yellow, and after purging with nitrogen, the mixture was heated to 70°C and reacted for 4 hours. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The combined organic phases were washed sequentially with saturated aqueous ammonium chloride (10 mL) and saturated aqueous sodium chloride (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 50% to 100%) and concentrated again to obtain the title compound (346 mg, 762.97 μmol).

[0765] Its structural characterization data are as follows:

[0766] MS m / z(ESI):471.2[M+H2O+H] +

[0767] Step 4: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14 acid (A-40-5)

[0768] (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundec-11-yl acetate (80 mg, 176.41 μmol) was dissolved in THF (2 mL). Glycolic acid (67.08 mg, 882.05 μmol) was added, followed by TsOH (3.04 mg, 17.64 μmol). The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the solvent was removed by vacuum drying. The residue was purified by preparative HPLC and freeze-dried to obtain 45 mg of the title compound.

[0769] Its structural characterization data are as follows:

[0770] MS m / z(ESI):492.1[M+Na] +

[0771] The separation and purification method is as follows:

[0772] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0773] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0774] Step 5: Preparation of (9H-fluoren-9-yl)methyl((S)-1-((S)-1-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino (A-40-6)

[0775] (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradec-14-oic acid (25 mg, 42.60 μmol) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H 1-Benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (23.34 mg, 42.60 μmol) was dissolved in DMF (1.5 mL). DIPEA (11.01 mg, 85.20 μmol) and HATU (19.44 mg, 51.12 μmol) were added and stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction mixture was used directly in the next step without further treatment.

[0776] Its structural characterization data are as follows:

[0777] MS m / z(ESI):903.3[M+H] +

[0778] Step 6: Preparation of (S)-2-amino-N-((S)-1-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)propanamide (A-40-7)

[0779] To the reaction mixture containing (9H-fluoren-9-yl)methyl((S)-1-((S)-1-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino (38 mg, 42.06 μmol) in the previous step was added DMF (1 mL) and diethylamine (0.2 mL), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly evaporated to remove diethylamine under reduced pressure, and the residue was used directly in the next reaction.

[0780] Its structural characterization data are as follows:

[0781] MS m / z(ESI):699.3[M+H2O+H] +

[0782] Step 7: Preparation of N-(((S)-1-(((S)-1-(2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-40)

[0783] (S)-2-amino-N-((S)-1-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino 2-(1-(2-((2-(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((-((-1-OXOPROPAN-2-YL)propanamide (((28 mg, 41.11 μmol,((((((((((((((((((((((((((((((-(((-1-OXOPROPAN-2-YL)propanamide ((((2-( ...-1-OXOPROPAN-2-YL)propanamide ((((2-((((((((-1-OXOPROPAN-2-YL)propanamide ((((2-((((((((-1-OXOPROPAN-2-YL)propanamide ((((2-(((((((-1-OXOPROPAN-2-YL)propanamide ((((2-

[0784] Its structural characterization data are as follows:

[0785] MS m / z(ESI):931.3[M+H] +

[0786] The separation and purification method is as follows:

[0787] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0788] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0789] Example 6: N-(1-(1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatridec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-42)

[0790] Step 1: Preparation of 13-amino-6,9,12-trioxo-3-oxa-5,8,11-triazatridecanoic acid (A-42-1)

[0791] Dissolve 1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (50 mg, 0.1 mol) and diethylamine (0.5 mL) in DMF (2.5 mL) and stir at room temperature for 1 hour. Remove the diethylamine and use the crude product directly in the next step.

[0792] Step 2: Preparation of 20-(2-methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraaza-19-ynoic acid (A-42-2)

[0793] 2,5-Dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (71.42 mg, 195.48 μmol) was added to the reaction mixture and stirred at 25°C for 1 hour. Methyl tert-butyl ether (30 mL) was added to the reaction mixture, causing solid to precipitate. The crude product (50 mg) was filtered and used directly in the next step without purification.

[0794] Step 3: Preparation of N-(1-(1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatridec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-42)

[0795] 20-(2-Methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraaza-19-ynoic acid (50 mg) and 1S,9S-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 56.38 μmol) were dissolved in DMF (1 mL), followed by the addition of HATU (21.65 mg, 56.98 μmol) and N,N-diisopropylethylamine (21.23 mg, 164.23 μmol) and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain the title compound (11 mg, 11.36 μmol).

[0796] Its structural characterization data are as follows:

[0797] MS m / z(ESI):961.3[M+H] +

[0798] The separation and purification method is as follows:

[0799] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0800] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0801] Example 7: N-(R)-1-((R)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-carboxamide (A-44)

[0802] Step 1: Preparation of (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (A-44-1)

[0803] Methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate (60 g, 156 mmol) and benzyl 2-hydroxyacetate (52.1 g, 313 mmol, 44.5 mL) were dissolved in dichloromethane (500 mL). PPTS (19.71 g, 78.45 mmol) was added, and the mixture was heated to 50°C and stirred for 24 hours. Water (500 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (500 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (45 g, 92.11 mmol).

[0804] Its structural characterization data are as follows:

[0805] MS m / z(ESI):506.2[M+H2O+H] +

[0806] Step 2: Preparation of (S)-2-((2-aminopropionamido)methoxy)benzyl acetate (A-44-2)

[0807] Dissolve (S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (40 g, 81.88 mmol) in DMF (500 mL). Add DBU (9.97 g, 65.50 mmol, 9.87 mL) and stir at 25°C for 1 hour. The reaction mixture was used directly in the next step without further treatment.

[0808] Its structural characterization data are as follows:

[0809] MS m / z(ESI):267.1[M+H] +

[0810] Step 3: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14 acid benzyl ester (A-44-3)

[0811] (S)-2-((2-Aminopropionamido)methoxy)benzyl acetate (2.18 g, 8.19 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (2.78 g, 8.19 mmol) were dissolved in DMF (20 mL). EDCI (1.88 g, 9.82 mmol) and HOBt (1.33 g, 9.82 mmol) were added sequentially, and the mixture was stirred at 25°C for 1 hour. Water (300 mL) was added to the reaction solution, and a large amount of solid precipitated. The crude product was filtered to obtain the crude product. The crude product was slurried (added to a 3 / 1 mixture of petroleum ether and ethyl acetate (50 mL x 2) and stirred at 25°C for 8 hours) to obtain the title compound (4.50 g, 7.66 mmol).

[0812] Its structural characterization data are as follows:

[0813] MS m / z(ESI):610.4[M+Na] +

[0814] Step 4: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14 acid (A-44-4)

[0815] Benzyl (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradec-14-oate (2 g, 3.40 mmol) and 10% Pd / C (500 mg, 3.40 mmol) were added to DMF (20 mL). The mixture was replaced with hydrogen three times and then hydrogenated for 5 hours. The reaction mixture was filtered to obtain a crude product, which was then slurried in a 3 / 1 mixture of petroleum ether and ethyl acetate (30 mL x 2) and stirred at 25°C for 8 hours to obtain the title compound (1.45 g, 2.60 mmol).

[0816] Its structural characterization data are as follows:

[0817] MS m / z(ESI):515.1[M+H2O+H] +

[0818] Step 5: Preparation of (9H-fluoren-9-yl)methyl((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)carbamate (A-44-5)

[0819] (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (30 mg, 54.74 μmol) and (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8 1,4-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-1,4-dicarboxylic acid (29.96 mg, 60.22 μmol) was dissolved in N,N-dimethylformamide (1 mL). HATU (24.96 mg, 65.69 μmol) and N,N-diisopropylethylamine (21.23 mg, 164.23 μmol) were then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain the title compound (50 mg, 53.68 μmol).

[0820] The separation and purification method is as follows:

[0821] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0822] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0823] Step 6: Preparation of (S)-2-amino-N-((S)-1-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (A-44-6)

[0824] (9H-Fluoren-9-yl)methyl((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)carbamate (50 mg, 53.68 μmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (1 mL, 53.68 μmol) was added. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was preliminarily concentrated to obtain a DMF solution of the crude title compound, which was used directly in the next step without purification.

[0825] Step 7: Preparation of N-(R)-1-((R)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-carboxamide (A-44)

[0826] (S)-2-Amino-N-((S)-1-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (38 mg, 53.58 μmol) was dissolved in To DMF (1 mL), N,N-diisopropylethylamine (20.78 mg, 160.75 μmol) and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (39.15 mg, 107.16 μmol) were added sequentially and dissolved in the concentrate from the previous step. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain the title compound (5 mg, 5.11 μmol).

[0827] Its structural characterization data are as follows:

[0828] MS m / z(ESI):959.3[M+H]+

[0829] The separation and purification method is as follows:

[0830] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0831] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0832] Example 8: N-(((S)-1-(((S)-6-amino-1-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro))-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxohexan-2-yl)amino]-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamide (A-52)

[0833] Step 1: (N 6 Preparation of -((4-methoxyphenyl)diphenylmethyl)-L-lysine (A-52-2)

[0834] N 2 -((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 4-((4-Methoxyphenyl)diphenylmethyl)-L-lysine (10.0 g, 15.0 mmol) was dissolved in MeCN (50.0 mL). Diethylamine (5.86 g, 15.61 mmol, 10.0 mL) was added and stirred at 25°C for 1 hour. The reaction solution was concentrated to obtain the crude product, which was purified on a silica gel column (petroleum ether / ethyl acetate = 2:1; 100 mL x 2) and concentrated again to obtain the title compound (5.50 g, 13.1 mmol).

[0835] Its structural characterization data are as follows:

[0836] MS m / z(ESI):373.2[M-MMT+H] +

[0837] Step 2: N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of -((4-methoxyphenyl)diphenylmethyl)-L-lysine (A-52-3)

[0838] 2,5-Dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (6.88 g, 15.8 mmol) and (N6-((4-methoxyphenyl)diphenylmethyl)-L-lysine (5.50 g, 13.1 mmol) were dissolved in dichloromethane (100 mL). Triethylamine (2.66 g, 26.3 mmol, 3.66 mL) was added and stirred at 25°C for 1 hour. The pH of the reaction solution was adjusted to 3-4 with 1N dilute hydrochloric acid and extracted three times with dichloromethane (60.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (10.1 g, 12.3 mmol).

