Antibody-drug conjugates

Abstract

An antibody-drug conjugate, a method for preparing the same, and its use, wherein the hydrophilic linker in the conjugate can enable linking of most active molecules, and furthermore, the obtained antibody-drug conjugate has relatively good stability, hydrophilicity, and pharmacokinetic properties.

Description

Detailed description of the invention

[0001] [Cross-reference of related applications] This application claims priority to two prior applications: one filed with the China National Intellectual Property Administration on April 27, 2023, with patent application number 2023104680075 and title "Antibody-Drug Conjugate," and another filed with the China National Intellectual Property Administration on January 30, 2024, with patent application number 2024101290645 and title "Antibody-Drug Conjugate." The full texts of these two prior applications are incorporated into this invention by reference.

[0002] [Technical Field] This invention belongs to the fields of biotechnology and pharmaceuticals, and more particularly to antibody-drug conjugates, methods for preparing them, and their use.

[0003] [Background technology] Antibody-conjugated drugs or antibody-drug conjugates (ADCs) are one of the biggest hotspots in current research and development of antitumor drugs. Currently, 14 antibody-conjugated drugs are marketed. These include Mylotarg, approved in 2000 for the treatment of CD33-positive acute myeloid leukemia (AML); Adcetris, approved in 2011 for Hodgkin lymphoma; Kadcyla, approved in 2013 for HER2-positive breast cancer; Besponsa, approved in 2017 for B-cell lymphoblastic leukemia; Lumoxiti, approved in 2018 for relapsed or refractory hairy cell leukemia; Polivy, approved in 2019 for relapsed or refractory diffuse large B-cell lymphoma; and urinary tract These include Padcev, approved in 2019 for skin cancer; Enhertu, approved in 2019 for HER2-positive breast cancer; Trodelvy, approved in 2020 for triple-negative breast cancer; Akalux, a photoimmunotherapy drug approved in 2020 for head and neck cancer; Zynlonta, approved in 2021 for large B-cell lymphoma; Aidixi, approved in 2021 for HER2-positive gastric cancer; Tivdak, approved in 2022 for cervical cancer; and Elaher, approved in 2022 for ovarian cancer. Mylotarg was withdrawn from the market in 2010 due to its high toxicity, but was remarketed in 2017 with an adjusted clinical dose. Blenrep's approval process for multiple myeloma was expedited in 2020, but it was withdrawn from the market due to insufficient subsequent clinical results.

[0004] Antibody-conjugated drugs (ADCs) consist of three parts: the antibody, the linker, and the biologically active molecule (drug). The antibody is closely related to the disease target, while the linker and drug are core components that determine the efficacy and safety of ADCs. Most commercially available ADCs use a technology developed decades ago by Seattle Genetics, which uses a microtubule inhibitor as the active molecule, combined with a cathepsin hydrolysis linker to conjugate to the antibody's cysteine ​​via maleimide. ADCs obtained using this technology are often highly toxic, hydrophobic, and have poor pharmacokinetic properties. Daiichi Sankyo's ADCs, obtained using a hydrophilic tetrapeptide linker, have shown significant improvement, but their properties differ considerably from those of naked antibodies. Trodelvy uses a linker containing polyethylene glycol and salt-forming lysine. The resulting ADCs have good hydrophilicity but low stability. According to the literature, linkers containing polysarcosine can significantly improve the hydrophilicity of ADCs. However, this linker is only suitable for active molecules containing a secondary amine structure, such as MMAE. In the case of active molecules with a primary amine structure, such as exatecan, the carbonamide bond formed by this linker is less stable.

[0005] The object of the present invention is to solve the above problems, enable linking of most active molecules, and provide a hydrophilic linker in which the resulting ADC has excellent stability and hydrophilicity, as well as good pharmacokinetic properties.

[0006] [Summary of the Invention] [Problems the invention aims to solve] The present invention provides antibody-drug conjugates having the structure of formula (I), and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof.

[0007] A-(LD) m (I) (Here, A is a targeted ligand selected from antibodies (e.g., monoclonal antibodies), antigen-binding fragments, small molecule ligands, polypeptides, L is a linker portion, with one end linked to ligand A and the other end linked to the biologically active molecule D. D is a biologically active molecule containing an amino group, or a tautomer, meso, racemic, enantiomer, diastereomer, or isotope-labeled molecule thereof, which is covalently bonded to the linker moiety L via the amino group in its molecular structure. m represents the molar ratio (DAR, also known as the drug-antibody binding ratio) between the cytotoxic drug molecule and antibody A.

[0008] m is an integer or decimal number between 1 and 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0009] If m is a decimal, it represents the average number of linker-drug molecules (LDs) conjugated to each antibody unit (A). In some embodiments, A is selected from antibodies or antigen-binding fragments targeting 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, VEGF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, FRα, or Mesothelin.

[0010] In some embodiments, A can be modified, for example, by altering, increasing, or decreasing one or more amino acids.

[0011] In some embodiments, A is selected from antibodies or antigen-binding fragments that target HER2, DLL3, B7H3, or FRα.

[0012] In some embodiments, A is an antibody or antigen-binding fragment targeting B7H3, the antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the heavy chain comprising three complementarity-determining regions (CDRs), where the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:2, the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:3, the light chain comprising three light chain complementarity-determining regions (CDRs), where the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID As shown in NO:6, the CDR is determined according to the Kabat numbering rules.

[0013] In some embodiments, A is an antibody or antigen-binding fragment that targets B7H3, wherein the anti-B7H3 antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, 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.

[0014] 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.

[0015] In some embodiments, A is an antigen-binding fragment that targets B7H3, and the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, or F(ab')2.

[0016] In some embodiments, A is an antibody or antigen-binding fragment that targets B7H3, and the anti-B7H3 antibody or antigen-binding fragment is a humanized or fully human antibody or its antigen-binding fragment.

[0017] In some embodiments, A is an antibody or antigen-binding fragment that targets B7H3, the anti-B7H3 antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO:9 or SEQ ID NO:10, and the amino acid sequence of the light chain being shown in SEQ ID NO:11.

[0018] In some embodiments, A is an antibody or antigen-binding fragment that targets DLL3, the antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, the heavy chain comprises three complementarity-determining regions (CDRs), where the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:12, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:13, the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:14, the light chain comprises three light chain complementarity-determining regions (CDRs), where the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:15, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:16, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID As shown in NO:17, the CDR is determined according to the Kabat numbering rules.

[0019] In some embodiments, A is an antibody or antigen-binding fragment that targets DLL3, the anti-DLL3 antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain shown in SEQ ID NO:18, and the variable region (LV) sequence of the light chain shown in SEQ ID NO:19.

[0020] 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.

[0021] 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.

[0022] In some embodiments, A is an antibody or antigen-binding fragment that targets DLL3, and the anti-DLL3 antibody or antigen-binding fragment is a humanized or fully human antibody or its antigen-binding fragment.

[0023] In some embodiments, A is an antibody or antigen-binding fragment that targets DLL3, and 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.

[0024] In some embodiments, A is an antibody or antigen-binding fragment that targets FRα, and the antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, the heavy chain comprising three complementarity-determining regions (CDRs), where the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:22, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:23, the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:24, the light chain comprising three complementarity-determining regions (CDRs), where the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:25, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:26, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID As shown in NO:27, the CDR is determined according to the Kabat numbering rules.

[0025] In some embodiments, A is an antibody or antigen-binding fragment that targets FRα, the anti-FRα antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain shown in SEQ ID NO:28, and the variable region (LV) sequence of the light chain shown in SEQ ID NO:29.

[0026] 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.

[0027] In some embodiments, A is an antigen-binding fragment that targets FRα, 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 antigen-binding fragment that targets FRα, and the anti-FRα antibody or antigen-binding fragment is a humanized or fully human antibody or its antigen-binding fragment.

[0029] In some embodiments, A is an antibody or antigen-binding fragment that targets FRα, the anti-FRα antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO:30 or SEQ ID NO:32, and the amino acid sequence of the light chain being shown in SEQ ID NO:31.

[0030] In some embodiments, A is an antibody or antigen-binding fragment that targets HER2, and the antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, the heavy chain comprising three complementarity-determining regions (CDRs), where the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:37, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:38, the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:39, and the light chain comprising three light chain complementarity-determining regions (CDRs), where the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:40, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:41, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID As shown in NO:42, the CDR is determined according to the Kabat numbering rules.

[0031] In some embodiments, A is an antibody or antigen-binding fragment that targets HER2, the anti-HER2 antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain shown in SEQ ID NO:35, and the variable region (LV) sequence of the light chain shown in SEQ ID NO:36.

[0032] 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.

[0033] In some embodiments, A is an antigen-binding fragment that targets HER2, and 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 antigen-binding fragment that targets HER2, and the anti-HER2 antibody or antigen-binding fragment is a humanized or fully human antibody or its antigen-binding fragment.

[0035] In some embodiments, A is an antibody or antigen-binding fragment that targets HER2, and 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.

[0036] In some embodiments, A is selected from 43A or 43B targeting B7H3, 33B targeting DLL3, farletuzumab or farletuzumab-FcS targeting FRα, or trastuzumab or its biosimilar targeting HER2.

[0037] 43A contains two identical heavy and light chains; the heavy chain amino acid sequence is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:11.

[0038] >43A heavy chain sequence (SEQ ID NO:9)

[0039] [ka]

[0040] >43A light chain sequence (SEQ ID NO:11)

[0041] [ka]

[0042] Here, the amino acid sequences shown in bold are heavy / 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.

[0043] [Table 1]

[0044] Here, the underlined amino acid sequences are heavy / light chain variable region sequences, and HV and LV are named SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0045] 43A heavy chain variable region (HV) sequence (SEQ ID NO: 7): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYFMNWVRQAPGQGLEWMGDVNPKTGSPSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRYGFLYSMDYWGQGTSVTVSS 43A Light Chain Variable Region (LV) Sequence (SEQ ID NO: 8): DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKVLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTHPFTFGGGTKVEIK 43B contains two identical heavy and light chains; the heavy chain amino acid sequence is shown in SEQ ID NO:10, and the light chain amino acid sequence is shown in SEQ ID NO:11.

[0046] >43B heavy chain sequence (SEQ ID No: 10)

[0047] [ka]

[0048] >43B light chain sequence (SEQ ID NO:11)

[0049] [ka]

[0050] Here, the amino acid sequences shown in bold are heavy / 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.

[0051] [Table 2]

[0052] The underlined amino acid sequences are heavy / light chain variable region sequences, and HV and LV are named SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0053] 43B heavy chain variable region (HV) sequence (SEQ ID NO:7): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYFMNWVRQAPGQGLEWMGDVNPKTGSPSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRYGFLYSMDYWGQGTSVTVSS 43B light chain variable region (LV) sequence (SEQ ID NO: 8): DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKVLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTHPFTFGGGTKVEIK 33B contains two identical heavy and light chains; the heavy chain amino acid sequence is shown in SEQ ID NO:20, and the light chain amino acid sequence is shown in SEQ ID NO:21.

[0054] >33B heavy chain sequence (SEQ ID NO:20)

[0055] [ka]

[0056] >33B light chain sequence (SEQ ID NO:21)

[0057] [ka]

[0058] Here, 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.

[0059] >33B heavy chain variable region (HV) sequence (SEQ ID NO: 18)

[0060] [ka]

[0061] >33B light chain variable region (LV) sequence (SEQ ID NO: 19)

[0062] [ka]

[0063] Here, 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.

[0064] [Table 3]

[0065] Farletuzumab contains two identical heavy and light chains (WO 2023 / 170247 Al), 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.

[0066] >Farletuzumab heavy chain sequence (SEQ ID NO:30):

[0067] [ka]

[0068] >Farletuzumab light chain sequence (SEQ ID NO:31):

[0069] [ka]

[0070] Here, the amino acid sequences shown in bold are heavy / 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.

[0071] [Table 4]

[0072] Here, the heavy / light chain variable region sequences, HV and LV, are named SEQ ID NO:28 and SEQ ID NO:29, respectively.

[0073] Farletuzumab heavy chain variable region (HV) sequence (SEQ ID NO:28):

[0074] [ka]

[0075] Farletuzumab light chain variable region (LV) sequence (SEQ ID NO:29):

[0076] [ka]

[0077] Farletuzumab-FcS contains two identical heavy and light chains; the heavy chain amino acid sequence is shown in SEQ ID NO:32, and the light chain amino acid sequence is shown in SEQ ID NO:31.

[0078] >Farletuzumab-FcS heavy chain sequence (SEQ ID NO:32):

[0079] [ka]

[0080] >Farletuzumab-FcS light chain sequence (SEQ ID NO:31):

[0081] [ka]

[0082] Here, the amino acid sequences shown in bold are heavy / 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.

[0083] [Table 5]

[0084] Here, the heavy / light chain variable region sequences, HV and LV, are named SEQ ID NO:28 and SEQ ID NO:29, respectively.

[0085] Farletuzumab-FcS heavy chain variable region (HV) sequence (SEQ ID NO:28):

[0086] [ka]

[0087] Farletuzumab-FcS light chain variable region (LV) sequence (SEQ ID NO:29):

[0088] [ka]

[0089] Trastuzumab contains two identical heavy and light chains; the heavy chain amino acid sequence is shown in SEQ ID NO:33, and the light chain amino acid sequence is shown in SEQ ID NO:34.

[0090] >Trastuzumab heavy chain (SEQ ID NO:33)

[0091] [ka]

[0092] >Trastuzumab light chain (SEQ ID NO:34)

[0093] [ka]

[0094] Here, the heavy chain variable region sequence is shown as SEQ ID NO:35, and the light chain variable region sequence is shown as SEQ ID NO:36.

[0095] >Trastuzumab heavy chain variable region (HV) sequence (SEQ ID NO:35)

[0096] [ka]

[0097] >Trastuzumab light chain variable region (LV) sequence (SEQ ID NO:36)

[0098] [ka]

[0099] Here, 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.

[0100] [Table 6]

[0101] In some embodiments, the linker portion -L- is represented by the following formula.

[0102] -L1-L2-L3-L4- (Here, L1 is the part linked to the ligand A of L, and preferably, L1 is

[0103]

Chemical formula

[0104] (or its ring-opened form

[0105]

Chemical formula

[0106] ),

[0107]

Chemical formula

[0108] selected from, where * represents being linked to the mercapto group of A (e.g., monoclonal antibody), ** represents being linked to L2, L2 is a spacer, and preferably, -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)-, or -L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)- is selected, where L 2a -C1~C8 alkylene-, -C1~C8 alkylene-C3~C8 cycloalkylene-, -C6~C 14 Arirene-, -C6~C 14Arylene-C1~C8 alkylene-, -5~6 member heteroarylene-, -5~6 member heteroarylene-C1~C8 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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, 4 A linear or branched heteroalkylene group having 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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 The alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclylene group are selected from linear or branched heteroalkylene groups having 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 atoms, and the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclylene group are, respectively, C1-C6 alkyl group, C1-C6 heteroalkyl group, C1-C6 alkoxy group, hydroxyl group, amino group, carboxyl group, or C3-C8 cycloalkyl group. The heteroalkylene group, heterocyclylene group, or heteroalkyl group is optionally substituted by one or more substituents independently selected from the above, and the heteroalkylene group, heterocyclylene group, or heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more (e.g., two or three) selected from N, O, or S, and L 2bR is selected from linear or branched heteroalkylene groups having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, 1 C1-C6 alkyl groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclyl groups, C1-C6 haloalkyl groups, heteroalkyl groups with 2-8 atoms, C6-C 14 The group is selected from an aryl group or a 5-6 membered heteroaryl group, and the alkyl group, heterocyclyl group, heteroalkyl group, aryl group, heteroaryl group are each optionally substituted with one or more substituents independently selected from a C1-C6 alkyl group, a heteroalkyl group having 2-6 atoms, a C1-C6 alkoxy group, an amino group, or a carboxyl group, and the heteroalkyl group or heteroalkylene group contains 1-12 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group, or heteroarylene group are one or more (e.g., two or three) selected from N, O, or S. L3 is a polypeptide sequence, preferably selected from peptide residues consisting of 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, 8) native or unnatural amino acids, where the amino acids are further optionally substituted with one or more substituents selected from C1-C6 alkyl groups, heteroalkyl groups having 2 to 6 atoms, C1-C6 alkoxy groups, hydroxyl groups, amino groups, carboxyl groups, or C3-C8 cycloalkyl groups. L4 is a self-cleaving fragment modified with a hydrophilic group. The self-cleaving fragment is selected from the following:

[0109] [ka]

[0110] Here, * indicates linkage to the carboxyl group of L3 via an amide bond, ** indicates linkage to the amino group of the biologically active molecule D, and X indicates absence or

[0111] [ka]

[0112] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and the arrow indicates the hydrophilic group modification site. More preferably, the self-cleaving fragment is selected from the following structures before being modified with a hydrophilic group:

[0113] [ka]

[0114] Here, Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 n is a C1-C6 alkylene group, or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, where n is an integer from 0 to 6. The hydrophilic group has at least one azide group and includes a polyethylene glycol group, a polynatural amino acid or unnatural amino acid group, a monosaccharide, an oligosaccharide or polysaccharide, or a combination of the above groups. Preferably, L4 is selected from the following:

[0115] [ka]

[0116] Here, * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the biologically active molecule D. R 2 The is a hydrophilic fragment, preferably a linear or branched heteroalkyl group containing 4 to 50 (preferably 4 to 24, more preferably 6 to 24, most preferably 8 to 24, for example 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, 4 to 50 (preferably 4 to 24, more preferably 8 to 2 4. Most preferably 10 to 24, for example 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) peptide chains containing protein amino acids (e.g., glycine) or non-protein amino acids (e.g., sarcosine), monosaccharides, oligosaccharides, or polysaccharides, selected from linear or branched heteroalkyl groups, where X is absent or

[0117] [ka]

[0118] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 This is a C1-C6 alkylene group, or a heteroalkylene group containing 1-24 (preferably 1-8, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)-OCH2CH2- structural units. n is an integer between 0 and 6 (for example, 0, 1, 2, 3, 4, 5, 6). In some embodiments, R 2 The following structure is selected.

