Antibody drug conjugate

Abstract

The invention discloses an antibody drug conjugate as well as a preparation method and application thereof. The hydrophilic linker in the conjugate can meet the connection of most active molecules, and the obtained antibody drug conjugate has good stability, hydrophilicity and pharmacokinetic properties.

Description

An antibody-drug conjugate

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to two prior applications, Patent Application No. 2023104680075, filed with the State Intellectual Property Office of China on April 27, 2023, and Patent Application No. 2024101290645, filed with the State Intellectual Property Office of China on January 30, 2024, both entitled “Antibody Drug Conjugate.” The entire contents of both prior applications are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology and medicine, and particularly relates to an antibody-drug conjugate, a preparation method and application thereof. Background Art

[0004] Antibody-drug conjugates or antibody-drug conjugates (ADCs) are one of the biggest hotspots in the current research and development of anti-tumor drugs. As of now, there are 14 antibody-drug conjugates on the market. They are: Mylotarg, approved in 2000 for the treatment of CD33-positive acute myeloid leukemia (AML); Adcetris, approved in 2011 for Hodgkin's lymphoma; Kadcyla, approved in 2013 for Her2-positive breast cancer; Besponsa, approved in 2017 for B-cell lymphocytic leukemia; Lumoxiti, approved in 2018 for relapsed or refractory capillary leukemia; Polivy, approved in 2019 for relapsed or refractory diffuse large B-cell lymphoma. Padcev for urothelial carcinoma, approved in 2019; Enhertu for HER2-positive breast cancer, approved in 2019; Trodelvy for triple-negative breast cancer, approved in 2020; Akalux, a photoimmunotherapy drug for head and neck cancer, approved in 2020; Zynlonta for large B-cell lymphoma, approved in 2021; Adixit for HER2-positive gastric cancer, approved in 2021; Tivdak for cervical cancer, approved in 2022; and Elahere for ovarian cancer, approved in 2022. It's worth noting that Mylotarg was withdrawn from the market in 2010 due to its high toxicity, but was re-launched in 2017 after adjusting its clinical dosage. Meanwhile, Blenrep received accelerated approval for multiple myeloma in 2020, but was withdrawn from the market due to unsatisfactory subsequent clinical results.

[0005] Antibody-drug conjugates consist of three parts: antibodies, linkers, and bioactive molecules (drugs). Among them, antibodies are closely related to disease targets. Linkers and drugs determine the effectiveness and safety of ADCs and are the most core parts. Among the ADC drugs that have been launched on the market, most use the technology developed by Seattle Genetics decades ago, which uses microtubule inhibitors as active molecules, coupled with tissue protease hydrolysis linkers, and conjugated to antibody cysteine ​​through maleimide. The ADCs obtained by this technology are often highly toxic, highly hydrophobic, and have poor pharmacokinetic properties. Although the ADCs obtained by Daiichi Sankyo using a hydrophilic tetrapeptide linker have been greatly improved, they are still quite different from the properties of naked antibodies. Trodelvy uses a lysine linker containing polyethylene glycol and a salt-forming lysine. Although the resulting ADC has good hydrophilicity, it has poor stability. Literature reports that a linker containing polysarcosine can greatly improve the hydrophilicity of ADCs. However, this linker is only suitable for active molecules containing secondary amine structures, such as MMAE. For active molecules with primary amine structures such as Exatecan, the carbonamide bond formed by this linker is less stable.

[0006] The purpose of the present invention is to solve the above problems and provide a hydrophilic linker that can meet the connection requirements of most active molecules, and the obtained ADC has good stability and hydrophilicity as well as good pharmacokinetic properties.

[0007] Summary of the Invention

[0008] The present invention provides an antibody drug conjugate having a structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels: A-(LD) m (I)

[0009] wherein A is a targeting ligand selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment, a small molecule ligand, or a polypeptide;

[0010] L is a linker moiety, one end of which is connected to ligand A and the other end is connected to the bioactive molecule D;

[0011] D is a biologically active molecule containing an amino group or a tautomer, mesomer, racemate, enantiomer, diastereomer, or isotope-labeled substance thereof, and is covalently linked to the linker portion L through the amino group in its molecular structure;

[0012] m represents the molar ratio of cytotoxic drug molecules to antibody A (also known as DAR, i.e. drug-antibody coupling ratio).

[0013] m is an integer or decimal from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0014] When m is a decimal, it refers to the average number of linker-drug molecules (LD) conjugated per antibody unit (A).

[0015] In some embodiments, A is selected from an antibody or antigen-binding fragment thereof that targets HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, DLL3, TROP2, B7H3, c-Met, CD20, CD22, CD30, CD33, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Y, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, FRα, or Mesothelin.

[0016] In some embodiments, A can be modified, such as by changing, adding, or removing one or more amino acids.

[0017] In some embodiments A is selected from antibodies or antigen-binding fragments thereof targeting Her2, DLL3, B7H3, or FRα.

[0018] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 3; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein CDRs are determined according to the Kabat numbering convention.

[0019] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO:8.

[0020] In some embodiments, A is an antibody targeting B7H3, and the anti-B7H3 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0021] In some embodiments, A is an antigen-binding fragment targeting B7H3, and the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0022] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, and the anti-B7H3 antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0023] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:9 or SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0024] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 12, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 13, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 14; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 15, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 17, wherein the CDRs are determined according to the Kabat numbering convention.

[0025] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the anti-DLL3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO: 18, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO: 19.

[0026] In some embodiments, A is an antibody targeting DLL3, and the anti-DLL3 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0027] In some embodiments, A is an antigen-binding fragment that targets DLL3, and the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0028] In some embodiments, A is an antibody or an antigen-binding fragment thereof that targets DLL3, and the anti-DLL3 antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0029] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the anti-DLL3 antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21.

[0030] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting FRα, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 22, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 23, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 24; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 25, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 27, wherein the CDRs are determined according to the Kabat numbering convention.

[0031] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting FRα, wherein the anti-FRα antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO: 28, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO: 29.

[0032] In some embodiments, A is an antibody targeting FRα, and the anti-FRα antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0033] In some embodiments, A is an antigen-binding fragment targeting FRα, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0034] In some embodiments, A is an antibody or an antigen-binding fragment thereof that targets FRα, and the anti-FRα antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0035] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting FRα, wherein the anti-FRα antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 30 or SEQ ID NO: 32, and the amino acid sequence of the light chain is shown in SEQ ID NO: 31.

[0036] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting HER2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 37, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 39; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 40, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 42, wherein the CDRs are determined according to the Kabat numbering convention.

[0037] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting HER2, wherein the anti-HER2 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO: 35, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO: 36.

[0038] In some embodiments, A is an antibody targeting HER2, and the anti-HER2 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0039] In some embodiments, A is an antigen-binding fragment targeting HER2, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0040] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting HER2, and the anti-HER2 antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0041] In some embodiments, A is an antibody targeting HER2 or an antigen-binding fragment thereof, wherein the anti-HER2 antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is shown in SEQ ID NO: 34.

[0042] In some embodiments, A is selected from 43A or 43B targeting B7H3, 33B targeting DLL3, farletuzumab or farletuzumab-FcS targeting FRα, trastuzumab targeting Her2, or a biosimilar thereof.

[0043] 43A includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0044] >43A heavy chain sequence (SEQ ID NO:9)

[0045] >43A light chain sequence (SEQ ID NO: 11)

[0046] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively:

[0047] HCDR1: DYFMN SEQ ID NO: 1

[0048] HCDR2:DVNPKTGSPSYNQKFKG SEQ ID NO:2

[0049] HCDR3:RYGFLYSMDY SEQ ID NO:3

[0050] LCDR1:RASQDISNYLN SEQ ID NO:4

[0051] LCDR2: YTSRLHS SEQ ID NO: 5

[0052] LCDR3: QQGNTHPFT SEQ ID NO: 6

[0053] Among them, the underlined amino acid sequences are the heavy chain / light chain variable region sequences, and HV and LV are named SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0054] 43A heavy chain variable region (HV) sequence (SEQ ID NO: 7):

[0055] 43A light chain variable region (LV) sequence (SEQ ID NO: 8):

[0056] 43B includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain is shown in SEQ ID NO: 11.

[0057] >43B heavy chain sequence (SEQ ID No: 10)

[0058] >43B light chain sequence (SEQ ID NO: 11)

[0059] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively:

[0060] HCDR1: DYFMN SEQ ID NO: 1

[0061] HCDR2:DVNPKTGSPSYNQKFKG SEQ ID NO:2

[0062] HCDR3:RYGFLYSMDY SEQ ID NO:3

[0063] LCDR1:RASQDISNYLN SEQ ID NO:4

[0064] LCDR2: YTSRLHS SEQ ID NO: 5

[0065] LCDR3: QQGNTHPFT SEQ ID NO: 6

[0066] The underlined amino acid sequences are the heavy chain / light chain variable region sequences, HV and LV are designated as SEQ ID NO: 7 and SEQ ID NO: 8, respectively. 43B heavy chain variable region (HV) sequence (SEQ ID NO: 7):

[0067] 43B light chain variable region (LV) sequence (SEQ ID NO: 8):

[0068] 33B includes two identical heavy chains and light chains, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21.

[0069] >33B heavy chain sequence (SEQ ID NO: 20)

[0070] >33B light chain sequence (SEQ ID NO:21)

[0071] The heavy chain variable region sequence is shown in SEQ ID NO: 18, and the light chain variable region sequence is shown in SEQ ID NO: 19.

[0072] >33B heavy chain variable region (HV) sequence (SEQ ID NO: 18)

[0073] >33B light chain variable region (LV) sequence (SEQ ID NO: 19)

[0074] Among them, the heavy chain CDR sequences HCDR1, HCDR2, and HCDR3 are named SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the light chain CDR sequences LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively.

[0075] HCDR1: DYYMK SEQ ID NO: 12

[0076] HCDR2:AFNLNNGDTFYNQKFKG SEQ ID NO:13

[0077] HCDR3: DVYGYGDY SEQ ID NO: 14

[0078] LCDR1:RASKSVSTSGYSYMH SEQ ID NO:15

[0079] LCDR2: LASNLDS SEQ ID NO: 16

[0080] LCDR3:QHSRELPYT SEQ ID NO:17

[0081] Farletuzumab includes two identical heavy chains and light chains (WO 2023 / 170247 A1), the heavy chain amino acid sequence is shown in SEQ ID NO: 30, and the light chain amino acid sequence is shown in SEQ ID NO: 31.

[0082] >Farletuzumab heavy chain sequence (SEQ ID NO: 30):

[0083] Farletuzumab light chain sequence (SEQ ID NO: 31):

[0084] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively:

[0085] HCDR1: GYGLS SEQ ID NO: 22

[0086] HCDR2: MISSGGSYTYYADSVKG SEQ ID NO:23

[0087] HCDR3:HGDDPAWFAY SEQ ID NO:24

[0088] LCDR1:SVSSSISSNNLH SEQ ID NO:25

[0089] LCDR2: GTSNLAS SEQ ID NO: 26

[0090] LCDR3:QQWSSYPYMYT SEQ ID NO:27

[0091] Among them, the heavy chain / light chain variable region sequences, HV and LV are named SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

[0092] Farletuzumab heavy chain variable region (HV) sequence (SEQ ID NO: 28):

[0093] Farletuzumab light chain variable region (LV) sequence (SEQ ID NO: 29):

[0094] Farletuzumab-Fc5 includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 32, and the amino acid sequence of the light chain is shown in SEQ ID NO: 31.

[0095] >Farletuzumab-Fc5 heavy chain sequence (SEQ ID NO: 32):

[0096] >Farletuzumab-Fc5 light chain sequence (SEQ ID NO: 31):

[0097] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively:

[0098] HCDR1: GYGLS SEQ ID NO: 22

[0099] HCDR2: MISSGGSYTYYADSVKG SEQ ID NO:23

[0100] HCDR3:HGDDPAWFAY SEQ ID NO:24

[0101] LCDR1:SVSSSISSNNLH SEQ ID NO:25

[0102] LCDR2: GTSNLAS SEQ ID NO: 26

[0103] LCDR3:QQWSSYPYMYT SEQ ID NO:27

[0104] Among them, the heavy chain / light chain variable region sequences, HV and LV are named SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

[0105] Farletuzumab-Fc5 heavy chain variable region (HV) sequence (SEQ ID NO: 28):

[0106] Farletuzumab-Fc5 light chain variable region (LV) sequence (SEQ ID NO: 29):

[0107] Trastuzumab includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is shown in SEQ ID NO: 34.

[0108] >Trastuzumab heavy chain (SEQ ID NO: 33)

[0109] >Trastuzumab light chain (SEQ ID NO: 34)

[0110] The heavy chain variable region sequence is shown in SEQ ID NO: 35, and the light chain variable region sequence is shown in SEQ ID NO: 36.

[0111] >Trastuzumab heavy chain variable region (HV) sequence (SEQ ID NO: 35)

[0112] Trastuzumab light chain variable region (LV) sequence (SEQ ID NO: 36)

[0113] Among them, the heavy chain CDR sequences HCDR1, HCDR2, and HCDR3 are named SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and the light chain CDR sequences LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively.

[0114] HCDR1: DTYIH SEQ ID NO: 37

[0115] HCDR2:RIYPTNGYTRYADSVKG SEQ ID NO:38

[0116] HCDR3:WGGDGFYAMDY SEQ ID NO:39

[0117] LCDR1:RASQDVNTAVA SEQ ID NO:40

[0118] LCDR2: SASFLYS SEQ ID NO:41

[0119] LCDR3:QQHYTTPPT SEQ ID NO:42

[0120] In some embodiments, the linker moiety -L- is represented by the formula:

[0121] -L1-L2-L3-L4-,

[0122] Wherein, L1 is the portion connecting L to ligand A; preferably, L1 is selected from (or its open-loop form ), or Where * indicates connection to the sulfhydryl group of A (such as a monoclonal antibody), and ** indicates connection to L2;

[0123] L2 is a spacer; preferably, it is selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-、-L 2a -L 2b -NH-C(O)-、-L 2a -L 2b -C(O)-NH-、-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -C(O)-NH-L 2b -C(O)-NH-、-L 2a -NH-C(O)-L 2b -NH-C(O)-、-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 14arylene-C1-C8 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from C1-C6 alkyl, C1 -C6 heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, wherein the heteroalkylene, heterocyclylene or heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene or heteroalkylene are selected from one or more (such as two or three) of N, O or S; L 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14Aryl, 5-6 membered heteroaryl, the alkyl, heterocyclic, heteroalkyl, aryl, heteroaryl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclic, heteroaryl, heteroalkylene, heteroarylene are selected from one or more (such as two or three) of N, O or S;

[0124] L3 is a polypeptide sequence; preferably, selected from peptide residues consisting of 2-8 (e.g., 2, 3, 4, 5, 6, 7, 8) natural or unnatural amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from C1-C6 alkyl, 2-6 atom heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl;

[0125] L4 is a self-cleaving fragment modified with a hydrophilic group,

[0126] The self-cleaving fragment is selected from:

[0127] or

[0128] Where * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups;

[0129] Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group:

[0130] or

[0131] Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6;

[0132] The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups;

[0133] Preferably, L4 is selected from:

[0134] or

[0135] Where * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D;

[0136] R 2 is a hydrophilic segment, preferably, selected from a linear or branched heteroalkyl group comprising 4-50 (preferably 4-24, more preferably 6-24, most preferably 8-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- structural units, a linear or branched heteroalkyl group comprising 4-50 (preferably 4-24, more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) protein amino acids (such as glycine) or non-protein amino acids (such as sarcosine), linear or branched heteroalkyl groups containing monosaccharides, oligosaccharides or polysaccharides; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 It is a C1-C6 alkylene group or a heteroalkylene group containing 1-24 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) -OCH2CH2- structural units;

[0137] n is an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, 6).

