Heterocyclic compound, method for preparing same, and use thereof

Abstract

The present invention relates to the field of pharmaceutical biology, and particularly relates to a heterocyclic compound, a method for preparing same, and use thereof. More particularly, the present invention relates to a pyrrolo-benzodiazepine compound, a drug linker thereof, a drug conjugate thereof, or a pharmaceutically acceptable salt, an ester, a stereoisomer, a polymorph, a solvate, a nitrogen oxide, an isotopically labeled form, a metabolite or prodrug, or a pharmaceutical composition thereof, a method for preparing same, and use thereof as a medicament for treating diseases associated with abnormal cell proliferation.

Description

Heterocyclic compounds, their preparation methods and uses

[0001] This application is based on and claims priority to Chinese patent applications No. 202411654443.2 filed on November 19, 2024, and No. 202411989594.3 filed on December 31, 2024, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the pharmaceutical field, specifically to a pyrrolobenzodiazepine with antitumor activity. Class of compounds, their preparation methods, and uses. Background Technology

[0003] In recent years, antibody-drug conjugates (ADCs) have become a hot research topic in the pharmaceutical field due to their ability to target and kill tumor cells. ADCs typically consist of a monoclonal antibody, a bioactive molecule primarily composed of a tumor-killing cytotoxic agent, and a linker. The bioactive molecule is covalently coupled to the antibody via the linker; the antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells, and then allowing the ADC to enter the cancer cells through endocytosis; subsequently, the bioactive molecule is released within the cancer cells, and through actions such as inhibiting microtubule proteins or damaging cancer cell DNA, it achieves the goal of killing cancer cells while minimizing damage to normal tissue cells.

[0004] Pyrrolobenzodiazepine Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor groove binders and members of the anthraxmycin family of antibiotics. Scientists have discovered that PBDs bind two pyrrolobenzodiazepines via alkylene chains. Units connected together can make pyrrolobenzodiazepine The dimer forms highly lethal interchain crosslinks or alkylation with DNA. Furthermore, this pyrrolobenzodiazepine... The activity of the dimer can reach pmol / L in various tumor cell lines.

[0005] However, currently, pyrrolobenzodiazepines are used... There are still many problems with using PBD-like compounds as antibody-drug conjugates as payloads. Therefore, there is still a need in the field to develop PBD-like drugs with ideal safety and efficacy so that they can be used in antibody-drug conjugates. Summary of the Invention

[0006] The present invention aims to provide a class of novel heterocyclic compounds that exhibit good tumor cell proliferation inhibition ability. When conjugated with antibodies, the compounds have good anti-tumor activity and good safety, and are expected to be used to treat diseases such as solid tumors.

[0007] compound

[0008] On the one hand, this application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the structure shown in formula (I):

[0009] in,

[0010] R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally and independently selected from H, deuterium (D), halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; or R 1 and R 2 It is linked to adjacent atoms to form a ring, wherein the ring is optionally selected by one or more atoms independently selected from H, deuterium (D), halogen, -OH, -NH2, C 1- 6-alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0011] X is selected from C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl (e.g., C 1-6 hydroxyalkyl), amino, aminoalkyl (e.g., C 1-6 Aminoalkyl), C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group, C 1-6 Amino-amide group, C 2-6 alkenyl, C 2-6 Alkyne group, substituted or unsubstituted C 3-10 Cycloalkyl, substituted or unsubstituted 3-10 membered heterocyclic groups, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of 5-10 heteroaryl groups, both substituted and unsubstituted.

[0012] In some implementation schemes, R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected independently by one or more of H, deuterium (D), halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; or R 1 and R 2 It is linked to adjacent atoms to form a ring, wherein the ring is optionally selected by one or more atoms independently selected from H, deuterium (D), halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0013] X is selected from C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl, amino, aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 amine alkyl-amide group, C 1-6 Amine alkoxy-amide group, C 1-6 Amino-amino-amide group, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0014] In some implementation schemes, R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 cycloalkyl; the C 1-6 Alkyl and C 3-6 The cycloalkyl group may be optionally separated by one or more elements independently selected from H, deuterium (D), halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2- 6-acetylinyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups.

[0015] In some implementations, the R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 Cycloalkyl.

[0016] In some implementations, the R 1 and R 2 Each is independently selected from hydrogen, methyl, and cyclopropyl.

[0017] In some implementations, the R 1 It is methyl.

[0018] In some implementations, the R 2 It is methyl or cyclopropyl.

[0019] In some implementations, the R 2 It is methyl.

[0020] In some implementations, the R 1 and R 2 It is linked with adjacent atoms to form a 5-6 member oxygen-containing heterocycle, wherein the ring is optionally selected by one or more independently chosen from H, deuterium (D), halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The substituents are aryl and 5-10 heteroaryl, preferably substituted by one or more substituents independently selected from H and deuterium (D).

[0021] In some implementations, the R 1 and R 2 Connected to adjacent atoms

[0022] In some implementations, the R 1 and R 2 Connected to adjacent atoms

[0023] In some implementations, the R 1 and R 2 Connected to adjacent atoms

[0024] In some implementations, X is selected from C. 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl (e.g., C 1-6 hydroxyalkyl), amino, aminoalkyl (e.g., C1-6 Aminoalkyl), C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group, C 1-6 Amino-amide group, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of substituted or unsubstituted 5-10 heteroaryl groups.

[0025] In some implementation schemes, X is Where X A Selected from chemical bonds, C 2-6 imide and C 2-6 Idemyne ​​group, the C 2-6 imide and C 2-6 The alkynyl group is optionally selected by one or more independently chosen from H, halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups;

[0026] Ring A is selected from 3-10 member heterocyclic groups, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl groups;

[0027] R 3 Selected from H, hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1- 6-hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group;

[0028] R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups;

[0029] t can be 0, 1, 2, 3, 4 or 5.

[0030] In some implementation schemes, X A Selected from chemical bonds, C 2-6 imide and C 2-6 Alynyl group.

[0031] In some implementation schemes, X A Selected from C 2-6 imide and C 2-6 Alynyl group.

[0032] In some implementation schemes, X A Selected from C 2-6 Alkenyl groups, such as vinylene groups.

[0033] In some implementations, ring A is selected from C. 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl compounds.

[0034] In some implementations, ring A is selected from C. 6-10 Aryl and 5-10 heteroaryl compounds.

[0035] In some implementations, ring A is selected from C. 10 Aryl (i.e. naphthyl) and 5-10 heteroaryl.

[0036] In some embodiments, ring A is selected from phenyl, naphthyl, ...

[0037] In some embodiments, the X group contains a primary amino group, a secondary amino group, and / or a hydroxyl group.

[0038] In some implementation schemes, R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group.

[0039] In some implementation schemes, R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C1-6 Hydroxyalkylamine-amide group.

[0040] In some implementation schemes, R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino and C 1-6 Aminoalkyl.

[0041] In some implementation schemes, R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl and amino.

[0042] In some implementation schemes, R 3 Selected from hydroxyl, hydroxymethyl and amino.

[0043] In some implementation schemes, R 3 Selected from C 1-6 Hydroxyalkyl and amino.

[0044] In some implementation schemes, R 3 Selected from hydroxymethyl and amino.

[0045] In some implementation schemes, R 3 It is an amino group.

[0046] In some implementation schemes, R 4 Each is independently selected from H, halogens, and C. 1-6 alkyl.

[0047] In some implementation schemes, R 4 Each is independently selected from H and halogens.

[0048] In some implementation schemes, R 4 For H.

[0049] In some implementations, t is 0, 1, or 2.

[0050] In some implementation schemes, X is Where X A Selected from C 2-6 imide and C 2-6 Ethyne group; ring A is selected from 3-10 membered heterocyclic groups, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2- 6-olefin, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t is 0, 1, 2, 3, 4 or 5.

[0051] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl group; ring A is selected from C 6-10 Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0052] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl group; ring A is selected from C 6-10 Aryl (e.g., phenyl or naphthyl); R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0053] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl group; ring A is selected from 5-10 membered heteroaryl groups; R 3 Selected from H, hydroxyl, C1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0054] In some implementation schemes, X is Where X A Selected from C 2-6 imide and C 2-6 Ethyne group; ring A is selected from 3-10 membered heterocyclic groups, C 3-10 cycloalkyl, C 10 Aryl and 5-10 heteroaryl groups; R 3 Selected from H, hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2- 6-olefin, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t is 0, 1, 2, 3, 4 or 5.

[0055] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl group; ring A is selected from C 10 Aryl and 5-10 heteroaryl groups; R 3 Selected from H, hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group; R 4 Each is independently selected from H, halogens, and C.1-6 Alkyl; t is 0, 1 or 2.

[0056] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl; ring A is phenylene; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0057] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl; ring A is phenylene; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group; R 4 H is a variable; t is 0, 1, or 2.

[0058] In some implementation schemes, X is Where X A Selected from C 2-6 alkenyl; ring A is phenylene; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino and C 1-6 Aminoalkyl; R 4 H is a variable; t is 0, 1, or 2.

[0059] In some implementation schemes, X is Where X A It is vinylene; ring A is phenylene; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino and C 1-6 Aminoalkyl; R 4 H is a variable; t is 0, 1, or 2.

[0060] In some implementation schemes, X is Where X A When it is a chemical bond, ring A is selected from 3-10 membered heterocyclic groups, C 3-10 cycloalkyl, C 10Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t is 0, 1, 2, 3, 4 or 5.

[0061] In some implementation schemes, X is Where X A When it is a chemical bond, ring A is selected from C. 10 Aryl and 5-10 membered heteroaryl groups (e.g., 9-10 membered heteroaryl groups, such as quinolinyl); R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0062] In some implementation schemes, X is X A For chemical bonds, ring A is selected from naphthyl and 5-10 membered heteroaryl groups (e.g., quinolinyl); R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

[0063] In some implementation schemes, X is X A For chemical bonds, ring A is selected from naphthyl and 5-10 membered heteroaryl groups (e.g., quinolinyl); R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 H is a variable; t is 0, 1, or 2.

[0064] In some implementation schemes, X is X A For chemical bonds, ring A is selected from naphthyl and quinolinyl; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino and C 1-6 Aminoalkyl; R 4 H is a variable; t is 0, 1, or 2.

[0065] In some implementation schemes, X is X A For chemical bonds, ring A is selected from naphthyl and quinolinyl; R 3 Selected from hydroxyl, hydroxymethyl, amino, and aminomethyl; R 4 H is a variable; t is 0, 1, or 2.

[0066] In some implementations, X is selected from

[0067] In some implementations, X is selected from

[0068] In some implementations, X is selected from

[0069] In some implementations, X is selected from

[0070] In some implementations, X is selected from

[0071] In some implementations, X is selected from

[0072] In some implementation schemes, X is

[0073] In some implementation schemes, X is

[0074] In some implementation schemes, X is

[0075] On the other hand, this application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the structure shown in formula (II):

[0076] Among them, R 1 R 2 X is as defined in equation (I) above.

[0077] In some embodiments, the carbon atom to which the hydroxyl group is attached in formula (II) has an R or S configuration.

[0078] In some implementations, the carbon atom to which the hydroxyl group is attached in formula (II) has an S configuration.

[0079] On the other hand, this application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the structure shown in formula (III):

[0080] Among them, R 1 R 2 X is as defined in equation (I) above;

[0081] M is selected from H and alkali metals.

[0082] In some implementations, the carbon atom attached to -SO3M in formula (III) has an R or S configuration.

[0083] In some implementations, the carbon atom attached to -SO3M in formula (III) has an S configuration.

[0084] In some implementations, M is selected from hydrogen, sodium, and potassium.

[0085] In some implementations, M is hydrogen.

[0086] On the other hand, this application provides the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, or prodrugs, wherein the compounds have the following structures:

[0087] In some embodiments, the carbon atoms connected to the hydroxyl, sulfonic acid, or sodium sulfonate groups in P-2 to P-4, P-6 to P-8, P-10 to P-12, P-14 to P-16, P-18 to P-20, P-22 to P-24, P-26 to P-28, P-30 to P-32, P-34 to P-36, and P-38 to P-40 have an R configuration.

[0088] In some embodiments, the carbon atoms connected to the hydroxyl, sulfonic acid, or sodium sulfonate groups in P-2 to P-4, P-6 to P-8, P-10 to P-12, P-14 to P-16, P-18 to P-20, P-22 to P-24, P-26 to P-28, P-30 to P-32, P-34 to P-36, and P-38 to P-40 are in the S configuration.

[0089] In some implementations, the carbon atom to which the hydroxyl group is attached in P-2 or P-6 has an S configuration.

[0090] On the other hand, this application provides drug linker compounds or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrides, isotope labels, metabolites, or prodrugs thereof, said compounds having the structure shown in formula (IV):

[0091] in,

[0092] R 1 and R 2 As defined above;

[0093] X' is the divalent structure formed after removing a hydrogen atom from the X group as defined above;

[0094] Y is selected from single bonds, -CH2NH-, and self-elimination structures;

[0095] Z is a structure connecting Y and CM;

[0096] CM is selected from structures that can react with specific functional groups in the target region (such as antibodies or their antigen-binding fragments).

[0097] In some implementation schemes, R 1 and R 2 As defined above;

[0098] X' is the divalent structure formed after removing a hydrogen atom from the X group as defined above;

[0099] Y is selected from a self-elimination structure;

[0100] Z is a structure connecting Y and CM;

[0101] CM is selected from structures that can react with specific functional groups in the target region (such as antibodies).

[0102] In some implementations, X' is a divalent structure formed by removing a hydrogen atom from the primary amino, secondary amino, or hydroxyl group of the X group as defined above.

[0103] In some implementations, X' is Where X A Ring A, R 4 And t as defined above; R 3A R as defined above 3 A divalent structure formed after a group loses a hydrogen atom.

[0104] In some implementations of this case, R 3A Selected from chemical bonds, -O-, -C 1-6 Alkylenes -O-, -NH-, -C 1-6 Alkylene -NH-, -NH-C(=O)-C 1-6 Alkylene -O-, -NH-C(=O)-OC 1-6 Alkylene-O-, -NH-C(=O)-NC 1-6 Alkylene -O-, -NH-C(=O)-C 1-6 Alkylene -NH-, -NH-C(=O)-OC 1-6 Alkylenes -NH- and -NH-C(=O)-NC 1-6 Alkylene-NH-.

[0105] In some implementations of this case, R 3A Selected from -O-, -C 1-6 Alkylenes -O-, -NH-, -C 1-6 Alkylene -NH-, -NH-C(=O)-C 1-6 Alkylene -O-, -NH-C(=O)-OC 1-6 Alkylene-O-, -NH-C(=O)-NC 1-6 Alkylene -O-, -NH-C(=O)-C 1-6 Alkylene -NH-, -NH-C(=O)-OC 1-6 Alkylenes -NH- and -NH-C(=O)-NC 1-6 Alkylene-NH-.

[0106] In some implementations of this case, R 3A Selected from -O-, -C 1-6 Alkylenes -O-, -NH-, -C 1-6 Alkylene -NH-, -NH-C(=O)-C 1-6 Alkylene -O-, -NH-C(=O)-OC 1-6 Alkylene-O- and -NH-C(=O)-NC 1-6 Alkylene-O-.

[0107] In some implementations of this case, R 3A Selected from -O-, -C 1-6 Alkyl-O-, -NH- and -C 1-6 Alkylene-NH-.

[0108] In some implementations of this case, R 3A Selected from -O-, -CH2O-, -NH- and -CH2NH-.

[0109] In some implementations of this case, R 3A Selected from -O-, -C 1-6 Alkylenes -O- and -NH-.

[0110] In some implementations of this case, R 3A Selected from -O-, -CH2O- and -NH-.

[0111] In some implementations of this case, R 3A Selected from -C 1-6 Alkylenes -O- and -NH-.

[0112] In some implementations of this case, R 3A Selected from -CH2O- and -NH-.

[0113] In some implementation schemes, R 3A It is -NH-.

[0114] In some implementations, -X'- is selected from

[0115] In some implementations, -X'- is selected from

[0116] In some implementations, X' is selected from

[0117] In some implementations, X is selected from

[0118] In some implementations, X' is selected from

[0119] In some implementations, X' is selected from

[0120] In some implementations, X' is

[0121] In some implementations, X' is

[0122] In some implementations, X' is

[0123] In some implementations, Y is selected from the self-elimination structure.

[0124] In some embodiments, Y is selected from single bonds, -CH2NH-, p-aminobenzyloxycarbonyl (PABC, i.e.) ) and carbonate group (i.e. -C(=O)-O-).

[0125] In some implementations, Y in equation (IV) is a single bond.

[0126] In some embodiments, Z is selected from a structure composed of one or more of the following groups, including but not limited to C. 1-6 Alkylene, C 2- 6-eneyl, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts.

[0127] In some embodiments, Z is selected from a substituted or unsubstituted structure consisting of one or more of the following groups: C 1-6 Alkylene, C 6-10Arylidene, 5-6 membered heteroarylene, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclic groups (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Lys(R')2, Glu(R'), Lys(COCH2CH2(OCH2CH2)) r OCH3), and short peptides composed of amino acids (such as Gly-Lys, Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, A la-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu -Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:41), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:42), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:43), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:44), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:45), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:46), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:47)), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O)r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r- C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), EDTA (ethylenediaminetetraacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-EDTA and R” group, wherein the R” group is a hydrophilic structure containing hydroxyl, glycosyl, amino and carboxylic acid and its derivatives; r is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0128] In some implementations, "-EDTA" refers to

[0129] In some implementations, "-NOTA" refers to

[0130] In some implementations, "-DOTA" refers to

[0131] In some implementations, "-DOTAGA" refers to

[0132] In some implementations, Lys(R') refers to the following structure

[0133] In some implementations, Lys(R')2 refers to the following structure

[0134] In some implementations, Glu(R') refers to the following structure

[0135] In some implementations, R' is R", such as those described below.

[0136] In some implementations, R” is selected from structures containing multiple hydroxyl groups and their derivatives.

[0137] In some implementations, R” is selected from structures containing glycosyl groups and their derivatives.

[0138] In some implementations, R” is selected from structures containing amino and carboxylic acids and their derivatives.

[0139] In some implementations, R” is selected from structures containing amino groups and their derivatives.

[0140] In some implementations, R” is selected from structures containing carboxylic acids and their derivatives.

[0141] In some implementations, "R" refers to the following structure: Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0142] In some implementations, "R" refers to the following structure: Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0143] In some implementations, "R" refers to the following structure:

[0144] In some implementations, Z is selected from short peptides composed of amino acids.

[0145] In some implementations, Z is selected from Ala-Val, Cit-Val, Lys-Val, Lys(R')-Val, Gly-Lys-Val, Ala-Val-Glu, and Ala-Val-Glu(R');

[0146] R' is selected from Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0147] Preferably, R' is selected from (For example ), (For example ), (For example )and Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0148] In some implementations, Z is selected from Ala-Val, Cit-Val, Lys-Val, and Gly-Lys-Val.

[0149] In some embodiments, CM is selected from functional groups or structures that can react with specific functional groups (e.g., cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs) contained in the targeting portion (e.g., antibody or its antigen-binding fragment).

[0150] In some embodiments, CM is selected from haloalkyl acyl, substituted or unsubstituted maleimide, alkylsulfonyl heteroaryl, and fluorophenol ester.

[0151] In some embodiments, CM is selected from iodoacetyl, maleimide, bromomaleimide, thiomaleimide, alkylsulfonylpyrimidinyl, and fluorophenol ester.

[0152] In some implementations, CM is selected from the following substituted or unsubstituted structures: (For example ), (For example ), (For example ), (For example ), Where R a Selected from hydroxyl, cyano, amino, halogen, nitro, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 1-6 Halogenated alkyl and C 1-6 Alkyl-C(=O)-; Lg is a leaving group for nucleophilic substitution reactions, such as those selected from halogens (e.g., F, Cl, Br, or I), halogenated C 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structures, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structures may optionally be replaced by one or more suitable substituents.

[0153] In some implementation schemes, R a Selected from halogen, cyano, nitro, C 2-6 alkynyl group, C 1-6 Halogenated alkyl and C1-6 Alkyl-C(=O)-.

[0154] In some implementation schemes, R a It is a cyano group.

[0155] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, or I), halogenated C 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structures.

[0156] In some implementations, Lg is selected from halogens, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH2).

[0157] In some implementations, Lg is selected from halogens, C 1-6 Alkyl sulfonyl and halophenoxy groups.

[0158] In some implementations, Lg is selected from C 1-6 Alkyl sulfonyl, such as methyl sulfonyl.

[0159] In some implementations, CM is selected from the following substituted or unsubstituted structures:

[0160] In some implementations, CM is selected from the following substituted or unsubstituted structures:

[0161] In some implementation schemes, CM is For example

[0162] In some embodiments, the drug linker compound is as follows:

[0163] DL-1:

[0164] DL-2:

[0165] DL-3:

[0166] DL-4:

[0167] DL-5:

[0168] DL-6:

[0169] On the other hand, this application provides drug linker compounds or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, or prodrugs thereof, said compounds having the structure shown in formula (V):

[0170] in,

[0171] R 1 R 2 And X is as defined in equation (I) above;

[0172] Y, Z and CM are as defined in formula (IV) above.

[0173] In some implementations, the carbon atom to which the hydroxyl group is attached in formula (V) has an R or S configuration.

[0174] In some implementations, the carbon atom to which the hydroxyl group is attached in formula (V) has an S configuration.

