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

Abstract

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

Description

Seaweed extract compounds, their conjugates, preparation methods, and uses Technical Field

[0001] This invention relates to a sucrose compound, its conjugates, its preparation method, and its uses. Background Technology

[0002] 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 minor groove of DNA, 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.

[0003] 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 both normal and tumor cells, as well as the high efficiency of cytotoxic drugs, while avoiding the drawbacks of lower efficacy with antibodies and excessive toxicity with cytotoxic drugs. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells, thus exhibiting greater efficacy and a wider therapeutic window.

[0004] By leveraging the differences in surface antigen expression between tumor cells and normal cells, ecteinascidin compounds can be combined with antibodies that bind to tumor cell surface antigens to construct antibody-drug conjugates. This not only improves the clinical efficacy and tolerability of ecteinascidin 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.

[0005] Therefore, it is necessary to develop new seasorghum ligand conjugates for the treatment of tumor-related diseases. Summary of the Invention

[0006] 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.

[0007] In a first aspect, the present 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 and a structure as shown in Formula I:

[0008] in, for The * terminal is connected to the carbonyl group, and the ** terminal is connected to the methylene group;

[0009] R is independently selected from -VC 0-6 Alkylene-UC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkylene-OC 0-6 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 3-8 Cycloalkyl-U-,-VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-(4- to 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-, -VOC 0-6 Alkylene-C(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 0-6 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-, -V-(4- to 8-membered heterocycloalkylene)-N(R) 6 )C(O)-C 0-6 Alkylene-U-, -V-(4- to 8-membered heterocyclic alkylene)-N(R) 6 )C(O)-C 3-8 Alkylene-U-, -V-(4- to 8-membered heterocyclic alkylene)-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-(4- to 8-membered heterocyclic alkylene)-C(O)-U- or -VC 0-6 Alkylene-S(O)2N(R) 6 )-C 0-6 alkylene-U-; the C 0-6 Alkylene, C 3-8The 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 group, C 1-6 Halogenated alkyl groups and C 3-6 Substituents of cycloalkyl groups;

[0010] U and V are independently -O-, -S-, and -NR, respectively. 6 -or chemical bonds;

[0011] R 6 It is hydrogen, deuterium, hydroxyl, C 1-6 Alkyl, C 3-8 Cycloalkyl or 4- to 8-membered heterocyclic alkyl; the C 1-6 Alkyl, 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, Halogenated C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups;

[0012] R 4 For hydrogen, deuterium, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)C 3-12 Cycloalkyl or -C(O) (4- to 12-membered heterocycloalkyl), wherein the C 1- 6-alkyl, 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;

[0013] Or when for At that time, R 4 R on R 6 The group, together with the atoms attached to it, forms a 5-12 membered heterocyclic alkyl group; the 5-12 membered heterocyclic alkyl group 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 1-6Alkylene -OH, -C(O)C 1-6 Alkylene -OH, -C(O)-C 3-12 Substitution of cyclohexene alkyl-OH and -C(O)-(4-12 membered heterocyclohexene alkyl)-OH;

[0014] R 5 Hydrogen, deuterium, halogen, -OH, -CN, -NH2, C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2, -C 3-6 cycloalkyl, -OC 1-6 Alkyl, 4-6 membered heterocyclic alkyl, -O (4-6 membered heterocyclic alkyl), -CONH2, -CONH (C 1-6 Alkyl), -CON(C) 1- 6-alkyl)2, -OCONH2, -OCONH(C 1-6 Alkyl), -OCON(C) 1-6 Alkyl)2、-NHCOO(C 1-6 alkyl) or -N(C) 1-6 Alkyl)COO(C 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;

[0015] Or when for At that time, two R 5 Together with the atoms attached to it, they form C 3-6 cycloalkyl; the C 3-6 The cycloalkyl group is optionally separated by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, and C. 1-6 Alkyl substituents;

[0016] Y is either -OH or -CN;

[0017] The heteroatoms in the heterocyclic alkyl and heterocyclic alkyl groups are selected from one, two, or three of N, O, and S; the number of heteroatoms is one, two, or three.

[0018] In some implementation schemes, R 6 It is hydrogen, hydroxyl or C 1-6 Alkyl; the C 1-6Each alkyl group is optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, -NHC. 1-6 Alkyl, -N(C) 1-6 Alkyl)2-substituent.

[0019] In some implementation schemes, R 6 It is hydrogen or C 1-3 alkyl.

[0020] In some implementation schemes, R 4 It is hydrogen or C 1-6 Alkyl, the C 1-6 Each alkyl group is optionally surrounded by one or more elements selected from halogens, -OH, -SH, -NH2, and -NHC. 1-6 Alkyl and -SC 1-6 Alkyl substituents.

[0021] In some implementation schemes, R 4 It is hydrogen or C 1-3 alkyl.

[0022] In some implementation schemes, R 4 It is hydrogen or -CH3.

[0023] In some implementation schemes, R 5 Hydrogen, halogen, -OH, C 1-6 Alkyl or -OC 1-6 Alkyl, the C 1-6 Alkyl groups are each optionally surrounded by one or more elements selected from deuterium, halogen, -OH, -CN, -NH2, C 1-6 Alkyl and C 3-6 Substitution of cycloalkyl groups.

[0024] In some implementation schemes, R 5 Hydrogen, halogen, or -OC 1-3 Alkyl group, preferably hydrogen.

[0025] In some implementation schemes, R 5 It can be hydrogen, -OH, or -OCH3.

[0026] In some implementations, the structure shown in Formula I is a structure shown in Formula Ia, Formula Ib, or Formula Ic:

[0027] Among them, R 1a For -VC 0-6 Alkylene-UC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U-, -VC 0- 6-alkylene-C(O)N(R)6 )-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6 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-, -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-S(O)2N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-(4- to 8-membered heterocyclic alkylene)-U-, -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6 Alkylene-U- or -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-U-, wherein U is attached to a benzene ring;

[0028] R 2a For -VC 0-6 Alkylene-UC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 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 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6Alkylene-U-, -VC 0-6 Alkylene-S(O)2N(R) 6 )-C 0-6 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)-C 0-6 Alkylene-U- or -V- (4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6 Alkylene-U-, wherein U is attached to a piperidine ring;

[0029] R 3a For -VC 0-6 Alkylene-UC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkylene-OC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 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 0-6 Alkylene-U-, -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-S(O)2N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)N(R 6 )-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C0-6 Alkylene-U- or -V- (4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6 Alkylene-U-, wherein U is attached to a piperidine ring;

[0030] Or, R 4 R 3a R on 6 The group and the atoms attached to it together form 5-8 membered heterocyclic alkyl groups;

[0031] The C mentioned 0-6 Alkylene, C 3-8 Cycloalkylene, 5-8 membered heterocyclic alkylene, and 4-8 membered heterocyclic alkylene are each optionally separated by one or more elements selected from halogen, -OH, and C. 3-6 Substituents of cycloalkyl groups;

[0032] U, V, R 4 R 5 R 6 The definitions of Y are as described above.

[0033] In some implementation schemes, R 1a For -VC 0-6 Alkylene-UC 0-6 Alkylene-U- or -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, where U is attached to a benzene ring.

[0034] In some implementation schemes, R 1a -N(CH3)-, -NH-, -O- or The a-terminus is connected to the benzene ring.

[0035] In some implementation schemes, R 1a -NH-, -O- or The a-terminus is connected to a benzene ring; preferably -NH-.

[0036] In some implementation schemes, R 1a It is -N(CH3)-.

[0037] In some implementation schemes, R 1a It is -NH- or -N(CH3)-.

[0038] In some implementation schemes, R 1a For -VC 0-6 Alkylene-C(O)-(4- to 8-membered heterocyclic alkylene)-U-, -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 -(4- to 8-membered heterocyclic alkylene)-U- or -V-(4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6 Alkylene-U-, where U is attached to a benzene ring.

[0039] In some implementation schemes, R 2a For -VC 0-6 Alkylene-U- or -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, wherein U is attached to a piperidine ring.

[0040] In some implementation schemes, R 2a -OC 1-6 Alkylene-C(O)N(R) 6 )-U-, where U is a chemical bond; where U is connected to the piperidine ring.

[0041] In some implementation schemes, R 3a For -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0- 6-alkylene-U-,-VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6 Alkylene-U- or -V- (4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6 alkylene-U-, wherein U is linked to a piperidine ring; the C 0-6 Alkylene is optionally C 3-6 Cycloalkyl substitution.

[0042] In some implementation schemes, R 3a For -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6 Alkylene-U- or -V- (4- to 8-membered heterocyclic alkylene)-C(O)-C 0-6alkylene-U-, wherein U is linked to a piperidine ring; the C 0-6 Alkylene is optionally C 3-6 Cycloalkyl substitution.

[0043] In some implementation schemes, R 3a For -VC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-.

[0044] In some implementation schemes, R 3a For -VC 0-6 Alkylene-N(R) 6 )C(O)-C 0-6 Alkylene-U- or -VOC 0-6 Alkylene-C(O)N(R) 6 )-C 0-6 Alkylene-U-, wherein U is attached to a piperidine ring.

[0045] In some implementation schemes, R 3a -OC 1-6 Alkylene-C(O)N(R) 6 )-C 1-6 alkylene-U- or -NR 6 -OC 1-6 Alkylene-C(O)N(R) 6 )-C 1-6 Alkylene-U-, where U is an independent chemical bond; wherein U is connected to a piperidine ring.

[0046] In some implementation schemes, R 4 R 3a R on 6 The group and the atoms attached to it together form 5-8 membered heterocyclic alkyl groups.

[0047] In some implementation schemes, for Or a combination thereof.

[0048] In some implementation schemes, for Or a combination thereof.

[0049] In some implementation schemes, R 1a for The a-terminus is connected to the benzene ring.

[0050] In some implementation schemes, R 1a for The a-terminus is connected to a benzene ring; preferably, R 1a for The a-terminus is connected to the benzene ring.

[0051] In some implementation schemes, R 2a -NH-, -O-, -OCH2- b , The b-end is connected to the piperidine ring.

[0052] In some implementation schemes, R 2a -NH-, -O-, -OCH2- b , The b-end is connected to the piperidine ring.

[0053] In some implementation schemes, R 2a for The b-end is connected to the piperidine ring.

[0054] In some implementation schemes, R 3a for The c-terminus is connected to the piperidine ring.

[0055] In some implementation schemes, R 3a for The c-terminus is connected to the piperidine ring.

[0056] In some implementation schemes, R 3a for The c-terminus is connected to the piperidine ring.

[0057] In some implementation schemes, R 3a for The c-terminus is connected to the piperidine ring.

[0058] In some implementation schemes, R 3a for The c-terminus is connected to the piperidine ring.

[0059] 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 A group is formed together with the atoms it is attached to.

[0060] In some implementations, the structure shown in Formula I is any of the following structures:

[0061] In some implementations, the structure shown in Formula I is any of the following structures:

[0062] In some embodiments, the ligand-drug conjugate comprises a structure as shown in Formula II:

[0063] Among them, L 1a The end is connected to the ligand;

[0064] A, R, and Y are defined as described in any of the schemes in Equation I;

[0065] L 1a for

[0066] L 2 -(C(R) L21 )2) n -;

[0067] n is a natural number from 0 to 50;

[0068] 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-,

[0069] -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;

[0070] RL21 R L22 and R cx Each independently is -(C(R) L2a )2) m -R L2b m is a natural number from 0 to 50;

[0071] 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(CH3)-, -O-, -S-, -SO- or -SO2-;

[0072] R L2a -(CH2) y -R L2b y is a natural number from 0 to 50;

[0073] 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-;

[0074] 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;

[0075] L 3 It is a short peptide consisting of 2-10 amino acid residues that does not exist.

[0076] 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;

[0077] Tr represents non-existent Or any combination of the above groups;

[0078] R TrIndependently 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;

[0079] 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,

[0080] z is an independent natural number from 0 to 50;

[0081] 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.

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

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

[0084] In some implementations, y is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0085] In some implementations, z is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0086] 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.

[0087] 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.

[0088] In some embodiments, the glycosyl group or its derivative is

[0089] In some implementations, L 1a for e-end L 2 Connection; preferably, L 1a for e-end L 2 Connection; more preferably, L 1a for

[0090] In some implementations, L 2 -(CHR) L21 ) n -; n is a natural number from 0 to 50;

[0091] L 2 Any CHR in L21 Each unit can be independently replaced by the following structural units: -Cy-, -C(O)-, -NH-, -O- or

[0092] -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;

[0093] R L21 and R cx Each independently is -(CHR) L2a )2) m -R L2b m is a natural number from 0 to 50;

[0094] RL21 and R cx Any CHR L2a Each unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, -O-, -S-, -SO-, or -SO2-;

[0095] R L2a -(CH2) y -R L2b y is a natural number from 0 to 50;

[0096] 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-;

[0097] 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;

[0098] 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...

[0099] 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.

[0100] 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,

[0101] 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;

[0102] L 2 Any CHR in L21Each element can be independently replaced by the following structural elements: -C(O)-, -NH-, -O- or

[0103] 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;

[0104] R L21 Any CHR L2a Each unit can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, or -O-;

[0105] 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;

[0106] R L2a Any CH2 unit in the structure can be independently replaced by the following structural units: -C(O)-, -NH-, -N(CH3)-, or -O-;

[0107] 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,

[0108] In some implementations, L 2 for f end and L 3 connect;

[0109] n1, n2, and n3 are each independent natural numbers from 0 to 8;

[0110] m1 is a natural number from 0 to 16;

[0111] m2 is a natural number from 1 to 5;

[0112] y1 is a natural number from 1 to 5;

[0113] G is non-existent. Or -NH-, g end with L 1a connect.

[0114] In some implementations, L 2 for f end and L 3 Connect; n1, n2, and n3 are each an independent natural number 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, n2, and n3 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 f end and L 3 connect.

[0124] In some implementations, L 2 for f end and L 3 connect.

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

[0126] In some implementation schemes, for Among them, the f end and L 3 Connected.

[0127] In some implementation schemes, for Among them, the f end and L 3 Connected.

[0128] In some implementation schemes, for f end and L 3 Connected.

[0129] In some implementations, L 3 Independently

[0130] In some implementations, L 3 Does not exist or L 3a -L 3b ;

[0131] L 3a and L 3b Independently, it is a short peptide composed of 2-10 amino acid residues, which is absent.

[0132] In some implementations, L 3 Does not exist or L 3a -L 3b ;

[0133] L 3a and L 3b Independently, it is a short peptide composed of 2-10 amino acid residues, which is absent.

[0134] In some implementations, L 3ais 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, (Ala)2-Pro-Nva or (Gly)5-Phe-Gly.

[0135] In some embodiments, 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.

[0136] In some implementations, L 3a It is (Gly)5-Phe-Gly.

[0137] In some implementations, L 3b For non-existent The methylene terminus is connected to Tr.

[0138] In some implementations, L 3b for The j-end is connected to the Tr-end.

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

[0140] In some implementations, L 3a The following amino acid residues or short peptides are absent: Gly, Ser, Ala-Ala, Ala-Asn, Ala-Ser, Gly-Glu, Glu-Gly, Gly-Gln, Gly-Ser, Glu-Ser, Ala-Ala-Asn, Ala-Ala-Ala, Glu-Ala-Ser, Gly-Glu-Gly, Glu-Gly-Ser, (Gly)2-Phe-Gly, or (Gly)5-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 The j-end is connected to the Tr-end.

[0141] In some implementations, L 3a For the absence of Ser, Ala-Ala, Ala-Asn, Ala-Ser, Gly-Glu, Glu-Gly, Gly-Gln, Gly-Ser, Glu-Ser, Ala-Ala-Asn, Ala-Ala-Ala, Glu-Ala-Ser, Gly-Glu-Gly, Glu-Gly-Ser, or (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 amino acid residues or short peptides mentioned above, or (Gly)2-Phe-Gly, are associated with the carbonyl terminus of the following amino acid residues or short peptides: Ser, Ala-Ala, Ala-Asn, Glu-Gly, Ala-Ser, Gly-Ser, Gly-Glu-Gly, Ala-Ala-Ala, Glu-Ala-Ser, Glu-Gly-Ser, and (Gly)2-Phe-Gly. 3b Connection; L 3b It does not exist.

[0143] In some implementations, L 3a The amino acid residues or short peptides are Ala-Ala, Glu-Gly, Gly-Glu-Gly, Ala-Ala-Ala, Ala-Ala-Asn, (Gly)2-Phe-Gly, or (Gly)5-Phe-Gly, wherein the carbonyl terminus of the above amino acid residues or short peptides is associated with L. 3b Connection; L3b It does not exist.

[0144] In some implementations, L 3a It does not exist; L 3b for The j-end is connected to the Tr-end.

[0145] In some implementation schemes, for The h terminal is connected to the Tr terminal.

[0146] In some implementation schemes, for The h terminal is connected to the Tr terminal.

[0147] In some implementation schemes, for The h terminal is connected to the Tr terminal.

[0148] In some implementations, Tr represents non-existence. 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;

[0149] z is a natural number from 0 to 8; for example, 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0150] In some implementations, Tr represents non-existence. i-end and L 3 connect.

[0151] In some embodiments, the ligand-drug conjugate comprises a structure as shown in formula IIa, formula IIb, or formula IIc:

[0152] Among them, L 1a The end is connected to the ligand;

[0153] R 1a R 2a R 3a R 4 R 5 Y is defined as described in any of the following schemes: I, Ia, Ib, and Ic.

[0154] L 1a L 2 L 3 The definitions of Tr are as described in any of the schemes in Equation II;

[0155] In some embodiments, the structure shown as in Formula II, Formula IIa, Formula IIb, or Formula IIc is any one of the structures in Table 1 below:

[0156] Table 1

[0157] In some embodiments, the ligand-drug conjugate is a ligand-drug conjugate as shown in Formula III:

[0158] Among them, L 1a The end is connected to the ligand;

[0159] Ab is the ligand that binds to the target.

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

[0161] The definitions of A, R, and Y are as described in any of the embodiments of Formula I in this invention;

[0162] L 1a L 2 L 3 The definitions of Tr are as described in any of the embodiments of formula II, IIa, IIb and IIc 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 in Ab 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 embodiments, the Ab is a monoclonal antibody, particularly a monoclonal antibody with a thiol group as the coupling site, or a site-directed mutated or modified monoclonal antibody with a thiol group as the coupling site.

[0174] 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.

[0175] In some implementations, the Ab is an antibody or antigen-binding fragment thereof targeting HER3, B7H3, Claudin18.2, CD30, CD33, CD70, BCMA, GPC-3, ADAM9 and EGFR.

[0176] 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.

[0177] In some preferred embodiments, in the ligand drug conjugate as shown in Formula III, 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.

[0178] In some preferred embodiments, in the ligand drug conjugate as shown in Formula III, Ab is an antibody targeting BCMA or an antigen-binding fragment thereof; preferably an anti-BCMA antibody or an antigen-binding fragment thereof, for example, DB1004 or a variant thereof.

[0179] In some preferred embodiments, in the ligand drug conjugate as shown in Formula III, Ab is an antibody targeting ADAM9 or an antigen-binding fragment thereof; preferably an antibody against ADAM9 or an antigen-binding fragment thereof, such as DB1001 or a variant thereof.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[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: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.

[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: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.

[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: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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] In some embodiments, in the ligand-drug conjugate shown in Formula III, 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.

[0202] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment as shown in Formula III 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.

[0203] In some embodiments, the anti-BCMA antibody or its antigen-binding fragment as shown in Formula III 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.

[0204] In some implementations, q is an integer or decimal of 1-32, preferably an integer or decimal of 1-16, and more preferably an integer or decimal of 2-8, such as 2, 3, 4, 5, 6, 7, 8, 16, 3.01, 3.22, 3.75, 3.76, 3.87, 3.89, 3.95, 3.98, 4.05, 4.06, 4.08, 4.11, 4.16, 4.22, 4.29, 5.11, 5.58, 6.17, 6.46, 6.56, 6.82, 6.88, 6.98, 7.02, 7.06, 7.08, 7.12, 7.18, 7.22, 7.25, 7.28, 7.31, 7.32, 7.33, 7.41, 7.42, 7.43, 7.45, 7.48, 7.52, 7.53, 7.56, 7.57, 7.58, 7.62, 7.64, 7.65, 7.71, 7.72, 7.73, 7.78, 7.81, 7.82, 7.87, 7.88, or 7.93.

[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, 3.86, 3.95, 3.96, 4.08, 4.12, 4.13, 4.17, 4.20, 4.27, 4.28, 4.31, 4.32, 4.34, 4.36, 4.41, 4.43, or 7.74.

[0206] 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.

[0207] In some embodiments, the ligand-drug conjugate as shown in Formula III is a ligand-drug conjugate as shown in Formula IIIa, Formula IIIb, or Formula IIIc.

[0208] Wherein, Ab and q are defined as described in any of the embodiments of Formula III of this invention;

[0209] R 1a R 2a R 3a R 4 R 5 The definition of Y is as described in any of the embodiments of formula I, formula Ia, formula Ib and formula Ic of this invention;

[0210] L 1a L 2 L 3The definitions of Tr are as described in any of the embodiments of formula II, IIa, IIb and IIc of this invention.

[0211] In some embodiments, the ligand-drug conjugate represented by Formula III is a ligand-drug conjugate as shown in Formula IIIb or Formula IIIc:

[0212] Among them, R 2a -OC 1-6 Alkylene-C(O)N(R) 6 )-U-, where U is connected to the piperidine ring;

[0213] R 3a -OC 1-6 Alkylene-C(O)N(R) 6 )-C 1-6 alkylene-U- or -NR 6 -OC 1-6 Alkylene-C(O)N(R) 6 )-C 1-6 Alkylene-U-, wherein U is attached to a piperidine ring;

[0214] U is an independent chemical bond;

[0215] R 6 It is hydrogen or C 1-6 alkyl;

[0216] R 4 and R 5 Independently hydrogen or C 1-6 alkyl;

[0217] Tr is i-end and L 3 connect;

[0218] L 3 For L 3a -L 3b L 3a It is a short peptide composed of 2-4 amino acid residues; L 3b It does not exist;

[0219] L 2 for f end and L 3 connect;

[0220] n1 and n2 are each independent natural numbers from 0 to 8;

[0221] m1 is a natural number from 0 to 16;

[0222] y1 is a natural number from 1 to 5;

[0223] G is non-existent. Or -NH-, g end with L 1a connect;

[0224] L 1a for e-end L 2 connect;

[0225] q is an integer or decimal between 2 and 8;

[0226] Ab represents an antibody or its antigen-binding fragment.

[0227] In some embodiments, the ligand-drug conjugates represented by formula III, formula (IIIa), formula (IIIb), or formula (IIIc) are any of the structures in Table 2 below:

[0228] Table 2

[0229] Wherein, Ab and q are as described in any of the embodiments of formula IIIa, IIIb and IIIc of the present invention.

[0230] In some embodiments, the ligand-drug conjugate is any of the structures in Table 3 below:

[0231] Table 3

[0232] 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.

[0233] In some embodiments, the average drug-to-ligand ratio (Dar) of the ligand-drug conjugate mixture 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, for example 2, 3, 4, 5, 6, 7, 8, 3.01, 3.22, 3.75, 3.76, 3.87, 3.89, 3.95, 3.98, 4.05, 4.06, 4.08, 4.11, 4.16, 4.22, 4.29, 5.11, 5.58, 6.17, 6.4 6, 6.56, 6.82, 6.88, 6.98, 7.02, 7.06, 7.08, 7.12, 7.18, 7.22, 7.25, 7.28, 7.31, 7.32, 7.33, 7.41, 7.42, 7.43, 7.45, 7.48, 7.52, 7.53, 7.56, 7.57, 7.58, 7.62, 7.64, 7.65, 7.71, 7.72, 7.73, 7.78, 7.81, 7.82, 7.87, 7.88, or 7.93.

[0234] 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, 3.86, 3.95, 3.96, 4.08, 4.12, 4.13, 4.17, 4.20, 4.27, 4.28, 4.31, 4.32, 4.34, 4.36, 4.41, 4.43, or 7.74.

[0235] In some implementations, the ligand-drug conjugate is any of the structures shown in Table 3.

[0236] Thirdly, the present invention provides a compound of formula (IV), its tautomers, its meso compound, its racemic compound, its enantiomers, its diastereomers, or a pharmaceutically acceptable salt thereof:

[0237] Among them, L 1 for

[0238] The definitions of A, R, and Y are as described in any of the embodiments of Formula I in this invention;

[0239] L 2 L 3 The definitions of Tr are as described in any of the embodiments of formula II, IIa, IIb and IIc of this invention.

[0240] In some embodiments, the compound represented by formula IV is a compound represented by formula IVa, formula IVb, or formula IVc:

[0241] Among them, L 1 The definition is as described in any embodiment of Formula IV of this invention;

[0242] R 1a R 2a R 3a R 4 R 5 The definition of Y is as described in any of the embodiments of formula I, formula Ia, formula Ib and formula Ic of this invention;

[0243] L 2 L 3 The definitions of Tr are as described in any of the embodiments of formula II, IIa, IIb and IIc of this invention.

[0244] In some implementations, L 1 for Preferably, L 1 for

[0245] In some implementation schemes, for

[0246] In some implementation schemes, for

[0247] In some implementation schemes, for

[0248] In some implementation schemes, for

[0249] In some implementation schemes, for

[0250] In some implementation schemes, for

[0251] In some implementation schemes, for

[0252] In some implementation schemes, for

[0253] In some implementation schemes, for

[0254] In some embodiments, the compound shown in Formula IV is any one of the compounds in Table 4 below:

[0255] Table 4

[0256] Fourthly, the present invention also provides a method for preparing a ligand-drug conjugate as shown in Formula III, comprising the following steps: reacting a heterocyclic compound as shown in Formula IV with Ab-SH via a substitution or addition reaction to obtain the ligand-drug conjugate as shown in Formula III.

[0257] in,

[0258] The definitions of A, R, and Y are as described in any of the embodiments of Formula I of this invention;

[0259] L 1a L 2 L 3 The definitions of Tr are as described in any embodiment of this invention II;

[0260] L 1 As described in any of the embodiments in IV of this invention;

[0261] Ab and q are as described in any of the embodiments in Formula III of this invention;

[0262] The Ab-SH is a product in which the interchain disulfide bonds of Ab are reduced to thiol groups.

[0263] In another aspect, the present invention provides a compound with the following structure:

[0264] Fifthly, the present invention also provides a pharmaceutical composition 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, and pharmaceutically acceptable excipients.

[0265] In a sixth aspect, 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.

[0266] In a seventh aspect, the present 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, a mixture of the aforementioned ligand-drug conjugates, the aforementioned pharmaceutical composition, or the aforementioned pharmaceutical preparation. The cancer is preferably a solid tumor or a non-solid tumor; for example, 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.

[0267] 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.

[0268] Eighthly, the present 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, 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.

[0269] Terminology definition:

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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 possess the desired biological activity. Such salts include: salts formed by addition to inorganic acids or acids formed with organic acids, or salts containing acidic protons on the parent compound but surrounded by metal ions, or coordination compounds formed with organic bases.

[0275] As used herein, the term "solvent" refers to a substance formed by the association of the compound of the present invention with solvent molecules. The solvent may be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.), for example, the compound of the present invention can form an ethanolide with ethanol. The compound of the present invention can also form a hydrate with water. The amount of the solvent may be stoichiometric or non-stoichiometric.

[0276] As used in this application, the term "and / or" should be considered as a specific disclosure of each of two or more specified features or elements, and any combination of two or more features or elements. Therefore, the term "and / or" as used in phrases, such as "A and / or B" herein, is intended to include "A and B", "A or B", "A" (alone), and "B" (alone).

[0277] 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.

[0278] 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), which can refer to a monoclonal antibody or antigen-binding fragment linked to a biologically active cytotoxic drug via a stable linker unit.

[0279] 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.

[0280] 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 Antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-ADAM9 antibody, anti-GPC-3 antibody and anti-Mesothelin antibody, such as DB1001, DB1002, DB1003 and / or DB1004.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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;

[0288] 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 C 1-4 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 C6-7 C 7-10 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] In this application, the term "C" 0-6 In the term "alkylene", when the value is 0, C0 alkylene is a connecting bond.

