Ecteinascidin compound, conjugate thereof, preparation method therefor, and use thereof

Abstract

Disclosed are an ecteinascidin compound, a conjugate thereof, a preparation method therefor, and use thereof. Specifically, disclosed is a ligand-drug conjugate comprising a ligand and a structure represented by formula Ib. The ligand-drug conjugate possesses good inhibitory activity on the proliferation of tumor cells or has a good in vivo anti-tumor effect.

Description

Sea tunicate compounds, their conjugates, their preparation methods and uses

[0001] This application claims priority to Chinese Patent Application No. 2024115960267, filed November 8, 2024, and Chinese Patent Application No. 2025110882340, filed August 4, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to a sea tunicate compound, its coupling compound, its preparation method, and its uses. Background Technology

[0003] Ecteinascidin compounds are alkaloids that exert anti-tumor effects through unique and diverse mechanisms of action: 1. Ecteinascidin compounds can inhibit cell division by binding to the DNA minor groove, affecting the cell cycle and transcription factor expression, leading to tumor cell apoptosis. 2. Ecteinascidin compounds can also act as tumor microenvironment modulators, activating the tumor immune system by regulating immune checkpoints, thus playing an immunotherapeutic role in tumors. 3. Low doses of ecteinascidin compounds can also inhibit the production of many inflammatory cytokines and chemokines, such as CCL2, CXCL8, IL6, and VEGF.

[0004] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxic drugs via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxic drugs, while avoiding the drawbacks of low efficacy of the former and excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce their impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814), thus exhibiting greater efficacy and a wider therapeutic window.

[0005] By leveraging the differences in surface antigen expression between tumor cells and normal cells, antibody-drug conjugates can be constructed by combining sea tunicate compounds with antibodies that bind to tumor cell surface antigens. This not only allows for the utilization of the clinical efficacy and tolerability of sea tunicate compounds but also expands their potential therapeutic range, such as for the treatment of breast cancer, lung cancer, liver cancer, gastric cancer, and prostate cancer, thereby broadening their application scope and providing patients with more options.

[0006] Therefore, it is necessary to develop new seasorghum ligand conjugates for the treatment of tumor-related diseases. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the deficiency of the limited variety of existing antibody-drug conjugates, thereby providing a tunicate compound, its conjugate, its preparation method, and its uses. The tunicate compound and its conjugate of this invention have one or more effects selected from the group consisting of: (1) inhibitory activity against the in vitro proliferation of tumor cells; (2) plasma stability; (3) in vivo tumor-suppressive effect; (4) anti-transporter transport capability; (5) in vivo tumor-targeting capability; and (6) good in vivo safety.

[0008] This invention provides a ligand-drug conjugate, its tautomers, its enantiomers, its diastereomers, its pharmaceutically acceptable salts, or solvates thereof, wherein the ligand-drug conjugate comprises a ligand with a structure shown in formula Ia, Ib, Ic, or Id:

[0009] in,

[0010] R 1a R 5a R 2a and R 3a Each independently selected from -VC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-(4- to 8-membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-OU-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-,-VC 0-6 Alkylene-N(R) 6 C(O)-(4-8 membered heterocyclic alkylene)-U-,-VC 0-6 Alkylene-N(R) 6 )C(O)OC 0-6 Alkylene-U-, -VC 0-6 Alkylene-OC(O)N(R) 6 )-C0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)OC 3-8 Cycloalkyl-U-,-VC 0-6 Alkylene-N(R) 6 C(O)O-(4-8 membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 3-8 Cycloalkyl-U-,-VC 0- 6-alkylene-N(R) 6 )C(O)N(R 6 )-(4- to 8-membered heterocyclic alkylene)-U-, -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 1-6 Alkylene-U-, -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 3-8 Cycloalkylene-U-, -V-(4- to 8-membered heterocycloalkylene)-C(O)-(4- to 8-membered heterocycloalkylene)-U-, -VC 3-8 Cycloalkyl-N(R) 6 )C(O)-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-,-VC 3-8 Cycloalkyl-N(R) 6 C(O)-(4-8 membered heterocyclic alkylene)-U-,-VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-C 3-8 Cycloalkyl-U,-VC 0-6 Alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 S(O)2-C 0-6 Alkylene-U- and -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 alkylene-U-; the C0-6 Alkylene, C 3-8 The cycloalkyl group and the 4- to 8-membered heteroalkyl group are each optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 2- 6-olefin, C 2-6 alkynyl, halogenated C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups;

[0011] U and V are each independently selected from -O-, -S-, and -NR-. 6 -or chemical bonds;

[0012] R 6 It is hydrogen, deuterium, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 4- to 8-membered heterocyclic alkyl; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 The cycloalkyl group and the 4- to 8-membered heterocycloalkyl group are each optionally separated by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, halogenated C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups;

[0013] R 4 For hydrogen, deuterium, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, -C(O)C 1-6 Alkyl, -C(O)C 3-12 Cycloalkyl or -C(O) (4- to 8-membered heterocycloalkyl), wherein the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 The cycloalkyl group and the 4- to 12-membered heterocycloalkyl group are each optionally separated by one or more elements selected from halogen, -OH, -SH, -NH2, -NHC. 1-6 Alkyl and -SC 1-6 Alkyl substituents;

[0014] Or R4 R 3a R on 6 The group and the atoms attached thereto form a 5- to 8-membered heterocyclic alkyl group; each of the 5- to 8-membered heterocyclic alkyl groups is optionally bonded by one or more groups selected from halogens, oxo groups, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, -C 1-6 Alkylene -OH, -C(O)C 1-6 Alkylene -OH, -C(O)-C 3-12 Substitution of cyclohexene alkyl-OH and -C(O)-(4- to 8-membered heterocyclohexene alkyl)-OH groups;

[0015] R 1b Hydrogen, deuterium, halogen, -OH, -NH2, -SH, C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -SC 1-6 Alkyl, C 3-6 cycloalkyl, -OC 3-6 cycloalkyl, -NHC 3-6 Cycloalkyl, -NH (4-6 membered heterocycloalkyl), -NHC(O)C 1-6 Alkyl or -NHS(O)2C 1-6 Alkyl, the C 1-6 Alkyl, C 3-6 The cycloalkyl group and the 4- to 6-membered heterocycloalkyl group are each optionally separated by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, C. 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups;

[0016] Alternatively, when the ligand-drug conjugate includes formula Ia, the two R... 1b Together with the atoms attached to it, they form C 3-6 cycloalkyl; the C 3-6 The cycloalkyl group is optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, C. 1-6 Alkyl substituents;

[0017] Q 1 and Q 2 Each is independently N or CR 5b ;

[0018] R 5b Hydrogen, deuterium, halogen, -OH, -CN, -NO2, C 1-6 Alkyl, -OC 1-6 Alkyl, C 3-6cycloalkyl, -OC 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, or -O (4-6 membered heterocycloalkyl), wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl group and the 4- to 6-membered heterocycloalkyl group are each optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, C. 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups;

[0019] Y can be either -OH or -CN.

[0020] In some implementation schemes, R 1a -NH-, -O-, a -NH-CH2-CH2-O-, -O-CH2-CH2-O-, a -NH-C(O)-CH2-O-、 a -NH-C(O)-CH2-NH- or a -O-CH2-C(O)-O-, α-terminus and connect.

[0021] In some implementation schemes, R 1a -N(CH3)- a -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-U- a -V-(4-8 membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U- a -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-OU- a -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-, a -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 alkylene-U- a -VC 0-6 Alkylene-N(R) 6 )C(O)OC 0-6 alkylene-U- a -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 alkylene-U-a -VC 0-6 Alkylene-N(R) 6 S(O)2-C 0-6 alkylene-U- a -VC 0-6 Alkylene-N(R) 6 )C(O)-(4-8 membered heterocyclic alkylene)-U- or a -VC 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U-, α-terminus with Connection; the C 0-6 Alkylene is optionally surrounded by one or more elements selected from -OH and C. 3-6 Substituents of cycloalkyl groups;

[0022] R 6 It is hydrogen or C 1-6 alkyl.

[0023] In some implementation schemes, R 1a -N(CH3)- a -N(R 6 )C(O)-C 1-6 alkylene-U- a -O-(4-8 membered heterocyclic alkylene)-C(O)-C 1- 6-alkylene-U, a -NHC(O)-(C 1-6 alkylene)-ON(C 1-6 alkyl)-, -N(R 6 )C(O)-C 3-8 Cycloalkyl-U-, a -NH-(4-8 membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U- a -NH-C 3-8 Cycloalkyl-U, a -N(R 6 )C(O)OC 1-6 alkylene-U- a -N(R 6 )C(O)N(R 6 )-C 1- 6-alkylene-U- a -N(R 6 S(O)2-C 1-6 alkylene-U- a -N(R 6 )C(O)-(4-8 membered heterocyclic alkylene)-U- or a -OC(O)N(R6 )-C 1-6 alkylene-U-, α-terminus with Connection; the C 1-6 Alkylene is optionally surrounded by -OH or C 3-6 Cycloalkyl substitution.

[0024] In some implementation schemes, R 1a for a -N(R 6 )C(O)-(C 1-6 alkylene)-ON(C 1-6 alkyl)-, a-terminus with Connection; R 6 It is hydrogen or C 1-6 alkyl.

[0025] In some implementation schemes, R 1a for a end and connect.

[0026] In some implementation schemes, R 5a -NH-, -O-, b -NH-CH2-CH2-O-, -O-CH2-CH2-O-, b -NH-C(O)-CH2-O-、 b -NH-C(O)-CH2-NH- or b -O-CH2-C(O)-O-, b-terminus and connect.

[0027] In some implementation schemes, R 5a for b -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-U- b -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0- 6-alkylene-OU- b -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-, b -VC 0-6 Alkylene-N(R) 6 )C(O)OC 0-6 alkylene-U- b -VC 0-6Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U- b -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 alkylene-U- b -VC 0-6 alkylene-(4- to 8-membered heterocyclic alkylene)-U- or b -V-(4-8 membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U-, b-terminus with Connection; the C 0-6 Alkylene is optionally surrounded by one or more elements selected from -OH and C. 3-6 Substituents of cycloalkyl groups;

[0028] R 6 It is hydrogen or C 1-6 alkyl.

[0029] In some implementation schemes, R 5a -N(R) 6 )C(O)-C 1-6 Alkylene-U-, -NHC(O)-(C 1-6 alkylene)-ON(C 1-6 alkyl)-, -N(R 6 )C(O)-C 3-8 Cycloalkylene-U-,-N(R) 6 )C(O)OC 1-6 Alkylene-U-, -OC(O)N(R) 6 )-C 1-6 Alkylene-U-, -OC 3-8 Cycloalkylene -U-, -O-(4- to 8-membered heterocycloalkylene)-U-, -O-(4- to 8-membered heterocycloalkylene)-C(O)-C 1-6 Alkylene-U- or -NH- (4- to 8-membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U-, b-terminus with Connection; the C 1-6 Alkylene is optionally C 3-6 Cycloalkyl substitution;

[0030] R 6 It is hydrogen or C 1-6 alkyl.

[0031] In some implementation schemes, R 5a for B-end and connect.

[0032] In some implementation schemes, R 2a for c -VC 0-6 alkylene-U- c -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-U- c -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 alkylene-U- c -VC 0-6 Alkylene-N(R) 6 )C(O)OC 0-6 alkylene-U- c -VC 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U- c -VC 0-6 Alkylene-N(R) 6 S(O)2-C 0-6 alkylene-U- c -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-, c -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 alkylene-U- c -VC 0-6 alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U- or c -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-OU-, with the c-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C 3-6 Cycloalkyl substitution.

[0033] In some implementation schemes, R 2a for c -C 0-6 alkylene-U- c -N(R 6 )C(O)-C 0-6 alkylene-U- c -N(R6 )C(O)N(R 6 )-C 0-6 alkylene-U- c -N(R 6 )C(O)OC 0-6 alkylene-U- c -OC(O)N(R 6 )-C 0-6 alkylene-U- c -N(R 6 S(O)2-C 0-6 alkylene-U- c -N(R 6 )C(O)-C 3-8 Cycloalkyl-U-, c -C(O)N(R 6 )-C 0-6 alkylene-U- c -C(O)-(4-8 membered heterocyclic alkylene)-U- or c -N(R 6 )C(O)-C 0-6 alkylene-OU-, with the c-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C 3-6 Cycloalkyl substitution.

[0034] In some implementation schemes, R 2a for c -C 0-6 alkylene-U- or c -N(R 6 )C(O)-C 0-6 Alkylene-U-, with the c-terminus connected to a piperidine ring.

[0035] In some implementation schemes, R 2a for c -N(R 6 )C(O)-C 1-6 Alkylene-U-, with the c-terminus connected to a piperidine ring; U is -O-.

[0036] In some implementation schemes, R 2a -NH-, -O-, c -CH2O-、 The c-terminus is connected to the piperidine ring.

[0037] In some implementation schemes, R 3a for d -VC 0-6 Alkylene-N(R)6 )C(O)-C 0-6 alkylene-U- d -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3- 8-cycloalkyl-U- d -VC 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U- d -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 alkylene-U- d -VC 0-6 alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U- or d -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 The alkylene group (OU-) has a d-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C 3-6 Cycloalkyl substitution.

[0038] In some implementation schemes, R 3a for d -C 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-U- d -C 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-, d -C 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U- d -C 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 alkylene-U- d -C(O)-(4-8 membered heterocyclic alkylene)-U- or d -C 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 The alkylene group (OU-) has a d-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C3-6 Cycloalkyl substitution.

[0039] In some implementation schemes, R 3a for d -C 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 The alkylene group (-U-) is connected to a piperidine ring at its d-terminus.

[0040] In some implementation schemes, R 3a for The d-end is connected to the piperidine ring.

[0041] In some implementation schemes, R 4 R 3a R on 6 The group and the atoms attached to it together form 5- to 8-membered heterocyclic alkyl groups.

[0042] In some implementation schemes, R 3a For -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 When alkylene-U-, R 4 R 3a R on 6 Groups and the atoms attached to them together form

[0043] In some implementation schemes, R 6 It is hydrogen or C 1-6 alkyl.

[0044] In some implementation schemes, R 6 C 1-6 alkyl.

[0045] In some implementation schemes, R 4 It is hydrogen or C 1-6 alkyl.

[0046] In some implementation schemes, R 4 It is hydrogen.

[0047] In some implementation schemes, R 1b For hydrogen, -OH, -NH2, C 1-6 Alkyl or -OC 1-6 Alkyl; the C 1-6 Alkyl groups may be optionally substituted with -OH.

[0048] In some implementation schemes, R 5b For hydrogen or -OC 1-6 alkyl.

[0049] In some implementations, Y is -CN.

[0050] In some implementation schemes, for Or a combination thereof.

[0051] In some implementation schemes, for Or a combination thereof.

[0052] In some embodiments, the structures represented by formulas Ia, Ib, Ic, and Id are the structures represented by formulas Ia-1, Ib-1, Ic-1, and Id-1:

[0053] Among them, R 1a R 5a R 2a R 3a R 4 R 5b The definition of Y is as described in any one of formulas Ia, Ib, Ic and Id of this invention.

[0054] In some embodiments, the ligand-drug conjugate comprises a ligand with the structure shown in Formula Ib-1:

[0055] Among them, R 5b -OC 1-6 alkyl;

[0056] R 4 It is hydrogen or C 1-6 alkyl;

[0057] R 2a for c -N(R 6 )C(O)-C 1-6 Alkylene-U-, with the c-terminus connected to a piperidine ring; U is -O-;

[0058] R 6 It is hydrogen or C 1-6 alkyl;

[0059] Y can be either -OH or -CN.

[0060] In some embodiments, the structure represented by formula Ia, formula Ib, formula Ic, formula Id, Ia-1, formula Ib-1, formula Ic-1, or formula Id-1 is any of the following structures:

[0061] In some embodiments, the ligand-drug conjugate comprises the structure shown in formula IIa, IIb, IIc, or IId:

[0062] Among them, R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definitions of Y and L are as described in any one of formulas Ia, Ib, Ic, and Id of this invention; 1a The end is connected to the ligand;

[0063] L 1a for

[0064] L 2 -(C(R) L21 )2) n -; n is a natural number from 0 to 50;

[0065] L 2 Any C(R) in L21 The )2 unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -, -O-, -S-, -SO-, -SO2-, -P(R L22 )-、-P(=O)(R L22 -, -C(=S)-, -C(=NR) L22 )-, -N=N-, -C=N-, -N=C-,

[0066] -Cy- is a phenylene, a 5- to 8-membered heteroaryl, a 3- to 10-membered heterocyclic alkyl, or a 3- to 10-membered cycloalkyl, wherein -Cy- is optionally surrounded by one or more R- cx replace;

[0067] R L21 R L22 and R cx Each independently is -(C(R) L2a )2) m -R L2b m is a natural number from 0 to 50;

[0068] R L21 R L22 and R cxAny C(R) in L2a The )2 unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(R)-. L2a )-, -N(CH3)-, -O-, -S-, -SO- or -SO2-;

[0069] R L2a -(CH2) y -R L2b y is a natural number from 0 to 50;

[0070] R L2a Any CH2 unit in the structure can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, -N(R)-. L2a )-, -O-, -S-, -SO- or -SO2-;

[0071] R L2b Each can be independently represented as hydrogen, deuterium, halogen, -NO2, -CN, -N(Me)2, -N + (Me)3, -COOH, -S(O)2OH, -P(O)(OH)2, glycosyl or its derivatives, C 2-6 alkenyl, C 2-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl;

[0072] L 3 For L 3a -L 3b ;

[0073] L 3a and L 3b Independently, it is a short peptide composed of 2-10 amino acid residues, which is absent.

[0074] Or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue;

[0075] Tr represents non-existent Or any combination of the above groups;

[0076] R Tr Independently selected from hydrogen, deuterium, halogens, -NO2, -CN, -OH, -SH, -NH2, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -OC(O)NH2, -CH2CO-(N(Me)CH2C(O))z -OR Tra -CH2CO-(N(Me)CH2C(O)) z -NHR Tra -(CH2CH2O) z -R Tra -CONH-(CH2CH2O) z -R Tra C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne group, 3-8 membered cycloalkyl group, 4-10 membered heterocycloalkyl group, 6-10 membered aryl group or 5-10 membered heteroaryl group, wherein C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl, and 5-10 membered heteroaryl are optionally surrounded by one or more R Tra replace;

[0077] R Tra Independently, it can be hydrogen, deuterium, halogen, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl,

[0078] z is an independent natural number from 0 to 50;

[0079] The heteroatoms in the heterocyclic alkyl, heteroaryl, heterocyclic alkyl and heteroaryl groups are selected from one, two or three of N, O and S; the number of heteroatoms is 1, 2 or 3.

[0080] In some implementations, n is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0081] In some implementations, m is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0082] In some implementations, y is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0083] In some implementations, z is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0084] In some embodiments, the glycosyl group or its derivative is a hexose or its derivative, a pentose or its derivative, a disaccharide or its derivative, or a polysaccharide or its derivative.