[0119] [ka]

[0120] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), R a The bond is selected from C1-C3 alkylene groups, such as methylene group, ethylene group, n-propylene group, and isopropylene group, R b (The C1-C3 alkyl group is selected from, for example, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group.) In some embodiments, R 2 The following structure is selected.

[0121] [ka]

[0122] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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).) In some embodiments, R 2 The following structure is selected.

[0123] [ka]

[0124] In some embodiments, Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 This is a C1-C6 alkylene group, or -C1-C6 alkylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0125] In some embodiments, Y is a C1-C3 alkylene group, or -R 3 -C(O)-, where R 3 This is a C1-C3 alkylene group, or -C1-C3 alkylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0126] In some embodiments, Y is a methylene group, an ethylene group, an n-propylene group, an isopropylene group, or -R 3 -C(O)-, where R 3 is methylene-(OCH2CH2) v -, ethylene-(OCH2CH2) v -, n-propylene-(OCH2CH2) v -, isopropylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0127] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0128] In some embodiments, L2 is -L 2a -C(O)-, -L 2a -NH-C(O)-L 2b -C(O)-, or -L 2a -NR1 -SO2-NH-C(O)-OL 2b -C(O)- is selected, where L 2a -C1~C6 alkylene-, -C1~C6 alkylene-C3~C6 cycloalkylene-, -C6~C 10 Arirene-, -C6~C 10 Arylene-C1~C6 alkylene-, -5~6 member heteroarylene-, -5~6 member heteroarylene-C1~C6 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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, A linear or branched heteroalkylene group having 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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, 4 Selected from linear or branched heteroalkylene-3-6 membered heterocyclene groups having 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclene group are, respectively, halogen, C1-C3 alkyl group, heteroalkyl group having 1-3 atoms, C1-C3 alkoxy group, hydroxyl group, amino group, carboxyl group, or The heteroalkylene group, heterocyclylene group, or heteroalkyl group is optionally substituted with one or more substituents independently selected from C3-C6 cycloalkyl groups, and the heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more selected from N, O, or S, and L 2bis selected from -C1-C6 alkylene-, a linear or branched heteroalkylene group having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, R 1 is hydrogen, a C1-C3 alkyl group, a C3-C6 cycloalkyl group, a 3-6 member heterocyclyl group, a C1-C3 haloalkyl group, a heteroalkyl group having 2 to 6 atoms, C6-C 10 an aryl group, a 5-6 member heteroaryl group, and the alkyl group, heteroalkyl group, aryl group, heteroaryl group, cycloalkyl group, heterocyclyl group are each optionally substituted by one or more substituents independently selected from a C1-C3 alkyl group, a heteroalkyl group having 2 to 3 atoms, a C1-C3 alkoxy group, an amino group, or a carboxy group, and the heteroalkyl group, heteroalkylene group contain 1 to 12 (preferably 1 to 8, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group are one or more selected from N, O or S.

[0129] In some embodiments, L2 is -L 2a -C(O)-, -L 2a -NH-C(O)-L 2b -C(O)-, or -L 2a -NR 1 -SO2-NH-C(O)-O-L 2b -C(O)-, where L 2aThis includes methylene group, ethylene group, n-propylene group, n-butylene group, n-pentylene group, n-hexylene group, -CH(CH3)CH2CH2CH2CH2-, -C1~C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1~C3 alkylene-, -5~6 member heteroarylene-C1~C3 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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) A linear or branched heteroalkylene group having 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, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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 Selected from linear or branched heteroalkylene groups having 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms and 3-6 membered heterocyclylene groups, the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclylene group are, respectively, halogen, methyl group, ethyl group, n-propyl group, isopropyl group, heteroalkyl group having 1-3 atoms, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, hydroxyl group, and The heteroalkylene group, heterocyclylene group, heteroalkyl group is optionally substituted with one or more substituents independently selected from a mino group, carboxyl group, or cyclopropyl group, cyclobutyl group, cyclopentyl group, or cyclohexyl group, and the heteroalkylene group, heterocyclylene group, or heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more selected from N, O, or S, and L 2bR is selected from a linear or branched heteroalkylene group having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, 1 The alkyl group is selected from hydrogen, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, 3-6 membered heterocyclyl group, -CF3, -CF2CF3, 2-3 atom heteroalkyl group, phenyl group, and 5-6 membered heteroaryl group, and the alkyl group, heteroalkyl group, phenyl group, heteroaryl group, cycloalkyl group, and heterocyclyl group are respectively methyl group, ethyl group, n-propyl group, isopropyl group, and 2-3 atom heteroalkyl group The heteroalkyl group, heteroalkylene group is optionally substituted with one or more substituents independently selected from a carboxyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, amino group, or carboxyl group, and the heteroalkyl group, heteroalkylene group contains 1 to 12 (preferably 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group are one or more selected from N, O, or S.

[0130] In some embodiments, L 2a These are -C1~C8 alkylene-, -C1~C8 alkylene-C3~C8 cycloalkylene group, C6~C 14Selected from an arylene-C1-C8 alkylene group, a linear or branched heteroalkylene group having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 8) atoms, wherein the alkylene group, cycloalkylene group, arylene group, and heteroalkylene group are each independently selected from a halogen, a C1-C6 alkyl group, a heteroalkyl group having 2 to 6 atoms, a C1-C6 alkoxy group, a hydroxy group, an amino group, a carboxy group, or a C3-C8 cycloalkyl group by one or more substituents that are optionally substituted, the heteroalkylene group and heteroalkyl group contain 1 to 12 (preferably 1 to 8, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene group and heteroalkyl group are one or more selected from N, O, or S, L 2b is selected from -C1-C8 alkylene-, a linear or branched heteroalkylene group having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 8) atoms, R 1 is hydrogen, a C1-C6 alkyl group, a heteroalkyl group having 2 to 8 atoms, a C6-C 14 aryl group, wherein the alkyl group, heteroalkyl group, and aryl group are each independently selected from a C1-C6 alkyl group, a heteroalkyl group having 2 to 6 atoms, a C1-C6 alkoxy group, an amino group, or a carboxy group by one or more substituents that are optionally substituted, the heteroalkyl group contains 1 to 12 (preferably 1 to 8, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group and heteroalkylene group are one or more selected from N, O, or S.

[0131] In some embodiments, L 2a is -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p -, -(OCH2CH2) p -, -CH2(OCH2CH2) p -, -CH2CH2(OCH2CH2) pCH2-, -CH2CH2(OCH2CH2) p CH2CH2-, -CH2(OCH2CH2) p CH2CH2-,

[0132]

Chem.

[0133] selected from, where p is an integer from 1 to 12 (preferably 2 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), and L 2b is -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, - (OCH2CH2) q -, -CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -, -CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-, -CH2(OCH2CH2) q CH2CH2- selected from, and R 1 is selected from hydrogen, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, where q is an integer from 1 to 12 (preferably 2 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0134] In some embodiments, L2 is selected from the following structures.

[0135]

Table 7

[0136] Here, w is selected from integers between 1 and 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates that it will be concatenated to L1, and ** indicates that it will be concatenated to L3.

[0137] In some embodiments, L3 is selected from peptide residues formed by 2 to 8 amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), and lysine substituted with C1-C6 alkyl groups (e.g., lysine substituted with C1-C3 alkyl groups, e.g., lysine substituted with a methyl group, lysine substituted with an ethyl group, lysine substituted with an n-propyl group, lysine substituted with an isopropyl group). For example, -ValCit-, -CitVal-, -AlaAla-, -AlaCit-, -CitAla-, -AsnCit-, -CitAsn-, -CitCit-, -ValGlu-, -GluVal-, -SerCit-, -CitSer-, -LysCit-, -CitLys-, -AspCit-, - CitAsp-, -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-, -DLysPhePhe-, -GlyPheLys-, -LysPheGly-, -GlyPh eLeuGly-, -GlyLeuPheGly-, -GluValCit-, -AlaLeuAlaLeu-, -GlyGlyGly-, -GlyGlyGlyGly-, -GlyPheValGly-, -GlyValPheGly-, -GlyGlyPheGly-, -GlyGlyValGly-.

[0138] In some embodiments, L3 is selected from the following structures.

[0139]

Chem.

[0140] (Here, * represents being linked to L2, and ** represents being linked to L4.) In some embodiments, L4 is selected from the following.

[0141]

Chem.

[0142] (Here, v is selected from integers from 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * represents being linked to the carboxy group of L3 via an amide bond, and ** represents being linked to the amino group of the biologically active molecule D.) In some embodiments, L4 is selected from the following.

[0143]

Chem.

[0144] In some embodiments, L4 is selected from the following.

[0145]

Chem.

[0146] In some embodiments, L4 is selected from the following.

[0147]

Chem.

[0148] In some embodiments, L4 is selected from the following.

[0149] [ka]

[0150] TIFF2026516826000047.tif32169

[0151] (Here, r, s, t, and u are each independent integers from 1 to 50 (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) (Selected from 42, 43, 44, 45, 46, 47, 48, 49, 50), v is an integer from 1 to 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 biologically active molecule D.) In some embodiments, L4 is selected from the following:

[0152] [ka]

[0153] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the biologically active molecule D.) In some embodiments, L is selected from the following structures.

[0154] [ka]

[0155] TIFF2026516826000050.tif219169

[0156] TIFF2026516826000051.tif199169

[0157] TIFF2026516826000052.tif224169

[0158] TIFF2026516826000053.tif207169

[0159] TIFF2026516826000054.tif216169

[0160] TIFF2026516826000055.tif202169

[0161] TIFF2026516826000056.tif122169

[0162] (Here, v and w are independently selected from integers between 1 and 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates linkage to the mercapto group of antibody A, and ** indicates linkage to the amino group of biologically active molecule D.) In some embodiments, L is selected from the following structures.

[0163] [ka]

[0164] TIFF2026516826000058.tif218169

[0165] TIFF2026516826000059.tif224169

[0166] TIFF2026516826000060.tif217169

[0167] TIFF2026516826000061.tif198169

[0168] TIFF2026516826000062.tif205169

[0169] TIFF2026516826000063.tif201169

[0170] TIFF2026516826000064.tif193169

[0171] TIFF2026516826000065.tif224169

[0172] TIFF2026516826000066.tif224169

[0173] TIFF2026516826000067.tif207169

[0174] TIFF2026516826000068.tif208169

[0175] TIFF2026516826000069.tif218169

[0176] TIFF2026516826000070.tif223169

[0177] TIFF2026516826000071.tif201169

[0178] TIFF2026516826000072.tif208169

[0179] TIFF2026516826000073.tif214169

[0180] TIFF2026516826000074.tif193169

[0181] TIFF2026516826000075.tif192169

[0182] TIFF2026516826000076.tif154169

[0183] (Here, v and w are independently selected from integers 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates linkage to the mercapto group of antibody A, ** indicates linkage to the amino group of biologically active molecule D, and r, s, t, and u are independently selected from integers 1 to 50 (e.g.) (The number is selected from 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.) In some embodiments, L is selected from the following structures.

[0184] [ka]

[0185] JPEG2026516826000078.jpg225169

[0186] JPEG2026516826000079.jpg192169

[0187] JPEG2026516826000080.jpg218169

[0188] JPEG2026516826000081.jpg217169

[0189] JPEG2026516826000082.jpg201169

[0190] TIFF2026516826000083.tif212169

[0191] TIFF2026516826000084.tif224169

[0192] TIFF2026516826000085.tif225169

[0193] TIFF2026516826000086.tif193169

[0194] TIFF2026516826000087.tif211169

[0195] TIFF2026516826000088.tif197169

[0196] TIFF2026516826000089.tif188169

[0197] TIFF2026516826000090.tif214169

[0198] TIFF2026516826000091.tif211169

[0199] TIFF2026516826000092.tif217169

[0200] TIFF2026516826000093.tif213169

[0201] TIFF2026516826000094.tif206169

[0202] TIFF2026516826000095.tif225169

[0203] TIFF2026516826000096.tif207169

[0204] TIFF2026516826000097.tif85169

[0205] (Here, * indicates linkage to the mercapto group of antibody A, and ** indicates linkage to the amino group of biologically active molecule D.) In some embodiments, the biologically active molecule D is selected from camptothecin derivatives, eribulin, and molecular glues.

[0206] In some embodiments, the biologically active molecule D is selected from compounds of the following formula.

[0207] [ka]

[0208] In some embodiments, the biologically active molecule D is selected from the following:

[0209] [ka]

[0210] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0211] [ka]

[0212] TIFF2026516826000101.tif204169

[0213] TIFF2026516826000102.tif223169

[0214] TIFF2026516826000103.tif212169

[0215] TIFF2026516826000104.tif199169

[0216] TIFF2026516826000105.tif197169

[0217] TIFF2026516826000106.tif210169

[0218] TIFF2026516826000107.tif219169

[0219] TIFF2026516826000108.tif220169

[0220] TIFF2026516826000109.tif110169

[0221] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0222] [ka]

[0223] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0224] [ka]

[0225] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0226] [ka]

[0227] TIFF2026516826000113.tif219169

[0228] TIFF2026516826000114.tif213169

[0229] TIFF2026516826000115.tif217169

[0230] TIFF2026516826000116.tif220169

[0231] TIFF2026516826000117.tif217169

[0232] TIFF2026516826000118.tif227169

[0233] TIFF2026516826000119.tif186169

[0234] TIFF2026516826000120.tif194169

[0235] TIFF2026516826000121.tif195169

[0236] TIFF2026516826000122.tif195169

[0237] TIFF2026516826000123.tif194169

[0238] TIFF2026516826000124.tif208169

[0239] TIFF2026516826000125.tif223169

[0240] TIFF2026516826000126.tif225169

[0241] TIFF2026516826000127.tif204169

[0242] TIFF2026516826000128.tif224169

[0243] TIFF2026516826000129.tif212169

[0244] TIFF2026516826000130.tif193169

[0245] TIFF2026516826000131.tif227169

[0246] TIFF2026516826000132.tif186169

[0247] TIFF2026516826000133.tif219169

[0248] TIFF2026516826000134.tif225169

[0249] TIFF2026516826000135.tif184169

[0250] TIFF2026516826000136.tif213169

[0251] TIFF2026516826000137.tif205169

[0252] TIFF2026516826000138.tif222169

[0253] TIFF2026516826000139.tif207169

[0254] TIFF2026516826000140.tif198169

[0255] TIFF2026516826000141.tif199169

[0256] TIFF2026516826000142.tif199169

[0257] TIFF2026516826000143.tif208169

[0258] TIFF2026516826000144.tif193169

[0259] TIFF2026516826000145.tif187169

[0260] TIFF2026516826000146.tif191169

[0261] TIFF2026516826000147.tif206169

[0262] TIFF2026516826000148.tif211169

[0263] TIFF2026516826000149.tif209169

[0264] TIFF2026516826000150.tif204169

[0265] TIFF2026516826000151.tif194169

[0266] TIFF2026516826000152.tif212169

[0267] TIFF2026516826000153.tif200169

[0268] TIFF2026516826000154.tif219169

[0269] TIFF2026516826000155.tif218169

[0270] TIFF2026516826000156.tif214169

[0271] TIFF2026516826000157.tif220169

[0272] TIFF2026516826000158.tif217169

[0273] TIFF2026516826000159.tif185169

[0274] TIFF2026516826000160.tif219169

[0275] TIFF2026516826000161.tif200169

[0276] TIFF2026516826000162.tif220169

[0277] TIFF2026516826000163.tif42169

[0278] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), and v and w are each independently selected from integers between 1 and 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).) In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0279] [Table 8]

[0280] TIFF2026516826000165.tif243169

[0281] TIFF2026516826000166.tif233169

[0282] TIFF2026516826000167.tif221169

[0283] TIFF2026516826000168.tif241169

[0284] TIFF2026516826000169.tif248169

[0285] TIFF2026516826000170.tif213169

[0286] TIFF2026516826000171.tif254169

[0287] TIFF2026516826000172.tif235169

[0288] TIFF2026516826000173.tif243169

[0289] TIFF2026516826000174.tif243169

[0290] TIFF2026516826000175.tif255168

[0291] TIFF2026516826000176.tif129169

[0292] In some embodiments, the antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from Trastuzumab.

[0293] In some embodiments, the antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from Farletuzumab.

[0294] In some embodiments, the antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from Farletuzumab FcS.

[0295] In some embodiments, antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from 33B.

[0296] In some embodiments, antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from 43A.

[0297] In some embodiments, antibodies for ADC1-ADC55 and ADC2-1, ADC2-2, ADC7-1, ADC7-2, ADC12-1, ADC12-2, ADC55-1, and ADC55-2 are selected from 43B.

[0298] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0299] [Table 9]

[0300] TIFF2026516826000178.tif255167

[0301] TIFF2026516826000179.tif225169

[0302] Note: In the table, T-ADC1 refers to an ADC in which antibody A is trastuzumab, and the same applies to other ADCs.

[0303] In some embodiments, the antibody-drug conjugate represented by Formula I, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures.

[0304] [Table 10]

[0305] TIFF2026516826000181.tif235169

[0306] TIFF2026516826000182.tif210169

[0307] TIFF2026516826000183.tif240169

[0308] TIFF2026516826000184.tif244169

[0309] TIFF2026516826000185.tif222169

[0310] TIFF2026516826000186.tif240169

[0311] TIFF2026516826000187.tif241169

[0312] TIFF2026516826000188.tif251169

[0313] TIFF2026516826000189.tif225169

[0314] (Here, m is selected from 2, 4, 6, and 8.) In some embodiments, the present invention provides for the use of antibody-drug conjugates of the structure of formula (I), and their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds in the preparation of cancer therapeutics. The aforementioned 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, hematological cancer or glioblastoma multiforme, 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 lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, and others.

[0315] In some embodiments, the present invention provides for the use of antibody-drug conjugates of the structure of formula (I), and their tautomers, meso-molecules, racemics, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products for the treatment of cancer. The aforementioned 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, hematological cancer or glioblastoma multiforme, 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 lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, and others.

[0316] In some embodiments, the cancer is a tumor that expresses HER2 (HER2+), DLL3 (DLL3+), FRα (FRα+), and B7H3 (B7H3+) on the surface of its tumor cells.