[0175] In some embodiments, Y in formula (V) is -CH2NH- or p-aminobenzyloxycarbonyl (PABC).

[0176] In some implementations, Y in formula (V) is p-aminobenzyloxycarbonyl (PABC).

[0177] In some embodiments, the drug linker compound is as follows:

[0178] PL-1:

[0179] PL-2:

[0180] PL-3:

[0181] PL-4:

[0182] PL-5:

[0183] PL-6:

[0184] PL-7:

[0185] PL-8:

[0186] PL-9:

[0187] PL-10:

[0188] PL-11:

[0189] PL-12:

[0190] PL-13:

[0191] PL-14:

[0192] On the other hand, this application provides the drug conjugate of formula (VI) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug:

[0193] in,

[0194] R 1 R 2 X', Y and Z are as defined above;

[0195] n is selected from 1-10;

[0196] A' represents the target region;

[0197] CM' is selected from the structure obtained by reacting with a specific functional group (such as cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs) contained in the target moiety (such as an antibody or its antigen-binding fragment).

[0198] In some implementations, CM' is the functional group or structure of CM after Lg removal as defined above, or the functional group or structure of CM after an addition reaction as defined above.

[0199] In some implementations, CM' is selected from C 1-6 Ionyl, substituted or unsubstituted succinimide, substituted or unsubstituted heteroaryl and 3-6 membered heterocyclic acyl groups.

[0200] In some embodiments, CM' is selected from acyl, succinimide, substituted or unsubstituted pyrimidinyl and piperidinyl.

[0201] In some implementations, CM' is selected from the following substituted or unsubstituted structures: (For example ), (For example ), (For example ), (For example ), Where R a As defined above.

[0202] In some implementation schemes, CM' is

[0203] In some implementations, A' is selected from antibodies targeting tumor antigens or their antigen-binding fragments, peptides, or small molecule fragments.

[0204] In some implementations, A' is selected from antibodies targeting tumor antigens or their antigen-binding fragments, such as monoclonal or bispecific antibodies targeting Her2, Her3, EGFR, TROP2, B7H3, c-Met, CEACAM5, CLDN18.2, FRa, CDH6, CDH3, PTK7, DLL3, or GPC3.

[0205] In some implementations, A' is selected from antibodies targeting Her2 or their antigen-binding fragments, such as trastuzumab antibody or patocilizumab antibody.

[0206] In some implementations, A' is selected from peptides that target tumor antigens, such as somatostatin analogs, GnRH / LHRH analogs, vascular peptide-2, heptaarginine, TAT47-57, DPV1047 Vectocell peptide, peptides that target EphA2, GPC3, or Nectin-4, etc.

[0207] In some implementations, A' is selected from small molecule fragments that target tumor antigens, such as N-acetylgalactosamine (GalNAc) fragments, bisphosphonate fragments with bone targeting, fragments that selectively bind to prostate-specific membrane antigen (PSMA), fragments that selectively bind to somatostatin receptors, etc.

[0208] In some embodiments, when A' is an antibody or its antigen-binding fragment, the drug conjugate is an antibody-drug conjugate (ADC).

[0209] In some implementation schemes, for Wherein A-[Q- is the targeting portion, preferably an antibody or its antigen-binding fragment; Q is the portion in the targeting portion where an amino acid residue is linked to CM'.

[0210] In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

[0211] In some implementations, Q is S, NH, or O.

[0212] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment targeting Her2.

[0213] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0214] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0215] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0216] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0217] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0218] or,

[0219] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0220] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0221] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0222] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0223] or,

[0224] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0225] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0226] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0227] Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0228] or,

[0229] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0230] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0231] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0232] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0233] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0234] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0235] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0236] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0237] or,

[0238] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0239] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0240] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0241] or,

[0242] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0243] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0244] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0245] or,

[0246] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0247] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

[0248] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0249] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0250] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0251] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0252] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0253] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0254] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0255] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0256] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0257] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0258] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0259] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0260] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0261] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0262] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0263] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0264] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0265] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0266] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0267] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0268] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0269] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or may lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0270] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0271] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0272] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0273] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0274] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

[0275] In some embodiments, the antibody or its antigen-binding fragment is selected from antibodies or their antigen-binding fragments that specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0276] In some implementations, the antibody or its antigen-binding fragment is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or its antigen-binding fragment constructed from trastuzumab and pertuzumab.

[0277] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0278] Another aspect of the present invention provides drug conjugates as shown below:

[0279] A-DL-1:

[0280] A-DL-2:

[0281] A-DL-3:

[0282] A-DL-4:

[0283] A-DL-5:

[0284] A-DL-6:

[0285] Where A-(S) is the target portion defined above, preferably the antibody or its antigen-binding fragment defined above; This indicates the specific connection method between the thiol group and the pyrimidine group in the targeted portion.

[0286] On the other hand, this application provides the drug conjugate of formula (VII) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug:

[0287] in,

[0288] R 1 R 2 The definitions of X, Y, Z, CM', A' and n are as described above.

[0289] In some implementations, the carbon atom to which the hydroxyl group is attached in formula (VII) has an R or S configuration.

[0290] In some implementations, the carbon atom to which the hydroxyl group is attached in formula (VII) has an S configuration.

[0291] In some implementations, Y is defined as in equation (V).

[0292] In some implementation schemes, for A and Q are as defined above.

[0293] Another aspect of the present invention provides drug conjugates as shown below:

[0294] A-PL-1:

[0295] A-PL-2:

[0296] A-PL-3:

[0297] A-PL-4:

[0298] A-PL-5:

[0299] A-PL-6:

[0300] A-PL-7:

[0301] A-PL-8:

[0302] A-PL-9:

[0303] A-PL-10:

[0304] A-PL-11:

[0305] A-PL-12:

[0306] A-PL-13:

[0307] A-PL-14:

[0308] Where A-(S) is the target portion defined above, preferably the antibody or its antigen-binding fragment defined above; This indicates the specific connection method between the thiol group and the pyrimidine group in the targeted portion.

[0309] All technical features disclosed in this specification, except for mutually exclusive features, can be combined in any way. This invention covers compounds and conjugates obtained by arbitrary combinations of various embodiments, wherein the compounds and conjugates of this invention may be optionally substituted with suitable substituents at suitable substitution positions.

[0310] intermediate

[0311] In some embodiments, this application provides intermediate compounds having the following structures, or salts, esters, stereoisomers, polymorphs, solvates, nitrides, or isotopically labeled compounds thereof:

[0312] Where R 1 R 2 Lg, X, and X' are as defined above;

[0313] PG 1 Each group is independently an H or amino protecting group. The amino protecting group can be an alkoxycarbonyl group, such as benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); an acyl group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), pentanoyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; or an alkyl group, such as triphenylmethyl (Trt), C 1-6 Alkyl-substituted triphenylmethyl, p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn), or (trimethylsilyl)ethoxymethyl (SEM);

[0314] PG 2 Each is independently an H or hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, triphenylmethyl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, trifluoromethanesulfonyl (Tf), or p-nitrobenzoyl.

[0315] PG 3Each is an independent H or carboxyl protecting group, and the carboxyl protecting groups are, for example, each independently selected from C. 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl.

[0316] In some embodiments, this application provides intermediate compounds having the following structures, or salts, esters, stereoisomers, polymorphs, solvates, nitrides, or isotopically labeled compounds thereof:

[0317] In another aspect, this application provides the use of the intermediate compound as described above, or its salt, stereoisomer, or isotopically labeled compound, in the preparation of the compound of the present invention, drug conjugate, or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotopically labeled compound, metabolite, or prodrug.

[0318] Connector

[0319] On the other hand, this application provides a linker structure as shown in -YZ-CM'-, wherein Y can be linked to a bioactive molecule, and CM' can be linked to A', wherein Y, Z, CM' and A' are as described in any of the preceding statements, and the bioactive molecule can be a compound as shown in formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug.

[0320] In some implementations, the connector structure is as follows:

[0321] It is connected to the bioactive molecule through position 1 and to A' through position 2, and the bioactive molecule and A' are as defined in any of the preceding terms.

[0322] On the other hand, this application provides drug linker structures represented by DY-, DYZ-, and DYZ-CM'-, wherein Y, Z, and CM' are as described in any of the preceding descriptions, and D is a bioactive molecule portion, such as a compound of formula (I), (II), or (III) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug. In some embodiments, D is the structure formed by removing a hydrogen atom from the X group of the bioactive molecule.

[0323] Preparation method

[0324] In some embodiments, a method for preparing a compound of formula (IA) includes one or more of the following steps:

[0325] Step 1: React the compound of formula (IA-1) with the compound of formula (IA-2) to generate the compound of formula (IA-3);

[0326] Step two involves reducing the compound of formula (IA-3) to produce the compound of formula (IA).

[0327] Among them, R 1 R 2 R 3 R 4 X A , ring A, t, PG 1 and PG 2 As defined in any of the preceding implementation schemes;

[0328] Q is a hydrogen, boric acid, or borate ester group (e.g., ).

[0329] In some embodiments, step one is carried out in a suitable solvent in the presence of a suitable catalyst and a base. In some embodiments, the suitable solvent is selected from toluene, methanol, ethanol, 1,4-dioxane, water, and any combination thereof, such as a mixture of toluene, ethanol, and water, or a mixture of 1,4-dioxane and water. In some embodiments, the suitable catalyst is selected from tetrakis(triphenylphosphine)palladium, tris(dibenzylacetone)dipalladium, Pd(dppf)₂Cl₂, palladium acetate, BINAP, BrettPhos Pd G₃, cataCXium A Pd G₃, and any combination thereof. In some embodiments, the base is selected from sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, DBU, potassium phosphate, sodium tert-butoxide, and any combination thereof. In some embodiments, the reaction in step one is carried out at 0-150°C for 0.5-24 hours.

[0330] In some embodiments, step two is carried out in a suitable solvent in the presence of a suitable reducing agent. In some embodiments, the suitable solvent is selected from tetrahydrofuran, dichloromethane, chloroform, toluene, and any combination thereof, such as tetrahydrofuran. In some embodiments, the suitable reducing agent is selected from lithium triethylborohydride, sodium borohydride, potassium borohydride, lithium tri-tert-butoxyaluminum hydride, and any combination thereof. In some embodiments, the reaction in step two is carried out at low temperatures, for example from -100°C to 25°C, for 10 minutes to 24 hours.

[0331] Composition

[0332] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug linker as described herein, and n independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug linkers. Therefore, the compositions are characterized by a drug-to-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0333] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 To 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0334] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 5.5, about 3.0 to 6.0, about 3.0 to 6.5, about 3.0 to 7.0, about 3.0 to 7.5, about 3.0 to 8.0, about 3.0 to 8.5, about 3.0 to 9.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, and about 3.5 to 6.0. Approximately 3.5 to 6.5, approximately 3.5 to 7.0, approximately 3.5 to 7.5, approximately 3.5 to 8.0, approximately 3.5 to 8.5, approximately 3.5 to 9.0, approximately 4.0 to 4.5, approximately 4.0 to 5.0, approximately 4.0 to 5.5, approximately 4.0 to 6.0, approximately 4.0 to 6.5, approximately 4.0 to 7.0, approximately 4.0 to 7.5, approximately 4.0 to 8.0, approximately 4.0 to 8.5, approximately 4.0 to 9.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4 .5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 4.5 to 8.5, approximately 4.5 to 9.0, approximately 5.0 to 5.5, approximately 5.0 to 6.0, approximately 5.0 to 6.5, approximately 5.0 to 7.0, approximately 5.0 to 7.5, approximately 5.0 to 8.0, approximately 5.0 to 8.5, approximately 5.0 to 9.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 5.5 to 8.5, approximately 5.5 to 9.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.0 to 8.5, approximately 6.0 to 9.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 6.5 to 8.5, approximately 6.5 to 9.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.0 to 8.5, approximately 7.0 to 9.0, approximately 7.5 to 8.0, approximately 7.5 to 8.5, approximately 7.5 to 9.0, approximately 8.0 to 8.5, approximately 8.0 to 9.0, approximately 8.5 to 9.0.

[0335] In some embodiments, the DAR of the ADC composition described herein is about 5.0 to 9.0, preferably about 5.0-9.0, for example about 5.0, about 5.01, about 5.02, about 5.03, about 5.04, about 5.05, about 5.06, about 5.07, about 5.08, about 5.09, about 5.1, about 5.11, about 5.12, about 5.13, about 5.14, about 5.15, about 5.16, about 5.17, about 5.18, about 5.19, about 5.2, about 5.21, about 5.22, about 5.23, about 5.24, about 5.25, about 5.26, about 5.27, about 5.28, about 5.29, about 5.3, about 5.31, about 5.32, about 5.33, about 5. .34, about 5.35, about 5.36, about 5.37, about 5.38, about 5.39, about 5.4, about 5.41, about 5.42, about 5.43, about 5.44, about 5.45, about 5.46, about 5.47, about 5.48, about 5.49, about 5.5, about 5.51, about 5.52, about 5.53, about 5.54, about 5. 55, approximately 5.56, approximately 5.57, approximately 5.58, approximately 5.59, approximately 5.6, approximately 5.61, approximately 5.62, approximately 5.63, approximately 5.64, approximately 5.65, approximately 5.66, approximately 5.67, approximately 5.68, approximately 5.69, approximately 5.7, approximately 5.71, approximately 5.72, approximately 5.73, approximately 5.74, approximately 5.75, approximately 5.7 6. Approximately 5.77, 5.78, 5.79, 5.8, 5.81, 5.82, 5.83, 5.84, 5.85, 5.86, 5.87, 5.88, 5.89, 5.9, 5.91, 5.92, 5.93, 5.94, 5.95, 5.96, 5.97 Approximately 5.98, 5.99, 6.0, 6.01, 6.02, 6.03, 6.04, 6.05, 6.06, 6.07, 6.08, 6.09, 6.1, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18 Approximately June 19, June 2, June 21, June 22, June 23, June 24, June 25, June 26, June 27, June 28, June 29, June 3, June 31, June 32, June 33, June 34, June 35, June 36, June 37, June 38, June 39, approximately 6.4, approximately 6.41, approximately 6.42, approximately 6.43, approximately 6.44, approximately 6.45, approximately 6.46, approximately 6.47, approximately 6.48, approximately 6.49, approximately 6.5, approximately 6.51, approximately 6.52, approximately 6.53, approximately 6.54, approximately 6.55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.76, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6. 82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.0, approximately 7.01, approximately 7.02, approximately 7.0 3. Approximately 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.1, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 7.2, 7.21, 7.22, 7.23, 7.24 Approximately 7.25, 7.26, 7.27, 7.28, 7.29, 7.3, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38, 7.39, 7.4, 7.41, 7.42, 7.43, 7.44, 7.45. Approximately 7.46, 7.47, 7.48, 7.49, 7.5, 7.51, 7.52, 7.53, 7.54, 7.55, 7.56, 7.57, 7.58, 7.59, 7.6, 7.61, 7.62, 7.63, 7.64, 7.65, 7.66. Approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0, approximately 8.01, approximately 8.02, approximately 8.03, approximately 8.04, approximately 8.05, approximately 8.06, approximately 8.07, approximately 8.08, approximately 8 .09, approximately 8.1, approximately 8.11, approximately 8.12, approximately 8.13, approximately 8.14, approximately 8.15, approximately 8.16, approximately 8.17, approximately 8.18, approximately 8.19, approximately 8.2, approximately 8.21, approximately 8.22, approximately 8.23, approximately 8.24, approximately 8.25, approximately 8.26, approximately 8.27, approximately 8.28, approximately 8.29, approximately 8.3. Approximately 8.31, 8.32, 8.33, 8.34, 8.35, 8.36, 8.37, 8.38, 8.39, 8.4, 8.41, 8.42, 8.43, 8.44, 8.45, 8.46, 8.47, 8.48, 8.49, 8.5, 8.51, 8.52, 8.53, 8.54, 8.55, 8.56, 8.57, 8.58, 8.59, 8.6, 8.61, 8.62, 8.63, 8.64, 8.6 5. Approximately 8.66, 8.67, 8.68, 8.69, 8.7, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.0.

[0336] In some embodiments, the composition comprises a drug conjugate as shown in formula (VI) or (VII), wherein A' is an antibody or an antigen-binding fragment thereof. In some embodiments, the DAR of the composition is 1-8, for example 5-8, such as about 5.47. In some embodiments, the DAR of the composition is 1-9, for example 5-9, such as about 5.11, about 5.47, about 5.80, about 6.22, about 6.25, about 6.45, about 6.90, about 7.08, about 7.36, about 7.61, about 7.85, about 8.0, about 8.08, about 8.12, or about 8.15. In some embodiments, the antibody-drug conjugate with x being 8 constitutes more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%.

[0337] Pharmaceutical Composition

[0338] In another aspect, the present invention provides a pharmaceutical composition comprising the compound described herein, a pharmaceutical conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug, and one or more pharmaceutically acceptable carriers.

[0339] The term "pharmaceutical composition" refers to a composition that can be used as a medicine, comprising a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers. The term "pharmaceuticalally acceptable carrier" refers to an excipient administered co-administered with the therapeutic agent, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0340] The above-described pharmaceutical compositions can act systemically and / or locally, which can be achieved through suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0341] The above-mentioned pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one of the compounds of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof.

[0342] The present invention also provides a method for preparing the above-described pharmaceutical composition or its corresponding formulation, comprising combining at least one compound of the present invention, a pharmaceutical conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug with one or more pharmaceutically acceptable carriers.

[0343] Pillbox products

[0344] In another aspect, the present invention provides a medicine box comprising:

[0345] a) at least one compound of the present invention, a drug conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, or a pharmaceutical composition as a first therapeutic agent;

[0346] b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a pharmaceutical composition comprising another therapeutic agent as a second pharmaceutical composition; and

[0347] c) Optional packaging and / or instructions.

[0348] The aforementioned kit products may contain 0.01 mg to 1000 mg of at least one compound of the present invention, a drug conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug.

[0349] The present invention also provides a method for preparing the above-mentioned medicine box, which includes combining at least one compound of the present invention, a drug conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug or the above-mentioned pharmaceutical composition with at least one other therapeutic agent or a pharmaceutical composition containing other therapeutic agents, packaging and / or instructions.

[0350] Medical Use

[0351] The compounds of formula (I), formula (II) or formula (III) provided in this application can be used to prepare drug linker compounds of formula (IV) or formula (V), and / or to prepare drug conjugates of formula (VI) or formula (VII). The drug linker compounds of formula (IV) or formula (V) provided in this application can also be used to prepare drug conjugates of formula (VI) or formula (VII).

[0352] The compounds, drug conjugates, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs of the present invention exhibit strong inhibitory effects on abnormal cell proliferation.

[0353] This application provides the compounds, drug conjugates, or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs described herein, the pharmaceutical compositions described above, or the kit products described above, for the treatment of diseases, particularly those involving abnormal cell proliferation.

[0354] This application also provides the use of the compounds described herein or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, and prodrugs thereof, the pharmaceutical compositions described above, or the cassettes described above, in the preparation of medicaments for treating diseases involving abnormal cell proliferation.

[0355] In some implementations, the diseases involving abnormal cell proliferation include, but are not limited to, tumors, such as advanced solid tumors.

[0356] In some implementations, the disease involving abnormal cell proliferation is selected from tumors, such as advanced solid tumors.

[0357] This application also provides the use of the compounds described herein or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, and prodrugs, or the pharmaceutical compositions described above in the present invention, in the preparation of formulations for inhibiting the proliferation of tumor cells.

[0358] In some embodiments, the formulation is for in vivo or in vitro administration. For example, the formulation may be administered to a subject to inhibit the proliferation of tumor cells in the subject; or, the formulation may be administered to in vitro cells (e.g., cell lines or cells derived from the subject) to inhibit the proliferation of tumor cells in vitro.

[0359] The tumors described in this invention include, but are not limited to, or selected from: brain tumors, lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, and sarcoma.

[0360] Treatment

[0361] In another aspect, the present invention provides a method for treating a disease involving abnormal cell proliferation, comprising the steps of administering a therapeutically effective amount of the compound, drug conjugate, or pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, and prodrug, or the pharmaceutical composition described above, to an individual in need of it.

[0362] In some implementations, the diseases involving abnormal cell proliferation include, but are not limited to, tumors, such as advanced solid tumors.

[0363] In some implementations, the disease involving abnormal cell proliferation is selected from tumors, such as advanced solid tumors.

[0364] In some implementations, the method is used to inhibit the proliferation of tumor cells in a subject's body.

[0365] The tumors described in this invention include, but are not limited to, or selected from: brain tumors, lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, and sarcoma.

[0366] The term "effective dose" refers to a dose that is sufficient to induce a biological or medical response in cells, tissues, organs, or organisms (e.g., individuals) and to achieve the desired preventive and / or therapeutic effects.

[0367] The dosing regimen can be adjusted to provide the optimal required response. For example, it can be administered as a single dose, divided into doses over time, or the dose can be reduced or increased proportionally as needed. It is understood that, for any given individual, the specific dosing regimen should be adjusted as required and with the professional judgment of the person administering the composition or supervising the administration of the composition.

[0368] The dosage of the compound or drug conjugate of the present invention will depend on individual circumstances, the severity of the disease or condition, the rate of administration, the disposal of the compound, and the prescribing physician's judgment. Generally, the effective dose is about 0.001-10000 mg / kg body weight / day. Where appropriate, the effective dose is about 0.01-1000 mg / kg body weight / day. The dosage and frequency of administration may vary depending on the half-life of the drug in the subject and may also vary depending on whether it is for prophylactic or therapeutic use. In prophylactic use, a relatively low dose is administered for a long period at relatively low frequency intervals; in therapeutic use, a relatively high dose may sometimes be administered at shorter intervals until the progression of the disease is slowed or stopped, preferably until the individual shows partial or complete improvement in the symptoms of the disease, after which prophylactic use may be adopted.