[0085] In some embodiments, the glycosyl group or its derivative is D / L-glucosyl, D / L-mannosyl, D / L-galactosyl, D / L-tarosyl, D / L-gulosyl, D / L-azolosyl, D / L-idurosyl, D / L-allosyl, D / L-mannohepanosyl, D / L-ribosyl, D / L-arabinosyl, D / L-xylose, sucrose, maltose, lactose, or derivatives of the above glycosyl groups.

[0086] In some embodiments, the glycosyl group or its derivative is

[0087] In some implementations, L 1a for e-end and L 2 Connection; preferably, L 1a for e-end and L 2 Connection; more preferably, L 1a for e-end and L 2 connect.

[0088] In some implementations, L 2 -(CHR) L21 ) n -; n is a natural number from 0 to 50;

[0089] L 2 Any CHR in L21 Each unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NH-, -O- or

[0090] -Cy- is a phenylene, a 4- to 6-membered heterocyclic alkylene, or a 3- to 6-membered heterocyclic alkylene, wherein -Cy- is optionally surrounded by one, two, or three R-. cx replace;

[0091] R L21 and R cx Each independently is -(CHR) L2a )2) m -R L2b m is a natural number from 0 to 50;

[0092] R L21 and R cx Any CHR L2aEach unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, -O-, -S-, -SO-, or -SO2-;

[0093] R L2a -(CH2) y -R L2b y is a natural number from 0 to 50;

[0094] R L2a Any CH2 unit in the structure can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, -O-, -S-, -SO-, or -SO2-;

[0095] R L2b Each can be independently represented as hydrogen, deuterium, halogen, -NO2, -CN, -N(Me)2, -N + (Me)3, or a sugar moiety or its derivative;

[0096] The glycosyl group or its derivative is a hexose glycosyl group or its derivative, or a pentose glycosyl group or its derivative, including but not limited to derivatives of D / L-glucose, D / L-mannose, D / L-galactose, D / L-tarose, D / L-ribose, etc.; preferably, the glycosyl group or its derivative is...

[0097] The heteroatoms in the 4- to 6-membered heterocyclic alkyl groups are selected from one, two, or three of N, O, and S; the number of heteroatoms is 1, 2, or 3.

[0098] In some implementation schemes, R L2b Hydrogen, deuterium, -N(Me)2, -N + (Me)3 or a glycosyl group or its derivative; preferably, R L2b For hydrogen, More preferably, R L2b For hydrogen,

[0099] In some implementations, L 2 -(CHR) L21 ) n -; n is a natural number from 0 to 50; for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0100] L 2 Any CHR in L21 Each element can be independently replaced by the following structural elements: -C(O)-, -NH-, -O- or

[0101] R L21 Each independently is -(CHR) L2a )2) m -R L2b m is a natural number from 0 to 50, for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0102] R L21 Any CHR L2a Each unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, or -O-;

[0103] R L2a -(CH2) y -R L2b y is a natural number from 0 to 50, for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0104] R L2a Any CH2 unit in the structure can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, or -O-;

[0105] R L2b Hydrogen, deuterium, -N(Me)2, -N + (Me)3, or a glycosyl group or its derivative; preferably, R L2b For hydrogen, More preferably, R L2b For hydrogen,

[0106] In some implementations, L 2 for

[0107] f end and L 3 connect;

[0108] n1, n2, and n3 are each independent natural numbers from 0 to 8;

[0109] m1 is a natural number from 0 to 16;

[0110] m2 is a natural number from 1 to 5;

[0111] y1 is a natural number from 1 to 5;

[0112] G is non-existent. Or -NH-, g end with L1a connect.

[0113] In some implementations, L 2 for f end and L 3 connect;

[0114] n1, n2, and n3 are each independent natural numbers from 0 to 8;

[0115] m1 is a natural number from 0 to 16;

[0116] y1 is a natural number from 1 to 5;

[0117] G is non-existent. Or -NH-, g end with L 1a connect.

[0118] In some implementations, L 2 for f end and L 3 connect;

[0119] n1 and n2 are each independent natural numbers from 0 to 8;

[0120] m1 is a natural number from 0 to 16;

[0121] m2 is a natural number from 1 to 5;

[0122] G is non-existent. g end and L 1a connect.

[0123] In some implementations, L 2 for

[0124] f end and L 3 connect.

[0125] In some implementations, L 2 for f end and L 3 connect.

[0126] In some implementations, L 2 for

[0127] f end and L 3 connect.

[0128] In some implementations, L 2 for f end and L 3 connect.

[0129] In some implementation schemes, for

[0130] f end and L 3 connect.

[0131] In some implementation schemes, for Among them, the f end and L 3 Connected.

[0132] In some implementation schemes, for f end and L 3 connect.

[0133] In some implementation schemes, for

[0134] f end and L 3 connect.

[0135] In some implementations, L 3a and L 3b Independently

[0136] In some implementations, L 3a and L 3b Independently, it is a short peptide composed of 2-10 amino acid residues, which is absent.

[0137] In some implementations, L 3aNot present, Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Ser, Val-Cit, Val-Ala, Val-Lys, Val -Lys(Ac), Val-Glu, Val-Asp, Val-Ser, Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Lys, Ala-Asn ,Ala-Gln,Ala-Gly,Ala-Ser,D-Ala-Ala,Gly-Glu,Gly-Asp,Gly-Asn,Gly-Ser,Gly-Gln,Glu-Gly,Glu-Gln,Glu-S er, Glu-Asn, Gln-Gly, Gln-Ser, Asp-Gly, Asn-Asn, Asp-Glu, Asp-Ser, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gl y-Ala-Ala, Gly-Val-Ala, Gly-Val-Cit, Glu-Val-Cit, Ala-Ala-Ala, Ala-(D-Ala)-Ala, Ala-Ala-Asn, Ala-(D-Ala )-Asn, Ala-Ala-Asp, Val-Lys-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Gly-Gly-Gly, Lys-Ala-Asn, Gly-Phe-Gly, Gly- Gly-Phe, Asn-Pro-Val, Ala-Lys-Gly, Gly-Lys-Gly, Gly-Glu-Gly, Glu-Gly-Ser, Glu-Ala-Ser, (Gly)4, (Gly)2-Ph e-Gly, (Gly)2-Glu-Gly, Lys-(Ala)2-Asn, Lys-(Ala)2-Asp, Gly-Leu-Gly-Lys, (Ala)2-Pro-Val or (Ala)2-Pro-Nva.

[0138] In some implementations, L 3a It is a short peptide that is absent or consists of 2-4 amino acid residues, preferably a short peptide consisting of 2-4 amino acid residues.

[0139] In some implementations, L 3b For non-existent The methylene terminus is connected to Tr.

[0140] In some implementations, L 3b For non-existent The j-end is connected to the Tr-end.

[0141] In some implementations, L 3a For those not present, Ala-Ala, Gly-Glu-Gly, Ala-Ala-Ala, Ala-Ala-Asn, and (Gly)2-Phe-Gly, the carbonyl terminus of the above amino acid residues or short peptides is associated with L. 3b Connection; L 3b For non-existent or The methylene terminus is connected to Tr.

[0142] In some implementations, L 3a The peptides are Gly-Glu-Gly, Ala-Ala-Ala, or (Gly)2-Phe-Gly, with the carbonyl terminus of the above short peptides being L... 3b Connection; L 3b It does not exist.

[0143] In some implementation schemes, for

[0144] The h terminal is connected to the Tr terminal.

[0145] In some implementation schemes, for

[0146] The h terminal is connected to the Tr terminal.

[0147] In some implementation schemes, for

[0148] The h terminal is connected to the Tr terminal.

[0149] In some implementation schemes, for The h terminal is connected to the Tr terminal.

[0150] In some implementations, Tr represents non-existence. i-end and L 3 Connection; R TrIndependently, it can be hydrogen, halogen, -NO2, -CN, -OH, -NH2, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -OC(O)NH2, or -CH2CO-(N(Me)CH2C(O)). z -NHMe, -(CH2CH2O) z -H or -CONH-(CH2CH2O) z -H;

[0151] z is a natural number from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0152] In some implementations, Tr represents non-existence. i-end and L 3 Connection; preferably, Tr is i-end and L 3 connect.

[0153] In some implementations, Tr is i-end and L 3 connect.

[0154] In some embodiments, the structures represented by formulas IIa, IIb, IIc, and IId are the structures represented by formulas IIa-1, IIb-1, IIc-1, and IId-1:

[0155] Among them, R 1a R 5a R 2a R 3a R 4 R 5b The definition of Y is as described in any one of formulas Ia, Ib, Ic, and Id of this invention; L 1a L 2 L 3 The definition of Tr is as described in any one of formulas IIa, IIb, IIc and IId of this invention.

[0156] In some embodiments, the structure represented by formula IIa, IIb, IIc, IId, IIa-1, IIb-1, IIc-1, or IId-1 is any one of the structures in Table A below:

[0157] Table A

[0158] In some embodiments, the ligand-drug conjugate is a ligand-drug conjugate as shown in formula IIIa, IIIb, IIIc, or IIId:

[0159] Wherein, Ab is the ligand that binds to the target;

[0160] q represents the drug loading (drug-ligand coupling ratio);

[0161] R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definitions of Y are as described in any one of formulas Ia, Ib, Ic and Id of this invention;

[0162] L 1a L 2 L 3 The definitions of Tr are as described in any one of formulas IIa, IIb, IIc and IId of this invention.

[0163] In some implementations, Ab is a target-binding polypeptide, antibody, or antigen-binding fragment thereof.

[0164] In some preferred embodiments, Ab is an antibody or its antigen-binding fragment.

[0165] In some implementations, the antibody is selected from one or more of the following:

[0166] (1) Fully human antibodies, humanized antibodies, murine antibodies, and chimeric antibodies;

[0167] (2) Probody;

[0168] (3) Bispecific antibodies and multispecific antibodies;

[0169] (4) Monoclonal antibodies and polyclonal antibodies;

[0170] (5)IgG antibodies.

[0171] In some implementations, the antigen-binding fragment in Ab is selected from: Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, VHH, and complementarity-determining region (CDR) fragments.

[0172] In some implementations, Ab is a monoclonal antibody.

[0173] In some preferred embodiments, the Ab targets antigens selected from the group consisting of: HER2, HER3, B7H3, B7H4, DLL3, TROP2, Claudin18.2, CD30, CD33, CD70, BCMA, GPC-3, ADAM9, and EGFR.

[0174] In some embodiments, the ligand-drug conjugate of formula (III) is wherein Ab is an antibody or antigen-binding fragment thereof targeting HER3, B7H3, Claudin18.2, CD30, CD33, CD70, BCMA, GPC-3, ADAM9 and EGFR.

[0175] In some embodiments, Ab is an antibody or antigen-binding fragment targeting GPC-3, preferably an anti-GPC-3 antibody or its antigen-binding fragment, such as DB1002 or a variant thereof.

[0176] In some preferred embodiments, Ab is an antibody targeting EGFR or an antigen-binding fragment thereof; preferably an anti-EGFR antibody or an antigen-binding fragment thereof, such as DB1003 or a variant thereof.

[0177] In some preferred embodiments, Ab is an antibody targeting BCMA or an antigen-binding fragment thereof; preferably, it is an anti-BCMA antibody or an antigen-binding fragment thereof, such as DB1004 or a variant thereof.

[0178] In some preferred embodiments, Ab is an antibody targeting ADAM9 or an antigen-binding fragment thereof; preferably, it is an antibody against ADAM9 or an antigen-binding fragment thereof, such as DB1001 or a variant thereof.

[0179] In this invention, the amino acid sequence of the DB1001 heavy chain is shown in SEQ ID NO:1, and the amino acid sequence of the DB1001 light chain is shown in SEQ ID NO:2.

[0180] In this invention, the amino acid sequence of the DB1002 heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of the DB1002 light chain is shown in SEQ ID NO:4.

[0181] In this invention, the amino acid sequence of the DB1003 heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the DB1003 light chain is shown in SEQ ID NO:6.

[0182] In this invention, the amino acid sequence of the DB1004 heavy chain is shown in SEQ ID NO:7, and the amino acid sequence of the DB1004 light chain is shown in SEQ ID NO:8.

[0183] In some embodiments, the Ab contains the sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO:9, and / or the sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:10.

[0184] In some embodiments, the Ab contains the sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO:17, and / or the sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:18.

[0185] In some embodiments, the Ab contains the sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO:25, and / or the sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:26.

[0186] In some embodiments, the Ab contains the sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO:33, and / or the sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:34.

[0187] In some embodiments, the Ab comprises sequences of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and / or sequences of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0188] In some embodiments, the Ab comprises sequences of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, and / or sequences of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0189] In some embodiments, the Ab comprises sequences of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, and / or sequences of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively.

[0190] In some embodiments, the Ab comprises sequences of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively, and / or sequences of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively.

[0191] In some embodiments, the Ab comprises a VH sequence as shown in SEQ ID NO:9 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:9, and / or a VL sequence as shown in SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:10.

[0192] In some embodiments, the Ab comprises a VH sequence as shown in SEQ ID NO:17 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:17, and / or a VL sequence as shown in SEQ ID NO:18 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:18.

[0193] In some embodiments, the Ab comprises a VH sequence as shown in SEQ ID NO:25 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:25, and / or a VL sequence as shown in SEQ ID NO:26 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:26.

[0194] In some embodiments, the Ab comprises a VH sequence as shown in SEQ ID NO:33 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:33, and / or a VL sequence as shown in SEQ ID NO:34 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:34.

[0195] In some embodiments, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the Ab is shown in SEQ ID NO:10.

[0196] In some embodiments, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region of the Ab is shown in SEQ ID NO:18.

[0197] In some embodiments, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region of the Ab is shown in SEQ ID NO:26.

[0198] In some embodiments, the amino acid sequence of the heavy chain variable region of the Ab is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region of the Ab is shown in SEQ ID NO:34.

[0199] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:2 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0200] In some embodiments, the anti-GPC-3 antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:4 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0201] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:6 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0202] In some embodiments, the anti-BCMA antibody or its antigen-binding fragment comprises a heavy chain and a light chain of the antibody, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:7 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO:8 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0203] In some implementations, q is an integer or decimal of 1-32, preferably an integer or decimal of 1-16, more preferably an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 4.32, 5.42, 6.12, 6.84, 6.93, 7.05, 7.22, 7.23, 7.25, 7.29, 7.31, 7.34, 7.36, 7.38, 7.45, 7.48, 7.51, 7.52, 7.53, 7.57, 7.58, 7.59, 7.62, 7.65, 7.68, 7.71, 7.78, or 7.86.

[0204] In some implementations, q is an integer from 1 to 32, preferably an integer from 1 to 16, and more preferably an integer from 2 to 8, such as 2, 3, 4, 5, 6, 7 or 8.

[0205] In some implementations, q is an integer or decimal of 1-32, preferably an integer or decimal of 1-16, more preferably an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 3.87, 3.97, 4.05, 4.11, 4.23, 4.26, 4.31, 7.57, 7.75 or 7.82.

[0206] In some embodiments, the ligand drug conjugates represented by Formulas IIIa, IIIb, IIIc, and IIId are ligand drug conjugates represented by Formulas IIIa-1, IIIb-1, IIIc-1, and IIId-1:

[0207] Among them, R 1a R 5a R 2a R 3a R 4 R 5b The definitions of L and Y are as described in any one of formulas Ia, Ib, Ic, and Id of this invention. 1a L 2 L 3 The definitions of Tr and are as described in any one of formulas IIa, IIb, IIc and IId of this invention, and the definitions of Ab and q are as described in any one of formulas IIIa, IIIb, IIIc and IIId of this invention.

[0208] In some embodiments, the ligand-drug conjugate is a ligand-drug conjugate of formula IIIb-1:

[0209] Among them, R 5b -OC 1-6 alkyl;

[0210] R4 It is hydrogen or C 1-6 alkyl;

[0211] R 2a for c -N(R 6 )C(O)-C 1-6 Alkylene-U-, with the c-terminus connected to a piperidine ring; U is -O-;

[0212] R 6 It is hydrogen or C 1-6 alkyl;

[0213] Y is either -OH or -CN;

[0214] L 1a for e-end and L 2 connect;

[0215] L 2 for f end and L 3 connect;

[0216] G is non-existent. Or -NH-, g end with L 1a connect;

[0217] n1 and n2 are each independent natural numbers from 0 to 8;

[0218] m1 is a natural number from 0 to 16;

[0219] y1 is a natural number from 1 to 5;

[0220] L 3 For L 3a -L 3b ;

[0221] L 3a It is a short peptide composed of 2-4 amino acid residues; L 3b It does not exist;

[0222] Tr is i-end and L 3 connect;

[0223] Ab represents an antibody or its antigen-binding fragment;

[0224] q is an integer or decimal between 2 and 8.

[0225] In some implementations, L 2 for

[0226] In some implementation schemes, for f end and L 3 connect.

[0227] In some implementation schemes, for

[0228] The h terminal is connected to the Tr terminal.

[0229] In some implementations, the ligand-drug conjugate is any one of the ligand-drug conjugates in Table B below:

[0230] Table B

[0231] Wherein, Ab and q are as described in any one of formulas IIIa, IIIb, IIIc and IIId of the present invention.

[0232] In some embodiments, the ligand-drug conjugate is any one of the ligand-drug conjugates in Table C below:

[0233] Table C

[0234] In a second aspect, the present invention provides a mixture of ligand-drug conjugates comprising the aforementioned ligand-drug conjugates, their tautomers, their enantiomers, their diastereomers, their pharmaceutically acceptable salts or solvates thereof, wherein the ligand-drug conjugates have one, two or more q values.

[0235] In some embodiments, the average drug-to-ligand ratio (Dar) of the mixture of ligand-drug conjugates is selected from an integer or decimal of 1-32, preferably an integer or decimal of 1-16, more preferably an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 4.32, 5.42, 6.12, 6.84, 6.93, 7.05, 7.22, 7.23, 7.25, 7.29, 7.31, 7.34, 7.36, 7.38, 7.45, 7.48, 7.51, 7.52, 7.57, 7.62, 7.65, 7.68, 7.78, or 7.86.

[0236] In some implementations, q is an integer or decimal of 1-32, preferably an integer or decimal of 1-16, more preferably an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 3.87, 3.97, 4.05, 4.11, 4.23, 4.26, 4.31, 7.57, 7.75, 7.82.

[0237] Thirdly, the present invention also provides compounds represented by formulas IVa, IVb, IVc and IVd, their tautomers, their enantiomers, their diastereomers or pharmaceutically acceptable salts thereof:

[0238] Among them, R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definitions of Y and L are as described in any one of formulas Ia, Ib, Ic, and Id of this invention; 2 L 3 The definitions of Tr are as described in any one of formulas IIa, IIb, IIc and IId of this invention;

[0239] L 1 for

[0240] In some embodiments, in the compounds with the structures shown in formulas IVa, IVb, IVc and IVd, L 1 for Preferably, L 1 for

[0241] In some embodiments, in the compounds with the structures shown in formulas IVa, IVb, IVc and IVd, for

[0242] In some implementation schemes, for

[0243] In some implementation schemes, for

[0244] In some implementation schemes, for

[0245] In some embodiments, in the compounds with the structures shown in formulas IVa, IVb, IVc and IVd, for

[0246] In some implementation schemes, for

[0247] The h terminal is connected to the Tr terminal.

