Compound capable of being used for coupling reaction and conjugate thereof

Abstract

The present application provides compounds useful in coupling reactions and conjugates thereof. The invention also provides synthetic intermediates and synthetic methods of the compound and the conjugate thereof, and application of the conjugate in preparation of drugs for preventing or treating tumor diseases.

Description

Compounds and conjugates thereof that can be used in coupling reactions

[0001] This application is based on the application with CN application number 202211509417.1 and application date of November 29, 2022, the application with CN application number 202211536550.6 and application date of December 2, 2022, and the application with CN application number 202211573193.0 and application date of December 8, 2022, and claims their priority. The disclosed contents of these CN applications are hereby introduced into this application as a whole. Technical Field

[0002] The present application relates to a class of compounds that can be used for coupling reactions and their conjugates, methods for preparing the compounds and conjugates, pharmaceutical compositions of the conjugates, and applications of the conjugates in treating tumor-related diseases. Background Art

[0003] In recent years, antibody-drug conjugates (ADCs) have become a hot topic in precision cancer treatment, offering hope for the future. Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies targeting specific antigens and small molecule cytotoxic drugs linked via a linker. They combine the powerful anti-tumor effects of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. As of the approval of Zynlonta in April 2021, 12 ADCs have been approved for marketing worldwide, including seven for the treatment of hematologic malignancies and five for the treatment of solid tumors.

[0004] Antibody-drug conjugates consist of antibodies, linkers, and payloads. The conjugation methods for antibodies and drug-linkers (payload-linkers) are mainly divided into non-site-specific conjugation and site-specific conjugation. In the early days, non-site-specific conjugation methods were used, mainly consisting of lysine conjugation and cysteine ​​conjugation. The drug was directly conjugated to the amino acid residues on the antibody using chemical methods, without involving the transformation or modification of the antibody. The number of conjugated toxin molecules and the conjugation sites could not be determined, and the uniformity was poor. The currently commonly used site-specific conjugation method is to perform specific conjugation through genetic engineering sites or special linkers to achieve more uniform conjugation and connect cytotoxins at specific sites. Antibody-drug conjugates produced by site-specific conjugation can reduce fluctuations in efficacy, pharmacokinetics, and CMC quality control caused by different conjugation sites and the number of conjugations.

[0005] Currently, common site-specific coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and ThioBridge technology. Among them, the modification of antibodies by antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody, and at the same time have certain requirements for CMC. In addition, some ThioBridge technologies that use chemical coupling also have certain defects. For example, DBM (dibromomaleimides)-type linkers and other sulfhydryl-containing biological groups undergo replacement effects, which are unstable in plasma, resulting in reduced efficacy and increased toxic side effects (Chem.-Eur.J., 2019, 25, 43-59.). Therefore, the development of new linker structures is still of great significance for the development of antibody-drug conjugates with good efficacy and safety.

[0006] Summary of the Invention

[0007] One purpose of the present application is to provide a new type of linker for chemical coupling. This type of linker has high reactivity, mild coupling conditions, simple operation, can achieve site-specific coupling, and the resulting bioactive conjugate has good uniformity and plasma stability, and clear in vitro and in vivo efficacy.

[0008] Compound

[0009] In a first aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula I:

[0010] in:

[0011] X is a leaving group, such as F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenol ester, fluorophenol ester, C 1-6 Alkylsulfonyl or

[0012] Y is absent or selected from substituted or unsubstituted C 1-6 Alkylene, sulfonyl and carbonyl, when substituted, the C 1-6 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0013] Ring A is selected from substituted or unsubstituted C 6-10Aromatic ring, substituted or unsubstituted 5-12 membered aromatic heterocyclic ring and substituted or unsubstituted 5-12 membered heterocyclic ring, when substituted, the C 6-10 Aromatic rings, 5-12 membered aromatic heterocyclic rings and 5-12 membered heterocyclic rings are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Substitution with haloalkyl, carboxyl, polyethylene glycol, amino acid, phosphate, sulfonic acid, amino, azide, and alkynyl substituents;

[0014] Q is absent or consists of one or more of the following substituted or unsubstituted groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of which is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0015] Z1 is absent or selected from substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl, amide, substituted or unsubstituted -CH2- and substituted or unsubstituted C 2-6 Alkyne, when substituted, the phenyl, 5-6 membered heteroaryl, -CH2- and C 2-6 Alkyne groups are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0016] W1 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O)p- and -(OCH2CH2)p- one or more, p is an integer of 1-20, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0017] J1 is selected from -COOH, -NH2, substituted -NH2, 3-10 membered nitrogen-containing heterocyclic group, substituted 3-10 membered nitrogen-containing heterocyclic group, alkynyl, 8-16 membered alkynyl ring group, substituted 8-16 membered alkynyl ring group, azido, tetrazine, hydroxylamine, aldehyde, ketone, sulfonylurea, isocyanate, thioisocyanate, maleimide and hydroxyl, wherein "substituted" refers to being independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 The substituents of the haloalkyl group are substituted.

[0018] In some embodiments, Ring A is selected from substituted or unsubstituted C 6-10 Aromatic ring, substituted or unsubstituted 5-12 membered aromatic heterocyclic ring and substituted or unsubstituted 5-12 membered heterocyclic ring, when substituted, the C 6-10 The aromatic ring, the 5- to 12-membered aromatic heterocyclic ring, and the 5- to 12-membered heterocyclic ring are substituted with substituents independently selected from carboxyl, polyethylene glycol, amino acid, phosphoric acid, sulfonic acid, amino group, azide, and alkynyl groups.

[0019] In some embodiments, X is a leaving group, such as Cl, Br, I, OMs, OTs, OTf, or

[0020] Y is absent or is a carbonyl group;

[0021] Ring A is selected from substituted or unsubstituted C 6-10 Aromatic ring, substituted or unsubstituted 5-12 membered aromatic heterocyclic ring and substituted or unsubstituted 5-12 membered heterocyclic ring, when substituted, the C 6-10 Aromatic rings, 5-12 membered aromatic heterocyclic rings and 5-12 membered heterocyclic rings are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0022] Q is absent or is substituted or unsubstituted -C(=O)NH-, when substituted, said -C(O)-NH- is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0023] Z1 is absent or is substituted or unsubstituted -CH2- or substituted or unsubstituted C 2-6 Alkynylidene, when substituted, the -CH2- or C2-6 Alkyne groups are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0024] W1 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O)p- and -(OCH2CH2)p- one or more, p is an integer of 1-10, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0025] J1 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group.

[0026] In some embodiments, X is selected from a leaving group such as F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenolate, fluorophenolate, C 1-6 Alkylsulfonyl or

[0027] Y is absent or selected from C 1-6 Alkylene, sulfonyl, carbonyl;

[0028] Ring A is selected from substituted or unsubstituted C 6-10 Aromatic ring, 5-12 membered aromatic heterocycle or 5-12 membered heterocycle; substituents are selected from carboxyl, polyethylene glycol, amino acid, phosphoric acid, sulfonic acid, amino, azide, alkynyl;

[0029] Q is absent or is a fragment consisting of one or more of the following groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne; preferably, Z2 is absent or selected from a fragment consisting of one or more of the following groups: -NH-, -CH2-, carbonyl or C 2-6 Alkynylidene;

[0030] Z1 is absent or selected from phenyl, 5-6 membered heteroaryl, amide, -CH2- or C 2-6 Alkynylidene;

[0031] W1 does not exist or is selected from C1-10 One or more of alkylene, -(CH2CH2O)p-, and -(OCH2CH2)p-; p is an integer from 1 to 20;

[0032] J1 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, an alkynyl group, an 8-16 membered alkynyl-containing ring group, an azido group, a tetrazine group, a hydroxylamine group, an aldehyde group, a ketone group, a sulfonylurea group, an isocyanate group, a thioisocyanate group, a maleimide group or a hydroxyl group;

[0033] p is an integer from 1 to 10.

[0034] In some embodiments, X is selected from one or more of Cl, Br, I, OMs, OTs, and OTf.

[0035] In some embodiments, Y is absent or is C 1-6 Alkylene.

[0036] In some embodiments, Q is absent or is -C(=O)-NH- or -NH-C(=O)-.

[0037] In some embodiments, Z1 is absent or is C 2-6 Alkynylidene.

[0038] In some embodiments, W1 is absent or selected from C 1-10 One or more of alkylene and -(CH2CH2O)p-.

[0039] In some embodiments, W1 is C 1-10 Alkylene; preferably C 1-6 Alkylene, more preferably C 1-3 Alkylene.

[0040] In some embodiments, J1 is -COOH, alkynyl, an 8-16 membered alkynyl-containing ring, an azide group, a tetrazine group, a hydroxylamine group, an aldehyde group, a ketone group, an isocyanate group, a thioisocyanate group, or a maleimide group.

[0041] In some embodiments, ring A is selected from a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle or a 5-12 membered nitrogen-containing heterocycle. When substituted, the 5-12 membered nitrogen-containing aromatic heterocycle and the 5-12 membered nitrogen-containing heterocycle are substituted by substituents independently selected from carboxyl, polyethylene glycol, amino acid, phosphoric acid, sulfonic acid, amino group, azide and alkynyl groups; preferably, ring A is selected from a 5-12 membered nitrogen-containing aromatic heterocycle or a 5-12 membered nitrogen-containing heterocycle that is unsubstituted or substituted with an oxo group or -COOH.

[0042] In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0043] In some embodiments, X is selected from Cl, Br, I, OMs, OTs, OTf, C 1-6 Alkylsulfonyl or

[0044] Y does not exist;

[0045] Ring A is selected from a 5-12 membered nitrogen-containing aromatic heterocycle or a 5-12 membered nitrogen-containing heterocycle which is unsubstituted or substituted with an oxo group or -COOH;

[0046] Q is absent or is -C(=O)-NH-;

[0047] Z1 does not exist or is -CH2-;

[0048] W1 does not exist or is selected from C 1-10 One or more of alkylene and -(CH2CH2O)p-;

[0049] J1 is -COOH;

[0050] p is an integer from 1 to 10.

[0051] In some embodiments, Formula I is selected from the following structures:

[0052] In some embodiments, the compound of Formula I has the following structure:

[0053] wherein p is an integer of 1-10, and J1 is -COOH or -NH2.

[0054] In some embodiments, the compound of Formula I has the following structure:

[0055] In a second aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula II:

[0056] Wherein, B1 and B2 are each independently a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle. When substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is substituted by a substituent consisting of one or more of the following groups: hydrogen, halogen, hydroxyl, -CN, substituted or unsubstituted C 1-10 Alkylene, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, carboxyl, substituted or unsubstituted amide, substituted or unsubstituted carbamoyl, substituted or unsubstituted polyethylene glycol, alkynyl and azido, when substituted, the C1-10 Alkylene, amido, carbamoyl and polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0057] Y1, Y2 and Y3 are independently selected from C(R) and N;

[0058] Z2 is absent or selected from the group consisting of one or more of the following substituted or unsubstituted functional groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0059] Preferably, Z2 is absent or selected from the group consisting of one or more substituted or unsubstituted functional groups: -NH-, -CH2-, carbonyl or C 2-6 Alkyne, when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0060] W2 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 The substituent of the haloalkyl group is substituted; p is an integer of 1 to 10;

[0061] J2 is selected from -COOH, -N(R)(R'), a substituted or unsubstituted 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, a substituted or unsubstituted 8-16 membered alkynyl ring group, an azido group, a tetrazine group, a substituted or unsubstituted hydroxylamine group, an aldehyde group, a ketone group, an isocyanate group, a thioisocyanate group, a maleimide group and a hydroxyl group, and when substituted, the 3-10 membered nitrogen-containing heterocyclic group, the 8-16 membered alkynyl ring group and the hydroxylamine group are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0062] R and R' are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Halogenated alkyl.

[0063] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5-12 membered nitrogen-containing aromatic heterocycle; the 5-12 membered nitrogen-containing aromatic heterocycle is optionally substituted by one or more fragments consisting of the following functional groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, azide;

[0064] Y1, Y2 and Y3, at each occurrence, are independently selected from CH and N;

[0065] Z2 is absent or is selected from a fragment consisting of one or more of the following groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne; preferably, Z2 is absent or selected from a fragment consisting of one or more of the following groups: -NH-, -CH2-, carbonyl or C 2-6 Alkynylidene;

[0066] W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p -or-(OCH2CH2) p - one or more of;

[0067] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, an 8-16 membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxylamine group, an aldehyde group, a ketone group, an isocyanate group, a thioisocyanate group, a maleimide group or a hydroxyl group;

[0068] p is an integer from 1 to 10.

[0069] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle, when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is substituted with a substituent consisting of one or more of the following groups: hydrogen, substituted or unsubstituted C 1-10 Alkylene, carboxyl, substituted or unsubstituted amide, substituted or unsubstituted carbamoyl, substituted or unsubstituted polyethylene glycol, alkynyl, azido, when substituted, the C 1-10 Alkylene, amide, carbamoyl, polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 The substituents of the haloalkyl group are substituted.

[0070] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle; when substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted with a substituent selected from the group consisting of hydrogen, halogen, hydroxyl, -CN, C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, or -OC 1-6 Halogenated alkyl.

[0071] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5-12 membered nitrogen-containing aromatic heterocycle; the 5-12 membered nitrogen-containing aromatic heterocycle is optionally substituted with one or more fragments consisting of the following functional groups: hydrogen, C 1-10 Alkylene group, carboxyl group, amide group, carbamoyl group, polyethylene glycol, alkynyl group, azide group.

[0072] Y1, Y2 and Y3, at each occurrence, are independently selected from CH and N;

[0073] Z2 is absent or is selected from a fragment consisting of one or more of the following groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne; preferably, Z2 is absent or selected from a fragment consisting of one or more of the following groups: -NH-, -CH2-, carbonyl or C 2-6 Alkynylidene;

[0074] W2 does not exist or is selected from C 1-10Alkylene, -(CH2CH2O) p -or-(OCH2CH2) p - one or more of;

[0075] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, an 8-16 membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxylamine group, an aldehyde group, a ketone group, an isocyanate group, a thioisocyanate group, a maleimide group or a hydroxyl group;

[0076] p is an integer from 1 to 10.

[0077] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle. When substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted by a substituent selected from the following: hydrogen, halogen, hydroxyl, -CN, substituted or unsubstituted C 1-10 Alkylene, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, carboxyl, substituted or unsubstituted amide, substituted or unsubstituted carbamoyl, substituted or unsubstituted polyethylene glycol, alkynyl and azido, when substituted, the C 1-10 Alkylene, amido, carbamoyl and polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Preferably, B1 and B2 are each independently a single bond or a substituted or unsubstituted pyrimidine ring, and when substituted, the pyrimidine ring is selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0078] Y1, Y2 and Y3 are each independently selected from CH and N;

[0079] Z2 is absent or selected from the group consisting of one or more of the following substituted or unsubstituted functional groups: -NH-, -CH2-, carbonyl, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0080] W2 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups;

[0081] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group; and

[0082] p is an integer from 1 to 10.

[0083] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle, wherein the substituents are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 Preferably, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted pyrimidine ring, and the substituents are independently selected from: hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 Halogenated alkyl.

[0084] Each of Y1, Y2 and Y3 is independently selected from CH and N;

[0085] Z2 is absent or consists of one or more of the following substituted or unsubstituted groups: -NH-, -CH2-, carbonyl, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, the substituents are independently selected from: hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 alkyl halide;

[0086] W2 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O) p -or-(OCH2CH2) p-, wherein one or more of the substituents are independently selected from the group consisting of hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 alkyl halide;

[0087] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group or a hydroxyl group; and

[0088] p is an integer from 1 to 10.

[0089] In some embodiments, B1 and B2 are each independently selected from a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle. When substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted by a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, azide;

[0090] Y1, Y2 and Y3 are independently selected from CH and N;

[0091] Z2 is absent or selected from a group consisting of one or more of the following functional groups: -NH-, -CH2-, carbonyl and C 2-6 Alkynylidene;

[0092] W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of;

[0093] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group; and

[0094] p is an integer from 1 to 10.

[0095] In some embodiments, B1 and B2 are each independently selected from substituted or unsubstituted pyridyl and pyrimidinyl, and when substituted, the pyridyl and pyrimidinyl are substituted with a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0096] Y1, Y2 and Y3 are each independently selected from CH and N;

[0097] Z2 is absent or selected from a group consisting of one or two of the following functional groups: -NH- and carbonyl;

[0098] W2 does not exist or is selected from C 1-10Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of;

[0099] J2 is -COOH or -NH2; and

[0100] p is an integer from 1 to 10.