[0293] In this application, the term "C" 0-6 In the alkyl group, when C0 is 0, C0 alkyl is hydrogen.

[0294] 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.).

[0295] 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.).

[0296] 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.).

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, iodine, such as fluorine and chlorine.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] When used alone or in combination with other groups in this document, the term "oxo" refers to =O.

[0309] 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.

[0310] 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-A Synthesis (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.

[0311] 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;

[0312] 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.

[0313] 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%).

[0314] 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).

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] As used herein, the term "treatment" aims to alleviate, reduce, improve, or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of the ligand-conjugated drug or its racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture of the foregoing forms is received, and one or more indications and symptoms exhibit an observable and / or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0321] As used in this application, the term "prevention" aims to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms before the onset of the relevant drug. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after the administration of the relevant drug, or lessening the severity of subsequently occurring related symptoms, can be considered as "prevention" of the onset or development of the disease.

[0322] 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.).

[0323] 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).

[0324] 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.

[0325] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0326] As used herein, the term "ester" means an ester derived from the compounds described herein, including physiologically hydrolyzable esters (the compounds of the present invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the present invention may themselves also be esters.

[0327] The compounds of the present invention can exist as solvates (preferably hydrates). The amount of polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0328] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body upon administration of the compounds of this invention. Metabolites of the compounds can be identified using techniques known in the art, and their activity can be characterized by experimental methods. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0329] The present invention further includes, within its scope, prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the present invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the desired therapeutically active compounds.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] The reagents and raw materials used in this invention are all commercially available.

[0334] The positive and progressive effects of this invention are that the compounds of this invention have one or more of the following advantages:

[0335] (1) It has inhibitory activity against the in vitro proliferation of tumor cells;

[0336] (2) It has plasma stability;

[0337] (3) It has an in vivo tumor-suppressing effect;

[0338] (4) It has the ability to resist transport by transporters;

[0339] (5) It has the ability to target tumors in vivo;

[0340] (6) It has good in vivo safety;

[0341] 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

[0342] Figure 1 shows the killing effect of ADC3.1 on MDA-MB-468 cells.

[0343] Figure 2 shows the killing effect of ADC3.1 on MDA-MB-468 cells and Raji-Luc cells.

[0344] Figure 3 shows the killing effect of ADC3.1 on Raji-Luc cells.

[0345] Figure 4 shows the in vivo tumor inhibition effect of ADC4.1 on NCI-H929 tumor-bearing mice.

[0346] Figure 5 shows the in vivo tumor inhibition effect of ADC2.6 on Huh7 tumor-bearing mice.

[0347] Figure 6 shows the in vivo tumor inhibition effect of ADC3.6 on HT-1376 tumor-bearing mice. Detailed Implementation

[0348] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0349] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0350] The preparative high performance liquid chromatography (HPLC) method was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250×20mm column).

[0351] Thin-layer chromatography purification was performed using GF 254 (0.4–0.5 nm) silica gel plates produced in Yantai.

[0352] 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.

[0353] 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.

[0354] Unless otherwise specified in the examples, the reaction temperature is room temperature (20℃~30℃).

[0355] 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. In the conventional synthesis methods, preparation examples, and intermediate synthesis examples, the starting materials were commercially available and purchased from Shanghai Leyan, Shanghai Shaoyuan, Bid Biotechnology, Aladdin Reagents, etc. The key starting material M24 and the control compound rupettedine were both purchased from Zhejiang Zhongke Chuangyue.

[0356] 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.

[0357] The meanings of each abbreviation are shown in the table below.

[0358] Example 1: Preparation of intermediates

[0359] Preparation of compound Int1

[0360] Step 1: Preparation of compound Int1-2

[0361] Compound Int1-1 (2.00 g, 11.35 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and Boc2O (6.40 g, 29.5 mmol), triethylamine (2.50 g, 25 mmol), and DMAP (0.70 g, 5.7 mmol) were added. The reaction was carried out at room temperature for two hours. After the reaction was completed under TLC monitoring, the tetrahydrofuran was removed by concentration under reduced pressure. Ethyl acetate and water were added to the residue, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude Int1-2 (3 g, 70% yield), which was used directly in the next reaction.

[0362] Step 2: Preparation of compound Int1-3

[0363] Lithium hydroxide (1.3 g, 54.27 mmol) was dissolved in a mixture of methanol (30 mL) and water (10 mL) and stirred until dissolved. Then, compound Int1-2 (3.0 g, 6.3 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by TLC, the pH was adjusted to 6-7 with 1 M dilute hydrochloric acid solution, ethyl acetate was 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 Int1-3 (1.5 g, 63% yield).

[0364] Step 3: Preparation of compound Int1-4

[0365] Compounds Int1-3 (1.5 g, 4.0 mmol, 1.0 eq) and Int1-7 (1.6 g, 4.38 mmol) were dissolved in DMF (30 mL), and cesium carbonate (2.60 g, 8.00 mmol) was added. The reaction mixture was heated to 65 °C with stirring for 6 hours. After the reaction was completed under LCMS monitoring, the reaction mixture was cooled to room temperature, 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 column chromatography to give Int1-4 (2.0 g, 89% yield).

[0366] Step 4: Preparation of compound Int1-5

[0367] Compound Int1-4 (2.0 g, 3.54 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and 10% Pd / C (0.2 g) was added. After hydrogen purging, the mixture was heated to 35 °C and stirred overnight. The reaction was monitored by LCMS, cooled to room temperature, and the reaction solution was directly filtered. The filter cake was washed with methanol, and the organic phase was concentrated to obtain Int1-5 (1.4 g, 93% yield), which was directly used in the next reaction.

[0368] Step 5: Preparation of compound Int1-6

[0369] Compound Int1-5 (1.1 g, 2.55 mmol), glycolic acid (0.21 g, 2.8 mmol), HATU (1.14 g, 3.01 mmol), and DIEA (0.98 g, 7.65 mmol) were dissolved in DMF (20 mL) and the mixture was stirred at room temperature for 2 h. After the reaction was completed by LCMS, ethyl acetate and water were added to the reaction solution, 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 Int1-6 (1.1 g, 63% yield).

[0370] Step 6: Preparation of compound Int1 hydrochloride

[0371] Compound Int1-6 (1.1 g, 2.24 mmol) was dissolved in 1,4-dioxane (3 mL), and hydrogen chloride / 1,4-dioxane (3 mL, 10 mmol) was added. The mixture was stirred at room temperature for 2 h. After monitoring the reaction by LCMS, the reaction solution was concentrated under reduced pressure to give Int1 hydrochloride (0.67 g, 92% yield).

[0372] MS m / z(ESI): 290.0 [M+H] +

[0373] 1H NMR (400MHz, DMSO-d6) δ10.90(d,J=2.5Hz,1H),8.03(s,3H),7.31(dd,J=8.7,5.0Hz,1H),7.23(d,J=2.4Hz, 1H),6.97(d,J=2.4Hz,1H),6.73(dd,J=8.7,2.5Hz,1H),5.10–5.05(m,1H),4.70–4.65(m,1H),4.42–4.38(m 1H),4.18–4.14(m,1H),3.96(s,2H),3.83–3.80(m,1H),3.10–2.97(m,4H).

[0374] Preparation of compound Int2

[0375] Step 1: Preparation of compound Int2-2

[0376] Compound Int2-1 (4.0 g, 13.78 mmol) was dissolved in a mixed solvent of DMSO (20 mL) and DCM (10 mL), and TEA (3.2 g, 31.4 mmol) was added. After purging the reaction solution with nitrogen, the temperature was lowered to 0 °C, and SO3·Py (4.4 g, 27.56 mmol) in DMSO (20 mL) solution was slowly added dropwise. The reaction was carried out at 0 °C for 2 h. After the reaction was monitored by LCMS until complete, ice water and ethyl acetate 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 obtain Int2-2 (3.8 g, 90% yield).

[0377] Step 2: Preparation of compound Int2-3

[0378] Compound Int2-2 (3.50 g, 0.012 mol), NaOAc (4.92 g, 0.060 mol), and methylamine hydrochloride (3.96 g, 0.060 mol) were added to MeOH (40 mL) and reacted at room temperature for 2 h. Then, NaCNBH3 (1.50 g, 0.024 mmol) was added and stirring continued for another 2 h. After monitoring the reaction by TLC, the reaction solution was concentrated under reduced pressure. The residue was added to saturated sodium bicarbonate and DCM, 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 Int2-3 (1.9 g, yield 52.7%).

[0379] Step 3: Preparation of compound Int2-4

[0380] Compound Int2-3 (1.2 g, 3.96 mmol), glycolic acid (0.34 g, 4.35 mmol), HATU (1.65 g, 4.35 mmol), and DIEA (0.78 g, 9.90 mmol) were dissolved in DMF (8 mL) and reacted at room temperature for 2 h. After the reaction was complete as monitored by LCMS, saturated sodium bicarbonate and ethyl acetate were added to the reaction solution, 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 Int2-4 (1.0 g, yield 71.4%).

[0381] Step 4: Preparation of compound Int2

[0382] Compound Int2-4 (500 mg, 1.38 mmol) was dissolved in ethyl acetate (5 mL), cooled to 0 °C, and then added to a mixture of hydrogen chloride / ethyl acetate (2 N, 10 mL). After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 2 h. The reaction was monitored by LCMS until complete. The reaction solution was concentrated under reduced pressure to dryness to obtain the hydrochloride salt of compound Int2 (400 mg), which was used directly in the next step without purification.

[0383] MS m / z(ESI): 261.9 [M+H] +

[0384] 1 H NMR (400MHz, DMSO-d6) δ11.05(s,1H),7.97(s,3H),7.61(d,J=7.9Hz,1H),7.39(d,J=8.1Hz,1H),7.30(d,J=2.4Hz,1H),7.11(t,J=7.5Hz, 1H),7.03(t,J=7.5Hz,1H),4.06(s,2H),4.03(t,J=7.1Hz,1H),3.63–3.64(m,1H),3.39(d,J=10.0Hz,1H),3.11–2.92(m,2H),2.83(s,3H).

[0385] Preparation of compound Int3

[0386] Step 1: Preparation of compound Int3-2

[0387] Compound Int3-1 (5.0 g, 0.034 mol) was dissolved in methanol (50 mL), cooled to 0 °C, and sodium acetate (14.0 g, 0.170 mol) and methylamine hydrochloride (11.0 g, 0.170 mol) were added sequentially. The reaction mixture was then heated to 45 °C and reacted for 4 h. The reaction was monitored by LCMS until complete. After cooling, the reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in dichloromethane (60 mL), and acetic acid (1.9 g, 0.032 mol) was added. The mixture was cooled to 0 °C, and TMSCN (9.5 g, 0.096 mol) was slowly added dropwise with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 h. The reaction was monitored by LCMS until complete. The reaction mixture was extracted with saturated sodium bicarbonate and DCM, 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 obtain Int3-2 (4 g, yield 63.4%).

[0388] Step 2: Preparation of compound Int3-3

[0389] Compound Int3-2 (4.0 g, 0.021 mol) was dissolved in THF (40 mL), and DIEA (5.4 g, 0.042 mol) and (Boc)2O (5.5 g, 0.025 mol) were added. The mixture was reacted at 60 °C for 16 h with stirring. The reaction solution was then cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography to give Int3-3 (0.6 g, 10% yield).

[0390] Step 3: Preparation of compound Int3

[0391] Compound Int3-3 (600 mg, 2.1 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 Int3 (410 mg, 67.7%).

[0392] MS m / z(ESI): 290.2 [M+H] + .

[0393] Preparation of compound Int4

[0394] Compound Int4 was synthesized using a method similar to that used for compound Int3.

[0395] MS m / z(ESI): 276.4 [M+H] +

[0396] 1 H NMR (400MHz, CDCl3) δ8.32(s,1H),7.65(d,J=7.9Hz,1H),7.37(d,J=8.1Hz,1H),7.24–7.18(m ,1H),7.15–7.08(m,2H),5.09(s,1H),4.98(d,J=7.4Hz,1H),3.21–3.13(m,2H),1.45(s,9H).

[0397] Preparation of compound Int5

[0398] Step 1: Preparation of compound Int5-1

[0399] Compound Int2-2 (2.5 g, 8.6 mmol) was dissolved in EtOH (40 mL), and AcOH (1.0 g, 17.2 mmol), ethanolamine (1.1 g, 17.2 mol), and NaCNBH3 (1.6 g, 25.8 mmol) were added sequentially. The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC until complete. The reaction solution was concentrated under reduced pressure, and the residue was added back to the reaction solution along with saturated sodium bicarbonate and ethyl acetate. After stirring, the mixture was separated into liquid and liquid phases. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give Int5-1 (2.6 g, 91% yield).

[0400] Step 2: Preparation of compound Int5-2

[0401] Compounds Int5-1 (2.6 g, 7.80 mmol) and DIEA (2.0 g, 15.61 mmol) were dissolved in ACN (30 mL). Under nitrogen protection, the mixture was cooled to 0°C, and AllocCl (1.8 g, 15.61 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for 16 h, and the reaction was monitored for completeness by LC-MS. Saturated sodium bicarbonate and ethyl acetate were added to the reaction mixture, which was stirred and then separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to give Int5-2 (1 g, 71% yield).

[0402] MS m / z (ESI): 418.2 [M+H] +

[0403] 1 H NMR(400MHz, CDCl3)δ8.10(s,1H),7.60(d,J=7.9Hz,1H),7.38–7.36(s,1H),7.22–7.17(m,1H),7.16–7.04(m,2H),5.94–5.79(m,1 H),5.25–5.12(m,2H),4.60–4.45(m,2H),4.37–4.25(m,1H),3.79–3.56(m,4H),3.28–2.97(m,2H),2.99–2.88(m,2H),1.40(s,9H).

[0404] Step 3: Preparation of compound Int5

[0405] Compound Int5-2 (0.5 g, 1.57 mmol) was dissolved in DCM (3 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 1 h. After the reaction was monitored by LCMS until complete, the reaction solution was concentrated under reduced pressure to obtain trifluoroacetate of compound Int5 (0.33 g), which was directly used in the next reaction.

[0406] Preparation of compound Int6

[0407] Step 1: Preparation of compound Int6-2

[0408] LiHMDS (1.12 mL, 6.06 mmol, 2N) was added to the reaction flask under nitrogen protection and cooled to -78 °C. Then, a tetrahydrofuran solution (5 mL) of Int6-1 (776.1 mg, 3.03 mmol) was added dropwise, and the reaction was maintained at this temperature for 1.5 h. Subsequently, a tetrahydrofuran solution (3 mL) of isobutyl chloroformate (0.32 mL, 3.64 mmol) was added dropwise, and the reaction was allowed to proceed naturally to room temperature for 2 h. LC-MS showed the reaction was complete. The mixture was cooled to 0 °C, and a saturated NH4Cl solution and ethyl acetate were added to the reaction solution. After stirring, the mixture was separated into liquid and liquid phases. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain Int6-2 (0.82 g, 76% yield).

[0409] Step 2: Preparation of compound Int6-3

[0410] Compound Int6-2 (500 mg, 1.40 mmol) was dissolved in a mixed solution of dichloromethane / trifluoroacetic acid (5 ml, dichloromethane / trifluoroacetic acid = 4 / 1) and reacted at room temperature for 2 h. After the reaction was complete, the mixture was concentrated under reduced pressure. A saturated sodium bicarbonate solution and DCM were added to the residue, 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 to give Int6-3 (250 mg, yield 69%).

[0411] Step 3: Preparation of compound Int6

[0412] Compound Int6-3 (300 mg, 1.17 mmol) was dissolved in tetrahydrofuran (4 mL), cooled to 0 °C, and lithium aluminum hydride (2.3 mL, 5.85 mmol) tetrahydrofuran solution was added dropwise. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was confirmed to be complete by LCMS, water was added to quench the reaction, followed by filtration, washing with methanol, concentration, and purification of the residue by preparative HPLC to obtain compound Int6 (50 mg, yield 22%).

[0413] MS m / z(ESI): 191.1 [M+H] + .

[0414] Preparation of compound Int7

[0415] Compound Int7 was synthesized using a method similar to that used for compound Int2.

[0416] Preparation of compound Int8

[0417] Compound Int8 was synthesized using the same method as compound Int2.

[0418] Preparation of compound Int9

[0419] Compound Int9 was synthesized using the same method as compound Int2.

[0420] Preparation of compound Int10

[0421] Step 1: Preparation of compound In10-2

[0422] Add Boc2O (11.46 g, 52.56 mmol) to acetonitrile (100 mL) containing In10-1 (5.0 g, 26.28 mmol) and stir at room temperature for 3 hours. Concentrate the reaction solution and purify by silica gel column chromatography to give compound In10-2 (5.5 g, yield 72.84%).

[0423] Step 2: Preparation of compound In10-3

[0424] N-hydroxyphthalimide (4.76 g, 32.37 mmol), PPh3 (8.48 g, 32.37 mmol), and DEAD (2.82 g, 16.18 mmol) were added to 80 mL of dichloromethane containing In10-2 (4.7 g, 16.18 mmol), and the mixture was stirred at 20 °C for 2 hours. The solution was diluted with water and extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography to obtain crude In10-3 (10.0 g).

[0425] Step 3: Preparation of compound In10-4

[0426] At room temperature, hydrazine hydrate (6 mL) was added to an ethanol (80 mL) solution of In10-3 (3.0 g, 7.15 mmol), and the mixture was stirred at 80 °C for 2 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain crude In10-4 (1.3 g).

[0427] Step 4: Preparation of compound In10-5

[0428] Alloc-Cl (749.7 mg, 6.22 mmol) was added to a solution of In10-4 (1.2 g), DIEA (1.6 g, 12.44 mmol) in acetonitrile (40 mL) / N,N-dimethylformamide (4 mL), and the mixture was stirred at 20 °C for 1 hour. The solution was diluted with water, extracted with ethyl acetate, concentrated in the organic phase, and purified by silica gel column chromatography to obtain the target compound In10-5 (1.1 g, 71% yield).

[0429] Step 5: Preparation of compound In10-6

[0430] Trifluoroformic acid (5 mL) was added to a solution of In10-5 (400.00 mg, 1.07 mmol) in dichloromethane (10 mL), and the mixture was stirred at 20 °C for 1 hour. In10-6 (200.0 mg, yield 48.3%) was obtained by reverse preparative column purification.

[0431] Step 6: Preparation of compound In10-7

[0432] M24 (50.0 mg, 0.08 mmol), In10⁻⁶ (100.0 mg, 0.26 mmol), and sodium acetate (100.0 mg, 1.21 mmol) were dissolved in acetic acid (10 mL), and the reaction mixture was stirred at 65 °C for 2 hours. The solution was concentrated, and the crude product was diluted with ethyl acetate and water, and the pH was adjusted to 9 with aq. NaHCO₃. The aqueous phase was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and purified by silica gel column chromatography to obtain the target compound In10⁻⁷ (60.0 mg, yield 85.0%).

[0433] Step 7: Preparation of compound Int10

[0434] Under nitrogen atmosphere, tributyltin hydrogen (270 mg, 0.93 mmol) was added to a solution of In10⁻⁷ (50.0 mg, 0.057 mmol), Pd(PPh₃)₄ (19.76 mg, 0.018 mmol), and acetic acid (120.0 mg, 1.14 mmol) in dichloromethane (25 mL), and the mixture was stirred at 20 °C for 1 hour. The reaction solution was diluted with water, adjusted to pH 9 with saturated NaHCO₃, and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain Int₁₀ (25.0 mg, yield 55.5%).

[0435] MS m / z(ESI): 793.2 [M+H] + .

[0436] Preparation of compound Int11

[0437] Step 1: Preparation of compound Int11-1

[0438] Boc2O (9.2 g, 42.06 mmol) was added to an ACN (82 mL) solution of In10-1 (4.0 g, 21.03 mmol), and the mixture was stirred at 20 °C for 1 hour. The solution was concentrated, and the residue was purified by column chromatography to obtain Int11-1 (5.0 g, 81% yield).

[0439] Step 2: Preparation of compound Int11-2

[0440] CBr4 (4.1 g, 12.40 mmol) was added to a DCM (30 mL) solution containing Int11-1 (3.0 g, 10.33 mmol) and PPh3 (3.3 g, 12.40 mmol). The mixture was stirred at 20 °C for 1 hour. The mixture was diluted with DCM (100 mL), washed with water (50 mL) and brine (20 mL), and the organic phase was dried and concentrated. Int11-2 (2.0 g, 54% yield) was purified by silica gel column chromatography.

[0441] Step 3: Preparation of compound Int11-3

[0442] Int11-3 (1.9 g, 5.38 mmol) was dissolved in 10 mL of 28% methylamine in ethanol and stirred at 22 °C for 16 hours. The solution was concentrated, and the residue was purified by column chromatography to give Int11-3 (1.5 g, 91% yield).

[0443] Steps 4 to 7: Preparation of compound Int11

[0444] Compound Int11 was synthesized using a method similar to steps 4 to 7 of compound Int10.

[0445] MS m / z (ESI): 807.2 [M+H] +

[0446] 1 HNMR (400MHz, CDCl3) δ8.44(s,1H),7.57(s,1H),7.38(d,J=7.9Hz,1H),7.32(d,J=8.2Hz,1H),7.14(t,J=7. 2Hz,1H),7.03(t,J=7.4Hz,1H),6.60(s,1H),6.22(d,J=1.1Hz,1H),6.02(d,J=1.1Hz,1H),4.82(d,J=11.7H z,1H),4.64(s,1H),4.34-4.22(m,2H),4.10(d,J=2.5Hz,1H),3.91(d,J=11.5Hz,1H),3.79(s,3H),3.52-3. 40(m,4H),2.98-2.82(m,6H),2.80-2.70(m,5H),2.42-2.32(m,4H),2.27(s,3H),2.14(s,3H),2.09(s,3H).

[0447] Preparation of compound Int12

[0448] Compound Int12 was synthesized using the same method as compound Int10.

[0449] Preparation of compound Int13

[0450] Compound Int13 was synthesized using the same method as compound Int11.

[0451] MS m / z (ESI): 807.2 [M+H] +

[0452] Preparation of compound Int14

[0453] Step 1: Preparation of compound Int14-2

[0454] Ammonium acetate (13.50 g, 176.47 mmol) was dissolved in nitromethane (200 mL), and compound Int14-1 (10.00 g, 58.80 mmol) was added. The mixture was heated to 105 °C for 2 hours under nitrogen protection. After the reaction was completed by TLC monitoring, it was cooled to room temperature and filtered. The filter cake was washed with H2O / MeOH (V / V = 1 / 1), and the solid was evaporated to dryness to give compound Int14-2 (10.30 g, yield 82%).

[0455] Step 2: Preparation of compound Int14-3

[0456] Compound Int14-2 (5.00 g, 23.47 mmol) was dissolved in a mixed solution of MeOH (25 ml) and DMF (25 ml). Sodium borohydride (4.30 g, 117.30 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed by TLC, the pH was adjusted to 7 with 2N dilute hydrochloric acid solution, DCM was 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 Int14-3 (1.77 g, 35% yield).

[0457] Step 3: Preparation of compound Int14-4

[0458] Compound Int14-3 (1.77 g, 8.20 mmol) was dissolved in MeOH (20 mL), and then a suspension of zinc powder (5.00 g, 82.00 mmol) in 2N HCl (20 mL) was added. The reaction mixture was heated to 85 °C for 2 hours with stirring. After the reaction was completed under LCMS monitoring, the reaction mixture was cooled to room temperature, and 1N NaOH solution was added to adjust the pH to 11. DCM was added, and the mixture was stirred and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound Int14-4 (800 mg, yield 53%), which was used directly in the next step.

[0459] Step 4: Preparation of compound Int14-5

[0460] Compound Int14-4 (750 mg, 4.00 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL), and an aqueous solution of NaCO3 (510 mg, 4.86 mmol) (6 mL) was added. After nitrogen purging, benzyl chloroformate (884 mg, 5.20 mmol) was added at 0 °C, and the mixture was stirred overnight at room temperature. After monitoring the reaction by LCMS, ethyl acetate was 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 Int14-5 (700 mg, 55% yield).

[0461] Step 5: Preparation of compound Int14-6

[0462] Compound Int14-5 (650 mg, 2.00 mmol) was dissolved in pyridine (8 mL), and acetic acid (4 mL), sodium hypophosphite aqueous solution (4 mL, 10 g / 35 mL), and Raney nickel (4 g) were added. The mixture was stirred at room temperature for 3 h under nitrogen protection. After the reaction was monitored by LCMS until complete, the reaction solution was filtered. Ethyl acetate and copper sulfate aqueous solution were added to the filtrate. After separation, the organic phase was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound Int14-6 (600 mg, 91% yield).

[0463] Step 6: Preparation of compound Int14-7

[0464] Compound Int14-6 (550 mg, 1.71 mmol) was dissolved in MeOH (5 mL), followed by the addition of methylamine hydrochloride (572 mg, 8.54 mmol) and sodium acetate (700 mg, 8.54 mmol). The mixture was stirred at room temperature for 1 h, then sodium cyanoborohydride (211 mg, 3.41 mmol) was added, and the mixture was stirred at room temperature for 1 h. After monitoring the reaction by LCMS, water and DCM were added to the reaction mixture, 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 Int14-7 (240 mg, 38% yield).

[0465] Step 7: Preparation of compound Int14-8

[0466] Compound Int14-7 (220 mg, 0.65 mmol) was dissolved in DMF (2 mL), and glycolic acid (54 mg, 0.70 mmol), DMAP (39 mg, 0.32 mmol), HOAT (442 mg, 3.25 mmol), and EDCI (620 mg, 3.25 mmol) were added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the LCMS was used to monitor the reaction. The reaction solution was purified by reverse-phase medium-pressure preparative column chromatography to obtain compound Int14-8 (200 mg, yield 78%).

[0467] Step 7: Preparation of compound Int14

[0468] Compound Int14-8 (170 mg, 0.43 mmol) was dissolved in trifluoroethanol (2 mL), and acetic acid (25 mg, 0.43 mmol) and Pd / C (85 mg, 10%) were added. The mixture was stirred overnight at room temperature under hydrogen protection. After the reaction was monitored by LCMS until complete, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound Int14 (100 mg, yield 89%), which was directly used in the next reaction.

[0469] Preparation of compound LK1

[0470] Step 1: Synthesis of compound LK1-C

[0471] LK1-B (4.0 g, 19.79 mmol) was dissolved in acetone / water (40 mL / 40 mL), and NaHCO3 (2.0 g, 23.74 mmol) and LK1-A (7.3 g, 23.74 mmol) were added. The mixture was stirred at 30°C for 16 hours. Citric acid aqueous solution was added to the reaction mixture, followed by ethyl acetate. The organic phase was concentrated under reduced pressure, and purified by column chromatography to obtain LK1-C (4.6 g, yield 59%).

[0472] Step 2: Synthesis of compound LK1-E

[0473] LK1-C (4.6 g, 11.6 mmol) was dissolved in DMF (150 mL), and HATU (4.4 g, 11.6 mmol), HOAT (1.6 g, 11.6 mmol), and TMP (2.4 g, 23.2 mmol) were added. LK1-D (1.7 g, 11.6 mmol) was slowly added under ice bath conditions, and the mixture was stirred at 20°C for 16 hours. Water and ethyl acetate were added to the reaction solution, and the organic phase was concentrated under reduced pressure. The solution was purified by column chromatography to obtain LK1-E (4.8 g, 79% yield).

[0474] Step 3: Synthesis of compound LK1

[0475] LK1-E (1.0 g, 1.91 mmol) was dissolved in ACN (24 mL) and water (6 mL). Pd(OAc)2 (0.2 g, 0.96 mmol), sodium triphenylphosphine tris(m-sulfonate) (1.1 g, 1.91 mmol), and N-methylmorpholine (0.8 g, 7.64 mmol) were added, and the mixture was stirred at 25°C for 2 hours. The reaction solution was filtered, concentrated under reduced pressure, and the residue was purified by reverse column chromatography to obtain LK1 (500 mg, yield 54%).

[0476] Preparation of compound LK2

[0477] Compound LK2 was synthesized using the same method as compound LK1.