[0248] In some implementation schemes, for

[0249] In some implementation schemes, for

[0250] In some embodiments, the compounds with structures represented by formulas IVa, IVb, IVc, and IVd are compounds with structures represented by formulas IVa-1, IVb-1, IVc-1, and IVd-1.

[0251] in,

[0252] R 1a R 5a R 2a R 3a R4 R 5b The definitions of Y are as described in any one of formulas Ia, Ib, Ic and Id of this invention;

[0253] L 2 L 3 The definitions of Tr are as described in any one of formulas IIa, IIb, IIc and IId of this invention;

[0254] L 1 The definition is as described in any one of formulas IVa, IVb, IVc and IVd of this invention.

[0255] The present invention also provides a compound as shown in Formula IVb-1, its tautomers, its enantiomers, its diastereomers, or pharmaceutically acceptable salts thereof:

[0256] Among them, R 5b -OC 1-6 alkyl;

[0257] R 4 It is hydrogen or C 1-6 alkyl;

[0258] R 2a for c -N(R 6 )C(O)-C 1-6 Alkylene-U-, with the c-terminus connected to a piperidine ring; U is -O-;

[0259] R 6 It is hydrogen or C 1-6 alkyl;

[0260] Y is either -OH or -CN;

[0261] L 1 for

[0262] L 2 for f end and L 3 connect;

[0263] G is non-existent. Or -NH-, g end with L 1a connect;

[0264] n1 and n2 are each independent natural numbers from 0 to 8;

[0265] m1 is a natural number from 0 to 16;

[0266] y1 is a natural number from 1 to 5;

[0267] L3 For L 3a -L 3b ;

[0268] L 3a It is a short peptide composed of 2-4 amino acid residues; L 3b It does not exist;

[0269] Tr is i-end and L 3 connect.

[0270] In some implementation schemes, for

[0271] In some implementation schemes, for

[0272] In some embodiments, the compound represented by formula IVa, IVb, IVc, IVd, IVa-1, IVb-1, IVc-1, or IVd-1 is any one of the compounds in Table D below:

[0273] Table D

[0274] The present invention also provides a method for preparing ligand-drug conjugates as shown in formulas IIIa, IIIb, IIIc and IIId, comprising the following steps: reacting compounds as shown in formulas IVa, IVb, IVc or IVc with Ab-SH via substitution or addition reactions to obtain the ligand-drug conjugates as shown in formulas IIIa, IIIb, IIIc or IIId.

[0275] Among them, R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2The definition of Y is as described in any one of formulas Ia, Ib, Ic and Id of this invention;

[0276] L 1a L 2 L 3 The definition of Tr is as described in any one of formulas IIa, IIb, IIc and IId of this invention;

[0277] L 1 The definition is as described in any one of formulas IVa, IVb, IVc and IVd of this invention;

[0278] The definitions of Ab and q are as described in any one of formulas IIIa, IIIb, IIIc and IIId of this invention.

[0279] The Ab-SH is a product in which the interchain disulfide bonds of Ab are reduced to thiol groups.

[0280] The present invention also provides a pharmaceutical composition comprising the above-described ligand drug conjugates, their tautomers, their enantiomers, their diastereomers, their pharmaceutically acceptable salts or solvates thereof, or mixtures of the above-described ligand drug conjugates, and pharmaceutically acceptable excipients.

[0281] The present invention also provides a pharmaceutical formulation comprising the above-described ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt or solvate thereof, or a mixture of the above-described ligand-drug conjugates.

[0282] This invention also provides the use of substance S in the preparation of a medicament for the prevention or treatment of cancer; said substance S is the aforementioned ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt or solvate thereof, or a mixture of the aforementioned ligand-drug conjugates, the aforementioned pharmaceutical composition, or the aforementioned pharmaceutical preparation. The cancer is a solid tumor or a non-solid tumor. Examples include esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumors, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.

[0283] In some embodiments, the cancer is EGFR, BCMA, ADAM9, or GPC-3 mediated or treated cancer; preferably a solid tumor or non-solid tumor, such as esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.

[0284] This invention also provides the use of substance S in the preparation of a drug for the prevention or treatment of diseases associated with abnormal cellular activity; said substance S is the aforementioned ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt or solvate thereof, or a mixture of the aforementioned ligand-drug conjugates, the aforementioned pharmaceutical composition, or the aforementioned pharmaceutical preparation. The disease associated with abnormal cellular activity may be cancer. The definition of cancer is as described above.

[0285] Terminology definition:

[0286] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0287] In this application, the term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. These are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0288] In this application, the term "pharmaceutical composition" refers to a dosage form that can be formulated according to various suitable routes of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, sprays, etc.

[0289] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0290] In this application, the terms "pharmaceutically acceptable salt" or "medicinal salt" generally refer to salts of compounds or ligand-drug conjugates of this application that are safe and / or effective when used in mammals and have the desired biological activity.

[0291] The term "drug loading" typically refers to the average amount of cytotoxic drug loaded onto each ligand, or it can be expressed as the ratio of cytotoxic drug to ligand. For example, the drug / antibody ratio is denoted as q, which can be an integer or decimal from 0 to 20; the average drug / antibody ratio is denoted as Dar, which can be an integer or decimal of 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. For example, Dar is 7.8 or 7.9. The drug loading of each ADC molecule after the coupling reaction can be characterized using conventional methods such as UV / Vis spectroscopy, mass spectrometry, HIC, ELISA assays, and HPLC.

[0292] In this application, the term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linker unit. In some embodiments of this application, "ligand-drug conjugate" can be an antibody-drug conjugate (ADC), whereby an ADC can refer to a monoclonal antibody or antigen-binding fragment linked to a biologically active cytotoxic drug via a stable linker unit.

[0293] In this application, the term "ligand" generally refers to small molecules, peptides, RNA, DNA, carbohydrates, and macromolecules that can recognize and bind to antigens or receptors associated with target cells. The role of a ligand can be to present a drug to a target cell population that has bound the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In this application, a ligand can be represented as Ab. The ligand antigen forms a linker bond with a linker unit (also called a "linker" or "connector") through a heteroatom on the ligand. The ligand can be an antibody or its antigen-binding fragment. The antibody can be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody targeting a subset of the following targets: HER2, HER3, B7H3, TROP2, Claudin 18.2, CD30, CD33, CD70, and EGFR. For example, the antibody may be an antibody targeting the following targets: 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, O772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1lc, CD123, CD138, CD142, CD147, CD166, CD19, CD19.CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4 , DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, GD2 , GEDA, GPC-1, GPC-3, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RN F43, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, short proteoglycans, mesothelin, sodium phosphate cotransporter 2B, Claudin18.2, endothelial peptide receptors, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin αvβ6, trophoblast cell glycoproteins, and tissue factor.

[0294] In this application, the term "antibody or antigen-binding fragment thereof" generally refers to an immunological conjugate, extending to all antibodies from all species, including dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and modified antibodies, and fragments thereof. The term "antibody or antigen-binding fragment thereof" can refer to any antibody-like molecule having an antigen-binding region, including small molecule fragments such as Fab′, Fab, F(ab′)2, single-domain antibodies (DABs), Fv, scFv (single-chain Fv), linear antibodies, diabody antibodies, etc. The term "antigen-binding fragment" can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The antibodies may include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TROP2 antibody, and anti-Claudin antibody. 18.2 Antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-ADAM9 antibodies, anti-GPC-3 antibodies, anti-BCMA antibodies, and anti-Mesothelin antibodies, such as DB1001, DB1002, DB1003, and DB1004.

[0295] In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. Methods for establishing chimeric antibodies include, for example, constructing a hybridoma that secretes murine-specific monoclonal antibodies, then cloning the variable region gene from the murine hybridoma cells, cloning the constant region gene of the human antibody as needed, and then linking the murine variable region gene and the human constant region gene to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in eukaryotic or prokaryotic systems.

[0296] In this application, the term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., grafting it into a different type of human germline antibody framework sequence. Humanized antibodies can overcome the problem of chimeric antibodies inducing a strong heterologous response due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy chain variable region and light chain variable region genes can be found in the VBase human germline sequence database.

[0297] In this application, the terms "fully human antibody", "human antibody", "fully human antibody" or "completely human antibody" are used interchangeably, and the variable region and constant region of the antibody may both be of human origin, with immunogenicity and toxic side effects removed.

[0298] The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0299] In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. The most common definitions of the six CDRs are provided, for example, by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242; Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987); and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography," J. Mol. Biol. 262:732 (1996). As used in this application, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3 or L1, L2, L3) of light chain variable structural domains, and CDR1, CDR2, and CDR3 (CDR H1, CDRH2, CDRH3 or H1, H2, H3) of heavy chain variable structural domains.

[0300] The term "one or more species" or similar expression "at least one species" can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more species.

[0301] When the lower and upper limits of a numerical range are disclosed, any numerical value falling within that range and any included range are specifically disclosed. In particular, each range of values ​​disclosed herein should be understood to represent each numerical value and range encompassed within a wider range;

[0302] For example, the expression "C" 1-6 "This should be understood as encompassing any subrange and each point value, such as C." 2-5 C 3-4 C 1-2 C 1-3 C1-4, C 1-5 And so on, as well as C1, C2, C3, C4, C5, C6, etc. For example, the expression "C 3-10 "It should also be understood in a similar way, for example, it can encompass any subrange and point value contained within it, such as C." 3-9 C 6-9 C 6-8 C 6-7 C7-10 C 7-9 C 7-8 C 8-9 And C3, C4, C5, C6, C7, C8, C9, C 10 For example, the expression "3-10 yuan" should be understood as encompassing any sub-range and each point value within it, such as 3-4 yuan, 3-5 yuan, 3-6 yuan, 3-7 yuan, 3-8 yuan, 3-9 yuan, 4-5 yuan, 4-6 yuan, 4-7 yuan, 4-8 yuan, 5-7 yuan, 5-8 yuan, 6-7 yuan, etc., as well as 3, 4, 5, 6, 7, 8, 9, 10 yuan, etc. Similarly, the expression "5-10 yuan" should also be understood in a similar way, for example, it can encompass any sub-range and point value included within it, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 5-10 yuan, 6-7 yuan, 6-8 yuan, 6-9 yuan, 6-10 yuan, 7-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

[0303] In this application, the term "natural number" is used for example, 0-50, 0-40, 0-30, 0-20, 0-10, 0-8, 0-6, 0-6, 0-4, or 0-2; and for example, 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0304] In this application, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group. As used herein, the term "C" refers to a saturated straight-chain or branched hydrocarbon group. 1-6 "Alkyl" refers to a saturated straight-chain or branched hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms). "C" 1-6 "alkyl" can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl, etc.

[0305] In this application, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. For example, the term "C 1-6 "Alkylene" refers to a saturated, straight-chain or branched divalent hydrocarbon group having 1-6 carbon atoms. 1-6 "Alkylene" includes, but is not limited to, methylene, ethylene, propylene, or butylene.

[0306] In this application, the term "C" 0-6 In the term "alkylene", when C0 is 0, the alkylene bond is a connecting bond.

[0307] In this application, the term "C" 0-6 In the alkyl group, when C0 is 0, alkyl is hydrogen.

[0308] In this application, the term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon double bonds. For example, the term "C" as used herein... 2-6"Alkenyl" refers to an alkenyl group having 2-6 carbon atoms and one, two, or three (preferably one) carbon-carbon double bonds (such as vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, etc.).

[0309] In this application, the term "alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more carbon-carbon triple bonds. For example, the term "C" as used herein... 2-6 "Alynyl" refers to an alkynyl group having 2-6 carbon atoms and one, two, or three (preferably one) carbon-carbon triple bonds (such as ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc.).

[0310] In this application, the term "aryl" refers to a monocyclic or fused-ring aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used herein... 6-10 "Aryl" refers to an aryl group having 6-10 carbon atoms (such as phenyl, naphthyl, etc.).

[0311] In this application, the term "heteroaryl" or "heteroary ring" refers to a monocyclic and fused heterocyclic system having one or more conjugated π-electron systems, wherein one or more (e.g., 1, 2, or 3) ring atoms are heteroatoms selected from N, O, P, and S, and the remaining ring atoms are C. Heteroaryl or heteroary rings can be characterized by the number of ring atoms. For example, a 5-12 membered heteroaryl may contain 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) ring atoms, particularly 5, 6, 9, or 10 ring atoms. Examples of heteroaryl groups include thiophene, furanyl, pyrrole, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, indole, etc.

[0312] In this application, the term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group. For example, "C 3-12"Cycloalkyl" or "3-12 membered cycloalkyl" refers to a cycloalkyl group having 3-12 ring carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Common cycloalkyl groups include (but are not limited to) monocyclic cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkyl groups, including fused rings, bridged rings, or spiro rings, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, bicyclic [3.2.1]octyl, bicyclic [5.2.0]nonyl, decahydronaphthyl, etc.

[0313] In this application, the term "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic dicyclic group. For example, "C 3-12 "Cycloalkylene" or "3-12-membered cycloalkylene" refers to cycloalkylene compounds having 3-12 ring carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Common cycloalkylene compounds include (but are not limited to) monocyclic cycloalkylene compounds, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkylene compounds, including fused rings, bridged rings, or spirocyclic compounds, such as bicyclic[1.1.1]pentylene, bicyclic[2.2.1]heptylene, bicyclic[3.2.1]octylene, bicyclic[5.2.0]nonylene, decahydronaphthylene, etc.

[0314] The term "heterocyclic alkyl" refers to a saturated or partially saturated non-aromatic cyclic group containing at least one heteroatom selected from N, O, P, and S as a ring member, preferably 1, 2, 3, or 4 heteroatoms. Examples include 3-8 membered and 3-6 membered heterocyclic alkyl groups. Specific examples include, but are not limited to, ethylene oxide, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, and homopiperazinyl.

[0315] The term "heterocyclic alkylene" refers to a saturated or partially saturated, non-aromatic divalent cyclic group containing at least one heteroatom selected from N, O, P, and S as a ring member. Preferably, the number of heteroatoms is 1, 2, 3, or 4. Examples include 3-8 membered and 3-6 membered heterocyclic alkylenes. Specific examples include, but are not limited to, ethylene oxide, cyclobutane, pyrroleyl, tetrahydrofuranyl, piperidinyl, piperazineyl, tetrahydropyranyl, and homopiperazineyl.

[0316] The term "fused ring (fused ring system)" refers to a chemically feasible polycyclic structure formed by two or more (e.g., 3, 4, or 5) carbon rings or heterocycles sharing common ring edges or common atoms, wherein the carbon rings include cycloalkyl and aryl groups, and the heterocycles include heteroaromatic and heterocyclic alkyl groups. Fused ring systems include, but are not limited to: fused ring systems formed by cycloalkyl groups with cycloalkyl groups, fused ring systems formed by cycloalkyl groups with heterocyclic alkyl groups, fused ring systems formed by cycloalkyl groups with aromatic rings, fused ring systems formed by cycloalkyl groups with heteroaromatic rings, fused ring systems formed by heterocyclic alkyl groups with aromatic rings, fused ring systems formed by heteroaromatic rings with heteroaromatic rings, and fused ring systems formed by heteroaromatic rings with aromatic rings.

[0317] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, iodine, such as fluorine and chlorine.

[0318] In this application, the term "each independently" means that at least two groups (or segments) in the structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0319] In this application, the terms “optional” or “optionally” generally mean that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0320] In this application, the term "substitution" and its other variant forms herein refer to the replacement of one or more (e.g., 1, 2, 3, or 4) atoms or groups of atoms (e.g., hydrogen atoms) on a specified atom with other equivalents, provided that the replacement does not exceed the normal valence of the specified atom or group of atoms in the present case and is capable of forming a stable compound. If an atom or group of atoms is described as "optionally substituted," it may or may not be substituted. Unless otherwise stated, the linking site of a substituent herein may be derived from any suitable position of the substituent. When the linking bond in a substituent is shown as a chemical bond through two atoms connected to each other in a ring system, it indicates that the substituent may be linked to any one of the cyclic atoms in the ring system.

[0321] This article uses wavy lines. The bonds in the structural formula are intended to indicate that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any proportion.

[0322] When used alone or in combination with other groups in this document, the term "oxo" refers to =O.

[0323] In this application, one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, are independently substituted by a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (e.g., alkene) bond.

[0324] In this application, the term "amino acid" includes both natural and non-natural amino acids, and the naming of conventional amino acids follows conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this application, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine may be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; and glutamine by Q or Gln.

[0325] In this application, the term "non-natural amino acid" has the following structure: Where r is selected from 0, 1, 2, 3, 4, and 5; where R a R b Each is independently selected from -C 1-6 Alkylene -NH2, -C 1-6 Alkylene-NH-C 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 Alkyl)2, -C 1-6 Alkylene-NH-C 3-10 cycloalkyl, -C 1-6 alkylene-N(3-10 membered cycloalkyl)(C 1-6 Alkyl), -C 1-6 Alkylene-C 3-10 cycloalkyl, -C 1-6 Alkylene (3-10 membered heterocyclic alkyl), -C 1-6Alkylene-NHCOC 1-6 Alkyl, -C 1-6 Alkylene-NHCOOC 1-6 Alkyl, -C 1-6 Alkylene-NHS(O)2C 1-6 Alkyl, -C 1-6 Alkylene-S(O)2-C 1-6 Alkyl, -C 1-6 Alkylene-S(O)2-C 3-10 cycloalkyl, -C 1-6 Alkylene -S(O)2-NH2, -C 1-6 Alkylene -COOH, -C 1-6 Alkylene -CONH2, -C 1-6 Alkylene-CONHC 1-6 Alkyl, -C 1-6 Alkylene-CO (3-10 membered heterocyclic alkyl), The alkyl, alkylene, cycloalkyl, and heterocycloalkyl groups are each optionally and independently substituted by one or more substituents selected from H, halogens, -OH, -NH2, -SH, -NO2, CN, -COOH, and oxo groups; or any R a R b Together with the atoms attached thereto, they form 3-10 membered heterocyclic alkyl groups and 3-10 membered cycloalkyl groups; each of the cycloalkyl groups and heterocyclic alkyl groups is optionally substituted by one or more substituents selected from H, halogens, -OH, -NH2, -SH, -NO2, CN, -COOH and oxo groups;

[0326] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound in this application can be an organic compound, a compound with a molecular weight of less than 500 Daltons, a compound with a molecular weight of less than 1000 Daltons, a compound with a molecular weight of more than 1000 Daltons, or a compound with a molecular weight of more than 10,000 Daltons or more than 100,000 Daltons. In this application, a compound can also refer to a compound linked by chemical bonds. For example, it can be a compound in which one or more molecules with a molecular weight of less than 1000 Daltons are linked by chemical bonds to a biological macromolecule, which can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound in this application can include a compound in which a protein is linked to one or more molecules with a molecular weight of less than 1000 Daltons, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000 Daltons, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000 Daltons.

[0327] In this application, the term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0328] Solid lines may be used in this article. solid wedge Or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0329] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the connection of the corresponding group R to other segments or groups in the compound through this site. The "-" at the end of the group indicates that the group is connected to other segments in the molecule through this site. For example, CH3-C(=O)- means that the C(=O) in the acetyl group is connected to other segments in the molecule.

[0330] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. The terms "above" and "below" generally refer to situations that include the stated number.