[0369] The term "treatment" refers to the reduction or elimination of a targeted disease or symptom. If a subject receives a therapeutic amount of a compound of the present invention or its pharmaceutically acceptable form, or a pharmaceutical composition of the present invention, and at least one indicator and symptom of the subject shows observable and / or detectable relief and / or improvement, the subject is considered to have been successfully "treated." It is understood that treatment includes not only complete cure but also the achievement of some biological or medically relevant outcome without achieving complete cure.

[0370] The term "administrate / administrating / administration" (or "drug administration") refers to the process of applying an active pharmaceutical ingredient (such as the compound of the present invention) or a pharmaceutical composition containing an active pharmaceutical ingredient (such as the pharmaceutical composition of the present invention) to an individual or its cells, tissues, organs, biological fluids, etc., so as to bring the active pharmaceutical ingredient or pharmaceutical composition into contact with the individual or its cells, tissues, organs, biological fluids, etc. Common methods of administration include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, and vaginal administration.

[0371] The term “needs” refers to the judgment of a physician or other caregiver regarding an individual’s need for or potential benefit from preventive and / or treatment processes, which is based on various factors within the physician’s or other caregiver’s area of ​​expertise.

[0372] The term "individual" (or subject) refers to a human or non-human animal. Individuals in this invention include individuals suffering from diseases and / or conditions (patients) and healthy individuals. Non-human animals in this invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0373] Beneficial effects of the invention

[0374] The compounds and drug conjugates of the present invention have good antitumor activity and can be used to treat diseases of abnormal cell proliferation, including but not limited to advanced solid tumors. Detailed Implementation

[0375] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of the application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its fundamental ideas and scope.

[0376] definition

[0377] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0378] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0379] The term "antibody" also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0380] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0381] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0382] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0383] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0384] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0385] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0386] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0387] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:48) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:49) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0388] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0389] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0390] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0391] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0392] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0393] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to enhance the immune response. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with the intended properties (e.g., antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and / or the ability to enhance the immune response).

[0394] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0395] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0396] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0397] The term "linker" refers to a structure that connects a bioactive molecule to a target moiety (such as an antibody or its antigen-binding fragment, peptide, or small molecule fragment that targets a tumor antigen). For example, it refers to the -YZ-CM'- structural portion in the general formula described herein.

[0398] The term "drug conjugate" refers to the structure of the bioactive molecule and the conjugate before they are covalently linked to the target moiety, as described in this invention. For example, "drug conjugate" refers to the DYZ-CM moiety, where CM is the structural form of CM' before it is covalently linked to the target moiety, and D is a bioactive molecule, such as a compound represented by formula (I), (II), or (III) herein, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug. The covalent linking of the "drug conjugate" to the target moiety yields the drug conjugate described in this application.

[0399] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0400] As used herein, an asterisk (*) in a compound structural formula indicates that the labeled carbon atom is a chiral carbon atom, and the invention includes a pair of enantiomers formed from that chiral carbon atom. If a compound contains two different chiral carbon atoms, the invention includes four optical isomers formed from that chiral carbon atom.

[0401] As used in this article, This indicates the location where a structural segment connects to other parts of the molecule.

[0402] The term "alkyl" is defined as a straight-chain or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 carbon atoms. For example, as used herein, the term "C 1-6 "Alkyl" refers to a linear or branched group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl), which is optionally substituted by one or more (such as 1, 2, or 3) suitable substituents.

[0403] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkyl groups, etc. Common C 1-6 Alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention may optionally be substituted by one or more substituents described in this invention.

[0404] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxyl groups, wherein the alkyl group is defined as described above. For example, the term "C" in this invention...1- "6-hydroxyalkyl" refers to a hydroxyalkyl group having 1-6 carbon atoms, i.e., -C 1-6 alkylene -OH, for example -C 1-4 Alkylene-OH, etc. Common C 1-6 Hydroxyalkyl groups include (but are not limited to) -CH2OH, -CH2CH2OH, -CH2CH(OH)2, and -(CH2)3OH.

[0405] The term "amino" or "amine group" refers to -NH2.

[0406] The term "amide group" or "amide group" refers to -C(=O)-NH-.

[0407] The term "aminoalkyl" or "aminealkyl" refers to an alkyl group substituted with one or more amino groups, wherein the alkyl group is defined as described above. For example, the term "C" in this invention... 1-6 "Aminoalkyl" or "C" 1-6 "Aminoalkyl" refers to an aminoalkyl group having 1-6 carbon atoms, i.e., -C 1-6 Alkylene -NH2, for example -C 1-4 Alkylene-NH2, etc. Common C 1-6 Aminoalkyl groups include (but are not limited to) -CH2-NH2, -CH2CH2-NH2, -CH2CH(NH2)2, and -(CH2)3-NH2.

[0408] The term "alkylamine" refers to an amino group substituted with an alkyl group, as defined above, such as -NH-C. 1-6 Alkyl and -N(C) 1-6 Alkyl group 2, for example -NH-C 1-4 Alkyl and -N(C) 1-4 Alkyl)2.

[0409] The term "hydroxyalkylamine" refers to an amino group substituted with a hydroxyl group or an alkylamine group substituted with a hydroxyl group, wherein the hydroxyl and alkylamine groups are as defined above. The term "C" 1-6 "Hydroxyalkylamine group" refers to a hydroxyalkylamine group with 1-6 carbon atoms, such as OH-C. 1-6 Alkylene-N-.

[0410] The term "hydroxyalkoxy" refers to an alkoxy group substituted with a hydroxyl group, as defined above. The term "C"... 1-6 "Hydroxyalkoxy" refers to a hydroxyalkoxy group having 1-6 carbon atoms, such as OH-C. 1-6 Alkylene-O-.

[0411] The term "aminoalkylamine" or "aminoalkylamine" refers to an amino group substituted with an aminoalkyl group or an alkylamine group substituted with an amino group, wherein the aminoalkyl and alkylamine groups are as defined above. The term "C"1-6 "aminoalkylamine" or "C" 1-6 "Aminoalkylamine group" refers to an aminoalkylamine group with 1-6 carbon atoms, such as NH2-C. 1-6 Alkylene-NH-.

[0412] The term "aminealkoxy" or "aminoalkoxy" refers to an alkoxy group substituted with an amino group, as defined above. The term "C" 1-6 "amine alkoxy" or "C" 1-6 "Aminoalkoxy" refers to an aminoalkoxy group having 1-6 carbon atoms, such as NH2-C. 1-6 Alkylene-O-.

[0413] The term "hydroxyalkyl-amide group" refers to an amide group substituted with a hydroxyalkyl group, wherein the hydroxyalkyl group is as defined above, for example, OH-C. 1-6 Alkylene -C(=O)-NH-, OH-C 1-4 Alkylene-C(=O)-NH-, etc.

[0414] The term "hydroxyalkylamine-amide group" refers to an amide group substituted with a hydroxyalkylamine group, wherein the hydroxyalkylamine group is as defined above, for example, C 1-6 Hydroxyalkylamino group -C(=O)-NH-, etc.

[0415] The term "hydroxyalkoxy-amide group" refers to an amide group substituted with a hydroxyalkoxy group, wherein the hydroxyalkoxy group is as defined above, for example, C 1-6 Hydroxyalkoxy-C(=O)-NH-, etc.

[0416] The term "aminoalkylamine-amide" or "aminoalkylamine-amide" refers to an amide group substituted with an aminoalkylamine group, wherein the aminoalkylamine group is as defined above, for example, C 1-6 Amino-amino group -C(=O)-NH-, etc.

[0417] The term "aminealkoxy-amide" or "aminealkoxy-amide" refers to an amide group substituted with an aminealkoxy group, wherein the aminealkoxy group is as defined above, for example, C 1-6 Aminoalkoxy-C(=O)-NH-, etc.

[0418] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C..." 2-6 "alkenyl", "C" 2-4 "Alkenyl", etc. C 2-6Examples of alkenyl groups include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0419] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Alkyne", "C" 4-6 "Alkyne group", etc. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, etc.

[0420] The term "cycloalkyl" refers to a saturated or partially saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-10 "Cycloalkyl" refers to a cycloalkyl group having 3 to 10 cyclic carbon atoms. The term "C" is used in this context. 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, C 3-10 cycloalkyl (e.g., C10) 3-6 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and its benzo[a] derivatives, for example wait.

[0421] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic groups used in this invention are preferably 3-10 membered, 3-8 membered, or 3-6 membered heterocyclic groups. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, such as 4-6 membered nitrogen-containing heterocyclic groups, 4-6 membered oxygen-containing heterocyclic groups, 5-6 membered oxygen-containing heterocyclic groups, and 5 membered oxygen-containing heterocyclic groups. Common 3-10 membered heterocyclic groups, 3-8 membered heterocyclic groups, and 3-6 membered heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0422] The term "oxygen-containing heterocycle" refers to a heterocycle as described above that has one or more (e.g., 1, 2, or 3) ring atoms of oxygen, such as 5-6 membered oxygen-containing heterocycles, five membered oxygen-containing heterocycles, and specific examples including but not limited to ethylene oxide rings, tetrahydrofuran rings, furan rings, tetrahydropyran rings, pyran rings, 1,3-dioxolane rings, etc. wait.

[0423] The "nitrogen-containing heterocycle" as described in this invention refers to the heterocycle described above, in which one or more (e.g., 1, 2 or 3) ring atoms are nitrogen atoms.

[0424] The term "halogenated alkyl" refers to an alkyl group substituted with one or more (such as 1, 2, or 3) identical or different halogen atoms, wherein the alkyl group is defined as described above. For example, the term "C" as used in this invention... 1-6"Halogenated alkyl" refers to an alkyl halogroup having 1 to 6 carbon atoms. Common alkyl halogroups include (but are not limited to) -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, etc. The alkyl halogroups in this invention are optionally substituted by one or more substituents described in this invention.

[0425] The term "alkoxyalkyl" refers to an alkyl group substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups, wherein the definitions of alkoxy and alkyl groups are as described above. For example, the term "C" as used in this invention... 1-6 "Alkoxyalkyl" refers to an alkyl group having 1-6 carbon atoms that is substituted with one or more (e.g., 1, 2, 3, or 4) alkoxy groups. Common alkoxyalkyl groups include (but are not limited to) CH3O-CH2-, C2H5-O-CH2-, C2H5-O-CH2CH2-, etc.

[0426] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0427] The term "aryl" refers to a C group with a conjugated π-electron system. 6-14 All-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) groups, preferably C 6-10 Aryl groups, such as phenyl and naphthyl, more preferably phenyl.

[0428] The term "5-10-membered heteroaryl" refers to an aromatic monocyclic or polycyclic system containing about 5 to about 10 ring atoms, wherein 1 to 4 ring atoms are independently O, N, S, or Se, and the remaining ring atoms are carbon atoms. In some embodiments, the heteroaryl is a monocyclic heteroaryl and has 5 or 6 ring atoms (i.e., a 5-6-membered heteroaryl). In some embodiments, the heteroaryl is a bicyclic heteroaryl, for example, having 9 or 10 ring atoms (i.e., a 9-10-membered heteroaryl). The heteroaryl may optionally be substituted by one or more "cyclic substituents," which may be the same or different, and are defined herein. The heteroaryl is linked by ring carbon atoms, and any nitrogen atom of the heteroaryl may optionally be oxidized to the corresponding N-oxide. Any C atom of the heteroaryl may optionally be oxidized or thiolated. The term "heteroaryl" also includes heteroaryls as defined above fused with heterocyclic groups or cycloalkyl groups. Non-limiting examples of 5-10 membered heteroaryl groups (e.g., 5-6 membered heteroaryl or 9-10 membered heteroaryl) include: pyridyl, pyrazinyl, furanyl, thiopheneyl, selenophenolyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiazolyl, pyrazolyl, furanyl, pyrroleyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxinylindolyl dolyl), imidazo[1,2-a]pyridyl, imidazo[2,1-b]thiazolyl, benzofuranyl, indolyl, azaindolyl, benzimidazolyl, benzothiophene, quinolinyl, imidazolyl, benzimidazolyl, thiophenepyridyl, quinazolinyl, thiophenepyrimidinyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, benzothiazolyl, benzooxazolyl, etc., and all their isomers.

[0429] If a substituent is described as “optionally substituted,” then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together by independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted by one or more substituents from the list of substituents, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted by independently selected optional substituents.

[0430] If a functional group or structure is described as “substituted or unsubstituted”, then the functional group or structure may be (1) unsubstituted or (2) substituted.

[0431] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms in a specified compound or structure by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. For example, the substituent or suitable substituent may each independently consist of one or more of the following structures: H, -O-, -S-, -NR. 8 - Halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C 1-6 (alkylene) group, C 1-6 Halogenated (alkylene) group, C 1-6 Alkoxy, C 2-6 (imide)alkenyl, C 2-6 (Immune)alkynyl, C3-8 (imune)cycloalkyl, 3-8 membered (imune)heterocyclic, C 6-10 (sub-)aryl and 5-10 quinone (sub-)heteroaryl, etc., among which R 8 Selected from H, C 1- 6-alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, halogens, hydroxyl groups, carboxyl groups, and ester groups (e.g., -C) 1-6 Alkylene-C(=O)-OC 1-6 Alkyl). For example, the substituent or suitable substituent is each independently composed of one or more of the following structures: F, Cl, Br, methyl, ethyl, propyl, halogenated C. 1-6 Alkyl, Halogenated C 1-6 Alkoxy, -O-, -S-, CN, =O, methoxy, ethoxy, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, -OH, 3-6 member nitrogen heterocyclic groups, phenyl, naphthyl, pyridyl, and phenolyl, etc. In some embodiments, the substituent or suitable substituent is independently selected from H, deuterium (D), halogen, -OH, -NH2, C, etc. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2- 6-acetylinyl, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl, 5-10 membered heteroaryl, cyano, nitro, C 1-6 Haloalkyl, C 1-6Alkyl-C(=O)-. If a substituent is described as being "independently selected" from a set of functional groups, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0432] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, six, seven, eight, nine, or ten.

[0433] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0434] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0435] Solid lines may be used in this article. solid wedge Or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0436] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0437] It should also be understood that certain compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to encompass the various derivative forms of the compounds described above.

[0438] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0439] Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts.

[0440] For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts for the compounds of the present invention are known to those skilled in the art.

[0441] The term "ester" refers to esters derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (compounds of the present invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the present invention may themselves be esters.

[0442] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0443] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0444] This invention further includes, within its scope, prodrugs of the compounds of the invention. Typically, such prodrugs are functional group derivatives of the compounds that readily convert in vivo into the desired therapeutically active compound. Therefore, in these cases, the term "administration" for the treatment methods of the invention should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but after administration to an individual, the prodrug form is converted in vivo into the aforementioned compound. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug," ed. H. Bundgaard, Elsevier, 1985.

[0445] This invention further includes, within its scope, isotopic labels of the compounds of this invention, which are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in this invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0446] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in *Protective Groups in Organic Chemistry*, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P. ​​G.W. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0447] The term "self-immolative groups" refers to structures that spontaneously rearrange or degrade upon triggering by specific stimuli (such as enzymatic cleavage, pH changes, or reducing environments), thereby releasing the bioactive molecules they are linked to. Examples of self-immolative groups include, but are not limited to, chemical bonds, -CH2NH-, p-aminobenzyloxycarbonyl (PABC), carbonate groups, p-aminobenzyl quaternary ammonium salts (PABQ), p-aminobenzyl ethers (PABE), N-methylallyloxy groups, and decaramate.

[0448] As used herein, the term “DAR” or “drug-antibody ratio” refers to: (a) the number of linker / drug moieties linked to the antibody in a single antibody-drug conjugate molecule, which is an integer from 0 to 10, such as an integer from 1 to 10; or (b) the average number of linker / drug moieties linked to the antibody in a composition comprising more than one antibody-drug conjugate molecule, which is an integer or decimal from 0 to 10, such as an integer or decimal from 1 to 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0449] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated value.

[0450] The abbreviations used in this invention have the following meanings:

[0451] Information about the sequences involved in this invention is described in the table below:

[0452] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0453] Nuclear magnetic resonance (NMR) 1 The H NMR (H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; hexadeuterated dimethyl sulfoxide (DMSO-d6) was used; and tetramethylsilane (TMS) was used as the internal standard.

[0454] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0455] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0456] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0457] Pyrrolobenzodiazepine Example of intermediate synthesis and preparation of monomer-like substances

[0458] Example 1 of intermediate preparation: N 6 -((allyloxy)carbonyl)-N 2 Preparation of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-1)

[0459] Step 1: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynyl)-L-valine tert-butyl ester (INT-1-2) 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (10 g, 37.27 mmol) and L-valine tert-butyl ester (7.75 g, 44.73 mmol) were dissolved in DMF (50 mL). HATU (21.25 g, 55.91 mmol) and DIPEA (14.45 g, 111.82 mmol, 19.48 mL) were added, and the mixture was stirred at room temperature for 5 hours. Water (500 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain the crude title compound (15.7 g, 37.07 mmol), which was used directly in the next step without purification.

[0460] Its structural characterization data are as follows:

[0461] MS m / z (ESI): 424.2 [M+H] +

[0462] Step 2: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (INT-1-3)

[0463] (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine tert-butyl ester (15.7 g, 37.07 mmol) was dissolved in DCM (70 mL), and TFA (20 mL) was added dropwise. The mixture was reacted at room temperature for 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50 mL). The organic phases were combined, washed seven times with purified water (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (13.6 g, 37.01 mmol), which was used directly in the next step without purification.

[0464] Its structural characterization data are as follows:

[0465] MS m / z (ESI): 368.1 [M+H] +

[0466] Step 3: N 6 -((allyloxy)carbonyl)-N 2 Preparation of -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-1)

[0467] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (1 g, 2.72 mmol) and N 6 -((allyloxy)carbonyl)-L-lysine (689.37 mg, 2.99 mmol) was dissolved in DMF (15 mL), and HATU (1.09 g, 2.86 mmol) was added. The mixture was reacted at room temperature for 1 h. Saturated brine (150 mL) and ethyl acetate (150 x 2) were added to the reaction solution for extraction. The combined organic phases were dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (MeOH / DCM = 0–10%, 50 min) and then concentrated again to obtain the title compound (1.3 g, 2.24 mmol).

[0468] Its structural characterization data are as follows:

[0469] MS m / z (ESI): 580.2 [M+H] +

[0470] Example 2 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (INT-2):

[0471] Step 1: Preparation of (2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carboxylic acid benzyl ester (INT-2-2):

[0472] (2S,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidine-1-carboxylate (10 g, 39.80 mmol) was dissolved in dichloromethane (100 mL), and triethylamine (6.04 g, 59.69 mmol, 8.30 mL) and DBU (1.21 g, 7.96 mmol, 1.19 mL) were added dropwise with stirring. Tert-butyldimethylchlorosilane (6.60 g, 43.78 mmol) was added in portions, and the reaction was continued for 18 hours. A saturated ammonium chloride aqueous solution was added with stirring, and the mixture was allowed to stand and separated. The aqueous phase was extracted with dichloromethane, the organic phases were combined, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 20-60% ethyl acetate / petroleum ether) to give the title compound (11.3 g, 30.91 mmol).

[0473] Step 2: Preparation of (3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-3-ol (INT-2-3):

[0474] (2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-carboxylic acid benzyl ester (11.3 g, 30.91 mmol) was dissolved in ethanol (100 mL), and 10% Pd / C (1.1 g, 3.09 mmol) was added. The mixture was stirred for 16 hours under hydrogen purging and protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the crude title compound (7.02 g, 30.34 mmol), which was directly used for the next reaction.

[0475] Step 3: Preparation of ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-yl)(4,5-dimethoxy-2-nitrophenyl)methyl ketone (INT-2-4):

[0476] 4,5-Dimethoxy-2-nitrobenzoic acid (6.53 g, 28.74 mmol), (3R,5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-3-ol (7 g, 30.25 mmol), and HATU (12.64 g, 33.27 mmol) were dissolved in DMF (140 mL). DIPEA (11.73 g, 90.75 mmol, 15.81 mL) was added dropwise with stirring, and the reaction was continued for 1 hour. Water and ethyl acetate were added, the mixture was stirred, allowed to stand, and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed three times with saturated brine, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (13.01 g, 29.53 mmol).

[0477] Its structural characterization data are as follows:

[0478] MS m / z (ESI): 441.3 [M+H] +

[0479] Step 4: Preparation of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-3-one (INT-2-5):

[0480] ((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidine-1-yl)(4,5-dimethoxy-2-nitrophenyl)methyl ketone (5.4 g, 12.26 mmol) was dissolved in dichloromethane (50 mL). Tetrapropylammonium perruthenate (1.29 g, 3.68 mmol) and N-methylmorpholine oxide (4.31 g, 36.77 mmol) were added with stirring, and the reaction was continued for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (3.3 g, 7.52 mmol).

