Compounds that can be used in conjugation reactions and their complexes

Abstract

The present application provides compounds and conjugates thereof that can be used in conjugation reactions, as well as synthetic intermediates and methods for the compounds and conjugates, and the use of the conjugates in the preparation of drugs for preventing or treating tumor diseases.

Description

[Technical Field]

[0001] This application is based on and claims priority from CN Application No. 202211509417.1, filed November 29, 2022, CN Application No. 202211536550.6, filed December 2, 2022, and CN Application No. 202211573193.0, filed December 8, 2022, the disclosures of which are hereby incorporated by reference in their entireties.

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

[0003] In recent years, antibody-drug conjugates have become a hot topic in the field of precision tumor treatment, bringing hope to tumor treatment. Antibody-drug conjugates (ADCs) are formed by linking a target-specific antigen monoclonal antibody drug with a small molecule cytotoxic drug via a linker, combining the potent killing effect of traditional small molecule chemotherapy with the tumor targeting ability of antibody drugs. By the time of the approval of Zynlonta in April 2021, 12 ADC drugs had already been approved and launched worldwide, of which seven were used to treat hematological malignancies and five were used to treat solid tumors.

[0004] Antibody-conjugated drugs consist of an antibody, a linker, and a payload. Conjugation methods for antibodies and drug-linkers (payload-linkers) can be broadly divided into nonspecific and specific conjugations. Early nonspecific conjugation methods, primarily lysine and cysteine ​​conjugation, used chemical methods to directly conjugate drugs to amino acid residues on antibodies. Regardless of antibody modification, the number of toxin molecules conjugated and the conjugation sites were undetermined, resulting in a lack of uniformity. Currently, the commonly used specific conjugation method uses genetically engineered sites or special connectors for specific conjugation, resulting in more uniform conjugation and enabling the attachment of cytotoxins at specific sites. Antibody-conjugated drugs produced using specific conjugation methods can reduce variations in drug efficacy, drug metabolism, and CMC quality control due to differences in conjugation sites and number of conjugates.

[0005] Currently commonly used specific conjugation methods include THIOMAB technology, unnatural amino acid conjugate technology, glutaminase-induced conjugate technology, sortase transpeptidase conjugate technology, and ThioBridge technology. Among these, antibody modification using antibody engineering or enzyme-induced conjugates can affect the structural stability of the antibody and place certain requirements on CMC. Furthermore, some ThioBridge technologies that use chemical conjugates also have certain drawbacks. For example, linkers such as DBM (dibromomaleimides) can be substituted with other sulfhydryl-containing biomolecules, resulting in instability in plasma, reduced efficacy, and increased toxic side effects (Chem.-Eur.J., 2019, 25, 43-59). Therefore, the development of new linker structures remains important for the development of antibody-based drug conjugates with good efficacy and safety. Summary of the Invention

[0006] One objective of this application is to provide a new type of linker for use in chemical conjugation, which has high reactivity, mild conjugation conditions, simple operation, and can realize specific conjugates. The resulting bioactive complexes have relatively good homogeneity and plasma stability, and have clear medicinal effects both in vivo and in vivo.

[0007] compound In a first aspect, the present application provides 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 I: [ka] During the ceremony, X is a leaving group, e.g., F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenol ester, fluorophenol ester, C 1-6 an alkylsulfonyl group, or [ka] and Y is absent or is a substituted or unsubstituted C 1-6 alkylene group, sulfonyl group, and carbonyl group, and when substituted, 1-6 The alkylene group can contain hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Ring A is a substituted or unsubstituted C 6-10 selected from aromatic rings, substituted or unsubstituted 5- to 12-membered aromatic heterocycles and substituted or unsubstituted 5- to 12-membered heterocycles, and when substituted, 6-10 The aromatic ring, the 5- to 12-membered aromatic heterocycle, and the 5- to 12-membered heterocycle are independently hydrogen, halogen, a hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC1-6 Alkyl group, -OC 1-6 substituted with a substituent selected from a haloalkyl group, a carboxy group, a polyethylene glycol, an amino acid, a phosphoric acid, a sulfonic acid, an amino group, an azide group, and an alkynyl group; Q is absent or is -NH-, -O-, -CH2-, a hydroxy group, a carbonyl group, an amide group, a sulfonyl group, a sulfonylurea group, an amidoformyl group, an oxime group, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 It consists of one or more substituted or unsubstituted groups of alkynylene groups, and when substituted, each such group independently is selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Z1 is absent or is a substituted or unsubstituted phenyl group, a substituted or unsubstituted 5- or 6-membered heteroaryl group, an amido group, a substituted or unsubstituted -CH2- or a substituted or unsubstituted C 2-6 alkynylene groups, when substituted, selected from the phenyl group, 5- to 6-membered heteroaryl groups, -CH2- and C 2-6 The alkynylene groups are independently hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; W1 is absent or a substituted or unsubstituted C 1-10 is one or more selected from the group consisting of an alkylene group, -(CH2CH2O)p-, and -(OCH2CH2)p-, where p is an integer of 1 to 20; and when substituted, 1-10 The alkylene group can contain hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6substituted by a substituent selected from haloalkyl groups; J1 is selected from -COOH, -NH2, substituted -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a substituted 3- to 10-membered nitrogen-containing heterocyclic group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, a substituted 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, a sulfonylurea group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxy group, and the "substituted" is independently selected from hydrogen, halogen, a hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 It refers to being substituted with a substituent selected from haloalkyl groups.

[0008] In some embodiments, the A ring is a substituted or unsubstituted C 6-10 selected from aromatic rings, substituted or unsubstituted 5- to 12-membered aromatic heterocycles and substituted or unsubstituted 5- to 12-membered heterocycles, and when substituted, 6-10 The aromatic ring, the 5- to 12-membered aromatic heterocycle, and the 5- to 12-membered heterocycle are independently substituted with a substituent selected from a carboxy group, a polyethylene glycol, an amino acid, a phosphoric acid, a sulfonic acid, an amino group, an azide group, and an alkynyl group.

[0009] In some embodiments, X is a leaving group, e.g., Cl, Br, I, OMs, OTs, OTf, or [ka] and Y is absent or a carbonyl group; Ring A is a substituted or unsubstituted C 6-10 selected from aromatic rings, substituted or unsubstituted 5- to 12-membered aromatic heterocycles and substituted or unsubstituted 5- to 12-membered heterocycles, and when substituted, 6-10 The aromatic ring, the 5- to 12-membered aromatic heterocycle, and the 5- to 12-membered heterocycle are independently hydrogen, halogen, a hydroxy group, -CN, -C 1-6 Alkyl group, -C1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Q is absent or is a substituted or unsubstituted —C(═O)NH—, and when substituted, the —C(O)—NH— is hydrogen, halogen, a hydroxy group, —CN, —C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Z1 is absent or is a substituted or unsubstituted -CH2- or a substituted or unsubstituted C 2-6 an alkynylene group, which, when substituted, is —CH— or C 2-6 The alkynylene groups are independently hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; W1 is absent or a substituted or unsubstituted C 1-10 alkylene group, -(CH2CH2O)p- and -(OCH2CH2)p-, where p is an integer of 1 to 10; 1-10 The alkylene group can contain hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; J1 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, and a hydroxy group.

[0010] In some embodiments, X is a leaving group, such as F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenol esters, fluorophenol esters, C 1-6alkylsulfonyl group or [ka] is selected from Y is absent or C 1-6 selected from an alkylene group, a sulfonyl group, and a carbonyl group; Ring A is a substituted or unsubstituted C 6-10 an aromatic ring, a 5- to 12-membered aromatic heterocycle, or a 5- to 12-membered heterocycle, wherein the substituent is selected from a carboxy group, a polyethylene glycol, an amino acid, a phosphoric acid, a sulfonic acid, an amino group, an azide group, and an alkynyl group; Q is absent or is -NH-, -O-, -CH2-, a hydroxy group, a carbonyl group, an amide group, a sulfonyl group, a sulfonylurea group, a carbamoyl group, an oxime group, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)-, or C 2-6 A fragment consisting of one or more groups of an alkynylene group, and preferably Z2 is absent or is -NH-, -CH2-, a carbonyl group or C 2-6 is a fragment consisting of one or more groups selected from alkynylene groups, Z1 is absent or is a phenyl group, a 5- or 6-membered heteroaryl group, an amide group, -CH2- or C 2-6 alkynylene groups, W1 does not exist or C 1-10 one or more selected from an alkylene group, -(CH2CH2O)p-, and -(OCH2CH2)p-, where p is an integer of 1 to 20; J1 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, a sulfonylurea group, an isocyanate, a thioisocyanate, a maleimide group, or a hydroxy group; p is an integer from 1 to 10.

[0011] In some embodiments, X is one or more selected from Cl, Br, I, OMs, OTs, and OTf.

[0012] In some embodiments, Y is absent or C 1-6 It is an alkylene group.

[0013] In some embodiments, Q is absent, -C(=O)-NH-, or -NH-C(=O)-.

[0014] In some implementations, Z1 is not present and C 2-6 It is an alkynylene group.

[0015] In some implementations, W1 does not exist or C 1-10 The alkylene group is one or more selected from the group consisting of an alkylene group and -(CH2CH2O)p-.

[0016] In some implementations, W1 is C 1-10 is an alkylene group, preferably C 1-6 is an alkylene group, more preferably C 1-3 It is an alkylene group.

[0017] In some embodiments, J1 is -COOH, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, or a maleimide group.

[0018] In some embodiments, the A ring is selected from a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle or a 5-12 membered nitrogen-containing heterocycle, and when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle and the 5-12 membered nitrogen-containing heterocycle are independently substituted with a substituent selected from a carboxy group, polyethylene glycol, amino acid, phosphoric acid, sulfonic acid, amino group, azide group, and alkynyl group, and preferably the A ring 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.

[0019] In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0020] In some embodiments, X is Cl, Br, I, OMs, OTs, OTf, C 1-6 alkylsulfonyl group or [ka] is selected from Y does not exist, Ring A is selected from a 5- to 12-membered nitrogen-containing aromatic heterocycle or a 5- to 12-membered nitrogen-containing heterocycle unsubstituted or substituted by an oxo group or —COOH; Q is absent or is -C(=O)-NH-; Z1 is absent or -CH2-; W1 does not exist or C 1-10 one or more selected from an alkylene group and -(CH2CH2O)p-; J1 is -COOH, p is an integer from 1 to 10.

[0021] In some embodiments, Formula I is selected from the following structures: [ka]

[0022] In some embodiments, the compound of Formula I has the following structure: [ka] In the formula, p is an integer of 1 to 10, and J1 is —COOH or —NH2.

[0023] In some embodiments, the compound of Formula I has the following structure: [ka]

[0024] In a second aspect, the present application provides 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 II: [ka] In the formula, B1 and B2 each independently represent a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle, and when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle may be hydrogen, halogen, a hydroxy group, -CN, a substituted or unsubstituted C 1-10 Alkylene group, -C 1-16 Haloalkyl group, -OC 1-6 Alkyl group, -OC 1-6 and when substituted by one or more substituents selected from the group consisting of haloalkyl groups, carboxy groups, substituted or unsubstituted amido groups, substituted or unsubstituted carbamoyl groups, substituted or unsubstituted polyethylene glycol, alkynyl groups, and azido groups, the C 1-10 The alkylene group, amide group, carbamoyl group, and polyethylene glycol are independently selected from hydrogen, halogen, hydroxyl group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Y1, Y2, and Y3 are independently selected from C(R) and N; Z2 is absent or is -NH-, -O-, -CH2-, a hydroxy group, a carbonyl group, an amide group, a sulfonyl group, a sulfonylurea group, a carbamoyl group, an oxime group, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O) or C 2-6 An alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Preferably, Z2 is absent or is -NH-, -CH2-, a carbonyl group or C 2-6 A group consisting of one or more functional groups of alkynylene groups, and when substituted, each of said functional groups independently is hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; W2 is absent or is a substituted or unsubstituted C 1-10 Alkylene group, -(CH2CH2O) p - and -(OCH2CH2) p -, and when substituted, said C 1-10 The alkylene group can contain hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups, and p is an integer of 1 to 10; J2 is selected from -COOH, -N(R)(R'), a substituted or unsubstituted 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, a substituted or unsubstituted 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a substituted or unsubstituted hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxyamide group, and when substituted, the 3- to 10-membered nitrogen-containing heterocyclic group, the 8- to 16-membered alkynyl-containing ring group, and the hydroxyamide group are independently hydrogen, halogen, a hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; R and R' are independently hydrogen, halogen, hydroxyl group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 haloalkyl groups.

[0025] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5-12 membered nitrogen-containing aromatic heterocycle, and the 5-12 membered nitrogen-containing aromatic heterocycle is optionally selected from hydrogen, C 1-10 substituted with a fragment consisting of one or more functional groups selected from the group consisting of alkylene, carboxy, amide, carbamoyl, polyethylene glycol, alkynyl, and diazo groups; each occurrence of Y1, Y2, and Y3 is independently selected from CH and N; Z2 is absent or is -NH-, -O-, -CH2-, a hydroxy group, a carbonyl group, an amide group, a sulfonyl group, a sulfonylurea group, a carbamoyl group, an oxime group, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Preferably, Z2 is absent or is selected from the group consisting of -NH-, -CH2-, a carbonyl group or C 2-6is a fragment selected from one or more groups of alkynylene groups, W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p -or-(OCH2CH2) p - one or more selected from J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxy group; p is an integer from 1 to 10.

[0026] 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, and if substituted, the 5-12 membered nitrogen-containing aromatic heterocycle is hydrogen, substituted or unsubstituted C 1-10 When substituted by one or more substituents selected from the group consisting of an alkylene group, a carboxy group, a substituted or unsubstituted amide group, a substituted or unsubstituted carbamoyl group, a substituted or unsubstituted polyethylene glycol, an alkynyl group, and an azide group, the C 1-10 Alkylene group, amide group, carbamoyl group, and polyethylene glycol are independently hydrogen, halogen, hydroxyl group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 It is substituted with a substituent selected from haloalkyl groups.

[0027] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5- to 12-membered nitrogen-containing aromatic heterocycle, and when substituted, the 5- to 6-membered nitrogen-containing aromatic heterocycle may be selected from hydrogen, halogen, a hydroxy group, —CN, C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group, or -OC1-6 It is substituted by a substituent selected from haloalkyl groups.

[0028] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5-12 membered nitrogen-containing aromatic heterocycle, and the 5-12 membered nitrogen-containing aromatic heterocycle is optionally selected from hydrogen, C 1-10 It is substituted with a fragment consisting of one or more functional groups selected from the group consisting of alkyl groups, carboxy groups, amide groups, carbamoyl groups, polyethylene glycol, alkynyl groups, and azide groups.

[0029] each occurrence of Y1, Y2, and Y3 is independently selected from CH and N; Z2 is absent or is -NH-, -O-, -CH2-, a hydroxy group, a carbonyl group, an amide group, a sulfonyl group, a sulfonylurea group, a carbamoyl group, an oxime group, -NH-S(=O)2-NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C 2-6 Preferably, Z2 is absent or is selected from the group consisting of -NH-, -CH2-, a carbonyl group or C 2-6 is a fragment selected from one or more groups of alkynylene groups, W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p -or-(OCH2CH2) p - one or more selected from J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, a maleimide group, or a hydroxy group; p is an integer from 1 to 10.

[0030] In some embodiments, B1 and B2 are each independently selected from a single bond or a substituted or unsubstituted 5- to 6-membered nitrogen-containing aromatic heterocycle, and when substituted, the 5- to 6-membered nitrogen-containing aromatic heterocycle is selected from hydrogen, halogen, a hydroxy group, —CN, a substituted or unsubstituted C 1-10 Alkylene group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group, -OC 1-6 and when substituted by a substituent selected from haloalkyl groups, carboxy groups, substituted or unsubstituted amide groups, substituted or unsubstituted carbamoyl groups, substituted or unsubstituted polyethylene glycol, alkynyl groups, and azido groups, the C 1-10 The alkylene group, amide group, carbamoyl group, and polyethylene glycol are independently selected from hydrogen, halogen, hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group 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 substituted with hydrogen, halogen, a hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; Y1, Y2, and Y3 are each independently selected from CH and N; Z2 is absent or is -NH-, -CH2-, a carbonyl group, -C(=O)NH-, -NHC(=O)- or C 2-6 An alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; W2 is absent or is a substituted or unsubstituted C 1-10 Alkylene group, -(CH2CH2O) p - and -(OCH2CH2) p -, and when substituted, said C 1-10 The alkylene group can contain hydrogen, halogen, hydroxyl, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group and -OC 1-6 substituted by a substituent selected from haloalkyl groups; J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, and a hydroxy group; and p is an integer from 1 to 10.

[0031] 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 hydrogen, halogen, a hydroxy group, —CN, —C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group 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 group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group or -OC 1-6 haloalkyl groups.

[0032] each Y1, Y2, and Y3 is independently selected from CH and N; Z2 is absent or is -NH-, -CH2-, a carbonyl group, -C(=O)NH-, -NHC(=O)- or C 2-6 The alkynylene group consists of one or more substituted or unsubstituted groups, the substituents being independently selected from hydrogen, halogen, hydroxyl, -CN, -C1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group or -OC 1-6 haloalkyl groups, W2 is absent or is a substituted or unsubstituted C 1-10 Alkylene group, -(CH2CH2O) p -or-(OCH2CH2) p -, wherein the substituents are independently one or more selected from hydrogen, halogen, hydroxy group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group or -OC 1-6 haloalkyl groups, J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, or a hydroxy group; and p is an integer from 1 to 10.

[0033] In some embodiments, B1 and B2 are each independently selected from a substituted or unsubstituted 5-6 membered nitrogen-containing heteroaromatic ring, and when substituted, the 5-6 membered nitrogen-containing heteroaromatic ring is independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from the group consisting of alkylene, carboxy, amide, carbamoyl, polyethylene glycol, alkynyl, and azide; Y1, Y2, and Y3 are independently selected from CH and N; Z2 is absent or is -NH-, -CH2-, a carbonyl group, or C 2-6 selected from the group consisting of one or more functional groups of alkynylene groups, W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p - and -(OCH2CH2) p - one or more selected from J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, and a hydroxy group; and p is an integer from 1 to 10.

