Antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

Disclosed are an antibody-drug conjugate, a preparation method therefor, and pharmaceutical use thereof. The antibody-drug conjugate has a structure represented by a formula Ab-[M-L-E-D]x, and has a targeted killing effect on various cancers or tumors.

Description

Antibody-drug conjugates, their preparation methods and uses

[0001] This application is based on and claims priority to Chinese patent applications No. 202411554571.X, filed on November 1, 2024, and No. 202510163776.3, filed on February 14, 2025, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application relates to the field of targeted therapy, specifically to an antibody-drug conjugate, its preparation method, and its uses. Background Technology

[0003] Antibody-drug conjugates (ADCs) for cancer treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a cytotoxic agent that kills tumor cells), 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. The ADC then enters the cancer cells via endocytosis. The bioactive molecule is then released within the cancer cells, killing them by inhibiting microtubules or damaging their DNA, thereby minimizing damage to normal tissue cells.

[0004] ErbB family receptor tyrosine kinases are important mediators 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). Clinically, the anti-ErbB2 antibody trastuzumab (trade name Herceptin) is commonly used to treat breast cancer with high ErbB2 expression, but the clinical response rate is low. To improve treatment efficacy, in recent years, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansine (e.g., DM1), aurestatins (e.g., MMAE), or DNA topoisomerase I inhibitors (e.g., Dxd) have been used in clinical treatment (Trastuzumab emtansine, Disitamab vedotin, Trastuzumab deruxtecan).

[0005] With the widespread clinical application of the aforementioned ADC drugs in the treatment of Her2-expressing tumors, safety issues or drug resistance problems, including neurotoxicity, hematologic toxicity, 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, both Disitamab vedotin and Trastuzumab deruxtecan use maleimide linkers (MC) as the linker portion. Literature reports that under physiological conditions, MC linkers are prone to reverse Michael reaction and thiol exchange, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). Regarding the hydrophilicity and hydrophobicity of linkers, Disitamab vedotin and Trastuzumab deruxtecan used valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), respectively, which are relatively hydrophobic. Literature reports that the hydrophilicity of linkers significantly affects the hydrophilicity of ADCs, thereby affecting the aggregation, pharmacokinetics and toxicity of ADCs (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3.). In terms of the bioactive molecules that kill cancer cells, Trastuzumab emtansine uses the microtubule inhibitor DM1 as a cytotoxin, which results in a weak bystander effect when combined with a non-cleavable linker; Disitamab vedotin uses the aurorastin toxin MMAE as a bioactive molecule, which is prone to problems such as neurotoxicity accumulation after continuous and repeated use; and both of the above ADC drugs are non-site-specific random conjugations with a drug loading ratio (DAR) of about 4, resulting in poor homogeneity. Summary of the Invention

[0007] This application aims to improve upon the aforementioned problems in existing pharmaceutical technologies, specifically by providing a class of anti-Her2 antibody-drug conjugates for the treatment of Her2-expressing tumors. The antibody-drug conjugates exhibit good stability, homogeneity, hydrophilicity, efficacy, and safety.

[0008] This application relates to an antibody-drug conjugate and, by way of example, discloses an antibody-drug conjugate with the general formula Ab-[MLED] targeting trastuzumab. x The antibody-drug conjugate shown has the indicated structure. Results showed that the conjugate has a superior drug-to-antibody ratio (e.g., 6.5–8.5), and exhibits excellent binding activity and inhibitory effect on Her2-positive cells, demonstrating good targeted killing effects against Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, this 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.

[0009] Antibody-drug conjugates

[0010] In one aspect, this application provides an antibody-drug conjugate having the formula Ab-[MLED] x The structure shown is as follows, wherein:

[0011] Ab is an antibody or its antigen-binding fragment:

[0012] M is a linker that is attached to an antibody or its antigen-binding fragment;

[0013] L is the structure connecting connectors M and E;

[0014] E is a structure that connects L and D;

[0015] D represents the cytotoxic drug component;

[0016] x is between 1 and 10.

[0017] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or its antigen-binding fragment via the "-MLE-" structure shown in this application.

[0018] In some implementation schemes, Ab-[MLED] x Further, it is HA-(ZMLED)x, where HA-(Z-) represents the antibody or its antigen-binding fragment as described above, and -(Z-) represents the linking portion of the amino acid residues in the antibody or its antigen-binding fragment with M.

[0019] In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

[0020] In some implementations, Z is S, NH, or O.

[0021] In some implementations, M is selected from the following substituted or unsubstituted structures:

[0022] In some implementations, M is selected from the following substituted or unsubstituted structures:

[0023] In some implementations, M is selected from the following substituted or unsubstituted structures:

[0024] In some implementations, M is selected from the following substituted or unsubstituted structures:

[0025] In some embodiments, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic groups (e.g., substituted with one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., 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, 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 (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), Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:46), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:47)),

[0026] Each R' is independently composed of one or more of the following groups (e.g., 1, 2, 3, 4, 5, 6, or 7), including but not limited to hydrogen, C, etc. 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, C 1-6 Acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups 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-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6 Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -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 group 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 Alkylenes -CO2H, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 ethoxy (EO) units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), EDTA (ethylenediaminetetraacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA,

[0027] Where r is selected from integers from 1 to 20, such as 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 integers from 1 to 20, such as 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.

[0028] In some embodiments, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic groups (e.g., substituted with one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., 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, 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 (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), Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:46), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:47)),

[0029] Wherein R' is composed of one or more of the following groups (e.g., 1, 2, 3, 4, 5, 6 or 7), including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups 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-heterocyclic, -C 1- 6-alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1-6 Alkylene (-SO3H)3, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -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 group 3, -CH2NH-C(=O)C 1-6 Alkylene-N + (C 1-6 alkyl)3、-CH2N(C 1- 6alkyl)-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 Alkylenes -CO2H, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1- 6-alkyl)-C(=O)-(OCH2CH2) r -OC 1-6Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 ethoxy (EO) units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), EDTA (ethylenediaminetetraacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA, wherein r is selected from integers 1-20, such as 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 integers 1-20, such as 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.

[0030] In some embodiments, R' is composed of one or more of the following groups (e.g., 1, 2, 3, 4, 5, 6, or 7), including but not limited to hydroxyl, amino, glucosyl, galactosyl, glucuronic acid, galacturonic acid, DOTA, DOTAGA, Nota, EDTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA,

[0031] In some embodiments, R' is composed of one or more of the following groups (e.g., 1, 2, 3, 4, 5, 6, or 7), including but not limited to hydroxyl, amino, glucosyl, galactosyl, glucuronic acid, galacturonic acid, etc.

[0032] In some implementations, "-EDTA" refers to

[0033] In some implementations, "-NOTA" refers to

[0034] In some implementations, "-DOTA" refers to

[0035] In some implementations, "-DOTAGA" refers to

[0036] In some implementations, Lys(R') refers to the following structure

[0037] In some implementations, Glu(R') refers to the following structure

[0038] In some implementations, R' is selected from glucuronic acid, -DOTA, -CH2-N(CH3)-DOTA,

[0039] In some implementations, R' is selected from

[0040] In some implementations, s is denoted as n.

[0041] In some implementations, the structure of L contains segments selected from the following:

[0042] In some implementations, the structure of L contains segments selected from the following:

[0043] In some implementations, the structure of L contains segments selected from the following:

[0044] In some implementations, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: 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(R'). Where R' represents glucosyl, galactosyl, glucuronic acid, galacturonic acid, 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), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA, Where s is selected from integers from 1 to 20, such as 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.

[0045] In some implementations, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: 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(R'). Where R' represents glucosyl, galactosyl, glucuronic acid, galacturonic acid, 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), EDTA (ethylenediaminetetraacetic acid residue), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA, wherein s is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0046] In some embodiments, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: 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,

[0047] In some embodiments, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: 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,

[0048] In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

[0049] s is selected from integers from 1 to 20, such as 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.

[0050] In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

[0051] s is selected from integers from 1 to 20, such as 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.

[0052] In some embodiments, L is formed by linking one or more substituted or unsubstituted structures selected from group I with one or more substituted or unsubstituted structures selected from group II:

[0053] Group I:

[0054] Group II:

[0055] s is selected from integers from 1 to 20, such as 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.

[0056] In some embodiments, L is formed by connecting one or more substituted or unsubstituted structures selected from group II above, and one or more substituted or unsubstituted structures optionally selected from group I above.

[0057] In some implementation schemes, group I consists of composition.

[0058] In some implementation schemes, group I consists of composition.

[0059] In some implementation schemes, group I consists of composition.

[0060] In some implementation schemes, group II consists of

[0061] Composition; s is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, and 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0062] In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

[0063] s is selected from integers from 1 to 20, such as 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.

[0064] In some embodiments, L is selected from a substituted or unsubstituted structure consisting of one or more of the following groups:

[0065] s is selected from integers from 1 to 20, such as 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.

[0066] In some implementations, L is selected from the following substituted or unsubstituted structures:

[0067] (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ); s is selected from integers from 1 to 20, such as integers from 1 to 15, 1 to 12, 3 to 12, 5 to 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 5, 8, 10.

[0068] In some implementations, L is selected from the following substituted or unsubstituted structures:

[0069] (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ); s is selected from integers from 1 to 20, such as integers from 1 to 15, 1 to 12, 3 to 12, 5 to 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 5, 8, 10.

[0070] In some implementations, L is selected from the following substituted or unsubstituted structures:

[0071] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2-, or selected from the following substituted or unsubstituted structures:

[0072] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2-,

[0073] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2- or

[0074] In some implementations, E is a single bond, -NH-CH2- or

[0075] In some implementations, E is substituted or unsubstituted -NH-CH2- or

[0076] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0077] n is an integer selected from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, 8 to 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 5, 8 or 10.

[0078] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0079] (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example );

[0080] s is selected from an integer from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, or an integer from 8 to 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 5, 8, or 10.

[0081] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0082] (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example );

[0083] s is selected from an integer from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, or an integer from 8 to 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 5, 8, or 10.

[0084] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0085] In some embodiments, the cytotoxic agent is selected from microtubule inhibitors, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors. In some embodiments, the microtubule inhibitor is an oliguriatin class of compounds, maytansine class of compounds, hammetrine class of compounds, or eribulin class of compounds. In some embodiments, the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) class of compounds, anthracycline class of compounds, trabectedin or rupettedin, or their derivatives or analogues. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, ixenonotecan, topotecan, belototecan, rubotecan, diflomotecan, lurtotecan, Karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, 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 analogues.

[0086] In some embodiments, the cytotoxic agent is selected from topoisomerase I inhibitors (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, ixotecan, topotecan, belototecan, rubotecan, diflomotecan, lurtotecan, Karenitecin, gimatecan, namitecan, simmitecan, chimmitecan, silatecan, or elomotecan).

[0087] The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amino (-NR1H), tertiary amino (-NR2R3), or mercapto (-SH), wherein R1, R2, and R3 represent only non-hydrogen substituents on N. These functional groups can react with suitable functional groups in the remainder of the conjugate to achieve linkage.

[0088] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary or tertiary amino group, or -SH group. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amino group on the cytotoxic drug.

[0089] In some embodiments, the cytotoxic agent is selected from compounds with the following structures or pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0090] in,

[0091] Existence or non-existence;

[0092] R1 is either fluorine or chlorine;

[0093] R2 is selected from methyl, chlorine, hydroxyl, or amino;

[0094] Alternatively, R1 and R2 form with the bonded carbon atoms

[0095] Each R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, C 1-6 Alkoxy or C 1- 6-Hydroalkyl group, or two R3 atoms on adjacent atoms connected to the linked atoms to form a ring;

[0096] Ring A is selected from:

[0097] X is selected from oxygen or sulfur;

[0098] m is selected from 1-5;

[0099] when When R1 is fluorine, R2 is methyl, R3 is hydrogen, and X is oxygen, ring A is not morpholino.

[0100] In some embodiments, the cytotoxic agent is a compound with the following structure or a pharmaceutically acceptable salt, stereoisomer, or isotopically labeled compound thereof:

[0101] in,

[0102] R1 is either fluorine or chlorine;

[0103] R2 is selected from methyl, chlorine, hydroxyl, or amino;

[0104] Alternatively, R1 and R2 form with the bonded carbon atoms

[0105] Each R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, C 1-6 Alkoxy or C 1- 6-Hydroalkyl group, or two R3 atoms on adjacent atoms connected to the linked atoms to form a ring;

[0106] Ring A is selected from: Preferred

[0107] X is selected from oxygen or sulfur;

[0108] m is selected from 1-5;

[0109] When R1 is fluorine, R2 is methyl, R3 is hydrogen, and X is oxygen, ring A is not morpholino.

[0110] In some embodiments, X is oxygen in the compound of formula (I) or formula (II) above.

[0111] In some embodiments, X is sulfur in the compound of formula (I) or formula (II) above.

[0112] In some embodiments, in the compounds of formula (I) or formula (II) above, each R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, C 1-6 Alkoxy or C1-6 Halogenated alkyl groups, or two R3 atoms on adjacent atoms connected to a 3-6 membered carbon ring or a 3-6 membered heterocycle.

[0113] In some embodiments, in the compounds of formula (I) or formula (II) above, each R3 is independently selected from hydrogen, C 1-4 Alkyl or halogen.

[0114] In some embodiments, in the compounds of formula (I) or formula (II) above, each R3 is independently selected from hydrogen, methyl, ethyl, isopropyl, fluorine, chlorine, bromine or iodine.

[0115] In some embodiments, in the compounds of formulas (I) to (II) above, ring A is selected from...

[0116] In some embodiments, in the compounds of formula (I) or formula (II) above, ring A is selected from... Preferred

[0117] In some embodiments, in the compounds of formula (I) or formula (II) above, Selected from

[0118] In some embodiments, in the compounds of formula (I) or formula (II) above, Selected from Preferred

[0119] In other embodiments, the cytotoxic agent is selected from compounds with the following structures or their pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0120] Among them, R5 and R6 are each independently selected from -H, -OH, -NH2, and -NH(C) 1-6 Alkyl), C 1-6 Alkyl and halogen; the C 1-6 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6 Haloalkyl, C 3-6 The cycloalkyl group is substituted; or, R5 and R6 together with the adjacent carbon atoms (connected to the cycloforming carbon atoms of R5 and R6, respectively) form a five-membered oxygen-containing heterocycle.

[0121] R7 is independently selected from -H, halogen, -OH, -NH2, -NH(C) 1-6alkyl), and -NH-CO-(C 1-6 alkylene)-OH; the C 1-6 Alkyl and C 1-6 Alkylene is optionally further subjected to one or more elements selected from halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0122] p is 1 or 2;

[0123] q is 0 or 1;

[0124] X represents oxygen or sulfur;

[0125] Y represents carbon or oxygen.

[0126] In some implementations, R5 and R6 are each independently selected from -H, -OH, -NH2, and C. 1-4 Alkyl and halogen; the C 1-4 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-4 Haloalkyl, C 3-6 The cycloalkyl groups are substituted. In some embodiments, R5 and R6 together with adjacent carbon atoms (connected to the cycloforming carbon atoms of R5 and R6, respectively) form a five-membered oxygen-containing heterocycle.

[0127] In some implementations, R7 is selected from H, halogens, -NH2, -NH(C) 1-4 alkyl), and -NH-CO-(C 1-4 alkylene)-OH; the C 1-4 Alkyl and C 1-4 Alkylene is optionally further subjected to one or more elements selected from halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3- It is substituted by a 6-cycloalkyl substituent.

[0128] In some implementations, R5 and R6 are each independently selected from -H, -OH, -NH2, and C. 1-6 Alkyl and halogen;

[0129] R7 is selected from -H, halogen, -NH2, -NH(C) 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; the C 1-6 Alkylene is optionally surrounded by hydroxyl, C 1-4 Alkyl, C 3-6 Cycloalkyl substitution;

[0130] Y represents carbon; p is 1; and q is 0.

[0131] In some implementations, X in Formula III is sulfur.

[0132] In some implementations, R5 and R6 are each independently selected from -H, -OH, -NH2, and C. 1-4 Alkyl groups and halogens.

[0133] In some implementations, R7 is selected from -H, -F, -NH2, -NH-CO-(C 1-4 alkylene)-OH, wherein the C 1-4 Alkylene is optionally surrounded by hydroxyl, C 1-4 Alkyl, C 3-6 Cycloalkyl substitution. In some embodiments, R7 is selected from -H, -F, -NH2, -NH-CO-CH2-OH, -NH-CO-CH(CH3)-OH, -NH-CO-CH(cyclopropyl)-OH, -NH-CO-C(CH3)2CH2-OH, and -NH-CO-CH(OH)CH2-OH.

[0134] In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds:

[0135] In some embodiments, the cytotoxic drug is selected from compound 3-1 or its pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds.

[0136] In some embodiments, the cytotoxic agent is selected from compound 5-1 or its pharmaceutically acceptable salt, stereoisomer, or isotopically labeled compound.

[0137] In some embodiments, the cytotoxic drug is selected from compound 7-1 or its pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds.

[0138] In some embodiments, the cytotoxic agent is selected from compounds 1-1, 1-2, or pharmaceutically acceptable salts, stereoisomers, or isotopically labeled compounds thereof.

[0139] In some embodiments, the cytotoxic drug is selected from compound 2-2 or its pharmaceutically acceptable salt, stereoisomer, or isotopically labeled compound.

[0140] In some implementations, D is selected from the following structures:

[0141] Among them, R 2ASelected from -O- or -N-;

[0142] R1, R2, R3, ring A, m, and X are as defined in the preceding term.

[0143] In some implementations, D is selected from the following structures:

[0144] Where 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 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0145] R5, R6, R7, X, Y, p, and q are defined as any of the above.

[0146] In some implementation schemes, R 7A Selected from chemical bonds, -NH-, and -NH-CO-(C 1-4 (alkylene)-O-; the C 1-4 The alkylene group is optionally further divided by one or more molecules selected from hydroxyl, C 1-4 Alkyl, C 3-6 The cycloalkyl group is substituted. In some embodiments, R 7A Selected from chemical bonds, -NH-, -NH-CO-CH2-O-, -NH-CO-CH(CH3)-O-, -NH-CO-CH(cyclopropyl)-O-, -NH-CO-C(CH3)2CH2-O-, -NH-CO-CH(OH)CH2-O-.