[0101] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5-6 membered nitrogen-containing aromatic heterocycle; preferably, B1 and B2 are each independently selected from a single bond, an oxazole ring, a thiazole ring or a pyrimidine ring.

[0102] In some embodiments, B1, at each occurrence, is independently selected from a single bond or a 5-6 membered nitrogen-containing aromatic heterocycle; preferably, B1, at each occurrence, is independently selected from a single bond or a 5-6 membered nitrogen-containing aromatic heterocycle; further preferably, B1, at each occurrence, is independently selected from a single bond, an oxazole ring, a thiazole ring or a pyrimidine ring.

[0103] In some embodiments, Y1, Y2, and Y3 are all N; or Y1 is CH, Y2, and Y3 are all N; or Y1 is N, Y2, and Y3 are all CH; or Y1, Y2, and Y3 are all CH.

[0104] In some embodiments, Z2 is absent or is selected from the group consisting of one or more substituted or unsubstituted functional groups: -NH-, -CH2-, carbonyl or C 2-6 Alkyne, when substituted, each of said functional groups is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 The substituents of the haloalkyl group are substituted.

[0105] In some embodiments, Z2 is absent or is selected from the group consisting of one or two of the following functional groups: -NH- and carbonyl.

[0106] In some embodiments, Z2 is absent or is an amide group (-C(=O)NH-).

[0107] In some embodiments, Z2 is absent or is carbamoyl.

[0108] In some embodiments, Z2 is absent or is C 2-6 Alkynylidene.

[0109] In some embodiments, W2 is absent or is C 1-10 Alkylene.

[0110] In some embodiments, J2 is selected from -COOH or -NH2.

[0111] In some embodiments, J2 is -COOH.

[0112] In some embodiments, B1, at each occurrence, is independently selected from a single bond or a pyrimidine ring;

[0113] Y1, Y2 and Y3 are all CH;

[0114] Z2 does not exist or is -C(=O)NH-;

[0115] W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p One or more of;

[0116] J2 is -COOH;

[0117] p is an integer from 1 to 10.

[0118] In some embodiments, B1 and B2 are each independently selected from a single bond or a pyrimidine ring;

[0119] Y1, Y2 and Y3 are all CH;

[0120] Z2 does not exist or is -C(=O)NH-;

[0121] W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p One or more of;

[0122] J2 is -COOH;

[0123] p is an integer from 1 to 10.

[0124] In some embodiments, B1 and B2 are each independently selected from a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle; when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is substituted by a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0125] Preferably, B1 and B2 are each independently selected from a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle; when substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted by a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0126] Preferably, B1 and B2 are each independently selected from substituted or unsubstituted pyridyl and pyrimidinyl; when substituted, the pyridyl or pyrimidinyl is substituted by a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0127] Preferably, B1 and B2 are each independently selected from pyridyl or pyrimidinyl;

[0128] and / or

[0129] Z2 is absent or is selected from a group consisting of one or more of the following functional groups: -NH-, -CH2- and carbonyl;

[0130] Preferably, Z2 is absent or is selected from a group consisting of one or more of the following functional groups: -NH- or carbonyl;

[0131] Preferably, Z2 is absent or is -C(=O)NH-;

[0132] and / or

[0133] J2 is -COOH or -NH2;

[0134] Preferably, J2 is -COOH;

[0135] and / or

[0136] p is an integer from 3 to 8.

[0137] In some embodiments, B1 and B2 are each independently selected from a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle; when substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted by a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0138] Each of Y1, Y2 and Y3 is independently selected from CH and N;

[0139] Z2 is absent or is selected from a fragment consisting of one or more of the following groups: -NH-, -CH2-, carbonyl or C 2-6 Alkynylidene;

[0140] W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p -or-(OCH2CH2) p - one or more of;

[0141] J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group or a hydroxyl group; and

[0142] p is an integer from 1 to 10;

[0143] Preferably,

[0144] B1 and B2 are each independently selected from substituted or unsubstituted pyridyl, pyrimidinyl; when substituted, the pyridyl or pyrimidinyl is substituted by one or more substituents independently selected from the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups;

[0145] Y1, Y2 and Y3 are each independently selected from CH and N;

[0146] Z2 is absent or selected from a group consisting of one or more of the following groups: -NH- or carbonyl;

[0147] J2 is selected from -COOH or -NH2; and

[0148] p is an integer from 1 to 10.

[0149] In some embodiments, the compound of Formula II has the following structure:

[0150] p is an integer from 1 to 10.

[0151] In some embodiments, p is an integer from 1 to 8; more preferably, p is an integer from 1 to 5.

[0152] In some embodiments, the compound of Formula II has the following structure:

[0153] In some embodiments, the compound of formula II has the following structure, and p is an integer from 1 to 10:

[0154] In some embodiments, the compound of Formula II has the following structure:

[0155] In some embodiments, the compound of formula II has the following structure:

[0156] In some embodiments, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula IIA:

[0157] Wherein, T1 and T2 are leaving groups; T1 and T2 may be the same or different;

[0158] J3 is selected from -COOR3, -NH2, 3-10 membered nitrogen-containing heterocyclic group, sulfonylurea group, alkynyl group, 8-16 membered alkynyl ring group, azido group, tetrazine group, hydroxylamine group, aldehyde group, ketone group, isocyanate group, thioisocyanate group, maleimide group and hydroxyl group; R3 is selected from H, C 1-6 Alkyl, C 6-10 Aryl, 3-8 membered heterocyclic group and 5-10 membered heterocyclic group; the C 1-6 Alkyl, C 6-10 Aryl, 3-8 membered heterocyclic group and 5-10 membered heterocyclic group are optionally substituted by one or more selected from hydroxyl, halogen, C 1-6 Substitution of alkoxy groups;

[0159] B1, B2, Y1, Y2, Y3, Z2, and W2 are as described in any one of the above aspects.

[0160] In some embodiments, T1 and T2 are each independently selected from halogen, OMs, OTs, OTf, nitro, and the following groups optionally substituted with one or more R4: alkyl sulfide, aryl sulfide, heteroaryl sulfide, alkyl sulfoxide, aryl sulfoxide, heteroaryl sulfoxide, alkyl sulfonyl, aryl sulfonyl and heteroaryl sulfonyl; wherein R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 membered aryl, and 5-12 membered heteroaryl;

[0161] Preferably, T1 and T2 are each independently selected from the following groups optionally substituted by one or more R4: alkyl sulfide, aryl sulfide, heteroaryl sulfide, alkyl sulfonyl, aryl sulfonyl and heteroaryl sulfonyl; wherein R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 membered aryl, and 5-12 membered heteroaryl;

[0162] Preferably, T1 and T2 are each independently selected from alkyl sulfide and alkyl sulfonyl groups optionally substituted by one or more R4; R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl and C 1-6 alkoxy;

[0163] Preferably, said T1 and T2 are each independently selected from C optionally substituted by one or more R4 1-6 Alkyl sulfide or C 1-6 Alkylsulfonyl; R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl and C 1-6 alkoxy;

[0164] Preferably, T1 and T2 are each independently C 1-6 Alkyl sulfide or C 1-6 alkylsulfonyl;

[0165] Preferably, T1 and T2 are each independently -S(O)2-CH3 or -S-CH3.

[0166] In some embodiments, J3 is -COOR3 or -NH2; R3 is selected from H, D, C 1-6 Alkyl, C 6-10 Aryl, 3-8 membered heterocyclic group and 5-10 membered heterocyclic group; the C 1-6 Alkyl, C 6-10 Aryl, 3-8 membered heterocyclic group and 5-10 membered heterocyclic group are optionally substituted by one or more selected from hydroxy, halogen and C 1-6 Substitution of alkoxy groups;

[0167] Preferably, J3 is -COOR3 or -NH2; R3 is selected from H, D and C 1-6 Alkyl; the C 1-6 The alkyl group is optionally substituted with one or more radicals selected from hydroxy, halogen and C 1-6 Substitution of alkoxy groups;

[0168] Preferably, the J3 is selected from -COOC 1-6 Alkyl, -COOH and -NH2; the C 1-6 The alkyl group is optionally substituted with one or more radicals selected from hydroxy, halogen and C 1-6 Substitution of alkoxy groups;

[0169] Preferably, J3 is selected from -COOCH3, -COOH and -NH2.

[0170] In some embodiments, the compound of Formula IIA has the following structure:

[0171] In some embodiments, the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound is selected from:

[0172] (1) 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoic acid;

[0173] (2) 3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzoic acid;

[0174] (3) tert-Butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleate;

[0175] (4) 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid;

[0176] (5) 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid;

[0177] (6) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oic acid;

[0178] (7) 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid;

[0179] (8) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid;

[0180] (9) 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid;

[0181] (10) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid;

[0182] (11) 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinic acid;

[0183] (12) 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid;

[0184] (13) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid;

[0185] (14) 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid;

[0186] (15) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid;

[0187] (16) 2,2″-bis(methylthio)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid;

[0188] (17) 2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid;

[0189] (18) 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidinyl]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; or

[0190] (19) 1-(2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid.

[0191] In a third aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula III:

[0192] in:

[0193] V1 is a group formed when J1 and L are connected in the compound of formula I of the first aspect of the present application; preferably, V1 is selected from -C(=O)-, -N(R1)-, -O-, a 3-10 membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; further preferably, V1 is -C(O)- or -N(R1)-, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl;

[0194] L is a linker connecting V1 and E';

[0195] E' is selected from H, -NHCH2-Lg, -COOH, Wherein, Lg represents a leaving group, such as Cl, Br, I, OMs, OTs, OTf or

[0196] X, Y, A, Q, Z1, and W1 are as defined in any one of the first aspects above.

[0197] In some embodiments, L is selected from one or more of the following groups: 1-6 Alkylene, -N(R6)-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Phe, Ala, Asn, D-Val-Leu-Lys, Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pr o, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu , D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Ly s, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Where R6 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl, s is an integer of 1-10.

[0198] In some embodiments, L is selected from one or more of the following groups: Val, Cit, Gly, Phe, Ala, Val-Cit, Val-Ala, Gly-Gly-Phe-Gly,

[0199] In some embodiments, L is selected from a structure consisting of one or more of the following:

[0200] s is an integer from 1 to 10;

[0201] In some embodiments, L is selected from

[0202] In some embodiments, L is selected from the following structures:

[0203] In some embodiments, L is selected from

[0204] In a fourth aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula IV:

[0205] in,

[0206] V2 is a group obtained by connecting J2 and L in the compound of formula II of the second aspect of the present application; preferably, V2 is selected from -C(=O)-, -N(R2)-, -O-, a 3-10 membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; further preferably, V2 is -C(O)- or -N(R2)-, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl;

[0207] B1, B2, Y1, Y2, Y3, Z2 and W2 are as defined in any one of the second aspects above;

[0208] L and E' are as defined in any one of the third aspects above.

[0209] In a fifth aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula V:

[0210] in:

[0211] E is selected from a single bond, -NH-CH2-,

[0212] D is a fragment of a biologically active molecule (e.g., a cytotoxic drug);

[0213] X, Y, A, Q, Z1, W1 are as defined in any one of the first aspects above;

[0214] V1 and L are as defined in any one of the third aspects.

[0215] In some embodiments, the biologically active molecule is selected from the group consisting of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0216] In some embodiments, the biologically active molecule is selected from the following group: microtubule inhibitors auristatins, maytansines; DNA intercalators pyrrolobenzodiazepines (PBDs); DNA topoisomerase inhibitors, for example, topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or topoisomerase II inhibitors (such as adriamycin, doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); RNA polymerase inhibitors α-amanitin, etc.; and pharmaceutically acceptable salts, esters and analogs thereof.

[0217] In some embodiments, the biologically active molecule is selected from the group consisting of: topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan), MMAE, and MMAE derivatives.

[0218] In some embodiments, the biologically active molecule is selected from the group consisting of: MMAE and MMAE derivatives.

[0219] In some embodiments, D is selected from:

[0220] In some embodiments, the compound of formula V is selected from:

[0221] In a sixth aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula VI:

[0222] B1, B2, Y1, Y2, Y3, Z2 and W2 are as defined in any one of the second aspects above;

[0223] L is as defined in any one of the third aspects above;

[0224] V2 is as defined in any one of the fourth aspects above;

[0225] E and D are as defined in any one of the fifth aspects above.

[0226] In some embodiments, the compound of formula VI is selected from:

[0227] In a seventh aspect, the present application provides a bioactive conjugate, the structure of which is shown in Formula VII:

[0228] Wherein, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); n is an integer or decimal selected from 1-10;

[0229] V1 is -C(O)- or -N(R1)-, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl;

[0230] L is a linker between V1 and E;

[0231] E is a structural fragment connecting L and D;

[0232] D is a fragment of a biologically active molecule (e.g., a cytotoxic drug);

[0233] The conjugate When the targeting moiety is an antibody, it indicates the specific connection method between the sulfhydryl group in the antibody and the rest of the conjugate;

[0234] The remaining groups are as defined in any of the preceding aspects.

[0235] Another aspect of the present invention provides a bioactive conjugate, the structure of which is shown in Formula VIII:

[0236] Wherein, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); n is an integer or decimal selected from 1-10;

[0237] V2 is -C(O)- or -N(R2)-, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl;

[0238] L is a linker between V2 and E;

[0239] E is a structural fragment connecting L and D;

[0240] D is a fragment of a biologically active molecule (e.g., a cytotoxic drug);

[0241] The conjugate When the targeting moiety is an antibody, it indicates the specific connection method between the sulfhydryl group in the antibody and the rest of the conjugate;

[0242] The remaining groups are as defined in any of the preceding aspects.

[0243] In some embodiments, the Ab is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receoptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2. BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-D OB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, C CR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANG PTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA, B7H3 and TENB2;.

[0244] Preferably, the Ab is selected from anti-Her2 antibody (eg, trastuzumab), anti-Trop2 antibody (eg, sacituzumab), anti-ROR1 antibody (eg, 19F6_Hu35V1), or anti-B7H3 antibody (eg, 2#8890).

[0245] In some embodiments, L is selected from one or more of the following groups: 1-6Alkylene, -N(R6)-, Carbonyl, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Phe, Ala, Asn, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gl y, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, D-Leu-Ala- Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val- Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Where R6 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl, s is an integer of 1-10;

[0246] Preferably, L is selected from the group consisting of one or more of the following:

[0247] s is an integer from 1 to 10;

[0248] Preferably, L is selected from the following structures:

[0249] Preferably, L is selected from the following structures:

[0250] In some embodiments, E is a single bond, -NH-CH2-,

[0251] In some embodiments, E is -NH-CH2-.

[0252] In some embodiments, the biologically active molecule is selected from the group consisting of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0253] In some embodiments, the biologically active molecule is selected from the following group: microtubule inhibitors auristatins, maytansines; DNA intercalators pyrrolobenzodiazepines (PBDs); DNA topoisomerase inhibitors, for example, topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or topoisomerase II inhibitors (such as adriamycin, doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); RNA polymerase inhibitors α-amanitin, etc.; and pharmaceutically acceptable salts, esters and analogs thereof.

[0254] In some embodiments, the biologically active molecule is selected from the group consisting of: topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan), MMAE, and MMAE derivatives.

[0255] In some embodiments, the biologically active molecule is selected from the group consisting of: MMAE and MMAE derivatives.

[0256] In some embodiments, D is selected from the group consisting of:

[0257] In some embodiments, n is 1-8; more preferably, n is 3-5.