[0839] Its structural characterization data are as follows:

[0840] MS m / z(ESI):738.3[MH] -

[0841] Step 3: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-lysine (A-52-4)

[0842] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Dissolve 4-((4-Methoxyphenyl)diphenylmethyl)-L-lysine (2.50 g, 3.38 mmol) in dichloromethane (20.0 mL). Add a 4 M solution of hydrogen chloride in 1,4-dioxane (7.00 mL). Stir at 25°C for 2 hours. The reaction mixture was concentrated and the crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 1:1; 50.0 mL x 2) and concentrated again to obtain the hydrochloride salt of the title compound (1.80 g, 3.02 mmol).

[0843] Its structural characterization data are as follows:

[0844] MS m / z(ESI):466.2[MH] -

[0845] Step 4: N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of -((allyloxy)carbonyl)-L-lysine (A-52-5)

[0846] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-lysine (1.6 g, 2.69 mmol) and allyl chloroformate (971 mg, 8.06 mmol, 855 μL) in 1,4-dioxane (30.0 mL). Add 5% aqueous potassium carbonate solution and stir at 25°C for half an hour. Adjust the pH to 3 with 1N dilute hydrochloric acid. Add water (100 mL) and extract three times with ethyl acetate (60.0 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify on a silica gel column (dichloromethane / methanol = 100 / 1 to 20 / 1) and reconcentrate to obtain the title compound (1.2 g, 2.04 mmol).

[0847] Its structural characterization data are as follows:

[0848] MS m / z(ESI):552.2[M+H] +

[0849] Step 5: N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of -((allyloxy)carbonyl)-L-lysine-L-glycine tert-butyl ester (A-52-6)

[0850] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N6-((allyloxy)carbonyl)-L-lysine (1.20 g, 2.18 mmol) and glycine tert-butyl ester (342 mg, 2.61 mmol) were dissolved in DMF (20.0 mL). HOBT (881 mg, 6.53 mmol), EDCI (1.25 g, 6.53 mmol), and DIPEA (1.12 g, 8.70 mmol, 1.52 mL) were added sequentially, and the mixture was stirred at 25°C for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (70.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 20 / 1) and concentrated again to obtain the title compound (1.10 g, 1.49 mmol).

[0851] Its structural characterization data are as follows:

[0852] MS m / z(ESI):665.2[M+H] +

[0853] Step 6: N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6Preparation of -((allyloxy)carbonyl)-L-lysine-L-glycine (A-52-7)

[0854] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Tert-butyl 1-((allyloxy)carbonyl)-L-lysine-L-glycine ester (1.00 g, 1.50 mmol) was dissolved in dichloromethane (30.0 mL). Trifluoroacetic acid (12.3 g, 107 mmol, 8.00 mL) was added and stirred at 25°C for 3 hours. The reaction mixture was concentrated to obtain the crude title compound (1.10 g), which was used in the next step without purification.

[0855] Its structural characterization data are as follows:

[0856] MS m / z(ESI):609.2[M+H] +

[0857] Step 7: Preparation of (5S,8S)-8-(4-(allyloxy)carbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundec-11-yl acetate (A-52-8)

[0858] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N6-((allyloxy)carbonyl)-L-lysine-L-glycine (910 mg, 1.50 mmol) was dissolved in DMF (15.0 mL). Lead tetraacetate (795 mg, 1.79 mmol), AcOH (197 mg, 3.29 mmol, 188 μL), and copper acetate (108 mg, 598 μmol) were added. The mixture was heated to 60°C and stirred for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (70.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) and concentrated again to obtain the title compound (890 mg, 1.02 mmol).

[0859] Its structural characterization data are as follows:

[0860] MS m / z(ESI):645.4[M+Na] +

[0861] Step 8: Preparation of (5S)-8-(4-(((allyloxy)carbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-oic acid (A-52-9)

[0862] (5S,8S)-8-(4-(allyloxy)carbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundec-11-yl acetate (880 mg, 1.41 mmol) and 2-hydroxyacetic acid (214 mg, 2.83 mmol, 172 μL) were dissolved in dichloromethane (10.0 mL). 4-Methylbenzenesulfonic acid pyridinium chloride (284 mg, 1.13 mmol) was added, and the mixture was stirred at 50°C for 12 hours. The reaction mixture was concentrated under a nitrogen atmosphere to obtain the crude product, which was purified by preparative HPLC and freeze-dried to obtain the title compound (250 mg, 387 μmol).

[0863] Its structural characterization data are as follows:

[0864] MS m / z(ESI):656.4[M+H2O+H] +

[0865] The separation and purification method is as follows

[0866] Chromatographic column: Phenomenex luna (10μm*25mm*150mm)

[0867] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0868] Step 9: Preparation of (9H-fluoren-9-yl)methyl((S)-1-((((S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,13-trioxo-3,14-dioxa-5,12-diazaheptadecan-16-en-7-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (A-52-10)

[0869] (5S)-8-(4-(((allyloxy)carbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-oic acid (40 mg, 62.63 μmol), (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-Benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (34.32 mg, 62.63 μmol), DIPEA (40.47 mg, 313.13 μmol), and DMTMM (36.92 mg, 125.25 μmol) were added sequentially to DMF (2 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, water (12 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (8 mL x 3). The combined organic phases were washed once with saturated brine (6 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (63 mg, 55.21 μmol).

[0870] Its structural characterization data are as follows:

[0871] MS m / z(ESI):1072.4[M+H] +

[0872] Step 10: Preparation of (((S)-5-((S(2-amino-3-methylbutyramido))-6-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-6-oxohexyl)aminoallyl ester (A-52-11)

[0873] To DMF (2 mL) were added (9H-fluoren-9-yl)methyl ((S)-1-((((S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,13-trioxo-3,14-dioxa-5,12-diazaheptadecan-16-en-7-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (63 mg, 94% purity, 55.21 μmol) and diethylamine (32 mg, 437 μmol). The reaction was stirred at room temperature for 1 hour. After the reaction was completed, petroleum ether (6 mL*3) was added to extract the liquid. The organic phase was diluted with water (4 mL) and freeze-dried to obtain the title compound (30 mg, 35.28 μmol).

[0874] Its structural characterization data are as follows:

[0875] MS m / z(ESI):850.3[M+H] +

[0876] Step 11: Preparation of ((S)-6-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-(S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)butyramido)-6-oxohexyl)aminoallyl ester (A-52-12)

[0877] To DMF (2 mL) were added (((S)-5-((S(2-amino-3-methylbutanamido))-6-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy A mixture of 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (30 mg, 35.28 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (12.89 mg, 35.28 μmol), and DIPEA (9.12 mg, 70.56 μmol) was stirred at room temperature for 1 hour. Water (8 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (6 mL x 3). The combined organic phases were washed once with saturated brine (6 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to yield the title compound (38 mg, 31.07 μmol), which was used directly in the next step without purification.

[0878] Its structural characterization data are as follows:

[0879] MS m / z(ESI):1100.4[M+H] +

[0880] Step 12: Preparation of N-(((S)-1-(((S)-6-amino-1-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro))-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxohexan-2-yl)amino]-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamide (A-52)

[0881] To DCM (3 mL) was added ((S)-6-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl) A mixture of 5-(S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynylamino)butyramido)-6-oxohexyl)aminoallyl ester (20 mg, 18.17 μmol), tetrakistriphenylphosphine palladium (4.75 mg, 4.09 μmol), formic acid (20 μL), and N-methylmorpholine (40 μL) was reacted at room temperature for 1 hour under nitrogen. After completion of the reaction, the system was concentrated to obtain a crude product. The crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (13.41 mg, 12.66 μmol).

[0882] Its structural characterization data are as follows:

[0883] MS m / z(ESI):1016.3[M+H] +

[0884] The separation and purification method is as follows

[0885] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0886] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0887] Example 9: N-((10S,13S)-10-(4-aminobutyl)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-14-methyl-1,6,9,12-tetraoxo-5,8,11-triazapentadecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-54)

[0888] Step 1: Preparation of methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (A-54-2)

[0889] Dissolve (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (20.0 g, 56.5 mmol) in DMF (200 mL). Add AcOH (7.46 g, 124 mmol, 7.10 mL), lead tetraacetate (27.5 g, 62.2 mmol), and copper acetate (4.10 g, 22.6 mmol). Heat to 60°C and stir for 2 hours. Add water (500 mL) to the reaction mixture, and extract with ethyl acetate three times (200 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (16.0 g, 42.22 mmol).

[0890] Step 2: Preparation of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (A-54-3)

[0891] Methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (2.00 g, 5.43 mmol) was dissolved in dichloromethane (10.0 mL). PPTS (683 mg, 2.71 mmol) and glycolic acid (826 mg, 10.9 mmol, 661 μL) were added, and the mixture was heated to 43°C and stirred for 4 hours. The reaction mixture was filtered and concentrated to obtain the crude product, which was purified on a silica gel column (dichloromethane / methanol = 10 / 1) and concentrated again to obtain the title compound (1.48 g, 3.51 mmol).

[0892] Its structural characterization data are as follows:

[0893] MS m / z(ESI):385.1[M+H] +

[0894] Step 3: Preparation of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid allyl ester (A-54-4)

[0895] 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (1.1 g, 2.86 mmol) was dissolved in DMF (10.0 mL), potassium carbonate (593 mg, 4.29 mmol) and 3-bromoprop-1-ene (692 mg, 5.72 mmol) were added, and the mixture was stirred at 25°C for 6 hours. After the reaction solution was cooled to room temperature, water (10.0 mL) was added and extracted with ethyl acetate three times (10.0 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain 1.10 g of the crude title compound.

[0896] Its structural characterization data are as follows:

[0897] MS m / z(ESI):425.2[M+H] +

[0898] Step 4: Preparation of allyl 2-(2-aminoacetamido)methoxy)acetate (A-54-5)

[0899] 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid allyl ester (1.10 g) was dissolved in DMF (10 mL), and DBU (316 mg, 2.07 mmol, 313 μL) was added and stirred at 25 ° C for 1 hour. The reaction solution was used directly in the next step without purification. Step 5: Preparation of allyl (5S, 8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-acid ester (A-54-6)

[0900] To the DMF (10.0 mL) solution of allyl 2-(2-aminoacetamido)methoxy)acetate (524 mg, 2.59 mmol) in the previous step were added EDCI (746 mg, 3.89 mmol), HOBt (525 mg, 3.89 mmol) and N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 4-((4-methoxyphenyl)diphenylmethyl)-L-lysine (1.92 g, 2.59 mmol) was added and stirred at 25°C for 1 hour. Water (20.0 mL) was added to the reaction system, and a solid precipitated. This was filtered and the filter cake dried under vacuum to obtain the crude product. Purification on a silica gel column (dichloromethane / methanol = 20 / 1 to 10 / 1) followed by concentration yielded the title compound (1.20 g, 909 μmol).