[0138] In some embodiments, R 2 Selected from the following structures:

[0139] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R b Selected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

[0140] In some embodiments, R 2 Selected from the following structures:

[0141] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0142] In some embodiments, R 2 Selected from the following structures:

[0143] In some embodiments, Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 C1-C6 alkylene or -C1-C6 alkylene-(OCH2CH2) v -, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0144] In some embodiments, Y is C1-C3 alkylene or -R 3 -C(O)-, where R 3 C1-C3 alkylene or -C1-C3 alkylene-(OCH2CH2) v -, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0145] In some embodiments, Y is methylene, ethylene, n-propylene, isopropylene, or -R 3-C(O)-, where R 3 Methylene -(OCH2CH2) v -, ethylene-(OCH2CH2) v -, n-propylene-(OCH2CH2) v -, isopropylidene-(OCH2CH2) v -, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0146] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0147] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2a Selected from -C1-C6 alkylene-, -C1-C6 alkylene-C3-C6 cycloalkylene-, -C6-C 10 Arylene-, -C6-C 10arylene-C1-C6 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C6 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C3 L is substituted by one or more substituents of alkyl, 1-3 atoms of heteroalkyl, C1-C3 alkoxy, hydroxy, amino, carboxyl or C3-C6 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl are selected from one or more of N, O or S; 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C1-C3 haloalkyl, 2-6 membered heteroalkyl, C6-C 10Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, 2-3 atoms of heteroalkyl, C1-C3 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclic radical, heteroaryl, heteroalkylene are selected from one or more of N, O or S.

[0148] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2aSelected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, -C1-C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1-C3 alkylene-, -5-6 membered heteroaryl-C1-C3 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched heteroalkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 47,48,49,50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, methyl, ethyl , n-propyl, isopropyl, 1-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, hydroxy, amino, carboxyl or cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl one or more substituents, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl heteroatoms are selected from one or more of N, O or S; L 2b is selected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, a straight or branched chain heteroalkylene radical of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, R 1Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-6 membered heterocyclyl, -CF3, -CF2CF3, 2-3 atoms of heteroalkyl, phenyl, 5-6 membered heteroaryl, said alkyl, heteroalkyl, phenyl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from methyl, ethyl, n-propyl, isopropyl, 2-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, amino or carboxyl, said heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of said heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S.

[0149] In some embodiments, L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene, C6-C 14 Arylene-C1-C8 alkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms of straight or branched heteroalkylene, the alkylene, cycloalkylene, arylene, heteroalkylene are each optionally substituted by one or more substituents independently selected from halogen, C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkylene, heteroalkylene are selected from one or more of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms, R 1 Selected from hydrogen, C1-C6 alkyl, 2-8 atoms of heteroalkyl, C6-C 14 Aryl, the alkyl, heteroalkyl, and aryl groups are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl and heteroalkylene groups are selected from one or more of N, O or S.

[0150] In some embodiments, L 2a Selected from -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p -、-(OCH2CH2) p-, -CH2(OCH2CH2) p -、-CH2CH2(OCH2CH2) p CH2-, -CH2CH2(OCH2CH2) p CH2CH2-, -CH2(OCH2CH2) p CH2CH2-, wherein p is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L 2b Selected from -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -(OCH2CH2) q -、-CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -、-CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-、-CH2(OCH2CH2) q CH2CH2-, R 1 Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, wherein q is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0151] In some embodiments, L2 is selected from the following structures:

[0152] Wherein, w is selected from an integer of 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection with L1, **

[0153] Indicates connection with L3.

[0154] In some embodiments, L3 is selected from 2-8 peptide residues formed by amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine), etc. For example -ValCit-;-CitVal-;-AlaAla-;-AlaCit-;-CitAla-;-AsnCit-;-CitAsn-;-CitCit-;-ValGlu-; itAsp--AlaVal--ValAla--PheAla--AlaPhe--PheLys--LysPhe--ValLys--LysVal--AlaLys--LysAla--PheCit--CitPhe--LeuCit--CitLeu--IleCi t-;-CitIle-;-PheArg-;-ArgPhe-;-CitTrp-;-TrpCit-;-AlaAlaAla-;-PhePheLys-;-LysPhePhe-;-DPhePheLys-; heLeuGly- -GlyLeuPheGly- -GluValCit- -AlaLeuAlaLeu- -GlyGlyGly- -GlyGlyGlyGly- -GlyPheValGly- -GlyValPheGly- -GlyGlyPheGly- -GlyGlyValGly-.

[0155] In some embodiments, L3 is selected from the following structures:

[0156] * indicates connection with L2, and ** indicates connection with L4.

[0157] In some embodiments, L4 is selected from:

[0158] wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D.

[0159] In some embodiments, L4 is selected from:

[0160] In some embodiments, L4 is selected from:

[0161] In some embodiments, L4 is selected from:

[0162] In some embodiments, L4 is selected from:

[0163] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the bioactive molecule D.

[0164] In some embodiments, L4 is selected from:

[0165] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); and * indicates connection to the carboxyl group of L3 via an amide bond. ** indicates connection to the amino group of the bioactive molecule D.

[0166] In some embodiments, L is selected from the following structures:

[0167] Wherein, v and w are each independently selected from integers of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection to the sulfhydryl group of antibody A, and ** indicates connection to the amino group of bioactive molecule D.

[0168] In some embodiments, L is selected from the following structures:

[0169] wherein v and w are each independently selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); * indicates linkage to the sulfhydryl group of antibody A; ** indicates linkage to the amino group of biologically active molecule D; and r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0170] In some embodiments, L is selected from the following structures:

[0171] Where * indicates connection with the sulfhydryl group of antibody A, and ** indicates connection with the amino group of bioactive molecule D.

[0172] In some embodiments, the bioactive molecule D is selected from camptothecin derivatives, eribulin, and molecular glues. In some embodiments, the bioactive molecule D is selected from compounds of the following formula:

[0173] In some embodiments, the biologically active molecule D is selected from:

[0174] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0175] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0176] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0177] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0178] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v and w are each independently selected from an integer of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0179] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0180] In some embodiments, the antibodies ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 are selected from Trastuzumab.

[0181] In some embodiments, the antibody ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 is selected from Farletuzumab.

[0182] In some embodiments, the antibodies ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 are selected from Farletuzumab FcS.

[0183] In some embodiments, the antibodies ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 are selected from 33B.

[0184] In some embodiments, the antibodies ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 are selected from 43A.

[0185] In some embodiments, the antibodies ADC1 to ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, ADC55-2 are selected from 43B.

[0186] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures: Note: In the table, T-ADC1 refers to ADC1 in which antibody A is trastuzumab. Other ADCs are similar.

[0187] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0188] Wherein m is selected from 2, 4, 6, and 8.

[0189] In some embodiments, the present invention provides use of an antibody-drug conjugate of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products in the preparation of drugs for treating cancer. The cancers include liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), gastric cancer, esophageal cancer, oral cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumors or multifocal glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

[0190] In some embodiments, the present invention provides an antibody drug conjugate of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products for use in treating cancer. The cancers include liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), stomach cancer, esophageal cancer, oral cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood cancer or multiforme glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

[0191] In some embodiments, the cancer is a tumor in which tumor cells express HER2 (HER2+), DLL3 (DLL3+), FRα (FRα+), and B7H3 (B7H3+).

[0192] In some embodiments, the present invention provides an antibody drug conjugate of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled forms for use in treating cancer. The cancer is preferably a cancer associated with abnormal expression of FRα, DLL3, B7H3, or HER2. Cancers associated with abnormal expression of FRα include lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer, ovarian cancer, cervical cancer, uterine cancer, and endometrial cancer. Cancers associated with abnormal expression of DLL3 include lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, prostate cancer, pancreatic cancer, breast cancer, and ovarian cancer. Cancers associated with abnormal expression of HER2 include lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer, ovarian cancer, endometrial cancer, gastric cancer, and prostate cancer.

[0193] In some embodiments, the present invention provides a pharmaceutical composition comprising an antibody drug conjugate having a structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels and one or more pharmaceutically acceptable excipients.

[0194] In some embodiments, the present invention provides a linker represented by the following formula, which is used to obtain an antibody drug conjugate formed by connecting a drug and an antibody via a linker: -L1-L2-L3-L4-,

[0195] The left end is the linking site with the antibody, and the right end is the linking site with the anti-tumor compound. The definitions of L1, L2, L3, and L4 are the same as those in formula (I).

[0196] Another aspect of the present invention provides a linker-drug compound represented by formula (II) or its tautomers, mesomers, racemates, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products: L'-D (II)

[0197] Wherein L' is a linker moiety;

[0198] D is a biologically active molecule containing an amino group or a pharmaceutically acceptable salt thereof, which is covalently linked to the linker portion L' through the amine group in its molecular structure;

[0199] In some embodiments, the linker moiety L' is represented by the formula:

[0200] L1'-L2-L3-L4-

[0201] Wherein, L1' is selected from or Among them, ** indicates connection with L2;

[0202] L2, L3, L4, and D are defined the same as in formula (I).

[0203] L2 is a spacer; preferably, it is selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-、-L 2a -C(O)-NH-、-L 2a -L 2b -NH-C(O)-、-L 2a -L 2b -C(O)-NH-、-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -C(O)-NH-L 2b -C(O)-NH-、-L 2a -NH-C(O)-L 2b -NH-C(O)-、-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 141-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C6 alkyl, 1-6 The heteroalkylene group, heterocyclylene group, heteroalkylene group or heteroalkylene group is substituted by one or more substituents of heteroalkylene, C1-C6 alkoxy, hydroxyl, amino, carboxyl or C3-C8 cycloalkyl, wherein the heteroalkylene group, heterocyclylene group or heteroalkylene group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene group, heterocyclylene group, heteroarylene group or heteroalkylene group are selected from one or more (such as two or three) of N, O or S; L 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14Aryl, 5-6 membered heteroaryl, the alkyl, heterocyclic, heteroalkyl, aryl, heteroaryl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclic, heteroaryl, heteroalkylene, heteroarylene are selected from one or more of N, O or S;

[0204] L3 is a polypeptide sequence; preferably, selected from peptide residues consisting of 2-8 (e.g., 2, 3, 4, 5, 6, 7, 8) natural or unnatural amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from C1-C6 alkyl, 2-6 atom heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl;

[0205] L4 is a self-cleaving fragment modified with a hydrophilic group,

[0206] The self-cleaving fragment is selected from:

[0207] or

[0208] Where * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups;

[0209] Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group:

[0210] or

[0211] Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6;

[0212] The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups;

[0213] Preferably, L4 is selected from:

[0214] or

[0215] Where * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D;

[0216] R 2 is a hydrophilic segment, preferably, selected from a linear or branched heteroalkyl group comprising 4-50 (preferably 4-24, more preferably 6-24, most preferably 8-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) (OCH2CH2) structural units, 24, more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) protein amino acids (such as glycine) or non-protein amino acid peptide chains (such as sarcosine), linear or branched heteroalkyl groups containing monosaccharides, oligosaccharides or polysaccharides; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 It is a C1-C6 alkylene group or a heteroalkylene group containing 1-24 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) -OCH2CH2- structural units, and n is an integer from 0 to 6 (such as 0, 1, 2, 3, 4, 5, 6).

[0217] In some embodiments, R 2 Selected from the following structures:

[0218] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50);a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R b Selected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

[0219] In some embodiments, R 2 Selected from the following structures:

[0220] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0221] In some embodiments, R 2 Selected from the following structures:

[0222] In some embodiments, Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 C1-C6 alkylene or -C1-C6 alkylene-(OCH2CH2) v -, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0223] In some embodiments, Y is C1-C3 alkylene or -R 3 -C(O)-, where R 3 C1-C3 alkylene or -C1-C3 alkylene-(OCH2CH2) v -, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0224] In some embodiments, Y is methylene, ethylene, n-propylene, isopropylene, or -R 3 -C(O)-, where R 3 Methylene -(OCH2CH2) v -, ethylene-(OCH2CH2) v -, n-propylene-(OCH2CH2) v -, isopropylidene-(OCH2CH2) v-, wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0225] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0226] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2a Selected from -C1-C6 alkylene-, -C1-C6 alkylene-C3-C6 cycloalkylene-, -C6-C 10 Arylene-, -C6-C 10 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 3-6 membered heterocyclylene, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C3 alkyl, 1-3 The heteroalkylene group, heterocyclylene group, heteroalkylene group or heteroalkylene group is substituted by one or more substituents of heteroalkylene, C1-C3 alkoxy, hydroxyl, amino, carboxyl or C3-C6 cycloalkyl, wherein the heteroalkylene group, heterocyclylene group or heteroalkylene group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene group, heterocyclylene group, heteroarylene group or heteroalkylene group are selected from one or more (such as two or three) of N, O or S; L2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C1-C3 haloalkyl, 2-6 membered heteroalkyl, C6-C 10 Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, 2-3 atoms of heteroalkyl, C1-C3 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more (such as two or three) of N, O or S.