[0258] In some embodiments, the bioactive conjugate structure is as follows, wherein Ab is selected from an anti-Her2 antibody (e.g., trastuzumab), an anti-Trop2 antibody (e.g., sacituzumab), or an anti-ROR1 antibody (e.g., 19F6_Hu35V1), and n1 is 1-8; more preferably 3-5:

[0259] In some embodiments, the structure of the bioactive conjugate is as follows, wherein Ab is selected from anti-Her2 antibody (e.g., trastuzumab), anti-Trop2 antibody (e.g., sacituzumab), anti-ROR1 antibody (e.g., 19F6_Hu35V1) or anti-B7H3 antibody (e.g., 2#8890), n1 is 1-8; more preferably 3-5; x is 1-10; more preferably 3-5:

[0260] In some embodiments, the anti-Her2 antibody described in any of the above items is trastuzumab, an antibody comprising a heavy chain variable region of the heavy chain complementarity determining region of trastuzumab and a light chain variable region of the light chain complementarity determining region of trastuzumab, or an antibody comprising a heavy chain variable region sequence of trastuzumab and a light chain variable region sequence of trastuzumab;

[0261] The anti-Trop2 antibody described in any of the above items is sacituzumab, an antibody comprising a heavy chain variable region of the sacituzumab heavy chain complementarity determining region and a light chain variable region of the sacituzumab light chain complementarity determining region, or an antibody comprising a sacituzumab heavy chain variable region sequence and a sacituzumab light chain variable region sequence; and / or

[0262] The anti-ROR1 antibody described in any one of the above items is:

[0263] i) an antibody defined according to the Chothia numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 3, CDR-H2 of SEQ ID NO: 4, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8;

[0264] ii) an antibody defined according to the AbM numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:10, and CDR-H3 of SEQ ID NO:5, and a light chain variable region comprising CDR-L1 of SEQ ID NO:6, CDR-L2 of SEQ ID NO:7, and CDR-L3 of SEQ ID NO:8;

[0265] iii) an antibody defined according to the Kabat numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8;

[0266] iv) an antibody comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 8, as defined by the IMGT numbering system;

[0267] v) an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2; or

[0268] vi) 19F6_Hu35V1, an antibody containing the heavy chain variable region shown in SEQ ID NO: 1, the light chain variable region shown in SEQ ID NO: 2, the heavy chain constant region shown in SEQ ID NO: 18, and the light chain constant region shown in SEQ ID NO: 19.

[0269] In some embodiments, n1 is 1-6, such as 3-5.

[0270] In some embodiments, x is 1-6, such as 3-5.

[0271] definition

[0272] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of technology or substitutions of equivalent technology that would be apparent to those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

[0273] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methylene, ethylene, propylene or butylene.

[0274] As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, such as 1 to 6, carbon atoms. For example, as used herein, the term "C 1-6 "Alkyl" refers to a linear or branched aliphatic hydrocarbon group of 1 to 6 carbon atoms (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents (such as halogen) (in this case, the group is referred to as "haloalkyl") (for example, CH2F, CHF2, CF3, CCl3, C2F5, C2Cl5, CH2CF3, CH2Cl or -CH2CH2CF3, etc.). The term "C 1-4 "Alkyl" refers to a linear or branched aliphatic hydrocarbon group of 1 to 4 carbon atoms (ie, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl).

[0275] As used herein, the term "alkoxy" is defined as -O-alkyl, wherein alkyl is as defined above. For example, as used herein, the term "C 1-6 "Alkoxy" refers to -OC 1-6 alkyl.

[0276] As used herein, the term "alkoxyalkyl" is defined as an alkyl group substituted with an alkoxy group, said alkyl group being as defined above. For example, as used herein, the term "C 2-6 "Alkoxyalkyl" refers to an alkyl group having 2 to 6 carbon atoms substituted by an alkoxy group.

[0277] As used herein, the term "alkynylene" refers to a divalent hydrocarbon group comprising at least one carbon-carbon triple bond, preferably having 1, 2, 3, 4, 5 or 6 carbon atoms, such as ethynylene, propynylene or butynylene.

[0278] As used herein, the terms "heterocyclyl" and "heterocycle" refer to a saturated or partially unsaturated (i.e., having one or more double bonds and / or triple bonds within the ring) cyclic group wherein at least one ring atom is a heteroatom selected from N, O, and S and the remaining ring atoms are C. For example, a "5-12 membered heterocyclyl" is a saturated or partially unsaturated heterocyclyl having 4-11 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) heteroatoms independently selected from N, O, and S. A "5-12 membered nitrogen-containing heterocyclyl" is a heterocyclyl in which at least one of the ring atoms is N. Examples of heterocyclic groups include, but are not limited to, oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolinyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl. The heterocyclic group may be optionally substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents, and may optionally form a ring structure with one or more aromatic rings or heteroaromatic rings.

[0279] As used herein, the term "aromatic ring" or "aryl" refers to a monocyclic or polycyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring-forming carbon atoms, particularly 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms.

[0280] As used herein, the term "aromatic heterocycle" or "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, and which contains at least one heteroatom which may be identical or different (the heteroatom being, for example, oxygen, nitrogen or sulfur) and, in addition, in each case may be benzo-fused.

[0281] As used herein, the term "halogen" includes F, Cl, Br or I.

[0282] As used herein, the term "sulfonylurea" refers to -SO2-NH-(C=O)-NH2 or -NH-(C=O)-NH-SO2H.

[0283] The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogen atoms on the designated atom are replaced with a group selected from the indicated group, provided that the designated atom's normal valence is not exceeded in the current context and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0284] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or collectively, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

[0285] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0286] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

[0287] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0288] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0289] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( 2 H), tritium ( 3 H)); carbon isotopes (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S).

[0290] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0291] In this article, solid lines (——), solid wedges or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0292] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0293] It should also be understood that certain compounds of the present invention may be used therapeutically in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, or prodrugs that, upon administration to a patient in need thereof, are capable of directly or indirectly providing a compound of the present invention or a metabolite or residue thereof. Therefore, when reference is made herein to a "compound of the present invention," such various derivative forms of the compound are also intended to be encompassed.

[0294] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.

[0295] Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, benzoate, bicarbonate / carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts.

[0296] Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts.

[0297] For a review of suitable salts see Stahl and Wermuth, “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0298] As used herein, the term "ester" refers to esters derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in the form of free acid or alcohol). The compounds of the present invention themselves may also be esters.

[0299] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0300] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0301] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of the compounds of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0302] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity and can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage when administered to the body or thereon. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compounds in vivo. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella). Prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

[0303] The present invention also encompasses compounds of the present invention that contain protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in TW Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0304] Preparation method

[0305] Another aspect of the present invention provides a method for preparing a compound of formula I, comprising the steps of:

[0306] When Y is absent, the compounds of formula I-TM1 and formula I-TM2 of the present invention can be synthesized by the following synthetic routes:

[0307] in:

[0308] X, Z1, W1, J1 are as defined in the general formula above;

[0309] M is a leaving group that undergoes substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, preferably halogen.

[0310] Step 1

[0311] The compound of formula I-SM1 undergoes a substitution reaction with the compound of formula M-Z1-W1-J1 to obtain the compound of formula I-IM1;

[0312] In some embodiments, the reaction is carried out under alkaline conditions;

[0313] In some embodiments, the reaction is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0314] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably acetone.

[0315] Step 2

[0316] The compound of formula I-IM1 undergoes a substitution reaction with sodium azide to obtain a compound of formula I-IM2;

[0317] In some embodiments, the reaction is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0318] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, preferably acetone.

[0319] Step 3

[0320] The compound of formula I-IM2 is subjected to a reduction reaction to obtain a compound of formula I-IM3;

[0321] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from palladium catalysts, platinum catalysts, and rhodium catalysts, preferably palladium catalysts;

[0322] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0323] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.

[0324] Step 4

[0325] By the compound of formula I-IM3 and A dehydration ring-closure reaction occurs to obtain a compound of formula I-TM1;

[0326] In some embodiments, this step is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0327] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from acetonitrile, ethanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably acetonitrile.

[0328] Step 5

[0329] The compound of formula I-TM1 is hydrolyzed to obtain the compound of formula I-TM2;

[0330] In some embodiments, the reaction is carried out under alkaline conditions, including but not limited to PB buffer at pH 7.0, 7.4 or 8.0;

[0331] In some embodiments, the reaction is carried out at a suitable temperature, which is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;

[0332] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to acetonitrile, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, preferably acetonitrile.

[0333] When Y is absent, the compound of formula I-TM3 of the present invention can be synthesized by the following synthetic route:

[0334] in:

[0335] X, Z1, W1, J1 are as defined in the general formula above;

[0336] L is a leaving group that undergoes a substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, and p-toluenesulfonate, preferably halogen or OTf.

[0337] Step 1

[0338] The compound of formula I-IM4 is obtained by coupling reaction of the compound of formula I-IM1;

[0339] In some embodiments, the coupling reaction reagent includes but is not limited to methylboronic acid, trimethylcyclotriboroxine, preferably trimethylcyclotriboroxine;

[0340] In some embodiments, the coupling reaction is carried out under alkaline conditions, and the base includes but is not limited to triethylamine, DIPEA, NMM, sodium tert-butoxide, potassium acetate, sodium acetate, cesium fluoride, potassium fluoride, potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium dihydrogen phosphate, preferably cesium fluoride.

[0341] In some embodiments, the coupling reaction is carried out in the presence of a catalyst, including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd2(dba)3, Pd(PPh3)2Cl2, Pd(PPh3)2Cl2 dichloromethane complex, Pd(dppf)Cl2, Pd(Amphos)Cl2, preferably tetrakistriphenylphosphine palladium.

[0342] In some embodiments, the coupling reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.

[0343] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide and any combination thereof, preferably 1,4-dioxane.

[0344] Step 2

[0345] A compound of formula I-TM3 is obtained by halogenating a compound of formula I-IM4;

[0346] In some embodiments, the halogenating agent includes but is not limited to bromine, N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide, preferably N-bromosuccinimide;

[0347] In some embodiments, the halogenation reaction is carried out in the presence of a catalyst, and the catalyst is benzoyl peroxide.

[0348] In some embodiments, the halogenation reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.

[0349] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (such as carbon tetrachloride, dichloromethane, chloroform, 1,2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (such as toluene, benzene, xylene), water and any combination thereof, preferably carbon tetrachloride.

[0350] When Z2 and W2 do not exist, the compound of formula II-TM1 in the present invention can be synthesized by the following synthetic route:

[0351] in:

[0352] Y1, Y2, Y3, B1, B2, Z2, W2 and J2 are as defined in the general formula above;

[0353] LG is a leaving group for coupling reaction, including but not limited to halogen and trifluoromethanesulfonate, preferably halogen.

[0354] Step 1

[0355] The compound of formula II-IM2 is obtained by coupling reaction of the compound of formula II-IM1;

[0356] In some embodiments, the coupling reaction reagent is

[0357] In some embodiments, the coupling reaction is carried out in the presence of a catalyst, including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd2(dba)3, Pd(PPh3)2Cl2, Pd(PPh3)2Cl2 dichloromethane complex, Pd(dppf)Cl2, Pd(Amphos)Cl2, preferably tetrakistriphenylphosphine palladium.

[0358] In some embodiments, the coupling reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.

[0359] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide and any combination thereof, preferably 1,4-dioxane.

[0360] Step 2

[0361] The compound of formula II-IM2 is subjected to oxidation reaction to obtain the compound of formula II-TM1;

[0362] In some embodiments, the oxidation reaction is carried out in the presence of an oxidizing agent, and the catalyst is meta-chloroperbenzoic acid.

[0363] In some embodiments, the oxidation reaction is carried out at a temperature of 0-120°C, preferably at a temperature of 50-80°C.

[0364] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (such as carbon tetrachloride, dichloromethane, chloroform, 1,2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (such as toluene, benzene, xylene), water and any combination thereof, preferably methanol.

[0365] In addition, the compounds of the present invention can also be prepared in a variety of ways known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below and synthetic methods known in the field of synthetic organic chemistry or variations thereof known to those skilled in the art. Preferred methods include, but are not limited to, those described above. The reaction can be carried out in a solvent or solvent mixture suitable for the reagents and materials used and suitable for achieving the transformation. Those skilled in the art of organic synthesis will understand that the functional groups present on the molecule should be consistent with the proposed transformation. This will sometimes require the following judgment: modifying the order of the synthesis steps or selecting another specific method route relative to one method route to obtain the desired compound of the present invention.

[0366] It will also be appreciated that another major consideration in designing any synthetic route in this area is the proper selection of protecting groups for use in protecting the reactive functional groups present in the compounds described herein. An authoritative description of many alternatives to the trained eye is provided by Greene et al. (Protective Groups in Organic Synthesis, 4th edition, Wiley-Interscience (2006)).

[0367] Unless otherwise indicated, the substituents of the compounds in the above routes are as defined herein. Those skilled in the art will appreciate that one or more steps in the above routes may be omitted depending on the desired product structure. Those skilled in the art may also adjust the order of the reaction steps as needed.

[0368] In some embodiments, the present invention also provides the use of a compound for preparing a drug linker compound;

[0369] Preferably, the drug linker compound is the compound described above or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof;

[0370] The compound described above is prepared by the following steps to prepare the drug linker compound:

[0371] Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, and D are as defined above; LG1 is selected from a group that undergoes a condensation reaction with J1, preferably, LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0372] In some embodiments, the present invention also provides the use of a compound for preparing a drug linker compound;

[0373] Preferably, the drug linker is selected from the compounds described above or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides or isotope-labeled compounds thereof;

[0374] Preferably, the compound described above is prepared by the following steps to prepare the drug linker compound:

[0375] Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, and D are as defined above; LG2 is selected from a group that undergoes a condensation reaction with J2, preferably, LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0376] In some embodiments, the present invention also provides the use of a compound for preparing a biologically active conjugate;

[0377] Preferably, the bioactive conjugate is selected from the bioactive conjugates described above;

[0378] Preferably, the compound described above is prepared into the bioactive conjugate by following steps 1a and 1b:

[0379] Step 1a:

[0380] Step 1b:

[0381] Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined above; LG1 is selected from a group that undergoes a condensation reaction with J1, preferably, LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0382] In some embodiments, the present invention also provides the use of a compound for preparing a biologically active conjugate;

[0383] The bioactive conjugate is selected from the bioactive conjugates described above;

[0384] Preferably, the compound described above is prepared into the bioactive conjugate by following steps 2a and 2b:

[0385] Step 2a:

[0386] Step 2b:

[0387] Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are as defined above; LG2 is selected from a group that undergoes a condensation reaction with J2, preferably, LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0388] In some embodiments, the present invention also provides a method for preparing a compound, comprising the following steps:.

[0389] Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, and D are as defined above; LG1 is selected from a group that undergoes a condensation reaction with J1, preferably, LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0390] In some embodiments, the present invention also provides a method for preparing a compound, comprising the steps of:

[0391] Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, and D are as defined above; LG2 is selected from a group that undergoes a condensation reaction with J2, preferably, LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0392] In some embodiments, the present invention also provides a method for preparing an antibody drug conjugate, comprising the following steps:

[0393] Step 1a:

[0394] Step 1b:

[0395] Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined above; LG1 is selected from a group that undergoes a condensation reaction with J1, preferably, LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0396] In some embodiments, the present invention also provides a method for preparing an antibody drug conjugate, comprising the following steps:

[0397] Step 2a:

[0398] Step 2b:

[0399] Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are as defined above; LG2 is selected from a group that undergoes condensation reaction with J2, preferably, LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

[0400] Pharmaceutical composition

[0401] The present invention also provides a pharmaceutical composition comprising the bioactive conjugate of the present invention and one or more pharmaceutically acceptable carriers.

[0402] In some embodiments, the drug / antibody ratio (DAR value) of the pharmaceutical composition is 1.0-6.0, such as 1, 2, 3, 4, 5 or 6, and further such as 1.0-1.5, 1.0-2.0, 1.0-2.5, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 1.0-5.5, 1.0-6.0, 1.5-2.0, 1.5-2.5, 1.5-3.0, 1.5-3.5, 1.5-4.0, 1.5-4.5, 1.5-5.0, 1.5-5.5, 1.5-6.0, 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 2.0-5.5, 2.0-6.0, 2.5-3.0, 2.5-3.5, 2.5-4.0, 2.5-4.5, 2.5-5.0, 2.5-5.5, 2.5-6.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0- 6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 5.0-5.5, 5.0-6.0 or 5.5-6.0.

[0403] The pharmaceutical excipients mentioned in this article refer to the excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances other than active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations.

[0404] The pharmaceutical composition can be administered in any form, as long as it prevents, alleviates, prevents or cures the symptoms of a human or animal patient. For example, it can be prepared into various suitable dosage forms according to the administration route.

[0405] The present application also provides a drug kit product, which contains the bioactive conjugate or the pharmaceutical composition of the present invention, and optional drug instructions.

[0406] Treatment methods and uses

[0407] Another aspect of the present application provides use of the bioactive conjugate in preparing a medicament for preventing or treating tumor diseases.

[0408] Another aspect of the present application provides the bioactive conjugate for use in preventing or treating tumor diseases.

[0409] Another aspect of the present application provides a method for preventing or treating tumor diseases, comprising administering an effective amount of the bioactive conjugate or a pharmaceutical composition comprising the bioactive conjugate to a subject in need thereof.

[0410] In one embodiment of the present invention, the tumor disease is a solid tumor or a hematological malignancy; for example, selected from colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), lymphoma.

[0411] As used herein, the term "effective amount" refers to that amount of the conjugate which, when administered, will relieve to some extent one or more symptoms of the condition being treated.

[0412] As used herein, unless otherwise indicated, the term "treat," ...