[0901] Its structural characterization data are as follows:

[0902] MS m / z(ESI):924.4[M+H] +

[0903] Step 6: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (A-54-7)

[0904] Allyl (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oate (1.20 g, 1.30 mmol) was dissolved in THF (10.0 mL). Morpholine (226 mg, 2.60 mmol, 229 μL) and Pd(PPh3)4 (45.1 mg, 38.9 μmol) were added, and the mixture was stirred at 25°C for 1 hour. The pH of the reaction solution was adjusted to 2 with 1N dilute hydrochloric acid, and the mixture was extracted three times with dichloromethane (30.0 mL x 3). The combined organic phases were washed with saturated aqueous sodium chloride (50.0 mL) and dried over anhydrous sodium sulfate. The crude product was filtered and concentrated, which was purified by preparative HPLC and freeze-dried to give the title compound (358 mg, 388 μmol).

[0905] Its structural characterization data are as follows:

[0906] MS m / z(ESI):884.4[M+H] +

[0907] The separation and purification method is as follows

[0908] Chromatographic column: Phenomenex luna (10μm*50mm*250mm)

[0909] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0910] Step 7: Preparation of (9H-fluoren-9-yl)methyl((10S,13S)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-14-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)carbamate (A-54-8)

[0911] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (200 mg, 226.24 μmol), (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,1 2,15-Hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (130 mg, 237.55 μmol), DIPEA (146.20 mg, 1.13 mmol), and DMTMM (133.36 mg, 452.48 μmol) were added sequentially to DMF (3.5 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, water (12 mL) and ethyl acetate (12 mL) were added to the reaction mixture, which was then filtered through Celite. The aqueous phase was extracted twice with ethyl acetate (12 mL x 2). The combined organic phases were washed with saturated sodium chloride (6 mL), filtered, and concentrated to yield 324 mg of the crude title compound.

[0912] Its structural characterization data are as follows:

[0913] MS m / z(ESI):1317.6[M+H] +

[0914] Step 8: Preparation of (S)-2-((S)-2-amino-3-methylbutyramido)-N-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)-6-((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (A-54-9)

[0915] To DMF (3 mL) were added (9H-fluoren-9-yl)methyl((10S,13S)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-14-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)carbamate (278 mg, 210.94 μmol) and diethylamine (15 mg). The mixture was stirred at room temperature for 1 hour. Petroleum ether (4 mL*2) was added to extract the separated liquids, and the aqueous phase was concentrated under reduced pressure to remove diethylamine to obtain 180 mg of a crude product of the title compound.

[0916] Its structural characterization data are as follows:

[0917] MS m / z(ESI):1095.5[M+H] +

[0918] Step 9: Preparation of N-((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-(4-((((4-methoxyphenyl)diphenylmethyl)amino)butyl)-14-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazapentadecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-54-10)

[0919] To DMF (2 mL) were added (S)-2-((S)-2-amino-3-methylbutyramido)-N-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy After addition of 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynoate (70 mg, 191.59 μmol), and DIPEA (37.75 mg, 292.06 μmol), the reaction mixture was stirred at room temperature for 1 h. Water (15 mL) was added to the reaction system, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phase was washed once with saturated sodium chloride aqueous solution (6 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 156 mg of the crude title compound.

[0920] Its structural characterization data are as follows:

[0921] MS m / z(ESI):1359.5[M+H] +

[0922] Step 10: Preparation of N-((10S,13S)-10-(4-aminobutyl)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-14-methyl-1,6,9,12-tetraoxa-5,8,11-triazapentadecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-54)

[0923] To glacial acetic acid (2 mL) and water (0.5 mL) was added N-((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-(4-((((4-methoxyphenyl)diphenylmethane After addition, the mixture was heated to 45°C and reacted for 1 hour. The solvent was removed by concentration and the residue was directly purified by preparative HPLC and freeze-dried to obtain the title compound (72.72 mg, 58.78 μmol).

[0924] Its structural characterization data are as follows:

[0925] MS m / z(ESI):1073.4[M+H] +

[0926] The separation and purification method is as follows

[0927] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0928] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0929] Example 10: (S)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indol[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-4-((S)-2-((R)-4-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)pentanamido)-5-oxopentanoic acid (A-62)

[0930] Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine (A-62-2)

[0931] (((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine (1.5 g, 4.24 mmol) was dissolved in THF (15 mL), and HOSu (586.16 mg, 5.09 mmol) and DCC (1.05 g, 5.09 mmol) were added. Stirring was continued at room temperature for approximately 1 hour. The system gradually became turbid, and a white solid precipitated. After completion of the reaction, the reaction mixture was filtered, and the filter cake was washed with a small amount of THF. The mixture was then concentrated under reduced pressure to dryness to obtain 1.91 g of the crude title compound, which was used directly in the next reaction without purification.

[0932] Its structural characterization data are as follows:

[0933] MS m / z(ESI):468.3[M+H+H2O] + ,923.3[2M+Na] +

[0934] Step 2: Preparation of ((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine-L-alanine (A-62-3)

[0935] 5-Dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine (3.1 g, 6.88 mmol) and L-alanine (735.71 mg, 8.26 mmol) were dissolved in acetone (50 mL) and water (25 mL). NaHCO₃ (1.16 g, 13.76 mmol) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, water was added to the reaction solution, and the pH was adjusted to 3-4 with 2N hydrochloric acid. The resulting solution formed a milky white slurry, which was extracted twice with ethyl acetate (20 mL x 2). The organic phase was washed with brine and concentrated to obtain 2.92 g of the crude title compound, which was used directly in the next step without purification.

[0936] Its structural characterization data are as follows:

[0937] MS m / z(ESI):425.3[M+H] +

[0938] Step 3: Preparation of 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine-L-alanine (A-62-4)

[0939] ((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine-L-alanine (2.92 g, 6.88 mmol) and HOSu (950.00 mg, 8.25 mmol) were dissolved in THF (30 mL). DCC (1.70 g, 8.25 mmol) was added portionwise. A white solid gradually precipitated. After completion of the reaction, the reaction mixture was filtered, the filter cake washed with a small amount of THF, and then concentrated under reduced pressure to dryness to obtain 4.76 g of the crude title compound, which was used directly in the next reaction without purification.

[0940] Its structural characterization data are as follows:

[0941] MS m / z(ESI):439.3[M+H+H2O] +

[0942] Step 4: Preparation of 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid (A-62-6)

[0943] HOSu (674.62 mg, 5.86 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic acid (2 g, 4.88 mmol) were dissolved in THF (20 mL). DCC (1.21 g, 5.86 mmol) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was filtered, the filter cake was washed with a small amount of THF, and then concentrated under reduced pressure to obtain 2.47 g of the crude title compound, which was used directly in the next reaction without purification.

[0944] Its structural characterization data are as follows:

[0945] MS m / z(ESI):524.3[M+H+H2O] +

[0946] Step 5: Preparation of (S)-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine (A-62-7)

[0947] Dissolve 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid (2.47 g, 4.88 mmol) and glycine (732.13 mg, 9.75 mmol) in water (20 mL) and acetone (40 mL). Add NaHCO₃ (819.33 mg, 9.75 mmol) at room temperature, and stir at room temperature for 1 hour. After the reaction is complete, water is added to the reaction solution, and the pH is adjusted to 3-4 with 2N hydrochloric acid. The system forms a milky white slurry. Extract with ethyl acetate twice (50 mL x 2). The combined organic phases are washed with brine and concentrated to obtain 2.27 g of the crude title compound, which is used directly in the next reaction without purification.

[0948] Its structural characterization data are as follows:

[0949] MS m / z(ESI):467.3[M+H] +

[0950] Step 6: Preparation of (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine (A-62-8)

[0951] Dissolve (S)-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine (2.27 g, 4.87 mmol) in DMF (20 mL), add diethylamine (2 mL), and stir at room temperature for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain 1.19 g of the crude title compound, which was used directly in the next reaction without purification.

[0952] Its structural characterization data are as follows:

[0953] MS m / z(ESI):245.1[M+H] +

[0954] Step 7: Preparation of ((S)-2-((S)-2-((R)-2-(4-(9H-fluoro-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)propionamido)-5-(allyloxy)-5-oxopentanoyl)glycine (A-62-9)

[0955] Dissolve 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-D-leucine-L-alanine (3.07 g, 5.90 mmol) and (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine (1.2 g, 4.91 mmol) in DMF (30 mL). Add DIPEA (1.27 g, 9.83 mmol) and stir at room temperature for 1 hour. After completion of the reaction, add 60 mL of water and extract with ethyl acetate three times (30 mL x 3). The ethyl acetate extractions are then dried by spin drying. Add 30 mL of methyl tert-butyl ether, filter to remove insoluble matter, and concentrate the filtrate to dryness. The crude product is purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 100%) and concentrated again to obtain the title compound (2.8 g, 4.30 mmol).

[0956] Its structural characterization data are as follows:

[0957] MS m / z(ESI):651.3[M+H] +

[0958] Step 8: Preparation of allyl (5R, 8S, 11S) -11- ((acetoxymethyl) carbamoyl) -1- (9H-fluoren-9-yl) -5-isobutyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-oic acid (A-62-10)

[0959] ((S)-2-((S)-2-((R)-2-(4-(9H-fluoro-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)propionamido)-5-(allyloxy)-5-oxopentanoyl)glycine (2.8 g, 4.30 mmol) was dissolved in THF (30 mL) and toluene (10 mL). Pyridine (1.02 g, 12.91 mmol) and lead tetraacetate (5.72 g, 12.91 mmol) were then added. The atmosphere was replaced with nitrogen and the temperature was raised to 85°C and refluxed for 16 hours. After the reaction was complete, the reaction solution was cooled to room temperature and filtered to remove the white solid. 40 mL of water and 20 mL of ethyl acetate were added to the filtrate, but no solid precipitated. 20 mL of saturated brine was then added, resulting in the precipitation of a large amount of white solid, which was then removed by filtration. The filtrate was separated and extracted with ethyl acetate three times (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 50%) and concentrated again to obtain the title compound (720 mg, 1.08 mmol).