[0227] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2aSelected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, -C1-C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1-C3 alkylene-, -5-6 membered heteroaryl-C1-C3 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein each of the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, and heterocyclylene groups is optionally independently selected from halogen, methyl, ethyl, n-propyl, isopropyl, thiazolinyl ... L is substituted by one or more substituents of propyl, 1-3 heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, hydroxy, amino, carboxyl or cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wherein the heteroalkylene, heterocyclylene, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkylene are selected from one or more (such as two or three) of N, O or S; 2b is selected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, a straight or branched chain heteroalkylene radical of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, R 1selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-6 membered heterocyclyl, -CF3, -CF2CF3, 2-3 membered heteroalkyl, phenyl, 5-6 membered heteroaryl, said alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from methyl, ethyl, n-propyl, isopropyl, 2-3 membered heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, amino or carboxyl, said heteroalkyl, heteroalkylene group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene group heteroatoms are selected from one or more (such as two or three) of N, O or S.

[0228] In some embodiments, L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene, C6-C 14 Arylene-C1-C8 alkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms of straight or branched heteroalkylene, the alkylene, cycloalkylene, arylene, heteroalkylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkylene, heteroalkylene are selected from one or more (such as two or three) of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms, R 1 Selected from hydrogen, C1-C6 alkyl, 2-8 atoms of heteroalkyl, C6-C 14 Aryl, the alkyl, heteroalkyl and aryl groups are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl and heteroalkylene groups are selected from one or more (such as two or three) of N, O or S.

[0229] In some embodiments, L 2a Selected from -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p-、-(OCH2CH2) p -, -CH2(OCH2CH2) p -、-CH2CH2(OCH2CH2) p CH2-, -CH2CH2(OCH2CH2) p CH2CH2-、-CH2(OCH2CH2) p CH2CH2-, wherein p is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L 2b Selected from -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -(OCH2CH2) q -、-CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -、-CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-、-CH2(OCH2CH2) q CH2CH2-, R 1 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, and cyclopropyl, and q is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0230] In some embodiments, L2 is selected from the following structures:

[0231] Wherein, w is selected from an integer of 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection with L1, and ** indicates connection with L3.

[0232] In some embodiments, L3 is selected from 2-8 peptide residues formed by amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine), etc. For example -ValCit-;-CitVal-;-AlaAla-;-AlaCit-;-CitAla-;-AsnCit-;-CitAsn-;-CitCit-;-ValGlu-; itAsp--AlaVal--ValAla--PheAla--AlaPhe--PheLys--LysPhe--ValLys--LysVal--AlaLys--LysAla--PheCit--CitPhe--LeuCit--CitLeu--IleCi t-;-CitIle-;-PheArg-;-ArgPhe-;-CitTrp-;-TrpCit-;-AlaAlaAla-;-PhePheLys-;-LysPhePhe-;-DPhePheLys-; heLeuGly- -GlyLeuPheGly- -GluValCit- -AlaLeuAlaLeu- -GlyGlyGly- -GlyGlyGlyGly- -GlyPheValGly- -GlyValPheGly- -GlyGlyPheGly- -GlyGlyValGly-.

[0233] In some embodiments, L3 is selected from the following structures:

[0234] * indicates connection with L2, and ** indicates connection with L4.

[0235] In some embodiments, L4 is selected from:

[0236] wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D.

[0237] In some embodiments, L4 is selected from:

[0238] In some embodiments, L4 is selected from:

[0239] In some embodiments, L4 is selected from:

[0240] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the bioactive molecule D.

[0241] In some embodiments, L4 is selected from:

[0242] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the bioactive molecule D.

[0243] In some embodiments, L' is selected from the following structures:

[0244] In some embodiments, L' is selected from the following structures:

[0245] wherein v and w are each independently selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); ** represents linkage to the amino group of the biologically active molecule D; and r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0246] In some embodiments, L' is selected from the following structures:

[0247] Where ** indicates connection with the amino group of the biologically active molecule D.

[0248] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope labels, wherein the biologically active molecule D is selected from camptothecin derivatives, eribulin, and molecular glues.

[0249] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein the biologically active molecule D is selected from the compounds of the following formula:

[0250] In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, mesomers, racemates, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein the biologically active molecule D is selected from:

[0251] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0252] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0253] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0254] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0255] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v and w are each independently selected from an integer of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0256] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0257] The present invention also provides a compound of formula (III) or its tautomers, mesomers, racemates, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances:

[0258] The formula (III) has the following structure:

[0259] or

[0260] Where X does not exist or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 It is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6.

[0261] The present invention also provides a compound of formula (IV) or its tautomers, mesomers, racemates, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances:

[0262] or

[0263] Where X does not exist or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6;

[0264] Wherein AA is L3 or a subfragment consisting of the first, second or more amino acids from the C-terminus of L3, N is H or an amino protecting group such as Boc or Fmoc, P C is H, a hydroxyl protecting group or a carbonate active ester group such as p-nitrophenol carbonate;

[0265] The compound of formula (IV) is preferably:

[0266] Abbreviations and definitions

[0267] Unless otherwise indicated, the following terms used in this application have the following meanings.

[0268] When a trade name is used in this application, unless the context indicates otherwise, the trade name includes the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product.

[0269] The term "antibody-drug conjugate" or "antibody-drug conjugate" (ADC) refers to a targeting ligand such as an antibody (eg, monoclonal antibody) or antibody fragment linked to a biologically active molecule through a stable chemical linker compound.

[0270] The term "linker-drug compound" or "linker-drug compound" refers to the partial structure of an antibody-drug conjugate consisting of a linker compound and a biologically active compound.

[0271] The term "linker" refers to a chemical structure fragment, represented by L, that is linked to an antibody at one end and to a cytotoxic drug at the other. It is formed by linking a "linker compound" to the antibody and a bioactive compound, respectively. In certain embodiments of the present invention, a portion of the linker compound is first linked to the antibody, and another portion is linked to the bioactive compound. The "linker" is then formed through a chemical reaction between the different portions of the linker. The linker herein comprises a portion that connects to the antibody, a spacer, a polypeptide sequence, and a self-cleaving fragment.

[0272] The linker-drug compound of the present invention is connected to the antibody by conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reductive disulfide bond coupling and directional coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6: 15-17; McCombs JR, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17: 339-351). The present invention preferably couples the linker-drug compound to the antibody by reductive disulfide bond coupling between the light and heavy chains, that is, one or more of the inter-light and heavy chain disulfide bond sites (two sites between the heavy chain and two sites between the heavy and light chains) are reduced to form a thiol group (sulfur atom of a cysteine ​​residue) for reaction. In the present invention, the connecting portion of the linker L and the antibody is represented by L1, which is formed by the reaction of the L1' group in the linker compound with the antibody, for example or Wherein, * indicates connection with the sulfhydryl group of the antibody, and ** indicates connection with the spacer.

[0273] The term "spacer" refers to the linker L and the connecting portion of the antibody and the polypeptide sequence, which can be any divalent organic group, such as a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; said C 1-10Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof and optionally interrupted by carbonyl, O, S, or N atoms; 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group may be optionally replaced by C 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6 Alkyl substituted; preferably, as defined for L2.

[0274] The term "polypeptide sequence" is well known in the art and is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or non-natural amino acid residues, L-form or D-form, each of which is the same or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle) or analogs of the above amino acids.

[0275] The term "self-cleaving fragment" is well known in the art, such as the p-aminobenzyl carbonate fragment commonly used in the art. In a specific embodiment of the present invention, the "self-cleaving fragment" of the linker of the present invention comprises a modification of the "hydrophilic fragment".

[0276] The term "hydrophilic segment" is well known in the art, and the hydrophilic segment may comprise a polyethylene glycol group, such as a polyethylene glycol group with a methoxy terminal group, or further connected to other hydrophilic segments via polyethylene glycol groups. The hydrophilic segment may also comprise a polyamino acid segment, such as polyglycine or polysarcosine, which may be combined with a polyethylene glycol group. The hydrophilic segment may also be a monosaccharide, a disaccharide or an oligosaccharide, which may be a sugar in the form of a chain molecule or a cyclic molecule, and may comprise a glycosamine, a sugar acid or a phosphate sugar. Preferably, the sugar group is combined with a polyethylene glycol segment or a polyamino acid segment; further preferably, at least two sugar groups are introduced via a multivalent linking group, such as a linking group based on aspartic acid, glutamic acid or lysine. It may also comprise a polycarboxylic acid group, a polysulfonic acid group or a chelating group, etc.; the structure of the polycarboxylic acid group may be as follows:

[0277] The structure of the polysulfonic acid group is as follows:

[0278] The chelating group may be, for example, a DOTA group or a NOTA group.

[0279] The terms "solvate" and "solvate" mean a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain circumstances, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0280] The term "stereoisomer" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0281] The term "tautomer" refers to isomers with different energies that are interconvertible across a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include tautomers that interconvert via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include tautomers that interconvert via reorganization of bonding electrons.

[0282] The term "plurality" in "one or more" includes two or more (e.g., 3, 4, 5, etc.);

[0283] The term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0284] The term "C m -C n " means that the moiety has an integer number of carbon atoms ranging from m to n. For example, "C1-C3" means that the group can have 1 carbon atom, 2 carbon atoms or 3 carbon atoms.

[0285] The term "-(CH2) n -" means that the part has n -CH2- connected groups. For example, when the number of a connecting group is 0, such as "-(CH)0-", it means that the connecting group is a covalent bond.

[0286] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C 1-10 Alkyl), further preferably containing 1 to 8 carbon atoms (C 1-8 Alkyl), more preferably containing 1-6 carbon atoms (ie C 1-6 alkyl). For example, "C 1-6 The term "alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl and the like.

[0287] The term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above, i.e., containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, and the like.

[0288] The term "halogen" or "halo" refers to F, Cl, Br, I.

[0289] The term "haloalkyl" refers to that one, two or more hydrogen atoms or all hydrogen atoms are replaced by halogen in the alkyl as defined above. Representative examples of haloalkyl include CCl , CHCl , CH Cl, CF , CHF , CH F, CBr , CHBr , CH Br, CI , CHI , CH I, CH CF , CF CF Deng.

[0290] The term "aryl" refers to an aromatic carbocyclic ring system containing 6-16 carbon atoms, or 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms, preferably 6-10 carbon atoms. Examples of aryl groups may include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, or pyrenyl. The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing 5-14 membered structures, or preferably 5-10 membered structures, or preferably 5-8 membered structures, more preferably 5-6 membered structures, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N or S, and the number of heteroatoms being preferably 1, 2 or 3. Examples of heteroaryl groups include, but are not limited to, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, etc.

[0291] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocycle, a bridged ring, or a spirocycle. Preferably, it comprises 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0292] The term "-alkyl-" or "alkylene" refers to a saturated straight or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight or branched alkylene group having 1 to 8 carbon atoms.

[0293] The term "heterocyclic radical" refers to a non-aromatic ring that is fully saturated or partially undersaturated (non-completely unsaturated heteroaromatic) and can exist as a monocycle, a bridged ring or a spirocycle. Unless otherwise indicated, the heterocycle is typically a 3 to 7 ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms, but excluding -OO-, -OS- or -SS- moieties) independently selected from sulphur, oxygen and / or nitrogen. Non-limiting examples of heterocyclic radicals include but are not limited to oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl etc.

[0294] The term "heteroalkyl" by itself or in combination with another term refers to a stable linear or branched alkyl radical or combinations thereof, consisting of a certain number of carbon atoms and at least one heteroatom. The number of carbon atoms can be 1-50 (preferably 1-20, more preferably 1-12, and most preferably 1-8), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. The heteroalkyl group may optionally contain one, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from N, O, and S (or interpreted as optionally heteroatoms inserted into the alkyl group, optionally C-C bonds and C-H bonds). The heteroatoms O, N, and S may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0295] Unless otherwise stated, the definition of a term herein also applies to the group containing that term, for example the definition of C1-6 alkyl also applies to C1-6 alkyloxy.

[0296] In various parts of the present invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for the group should be understood as linking groups. For example, if the structure requires a linking group and the Markush group definition for the variable lists "heteroalkyl", "aryl", etc., it should be understood that the "heteroalkyl" and "aryl" represent the heteroalkylene group or arylene group connected, respectively. And it should also be understood that when the Markush variables listed for the group are understood as linking groups, whether they are defined as "alkylene" or "heteroalkylene", the definition scope of the two is the same. Therefore, for example, the definitions of the terms "alkylene", "cycloalkylene", "arylene", "heteroalkylene", "heteroarylene", and "heterocyclylene" are equivalent or refer to the "alkyl", "cycloalkyl", "aryl", "heteroalkyl", "heteroaryl", and "heterocyclyl" as defined above.

[0297] The term "derivative" refers to a compound formed by replacing an atom or an atomic group in a parent compound molecule with another atom or an atomic group, which is called a derivative of the parent compound.

[0298] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a drug-linker or an antibody-linker-drug conjugate). The compound may contain at least one amino, imino, hydroxyl or carboxyl group and may therefore form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodides, nitrates, bisulfates, phosphates, acid phosphates (-H2PO4) salts, phosphites, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, and the like. In addition, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter atoms. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0299] The term "amino acid" refers to naturally occurring amino acids or non-naturally occurring amino acids, represented by NH2-C(R'R")-C(=O)OH, wherein R' and R" are each independently hydrogen, optionally substituted straight chain, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl, or R" and the N-terminal nitrogen atom can be combined to form a heterocycle, for example, proline.

[0300] The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or one hydroxyl group is removed from the carboxyl terminus of an amino acid, for example -NH-C(R'R")-C(O)-.

[0301] The term "peptide" refers to a short chain of amino acid monomers connected by peptide (amide) bonds.

[0302] The term "tumor" refers to a new organism formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation.

[0303] In a broad sense, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, via at least one antigen recognition site within the variable region of the immunoglobulin molecule, represented by A. This term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (e.g., bispecific antibodies, biparatopic antibodies, etc.), multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies having three, four, or more antigen-binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site, as long as the antibody exhibits the desired biological activity. When the terms "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" should be understood as referring to the intact antibody relative to the "antigen-binding fragment / antigen-binding portion," both of which collectively correspond to the broad concept of an antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0304] Figure 1 is the single crystal diffraction pattern of compound 82. DETAILED DESCRIPTION

[0305] The present application is further illustrated by examples, but these examples are not intended to limit the scope of this application. The test methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, ratios, proportions, or parts are by weight.

[0306] Abbreviation Description

[0307] Table 1

[0308] Example 1: Synthesis of Compounds 8-10

[0309] Compound 1 (50 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The temperature was raised to 70°C and the reaction was refluxed for 6 hours. After cooling, white crystals precipitated. The remaining solid was filtered and washed with methanol (20 mL x 3) to obtain a white solid 2 (50 g, 100% yield). MS (ESI): (M+H) + Calculated value 266.1, experimental value 266.2.

[0310] Compound 2 (50 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol) and ethanol (400 mL) were added to a reaction flask and stirred at room temperature to dissolve. After slowly adding carbon disulfide (17 g, 283 mmol), the temperature was raised to 100°C and the reaction was refluxed for 5 hours to complete. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 3 (54 g, yield 93%). MS (ESI): (M+H) + Calculated value 308.1, experimental value 308.2.