[0317] In some embodiments, the present invention provides the use of antibody-drug conjugates of the structure of formula (I), and their tautomers, meso-molecules, racemics, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products for the treatment of cancer. The cancers are preferably those associated with the aberration of FRα, DLL3, B7H3, or HER2. Cancers associated with the aberration 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 the aberration of DLL3 include lung cancer (e.g., small cell lung cancer and non-small cell lung cancer). Cancers associated with the aberration of B7H3 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 HER2 expression 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.

[0318] In some embodiments, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of the structure of formula (I), its tautomers, meso-molecules, racemics, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products, and one or more pharmaceutically acceptable auxiliary materials.

[0319] In some embodiments, the present invention provides a linker represented by the following formula for obtaining an antibody-drug conjugate in which a drug and an antibody are linked via a linker.

[0320] -L1-L2-L3-L4- (Here, the left end is the binding site for the antibody, and the right end is the binding site for the antitumor compound. The definitions of L1, L2, L3, and L4 are the same as in formula (I).) In another aspect of the present invention, linker-drug compounds represented by formula (II) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof are provided.

[0321] L’-D (II) (Here, L’ is the linker part, D is a biologically active molecule containing an amino group or a pharmaceutically acceptable salt thereof, which is covalently bonded to the linker part L’ via the amine group in its molecular structure.) In some embodiments, the linker part L’ is represented by the following formula.

[0322] L1’-L2-L3-L4- (Here, L1’ is

[0323]

Chemical formula

[0324] selected from, where ** represents being linked to L2, The definitions of L2, L3, L4, and D are the same as those in formula (I).

[0325] L2 is a spacer, preferably -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)-O-L 2b -C(O)-, or -L 2a-NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)- is selected, where L 2a -C1~C8 alkylene-, -C1~C8 alkylene-C3~C8 cycloalkylene-, -C6~C 14 Arirene-, -C6~C 14Arylene-C1~C8 alkylene-, -5~6 member heteroarylene-, -5~6 member heteroarylene-C1~C8 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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, A linear or branched heteroalkylene group having 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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, 4 Selected from linear or branched heteroalkylene groups having 2, 43, 44, 45, 46, 47, 48, 49, 50) atoms and 3-8 membered heterocyclene groups, wherein the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclene group are, respectively, halogen, C1-C6 alkyl group, heteroalkyl group having 1-6 atoms, C1-C6 alkoxy group, hydroxyl group, amino group, carboxyl group, or C3-C8 cycloalkyl group The heteroalkylene group, heterocyclylene group, or heteroalkyl group is optionally substituted with one or more substituents independently selected from the kill group, and the heteroalkylene group, heterocyclylene group, or heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more (e.g., two or three) selected from N, O, or S, and L 2bR is selected from linear or branched heteroalkylene groups having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, 1 C1-C6 alkyl groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclyl groups, C1-C6 haloalkyl groups, heteroalkyl groups with 2-8 atoms, C6-C 14 The group is selected from an aryl group or a 5-6 membered heteroaryl group, and the alkyl group, heterocyclyl group, heteroalkyl group, aryl group, heteroaryl group are each optionally substituted with one or more substituents independently selected from a C1-C6 alkyl group, a heteroalkyl group having 2-6 atoms, a C1-C6 alkoxy group, an amino group, or a carboxyl group, and the heteroalkyl group or heteroalkylene group contains 1-12 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group, or heteroarylene group are one or more selected from N, O, or S. L3 is a polypeptide sequence, preferably selected from peptide residues consisting of 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, 8) native or unnatural amino acids, where the amino acids are further optionally substituted with one or more substituents selected from C1-C6 alkyl groups, heteroalkyl groups having 2 to 6 atoms, C1-C6 alkoxy groups, hydroxyl groups, amino groups, carboxyl groups, or C3-C8 cycloalkyl groups. L4 is a self-cleaving fragment modified with a hydrophilic group. The self-cleaving fragment is selected from the following:

[0326] [ka]

[0327] Here, * indicates linkage to the carboxyl group of L3 via an amide bond, ** indicates linkage to the amino group of the biologically active molecule D, and X indicates absence or

[0328] [ka]

[0329] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and the arrow indicates the hydrophilic group modification site. More preferably, the self-cleaving fragment is selected from the following structures before being modified with a hydrophilic group:

[0330] [ka]

[0331] Here, Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 n is a C1-C6 alkylene group, or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, where n is an integer from 0 to 6. The hydrophilic group has at least one azide group and includes a polyethylene glycol group, a polynatural amino acid or unnatural amino acid group, a monosaccharide, an oligosaccharide or polysaccharide, or a combination of the above groups. Preferably, L4 is selected from the following:

[0332] [ka]

[0333] Here, * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the biologically active molecule D. R 2 The hydrophilic fragment is preferably a linear or branched heteroalkyl group containing 4 to 50 (preferably 4 to 24, more preferably 6 to 24, most preferably 8 to 24, for example 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, and 4 to 50 (preferably 4 to 24, more preferably 8 to 2 4. Most preferably 10 to 24, for example 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) selected from peptide chains containing protein amino acids (e.g., glycine) or non-protein amino acids (e.g., sarcosine), monosaccharides, oligosaccharides or polysaccharides, and X is absent or

[0334] [ka]

[0335] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 n is a C1-C6 alkylene group, or a heteroalkylene group containing 1-24 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) -OCH2CH2- structural units, where n is an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, 6). In some embodiments, R 2 The following structure is selected.

[0336] [ka]

[0337] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), R a The bond is selected from C1-C3 alkylene groups, such as methylene group, ethylene group, n-propylene group, and isopropylene group, R b (The C1-C3 alkyl group is selected from, for example, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group.) In some embodiments, R 2 The following structure is selected.

[0338] [ka]

[0339] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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).) In some embodiments, R 2 The following structure is selected.

[0340] [ka]

[0341] In some embodiments, Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 This is a C1-C6 alkylene group, or -C1-C6 alkylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0342] In some embodiments, Y is a C1-C3 alkylene group, or -R 3 -C(O)-, where R 3 This is a C1-C3 alkylene group, or -C1-C3 alkylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0343] In some embodiments, Y is a methylene group, an ethylene group, an n-propylene group, an isopropylene group, or -R 3 -C(O)-, where R 3 is methylene-(OCH2CH2) v -, ethylene-(OCH2CH2) v -, n-propylene-(OCH2CH2) v -, isopropylene-(OCH2CH2) v -where v is selected from integers 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0344] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0345] In some embodiments, L2 is -L 2a -C(O)-, -L 2a -NH-C(O)-L 2b -C(O)-, or -L 2a -NR1 -SO2-NH-C(O)-OL 2b -C(O)- is selected, where L 2a -C1~C6 alkylene-, -C1~C6 alkylene-C3~C6 cycloalkylene-, -C6~C 10 Arirene-, -C6~C 10Arylene-C1~C6 alkylene-, -5~6 member heteroarylene-, -5~6 member heteroarylene-C1~C6 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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, A linear or branched heteroalkylene group having 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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, 4 Selected from linear or branched heteroalkylene groups having 2, 43, 44, 45, 46, 47, 48, 49, 50) atoms and 3-6 membered heterocyclene groups, wherein the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclene group are, respectively, halogen, C1-C3 alkyl group, heteroalkyl group having 1-3 atoms, C1-C3 alkoxy group, hydroxyl group, amino group, carboxyl group, or C3-C6 cycloalkyl group The heteroalkylene group, heterocyclylene group, or heteroalkyl group is optionally substituted with one or more substituents independently selected from the kill group, and the heteroalkylene group, heterocyclylene group, or heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more (e.g., two or three) selected from N, O, or S, and L 2bR is selected from linear or branched heteroalkylene groups having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, 1 C1-C3 alkyl groups, C3-C6 cycloalkyl groups, 3-6 membered heterocyclyl groups, C1-C3 haloalkyl groups, heteroalkyl groups with 2-6 atoms, C6-C 10 The alkyl group is selected from an aryl group or a 5-6 membered heteroaryl group, and the alkyl group, heteroalkyl group, aryl group, heteroaryl group, cycloalkyl group, or heterocyclyl group is optionally substituted with one or more substituents independently selected from a C1-C3 alkyl group, a 2-3 atom heteroalkyl group, a C1-C3 alkoxy group, an amino group, or a carboxyl group, and the heteroalkyl group or heteroalkylene group contains 1 to 12 (preferably 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, or heteroalkylene group are one or more (e.g., two or three) selected from N, O, or S.

[0346] In some embodiments, L2 is -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)- is selected, where L 2aThese are methylene groups, ethylene groups, n-propylene groups, n-butylene groups, n-pentylene groups, n-hexylene groups, -CH(CH3)CH2CH2CH2CH2-, -C1~C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1~C3 alkylene-, -5~6 member heteroarylene-C1~C3 alkylene-, 1~50 (preferably 1~20, more preferably 1~12, 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, A linear or branched heteroalkylene group having 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, 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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, Selected from linear or branched heteroalkylene groups having 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms and 3-6 membered heterocyclylene groups, wherein the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclylene group are, respectively, halogen, methyl group, ethyl group, n-propyl group, isopropyl group, heteroalkyl group having 1-3 atoms, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, hydroxyl group, amino group, and carboxyl group. or optionally substituted with one or more substituents independently selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups, wherein the heteroalkylene group, heterocyclylene group, or heteroalkyl group contains 1 to 12 (preferably 1 to 8, e.g., 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 heteroalkyl group are one or more (e.g., two or three) selected from N, O, or S, and L 2bR is selected from a linear or branched heteroalkylene group having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 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) atoms, 1 The alkyl group is selected from hydrogen, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, 3-6 membered heterocyclyl group, -CF3, -CF2CF3, 2-3 atom heteroalkyl group, phenyl group, and 5-6 membered heteroaryl group, and the alkyl group, heteroalkyl group, aryl group, heteroaryl group, cycloalkyl group, and heterocyclyl group are respectively methyl group, ethyl group, n-propyl group, isopropyl group, 2-3 atom heteroalkyl group, methyl group The heteroalkyl group, heteroalkylene group is optionally substituted with one or more substituents independently selected from a cy group, ethoxy group, n-propoxy group, isopropoxy group, amino group, or carboxyl group, and the heteroalkyl group, heteroalkylene group contains 1 to 12 (preferably 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group are one or more (e.g., two or three) selected from N, O, or S.

[0347] In some embodiments, L 2a These are -C1~C8 alkylene-, -C1~C8 alkylene-C3~C8 cycloalkylene group, C6~C 14Arylene is selected from C1-C8 alkylene groups and linear or branched heteroalkylene groups having 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms, and each alkylene group, cycloalkylene group, arylene group, and heteroalkylene group is optionally substituted with one or more substituents independently selected from C1-C6 alkyl groups, heteroalkyl groups having 2-6 atoms, C1-C6 alkoxy groups, hydroxyl groups, amino groups, carboxyl groups, or C3-C8 cycloalkyl groups, and each heteroalkylene group and heteroalkyl group contains 1-12 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and each heteroatom of the heteroalkylene group and heteroalkyl group is one or more (e.g., 2 or 3) selected from N, O, or S, and L 2b R is selected from linear or branched heteroalkylene groups having 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 8) atoms of -C1 to C8 alkylene-, 1 These are hydrogen, C1-C6 alkyl groups, heteroalkyl groups with 2-8 atoms, and C6-C 14 The groups are selected from aryl groups, and the alkyl group, heteroalkyl group, and aryl group are each optionally substituted with one or more substituents independently selected from C1-C6 alkyl groups, heteroalkyl groups having 2-6 atoms, C1-C6 alkoxy groups, amino groups, or carboxyl groups, wherein the heteroalkyl group contains 1-12 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl group and heteroalkylene group are one or more (e.g., two or three) selected from N, O, or S.

[0348] In some embodiments, L 2a -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p -,-(OCH2CH2) p -, -CH2(OCH2CH2) p -, -CH2CH2(OCH2CH2)p CH2-, -CH2CH2(OCH2CH2) p CH2CH2-, -CH2(OCH2CH2) p CH2CH2-,

[0349] [ka]

[0350] Selected from, where p is an integer from 1 to 12 (preferably 2 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), and L 2b is -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, - (OCH2CH2) q -, -CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -, -CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-, -CH2(OCH2CH2) q Selected from CH2CH2-, R 1 q is selected from hydrogen, methyl group, ethyl group, n-propyl group, isopropyl group, and cyclopropyl group, and q is an integer from 1 to 12 (preferably 2 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0351] In some embodiments, L2 is selected from the following structures.

[0352] [Table 11]

[0353] (Here, w is selected from integers between 1 and 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates concatenation to L1, and ** indicates concatenation to L3.) In some embodiments, L3 is selected from peptide residues formed by 2 to 8 amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), and lysine substituted with C1-C6 alkyl groups (e.g., lysine substituted with C1-C3 alkyl groups, lysine substituted with methyl groups, lysine substituted with ethyl groups, lysine substituted with n-propyl groups, lysine substituted with isopropyl groups). For example -ValCit-, -CitVal-, -AlaAla-, -AlaCit-, -CitAla-, -AsnCit-, -CitAsn-, -CitCit-, -ValGlu-, -GluVal-, -SerCit-, -CitSer-, -LysCit-, -CitLys-, -AspCit-, -C itAsp-, -AlaVal-, -ValAla-, -PheAla-, -AlaPhe-, -PheLys-, -LysPhe-, -ValLys-, -LysVal-, -AlaLys-, -LysAla-, -PheCit-, -CitPhe-, -LeuCit-, -CitLeu-, -IleCit -, -CitIle-, -PheArg-, -ArgPhe-, -CitTrp-, -TrpCit-, -AlaAlaAla-, -PhePheLys-, -LysPhePhe-, -DPhePheLys-, -DLysPhePhe-, -GlyPheLys-, -LysPheGly-, -GlyPhe LeuGly-, -GlyLeuPheGly-, -GluValCit-, -AlaLeuAlaLeu-, -GlyGlyGly-, -GlyGlyGlyGly-, -GlyPheValGly-, -GlyValPheGly-, -GlyGlyPheGly-, -GlyGlyValGly-.

[0354] In some embodiments, L3 is selected from the following structures.

[0355] [ka]

[0356] (Here, * indicates that it will be linked to L2, and ** indicates that it will be linked to L4.) In some embodiments, L4 is selected from the following:

[0357] [ka]

[0358] (Here, v is selected from an integer between 1 and 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 linkage of the biologically active molecule D.) In some embodiments, L4 is selected from the following:

[0359] [ka]

[0360] In some embodiments, L4 is selected from the following:

[0361] [ka]

[0362] In some embodiments, L4 is selected from the following:

[0363] [ka]

[0364] TIFF2026516826000206.tif110169

[0365] (Here, r, s, t, and u are each independent integers from 1 to 50 (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) (Selected from 42, 43, 44, 45, 46, 47, 48, 49, 50), v is an integer from 1 to 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 biologically active molecule D.) In some embodiments, L4 is selected from the following:

[0366] [ka]

[0367] (Here, r, s, t, and u are each independent integers from 1 to 50 (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, (Selected from 42, 43, 44, 45, 46, 47, 48, 49, 50), v is an integer from 1 to 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 linkage of the biologically active molecule D.) In some embodiments, L' is selected from the following structures.

[0368] [ka]

[0369] TIFF2026516826000209.tif160169

[0370] In some embodiments, L' is selected from the following structures.

[0371] [ka]

[0372] JPEG2026516826000211.jpg199169

[0373] JPEG2026516826000212.jpg225169

[0374] JPEG2026516826000213.jpg206169

[0375] JPEG2026516826000214.jpg183169

[0376] JPEG2026516826000215.jpg206169

[0377] JPEG2026516826000216.jpg178169

[0378] JPEG2026516826000217.jpg180169

[0379] TIFF2026516826000218.tif174169

[0380] TIFF2026516826000219.tif212169

[0381] TIFF2026516826000220.tif220169

[0382] TIFF2026516826000221.tif213169

[0383] TIFF2026516826000222.tif227169

[0384] TIFF2026516826000223.tif214169

[0385] TIFF2026516826000224.tif209169

[0386] TIFF2026516826000225.tif208169

[0387] TIFF2026516826000226.tif200169

[0388] TIFF2026516826000227.tif201169

[0389] TIFF2026516826000228.tif201169

[0390] TIFF2026516826000229.tif194169

[0391] TIFF2026516826000230.tif198169

[0392] TIFF2026516826000231.tif72169

[0393] (Here, v and w are independently selected from integers 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), ** indicates linkage to the amino group of the biologically active molecule D, and r, s, t, and u are independently selected from integers 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).) In some embodiments, L' is selected from the following structures.

[0394] [ka]

[0395] TIFF2026516826000233.tif195169

[0396] TIFF2026516826000234.tif219169

[0397] TIFF2026516826000235.tif223169

[0398] TIFF2026516826000236.tif228169

[0399] TIFF2026516826000237.tif186169

[0400] TIFF2026516826000238.tif191169

[0401] TIFF2026516826000239.tif182169

[0402] TIFF2026516826000240.tif223169

[0403] TIFF2026516826000241.tif187169

[0404] TIFF2026516826000242.tif206169

[0405] TIFF2026516826000243.tif206169

[0406] TIFF2026516826000244.tif225169

[0407] TIFF2026516826000245.tif227169

[0408] TIFF2026516826000246.tif218169

[0409] TIFF2026516826000247.tif207169

[0410] TIFF2026516826000248.tif198169

[0411] TIFF2026516826000249.tif226169

[0412] TIFF2026516826000250.tif213169

[0413] TIFF2026516826000251.tif225169

[0414] TIFF2026516826000252.tif217169

[0415] TIFF2026516826000253.tif223169

[0416] (Here, ** indicates that it is linked to the amino group of biologically active molecule D.) In some embodiments, in the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof, the biologically active molecule D is selected from camptothecin derivatives, eribulins, and molecular glues.

[0417] In some embodiments, in the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof, the biologically active molecule D is selected from the compounds of the following formulas.

[0418] [ka]

[0419] TIFF2026516826000255.tif25169

[0420] In some embodiments, in the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof, the biologically active molecule D is selected from the following:

[0421] [ka]

[0422] In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof have the following structures.