[0960] Its structural characterization data are as follows:

[0961] MS m / z(ESI):684.4[M+H+H2O]+ ,687.4[M+Na] +

[0962] Step 9: Preparation of (5R,8S,11S)-11-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-5-isobutyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (A-62-11)

[0963] Allyl (5R,8S,11S)-11-((acetoxymethyl)carbamoyl)-1-(9H-fluoren-9-yl)-5-isobutyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradec-14-oic acid (720 mg, 1.08 mmol) and glycolic acid (247.12 mg, 3.25 mmol) were dissolved in THF (10 mL). TsOH (37.30 mg, 216.62 μmol) was added, and the mixture was heated to 45°C and stirred for 16 hours. After completion of the reaction, the reaction solution was concentrated to dryness, 5 mL of methanol was added, and the insoluble material was removed by filtration. The filtrate was concentrated and purified on a reverse phase column (acetonitrile / 0.05% aqueous ammonium bicarbonate solution = 0% to 50%) to obtain the title compound (230 mg, 337.86 μmol).

[0964] Its structural characterization data are as follows:

[0965] MS m / z(ESI):698.3[M+H+H2O] +

[0966] Step 10: Preparation of allyl (5R, 8S, 11S) -11- ((2- (((1S, 9S) -5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyrano [3', 4': 6,7] indolizino [1,2-b] quinolin-1-yl) amino) -2-oxoethoxy) methyl) carbamoyl) -1- (9H-fluoren-9-yl) -5-isobutyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14 acid (A-62-12)

[0967] (5R,8S,11S)-11-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-5-isobutyl-8-methyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (37.27 mg, 54.74 μmol), (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3, 9,12,15-Hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (30 mg, 54.74 μmol) was dissolved in DMF (0.5 mL). DIPEA (42.45 mg, 328.46 μmol) was added, followed by DMTMM (48.40 mg, 164.23 μmol). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was used directly in the next step without further treatment.

[0968] Its structural characterization data are as follows:

[0969] MS m / z(ESI):1114.3[M+H] +

[0970] Step 11: Preparation of allyl (S)-4-((S)-2-((R)-2-amino-4-methylpentanamido)propionamido)-5-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolinyl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate formate (A-62-13)

[0971] The reaction mixture from the previous step containing allyl (5R,8S,11S)-11-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)carbamoyl)-1-(9H-fluoren-9-yl)-5-isobutyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradec-14-oic acid was transferred to a one-necked flask with dichloromethane (1 mL), and then diethylamine (1 mL) was added and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was evaporated under reduced pressure to remove diethylamine and dichloromethane. The residue was purified by reverse phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0% to 50%) and then freeze-dried to obtain the formate salt of the title compound (100 mg, 88.45 μmol).

[0972] Its structural characterization data are as follows:

[0973] MS m / z(ESI):892.4[M+H] +

[0974] Step 12: Preparation of (S)-4-((S)-2-((R)-2-amino-4-methylpentanamido)propionamido)-5-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoic acid formate (A-62-14)

[0975] Allyl (S)-4-((S)-2-((R)-2-amino-4-methylpentanamido)propionamido)-5-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizine[1,2-b]quinolinyl) (4-(amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate formate (71.31 mg, 79.91 μmol, formate) was dissolved in THF (6 mL), and morpholine (13.92 mg, 159.83 μmol) was added. After nitrogen purge, Pd(PPh3)4 (9.23 mg, 7.99 μmol) was added. The nitrogen purge was repeated three times, and the mixture was stirred at room temperature for 5 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain the title compound (25 mg, 27.27 μmol).

[0976] Its structural characterization data are as follows:

[0977] MS m / z(ESI):852.3[M+H] +

[0978] The separation and purification method is as follows:

[0979] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0980] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0981] Step 13: Preparation of (S)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-4-((S)-2-((R)-4-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)pentanamido)-5-oxopentanoic acid (A-62)

[0982] (S)-4-((S)-2-((R)-2-amino-4-methylpentanamido)propionamido)-5-(((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoyl Acid formate (7 mg, 7.79 μmol) was dissolved in DMF (1 mL), and DIPEA (2.01 mg, 15.58 μmol) was added, followed by 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (3.42 mg, 9.35 μmol). The mixture was stirred at room temperature for 4 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain the title compound (6 mg, 5.17 μmol).

[0983] Its structural characterization data are as follows:

[0984] MS m / z(ESI):1102.4[M+H] +

[0985] The separation and purification method is as follows:

[0986] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0987] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0988] Example 11: (S)-5-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)acetamido)-5-oxopentanoic acid (A-66)

[0989] Step 1: Preparation of (S)-5-(allyloxy)-2-amino-5-oxopentanoic acid (A-66-2)

[0990] Dissolve (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic acid (1.5 g, 3.66 mmol) in DMF (15 mL). Add diethylamine (2 mL) and stir at room temperature for 2 hours. After completion of the reaction, lyophilize to remove diethylamine and DMF to obtain the crude title compound, which is used directly in the next reaction without purification.

[0991] Its structural characterization data are as follows:

[0992] MS m / z(ESI):188.1[M+H] +

[0993] Step 2: Preparation of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycine (A-66-4)

[0994] Dissolve ((9H-fluoren-9-yl)methoxy)carbonyl)glycine (1 g, 3.36 mmol) in THF (30 mL) and add HOSu (425.81 mg, 3.70 mmol) and DCC (763.40 mg, 3.70 mmol). Stir at room temperature for approximately 1 hour. The system gradually becomes turbid, and a white solid precipitates. After completion of the reaction, the reaction mixture is filtered, the filter cake is washed with an appropriate amount of THF, and then concentrated under reduced pressure to dryness to obtain 1.27 g of the crude title compound, which is used directly in the next reaction without purification.

[0995] Its structural characterization data are as follows:

[0996] MS m / z(ESI):412.1[M+H2O+H] +

[0997] Step 3: Preparation of (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(allyloxy)-5-oxopentanoic acid (A-66-5)

[0998] (S)-5-(Allyloxy)-2-amino-5-oxopentanoic acid (680 mg, 3.63 mmol) was dissolved in DMF (15 mL). DIPEA (469.49 mg, 3.63 mmol) was added, followed by 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)glycine (1.43 g, 3.63 mmol). The mixture was stirred at room temperature for 2 hours. After completion, the reaction mixture was poured into 30 mL of water, and the pH was adjusted to 3-4 with 2N hydrochloric acid. The mixture was extracted three times with ethyl acetate (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1.5 g of the crude title compound, which was used directly in the next step without purification.

[0999] Its structural characterization data are as follows:

[1000] MS m / z(ESI):467.2[M+H] +

[1001] Step 4: Preparation of 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-glutamic acid (A-66-6)

[1002] (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(allyloxy)-5-oxopentanoic acid (1.69 g, 3.62 mmol) and HOSu (500.33 mg, 4.35 mmol) were suspended in dichloromethane (20 mL). DCC (896.99 mg, 4.35 mmol) was added and stirred at room temperature for 2 hours. A large amount of insoluble material appeared. After completion of the reaction, the reaction mixture was filtered to remove the insoluble material. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20% to 60%). The mixture was then concentrated again to obtain 1.38 g of the title compound.

[1003] Its structural characterization data are as follows:

[1004] MS m / z(ESI):564.2[M+H] + ,591.3[M+H2O+H] +

[1005] Step 5: Preparation of (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(allyloxy)-5-oxopentanoyl)glycylglycine (A-66-7)

[1006] Dissolve glycylglycine (154.73 mg, 1.17 mmol) in acetone (3 mL), add sodium bicarbonate (98.39 mg, 1.17 mmol), and then add 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-glutamate (330 mg, 585.57 μmol). Stir at room temperature for 2 hours. After completion of the reaction, pour the reaction solution into 10 mL of water, adjust the pH to 3-4 with 2N dilute hydrochloric acid, and extract with ethyl acetate three times (10 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (270 mg, 465.05 μmol), which is used directly in the next reaction without purification.

[1007] Its structural characterization data are as follows:

[1008] MS m / z(ESI):581.3[M+H] +

[1009] Step 6: Preparation of allyl (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((2-(acetoxymethyl)amino)-2-oxoethyl)amino)-5-oxopentanoate (A-66-8)

[1010] (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(allyloxy)-5-oxopentanoyl)glycylglycine (270 mg, 465.05 μmol) was dissolved in THF (3.0 mL) and toluene (1.0 mL), and pyridine (110.36 mg, 1.40 mmol) and lead tetraacetate (618.57 mg, 1.40 mmol) were added. The system turned yellow and the atmosphere was purged with nitrogen before heating to 70 ° C for 4 hours. After completion of the reaction, water (5 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed sequentially with saturated ammonium chloride (5 mL) and brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 50% to 100%) and concentrated again to obtain the title compound (120 mg, 201.8 μmol).

[1011] Its structural characterization data are as follows:

[1012] MS m / z(ESI):612.3[M+H2O+H] +

[1013] Step 7: Preparation of allyl (S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (A-66-9)

[1014] Allyl (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((2-(acetoxymethyl)amino)-2-oxoethyl)amino)-5-oxopentanoate (40 mg, 67.27 μmol) and glycolic acid (25.58 mg, 336.35 μmol) were dissolved in THF (1 mL), and p-toluenesulfonic acid (1.16 mg, 6.73 μmol) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, water (5 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (5 mL x 3). The combined organic phases were washed twice with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (30 mg, 39.30 μmol), which was used directly in the next reaction without purification.

[1015] Its structural characterization data are as follows:

[1016] MS m / z(ESI):609.3[MH] -

[1017] Step 8: Preparation of allyl (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-5-oxopentanoate (A-66-10)

[1018] Allyl (S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (30 mg, 39.30 μmol) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-Benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (21.54 mg, 39.30 μmol) was dissolved in DMF (1.5 mL). DIPEA (30.48 mg, 235.83 μmol) and DMTMM (34.75 mg, 117.91 μmol) were added and stirred at room temperature for 0.5 hour. After the reaction was complete, the reaction mixture was used directly in the next step without further treatment.

[1019] Its structural characterization data are as follows:

[1020] MS m / z(ESI):1045.4[M+H] +

[1021] Step 9: Preparation of allyl (S)-4-(2-aminoacetamido)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-5-oxopentanoic acid (A-66-11)

[1022] To the reaction mixture containing allyl (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-5-oxopentanoate (41 mg, 39.25 μmol) was added DMF (1.5 mL) and ethylenediamine (0.5 mL), and the mixture was stirred at room temperature for 10 min. After the reaction was completed, diethylamine was removed by rotary evaporation under reduced pressure. The residue was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (20 mg, 24.32 μmol).