[0331] Unless otherwise specified, the structures described in this application may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose structures are identical to those of this application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of this application.

[0332] The terms “active ingredient,” “therapeutic agent,” “active substance,” or “active agent” refer to a chemical entity that can effectively treat one or more symptoms of a target condition or disease.

[0333] Unless otherwise stated, as used herein, the term “treatment” means to reverse, alleviate, or inhibit the progression of a disease or condition to which such term applies, or one or more symptoms of such a disease or condition, or to prevent such a disease or condition, or one or more symptoms of such a disease or condition.

[0334] As used herein, “individual” includes both human and non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. In this invention, “non-human animals” includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0335] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form nitrogen oxides because nitrogen requires available lone pairs of electrons to be oxidized. Those skilled in the art will identify nitrogen-containing heterocycles capable of forming nitrogen oxides. They will also recognize that tertiary amines can form nitrogen oxides. Synthetic methods for preparing nitrogen oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (m-CPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing nitrogen oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol.7, pp. 748-750 (AR. Katritzky and A.J. Boulton, Eds., Academic Press); and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol.22, pp. 390-392 (AR. Katritzky and A.J. Boulton, Eds., Academic Press).

[0336] Those skilled in the art will understand that all compounds covered by this invention are chemically feasible compounds; and all chemical bonds are linked in a chemically feasible manner.

[0337] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 2006, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0338] This invention also covers methods for preparing the compounds described herein. It should be understood that the compounds of this invention can be synthesized using the methods described below, as well as synthetic methods known in the field of synthetic organic chemistry or variations thereof understood by those skilled in the art. Preferred methods include (but are not limited to) those described below. The reaction can be carried out in a solvent or solvent mixture suitable for the reagents and materials used and suitable for achieving the conversion.

[0339] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0340] The reagents and raw materials used in this invention are all commercially available.

[0341] The positive and progressive effects of this invention are that the compounds of this invention have one or more of the following advantages:

[0342] (1) It has inhibitory activity against the in vitro proliferation of tumor cells;

[0343] (2) It has plasma stability;

[0344] (3) It has an in vivo tumor-suppressing effect;

[0345] (4) It has the ability to resist transport by transporters;

[0346] (5) It has the ability to target tumors in vivo;

[0347] (6) It has good in vivo safety;

[0348] Furthermore, the conjugation methods described in this disclosure have a wide range of applications and can be widely used for conjugation with bioactive molecules such as antibodies or targeting small molecule ligands. In summary, the protein degrading agents, linkers, antibodies, and ADCs of this invention have significant clinical value. Attached Figure Description

[0349] Figure 1 shows the killing effect of ADC3.1 on MDA-MB-468 cells.

[0350] Figure 2 shows the killing effect of ADC3.1 on MDA-MB-468 cells and Raji-Luc cells.

[0351] Figure 3 shows the killing effect of ADC3.1 on Raji-Luc cells.

[0352] Figure 4 shows the in vivo tumor inhibition effect of ADC3.1 on Huh7 tumor-bearing mice.

[0353] Figure 5 shows the in vivo tumor inhibition effect of ADC3.3-P2 on HCC827 tumor-bearing mice. Detailed Implementation

[0354] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in quotation marks, are incorporated herein by reference in their entirety. The invention is further illustrated below by way of examples, but this does not limit the invention to the scope of the examples described. Experimental methods in the following examples, unless specific conditions are specified, are performed according to conventional methods and conditions, or as selected according to the trade specification.

[0355] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0356] The preparative high performance liquid chromatography (HPLC) method was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250×20mm column).

[0357] Thin-layer chromatography purification was performed using GF 254 (0.4–0.5 nm) silica gel plates produced in Yantai.

[0358] The reaction was monitored using thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). The developing solvent systems used included, but were not limited to, dichloromethane and methanol systems, n-hexane and ethyl acetate systems, and petroleum ether and ethyl acetate systems. The volume ratio of the solvent was adjusted according to the polarity of the compound, or by adding triethylamine, etc.

[0359] Column chromatography typically uses 200-300 mesh silica gel from Qingdao Ocean as the stationary phase. Eluent systems include, but are not limited to, dichloromethane and methanol systems and n-hexane and ethyl acetate systems. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0360] Unless otherwise specified in the examples, the reaction temperature is room temperature (20℃~30℃).

[0361] Unless otherwise specified, the reagents used in the examples were purchased from Acros Organics, Aldrich Chemical Company, Nanjing Yaoshi Technology, Anaiji, or Shanghai Shuya Pharmaceutical Technology, etc.

[0362] The above embodiments do not limit the scope of this application in any way. In addition to those described herein, various modifications to the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

[0363] In the conventional synthesis methods, preparation examples, and intermediate synthesis examples, the starting materials were commercially feasible and purchased from Shanghai Leyan, Shanghai Shaoyuan, Bid Biotechnology, Aladdin Reagent, etc. The key starting material M24 and the control compound rupettedine were both purchased from Zhejiang Zhongke Chuangyue. The meanings of the abbreviations are shown in the table below.

[0364] Example 1: Preparation of intermediates

[0365] Preparation of compound Int7

[0366] Step 1: Preparation of compound Int7-2

[0367] Compound Int7-1 (5.0 g, 32.7 mmol) was dissolved in anhydrous methanol (80 mL), and a 30% aqueous solution of methylamine (6.8 g, 65.4 mmol) was added. After stirring for 30 min, trimethylcyanosilane (4.9 g, 49.3 mmol) was added, and the mixture was stirred at room temperature for another 30 min. The reaction was monitored by LCMS until complete. The reaction solution was concentrated under reduced pressure, and the residue was extracted with saturated sodium bicarbonate and DCM. After stirring, the mixture was separated into liquid and liquid phases, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain Int7-2 (4.12 g, 64% yield).

[0368] Step 2: Preparation of compound Int7-3

[0369] Compound Int7-2 (1.36 g, 7.08 mmol) was dissolved in THF (40 mL), and DIEA (1.37 g, 10.62 mol) and (Boc)₂O (3.2 g, 17 mmol) were added. The reaction mixture was stirred at room temperature for 16 h, cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography to give Int7-3 (1.2 g, yield 58%).

[0370] MS m / z(ESI): 293.1 [M+H] +

[0371] 1H NMR (400MHz, CDCl3) δ7.28 (dd, J=8.5, 2.4Hz, 1H), 7.15 (d, J=2.4Hz, 1H), 6.98 (d ,J=8.5Hz,1H),6.40(brs,1H),3.86(s,3H),2.73(s,3H),1.52(d,J=16.5Hz,9H).

[0372] Step 3: Preparation of compound Int7

[0373] Compound Int7-3 (0.5 g, 1.28 mmol) was dissolved in a mixed solution of EtOH (40 mL) and ammonia (30%, 10 mL), and RanyNi (100 mg) was added. After three hydrogen purgings, the pressure was increased to 1.5 MPa and the reaction was carried out at 50 °C for 16 h. The reaction was monitored by LC-MS to ensure complete reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain Int7 (0.26 g, yield 69%).

[0374] Preparation of compound Int1

[0375] Step 1: Preparation of compound Int1-2

[0376] Int1-1 (450.0 mg, 2.13 mmol) was dissolved in MeOH (10 mL), 10% Pd / C (50 mg) was added, and the mixture was purged with hydrogen and stirred at 20 °C under hydrogen for 2 hours. The resulting mixture was filtered through diatomaceous earth, concentrated, and dried to obtain a colorless oily substance, Int1-2 (385 mg crude), which was then used in the next reaction.

[0377] Step 2: Preparation of compound Int1-3

[0378] Int1-2 (385 mg, 2.13 mmol) was dissolved in THF (10 mL), followed by the addition of saturated NaHCO3 (3 mL) and Boc2O (928.68 mg, 4.26 mmol). The mixture was stirred overnight at 20 °C. The resulting mixture was adjusted to pH 5-6 with 1 N HCl, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound Int1-3 (280.0 mg, 47% yield).

[0379] Step 3: Preparation of compound Int1-4

[0380] Int1-3 (270.0 mg, 0.96 mmol) was dissolved in DCM (10 mL) and DMF (1 drop), cooled to 0 °C, and oxaloyl chloride (243.7 mg, 1.92 mmol) was added under nitrogen protection. After the addition was complete, the mixture was allowed to warm naturally to room temperature for 30 minutes. The mixed solution was cooled to 0 °C, and ammonia was slowly added to adjust the pH to approximately 8. The solution was then extracted with ethyl acetate, separated, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound Int1-4 (230.0 mg, yield 85.48%).

[0381] Step 4: Preparation of compound Int1

[0382] Int1-4 (230.0 mg, 0.82 mmol) was dissolved in THF (5 mL), followed by the addition of BH3 / THF (6 mL, 1N), and the mixture was heated to 70 °C and stirred for 2 hours. The reaction solution was then cooled to 0 °C, and the reaction was quenched by slow dropwise addition of methanol. HCl / MeOH solution was then added, and the mixture was heated to 70 °C and stirred for 2 hours. The reaction solution was cooled to 40 °C and concentrated under reduced pressure. The residue was purified by reversed-phase HPLC to give compound Int1 (70.0 mg, yield 51.33%).

[0383] Preparation of compound Int2

[0384] Intermediate Int2 was synthesized following steps 3 to 4 of compound Int1.

[0385] Preparation of compound Int3

[0386] Intermediate Int3 was synthesized following steps 3 to 4 of compound Int1.

[0387] Preparation of compound Int4

[0388] Intermediate Int4 was synthesized following steps 3 to 4 of compound Int1.

[0389] Preparation of compound Int6

[0390] Intermediate Int6 was synthesized following steps 3 to 4 of compound Int1.

[0391] Preparation of compound LK3

[0392] Step 1: Preparation of compound LK3-D

[0393] LK3-C (1.02 g, 2.11 mmol), HATU (0.88 g, 2.32 mmol), HOAt (0.32 g, 2.32 mmol), and TMP (1.00 g, 8.43 mmol) were dissolved in DMF (10 mL) and stirred for 10 min. Then, LK3-B (0.53 g, 2.11 mmol) was added to the reaction system and stirred for 2 h. After the reaction was confirmed to be complete by LCMS, the solution was diluted with water and saturated brine and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain LK3-D (1.2 g, 70% yield).

[0394] Step 2: Preparation of compound LK3-E

[0395] Compound LK3-D (850 mg, 1.19 mmol, 1.0 eq) and 10% Pd / C (170 mg) were dissolved in MeOH / EA (15 ml / 15 ml) and stirred at 25 °C for 2 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated to obtain crude product LK3-E (0.6 g).

[0396] Step 3: Preparation of compound LK3-F

[0397] Compound LK3-E (200 mg, 0.32 mmol) and TEA (81.5 mg, 0.80 mmol) were dissolved in DMF (1 ml), and the solution was stirred at 20 °C for 16 hours. The reaction was confirmed to be complete by LCMS, and the reaction solution was used directly in the next step.

[0398] Step 4: Preparation of compound LK3

[0399] Compound LK1-A (93 mg, 0.32 mmol) and TEA (65 mg, 0.64 mmol) were dissolved in DMF (3 mL). LK3-F (129 mg, 0.32 mmol, from the reaction solution in step 3) was added to the solution, and the mixture was stirred at 20 °C for 2 hours. The reaction was confirmed to be complete by LCMS. The reaction solution was concentrated under reduced pressure and purified by reverse-phase column chromatography to give compound LK3 (80 mg, yield 42%).

[0400] Synthesis of compound LK18

[0401] Step 1: Synthesis of compound LK18-B

[0402] LK18-A ​​(2 g, 4.7 mmol) was dissolved in THF (10 mL), followed by the addition of N-hydroxysuccinimide (541 mg, 4.7 mmol) and DCC (136 mg, 4.7 mmol). The reaction solution was stirred at 25 °C for 16 hours. The reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated to obtain crude LK18-B (2.7 g), which was used directly in the next step without further purification.

[0403] Step 2: Synthesis of compound LK18-D

[0404] LK18-B (1.36 g, 2.61 mmol) was dissolved in acetone / H2O (7 mL / 7 mL), followed by the addition of LK18-C (900 mg, 1.57 mmol) and NaHCO3 (251.31 mg, 3.14 mmol). The solution was then stirred at 25 °C for 3 hours. The reaction was confirmed by LCMS. The solution was purified by reverse-phase silica gel column chromatography to obtain LK18-D (1.4 g, 90% yield).

[0405] Step 3: Synthesis of compound LK18-F

[0406] LK18-E (2 g, 5.41 mmol) was added to DMF (20 mL), followed by D-glucosamine (2 g, 11.04 mmol), HATU (5 g, 13.15 mmol), HOAt (3 g, 22.04 mmol), and TMP (3 g, 22.36 mmol). The solution was then stirred at 25 °C for 16 hours. The reaction was confirmed by LCMS. The solution was purified by reverse-phase silica column chromatography to obtain LK18-F (3 g, 90% yield).

[0407] Step 4: Synthesis of compound LK18-G

[0408] LK18-F (2g, 2.88mmol) was dissolved in DMF (20mL), and TEA (582mg, 5.76mmol) was added. The mixture was then stirred at 25°C for 16 hours. LCMS analysis confirmed the reaction was complete, yielding LK18-G. This reaction solution was used directly in the next step without any further processing.

[0409] Step 5: Synthesis of compound LK18-H

[0410] HATU (991 mg, 2.61 mmol), HOAt (467 mg, 3.43 mmol), and TMP (835 mg, 6.22 mmol) were added to 10 mL of DMF containing compound LK18-D (1.4 g, 1.43 mmol), and the mixture was stirred at 25 °C for 10 min. Then, LK18-G (the reaction solution from step 4) was added dropwise to this reaction solution, and the reaction was continued for 0.5 h. The reaction was monitored for completion by LCMS. The solution was purified by reverse-phase silica gel column chromatography to give LK18-H (1.8 g, 88% yield).

[0411] Step 6: Synthesis of compound LK18-I

[0412] TFA (20 mL) was added to a DCM (20 mL) solution of compound LK18-H (1.8 g, 1.25 mmol), and the solution was stirred at 25 °C for 0.5 h. The reaction solution was then rapidly dried using an oil pump and dissolved in a small amount of DMF. The solution was purified by reverse silica gel column chromatography to give LK18-I (1 g, 57.8% yield).

[0413] Steps 7 and 8: Synthesis of compound LK18-K

[0414] Dissolve LK3-D (700.0 mg, 0.98 mmol) in DMF (7.0 mL), and add triethylamine (247.5 mg, 2.45 mmol). Stir the reaction mixture overnight at 35°C. The resulting reaction mixture of LK18-J is used for the next step and requires no purification.

[0415] LK18-I (300.0 mg, 0.22 mmol), HATU (350.0 mg, 0.92 mmol), HOAt (350.0 mg, 2.6 mmol), and TPM (0.24 mL, 1.76 mmol) were dissolved in DMF (5.0 mL), and LK18-J (300.0 mg, 0.61 mmol) was added. The reaction mixture was stirred at 35 °C for 10 minutes. LK18-K ​​(220.0 mg, yield 36.7%) was purified by reversed-phase chromatography.

[0416] Step 9: Synthesis of compound LK18

[0417] LK18-K ​​(200.0 mg, 0.11 mmol) was dissolved in DMF (8.0 mL), and Pd / C (80.0 mg, 0.75 mmol) was added. The reaction mixture was stirred at 35 °C for one hour under hydrogen protection. The mixture was filtered and evaporated to dryness, and purified by reversed-phase chromatography to obtain LK18 (60.0 mg, yield 28.5%).

[0418] MS m / z (ESI): 844.6 [M+H] +

[0419] Synthesis of compound LK25

[0420] Following a similar method to steps 3 to 6 of compound LK18, compound LK25 was synthesized using 1-amino-1-deoxy-D-mannitol.

[0421] Synthesis of compound LK26

[0422] Following a similar method to steps 3 to 6 of compound LK18, compound LK26 was synthesized using 1-amino-1-deoxy-D-galactitol.

[0423] Preparation of compound LK27

[0424] Compound LK27 was synthesized following steps 2, 5, and 6 of compound LK18.

[0425] MS m / z (ESI): 1248.3 [M+H] + .

[0426] Example 2: Payload Synthesis

[0427] Synthesis of Compounds 18, 10, 10R and 10S

[0428] Step 1: Preparation of compound 18-1

[0429] Compound M24 (100 mg, 0.16 mmol) and compound Int7 (0.80 mmol) were dissolved in anhydrous ethanol (5 mL), and acetic acid (120 mg, 2.00 mmol) was added. The mixture was stirred overnight at 60 °C. After the reaction was monitored by LCMS until complete, the mixture was cooled to room temperature and concentrated under reduced pressure. The residue was added to ethyl acetate and saturated sodium bicarbonate aqueous solution, stirred, and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by Pre-TLC to give compound 18-1 (112 mg, 78% yield).

[0430] Step 2: Preparation of Compound 18

[0431] Compound 18-1 (112 mg, 0.12 mmol) was dissolved in DCM (2 mL) and added to TFA (0.5 mL). The reaction was carried out at room temperature for 2 h, and the reaction was monitored for completeness by LCMS. The reaction solution was concentrated to dryness under reduced pressure, the pH was adjusted to 8 with saturated sodium bicarbonate solution, and then extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 53 (90 mg, 93% yield). Further purification by reverse HPLC yielded compound 18 (70 mg, 72% yield).

[0432] MS m / z (ESI): 800.1 [M+H] + .

[0433] 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),8.95–8.65(m,1H),8.50(s,1H),8.43–8.10(m,1H),6.85–6.65(m,1H) ,6.55–6.35(m,2H),6.23–6.11(m,2H),5.95–5.15(brs,4H),5.06(t,J=12.2Hz,1H),4.53(s,2H),4.30–4.0 5(m,3H),4.01–3.88(m,1H),3.68–3.60(m,3H),3.60–3.50(m,3H),3.50–3.24(m,2H),3.24–3.03(m,1H),3. 01–2.61(m,3H),2.43(t,J=5.3Hz,2H),2.29(s,2H),2.24(s,2H),2.14–2.05(m,3H),1.97(d,J=6.8Hz,3H).

[0434] Step 3: Preparation of Compound 10, Compound 10R and Compound 10S

[0435] Compound 18 (60.0 mg, 0.075 mmol) was dissolved in DMF (1.5 mL), and glycolic acid (5.7 mg, 0.075 mol), HATU (28.7 mg, 0.075 mmol), and DIEA (11.6 mg, 0.090 mmol) were added sequentially. The reaction was carried out at room temperature with stirring for 2 h, and the reaction was monitored for completeness by LC-MS. Ethyl acetate and saturated sodium bicarbonate aqueous solution were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative HPLC to give compound 10 (50 mg, yield 64%).

[0436] MS m / z (ESI): 858.3 [M+H] + .

[0437] The compounds were further purified by Pre-TLC using DCM:MeOH as the developing solvent (10:1) to yield two compounds. Peak 1 had a retention time of 1.557 min on LCMS, and peak 2 had a retention time of 1.543 min on LCMS. Characterization is as follows:

[0438] Peak 1: Compound 10R or Compound 10S

[0439] MS m / z (ESI): 858.3 [M+H] + .