[0423] [ka]

[0424] TIFF2026516826000258.tif192169

[0425] TIFF2026516826000259.tif220169

[0426] TIFF2026516826000260.tif226169

[0427] TIFF2026516826000261.tif213169

[0428] TIFF2026516826000262.tif221169

[0429] TIFF2026516826000263.tif226169

[0430] TIFF2026516826000264.tif212169

[0431] TIFF2026516826000265.tif198169

[0432] TIFF2026516826000266.tif104169

[0433] In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof have the following structures.

[0434] [ka]

[0435] In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof have the following structures.

[0436] [ka]

[0437] In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof have the following structures.

[0438] [ka]

[0439] TIFF2026516826000270.tif206169

[0440] TIFF2026516826000271.tif215169

[0441] TIFF2026516826000272.tif206169

[0442] TIFF2026516826000273.tif228169

[0443] TIFF2026516826000274.tif199169

[0444] TIFF2026516826000275.tif186169

[0445] TIFF2026516826000276.tif208169

[0446] TIFF2026516826000277.tif223169

[0447] TIFF2026516826000278.tif186169

[0448] TIFF2026516826000279.tif170169

[0449] TIFF2026516826000280.tif218169

[0450] TIFF2026516826000281.tif199169

[0451] TIFF2026516826000282.tif215169

[0452] TIFF2026516826000283.tif193169

[0453] TIFF2026516826000284.tif195169

[0454] TIFF2026516826000285.tif187169

[0455] TIFF2026516826000286.tif210169

[0456] TIFF2026516826000287.tif227169

[0457] TIFF2026516826000288.tif200169

[0458] TIFF2026516826000289.tif192169

[0459] TIFF2026516826000290.tif213169

[0460] TIFF2026516826000291.tif225169

[0461] TIFF2026516826000292.tif225169

[0462] TIFF2026516826000293.tif196169

[0463] TIFF2026516826000294.tif206169

[0464] TIFF2026516826000295.tif219169

[0465] TIFF2026516826000296.tif219169

[0466] TIFF2026516826000297.tif195169

[0467] TIFF2026516826000298.tif227169

[0468] TIFF2026516826000299.tif182169

[0469] TIFF2026516826000300.tif207169

[0470] TIFF2026516826000301.tif219169

[0471] TIFF2026516826000302.tif219169

[0472] TIFF2026516826000303.tif188169

[0473] TIFF2026516826000304.tif211169

[0474] TIFF2026516826000305.tif226169

[0475] TIFF2026516826000306.tif201169

[0476] TIFF2026516826000307.tif219169

[0477] TIFF2026516826000308.tif219169

[0478] TIFF2026516826000309.tif213169

[0479] TIFF2026516826000310.tif201169

[0480] TIFF2026516826000311.tif212169

[0481] TIFF2026516826000312.tif216169

[0482] (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), and v and w are each independently selected from integers between 1 and 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).) In some embodiments, the linker-drug compound represented by formula (II) or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof have the following structures.

[0483] [Table 12]

[0484] TIFF2026516826000314.tif233169

[0485] TIFF2026516826000315.tif228169

[0486] TIFF2026516826000316.tif230169

[0487] TIFF2026516826000317.tif255165

[0488] TIFF2026516826000318.tif228169

[0489] TIFF2026516826000319.tif253169

[0490] TIFF2026516826000320.tif218169

[0491] TIFF2026516826000321.tif225169

[0492] TIFF2026516826000322.tif246169

[0493] TIFF2026516826000323.tif243169

[0494] TIFF2026516826000324.tif236169

[0495] TIFF2026516826000325.tif131169

[0496] The present invention also provides compounds of formula (III) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof.

[0497] [ka]

[0498] ((III), the above formula (III) has the following structure,

[0499] [ka]

[0500] Here, X does not exist, or

[0501] [ka]

[0502] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3(where n 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.) The present invention also provides compounds of formula (IV) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products thereof.

[0503] [ka]

[0504] (Here, X does not exist, or

[0505] [ka]

[0506] Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and Y is a C1-C6 alkylene group, or -R 3 -C(O)-, where R 3 n is a C1-C6 alkylene group, or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, where n is an integer from 0 to 6. Here, AA is a subfragment consisting of L3 or the first, second, or more amino acids from the C-terminus of L3, and P N is H or an amino protecting group, for example, Boc or Fmoc, and 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 as follows:

[0507] [ka]

[0508] (Abbreviations and definitions) Unless otherwise specified, the following terms used in this application have the meanings set forth below.

[0509] Where a trademark name is used in this application, unless otherwise indicated by the context, the trademark name includes the product formulation of the trademarked product, generic drugs, and active drug components.

[0510] The term "antibody-drug conjugate" or "antibody-conjugated drug" (ADC) refers to a biologically active molecule in which a targeted ligand, such as an antibody (e.g., a monoclonal antibody) or antibody fragment, is linked to a biologically active molecule via a stable chemical linker compound.

[0511] The term "linker-drug" refers to a substructure of an antibody-drug conjugate consisting of a linker compound and a biologically active compound.

[0512] The term "linker" refers to a chemical structural fragment, denoted by L, in which one end is linked to an antibody and the other end to a cytotoxic drug, and is formed by linking the antibody and the biologically active compound with "linker compounds," respectively. In some embodiments of the present invention, a portion of the linker compound is first linked to the antibody, and the other portion is linked to the biologically active compound, after which the "linker" is formed by chemical reactions between different parts of the linker. The linker in the present invention includes a portion that links to the antibody, a spacer, a polypeptide sequence, and a self-cleaving fragment.

[0513] The linker-drug compounds described in the present invention are linked to antibodies by conventional linkage methods in the art, including linkage via lysine, linkage via reductive disulfide bonds between light and heavy chains, and directional linkage (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). Preferably, the linkage is achieved by reductive disulfide bonds between light and heavy chains, that is, by the reaction of thiol groups (sulfur atoms of cysteine ​​residues) formed by the reduction of one or more disulfide bond sites between light and heavy chains (two sites between heavy chains and two sites between heavy and light chains). In this invention, the linkage portion between linker L and antibody is represented by L1, which is L1 in the linker compound. ’ It is formed by the reaction of a group with an antibody.

[0514] [ka]

[0515] Here, * indicates that it is linked to the mercapto group of the antibody, and ** indicates that it is linked to the spacer.

[0516] The term "spacer" refers to the linking group between the linkage portion of linker L and the antibody and the polypeptide sequence, and can be any divalent organic group, such as a chemical bond or C. 1~10 Alkylene group, C 2~10 Alkenylene group, C 2~10 Alkylene group, C 3~10 Cycloalkyl groups, C 6~12 The C may be an aryl group, a 5-12 membered heteroaryl group, a 5-12 membered heterocyclyl group, or a combination of two or more of these groups.1~10 Alkylene group, C 2~10 Alkenylene group, C 2~10 Alkylene group, C 3~10 Cycloalkyl groups, C 6~12 The aryl group, 5-12 membered heteroaryl group, 5-12 membered heterocyclyl group, or combination of two or more of these groups is arbitrarily separated by a carbonyl group, O, S, N atom, and the C 1~10 Alkylene group, C 2~10 Alkenylene group, C 2~10 Alkylene group, C 3~10 Cycloalkyl groups, C 6~12 Aryl groups, 5-12 membered heteroaryl groups, and 5-12 membered heterocyclyl groups are C 1~6 Alkyl alkyl group, C 3~6 Cycloalkyl groups, halogen atoms, halo C 1~6 It may be optionally substituted with an alkyl group, preferably as defined in L2.

[0517] The term "polypeptide sequence" is known in the field and is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or unnatural, L-type or D-type amino acid residues, each of which may be identical 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), norvaline (Nva), norleucine (Nle), or analogues of the above amino acids.

[0518] The term “self-cleaving fragment” is known in the field and is, for example, a p-aminobenzyl alcohol carbonate fragment commonly used in the field. In specific embodiments of the present invention, the “self-cleaving fragment” of the linker of the present invention includes “hydrophilic fragment” modification.

[0519] The term "hydrophilic fragment" is known in the art, and the hydrophilic fragment contains polyethylene glycol groups, for example, polyethylene glycol groups with a methoxy terminal group, or other hydrophilic fragments are further linked via polyethylene glycol groups. The hydrophilic fragment may further contain polyamino acid fragments, for example, polyglycine or polysarcosine, and the polyamino acid fragments may be combined with polyethylene glycol groups. The hydrophilic fragment may be a monosaccharide, disaccharide or oligosaccharide, and the monosaccharide, disaccharide or oligosaccharide may be a chain-like or cyclic sugar, and may also contain sugar amines, sugar acids or sugar phosphates. Preferably, the sugar group is combined with a polyethylene glycol fragment or a polyamino acid fragment. More preferably, at least two sugar groups are introduced via a polyvalent linking group, such as a linking group based on aspartic acid, glutamic acid or lysine. Polycarboxylic acid groups, polysulfonic acid groups or chelating groups may be included. The structure of the polycarboxylic acid group is as follows:

[0520] [ka]

[0521] The structure of the polysulfonic acid group is as follows:

[0522] [ka]

[0523] The chelating group may be, for example, a DOTA group or a NOTA group.

[0524] The terms “solvate” and “solvated compound” refer to a physical bond between the compound of the present invention and one or more solvent molecules (whether organic or inorganic). This physical bond includes hydrogen bonds. In certain cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvated compound becomes separable. The solvent molecules in the solvated compound may exist in regular and / or irregular arrangements. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. “Solvate” includes both the solution phase and the separable solvate. Exemplary solvates include, but are not limited to, hydrates, ethanol hydrates, methanol hydrates, and isopropanol hydrates. Solvation methods are known in the art.

[0525] The term "stereoisomer" refers to compounds that have the same chemical structure but differ in the spatial arrangement of their atoms or groups. Stereoiomers include enantiomers, diastereomers, conformers (rotational isomers), geometric isomers (cis / trans isomers), and atrop isomers. A mixture of all resulting stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physical and chemical properties of the components, such as by chromatography and / or fractional crystallization.

[0526] The term "tautomer" refers to isomers of different energies that can interconvert across low energy barriers. When tautomerization can occur (such as in solution), a chemical equilibrium of tautomers is achieved. For example, proton tautomers (also called prototropic tautomers) include tautomers that interconvert by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include tautomers that interconvert by rearrangement of bonded electrons.

[0527] In the term "one or more," "plural" includes two or more (for example, 3, 4, 5, etc.).

[0528] The term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0529] "C m ~C n The term "C1-C3" means that the part has an integer number of carbon atoms in the range of m to n. For example, "C1-C3" means that the group can contain one carbon atom, two carbon atoms, or three carbon atoms.

[0530] "-(CH2)" n The term "-" indicates that the part has n linked -CH2- groups. For example, if the number of linked groups is 0, such as "-(CH)0-", it means that the linked group is covalent.

[0531] The term "alkyl group" refers to a monovalent saturated aliphatic hydrocarbon group, which is a straight or branched chain containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms (i.e., C 1~10 Alkyl alkyl groups), more preferably 1 to 8 carbon atoms (C 1~8 Alkyl alkyl groups), more preferably 1 to 6 carbon atoms (i.e., C 1~6 Contains alkyl groups. For example, "C 1~6 The term "alkyl group" means that its group is an alkyl group, and the number of carbon atoms on 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, and n-hexyl groups.

[0532] The term "alkoxy group" refers to an -O-alkyl group, where the alkyl group is as defined above, that is, it contains 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and even more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5, or 6). Typical 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, and 1-ethylpropoxy groups.

[0533] The term "halogen" refers to F, Cl, Br, and I.

[0534] The term "haloalkyl group" refers to an alkyl group defined above in which one, two, or more hydrogen atoms, or all of the hydrogen atoms, are substituted with a halogen. Typical examples of haloalkyl groups include CCl3, CHCl2, CH2Cl, CF3, CHF2, CH2F, CBr3, CHBr2, CH2Br, CI3, CHI2, CH2I, CH2CF3, and CF2CF3.

[0535] The term "aryl group" refers to monocyclic, bicyclic, and tricyclic aromatic carbocyclic systems containing 6 to 16 carbon atoms, or 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms, preferably containing 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenantrenyl, or pyrenyl groups. The term "heteroaryl group" refers to aromatic monocyclic or polycyclic ring systems containing a 5 to 14-membered structure, preferably a 5 to 10-membered structure, preferably a 5 to 8-membered structure, more preferably a 5 to 6-membered structure, where one, two, or three or more ring atoms are heteroatoms, the remaining atoms are carbon, the heteroatoms are independently selected from O, N, or S, and the number of heteroatoms is preferably one, two, or three. Examples of heteroaryl groups include furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, prinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, and benzopiryl groups. This includes, but is not limited to, lysinyl groups, benzopyrimidinyl groups, benzopyradinyl groups, benzimidazolyl groups, benzophthalazinyl groups, pyrrolo[2,3-b]pyridinyl groups, imidazo[1,2-a]pyridinyl groups, pyrazolo[1,5-a]pyridinyl groups, pyrazolo[1,5-a]pyridinyl groups, imidazo[1,2-b]pyridadinyl groups, [1,2,4]triazolo[4,3-b]pyridadinyl groups, [1,2,4]triazolo[1,5-a]pyridinyl groups, and [1,2,4]triazolo[1,5-a]pyridinyl groups.

[0536] The term "cycloalkyl group" refers to a fully saturated carbon ring that can exist as a monocyclic, bridging, or spirocyclic group. Preferably, it includes 3 to 12 carbon atoms (i.e., C3-12 cycloalkyl groups), more preferably 3 to 10 carbon atoms (C3-10 cycloalkyl groups), even more preferably 3 to 7 carbon atoms (C3-7 cycloalkyl groups), 4 to 6 carbon atoms (C4-6 cycloalkyl groups), and 5 to 6 carbon atoms (C5-6 cycloalkyl groups). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethylcyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, and adamantyl groups.

[0537] The terms "-alkyl-" or "alkylene group" refer to saturated straight-chain or branched-chain divalent hydrocarbon groups. For example, C1-C8 alkylene groups refer to straight-chain or branched-chain alkylene groups having 1 to 8 carbon atoms.

[0538] The term "heterocyclyl group" refers to a non-aromatic ring that is either fully saturated or partially unsaturated (heteroaromatic but not fully unsaturated), and may exist as a monocyclic, bridging, or spirocyclic ring. Unless otherwise specified, the heterocyclic ring is typically a 3- to 7-membered ring containing 1-3 heteroatoms (preferably 1 or 2 heteroatoms, but excluding -OO-, -OS-, or -SS- portions) independently selected from sulfur, oxygen, and / or nitrogen. Non-limiting examples of heterocyclyl groups include, but are not limited to, oxyranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, and tetrahydrothiophenyl groups.

[0539] The term "heteroalkyl group," either alone or in combination with other terms, refers to a stable linear, branched alkyl group, or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. The number of carbon atoms is 1 to 50 (preferably 1 to 20, more preferably 1 to 12, and most preferably 1 to 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 any heteroatom may be interpreted as being inserted into any CC and CH bond of the alkyl group). The heteroatoms O, N, and S can be located at any position within the heteroalkyl group or at a position where the alkyl group is attached to other parts 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, and -CH2-CH2-S(O)2-CH3. There may be up to two heteroatoms in a row, for example, -CH2-NH-OCH3.

[0540] Unless otherwise specified, the definitions of terms herein also apply to groups containing those terms; for example, the definition of a C1-6 alkyl group also applies to a C1-6 alkyloxy group.

[0541] Linking substituents are described in various parts of this disclosure. Where a linking group is clearly structurally required, the Markousse variable given for that group shall be understood as a linking group. For example, if a linking group is required in a structure and the definition of the Markousse variable for that group includes "heteroalkyl group," "aryl group," etc., then "heteroalkyl group" and "aryl group" should be understood to represent the heteroalkylene group or arylene group to be linked, respectively. Furthermore, when a Markousse variable given for a group is understood as a linking group, it should be understood that the same definition applies to both, regardless of whether it is defined as "alkylene group" or "heteroalkylene group." Accordingly, in this specification, the definitions of terms such as "alkylene group," "cycloalkylene group," "arylene group," "heteroalkylene group," "heteroarylene group," and "heterocyclylene group" are equivalent to, or refer to, the definitions of "alkyl group," "cycloalkyl group," "aryl group," "heteroalkyl group," "heteroaryl group," and "heterocyclyl group" as defined above.

[0542] The term "derivative" refers to a compound formed by replacing an atom or group of atoms within the molecule of a parent compound with another atom or group of atoms, and is called a derivative of that parent compound.

[0543] 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 has at least one amino, imino, hydroxyl, or carboxyl group and can form addition salts with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobroms, hydroiodides, nitrates, bisulfates, phosphates, acidic phosphates (-H2PO4), phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid, formate, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, potassium salts, sodium salts, ammonium salts, and calcium salts. Furthermore, pharmaceutically acceptable salts have multiple charged atoms in their structure. Multiple charged atoms are some examples of pharmaceutically acceptable salts, and they may have multiple counterions. For example, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter atoms.

[0544] The term "amino acid" refers to a natural or unnatural amino acid represented by NH2-C(R'R'')-C(=O)OH. Here, R' and R'' are independently a hydrogen atom, an optionally substituted linear, branched, or cyclic alkyl group having 1 to 10 carbon atoms, an alkenyl group or alkynyl group, an aryl group, a heteroaryl group or heterocyclyl group, or R'' may be bonded to the N-terminal nitrogen atom to form a heterocycle, such as proline.

[0545] The term "amino acid residue" refers to the corresponding residue obtained when one hydrogen atom is removed from the amine of an amino acid and / or one hydroxyl group is removed from the carboxyl terminus, e.g., -NH-C(R'R'')-C(O)-.

[0546] The term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds.

[0547] The term "tumor" refers to a new organism that forms when cells in a local tissue lose normal growth control at the genetic level due to the action of various oncogenic factors, leading to abnormal proliferation of clones.