[0413] As used herein, "individual" or "subject" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. "Non-human animals" herein include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). DETAILED DESCRIPTION

[0414] The present invention will be described in detail below with reference to Examples and Test Examples. However, these examples are not intended to limit the scope of the present invention, and variations are possible without departing from the scope of the present invention.

[0415] 1. Antibody Preparation

[0416] 1.1 Preparation of the anti-ROR1 antibody 19F6_Hu35V1 involved in the present invention

[0417] In the initial stage, the murine antibody 19F6 was obtained by immunizing Balb / c, C57Bl / 6, NZB, and A / J mice and screening hybridomas. After humanization, the humanized antibody sequence 19F6_Hu35V1 was obtained: heavy chain variable region, SEQ ID NO: 1; light chain variable region, SEQ ID NO: 2; heavy chain constant region, human IgG1 heavy chain constant region (SEQ ID NO: 18); light chain constant region, human kappa light chain constant region (SEQ ID NO: 19). The DNA sequence encoding the humanized antibody was synthesized and codon-optimized, then cloned into the pcDNA3.4 plasmid. The pcDNA3.4 plasmids corresponding to the heavy and light chains of the humanized antibody were simultaneously transfected into Expi293F cells. The expressed antibody in the supernatant was purified using protein A to obtain the corresponding antibody.

[0418] Sequence information of 19F6_Hu35V1 Table-1:

[0419] Sequence information of 19F6_Hu35V1 Table-2:

[0420] 1.2 Preparation of the B7-H3 Antibody 2#8890 Concerning the Present Invention

[0421] Fully human mice were immunized with human B7-H3-4Ig-His protein, and serum titers were assessed by ELISA and flow cytometry. Based on the titer results, the optimal mice were selected for spleen cell fusion, screening, and subcloning. Different monoclonal clones were tested for binding to human and monkey proteins and cells, resulting in the preferred clone 20G11G6. The antibody sequence was then modified to remove PTM sites, reduce PI, and remove ADCC from the heavy chain constant region, ultimately yielding the fully human antibody 2#8890. Nanjing GenScript Biotechnology Co., Ltd. performed codon optimization and gene synthesis, and the antibody was constructed into the pTT5 plasmid. The heavy and light chain plasmids were simultaneously transfected into CHO-S cells, and the expressed antibody in the supernatant was purified using Protein A to obtain the corresponding antibody protein 2#8890. The heavy and light chain amino acid sequences of 2#8890 are shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0422] 2#8890 sequence information table:

[0423] 2. Synthesis of Drug-Linker

[0424] As used herein, the abbreviations have the following meanings:

[0425] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0426] Nuclear magnetic resonance (NMR) 1 H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance spectrometer; deuterated chloroform (CDCl 3 ) was used; and tetramethylsilane (TMS) was used as the internal standard.

[0427] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0428] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, CDCl3: deuterated chloroform. δ values ​​are expressed in ppm.

[0429] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0430] Example 1 2-(2,3-bis(bromomethyl)-5,7-dioxy-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)acetic acid (I-1)

[0431] Step 1: Synthesis of tert-butyl 2-(3,4-diazide-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-2)

[0432] Compound 1-1 (0.40 g, 1.08 mmol, synthesis method referenced in patent WO2019057964) and sodium azide (141.00 mg, 2.17 mmol) were dissolved in acetone (10 mL) and reacted at 25°C for 8 h. The reaction was monitored by HPLC-MS / MS. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 0.28 g of the crude title compound, which was used directly in the next step without purification.

[0433] The structural characterization data are as follows:

[0434] ESI-MS (m / z): 311.0 [M+18] + .

[0435] Step 2: Synthesis of tert-butyl 2-(3,4-diamino-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-3)

[0436] Compound 1-2 (0.15 g, 0.51 mmol) and 10% palladium on carbon (15.00 mg) were dissolved in ethanol (20 mL). The mixture was replaced with hydrogen three times and allowed to react at 25°C for 3 h under a hydrogen atmosphere. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered through celite and the filtrate was concentrated under reduced pressure to obtain 101.00 mg of the crude title compound, which was used directly in the next step without purification.

[0437] The structural characterization data are as follows:

[0438] ESI-MS (m / z): 185.9 [M-56] + .

[0439] Step 3: Synthesis of tert-butyl 2-(2,3-bis(bromomethyl)-5,7-dioxy-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)acetate (1-5)

[0440] Compound 1-3 (50.00 mg, 0.21 mmol) and compound 1-4 (54.00 mg, 0.21 mmol) were dissolved in acetonitrile (5 mL) and reacted at 25°C for 3 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified on a flash silica gel column (petroleum ether:ethyl acetate = 5:1) to obtain 40.00 mg of the title compound.

[0441] The structural characterization data are as follows:

[0442] ESI-MS (m / z): 466.8 [M+18] + .

[0443] 1 H NMR (400MHz, CDCl3): δ4.88(s,4H),4.47(s,2H),1.48(s,9H).

[0444] Step 4: Synthesis of 2-(2,3-bis(bromomethyl)-5,7-dioxy-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)acetic acid (I-1)

[0445] Compound 1-5 (20.00 mg, 0.05 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1 mL). The reaction was allowed to react at 25°C for 5 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure to obtain 15.00 mg of the crude title compound, which was used directly in the next step without purification.

[0446] The structural characterization data are as follows:

[0447] ESI-MS (m / z): 410.8 [M+18] + .

[0448] Example 2 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (I-2)

[0449] Step 1: Synthesis of tert-butyl 2-(3,4-dimethyl-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-1)

[0450] Under nitrogen, compound 1-1 (0.20 g, 0.54 mmol), trimethylboroxine (0.34 g, 2.71 mmol), cesium fluoride (0.41 g, 2.71 mmol), and tetrakistriphenylphosphine palladium (63.00 mg, 0.05 mmol) were dissolved in 1,4-dioxane (20 mL) and reacted at 110°C for 3 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified on a flash silica gel column (petroleum ether:ethyl acetate = 5:1) to obtain 80.00 mg of the title compound.

[0451] The structural characterization data are as follows:

[0452] ESI-MS (m / z): 184.0 [M+H-56] + .

[0453] Step 2: Synthesis of tert-butyl 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-2)

[0454] Under nitrogen, compound 2-1 (72.00 mg, 0.30 mmol), NBS (118.00 mg, 0.66 mmol), and dibenzoyl peroxide (7.00 mg, 0.03 mmol) were dissolved in carbon tetrachloride (8 mL) and reacted at 85°C for 12 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified on a flash silica gel column (petroleum ether:ethyl acetate = 10:1) to obtain 65.00 mg of the title compound.

[0455] The structural characterization data are as follows:

[0456] ESI-MS (m / z): 414.8 [M+18] + .

[0457] 1H-NMR (400MHz, CDCl3): δ4.28(s,4H),4.20(s,2H),1.45(s,9H).

[0458] Step 3: Synthesis of 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (I-2)

[0459] Compound 2-2 (60.00 mg, 0.15 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (2 mL) and reacted at 25°C for 5 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly concentrated under reduced pressure to obtain 50.00 mg of the crude title compound, which was used directly in the next step without purification.

[0460] The structural characterization data are as follows:

[0461] ESI-MS (m / z): 358.9 [M+18] + .

[0462] Example 3 4-((S)-2-((S)-2-(2-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S),2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 3)

[0463] Step 1: Synthesis of 4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutylamino)-5-ureidopentanamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3-2)

[0464] At 25° C., (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide (30.00 mg, 0.04 mmol) and compound 3-1 (38.50 mg, 0.05 mmol) were dissolved in DMF (3 mL), and HOBt (8.46 mg, 0.06 mmol) and DIPEA (10.80 mg, 0.08 mmol) were added. The reaction was maintained at 25°C for 2 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was used directly in the next reaction without further treatment.

[0465] The structural characterization data are as follows:

[0466] ESI-MS (m / z): 1345.2 [M+H] + .

[0467] Step 2: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureidopentanamido)benzyl((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate (3-3)

[0468] Diethylamine (0.30 mL) was added to the reaction mixture of compound 3-2 at 25°C. The reaction was maintained at 25°C for 2 h, and the reaction was monitored by HPLC-MS / MS. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 36.30 mg of the formate salt of the title compound.

[0469] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0470] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0471] The structural characterization data are as follows:

[0472] ESI-MS (m / z): 1123.2 [M+H] +.

[0473] Step 3: Synthesis of 4-((S)-2-((S)-2-(2-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3-methylbutylamino)-5-ureidopentaamido)benzyl((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S),2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3)

[0474] Compound I-2 (12.75 mg, 0.04 mmol) was dissolved in dichloromethane (3 mL), and DIC (2.36 mg, 0.02 mmol) and compound 3-3 (21.00 mg, 0.02 mmol) were added. The mixture was reacted at 25°C for 1 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 14.30 mg of the title compound.

[0475] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0476] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0477] The structural characterization data are as follows:

[0478] ESI-MS (m / z): 1445.9 [M+H] + .

[0479] Example 4 (S)-2-((2S,13S)-13-benzyl-22-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-isopropyl-3-methyl-4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazadocosamil)-N -((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethylbutanamide (Drug-Linker 4)

[0480] Step 1: Synthesis of (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptacosa-27-yl)carbamate (4-2)

[0481] Compound 4-1 (50.00 mg, 0.08 mmol) and (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3- ... 3-Dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide (55.60 mg, 0.08 mmol) was dissolved in DMF (1 mL), followed by the addition of HATU (32.37 mg, 85.18 μmol) and DIPEA (20.02 mg, 154.88 μmol). The reaction was allowed to react at room temperature for 1 h, monitored by HPLC-MS / MS. The reaction solution was directly purified by preparative HPLC to afford 35.00 mg of the title compound.

[0482] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0483] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0484] The structural characterization data are as follows:

[0485] ESI-MS (m / z): 1345.1 [M+H] + .

[0486] Step 2: Synthesis of (S)-2-((2S,13S)-19-amino-13-benzyl-2-isopropyl-3-methyl-4,9,12,15,18-pentaoxo-6-oxa-3,8,11,14,17-pentazanonadecanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethylbutanamide (4-3)

[0487] Compound 4-2 (20.00 mg, 0.02 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of diethylamine (1 mL). The reaction was allowed to react at room temperature for 1 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 15.00 mg of the formate salt of the title compound.

[0488] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0489] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0490] The structural characterization data are as follows:

[0491] ESI-MS (m / z): 1123.1 [M+H] + .

[0492] Step 3: (S)-2-((2S,13S)-13-benzyl-22-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-isopropyl-3-methyl-4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazadocosamil) Synthesis of N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethylbutanamide (4)

[0493] Compound I-2 (4.00 mg, 12.0 μmol) was dissolved in dichloromethane (3 mL), and DIC (1.12 mg, 8.00 μmol) and compound 4-3 (6.58 mg, 6.00 μmol) were added. The reaction was allowed to proceed at 25°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 4.77 mg of the title compound.

[0494] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0495] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0496] The structural characterization data are as follows:

[0497] ESI-MS (m / z): 1445.9 [M+H] + .

[0498] Example 5 N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11 -dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptacosan-27-yl)-1-(2-(3,4-bis(bromomethyl)-2,5-dioxa-2-,5-dihydro-1H-pyrrol-1-yl)acetamido)-3,6,9,12-tetraoxopentadecanepentadecanamide (Drug-Linker 5)

[0499] Step 1: Synthesis of (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-(((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26,29-octaoxo-2,14,32,35,38,41-hexaoxa-5,8,11,16,19,22,25,28-octaazatricarboxan-43-yl)carbamate (5-2)

[0500] Compounds 5-1 (5.21 mg, 11.00 μmol) and 4-3 (10.00 mg, 9.00 μmol) were weighed and dissolved in DMF (1 mL). HATU (4.06 mg, 11.00 μmol) and DIPEA (2.30 mg, 18.00 μmol) were then added. The mixture was reacted at room temperature for 1 h and monitored by HPLC-MS / MS. The reaction solution was directly purified by preparative HPLC to yield 9.00 mg of the title compound.

[0501] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0502] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0503] The structural characterization data are as follows:

[0504] ESI-MS (m / z): 1592.8 [M+H] + .

[0505] Step 2: Synthesis of 1-amino-N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((((S,2R)-1-hydroxy-1-phenylpropanyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptacosan-27-yl)-3,6,9,12-tetraoxapentadecan-15-amide (5-3)

[0506] Compound 5-2 (10.00 mg, 6.00 μmol) was dissolved in dichloromethane (2 mL), followed by the addition of diethylamine (1 mL). The reaction was allowed to react at room temperature for 1 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 7.00 mg of the formate salt of the title compound.

[0507] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0508] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0509] The structural characterization data are as follows:

[0510] ESI-MS (m / z): 1370.9 [M+H] + .

[0511] Step 3: N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,1 Synthesis of 1-dimethyl-6,9,12,17,20,23,26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazacosanoic-27-yl)-1-(2-(3,4-bis(bromomethyl)-2,5-dioxa-2-,5-dihydro-1H-pyrrol-1-yl)acetamido)-3,6,9,12-tetraoxopentadecanoic acid pentadecanamide (5)

[0512] Compound I-2 (4.00 mg, 12.00 μmol) was dissolved in dichloromethane (2 mL), and DIC (1.12 mg, 8.00 μmol) and compound 5-3 (8.04 mg, 6.00 μmol) were added. The reaction was allowed to proceed at 25°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 4.68 mg of the title compound.

[0513] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0514] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0515] The structural characterization data are as follows:

[0516] ESI-MS (m / z): 1693.7 [M+H] + .

[0517] Example 6 (S)-2-((2S,13S)-13-benzyl-22-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)-2-isopropyl-3-methyl-4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17,20-hexaazadocosapentaenoic acid N-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino))-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-N,3-dimethylbutanamide (Drug-Linker 6)

[0518] Compound I-1 (6.03 mg, 15.00 μmol) was dissolved in dichloromethane (2 mL), and DIC (1.45 mg, 12.00 μmol) and compound 4-3 (8.62 mg, 8.00 μmol) were added. The reaction was allowed to proceed at 25°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 4.21 mg of the title compound.

[0519] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0520] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0521] The structural characterization data are as follows:

[0522] ESI-MS (m / z): 1498.5 [M+H] + .

[0523] Example 7 N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl- 6,9,12,17,20,23,26-Heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptacosan-27-yl)-1-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)acetamido)-3,6,9,12-tetraoxapentadecan-15-amide (Drug-Linker 7)

[0524] Compound I-1 (12.06 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL), and DIC (3.00 mg, 0.02 mmol) and compound 5-3 (20.00 mg, 15.00 μmol) were added. The reaction was incubated at 25°C for 1 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain 12.00 mg of the title compound.

[0525] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0526] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0527] The structural characterization data are as follows:

[0528] ESI-MS (m / z): 1745.8 [M+H] + .

[0529] Example 8 4-((2S,5S)-41-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,11,40-tetraoxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12,39-tetraazatetracenadecanoyl)benzyl ((S)-1-(((S)-1-(((3R,4S,5S )-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 8)

[0530] Step 1: Synthesis of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontamide)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (8-3)

[0531] Compound 8-2 (3.60 g, 12.27 mmol) was dissolved in dichloromethane (30 mL), and compound 8-1 (7.49 g, 13.50 mmol) and EEDQ (6.07 g, 24.54 mmol) were added. The mixture was allowed to react at 25°C for 4 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was separated on a reverse-phase C18 column (70% acetonitrile / 0.1% formic acid aqueous solution) to obtain the title compound 8-3 (7.80 g).

[0532] The structural characterization data are as follows:

[0533] ESI-MS (m / z): 830.4 [M+H] + .

[0534] Step 2: Synthesis of 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontanoyl)benzyl(4-nitrophenyl)carbonate (8-4)

[0535] Compound 8-3 (2.50 g, 3.01 mmol) was dissolved in dichloromethane (20 mL), and di(p-nitrobenzene) carbonate (3.66 g, 12.05 mmol) and DIPEA (1.56 g, 12.05 mmol) were added. The mixture was reacted at 25°C for 4 hours. The reaction solution was directly purified on a silica gel column (ethyl acetate-dichloromethane:methanol = 84:16) to obtain the title compound 8-4 (2.07 g).

[0536] The structural characterization data are as follows:

[0537] ESI-MS (m / z): 995.4 [M+H] + .