[0034] In some embodiments, B1 and B2 are each independently selected from substituted or unsubstituted pyridine and pyrimidine groups, and when substituted, the pyridine and pyrimidine groups are independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from the group consisting of alkylene, carboxy, amide, carbamoyl, polyethylene glycol, alkynyl, and azide; Y1, Y2, and Y3 are each independently selected from CH and N; Z2 is absent or is selected from the group consisting of one or up to two functional groups selected from -NH- and carbonyl groups; W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p - and -(OCH2CH2) p - one or more selected from J2 is -COOH or -NH2, and p is an integer from 1 to 10.

[0035] In some embodiments, B1 and B2 are each independently selected from a single bond or a 5- or 6-membered nitrogen-containing aromatic heterocycle, and preferably B1 and B2 are each independently selected from a single bond, an oxazole ring, a thiazole ring, or a pyrimidine ring.

[0036] In some embodiments, each occurrence of B1 is independently selected from a single bond or a 5- or 6-membered nitrogen-containing aromatic heterocycle; preferably, each occurrence of B1 is independently selected from a single bond or a 5- or 6-membered nitrogen-containing aromatic heterocycle; more preferably, each occurrence of B1 is independently selected from a single bond, an oxazole ring, a thiazole ring, or a pyrimidine ring.

[0037] In some embodiments, Y1, Y2, and Y3 are all N, or Y1 is CH, and Y2 and Y3 are all N, or Y1 is N, and Y2 and Y3 are all CH, or Y1, Y2, and Y3 are all CH.

[0038] In some embodiments, Z2 is absent or is -NH-, -CH2-, a carbonyl group, or C 2-6 The alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is selected from the group consisting of hydrogen, halogen, hydroxyl group, -CN, -C 1-6 Alkyl group, -C 1-6 Haloalkyl group, -OC 1-6 Alkyl group or -OC 1-6 It is substituted with a substituent selected from haloalkyl groups.

[0039] In some embodiments, Z2 is absent or is selected from the group consisting of one or up to two of the following functional groups: -NH- and a carbonyl group.

[0040] In some embodiments, Z2 is absent or is an amide group (-C(=O)NH-).

[0041] In some embodiments, Z2 is absent or a carbamoyl group.

[0042] In some implementations, Z2 is absent or C 2-6 It is an alkynylene group.

[0043] In some implementations, W2 does not exist or 1-10 It is an alkylene group.

[0044] In some embodiments, J2 is selected from -COOH or -NH2.

[0045] In some embodiments, J2 is -COOH.

[0046] In some embodiments, each occurrence of B1 is independently selected from a single bond or a pyrimidine ring; Y1, Y2 and Y3 are all CH; Z2 is absent, - or C(=O)NH-; W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p and one or more selected from J2 is -COOH, p is an integer from 1 to 10.

[0047] In some embodiments, B1 and B2 are each independently selected from a single bond or a pyrimidine ring; Y1, Y2 and Y3 are all CH; Z2 is absent or -C(=O)NH-; W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p and one or more selected from J2 is -COOH, p is an integer from 1 to 10.

[0048] In some embodiments, B1 and B2 are each independently selected from a substituted or unsubstituted 5-12 membered nitrogen-containing heteroaromatic ring, and when substituted, the 5-12 membered nitrogen-containing heteroaromatic ring is independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol, an alkynyl group, and an azide group; Preferably, B1 and B2 are each independently selected from substituted or unsubstituted 5-6 membered nitrogen-containing aromatic heterocycles, and when substituted, the 5-6 membered nitrogen-containing aromatic heterocycles are independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol, an alkynyl group, and an azide group; Preferably, B1 and B2 are each independently selected from substituted or unsubstituted pyridine and pyrimidine groups, and when substituted, the pyridine or pyrimidine groups are independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol, an alkynyl group, and an azide group; Preferably, B1 and B2 are each independently selected from a pyridine group or a pyrimidine group; and / or Z2 is absent or is selected from the group consisting of one or more functional groups selected from -NH-, -CH2- and a carbonyl group; Preferably, Z2 is absent or is selected from the group consisting of one or more of the following functional groups: -NH- or carbonyl group; Preferably, Z2 is absent or -C(=O)NH-; and / or J2 is -COOH or -NH2, Preferably, J2 is -COOH; and / or p is an integer of 3 to 8.

[0049] In some embodiments, B1 and B2 are each independently selected from substituted or unsubstituted 5- to 6-membered nitrogen-containing aromatic heterocycles, and when substituted, the 5- to 6-membered nitrogen-containing aromatic heterocycles are independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol, an alkynyl group, and an azide group; each Y1, Y2, and Y3 is independently selected from CH and N; Z2 is absent or is -NH-, -CH2-, a carbonyl group or C 2-6 selected from the group consisting of one or more alkynylene groups, W2 does not exist or C 1-10 Alkylene group, -(CH2CH2O) p-or-(OCH2CH2) p - one or more selected from J2 is selected from -COOH, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, or a hydroxy group; and p is an integer from 1 to 10, Preferably, B1 and B2 are each independently selected from a substituted or unsubstituted pyridine group and a pyrimidine group, and when substituted, the pyridine group or pyrimidine group is independently selected from hydrogen, C 1-10 substituted with one or more substituents selected from an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol group, an alkynyl group, and an azide group; Y1, Y2, and Y3 are each independently selected from CH and N; Z2 is absent or is selected from the group consisting of one or more of -NH- or carbonyl groups; J2 is selected from -COOH or -NH2, and p is an integer from 1 to 10.

[0050] In some embodiments, the compound of Formula II has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0051] p is an integer from 1 to 10.

[0052] In some embodiments, p is an integer from 1 to 8, and more preferably, p is an integer from 1 to 5.

[0053] In some embodiments, the compound of Formula II has the following structure: [ka]

[0054] In some embodiments, the compound of Formula II has the following structure, where p is an integer from 1 to 10: [ka] [ka]

[0055] In some embodiments, the compound of Formula II has the following structure: [ka] [ka] [ka] [ka]

[0056] In some embodiments, the compound of Formula II has the following structure: [ka]

[0057] In some embodiments, the present application provides 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 IIA. [ka] In the formula, T1 and T2 are leaving groups, and T1 and T2 may be the same or different. J3 is selected from -COOR3, -NH2, a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxy group; R3 is H, C 1-6 Alkyl group, C 6-10 an aryl group, a 3- to 8-membered heterocyclic group, and a 5- to 10-membered heterocyclic group; 1-6 Alkyl group, C 6-10 The aryl group, the 3- to 8-membered heterocyclic group and the 5- to 10-membered heterocyclic group may optionally contain one or more hydroxy groups, halogens, C 1-6 substituted by a substituent selected from an alkoxy group; B1, B2, Y1, Y2, Y3, Z2, and W2 are as described in any of the above documents.

[0058] In some embodiments, T1 and T2 are each independently selected from the group consisting of halogen, OMs, OTs, OTf, nitro, and the following groups, optionally substituted with one or more R4: alkyl thioether, aryl thioether, heteroaryl thioether, alkyl sulfone, aryl sulfone, heteroaryl sulfone, alkyl sulfonyl, aryl sulfonyl, and heteroaryl sulfonyl, wherein R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl group, halo C 1-6 Alkyl group, C 1-6 selected from an alkoxy group, a 6- to 10-membered aryl group, and a 5- to 12-membered heteroaryl group; Preferably, T1 and T2 are each independently selected from the following groups, optionally substituted by one or more R4: alkyl thioether group, aryl thioether group, heteroaryl thioether group, alkylsulfonyl group, arylsulfonyl group, and heteroarylsulfonyl group, wherein R4 is independently H (hydrogen), D (deuterium), halogen, CN, nitro group, C 1-6 Alkyl group, halo C 1-6 Alkyl group, C 1-6 selected from an alkoxy group, a 6- to 10-membered aryl group, and a 5- to 12-membered heteroaryl group; Preferably, T1 and T2 are each independently selected from alkylthioether groups and alkylsulfonyl groups, optionally substituted with one or more R4, where R4 is independently H (hydrogen), D (deuterium), halogen, CN, a nitro group, C 1-6 Alkyl group, halo C 1-6 Alkyl groups and C 1-6 alkoxy groups, Preferably, T1 and T2 are each independently selected from C optionally substituted by one or more R4. 1-6 Alkyl thioether group or C 1-6 alkylsulfonyl groups, and R4 is independently selected from H (hydrogen), D (deuterium), halogen, CN, a nitro group, C 1-6 Alkyl group, halo C 1-6 Alkyl groups and C 1-6 alkoxy groups, Preferably, T1 and T2 are each independently C 1-6 Alkyl thioether group or C 1-6 an alkylsulfonyl group, Preferably, T1 and T2 are each independently -S(O)2-CH3 or -S-CH3.

[0059] In some embodiments, J3 is -COOR3 or -NH2, and R3 is H, D, C 1-6 Alkyl group, C 6-10 an aryl group, a 3- to 8-membered heterocyclic group, and a 5- to 10-membered heterocyclic group; 1-6 Alkyl group, C 6-10The aryl group, the 3- to 8-membered heterocyclic group and the 5- to 10-membered heterocyclic group may optionally contain one or more hydroxy groups, halogens and C 1-6 Preferably, J3 is -COOR3 or -NH2, and R3 is H, D, or C. 1-6 alkyl group, 1-6 The alkyl group may optionally contain one or more hydroxy groups, halogens and C 1-6 substituted by a substituent selected from an alkoxy group; Preferably, J3 is -COOC 1-6 alkyl group, -COOH, and -NH2; 1-6 The alkyl group may optionally contain one or more hydroxy groups, halogens and C 1-6 substituted by a substituent selected from an alkoxy group; Preferably, J3 is selected from -COOCH3, -COOH and -NH2.

[0060] In some embodiments, the compound of Formula IIA has the following structure: [ka] [ka] [ka] [ka]

[0061] In some embodiments, the present invention provides a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, or isotopically labeled compound thereof, wherein the compound is selected from: (1) 3,5-di(2-(methylsulfide)pyrimidin-4-yl)benzoic acid, (2) 3,5-di(2-(mesyl)pyrimidin-4-yl)benzoic acid, (3) tert-butyl 1-(3,5-di(2-(mesyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleate, (4) 3,5-di(2-(methylsulfide)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-azatridecan-13-oic acid, (7) 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (8) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (9) 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonacosan-29-oic acid, (10) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonacosan-29-oic acid, (11) 2,6-di(2-(methylsulfido)pyrimidin-5-yl)isonicotinic acid, (12) 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (13) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (14) 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonacosan-29-oic acid, (15) 1-(2,6-di(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonacosan-29-oic acid, (16) 2,2″-bis(methylsulfide)-[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(methylsulfide)-[5,4':6',5"-tripyrimidin]-2"-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, or (19) 1-(2,2″-bis(methylsulfonyl)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid.

[0062] In a third aspect, the present application provides 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 III. [ka] Here, V1 is a group formed when J1 and L are linked in the compound of formula I in the first aspect of the present application, and preferably V1 is selected from -C(=O)-, -N(R1)-, -O-, a 3- to 10-membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R1 is H, C 1-6 Alkyl group or C 2-6 More preferably, V1 is -C(O)- or -N(R1)-, where R1 is H, C 1-6 Alkyl group or C 2-6is an alkoxyalkyl group, L is a linker connecting V1 and E', E' is H, -NHCH2-Lg, -COOH, [ka] wherein Lg is a leaving group, e.g., Cl, Br, I, OMs, OTs, OTf, or [ka] indicates, X, Y, A, Q, Z1, W1 are as defined in any one of the first aspects above.

[0063] In some embodiments, L is the following group: C 1-6 Alkylene group, -N(R6)-, carbonyl group, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Phe, Ala, Asn, D-Val-Leu-Lys, Ala-Ala, Ala-Lys, Ala-Lys(Ac), A la-Pro, 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-L ys, 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, [ka] wherein R6 is H, C 1-6 Alkyl group or C 2-6It is an alkoxyalkyl group, and s is an integer of 1-10.

[0064] In some embodiments, L is Val, Cit, Gly, Phe, Ala, Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, [ka] The one or more are selected from the following:

[0065] In some embodiments, L is selected from the structure consisting of one or more of the following: [ka] [ka] [ka] s is an integer ~10.

[0066] In some embodiments, L is selected from the following: [ka] [ka] [ka]

[0067] In some embodiments, L is selected from the following structures: [ka] [ka]

[0068] In some implementations, L is [ka] is selected from.

[0069] In a fourth aspect, the present application provides 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 IV: [ka] During the ceremony, V2 is a group obtained by linking J2 and L in the compound of formula II according to the second aspect of the present application, and preferably V2 is selected from -C(=O)-, -N(R2)-, -O-, a 3- to 10-membered nitrogen-containing heterocyclic group and a sulfonylurea group, wherein R2 is H, C 1-6 Alkyl group or C 2-6 More preferably, V2 is -C(O)- or -N(R2)-, where R2 is H, C 1-6 Alkyl group or C 2-6 is an alkoxyalkyl group, B1, B2, Y1, Y2, Y3, Z2 and W2 are as defined in any one of the above second aspects; L and E' are as defined in any one of the third aspects above.

[0070] In a fifth aspect, the present application provides 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: [ka] During the ceremony, E is a single bond, -NH-CH2-, [ka] is selected from 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 the first aspects above; V1 and L are as defined in any one of the items in the third aspect.

[0071] In some embodiments, the biologically active molecule is selected from the following group: antitubulin agents, DNA intercalating agents, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0072] In some embodiments, the biologically active molecule is selected from the following group: tubulin inhibitors, olestatin-based compounds, medellin-based compounds; DNA intercalating agents, pyrrolidinedibenzodiazepines (PBDs); DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, veroticin, ruboticin, etc.) or topoisomerase II inhibitors (doxorubicin, PNU-159682, dokamycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); RNA polymerase inhibitors, such as α-amanitin, and pharmaceutically acceptable salts, esters, and analogs thereof.

[0073] In some embodiments, the biologically active molecule is selected from topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, verotican, rubotican, etc.), MMAE, and MMAE derivatives.

[0074] In some embodiments, the biologically active molecule is selected from MMAE and MMAE derivatives.

[0075] In some embodiments, D is: [ka] is selected from.

[0076] In some embodiments, the compound of formula V is [ka] [ka] [ka] [ka] [ka] is selected from.

[0077] In a sixth aspect, the present application provides 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: [ka]

[0078] B1, B2, Y1, Y2, Y3, Z2 and W2 are as defined in any one of the above second aspects; L is as defined in any one of the third aspects above; V2 is as defined in any one of the fourth aspects above; E and D are as defined in any one of the fifth aspect above.

[0079] In some embodiments, the compound of Formula VI is selected from: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0080] In a seventh aspect, the present application provides a bioactive conjugate, the structure of which is shown in Formula VII. [ka] wherein Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), and n is selected from integers or decimals between 1 and 10; V1 is -C(O)- or -N(R1)-, where R1 is H, C 1-6 Alkyl group or C 2-6 is an alkoxyalkyl group, L is a linker connecting V1 and E, E is a structural fragment connecting L and D, D is a fragment of a biologically active molecule (e.g., a cytotoxic drug); In the complex [ka] indicates a specific linking method between a sulfhydryl group in the antibody and another part of the conjugate when the targeting moiety is an antibody, The remaining groups are as defined in any one of the preceding aspects.

[0081] In another aspect of the present invention, a bioactive conjugate is provided, the structure of which is shown in Formula VIII. [ka] wherein Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), and n is selected from integers or decimals between 1 and 10; V2 is -C(O)- or -N(R2)-, where R2 is H, C 1-6 Alkyl group or C 2-6 is an alkoxyalkyl group, L is a linker connecting V2 and E, E is a structural fragment connecting L and D, D is a fragment of a biologically active molecule (e.g., a cytotoxic drug); In the complex [ka] indicates a specific linking method between a sulfhydryl group in the antibody and another part of the conjugate when the targeting moiety is an antibody, The remaining groups are as defined in any one of the preceding aspects.

[0082] In some embodiments, the antibody is directed against epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, mesothelin, folate receptor 1, mucin 1, CD138, CD20, CD19, CD30, SLTRK6, nectin 4, tissue factor, mucin 16, endothelin receptor, 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, and 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, FcR H1, 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-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LO X, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KIS S1R, 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. Preferably, the Ab is selected from an anti-Her2 antibody (e.g., trastuzumab), an anti-Trop2 antibody (e.g., sacituzumab), an anti-ROR1 antibody (e.g., 19F6_Hu35V1), or an anti-B7H3 antibody (e.g., 2#8890).

[0083] In some embodiments, L is the following group: C 1-6Alkylene group, -N(R6)-, carbonyl group, -O-, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Phe, Ala, Asn, D-Val-Leu-Lys, Ala-Ala, Ala-Lys, Ala-Lys(Ac), A la-Pro, 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-L ys, 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, [ka] wherein R6 is H, C 1-6 Alkyl group or C 2-6 an alkoxyalkyl group, and s is an integer of 1 to 10; Preferably, L is [ka] [ka] [ka] [ka] wherein s is an integer of 1 to 10; Suitably, L has the following structure: [ka] is selected from Suitably, L has the following structure: [ka] [ka] is selected from.

[0084] In some embodiments, E is a single bond, —NH—CH—, [ka] is.

[0085] In some embodiments, E is —NH—CH 2 —.

[0086] In some embodiments, the biologically active molecule is selected from the following group: antitubulin agents, DNA intercalating agents, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0087] In some embodiments, the biologically active molecule is selected from the following group: tubulin inhibitors, olestatin-based compounds, medellin-based compounds; DNA intercalating agents, pyrrolidinedibenzodiazepines (PBDs); DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, veroticin, ruboticin, etc.) or topoisomerase II inhibitors (doxorubicin, PNU-159682, dokamycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); RNA polymerase inhibitors, such as α-amanitin, and pharmaceutically acceptable salts, esters, and analogs thereof.

[0088] In some embodiments, the biologically active molecule is selected from topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, verotican, rubotican, etc.), MMAE, and MMAE derivatives.

[0089] In some embodiments, the biologically active molecule is selected from MMAE and MMAE derivatives.

[0090] In some embodiments, D is: [ka] is selected from.

[0091] In some embodiments, n is 1-8, more preferably n is 3-5.