[0548] 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 combination thereof via at least one antigen-recognizing site within the variable region of the immunoglobulin molecule, denoted by A. Antibodies include, but are not limited to, fully polyclonal antibodies, fully monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv(scFv) variants, multispecific antibodies (e.g., bispecific antibodies, biparatopic antibodies, etc.), multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies with three, four, or more antigen-binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigenic determinant portion of an antibody, and other modified immunoglobulin molecules containing an antigen-recognizing site. When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" is understood as the complete entity relative to the "antigen-binding fragment / antigen-binding portion," and these two together constitute the broader concept of antibody.

[0549] [Brief explanation of the drawing] [Figure 1] This is the single-crystal diffraction pattern of compound 82.

[0550] [Modes for carrying out the invention] The present invention will be further described below with reference to examples, but these examples are not intended to limit the scope of the invention. In the following examples, test methods that do not specify particular conditions are usually carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all percentages, proportions, ratios, or parts are by weight.

[0551] Explanation of abbreviations

[0552] [Table 13]

[0553] Example 1: Synthesis of Compounds 8-10

[0554] [ka]

[0555] 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, and the reaction was terminated by refluxing to 70°C for 6 hours. The mixture was cooled to precipitate white crystals, which were then filtered by suction. The remaining solid was washed with methanol (20 mL x 3) to obtain white solid 2 (50 g, 100% yield). MS(ESI): (M+H) + Calculated value: 266.1, experimental value: 266.2.

[0556] Compound 2 (50 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol), and ethanol (400 mL) were added to a reaction flask and dissolved by stirring at room temperature. Carbon disulfide (17 g, 283 mmol) was slowly added, and the mixture was heated to 100 °C and refluxed for 5 hours to complete the reaction. After removing the solvent under reduced pressure, water (50 mL) was added, the pH was adjusted to 6 with dilute hydrochloric acid, and the mixture was extracted with dichloromethane (100 mL x 3). The organic phase was 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, 93% yield). MS(ESI): (M+H) + Calculated value: 308.1, experimental value: 308.2.

[0557] Compound 3 (5 g, 16.3 mmol), triethylamine (2 g, 19.6 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask. Iodomethane (2.5 g, 17.9 mmol) was then added to the reaction mixture. The reaction was carried out at 25°C for 1.5 hours with stirring. After the reaction was terminated, 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.

[0558] Compound 4 (3.4 g, 10.6 mmol) and ethyl acetate (20 mL) were added to the reaction flask, and ethyl acetate hydrogen chloride solution (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours, and the solvent was removed under reduced pressure to obtain crude product 5, which was then used directly in the next step of the reaction.

[0559] Crude product 5, diglycolic acid 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, ethyl ether was added to the residue, and after suction filtration, the mixture was washed with water and ethyl ether to obtain compound 7 (3.5 g, 99% yield). MS(ESI): (M+H)+ Calculated value: 338.0, experimental value: 338.2.

[0560] 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 mixture was allowed to react for 1 hour at 25°C. Saturated sodium sulfite solution was added until the solution became colorless, and the mixture was extracted with ethyl acetate (100 mL x 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.

[0561] [Table 14]

[0562] The synthesis of compounds 9 and 10 follows the synthetic route of compound 8.

[0563] Example 2. Synthesis of Compound 18

[0564] [ka]

[0565] 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, and the reaction was completed by refluxing to 70°C for 6 hours. The mixture was cooled to precipitate white crystals, which were then filtered by suction. The remaining solid was washed with methanol (20 mL x 3) to obtain white solid 12 (47.47 g, 100% yield). MS(ESI): (M+H) + Calculated value: 252.1, experimental value: 252.2.

[0566] 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 dissolved by stirring at room temperature. 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, the pH was adjusted to 6 with dilute hydrochloric acid, and the mixture was extracted with dichloromethane (100 mL x 3). The organic phase was combined and 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.

[0567] Compound 13 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to a reaction flask. Iodomethane (3.12 g, 22 mmol) was then added to the reaction mixture. The reaction was carried out at 25°C for 1.5 hours with stirring. After the reaction was terminated, 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.

[0568] Compound 14 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to the reaction flask, and ethyl acetate hydrogen chloride solution (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours, and the solvent was removed under reduced pressure to obtain crude product 15, which was then used directly in the next step of the reaction.

[0569] Crude product 15, diglycolic acid 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, ethyl ether was added to the residue, and after suction filtration, the mixture was washed with water and ethyl ether to obtain compound 17 (3.4 g, 99% yield). MS(ESI): (M+H) + Calculated value: 324.1, experimental value: 324.2.

[0570] 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 mixture was allowed to react for 1 hour at 25°C. Saturated sodium sulfite solution was added until the solution became colorless, and the mixture was extracted with ethyl acetate (100 mL x 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.

[0571] Example 3: Synthesis of Compound 21

[0572] [ka]

[0573] t-butyl glycolate (2.57 g, 19.5 mmol) was slowly added dropwise at 0°C to a chlorosulfonyl isocyanate (2.75 g, 19.5 mmol) dichloromethane (40 mL) solution. The mixture was then allowed to react for 1 hour at 25°C. Compound 5 (5 g, 19.5 mmol) in dichloromethane (10 mL) solution was added dropwise to the reaction system and allowed to react for 6 hours. After adding water (40 mL), the mixture was extracted with ethyl acetate (100 mL x 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, yield 86%). MS(ESI): (M+H) + Calculated value: 459.1, experimental value: 459.0.

[0574] Compound 19 (7.68 g, 16.8 mmol) and ethyl acetate (20 mL) were added to the reaction flask, and ethyl acetate hydrogen chloride solution (4.2 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours, and the solvent was removed under reduced pressure to obtain crude product 20, which was then used directly in the next step of the reaction.

[0575] 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 mixture was allowed to react for 1 hour at 25°C. Saturated sodium sulfite solution was added until the solution became colorless, and the mixture was extracted with ethyl acetate (100 mL x 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, yield 45%). MS(ESI): (M+H) + Calculated value: 435.0, experimental value: 435.1.

[0576] Example 4: Synthesis of Compound 25

[0577] [ka]

[0578] For the synthesis of compound 25, refer to the synthesis method for compound 21.

[0579] Example 5: Synthesis of compounds 29 and 37

[0580] [ka]

[0581] 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 was completed, the mixture was cooled, water was added, and the mixture was extracted with ethyl acetate (100 mL x 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.

[0582] Zinc powder (7.3 g, 113 mmol) and tetrahydrofuran (50 mL) were added to a reaction flask, and a solution of propargyl 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 then compound 26 (2.75 g, 18.7 mmol) in tetrahydrofuran (30 mL) was slowly added dropwise, followed by stirring for 2 hours. The reaction mixture was poured into a 1N HCl aqueous solution at 0°C, extracted with ethyl acetate (100 mL x 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.

[0583] Compound 27 (1 g, 5.3 mmol) and dichloromethane (5 mL) were added to the reaction flask, and thionyl chloride (1.9 g, 16 mmol) was added dropwise at 0°C. After the addition was complete, the mixture was returned to 25°C and reacted for 2 hours. The solvent and remaining thionyl chloride were removed under reduced pressure, and potassium thioacetate (906 mg, 8 mmol) and N,N-dimethylformamide (10 mL) were added. The mixture was reacted at 25°C for 12 hours, water was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined and saturated salt solution was added. The compound was washed with water, 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, and the mixture was reacted at 25°C for 2 hours. Water was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was 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.

[0584] Cyclohexene oxide (1.47 g, 15 mmol) and 40% trimethylbenzylammonium hydroxide (2.5 g, 6 mmol) were added to the reaction flask. Compound 28 (612 mg, 3 mmol) was dissolved in N,N-dimethylformamide (6 mL) and added dropwise to the reaction system. The reaction was carried out at 25°C for 3 hours. Water was added, and the mixture was extracted with ethyl acetate (20 mL x 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.

[0585] 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, and 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, yield 66%). MS(ESI): (M+H) + Calculated value: 254.0, experimental value: 254.1.

[0586] Compound 31 (5.4 g, 21.5 mmol) was added to tetrahydrofuran, and a solution of boranetetrahydrofuran (21.5 mL, 1 M) was added dropwise at 0°C. The reaction was then carried out at 25°C for 16 hours, followed by quenching with methanol. The solvent was removed under reduced pressure, and 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.

[0587] Compound 32 (4.1 g, 15.9 mmol) and triethylamine (3.2 g, 31.8 mmol) were added to dichloromethane (30 mL). Trifluoroacetic anhydride (5 g, 23.8 mmol) was added dropwise at 0°C, and the mixture was reacted at 25°C for 1 hour. The mixture was diluted with water, extracted with dichloromethane (20 mL x 3), and the organic phases were washed with saturated brine. The mixture was 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.

[0588] 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 mixture was poured into ice water and extracted with ethyl acetate (20 mL x 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, 80% yield). MS(ESI): (M+H) + Calculated value: 366.0, experimental value: 366.1.

[0589] Compound 34 (2.77 g, 7.6 mmol) was added to tetrahydrofuran, and diisobutylaluminum hydride was added dropwise at 0°C. The reaction was then carried out at 25°C for 2 hours, water was added to quench the reaction, and after filtration, the filtrate was 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.

[0590] 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). 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 x 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. 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, 80% yield). MS(ESI): (M+H) + Calculated value: 447.2, experimental value: 447.1.

[0591] Example 6: Synthesis of Compound 45

[0592] [ka]

[0593] Compound 38 (10 g, 50 mmol) was dissolved in tetrahydrofuran (120 mL), and sodium hydride (4 g, 100 mmol) was added at 0°C. The mixture was stirred for 0.5 hours. Then, allyl bromide (12.1 g, 100 mmol) was added, and the mixture was reacted for 2 hours. The reaction was then quenched with saturated ammonium chloride solution. Extraction was performed with ethyl acetate (10 mL x 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 39 (4.65 g, yield 66%). MS(ESI): (M+H) + Calculated value: 142.2, experimental value: 142.3.

[0594] Compound 39 (4.65 g, 33 mmol) was added to tetrahydrofuran (100 mL), and a solution of 9-borabicyclo[3.3.1]nonane (80 mL, 0.5 M) in tetrahydrofuran was added. The mixture was reacted at 80°C for 2 hours, cooled to room temperature, and the reaction solution was used directly in the next step.

[0595] To the reaction solution of compound 40, N,N-dimethylformamide (100 mL) and potassium phosphate (8.7 g, 41 mmol) aqueous solution were added and stirred for 10 minutes. Then, 3-chloro-4-bromoaniline (6.8 g, 33 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (2.4 g, 3.3 mmol) were added and the mixture was reacted at 90°C for 3 hours. After cooling to room temperature, the mixture was extracted with ethyl acetate (10 mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 41 (3.7 g, yield 42%). MS(ESI): (M+H) + Calculated value: 269.1, experimental value: 269.1.

[0596] 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 sequentially added to the solution. The reaction was carried out at 25°C for 0.5 hours, the reaction mixture 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, experimental value: 389.2.

[0597] Compound 42 (4.66 g, 12 mmol) was dissolved in N,N-dimethylformamide (50 mL), then compound 43 (4.08 g, 13.2 mmol) was added. The solid was dispersed in the solution by sonication, then diisopropylethylamine (48 mmol) was added. The mixture was reacted at 50°C for 3 hours, cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 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.

[0598] Compound 44 (1.6 g, 2.8 mmol) was added to dichloromethane (19 mL), and ethyl hydrogen chloride solution (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 vacuum-dried to obtain compound 45 (1.6 g, 94% yield). MS(ESI): (M+H) + Calculated value: 568.2, experimental value: 568.3.

[0599] Example 7: Synthesis of Compounds 46-49

[0600] [Table 15]

[0601] The synthesis of compounds 46-49 follows the synthetic route of compound 45.

[0602] Example 8: Synthesis of compounds 52 and 55

[0603] [ka]

[0604] Compound 50 (1.1 g, 1.06 mmol, obtained by solid-phase peptide synthesis) and diethylamine (3.89 g, 53.24 mmol) were dissolved in dichloromethane (5 mL). The reaction mixture was purged three times with N2, and then reacted at 25°C for 1 hour until the starting materials were completely converted. After the solvent was removed from the reaction mixture under reduced pressure, the low-boiling point compounds were removed by aspirate under high vacuum. Compound 51 (3.15 g, 1.6 mmol) and N,N-dimethylformamide (4 mL) were then added, and the mixture was stirred. Triethylamine (3.23 g, 3.2 mmol) was added dropwise at 25°C and reacted for 6 hours. LC-MS detection confirmed that no starting materials remained. Excess triethylamine was removed under reduced pressure, and the residue was separated by reverse-phase chromatography to obtain compound 52 (810 mg, yield 10%). MS(ESI): (M+H) + Calculated value: 812.4, experimental value: 812.3.

[0605] 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 by solid-phase peptide synthesis) were added to N,N-dimethylformamide (40 mL). Subsequently, 1-hydroxybenzotriazole (40 mg, 0.26 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (285 mg, 1.46 mmol) were added, and the mixture was reacted at 25°C for 16 hours with stirring. After stopping 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.

[0606] Example 9: Synthesis of compound LD1

[0607] [ka]

[0608] 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. Triethylamine (2.32 g, 23 mmol) was slowly added dropwise while stirring, and the mixture was reacted 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).

[0609] Compound 56 (176 mg, 0.5 mmol), exatecan mesylate (243 mg, 0.45 mmol), and N,N-dimethylformamide (2 mL) were added to a reaction flask. Triethylamine (101 mg, 1 mmol) was slowly added dropwise while stirring, and the reaction was carried out 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.

[0610] Compound 57 (259 mg, 0.4 mmol) and compound 58 (670 mg, 1 mmol, synthesized referring to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, substituted three times with nitrogen, tetrahydrofuran (10 mL) and water (4 mL) were added, and the mixture was reacted at 25°C for 24 hours. The mixture was diluted with water (10 mL), extracted with ethyl acetate (20 mL x 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 59 (239 mg, yield 59%). MS(ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.3.

[0611] Compound 59 (239 mg, 0.24 mmol) and N,N-dimethylformamide (2 mL) were added to the reaction flask, and 1,8-diazabicyclo[5.4.0]undeca-7-ene (18 mg, 0.12 mmol) was added dropwise, and the mixture was reacted at 25°C for 0.5 hours. Compound 8 (107 mg, 0.29 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (890 mg, 0.36 mmol), and di Sopropylethylamine (48 mg, 0.48 mmol) and N,N-dimethylformamide (10 mL) were added, and the mixture was reacted at 25°C for 24 hours. The mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL x 3), and the organic phases were combined. The mixture was 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 60 (192 mg, 70% yield). MS(ESI): (M+H) + Calculated value: 1144.4, experimental value: 1144.3.

[0612] 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, substituted three times with nitrogen, 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), extracted with ethyl acetate (20 mL x 3), and the organic phases were washed with saturated brine. The mixture was 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, yield 62%). MS(ESI): (M+H) + Calculated value: 2258.0, experimental value: 2258.2.

[0613] Example 10: Synthesis of compounds LD2-19, LD23-28, and LD32-54

[0614] [ka]

[0615] [Table 16]

[0616] TIFF2026516826000348.tif247169

[0617] The synthetic routes for compounds LD2-19, LD23-28, and LD32-54 refer to the synthesis of compound LD1.

[0618] The structures of Con1-5, Pep1-5, Trig1-3, HDP1-3, and Pay1-13 are shown below.

[0619] [ka]

[0620] Here, the nitrogen terminus is connected to Fmoc.

[0621] [ka]

[0622] Here, an -N3 group is attached at position #, an -OH group at position ##, and an alkynyl group at position ###.

[0623] [ka]

[0624] Synthesis of compound LD17 Compound LD17' was synthesized by referring to the synthesis route of compound LD1. Compound LD17' (130 mg, 0.05 mmol) was dissolved in dichloromethane (2 mL), and dichloroacetic acid (2 mL) and a small amount of triethylsilane were added to the reaction system at 0°C. After reacting for 3 hours, the reaction was terminated, 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.

[0625] [ka]

[0626] Example 11: Synthesis of compound LD20-22

[0627] [ka]

[0628] [Table 17]

[0629] [ka]

[0630] Here, an -N3 group is attached at position #, an -OH group at position ##, and an alkynyl group at position ###.

[0631] 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. Triethylamine (2 g, 20 mmol) was slowly added dropwise while stirring, and the mixture was reacted 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, 96% yield).

[0632] Compound 62 (233 mg, 0.5 mmol), exatecan mesylate (243 mg, 0.45 mmol), and N,N-dimethylformamide (2 mL) were added to a reaction flask. Triethylamine (101 mg, 1 mmol) was slowly added dropwise while stirring, and the reaction was carried out 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.

[0633] Compound 63 (343 mg, 0.45 mmol) and compound 58 (670 mg, 1 mmol, synthesized referring to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, replaced three times with nitrogen, tetrahydrofuran (10 mL) and water (4 mL) were added, and the mixture was reacted at 25°C for 24 hours. The mixture was diluted with water (10 mL), extracted with ethyl acetate (20 mL x 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 64 (304 mg, yield 60%). MS(ESI): (M+H) + Calculated value: 1129.4, experimental value: 1129.3.

[0634] Compound 64 (304 mg, 0.27 mmol) and N,N-dimethylformamide (2 mL) were added to the reaction flask, and 1,8-diazabicyclo[5.4.0]undeca-7-ene (18 mg, 0.13 mmol) was added dropwise, and the mixture was reacted at 25°C for 0.5 hours. Compound 8 (107 mg, 0.3 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (890 mg, 0.36 mmol), and diiso were added to the reaction system. Propylethylamine (54 mg, 0.54 mmol) and N,N-dimethylformamide (10 mL) were added, and the mixture was reacted at 25°C for 24 hours. The mixture was diluted with water (20 mL x 3), extracted with ethyl acetate (20 mL x 3), and the organic phases were combined. The mixture was 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 65 (170 mg, 50% yield). MS(ESI): (M+H) + Calculated value: 1258.4, experimental value: 1258.3.

[0635] 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, substituted three times with nitrogen, 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), extracted with ethyl acetate (20 mL x 3), and the organic phases were washed with saturated brine. The mixture was 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, yield 56%). MS(ESI): (M+H) + Calculated value: 2372.1, experimental value: 2372.2.

[0636] The synthetic route for compounds LD21-22 follows the synthesis of compound LD20.