[0538] Step 3: 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontanoyl)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 Synthesis of (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (8-5)

[0539] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide (100 mg, 139.28 μmol) 1), compound 8-4 (180.17 mg, 181.06 μmol) and HOBt (56.46 mg, 417.84 μmol) were dissolved in DMF (3 mL), and DIPEA (54.00 mg, 417.84 μmol) was added. The mixture was reacted at 25°C for 6 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was separated on a reverse-phase C18 column (55% acetonitrile / 0.1% formic acid aqueous solution) to obtain the title compound 8-5 (60.00 mg).

[0540] The structural characterization data are as follows:

[0541] ESI-MS (m / z): 1573.9 [M+H] + .

[0542] Step 4: 4-((2S,5S)-38-amino-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontanoyl)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 Synthesis of (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (8-6)

[0543] Compound 8-5 (0.60 g, 381.22 μmol) was dissolved in THF (10 mL), and triphenylphosphine (232.02 mg, 762.44 μmol) was added. Water (10 mL) was then added and the mixture was reacted at 50°C for 6 hours. Water was added and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purification on a silica gel column (dichloromethane:methanol = 84:16) afforded the title compound 8-6 (50.00 mg).

[0544] The structural characterization data are as follows:

[0545] ESI-MS (m / z): 1548.9 [M+H] + .

[0546] Step 5: 4-((2S,5S)-41-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,11,40-tetraoxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12,39-tetraazatetradecanoylamido)phenyl((S)-1-(((S)-1-(((3R,4S, Synthesis of 5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (8)

[0547] Compound I-2 (10.00 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL), and DIC (3.70 mg, 0.03 mmol) and compound 8-6 (23.37 mg, 15.00 μmol) were added. The reaction was incubated at 25°C for 1 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain the title compound 8 (12.88 mg).

[0548] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0549] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0550] The structural characterization data are as follows:

[0551] ESI-MS (m / z): 1871.8 [M+H] + .

[0552] Example 9 2-(2-(2-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)ethoxy)ethoxy)acetic acid (I-3)

[0553] Step 1: Synthesis of tert-butyl 2-(2-(2-(2-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetate (9-2)

[0554] Compound 9-1 (523.00 mg, 1.98 mmol) and triphenylphosphine (518.97 mg, 1.98 mmol) were dissolved in tetrahydrofuran (8 mL) at 0°C. DIAD (400.11 mg, 1.98 mmol, 389.59 μL) was added and stirred for 5 minutes. 3,4-Dibromopyrrole-2,5-dione (504.30 mg, 1.98 mmol) was then added and allowed to react at 0°C for 3 hours. Saturated aqueous ammonium chloride was added and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the title compound 9-2 (0.90 g).

[0555] The structural characterization data are as follows:

[0556] ESI-MS (m / z): 519.0 [M+18] + .

[0557] Step 2: Synthesis of tert-butyl 2-(2-(2-(2-(3,4-diazide-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetate (9-3)

[0558] Compound 9-2 (0.20 g, 399.07 μmol) was dissolved in acetone (8 mL), and sodium azide (51.89 mg, 798.14 μmol) was added. The mixture was reacted at 25°C for 8 hours. Water was added and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product of the title compound 9-3 (160.00 mg).

[0559] The structural characterization data are as follows:

[0560] ESI-MS (m / z): 443.1 [M+18] + .

[0561] Step 3: Synthesis of tert-butyl 2-(2-(2-(2-(3,4-diamino-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetate (9-4)

[0562] Compound 9-3 (170.00 mg, 399.63 μmol) and 10% palladium on carbon (85.00 mg) were dissolved in ethanol (34 mL) and reacted under a hydrogen atmosphere at 25°C for 8 hours. The reaction solution was filtered and the filtrate was dried under reduced pressure to obtain a crude product of the title compound 9-4 (140.00 mg).

[0563] The structural characterization data are as follows:

[0564] ESI-MS (m / z): 391.3 [M+18] + .

[0565] Step 4: Synthesis of 2-(2-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)ethoxy)ethoxy)acetic acid (I-3)

[0566] Compound 9-4 (70.00 mg, 187.47 μmol) and compound 1-4 (45.72 mg, 187.47 μmol) were dissolved in acetonitrile (5 mL) and reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the title compound I-3 (26.00 mg).

[0567] The structural characterization data are as follows:

[0568] ESI-MS (m / z): 391.3 [M+18]+ .

[0569] Example 10 4-((2S,5S)-17-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diazaheptadecanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)- 2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 10)

[0570] Compound I-3 (13.47 mg, 25.65 μmol) was dissolved in dichloromethane (4 mL), and DIC (2.43 mg, 19.24 μmol, 2.98 μL) was added. After stirring for 20 min, the formate salt of compound 10-1 (15.00 mg, 12.83 μmol) was added and reacted at 20°C for 2 hours. The solvent was evaporated under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was freeze-dried to obtain the title compound 10 (9.00 mg).

[0571] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0572] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0573] The structural characterization data are as follows:

[0574] ESI-MS (m / z): 1630.4 [M+H] + .

[0575] Example 11: 3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzoic acid (II-19)

[0576] Step 1: Synthesis of methyl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (11-3)

[0577] Methyl 3,5-dibromobenzoate (1.00 g, 3.40 mmol), 4-tributylstannyl-2-thiomethylpyrimidine (3.11 g, 7.48 mmol), and tetrakis(triphenylphosphine)palladium (393.00 mg, 0.034 mmol) were dissolved in 1,4-dioxane (10 mL). After nitrogen replacement, the mixture was reacted in a microwave oven at 110°C for 6 hours. The reaction solution was concentrated under reduced pressure and the concentrate was purified on a silica gel column (petroleum ether:ethyl acetate = 5:1) to obtain compound 11-3 (398.00 mg).

[0578] The structural characterization data are as follows:

[0579] ESI-MS (m / z): 385.0 [M+H] + .

[0580] Step 2: Synthesis of 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoic acid (11-4)

[0581] Compound 11-3 (398.00 mg, 1.04 mmol) was dissolved in methanol (5 mL), tetrahydrofuran (5 mL), and water (1 mL). Sodium hydroxide (166.00 mg, 4.14 mmol) was added and stirred for 1 hour. A 3N aqueous hydrochloric acid solution was added dropwise to neutralize the reaction. The mixture was concentrated under reduced pressure, stirred with water, and filtered. The solid was washed with water and dried under vacuum to obtain the title compound 11-4 (0.38 g).

[0582] The structural characterization data are as follows:

[0583] ESI-MS (m / z): 371.1 [M+18] + .

[0584] Step 3: Synthesis of 3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzoic acid (II-19)

[0585] Compound 11-4 (0.38 g, 1.03 mmol) was dissolved in methanol (20 mL), and m-chloroperbenzoic acid (1.25 g, 80%, 6.15 mmol) was added with stirring. The temperature was raised to 60°C and the reaction was allowed to react for 4 hours. The solvent was dried with nitrogen, and the solid was dissolved in dichloromethane and directly purified on a silica gel column (dichloromethane:methanol = 10:1) to obtain the title compound II-19 (0.25 g).

[0586] The structural characterization data are as follows:

[0587] ESI-MS (m / z): 452.0 [M+18] + .

[0588] Example 12 4-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32-trioxo-34-(3-ureidopropyl))-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatriacontane-35-amido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1- ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 12)

[0589] Step 1: 4-((34S,37S)-34-isopropyl-2,2-dimethyl-4,32,35-trioxo-37-(3-ureidopropyl)-3,8,11,14,17,20,23,26,29-nonaoxa-5,33,36-triazatriacontane-38-amido)benzyl((S)-1-(((S)-1-((((3R,4S,5S)-1-((S)-)-2- Synthesis of ((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (12-2)

[0590] The formate salt of compound 3-3 (200.00 mg, 171.02 μmol), HATU (91.04 mg, 239.43 μmol), and compound 12-1 (120.42 mg, 222.33 μmol) were dissolved in DMF (6 mL). DIPEA (66.31 mg, 513.06 μmol) was then added and the mixture was allowed to react at room temperature for 0.5 h. The solvent was removed under reduced pressure, and the concentrate was directly purified by preparative HPLC. The preparative solution was lyophilized to obtain the title compound 12-2 (210.00 mg).

[0591] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0592] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0593] The structural characterization data are as follows:

[0594] ESI-MS (m / z): 824.0 [(M+H) / 2] + .

[0595] Step 2: 4-((29S,32S)-1-amino-29-isopropyl-27,30-dioxo-32-(3-ureidopropyl)-3,6,9,12,15,18,21,24-octaoxa-28,31-diazatricarboxane-33-amido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R Synthesis of 3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (12-3)

[0596] Compound 12-2 (140.00 mg, 85.00 μmol) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (5 mL) and reacted at 0° C. for 2 hours. The solvent was evaporated under reduced pressure to obtain 73.00 mg of crude formate salt of the title compound 12-3.

[0597] The structural characterization data are as follows:

[0598] ESI-MS (m / z): 774.1 [(M+H) / 2] + .

[0599] Step 3: 4-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32-trioxo-34-(3-ureidopropyl))-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatriacontane-35-amido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1 Synthesis of ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (12)

[0600] Compound II-19 (5.45 mg, 12.56 μmol), HATU (14.32 mg, 37.67 μmol), and the formate salt of compound 12-3 (20.00 mg, 12.56 μmol) were dissolved in DMF (2 mL). DIPEA (8.11 mg, 62.78 μmol, 11.18 μL) was then added and the mixture was allowed to react at room temperature for 2 hours. The solvent was then removed under reduced pressure. The concentrate was directly purified by preparative HPLC, and the preparative solution was freeze-dried to give the title compound 12 (6.50 mg).

[0601] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0602] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0603] The structural characterization data are as follows:

[0604] ESI-MS (m / z): 982.5 [(M+H) / 2] + .

[0605] Example 13: N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-1-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3,6,9,12-tetraoxapentadecanamide (Drug-Linker 13)

[0606] Step 1: Synthesis of (9H-fluoro-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl))carbamate (13-3)

[0607] Compound 13-1 (1.00 g, 1.55 mmol) was dissolved in DMF (5 mL). HATU (647.40 mg, 1.70 mmol), the mesylate salt of isotecan 13-2 (1.00 g, 1.55 mmol), and DIPEA (400.34 mg, 3.10 mmol) were added sequentially. The mixture was reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly purified by HPLC and freeze-dried to obtain the title compound 13-3 (1.05 g).

[0608] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0609] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0610] The structural characterization data are as follows:

[0611] ESI-MS (m / z): 1063.4 [M+H] + .

[0612] Step 2: Synthesis of (S)-2-(2-aminoacetamido)acetamido)-N-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dihydro-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-2-oxo-3-phenylpropanamide (13-4)

[0613] Compound 13-3 (1.05 g, 987.69 μmol) was dissolved in dichloromethane (100 mL), and diethylamine (20 mL) was added. The mixture was reacted at 25°C for 1 hour. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the formate salt of the title compound 13-4 (285.00 mg).

[0614] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0615] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0616] The structural characterization data are as follows:

[0617] ESI-MS (m / z): 841.2 [M+H] + .

[0618] Step 3: Synthesis of (9H-fluoren-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-1,2,3,9,10,12,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,9,15,18-hexaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17-pentaazatricarboxylate (13-6)

[0619] The formate salt of compound 13-4 (83.00 mg, 98.71 μmol), HATU (45.04 mg, 118.45 μmol), and compound 13-5 (57.75 mg, 118.45 μmol) were dissolved in DMF (2 mL). DIPEA (25.51 mg, 197.42 μmol, 35.14 μL) was then added and the mixture was allowed to react at room temperature for 0.5 h. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly purified by HPLC and freeze-dried to obtain the title compound 13-6 (30.00 mg).

[0620] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0621] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0622] The structural characterization data are as follows:

[0623] ESI-MS (m / z): 1310.5 [M+H] + .

[0624] Step 4: Synthesis of 1-amino-N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-3,6,9,12-tetraoxa-15-decaneamide (13-7)

[0625] Compound 13-6 (30.00 mg, 22.89 μmol) was dissolved in diethylamine (1 mL) and DMF (2 mL) and reacted at room temperature for 1 hour. The solvent in the reaction solution was removed by vacuum extraction, and the concentrate was directly purified by HPLC and freeze-dried to obtain the formate salt of the title compound 13-7 (16.00 mg).

[0626] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0627] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0628] The structural characterization data are as follows:

[0629] ESI-MS (m / z): 1088.4 [M+H] + .

[0630] Step 5: Synthesis of N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-1-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3,6,9,12-tetraoxapentadecanamide (13)

[0631] The formate salt of compound 13-7 (16.00 mg, 14.70 μmol), compound I-3 (11.56 mg, 29.41 μmol), and DIC (2.60 mg, 20.59 μmol, 3.19 μL) were dissolved in dichloromethane (4 mL) and reacted at 20°C for 1 hour. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly purified by HPLC and freeze-dried to obtain the title compound 13 (5.33 mg).

[0632] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0633] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0634] The structural characterization data are as follows:

[0635] ESI-MS (m / z): 1463.3 [M+H] + .

[0636] Example 14 (S)-2-(17-(2,3-bis(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptanecarboxamide)-N-(2-((2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo)-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy(methyl)amino)-2-oxoethyl)-3-phenylpropanamide (Drug-Linker 14)

[0637] Compound I-3 (40.26 mg, 76.67 μmol) was dissolved in dichloromethane (5 mL), and DIC (9.68 mg, 76.67 μmol) was added. After stirring for 20 minutes, the formate salt of compound 13-4 (34.00 mg, 38.34 μmol) was added and reacted at 20°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the title compound 14 (5.33 mg).

[0638] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0639] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0640] The structural characterization data are as follows:

[0641] ESI-MS (m / z): 1348.3 [M+H] + .

[0642] Example 15 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleic acid (15)

[0643] Step 1: Synthesis of tert-butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleate (15-2)

[0644] Compound II-9 (50.00 mg, 115.09 μmol) was dissolved in DMF (2 mL), and HATU (65.60 mg, 172.53 μmol) was added. After stirring, compound 15-1 (36.37 mg, 138.11 μmol) and DIPEA (44.62 mg, 345.27 μmol, 61.46 μL) were added. The mixture was reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the title compound 15-2 (25.00 mg).

[0645] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0646] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0647] The structural characterization data are as follows:

[0648] ESI-MS (m / z): 697.2 [M+18] + .

[0649] Step 2: Synthesis of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleic acid (15)

[0650] Compound 15-2 (20.00 mg, 29.42 μmol) was dissolved in trifluoroacetic acid (1 mL) and dichloromethane (5 mL) and reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure and the concentrated product obtained the crude product of the title compound 15 (15.00 mg), which was directly used for the next step without purification.

[0651] The structural characterization data are as follows:

[0652] ESI-MS (m / z): 641.1 [M+18] + .

[0653] Example 16: N-((S)-10-Benzyl-1-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18,46-heptaoxo-3,21,24,27,30,33,36,39,42,48,51,54-dodecanooxa-5,8,11,14,17,45-hexaazahexapentan-56-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (Drug-Linker 16)

[0654] Step 1: Synthesis of (9H-fluoro-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonanoxa-5,8,11,14,17-pentaazatetradec-44-yl)carbamate (16-2)

[0655] The mesylate salt of compound 13-4 (132.00 mg, 148.84 μmol) was dissolved in DMF (5 mL), and HATU (90.55 mg, 238.14 μmol), compound 16-1 (148.19 mg, 223.26 μmol), and DIPEA (96.18 mg, 744.19 μmol, 132.48 μL) were added. The mixture was reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the title compound 16-2 (110.00 mg).

[0656] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0657] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0658] The structural characterization data are as follows:

[0659] ESI-MS (m / z): 1486.5 [M+1] + .

[0660] Step 2: Synthesis of 1-amino-N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-3,6,9,12,15,18,21,2-octaoxaheptane-27-amide (16-3)

[0661] Compound 16-2 (110.00 mg, 74.00 μmol, FR) was dissolved in diethylamine (1 mL) and dichloromethane (5 mL) and reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the formate salt of the title compound 16-3 (40.00 mg).

[0662] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0663] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0664] The structural characterization data are as follows:

[0665] ESI-MS (m / z): 1264.6 [M+1] + .

[0666] Step 3: Synthesis of N-((S)-10-benzyl-1-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18,46-heptaoxo-3,21,24,27,30,33,36,39,42,48,51,54-dodecanooxa-5,8,11,14,17,45-hexaazahexapentan-56-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (16)

[0667] Compound 15 (7.00 mg, 11.22 μmol) was dissolved in DMF (3 mL), and HATU (7.76 mg, 20.41 μmol), the formate salt of compound 16-3 (13.37 mg, 10.20 μmol) and DIPEA (3.96 mg, 30.61 μmol, 5.45 μL) were added, and the mixture was reacted at 25°C for 2 hours. The reaction solvent was evaporated under reduced pressure, and the concentrate was directly subjected to HPLC and freeze-dried to obtain the title compound 16 (7.00 mg).