[1023] Its structural characterization data are as follows:

[1024] MS m / z(ESI):840.3[M+H2O+H] +

[1025] Step 10: Preparation of (S)-4-(2-aminoacetamide)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-5-oxopentanoic acid (A-66-12)

[1026] Allyl (S)-4-(2-aminoacetamido)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-5-oxopentanoic acid (20 mg, 24.32 μmol) was dissolved in THF (1 mL), and morpholine (2.12 mg, 24.32 μmol) and Pd(PPh3)4 (2.81 mg, 2.43 μmol) were added. The atmosphere was replaced with nitrogen three times, and the reaction was carried out under nitrogen protection for 2 hours. After the reaction was completed, the reaction solution was directly dried under reduced pressure, dissolved in DMF, and filtered. The filtrate was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (3 mg, 3.84 μmol).

[1027] Its structural characterization data are as follows:

[1028] MS m / z(ESI):782.2[M+H] +

[1029] The separation and purification method is as follows:

[1030] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1031] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1032] Step 11: Preparation of (S)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)acetamido)-5-oxopentanoic acid (A-66)

[1033] (S)-4-(2-aminoacetamide)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino To DMF (1 mL) were added 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (3 mg, 3.84 μmol) and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (1.40 mg, 3.84 μmol), followed by the addition of DIPEA (991.38 μg, 7.67 μmol). The mixture was stirred at room temperature for 5 hours. After completion of the reaction, formic acid was added dropwise to the reaction solution until the system became weakly acidic at pH 5-6. The mixture was then purified by preparative HPLC and freeze-dried to obtain the title compound (0.7 mg, 0.67 μmol).

[1034] Its structural characterization data are as follows:

[1035] MS m / z(ESI):1034.3[M+H] +

[1036] The separation and purification method is as follows:

[1037] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1038] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1039] Example 12: (S)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamido)-5-oxopentanoic acid (A-68)

[1040] Step 1: Preparation of (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(allyloxy)-5-oxopentanoyl)glycine (A-68-1)

[1041] Dissolve glycine (87.91 mg, 1.17 mmol) in acetone (3 mL), add sodium bicarbonate (98.39 mg, 1.17 mmol), and then add 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-glutamate (330 mg, 585.57 μmol). Stir at room temperature for 2 hours. After completion of the reaction, pour the reaction solution into 10 mL of water, add 2N hydrochloric acid to adjust the pH to 3-4, and extract with ethyl acetate three times (10 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 230 mg of the crude title compound, which is used directly in the next reaction without purification.

[1042] Its structural characterization data are as follows:

[1043] MS m / z(ESI):524.3[M+H] +

[1044] Step 2: Preparation of allyl (S)-4-(2-(3-(9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((acetoxymethyl)amino)-5-oxopentanoate (A-68-2)

[1045] (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(allyloxy)-5-oxopentanoyl)glycine (230 mg, 439.32 μmol) was dissolved in THF (3 mL) and toluene (1 mL). Pyridine (104.25 mg, 1.32 mmol) and lead tetraacetate (584.35 mg, 1.32 mmol) were added. The atmosphere was purged with nitrogen and heated to 70°C for 4 hours. After completion of the reaction, 5 mL of water was added, and the mixture was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed sequentially with 5 mL of saturated ammonium chloride and 5 mL of brine. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 30% to 70%) to obtain the title compound (63 mg, 117.2 μmol).

[1046] Its structural characterization data are as follows:

[1047] MS m / z(ESI):555.3[M+H2O+H] +

[1048] Step 3: Preparation of (S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxy-2,12-dioxy-4,7,10-triazatetradecane-14 acid (A-68-3)

[1049] Allyl (S)-4-(2-(3-(9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((acetoxymethyl)amino)-5-oxopentanoate (30 mg, 55.81 μmol) and glycolic acid (21.22 mg, 279.04 μmol) were dissolved in THF (1 mL). p-Toluenesulfonic acid (961.02 μg, 5.58 μmol) was added and stirred at room temperature for 3 hours. Water (5 mL) was added to the reaction solution, which was then extracted three times with dichloromethane (5 mL x 3). The combined organic phases were washed twice with saturated brine (5 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (35 mg, 50.58 μmol), which was used directly in the next step without purification.

[1050] Its structural characterization data are as follows:

[1051] MS m / z(ESI):552.2[MH] -

[1052] Step 4: Preparation of allyl (S)-4-(2-aminoacetamide)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate (A-68-4)

[1053] (S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxy-2,12-dioxo-4,7,10-triazatetradec-14-oic acid (35 mg, 50.58 μmol) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,1 3H-Benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (27.72 mg, 50.58 μmol) was dissolved in DMF (1.5 mL). DIPEA (39.22 mg, 303.49 μmol) and DMTMM (44.72 mg, 151.75 μmol) were added and stirred at room temperature for 0.5 hours. After completion of the reaction, the reaction mixture was used directly in the next step without further treatment.

[1054] Its structural characterization data are as follows:

[1055] MS m / z(ESI):987.2[M+H] +

[1056] Step 5: Preparation of allyl (S)-4-(2-aminoacetamido)-5-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate (A-68-5)

[1057] To the reaction mixture containing allyl (S)-4-(2-aminoacetamide)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate (49 mg, 49.62 μmol) was added DMF (1.5 mL) and diethylamine (0.5 mL), and the mixture was stirred at room temperature for 10 minutes. After completion of the reaction, the reaction mixture was directly purified by reverse phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (30 mg, 39.21 μmol).

[1058] Its structural characterization data are as follows:

[1059] MS m / z(ESI):765.3[M+H] +

[1060] Step 6: Preparation of (S)-4-(2-aminoacetamido)-5-((2-((((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoic acid (A-68-6)

[1061] Allyl (S)-4-(2-aminoacetamido)-5-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-5-oxopentanoate (30 mg, 39.21 μmol) was dissolved in THF (1 mL), and morpholine (3.42 mg, 39.21 μmol) and Pd(PPh3)4 (4.53 mg, 3.92 μmol) were added. The atmosphere was replaced with nitrogen three times, and the reaction was carried out at room temperature under nitrogen protection for 2 hours. The reaction mixture was concentrated and the crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (3 mg, 4.14 μmol).

[1062] Its structural characterization data are as follows:

[1063] MS m / z(ESI):725.3[M+H] +

[1064] The separation and purification method is as follows:

[1065] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1066] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1067] Step 7: Preparation of (S)-5-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamido)-5-oxopentanoic acid (A-68)

[1068] (S)-4-(2-aminoacetamido)-5-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino To a solution of 1 mL of DMF, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (1.51 mg, 4.14 μmol) was added, followed by DIPEA (1.07 mg, 8.27 μmol). The mixture was stirred at room temperature for 5 hours. Formic acid was added dropwise to a slightly acidic pH of 5-6. The mixture was then purified by preparative HPLC and freeze-dried to afford the title compound (3.49 mg, 3.45 μmol).

[1069] Its structural characterization data are as follows:

[1070] MS m / z(ESI):975.2[M+H] +

[1071] The separation and purification method is as follows:

[1072] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1073] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1074] Example 13: (S)-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[D]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-1-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynyl)-L-serine-D-propylamino)pyrrolidine-2-carboxamide (A-72)

[1075] Step 1: Preparation of (2S)-3-hydroxy-2-(6-(2-methylsulfonylpyrimidin-5-yl)hexyl-5-ynylamino)propanoic acid (A-72-2)

[1076] (2,5-Dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl) hexyl-5-ynoate (200.0 mg, 0.547 mmol), L-serine (63.3 mg, 0.602 mmol) and DIPEA (106.1 mg, 0.821 mmol) were added to DMF (4 mL) in sequence and reacted at 25 ° C for 16 hours. The reaction solution was directly purified by C18 reverse phase column (H2O / ACN = 20-50%, 0.1% FA) and lyophilized to obtain compound 2 (110 mg, 294.06 μmol).

[1077] Its structural characterization data are as follows:

[1078] MS m / z(ESI):356.1[M+H] +

[1079] Step 2: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-proline (A-72-4)

[1080] (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propionic acid (2.00 g, 6.42 mmol), 1-hydroxypyrrolidine-2,5-dione (887.2 mg, 7.71 mmol) and DCC (1.59 g, 7.71 mmol) were added to DMF (30 mL) in sequence and reacted at 25°C for 2 hours. L-proline (738.6 mg, 6.42 mmol) was added all at once, and DIPEA (829.1 mg, 6.42 mmol) was added dropwise. The reaction was continued at 25°C for 4 hours. The reaction solution was poured into water (90 mL), and the insoluble solid was filtered off. The impurities in the filtrate were extracted with ethyl acetate (50 ml + 30 ml), and the pH of the aqueous phase was adjusted with hydrochloric acid. 3-4, and then extracted twice with ethyl acetate (100 ml + 50 ml). The combined organic phases were washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude title compound (2.40 g, 4.70 mmol), which was used directly in the next step without purification.

[1081] Its structural characterization data are as follows:

[1082] MS m / z(ESI):409.2[M+H] +

[1083] Step 3: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-alanylglycine (A-72-5)

[1084] (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-proline (1.60 g, 3.92 mmol), 1-hydroxypyrrolidine-2,5-dione (541.00 mg, 4.70 mmol), and DCC (969.90 mg, 4.70 mmol) were added to DMF (24 mL) and reacted at 25°C for 2 hours; glycine (352.8 mg, 4.70 mmol) was added, and DIPEA (506.20 mg, 3.92 mmol) was added dropwise, and the system was reacted at 25°C for 4 hours. The reaction solution was poured into water (80 mL), the insoluble solid was filtered off, and the impurities were extracted with ethyl acetate (80 mL). The aqueous phase was adjusted to pH 3 with hydrochloric acid, and then extracted with ethyl acetate (100 mL). After drying over anhydrous sodium sulfate, it was filtered and concentrated to give the crude product, which was purified on a silica gel column (MeOH / DCM = 5-15%) to give the title compound (560 mg, 1.08 mmol).

