Pharmaceutical compound and application thereof

Abstract

Relates to a compound or a ligand-drug conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof. The invention also relates to a preparation method and application of the compound or the ligand-drug conjugate thereof.

Description

A pharmaceutical compound and its use Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a drug compound and a ligand-drug conjugate, as well as their uses. Background Art

[0002] The Toll-like receptor (TLR) family of highly conserved pattern recognition receptor proteins is believed to participate in innate immunity by acting as receptors for pathogen-associated molecular patterns (PAMPs). Compounds that affect TLR activity can impact treatments for conditions such as autoimmunity, inflammation, allergies, asthma, transplant rejection, graft-versus-host disease, infection, cancer, or immunodeficiency. Therefore, there is an urgent need in the art for compounds or drug conjugates that can influence TLR activity.

[0003] Summary of the Invention

[0004] The present application provides a compound or its ligand-drug conjugate, or its isomers, or its mixture form, or its pharmaceutically acceptable salt, which can have an effect selected from the following groups: inhibiting tumor growth, affecting Toll-like receptor (TLR) function, and affecting immune system function.

[0005] In one aspect, the present application provides a compound or a conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound structure is as shown in formula (II-a):

[0006] in,

[0007] R1 is an optionally substituted amino group;

[0008] X1 is selected from optionally substituted -CH2-;

[0009] X3 is selected from optionally substituted -CH= or -N=;

[0010] R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure;

[0011] W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure;

[0012] B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

[0013] Said B is substituted with one or more optionally substituted amino groups, wherein said one or more amino groups are each independently substituted with hydrogen or an optionally substituted C1-C6 alkyl group;

[0014] Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group;

[0015] Ring A can be represented by m R A-1 Substituted, the R A-1 are each independently selected from any group; said m is selected from 1, 2, 3, 4 or 5.

[0016] In another aspect, the present application provides a ligand-drug conjugate, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (II-2A):

[0017] in,

[0018] R1 is an optionally substituted amino group;

[0019] X1 is selected from optionally substituted -CH2-;

[0020] X3 is selected from optionally substituted -CH= or -N=;

[0021] R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure;

[0022] W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure;

[0023] B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

[0024] Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl;

[0025] Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group;

[0026] R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1The methylene units are each independently not replaced, or are each independently replaced by any structure;

[0027] m is selected from 1, 2 or 3;

[0028] Wavy lines in the general formula It indicates that the Z group is directly connected to the ligand through the nitrogen atom on the Z group, or is connected to the ligand through a linker unit.

[0029] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (II-2B):

[0030] in,

[0031] R1 is an optionally substituted amino group;

[0032] X1 is selected from optionally substituted -CH2-;

[0033] X3 is selected from optionally substituted -CH= or -N=;

[0034] R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure;

[0035] W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure;

[0036] B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

[0037] Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl;

[0038] Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group;

[0039] R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure;

[0040] m is selected from 1, 2 or 3;

[0041] Wavy lines in the general formula It means that it is connected to the ligand through the nitrogen atom or carbon atom on the L group;

[0042] L is a connector unit.

[0043] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate has a structure represented by formula (II-2C):

[0044] in,

[0045] R1 is an optionally substituted amino group;

[0046] X1 is selected from optionally substituted -CH2-;

[0047] X3 is selected from optionally substituted -CH= or -N=;

[0048] R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure;

[0049] W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure;

[0050] B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

[0051] Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl;

[0052] Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group;

[0053] R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure;

[0054] m is selected from 1, 2 or 3;

[0055] L is the joint unit;

[0056] n is an integer or decimal from 1 to 10;

[0057] Pc is a ligand.

[0058] In certain preferred embodiments of the present invention, certain groups in the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C) are defined as follows, and the groups not mentioned are the same as those described in any scheme of this application.

[0059] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R1 is -NH2.

[0060] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein X1 is selected from -CH2- or

[0061] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein X3 is selected from -CH= or -C(CH3)=.

[0062] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R2 is selected from the group consisting of 1, 2, or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R 2-2 Substituted, the R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; wherein the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution, R 2-3 Selected from: hydrogen or C1-C3 alkyl.

[0063] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R 2-1 Each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, -CN, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3 or

[0064] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R2 is selected from wherein the methylene units of R2 are each independently unsubstituted, substituted by -O-, or substituted by -NH-.

[0065] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R2 is selected from: For example, R2 is selected from

[0066] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein,

[0067] W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Substituted, the R w-2 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0068] wherein the methylene units of the W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl.

[0069] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R w-1 Each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, -CN, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3 or

[0070] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein W is selected from wherein the methylene units of W are each independently unsubstituted, substituted by -O-, or substituted by -NH-.

[0071] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein W is selected from For example, W is selected from

[0072] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein B is selected from phenyl and pyridyl, and the phenyl and pyridyl are each independently optionally substituted with 1, 2, or 3 R B-1 Substituted, the R B-1 Each is independently selected from hydrogen, halogen, hydroxy, amino, C1-C3 alkyl and C1-C3 alkoxy.

[0073] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein B is selected from Preferably, the B is selected from For example, B is selected from

[0074] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein A is selected from: phenyl, pyridyl, pyrrolyl, thienyl, furyl, pyridazinyl, pyrimidinyl, and pyrazinyl.

[0075] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein A is selected from phenyl and pyridyl. For example, ring A is selected from phenyl. For example, ring A is selected from pyridyl.

[0076] In some embodiments, the structures represented by formula (II-a), (II-2A), (II-2B), and (II-2C), wherein,

[0077] R A-1 R A2-1 -Lx-;

[0078] Lx is selected from: -(CH2) t -、-(CH2) t O-, -O(CH2) t -、-(CH2) t N(R L-1 )-、-(CH2) t S-, -(CH2) t C(=O)-、-(CH2) t N(R L-1 )C(=O)-、 where R L-1 is selected from H or C1-C3 alkyl, t is selected from 0, 1, 2 or 3;

[0079] R A2-1is selected from hydrogen, halogen, hydroxy, amino, cyano, C1-C6 alkyl, alicyclic, alicyclic heterocyclic, aryl and heteroaryl, wherein the C1-C6 alkyl, alicyclic, alicyclic heterocyclic, aryl and heteroaryl are each independently optionally substituted by 1, 2 or 3 R A2-2 Replacement; R A2-2 is selected from hydrogen, halogen, hydroxy, amino, and optionally substituted by 1, 2 or 3 R A2-3 Substituted C1-C6 alkyl; R A2-3 Each is independently selected from: hydrogen, halogen, hydroxy, amino and C1-C3 alkyl;

[0080] Among them, R A2-1 Any methylene unit of can be replaced by the following structures: -O-, -S-, -S(=O)2-, -NH-, -CO-,

[0081] m is selected from 1, 2 or 3.

[0082] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1 R A2-1 -Lx-; Lx is a single bond or -NHC(=O)-.

[0083] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1 R A2-1 -Lx-;R A2-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, or optionally substituted by 1, 2 or 3 R A2-2 Substituted: C1-C6 alkyl, Among them, R A2-1 Any methylene unit of can be replaced by the following structures: -O-, -S-, -S(=O)-, -S(=O)2-, -NH-, -C(O)-,

[0084] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1Selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, cyano, mercapto, -CH3, -CH(=O), -C(=O)OH, -OCH(=O), -SH(=O), -SH(=O)2, -CH2CH3, -CH2OH, -OCH3, -NHCH3, -CH2NH2, -S(=O)2NH2, -NHSH(=O)2, -SCH3, -CH2SH, -S(=O)CH3, -CH2SH(=O), -S(=O)2CH3, -CH2SH(=O)2, -NHCH(=O), -C(=O)NH2, -CH2CH(=O), -CH2NHCH(=O), -C(=O)NHCH3,

[0085]

[0086] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1 R A2-1 -Lx-, Lx is a single bond, R A2-1 Each independently selected from optionally 1, 2 or 3 R A2-2 Substituted: phenyl and pyridyl; R A2-2 is selected from hydrogen, halogen, hydroxy, amino, and optionally substituted by 1, 2 or 3 R A2-3 Substituted C1-C6 alkyl; R A2-3 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino;

[0087] Preferably, R 2A-1 Selected from:

[0088] More preferably, R A-1 Selected from:

[0089] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1 R A2-1 -Lx-, Lx is a single bond, R A2-1 Selected from Preferably, R A2-2 It is -CH2OH.

[0090] In some embodiments, the structures shown in formula (II-a), (II-2A), (II-2B), and (II-2C), wherein R A-1 RA2-1 -Lx-, Lx is -NHC(=O)-, R A2-1 Each independently selected from optionally 1, 2 or 3 R A2-2 Substituted: phenyl and pyridyl; R A2-2 is selected from hydrogen, halogen, hydroxy, amino, and optionally substituted by 1, 2 or 3 R A2-3 Substituted C1-C6 alkyl; R A2-3 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino;

[0091] Preferably, R 2A-1 Selected from:

[0092] More preferably, R 2A-1 Selected from:

[0093] In some embodiments, the structures shown in formula (II-2A), (II-2B), and (II-2C), wherein Z is selected from -N(R Z-1 )-, the R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups; preferably, R Z-1 is selected from H, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3; more preferably, R Z-1 Selected from H.

[0094] In some embodiments, the structures shown in (II-2A), (II-2B), and (II-2C), wherein the structural unit Selected from:

[0095] Among them, R Z-1 , R2, W, R A-1 As described in any one of the schemes (II-2A), (II-2B), and (II-2C) of this application.

[0096] In some embodiments, the present application provides a compound, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound may comprise a structure represented by formula (II-a):

[0097] R1 may be an optionally substituted amino group, X1 may be selected from an optionally substituted -CH2-; X3 may be selected from an optionally substituted -CH- or -N-;

[0098] The R2 may be selected from the following groups: optionally substituted Optionally substituted Optionally substituted and optionally substituted The R2 can be independently one or more R 2-1 Substituted, said one or more R 2-1 may be selected from the group consisting of hydrogen, fluorine, optionally substituted methyl, optionally substituted cyclopropyl, and optionally substituted phenyl;

[0099] W may be selected from the following group: optionally substituted Optionally substituted Optionally substituted Optionally substituted and optionally substituted

[0100] B may be selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted pyridyl, optionally substituted pyrrolyl, optionally substituted thienyl, optionally substituted furanyl, and optionally substituted pyrazinyl, and B may be substituted by one or more optionally substituted amino groups;

[0101] A may be selected from the group consisting of an optionally substituted phenyl group, an optionally substituted pyridyl group, an optionally substituted pyrrolyl group, an optionally substituted thienyl group, an optionally substituted furanyl group, and an optionally substituted pyrazinyl group, and A may be substituted by one or more R A-1 Substituted, the R A-1 does not exist or may contain: optionally substituted CH3-, optionally substituted CH(=CH2)-, optionally substituted HC(=O)-, optionally substituted HOC(=O)-, optionally substituted HC(=O)O-, optionally substituted NH2-, optionally substituted HO-, optionally substituted HS-, optionally substituted HS(=O)-, optionally substituted HS(=O)2-, optionally substituted CH3CH2-, optionally substituted CH2=CH-, optionally substituted HC≡C-, optionally substituted HOCH2-, optionally substituted CH3O-, optionally substituted CH3NH-, optionally substituted NH2CH2-, optionally substituted HS(=O)2-NH-, optionally substituted NH2-S(=O)2-, optionally substituted HS-CH2-, optionally substituted CH3S-, optionally substituted HS(=O)-CH2-, optionally substituted CH3-S(= O)-, optionally substituted HS(=O)2-CH2-, optionally substituted CH3-S(=O)2-, optionally substituted NH2C(=O)-, optionally substituted HC(=O)NH-, optionally substituted HC(=O)CH2-, optionally substituted HC(=O)NHCH2-, optionally substituted CH3NHC(=O)-, optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted and / or optionally substituted One or more R A-1 Each independently can be represented by one or more R A-2 Replacement, R A-2 may contain hydrogen, halogen, optionally substituted methyl and / or optionally substituted hydroxy, said one or more R A-2 Each independently can be represented by one or more R A-3 Substituted, the R A-3 An optionally substituted methyl group and / or an optionally substituted hydroxy group may be contained.

[0102] In some embodiments, the structures represented by formula (II-2B) and (II-2C), wherein the linker unit L is -Tr-L3-L2-L 1c -,

[0103] Tr is selected from and single bonds;

[0104] L 1c Selected from

[0105] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0106] L3 is selected from a single bond or a peptide residue consisting of 1-4 amino acids, wherein the peptide residue is a peptide residue formed by an amino acid selected from the following group: valine, lysine, cysteine, alanine, glycine, glutamic acid, glutamine, phenylalanine, citrulline, aspartic acid, asparagine, and serine.

[0107] In some embodiments, the structures shown in formula (II-2B) and (II-2C), wherein L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

[0108] In some embodiments, the structures represented by formula (II-2B) and (II-2C), wherein the linker unit L is -L3-L2-L 1c -,

[0109] L 1c Selected from

[0110] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0111] L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

[0112] In some embodiments, the structures represented by formula (II-2B) and (II-2C), wherein the linker unit L is -L3-L2-L 1c-or-Tr-L3-L2-L 1c -, the L3 end or the Tr end is connected to the nitrogen atom of the Z group, L 1c end connected to the ligand.

[0113] In some embodiments, the structures represented by formula (II-2B) and (II-2C), wherein the linker unit L is selected from:

[0114] For example, the linker unit L is selected from:

[0115] In some embodiments, the ligand-drug conjugate, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is represented by formula (II-2C-1) and (II-2C-2):

[0116] in,

[0117] R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups;

[0118] X4 and X5 are each independently selected from -CH= and -N=;

[0119] R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R 2-2 Replacement; R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution; R 2-3 Selected from: hydrogen or C1-C3 alkyl;

[0120] W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Replacement; R w-2Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of said W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl;

[0121] R A-1 Selected from R A2-1 -Lx-;

[0122] Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-,

[0123] R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by

[0124] where R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2;

[0125] L 1c Selected from

[0126] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0127] L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-;

[0128] n is an integer or decimal from 1 to 10;

[0129] Pc is a ligand.

[0130] In certain preferred embodiments of the present invention, the structures shown in formula (II-2C-1) and (II-2C-2), wherein X4 and X5 are each independently selected from -CH= and -N=, R Z-1 , R2, W, R A-1 , L 1c , L2, L3, Pc and n are as described in any one of the schemes (Ⅱ-2C) of this application.

[0131] In some embodiments, the ligand-drug conjugate of the present invention, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is selected from the following structural formulas:

[0132] n is an integer or decimal from 1 to 10;

[0133] Pc is a ligand.

[0134] In some embodiments, the ligand-drug conjugate of the present invention, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is selected from the following structural formulas:

[0135] Where n is an integer or decimal from 1 to 10.

[0136] In some embodiments, the compound of the present invention or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt is selected from:

[0137] in,

[0138] R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups;

[0139] R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R 2-2 Replacement; R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution; R 2-3 Selected from: hydrogen or C1-C3 alkyl;

[0140] W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Replacement; R w-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of said W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl;

[0141] R A-1 Selected from R A2-1 -Lx-;

[0142] Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-,

[0143] R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by

[0144] where R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, and the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2.

[0145] In certain preferred embodiments of the present invention, the structures represented by formulas (II-1A-1), (II-1A-2), (II-1A-3), (II-1A-4), (II-1A-5) and (II-1A-6), wherein R Z-1 , R2, W, R A-1 As described in any one of the schemes (II-1A) or (II-2A) of this application.

[0146] In another aspect, the present invention provides a compound or a conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (II-3A):

[0147] in,

[0148] R1 is optionally substituted NH2;

[0149] X1 is selected from optionally substituted -CH2-;

[0150] X3 is selected from optionally substituted -CH= or -N=;

[0151] R2 is selected from hydrogen and optionally substituted C1-C6 alkyl; when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure;

[0152] W is absent, or W is selected from an optionally substituted C1-C6 alkyl group; when W comprises a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure;

[0153] B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

[0154] Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl;

[0155] Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group;

[0156] R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure;

[0157] m is selected from 1, 2 or 3;

[0158] L w is -L3-L2-L1;

[0159] L1 is selected from

[0160] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0161] L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-cysteine-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

[0162] In some embodiments, the structure shown in formula (II-3C), wherein L w Selected from

[0163] In certain preferred embodiments of the present invention, the structure represented by formula (II-3A), wherein R1, X1, X3, R2, W, B, Z, R A-1 and m are as described in any one of the schemes (II-2B) or (II-2C) of this application.

[0164] In some embodiments, the compound of the present invention or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein the compound is represented by formula (II-3A-1) and (II-3A-2):

[0165] in,

[0166] R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups;

[0167] X4 and X5 are each independently selected from -CH= and -N=;

[0168] R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R 2-2 Replacement; R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution; R 2-3 Selected from: hydrogen or C1-C3 alkyl;

[0169] W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Replacement; R w-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of said W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl;

[0170] R A-1Selected from R A2-1 -Lx-;

[0171] Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-,

[0172] R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by

[0173] where R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2;

[0174] L w Selected from

[0175] In certain preferred embodiments of the present invention, the structures represented by formula (II-3A-1) and (II-3A-2), wherein X4 and X5 are each independently selected from -CH= and -N=; R2, W, R Z-1 and R A-1 As described in any one of the schemes (II-2B) or (II-2C) of this application.

[0176] In another aspect, the present invention provides a compound or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, selected from:

[0177] In another aspect, the present invention provides a compound or a conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a structure represented by formula (III-1A):

[0178] in,

[0179] X is selected from -S- and -O-;

[0180] R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl;

[0181] Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl;

[0182] Ring A is selected from phenyl or heteroaryl;

[0183] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0184] V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0185] Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and

[0186] Y is selected from -N(R Y1 )-、-O-、-S-、

[0187] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0188] q is selected from 0, 1, 2 or 3;

[0189] p is selected from the group consisting of 1, 2, 3, 4, 5, 7 and 8.

[0190] In another aspect, the present invention provides a ligand-drug conjugate, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (III-2A):

[0191] in,

[0192] X is selected from -S- and -O-;

[0193] R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl;

[0194] Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl;

[0195] Ring A is selected from phenyl or heteroaryl;

[0196] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0197] V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0198] Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and

[0199] Y is selected from -N(R Y1 )-、-O-、-S-、

[0200] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0201] q is selected from 0, 1, 2 or 3;

[0202] p is selected from 1, 2, 3, 4, 5, 7 and 8;

[0203] Wavy lines in the general formula It indicates that the Y group is directly connected to the ligand through the nitrogen atom or oxygen atom on the Y group, or is connected to the ligand through a linker unit.

[0204] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (III-2B):

[0205] in,

[0206] X is selected from -S- and -O-;

[0207] R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl;

[0208] Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl;

[0209] Ring A is selected from phenyl or heteroaryl;

[0210] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0211] V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0212] Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and

[0213] Y is selected from -N(R Y1 )-、-O-、-S-、

[0214] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0215] q is selected from 0, 1, 2 or 3;

[0216] p is selected from 1, 2, 3, 4, 5, 7 and 8;

[0217] L is the joint unit;

[0218] Wavy lines in the general formula It means that the ligand is connected through the nitrogen atom or carbon atom on the L group.

[0219] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (III-2C):

[0220] in,

[0221] X is selected from -S- and -O-;

[0222] R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl;

[0223] Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl;

[0224] Ring A is selected from phenyl or heteroaryl;

[0225] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0226] V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0227] Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4)-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and

[0228] Y is selected from -N(R Y1 )-、-O-、-S-、

[0229] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0230] q is selected from 0, 1, 2 or 3;

[0231] p is selected from 1, 2, 3, 4, 5, 7 and 8;

[0232] L is the joint unit;

[0233] n is an integer or decimal from 1 to 10;

[0234] Pc is a ligand.

[0235] In certain preferred embodiments of the present invention, certain groups in the structures shown in formula (III-1A), (III-2A), (III-2B) or (III-2C) are defined as follows, and the groups not mentioned are the same as those described in any scheme of this application.

[0236] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein the R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each independently selected from: hydrogen, chlorine, bromine, iodine, hydroxyl, amino, -CH3, -CH2CH3 and wherein the methylene units of R1 are each independently unsubstituted or replaced by a group selected from the group consisting of: -O-, -N(CH3)-, and -NH-.

[0237] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein R1 is selected from wherein the methylene units of R1 are each independently not substituted or replaced by a group selected from the group consisting of: -O-, -N(CH3)-, and -NH-.

[0238] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein R1 is selected from For example, R1 is selected from

[0239] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, thiol, C1-C3 alkyl and C1-C3 alkoxy, and the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally replaced by 1, 2 or 3 R 2-1 Substituted, the R 2-1 Each is independently selected from the group consisting of: hydrogen, halogen, hydroxyl and amino.

[0240] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, thiol, -CH3, -CH2CH3 and -OCH3. For example, R2 is selected from H.

[0241] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein the ring A is selected from phenyl, pyridyl, pyrrolyl, thienyl, furyl, pyridazinyl, pyrimidinyl and pyrazinyl.

[0242] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein the ring A is selected from phenyl or pyridyl. For example, the ring A is selected from phenyl.

[0243] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, -CH3, -CH2CH3 and Wherein, the methylene units of V are each independently not substituted or replaced by a group selected from the following groups: -O-, -NH-, -CO-, -NHCO-,

[0244] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein V is selected from Wherein, the methylene units of V are each independently not substituted or replaced by a group selected from the following groups: -O-, -NH-, -CO-, -NHCO-,

[0245] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein V is selected from For example, V is selected from

[0246] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein Y is selected from -NH-, -O-, -S-,

[0247] In some embodiments, the structure represented by formula (III-1A), (III-2A), (III-2B) or (III-2C), wherein the structural unit Selected from:

[0248] Preferably, it is selected from

[0249] Wherein, X is selected from -S- and -O-; R1, R2, V, and Y are as described in any one of the schemes (III-1A), (III-2A), (III-2B), and (III-2C) of the present application.

[0250] In some embodiments, the structures represented by formula (III-2B) and (III-2C), wherein the linker unit L is -Tr-L3-L2-L 1c -,

[0251] Tr is selected from and single bonds;

[0252] L 1c Selected from

[0253] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0254] L3 is selected from a single bond or a peptide residue consisting of 1-4 amino acids, wherein the peptide residue is a peptide residue formed by an amino acid selected from the following group: valine, lysine, cysteine, alanine, glycine, glutamic acid, phenylalanine, and serine.

[0255] In some embodiments, the structures represented by formula (III-2B) and (III-2C), wherein L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-.

[0256] In some embodiments, the structures shown in formula (III-2B) and (III-2C), wherein the linker unit L is -L3-L2-L 1c -,

[0257] L 1c Selected from

[0258] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0259] L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

[0260] In some embodiments, the structures represented by formula (III-2B) and (III-2C), wherein the linker unit L is -L3-L2-L 1c -or-Tr-L3-L2-L 1c -, the L3 end or the Tr end is connected to the nitrogen atom of the Z group, L 1c end connected to the ligand.

[0261] In some embodiments, the structures represented by formula (III-2B) and (III-2C), wherein the linker unit L is selected from:

[0262] For example, L is selected from

[0263] In some embodiments, the ligand-drug conjugate of the present invention, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is represented by formula (III-2C-1) and (III-2C-2):

[0264] in,

[0265] X is selected from -S- and -O-;

[0266] R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; wherein the methylene units of R1 are each independently not replaced or replaced by a group selected from the group consisting of -O-, -N(CH3)- and -NH-;

[0267] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted; said R 2-1 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino;

[0268] V is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the group consisting of: -NH(C1-C6 alkyl), -N(C1-C6 alkyl), C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halo-C1-C6 alkyl or halo-C1-C6 alkoxy;

[0269] Y is selected from -N(R Y1 )-;

[0270] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0271] L is -L3-L2-L 1c -or-Tr-L3-L2-L 1c -;

[0272] Tr is selected from:

[0273] L 1c Selected from

[0274] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0275] L3 is selected from the group consisting of peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine-, and -glycine-glycine-glycine-glycine-;

[0276] n is an integer or decimal from 1 to 10;

[0277] Pc is a ligand.

[0278] In certain preferred embodiments of the present invention, the structures shown in formulas (III-2C-1) and (III-2C-2), wherein R1, R2, V, Y, L, n and Pc are respectively as described in any one of the schemes (II-2C) of this application.

[0279] In some embodiments, the ligand-drug conjugate of the present invention, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is selected from the following structural formulas:

[0280] n is an integer or decimal from 1 to 10;

[0281] Pc is a ligand.

[0282] In some embodiments, the ligand-drug conjugate of the present invention, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate is selected from the following structural formulas:

[0283] n is an integer or decimal from 1 to 10.

[0284] In some embodiments, the compound of the present invention or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt is selected from:

[0285] in,

[0286] X is selected from -S- and -O-;

[0287] R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; wherein the methylene units of R1 are each independently not replaced or replaced by a group selected from the group consisting of -O-, -N(CH3)- and -NH-;

[0288] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted, the R 2-1 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino;

[0289] V is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halo-C1-C6 alkyl or halo-C1-C6 alkoxy;

[0290] Y is selected from -N(R Y1 )-;

[0291] R Y1 is selected from hydrogen and C1-C6 alkyl.

[0292] In certain preferred embodiments of the present invention, the structures shown in formulas (III-2A-1), (III-2A-2), and (III-2A-3), wherein R1, R2, V, and Y are respectively as described in any one of the schemes (II-2A) of this application.

[0293] In another aspect, the present invention provides a compound or a conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (III-3A):

[0294] in,

[0295] X is selected from -S- and -O-;

[0296] R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl;

[0297] Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl;

[0298] Ring A is selected from phenyl or heteroaryl;

[0299] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0300] V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino;

[0301] Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and

[0302] Y is selected from -N(R Y1 )-、-O-、-S-、

[0303] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0304] q is selected from 0, 1, 2 or 3;

[0305] p is selected from 1, 2, 3, 4, 5, 7 and 8;

[0306] L W -Tr-L3-L2-L1,

[0307] Tr is selected from and single bonds;

[0308] ;

[0309] L1 is selected from

[0310] L2 is selected from and a single bond, wherein q is an integer from 1 to 20;

[0311] L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, glycine-glutamine, glycine-aspartic acid-, glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-.

[0312] In certain preferred embodiments of the present invention, the structure shown in formula (III-3A), wherein R1, X2, ring A, V, Y, Lw and q are respectively as described in any one of the schemes (III-1A), (III-2A), (III-2B) or (III-2C) in this application.

[0313] In some embodiments, the compound of the present invention or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein the compound is represented by formula (III-3A-1) and (III-3A-2):

[0314] in,

[0315] X is selected from -S- and -O-;

[0316] R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; wherein the methylene units of R1 are each independently not replaced or replaced by a group selected from the group consisting of -O-, -N(CH3)- and -NH-;

[0317] R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted; said R 2-1 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino;

[0318] V is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halo-C1-C6 alkyl or halo-C1-C6 alkoxy;

[0319] Y is selected from -N(R Y1 )-;

[0320] R Y1 is selected from hydrogen and C1-C6 alkyl;

[0321] L W Selected from

[0322] In certain preferred embodiments of the present invention, the structures shown in formula (Ⅲ-3A-1) and (Ⅲ-3A-2), wherein R1, R2, V, Y and Lw are respectively as described in any one of the schemes (Ⅲ-1A), (Ⅲ-2A), (Ⅲ-2B) or (Ⅲ-2C) in this application.

[0323] In another aspect, the present invention provides a compound or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, selected from:

[0324] On the other hand, the present invention provides a ligand-drug conjugate comprising a ligand and a drug connected to the ligand, wherein the drug is a compound described in any one of the present application, preferably the drug is connected to the ligand through a linker, and preferably the ligand is an antibody or an antigen-binding fragment thereof.

[0325] In some embodiments, in the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, n is an integer or decimal from 2 to 8. For example, the average number of connections n can be an integer or decimal from 3 to 8. For example, the average number of connections n can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0326] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the Pc is selected from protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells, receptors and / or antigens.

[0327] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein Pc is an antibody or an antigen-binding fragment thereof.

[0328] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein Pc is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0329] In some embodiments, the ligand-drug conjugate of the present invention, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein the Pc is an antibody targeting the following targets: 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 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, EGF RvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, GD2, GEDA, GPC-1, 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, LY6 4, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1,PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PSMA, PTK7, P-cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, brevican, mesothelin, sodium phosphate cotransporter 2B, claudin 18.2, endothrin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin a4p7, integrin a5p6, trophoblast glycoprotein, and tissue factor. ,

[0330] In some embodiments, the ligand-drug conjugate of the present invention, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the Pc is an antibody or antigen-binding fragment thereof targeting GPC3, Trop2 and HER2; preferably, the antibody is selected from: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

[0331] In the present application, the CDRs of the antibody or antigen-binding fragment thereof may be defined according to Kabat.

[0332] In the present application, the antibody or antigen-binding fragment thereof may comprise LCDR1-3 and HCDR1-3. The LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-4; the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-8; the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 9-12; the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 13-16; the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 17-20; and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 21-24.

[0333] For example, the antibodies or antigen-binding fragments thereof described herein comprise the same LCDR1-3 and HCDR1-3 as trastuzumab, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 5; the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 9; the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 17; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 21.

[0334] For example, the antibodies or antigen-binding fragments thereof described herein may comprise LCDR1-3 and HCDR1-3 identical to those of Pertuzumab, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 2; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 6; the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 10; the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 14; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 22.