[0478] MS m / z (ESI): 356.2 [M+H] +

[0479] 1 HNMR(400MHz,DMSO-d6)δ12.56(s,1H),8.19–7.82(m,2H),7.00(s,2H),4.98(s,1H),4.31–4.22(m,1H ),3.86–3.48(m,4H),3.42–3.34(m,2H),2.10(t,J=7.5Hz,2H),1.54–1.40(m,4H),1.23–1.13(m,2H).

[0480] Preparation of compound LK3

[0481] Step 1: Preparation of compound LK3-D

[0482] 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).

[0483] Step 2: Preparation of compound LK3-E

[0484] 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).

[0485] Step 3: Preparation of compound LK3-F

[0486] 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.

[0487] Step 4: Preparation of compound LK3

[0488] 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%).

[0489] Preparation of compound LK4

[0490] Step 1: Preparation of compound LK4-B

[0491] Glycine tert-butyl ester (6.1 g, 46.09 mmol), HATU (19.2 g, 50.28 mmol), HOAT (6.9 g, 50.28 mmol), and TMP (10.2 g, 83.8 mmol) were added to a DMF (300 mL) solution of LK4-A (25.1 g, 41.9 mmol), and the mixture was stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure, water was added to the residue, the mixture was slurried, filtered, and the solid was washed with acetonitrile to give LK4-B (28.9 g, 95% yield).

[0492] Step 2: Preparation of compound LK4-C

[0493] In a DCM solution of LK4-B (28 g, 39.45 mmol) in 13 mL, TFA (30 mL) was added and the mixture was stirred at room temperature for 16 h. The solvent was removed by concentration under reduced pressure, and the remaining oily substance was further treated with TFA (30 mL) and stirred at room temperature for 1 h. The solvent was then removed by concentration under reduced pressure, and the residue was treated with ethyl acetate. After stirring, the mixture was filtered to obtain compound LK4-C (16.1 g, 89% yield).

[0494] Step 3: Preparation of compound LK4-D

[0495] Cu(OAc)₂ (1.94 g, 10.69 mmol), acetic acid (3.36 g, 55.91 mmol), and Pb(OAc)₄ (12.93 g, 29.17 mmol) were added to a DMF (50 mL) solution of LK₄-C (10.00 g, 24.31 mmol) at room temperature, and the mixture was stirred at 60 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature, filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was slurried with ethyl acetate, filtered, and dried to obtain the crude compound LK₄-D (11.35 g).

[0496] Step 4: Preparation of compound LK4-E

[0497] Benzyl glycolate (19.66 g, 117.53 mmol) was added to a THF (200 mL) solution of LK4-D (11.35 g, 23.51 mmol) and p-toluenesulfonic acid monohydrate (1.22 g, 5.88 mmol), and the mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure, and the residue was purified by reversed-phase column chromatography to give LK4-E (1.11 g, yield 8.91%).

[0498] Step 5: Preparation of compound LK4-F

[0499] TEA (1.06 g, 10.48 mmol) was added to a DMF (10 mL) solution of LK4-E (1.11 g, 2.10 mmol), and the mixture was stirred at room temperature for 1 h. The solution was then subjected to reversed-phase column chromatography to obtain LK4-F (0.61 g, yield 94.5%).

[0500] Step 6: Preparation of compound LK4-G

[0501] Fmoc-L-alanine (336 mg, 1.07 mmol), HATU (447 mg, 1.16 mmol), HOAT (161 mg, 1.16 mmol), and TMP (238 mg, 1.94 mmol) were added to a DMF (6 mL) solution of LK4-F (304 mg, 0.97 mmol) at room temperature and stirred at room temperature for 3 h. After the reaction was complete, the solution was purified by reverse-phase column chromatography to obtain LK4-G (506 mg, yield 86.6%).

[0502] Step 7: Preparation of compound LK4-H

[0503] In a 5 mL solution of LK4-G (256 mg, 0.41 mmol) in AcOH, Pd / C (10%, 200 mg) was added under nitrogen protection. The suspension was degassed under vacuum and purged with hydrogen three times. Finally, the mixture was stirred at room temperature under pressurized hydrogen for 16 h. The reaction was monitored by TLC until the starting material disappeared. The reaction solution was then filtered to remove the solid, washed with acetic acid, and the filtrate was collected, concentrated under reduced pressure, and dried to obtain crude LK4-H (102 mg, yield 85%).

[0504] Step 8: Preparation of compound LK4-I

[0505] TEA (60.5 mg, 0.6 mmol) was added to a DMF (5 mL) solution of LK4-H (102 mg, 0.20 mmol), and the mixture was stirred at room temperature for 16 hours. The resulting LK4-I reaction solution was used directly in the next step without any further treatment.

[0506] Step 9: Preparation of compound LK4

[0507] Add 1 mL of DMF solution of LK1-A (152 mg, 0.50 mmol) to the DMF (5 mL) reaction solution of LK4-I in step 8, and then stir at room temperature for 2 h. After the reaction is complete, concentrate the solution under reduced pressure, and purify the residue by reverse-phase column chromatography to obtain LK4 (17 mg, yield 8.5%).

[0508] MS m / z(ESI): 482.2 [M+H] +

[0509] 1 H NMR(400MHz,DMSO-d6)δ12.38(s,1H),8.45(t,J=6.6Hz,1H),8.21–7.99(m,2H),7.36 (s,1H),7.00(s,2H),6.91(s,1H),4.57(d,J=6.6Hz,1H),4.56–4.46(m,1H),4.43(dd ,J=13.8,6.1Hz,1H),4.18(p,J=7.1Hz,1H),3.92(s,1H),3.37(t,J=7.1Hz,3H),2.49 –2.44(m,2H),2.10(t,J=7.5Hz,2H),1.48(dq,J=14.4,7.1Hz,4H),1.22–1.14(m,5H).

[0510] Preparation of compound LK5

[0511] Compound LK5-A (500 mg, 2.46 mmol) was dissolved in DMF (10 mL), followed by the addition of compound LK1-A (757 mg, 2.46 mmol) and DIEA (800 mg, 6.15 mmol). The mixture was stirred overnight at 25 °C. The resulting mixture was concentrated and purified by reversed-phase column chromatography to give LK5 (420 mg, yield 43.1%).

[0512] Preparation of compound LK6

[0513] Compound LK6 was synthesized using the same method as compound LK5.

[0514] Preparation of compound LK19

[0515] Step 1: Preparation of compound LK19-B

[0516] To a DMF (50 mL) solution containing LK19-A (5.0 g, 11.99 mmol), HATU (5.0 g, 13.19 mmol), HOAT (1.8 g, 13.19 mmol), TMP (4.3 g, 35.97 mmol), and glycine tert-butyl ester (1.6 g, 11.99 mmol) were added. The mixture was stirred at 20 °C for 2 hours. EA was added, and the mixture was washed with water and brine. The organic phase was dried and concentrated. LK19-B (5.5 g, 86% yield) was purified by column chromatography.

[0517] Step 2: Preparation of compound LK19-C

[0518] TEA (1.7 g, 16.96 mmol) was added to a 20 mL solution of DMF containing LK19-B (4.5 g, 8.48 mmol). The mixture was stirred at room temperature for 16 hours. Then, N-benzyloxycarbonyl-L-alanine N-hydroxysuccinimide ester (2.7 g, 8.48 mmol) was added, and the mixture was stirred at 20 °C for 30 minutes. EA and brine were added, and the mixture was stirred, separated, dried, and concentrated. LK19-C (2.5 g, 57% yield) was purified by column chromatography.

[0519] Step 3: Preparation of compound LK19-D

[0520] TFA (15 mL) was added to a mixture of LK19-C (2.5 g, 4.87 mmol) and DCM (40 mL). The mixture was stirred at 22 °C for 3 hours under a hydrogen atmosphere. The mixture was concentrated and purified by column chromatography to give LK19-D (2.0 g, 89% yield).

[0521] Step 4: Preparation of compound LK19-E

[0522] Cu(OAc)₂ (190.9 mg, 1.10 mmol), AcOH (391.9 mg, 6.53 mmol), and Pb(OAc)₄ (1510.0 mg, 3.41 mmol) were added to a DMF (22 mL) mixture of LK19-D (1.3 g, 2.84 mmol), and the mixture was stirred at 60 °C for 30 min. LK19-E (1.2 g, 88.8% yield) was obtained by column chromatography.

[0523] Step 5: Preparation of compound LK19-F

[0524] To a mixture of LK19-E (1.2 g, 2.55 mmol) and TsOH·H2O (49.4 mg, 0.26 mmol) and benzyl glycolate (2110.0 mg, 12.73 mmol), the mixture was stirred at 25 °C for 30 minutes. The mixture was concentrated and purified by reverse chromatography to obtain LK19-F (1.0 g, 68% yield).

[0525] Step 5: Preparation of compound LK19-G

[0526] Pd / C (100.0 mg) was added to a solution of LK19-F (0.2 g, 0.35 mmol) in AcOH (5 mL). The mixture was stirred at 20 °C under a hydrogen atmosphere for 6 hours. After filtration and lyophilization, LK19-G (90.0 mg, 98% yield) was obtained.

[0527] Step 7: Preparation of compound LK19

[0528] DIEA (28.4 mg, 0.22 mmol) and LKI-A (35.1 mg, 0.11 mmol) were added to a DMF (1 mL) mixture of LK19-G (30.0 mg, 0.11 mmol), and the mixture was stirred at 20 °C for 1 hour. LK19 (30.0 mg, 57% yield) was purified by reverse chromatography.

[0529] MS m / z (ESI): 479.3 [M+H] +

[0530] Preparation of compound LK8

[0531] Step 1: Preparation of compound LK8-B

[0532] DIEA (0.13 g, 1 mmol) was added to a 2 mL solution of LK8-A (0.26 g, 0.5 mmol) and LK13-G (0.13 g, 0.5 mmol) in DMF. The mixture was stirred at 25 °C for 30 minutes. After the reaction was confirmed to be complete by LMCS, the solution was concentrated under reduced pressure and purified by reverse column chromatography to obtain LK8-B (301 mg, 90% yield).

[0533] Steps 2 to 3: Preparation of compound LK8-C

[0534] Compound LK8 was synthesized using a method similar to steps 8 and 9 in the synthesis of compound LK4.

[0535] MS m / z (ESI): 642.3 [M+H] +

[0536] Preparation of compound LK9

[0537] Compound LK9 was synthesized using the same method as compound LK19.

[0538] MS m / z (ESI): 465.2 [M+Na] +

[0539] 1 HNMR (400MHz, DMSO-d6) δ8.68(t,J=8.0Hz,1H),8.07(t,J=4.0Hz,1H),7.95(d,J=8.0Hz,1H),7.00(s,2H),4.60(d,J=8.0Hz,2H),4.25-4.21( m,1H),3.96(s,2H),3.75-3.73(m,2H),3.62-3.55(m,2H),3.38(d,J=8 .0Hz,3H),2.11(t,J=8.0Hz,2H),1.53-1.44(m,4H),1.24–1.15(m,2H).

[0540] Preparation of compound LK14

[0541] Compound LK14 was synthesized using the same method as compound LK19.

[0542] MS m / z (ESI): 386.3 [M+H] +

[0543] Preparation of compound LK11

[0544] Compound LK11 was synthesized following steps 6 to 9 of the synthesis of compound LK4.

[0545] MS m / z (ESI): 563.2 [M+Na] + .

[0546] Preparation of compound LK12

[0547] Step 1: Synthesis of compound LK12-B

[0548] LK12-A (1.5 g, 3.08 mmol) was added to a DMF (20 mL) solution of LK1-A (949.5 mg, 3.08 mmol) and TEA (0.63 g, 6.15 mmol), and the mixture was stirred at 20 °C for 1 hour. The target compound LK12-B (1.3 g, yield 62.2%) was obtained by reverse preparative column purification.

[0549] Step 2: Synthesis of compound LK12

[0550] TFA (15 ml) was added to 30 ml of DCM containing compound LK12-B (1.2 g, 1.76 mmol), and the mixture was stirred at 20 °C for 1 hour. The reaction solution was concentrated and purified by reverse preparative column chromatography to obtain the target compound LK12 (100 mg, yield 14.85%).

[0551] MS m / z (ESI): 405.1 [M+Na] +

[0552] Preparation of compound LK13

[0553] Compound LK13 was synthesized using the same method as compound LK8.

[0554] MS m / z (ESI): 650.2 [M+Na] +

[0555] Preparation of compound LK15

[0556] To a mixture of LK15-A (100.0 mg, 0.23 mmol) and DMF (1.5 mL), DIEA (59.5 mg, 0.46 mmol) and 3-maleimide propionic acid hydroxysuccinimide ester (61.2 mg, 0.23 mmol) were added. The mixture was stirred at 25 °C for 30 min. The residue was purified by reverse column chromatography to give LK15 (60.0 mg, 44% yield).

[0557] MS m / z (ESI): 593.6 [M+Na] +

[0558] Preparation of compound LK16

[0559] Steps 1 and 2: Synthesis of compound LK16-D

[0560] K₂CO₃ (167.5 mg, 1.21 mmol) was added to a DMF (10 mL) solution of compound LK16-B (650 mg, 1.01 mmol), and the reaction mixture was reacted at 20 °C for 1 hour. Then, allyl bromide (146.4 mg, 1.21 mmol) was dissolved in DMF (2 mL) and added dropwise to the above reaction mixture, and the mixture was stirred at 20 °C for another 10 hours. Water was then added, and the mixture was extracted with DCM. The DCM layer was dried and concentrated to obtain crude LK16-C, which was used directly in the next step.

[0561] Add 10 mL of DMF solution to the crude LK16-C obtained in step 1, then add TEA (511.0 mg, 5.05 mmol). Stir the reaction mixture at 60 °C for 1 hour. After cooling, purify directly by reverse column chromatography to obtain LK16-D (220 mg, yield 47.0%).

[0562] Step 3: Synthesis of compound LK16-F

[0563] LK16-E (234.3 mg, 0.56 mmol), HATU (212.8 mg, 0.56 mmol), HOAT (76.2 mg, 0.56 mmol), and TMP (172.0 mg, 1.41 mmol) were added to 5 mL of DMF containing compound LK16-D (220.0 mg, 0.47 mmol). The reaction solution was stirred at 25 °C for 16 hours. After concentration to remove most of the DMF, LK16-F (200 mg, yield 49.3%) was purified by reverse column chromatography.

[0564] Step 4: Synthesis of Compound LK16 Pd(OAc)2 (23.6 mg, 0.11 mmol), trisodium triphenylphosphine tris(m-sulfonate) (119.4 mg, 0.21 mmol), and N-methylmorpholine (85.0 mg, 0.84 mmol) were added to a solution of compound LK16-F (180 mg, 0.21 mmol) in ACN (12 mL) and H2O (3 mL). The reaction mixture was then stirred at 20 °C under nitrogen protection for 2 hours. The mixture was filtered, and the filter cake was washed with ACN. The combined filtrates were concentrated and purified by reverse HPLC to obtain LK16 (25 mg, yield 14.5%).

[0565] MS m / z (ESI): 822.3 [MH] -

[0566] Preparation of compound LK17

[0567] Step 1: Synthesis of compound LK17-B

[0568] To a DMF (10 mL) solution of compound LK17-A (930 mg, 5.13 mmol), N-fluorenylmethoxycarbonyl-L-glutamic acid 1-allyl ester (1.4 g, 3.42 mmol), DMTMM (1.32 g, 4.79 mmol), and DIEA (880 mg, 6.84 mmol) were added, and the mixture was stirred at 25 °C for 1 hour. The reaction solution was concentrated to obtain the crude product. LK17-B (1.5 g, 77% yield) was purified by reversed-phase column chromatography.

[0569] Step 2: Synthesis of compound LK17-C Ac2O (5 mL) and pyridine (2 mL) were added to a DMF (5 mL) solution of compound LK17-B (1.5 g, 2.6 mmol), and the mixture was stirred at 25 °C for 48 hours. The reaction solution was directly purified by reversed-phase column chromatography to obtain LK17-C (2 g, 85% yield).

[0570] Step 3: Synthesis of compound LK17-D

[0571] Pd(OAc)₂ (290 mg, 1.3 mmol), sodium triphenylphosphine tris(m-sulfonate) (1.5 g, 2.6 mmol), N-methylmorpholine (1.1 g, 10.4 mmol), and HOAc (781.0 mg, 12.9 mmol) were added to a solution of compound LK17-C (2.0 g, 2.6 mmol) in ACN (40 mL) and H₂O (10 mL). The reaction was then carried out under N₂ protection at 25 °C for 2 h. The reaction solution was filtered, the filter cake was washed with ACN, the combined filtrates were concentrated, and then purified by reversed-phase column chromatography to obtain LK17-D (1.1 g, yield 57.9%).

[0572] Steps 4 and 5: Synthesis of compound LK17-F

[0573] 7M NH3 / MeOH (20 mL) solution was added to LK17-D (1.1 g, 1.48 mmol) and stirred at 20 °C under N2 protection for 48 hours. After the reaction was completed as detected by LCMS, the solution was evaporated to dryness to obtain LK17-E, which was then used in the next step.

[0574] To a 10 mL DMF solution of crude compound LK17-E, hydroxysuccinimide 3-maleimide propionate (472.8 mg, 1.78 mmol) and DIPEA (572.38 mg, 4.44 mmol) were added, and the mixture was reacted at 25 °C for 6 hours. The reaction solution was purified by reversed-phase column chromatography to give LK17-F (550 mg, yield 80.6%).

[0575] Step 6: Synthesis of compound LK17

[0576] LK17-G (96.4 mg, 0.23 mmol), HATU (98.9 mg, 0.26 mmol), HOAT (35.4 mg, 0.26 mmol), and TMP (78.8 mg, 0.65 mmol) were added to a DMF (5 mL) solution of LK17-F (100.0 mg, 0.22 mmol). The reaction solution was reacted at 20 °C for 16 hours, and then purified by reversed-phase column chromatography to obtain LK17 (100.0 mg, yield 52.9%).

[0577] MS m / z (ESI): 867.3 [M+H] +

[0578] Synthesis of compound LK18

[0579] Step 1: Synthesis of compound LK18-B

[0580] 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.

[0581] Step 2: Synthesis of compound LK18-D

[0582] 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).

[0583] Step 3: Synthesis of compound LK18-F

[0584] 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).

[0585] Step 4: Synthesis of compound LK18-G

[0586] 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.

[0587] Step 5: Synthesis of compound LK18-H

[0588] 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).

[0589] Step 6: Synthesis of compound LK18-I

[0590] 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).

[0591] Steps 7 and 8: Synthesis of compounds LK18-J and LK18-K

[0592] 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.

[0593] 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.

[0594] Step 9: Synthesis of compound LK18

[0595] 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%).

[0596] MS m / z (ESI): 883.9 [M / 2+H] +

[0597] Synthesis of compound LK20

[0598] Compound LK20 was synthesized using a method similar to step 6 in the synthesis of compound LK17.

[0599] MS m / z (ESI): 1766.9 [M+H] + .

[0600] Synthesis of compound LK21

[0601] Compound LK21 was synthesized using a method similar to steps 5 and 6 in the synthesis of compound LK17.

[0602] MS m / z (ESI): 749.6 [M+H] +

[0603] Synthesis of compound LK22

[0604] Step 1: Synthesis of compound LK22-B

[0605] DIEA (1.18 g, 9.2 mmol) was added to a DMF (10 mL) solution of LK22-A (1.0 g, 3.1 mmol) and 3-bromopropene (1.1 g, 9.2 mmol). The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was extracted with water and ethyl acetate, the organic phases were combined and washed with saturated brine, and then dried over anhydrous sodium sulfate. The resulting organic phase was evaporated under vacuum and purified by normal-phase chromatography to give LK22-B (0.96 g, 85% yield).

[0606] Step 2: Synthesis of compound LK22-D

[0607] Silver oxide (2 g, 7.8 mmol) was added to a solution of LK22-B (0.96 g, 2.6 mmol), LK22-C (2.15 g, 5.2 mmol), and anhydrous acetonitrile (9 mL) under light-protected conditions. The reaction mixture was stirred overnight at room temperature in the dark. The reaction mixture was filtered, and the filtrate was concentrated to dryness and purified by column chromatography to obtain LK22-D (0.56 g, yield 31%).

[0608] Step 3: Synthesis of compound LK22-E

[0609] To a solution of LK22-D (560 mg, 0.8 mmol) in 20 mL of ACN and 5 mL of water, Pd(OAc)2 (90 mg, 0.4 mmol), sodium triphenylphosphine tris(m-sulfonate) (456 mg, 0.8 mmol), N-methylmorpholine (325 mg, 3.21 mmol), and HOAc (241 mg, 4.01 mmol) were added. The reaction mixture was reacted at room temperature for 16 hours. The reaction mixture was filtered, and the filter cake was purified by reversed-phase column chromatography to obtain LK22-E (430 mg, yield 81%).

[0610] Steps 4 to 6: Synthesis of compound LK22

[0611] Compound LK22 was synthesized using a method similar to steps 4 to 6 in the synthesis of compound LK17.

[0612] MS m / z (ESI): 706.3 [M+H] +

[0613] Synthesis of compound LK23

[0614] Compound LK23 was synthesized using a method similar to steps 5 and 6 in the synthesis of compound LK17.

[0615] MS m / z (ESI): 664.3 [M+H] +

[0616] Preparation of compound LK24

[0617] Compound LK24 was synthesized using a method similar to steps 4 to 7 in the synthesis of compound LK19.

[0618] MS m / z (ESI): 441.3 [M+H] + .

[0619] Synthesis of compound LK25

[0620] Following a similar method to steps 3 to 6 of compound LK18, compound LK25 was synthesized using 1-amino-1-deoxy-D-mannitol.

[0621] Synthesis of compound LK26

[0622] Following a similar method to steps 3 to 6 of compound LK18, compound LK26 was synthesized using 1-amino-1-deoxy-D-galactitol.

[0623] Preparation of compound LK27

[0624] Compound LK27 was synthesized following steps 2, 5, and 6 of the procedure for compound LK18. MS m / z (ESI): 1248.3

[0625] [M+H] + .

[0626] Preparation of compound LK28

[0627] Step 1: Synthesis of compound LK28-2

[0628] To a DMF (10 mL) solution of LK28-1 (1.00 g, 2.81 mmol), HATU (1.28 g, 3.38 mmol), HOAt (0.77 g, 5.63 mmol), and TMP (1.37 g, 11.26 mmol) were added, and the reaction was allowed to proceed for 10 minutes. Then, D-glucosamine (1.02 g, 5.62 mmol) was added, and the reaction was allowed to proceed for 30 minutes at room temperature. LCMS was used to monitor the reaction until complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to obtain compound LK28-2 (1.7 g, yield 88.5%).

[0629] Step 2: Synthesis of compound LK28-3

[0630] To a DMF (8 mL) solution of Fmoc-O-tert-butyl-L-glutamic acid (0.96 g, 2.26 mmol), HATU (1.03 g, 2.71 mmol), HOAt (0.62 g, 4.52 mmol), and TMP (1.10 g, 9.04 mmol) were added, and the mixture was reacted at room temperature for 10 minutes. Then, the previously prepared reaction solution was added, and the reaction was continued at room temperature for 30 minutes. The reaction mixture was monitored by LC-MS to ensure complete reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound LK28-3 (1 g, yield 56.3%).

[0631] Step 3: Synthesis of compound LK28-4

[0632] TFA (5 mL) was added to a DMF (5 mL) solution of compound LK28-3 (1.0 g, 1.15 mmol), and the mixture was stirred at 25 °C for 3 hours. After the reaction was complete, the solution was concentrated and slurried with anhydrous diethyl ether (20 mL x 5) to obtain a TFA salt of compound LK28-4 (1.0 g), which was used directly in the next reaction.

[0633] Step 4: Synthesis of compound LK28-5

[0634] At room temperature, HATU (437.0 mg, 1.15 mmol), HOAT (156.5 mg, 1.15 mmol), and TMP (420.9 mg, 3.45 mmol) were added to a DMF (10 mL) solution of compound LK28-4 (1.0 g, 1.15 mmol) and D-glucosamine (195.4 mg, 208.4 mmol). The reaction solution was reacted at this temperature for 20 min. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound LK28-5 (1.1 g, yield 91.7%).

[0635] Step 5: Synthesis of compound LK28-6

[0636] At room temperature, TEA (45.5 mg, 0.45 mmol) was added to a 2 mL DMF solution of compound LK28-5 (150 mg, 0.15 mmol), and the reaction was allowed to proceed for 8 hours. The reaction was monitored by LCMS until the starting material was completely reacted, and the mixture was stored for later use.

[0637] HATU (58.52 mg, 0.15 mmol) and HOAt (20.96 mg, 0.15 mmol) were added to a 2 mL DMF solution of compound LK18-D (151.10 mg, 0.15 mmol). The reaction mixture was then added dropwise to this solution, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound LK28-6 (0.16 g, yield 60.6%).

[0638] Step 6: Synthesis of compound LK28

[0639] TFA (2 mL) was added to a DCM (2 mL) solution of compound LK28-6 (160 mg, 0.09 mmol) and stirred at 25 °C for 3 hours. After the reaction was complete, the solution was concentrated and slurried with anhydrous diethyl ether (10 mL x 5) to obtain a trifluoroacetate of compound LK28 (120 mg), which was directly used in the next reaction. MS m / z (ESI): 829.7 [M / 2+H] + .

[0640] Preparation of compound LK29

[0641] Compound LK29 was synthesized using a method similar to steps 4 to 6 in the synthesis of compound LK28.

[0642] MS m / z (ESI): 969.4 [M / 2+H] +

[0643] Preparation of compound LK30

[0644] Step 1: Synthesis of compound LK30-1

[0645] LK18-H (0.40 g, 0.28 mmol) was dissolved in DMF (2 mL), and TEA (0.14 g, 1.4 mmol) was added. The mixture was then stirred at 25 °C for 16 hours. LCMS analysis confirmed the reaction was complete, yielding LK30-1. This reaction solution was used directly in the next step without any further processing.

[0646] Step 2: Synthesis of compound LK30-3

[0647] To a DMF (10 mL) solution of LK30-2 (0.1 g, 0.28 mmol), HATU (0.13 g, 0.34 mmol), HOAt (0.08 g, 0.57 mmol), and TMP (0.14 g, 1.13 mmol) were added, and the mixture was reacted for 10 minutes. Then, the reaction solution of LK30-1 from step 1 was added, and the mixture was reacted at room temperature for 40 minutes. The reaction mixture was monitored by LCMS to ensure complete reaction of the starting materials. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to obtain compound LK30-3 (0.26 g, 60% yield).

[0648] Steps 3 to 5: Synthesis of compound LK30

[0649] Compound LK30 was synthesized using a method similar to steps 6, 8, and 9 in the synthesis of compound LK28.

[0650] MS m / z (ESI): 941.1 [M / 2+H] + .

[0651] Preparation of compound LK31

[0652] Referring to the synthesis method of LK30, using To replace LK30-2, compound LK31 was synthesized.

[0653] MS m / z (ESI): 969.6 [M / 2+H] + .

[0654] Preparation of compound LK32

[0655] Step 1: Synthesis of compound LK32-2

[0656] LK32-1 (7.00 g, 15.23 mmol) was dissolved in DMF (140 mL), and allyl bromide (4.58 g, 38.08 mmol) was added at room temperature and stirred for 16 hours. The reaction solution was quenched with saturated sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give LK32-2 (4.1 g, yield 53.8%).

[0657] Step 2: Synthesis of compound LK32-3

[0658] Compound LK32-2 (4.1 g, 8.21 mmol) was dissolved in dichloromethane (200 mL). Tetramethylethylenediamine (4.76 g, 40.96 mmol) and allyl chloroformate (2.92 g, 24.23 mmol) were added under ice bath conditions, and the mixture was stirred for 3 hours under nitrogen protection. The reaction solution was filtered, the filtrate was concentrated, and the residue was purified by column chromatography to give compound LK32-3 (4.6 g, yield 74.5%).