[0637] Example 12: Synthesis of compound LD29-31

[0638] [ka]

[0639] [Table 18]

[0640] [ka]

[0641] 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. Triethylamine (101 mg, 1 mmol) was slowly added dropwise while stirring, and 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.

[0642] 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, after which it was 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.

[0643] Compound 69 (185 mg, 0.3 mmol) and compound 58 (503 mg, 0.75 mmol, synthesized referring to the reaction route reported in patent WO 2021252708 A1) were added to a reaction flask, substituted three times with nitrogen, tetrahydrofuran (10 mL) and water (4 mL) were added, and the mixture was reacted at 25°C for 24 hours. The mixture was diluted with water (10 mL), extracted with ethyl acetate (20 mL x 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 70 (177 mg, yield 60%). MS(ESI): (M+H) + Calculated value: 983.4, experimental value: 983.3.

[0644] Compound 70 (177 mg, 0.18 mmol) and N,N-dimethylformamide (2 mL) were added to the reaction flask, and 1,8-diazabicyclo[5.4.0]undeca-7-ene (14 mg, 0.09 mmol) was added dropwise, and the mixture was reacted at 25°C for 0.5 hours. Compound 11 (82 mg, 0.22 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (666 mg, 0.27 mmol), and dihydroquinoline were added to the above reaction system. Sopropylethylamine (36 mg, 0.36 mmol) and N,N-dimethylformamide (10 mL) were added and reacted at 25°C for 24 hours. The mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL x 3), and 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 71 (150 mg, 75% yield). MS(ESI): (M+H) + Calculated value: 1112.4, experimental value: 1112.3.

[0645] 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, substituted three times with nitrogen, 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), extracted with ethyl acetate (20 mL x 3), and the organic phases were washed with saturated brine. The mixture was 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, yield 60%). MS(ESI): (M+H) + Calculated value: 2212.0, experimental value: 2212.2.

[0646] The synthetic route for compounds LD30-31 refers to the synthesis of compound LD29.

[0647] The compounds shown in Table 7 may be synthesized by referring to the examples described above (where the final product containing the Pep3 structure is obtained by treatment with acid with reference to LD17).

[0648] [Table 19]

[0649] TIFF2026516826000360.tif225169

[0650] [ka]

[0651] Example 13: Synthesis of compound LD55 and separation of LD55-1 and LD55-2

[0652] [ka]

[0653] 1-(4-aminophenyl)-3-buty-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultra-dried DCM (90 mL) were added to a reaction flask at 0°C. After reacting for 3 hours, the solvent was removed under reduced pressure, and 500 mL of MTBE was added to the residue to form a slurry, yielding compound 72 (9007 mg, yield 64%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 455.2, experimental value: 455.2.

[0654] Compound 72 (4000 mg, 8.8 mmol, dr = 1:1) and 100 mL of commercially available THF were added to a reaction flask at 0°C. While stirring, DBU (1337 mg, 8.8 mmol) was slowly added, and the mixture was reacted for half an hour. The mixture was then transferred to room temperature. Once the starting materials disappeared under TLC monitoring, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 73 (1776 mg, yield 87%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 233.1, experimental value: 233.2.

[0655] 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. DIEA (1158 μL, 7 mmol) was slowly added dropwise while stirring, and the mixture was reacted 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 to form a slurry. A white solid precipitated, and this process was repeated three times to obtain compound 74 (dr = 1:1). MS(ESI): (M+H) + Calculated value: 554.3, experimental value: 554.4. 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. While stirring, Py (382.8 μL, 4.8 mmol) was added dropwise, and 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 75 (1430 mg, yield 83%, dr = 1:1). MS(ESI): (M+Na) + Calculated value: 741.3, experimental value: 741.5.

[0656] Compound 75 (1554 mg, 2.2 mmol, dr = 1:1), exatecan mesylate 76 (520 mg, 1.8 mmol), and N,N-dimethylformamide (80 mL) were added to a reaction flask. DIEA (740 μL, 4.5 mmol) was slowly added dropwise while stirring, and 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, yield 79%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.1.

[0657] Compound 77 (1400 mg, 1.37 mmol, dr = 1:1) and 35 mL of commercially available THF were added to a reaction flask at 0°C. While stirring, DBU (209 mg, 1.37 mmol) was slowly added, and the mixture was reacted for half an hour. The mixture was then transferred to room temperature and the reaction was continued for 40 minutes until the starting materials disappeared by TLC. The reaction mixture was concentrated under vacuum to remove the THF, and DCM / PE (20 mL / 200 mL) was added to the residue to form a slurry. The slurry was filtered by suction and then through a funnel to obtain compound 78 (1032 mg, yield 95%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 793.3, experimental value: 793.6.

[0658] Compound 78 (793 mg, 1 mmol, dr = 1:1), Compound 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 with C18 (60% ACN / 0.05% formic acid in H2O). The mixture was then lyophilized to obtain Compound 79 (960 mg, yield 85%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 1130.4, experimental value: 1130.7.

[0659] 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, substituted three times with nitrogen, and THF / DMF / H2O (3.5 mL: 0.6 mL: 0.4 mL) was added. The mixture was reacted at 25°C for 0.5 hours, and the residue was separated and purified using C18 (62% ACN / 0.05% formic acid in H2O) to obtain Compound LD55 (1180 mg, yield 86%, dr = 1:1). MS(ESI): (M+H) + Calculated value: 1715.7, experimental value: 1716.2.

[0660] LD55 (1180 mg, dr = 1:1) was separated by preparative column (Tsuki Asahi Xtimate C18 30 × 250 mm × 10 μm) using ACN / H2O (0.05% formic acid) to obtain LD55-1 (550 mg, HPLC retention time 18.397 min) and LD55-2 (520 mg, HPLC retention time 18.163 min).

[0661] Note: HPLC equipment information: Thermo liquid chromatograph (ADC-U3000-01); chromatographic column: Hypersil GOLD (trademark) (4.6*250mm, 5μm); column temperature: 30℃; sample tray temperature: 10℃; mobile phase: A: Water + 0.05% TFA; B: ACN; flow rate: 1.0 mL / min; detection wavelength: 254 nm; injection volume: 10 μL;

[0662] [Table 20]

[0663] Compounds LD1-LD54, LD56-LD144, LD2-1, LD2-2, LD7-1, LD7-2, LD12-1, and LD12-2 can also be synthesized by referring to the same method.

[0664] Example 14: Synthesis of LD55-1 using chiral raw material 82 separated from racemic raw material 72 as a starting material.

[0665] [ka]

[0666] Chiral resolution of intermediate 72: Compound 72 (50g) was subjected to SFC resolution to obtain compound 81 (19g, retention time 13.55min) and compound 82 (20g, retention time 16.29min). SFC resolution method: Column model: DAIEL CHIRALCEL OD (250mm~50mm, 10um); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200mL / min; Detector: PDA; Column temperature: 25 o C; Back pressure: 100Bar; HPLC Method: Equipment Information: Thermo Liquid Chromatograph (ADC-U3000-01); Chromatography Column: CHIRALPAK® ID (4.6*150mm, 5μm); Column Temperature: 25℃; Sample Tray Temperature: 25℃; Mobile Phase: A: 10 mM NH4FA; B: ACN; Flow Rate: 0.8 mL / min; Detection Wavelength: 254 nm; Injection Volume: 2 μl

[0667] [Table 21]

[0668] X-ray analysis revealed the following structure and configuration of compound 82. See Figure 1 for its single-crystal diffraction pattern.

[0669] [ka]

[0670] Compound 82 (3632 mg, 8 mmol) and 90 mL of THF (commercial) were added to a reaction flask at 0°C. While stirring, DBU (1215 mg, 8 mmol) was slowly added, and the mixture was reacted for half an hour. The mixture was then transferred to room temperature. Once the starting materials disappeared under TLC monitoring, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 83 (1707 mg, 92% yield). MS(ESI): (M+H) + Calculated value: 233.1, experimental value: 233.2.

[0671] 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. DIEA (1158 μL, 7 mmol) was slowly added dropwise while stirring, and the mixture was reacted 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 to form a slurry. A white solid precipitated, and this process was repeated three times to obtain compound 84. MS(ESI): (M+H) +Calculated value: 554.3, experimental value: 554.4.

[0672] Compound 84 (1661 mg, 3 mmol), p-nitrophenyl chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask. Py (474 ​​μL, 6 mmol) was added dropwise while stirring, and 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.

[0673] Compound 85 (1436 mg, 2 mmol), exatecan mesylate 86 (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask. DIEA (695 μL, 4 mmol) was slowly added dropwise while stirring, and 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 87 (1664 mg, 82% yield). MS(ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.1.

[0674] Compound 87 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask at 0°C. While stirring, DBU (228 mg, 1.5 mmol) was slowly added, and the mixture was reacted for half an hour. The mixture was then transferred to room temperature and allowed to continue for 40 minutes until the starting materials disappeared by TLC. The reaction mixture was vacuum concentrated to remove THF, and 15 mL of DCM and 300 mL of PE were added to produce a large quantity of yellowish-green solid. Solid 88 (1177 mg, 99% yield) was filtered using a Buchner funnel. MS(ESI): (M+H) + Calculated value: 793.3, experimental value: 793.6.

[0675] Compound 88 (1031 mg, 1.3 mmol), Compound 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 with C18 (60% ACN / 0.05% formic acid in H2O). The mixture was then lyophilized to obtain Compound 89 (1322 mg, 90% yield). MS(ESI): (M+H) + Calculated value: 1130.4, experimental value: 1130.7.

[0676] 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, substituted three times with nitrogen, and THF / DMF / H2O (3.5 mL: 0.6 mL: 0.4 mL) was added. The mixture was reacted at 25°C for 0.5 hours, and the residue was separated and purified using C18 (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 (600 MHz, DMSO) δ 10.71 (s, 1H), 10.08 (s, 1H), 8.48 (d, J = 6.7 Hz, 1H), 8.17 (d, J = 8.6 Hz, 3H), 8.09 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.7 Hz, 2H), 7.89 (s, 1H), 7.81 (d, J = 10.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.42 - 7.36 (m, 3H), 6.61 (s, 1H), 6.02 (t, J = 6.8 Hz, 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 = 15.6 Hz, 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 = 7.0 Hz, 3H), 1.02 (d, J = 6.7 Hz, 3H), 0.98 (t, J = 7.2 Hz, 6H).

[0677] Compared with the HPLC method for LD55-1 in Example 13, the retention time of the LD55-1 product separated in this step is the same as that of LD55-1 in Example 13.

[0678] Example 15: Synthesis of LD2-1

[0679] [ka]

[0680] 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, substituted three times with nitrogen, and THF / DMF / H2O (1 mL: 0.2 mL: 0.2 mL) was added. The mixture was reacted at 25°C for 0.5 hours. The residue was separated and purified using C18 (60% ACN / 0.05% formic acid in H2O) to obtain Compound LD2-1 (199.7 mg, yield 89%). MS(ESI): (1 / 2 M+H) + Calculated value: 1123.0, experimental value: 1123.0.

[0681] Example 16: Synthesis of LD7-1

[0682] [ka]

[0683] 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, substituted three times with nitrogen, and THF / DMF / H2O (1 mL: 0.2 mL: 0.2 mL) was added. The mixture was reacted at 25°C for 0.5 hours, and the residue was separated and purified using C18 (55% ACN / 0.05% formic acid in H2O) to obtain Compound LD7-1 (180 mg, 93% yield). (1 / 2 M+H) + Calculated value: 971.4, experimental value: 971.5.

[0684] Example 17: Synthesis of LD12-1

[0685] [ka]

[0686] 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, substituted three times with nitrogen, and THF / DMF / H2O (1 mL: 0.2 mL: 0.2 mL) was added. The mixture was reacted at 25°C for 0.5 hours. The residue was separated and purified using C18 (55% ACN / 0.05% formic acid in H2O) to obtain Compound LD12-1 (229 mg, 98% yield). MS(ESI): (1 / 2 M+H) + Calculated value: 1170.0, experimental value: 1170.0.

[0687] Example 18: General method for preparing ADC samples and measuring DAR Antibodies with known sequences can be purified by first obtaining a fermentation solution using a protein expression method well known to those skilled in the art, and then subjecting it to steps such as affinity chromatography or ion chromatography to obtain a sample of higher purity.

[0688] The antibody sample was diluted to approximately 10 mg / mL with a suitable buffer (matching the sample buffer), and an appropriate amount of reducing agent TCEP was added, adjusting the number of equivalents according to the target DAR; for a target DAR of 8, 8 to 10 equivalents were added. Then, the pH was adjusted to 7 to 7.4 with Tris buffer, and reduction was carried out at room temperature for 1 to 1.5 hours. The intermediate state of antibody reduction could be monitored with CE-SDS. After the antibody was completely reduced, an appropriate amount of saturated citrate solution was added to adjust the pH to approximately 6.5, and then an excess amount of DMSO solution of linker-drug was added to bring the equivalent amount of linker-drug to 15 to 20 times the equivalent amount of antibody. The binding reaction was carried out at room temperature for approximately 30 minutes. After binding was complete, the reaction solution was first filtered, and then ultrafiltration was performed using a centrifugal concentrator tube to remove excess linker-drug and other small molecule impurities. After purification, the obtained sample was subjected to DAR measurement by reverse-phase chromatography.

[0689] Method for preparing T-ADC The trastuzumab concentration was adjusted to 10 g / L using phosphate buffer (pH 7.0). The antibody solution, after fluid exchange, was placed in a centrifuge tube, and 8.0 equivalents of 5 mM TCEP (Adamas Beta) were added. The mixture was reacted at room temperature for 1 hour to reduce the disulfide bonds between antibody chains to mercapto groups. Next, 20 mM linker-drug solution (15 equivalents of toxin per antibody) dissolved in DMSO was added. The mixture was reacted at 10°C for 1 hour. Residual linker toxin was removed from the conjugated solution using an ultrafiltration membrane package (Cobetter), and the solution was stored in PBS at pH 7.4. The average drug load of each antibody was measured by reverse-phase chromatography.

[0690] Preparation method for F-ADC and FS-ADC The concentration of farletuzumab or farletuzumabFcS antibody was adjusted to 10 g / L using phosphate buffer (pH 7.0). After liquid exchange, the antibody solution was placed in a centrifuge tube, and 7.0 equivalents of 5 mM TCEP (Adamas Beta) were added. The mixture was reacted at 37°C for 1 hour to reduce the disulfide bonds between antibody chains to mercapto groups. Next, 20 mM linker-drug (10 equivalents of toxin per antibody) dissolved in DMSO was added. The mixture was reacted at 10°C for 1 hour. Residual linker toxin was removed from the conjugated solution using an ultrafiltration tube, and the solution was stored in PBS at pH 7.4. The average drug load of each antibody was measured by reverse-phase chromatography.

[0691] Method for preparing D-ADC The concentration of antibody 33B was adjusted to 10 g / L using phosphate buffer (pH 7.0). After liquid exchange, the antibody solution was placed in a centrifuge tube, and 10.0 equivalents of 5 mM TCEP (Adamas Beta) were added. The mixture was reacted at 37°C for 1 hour to reduce the disulfide bonds between antibody chains to mercapto groups. Next, a 20 mM linker-drug solution (12 equivalents of toxin per antibody) dissolved in DMSO was added. The mixture was reacted at 25°C for 1 hour. The residual linker toxin was removed from the conjugated solution using an ultrafiltration membrane package (Cobetter), and the solution was stored in PBS at pH 7.4. The average drug load of each antibody was measured by reverse-phase chromatography.

[0692] Example B - Method for preparing ADC The concentration of antibody 43B was adjusted to 10 g / L using phosphate buffer (pH 7.0). After liquid exchange, the antibody solution was placed in a double-layer glass reaction vessel, and 12.0 equivalents of 100 mM TCEP (Adamas Beta) were added. The reaction was carried out at 37°C for 3 hours to reduce the disulfide bonds between antibody chains to mercapto groups. Next, 10 mM linker-drug solution (10 equivalents of toxin per antibody) dissolved in DMSO was added at room temperature. The reaction was carried out at 25°C for 30 minutes. The residual linker toxin was removed from the conjugated solution with protein A and stored in PBS at pH 7.4. The average drug load of each antibody was measured by reverse-phase chromatography.

[0693] By adjusting the usage of TCEP and linker-drug, it is possible to obtain ADCs with different Dar values.

[0694] The data statistics for all ADC samples are shown in the following table.

[0695] [Table 22]

[0696] TIFF2026516826000371.tif255165

[0697] TIFF2026516826000372.tif255167

[0698] TIFF2026516826000373.tif116169

[0699] Example 19: Preparation of control ADC samples ORM-5029 was obtained as a control for HER-2 ADCs by referring to the method in reference WO 2021 / 198965 A1. DAR was measured by reverse-phase chromatography and was 3.5. Furthermore, low-DAR samples of ADC38-ADC44 were obtained under the same binding conditions and named ADC38-4-ADC44-4, respectively. DAR values ​​measured by LC-MS were all 3.5±0.1. In particular, when the linker-drug used in ORM-5029 was bound to the antibody, the high aggregate ratio prevented the successful acquisition of qualified ADC samples with high Dar values ​​(>7). Enhertu is a commercially available product.

[0700] [ka]

[0701] BATLD was synthesized according to the method in reference CN116333135A. Using BATLD as a linker drug and Farletuzumab, 33B, and 43B as antibodies, F-BATADC, D-BATADC, and B-BATADC were synthesized according to the ADC preparation method described above. The DAR values ​​measured by reverse-phase chromatography were 7.9, 7.7, and 5.5, respectively.

[0702] [ka]

[0703] TIFF2026516826000376.tif92169

[0704] DSADC was obtained by following the method described in reference CN104755494A, and the DAR was measured by reverse-phase chromatography, resulting in a value of 3.9.

[0705] [ka]

[0706] The M30 antibody sequence is as follows:

[0707] >M30-H1 (SEQ ID NO:43) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >M30-L4 (SEQ ID NO: 44) EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Effect Example 1: In vitro activity test of ADC samples As test cells, SK-BR-3 cells highly expressing HER2 were selected. After culturing the cells according to standard procedures, they were inoculated into 96-well plates at an appropriate density. After confirming that the cells were in a normal state, the test sample was added, the concentration gradient was set to 0-100 nM, and after 144 hours of incubation, cell viability was measured using the CTG method, and IC was obtained. 50 The value was calculated.