[0335] For example, the antibodies or antigen-binding fragments thereof described herein may comprise LCDR1-3 and HCDR1-3 identical to those of sacituzumab, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:3; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:7; the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:11; the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:15; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:19; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:23.

[0336] For example, the antibodies or antigen-binding fragments thereof described herein may comprise LCDR1-3 and HCDR1-3 identical to those of codrituzumab, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:4; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:8; the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:12; the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:16; the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:20; and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:24.

[0337] In the present application, the antibody or antigen-binding fragment thereof may comprise a light chain variable region VL and a heavy chain variable region VH, wherein the VL may comprise the amino acid sequence shown in any one of SEQ ID NOs: 25-28, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 29-32.

[0338] For example, the antibody or antigen-binding fragment thereof described herein may comprise the same light chain variable region VL and heavy chain variable region VH as trastuzumab, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO: 25, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 29.

[0339] For example, the antibody or antigen-binding fragment thereof described herein may comprise the same light chain variable region VL and heavy chain variable region VH as pertuzumab, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO: 26, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 30.

[0340] For example, the antibody or antigen-binding fragment thereof described herein may comprise the same light chain variable region VL and heavy chain variable region VH as sacituzumab, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO: 27, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 31.

[0341] For example, the antibody or antigen-binding fragment thereof described herein may comprise the same light chain variable region VL and heavy chain variable region VH as codrituzumab, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO: 28, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 32.

[0342] In the present application, the antibody or antigen-binding fragment thereof may comprise an antibody light chain and an antibody heavy chain, wherein the light chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 33-36, and the heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 37-40.

[0343] For example, the antibody or antigen-binding fragment thereof described in the present application may comprise the same antibody light chain and antibody heavy chain as Trastuzumab, wherein the light chain may comprise the amino acid sequence shown in SEQ ID NO: 33, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 37.

[0344] For example, the antibody or antigen-binding fragment thereof described in the present application may comprise the same antibody light chain and antibody heavy chain as Pertuzumab, wherein the light chain may comprise the amino acid sequence shown in SEQ ID NO: 34, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 38.

[0345] For example, the antibody or antigen-binding fragment thereof described in the present application may comprise the same antibody light chain and antibody heavy chain as Sacituzumab, wherein the light chain may comprise the amino acid sequence shown in SEQ ID NO: 35, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 39.

[0346] For example, the antibody or antigen-binding fragment thereof described in the present application may comprise the same antibody light chain and antibody heavy chain as Codrituzumab, wherein the light chain may comprise the amino acid sequence shown in SEQ ID NO: 36, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 40.

[0347] In certain preferred embodiments of the present invention, the present invention provides a compound of the following formula (A), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0348] in:

[0349] R A-1 Selected from R A2-1 -Lx-;

[0350] Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-, preferably, Lx is selected from a single bond, -C(O)NH- or -NHC(O)-;

[0351] R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by

[0352] where R A2-2 Each is independently selected from C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2,

[0353] R2 is selected from -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 alkylenehydroxy or halogenated C1-C6 alkyl;

[0354] W is selected from -C1-C6 alkylene-, -O-C1-C6 alkylene-;

[0355] B is phenyl, and B is optionally substituted by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, and

[0356] Z is -NH(R Z-1 ), the R Z-1is selected from H, -C1-C6 alkyl, halogenated C1-C6 alkyl. In a particularly preferred embodiment, the present invention provides a compound of formula (A) selected from the following, or a mixture of its tautomers, enantiomers, diastereomers, or isomers, or a pharmaceutically acceptable salt or solvate thereof:

[0357] In certain preferred embodiments of the present invention, the present invention provides a compound of the following formula (A-1), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0358] Among them, R A-1 , W, B, Z and R2 are as defined above, L is a linker unit, preferably L is a linker unit as defined in any one of claims 27 to 31, wherein the wavy line indicates that it is connected to the ligand through a nitrogen atom or a carbon atom on the L group.

[0359] In a more preferred embodiment, the linker unit L is selected from:

[0360] In a particularly preferred embodiment, the compound of the present invention is selected from the following compounds, or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0361] In certain preferred embodiments of the present invention, the present invention provides a ligand-drug conjugate of the following formula (A-2), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0362] Among them, R A-1 , W, B, Z and R2 are as defined above, L is a linker unit as defined above, n is an integer or decimal of 1-10, preferably n is an integer or decimal of 2-8, Pc is a ligand, preferably Pc is an antibody or an antigen-binding fragment thereof.

[0363] In a more preferred embodiment, Pc is an antibody or an antigen-binding fragment thereof targeting GPC3, Trop2 and HER2; preferably, the antibody is selected from the group consisting of: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

[0364] In certain preferred embodiments of the present invention, the present invention provides a compound of the following formula (B), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0365] in:

[0366] R Y1 Selected from hydrogen and -C1-C6 alkyl;

[0367] X e is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl or halogenated C1-C6 alkoxy,

[0368] X is selected from -O- or -S-, and

[0369] R f Selected from C1-C6 alkyl, wherein one or more carbon atoms in the C1-C6 alkyl can be optionally replaced by a heteroatom selected from N, O or S, and the C1-C6 alkyl is optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, -C1-C6 alkyl-NH(C1-C6 alkyl), or -C1-C6 alkyl-N(C1-C6 alkyl)2.

[0370] In a more preferred embodiment, the present invention provides a compound of formula (B) selected from the following, or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0371] In certain preferred embodiments of the present invention, the present invention provides a compound of the following formula (B-1), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0372] Among them, X, X e 、R f and R Y1 As defined above, L is a linker unit, preferably L is a linker unit as defined in any one of claims 27 to 31, wherein the wavy line indicates that the linker unit is connected to the ligand through the nitrogen atom or carbon atom on the L group.

[0373] In a more preferred embodiment, the linker unit L is selected from:

[0374] For example, L is selected from

[0375] In a particularly preferred embodiment, the compound of the present invention is selected from the following compounds, or tautomers, enantiomers, diastereomers, or mixtures of isomers thereof, or pharmaceutically acceptable salts or solvates thereof:

[0376] In certain preferred embodiments of the present invention, the present invention provides a ligand-drug conjugate of the following formula (B-2), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof,

[0377] Among them, X, X e 、R f and R Y1 As defined above, L is a linker unit as defined above, n is an integer or decimal of 1-10, preferably n is an integer or decimal of 2-8, Pc is a ligand, preferably Pc is an antibody or an antigen-binding fragment thereof.

[0378] In a more preferred embodiment, Pc is an antibody or an antigen-binding fragment thereof targeting GPC3, Trop2 and HER2; preferably, the antibody is selected from the group consisting of: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

[0379] Pharmaceutical composition

[0380] On the other hand, the present invention provides a pharmaceutical composition comprising the compound of the present application or its conjugate, or its isomers, or its mixture form, or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient; or, comprising the ligand-drug conjugate of the present application, or its isomers, or its mixture form, or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0381] The pharmaceutical compositions described herein may contain, in addition to the active compound, one or more excipients, which may be selected from the following groups: fillers (diluents), binders, wetting agents, disintegrants, and excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0382] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as a tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granules, emulsion, hard or soft capsule, or syrup. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and may contain a binder, a filler, a lubricant, a disintegrant, or a pharmaceutically acceptable wetting agent. The composition may also contain one or more ingredients selected from the group consisting of a sweetener, a flavoring agent, a coloring agent, and a preservative.

[0383] Aqueous suspensions may contain the active ingredient and excipients suitable for preparing aqueous suspensions for mixing. Aqueous suspensions may also contain one or more preservatives, such as one or more colorants, one or more flavorings, and one or more sweeteners. Oil suspensions may be prepared by suspending the active ingredient in a vegetable oil. Oil suspensions may contain a thickening agent. The sweeteners and flavorings mentioned above may also be added.

[0384] The pharmaceutical composition may also be provided as a dispersible powder or granule for preparing an aqueous suspension, wherein the active ingredient is mixed with water to form one or more of a dispersant, a wetting agent, a suspending agent, or a preservative. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. The pharmaceutical composition of the present application may also be in the form of an oil-in-water emulsion.

[0385] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or vehicles that may be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution may then be added to a mixture of water and glycerol to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream via local bolus injection. Alternatively, the solution or microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous drug delivery device may be used. For example, the device may be a Deltec CADD-PLUS™ 5400 intravenous pump.

[0386] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. Such suspensions may be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents as described herein. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in a parenterally acceptable nontoxic diluent or solvent. Alternatively, a sterile fixed oil may conveniently be used as a solvent or suspending medium.

[0387] The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum, thereby dissolving and releasing the drug in the rectum. Such substances include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.

[0388] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the time of administration, the route of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal treatment mode, such as the mode of treatment, the daily dosage of the compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and / or the compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the type of pharmaceutically acceptable salt thereof can be verified according to traditional treatment regimens.

[0389] Prevent and / or treat tumors

[0390] On the other hand, the present application provides a method for affecting the function of Toll-like receptors (TLRs), comprising administering the compound of the present application or its conjugate, or its isomer, or its mixture form, or its pharmaceutically acceptable salt; or, administering the ligand-drug conjugate of the present application, or its isomer, or its mixture form, or its pharmaceutically acceptable salt, or, administering the pharmaceutical composition of the present application.

[0391] On the other hand, the present application provides a method for regulating immune system function, comprising administering the compound of the present application or its conjugate, or its isomer, or its mixture form, or its pharmaceutically acceptable salt; or, administering the ligand-drug conjugate of the present application, or its isomer, or its mixture form, or its pharmaceutically acceptable salt, or, administering the pharmaceutical composition of the present application.

[0392] On the other hand, the present application provides the use of the compound of the present application or its conjugate, or its isomer, or its mixture form, or its pharmaceutically acceptable salt; or, the ligand-drug conjugate of the present application, or its isomer, or its mixture form, or its pharmaceutically acceptable salt, or, the pharmaceutical composition of the present application in the preparation of a drug for preventing and / or treating a disease and / or symptom.

[0393] In some embodiments, the uses or methods described herein, the diseases and / or symptoms comprise diseases and / or symptoms associated with Toll-like receptor (TLR) signal transduction.

[0394] In some embodiments, the use or method described herein, the disease and / or symptom is selected from the group consisting of tumors, autoimmune diseases, inflammation, sepsis, allergies, asthma, transplant rejection, graft-versus-host disease, immunodeficiency, and viral infections.

[0395] In some embodiments, the uses or methods described herein, the disease and / or symptom is a tumor selected from the group consisting of: GPC3, Trop2, and HER2. In some embodiments, the disease and / or symptom is a tumor selected from the group consisting of: melanoma, lung cancer, liver cancer, basal cell carcinoma, kidney cancer, myeloma, biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, rectal cancer, head and neck cancer, peritoneal tumor, fallopian tube cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, lymphoma, sarcoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, skin cancer, and thyroid cancer.

[0396] In some embodiments, the uses or methods described herein, the diseases and / or symptoms are selected from infections caused by the following viruses: dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HPV (human papillomavirus), RSV (respiratory syncytial virus), SARS-CoV (severe acute respiratory syndrome coronavirus), SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2), MERS-CoV (Middle East respiratory syndrome coronavirus) and influenza virus.

[0397] The compounds described in this application can affect the activity of TLR. The effect of the activity can be that when the compound of the present application is added to the culture medium, the activity of the TLR is increased by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%. For example, the effect of the activity can be on the EC of TLR. 50 The value (nM) is 10000 or less, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 150 or less, 120 or less, 110 or less, 100 or less, 99 or less, 98 or less, 97 or less, 95 or less, 90 or less, 80 or less, 75 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less , 8.5 or less, 7 or less, 6.7 or less, 6 or less, 5.9 or less, 5.5 or less, 5.0 or less, 4.8 or less, 4.5 or less, 4.4 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0 or less, 0.5 or less, 0.3 or less, 0.29 or less, 0.25 or less, 0.21 or less, 0.20 or less, 0.18 or less, 0.17 or less, 0.15 or less, 0.12 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. For example, the immune cells may include but are not limited to granulocytes and / or agranulocytes, for example, the immune cells include but are not limited to B cells, T cells, natural killer cells, monocytes, macrophages, mast cells and / or dendritic cells. For example, the immune cells include PBMC cells. For example, the TLR includes human TLR. For example, the TLR includes TLR7 and / or TLR8.

[0398] The compounds described herein can affect the ability of immune cells to express and / or release cytokines. The effect of the activity can be that when the compound of the present application is added to the culture medium of immune cells or administered to a subject, the ability of the immune cells to express and / or release cytokines is increased by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%. For example, the immune cells can include but are not limited to granular leukocytes and / or agranular leukocytes, for example, the immune cells include but are not limited to B cells, T cells, natural killer cells, monocytes, macrophages, mast cells and / or dendritic cells. For example, the immune cells include PBMC cells. For example, the cytokine can be an immune cytokine. For example, the cytokine can be TNF-α and / or IFN-α

[0399] The compounds described in this application can affect the activity of immune cells. The effect of the activity can be that the compounds of this application are added to the culture medium of immune cells, and the activity of the immune cells is increased by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% compared to the addition of negative control or control drug. For example, the effect of the activity can be on the EC of immune cells. 50The value (nM) is 10000 or less, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 150 or less, 120 or less, 110 or less, 100 or less, 99 or less, 98 or less, 97 or less, 95 or less, 90 or less, 80 or less, 75 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 0.5, 0.6, 0.7, 0.8, 0.9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 40000, 50000, 60000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000, 80000 For example, the immune cells include PBMC cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0400] Figure 1: (a) TNF-α release from human whole blood in the presence of different concentrations of antibody-drug conjugates; (b) IFN-α release from human whole blood in the presence of different concentrations of antibody-drug conjugates.

[0401] Figure 2: (a) Results of the co-incubation assay of control ADC1, tumor cells, and human peripheral blood mononuclear cells; (b) Results of the co-incubation assay of ADC6, tumor cells, and human peripheral blood mononuclear cells.

[0402] Figure 3: IFN-α release in the co-incubation experiment of ADC-13 with HepG2 and PBMC.

[0403] Figure 4: IL-6 release in the co-incubation experiment of ADC-13 with HepG2 and PBMC.

[0404] Figure 5: TNF-α release in the co-incubation experiment of ADC-13 with HepG2 and PBMC.

[0405] Figure 6: Experimental results of in vivo efficacy experiment 1 in the hGPC3-MC38 colon cancer animal model.

[0406] Figure 7: Experimental results of in vivo efficacy experiment 2 in the hGPC3-MC38 colon cancer animal model.

[0407] FIG8 : Results of tumor cell re-inoculation experiments in the MC38 / hTLR8KI mouse model that does not express hGPC3.

[0408] Definition of terms

[0409] As used herein, the terms "Toll-like receptor" and "TLR" generally refer to any member of the highly conserved mammalian pattern recognition receptor family that recognize pathogen-associated molecular patterns (PAMPs) and serve as key signaling components in innate immunity. TLR polypeptides share a characteristic structure, including an extracellular domain with leucine-rich repeats, a transmembrane domain, and an intracellular domain involved in TLR signaling. TLRs include, but are not limited to, human TLRs.

[0410] In this application, the terms "Toll-like receptor 7" and "TLR7" refer to nucleic acids or polypeptides that share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a publicly available TLR7 sequence, such as GenBank Accession No. AAZ99026 for human TLR7 polypeptide, or GenBank Accession No. AAK62676 for mouse TLR7 polypeptide.

[0411] In this application, the terms "Toll-like receptor 8" and "TLR8" refer to nucleic acids or polypeptides that share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a publicly available TLR8 sequence, such as GenBank Accession No. AAZ95441 for human TLR8 polypeptide, or GenBank Accession No. AAK62677 for murine TLR8 polypeptide.

[0412] In the present application, the term "TLR agonist" is generally a substance that directly or indirectly binds to a TLR (e.g., TLR7 and / or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling can indicate that the agonist stimulates or activates the TLR. Signaling differences can be manifested as changes in target gene expression, changes in the phosphorylation of signal transduction components, changes in the intracellular localization of downstream elements such as nuclear factor-κB (NF-κB), changes in the association of certain components (e.g., IL-1 receptor-associated kinase (IRAK)) with other proteins or intracellular structures, or changes in the biochemical activity of components such as kinases (e.g., mitogen-activated protein kinases (MAPK)). In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine, iodine, and can be, for example, fluorine or chlorine.

[0413] In the present application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. An alkyl group can be substituted or non-substituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane by removing a hydrogen atom, and can be a straight or branched group containing 1 to 20 carbon atoms, for example, a chain alkyl group containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl group may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic group.

[0414] In this application, the term "alkoxy" generally refers to an alkyl group as described above with a specific number of carbon atoms attached through an oxygen bridge, unless otherwise specified. 1-6 Alkoxy groups include C1, C2, C3, C4, C5 and C6 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy.

[0415] In this application, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group, and can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from a group containing 1 carbon atom by removing two hydrogen atoms. The methylene group can be substituted or unsubstituted, substituted or unsubstituted; for example, an alkylene group containing 1 to 12 carbon atoms, for example, an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), and the like. Alkylene may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, or C 1-6 Aliphatic groups. Methylene or alkylene groups may be substituted or unsubstituted.

[0416] In this application, the term "alkenyl" generally refers to a straight or branched hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. Illustrative examples of C2-6 alkenyl groups with one or more double bonds include butadienyl, pentadienyl, hexadienyl, and hexatrienyl, as well as branched forms thereof. The position of the unsaturated bond (double bond) can be at any position of the carbon chain. Alkenyl groups can be substituted or unsubstituted.

[0417] In this application, the term "alkenylene" generally refers to a residue derived from an alkene carbon atom by removing two hydrogen atoms. For example, it can be allylene, vinylene, butenylene, pentenylene and hexenylene. Alkenylene can be substituted or unsubstituted.

[0418] In the present application, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl groups can be substituted or unsubstituted.

[0419] In this application, the term "alkynylene" generally refers to a residue derived from a carbon atom of an alkyne by removing two hydrogen atoms. For example, it may be ethynylene, propynylene, propargylene, butynylene, etc. Alkynylenes may be substituted or unsubstituted.

[0420] In this application, the term "aryl" generally refers to a residue derived from an aromatic ring by removing a hydrogen atom. The term "aromatic ring" can refer to a 6- to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring sharing adjacent pairs of carbon atoms) with a conjugated π electron system, which can be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring connected to the parent structure is the aryl ring. Aryl groups can be substituted or unsubstituted. When substituted, the substituents can be one or more of the following groups independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Aryl groups can be substituted or unsubstituted.

[0421] In this application, the term "arylene" generally refers to a residue derived from a carbon atom in an aromatic ring by removing two hydrogen atoms. Examples include phenylene and naphthylene. Arylene can be substituted or unsubstituted.

[0422] As used herein, the term "heteroaryl" generally refers to a residue derived from a carbon atom in a heteroaromatic ring by removing a hydrogen atom. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. Heteroaryl groups may be 5 to 10-membered, and may be 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl group may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. Heteroaryl can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl can be substituted or unsubstituted.

[0423] In this application, the term "heteroarylene" generally refers to a residue derived from a carbon atom of a heteroaromatic ring by removing two hydrogen atoms. For example, it may be furanylene, thienylene, pyridylene, pyrrolylene, pyrimidylene, pyrazinylene, imidazolylene, tetrazolylene, etc. The heteroarylene group may be substituted or unsubstituted.

[0424] As used herein, the term "alicyclic group" generally refers to a residue derived by removing a hydrogen atom from the same carbon atom or multiple different carbon atoms of an aliphatic ring. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon, wherein the carbocyclic ring contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, or may contain 3 to 8 carbon atoms. Non-limiting examples of alicyclic groups include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctanyl, and the like; polycyclic carbocyclic rings may include spiro, fused, and bridged carbocyclic rings. Alicyclic groups may be substituted or unsubstituted. As used herein, the term "carbocyclic group" generally refers to a residue derived by removing a hydrogen atom from a carbon atom of a carbocyclic ring. The term "carbocycle" generally refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon ring, wherein the carbocycle contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, and may contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, and the like; polycyclic carbocycles may include spirocyclic, fused, and bridged carbocycles. Carbocyclyl groups may be substituted or unsubstituted. In some cases, alicyclic and carbocyclic rings may be used interchangeably.

[0425] As used herein, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" refers to a moiety having one or more degrees of unsaturation.

[0426] In this application, the term "alicyclic group" generally refers to a residue derived from a carbon atom of an alicyclic ring by removing two hydrogen atoms. Examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, cyclohexenylene, cyclohexadienylene, cycloheptanylene, cycloheptatrienylene, and cyclooctanylene. Polycyclic carbocycles may include spiro, fused, and bridged carbocycles. Alicyclic groups may be substituted or unsubstituted.

[0427] In this application, the term "alicyclic group" generally refers to a stable, non-aromatic, 3- to 7-membered monocyclic carbocyclic ring structure, a fused (or fused) 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. These ring structures may be saturated or partially saturated and, in addition to carbon atoms, may contain one or more heteroatoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, the ring structure may contain 1 to 4 heteroatoms as defined above. When used to refer to atoms in an alicyclic heterocyclic ring structure, the term "nitrogen" may include substituted nitrogen. For example, an alicyclic heterocyclic group may include a "heterocycloalkyl" group, which may refer to a stable, non-aromatic, 3- to 7-membered monocyclic carbocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. In addition to carbon atoms, these ring structures may contain one or more heteroatoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, it contains 1 to 4 heteroatoms as defined above. Heterocycloalkyl groups may be substituted or unsubstituted. Alicyclic heterocyclic groups may be substituted or unsubstituted.

[0428] In the present application, the term "aliphatic heterocyclic group" generally refers to a residue derived from a carbon atom of an aliphatic heterocyclic ring by removing two hydrogen atoms. The aliphatic heterocyclic group may be substituted or unsubstituted.

[0429] In this application, the term "ring-forming atoms" generally refers to atoms contained in a ring structure. For example, ring-forming atoms can be carbon atoms in a benzene ring or nitrogen atoms in a pyridine ring. When a ring-forming atom is attached to a hydrogen atom, the ring-forming atom can be substituted or unsubstituted.

[0430] As used herein, the term "each independently" generally refers to a variable being applicable in any instance, regardless of the presence or absence of variables with the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in a compound, or the type of atoms in the compound. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen," both R may be carbon, both R may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0431] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group can but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0432] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are independently replaced 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) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0433] In this application, the term 0 or more (e.g., 0 or more, 0 or 1, 0) methylene units are "replaced" generally means that when the structure contains 1 or more methylene units, the one or more methylene units may be unsubstituted or replaced by one or more groups that are not methylene (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SONH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C- or -C(=N2)-).

[0434] In this application, the "connection" of group X to group Y can generally be in any orientation, which generally means that when group X is used to connect group Y and group Z, the two or more connection sites of group X can be connected to group Y or group Z at will.

[0435] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 1000 or more than 10000. In this application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by a chemical bond, and the biomacromolecule may be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000.

[0436] In this application, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In the examples, the members of the group may have any number of carbon atoms falling within the range of 1-10.

[0437] One or more hydrogen atoms, for example up to 5, for example 1 to 3, in a group are independently replaced by a corresponding number of substituents. The substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0438] In the present application, the compound of the present application includes its tautomer, mesomorph, racemate, enantiomer and / or diastereomer of compound.In the present application, the term "diastereomer" generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, for example, melting point, boiling point, spectral properties and reactivity. In the present application, the term "tautomer" or "tautomeric form" is used interchangeably, generally referring to structural isomers of different energies that can be converted to each other by low energy barriers (low energy barrier). For example, proton tautomers (protontautomers) (also referred to as prototropic tautomers (prototropic tautomers)) include the mutual conversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valence tautomers) include the mutual conversions carried out by the reorganization of some bonding electrons. As used herein, the term "meso" generally refers to a molecule containing asymmetric atoms but possessing symmetry such that the total optical rotation is zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric species.

[0439] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon may exist in three different isotopes ( 12C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125 I, or similar isotopes. Therefore, the compounds of the present application may be enriched in one or more of these isotopes relative to the natural abundance of these isotopes. As known to those skilled in the art, such isotopically enriched compounds can be used for a variety of purposes. For example, using heavy isotopes such as deuterium ( 2 Deuterium (H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. 2 The natural abundance of deuterium (H) is about 0.015%. Therefore, there is one deuterium atom for approximately every 6500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds of the present application have a deuterium abundance greater than 0.015% at one or more positions (as the case may be). Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose remaining portions are consistent with the present structure 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 the present application.

[0440] In this application, the term "isomer" of a compound or ligand-drug conjugate generally encompasses its tautomers, mesomers, racemates, stereoisomers, enantiomers, diastereomers, or mixtures thereof.

[0441] In this application, the term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linker. In this application, a "ligand-drug conjugate" may be an antibody-drug conjugate (ADC), which may be a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a stable linker.

[0442] In this application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of the ligand can be to present the drug to the target cell population bound to 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, the ligand can be represented by Pc, and the ligand antigen forms a connection bond with the linker through the heteroatom on the ligand, which can be an antibody or an antigen-binding fragment thereof (Ab). The antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody targeting the following targets: HER2, TROP2 or GPC3. For example, the antibody can be an antibody targeting the following targets: 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 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, C Dllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E1 6. EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3 , FOLR-α, GD2, GEDA, GPC-1, 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, M SLN, MUC-1, NaPi2b, Napi3b, Nectin-4, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, P SMA, PSMA, PTK7, P-cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, epidermal growth factor, brevican, mesothelin, sodium phosphate cotransporter 2B, claudin 18.2, endothrin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin a4p7, integrin a5p6, trophoblast glycoprotein, or tissue factor.

[0443] In this application, the term "cytotoxic drug" generally refers to a toxic drug that has a strong chemical molecule that destroys the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at a sufficiently high concentration. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, for example, can be toxic drugs, including but not limited to camptothecin derivatives, for example, can be the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0444] In this application, the term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. It can also be connected to a cytotoxic drug after being connected to other linkers. The direct or indirect connection to the ligand can refer to the group being directly connected to the ligand through a covalent bond, or it can be connected to the ligand through a linker structure. For example, the linker structure can be the -L described in this application. ax -L b -L c -and-or-L a -L b -L c For example, a chemical structure fragment or bond comprising an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as the linker structure.

[0445] In this application, the term "optionally linked to other molecular moieties" generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures (such as the ligands described in this application) that are different from the structure (for example, linked by a chemical bond or linked by a linker structure).

[0446] In this application, the term "antibody or its antigen-binding fragment" generally refers to immunological binding agents extending to all antibodies from all species, including dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and reconstructed antibodies and their fragments. The term "antibody or its fragment" can refer to any antibody-like molecule with an antigen-binding region, and the term includes small molecule fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies, 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, a fragment of a full-length antibody can be used to perform the antigen-binding function of an antibody. The 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-B7-H3 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, anti-Claudin 18.2 antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody. One or more.

[0447] The term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are designated HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each heavy and light chain variable region. The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), based on the variability of antibody sequences; AbM (University of Bath); Contact (University College London); the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be defined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations thereof).

[0448] The term "drug loading" generally refers to the average number of cytotoxic drugs loaded on each ligand, and can also be expressed as the ratio of the amount of cytotoxic drug to the amount of antibody. The range of cytotoxic drug loading can be 0-12, for example, 1-10 cytotoxic drugs connected to each ligand (Ab). In the embodiment of the present application, the drug loading is expressed as N a , which can be exemplified by the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC characterization can be used to identify the drug loading of each ADC molecule after the coupling reaction.

[0449] As used herein, the term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical composition preparations can be found in the Chinese Pharmacopoeia. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions prepared in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Furthermore, sterile fixed oils can conveniently be used as solvents or suspending media. For example, any blended fixed oil, including synthetic mono- or diglycerides, can be used. Fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0450] In the present application, the term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refers to a salt of the compound or ligand-drug conjugate of the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The ligand-drug conjugate of the present application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0451] In the present application, the term "conjugate" generally refers to a compound prepared by one or more chemical reactions of the compounds of the present application, or connected to each other through one or more connecting structures such as a bridge, a spacer, or a connecting part.

[0452] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier or vehicle for administering therapeutic agents, such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers.

[0453] In this application, the terms "treatment" and "treating" generally refer to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefit includes but is not limited to eradication, inhibition, reduction or improvement of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradication, inhibition, reduction or improvement of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the patient, but the patient may still suffer from the underlying disorder.

[0454] As used herein, the terms "prevention" and "preventing" generally refer to an approach to obtaining a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk for developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0455] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a ligand-drug conjugate of the invention, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions, when administered to a cell, tissue, or subject. A therapeutically effective dose also refers to a dose sufficient to result in amelioration of symptoms, e.g., an amount that treats, cures, prevents, or ameliorates a related medical condition, or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to a subject, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0456] In this application, the term "subject" or "patient" generally refers to humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys.

[0457] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0458] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. DETAILED DESCRIPTION

[0459] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the specification of the present application is merely exemplary and not restrictive.

[0460] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0461] Synthesis technology solutions

[0462] In order to achieve the synthesis purpose of this application, the compounds provided in this application can be prepared by the following technical scheme.