[0659] Step 3: Synthesis of compound LK32-4

[0660] Compound LK32-3 (4.6 g, 6.12 mmol) was dissolved in tetrahydrofuran / acetic acid solution (92 mL / 18.4 mL), and zinc powder (40.0 g, 330.66 mmol) was added at room temperature and stirred for 16 hours. After the reaction was confirmed to be complete by LMCS, the mixture was filtered, the filtrate was quenched with sodium bicarbonate aqueous solution, extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give compound LK32-4 (3.9 g, yield 88.3%).

[0661] Step 4: Synthesis of compound LK32-5

[0662] Compound LK32-4 (3.60 g, 4.99 mmol) was dissolved in DMF (90 mL). FMOC-β-alanine (2.32 g, 7.48 mmol), HATU (2.85 g, 7.481 mmol), HOAt (1.05 g, 7.48 mmol), and TMP (1.22 g, 9.98 mmol) were added at room temperature, and the mixture was stirred for 16 hours. The reaction solution was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by column chromatography to give compound LK32-5 (4.0 g, 79% yield).

[0663] Step 5: Synthesis of compound LK32-6

[0664] Compound LK32-5 (3.50 g, 3.45 mmol) was dissolved in tetrahydrofuran (38 mL), and 70% HF·Pyridime (12.7 mL) was added at 0°C. The mixture was stirred at 0°C for 2 hours. The reaction solution was quenched with aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LK32-6 (2.5 g, 80.4% yield).

[0665] Step 6: Synthesis of compound LK32

[0666] Compound LK32-6 (2.50 g, 2.78 mmol) was dissolved in DMF (32 mL), and LK32-7 (1.71 g, 5.55 mmol) and DMAP (0.34 g, 2.775 mmol) were added at room temperature and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound LK32 (1.6 g, 54% yield).

[0667] MS m / z (ESI): 1066.3 [M+H] +

[0668] 1H NMR (400MHz, DMSO-d6) δ8.82(s,1H),8.33-8.29(m,2H),8.10(s,1H),7.88(d,J=7.5Hz,2H),7.68(d,J=7.4Hz,2H ),7.59-7.55(m,2H),7.40(t,J=7.2Hz,3H),7.31(t,J=7.3Hz,2H),7.23(d,J=10.2Hz,1H),7.14(d,J=8.5Hz,1H), 5.94-5.80(m,4H),5.73(d,J=7.7Hz,1H),5.45(t,J=9.5Hz,1H),5.34-5.16(m,11H),5.10(t,J=9.7Hz,1H),4.85( d,J=10.0Hz,1H),4.69-4.51(m,8H),4.32-4.24(m,2H),4.22-4.18(m,1H),3.30-3.27(m,2H),2.60-2.53(m,2H).

[0669] Preparation of compound LK33

[0670] Step 1: Synthesis of compound LK33-1

[0671] At 0°C, an aqueous solution (2 mL) of Int15 (125 mg, 0.33 mmol) was added to a DMF (3 mL) solution of compound LK30-1 (400 mg, 0.33 mmol), and the resulting mixture was then transferred to room temperature and stirred for 5 minutes. The reaction mixture was purified by reverse-phase chromatography to give compound LK33-1 (300 mg, 61% yield).

[0672] Step 2: Synthesis of compound LK33-2

[0673] TFA (5 mL) was added to a DMF (5 mL) solution of compound LK33-1 (300 mg, 0.20 mmol), and the mixture was stirred at 25 °C for 3 hours. After the reaction was complete, the mixture was concentrated, and the residue was purified by reverse chromatography to obtain compound LK33-2 (150 mg, yield 52%).

[0674] Step 3: Synthesis of compound LK33

[0675] At 0 °C, an aqueous solution (2 mL) of oxygen (676.3 mg, 1.1 mmol, 10 eq) was added dropwise to a THF (2 mL) solution of compound LK33-2 (150 mg, 0.11 mmol), and the mixture was stirred at room temperature for 10 hours. The solid was removed by filtration, and the filtrate was directly purified by reverse-phase chromatography to give compound LK33 (70.5 mg, yield 43%).

[0676] MS m / z (ESI): 728.4 [M+H] +

[0677] Preparation of compound LK34

[0678] Compound LK34 was synthesized using a method similar to that used for LK2.

[0679] Preparation of compound LK35

[0680] Step 1: Synthesis of compound LK35-2

[0681] LK35-1 (2.0 g, 4.70 mmol) and LK17-1 (1.0 g, 5.64 mmol) were dissolved in DMF (15 mL), and HATU (2.1 g, 5.64 mmol), HOAT (757.8 mg, 5.64 mmol), and TMP (1.7 g, 14.1 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by reverse-phase chromatography to give compound LK35-2 (2.5 g, 90.4% yield).

[0682] Step 2: Synthesis of compound LK35-3

[0683] Compound LK35-2 (2.5 g, 4.25 mmol) was added to DMF (20 mL) with TEA (1.8 mL, 12.74 mmol), and the reaction mixture was stirred overnight at room temperature. After the reaction was confirmed to be complete by LCMS, the reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound LK35-3 (1.3 g, yield 83.5%).

[0684] Step 3: Synthesis of compound LK35-4

[0685] Compound LP76-A (1.12 g, 3.54 mmol) was dissolved in anhydrous DMF (5 mL) under ice bath conditions. HATU (2.0 g, 5.24 mmol) and HOAT (713.2 mg, 5.24 mmol) were added, and the mixture was stirred for 15 min. Then, an anhydrous DMF solution of LK35-3 (1.3 g, 3.54 mmol) was added (4 mL). The mixture was allowed to warm to room temperature and reacted for 0.5 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound LK35-4 (1.12 g, 48% yield).

[0686] Step 4: Synthesis of compound LK35

[0687] TFA (2 mL) was added to a DCM (2 mL) solution of LK35-4 (200.0 mg, 0.3 mmol), the reaction mixture was stirred at 25 °C for 1 hour, and then evaporated to dryness to obtain crude white solid LK35 (180.0 mg, yield 98.3%), which was directly used in the next step of the reaction.

[0688] Example 2: Payload Synthesis

[0689] Synthesis of Compound 3

[0690] Compound M24 (100 mg, 0.16 mmol) and compound Int1 hydrochloride (260 mg, 0.80 mmol) were dissolved in anhydrous ethanol (5 mL), and sodium acetate (164 mg, 2.00 mmol) and acetic acid (120 mg, 2.00 mmol) were 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 3 (65 mg, 45% yield).

[0691] MS m / z (ESI): 893.4 [M+H] +

[0692] 1H NMR (400MHz, CDCl3) δ7.74(s,1H),7.17(d,J=8.8Hz,1H),6.66(d,J=8.7Hz,2H),6.59(d,J=2.4Hz,1H),6.22( s,1H),6.02(s,1H),5.81(s,1H),5.08(d,J=11.7Hz,1H),5.00(d,J=7.4Hz,1H),4.56(s,1H),4.48–4.36(m,2 H),4.33(s,1H),4.27(d,J=4.6Hz,1H),4.17–4.07(m,3H),4.00(s,2H),3.81(s,3H),3.42(d,J=9.9Hz,2H),3 .15–3.05(m,2H),2.97–2.75(m,3H),2.58–2.52(m,4H),2.37(s,3H),2.26(s,3H),2.22(s,3H),2.06(s,3H).

[0693] Synthesis of Compound 4

[0694] Compound 3 (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 4 (1.5 mg, 4% yield).

[0695] MS m / z (ESI): 866.3 [M-OH] +

[0696] 1H NMR (400MHz, CDCl3) δ7.71(s,1H),7.19(d,J=8.7Hz,1H),6.68(d,J=8.7Hz,2H),6.61(s,1H) ,6.22(s,1H),6.01(s,1H),5.22(d,J=11.5Hz,1H),5.04–5.02(m,1H),4.86(s,1H),4.52–4. 43(m,4H),4.23–4.09(m,4H),4.03(s,2H),3.84(s,3H),3.55–3.52(m,1H),3.25–3.17(m,2H ),3.05–2.83(m,3H),2.62–2.53(m,4H),2.40(s,3H),2.28(s,3H),2.23(s,3H),2.07(s,3H).

[0697] Synthesis of Compound 25

[0698] Compound M24 (40 mg, 0.06 mmol) and compound Int2 hydrochloride (100 mg, 0.35 mmol) were dissolved in anhydrous ethanol (1 mL) and acetic acid (2 mL). Sodium acetate (7.38 mg, 0.09 mmol) was added, and the mixture was stirred overnight at 60 °C. After the reaction was monitored by LCMS until complete, the reaction solution was cooled to room temperature. 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 25 (20 mg, 43% yield).

[0699] MS m / z (ESI): 865.3 [M+H] +

[0700] 1H NMR(400MHz, CDCl3)δ7.78(s,1H),7.36–7.32(m,1H),7.25–7.20(m,1H),7.16–7.08(m,1H),7.04–6.99(m,1 H),6.59(d,J=8.0Hz,1H),6.25(d,J=4.4Hz,1H),6.04(s,1H),5.82(s,1H),5.15–5.09(m,1H),4.63–4.58(m ,1H),4.43–4.15(m,5H),4.11–4.05(m,1H),3.83(d,J=13.1Hz,3H),3.64–3.62(m,1H),3.55–3.41(m,3H),3 .40–3.20(m,1H),3.04(s,2H),2.96–2.91(m,4H),2.63–2.35(m,4H),2.30(s,6H),2.15(s,3H),2.05(s,3H).

[0701] Synthesis of Compound 26

[0702] Compound 25 (20 mg, 0.023 mmol) was dissolved in ACN (3 mL) and water (2 mL), and then silver nitrate (98 mg, 0.577 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 26 (3 mg, yield 15.6%).

[0703] MS m / z (ESI): 838.3 [M-OH] +

[0704] 1H NMR (400MHz, CDCl3) δ7.34(t,J=7.2Hz,1H),7.22(d,J=6.3Hz,1H),7.13–7.06(m,1H),7.06–6.94(m,1H),6.65–6.54(m,1H),6.24–6.18(m,1H) ,6.01(t,J=1.7Hz,1H),5.22(d,J=11.8Hz,1H),4.90(s,1H),4.51(s,2H),4.26(dd,J=15.8,11.4Hz,2H),4.17(d,J=7.5Hz,2H),4.11(s,1H),3 .84(d,J=2.6Hz,1H),3.81(s,2H),3.56(s,1H),3.24(d,J=9.0Hz,1H),3.04(d,J=3.3Hz,2H),2.99(d,J=11.7Hz,1H),2.93(d,J=4.0Hz,2H),2. 86(d,J=9.1Hz,1H),2.64–2.46(m,3H),2.43–2.32(m,3H),2.30(d,J=3. 0Hz, 6H), 2.27–2.19 (m, 2H), 2.16 (d, J = 11.4Hz, 1H), 2.13-2.08 (m, 3H).

[0705] Synthesis of Compound 17

[0706] Step 1: Preparation of Compound 17-1

[0707] Compound 17-1 was synthesized using a method similar to that used for compound 3.

[0708] MS m / z (ESI): 893.6 [M+H] + .

[0709] Step 2: Preparation of compound 17-2

[0710] Compound 17-1 (48 mg, 2.1 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 obtain crude product 17-2 (40 mg), which was used directly in the next reaction.

[0711] Step 3: Preparation of Compound 17

[0712] Compound 17-2 (23.8 mg, 0.030 mmol) was dissolved in DMF (1.5 mL), and glycolic acid (3.0 mg, 0.039 mol), HATU (14.9 mg, 0.039 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 Pre-TLC to give compound 17 (16 mg, 62% yield).

[0713] MS m / z (ESI): 851.5 [M+H] +

[0714] 1 H NMR (400MHz, CDCl3) δ6.56(d,J=8.3Hz,1H),6.47–6.42(m,1H),6.05(d,J=5.0Hz,1H),5.98(s,1H),5.75(d,J=9.5Hz,1H), 5.49(d,J=9.8Hz,1H),5.35(s,1H),5.03–4.95(m,1H),4.59(s,1H),4.33(d,J=7.7Hz,1H),4.28–4.20(m,2H),4.16–4.09( m,3H),3.81–3.77(m,3H),3.74–3.70(m,1H),3.65(d,J=3.2Hz,3H),3.52–3.49(m,1H),3.43–3.39(m,1H),2.93–2.82(m,3 H),2.61–2.58(m,1H),2.48(s,1H),2.45(s,1H),2.31(dd,J=6.5,2.9Hz,4H),2.29–2.23(m,3H),2.21(s,3H),2.05(s,3H).

[0715] Synthesis of Compound 18

[0716] Compound 18 was synthesized using a method similar to that used for compound 4.

[0717] MS m / z (ESI): 824.4 [M-OH] +

[0718] 1H NMR (400MHz, CDCl3) δ7.27–7.21(m,2H),7.07(t,J=7.3Hz,1H),6.95(t,J=7.5Hz,1H),6.56(d,J=8.8Hz,1H),6.15(s,1H),5.93 (s,1H),5.73–5.68(m,1H),5.10–5.05(m,1H),4.79–4.74(m,1H),4.44–4.37(m,2H),4.26–4.22(m,1H),4.12–4.01(m,3H),3.77 –3.70(m,3H),3.62–3.59(m,1H),3.43(d,J=7.9Hz,1H),3.19–3.12(m,1H),3.08–2.96(m,1H),2.96–2.72(m,4H),2.60(d,J=2.6 Hz,2H),2.48(d,J=3.8Hz,2H),2.34–2.27(m,3H),2.27–2.22(m,3H),2.19(d,J=6.3Hz,2H),2.13(d,J=2.6Hz,2H),2.00(s,3H).

[0719] Synthesis of compounds 13, 13-P1, and 13-P2

[0720] Step 1: Preparation of compound 13-1

[0721] Compound 13-1 was synthesized using a method similar to that used for compound 3.

[0722] Step 2: Preparation of Compound 13

[0723] Crude compound 13 was synthesized using a method similar to step 2 of compound 17. Crude compound 13 was purified by pre-TLC to obtain compound 13.

[0724] MS m / z (ESI): 779.3 [M+H] +

[0725] 1H NMR (400MHz, CDCl3) δ8.47(s,1H),7.82(s,1H),7.56(d,J=7.8Hz,1H),7.22(d,J=8.2Hz,1H),7.11(t,J=7.6Hz,1H) ,7.03(t,J=7.4Hz,1H),6.65(s,1H),6.25(d,J=1.4Hz,1H),6.03(d,J=1.4Hz,1H),5.10(d,J=11.8Hz,1H),4.53(s,1 H),4.35(s,1H),4.33–4.24(m,2H),4.17(s,2H),3.78(s,4H),3.43(d,J=4.7Hz,1H),3.39(s,1H),2.90(d,J=5.1Hz ,4H),2.78(d,J=11.9Hz,2H),2.46(d,J=16.0Hz,2H),2.37(s,3H),2.25(s,3H),2.21(d,J=3.3Hz,3H),2.04(s,3H).

[0726] The pure compound 13 was then purified using a reverse-phase medium-pressure preparative column under the following purification conditions:

[0727] Column: Agela Technologies C18, 20-35 μM, 100A, 120g

[0728] Mobile phase: pure water (0.1% hydrochloric acid) / acetonitrile

[0729] Gradient: 100% pure water - 100% acetonitrile, 50% acetonitrile product

[0730] The retention times of the first and second peaks on LCMS were 1.415 min and 1.392 min, respectively.

[0731] The first peak (1.415 min) is characterized as follows: 13-P1 (13R or 13S):

[0732] MS m / z (ESI): 779.3 [M+H] +

[0733] 1H NMR (400MHz, DMSO-d6) δ10.81(s,1H),8.95(s,1H),7.93(s,3H),7.64(d,J=6.4Hz,1H),7.40(d,J=6.4Hz,1H),7.11(t,J=6. 4Hz,1H),7.02(t,J=6.4Hz,1H),6.53(s,1H),6.25(d,J=16.8Hz,2H),5.06(d,J=11.2Hz,1H),4.71-4.57(m,1H),4.55-4.47 (m,1H),4.44-4.38(m,1H),4.37-4.28(m,1H),4.09(s,1H),4.07-4.04(m,1H),3.67(s,3H),3.43-3.40(m,1H),3.35-3.25( m,1H),2.99-2.92(m,3H),2.73–2.69(m,1H),2.32(s,3H),2.30(s,3H),2.22-2.08(m,4H),2.00(s,3H),1.99-1.94(m,1H).

[0734] The second peak (1.392 min) is characterized as follows: 13-P2 (13R or 13S):

[0735] MS m / z (ESI): 779.3 [M+H] +

[0736] 1 H NMR (400MHz, DMSO) δ10.67(s,1H),9.02(s,1H),8.24(s,3H),7.74(d,J=6.4Hz,1H),7.42(d,J=6.4Hz,1H),7.12(t,J= 6.4Hz,1H),7.02(t,J=6.4Hz,1H),6.49(s,1H),6.25(d,J=16.8Hz,2H),5.15(d,J=11.2Hz,1H),4.68-4.57(m,1H),4.5 4-4.42(m,1H),4.38-4.26(brs,2H),4.23-4.20(m,1H),4.14(s,1H),3.66(s,3H),3.34-3.26(m,1H),3.14-3.03(m,1H ),3.01-2.84(m,3H),2.56–2.53(m,1H),2.31(s,3H),2.29(s,3H),2.22-2.11(m,3H),2.10-2.01(m,2H),1.97(s,3H).

[0737] Synthesis of Compound 14

[0738] Compound 14 was synthesized using a method similar to that used for compound 4.

[0739] MS m / z (ESI): 770.9 [M+H] +

[0740] 1 H NMR (400MHz, CDCl3) δ7.48(d,J=25.9Hz,1H),7.15(s,1H),7.08–6.94(m,1H),6.51(s,1H),6.16(d,J=11.4Hz,1H),5.92 (d,J=6.9Hz,1H),5.00(d,J=10.9Hz,1H),4.76(d,J=26.5Hz,1H),4.37(d,J=13.9Hz,1H),4.21(s,1H),4.14(s,1H),4.1 0(s,1H),4.01(d,J=11.2Hz,1H),3.74(d,J=5.5Hz,3H),3.62(d,J=18.1Hz,1H),3.42(s,1H),3.23(s,1H),3.13(s,1H), 2.99–2.69(m,7H),2.34(s,2H),2.32(s,2H),2.28(s,2H),2.21(s,2H),2.13(d,J=5.5Hz,3H),2.05(s,3H),1.96(s,2H).

[0741] Synthesis of Compound 33

[0742] Step 1: Preparation of compound 33-1

[0743] Compound 33-1 was synthesized using a method similar to that used for compound 3.

[0744] Step 2: Preparation of compound 33-2

[0745] Compound 33-1 (85 mg, 0.09 mmol) and TEA (27 mg, 0.27 mmol) were dissolved in anhydrous DCM (4 mL) and cooled to 0 °C. Ms₂O (32.5 mg, 0.184 mmol) was then added, and the mixture was allowed to warm naturally to room temperature for 2 h. The mixture was quenched with saturated sodium bicarbonate solution (3 mL) and heated to 45 °C overnight. The reaction was monitored for completeness by LC-MS. The reaction solution was directly extracted with DCM, and the organic phase was separated, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by Pre-TLC to give compound 33-2 (28 mg, 25% yield).

[0746] Step 3: Preparation of compound 33

[0747] Compound 33-2 (16 mg, 0.018 mmol), acetic acid (5.4 mg, 0.09 mmol), and Pd(PPh3)4 (2.0 mg, 0.002 mmol) were added to DCM (2 mL). Bu3SnH (52 mg, 0.18 mmol) was added under nitrogen protection, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by LCMS until complete. Saturated ammonium chloride and dichloromethane were added to the reaction mixture, and after stirring, the mixture was separated into liquid and liquid phases. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain compound 33 (8 mg, 50% yield).

[0748] MS m / z (ESI): 819.4 [M+H] +

[0749] H NMR(400MHz, CDCl3)δ7.36(d,J=7.9Hz,1H),7.19–6.99(m,4H),6.50(s,1H),6.33(s,1H),6.14( s,1H),5.03(d,J=11.2Hz,1H),4.48–4.41(m,2H),4.28(d,J=4.7Hz,1H),4.21–4.18(m,1H),4.05 –4.02(m,1H),3.84(s,3H),3.69(d,J=4.7Hz,1H),3.44(d,J=9.2Hz,1H),3.18(d,J=12.0Hz,1H), 3.00–2.69(m,7H),2.61–2.55(m,2H),2.49–2.36(m,2H),2.32(s,6H),2.20(s,3H),2.02(s,3H).

[0750] Synthesis of Compound 34

[0751] Compound 34 was synthesized using a method similar to that used for compound 4.

[0752] MS m / z (ESI): 810.3 [M+H] +

[0753] 1H NMR (400MHz, CDCl3) δ7.25(d,J=7.9Hz,1H),7.05(d,J=7.3Hz,2H),7.00(d,J=7.4Hz,2H),6.95–6.92(m,1H),6.40( s,1H),6.20(d,J=3.6Hz,1H),6.00(d,J=3.6Hz,1H),5.04–4.96(m,1H),4.29–4.23(m,1H),4.16–3.98(m,2H),3.87 –3.80(m,1H),3.75(s,3H),3.71(s,1H),3.65(d,J=4.9Hz,1H),3.42(s,1H),3.19–3.03(m,3H),2.90–2.82(m,3H), 2.82–2.44(m,7H),2.36–2.25(m,3H),2.22(s,3H),2.21(s,3H),2.19–2.14(m,1H),2.09(s,3H),2.06–1.96(m,1H).

[0754] Synthesis of Compound 21

[0755] Compound 21 was synthesized using a method similar to that used for compound 3.

[0756] MS m / z (ESI): 794.3 [M+H] +

[0757] 1 H-NMR (400MHz, DMSO-d6): δ10.07(s,1H),8.77(s,1H),7.42(d,J=7.9Hz,1H),7.33(d,J=8.1Hz,1H),7.00(t,J=7.5Hz, 1H),6.89(t,J=7.5Hz,1H),6.48(s,1H),6.23(d,J=19.4Hz,2H),5.11(d,J=11.6Hz,1H),4.69(s,1H),4.51–4.37(m,2H) ,4.20(d,J=4.6Hz,1H),4.14(d,J=11.7Hz,1H),4.09(s,1H),3.82(d,J=9.8Hz,2H),3.66(s,3H),2.98–2.92(m,1H),2.8 2–2.71(m,5H),2.67(s,1H),2.33(d,J=4.6Hz,1H),2.27(d,J=7.2Hz,6H),2.06(s,3H),2.00–1.98(m,1H),1.96(s,3H).

[0758] Synthesis of Compound 22

[0759] Compound 22 was synthesized using a method similar to that used for compound 4.

[0760] MS m / z (ESI): 785.4 [M+H] +

[0761] 1 H-NMR (400MHz, DMSO-d6): δ10.05(d,J=8.8Hz,1H),8.65(s,1H),7.56–7.19(m,2H),6.99–6.90(m,2 H),6.48(s,1H),6.23(d,J=7.0Hz,1H),6.13(s,1H),5.04(dd,J=22.9,11.4Hz,1H),4.68(d,J=13.7H z,1H),4.35–4.30(m,2H),4.12–4.09(m,2H),3.83–3.81(m,1H),3.66(s,3H),3.64–3.51(m,2H),3. 11–2.99(m,4H),2.78–2.69(m,4H),2.29(s,3H),2.27(s,3H),2.07–2.03(m,4H),1.96–1.92(m,4H).

[0762] Synthesis of Compound 1

[0763] Compound 1 was synthesized using a method similar to that used for compound 3.

[0764] MS m / z(ESI): 779.4 [M+H] + ;

[0765] 1H NMR(400MHz, DMSO-d6)δ9.62(s,1H),8.80(s,1H),7.03(d,J=8.6Hz,1H),6.52–6.46(m,2H),6.42(d d,J=8.6,2.1Hz,1H),6.23(d,J=7.8Hz,2H),5.34(t,J=4.7Hz,1H),5.07(d,J=11.2Hz,1H),4.47(d,J =2.7Hz,2H),4.22–4.19(m,1H),4.10(s,1H),4.07(s,2H),3.67(s,3H),3.21–3.08(m,2H),2.85–2.6 9(m,6H),2.36–2.33(m,2H),2.30(s,3H),2.27(s,3H),2.06(s,3H),2.04–2.02(s,1H),1.99(s,3H).

[0766] Synthesis of Compound 2

[0767] Compound 2 was synthesized using a method similar to that used for compound 4.

[0768] MS m / z (ESI): 770.4 [M+H] + .

[0769] 1 H NMR (400MHz, CDCl3) δ7.03 (s, 1H), 6.68 (s, 2H), 6.56 (d, J = 8.8Hz, 1H), 6.19 (s ,1H),5.99(s,1H),5.34–5.28(m,2H),5.19(d,J=11.5Hz,1H),4.89–4.84(m,1H ),4.52–4.50(m,2H),4.22–4.08(m,2H),3.82(s,3H),3.23–3.08(m,2H),2.92 –2.42(m,6H),2.37(s,3H),2.26–2.22(m,7H),2.05(s,3H),2.04–2.02(m,1H).

[0770] Synthesis of compounds 15, 15R, and 15S

[0771] Compound 13 (50 mg, 0.064 mmol) was dissolved in DMF (1.5 mL), and glycolic acid (5.8 mg, 0.077 mol), HATU (29.4 mg, 0.077 mmol), and DIEA (16.5 mg, 0.128 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 Pre-TLC to obtain compound 15.

[0772] Compound 15: MS m / z (ESI): 837.2 [M+H] + ;

[0773] Compound 15 was then purified using a reverse-phase medium-pressure preparative column under the following purification conditions:

[0774] Column: Agela Technologies C18, 20-35 μM, 100A, 120g

[0775] Mobile phase: pure water (0.1% formic acid) / acetonitrile

[0776] Gradient: 100% pure water - 100% acetonitrile, 50% acetonitrile product

[0777] The retention times of the first and second peaks on LCMS were 1.651 min and 1.587 min, respectively.

[0778] The first peak (1.651 min) is characterized as follows: 15R or 15S:

[0779] MS m / z (ESI): 837.2 [M+H] + ;

[0780] 1H NMR (400MHz, DMSO) δ10.49(s,1H),8.82(s,1H),7.43(d,J=6.4Hz,1H),7.34(d,J=6.4Hz,1H),7.29(d,J=8.8Hz,1H),7.04(t,J=6.4Hz,1H), 6.92(t,J=6.4Hz,1H),6.49(s,1H),6.24(d,J=16.8Hz,2H),5.43(t,J=5.6Hz,1H),5.10-5.07(m,1H),4.98-4.96(m,1H),4.48-4.47(m,2H) ,4.21-4.20(m,1H),4.08-4.05(m,3H),3.81-3.68(m,2H),3.65(s,3H ),3.45-3.42(m,1H),3.38-3.35(m,1H),3.23-3.19(m,1H),2.90-2.7 8(m,2H),2.76-2.72(m,1H),2.69-2.66(m,2H),2.31(s,3H),2.23(s, 3H),2.06(s,3H),1.99(s,3H),1.98-1.96(m,1H),1.85-1.83(m,1H).

[0781] The second peak (1.587 min) is characterized as follows: 15R or 15S:

[0782] MS m / z (ESI): 837.2 [M+H] + ;

[0783] 1H NMR (400MHz, DMSO) δ10.39(s,1H),8.81(s,1H),7.50(d,J=6.4Hz,1H),7.36(d,J=6.4Hz,1H),7.30(d,J=8.8Hz,1H),7.04(t,J=6. 4Hz,1H),6.90(t,J=6.4Hz,1H),6.48(s,1H),6.24(d,J=16.8Hz,2H),5.39(t,J=5.6Hz,1H),5.13-5.10(m,1H),4.97-4.92(m,1H) ,4.48-4.47(m,2H),4.21-4.20(m,1H),4.14-4.09(m,2H),3.86-3.85(m,2H),3.65(s,3H),3.40-3.38(m,1H),3.21-3.20(m,1H), 2.95-2.76(m,4H),2.63-2.59(m,1H),2.30(s,3H),2.24(s,3H),2.06(s,3H),2.05-2.02(m,1H),1.98(s,3H),1.93-1.82(m,1H).

[0784] Synthesis of Compound 16

[0785] Compound 16 was synthesized using a method similar to that used for compound 4.