[0147] In some implementations, D is selected from the following structures:

[0148] In some implementations, D is selected from the following structures:

[0149] In some implementations, D is selected from the following structures:

[0150] In some embodiments, the 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 receptor tyrosine kinase family.

[0151] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0152] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0153] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0154] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0155] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0156] or,

[0157] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0158] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0159] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0160] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0161] or,

[0162] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0163] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0164] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0165] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0166] or,

[0167] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0168] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0169] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0170] 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 with respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions.

[0171] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0172] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0173] (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,

[0174] (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;

[0175] or,

[0176] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0177] (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,

[0178] (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;

[0179] or,

[0180] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0181] (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,

[0182] (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;

[0183] or,

[0184] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0185] (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,

[0186] (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;

[0187] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0188] (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

[0189] (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;

[0190] 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 with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0191] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0192] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0193] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0194] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0195] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0196] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (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).

[0197] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0198] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0199] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0200] In some embodiments, the antibody or its antigen-binding fragment 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.

[0201] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0202] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0203] (2) A heavy chain comprising the VH of the 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 of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0204] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0205] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0206] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0207] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or may lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0208] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0209] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments 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, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0210] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0211] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0212] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

[0213] In some implementations, the Ab is selected from antibodies or antigen-binding fragments thereof that specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0214] In some implementations, Ab is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or antigen-binding fragment thereof constructed from trastuzumab and pertuzumab.

[0215] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0216] In some embodiments, the antibody-drug conjugate has a structure as shown in formula IV-A or IV-A':

[0217] Wherein HA-(S-) is the antibody or its antigen-binding fragment as described in any of the preceding descriptions, preferably trastuzumab, -(S-) is the specific connection mode between the thiol group from the antibody or its antigen-binding fragment and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and M, L, and E are as defined in any of the preceding descriptions.

[0218] In some embodiments, the antibody-drug conjugate has a structure as shown in formula IV-A1 or IV-A2:

[0219] Wherein HA-(S-) is the antibody or its antigen-binding fragment as described in any of the preceding descriptions, preferably trastuzumab, -(S-) is the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and L and E are as defined in any of the preceding descriptions.

[0220] In some embodiments, the antibody-drug conjugate has a structure as shown in formula IV-B1 or formula IV-B2:

[0221] Wherein HA-(S-) is the antibody or its antigen-binding fragment as described in any of the preceding descriptions, preferably trastuzumab, -(S-) is the specific connection mode between the thiol group from the antibody or its antigen-binding fragment and M, x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8, and M, L, and E are as defined in any of the preceding descriptions.

[0222] In some embodiments, the antibody-drug conjugate is selected from ADC B-1 to ADC B-21 and ADC D-1 to ADC D-15: ADC B-1:

[0223] ADC B-2:

[0224] ADC B-3:

[0225] ADC B-4:

[0226] ADC B-5:

[0227] ADC B-6:

[0228] ADC B-7:

[0229] ADC B-8:

[0230] ADC B-9:

[0231] ADC B-10:

[0232] ADC B-11:

[0233] ADC B-12:

[0234] ADC B-13:

[0235] ADC B-14:

[0236] ADC B-15:

[0237] ADC B-16:

[0238] ADC B-17:

[0239] ADC B-18:

[0240] ADC B-19:

[0241] ADC B-20:

[0242] ADC B-21:

[0243] ADC D-1:

[0244] ADC D-2:

[0245] ADC D-3:

[0246] ADC D-4:

[0247] ADC D-5:

[0248] ADC D-6:

[0249] ADC D-7:

[0250] ADC D-8:

[0251] ADC D-9:

[0252] ADC D-10:

[0253] ADC D-11:

[0254] ADC D-12:

[0255] ADC D-13:

[0256] ADC D-14:

[0257] ADC D-15:

[0258] Wherein HA-(S-) refers to any of the antibodies or antigen-binding fragments described above;

[0259] This indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0260] In some implementations, x in the coupling shown by Ab-[MLED]x is 1-10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 6-8.

[0261] In some implementations, x is an integer.

[0262] In some implementations, x in the coupling shown as Ab-[MLED]x is approximately 8.

[0263] In some implementations, the antibody-drug conjugate is selected from ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, and ADC B-12a.

[0264] ADC B-1a:

[0265] ADC B-2a:

[0266] ADC B-5a:

[0267] ADC B-6a:

[0268] ADC B-9a:

[0269] ADC B-11a:

[0270] ADC B-12a:

[0271] Wherein HA-(S- is the antibody or its antigen-binding fragment as described in any of the preceding items, and -(S- is derived from the specific connection between the thiol group in the antibody or its antigen-binding fragment and the pyrimidine group in ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, ADC B-12a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

[0272] In some embodiments, the antibody-drug conjugate is selected from ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, and ADC B-12a. HA-(S-) comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4. -(S-) represents the specific linkage between the thiol group in the antibody or its antigen-binding fragment and the pyrimidine group in ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, and ADC B-12a, where x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8. In some embodiments, -(S-) represents the thiol group of the cysteine ​​residue in the antibody or its antigen-binding fragment and the pyrimidine group in ADC B-1a, ADC B-2a, ADC B-5a, and ADC B-12a. The specific connection method of the pyrimidine group in B-6a, ADC B-9a, ADC B-11a, and ADC B-12a.

[0273] In some embodiments, the antibody-drug conjugate is selected from ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, and ADC B-12a. HA-(S-) comprises: a heavy chain including the VH shown in SEQ ID NO:1 and the heavy chain constant region (CH) shown in SEQ ID NO:35, and a light chain including the VL shown in SEQ ID NO:2 and the light chain constant region (CL) shown in SEQ ID NO:36; or, a heavy chain including the VH shown in SEQ ID NO:3 and the heavy chain constant region (CH) shown in SEQ ID NO:35, and a light chain including the VL shown in SEQ ID NO:4 and the light chain constant region (CL) shown in SEQ ID NO:36. -(S-) represents the thiol group in the antibody or its antigen-binding fragment and its association with ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, and ADC B-12a. The specific connection mode of the pyrimidine group in B-12a is x, which can be 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

[0274] In some implementations, the antibody-drug conjugate is selected from ADC B-1b, ADC B-2b, ADC B-5b, ADC B-6b, ADC B-9b, ADC B-11b, and ADC B-12b.

[0275] ADC B-1b:

[0276] ADC B-2b:

[0277] ADC B-5b:

[0278] ADC B-6b:

[0279] ADC B-9b:

[0280] ADC B-11b:

[0281] ADC B-12b:

[0282] Where Ab is an antibody or its antigen-binding fragment as described in any of the preceding items, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

[0283] In some embodiments, the antibody-drug conjugate is selected from ADC B-1b, ADC B-2b, ADC B-5b, ADC B-6b, ADC B-9b, ADC B-11b, and ADC B-12b, wherein the Ab contains VH of the sequence shown in SEQ ID NO:1 and VL of the sequence shown in SEQ ID NO:2, or VH of the sequence shown in SEQ ID NO:3 and VL of the sequence shown in SEQ ID NO:4, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

[0284] In some embodiments, the antibody-drug conjugate is selected from ADC B-1b, ADC B-2b, ADC B-5b, ADC B-6b, ADC B-9b, ADC B-11b, and ADC B-12b. The Ab comprises: a heavy chain including the VH region shown in SEQ ID NO:1 and the heavy chain constant region (CH) shown in SEQ ID NO:35, and a light chain including the VL region shown in SEQ ID NO:2 and the light chain constant region (CL) shown in SEQ ID NO:36; or, a heavy chain including the VH region shown in SEQ ID NO:3 and the heavy chain constant region (CH) shown in SEQ ID NO:35, and a light chain including the VL region shown in SEQ ID NO:4 and the light chain constant region (CL) shown in SEQ ID NO:36, where x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

[0285] In some embodiments, the Ab is linked by one or more thiol groups of cysteine ​​residues, one or more amino groups of lysine residues, one or more hydroxyl groups of threonine residues, or one or more hydroxyl groups of serine residues in the antibody to form a conjugate.

[0286] In some implementations, the Ab is linked to one or more thiol groups of cysteine ​​residues in the antibody to form a conjugate.

[0287] In some implementations, the Ab is linked to one or more amino groups of lysine residues in the antibody to form a conjugate.

[0288] In some implementations, the Ab is linked to one or more hydroxyl groups of threonine residues in the antibody to form a conjugate.

[0289] In some implementations, the Ab is linked to one or more hydroxyl groups of serine residues in the antibody to form a conjugate.

[0290] In some implementations, the Ab is linked by x thiol groups of cysteine ​​residues, x amino groups of lysine residues, x hydroxyl groups of threonine residues, or x hydroxyl groups of serine residues in the antibody to form a conjugate.

[0291] In some implementations, the Ab is linked by x thiol groups of cysteine ​​residues in the antibody.

[0292] In some implementations, the Ab is linked by x amino groups of lysine residues in the antibody.

[0293] In some implementations, the Ab is linked by x hydroxyl groups of threonine residues in the antibody.

[0294] In some implementations, the Ab is linked by x hydroxyl groups of serine residues in the antibody.

[0295] In some embodiments, the antibody-drug conjugates of the present invention are optionally substituted with one or more suitable substituents.

[0296] Drug linker

[0297] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, a pre-conjugation form of the "drug linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking with the antibody or its antigen-binding fragment) can be obtained, and then covalently linked with the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application. The pre-conjugation form of the "drug linker" can be a free form of the "drug linker". Accordingly, in the pre-conjugation form of the "drug linker", M' is linked to one or more thiol (-SH), amino (-NH2), or carboxyl (-COOH) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or -Br structure) or an addition reaction.

[0298] In one aspect, this application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof having a structure represented by formula M'-LED, wherein:

[0299] M' is -M-Lg, where Lg is a leaving group or an addition group for nucleophilic substitution, and M is a structure that binds to the antibody or its antigen-binding fragment.

[0300] L is the structure connecting M and E;

[0301] E is a structure that connects L and D;

[0302] D represents the cytotoxic drug component.

[0303] In some implementations, L, E, and D are as defined in any of the preceding text.

[0304] In some implementations, M' is selected from:

[0305] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structures, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structures may optionally be replaced by one or more suitable substituents.

[0306] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structures.

[0307] In some implementations, Lg is selected from halogenated, substituted, or unsubstituted C. 1-6 Alkylsulfonyl (C 1-6 Alkyl (-SO2-), halogenated phenoxy, hydroxyl (-OH), mercapto (-SH), or amino (-NH2).

[0308] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methanesulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH) or amino (-NH2).

[0309] In some implementations, Lg is selected from C 1-6 Alkyl sulfonyl and halophenoxy groups.

[0310] In some implementations, Lg is selected from methanesulfonyl.

[0311] In some implementations, M' is selected from (For example ).

[0312] In some embodiments, the free-form "drug conjugate" is selected from B-1 to B-21 and D-1 to D-15 as shown below: B-1:

[0313] B-2:

[0314] B-3:

[0315] B-4:

[0316] B-5:

[0317] B-6:

[0318] B-7:

[0319] B-8:

[0320] B-9:

[0321] B-10:

[0322] B-11:

[0323] B-12:

[0324] B-13:

[0325] B-14:

[0326] B-15:

[0327] B-16:

[0328] B-17:

[0329] B-18:

[0330] B-19:

[0331] B-20:

[0332] B-21:

[0333] D-1:

[0334] D-2:

[0335] D-3:

[0336] D-4:

[0337] D-5:

[0338] D-6:

[0339] D-7:

[0340] D-8:

[0341] D-9:

[0342] D-10:

[0343] D-11:

[0344] D-12:

[0345] D-13:

[0346] D-14:

[0347] D-15:

[0348] In some embodiments, the drug linker of the present invention may optionally be replaced by one or more suitable substituents.

[0349] Connector / Connecting Unit

[0350] In some embodiments, the present invention provides a linker unit of formula -MLE-, wherein M, L, and E are as described in any of the preceding embodiments. In some embodiments, the linker unit is used to link a cytotoxic drug and an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate. In some embodiments, in the linker unit, E is a linking portion for linking the cytotoxic drug, and M is a connector for linking to an antibody or an antigen-binding fragment thereof. In some embodiments, in the linker unit, E is used to link to D as described in any of the present application, and M is used to link to an antibody or an antigen-binding fragment thereof as described in any of the present application.

[0351] In some implementations, the connection unit shown by -MLE- is selected from the following structures:

[0352] (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ), (For example ),

[0353] s is selected from an integer from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, or an integer from 8 to 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 5, 8, or 10.

[0354] In some implementations, the left end of the -MLE----- is connected to an antibody or its antigen-binding fragment, and the right end is connected to a cytotoxic drug.

[0355] In some embodiments, the antibody or its antigen-binding fragment is as defined in any of the preceding descriptions, and the cytotoxic drug is as defined in any of the preceding descriptions.

[0356] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising a linker unit of the structure shown in -MLE-.

[0357] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising a linking unit of the structure shown in -MLE- and Ab and / or D, said linking unit being connected to Ab and / or D; wherein Ab or D is as defined in any of the preceding embodiments.

[0358] In some embodiments, a linker unit of the formula M'-LE- is provided, wherein M', L, and E are as described in any of the preceding embodiments. In some embodiments, the linker unit is used to link a cytotoxic drug and an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate. In some embodiments, in the linker unit, E is a linking portion for linking the cytotoxic drug, and M' is a structure capable of reacting with and linking to an antibody or an antigen-binding fragment thereof. In some embodiments, in the linker unit, E is used to link with D as described in any of the present application, and M' is used to react with and link with an antibody or an antigen-binding fragment thereof as described in any of the present application.

[0359] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising a linker unit of the structure shown in M'-LE-.

[0360] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising a linker unit of the structure shown in M'-LE- and D, said linker unit being connected to D; wherein D is as defined in any of the preceding embodiments.

[0361] In some embodiments, the present invention provides a connecting unit as shown in Formula-LED, wherein L, E, and D are as described in any of the preceding embodiments. In some embodiments, the connecting unit is used to connect an antibody or its antigen-binding fragment via a connector to obtain an antibody-drug conjugate.

[0362] In some embodiments, the present invention provides a linker unit of formula -MLED, wherein M, L, E, and D are as described in any of the preceding embodiments. In some embodiments, the linker unit is used to link an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate.

[0363] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising a linker unit with a structure shown in -LED or -MLED.

[0364] All technical features disclosed in this specification, except for mutually exclusive features, can be combined in any way. This invention covers compounds and conjugates obtained by arbitrary combinations of various embodiments, wherein the compounds and conjugates of this invention may be optionally substituted with suitable substituents at suitable substitution positions.

[0365] definition

[0366] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0367] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0368] The term "antibody" also includes embodiments in which the heavy chain constant region contains 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 into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0369] 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. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), 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, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0370] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0371] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0372] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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 (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0373] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0374] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0375] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0376] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, 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, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:48) amino acid sequence or a variant 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 this 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 exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0377] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0378] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0379] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0380] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0381] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0382] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0383] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage 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 × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0384] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues 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 or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0385] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this 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.

[0386] The term "linker" or "linker unit" refers to a structural segment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, it refers to the formula Ab-[MLED]. x The -MLE- structure fragment in the text.

[0387] The term "drug conjugate" refers to the structure of the cytotoxic drug and conjugate described in this invention before they are covalently linked to an antibody or its antigen-binding fragment. For example, "drug conjugate" refers to M'-LED, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. Covalent linking of the "drug conjugate" with an antibody or its antigen-binding fragment yields the antibody-drug conjugate described in this application.

[0388] The "drug linker" also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the "drug linker" compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium (D or 1000 ppm)). 2 H), tritium ( 3 H or T); carbon isotopes (e.g. 11 C 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0389] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0390] As used herein, an asterisk (*) in a compound structural formula indicates that the labeled carbon atom is a chiral carbon atom, and the invention includes a pair of enantiomers formed from that chiral carbon atom. If a compound contains two different chiral carbon atoms, the invention includes four optical isomers formed from that chiral carbon atom.

[0391] As used in this article, This indicates the location where the structure connects to other parts of the molecule.

[0392] The term "alkyl" is defined as a straight-chain or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 carbon atoms. For example, as used herein, the term "C 1-6 "alkyl" and "C"1-4 "alkyl" refers to a linear or branched group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl) having 1 to 6 carbon atoms and 1 to 4 carbon atoms respectively, which is optionally substituted by one or more (such as 1, 2, or 3) suitable substituents.

[0393] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxyl groups, wherein the alkyl group is defined as described above. For example, the term "C" in this invention... 1-6 "Hydroxyalkyl" refers to hydroxyalkyl groups having 1-6 carbon atoms. Common hydroxyalkyl groups include (but are not limited to) -CH2OH, -CH2CH2OH, -CH2CH(OH)2, and -(CH2)3OH.

[0394] The term "aminoalkyl" refers to an alkyl group substituted with one or more amino groups, wherein the alkyl group is defined as described above. For example, the term "C" in this invention... 1-6 "Aminoalkyl" refers to an aminoalkyl group having 1-6 carbon atoms. Common aminoalkyl groups include (but are not limited to) -CH2-NH2, -CH2CH2-NH2, -CH2CH(NH2)2, and -(CH2)3-NH2.

[0395] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C..." 2-6 "alkenyl", "C" 2-4 Examples of these include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0396] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Alkyne", "C" 4-6 Examples of "alkynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, etc.

[0397] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-6"Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by one or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0398] The term "carbocyclic group" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic hydrocarbon group linked by a ring carbon. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0399] The term "carbocyclic ring" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic rings such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, or bicyclic rings, including spirocyclic, fused, or bridged systems (such as bicyclic [1.1.1]pentane, bicyclic [2.2.1]heptane, bicyclic [3.2.1]octane, or bicyclic [5.2.0]nonane, decahydronaphthalene, etc.), which may optionally be substituted by one or more (such as one, two, or three) suitable substituents. The term "3-6 membered carbocyclic ring" refers to a carbocyclic ring containing 3, 4, 5, or 6 cyclic carbon atoms.