[0481] Step 5: Preparation of (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (INT-2-6):

[0482] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-3-one (2.8 g, 6.38 mmol) was dissolved in dry dichloromethane (30 mL). The solution was cooled and stirred to -35 °C under nitrogen purging and protection. 2,6-Lutidine (1.37 g, 12.77 mmol, 1.49 mL) was added dropwise, followed by a slow dropwise addition of a dichloromethane (10 mL) solution of trifluoromethanesulfonic anhydride (2.70 g, 9.58 mmol, 1.61 mL). The reaction was maintained at this temperature for 0.5 hours. The reaction was quenched with 40 mL of water, and the mixture was allowed to stand and separated. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-5% methanol / dichloromethane) to give the title compound (2.2 g, 3.86 mmol).

[0483] Step Six: Preparation of (S,E)-(4-(4-aminostyryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (INT-2-7):

[0484] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (4.57 g, 8.01 mmol), [(E)-2-(4-aminophenyl)vinyl]boronic acid (2.61 g, 16.02 mmol), Pd(dppf)₂Cl₂ (1.31 g, 1.60 mmol), and K₂CO₃ (2.21 g, 16.02 mmol) were dissolved in a mixed solvent of tetrahydrofuran (60 mL) and water (6 mL). The reaction mixture was heated to 75 °C for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 10-50% ethyl acetate / petroleum ether) to give the title compound (1.25 g, 2.32 mmol).

[0485] Its structural characterization data are as follows:

[0486] MS m / z (ESI): 540.2 [M+H] +

[0487] Step 7: Preparation of (S,E)-(4-(2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (INT-2-8):

[0488] (S,E)-(4-(4-aminostyryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (1.2 g, 2.22 mmol) was dissolved in dry dichloromethane (60 mL), cooled and stirred to 0 °C, and DIPEA (862.09 mg, 6.67 mmol, 1.16 mL) was added dropwise, followed by allyl chloroformate (536.01 mg, 4.45 mmol, 472.67 μL). The reaction was stirred for 1 hour. The reaction was quenched with saturated brine, allowed to stand and separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried and concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (1.14 g, 1.83 mmol).

[0489] Step 8: Preparation of (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (INT-2-9):

[0490] (S,E)-(4-(2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (1.14 g, 1.83 mmol) was dissolved in methanol (50 mL), and saturated ammonium chloride aqueous solution (10 mL) was added dropwise. Zinc powder (1.20 g, 18.28 mmol) was added with stirring, and the reaction was continued for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and dissolved in dichloromethane and water. After standing, the liquid was separated, and the organic phase was dried, concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (927 mg, 1.56 mmol).

[0491] Its structural characterization data are as follows:

[0492] MS m / z (ESI): 594.2 [M+H] +

[0493] Step Nine: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-2-10):

[0494] (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (400 mg, 673.65 μmol) was dissolved in dry tetrahydrofuran (8 mL). Triethylamine (95.43 mg, 943.10 μmol, 131.09 μL) was added dropwise under nitrogen purging and protection. The mixture was cooled and stirred to -10 °C, and triphosgene (85.96 mg, 28 μL) was slowly added dropwise. A solution of 9.67 μmol) tetrahydrofuran (2 mL) was stirred for about 10 minutes. Then, a mixed solution of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (228.83 mg, 606.28 μmol) and DMAP (115.22 mg, 943.10 μmol) in tetrahydrofuran (8 mL) and DMF (2.5 mL) was slowly added dropwise. The mixture was heated slowly to 40 °C and reacted for 1 hour. The reaction was quenched by adding 1 mL of water. After concentration under reduced pressure, water and dichloromethane were added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated. The solution was purified by silica gel column chromatography (eluent: 0-3% methanol / dichloromethane) to give the title compound (486 mg, 487.36 μmol).

[0495] Its structural characterization data are as follows:

[0496] MS m / z (ESI): 998.4 [M+H] +

[0497] Step 10: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-2-11):

[0498] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-((E)-4-(((allyloxy)carbonyl)amino)styryl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (486 mg, 487.36 μmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and water (2 mL). Glacial acetic acid (10 mL) was added dropwise with stirring, and the mixture was heated to 40 °C and reacted for 2 hours. After concentration under reduced pressure, the mixture was purified by reverse-phase column chromatography (eluent: 0-60% acetonitrile / 0.5% formic acid water) and lyophilized to give the title compound (219 mg, 248.03 μmol).

[0499] Its structural characterization data are as follows:

[0500] MS m / z (ESI): 883.3 [M+H] +

[0501] Step 11: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carbonate (INT-2-12):

[0502] Dry DMSO (66.37 mg, 849.42 μmol, 60.33 μL) was dissolved in dry dichloromethane (5 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (51.75 mg, 407.72 μmol, 34.50 μL) was added dropwise. After stirring for 0.5 hours, 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl (2... A solution of 300 mg (339.77 μmol) of 4-((S)-4-(((allyloxy)carbonyl)amino)styryl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate in dichloromethane (5 mL) was added, and the reaction was continued for 1 hour. Then, triethylamine (171.91 mg, 1.70 mmol, 236.14 μL) was added dropwise, and the reaction was allowed to return to room temperature for 1 hour. The reaction was quenched with water, allowed to stand, and separated. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, dried, concentrated, and purified by reverse-phase column chromatography (eluent: 0-55% acetonitrile / 0.5% formic acid in water). The purified compound was lyophilized to give the title compound (110 mg, 124.87 μmol).

[0503] Its structural characterization data are as follows:

[0504] MS m / z (ESI): 881.3 [M+H] +

[0505] Step Twelve: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (INT-2):

[0506] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carbonate (100 mg, 113.52 μmol), tetra(triphenylphosphine)palladium (26.23 mg, 22.70 μmol), and 1,3-dimethylbarbituric acid (26.59 mg, 170.27 μmol) were dissolved in DMF (2 mL) and reacted with stirring for 1 hour under nitrogen purging and protection. Reversed-phase column purification (eluent: 0-70% acetonitrile / 0.5% ammonium bicarbonate in water), followed by lyophilization to obtain the title compound (65 mg, 91.19 μmol).

[0507] Its structural characterization data are as follows:

[0508] MS m / z (ESI): 713.2 [M+H] +

[0509] Example 3 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (INT-3):

[0510] Step 1: Preparation of (S)-(4-(6-aminonaphthyl-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (INT-3-1):

[0511] (S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrole-3-yltrifluoromethanesulfonate (2.0 g, 3.51 mmol), 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (A-6-2) (1.42 g, 5.26 mmol), Pd(dppf)2Cl2 (256.57 mg, 350.50 μmol), K2CO3 (1.21 g, 8.76 mmol) were dissolved in a mixed solvent of dioxane (30 mL) and water (10 mL), and the mixture was heated to 75 °C for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 10-50% ethyl acetate / petroleum ether) to give the title compound (1.4 g, 2.48 mmol).

[0512] Its structural characterization data are as follows:

[0513] MS m / z (ESI): 564.2 [M+H] +

[0514] Step 2: Preparation of (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (INT-3-2):

[0515] (S)-(4-(6-aminonaphthyl-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrolo-1-yl)(4,5-dimethoxy-2-nitrophenyl) ketone (2.2 g, 3.90 mmol) was dissolved in dry dichloromethane (30 mL), cooled and stirred to 0 °C, and DIPEA (1.51 g, 11.71 mmol) was added dropwise, followed by allyl chloroformate (564.49 mg, 4.68 mmol). The reaction was stirred for 1 hour. The reaction was quenched with saturated brine, allowed to stand and separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried and concentrated, and purified by silica gel column chromatography (eluent: 0-35% ethyl acetate / petroleum ether) to give the title compound (2.12 g, 3.27 mmol).

[0516] Its structural characterization data are as follows:

[0517] MS m / z (ESI): 648.2 [M+H] +

[0518] Step 3: Preparation of (S)-(6-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (INT-3-3):

[0519] (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (2.12 g, 3.27 mmol) was dissolved in methanol (70 mL), and saturated ammonium chloride aqueous solution (20 mL) was added dropwise. Zinc powder (2.14 g, 32.73 mmol) was added with stirring, and the reaction was continued for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and dissolved in dichloromethane and water. After standing, the liquid was separated, and the organic phase was dried, concentrated, and purified by silica gel column chromatography (eluent: 0-55% ethyl acetate / petroleum ether) to give the title compound (1.78 g, 2.88 mmol).

[0520] Its structural characterization data are as follows:

[0521] MS m / z (ESI): 618.3 [M+H] +

[0522] Step 4: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-3-4):

[0523] (S)-(6-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (1 g, 1.62 mmol) was dissolved in dry tetrahydrofuran (8 mL). Triethylamine (229.31 mg, 2.27 mmol) was added dropwise under nitrogen purging and protection. The mixture was cooled and stirred to -10 °C, and triphosgene (158.51 mg, 534.15 μmol) was slowly added dropwise. A solution of tetrahydrofuran (2 mL) was stirred for about 10 minutes, and then a mixture of allyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (610.93 mg, 1.62 mmol) and DMAP (276.85 mg, 2.27 mmol) in tetrahydrofuran (8 mL) and DMF (2.5 mL) was slowly added dropwise. The mixture was heated slowly to 40 °C and reacted for 1 hour. The reaction was quenched by adding 1 mL of water. After concentration under reduced pressure, water and dichloromethane were added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent: 0-65% ethyl acetate / petroleum ether) to give the title compound (1.58 g, 1.55 mmol).

[0524] Its structural characterization data are as follows:

[0525] MS m / z (ESI): 1022.4 [M+H] +

[0526] Step 5: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (INT-3-5):

[0527] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate (1.58 g, 1.55 mmol) was dissolved in tetrahydrofuran (10 mL), and a THF solution of TBAF (1 M, 8.37 mL) was added dropwise with stirring. The mixture was concentrated under reduced pressure and purified by normal-phase column chromatography (0-90% ethyl acetate / petroleum ether). The solution was then lyophilized to give the title compound (998 mg, 1.10 mmol).

[0528] Its structural characterization data are as follows:

[0529] MS m / z (ESI): 907.3 [M+H] +

[0530] Step Six: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (INT-3-6):

[0531] Dry DMSO (116.29 mg, 1.49 mmol, 105.72 μL) was dissolved in dry dichloromethane (5 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (94.46 mg, 744.23 μmol, 62.98 μL) was added dropwise. After stirring for 5 minutes, 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl (2... A solution of 450 mg (496.16 μmol) of 4-((S)-4-(6-((((allyloxy)carbonyl)amino)naphth-2-yl)-2-(hydroxymethyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4,5-dimethoxyphenyl)carbamate in dichloromethane (5 mL) was added. After reacting for 1 hour, triethylamine (251.03 mg, 2.48 mmol, 344.82 μL) was added dropwise, and the reaction was allowed to return to room temperature for 1 hour. The reaction was quenched with water, allowed to stand, and separated. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, dried, concentrated, and purified by reverse-phase column chromatography (eluent: 0-18% 30% methanol / dichloromethane). The purified compound was lyophilized to give the title compound (324 mg, 358.03 μmol).

[0532] Its structural characterization data are as follows:

[0533] MS m / z (ESI): 906.3 [M+H] +

[0534] Step 7: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (INT-3):

[0535] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (324 mg, 358.03 μmol), tetrakis(triphenylphosphine)palladium (76.58 mg, 66.30 μmol), and 1,3-dimethylbarbituric acid (155.15 mg, 994.52 μmol) were dissolved in DMF (2 mL) and reacted with stirring for 1 hour under nitrogen purging and protection. Reversed-phase column purification (eluent: 0-40% acetonitrile / 0.5% ammonium bicarbonate in water), followed by lyophilization to obtain the title compound (228 mg, 309.44 μmol).

[0536] Its structural characterization data are as follows:

[0537] MS m / z (ESI): 737.3 [M+H] +

[0538] Example 4 of intermediate preparation: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxovalerate (INT-4)

[0539] Step 1: Preparation of perfluorophenyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (INT-4-2)

[0540] 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (3 g, 6.36 mmol) and pentafluorophenol (4.69 g, 25.45 mmol) were dissolved in dichloromethane (50 mL), and EDCI (4.88 g, 25.45 mmol) was added. The mixture was stirred at room temperature for one hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether, 30-100%), concentrated, and dried to obtain the title compound (5.0 g, 5.16 mmol).

[0541] Its structural characterization data are as follows:

[0542] MS m / z (ESI): 970.6 [M+H] +

[0543] Step 2: Preparation of ((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)benzyl carbamate (INT-4-3)

[0544] Perfluorophenyl 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (5.0 g, 5.16 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentanol (4.03 g, 20.63 mmol) were dissolved in DMF (15 mL). DIPEA (2.00 g, 15.47 mmol, 2.69 mL) was added, and the mixture was reacted at room temperature for 2 hours. After removing most of the solvent from the reaction solution under vacuum, the solution was purified by C18 column chromatography (acetonitrile: 0.05% formic acid aqueous solution, 10%–60%) and lyophilized to obtain the title compound (5 g, 4.98 mmol).

[0545] Its structural characterization data are as follows:

[0546] MS m / z (ESI): 1003.4 [M+H] +

[0547] Step 3: Preparation of 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(INT-4-4)

[0548] 5 g (4.98 mmol) of benzyl carbamate ((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)carbamate was dissolved in a mixed solvent of ethanol (20 mL) and water (20 mL), 10% Pb / C (0.1 g) was added, and the mixture was purged with nitrogen three times. The mixture was then subjected to catalytic hydrogenation for 4 h. The reaction solution was filtered and concentrated to obtain the crude title compound (4.0 g, 4.60 mmol), which was used directly in the next step without purification.

[0549] Its structural characterization data are as follows:

[0550] MS m / z (ESI): 869.9 [M+H] +

[0551] Step 4: Preparation of (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentanoic acid allyl ester (INT-4-5)

[0552] Add 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(910 mg, 1.05 mm (ol) and (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxovaleric acid (471.67 mg, 1.15 mmol) were dissolved in DMF (2 mL), and DIPEA (541.40 mg, 4.19 mmol) and HATU (597.27 mg, 1.57 mmol) were added. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction solution was directly purified by C18 column (acetonitrile: 0.05% formic acid aqueous solution, 10%–65%), and lyophilized to obtain the crude product. It was then purified again by C18 column (acetonitrile: 0.05% formic acid aqueous solution, 10%–50%), and lyophilized to obtain the title compound (843 mg, 601.98 μmol).

[0553] Its structural characterization data are as follows:

[0554] MS m / z (ESI): 1261.4 [M+H] +

[0555] Step 5: Preparation of (S)-4-amino-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentanoic acid allyl ester (INT-4-6)

[0556] The (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy-13-dimethyl ... Hydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-5-oxopentaic acid allyl ester (833 mg, 660.94 μmol, FR) was dissolved in DMF (3 mL), and diethylamine (146.73 mg, 1.98 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution under vacuum, the solution was purified by C18 column chromatography (acetonitrile: 0.05% trifluoroacetic acid aqueous solution, 10%–70%), and lyophilized to give the trifluoroacetate of the title compound (699 mg, 606.71 μmol).

[0557] Its structural characterization data are as follows:

[0558] MS m / z(ESI): 1038.1 [M+H] +

[0559] Step Six: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid allyl ester (INT-4-7)

[0560] The (S)-4-amino-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazadi Allyl pentadecane-13-yl)amino)-5-oxopentanoate (620 mg, 538.14 μmol, TF) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (158.81 mg, 591.95 μmol, FR) were dissolved in DMF (6 mL), and DIPEA (278.20 mg, 2.15 mmol) and DMTMM (297.51 mg, 1.08 mmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by C18 column chromatography (acetonitrile: 0.05% trifluoroacetic acid aqueous solution, 10%–70%) and lyophilized to give the title compound (569 mg, 388.65 μmol).

[0561] Its structural characterization data are as follows:

[0562] MS m / z (ESI): 1288.4 [M+H] +

[0563] Step 7: Preparation of (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (INT-4)

[0564] The (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane Allyl 1,3-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)-5-oxovalerate (520 mg, 403.61 μmol) was dissolved in a mixed solvent of dichloromethane (5 mL) and DMF (5 mL). Under nitrogen protection, tetrakis(triphenylphosphine)palladium (233.09 mg, 201.81 μmol), formic acid (0.4 mL), and N-methylmorpholine (0.8 mL) were added, and the reaction was stirred at room temperature for 2 hours. The reaction solution was directly purified by C18 column chromatography (acetonitrile: 0.05% formic acid aqueous solution, 10%–50%), and lyophilized to give the title compound (340 mg, 245.13 μmol).

[0565] Its structural characterization data are as follows:

[0566] MS m / z (ESI): 1248.3 [M+H] +

[0567] Example 5 of intermediate preparation: N 5 Preparation of -((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-5)

[0568] Step 1: Preparation of 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (INT-5-2)

[0569] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (6.00 g, 14.10 mmol) and lithium hydroxide monohydrate (1.18 g, 28.20 mmol) were added sequentially to DMF (30 mL). After the addition was complete, the mixture was stirred at 25 °C for 10 min, and then allyl bromide (5.12 g, 42.31 mmol) was added. The reaction mixture was reacted at 25 °C for 1 h. The reaction solution was poured into dilute hydrochloric acid (100 mL of 0.5 N HCl), extracted with EA (40 mL * 2), washed with water (20 mL), dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO2, EA / PE = 0–45%). The title compound (4.67 g, 10.03 mmol) was obtained.

[0570] Its structural characterization data are as follows:

[0571] ESI-MS (m / z): 410.3 [M+H-56] + .

[0572] Step 2: Preparation of 5-(tert-butyl)-L-glutamic acid allyl ester (INT-5-3)

[0573] 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (4.40 g, 9.45 mmol) and DBU (4.32 g, 28.35 mmol) were added sequentially to DMF (22 mL). After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude title compound (2.29 g, 9.41 mmol), which was used directly in the next step without purification.

[0574] Its structural characterization data are as follows:

[0575] ESI-MS (m / z): 244.2 [M+H] + .

[0576] Step 3: Preparation of 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (INT-5-4)

[0577] Crude 5-(tert-butyl)-L-glutamic acid allyl ester (2.29 g, 9.41 mmol), 6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (3.03 g, 11.29 mmol), DIPEA (4.87 g, 37.65 mmol), and HATU (5.37 g, 14.12 mmol) were added sequentially to DMF (22 mL). After addition, the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LC-MS until complete. The crude product was concentrated and purified by column chromatography (ACN / H2O = 0.75%, 0.05% formic acid). The product was then lyophilized to obtain the title compound (1.42 g, 2.87 mmol).

[0578] Its structural characterization data are as follows:

[0579] ESI-MS (m / z): 511.2 [M+H2O] + .

[0580] Step 4: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovalerate (INT-5-5)

[0581] 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (830.00 mg, 1.48 mmol) was dissolved in DCM (10 mL), and TFA (5 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was subjected to reduced pressure to remove DCM and TFA to obtain the crude product, which was purified by high performance liquid chromatography to obtain the title compound (361.00 mg, 825.21 μmol).

[0582] The purification method is as follows:

[0583] Column: Phenomenex C18 250mm×50mm×10μm

[0584] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0585] Its structural characterization is as follows:

[0586] ESI-MS (m / z): 438.1 [M+H] + .

[0587] Step 5: N 5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane-13-yl)-N 2 Preparation of -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (INT-5-6):

[0588] (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxovaleric acid (200 mg, 457.18 μmol), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl) Di(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (436.97 mg, 502.90 μmol) was dissolved in DMF (5 mL), and HATU (208.47 mg, 548.61 μmol) was added with stirring. DIPEA (177.26 mg, 1.37 mmol) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-35% acetonitrile / 0.5% formic acid water), and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0589] Its structural characterization data are as follows:

[0590] MS m / z (ESI): 1338.6 [M+H] +

[0591] Step Six: N 5 Preparation of -((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-5):

[0592] Allyl N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19- (diazapentadecane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (311 mg, 241.39 μmol), tetra(triphenylphosphine)palladium (27.89 mg, 24.14 μmol), and 1,3-dimethylbarbituric acid (37.69 mg, 241.39 μmol) were dissolved in DMF (6 mL). The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-30% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (292 mg, 233.92 μmol).

[0593] Its structural characterization data are as follows:

[0594] MS m / z (ESI): 1249.4 [M+H] +

[0595] Example 6 of intermediate preparation: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0596] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (INT-6-2)

[0597] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-1-methylpyridine trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80–85 °C for 16 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10–70%) and lyophilized to give the title compound (0.78 g, 1.51 mmol).

[0598] Its structural characterization data are as follows:

[0599] ESI-MS (m / z): 515.2 (M+H) +

[0600] Step 2: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-6-3)

[0601] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.78 g, 1.51 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) were dissolved in DMF (15 mL), DIPEA (1.17 g, 9.07 mmol) was added, and HATU (2.59 g, 6.80 mmol) was added in portions. The reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.74 g, 0.90 mmol).