[0092] In some embodiments, the structure of the biologically active conjugate is as follows, in which 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 to 8, more preferably 3 to 5. [ka] [ka] [ka] [ka]

[0093] In some embodiments, the structure of the bioactive conjugate is as follows: Ab is selected from an anti-Her2 antibody (e.g., trastuzumab), an anti-Trop2 antibody (e.g., sacituzumab), an anti-ROR1 antibody (e.g., 19F6_Hu35V1), or an anti-B7H3 antibody (e.g., 2#8890); n1 is 1 to 8, more preferably 3 to 5; and x is 1 to 10, more preferably 3 to 5. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0094] In some embodiments, the anti-Her2 antibody described in any one of the above is trastuzumab, an antibody containing a heavy chain variable region of a trastuzumab heavy chain complementarity determining region and a light chain variable region of a trastuzumab light chain complementarity determining region, or an antibody containing a trastuzumab heavy chain variable region sequence and a trastuzumab light chain variable region sequence; The anti-Trop2 antibody according to any one of the preceding claims is sacituzumab, an antibody comprising a heavy chain variable region of a sacituzumab heavy chain complementarity determining region and a light chain variable region of a 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 according to any one of the above is any one of the following: i) An antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:3, CDR-H2 represented by SEQ ID NO:4, and CDR-H3 represented by SEQ ID NO:5, and a light chain variable region of CDR-L1 represented by SEQ ID NO:6, CDR-L2 represented by SEQ ID NO:7, and CDR-L3 represented by SEQ ID NO:8, as defined by the Chothia numbering system. ii) An antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:9, CDR-H2 represented by SEQ ID NO:10, and CDR-H3 represented by SEQ ID NO:5, and a light chain variable region of CDR-L1 represented by SEQ ID NO:6, CDR-L2 represented by SEQ ID NO:7, and CDR-L3 represented by SEQ ID NO:8, as defined by the AbM numbering system. iii) An antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:11, CDR-H2 represented by SEQ ID NO:12, and CDR-H3 represented by SEQ ID NO:5, and a light chain variable region of CDR-L1 represented by SEQ ID NO:6, CDR-L2 represented by SEQ ID NO:7, and CDR-L3 represented by SEQ ID NO:8, as defined by the Kabat numbering system. iv) An antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:13, CDR-H2 represented by SEQ ID NO:14, and CDR-H3 represented by SEQ ID NO:15, and a light chain variable region of CDR-L1 represented by SEQ ID NO:16, CDR-L2 represented by SEQ ID NO:17, and CDR-L3 represented by SEQ ID NO:8, as defined by the IMGT numbering system. v) an antibody comprising a heavy chain variable region set forth in SEQ ID NO:1 and a light chain variable region set forth in SEQ ID NO:2, or vi) 19F6_Hu35V1, which contains an antibody having a heavy chain variable region represented by SEQ ID NO:1, a light chain variable region represented by SEQ ID NO:2, a heavy chain constant region represented by SEQ ID NO:18, and a light chain constant region represented by SEQ ID NO:19.

[0095] In some embodiments, n1 is 1-6, for example, 3-5.

[0096] In some embodiments, x is 1-6, for example, 3-5. DETAILED DESCRIPTION OF THE INVENTION

[0097] definition Unless otherwise defined in the following text, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. References to technical meanings used herein refer to techniques commonly understood by those skilled in the art, including modifications of the techniques or replacement of equivalent techniques that are obvious to those skilled in the art. Although it is believed that the following terms are easily understandable to those skilled in the art, the following definitions are restated below to better understand the present invention.

[0098] As used herein, the term "alkylene group" refers to a saturated divalent hydrocarbon group, preferably having 1, 2, 3, 4, 5, or 6 carbon atoms, such as a methylene group, ethylene group, propylene group, or butylidene group.

[0099] As used herein, the term "alkyl group" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, e.g., 1 to 6, carbon atoms. For example, as used herein, the term "C 1-6 The term "alkyl group" refers to a linear or branched aliphatic hydrocarbon group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, p-butyl, tert-butyl, n-amyl, isoamyl, neoperyl, or n-hexyl), which is optionally substituted with one or more (e.g., 1 to 3) suitable substituents (e.g., halogen) (wherein the group is referred to as a "haloalkyl group") (e.g., CH2F, CHF2, CF3, CCl3, C2F5, C2Cl5, CH2CF3, CH2Cl, or -CH2CH2CF3, etc.). 1-4 "Alkyl group" refers to a linear or branched aliphatic hydrocarbon group of 1 to 4 carbon atoms (i.e., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, p-butyl, or tert-butyl).

[0100] As used herein, the term "alkoxy group" is defined as an -O-alkyl group, wherein said alkyl group is defined above. For example, as used herein, the term "C 1-6 Alkoxy group is -OC 1-6 It refers to an alkyl group.

[0101] As used herein, the term "alkoxyalkyl group" is defined as an alkyl group substituted with an alkoxy group, said alkyl group being defined above. For example, as used herein, the term "C 2-6 "Alkoxyalkyl group" refers to an alkyl group substituted with an alkoxy group having from 2 to 6 carbon atoms.

[0102] As used herein, the term "alkynylene group" refers to a divalent hydrocarbon group containing at least one carbon-carbon triple bond, preferably having 1, 2, 3, 4, 5, or 6 carbon atoms, such as an ethynylene group, a propynylene group, or a butynylene group.

[0103] As used herein, the terms "heterocyclic group" and "heterocycle" refer to saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) cyclic groups in which 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 heterocyclic group" is a saturated or partially unsaturated heterocyclic group having 4 to 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 heterocyclic group" is a heterocyclic group in which at least one ring-forming atom is N. Examples of heterocyclic groups include, but are not limited to, ethylene oxide, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuran, dioxolinyl, pyrrolidine, pyrrolidone, imidazolidinyl, pyrazolidine, pyrazoline, tetrahydropyran, piperidyl, morpholine, dithianyl, thymorpholine, piperazine, or trithianyl. The heterocyclic groups may be optionally substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents and may optionally form a parallel ring structure with one or more aromatic or heteroaromatic rings.

[0104] As used herein, the term "aromatic ring" or "aryl group" 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.

[0105] As used herein, the term "heteroaromatic ring" or "heteroaryl group" refers to a monocyclic or polycyclic aromatic ring system, which has, 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 may contain at least one identical or different heteroatom (said heteroatom being, for example, oxygen, nitrogen or sulfur), and which, in each case, is capable of benzo-fusion.

[0106] As used herein, the term "halogen" includes F, Cl, Br, or I.

[0107] As used herein, the term "sulfonylurea group" refers to -SO2-NH-(C=O)-NH2 or -NH-(C=O)-NH-SO2H.

[0108] The term "substituted" refers to the selective replacement of one or more (e.g., 1, 2, 3, or 4) hydrogens on a designated atom from a designated group, provided that the substitution does not exceed the normal valence of the designated atom under the circumstances, 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.

[0109] When a substituent is described as being "optionally substituted with," the substituent is either (1) unsubstituted or (2) substituted. When a carbon of a substituent is described as being optionally substituted with one or more in a list of substituents, one or more hydrogens on the carbon (to any extent that hydrogens are present) are singly and / or collectively replaced with any independently selected substituents. When a nitrogen of a substituent is described as being optionally substituted with one or more in a list of substituents, one or more hydrogens on the nitrogen (to any extent that hydrogens are present) are each replaced with any independently selected substituent.

[0110] When substituents are described as being "independently selected from a set of groups," each substituent is selected independently of the others. Thus, each substituent can be the same or different from another (other) substituent.

[0111] As used herein, the term "one or more" refers to one or more than one, under reasonable conditions, for example, 2, 3, 4, 5 or 10.

[0112] Unless otherwise specified, as used herein, the point of attachment of a substituent may be from any suitable position on the substituent.

[0113] When a substituent bond is shown as a bond that passes through a ring to connect two atoms, then such substituent may be bonded to any ring-forming atom of the substitutable ring.

[0114] The present invention further includes all pharmaceutically acceptable isotopically labeled compounds that are similar 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 predominant in nature. Illustrative examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen (e.g., deuterium ( 2 H), tritium ( 3 H), isotopes of carbon (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 (e.g. 123 I and 125 I), nitrogen isotopes (e.g. 13 N and 15 N), isotopes of oxygen (e.g. 15 O. 17 O. 18 O), isotopes of phosphorus (e.g. 32 P), and sulfur isotopes (e.g. 35 S), including (but not limited to).

[0115] The term "stereoisomer" refers to an isomer formed by at least one asymmetric center. Compounds with one or more (e.g., one, two, three, or four) asymmetric centers can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. Certain individual molecules can exist as geometric isomers (cis / trans). Similarly, compounds of the present invention can exist as mixtures of two or more rapidly equilibrating structurally distinct forms (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. As will be understood, the scope of this application covers all isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) of the isomers.

[0116] In this specification, the solid line ( [ka] Solid wedge [ka] or imaginary wedge [ka] The carbon-carbon bonds of the compounds of the present invention can be depicted using a solid line to depict a bond connected to an asymmetric carbon atom, which expresses the inclusion of all possible stereoisomers at that carbon atom (e.g., specific enantiomers, racemic mixtures, etc.). The use of a real or imaginary wedge to depict a bond connected to an asymmetric carbon atom expresses the existence of the depicted stereoisomer. When present in a racemic mixture, the real and imaginary wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist in the form of stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformational isomers, retardation isomers, and mixtures thereof. The compounds of the present invention can exhibit more than one type of isomerism and can consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0117] The present invention covers all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of multiple polymorphs in any ratio.

[0118] It should be further understood that certain compounds of the present invention can exist in free form and be used in therapy, or, where appropriate, in pharmaceutically acceptable derivative form. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, or prodrugs, which, upon administration to a patient in need thereof, can directly or indirectly provide the compound of the present invention, or a metabolite or residue thereof. Thus, when reference is made herein to a "compound of the present invention," it is intended to encompass the various derivative forms of the compound.

[0119] Pharmaceutically acceptable salts of the compounds of the present invention include the acid addition and alkali addition salts thereof.

[0120] Suitable acid addition salts are formed with acids which form pharmaceutically acceptable salts. Examples include aspartate, benzoate, bicarbonate / carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts.

[0121] Suitable alkali addition salts are formed with alkalis which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts.

[0122] A review of suitable salts can be found in 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.

[0123] As used herein, the term "ester" refers to esters derived from various compounds of the general formula herein, including physiologically hydrolyzable esters (forms of the compounds of the invention that are hydrolyzed under physiological conditions to release the free acid or alcohol). The compounds of the invention themselves can also be esters.

[0124] The compounds of the present invention can exist in the form of solvates, preferably hydrates, which contain polar solvents as structural elements of the compound lattice, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, can be present in a stoichiometric or non-stoichiometric ratio.

[0125] As those skilled in the art will understand, not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires an available lone pair of electrons to be oxidized to an oxide, and those skilled in the art will be able to identify nitrogen-containing heterocycles that can form N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Those skilled in the art are familiar with N-oxide synthesis methods for preparing heterocycles and tertiary amines, including oxidizing heterocycles and tertiary amines with peroxy acids such as peroxyacetic acid and metachloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydrogen peroxides such as tertiary butyl hydroperoxide, sodium perborate, and dioxiranes such as dimethyldioxirane. Methods for preparing these N-oxides have already been extensively described and reviewed in the literature, see, for example, T.L.G. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750, A.R.Katritzky and A.J.Boulton, Eds., Academic Press, and G.W.H. Cheeseman and E.S.G. Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R.Katritzky and A.J.Boulton, Eds., Academic Press.

[0126] Also included within the scope of this invention are metabolites of the compounds of this invention, i.e., substances formed in the body upon administration of a compound of this invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic degradation, etc. of the administered compound. Accordingly, the invention includes metabolites of the compounds of this invention, which are compounds prepared by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0127] 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 relatively little or no pharmacological activity and that, upon administration to or into the body, can be converted, for example, by hydrolysis, into compounds of the present invention having the desired activity. Typically, such prodrugs are functional derivatives of the compounds, which are readily converted into the desired therapeutically active compound in the body. Further information regarding the use of prodrugs is provided in "Prodrugs as Novel Delivery Systems," Vol. 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" (see, for example, "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0128] The present invention further covers compounds of the present invention that contain protecting groups. During any process for preparing compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any relevant molecule, thereby forming a form of chemical protection for the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in T.W. Greene & P. ​​G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. The protecting groups can be removed at a suitable subsequent stage using methods known in the art.

[0129] Preparation method In another aspect of the present invention, there is provided a process for preparing a compound of formula I, said process comprising the steps of:

[0130] When Y is absent, the compounds of formula I-TM1 and formula I-TM2 of the present invention can be synthesized and prepared by the following synthetic route. [ka] During the ceremony, X, Z1, W1, and J1 are as defined in the general formula above; M is a leaving group that causes a substitution reaction, and includes, but is not limited to, halogen, trifluoromethanesulfonate, and p-toluenesulfonate, and is preferably halogen.

[0131] Step 1 The compound of formula I-SM1 is subjected to a substitution reaction with the compound M-Z1-W1-J1 to obtain the compound of formula I-IM1.

[0132] In some embodiments, the reaction is carried out under alkaline conditions.

[0133] In some embodiments, the reaction is carried out at a suitable temperature, which may be 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 20°C. 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, and dimethyl sulfoxide, preferably acetone.

[0134] Step 2 The compound of formula I-IM1 is subjected to a substitution reaction with sodium adoxide to give the compound of formula I-IM2.

[0135] In some embodiments, the reaction is carried out at a suitable temperature, which may be 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 20°C.

[0136] 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, and dimethyl sulfoxide, preferably acetone.

[0137] Step 3 The compound of formula I-IM3 is obtained by reduction of the compound of formula I-IM2.

[0138] In some embodiments, this step is carried out in the presence of a suitable reducing agent, which may be selected from a palladium catalyst, a platinum catalyst, a rhodium catalyst, and preferably a palladium catalyst.

[0139] In some embodiments, this step is carried out at a suitable temperature, which may be 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C.

[0140] 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, and ethyl acetate, preferably ethyl acetate.

[0141] Step 4 Compounds of formula I-IM3 and [ka] Dehydration ring closure with the compound of formula I-TM1 is then carried out.

[0142] In some embodiments, this step is carried out at a suitable temperature, which may be 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C.

[0143] 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, and preferably acetonitrile.

[0144] Step 5 The compound of formula I-TM1 is hydrolyzed to give the compound of formula I-TM2.

[0145] In some embodiments, the reaction is carried out under alkaline conditions, including but not limited to PB buffer at a pH of 7.0, 7.4, or 8.0.

[0146] In some embodiments, the reaction is carried out at a suitable temperature, which may be 20°C, 25°C, 50°C, 60°C, or 100°C, preferably 20°C.

[0147] 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.

[0148] When Y is absent, the compound of formula I-TM3 in the present invention can be synthesized and prepared by the following synthetic route. [ka] During the ceremony, X, Z1, W1, and J1 are as defined in the general formula above; L is a leaving group that causes a substitution reaction, and includes, but is not limited to, halogen, trifluoromethanesulfonate, and p-toluenesulfonate, and is preferably halogen or OTf.

[0149] Step 1 The compound of formula I-IM4 is obtained from the compound of formula I-IM1 by a conjugation reaction.

[0150] In some embodiments, the conjugate reaction reagent includes, but is not limited to, methyl boric acid and trimethylcyclotriboroxane, preferably trimethylcyclotriboroxane.

[0151] In some embodiments, the conjugation reaction is carried out under alkaline conditions, and the alkali 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, and potassium dihydrogen phosphate, preferably cesium fluoride.

[0152] In some embodiments, the conjugation reaction is carried out in the presence of a catalyst, including, but not limited to, tetratriphenylphosphine palladium, palladium acetate, Pd(dba), Pd(PPh)Cl, Pd(PPh)Cl dichloromethane complex, Pd(dppf)Cl, and Pd(Amphos)Cl, preferably tetratriphenylphosphine palladium.

[0153] In some embodiments, the conjugation reaction is carried out at a temperature of 0 to 200°C, preferably at a temperature of 50 to 150°C.

[0154] 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.

[0155] Step 2 The compound of formula I-IM4 is subjected to a halogenation reaction to give the compound of formula I-TM3.

[0156] In some embodiments, the halogenating reagent includes, but is not limited to, bromine, N-iodobutanediimide, N-bromobutanediimide, and N-chlorobutanediimide, preferably N-bromobutanediimide.

[0157] In some embodiments, the halogenation reaction is carried out in the presence of a catalyst, and the catalyst is benzoyl peroxide.

[0158] In some embodiments, the halogenation reaction is carried out at a temperature of 0 to 200°C, preferably at a temperature of 50 to 150°C.

[0159] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (e.g., carbon tetrachloride, dichloromethane, chloroform, 1,2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (e.g., ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (e.g., toluene, benzene, xylene), water, and any combination thereof, preferably carbon tetrachloride.

[0160] When Z2 and W2 are not present, the compound of formula II-TM1 of the present invention can be synthesized and prepared by the following synthetic route. [ka] During the ceremony, Y1, Y2, Y3, B1, B2, Z2, W2 and J2 are as defined in the general formula above; LG is a leaving group that generates the conjugation reaction, and includes, but is not limited to, halogen, trifluoromethanesulfonate, and is preferably halogen.

[0161] Step 1 The compound of formula II-IM2 is obtained from the compound of formula II-IM1 by a conjugation reaction.

[0162] In some embodiments, the conjugation reaction reagent is [ka] is.

[0163] In some embodiments, the conjugation reaction is carried out in the presence of a catalyst, including, but not limited to, tetratriphenylphosphine palladium, palladium acetate, Pd(dba), Pd(PPh)Cl, Pd(PPh)Cl dichloromethane complex, Pd(dppf)Cl, and Pd(Amphos)Cl, preferably tetratriphenylphosphine palladium.

[0164] In some embodiments, the conjugation reaction is carried out at a temperature of 0 to 200°C, preferably at a temperature of 50 to 150°C.

[0165] 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.

[0166] Step 2 The compound of formula II-IM2 is subjected to an oxidation reaction to obtain the compound of formula II-TM1.

[0167] In some embodiments, the oxidation reaction is carried out in the presence of an oxidizing agent, and the catalyst is metachloroperbenzoic acid.

[0168] In some embodiments, the oxidation reaction is carried out at a temperature of 0 to 120°C, preferably at a temperature of 50 to 80°C.

[0169] In some embodiments, the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (e.g., carbon tetrachloride, dichloromethane, chloroform, 1,2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (e.g., ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (e.g., toluene, benzene, xylene), water, and any combination thereof, preferably methanol.