[0440] 1 H NMR (400MHz, DMSO) δ9.21 (s, 1H), 8.75 (s, 1H), 6.51–6.29 (m, 3H), 6.16 (d, J = 9.1Hz, 2H) ,5.14(s,1H),5.04(d,J=11.6Hz,1H),4.54–4.42(m,3H),4.21–4.16(m,1H),4.13–4.08 (m,1H),4.07–4.01(m,2H),3.62(s,3H),3.52(s,3H),2.83–2.67(m,4H),2.47–2.30(m, 3H),2.36–2.29(m,3H),2.22–2.16(m,3H),2.04(s,3H),1.98(s,3H),1.28–1.20(m,6H).

[0441] Peak 2: Compound 10R or Compound 10S

[0442] MS m / z (ESI): 858.3 [M+H] + .

[0443] 1H NMR(400MHz,DMSO-d6)δ9.26(s,1H),8.79(s,1H),6.50–6.40(m,1H),6.40–6.23(m,2H),6.20– 6.10(m,2H),5.40–5.25(m,1H),5.10–5.00(m,1H),4.70–4.60(m,1H),4.53–4.43(m,1H),4.24 –4.19(m,2H),4.15–4.08(m,3H),3.63(s,3H),3.21–3.13(d,J=4.4Hz,3H),2.82–2.64(m,4H), 2.34–2.28(m,3H),2.25–2.16(m,3H),2.08–2.03(m,3H),2.01–1.96(m,3H),1.29–1.21(m,6H).

[0444] Synthesis of Compound 18R and Compound 18S

[0445] Step 1: Synthesis of compound 18R-1 or 18S-1

[0446] A (94.8 mg, 0.32 μmol) and NaOAc (200 mg, 1.8 mmol) were added to a 10 mL solution of acetic acid containing M24 (100.0 mg, 0.16 mmol). The reaction solution was reacted at 60 °C for 2 hours. After concentration, the product was purified by normal column chromatography (petroleum ether: ethyl acetate = 1:1) to give a white solid product 18R-1 or 18S-1. The first peak on LCMS had an elution time of 1.810 min and was 18R-1 or 18S-1 (65.0 mg, yield: 45%). The second peak had an elution time of 1.793 min and was 18R-1 or 18S-1 (65.0 mg, yield: 45%).

[0447] The compound with the first peak (t = 1.810 min), MS m / z (ESI): 900.2 [M+H] + ;

[0448] The compound with the second peak (t = 1.793 min), MS m / z (ESI): 900.2 [M+H] + .

[0449] Step 2: Synthesis of compound 18R or 18S

[0450] TFA (2 mL) was added to DCM (2 mL) containing either compound 18R-1 or 18S-1 (65.0 mg, 0.07 mmol), and the mixture was stirred at 20 °C for 2 hours. The reaction mixture was purified by reverse HPLC and lyophilized to give a white solid product 18R or 18S. The first peak on LCMS had an elution time of 1.527 min, which was 18R or 18S (33.6 mg, yield: 60%). The second peak had an elution time of 1.515 min, which was 18R or 18S (38.0 mg, yield: 68%).

[0451] Peak P1 compound (18R or 18S, t = 1.527 min), MS m / z (ESI): 800.2 [M+H] + ;

[0452] Peak P2 compound (18R or 18S, t = 1.515 min), MS m / z (ESI): 800.2 [M+H] + .

[0453] Synthesis of Compound 11

[0454] Compound 10 (30 mg, 0.034 mmol) was dissolved in ACN (3 mL) and water (2 mL), and then silver nitrate (143 mg, 0.840 mmol) was added. The mixture was stirred at room temperature in the dark for 12 hours. After the reaction was monitored by LCMS until complete, saturated sodium bicarbonate aqueous solution was added, and the mixture was stirred and separated. The organic phase was retained, and the aqueous phase was extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative HPLC to give compound 11 (5 mg, yield 17%).

[0455] MS m / z (ESI): 849.4 [M+H] + .

[0456] 1H NMR (400MHz, CDCl3) δ6.67-6.29(m,3H),6.08-5.91(m,2H),5.84-5.80(m,1H),5.63-5.50(m ,1H),5.40-5.30(m,1H),5.20-5.04(m,1H),4.88-4.84(m,1H),4.60-4.45(m,2H),4.45-4.0 0(m,5H),3.79(d,J=3.6Hz,3H),3.75-3.70(m,1H),3.67-3.62(m,3H),3.58-3.15(m,3H),3. 00-2.78(m,3H),2.60-2.58(m,1H),2.49-2.45(m,2H),2.38-2.20(m,9H),2.10-2.00(m,4H).

[0457] Synthesis of Compound 22, Compound 22R and Compound 22S

[0458] Step 1: Preparation of compound 22-1

[0459] At room temperature, Cs₂CO₃ (57.9 mg, 0.18 mmol) and iodomethane (25.2 mg, 0.18 mmol) were added to a DMF (12 mL) solution of compound 18-1 (80.0 mg, 0.09 mmol). The reaction mixture was stirred at room temperature for 1 minute. Ethyl acetate and a saturated aqueous solution of sodium bicarbonate were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound 22-1 (30.0 mg, 37% yield).

[0460] Step 2: Preparation of compound 22-2

[0461] At room temperature, HCl / dioxane (4 mmol, 1 mL, 4 M) was added to a DCM (1 mL) solution of compound 22-1 (30.0 mg, 0.03 mmol). The reaction mixture was stirred at room temperature for half an hour. After concentration, the residue was purified by column chromatography to give compound 22-2 (20.0 mg, 74% yield).

[0462] Steps 3 to 4: Preparation of compounds 22R and 22S

[0463] At room temperature, glycolic acid (13.1 mg, 0.17 mmol), HATU (31.9 mg, 0.08 mmol), HOAT (31.9 mg, 0.24 mmol), and TMP (38.3 mg, 0.32 mmol) were added to a DMF (4 mL) solution of compound 22-2 (20.0 mg, 0.03 mmol). The reaction solution was stirred at room temperature for 1 hour. The reaction was confirmed to be complete by LCMS. Ethyl acetate and saturated ammonium chloride aqueous solution were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by Pre-TLC. Two compounds were obtained using DCM:MeOH = 10:1 as the developing solvent. Peak 1 had a retention time of 1.649 min on LCMS, and peak 2 had a retention time of 1.622 min on LCMS. Characterization is as follows:

[0464] Peak 1: Compound 22R or 22S, 6 mg, yield 30%.

[0465] MS m / z (ESI): 872.2 [M+H] +

[0466] 1 H NMR (400MHz, DMSO-d6) δ8.74 (s, 1H), 6.74-6.30 (m, 3H), 6.15 (d, J = 9.5Hz, 2H), 5.44-4.90 (m, 2H), 4.40-4.34 (m, 3H), 4.22-4.05 (m, 5H), 3.68-3. 57(m,6H),3.51(s,3H),3.28-3.27(m,3H),3.00-2.62(m,3H),2.42(d,J= 3.1Hz, 3H), 2.32 (d, J = 2.3Hz, 3H), 2.22-2.18 (m, 4H), 2.12-1.87 (m, 8H).

[0467] Peak 2: Compound 22R or 22S, 6 mg, yield 30%.

[0468] MS m / z (ESI): 872.2 [M+H] +

[0469] 1H NMR (400MHz, DMSO-d6) δ8.74 (d, J=6.3Hz, 1H), 6.49-6.29 (m, 3H), 6.14 (d, J= 4.1Hz,2H),5.40-5.27(m,1H),5.05(d,J=11.4Hz,1H),4.67-4.42(m,3H),4. 30-4.03(m,5H),3.63(t,J=2.4Hz,6H),3.49(s,3H),3.27(d,J=12.1Hz,3H), 2.86-2.65(m,3H),2.42-2.28(m,6H),2.22-2.18(m,4H),2.06-1.94(m,8H).

[0470] Synthesis of Compound 23

[0471] 37% formic acid (4 drops) and NaBH3CN (20.0 mg, 0.32 mmol) were added to a 1 mL ethanol solution of compound 10 (25.0 mg, 0.006 mmol) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse HPLC to give compound 23 (10.0 mg, 40% yield).

[0472] MS m / z (ESI): 872.8 [M+H] +

[0473] 1 H NMR(400MHz,DMSO-d6)δ9.15(d,J=14.0Hz,1H),8.71(d,J=10.2Hz,1H),6.4 5-6.19(m,3H),6.11(d,J=9.7Hz,2H),5.39-4.78(m,2H),4.68-4.43(m,2H), 4.36-3.98(m,4H),3.79-3.54(m,4H),3.52-3.34(m,4H),3.30-3.20(m,3H), 2.87-2.80(m,2H),2.40-2.28(m,7H),2.22-2.06(m,6H),2.01-1.98(m,8H).

[0474] Synthesis of Compound 32

[0475] Compound 32 was synthesized following steps 2 and 3 of compound 10.

[0476] MS m / z (ESI): 858.2 [M+H] + .

[0477] Synthesis of Compound 34

[0478] Compound 34 was synthesized following steps 2 and 3 of compound 10.

[0479] MS m / z (ESI): 858.2 [M+H] + .

[0480] Synthesis of Compound 24

[0481] Compound 24 was synthesized following steps 2 and 3 of compound 10.

[0482] MS m / z (ESI): 828.2 [M+H] + .

[0483] 1 H NMR (400MHz, DMSO-d6) δ9.32(s,1H),8.73(s,1H),7.67(t,J=6.0Hz,1H),6.83(d,J=8.0Hz,1H),6.48-6.39(m,2H),6.34(d,J=2.4Hz,1H),6. 25(s,1H),6.16(s,1H),5.56(t,J=5.6Hz,1H),5.09(d,J=11.2Hz,1H),4.54-4.50(m,1H),4.43(d,J=2.4Hz,1H),4.19(d,J=4.0Hz,1H),4.14 -4.12(m,1H),4.08-4.05(m,1H),3.85(d,J=3.6Hz,2H),3.65-3.64(m,3H),3.40(d,J=10.4Hz,1H),3.22(d,J=4.4Hz,1H),3.15-3.08(m,1H) ,2.89-2.77(m,2H),2.72–2.64(m,1H),2.58-2.54(m,1H),2.38-2.33 (m,1H),2.29(s,3H),2.15(s,3H),1.97(d,J=6.4Hz,6H),1.80(s,1H).

[0484] Synthesis of Compound 26

[0485] Compound 26 was synthesized following steps 2 to 3 of compound 10.

[0486] MS m / z (ESI): 828.2 [M+H] + .

[0487] 1 H NMR (400MHz, DMSO-d6) δ9.34(s,1H),8.73(s,1H),7.47(t,J=4.0Hz,1H),6.68(d,J=8.8Hz,1H),6.49(s,1H),6.45(dd,J=8.8,2.4Hz,1H),6 .34(d,J=2.4Hz,1H),6.26(s,1H),6.15(s,1H),5.54(t,J=5.6Hz,1H),5.05(d,J=11.2Hz,1H),4.55-4.51(m,1H),4.46(d,J=2.4Hz,1H),4.1 9(d,J=4.0Hz,1H),4.10(s,1H),4.04-3.94(m,1H),3.85(d,J=5.2Hz,2H),3.63(s,3H),3.50-3.45(m,1H),3.42-3.38(m,1H),3.21(d,J=4. 8Hz,1H),2.91-2.75(m,3H),2.39-2.28(m,3H),2.29(s,3H),2.22-2.14(m,4H),2.02(d,J=7.6Hz,6H),1.97(s,1H),1.70(d,J=14.4Hz,1H).

[0488] Synthesis of Compound 37

[0489] Step 1: Preparation of compound 37-2

[0490] M24 (45.0 mg, 72.4 μmol) and compound 37-1 (45.0 mg, 330.4 μmol) were dissolved in acetic acid (2 mL), and NaOAc (80 mg, 975.3 μmol) was added. The mixture was stirred at 65 °C for 1 hour. The reaction was confirmed to be complete by LCMS. The mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase HPLC to obtain compound 37-2 (39.8 mg, yield 74.25%).

[0491] MS m / z (ESI): 740.7 [M+H] + .

[0492] 1H NMR(400MHz,DMSO-d6)δ8.73(s,1H),6.55(d,J=8.5Hz,1H),6.44(s,1H),6.25(s,1H),6.21(dd,J=8 .5,2.3Hz,1H),6.15-6.12(m,2H),5.04(d,J=11.3Hz,1H),4.91(s,2H),4.44(d,J=2.6Hz,2H),4.19 (d,J=3.6Hz,1H),4.05(s,1H),3.94(d,J=9.8Hz,1H),3.63(s,3H),3.24-3.06(m,2H),2.78(d,J=5. 0Hz,2H),2.69-2.57(m,1H),2.45-2.31(m,3H),2.28(s,3H),2.22(s,3H),2.03(s,3H),1.98(s,4H).

[0493] Step 2: Preparation of compound 37-3

[0494] Compound 37-2 (30.0 mg, 0.041 mmol) was dissolved in DMF (1 mL), and tert-butoxycarbonylaminooxyacetic acid (7.8 mg, 0.041 mol), HATU (15.7 mg, 0.041 mmol), and DIEA (11.6 mg, 0.090 mmol) were added sequentially. The reaction was carried out at room temperature with stirring for 2 h, and the reaction was monitored by LC-MS until complete. Ethyl acetate and saturated sodium bicarbonate aqueous solution were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give compound 37-3 (26.5 mg, 70% yield).

[0495] Step 3: Preparation of Compound 37

[0496] Compound 37-3 (26.5 mg, 0.028 mmol) was dissolved in DCM (2 mL) and added to TFA (0.5 mL). The reaction was carried out at room temperature for 2 h, and the reaction was monitored for completeness by LC-MS. The reaction solution was concentrated to dryness under reduced pressure, the pH was adjusted to 8 with saturated sodium bicarbonate solution, and then extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Compound 37 (3.4 mg, 15% yield) was purified by reverse HPLC. MS m / z (ESI): 827.2 [M+H] + .

[0497] 1H NMR (400MHz, DMSO-d6) δ9.55(s,1H),8.76(s,1H),7.32(s,1H),7.27(d,J=8.7Hz,1H),6.90-6.80(m,2H),6.46(s,1H),6.27(s, 1H),6.17(s,1H),5.06(d,J=11.3Hz,1H),4.46(d,J=2.5Hz,1H),4.20(d,J=4.0Hz,1H),4.08(d,J=9.1Hz,3H),4.01(d,J=10.2H z,1H),3.63(s,3H),3.22(d,J=3.7Hz,1H),3.16-3.11(m,1H),2.79(d,J=4.7Hz,2H),2.69-2.65(m,1H),2.56(d,J=6.4Hz,4H), 2.43(d,J=14.8Hz,2H),2.36–2.31(m,1H),2.28(s,3H),2.23(s,3H),2.04(s,3H),1.99(s,3H),1.77-1.67(s,1H),1.23(s,4H).

[0498] Synthesis of Compound 36

[0499] Step 1: Preparation of compound 36-2

[0500] At room temperature, compounds Int1 (50.0 mg, 0.29 mmol) and NaOAc (200 mg, 2.46 mmol) were added to a solution of compound M24 (90.0 mg, 0.14 mmol) in acetic acid (3.0 mL). The reaction mixture was heated to 65 °C and stirred for 3 h. The resulting mixture was then concentrated, and the residue was purified by reversed-phase HPLC to give compound 36-2 (75 mg, 69% yield).

[0501] MS m / z (ESI): 770.2 [M+H] + .

[0502] 1H NMR (400MHz, DMSO-d6) δ8.69(s,1H),6.43(s,1H),6.20(s,1H),6.17(s,1H),6.15(s,1H),6.14(s,1H),5.02(d,J=11.6H z,1H),4.61(s,2H),4.47(d,J=2.8Hz,2H),4.18(d,J=3.6Hz,1H),4.09(s,1H),4.00(dd,J=2.0,11.2Hz,1H),3.63(s,3H ),3.47(s,3H),3.38-3.34(m,1H),3.32-3.26(m,1H),2.99-2.93(m,1H),2.83-2.75(m,2H),2.71-2.67(m,1H),2.40-2. 32(m,2H),2.29(s,3H),2.24(s,1H),2.20(s,3H),2.10(d,J=14.4Hz,1H),2.04(s,3H),1.97(s,3H),1.79-1.68(m,1H).

[0503] Steps 2 to 3: Preparation of compound 36

[0504] Compound 36 was synthesized following steps 2 to 3 of compound 37.

[0505] MS m / z (ESI): 857.2 [M+H] + .

[0506] 1 H NMR(400MHz,DMSO-d6)δ9.18(s,1H),8.91(s,1H),7.90(s,1H),6.52(s,1H),6.32(s,1H),6.22(s,1 H),6.19(s,1H),5.18(d,J=10.4Hz,1H),4.66-4.57(m,2H),4.25-4.19(m,6H),3.65(s,4H),3.62(s, 4H),3.46(d,J=8.0Hz,1H),3.35(d,J=4.8Hz,2H),3.14(s,1H),2.90-2.84(m,1H),2.76(s,1H),2.6 9-2.66(m,1H),2.62(s,3H),2.43-2.36(s,2H),2.31(s,3H),2.25(s,3H),2.01(s,3H),1.98(s,3H).

[0507] Synthesis of Compound 19

[0508] Step 1: Preparation of compound 18-2

[0509] Compound 18-2 was synthesized using the same method as compound 11.

[0510] Step 2: Preparation of Compound 19

[0511] Compound 18-2 (80 mg, 0.09 mmol) was dissolved in DCM (2 mL) and added to TFA (0.5 mL). The reaction was carried out at room temperature for 2 h, and the reaction was monitored for completeness by LCMS. The reaction solution was concentrated to dryness under reduced pressure, the pH was adjusted to 8 with saturated sodium bicarbonate solution, and then extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by reverse HPLC to obtain compound 19 (50 mg, 70% yield).

[0512] MS m / z (ESI): 791.4 [M+H] + .

[0513] Example 3: Preparation of linker-payload

[0514] Synthesis of compound LP1

[0515] At room temperature, compounds LP1-1 (15.4 mg, 0.02 mmol), HATU (3.8 mg, 0.01 mmol), HOAT (1.7 mg, 0.01 mmol), and TMP (3.8 mg, 0.03 mmol) were added to a DMF (1 mL) solution of compound 18 (10 mg, 0.01 mmol). The reaction mixture was stirred for 2 hours, and the reaction was confirmed to be complete by LC-MS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse HPLC to obtain compound LP1 (5 mg, yield 28.6%).

[0516] MS m / z (ESI): 1397.0 [MH] - .

[0517] Synthesis of compound LP5

[0518] Step 1: Synthesis of compound LP5-A

[0519] LK3-E (62.7 mg, 0.10 mmol) and compound 18 (40.0 mg, 0.05 mmol) were dissolved in DMF (3 mL) and stirred. HATU (40.0 mg, 0.10 mmol), HOAT (40.0 mg, 0.30 mmol), and TMP (80.0 mg, 0.66 mmol) were added at 30 °C, and the mixture was stirred at 30 °C for 30 minutes. The reaction was confirmed to be complete by LCMS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP5-A (30.0 mg, yield 42%).