[0311] Compound 3 (5 g, 16.3 mmol), triethylamine (2 g, 19.6 mmol), and tetrahydrofuran (30 mL) were added to the reaction flask. Methyl iodide (2.5 g, 17.9 mmol) was then added to the reaction solution. The reaction was stirred at 25°C for 1.5 hours until completion. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 4 (3.4 g, 65% yield). MS (ESI): (M+H) + Calculated value 322.1, experimental value 322.2.

[0312] Compound 4 (3.4 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 5, which was directly used for the next reaction.

[0313] The crude product 5, diglycolic anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and ether was added to the residue. After filtration, the residue was washed with water and ether to obtain compound 7 (3.5 g, yield 99%). MS (ESI): (M+H) + Calculated value 338.0, experimental value 338.2.

[0314] Compound 7 (3.5 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 8 (2.3 g, yield 60%). MS (ESI): (M+H)+ Calculated value 370.0, experimental value 370.1.

[0315] Table 2

[0316] The synthesis of compounds 9-10 refers to the synthetic route of compound 8.

[0317] Example 2. Synthesis of Compound 18

[0318] Compound 11 (47.5 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The temperature was raised to 70°C and the reaction was refluxed for 6 hours. After cooling, white crystals precipitated. The remaining solid was filtered and washed with methanol (20 mL × 3) to obtain a white solid 12 (47.47 g, 100% yield). MS (ESI): (M+H) + Calculated value 252.1, experimental value 252.2.

[0319] Compound 12 (47.4 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol), and ethanol (400 mL) were added to a reaction flask and stirred at room temperature to dissolve. Carbon disulfide (17 g, 283 mmol) was slowly added, and the temperature was raised to 100°C and refluxed for 5 hours to complete the reaction. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 13 (49.9 g, 90% yield). MS (ESI): (M+H) + Calculated value 294.1, experimental value 294.3.

[0320] Compound 13 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to the reaction flask. Methyl iodide (3.12 g, 22 mmol) was then added to the reaction solution. The reaction was stirred at 25°C for 1.5 hours until completion. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 14 (4.05 g, 66% yield). MS (ESI): (M+H) + Calculated value 308.1, experimental value 308.2.

[0321] Compound 14 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 15, which was directly used for the next reaction.

[0322] The crude product 15, diglycolic anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and ether was added to the residue. After filtration, the residue was washed with water and ether to obtain compound 17 (3.4 g, yield 99%). MS (ESI): (M+H) + Calculated value 324.1, experimental value 324.2.

[0323] Compound 17 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 18 (2.4 g, yield 65%). MS (ESI): (M+H) + Calculated value 356.0, experimental value 356.0.

[0324] Example 3: Synthesis of Compound 21

[0325] Tert-butyl glycolate (2.57 g, 19.5 mmol) was slowly added dropwise to a solution of chlorosulfonyl isocyanate (2.75 g, 19.5 mmol) in dichloromethane (40 mL) at 0°C. The temperature was restored to 25°C and the reaction was allowed to proceed for 1 hour. A solution of compound 5 (5 g, 19.5 mmol) in dichloromethane (10 mL) was added dropwise to the above reaction system. After 6 hours of reaction, water (40 mL) was added and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 19 (7.68 g, 86% yield). MS (ESI): (M+H) + Calculated value 459.1, experimental value 459.0.

[0326] Compound 19 (7.68 g, 16.8 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (4.2 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 20, which was directly used for the next reaction.

[0327] The crude product 20 and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (4 g, 25.2 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 21 (3.28 g, 45% yield). MS (ESI): (M+H) + Calculated value 435.0, experimental value 435.1.

[0328] Example 4: Synthesis of Compound 25

[0329] The synthesis of compound 25 was carried out according to the synthesis method of compound 21.

[0330] Example 5: Synthesis of Compounds 29 and 37

[0331] p-Bromobenzaldehyde (7.97 g, 43 mmol), sodium azide (5.57 g, 86 mmol), cuprous iodide (817 mg, 4.3 mmol), L-proline (1.48 g, 12.9 mmol), sodium hydroxide (515 mg, 12.9 mmol), ethanol (70 mL), and water (20 mL) were added to a reaction flask and reacted at 95°C for 24 hours. After the reaction, the mixture was cooled, water was added, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 26 (5.5 g, yield 87%). MS (ESI): (M+H) + Calculated value 148.0, experimental value 148.1.

[0332] Zinc powder (7.3 g, 113 mmol) and tetrahydrofuran (50 mL) were added to a reaction flask, and a solution of propyne bromide (14.9 g, 125.5 mmol) in tetrahydrofuran (30 mL) was added dropwise at 10°C. The mixture was reacted at 10°C for 2 hours, and a solution of 26 (2.75 g, 18.7 mmol) in tetrahydrofuran (30 mL) was slowly added dropwise, and stirred for another 2 hours. The reaction solution was poured into a 1N HCl aqueous solution at 0°C and extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 27 (3.15 g, 90% yield). MS (ESI): (M+H) + Calculated value 188.0, experimental value 188.1.

[0333] Compound 27 (1 g, 5.3 mmol) and dichloromethane (5 mL) were added to a reaction flask, and dichlorothionyl (1.9 g, 16 mmol) was added dropwise at 0°C. After the addition, the temperature was restored to 25°C and the reaction was continued for 2 hours. The solvent and the remaining dichlorothionyl were removed under reduced pressure, and potassium thioacetate (906 mg, 8 mmol) and N,N-dimethylformamide (10 mL) were added. The reaction was continued at 25°C for 12 hours. Water was added and the mixture was extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Tetrahydrofuran (10 mL) and potassium hydroxide (594 mg, 10.6 mmol) solution were added, the reaction was continued at 25°C for 2 hours, water was added, extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 28 (612 mg, yield 56%). MS(ESI):(M+H) + Calculated value 204.0, experimental value 204.1.

[0334] Cyclohexene oxide (1.47 g, 15 mmol) and 40% trimethylbenzylammonium hydroxide (2.5 g, 6 mmol) were added to a reaction flask. Compound 28 (612 mg, 3 mmol) was dissolved in N,N-dimethylformamide (6 mL) and added dropwise to the above reaction system. The mixture was reacted at 25°C for 3 hours. Water was added and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 29 (813 mg, 90% yield). MS (ESI): (M+H) + Calculated value 302.1, experimental value 302.1.

[0335] Trimethylsilyl cyanide (3.86 g, 39 mmol) was added to tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (35.7 mL, 1 M) was added dropwise at 0°C. After reacting for 1 hour, a solution of compound 30 (10 g, 32.5 mmol) in acetonitrile (30 mL) was added. The mixture was reacted at 80°C for 1 hour. After cooling, the solvent was removed, and the residue was purified by silica gel chromatography to obtain compound 31 (5.4 g, 66% yield). MS (ESI): (M+H) + Calculated value 254.0, experimental value 254.1.

[0336] Compound 31 (5.4 g, 21.5 mmol) was added to tetrahydrofuran, and a borane tetrahydrofuran solution (21.5 mL, 1 M) was added dropwise at 0°C. The mixture was then reacted at 25°C for 16 hours. Methanol was added to quench the reaction, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 32 (4.1 g, 74% yield). MS (ESI): (M+H) + Calculated value 258.0, experimental value 258.1.

[0337] Compound 32 (4.1 g, 15.9 mmol) and triethylamine (3.2 g, 31.8 mmol) were added to dichloromethane (30 mL), and trifluoroacetic anhydride (5 g, 23.8 mmol) was added dropwise at 0°C. The mixture was then reacted at 25°C for 1 hour, diluted with water, and extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 33 (5.6 g, 60% yield). MS (ESI): (M+H) + Calculated value 354.0, experimental value 354.1.

[0338] Compound 33 (5.6 g, 9.5 mmol) and paraformaldehyde (0.37 g, 12.4 mmol) were added to sulfuric acid and reacted at 25°C for 1 hour. The reaction solution was poured into ice water and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 34 (2.77 g, yield 80%). MS (ESI): (M+H) + Calculated value 366.0, experimental value 366.1.

[0339] Compound 34 (2.77 g, 7.6 mmol) was added to tetrahydrofuran, and diisobutylaluminum hydride was added dropwise at 0°C. The mixture was then reacted at 25°C for 2 hours. Water was added to quench the reaction, and the filtrate was filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 35 (1.65 g, 90% yield). MS (ESI): (M+H) + Calculated value 242.0, experimental value 242.1.

[0340] Compound 35 (1.65 g, 6.8 mmol), sodium azide (3.5 g, 54.4 mmol), cuprous iodide (130 mg, 0.68 mmol), L-proline (0.4 g, 2 mmol), and sodium hydroxide (80 mg, 2 mmol) were added to ethanol (20 mL) and water (10 mL), and the mixture was reacted at 95°C for 24 hours. After cooling, water was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 36 (0.97 g, 70% yield). MS (ESI): (M+H) + Calculated value 205.1, experimental value 205.1.

[0341] Compound 36 (0.97 g, 4.8 mmol), compound 16 (1.8 g, 5.04 mmol), and triethylamine (0.76 g, 7.2 mmol) were added to tetrahydrofuran (10 mL) and reacted at 25°C for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 37 (1.7 g, yield 80%). MS (ESI): (M+H) + Calculated value 447.2, experimental value 447.1.

[0342] Example 6: Synthesis of Compound 45

[0343] Compound 38 (10 g, 50 mmol) was dissolved in tetrahydrofuran (120 mL), and sodium hydride (4 g, 100 mmol) was added at 0°C and stirred for 0.5 hours. Allyl bromide (12.1 g, 100 mmol) was then added and allowed to react for 2 hours. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted with ethyl acetate (10 mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 39 (4.65 g, 66% yield). MS (ESI): (M+H) + Calculated value 142.2, experimental value 142.3.

[0344] Compound 39 (4.65 g, 33 mmol) was added to tetrahydrofuran (100 mL) and a tetrahydrofuran solution of 9-borabicyclo[3.3.1]nonane (80 mL, 0.5 M) was added. The mixture was reacted at 80° C. for 2 hours, cooled to room temperature, and the reaction solution was directly used for the next reaction.

[0345] To the reaction mixture of compound 40, N,N-dimethylformamide (100 mL) and an aqueous solution of potassium phosphate (8.7 g, 41 mmol) were added, stirred for 10 minutes, and 3-chloro-4-bromoaniline (6.8 g, 33 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (2.4 g, 3.3 mmol) were added. The reaction was carried out at 90°C for 3 hours, cooled to room temperature, and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 41 (3.7 g, 42% yield). MS (ESI): (M+H) + Calculated value 269.1, experimental value 269.1.

[0346] Compound 41 (3.7 g, 13.8 mmol) was added to dichloromethane (90 mL), and diisopropylethylamine (3.5 g, 27 mmol) and phenyl chloroformate (2.5 g, 16 mmol) were added to the solution in sequence. The mixture was reacted at 25°C for 0.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 42 (4.66 g, yield 89%). MS (ESI): (M+H) + Calculated value 389.2, actual value 389.2.

[0347] Compound 42 (4.66 g, 12 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of compound 43 (4.08 g, 13.2 mmol). The solid was dispersed in the solution by ultrasound, and diisopropylethylamine (48 mmol) was added. The reaction was carried out at 50°C for 3 hours. The mixture was cooled to room temperature, and water was added. The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 44 (6.4 g, yield 94%). MS (ESI): (M+H) + Calculated value 568.2, experimental value 568.4.

[0348] Compound 44 (1.6 g, 2.8 mmol) was added to dichloromethane (19 mL), and a solution of hydrogen chloride in ethyl acetate (11 mL, 4 M) was added dropwise at 0°C. The reaction was continued for 2 hours. The solvent was removed under reduced pressure and the mixture was dried in vacuo to obtain compound 45 (1.6 g, 94% yield). MS (ESI): (M+H) +Calculated value 568.2, experimental value 568.3.

[0349] Example 7: Synthesis of Compounds 46-49

[0350] Table 3

[0351] The synthesis of compounds 46-49 followed the synthetic route of compound 45.

[0352] Example 8: Synthesis of Compounds 52 and 55

[0353] Compound 50 (1.1 g, 1.06 mmol, obtained from solid-phase peptide synthesis) and diethylamine (3.89 g, 53.24 mmol) were dissolved in dichloromethane (5 mL). The reaction solution was purged with N2 three times and then reacted at 25°C for 1 hour. After complete conversion of the starting material, the solvent was removed under reduced pressure, and low-boiling point compounds were removed under high vacuum. Compound 51 (3.15 g, 1.6 mmol) and N,N-dimethylformamide (4 mL) were then added and stirred. Triethylamine (3.23 g, 3.2 mmol) was added dropwise at 25°C. After 6 hours of reaction, no starting material remained as determined by LCMS. Excess triethylamine was removed under reduced pressure, and the residue was directly separated by reverse phase chromatography to obtain compound 52 (810 mg, 10% yield). MS (ESI): (M+H) + Calculated value 812.4, experimental value 812.3.

[0354] Compound 53 (1.2 g, 1.36 mmol, synthesized according to the synthetic route reported in patent CN 111757757 A) and compound 54 (447 mg, 1.36 mmol, obtained from solid-phase peptide synthesis) were added to N,N-dimethylformamide (40 mL), followed by the addition of 1-hydroxybenzotriazole (40 mg, 0.26 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (285 mg, 1.46 mmol). The reaction was stirred at 25°C for 16 hours. After terminating the reaction, the solvent was removed under reduced pressure, and the residue was separated by reverse phase chromatography to obtain compound 55 (820 mg, 50% yield). MS (ESI): (M+H) + Calculated value 1208.5, experimental value 1208.6.

[0355] Example 9: Synthesis of Compound LD1

[0356] Compound 27 (2.15 g, 11.5 mmol), p-nitrophenyl chloroformate (3 g, 15 mmol) and N,N-dimethylformamide (15 mL) were added to a reaction flask, stirred and triethylamine (2.32 g, 23 mmol) was slowly added dropwise. The reaction was carried out at 25°C for 4 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography to obtain compound 56 (4.05 g, 100% yield).

[0357] Compound 56 (176 mg, 0.5 mmol), isotecan mesylate (243 mg, 0.45 mmol), and N,N-dimethylformamide (2 mL) were added to a reaction flask, stirred, and triethylamine (101 mg, 1 mmol) was slowly added dropwise. The mixture was reacted at 25°C for 6 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 57 (259 mg, 89% yield). MS (ESI): (M+H) + Calculated value 649.2, experimental value 649.1.