[0668] Column: SunFire Prep C18 OBD 5μm 19x150mm

[0669] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0670] The structural characterization data are as follows:

[0671] ESI-MS (m / z): 1869.7 [M+1] + .

[0672] Example 17: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-10)

[0673] Step 1:

[0674] The raw materials, methyl 3,5-dibromobenzoate (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and H2O (4 mL). The reaction system was stirred at 90°C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through celite, and water and ethyl acetate were added to the filtrate. The mixture was extracted and concentrated to give a crude product, which was purified by column chromatography (EA / PE = 0-25%) to give 710 mg of methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate.

[0675] Its structural characterization data are as follows:

[0676] ESI-MS (m / z): 385.1 [M+H] + .

[0677] Step 2:

[0678] Methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and water (2 mL). The reaction was stirred at 25°C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1N HCl, resulting in the precipitation of a large amount of solid. The filter cake was collected by filtration and dried to yield 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

[0679] Its structural characterization data are as follows:

[0680] ESI-MS (m / z): 371.1 [M+H] + .

[0681] Step 3:

[0682] 3,5-Bis(2-(methylsulfanyl)pyrimidin-5-yl)benzoic acid (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL). m-CPBA (2.46 g, 12.1 mmol, 85% purity) was added to the reaction system. The reaction was allowed to proceed at 25°C for 12 hours and monitored by LC-MS. The solvent was evaporated under a stream of nitrogen to afford a crude product, which was purified by preparative HPLC and freeze-dried to yield 153 mg of 3,5-bis(2-(methylsulfanyl)pyrimidin-5-yl)benzoic acid.

[0683] Its structural characterization data are as follows:

[0684] ESI-MS (m / z): 435.0 [M+H] + .

[0685] The preparation method is as follows:

[0686] Chromatographic column: Phenomenex Luna C18 200*40mm*10um.

[0687] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid)

[0688] Mobile phase: [water (HCl) -ACN]; B%: 13% - 43%, 10 min).

[0689] Step 4:

[0690] 3,5-Bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid (140 mg, 322.25 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (84.86 mg, 322.25 μmol), HATU (183.80 mg, 483.37 μmol), and DIPEA (124.94 mg, 966.75 μmol) were added to DMF (4 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to yield 51 mg of tert-butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate.

[0691] Its structural characterization data are as follows:

[0692] ESI-MS (m / z): 694.2 [M+H] + .

[0693] The preparation method is as follows:

[0694] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0695] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0696] Step 5:

[0697] Compound 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridec-13-oic acid tert-butyl ester (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL), and the reaction was carried out at 25 ° C. for 1 h. The reaction was monitored by LC-MS. The reaction system was concentrated to dryness to obtain 45 mg of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oic acid compound.

[0698] Its structural characterization data are as follows:

[0699] ESI-MS (m / z): 637.2 [M+H] + .

[0700] Step 6:

[0701] Compounds 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-oic acid (31.91 mg, 51.17 μmol), (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-fluoro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxin-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl) Propionamide (C-07-8, 40 mg, 51.17 μmol), HATU (29.18 mg, 76.75 μmol), and DIPEA (19.84 mg, 153.50 μmol) were added to DMF (3 mL) and reacted at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 13 mg of N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacarbonyl-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide.

[0702] Its structural characterization data are as follows:

[0703] ESI-MS (m / z): 1342.5 [M+H] + .

[0704] The preparation method is as follows:

[0705] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0706] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0707] Example 18 N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacarbonyl-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-17)

[0708] Step 1:

[0709] 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (2.25 g, 8.10 mmol) were added to DMF (3 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol) and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in sequence, and the temperature was raised to 60 ° C for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (3.8 g, 4.75 mmol), which was used directly in the next step without purification.

[0710] Step 2:

[0711] Tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (3.40 g, 5.40 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL) was added. The reaction system was stirred at 25°C for 2 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (2.09 g, 3.64 mmol).

[0712] Its structural characterization data are as follows:

[0713] ESI-MS (m / z): 574.2 [M+H] + .

[0714] The preparation method is as follows:

[0715] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0716] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0717] Step 3:

[0718] 1-(3,5-bis(2-(methylsulfanyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oic acid (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), and then sodium periodate (208.79 mg, 976.15 μmol) and ruthenium trichloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system. The reaction was stirred at 25°C for 30 minutes and monitored by LC-MS. Water and ethyl acetate were added for extraction, and the mixture was concentrated to give 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oic acid (60 mg).

[0719] Its structural characterization data are as follows:

[0720] ESI-MS (m / z): 638.2 [M+H] + .

[0721] Step 4:

[0722] (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxolane-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propane Amide (IM-6, 20 mg, 25.06 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid (16 mg, 25.06 μmol), HATU (19.05 mg, 50.11 μmol) and DIPEA (16.19 mg, 125.28 μmol) were added to DMF (3 mL) in sequence, and the reaction system was reacted at 25 ° C for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to give N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacarbonyl-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (16 mg).

[0723] Its structural characterization data are as follows:

[0724] ESI-MS (m / z): 1371.4 [M+H] + .

[0725] The preparation method is as follows:

[0726] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0727] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0728] Example 19: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazacyclotetradecane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21)

[0729] Step 1:

[0730] 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxacosanoic acid-27-decanedate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol) and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in sequence, and the reaction system was stirred at 60 ° C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oate (4.20 g, 4.14 mmol), which was used in the next step without purification.

[0731] Step 2:

[0732] Dissolve tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosadecane-29-oate (3.60 g, 4.24 mmol) in dichloromethane (30 mL). Add TFA (15.3 g, 134 mmol, 10 mL). Stir the reaction system at 25°C for 6 hours. Add water (60 mL) and extract with ethyl acetate (40 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to give 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (2.93 g, 3.63 mmol).

[0733] Its structural characterization data are as follows:

[0734] ESI-MS (m / z): 794.3 [M+H] + .

[0735] The preparation method is as follows:

[0736] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0737] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0738] Step 3:

[0739] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetan-29-oic acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride were added. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system and stirred at 25 ° C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to give 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (155 mg).

[0740] Its structural characterization data are as follows:

[0741] ESI-MS (m / z): 858.3 [M+H] +.

[0742] Step 4:

[0743] (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxolane-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (IM-6, To the mixture of 4-nitro-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonaconicoside-29-oic acid (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol) and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL), and the reaction system was reacted at 25 ° C for 1 hour. The reaction solution was purified by high performance liquid chromatography and freeze-dried to give N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazapyrimidin-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (15 mg).

[0744] Its structural characterization data are as follows:

[0745] ESI-MS (m / z): 1591.7 [M+H] + .

[0746] The preparation method is as follows:

[0747] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0748] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0749] Example 20 N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexacan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19)

[0750] Step 1:

[0751] Methyl 2,6-dibromoisonicotinate (5.00 g, 16.9 mmol), (2-(methylthio)pyrimidin-5-yl)boric acid (6.34 g, 37.3 mmol), XPhos Pd G3 (1.44 g, 1.70 mmol), and potassium phosphate (10.80 g, 50.9 mmol) were added to 1,4-dioxane (51.0 mL) and water (17.0 mL). The reaction system was purged with nitrogen three times and then reacted at 100°C for 5 hours. After the reaction system was cooled to room temperature, water (50.0 mL) was added to the reaction solution, which was filtered and concentrated to obtain the crude product. The crude product was slurried with petroleum ether and filtered again. The filter cake was dried under vacuum to obtain 5.65 g of methyl 2,6-di(2-(methylthio)pyrimidin-5-yl)isonicotinate.

[0752] Step 2:

[0753] Methyl 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinate (5.26 g, 13.7 mmol) was dissolved in THF (30.0 mL), MeOH (30.0 mL), and water (30.0 mL). LiOH·H₂O (1.72 g, 40.9 mmol) was added and stirred at 25°C for 2 hours. The reaction mixture was adjusted to pH 3 with 1N aqueous hydrochloric acid. Solids precipitated and were filtered. The filter cake was dried under vacuum to yield 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinoic acid (4.20 g).

[0754] Its structural characterization data are as follows:

[0755] ESI-MS (m / z): 372.1 [M+H] + Step 3:

[0756] 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinic acid (1.50 g, 4.04 mmol) and tert-butyl 3-(2-(2-aminoethoxy)ethoxyethoxyethyl)propionate (1.12 g, 4.04 mmol) were dissolved in DMF (20.0 mL), and HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol) and DIPEA (2.09 g, 16.2 mmol) were added in sequence. The temperature was raised to 60 °C and stirred for 2 hours. After the reaction system was cooled to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution, and the aqueous phase was extracted twice with ethyl acetate (25.0 mL*2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product of tert-butyl 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (2.50 g), which was used directly in the next step without purification.

[0757] Step 4:

[0758] Tert-butyl 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (2.50 g, 3.96 mmol) was dissolved in dichloromethane (3.00 mL). TFA (4.61 g, 40.4 mmol) was added, and the reaction system was stirred at 25°C for 12 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradec-14-oate (1.20 g).

[0759] Its structural characterization data are as follows:

[0760] ESI-MS (m / z): 575.2 [M+H] + .

[0761] The preparation method is as follows:

[0762] Chromatographic column: Phenomenex luna C18 (150mm*25mm*10μm)

[0763] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0764] Step 5:

[0765] 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxo-2-azatetradecane-14-oic acid (1.10 g, 1.91 mmol) was dissolved in a mixed solvent of acetonitrile (30 mL) and water (15 mL), and ruthenium trichloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. l), the reaction system was reacted at 25 ° C for 1 hour, extracted with water (50 mL) and ethyl acetate (80 mL), and the organic phase was concentrated to obtain a crude product, which was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxo-2-azatetradecane-14-oic acid (130 mg).

[0766] Its structural characterization data are as follows:

[0767] ESI-MS (m / z): 639.2 [M+H] + .

[0768] Step 6:

[0769] (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxolane-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino )-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propionamide (IM-6, 20.0 mg, 0.025 mmol) and 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxo-2-azatetradecane-14-oic acid (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and stirred to dissolve. H ATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexacan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (20.4 mg).

[0770] Its structural characterization data are as follows:

[0771] ESI-MS (m / z): 1372.4 [M+H] + .

[0772] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0773] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0774] Example 21: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazatetracan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23)

[0775] Step 1:

[0776] 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinic acid (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxacosanoic-27-acid ester (2.01 g, 4.04 mmol) were added to DMF (20.0 mL), and HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol) and DIEA (2.09 g, 16.2 mmol) were added in sequence. The temperature was raised to 60°C and stirred for 2 hours. After cooling to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution for separation. The aqueous phase was washed with ethyl acetate (25.0 mL x 40 mL). 2) Extract twice, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate to give crude 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid tert-butyl ester (3.00 g), which was used directly in the next step.

[0777] Step 2:

[0778] Tert-butyl 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oate (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), followed by the addition of TFA (15.4 g, 134 mmol) and stirring at 25°C for 12 hours. The reaction mixture was concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to afford 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oate (1.20 g).

[0779] Its structural characterization data are as follows:

[0780] ESI-MS (m / z): 795.3 [M+H] + .

[0781] The preparation method is as follows:

[0782] Chromatographic column: Welch Ultimate C18 (150mm*25mm*5μm)

[0783] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0784] Step 3:

[0785] 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetan-29-oic acid (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), and ruthenium trichloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol) were added. The system was reacted at 25°C for 1 hour and then extracted with water (10 ml) and ethyl acetate (40 ml). The organic phase was concentrated to give a crude product, which was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonaconicoside-29-oic acid (350 mg).

[0786] Its structural characterization data are as follows:

[0787] ESI-MS (m / z): 859.3 [M+H] + .

[0788] Step 4:

[0789] (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxolane-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL) and HA was added. TU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H 1-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazatetracan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (17.0 mg).

[0790] Its structural characterization data are as follows:

[0791] ESI-MS (m / z): 1592.6 [M+H] + .

[0792] The preparation method is as follows:

[0793] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0794] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0795] Example 22: 2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid (II-9)

[0796] Step 1:

[0797] Methyl 4,6-dichloropyrimidine-2-carboxylate (II-9-1, 1.00 g, 4.83 mmol), (2-methylthiopyrimidin-5-yl)boronic acid (1.81 g, 10.6 mmol), Xphos-Pd-G3 (409 mg, 483 μmol), and K3PO4 (3.08 g, 14.5 mmol) were added to 1,4-dioxane (9.00 mL) and water (3.00 mL). The atmosphere was replaced with nitrogen three times, and the temperature was raised to 100°C and stirred for 5 hours. After cooling to room temperature, water (60.0 mL) was added to the reaction mixture and stirring was continued for 1 hour. Filter and concentrate the filtrate to obtain the crude product. The crude product was slurried with petroleum ether / ethyl acetate (1:1, 40 mL) and filtered again. The filter cake was dried under vacuum to give methyl 2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-carboxylate (1.39 g, 3.60 mmol).

[0798] Step 2:

[0799] Methyl 2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-carboxylate (1.39 g, 3.60 mmol) was dissolved in THF (10.0 mL), methanol (10.0 mL), and water (10.0 mL). Lithium hydroxide (258 mg, 11 mmol) was added, and the mixture was stirred at 25°C for 2 hours. The pH of the reaction solution was adjusted to 2 with 1N aqueous hydrochloric acid. Solids precipitated and were filtered. The filter cake was dried under vacuum to obtain crude 2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-carboxylic acid (1.06 g).

[0800] Its structural characterization data are as follows:

[0801] ESI-MS (m / z): 373.1 [M+H] + .

[0802] Step 3:

[0803] Using the same method as Example 21 to prepare C-23-3, and replacing C-23-2 with II-9-3 (2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-carboxylic acid), compound II-9 can be obtained.

[0804] Its structural characterization data are as follows:

[0805] ESI-MS (m / z): 437.0 [M+H] + .

[0806] Example 23 1-(2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid (C-29-3)

[0807] Step 1:

[0808] 2,2"-Bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-carboxylic acid (350 mg, 940 μmol) was dissolved in DMF (5.00 mL), and T3P (1.91 g, 3.00 mmol, 1.79 mL), DIPEA (365 mg, 2.82 mmol, 491 μL), and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (261 mg, 940 μmol) were added. The mixture was stirred at 25°C for 6 hours. Water (60.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (40 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude tert-butyl 1-(2,2″-bis(methylthio)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-ate (850 mg).

[0809] Step 2:

[0810] Tert-butyl 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidinyl]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate (800 mg, 1.27 mmol) was dissolved in dichloromethane (2.00 mL). TFA (3.07 g, 26.9 mmol, 2.00 mL) was added and stirred at 25°C for 2 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidinyl]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate (145 mg, 249 μmol).

[0811] Its structural characterization data are as follows:

[0812] ESI-MS (m / z): 576.2 [M+H] + .

[0813] The preparation method is as follows:

[0814] Chromatographic column: Phenomenex luna C18 (150mm*25mm*10μm)

[0815] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0816] Step 3:

[0817] The same method as that used in Example 21 to prepare C-23-3 is used, and C-23-2 is replaced by C-29-2 (1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid) to obtain compound C-29-3.

[0818] Its structural characterization data are as follows:

[0819] ESI-MS (m / z): 640.1 [M+H] + .

[0820] Example 24: 1-(2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (C-30-3)

[0821] Step 1:

[0822] Dissolve 4,6-bis(2-methylsulfanylpyrimidin-5-yl)pyrimidine-2-carboxylic acid (0.35 g, 940 μmol) in DMF (6.00 mL), add T3P (2.04 g, 3.21 mmol, 1.91 mL), DIPEA (364 mg, 2.82 mmol, 491 μL), and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (468 mg, 940 μmol). Stir at 25°C for 12 hours. Add water (60.00 mL) to the reaction mixture, and extract with ethyl acetate three times (40.00 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product of tert-butyl 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oate (800 mg).

[0823] Step 2:

[0824] tert-Butyl 1-(2,2″-bis(methylthio)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oate (800 mg, 939 μmol) was dissolved in dichloromethane (10.0 mL). TFA (3.07 g, 26.9 mmol, 2 mL) was added and the mixture was stirred at 25°C for 2 hours. The reaction solution was directly concentrated, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid (389 mg, 483 μmol).

[0825] Its structural characterization data are as follows:

[0826] ESI-MS (m / z): 796.3 [M+H] + .