[0463] Reagents providing bromination conditions include, but are not limited to, bromine water, N-bromosuccinimide, dibromohydantoin, phosphorus tribromide, liquid bromine, liquid bromine / triphenylphosphine, hydrobromic acid, and carbon tetrabromide;

[0464] Titanium catalysts include, but are not limited to, tetraisopropyl titanate, triisopropoxytitanium chloride, titanium tetrachloride, and methyl titanium triisopropoxide;

[0465] Palladium catalysts include, but are not limited to, tetrakistriphenylphosphine palladium, palladium acetate, palladium chloride, bis(triphenylphosphine)palladium dichloride, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)dipalladium, bis(acetonitrile)palladium dichloride, [1,1'-bis{diphenylphosphino}ferrocene]palladium dichloride, [1,1'-bis{diphenylphosphino}ferrocene]palladium dichloride dichloromethane complex, dibenzonitrilepalladium dichloride, 1,4-bis(diphenylphosphino)butane-palladium chloride, allylpalladium chloride dimer, and allylcyclopentadienylpalladium;

[0466] Boronate dimers include, but are not limited to, bis(2-methyl-2,4-pentanediol) borate, bis(diisopropyl-L-tartrate diethyl) borate, bis[(-)pinanediol]diboronate, bis(1S,2S,3R,5S)(+)-pinanediol diboronate, tetramethylaminodiborane, bis(N,N,N',N'-tetramethyl-D-tartrate)amide diboron, tetrahydroxydiboron, bis(N,N,N',N'-tetramethyl-L-tartrate diolate)diboron, bis(diisopropyl-D-tartaric acid glycolate)diboron, bis(D-tartrate diethyl)borate, bis(2,4-dimethyl-2,4-pentanediol)borate, bis(L-tartrate diethyl)borate, 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-ylboronic acid;

[0467] Metal copper salts include, but are not limited to, copper sulfate, copper sulfate pentahydrate, cuprous sulfate, cupric chloride, cuprous chloride, copper carbonate, copper phosphate, copper acetate and its hydrates, copper oxalate, copper fluoroborate and its hydrates, copper dimethoxide, copper tartrate, copper formate, cuprous iodide, copper trifluoroacetate, copper trifluoromethanesulfonate, basic copper carbonate, copper bromide, cuprous bromide, and cuprous oxide;

[0468] The ligand can be selected from any ligand commonly used in the Ullmann reaction, including but not limited to L-proline, tyrosine, phenylalanine, 1,10-phenanthroline, N,N'-dimethylethylenediamine, ethylene glycol, 1,1'-binaphthyl-2,2'-diol, ethyl 2-carbonylcyclohexylcarboxylate, and salicylaldehyde hydrazone;

[0469] The condensing agent can be selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N, N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole))-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0470] Reagents providing alkaline conditions include organic bases and inorganic bases, the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, potassium tert-butoxide, etc.; the inorganic bases include but are not limited to sodium hydride, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide, lithium hydroxide, sodium phosphate, potassium phosphate;

[0471] Reagents providing acidic conditions include protonic acids and Lewis acids, the protonic acids include but are not limited to hydrochloric acid, sulfuric acid, nitric acid, nitrous acid, sulfurous acid, phosphoric acid, phosphorous acid, formic acid, acetic acid, propionic acid, butyric acid, citric acid, benzoic acid, p-toluenesulfonic acid, p-nitrobenzoic acid, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid; the Lewis acids include but are not limited to boron trifluoride, zinc chloride, magnesium chloride, aluminum chloride, tin chloride, ferric chloride;

[0472] Hydrogenation conditions include, but are not limited to: Pb / C / hydrogen, Pt / C / hydrogen, palladium chloride / hydrogen, Raney nickel / hydrogen, palladium hydroxide on carbon / hydrogen, palladium hydroxide / hydrogen;

[0473] Reagents providing oxidation conditions include, but are not limited to, Dess-Martin periodinane, hydrogen peroxide, sodium chlorite, sodium hypochlorite, and potassium perchlorate;

[0474] Reagents providing reducing conditions include, but are not limited to, sodium hydride, calcium hydride, lithium hydride, lithium aluminum hydride, sodium borohydride, lithium borohydride, sodium triethylborohydride, sodium triacetoxyborohydride, and sodium cyanoborohydride;

[0475] Reagents providing oxidation conditions include, but are not limited to, Dess-Martin periodinane, hydrogen peroxide, sodium chlorite, sodium hypochlorite, and potassium perchlorate;

[0476] Reagents providing nitration conditions include, but are not limited to, dilute nitric acid, concentrated nitric acid, concentrated sulfuric acid / nitric acid, and nitric acid / acetic anhydride;

[0477] Reagents providing hydroboration include, but are not limited to, borane-tetrahydrofuran, borane-dimethyl sulfide, catechol borane, pinacol borane, 9-borabicyclo[3.3.1]nonane, diisopentylborane, dicyclohexylborane, 1,1,2-trimethylpropylborane, monochloroborane, dichloroborane, monobromoborane, and dibromoborane; reagents providing oxidation conditions include, but are not limited to, Dess-Martin periodinane, hydrogen peroxide, sodium chlorite, sodium hypochlorite, and potassium perchlorate;

[0478] The alkaline buffer is selected from the following buffers with a pH of 7 to 11, citric acid-sodium citrate buffer, phosphoric acid-sodium phosphate buffer, phosphoric acid-potassium phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, succinic acid-sodium succinate buffer, acetic acid-sodium acetate buffer, boric acid-borax buffer, boric acid-potassium borate buffer, borax-sodium hydroxide buffer, histidine-hydrochloric acid buffer, glycine-sodium hydroxide buffer, arginine-hydrochloric acid buffer, sodium bicarbonate-sodium carbonate buffer, potassium bicarbonate-potassium carbonate buffer, Tris-hydrochloric acid buffer, ammonia-ammonium chloride buffer, sodium barbital-hydrochloric acid buffer, borax-sodium carbonate buffer, boric acid-potassium chloride buffer, or a combination of two or more of the above buffers.

[0479] In addition, the structures of the compounds in this application are determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Quantum-1 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-D), deuterated chloroform (CDC13), or deuterated methanol (CD3OD) as the solvents, and tetramethylsilane (TMS) as the internal standard. Chemical shifts are given in units of 10_6 (ppm).

[0480] MS was measured using an Angilent 6230 ESI-TOF mass spectrometer (manufacturer: Agilent, model: 6230).

[0481] UPLC analysis was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometer (Poroshell 120 EC-C18, 2.1 mm x 50 mm, 1.9 μm column).

[0482] HPLC analysis was performed using an Agilent 1260 high pressure liquid chromatograph (TOSOH G3000 SW SEC column).

[0483] UV was measured using a Thermonanodrop 2000 UV spectrophotometer.

[0484] Enzyme-linked immunosorbent assay was performed using EnVision microplate reader (PerkinElmer).

[0485] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specifications of the silica gel plate used in thin layer chromatography (TLC) are 0.15mm0.2mm, and the specifications used for thin layer chromatography separation and purification products are 0.4mm0.5mm silica gel plates.

[0486] Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0487] The known starting materials of the present application can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Accela Chem Bio Inc, Darui Chemicals and other companies.

[0488] Unless otherwise specified, reactions in the Examples were carried out under an argon or nitrogen atmosphere. Argon or nitrogen atmosphere refers to a reaction flask connected to an approximately 1 L argon or nitrogen balloon. Hydrogen atmosphere refers to a reaction flask connected to an approximately 1 L hydrogen balloon.

[0489] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0490] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is the most suitable reaction temperature and ranges from 20°C to 30°C.

[0491] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used for purifying compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.

[0492] Some compounds of the present disclosure were characterized by TOF-LC / MS using an Agilent 6230 time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-performance liquid chromatograph.

[0493] The exemplary preparation route of the present application is as follows:

[0494] Technical Solution 1

[0495] The first step: reacting the compound of formula (P1) with bromoacetonitrile under heating conditions to obtain a compound of formula (P2);

[0496] Step 2: The compound of formula (P2) reacts with the compound of formula (Y1) at room temperature to obtain the compound of formula (P3);

[0497] Step 3: The compound of formula (P3) is subjected to reduction conditions to obtain the compound of formula (P4);

[0498] Step 4: removing the protective group PG from the compound of formula (P4) to obtain the compound of formula (P5);

[0499] Step 5: reacting the compound of formula (P5) with the compound of formula (Y2) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P6);

[0500] Step 6: reacting the compound of formula (P6) with the compound of formula (Y3) under optional palladium reagent catalysis conditions to obtain the compound of formula (P7);

[0501] in:

[0502] PG can be a common carboxyl protecting group;

[0503] X is selected from: -O-, -S-, and -NR-;

[0504] Ring B is an optionally substituted aryl or heteroaryl group;

[0505] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0506] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0507] R 1 Independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy.

[0508] Technical Solution 2

[0509] Step 1: reacting a compound of formula (P1) with a compound of formula (Y1) under optional palladium catalysis to obtain a compound of formula (P2);

[0510] Step 2: removing the protective group PG from the compound of formula (P2) to obtain the compound of formula (P3);

[0511] in:

[0512] PG can be a protecting group for common amino groups;

[0513] X is selected from: -O-, -S-, and -NR-;

[0514] Ring B is an optionally substituted aryl or heteroaryl group;

[0515] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0516] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0517] R 1 Independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy.

[0518] Technical Solution 3

[0519] Step 1: Adding a protective group PG to the compound of formula (P1) under optional acidic or basic conditions to obtain a compound of formula (P2);

[0520] Step 2: The compound of formula (P2) reacts with a borate dimer under optional palladium reagent catalysis to obtain a compound of formula (P3);

[0521] Step 3: reacting the compound of formula (P3) with the compound of formula (Y1) under optional palladium reagent catalysis conditions to obtain a compound of formula (P4);

[0522] Step 4: removing the protective group PG from the compound of formula (P4) to obtain the compound of formula (P5);

[0523] in:

[0524] PG can be a protecting group for common amino groups;

[0525] R 2 It can be a protecting group for hydrogen or a common amino group;

[0526] X is selected from: -O-, -S-, and -NR-;

[0527] Ring B is an optionally substituted aryl or heteroaryl group;

[0528] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0529] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0530] R 1 Independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy.

[0531] Technical Solution 4

[0532] The first step: the compound of formula (P1) undergoes carbonyl insertion reaction under optional palladium reagent catalysis conditions to obtain a compound of formula (P2);

[0533] Step 2: The compound of formula (P2) is treated with a protective group PG1 under optional acidic or basic conditions to obtain a compound of formula (P3);

[0534] Step 3: hydrolyzing the ester group of the compound of formula (P3) under optional alkaline conditions to obtain a compound of formula (P4);

[0535] Step 4: reacting the compound of formula (P4) with the compound of formula (Y1) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P5);

[0536] Step 4: removing the protecting groups PG1 and PG2 from the compound of formula (P5) to obtain the compound of formula (P6);

[0537] in:

[0538] PG1 and PG2 can be common amino protecting groups;

[0539] X is selected from: -O-, -S-, and -NR-;

[0540] Y is selected from: -O-, -S-, and -NR-;

[0541] R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0542] R 1 independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0543] R 2 Optionally selected from C1-C6 alkyl;

[0544] Ring B is independently selected from optionally substituted aryl or heteroaryl;

[0545] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0546] Technical Solution 5

[0547] Step 1: Reductive amination reaction of the compound of formula (P1) with the compound of formula (Y1) under optional reducing conditions to obtain a compound of formula (P2);

[0548] Step 2: The compound of formula (P2) reacts with the compound of formula (Y2) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P3);

[0549] Step 3: reacting the compound of formula (P3) with the compound of formula (Y3) under optional palladium reagent catalysis conditions to obtain the compound of formula (P4);

[0550] Step 4: removing the protective group PG from the compound of formula (P4) to obtain the compound of formula (P5);

[0551] in:

[0552] PG can be a protecting group for common amino groups;

[0553] Ring B is an optionally substituted aryl or heteroaryl group;

[0554] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0555] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0556] R 1 independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0557] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0558] Technical Solution 6

[0559] Step 1: reacting the compound of formula (P1) under nitration conditions to obtain the compound of formula (P2);

[0560] Step 2: Compound (P2) is subjected to reduction conditions to obtain compound (P3);

[0561] Step 3: reacting the compound of formula (P3) with the compound of formula (Y1) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P4);

[0562] Step 4: The compound of formula (P4) undergoes a ring-closure reaction under heating conditions to obtain a compound of formula (P5);

[0563] Step 5: The compound of formula (P5) is oxidized to obtain the compound of formula (P6);

[0564] Step 6: The compound of formula (P6) is treated in the presence of phosphorus oxychloride to obtain a compound of formula (P7);

[0565] Step 7: reacting the compound of formula (P7) with the compound of formula (Y2) under heating conditions to obtain a compound of formula (P8);

[0566] Step 8: reacting the compound of formula (P8) with the compound of formula (Y3) under optional palladium reagent catalysis conditions to obtain a compound of formula (P9);

[0567] Step 9: The compound of formula (P9) is subjected to reduction conditions to obtain the compound of formula (P10);

[0568] Step 10: removing the protecting groups PG1 and PG2 from the compound of formula (P10) to obtain the compound of formula (P11);

[0569] in:

[0570] PG1 and PG2 can be common amino protecting groups;

[0571] X is selected from: -O- and -S-;

[0572] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0573] R 1 are independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy; wherein, when R 1 When containing a methylene unit, the R 1 The methylene units are each independently not substituted, or the R 1 The methylene units of are each independently substituted by -O-, -S- and optionally substituted -NH-.

[0574] Technical Solution 7

[0575] Step 1: reacting a compound of formula (P1) with a compound of formula (Y1) under optional palladium catalysis to obtain a compound of formula (P2);

[0576] Step 2: removing the protecting groups PG1 and PG2 from the compound of formula (P2) to obtain the compound of formula (P3);

[0577] in:

[0578] PG1 and PG2 can be common amino protecting groups;

[0579] X is selected from: -O- and -S-;

[0580] Y is selected from: -O- and -NH-;

[0581] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0582] R 1 are independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy; wherein, when R 1 When containing a methylene unit, the R 1The methylene units are each independently not substituted, or the R 1 The methylene units of are each independently substituted by -O-, -S- and optionally substituted -NH-.

[0583] Technical Solution 8

[0584] Step 1: The compound of formula (P1) undergoes carbonyl insertion reaction under optional palladium reagent catalysis to obtain a compound of formula (P2);

[0585] Step 2: Compound (P2) is subjected to reduction conditions to obtain compound (P3);

[0586] Step 3: The compound of formula (P3) undergoes a substitution reaction with the compound of formula (Y1) under alkaline conditions to obtain a compound of formula (P4);

[0587] Step 4: removing the protecting groups PG1 and PG2 from the compound of formula (P4) to obtain the compound of formula (P5);

[0588] in:

[0589] PG1 and PG2 can be common amino protecting groups;

[0590] X is selected from: -O- and -S-;

[0591] R 2 Selected from: halogen, methanesulfonyloxy, p-toluenesulfonyloxy;

[0592] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0593] R 1 are independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy; wherein, when R 1 When containing a methylene unit, the R 1 The methylene units are each independently not substituted, or the R 1 The methylene units of are each independently substituted by -O-, -S- and optionally substituted -NH-.

[0594] Technical Solution 9

[0595] Step 1: The compound of formula (P1) undergoes Heck reaction under the catalytic conditions of an optional palladium reagent to obtain a compound of formula (P2);

[0596] Step 2: The compound of formula (P2) is subjected to hydroboration and oxidation conditions to obtain the compound of formula (P3);

[0597] Step 3: The compound of formula (P3) undergoes a substitution reaction with the compound of formula (Y1) under alkaline conditions to obtain a compound of formula (P4);

[0598] Step 4: removing the protecting groups PG1 and PG2 from the compound of formula (P4) to obtain the compound of formula (P5);

[0599] in:

[0600] PG1 and PG2 can be common amino protecting groups;

[0601] X is selected from: -O- and -S-;

[0602] R 2 Selected from: halogen, methanesulfonyloxy, p-toluenesulfonyloxy;

[0603] Z is selected from the group consisting of absent, C1-C6 alkylene, arylene, heteroarylene, cycloalkylene, heterocycloalkylene, and halogen-substituted C1-C6 alkylene.

[0604] R 1 are independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy; wherein, when R 1 When containing a methylene unit, the R 1 The methylene units are each independently not substituted, or the R 1 The methylene units of are each independently substituted by -O-, -S- and optionally substituted -NH-.

[0605] Technical Solution 10

[0606] Step 1: reacting a compound of formula (P1) with aminoacetonitrile under heating conditions to obtain a compound of formula (P2);

[0607] Step 2: The compound of formula (P2) reacts with the compound of formula (Y1) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound represented by formula (P3);

[0608] Step 6: reacting the compound of formula (P3) with the compound of formula (Y2) under optional palladium reagent catalysis conditions to obtain a compound of formula (P4);

[0609] in:

[0610] Ring B is an optionally substituted aryl or heteroaryl group;

[0611] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0612] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0613] R 1 、R 2 and R 3 Independently selected from hydrogen, protium, deuterium, tritium, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl.

[0614] Technical Solution 11

[0615] Step 1: reacting a compound of formula (P1) with a compound of formula (Y1) under optional palladium catalysis to obtain a compound of formula (P2);

[0616] Step 2: removing the protecting groups PG1 and PG2 from the compound of formula (P2) to obtain the compound of formula (P3);

[0617] in:

[0618] PG1 can be a common amino protecting group;

[0619] Ring B is an optionally substituted aryl or heteroaryl group;

[0620] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0621] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0622] R 1 、R 2 and R 3Independently selected from hydrogen, protium, deuterium, tritium, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl.

[0623] Technical Solution 12

[0624] Step 1: reacting a compound of formula (P1) with a compound of formula (Y1) under optionally alkaline conditions to obtain a compound of formula (P2);

[0625] Step 2: The compound of formula (P2) reacts with the compound of formula (Y2) under optional alkaline conditions to obtain a compound of formula (P3);

[0626] Step 3: removing the protecting group PG2 from the compound of formula (P3) to obtain the compound of formula (P4);

[0627] Step 4: reacting the compound of formula (P5) with the compound of formula (P4) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P6);

[0628] Step 5: reacting the compound of formula (P6) with the compound of formula (Y3) under optional palladium reagent catalysis conditions to obtain the compound of formula (P7);

[0629] Step 6: removing the protecting group PG1 from the compound of formula (P7) to obtain the compound of formula (P8);

[0630] in:

[0631] PG1 and PG2 can be common amino protecting groups;

[0632] X is independently selected from halogen, methanesulfonyloxy and p-toluenesulfonyloxy;

[0633] Ring A and Ring B are independently selected from optionally substituted aryl or heteroaryl;

[0634] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0635] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0636] R 1 is an optionally substituted C1-C6 alkyl group;

[0637] Z is selected from optionally substituted C1-C6 alkylene, optionally substituted cycloalkylene, and optionally substituted heterocycloalkylene.

[0638] Technical Solution 13

[0639] Step 1: reacting a compound of formula (P1) with a compound of formula (Y1) under optionally alkaline conditions to obtain a compound of formula (P2);

[0640] Step 2: The compound of formula (P2) reacts with the compound of formula (Y2) under optional alkaline conditions to obtain a compound of formula (P3);

[0641] Step 3: removing the protecting group PG2 from the compound of formula (P3) to obtain the compound of formula (P4);

[0642] Step 4: reacting the compound of formula (P5) with the compound of formula (P4) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P6);

[0643] Step 5: reacting the compound of formula (P6) with the compound of formula (Y3) under optional palladium reagent catalysis conditions to obtain the compound of formula (P6);

[0644] Step 6: removing the protecting group PG1 from the compound of formula (P4) to obtain the compound of formula (P7);

[0645] in:

[0646] PG2 can be a common amino protecting group;

[0647] X is independently selected from halogen, methanesulfonyloxy and p-toluenesulfonyloxy;

[0648] Ring A and Ring B are independently selected from optionally substituted aryl or heteroaryl;

[0649] -B(OR)2 is a borate-based monomer, wherein two Rs may be linked to form a heterocyclic ring, a heterobridged ring, or a heterospirocyclic ring, and the ring may be optionally substituted with a C1-C6 alkyl group, an aryl group, a heteroaryl group, a carboxyl group, or an acyloxy C1-C6 alkyl group;

[0650] wherein each R is independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxyl, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0651] R 1 is an optionally substituted C1-C6 alkyl group;

[0652] Z is selected from optionally substituted C1-C6 alkylene, optionally substituted cycloalkylene, and optionally substituted heterocycloalkylene.

[0653] Technical Solution 14

[0654] The first step: the compound of formula (P1) undergoes carbonyl insertion reaction under optional palladium reagent catalysis conditions to obtain a compound of formula (P2);

[0655] Step 2: hydrolyzing the ester group of the compound of formula (P2) under optional alkaline conditions to obtain a compound of formula (P3);

[0656] Step 3: reacting the compound of formula (P3) with the compound of formula (Y1) in the presence of an optional condensing agent, optionally under alkaline conditions, to obtain a compound of formula (P4);

[0657] Step 4: Reducing the nitro group of the compound of formula (P4) under reducing conditions, or removing the protecting group PG under optional alkaline or acidic conditions to obtain a compound of formula (P5);

[0658] in:

[0659] Ring B is an optionally substituted aryl or heteroaryl group;

[0660] Z is selected from optionally substituted C1-C6 alkylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene;

[0661] R 1 independently selected from hydrogen, protium, deuterium, tritium, oxygen, hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy;

[0662] R 2 Optional from nitro or -NH-PG, PG can be a common amino protecting group;

[0663] R is optionally selected from C1-C6 alkyl;

[0664] Y is selected from: -O-, -S-, and -NR-;

[0665] R 3 Optionally selected from C1-C6 alkyl, C1-C6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halogen-substituted C1-C6 alkyl, and halogen-substituted C1-C6 alkoxy.

[0666] Without intending to be bound by any theory, the following examples are merely for illustrating the compounds, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0667] Example

[0668] Example 1

[0669] Compound 1

[0670] Step 1: Add EA (180 mL) and 1A (30 g, 79.7 mmol, 1 eq) to a 500 mL three-necked flask. Then, add bromoacetonitrile (5.73 g, 47.7 mmol, 0.6 eq) dropwise at room temperature. After completion, heat to 85°C and react overnight. Cool the reaction mixture and filter directly. Wash the filter cake with 50 mL of EA. The filtrate is then dried to give a dark red solid, crude 1B (9 g), which is used directly in the next step without calculating the yield.

[0671] Step 2: Dissolve 1B (9 g, 21.74 mmol, 1 eq) obtained in the previous step in toluene (90 mL) (not clear). Add 1C (5 g, 21.74 mmol, 1 eq) at room temperature and stir overnight. Dried toluene was evaporated, and the mixture was purified by column chromatography (PE:EA = 20:1 to 10:1). The mixture was then dried under reduced pressure and slurried with PE (50 mL) for filtration to afford 1D (5 g, 62.5% yield) as pale yellow flaky crystals.

[0672] Step 3 In a 250mL three-necked flask, 1D (5g, 13.6mmol, 1eq) was added to glacial acetic acid (50mL), and the internal temperature was raised to about 60-65°C. Iron powder (3.8g, 68mmol, 5eq) was slowly added in batches. After the addition was complete, the internal temperature was raised to 85°C and the reaction was reacted for 2 hours. The mixture was cooled to about 60°C, and DCM (50mL) was added to dilute the reaction solution. The mixture was then filtered while hot through a pad of diatomaceous earth. The filter cake was washed with DCM (100mL), and the filtrate was dried under reduced pressure to obtain a dark red oil. The mixture was diluted with DCM (50mL) and poured into a saturated aqueous sodium bicarbonate solution (200mL). The mixture was extracted with DCM (100mL×4), and the organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. After being dried, PE:EA=2:1 (30mL) was added and pulped and filtered to obtain an off-white powder 1E (4.5g, 97% yield). MS-ESI: m / z 337.1[M+H] + .

[0673] Step 4: Add 4N HCl / Dioxane (40 mL) to a 100 mL single-necked flask, followed by 1E (4.5 g, 13.4 mmol, 1 eq), and stir overnight at room temperature. LCMS confirmed the complete reaction, and the mixture was directly dried to afford 1F (4.2 g, 99% yield), an off-white powder. MS-ESI: m / z 281.0 [M+H] + .

[0674] Step 5: In a 250 mL three-necked flask, DMF (60 mL) and 1F (3 g, 9.45 mmol, 1 eq) were added. HBTU (4.3 g, 11.34 mmol, 1.2 eq) and DIEA (4.88 g, 37.79 mmol, 4 eq) were then added at 0°C. After complete addition, the mixture was stirred for 10 minutes. Dipropylamine (1.86 g, 18.42 mmol, 1.95 eq) was added dropwise. After complete addition, the mixture was warmed to room temperature and stirred overnight. The reaction mixture was poured into water (240 mL), extracted with EA (100 mL × 5), backwashed with saturated brine (50 mL × 2), dried over sodium sulfate, spin-dried, and filtered through a silica gel column to obtain 1G (3 g, 87.5% yield) as a dark red solid. MS-ESI: m / z 364.2 [M+H] + .

[0675] Step 6: A 25 mL three-necked flask was charged with a mixed solvent of dioxane (5 mL) and water (0.5 mL). 1G (200 mg, 0.55 mmol, 1 eq), 1H (121 mg, 0.55 mmol, 1 eq), and anhydrous potassium phosphate (350 mg, 1.65 mmol, 3 eq) were then added. The atmosphere was then replaced with argon twice. Pd(PPh3)4 (50 mg) was added, the atmosphere was replaced with argon twice again, and the temperature was raised to 85°C for 3 hours. After cooling, the reaction solution was dried over sodium sulfate, the water was removed, and the product was directly purified by thin-layer chromatography to obtain 1 (25 mg, 12% yield), a dark yellow powder.

[0676] MS-ESI: m / z 377.3[M+H] + .

[0677] 1 H NMR (400MHz, DMSO-d6) δ7.36(d,J=8.6Hz,2H),7.25(d,J=8.2Hz,1H),7.15(d,J=1.9Hz,1H),7.11(dd,J=8.2,2.0Hz,1H), 6.79–6.67(m,3H),6.62(d,J=8.6Hz,2H),5.22(s,2H),3.30–3.25(m,4H),2.70(s,2H),1.63–1.48(m,4H),0.82(brs,6H).

[0678] Compound 2

[0679] Step 1: A 25 mL three-necked flask was charged with a mixed solvent of dioxane (5 mL) and water (0.5 mL). 1G (200 mg, 0.55 mmol, 1 eq), 2A (121 mg, 0.55 mmol, 1 eq), and anhydrous potassium phosphate (350 mg, 1.65 mmol, 3 eq) were then added. The atmosphere was then replaced with argon twice. Pd(PPh3)4 (50 mg) was added, and the atmosphere was replaced with argon twice more. The temperature was raised to 85°C and the reaction mixture was reacted for 3 hours. After cooling, the reaction mixture was dried over sodium sulfate, the water was removed, and column chromatography (DCM:MeOH = 100:0 to 10:1) afforded 2 (23 mg, 11% yield) as a light yellow powder.

[0680] MS-ESI: m / z 378.3[M+H] + .

[0681] 1 H NMR (400MHz, DMSO-d6) δ8.04(d,J=2.0Hz,1H),7.93(d,J=2.5Hz,1H),7.44(d,J=8.0Hz,1H),7.34–7.23(m,2H),7. 16(t,J=2.3Hz,1H),6.82(s,1H),5.43(s,2H),3.31–3.26(m,4H),2.89(s,2H),1.61–1.51(m,4H),0.83(brs,6H).

[0682] Compound 3

[0683] Step 1: A 25 mL three-necked flask was charged with a mixed solvent of dioxane (10 mL) and water (1 mL). 1G (400 mg, 1.1 mmol, 1 eq), 3A (151 mg, 1.1 mmol, 1 eq), and anhydrous potassium phosphate (700 mg, 3.29 mmol, 3 eq) were then added. The atmosphere was then replaced with argon twice, and Pd(PPh3)4 (100 mg) was added. The atmosphere was again replaced with argon twice, and the temperature was raised to 85°C for 3 hours. After cooling, the reaction solution was dried over 5 g of sodium sulfate, directly mixed with silica gel, and purified by column chromatography to obtain a pale yellow powder 3 (45 mg, 11% yield).

[0684] MS-ESI: m / z 377.2[M+H] + .

[0685] 1H NMR(400MHz, Methanol-d4)δ7.72–7.60(m,3H),7.59–7.37(m,3H),7.37–7.12(m, 1H),7.08(s,1H),3.47(brs,4H),3.36(s,2H),1.77–1.63(m,4H),0.95(brs,6H).

[0686] Compound 4

[0687] Step 1: Dissolve 4A (2 g, 9.66 mmol, 1 eq) in toluene (40 mL). Add triethylamine (2.44 g, 24.15 mmol, 2.5 eq), DPPA (3.19 g, 11.59 mmol, 1.2 eq), and tert-butanol (7.16 g, 96.6 mmol, 10 eq) at room temperature. After complete addition, heat the temperature to 100°C and stir overnight. The reaction mixture was poured into 50 mL of water and extracted with EA (50 mL x 3). The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and filtered through silica gel column chromatography (PE:EA = 5:1) to afford 4B (0.6 g, 22.3% yield) as an off-white solid.

[0688] Step 2: 1G (2 g, 5.49 mmol, 1 eq) was dissolved in DCM (40 mL). Triethylamine (1.67 g, 16.47 mmol, 3 eq) was added at room temperature, followed by the dropwise addition of Boc2O (1.8 g, 8.24 mmol, 1.5 eq). The mixture was stirred overnight at room temperature. The reaction mixture was poured into 50 mL of water and extracted with DCM (50 mL x 3). The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography on silica gel (PE:EA = 5:1) to afford 4C (1.2 g, 49% yield) as a light yellow solid.