[0170] In addition, the compounds of the present invention can be prepared by several methods known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods depicted below and synthetic methods known in the art of synthetic organic chemistry, or variations thereof recognized by those skilled in the art. Suitable methods include, but are not limited to, those described in the text above. The reactions can be carried out in a solvent or solvent mixture appropriate to the reagents and materials used and compatible with the transformations achieved. As understood by those skilled in the art of organic synthesis, the functional groups present on the molecule shall be consistent with the proposed transformations. In some cases, it may be necessary to modify the order of synthetic steps or to determine whether one particular process route over another will yield the desired compounds of the present invention.

[0171] It should also be recognized that another major consideration for designing any synthetic route in this field is the correct selection of protecting groups to protect reactive functional groups present in the compounds described in this invention. An authoritative account that describes many alternatives for the trained practitioner is Greene et al. (Protective Groups in Organic Synthesis, 4th ed., Wiley-Interscience (2006)).

[0172] Unless otherwise specified, the substituents of the compounds in the above pathways are as defined in the present invention.It is obvious to those skilled in the art that one or more steps in the above pathways can be omitted depending on the structure of the product that is desired to be obtained.Those skilled in the art can also adjust the order of reaction steps as needed.

[0173] In some embodiments, the present invention further provides the use of the compounds in the preparation of drug-linker compounds.

[0174] Preferably, the Drug Linker Compound is a compound described in the preceding sentence or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof; The compounds described in the above paragraphs are prepared by the following steps to prepare the drug linker compounds. [ka]

[0175] In the formula, X, Y, A, Q, Z1, J1, W1, V1, L, E, D are as defined in the above sentence, and LG1 is selected from groups that undergo a condensation reaction with J1, preferably LG1 is selected from -COOH or -NH(R1), where R1 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0176] In some embodiments, the present invention further provides the use of the compounds in the preparation of drug-linker compounds.

[0177] Preferably, the Drug Linker is selected from a compound described in the preceding sentence or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof; Preferably, the compounds described in the above sentence are prepared by the following steps to prepare the drug linker compound: [ka] In the formula, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D are as defined in the above sentence, and LG2 is selected from groups that undergo a condensation reaction with J2, preferably LG2 is selected from -COOH or -NH(R2), where R2 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0178] In some embodiments, the present invention further provides the use of the compounds in the preparation of bioactive conjugates.

[0179] Preferably, the bioactive conjugate is selected from the bioactive conjugates described in the preceding sentences, Preferably, the compounds described in the above paragraphs are prepared into the bioactive conjugates by the following steps 1a and 1b: Step 1a: [ka] Step 1b: [ka] In the formula, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined in the preceding sentence, and LG1 is selected from groups that undergo a condensation reaction with J1, preferably LG1 is selected from -COOH or -NH(R1), where R1 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0180] In some embodiments, the present invention further provides the use of the compounds in the preparation of bioactive conjugates.

[0181] The bioactive conjugate is selected from the bioactive conjugates described in the preceding sentences, Preferably, the compounds described in the above paragraphs are prepared into the bioactive conjugates by the following steps 2a and 2b: Step 2a: [ka] Step 2b: [ka] In the formula, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are as defined in the preceding sentence, and LG2 is selected from groups that undergo a condensation reaction with J2, preferably LG2 is selected from -COOH or -NH(R2), where R2 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0182] In some embodiments, the present invention further provides a method for preparing a compound, the method comprising the steps of: [ka] In the formula, X, Y, A, Q, Z1, J1, W1, V1, L, E, D are as defined in the above sentence, and LG1 is selected from groups that undergo a condensation reaction with J1, preferably LG1 is selected from -COOH or -NH(R1), where R1 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0183] In some embodiments, the present invention further provides a method for preparing a compound, the method comprising the steps of: [ka] In the formula, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D are as defined in the above sentence, and LG2 is selected from groups that undergo a condensation reaction with J2, preferably LG2 is selected from -COOH or -NH(R2), where R2 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0184] In some embodiments, the present invention further provides a method for preparing an antibody-drug conjugate, the method comprising the steps of: Step 1a: [ka] Step 1b: [ka] In the formula, X, Y, A, Q, Z1, J1, W1, V1, L, E, D, Ab and n are as defined in the preceding sentence, and LG1 is selected from groups that undergo a condensation reaction with J1, preferably LG1 is selected from -COOH or -NH(R1), where R1 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0185] In some embodiments, the present invention further provides a method for preparing an antibody-drug conjugate, the method comprising the steps of: Step 2a: [ka] Step 2b: [ka] In the formula, B1, B2, Y1, Y2, Y3, Z2, J2, W2, V2, L, E, D, Ab and n1 are as defined in the preceding sentence, and LG2 is selected from groups that undergo a condensation reaction with J2, preferably LG2 is selected from -COOH or -NH(R2), where R2 is H, C 1-6 Alkyl group or C 2-6 It is an alkoxyalkyl group.

[0186] Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising a bioactive conjugate according to the present invention and one or more pharmaceutically acceptable carriers.

[0187] In some embodiments, the drug-to-antibody ratio (DAR value) of the pharmaceutical composition is 1.0-6.0, for example, 1, 2, 3, 4, 5, or 6, or for example, 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.

[0188] The medicinal auxiliary materials referred to in this specification refer to excipients and additives used in producing and formulating drugs, and refer to substances other than active ingredients that have been reasonably evaluated in terms of safety and are included in drug formulations.

[0189] The pharmaceutical composition can be administered in any form, as long as it achieves the prevention, alleviation, prevention or cure of symptoms in a human or animal patient. For example, various suitable dosage forms can be prepared depending on the route of administration.

[0190] The present application further provides a medicine case product containing a bioactive conjugate according to the present invention or said pharmaceutical composition, and an optional medicine leaflet.

[0191] Treatments and Uses In another aspect of the present application, there is provided the use of said bioactive conjugate in the preparation of a medicament for preventing or treating a tumor disease.

[0192] In another aspect of the present application, there is provided the bioactive conjugate, which is used in the prevention or treatment of a tumor disease.

[0193] In another aspect of the present application, there is provided a method for preventing or treating a tumor disease, comprising administering to a subject in need thereof an effective amount of said chemical-bioactive conjugate, or a pharmaceutical composition comprising said bioactive conjugate.

[0194] In one embodiment of the present invention, the tumor disease is a solid cancer or a hematological malignancy, for example, selected from colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), and lymphatic cancer.

[0195] As used herein, the term "effective amount" refers to the amount of conjugate that, after administration, relieves to some extent one or more of the symptoms being treated.

[0196] Unless otherwise specified, as used herein, the term "treatment" refers to reversing or alleviating the progression of the applicable condition or pathology, or one or more symptoms of such condition or pathology.

[0197] As used herein, an "individual" or "subject" includes a human or non-human animal. Illustratively, a human individual includes a human individual (referred to as a patient) suffering from a disease (e.g., a disease described herein) or a normal individual. In the present invention, a "non-human animal" includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, and livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0198] Specific Embodiments The present invention will be explained in more detail below by listing examples and test examples, but these do not limit the scope of the present invention and can be modified within the scope of the present invention.

[0199] 1. Antibody Preparation 1.1 Preparation of the anti-ROR1 antibody 19F6_Hu35V1 of the present invention Initially, Balb / c, C57Bl / 6, NZB, and A / J mice were immunized, and the mouse-derived antibody 19F6 was isolated through hybridoma screening. After humanization, the humanized antibody sequence 19F6_Hu35V1 (heavy chain variable region, SEQ ID NO: 1; light chain variable region, SEQ ID NO: 2; heavy chain constant region is human IgG1 heavy chain constant region (SEQ ID NO: 18), and light chain constant region is human kappa light chain constant region (SEQ ID NO: 19)) was obtained. The coding DNA sequence for this 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, and the expressed antibody in the supernatant was purified using protein A to obtain the corresponding antibody. [Table 1] [Table 2]

[0200] 1.2 Preparation of B7-H3 antibody 2#8890 according to the present invention Fully humanized mice were immunized with human B7-H3-4Ig-His protein, and serum titers were monitored by ELISA and flow cytometry. The optimal mice were selected based on the titer results. Spleen cells were fused, screened, and subcloned. The activity of different monoclonal antibodies binding human / monkey proteins and cells was tested and measured. The optimal clone, 20G11G6, was obtained. The antibody sequence was modified to remove PTM sites, reduce PI, and remove ADCC in the heavy chain constant region, resulting in the fully humanized antibody 2#8890. The antibody was codon-optimized and gene-synthesized by Nanjing Jinsirui Biotechnology Co., Ltd., and constructed into pTT5 plasmid. The heavy and light chain plasmids were co-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. [Table 3]

[0201] 2. Drug-linker Synthesis The abbreviations used herein have the following meanings: [Table 4]

[0202] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 The identity was confirmed by 1 H NMR or mass spectrometry (MS).

[0203] Nuclear magnetic resonance ( 1 The H NMR measurement equipment used was a Bruker 400 MHz nuclear magnetic resonance spectrometer, deuterated chloroform (CDCl3), and the internal standard substance was tetramethylsilane (TMS).

[0204] The abbreviations used in the examples for nuclear magnetic resonance (NMR) spectra are as follows:

[0205] 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.

[0206] The mass spectrometry (MS) measurement equipment used was an Agilent (ESI) mass spectrometer, model number Agilent 6120B.

[0207] 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) [ka]

[0208] Step 1: Synthesis of tert-butyl 2-(3,4-diazido-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-2) Compound 1-1 (0.40 g, 1.08 mmol; see Patent WO 2019057964 for synthesis method) 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 using high-performance liquid chromatography / mass spectrometry. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined, 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 reaction without purification.

[0209] The structural property data are as follows: ESI-MS(m / z):311.0[M+18] + .

[0210] Step 2: Synthesis of tert-butyl 2-(3,4-diamino-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-3) Compound 1-2 (0.15 g, 0.51 mmol) and 10% palladium carbon (15.00 mg) were dissolved in ethanol (20 mL), purged with hydrogen gas three times, and reacted under hydrogen gas at 25 °C for 3 h. The reaction was monitored using high-performance liquid chromatography / mass spectrometry. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give 101.00 mg of the crude title compound, which was used directly in the next reaction without purification.

[0211] The structural property data are as follows: ESI-MS(m / z):185.9[M-56] + .

[0212] 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) 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 using high-performance liquid chromatography-mass spectrometry. The reaction mixture was directly purified using a flash silica gel column (petroleum ether:ethyl acetate = 5:1) to obtain 40.00 mg of the title compound.

[0213] The structural property data are as follows: ESI-MS(m / z):466.8[M+18] + . 1 H NMR (400MHz, CDCl3): δ 4.88(s,4H),4.47(s,2H),1.48(s,9H).

[0214] 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) Compound 1-5 (20.00 mg, 0.05 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1 mL) and reacted at 25 °C for 5 h. The reaction was monitored using high-performance liquid chromatography-mass spectrometry. The reaction mixture was directly concentrated under reduced pressure to give 15.00 mg of the crude title compound, which was used directly in the next reaction without further purification.

[0215] The structural property data are as follows: ESI-MS(m / z):410.8[M+18] + .

[0216] Example 2 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (I-2) [ka]

[0217] Step 1: Synthesis of tert-butyl 2-(3,4-dimethyl-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-1) Under nitrogen gas protection, compound 1-1 (0.20 g, 0.54 mmol), trimethylcyclotriboroxane (0.34 g, 2.71 mmol), cesium fluoride (0.41 g, 2.71 mmol), and tetratriphenylphosphine 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 high-performance liquid chromatography (HPLC-MS). The reaction mixture was directly purified using a flash silica gel column (petroleum ether:ethyl acetate = 5:1) to give 80.00 mg of the title compound.

[0218] The structural property data are as follows: ESI-MS(m / z): 184.0 [M+H-56] + .

[0219] Step 2: Synthesis of tert-butyl 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-2) Compound 2-1 (72.00 mg, 0.30 mmol), NBS (118.00 mg, 0.66 mmol), and benzoyl peroxide (7.00 mg, 0.03 mmol) were dissolved in carbon tetrachloride (8 mL) under nitrogen gas protection and reacted at 85 °C for 12 h. The reaction was monitored by high-performance liquid chromatography (HPLC-MS). The reaction mixture was directly purified using a flash silica gel column (petroleum ether:ethyl acetate = 10:1) to give 65.00 mg of the title compound.

[0220] The structural property data are as follows: ESI-MS(m / z):414.8[M+18] + . 1 H-NMR (400MHz, CDCl3): δ 4.28(s,4H),4.20(s,2H),1.45(s,9H).

[0221] Step 3: Synthesis of 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (I-2) 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 using high-performance liquid chromatography-mass spectrometry. The reaction mixture was directly concentrated under reduced pressure to give 50.00 mg of the crude title compound, which was used directly in the next reaction without further purification.

[0222] The structural property data are as follows: ESI-MS(m / z):358.9[M+18] + .

[0223] Example 3 4-((S)-2-((S)-2-(2-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3-methylbutamido)-5-ureapranil)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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 3) [ka]

[0224] Step 1: Synthesis of 4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutylamino)-5-ureapranyl)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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3-2) 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-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino) Butylamide (30.00 mg, 0.04 mmol) and compound 3-1 (38.50 mg, 0.05 mmol) were dissolved in DMF (3 mL), HOBt (8.46 mg, 0.06 mmol) and DIPEA (10.80 mg, 0.08 mmol) were added, and the mixture was maintained at 25 °C for 2 h. The reaction was monitored by high-performance liquid chromatography-mass spectrometry. The reaction mixture was used directly in the next reaction without further treatment.

[0225] The structural property data are as follows: ESI-MS(m / z):1345.2[M+H] + .

[0226] Step 2: Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureapranil)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-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate (3-3) Diethylamine (0.30 mL) was added to the reaction mixture containing compound 3-2 at 25°C, and the mixture was allowed to react for 2 hours while maintaining the temperature at 25°C. The reaction was monitored using high-performance liquid chromatography mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was freeze-dried to obtain 36.30 mg of the formate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 5]

[0227] The structural property data are as follows: ESI-MS(m / z): 1123.2 [M + H] + .

[0228] 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-ureylpentamido)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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3) Compound I-2 (12.75 mg, 0.04 mmol) was dissolved in dichloromethane (3 mL), DIC (2.36 mg, 0.02 mmol) and compound 3-3 (21.00 mg, 0.02 mmol) were added, and the reaction was carried out at 25 °C for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The preparation was lyophilized to give 14.30 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 6]

[0229] The structural property data are as follows: ESI-MS(m / z):1445.9[M+H] + .

[0230] 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-hexaazadocosamide)-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-oxoheptan-4-yl)-N,3-dimethylbutyramide (drug-linker 4) [ka]

[0231] Step 1: Synthesis of (9H-fluoren-9-yl)methyl ((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-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)carbamate (4-2) 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-oxoheptan-4-yl)-N,3-dimethyl-2-(( S)-3-Methyl-2-(methylamino)butyrylamino)butyramide (55.60 mg, 0.08 mmol) was weighed and dissolved in DMF (1 mL). HATU (32.37 mg, 85.18 μmol) and DIPEA (20.02 mg, 154.88 μmol) were then added. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was directly purified using preparative high-performance liquid chromatography (HPLC) to yield 35.00 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 7]

[0232] The structural property data are as follows: ESI-MS(m / z):1345.1[M+H] + .

[0233] 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-pentaazadodecanoamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutyramide (4-3) Compound 4-2 (20.00 mg, 0.02 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of diethylamine (1 mL). After the addition was complete, the mixture was allowed to react at room temperature for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was lyophilized to obtain 15.00 mg of the formate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 8]

[0234] The structural property data are as follows: ESI-MS(m / z): 1123.1 [M + H] + .

[0235] 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-hexaazadocosanamide)-N Synthesis of -((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)-N,3-dimethylbutyramide (4) Compound I-2 (4.00 mg, 12.0 μmol) was dissolved in dichloromethane (3 mL), DIC (1.12 mg, 8.00 μmol) and compound 4-3 (6.58 mg, 6.00 μmol) were added, and the mixture was reacted at 25° C. for 1 hour. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was lyophilized to give 4.77 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 9]

[0236] The structural property data are as follows: ESI-MS(m / z):1445.9[M+H] + .

[0237] Example 5 N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-butyl)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-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)acetylamino)-3,6,9,12-tetraoxypentadecane pentadecanoamide (drug-linker 5) [ka]

[0238] Step 1: (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-butyl)-3-(2-((S)-2-(((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pi Synthesis of (roridin-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-octaazatritetracontan-43-yl)carbamate (5-2) 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. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was directly purified using preparative high-performance liquid chromatography (HPLC) to give 9.00 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 10]

[0239] The structural property data are as follows: ESI-MS(m / z): 1592.8 [M + H] + .

[0240] Step 2: Synthesis of 1-amino-N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-butyl)-3-(2-((S)-2-((1R,2R)-3-((((S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-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) Compound 5-2 (10.00 mg, 6.00 μmol) was dissolved in dichloromethane (2 mL), followed by the addition of diethylamine (1 mL). After the addition was complete, the mixture was allowed to react at room temperature for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was lyophilized to obtain 7.00 mg of the formate salt of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 11]

[0241] The structural property data are as follows: ESI-MS(m / z):1370.9[M+H] + .

[0242] Step 3: N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-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-oxyethyl)-7,10-diisopropyl-5,11- Synthesis of 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)acetylamino)-3,6,9,12-tetraoxypentadecane pentadecanoamide (5) Compound I-2 (4.00 mg, 12.00 μmol) was dissolved in dichloromethane (2 mL), DIC (1.12 mg, 8.00 μmol) and compound 5-3 (8.04 mg, 6.00 μmol) were added, and the mixture was reacted at 25° C. for 1 hour. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was lyophilized to give 4.68 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 12]

[0243] The structural property data are as follows: ESI-MS(m / z):1693.7[M+H] + .

[0244] 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-hexaazadocosamide)- N-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino))-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptyl-4-yl)-N,3-dimethylbutyramide (Drug-Linker 6) [ka]

[0245] Compound I-1 (6.03 mg, 15.00 μmol) was dissolved in dichloromethane (2 mL), DIC (1.45 mg, 12.00 μmol) and compound 4-3 (8.62 mg, 8.00 μmol) were added, and the mixture was reacted at 25° C. for 1 hour. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting mixture was lyophilized to give 4.21 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 13]

[0246] The structural property data are as follows: ESI-MS(m / z):1498.5[M+H] + .