[0827] The preparation method is as follows:

[0828] Chromatographic column: Phenomenex luna C18 (150mm*25mm*10μm)

[0829] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0830] Step 3:

[0831] The same method as that used in Example 21 to prepare C-23-3 is used, and C-23-2 is replaced by C-30-2 (1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidine]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-oic acid) to obtain compound C-30-3.

[0832] Its structural characterization data are as follows:

[0833] ESI-MS (m / z): 860.3 [M+H] + .

[0834] 3. Preparation of Bioactive Conjugates

[0835] 3.1 Preparation of anti-ROR1 antibody bioactive conjugates

[0836] Coupling method A:

[0837] 0.5 mL of antibody (anti-ROR1 antibody 19F6_Hu35V1, 3-20 mg / mL, referred to as 19F6 in Table 1) was diluted with 0.1 M disodium edetate solution (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed, and allowed to stand at room temperature for 2 hours. A 10 mM amount of drug-linker dissolved in DMSO (5-10 times the amount of the antibody) was added to the solution, mixed, and allowed to stand at room temperature for 20 hours. After completion, the buffer was exchanged with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). The resulting ADC product is shown in Table 1.

[0838] Coupling method B:

[0839] 0.5 mL of antibody (anti-ROR1 antibody 19F6_Hu35V1, 3-20 mg / mL, referred to as 19F6 in Table 1) was diluted with 0.1 M disodium edetate solution (pH 7.60), then adjusted to pH 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added and mixed thoroughly. The solution was allowed to stand at room temperature for 2 hours. TCEP was removed using a NAP-5 gel column (Cytiva), and the resulting solution was added with a 10 mM drug-linker dissolved in DMSO at a volume 5-10 times that of the antibody. The solution was mixed thoroughly and allowed to stand at room temperature for 20 hours. After completion, the buffer was exchanged with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). The resulting ADC product is shown in Table 1.

[0840] Coupling method C: (applicable to the preparation of 19F6-MC-VC-PABC-MMAE)

[0841] 1.8 mL of 19F6_Hu35V1 antibody (22.7 g / L) was diluted with 90 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjusted to pH 7.66 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 66.2 μL, pH 7.60) solution was added and mixed, and allowed to stand at room temperature for 1.5 h. A 4.8-fold amount of drug-linker (MC-VC-PABC-MMAE, 10 mM) solution was added to the above solution system, mixed, and allowed to stand at room temperature for 2 h. After completion, the buffer was exchanged with a 10 mM histidine-histidine hydrochloride buffer solution at pH 6.0 using a NAP-25 gel column (Cytiva) to obtain an antibody-drug conjugate, i.e., ADC (19F6-MC-VC-PABC-MMAE). The DAR value was determined by mass spectrometry to be 4.15.

[0842] Table 1: ADC preparation method, number and DAR

[0843] Determination of the Drug-Antibody Ratio (DAR) of Bioactive Conjugates

[0844] The molecular weight of ADC samples was determined by SEC-MS, and the drug / antibody ratio (DAR) was calculated.

[0845] Chromatographic determination conditions:

[0846] Chromatographic column: ACQUITY UPLC Protein BEH SEC Column, BTQR-18-016;

[0847] Sample chamber temperature: 8°C; column temperature: no temperature control; UV: 280nm;

[0848] Mobile phase: 20 mM ammonium acetate, flow rate: 0.1 ml / min, 20 min, sample injection volume: 50 μg;

[0849] Mass spectrometry conditions:

[0850] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0851] GS1 55; GS2 55; CUR 30; TEM 450; ISVF 5500; DP 75; CE 5;

[0852] m / z 900-7000; Time bins to sum 100.

[0853] (1) SEC-MS determination of the molecular weight of 19F6-ADC III-5-A and calculation of the drug / antibody ratio

[0854] The molecular weight analysis of the coupled 19F6-ADC III-5-A by SEC-MS is shown in Table 2, and the DAR is 3.87.

[0855] Table 2: Measured molecular weight and DAR calculation for 19F6-ADC III-5-A

[0856] (2) SEC-MS determination of the molecular weight of 19F6-ADC III-6-A and calculation of the drug / antibody ratio

[0857] The molecular weight analysis of the coupled 19F6-ADC III-6-A by SEC-MS is shown in Table 3, and the DAR is 4.42.

[0858] Table 3: Measured molecular weight and DAR calculation of 19F6-ADC III-6-A

[0859] (3) SEC-MS determination of the molecular weight of 19F6-ADC IV-1-A and calculation of the drug / antibody ratio

[0860] The molecular weight analysis of the coupled 19F6-ADC IV-1-A by SEC-MS is shown in Table 4, and the DAR is 3.74.

[0861] Table 4: Measured molecular weight and DAR calculation of 19F6-ADC IV-1-A

[0862] (4) SEC-MS determination of the molecular weight of 19F6-ADC IV-1-B and calculation of the drug / antibody ratio

[0863] The molecular weight analysis of the coupled 19F6-ADC IV-1-B by SEC-MS is shown in Table 5, and the DAR is 3.75.

[0864] Table 5: Measured molecular weight and DAR calculation for 19F6-ADC IV-1-B

[0865] (5) SEC-MS determination of the molecular weight of 19F6-ADC IV-1-C and calculation of the drug / antibody ratio

[0866] The molecular weight analysis of the coupled 19F6-ADC IV-1-C by SEC-MS is shown in Table 6, and the DAR is 3.82.

[0867] Table 6: Measured molecular weight and DAR calculation of 19F6-ADC IV-1-C

[0868] (6) SEC-MS determination of the molecular weight of 19F6-ADC IV-1-D and calculation of the drug / antibody ratio

[0869] The molecular weight analysis of the coupled 19F6-ADC IV-1-D by SEC-MS is shown in Table 7, and the DAR is 3.87.

[0870] Table 7: Measured molecular weight and DAR calculation of 19F6-ADC IV-1-D

[0871] (7) SEC-MS determination of the molecular weight of 19F6-ADC IV-7-A and calculation of the drug / antibody ratio

[0872] The molecular weight analysis of the coupled 19F6-ADC IV-7-A by SEC-MS is shown in Table 8, and the DAR is 3.98.

[0873] Table 8: Measured molecular weight and DAR calculation for 19F6-ADC IV-7-A

[0874] 3.2 Preparation of B7-H3 Antibody Bioactive Conjugates

[0875] 3.2.1 Preparation of 2#8890ADC C-10 (DAR 4)

[0876] 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 9.5 μL, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. An 8-fold amount of C-10 dissolved in dimethyl sulfoxide (14 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, 2#8890 ADC C-10. The DAR value was 4.17 as determined by mass spectrometry.

[0877] The ADC samples after coupling were subjected to LC-MS molecular weight analysis.

[0878] Chromatographic determination conditions:

[0879] Liquid chromatography column: ACQUITY UPLC MAbPac BEH SEC;

[0880] Mobile phase A: 20 mM NH4Ac;

[0881] Flow rate: 0.1 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0882] Mass spectrometry conditions:

[0883] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0884] GS1 55; GS2 55; CUR 30; TEM 450; ISVF 5500; DP 75; CE 5; Accumulation time 0.5s;

[0885] m / z 900-7000; Time bins to sum 40.

[0886] 3.2.2 Preparation of 2#8890ADC C-17 (DAR 4)

[0887] 0.292 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 14.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 4.77 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 5-fold amount of C-17 dissolved in dimethyl sulfoxide (8.76 μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, 2#8890 ADC C-17. The DAR value, determined by mass spectrometry, was 3.86.

[0888] Table 9: Measured molecular weight and DAR calculation of 2#8890ADC C-17

[0889] 3.2.3 Preparation of 2#8890ADC C-19 (DAR 4)

[0890] 0.584 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. A 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-19 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, 2#8890 ADC C-19. The DAR value was 4.05 as determined by mass spectrometry.

[0891] Table 10: Measured molecular weight and DAR calculation of 2#8890ADC C-19

[0892] 3.2.4 Preparation of 2#8890ADC C-21 (DAR 4)

[0893] 0.584 mL of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na2HPO4 solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-21 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, 2#8890 ADC C-21. The DAR value was 3.92 as determined by mass spectrometry.

[0894] Table 11: Measured molecular weight and DAR calculation of 2#8890ADC C-21

[0895] 4. Detection of the inhibitory effect of bioactive conjugates on in vitro cell activity

[0896] 4.1 Cell proliferation inhibition effect of ADC

[0897] (1) Cell plating: First, culture NCI-N87 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The tumor cell sources are shown in Table 9.

[0898] Table 9: Tumor cell sources

[0899] Co-incubation of the ADC of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (ADC of the present invention) is added to the above plate wells and incubated for 96 hours.

[0900] In vitro cell activity assay: After incubation, add Cell Counting-Lite TM2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes and then detect, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM Obtain background RLU, Cell Counting-Lite of culture medium containing cells TM Obtain vehicle RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (vehicle RLU-background RLU) × 100%, and calculate the half-maximal inhibitory concentration (IC) of the compound according to the four-parameter model fitting curve. 50 ).

[0901] (2) Data results: The test results are shown in Table 10.

[0902] Table 10: ADC killing results on NCI-N87 cell line

[0903] This study demonstrates that the ADCs (19F6-ADC III-6-A and 19F6-ADC IV-1-D) prepared using the novel conjugation method can kill tumor cells and demonstrates the effectiveness of the novel conjugation method in ADC molecules. Furthermore, compared to the randomly conjugated ADC (19F6-MC-VC-PABC-MMAE), the ADCs prepared using the novel conjugation method (19F6-ADC III-6-A and 19F6-ADC IV-1-D) exhibited superior cell-killing activity.

[0904] 4.2 Inhibitory effect of ADC on HT29 cell proliferation

[0905] (1) Cell plating: First, culture HT29 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 11.

[0906] Table 11: Tumor cell sources

[0907] Co-incubation of the ADC of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (ADC of the present invention) is added to the above plate wells and incubated for 96 hours.

[0908] In vitro cell activity assay: After incubation, add Cell Counting-Lite TM2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes and then detect, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM Obtain background RLU, Cell Counting-Lite of culture medium containing cells TM Obtain vehicle RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (vehicle RLU-background RLU) × 100%, and calculate the half-maximal inhibitory concentration (IC) of the compound according to the four-parameter model fitting curve. 50 ).

[0909] (2) Data results: The test results are shown in Table 12.

[0910] Table 12: ADC killing results on HT29 cell line

[0911] This shows that the ADC (19F6-ADC IV-1-D) formed using the new coupling method can have a killing effect on tumor cells, and the new coupling method is effective when applied to ADC molecules.

[0912] 4.3 Inhibitory Effect of ADC on NCI-H1975 Cell Proliferation

[0913] (1) Cell plating: First, culture NCI-H1975 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The tumor cell sources are shown in Table 13.

[0914] Table 13: Tumor cell sources

[0915] Co-incubation of the ADC of the present invention and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (ADC of the present invention) is added to the above plate wells and incubated for 96 hours.

[0916] In vitro cell activity assay: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes and then detect, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM Obtain background RLU, Cell Counting-Lite of culture medium containing cells TMObtain vehicle RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (vehicle RLU-background RLU) × 100%, and calculate the half-maximal inhibitory concentration (IC) of the compound according to the four-parameter model fitting curve. 50 ).

[0917] (2) Data results: The test results are shown in Table 14.

[0918] Table 14: ADC killing results on NCI-H1975 cell line

[0919] This shows that the ADC (19F6-ADC IV-1-D) formed using the new coupling method can have a killing effect on tumor cells, and the new coupling method is effective when applied to ADC molecules.

[0920] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has the structure of Formula I: in: X is a leaving group, such as F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenol ester, fluorophenol ester, C 1-6 Alkylsulfonyl or Y is absent or selected from substituted or unsubstituted C 1-6 Alkylene, sulfonyl and carbonyl, when substituted, the C 1-6 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Ring A is selected from substituted or unsubstituted C 6-10 Aromatic ring, substituted or unsubstituted 5-12 membered aromatic heterocyclic ring and substituted or unsubstituted 5-12 membered heterocyclic ring, when substituted, the C 6-10 Aromatic rings, 5-12 membered aromatic heterocyclic rings and 5-12 membered heterocyclic rings are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Substitution with haloalkyl, carboxyl, polyethylene glycol, amino acid, phosphate, sulfonic acid, amino, azide, and alkynyl substituents; Q is absent or consists of one or more of the following substituted or unsubstituted groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of which is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Z1 is absent or selected from substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl, amide, substituted or unsubstituted -CH2- and substituted or unsubstituted C 2-6 Alkyne, when substituted, the phenyl, 5-6 membered heteroaryl, -CH2- and C 2-6 Alkyne groups are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; W1 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O)p- and -(OCH2CH2)p- one or more, p is an integer of 1-20, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; J1 is selected from -COOH, -NH2, substituted -NH2, 3-10 membered nitrogen-containing heterocyclic group, substituted 3-10 membered nitrogen-containing heterocyclic group, Alkynyl, 8-16 membered alkynyl-containing ring group, substituted 8-16 membered alkynyl-containing ring group, azido group, tetrazine group, hydroxylamine group, aldehyde group, ketone group, sulfonylurea group, isocyanate group, thioisocyanate group, maleimide group and hydroxyl group, wherein the “substituted” refers to being independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 The substituents of the haloalkyl group are substituted.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein: X is a leaving group, such as Cl, Br, I, OMs, OTs, OTf or Y is absent or is a carbonyl group; Ring A is selected from substituted or unsubstituted C 6-10 Aromatic ring, substituted or unsubstituted 5-12 membered aromatic heterocyclic ring and substituted or unsubstituted 5-12 membered heterocyclic ring, when substituted, the C 6-10 Aromatic rings, 5-12 membered aromatic heterocyclic rings and 5-12 membered heterocyclic rings are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Q is absent or is substituted or unsubstituted -C(=O)NH-, when substituted, said -C(O)-NH- is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Z1 is absent or is substituted or unsubstituted -CH2- or substituted or unsubstituted C 2-6 Alkynylidene, when substituted, the -CH2- or C 2-6 Alkyne groups are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; W1 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O)p- and -(OCH2CH2)p- one or more, p is an integer of 1-10, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; J1 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group.

3. The compound of formula I according to claim 1 or 2, wherein the structure is selected from: wherein p is an integer of 1-10, and J1 is -COOH or -NH2.

4. The compound of formula I according to any one of claims 1 to 3, wherein the structure is selected from:

5. A compound of formula II or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound of formula II has the following structure: in, B1 and B2 are each independently a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle. When substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is substituted by a substituent consisting of one or more of the following groups: hydrogen, halogen, hydroxyl, -CN, substituted or unsubstituted C 1-10 Alkylene, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, carboxyl, substituted or unsubstituted amide, substituted or unsubstituted carbamoyl, substituted or unsubstituted polyethylene glycol, alkynyl and azido, when substituted, the C 1-10 Alkylene, amido, carbamoyl and polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Y1, Y2 and Y3 are independently selected from C(R) and N; Z2 is absent or selected from the group consisting of one or more of the following substituted or unsubstituted functional groups: -NH-, -O-, -CH2-, hydroxyl, carbonyl, amide, sulfonyl, sulfonylurea, carbamoyl, oxime, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; W2 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 The substituent of the haloalkyl group is substituted; p is an integer of 1 to 10; J2 is selected from -COOH, -N(R)(R'), a substituted or unsubstituted 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, a substituted or unsubstituted 8-16 membered alkynyl ring group, an azido group, a tetrazine group, a substituted or unsubstituted hydroxylamine group, an aldehyde group, a ketone group, an isocyanate group, a thioisocyanate group, a maleimide group and a hydroxyl group, and when substituted, the 3-10 membered nitrogen-containing heterocyclic group, the 8-16 membered alkynyl ring group and the hydroxylamine group are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; R and R' are independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Halogenated alkyl.

6. The compound of formula II according to claim 5, wherein: B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycle, when substituted, the 5-6 membered nitrogen-containing aromatic heterocycle is substituted by a substituent selected from the following: hydrogen, halogen, hydroxyl, -CN, substituted or unsubstituted C 1-10 Alkylene, -C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, carboxyl, substituted or unsubstituted amide, substituted or unsubstituted carbamoyl, substituted or unsubstituted polyethylene glycol, alkynyl and azido, when substituted, the C 1-10 Alkylene, amido, carbamoyl and polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Preferably, B1 and B2 are each independently a single bond or a substituted or unsubstituted pyrimidine ring, and when substituted, the pyrimidine ring is selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; Y1, Y2 and Y3 are each independently selected from CH and N; Z2 is absent or selected from the group consisting of one or more of the following substituted or unsubstituted functional groups: -NH-, -CH2-, carbonyl, -C(=O)NH-, -NHC(=O)- or C 2-6 Alkyne, when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; W2 is absent or selected from substituted or unsubstituted C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of, when substituted, the C 1-10 Alkylene is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl and -OC 1-6 Substitution of haloalkyl groups; J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group; and p is an integer from 1 to 10.