[0689] Step 3: 4C (1.2 g, 2.58 mmol, 1 eq) was dissolved in Dioxane (20 mL), and diboronic acid pinacol ester (722 mg, 2.84 mmol, 1.1 eq) and AcOK (507 mg, 5.17 mmol, 2 eq) were added. The atmosphere was purged with argon twice, and then Pd(dppf)Cl2 (0.2 g) was added all at once. After the addition, the atmosphere was purged with argon twice more, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature and directly poured onto a silica gel column. The product was eluted with PE:EA = 2:1 to obtain 4D (600 mg, 45% yield) as a yellow solid. MS-ESI: m / z 512.3 [M+H] + .

[0690] Step 4: 4B (359 mg, 1.29 mmol, 1.2 eq) and 4D (550 mg, 1.08 mmol, 1 eq) were added to a mixed solvent of dioxane (10 mL) and water (1 mL). Sodium carbonate (285 mg, 2.69 mmol, 2.5 eq) was added, and the atmosphere was replaced with argon twice. Pd(dppf)Cl2 (100 mg) was then added all at once. After the addition was complete, the atmosphere was replaced with argon twice more, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature, dried over sodium sulfate, and directly purified by silica gel column chromatography to obtain 4E (100 mg, 15.9% yield) as a yellow solid. MS-ESI: m / z 583.3 [M+H] + .

[0691] Step 5: Dissolve 4E (100 mg, 0.17 mmol, 1 eq) in EA (5 mL), add 4N dioxane hydrochloride (1 mL) under ice-water bath, stir at room temperature overnight, concentrate under reduced pressure, and freeze-dry to obtain a yellow solid 4 (22 mg, 27% yield).

[0692] MS-ESI: m / z 383.2[M+H] + .

[0693] 1 H NMR (400MHz, Methanol-d4) δ7.35(d,J=1.7Hz,1H),7.30(d,J=8.1Hz,1H),7.26(dd,J=8.2,1.8Hz,1H),7.05(d,J=1.5 Hz,1H),6.81(s,1H),6.25(d,J=1.6Hz,1H),4.52–4.38(m,2H),3.46–3.37(m,4H),1.73–1.60(m,4H),0.91(brs,6H).

[0694] Compound 5

[0695] Step 1: 5A (280 mg, 0.98 mmol, 1 eq) and 4D (500 mg, 0.98 mmol, 1 eq) were added to a mixed solvent of dioxane (10 mL) and water (2 mL). Sodium carbonate (259 mg, 2.44 mmol, 2.5 eq) was added, and the atmosphere was replaced with argon twice. Pd(dppf)Cl2 (100 mg) was then added all at once. After the addition was complete, the atmosphere was replaced with argon twice more. The temperature was raised to 95°C and the reaction mixture was reacted for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature, dried over sodium sulfate, and purified directly by thin-layer chromatography to obtain 5B (200 mg, 35% yield) as a yellow solid. MS-ESI: m / z 591.4 [M+H]+ .

[0696] Step 2: Dissolve 5B (200 mg, 0.33 mmol, 1 eq) in EA (5 mL). Add 4N dioxane hydrochloride (0.5 mL) under ice-water bath and stir at room temperature overnight. LCMS confirmed the complete reaction. The mixture was concentrated under reduced pressure, sent to a preparative step, and lyophilized to afford 5 (25 mg, 19% yield) as a yellow solid.

[0697] MS-ESI: m / z 391.3[M+H] + .

[0698] 1 H NMR(400MHz,Methanol-d4)δ7.20(d,J=7.9Hz,1H),7.05–6.96(m,2H),6.90(dd,J=8.0,1.9Hz,1H),6.78(s,1 H),6.63–6.53(m,3H),4.69–4.48(m,2H),3.85(s,2H),3.45–3.36(m,4H),1.75–1.57(m,4H),0.90(brs,6H).

[0699] Compound 6

[0700] Step 1: A 50 mL autoclave was charged with methanol (20 mL), 1G (1.2 g, 3.29 mmol, 1 eq), and triethylamine (1 g, 9.88 mmol, 3 eq). After purging with argon, Pd(dppf)Cl2 (0.2 g) was added. The autoclave was then quickly sealed and replaced with argon twice. CO (60 psi) was introduced and the temperature was raised to 80°C for 40 hours. LCMS analysis indicated a small amount of residual starting material. The product was filtered and concentrated under reduced pressure to obtain a dark red oily crude product 6A (1.5 g), which was directly used in the next step without considering the yield. MS-ESI: m / z 344.2 [M+H] + .

[0701] Step 2: The crude product 6A (1.5 g, 4.37 mmol, 1 eq) obtained in the previous step was dissolved in DCM (30 mL). Triethylamine (1.33 g, 13.10 mmol, 3 eq) was added at room temperature, followed by dropwise addition of Boc2O (1.43 g, 6.55 mmol, 1.5 eq). The mixture was stirred overnight at room temperature. The reaction mixture was poured into water (50 mL) and extracted with DCM (50 mL x 3). The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 5:1) to afford 6B (0.4 g, 27.6% yield for both steps) as a light yellow solid. MS-ESI: m / z 444.3 [M+H] + .

[0702] Step 3: 6B (400 mg, 0.9 mmol, 1 eq) was added to a mixed solvent of water (5 mL) and THF (5 mL). Lithium hydroxide monohydrate (56.77 mg, 1.35 mmol, 1.5 eq) was added at room temperature, and the mixture was stirred overnight. TLC confirmed the reaction completion. The pH was adjusted to 5 with saturated citric acid, and the reaction solution was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure to afford 6C (350 mg, 90% yield), a yellow powder.

[0703] Step 4: 6C (150 mg, 0.35 mmol, 1 eq) was added to DCM (5 mL). HATU (159 mg, 0.42 mmol, 1.2 eq) was added at room temperature and stirred for 10 minutes. NMM (89 mg, 0.873 mmol, 2 eq) and 6D (73 mg, 0.35 mmol, 1 eq) were then added and stirred at room temperature overnight. TLC confirmed the complete reaction. The reaction solution was poured into water (15 mL) and extracted with EA (30 mL x 4). The organic phases were combined, concentrated under reduced pressure, and purified directly by thin-layer chromatography to obtain 6E (50 mg, 23% yield) as an off-white powder. MS-ESI: m / z 620.4 [M+H] + .

[0704] Step 5: Dissolve 6E (50 mg, 0.08 mmol, 1 eq) in ethyl acetate (5 mL). Add dioxane hydrochloride (4N, 0.5 mL) under ice-water bath, and stir at room temperature overnight. LCMS confirmed the complete reaction. The product was concentrated under reduced pressure and directly purified by thin-layer chromatography to afford 6 (24 mg, 70.8% yield), a light yellow powder.

[0705] MS-ESI: m / z 420.6[M+H] + .

[0706] 1 H NMR (400MHz, Methanol-d4) δ7.66(d,J=2.0Hz,1H),7.55(dd,J=8.1,1.9Hz,1H),7.43(d,J=8.2Hz,1H),7.41–7.3 6(m,2H),6.89(s,1H),6.78–6.71(m,2H),4.58(s,2H),3.47–3.38(m,4H),1.76–1.60(m,4H),1.07–0.77(m,6H).

[0707] Compound 7

[0708] Step 1: 6C (300 mg, 0.70 mmol) was added to a 100 mL vial. DMF (10 mL), 7A (174 mg, 0.84 mmol), DIEA (271 mg, 2.10 mmol), and HATU (372 mg, 0.98 mmol) were added at 0°C. The reaction was stirred at room temperature for 16 hours. LCMS showed the disappearance of the starting material. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with EA (100 mL × 3). The organic phase was dried and purified by column chromatography (EA:PE = 0-20%) to obtain 7B (300 mg, 69% yield) as a light yellow oil. MS-ESI: m / z 620.4 [M+H] + .

[0709] Step 2: 7B (300 mg, 0.48 mmol) was added to a 100 mL vial. DCM (8 mL) and TFA (2 mL) were added at 0°C. The reaction was stirred at room temperature for 16 hours. LCMS showed the disappearance of the starting material. The reaction mixture was evaporated to dryness and purified by reverse-phase column chromatography (NH4HCO3) to afford 7 (70 mg, 34% yield) as a pale yellow solid.

[0710] MS-ESI: m / z 420.2[M+H] + .

[0711] 1 H NMR (400MHz, DMSO-d6) δ9.90(s,1H),7.60(d,J=1.8Hz,1H),7.43(dd,J=8.1,1.9Hz,1H),7.38(d,J=8.2Hz,1H),7.14(t,J=2.0Hz,1H),6.94(t,J =7.9Hz,1H),6.91–6.81(m,3H),6.77(s,1H),6.33–6.26(m,1H),5.04(s ,2H),3.31–3.26(m,4H),2.71(s,2H),1.64–1.49(m,4H),0.83(brs,6H).

[0712] Compound 8

[0713] Step 1: Dissolve 8A (1 g, 8.4 mmol) in DCM (20 mL), add DMAP (102 mg, 0.84 mmol) and Boc2O (2.2 g, 10.1 mmol), and stir at room temperature overnight. After concentration, purification by column chromatography (PE:EA = 1:1) afforded 8B as a white solid (1.2 g, 65% yield, 95% purity). MS-ESI: m / z 220.0 [M+H] + .

[0714] Step 2 8B (500 mg, 2.27 mmol) was dissolved in MeOH / NH3 . To a solution of H2O (5 mL, v / v = 4 / 1) was added Raney Ni (100 mg) at room temperature, and the H2 was exchanged three times. The mixture was stirred at room temperature overnight. After filtration and concentration, a green solid 8C (450 mg, 85% yield, 84% purity) was obtained. MS-ESI: m / z 224.2 [M+H] + .

[0715] Step 1: Dissolve 6C (300 mg, 0.7 mmol) and 8C (156 mg, 0.7 mmol) in DMF (3 mL). Add DIEA (271 mg, 2.1 mmol) and HATU (320 mg, 0.84 mmol) and stir at room temperature overnight. The reaction mixture is directly purified by HPLC (MeCN / H2O) to give 8D as a pale yellow solid (200 mg, 45% yield, 85% purity). MS-ESI: m / z 635.4 [M+H] + .

[0716] Step 4: 8D (200 mg, 0.3 mmol) was dissolved in DCM (5 mL), and TFA (180 mg, 1.5 mmol) was added. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with water, and the pH was adjusted to 8-9 with NaHCO₃. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and dried. HPLC (MeCN / H₂O) was used to obtain 8 (52.8 mg, 35% yield, 95.23% purity) as a yellow solid.

[0717] MS-ESI: m / z 435.3[M+H] + .

[0718] 1 H NMR (400MHz, DMSO-d6) δ7.90–7.77(m,2H),7.64–7.58(m,1H),7.52–7.44(m,1H),7.40(dd,J=8.1,3.3Hz,1H),7.10(s, 1H),6.90–6.84(m,1H),4.55–4.47(m,2H),3.47–3.36(m,4H),3.34–3.32(m,2H),1.78–1.56(m,4H),1.11–0.71(m,6H).

[0719] Compound 9

[0720] Step 1: Dissolve 6C (300 mg, 0.7 mmol) in DMF (3 mL), add 9A (155 mg, 0.7 mmol), DIEA (211 mg, 2.1 mmol), and HATU (320 mg, 0.84 mmol), and stir at room temperature overnight. The reaction mixture was directly purified by HPLC (MeCN / H2O) to give 9B as a white solid (250 mg, 55% yield, 90% purity). MS-ESI: m / z 634.4 [M+H] + .

[0721] Step 2: 9B (250 mg, 0.4 mmol) was dissolved in DCM (5 mL), and TFA (135 mg, 1.2 mmol) was added. The mixture was allowed to react at room temperature for 3 hours. The mixture was diluted with water, and the pH was adjusted to 8-9 with NaHCO₃. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and dried. HPLC (MeCN / H₂O) was used to obtain 9 as a light yellow solid (26.3 mg, 15% yield, 99.59% purity).

[0722] MS-ESI: m / z 434.3[M+H] + .

[0723] 1 H NMR (400MHz, Methanol-d4) δ7.61(d,J=1.8Hz,1H),7.48(dd,J=8.1,1.9Hz,1H),7.39(d,J=8.2Hz,1H),7.06(t,J=7.7Hz,1H),6.87(s,1H),6.7 4(t,J=2.0Hz,1H),6.71–6.66(m,1H),6.62(ddd,J=8.0,2.4,1.0Hz,1H) ,4.49(s,2H),3.46–3.37(m,4H),1.76–1.57(m,4H),1.11–0.72(m,6H).

[0724] Compound 10

[0725] Step 1: 10A (5 g, 22.60 mmol, 1 eq) was added to DCM (100 mL). n-Propylamine (4.01 g, 67.80 mmol, 3 eq) was added at room temperature and stirred overnight. Sodium cyanoborohydride (2.84 g, 45.20 mmol, 2 eq) was slowly added at room temperature and stirred overnight. The reaction mixture was poured into 100 mL of water, separated, and the aqueous phase was extracted with DCM (100 mL x 2). The organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and filtered through a silica gel chromatography column (DCM:MeOH = 50:1 to 10:1) to obtain 10B (3 g, 50.3% yield) as a white solid.

[0726] Step 2: 1F (1 g, 3.15 mmol, 1 eq) was dissolved in DMF (20 mL), cooled to approximately 0°C, and HBTU (1.43 g, 3.78 mmol, 1.2 eq) and DIEA (1.63 g, 12.60 mmol, 4 eq) were added. The mixture was stirred at 0°C for 10 minutes. 10B (1 g, 3.78 mmol, 1.2 eq) was added to the reaction mixture and stirred at room temperature overnight. The reaction mixture was poured into water (60 mL) and extracted with EA (50 mL x 5). The organic phases were combined, backwashed with saturated brine (50 mL x 2), dried over sodium sulfate, concentrated under reduced pressure, and filtered on silica gel for column chromatography (DCM:MeOH = 20:1 to 10:1). The mixture was concentrated under reduced pressure to afford 10C (550 mg, 30% yield) as a yellow solid. MS-ESI: m / z 527.2 [M+H] + .

[0727] Step 3: 10C (550 mg, 1.04 mmol, 1 eq) was added to a mixed solvent of dioxane (20 mL) and water (2 mL). 10D (243 mg, 1.04 mmol, 1 eq) and anhydrous potassium phosphate (664 mg, 3.13 mmol, 3 eq) were then added. The atmosphere was purged with argon twice, and then Pd(PPh3)4 (110 mg) was added. The atmosphere was purged with argon twice, and the temperature was raised to 90°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The reaction solution was poured into 50 mL of water and extracted with EA (50 mL x 3). The organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 2:1 to 0:1) to afford 10E (200 mg, 34.7% yield) as a white solid. MS-ESI: m / z 554.4 [M+H] + .

[0728] Step 4: 10E (200 mg, 0.36 mmol, 1 eq) was added to methanol (10 mL). Sodium borohydride (41 mg, 1.08 mmol, 3 eq) was slowly added portionwise at room temperature. The reaction was maintained at room temperature for 2 hours. LCMS confirmed the complete reaction of the starting material. The reaction solution was quenched with saturated aqueous ammonium chloride (5 mL), concentrated under reduced pressure to remove the solvent, and directly purified by thin-layer chromatography to obtain 10F (160 mg, 80% yield) as a white solid. MS-ESI: m / z 556.3 [M+H] + .

[0729] Step 5: 10F (160 mg, 0.28 mmol, 1 eq) was dissolved in EA (5 mL). 4N dioxane hydrochloride (0.5 mL) was added under ice-water bath and stirred at room temperature overnight. LCMS confirmed the reaction completion. The mixture was concentrated under reduced pressure, sent to a preparative step, and lyophilized to afford 10 (30 mg, 23% yield) as a white powder.

[0730] MS-ESI: m / z 456.3[M+H] + .

[0731] 1 H NMR(400MHz, Methanol-d4)δ8.88(d,J=2.2Hz,1H),8.66(d,J=1.9Hz,1H),8.34(s,1H),7.84–7.56(m,3H),7.56–7.18 (m,4H),7.16(s,1H),4.81(s,2H),4.78(s,2H),3.55–3.45(m,2H),3.45–3.33(m,2H),1.76–1.63(m,2H),0.92(s,3H).

[0732] Compound 11

[0733] Step 1: 11A (1 g, 4.97 mmol, 1 eq) was dissolved in DMF (20 mL). DIEA (1.93 g, 14.92 mmol, 3 eq) and HATU (2.08 g, 5.47 mmol, 1.1 eq) were added at room temperature. After addition, the mixture was stirred at room temperature for 5 minutes. 11B (0.434 g, 4.97 mmol, 1 eq) was then added and stirred at room temperature overnight. The reaction mixture was poured into 60 mL of water and extracted with EA (50 mL x 3). The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and filtered on silica gel. The mixture was then chromatographed on a column (PE:EA = 5:1) to afford 11C (1 g, 74.6% yield) as a white solid. MS-ESI: m / z 270.0 [M+H] + .

[0734] Step 2: 11C (0.9 g, 3.33 mmol, 1 eq) was dissolved in Dioxane (20 mL), and diboronic acid pinacol ester (931 mg, 3.67 mmol, 1.1 eq) and AcOK (654 mg, 6.66 mmol, 2 eq) were added. The atmosphere was purged with argon twice, and then Pd(dppf)Cl2 (0.2 g) was added all at once. After the addition, the atmosphere was purged with argon twice more, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature and directly loaded onto a silica gel column (PE:EA = 2:1) for purification to afford 11D (300 mg, 28.3% yield) as a light yellow solid. MS-ESI: m / z 318.3 [M+H] + .

[0735] Step 3: 10C (300 mg, 0.569 mmol, 1 eq) and 11D (180.4 mg, 0.569 mmol, 1 eq) were added to a mixed solvent of dioxane (10 mL) and water (1 mL). Sodium carbonate (150.71 mg, 1.42 mmol, 2.5 eq) was added, and the atmosphere was replaced with argon twice. Pd(dppf)Cl2 (50 mg) was then added all at once. After the addition, the atmosphere was replaced with argon twice more, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature, dried over sodium sulfate, and directly purified by silica gel column chromatography to obtain 11E (100 mg, 27.6% yield) as a yellow solid. MS-ESI: m / z 638.4 [M+H] + .

[0736] Step 4: Dissolve 11E (100 mg, 0.157 mmol, 1 eq) in EA (5 mL). Add 4N dioxane hydrochloride (1 mL) under ice-water bath and stir at room temperature overnight. LCMS confirmed the complete reaction. The mixture was concentrated under reduced pressure, sent to a preparative step, and lyophilized to afford 11 (30 mg, 35.7% yield) as a yellow solid.

[0737] MS-ESI: m / z 538.3[M+H] + .

[0738] 1H NMR(400MHz, Methanol-d4)δ7.80(d,J=7.9Hz,2H),7.76–7.66(m,4H),7.66–7.30(m,3H),7.25(s,2H),7.14(s,1H),4.78 (s,2H),4.45(d,J=47.8Hz,1H),3.84–3.58(m,3H),3.58–3.32(m,5H),2.19–1.90(m,2H),1.75–1.63(m,2H),0.91(s,3H).

[0739] Compound 12

[0740] Step 1: Dissolve 12A (1 g, 4.69 mmol, 1 eq) in dioxane (20 mL), add pinacol diboronate (1.31 g, 5.16 mmol, 1.1 eq) and AcOK (921.4 mg, 9.39 mmol, 2 eq), and replace the atmosphere with argon twice. Then, add Pd(dppf)Cl2 (0.1 g) all at once. After addition, replace the atmosphere with argon twice more, and heat to 85°C for 3 hours. LCMS analysis confirmed the complete reaction of the starting material. After cooling to room temperature, the reaction mixture was directly loaded and purified on a silica gel column (PE:EA = 10:1) to obtain 12B (300 mg, 24.6% yield) as a white solid. MS-ESI: m / z 261.2 [M+H] + .

[0741] Step 2: 10C (250 mg, 0.474 mmol, 1 eq) and 12B (247 mg, 0.948 mmol, 2 eq) were added to a mixed solvent of dioxane (10 mL) and water (1 mL). Potassium phosphate (302 mg, 1.42 mmol, 3 eq) was added, and the atmosphere was replaced with argon twice. Pd(dppf)Cl2 (50 mg) was then added all at once. After the addition, the atmosphere was replaced with argon twice more, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The mixture was cooled to room temperature, dried over sodium sulfate, and directly purified by silica gel column chromatography to obtain 12C (100 mg, 36.4% yield) as a yellow solid. MS-ESI: m / z 581.2 [M+H] + .

[0742] Step 3: Dissolve 12C (100 mg, 0.172 mmol, 1 eq) in EA (5 mL). Add 4N dioxane hydrochloride (1 mL) under ice-water bath and stir at room temperature overnight. LCMS confirmed the complete reaction. The mixture was concentrated under reduced pressure, sent to a preparative step, and lyophilized to afford 12 (23 mg, 28% yield) as a yellow solid.

[0743] MS-ESI: m / z 481.3[M+H] + .

[0744] 1 H NMR(400MHz, Methanol-d4)δ8.17(s,1H),8.10(d,J=8.1Hz,1H),7.84–7.70(m,3H),7.70–7.27(m,3H),7.27– 7.06(m,3H),5.47(s,2H),4.77(s,2H),3.54–3.44(m,2H),3.44–3.32(m,2H),1.77–1.64(m,2H),0.92(s,3H).

[0745] Compound 13

[0746] Step 1: 13A (5 g, 22.60 mmol, 1 eq) was added to DCM (100 mL). n-Propylamine (4.01 g, 67.80 mmol, 3 eq) was added at room temperature and stirred overnight. Sodium cyanoborohydride (2.84 g, 45.20 mmol, 2 eq) was slowly added at room temperature and stirred overnight. The reaction mixture was poured into 100 mL of water, separated, and the aqueous phase was extracted with DCM (100 mL x 2). The organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and passed through a silica gel chromatography column (DCM:MeOH = 50:1-10:1) to obtain 13B (1.5 g, 25.1% yield) as a white solid. MS-ESI: m / z 265.2 [M+H] + .

[0747] Step 2: 1F (2 g, 6.30 mmol, 1 eq) was dissolved in DMF (40 mL), cooled to approximately 0°C, and HBTU (2.86 g, 7.56 mmol, 1.2 eq) and DIEA (3.26 g, 25.20 mmol, 4 eq) were added. The mixture was stirred at 0°C for 10 minutes. 13B (2 g, 7.56 mmol, 1.2 eq) was added to the reaction mixture, and the mixture was stirred at room temperature overnight. LCMS confirmed the complete reaction of 1F. The reaction mixture was poured into water (120 mL) and extracted with EA (100 mL × 5). The organic phases were combined, backwashed with saturated brine (100 mL × 2), dried over sodium sulfate, and concentrated under reduced pressure. The mixture was then filtered on silica gel and purified by column chromatography (DCM:MeOH = 20:1-10:1). The mixture was then concentrated under reduced pressure to afford 13C (1.2 g, 36.1% yield) as a yellow solid. MS-ESI: m / z 527.2[M+H] + .

[0748] Step 3: 13C (1.2 g, 2.28 mmol, 1 eq) was added to a mixed solvent of dioxane (40 mL) and water (4 mL). 10D (530.3 mg, 2.28 mmol, 1 eq) and anhydrous potassium phosphate (1.45 g, 6.83 mmol, 3 eq) were added. The atmosphere was purged with argon twice, and then Pd(PPh3)4 (220 mg) was added. The atmosphere was purged with argon twice, and the temperature was raised to 90°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The reaction solution was poured into 100 mL of water and extracted with EA (100 mL × 3). The organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 2:1–0:1) to afford 13D (250 mg, 19.9% ​​yield) as a white solid. MS-ESI: m / z 554.3 [M+H] + .

[0749] Step 4: 13D (250 mg, 0.45 mmol, 1 eq) was added to methanol (10 mL). Sodium borohydride (51.3 mg, 1.35 mmol, 3 eq) was slowly added portionwise at room temperature. The reaction was maintained at room temperature for 2 hours after the addition was complete. The reaction solution was quenched with 5 mL of saturated aqueous ammonium chloride solution, concentrated under reduced pressure to remove the solvent, and directly purified by thin-layer chromatography to obtain 13E (124 mg, 49.4% yield) as a white solid. MS-ESI: m / z 556.2 [M+H] + .

[0750] Step 5: 13E (124 mg, 0.22 mmol, 1 eq) was dissolved in EA (5 mL). 4N dioxane hydrochloride (0.5 mL) was added under ice-water bath and stirred at room temperature overnight. LCMS confirmed the reaction was complete. The mixture was concentrated under reduced pressure and the pH was adjusted to 8 with saturated NaHCO₃ solution. EA (10 ml x 3) was added. After concentration, the sample was passed through a column (DCM:MeOH = 30:1–10:1) and lyophilized to afford 13 (25 mg, 24.6% yield) as a white powder.

[0751] MS-ESI: m / z 456.2[M+H] + .

[0752] 1H NMR(400MHz,DMSO-d6)δ8.76(d,J=2.0Hz,1H),8.50(s,1H),7.99(s,1H),7.3 2(s,1H),7.28–7.26(d,J=7.7Hz,1H),6.99–6.97(d,J=7.6Hz,1H),6.86(s,2H ),6.47–6.45(d,J=8.1Hz,3H),5.08(s,2H),4.61(s,2H),4.48(s,2H),3.32– 3.21(m,2H),2.79(s,2H),1.56–1.52(m,2H),1.23(s,1H),0.85–0.81(m,3H).

[0753] Compound 14

[0754] Step 1: To a 250 mL three-necked flask at room temperature, 14A (4.5 g, 20.2 mmol) and propionic acid (100 mL) were added. The reaction mixture was heated to 125°C and HNO3 (3.2 g, 50.8 mmol) was added dropwise while maintaining the internal temperature at 120-130°C. The reaction was stirred at 125°C for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was cooled to room temperature, slowly poured into ethanol (500 mL), filtered, and the filter cake was rinsed with ethanol (100 mL), water (100 mL), and ethanol (100 mL) in sequence. The filter cake was collected and dried to afford 14B as a yellow solid (3 g, 55.6% yield). MS-ESI: m / z 269.0 [M+H] + .

[0755] Step 2: At room temperature, 14B (3 g, 11.2 mmol) was added to a 250 mL single-necked flask. EtOH (50 mL) and H₂O (10 mL) were added to dissolve the mixture. NH₄Cl (1.8 g, 33.6 mmol) and iron powder (3.1 g, 56 mmol) were then added sequentially. The reaction was stirred at 80°C for 2 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered while hot, and the filtrate was added with ethyl acetate (200 mL) and water (100 mL). The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (100 mL x 3). The organic phases were collected, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and dried. The crude product was purified by column chromatography (PE / EA = 10:1 to 3:1) to afford 14C (2 g, 75% yield) as a yellow solid. MS-ESI: m / z 241.0 [M+H] + .

[0756] Step 3: To a 100 mL three-necked flask, 14C (2 g, 8.4 mmol), DCM (20 mL), and TEA (1.3 g, 12.6 mmol) were added. Under an ice bath, n-valeryl chloride (1.2 g, 10.1 mmol) was slowly added, and the reaction was stirred at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was poured into water (20 mL) and extracted with EA (50 mL × 3). The organic phase was collected, washed with saturated brine (20 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by column chromatography (PE / EA = 3 / 1) to isolate a yellow oily liquid 14D (1.5 g, yield 55.6%). MS-ESI: m / z 323.0 [M+H] + .

[0757] Step 4: To a 250 mL single-necked flask at room temperature, add 14D (1.5 g, 4.7 mmol) and pyridine (50 mL), followed by P2S5 (9 g, 47 mmol). The reaction was stirred at 120°C overnight. The reaction solution was spin-dried and poured into water (100 mL). Extraction was performed with EA (3 × 100 mL). The organic phase was collected, washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by column chromatography (PE / EA = 10 / 1) to obtain 14E as a yellow solid (1 g, yield 66.7%). MS-ESI: m / z 321.0 [M+H] + .

[0758] Step 5: To a 250 mL single-necked flask at room temperature, 14E (2.6 g, 8.1 mmol) and CHCl₃ (50 mL) were added. m-CPBA (3.3 g, 16.2 mmol) was slowly added under ice-cooling, and the reaction was stirred at 25°C overnight. The reaction solution was poured into DCM (200 mL) and washed sequentially with 5% sodium thiosulfate (50 mL), saturated sodium bicarbonate (50 mL), and saturated brine (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by column chromatography (PE:EA = 3:1) to obtain 14F (2 g, 73.5% yield) as a yellow oily liquid. MS-ESI: m / z 337.0 [M+H] + .

[0759] Step 6: 14F (2 g, 5.95 mmol) was added to a 100 mL three-necked flask, and POCl3 (27 g, 178.5 mmol) was slowly added under an ice bath. The internal temperature was maintained at 15°C, and DIEA (2.3 g, 17.85 mmol) was slowly added dropwise. The reaction was stirred at 100°C overnight. LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and then slowly poured into ice water (100 mL). The pH was adjusted to 9 with potassium carbonate solid, and the aqueous phase was extracted with ethyl acetate (100 mL × 3). The organic phase was collected, washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. After filtration and drying, the product was purified by column chromatography (PE / EA = 10:1) to obtain a yellow oily liquid 14G (1.8 g, yield 85.7%). MS-ESI: m / z 355.0 [M+H] + .