[0520] Step 2: Synthesis of compound LP5-B

[0521] LP5-A (30.0 mg, 0.02 mmol) was dissolved in DMF (1 mL), and TEA (26.0 mg, 0.26 mmol) was added at room temperature and stirred overnight. The reaction was confirmed to be complete by LCMS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse column chromatography to obtain compound LP5-B (20.0 mg, yield 79%).

[0522] Step 3: Synthesis of compound LP5-D

[0523] LP5-C (8.2 mg, 0.03 mmol) and LP5-B (18.0 mg, 0.02 mmol) were dissolved in DMF (1.0 mL) and stirred. HATU (8.0 mg, 0.02 mmol), HOAT (8.0 mg, 0.06 mmol), and TMP (20.0 mg, 0.17 mmol) were added at room temperature, and stirring continued for 30 minutes after the addition was complete. The reaction was confirmed to be complete by LCMS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP5-D (15.0 mg, yield 71%).

[0524] Step 4: Synthesis of compound LP5

[0525] LP5-D (13.0 mg, 0.01 mmol) was dissolved in nitromethane (1 mL), and zinc bromide (100.0 mg) was added at room temperature and stirred for 30 minutes. The reaction was confirmed to be complete by LCMS, and the reaction solution was directly purified by reverse column chromatography to obtain compound LP5 (11.0 mg, yield 88%).

[0526] MS m / z (ESI): 1380.7 [M+H] +

[0527] 1 H NMR(400MHz,DMSO-d6)δ12.12(s,1H),9.23(s,1H),9.11(s,2H),8.75-8.63(m ,2H),8.25(t,J=5.7Hz,1H),8.18-8.00(m,2H),6.42(d,J=2.5Hz,1H),6.37-6. 26(m,2H),6.25-6.03(m,2H),5.34-5.12(m,1H),5.03(d,J=11.7Hz,1H),4.62- 4.56(m,2H),4.48(s,2H),4.29-4.15(m,3H),4.12-3.96(m,3H),3.75-3.70(m, 4H),3.62(d,J=4.1Hz,3H),3.50(d,J=5.3Hz,3H),3.41(s,3H),3.30-3.25(m, 3H),3.15-3.05(m,1H),2.92-2.63(m,3H),2.57(t,J=7.1Hz,2H),2.49-2.45(m ,2H),2.37(d,J=2.0Hz,1H),2.35-2.28(m,5H),2.24(dd,J=14.7,7.2Hz,3H),2 .19(d,J=2.5Hz,3H),2.06-2.03(m,3H),2.01-1.88(m,5H),1.87-1.67(m,3H).

[0528] Synthesis of compound LP8

[0529] Step 1: Synthesis of compound LP8-2

[0530] To a DMF (2 mL) solution of LP8-1 (48.5 mg, 0.08 mmol), HATU (30.4 mg, 0.08 mmol), compound 19 (50.0 mg, 0.06 mmol), HOAT (20.4 mg, 0.15 mmol), and TMP (54.5 mg, 0.45 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. The reaction was confirmed to be complete by LC-MS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP8-2 (40.0 mg, 56% yield).

[0531] Steps 2 to 3: Synthesis of compound LP8

[0532] LP8 was synthesized using a method similar to steps 3 and 4 of compound LP5.

[0533] MS m / z (ESI): 1429.2 [M-OH] +

[0534] 1 H NMR(400MHz,DMSO-d6)δ9.16-9.07(m,1H),9.10(s,2H),8.64-8.53(m,2H),8.3 3(dd,J=9.5,2.7Hz,1H),8.19(t,J=5.7Hz,1H),8.14(d,J=8.1Hz,1H),8.06(t, J=4.0Hz,1H),7.28-7.11(m,6H),6.49-6.23(m,3H),6.15-6.08(m,2H),5.32(t ,J=4.7Hz,1H),5.14-4.92(m,1H),4.83(d,J=10.4Hz,2H),4.73-4.58(m,3H),4 .50-4.48(m,1H),4.40-4.30(m,2H),4.27-4.03(m,3H),4.00-3.89(m,1H),3.7 7–3.70(m,5H),3.60(dd,J=17.7,5.3Hz,4H),3.49(d,J=3.4Hz,3H),3.41(s,3H ),3.09-3.03(m,3H),2.80-2.76(m,1H),2.71-2.61(m,3H),2.57(t,J=7.4Hz,3 H),2.43-2.26(m,9H),2.21-2.18(m,2H),2.10-1.91(m,7H),1.88-1.78(m,2H).

[0535] Synthesis of compound LP33

[0536] Step 1: Synthesis of compound LP33-2

[0537] At room temperature, 12 drops of TMP were added to a 2 mL solution of LP33-1 (24.3 mg, 45.0 μmol), HATU (142.5 mg, 375.0 μmol), and HOAT (127.5 mg, 937.6 μmol) in DMF, and the mixture was stirred for 5 minutes. Then, compound 18 (30.0 mg, 37.5 μmol) was added, and the mixture was stirred for another 30 minutes. The reaction was confirmed to be complete by LCMS. Ethyl acetate and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP33-2 (30.0 mg, 60% yield).

[0538] Steps 2 to 3: Synthesis of compound LP33-4

[0539] Add 2 drops of triethylamine to 2 mL of DMF containing 25.0 mg (18.9 μmol) of LP33-2 at room temperature and stir overnight. The reaction solution is used directly for the next step.

[0540] At 20°C, TMP (8 drops) was added to 2 mL of DMF containing LK18-I (29.8 mg, 21.6 μmol), HATU (68.4 mg, 180.0 μmol), and HOAT (48.9 mg, 359.9 μmol), and the mixture was stirred for 5 minutes. Then, the reaction solution from step 2 (LP33-3) was added, and the mixture was stirred for another 30 minutes. The reaction was confirmed to be complete by LC-MS. Dichloromethane and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP33-4 (20.0 mg, 45% yield).

[0541] Steps 4 to 5: Synthesis of compound LP33

[0542] At room temperature, add 4 drops of triethylamine to 2 mL of DMF containing 20.0 mg (8.1 μmol) of LP33-4 and stir overnight. The reaction solution is used directly for the next step.

[0543] At room temperature, TMP (8 drops) was added to 3 mL of DMF containing LP5-C (3.28 mg, 12.2 μmol), HATU (30.9 mg, 81.5 μmol), and HOAT (22.2 mg, 163.0 μmol), and the mixture was stirred for 5 minutes. Then, the reaction solution of LP33-5 from step 4 was added, and the mixture was stirred for another 30 minutes. The reaction was confirmed to be complete by LCMS. Dichloromethane and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse HPLC to obtain compound LP33 (7.0 mg, yield 34.6%).

[0544] MS m / z(ESI):1245.6, 1246.8[M / 2+H] +

[0545] 1H NMR(400MHz,DMSO-d6)δ9.17-9.09(m,3H),8.74-8.72(m,1H),8.68-8.64(m,1H),8.09-7.93(m,6H),7.90-7.87(m,2H),7.76-7.73(m,1H),7.7 1-7.68(m,1H),6.43-6.41(m,1H),6.35-6.26(m,2H),6.17-6.14(m,2H) ,5.06-5.03(m,1H),4.76-4.75(m,1H),4.69-4.47(m,6H),4.38-4.02(m ,16H),3.63-3.54(m,10H),3.52-3.48(m,44H),3.40(s,3H),3.39-3.3 4(m,10H),3.31-3.15(m,8H),3.06-2.98(m,3H),2.83-2.74(m,2H),2.5 7-2.54(m,4H),2.46-2.37(m,4H),2.31-2.27(m,5H),2.21-2.19(m,3H) ,2.14-2.04(m,7H),1.98(s,3H),1.91-1.66(m,7H),1.23-1.18(m,9H).

[0546] Synthesis of compound LP34

[0547] Step 1: Synthesis of compound LP34-1

[0548] LK18 (50.0 mg, 28.0 μmol) was dissolved in DMF (1.5 mL), and HATU (80.0 mg, 210.0 μmol), HOAt (80.0 mg, 588.0 μmol), TMP (0.1 mL), and compound 18 (44.0 mg, 56.0 μmol) were added sequentially. The reaction mixture was stirred at 35 °C for 10 min. LP34-1 (45.0 mg, yield 63.3%) was obtained by reverse HPLC purification.

[0549] Step 2: Synthesis of compound LP34-2

[0550] LP34-1 (40.0 mg, 15.7 μmol) was dissolved in DMF (1 mL), and TEA (27.0 mg, 266.8 μmol) was added at 20 °C and stirred overnight. The reaction was confirmed to be complete by LCMS. Dichloromethane and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to obtain compound LP34-2 (30.0 mg, yield 82%).

[0551] Step 3: Synthesis of compound LP34-3

[0552] LP34-2 (30.0 mg, 12.9 μmol) and LP5-C (7.0 mg, 26.1 μmol) were dissolved in DMF (1 mL), and HATU (10.0 mg, 26.3 μmol), HOAT (10.0 mg, 73.5 μmol), and TMP (3 drops) were added at room temperature. The resulting mixture was then stirred at room temperature for 1 minute. The reaction was confirmed to be complete by LCMS, and the reaction solution was purified by reverse column chromatography to obtain LP34-3 (20.0 mg, 39.57%).

[0553] Step 4: Synthesis of compound LP34

[0554] LP34-3 (20.0 mg, 7.8 μmol) was dissolved in nitromethane (2 mL), and zinc bromide (160.0 mg, 710.4 μmol) was added at room temperature and stirred for 30 minutes. The reaction solution was purified by reverse HPLC to obtain LP34 (5.0 mg, yield 25.56%).

[0555] MS m / z (ESI): 1260.5 [M / 2+H] +

[0556] 1H NMR (400MHz, DMSO-d6) δ9.11(s,3H),8.74(d,J=6.0Hz,1H),8.20(s,3H),8.04(d,J=7.0Hz,2H),7.75-7.7(m,2H),7.21(s,1 H),6.67(s,1H),6.42-6.34(m,3H),6.15(d,J=10.0Hz,3H),5.35-5.30(m,2H),5.10-5.00(m,2H),4.8-4.75(m,2H),4.48(s ,5H),4.40-4.31(m,6H),4.23-4.18(m,6H),4.10(s,2H),3.64-3.61(m,6H),3.50(s,44H),3.20-3.15(m,8H),3.05-3.00(m ,6H),2.70-2.65(m,5H),2.35-2.28(m,13H),2.28-2.18(m,7H),2.07-1.96(m,17H),1.88-1.77(m,10H),1.50-1.42(m,3H).

[0557] Synthesis of compounds LP30-P1 and LP30-P2

[0558] Using isomers of compound 18 (LCMS peak times 1.527 min and 1.515 min) as starting materials, compounds LP30-P1 (corresponding to P1 of compound 18) and LP30-P2 (corresponding to P2 of compound 18) were obtained by a similar synthetic method to that of compound LP33. Note: P1 is defined as LCMS peak time 1.557 min, and P2 is defined as LCMS peak time 1.556 min.

[0559] LP30-P1: MS m / z(ESI):1299.4, 1298.6[M / 2+H] +

[0560] 1H NMR(400MHz,DMSO-d6)δ9.33–9.21(m,1H),9.11(s,2H),8.74(s,1H),8.67–8.57(m,1H),8.45(s,1H),8.40-8.33(m,1H),8.25–8.12(m,4H),8.10-8.03(m,2H),7.92-7.87(s,1H),7.80-7.70(m,2H),7.29–7.12(m,7H),6.41(d,J=6.4Hz,1H),6.35(d,J=8.1Hz,2H),6.16(d,J=9.9Hz,2H),5.15-5.11(m,1H),5.07-5.00(m,1H),4.83-4.77(m,3H),4.65-4.57(m,3H),4.55-4.45(m,7H),4.43-4.15(m,13H),4.10(s,3H),4.03(d,J=10.2Hz,2H),3.74-3.69(m,5H),3.61(s,5H),3.60–3.54(m,9H),3.50(s,42H),3.21–3.13(m,3H),3.07-2.97(m,4H),2.84–2.65(m,7H),2.42-2.36(m,3H),2.35–2.30(m,6H),2.18(s,4H),2.11(dd,J=16.8,8.8Hz,5H),2.04(s,3H),1.98(s,4H),1.93–1.66(m,9H),1.50–1.41(m,1H).

[0561] LP30-P2:MS m / z(ESI):1299.4,1298.6[M / 2+H] +

[0562] 1H NMR(400MHz,DMSO-d6)δ9.23–9.14(m,1H),9.11(s,2H),8.78-8.73(m,1H),8.67–8.56(m,1 H),8.43–8.31(m,1H),8.23-8.13(s,3H),8.08-8.02(m,2H),7.92-7.85(m,1H),7.78-7.67( m,2H),7.27-7.15(m,6H),6.44-6.39(m,1H),6.35-6.27(m,2H),6.15(d,J=3.9Hz,2H),5.36 –5.25(m,2H),5.09–4.99(m,1H),4.78(d,J=3.9Hz,2H),4.66-4.58(m,3H),4.53–4.44(m,5H ),4.42–4.35(m,4H),4.34-4.27(m,3H),4.25–4.15(m,6H),4.13-4.03(m,3H),3.75-3.69(m ,8H),3.65-3.60(m,12H),3.50(s,42H),3.20-3.15(m,2H),3.08-2.97(m,4H),2.83-2.72(m ,3H),2.69-2.66(m,1H),2.41-2.37(m,3H),2.35-2.32(m,2H),2.31-2.27(m,4H),2.23-2.1 7(m,3H),2.15-2.07m,4H),2.04(s,3H),1.98(s,4H),1.92-1.65(m,7H),1.51–1.39(m,1H).

[0563] Synthesis of compound LP30

[0564] Using compound 18 as the starting material, compound LP30 was obtained by referring to a similar synthesis method as compound LP33.

[0565] MS m / z(ESI):1299.4,1298.6[M / 2+H] +

[0566] Synthesis of compound LP63

[0567] Compound LP63 was synthesized using a method similar to steps 4 and 5 of compound LP33.

[0568] MS m / z(ESI):1218.5,1217.6[M / 2+H] + .

[0569] Synthesis of compound LP4

[0570] Compound LP4 was synthesized using the same method as compound LP1.

[0571] MS m / z(ESI): 1222.2 [M+H] + .

[0572] Synthesis of compound LP61

[0573] To a DMF (2 mL) solution of LP1-1 (14.0 mg, 0.01 mmol), HATU (20.4 mg, 0.05 mmol), compound 19 (10.0 mg, 0.01 mmol), HOAT (20.4 mg, 0.15 mmol), and TMP (54.5 mg, 0.45 mmol) were added, and the mixture was stirred at 29 °C for 30 minutes. The reaction was confirmed to be complete by LC-MS. Dichloromethane and water were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by reverse HPLC to obtain compound LP61 (4.0 mg, yield 23%).

[0574] MS m / z(ESI):1373.1,1371.1[M-OH] +

[0575] 1 H NMR(400MHz,DMSO-d6)δ9.29-8.95(m,1H),8.65-8.58(m,2H),8.35-8.25(m,1H),8.13-8.07(m ,3H),7.23-7.19(m,6H),6.99(s,2H),6.53-5.96(m,6H),5.36-5.30(m,1H),5.13-5.02(m,1H), 4.84-4.80(m,1H),4.68-4.60(m,3H),4.55-4.45(m,2H),4.40-3.91(m,6H),3.72-3.50(m,13H) ,3.11-3.03m,5H),2.88-2.63(m,5H),2.39-1.96(m,16H),1.52-142(m,4H),1.24-1.19(m,2H).

[0576] Synthesis of compound LP62

[0577] Step 1: Synthesis of compound LP62-1

[0578] Compound LP30-3 (50.0 mg, 0.02 mmol) was dissolved in ACN / H2O (1 mL / 1 mL), and then nitric acid (80.0 mg) was added. The mixture was stirred overnight at room temperature in the dark. LCMS showed that the reaction was complete. Buffer solvent (2 mL, saturated solution of NaCl / NaHCO3, V / V 1 / 1) was added, and the mixture was stirred at room temperature for about ten minutes. The mixture was then filtered, and the filtrate was purified by reverse HPLC to obtain compound LP62-1 (30.0 mg, yield 61%).

[0579] Steps 2 to 3: Synthesis of compound LP62

[0580] Compound LP62 was synthesized using a method similar to steps 3 and 4 of compound LP30.

[0581] MS m / z(ESI): 1285.3 [(M-16) / 2] +

[0582] 1 HNMR(400MHz,DMSO-d6)δ9.18(d,J=4.4Hz,1H),9.10(d,J=6.1Hz,2H),8.68-8. 57(m,2H),8.34(t,J=7.4Hz,1H),8.18(d,J=5.8Hz,1H),8.14-8.12(m,2H),8.0 4(d,J=8.0Hz,2H),7.89(t,J=5.6Hz,1H),7.76-7.69(m,2H),7.30-7.03(m,5H) ,6.48-6.27(m,3H),6.15-6.08(m,2H),5.32(dd,J=12.2,7.1Hz,1H),5.17-4.94 (m,2H),4.77(d,J=3.6Hz,2H),4.68-4.60(m,3H),4.49(d,J=3.3Hz,3H),4.42- 3.92(m,5H),3.79-3.47(m,73H),3.41(s,3H),3.25-3.15(m,5H),3.09-2.95(m ,4H),2.81-2.67(m,4H),2.57-2.40(m,6H),2.43-2.39(m,3H),2.35-2.26(m,6 H),2.22-2.19(m,4H),2.14-2.08(m,7H),2.03-1.95(m,4H),1.93-1.76(m,3H).

[0583] Synthesis of compound LP38

[0584] Compound LP38 was synthesized using a similar method to that used for compound LP30. MS m / z (ESI): 1299.4, 1298.6 [M / 2+H] + .

[0585] Synthesis of compound LP29

[0586] Compound LP29 was synthesized using a method similar to step 5 of compound LP33. MS m / z (ESI): 1270.2 [M / 2+H] + .

[0587] Synthesis of compound LP35

[0588] Compound LP35 was synthesized using a method similar to step 5 of compound LP33.

[0589] MS m / z (ESI): 1322.2 [M / 2+H] + .

[0590] Synthesis of compound LP75

[0591] Step 1: Synthesis of compound LP75-2

[0592] Compound LP75-1 (23.7 mg, 36.32 μmol) and compound 37 (25.0 mg, 30.27 μmol) were dissolved in acetonitrile / water (2.1 mL, v / v 3 / 1), and K2CO3 (16.0 mg, 6.80 μmol) was added. The reaction mixture was stirred at 25 °C for 30 min. After the reaction was confirmed to be complete by LCMS, compound LP75-2 (16.0 mg, yield 53.3%) was purified by reversed-phase chromatography.

[0593] Steps 2 to 5: Synthesis of compound LP75

[0594] Compound LP75 was synthesized using a method similar to steps 2 through 5 of compound LP30.

[0595] MS m / z (ESI): 1282.7 [M / 2+H] + .