[0247] Example 7 N-((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-p-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)acetylamino)-3,6,9,12-tetraoxapentadecan-15-amide (Drug-Linker 7) [ka]

[0248] Compound I-1 (12.06 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL), DIC (3.00 mg, 0.02 mmol) and compound 5-3 (20.00 mg, 15.00 mmol) were added, and the reaction was carried out at 25 °C for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The preparation was lyophilized to obtain 12.00 mg of the title compound. Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 14]

[0249] The structural property data are as follows: ESI-MS(m / z):1745.8[M+H] + .

[0250] 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-nonaoxo-3,6,12,39-tetraazatetraundecanoamido)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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 8) [ka]

[0251] Step 1: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-azadotriacontanamido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutamide (8-3) 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, followed by reaction for 4 hours at 25° C. The reaction mixture 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).

[0252] The structural property data are as follows: ESI-MS(m / z):830.4[M+H] + .

[0253] 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-oxy-3,6,12-triazetrioctadecylamido)benzyl(4-nitrophenyl)carbonate (8-4) Compound 8-3 (2.50 g, 3.01 mmol) was dissolved in dichloromethane (20 mL), and di(p-nitrophenyl)carbonate (3.66 g, 12.05 mmol) and DIPEA (1.56 g, 12.05 mmol) were added, followed by reaction for 4 hours at 25° C. The reaction mixture was directly purified using a silica gel column (ethyl acetate-dichloromethane:methanol=84:16) to obtain the title compound 8-4 (2.07 g).

[0254] The structural property data are as follows: ESI-MS(m / z):995.4[M+H] + .

[0255] Step 3: 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-oxy-3,6,12-triazatrioctadecylamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3)-(((1S,2R Synthesis of )-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (8-5) (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-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyramide (100 mg, 139 28 μmol), compound 8-4 (180.17 mg, 181.06 μmol), and HOBt (56.46 mg, 417.84 μmol) were dissolved in DMF (3 mL), DIPEA (54.00 mg, 417.84 μmol) was added, and the reaction was carried out 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).

[0256] The structural property data are as follows: ESI-MS(m / z):1573.9[M+H] + .

[0257] Step 4: 4-((2S,5S)-38-amino-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxy-3,6,12-triazatrioctadecylamide)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3)-(((1S,2R Synthesis of )-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (8-6) Compound 8-5 (0.60 g, 381.22 μmol) was dissolved in THF (10 mL), triphenylphosphorus (232.02 mg, 762.44 μmol) was added, and water (10 mL) was added. The mixture was then 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. The mixture was purified using a silica gel column (dichloromethane:methanol = 84:16) to give the title compound 8-6 (50.00 mg).

[0258] The structural property data are as follows: ESI-MS(m / z):1548.9[M+H] + .

[0259] Step 5: 4-((2S,5S)-41-(3,4-di(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-oxy-3,6,12,39-tetraazatetraundecanoamido)phenyl((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-oxopropane)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (8) Compound I-2 (10.00 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL), DIC (3.70 mg, 0.03 mmol) and compound 8-6 (23.37 mg, 15.00 μmol) were added, and the reaction was carried out at 25 °C for 1 h. The reaction was monitored using high-performance liquid chromatography (HPLC) mass spectrometry. The reaction mixture was concentrated under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The preparation was lyophilized to give the title compound 8 (12.88 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 15]

[0260] The structural property data are as follows: ESI-MS(m / z):1871.8[M+H] + .

[0261] Example 9 2-(2-(2-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrole[3,4-b]pyrazin-6-yl)ethoxy)ethoxy)acetic acid (I-3) [ka]

[0262] 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) 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, and 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 added and the mixture was 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 purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give the title compound 9-2 (0.90 g).

[0263] The structural property data are as follows: ESI-MS(m / z):519.0[M+18]+ .

[0264] Step 2: Synthesis of tert-butyl 2-(2-(2-(2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetate (9-3) Compound 9-2 (0.20 g, 399.07 μmol) was dissolved in acetone (8 mL), sodium azide (51.89 mg, 798.14 μmol) was added, and the mixture was reacted for 8 hours at 25° C. 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 give the crude product of the title compound 9-3 (160.00 mg).

[0265] The structural property data are as follows: ESI-MS(m / z):443.1[M+18] + .

[0266] 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) Compound 9-3 (170.00 mg, 399.63 μmol) and 10% palladium carbon (85.00 mg) were dissolved in ethanol (34 mL) and reacted for 8 hours in a hydrogen gas atmosphere at 25° C. The reaction mixture was filtered, and the filtrate was extracted under reduced pressure to obtain the crude product of the title compound 9-4 (140.00 mg).

[0267] The structural property data are as follows: ESI-MS(m / z):391.3[M+18] + .

[0268] Step 4: Synthesis of 2-(2-(2-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrole[3,4-b]pyrazin-6-yl)ethoxy)ethoxy)acetic acid (I-3) 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 for 2 hours at 25 °C. The reaction solvent was extracted 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).

[0269] The structural property data are as follows: ESI-MS(m / z):391.3[M+18] + .

[0270] Example 10 4-((2S,5S)-17-(2,3-di(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrole[3,4-b]pyrazin-6-yl)-5-isopropyl-4,7-dioxo-2-(3-ureylpropyl)-9,12,15-trioxa-3,6-diazacetadecylamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2 R)-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 10) [ka]

[0271] Compound I-3 (13.47 mg, 25.65 μmol) was dissolved in dichloromethane (4 mL), DIC (2.43 mg, 19.24 μmol, 2.98 μL) was added, and the mixture was stirred for 20 min. Compound 10-1 formate (15.00 mg, 12.83 μmol) was added, and the mixture was allowed to react for 2 h at 20 ° C. The solvent was extracted under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The preparation was lyophilized to give the title compound 10 (9.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 16]

[0272] The structural property data are as follows: ESI-MS(m / z):1630.4[M+H] + .

[0273] Example 11 3,5-Di(2-(mesyl)pyrimidin-4-yl)benzoic acid (II-19) [ka]

[0274] Step 1: Synthesis of methyl 3,5-di(2-(methylsulfido)pyrimidin-4-yl)benzoate (11-3) Methyl 3,5-dibromobenzoate (1.00 g, 3.40 mmol), 4-tributylstannyl-2-thiomethylpyrimidine (3.11 g, 7.48 mmol), and tetra(triphenylphosphine)palladium (393.00 mg, 0.034 mmol) were dissolved in 1,4-dioxane (10 mL). The atmosphere was purged with nitrogen gas, and the mixture was microwaved at 110° C. for 6 hours. The reaction mixture was concentrated under reduced pressure, and the concentrate was purified on a silica gel column (petroleum ether:ethyl acetate=5:1) to give compound 11-3 (398.00 mg).

[0275] The structural property data are as follows: ESI-MS(m / z):385.0[M+H] + .

[0276] Step 2: Synthesis of 3,5-di(2-(methylsulfido)pyrimidin-4-yl)benzoic acid (11-4) Compound 11-3 (398.00 mg, 1.04 mmol) was dissolved in methanol (5 mL), tetrahydrofuran (5 mL), and water (1 mL), and sodium hydroxide (166.00 mg, 4.14 mmol) was added and stirred for 1 hour. The mixture was then added dropwise to a 3N aqueous hydrochloric acid solution to neutralize the mixture. After concentrating under reduced pressure, water was added and the mixture was stirred. The mixture was filtered, washed with water, and dried under vacuum to give the title compound 11-4 (0.38 g).

[0277] The structural property data are as follows: ESI-MS(m / z):371.1[M+18] + .

[0278] Step 3: Synthesis of 3,5-di(2-(mesyl)pyrimidin-4-yl)benzoic acid (II-19) Compound 11-4 (0.38 g, 1.03 mmol) was dissolved in methanol (20 mL), stirred, and metachloroperbenzoic acid (1.25 g, 80%, 6.15 mmol) was added. The mixture was heated to 60 °C and reacted for 4 hours. Nitrogen gas was sprayed onto the solvent to dry it, and the solid was dissolved in dichloromethane and then directly purified on a silica gel column (dichloromethane:methanol = 10:1) to obtain the title compound II-19 (0.25 g).

[0279] The structural property data are as follows: ESI-MS(m / z):452.0[M+18] + .

[0280] Example 12 4-((31S,34S)-1-(3,5-bis(2-(mesyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32-trioxo-34-(3-ureylpropyl))-5,8,11,14,17,20,23,26-octaoxo-2,30,33-triazapentacontan-35-amide)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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (Drug-Linker 12) [ka]

[0281] Step 1: 4-((34S,37S)-34-isopropyl-2,2-dimethyl-4,32,35-trioxo-37-(3-ureylpropyl)-3,8,11,14,17,20,23,26,29-oxy-5,33,36-triazaoctatriacontan-38-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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (12-2) 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. After the addition was complete, the mixture was allowed to react at room temperature for 0.5 hours. The solvent was extracted under reduced pressure, and the concentrate was directly purified using preparative high-performance liquid chromatography. The resulting solution was lyophilized to give the title compound 12-2 (210.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 17]

[0282] The structural property data are as follows: ESI-MS (m / z): 824.0 [(M + H) / 2] + .

[0283] Step 2: 4-((29S,32S)-1-amino-29-isopropyl-27,30-dioxo-32-(3-ureylpropyl)-3,6,9,12,15,18,21,24-octaoxy-28,31-diazatritriacontan-33-amido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1 Synthesis of S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamine (12-3) Compound 12-2 (140.00 mg, 85.00 μmol) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (5 mL), and the mixture was reacted for 2 hours at 0° C. The solvent was extracted under reduced pressure to obtain 73.00 mg of a crude formate of the title compound 12-3.

[0284] The structural property data are as follows: ESI-MS(m / z):774.1[(M+H) / 2] + .

[0285] Step 3: 4-((31S,34S)-1-(3,5-bis(2-(mesyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32-trioxo-34-(3-ureylpropyl))-5,8,11,14,17,20,23,26-octaoxy-2,30,33-triazapentatriacontan-35-amido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S Synthesis of )-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (12) 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. After the addition was complete, the mixture was allowed to react at room temperature for 2 hours. The solvent was extracted under reduced pressure. The concentrate was directly purified using preparative high-performance liquid chromatography, and the resulting solution was lyophilized to give the title compound 12 (6.50 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 18]

[0286] The structural property data are as follows: ESI-MS(m / z):982.5[(M+H) / 2] + .

[0287] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1-(2-(2,3-di(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3,6,9,12-tetraoxapentadecanamide (Drug-Linker 13) [ka]

[0288] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)carbamate (13-3) Compound 13-1 (1.00 g, 1.55 mmol) was dissolved in DMF (5 mL), and HATU (647.40 mg, 1.70 mmol), mesylate of compound esetican 13-2 (1.00 g, 1.55 mmol), and DIPEA (400.34 mg, 3.10 mmol) were added sequentially, followed by reaction at 25 ° C. for 2 hours. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to obtain the title compound 13-3 (1.05 g). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 19]

[0289] The structural property data are as follows: ESI-MS(m / z): 1063.4 [M + H] + .

[0290] Step 2: Synthesis of (S)-2-(2-aminoacetamido)acetamido)-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]indoxazino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-2-oxo-3-phenylpropamide (13-4) Compound 13-3 (1.05 g, 987.69 μmol) was dissolved in dichloromethane (100 mL), and diethylamine (20 mL) was added and reacted at 25° C. for 1 hour. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to obtain the formate salt of the title compound 13-4 (285.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 20]

[0291] The structural property data are as follows: ESI-MS(m / z):841.2[M+H] + .

[0292] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,9,15,18-hexaoxo-3,21,24,27,30-pentaoxo-5,8,11,14,17-pentaazatricarboxylic acid ester (13-6) 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. After the addition was complete, the mixture was allowed to react at room temperature for 0.5 h. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the title compound 13-6 (30.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 21]

[0293] The structural property data are as follows: ESI-MS(m / z):1310.5[M+H] + .

[0294] 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]indoxazino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3,6,9,12-tetraoxa-15-decaneamide (13-7) 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 reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the formate salt of the title compound 13-7 (16.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 22]

[0295] The structural property data are as follows: ESI-MS(m / z): 1088.4 [M + H] + .

[0296] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1-(2-(2,3-di(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3,6,9,12-tetraoxapentadecanamide (13) 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 extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the title compound 13 (5.33 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 23]

[0297] The structural property data are as follows: ESI-MS(m / z):1463.3[M+H] + .

[0298] Example 14 (S)-2-(17-(2,3-di(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)-4,7-dioxo-9,12,15-trioxa-3,6-diazocetylamide)-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]indoxazino[1,2-b]quinolin-1-yl)amino)-2-oxyethoxy(methyl)amino)-2-oxyethyl)-3-phenylpropamide (Drug-Linker 14) [ka]

[0299] Compound I-3 (40.26 mg, 76.67 μmol) was dissolved in dichloromethane (5 mL), DIC (9.68 mg, 76.67 μmol) was added, and the mixture was stirred for 20 minutes. Compound 13-4 formate (34.00 mg, 38.34 μmol) was added, and the mixture was allowed to react for 2 hours at 20° C. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the title compound 14 (5.33 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 24]

[0300] The structural property data are as follows: ESI-MS(m / z):1348.3[M+H] + .

[0301] Example 15 1-(3,5-di(2-(mesyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxo-2-azatridecane-13-oleic acid (15) [ka]

[0302] Step 1: Synthesis of tert-butyl 1-(3,5-di(2-(mesyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxo-2-azatridecane-13-oleate (15-2) Compound II-9 (50.00 mg, 115.09 μmol) was dissolved in DMF (2 mL), HATU (65.60 mg, 172.53 μmol) was added, and the mixture was stirred. Compound 15-1 (36.37 mg, 138.11 μmol) and DIPEA (44.62 mg, 345.27 μmol, 61.46 μL) were then added, and the mixture was allowed to react for 2 hours at 25° C. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to obtain the title compound 15-2 (25.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 25]

[0303] The structural property data are as follows: ESI-MS(m / z):697.2[M+18] + .

[0304] Step 2: Synthesis of 1-(3,5-di(2-(mesyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxo-2-azatridecane-13-oleic acid (15) Compound 15-2 (20.00 mg, 29.42 μmol) was dissolved in trifluoroacetic acid (1 mL) and dichloromethane (5 mL) and reacted for 2 hours at 25° C. The reaction solvent was extracted under reduced pressure, and the crude product of the title compound 15 (15.00 mg) was obtained from the concentrate, which was directly used in the next step without purification.

[0305] The structural property data are as follows: ESI-MS(m / z):641.1[M+18] + .

[0306] 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]indoxazino[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-dodecyloxo-5,8,11,14,17,45-hexaazahexapentan-56-yl)-3,5-bis(2-(mesyl)pyrimidin-4-yl)benzamide (Drug-Linker 16) [ka]

[0307] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonoxy-5,8,11,14,17-pentaazatetradecan-44-yl)carbamate (16-2) 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 and reacted for 2 hours at 25° C. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the title compound 16-2 (110.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 26]

[0308] The structural property data are as follows: ESI-MS(m / z):1486.5[M+1] + .

[0309] 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]indoxazino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazohexadecan-16-yl)-3,6,9,12,15,18,21,2-octaoxaheptan-27-amide (16-3) Compound 16-2 (110.00 mg, 74.00 μmol, FR) was dissolved in diethylamine (1 mL) and dichloromethane (5 mL) and reacted for 2 hours at 25° C. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to obtain the formate salt of the title compound 16-3 (40.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 27]

[0310] The structural property data are as follows: ESI-MS(m / z): 1264.6[M+1] + .

[0311] 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]indoxazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18,46-heptaoxo-3,21,24,27,30,33,36,39,42,4851,54-12alkoxy-5,8,11,14,17,45-hexaazahexapentan-56-yl)-3,5-bis(2-(mesyl)pyrimidin-4-yl)benzamide (16) 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 reacted at 25 °C for 2 h. The reaction solvent was extracted under reduced pressure, and the concentrate was directly purified by high-performance liquid chromatography and lyophilized to give the title compound 16 (7.00 mg). Chromatography column: SunFire Prep C18 OBD 5μm 19x150mm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 28]

[0312] The structural property data are as follows: ESI-MS(m / z):1869.7[M+1] + .

[0313] Example 17 N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxy-3,17,20,23-tetraoxy-5,8,11,14-tetraazopentadecan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-10) [ka]

[0314] Step 1: 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 HO (4 mL). The reaction mixture was stirred at 90 °C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through a pad of diatomaceous earth, and the filtrate was extracted with water and ethyl acetate. The crude product was purified by column chromatography (EA / PE = 0-25%) to give 710 mg of methyl 3,5-di(2-(methylsulfido)pyrimidin-5-yl)benzoate.

[0315] Its structural property data are as follows: ESI-MS(m / z):385.1[M+H] + .

[0316] Step 2: The compound methyl 3,5-di(2-(methylsulfido)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 mixture was stirred at 25° C. for 2 hours and monitored by LC-MS. The pH of the mixture was adjusted to about 2 with 1N HCl to precipitate a large amount of solid. The cake was collected by filtration and dried to obtain 560 mg of 3,5-di(2-(methylsulfido)pyrimidin-5-yl)benzoic acid.

[0317] Its structural property data are as follows: ESI-MS(m / z):371.1[M+H] + .

[0318] Step 3: The compound 3,5-di(2-(methylsulfido)pyrimidin-5-yl)benzoic acid (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL), and 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 the reaction was monitored by LC-MS. The solvent was dried by blowing with a stream of nitrogen gas to obtain a crude product, which was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 153 mg of 3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid.

[0319] Its structural property data are as follows: ESI-MS(m / z):435.0[M+H] + .

[0320] The preparation method is as follows. Chromatography column: Phenomenex Luna C18 200 x 40 mm x 10 um. Mobile phase A: acetonitrile, Mobile phase B: water (0.05% hydrochloric acid) Mobile phase: [water(HCl)-ACN]; B%:13%-43%, 10min). [Table 29]

[0321] Step 4: The compound 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 mixture was purified using preparative high-performance liquid chromatography and then lyophilized to yield 51 mg of tert-butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-ate.

[0322] Its structural property data are as follows: ESI-MS(m / z):694.2[M+H] + .

[0323] The preparation method is as follows. Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 30]

[0324] Step 5: Compound tert-butyl 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-oate (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL) and reacted at 25 °C for 1 h. LC-MS reaction was monitored and the reaction system was concentrated to dryness to give compound 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-14-oate (45 mg).