[0760] Step 7: At room temperature, 14G (1.8 g, 5.1 mmol) and toluene (20 mL) were added to a 100 mL single-necked bottle. 14H (8.5 g, 51 mmol) and 2-Chlorobenzoic acid (799 mg, 5.1 mmol) were then added sequentially at room temperature. The reaction was stirred at 120°C overnight. LCMS showed that the reaction was complete. The solvent was evaporated under reduced pressure, 100 mL of water was added, and EA was extracted (3×100 mL). The organic phase was collected, washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse phase column chromatography (neutral separation system) to obtain a yellow solid 14I (2 g, yield 80%). MS-ESI: m / z 486.1 [M+H] + .

[0761] Step 8: To a 100 mL single-necked flask at room temperature, 14I (0.5 g, 1.03 mmol) and DMF (20 mL) were added. 14J (377 mg, 2.06 mmol), Pd(PPh3)2Cl2 (75 mg, 0.103 mmol), and Cu2O (442 mg, 3.09 mmol) were then added sequentially at room temperature. The reaction was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 100 mL of water was added to the reaction solution, and extraction was performed with EA (100 mL x 3). The organic phase was collected, washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was separated by column chromatography (PE:EA = 3:1) to afford 14K (0.3 g, 49.5% yield) as a yellow oil. MS-ESI: m / z 589.3 [M+H] + .

[0762] Step 9: To a 100 mL single-necked flask at room temperature, 14K (0.3 g, 0.51 mmol) and THF (20 mL) were added, followed by Pd / C (50 mg) at room temperature. After H2 displacement three times, the reaction was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was filtered through celite, rinsed with THF (3 × 20 mL), and the filtrate was evaporated to dryness under reduced pressure. The crude product was separated by chromatography on a PE / EA = 5 / 1 column to afford 14L (0.2 g, 66.7% yield) as a yellow oily liquid. MS-ESI: m / z 593.3 [M+H] + .

[0763] Step 10: To a 100 mL single-necked bottle at room temperature was added 14L (200 mg, 0.34 mmol), followed by the slow addition of TFA (5 mL) under ice-cooling. The reaction was stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete, and the solvent was evaporated under reduced pressure. The product was purified by reverse phase column chromatography (neutral separation system) to afford 14 as a white solid (77.6 mg, 66.9% yield).

[0764] MS-ESI: m / z 343.2[M+H] + .

[0765] 1 H NMR (400MHz, Methanol-d4) δ7.57(d,J=8.3Hz,1H),7.49–7.42(m,1H),7.18(d,J=7.2Hz,1H),3.23–3.15(m,2H),3.11 –3.02(m,2H),2.72(t,J=6.7Hz,2H),1.97–1.85(m,2H),1.76–1.65(m,2H),1.64–1.46(m,6H),1.02(t,J=7.4Hz,3H).

[0766] Compound 15

[0767] Step 1: Dissolve 15A (5 g, 23.14 mmol, 1 eq) in acetonitrile (100 mL). Add tert-butyl N-hydroxycarbamate 15B (4.01 g, 30.09 mmol, 1.3 eq) at 0°C, followed by slow dropwise addition of DBU (3.52 g, 23.14 mmol, 1 eq). After complete addition, warm the mixture to room temperature and stir for 3 hours. LCMS confirmed the complete reaction of 15A. The mixture was concentrated under reduced pressure, then added with saturated potassium carbonate solution (100 mL). Extraction was performed with DCM (50 mL x 3). The organic phases were combined and backwashed once with saturated brine (50 mL). The organic phases were dried over sodium sulfate, concentrated under reduced pressure, and filtered on silica gel. The mixture was then purified by column chromatography (PE:EA = 5:1) to afford 15C (4.5 g, 72.5% yield) as a white solid. MS-ESI: m / z 291.0 [M+Na] + .

[0768] Step 2: 15C (4.5 g, 16.77 mmol, 1 eq) was dissolved in DMF (50 mL), cooled to 0°C, and NaH (671 mg, 16.77 mmol, 1 eq, 60%) was added all at once. The mixture was stirred at 0°C for 10 minutes, followed by dropwise addition of iodopropane (3.14 g, 18.45 mmol, 1.1 eq). After complete addition, the mixture was allowed to warm to room temperature and react overnight. LCMS confirmed the complete reaction of 15C. The reaction solution was poured into ice water (150 mL) and extracted with EA (100 mL × 3). The organic phases were combined and washed once with 10% aqueous citric acid (100 mL), once with saturated sodium bicarbonate (100 mL), and once with saturated brine (100 mL). The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and filtered through silica gel column chromatography (PE:EA = 5:1) to afford 15D (4.5 g, 86.4% yield) as a yellow solid. MS-ESI: m / z 333.2[M+Na] + .

[0769] Step 3: Dissolve 15D (4.5 g, 14.52 mmol, 1 eq) in EA (50 mL). Add 4N dioxane hydrochloride (50 mL) under ice-water bath and stir at room temperature overnight. LCMS confirmed the complete reaction of 15D. Filter directly, rinse the filter cake with EA (100 mL), transfer the filter cake to a bottle, and dry in vacuo to obtain a yellow solid 15E (2.5 g, 83.3% yield). MS-ESI: m / z 211.2 [M+H] + .

[0770] Step 2 Step 4 1F (3 g, 9.45 mmol, 1 eq) and 15E (2.5 g, 10.39 mmol, 1.1 eq) were added to a mixed solvent of DMA (30 mL) and DCM (30 mL). The atmosphere was replaced with argon twice, and then EDCI (7.24 g, 37.79 mmol, 4 eq) was added all at once. After the addition was complete, the atmosphere was replaced with argon twice more and the mixture was allowed to react at room temperature overnight. LCMS confirmed the complete reaction of the starting materials. The reaction solution was poured into 100 mL of water and extracted with DCM (100 mL x 3). The organic phases were combined and backwashed with saturated brine (100 mL). The organic phase was dried over sodium sulfate, spin-dried, and purified by silica gel column chromatography to obtain 15F (1.9 g, 42.5% yield) as a yellow solid. MS-ESI: m / z 473.1 [M+H] + .

[0771] Step 5: 15F (1.9 g, 4.01 mmol, 1 eq) was added to a mixed solvent of dioxane (50 mL) and water (5 mL). 10D (936 mg, 4.01 mmol, 1 eq) and anhydrous potassium phosphate (2.56 g, 12.04 mmol, 3 eq) were then added. The atmosphere was purged with argon twice, and then Pd(PPh3)4 (200 mg) was added. The atmosphere was purged with argon twice, and the temperature was raised to 85°C for 3 hours. LCMS confirmed the complete reaction of the starting material. The reaction solution was poured into water (150 mL) and extracted with EA (150 mL x 3). The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to afford 15G (900 mg, 45% yield) as a dark red solid. MS-ESI: m / z 500.2 [M+H] + .

[0772] Step 6: 15G (700 mg, 1.4 mmol, 1 eq) was added to methanol (20 mL). Sodium borohydride (32 mg, 0.84 mmol, 0.6 eq) was slowly added portionwise at room temperature. The reaction was maintained at room temperature for 2 hours after the addition was complete. The reaction solution was quenched with saturated aqueous ammonium chloride (50 mL) and extracted with DCM (50 mL x 3). The organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 20:1) to afford 15H (200 mg, 22.2% yield) as a light yellow solid. MS-ESI: m / z 502.2 [M+H] + .

[0773] Step 7: 15H (200 mg, 0.31 mmol, 1 eq) was dissolved in methanol (10 mL), and Raney Ni (0.1 g) was added. The atmosphere was replaced with hydrogen twice, and the mixture was stirred at room temperature for 3 hours. LCMS confirmed the completion of the reaction. The mixture was filtered through a pad of Celite, dried, and slurried with petroleum ether (10 mL) and methanol (2 mL). The mixture was filtered to afford 15 (28 mg, 14.9% yield) as a yellow solid.

[0774] MS-ESI: m / z 472.2[M+H] + .

[0775] 1 H NMR (400MHz, DMSO-d6) δ8.80(d,J=2.2Hz,1H),8.52(d,J=2.0Hz,1H),8.02(s,1H),7.41(d ,J=8.2Hz,1H),7.36(d,J=1.9Hz,1H),7.29(dd,J=8.0,2.0Hz,1H),7.09(s,1H),6.89(d,J= 8.1Hz,2H),6.83(s,2H),6.41(d,J=8.2Hz,2H),5.37(t,J=5.8Hz,1H),5.16(s,2H),4.69– 4.52(m,4H),3.63(t,J=7.0Hz,2H),2.77(s,2H),1.71–1.58(m,2H),0.90(t,J=7.4Hz,3H).

[0776] Compound 16

[0777] Step 1: To a 100 mL single-necked flask at room temperature, 16A (0.5 g, 1.03 mmol) and DMF (20 mL) were added. 16B (348 mg, 2.06 mmol), Pd(PPh3)2Cl2 (75 mg, 0.103 mmol), and Cu2O (442 mg, 3.09 mmol) were then added sequentially at room temperature. The mixture was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 100 mL of water was added to the reaction solution, and the mixture was extracted with EA (3 × 100 mL). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to afford 16C (0.3 g, yield: 50.8%) as a yellow oil. MS-ESI: m / z 575.3 [M+H] + .

[0778] Step 2: To a 100 mL single-necked flask at room temperature, 16C (0.3 g, 0.52 mmol) and THF (20 mL) were added. Pd / C (50 mg) was then added at room temperature. After H2 displacement three times, the mixture was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was filtered through celite and rinsed with THF (3 × 20 mL). The filtrate was evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (PE / EA = 5 / 1) to afford 16D (0.2 g, yield: 66.2%) as a yellow oil. MS-ESI: m / z 579.4 [M+H] + .

[0779] Step 3: 16D (200 mg, 0.35 mmol) was added to a 100 mL single-necked bottle at room temperature. TFA (5 mL) was slowly added under ice-cooling and stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete. The solvent was evaporated under reduced pressure and purified by reverse phase column chromatography (neutral separation system) to give 16 (59.4 mg, yield: 52.6%) as a white solid. MS-ESI: m / z 329.2 [M+H] + .

[0780] 1 H NMR (400MHz, Methanol-d4) δ7.58(dd,J=8.4,1.3Hz,1H),7.47(dd,J=8.4,7.2Hz,1H),7.22(dd,J=7.3,1.3Hz,1H),3.24–3.17 (m,2H),3.17–3.10(m,2H),2.88–2.81(m,2H),1.97–1.87(m,2H),1.83–1.67(m,4H),1.58–1.46(m,2H),1.02(t,J=7.4Hz,3H).

[0781] Compound 17

[0782] Step 1: To a 100 mL single-necked flask at room temperature, 17A (0.5 g, 1.03 mmol) and DMF (20 mL) were added. 17B (203 mg, 2.06 mmol), Pd(PPh3)2Cl2 (75 mg, 0.103 mmol), and Cu2O (442 mg, 3.09 mmol) were then added sequentially at room temperature. The mixture was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 100 mL of water was added to the reaction solution, and the mixture was extracted with EA (3 × 100 mL). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to afford 17C (0.3 g, yield: 48.4%) as a yellow oil. MS-ESI: m / z 603.4 [M+H]+ .

[0783] Step 2: To a 100 mL single-necked bottle at room temperature, 17C (0.3 g, 0.50 mmol) and THF (20 mL) were added. Pd / C (50 mg) was then added at room temperature. After H2 displacement three times, the mixture was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was filtered through celite and washed with THF (3 × 20 mL). The filtrate was evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (PE / EA = 5 / 1) to afford 17D (0.2 g, yield: 66.2%) as a yellow oil. MS-ESI: m / z 607.4 [M+H] + .

[0784] Step 3: 17D (200 mg, 0.33 mmol) was added to a 100 mL single-necked bottle at room temperature. TFA (5 mL) was slowly added under ice-cooling and stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete. The mixture was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (neutral separation system) to give 17 as a white solid (51.4 mg, yield: 43.7%). MS-ESI: m / z 357.2 [M+H] + .

[0785] 1 H NMR (400MHz, Methanol-d4) δ7.58(dd,J=8.3,1.3Hz,1H),7.46(dd,J=8.4,7.2Hz,1H),7.20(dd,J=7.3,1.3Hz,1H),3.25–3.18 (m,2H),3.13–3.07(m,2H),2.78–2.71(m,2H),1.98–1.87(m,2H),1.78–1.67(m,2H),1.62–1.39(m,8H),1.02(t,J=7.4Hz,3H).

[0786] Compound 18

[0787] Step 1: To a 250 mL three-necked flask at room temperature, 18A (10 g, 44.8 mmol) and propionic acid (200 mL) were added. The reaction solution was heated to 125°C and HNO3 (8.6 g, 136.6 mmol) was added dropwise, maintaining the internal temperature at 120-130°C. The mixture was stirred at 125°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was cooled to room temperature and slowly poured into ethanol (200 mL). The filter cake was rinsed sequentially with ethanol (100 mL), water (100 mL), and ethanol (100 mL). The filter cake was collected and dried to obtain 18B (7 g, yield: 58%) as a yellow solid. MS-ESI: m / z 269.0 [M+H] +.

[0788] Step 2: At room temperature, 18B (7 g, 26.1 mmol) was added to a 250 mL single-necked flask. EtOH (100 mL) and H₂O (20 mL) were added to dissolve the mixture. NH₄Cl (4.23 g, 78.3 mmol) and Fe powder (7.31 g, 130.5 mmol) were then added sequentially. The mixture was stirred at 80°C for 2 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered while hot, and the filtrate was added with ethyl acetate (200 mL) and water (100 mL). The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (3 × 100 mL). The organic phase was collected, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and dried. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to obtain 18C (4.5 g, yield: 72%) as a yellow solid. MS-ESI: m / z 239.0 [M+H] + .

[0789] Step 3: To a 250 mL three-necked flask, 18C (2.2 g, 9.2 mmol) and DCM (100 mL) were added, followed by TEA (1.4 g, 13.8 mmol). 18D (1.35 g, 11.04 mmol) was slowly added under ice-cooling and stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was poured into water (20 mL) and extracted with EA (3×50 mL). The organic phase was collected, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA=3 / 1) to obtain 18E (1.6 g, yield: 53.5%) as a yellow solid. MS-ESI: m / z 325.0 [M+H] + .

[0790] Step 4: To a 250 mL single-necked flask at room temperature, 18E (2.8 g, 8.6 mmol) and pyridine (120 mL) were added, followed by P2S5 (16.4 g, 86 mmol). The mixture was stirred at 120°C overnight. LCMS indicated the reaction was complete. The reaction mixture was dried to remove the solvent and poured into water (100 mL). The mixture was extracted with EA (3 × 100 mL). The organic phase was collected, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by chromatography (PE / EA = 10 / 1) to obtain 18F as a yellow solid (0.5 g, yield: 18.5%). MS-ESI: m / z 323.0 [M+H] + .

[0791] Step 5: At room temperature, 18F (0.5 g, 1.55 mmol) and CHCl (10 mL) were added to a 250 mL single-necked flask. m-CPBA (0.53 g, 3.1 mmol) was slowly added under ice-cooling and stirred at 25°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was poured into DCM (50 mL) and washed sequentially with 5% sodium thiosulfate (50 mL), saturated sodium bicarbonate (50 mL), and saturated brine (50 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by chromatography on a PE / EA = 3 / 1 column to obtain 18G (0.43 g, yield: 82.6%) as a yellow oily liquid. MS-ESI: m / z 339.0 [M+H] + .

[0792] Step 6: 18G (0.43 g, 1.27 mmol) was added to a 50 mL single-necked bottle. POCl3 (5.1 g, 33.1 mmol) was slowly added under an ice bath. Maintaining the internal temperature at 15°C, DIPEA (0.43 g, 3.31 mmol) was slowly added dropwise and stirred at 100°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and then slowly poured into ice water (50 mL). The pH was adjusted to 9 with solid potassium carbonate, and the aqueous phase was extracted with ethyl acetate (3×30 mL). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography (PE / EA=10:1) to obtain a yellow oil 18H (0.32 g, yield: 71.1%). MS-ESI: m / z 357.0 [M+H] + .

[0793] Step 7: At room temperature, 18H (0.32 g, 0.9 mmol) and toluene (5 mL) were added to a 50 mL single-necked bottle. 2,4-dimethoxybenzylamine (1.5 g, 9 mmol) and 2-chlorobenzoic acid (0.14 g, 0.9 mmol) were added in sequence at room temperature. The mixture was stirred at 120°C overnight. LCMS showed that the reaction was complete. The solvent was evaporated under reduced pressure, 10 mL of water was added, and EA was extracted (3×30 mL). The organic phase was collected, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by reverse phase column chromatography (neutral separation system) to obtain a yellow solid 18I (0.14 g, yield: 32.5%). MS-ESI: m / z 488.0 [M+H] + .

[0794] Step 8: To a 50 mL single-necked flask at room temperature, 18I (0.14 g, 0.28 mmol) and DMF (5 mL) were added. 18J (0.102 g, 0.56 mmol), Pd(PPh3)2Cl2 (0.02 g, 0.028 mmol), and Cu2O (0.12 g, 0.84 mmol) were then added sequentially at room temperature. The mixture was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 20 mL of water was added to the reaction solution, and the mixture was extracted with EA (3 × 30 mL). The organic phase was collected, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to obtain 18K (0.12 g, yield: 70.5%) as a yellow oily liquid. MS-ESI: m / z 591.2 [M+H] + .

[0795] Step 9: To a 50 mL single-necked flask at room temperature, 18K (0.12 g, 0.2 mmol) and THF (10 mL) were added, followed by Pd / C (50 mg) at room temperature. After H2 replacement three times, the mixture was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was filtered through celite, rinsed with THF (3 × 20 mL), and the filtrate was evaporated to dryness under reduced pressure. The crude product was separated by chromatography on a PE / EA = 5 / 1 column to afford 18L (0.1 g, yield: 83.3%) as a yellow oily liquid. MS-ESI: m / z 595.3 [M+H] + .

[0796] Step 10: 18L (0.1 g, 0.17 mmol) was added to a 50 mL single-necked bottle at room temperature. TFA (3 mL) was slowly added under ice-cooling and stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete. The solvent was evaporated under reduced pressure and purified by reverse phase column chromatography (neutral separation system) to give 18 as a white solid (18 mg, yield: 31.1%). MS-ESI: m / z 345.2 [M+H] + .

[0797] 1 H NMR (400MHz, Methanol-d4) δ7.59(dd,J=8.3,1.3Hz,1H),7.49(dd,J=8.4,7.2Hz,1H),7.23(dd,J=7.2,1.3Hz,1H),4.98(s,2H) ,3.77(q,J=7.0Hz,2H),3.19–3.11(m,2H),2.78(t,J=6.9Hz,2H),1.82–1.70(m,2H),1.69–1.55(m,4H),1.33(t,J=7.0Hz,3H).

[0798] Compound 19

[0799] Step 1: To a 250 mL three-necked flask, 19A (2 g, 8.4 mmol) and DCM (100 mL) were added, followed by TEA (1.27 g, 12.6 mmol). Under ice-cooling, 19B (1.21 g, 10.08 mmol) was slowly added and stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was poured into water (20 mL) and extracted with EA (3 × 50 mL). The organic phase was collected, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to obtain 19C (1.4 g, yield: 51.8%) as a yellow solid. MS-ESI: m / z 323.0 [M+H] + .

[0800] Step 2: To a 100 mL single-necked flask at room temperature, 19C (1.4 g, 4.3 mmol) and pyridine (60 mL) were added, followed by P2O5 (6.1 g, 43 mmol). The mixture was stirred at 120°C overnight. LCMS indicated the reaction was complete. The reaction solution was dried to remove the solvent and poured into water (100 mL). The mixture was extracted with EA (3 × 50 mL). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by chromatography (PE / EA = 10 / 1) to give 19D (0.5 g, yield: 37.8%) as a yellow solid. MS-ESI: m / z 305.1 [M+H] + .

[0801] Step 3: At room temperature, 19D (0.5 g, 1.6 mmol) and CHCl3 (10 mL) were added to a 50 mL single-necked bottle. m-CPBA (0.65 g, 3.2 mmol) was slowly added under ice bath and stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was poured into DCM (50 mL) and washed with 5% sodium thiosulfate (50 mL), saturated sodium bicarbonate (50 mL), and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to obtain 19E (0.4 g, yield: 76%) as a yellow oily liquid. MS-ESI: m / z 321.0 [M+H] + .

[0802] Step 4: 19E (0.4 g, 1.25 mmol) was added to a 250 mL three-necked flask. POCl3 (5.75 g, 37.5 mmol) was slowly added under an ice bath. Maintaining the internal temperature at 15°C, DIPEA (0.48 g, 3.75 mmol) was slowly added dropwise. The mixture was stirred at 100°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and then slowly poured into ice water (50 mL). The pH was adjusted to 9 with solid potassium carbonate. The aqueous phase was extracted with ethyl acetate (3×30 mL). The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography (PE / EA = 10:1) to obtain 19F (0.35 g, yield: 83.3%) as a yellow oily liquid. MS-ESI: m / z 339.0 [M+H] + .

[0803] Step 5: At room temperature, 19F (0.35 g, 1.04 mmol) and toluene (5 mL) were added to a 50 mL single-necked bottle. 2,4-dimethoxybenzylamine (1.74 g, 10.4 mmol) and 2-chlorobenzoic acid (0.16 g, 1.04 mmol) were added in sequence at room temperature. The mixture was stirred at 120°C overnight. LCMS showed that the reaction was complete. The solvent was evaporated under reduced pressure, 10 mL of water was added, and EA was extracted (3×30 mL). The organic phase was collected, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by reverse phase column chromatography (neutral separation system) to obtain a yellow solid 19G (0.2 g, yield 41.1%). MS-ESI: m / z 470.0 [M+H] + .

[0804] Step 6: To a 50 mL single-necked flask at room temperature, 19G (0.2 g, 0.43 mmol) and DMF (5 mL) were added. 19H (0.16 g, 0.86 mmol), Pd(PPh3)2Cl2 (0.03 g, 0.043 mmol), and Cu2O (0.18 g, 1.29 mmol) were then added sequentially at room temperature. The mixture was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 20 mL of water was added to the reaction solution, and the mixture was extracted with EA (3 × 30 mL). The organic phase was collected, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to obtain 19I (0.12 g, 49.4% yield) as a yellow oily liquid. MS-ESI: m / z 573.4 [M+H] + .

[0805] Step 7: To a 50 mL single-necked bottle at room temperature, 19I (0.12 g, 0.21 mmol) and THF (10 mL) were added, followed by the addition of Pd / C (50 mg) at room temperature. After H2 replacement three times, the mixture was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was filtered through celite, rinsed with THF (3 × 20 mL), and the filtrate was evaporated to dryness under reduced pressure. The crude product was separated by chromatography on a PE / EA = 5 / 1 column to afford 19J (96 mg, 80% yield) as a yellow oil. MS-ESI: m / z 577.4 [M+H] + .

[0806] Step 8: 19J (0.096 g, 0.17 mmol) was added to a 50 mL single-necked bottle at room temperature. TFA (3 mL) was slowly added under ice-cooling and stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete. The solvent was evaporated under reduced pressure and purified by reverse phase column chromatography (neutral separation system) to give 19 as a white solid (22 mg, yield: 40.5%). MS-ESI: m / z 327.3 [M+H] + .

[0807] 1 H NMR (400MHz, Methanol-d4) δ7.54 (dd, J=8.5, 1.2Hz, 1H), 7.45 (dd, J=8.5, 7.1Hz, 1H),7.16(d,J=7.1Hz,1H),3.25–3.17(m,2H),3.08(t,J=7.4Hz,2H),2.69(t,J=7.0Hz ,2H),1.99–1.88(m,2H),1.79–1.68(m,2H),1.64–1.44(m,6H),1.02(t,J=7.4Hz,3H).

[0808] Compound 20

[0809] Step 1: To a 50 mL single-necked flask at room temperature, 19G (0.28 g, 0.6 mmol) and DMF (6 mL) were added. 20A (0.254 g, 1.5 mmol), Pd(PPh3)2Cl2 (0.042 g, 0.06 mmol), and Cu2O (0.257 g, 1.8 mmol) were then added sequentially at room temperature. The mixture was stirred at 110°C under N2 protection overnight. LCMS indicated the reaction was complete. 20 mL of water was added to the reaction solution, and the mixture was extracted with EA (3 × 30 mL). The organic phase was collected, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (PE / EA = 3 / 1) to afford 20B (140 mg, yield: 42%) as a yellow oil. MS-ESI: m / z 559.2 [M+H]+ .

[0810] Step 2: To a 50 mL single-necked flask at room temperature, 20B (0.14 g, 0.25 mmol) and THF (10 mL) were added. Pd / C (50 mg) was then added at room temperature. After H2 replacement three times, the mixture was stirred at 25°C overnight. LCMS indicated complete reaction. The reaction mixture was filtered through celite and rinsed with THF (3 x 20 mL). The filtrate was then dried under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5 / 1) to afford 20C (120 mg, 85% yield) as a yellow oil. MS-ESI: m / z 563.4 [M+H] + .

[0811] Step 3: 20C (0.12 g, 0.21 mmol) was added to a 50 mL single-necked bottle at room temperature. TFA (4 mL) was slowly added under ice-cooling and stirred at 80°C for 15 minutes. LCMS indicated the reaction was complete. The reaction solution was evaporated to dryness under reduced pressure and separated by reverse phase preparation (TFA separation method) to obtain 20 (4.2 mg, yield: 6.3%) as a white solid. MS-ESI: m / z 313.2 [M+H] + .

[0812] 1 H NMR (400MHz, Methanol-d4) δ7.76–7.66(m,2H),7.47(dd,J=6.9,1.5Hz,1H),3.36–3.32(m,2H),3.15(t,J=7. 5Hz,2H),3.04–2.95(m,2H),2.02–1.92(m,2H),1.90–1.77(m,4H),1.61–1.49(m,2H),1.03(t,J=7.4Hz,3H).

[0813] Compound 21

[0814] Step 1: Dissolve 21A (3.3 g, 20.60 mmol, 1 eq) and 21B (7 g, 22.66 mmol, 1.1 eq) in a mixture of acetonitrile (30 mL) and water (60 mL). Stir at room temperature overnight under argon. LCMS analysis indicates that the remaining starting material has not reacted completely. The reaction mixture is directly applied to a reverse-phase column (mobile phase: water and acetonitrile). The product is filtered under 50% acetonitrile and lyophilized to obtain 21C as a white solid (2 g, yield: 27.5%). MS-ESI: m / z 354.2 [M+H] + .

[0815] Step 2: 10 (180 mg, 0.366 mmol, 1 eq) and 21C (130 mg, 0.366 mmol, 1 eq) were added to a mixed solvent of DCM (5 mL) and DMA (5 mL). The atmosphere was replaced with argon twice. EDCI (281 mg, 1.46 mmol, 4 eq) was added in one portion at room temperature. The atmosphere was replaced with argon twice and stirred at room temperature overnight. LCMS confirmed the complete reaction of the starting material. The mixture was directly sent to the preparation and lyophilized to obtain 21 (30 mg, yield: 11.5%) as a white solid. MS-ESI: m / z 791.4 [M+H] + .

[0816] 1 H NMR(400MHz,Chloroform-d)δ8.72(d,J=2.3Hz,1H),8.48(d,J=1.9Hz,1H),8.00–7.95(m,1H),7. 58(d,J=8.4Hz,2H),7.38(s,1H),7.33–7.15(m,4H),6.91(s,1H),6.84(s,1H),4.63–4.51(m,4H), 4.32(q,J=7.1Hz,1H),4.19(q,J=7.1Hz,1H),3.34(t,J=7.0Hz,2H),3.30–3.10(m,3H),2.77(s,2H ),2.15–1.98(m,3H),1.59–1.38(m,6H),1.29(d,J=7.1Hz,3H),1.24–1.06(m,6H),0.75(brs,3H).

[0817] Compound 22

[0818] Step 1: 11 (130 mg, 0.226 mmol, 1 eq) and 21C (80 mg, 0.226 mmol, 1 eq) were added to a mixed solvent of DCM (5 mL) and DMA (5 mL). The atmosphere was replaced with argon twice. EDCI (174 mg, 0.906 mmol, 4 eq) was added in one portion at room temperature. The atmosphere was replaced with argon twice and stirred at room temperature overnight. LCMS confirmed the complete reaction of the starting materials. The mixture was directly sent to the preparation and lyophilized to obtain 22 (13 mg, yield: 6.6%) as a white solid. MS-ESI: m / z 873.4 [M+H] + .

[0819] 1H NMR(400MHz, DMSO-d6)δ9.87(s,1H),8.06(d,J=7.1Hz,1H),7.99(d,J=7.0Hz,1H),7.73(d,J=8.1Hz,2H),7.66–7.55(m,4H),7 .42–7.34(m,1H),7.34–7.29(m,1H),7.29–7.17(m,3H),6.99(s,2H),6.92–6.80(m,3H),5.85–5.72(m,1H),4.97(dd,J=33.8,3 .4Hz,1H),4.58(s,2H),4.44–4.19(m,3H),3.67–3.51(m,3H),3.42–3.37(m,2H),3.24–3.17(m,2H),3.03–2.91(m,2H),2.84–2 .75(m,2H),2.27–2.02(m,5H),2.01–1.74(m,2H),1.59–1.41(m,7H),1.30(d,J=7.1Hz,3H),1.24–1.15(m,5H),0.77(brs,3H).