[0358] Compound 57 (259 mg, 0.4 mmol) and compound 58 (670 mg, 1 mmol, synthesized according to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, the nitrogen atmosphere was replaced three times, tetrahydrofuran (10 mL) and water (4 mL) were added, and the reaction was carried out at 25°C for 24 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 59 (239 mg, 59% yield). MS (ESI): (M+H) + Calculated value 1015.4, experimental value 1015.3.

[0359] Compound 59 (239 mg, 0.24 mmol) and N,N-dimethylformamide (2 mL) were added to a reaction flask, and 1,8-diazobispiro[5.4.0]undec-7-ene (18 mg, 0.12 mmol) was added dropwise. The mixture was reacted at 25°C for 0.5 h. Compound 8 (107 mg, 0.29 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (890 mg, 0.36 mmol), diisopropylethylamine (48 mg, 0.48 mmol) and N,N-dimethylformamide (10 mL) were added to the above reaction system. The mixture was reacted at 25°C for 24 h, diluted with water (20 mL), and extracted with ethyl acetate (20 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 60 (192 mg, 70% yield). MS(ESI):(M+H) +Calculated value 1144.4, experimental value 1144.3.

[0360] Compound 60 (192 mg, 0.168 mmol), compound 61 (280 mg, 0.252 mmol), sodium ascorbate (7 mg, 0.034 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (15 mg, 0.034 mmol), and cuprous bromide (5 mg, 0.034 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and tert-butanol (4 mL) and water (1 mL) were added. The mixture was reacted at 25°C for 12 hours, diluted with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound LD1 (220 mg, 62% yield). MS (ESI): (M+H) + Calculated value 2258.0, experimental value 2258.2.

[0361] Example 10: Synthesis of compounds LD2-19, LD23-28, and LD32-54

[0362] Table 4

[0363] The synthetic routes of compounds LD2-19, LD23-28, and LD32-54 refer to the synthesis of compound LD1.

[0364] The structures of Con1-5, Pep1-5, Trig1-3, HDP1-3, and Pay1-13 are as follows:

[0365] The nitrogen end was connected to Fmoc.

[0366] Among them, the # position is connected to -N3, the ## position is connected to -OH, and the ### position is connected to alkynyl.

[0367] Synthesis of compound LD17

[0368] Compound LD17' was synthesized using the synthetic route of compound LD1. Compound LD17' (130 mg, 0.05 mmol) was dissolved in dichloromethane (2 mL). Dichloroacetic acid (2 mL) and a small amount of triethylsilane were added to the reaction system at 0°C. The reaction was allowed to proceed for 3 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound LD17 (70 mg, 60% yield). MS (ESI): (M+H) +Calculated value 2363.1, experimental value 2363.2.

[0369] Example 11: Synthesis of Compound LD20-22

[0370] Table 5

[0371] Among them, the # position is connected to -N3, the ## position is connected to -OH, and the ### position is connected to alkynyl.

[0372] Compound 29 (3 g, 10 mmol), p-nitrophenyl chloroformate (3 g, 15 mmol) and N,N-dimethylformamide (12 mL) were added to a reaction flask, stirred and triethylamine (2 g, 20 mmol) was slowly added dropwise. The reaction was carried out at 25°C for 4 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography to obtain compound 62 (4.5 g, yield 96%).

[0373] Compound 62 (233 mg, 0.5 mmol), isotecan mesylate (243 mg, 0.45 mmol), and N,N-dimethylformamide (2 mL) were added to a reaction flask, stirred, and triethylamine (101 mg, 1 mmol) was slowly added dropwise. The mixture was reacted at 25°C for 6 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 63 (343 mg, 90% yield). MS (ESI): (M+H) + Calculated value 763.2, experimental value 763.3.

[0374] Compound 63 (343 mg, 0.45 mmol) and compound 58 (670 mg, 1 mmol, synthesized according to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, the nitrogen atmosphere was replaced three times, tetrahydrofuran (10 mL) and water (4 mL) were added, and the reaction was carried out at 25°C for 24 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 64 (304 mg, 60% yield). MS (ESI): (M+H) + Calculated value 1129.4, experimental value 1129.3.

[0375] Compound 64 (304 mg, 0.27 mmol) and N,N-dimethylformamide (2 mL) were added to a reaction flask, and 1,8-diazobispiro[5.4.0]undec-7-ene (18 mg, 0.13 mmol) was added dropwise. The mixture was reacted at 25°C for 0.5 h. Compound 8 (107 mg, 0.3 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (890 mg, 0.36 mmol), diisopropylethylamine (54 mg, 0.54 mmol) and N,N-dimethylformamide (10 mL) were added to the above reaction system. The mixture was reacted at 25°C for 24 h, diluted with water (20 mL), and extracted with ethyl acetate (20 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 65 (170 mg, yield 50%). MS(ESI):(M+H) + Calculated value 1258.4, experimental value 1258.3.

[0376] Compound 65 (170 mg, 0.135 mmol), compound 61 (235 mg, 0.21 mmol), sodium ascorbate (6 mg, 0.027 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (11 mg, 0.027 mmol), and cuprous bromide (4 mg, 0.027 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and tert-butyl alcohol (4 mL) and water (1 mL) were added. The mixture was reacted at 25°C for 12 hours, diluted with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound LD20 (180 mg, 56% yield). MS (ESI): (M+H) + Calculated value 2372.1, experimental value 2372.2.

[0377] The synthetic routes of compounds LD21-22 refer to the synthesis of compound LD20.

[0378] Example 12: Synthesis of Compound LD29-31

[0379] Table 6

[0380] Compound 56 (176 mg, 0.5 mmol), compound 66 (88 mg, 0.5 mmol), and N,N-dimethylformamide (2 mL) were added to a reaction flask, stirred, and triethylamine (101 mg, 1 mmol) was slowly added dropwise. The mixture was reacted at 25°C for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 67 (128 mg, 66% yield). MS (ESI): (M+H) + Calculated value 390.1, experimental value 390.1.

[0381] Compound 67 (128 mg, 0.33 mmol), compound 68 (95 mg, 0.36 mmol), pyridinium p-toluenesulfonate (8 mg, 0.03 mmol), and toluene (5 mL) were added to a reaction flask and reacted at 100°C for 12 hours, then cooled to room temperature. The mixture was filtered and the solid was washed with toluene (5 ml) to obtain compound 69 (185 mg, 90% yield). MS (ESI): (M+H) + Calculated value 617.2, experimental value 617.3.

[0382] Compound 69 (185 mg, 0.3 mmol) and compound 58 (503 mg, 0.75 mmol, synthesized according to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, the nitrogen atmosphere was replaced three times, tetrahydrofuran (10 mL) and water (4 mL) were added, and the reaction was carried out at 25°C for 24 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 70 (177 mg, 60% yield). MS (ESI): (M+H) + Calculated value 983.4, experimental value 983.3.

[0383] Compound 70 (177 mg, 0.18 mmol) and N,N-dimethylformamide (2 mL) were added to a reaction flask, and 1,8-diazobispiro[5.4.0]undec-7-ene (14 mg, 0.09 mmol) was added dropwise. The mixture was reacted at 25°C for 0.5 h. Compound 11 (82 mg, 0.22 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (666 mg, 0.27 mmol), diisopropylethylamine (36 mg, 0.36 mmol) and N,N-dimethylformamide (10 mL) were added to the above reaction system. The mixture was reacted at 25°C for 24 h, diluted with water (20 mL), and extracted with ethyl acetate (20 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 71 (150 mg, yield 75%). MS(ESI):(M+H) + Calculated value 1112.4, experimental value 1112.3.

[0384] Compound 71 (150 mg, 0.135 mmol), compound 61 (224 mg, 0.202 mmol), sodium ascorbate (5 mg, 0.027 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (12 mg, 0.027 mmol), and cuprous bromide (4 mg, 0.027 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and tert-butanol (4 mL) and water (1 mL) were added. The mixture was reacted at 25°C for 12 hours, diluted with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound LD29 (155 mg, 60% yield). MS (ESI): (M+H) + Calculated value 2212.0, experimental value 2212.2.

[0385] The synthetic routes of compounds LD30-31 refer to the synthesis of compound LD29.

[0386] The compounds shown in Table 7 can also be synthesized with reference to the aforementioned examples (wherein the final product containing the Pep3 structure is obtained by acid treatment with reference to LD17).

[0387] Table 7

[0388] Example 13: Synthesis of compound LD55 and separation of LD55-1 and LD55-2

[0389] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultra-dry DCM (90 mL) were added to a reaction flask. After 3 hours of reaction, the solvent was removed under reduced pressure, and the residue was slurried in 500 mL of MTBE to obtain compound 72 (9007 mg, 64% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 455.2, experimental value 455.2.

[0390] At 0°C, 72 (4000 mg, 8.8 mmol, dr = 1:1) and 100 mL of commercially available THF were added to a reaction flask. DBU (1337 mg, 8.8 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was allowed to cool to room temperature. After TLC monitoring of the disappearance of the starting material, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to afford compound 73 (1776 mg, 87% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 233.1, experimental value 233.2.

[0391] Compound 73 (1624 mg, 7 mmol, dr = 1:1), Fmoc-Val-OSu (362 mg, 8.4 mmol, CASRN: 688585-20-8), and N,N-dimethylformamide (150 mL) were added to a reaction flask, stirred, and DIEA (1158 uL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25°C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added for slurrying to precipitate a white solid. This reaction was repeated three times to obtain compound 74 (dr = 1:1). MS (ESI): (M+H) + Calculated value 554.3, experimental value 554.4.

[0392] Compound 74 (1303 mg, 2.4 mmol, dr = 1:1), p-nitrophenyl chloroformate (964.8 mg, 4.8 mmol), and THF (120 mL) were added to a reaction flask, stirred, and Py (382.8 uL, 4.8 mmol) was added dropwise. The reaction was continued at 65°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to obtain compound 75 (1430 mg, 83% yield, dr = 1:1). MS (ESI): (M+Na) + Calculated value 741.3, experimental value 741.5.

[0393] Compound 75 (1554 mg, 2.2 mmol, dr = 1:1), isotecan mesylate 76 (520 mg, 1.8 mmol), and N,N-dimethylformamide (80 mL) were added to a reaction flask, stirred, and DIEA (740 uL, 4.5 mmol) was slowly added dropwise. The mixture was reacted at 25°C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 77 (1442 mg, 79% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 1015.4, experimental value 1015.1.

[0394] At 0 degrees Celsius, 77 (1400 mg, 1.37 mmol, dr = 1:1) and 35 mL of THF (commercially available) were added to the reaction flask. DBU (209 mg, 1.37 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was moved to room temperature and the reaction was continued for 40 minutes until the starting material disappeared on TLC. The reaction solution was concentrated in vacuo to remove THF, and DCM / PE (20 mL / 200 mL) was added to the residue for slurrying. 78 (1032 mg, 95% yield, dr = 1:1) was obtained by filtration through a suction funnel. MS (ESI): (M+H) + Calculated value 793.3, experimental value 793.6.

[0395] Compound 78 (793 mg, 1 mmol, dr = 1:1), 18 (426 mg, 1.2 mmol), EDCI (382 mg, 2 mmol), HOBT (202 mg, 1.5 mmol), and N,N-dimethylformamide (10 mL) were added to a reaction flask and reacted at 25°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by C18 purification (60% ACN / 0.05% formic acid in H2O) and lyophilized to obtain compound 79 (960 mg, 85% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 1130.4, experimental value 1130.7.

[0396] Compound 79 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol), and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and THF / DMF / H2O (3.5 mL:0.6 mL:0.4 mL) was added. The reaction was allowed to proceed at 25°C for 0.5 h. The residue was purified by C18 preparative purification (62% ACN / 0.05% formic acid in H2O) to afford compound LD55 (1180 mg, 86% yield, dr = 1:1). MS (ESI): (M+H)+ Calculated value 1715.7, experimental value 1716.2.

[0397] LD55 (1180 mg, dr = 1:1) was separated using ACN / H2O (0.05% formic acid) on a preparative column (Yuexu Xtimate C18 30×250 mm×10 um) to give LD55-1 (550 mg, HPLC retention time 18.397 min) and LD55-2 (520 mg, HPLC retention time 18.163 min).

[0398] Note: HPLC instrument information: Thermo liquid chromatograph (ADC-U3000-01); chromatographic column: Hypersil GOLD TM (4.6*250mm, 5μm); column temperature: 30°C; injection plate temperature: 10°C; mobile phase: A: Water + 0.05% TFA; B: ACN; flow rate: 1.0mL / min; detection wavelength: 254nm; injection volume: 10μL;

[0399] Gradient conditions:

[0400] Compounds LD1 to LD54, LD56-LD144, LD2-1, LD2-2, LD7-1, LD7-2, LD12-1 and LD12-2 can also be synthesized by the same method.

[0401] Example 14: Synthesis of LD55-1 from chiral raw material 82 obtained by resolution of racemic raw material 72

[0402] Chiral resolution of intermediate 72: Compound 72 (50 g) was subjected to SFC to afford 81 (19 g, retention time 13.55 min) and 82 (20 g, retention time 16.29 min). SFC separation method: Column: DAICEL CHIRALCEL OD (250 mm-50 mm, 10 μm); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25°C; Back pressure: 100 bar.

[0403] HPLC method: Instrument information: Thermo liquid chromatograph (ADC-U3000-01); Chromatographic column: ID (4.6*150mm, 5μm); column temperature: 25°C; injection plate temperature: 25°C; mobile phase: A: 10mM NH4FA; B: ACN; flow rate: 0.8mL / min; detection wavelength: 254nm; injection volume: 2μl;

[0404] Gradient conditions:

[0405] The structure and configuration of compound 82 were confirmed by X-RAY. The single crystal diffraction pattern of compound 82 is shown in Figure 1:

[0406] At 0°C, compound 82 (3632 mg, 8 mmol) and 90 mL of commercially available THF were added to a reaction flask. DBU (1215 mg, 8 mmol) was slowly added with stirring, and the reaction was allowed to proceed for half an hour before being allowed to cool to room temperature. After TLC monitoring of the disappearance of the starting material, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to afford compound 83 (1707 mg, 92% yield). MS (ESI): (M+H) + Calculated value 233.1, experimental value 233.2.

[0407] Compound 83 (1624 mg, 7 mmol), Fmoc-Val-OSu (362 mg, 8.4 mmol), and N,N-dimethylformamide (150 mL) were added to a reaction flask, stirred, and DIEA (1158 uL, 7 mmol) was slowly added dropwise. The reaction was continued at 25°C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added for slurrying to precipitate a white solid. This reaction was repeated three times to obtain compound 84. MS (ESI): (M+H) + Calculated value 554.3, experimental value 554.4.