[0602] Its structural characterization data are as follows:

[0603] ESI-MS (m / z): 824.4 (M+H) +

[0604] Step 3: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6) Benzyl methylpropoxy-2-ylamino-3-oxopropoxy-methylpropoxy-propanoate (0.74 g, 0.90 mmol) was dissolved in ethanol (30 mL), and 10% palladium on carbon (0.15 g) and acetic acid (0.14 g, 2.42 mmol) were added. The air was removed, and hydrogen gas (balloon) was introduced and the mixture was heated to 40 °C for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0605] Its structural characterization data are as follows:

[0606] ESI-MS (m / z): 734.3 (M+H) +

[0607] Example 7 of intermediate preparation: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0608] Step 1: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-7-1)

[0609] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1.07 g, 2.08 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, and HATU (3.56 g, 9.36 mmol) was added in portions. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0610] Its structural characterization data are as follows:

[0611] ESI-MS (m / z): 1046.5 (M+H) +

[0612] Step 2: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0613] Benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 mL). 10% palladium on carbon (0.13 g) and acetic acid (0.10 g, 1.70 mmol) were added. The air was removed, and hydrogen gas (in a balloon) was introduced. The mixture was heated to 40 °C and reacted for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0614] Its structural characterization data are as follows:

[0615] ESI-MS (m / z): 956.4 (M+H) +

[0616] Example 8 of intermediate preparation: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (INT-8)

[0617] Step 1: Preparation of 1-amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azacosanodecane-22-acid (INT-8-2):

[0618] 17,17-bis((2-carboxyethoxy)methyl)-15-oxo-1-triazyl-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (670 mg, 1.10 mmol) was dissolved in tetrahydrofuran (5 mL), and 10% palladium on carbon (50 mg, 109.73 μmol) was added. The reaction mixture was stirred for 18 hours under hydrogen purging and protection. The reaction solution was filtered through a diatomaceous earth filter, and the filtrate was concentrated under reduced pressure to give the crude title compound (640 mg, 1.09 mmol).

[0619] Its structural characterization data are as follows:

[0620] MS m / z (ESI): 585.2 [M+H] +

[0621] Step 2: Preparation of 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (INT-8-3):

[0622] 1-Amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (950 mg, 1.63 mmol) was dissolved in a mixture of acetonitrile (10 mL) and water (10 mL). Sodium bicarbonate (682.51 mg, 8.13 mmol) was added with stirring, followed by 9-fluorenemethyl-N-succinimide carbonate (657.74 mg, 1.95 mmol). The reaction mixture was stirred for 5 hours. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-50% acetonitrile / 0.5% formic acid in water) and lyophilized to give the title compound (870 mg, 1.08 mmol).

[0623] Its structural characterization data are as follows:

[0624] MS m / z (ESI): 807.3 [M+H] +

[0625] Step 3: Preparation of (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (INT-8-4):

[0626] 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (870 mg, 1.08 mmol), meglumine (841.97 mg, 4.31 mmol), and HATU (2.05 g, 5.39 mmol) were dissolved in DMF (10 mL). DIPEA (696.78 mg, 5.39 mmol, 939.05 μL) was added dropwise with stirring, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-45% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0627] Its structural characterization data are as follows:

[0628] MS m / z (ESI): 1338.6 [M+H] +

[0629] Step 4: Preparation of 3,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(INT-8-5):

[0630] (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (200 mg, 149.43 μmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The solvent in the reaction solution was evaporated under reduced pressure, ethyl acetate and water were added and stirred, and the mixture was allowed to stand and separated. The aqueous phase was lyophilized to give the crude product of the title compound (166 mg, 148.72 μmol).

[0631] Its structural characterization data are as follows:

[0632] MS m / z (ESI): 1116.6 [M+H] +

[0633] Step 5: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid allyl ester (INT-8-6):

[0634] (S)-5-(allyloxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (65.06 mg, 148.72 μmol), 33,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl 166 mg (148.72 μmol) of propane-1,3-diyl)bis(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (2 mL) was dissolved in DMF. HATU (84.99 mg, 223.66 μmol) was added with stirring, followed by dropwise addition of DIPEA (57.66 mg, 446.16 μmol, 77.71 μL). The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (145 mg, 94.42 μmol).

[0635] Its structural characterization data are as follows:

[0636] MS m / z (ESI): 1535.5 [M+H] +

[0637] Its preparation method is as follows:

[0638] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

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

[0640] Step Six: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (INT-8):

[0641] (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28 -trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid allyl ester (145 mg, 94.42 μmol) was dissolved in DMF (3 mL), and tetrakis(triphenylphosphine)palladium (10.91 mg, 9.44 μmol) and 1,3-dimethylbarbituric acid (14.74 mg, 94.42 μmol) were added. The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (108 mg, 72.21 μmol).

[0642] Its structural characterization data are as follows:

[0643] MS m / z (ESI): 1496.5 [M+H] +

[0644] Its preparation method is as follows:

[0645] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

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

[0647] Pyrrolobenzodiazepine Synthesis and preparation examples of monomers

[0648] Preparation Example 1: (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-1)

[0649] Step 1: Preparation of (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (P-1-2)

[0650] 4,5-Dimethoxy-2-nitrobenzoic acid (5 g, 22.01 mmol) and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (4.4 g, 24.21 mmol) were dissolved in DMF (80 mL). HATU (8.78 g, 23.11 mmol) and DIPEA (8.53 g, 66.03 mmol) were added with stirring, and the reaction was continued for 16 hours. Saturated brine (100 mL) and ethyl acetate (100 mL) were added to the reaction solution, stirred, allowed to stand, and separated. The aqueous phase was extracted twice with ethyl acetate (100 mL). The organic phases were combined, washed three times with saturated brine, dried, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%) and concentrated again to obtain the title compound (3.6 g, 10.16 mmol).

[0651] Its structural characterization data are as follows:

[0652] ESI-MS (m / z): 355.1 [M+H] +

[0653] Step 2: (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (P-1-3)

[0654] (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (3.6 g, 10.16 mmol) was dissolved in methanol (60 mL), and zinc powder (6.6 g, 101.6 mmol) and saturated ammonium chloride aqueous solution (20 mL) were added with stirring. The mixture was heated to 80 °C and reacted for 16 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%), and concentrated again to give the title compound (2.1 g, 7.18 mmol).

[0655] Its structural characterization data are as follows:

[0656] ESI-MS (m / z): 293.1 [M+H] +

[0657] Step 3: (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -5,11(10H)-dione (P-1-4)

[0658] (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepines -5,11(10H)-dione (2.1 g, 7.18 mmol) was dissolved in DMF (30 mL), and imidazole (2.45 g, 35.92 mmol) and tert-butyldimethylchlorosilane (2.38 g, 15.81 mmol) were added with stirring. The reaction was continued for 16 hours. The reaction solution was quenched with water, extracted three times with ethyl acetate (30 mL x 3), the organic phases were combined, washed three times with saturated brine, dried and concentrated. Purification by silica gel column chromatography (methanol / dichloromethane = 0%–15%) gave the title compound (1 g, 2.46 mmol).

[0659] Its structural characterization data are as follows:

[0660] ESI-MS (m / z): 407.2 [M+H] +

[0661] Step 4: (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -5,11(10H)-dione (P-1-5)

[0662] (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (1 g, 2.46 mmol) was dissolved in dry DMF (30 mL), cooled and stirred to 0 °C, and NaH (147.58 mg, 3.69 mmol) was added in portions. After the addition was complete, the reaction was maintained at this temperature for 30 minutes under nitrogen protection, and then 2-(trimethylsilyl)ethoxymethyl chloride (616.17 mg, 2.46 mmol) was slowly added dropwise, and the reaction was continued for 1 hour. The reaction solution was poured into ice water and stirred, and a solid precipitated. The solid was filtered and dried to obtain the crude product of the title compound (1.3 g, 2.42 mmol), which was used directly in the next reaction.

[0663] Its structural characterization data are as follows:

[0664] ESI-MS (m / z): 537.3 [M+H] +

[0665] Step 5: (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (P-1-6)

[0666] (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (1.3 g, 2.42 mmol) was dissolved in tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (1.26 g, 4.84 mmol) was added with stirring. The reaction was continued for 2 hours. Saturated brine was added, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, dried, and concentrated. Purification by silica gel column chromatography (methanol / dichloromethane = 0%–10%) yielded the title compound (0.8 g, 1.89 mmol).

[0667] Its structural characterization data are as follows:

[0668] ESI-MS (m / z): 423.1 [M+H] +

[0669] Step 6: (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -2,5,11(3H,10H)-trione (P-1-7)

[0670] (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (0.8 g, 1.89 mmol) was dissolved in dichloromethane (20 mL), and Dysmartin oxidant (2.01 g, 4.73 mmol) was added with stirring. The reaction was continued for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, washed three times with saturated brine, and then dried and concentrated. The crude product was used directly in the next reaction (0.65 g, 1.55 mmol).

[0671] Its structural characterization data are as follows:

[0672] ESI-MS (m / z): 421.1 [M+H] +

[0673] Step 7: (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of 2-yltrifluoromethanesulfonate (P-1-8)

[0674] (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -2,5,11(3H,10H)-trione (0.65 g, 1.55 mmol, 57.46 μmol) was dissolved in dichloromethane (20 mL), cooled and stirred to -40 °C under nitrogen purging and protection, and 2,6-dimethylpyridine (1.32 g, 12.37 mmol) was added dropwise, followed by slow dropwise addition of trifluoromethanesulfonic anhydride (3.05 g, 10.82 mmol). The reaction mixture was kept at this temperature for 1 hour. The reaction solution was quenched with water, extracted three times with dichloromethane (20 mL x 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (methanol / dichloromethane = 0%–5%) yielded the title compound (0.6 g, 1.09 mmol).

[0675] Its structural characterization data are as follows:

[0676] ESI-MS (m / z): 553.3 [M+H] +

[0677] Step 8: (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -5,11(10H)-dione (P-1-9)

[0678] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide 2-yltrifluoromethanesulfonate (0.6 g, 1.09 mmol) and (E)-(4-aminostyryl)boronic acid (210.88 mg, 1.29 mmol) were dissolved in a mixed solvent of toluene (20 mL), ethanol (5 mL), and water (5 mL). Tetra(triphenylphosphine)palladium (62.95 mg, 54.5 μmol) and sodium carbonate (346.62 mg, 3.27 mmol) were added, and the mixture was stirred at 80 °C for 2 hours under nitrogen purging and protection. After adding saturated brine (30 mL), the mixture was extracted twice with ethyl acetate (20 mL x 2). The organic phases were combined, dried, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (0.35 g, 670.91 μmol).

[0679] Its structural characterization data are as follows:

[0680] ESI-MS (m / z): 521.2 [M+H]+

[0681] Step Nine: (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of 5-one (P-1)

[0682] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (0.35 g, 670.91 μmol) was dissolved in dry tetrahydrofuran (10 mL), cooled and stirred to -78 °C under nitrogen purging and protection, and then triethyllithium borohydride solution (1.34 mL, 1.34 mmol, 1 M tetrahydrofuran solution) was added dropwise. The reaction was maintained at this temperature for 30 min. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by high performance liquid chromatography, the solution was freeze-dried to give the title compound (7.3 mg, 19.25 μmol).

[0683] Its structural characterization data are as follows:

[0684] ESI-MS (m / z): 376.2 [M+H]+

[0685] 1 H NMR (400MHz, CDCl3) δ7.88(d,J=3.8Hz,1H),7.52(s,1H),7.24(s,2H),7.09(s,1H),6.83(s,1H),6.81(d,J=18.2Hz,1H),6.66(d,J =8.2Hz,2H),6.34(d,J=16.0Hz,1H),4.41-4.34(m,1H),3.97(s,3H),3.95(s,3H),3.49-3.41(m,1H),3.27(dd,J=16.0,4.4Hz,1H).

[0686] Its preparation method is as follows:

[0687] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0688] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0689] Preparation Example 2: (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-5)

[0690] Step 1: Preparation of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)naphthyl-2-amine (P-5-2)

[0691] tert-butyl(6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)naphth-2-yl)carbamate (100 mg, 270.81 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure to give the crude title compound (72.89 mg, 270.81 μmol).

[0692] Its structural characterization data are as follows:

[0693] ESI-MS (m / z): 270.1 [M+H]+

[0694] Step 2: (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (P-5-3)

[0695] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide 2-yltrifluoromethanesulfonate (149.65 mg, 270.82 μmol) and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (72.89 mg, 270.81 μmol) were dissolved in a mixed solvent of 1,4-dioxane (4 mL) and water (1 mL). Pd(dppf)2Cl2 (7.64 mg, 27.08 μmol) and potassium carbonate (112.29 mg, 812.46 μmol) were added. The mixture was purged with nitrogen and protected under a stirred atmosphere at 80 °C for 2 hours. After the reaction solution was concentrated under reduced pressure, saturated saline (10 mL) and ethyl acetate (10 mL) were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, dried and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (110 mg, 201.58 μmol).

[0696] Its structural characterization data are as follows:

[0697] ESI-MS (m / z): 546.3 [M+H]+

[0698] Step 3: (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-5)

[0699] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (110 mg, 201.58 μmol) was dissolved in dry tetrahydrofuran (5 mL), cooled and stirred to -78 °C under nitrogen purging and protection, and triethyllithium borohydride (403.15 μL, 403.15 μmol, 1 M tetrahydrofuran solution) was slowly added dropwise. The reaction was maintained at this temperature for 30 minutes. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by high performance liquid chromatography, the mixture was freeze-dried to give the title compound (7.6 mg, 18.84 μmol).

[0700] Its structural characterization data are as follows:

[0701] ESI-MS (m / z): 400.3 [M+H]+

[0702] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=4.0Hz,1H),7.63(d,J=8.6Hz,1H),7.62-7.44(m,5H),7.01-6.93(m, 2H),6.85(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0703] Its preparation method is as follows:

[0704] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0705] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0706] Preparation Example 3: (S)-2-(7-aminoquinolin-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-9)

[0707] Step 1: Preparation of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)quinoline-7-amine (P-9-2)

[0708] 3-Bromoquinoline-7-amine (100.00 mg, 448.29 μmol) and pinacol diborate (227.68 mg, 896.58 μmol) were dissolved in 1,4-dioxane (5 mL). Pd(dppf)₂Cl₂ (32.80 mg, 44.83 μmol) and potassium acetate (131.99 mg, 1.34 mmol) were added. The mixture was purged with nitrogen for protection, and the temperature was raised to 90 °C with stirring for 2 hours. The reaction solution was then cooled to room temperature and proceeded directly to the next step of the reaction.

[0709] Its structural characterization data are as follows:

[0710] ESI-MS (m / z): 271.2 [M+H]+

[0711] Step 2: (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -5,11(10H)-dione (P-9-3)

[0712] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide 2-Methyltrifluoromethanesulfonate (149.65 mg, 270.82 μmol), Pd(dppf)₂Cl₂ (7.64 mg, 27.08 μmol), potassium carbonate (112.29 mg, 812.46 μmol), and water (1 mL) were added to the reaction system from the previous step. The mixture was purged with nitrogen and protected, and the temperature was raised to 80 °C with stirring for 2 hours. The reaction solution was concentrated under reduced pressure, and after adding saturated brine (10 mL), it was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (132 mg, 144.87 μmol).

[0713] Its structural characterization data are as follows:

[0714] ESI-MS (m / z): 548.3 [M+H]+

[0715] Step 3: (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-9)

[0716] (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (132 mg, 144.87 μmol) was dissolved in dry tetrahydrofuran (5 mL), purged with nitrogen and protected, and cooled and stirred to -78 °C. Triethyllithium borohydride (403.15 μL, 403.15 μmol, 1 M tetrahydrofuran solution) was added dropwise, and the reaction was continued for 30 minutes. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by high performance liquid chromatography, the mixture was freeze-dried to give the title compound (5.3 mg, 12.97 μmol).

[0717] Its structural characterization data are as follows:

[0718] ESI-MS (m / z): 401.2 [M+H]+

[0719] 1 H NMR (400MHz, CDCl3) δ7.96 (d, J = 4.0Hz, 1H), 7.63 (m, 3H), 7.55-7.52 (m, 2H), 7.00 (s, 2H), 6.8 6(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0720] Its preparation method is as follows:

[0721] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0722] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0723] Preparation Example 4: (S)-2-(6-hydroxynaphth-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-29)

[0724] Step 1: (S)-2-(6-hydroxynaphth-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -5,11(10H)-dione (P-29-1)

[0725] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide 2-yltrifluoromethanesulfonate (200.1 mg, 0.36 mmol) and (6-hydroxynaphth-2-yl)boronic acid (101.5 mg, 0.54 mmol) were dissolved in 1,4-dioxane (5 mL) and water (1 mL). Pd(dppf)₂Cl₂ (52.7 mg, 0.07 mmol) and potassium carbonate (99.5 mg, 0.72 mmol) were added. The mixture was heated to 80 °C and stirred for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (eluent: 0-35% methanol / dichloromethane) and then concentrated under reduced pressure to obtain the title compound (106 mg, 0.19 mmol).

[0726] Its structural characterization data are as follows:

[0727] MS m / z (ESI): 547.2 [M+H] +

[0728] Step 2: (S)-2-(6-hydroxynaphth-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-29)

[0729] (S)-2-(6-hydroxynaphth-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-dione (52 mg, 0.1 mmol) was dissolved in THF (2 mL), purged with nitrogen and protected, cooled and stirred to -78 °C, and then 1 M triethyllithium borohydride solution (192.60 μL) was added dropwise. The mixture was kept at this temperature and stirred for 1 hour. After adding 20 mL of water, the mixture was extracted twice with 15 mL of ethyl acetate. The organic phases were combined, dried, and concentrated to obtain the crude product. The crude product was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (7.41 mg, 18.2 μmol, 98.24% purity).

[0730] Its structural characterization data are as follows:

[0731] MS m / z (ESI): 401.1 [M+H] +

[0732] 1 H NMR (400MHz, CDCl3) δ7.95(d,J=3.3Hz,1H),7.71(d,J=8.6Hz,1H),7.63(d,J=8.4Hz,1H),7.57(d,J=10.1Hz,4H),7 .19-7.09(m,2H),6.87(s,1H),4.53-4.44(m,1H),3.98(s,3H),3.96(s,3H),3.76-3.66(m,1H),3.58-3.48(m,1H).

[0733] Its preparation method is as follows:

[0734] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0735] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0736] Preparation Example 5: (S,E)-2-(4-hydroxystyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-33)

[0737] Step 1: Preparation of 4-ethynylphenol (P-33-2)

[0738] 4-((trimethylsilyl)ethynyl)phenol (1 g, 5.25 mmol) was dissolved in THF (10 mL), and tetrabutylammonium fluoride solution (6.31 mmol, 6.31 mL, 1 M tetrahydrofuran solution) was added dropwise with stirring. Saturated ammonium chloride aqueous solution was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, washed three times with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–70%) yielded the title compound (0.52 g, 4.40 mmol).

[0739] Its structural characterization data are as follows:

[0740] ESI-MS (m / z): 117.1 [MH]

[0741] Step 2: Preparation of (E)-4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)vinyl)phenol (P-33-3)

[0742] 4-Ethynylphenol (0.2 g, 1.69 mmol) was dissolved in pinacolborane (2 mL), purged with nitrogen for protection, and the mixture was heated to 110 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature, quenched with water, and extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–90%) yielded the title compound (302 mg, 416.67 mg).

[0743] Its structural characterization data are as follows:

[0744] ESI-MS (m / z): 245.1 [MH]

[0745] Step 3: (S,E)-2-(4-hydroxystyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -5,11(10H)-dione (P-33-4)

[0746] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide 2-yltrifluoromethanesulfonate (280.66 mg, 406.32 μmol) and (E)-4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)vinyl)phenol (100 mg, 406.32 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL). Pd(dppf)₂Cl₂ (29.73 mg, 40.63 mmol) and potassium carbonate (168.47 mg, 1.22 mmol) were added. The mixture was heated to 80 °C and stirred for 2 hours under nitrogen purging and protection. The reaction solution was concentrated under reduced pressure, and after adding saturated brine (10 mL), it was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (130 mg, 248.73 μmol).

[0747] Its structural characterization data are as follows:

[0748] ESI-MS (m / z): 523.3 [M+H]+

[0749] Step 4: (S,E)-2-(4-hydroxystyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 5-one (P-33)

[0750] The (S,E)-2-(4-hydroxystyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide -5,11(10H)-diketone (130 mg, 248.73 μmol) was dissolved in dry tetrahydrofuran (10 mL), purged with nitrogen and protected, cooled and stirred to -78 °C, and triethyllithium borohydride (497.45 μL, 497.45 μmol, 1 M tetrahydrofuran solution) was added dropwise, and the reaction was continued for 30 minutes. The reaction was quenched with water, extracted three times with ethyl acetate (10 mL x 3), the organic phases were combined, washed with saturated brine, dried and concentrated. After purification by preparative high-performance liquid chromatography, the solution was freeze-dried to give the title compound (6.54 mg, 17.20 μmol).

[0751] Its structural characterization data are as follows:

[0752] ESI-MS (m / z): 377.1 [M+H]+

[0753] 1 H NMR (400MHz, CDCl3) δ7.90 (d, J = 4.0Hz, 1H), 7.52 (s, 1H), 7.34-7.29 (m, 2H), 7.12 (s, 1H), 6.91-6.85 (m, 2H), 6.83-6. 78(m,2H),6.36(d,J=4.0Hz,1H),4.42-4.37(m,1H),3.95(s,3H),3.93(s,3H),3.51-3.41(m,1H),3.31-3.24(m,1H).

[0754] Its preparation method is as follows:

[0755] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0756] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0757] Pyrrolobenzodiazepine Synthesis Examples of Compound Linkers

[0758] Example 1: N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-1 (-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0759] Step 1: (9H-fluorene-9-yl)methyl[(S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide] Preparation of 2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)carbamate (DL-1-1)

[0760] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide 5-ketoone (20 mg, 53.27 μmol) was dissolved in DMF (1 mL), and DIPEA (20.66 mg, 159.82 μmol), HATU (24.29 mg, 63.93 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (19.90 mg, 63.93 μmol) were added sequentially. The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18 column, water / acetonitrile = 1 / 2) and then freeze-dried to give the title compound (11 mg, 16.45 μmol). Its structural characterization data are as follows:

[0761] MS m / z (ESI): 669.3 [M+H] +

[0762] Step 2: (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of 2-yl)vinyl)phenyl)propionamide (DL-1-2)

[0763] The (9H-fluorene-9-yl)methyl[(S)-1-((4-(((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide] 2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)carbamate (11 mg, 16.45 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (6.68 mg, 14.96 μmol).