[1085] Its structural characterization data are as follows:

[1086] MS m / z(ESI):466.2[M+H] +

[1087] Step 4: Preparation of methyl ((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)-D-propylamino)pyrrolidine-2-carboxamido)acetate (A-72-6)

[1088] (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanyl-L-alanylglycine (560 mg, 1.20 mmol) and Pb(OAc)4 (1.17 g, 3.61 mmol) were dissolved in a mixed solvent of THF (15 mL) and toluene (5 mL), and pyridine (9.52 mg, 0.120 mmol) was added. The temperature was raised to 70°C and the mixture was refluxed for 4 hours. The mixture was filtered through celite and the filtrate was concentrated to obtain a crude product, which was purified by silica gel column chromatography (MeOH / DCM = 5-10%) to give the title compound (530 mg, 750.74 μmol).

[1089] Its structural characterization data are as follows:

[1090] MS m / z(ESI):502.2[M+Na] +

[1091] Step 5: Preparation of 2-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)-D-propylamino)pyrrolidine-2-carboxamido)methyl)acetic acid (A-72-7)

[1092] Compound A-72-6 (480 mg, 1.00 mmol) and glycolic acid (228.38 mg, 3.00 mmol) were dissolved in THF (10 mL), and p-toluenesulfonic acid (34.5 mg, 0.200 mmol) was added. The system was reacted at 25°C for 4 hours. The reaction solution was concentrated to obtain a crude product, which was purified by column chromatography (MeOH / DCM = 5-10%) and concentrated again to obtain the title compound (485 mg, 783.01 μmol).

[1093] Its structural characterization data are as follows:

[1094] MS m / z(ESI):496.2[M+H] +

[1095] Step 6: Preparation of (9H-fluoren-9-yl)methyl ((R)-1-((S)-2-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)carbamoyl)pyrrolidin-1-yl)-1-oxopropan-2-yl)carbamate (A-72-8)

[1096] (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (100 mg, 0.182 mmol) and 2-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)-D-propylamino)pyrrolidine-2-carboxamido)methyl)acetic acid (135.6 mg, 0.218 mmol) were added to DMF (2 mL), and DMTMM (100.99 mg, 0.365 mmol) was added, and DIPEA (70.8 mg, 0.547 mmol) was added dropwise. The reaction system was reacted at 25°C for 4 hours. Water (6 mL) was added to the reaction solution, extracted with ethyl acetate (20 mL), and concentrated to give a crude product, which was purified by preparative HPLC (MeOH / DCM = 5%) to give the title compound (37 mg, 39.81 μmol).

[1097] Its structural characterization data are as follows:

[1098] MS m / z(ESI):929.2[M+H] +

[1099] Step 7: Preparation of (S)-1-(D-alanyl)-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[D]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)pyrrolidine-2-carboxamide (A-72-9)

[1100] Under nitrogen protection, (9H-fluoren-9-yl)methyl((R)-1-((S)-2-((2-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl To the mixture of 1-hydroxy-2-nitropropane-1-yl)-2-nitropropane-2-yl)-1-nitropropane-3-yl)-1-nitropropane-3-yl)-1-nitropropane-4-yl)-1-nitropropane-5-yl)-1-nitropropane-6-yl)-1-nitropropane-7-yl)-1-nitropropane-8-yl)-1-nitropropane-1-yl)-1-nitropropane-2-yl)-1-nitropropane-3 ...1-yl)-1-nitropropane-2-yl)-1-nitropropane-3-yl)-1-nitropropane

[1101] Its structural characterization data are as follows:

[1102] MS m / z(ESI):707.3[M+H] +

[1103] The separation and purification method is as follows:

[1104] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1105] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1106] Step 8: Preparation of (S)-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[D]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-1-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynyl)-L-serine-D-propylamino)pyrrolidine-2-carboxamide (A-72)

[1107] (S)-1-(D-alanyl)-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[D]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)pyrrolidine-2-carboxamide (11.0 mg, 0.0156 mmol), (2S)-3-hydroxy- 2-(6-(2-methylsulfonylpyrimidin-5-yl)hexyl-5-ynylamino)propanoic acid (6.63 mg, 0.0187 mmol) was dissolved in DMF (0.5 mL), and HATU (11.8 mg, 0.0310 mmol) and DIPEA (6.00 mg, 0.0464 mmol) were added. The reaction system was reacted at 25°C for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (6.20 mg, 5.64 μmol).

[1108] Its structural characterization data are as follows:

[1109] MS m / z(ESI):1044.3[M+H] +

[1110] The separation and purification method is as follows:

[1111] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1112] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1113] Example 14: (S)-N-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-1-((4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)benzoyl)-D-alanyl)pyrrolidine-2-carboxamide (A-74)

[1114] Step 1: Preparation of 4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)benzoic acid (A-74-2)

[1115] (2,5-Dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl) hexyl-5-ynoate (200.0 mg, 0.547 mmol) and 4-(aminomethyl)benzoic acid (86.9 mg, 0.575 mmol) were added to DMF (4 mL), the temperature was raised to 30°C and the reaction was carried out for 16 hours. The reaction solution was poured into water (15 mL) and stirred for 1 hour. The precipitated solid was filtered, and the filter cake was washed with water (5 mL) and methyl tert-butyl ether (5 mL) in sequence, and then dried to give the title compound (169 mg, 399.94 μmol).

[1116] Its structural characterization data are as follows:

[1117] MS m / z(ESI):402.2[M+H] +

[1118] Step 2: Preparation of (S)-N-(2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-1-((4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)benzoyl)-D-alanyl)pyrrolidine-2-carboxamide (A-74)

[1119] Under nitrogen protection, (S)-1-(D-alanyl)-N-((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[D]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)pyrrolidine-2-carboxamide (16.0 mg, 0.0195 mmol), 4-(( 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)benzoic acid (9.4 mg, 0.0238 mmol) was added to DMF (0.5 mL), followed by HATU (14.8 mg, 0.039 mmol) and DIPEA (7.6 mg, 0.058 mmol). The reaction system was reacted at 25°C for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (6.00 mg, 5.34 μmol).

[1120] Its structural characterization data are as follows:

[1121] MS m / z(ESI):1090.3[M+H] +

[1122] The separation and purification method is as follows:

[1123] Column: Waters SunFire Prep C18 OBD (5μm*19mm*250mm)

[1124] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1125] Example 15: N-((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-76)

[1126] Step 1: Preparation of ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine glycylglycine (A-76-1)

[1127] Dissolve 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (1.0 g, 2.09 mmol) and glycylglycine (551.09 mg, 4.17 mmol) in acetone (12 mL) and water (6 mL). Then add NaHCO₃ (350.41 mg, 4.17 mmol) and stir at room temperature for 2 hours. Add water (20 mL) to the reaction mixture, adjust the pH to 3-4 with concentrated hydrochloric acid, and extract with ethyl acetate three times (15 mL x 3). The combined organic phases are washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford 3.4 g of the crude title compound, which is used directly in the next step without purification.

[1128] Its structural characterization data are as follows:

[1129] MS m / z(ESI):497.2[M+H] + ,515.2[M+H+H2O] +

[1130] Step 2: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradec-14-yl acetate (A-76-2)

[1131] ((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine glycylglycine (720 mg, 1.45 mmol) was dissolved in THF (10 mL). Pyridine (344.11 mg, 4.35 mmol) and lead tetraacetate (1.93 g, 4.35 mmol) were added. The system turned yellow. After purging with nitrogen, the mixture was heated to 70°C and reacted for 4 hours. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phase was washed sequentially with 10 mL of saturated ammonium chloride and 10 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 50% to 100%) to obtain the title compound (270 mg, 528.8 μmol).

[1132] Its structural characterization data are as follows:

[1133] MS m / z(ESI):528.2[M+H+H2O] +

[1134] Step 3: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (A-76-3)

[1135] (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9,12-tetraoxo-2-oxo-4,7,10,13-tetraazatetradec-14-yl acetate (80 mg, 156.70 μmol) was suspended in THF (2 mL). Glycolic acid (59.59 mg, 783.49 μmol) was added, followed by TsOH (2.70 mg, 15.67 μmol). The mixture was stirred at room temperature for 6 hours, and the insoluble matter gradually dissolved. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent. The concentrate was purified by preparative HPLC and freeze-dried to obtain 45 mg of the title compound.

[1136] Its structural characterization data are as follows:

[1137] MS m / z(ESI):535.3[M+Na] +

[1138] The separation and purification method is as follows:

[1139] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1140] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1141] Step 4: Preparation of ((9H-fluoren-9-yl)methyl((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-methyl-1,6,9,12-tetraoxo-5,8,11-triazatetradec-13-yl)carbamate (A-76-4)

[1142] (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (26.02 mg, 47.48 μmol) and (5S,8S)-1-(9H-fluoren-9-yl)- 5,8-Dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (25.00 mg, 47.48 μmol) was dissolved in DMF (1.5 mL). DIPEA (12.27 mg, 94.96 μmol) and HATU (21.66 mg, 56.98 μmol) were added and stirred at room temperature for 0.5 hour. After completion of the reaction, the reaction mixture was used directly in the next step without further treatment.

[1143] Its structural characterization data are as follows:

[1144] MS m / z(ESI):960.3[M+H] +

[1145] Step 5: Preparation of ((S)-2-amino-N-((S)-1-((2-((2-)(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)amino)-1-oxopropan-2-yl)propanamide (A-76-5)

[1146] To the reaction mixture containing ((9H-fluoren-9-yl)methyl((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-methyl-1,6,9,12-tetraoxo-5,8,11-triazatetradec-13-yl)carbamate (45 mg, 46.85 μmol) in the previous step was added DMF (1 mL) and diethylamine (0.2 mL), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was directly evaporated to remove diethylamine under reduced pressure, and the residue was used directly in the next reaction.

[1147] Its structural characterization data are as follows:

[1148] MS m / z(ESI):757.3[M+H+H2O] +

[1149] Step 6: Preparation of (N-((10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-76)

[1150] ((S)-2-amino-N-((S)-1-((2-((2-)(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2 To the mixture was dissolved 1-oxoethyl)amino)-1-oxopropan-2-yl)propionamide (34 mg, 46.06 μmol) in DMF (1 mL). DIPEA (5.95 mg, 46.06 μmol) was added, followed by 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (16.83 mg, 46.06 μmol). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the residue was directly purified by preparative HPLC and freeze-dried to obtain the title compound (17.24 mg, 16.35 μmol).