[0408] Compound 84 (1661 mg, 3 mmol), p-nitrophenyl chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask, stirred, and Py (474 ​​uL, 6 mmol) was added dropwise. The mixture was reacted at 65°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to obtain compound 85 (1831 mg, 85% yield). MS (ESI): (M+H) + Calculated value 719.8, experimental value 719.9.

[0409] Compound 85 (1436 mg, 2 mmol), isotecan mesylate 86 (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask, stirred, and DIEA (695 uL, 4 mmol) was slowly added dropwise. The reaction was continued at 25°C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 87 (1664 mg, 82% yield). MS (ESI): (M+H) + Calculated value 1015.4, experimental value 1015.1.

[0410] At 0°C, 87 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask. DBU (228 mg, 1.5 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was brought to room temperature and the reaction continued for 40 minutes until the starting material disappeared on TLC. The reaction solution was concentrated in vacuo to remove THF, and 15 mL of DCM and 300 mL of PE were added to produce a large amount of yellow-green solid. Solid 88 (1177 mg, 99% yield) was filtered off using a Buchner funnel. MS (ESI): (M+H) + Calculated value 793.3, experimental value 793.6.

[0411] Compound 88 (1031 mg, 1.3 mmol), 18 (554 mg, 1.56 mmol), EDCI (498 mg, 2.6 mmol), HOBT (263 mg, 1.95 mmol), and N,N-dimethylformamide (20 mL) were added to a reaction flask and reacted at 25°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by C18 purification (60% ACN / 0.05% formic acid in H2O) and lyophilized to obtain compound 89 (1322 mg, 90% yield). MS (ESI): (M+H) + Calculated value 1130.4, experimental value 1130.7.

[0412] Compound 89 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol), and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and THF / DMF / H2O (3.5 mL:0.6 mL:0.4 mL) was added. The reaction was continued at 25°C for 0.5 h. The residue was purified by C18 preparative purification (62% ACN / 0.05% formic acid in H2O) to obtain compound LD55-1 (1276 mg, 93% yield). MS (ESI): (M+H) + Calculated value 1715.7, experimental value 1716.2. 1H NMR (600MHz, DMSO) δ10.71(s,1H),10.08(s,1H),8.48(d,J=6.7Hz,1H),8.17(d,J=8.6Hz,3H),8.09(d,J=8.8Hz,1H),8.03(d,J=8.7Hz,2 H),7.89(s,1H),7.81(d,J=10.8Hz,1H),7.66(d,J=8.4Hz,2H),7.42–7.36(m,3H),6.61(s,1H),6.02(t,J=6.8Hz,1H),5.55(s,2H),5.38 -5.26(m,3H),4.55–4.47(m,3H),4.46–4.41(m,1H),4.36(q,J=15.6Hz,2H),4.28( s,2H),3.80(s,5H),3.62–3.55(m,36H),3.52–3.47(m,9H),3.44–3.36(m,2H),3.30 -2.29(m,1H),3.19–3.09(m,1H),2.42(s,3H),2.28–2.19(m,1H),2.17(s,3H),2.17–2.10(m ,1H),2.04–1.89(m,2H),1.42(d,J=7.0Hz,3H),1.02(d,J=6.7Hz,3H),0.98(t,J=7.2Hz,6H).

[0413] After comparison with the HPLC method of LD55-1 in Example 13, the retention time of the product LD55-1 separated in this step is consistent with that of LD55-1 in Example 13.

[0414] Example 15: Synthesis of LD2-1

[0415] Compound 89 (113 mg, 0.1 mmol), compound 61 (134 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (4.3 mg, 0.01 mmol), and cuprous bromide (14 mg, 0.01 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and THF / DMF / H2O (1 mL:0.2 mL:0.2 mL) was added. The reaction was continued at 25°C for 0.5 h. The residue was purified by C18 preparative purification (60% ACN / 0.05% formic acid in H2O) to afford compound LD2-1 (199.7 mg, 89% yield). MS (ESI): (1 / 2 M+H) + Calculated value 1123.0, experimental value 1123.0.

[0416] Example 16: Synthesis of LD7-1

[0417] Compound 89 (113 mg, 0.1 mmol), compound 52 (99.3 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (4.3 mg, 0.01 mmol) and cuprous bromide (14 mg, 0.01 mmol) were added to a reaction flask, the atmosphere was purged with nitrogen three times, THF / DMF / H2O (1 mL:0.2 mL:0.2 mL) was added, and the mixture was reacted at 25°C for 0.5 h. The residue was purified by C18 preparative purification (55% ACN / 0.05% formic acid in H2O) to give compound LD7-1 (180 mg, 93% yield). (1 / 2M+H) + Calculated value 971.4, experimental value 971.5.

[0418] Example 17: Synthesis of LD12-1

[0419] Compound 89 (113 mg, 0.1 mmol), compound 55 (147 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (4.3 mg, 0.01 mmol), and cuprous bromide (14 mg, 0.01 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and THF / DMF / H2O (1 mL:0.2 mL:0.2 mL) was added. The reaction was continued at 25°C for 0.5 h. The residue was purified by C18 preparative purification (55% ACN / 0.05% formic acid in H2O) to afford compound LD12-1 (229 mg, 98% yield). MS (ESI): (1 / 2 M+H) + Calculated value 1170.0, experimental value 1170.0.

[0420] Example 18: General method for ADC sample preparation and DAR determination

[0421] Antibodies with known sequences can be obtained by protein expression methods well known to those skilled in the art, and then the fermentation broth can be subjected to affinity chromatography, ion chromatography and other steps to obtain a sample with higher purity.

[0422] Dilute the antibody sample to approximately 10 mg / mL in a suitable buffer (the same as the sample buffer). Add an appropriate amount of the reducing agent, TCEP, with the equivalents adjusted based on the target DAR (e.g., 8-10 equivalents for a DAR of 8). Adjust the pH to 7-7.4 with Tris buffer and reduce the sample at room temperature for 1-1.5 hours. The antibody reduction intermediate can be monitored by CE-SDS. Once the antibody is fully reduced, adjust the pH to approximately 6.5 with an appropriate amount of saturated citric acid solution. Then, add an excess of the linker-drug solution in DMSO to a linker-drug equivalent ratio of 15-20 times that of the antibody. The coupling reaction is carried out at room temperature for approximately 30 minutes. After coupling, filter the reaction solution and then perform ultrafiltration using a centrifugal concentrator to remove excess linker-drug and other small molecule impurities. After purification, the resulting sample is subjected to reverse-phase chromatography for DAR determination.

[0423] Preparation method of T-ADC

[0424] The concentration of the antibody trastuzumab was adjusted to 10 g / L using phosphate buffer solution (pH 7.0). The antibody solution after the liquid exchange was placed in a centrifuge tube, and 8.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was allowed to proceed at room temperature for 1 hour to reduce the disulfide bonds between the antibody chains to sulfhydryl groups. Subsequently, a 20 mM linker-drug solution dissolved in DMSO was added (15 equivalents of toxin per antibody). The reaction was continued at 10°C for 1 hour. After the coupling solution was passed through an ultrafiltration membrane bag (Cobetter, Ltd.) to remove the residual linker toxin and stored in PBS solution at pH 7.4. The average drug loading of each antibody was measured by reversed-phase chromatography.

[0425] Preparation methods of F-ADC and FS-ADC

[0426] The concentration of the antibody farletuzumab or farletuzumab FcS was adjusted to 10 g / L using phosphate buffer solution (pH 7.0). The antibody solution after the liquid exchange was placed in a centrifuge tube, and 7.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added and reacted at 37°C for 1 hour to reduce the disulfide bonds between the antibody chains to sulfhydryl groups. Subsequently, 20 mM linker-drug dissolved in DMSO was added (10 equivalents of toxin for each antibody). The reaction was carried out at 10°C for 1 hour. After the coupling solution was passed through an ultrafiltration tube to remove the residual linker toxin, it was stored in a PBS solution at pH 7.4. The average drug loading of each antibody was measured by reverse chromatography.

[0427] Preparation method of D-ADC

[0428] The concentration of antibody 33B was adjusted to 10 g / L using phosphate buffer solution (pH 7.0). The antibody solution after the liquid exchange was placed in a centrifuge tube, and 10.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was carried out at 37°C for 1 hour to reduce the disulfide bonds between the antibody chains to sulfhydryl groups. Subsequently, 20 mM linker-drug solution dissolved in DMSO was added (12 equivalents of toxin per antibody). The reaction was carried out at 25°C for 1 hour. After the coupling solution was passed through an ultrafiltration membrane bag (Cobetter, Ltd.) to remove the residual linker toxin and stored in PBS solution at pH 7.4. The average drug loading of each antibody was measured by reverse chromatography.

[0429] Example B-ADC preparation method

[0430] The concentration of antibody 43B was adjusted to 10 g / L using phosphate buffer solution (pH 7.0). The antibody solution after the solution exchange was placed in a double-layer glass reactor, and 12.0 equivalents of 100 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was carried out at 37°C for 3 hours to reduce the disulfide bonds between the antibody chains to sulfhydryl groups. Subsequently, 10 mM linker-drug solution dissolved in DMSO was added (10 equivalents of toxin per antibody) at room temperature. The reaction was carried out at 25°C for 30 minutes. After the coupling solution was removed by protein A, the residual linker toxin was removed and stored in PBS solution at pH 7.4. The average drug loading of each antibody was measured by reversed-phase chromatography.

[0431] By adjusting the dosage of TCEP and linker-drug, ADCs with different Dar values ​​can be obtained.

[0432] The data statistics of all ADC samples are shown in the following table.

[0433] Table 8

[0434] Example 19: Preparation of control ADC samples

[0435] ORM-5029 was obtained as a control example of HER-2 ADC by referring to the method of document WO 2021 / 198965 A1, and the DAR was determined to be 3.5 by reverse phase chromatography. In addition, low DAR samples of ADC38-ADC44 were obtained under the same coupling conditions, respectively recorded as ADC38-4 to ADC44-4, and the DAR determined by LC-MS were also 3.5±0.1. In particular, when the linker-drug used by ORM-5029 was coupled to the antibody, due to the high proportion of aggregates, it was not possible to successfully obtain qualified ADC samples with high Dar values ​​(>7). Enhertu is a commercial product.

[0436] BATLD was synthesized according to the method of CN116333135A, and referring to the aforementioned ADC preparation method, F-BATADC, D-BATADC, and B-BATADC were synthesized using BATLD as the linker-drug and farletuzumab, 33B, and 43B as antibodies. The DAR values ​​determined by reverse phase chromatography were 7.9, 7.7, and 5.5, respectively.

[0437] DSADC was obtained by referring to the method of document CN104755494A, and the DAR was determined to be 3.9 by reverse phase chromatography.

[0438] The sequence of the M30 antibody is as follows:

[0439] >M30-H1 (SEQ ID NO:43)

[0440] >M30-L4 (SEQ ID NO: 44)

[0441] Effect Example 1: In vitro activity test of ADC samples

[0442] SK-BR-3 cells, which overexpress HER2, were selected as test cells. Cells were cultured according to standard methods and seeded at an appropriate density in 96-well plates. After observation, the test sample was added using a concentration gradient of 0-100 nM. After 144 hours of culture, cell viability was determined using the CTG assay to calculate the IC50 value.

[0443] Table 9 Note: A in the table represents IC 50 Value <15 pM, B indicates IC 50 The value is between 15pM and 25pM, and C represents IC 50 >25pM; Enhertu was purchased as a commercial product.

[0444] From the activity test results, it can be seen that the ADC in the present invention has significantly improved activity compared with the same type ADC ORM-5029, and also has significantly improved activity compared with Enhertu with the same target.

[0445] Effect Example 2: In vivo activity test of ADC samples

[0446] JIMT-1 cells with low expression of her2 were selected and inoculated into NUNU mice. After tumor formation, mice with a tumor volume of approximately 150 cubic millimeters were selected and divided into different groups, with 5 mice in each group. Different drugs were administered through tail vein injection at pre-set doses. After administration, the tumor size and mouse weight were observed and measured regularly, and the frequency of administration was increased as appropriate. The experiment was terminated after meeting certain conditions, and the final tumor size and mouse weight were measured to calculate the tumor inhibition rate. The test results are shown in the table below:

[0447] Table 10: In vivo efficacy of non-hydrophilic linker ADCs

[0448] Among them, T-ADC is prepared according to the method of document WO 2021 / 198965 A1, and the following linker drug compound is first prepared:

[0449] Then conjugate with the antibody to obtain the target ADC:

[0450] T-ADC

[0451] Table 11: In vivo efficacy of hydrophilic linker ADCs

[0452] The in vivo efficacy results in Table 10 show that, for ADCs using non-hydrophilic linkers, T-ADC is significantly more effective than ORM-5029, with the same linker, and slightly less effective than Enhertu. The in vivo efficacy results in Table 11 show that ADCs using hydrophilic linkers are more effective than Enhertu. Combining the results in Tables 10 and 11, we can conclude that ADCs using hydrophilic linkers are significantly more effective than those using non-hydrophilic linkers.

[0453] Effect Example 3: In vitro cytotoxicity of Her2-ADC

[0454] The HER2-ADC-mediated in vitro cytotoxicity was evaluated using the HER2-positive cell line SK-BR-3. Cells were harvested by trypsin, incubated with serially diluted HER2-ADCs, and then incubated at 37°C. Viability was determined after 6 days using CTL Plus. The cells were read and analyzed on an EnVision 2105 (PerkinElmer) to determine the IC 50 (half-maximal inhibitory concentration) value. SK-BR-3 cells were adjusted to a cell density of 40,000 cells / mL using McCoy's 5A + 15% FBS, and 75uL was seeded into each well of a 96-well plate (Manufacturer: Corning, Catalog No.: 3599) and placed in a Forma TMSteri-Cycle TM Incubate overnight in an i160 CO2 incubator at 37°C and 5% CO2. Dilute the HER2-ADC with the culture medium of the corresponding cells, starting from 100nM and diluting 5-fold for a total of 9 concentration gradients. Then add 75uL to each well of the cells. The blank control is the culture medium of the corresponding cells. Set up two replicates for each concentration. SK-BR-3 cells are placed in Forma TM Steri-Cycle TM Cells were cultured in an i160 CO2 incubator at 37°C and 5% CO2 for 6 days. After 6 days, 50 μL of CTL Plus (Manufacturer: Beyotime, Catalog No.: C0068XL) luminescent reagent was added and incubated at room temperature in the dark for 10 minutes. Chemiluminescence detection was performed on an EnVision 2105 microplate reader. Data Analysis and Management: Using the blank control as the zero-killing control, the inhibition rate was calculated as follows: Inhibition rate (%) = (1 - experimental group / blank control) x 100. Data were processed and analyzed using GraphPad Prism. The results are shown in the table. The results demonstrate that the HER2-ADC exhibits high in vitro cytotoxicity against HER2-positive cell lines.