[0764] Its structural characterization data are as follows:

[0765] MS m / z (ESI): 447.1 [M+H] +

[0766] Step 3: (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) Preparation of DL-1-(2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (DL-1-3)

[0767] The (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) (-2-yl)vinyl)phenyl)propionamide (6.68 mg, 14.96 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (10.62 mg, 82.20 μmol), HATU (18.74 mg, 49.32 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (16.74 mg, 39.64 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (10 mg, 13.02 μmol). Its structural characterization data are as follows:

[0768] MS m / z (ESI): 768.3 [M+H] +

[0769] Step 4: (S)-2-amino-N-((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-1-(2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (DL-1-4)

[0770] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) 2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (10 mg, 13.02 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction mixture was reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (7.11 mg, 13.02 μmol).

[0771] Its structural characterization data are as follows:

[0772] MS m / z (ESI): 546.1 [M+H] +

[0773] Step 5: N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of DL-1 (-2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0774] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) 2-yl)vinyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (7.11 mg, 13.02 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (5.05 mg, 39.09 μmol) and 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (5.71 mg, 15.64 μmol). The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (2.36 mg, 2.92 μmol).

[0775] Its structural characterization data are as follows:

[0776] MS m / z (ESI): 796.3 [M+H] +

[0777] Its preparation method is as follows:

[0778] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0779] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0780] Example 2: N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-2 (-2-yl)vinyl)phenyl)amino)-1-oxo-5-ureapre-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0781] Step 1: (9H-fluorene-9-yl)methyl[(S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza] Preparation of [-2-yl)vinyl)phenyl)amino]-1-oxo-5-ureidopentane-2-yl)carbamate (DL-2-1)

[0782] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide 5-one (50 mg, 133.18 μmol) was dissolved in DMF (1 mL), and DIPEA (51.64 mg, 399.55 μmol), HATU (126.53 mg, 332.96 μmol), and (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidovalerate (158.79 mg, 399.55 μmol) were added sequentially with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-50% acetonitrile / 0.05% formic acid water) and then freeze-dried to give the title compound (10 mg, 13.25 μmol).

[0783] Its structural characterization data are as follows:

[0784] MS m / z (ESI): 755.3 [M+H] +

[0785] Step 2: (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of 2-yl)vinyl)phenyl)-5-ureidopentanamide (DL-2-2)

[0786] (9H-fluorene-9-yl)methyl[(S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza] [-2-yl)vinyl)phenyl)amino]-1-oxo-5-ureidopentan-2-yl)carbamate (10 mg, 13.25 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The solvent was removed from the reaction solution under vacuum to obtain the crude product of the title compound (theoretical amount: 7 mg, 13.14 μmol), which was directly used for the next reaction.

[0787] Its structural characterization data are as follows:

[0788] MS m / z (ESI): 533.4 [M+H] +

[0789] Step 3: N-((S)-1-(((S)-1-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-2 (-2-yl)vinyl)phenyl)amino)-1-oxo-5-ureapre-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0790] (S)-2-amino-N-(4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) (-2-yl)vinyl)phenyl)-5-ureidopentanamide crude product (theoretical mass: 7 mg, 13.14 μmol) was dissolved in DMF (1 mL), and DIPEA (8.49 mg, 65.72 μmol), HATU (12.49 mg, 32.86 μmol), and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (14.49 mg, 39.43 μmol) were added sequentially with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (10 mg, 13.02 μmol).

[0791] Its structural characterization data are as follows:

[0792] MS m / z (ESI): 882.4 [M+H] +

[0793] Its preparation method is as follows:

[0794] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0795] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0796] Example 3: N-((S)-1-(((S)-6-amino-1-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-4 (2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0797] Step 1: (9H-fluorene-9-yl)methyl(S,E)-(2-((4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) Preparation of DL-4-1 (2-yl)vinyl)phenyl)amino)-2-oxoethyl)carbamate

[0798] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide 5-one (50 mg, 133.18 μmol) was dissolved in DMF (1 mL), and DIPEA (51.64 mg, 399.55 μmol), HATU (126.53 mg, 332.96 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (118.79 mg, 399.55 μmol) were added sequentially with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-70% acetonitrile / 0.05% formic acid water) and then freeze-dried to give the title compound (43 mg, 13.25 μmol).

[0799] Its structural characterization data are as follows:

[0800] MS m / z (ESI): 655.3 [M+H] +

[0801] Step 2: (S,E)-2-amino-N-(4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) Preparation of 2-yl)vinyl)phenyl)acetamide (DL-4-2)

[0802] (9H-fluorene-9-yl)methyl(S,E)-(2-((4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) 2-yl)vinyl)phenyl)amino)-2-oxoethyl)carbamate (43 mg, 13.25 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The solvent was removed from the reaction solution under vacuum to obtain the crude product of the title compound (theoretical amount: 28 mg, 64.74 μmol).

[0803] Its structural characterization data are as follows:

[0804] MS m / z(ESI): 433.4 [M+H] +

[0805] Step 3: Allyl((S)-6-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-4-3 (-2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-6-oxohexyl)carbamate (DL-4-3)

[0806] (S,E)-2-amino-N-(4-(2-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) Crude acetamide (theoretical amount: 28 mg, 64.74 μmol) was dissolved in DMF (1 mL). Under stirring, DIPEA (25.10 mg, 194.23 μmol), HATU (27.06 mg, 71.22 μmol), and N6-((allyloxy)carbonyl)-N2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (45.04 mg, 71.22 μmol) were added sequentially. The reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-70% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (11 mg, 11.07 μmol).

[0807] Its structural characterization data are as follows:

[0808] MS m / z (ESI): 994.7 [M+H] +

[0809] Step 4: N-((S)-1-(((S)-6-amino-1-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-4 (2-yl)vinyl)phenyl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0810] Allyl((S)-6-((2-((4-((E)-2-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) 11 mg (11.07 μmol) of 2-(s)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)butamido)-6-oxohexyl)carbamate was dissolved in DMF (1 mL), and 1,3-dimethylbarbituric acid (5.18 mg, 33.20 μmol) and tetrakis(triphenylphosphine)palladium (1.28 mg, 1.11 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to give the title compound (2.23 mg, 2.43 μmol).

[0811] Its structural characterization data are as follows:

[0812] MS m / z (ESI): 929.2 [M+H2O+H] +

[0813] Its preparation method is as follows:

[0814] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

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

[0816] Example 4: N-((S)-1-(((S)-6-amino-1-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-5 (2-yl)naphth-2-yl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0817] Step 1: (9H-fluorene-9-yl)methyl(S)-(2-((6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) Preparation of 2-yl)naphth-2-yl)amino)-2-oxoethyl)carbamate (DL-5-1)

[0818] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide 5-one (30 mg, 60.08 μmol) was dissolved in DMF (1 mL), and pyridine (0.5 mL), EDCI (57.68 mg, 300.42 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (35.73 mg, 120.17 μmol) were added sequentially with stirring. The reaction was continued for 1 hour. After the solvent was removed by vacuum evacuation, the reaction solution was purified by reverse-phase column chromatography (eluent: 0-70% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (10 mg, 14.73 μmol).

[0819] Its structural characterization data are as follows:

[0820] MS m / z (ESI): 693.3 [M+H] +

[0821] Step 2: (S)-2-amino-N-(6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) Preparation of 2-yl)naphth-2-yl)acetamide (DL-5-2)

[0822] (9H-fluorene-9-yl)methyl(S)-(2-((6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) 2-yl)naphth-2-yl)amino)-2-oxoethyl)carbamate (10 mg, 14.73 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After removing the solvent from the reaction solution under vacuum, the crude product of the title compound was obtained (theoretical amount: 6.5 mg, 14.24 μmol).

[0823] Its structural characterization data are as follows:

[0824] MS m / z (ESI): 457.4 [M+H] +

[0825] Step 3: Allyl((S)-6-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-6-oxohexyl)carbamate (DL-5-3)

[0826] (S)-2-amino-N-(6-(7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide) (-2-yl)naphth-2-yl)acetamide (6.5 mg, 14.24 μmol) was dissolved in DMF (1 mL). While stirring, DIPEA (5.10 mg, 39.43 μmol), HATU (5.99 mg, 15.77 μmol), and N6-((allyloxy)carbonyl)-N2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (11.43 mg, 19.72 μmol) were added sequentially. The reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-60% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (11 mg, 10.80 μmol).

[0827] Its structural characterization data are as follows:

[0828] MS m / z (ESI): 1018.4 [M+H] +

[0829] Step 4: N-((S)-1-(((S)-6-amino-1-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-5 (2-yl)naphth-2-yl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)

[0830] Allyl((S)-6-((2-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) (-2-yl)naphth-2-yl)amino)allyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-6-oxohexyl)carbamate (11 mg, 10.80 μmol) was dissolved in DMF (1 mL), and 1,3-dimethylbarbituric acid (5.06 mg, 32.41 μmol) and tetrakis(triphenylphosphine)palladium (1.25 mg, 1.08 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to give the title compound (2.14 mg, 2.14 μmol).

[0831] Its structural characterization data are as follows:

[0832] MS m / z (ESI): 934.5 [M+H2O+H] +

[0833] Its preparation method is as follows:

[0834] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0835] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0836] Example 5: N-((S)-1-(((S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-6 (2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-6)

[0837] Step 1: (9H-fluorene-9-yl)methyl[(S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza] Preparation of 2-yl)naphth-2-yl)amino]-1-oxopropyl-2-yl)carbamate

[0838] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazide 5-one (30 mg, 60.08 μmol) was dissolved in DMF (1 mL), and pyridine (0.5 mL), EDCI (57.68 mg, 300.42 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (28.06 mg, 90.13 μmol) were added sequentially with stirring. The reaction was continued for 1 hour. After the solvent was removed by vacuum evacuation, the reaction solution was purified by reverse-phase column chromatography (eluent: 0-65% acetonitrile / 0.05% formic acid water) and then freeze-dried to obtain the title compound (19 mg, 27.43 μmol).

[0839] Its structural characterization data are as follows:

[0840] MS m / z (ESI): 693.3 [M+H]+

[0841] Step 2: (S)-2-amino-N-(6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 2-yl)naphth-2-yl)propionamide (DL-6-2)

[0842] (9H-fluorene-9-yl)methyl[(S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza] [-2-yl)naphthyl-2-yl)amino]-1-oxopropyl-2-yl)carbamate (19 mg, 27.43 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After removing the solvent from the reaction solution under vacuum, the crude product of the title compound was obtained (theoretical amount: 12 mg, 25.50 μmol).

[0843] Its structural characterization data are as follows:

[0844] MS m / z (ESI): 471.3 [M+H] +

[0845] Step 3: N-((S)-1-(((S)-1-((6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of DL-6 (2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-6)

[0846] (S)-2-amino-N-(6-((S)-7,8-dimethoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza) (-2-yl)naphth-2-yl)propionamide (6 mg, 12.75 μmol) was dissolved in DMF (1 mL). With stirring, DIPEA (4.94 mg, 38.26 μmol), HATU (7.27 mg, 19.13 μmol), and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (5.62 mg, 15.30 μmol) were added in succession, and the reaction was continued for 2 hours. The reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound (1.3 mg, 1.57 μmol).

[0847] Its structural characterization data are as follows:

[0848] MS m / z (ESI): 820.3 [M+H] +

[0849] Its preparation method is as follows:

[0850] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0851] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0852] Example 6: 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -10(5H)-carbonate (PL-1):

[0853] 2,5-Dioxopyrrolidine-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (5.13 mg, 14.03 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (10 mg, 14.03 μmol) was dissolved in DMF (1 mL), and DIPEA (5.44 mg, 42.09 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (1.58 mg, 1.56 μmol).

[0854] Its structural characterization data are as follows:

[0855] MS m / z (ESI): 963.4 [M+H] +

[0856] Its preparation method is as follows:

[0857] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0858] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0859] Example 7: 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-3)

[0860] Step 1: Preparation of allyl (4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrole-3-yl)vinyl)phenyl)carbamate (PL-3-2)

[0861] Allyl (S,E)-(4-(2-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (0.5 g, 842.06 μmol) was dissolved in dry tetrahydrofuran (10 mL), cooled and stirred to -10 °C under nitrogen purging and protection, and then triethylamine (119.29 mg, 1.18 mmol) was added dropwise, followed by slow dropwise addition of triphosgene (84.96 mg, 286.30 μmol, dissolved in 5 mL of dry tetrahydrofuran). A solution of 2 mL of dry tetrahydrofuran was reacted, and after 10 minutes, a mixture of allyl ((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (581.75 mg, 842.06 μmol) and DMAP (144.02 mg, 1.18 mmol) was slowly added dropwise (dissolved in 2 mL of dry tetrahydrofuran and 2 mL of dry DMF). The mixture was allowed to return to room temperature for 1 hour. The reaction solution was quenched with water, and ethyl acetate and saturated brine were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (1 g, 762.98 μmol).

[0862] Its structural characterization data are as follows:

[0863] MS m / z (ESI): 1311.7 [M+H] +

[0864] Step 2: Preparation of allyl (4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrole-3-yl)vinyl)phenyl)carbamate (PL-3-3)

[0865] Allyl (4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (1 g, 762.98 μmol) was dissolved in tetrahydrofuran (5 mL), and a tetrabutylammonium fluoride tetrahydrofuran solution (1 M, 1.52 mL) was added dropwise with stirring, and the reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 20-75% ethyl acetate / petroleum ether) to obtain the title compound (820 mg, 685.4 μmol).

[0866] Its structural characterization data are as follows:

[0867] MS m / z (ESI): 1197.4 [M+H] +

[0868] Step 3: 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-3-4)

[0869] Dry DMSO (165.00 mg, 2.11 mmol) was dissolved in dry dichloromethane (10 mL), cooled and stirred to -78 °C under nitrogen purging and protection, and oxaloyl chloride (112.50 mg, 886.32 μmol) was slowly added dropwise. After reacting for 5 minutes, allyl(4-((E)-2-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-(( Diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)vinyl)phenyl)carbamate (820 mg, 685.4 μmol, dissolved in 5 mL of dry dichloromethane) solution was added and the reaction was continued at this temperature for 0.5 hours. Triethylamine (218.40 mg, 2.16 mmol) was added dropwise, and the mixture was allowed to cool naturally to -35 °C for 0.5 hours. The reaction solution was quenched with water, and after returning to room temperature, ethyl acetate and saturated brine were added. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (400 mg, 334.9 μmol).

[0870] Its structural characterization data are as follows:

[0871] MS m / z (ESI): 1195.4 [M+H] +

[0872] Step 4: 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-3-5)

[0873] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-(((allyloxy)carbonyl)amino)styryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (400 mg, 334.9 μmol) was dissolved in DMF (4 mL), and 1,3-dimethylbarbituric acid (78.37 mg, 502.36 μmol) and tetrakis(triphenylphosphine)palladium (19.34 mg, 16.75 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% formic acid aqueous solution) and then freeze-dried to give the title compound (310 mg, 302.08 μmol).

[0874] Its structural characterization data are as follows:

[0875] MS m / z (ESI): 1026.8 [M+H] +

[0876] Step 5: 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-3-6)

[0877] 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (310 mg, 302.08 μmol) was dissolved in DMF (5 mL), and DIPEA (58.56 mg, 453.12 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (121.4 mg, 166.14 μmol) were added sequentially. The reaction was continued for 1 hour. Ethyl acetate and water were added to the reaction solution and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine, dried, and concentrated under reduced pressure to obtain the crude product of the title compound (380 mg, 297.68 μmol), which was directly used for the next reaction.

[0878] Its structural characterization data are as follows:

[0879] MS m / z (ESI): 1277.2 [M+H] +

[0880] Step Six: 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-3)

[0881] 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (380 mg, 297.68 μmol) was dissolved in dichloromethane (5 mL), and formic acid (4 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure, purified by high performance liquid chromatography, and then freeze-dried to give the title compound (120 mg, 117.62 μmol).

[0882] Its structural characterization data are as follows:

[0883] MS m / z(ESI): 1021.3 [M+H] +

[0884] Its preparation method is as follows:

[0885] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0886] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0887] Example 8: N5-((S)-1-(((S)-1-((4-((((11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (PL-5):

[0888] Step 1: Preparation of 1-allyl-5-(tert-butyl)[((9H-fluorene-9-yl)methoxy)carbonyl]-L-glutamate ester (PL-5-2):

[0889] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (20 g, 47.01 mmol) and 3-bromoprop-1-ene (7.39 g, 61.11 mmol, 5.29 mL) were dissolved in DMF (100 mL). The mixture was cooled and stirred to 0 °C, and DIPEA (8.51 g, 65.81 mmol, 11.46 mL) was added dropwise. The mixture was allowed to return to room temperature naturally for 16 hours. Water and ethyl acetate were added, and the mixture was allowed to stand and separate. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-25% ethyl acetate / petroleum ether) to give the title compound (20.8 g, 44.68 mmol).

[0890] Step 2: Preparation of 1-allyl-5-(tert-butyl)-L-glutamate (PL-5-3):

[0891] 1-Allyl-5-(tert-butyl)[((9H-fluorene-9-yl)methoxy)carbonyl]-L-glutamate (20.8 g, 44.68 mmol) was dissolved in DMF (200 mL), and diethylamine (20 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. Water and ethyl acetate were added, and the mixture was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, and concentrated under reduced pressure. The residue was added to methyl tert-butyl ether with stirring, filtered, and the filtrate was concentrated under reduced pressure to give the crude product of the title compound (10 g, 41.10 mmol), which was directly used for the next reaction.

[0892] Its structural characterization data are as follows:

[0893] MS m / z(ESI): 244.2 [M+H] +

[0894] Step 3: Preparation of 1-allyl-5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamate (PL-5-4):

[0895] 6-(2-Methylsulfonylmyn-5-yl)hex-5-alkynic acid (11.03 g, 41.10 mmol) and 1-allyl-5-(tert-butyl)-L-glutamate (10 g, 41.10 mmol) were dissolved in DMF (100 mL). HATU (20.30 g, 53.43 mmol) was added with stirring, followed by dropwise addition of DIPEA (15.94 g, 123.31 mmol, 21.48 mL). The reaction was continued for 1 hour. Water and ethyl acetate were added, and the mixture was filtered. The filtrate was allowed to stand and separated. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, and concentrated. The purified compound (20.2 g, 40.93 mmol) was obtained by silica gel column chromatography (eluent: 0-65% ethyl acetate / petroleum ether).

[0896] Step 4: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxovaleric acid (PL-5-5):

[0897] 1-Allyl-5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamate (20 g, 40.52 mmol) was dissolved in dichloromethane (200 mL), and trifluoroacetic acid (50 mL) was added dropwise with stirring. The reaction was continued for 2 hours. The reaction solution was concentrated under reduced pressure, purified by reverse-phase column chromatography (eluent: 0-36% acetonitrile / 0.5% trifluoroacetic acid in water), and lyophilized to give the title compound (13.88 g, 31.73 mmol).

[0898] Its structural characterization data are as follows:

[0899] MS m / z (ESI): 438.1 [M+H] +

[0900] Step 5: 4-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxopentanamido)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyrene)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of -10(5H)-carbonate (PL-5-6):

[0901] (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxovaleric acid (14.73 mg, 33.67 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (24 mg, 33.67 μmol) was dissolved in DMF (1 mL), and HATU (19.19 mg, 50.51 μmol) was added with stirring, followed by dropwise addition of DIPEA (13.05 mg, 101.01 μmol, 18.03 μL). The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (15 mg, 13.25 μmol).

[0902] Its structural characterization data are as follows:

[0903] MS m / z(ESI): 1133.3 [M+H] +

[0904] Its preparation method is as follows:

[0905] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0906] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0907] Step Six: N5-((S)-1-(((S)-1-((4-((((11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazide Preparation of -10-carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (PL-5):

[0908] 4-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxopentanamido)-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyrene)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (15 mg, 13.25 μmol), tetrakis(triphenylphosphine)palladium (1.53 mg, 1.32 μmol), and 1,3-dimethylbarbituric acid (2.07 mg, 13.25 μmol) were dissolved in DMF (1 mL). The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (3.15 mg, 2.77 μmol).

[0909] Its structural characterization data are as follows:

[0910] MS m / z(ESI): 1092.3 [M+H] +

[0911] Its preparation method is as follows:

[0912] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0913] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0914] Example 9: 4-((2S,5S,10S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)) (I)pyrimidin-5-yl)hex-5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapentacarbamate)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-7):

[0915] (S)-5-(((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane-13-yl)amino)-4 -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)-5-oxopentanoic acid (35.03 mg, 28.06 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (20 mg, 28.06 μmol) and HATU (12.79 mg, 33.67 μmol) were dissolved in DMF (1 mL), and DIPEA (7.25 mg, 56.12 μmol) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (13.67 mg, 6.89 μmol).