[0786] MS m / z (ESI): 828.7 [M+H] + ;

[0787] 1 H NMR (400MHz, CDCl3) δ7.96 (d, J = 24.5Hz, 1H), 7.61-7.43 (m, 1H), 7.30 (s, 1H), 7.14 (t, J = 7. 7Hz,1H),7.09-6.97(m,1H),6.88(s,1H),6.64(s,1H),6.23(d,J=3.2Hz,1H),6.01(s,1H),5 .97-5.64(m,1H),5.46-5.08(m,3H),4.94(s,1H),4.70-4.44(m,2H),4.29-4.03(m,4H),3.9 2-3.79(m,5H),3.53-3.40(m,2H),2.94-2.67(m,4H),2.58-2.16(m,9H),2.18-1.94(m,5H).

[0788] Synthesis of Compounds 23 and 24

[0789] Compounds 23 and 24 were synthesized using a similar method to that used for compounds 3 and 4.

[0790] Compound 23

[0791] MS m / z (ESI): 851.2 [M+H] +

[0792] 1 H NMR(400MHz, DMSO-d6)δ9.92(s,1H),8.77(s,1H),7.74(t,J=5.6Hz,1H),7.36–7.25(m,2H),7.02(t,J=7.6Hz,1H),6 .91(t,J=7.4Hz,1H),6.47(s,1H),6.29–6.20(m,2H),5.58(t,J=5.7Hz,1H),5.17(d,J=10.8Hz,1H),4.60–4.43(m,2H ),4.30–4.18(m,2H),4.15(s,1H),3.90–3.84(m,2H),3.69(s,3H),3.46–3.39(m,2H),3.26–3.19(m,2H),3.00–2.88( m,1H),2.85–2.70(m,3H),2.42–2.34(m,1H),2.29(s,3H),2.24–2.14(m,5H),2.01(s,3H),1.95(s,3H),1.86(s,1H).

[0793] Compound 24

[0794] MS m / z (ESI): 842.2 [M+H] + .

[0795] Synthesis of Compounds 27 and 28

[0796] Compounds 27 and 28 were synthesized using a similar method to that used for compounds 3 and 4.

[0797] Compound 27:

[0798] MS m / z (ESI): 851.2 [M+H] +

[0799] 1H NMR (400MHz, DMSO-d6) δ10.16(s,1H),8.77(s,1H),7.60–7.50(m,1H),7.40–7.25(m,2H),7.03(t,J=7.6Hz,1H),6.91(t,J=7 .8Hz,1H),6.53(s,1H),6.25(d,J=11.4Hz,2H),5.55(t,J=5.5Hz,1H),5.08(d,J=11.0Hz,1H),4.60–4.43(m,2H),4.20(d,J=3 .9Hz,1H),4.15–4.04(m,2H),3.87(d,J=5.5Hz,2H),3.69–3.56(m,4H),3.46–3.40(m,1H),3.25–3.15(m,1H),3.05–2.93(m,1 H),2.95–2.80(m,2H),2.63–2.57(m,1H),2.40–2.28(m,4H),2.19(s,3H),2.15–2.05(m,2H),2.04–1.99(m,7H),1.81(s,1H).

[0800] Compound 28:

[0801] MS m / z (ESI): 842.2 [M+H] +

[0802] 1 H NMR(400MHz, CDCl3)δ12.52(s,1H),7.66-7.52(m,1H),7.35-7.26(m,2H),7.18-6.97(m,2H) ,6.75(s,2H),6.25(s,1H),6.07-6.01(m,2H),5.15(d,J=11.5Hz,2H),4.90-4.89(m,2H),4. 65(s,1H),4.33(s,1H),4.17-4.09(m,4H),3.97-3.83(m,6H),3.51-3.50(m,2H),3.18-3.14 (m,2H),2.65-2.58(m,4H),2.38-2.35(m,4H),2.31-2.14(m,4H),2.08(s,3H),2.02(m,1H).

[0803] Synthesis of Compound 39

[0804] Step 1: Preparation of compound 39-1

[0805] To a DMF (2 mL) solution of compound 13 (95 mg, 0.12 μmol), Boc-glycine (64 mg, 0.36 μmol), HATU (45.6 mg, 0.12 μmol), and HOAt (27.2 mg, 0.12 μmol) were added, and the mixture was stirred at room temperature. Then, TMP (43.6 mg, 0.36 μmol) was added. The reaction mixture was stirred at room temperature for one hour. The reaction mixture was purified directly by reverse-phase preparative column chromatography and lyophilized to give compound 39-1 (65 mg, yield 58.0%).

[0806] Step 2: Preparation of compound 39

[0807] At room temperature, ZnBr2 (40.53 mg, 0.18 mmol) was added to a solution of 39-1 (60 mg, 0.06 mmol) in 1.0 mL of MeNO2. The reaction mixture was stirred at room temperature for 3 hours and then concentrated. The reaction mixture was purified by reverse HPLC and lyophilized to give compound 39 (45 mg, 84.9% yield) as a white solid.

[0808] MS m / z (ESI): 836.2 [M+H] +

[0809] 1 HNMR(400MHz,DMSO-d6)δ10.49–10.37(m,1H),8.82(s,1H),8.33(s,1H),8.07–7.89(m,1H),7.49–7.26(m,2H), 7.04(t,J=7.6Hz,1H),6.95–6.85(m,1H),6.48(d,J=5.0Hz,1H),6.36–6.17(m,2H),5.17–5.01(m,1H),4.94(s, 1H),4.48(s,2H),4.20(s,1H),4.13–4.01(m,2H),3.65(s,3H),3.21(s,3H),3.10(s,1H),3.00–2.60(m,5H),2. 36–2.26(m,3H),2.26–2.20(m,3H),2.12–2.07(m,1H),2.08–2.02(m,3H),2.02–1.95(m,3H),1.94–1.75(m,2H).

[0810] Synthesis of Compound 29

[0811] Compound M24 (150 mg, 0.24 mmol) and compound Int9 (213 mg, 0.7 mmol) were dissolved in anhydrous ethanol (4 mL) and acetic acid (4 mL) and stirred overnight at 70 °C. After the reaction was monitored by LCMS until complete, the reaction solution was cooled to room temperature. 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 reverse HPLC to give compound 29 (18 mg, yield 8.6%).

[0812] MS m / z (ESI): 865.3 [M+H] +

[0813] 1 H NMR (400MHz, CDCl3) δ7.62(d,J=12.4Hz,1H),7.30(t,J=7.3Hz,1H),7.27–7.20(m,1H),7.10–7.01(m,1H),6.96(q,J=8.0Hz,1H),6.49(d,J=22.3Hz ,1H),6.17(dd,J=2.8,1.3Hz,1H),5.96(d,J=1.2Hz,1H),5.71(d,J=3.2H z,1H),4.96(d,J=11.7Hz,1H),4.57(s,1H),4.31(d,J=4.8Hz,1H),4.26–4 .18(m,2H),4.16(d,J=2.7Hz,2H),4.13–4.06(m,1H),3.74(d,J=1.0Hz,3 H),3.66–3.54(m,2H),3.44(s,1H),3.40(d,J=5.1Hz,3H),3.09(s,1H),3. 07–2.94(m,1H),2.93(s,3H),2.54(ddd,J=31.3,14.7,3.1Hz,1H),2.43–2 .33(m,1H),2.25(s,3H),2.19(s,3H),2.10(d,J=1.3Hz,5H),2.02(s,3H).

[0814] Synthesis of Compound 7

[0815] Compound Int14 (180 mg, 0.43 mmol) was dissolved in trifluoroethanol (2 mL), and M24 (89 mg, 0.14 mmol) was added. The mixture was stirred at 60 °C for 2 h. After the reaction was monitored by LCMS until complete, the reaction solution was sent to reversed-phase HPLC for preparation. The prepared solution was lyophilized to give compound 7 (38.79 mg, yield 33%).

[0816] MS m / z (ESI): 865.4 [M+H] + .

[0817] 1 H NMR (400MHz, CDCl3) δ10.19(d,J=14.8Hz,1H),8.76(s,1H),7.28–7.34(m,1H),7.19(d,J=15. 6,1H),6.89-6.93(m,1H),6.49(s,1H),6.24(d,J=15.2,2H),5.08(d,J=10.8,1H),4.52(s,1H ),4.46(d,J=12.8,3H),4.17-4.21(m,2H),4.08(d,J=11.2Hz,3H),3.66(s,3H),3.12-3.20(m ,4H),2.62-2.90(m,9H),2.39-2.47(m,1H),2.28(d,J=12.4Hz,6H),2.06(s,4H),1.99(s,3H).

[0818] Synthesis of compound 124R

[0819] Step 1: Preparation of compound 124R-2

[0820] Compound 124R-1 (2.0 g, 9.1 mmol) was dissolved in THF (40 mL), and 2N sodium bicarbonate aqueous solution (30 mL) was added. After stirring at room temperature, di-tert-butyl dicarbonate (2.4 g, 10.9 mmol) was added, and the reaction was continued for 2 h. After the reaction was monitored by LCMS until complete, the reaction solution was concentrated under reduced pressure to remove THF. The residue was added to ethyl acetate, and the pH was adjusted to about 6 with saturated citric acid aqueous solution while 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 124R-2 (2.5 g, 86% yield).

[0821] Step 2: Preparation of compound 124R-3

[0822] Compound 124R-2 (2.50 g, 7.8 mmol) and ammonium chloride (0.54 g, 10.1 mmol) were dissolved in DMF (20 mL), and HATU (3.56 g, 9.36 mmol) and DIEA (2.6 g, 20.2 mmol) were added sequentially. The reaction was carried out at room temperature with stirring for 4 h, and the reaction was monitored for completeness 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 column chromatography to give 124R-3 (2 g, 80% yield).

[0823] Step 3: Preparation of compound 124R-4

[0824] Compound 124R-3 (2 g, 6.26 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), cooled to 0 °C, and a borane tetrahydrofuran solution (1 N, 50 mL) was slowly added. After the addition was complete, the mixture was allowed to warm to room temperature and the reaction continued for 4 h. The reaction was monitored by LCMS until complete. The reaction solution was cooled to 0 °C, and anhydrous methanol was slowly added under controlled temperature until the borane was completely quenched. Subsequently, 2 N hydrochloric acid aqueous solution was slowly added under controlled temperature until the pH of the mixture was approximately 3. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography to give compound 124R-4 (0.83 g, 65% yield).

[0825] Step 4: Preparation of compound 124R-5

[0826] Compound M24 (100 mg, 0.16 mmol), compound 124R-4 (65 mg, 0.32 mmol), and sodium acetate (138 mg, 1.60 mmol) were dissolved in acetic acid (4 mL). The reaction mixture was stirred at 65 °C for 2 hours. The solution was concentrated, and the crude product was diluted with ethyl acetate and water. The pH was adjusted to 9 with saturated sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and purified by silica gel column chromatography to give compound 124R-5 (72 mg, 60% yield).

[0827] Step 5: Preparation of compound 124R

[0828] Compound 124R-5 (27 mg, 0.033 mmol) was dissolved in DMF (1.5 mL), and glycolic acid (3.0 mg, 0.039 mol), HATU (14.9 mg, 0.039 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 LCMS 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 reverse HPLC to obtain compound 124R (10 mg, 35% yield).

[0829] MS m / z (ESI): 867.1 [M+H] +

[0830] 1H NMR (400MHz, DMSO-d6) δ9.81(s,1H),8.79(s,1H),8.62(s,1H),7.55(s,1H),7.15(d,J=8.6Hz,1H),6.58(s,1H) ,6.57-7.51(m,2H),6.24(d,J=7.7Hz,2H),5.08(d,J=11.1Hz,1H),4.63-4.44(m,2H),4.21(s,1H),4.14-4.02(m ,2H),3.88(s,2H),3.65(s,3H),3.63-3.54(m,1H),3.50-3.41(m,3H),3.23-3.17(m,1H),2.96(s,1H),2.90-2.7 8(m,2H),2.6102.54(m,1H),2.46-2.36(m,1H),2.30(s,3H),2.19(s,3H),2.08-1.92(m,8H),1.86-1.71(m,1H).

[0831] Synthesis of Compound 125R

[0832] Compound 125R was synthesized using the same method as compound 124R.

[0833] MS m / z (ESI): 881.4 [M+H] +

[0834] 1H NMR (400MHz, DMSO-d6) δ9.97(s,1H),8.77(s,1H),7.53(t,J=6.8Hz,1H),7.24(d,J=8.8Hz,1H),6.78(d,J=2.3Hz,1H),6.66(dd,J=8.8 ,2.4Hz,1H),6.53(s,1H),6.24(d,J=9.4Hz,2H),5.56(s,1H),5.08(d,J=11.1Hz,1H),4.53(s,1H),4.47(d,J=2.4Hz,1H),4.20(d,J=4. 5Hz,1H),4.13-4.03(m,2H),3.88(s,2H),3.69(s,3H),3.65(s,3H),3.62-3.57(m,1H),3.44-3.40(m,2H),3.20(d,J=4.2Hz,1H),3.03– 2.93(m,1H),2.89-2.80(m,2H),2.58-2.54(m,2H),2.30(s,3H),2.19(s,3H),2.11–2.05(m,1H),2.05-1.96(m,7H),1.84-1.72(m,1H).

[0835] Synthesis of compound 124S

[0836] Referring to the method of compound 124R, using Compound 124S was synthesized using these as starting materials.

[0837] MS m / z (ESI): 867.3 [M+H] +

[0838] 1H NMR (400MHz, DMSO-d6) δ9.58(s,1H),8.75(s,1H),8.60(s,1H),7.73(t,J=6.1Hz,1H),7.10(d,J=8.6Hz,1H),6.57–6.50(m,2H),6.46 (s,1H),6.23(d,J=11.1Hz,2H),5.57(t,J=4.8Hz,1H),5.15(d,J=11.1Hz,1H),4.52(brs,1H),4.45(d,J=2.4Hz,1H),4.24(d,J=10.4H z,1H),4.21(d,J=4.4Hz,1H),4.14(s,1H),3.87(s,2H),3.68(s,3H),3.45-3.39(m,1H),3.26-3.18(m,2H),2.98-2.86(m,1H),2.82– 2.73(m,3H),2.39–2.31(m,2H),2.29(s,3H),2.25-2.15(m,4H),2.11(dd,J=15.1,9.9Hz,1H),2.00(s,3H),1.95(s,3H),1.82(s,1H).

[0839] Synthesis of compound 89R

[0840] Step 1: Preparation of compound 89R-2

[0841] At room temperature, HATU (133.0 mg, 0.35 mmol), HOAT (47.0 mg, 0.35 mmol), and TMP (30 drops) were added to a DMF (5 mL) solution of compound Int10 (100.0 mg, 0.13 mmol) and compound 89R-1 (60.0 mg, 0.29 mmol). The resulting mixture was then stirred at 20 °C for 5 minutes. After the reaction was confirmed to be complete by LCMS, 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 reverse HPLC to obtain compound 89R-2 (70 mg, yield 54.6%).

[0842] Step 3: Preparation of compound 89R

[0843] At room temperature, TFA (5 mL) was added to a solution of compound 89R-2 (70.0 mg, 0.07 mmol) in DCM (5 mL), and the reaction was stirred at 20 °C for 1 hour. The reaction solution was evaporated to dryness and dissolved in DMF (2 mL), and then purified by reverse HPLC to obtain compound 89R (50 mg, yield 79%).

[0844] MS m / z (ESI): 880.3 [M+H] +

[0845] 1 HNMR (400MHz, DMSO-d6) δ10.14(s,1H),8.78(s,1H),7.61(t,J=5.9Hz,1H),7.37(d,J=8.3Hz,1H),7.29(d,J=7.1Hz ,1H),7.03(t,J=7.2Hz,1H),6.95–6.84(m,2H),6.52(s,1H),6.25(d,J=11.7Hz,2H),5.08(d,J=11.2Hz,1H),4.59– 4.44(m,2H),4.20(d,J=3.8Hz,1H),4.14-4.02(m,3H),3.66(s,3H),3.56–3.40(m,3H),3.22-3.18(m,1H),3.05-2. 95(m,1H),2.90–2.78(m,2H),2.62–2.54(m,4H),2.30(s,3H),2.20(s,3H),2.09–1.96(m,10H),1.85–1.74(m,1H).

[0846] Synthesis of compound 89S

[0847] Compound 89S was synthesized using a method similar to that used for compound 89R.

[0848] MS m / z (ESI): 880.3 [M+H] + .

[0849] Synthesis of compound 127R

[0850] At room temperature, HATU (115.02 mg, 0.30 mmol), HOAt (41.16 mg, 0.30 mmol), and TMP (91.56 mg, 0.76 mmol) were added to a DMF (6 mL) solution of compound Int10 (200.00 mg, 0.25 mmol) and compound 127R-1 (105.04 mg, 1.01 mmol). The reaction mixture was stirred at room temperature for 5 minutes. LC-MS showed that the reaction was 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 reverse HPLC to obtain compound 127R (120 mg, yield 54%).

[0851] MS m / z (ESI): 879.2 [M+H]+

[0852] 1 HNMR (400MHz, DMSO-d6) δ10.15(s,1H),8.78(s,1H),7.50(dd,J=7.0,4.3Hz,1H),7.37(d,J=8.1Hz,1H),7.29(d,J=8.0Hz,1H), 7.03(t,J=7.3Hz,1H),6.91(t,J=7.5Hz,1H),6.54(s,1H),6.25(d,J=8.5Hz,2H),5.45(s,1H),5.08(d,J=11.2Hz,1H),4.54-4.4 8(m,2H),4.20(d,J=4.6Hz,1H),4.10-4.07(m,2H),3.66(s,3H),3.64-3.52(m,1H),3.46-3.31(m,2H),3.20(d,J=4.3Hz,1H),3 .01-2.77(m,3H),2.70-2.53(m,2H),2.31(s,3H),2.20(s,3H),2.16-2.09(m,1H),2.02-1.97(m,8H),1.31(s,3H),1.27(s,3H).

[0853] Example 3: Preparation of linker-payload

[0854] Synthesis of compound LP1

[0855] Step 1: Synthesis of compound LP1-A

[0856] TMP (10 mg, 0.08 mmol) was added to a DMF (1.5 mL) solution of compound 1 (20.0 mg, 0.03 mmol), LP1-B (13.0 mg, 0.03 mmol), HATU (22.0 mg, 0.05 mmol), and HOAT (8.0 mg, 0.05 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction was purified by reversed-phase column chromatography to give LP1-A (17 mg, yield 35.7%).

[0857] Step 2: Synthesis of compound LP1

[0858] TFA (1 mL) was added to a DCM (1 mL) solution of LP1-A (17 mg, 0.02 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness and purified by reversed-phase HPLC to obtain LP1 (10.6 mg, yield 55.8%).

[0859] MS m / z (ESI): 1159.3 [M+H] +

[0860] Synthesis of compound LP3

[0861] Step 1: Synthesis of compound LP3-A

[0862] Compounds LK1 (24.0 mg, 0.06 mmol), HATU (11.4 mg, 0.03 mmol), HOAT (5.0 mg, 0.03 mmol), and TMP (11.3 mg, 0.09 mmol) were added to a 1 mL DMF solution of compound 1 (20.0 mg, 0.03 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was purified directly by reverse preparative column chromatography to give LP3-A (12 mg, yield 37.5%).

[0863] Step 2: Synthesis of compound LP3

[0864] At room temperature, TFA (1 mL) was added to a DCM (1 mL) solution of compound LP3-A (12.0 mg, 0.01 mmol), and the reaction mixture was stirred for 1 hour. After the reaction was complete, the reaction mixture was concentrated and purified by reversed-phase HPLC to obtain LP3 (5.0 mg, yield 43.5%).

[0865] MS m / z (ESI): 1189.6 [M+H] +

[0866] Synthesis of compound LP4

[0867] Compound LP4 was synthesized using a method similar to that used for compound LP3.

[0868] MS m / z (ESI): 1158.6 [M+H] +

[0869] 1HNMR(400MHz,DMSO-d6)δ12.05(s,1H),10.09(s,1H),9.64(s,1H),8.77(s,1H),8.12- 8.08(m,2H),7.64(s,1H),7.23(d,J=11.6Hz,1H),7.17(dd,J=1.2,8.8Hz,1H),7.09-6. 98(m,2H),6.43(s,1H),6.24(s,1H),6.21(s,1H),5.07(d,J=10.8Hz,1H),4.46(d,J=1 .6Hz,1H),4.45-4.37(m,1H),4.20(d,J=3.6Hz,1H),4.08(d,J=10.8Hz,1H),4.06(s,1H ),3.71(d,J=5.6Hz,2H),3.65(s,3H),3.37-3.31(m,2H),3.19(d,J=7.2Hz,1H),3.17- 3.12(m,1H),2.85-2.75(m,4H),2.69-2.59(m,2H),2.48-2.41(m,2H),2.37-2.31(m,1H ),2.29(s,3H),2.26(s,3H),2.25-2.21(m,1H),2.11(t,J=14.8Hz,1H),2.05(s,3H),1. 98(s,3H),1.98-1.90(m,1H),1.83-1.78(m,2H),1.50-1.41(m,4H),1.22-1.16(m,2H).

[0870] Synthesis of compound LP11

[0871] Compound LP11 was synthesized using a method similar to step 1 in the synthesis of compound LP1.

[0872] MS m / z (ESI): 1157.7 [M+H] + .

[0873] Synthesis of compound LP12

[0874] At 20°C, compounds Int13 (10 mg, 0.01 mmol), HATU (5.05 mg, 0.01 mmol), HOAT (1.81 mg, 0.01 mmol), and TMP (4.02 mg, 0.03 mmol) were added to a DMF (1 mL) solution of compound LP12-1 (7.16 mg, 0.01 mmol). After stirring at 20°C for 2 hours, LP12 (7.1 mg, 46% yield) was directly purified by reverse HPLC.

[0875] MS m / z (ESI): 1406.4 [M+H] +

[0876] 1 HNMR(400MHz,DMSO-d6)δ9.89(s,1H),8.81-8.79(m,1H),8.60-8.53(m 1H),8.35-8.28(m,1H),8.15-8.13(m,1H),8.09-8.05(m,1H),8.03-8.00(m,1H),7.35-7.28(m,2H),7.25-7.21(m,6H),7.03-6.99 (m,3H),6.89(t,J=7.4Hz,1H),6.47-6.45(m,1H),6.28-6.23(m,2H),5.15(d,J=12.8Hz,1H),4.65-4.60(m,2H),4.53-4.45(m,3H), 4.27-4.15(m,5H),3.75-3.56(m,10H),3.43-3.37(m,1H),3.23-3.20(m,1H),3.17-3.11(m,1H),3.09-3.02(m,2H),2.94(s,3H),2 .83-2.77(m,4H),2.29(s,3H),2.22-2.19(m,5H),2.10(t,J=15.2Hz,3H),1.99-1.95(m,7H),1.49-1.44(m,5H),1.21-1.16(m,3H).

[0877] Synthesis of compound LP14

[0878] At room temperature, HATU (11.5 mg, 0.03 mmol), HOAT (4.1 mg, 0.03 mmol), and Int13 (26.4 mg, 0.03 mmol) were added to 1 mL of DMF containing compound LK16 (25 mg, 0.03 mmol). TMP (11.0 mg, 0.09 mmol) was then slowly added after cooling to 0 °C. The reaction solution was reacted at 20 °C for 2 hours. After the reaction was complete, the reaction solution was purified by reverse HPLC to obtain LP14 (16.5 mg, yield 38.4%).

[0879] MS m / z (ESI): 1612.5 [M+H] +

[0880] Synthesis of compound LP16

[0881] Step 1: Synthesis of compound LP16-A

[0882] To a DMF (2 mL) solution of Int11 (25.0 mg, 30.98 μmol), HATU (14.1 mg, 37.18 μmol), LK3 (18.5 mg, 30.98 μmol), HOAT (5.1 mg, 37.18 μmol), and TMP (11.3 mg, 92.92 μmol) were added, and the mixture was stirred at 15 °C for 30 min. LP16-A (18.0 mg, yield 41%) was purified by reverse column chromatography.

[0883] Step 2: Synthesis of compound LP16

[0884] Zinc bromide (87.7 mg, 389.46 μmol) was added to a solution of 1.5 mL of nitromethane containing LP16-A (18.0 mg, 12.98 μmol), and the mixture was stirred at 20 °C for 5 hours. LP16 (12.0 mg, yield 69%) was purified by reverse HPLC. MS m / z (ESI): 1330.2 [M+H] +

[0885] Synthesis of compound LP17

[0886] Compound LP17 was synthesized using a method similar to that used for compound LP14.

[0887] MS m / z (ESI): 1641.6 [M+H] +

[0888] Synthesis of compound LP18

[0889] Step 1: Synthesis of compound LP18-A

[0890] HATU (11.4 mg, 0.03 mmol), Int12 (15.0 mg, 18.92 μmol), HOAT (4.1 mg, 0.03 mmol), and TMP (7.3 mg, 0.1 mmol) were added to a DMF (1 mL) solution of LK8 (24.3 mg, 37.84 μmol). The mixture was stirred at 20 °C for 30 min. After the reaction was confirmed to be complete by LCMS, LP18-A (12.0 mg, 44% yield) was purified by reverse column chromatography.

[0891] Step 2: Synthesis of compound LP18

[0892] Zinc bromide (95.4 mg, 0.42 mmol) was added to a solution of 1.0 mL of nitromethane containing LP18-A (12.0 mg, 8.47 μmol), and the mixture was stirred at 25 °C for 3 hours. LP18 (9.0 mg, 78% yield) was purified by reverse chromatography.

[0893] MS m / z (ESI): 1360.7 [M+H] + .

[0894] Synthesis of compound LP19

[0895] Compound LP19 was synthesized using a method similar to that used for compound LP18.

[0896] MS m / z (ESI): 1346.4 [M+H] +

[0897] Synthesis of compound LP20

[0898] Compound LP20 was synthesized using a method similar to that used for compound LP16.

[0899] MS m / z (ESI): 1316.5 [M+H] +

[0900] 1HNMR(400MHz, DMSO-d6)δ9.92(s,1H),8.76(s,1H),8.68(t,J=6.4Hz,1H),8.26(t,J=5.6Hz,1H),8.14(s,1H),8.04(t,J=5.6Hz,1H),7 .81(m,1H),7.31(dd,J=13.3,8.0Hz,2H),7.03(d,J=7.6Hz,3H),6.91(m,1H),6.46(s,1H),6.25(m,2H),5.16(d,J=11.2Hz,1H),4.71– 4.59(m,2H),4.48(m,2H),4.31–4.19(m,3H),4.15(s,1H),3.92(s,2H),3.71(m7H),3.48–3.40(m,2H),3.26–3.09(m,4H),3.03–2.95( m,1H),2.78(t,J=9.2Hz,3H),2.41–2.15(m,12H),2.10(m,2H),1.96(m,6H),1.86(s,1H),1.81–1.72(m,1H),1.47(m,4H),1.19(m,3H).

[0901] Synthesis of compound LP21

[0902] Compound LP21 was synthesized using a method similar to that used for compound LP14.

[0903] MS m / z(ESI): 1231.8 [M+H] + .

[0904] Synthesis of compound LP22

[0905] Compound LP22 was synthesized using a method similar to that used for compound LP14.

[0906] MS m / z (ESI): 1160.7 [M+H] +

[0907] Synthesis of compound LP25

[0908] Compound LP25 was synthesized using a method similar to that used for compound LP14.

[0909] MS m / z (ESI): 1481.5 [M+H] +

[0910] Synthesis of compound LP23

[0911] Compound LP23 was synthesized using a method similar to that used for compound LP14.

[0912] MS m / z (ESI): 1523.6 [M+H] +

[0913] Synthesis of compound LP24

[0914] Compound LP24 was synthesized using a method similar to that used for compound LP14.

[0915] MS m / z (ESI): 1480.7 [M+H] +

[0916] Synthesis of compound LP26

[0917] Compound LP26 was synthesized using a method similar to that used for compound LP25.

[0918] MS m / z (ESI): 719.8 [M / 2+H] +

[0919] Synthesis of compound LP27

[0920] Compound LP27 was synthesized using a method similar to that used for compound LP16.