[0455] Table 12: HER2-ADC in vitro cytotoxicity

[0456] Effect Example 4: Determination of ADC hydrophilicity by hydrophobic interaction chromatography (HIC)

[0457] HIC-HPLC conditions: ① Column model: TSKgel Butyl-NPR, 2.5 μm, 4.6*100 mm, PN: 0042168; ② Column temperature: 30°C; ③ UV: 280 nm; ④ Mobile phase: A: 1.5 M (NH₄)₂SO₄, 25 mM NaPi, pH 6.73; B: 12.5 mM NaPi (pH 7.28): 20% IPA = 80:20 (v:v); ⑤ Flow rate: 0.7 mL / min; ⑥ Elution gradient: 0 min → 1.01 min (0% B → 20% B), 1.01 min → 10 min (20% B → 100% B), 10 min → 11 min (100% B → 100% B), 11 min → 11.01 min (100% B → 0% B), 11.01 min → 17 min (0% B)

[0458] Table 13: ADC hydrophilicity

[0459] Highly hydrophilic linkers not only overcome the difficulty of conjugation due to poor water solubility of many toxins, but also reduce the formation of aggregates during the conjugation process, ensuring that the ADC has lower clearance and better PK. The data in the table show that different hydrophilic side chains can increase the hydrophilicity of the ADC to varying degrees. Among them, naked farletuzumab has the highest hydrophilicity. The hydrophilicity of the ADCs of the present invention is superior to that of the control F-BAT ADC.

[0460] 1. Antigen coating: Dilute FOLR1 (Sino Biological, Cat. No. 11241-H08H) antigen to 0.1 μg / ml using coating solution (0.05 mol / L carbonate buffer, pH 9.6). Add 100 μl of the solution to each well of an ELISA-specific polystyrene microplate (Corning, Cat. No. 3590) and incubate at 4°C overnight for antigen coating.

[0461] 2. Blocking: Discard the coating solution in the microplate and wash three times with PBST (PBS containing 0.05% Tween-20 10mM, pH 7.4). Add 300ul of 2% BSA to each well and block at 37°C for 1 hour.

[0462] 3. Washing: Wash the microplate three times with washing buffer, perform two replicates for each sample, and add F-ADC55-1-8 with a maximum concentration of 1000 nM in a volume of 100 μL. ADC samples were serially diluted 11 times with 2% BSA and incubated at 37°C for 2 h.

[0463] 4. Secondary antibody: Discard the FRα primary antibody dilution in the microplate and wash three times with washing buffer. Add secondary antibody (Goat anti-human IgG (L&H) secondary, HRP) to each well and incubate at 37°C for 1 hour.

[0464] 5. Color development: discard the secondary antibody diluent, wash with detergent 6 times, then add 100ul TMB color development solution. The color development time is about 6-8min, and then add 100ul 2M dilute hydrochloric acid to terminate the reaction.

[0465] 6. Plate reading: After the reaction is terminated, read the absorbance at OD450 on a microplate reader (manufacturer: BioTek, model: Synergy LX);

[0466] 7. Calculate EC50

[0467] Table 14: ADC Binding Activity

[0468] Effect Example 6: Pharmacodynamic Evaluation in Human Oral Cancer KB Xenograft Mouse Model

[0469] 10 cells were collected from FRα-positive cell line KB 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). When the average tumor volume of the mice reached 100 mm 3 (1 to 6 groups) and 145mm 3 (7-8 groups) were randomly divided into groups and administered by single intravenous injection. The day of grouping was set as day 0, and administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of animals, including the activity of experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0470] Table 15: Drug efficacy of groups 1-6 in KB xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 30, respectively; c. Compared with the tumor volume of the vehicle control group.

[0471] Table 16: Efficacy of Groups 7-8 in the KB Xenograft Tumor Model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 20, respectively; c. Compared with the tumor volume of the vehicle control group.

[0472] The ADC of the present invention in the 3 mg / kg test group showed significantly better efficacy than the control F-BAT ADC (TGI 92.0%), with tumor inhibition rates (TGI) of 104.7%, 104.7%, 104.6%, and 104.6% in each group, respectively. Among them, 4, 4, 3, and 3 mice in the test groups achieved CR, respectively. Furthermore, there were no significant differences in the efficacy of ADCs with different chiral linker configurations. When the dose of F-ADC55-1-8 was reduced to 1.5 mg / kg, the tumor inhibition rate (TGI) still reached 105.6%. There were no animal deaths or significant weight loss in any of the treatment groups, and no obvious drug toxicity was observed. During treatment, mice tolerated the ADC of the present invention well. Specific results are shown in the table.

[0473] Effect Example 7: Pharmacodynamic Evaluation of the Test Drug in the Human Lung Adenocarcinoma NCI-H441 Xenograft Tumor Mouse Model

[0474] 10 cells were collected from the FRα-positive cell line NCI-H441 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). When the average tumor volume of the mice reached 100 mm 3 The mice were randomly divided into groups (1 to 6 groups) and administered by a single intravenous injection. The day of grouping was set as day 0, and administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. Specifically, the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b) 2 (where a represents the major diameter and b represents the minor diameter.) The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0475] Table 17: Drug efficacy of each group in the NCI-H441 xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 32, respectively; c. Compared with the tumor volume of the vehicle control group.

[0476] Compared to the vehicle control group, the ADC of the present invention significantly inhibited tumor growth in the NCI-H441 model at a 1 mg / kg dose, demonstrating significantly superior tumor inhibition compared to the F-BAT ADC control group. No animal mortality or significant weight loss was observed in any of the treatment groups, and no overt drug toxicity was observed. The ADC of the present invention was well tolerated by mice during treatment. Specific results are shown in the table.

[0477] Effect Example 8: Pharmacodynamic evaluation of the test drug in a human colon cancer SW620 xenograft mouse model

[0478] 10 cells were collected from FRα-positive cell line SW620 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). 3 (1-2 groups), the mice were randomly divided into groups and dosed by a single intravenous injection. The day of grouping was set as day 0, and the dosing began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0479] Table 18: Drug efficacy of each group in SW620 xenograft tumor model

[0480] Compared with the vehicle control group, the ADC of the present invention in the 1.5 mg / kg test group significantly inhibited tumor growth in the SW620 model. There were no animal deaths or significant animal weight loss in each treatment group, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC of the present invention well. Specific results are shown in the table.

[0481] Effect Example 9: Pharmacodynamic Evaluation of the Test Drug in a Human Ovarian Cancer OV-90 Xenograft Mouse Model

[0482] 10 cells were collected from FRα-positive cell line OV-90 7 The cells were inoculated into the right forelimb axilla of 4-6 week old NCG and NOD SCID immunodeficient mice (3 mice per group). 3 (1 to 3 groups) and 145mm 3 When the mice were randomly divided into groups (4-5 groups), they were given drugs. The day of grouping was set as day 0, and drug administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. Specifically, the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0483] Table 19: Drug efficacy of each group in OV-90 xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 11, respectively; c. Compared with the tumor volume of the vehicle control group; d. NCG immunodeficient mice e. NOD SCID immunodeficient mice

[0484] Compared with the vehicle control group, the ADC of the present invention significantly inhibited tumor growth in the OV-90 model at 1, 3, and 5 mg / kg doses, with tumor growth inhibition rates (TGI) of 110.9%, 126.1%, and 120.7%, respectively. No animal mortality or significant weight loss was observed in any of the treatment groups, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during treatment. Specific results are shown in the table.

[0485] Effect Example 10: Flow cytometry determination of DLL3 expression cell positive rate

[0486] Binding of DLL3 antibodies to surface DLL3 in NCI-H209, SHP77, NCI-H526, NCI-H82, and NCI-H69 cells was determined by flow cytometry.

[0487] Experimental procedure: NCI-H209, SHP77, NCI-H526, NCI-H82 and NCI-H69 cells were collected and prepared into 5×10 5 / mL cell suspension and add 100uL per well to a 96-well round-bottom plate (Manufacturer: Corning, Catalog No.: 3795). After centrifugation to remove the supernatant, add 100uL / well of different concentrations of D-ADC55-1-8 diluted with FACS buffer (final concentration 15ug / ml) and incubate in a 4°C refrigerator in the dark for 2 hours. After centrifugation and washing three times with FACS buffer, add working concentrations of Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488 (Manufacturer: Invitrogen, Catalog No.: A11013), placed in a 4°C refrigerator in the dark for 1 hour. The geometric mean fluorescence intensity was measured on a CytoFLEX flow cytometer, and DLL3 was calculated. + %.

[0488] Table 20: Flow cytometry determination of DLL3 expression cell positive rate

[0489] Effect Example 11: Enzyme-linked immunosorbent assay to determine the binding activity of DLL3-ADC

[0490] The affinity of the antibody for recombinant human DLL3 protein was determined using an ELISA method. Human DLL3 protein (Kaixia Biotechnology, Cat. No. DLL-HM103) was diluted to a final concentration of 0.1 μg / ml and plated onto 96-well microtiter plates at 4°C overnight. The supernatant was discarded the next day. After blocking, the wells were added with a 4-fold serial dilution of the antibody (0-100 nM). After incubation with a secondary antibody, color was developed with TMB colorimetric solution for 10 minutes. After terminating the reaction with 2M HCl, the absorbance at 450 nm was read on a microplate reader, and the EC50 was calculated. The results, as shown in the table, show that the affinity of the ADC for human DLL3 protein is essentially equivalent to that of the unconjugated antibody.

[0491] Table 21: ELISA assay for DLL3 ADC binding activity

[0492] Effect Example 12: In vitro cytotoxicity of DLL3-ADC

[0493] D-ADC55-1-8-mediated in vitro cytotoxicity was evaluated in DLL3-positive cell lines NCI-H82, SHP77, NCI-H526, and NCI-H209, and DLL3-negative cell lines A431 and NCI-H69. Cells were harvested, incubated with serial dilutions of D-ADC55-1-8, and then incubated at 37°C. Viability was determined after 6 days using CTL Plus. The cells were read and analyzed on an EnVision 2105 (PerkinElmer) to determine the IC 50 (half-maximal inhibitory concentration) value.

[0494] Experimental process: The cell density of the test cells was adjusted to 6.7×10 4 / mL, and 75μL per well was inoculated into a 96-well plate and incubated overnight in a 37°C, 5% CO2 incubator. A five-fold serial dilution of D-ADC55-1-8 (0-100nM) was added to the cells, with 75μL added to each well. The blank control consisted of the culture medium of the corresponding cells. Two replicate wells were set for each concentration. After culturing the cells in a 37°C, 5% CO2 incubator for 6 days, 50μL of CTL Plus (Manufacturer: Biyuntian, Catalog No.: C0068XL) luminescent reagent was added and incubated at room temperature in the dark for 10 minutes. Chemiluminescence detection was performed on an EnVision 2105 microplate reader. Data analysis and organization: The blank control was used as the zero-killing control, and the inhibition rate was calculated as follows: Inhibition rate (%) = (1-test group / blank control group) × 100%. GraphPad Prism was used to process and analyze the data and calculate the IC50. The results are shown in the table. The results showed that D-ADC55-1-8 had good in vitro cytotoxicity against DLL3-positive cell lines.

[0495] Table 22: DLL3-ADC in vitro cytotoxicity

[0496] Effect Example 13: Pharmacodynamic Evaluation of the Test Drug in a Human Small Cell Lung Cancer SHP77 Xenograft Mouse Model

[0497] 10 cells were collected from the DLL3-positive cell line SHP77 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (7 mice per group). When the average tumor volume of the mice reached 100 mm 3 The mice were randomly divided into groups (1 to 6 groups) and administered by a single intravenous injection. The day of grouping was set as day 0, and administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. Specifically, the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0498] Table 23: Pharmacodynamic evaluation of the test drug in the human small cell lung cancer xenograft SHP-77 mouse model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 22, respectively; c. Compared with the tumor volume of the vehicle control group.

[0499] Compared to the vehicle control group, the ADC of the present invention significantly inhibited tumor growth in the SHP-77 model at a dose of 1 mg / kg, with tumor growth inhibition rates (TGI) of 98.1%, 96.6%, 97.1%, and 97.3%, respectively, significantly superior to the control group (TGI 79.9%), D-BAT ADC. No animal mortality or significant weight loss was observed in any treatment group, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during treatment. Specific results are shown in the table.

[0500] Effect Example 14: Pharmacodynamic Evaluation of the Test Drug in the Human Small Cell Lung Cancer NCI-H526 Xenograft Mouse Model

[0501] 10 cells were collected from the DLL3-positive cell line NCI-H526 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (3 mice per group). 3 (1-2 groups), the mice were randomly divided into groups and dosed by a single intravenous injection. The day of grouping was set as day 0, and the dosing began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0502] Table 24: Pharmacodynamic evaluation of the investigational drug in the human small cell lung cancer xenograft NCI-H526 mouse model Note: a. Data are expressed as "mean ± standard error"; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the average tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the average tumor volumes of the drug-treated group and the vehicle control group on Day 21, respectively.

[0503] Effect Example 15: Pharmacodynamic Evaluation of the Test Drug in the Human Small Cell Lung Cancer NCI-H69 Xenograft Mouse Model

[0504] 10 cells were collected from the DLL3-positive cell line NCI-H69 7 The cells were inoculated into the right forelimb axilla of nu / nu mice (3 mice per group). When the average tumor volume of the mice reached 140 mm 3 The mice were randomly divided into groups and administrated by single intravenous injection. The day of grouping was set as day 0, and the administration started on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the experimental drugs is shown in the following table.

[0505] Table 25: Pharmacodynamic evaluation of the experimental drug in the human small cell lung cancer xenograft NCI-H69 mouse model Note: a. Data are expressed as "mean ± standard error"; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100%, where T0 and C0 are the average tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the average tumor volumes of the drug-treated group and the vehicle control group on Day 21, respectively.

[0506] In the NCI-H526 and NCI-H69 models, the ADC of the present invention significantly inhibited tumor growth in the NCI-H526 and NCI-H69 models at a dose of 3 mg / kg, compared with the vehicle control group, with tumor growth inhibition rates (TGI) of 113.0% and 76.5%, respectively. There were no animal deaths or significant animal weight loss in each treatment group, and no obvious drug toxicity reactions were observed. During the treatment period, mice tolerated the ADC of the present invention well. Specific results are shown in the table.