[0400] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic group in this invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, such as 4-6 membered nitrogen-containing heterocyclic groups, 4-6 membered oxygen-containing heterocyclic groups, 5-6 membered oxygen-containing heterocyclic groups, and 5 membered oxygen-containing heterocyclic groups. Common heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0401] The term "oxygen-containing heterocycle" refers to a heterocycle as described above that has one or more (e.g., 1, 2, or 3) ring atoms of oxygen atoms, such as 5-6 membered oxygen-containing heterocycles, five membered oxygen-containing heterocycles, and specific examples include, but are not limited to, ethylene oxide rings, tetrahydrofuran rings, furan rings, tetrahydropyran rings, pyran rings, 1,3-dioxolane rings, etc.

[0402] The "nitrogen-containing heterocycle" as described in this invention refers to the heterocycle described above, in which one or more (e.g., 1, 2 or 3) ring atoms are nitrogen atoms.

[0403] The term "halogenated alkyl" refers to an alkyl group substituted with one or more (such as 1, 2, or 3) identical or different halogen atoms, wherein the alkyl group is defined as described above. For example, the term "C" as used in this invention... 1-6 "Halogenated alkyl" refers to an alkyl halogroup having 1 to 6 carbon atoms. Common alkyl halogroups include (but are not limited to) -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, etc. The alkyl halogroups in this invention are optionally substituted by one or more substituents described in this invention.

[0404] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy groups, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention are optionally substituted by one or more substituents described in this invention.

[0405] The term "alkoxyalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups, wherein the definitions of alkoxy and alkyl groups are as described above. For example, the term "C" as used in this invention... 1-6 "Alkoxyalkyl" refers to an alkyl group having 1-6 carbon atoms that is substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups. Common alkoxyalkyl groups include (but are not limited to) CH3O-CH2-, C2H5-O-CH2-, C2H5-O-CH2CH2-, etc.

[0406] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0407] The term "nitrogen oxide" refers to an oxide (e.g., a mono- or di-oxide) of at least one nitrogen atom in the structure of the compounds of this application. Mono-oxides of nitrogen may exist as a single positional isomer or a mixture of positional isomers.

[0408] If a substituent is described as “optionally substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together by independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted by independently selected optional substituents.

[0409] If a functional group or structure is described as “substituted or unsubstituted”, then the functional group or structure may be (1) unsubstituted or (2) substituted.

[0410] As used herein, the term "suitable substituent" refers to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents. "Suitable substituents" include oxo (=O), halogen, cyano, and NR. 8 R 9 Carboxyl, thiol, hydroxyl, ester group (e.g., -C) 1-6 Alkylene-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, -C 1-6 Alkylene-OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-8 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 R 9 Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, halogens, hydroxyl groups, carboxyl groups, and ester groups (e.g., -C) 1-6 Alkylene-C(=O)-OC 1-6 alkyl).

[0411] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms in a specified compound or structure by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. For example, each substituent may independently consist of one or more of the following structures: NR 8 R 9 -O-, -S-, -NR'-, halogens, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C 1-6 (alkylene) group, C 1-6 Halogenated (alkylene) group, C 1-6 Alkoxy, C 2-6 (imide)alkenyl, C 2-6 (Asyl) ynyl, C 3-8 (Hypo-cycloalkylene), 3-8 membered (hetero-cycloalkylene), C 6-10 (sub-)aryl and 5-10 quinone (sub-)heteroaryl, etc., among which R 8 R 9 R' is as defined above. For example, the substituent can be a suitable substituent as described above. If a substituent is described as being "independently selected" from a set of functional groups, then each substituent is selected independently of the others. Therefore, each substituent can be the same as or different from another (other) substituent.

[0412] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

[0413] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0414] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0415] Solid lines (—) and solid wedges may be used in this article. Or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0416] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0417] It should also be understood that certain compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to encompass the various derivative forms of the compounds described above.

[0418] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0419] The term "ester" refers to esters derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (compounds of the present invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the present invention may themselves be esters.

[0420] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0421] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0422] This invention further includes, within its scope, prodrugs of the compounds of the invention. Typically, such prodrugs are functional group derivatives of the compounds that readily convert in vivo into the desired therapeutically active compound. Therefore, in these cases, the term "administration" for the treatment methods of the invention should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but after administration to an individual, the prodrug form is converted in vivo into the aforementioned compound. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug," ed. H. Bundgaard, Elsevier, 1985.

[0423] The invention further includes, within its scope, isotopic labels of the compounds of the invention, which are identical to the compounds of the invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature.

[0424] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in *Protective Groups in Organic Chemistry*, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P. ​​G.W. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0425] As used herein, the term “DAR” or “drug-antibody ratio” refers to: (a) the number of linker / drug moieties linked to the antibody in a single antibody-drug conjugate molecule, which is an integer from 0 to 10, such as an integer from 1 to 10; or (b) the average number of linker / drug moieties linked to the antibody in a composition comprising more than one antibody-drug conjugate molecule, which is an integer or decimal from 0 to 10, such as an integer or decimal from 1 to 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0426] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated value.

[0427] intermediate

[0428] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0429] in,

[0430] Each PG1 is independently protected by an H or amino group, such as alkoxycarbonyl amino groups, including benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); acyl amino groups, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pentanoyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; and alkyl amino groups, such as triphenylmethyl (Trt), C 1-6 Alkyl-substituted triphenylmethyl, p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0431] Each PG2 group is independently protected by an H or a carboxyl group, such as a C group. 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl;

[0432] Each PG3 is independently protected by an H or hydroxyl group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, triphenylmethyl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl.

[0433] s1 is selected from integers from 1 to 20, such as 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;

[0434] Lg is as defined above.

[0435] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0436] In another aspect, this application provides the use of the intermediate compound as described above, or its salts, stereoisomers, tautomers, or isotopically labeled compounds, in the preparation of the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, nitrides, isotopically labeled substances, metabolites, or prodrugs or antibody-drug conjugates.

[0437] Composition

[0438] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug linkers. Therefore, the compositions are characterized by a drug-to-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0439] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 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.

[0440] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, for example, 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 5.5, about 3.0 to 6.0, about 3.0 to 6.5, about 3.0 to 7.0, about 3.0 to 7.5, about 3.0 to 8.0, about 3.0 to 8.5, about 3.0 to 9.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, and about 3.5 to 6.0. Approximately 3.5 to 6.5, approximately 3.5 to 7.0, approximately 3.5 to 7.5, approximately 3.5 to 8.0, approximately 3.5 to 8.5, approximately 3.5 to 9.0, approximately 4.0 to 4.5, approximately 4.0 to 5.0, approximately 4.0 to 5.5, approximately 4.0 to 6.0, approximately 4.0 to 6.5, approximately 4.0 to 7.0, approximately 4.0 to 7.5, approximately 4.0 to 8.0, approximately 4.0 to 8.5, approximately 4.0 to 9.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4. 5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 4.5 to 8.5, approximately 4.5 to 9.0, approximately 5.0 to 5.5, approximately 5.0 to 6.0, approximately 5.0 to 6.5, approximately 5.0 to 7.0, approximately 5.0 to 7.5, approximately 5.0 to 8.0, approximately 5.0 to 8.5, approximately 5.0 to 9.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 5.5 to 8.5, approximately 5.5 to 9.0, approximately 6.0 to 6.0 .5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.0 to 8.5, approximately 6.0 to 9.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 6.5 to 8.0, approximately 6.5 to 9.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.0 to 8.5, approximately 7.0 to 9.0, approximately 8.5 to 9.0.

[0441] In some embodiments, the DAR of the ADC composition described herein is about 3 to 8.

[0442] In some embodiments, the DAR of the ADC compositions described herein is 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, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.7 6, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97 Approximately 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.1, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18 Approximately 7.19, 7.2, 7.21, 7.22, 7.23, 7.24, 7.25, 7.26, 7.27, 7.28, 7.29, 7.3, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38, 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0, approximately 8.01, approximately 8.02, approximately 8.03, approximately 8.04, approximately 8.05, approximately 8.06, approximately 8.07, approximately 8.08, approximately 8.09, approximately 8.1, approximately 8.11, approximately 8.12, approximately 8.13, approximately 8.14, approximately 8.15, approximately 8.16, approximately 8.17, approximately 8.18, approximately 8.19, approximately 8.2, approximately 8.21, approximately 8.22, approximately 8.23, approximately 8.24, approximately 8.25, approximately 8.26, approximately 8.27, approximately 8.28, approximately 8.29, approximately 8.3, approximately 8.31, approximately 8.32, approximately 8.33, approximately 8.34, approximately 8.35, approximately 8.36, approximately 8.37, approximately 8.38, approximately 8.39, approximately 8.4, approximately 8.41, approximately 8.42, approximately 8.43, approximately 8.44, approximately 8.45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, approximately 8.55, approximately 8.56, approximately 8.57, approximately 8.58, approximately 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, Approximately 8.66, 8.67, 8.68, 8.69, 8.7, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.0.

[0443] In some embodiments, the composition is composed of, for example, Ab-[MLED]. xThe antibody-drug conjugate composition is shown. In some embodiments, the DAR of the composition is 1-8, for example 5-8, such as about 6.4, 6.68, 7.08, 7.4, 7.5, 7.55, 7.68, 7.78, 7.87, 7.9, 8.0. In some embodiments, the antibody-drug conjugate with x=8 constitutes 60%, 70%, 80%, or 90% or more.

[0444] In some specific embodiments, the composition comprises ADC B-1a or ADC B-1b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the DAR of the composition is about 6 to 8, for example about 7.5.

[0445] In some specific embodiments, the composition comprises ADC B-2a or ADC B-2b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the DAR of the composition is about 6 to 8, for example about 7.4.

[0446] In some specific embodiments, the composition comprises ADC B-5a or ADC B-5b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the composition has a DAR of about 7.5 to 8.5, for example about 8.0, for example 8.03.

[0447] In some specific embodiments, the composition comprises ADC B-6a or ADC B-6b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the composition has a DAR of about 7.5 to 8.5, for example about 8.0.

[0448] In some specific embodiments, the composition comprises ADC B-9a or ADC B-9b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the composition has a DAR of about 7.5 to 8.5, for example about 8.0, for example 8.05.

[0449] In some specific embodiments, the composition comprises ADC B-11a or ADC B-11b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the composition has a DAR of about 7.5 to 8.5, for example about 8.0, for example 8.01.

[0450] In some specific embodiments, the composition comprises ADC B-12a or ADC B-12b, wherein the antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or comprises HC as shown in SEQ ID NO:37 and LC as shown in SEQ ID NO:38, or HC as shown in SEQ ID NO:39 and LC as shown in SEQ ID NO:40; the composition has a DAR of about 7.5 to 8.5, for example about 8.0.

[0451] Pharmaceutical Composition

[0452] In another aspect, the present invention provides a pharmaceutical composition comprising any of the antibody-drug conjugates or compositions thereof described in any of the preceding claims, or any of the drug linkers or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrides, isotope labels, metabolites or prodrugs thereof, and one or more pharmaceutically acceptable carriers.

[0453] The term "pharmaceutical composition" refers to a composition that can be used as a medicine, comprising a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceuticalally acceptable carrier" refers to an excipient administered co-administered with the therapeutic agent, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0454] The above-described pharmaceutical compositions can act systemically and / or locally, which can be achieved through suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0455] The above-mentioned pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof.

[0456] The present invention also provides a method for preparing the above-described pharmaceutical composition or its corresponding formulation, comprising combining at least one antibody-drug conjugate or drug linker of the present invention, or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug, with one or more pharmaceutically acceptable carriers.

[0457] Pillbox products

[0458] In another aspect, the present invention provides a medicine box product comprising:

[0459] a) at least one antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, or a composition or pharmaceutical composition thereof, as a first therapeutic agent.

[0460] b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a composition or pharmaceutical composition comprising another therapeutic agent; and

[0461] c) Optional packaging and / or instructions.

[0462] The aforementioned kit products may contain 0.01 mg to 1000 mg of at least one antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof.

[0463] The present invention also provides a method for preparing the above-mentioned medicine box, which includes combining at least one antibody-drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, or the above-mentioned composition, with at least one other therapeutic agent optionally present or a composition containing other therapeutic agents, packaging and / or instructions.

[0464] Medical Use

[0465] The antibody-drug conjugates or drug linkers of the present invention, or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs, can exhibit strong inhibitory effects on abnormal cell proliferation.

[0466] Therefore, this application provides the antibody-drug conjugate or drug linker of the present invention, or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite and prodrug, the above-mentioned composition, pharmaceutical composition or the above-mentioned kit product, for the treatment of diseases, particularly diseases of abnormal cell proliferation.

[0467] In addition, this application also provides the use of the antibody-drug conjugate or drug linker of the present invention, or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug, the above-mentioned composition, pharmaceutical composition or the above-mentioned kit product in the preparation of a medicament for treating diseases, particularly diseases of abnormal cell proliferation.

[0468] In some implementations, the diseases involving abnormal cell proliferation include (but are not limited to) cancer or tumors, such as advanced solid tumors.

[0469] This application also provides the use of the antibody-drug conjugates or drug linkers of the present invention, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrides, isotope-labeled substances, metabolites, and prodrugs thereof, or compositions or pharmaceutical compositions of the present invention, in the preparation of formulations for inhibiting the proliferation of tumor cells. In some embodiments, the formulations are for in vivo or in vitro administration. For example, the formulations may be administered to a subject to inhibit the proliferation of tumor cells in the subject; or, the formulations may be administered to in vitro cells (e.g., cell lines or cells derived from the subject) to inhibit the proliferation of tumor cells in vitro.

[0470] The cancers or tumors described in this invention include (but are not limited to): brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

[0471] Treatment

[0472] In another aspect, the present invention provides a method for treating diseases, particularly those involving abnormal cell proliferation, comprising the steps of: administering a therapeutically effective amount of the antibody-drug conjugate or drug linker of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, or a combination thereof, or a pharmaceutical composition thereof, to an individual in need of it.

[0473] In some implementations, the disease is as described in the previous one.

[0474] The term "effective dose" refers to a dose that is sufficient to induce a biological or medical response in cells, tissues, organs, or organisms (e.g., individuals) and to achieve the desired preventive and / or therapeutic effects.

[0475] The dosing regimen can be adjusted to provide the optimal required response. For example, it can be administered as a single dose, divided into doses over time, or the dose can be reduced or increased proportionally as needed. It is understood that, for any given individual, the specific dosing regimen should be adjusted as required and with the professional judgment of the person administering the composition or supervising the administration of the composition.

[0476] The dosage of the antibody-drug conjugate or drug linker of the present invention, or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs, will depend on individual circumstances, the severity of the disease or condition, the rate of administration, the disposal of the compound, and the prescribing physician's judgment. Generally, the effective dose is about 0.001-10000 mg / kg body weight per day. Where appropriate, the effective dose is about 0.01-1000 mg / kg body weight per day. About 0.01-1000 mg / kg body weight can be administered daily, every two days, or every three days, typically about 0.1-500 mg / kg body weight. Exemplary dosing regimens are once or more daily, once or more weekly, or once or more monthly. With multiple administrations, the interval between single doses can typically be daily, weekly, monthly, or yearly. Alternatively, it can be administered in the form of a sustained-release formulation, in which case a lower dosing frequency is required. Dosage and frequency may vary depending on the drug's half-life in the subject and whether it is for prophylactic or therapeutic use. In prophylactic use, a relatively low dose is administered at relatively low intervals over a long period; in therapeutic use, a relatively high dose may be administered at shorter intervals until disease progression is slowed or stopped, preferably until the individual shows partial or complete improvement in disease symptoms, after which prophylactic use may be used.

[0477] The term "treatment" refers to the reduction or elimination of a targeted disease or symptom. A subject is considered successfully "treated" if, after receiving a therapeutic amount of the antibody-drug conjugate or drug linker of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrides, isotope-labeled substances, metabolites, and prodrugs, or the compositions or pharmaceutical compositions of the present invention, at least one indicator and symptom of the subject shows observable and / or detectable relief and / or improvement. It is understood that treatment includes not only complete treatment but also the achievement of some biologically or medically relevant outcome without achieving complete treatment.

[0478] The term "administrate / administrating / administration" (or "drug administration") refers to the process of applying an active pharmaceutical ingredient (such as the antibody-drug conjugate or drug linker of the present invention, or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs) or a pharmaceutical composition containing the active pharmaceutical ingredient (such as the pharmaceutical composition of the present invention) to an individual or its cells, tissues, organs, biological fluids, etc., so as to bring the active pharmaceutical ingredient or pharmaceutical composition into contact with the individual or its cells, tissues, organs, biological fluids, etc. Common methods of administration include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, etc.

[0479] The term “needs” refers to the judgment of a physician or other caregiver regarding an individual’s need for or potential benefit from preventive and / or treatment processes, which is based on various factors within the physician’s or other caregiver’s area of ​​expertise.

[0480] The term "individual" (or subject) refers to a human or non-human animal. Individuals in this invention include individuals suffering from diseases and / or conditions (patients) and healthy individuals. Non-human animals in this invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0481] Beneficial effects of the invention

[0482] The antibody-drug conjugates and / or drug linkers of the present invention have good antitumor activity, high safety, and good pharmacokinetic properties (e.g., suitable half-life and duration of action), and can be used to treat diseases of abnormal cell proliferation, including but not limited to advanced solid tumors. Detailed Implementation

[0483] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of the application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its fundamental ideas and scope.

[0484] Information about the sequences involved in this invention is described in the table below:

[0485] The abbreviations used in this invention have the following meanings:

[0486] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0487] Nuclear magnetic resonance (NMR) 1 The H NMR (H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; hexadeuterated dimethyl sulfoxide (DMSO-d6) was used; and tetramethylsilane (TMS) was used as the internal standard.

[0488] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0489] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0490] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0491] Example 1 of intermediate preparation: Preparation of 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (INT-1)

[0492] Step 1: Preparation of 1-amino-3,6,9,12,15-pentaoxoctadecane-18-acid (INT-1-2)

[0493] 0.50 g (1.22 mmol) of 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatocetane-23-acid was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to give the title compound as crude trifluoroacetate (650 mg, 1.21 mmol).