[0596] 1HNMR(400MHz,DMSO-d6)δ9.80(s,1H),9.11(s,2H),8.76(s,1H),8.50–8.47(m,1H),8.41-8.35(m,2H),8. 23–8.12(m,3H),8.08-8.03(m,2H),7.91–7.86(m,1H),7.79–7.69(m,2H),7.35(s,1H),7.32–7.28(m,1H) ,7.24-7.20(m,4H),7.18–7.13(m,1H),6.86(d,J=9.2Hz,1H),6.46(s,1H),6.25(s,1H),6.16(s,1H),5.0 9–5.01(m,1H),4.82(s,2H),4.55-4.42(m,2H),4.40(d,J=5.8Hz,2H),4.37-4.27(m,4H),4.25-4.18(m,5 H),4.16(s,2H),4.07(s,1H),4.03–3.97(m,1H),3.77(d,J=6.3Hz,2H),3.71(d,J=5.6Hz,3H),3.63(s,3H ),3.61–3.54(m,9H),3.50(s,36H),3.41(s,5H),3.24–3.10(m,7H),3.08-2.97(m,4H),2.82-2.75(m,3H) ,2.69–2.61(m,2H),2.43–2.37(m,3H),2.35-2.32(m,3H),2.28(s,3H),2.22(s,3H),2.15-2.07(m,5H),2 .04(s,4H),2.01(s,1H),1.98(s,3H),1.92-1.78(d,J=7.3Hz,5H),1.77–1.67(m,3H),1.52–1.39(m,1H).

[0597] Synthesis of compound LP56

[0598] Compound LP56 was synthesized using a method similar to that used for compound LP75.

[0599] MS m / z (ESI): 1297.6 [M / 2+H] + .

[0600] Synthesis of compound LP77

[0601] Step 1: Synthesis of compound LP77-2

[0602] Compounds LP77-1 (55.0 mg, 84.36 μmol), HOAt (55.0 mg, 404.41 μmol), and compound 19 (55.0 mg, 68.84 μmol) were dissolved in DMF (2.5 mL), and DIEA (25 drops) was added. The reaction mixture was stirred at 40 °C for one hour. The product was purified by reversed-phase chromatography to give compound LP77-2 (35.0 mg, yield 58.3%).

[0603] Steps 2 to 5: Synthesis of compound LP77

[0604] Compound LP77 was synthesized using a method similar to steps 2 through 5 of compound LP30.

[0605] MS m / z (ESI): 1241.1 [M / 2+H] + .

[0606] 1 HNMR(400MHz,DMSO-d6)δ8.72–8.56(m,2H),8.28–8.02(m,4H),7.94–7.56(m,5H),7.51–7.19(m,3H),6.70-6.55(m,1H),6.45-6.30(m,2H) ,6.20-6.08(m,2H),5.33(t,J=4.6Hz,1H),5.10-5.05(d,J=13.8Hz,2H),4.82-4.72(m,2H),4.55-4.45(m,4H),4.43–4.24(m,7H),4.23–4.0 0(m,5H),3.65–3.54(m,10H),3.50(s,42H),3.20-3.13(m,4H),3.06- 2.97(m,3H),2.41-2.37(m,4H),2.36–2.26(m,8H),2.23(s,2H),2.21– 2.02(m,12H),2.01-1.95(m,7H),1.91-1.80(m,3H),1.77(s,4H),1.7 6–1.64(m,4H),1.49-1.42(m,1H),1.34-1.1.28(m,4H),1.24(s,12H).

[0607] Synthesis of compound LP39

[0608] Compound LP39 was synthesized using a method similar to that used for compound LP30.

[0609] MS m / z (ESI): 1298.7 [M / 2+H] + .

[0610] Example 4: Preparation of ligand-drug conjugates

[0611] The antibodies used as ligands are prepared using conventional methods, such as vector construction followed by transfection into eukaryotic cells like HEK293 or CHO cells for purification and expression. Ligand-drug conjugates were prepared using anti-ADAM9 antibody DB1001 (prepared according to WO2024193692), anti-GPC-3 antibody DB1002 (prepared according to WO2006006693), anti-EGFR antibody DB1003 (prepared according to WO2002100348), and anti-BCMA antibody DB1004 (prepared according to US patent US9,273,141) as examples.

[0612] Amino acid sequence of anti-ADAM9 antibody DB1001

[0613] Heavy chain (SEQ ID NO:1)

[0614] Light chain (SEQ ID NO:2)

[0615] The complementarity-determining region and variable region sequences of antibody DB1001 are shown in the table below.

[0616] The amino acid sequence of the anti-GPC-3 antibody DB1002:

[0617] Heavy chain (SEQ ID NO:3)

[0618] Light chain (SEQ ID NO:4)

[0619] The complementarity-determining region and variable region sequences of antibody DB1002 are shown in the table below.

[0620] The amino acid sequence of the anti-EGFR antibody DB1003 is as follows:

[0621] Heavy chain (SEQ ID NO:5)

[0622] Light chain (SEQ ID NO:6)

[0623] The complementarity-determining region and variable region sequences of antibody DB1003 are shown in the table below.

[0624] The amino acid sequence of anti-BCMA antibody DB1004:

[0625] Heavy chain (SEQ ID NO:7)

[0626] Light chain (SEQ ID NO:8)

[0627] The complementarity-determining region and variable region sequences of antibody DB1004 are shown in the table below.

[0628] Preparation of ADC1.3

[0629] At 37°C, the prepared TCEP (10mM, 0.034mL, 0.337μmol) was added to the buffer solution of DB1001 monoclonal antibody (14.0mM succinate-sodium hydroxide + 108mM NaCl pH 7.4; 5mg, 10.0mg / mL, 0.034μmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours, and then cooled to room temperature.

[0630] Compound LP8 (0.59 mg, 0.408 μmol) was dissolved in 25 μL DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 22 °C for 2 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 20 mM histidine-acetic acid, pH 6.0) to obtain a solution of the exemplary product ADC1.1 (20 mM histidine-acetic acid, pH 6.0; 3.1 mg, 6.4 mg / mL, yield: 60%), which was stored at 4 °C.

[0631] LC-MS analysis and calculation yielded a Dar value of 5.42.

[0632] Following the preparation method of ADC1.3, the following compounds were synthesized using appropriate linker-payload.

[0633] Preparation of ADC2.1

[0634] At 37°C, the prepared TCEP (10mM, 0.034mL, 0.337μmol) was added to the buffer solution of DB1002 monoclonal antibody (14.0mM succinate-sodium hydroxide + 108mM NaCl pH 6.5; 5mg, 10.0mg / mL, 0.034μmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours, and then cooled to room temperature.

[0635] Compound LP5 (0.54 mg, 0.413 μmol) was dissolved in 25 μL DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 25 °C for 6 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 20 mM histidine-acetic acid, pH 6.0) to obtain a solution of the exemplary product ADC2.1 (20 mM histidine-acetic acid, pH 6.0; 3.5 mg, 5.4 mg / mL, yield: 70%), which was stored at 4 °C.

[0636] LC-MS analysis and calculation yielded a Dar value of 7.05.

[0637] Following the preparation method in AD2.1, the following compounds were synthesized using suitable linker-payload.

[0638] Preparation of ADC3.1

[0639] At 37°C, the prepared TCEP (10mM, 0.034mL, 0.337μmol) was added to the buffer solution of DB1003 monoclonal antibody (14.0mM succinate-sodium hydroxide + 108mM NaCl pH 6.5; 5mg, 10.0mg / mL, 0.034μmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours, and then cooled to room temperature.

[0640] Compound LP30 (1.07 mg, 0.413 μmol) was dissolved in 25 μL DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 25 °C for 6 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 20 mM histidine-acetic acid, pH 6.0) to obtain a solution of the exemplary product ADC3.1 (20 mM histidine-acetic acid, pH 6.0; 3.8 mg, 5.8 mg / mL, yield: 75%), which was stored at 4 °C.

[0641] LC-MS analysis and calculation yielded a Dar value of 7.68.

[0642] Following the preparation method in AD3.1, the following compounds were synthesized using suitable linker-payload.

[0643] Following the preparation method of AD3.1, the following control compounds were synthesized using Zalutumumab and IgG1 antibodies, respectively, and commercially available MC-GGFG-AM-DXD.

[0644] Preparation of ADC4.1

[0645] At 37°C, TCEP (10mM, 0.034mL, 0.337μmol) was added to the buffer solution of DB1004 monoclonal antibody (14.0mM succinate-sodium hydroxide + 108mM NaCl pH 6.5; 5mg, 10.0mg / mL, 0.034μmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours, and then cooled to room temperature.

[0646] Compound LP30 (1.03 mg, 0.413 μmol) was dissolved in 25 μL DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 25 °C for 6 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 20 mM histidine-acetic acid, pH 6.5) to obtain a solution of the exemplary product ADC4.1 (20 mM histidine-acetic acid, pH 6.5; 4.1 mg, 4.6 mg / mL, yield: 80%), which was stored at 4 °C.

[0647] LC-MS analysis and calculation yielded a Dar value of 7.22.

[0648] Following the preparation method in AD4.1, the following compounds were synthesized using suitable linker-payloads.

[0649] Example 5: In vitro proliferation inhibition test of compounds on OVCAR-3, BT474, LOVO and HCC1806 tumor cells

[0650] Test objective

[0651] To detect the inhibitory activity of drug compounds on the in vitro proliferation of OVCAR-3, BT474, LOVO, and HCC1806 tumor cells, cells were treated with different concentrations of the compounds in vitro and cultured for 6 days. CTG (Cellular Transmission Therapy) was then used to analyze the cell proliferation. The Luminescent Cell Viability Assay (Promega, catalog number: G7558) uses reagents to detect cell proliferation based on IC50. 50 The value was used to evaluate the in vitro activity of the compound.

[0652] Experimental methods (using OVCAR3 as an example)

[0653] 1. Cell culture: OVCAR-3 cells were cultured in 10% FBS RPMI-1640 medium.

[0654] 2. Cell preparation: Take OVCAR-3 cells in the logarithmic growth phase, wash them once with PBS, add 2-3 mL of trypsin to digest for 2-3 min. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to prepare a single-cell suspension.

[0655] 3. Cell plating: Mix the OVCAR-3 single-cell suspension thoroughly, and adjust the viable cell density to 2x10⁻⁶ cells / cells using cell culture medium. 3 Cells / ml: After adjusting the cell density, mix the cell suspension thoroughly and add 50 μL / well to a 96-well cell culture plate. Incubate the plate in an incubator for 18 hours (37°C, 5% CO2).

[0656] 4. Compound preparation: Dissolve the compound in DMSO to prepare a stock solution with an initial concentration of 10 mM. There are 9 concentrations of small molecule compounds, with the highest concentration being 1 μM, diluted 3 times.

[0657] 5. Sample addition procedure: Add the prepared test samples at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2).

[0658] 6. Color development procedure: Take out the 96-well cell culture plate, add 50 μL of CTG reagent to each well, and incubate at room temperature for 10 minutes.

[0659] 7. Plate reading procedure: Take out the 96-well cell culture plate, place it in the microplate reader, and use the microplate reader to measure the chemiluminescence.

[0660] Data analysis: The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[0661] Table 1 shows the IC50 values ​​of the compounds in this application for inhibiting the in vitro cell proliferation of the aforementioned cells. 50 value

[0662] Note: " / " indicates that it was not detected.

[0663] Conclusion: The results show that the compound in this application has strong inhibitory activity against the proliferation of OVCAR-3, BT474, LOVO and HCC1806 cells, and its activity is not weaker than or better than that of the control compound trabectedin.

[0664] Example 6: Toxicological study of different compounds administered intravenously to SD rats

[0665] Experimental Objective

[0666] Sprague-Dawley rats were given a single intravenous injection of three test products. The acute toxicity of the three test products in SD rats was observed to compare their toxic effects and provide data support for subsequent safety evaluation.

[0667] Table 2 Experimental Scheme

[0668] Under the conditions of this experiment, SD rats were given a single intravenous administration of 0.3 mg / kg of trabectin and 3.6 mg / kg of compound 10R or 10S (peak 2).

[0669] Phenomena: On the second day after administration, male rats in the G1 group showed perianal soiling due to loose stools, while female rats exhibited piloerection, arched backs, reduced activity, and reddish discharge around the eyes and nose. All animals in the G1 group died on the second day after administration. Clinical pathological results showed that the G1 group exhibited decreased levels of WBC, PLT, LYMP, MONO, and RET; elevated serum ALT, AST, GGT, DBIL, and TBIL; and decreased K and Cl. Autopsy revealed yellowing in multiple tissues (inner skin, thymus, gastrointestinal tract, and uterus). Female animals also showed hepatomegaly, indicating potential hepatotoxicity of trabectin. Autopsy revealed intestinal hemorrhage, perianal soiling, and thin stomach walls. Male animals also showed punctate protrusions on the intestinal surface; the thymus and spleen were smaller; female animals also showed pulmonary hemorrhages and patchy gray spots on the heart.

[0670] The G2 administration group was well tolerated, with no significant changes in body weight or food intake.

[0671] Conclusion: Trabectin was intolerable in both male and female SD rats at 0.3 mg / kg. Compounds 10R or 10S (peak 2) were well tolerated at 3.6 mg / kg, and their toxicity was significantly better than that of trabectin.

[0672] Example 7: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

[0673] use Chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of antibody-drug conjugates against ADAM9, GPC-3, EGFR, and BCMA on cell proliferation after incubation for 6 days in ADAM9, GPC-3, EGFR, and BCMA-positive cells, respectively.

[0674] Logarithmic growth phase cells were collected and cultured at a density of 6000 cells / well in 96-well cell culture plates. The plates were incubated overnight at 37°C with 5% CO2. On the second day of the experiment, the ADC drug of camptothecin was diluted 3-fold with complete culture medium to obtain 9 concentration gradients (starting from the highest concentration of 300 nM). 100 μL of the drug was added to each well of the cell culture plate, with complete culture medium used as a blank control. Three replicates were set up. The plates were incubated at 37°C with 5% CO2 for another 6 days. After incubation, the cell culture plates were removed and equilibrated to room temperature. 50 μL of CTG assay reagent (Promega, Cat#: G7573) was added to each well. After vortexing and incubation in the dark for 10 minutes, the signal value was read using a microplate reader. GraphPad Prism software was used to plot the sigmoid dose-response curve using a nonlinear regression model and the IC50 was calculated. 50 Value. Cell viability calculation formula = (Lum) 待测药 -Lum 空白对照 ) / (Lum 溶剂空白对照 -Lum 空白对照 )×100%.

[0675] Table 3: Inhibitory activity of antibody-drug conjugates on the proliferation of human HCT116 cells

[0676] Note: +++ indicates IC 50 ≤15nM; ++ means 15 < IC 50 <50nM; + indicates IC 50 ≥50nM.

[0677] Table 4: Inhibitory activity of antibody-drug conjugates on the proliferation of human Huh7 cells

[0678] Note: ++++ indicates IC 50 ≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[0679] Table 5: Inhibitory activity of antibody-drug conjugates on the proliferation of human MDA-MB-468, HCC827, and MDA-MB-231 cells.

[0680] Note: ++++ indicates IC 50 ≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[0681] Table 6: Inhibitory activity of antibody-drug conjugates on the proliferation of human MM.1R cells

[0682] Note: ++++ indicates IC 50 ≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[0683] Experimental conclusion: The antibody-drug conjugates of this application have significant inhibitory activity against human cancer cells that are positive for ADAM9, GPC-3, EGFR and BCMA.

[0684] Example 8: Bystander Killing Effect

[0685] Experimental objective: To investigate the killing effect of ADC3.1 under co-culture conditions of EGFR-positive and EGFR-negative tumor cells.

[0686] Test methods

[0687] 1. Fluorescently labeled cells

[0688] 1) Collect cells in the exponential growth phase and count live cells using a cell counter.

[0689] 2) Adjust the cell suspension to a certain cell density using the appropriate culture medium.

[0690] 3) Prepare the fluorescent labeling solution by premixing 1 μL of tracker@components A and B in a 1.5 ml EP tube. Incubate at room temperature and immediately proceed to step 4).

[0691] 4) Add 0.2 mL of fresh complete culture medium to the EP tube and vortex for 30 seconds.

[0692] 5) Add 1x10 6 The cells were added to the fluorescently labeled solution prepared above.

[0693] 6) Incubate at 37℃ for 60 minutes.

[0694] 7) Detect cell labeling under a fluorescence microscope.

[0695] 8) Wash twice with complete culture medium. Set aside for use.

[0696] 2. Cell inoculation

[0697] 1) Set up the following three cell combinations in a 96-well cell culture plate as shown in Table 7:

[0698] Table 7 Cell Combinations (+ indicates that this type of cell is present in the cell combination).

[0699] 2) Add 90 μL of cell suspension to each well of a 96-well plate.

[0700] 3) Incubate the 96-well plate overnight in a 37°C, 5% CO2 incubator.

[0701] 3. Chemical treatment

[0702] 1) Prepare 10× drug diluent.

[0703] 2) Add diluted candidate molecule and positive control to each well, with 3 replicates for each.

[0704] 3) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 4 days.

[0705] 4. Testing

[0706] 1) Observe and record the images of each group under a fluorescence microscope.

[0707] 2) Following the CTG operating instructions, add pre-melted and equilibrated CTG solution to each well, mix well with a microplate shaker, and measure the fluorescence signal value using a plate reader after standing at room temperature for a period of time.

[0708] 3) Cell viability is expressed as mean fluorescence signal value (treatment group) / mean light signal value (control group) × 100%.

[0709] Experimental Conclusions: As shown in Figures 1-3, under the experimental conditions, the ADC3.1 molecule of this application exhibits significant killing activity against EGFR-positive cells, but no significant inhibitory effect on EGFR-negative cells. In cells co-cultured with EGFR-positive and EGFR-negative cells, the ADC molecule of this application can simultaneously and significantly inhibit both EGFR-positive and EGFR-negative cells, demonstrating a significant bystander killing effect. Other ADCs of this application also exhibit similar bystander killing effects.

[0710] Example 8: In vivo tumor suppression test of antibody-drug conjugates

[0711] 8.1 Evaluation of the pharmacodynamics of ADC drugs in a subcutaneous transplantation of Huh7 cell line model in NCG mice

[0712] 1. Test drug and materials

[0713] Blank control group (control group): physiological saline

[0714] ADC (treatment group): 2 mg / kg, administered twice.

[0715] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0716] 3. Experimental animals: 6-8 week old female NPG mice, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0717] 4. Test methods:

[0718] 5×10 6 One Huh7 cell was subcutaneously injected into the right anterior back of 6-8 week old female NPG mice. When the tumor grew to approximately 130 mm... 3 StudyDirector TM Patients were randomly assigned to groups and began receiving intravenous (iv) injections of the ADC drug on day 0 (day 0), once a week for a total of two injections, at a dose of 2 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0719] Five mice were included in each of the solvent control group and the treatment group. The tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).

[0720] Table 8: In vivo tumor-inhibiting effect of antibody-drug conjugate ADC2.3 on Huh7 tumor-bearing mice

[0721] The experimental results are shown in Table 8 and Figure 4. The antibody-drug conjugate ADC2.3 showed significant antitumor activity after administration, and there was no significant change in body weight.

[0722] 8.2 Evaluation of the pharmacodynamics of ADC drugs in the subcutaneous transplantation of HCC827 cell line model in CB-17 severely immunodeficient mice

[0723] 1. Test drug and materials

[0724] Blank control group (control group): physiological saline

[0725] ADC (treatment group): 5 mg / kg, administered once.