[0325] Its structural property data are as follows: ESI-MS(m / z):637.2[M+H] + .

[0326] Step 6: The compound 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[a]thiazolinone]-2-yl) ... Zo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxypropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (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 mixture 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-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxy-3,17,20,23-tetraoxy-5,8,11,14-tetraazopentadecan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide.

[0327] Its structural property data are as follows: ESI-MS(m / z):1342.5[M+H] + .

[0328] The preparation method is as follows. Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 31]

[0329] Example 18 N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxy-3,17,20,23-tetraoxy-5,8,11,14-tetraazopentadecan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-17) [ka]

[0330] Step 1: 3,5-Di(2-(methylsulfido)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 sequentially. The mixture was heated to 60°C and reacted for 2 hours. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude tert-butyl 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oate (3.8 g, 4.75 mmol), which was used directly in the next step without further purification.

[0331] Step 2: Tert-butyl 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-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 was stirred at 25 °C for 2 hours, and the reaction mixture was directly concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to give 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oate (2.09 g, 3.64 mmol).

[0332] Its structural property data are as follows: ESI-MS(m / z):574.2[M+H] + .

[0333] The preparation method is as follows. Chromatography column: Phenomenex luna C18 (250 x 70 mm x 10 μm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 32]

[0334] Step 3: 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecanoic acid (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), and sodium periodate (208.79 mg, 976.15 μmol) and ruthenium trichloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system, and the mixture was stirred at 25 ° C. for 30 minutes. The reaction was monitored by LC-MS, and the mixture 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-trioxa-2-azatetradecanoic acid (60 mg).

[0335] Its structural property data are as follows: ESI-MS(m / z):638.2[M+H] + .

[0336] Step 4: (2S)-2-amino-N-((2S)-1-(((2S)-1-((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxin-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxyethoxy)methyl)amino)-1-oxypropan-2-yl)amino)-1-oxypropane- 2-yl)propanamide (IM-6, 20 mg, 25.06 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-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 sequentially to DMF (3 mL), and the reaction system was reacted at 25° C. for 1 hour. The reaction mixture was purified directly using preparative high-performance liquid chromatography and then lyophilized to give N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxy-3,17,20,23-tetraoxy-5,8,11,14-tetraazopentadecan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (16 mg).

[0337] Its structural property data are as follows: ESI-MS(m / z):1371.4[M+H] + .

[0338] The preparation method is as follows. Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 33]

[0339] 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]indolizine[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-nonoxy-5,8,11,14-tetraazatetraalkane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21) [ka]

[0340] Step 1: 3,5-Di(2-(methylsulfido)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), followed by the addition of 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), and the reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was extracted with water (100 mL) and ethyl acetate (60 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxo-2-azanonan-29-oate (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[0341] Step 2: Tert-Butyl 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oate (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), TFA (15.3 g, 134 mmol, 10 mL) was added, and the reaction was stirred at 25° C. for 6 hours. The reaction mixture was extracted with water (60 mL) and ethyl acetate (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by preparative high-performance liquid chromatography and then lyophilized to give 1-(3,5-bis(2-(methylsulfido)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid (2.93 g, 3.63 mmol).

[0342] Its structural property data are as follows: ESI-MS(m / z):794.3[M+H] + .

[0343] The preparation method is as follows. Chromatography column: Phenomenex luna C18 (250 x 70 mm x 10 μm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 34]

[0344] Step 3: 1-(3,5-bis(2-(methylsulfide)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-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 trichloride were added. Ruthenium hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, and the reaction was carried out with stirring 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-octaoxy-2-azanonacosan-29-oic acid (155 mg).

[0345] Its structural property data are as follows: ESI-MS(m / z):858.3[M+H] + .

[0346] Step 4: (2S)-2-amino-N-((2S)-1-(((2S)-1-((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxin-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxyethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)pro Panamide (IM-6, 27.91 mg, 34.97 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-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 mixture was reacted at 25°C for 1 hour. The reaction mixture was purified by high-performance liquid chromatography and then 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]indolizine [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-tetraazatetraalkane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (15 mg).

[0347] Its structural property data are as follows: ESI-MS(m / z):1591.7[M+H] + .

[0348] The preparation method is as follows. Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 35]

[0349] 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]indolizine[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-tetraazahexadecan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19) [ka]

[0350] Step 1: Methyl 2,6-dibromoisonicotinate (5.00 g, 16.9 mmol), (2-(methylsulfido)pyrimidin-5-yl)boronic 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 mL) and water (17 mL). The reaction mixture was purged with nitrogen gas three times and then reacted at 100 °C for 5 hours. After allowing the reaction mixture to cool to room temperature, water (50 mL) was added to the reaction mixture, filtered, and the filtrate was concentrated to give the crude product. The product was triturated with petroleum ether, filtered, and the cake was dried under vacuum to give 5.65 g of methyl 2,6-di(2-(methylsulfido)pyrimidin-5-yl)isonicotinate.

[0351] Step 2: Methyl 2,6-di(2-(methylsulfido)pyrimidin-5-yl)isonicotinate (5.26 g, 13.7 mmol) was dissolved in THF (30 mL), MeOH (30 mL), and water (30 mL). LiOH·HO (1.72 g, 40.9 mmol) was added and the mixture was stirred at 25°C for 2 hours. The pH of the reaction mixture was adjusted to 3 with 1N aqueous hydrochloric acid. A solid precipitate was formed, which was filtered, and the cake was dried under vacuum to give 2,6-di(2-(methylsulfido)pyrimidin-5-yl)isonicotinic acid (4.20 g).

[0352] Its structural property data are as follows: ESI-MS(m / z):372.1[M+H] + .

[0353] Step 3: 2,6-Di(2-(methylsulfido)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 successively. The mixture was heated to 60°C and stirred for 2 hours. After allowing the reaction to cool to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction mixture, 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 give crude 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid tert-butyl ester (2.50 g), which was used directly in the next step without further purification.

[0354] Step 4: Tert-butyl 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-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 mixture was stirred at 25 ° C. for 12 hours. The reaction mixture was directly concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to give 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecane-14-oate (1.20 g).

[0355] Its structural property data are as follows: ESI-MS(m / z):575.2[M+H] + .

[0356] The preparation method is as follows. Chromatography column: Phenomenex Luna C18 (150 mm x 25 mm x 10 μm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 36]

[0357] Step 5: 1-(2,6-bis(2-(methylsulfide)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxy-2-azatetradecan-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. The reaction mixture was reacted at 25°C for 1 hour, then extracted with water (50 mL) and ethyl acetate (80 mL). The organic phase was concentrated to obtain a crude product, which was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxy-2-azatetradecan-14-oic acid (130 mg).

[0358] Its structural property data are as follows: ESI-MS(m / z):639.2[M+H] + .

[0359] Step 6: (2S)-2-amino-N-((2S)-1-(((2S)-1-((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxin-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxypropan-1 (2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxy-2-azatetradecan-14-oic acid) (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and dissolved with stirring. 1.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours. The reaction mixture was directly purified using preparative high performance liquid chromatography, and then 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,1 2H-Benzo[de]pyrano[3',4':6,7]indolizine[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-tetraazahexadecan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (20.4 mg) was obtained.

[0360] Its structural property data are as follows: ESI-MS(m / z):1372.4[M+H] + . Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 37]

[0361] 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]indolizine[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-nonoxy-5,8,11,14-tetraazatetradecan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23) [ka]

[0362] Step 1: 2,6-Di(2-(methylsulfide)pyrimidin-5-yl)isonicotinic acid (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate (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 successively. The mixture was heated to 60° C. and stirred for 2 hours. The reaction mixture was cooled to room temperature, and water (10.0 mL) and ethyl acetate (20.0 mL) were added to separate the layers. 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 concentrated to give crude tert-butyl 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonacosan-29-oate (3.00 g), which was used directly in the next step.

[0363] Step 2: Tert-butyl 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oate (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), and TFA (15.4 g, 134 mmol) was added, followed by stirring at 25° C. for 12 hours. The reaction mixture was concentrated directly to give a crude product, which was purified by preparative high-performance liquid chromatography and then lyophilized to give 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid (1.20 g).

[0364] Its structural property data are as follows: ESI-MS(m / z):795.3[M+H] + .

[0365] The preparation method is as follows. Chromatography column: Welch Ultimate C18 (150 mm x 25 mm x 5 μm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 38]

[0366] Step 3: 1-(2,6-Di(2-(methylsulfide)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-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, and the system was heated at 25°C. After reacting for 1 hour, the mixture was extracted with water (10 ml) and ethyl acetate (40 ml), and the organic phase was concentrated to obtain a crude product. The crude product was purified by column chromatography (MeOH / DCM=10-20%) and concentrated to obtain 1-(2,6-di(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (350 mg).

[0367] Its structural property data are as follows: ESI-MS(m / z):859.3[M+H] + .

[0368] Step 4: (2S)-2-Amino-N-((2S)-1-(((2S)-1-((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxin-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-2-oxyethoxy)methyl)amino)-1-oxypropan-2-yl (2,6-Di(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid (21.5 mg, 0.025 mmol) was dissolved in DMF (1 mL), and HATU (19.0 mL) was added. g, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added, and the mixture was reacted at room temperature for 2 hours. The reaction mixture was directly purified using preparative high-performance liquid chromatography, and then 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]indolizine[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-tetraazatetradecan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (17.0 mg).

[0369] Its structural property data are as follows: ESI-MS(m / z):1592.6[M+H] + .

[0370] The preparation method is as follows. Chromatography column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 39]

[0371] Example 22 2,2"-Bis(methylsulfonyl)-[5,4':6',5"-tripyrimidine]-2'-carboxylic acid (II-9) [ka]

[0372] Step 1: 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 mixture was purged with nitrogen gas three times, then heated to 100 °C and stirred for 5 hours. After cooling to room temperature, water (60.0 mL) was added to the reaction mixture and stirred for an additional 1 hour. After filtration and concentration of the filtrate, the crude product was obtained. The crude product was triturated with petroleum ether / ethyl acetate (1:1, 40 mL), filtered again, and the cake was dried under vacuum to give methyl 2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylate (1.39 g, 3.60 mmol).

[0373] Step 2: Methyl 2,2"-bis(methylsulfide)-[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 mixture was adjusted to 2 with 1N aqueous hydrochloric acid. A solid precipitate was formed, so the mixture was filtered, and the cake was dried under vacuum to obtain crude 2,2"-bis(methylsulfide)-[5,4':6',5"-tripyrimidine]-2'-carboxylic acid (1.06 g).

[0374] Its structural property data are as follows: ESI-MS(m / z):373.1[M+H] + .

[0375] Step 3: Using the same method as in the preparation of C-23-3 in Example 21, and replacing C-23-2 with II-9-3 (2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidine]-2′-carboxylic acid), compound II-9 could be obtained.

[0376] Its structural property data are as follows: ESI-MS(m / z):437.0[M+H] + .

[0377] Example 23 1-(2,2"-bis(methylsulfonyl)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid (C-29-3) [ka]

[0378] Step 1: 2,2"-Bis(methylsulfide)-[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 and 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 organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude tert-butyl 1-(2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecanoate (850 mg).

[0379] Step 2: Tert-butyl 1-(2,2"-bis(methylsulfide)-[5,4':6',5"-tripyrimidin]-2"-yl)-1-oxo-5,8,11-trioxa-2-azatetradecanoate (800 mg, 1.27 mmol) was dissolved in dichloromethane (2.00 mL), and TFA (3.07 g, 26.9 mmol, 2.00 mL) was added, followed by stirring at 25°C for 2 hours. The reaction mixture was directly concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to give 1-(2,2"-bis(methylsulfide)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecanoate (145 mg, 249 µmol).

[0380] Its structural property data are as follows: ESI-MS(m / z):576.2[M+H] + .

[0381] The preparation method is as follows. Chromatography column: Phenomenex luna C18 (150 mm x 25 mm x 10 μm) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 40]

[0382] Step 3: Using the same method as in the preparation of C-23-3 in Example 21, and substituting C-29-2 (1-(2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid) for C-23-2, compound C-29-3 could be obtained.

[0383] Its structural property data are as follows: ESI-MS(m / z):640.1[M+H] + .

[0384] Example 24 1-(2,2"-bis(methylsulfonyl)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonan-29-oic acid (C-30-3) [ka]

[0385] Step 1: 4,6-Bis(2-methylthiopyrimidin-5-yl)pyrimidine-2-carboxylic acid (0.35 g, 940 μmol) was dissolved in DMF (6.00 mL), and 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-octaoxaheptan-27-oate (468 mg, 940 μmol) were added, followed by stirring for 12 hours at 25° C. Water (60.00 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (40.00 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude tert-butyl 1-(2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonan-29-oate (800 mg).

[0386] Step 2: tert-Butyl 1-(2,2"-bis(methylsulfide)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonan-29-oate (800 mg, 939 μmol) was dissolved in dichloromethane (10.0 mL), and TFA (3.07 g, 26.9 mmol, 2 mL) was added, followed by stirring at 25°C for 2 hours. The reaction mixture was directly concentrated, purified by preparative high-performance liquid chromatography, and then lyophilized to give 1-(2,2"-bis(methylsulfide)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxy-2-azanonan-29-oic acid (389 mg, 483 μmol).

[0387] Its structural property data are as follows: ESI-MS(m / z):796.3[M+H] + .

[0388] The preparation method is as follows. Chromatography column: Phenomenex luna C18 (150mm x 25mm x 10um) Mobile phase A: acetonitrile, Mobile phase B: water (0.05% formic acid) [Table 41]

[0389] Step 3: The same method as in the preparation of C-23-3 in Example 21 was employed, but C-23-2 was replaced with C-30-2 (1-(2,2″-bis(methylsulfide)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonan-29-oic acid) to obtain compound C-30-3.

[0390] Its structural property data are as follows: ESI-MS(m / z):860.3[M+H] + .

[0391] 3. Preparation of Bioactive Conjugates 3.1 Preparation of anti-ROR1 antibody bioactive conjugate Conjugation Method A: 0.5 mL of antibody (anti-ROR1 antibody 19F6_Hu35V1,3-20 mg / mL, referred to as 19F6 in Table 1) was taken and diluted with 0.1 M edetate disodium solution (pH 7.60). The pH was then adjusted to 7.60 with 1 M NaHPO solution, and 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed uniformly, and left at room temperature for 2 hours. A 5-10-fold excess of the antibody in DMSO (10 mM drug-linker) was added to the above solution, mixed uniformly, and left at room temperature for 20 hours. After completion, the buffer was replaced with 10 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva). The resulting ADC product is shown in Table 1.

[0392] Conjugation Method B: 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 edetate disodium solution (pH 7.60). The pH was then adjusted to 7.60 with 1 M NaHPO solution. 10 mM TCEP (tri(2-carboxyethyl)phosphine) solution (pH 7.60) was added and mixed uniformly. The mixture was then incubated at room temperature for 2 hours. TCEP was removed using a NAP-5 gel column (Cytiva). A 5-10-fold excess of the antibody was added to the resulting solution, followed by mixing uniformly and incubation at room temperature for 20 hours. The buffer was then replaced with 10 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva). The resulting ADC product is shown in Table 1.

[0393] Conjugation Method C: (applied for the preparation of 19F6-MC-VC-PABC-MMAE) 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). The pH was adjusted to 7.66 with 1 M NaHPO solution, and 66.2 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added. The mixture was mixed uniformly and left at room temperature for 1.5 hours. A 4.8x equivalent of drug-linker (MC-VC-PABC-MMAE, 10 mM) solution was added to the above solution, mixed uniformly, and left at room temperature for 2 hours. After completion, the buffer was replaced with 10 mM histidine-HCl buffer, pH 6.0, using a NAP-25 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC (19F6-MC-VC-PABC-MMAE). The DAR value determined by mass spectrometry was 4.15. [Table 42]

[0394] Determining the Drug / Antibody Ratio (DAR) of Bioactive Conjugates The molecular weight of the ADC samples was measured by SEC-MS, and the drug / antibody ratio (DAR) was calculated.

[0395] Chromatography conditions: Chromatography column: ACQUITY UPLC Protein BEH SEC Column, BTQR-18-016. Sample chamber temperature: 8°C, column temperature: column temperature not controlled, UV: 280 nm. Mobile phase: 20 mM ammonium acetate, flow rate: 0.1 ml / min, 20 min, sample injection amount: 50 μg. Mass spectrometry measurement conditions: Mass spectrometry model number: AB Sciex Triple TOF 5600+; GS1 55;GS2 55;CUR 30;TEM 450;ISVF 5500;DP 75;CE 5; m / z 900-7000; Time bins to sum 100.

[0396] (1) Measure the molecular weight of 19F6-ADC III-5-A by SEC-MS and calculate the drug / antibody ratio The conjugated 19F6-ADC III-5-A was subjected to molecular weight analysis by SEC-MS, and the results are shown in Table 2, with a DAR of 3.87. [ka] [Table 43]

[0397] (2) Measure the molecular weight of 19F6-ADC III-6-A by SEC-MS and calculate the drug / antibody ratio The conjugated 19F6-ADC III-6-A was subjected to molecular weight analysis by SEC-MS, and the results are shown in Table 3, with a DAR of 4.42. [ka] [Table 44]

[0398] (3) Measure the molecular weight of 19F6-ADC IV-1-A by SEC-MS and calculate the drug / antibody ratio Molecular weight analysis of the conjugated 19F6-ADC IV-1-A was performed by SEC-MS, and the results are shown in Table 4, with a DAR of 3.74. [ka] [Table 45]

[0399] (4) Measure the molecular weight of 19F6-ADC IV-1-B by SEC-MS and calculate the drug / antibody ratio Molecular weight analysis of the conjugated 19F6-ADC IV-1-B was performed by SEC-MS, and the results are shown in Table 5, with a DAR of 3.75. [ka] [Table 46]

[0400] (5) Measure the molecular weight of 19F6-ADC IV-1-C by SEC-MS and calculate the drug / antibody ratio Molecular weight analysis of the conjugated 19F6-ADC IV-1-C was performed by SEC-MS, and the results are shown in Table 6, with a DAR of 3.82. [ka] [Table 47]

[0401] (6) Measure the molecular weight of 19F6-ADC IV-1-D by SEC-MS and calculate the drug / antibody ratio Molecular weight analysis of the conjugated 19F6-ADC IV-1-D was performed by SEC-MS, and the results are shown in Table 7, with a DAR of 3.87. [ka] [Table 48]

[0402] (7) Measure the molecular weight of 19F6-ADC IV-7-A by SEC-MS and calculate the drug / antibody ratio Molecular weight analysis of the conjugated 19F6-ADC IV-7-A was performed by SEC-MS, and the results are shown in Table 8, with a DAR of 3.98. [ka] [Table 49]

[0403] 3.2 Preparation of B7-H3 antibody bioactive conjugate 3.2.1 Preparation of 2#8890ADC C-10 (DAR4) 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was taken and 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 NaHPO solution. 9.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed uniformly, and left at room temperature for 1.5 h. An 8x volume of C-10 (14 μL, 10 mM) solution dissolved in dimethyl sulfoxide was slowly added, mixed uniformly, and left at room temperature overnight. After completion of the dilution, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., 2#8890ADC C-10. The DAR measured by mass spectrometry was 4.17.