[0921] MS m / z (ESI): 658.8 [M / 2+H] +

[0922] Synthesis of compound LP31

[0923] Step 1: Synthesis of compound LP31-A

[0924] In10 (40.0 mg, 50.5 μmol) and LK3-E (37.9 mg, 60.5 μmol) were dissolved in DMF (1.2 mL) and stirred. Then, HATU (40.0 mg, 105.3 μmol), HOAT (40.0 mg, 293.9 μmol), and TMP (12 drops) were added at 25 °C and stirred for 5 minutes at 25 °C. LP31-A (40.0 mg, yield 56.57%) was obtained by reverse column chromatography.

[0925] Step 2: Synthesis of compound LP31-B

[0926] LP31-A (40.0 mg, 28.5 μmol) was dissolved in DMF (1 mL), and TEA (27.0 mg, 266.8 μmol) was added at 25 °C, and the mixture was stirred overnight at 25 °C. The LP31-B reaction mixture was then used directly in the next step.

[0927] Step 3: Synthesis of compound LP31-D

[0928] LP31-C (15.0 mg, 55.9 μmol) was dissolved in DMF (1 mL), and HATU (15.0 mg, 39.5 μmol), HOAT (15.0 mg, 110.2 μmol), and TMP (5 drops) were added at 25 °C. The resulting mixture was then stirred at 20 °C for 1 minute, followed by the addition of the reaction solution of LP31-B (1 mL) obtained in step 2 at 25 °C, and stirred at 25 °C for 2 minutes. LP31-D (35.0 mg, yield 85.78%) was purified by reverse column chromatography.

[0929] Step 4: Synthesis of compound LP31

[0930] LP31-D (35.0 mg, 24.5 μmol) was dissolved in nitromethane (1.2 mL), and zinc bromide (200.0 mg, 888.1 μmol) was added at 20 °C and stirred for 30 minutes. LP31 (15.0 mg, 45.13%) was obtained by reverse HPLC purification.

[0931] MS m / z (ESI): 1373.6 [M+H] +

[0932] 1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),9.10(s,2H),8.77(s,1H),8.69(s,1H),8.27-8.15(m,4H),7.70(s,1H),7.37-7. 30(m,2H),7.04-6.89(m,2H),6.53(s,1H),6.24(d,J=12.0Hz,2H),5.08(d,J=11.0Hz,1H),4.64-4.62(m,2H),4.47(s,1H ),4.32-4.17(m,2H),4.10-4.07(m,2H),3.94(s,2H),3.79-3.71(m,4H),3.65(s,3H),3.40(s,4H),3.20(s,1H),2.98(s, 1H),2.89-2.82(m,2H),2.60-2.55(m,5H),2.39-2.29(m,6H),2.28-2.18(m,6H),2.02-2.00(m,10H),1.86-1.72(m,4H).

[0933] Synthesis of compound LP33

[0934] Compound LP33 was synthesized using a method similar to that used for compound LP16.

[0935] MS m / z (ESI): 651.8 [M / 2+H] +

[0936] 1HNMR(400MHz,DMSO-d6)δ12.05(s,1H),10.46(s,1H),8.81(s,3H),8.61-8.55(m,1H),8.23-8.16(m,1H),8.08-8.01(m,1H),7.38-7.33 (m,2H),7.29-7.20(m,1H),7.05-7.01(m,1H),6.99(s,2H),6.93-6.90(m,1H),6.48(s,1H),1.23(d,J=16.4Hz,2H),5.34-5.31(m,1H), 5.12-5.07(m,1H),5.05-4.94(m,1H),4.59-4.48(m,4H),4.27-4.20(m,2H),4.09-4.07(m,2H),3.92(s,1H),3.82(s,1H),3.73-3.67(m ,5H),3.66(s,3H),3.06-2.96(m,1H),2.90-2.80(m,2H),2.71-2.63(m,2H),2.33-2.22(m,11H),2.12-1.96(m,11H),1.51-1.42(m,6H).

[0937] Synthesis of compound LP36

[0938] Compound LP36 was synthesized using a method similar to that used for compound LP16.

[0939] MS m / z (ESI): 666.8 [M / 2+H] +

[0940] Synthesis of compound LP37

[0941] Compound LP37 was synthesized using a method similar to that used for compound LP16.

[0942] MS m / z(ESI): 623.3 [M / 2+H] + .

[0943] Synthesis of compound LP38

[0944] Compound LP38 was synthesized using a method similar to that used for compound LP19.

[0945] MS m / z (ESI): 1217.7 [M+H] +

[0946] Synthesis of compounds LP39 and LP40

[0947] A mixture of crude compounds LP39 and LP40 was synthesized using a method similar to that used for compound LP14. The crude compounds were initially purified by reverse-phase column chromatography, followed by pre-TLC purification to obtain two peaks. The retention times of the first and second peaks on LCMS were 1.504 min and 1.454 min, respectively, corresponding to LP39 or LP40.

[0948] First peak: MS m / z (ESI): 601.4 [M / 2+H] +

[0949] Second peak: MS m / z (ESI): 601.4 [M / 2+H] + .

[0950] Synthesis of compound LP41

[0951] Compound LP41 was synthesized using the same method as compound LP31.

[0952] MS m / z (ESI): 680.6 [M / 2+H] +

[0953] 1 HNMR(400MHz,DMSO-d6)δ10.50-10.40(m,1H),9.11(d,J=3.1Hz,2H),8.90–8.78(m,1H),8.65-8.60(m,1H),8.50–8.39(m,1H),8.25–8.14(m,2H ),7.35(dd,J=14.0,8.0Hz,3H),7.03(t,J=7.5Hz,1H),6.94-6.89(m,1H),6.48(s,1H),6.23(d,J=16.6Hz,2H),5.08(d,J=10.8Hz,1H),4.98(d,J =8.7Hz,1H),4.55–4.44(m,4H),4.24-4.18(m,2H),4.11-4.04(m,3H),3 .82(d,J=3.7Hz,2H),3.74(d,J=5.4Hz,2H),3.66(s,3H),3.20(s,3H),2. 90–2.78(m,2H),2.77–2.62(m,3H),2.60-2.54(m,3H),2.36–2.29(m,5H ),2.24–2.16(m,5H),2.09–1.93(m,9H),1.90–1.71(m,4H),1.23(s,3H).

[0954] Synthesis of compound LP43

[0955] Compound LP43 was synthesized using a method similar to that used for compound LP14.

[0956] MS m / z(ESI): 1203.6 [M+H] +

[0957] Synthesis of compound LP44

[0958] Compound LP44 was synthesized using a method similar to that used for compound LP14.

[0959] MS m / z (ESI): 1244.7 [M+H] +

[0960] Synthesis of compound LP46

[0961] Step 1: Synthesis of compound LP46-1

[0962] Compound Int1-5 (100 mg, 0.23 mmol) was dissolved in DCM (5 mL), cooled to 0 °C, and 3 mL of hydrochloric acid / dioxane solution (4 N) was added. The mixture was then allowed to warm naturally to room temperature and the reaction continued for 1 h. LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure to give compound LP46-1 (70 mg, 100% yield).

[0963] Step 2: Synthesis of compound LP46-2

[0964] Compound M24 (40 mg, 0.06 mmol) and compound LP46-1 (60 mg, 0.20 mmol) were dissolved in anhydrous ethanol (1 mL) and acetic acid (2 mL), and sodium acetate (7.38 mg, 0.09 mmol) was added. The mixture was stirred overnight at 60 °C. After the reaction was monitored by LCMS until complete, the reaction solution was cooled to room temperature. 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 LP46-2 (15 mg, 30% yield).

[0965] MS m / z (ESI): 835.3 [M+H] +

[0966] Step 3: Synthesis of compound LP46

[0967] Compound LP46 was synthesized using the same method as compound LP14.

[0968] MS m / z (ESI): 1433.3 [M+H]+

[0969] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),8.77(s,1H),8.55(t,J=6.7Hz,1H),8.30(d,J= 3.6Hz,1H),8.13(d,J=8.0Hz,1H),8.07(t,J=5.7Hz,1H),8.01(t,J=5.8Hz,1H),7.26 –7.21(m,5H),7.21–7.12(m,2H),6.98(s,2H),6.67–6.60(m,2H),6.48(s,1H),6.22( d,J=14.3Hz,2H),5.07(d,J=11.3Hz,1H),4.96(d,J=6.8Hz,1H),4.56(d,J=6.7Hz,4H) ,4.46(q,J=5.4,4.2Hz,3H),4.30(s,1H),4.20(d,J=4.5Hz,1H),4.09(s,3H),3.96(s ,2H),3.83–3.67(m,5H),3.65(s,4H),3.59(dd,J=16.7,5.5Hz,2H),3.19(s,2H),3.1 3–3.01(m,2H),2.92–2.72(m,4H),2.69–2.55(m,2H),2.46–2.31(m,2H),2.29(s,3H) ,2.25(s,3H),2.10(t,J=7.4Hz,3H),2.05(s,4H),1.98(s,3H),1.46(p,J=7.4Hz,4H).

[0970] Synthesis of compound LP47

[0971] Compound LP47 was synthesized using a method similar to that used for compound LP34.

[0972] MS m / z (ESI): 594.3 [M / 2+H] +

[0973] Synthesis of compound LP48

[0974] Compound LP48 was synthesized using a method similar to that used for compound LP14.

[0975] MS m / z (ESI): 587.3 [M / 2+H] +

[0976] Synthesis of compound LP60

[0977] Step 1: Synthesis of compound LP60-B

[0978] Compound 13 (83.0 mg, 106.6 μmol) and LP60-A (82.6 mg, 127.9 μmol) were dissolved in DMF (4 mL) and stirred. Then, HATU (80.0 mg, 210.4 μmol), HOAT (80.0 mg, 587.8 μmol), and TMP (24 drops) were added at 20 °C, and the mixture was stirred at 20 °C for 30 minutes. LP60-B (65.0 mg, 43.37% yield) was purified by reverse column chromatography.

[0979] Step 2: Synthesis of compound LP60-C

[0980] Compound LP60-B (65.0 mg, 46.2 μmol) was dissolved in DMF (1 mL), and TEA (26.0 mg, 256.9 μmol) was added at 20 °C. The mixture was stirred at 20 °C for 12 hours, and the resulting LP60-C reaction mixture was used for the next step.

[0981] MS m / z (ESI): 1184.2 [M+H] +

[0982] Step 3: Synthesis of compound LP60-D

[0983] LK18-I (85.0 mg, 61.57 μmol) was dissolved in DMF (3 mL), and HATU (80.0 mg, 210.4 μmol), HOAT (80.0 mg, 587.8 μmol), and TMP (24 drops) were added at 20 °C. The resulting mixture was then stirred at 20 °C for 5 minutes, followed by the addition of a DMF solution of LP60-C (the reaction solution from the previous step, 1 mL) at 20 °C, followed by stirring at 20 °C for 10 minutes. LP60-D (59.0 mg) was purified by reverse column chromatography.

[0984] Step 4: Synthesis of compound LP60-E

[0985] Compound LP60-D (14.0 mg, 5.5 μmol) was dissolved in DMF (1 mL), and TEA (26.0 mg, 256.9 μmol) was added at 20 °C and stirred overnight. LP60-E (8.0 mg, yield 62.61%) was obtained by reverse column chromatography.

[0986] MS m / z (ESI): 1163.8 [M / 2+H] +

[0987] Step 5: Synthesis of compound LP60

[0988] LK1-A (3.0 mg, 9.7 μmol) was dissolved in DMF (0.5 mL) with stirring. DIEA (1 drop) was added at 20 °C. The mixture was then stirred at 20 °C for 5 minutes. LP60-D (7.0 mg, 3.0 μmol) dissolved in DMF (0.5 mL) was added to the reaction mixture, and the mixture was stirred at 20 °C for 1 hour. The solution was purified by reverse HPLC to obtain yellow LP60 (3.5 mg, yield 46.16%).

[0989] MS m / z (ESI): 1281.3 [M / 2 + Na] +

[0990] Synthesis of compound LP61

[0991] Compound LP61 was synthesized using a method similar to that used for compound LP60.

[0992] MS m / z (ESI): 1223.3 [M / 2+H] + .

[0993] Synthesis of compound LP63

[0994] Step 1: Synthesis of compound LP63-B

[0995] LP63-A (30.0 mg, 78.0 μmol), HATU (50.0 mg, 132.0 μmol), HOAt (50.0 mg, 368.0 μmol), and TMP (0.15 mL) were dissolved in DMF (2.0 mL), and compound 1 (30.0 mg, 39.0 μmol) was added. The reaction mixture was stirred at 35 °C for 10 min. LP63-B (28.0 mg, 46.7%) was purified by reverse column chromatography.

[0996] Step 2: Synthesis of compound LP63-C

[0997] Dissolve LP63-B (28.0 mg, 24.0 μmol) in DMF (1.0 mL) and add triethylamine (26.0 mg). Stir the reaction mixture overnight at 35 °C to obtain the LP63-C reaction mixture for the next step; no purification is required.

[0998] Step 3: Synthesis of compound LP63-D

[0999] LK18-I (35.0 mg, 25.0 μmol), HATU (50.0 mg, 132.0 μmol), HOAt (50.0 mg, 368.0 μmol), and TMP (0.1 mL) were dissolved in DMF (2.5 mL), and LP63-C (28.0 mg, 25.0 μmol) was added. The reaction mixture was stirred at 35 °C for 10 min. LP63-D (30.0 mg, yield 47.6%) was purified by reverse column chromatography.

[1000] Step 4: Synthesis of compound LP63-E

[1001] Dissolve LP63-D (30.0 mg, 13.0 μmol) in DMF (1.0 mL) and add triethylamine (26.0 mg). Stir overnight at 35 °C to obtain the LP63-E reaction mixture for the next step; no purification is required.

[1002] Step 5: Synthesis of compound LP63

[1003] LP31-C (15.0 mg, 56.0 μmol), HATU (50.0 mg, 132.0 μmol), HOAt (50.0 mg, 368.0 μmol), and TMP (0.1 mL) were dissolved in DMF (2.5 mL), and LP63-E (30.0 mg, 13.0 μmol) was added. The reaction mixture was stirred at 35 °C for 10 min. LP63 (16.0 mg, 35.6%) was obtained by reverse HPLC purification.

[1004] MS m / z (ESI): 1157.4 [M / 2+H] +

[1005] 1HNMR(400MHz,DMSO-d6)δ10.08(s,1H),9.65(s,1H),9.11(s,2H),8.79(s,1 H),8.12-8.02(m,4H),7.90–7.85(m,1H),7.79–7.68(m,2H),7.65(s,1H),7. 23(d,J=8.8Hz,1H),7.16–7.11(m,1H),6.48(s,1H),6.22(d,J=12.0Hz,2H) ,5.11–5.03(m,1H),4.77(d,J=3.7Hz,2H),4.51-4.45(m,4H),4.41–4.17(m, 12H),4.06(d,J=12.0Hz,2H),3.65(s,3H),3.62–3.54(m,10H),3.52–3.48( m,36H),3.20–3.13(m,5H),3.07–2.97(m,3H),2.86–2.75(m,3H),2.55(s,4H) ),2.42-2.36(m,4H),2.34-2.31(m,2H),2.27(d,J=12.7Hz,7H),2.16–2.08( m,4H),2.05(s,4H),1.98(s,4H),1.82(d,J=7.2Hz,8H),1.30-1.20(m,12H).

[1006] Synthesis of compound LP65

[1007] LP31-C (6.0 mg, 22.4 μmol) was dissolved in DMF (0.8 mL). HATU (15.0 mg, 39.5 μmol), HOAT (15.0 mg, 110.2 μmol), and TMP (5 drops) were added at 20 °C and stirred for 5 minutes. Then, a DMF solution of LP60-E (41.1 mg, 17.7 μmol) was added dropwise to the reaction mixture, and the mixture was stirred at 20 °C for 5 minutes. LP65 (30.0 mg, 66.67% yield) was obtained by reverse HPLC purification.

[1008] MS m / z (ESI): 1288.9 [M / 2+H] +

[1009] 1HNMR(400MHz,DMSO-d6)δ10.43(d,J=23.6Hz,1H),9.11(s,2H),8.81(s,1H),8.58-8.48(m,1H ),8.39-8.28(m,1H),8.23-8.11(m,3H),8.05(d,J=7.9Hz,2H),7.89-7.87(m,1H),7.78-7.65( m,2H),7.42-7.14(m,8H),7.05-7.01(m,1H),6.96-6.86(m,1H),6.48-6.47(m,1H),6.23(d,J= 16.4Hz,2H),5.09(d,J=11.7Hz,1H),4.99-4.95(m,1H),4.77(d,J=3.6Hz,2H),4.61-4.53(m,2 H),4.49-4.46(m,4H),4.40-4.18(m,10H),4.11-4.06(m,2H),3.95-3.92(m,1H),3.84-3.82( m,1H),3.74-3.69(m,4H),3.67-3.64(m,4H),3.61-3.55(m,10H),3.5-3.47(m,44H),3.41-3.3 9(m,6H),3.23-3.16(m,4H),3.07-2.98(m,4H),2.87-2.63(m,6H),2.57-2.54(m,2H),2.42-2. 28(m,8H),2.23(d,J=6.1Hz,3H),2.16-2.03(m,8H),1.98(d,J=1.5Hz,3H),1.91-1.65(m,7H).

[1010] Synthesis of compounds LP65-P1 and LP65-P2

[1011] Using isomers of compound 13 (LCMS elution times 1.415 min and 1.392 min) as starting materials, compounds LP65-P1 (corresponding to compound 13-P1) and LP65-P2 (corresponding to compound 13-P2) were obtained by following a similar synthetic method to compound LP60.

[1012] Note: The bond indicates that the chirality of the isolated 13 isomer is uncertain. The peak time at LCMS 1.415 min is defined as 13-P1, and 1.392 min as 13-P2.

[1013] LP65-P1:MS m / z(ESI):1288.9[M / 2+H]+ ;

[1014] LP65-P2: MS m / z(ESI):1288.9[M / 2+H] + .

[1015] Synthesis of compound LP66

[1016] Step 1: Synthesis of compound LP66-B

[1017] At room temperature, TMP (97.9 mg, 0.81 mmol) was added to a DMF (2.5 mL) solution of LP66-A (35.0 mg, 90.00 μmol), HATU (25.6 mg, 67.00 μmol), and HOAT (18.3 mg, 135.00 μmol), and the mixture was stirred for 5 minutes. Then, 13 (35.0 mg, 45.0 μmol) was added, and the mixture was stirred for 30 minutes at room temperature. The reaction solution was purified by reverse-phase column chromatography to obtain LP66-B (40.0 mg, 68% yield).

[1018] Step 2: Synthesis of compound LP66-C

[1019] At room temperature, triethylamine (24.9 mg, 0.25 mmol) was added to 1 mL of DMF containing LP66-B (40.0 mg, 0.03 mmol), and the mixture was stirred overnight to obtain LP66-C reaction solution, which was directly used in the next step.

[1020] Step 3: Synthesis of compound LP66-D

[1021] At room temperature, TMP (36.3 mg, 0.30 mmol) was added to 2.5 mL of DMF containing LK18-I (46.4 mg, 0.03 mmol), HATU (17.4 mg, 0.05 mmol), and HOAT (12.5 mg, 0.09 mmol), and the mixture was stirred for 5 minutes. Then, LP66-C (33.0 mg, 0.03 mmol) was added and the mixture was stirred for 30 minutes. The reaction solution was purified by reverse-phase column chromatography to obtain LP66-D (60.0 mg, 80% yield).

[1022] Step 4: Synthesis of compound LP66-E

[1023] At room temperature, triethylamine (27.1 mg, 0.27 mmol) was added to 1 mL of DMF containing LP66-D (30.0 mg, 0.01 mmol) and stirred overnight to obtain LP66-E reaction solution, which was directly used in the next step.

[1024] Step 5: Synthesis of compound LP66

[1025] At 35°C, TMP (20.4 mg, 0.17 mmol) was added to 4 mL of DMF containing LP31-C (10.5 mg, 0.04 mmol), HATU (17.0 mg, 0.05 mmol), and HOAT (16.9 mg, 0.13 mmol), and the mixture was stirred for 5 minutes. Then, 1 mL of DMF containing LP66-E (29.0 mg, 0.01 mmol) was added and the mixture was stirred for 30 minutes. The reaction solution was purified by preparative reverse-phase column chromatography to obtain LP66 (11.0 mg, 34% yield).

[1026] MS m / z (ESI): 1235.1 [M / 2+H] +

[1027] 1 HNMR(400MHz,DMSO-d6)δ10.47(s,1H),9.12(s,2H),8.84(s,1H),8.63(d, J=7.0Hz,1H),8.09-8.03(m,4H),7.98-7.85(m,2H),7.74-7.69(m,2H),7. 37-7.21(m,3H),7.04(s,1H),6.92-6.67(m,1H),6.49(s,1H),6.24(d,J=1 6.0Hz,2H),5.11-4.99(m,2H),4.61-4.50(m,6H),4.27-4.10(m,12H),3.9 1-3.81(m,4H),3.66(s,3H),3.59-3.50(m,62H),3.45-3.42(m,4H),3.23- 3.17(m,4H),3.05-3.00(m,3H),2.87-2.83(m,2H),2.70-2.63(m,3H),2.6 0-2.56(m,2H),2.43-2.37(m,3H),2.34-2.28(m,7H),2.25-2.22(m,3H),2 .15-2.05(m,8H),2.03-1.98(m,6H),1.85-1.79(m,4H),1.75-1.65(m,2H).

[1028] Synthesis of compound LP68

[1029] Step 1: Synthesis of compound LP68-B

[1030] The synthesis of compound LP66 involved dissolving Int10 (200.0 mg, 252.2 μmol) and LP60-A (195.4 mg, 302.7 μmol) in DMF (5 mL) and stirring. Then, HATU (200.0 mg, 526.3 μmol), HOAT (200.0 mg, 1.5 mmol), and TMP (50 drops) were added at 20 °C, and the mixture was stirred at 20 °C for 5 minutes. Pre-HPLC purification yielded LP68-B (200.0 mg, yield 55.82%).

[1031] Step 2: Synthesis of compound LP68-C

[1032] Dissolve LP68-B (200.0 mg, 140.8 μmol) in DMF (2 mL), add TEA (54.0 mg, 533.6 μmol) at room temperature, and stir overnight at room temperature to obtain the LP68-C reaction mixture, which can be used directly in the next step.

[1033] Step 3: Synthesis of compound LP68-D

[1034] LK18-I (240.0 mg, 173.9 μmol) was dissolved in DMF (5 mL), and HATU (240.0 mg, 631.6 μmol), HOAT (240.0 mg, 1.8 mmol), and TMP (60 drops) were added at room temperature. The resulting mixture was then stirred at room temperature for 1 minute, followed by the addition of LP68-C (2 mL), and stirring was continued for 2 minutes. LP68-D (250.0 mg, yield 69.44%) was purified by reverse column chromatography.

[1035] Steps 4 to 5: Synthesis of compound LP68

[1036] LP68-D (250.0 mg, 97.6 μmol) was dissolved in DMF (2 mL), and TEA (54.0 mg, 533.6 μmol) was added at room temperature and stirred overnight. The resulting LP68-E reaction mixture was then used directly in the next step.

[1037] LP31-C (52.0 mg, 193.8 μmol) was dissolved in DMF (4.5 mL), and HATU (90.0 mg, 236.8 μmol), HOAT (90.0 mg, 661.2 μmol), and TMP (23 drops) were added at 20 °C. The resulting mixture was then stirred at room temperature for 1 minute, followed by the addition of the LP68-E reaction mixture (2 mL), and stirred at 20 °C for 1 minute. LP68 (100.0 mg, yield 39.57%) was purified by reverse HPLC.

[1038] MS m / z (ESI): 1294.8 [M / 2+H] +

[1039] 1 HNMR(400MHz,DMSO-d6)δ10.15(s,1H),9.10(s,2H),8.78(s,1H),8.65-8.62(m,1H) ,8.39-8.35(m,1H),8.25-8.00(m,5H),7.9-7.87(m,1H),7.79-7.67(m,3H),7.40-7. 13(m,7H),7.04-7.00(m,1H),6.92-6.88(m,1H),6.53(s,1H),6.24(d,J=11.5Hz,2H) ,5.07(d,J=11.0Hz,1H),4.77-4.75(m,1H),4.69-4.59(m,2H),4.51-4.43(m,4H),4. 41-4.18(m,9H),4.09(d,J=11.6Hz,2H),3.94(s,2H),3.76-3.71(m,5H),3.65(s,4H ),3.62-3.54(m,8H),3.50(s,44H),3.41(s,7H),3.33-3.30(m,4H),3.19-3.15(m,4H ),3.09-2.96(m,4H),2.85-2.77(m,3H),2.67(s,1H),2.55(d,J=7.4Hz,3H),2.43-2. 28(m,8H),2.20(s,3H),2.14-2.11(m,4H),2.01(d,J=7.4Hz,7H),1.82-1.71(m,8H).

[1040] Synthesis of compound LP69

[1041] Step 1: Synthesis of compound LP69-A

[1042] LK18 (25.0 mg, 14.0 μmol) was dissolved in DMF (1.5 mL), and HATU (40.0 mg, 105.0 μmol), HOAt (40.0 mg, 294.0 μmol), TMP (0.1 mL), and Int10 (22.0 mg, 28.0 μmol) were added sequentially. The reaction mixture was stirred at 35 °C for 10 min. LP69-A (30.0 mg, yield 63.7%) was obtained by reverse HPLC purification.

[1043] Step 2: Synthesis of compound LP69-B

[1044] Dissolve LP69-A (30.0 mg, 12.0 μmol) in DMF (1.3 mL) and add triethylamine (26.0 mg). Stir the reaction mixture overnight at 35 °C. The resulting LP69-B reaction mixture can be used directly in the next step without purification.

[1045] Step 3: Synthesis of compound LP69-C

[1046] LP31-C (15.0 mg, 56.0 μmol), HATU (50.0 mg, 132.0 μmol), HOAt (50.0 mg, 368.0 μmol), and TMP (0.1 mL) were dissolved in DMF (2.5 mL), and the reaction solution from step 2 (LP69-B) was added. The mixture was stirred at 35 °C for 10 minutes. LP69-C (16.0 mg, yield 35.6%) was obtained by reverse HPLC purification.

[1047] Step 4: Synthesis of compound LP69

[1048] LP69-C (16.0 mg, 6.0 μmol) was dissolved in nitromethane (1.5 mL), and zinc bromide (200.0 mg) was added. The reaction mixture was stirred at 35 °C for 30 minutes. LP69 (7.0 mg, yield 43.8%) was obtained by reverse HPLC purification.

[1049] MS m / z (ESI): 1257.4 [M / 2+H] +

[1050] 1HNMR(400MHz,DMSO-d6)δ10.14(brs,1H),9.10(s,2H),8.78(s,1H),8.70–8.63 (m,1H),8.28–8.16(m,3H),8.07–8.02(m,2H),7.92–7.86(m,1H),7.70(s,3H), 7.39–7.29(m,2H),7.07–6.99(m,1H),6.95–6.88(m,1H),6.53(s,1H),6.24(d, J=11.6Hz,2H),5.36–5.29(m,1H),5.11–5.04(m,1H),4.76(d,J=3.2Hz,2H),4. 67–4.60(m,2H),4.51-4.46(m,4H),4.40-4.34(m,4H),4.23-4.17(m,3H),4.20 (s,3H),4.12-4.08(m,2H),3.93(s,2H),3.78-3.72(m,5H),3.65(s,4H),3.63– 3.49(m,60H),3.21–3.15(m,6H),3.05-2.97(m,5H),2.89–2.80(m,3H),2.69–2 .66(m,2H),2.34–2.30(m,6H),2.20(s,3H),2.05–1.95(m,14H),1.23(s,20H).

[1051] Synthesis of compound LP70

[1052] Compound LP70 was synthesized using a method similar to that used for compound LP66.