[0916] Its structural characterization data are as follows:

[0917] MS m / z(ESI): 972.0 [1 / 2M+H] +

[0918] Its preparation method is as follows:

[0919] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0920] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0921] Example 10: 4-((2S,5S,10S,37S,38R,39R,40R)-37,38,39,40,41-pentahydroxy-5-isopropyl-2,35-dimethyl-29,29-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl) (hexyl-5-acetylamido)-4,7,11,27,34-pentoxo-15,18,21,24,31-pentoxa-3,6,12,28,35-pentazanonadecanamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-8):

[0922] Step 1: Preparation of 1-amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azacosanodecane-22-acid (PL-8-2):

[0923] 17,17-bis((2-carboxyethoxy)methyl)-15-oxo-1-triazyl-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (670 mg, 1.10 mmol) was dissolved in tetrahydrofuran (5 mL), and 10% palladium on carbon (50 mg, 109.73 μmol) was added. The reaction mixture was stirred for 18 hours under hydrogen purging and protection. The reaction solution was filtered through a diatomaceous earth filter, and the filtrate was concentrated under reduced pressure to give the crude title compound (640 mg, 1.09 mmol).

[0924] Its structural characterization data are as follows:

[0925] MS m / z (ESI): 585.2 [M+H] +

[0926] Step 2: Preparation of 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (PL-8-3):

[0927] 1-Amino-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocoadecan-22-acid (950 mg, 1.63 mmol) was dissolved in a mixture of acetonitrile (10 mL) and water (10 mL). Sodium bicarbonate (682.51 mg, 8.13 mmol) was added with stirring, followed by 9-fluorenemethyl-N-succinimide carbonate (657.74 mg, 1.95 mmol). The reaction mixture was stirred for 5 hours. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-50% acetonitrile / 0.5% formic acid in water) and lyophilized to give the title compound (870 mg, 1.08 mmol).

[0928] Its structural characterization data are as follows:

[0929] MS m / z (ESI): 807.3 [M+H] +

[0930] Step 3: Preparation of (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (PL-8-4):

[0931] 21,21-bis((2-carboxyethoxy)methyl)-1-(9H-fluorene-9-yl)-3,19-dioxo-2,7,10,13,16,23-hexaoxa-4,20-diazahexacosane-26-acid (870 mg, 1.08 mmol), meglumine (841.97 mg, 4.31 mmol), and HATU (2.05 g, 5.39 mmol) were dissolved in DMF (10 mL). DIPEA (696.78 mg, 5.39 mmol, 939.05 μL) was added dropwise with stirring, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-45% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0932] Its structural characterization data are as follows:

[0933] MS m / z (ESI): 1338.6 [M+H] +

[0934] Step 4: Preparation of 3,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(PL-8-5):

[0935] (9H-fluorene-9-yl)methyl[(25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis[(3-(methyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino]-3-oxopropoxy]methyl]-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazaeicosyl]carbamate (200 mg, 149.43 μmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The solvent in the reaction solution was evaporated under reduced pressure, ethyl acetate and water were added and stirred, and the mixture was allowed to stand and separated. The aqueous phase was lyophilized to give the crude product of the title compound (166 mg, 148.72 μmol).

[0936] Its structural characterization data are as follows:

[0937] MS m / z (ESI): 1116.6 [M+H] +

[0938] Step 5: Preparation of allyl(4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoate (PL-8-6):

[0939] (S)-5-(allyloxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxopentanoic acid (65.06 mg, 148.72 μmol), 33,3'-((2-(1-amino-3,6,9,12-tetraoxapentadecano-15-amido)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl 166 mg (148.72 μmol) of propane-1,3-diyl)bis(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (2 mL) was dissolved in DMF. HATU (84.99 mg, 223.66 μmol) was added with stirring, followed by dropwise addition of DIPEA (57.66 mg, 446.16 μmol, 77.71 μL). The reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to obtain the title compound (145 mg, 94.42 μmol).

[0940] Its structural characterization data are as follows:

[0941] MS m / z (ESI): 1535.5 [M+H] +

[0942] Its preparation method is as follows:

[0943] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0944] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0945] Step Six: Preparation of (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoic acid (PL-8-7):

[0946] Allyl(4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,2 1,28-trioxo-9,12,15,18,25-pentaoxa-6,22,29-triazapentadecanoate (145 mg, 94.42 μmol) was dissolved in DMF (3 mL), and tetrakis(triphenylphosphine)palladium (10.91 mg, 9.44 μmol) and 1,3-dimethylbarbituric acid (14.74 mg, 94.42 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (108 mg, 72.21 μmol).

[0947] Its structural characterization data are as follows:

[0948] MS m / z (ESI): 1496.5 [M+H] +

[0949] Its preparation method is as follows:

[0950] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0951] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0952] Step 7: 4-((2S,5S,10S,37S,38R,39R,40R)-37,38,39,40,41-pentahydroxy-5-isopropyl-2,35-dimethyl-29,29-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl) -5-acetylamido)-4,7,11,27,34-pentoxo-15,18,21,24,31-pentoxa-3,6,12,28,35-pentazanonadecanamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-8):

[0953] (4S,31S,32R,33R,34R)-31,32,33,34,35-pentahydroxy-29-methyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5,21,28-trioxo-9,12,15,18, 25-Pentaoxa-6,22,29-triazapentapentadecanoic acid (27.28 mg, 18.24 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (13 mg, 18.24 μmol) was dissolved in DMF (1 mL), and HATU (8.32 mg, 21.89 μmol) was added with stirring. DIPEA (4.71 mg, 36.48 μmol, 6.35 μL) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (2.57 mg, 1.16 μmol).

[0954] Its structural characterization data are as follows:

[0955] MS m / z (ESI): 1095.5 [1 / 2M+H] +

[0956] Its preparation method is as follows:

[0957] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0958] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0959] Example 11: 4-((2S,5S,8S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl) )pyrimidin-5-yl)hex-5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapentacarbamate)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-9):

[0960] Step 1: Preparation of allyl N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (PL-9-2):

[0961] (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-5-oxovaleric acid (200 mg, 457.18 μmol), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl) Di(oxy)bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide (436.97 mg, 502.90 μmol) was dissolved in DMF (5 mL), and HATU (208.47 mg, 548.61 μmol) was added with stirring. DIPEA (177.26 mg, 1.37 mmol) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-35% acetonitrile / 0.5% formic acid water), and lyophilized to give the title compound (1.05 g, 781.51 μmol).

[0962] Its structural characterization data are as follows:

[0963] MS m / z (ESI): 1338.6 [M+H] +

[0964] Step 2: Preparation of N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (PL-9-3):

[0965] Allyl N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19- (diazapentadecane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (311 mg, 241.39 μmol), tetra(triphenylphosphine)palladium (27.89 mg, 24.14 μmol), and 1,3-dimethylbarbituric acid (37.69 mg, 241.39 μmol) were dissolved in DMF (6 mL). The reaction mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-30% acetonitrile / 0.5% trifluoroacetic acid in water) and lyophilized to give the title compound (292 mg, 233.92 μmol).

[0966] Its structural characterization data are as follows:

[0967] MS m / z (ESI): 1249.4 [M+H] +

[0968] Step 3: 4-((2S,5S,8S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)) (pyrimidin-5-yl)hex-5-ynamide-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapentacarbamate-)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza Preparation of 10(5H)-carbonate (PL-9):

[0969] N5-((2R,3R,4R,5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazatocetane-13-yl)-N2- (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (15 mg, 12.02 μmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza -10(5H)-carbonate (8.57 mg, 12.02 μmol) was dissolved in DMF (1 mL), and HATU (5.02 mg, 13.22 μmol) was added with stirring. DIPEA (3.11 mg, 24.03 μmol, 4.19 μL) was added dropwise, and the reaction was continued for 1 hour. The reaction solution was directly purified by rapid column chromatography and then freeze-dried to give the title compound (2.57 mg, 1.16 μmol).

[0970] Its structural characterization data are as follows:

[0971] MS m / z (ESI): 1095.5 [1 / 2M+H] +

[0972] Its preparation method is as follows:

[0973] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0974] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0975] Example 12: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)) (pyrimidin-5-yl)hex-5-ynamide)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentacarbamate)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapeco ... Preparation of 10(5H)-carbonate (PL-10)

[0976] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza 10(5H)-carbonate (50 mg, 49.01 μmol) was dissolved in DMF (1 mL). While stirring, DIPEA (18.99 mg, 147.03 μmol), HATU (22.35 mg, 58.81 μmol), and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (70.28 mg, 73.52 μmol) were added sequentially. The reaction was continued for 2 hours. The reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound formate (24.7 mg, 11.95 μmol).

[0977] Its structural characterization data are as follows:

[0978] MS m / z(ESI): 1958.7 [M+H] +

[0979] Its preparation method is as follows:

[0980] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0981] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0982] Example 13: 4-((S)-9,9-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-1-hydroxy-2,2-bis(hydroxymethyl)-20-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)butamido)-4,14-dioxo-7,11-dioxa-3,15-diazaeicosico-21-amido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazae Preparation of 10(5H)-carbonate (PL-11)

[0983] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-((E)-4-aminostyryl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza 10(5H)-carbonate (10 mg, 9.80 μmol) was dissolved in DMF (1 mL). While stirring, DIPEA (3.80 mg, 29.41 μmol), HATU (4.10 mg, 10.78 μmol), and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (7.91 mg, 10.78 μmol) were added sequentially. The reaction was continued for 2 hours. The reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound formate (3.52 mg, 2.01 μmol).

[0984] Its structural characterization data are as follows:

[0985] MS m / z (ESI): 1736.6 [M+H] +

[0986] Its preparation method is as follows:

[0987] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0988] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0989] Example 14: 4-((2S,5S,10S,37S,38R,39R,40R)-37,38,39,40,41-pentahydroxy-5-isopropyl-2,35-dimethyl-29,29-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)- Preparation of 4,7,11,27,34-pentoxa-15,18,21,24,31-pentoxa-3,6,12,28,35-pentazatetradecanoamide)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (PL-12)

[0990] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (12 mg, 16.29 μmol) was dissolved in DMF (1 mL), and HATU (6.81 mg, 17.92 μmol), DIPEA (6.31 mg, 48.86 μmol), and (S)-2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-N1-((25S,26R,27R,28R)-25,26,27,28,29-pentahydroxy-23-methyl-17,17-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-15,22-dioxo-3,6,9,12,19-pentaoxa-16,23-diazanonyl)pentanediamide (26.78 mg, 17.92 μmol, INT-8), reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to give the title compound (7.84 mg, 3.40 μmol).

[0991] Its structural characterization data are as follows:

[0992] ESI-MS (m / z): 1107.6 [M / 2+H] +

[0993] Its preparation method is as follows:

[0994] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0995] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0996] Example 15: 4-((2S,5S,8R,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-5-isopropyl-2,19-dimethyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex- Preparation of 5-acetylamido)-4,7,11,18-tetraoxo-15-oxa-3,6,12,19-tetraazapecopentacarbamate)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-10(5H)-carboxylic acid ester (PL-13)

[0997] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (12 mg, 16.29 μmol) was dissolved in DMF (1 mL), and HATU (6.81 mg, 17.92 μmol), DIPEA (6.31 mg, 48.86 μmol), and N5-((2R,3R,4R, 5S,21S,22R,23R,24R)-1,2,3,4,5,21,22,23,24,25-Decahydroxy-7,19-dimethyl-13-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapecopentane-13-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-D-glutamine (22.36 mg, 17.92 μmol, INT-5), reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to give the title compound (4.86 mg, 2.32 μmol).

[0998] Its structural characterization data are as follows:

[0999] ESI-MS (m / z): 1967.6 [M+H] +

[1000] Its preparation method is as follows:

[1001] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1002] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1003] Example 16: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5 Preparation of (-acetylamido)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazatenecapentanoamide)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazatene-10(5H)-carboxylic acid ester (PL-14)

[1004] Step 1: Preparation of allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (PL-14-1)

[1005] Allyl (S)-(6-(1-(2-amino-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (200 mg, 323.73 μmol) was dissolved in dry tetrahydrofuran (5 mL), cooled and stirred to -10 °C under nitrogen purging and protection, and then triethylamine (45.86 mg, 453.22 μmol) was added dropwise, followed by the slow addition of triphosgene (31.70 mg, 106.83 μmol, dissolved in 5 mL dry tetrahydrofuran). A solution of dry tetrahydrofuran was reacted, and after 10 minutes, a mixed solution of allyl ((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (223.65 mg, 323.73 μmol) and DMAP (55.37 mg, 453.22 μmol) was slowly added dropwise (dissolved in 1 mL of dry tetrahydrofuran and 1 mL of dry DMF). The mixture was allowed to return to room temperature naturally for 1 hour. The reaction solution was quenched with water, and ethyl acetate and saturated brine were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate, and the organic phases were combined, washed three times with saturated brine, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain the title compound (340 mg, 203.80 μmol).

[1006] Its structural characterization data are as follows:

[1007] MS m / z (ESI): 1336.5 [M+H] +

[1008] Step 2: Preparation of allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (PL-14-2)

[1009] Allyl (6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (340 mg, 203.80 μmol) was dissolved in tetrahydrofuran (5 mL), and a tetrabutylammonium fluoride tetrahydrofuran solution (1 M, 1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: 20-75% ethyl acetate / petroleum ether) to obtain the title compound (180 mg, 120.94 μmol).

[1010] Its structural characterization data are as follows:

[1011] MS m / z(ESI): 1221.4 [M+H] +

[1012] Step 3: Preparation of 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (PL-14-3)

[1013] Dry DMSO (34.57 mg, 442.48 μmol) was dissolved in dry dichloromethane (10 mL). The solution was cooled and stirred to -78 °C under nitrogen purging and protection. Oxaloyl chloride (28.08 mg, 221.24 μmol) was slowly added dropwise. After reacting for 5 minutes, allyl(6-((S)-1-(2-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl( A solution of p-tolyl)methyl)amino)hexamido)benzyl)oxy)carbonyl)amino)-4,5-dimethoxybenzoyl)-5-(hydroxymethyl)-4,5-dihydro-1H-pyrrolo-3-yl)naphth-2-yl)carbamate (180 mg, 120.94 μmol, dissolved in 5 mL of dry dichloromethane) was prepared and the reaction was continued at this temperature for 0.5 h. Triethylamine (74.62 mg, 737.46 μmol) was added dropwise, and the mixture was allowed to cool naturally to -35 °C for 0.5 h. The reaction solution was quenched with water, and after returning to room temperature, ethyl acetate and saturated brine were added. The mixture was allowed to stand and separated. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, dried, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: 0-70% ethyl acetate / petroleum ether) to obtain the title compound (112 mg, 91.92 μmol).

[1014] Its structural characterization data are as follows:

[1015] MS m / z (ESI): 1219.5 [M+H] +

[1016] Step 4: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (PL-14-4)

[1017] 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-(((allyloxy)carbonyl)amino)naphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (40 mg, 32.83 μmol) was dissolved in DMF (1 mL), and 1,3-dimethylbarbituric acid (15.36 mg, 98.49 μmol) and tetrakis(triphenylphosphine)palladium (3.79 mg, 3.28 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% formic acid aqueous solution) and then freeze-dried to give the title compound (26 mg, 22.28 μmol).

[1018] Its structural characterization data are as follows:

[1019] MS m / z (ESI): 1050.5 [M+H] +

[1020] Step 5: Preparation of 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-10(5H)-carboxylic acid ester (PL-14-5)

[1021] 4-((S)-2-((S)-2-amino-3-methylbutamido)-6-((diphenyl(p-tolyl)methyl)amino)hexamido)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (26 mg, 22.28 μmol) was dissolved in DMF (1 mL), and HATU (11.29 mg, 29.71 μmol), DIPEA (9.60 mg, 74.27 μmol), and 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-alkynic acid (7.97 mg, 29.71 μmol) were added sequentially. The reaction was carried out for 1 hour. Ethyl acetate and water were added to the reaction solution and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine and dried. The solution was concentrated under reduced pressure to obtain the crude product of the title compound (34 mg, 23.84 μmol), which was then directly used for the next reaction.

[1022] Its structural characterization data are as follows:

[1023] MS m / z (ESI): 1301.4 [M+H] +

[1024] Step Six: Preparation of 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (PL-14-6)

[1025] 4-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (34 mg, 23.84 μmol) was dissolved in dichloromethane (1 mL), and formic acid (1 mL) was added dropwise with stirring. The reaction was continued for 1 hour. The reaction solution was concentrated and purified by reverse-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% trifluoroacetic acid in water), followed by freeze-drying to obtain the title compound (14 mg, 13.41 μmol).

[1026] Its structural characterization data are as follows:

[1027] MS m / z (ESI): 1044.4 [M+H] +

[1028] Step 7: 4-((2S,21S,22R,23R,24R)-21,22,23,24,25-pentahydroxy-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5- Preparation of acetylamido)butamido)-8,18-dioxo-11,15-dioxa-7,19-diazatenecapentanoamide)benzyl(11S,11aS)-2-(6-aminonaphth-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazatene-10(5H)-carboxylic acid ester (PL-14)

[1029] 4-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)hexamido)benzyl(11S,11aS)-2-(6-aminonaphthyl-2-yl)-11-hydroxy-7,8-dimethoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-10(5H)-carboxylic acid ester (14 mg, 13.41 μmol) was dissolved in DMF (1 mL), and then added sequentially... HATU (5.60 mg, 14.75 μmol), DIPEA (5.20 mg, 40.22 μmol), and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (15.38 mg, 16.09 μmol) were reacted for 1 hour. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (7.56 mg, 3.74 μmol).

[1030] Its structural characterization data are as follows:

[1031] ESI-MS (m / z): 1982.8 [M+H] +

[1032] Its preparation method is as follows:

[1033] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1034] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1035] Coupling Examples

[1036] 1. Preparation of the antibody-drug conjugate Trastuzumab-DL-1

[1037] Take 0.65 mL of Trastuzumab antibody (24 mg / mL), dilute with 47.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 59.1 μL, pH 7.60) solution, mix well, and incubate at room temperature for 1.5 h. Add DL-1 solution dissolved in dimethyl sulfoxide (109.2 μL, 10 mM, 10 molar equivalents of the antibody), mix well, and incubate at room temperature for 3 h. After completion, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-1). The DAR value was determined by mass spectrometry to be 5.47.

[1038] 2. Preparation of the antibody-drug conjugate Trastuzumab-DL-2

[1039] 0.476 mL of Trastuzumab antibody (22.5 mg / mL) was diluted with 23.8 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 40.6 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 112.2 μL of DL-2 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-2). The DAR value was determined by mass spectrometry to be 5.80.

[1040] 3. Preparation of the antibody-drug conjugate Trastuzumab-DL-4

[1041] 0.667 mL of Trastuzumab antibody (22.5 mg / mL) was diluted with 33.3 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 156.6 μL of DL-4 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-4). The DAR value was 6.22 as determined by mass spectrometry.

[1042] 4. Preparation of the antibody-drug conjugate Trastuzumab-DL-5

[1043] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 166.0 μL of DL-5 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-5). The DAR value was determined by mass spectrometry to be 6.90.

[1044] 5. Preparation of the antibody-drug conjugate Trastuzumab-DL-6

[1045] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 156.6 μL of DL-6 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-6). The DAR value was determined by mass spectrometry to be 5.11.

[1046] 6. Preparation of the antibody-drug conjugate Trastuzumab-PL-1

[1047] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. PL-1 solution dissolved in dimethyl sulfoxide (163.2 μL, 10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and incubated at room temperature for 2 h. Afterward, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-1). The DAR value was 6.45 as determined by mass spectrometry.

[1048] 7. Preparation of the antibody-drug conjugate Trastuzumab-PL-3

[1049] 0.985 mL of Trastuzumab antibody (20.3 mg / mL) was diluted with 49.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 208.8 μL of PL-3 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-3). The DAR value was determined by mass spectrometry to be 8.08.

[1050] 8. Preparation of the antibody-drug conjugate Trastuzumab-PL-5

[1051] 0.781 mL of Trastuzumab antibody (19.2 mg / mL) was diluted with 39.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 161.5 μL of PL-5 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-5). The DAR value was determined by mass spectrometry to be 6.25.

[1052] 9. Preparation of the antibody-drug conjugate Trastuzumab-PL-7

[1053] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 55.7 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 134.4 μL of PL-7 solution dissolved in dimethyl sulfoxide (10 mM, 13 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-7). The DAR value was determined by mass spectrometry to be 7.85.

[1054] 10. Preparation of the antibody-drug conjugate Trastuzumab-PL-8

[1055] 0.36 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 18.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 28.1 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 75.3 μL of PL-8 solution dissolved in dimethyl sulfoxide (10 mM, 14 molar equivalents of the antibody) was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-8). The DAR value was determined by mass spectrometry to be 8.15.

[1056] 11. Preparation of the antibody-drug conjugate Trastuzumab-PL-9

[1057] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 55.7 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 133.0 μL of PL-9 solution dissolved in dimethyl sulfoxide (10 mM, 13 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-9). The DAR value was 7.08 as determined by mass spectrometry.

[1058] 12. Preparation of the antibody-drug conjugate Trastuzumab-PL-10

[1059] 0.728 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 36.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 146.1 μL of PL-10 solution dissolved in dimethyl sulfoxide (10 mM, 14 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-10). The DAR value was determined by mass spectrometry to be 8.0.

[1060] 13. Preparation of the antibody-drug conjugate Trastuzumab-PL-11

[1061] 0.728 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 36.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 146.1 μL of PL-11 solution dissolved in dimethyl sulfoxide (10 mM, 14 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-11). The DAR value was determined to be 8.0 by mass spectrometry.