[0494] Its structural characterization data are as follows:

[0495] MS m / z (ESI): 310.2 [M+H] +

[0496] Step 2: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynical acid (INT-1)

[0497] 1-Amino-3,6,9,12,15-pentoxaoctadecanoic acid trifluoroacetate (650 mg, 1.21 mmol) was dissolved in DMF (5.0 mL), and DIPEA (781.61 mg, 6.05 mmol) was added. Then, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (441.91 mg, 1.21 mmol) was added in portions, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile / 0.05% ammonium bicarbonate aqueous solution = 0–60%) and then freeze-dried to give the title compound (646 mg, 1.15 mmol).

[0498] Its structural characterization data are as follows:

[0499] MS m / z (ESI): 560.3 [M+H] +

[0500] Example 2 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0501] Step 1: Preparation of L-alanyl-L-alanine (INT-2-1)

[0502] (tert-butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, yielding the pale yellow title compound (160 mg, 370.93 μmol).

[0503] Its structural characterization data are as follows:

[0504] MS m / z (ESI): 161.1 [M+H] +

[0505] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0506] L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester (150.58 mg, 412.14 μmol) were dissolved in N,N-dimethylacetamide (2 mL), and N,N-diisopropylethylamine (266.33 mg, 2.06 mmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (125 mg, 304.55 μmol).

[0507] Its structural characterization data are as follows:

[0508] MS m / z (ESI): 428.1 [M+H2O] +

[0509] Example 3 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (INT-3)

[0510] Glycylglycine (1 g, 7.57 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (2.77 g, 7.57 mmol) were dissolved in dimethyl sulfoxide (10 mL). The reaction mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (2.52 g, 6.59 mmol).

[0511] Its structural characterization data are as follows:

[0512] MS m / z (ESI): 383.2 [M+H] +

[0513] Example 4 of intermediate preparation: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0514] Step 1: Preparation of glycyl-glycyl-L-phenylalanine (INT-4-2)

[0515] ((benzyloxy)carbonyl)glycylglycyl-L-phenylalanine (200 mg, 483.77 μmol) was dissolved in methanol (8 mL). After purging with nitrogen, palladium on carbon (20 mg) was added. The gas was purged three times with hydrogen, and the reaction was stirred at room temperature for 3 hours. After the reaction was complete, palladium on carbon was removed by filtration. The filtrate was then concentrated to remove the solvent, giving the title compound (135 mg, 483.37 μmol).

[0516] Its structural characterization data are as follows:

[0517] MS m / z (ESI): 280.2 [M+H] +

[0518] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0519] Glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (170.06 mg, 465.46 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (120.32 mg, 930.93 μmol) was added. The reaction was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (60 mg, 0.11 mmol).

[0520] Its structural characterization data are as follows:

[0521] MS m / z (ESI): 530.3 [M+H] +

[0522] Example 5 of intermediate preparation: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0523] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-5-2)

[0524] 50.0 g (279 mmol) of 6-nitroisobenzofuran-1(3H)-one was added to a tetrahydrofuran solution of methylamine (2 M, 500 mL), and the mixture was heated to 75 °C and stirred for 5 hours. The reaction solution was directly concentrated to obtain the crude title compound (58.0 g), which was used directly in the next step without purification.

[0525] Its structural characterization data are as follows:

[0526] ESI-MS (m / z): 211.0 [M+H] + .

[0527] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-5-3)

[0528] 2-Hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) was dissolved in tetrahydrofuran (500 mL). After cooling to 0 °C, a borane-dimethyl sulfide solution (10 M, 89.3 mL) was added dropwise to the reaction system. After the addition was complete, the temperature was raised to 70 °C and stirred for 5 hours. The temperature was lowered to 0 °C again, and a hydrogen chloride-methanol solution (2 M, 100 mL) was added dropwise to the above reaction solution. The temperature was raised again to 65 °C and stirred for 12 hours. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound (44.3 g), which was used directly in the next step without purification.

[0529] Its structural characterization data are as follows:

[0530] ESI-MS (m / z): 197.0 [M+H] + .

[0531] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (INT-5-4)

[0532] (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) was dissolved in dichloromethane (800 mL), cooled to 0 °C, and then imidazole (55.9 g, 822 mmol) and tert-butyldiphenylchlorosilane (84.0 g, 308 mmol, 78.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (200 mL), and the mixture was extracted three times with dichloromethane (300 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 5 / 1), the product was concentrated again to obtain the title compound (59.7 g, 137 mmol).

[0533] Its structural characterization data are as follows:

[0534] ESI-MS (m / z): 435.1 [M+H] + .

[0535] Step 4: Preparation of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-5-5)

[0536] 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (54.9 g, 126 mmol) was dissolved in dichloromethane (550 mL), cooled to 0 °C, and DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (300 mL), and the mixture was extracted three times with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1), the product was concentrated again to obtain the title compound (65.5 g, 126 mmol).

[0537] Its structural characterization data are as follows:

[0538] ESI-MS (m / z): 519.1 [M+H] + .

[0539] Step 5: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5) Allyl (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80 °C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0540] Its structural characterization data are as follows:

[0541] ESI-MS (m / z): 511.7 [M+H] + .

[0542] Example 6: Preparation of intermediate (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0543] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-1)

[0544] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solution was concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0545] Its structural characterization data are as follows:

[0546] MS m / z (ESI): 782.2 [M+H] +

[0547] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-6-2)

[0548] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0549] Its structural characterization data are as follows:

[0550] MS m / z (ESI): 566.1 [M+Na] +

[0551] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-3)

[0552] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, followed by p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (134.50 mg, 0.19 mmol).

[0553] Its structural characterization data are as follows:

[0554] MS m / z (ESI): 731.2 [M+Na] +

[0555] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-4)

[0556] Allyl carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H 165.50 mg, 0.38 mmol of benzo[de]pyran[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and lyophilized to give the title compound (114.70 mg, 0.12 mmol).

[0557] Its structural characterization data are as follows:

[0558] MS m / z (ESI): 1005.2 [M+H] +

[0559] Step 5: Preparation of (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-5)

[0560] Dissolve (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) in DMF (3 mL), add diethylamine (87.8 mg, 1.2 mmol), and stir at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) after most of the solvent was removed, and then freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

[0561] Its structural characterization data are as follows:

[0562] MS m / z(ESI): 783.1 [M+H] +

[0563] Step Six: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-6)

[0564] (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl) Allyl carbamate (1.0 g, 1.28 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (476.90 mg, 1.41 mmol) were dissolved in DMF (5 mL). HATU (631.06 mg, 1.66 mmol) and DIPEA (495.29 mg, 3.83 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was rapidly purified and then freeze-dried to give the title compound (950 mg, 860.37 μmol).

[0565] Its structural characterization data are as follows:

[0566] MS m / z (ESI): 1104.5 [M+H] +

[0567] Its preparation method is as follows:

[0568] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0569] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0570] Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0571] (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]) Quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (950 mg, 860.37 μmol) was dissolved in DMF (3.5 mL), and diethylamine (860.37 μmol) was added. The mixture was stirred at room temperature for 20 min. The reaction solution was concentrated and then added to 100 mL of a mixed solvent (EA / PE = 1 / 3) and stirred overnight. The mixture was filtered and dried to obtain the title compound (750 mg, 850.4 μmol).

[0572] Its structural characterization data are as follows:

[0573] MS m / z (ESI): 882.5 [M+H] +

[0574] Example 7 of intermediate preparation: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-7)

[0575] Step 1: Preparation of 3-((tert-Butoxycarbonyl)amino)propionate allyl ester (INT-7-2)

[0576] 3-((tert-Butoxycarbonyl)amino)propionic acid (2.3 g, 12.16 mmol) and 3-bromopropene (1.62 g, 13.37 mmol) were dissolved in DMF (10 mL), and potassium carbonate (5.04 g, 36.47 mmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1), the crude compound was concentrated again to obtain the title compound (2.2 g, 9.6 mmol).

[0577] Its structural characterization data are as follows:

[0578] ESI-MS (m / z): 252.1 [M+Na] + .

[0579] Step 2: Preparation of allyl 3-aminopropionate (INT-7-3)

[0580] Allyl 3-((tert-Butoxycarbonyl)amino)propionate (2.2 g, 9.6 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (5 mL). The mixture was stirred at room temperature for 2 hours. The reaction solution was directly concentrated to give the crude title compound (2.3 g), which was used directly in the next step without purification.

[0581] Its structural characterization data are as follows:

[0582] ESI-MS (m / z): 130.1 [M+H] + .

[0583] Step 3: Preparation of (S)-3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexamamide)allyl propionate (INT-7-4)

[0584] Allyl 3-aminopropionate (2.0 g, 8.22 mmol) and N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (5.14 g, 8.22 mmol) was dissolved in DMF (20 mL), and DIPEA (5.31 g, 41.12 mmol, 7.38 mL) and HATU (6.25 g, 16.45 mmol) were added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (4.5 g, 6.11 mmol).

[0585] Its structural characterization data are as follows:

[0586] ESI-MS (m / z): 436.4 [M+H] + .

[0587] Step 4: Preparation of (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)propionate (INT-7-5)

[0588] (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)propionate (3.0 g, 4.08 mmol) was dissolved in DMF (10 mL), and diethylamine (596.30 mg, 8.15 mmol, 828.20 μL) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to give the title compound (1.8 g, 3.5 mmol).

[0589] Its structural characterization data are as follows:

[0590] ESI-MS (m / z): 514.3 [M+H] + .

[0591] Step 5: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynyl ester (INT-7-6)

[0592] (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamamide)propionate (1.8 g, 3.5 mmol) and 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azatetradecane-24-alkynic acid (1.69 g, 3.01 mmol) were dissolved in DMF (5 mL). DIPEA (1.85 g, 14.34 mmol, 2.57 mL) and HATU (2.18 g, 5.74 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (960 mg, 909.71 μmol).

[0593] Its structural characterization data are as follows:

[0594] MS m / z (ESI): 1055.5 [M+H] +

[0595] Step Six: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-7)

[0596] (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triaza-tetane-31-alkynyl ester (960 mg, 909.71 μmol) was dissolved in DMF (5 mL), purged three times with nitrogen, and then tetratetraphenylphosphine palladium (525.61 mg, 454.85 μmol) and 1,3-dimethylbarbituric acid (710.21 mg, 4.55 mmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was purified by preparative high-performance liquid chromatography and then freeze-dried to give the title compound (390.0 mg, 384.15 mmol).

[0597] Its structural characterization data are as follows:

[0598] MS m / z (ESI): 1015.4 [M+H] +

[0599] Its preparation method is as follows:

[0600] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0601] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0602] Example 8 of intermediate preparation: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8)

[0603] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-1)

[0604] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.0 g, 2.05 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-citrulline (1.22 g, 3.07 mmol) were dissolved in DCM (28 mL) and MeOH (7 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.52 g, 6.15 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to obtain the crude product. After purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%), the crude product was concentrated again under reduced pressure to obtain the title compound (1.07 g, 1.23 mmol, 90% purity).

[0605] Its structural characterization data are as follows:

[0606] MS m / z (ESI): 868.2 [M+H] +

[0607] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-8-2)

[0608] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.07 g, 1.23 mmol, 90% purity) was dissolved in DMF (8 mL), and pyridine hydrofluoric acid salt (585.2 mg, 5.9 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 50 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3), washed with 30 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (418.5 mg, 0.67 mmol, 92% purity).

[0609] Its structural characterization data are as follows:

[0610] MS m / z (ESI): 652.1 [M+Na] +

[0611] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-3)

[0612] (S)-(5-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(hydroxymethyl)benzyl)(methyl)carbamate (418.5 mg, 0.67 mmol, 92% purity) was dissolved in DMF (5 mL), DIPEA (172.3 mg, 1.34 mmol) was added, and p-nitrophenyl chloroformate (161.8 mg, 0.81 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 3 / 2) and then freeze-dried to give the title compound (416.10 mg, 0.52 mmol, 90% purity).

[0613] Its structural characterization data are as follows:

[0614] MS m / z (ESI): 817.2 [M+Na] +

[0615] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-4)

[0616] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (416.10 mg, 0.52 mmol, 90% purity), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 H,13H-benzo[de]pyran[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (331.50 mg, 0.78 mmol) was dissolved in DMF (8 mL), and 1-hydroxybenzotriazole (70.20 mg, 0.52 mmol), pyridine (61.5 mg, 0.78 mmol), and DIPEA (201.5 mg, 1.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent from the reaction solution, it was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and lyophilized to give the title compound (357.10 mg, 0.33 mmol, 89% purity).

[0617] Its structural characterization data are as follows:

[0618] MS m / z (ESI): 1091.2 [M+H] +

[0619] Step 5: Preparation of (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-5)

[0620] (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (357.10 mg, 0.33 mmol, 89% purity) was dissolved in DMF (3 mL), and diethylamine (73.8 mg, 1.0 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution, it was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (242.5 mg, 0.28 mmol, 95% purity).

[0621] Its structural characterization data are as follows:

[0622] MS m / z (ESI): 869.1 [M+H] +

[0623] Step Six: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-6)

[0624] (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)allyl)carbamate (242.5) (mg, 0.28mmol, 95% purity) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (113.6mg, 0.34mmol) were dissolved in DMF (3mL), and HATU (160.3mg, 0.42mmol) and DIPEA (108.4mg, 0.84mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, 80mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30mL x 3). The mixture was washed with 50mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. After purification by rapid column chromatography (silica gel column, dichloromethane / methanol = 10 / 1), the crude product of the title compound (266.6mg, 0.23mmol, 89% purity) was obtained by concentration.

[0625] Its structural characterization data are as follows:

[0626] MS m / z(ESI): 1190.9 [M+H] +

[0627] Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8)

[0628] (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (266.6 mg, 0.23 mmol, 89% purity) was dissolved in DMF (3 mL), and diethylamine (50.4 mg, 0.69 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution, it was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (211.5 mg, 0.22 mmol, 92% purity).

[0629] Its structural characterization data are as follows:

[0630] MS m / z (ESI): 968.3 [M+H] +

[0631] Example 9 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

[0632] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-9-2)

[0633] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (150.0 mg, 0.24 mmol) was dissolved in DMF (2 mL), and diethylamine (182.3 mg, 2.5 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 10 mL of a mixed solvent (EA / PE = 1 / 3) was added for recrystallization, and the solution was filtered to give the title compound (90.1 mg, 0.22 mmol).

[0634] Step Two: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

[0635] N6-(diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), DIPEA (86.3 mg, 0.66 mmol) was added, followed by 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate (160.6 mg, 0.44 mmol). After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to obtain the title compound (75.2 mg, 0.11 mmol).

[0636] Its structural characterization data are as follows:

[0637] MS m / z (ESI): 653.2 [M+H] +

[0638] Example 10: Preparation of intermediate 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexahexadecyl-35-alkynical acid (INT-10)

[0639] Step 1: 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-decaazatrione-31-acid (INT-10-2)

[0640] 1-(9H-fluorene-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-decaazatritane-33-acid (1.0 g, 1.05 mmol) was dissolved in DMF (5.0 mL), and diethylamine (0.5 mL) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was removed using a lyophilizer, and 50 mL of ethyl acetate / petroleum ether (1 / 3) was added and stirred for 1 hour. The solvent was then removed to obtain the title compound (688.9 mg, 0.95 mmol, 85% purity).

[0641] Its structural characterization data are as follows:

[0642] MS m / z (ESI): 729.2 [M+H] +

[0643] Step 2: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexadecane-35-alkynical acid (INT-10)

[0644] 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-decazaneco-31-acid (688.9 mg, 0.95 mmol, 85% purity) was dissolved in DMF (8 mL), and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetic acid ester (520.2 mg, 1.43 mmol) and DIPEA (367.7 mg, 2.9 mmol) were added. The mixture was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the product was rapidly purified and then freeze-dried to obtain the title compound (465.1 mg, 0.48 mmol, 98.0% purity).

[0645] Its structural characterization data are as follows:

[0646] MS m / z (ESI): 979.3 [M+H] +

[0647] Its preparation method is as follows:

[0648] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0649] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0650] Example 11: Preparation of intermediate (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynyl)-L-valine (INT-11)

[0651] Step 1: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-ynyl)-L-valine tert-butyl ester (INT-11-1)

[0652] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynyl acid (0.23 g, 0.41 mmol) and L-valine tert-butyl ester (0.95 g, 0.45 mmol) were dissolved in DMF (3 mL), and HATU (305.3 mg, 1.23 mmol) and DIPEA (106.2 mg, 0.82 mmol) were added. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (142.0 mg, 198.6 μmol).

[0653] Its structural characterization data are as follows:

[0654] MS m / z (ESI): 715.8 [M+H] +

[0655] Its preparation method is as follows:

[0656] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0657] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0658] Step 2: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecosano-24-ynyl)-L-valine (INT-11)

[0659] (25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-ynyl)-L-valine tert-butyl ester (142.0 mg, 198.6 μmol) was dissolved in DCM (5.0 mL), and TFA (5.0 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by reversed-phase column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (60 mg, 91.08 mmol).

[0660] Its structural characterization data are as follows:

[0661] MS m / z (ESI): 659.7 [M+H] +

[0662] Example 12 of intermediate preparation: Preparation of (S)-6-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triaza-tetane-31-alkynic acid (INT-12):

[0663] Step 1: tert-butyl N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2 Preparation of -(25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-aza-tetradecane-24-alkynyl)-L-lysine ester (INT-12-1)

[0664] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (800 mg, 1.43 mmol), N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine tert-butyl ester (724.90 mg, 1.57 mmol), DIPEA (739 mg, 5.72 mmol), and HATU (815.26 mg, 2.14 mmol) were added to DMF (2 mL). After addition, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (1.3 g, 1.14 mmol, 85% purity).

[0665] Its structural characterization data are as follows:

[0666] MS m / z (ESI): 966.4 [M+H] +

[0667] Step Two: N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2 Preparation of -(25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynyl)-L-lysine (INT-12-2)

[0668] Add tert-butyl N to DCM (10 mL) and TFA (5 mL) 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2-(25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-penta-19-aza-peco-24-acetylacetyl)-L-lysine ester (1.3 g, 1.14 mmol, 85% purity), after addition, the reaction mixture was stirred at 25 °C for 3 h. The reaction solution was directly evaporated to dryness to give the crude title compound (1.2 g).