[1053] MS m / z (ESI): 1242.4 [M / 2+H] +

[1054] 1HNMR(400MHz,DMSO-d6)δ10.15(s,1H),9.12(s,2H),9.11(s,2H),8.78(s,1H),8.72 -8.69(m,1H),8.09-8.02(m,4H),7.98-7.96(m,1H),7.90-7.87(m,1H),7.74-7.69( m,3H),7.37-7.35(m,1H),7.32-7.30(m,1H),7.05-7.01(m,1H),6.93-6.89(m,1H), 6.53(s,1H),6.26-6.23(m,2H),5.10-5.07(m,1H),4.68-4.58(m,3H),4.53-4.48(m ,2H),4.27-4.18(m,7H),4.11-4.07(m,2H),3.92(s,2H),3.65(s,3H),3.60-3.55(m ,9H),3.50(s,44H),3.45-3.41(m,17H),3.21-3.16(m,5H),3.05-2.98(m,3H),2.88 -2.81(m,2H),2.61-2.56(m,6H),2.41-2.37(m,2H),2.32-2.30(m,5H),2.20(s,3H) ,2.14-2.07(m,4H),2.02(s,3H),2.00(s,3H),1.91-1.67(m,7H),1.24-1.19(m,9H).

[1055] Synthesis of compound LP76

[1056] At room temperature, diisopropylethylamine (5 drops) was added to 2 mL of DMF containing LP76-A (4.1 mg, 12.9 μmol), LP60-E (30.0 mg, 12.9 μmol), EDCI (25.4 mg, 129.1 μmol), and HOBT (35.1 mg, 258.1 μmol), and the mixture was stirred for 30 minutes. The reaction solution was purified by reverse HPLC to obtain LP76 (9.0 mg, yield 26.6%).

[1057] MS m / z (ESI): 1311.1 [M / 2+H] +

[1058] 1HNMR(400MHz,DMSO-d6)δ10.46-10.40(m,1H),9.26(s,2H),8.80(s,1H),8.59-8. 50(m,1H),8.37-8.27(m,2H),8.15-8.12(m,1H),8.09-8.01(m,3H),7.91-7.89(m ,1H),7.76-7.66(m,3H),7.38-7.16(m,8H),7.05-7.01(m,1H),6.93-6.88(m,1H) ,6.47(m,1H),6.25-6.21(m,2H),5.10-5.07(m,1H),5.00-4.97(m,1H),4.76-4.75 (m,2H),4.61-4.43(m,10H),4.38-4.19(m,12H),4.11-3.90(m,7H),3.78-3.65(m ,10H),3.60-3.55(m,8H),3.50(s,44H),3.45(s,3H),3.23-3.14(m,4H),2.08-2.9 8(m,5H),2.88-2.73(m,5H),2.65-2.61(m,1H),2.41-2.37(m,2H),2.30(s,3H),2 .23-2.22(m,3H),2.14-2.07(m,5H),2.05(s,3H),1.98(s,3H),1.95-1.64(m,6H).

[1059] Synthesis of compound LP73

[1060] At room temperature, diisopropylethylamine (5 drops) was added to 2 mL of DMF containing LP76-A (4.9 mg, 14.1 μmol), LP68-E (32.9 mg, 14.1 μmol), EDCI (25.4 mg, 129.1 μmol), and HOBT (35.1 mg, 258.1 μmol), and the mixture was stirred for 30 minutes. The reaction solution was purified by reverse HPLC to obtain LP73 (9.0 mg, yield 26.6%).

[1061] MS m / z (ESI): 1318.1 [M / 2+H] +

[1062] 1HNMR (400MHz, DMSO-d6) δ10.15(s,1H),9.26(s,2H),8.78(s,1H),8.65(t,J=6.6Hz,1H),8.38( t,J=5.6Hz,1H),8.29(d,J=8.2Hz,2H),8.16(d,J=7.9Hz,1H),8.09-8.00(m,3H),7.91(t,J=5.4 Hz,1H),7.77-7.70(m,3H),7.36(d,J=8.2Hz,1H),7.31(d,J=7.9Hz,1H),7.26-7.21(m,4H),7. 17(d,J=5.8Hz,1H),7.02(t,J=7.6Hz,1H),6.90(t,J=7.5Hz,1H),6.53(s,1H),6.24(d,J=12.0H z,2H),5.08-5.05(m,1H),4.78(s,2H),4.68-4.59(m,2H),4.60-4.45(m,8H),4.42-4.27(m,9H ),4.22-4.19(m,3H),4.10-4.00(m,4H),3.95(s,4H),3.80-3.69(m,6H),3.65(s,4H),3.60-3.5 6(m,10H),3.50-3.45(m,51H),3.25-3.12(m,4H),3.12-2.96(m,4H),2.89-2.74(m,3H),2.72-2 .55(m,3H),2.43-2.30(m,3H),2.30(s,3H),2.20(s,3H),2.14-2.05(m,4H),2.02-2.00(m,8H).

[1063] Synthesis of compound LP8

[1064] Compound LP8 was synthesized using a method similar to that used for compound LP69.

[1065] MS m / z (ESI): 1215.6 [M+H] +

[1066] 1HNMR (400MHz, DMSO-d6) δ10.09(s,1H),9.51(s,1H),9.09(s,2H),8.78(s,1H),8.32(t,J=5.5Hz,2H),7.66(s,1H ),7.31-7.08(m,2H),6.48(s,1H),6.22(d,J=12.7Hz,2H),5.07(d,J=10.5Hz,1H),4.46(d,J=2.1Hz,2H),4.25-4. 19(m,2H),4.09-4.06(m,2H),3.83(d,J=5.4Hz,2H),3.65(s,3H),3.41(s,3H),3.20-3.14(m,3H),2.89-2.75(m, 4H),2.67-2.54(m,4H),2.42-2.32(m,4H),2.29-2.25(m,8H),2.07-2.03(m,4H),1.98(s,3H),1.87-1.76(m,3H).

[1067] Synthesis of compound LP67

[1068] Compound LP67 was synthesized using a method similar to that used for compound LP69.

[1069] MS m / z (ESI): 1251.4 [M / 2+H] +

[1070] 1HNMR(400MHz,DMSO-d6)δ12.03(s,1H),10.46-10.40(m,1H),9.11(s,2H),8.81-8.8 0(m,1H),8.66-8.57(m,1H),8.30-8.12(m,3H),8.05-8.03(m,1H),8.01-7.83(m,3H ),7.76-7.65(m,2H),7.39-7.26(m,3H),7.05-7.01(m,1H),6.94-6.89(m,1H),6.48 (s,1H),6.25-6.21(m,2H),5.12-5.07(m,1H),4.99-4.96(m,1H),4.80-4.74(m,2H), 4.58-4.45(m,6H),4.39-4.21(m,10H),4.08-4.06(m,2H),3.95-3.91(m,1H),3.82- 3.65(m,8H),3.60-3.55(m,9H),3.50(s,44H),3.41-3.38(m,7H),3.23-3.15(m,4H) ,3.05-2.98(m,3H),2.89-2.75(m,3H),2.61-2.55(m,2H),2.41-2.37(m,3H),2.31- 2.30(m,3H),2.24-2.22(m,6H),2.15-2.07(m,5H),2.05(s,3H),1.95-1.67(m,11H).

[1071] Synthesis of compound LP62

[1072] Compound LP62 was synthesized using a method similar to that used for compound LP69.

[1073] MS m / z(ESI):1178.6,1179.2[M / 2+H] +

[1074] 1HNMR(400MHz,DMSO-d6)δ10.09(s,1H),9.55(s,1H),9.08(s,2H),8.78(s,1H), 8.25(s,1H),8.16(s,1H),8.08(d,J=7.8Hz,1H),7.90(s,1H),7.80(s,1H),7.73 (s,1H),7.65(s,1H),7.25-7.19(m,3H),6.48(s,1H),6.22(d,J=13.7Hz,2H),5 .34-5.29(m,1H),5.07(d,J=10.6Hz,1H),4.46-4.40(m,6H),4.24-4.19(m,5H), Synthesis of compound LP35: 4.07 (d, J = 10.1 Hz, 2H), 3.87-3.82 (m, 2H), 3.65 (s, 4H), 3.60-3.55 (m, 11H), 3.50 (s, 44H), 3.23-3.10 (m, 9H), 3.06-2.98 (m, 4H), 2.91-2.74 (m, 5H), 2.71-2.58 (m, 4H), 2.43-2.38 (m, 4H), 2.35-2.32 (m, 3H), 2.27 (d, J = 13.3 Hz, 7H), 2.20 (s, 2H), 2.14-2.10 (m, 3H), 2.05 (s, 4H), 1.98 (s, 4H), 1.90-1.78 (m, 8H).

[1075] Compound LP35 was synthesized using a method similar to that used for compound LP12.

[1076] MS m / z (ESI): 1368.6 [M+H] +

[1077] Synthesis of compound LP79

[1078] HATU (11.4 mg, 0.03 mmol), HOAT (4.1 mg, 0.03 mmol), and TMP (50.0 mg, 0.2 mmol) were added to a DMF (0.5 mL) solution containing LK15 (9.9 mg, 18.0 μmol), and the mixture was stirred at 30 °C for 30 min. LP61-B (9.0 mg, 9.0 μmol) was added to the reaction mixture and stirred for 30 min. LP79 (3.2 mg, 21% yield) was purified by reverse HPLC.

[1079] MS m / z (ESI): 1582.6 [M+H] + .

[1080] Synthesis of compound LP82

[1081] LK1-A (9.88 mg, 32.08 μmol) was dissolved in 1 mL of DMF with stirring. DIEA (3 drops) was added at 250 °C. The mixture was then stirred at 25 °C for 5 minutes. LP68-E (50.0 mg, 21.39 μmol) dissolved in 1 mL of DMF was added to the reaction mixture, and the mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction mixture was purified by reverse HPLC to obtain LP82 (28.3 mg, yield 52.3%).

[1082] MS m / z (ESI): 1266.1 [M / 2+H] +

[1083] 1HNMR(400MHz,DMSO-d6)δ10.15(s,1H),8.79(s,1H),8.63(t,J=6.6Hz,1H),8.36(t,J=5.8Hz,1H),8.19- 8.11(m,2H),8.05-8.00(m,3H),7.87(t,J=5.6Hz,1H),7.79-7.67(m,3H),7.33(dd,J=20.9,8.1Hz,2H), 7.26-7.21(m,4H),7.21-7.13(m,1H),7.06-6.97(m,3H),6.91(t,J=7.3Hz,1H),6.53(s,1H),6.24(d,J= 11.5Hz,2H),5.07(d,J=10.7Hz,1H),4.78-4.75(m,2H),4.69-4.59(m,2H),4.50-4.48(m,3H),4.45(s,1 H),4.41-4.34(m,5H),4.30(dd,J=14.3,6.4Hz,2H),4.25-4.15(m,4H),4.09(d,J=11.1Hz,2H),3.95(s, 2H),3.78-3.7(m,6H),3.65(s,3H),3.62-3.54(m,13H),3.50(s,45H),3.25-3.13(m,4H),3.09-2.96(m, 4H),2.85-2.77(m,4H),2.6-2.57(m,2H),2.44-2.36(m,2H),2.30(s,2H),2.20(s,2H),2.10(t,J=7.4Hz ,7H),2.01(d,J=7.8Hz,7H),1.90-1.82(m,3H),1.75-1.66(m,3H),1.50-1.42(m,5H),1.26-1.12(m,4H).

[1084] Synthesis of compound LP72

[1085] Compound LP72 was synthesized using a method similar to steps 3 to 5 of compound LP68.

[1086] MS m / z (ESI): 1228.7 [M / 2+H] +

[1087] 1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),9.10(s,2H),8.77(s,1H),8.67-8.59(m,1H),8.40-8 .34(m,1H),8.22-8.10(m,3H),8.08-8.02(m,2H),7.91-7.86(m,1H),7.77–7.68(m,3H),7.33 (dd,J=20.7,8.3Hz,2H),7.25-7.21(m,4H),7.19-7.14(m,1H),7.02(t,J=7.7Hz,1H),6.90(t ,J=7.3Hz,1H),6.53(s,1H),6.24(d,J=11.7Hz,2H),5.07(d,J=11.2Hz,1H),4.78-4.74(m,2H ),4.67-4.61(m,2H),4.51–4.44(m,4H),4.38(d,J=6.1Hz,2H),4.35–4.18(m,7H),4.08(d,J =12.6Hz,2H),3.94(s,2H),3.77-3.70(m,5H),3.65(s,4H),3.61-3.55(m,9H),3.50(s,36H), 3.22-3.15(m,6H),3.07-2.97(m,6H),2.87-2.77(m,4H),2.69–2.65(m,2H),2.35-2.28(m,8H ),2.20(s,3H),2.15–2.06(m,5H),2.03-1.98(m,10H),1.90-1.70(m,8H),1.50-1.42(m,1H).

[1088] Synthesis of compound LP83

[1089] HATU (15.2 mg, 0.04 mmol), HOAT (5.5 mg, 0.04 mmol), and TMP (0.01 mL, 0.1 mmol) were added to a solution of LK35 (40.1 mg, 0.07 mmol) and LP68-C (40.0 mg, 0.03 mmol) in DMF (1 mL). The reaction mixture was stirred at 25 °C for 10 min. The reaction solution was subjected to reverse-phase chromatography to give compound LP83 (17.0 mg, yield 28.5%).

[1090] MS m / z (ESI): 895.4 [M / 2+H] +

[1091] 1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),9.26(s,2H),8.77(s,1H),8.64(s,1H),8.37(s,1H),8. 30(s,1H),8.16(s,1H),8.05(s,1H),8.00(s,1H),7.77(s,1H),7.71(s,1H),7.36(d,J=8.1Hz,1 H),7.31(d,J=7.7Hz,1H),7.26–7.21(m,4H),7.20–7.13(m,1H),7.02(t,J=7.9Hz,1H),6.90(t, J=7.4Hz,1H),6.53(s,1H),6.24(d,J=11.8Hz,2H),5.07(d,J=10.8Hz,1H),4.75(d,J=5.0Hz,1H ),4.68–4.61(m,2H),4.58-4.43(m,6H),4.42–4.25(m,6H),4.20(s,1H),4.12-3.98(m,3H),3.9 4(s,4H),3.80–3.70(m,5H),3.65(s,3H),3.61-3.53(m,3H),3.51-3.47(m,1H),3.45(s,3H),3. 43–3.36(m,3H),3.27(s,2H),3.19(d,J=6.8Hz,1H),3.09-2.92(m,5H),2.87-2.75(m,3H),2.60 (s,1H),2.45–2.42(m,5H),2.30(s,3H),2.20(s,3H),2.13(t,J=7.5Hz,2H),2.03-1.98(m,7H).

[1092] Synthesis of compound LP84

[1093] Following steps 1 to 2 of compound LP60, compound LP84 was synthesized using the same method as compound LP83.

[1094] MS m / z (ESI): 864.4 [M / 2+H] +

[1095] 1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),9.28-9.25(m,2H),8.77(s,1H),8.64-8.46(m ,1H),8.43-8.01(m,3H),7.97(dd,J=16.5,7.0Hz,1H),7.82-7.75(m,1H),7.72-7.63( m,1H),7.33(dd,J=19.0,7.9Hz,3H),7.02(t,J=7.5Hz,1H),6.91(t,J=7.2Hz,2H),6.5 3(s,1H),6.24(d,J=11.9Hz,2H),5.08(d,J=11.4Hz,1H),4.80-4.73(s,1H),4.68-4.4 3(m,8H),4.4-4.18(m,9H),4.13-4.05(m,2H),4.01(d,J=11.0Hz,1H),3.98-3.88(m,4 H),3.65(s,3H),3.63-3.37(m,11H),3.30-3.22(m,2H),3.20(d,J=4.8Hz,1H),3.07-2 .94(m,2H),2.91-2.80(m,2H),2.63-2.55(m,3H),2.45–2.38(m,1H),2.30(s,3H),2.2 0(s,3H),2.17-2.08(m,2H),2.07-1.97(m,8H),1.93-1.72(s,3H),1.28-1.15(m,6H).

[1096] Using methods similar to LP60 and LP69, the following compounds were synthesized using the intermediates listed in the table:

[1097] Synthesis of compound LP86

[1098] Step 1: Synthesis of compound LP86-1

[1099] HATU (22.1 mg, 0.06 mmol), HOAt (8.0 mg, 0.06 mmol), TMP (22.0 mg, 0.18 mmol), and compound LP68-C (71.9 mg, 0.06 mmol) were added to a 2 mL DMF solution of compound LK28 (99 mg, 0.06 mmol), and the reaction was carried out at room temperature for 10 minutes. The reaction solution was purified by reverse-phase chromatography to give compound LP86-1 (100 mg, yield 58.9%).

[1100] Step 2: Synthesis of compound LP86-2

[1101] At room temperature, TEA (36.1 mg, 0.357 mmol) was added to a 2 mL DMF solution of compound L1159P9023A-10 (100 mg, 0.035 mmol), and the reaction was allowed to proceed for 8 hours. The reaction solution was purified by reverse chromatography to give compound LP86-2 (60 mg, yield 65.2%).

[1102] Step 3: Synthesis of compound LP86

[1103] At 20°C, HATU (17.5 mg, 0.046 mmol), HOAt (5.7 mg, 0.08 mmol), and TMP (17.1 mg, 0.14 mmol) were added to 2 mL of DMF containing LP86-2 (70.5 mg, 0.023 mmol) and stirred for 10 minutes. Then, LP86-2 (60.0 mg, 0.023 mmol) was added to the reaction solution, and stirring was continued for 30 minutes. After the reaction was complete, compound LP86 (14 mg, yield 20.9%) was purified by reverse HPLC.

[1104] MS m / z (ESI): 1458.3 [M / 2+H] +

[1105] 1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),9.26(s,2H),8.77(s,1H),8.63(s,1H), 8.36(s,2H),8.29(s,1H),8.22(d,J=7.9Hz,1H),8.16(s,1H),8.09–7.93(m,4H ),7.90(s,1H),7.67(s,2H),7.33(dd,J=21.2,8.1Hz,3H),7.20(d,J=25.3Hz,7 H),7.02(s,1H),6.91(d,J=7.6Hz,1H),6.53(s,1H),6.24(d,J=11.7Hz,2H),5.3 2(s,1H),5.07(d,J=11.3Hz,1H),4.85–4.73(m,3H),4.64(s,2H),4.55–4.44(m ,10H),4.41–4.25(m,16H),4.10(s,3H),3.94(s,4H),3.73(s,5H),3.65(s,4H) ,3.59(s,14H),3.50(s,51H),3.17(d,J=4.5Hz,4H),3.04(s,4H),2.85–2.79(m ,3H),2.31(d,J=11.7Hz,7H),2.20(s,3H),2.12(s,3H),2.01(d,J=7.1Hz,13H).

[1106] Synthesis of compound LP105

[1107] Step 1: Synthesis of compound LP105

[1108] Compound LP85-1 (23.7 mg, 36.32 μmol), compound 89R (26.6 mg, 30.27 μmol), and potassium carbonate (16.0 mg, 6.80 μmol) were dissolved in acetonitrile / water (2.1 mL, v / v 3 / 1). The mixture was stirred at room temperature for 30 min, and the reaction was confirmed to be complete by LCMS. The reaction solution was directly purified by reverse HPLC to obtain compound LP105-2 (29.9 mg, yield 60%).

[1109] Steps 2 to 4: Synthesis of compound LP105

[1110] Compound LP105 was synthesized following steps 1 to 3 of LP86.

[1111] MS m / z (ESI): 1472.6 [M / 2+H] +

[1112] Synthesis of compound LP99

[1113] Step 1: Synthesis of compound LP99-1

[1114] At room temperature, HATU (148.9 mg, 0.38 mmol), HOAT (52.2 mg, 0.38 mmol), and TMP (40 drops) were added to a DMF (12 mL) solution of LP68-C (230.0 mg, 0.19 mmol) and compound LK31-1 (134.2 mg, 0.33 mmol). The resulting mixture was then stirred at 20 °C for 5 minutes. The solvent was removed by vacuum concentration, and the residue was purified by reverse-phase chromatography to obtain the synthesis of compound LP99-1 (150.0 mg, yield 49.8%).

[1115] Step 2: Synthesis of compound LP99-2

[1116] At room temperature, TEA (53.0 mg, 0.53 mmol) was added to a solution of LP99-1 (70.0 mg, 0.04 mmol) in 2 mL of DMF, and the reaction was stirred overnight at 20 °C. The reaction mixture was then used directly in the next step.

[1117] MS m / z (ESI): 1369.4 [M / 2+H] +

[1118] Steps 3 to 5: Synthesis of compound LP99

[1119] Compound LP99 was synthesized using a method similar to steps 1 to 3 of compound LP86.

[1120] MS m / z (ESI): 1543.1 [M / 2+H] +

[1121] 1HNMR (400MHz, DMSO-d6) δ10.15(s,1H),9.27(s,2H),8.78(s,1H),8.65(t,J=6.6Hz,1H),8.37(t,J=5.4Hz,2H),8.28( t,J=5.2Hz,1H),8.23(d,J=7.9Hz,1H),8.19-8.13(m,4H),8.08-8.02(m,2H),7.99(t,J=5.0Hz,1H),7.91(t,J=4.9Hz ,1H),7.75–7.65(m,2H),7.36(d,J=8.1Hz,1H),7.31(d,J=8.0Hz,1H),7.26–7.22(m,4H),7.20-7.16(m,1H),7.03(t, J=7.5Hz,1H),6.91(t,J=7.5Hz,1H),6.54(s,1H),6.25(d,J=11.7Hz,2H),5.07(d,J=11.0Hz,1H),4.82(d,J=4.5Hz,1 H),4.78(d,J=4.3Hz,1H),4.74(d,J=4.0Hz,1H),4.68–4.61(m,2H),4.59-4.43(m,10H),4.42–4.24(m,13H),4.20(d, J=3.9Hz,1H),4.14-4.00(m,5H),3.97-3.90(m,5H),3.85–3.71(m,13H),3.69-3.64(m,5H),3.63-3.55(m,12H),3.53 -3.44(m,52H),322-3.14(m,4H),3.11–2.96(m,5H),2.87–2.75(m,3H),2.65-2.55(m,3H),2.44–2.33(m,2H),2.31(s ,3H),2.20(s,3H),2.16–2.07(m,4H),2.2.05-1.96(m,9H),1.96–1.87(m,2H),1.85–1.75(m,2H),1.71-1.59(m,1H).

[1122] Using a method similar to LP86, the following compounds were synthesized using the intermediates listed in the table.

[1123] Synthesis of compound LP93

[1124] Step 1: Synthesis of compound LP93-2

[1125] Compound LP93-1 (50.0 mg, 76.69 μmol) and HOAt (70.0 mg, 514.71 μmol) were dissolved in DMF (2.0 mL), and compound 13 (50.0 mg, 64.27 μmol) was added. The reaction mixture was stirred at 25 °C for 20 min. The reaction mixture was purified by reverse-phase chromatography to give compound LP93-2 (25.0 mg, yield 41.5%).

[1126] Step 2: Synthesis of compound LP93-3

[1127] Compound LP93-1 (40.0 mg, 3.87 μmol) was dissolved in DMF (1.0 mL), and TEA (26.0 mg, 257.43 μmol) was added. The reaction mixture was stirred overnight at 25 °C. The reaction mixture was used for the next step without purification.

[1128] Steps 3 to 5: Synthesis of compound LP93

[1129] Compound LP93 was synthesized using a method similar to steps 2 to 4 of compound LP69.

[1130] MS m / z (ESI): 1230.7 [M / 2+H] +

[1131] 1HNMR(400MHz,DMSO-d6)δ10.36(s,1H),9.97(s,1H),9.11(s,2H),8.79(d,J=19.8Hz,1H),8.18–8 .02(m,4H),7.92-7.87(m,1H),7.79-7.69(m,2H),7.61(d,J=7.6Hz,2H),7.37-7.31(m,2H),7.28( d,J=7.7Hz,2H),7.18-7.08(m,1H),7.06-6.99(m,1H),6.94-6.80(m,1H),6.47(s,1H),6.23(d,J =18.0Hz,2H),5.11–4.99(m,2H),4.93(d,J=12.3Hz,1H),4.81-4.74(m,2H),4.69-4.62(m,1H),4. 48(d,J=18.7Hz,4H),4.42-4.35(m,5H),4.31(dd,J=13.9,6.2Hz,3H),4.25-4.17(m,3H),4.08(d ,J=11.1Hz,2H),3.68-3.56(m,14H),3.50(s,38H),3.22-3.14(m,6H),3.07-2.97(m,4H),2.89–2. 75(m,4H),2.72–2.59(m,4H),2.41-2.37(m,2H),2.35-2.27(m,6H),2.18(s,2H),2.11(dd,J=16.3 ,8.4Hz,5H),2.05(s,3H),1.98(s,3H),1.92-1.78(m,6H),1.76–1.67(m,2H),1.36–1.21(m,13H).

[1132] Synthesis of compound LP100

[1133] Step 1: Synthesis of compound LP100-1

[1134] At room temperature, HOAt (18.5 mg, 0.14 mmol) and DIEA (17.6 mg, 0.14 mmol) were added to a DMF (1.5 mL) solution of compound 89R (60.0 mg, 0.068 mmol) and compound LK32 (145.4 mg, 0.14 mmol). The resulting mixture was then stirred at room temperature for 5 minutes. The reaction mixture was purified by reverse-phase chromatography to give compound LP100-1 (85 mg, 69% yield).

[1135] Step 2: Synthesis of compound LP100-2

[1136] At 0 °C, TEA (12 drops), HCOOH (4 drops), and Pd(PPh3)4 (4.0 mg, 0.004 mmol) were added to a THF (4 mL) solution of compound LP100-1 (80.0 mg, 0.046 mmol). The reaction mixture was stirred at 0 °C for 5 minutes and then at room temperature for 2 hours. The reaction mixture was purified by reverse-phase chromatography to give compound LP100-2 (40 mg, yield 58%).

[1137] Step 3: Synthesis of compound LP100-3

[1138] At room temperature, TEA (52.0 mg, 0.50 mmol) was added to a DMF (5 mL) solution of compound LP100-2 (40.0 mg, 0.026 mmol), and the mixture was stirred overnight. The reaction mixture was then used directly in the next step.

[1139] Step 4: Synthesis of compound LP100-4

[1140] At room temperature, DCC (10.6 mg, 0.052 mmol) and HOSu (6.0 mg, 0.052 mmol) were added to a DMF (1 mL) solution of compound LK33 (50.0 mg, 0.034 mmol), and the reaction was stirred overnight at 20 °C. LC-MS showed that the reaction was complete and was used directly for the next reaction without further treatment. MS m / z (ESI): 1553.0 [M+H] +

[1141] Step 5: Synthesis of compound LP100

[1142] At room temperature, 1.5 mL of the reaction solution from step 3 was added to the reaction solution from step 4, and the resulting mixture was stirred at room temperature for 5 minutes. The reaction mixture was purified by reverse HPLC to obtain compound LP100 (22 mg, yield 42.1%).

[1143] MS m / z (ESI): 911.3 [M / 3+H] +

[1144] 1H NMR (400MHz, DMSO-d6) δ10.16(s,1H),9.26(s,1H),8.79(s,1H),8.79(s,1H),8.15(m,2H),8.09–8.01(m,2H),7.97(m,2H),7.73(m,2H),7. 36(d,J=8.0Hz,1H),7.30–7.19(m,3H),7.11–6.97(m,3H),6.91(t,J=7.3Hz,1H),6.67(s,1H),6.52(s,1H),6.24(m,2H),5.32(t,J=4.8Hz,3 H),5.11–5.00(m,3H),4.78(d,J=4.0Hz,2H),4.57–4.45(m,5H),4.45–4.16(m,13H),4.09(d,J=10.5Hz,2H),3.91(m,2H),3.65(s,4H),3.58 (m,11H),3.50(s,42H),3.18(m,15H),3.03(m,7H),2.87(m,7H),2.67(m,8H),2.39(d,J=4.6Hz,4H),2.29(m,8H),2.21(s,3H),2.07(m,6H).

[1145] Synthesis of compound LP102

[1146] Synthesize starting material LP102-1 according to patent WO2023170247;

[1147] Step 1: Synthesis of compound LP102-2

[1148] At room temperature, HOAT (14.9 mg, 0.11 mmol) and DIEA (14.1 mg, 0.11 mmol) were added to a DMF (3.5 mL) solution of compound 89R (80.0 mg, 0.09 mmol) and compound LP102-1 (141.8 mg, 0.18 mmol). The reaction was stirred at 25 °C for 20 min. The reaction solution was purified by reverse-phase chromatography to give compound LP102-2 (60.0 mg, yield 43.4%).