[0708] [Table 23]

[0709] Note: In the table, A represents IC. 50 If the value is less than 15 pM, B is IC 50 When the value is between 15 pM and 25 pM, C is IC 50 The value is greater than 25 pM, and Enhertu is a commercially available product.

[0710] Activity test results showed that the ADC of the present invention exhibits significantly improved activity compared to ORM-5029, a similar ADC, and also significantly improved activity compared to Enhertu, which targets the same organism.

[0711] Effect Example 2: In vivo activity test of ADC samples JIMT-1 cells expressing HER2 at low or moderate levels were selected and inoculated into NUNU mice. After tumor formation, the tumor volume was approximately 150 mm². 3 Selected mice were divided into two groups of five. Various drugs were administered via tail vein injection at predetermined doses. After administration, tumor size and mouse body weight were regularly observed and measured, and the administration frequency was increased as appropriate. Once certain conditions were met, the experiment was terminated, and the final tumor size and mouse body weight were measured to calculate the tumor inhibition rate. The test results are shown in the table below.

[0712] [Table 24]

[0713] Here, T-ADCs were prepared according to the method described in reference WO 2021 / 198965 A1. First, the following linker drug compounds were prepared.

[0714] [ka]

[0715] Next, the antibody was bound to obtain the desired ADC.

[0716] [ka]

[0717] [Table 25]

[0718] Table 10 shows the in vivo efficacy results, indicating that, for ADCs using non-hydrophilic linkers, the effect of T-ADC was significantly superior to that of ORM-5029, and slightly lower than that of Enhertu, given the same linker. Table 11 shows the in vivo efficacy results, indicating that ADCs using hydrophilic linkers were more effective than Enhertu. Combining the results from Tables 10 and 11, it can be concluded that ADCs using hydrophilic linkers are significantly more effective than ADCs using non-hydrophilic linkers.

[0719] Effect Example 3: In vitro cytotoxicity of HER2-ADC In vitro cytotoxicity mediated by HER2-ADC was evaluated using the HER2-positive cell line SK-BR-3. Cells were isolated with trypsin, cultured in gradient-diluted HER2-ADC, and incubated at 37°C. Viability was measured after 6 days using CTL Plus. Reading and analysis were performed using EnVision 2105 (PerkinElmer), and IC50 was obtained. 50The (median inhibitory concentration) values ​​were determined. SK-BR-3 cells were adjusted to a cell density of 40,000 cells / mL in McCoy's 5A + 15% FBS, and 75 μL per well was inoculated into a 96-well plate (manufacturer: Corning, catalog number: 3599). The plate was placed in a Forma® Steri-Cycle® i160 CO2 incubator and incubated overnight at 37°C and 5% CO2. Nine concentration gradients were created by diluting HER2-ADC fivefold from 100 nM in the medium of the corresponding cells. Next, 75 μL was added to the cells per well. The blank control was the medium of the corresponding cells. Two duplicate wells were set up for each concentration. SK-BR-3 cells were cultured for 6 days in a Forma® Steri-Cycle® i160 CO2 incubator at 37°C and 5% CO2. Six days later, 50 μL of CTL Plus (manufacturer: Beyotime, catalog number: C0068XL) luminescence reagent was added and incubated at room temperature in the dark for 10 minutes. Chemiluminescence detection was performed using an EnVision 2105 microplate reader. Data analysis and matching: The blank control was used as a zero-killing control, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - experimental group / blank control group) × 100. Data processing and analysis were performed using GraphPad Prism. The results are shown in the table below. These results indicate that HER2-ADC exhibits high in vitro cytotoxicity against HER2-positive cell lines.

[0720] [Table 26]

[0721] Effect Example 4: Measurement of ADC hydrophilicity by hydrophobic interaction chromatography (HIC) 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)

[0722] [Table 27]

[0723] Highly hydrophilic linkers can overcome the problem of difficulty in binding many toxins due to low water solubility, and can also reduce aggregate formation during the binding process, lower the clearance rate of ADCs, and ensure better PK. The data in the table shows that different hydrophilic side chains can improve the hydrophilicity of ADCs to different degrees, with the naked antibody farletuzumab exhibiting the best hydrophilicity. The hydrophilicity of the ADCs of this invention is superior to that of the control F-BATADC in all cases.

[0724] Effect Example 5: Experimental process for measuring the binding activity of ADCs by enzyme-linked immunosorbent method 1. Antigen Coating: The FOLR1 antigen (manufacturer: Sino Biological, catalog number: 11241-H08H) was diluted to 0.1 ug / ml with a coating solution (0.05 mol / L carbonate buffer, pH 9.6), and 100 μl was added per well to an ELISA-specific polystyrene microplate (manufacturer: Corning, catalog number: 3590). The antigen was coated overnight at 4°C.

[0725] 2. Blocking: The coating solution in the microplate was discarded, the plate was washed three times with washing solution PBST (PBS at pH 7.4 containing 0.05% Tween-20 10 mM), 300 μl of 2% BSA was added per well, and the plate was blocked at 37°C for 1 hour.

[0726] 3. Washing: The microplate was washed three times with washing solution, two duplicate wells were placed for each sample, the ADC sample was added, and the plate was incubated at 37°C for 2 hours. For the ADC samples, the maximum concentration of F-ADC55-1-8 was 1000 nM, and the volume was 100 μL in each case, obtained by diluting with 2% BSA in 11 gradients.

[0727] 4. Secondary antibody: The FRα primary antibody dilution in the microplate was discarded, the plates were washed three times with washing solution, and the secondary antibody (goat anti-human IgG (L&H) secondary, HRP) was added to each well. The plates were incubated at 37°C for 1 hour.

[0728] 5. Color Development: The secondary antibody diluent was discarded, and the sample was washed six times with washing solution. Then, 100 μL of TMB color development solution was added. The color development time was approximately 6-8 minutes. After that, 100 μL of 2M dilute hydrochloric acid was added to stop the reaction.

[0729] 6. Plate reading: Once the reaction was complete, the OD450 absorbance was read using a microplate reader (manufacturer: BioTek, model: Synergy LX).

[0730] 7.EC 50 Calculation

[0731] [Table 28]

[0732] Effect Example 6: Pharmacodynamic evaluation in a mouse model of human oral cancer using KB xenograft. 10 FRα-positive cell lines from KB 7Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). The average tumor volume of the mice was approximately 100 mm². 3 (Groups 1-6) and approximately 145 mm 3 When the mice reached groups 7-8, they were randomly divided into groups and administered the drug by a single intravenous injection. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular 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 measured twice a week), eyes, fur, and other abnormalities. Tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0733] [Table 29]

[0734] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 30, respectively. c. Compare with the tumor volume of the vehicle control group.

[0735] [Table 30]

[0736] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 20, respectively. c. Compare with the tumor volume of the vehicle control group.

[0737] The efficacy of the ADC in the 3 mg / kg test group of the present invention was significantly superior to that of the control group F-BATADC (TGI 92.0%), with tumor inhibition rates (TGI) of 104.7%, 104.7%, 104.6%, and 104.6% in each group, respectively. Within these groups, 4, 4, 3, and 3 mice achieved complete response (CR). Furthermore, there was no significant difference in the efficacy of ADCs using linkers with different chiral configurations. Even when the dose of F-ADC55-1-8 was reduced to 1.5 mg / kg, the tumor inhibition rate (TGI) still reached 105.6%. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. During the treatment period, mice showed good tolerability to the ADC of the present invention. Specific results are shown in the table.

[0738] Effect Example 7: Pharmacodynamic evaluation of test drug in a mouse model of human lung adenocarcinoma NCI-H441 xenograft tumor. 10 FRα-positive cell lines from NCI-H441 7 Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). The average tumor volume of the mice was approximately 100 mm². 3 At the point when the mice reached the required stage (groups 1-6), they were randomly divided into groups and administered the drug by a single intravenous injection. The group division day was set as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, this included 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0739] [Table 31]

[0740] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 32, respectively. c. Compare with the tumor volume of the vehicle control group.

[0741] The 1 mg / kg ADC in the present invention significantly inhibited tumor growth in the NCI-H441 model compared to the vehicle control group, and its tumor inhibitory effect was significantly superior to that of the control group F-BATADC. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. During the treatment period, mice showed good tolerability to the ADC of the present invention. Specific results are shown in the table.

[0742] Effect Example 8: Pharmacodynamic evaluation of test drug in a human colon cancer SW620 xenograft mouse model. FRα-positive cell line SW620 to 10 7 Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (6 mice per group). The average tumor volume of the mice was approximately 130 mm². 3 At the point when the mice reached the required stage (groups 1-2), they were randomly divided into groups and administered the drug by a single intravenous injection. The group change day was set as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, this included 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0743] [Table 32]

[0744] Compared to the vehicle control group, the 1.5 mg / kg ADC in the SW620 model significantly inhibited tumor growth. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. During the treatment period, mice showed good tolerability to the ADC of the present invention. Specific results are shown in the table.

[0745] Effect Example 9: Pharmacodynamic evaluation of test drug in a mouse model of human ovarian cancer (OV-90) xenograft tumor. FRα-positive cell line OV-90 to 10 7 Cells were collected and inoculated into the right forelimb axilla of 4-6 week old NCG and NOD SCID immunodeficient mice (3 mice per group). The average tumor volume of the mice was approximately 180 mm². 3 (Groups 1-3) and approximately 145 mm 3 When the mice reached groups 4-5, they were randomly divided into groups and administered drugs. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, this included 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0746] [Table 33]

[0747] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 11, respectively. c. Comparison with tumor volume of the vehicle control group, d. NCG immunodeficient mice e. NOD SCID immunodeficient mice The ADC of the present invention significantly inhibited tumor growth in the OV-90 model in the 1 / 3 / 5 mg / kg groups compared to the control group, with tumor growth inhibition rates (TGI) of 110.9%, 126.1%, and 120.7%, respectively. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. Mice showed good tolerability to the ADC of the present invention during the treatment period. Specific results are shown in the table.

[0748] Effect Example 10: Measurement of the positive rate of DLL3-expressing cells by flow cytometry The binding of DLL3 antibodies to surface DLL3 in NCI-H209, SHP77, NCI-H526, NCI-H82, and NCI-H69 cells was measured by flow cytometry.

[0749] Experimental process: Collect NCI-H209, SHP77, NCI-H526, NCI-H82, and NCI-H69 cells and inoculate them in FACS buffer (pH 7.4 PBS + 2% FBS) in 5 × 10⁶ solutions. 5A cell suspension was prepared, and 100 μL was added to each well of a 96-well round-bottom plate (manufacturer: Corning, catalog number: 3795). After centrifugation and removal of the supernatant, 100 μL / well of various concentrations of D-ADC55-1-8 diluted in FACS buffer was added (final concentration 15 μg / ml), and the mixture was incubated in the dark at 4°C for 2 hours. After centrifugation three times in FACS buffer, the goat anti-human IgG(H+L) cross-adsorbed secondary antibody Alexa Fluor® 488 (manufacturer: Invitrogen, catalog number: A11013) was added at working concentration, and the mixture was incubated in the dark at 4°C for 1 hour. Geometric mean fluorescence intensity was detected using a CytoFLEX flow cytometer, and DLL3 + The percentage was calculated.

[0750] [Table 34]

[0751] Effect Example 11: Experiment to measure the binding activity of DLL3-ADC by enzyme-linked immunosorbent assay. The affinity of the antibody to recombinant human DLL3 protein was measured using the ELISA method. Human DLL3 protein (Kactus Biosystems, catalog number: DLL-HM103) was diluted to a final concentration of 0.1 μg / ml, spread on a 96-well ELISA plate, and allowed to stand overnight at 4°C. The next day, the supernatant was discarded, and after blocking was complete, a 4-fold gradient diluted antibody (0-100 nM) was added. After incubation with the secondary antibody, the mixture was developed with TMB chromogenic solution for 10 minutes. After stopping the reaction by adding 2 M HCl, the absorbance at 450 nm was read using a microplate reader, and the EC was measured. 50 The following was calculated. From the results shown in the table, the affinity of ADC for human DLL3 protein was approximately equivalent to the binding ability of the unbound antibody.

[0752] [Table 35]

[0753] Effect Example 12: In vitro cytotoxicity of DLL3-ADC In vitro cytotoxicity mediated by D-ADC55-1-8 was evaluated using DLL3-positive cell lines NCI-H82, SHP77, NCI-H526, and NCI-H209, and DLL3-negative cell lines A431 and NCI-H69. Cells were harvested, cultured in gradient-diluted D-ADC55-1-8, and incubated at 37°C. Viability was measured after 6 days using CTL Plus. Readouts were performed on EnVision 2105 (PerkinElmer), analyzed, and IC50 was obtained. 50 The (median inhibitory concentration) value was determined.

[0754] Experimental process: The cell density of the target cells was set to 6.7 × 10⁻⁶. 4 The solution was adjusted to 1 / mL, and 75 μL was inoculated into each well of a 96-well plate. The cells were incubated overnight in a 37°C, 5% CO2 incubator. A 5-fold gradient dilution of D-ADC55-1-8 (0-100 nM) was added to create a total of nine concentration gradients, with 75 μL added to each well. The blank control was the culture medium for the corresponding cells. Two duplicate wells were set up for each concentration. After culturing the cells in a 37°C, 5% CO2 incubator for 6 days, 50 μL of CTL Plus (manufacturer: Beyotime, catalog number: C0068XL) luminescence reagent was added, and the cells were incubated at room temperature in the dark for 10 minutes. Chemiluminescence detection was performed using an EnVision 2105 microplate reader. Data analysis and matching: The blank control was used as a zero-killing control, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - test group / blank control group) × 100%. Data processing and analysis were performed using GraphPad Prism, and IC 50 The following was calculated. The results are shown in the table. The results indicate that D-ADC55-1-8 showed excellent in vitro cytotoxicity against DLL3-positive cell lines.

[0755] [Table 36]

[0756] Effect Example 13: Pharmacodynamic evaluation of test drug in a mouse model of human small cell lung cancer (SHP77) xenograft tumor. DLL3-positive cell line SHP77 to 10 7 Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (7 mice per group). The average tumor volume of the mice was approximately 100 mm². 3 Once the mice reached groups 1-6, they were randomly divided into groups and administered the drug by a single intravenous injection. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, this included 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0757] [Table 37]

[0758] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 22, respectively. c. Compare with the tumor volume of the vehicle control group.

[0759] The ADCs of the present invention significantly inhibited tumor growth in all SHP-77 models at a dose of 1 mg / kg compared to the vehicle control group, with tumor growth inhibition rates (TGI) of 98.1%, 96.6%, 97.1%, and 97.3%, respectively, which were significantly superior to the control group D-BATADC (TGI 79.9%). No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. Mice showed good tolerability to the ADCs of the present invention during the treatment period. Specific results are shown in the table.

[0760] Effect Example 14: Pharmacodynamic evaluation of test drug in a mouse model of human small cell lung cancer NCI-H526 xenograft tumor. 10 DLL3-positive cell lines from NCI-H526 7 Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (3 mice per group). The average tumor volume of the mice was approximately 160 mm². 3 Once the mice reached groups 1-2, they were randomly divided into groups and administered the drug by a single intravenous injection. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specifically, this included 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0761] [Table 38]

[0762] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 21, respectively.

[0763] Effect Example 15: Pharmacodynamic evaluation of test drug in a mouse model of human small cell lung cancer NCI-H69 xenograft tumor. 10 DLL3-positive cell lines from NCI-H69 7 Individual cells were collected and inoculated into the right forelimb axilla of nu / nu nude mice (3 mice per group). The average tumor volume of the mice was approximately 140 mm². 3 Once the target was reached, the mice were randomly divided into groups and administered the drug by a single intravenous injection. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals, specifically 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 (Here, a is the major axis and b is the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0764] [Table 39]

[0765] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 21, respectively.

[0766] 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 to the vehicle control group, with tumor growth inhibition rates (TGI) of 113.0% and 76.5%, respectively. No animal deaths or significant weight loss were observed in either treatment group, and no obvious drug toxicity reactions were observed. Mice showed good tolerability to the ADC of the present invention during the treatment period. Specific results are shown in the table.

[0767] Effect Example 16: FACS measurement of ADC affinity The affinity for B-ADC55-1-6, B-ADC2-1-6, B-ADC12-1-6, and DSADC was detected using the B7H3-positive human melanoma cell line A375. Samples were diluted to 15,000 ng / mL in PBS solution containing 2% BSA, and then serially diluted in 11 gradients to obtain sample concentrations ranging from 0.014 to 15,000 ng / mL. A375 cells were harvested, centrifuged at 500×g for 5 minutes, washed three times in PBS solution containing 2% BSA, incubated with samples of different dilutions at 4°C for 2 hours, washed, and incubated with goat anti-human Alexa Fluor488 fluorescent dye at 4°C in the dark for 1 hour. After washing and resuspending, the mean fluorescence intensity (MFI) of the cells was detected using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.).

[0768] [Table 40]

[0769] The above data shows that the affinity of the present invention's ADC for the B7H3 antigen on the A375 surface is significantly superior to that of the control group's DSADC.

[0770] Effect Example 17: Pharmacodynamic evaluation of test drug in a mouse model of human liver cancer Hep3B xenograft tumor. From the B7H3-positive cell line Hep3B, 10 7 Individual cells were collected and inoculated into the right forelimb axilla of 4-6 week old nu / nu nude mice (6 mice per group). The average tumor volume of the mice was approximately 150 mm². 3 Once the target was reached, the mice were randomly divided into groups and administered the drug by a single injection. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals, specifically 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 (Here, a is the major axis and b is the minor axis).

[0771] [Table 41]

[0772] 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 treatment group and vehicle control group on day 0, respectively, and Ti and Ci are the mean tumor volumes of the treatment group and vehicle control group on day 22, respectively. c. Compare with the tumor volume of the vehicle control group.

[0773] The ADC 2 mg / kg group of the present invention significantly inhibited tumor growth in all Hep3B models compared to the vehicle control group, with the experimental group being significantly superior to the control group. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity reactions were observed. During the treatment period, mice showed good tolerability to the ADC of the present invention. Specific results are shown in the table.