[0820] Compound 23

[0821] Step 1: 12 (130 mg, 0.251 mmol, 1 eq) and 21C (89 mg, 0.251 mmol, 1 eq) were added to a mixed solvent of DCM (5 mL) and DMA (5 mL). The atmosphere was replaced with argon twice. EDCI (193 mg, 1.01 mmol, 4 eq) was added in one portion at room temperature. The atmosphere was replaced with argon twice and stirred at room temperature overnight. LCMS analysis indicated that the reaction was complete. The mixture was directly transferred to the preparative preparation and lyophilized to afford 23 (15 mg, 7.3% yield) as a white solid.

[0822] MS-ESI: m / z 816.4[M+H] + .

[0823] 1H NMR(400MHz, DMSO-d6)δ9.87(s,1H),8.14–7.94(m,4H),7.75(d,J=8.0Hz,1H),7.61(d,J=8.6Hz,2H),7 .45–7.35(m,1H),7.35–7.29(m,2H),7.29–7.19(m,2H),6.99(s,2H),6.93–6.82(m,3H),5.46(s,2H),4. 58(s,2H),4.37(p,J=6.9Hz,1H),4.25(p,J=7.1Hz,1H),3.39–3.36(m,2H),3.21(d,J=7.8Hz,2H),2.80( s,2H),2.10(t,J=7.4Hz,2H),1.58–1.42(m,6H),1.30(d,J=7.1Hz,3H),1.25–1.13(m,5H),0.77(s,3H).

[0824] Compound 24

[0825] Step 1: 15 (100 mg, 0.212 mmol, 1 eq) was prepared as a hydrochloride salt and then added to a mixture of DCM (5 mL) and DMA (5 mL) with 21C (75 mg, 0.212 mmol, 1 eq). The mixture was purged with argon twice. EDCI (163 mg, 0.848 mmol, 4 eq) was added in one portion at room temperature. The atmosphere was purged with argon twice and stirred at room temperature overnight. The reaction was complete by LCMS. The mixture was directly transferred to the preparation and lyophilized to afford 24 (14 mg, 8.2% yield) as a white solid.

[0826] MS-ESI: m / z 807.4[M+H] + .

[0827] 1H NMR (400MHz, DMSO-d6) δ9.90(s,1H),8.79(d,J=2.3Hz,1H),8.51(d,J=1.9Hz,1H),8.08(d,J=7.2Hz,1H),8.04–7.97(m,2H),7.53(d ,J=8.4Hz,2H),7.39(d,J=8.2Hz,1H),7.35(d,J=1.9Hz,1H),7.28(dd,J=8.0,2.0Hz,1H),7.21(d,J=8.3Hz,2H),7.07(s,1H),6.99(s ,2H),6.87(s,2H),5.40(t,J=5.8Hz,1H),4.75(s,2H),4.61(d,J=5.5Hz,2H),4.39–4.29(m,1H),4.27–4.18(m,1H),3.64(t,J=7.0Hz ,2H),2.78(s,2H),2.08(t,J=7.5Hz,2H),1.71–1.59(m,2H),1.51–1.39(m,4H),1.27(d,J=7.1Hz,3H),1.21–1.11(m,5H),0.90(t,J= 7.4Hz,3H).

[0828] Compound 25

[0829] Step 1: To a 100 mL single-necked vial at room temperature was added 14 (65 mg, 0.19 mmol). DMF (10 mL) was added to dissolve the mixture, followed by the addition of DIEA (74 mg, 0.57 mmol) and 15A (140 mg, 0.19 mmol). The reaction was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction solution was directly purified by reverse-phase column chromatography (neutral separation system) to afford 25 (11 mg, 6.1% yield) as a white solid.

[0830] MS-ESI: m / z 941.4[M+H] + .

[0831] 1H NMR (400MHz, Methanol-d4) δ7.61–7.53(m,3H),7.49–7.43(m,1H),7.30(d,J=8.2Hz,2H),7.21(d,J= 7.1Hz,1H),6.77(s,2H),5.02(s,2H),4.49(dd,J=9.0,5.1Hz,1H),4.15(d,J=7.4Hz,1H),3.46(t,J= 7.1Hz,2H),3.24–3.04(m,8H),2.26(t,J=7.4Hz,2H),2.11–2.00(m,1H),1.97–1.82(m,3H),1.80–1. 67(m,3H),1.67–1.44(m,12H),1.34–1.24(m,2H),1.01(t,J=7.4Hz,3H),0.95(dd,J=6.8,3.2Hz,6H).

[0832] Compound 26

[0833] Step 1: To a 100 mL three-necked flask, add oxalyl chloride (129.5 mg, 1.02 mmol) and DCM (5 mL) at room temperature. The reaction mixture was cooled to -78°C under N2 protection, and DMSO (159.4 mg, 2.04 mmol, in 5 mL DCM) was slowly added. The reaction mixture was stirred at -78°C for 15 minutes, and then 26A (300 mg, 0.51 mmol, in 5 mL DCM) was slowly added. After stirring for an additional 15 minutes, Et3N (309.6 mg, 3.06 mmol, in 5 mL DCM) was slowly added. The reaction mixture was stirred at -78°C for 15 minutes, then slowly returned to 25°C over 30 minutes. TLC indicated complete reaction of the starting material with the formation of new spots. The reaction was quenched with saturated NH4Cl, the aqueous phase was extracted three times with DCM, and the organic phases were collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 26B (250 mg, crude product not included in yield) as a yellow oil.

[0834] 1 H NMR (400MHz, Chloroform-d) δ9.73 (s, 1H), 4.16 (d, J = 0.8Hz, 2H), 3.78–3.57 (m, 38H), 2.50 (t, J = 6.4Hz, 2H), 1.47–1.41 (m, 9H).

[0835] Step 2: At room temperature, 26B (250 mg, 0.43 mmol) was added to a 50 mL single-necked flask. DMF (10 mL) was added to dissolve the mixture, followed by the addition of 14 (100 mg, 0.29 mmol) and AcOH (0.1 mL). The reaction was stirred at 25°C for 2 hours, followed by the addition of Na(OAc)3BH (120 mg, 0.87 mmol). The reaction was stirred overnight. TLC indicated the reaction was complete. The reaction solution was evaporated to dryness and purified by column chromatography (EA / MeOH = 10:1 to 5:1) to afford 26C (200 mg, 75.7% yield) as a yellow oil. MS-ESI: m / z 911.5 [M+H] + .

[0836] Step 3: At room temperature, 26C (200 mg, 0.22 mmol) was added to a 50 mL single-necked bottle. DCM (10 mL) was added to dissolve the mixture, and TFA (2 mL) was slowly added in an ice bath. The reaction was stirred at 25°C for 2 hours. LCMS indicated the reaction was complete. The solvent was evaporated under reduced pressure and purified by reverse phase column chromatography (TFA / ACN system) to afford 26D as a colorless oil (100 mg, yield: 54.3%). MS-ESI: m / z 855.5 [M+H] + .

[0837] Step 4: 2,3,5,6-tetrafluorophenol (50 mg, 0.3 mmol) was added to a 50 mL single-necked flask at 25°C. DMF (10 mL) was added to dissolve the mixture, followed by EDCI (57.5 mg, 0.3 mmol). The reaction mixture was stirred for 5 minutes, followed by the addition of 26D (100 mg, 0.12 mmol). The reaction was stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was directly purified by reverse phase column chromatography (TFA / MeCN separation system) to afford 26 (25 mg, 20.8%) as an off-white oil.

[0838] MS-ESI: m / z 1003.5[M+H] + .

[0839] 1H NMR(400MHz, Methanol-d4)δ7.76–7.67(m,2H),7.54–7.48(m,1H),7.47–7.3 7(m,1H),3.85(t,J=5.9Hz,2H),3.82–3.75(m,2H),3.70–3.56(m,36H),3.29– 3.19(m,4H),3.16–3.07(m,2H),2.98–2.92(m,2H),1.96(p,J=7.7Hz,2H),1. 89–1.74(m,4H),1.71–1.59(m,2H),1.59–1.48(m,2H),1.03(t,J=7.4Hz,3H).

[0840] Compound 27

[0841] Step 1: To a 25 mL single-necked vial at room temperature was added 16 (42 mg, 0.13 mmol). Dissolved in DMF (10 mL) was then added DIEA (50 mg, 0.39 mmol) and 25A (96 mg, 0.13 mmol). The reaction was stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was directly purified by reverse-phase column chromatography (neutral reverse-phase separation system) to afford 27 (7.7 mg, 6.4%) as a white solid.

[0842] MS-ESI: m / z 927.5[M+H] + .

[0843] 1 H NMR (400MHz, Methanol-d4) δ7.58(d,J=8.3Hz,1H),7.54(d,J=8.2Hz,2H),7.47(t,J=7.8Hz,1H),7.26(d ,J=8.2Hz,2H),7.21(d,J=7.2Hz,1H),6.77(s,2H),5.00(s,2H),4.51(dd,J=9.1,5.1Hz,1H),4.16(d,J=7 .4Hz,1H),3.46(t,J=7.2Hz,2H),3.25–3.16(m,5H),3.16–3.05(m,3H),2.27(t,J=7.4Hz,2H),2.13–2.0 2(m,1H),1.96–1.84(m,3H),1.81–1.68(m,5H),1.68–1.43(m,9H),1.36–1.21(m,3H),1.05–0.93(m,9H).

[0844] Compound 28

[0845] Step 1: To a 25 mL single-necked vial at room temperature was added 17 (50 mg, 0.14 mmol). Dissolved in DMF (10 mL) was then added DIEA (54 mg, 0.42 mmol) and 25A (103 mg, 0.13 mmol). The reaction was stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was directly purified by reverse-phase column chromatography (neutral reverse-phase separation system) to afford 28 (9.2 mg, 6.9%) as a white solid.

[0846] MS-ESI: m / z 955.5[M+H] + .

[0847] 1 H NMR (400MHz, Methanol-d4) δ7.62–7.51(m,3H),7.47(t,J=7.7Hz,1H),7.29(d,J=8.2Hz, 2H),7.21(d,J=7.3Hz,1H),6.78(s,2H),5.01(s,2H),4.58(s,1H),4.53–4.44(m,1H),4. 15(d,J=7.5Hz,1H),3.50–3.44(m,2H),3.25–3.17(m,3H),3.17–3.05(m,6H),2.26(t,J= 7.5Hz,2H),2.13–2.00(m,1H),2.00–1.81(m,3H),1.81–1.67(m,3H),1.67–1.36(m,15H), 1.36–1.23(m,3H),1.05–0.89(m,9H).

[0848] Compound 29

[0849] Step 1: To a 50 mL single-necked vial at room temperature was added 20 (34 mg, 0.063 mmol). DMF (6 mL) was added to dissolve the mixture, followed by the addition of DIEA (20 mg, 0.157 mmol) and 25A (46 mg, 0.063 mmol). The reaction was stirred at 25°C overnight. LCMS indicated the reaction was complete. The reaction mixture was directly subjected to reverse phase purification (NH4HCO3 system) to afford 29 (27.1 mg, 27% yield) as a white solid.

[0850] MS-ESI: m / z 911.4[M+H] + .

[0851] 1H NMR (400MHz, Methanol-d4) δ7.59–7.51(m,3H),7.49–7.43(m,1H),7.27(d,J=8.2Hz,2H),7.16( d,J=7.0Hz,1H),6.77(s,2H),5.00(s,2H),4.51(dd,J=8.9,4.9Hz,1H),4.16(d,J=7.5Hz,1H),3. 49–3.44(m,2H),3.26–3.15(m,5H),3.15–3.03(m,3H),2.27(t,J=7.4Hz,2H),2.13–2.02(m,1H), 1.97–1.84(m,3H),1.80–1.54(m,11H),1.54–1.44(m,2H),1.35–1.27(m,2H),1.03–0.94(m,9H).

[0852] Compound 32

[0853] Step 1: Compound 15F (1.0 g, 2.11 mmol, 1 eq) was added to a mixed solvent of 1,4-dioxane (20 mL) and water (2 mL). Compound 32A (556 mg, 2.11 mmol, 1 eq) and anhydrous potassium phosphate (1.35 g, 6.34 mmol, 3 eq) were added. After argon replacement twice, Pd(PPh3)4 (100 mg) was added, and the atmosphere was replaced with argon twice. The temperature was raised to 90°C and the reaction was allowed to react for 2 hours. LCMS showed that the starting material was completely reacted. The reaction solution was poured into 100 mL of water and extracted three times with ethyl acetate (100 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by column chromatography (DCM:MeOH = 20:1) to obtain 32B (400 mg, 36% yield) as a yellow solid. MS-ESI: m / z 530.2 [M+H] + .

[0854] Step 2: Compound 32B (400 mg, 0.756 mmol, 1 eq) was dissolved in methanol (10 mL) and Raney nickel (0.1 g) was added. The mixture was replaced with hydrogen twice and stirred at room temperature for 3 hours. LCMS showed a clear product peak. The product was filtered through celite and the filtrate was directly used in the next reaction. MS-ESI: m / z 500.2 [M+H] + .

[0855] Step 3: The reaction solution obtained in the previous step was supplemented with THF (10 mL) and water (10 mL), and lithium hydroxide monohydrate (91 mg, 2.27 mmol, 3 eq) was added. The mixture was stirred at room temperature for 3 hours. LCMS confirmed that the reaction was complete. After concentration under reduced pressure, the pH was adjusted to 5-6 with 1N hydrochloric acid. Filter the mixture, and the resulting yellow filter cake was purified by HPLC to obtain 32 as a white solid (21 mg, 5.7% yield over two steps). MS-ESI: m / z 486.2 [M+H] + .

[0856] 1 H NMR (400MHz, DMSO) δ12.87–11.32(m,1H),9.87(s,1H),9.26(s,1H),9.20(d,J=2.2Hz,1H),9. 13–9.12(d,J=1.8Hz,1H),8.55–8.54(t,J=2.0Hz,1H),7.88–7.86(d,J=8.2Hz,1H),7.79(s,1H ),7.70–7.68(d,J=8.3Hz,1H),7.22(s,1H),7.01–6.99(d,J=8.2Hz,2H),6.59–6.57(d,J=8.1H z,2H),4.70(s,2H),3.70(s,2H),3.26(s,2H),1.73–1.60(m,2H),0.94–0.91(t,J=7.4Hz,3H).

[0857] Compound 46

[0858] Step 1: Compound 40A (100 mg, 0.228 mmol, 1 eq.) and compound 46A (19.87 mg, 0.228 mmol, 1 eq.) were dissolved in DMA (3 mL). EDCI (174.90 g, 0.912 mmol, 4 eq.) was added all at once and stirred at room temperature overnight. LCMS analysis indicated that compound 40A had reacted completely and was carried on to the next step without further treatment. MS-ESI: m / z 508.2 [M+H] + .

[0859] Step 2: Raney nickel (0.5 g) was added to the untreated solution of compound 46B from the previous step. The atmosphere was replaced with hydrogen twice and stirred overnight at room temperature. LCMS confirmed the complete reaction. The mixture was filtered through celite and purified by preparative HPLC to afford compound 46 (8.2 mg, 7.5% yield over two steps) as a yellow solid. MS-ESI: m / z 478.2 [M+H] + .

[0860] 1H NMR (400MHz, DMSO) δ8.14(s,1H),7.38(d,J=6.5Hz,2H),7.17(d,J=20.9Hz,2H ),7.06(s,1H),6.87(d,J=7.4Hz,2H),6.41(d,J=7.3Hz,2H),4.59(s,2H),4.28 (d,J=37.6Hz,2H),3.59(dd,J=23.4,7.9Hz,7H),3.25(d,J=10.5Hz,1H),2.82 (d,J=14.7Hz,1H),1.88(d,J=56.6Hz,3H),1.63(d,J=6.4Hz,2H),0.89(s,3H).

[0861] Compound 57

[0862] Step 1: Compound 10 (300 mg, 0.609 mmol, 1 eq) and compound 57A (301 mg, 0.731 mmol, 1.2 eq) were added to a mixed solvent of DCM (3 mL) and DMA (3 mL). The atmosphere was replaced with argon twice. EDCI (468 mg, 2.44 mmol, 4 eq) was added at room temperature, the atmosphere was replaced with argon twice, and the mixture was stirred at room temperature overnight. LCMS analysis indicated the formation of the desired product, which was directly purified by preparative HPLC to afford 57B as a white solid (50 mg, 9.7% yield). MS-ESI: m / z 849.4 [M+H] + .

[0863] Step 2: Compound 57B (50 mg, 0.0589 mmol, 1 eq) was dissolved in DCM (3 mL). TFA (0.5 mL) was added under ice-water bath and stirred at room temperature for 2 h. LCMS confirmed the complete reaction. The mixture was concentrated under reduced pressure and purified by preparative HPLC to afford 57 (17 mg, 37% yield) as a white solid.

[0864] MS-ESI: m / z 793.4[M+H] + .

[0865] 1H NMR (400MHz, DMSO) δ12.02(s,1H),10.05–9.75(m,2H),9.09(s,1H),8.86(s,1H),8.60(s,1H),8.32–8.30(t,J=5. 6Hz,1H),8.23–8.21(d,J=7.8Hz,1H),8.10(s,1H),7.85–7.59(m,5H),7.39–7.17(m,2H),7.13(s,1H),7.00(s,2H) ,4.65(s,4H),4.43–4.34(dd,J=13.4,8.1Hz,1H),3.72–3.71(d,J=5.6Hz,2H),3.63–3.52(m,3H),2.44–2.41(m,3H ),2.30–2.22(m,2H),2.06–1.93(m,1H),1.90–1.75(m,1H),1.59–1.54(dd,J=14.8,7.4Hz,2H),0.90–0.70(m,3H).

[0866] Compound 58

[0867] Step 1: Compound 10 (500 mg, 1.02 mmol, 1 eq) and compound 58A (360 mg, 1.02 mmol, 1 eq) were added to a mixed solvent of DCM (10 mL) and DMA (10 mL). The atmosphere was replaced with argon twice. EDCI (779 mg, 4.06 mmol, 4 eq) was added at room temperature, the atmosphere was replaced with argon twice, and the mixture was stirred at room temperature overnight. LCMS analysis indicated the formation of the desired product. The product was directly purified by preparative HPLC, lyophilized, and then repurified by preparative HPLC to obtain 58 (8 mg, 1% yield) as a white solid.

[0868] MS-ESI: m / z 792.4[M+H] + .

[0869] 1H NMR(400MHz,DMSO)δ11.93(s,1H),10.0–9.74(m,2H),9.02(s,1H),8.83(s,1H),8.59(s, 1H),8.31–8.28(t,J=5.6Hz,1H),8.25–8.23(d,J=7.7Hz,1H),8.06(s,1H),7.81–7.69(m ,3H),7.65–7.63(d,J=8.6Hz,2H),7.33-7.19(m,3H),7.13(s,1H),6.99(s,2H),6.78(s, 1H),4.74–4.57(m,4H),4.37–4.32(dd,J=13.5,7.8Hz,1H),3.71–3.70(d,J=5.6Hz,2H), 3.65–3.59(m,2H),3.04(s,2H),2.79–2.78(d,J=3.8Hz,2H),2.46–2.41(m,2H),2.19–2.10(m,2 H),2.01–1.94(m,1H),1.86–1.79(m,1H),1.59–1.54(dd,J=14.9,7.5Hz,2H),0.87–0.74(m,3H).

[0870] Compound 59

[0871] Step 1: Compound 10 (400 mg, 0.813 mmol, 1 eq) and compound 59A (420 mg, 1.06 mmol, 1.3 eq) were added to DMF (8 mL). DIEA (525.4 mg, 4.06 mmol, 5 eq) and T3P (1.55 g, 2.44 mmol, 3 eq, 50%) were added and stirred at room temperature overnight. LCMS analysis indicated the formation of the product, which was directly purified by preparative HPLC to afford 59B as a white solid (50 mg, 7.4% yield). MS-ESI: m / z 835.3 [M+H] + .

[0872] Step 2: Compound 59B (50 mg, 0.0599 mmol, 1 eq) was dissolved in DCM (3 mL). TFA (0.5 mL) was added under ice-water bath and stirred at room temperature for 2 h. LCMS confirmed the complete reaction and the product was purified directly by preparative HPLC to afford 59 as a white solid (15 mg, 33% yield).

[0873] MS-ESI: m / z 779.4[M+H] + .

[0874] 1H NMR(400MHz,DMSO)δ12.01(s,1H),10.01–9.64(m,2H),9.08(s,1H),8.86(s,1H),8.60(s,1H),8.45–8.26 (m,2H),8.09(s,1H),7.88–7.63(m,5H),7.51–7.15(m,3H),7.13(s,1H),7.00(s,2H),4.83–4.51(m,6H),3 .76–3.67(dd,J=15.3,9.8Hz,2H),3.64–3.61(t,J=7.4Hz,3H),2.80–2.75(dd,J=16.7,5.9Hz,1H),2.63–2 .57(dd,J=16.7,8.0Hz,1H),2.44–2.42(d,J=6.5Hz,2H),1.59–1.54(dd,J=14.7,7.7Hz,2H),0.80(s,3H).

[0875] Compound 60

[0876] Step 1: Compound 15 (450 mg, 0.954 mmol, 1 eq) and compound 57A (511 mg, 1.24 mmol, 1.3 eq) were added to a mixed solvent of DCM (5 mL) and DMA (5 mL). The atmosphere was replaced with argon twice. EDCI (732 mg, 3.82 mmol, 4 eq) was added at room temperature, the atmosphere was replaced with argon twice, and the mixture was stirred at room temperature overnight. LCMS analysis indicated the formation of the product, which was directly purified by preparative HPLC to afford 60A as a white solid (25 mg, 3% yield). MS-ESI: m / z 865.4 [M+H] + .

[0877] Step 2: Compound 60A (25 mg, 0.0289 mmol, 1 eq) was dissolved in DCM (3 mL). TFA (0.5 mL) was added under ice-water bath and stirred at room temperature (25°C) for 2 hours. LCMS confirmed the reaction was complete. The mixture was concentrated under reduced pressure and purified by preparative HPLC to afford 60 (7 mg, 30% yield) as a white solid.

[0878] MS-ESI: m / z 809.3[M+H] + .

[0879] 1H NMR (400MHz, DMSO) δ11.91(s,1H),9.92(s,1H),9.83(s,1H),8.93(s,1H),8.87–8.85(d,J=2.1Hz,1H),8.61–8.59(d,J=1.7Hz,1H),8.28 –8.25(t,J=5.7Hz,1H),8.19–8.17(d,J=7.8Hz,1H),8.08(s,1H),7.80–7.76(dd,J=8.2,1.7Hz,1H),7.73(s,1H),7.65–7.63(d,J=8.3Hz ,1H),7.57–7.55(d,J=8.5Hz,2H),7.28–7.15(m,3H),6.97(s,2H),4.81(s,2H),4.65(s,3H),3.72–3.68(dd,J=14.3,6.4Hz,4H),3.62–3 .56(m,2H),2.42–2.38(m,2H),2.30–2.22(m,2H),2.03–1.93(m,1H),1.85–1.75(m,1H),1.74–1.64(m,2H),0.94–0.92(t,J=7.4Hz,3H).

[0880] Compound 61

[0881] Step 1: Compound 15 (250 mg, 0.53 mmol, 1 eq) and compound 58A (188 mg, 0.53 mmol, 1 eq) were added to a mixed solvent of DCM (10 mL) and DMA (10 mL). The atmosphere was replaced with argon twice. EDCI (407 mg, 2.12 mmol, 4 eq) was added at room temperature, the atmosphere was replaced with argon twice, and the mixture was stirred at room temperature overnight. LCMS analysis indicated the formation of product, which was directly purified by preparative HPLC to afford 61 (20 mg, 4.7% yield) as a white solid.

[0882] MS-ESI: m / z 808.4[M+H] + .

[0883] 1H NMR(400MHz,DMSO)δ12.24(s,1H),9.96(s,1H),9.87(s,1H),9.25(s,1H),8. 92–8.91(d,J=2.0Hz,1H),8.65(s,1H),8.33–8.31(t,J=5.6Hz,1H),8.27–8. 26(d,J=7.6Hz,1H),8.18(s,1H),7.82–7.80(d,J=8.2Hz,1H),7.75(s,1H),7 .67–7.65(d,J=8.2Hz,1H),7.59–7.57(d,J=8.4Hz,2H),7.33(s,1H),7.26–7. 24(d,J=8.5Hz,2H),7.21(s,1H),7.00(s,2H),6.81(s,1H),4.83(s,2H),4.6 9(s,2H),4.36–4.31(m,1H),3.76–3.70(dd,J=16.3,6.2Hz,5H),3.63(s,1H), 3.32(s,2H),2.47–2.40(m,2H),2.24–2.09(m,2H),2.04–1.91(m,1H),1.89– 1.79(m,1H),1.73–1.68(dd,J=14.4,7.2Hz,2H),0.97–0.93(t,J=7.4Hz,3H).

[0884] Compound 62

[0885] Step 1: Compound 59A (375.5 mg, 0.945 mmol, 1 eq) was added to DMF (40 mL). NMI (258.6 mg, 258.6 mg, 4 eq) and TCFH (331.4 mg, 1.18 mmol, 1.5 eq) were added at room temperature. The mixture was stirred at room temperature for 3 minutes. The hydrochloride salt of compound 15 (400 mg, 0.787 mmol, 1 eq) was added. The atmosphere was replaced with argon once and the mixture was stirred at room temperature overnight. LCMS detected the formation of product, which was directly purified by preparative HPLC to obtain 62A as a yellow solid (1.1 g, crude product not included in yield). MS-ESI: m / z 851.4 [M+H] + .

[0886] Step 2: Compound 62A (1.1 g, 1.29 mmol, 1 eq) was dissolved in DCM (50 mL). TFA (10 mL) was added under ice-water bath and stirred at room temperature for 2 hours. LCMS confirmed the complete reaction. The mixture was concentrated under reduced pressure at 0°C to remove most of the DCM and purified by preparative HPLC to afford 62 as a light yellow solid (240 mg, 7.6% yield over two steps).

[0887] MS-ESI: m / z 795.3[M+H] + .

[0888] 1 H NMR(400MHz,DMSO)δ12.32(s,1H),9.88–9.81(d,J=25.1Hz,2H),9.36(s,1H),8.91(s, 1H),8.65(s,1H),8.39–8.36(t,J=7.2Hz,2H),8.17(s,1H),7.88–7.78(m,1H),7.75(s, 1H),7.68–7.61(dd,J=19.8,8.4Hz,3H),7.30–7.22(m,3H),7.00(s,2H),4.84(s, 2H),4.74–4.68(m,3H),3.76–3.71(dd,J=12.3,5.0Hz,3H),3.64–3.56(m,2H),3.3 3(s,2H),2.80–2.74(dd,J=16.4,5.8Hz,1H),2.63–2.57(dd,J=16.5,7.8Hz,1H),2 .48–2.27(m,2H),1.74–1.68(dd,J=14.4,7.1Hz,2H),0.97–0.93(t,J=7.4Hz,3H).

[0889] Compound 63

[0890] Step 1: Compound 21C (162.5 mg, 0.46 mmol, 1.2 eq) was added to DMF (20 ml). NMI (126 mg, 1.53 mmol, 4 eq) and TCFH (161.3 mg, 0.575 mmol, 1.5 eq) were added, and the mixture was stirred at room temperature for 2 minutes. The crude hydrochloride of compound 32 (200 mg, 0.383 mmol, 1 eq) was then added, and the mixture was stirred at room temperature overnight. LCMS analysis confirmed the complete reaction of the starting material to produce the desired product, which was directly purified by preparative HPLC to afford 63 (20 mg, 6.4% yield) as a white solid.

[0891] MS-ESI: m / z 821.4[M+H] + .

[0892] 1H NMR (400MHz, DMSO) δ11.96 (s, 1H), 9.87–9.85 (d, J = 6.7Hz, 2H), 9.20 (d, J = 2.2Hz, 1H),9.13(d,J=1.8Hz,1H),9.02(s,1H),8.55–8.54(t,J=2.0Hz,1H),8.07–8.05(d ,J=7.2Hz,1H),7.99–7.98(d,J=6.9Hz,1H),7.88–7.85(dd,J=8.1,1.5Hz,1H),7. 80(s,1H),7.66–7.64(d,J=8.3Hz,1H),7.56–7.54(d,J=8.5Hz,2H),7.24–7.21(d, J=9.5Hz,3H),6.99(s,2H),4.81(s,2H),4.38–4.31(p,J=6.9Hz,1H),4.26–4.19( p,J=7.0Hz,1H),3.74–3.71(t,J=6.9Hz,2H),3.36–3.33(d,J=7.1Hz,2H),3.30(s, 2H),2.11–2.07(t,J=7.3Hz,2H),1.76–1.63(m,2H),1.48–1.41(dt,J=14.6,7.3Hz ,4H),1.29–1.27(d,J=7.1Hz,4H),1.22–1.11(m,5H),0.95–0.92(t,J=7.4Hz,3H).

[0893] Compound 64

[0894] Step 1: Compound 21C (123.7 mg, 0.35 mmol, 1.2 eq) was added to DMF (5 ml). NMI (96 mg, 1.17 mmol, 4 eq) and TCFH (123 mg, 0.438 mmol, 1.5 eq) were added, and the mixture was stirred at room temperature for 2 minutes. The hydrochloride salt of compound 46 (150 mg, 0.292 mmol, 1 eq) was then added, and the mixture was stirred at room temperature overnight. LCMS analysis confirmed the complete reaction of the starting material to produce the desired product, which was purified by preparative HPLC to afford 64 (110 mg, 43% yield) as a white solid.

[0895] MS-ESI: m / z 813.4[M+H] + .