[0507] Effect Example 16: FACS determination of ADC affinity

[0508] The affinity of B-ADC55-1-6, B-ADC2-1-6, B-ADC12-1-6, and DSADC was tested using the B7H3-positive human melanoma cell line A375. The sample was diluted to 15,000 ng / mL with 2% BSA in PBS and then serially diluted 11 times to a concentration of 0.014 to 15,000 ng / mL. A375 cells were centrifuged at 500 × g for 5 minutes, washed three times with 2% BSA in PBS, and incubated with the various diluted samples at 4°C for 2 hours. After washing, the cells were incubated with goat anti-human Alexa Fluor 488 fluorescent dye for 1 hour at 4°C in the dark. After washing, the cells were resuspended and the mean fluorescence intensity (MFI) was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.).

[0509] Table 26: ADC affinity for B7H3 antigen on the surface of A375 detected by flow cytometry

[0510] From the above data, it can be seen that the affinity of the ADC of the present invention to the B7H3 antigen on the surface of A375 is significantly better than that of the control group DSADC.

[0511] Effect Example 17: Pharmacodynamic Evaluation of the Test Drug in a Human Hep3B Hepatocellular Carcinoma Xenograft Mouse Model

[0512] 10 cells were collected from the B7H3-positive cell line Hep3B 7 The cells were inoculated into the right forelimb axilla of 4-6 week old nu / nu nude mice (6 mice per group). 3 The mice were randomly divided into groups and given a single injection. The day of grouping was set as day 0, and the administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0513] Table 27: Pharmacodynamic evaluation of the experimental drugs in the Hep3B liver cancer xenograft mouse model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 22, respectively; c. Compared with the tumor volume of the vehicle control group.

[0514] Compared to the vehicle control group, the ADC of the present invention at 2 mg / kg significantly inhibited tumor growth in the Hep3B model; the experimental groups significantly outperformed the control group. No animal mortality or significant weight loss occurred in any treatment group, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during treatment. Detailed results are shown in the table.

[0515] Effect Example 18: Pharmacodynamic Evaluation of the Test Drug in a Human Malignant Melanoma Cell A375 Xenograft Mouse Model

[0516] 10 cells were collected from the B7H3-positive cell line A375 7 The cells were inoculated into the right forelimb axilla of 4-6 week old nu / nu nude mice (7 mice per group). 3 The mice were randomly divided into groups and given medication. The day of grouping was set as day 0, and medication began on day 0. After tumor inoculation, routine monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0517] Table 28: Grouping and Dosage Regimen

[0518] Table 29: Pharmacodynamic evaluation of the experimental drug in the human malignant melanoma cell xenograft A375 mouse model Note: a. Data are expressed as mean ± standard error; b. Compared with the tumor volume of the vehicle control group.

[0519] The data in the table show that on day 22, the mean tumor volume of the vehicle control group reached 3000 mm 3 , euthanasia was performed according to animal welfare. On day 26, the control group had a significantly lower efficacy than the equal-dose test group. Compared with the vehicle control group, the test groups all showed significant tumor inhibition effects. Effect Example 19: Antitumor Experiment of Antibody-Drug Conjugate on Human Lung Cancer Calu-6 Mouse Transplanted Tumors

[0520] In this experiment, NOD / SCID mice of appropriate age were inoculated with human lung cancer cells Calu-6. When the tumor volume grew to about 100-200 mm, 3 When the drug was administered, animals with well-growing tumors were selected and divided into groups evenly according to tumor volume. The animal groups and dosage regimens are shown in the table below.

[0521] Table 30 Animal grouping and dosage regimen

[0522] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows: Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0523] At the end of the experiment, the test group could significantly inhibit tumor growth compared with the vehicle group.

[0524] Table 31 Efficacy of ADC on Calu-6 xenograft tumors in tumor-bearing mice

[0525] Effect Example 20: Enzyme Digestion Rate Determination

[0526] 1.CTB activation:

[0527] Cathepsin B lyophilized powder (10 units) was diluted with the corresponding storage solution (pH 5.0, 25mM acetate buffer + 1.0mM EDTA) to a 10 UN / mL solution. The total volume was 1 mL. The solution was aliquoted into 50 μL tubes and stored at -80°C. For the enzymatic digestion reaction, 50 μL of CTB (10 UN / mL) stock solution was added to 860 μL of pH 5.0 buffer, followed by 100 μL of Cys (200mM) activation solution. Activation was performed for 15 minutes, and the total volume was 1.0 mL, resulting in a final concentration of 0.5 UN / mL of CTB activated solution.

[0528] 2. Blocking of substrate mother liquor:

[0529] Measure 5 μL of 10mM DMSO solution of LD55-1, LD55-2, LD2-1, and LD2-2, add 5 μL of 100mM (5.0equiv) cysteine ​​(Cys) aqueous solution, mix well, and carry out blocking reaction for 5-10 minutes to obtain the blocked substrate mother solution Cys-LD55-1, Cys-LD55-2, Cys-LD2-1, and Cys-LD2-2, whose substrate concentration is 10 μL 5mM.

[0530] 3. Perform enzyme digestion reaction:

[0531] The final concentration of the substrate is 40uM: Measure 2.0μL of 5mM substrate blocking solution, add 250μL of 0.5UN / mL CTB solution, the total volume is 0.25mL, incubate at 37℃, and sample and test every 30min for up to 24h.

[0532] 4. Test samples:

[0533] After the reaction time point, take an appropriate amount of sample, add an equal volume of ice-cold MeOH (HPLC grade) for dilution and precipitation, then centrifuge at more than 10000 rpm and 4°C for 10 min, and take the supernatant for detection.

[0534] 5. Data Results

[0535] The drug peak area detected without enzyme addition was set to 1, and the percentage obtained by comparing the drug peak area detected at the corresponding time point with the drug peak area was the drug release ratio at that time point. Table 28 records the remaining Cys-Linker-drug amount after enzymatic digestion of Cys-LD55-1, Cys-LD55-2, Cys-LD2-1, and Cys-LD2-2.

[0536] Table 32: Enzyme cleavage release assay

[0537] From the enzyme cleavage curves, it can be seen that Cys-LD55-1, Cys-55-2, Cys-LD2-1, and Cys-LD2-2 configurations can all be completely cleaved by enzymes. Cys-LD55-1 and Cys-LD2-1 are completely cleaved in about 30-60 minutes, and Cys-LD55-2 and Cys-LD2-2 are completely cleaved in about 24 hours.

[0538] Effect Example 21: Evaluation of the toxicokinetics and tolerability of ADC in cynomolgus monkeys

[0539] Cynomolgus monkeys (2 females) were selected and ADC (5 mg / mL, 30-minute injection) was administered intravenously at a dose level of 10-50 mg / kg every 2-3 weeks (3 doses in total). Parameters evaluated during the study included general observations, body weight, food intake, body temperature, electrocardiogram (lead II), clinical pathology (hematology, blood biochemistry, coagulation), and macroscopic and microscopic examinations of a large number of tissues. Toxicokinetic samples were collected at 0, 24, 72, 120, 336, and 504 hours after each dose. TK samples were analyzed using the MesoScale Discovery (MSD) electrochemiluminescence platform (total monoclonal antibody, total ADC) and LC-MS / MS (free exitecan). The experiment showed that the ADC of the present invention was well tolerated. In the dose study, after injection of ADC, no abnormalities were observed in the general observation of the cynomolgus monkeys, and no toxicological abnormalities were observed in body weight, body temperature, coagulation, or urine analysis. The ADC of the present invention was well tolerated in cynomolgus monkeys and exhibited stable pharmacokinetic characteristics.

Claims

1. An antibody drug conjugate having the structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: A-(LD) m (I) wherein A is a targeting ligand selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment, a small molecule ligand, or a polypeptide; L is a linker part, one end of which is connected to the ligand A and the other end is connected to the bioactive molecule D; D is an amino-containing biologically active molecule or its tautomer, mesomer, racemate, enantiomer, diastereomer, isotope-labeled substance, which is covalently linked to the linker portion L through the amino group in its molecular structure; m is an integer or decimal from 1 to 12; The linker moiety -L- is represented by the formula: -L1-L2-L3-L4-, in, L1 is the part of L that connects to ligand A; preferably, L1 is selected from (or its open loop form or )、 or Wherein * indicates connection with the sulfhydryl group of A (such as monoclonal antibody), and ** indicates connection with L2; L2 is a spacer; preferably, selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-, -L 2a -C(O)-NH-, -L 2a -L 2b -NH-C(O)-, -L 2a -L 2b -C(O)-NH-, -L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -NH-C(O)-, -L 2a -NH-C(O)-L 2b -NH-C(O)-, -L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 14 Arylene-C1-C8 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8 alkylene-, 1-50 straight chain or branched heteroalkylene, 1-50 straight chain or branched heteroalkylene-3-8 membered heterocyclylene, the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 1-6 heteroalkylene atoms, C1-C6 alkoxy, hydroxyl, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkylene contains 1-12 heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkylene are selected from one or more of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight chain or branched chain heteroalkylene of 1 to 50 atoms, R 1 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14 Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 heteroatoms, and the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene, heteroarylene are selected from one or more of N, O or S; L3 is a polypeptide sequence; preferably, selected from a peptide residue consisting of 2-8 natural or unnatural amino acids, wherein the optional amino acid is further substituted by one or more substituents selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxyl, amino, carboxyl or C3-C8 cycloalkyl; L4 is a self-cleaving fragment modified with a hydrophilic group. The self-cleaving fragment is selected from: or Wherein, * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups; Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group: Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6; The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or a non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups; Preferably, L4 is selected from: or Wherein * indicates connection with the carboxyl group of L3 by an amide bond, and ** indicates connection with the amino group of the bioactive molecule D; R 2 is a hydrophilic fragment, preferably, selected from a linear or branched heteroalkyl group containing 4-50 (OCH2CH2) structural units, a peptide chain containing 4-50 protein amino acids (such as glycine) or non-protein amino acids (such as sarcosine), a linear or branched heteroalkyl group containing a monosaccharide, an oligosaccharide or a polysaccharide; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6; D is selected from the following compounds:

2. The antibody drug conjugate having the structure of formula (I) according to claim 1 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels, wherein A is selected from the group consisting of targeting HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, DLL3, TROP2, B7-H3, c-Met, CD20, CD22, CD30, CD33, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Antibodies or antigen-binding fragments thereof to Y, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, FRα or Mesothelin.

3. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 2, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L2 is selected from the following structures: in, w is an integer selected from 1-12, * indicates connection with L1, and ** indicates connection with L3.

4. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 3, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L3 is selected from the following structures: Among them, * indicates connection with L2, and ** indicates connection with L4.

5. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 4, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L4 is selected from the following structures: in, v is an integer selected from 1-12, * indicates connection with the carboxyl group of L3 via an amide bond, and ** indicates connection with the amino group of the biologically active molecule D.

6. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 5, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L4 is selected from the following structures:

7. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 6, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein R 2 Select from the following structures: in, r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); R a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R b Selected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

8. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 7, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L is selected from the following structures: Wherein, * indicates connection with the sulfhydryl group of antibody A, and ** indicates connection with the amino group of biologically active molecule D.

9. An antibody-drug conjugate having a structure of formula (I) according to any one of claims 1 to 8, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting FRα, DLL3, B7H3, HER2.

10. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 9, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting FRα, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO: 22, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO: 23, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO: 24; the light chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO: 25, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO: 27, wherein the CDRs are determined according to the Kabat numbering convention; or A is an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO: 12, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO: 13, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO: 14; the light chain comprises three complementary determining regions (CDRs) of the light chain, wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO: 15, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO: 17, wherein the CDRs are determined according to the Kabat numbering convention; or A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO: 3; the light chain comprises three complementary determining regions (CDRs) of the light chain, wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering convention; or A is an antibody or an antigen-binding fragment thereof targeting HER2, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain The light chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO:37, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO:38, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO:39; the light chain comprises three complementary determining regions (CDRs) of the light chain, wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO:40, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO:41, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO:42, wherein CDRs are determined according to the Kabat numbering rules.

11. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 10, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting FRα, wherein the anti-FRα antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is as shown in SEQ ID NO: 28, and the variable region (LV) sequence of the light chain is as shown in SEQ ID NO: 29; or A is an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the anti-DLL3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is as shown in SEQ ID NO: 18, and the variable region (LV) sequence of the light chain is as shown in SEQ ID NO: 19; or A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is as shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is as shown in SEQ ID NO:8; or A is an antibody or an antigen-binding fragment thereof targeting HER2, wherein the anti-HER2 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO:35, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO:

36.

12. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 11, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody targeting FRα or an antigen-binding fragment thereof, wherein the anti-FRα antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 30 or SEQ ID NO: 32, and the amino acid sequence of the light chain is shown in SEQ ID NO: 31; or A is an antibody targeting DLL3 or an antigen-binding fragment thereof, wherein the anti-DLL3 antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21; or A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 9 or SEQ ID NO: 10, and the amino acid sequence of the light chain is shown in SEQ ID NO: 11; or A is an antibody targeting HER2 or an antigen-binding fragment thereof, wherein the anti-HER2 antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is shown in SEQ ID NO:

34.

13. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 12, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, which are selected from the following structures: in, m is an integer or decimal of 1 to 12, preferably an integer or decimal of 2 to 8, and more preferably 2, 4, 6 or 8.

14. A pharmaceutical composition comprising an antibody drug conjugate having a structure of formula (I) as described in any one of claims 1 to 13, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products, and one or more pharmaceutically acceptable excipients.

15. Use of an antibody drug conjugate having a structure of formula (I) as claimed in any one of claims 1 to 13, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products, or the pharmaceutical composition as claimed in claim 14 in the preparation of a drug for treating cancer.

16. The use of claim 15, wherein the cancer comprises liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), gastric cancer, esophageal cancer, oral cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumor or multiform glioma, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma or recurrent anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

17. A linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: L'-D (II) Wherein L' is a linker moiety; D is an amino-containing biologically active molecule or a pharmaceutically acceptable salt thereof, which is covalently linked to the linker portion L' via the amine group in its molecular structure; L' is represented by the following formula: L1'-L2-L3-L4- in, L1' is selected from or Among them, ** indicates connection with L2; The definitions of L2, L3, L4, and D are the same as those of claims 1-8.

18. A compound of formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: The formula (III) has the following structure: or in, X does not exist or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 It is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6.

19. A compound of formula (IV) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: or in, X does not exist or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6; wherein AA is L3 or a subfragment consisting of the first, second or more amino acids from the C-terminus of L3, N is H or an amino protecting group such as Boc or Fmoc, P C is H, a hydroxyl protecting group or a carbonate active ester group such as p-nitrophenol carbonate; The compound of formula (IV) is preferably:

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