[0669] Its structural characterization data are as follows:

[0670] MS m / z (ESI): 910.3 [M+H] +

[0671] Step 3: Preparation of tert-butyl(S)-6-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-acetylacetate (INT-12-3):

[0672] Add N to DMF (2 mL) 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2 -(25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-ynyl)-L-lysine crude product (600 mg), tert-butyl 3-aminopropionate hydrochloride (143.7 mg, 791.8 μmol), DIPEA (426.05 mg, 3.30 mmol), and HATU (426.14 mg, 1.12 mmol) were added, and the reaction mixture was stirred at 25 °C for 4 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (332 mg, 294.48 μmol).

[0673] Its structural characterization data are as follows:

[0674] MS m / z (ESI): 1037.4 [M+H] +

[0675] Step 4: Preparation of (S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-12)

[0676] 332 mg (320.09 μmol) of tert-butyl(S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triaza-tetane-31-acetylacetate was added to DCM (5 mL) and TFA (3 mL). After the addition was complete, the mixture was stirred at 25 °C for 2 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (250 mg, 234.43 μmol).

[0677] Its structural characterization data are as follows:

[0678] MS m / z (ESI): 981.5 [M+H] +

[0679] Example 13 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-13)

[0680] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-13-2)

[0681] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (10 g, 16.01 mmol) was dissolved in DMF (100 mL), and diethylamine (10 g, 16.01 mmol) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was diluted with water and extracted with ethyl acetate. The combined organic phases were dried and concentrated under reduced pressure to remove the solvent, giving the pale yellow title compound (6.44 g, 14.16 mmol).

[0682] Its structural characterization data are as follows:

[0683] MS m / z(ESI): 403 [M+H] +

[0684] Step Two: N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-13-3)

[0685] N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2 g, 4.97 mmol) and 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine ester (2.39 g, 5.47 mmol) were dissolved in N,N-dimethylacetamide (20 mL), and N,N-diisopropylethylamine (1.28 g, 9.94 mmol) was added. The reaction mixture was reacted at 20 °C for 2 hours. The reaction solution was subjected to rapid column chromatography (dichloromethane / methanol = 10 / 1) and then evaporated to dryness to give the title compound (2.4 g, 3.23 mmol).

[0686] Its structural characterization data are as follows:

[0687] MS m / z(ESI): 724 [M+H] +

[0688] Step 3: N 2 -(L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-13-4)

[0689] N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2.4 g, 3.32 mmol) was dissolved in N,N-dimethylacetamide (20 mL), and diethylamine (484.95 mg, 6.63 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and evaporated to dryness to give the title compound (1.98 g, 3.32 mmol).

[0690] Its structural characterization data are as follows:

[0691] MS m / z(ESI): 502 [M+H] +

[0692] Step 4: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-13)

[0693] N 2 -(L-valine)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (1.98 g, 3.25 mmol), 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester, and N,N-dimethylacetamide (20 mL) were dissolved in the solution, and DIPEA (1.26 g, 9.76 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, and the combined organic phases were dried and then evaporated to dryness to give the title compound (2.4 g, 3.03 mmol).

[0694] Its structural characterization data are as follows:

[0695] MS m / z(ESI): 752 [M+H] +

[0696] Example 14: Preparation of (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-yneamide)hexamide)propionic acid (INT-14)

[0697] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (1.15 g, 1.76 mmol), DIPEA (683.02 mg, 5.28 mmol), and HATU (736.83 mg, 1.94 mmol) were added to DMF (20 mL), and the mixture was stirred at 25 °C for 30 minutes. Then, 3-aminopropionic acid (235.42 mg, 2.64 mmol, FR) was added to the reaction mixture, and the mixture was stirred at 25 °C for 2.5 hours. The reaction mixture was concentrated to dryness and purified by lyophilization to obtain the title compound (170 mg, 234.85 μmol).

[0698] Its structural characterization data are as follows:

[0699] MS m / z(ESI): 724.1 [M+H] +

[0700] The purification method is as follows:

[0701] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0702] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0703] Example 15 of intermediate preparation: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-15)

[0704] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (INT-15-2)

[0705] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-1-methylpyridine trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80–85 °C for 16 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10–70%) and lyophilized to give the title compound (0.78 g, 1.51 mmol).

[0706] Its structural characterization data are as follows:

[0707] ESI-MS (m / z): 515.2 (M+H) +

[0708] Step 2: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-15-3)

[0709] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.78 g, 1.51 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) were dissolved in DMF (15 mL), DIPEA (1.17 g, 9.07 mmol) was added, and HATU (2.59 g, 6.80 mmol) was added in portions. The reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.74 g, 0.90 mmol).

[0710] Its structural characterization data are as follows:

[0711] ESI-MS (m / z): 824.4 (M+H) +

[0712] Step 3: Preparation of 3-(3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-15)

[0713] Benzyl 3-(3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.74 g, 0.90 mmol) was dissolved in ethanol (30 mL), 10% palladium on carbon (0.15 g) and acetic acid (0.14 g, 2.42 mmol) were added, the air was removed, hydrogen gas (balloon) was introduced, the temperature was raised to 40 °C and reacted for 4 hours, palladium on carbon was filtered off, the filtrate was concentrated, the residue was dissolved in water (15 mL) and acetonitrile (15 mL) and clarified, and lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0714] Its structural characterization data are as follows:

[0715] ESI-MS (m / z): 734.3 (M+H) + .

[0716] Example 16: Preparation of intermediate 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-16)

[0717] Step 1: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-16-1)

[0718] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1.07 g, 2.08 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, and HATU (3.56 g, 9.36 mmol) was added in portions. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0719] Its structural characterization data are as follows:

[0720] ESI-MS (m / z): 1046.5 (M+H) + .

[0721] Step 2: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-16)

[0722] Benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 mL). 10% palladium on carbon (0.13 g) and acetic acid (0.10 g, 1.70 mmol) were added. The air was removed, and hydrogen gas (in a balloon) was introduced. The mixture was heated to 40 °C and reacted for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0723] Its structural characterization data are as follows:

[0724] ESI-MS (m / z): 956.4 (M+H) + .

[0725] Preparation Example 1: (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide

[0726] Step 1: Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid tert-butyl ester (compound 7-1-2)

[0727] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione methanesulfonate (80 mg, 150.50 μmol), (S)-3-(tert-butoxycarbonyl)thiazolyl-4-carboxylic acid (70.22 mg, 301.01 μmol), HATU (171.68 mg, 451.5 μmol), and DIPEA (97.25 mg, 752.51 μmol) were added to a DMF (6 mL) reaction system. The reaction mixture was stirred at 25 °C for 18 hours. The reaction solution was prepared by high performance liquid chromatography and then freeze-dried to obtain the title compound (85 mg, 130.63 μmol).

[0728] Its structural characterization data are as follows:

[0729] MS m / z (ESI): 651.3 [M+H] +

[0730] Its preparation method is as follows:

[0731] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0732] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0733] Step 2: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (compound 7-1)

[0734] (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-carboxylic acid tert-butyl ester (60 mg, 92.21 μmol) was dissolved in HCl / 1,4-dioxane (5 mL) and stirred at 25 °C for 1 hour. The reaction solution was concentrated to obtain a crude product, which was then purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (25 mg, 42.16 μmol).

[0735] Its structural characterization data are as follows:

[0736] MS m / z (ESI): 551.2 [M+H] +

[0737] 1 H NMR(400MHz,DMSO)δ8.65(d,J=8.4Hz,1H),7.77(d,J=10.4Hz,1H),7.29(s,1H), 6.53(s,1H),5.60-5.50(m,1H),5.42(s,2H),5.28-5.08(m,2H),4.14(d,J=8.4H z,1H),4.00(d,J=7.6Hz,1H),3.90-3.80(m,1H),3.20-3.10(m,2H),3.08-2.90( m,2H),2.38(s,3H),2.27-2.05(m,2H),1.95-1.75(m,2H),0.87(t,J=6.0Hz,3H).

[0738] Its preparation method is as follows:

[0739] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0740] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0741] Preparation Example 2: Preparation of (S)-3-amino-8-ethyl-8-hydroxy-12-thio-2,8,12,14-tetrahydro-11H-cyclopentanopyrano[3',4':6,7]indoleazino[1,2-b]quinoline-9(1H)-one (5-1)

[0742] Step 1: Preparation of (S)-N-(8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide (5-1-2)

[0743] N-(7-amino-1-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (300.0 mg, 1.46 mmol), (S)-4-ethyl-4-hydroxy-10-thio-4,7,8,10-tetrahydro-1H-pyrano[3,4-f]indoleazine-3,6-dione (490.6 mg, 1.75 mmol), and p-toluenesulfonic acid pyridinium salt (440.3 mg, 1.75 mmol) were added to a single-necked flask, followed by toluene (50 mL). The mixture was heated to 130 °C and stirred for 8 hours under a reflux evaporator, then cooled to 100 °C and stirred for another 8 hours. After the reaction was complete, the mixture was concentrated to obtain a crude product, which was purified by rapid column chromatography (silica gel column, methanol / dichloromethane = 0.1) and dried to obtain the title compound (120.0 mg, 0.27 mmol, 85% purity).

[0744] Its structural characterization data are as follows:

[0745] MS m / z (ESI): 448.1 [M+H] +

[0746] Step 2: Preparation of (S)-3-amino-8-ethyl-8-hydroxy-12-thio-2,8,12,14-tetrahydro-11H-cyclopentanopyrano[3',4':6,7]indoleazino[1,2-b]quinoline-9(1H)-one (5-1)

[0747] (S)-N-(8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)acetamide (50.0 mg, 0.11 mmol, 85% purity) was added to a single-necked flask, followed by 6 mL of 50% hydrochloric acid aqueous solution. The mixture was heated to 80 °C and stirred for 4 hours. After the reaction was completed, the crude product was concentrated and purified by rapid column chromatography (silica gel column, methanol / dichloromethane = 0.2), and then dried to obtain the title compound (15.0 mg, 0.037 mmol, 93% purity).

[0748] Its structural characterization data are as follows:

[0749] MS m / z (ESI): 406.2 [M+H] +

[0750] Example 1: Preparation of (S)-3-((S)-7-benzyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosico-19-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-1)

[0751] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (B-1-1)

[0752] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (240 mg, 435.89 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (109.74 mg, 2.62 mmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (642.29 mg, 1.74 mmol) was added. After the addition was complete, the mixture was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (100 mg, 116.42 μmol).

[0753] Its structural characterization data are as follows:

[0754] MS m / z (ESI): 859.3 [M+H] +

[0755] Its preparation method is as follows:

[0756] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0757] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0758] Step 2: Preparation of (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-1-2)

[0759] (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (40 mg, 46.57 μmol) was dissolved in DMF (2 mL), and diethylamine (17.03 mg, 232.85 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (12 mg, 18.85 μmol).

[0760] Its structural characterization data are as follows:

[0761] MS m / z (ESI): 637.3 [M+H] +

[0762] Its preparation method is as follows:

[0763] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0764] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0765] Step 3: Preparation of (S)-3-((S)-7-benzyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosico-19-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-1)

[0766] (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (12 mg, 18 mg). 85 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (11.98 mg, 22.62 μmol) were dissolved in DMF (2 mL), and DIPEA (9.74 mg, 75.39 μmol) and HATU (14.32 mg, 37.69 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (18.25 mg, 15.42 μmol).

[0767] Its structural characterization data are as follows:

[0768] MS m / z (ESI): 1148.3 [M+H] +

[0769] Its preparation method is as follows:

[0770] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0771] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0772] Example 2: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (B-2)

[0773] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (B-2-1)

[0774] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (70 mg, 127.13 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (32.01 mg, 762.80 μmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (97.23 mg, 254.27 μmol) was added. After the addition was complete, the reaction was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (70 mg, 80.19 μmol).

[0775] Its structural characterization data are as follows:

[0776] MS m / z (ESI): 873.3 [M+H] +

[0777] Its preparation method is as follows:

[0778] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0779] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0780] Step 2: Preparation of (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-2-2)

[0781] (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (40 mg, 45.82 μmol) was dissolved in DMF (2 mL), and diethylamine (16.76 mg, 229.11 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (34 mg, 44.46 μmol).

[0782] Its structural characterization data are as follows:

[0783] MS m / z (ESI): 651.2 [M+H] +

[0784] Its preparation method is as follows:

[0785] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0786] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0787] Step 3: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (B-2)

[0788] (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (40mg, 52mg) (31 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (25.76 mg, 62.77 μmol) were dissolved in DMF (2 mL), and DIPEA (27.04 mg, 209.22 μmol) and HATU (39.75 mg, 104.61 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (41 mg, 38.52 μmol).

[0789] Its structural characterization data are as follows:

[0790] MS m / z (ESI): 1043.3 [M+H] +

[0791] Its preparation method is as follows:

[0792] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0793] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0794] Example 3: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl) Preparation of carbamoyl)thiazolidin-3-carboxyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-5)

[0795] Step 1: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-5-1)

[0796] INT-6-3 (6.18 g, 8.72 mmol) and (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (1.50 g, 2.18 mmol, 80% purity) were dissolved in DMF (20 mL) and pyridine (10 mL). 1-hydroxybenzotriazole (1.18 g, 0.93 mmol) was added, and the mixture was stirred at 85 °C for 1 hour. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (328 mg, 324.11 μmol).

[0797] Its structural characterization data are as follows:

[0798] MS m / z (ESI): 898.3 [M+H] +

[0799] Its preparation method is as follows:

[0800] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0801] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0802] Step 2: Preparation of 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-5-2)

[0803] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazole Alkyl-3-carboxylic acid ester (225 mg, 222.33 μmol) was dissolved in DMF (20 mL), and N,N-diisopropylethylamine (172.41 mg, 1.33 mmol), Fmoc-L-valine (264.10 mg, 778.17 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (262.12 mg, 889.34 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (125 mg, 102.51 μmol).

[0804] Its structural characterization data are as follows:

[0805] MS m / z (ESI): 1219.4 [M+H] +

[0806] Spectral column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0807] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0808] Step 3: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-5-3)

[0809] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (125 mg, 102.51 μmol) was dissolved in DMF (6 mL), and DBU (46.82 mg, 307.54 μmol) was added. The mixture was stirred at room temperature for 20 minutes. After the reaction was complete, the compound was purified by rapid column chromatography (C18, water / acetonitrile = 0-50%, 0.05% TFA) and then freeze-dried to obtain the title compound (91 mg, 81.90 μmol).

[0810] Its structural characterization data are as follows:

[0811] MS m / z(ESI): 998.0 [M+H]+

[0812] Step 4: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-5-4)

[0813] 2-(((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Acyl)thiazolidin-3-carboxylic acid ester (55 mg, 49.50 μmol) was dissolved in DMF (5 mL), and N,N-diisopropylethylamine (31.99 mg, 247.50 μmol), 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ethynic acid (26.56 mg, 99.00 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (43.77 mg, 148.50 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (45 mg, 36.08 μmol).

[0814] Its structural characterization data are as follows:

[0815] MS m / z (ESI): 1247.4 [M+H] +

[0816] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0817] Step 5: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-5-5)

[0818] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ [de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazoline-3-carboxylic acid ester (45 mg, 35.72 μmol) was dissolved in DMF (6 mL), followed by the addition of 1,3-dimethylbarbituric acid (27.88 mg, 178.58 μmol) and tetrakis(triphenylphosphine)palladium (20.64 mg, 17.86 μmol). The mixture was evacuated, purged with nitrogen three times, and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was analyzed by high performance liquid chromatography to obtain the title compound (29 mg, 24.68 μmol).

[0819] Its structural characterization data are as follows:

[0820] MS m / z (ESI): 1163.3 [M+H] +

[0821] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0822] Step Six: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl) Preparation of carbamoyl)thiazolidin-3-carboxyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-5)

[0823] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]in Dolino[1,2-b]quinoline-1-yl)carbamoyl)thiazoline-3-carboxylic acid ester (29 mg, 24.68 μmol) was dissolved in DMF (5 mL), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (37.13 mg, 74.04 μmol) and DIPEA (63.79 mg, 493.60 μmol) were added. The mixture was stirred at 40 °C for 3 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (8 mg, 4.57 μmol).

[0824] Its structural characterization data are as follows:

[0825] MS m / z (ESI): 1549.5 [M+H] +

[0826] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0827] Example 4: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino Preparation of (formyl)thiazolidin-3-carbonyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-6)

[0828] Step 1: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (B-6-1)

[0829] To a mixed solvent of dichloromethane (500 mL) and methanol (125 mL), (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (17.71 g, 36.25 mmol), (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidovalerate (15 g, 30.21 mmol), and EEDQ (14.94 g, 60.42 mmol) were added sequentially. After the addition was complete, the mixture was stirred at 30 °C for 4 hours. After removing most of the insoluble solvent by rotary evaporation, MTBE (350 mL) was added to the residue and stirred to induce crystallization. The crystals were then filtered and dried to obtain the crude product of the title compound (16.94 g).

[0830] Its structural characterization data are as follows:

[0831] ESI-MS (m / z): 990.3 [M+Na] + .

[0832] Step 2: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (B-6-2)

[0833] Add crude (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (16.90 g) and pyridine hydrofluoric acid salt (17.32 g, 174.73 mmol) to DMF (60 mL). After addition, heat to 50 °C and stir for 4 h. Cool to 25 °C, add water (350 mL) dropwise to the reaction solution, stir to induce crystallization, filter and dry to obtain crude (title compound) (13.4 g).

[0834] Its structural characterization data are as follows:

[0835] ESI-MS (m / z): 729.3 [M+H] + .

[0836] Step 3: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (B-6-3)

[0837] Add (12.9 g) crude allyl 5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate, 10.77 g (35.40 mmol), and DIPEA (9.15 g, 70.80 mmol) to DMF (100 mL). After addition, stir the mixture at 30 °C for 3 hours. Then, reduce the temperature to 0 °C and add the reaction mixture dropwise to water (400 mL) while stirring to induce crystallization. Filter and dry to obtain the crude title compound (13.5 g).

[0838] Its structural characterization data are as follows:

[0839] ESI-MS (m / z): 894.3 [M+H] + .