[1151] Its structural characterization data are as follows:

[1152] MS m / z(ESI):988.4[M+H] +

[1153] The separation and purification method is as follows:

[1154] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1155] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1156] Example 16: Preparation of N-((7S,10S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-70)

[1157] Step 1: Preparation of 2-(((S)-2-((S(-2-amino-3-methylbutyramido)propionamido)methoxy)benzyl acetate (A-70-1)

[1158] Dissolve (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradec-1-oic acid benzyl ester (2 g, 3.40 mmol) in DMF (20 mL). Add DBU (414 mg, 2.72 mmol, 410 μL) and stir at 25°C for 1 hour. The reaction mixture was used in the next step without purification.

[1159] Step 2: Preparation of (11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosanoic acid benzyl ester (A-70-2)

[1160] To the reaction mixture from the previous step, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (1.16 g, 3.28 mmol), EDCI (755 mg, 3.94 mmol), and HOBt (532 mg, 3.94 mmol) were added sequentially. Stir at 25°C for 2 hours. Water (100 mL) was added to the reaction mixture, and a solid precipitated. This solid was filtered. The filter cake was slurried (petroleum ether / ethyl acetate = 3 / 1, 40 mL x 2), filtered again, and dried to yield the title compound (2.2 g, 3.13 mmol).

[1161] Its structural characterization data are as follows:

[1162] MS m / z(ESI):719.4[M+H] +

[1163] Step 3: Preparation of (11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosanoic acid (A-70-3)

[1164] Benzyl (11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosanoate (2 g, 2.85 mmol) and 10% Pd / C (0.5 g) were added to DMF (30 mL) and the atmosphere was replaced with hydrogen three times (15 PSI). The reaction mixture was filtered, and water (30 mL) was added. The crude product was filtered, slurried (petroleum ether / ethyl acetate = 3 / 1, 40 mL x 2), filtered again, and dried to obtain the title compound (1.03 g, 1.51 mmol).

[1165] Its structural characterization data are as follows:

[1166] MS m / z(ESI):568.2[M+H] +

[1167] Step 4: Preparation of (7S,10S)-16-amino-10-isopropyl-7-methyl-6,9,12,15-tetraoxo-5,8,11,14-tetraazahexadecanoic acid (A-70-4)

[1168] Dissolve (11S,14S)-1-(9H-fluoren-9-yl)-11-isopropyl-14-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosateicosenoic acid (30 mg, 49.05 μmol) and diethylamine (0.5 mL) in DMF (2.5 mL) and stir at room temperature for 1 hour. Concentrate under reduced pressure to obtain the crude title compound, which is used in the next step without purification.

[1169] Step 5: Preparation of (7S,10S)-10-isopropyl-7-methyl-23-(2-methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazadocosa-22-ynoic acid (A-70-5)

[1170] To the reaction system from the previous step, (2,5-dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl) hexyl-5-ynoate (17.92 mg, 49.05 μmol) was added and stirred at 25°C for 1 hour. Methyl tert-butyl ether (30 mL) was added to the reaction solution, causing a large amount of solid to precipitate. The crude product (20 mg) was filtered and used directly in the next step without purification.

[1171] Step 6: Preparation of N-(7S,10S)-1-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzofurano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-imine (A-70)

[1172] (7S,10S)-10-isopropyl-7-methyl-23-(2-methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxa-3-oxa-5,8,11,14,17-pentaazadocosa-22-ynoic acid (20 mg, crude) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro- 10H,13H-Benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (17.13 mg, 31.27 μmol) was dissolved in DMF (1 mL). HATU (14.26 mg, 37.52 μmol) and N,N-diisopropylethylamine (12.12 mg, 93.80 μmol) were then added. The reaction was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC and freeze-dried to obtain the title compound (8.45 mg, 7.79 μmol).

[1173] Its structural characterization data are as follows:

[1174] MS m / z(ESI):1074.3[M+H] +

[1175] The preparation method is as follows:

[1176] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1177] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1178] Example 17: Preparation of N-((3S,8S,11S,14S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,8-11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazapentadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-36)

[1179] Step 1: Preparation of (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetic acid methyl ester (A-36-2)

[1180] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanineglycine (10.00 g, 27.15 mmol) and lead tetraacetate (26.49 g, 81.44 mmol) were dissolved in a mixed solvent of tetrahydrofuran (150 mL) and toluene (50 mL). Pyridine (214.7 mg, 2.71 mmol) was added, and the temperature was raised to 70°C and refluxed for 4 hours. The mixture was filtered through celite and the filtrate was concentrated to a residual amount of about 20 ml. A small amount of solid precipitated. Methyl tert-butyl ether (150 ml) was added and stirred for 1 hour. The mixture was filtered, and the filter cake was collected and dried to give the title compound (7.90 g, 18.59 mmol).

[1181] Its structural characterization data are as follows:

[1182] MS m / z(ESI):405.2[M+Na] +

[1183] Step 2: Preparation of (5S,10S)-1-(9H-fluoren-9-yl)-5,10-dimethyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid (A-36-3)

[1184] Methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate (500 mg, 1.31 mmol) and S-3-hydroxybutyric acid (204.2 mg, 1.96 mmol) were dissolved in tetrahydrofuran (10 mL), p-toluenesulfonic acid (45.0 mg, 0.261 mmol) was added, and the temperature was raised to 30°C for 6 hours. The crude product was concentrated to give a crude product, which was purified by column chromatography (MeOH / DCM = 5-10%) to give the title compound (260 mg, 0.488 mmol).

[1185] Its structural characterization data are as follows:

[1186] MS m / z(ESI):449.2[M+Na] +

[1187] Step 3: Preparation of (9H-fluoro-9-yl)methyl((S)-1-(S)-4-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-4-oxobutan-2-yl)oxy)methyl)amino)-1-oxopropyl-2-yl)carbamate (A-36-4)

[1188] (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (100.0 mg, 0.175 mmol, trifluoroacetate), (5S,10S)-1-(9H-fluoren-9-yl)-5,10-dimethyl-3,6-dioxo-2,9-dioxo-4,7-diazadecanoate Dioxane-12-oic acid (89.8 mg, 0.211 mmol) was dissolved in DMF (3 mL), and HATU (138.8 mg, 0.365 mmol) and DIPEA (68.0 mg, 0.526 mmol) were added. The mixture was reacted at 25°C for 1 hour. Water (10 ml) was added to the system, and the product was extracted with ethyl acetate (30 ml). The product was concentrated to obtain a crude product, which was purified by thin layer chromatography (MeOH / DCM = 5%) to give the title compound (40.0 mg, 0.037 mmol).

[1189] Its structural characterization data are as follows:

[1190] MS m / z(ESI):864.4[M+H] +

[1191] Step 4: Preparation of (S)-3-(((S)-2-aminopropionamido)methoxy)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (A-36-5)

[1192] (9H-Fluoro-9-yl)methyl((S)-1-(S)-4-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-4-oxobutan-2-yl)oxy)methyl)amino)-1-oxopropan-2-yl)carbamate (40.00 mg, 0.046 mmol) was dissolved in DMF (1 mL), and DIPEA (16.9 mg, 0.231 mmol) was added. The mixture was reacted at 25°C for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (11.0 mg, 0.017 mmol).

[1193] Its structural characterization data are as follows:

[1194] MS m / z(ESI):642.2[M+H] +

[1195] Its separation and purification method is as follows:

[1196] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1197] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate aqueous solution)

[1198] Step 5: Preparation of (9H-fluoren-9-yl)methyl((3S,8S,11S,14S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazapentadecan-14-yl)carbamate (A-36-6)

[1199] (S)-3-(((S)-2-Aminopropionamido)methoxy)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (11 mg, 0.017 nmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-L-alanine (7.9 mg, 0.20 mmol) were dissolved in DMF (1 mL), and HATU (13.0 mg, 0.034 mmol) and DIPEA (6.6 mg, 0.051 mmol) were added, and the mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC and freeze-dried to give the title compound (13.0 mg, 0.012 mmol).

[1200] Its structural characterization data are as follows:

[1201] MS m / z(ESI):1006.4[M+H] +

[1202] Its preparation method is as follows:

[1203] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1204] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% FA aqueous solution)

[1205] Step 6: Preparation of (S)-3-(((S)-2-((S-2-aminopropionamide)propionamido)methoxy)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (A-36-7)

[1206] (9H-fluoren-9-yl)methyl((3S,8S,11S,14S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,8,11-trimethyl-1,7 ,10,13-tetraoxo-4-oxa-6,9,12-triazapentadecan-14-yl)carbamate (13.0 mg, 0.012 mmol) was dissolved in DMF (0.5 mL), diethylamine (9.4 mg, 0.129 mmol) was added, and the reaction was carried out at 25 ° C for 1 hour; the reaction solution was directly purified by high performance liquid chromatography to obtain compound 7 (6.0 mg, 0.0069 mmol, formate).

[1207] Its structural characterization data are as follows:

[1208] MS m / z(ESI):784.3[M+H] +

[1209] Its separation and purification method is as follows:

[1210] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1211] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1212] Step 7: Preparation of N-((3S,8S,11S,14S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-3,8-11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazapentadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamide (A-36)

[1213] (S)-3-(((S)-2-((S-2-aminopropionamido)propionamido)methoxy)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)butanamide (6.0 mg, 0.0069 mmol) , formate), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (4.0 mg, 0.011 mmol) were dissolved in DMF (0.5 mL), DIPEA (1.9 mg, 0.014 mmol) was added, and the mixture was reacted at 25°C for 2 hours; the reaction solution was directly purified by preparative HPLC and freeze-dried to give the title compound (3.0 mg, 0.0028 mmol).

[1214] Its structural characterization data are as follows:

[1215] MS m / z(ESI):1034.3[M+H] +

[1216] Its preparation method is as follows:

[1217] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1218] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1219] Example 18: Preparation of N-(((S)-1-(((S)-1-(S)-4-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-4-oxobutan-2-yl(oxy)methyl)amino)-1-oxopropan-2-yl)amino-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-46)

[1220] Step 1: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-alanine-glycine (A-46-2)

[1221] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-alanine (1.00 g, 2.44 mmol), HOSU (336.5 mg, 2.92 mmol), and DCC (603.2 mg, 2.92 mmol) were added to DMF (15 mL), and the mixture was reacted at 25°C for 16 hours. Glycine (201.7 mg, 2.69 mmol) was added, and DIPEA (315.8 mg, 2.44 mmol) was added dropwise, and the mixture was reacted at 25°C for 5 hours. The reaction solution was poured into water (50 ml), and the insoluble solid was filtered out. The impurities were extracted with ethyl acetate (50 ml + 30 ml). The aqueous phase was adjusted to pH 3 with hydrochloric acid, and the product was extracted with ethyl acetate (80 ml + 50 ml). The organic phases were combined, washed with brine (10 ml), and concentrated to give the title compound (805 mg, 1.38 mmol).