7. The compound of formula II according to claim 5 or 6, wherein: B1 and B2 are each independently selected from substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycles, when substituted, the 5-6 membered nitrogen-containing aromatic heterocycles are substituted by substituents independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups; Y1, Y2 and Y3 are independently selected from CH and N; Z2 is absent or selected from a group consisting of one or more of the following functional groups: -NH-, -CH2-, carbonyl and C 2-6 Alkynylidene; W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of; J2 is selected from -COOH, -NH2, a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxyl group; and p is an integer from 1 to 10.

8. A compound of formula II according to any one of claims 5 to 7, wherein: B1 and B2 are each independently selected from substituted or unsubstituted pyridyl and pyrimidinyl, and when substituted, the pyridyl and pyrimidinyl are substituted with substituents independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups; Y1, Y2 and Y3 are each independently selected from CH and N; Z2 is absent or selected from a group consisting of one or two of the following functional groups: -NH- and carbonyl; W2 does not exist or is selected from C 1-10 Alkylene, -(CH2CH2O) p -and-(OCH2CH2) p - one or more of; J2 is -COOH or -NH2; and p is an integer from 1 to 10.

9. The compound of claim 5 or 6, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, or isotope-labeled compound thereof, wherein: B1 and B2 are each independently selected from substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycles, when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is substituted by a substituent consisting of one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups; and / or Z2 is absent or is selected from a group consisting of one or more of the following functional groups: -NH-, -CH2- and carbonyl; and / or J2 is -COOH or -NH2; and / or p is an integer from 3 to 8.

10. A compound of formula II according to any one of claims 5 to 9, wherein: B1 and B2 are each independently selected from substituted or unsubstituted pyridyl and pyrimidinyl, and when substituted, the pyridyl or pyrimidinyl is substituted with a substituent independently selected from one or more of the following groups: hydrogen, C 1-10 Alkylene, carboxyl, amide, carbamoyl, polyethylene glycol, alkynyl, and azide groups.

11. A compound of formula II according to any one of claims 5 to 10, wherein: B1 and B2 are each independently selected from pyridyl and pyrimidinyl.

12. A compound of formula II according to any one of claims 5 to 11, wherein: Z2 does not exist or is selected from the group consisting of one or more substituted or unsubstituted functional groups: -NH-, -CH2-, carbonyl or C 2-6 Alkyne, when substituted, each of said functional groups is selected from hydrogen, halogen, hydroxy, -CN, -C 1-6 Alkyl, -C 1-6 Haloalkyl, -OC 1-6 Alkyl or -OC 1-6 The substituents of the haloalkyl group are substituted.

13. A compound of formula II according to any one of claims 5 to 12, wherein: Z2 is absent or selected from the group consisting of one or two of the following functional groups: -NH- and carbonyl.

14. A compound of formula II according to any one of claims 5 to 13, wherein: Z2 is absent or is -C(=O)NH-.

15. A compound of formula II according to any one of claims 5 to 14, wherein: J2 is -COOH.

16. The compound of formula II according to any one of claims 5 to 15, which has the following structure: p is an integer from 1 to 10.

17. The compound of formula II according to any one of claims 5 to 16, wherein the compound has the following structure:

18. The compound of formula II according to any one of claims 5 to 17, wherein the compound has the following structure:

19. The following compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound is selected from: (1) 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoic acid; (2) 3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzoic acid; (3) tert-Butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleate; (4) 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid; (5) 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid; (6) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oic acid; (7) 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; (8) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; (9) 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxo Hetero-2-azanonacosane-29-oic acid; (10) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid; (11) 2,6-bis(2-(methylthio)pyrimidin-5-yl)isonicotinic acid; (12) 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; (13) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; (14) 1-(2,6-bis(2-(methylthio)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid; (15) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid; (16) 2,2″-bis(methylthio)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid; (17) 2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid; (18) 1-(2,2"-bis(methylthio)-[5,4':6',5"-tripyrimidinyl]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid; or (19) 1-(2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidine]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oic acid.

20. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein the compound has the structure of Formula V: in: E is selected from a single bond, -NH-CH2-, D is a fragment of a biologically active molecule (e.g., a cytotoxic drug); X, Y, A, Q, Z1, W1 are as defined in any one of claims 1 to 4; V1 is a group formed when J1 and L are connected in the compound of formula I according to any one of claims 1 to 4; preferably, V1 is selected from -C(=O)-, -N(R1)-, -O-, a 3-10 membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; further preferably, V1 is -C(=O)- or -N(R1)-, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; L is a linker connecting V1 and E.

21. The compound of formula V according to claim 20, which has the following structure:

22. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein the compound has the structure of Formula VI: B1, B2, Y1, Y2, Y3, Z2 and W2 are as defined in any one of claims 5 to 18; L is a linker between V2 and E; V2 is a group formed when J2 in any one of claims 5 to 18 is connected to L; preferably, V2 is selected from -C(=O)-, -N(R2)-, -O-, a 3-10 membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; further preferably, V2 is -C(=O)- or -N(R2)-, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; E is selected from a single bond, -NH-CH2-, D is a fragment of a biologically active molecule (eg, a cytotoxic drug).

23. The compound according to claim 22, wherein B1 and B2 are pyrimidinyl groups; Y1 is CH or C, Y2 and Y3 are both CH; Z2 is -C(=O)NH-; W2 is selected from -(CH2CH2O) p -C 1-10 Alkylene- or -(OCH2CH2) p -C 1-10 Alkylene-, p is an integer from 1 to 10; V2 is -C(=O)-; L is E is -NH-CH2-; D is selected from 24. The compound according to claim 23, wherein W2 is selected from -(CH2CH2O)3-CH2-, -(CH2CH2O)3-(CH2)2- and -(OCH2CH2)8-(CH2)2-.

25. according to the compound described in any one of claim 22-24, wherein D is 26. The compound according to any one of claims 22 to 25, wherein the structure is selected from:

27. A bioactive conjugate, the structure of which is shown in Formula VII: in, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a peptide, a non-protein agent (e.g., sugar, RNA, or DNA); n is selected from an integer or decimal between 1 and 10; V1 is -C(O)- or -N(R1)-, wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; L is a linker between V1 and E; E is a structural fragment connecting L and D, preferably, E is as defined in claim 20; D is a fragment of a biologically active molecule (e.g., a cytotoxic drug); The conjugate When the targeting moiety is an antibody, it indicates the specific connection method between the sulfhydryl group in the antibody and the rest of the conjugate; The remaining groups are as defined in any one of claims 1 to 4.

28. A bioactive conjugate, the structure of which is shown in Formula VIII: in, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); n is an integer or decimal selected from 1-10; V2 is selected from -C(O)- or -N(R2)-, wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl; L is a linker between V2 and E; E is a structural fragment connecting L and D, preferably, E is as defined in claim 22; D is a fragment of a biologically active molecule (e.g., a cytotoxic drug); The conjugate When the targeting moiety is an antibody, it indicates the specific connection method between the sulfhydryl group in the antibody and the rest of the conjugate; The remaining groups are as defined in any one of claims 5 to 18.

29. The bioactive conjugate according to claim 28, wherein Ab is selected from anti-Her2 antibody, anti-Trop2 antibody or anti-ROR1 antibody, and n1 is 1-8:

30. The bioactive conjugate according to claim 29, wherein: The anti-Her2 antibody is trastuzumab, an antibody comprising a heavy chain variable region of the trastuzumab heavy chain complementary determining region and a light chain variable region of the trastuzumab light chain complementary determining region, or an antibody comprising a trastuzumab heavy chain variable region sequence and a trastuzumab light chain variable region sequence; The anti-Trop2 antibody is sacituzumab, an antibody comprising a heavy chain variable region of the sacituzumab heavy chain complementarity determining region and a light chain variable region of the sacituzumab light chain complementarity determining region, or an antibody comprising a sacituzumab heavy chain variable region sequence and a sacituzumab light chain variable region sequence; and / or The anti-ROR1 antibody is: i) an antibody defined according to the Chothia numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 3, CDR-H2 of SEQ ID NO: 4, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8; ii) an antibody defined according to the AbM numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:10, and CDR-H3 of SEQ ID NO:5, and a light chain variable region comprising CDR-L1 of SEQ ID NO:6, CDR-L2 of SEQ ID NO:7, and CDR-L3 of SEQ ID NO:8; iii) an antibody defined according to the Kabat numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8; iv) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 8, as defined by the IMGT numbering system Antibody; v) an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2; or vi) 19F6_Hu35V1, an antibody containing the heavy chain variable region shown in SEQ ID NO: 1, the light chain variable region shown in SEQ ID NO: 2, the heavy chain constant region shown in SEQ ID NO: 18, and the light chain constant region shown in SEQ ID NO:

19.

31. The bioactive conjugate according to claim 29 or 30, wherein: n1 is 1-6, for example, 3-5.

32. The bioactive conjugate according to claim 28, wherein Ab is selected from anti-Her2 antibody, anti-Trop2 antibody or anti-ROR1 antibody; n1 is 1-8; and x is 1-10:

33. The bioactive conjugate according to claim 32, wherein: (1) The anti-Her2 antibody is trastuzumab, an antibody comprising a heavy chain variable region of the trastuzumab heavy chain complementary determining region and a light chain variable region of the trastuzumab light chain complementary determining region, or an antibody comprising a trastuzumab heavy chain variable region sequence and a trastuzumab light chain variable region sequence; (2) the anti-Trop2 antibody is sacituzumab, an antibody comprising a heavy chain variable region of the sacituzumab heavy chain complementary determining region and a light chain variable region of the sacituzumab light chain complementary determining region, or an antibody comprising a sacituzumab heavy chain variable region sequence and a sacituzumab light chain variable region sequence; (3) The anti-ROR1 antibody is: i) an antibody defined according to the Chothia numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 3, CDR-H2 of SEQ ID NO: 4, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8; ii) an antibody defined according to the AbM numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:10, and CDR-H3 of SEQ ID NO:5, and a light chain variable region comprising CDR-L1 of SEQ ID NO:6, CDR-L2 of SEQ ID NO:7, and CDR-L3 of SEQ ID NO:8; iii) an antibody defined according to the Kabat numbering system, comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 5, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 6, CDR-L2 of SEQ ID NO: 7, and CDR-L3 of SEQ ID NO: 8; iv) an antibody comprising a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15, and a light chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 8, as defined by the IMGT numbering system; v) an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2; or vi) 19F6_Hu35V1, an antibody containing the heavy chain variable region shown in SEQ ID NO: 1, the light chain variable region shown in SEQ ID NO: 2, the heavy chain constant region shown in SEQ ID NO: 18, and the light chain constant region shown in SEQ ID NO:

19.

34. The bioactive conjugate according to claim 31 or 32, wherein: n1 is 1-6, for example, 3-5.

35. The bioactive conjugate according to any one of claims 31 to 33, wherein: x is 1-6, for example, 3-5.

36. A pharmaceutical composition comprising the compound of any one of claims 20-26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, or the bioactive conjugate of any one of claims 27-35, and one or more pharmaceutically acceptable carriers; Preferably, the drug / antibody ratio (DAR value) of the pharmaceutical composition is 1.0-6.0, such as 1, 2, 3, 4, 5 or 6, and further such as 1.0-1.5, 1.0-2.0, 1.0-2.5, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 1.0-5.5, 1.0-6.0, 1.5-2.0, 1.5-2.5, 1.5-3.0, 1.5-3.5, 1.5-4.0, 1.5-4.5, 1.5-5.0, 1.5-5.5, 1.5-6.0, 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0 -5.0, 2.0-5.5, 2.0-6.0, 2.5-3.0, 2.5-3.5, 2.5-4.0, 2.5-4.5, 2.5-5.0, 2.5-5.5, 2.5-6.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 5.0-5.5, 5.0-6.0, or 5.5-6.

0.

37. A kit comprising the compound of any one of claims 20-26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, or the bioactive conjugate of any one of claims 27-35, or the pharmaceutical composition of claim 36, and optionally a package insert.

38. Use of the compound of any one of claims 20-26, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, the bioactive conjugate of any one of claims 27-35, or the pharmaceutical composition of claim 36, in the preparation of a medicament for preventing or treating a tumor disease.

39. A compound according to any one of claims 20-26, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, the bioactive conjugate according to any one of claims 27-35, or the pharmaceutical composition according to claim 36, for use in preventing or treating a tumor disease.

40. A method for preventing or treating a tumor disease, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 20 to 26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, the bioactive conjugate according to any one of claims 27 to 35, or the pharmaceutical composition according to claim 36.

41. A method for synthesizing a compound, characterized in that: The method comprises the following steps: in: X, Z1, W1, J1 are as defined in any one of claims 1 to 4; Y is absent; M is a leaving group that undergoes substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, preferably halogen; Alternatively, include the following steps: in: X, Z1, W1, J1 are as defined in any one of claims 1 to 4; L is a leaving group that undergoes substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, preferably halogen, OTf; Alternatively, include the following steps: in: Y1, Y2, Y3, B1, B2, Z2, W2 and J2 are as defined in any one of claims 5 to 18; LG is a leaving group for coupling reaction, including but not limited to halogen and trifluoromethanesulfonate, preferably halogen.

42. Use of the compound according to any one of claims 1 to 4 for preparing a drug linker compound, wherein: The compound according to any one of claims 1 to 4 is prepared by the following steps: Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, and D are as defined in any one of claims 1 to 4, 20, and 21; and LG1 is selected from a group that undergoes a condensation reaction with J1.

43. The use according to claim 42, wherein: The drug linker compound is selected from the compound of claim 20 or 21 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof.

44. The use according to claim 43, wherein: LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

45. Use of the compound according to any one of claims 5 to 18 for preparing a drug linker compound; The compound according to any one of claims 5 to 18 is prepared by the following steps: in, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, and D are as defined in any one of claims 5 to 18 and 22 to 26; and LG2 is selected from a group that undergoes a condensation reaction with J2.

46. ​​The use according to claim 45, wherein: The drug linker is selected from the compound of any one of claims 22 to 26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof.

47. The use according to claim 45, wherein: LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

48. Use of the compound according to any one of claims 1 to 4 for preparing a biologically active conjugate; The compound according to any one of claims 1 to 4 is prepared into the bioactive conjugate by following steps 1a and 1b: Step 1a: Step 1b: in, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined in any one of claims 1-4, 20, 21, 27, 29-31; LG1 is selected from a group that undergoes a condensation reaction with J1.

49. The use according to claim 48, wherein: The bioactive conjugate is selected from the bioactive conjugate according to any one of claims 27 and 29-31.

50. The use according to claim 48 or 49, wherein: LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

51. Use of the compound according to any one of claims 5 to 18 for preparing a biologically active conjugate; The compound according to any one of claims 5 to 18 is prepared into the bioactive conjugate by following steps 2a and 2b: Step 2a: Step 2b: in, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are as defined in any one of claims 5-18, 22-26, 28, 32-35; LG2 is selected from a group that undergoes condensation reaction with J2.

52. The use according to claim 51, wherein: The bioactive conjugate is selected from the bioactive conjugate according to any one of claims 28 and 32-35.

53. The use according to claim 51 or 52, wherein: LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

54. A method for preparing a compound, the method comprising the following steps:. Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, and D are as defined in any one of claims 1 to 4, 20, and 21; and LG1 is selected from a group that undergoes a condensation reaction with J1.

55. The method of claim 54, wherein: LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

56. A method for preparing a compound, comprising the steps of: Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, and D are as defined in any one of claims 5 to 18 and 22 to 26; and LG2 is selected from a group that undergoes a condensation reaction with J2.

57. The method of claim 56, wherein: LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

58. A method for preparing an antibody drug conjugate, the method comprising the following steps: Step 1a: Step 1b: Wherein, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined in any one of claims 1-4, 20, 21, 27, 29-31; LG1 is selected from a group that undergoes condensation reaction with J1.

59. The method of claim 58, wherein: LG1 is selected from -COOH or -NH(R1), wherein R1 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

60. A method for preparing an antibody drug conjugate, the method comprising the following steps: Step 2a: Step 2b: Wherein, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are defined as any one of claims 5-18, 22-26, 28, 32-35; LG2 is selected from a group that undergoes condensation reaction with J2.

61. The method of claim 60, wherein: LG2 is selected from -COOH or -NH(R2), wherein R2 is H, C 1-6 Alkyl or C 2-6 Alkoxyalkyl.

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