[0840] Step 4: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-6-4)

[0841] Add (6.11 g) of crude (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate crude product and (S)-N-((1S,9S)-9-ethyl- 5-Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (1 g, 1.45 mmol) and HOBt (785.30 mg, 5.81 mmol) were added, and the mixture was heated to 85 °C under nitrogen protection and stirred for 2 hours. The reaction solution was purified by rapid column chromatography (C18, 0.5% formic acid aqueous solution / acetonitrile = 10%–95%) and then freeze-dried to give the crude title compound (2.8 g, purity 15%, 321.73 μmol).

[0842] Its structural characterization data are as follows:

[0843] ESI-MS (m / z): 1306.4 [M+H] + .

[0844] Step 5: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-6-5)

[0845] Add 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxane to DMF (3 mL) Crude 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (1 g, 15% purity, 114.90 μmol) and diethylamine (25.51 mg, 344.71 μmol) were added, and the mixture was stirred at 30 °C for 3 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to give the trifluoroacetate of the title compound (84 mg, 70.16 μmol).

[0846] Its structural characterization data are as follows:

[0847] MS m / z (ESI): 1083.4 [M+H] +

[0848] Its preparation method is as follows:

[0849] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0850] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0851] Step Six: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-6-6)

[0852] Add 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1, [2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (42 mg, 35.08 μmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (11.29 mg, 42.10 μmol), DIPEA (22.67 mg, 175.41 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (19.37 mg, 70.16 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (39 mg, 29.25 μmol).

[0853] Its structural characterization data are as follows:

[0854] MS m / z (ESI): 1334.4 [M+H] +

[0855] Its preparation method is as follows:

[0856] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0857] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0858] Step 7: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-6-7)

[0859] Under nitrogen protection, 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1 0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (30 mg, 22.50 μmol), tetraphenylphosphine palladium (12.99 mg, 11.25 μmol), and 1,3-dimethylbarbituric acid (5.27 mg, 33.75 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate salt of the title compound (13 mg, 10.04 μmol).

[0860] Its structural characterization data are as follows:

[0861] MS m / z (ESI): 1250.4 [M+H] +

[0862] Its preparation method is as follows:

[0863] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0864] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0865] Step 8: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Preparation of acyl)thiazolidin-3-carbonyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-6)

[0866] Add 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3- The formate of the title compound (13 mg, 10.04 μmol), DIPEA (19.45 mg, 150.53 μmol), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (15.10 mg, 30.11 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to give the formate of the title compound (3.42 mg, 1.93 μmol).

[0867] Its structural characterization data are as follows:

[0868] MS m / z (ESI): 1636.5 [M+H] +

[0869] Its preparation method is as follows:

[0870] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0871] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0872] Example 5: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl Preparation of (2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-7)

[0873] Step 1: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-7-1)

[0874] 2-(((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (55mg, 49.5g) 0 μmol) was dissolved in DMF (5 mL), and N,N-diisopropylethylamine (31.99 mg, 247.50 μmol), 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (61.56 mg, 99.00 μmol, purity 90%) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (43.77 mg, 148.50 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (52 mg, 33.46 μmol).

[0875] Its structural characterization data are as follows:

[0876] MS m / z (ESI): 1538.5 [M+H] +

[0877] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0878] Step 2: Preparation of 4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-7-2)

[0879] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylenamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (52 mg, 33.46 μmol) was dissolved in DMF (6 mL), followed by the addition of 1,3-dimethylbarbituric acid (26.12 mg, 167.28 μmol) and tetrakis(triphenylphosphine)palladium (19.33 mg, 16.73 μmol). The mixture was evacuated under vacuum, purged three times with nitrogen, and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (41 mg, 27.05 μmol).

[0880] Its structural characterization data are as follows:

[0881] MS m / z (ESI): 1454.4 [M+H] +

[0882] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0883] Step 3: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl Preparation of (2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-7)

[0884] 4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] Pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazoline-3-carboxylic acid ester (41 mg, 27.05 μmol) was dissolved in DMF (3 mL), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (41.98 mg, 83.71 μmol) and N,N-diisopropylethylamine (72.13 mg, 558.08 μmol) were added. The mixture was stirred at 40 °C for 3 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (14 mg, 6.80 μmol).

[0885] Its structural characterization data are as follows:

[0886] MS m / z(ESI): 1841.6 [M+H]+

[0887] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0888] Example 6: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)- Preparation of 5-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-8)

[0889] Step 1: 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-8-1)

[0890] Add 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin- The trifluoroacetate of 3-carboxylic acid ester (42 mg, 35.08 μmol), DIPEA (22.67 mg, 175.41 μmol), 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (29.45 mg, 52.62 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (19.37 mg, 70.16 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high-performance liquid chromatography and then freeze-dried to give the title compound (37 mg, 20.95 μmol). Its structural characterization data are as follows:

[0891] MS m / z (ESI): 1625.5 [M+H] +

[0892] Its preparation method is as follows:

[0893] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0894] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0895] Step 2: Preparation of 4-((2S,5S)-5-isopropyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-8-2)

[0896] Under nitrogen protection, 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-) Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (30 mg, 18.46 μmol), tetraphenylphosphine palladium (10.66 mg, 9.23 μmol), and 1,3-dimethylbarbituric acid (4.32 mg, 27.70 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate salt of the title compound (19 mg, 11.02 μmol).

[0897] Its structural characterization data are as follows:

[0898] MS m / z (ESI): 1541.4 [M+H] +

[0899] Its preparation method is as follows:

[0900] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0901] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0902] Step 3: 2,2',2”-(10-(2-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)- Preparation of 5-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-8)

[0903] Add 4-((2S,5S)-5-isopropyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2- [b] Formate of quinolino-1-yl)carbamoyl)thiazolidin-3-carboxylic acid ester (19 g, 11.02 μmol), DIPEA (30.95 mg, 239.48 μmol), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (18.01 mg, 35.92 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate of the title compound (2.04 mg, 0.982 μmol).

[0904] Its structural characterization data are as follows:

[0905] MS m / z (ESI): 1927.7 [M+H] +

[0906] Its preparation method is as follows:

[0907] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0908] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0909] Example 7: 2,2',2”-(10-((2S,5S,12S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino Preparation of (B-9) thiazolyl-3-carboxylic acid alkynyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamide)-1,4,7,11,18-pentoxo-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0910] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (B-9-2)

[0911] (4-Aminophenyl)methanol (300.0 mg, 2.44 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (1.2 g, 2.93 mmol) were dissolved in DCM (28 mL) and MeOH (7 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.81 g, 7.32 mmol) was added. The mixture was stirred at room temperature for 15 hours and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (881.3 mg, 1.7 mmol, 90% purity).

[0912] Its structural characterization data are as follows:

[0913] MS m / z (ESI): 516.2 [M+H] +

[0914] Step 2: Preparation of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate (B-9-3)

[0915] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (200.0 mg, 0.39 mmol, 90% purity) was dissolved in DMF (3 mL), DIPEA (100.2 mg, 0.78 mmol) and p-nitrophenyl chloroformate (94.1 mg, 0.47 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 3 / 2), and lyophilized to give the title compound (185.6 mg, 0.27 mmol, 90% purity).

[0916] Its structural characterization data are as follows:

[0917] MS m / z (ESI): 703.2 [M+Na] +

[0918] Step 3: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-9-4)

[0919] Add (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate (247.26 mg, 363.24 μmol) and (S) to DMF (2 mL) and pyridine (1 mL) respectively. 3-Amino-8-ethyl-8-hydroxy-12-thio-2,8,12,14-tetrahydro-11H-cyclopentanopyrano[3',4':6,7]indolazino[1,2-b]quinoline-9(1H)-one (50 mg, 90.81 μmol) and HOBt (49.08 mg, 363.24 μmol) were added, and the mixture was heated to 85 °C and stirred for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (24 mg, 21.97 μmol).

[0920] Its structural characterization data are as follows:

[0921] MS m / z(ESI): 1092.3 [M+H] +

[0922] Its preparation method is as follows:

[0923] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0924] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0925] Step 4: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-9-5)

[0926] Add 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (24 mg, 21.97 μmol) and diethylamine (18.7 mg, 256.4 μmol) to DMF (2 mL). After the addition is complete, stir the mixture at 30 °C for 2 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried again to obtain the formate of the title compound (18 mg, 19.65 μmol).

[0927] Its structural characterization data are as follows:

[0928] MS m / z (ESI): 870.3 [M+H] +

[0929] Its preparation method is as follows:

[0930] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0931] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0932] Step 5: Preparation of 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-9-6)

[0933] Add 18 mg of formate of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester to DMF (2 mL). 19.65 μmol), DIPEA (12.70 mg, 98.26 μmol), (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)hexamide)propionic acid (21.34 mg, 29.48 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (10.85 mg, 39.30 μmol) were added, and the mixture was stirred at 30 °C for 3 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (18 mg, 10.51 μmol).

[0934] Its structural characterization data are as follows:

[0935] MS m / z (ESI): 1576.5 [M+H] +

[0936] Its preparation method is as follows:

[0937] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0938] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0939] Step Six: Preparation of 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-9-7)

[0940] Add 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)benzyl(S)-4-(((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (18 mg, 10.51 μmol), after addition, the reaction mixture was stirred at 30 °C for 3 hours. The reaction solution was evaporated to dryness to give crude formate of the title compound (15 mg).

[0941] Its structural characterization data are as follows:

[0942] MS m / z (ESI): 1319.6 [M+H] +

[0943] Step 7: 2,2',2”-(10-((2S,5S,12S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino Preparation of (B-9) thiazolyl-3-carboxylic acid alkynyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetamide)-1,4,7,11,18-pentoxo-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0944] Add 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline- Crude formate of 1-yl)carbamoyl)thiazolidin-3-carboxylic acid ester (15 mg), DIPEA (14.20 mg, 109.85 μmol), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (16.53 mg, 32.95 μmol) were added and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate of the title compound (11.38 mg, 6.17 μmol).

[0945] Its structural characterization data are as follows:

[0946] MS m / z(ESI): 1706.6 [M+H]+

[0947] Its preparation method is as follows:

[0948] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0949] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0950] Example 8: 2,2',2”-(10-((6S,9S,16S)-1-amino-6-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl) Preparation of carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,8,11,15,22-pentaoxo-2,7,10,14,21-pentazatoridecane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-10)

[0951] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-10-1)

[0952] Add (S)-3-amino-8-ethyl-8-hydroxy-12-thio-2,8,12,14-tetrahydro-11H-cyclopentanopyrano[3',4':6,7]indolazino[1,2-b]quinoline-9(1H)-one (500 mg, 908.10 μmol), N-(4-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)oxy)phenyl)-N-oxohydroxyammonium (2.79 g, 3.63 mmol) and HOBt (490.81 mg, 3.63 mmol) to DMF (15 mL) and pyridine (7.5 mL). After addition, heat to 75 °C and stir for 3 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried again to obtain the crude product of the title compound (200 mg, 84.87 μmol, 50% purity).

[0953] Its structural characterization data are as follows:

[0954] MS m / z (ESI): 1178.4 [M+H] +

[0955] Its preparation method is as follows:

[0956] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0957] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0958] Step 2: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-10-2)

[0959] Add crude 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (85 mg) and 1,8-diazobisspiro[5.4.0]undec-7-ene (21.96 mg, 144.28 μmol) to DMF (1 mL). After addition, stir the mixture at 30 °C for 15 min. The reaction solution is used directly in the next step.

[0960] Step 3: Preparation of 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-2-(3-ureopropyl)-3,6,10,13-tetraazanonadecan-18-acetylamamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-10-3)

[0961] DIPEA (55.15 mg, 426.76 μmol), (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionic acid (77.23 mg, 106.69 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (39.26 mg, 142.25 μmol) were added to the reaction solution from the previous step. After the addition was complete, the mixture was heated to 45 °C and stirred for 3 hours. The reaction solution was purified by preparative high-performance liquid chromatography and then freeze-dried again to obtain the title compound (32 mg, 17.33 μmol, 90% purity).

[0962] Its structural characterization data are as follows:

[0963] MS m / z (ESI): 1661.6 [M+H] +

[0964] Its preparation method is as follows:

[0965] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0966] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0967] Step 4: Preparation of 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-2-(3-ureopropyl)-3,6,10,13-tetraazanonadecan-18-acetylamidamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-10-4)

[0968] Add 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-2-(3-ureopropyl)-3,6,10,13-tetraazanonadecan-18-acetylamidamide)benzyl(S)-4-((( 1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (32 mg, 17.33 μmol, 90% purity) was added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was evaporated to dryness to give the formate salt (27 mg) of the crude title compound.

[0969] Its structural characterization data are as follows:

[0970] MS m / z (ESI): 1405.5 [M+H] +

[0971] Step 5: 2,2',2”-(10-((6S,9S,16S)-1-amino-6-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl) Preparation of carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,8,11,15,22-pentaoxo-2,7,10,14,21-pentazatoridecane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-10)

[0972] Add 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-2-(3-ureopropyl)-3,6,10,13-tetraazanonadecan-18-acetylamidamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline to DMF (3 mL) The crude formate of lin-1-yl)carbamoyl)thiazolidin-3-carboxylic acid ester (27 mg), DIPEA (24.04 mg, 186.00 μmol), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (27.98 mg, 55.80 μmol) were added, and the mixture was stirred at 30 °C for 3 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the trifluoroacetate of the title compound (18.22 mg, 9.08 μmol).

[0973] Its structural characterization data are as follows:

[0974] MS m / z (ESI): 896.5 [M / 2+H] +

[0975] Its preparation method is as follows:

[0976] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0977] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0978] Example 9: 2,2',2”-(10-((S)-8-((3-(((S)-1-(((S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy Preparation of methyl)phenyl)aamino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-oxopropyl)carbamoyl)-3,4-(2-(methylsulfonyl)pyrimidin-5-yl)-2,10,29-trioxo-13,16,19,22,25-pentaoxa-3,9,28-triazatetratetradecane-33-yn-1-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-11)

[0979] Step 1: 4-((2S,5S,12S)-12-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-5-isopropyl-2-methyl-38-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctacosane-3 Preparation of 7-acetylamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-11-1)

[0980] Add 18 mg (19.65 μmol) of formate of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester and 15.24 μmol of DIPEA to DMF (2 mL). (117.91 μmol g), (S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatriadodecane-31-alkynyl acid (28.92 mg, 29.48 μmol) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (10.85 mg, 39.30 μmol) were added, and the mixture was stirred at 30 °C for 3 hours. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (20 mg, 10.37 μmol).

[0981] Its structural characterization data are as follows:

[0982] MS m / z (ESI): 1832.6 [M / 2+H] +

[0983] Its preparation method is as follows:

[0984] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0985] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0986] Step 2: 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-38-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctadecane-37-acetylamide)benzyl(S Preparation of 4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-11-2)

[0987] Add 4-((2S,5S,12S)-12-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyl)-5-isopropyl-2-methyl-38-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctadecane-37-acetylamido)benzyl(S)-4-(((1S,9S)-9-ethyl) to DMF (2 mL). 5-Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (20 mg, 10.37 μmol) and 1,8-diazobisspirocyclic[5.4.0]undecyl-7-ene (5.50 mg, 21.82 μmol) were added, and the mixture was stirred at 30 °C for 15 min. The reaction solution was used directly in the next step.

[0988] Its structural characterization data are as follows:

[0989] MS m / z (ESI): 1610.7 [M / 2+H] +

[0990] Step 3: 2,2',2”-(10-((S)-8-((3-(((S)-1-(((S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy Preparation of methyl)phenyl)aamino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-oxopropyl)carbamoyl)-3,4-(2-(methylsulfonyl)pyrimidin-5-yl)-2,10,29-trioxo-13,16,19,22,25-pentaoxa-3,9,28-triazatetratetradecane-33-yn-1-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-11)

[0991] DIPEA (2.73 mg, 21.11 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (15.88 mg, 31.66 μmol) were added to the reaction solution from the previous step. After the addition was complete, the mixture was stirred at 30 °C for 3 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried again to obtain the formate of the title compound (12 mg, 5.58 μmol).

[0992] Its structural characterization data are as follows:

[0993] MS m / z(ESI): 999.2 [M / 2+H] +

[0994] Its preparation method is as follows:

[0995] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0996] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0997] Example 10: 2,2',2”-(10-((S)-8-((3-(((S)-1-(((S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl Preparation of B-12 (B-12) (B-12)-(2-(methylsulfonyl)pyrimidin-5-yl)-2,10,29-trioxo-13,16,19,22,25-pentaoxa-3,9,28-triazatetratetradecane-33-yn-1-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-12)

[0998] Step 1: 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-38-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-2-(3-ureopropyl)-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctacosane-3 Preparation of 7-acetylamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-12-1)

[0999] 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (32 mg, 33.47 μmol) was dissolved in DMF (5 mL), and (S)-6-(4 -((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatriadodecane-31-alkynyl acid (84.95 mg, 83.68 μmol) was added, along with N,N-diisopropylethylamine (25.95 mg, 200.83 μmol) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (39.46 mg, 133.88 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was subjected to high-performance liquid chromatography to obtain the title compound (16 mg, 8.19 μmol).

[1000] Its structural characterization data are as follows:

[1001] MS m / z (ESI): 1953.7 [M+H] +

[1002] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1003] Step 2: 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-38-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-2-(3-ureopropyl)-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctadecane-37-acetylamide) benzyl Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-12-2)

[1004] 4-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-38-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-2-(3-ureopropyl)-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctadecane-37-acetylamido)benzyl(S)-4-((( 1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (26 mg, 13.31 μmol) was dissolved in DCM (2 mL) and formic acid (6 mL) and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated to dryness to give the title compound (crude product) (23 mg, 13.20 μmol).

[1005] Its structural characterization data are as follows:

[1006] MS m / z (ESI): 1697.6 [M+H] +

[1007] Step 3: 2,2',2”-(10-((S)-8-((3-(((S)-1-(((S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl Preparation of B-12 (B-12) (B-12)-(2-(methylsulfonyl)pyrimidin-5-yl)-2,10,29-trioxo-13,16,19,22,25-pentaoxa-3,9,28-triazatetratetradecane-33-yn-1-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-12)

[1008] 4-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-38-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14,33-pentoxo-2-(3-ureopropyl)-17,20,23,26,29-pentoxa-3,6,10,13,32-pentazaoctadecane-37-acetylamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexadecane) Hydrogen-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (26 mg, 13.20 μmol) was dissolved in DMF (3 mL), followed by the addition of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (19.85 mg, 39.59 μmol), and then DIPEA (34.11 mg, 263.92 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (10.50 mg, 4.83 μmol).