[1149] Step 2: Synthesis of compound LP102-3

[1150] At room temperature, water (0.25 mL) and DIEA (0.25 mL) were added to a solution of compound LP102-2 (60.0 mg, 0.04 mmol) in MeOH (2 mL). The reaction was stirred at 25 °C for 10 minutes. The reaction solution was purified by reverse chromatography to give compound LP102-3 (30 mg, yield 55%).

[1151] Step 3: Synthesis of compound LP102-4

[1152] At room temperature, TFA (1 mL) was added to a solution of compound LP102-3 (20.0 mg, 0.01 mmol) in DCM (1 mL), and the reaction was stirred at 25 °C for 5 minutes. The reaction solution was evaporated to dryness, and the residue was purified by reverse-phase chromatography to give compound LP102-4 (15 mg, yield 81%).

[1153] Step 4: Synthesis of compound LP102

[1154] At room temperature, HOAt (7.6 mg, 0.06 mmol), HATU (21.3 mg, 0.06 mmol), and TMP (16 drops) were added to a solution of compound LP102-4 (15.0 mg, 0.01 mmol) and compound LP100-4 (81.9 mg, 0.06 mmol) in DMF (2 mL). The resulting mixture was then stirred at 25 °C for 5 minutes. LMCS showed that the reaction was complete, and compound LP102 (4 mg, 10% yield) was purified by reverse-phase column chromatography.

[1155] MS m / z (ESI): 1360.8 [M / 2+H] +

[1156] Synthesis of compound LP104

[1157] Step 1: Synthesis of compound LP104-3

[1158] DMAP (60.71 mg, 0.50 mmol) was added to a THF (6 mL) solution of compound LP104-1 (200.00 mg, 0.50 mmol) and compound LP104-2 (302.36 mg, 0.99 mmol) at 15 °C. The reaction solution was stirred at 15 °C for 10 minutes. The reaction solution was concentrated. The residue was purified by DCM slurry to give compound LP104-3 (150 mg, yield 53.2%).

[1159] Step 2: Synthesis of compound LP104-4

[1160] DMAP (30.51 mg, 0.25 mmol) was added to a 2 mL DMF solution of compound LP104-3 (141.74 mg, 0.25 mmol) and 2-hydroxyisobutyric acid (26.00 mg, 0.25 mmol) at 15 °C, and the reaction mixture was stirred at 15 °C for 10 minutes. The reaction mixture was purified by reverse-phase chromatography to give compound LP104-4 (60 mg, yield 45.1%).

[1161] Steps 3 to 4: Synthesis of compound LP104-6

[1162] Compound LP104-6 was synthesized following steps 1 and 2 of LP69.

[1163] Step 5: Synthesis of compound LP104

[1164] Compound LP104 was synthesized following step 4 of LP102.

[1165] MS m / z (ESI): 1262.2 [M / 2+H] +

[1166] 1 H NMR(400MHz,DMSO-d6)δ10.13(s,1H),9.91(s,1H),9.26(s,2H),8.78(s,1H),8.43-8.40(m,1H),8 .15(d,J=7.1Hz,1H),8.05-7.93(m,3H),7.75-7.68(m,2H),7.62(d,J=8.4Hz,2H),7.37-7.30(m,3 H),7.23(d,J=7.8Hz,1H),7.02(t,J=7.7Hz,1H),6.89(t,J=7.6Hz,1H),6.52(s,1H),6.26-6.23(m ,2H),5.14-4.94(m,3H),4.76(dd,J=4.5,1.8Hz,2H),4.61-4.44(m,6H),4.43-4.17(m,12H),4.14 -3.94(m,5H),3.89(d,J=5.6Hz,2H),3.65(s,3H),3.60-3.55(m,9H),3.51-3.43(m,51H),3.29-3.09(m,7H),3.06-2.79(m,6 H),2.67-2.60(m,2H),2.45-2.35(m,2H),2.30(s,3H),2.23-2.05(m,8H),2.02-1.96(m,8H),1.88-1.84(m,2H),1.55(s,6H).

[1167] Control compound 1 was synthesized according to the method described in patent WO2021214126; control compound DL1 was synthesized according to the method described in patent WO2020084115.

[1168] Example 4: Preparation of ligand-drug conjugates

[1169] 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.

[1170] The amino acid sequence of the anti-ADAM9 antibody DB1001:

[1171] Heavy chain (SEQ ID NO:1)

[1172] Light chain (SEQ ID NO:2)

[1173] The complementarity-determining region and variable region sequences of antibody DB1001 are shown in the table below.

[1174] The amino acid sequence of the anti-GPC-3 antibody DB1002:

[1175] Heavy chain (SEQ ID NO:3)

[1176] Light chain (SEQ ID NO:4)

[1177] The complementarity-determining region and variable region sequences of antibody DB1002 are shown in the table below.

[1178] The amino acid sequence of the anti-EGFR antibody DB1003 is as follows:

[1179] Heavy chain (SEQ ID NO:5)

[1180] Light chain (SEQ ID NO:6)

[1181] The complementarity-determining region and variable region sequences of antibody DB1003 are shown in the table below.

[1182] The amino acid sequence of anti-BCMA antibody DB1004:

[1183] Heavy chain (SEQ ID NO:7)

[1184] Light chain (SEQ ID NO:8)

[1185] The complementarity-determining region and variable region sequences of antibody DB1004 are shown in the table below.

[1186] Preparation of ADC1.14

[1187] 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.

[1188] Compound LP31 (0.61 mg, 1.65 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 ADC1.14 (20 mM histidine-acetic acid, pH 6.0; 3.1 mg, 6.4 mg / mL, yield: 60%), which was stored at 4 °C.

[1189] LC-MS analysis and calculation yielded a Dar value of 5.11.

[1190] Following the synthetic method of compound ADC1.14, the following compounds were synthesized using appropriate linker-payloads.

[1191] Preparation of ADC2.6

[1192] 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.

[1193] Compound LP68 (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 ADC2.6 (20 mM histidine-acetic acid, pH 6.0; 3.5 mg, 5.4 mg / mL, yield: 70%), which was stored at 4 °C.

[1194] LC-MS analysis and calculation yielded a Dar value of 7.48.

[1195] Following the synthesis method of compound ADC2.6, the following compounds were synthesized using appropriate linker-payload.

[1196] Preparation of ADC3.1

[1197] 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.

[1198] Compound LP65 (1.04 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 ADC-3.1 (20 mM histidine-acetic acid, pH 6.0; 3.8 mg, 5.8 mg / mL, yield: 75%), which was stored at 4 °C.

[1199] LC-MS analysis and calculation yielded a Dar value of 7.73.

[1200] SEC instrument detection method for antibody-drug conjugates

[1201] Following the synthesis method of compound ADC3.1, the following compounds were synthesized using appropriate linker-payloads.

[1202] HIC Instrumental Detection Methods for Antibody-Drug Conjugates

[1203] As can be seen from the data in the table, the ADC of the present invention is significantly superior to the control ADC3 and control ADC4 in terms of aggregation and hydrophilicity. Therefore, it can be concluded that the unique linker-payload combination of the ADC of the present invention has better drug-like properties and is significantly superior to the control ADC.

[1204] 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.

[1205] Preparation of ADC4.1

[1206] 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.

[1207] Compound LP65 (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 ADC-4.1 (20 mM histidine-acetic acid, pH 6.5; 4.1 mg, 4.6 mg / mL, yield: 80%), which was stored at 4 °C.

[1208] LC-MS analysis and calculation yielded a Dar value of 7.56.

[1209] Following the synthesis method of compound ADC4.1, the following compounds were synthesized using appropriate linker-payload.

[1210] Example 5: In vitro test of the compound's inhibitory effect on tumor cell proliferation

[1211] Test objective

[1212] To detect the inhibitory activity of drug compounds on the in vitro proliferation of NCI-H82, SKOV-3, OVCAR-3, NCI-H1781, MKN-45, LS174T, and BT474 tumor cells, cells were treated with different concentrations of the compounds in vitro. After 6 days of culture, the cells were analyzed using a CTG (CellTiter- ) assay. 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.

[1213] Experimental methods

[1214] The following example, using the in vitro proliferation inhibition assay for NCI-H82 cells, illustrates the method for testing the in vitro proliferation inhibition activity of the compounds in this application against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[1215] 1. Cell culture: NCI-H82 cells were cultured in 10% FBS RPMI-1640 medium.

[1216] 2. Cell preparation: Take NCI-H82 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.

[1217] 3. Cell plating: Mix the NCI-H82 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).

[1218] 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.

[1219] 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).

[1220] 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.

[1221] 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.

[1222] Data analysis: The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5.

[1223] Table 5 shows the IC50 values ​​of the compounds in this application for inhibiting the in vitro cell proliferation of the aforementioned cells. 50 value Note: " / " indicates that it was not detected.

[1224] Conclusion: Based on the results in Table 5, the compounds in this application exhibited proliferative inhibitory activity against at least one of NCI-H82, SKOV-3, OVCAR-3, NCI-H1781, MKN-45, LS174T, and BT474 tumor cells that was no weaker than or better than the control compound rubitinine.

[1225] Example 6: Toxicological study of different compounds administered intravenously to SD rats

[1226] Experimental Objective

[1227] Sprague-Dawley rats were given a single intravenous injection of seven test products. The acute toxicity of the seven test products in SD rats was observed to compare their toxic effects and provide data support for subsequent safety evaluation.

[1228] Table 6 Experimental Scheme

[1229] Phenomenon: In Sprague-Dawley (SD) rats, after a single intravenous administration of control compound 1, compound 27, compound 89R, compound 15R or 15S (peak 1), or compound 15R or 15S (peak 2) to either male or female rats in group G1, piloerection, arched back, reduced activity, and reddish discharge around the nose were observed, and all rats died between days 6 and 7 post-administration. No obvious clinical symptoms were observed in the other administered groups, and all rats survived to the planned dissection day.

[1230] Conclusion: All animals died during the experiment with compound 1 (control compound), and the maximum tolerated dose was <3.6 mpk. Compounds 27, 89R, 15R or 15S (peak 1), and 15R or 15S (peak 2) showed better tolerability, with a maximum tolerated dose ≥3.6 mpk, and their toxicity was significantly better than that of compound 1 (control compound). The clinical application of antibody-drug conjugates is mainly limited by the toxicity caused by toxins. Increasing the maximum tolerated dose of toxins is beneficial to antibody-drug conjugates, as it can reduce non-target side effects caused by toxins released outside the tumor and reduce systemic toxicity caused by free toxins, showing greater potential for conversion into safer antibody-drug conjugates.

[1231] Example 7: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

[1232] CellTiter-Glo 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.

[1233] 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 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 an S-shaped 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%.

[1234] Table 7: Inhibitory activity of antibody-drug conjugates on the proliferation of human HCT116 cells Note: +++ indicates IC 50 ≤15nM; ++ means 15 < IC 50 <50nM; + indicates IC 50 ≥50nM.

[1235] Table 8: Inhibitory activity of antibody-drug conjugates on the proliferation of human Huh7 cells Note: ++++ indicates IC 50 ≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[1236] Table 9: Inhibitory activity of antibody-drug conjugates on the proliferation of human MDA-MB-468, HCC827, and MDA-MB-231 cells. Note: ++++ indicates IC 50 ≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[1237] Table 10: Inhibitory activity of antibody-drug conjugates on the proliferation of human MM.1R cells Note: ++++ indicates IC 50≤0.1nM; +++ means 0.1 < IC 50 <1nM;++ indicates 1≤IC 50 ≤5nM; + indicates IC 50 >5nM.

[1238] 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.

[1239] Example 8: Bystander Killing Effect

[1240] Experimental objective: To investigate the killing effect of ADC3.1 under co-culture conditions of EGFR-positive and EGFR-negative tumor cells.

[1241] Test methods

[1242] 1. Adjusting cell density

[1243] Cells in the exponential growth phase were collected and viable cells were counted using a cell counter.

[1244] 2. Cell inoculation

[1245] 1) Set up the following three cell combinations in a 96-well cell culture plate as shown in Table 10:

[1246] Table 11 Cell Combinations (+ indicates that this type of cell is present in the cell combination)

[1247] 2) Add 90 μL of cell suspension to each well of a 96-well plate.

[1248] 3) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate overnight.

[1249] 3. Chemical treatment

[1250] 1) Prepare 10× drug diluent.

[1251] 2) Add diluted candidate molecule and control molecule to each well, with 3 replicates for each.

[1252] 3) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 5 days.

[1253] 4. Testing

[1254] 1) 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.

[1255] 2) Cell viability is expressed as mean fluorescence signal value (treatment group) / mean light signal value (control group) × 100%.

[1256] 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.

[1257] Example 9: In vivo tumor suppression test of antibody-drug conjugates

[1258] 9.1 To evaluate the antitumor effect of ADC drugs in a human multiple myeloma NCI-H929 subcutaneous xenograft NPG mouse model.

[1259] 1. Test drug and materials

[1260] Blank control group (control group): physiological saline

[1261] ADC (treatment group): 5 mg / kg, administered twice.

[1262] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[1263] 3. Experimental animals: 6-8 week old female NPG mice, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[1264] 4. Test methods:

[1265] 5×10 6 One NCI-H929 cell was subcutaneously instilled into the right anterior back of 6-8 week old female NPG mice. When the tumor grew to approximately 120 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 5 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded.

[1266] 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).

[1267] The experimental results are shown in Table 12 and Figure 4. The antibody-drug conjugate ADC4.1 showed significant antitumor activity after administration, and there was no significant change in body weight.

[1268] Table 12: In vivo tumor-inhibiting effect of antibody-drug conjugate ADC4.1 on NCI-H929 tumor-bearing mice

[1269] 9.2 Evaluation of the pharmacodynamics of ADC drugs in NCG mouse subcutaneous transplantation of Huh7 cell line model

[1270] 1. Test drug and materials

[1271] Blank control group (control group): physiological saline

[1272] ADC (treatment group): 2 mg / kg, administered twice.

[1273] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[1274] 3. Experimental animals: 6-8 week old female NPG mice, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[1275] 4. Test methods:

[1276] 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.

[1277] 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).

[1278] Table 13: In vivo tumor-inhibiting effect of antibody-drug conjugate ADC2.6 on Huh7 tumor-bearing mice

[1279] The experimental results are shown in Table 13 and Figure 5. The antibody-drug conjugate ADC2.6 showed significant antitumor activity after administration, and there was no significant change in body weight.

[1280] 9.3 Evaluation of the pharmacodynamics of ADC drugs in NPG mouse subcutaneous transplantation of HT-1376 cell line model

[1281] 1. Test drug and materials

[1282] Blank control group (control group): physiological saline

[1283] ADC (treatment group): 5 mg / kg, administered once.

[1284] ADC Control 1 (Control Group): 5 mg / kg, single dose

[1285] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[1286] 3. Experimental animals: 6-8 week old female NPG mice, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[1287] 4. Test methods:

[1288] 5×10 6 One HT-1376 cell was subcutaneously instilled into the right anterior back of 6-8 week old female NPG mice. When the tumor grew to approximately 110 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.

[1289] 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).

[1290] Table 14: In vivo tumor-inhibiting effect of antibody-drug conjugate ADC3.6 on HT-1376 tumor-bearing mice

[1291] The experimental results are shown in Table 14 and Figure 6. The antibody-drug conjugate ADC3.6 showed significant antitumor activity after administration, which was superior to ADC control 1, and there was no significant change in body weight.

Claims

1. A ligand-drug conjugate, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt, or a solvate thereof, wherein, The ligand drug conjugate comprises a ligand and a structure as shown in Formula I: wherein For The * terminal is connected to the carbonyl group, and the ** terminal is connected to the methylene group; R is independently selected from -VC 0-6 Alkylene-UC 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkyl-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C 3-8 Cycloalkylene-OC 0-6 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 3-8 Cycloalkyl-U-,-VC 3-8 Cycloalkyl-C(O)N(R) 6 )-C 0-6 Alkylene-U-, -VC 0-6 Alkylene-C(O)N(R) 6 )-(4- to 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-, -VOC 0-6 Alkylene-C(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 -(4-8 membered heterocycloalkylene)-U-, -V-(4-8 membered heterocycloalkylene)-C(O)-C 0-6 alkylene-U-, -V-(4-8 membered heterocycloalkylene)-C(O)-C 3-8 cycloalkylene-U-, -V-(4-8 membered heterocycloalkylene)-C(O)-(4-8 membered heterocycloalkylene)-U-, -V-(4-8 membered heterocycloalkylene)-N(R 6 )C(O)-C 0-6 alkylene-U-, -V-(4-8 membered heterocycloalkylene)-N(R 6 )C(O)-C 3-8 alkylene-U-, -V-(4-8 membered heterocycloalkylene)-N(R 6 )C(O)-(4-8 membered heterocycloalkylene)-U-, -V-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C(O)N(R 6 )-C 3-8 cycloalkylene-U-, -V-C 0- 6alkylene-(4-8 membered heterocycloalkylene)-C(O)-U- or -V-C 0-6 alkylene-S(O)2N(R 6 )-C 0-6 alkylene-U-; said C 0-6 alkylene, C 3-8 cycloalkylene and 4-8 membered heterocycloalkylene are each optionally substituted with one or more substituents selected from deuterium, halogen, -OH, -CN, -NH2, -NHC 1- 6alkyl, -N(C 1-6 alkyl)2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl and C 3-6 cycloalkyl; U and V are each independently -0-, -S-, -NR 6 - or a chemical bond; R 6 is hydrogen, deuterium, hydroxyl, C 1-6 alkyl, C 3-8 cycloalkyl, or 4- to 8-membered heterocycloalkyl; each of said C 1-6 alkyl, C 3-8 cycloalkyl, and 4- to 8-membered heterocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of deuterium, halogen, -OH, -CN, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2, C 1-6 alkyl, halogenated C 1-6 alkyl, and C 3-6 cycloalkyl; R 4 is hydrogen, deuterium, C 1-6 alkyl, -C(O)C 1-6 alkyl, -C(O)C 3-12 cycloalkyl or -C(O)(4- to 12-membered heterocycloalkyl), wherein the C 1- 6alkyl, C 3-12 cycloalkyl and 4- to 12-membered heterocycloalkyl are each optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -SH, -NH2, -NHC 1- 6alkyl and -SC 1-6 alkyl; or when For R 4 , R 6 groups together with the atom to which they are attached form a 5-12 membered heterocycloalkyl; said 5-12 membered heterocycloalkyl is optionally substituted with one or more substituents selected from halogen, oxo, -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 cycloalkylene-OH, and -C(O)-(4-12 membered heterocycloalkylene)-OH; R 5 Hydrogen, deuterium, halogen, -OH, -CN, -NH2, C 1-6 Alkyl, -OC 1-6 Alkyl, -NHC 1-6 Alkyl, -N(C) 1-6 Alkyl)2, -C 3-6 cycloalkyl, -OC 1-6 Alkyl, 4-6 membered heterocyclic alkyl, -O (4-6 membered heterocyclic alkyl), -CONH2, -CONH (C 1-6 Alkyl), -CON(C) 1- 6-alkyl)2, -OCONH2, -OCONH(C 1-6 Alkyl), -OCON(C) 1-6 Alkyl)2、-NHCOO(C 1-6 alkyl) or -N(C) 1-6 Alkyl)COO(C 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; or when For R2and R3are taken together to form a bivalent linking group selected from -CH2-CH2-, 5 with the atom to which it is attached to form C 3-6 cycloalkyl; said C 3-6 cycloalkyl is optionally substituted with one or more substituents selected from deuterium, halogen, -OH, -CN, -NH2, and C 1-6 alkyl; Y is either -OH or -CN; The heteroatoms in the heterocyclic alkyl and heterocyclic alkyl groups are selected from one, two, or three of N, O, and S; the number of heteroatoms is one, two, or three.

2. The ligand drug conjugate, a tautomer thereof, an enantiomer thereof, a diastereomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof according to claim 1, wherein, The structure shown in Formula I satisfies one or more of the following conditions: ​ (1) R 6 is hydrogen, hydroxyl, or C 1-6 alkyl; said C 1-6 alkyl is optionally substituted with one or more substituents selected from deuterium, halogen, -OH, -CN, -NH2, -NHC 1-6 alkyl, -N(C 1-6 alkyl)2substituents; (2) R 4 is hydrogen or C 1-6 alkyl, said C 1-6 alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -SH, -NH2, -NHC 1-6 alkyl and -SC 1-6 alkyl; (3) R 5 is hydrogen, halogen, -OH, C 1-6 alkyl or -OC 1-6 alkyl, said C 1-6 alkyl is optionally substituted with one or more substituents selected from deuterium, halogen, -OH, -CN, -NH2, C 1-6 alkyl and C 3-6 cycloalkyl; (4) the structure according to Formula I is a structure according to Formula la, Formula lb, or Formula Ic: wherein R 1a is -V-C 0-6 alkylene-U-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C(O)-(4-8 membered heterocycloalkylene)-U-, -V-C 0- 6alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 3-8 cycloalkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)O-C 0-6 alkylene-U-, -V-C 0-6 alkylene-OC(O)N(R 6 )-C 0-6 alkylene-U-, -V-O-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-S(O)2N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C(O)N(R 6 )-(4-8 membered heterocycloalkylene)-U-, -V-(4-8 membered heterocycloalkylene)-C(O)-C 0-6 alkylene-U- or -V-C 0-6 alkylene-N(R 6 )C(O)-C 0-6 alkylene-U-, wherein U is attached to the phenyl ring; R 2a -V-C 0-6 alkylene-U-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C 3-8 cycloalkylene-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-O-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-OC(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)O-C 0-6 alkylene-U-, -V-C 3-8 cycloalkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-S(O)2N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0- 6alkylene-N(R 6 )C(O)-C 0-6 alkylene-U- or -V-(4 to 8 membered heterocycloalkylene)-C(O)-C 0-6 alkylene-U-, wherein U is attached to the piperidine ring; R 3a -V-C 0-6 alkylene-U-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C 3-8 cycloalkylene-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C 3-8 cycloalkylene-O-C 0-6 alkylene-U-, -V-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)-C 0-6 alkylene-U-, -V-C 0-6 alkylene-OC(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)O-C 0-6 alkylene-U-, -V-O-C 0-6 alkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-S(O)2N(R 6 )-C 0-6 alkylene-U-, -V-C 0-6 alkylene-N(R 6 )C(O)N(R 6 )-C 0-6 alkylene-U-, -V-C 3-8 cycloalkylene-C(O)N(R 6 )-C 0-6 alkylene-U-, or -V-(4- to 8-membered heterocycloalkylene)-C(O)-C 0-6 alkylene-U-, wherein U is attached to the piperidine ring; or R 4 or R 3a or R 6 group together with the atom to which it is attached forms a 5-8 membered heterocycloalkyl; The C 0-6 alkylene, C 3-8 cycloalkylene, 5-8 membered heterocycloalkylene, and 4-8 membered heterocycloalkylene are each optionally substituted with one or more substituents selected from halogen, -OH, and C 3-6 substituents of the cycloalkyl.

3. The ligand drug conjugate, a tautomer thereof, an enantiomer thereof, a diastereomer thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof according to claim 2, wherein, The structures shown in equations Ia, Ib, and Ic satisfy one or more of the following conditions: ​ (1) R 1a -N(CH3)-, -NH-, -O- or The a-terminus is connected to the benzene ring; (2) R 1a To The a-terminus is connected to the benzene ring; (3) R 2a is -NH-, -O-, -OCH2- b , The b-end is connected to the piperidine ring; (4) R 3a To The c-terminus is connected to the piperidine ring; (5)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 A group is formed together with the atoms it is attached to.

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 shown in Formula I 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 a structure as shown in Formula II: in, The definitions of A, R, and Y are as described in any one of claims 1-3; L 1a ends are attached to ligands; 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 phenylene, 5-8 membered heteroarylene, 3-10 membered heterocycloalkylene, or 3-10 membered cycloalkylene, wherein said -Cy- is optionally substituted with 1 or more R cx substituents; 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 any C(R cx )2unit in R L2a may each independently be replaced with -C(O)-, -NH-, -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 CH2unit in any of the above structures can be replaced independently by the following structural units: -C(O)-, -NH-, -N(CH3)-, -0-, -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, sugar group or derivative thereof, 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 It is a short peptide consisting of 2-10 amino acid residues that does not exist. 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, halogen, -N02, -CN, -OH, -SH, -NH2, -C02H, -S(0)2OH, -C(0)NH2, -SO2NH2, -OC(0)NH2, -CH2CO-(N(Me)CH2C(0)) z -OR Tra , -CH2CO-(N(Me)CH2C(0)) z -NHR Tra , -(CH2CH20) z -R Tra , -CONH-(CH2CH20) z -R Tra , C 1-6 alkyl, 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, said C 1-6 alkyl, C 2-6 alkenyl, -C 2-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl, and 5-10 membered heteroaryl are optionally substituted with one or more R Tra ; R Tra independently hydrogen, deuterium, halogen, -N02, -CN, -OH, -SH, -NH2, -N(Me)2, -S(0)2Me, -C02H, -S(0)2OH, -C(0)NH2, -SO2NH2, C 1-6 alkyl, 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, z is an independent natural number from 0 to 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 structure shown in Formula II satisfies one or more of the following conditions: (1)L 1a for e end 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 to L 1a connected; (3)L 3 Absent or L 3a -L 3b ; wherein, 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, (Ala)2-Pro-Nva or (Gly)5-Phe-Gly; L 3b is absent, The j-end is connected to Tr; (4) Tr indicates that it does not exist. i end to L 3 Connection.

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 III: in, Ab is the ligand that binds to the target. q represents the drug loading capacity; The definitions of A, R, and Y are as described in any one of claims 1-3; L 1a , L 2 , L 3 and Tr are as defined 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 compound shown in Formula III satisfies one or more 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, for example, 2, 3, 4, 5, 6, 7, 8, 16, 3.01, 3.22, 3.75, 3.76, 3.86, 3.87, 3.89, 3.95, 3.96, 3.98, 4.05, 4.06, 4.08, 4.11, 4.12, 4.13, 4.16, 4.17, 4.20, 4.22, 4.27, 4.28, 4.29, 4.31, 4.32, 4.34, 4.41, 4.36, 4.43, 5.11, 5.58, 6.17, 6.46, 6.56, 6.82, 6.88, 6.98, 7.02, 7.06, 7.08, 7.12, 7.18, 7.22, 7.25, 7.28, 7.31, 7.32, 7.33, 7.41, 7.42, 7.43, 7.45, 7.48, 7.52, 7.53, 7.56, 7.57, 7.58, 7.62, 7.64, 7.65, 7.71, 7.72, 7.73, 7.74, 7.78, 7.81, 7.82, 7.87, 7.88, or 7.93; (3) The ligand-drug conjugate shown in Formula III is a ligand-drug conjugate shown in Formula IIIa, Formula IIIb, or Formula IIIc:

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 shown in Formula III are any of the structures in Table 2.

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 shown in Formula III are any of the structures in Table 3.

11. A mixture of ligand-drug conjugates comprising, as described in any one of claims 1-9, 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 q values.

12. A compound of formula (IV), its tautomer, its meso compound, its racemic compound, its enantiomer, its diastereomer, or a pharmaceutically acceptable salt thereof: in, L 1 for The definitions of A, R, and Y are as described in any one of claims 1-3; L 2 , L 3 and Tr are as defined in claim 5 or 6.

13. The compound of formula (IV) as claimed in claim 12, its tautomers, its meso compound, its racemic compound, its enantiomers, its diastereomers, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula (IV) is any one of the compounds in Table 4.

14. A pharmaceutical composition comprising a ligand drug conjugate as described in any one of claims 1-10, its tautomer, its enantiomer, its diastereomer, its pharmaceutically acceptable salt or solvate thereof, or a mixture of the ligand drug conjugate 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, 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 claimed in claim 11, or a pharmaceutical composition as claimed in claim 14; said cancer is preferably a solid tumor or a non-solid tumor, such as 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.

16. A compound with the structure shown below:

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