[0726] ADC Control 1 (Control Group): 5 mg / kg, single dose

[0727] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0728] 3. Experimental animals: 6-8 week old female CB-17 severely immunodeficient mice, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0729] 4. Test methods:

[0730] 5×10 6 One HCC827 cell was subcutaneously instilled into the right anterior back of 6-8 week old female CB-17 severely immunodeficient mice. When the tumor grew to approximately 170 mm... 3 StudyDirector TM Patients were randomly assigned to groups and began receiving intravenous (iv) injections of the ADC drug on day 0 (day 0), once a week for a total of one injection, at a dose of 5 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0731] Five mice were included in each of the solvent control group and the treatment group. The tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).

[0732] Table 9: In vivo tumor-inhibiting effect of antibody-drug conjugate ADC3.3-P2 on HCC827 tumor-bearing mice

[0733] The experimental results are shown in Table 9 and Figure 5. The antibody-drug conjugate ADC3.3-P2 showed significant antitumor activity after administration, which was slightly better than ADC control 1, and there was no significant change in body weight.

Claims

1. A ligand-drug conjugate, its tautomers, its enantiomers, its diastereomers, its pharmaceutically acceptable salts, or solvates thereof, characterized in that, The ligand-drug conjugate comprises a ligand with a structure shown in formula Ia, Ib, Ic, or Id: in, R 1a R 5a R 2a and R 3a Each independently selected from -VC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-(4- to 8-membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-OU-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-,-VC 0-6 Alkylene-N(R) 6 C(O)-(4-8 membered heterocyclic alkylene)-U-,-VC 0-6 Alkylene-N(R) 6 )C(O)OC 0-6 Alkylene-U-, -VC 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)OC 3-8 Cycloalkyl-U-,-VC 0-6 Alkylene-N(R) 6 C(O)O-(4-8 membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 3-8 Cycloalkyl-U-,-VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-(4- to 8-membered heterocyclic alkylene)-U-, -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 1-6 Alkylene-U-, -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 3-8 Cycloalkylene-U-, -V-(4- to 8-membered heterocycloalkylene)-C(O)-(4- to 8-membered heterocycloalkylene)-U-, -VC 3-8 Cycloalkyl-N(R) 6 )C(O)-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-,-VC 3- 8-cycloalkylene-N(R) 6 C(O)-(4-8 membered heterocyclic alkylene)-U-,-VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-C 3-8 Cycloalkyl-U,-VC 0-6 Alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U-, -VC 0-6 Alkylene-N(R) 6 S(O)2-C 0-6 Alkylene-U- and -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 alkylene-U-; the C 0-6 Alkylene, C 3-8 The cycloalkyl group and the 4- to 8-membered heteroalkyl group are each optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, halogenated C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups; U and V are each independently selected from -O-, -S-, and -NR-. 6 -or chemical bonds; R 6 It is hydrogen, deuterium, hydroxyl, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 Cycloalkyl or 4- to 8-membered heterocyclic alkyl; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 The cycloalkyl group and the 4- to 8-membered heterocycloalkyl group are each optionally separated by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, halogenated C 1-6 Alkyl and C 3- Substituents of 6-cycloalkyl groups; R 4 For hydrogen, deuterium, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, -C(O)C 1-6 Alkyl, -C(O)C 3-12 Cycloalkyl or -C(O) (4- to 8-membered heterocycloalkyl), wherein the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-12 The cycloalkyl group and the 4- to 12-membered heterocycloalkyl group are each optionally separated by one or more elements selected from halogen, -OH, -SH, -NH2, -NHC. 1-6 Alkyl and -SC 1-6 Alkyl substituents; Or R 4 R 3a R on 6 The group and the atoms attached thereto form a 5- to 8-membered heterocyclic alkyl group; each of the 5- to 8-membered heterocyclic alkyl groups is optionally bonded by one or more groups selected from halogens, oxo groups, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2, C 1-6 Alkyl, -C 1-6 Alkylene -OH, -C(O)C 1-6 Alkylene -OH, -C(O)-C 3-12 Substituents of cyclohexene alkyl-OH and -C(O)-(4- to 8-membered heterocyclohexene alkyl)-OH; R 1b Hydrogen, deuterium, halogen, -OH, -NH2, -SH, C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -SC 1-6 Alkyl, C 3- 6-cycloalkyl, -OC 3-6 cycloalkyl, -NHC 3-6 Cycloalkyl, -NH (4-6 membered heterocycloalkyl), -NHC(O)C 1-6 Alkyl or -NHS(O)2C 1-6 Alkyl, the C 1-6 Alkyl, C 3-6 The cycloalkyl group and the 4- to 6-membered heterocycloalkyl group are each optionally separated by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, C. 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups; Alternatively, when the ligand-drug conjugate includes formula Ia, the two R... 1b Together with the atoms attached to it, they form C 3-6 cycloalkyl; the C 3-6 The cycloalkyl group is optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, C. 1-6 Alkyl substituents; Q 1 and Q 2 Each is independently N or CR 5b ; R 5b Hydrogen, deuterium, halogen, -OH, -CN, -NO2, C 1-6 Alkyl, -OC 1-6 Alkyl, C 3-6 cycloalkyl, -OC 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, or -O (4-6 membered heterocycloalkyl), wherein the C 1-6 Alkyl, C 3-6 The cycloalkyl group and the 4- to 6-membered heterocycloalkyl group are each optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, C. 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups; Y can be either -OH or -CN.

2. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 1, is characterized in that... The structures represented by formula Ia, Ib, Ic, or Id satisfy one or more of the following conditions: (1)R 1a -N(CH3)- a -N(R 6 )C(O)-C 1-6 alkylene-U- a -O-(4-8 membered heterocyclic alkylene)-C(O)-C 1-6 Alkylene-U, a -NHC(O)-(C 1-6 alkylene)-ON(C 1-6 alkyl)-, -N(R 6 )C(O)-C 3-8 Cycloalkyl-U-, a -NH-(4-8 membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U- a -NH-C 3-8 Cycloalkyl-U, a -N(R 6 )C(O)OC 1-6 alkylene-U- a -N(R 6 )C(O)N(R 6 )-C 1-6 alkylene-U- a -N(R 6 S(O)2-C 1-6 alkylene-U- a -N(R 6 )C(O)-(4-8 membered heterocyclic alkylene)-U- or a -OC(O)N(R 6 )-C 1-6 alkylene-U-, α-terminus with Connection; the C 1-6 Alkylene is optionally surrounded by -OH or C 3-6 Cycloalkyl substitution; (2)R 5a -N(R) 6 )C(O)-C 1-6 Alkylene-U-, -NHC(O)-(C 1-6 alkylene)-ON(C 1-6 alkyl)-, -N(R 6 )C(O)-C 3-8 Cycloalkylene-U-,-N(R) 6 )C(O)OC 1-6 Alkylene-U-, -OC(O)N(R) 6 )-C 1-6 Alkylene-U-, -OC 3-8 Cycloalkylene -U-, -O-(4- to 8-membered heterocycloalkylene)-U-, -O-(4- to 8-membered heterocycloalkylene)-C(O)-C 1-6 Alkylene-U- or -NH- (4- to 8-membered heterocyclic alkylene)-C(O)-C 1-6 alkylene-U-, b-terminus with Connection; the C 1-6 Alkylene is optionally C 3-6 Cycloalkyl substitution; R 6 It is hydrogen or C 1-6 alkyl; (3)R 2a for c -C 0-6 alkylene-U- c -N(R 6 )C(O)-C 0-6 alkylene-U- c -N(R 6 )C(O)N(R 6 )-C 0-6 alkylene-U- c -N(R 6 )C(O)OC 0-6 alkylene-U- c -OC(O)N(R 6 )-C 0-6 alkylene-U- c -N(R 6 S(O)2-C 0-6 alkylene-U- c -N(R 6 )C(O)-C 3-8 Cycloalkyl-U-, c -C(O)N(R 6 )-C 0-6 alkylene-U- c -C(O)-(4-8 membered heterocyclic alkylene)-U- or c -N(R 6 )C(O)-C 0-6 alkylene-OU-, with the c-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C 3-6 Cycloalkyl substitution; (4)R 3a for d -C 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 alkylene-U- d -C 0-6 Alkylene-N(R) 6 )C(O)-C 3-8 Cycloalkyl-U-, d -C 0-6 Alkylene-OC(O)N(R) 6 )-C 0-6 alkylene-U- d -C 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 alkylene-U- d -C(O)-(4-8 membered heterocyclic alkylene)-U- or d -C 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 The alkylene group (OU-) has a d-terminus connected to a piperidine ring; the C 0-6 Each alkylene group is optionally C 3-6 Cycloalkyl substitution; (5)R 4 R 3a R on 6 The group and the atoms attached to it together form 5- to 8-membered heterocyclic alkyl groups; (6)R 6 It is hydrogen or C 1-6 alkyl; (7)R 4 It is hydrogen or C 1-6 alkyl; (8)R 1b For hydrogen, -OH, -NH2, C 1-6 Alkyl or -OC 1-6 Alkyl; the C 1-6 Alkyl groups may be optionally substituted with -OH; (9)R 5b For hydrogen or -OC 1-6 alkyl; (10) Y is -CN.

3. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 2, is characterized in that... The structures represented by formula Ia, Ib, Ic, or Id satisfy one or more of the following conditions: (1)R 1a -NH-, -O-, a -NH-CH2-CH2-O-, -O-CH2-CH2-O-, a -NH-C(O)-CH2-O-、 a -NH-C(O)-CH2-NH- or a -O-CH2-C(O)-O-, α-terminus and connect; (2)R 1a for a end and connect; (3)R 5a -NH-, -O-, b -NH-CH2-CH2-O-, -O-CH2-CH2-O-, b -NH-C(O)-CH2-O-、 b -NH-C(O)-CH2-NH- or b -O-CH2-C(O)-O-, b-terminus and connect; (4)R 5a for B-end and connect; (5)R 2a -NH-, -O-, c -CH2O-、 The c-terminus is connected to the piperidine ring; (6)R 3a for The d-end is connected to the piperidine ring; (7)R 3a For -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 When alkylene-U-, R 4 R 3a R on 6 Groups and the atoms attached to them together form 4. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 1, is characterized in that... The structure represented by formula Ia, formula Ib, formula Ic, or formula Id is any of the following structures:

5. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 1, characterized in that, The ligand-drug conjugate comprises the structure shown in formula IIa, IIb, IIc or IId: Among them, R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definitions of Y and L are as described in any one of claims 1-4; 1a The end is connected to the ligand; L 1a for L 2 -(C(R) L21 )2) n -; n is a natural number from 0 to 50; L 2 Any C(R) in L21 The )2 unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR L22 -, -O-, -S-, -SO-, -SO2-, -P(R L22 )-、-P(=O)(R L22 -, -C(=S)-, -C(=NR) L22 )-, -N=N-, -C=N-, -N=C-, -Cy- is a phenylene, a 5- to 8-membered heteroaryl, a 3- to 10-membered heterocyclic alkyl, or a 3- to 10-membered cycloalkyl, wherein -Cy- is optionally surrounded by one or more R- cx replace; R L21 R L22 and R cx Each independently is -(C(R) L2a )2) m -R L2b m is a natural number from 0 to 50; R L21 R L22 and R cx Any C(R) in L2a The )2 unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(R)-. L2a )-, -N(CH3)-, -O-, -S-, -SO- or -SO2-; R L2a -(CH2) y -R L2b y is a natural number from 0 to 50; R L2a Any CH2 unit in the structure can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, -N(R)-. L2a )-, -O-, -S-, -SO- or -SO2-; R L2b Each can be independently represented as hydrogen, deuterium, halogen, -NO2, -CN, -N(Me)2, -N + (Me)3, -COOH, -S(O)2OH, -P(O)(OH)2, glycosyl or its derivatives, C 2-6 alkenyl, C 2-6 Alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl; L 3 For L 3a -L 3b ; L 3a and L 3b Independently, it is a short peptide composed of 2-10 amino acid residues, which is absent. Or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; Tr represents non-existent Or any combination of the above groups; R Tr Independently selected from hydrogen, deuterium, halogens, -NO2, -CN, -OH, -SH, -NH2, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -OC(O)NH2, -CH2CO-(N(Me)CH2C(O)) z -OR Tra -CH2CO-(N(Me)CH2C(O)) z -NHR Tra -(CH2CH2O) z -R Tra -CONH-(CH2CH2O) z -R Tra C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne group, 3-8 membered cycloalkyl group, 4-10 membered heterocycloalkyl group, 6-10 membered aryl group or 5-10 membered heteroaryl group, wherein C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl, and 5-10 membered heteroaryl are optionally surrounded by one or more R Tra replace; R Tra Independently, it can be hydrogen, deuterium, halogen, -NO2, -CN, -OH, -SH, -NH2, -N(Me)2, -S(O)2Me, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl, z is an independent natural number between 0 and 50; The heteroatoms in the heterocyclic alkyl, heteroaryl, heterocyclic alkyl and heteroaryl groups are selected from one, two or three of N, O and S; the number of heteroatoms is 1, 2 or 3.

6. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 5, is characterized in that... The structures represented by formulas IIa, IIb, IIc, or IId satisfy one or more of the following conditions: (1)L 1a for e-end and L 2 connect; (2)L 2 for f end and L 3 connect; n1, n2, and n3 are each independent natural numbers from 0 to 8; m1 is a natural number from 0 to 16; m2 is a natural number from 1 to 5; y1 is a natural number from 1 to 5; G is non-existent. Or -NH-, g end with L 1a connect; (3)L 3a is absent, Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Ser, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Val-Glu, Val-Asp, Val-Ser, Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Lys, Ala-Asn, Ala-Gln, Ala-Gly, Ala-Ser, D-Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Gly-Ser, Gly-Gln, Glu-Gly, Glu-Gln, Glu-Ser, Glu-Asn, Gln-Gly, Gln-Ser, Asp-Gly, Asn-Asn, Asp-Glu, Asp-Ser, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gly-Ala-Ala, Gly-Val-Ala, Gly-Val-Cit, Glu-Val-Cit, Ala-Ala-Ala, Ala-(D-Ala)-Ala, Ala-Ala-Asn, Ala-(D-Ala)-Asn, Ala-Ala-Asp, Val-Lys-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Gly-Gly-Gly, Lys-Ala-Asn, Gly-Phe-Gly, Gly-Gly-Phe, Asn-Pro-Val, Ala-Lys-Gly, Gly-Lys-Gly, Gly-Glu-Gly, Glu-Gly-Ser, Glu-Ala-Ser, (Gly)4, (Gly)2-Phe-Gly, (Gly)2-Glu-Gly, Lys-(Ala)2-Asn, Lys-(Ala)2-Asp, Gly-Leu-Gly-Lys, (Ala)2-Pro-Val or (Ala)2-Pro-Nva; (4)L 3b For non-existent The j-end is connected to Tr; (5) Tr indicates that it does not exist. i-end and L 3 Connection; R Tr Independently, it can be hydrogen, halogen, -NO2, -CN, -OH, -NH2, -CO2H, -S(O)2OH, -C(O)NH2, -SO2NH2, -OC(O)NH2, or -CH2CO-(N(Me)CH2C(O)). z -NHMe, -(CH2CH2O) z -H or -CONH-(CH2CH2O) z -H; z is a natural number from 0 to 8.

7. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 1, is characterized in that... The ligand-drug conjugate is a ligand-drug conjugate as shown in Formula IIIa, Formula IIIb, Formula IIIc or Formula IIId: Wherein, Ab is the ligand that binds to the target; q represents the drug loading rate; R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definition of Y is as described in any one of claims 1-4; L 1a L 2 L 3 The definition of Tr is as described in claim 5 or 6.

8. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 7, is characterized in that... The ligand-drug conjugates represented by formula IIIa, IIIb, IIIc or IIId satisfy one or two of the following conditions: (1)Ab is an antibody or antigen-binding fragment thereof that targets HER3, B7H3, Claudin18.2, CD30, CD33, CD70, BCMA, GPC-3, ADAM9 and EGFR; (2) q is an integer or decimal from 1 to 32, such as 2, 3, 4, 5, 6, 7, 8, 16, 3.87, 3.97, 4.05, 4.11, 4.23, 4.26, 4.31, 4.32, 5.42, 6.12, 6.84, 6.93, 7.05, 7.22, 7.23, 7.25, 7.29, 7.31, 7.34, 7.36, 7.38, 7.45, 7.48, 7.51, 7.52, 7.53, 7.57, 7.58, 7.59, 7.62, 7.65, 7.68, 7.71, 7.75, 7.78, 7.82, or 7.

86.

9. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 7, is characterized in that... The ligand-drug conjugates represented by Formula IIIa, Formula IIIb, Formula IIIc or Formula IIId are any of the ligand-drug conjugates in Table B.

10. The ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or its solvate, as described in claim 7, characterized in that... The ligand-drug conjugates represented by Formula IIIa, Formula IIIb, Formula IIIc or Formula IIId are any of the ligand-drug conjugates in Table C.

11. A mixture of ligand-drug conjugates comprising, as described in any one of claims 1-10, a tautomer thereof, an enantiomer thereof, a diastereomer thereof, a pharmaceutically acceptable salt thereof, or a solvation thereof, wherein, The ligand-drug conjugate has one, two, or more of the aforementioned q values; preferably, the average drug-to-ligand ratio of the ligand-drug conjugate is selected from integers or decimals of 1-32, such as 2, 3, 4, 5, 6, 7, 8, 16, 3.87, 3.97, 4.05, 4.11, 4.23, 4.26, 4.31, 4.32, 5.42, 6.12, 6.84, 6.93, 7.05, 7.22, 7.23, 7.25, 7.29, 7.31, 7.34, 7.36, 7.38, 7.45, 7.48, 7.51, 7.52, 7.57, 7.62, 7.65, 7.68, 7.75, 7.78, 7.82, or 7.

86.

12. A compound represented by formula IVa, IVb, IVc and IVd, its tautomers, its enantiomers, its diastereomers or pharmaceutically acceptable salts thereof, in, L 1 for R 1a R 5a R 2a R 3a R 4 R 1b Q1, Q 2 The definitions of Y and L are as described in any one of claims 1-4; 2 L 3 The definition of Tr is as described in claim 5 or 6.

13. The compounds of formulas IVa, IVb, IVc, and IVd as claimed in claim 12, their tautomers, their enantiomers, their diastereomers, or their pharmaceutically acceptable salts, characterized in that, The compounds represented by formula IVa, IVb, IVc or IVd are any of the compounds in Table D.

14. A pharmaceutical composition comprising a ligand drug conjugate as described in any one of claims 1-10, a tautomer thereof, an enantiomer thereof, a diastereomer thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, or a mixture of ligand drug conjugates as described in claim 11, and a pharmaceutically acceptable excipient.

15. Use of a substance S in the preparation of a medicament for the prevention or treatment of cancer; said substance S is a ligand-drug conjugate as described in any one of claims 1-10, its tautomers, its enantiomers, its diastereomers, its pharmaceutically acceptable salts or solvates thereof, a mixture of ligand-drug conjugates as described in claim 11, or a pharmaceutical composition as described in claim 14; said cancer is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors, prostate cancer, or thyroid cancer.

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