[1009] Its structural characterization data are as follows:

[1010] MS m / z (ESI): 1042.1 [0.5M+H] +

[1011] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1012] Example 11: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-14)

[1013] 4-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3- A carboxylic acid ester (14 mg, 14.64 μmol) was dissolved in DMF (4 mL), and N,N-diisopropylethylamine (5.68 mg, 43.93 μmol), 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ethynic acid (3.93 mg, 14.64 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (8.63 mg, 29.29 μmol) were added. The mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (4.50 mg, 3.58 μmol).

[1014] Its structural characterization data are as follows:

[1015] MS m / z (ESI): 1206.4 [M+H] +

[1016] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1017] Example 12: Preparation of 4-((2S,5S)-5-isopropyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-30-acetylamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (B-16)

[1018] 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-15-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (28 mg, 29.29 μmol) was dissolved in... In DMF (6 mL), N,N-diisopropylethylamine (11.36 mg, 87.86 μmol), 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (27.32 mg, 29.29 μmol, purity 60%), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (17.26 mg, 58.57 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (15 mg, 9.61 μmol).

[1019] Its structural characterization data are as follows:

[1020] MS m / z (ESI): 1497.5 [M+H] +

[1021] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1022] Example 13: N-((35S,38S)-39-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-35-isopropyl-3,6,9,12,15,18,2 Preparation of 1,24,27,38-Decamethyl-2,5,8,11,14,17,20,23,26,29,33,36,39-Tribo-3,6,9,12,15,18,21,24,27,30,34,37-Dodecazanechoyl)-N-methyl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (D-1)

[1023] Step 1: Preparation of ((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentano[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)tert-butyl carbamate (D-1-1)

[1024] (S)-3-amino-8-ethyl-8-hydroxy-12-thio-2,8,12,14-tetrahydro-11H-cyclopentanopyrano[3',4':6,7]indolazino[1,2-b]quinoline-9(1H)-one (40.0 mg, 0.098 mmol, 93% purity) and (tert-butoxycarbonyl)-L-alanine (112.1 mg, 0.59 mmol) were dissolved in DMF (3 mL). HATU (112.6 mg, 0.30 mmol) and DIPEA (38.2 mg, 0.30 mmol) were added, and the mixture was heated to 50 °C and stirred for 15 hours. After removing most of the reaction solution, the mixture was purified by rapid column chromatography (silica gel column, methanol / dichloromethane = 0.2) and concentrated again to obtain the title compound (39.6 mg, 0.07 mmol, 90% purity).

[1025] Its structural characterization data are as follows:

[1026] MS m / z (ESI): 577.2 [M+H] +

[1027] Step 2: Preparation of (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)propionamide (D-1-2)

[1028] ((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentano[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)tert-butyl carbamate (39.6 mg, 0.07 mmol, 90% purity) was added to a single-necked flask, followed by the addition of dichloromethane (2 mL) and then trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated at room temperature to obtain the crude product of the title compound (30.7 mg, 0.064 mmol, 80% purity).

[1029] Its structural characterization data are as follows:

[1030] MS m / z (ESI): 477.2 [M+H] +

[1031] Step 3: Preparation of (9H-fluorene-9-yl)methyl(3-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-oxopropyl)carbamate (D-1-3)

[1032] (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)propionamide (30.7 mg, 0.064 mmol, 80% purity) and (3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionyl)-L-valine (26.2 mg, 0.064 mmol) were dissolved in DMF (3 mL), and HATU (29.2 mg, 0.077 mmol) and DIPEA (24.8 mg, 0.19 mmol) were added. The mixture was stirred at room temperature for 1 hour. After removing most of the reaction solution, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and then freeze-dried to obtain the title compound (22.2 mg, 0.025 mmol, 80% purity).

[1033] Its structural characterization data are as follows:

[1034] MS m / z (ESI): 869.3 [M+H] +

[1035] Step 4: Preparation of (S)-2-(3-aminopropamido)-N-((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (D-1-4)

[1036] (9H-fluorene-9-yl)methyl(3-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-oxopropyl)carbamate (22.2 mg, 0.025 mmol, 80% purity) was dissolved in DMF (1.5 mL), and diethylamine (22.1 mg, 0.3 mmol) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was concentrated, and 10 mL of (ethyl acetate / petroleum ether = 1 / 5) was added and stirred for 1 hour. The solvent was removed to obtain the title compound (14.6 mg, 0.022 mmol, 80% purity).

[1037] Its structural characterization data are as follows:

[1038] MS m / z (ESI): 647.2 [M+H] +

[1039] Step 5: N-((35S,38S)-39-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-35-isopropyl-3,6,9,12,15,18,21 Preparation of 24,27,38-Decamethyl-2,5,8,11,14,17,20,23,26,29,33,36,39-Tribo-3,6,9,12,15,18,21,24,27,30,34,37-Dodecazanechoyl)-N-methyl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (D-1)

[1040] (S)-2-(3-aminopropamido)-N-((S)-1-(((S)-8-ethyl-8-hydroxy-9-oxo-12-thio-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (14.6 mg, 0.022 mmol, 80% purity), 3,6,9,12,15,18,21,24,27 30-Decamethyl-36-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexadecane-35-alkynyl acid (22.1 mg, 0.026 mmol) was dissolved in DMF (2 mL), and HATU (12.5 mg, 0.033 mmol) and DIPEA (8.8 mg, 0.066 mmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (1.72 mg, 0.001 mmol, 95.1% purity).

[1041] Its structural characterization data are as follows:

[1042] MS m / z (ESI): 1607.9 [M+H] +

[1043] Its preparation method is as follows:

[1044] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1045] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1046] Example 14: Preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-3)

[1047] Step 1: Preparation of (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-3-1)

[1048] (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (100 mg, 133.21 μmol) and N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (110.18 mg, 146.53 μmol) was dissolved in DMF (2 mL), and DIPEA (68.86 mg, 532.82 μmol) and HATU (119.37 mg, 314.12 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (60 mg, 43.78 μmol).

[1049] Its structural characterization data are as follows:

[1050] MS m / z (ESI): 1370.5 [M+H] +

[1051] Its preparation method is as follows:

[1052] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1053] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1054] Step 2: Preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (B-3)

[1055] (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (80 mg, 58.37 μmol) was dissolved in DCM (5 mL), and formic acid (1 mL) was added. The mixture was stirred at 22 °C for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (11 mg, 8.51 μmol, purity 95%).

[1056] Its structural characterization data are as follows:

[1057] MS m / z (ESI): 1114.4 [M+H] +

[1058] Its preparation method is as follows:

[1059] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1060] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1061] Example 15: 2,2',2”-(10-(2-(((S)-6-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline) Preparation of -1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-4)

[1062] The (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxyl Amine (7 mg, 6.28 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9.45 mg, 18.85 μmol) were dissolved in DMF (2 mL). DIPEA (1.0 mg, 7.74 μmol) was added, and the mixture was stirred at 22 °C for 2 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (3.2 mg, 2.03 μmol, purity 95%).

[1063] Its structural characterization data are as follows:

[1064] MS m / z (ESI): 1500.6 [M+H] +

[1065] Its preparation method is as follows:

[1066] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1067] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1068] Example 16: (17S,20S)-6,6-bis((2-carboxyethoxy)methyl)-17-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7 Preparation of indoleazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-20-isopropyl-27-(2-(methylsulfonyl)pyrimidin-5-yl)-11,19,22-trioxo-4,8-dioxa-12,18,21-triazaheptadecane-26-alkynic acid (B-18)

[1069] The (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]in Dolino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μmol) and 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (27.64 mg, 65.13 μmol) were dissolved in DMF (2 mL). DIPEA (8.42 mg, 65.13 μmol) and HATU (13.64 mg, 35.90 μmol) were added, and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (5.0 mg, 3.09 μmol, purity 94%).

[1070] Its structural characterization data are as follows:

[1071] MS m / z (ESI): 1520.4 [M+H] +

[1072] Its preparation method is as follows:

[1073] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1074] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1075] Example 17: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazine) Preparation of [1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetradecane-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (B-19)

[1076] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)thiazolidin-4-carboxamide Dissolve 6 mg (4.85 μmol) and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (5.93 mg, 7.27 μmol) in DMF (1 mL), add 2,6-dimethylpyridine (2.08 mg, 19.38 μmol) and HATU (3.69 mg, 9.69 μmol), and stir at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.18 mg, 1.65 μmol, purity 95%).

[1077] Its structural characterization data are as follows:

[1078] MS m / z(ESI): 1829.7 [M+H] +

[1079] Its preparation method is as follows:

[1080] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1081] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1082] Example 18: 3,3'-((2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-2 Preparation of 1-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetradecane-20-yn-1-yl)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(B-20)

[1083] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μL) 20.24 mg (21.17 μmol) of methylpropoxy ((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (20.24 mg, 21.17 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (8.42 mg, 65.13 μmol) and HATU (12.37 mg, 32.57 μmol), and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (8.8 mg, 4.07 μmol, purity 95%).

[1084] Its structural characterization data are as follows:

[1085] MS m / z (ESI): 1026.9 [M / 2+H] +

[1086] Its preparation method is as follows:

[1087] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1088] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1089] Example 19: 4-((S)-2-(4-aminobutyl)-3S-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecanoyl) Preparation of (Amino)benzyl ((1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-21)

[1090] Step 1: Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (B-21-1)

[1091] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (800 mg, 1.45 mmol), allyl chloroformate (262.70 mg, 2.18 mmol), and DIPEA (375.56 mg, 2.91 mmol) were added to DMF (7 mL). After the addition was complete, the mixture was stirred at 28 °C for 2 h. The reaction solution was directly purified by reverse-phase chromatography (acetonitrile / water (0.05% formic acid) = 0-80%) and lyophilized to give the title compound (758 mg, 1.19 mmol).

[1092] Its structural characterization data are as follows:

[1093] ESI-MS(m / z): 635.4(M+H)+.

[1094] Step 2: Preparation of (S)-4-(((1S,9S)-9-ethyl-5-fluoro-4-methyl-9-(((4-nitrophenoxy)carbonyl)oxy)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (B-21-2)

[1095] Add (S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (758 mg, 1.19 mmol), DMAP (364.77 mg, 2.99 mmol), and 4-nitrophenyl chloroformate (601.83 mg, 2.99 mmol) to DCM (8 mL). After the addition is complete, stir the mixture at 28 °C for 3 h. The reaction mixture is evaporated to dryness, and MTBE:PE = (1:1) (15 mL) is added and stirred. The mixture is filtered, and the filter cake is dried to obtain the crude product of the title compound (1.32 g).

[1096] Its structural characterization data are as follows:

[1097] ESI-MS(m / z): 800.4(M+H)+.

[1098] Step 3: Preparation of (S)-4-(((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-nonoxa-3,9-diazapentadecanoamide)benzyl)oxy)carbonyl)oxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (B-21-3)

[1099] Add crude (S)-4-(((1S,9S)-9-ethyl-5-fluoro-4-methyl-9-(((4-nitrophenoxy)carbonyl)oxy)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (1.32 g) and DMAP (436.7 g) to anhydrous DCM (10 mL). 3 mg (3.57 mmol) was added, and after nitrogen purging, a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatridodecanoamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexamethyleneamide (1.26 g, 1.19 mmol) in DCM (31 mL) was slowly added under nitrogen protection. After the addition was complete, the mixture was stirred at 26 °C for 20 h. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography to obtain the title compound (953 mg, 498.40 μmol, 90% purity).

[1100] Column: Phenomenex C18 150mm×25mm×10μm

[1101] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate aqueous solution)

[1102] Its structural characterization is as follows:

[1103] ESI-MS (m / z): 1720.6 (M+H) + .

[1104] Step 4: Preparation of 4-((S)-35-azido-2-(4-((((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl[(1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-21-4)

[1105] Add (S)-4-(((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-nonazo-3,9-diazapentadecanoamide)benzyl)oxy)carbonyl)oxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid allyl ester (350 mg, 203.38 μmol), tetraphenylphosphine palladium (23.50 mg, 20.34 μmol), and 1,3-dimethylbarbituric acid (31.75 mg, 20.34 μmol) were added, purged with nitrogen, and stirred at 25 °C for 1 h. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (211 mg, 116.02 μmol, 90% purity).

[1106] Column: Phenomenex C18 150mm×25mm×10μm

[1107] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate aqueous solution)

[1108] Its structural characterization is as follows:

[1109] ESI-MS (m / z): 1637.6 (M+H) + .

[1110] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl[(1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-21-5)

[1111] Add 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecanoamide)benzyl[(1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13 -dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (100 mg, 61.09 mol), formic acid (0.15 mL), after addition, the mixture was purged with nitrogen and stirred at 25 °C for 1 h. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography to obtain the formate salt of the title compound (68 mg, 45.80 μmol).

[1112] Column: Phenomenex C18 150mm×25mm×10μm

[1113] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid aqueous solution)

[1114] Its structural characterization is as follows:

[1115] ESI-MS(m / z):1365.5(M+H)+.

[1116] Step Six: 4-((S)-2-(4-aminobutyl)-3S-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazatriapentadecanamide Preparation of benzyl((1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-21)

[1117] Add 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl[(1S,9S)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-1-((S)-thiazolidin-4-carboxamido)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] to DMSO (1.5 mL). The formate of pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (68 mg, 45.80 μmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (14.72 mg, 45.80 μmol), and water (0.15 mL) were reacted with nitrogen and then cuprous bromide (27.66 mg, 192.84 μmol). After the addition was complete, the mixture was stirred at 25 °C for 40 min. The reaction solution was purified by high performance liquid chromatography to obtain the formate of the title compound (12.80 mg, 6.86 μmol, 92% purity).

[1118] Column: Phenomenex C18 150mm×25mm×10μm

[1119] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid aqueous solution)

[1120] Its structural characterization is as follows:

[1121] ESI-MS (m / z): 1670.4 (M+H) + .

[1122] Coupling Examples

[1123] 1. Example: Preparation of Trastuzumab-B-1

[1124] Take 0.82 mL of Trastuzumab antibody (24.4 mg / mL), add 56.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide B-1 (147.0 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-1). The DAR value was determined by mass spectrometry to be 7.5.

[1125] 2. Example: Preparation of Trastuzumab-B-2

[1126] Take 0.82 mL of Trastuzumab antibody (24.4 mg / mL), add 56.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide B-2 (141.0 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-2). The DAR value was determined by mass spectrometry to be 7.4.

[1127] 3. Example: Preparation of Trastuzumab-B-3

[1128] Take 0.55 mL of Trastuzumab antibody (24.4 mg / mL), add 42.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 50.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide B-3 (97.3 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-3). The DAR value was determined by mass spectrometry to be 7.4.

[1129] 4. Example: Preparation of Trastuzumab-B-4

[1130] Take 1.013 mL of Trastuzumab antibody (14.8 mg / mL), add 51.8 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 15 mM TCEP (tris(2-carboxyethyl)phosphine, 38.0 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide B-4 (97.3 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-4). The DAR value was determined by mass spectrometry to be 6.4.

[1131] 5. Example: Preparation of Trastuzumab-B-5

[1132] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-5 (174.0 μL, 10 mM), mix well, and incubate at room temperature for 2 h. After that, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-5). The DAR value was determined by mass spectrometry to be 8.03.

[1133] 6. Example: Preparation of Trastuzumab-B-6

[1134] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-6 (174.0 μL, 10 mM), mix well, and incubate at room temperature for 2 h. After that, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-6). The DAR value was determined by mass spectrometry to be 8.0.

[1135] 7. Example: Preparation of Trastuzumab-B-7

[1136] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-7 (174.0 μL, 10 mM), mix well, and incubate at room temperature for 2 h. After that, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-7). The DAR value was determined by mass spectrometry to be 8.0.

[1137] 8. Example: Preparation of Trastuzumab-B-8

[1138] Take 0.534 mL of Trastuzumab antibody (20.9 mg / mL), add 27.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 42.3 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-8 solution (97.2 μL, 10 mM), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-8). The DAR value was determined by mass spectrometry to be 8.1.

[1139] 9. Example: Preparation of Trastuzumab-B-9

[1140] Take 0.82 mL of Trastuzumab antibody (24.4 mg / mL), add 56.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-9 (174.0 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-9). The DAR value was determined by mass spectrometry to be 8.05.

[1141] 10. Example: Preparation of Trastuzumab-B-10

[1142] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), add 48.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in B-10 (174.0 μL, 10 mM) in dimethyl sulfoxide solution, mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-10). The DAR value was determined by mass spectrometry to be 8.0.

[1143] 11. Example: Preparation of Trastuzumab-B-11

[1144] Take 0.82 mL of Trastuzumab antibody (24.4 mg / mL), add 41.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 76.0 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-11 (174.0 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-11). The DAR value was determined by mass spectrometry to be 8.01.

[1145] 12. Example: Preparation of Trastuzumab-B-12

[1146] Take 0.574 mL of Trastuzumab antibody (20.9 mg / mL), add 28.7 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), adjust the pH to 7.6 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 45.5 μL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Then add 12 times the amount of antibody dissolved in dimethyl sulfoxide B-12 (101.2 μL, 10 mM), mix well, and let stand at room temperature for 2 h. After completion, use a NAP gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 to obtain the antibody-drug conjugate (Trastuzumab-B-12). The DAR value was determined by mass spectrometry to be 8.0.

[1147] 13. Example: Preparation of Trastuzumab-B-14 (DAR8)

[1148] 0.819 mL of Trastuzumab antibody (24.4 mg / mL) was diluted with 66.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 143.5 μL of B-14 solution dissolved in dimethyl sulfoxide (10 mM, 10 molar equivalents of the antibody) was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-14). The DAR value was 7.78 as determined by mass spectrometry.

[1149] 14. Example: Preparation of Trastuzumab-B-16 (DAR8)

[1150] Take 0.819 mL of Trastuzumab antibody (24.4 mg / mL), dilute with 66.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Add B-16 solution dissolved in dimethyl sulfoxide (143.5 μL, 10 mM, 10 molar equivalents of the antibody), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-B-16). The DAR value was determined by mass spectrometry to be 6.68.