[1222] Its structural characterization data are as follows:

[1223] MS m / z(ESI):499.5[M+Na] +

[1224] Step 2: Preparation of (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundec-11-yl acetate (A-46-3)

[1225] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-alanine glycine (750 mg, 1.60 mmol) and lead tetraacetate (1.57 g, 4.81 mmol) were dissolved in a mixed solvent of tetrahydrofuran (30 mL) and toluene (10 mL), pyridine (25.4 mg, 0.32 mmol) was added, and the temperature was raised to 70°C and refluxed for 4 hours; filtered through celite, and the filtrate was concentrated to obtain a crude product, which was purified by column chromatography (MeOH / DCM = 5-10%) and concentrated again to obtain the title compound (230 mg, 0.043 mmol).

[1226] Its structural characterization data are as follows:

[1227] MS m / z(ESI):504.2[M+Na] +

[1228] Step 3: Preparation of (5S,8S,13S)-1-(9H-fluoren-9-yl)-5-isopropyl-8,13-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecan-15-oic acid (A-46-4)

[1229] (5S,8S)-1-(9H-fluoren-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundec-11-yl acetate (210.0 mg, 0.436 mmol) and (S)-3-hydroxybutyric acid (68.1 mg, 0.654 mmol) were dissolved in tetrahydrofuran (5 mL), p-toluenesulfonic acid (15.02 mg,...

Claims

1. An antibody-drug conjugate having a structure shown in the formula Ab-[MLED]x, wherein: Ab is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases: M is a linker site with an antibody or antigen-binding fragment thereof; L is the structural fragment between the linkers M and E; E is a structural fragment connecting L and D; D is the cytotoxic drug fragment; X is 1 to 10.

2. The antibody-drug conjugate according to claim 1, wherein: M is selected from the following substituted or unsubstituted structural fragments:

3. The antibody-drug conjugate according to claim 1, wherein: M is selected from the following substituted or unsubstituted structural fragments:

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclic group (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, T hr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly, Glu-Gly-Gly-Phe-Gly, Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pr o, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gl y, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gl y, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkylene, amine, hydroxyl, carboxyl, acyl, -O-, -C 1-6 AlkyleneCO2H, -C 1-6 Alkylene SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 Alkyl)2, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-heterocycle, -C 1-6 Alkylene-heterocycle, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C 1-6 Alkyl)-SO3H, -CH2NHC 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylene -SO3H, -CH2N(C 1-6 Alkylene -SO3H)2, -CH2N + (C 1-6 Alkylene -SO3H)3, -CH2N + (C 1-6 Alkyl)2-C 1-6 Alkylene -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkylene -SO3H)3, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)3, -CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N + (C 1-6 Alkyl)2-CH2CO2H, -CH2N(C 1-6 Alkyl)-C 1-6 Alkylene -CO2H, -CH2N + (C 1-6 Alkyl)2-C 1-6 Alkylene -CO2H, glucose, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl, -CH2N(C 1-6 alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) r -C 1-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), -C 1-6 Alkylene-N(C 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C 1-6 alkyl)-NOTA, wherein r is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is an integer selected from 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; L is selected from the group consisting of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) of the following substituted or unsubstituted structural fragments: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Ly s, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) of the following groups, including but not limited to hydrogen, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkyl)2, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), wherein s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more (e.g., 1, 2, 3, 4 or 5) of the following groups: s is selected from an integer of 1-20, such as an integer of 1-15, 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, L is selected from a substituted or unsubstituted structural fragment consisting of one or more (eg, 1, 2, 3, 4 or 5) of the following groups: s is selected from an integer of 1-20, for example an integer of 1-15, 1-12, 3-12, 5-10, 8-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 8, 10.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein: E is a single bond, a substituted or unsubstituted -NH-CH2-, or a substituted or unsubstituted structural fragment selected from the following: Preferably, E is a single bond, a substituted or unsubstituted -NH-CH2-, Preferably, E is a single bond, substituted or unsubstituted -NH-CH2-, or 6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein: The cytotoxic drug is selected from anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors, RNA polymerase inhibitors and gene transcription inhibitors; Preferably, the microtubule inhibitor is an auristatin compound, a maytansine compound or an eribulin compound; the DNA intercalator is a pyrrolobenzodiazepine (PBD) compound, trabectedin or rubectedin; the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belototecan, rubitecan, diflomotecan, lurtotecan, karenitecin, gim atecan, Namitecan, Simmitecan, Chimmitecan, Silatecan or Elomotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogs, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); the RNA polymerase inhibitor is α-amanitin; the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters and analogs; Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is selected from the following compounds or isotope-labeled compounds thereof: Preferably, the cytotoxic drug is connected to E in the antibody-drug conjugate via the -OH, primary amino, secondary amine or tertiary amine group on the cytotoxic drug; Preferably, D is selected from:

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: -MLED can be formed from the following structures: A-1 to A-204 and B-1 to B-24, for example, by a substitution reaction (for example, by removing the methylsulfonyl structure thereon): wherein n is selected from an integer of 1-20, such as an integer of 1-12, 3-12, 5-15, 5-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: The antibody or antigen-binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL): (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 5 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 6 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 20 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 21 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 23 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 24 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (2) the following heavy chain variable region (VH) and / or light chain variable region (VL): (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 18 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 19 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 33 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 34 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 23 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 24 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL): (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 9 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 10 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (4) the following heavy chain variable region (VH) and / or light chain variable region (VL): (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

9. The antibody-drug conjugate according to claim 1 or 8, wherein: The antibody or antigen-binding fragment thereof comprises: (a) VH or a variant thereof as shown in SEQ ID NO: 1, and / or VL or a variant thereof as shown in SEQ ID NO: 2; or (b) VH or a variant thereof as shown in SEQ ID NO: 3, and / or VL or a variant thereof as shown in SEQ ID NO: 4; Wherein, the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

10. The antibody-drug conjugate according to claim 8 or 9, wherein: The antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:35 and a light chain constant region (CL) as shown in SEQ ID NO:

36.

11. The antibody-drug conjugate according to any one of claims 1, 8 to 10, wherein: The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 35, and a light chain comprising a VL sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 36; or (2) A heavy chain comprising the VH of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 35, and a light chain comprising the VL of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO:

36.

12. The antibody-drug conjugate of any one of claims 1 to 11, wherein M is linked to a sulfhydryl group (-SH) on Ab.

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein: The antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof according to claim 11; -MLED is selected from the structural compound shown in claim 7; x is 1 to 10; preferably, x is 1 to 9, and more preferably x is 5 to 8.

14. The antibody drug conjugate according to any one of claims 8 to 13, which is selected from: in, HA in each antibody drug conjugate represents an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase; n is an integer selected from 1-20, preferably n is an integer selected from 1-15, such as 1-12, 3-12, 5-15, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; in, It indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the linker.

15. A composition comprising one or more antibody drug conjugates according to any one of claims 1 to 14, wherein the DAR value (drug antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8 ~10, or 9~10, preferably 3-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.0; or, the composition has a DAR of about 6.0 to 9.0, preferably about 6.0-8.0, for example, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6 .53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6.73, about 6 .74, about 6.75, about 6.76, about 6.77, about 6.78, about 6.79, about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.9, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, about 7.1, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7. 16, about 7.17, about 7.18, about 7.19, about 7.2, about 7.21, about 7.22, about 7.23, about 7.24, about 7.25, about 7.26, about 7.27, about 7.28, about 7.29, about 7.3, about 7.31, about 7.32, about 7.33, about 7.34, about 7.35, about 7.36, about 7.3 7, about 7.38, about 7.39, about 7.4, about 7.41, about 7.42, about 7.43, about 7.44, about 7.45, about 7.46, about 7.47, about 7.48, about 7.49, about 7.5, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.5 8, about 7.59, about 7.6, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.7, about 7.71, about 7.72, about 7.73, about 7.74, about 7.75, about 7.76, about 7.77, about 7.78, about 7.79 , about 7.8, about 7.81, about 7.82, about 7.83, about 7.84, about 7.85, about 7.86, about 7.87, about 7.88, about 7.89, about 7.9, about 7.91, about 7.92, about 7.93, about 7.94, about 7.95, about 7.96, about 7.97, about 7.98, about 7.99, about 8.0, about 8.01, about 8.02, about 8.03, about 8.04, about 8.05, about 8.06, about 8.07, about 8.08, about 8.09, about 8.1, about 8.11, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.23, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33, about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, about 8.42, about 8 .43, about 8.44, about 8.45, about 8.46, about 8.47, about 8.48, about 8.49, about 8.5, about 8.51, about 8.52, about 8.53, about 8.54, about 8.55, about 8.56, about 8.57, about 8.58, about 8.59, about 8.6, about 8.61, about 8.62, about 8.63, about 8.64, about 8.65, about 8.66, about 8.67, about 8.68, about 8.69, about 8.7, about 8.71, about 8.72, about 8.73, about 8.74, about 8.75, about 8.76, about 8.77, about 8.78, about 8.79, about 8.8, about 8.81, about 8.82, about 8.83, about 8.84, about 8.85, about 8.86, about 8.87, about 8.88, about 8.89, about 8.9, about 8.91, about 8.92, about 8.93, about 8.94, about 8.95, about 8.96, about 8.97, about 8.98, about 8.99, about 9.

0. .

16. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 14, the composition according to claim 15, and one or more pharmaceutical excipients.

17. Use of the antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating Her2-expressing cancer.

18. The use of claim 17, wherein the cancer is selected from solid tumors or hematological malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, in particular lung adenocarcinoma) and urothelial carcinoma.

19. The antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16, for use in treating Her2-expressing cancer.

20. The antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16, for use in treating solid tumors or hematological malignancies; for example, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) or urothelial carcinoma.

21. A method for treating Her2-expressing cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of the antibody drug conjugate of any one of claims 1 to 14, the composition of claim 15, or the pharmaceutical composition of claim 16.

22. The method of treatment of claim 21, wherein the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma) and urothelial carcinoma.

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