[0896] 1H NMR (400MHz, DMSO) δ12.14(s,1H),9.80–9.88(d,J=6.1Hz,1H),9.83(s,1H),9.19–9.18(d,J=6.3Hz,1H),8.07–8.06(d,J=7.2Hz,1H),8.0–7 .98(d,J=7.0Hz,1H),7.55–7.49(m,5H),7.22–7.20(d,J=7.2Hz,2H),7.15(d,J=0.7Hz,1H),6.99(s,2H),4.79(s,2H),4.37–4.18(m,3H),3. 72–3.68(t,J=7.1Hz,2H),3.65–3.56(m,3H),3.39–3.34(dd,J=14.3,7.3Hz,3H),3.31–3.19(m,3H),2.12–2.08(t,J=7.4Hz,2H),2.03–1.80 (m,2H),1.73–1.62(m,2H),1.51-1.42(dt,J=14.2,7.1Hz,4H),1.29–1.28(d,J=7.1Hz,3H),1.22–1.11(m,5H),0.94–0.90(t,J=7.4Hz,3H).

[0897] Compound 65

[0898] Synthesized according to patent WO2020056194A1.

[0899] Compound 66

[0900] Synthesized according to patent WO2020190725A1.

[0901] Example 2 Preparation of Ligand-Drug Conjugates

[0902] 2.1 Antibody Preparation

[0903] The following antibodies can be prepared according to conventional antibody methods, for example, after constructing a vector, transfecting eukaryotic cells such as HEK293 cells (Life Technologies Cat No. 11625019), and then purified and expressed.

[0904] Sequence of Trastuzumab:

[0905] Light chain (SEQ ID NO: 33)

[0906] Heavy chain (SEQ ID NO: 37)

[0907] Pertuzumab antibody sequence

[0908] Light chain (SEQ ID NO: 34)

[0909] Heavy chain (SEQ ID NO: 38)

[0910] hRS7 antibody (Sacituzumab) sequence

[0911] Light chain (SEQ ID NO: 35)

[0912] Heavy chain (SEQ ID NO: 39)

[0913] Sequence of the antibody Codrituzumab

[0914] Light chain (SEQ ID NO: 36)

[0915] Heavy chain (SEQ ID NO: 40)

[0916] Example 2.2 Preparation of ligand-drug conjugates

[0917] In the present application, the 20 mM succinate buffer solution at pH 5.0 is obtained by adjusting the pH of a 20 mM succinic acid aqueous solution to 5.0 with NaOH; the 20 mM Histidine buffer solution at pH 5.5 is obtained by adjusting the pH of a 20 mM histidine aqueous solution to 5.5 with HOAc.

[0918] The protein purity of ADC in this application was detected by SEC method, and the parameters are shown below.

[0919] The DAR values ​​of the ADCs in this application were determined by LC-MS. The experimental procedure was as follows: 75.0 μL of 8.0 mol / L guanidine hydrochloride (Gdn-HCl), 5.0 μL of 1.0 mol / L Tris-HCl, and 2.0 μL of 1 mol / L DTT were added to a 40.0 μg ADC sample solution. The solution was diluted to 100.0 μL with ultrapure water, mixed thoroughly, and incubated at 22°C for 30 minutes. LC-MS was then used to analyze the drug / antibody ratio (DAR). The LC parameters are shown below.

[0920] The MS parameters are shown below.

[0921] Preparation Example ADC-3

[0922] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 32 mg, 10 mg / mL, 0.220 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.158 mL, 0.792 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0923] 21 (2.3 mg, 2.860 μmol) was dissolved in 0.23 mL DMSO and added to the above solution. The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours. A solution of 21 (1.0 mg, 1.320 μmol) in 0.10 mL DMSO was added and shaken at 22°C for 15 hours. A solution of 21 (1.0 mg, 1.320 μmol) in 0.10 mL DMSO was added and shaken at 22°C for 2 hours. A solution of 21 (0.7 mg, 0.880 μmol) in 0.070 mL DMSO was added and shaken at 22°C for 1 hour to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-3 (20 mM Histidine buffer solution at pH = 5.5; 19.5 mg, 4.43 mg / mL, yield: 60.9%), which was stored at 4°C.

[0924] LC-MS detection and calculation of DAR value n = 3.63

[0925] Preparation Example ADC-4

[0926] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 21 mg, 10 mg / mL, 0.144 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.104 mL, 0.518 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0927] 22 (1.9 mg, 2.160 μmol) was dissolved in 0.19 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-4 (20 mM Histidine buffer solution at pH = 5.5; 14 mg, 5.62 mg / mL, yield: 66.7%), which was stored at 4° C.

[0928] LC-MS detection and calculation of DAR value n = 3.74

[0929] Preparation Example ADC-5

[0930] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 25 mg, 10 mg / mL, 0.172 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.124 mL, 0.619 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0931] 23 (2.1 mg, 2.58 μmol) was dissolved in 0.21 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH 5.5) to obtain a solution of the exemplary product ADC-5 (20 mM Histidine buffer solution at pH 5.5; 20 mg, 6.76 mg / mL, yield: 80.0%), which was stored at 4° C.

[0932] LC-MS detection and calculation of DAR value n = 3.71

[0933] Preparation Example ADC-6

[0934] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.071 mL, 0.356 μmol) was added to a buffered aqueous solution of the antibody trastuzumab (PB buffer, pH = 7.0; 20 mg, 6.25 mg / mL, 0.137 μmol), and the mixture was placed in a water bath shaker and shaken at 37°C for 2 hours to stop the reaction.

[0935] 24 (0.84 mg, 1.04 μmol) was dissolved in 0.084 mL of DMSO, added to the above solution, and placed in a water bath shaker for 1 hour at 4°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-6 (20 mM Histidine buffer solution at pH = 5.5; 18.94 mg, 4.96 mg / mL, yield: 94.7%), which was stored at 4°C.

[0936] LC-MS detection and calculation of DAR value n = 4.48

[0937] Preparation Example ADC-7

[0938] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 15 mg, 10 mg / mL, 0.103 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.062 mL, 0.309 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0939] 25 (1.0 mg, 1.03 μmol) was dissolved in 0.10 mL DMSO and added to the above solution, which was placed in a water bath shaker and shaken at 22° C. for 1 hour. A solution of 25 (1.0 mg, 1.03 μmol) in 0.10 mL DMSO was added and shaken at 22° C. for 1 hour to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-7 (20 mM Histidine buffer solution at pH = 5.5; 14 mg, 1.33 mg / mL, yield: 93.3%), which was stored at 4° C.

[0940] LC-MS detection and calculation of DAR value n = 3.87

[0941] Preparation Example ADC-8

[0942] Compound 0735 (2.0 mg, 2.03 μmol) was dissolved in 0.20 mL of DMSO and added to a buffered aqueous solution of the antibody trastuzumab (0.05 M PB buffer at pH 7.0; 25 mg, 5 mg / mL, 0.172 μmol). The mixture was placed in a water bath shaker and shaken at 37° C. for 2 hours. The reaction was quenched with 200 mM succinic acid (pH 2.356, 1 / 8 V). The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM succinate buffer at pH 5.0) to obtain an aqueous solution of the product ADC-8 (20 mM succinate buffer at pH 5.0; 20.27 mg, 5.08 mg / mL, yield: 81.1%), which was stored at 4° C.

[0943] LC-MS detection and calculation of DAR value n = 3.73

[0944] Preparation Example ADC-9

[0945] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 27 mg, 10 mg / mL, 0.186 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.134 mL, 0.670 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0946] 27 (2.6 mg, 2.79 μmol) was dissolved in 0.26 mL of a 6:14 mixed solution of DMSO and DMA, added to the above solution, and placed in a water bath shaker for 2 hours at 22°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH 5.5) to obtain a solution of the exemplary product ADC-9 (20 mM Histidine buffer solution at pH 5.5; 12 mg, 3.05 mg / mL, yield: 44.4%), which was stored at 4°C.

[0947] LC-MS detection and calculation of DAR value n=3.69

[0948] Preparation Example ADC-10

[0949] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 30 mg, 10 mg / mL, 0.206 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.157 mL, 0.783 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[0950] 28 (3.0 mg, 3.09 μmol) was dissolved in 0.30 mL of a 6:14 mixed solution of DMSO and DMA, added to the above solution, and placed in a water bath shaker for 2 hours at 22°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH 5.5) to obtain a solution of the exemplary product ADC-10 (20 mM Histidine buffer solution at pH 5.5; 20 mg, 3.96 mg / mL, yield: 66.7%), which was stored at 4°C.

[0951] LC-MS detection and calculation showed that the DAR value was n=3.54.

[0952] Preparation Example ADC-12

[0953] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 15 mg, 6.85 mg / mL, 0.103 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.058 mL, 0.288 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0954] 21 (1.2 mg, 1.54 μmol) was dissolved in 0.12 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 4°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-12 (20 mM Histidine buffer solution at pH = 5.5; 13 mg, 3.92 mg / mL, yield: 86.7%), which was stored at 4°C.

[0955] LC-MS detection and calculation of DAR value n = 4.24

[0956] Preparation Example ADC-13

[0957] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 25 mg, 6.85 mg / mL, 0.172 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.096 mL, 0.482 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0958] 24 (2.1 mg, 2.58 μmol) was dissolved in 0.21 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-13 (20 mM Histidine buffer solution at pH = 5.5; 20 mg, 4.80 mg / mL, yield: 80.0%), which was stored at 4° C.

[0959] LC-MS detection and calculation of DAR value n = 3.76

[0960] Preparation Example ADC-14

[0961] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 21.09 mg, 6.84 mg / mL, 0.145 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.087 mL, 0.435 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0962] 25 (2.0 mg, 2.18 μmol) was dissolved in 0.20 mL of a mixed solution of DMSO and DMA (3.9:16.1), added to the above solution, and placed in a water bath shaker for 2 hours at 4°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-14 (20 mM Histidine buffer solution at pH = 5.5; 15 mg, 2.34 mg / mL, yield: 71.1%), which was stored at 4°C.

[0963] LC-MS detection and calculation of DAR value n = 4.22

[0964] Preparation Example ADC-15

[0965] Compound 26 (1.6 mg, 1.64 μmol) was dissolved in 0.20 mL of DMSO and added to a buffered aqueous solution of the antibody codrituzumab (50 mM PB buffer, pH = 7.05, containing 2 mM EDTA; 17 mg, 4.22 mg / mL, 0.117 μmol). The mixture was placed in a water bath shaker and shaken at 22° C. for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM succinate buffer, pH = 5.0) to obtain an aqueous solution of the product ADC-15 (20 mM succinate buffer, pH = 5.0; 12.71 mg, 3.53 mg / mL, yield: 74.8%), which was stored at 4° C.

[0966] LC-MS detection and calculation of DAR value n = 3.67

[0967] Preparation Example ADC-16

[0968] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 25 mg, 8.34 mg / mL, 0.172 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.096 mL, 0.482 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0969] 27 (2.4 mg, 2.58 μmol) was dissolved in 0.24 mL of a mixed solution of DMSO and DMA (4.7:15.3), added to the above solution, and placed in a water bath shaker for 2 hours at 4°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-16 (20 mM Histidine buffer solution at pH = 5.5; 18.69 mg, 3.60 mg / mL, yield: 74.8%), which was stored at 4°C.

[0970] LC-MS detection and calculation of DAR value n = 4.13

[0971] Preparation Example ADC-17

[0972] At 22°C, a buffered aqueous solution of the antibody codrituzumab (Gibco PBS buffer, pH = 7.4; 18.7 mg, 6.84 mg / mL, 0.128 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.077 mL, 0.384 μmol), and the mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0973] 28 (1.8 mg, 1.92 μmol) was dissolved in 0.18 mL of a mixed solution of DMSO and DMA (4.2:15.8), added to the above solution, and placed in a water bath shaker for 2 hours at 4°C to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-17 (20 mM Histidine buffer solution at pH = 5.5; 13.69 mg, 3.47 mg / mL, yield: 73.2%), which was stored at 4°C.

[0974] LC-MS detection and calculation of DAR value n = 4.15

[0975] Preparation Example ADC-18

[0976] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 25 mg, 8.34 mg / mL, 0.172 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.089 mL, 0.447 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0977] Compound 29 (9.4 mg, 10.32 μmol) was dissolved in 0.94 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-18 (20 mM Histidine buffer solution at pH = 5.5; 19.0 mg, 3.32 mg / mL, yield: 76%), which was stored at 4° C.

[0978] LC-MS detection and calculation of DAR value n = 3.47

[0979] Preparation Example ADC-19

[0980] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 15 mg, 8.00 mg / mL, 0.101 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.067 mL, 0.333 μmol), and the mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0981] Compound 60 (2.6 mg, 3.23 μmol) was dissolved in 0.32 mL of DMSO, added to the above solution, and placed in a water bath shaker for 3 hours at 22° C. to stop the reaction. The reaction solution was purified using a HiTrap desalting column (5 mL*3, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-19 (20 mM Histidine buffer solution at pH = 5.5; 10.5 mg, 1.81 mg / mL, yield: 70.0%), which was stored at 4° C.

[0982] LC-MS detection and calculation of DAR value n = 4.28

[0983] Preparation Example ADC-20

[0984] At 22°C, a buffered aqueous solution of the antibody codrituzumab (Gibco PBS buffer, pH = 7.4; 19.1 mg, 8.00 mg / mL, 0.129 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.075 mL, 0.374 μmol), and the mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0985] Compound 61 (1.6 mg, 1.94 μmol) was dissolved in 0.19 mL of DMSO, added to the above solution, and placed in a water bath shaker for 3 hours at 22° C. to stop the reaction. The reaction solution was purified using a HiPrep desalting column (53 mL, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-20 (20 mM Histidine buffer solution at pH = 5.5; 16.9 mg, 4.43 mg / mL, yield: 88.5%), which was stored at 4° C.

[0986] LC-MS detection and calculation of DAR value n = 4.09

[0987] Preparation Example ADC-21

[0988] At 22°C, a buffered aqueous solution of the antibody codrituzumab (Gibco PBS buffer, pH = 7.4; 12 mg, 11.5 mg / mL, 0.081 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.042 mL, 0.211 μmol), and the mixture was placed in a water bath shaker and shaken at 22°C for 3 hours to stop the reaction.

[0989] Compound 62 (0.45 mg, 0.567 μmol) was dissolved in 0.057 mL of DMSO, added to the above solution, placed in a water bath shaker, and shaken at 4° C. for 2 hours to stop the reaction. The reaction solution was purified using a HiTrap desalting column (5 mL*2, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-21 (20 mM Histidine buffer solution at pH = 5.5; 6.5 mg, 3.78 mg / mL, yield: 54.2%), which was stored at 4° C.

[0990] The DAR value detected and calculated by LC-MS was n=4.05.

[0991] Preparation Example ADC-22

[0992] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 14 mg, 8.0 mg / mL, 0.094 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.053 mL, 0.263 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[0993] Compound 63 (1.16 mg, 1.41 μmol) was dissolved in 0.14 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2.5 hours at 4° C. to stop the reaction. The reaction solution was purified using a HiTrap desalting column (5 mL*3, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-22 (20 mM Histidine buffer solution at pH = 5.5; 7.95 mg, 3.95 mg / mL, yield: 56.8%), which was stored at 4° C.

[0994] The DAR value detected and calculated by LC-MS was n=3.70.

[0995] Preparation Example ADC-23

[0996] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 30.5 mg, 11.0 mg / mL, 0.206 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.062 mL, 0.309 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 3 hours to stop the reaction.

[0997] Compound 64 (2.5 mg, 3.09 μmol) was dissolved in 0.31 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was purified using a desalting column (53 mL, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-23 (20 mM Histidine buffer solution at pH = 5.5; 22.03 mg, 6.77 mg / mL, yield: 72.2%), which was stored at 4° C.

[0998] The DAR value detected and calculated by LC-MS was n=2.00.

[0999] Preparation Example ADC-24

[1000] At 22°C, a buffered aqueous solution of codrituzumab (Gibco PBS buffer, pH = 7.4; 25 mg, 8.34 mg / mL, 0.169 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.095 mL, 0.473 μmol). The mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[1001] Compound 57 (2.0 mg, 2.54 μmol) was dissolved in 0.20 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2.5 hours at 4°C to stop the reaction. The reaction solution was purified using a HiTrap desalting column (5 mL*2, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-24 (20 mM Histidine buffer solution at pH = 5.5; 19 mg, 5.30 mg / mL, yield: 76.0%), which was stored at 4°C.

[1002] The DAR value detected and calculated by LC-MS was n=4.03.

[1003] Preparation Example ADC-25 (Control ADC1)

[1004] At 22°C, to a buffered aqueous solution of the antibody trastuzumab (pH = 6.0, 1.65 g / L succinic acid, 1.02 g / L NaOH, 6.30 g / L NaCl buffer, containing 2 mM EDTA; 40 mg, 10 mg / mL, 0.270 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.432 mL, 2.160 μmol), placed in a water bath shaker, and shaken at 22°C for 3 hours to stop the reaction;

[1005] Compound 65 (4.9 mg, 4.752 μmol) was dissolved in 0.49 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 22° C. to stop the reaction. The reaction solution was purified using a Spin desalting column (10 mL, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-25 (20 mM Histidine buffer solution at pH = 5.5; 33 mg, 7.54 mg / mL, yield: 82.5%), which was stored at 4° C.

[1006] The DAR value detected and calculated by LC-MS was n=6.35.

[1007] Preparation Example ADC-26 (Control ADC2)

[1008] At 22°C, a buffered aqueous solution of the antibody codrituzumab (Gibco PBS buffer, pH = 7.4; 21.09 mg, 6.85 mg / mL, 0.142 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.080 mL, 0.398 μmol), and the mixture was placed in a water bath shaker and shaken at 22°C for 2 hours to stop the reaction.

[1009] Compound 65 (2.2 mg, 2.13 μmol) was dissolved in 0.22 mL of DMSO, added to the above solution, and placed in a water bath shaker for 2 hours at 4° C. to stop the reaction. The reaction solution was purified using a Spin desalting column (10 mL, elution phase: 20 mM Histidine buffer solution at pH = 5.5) to obtain a solution of the exemplary product ADC-26 (20 mM Histidine buffer solution at pH = 5.5; 15.3 mg, 2.77 mg / mL, yield: 72.5%), which was stored at 4° C.

[1010] The DAR value detected and calculated by LC-MS was n=4.09.

[1011] Preparation Example ADC-27 (Control ADC3)

[1012] Compound 66 (2.7 mg, 2.74 μmol) was dissolved in 0.27 mL of DMA and added to a buffered aqueous solution of the antibody trastuzumab (50 mM PB buffer, pH = 7.0; 30 mg, 5 mg / mL, 0.203 μmol). The mixture was placed in a water bath shaker and shaken at 37° C. for 2 hours to stop the reaction. 1 / 8 volume of aqueous succinic acid solution (200 mM, pH = 2.356) was added to quench the reaction. The reaction solution was purified using a spin desalting column (elution phase: 20 mM succinate buffer, pH = 5.0) to obtain an aqueous solution of the product ADC-27 (20 mM succinate buffer, pH = 5.0; 23 mg, 4.08 mg / mL, yield: 76.7%), which was stored at 4° C.

[1013] The DAR value detected and calculated by LC-MS was n=3.45.

[1014] Example 3

[1015] Agonist activity of compounds on hTLR7 (human Toll-like receptor 7) and hTLR8 (human Toll-like receptor 8)

[1016] This example evaluates the agonist activity of the compounds of the present application on hTLR7 (human Toll-like receptor 7) and hTLR8 (human Toll-like receptor 8); and in human PBMC (human peripheral blood mononuclear cells), evaluates the induction activity of the compounds on the production of TNF-α and IFN-α by human PBMC.

[1017] All compounds were prepared in DMSO to a 30 mM concentrated stock solution and stored at 4°C. Reference compound R848 was prepared in DMSO to a 5 mg / ml concentrated stock solution and stored at -20°C. HEK-Blue hTLR7 and HEK-Blue hTLR8 cell lines were purchased from InvivoGen, and human PBMCs were purchased from Shanghai Saili Biotechnology Co., Ltd. (Cat. No. XFB-HP100B).

[1018] Reference compounds R848 (CAS No.: 144875-48-9) and VTX-2337 (CAS No.: 926927-61-9) were purchased from Haoyuan Pharmaceuticals. Their structures are shown below:

[1019] Other commonly used reagents can be purchased commercially, for example from the following sources:

[1020] The main instruments used in this study are the multifunctional microplate reader Flexstation III (Molecular Device), Envision (Perkin Elmer), M2e (Molecular Device) and Echo555 (Labcyte).

[1021] Agonist activity of compounds on hTLR7 and hTLR8

[1022] Compound Dilution: Dilute the present compound to approximately 15 mM in DMSO, followed by the reference compound, and then add them to the cell plate. Add the compound to a 96-well plate in a 3-fold serial dilution series for a total of 10 concentrations, with two replicates for each concentration. Add 0.2 μl of DMSO to each negative control well, and add the reference compound to each positive control well, for a final DMSO concentration of 0.2%.

[1023] Cell plating: Observe the HEK Blue hTLR7&8 cells in the culture flask until they have reached 80% confluence under a microscope. Discard the culture medium, add 10 ml of assay medium, and dissect into single cells. Count the cells using a cell counter and dilute to 500,000 cells / mL. Plate 100 μl of cells into a 96-well plate pre-added with compound at a density of 50,000 cells / well. Incubate the cells and compound in a 37°C, 5% CO2 incubator for 24 hours.

[1024] Compound activity detection: Take 20 μl of cell supernatant from each well and add 180 μl of QUANTI-Blue TM After incubation at 37°C for 1 hour, the absorbance at 650 nm (OD 650 ).

[1025] Cell viability assay: The assay was performed according to the Celltiter-Glo instructions, and the chemiluminescence signal (RLU) was detected using a multifunctional microplate reader Flexstation III.

[1026] Compound activity data analysis: OD 650 The values ​​were analyzed using GraphPad Prism software, and the compound dose-effect curves were fitted to calculate the EC of the compound. 50 value.

[1027] See Table 1 for details.

[1028] The results showed that the compounds of the present application had significant activation activity on both TLR7 and TLR8, and the activation activity was better than that of the controls VTX-2337 and R848.

[1029] Induction activity of compounds on TNF-α and IFN-α in human PBMCs

[1030] PBMC cells were treated with compounds. The compound of the present application was diluted with DMSO to, for example, about 15 mM, and the reference compound was diluted to 5 mg / ml and then added to the cell plate in sequence. The compound was diluted 3-fold in a gradient and added to the cell plate, with a total of 8 concentrations, and each concentration was repeated in duplicate. 0.4 μl DMSO was added to each well of the negative control well, and 0.4 μl 5 mg / ml reference compound was added to each well of the positive control well. The final concentration of DMSO was 0.2%. The cryovial containing frozen human PBMC was quickly thawed in a 37°C water bath, and then transferred to a 50 ml centrifuge tube, preheated RPMI1640 culture medium was gradually added and mixed, centrifuged for 15 minutes, and the supernatant was removed. The cells were resuspended in culture medium and counted, and the cell suspension was adjusted to 2.5×10 6 Then, 200 μl of the diluted cells (500,000 cells / well) were added to each well of the 96-well plate containing the compound. The cell plate was placed at 37°C and 5% CO2 for 24 hours.

[1031] TNF-α content detection:

[1032] Add samples: Add 100 μl of serially diluted Cytokine standard to each well, add 100 μl of a 10-fold diluted test sample to each well, and add 100 μl of 1× Dilution Buffer R to each blank control well. Add detection antibody: Add 50 μl of Biotinylated antibody working solution to each well. Mix thoroughly, cover with a film sealer, incubate at room temperature for 3 hours, wash the plate three times, and add 100 μl of Streptavidin-HRP working solution to each well. Cover with a film sealer, incubate at room temperature for 20 minutes, and continue washing the plate three times. Color development: Add 100 μl of TMB solution to each well and incubate at room temperature (18-25°C) in the dark for 25 minutes. Stop the reaction: Immediately stop the reaction by adding 100 μl of Stop solution to each well. Read the plate: Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1033] IFN-α content detection:

[1034] Add samples: Add 100 μl of serially diluted Cytokine standard to each well, add 100 μl of a 2-fold diluted test sample to each well, and add 100 μl of 1× Dilution Buffer R to each blank control well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 2 hours. Wash the plate four times. Add detection antibody: Add 100 μl of Biotinylated antibody working solution to each well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 1 hour. Wash the plate four more times. Add enzyme: Add 100 μl of Streptavidin-HRP working solution to each well. Cover with a film sealer, and incubate at room temperature for 30 minutes. Wash the plate four more times. Color development: Add 100 μl of TMB solution to each well and incubate at room temperature (18-25°C) in the dark for 25 minutes. Stop the reaction: Immediately add 100 μl of Stop solution to each well. Reading the plate: Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1035] Cell viability assay:

[1036] The method was operated according to the instructions of Celltiter-Glo, and the chemiluminescence signal (RLU) was detected using the multifunctional microplate reader Envision.

[1037] Compound activity data analysis: GraphPad Prism software was used to analyze the OD 450 The values ​​were converted into the contents of TNF-α and IFN-α, and the compound dose-effect curve was fitted to calculate the EC of the compound. 50 value.

[1038] The results are shown in Table 2.

[1039] The results showed that the small molecule compound of the present application can activate PBMC TLR7 and TLR8 receptors, induce the secretion of immune cytokines TNF-α and / or IFN-α, and the activation activity is better than the control VTX-2337 and R848.

[1040] Table 1

[1041] Table 2

[1042] Example 4

[1043] Detection of agonist activity of antibody-drug conjugates in whole blood

[1044] This example evaluates the agonist activity of the antibody-drug conjugate of the present application on human whole blood.

[1045] The antibody-drug conjugate of the present application was diluted to 200 nM in PBS and then added to a 96-well plate in a 3-fold serial dilution series for a total of five concentrations, with two replicates per well. 0.2% DMSO was added to each negative control well, and 100 μl of 60 μM R848 was added to each positive control well. 100 μl of anticoagulated whole blood was added to each well of the 96-well plate, and the plate was incubated at 37°C, 5% CO2 for 24 hours. The cells were centrifuged at 2000g for 15 minutes at 4°C. The supernatant was then collected and assayed for TNF-α and IFN-α concentrations.

[1046] TNF-α concentrations were measured using the Dakoway TNF-α ELISA Kit (Cat. No. 1117202). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 10-fold diluted sample to be tested was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 3 hours. Wash the plate three times, remove all liquid from the wells, and add 300 μl of 1× Washing Buffer Working Solution to each well. Allow to stand for 1 minute, then discard all liquid. Repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with a film sealer, and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1047] IFN-α concentrations were measured using the Dakoway IFN-α ELISA Kit (Cat. No. 1110012). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 2-fold diluted test sample was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 3 hours. Wash the plate three times, remove all liquid from the wells, and add 300 μl of 1× Washing Buffer Working Solution to each well. Allow to stand for 1 minute, then discard all liquid. Repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with a film sealer, and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1048] GraphPad Prism software was used for analysis, and the OD450 values ​​were converted into the contents of TNF-α and IFN-α. The dose-effect curves of the compounds were fitted to calculate the EC50 values ​​of the compounds.

[1049] The results are shown in Figures 1(a) and (b).

[1050] The results showed that the antibody-drug conjugate of the present application was active in human whole blood at high concentrations, but did not induce cytokine production at physiological blood concentrations, indicating that the antibody-drug conjugate of the present application had good safety and low systemic toxicity, and was superior to the control ADC1.

[1051] Example 5

[1052] Co-incubation assay of antibody-drug conjugates, tumor cells, and human peripheral blood mononuclear cells (PBMCs)

[1053] The test antibody-drug conjugate was diluted to 400 nM with PBS buffer and added to a 96-well plate in a 4-fold serial dilution, for a total of 7 concentrations, with 2 replicates for each concentration. 0.2% DMSO was added to each negative control well.

[1054] The pre-cultured NCI-N87, JIM-1 and MDA-MB-231 cells (cells purchased from American Type Culture Collection) were digested and collected to obtain single cell suspensions, and the suspensions were adjusted to 2×10 6Cells / ml. Thaw frozen human PBMC cells, resuspend the cells in complete culture medium, count the cells, and adjust the cell suspension to 2×10 7 Tumor cells and PBMCs were mixed at a 1:1 ratio and 100 μl of the mixture was added to each well (500,000 cells / well). The cell plates were incubated at 37°C, 5% CO₂ for 24 hours, and the supernatant was collected to measure TNF-α and IFN-α concentrations, as well as dendritic cell (DC) and macrophage activation.

[1055] TNF-α concentrations were measured using the Dakoway TNF-α ELISA Kit (Cat. No. 1117202). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 10-fold diluted sample to be tested was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with film, and incubate at room temperature for 3 hours. Wash the plate three times: remove all liquid from the wells and add 300 μl of 1× Washing Buffer Working Solution to each well; allow to stand for 1 minute, then discard the liquid. Repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with film and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1056] IFN-α concentrations were measured using the Dakoway IFN-α ELISA Kit (Cat. No. 1110012). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 2-fold diluted test sample was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 3 hours. Wash the plate three times, remove all liquid from the wells, and add 300 μl of 1× Washing Buffer Working Solution to each well. After 1 minute, remove all liquid from the wells and repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with a film sealer, and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1057] To detect the activation of dendritic cells and macrophages, FACS was used to detect the activation of DC cells (HLA-DR+CD11c+) and macrophages (CD68+CD11b+) in PBMCs after co-incubation.

[1058] The results are shown in Table 3 and Figures 2(a) and (b).