[0774] Effect Example 18: Pharmacodynamic evaluation of test drug in a mouse model of human malignant melanoma cell A375 xenograft tumor. From B7H3-positive cell line A375 to 107 Individual cells were collected and inoculated into the right forelimb axilla of 4-6 week old nu / nu nude mice (7 mice per group). The average tumor volume of the mice was approximately 130 mm². 3 Once the target was reached, the mice were randomly divided into groups and administered the drug. The day of group division was designated as day 0, and drug administration began on day 0. After tumor inoculation, regular monitoring included tumor growth and the effects of treatment on the normal behavior of the animals, specifically 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 (Here, a is the major axis and b is the minor axis).

[0775] [Table 42]

[0776] [Table 43]

[0777] Note: a. Data are expressed as "mean ± standard error". b. Compared to tumor volume in the vehicle control group.

[0778] The data in the table shows that the average tumor volume in the vehicle control group on day 22 was 3000 mm². 3 This indicates that the condition was reached. Euthanasia was performed based on animal welfare. On day 26, the efficacy of the control group was significantly inferior to that of the test group at the same dose. Compared to the vehicle control group, all test groups showed significant tumor inhibitory effects.

[0779] Effect Example 19: Antitumor experiment of antibody-drug conjugate against human lung cancer tumors transplanted into Calu-6 mice In this experiment, NOD / SCID mice of appropriate age were inoculated with human lung cancer cells Calu-6. The tumor volume was approximately 100-200 mm². 3At the point of tumor growth, mice with favorable tumor development were selected and divided into groups according to tumor volume. The grouping and administration plan for the animals are shown in the table below.

[0780] [Table 44]

[0781] After dividing the mice into groups, drugs were administered, their body weight was measured, and the data was recorded. The diameter of the tumors was measured at different time points after drug administration to dynamically observe tumor growth, and the tumor volume was calculated using the following formula.

[0782] Tumor volume (mm 3 ) = 1 / 2 × major axis (mm) × minor axis (mm) 2 At the experimental endpoint, the test group was able to significantly inhibit tumor growth compared to the vehicle control group.

[0783] [Table 45]

[0784] Effect Example 20: Measurement of enzyme digestion rate 1. Activation of CTB: 10 units of lyophilized cathepsin B powder were diluted to a 10 UN / mL solution with the corresponding stock solution (pH=5.0, 25 mM acetate buffer + 1.0 mM EDTA) to a total volume of 1 mL. This solution was then dispensed into 50 μL / tube and stored at -80°C. For the enzyme digestion reaction, 50 μL of CTB (10 UN / mL) stock solution was taken, 860 μL of pH=5.0 buffer was added, and 100 μL of Cys (200 mM) activating solution was added. The solution was activated for 15 minutes. The total volume was adjusted to 1.0 mL to obtain a CTB activated solution with a final concentration of 0.5 UN / mL.

[0785] 2. Blocking of the substrate mother liquor: 5 μL of DMSO solutions of 10 mM LD55-1, LD55-2, LD2-1, and LD2-2 were taken, 5 μL of 100 mM (5.0 equivalent) cysteine ​​(Cys) aqueous solution was added, and the mixture was homogeneously mixed. A blocking reaction was carried out for 5 to 10 minutes to obtain the blocked substrate mother liquors Cys-LD55-1, Cys-LD55-2, Cys-LD2-1, and Cys-LD2-2, with a substrate concentration of 5 mM per 10 μL.

[0786] 3. Progression of the enzymatic digestion reaction: The final substrate concentration was 40 μM. 2.0 μL of 5 mM substrate blocking solution was measured, and 250 μL of 0.5 UN / mL CTB solution was added to make a total volume of 0.25 mL. The solution was incubated at 37°C, and samples were taken at 30-minute intervals for up to 24 hours for detection.

[0787] 4. Sample detection: When the reaction time arrived, an appropriate amount of sample was taken, diluted with an equal volume of ice-cold MeOH (HPLC grade), and precipitated. Then, the mixture was centrifuged at 10,000 rpm or higher at 4°C for 10 minutes, and the supernatant was collected and detected.

[0788] 5. Data Results The drug peak area detected without adding enzyme to the drug was set to 1, and the percentage obtained by comparing this to the drug peak area detected when a sample was taken at the corresponding time point was defined as the drug release rate at that time point. Table 28 shows the remaining amount of Cys-Linker-drug after enzymatic cleavage of Cys-LD55-1, Cys-LD55-2, Cys-LD2-1, and Cys-LD2-2.

[0789] [Table 46]

[0790] From the enzyme digestion curves, it was found that the arrangements of Cys-LD55-1, Cys-55-2, Cys-LD2-1, and Cys-LD2-2 can all be completely digested by enzymes. Cys-LD55-1 and Cys-LD2-1 are completely digested in about 30-60 minutes, while Cys-LD55-2 and Cys-LD2-2 are completely digested in about 24 hours.

[0791] Effect Example 21: Evaluation of the toxicological kinetics and tolerability of ADC in cynomolgus monkeys Two female cynomolgus monkeys were selected and intravenously administered ADC (5 mg / mL, 30-minute injection) at doses of 10–50 mg / kg every 2–3 weeks (total of 3 doses). Parameters evaluated during the study period included general observation, body weight, food intake, body temperature, electrocardiogram (lead II), clinicopathological examination (hematology, blood biochemistry, coagulation), and macroscopic and microscopic examination of numerous tissues. Toxicological samples were collected at 0, 24, 72, 120, 336, and 504 hours after each administration. TK samples were analyzed using a MesoScale Discovery (MSD) electrochemiluminescence platform (total monoclonal antibodies, total ADC) and LC-MS / MS (free exotecan). The experimental results demonstrated that the ADC of the present invention was well-tolerated. In dose consideration, general observation of cynomolgus monkeys after ADC injection was normal, and no toxicological abnormalities were observed in body weight, body temperature, coagulation, or urinalysis. The ADC of the present invention was well-tolerated and exhibited stable pharmacokinetic properties in cynomolgus monkeys. [Brief explanation of the drawing]

[0792] [Figure 1] This is the single-crystal diffraction pattern of compound 82.

Claims

1. Antibody-drug conjugates having the structure of formula (I), and their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds. A-(L-D) ｍ (I) (Here, A is a targeted ligand selected from antibodies (e.g., monoclonal antibodies), antigen-binding fragments, small molecule ligands, polypeptides, L is a linker portion, with one end linked to ligand A and the other end linked to the biologically active molecule D. D is a biologically active molecule containing an amino group or its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, or isotope-labeled compounds, which are covalently bonded to the linker moiety L via the amino group in its molecular structure. m is an integer or decimal number between 1 and 12. The linker portion -L- is given by the following formula: -8 １ -8 ２ -8 ３ -8 ４ - Here, L １ This is the portion of L that is linked to ligand A, and preferably L １ teeth, 【Chemistry 1】 (or its open ring form) 【Chemistry 2】 ）、 【Transformation 3】 Selected from, where * indicates that it is linked to the mercapto group of A (e.g., monoclonal antibody), and ** indicates L ２ This indicates that it is connected to, L ２ is a spacer, preferably, -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 -SO ２ -NH-C(O)-O-L ２ｂ -C(O)-, or -L ２ａ -NR １ -SO ２ -NH-C(O)-O-L ２ｂ is selected from -NH-C(O)-, where L ２ａ is -C １ ~C ８ alkylene-, -C １ ~C ８ alkylene-C ３ ~C ８ cycloalkylene-, -C ６ ~C １４ arylene-, -C ６ ~C １４ arylene-C １ ~C ８ alkylene-, -5- to 6-membered heteroarylene-, -5- to 6-membered heteroarylene-C １ ~C ８ Selected from alkylene-, a linear or branched heteroalkylene group having 1 to 50 atoms, and a linear or branched heteroalkylene-3 to 8-membered heterocyclene group having 1 to 50 atoms, wherein the alkylene group, cycloalkylene group, arylene group, heteroalkylene group, heteroarylene group, and heterocyclene group are each C １ ~C ６ Alkyl alkyl groups, heteroalkyl groups having 1 to 6 atoms, C １ ~C ６ Alkoxy group, hydroxyl group, amino group, carboxyl group, or C ３ ~C ８ The cycloalkyl group is optionally substituted with one or more substituents independently selected from the cycloalkyl group, wherein the heteroalkylene group, heterocyclylene group, and heteroalkyl group contain 1 to 12 heteroatoms, and the heteroatoms of the heteroalkylene group, heterocyclylene group, heteroarylene group, and heteroalkyl group are one or more selected from N, O, or S, and L ２ｂ is, -C １ ~C ８ Selected from alkylene-, a linear or branched heteroalkylene group having 1 to 50 atoms, R １ is hydrogen, C １ ~C ６ alkyl group, C ３ ~C ８ Cycloalkyl group, 3-8 membered heterocyclyl group, C １ ~C ６ Haloalkyl groups, heteroalkyl groups having 2 to 8 atoms, C ６ ~C １４ Selected from aryl groups and 5-6 membered heteroaryl groups, the alkyl group, heteroalkyl group, aryl group, and heteroaryl group are each C １ ~C ６ Alkyl alkyl groups, heteroalkyl groups having 2 to 6 atoms, C １ ~C ６ The heteroalkyl group, heteroalkylene group is optionally substituted with one or more substituents independently selected from an alkoxy group, an amino group, or a carboxyl group, and the heteroalkyl group contains 1 to 12 heteroatoms, and the heteroatoms of the heteroalkyl group, heterocyclyl group, heteroaryl group, heteroalkylene group, or heteroarylene group are one or more selected from N, O, or S. L ３ This is a polypeptide sequence, preferably selected from peptide residues consisting of 2 to 8 natural or non-natural amino acids, where the amino acids are further C １ ~C ６ Alkyl alkyl groups, heteroalkyl groups having 2 to 6 atoms, C １ ~C ６ Alkoxy group, hydroxyl group, amino group, carboxyl group, or C ３ ~C ８ Optionally substituted with one or more substituents selected from cycloalkyl groups, L ４ It is a self-cleaving fragment modified with a hydrophilic group, The self-cleaving fragment is selected from the following: 【Chemistry 4】 Here, * is L via an amide bond. ３ This indicates that it is linked to the carboxyl group of , ** indicates that it is linked to the amino group of biologically active molecule D, and X indicates that it is not present or 【Transformation 5】 Here, *** indicates bonding to a carbon atom, **** indicates bonding to an oxygen atom, and the arrow indicates the hydrophilic group modification site. More preferably, the self-cleaving fragment is selected from the following structures before being modified with a hydrophilic group: 【Transformation 6】 Here, Y is C １ ~C ６ Alkylene group, or -R ３ -C(O)-, where R ３ C １ ~C ６ Alkylene group, or 1 to 8 -OCH groups ２ CH ２ - A heteroalkylene group containing a structural unit, where n is an integer from 0 to 6. The hydrophilic group has at least one azide group and includes a polyethylene glycol group, a polynatural amino acid or unnatural amino acid group, a monosaccharide, an oligosaccharide or polysaccharide, or a combination of the above groups. Preferably, L ４ The following can be selected: 【Transformation 7】 Here, * represents being linked to the carboxy group of L via an amide bond, ** represents being linked to the amino group of the biologically active molecule D, ３ and R ２ These are hydrophilic fragments, preferably 4 to 50 (OCH) ２ CH ２ Selected from linear or branched heteroalkyl groups containing structural units, peptide chains containing 4 to 50 protein amino acids (e.g., glycine) or non-protein amino acids (e.g., sarcosine), monosaccharides, oligosaccharides, or polysaccharides, where X is absent or 【Transformation 8】 wherein *** represents being linked to a carbon atom, **** represents being linked to an oxygen atom, and Y is C １ -C ６ an alkylene group, or -R ３ -C(O)-, wherein R ３ is C １ -C ６ an alkylene group, or a heteroalkylene group containing 1 to 8 -OCH ２ CH ２ -structural units, and n is an integer from 0 to 6 D is selected from the compounds of the following formula. 【Chemistry 9】

2. A is HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, DLL3, TROP2, B7H3, c-Met, CD20, CD22, CD3 0, CD33, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis An antibody-drug conjugate having the structure of formula (I) as described in claim 1, selected from antibodies or antigen-binding fragments thereof that target Y, VEGFR, VEGF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, FRα, or Mesothelin, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds.

3. L ２ The following are antibody-drug conjugates having the formula (I) structure described in any one of claims 1 to 2, selected from the structures below, and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. Table 1 (Here, w is selected from integers between 1 and 12, and * is L) １ This indicates that it is connected to L ３ (This indicates that it will be connected to.)

4. L ３ The following are antibody-drug conjugates having the structure of formula (I) described in any one of claims 1 to 3, selected from the structures below, and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 10】 (Here, * is L ２ This indicates that it is connected to L ４ (This indicates that it will be connected to.)

5. L ４ The following are antibody-drug conjugates having the structure of formula (I) described in any one of claims 1 to 4, selected from the structures below, and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 11】 (Here, v is selected from an integer between 1 and 12, and * is L via an amide bond) ３ (This indicates that it is linked to the carboxyl group, and ** indicates that it is linked to the amino group of the biologically active molecule D.)

6. L ４ The following are antibody-drug conjugates having the structure of formula (I) described in any one of claims 1 to 5, selected from the structures below, and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 12】

7. R ２ The following are antibody-drug conjugates having the formula (I) structure described in any one of claims 1 to 6, selected from the structures below, and tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 13】 (Here, r, s, t, and u are each independently selected from integers between 1 and 50 (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), R ａ C １ ~C ３ An alkylene group, for example, selected from methylene group, ethylene group, n-propylene group, isopropylene group, R ｂ C １ ~C ３ Alkyl groups are selected from, for example, methyl, ethyl, n-propyl, and isopropyl groups.

8. L is an antibody-drug conjugate having the formula (I) structure described in any one of claims 1 to 7, selected from the structures described below, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds. 【Chemistry 14】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 (Here, * indicates linkage to the mercapto group of antibody A, and ** indicates linkage to the amino group of biologically active molecule D.)

9. A is an antibody-drug conjugate having the structure of formula (I) according to any one of claims 1 to 8, wherein A is an antibody or antigen-binding fragment targeting FRα, DLL3, B7H3, or HER2, and a tautomer, meso, racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate, or isotope-labeled thereof.

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

11. A is an antibody or antigen-binding fragment that targets FRα, wherein the anti-FRα antibody or antigen-binding fragment 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, or A is an antibody or antigen-binding fragment that targets DLL3, wherein the anti-DLL3 antibody or antigen-binding fragment 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, or A is an antibody or antigen-binding fragment that targets B7H3, wherein the anti-B7H3 antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, 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, or A is an antibody targeting HER2 or an antigen-binding fragment thereof, wherein the anti-HER2 antibody or antigen-binding fragment 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, and the antibody-drug conjugate having the structure of formula (I) as described in any one of claims 1 to 10, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products.

12. A is an antibody or antigen-binding fragment that targets 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, or A is an antibody or antigen-binding fragment that targets 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, or A is an antibody or antigen-binding fragment that targets B7H3, wherein the anti-B7H3 antibody comprises a heavy chain and / or a light chain, 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 being shown in SEQ ID NO: 33, and the amino acid sequence of the light chain being shown in SEQ ID NO: 34, and an antibody-drug conjugate having the formula (I) structure described in any one of claims 1 to 11, and a tautomer, meso, racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate, or isotope-labeled thereof.

13. An antibody-drug conjugate having the formula (I) structure described in any one of claims 1 to 12, selected from the structures below, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds. Table 2 (Here, m is an integer or decimal between 1 and 12, preferably an integer or decimal between 2 and 8, and more preferably 2, 4, 6, or 8.)

14. A pharmaceutical composition comprising an antibody-drug conjugate having the structure of formula (I) as described in any one of claims 1 to 13, a tautomer, meso, racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate, or isotope-labeled thereof, and one or more pharmaceutically acceptable auxiliary materials.

15. Use in the preparation of cancer therapeutic agents of an antibody-drug conjugate having the formula (I) structure described in any one of claims 1 to 13, and its tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds, or the pharmaceutical composition described in claim 14.

16. The use according to claim 15, wherein 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, hematological cancer or glioblastoma multiforme, 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 lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

17. Linker-drug compounds represented by formula (II) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotopically labeled products thereof. L'-D (II) (Here, L' is the linker part, D is a biologically active molecule containing an amino group or a pharmaceutically acceptable salt thereof, which is covalently bonded to the linker moiety L' via the amine group of its molecular structure. L' is shown in the following equation: L １ '-L ２ -8 ３ -8 ４ - Here, L １ 'teeth, 【Chemistry 15】 Selected from, where ** is L ２ This indicates that it is connected to, L ２ , L ３ , L ４ (D is equivalent to claims 1 to 8.)

18. Compounds of formula (III) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 16】 (III) Formula (III) has the following structure, 【Chemistry 17】 Here, X is an existence assault, or [Chemistry 18] And *** represents being bonded to a carbon atom, **** represents being bonded to an oxygen atom, and Y is C １ ~C ６ Alkylene group, or -R ３ -C(O)-, where R ３ C １ ~C ６ Alkylene group, or 1 to 8 -OCH groups ２ CH ２ (—A heteroalkylene group containing a structural unit, where n is an integer from 0 to 6.)

19. Compounds of formula (IV) or their tautomers, meso compounds, racemic compounds, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled compounds thereof. 【Chemistry 19】 (Here, X does not exist, or 【Chemistry 20】 And *** represents being bonded to a carbon atom, **** represents being bonded to an oxygen atom, and Y is C １ ~C ６ Alkylene group, or -R ３ -C(O)- represents, where R ３ C １ ~C ６ Alkylene group, or 1 to 8 -OCH groups ２ CH ２ - A heteroalkylene group containing a structural unit, where n is an integer from 0 to 6. Here, AA is L ３ or L ３ A subfragment consisting of the first, second, or more amino acids from the C-terminus, P Ｎ is H or an amino protecting group, for example, Boc or Fmoc, and 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 the following: 【Chemistry 21】

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