[0404] Molecular weight analysis was performed on the conjugated ADC samples by LC-MS. Chromatography conditions: Liquid chromatography column: ACQUITY UPLC MAbPac BEH SEC, Mobile phase A: 20mM NH4Ac, Flow rate: 0.1 ml / min, sample chamber temperature: 8°C, column temperature: 60°C, injection volume: 2 μl. [Table 50]

[0405] Mass spectrometry measurement conditions: Mass spectrometry model number: AB Sciex Triple TOF 5600+, GS1 55, GS2 55, CUR 30, TEM 450, ISVF 5500, DP 75, CE 5, Accumulation time 0.5s, m / z 900-7000, Time bins to sum 40.

[0406] 3.2.2 Preparation of 2#8890ADC C-17(DAR4) 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 NaHPO solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 4.77 μL, pH 7.60) solution was added, mixed uniformly, and left at room temperature for 1.5 h. A 5x volume of C-17 (8.76 μL, 10 mM) dissolved in dimethyl sulfoxide was then slowly added, mixed uniformly, and left at room temperature overnight. After completion of the dilution, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., 2#8890ADC C-17. The DAR determined by mass spectrometry was 3.86. [Table 51]

[0407] 3.2.3 Preparation of 2#8890ADC C-19(DAR4) 0.584 mL of 2#8890 antibody (8.565 mg / mL) was taken and 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 NaHPO solution. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed uniformly, and left at room temperature for 1.5 h. A 5.5-fold excess of C-19 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed uniformly, and left at room temperature overnight. The buffer was then replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., 2#8890ADC C-19. The DAR measured by mass spectrometry was 4.05. [Table 52]

[0408] 3.2.4 Preparation of 2#8890ADC C-21(DAR4) 0.584 mL of 2#8890 antibody (8.565 mg / mL) was taken and 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 NaHPO solution. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed uniformly, and left at room temperature for 1.5 h. A 5.5-fold excess of C-21 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed uniformly, and left at room temperature overnight. After completion of the dilution, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., 2#8890ADC C-21. The DAR measured by mass spectrometry was 3.92. [Table 53]

[0409] 4. Detection of the inhibitory effect of the bioactive complex on in vitro cellular activity 4.1 Cytostatic effect of ADC (1) Cell seeding: First, NCI-N87 tumor cells were cultured in the appropriate medium, digested with pancreatinine, and then centrifuged to count the resuspended cells. The cells were then seeded at an appropriate concentration. The tumor cell sources are listed in Table 9. [Table 54]

[0410] Co-incubation of the ADC of the present invention and tumor cells: After waiting for the cells to adhere to the wall, the medium inside the cells was removed, and diluted bioactive molecules (ADC of the present invention) were added to the wells of the plate and incubated for 96 hours.

[0411] In vitro cell activity assay: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme / Novisan) was added to each well, and the wells were shaken in the dark to mix evenly. After 10 minutes of incubation, assays were performed. The values ​​were read using a microplate reader (manufacturer: BMG, model number: PHERAStar-FS). Background RLU was obtained from the Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained from the Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%, and the compound's median inhibitory concentration (IC50) was calculated according to a four-parameter model fitting curve. 50 ) was calculated.

[0412] (2) Data Results: The test measurement results are shown in Table 10. [Table 55]

[0413] The ADCs (19F6-ADC III-6-A and 19F6-ADC IV-1-D) formed using the new conjugation method were able to produce tumor cell-killing activity, demonstrating the effectiveness of applying the new conjugation method to ADC molecules. Furthermore, compared with the randomly conjugated ADC (19F6-MC-VC-PABC-MMAE), the ADCs (19F6-ADC III-6-A and 19F6-ADC IV-1-D) prepared using the new conjugation method exhibited superior cell-killing activity.

[0414] 4.2 Antiproliferative effect of ADC on HT29 cells (1) Cell seeding: First, HT29 tumor cells were cultured in the appropriate medium, digested with pancreatinine, and then centrifuged to count the resuspended cells. The cells were then seeded at an appropriate concentration. The tumor cell sources are listed in Table 11. [Table 56]

[0415] Co-incubation of the ADC of the present invention and tumor cells: After waiting for the cells to adhere to the wall, the medium inside the cells was removed, and diluted bioactive molecules (ADC of the present invention) were added to the wells of the plate and incubated for 96 hours.

[0416] In vitro cell activity assay: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme / Novisan) was added to each well, and the wells were shaken in the dark to mix evenly. After 10 minutes of incubation, assays were performed. The values ​​were read using a microplate reader (manufacturer: BMG, model number: PHERAStar-FS). Background RLU was obtained from the Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained from the Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%, and the compound's median inhibitory concentration (IC50) was calculated according to a four-parameter model fitting curve. 50 ) was calculated.

[0417] (2) Data Results: The test measurement results are shown in Table 12. [Table 57]

[0418] The ADC (19F6-ADC IV-1-D) formed using the new conjugation method was able to produce killing effects against tumor cells, demonstrating the effectiveness of applying the new conjugation method to ADC molecules.

[0419] 4.3 Growth inhibitory effect of ADC on NCI-H1975 cells (1) Cell seeding: First, NCI-H1975 tumor cells were cultured in the appropriate medium, digested with pancreatinine, and then centrifuged to count the resuspended cells. The cells were then seeded at an appropriate concentration. The tumor cell sources are listed in Table 13. [Table 58]

[0420] Co-incubation of the ADC of the present invention and tumor cells: After waiting for the cells to adhere to the wall, the medium inside the cells was removed, and diluted bioactive molecules (ADC of the present invention) were added to the wells of the plate and incubated for 96 hours.

[0421] In vitro cell activity assay: After incubation, 50 μL of Cell Counting-Lite™ 2.0 Reagent (Vazyme / Novisan) was added to each well, and the wells were shaken in the dark to mix evenly. After 10 minutes of incubation, assays were performed. The values ​​were read using a microplate reader (manufacturer: BMG, model number: PHERAStar-FS). Background RLU was obtained from the Cell Counting-Lite™ in cell-free medium, and solvent RLU was obtained from the Cell Counting-Lite™ in cell-containing medium. Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) × 100%, and the compound's median inhibitory concentration (IC50) was calculated according to a four-parameter model fitting curve. 50 ) was calculated.

[0422] (2) Data Results: The test measurement results are shown in Table 14. [Table 59]

[0423] The ADC (19F6-ADC IV-1-D) formed using the new conjugation method was able to produce killing effects against tumor cells, demonstrating the effectiveness of applying the new conjugation method to ADC molecules.

[0424] Various modifications of the present invention in accordance with the foregoing description, except as described herein, will be apparent to those skilled in the art, and such modifications are 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 isotopically labeled compound thereof, wherein said compound has the structure of Formula I: 【Chemistry 1】 During the ceremony, X is a leaving group, for example, F, Cl, Br, I, OMs, OTs, OTf, p-nitrophenol ester, fluorophenol ester, C １－６ alkylsulfonyl, or 【Chemistry 2】 and Y is absent or is a substituted or unsubstituted C １－６ alkylene group, sulfonyl group, and carbonyl group, and when substituted, １－６ The alkylene group may contain hydrogen, halogen, hydroxyl, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; Ring A is a substituted or unsubstituted C ６－１０ selected from aromatic rings, substituted or unsubstituted 5- to 12-membered aromatic heterocycles and substituted or unsubstituted 5- to 12-membered heterocycles, and when substituted, ６－１０ The aromatic ring, the 5- to 12-membered aromatic heterocycle, and the 5- to 12-membered heterocycle are independently hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group, —OC １－６ substituted with a substituent selected from a haloalkyl group, a carboxy group, a polyethylene glycol, an amino acid, a phosphoric acid, a sulfonic acid, an amino group, an azide group, and an alkynyl group; Q is absent or is -NH-, -O-, -CH ２ -, hydroxy group, carbonyl group, amide group, sulfonyl group, sulfonylurea group, amidoformyl group, oxime group, -NH-S(=O) ２ -NH-C(=O)O-, -C(=O)NH-, -NHC(=O)- or C ２－６ The alkynylene group is composed of one or more substituted or unsubstituted groups, and when substituted, each of said groups is independently selected from hydrogen, halogen, hydroxyl, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and -OC １－６ substituted by a substituent selected from haloalkyl groups; Z １ is absent or is a substituted or unsubstituted phenyl group, a substituted or unsubstituted 5- or 6-membered heteroaryl group, an amido group, a substituted or unsubstituted —CH ２ - and substituted or unsubstituted C ２－６ alkynylene groups, and when substituted, the phenyl group, the 5- to 6-membered heteroaryl group, —CH ２ - and C ２－６ The alkynylene groups are independently selected from hydrogen, halogen, hydroxyl, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; W １ is absent or is a substituted or unsubstituted C １－１０ Alkylene group, -(CH ２ CH ２ O)p- and -(OCH ２ CH ２ )p-, where p is an integer of 1 to 20, and when substituted, １－１０ The alkylene group may contain hydrogen, halogen, hydroxyl, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; J １ -COOH, -NH ２ , substituted —NH ２ , a 3- to 10-membered nitrogen-containing heterocyclic group, a substituted 3- to 10-membered nitrogen-containing heterocyclic group, an alkynyl group, an 8- to 16-membered alkynyl-containing ring group, a substituted 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a hydroxyamide group, an aldehyde group, a keto group, a sulfonylurea group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxy group, wherein the "substituted" is independently selected from hydrogen, halogen, a hydroxy group, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ It refers to being substituted with a substituent selected from haloalkyl groups.

2. X is a leaving group, e.g., Cl, Br, I, OMs, OTs, OTf, or 【Transformation 3】 and Y is absent or a carbonyl group; Ring A is a substituted or unsubstituted C ６－１０ an aromatic ring, a substituted or unsubstituted 5- to 12-membered aromatic heterocycle, or a substituted or unsubstituted 5- to 12-membered heterocycle, and when substituted, ６－１０ The aromatic ring, the 5- to 12-membered aromatic heterocycle, and the 5- to 12-membered heterocycle are independently hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; Q is absent or is a substituted or unsubstituted —C(═O)NH—, and when substituted, the —C(O)—NH— is hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; Z １ is absent or is a substituted or unsubstituted —CH ２ - or substituted or unsubstituted C ２－６ an alkynylene group, and when substituted, the —CH ２ - or C ２－６ The alkynylene groups are independently selected from hydrogen, halogen, hydroxyl, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; W １ is absent or is a substituted or unsubstituted C １－１０ Alkylene group, -(CH ２ CH ２ O)p- and -(OCH ２ CH ２ )p-, where p is an integer from 1 to 10, and when substituted, １－１０ The alkylene group may contain hydrogen, halogen, hydroxyl, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; J １ -COOH, -NH ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein the N-membered heterocyclic group is selected from the group consisting of a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group and a hydroxy group.

3. 3. The compound of formula I of claim 1 or 2, wherein the structure is selected from the following: 【Chemistry 4】 In the formula, p is an integer from 1 to 10, and J １ is -COOH or -NH ２ is.

4. The compound of formula I according to any one of claims 1 to 3, wherein the structure is selected from: 【Transformation 5】

5. 1. A compound of Formula II or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein the structure of said compound of Formula II is as follows: 【Transformation 6】 During the ceremony, B １ and B ２ are each independently a single bond or a substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycle, and when substituted, the 5-12 membered nitrogen-containing aromatic heterocycle may be hydrogen, halogen, a hydroxy group, —CN, a substituted or unsubstituted C １－１０ Alkylene group, —C １－６ Haloalkyl group, —OC １－６ Alkyl group, —OC １－６ It is substituted by one or more substituents selected from the group consisting of haloalkyl groups, carboxy groups, substituted or unsubstituted amide groups, substituted or unsubstituted carbamoyl groups, substituted or unsubstituted polyethylene glycol, alkynyl groups, and azido groups. When substituted, the C １－１０ The alkylene group, the amide group, the carbamoyl group, and the polyethylene glycol are independently selected from hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by haloalkyl group substituents, Y １ , Y ２ and Y ３ is independently selected from C(R) and N; Z ２ is absent or is —NH—, —O—, —CH ２ -, hydroxy group, carbonyl group, amide group, sulfonyl group, sulfonylurea group, carbamoyl group, oxime group, -NH-S(=O) ２ -NH-C(=O)O-, -C(=O)NH-, -NHC(=O) or C ２－６ The alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxyl group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; W ２ is absent or is a substituted or unsubstituted C １－１０ Alkylene group, -(CH ２ CH ２ O) ｐ - and - (OCH ２ CH ２ ) ｐ -, and when substituted, １－１０ The alkylene group may contain hydrogen, halogen, hydroxyl, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups, and p is an integer from 1 to 10; J ２ is selected from -COOH, -N(R)(R'), a substituted or unsubstituted 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, an alkynyl group, a substituted or unsubstituted 8- to 16-membered alkynyl-containing ring group, an azide group, a tetrazine group, a substituted or unsubstituted hydroxyamide group, an aldehyde group, a keto group, an isocyanate, a thioisocyanate, a maleimide group, and a hydroxyamide group, and when substituted, the 3- to 10-membered nitrogen-containing heterocyclic group, the 8- to 16-membered alkynyl-containing ring group, and the hydroxyamide group are independently selected from hydrogen, halogen, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by haloalkyl group substituents, R and R' are independently hydrogen, halogen, a hydroxy group, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ haloalkyl groups.

6. During the ceremony, B １ and B ２ are each independently selected from a single bond or a substituted or unsubstituted 5- to 6-membered nitrogen-containing aromatic heterocycle, and when substituted, the 5- to 6-membered nitrogen-containing aromatic heterocycle is selected from hydrogen, halogen, a hydroxy group, —CN, a substituted or unsubstituted C １－１０ Alkylene group, —C １－６ Haloalkyl group, —OC １－６ Alkyl group, —OC １－６ It is substituted by a substituent selected from a haloalkyl group, a carboxy group, a substituted or unsubstituted amide group, a substituted or unsubstituted carbamoyl group, a substituted or unsubstituted polyethylene glycol, an alkynyl group and an azide group. When substituted, the C １－１０ The alkylene group, the amide group, the carbamoyl group, and the polyethylene glycol are independently selected from hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ Substituted by a substituent selected from haloalkyl groups, preferably B １ and B ２ are each independently a single bond or a substituted or unsubstituted pyrimidine ring, and when substituted, the pyrimidine ring may be substituted with hydrogen, halogen, a hydroxy group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; Y １ , Y ２ and Y ３ are each independently selected from CH and N; Z ２ is absent or is —NH—, —CH ２ -, a carbonyl group, -C(=O)NH-, -NHC(=O)- or C ２－６ The alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is independently selected from hydrogen, halogen, hydroxyl group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; W ２ is absent or is a substituted or unsubstituted C １－１０ Alkylene group, -(CH ２ CH ２ O) ｐ - or - (OCH ２ CH ２ ) ｐ -, and when substituted, １－１０ The alkylene group may contain hydrogen, halogen, hydroxyl, -CN, -C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group and —OC １－６ substituted by a substituent selected from haloalkyl groups; J ２ -COOH, -NH ２ , a 3-10 membered nitrogen-containing heterocyclic group, a sulfonylurea group, and a hydroxy group; 6. The compound of formula II according to claim 5, wherein p is an integer from 1 to 10.

7. During the ceremony, B １ and B ２ are each independently selected from substituted or unsubstituted 5- to 6-membered nitrogen-containing aromatic heterocycles, and when substituted, the 5- to 6-membered nitrogen-containing aromatic heterocycles are independently selected from hydrogen, C １－１０ substituted with one or more substituents selected from the group consisting of an alkyl group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol group, an alkynyl group, and an azide group; Y １ , Y ２ and Y ３ is independently selected from CH and N; Z ２ is absent or is —NH—, —CH ２ -, carbonyl group and C ２－６ selected from the group consisting of one or more functional groups of alkynylene groups, W ２ does not exist or C １－１０ Alkylene group, -(CH ２ CH ２ O) ｐ - and - (OCH ２ CH ２ ) ｐ - one or more selected from J ２ -COOH, -NH ２ , a 3- to 10-membered nitrogen-containing heterocyclic group, a sulfonylurea group, and a hydroxy group; 7. The compound of formula II according to claim 5 or 6, wherein p is an integer from 1 to 10.

8. During the ceremony, B １ and B ２ are each independently selected from substituted or unsubstituted pyridine and pyrimidine groups, and when substituted, the pyridine and pyrimidine groups are independently selected from hydrogen, C １－１０ substituted with one or more substituents selected from the group consisting of an alkylene group, a carboxy group, an amide group, a carbamoyl group, a polyethylene glycol group, an alkynyl group, and an azide group; Y １ , Y ２ and Y ３ are each independently selected from CH and N; Z ２ is absent or is selected from the group consisting of one or not more than two functional groups selected from the group consisting of —NH— and a carbonyl group; W ２ does not exist or C １－１０ Alkylene group, -(CH ２ CH ２ O) ｐ - and - (OCH ２ CH ２ ) ｐ - one or more selected from J ２ is -COOH or -NH ２ and 8. The compound of formula II according to any one of claims 5 to 7, wherein p is an integer from 1 to 10.

9. During the ceremony, B １ and B ２ are each independently selected from substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycles, and when substituted, the 5-12 membered nitrogen-containing aromatic heterocycles are each independently selected from hydrogen, C １－１０ substituted with one or more substituents selected from the group consisting of alkylene, carboxy, amide, carbamoyl, polyethylene glycol, alkynyl, and azide; and / or Z ２ is absent or is —NH—, —CH ２ - and a carbonyl group, and / or J ２ is -COOH or -NH ２ and and / or 7. The compound of claim 5 or 6, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein p is an integer of 3 to 8.