[1059] The results showed that the antibody-drug conjugate of the present application can induce the expression of human PBMC cytokines, such as immune cytokines (such as TNF-α and / or IFN-α), and induce the activation of immune cells, such as dendritic cells and macrophages, in the presence of tumor cells, and is superior to the control ADC1.

[1060] Table 3

[1061] Example 6

[1062] Co-incubation assay of antibody-drug conjugates, tumor cells, and human peripheral blood mononuclear cells (PBMCs)

[1063] The test antibody-drug conjugate was diluted to 100nM-400nM in PBS buffer and added to a 96-well plate in a 4-fold gradient, with 7 concentrations in total and 2 replicates for each concentration. 0.2% DMSO was added to each negative control well. HepG2 cells cultured in advance were digested and collected to obtain a single-cell suspension, which was adjusted to 2×10 6 Cells / ml. Thaw frozen human PBMC cells, resuspend the cells in complete culture medium, count the cells, and adjust the cell suspension to 2×107 Tumor cells and PBMCs were mixed at a 1:1 ratio and 100 μl of the mixture was added to each well (500,000 cells / well). The cell plates were incubated at 37°C, 5% CO2 for 18 hours. The supernatant was then collected to measure TNF-α and IFN-α concentrations, and to examine the activation of dendritic cells (DCs) and macrophages.

[1064] TNF-α concentrations were measured using the Dakoway TNF-α ELISA Kit (Cat. No. 1117202). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 10-fold diluted sample to be tested was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with film, and incubate at room temperature for 3 hours. Wash the plate three times: remove all liquid from the wells and add 300 μl of 1× Washing Buffer Working Solution to each well; allow to stand for 1 minute, then discard the liquid. Repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with film and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1065] IFN-α concentrations were measured using the Dakoway IFN-α ELISA Kit (Cat. No. 1110012). 100 μl of serially diluted Cytokine standard was added to each well. 100 μl of a 2-fold diluted test sample was added to the sample well. 100 μl of 1× Dilution Buffer R was added to the blank control well. 50 μl of Biotinylated Antibody Working Solution was added to each well. Mix thoroughly, cover with a film sealer, and incubate at room temperature for 3 hours. Wash the plate three times, remove all liquid from the wells, and add 300 μl of 1× Washing Buffer Working Solution to each well. After 1 minute, remove all liquid from the wells and repeat three times. Add 100 μl of Streptavidin-HRP Working Solution to each well. Cover with a film sealer, and incubate at room temperature for 20 minutes. Wash the plate three times, add 100 μl of TMB solution to each well, and incubate at room temperature (18-25°C) in the dark for 25 minutes. Immediately add 100 μl of Stop solution to each well to terminate the reaction. Within 10 minutes after termination, read the value at a wavelength of 450 nm using an M2e instrument.

[1066] To detect the activation of dendritic cells and macrophages, FACS was used to detect the activation of DC cells (HLA-DR+CD11c+) and macrophages (CD68+CD11b+) in PBMCs after co-incubation.

[1067] The results are shown in Figures 3, 4 and 5.

[1068] The results showed that the antibody-drug conjugates of the present application can induce human PBMC cytokines in the presence of tumor cells, such as significantly increased expression of immune cytokines (such as TNF-α and / or IFN-α). The antibody-drug conjugates of the present application can activate immune cells, such as dendritic cells and macrophages, and significantly increase their activity.

[1069] Example 7

[1070] Establishing hGPC3-MC38 colon cancer animal model using hTLR8 gene knock-in humanized mice and testing the in vivo efficacy of drugs

[1071] hGPC3-MC38 cells: A cell line constructed through site-directed transgenic modification technology that stably expresse...

Claims

1. A ligand-drug conjugate, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (II-2A): in, R1 is an optionally substituted amino group; X1 is selected from optionally substituted -CH2-; X3 is selected from optionally substituted -CH= or -N=; R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure; W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure; B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl; Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl; Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure; m is selected from 1, 2 or 3; Wavy lines in the general formula It indicates that the Z group is directly connected to the ligand through the nitrogen atom on the Z group, or is connected to the ligand through a linker unit.

2. The ligand-drug conjugate according to claim 1, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (II-2B): in, R1 is an optionally substituted amino group; X1 is selected from optionally substituted -CH2-; X3 is selected from optionally substituted -CH= or -N=; R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure; W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure; B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl; Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl; Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure; m is selected from 1, 2 or 3; Wavy lines in the general formula It means that it is connected to the ligand through the nitrogen atom or carbon atom on the L group; L is a connector unit.

3. The ligand-drug conjugate according to claim 1, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein the ligand-drug conjugate has a structure represented by formula (II-2C): in, R1 is an optionally substituted amino group; X1 is selected from optionally substituted -CH2-; X3 is selected from optionally substituted -CH= or -N=; R2 is selected from hydrogen and optionally substituted C1-C6 alkyl, and when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure; W is absent, or W is selected from an optionally substituted C1-C6 alkyl group. When W contains a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure; B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl; Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl; Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure; m is selected from 1, 2 or 3; L is the joint unit; n is an integer or decimal from 1 to 10; Pc is a ligand.

4. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The R1 is -NH2.

5. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: Said X1 is selected from -CH2- or 6. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: Said X3 is selected from -CH= or -C(CH3)=.

7. The compound or conjugate thereof, or its isomer, or its mixture, or its pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein: R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R 2-2 Substituted, the R 2-2 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; wherein the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution, R 2-3 Selected from: hydrogen or C1-C3 alkyl.

8. The ligand-drug conjugate according to claim 7, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: R 2-1 Each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, -CN, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3 or 9. The ligand-drug conjugate according to any one of claims 1 to 8, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: R2 is selected from wherein the methylene units of R2 are each independently unsubstituted, substituted by -O-, or substituted by -NH-.

10. The ligand-drug conjugate according to any one of claims 1 to 9, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: R2 is selected from:

11. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkylene; R w-1 are each independently selected from hydrogen, halogen, Hydroxyl, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Substituted, the R w-2 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; wherein the methylene units of the W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl.

12. The ligand-drug conjugate according to claim 11, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: R w-1 Each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, -CN, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3 or 13. The ligand-drug conjugate according to claim 12, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: W is selected from wherein the methylene units of W are each independently unsubstituted, substituted by -O-, or substituted by -NH-.

14. The ligand-drug conjugate according to claim 13, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: W is selected from 15. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: B is selected from phenyl and pyridyl, each of which is independently optionally substituted by 1, 2 or 3 R B-1 Substituted, the R B-1 Each is independently selected from hydrogen, halogen, hydroxy, amino, C1-C3 alkyl and C1-C3 alkoxy.

16. The ligand-drug conjugate according to any one of claims 1 to 15, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The B is selected from Preferably, the B is selected from 17. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The A is selected from the group consisting of phenyl, pyridyl, pyrrolyl, thienyl, furyl, pyridazinyl, pyrimidinyl and pyrazinyl.

18. The ligand-drug conjugate according to any one of claims 1 to 17, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The A is selected from phenyl and pyridyl.

19. The ligand-drug conjugate according to any one of claims 1 to 3, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: R A-1 R A2-1 -Lx-; Lx is selected from: -(CH2) t -、-(CH2) t O-, -O(CH2) t -、-(CH2) t N(R L-1 )-、-(CH2) t S-, -(CH2) t C(=O)-、-(CH2) t N(R L-1 )C(=O)-、 where R L-1 is selected from H or C1-C3 alkyl, t is selected from 0, 1, 2 or 3; R A2-1 is selected from hydrogen, halogen, hydroxy, amino, cyano, C1-C6 alkyl, alicyclic, alicyclic heterocyclic, aryl and heteroaryl, wherein the C1-C6 alkyl, alicyclic, alicyclic heterocyclic, aryl and heteroaryl are each independently optionally substituted by 1, 2 or 3 R A2-2 Replacement; R A2-2 is selected from hydrogen, halogen, hydroxy, amino, and optionally substituted by 1, 2 or 3 R A2-3 Substituted C1-C6 alkyl; R A2-3 Each is independently selected from: hydrogen, halogen, hydroxy, amino and C1-C3 alkyl; Among them, R A2-1 Any methylene unit of can be replaced by the following structures: -O-, -S-, -S(=O)2-, -NH-, -CO-, m is selected from 1, 2 or 3.

20. The ligand-drug conjugate according to claim 19, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: Lx is a single bond or -NHC(=O)-.

21. The ligand-drug conjugate according to claim 19 or 20, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: R A2-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, or optionally substituted by 1, 2 or 3 R A2-2 Substituted: C1-C6 alkyl, Among them, R A2-1 Any methylene unit of can be replaced by the following structures: -O-, -S-, -S(=O)-, -S(=O)2-, -NH-, -C(O)-, 22. The ligand-drug conjugate according to any one of claims 1 to 21, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: R A-1 Selected from: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, cyano, mercapto, -CH3, -CH(=O), -C(=O)OH, -OCH(=O), -SH(=O), -SH(=O)2, -CH2CH3, -CH2OH, -OCH3, -NHCH3, -CH2NH2, -S(=O)2NH2, -NHSH(=O)2, -SCH3, -CH2SH, -S(=O)CH3, -CH2SH(=O), -S(=O)2CH3, -CH2SH(=O)2, -NHCH(=O), -C(=O)NH2, -CH2CH(=O), -CH2NHCH(=O), -C(=O)NHCH3, 23. The ligand-drug conjugate according to any one of claims 1 to 22, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: R A-1 R A2-1 -Lx-, Lx is a single bond or an amide bond, R A2-1 Each independently selected from optionally 1, 2 or 3 R A2-2 Substituted: phenyl and pyridyl; R A2-2 is selected from hydrogen, halogen, hydroxy, amino and optionally substituted by 1, 2 or 3 R A2-3 Substituted C1-C6 alkyl; R A2-3 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino; Preferably, R A-1 Selected from: More preferably, R A-1 Selected from:

24. The ligand-drug conjugate according to any one of claims 1 to 23, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: Z is selected from -N(R Z-1 )-, the R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups; preferably, R Z-1 Selected from H, -CH3, -CH2F, -CHF2, -CF3, -CH2CH3.

25. The ligand-drug conjugate according to any one of claims 1 to 24, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The structural unit Selected from: in, R2 is as defined in any one of claims 7 to 10; R Z-1 As defined in claim 1 or 24; W is defined as any one of claims 11 to 14; R A-1 As defined in any one of claims 19 to 23, respectively.

26. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 2 to 25, wherein: The linker unit L is -Tr-L3-L2-L 1c -, Tr is selected from and single bonds; L 1c Selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or a peptide residue consisting of 1-4 amino acids, wherein the peptide residue is a peptide residue formed by an amino acid selected from the following group: valine, lysine, cysteine, alanine, glycine, glutamic acid, glutamine, phenylalanine, citrulline, aspartic acid, asparagine, and serine.

27. The ligand-drug conjugate according to claim 26, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

28. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 2 to 27, wherein: The connector unit L is -L3-L2-L 1c -, L 1c Selected from L2 is selected from and single bonds, where q is an integer from 1 to 20 number; L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

29. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 2 to 28, wherein: The connector unit L is -L3-L2-L 1c -or-Tr-L3-L2-L 1c -, the L3 end or the Tr end is connected to the nitrogen atom of the Z group, L 1c end connected to the ligand.

30. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 2 to 29, wherein: The linker unit L is selected from:

31. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 30, wherein: The ligand-drug conjugates are shown in formulas (II-2C-1) and (II-2C-2): in, R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups; X4 and X5 are each independently selected from -CH= and -N=; R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are are independently optionally substituted by 1, 2 or 3 R 2-2 Replacement; R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution; R 2-3 Selected from: hydrogen or C1-C3 alkyl; W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Replacement; R w-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of said W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl; R A-1 Selected from R A2-1 -Lx-; Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-; R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by,; where R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2; L 1c Selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-; n is an integer or decimal from 1 to 10; Pc is a ligand.

32. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 31, wherein: The ligand-drug conjugate is selected from the following structural formula: n is an integer or decimal from 1 to 10; Pc is a ligand.

33. The ligand-drug conjugate according to any one of claims 3 to 32, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: n is an integer or decimal from 2 to 8.

34. The ligand-drug conjugate according to any one of claims 3 to 33, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: Pc is an antibody or an antigen-binding fragment thereof.

35. The ligand-drug conjugate according to claim 3-34, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The Pc targets GPC3, Trop2 and HER2; preferably, the antibody is selected from: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

36. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 35, wherein: The ligand-drug conjugate is selected from the following structural formula: Where n is an integer or decimal from 1 to 10.

37. A compound or a conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (II-3A): in, R1 is optionally substituted NH2; X1 is selected from optionally substituted -CH2-; X3 is selected from optionally substituted -CH= or -N=; R2 is selected from hydrogen and optionally substituted C1-C6 alkyl; when said R2 contains a methylene unit, said methylene unit of said R2 is independently not replaced, or is independently replaced by any structure; W is absent, or W is selected from an optionally substituted C1-C6 alkyl group; when W comprises a methylene unit, the methylene units of W are each independently not substituted or are each independently substituted by any structure; B is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl; Z is selected from -N(R Z-1 )-, where R Z-1 is selected from H or optionally substituted C1-C6 alkyl; Ring A is selected from the group consisting of an optionally substituted alicyclic group, an optionally substituted alicyclic heterocyclic group, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R A-1 is selected from hydrogen, halogen, hydroxy, amino, optionally substituted C1-C6 alkyl, when said R A-1 When containing a methylene unit, the R A-1 The methylene units are each independently not replaced, or are each independently replaced by any structure; m is selected from 1, 2 or 3; L w is -L3-L2-L1; L1 is selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-cysteine-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

38. The compound according to claim 37, or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: The compounds are shown in formula (II-1B-1) and (II-1B-2): in, R Z-1 is selected from H or C1-C6 alkyl optionally substituted by 1, 2 or 3 fluorine, chlorine, bromine, iodine, hydroxyl or amino groups; X4 and X5 are each independently selected from -CH= and -N=; R2 is selected from 1, 2 or 3 R 2-1 Substituted C1-C6 alkyl; R 2-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are are independently optionally substituted by 1, 2 or 3 R 2-2 Replacement; R 2-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of R2 are each independently not substituted, substituted by -O-, or substituted by -N(R 2-3 )-substitution; R 2-3 Selected from: hydrogen or C1-C3 alkyl; W is selected from the group consisting of 1, 2 or 3 R w-1 Substituted C1-C6 alkyl; R w-1 Each independently selected from: hydrogen, halogen, hydroxy, amino, C1-C6 alkyl and C3-C6 cycloalkyl, wherein the C1-C6 alkyl and C3-C6 cycloalkyl are each independently optionally substituted by 1, 2 or 3 R w-2 Replacement; R w-2 Each independently selected from: hydrogen, halogen, hydroxyl or amino; the methylene units of said W are each independently not substituted, substituted by -O-, or substituted by -N(R w-3 )-substitution; R w-3 Selected from: hydrogen or C1-C3 alkyl; R A-1 Selected from R A2-1 -Lx-; Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-; R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by; R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2; L w Selected from 39. A compound or conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, selected from:

40. A ligand-drug conjugate, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises the structure represented by formula (III-2A): in, X is selected from -S- and -O-; R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl; Ring A is selected from phenyl or heteroaryl; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and Y is selected from -N(R Y1 )-、-O-、-S-、 R Y1 is selected from hydrogen and C1-C6 alkyl; q is selected from 0, 1, 2 or 3; p is selected from 1, 2, 3, 4, 5, 7 and 8; Wavy lines in the general formula It indicates that the Y group is directly connected to the ligand through the nitrogen atom or oxygen atom on the Y group, or is connected to the ligand through a linker unit.

41. The ligand-drug conjugate of claim 40, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (III-2B): in, X is selected from -S- and -O-; R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl; Ring A is selected from phenyl or heteroaryl; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 Selected from H, C1-C6 Alkyl and Y is selected from -N(R Y1 )-、-O-、-S-、 R Y1 is selected from hydrogen and C1-C6 alkyl; q is selected from 0, 1, 2 or 3; p is selected from 1, 2, 3, 4, 5, 7 and 8; L is the joint unit; Wavy lines in the general formula It means that the ligand is connected through the nitrogen atom or carbon atom on the L group.

42. The ligand-drug conjugate of claim 40, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (III-2C): in, X is selected from -S- and -O-; R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl; Ring A is selected from phenyl or heteroaryl; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and Y is selected from -N(R Y1 )-、-O-、-S-、 R Y1 is selected from hydrogen and C1-C6 alkyl; q is selected from 0, 1, 2 or 3; p is selected from 1, 2, 3, 4, 5, 7 and 8; L is the joint unit; n is an integer or decimal from 1 to 10; Pc is a ligand.

43. The ligand-drug conjugate according to any one of claims 40 to 42, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The R1 is selected from 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each independently selected from: hydrogen, chlorine, bromine, iodine, hydroxyl, amino, -CH3, -CH2CH3 and wherein the methylene units of R1 are each independently unsubstituted or replaced by a group selected from the group consisting of: -O-, -N(CH3)-, and -NH-.

44. The ligand-drug conjugate according to any one of claims 40 to 43, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The R1 is selected from wherein the methylene units of R1 are each independently unsubstituted or replaced by a group selected from the group consisting of: -O-, -N(CH3)-, and -NH-.

45. The ligand-drug conjugate according to any one of claims 40 to 44, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The R1 is selected from 46. ​​The ligand-drug conjugate according to any one of claims 40 to 42, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: The R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, and the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted, the R 2-1 Each is independently selected from the group consisting of: hydrogen, halogen, hydroxyl and amino.

47. The ligand-drug conjugate according to any one of claims 40 to 46, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: The R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, -CH3, -CH2CH3 and -OCH3.

48. The ligand-drug conjugate according to any one of claims 40 to 42, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: The ring A is selected from phenyl, pyridyl, pyrrolyl, thienyl, furyl, pyridazinyl, pyrimidinyl and pyrazinyl.

49. The ligand-drug conjugate according to any one of claims 40 to 48, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: The ring A is selected from phenyl or pyridyl.

50. The ligand-drug conjugate according to any one of claims 40 to 42, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, -CH3, -CH2CH3 and Wherein, the methylene units of V are each independently not substituted or replaced by a group selected from the following groups: -O-, -NH-, -CO-, -NHCO-, 51. The ligand-drug conjugate according to any one of claims 40 to 50, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The V is selected from Wherein, the methylene units of V are each independently not substituted or replaced by a group selected from the following groups: -O-, -NH-, -CO-, -NHCO-, 52. The ligand-drug conjugate according to any one of claims 40 to 51, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The V is selected from 53. The ligand-drug conjugate according to any one of claims 40 to 42, or its isomers, or its mixture, or its pharmaceutically acceptable salt, wherein: The Y is selected from -NH-, -O-, -S-, 54. The ligand-drug conjugate according to any one of claims 40 to 53, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: The structural unit Selected from: Preferably, it is selected from in, X is selected from -S- and -O-; R1 is as defined in any one of claims 40, 43-45; R2 is as defined in claim 40, 46 or 47; V as defined in any one of claims 40, 50-52; Y is as defined in claim 40 or 53.

55. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 42, wherein: The linker unit L is -Tr-L3-L2-L 1c -, Tr is selected from and single bonds; L 1c Selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or a peptide residue consisting of 1-4 amino acids, wherein the peptide residue is a peptide residue formed by an amino acid selected from the following group: valine, lysine, cysteine, alanine, glycine, glutamic acid, phenylalanine, and serine.

56. The ligand-drug conjugate according to claim 55, its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

57. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 56, wherein: The connector unit L is -L3-L2-L 1c -, L 1c Selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-glycine-.

58. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 57, wherein: The connector unit L is -L3-L2-L 1c -or-Tr-L3-L2-L 1c -, the L3 end or the Tr end is connected to the nitrogen atom of the Z group, L 1c end connected to the ligand.

59. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 58, wherein: The linker unit L is selected from:

60. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 59, wherein: The ligand-drug conjugates are shown in formulas (III-2C-1) and (III-2C-2): in, X is selected from -S- and -O-; R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; wherein the methylene units of R1 are each independently not replaced or replaced by a group selected from the group consisting of -O-, -N(CH3)- and -NH-; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted; said R 2-1 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino; V is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halo-C1-C6 alkyl or halo-C1-C6 alkoxy; Y is -N(R Y1 )-; R Y1 is selected from hydrogen and C1-C6 alkyl; L is -L3-L2-L 1c -or-Tr-L3-L2-L 1c -; Tr is selected from: L 1c Selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from the group consisting of peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, -glycine-glutamine-, -glycine-aspartic acid-, -glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine-, and -glycine-glycine-glycine-glycine-; n is an integer or decimal from 1 to 10; Pc is a ligand.

61. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 60, wherein: The ligand-drug conjugate is selected from the following structural formula: n is an integer or decimal from 1 to 10; Pc is a ligand.

62. The ligand-drug conjugate according to any one of claims 40 to 61, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: n is an integer or decimal from 2 to 8.

63. The ligand-drug conjugate according to any one of claims 40 to 62, or its isomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, wherein: Pc is an antibody or an antigen-binding fragment thereof.

64. The ligand-drug conjugate according to claim 63, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: The Pc targets GPC3, Trop2 or HER2; preferably, the antibody is selected from: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

65. The ligand-drug conjugate, its isomer, or mixture thereof, or pharmaceutically acceptable salt thereof according to any one of claims 40 to 64, wherein: The ligand-drug conjugate is selected from the following structural formula: n is an integer or decimal from 1 to 10.

66. A compound or conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (III-1B): in, X is selected from -S- and -O-; R1 is selected from the group consisting of one or more R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; Wherein, the methylene units of R1 are each independently not replaced, or are replaced by a group selected from the following groups: -O-, -N(R 1-2 )-, -C(=O)- and -NHC(=O)-, R 1-2 Selected from: hydrogen or C1-C6 alkyl; Ring A is selected from phenyl or heteroaryl; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by one or more R 2-1 Substituted, the R 2-1 are each independently selected from: hydrogen, halogen, hydroxyl or amino; V is selected from -(C(R V1 )(R V2 )) p -, where R A1 and R A2 Each independently selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl, the C1-C3 alkyl and C1-C3 cycloalkyl are each independently optionally substituted by one or more R V3 Substituted, the R V3 Each is independently selected from: hydrogen, halogen, hydroxyl or amino; Wherein, the methylene units of V are each independently not substituted, or are replaced by a group selected from the following groups: -O-, -N(R V4 )-、-C(=O)-、-N(R V4 )C(=O)-、 R V4 selected from H, C1-C6 alkyl and Y is selected from -N(R Y1 )-、-O-、-S-、 R Y1 is selected from hydrogen and C1-C6 alkyl; q is selected from 0, 1, 2 or 3; p is selected from 1, 2, 3, 4, 5, 7 and 8; L W For --Tr-L3-L2-L1, Tr is selected from and single bonds; L1 is selected from L2 is selected from and a single bond, wherein q is an integer from 1 to 20; L3 is selected from a single bond or the following peptide residues: -valine-cysteine-, -valine-citrulline-, -valine-lysine-, -valine-alanine-, -alanine-alanine-, -glycine-glycine-, -glycine-glutamate-, glycine-glutamine, glycine-aspartic acid-, glycine-asparagine-, -glycine-glycine-glycine-, -glycine-phenylalanine-glycine-, -alanine-alanine-alanine-, -glycine-glutamate-glycine-, -glycine-glutamate-serine-, -glycine-glycine-phenylalanine-glycine- and -glycine-glycine-glycine-glycine-.

67. The compound according to claim 66, or its conjugate, or its isomer, or its mixture, or its pharmaceutically acceptable salt, wherein: The compounds are shown in formula (III-3A-1) and (III-3A-2): in, X is selected from -S- and -O-; R1 is selected from the group consisting of 1, 2 or 3 R 1-1 Substituted C1-C6 alkyl, R 1-1 Each is independently selected from the group consisting of hydrogen, chlorine, bromine, iodine, hydroxyl, amino, C1-C3 alkyl and C3-C6 cycloalkyl; wherein the methylene units of R1 are each independently not replaced or replaced by a group selected from the group consisting of -O-, -N(CH3)- and -NH-; R2 is selected from hydrogen, chlorine, bromine, iodine, hydroxyl, amino, mercapto, C1-C3 alkyl and C1-C3 alkoxy, wherein the C1-C3 alkyl and C1-C3 alkoxy are each independently optionally substituted by 1, 2 or 3 R 2-1 Substituted; said R 2-1 are each independently selected from the group consisting of: hydrogen, halogen, hydroxyl, and amino; V is selected from C1-C8 alkylene, preferably C1-C6 alkylene, wherein one or more carbon atoms in the C1-C8 alkylene and C1-C6 alkylene may be optionally replaced by a heteroatom selected from N, O or S, and the C1-C8 alkylene and C1-C6 alkylene are optionally substituted by one or more substituents selected from the following: -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkyl, oxo, hydroxy, C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halo-C1-C6 alkyl or halo-C1-C6 alkoxy; Y is -N(R Y1 )-; R Y1 is selected from hydrogen and C1-C6 alkyl; L W Selected from 68. A compound or conjugate thereof, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, selected from:

69. The compound according to claim 37, which is a compound of formula (A-1), or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof, in, R A-1 Selected from R A2-1 -Lx-; Lx is selected from a single bond, C1-C6 alkylene, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, or -N(C1-C6 alkylene)-C(O)-, R A2-1 is an aryl, heteroaryl or heterocyclic group, preferably a phenyl, pyridyl or isobenzofuranone group, and R A2-1 Optionally substituted by one or more substituents R A2-2 replaced by where R A2-2 Each is independently selected from -C1-C6 alkyl, amino, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, -C1-C6 alkylamino, -C1-C6 alkyl-NH(C1-C6 alkyl), -C1-C6 alkyl-N(C1-C6 alkyl)2, hydroxy, -C1-C6 alkoxy, -C1-C6 alkylhydroxy, -C1-C6 alkyl-C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy or -C(O)NR e R f , and R e and R f are each independently selected from H or C1-C6 alkyl, or R e and R f Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered nitrogen-containing heterocyclic ring, such as tetrahydropyrrolyl or piperidinyl, wherein the 5- or 6-membered nitrogen-containing heterocyclic ring is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy, oxo, amino, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2, R2 is selected from -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 alkylenehydroxy or halogenated C1-C6 alkyl; W is selected from -C1-C6 alkylene-, -O-C1-C6 alkylene-; B is phenyl, and B is optionally substituted by one or more substituents selected from the group consisting of halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, and Z is -NH(R Z-1 ), the R Z-1 Selected from H, -C1-C6 alkyl or halogenated C1-C6 alkyl; L is a linker unit, preferably L is a linker unit as defined in any one of claims 26 to 30, wherein the wavy line indicates that the linker unit is connected to the ligand through the nitrogen atom or carbon atom on the L group.

70. The compound according to claim 69, wherein the linker unit L is selected from:

71. A compound selected from the group consisting of the following compounds, or tautomers, enantiomers, diastereomers, or mixtures of isomers thereof, or pharmaceutically acceptable salts or solvates thereof:

72. The ligand-drug conjugate according to claim 3, which is a ligand-drug conjugate of formula (A-2), or a tautomer, enantiomer, diastereomer, or isomer mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, in, R A-1 , W, B, Z and R2 are as defined in claim 69, L is a linker unit as defined in claim 70, n is an integer or decimal of 1-10, preferably n is an integer or decimal of 2-8, Pc is a ligand, preferably Pc is an antibody or an antigen-binding fragment thereof.

73. The ligand-drug conjugate of claim 72, wherein Pc is an antibody or antigen-binding fragment thereof targeting GPC3, Trop2 and HER2; preferably, the antibody is selected from the group consisting of: Trastuzumab, Pertuzumab, Sacituzumab and Codrituzumab.

74. A pharmaceutical composition comprising the ligand-drug conjugate of any one of claims 1-36 or 40-65, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, or comprising the compound or ligand-drug conjugate of any one of claims 69-73, or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof.

75. A method of modulating immune system function comprising administering a ligand-drug conjugate of any one of claims 1-36 or 40-65, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof; or administering a compound or ligand-drug conjugate of any one of claims 69-73, or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof; or administering the pharmaceutical composition of claim 74.

76. The ligand-drug conjugate of any one of claims 1-36 or 40-65, or an isomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof; or the compound or ligand-drug conjugate of any one of claims 69-73, or a tautomer, enantiomer, diastereomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt or solvate thereof; or the use of the pharmaceutical composition of claim 74 in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

77. The use according to claim 76, wherein the disease and / or condition comprises a disease and / or condition associated with Toll-like receptor (TLR) signal transduction.

78. The use according to claim 76 or 77, wherein the disease and / or symptom is selected from the group consisting of tumors, autoimmune diseases, inflammation, sepsis, allergies, asthma, transplant rejection, graft-versus-host disease, immunodeficiency, and viral infections.

79. The method of any one of claims 76 to 78, wherein the disease and / or condition is selected from the group consisting of melanoma, lung cancer, liver cancer, basal cell carcinoma, kidney cancer, myeloma, biliary tract cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, rectal cancer, head and neck cancer, peritoneal tumors, fallopian tube cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, lymphoma, sarcoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, skin cancer, and thyroid cancer.

80. The method of claim 76 , wherein the disease and / or symptom is selected from the group consisting of infections caused by viruses such as dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HPV (human papillomavirus), RSV (respiratory syncytial virus), SARS-CoV (severe acute respiratory syndrome coronavirus), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), MERS-CoV (Middle East respiratory syndrome coronavirus), and influenza virus.

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