[1151] 15. Example: Preparation of Trastuzumab-B-18 (DAR8)

[1152] Take 0.667 mL of Trastuzumab antibody (22.5 mg / mL), dilute with 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Add B-18 solution dissolved in dimethyl sulfoxide (121.0 μL, 10 mM, 11 molar equivalents of the antibody), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-B-18). The DAR value was determined by mass spectrometry to be 7.08.

[1153] 16. Example: Preparation of Trastuzumab-B-19

[1154] Take 0.667 mL of Trastuzumab antibody (22.5 mg / mL), add 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 57 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Then add 11 times the amount of antibody dissolved in dimethyl sulfoxide B-19 (120.0 μL, 10 mM) solution and mix well. Incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-19). The DAR value was determined by mass spectrometry to be 7.9.

[1155] 17. Example: Preparation of Trastuzumab-B-20 (DAR8)

[1156] Take 0.667 mL of Trastuzumab antibody (22.5 mg / mL), dilute with 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Add B-20 solution dissolved in dimethyl sulfoxide (119.7 μL, 10 mM, 11 molar equivalents of the antibody), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-B-20). The DAR value was determined by mass spectrometry to be 7.87.

[1157] 18. Example: Preparation of Trastuzumab-B-21 (DAR8)

[1158] 0.77 mL of Trastuzumab antibody (19.5 mg / mL) was diluted with 38.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 123.6 μL of B-21 solution dissolved in dimethyl sulfoxide (10 mM, 11 molar equivalents of the antibody) was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-B-21). The DAR value was determined by mass spectrometry to be 7.68.

[1159] 19. Example: Preparation of Trastuzumab-D-1 (DAR8)

[1160] Take 0.468 mL of Trastuzumab antibody (24.4 mg / mL), dilute with 30.1 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 43.3 μL, pH 7.6) solution, mix well, and incubate at room temperature for 1.5 h. Add D-1 solution dissolved in dimethyl sulfoxide (83.0 μL, 10 mM, 10 molar equivalents of the antibody), mix well, and incubate at room temperature for 2 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-D-1). The DAR value was determined by mass spectrometry to be 7.55.

[1161] Biological data

[1162] I. Evaluation of the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model

[1163] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human gastric cancer cell line NCI-N87 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[1164] test drug

[1165] Take an appropriate amount of ADC and administer it at a dose of 1 mg / kg, as detailed below. Dilute the stock solution to the administration solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[1166] Laboratory animals and cell lines

[1167] Balb / c Nude mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[1168] Human gastric cancer cells NCI-N87 (ATCC)

[1169] Experimental grouping and evaluation methods

[1170] Select tumors with an average volume of approximately 150 mm. 3 Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2, where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[1171] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[1172] V T末 >V T0 TGI(%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1-(V T末 -VT0) / VT0]*100%.

[1173] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[1174] V T0 Mean tumor volume at the start of treatment in the treatment group;

[1175] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[1176] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[1177] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[1178] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[1179] (1) Pharmacodynamic assay of anti-human Her2 antibody-drug conjugate in NCI-N87 model

[1180] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb / c Nude mice to establish a gastric cancer model. The tumors were cultured until the average volume reached approximately 150 mmHg. 3At approximately 10:00 AM, patients were randomly assigned to groups based on tumor size, in the following order: solvent control group (i.e., negative control, Vehicle group), and patients receiving the present invention Trastuzumab-B-9 1 mg / kg, Trastuzumab-B-1 1 mg / kg, Trastuzumab-B-2 1 mg / kg, and control DS8201 1 mg / kg (Note: DS8201 is an ADC targeting human HER2 developed by Daiichi Sankyo; the sample used in this experiment was prepared by Kelun Biotech). All groups received intravenous (iv) injection via tail vein on Day 0 and Day 7, for a total of two administrations.

[1181] The ADC of this invention significantly inhibited tumor growth in the NCI-N87 gastric cancer xenograft model. On Day 41, no animals died or experienced significant weight loss in any of the treatment groups, and no obvious drug toxicity was observed. Mice tolerated the ADC of this invention well during the treatment period. Specific results are shown in Table 1.

[1182] Table 1. Human gastric cancer cell NCI-N87 CDX model

[1183] II. Evaluation of the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model

[1184] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human breast cancer cell JIMT-1 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[1185] test drug

[1186] Take an appropriate amount of ADC and administer it at a dose of 1.5 mg / kg, as detailed below. Dilute the stock solution to the administration solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[1187] Laboratory animals and cell lines

[1188] NOD SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.)

[1189] Human breast cancer cell line JIMT-1 (Nanjing Kebai)

[1190] Experimental grouping and evaluation methods

[1191] Select tumors with an average volume of approximately 150 mm. 3Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[1192] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tu...

Claims

1. An antibody-drug conjugate having the formula Ab-[MLED] x The structure shown is as follows, wherein: Ab is an antibody or its antigen-binding fragment: M is a linker that is attached to an antibody or its antigen-binding fragment; L is the structure connecting connectors M and E; E is a structure that connects L and D; D represents the cytotoxic drug component; x is between 1 and 10.

2. The antibody-drug conjugate of claim 1, wherein M is selected from the following substituted or unsubstituted structures:

3. The antibody-drug conjugate of claim 1 or 2, wherein L is selected from one or more of the following substituted or unsubstituted structures: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, substituted or unsubstituted 9-12 nitrogen-containing heterocyclic groups (e.g., substituted with one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., 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)) r OCH3), Lys(R'), Glu(R'), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG), Glu-Gly-Gly-Phe-Gly (EGGFG), 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-Gl) y, 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, V al-Lys-Gly, Gly-Gly-Phe-Gly(GGFG), Gly-Gly-Val-Ala(GGVA), Gly-Phe-Leu-Gly(GFLG), Glu-Ala-Ala-Ala(EAAA), Gly-Gly-Gly-Gly-Gly(GGGGG)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkylene, amino, hydroxyl, carboxyl, C 1-6 Acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups 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-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N + (C 1- 6-alkylene-SO3H)3、-CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylenes -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 group 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 groups -CO2H, -CH2N + (C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1- 6-alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 ethoxy (EO) units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), EDTA (ethylenediaminetetraacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkylene-N(C) 1-6 Alkyl)-NOTA or -C 1-6 Alkylene-N(C) 1-6 Alkyl)-EDTA, or R' selected from Where r is selected from integers from 1 to 20, such as 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 integers from 1 to 20, such as 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; Preferably, L is selected from one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) substituted or unsubstituted structures: 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, Preferably, L is selected from a substituted or unsubstituted structure composed of one or more of the following groups: s is selected from integers from 1 to 20, such as 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 structure composed of one or more of the following groups: Preferably, L is selected from the following substituted or unsubstituted structures: (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )、 (For example )； s is selected from integers from 1 to 20, such as 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.

4. The antibody-drug conjugate according to any one of claims 1-3, wherein E is selected as a single bond, substituted or unsubstituted -NH-CH2-, or selected from the following substituted or unsubstituted structures:

5. The antibody-drug conjugate according to any one of claims 1-4, wherein the cytotoxic drug is selected from topoisomerase inhibitors, tubulin inhibitors, DNA intercalating agents, RNA polymerase inhibitors, and gene transcription inhibitors; preferably, the tubulin inhibitor is an oligurin-like compound, a maytansine-like compound, a hammetrine-like compound, or an eribulin-like compound; the DNA intercalating agent is a pyrrolobenzodiazepine (PBD)-like compound, anthracycline-like compound, trabectedin or rubotedin, or their derivatives or analogs; the topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, ixenonotecan, topotecan). The RNA polymerase inhibitor is α-amanitin; the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogues. Preferably, the cytotoxic drug is a compound with the following structure or a pharmaceutically acceptable salt, stereoisomer, or isotopically labeled compound thereof: in, R1 is either fluorine or chlorine; R2 is selected from methyl, chlorine, hydroxyl, or amino; Alternatively, R1 and R2 form with the bonded carbon atoms Each R3 is independently selected from hydrogen, C 1-6 Alkyl, halogen, hydroxyl, =O, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, C 1-6 Alkoxy or C 1- 6-Hydroalkyl group, or two R3 atoms on adjacent atoms connected to the linked atoms to form a ring; Ring A is selected from: Preferred X is selected from oxygen or sulfur; m is selected from 1-5; R5 and R6 are each independently selected from H, OH, -NH2, and -NH(C) 1-6 Alkyl), C 1-6 Alkyl and halogen; the C 1-6 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6 Haloalkyl, C 3-6 The cycloalkyl group is substituted; or, R5 and R6 together with the adjacent carbon atoms (connected to the cycloforming carbon atoms of R5 and R6, respectively) form a five-membered oxygen-containing heterocycle. R7 is independently selected from H, halogen, -OH, -NH2, -NH(C) 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; the C 1-6 Alkyl and C 1-6 Alkylene is optionally further subjected to one or more elements selected from halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups; p is 1 or 2; q is 0 or 1; Y represents carbon or oxygen; Preferably, the D is selected from the following structures:

6. The antibody-drug conjugate of claims 1-5, wherein the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase group, preferably the antibody or its antigen-binding fragment comprising: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the 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 the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; in, 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 respect to 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; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the 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 the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the 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 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence 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 with respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence 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 with respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (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 (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; 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 with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions; More preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (2) A heavy chain comprising the VH of the 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 of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; More preferably, the antibody or its antigen-binding fragment comprises: (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40; More preferably, the antibody is a trastuzumab antibody, a pertuzumab antibody, or a dual epitope antibody composed of these.

7. The antibody-drug conjugate of claim 1, wherein the conjugate is selected from ADC B-1 to ADC B-21 and ADC D-1 to ADC D-15: ADC B-1: ADC B-2: ADC B-3: ADC B-4: ADC B-5: ADC B-6: ADC B-7: ADC B-8: ADC B-9: ADC B-10: ADC B-11: ADC B-12: ADC B-13: ADC B-14: ADC B-15: ADC B-16: ADC B-17: ADC B-18: ADC B-19: ADC B-20: ADC B-21: ADC D-1: ADC D-2: ADC D-3: ADC D-4: ADC D-5: ADC D-6: ADC D-7: ADC D-8: ADC D-9: ADC D-10: ADC D-11: ADC D-12: ADC D-13: ADC D-14: ADC D-15: The aforementioned HA or HA-(S-) is the antibody or its antigen-binding fragment as described in claim 6, preferably trastuzumab antibody, patocilizumab antibody or a dual epitope antibody composed thereof.

8. The antibody-drug conjugate of claim 1, having the following structure: ADC B-1a: ADC B-2a: ADC B-5a: ADC B-6a: ADC B-9a: ADC B-11a: ADC B-12a: Wherein HA-(S- is the antibody or antigen-binding fragment thereof as described in any of the preceding items, preferably trastuzumab, and -(S- is derived from the specific linkage between the thiol group in the antibody or antigen-binding fragment and the pyrimidine group in ADC B-1a, ADC B-2a, ADC B-5a, ADC B-6a, ADC B-9a, ADC B-11a, ADC B-12a, and x is 1-10, 1-8, 4-8, 6-9, 7-9, or 6-8.

9. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, having the structure shown in formula M'-LED, wherein: M' is -M-Lg, where Lg is a leaving group or an addition group for nucleophilic substitution, and M is a structure that binds to the antibody or its antigen-binding fragment. L is the structure connecting M and E; E is a structure that connects L and D; D represents the cytotoxic drug component; Preferably, M, L, E, D are as defined in any one of claims 1-8.

10. The compound of claim 9 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, M' is selected from: Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structures, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated 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 The alkyl sulfoxide group, halophenoxy group, alkenyl group, alkynyl group, and alkynyl-containing structure are optionally substituted by one or more suitable substituents; preferably, Lg is selected from halogen, substituted or unsubstituted C. 1-6 Alkylsulfonyl (C 1-6 Alkyl (-SO2-), halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH2), more preferably selected from C 1-6 Alkyl sulfonyl (e.g., methyl sulfonyl); Preferably, M' is selected from (For example )。 11. The compound of claim 9 or 10, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, having a structure of B-1 to B-21 and D-1 to D-15: B-1: B-2: B-3: B-4: B-5: B-6: B-7: B-8: B-9: B-10: B-11: B-12: B-13: B-14: B-15: B-16: B-17: B-18: B-19: B-20: B-21: D-1: D-2: D-3: D-4: D-5: D-6: D-7: D-8: D-9: D-10: D-11: D-12: D-13: D-14: D-15:

12. A composition comprising one or more antibody-drug conjugates according to any one of claims 1-8, wherein the DAR value (drug-antibody conjugate ratio) of the composition is about 1-10, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 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 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10. Approximately 5 to 6, approximately 5 to 7, approximately 5 to 8, approximately 5 to 9, approximately 5 to 10, approximately 6 to 7, approximately 6 to 8, approximately 6 to 9, approximately 6 to 10, approximately 7 to 8, approximately 7 to 9, approximately 7 to 10, approximately 8 to 9, approximately 8 to 10, or approximately 9 to 10; for example, approximately 6.0, approximately 6.01, approximately 6.02, approximately 6.03, approximately 6.04, approximately 6.05, approximately 6.0 6, approximately 6.07, approximately 6.08, approximately 6.09, approximately 6.1, approximately 6.11, approximately 6.12, approximately 6.13, approximately 6.14, approximately 6.15, approximately 6.16, approximately 6.17, approximately 6.18, approximately 6.19, approximately 6.2, approximately 6.21, approximately 6.22, approximately 6.23, approximately 6.24, approximately 6.25, approximately 6.26, approximately 6.27 Approximately 6.28, 6.29, 6.3, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.4, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48 Approximately 6.49, approximately 6.5, approximately 6.51, approximately 6.52, approximately 6.53, approximately 6.54, approximately 6.55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69 Approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.76, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 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, approximately 7.34, approximately 7.35, approximately 7.36, approximately 7.37, approximately 7.38, approximately 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.5 4. Approximately 7.55, 7.56, 7.57, 7.58, 7.59, 7.6, 7.61, 7.62, 7.63, 7.64, 7.65, 7.66, 7.67, 7.68, 7.69, 7.7, 7.71, 7.72, 7.73, 7.74, 7.7 5. Approximately 7.76, 7.77, 7.78, 7.79, 7.8, 7.81, 7.82, 7.83, 7.84, 7.85, 7.86, 7.87, 7.88, 7.89, 7.9, 7.91, 7.92, 7.93, 7.94, 7.95, 7.96 Approximately 7.97, 7.98, 7.99, 8.0, 8.01, 8.02, 8.03, 8.04, 8.05, 8.06, 8.07, 8.08, 8.09, 8.1, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17 Approximately August 18, August 19, August 2, August 21, August 22, August 23, August 24, August 25, August 26, August 27, August 28, August 29, August 3, August 31, August 32, August 33, August 34, August 35, August 36, August 37, August 38 Approximately 8.39, 8.4, 8.41, 8.42, 8.43, 8.44, 8.45, 8.46, 8.47, 8.48, 8.49, 8.5, 8.51, 8.52, 8.53, 8.54, 8.55, 8.56, 8.57, 8.58, 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, approximately 8.66, approximately 8.67, approximately 8.68, approximately 8.69, approximately 8.7, approximately 8.71, approximately 8.72, approximately 8.73, approximately 8.74, approximately 8.75, approximately 8.76, approximately 8.77, approximately 8.78, approximately 8.79, approximately 8.8, approximately 8.81, approximately 8.82, approximately 8.83, approximately 8.84, approximately 8.85, approximately 8.86, approximately 8.87, approximately 8.88, approximately 8.89, approximately 8.9, approximately 8.91, approximately 8.92, approximately 8.93, approximately 8.94, approximately 8.95, approximately 8.96, approximately 8.97, approximately 8.98, approximately 8.99, approximately 9.

0.

13. A pharmaceutical composition comprising an antibody-drug conjugate of any one of claims 1-8, a compound of any one of claims 9-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, or a composition of claim 12, and one or more pharmaceutically acceptable carriers.

14. A medicine box product comprising: a) at least one antibody-drug conjugate according to any one of claims 1-8 as a first therapeutic agent, a compound according to any one of claims 9-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, or a composition according to claim 12 or a pharmaceutical composition according to claim 13. b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a pharmaceutical composition comprising another therapeutic agent; and c) Optional packaging and / or instructions.

15. The use of the antibody-drug conjugate of any one of claims 1-8, the compound of any one of claims 9-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, the composition of claim 12 or the pharmaceutical composition of claim 13 or the kit product of claim 14 in the preparation of a medicament for treating diseases, particularly diseases of abnormal cell proliferation; Preferably, the disease is cancer or tumor, such as advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

16. A method for treating a disease, particularly a disease involving abnormal cell proliferation, comprising the following steps: The therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-8, the compound of any one of claims 9-11 or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug, or the composition of claim 12 or the pharmaceutical composition of claim 13 or the kit product of claim 14 is applied to an individual in need of it. Preferably, the disease is cancer or tumor, such as advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

17. The antibody-drug conjugate of any one of claims 1-8 and the compound of any one of claims 9-11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug, the composition of claim 12 or the pharmaceutical composition of claim 13 or the kit product of claim 14, for the treatment of diseases, particularly diseases of abnormal cell proliferation; Preferably, the disease is cancer or tumor, such as advanced solid tumor; Preferably, the cancer or tumor is selected from brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

18. Intermediate compounds with the following structures, or their salts, stereoisomers, tautomers, or isotopically labeled compounds: in, Each PG1 is independently protected by an H or amino group, such as alkoxycarbonyl amino groups, including benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); acyl amino groups, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pentanoyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; and alkyl amino groups, such as triphenylmethyl (Trt), C 1-6 Alkyl-substituted triphenylmethyl, p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn); Each PG2 group is independently protected by either an H or a carboxyl group, such as a C group. 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl; Each PG3 is independently protected by an H or hydroxyl group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, triphenylmethyl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, or p-nitrobenzoyl. s1 is selected from integers from 1 to 20, such as 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; Lg is defined as in any one of claims 9-11; Preferably, the compound is selected from:

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