10. During the ceremony, B １ and B ２ are each independently selected from substituted or unsubstituted pyridine and pyrimidine groups, and when substituted, the pyridine or pyrimidine groups are independently selected from hydrogen, C １－１０ 10. The compound of formula II according to any one of claims 5 to 9, substituted with one or more substituents selected from alkylene groups, carboxy groups, amide groups, carbamoyl groups, polyethylene glycol, alkynyl groups and azide groups.

11. During the ceremony, B １ and B ２ The compound of formula II according to any one of claims 5 to 10, wherein each is independently selected from a pyridine group and a pyrimidine group.

12. During the ceremony, Z ２ is absent or is —NH—, —CH ２ -, carbonyl group or C ２－６ The alkynylene group is selected from the group consisting of one or more substituted or unsubstituted functional groups, and when substituted, each of said functional groups is selected from the group consisting of hydrogen, halogen, hydroxyl group, —CN, —C １－６ alkyl group, -C １－６ Haloalkyl group, —OC １－６ Alkyl group or —OC １－６ The compound of formula II according to any one of claims 5 to 11, substituted with a substituent selected from haloalkyl groups.

13. During the ceremony, Z ２ is absent or is selected from the group consisting of one or not more than two of the following functional groups: -NH- and a carbonyl group.

14. During the ceremony, Z ２ 14. The compound of formula II according to any one of claims 5 to 13, wherein is absent or is -C(=O)NH-.

15. During the ceremony, J ２ The compound of formula II according to any one of claims 5 to 14, wherein is -COOH.

16. A compound of formula II according to any one of claims 5 to 15, having the following structure: 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 【Chemistry 7-4】 【Transformation 7-5】 【Transformation 7-6】 【Transformation 7-7】 [Transformation 7-8] p is an integer from 1 to 10.

17. The compound of formula II according to any one of claims 5 to 16, having the following structure: 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】

18. The compound of formula II according to any one of claims 5 to 17, having the following structure: 【Chemistry 9】

19. 1. The compound below, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein said compound is (1) 3,5-di(2-(methylsulfido)pyrimidin-4-yl)benzoic acid, (2) 3,5-di(2-(mesyl)pyrimidin-4-yl)benzoic acid, (3) tert-butyl 1-(3,5-di(2-(mesyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-oleate, (4) 3,5-di(2-(methylsulfido)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-azatridecan-13-oic acid, (7) 1-(3,5-bis(2-(methylsulfide)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (8) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (9) 1-(3,5-bis(2-(methylsulfide)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-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-azanonacosan-29-oic acid, (11) 2,6-di(2-(methylsulfido)pyrimidin-5-yl)isonicotinic acid, (12) 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (13) 1-(2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, (14) 1-(2,6-di(2-(methylsulfido)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid, (15) 1-(2,6-di(2-(methylsulfonyl)pyrimidin-5-yl)pyridin-4-yl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid, (16) 2,2"-bis(methylsulfide)-[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(methylsulfide)-[5,4′:6′,5″-tripyrimidin]-2′-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, or (19) A compound selected from 1-(2,2"-bis(methylsulfonyl)-[5,4':6',5"-tripyrimidin]-2'-yl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oic acid, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, or isotopically labeled compound thereof.

20. 1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein said compound has the structure of Formula V: 【Chemistry 10】 During the ceremony, E is a single bond, -NH-CH ２ -, 【Chemistry 11】 is selected from D is a fragment of a biologically active molecule (e.g., a cytotoxic agent); X, Y, A, Q, Z １ , W １ is defined by any one of claims 1 to 4, V １ is a compound of formula I according to any one of claims 1 to 4, １ is a group formed when V is linked to L, and preferably １ is -C(=O)-, -N(R １ )-, —O—, a 3- to 10-membered nitrogen-containing heterocyclic group, and a sulfonylurea group, wherein R １ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, and more preferably V １ is -C(=O)- or -N(R １ )—, wherein R １ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, L is V １ and E.

21. 21. The compound of formula V of claim 20, having the following structure: 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】 【Chemistry 12-6】

22. 1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein said compound has the structure of Formula VI: 【Chemistry 13】 B １ , B ２ , Y １ , Y ２ , Y ３ , Z ２ and W ２ is defined in any one of claims 5 to 18, L is V ２ and E, V ２ In any one of claims 5 to 18, J ２ is a group formed when linked to L, and preferably V ２ is -C(=O)-, -N(R ２ )-, —O—, a 3- to 10-membered nitrogen-containing heterocycle, and a sulfonylurea group, wherein R ２ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, and more preferably V ２ is -C(=O)- or -N(R ２ )—, wherein R ２ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, E is a single bond, -NH-CH ２ -, 【Chemistry 14】 is selected from D is a fragment of a biologically active molecule (eg, a cytotoxic drug).

23. During the ceremony, B １ and B ２ is a pyrimidine group, Y １ is CH or C, and Y ２ and Y ３ are both CH, Z ２ is —C(═O)NH—, W ２ Ha-(CH ２ CH ２ O) ｐ -C １－１０ Alkylene group - or -(OCH ２ CH ２ ) ｐ -C １－１０ alkylene group, where p is an integer from 1 to 10; V ２ is -C(=0)-, L is 【Chemistry 15】 and E is -NH-CH ２ - and D is 【Chemistry 16】 23. The compound of claim 22, selected from:

24. During the ceremony, W ２ is -(CH ２ CH ２ O) ３ -CH ２ -, -(CH ２ CH ２ O) ３ - (CH ２ ) ２ - and - (OCH ２ CH ２ ) ８ - (CH ２ ) ２ 24. The compound of claim 23, wherein the compound is selected from:

25. In the formula, D is 【Chemistry 17】 The compound according to any one of claims 22 to 24,

26. The compound of any one of claims 22 to 25, wherein the structure is selected from: 【Chemistry 18-1】 【Chemistry 18-2】 【Chemistry 18-3】 【Chemistry 18-4】 【Chemistry 18-5】 【Chemistry 18-6】 【Chemistry 18-7】 【Chemistry 18-8】 【Chemistry 18-9】 【Chemistry 18-10】 【Chemistry 18-11】 【Chemistry 18-12】 [Chemistry 18-13] [Chemistry 18-14]

27. A bioactive conjugate, the structure of which is shown in Formula VII: 【Chemistry 19】 wherein Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), and n is selected from integers or decimal numbers between 1 and 10; V １ is -C(O)- or -N(R １ )—, wherein R １ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, L is V １ 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); In the complex 【Chemistry 20】 indicates a specific linking method between a sulfhydryl group in the antibody and another part of the conjugate when the targeting moiety is an antibody, The remaining radicals are as defined in any one of claims 1 to 4.

28. A bioactive conjugate, the structure of which is shown in Formula VIII: 【Chemistry 21】 wherein Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), and n is selected from integers or decimal numbers between 1 and 10; V ２ is -C(O)- or -N(R ２ )-, wherein R ２ is H, C １－６ Alkyl group or C ２－６ is an alkoxyalkyl group, L is V ２ 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); In the complex 【Chemistry 22】 indicates a specific linking method between a sulfhydryl group in the antibody and another part of the conjugate when the targeting moiety is an antibody, The remaining radicals are as defined in any one of claims 5 to 18.

29. wherein Ab is selected from an anti-Her2 antibody, an anti-Trop2 antibody, or an anti-ROR1 antibody; １ The bioactive conjugate of claim 28, wherein is 1 to 8. 【Chemistry 23-1】 【Chemistry 23-2】 【Chemistry 23-3】

30. During the ceremony, the anti-Her2 antibody is trastuzumab, an antibody comprising a heavy chain variable region of a trastuzumab heavy chain complementarity determining region and a light chain variable region of a trastuzumab light chain complementarity 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 a sacituzumab heavy chain complementarity determining region and a light chain variable region of a 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 comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:3, CDR-H2 represented by SEQ ID NO:4, and CDR-H3 represented by SEQ ID NO:5, and a light chain variable region of CDR-L1 represented by SEQ ID NO:6, CDR-L2 represented by SEQ ID NO:7, and CDR-L3 represented by SEQ ID NO:8, as defined by the Chothia numbering system; ii) an antibody comprising a heavy chain variable region of CDR-H1 set forth in SEQ ID NO:9, CDR-H2 set forth in SEQ ID NO:10, and CDR-H3 set forth in SEQ ID NO:5, and a light chain variable region of CDR-L1 set forth in SEQ ID NO:6, CDR-L2 set forth in SEQ ID NO:7, and CDR-L3 set forth in SEQ ID NO:8, as defined by the AbM numbering system; iii) an antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO:11, CDR-H2 represented by SEQ ID NO:12, and CDR-H3 represented by SEQ ID NO:5, and a light chain variable region of CDR-L1 represented by SEQ ID NO:6, CDR-L2 represented by SEQ ID NO:7, and CDR-L3 represented by SEQ ID NO:8, as defined by the Kabat numbering system; iv) an antibody comprising a heavy chain variable region of CDR-H1 represented by SEQ ID NO: 13, CDR-H2 represented by SEQ ID NO: 14, and CDR-H3 represented by SEQ ID NO: 15, and a light chain variable region of CDR-L1 represented by SEQ ID NO: 16, CDR-L2 represented by SEQ ID NO: 17, and CDR-L3 represented by SEQ ID NO: 8, as defined by the IMGT numbering system; v) an antibody comprising a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2, or vi) The bioactive conjugate of claim 29, which is either 19F6_Hu35V1, which is an antibody having a heavy chain variable region represented by SEQ ID NO: 1, a light chain variable region represented by SEQ ID NO: 2, a heavy chain constant region represented by SEQ ID NO: 18, and a light chain constant region represented by SEQ ID NO:

19.

31. During the ceremony, n １ A bioactive conjugate according to claim 29 or 30, wherein is 1 to 6, for example 3 to 5.

32. wherein Ab is selected from an anti-Her2 antibody, an anti-Trop2 antibody, or an anti-ROR1 antibody; １ The bioactive conjugate of claim 28, wherein x is 1 to 8 and x is 1 to 10. 【Chemistry 24-1】 【Chemistry 24-2】 【Chemistry 24-3】 【Chemistry 24-4】 【Chemistry 24-5】 【Chemistry 24-6】 【Chemistry 24-7】 【Chemistry 24-8】 【Chemistry 24-9】 【Chemistry 24-10】 【Chemistry 24-11】 【Chemistry 24-12】 【Chemistry 24-13】 【Chemistry 24-14】 【Chemistry 24-15】 [Chemistry 24-16] 【Chemistry 24-17】 【Chemistry 24-18】

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

19.

34. During the ceremony, n １ 33. The bioactive conjugate of claim 31 or 32, wherein is 1 to 6, for example 3 to 5.

35. During the ceremony, 34. A bioactive conjugate according to any one of claims 31 to 33, wherein x is 1 to 6, for example 3 to 5.

36. A pharmaceutical composition comprising a compound according to any one of claims 20 to 26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, or a bioactive conjugate according to any one of claims 27 to 35, and one or more pharmaceutically acceptable carriers, Preferably, the drug / antibody ratio (DAR value) of the pharmaceutical composition is 1.0-6.0, for example, 1, 2, 3, 4, 5, or 6, and further for example, 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 drug case product containing a compound according to any one of claims 20 to 26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, or a bioactive conjugate according to any one of claims 27 to 35, or a pharmaceutical composition according to claim 36, and an optional drug instruction leaflet.

38. Use of the compound according to any one of claims 20 to 26 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, the bioactive conjugate according to any one of claims 27 to 35, or the pharmaceutical composition according to 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 to 26, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, a biologically active conjugate according to any one of claims 27 to 35, or a pharmaceutical composition according to claim 36, for use in the prevention or treatment of 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 the compound according to any one of claims 20 to 26, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, the bioactive conjugate according to any one of claims 27 to 35, or the pharmaceutical composition according to claim 36.

41. 1. A method for synthesizing a compound, said method comprising the steps of: 【Chemistry 25】 During the ceremony, X, Z １ , W １ , J １ is defined according to any one of claims 1 to 4, Y is absent, M is a leaving group that causes a substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, and is preferably halogen or Or, the method includes the steps of: 【Chemistry 26】 During the ceremony, X, Z １ , W １ , J １ is defined according to any one of claims 1 to 4, L is a leaving group that causes a substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, and preferably halogen, OTf, or Or, the method includes the steps of: 【Chemistry 27】 During the ceremony, Y １ , Y ２ , Y ３ , B １ , B ２ , Z ２ , W ２ and J ２ is defined by any one of claims 5 to 18, LG is a leaving group that generates a conjugation reaction, and includes, but is not limited to, halogen, trifluoromethanesulfonate, and is preferably halogen.

42. A use of the compound according to any one of claims 1 to 4 for preparing a drug-linker compound, The compound according to any one of claims 1 to 4 is used for preparing a drug-linker compound, the drug-linker compound being prepared by the following steps: 【Chemistry 28】 In the formula, X, Y, A, Q, Z １ , J １ , W １ , V １ , L, E, D are defined in any one of claims 1 to 4, 20 and 21, and LG １ Is J １ The group is selected from groups that undergo a condensation reaction with

43. 43. The use of claim 42, wherein the drug-linker compound is selected from the compounds of claim 20 or 21, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides or isotopically labeled compounds thereof.

44. LG １ is -COOH or -NH(R １ ) wherein R １ is H, C １－６ Alkyl group or C ２－６ The use according to claim 43, wherein the alkyl group is an alkoxyalkyl group.

45. A 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 used for preparing a drug-linker compound, the drug-linker compound being prepared by the following steps: 【Chemistry 29】 In the formula, B １ , B ２ , Y １ , Y ２ , Y ３ , Z ２ , J ２ , W ２ , V ２ , L, E, D are defined in any one of claims 5 to 18, 22 to 26, and LG ２ Is J ２ The group is selected from groups that undergo a condensation reaction with

46. The use of claim 45, wherein the drug-linker is selected from the compounds of any one of claims 22 to 26, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides or isotopically labeled compounds thereof.

47. LG ２ is -COOH or -NH(R ２ ) wherein R ２ is H, C １－６ Alkyl group or C ２－６ The use according to claim 45, which is an alkoxyalkyl group.

48. Use of the compound according to any one of claims 1 to 4 for the preparation of a biologically active conjugate, The compound according to any one of claims 1 to 4 is used for preparing a bioactive conjugate, wherein the bioactive conjugate is prepared by the following steps 1a and 1b: Step 1a: 【Transformation 30】 Step 1b: 【Chemistry 31】 In the formula, X, Y, A, Q, Z １ , J １ , W １ , V １ , L, E, D, Ab and n are defined in any one of claims 1 to 4, 20, 21, 27, 29 to 31, and LG １ Is J １ The group is selected from groups that undergo a condensation reaction with

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

50. LG １ is -COOH or -NH(R １ ) wherein R １ is H, C １－６ Alkyl group or C ２－６ 50. The use according to claim 48 or 49, wherein the alkyl group is an alkoxyalkyl group.

51. Use of a compound according to any one of claims 5 to 18 for the preparation of a biologically active conjugate, The compound according to any one of claims 5 to 18 is used for preparing a bioactive conjugate, wherein the bioactive conjugate is prepared by the following steps 2a and 2b: Step 2a: 【Chemistry 32】 Step 2b: 【Transformation 33】 In the formula, B １ , B ２ , Y １ , Y ２ , Y ３ , Z ２ , J ２ , W ２ , V ２ , L, E, D, Ab and n1 are defined in any one of claims 5 to 18, 22 to 26, 28, 32 to 35, and LG ２ Is J ２ The group is selected from groups that undergo a condensation reaction with

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

53. LG ２ is -COOH or -NH(R ２ ) wherein R ２ is H, C １－６ Alkyl group or C ２－６ 53. The use according to claim 51 or 52, wherein the alkyl group is an alkoxyalkyl group.

54. 1. A method of preparing a compound, said method comprising the steps of: 【Transformation 34】 In the formula, X, Y, A, Q, Z １ , J １ , W １ , V １ , L, E, D are defined in any one of claims 1 to 4, 20 and 21, and LG １ Is J １ The group is selected from groups that undergo a condensation reaction with

55. LG １ is -COOH or -NH(R １ ) wherein R １ is H, C １－６ Alkyl group or C ２－６ 55. The method of claim 54, wherein the alkyl group is an alkoxyalkyl group.

56. 1. A method of preparing a compound, said method comprising the steps of: 【Chemistry 35】 In the formula, B １ , B ２ , Y １ , Y ２ , Y ３ , Z ２ , J ２ , W ２ , V ２ , L, E, D are defined in any one of claims 5 to 18, 22 to 26, and LG ２ Is J ２ The group is selected from groups that undergo a condensation reaction with

57. LG ２ is -COOH or -NH(R ２ ) wherein R ２ is H, C １－６ Alkyl group or C ２－６ 57. The method of claim 56, wherein the group is an alkoxyalkyl group.

58. 1. A method for preparing an antibody-drug conjugate, the method comprising the steps of: Step 1a: 【Transformation 36】 Step 1b: 【Chemistry 37】 In the formula, X, Y, A, Q, Z １ , J １ , W １ , V １ , L, E, D, Ab and n are defined in any one of claims 1 to 4, 20, 21, 27, 29 to 31, and LG １ Is J １ The group is selected from groups that undergo a condensation reaction with

59. LG １ is -COOH or -NH(R １ ) wherein R １ is H, C １－６ Alkyl group or C ２－６ 59. The method of claim 58, wherein the group is an alkoxyalkyl group.

60. 1. A method for preparing an antibody-drug conjugate, the method comprising the steps of: Step 2a: 【Transformation 38】 Step 2b: 【Chemistry 39】 In the formula, B １ , B ２ , Y １ , Y ２ , Y ３ , Z ２ , J ２ , W ２ , V ２ , L, E, D, Ab and n1 are defined in any one of claims 5 to 18, 22 to 26, 28, 32 to 35, and LG ２ Is J ２ The group is selected from groups that undergo a condensation reaction with

61. LG ２ is -COOH or -NH(R ２ ) wherein R ２ is H, C １－６ Alkyl group or C ２－６ 61. The method of claim 60, wherein the group is an alkoxyalkyl group.

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