[1062] 14. Preparation of the antibody-drug conjugate Trastuzumab-PL-12

[1063] 0.769 mL of Trastuzumab antibody (19.09 mg / mL) was diluted with 38.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 158 μL of PL-12 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-12). The DAR value was determined by mass spectrometry to be 7.61.

[1064] 15. Preparation of the antibody-drug conjugate Trastuzumab-PL-13

[1065] 0.769 mL of Trastuzumab antibody (19.09 mg / mL) was diluted with 38.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 161.5 μL of PL-13 solution dissolved in dimethyl sulfoxide (10 mM, 15 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-13). The DAR value was 7.36 as determined by mass spectrometry.

[1066] 16. Preparation of the antibody-drug conjugate Trastuzumab-PL-14

[1067] 1 mL of Trastuzumab antibody (14.9 mg / mL) was diluted with 50 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 147.6 μL of PL-14 solution dissolved in dimethyl sulfoxide (10 mM, 14 molar equivalents of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-PL-14). The DAR value was determined by mass spectrometry to be 8.12.

[1068] Biological evaluation

[1069] Experimental Example 1. Inhibitory effect of the compound on the proliferation of HT29 cells

[1070] Cell plating: First, HT29 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended, and counted. The cell concentration was then adjusted to a suitable level for plating. The source of tumor cells is shown in Table 1.

[1071] Table 1. Tumor cell origin

[1072] The present invention involves co-incubating the test compound and tumor cells: cell suspension is added to each well of a 96-well plate, and after the cells adhere, the test compound diluted with culture medium is added to the wells and incubated.

[1073] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent, mix thoroughly by shaking in the dark, and perform detection after a certain reaction time using an ELISA reader (manufacturer: BMG). The readings from cell-free culture wells are the background RLU, and the readings from wells containing cells but not the test compound are the cell control RLU. Cell inhibition rate = (1 - (Test compound RLU - Background RLU) / (Cell control RLU - Background RLU)) × 100%. Calculate the half-maximal inhibitory concentration (IC50) of the compound using a four-parameter model to fit the curve. 50 The test results are shown in Table 2.

[1074] Table 2. Inhibitory activity of compounds against HT29 cell proliferation

[1075] Test results show that the compound of the present invention has an inhibitory effect on the proliferation of HT29 human colon cancer cells.

[1076] Experimental Example 2. Inhibitory effect of the compound on the proliferation of HCC1954 cells

[1077] Cell plating: First, HCC1954 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cell concentration was adjusted to a suitable level for plating. The source of tumor cells is shown in Table 3.

[1078] Table 3. Tumor cell origin

[1079] The present invention involves co-incubating the test compound and tumor cells: after the cells adhere, the culture medium in the cells is removed, and the diluted test compound is added to the wells of the plate for incubation.

[1080] In vitro cell viability assay: After incubation, add Cell Counting-Lite™ 2.0 reagent to each well, mix thoroughly by shaking in the dark, and allow to react for a certain period before detection. Read the values ​​using a microplate reader (manufacturer: BMG). Obtain the background RLU using the readings from cell-free culture wells, and obtain the solvent RLU using the readings from culture wells containing cells but not the test compound. Cell inhibition rate = (1 - (analyte RLU - background RLU) / (solvent RLU - background RLU)) × 100%. Calculate the half-maximal inhibitory concentration (IC50) of the compound by fitting a curve using a four-parameter model. 50 RLU (relative light unit): The detection results are shown in Table 4.

[1081] Table 4. Inhibitory activity of compounds on the proliferation of HCC1954 cells

[1082] Test results show that the compound of the present invention has an inhibitory effect on the proliferation of HCC1954 human breast cancer cells.

[1083] Experimental Example 3. Inhibitory effect of the compound on the proliferation of NCI-N87 cells

[1084] Cell plating: First, NCI-N87 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cell concentration was adjusted to a suitable level for plating. The source of tumor cells is shown in Table 5.

[1085] Table 5. Tumor cell origin

[1086] The present invention involves co-incubation of the test compound and tumor cells: cell suspension is added to each well of a 96-well plate, and after the cells adhere, the test compound diluted with culture medium is added to the wells and incubated for 72 hours.

[1087] In vitro cell viability assay: After incubation, Cell Counting-Lite was added to each well. TM 2.0 Reagent, mix thoroughly by shaking in the dark, and perform detection after a certain reaction time using an ELISA reader (manufacturer: BMG). The readings from cell-free culture wells are the background RLU, and the readings from wells containing cells but not the test compound are the cell control RLU. Cell inhibition rate = (1 - (Test compound RLU - Background RLU) / (Cell control RLU - Background RLU)) × 100%. Calculate the half-maximal inhibitory concentration (IC50) of the compound using a four-parameter model to fit the curve. 50 The test results are shown in Table 6.

[1088] Table 6. Inhibitory activity of compounds against NCI-N87 cell proliferation

[1089] Test results show that the compound of the present invention has an inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.

[1090] Experimental Example 4. Inhibitory effect of the compound on the proliferation of NCI-H358 cells

[1091] Cell plating: First, NCI-H358 tumor cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and the cell concentration was adjusted to a suitable level for plating. The source of tumor cells is shown in Table 7.

[1092] Table 7. Tumor cell origin

[1093] The present invention involves co-incubating the test compound and tumor cells: after the cells adhere, the culture medium in the cells is removed, and the diluted test compound is added to the wells of the plate for incubation.

[1094] In vitro cell viability assay: After incubation, add Cell Counting-Lite™ 2.0 reagent to each well, mix thoroughly by shaking in the dark, and allow to react for a certain period before detection. Read the values ​​using a microplate reader (manufacturer: BMG). Obtain the background RLU using the readings from cell-free culture wells, and obtain the solvent RLU using the readings from culture wells containing cells but not the test compound. Cell inhibition rate = (1 - (analyte RLU - background RLU) / (solvent RLU - background RLU)) × 100%. Calculate the half-maximal inhibitory concentration (IC50) of the compound by fitting a curve using a four-parameter model. 50 RLU (relative light unit): The detection results are shown in Table 8.

[1095] Table 8. Inhibitory activity of compounds on the proliferation of NCI-H358 cells

[1096] Test results show that the compound of the present invention has an inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.

[1097] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof: in, R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally and independently selected from H, deuterium (D), halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; or R 1 and R 2 It is linked to adjacent atoms to form a ring, wherein the ring is optionally selected by one or more atoms independently selected from H, deuterium (D), halogen, -OH, -NH2, C 1- 6-alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The substituents are aryl and 5-10 heteroaryl, preferably substituted by one or more substituents independently selected from H and deuterium (D); X is selected from C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl, the C 2-6 alkenyl, C 2-6 Alkyne group, 3-10 membered heterocyclic group, C 3-10 cycloalkyl, C 6-10 The aryl group and the 5-10 heteroaryl group are optionally selected independently by one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxyalkyl (e.g., C 1-6 hydroxyalkyl), amino, aminoalkyl (e.g., C 1-6 Aminoalkyl), C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group, C 1-6 Amino-amide group, C 2-6 alkenyl, C 2-6 Alkyne, substituted or unsubstituted C 3-10 Cycloalkyl, substituted or unsubstituted 3-10 membered heterocyclic groups, substituted or unsubstituted C 6-10 The aryl group is substituted with substituents of 5-10 heteroaryl groups, both substituted and unsubstituted.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein, R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 cycloalkyl; the C 1-6 Alkyl and C 3-6 The cycloalkyl group may be optionally separated by one or more elements independently selected from H, deuterium (D), halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; Preferably, the R 1 and R 2 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 cycloalkyl; More preferably, the R 1 and R 2 Each is independently selected from hydrogen, methyl, and cyclopropyl; More preferably, the R 1 The R is methyl. 2 It is methyl; Or, the R mentioned above 1 and R 2 It is linked with adjacent atoms to form a 5-6 member oxygen-containing heterocycle, wherein the ring is optionally selected by one or more independently chosen from H, deuterium (D), halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 The substituents are aryl and 5-10 heteroaryl, preferably substituted by one or more substituents independently selected from H and deuterium (D); Preferably, the R 1 and R 2 Connected to adjacent atoms More preferably, the R 1 and R 2 Connected to adjacent atoms More preferably, the R 1 and R 2 Connected to adjacent atoms 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, X is Among them, X A Selected from chemical bonds, C 2-6 imide and C 2-6 Imyynyl group, the C 2-6 imide and C 2-6 The alkynyl group is optionally selected by one or more independently chosen from H, halogen, -OH, -NH2, C. 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Substituents of aryl and 5-10 heteroaryl groups; Ring A is selected from 3-10 member heterocyclic groups, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl groups; R 3 Selected from H, hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1- 6-hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t can be 0, 1, 2, 3, 4 or 5.

4. The compound according to claim 3, or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug, satisfies one or more of the following: (1)X A Selected from chemical bonds, C 2-6 imide and C 2-6 Idemynyl; preferably, X A Selected from C 2-6 imide and C 2-6 etyne group; more preferably, X A Selected from C 2-6 Alkenyl groups, such as vinylidenes; (2) Ring A is selected from C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl groups; (3)R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amino-amide group; preferably, R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group; preferably, R 3 Selected from C 1-6 Hydroxyalkyl and amino; (4)R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; preferably, R 4 Each is independently selected from H and halogens; (5) t is 0, 1 or 2.

5. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, X is Where X A Selected from C 2-6 imide and C 2-6 Ethyne group; ring A is selected from 3-10 membered heterocyclic groups, C 3-10 cycloalkyl, C 6-10 Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group and C 1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t is 0, 1, 2, 3, 4 or 5; Preferably, X A Selected from C 2-6 alkenyl group; ring A is selected from C 6-10 Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

6. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein, X is Where X A When it is a chemical bond, ring A is selected from 3-10 membered heterocyclic groups, C 3-10 cycloalkyl, C 10 Aryl and 5-10 heteroaryl groups; R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group, C 1-6 Hydroxyalkylamine-amide group, C 1-6 Aminoalkyl-amide group, C 1-6 Aminoalkoxy-amide group, C 1-6 Amino-amide group; R 4 Each is independently selected from H, halogen, -OH, -NH2, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 6-10 Aryl and 5-10 heteroaryl groups; t is 0, 1, 2, 3, 4 or 5; Preferably, when X A When it is a chemical bond, ring A is selected from C. 10 Aryl and 5-10 membered heteroaryl groups (e.g., 9-10 membered heteroaryl groups, such as quinolinyl); R 3 Selected from hydroxyl, C 1-6 Hydroxyalkyl, amino, C 1-6 Aminoalkyl, C 1-6 hydroxyalkyl-amide group, C 1-6 hydroxyalkoxy-amide group and C 1-6 Hydroxyalkylamine-amide group, preferably hydroxyl, C 1-6 Hydroxyalkyl, amino or C 1-6 Aminoalkyl; R 4 Each is independently selected from H, halogens, and C. 1-6 Alkyl; t is 0, 1 or 2.

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, in, X is selected from Preferably, X is selected from Preferably, X is selected from More preferably, X is selected from More preferably, X is selected from More preferably, X is More preferably, X is 8. The compound represented by formula (II) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug: in, R 1 R 2 And X as defined in any one of claims 1-7.

9. A compound of formula (III) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof: in, R 1 R 2 X is defined as described in any one of claims 1-7; M is selected from H and alkali metals; Preferably, M is selected from hydrogen, sodium, and potassium; More preferably, M is hydrogen.

10. The compound according to any one of claims 1-9, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the following structure:

11. The drug linker compound of formula (IV) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug: in, R 1 and R 2 As defined in any one of claims 1-10; X' is a divalent structure formed by removing a hydrogen atom from the X group as defined in any one of claims 1-10; Y is selected from single bonds, -CH2NH-, and self-elimination structures; Z is a structure connecting Y and CM; CM is selected from structures that can react with specific functional groups in the target region (such as antibodies or their antigen-binding fragments).

12. The compound of claim 11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, Y is selected from single bond, -CH2NH-, p-aminobenzyloxycarbonyl (PABC), and carbonate group; Preferably, Y is a single bond.

13. The compound of claim 11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, or prodrug thereof, wherein, Z is selected from a structure composed of one or more of the following groups: C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl, carbonyl, sulfonyl, amino, hydroxyl, -O-, heterocyclic, heteroaryl, aryl, natural or non-natural amino acids and their polypeptides, polyethylene glycol, polysarcosine, carboxylic acid, glycosyl and their derivatives or quaternary ammonium salts; Preferably, Z is selected from a substituted or unsubstituted structure composed of one or more of the following groups: C 1-6 Alkylene, C 6-10 Arylidene, 5-6 membered heteroarylene, substituted or unsubstituted 9-12 membered nitrogen-containing heterocyclic groups (e.g., substituted by one or more R'), -N(R')-, -NH(R'), -N(R')2, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(R'), Lys(R')2, Glu(R'), Lys(COCH2CH2(OCH2CH2)) r OCH3), and short peptides composed of amino acids (such as Gly-Lys, Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, A la-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu -Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:41), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:42), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:43), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:44), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:45), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:46), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:47)), R' is composed of one or more of the following groups: Hydrogen, C 1-6 Alkyl, C 1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C 1-6 Alkylene CO2H, -C 1-6 Alkylene groups SO3H, -SO3H, -PO3H2, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 alkyl)2、-CH2N(C 1-6 alkyl)-C(=O)C 1-6 alkylene-heterocyclic, -C 1-6 Alkylene-heterocyclic, -NHC 1-6 Alkylene-SO3H, -CH2NH-SO3H, -CH2N(C) 1-6 (alkyl)-SO3H,-CH2NHC 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkyl)C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 Alkylene (-SO3H)2, -CH2N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 (alkyl)2-C 1-6 Alkylenes -SO3H, -CH2N(C 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (SO3H)3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkylene (-SO3H)3, -CH2N(C) 1-6 alkyl)-C(=O)C 1-6 Alkylene-N(C) 1-6 Alkyl group 3, -CH2NH-C(=O)C 1-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)3、-CH2N(C 1-6 alkyl)-C(=O)OC 2-6 Alkylene-N(C) 1-6 alkyl)2-CH2CO2H, -CH2N(C 1-6 alkyl)-C 1-6 Alkylene -CO2H, -CH2N(C 1-6 (alkyl)2-C 1-6 Alkylene -CO2H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -CH2N(C) 1-6 Alkyl)-C(=O)-(OCH2CH2) r -OC 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). r -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), EDTA (ethylenediaminetetraacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-EDTA and R” group, wherein the R” group is a hydrophilic structure containing hydroxyl, glycosyl, amino or carboxylic acid and its derivatives; r is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from integers from 1 to 20, such as 1 to 15, 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Preferably, "R" is selected from the following structures: Alternatively, "R" can be selected from the following structure: Alternatively, "R" can be selected from the following structure: Each time m appears, it is independently selected from an integer between 1 and 30. For example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Preferably, Z is selected from Ala-Val, Cit-Val, Lys-Val, Lys(R')-Val, Gly-Lys-Val, Ala-Val-Glu, and Ala-Val-Glu(R'); R' is selected from Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Preferably, R' is selected from (For example )、 (For example )、 (For example )and Each time m appears, it is independently selected from an integer between 1 and 30; for example, each time m appears, it is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Preferably, Z is selected from Ala-Val, Cit-Val, Lys-Val, and Gly-Lys-Val.

14. The compound according to any one of claims 11-13, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, CM is selected from functional groups or structures that can react with specific functional groups (e.g., cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs) contained in the target portion (e.g., antibody or its antigen-binding fragment). Preferably, CM is selected from the following substituted or unsubstituted structures: (For example ), (For example ), (For example ), (For example ), Where R a Selected from hydroxyl, cyano, amino, halogen, nitro, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1- 6-alkoxy, C 1-6 Halogenated alkyl and C 1-6 Alkyl-C(=O)-, preferably cyano; Lg is a leaving group for nucleophilic substitution, for example selected from halogens (e.g., F, Cl, Br or I), halogenated C 1-6 Alkyl, maleimide, halogenated maleimide, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structures, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkyl sulfoxide group, halophenoxy group, alkenyl group, alkynyl group, and alkynyl-containing structure are optionally replaced by one or more suitable substituents; preferably, Lg is selected from halogens, C 1-6 Alkyl sulfonyl and halophenoxy groups; More preferably, CM is selected from the following substituted or unsubstituted structures: More preferably, CM is selected from the following substituted or unsubstituted structures: More preferably, CM is For example 15. The compound according to any one of claims 11-14, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein, X' is a divalent structure formed by removing a hydrogen atom from the X group as defined in any one of claims 1-10; Preferably, X' is Where X A Ring A, R 4 and t as defined in any one of claims 1-10; R 3A R as defined in any one of claims 1-10 3 A divalent structure formed after a group loses a hydrogen atom; Preferably, R 3A Selected from chemical bonds, -O-, -C 1-6 Alkylenes -O-, -NH-, -C 1-6 Alkylene -NH-, -NH-C(=O)-C 1-6 Alkylene -O-, -NH-C(=O)-OC 1-6 Alkylene-O-, -NH-C(=O)-NC 1-6 Alkylene -O-, -NH-C(=O)-C 1-6 Alkylene -NH-, -NH-C(=O)-OC 1-6 Alkylene-NH- and -NH-C(=O)-NC 1-6 Alkylene-NH-, more preferably, R 3A -O-, -C 1-6 Alkylene -O- or -NH-; More preferably, -X'- is selected from More preferably, X' is selected from More preferably, X' is selected from More preferably, X' is 16. The compound of claim 11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the structure shown below:

17. The drug linker compound represented by formula (V) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug: in, R 1 R 2 And X as defined in any one of claims 1-10; Z and CM are as defined in any one of claims 11-16; Y is selected from single bond, -CH2NH-, p-aminobenzyloxycarbonyl (PABC), carbonate group; preferably, Y is p-aminobenzyloxycarbonyl (PABC); Preferably, the drug linker compound has the following structure:

18. The drug conjugate of formula (VI) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug: in, R 1 and R 2 As defined in any one of claims 1-10; X', Y and Z are as defined in any one of claims 11-16; n is selected from 1-10; A' represents the target region; CM' is selected from the structure obtained by reacting with a specific functional group (e.g., cysteine, lysine, glutamic acid, glutamine, aspartic acid, asparagine, tyrosine, serine, threonine, methionine, histidine, arginine, non-natural amino acids, glycosyl groups and their derivatives or analogs) contained in the target moiety (e.g., antibody or its antigen-binding fragment). Preferably, CM' is the functional group or structure of CM after Lg removal as defined in claim 14, or the functional group or structure of CM after an addition reaction as defined in claim 14. More preferably, CM' is selected from the following substituted or unsubstituted structures: (For example )， (For example ), (For example ), (For example ), Where R a As defined in claim 14; More preferably, CM' is Preferably, A' is selected from antibodies targeting tumor antigens or their antigen-binding fragments, peptides, or small molecule fragments; More preferably, A' is selected from antibodies targeting tumor antigens or their antigen-binding fragments, such as monoclonal or bispecific antibodies targeting Her2, Her3, EGFR, TROP2, B7H3, c-Met, CEACAM5, CLDN18.2, FRa, CDH6, CDH3, PTK7, DLL3, or GPC3, such as antibodies targeting Her2 or their antigen-binding fragments, such as trastuzumab antibody or patocilizumab antibody; A' is selected from peptides that target tumor antigens, such as somatostatin analogs, GnRH / LHRH analogs, vascular peptide-2, Heptaarginine, TAT47-57, DPV1047 Vectocell peptide, and peptides that target EphA2, GPC3, or Nectin-4. A' is selected from small molecule fragments that target tumor antigens, such as N-acetylgalactosamine (GalNAc) fragments, bisphosphonate fragments with bone targeting, fragments that selectively bind to prostate-specific membrane antigen (PSMA), or fragments that selectively bind to somatostatin receptors. More preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25; or, (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25; or, (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25; or, (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25; Preferably, the antibody or its antigen-binding fragment comprises: (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; The variant has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions. Preferably, the antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4; Preferably, the antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO:

40.

19. The drug conjugate of claim 18 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug thereof, wherein the drug conjugate has the following structure: Wherein A-(S) is the target portion as described in claim 18, preferably the antibody or its antigen-binding fragment as described in claim 18; This indicates the specific connection method between the thiol group and the pyrimidine group in the targeted portion.

20. The drug conjugate of formula (VII) or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug: in, R 1 R 2 X is as defined in any one of claims 1-10; Y and Z are as defined in claim 17; CM', A' and n are as defined in claim 18 or 19.

21. The drug conjugate of claim 20, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the drug conjugate has the following structure: Wherein A-(S) is the target portion as described in claim 18, preferably the antibody or its antigen-binding fragment as described in claim 18; This indicates the specific connection method between the thiol group and the pyrimidine group in the targeted portion.

22. A composition comprising one or more drug conjugates according to any one of claims 18-21, wherein the DAR value (drug-antibody conjugate ratio) of the composition is about 1-10, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 4 to 8, about 4 to 9, about 4 Up to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10, for example about 5.0, about 5.01, about 5.02, about 5.03, about 5.04, about 5.05, about 5. 06, approximately 5.07, approximately 5.08, approximately 5.09, approximately 5.1, approximately 5.11, approximately 5.12, approximately 5.13, approximately 5.14, approximately 5.15, approximately 5.16, approximately 5.17, approximately 5.18, approximately 5.19, approximately 5.2, approximately 5.21, approximately 5.22, approximately 5.23, approximately 5.24, approximately 5.25, approximately 5.26, approximately 5.2 7, approximately 5.28, approximately 5.29, approximately 5.3, approximately 5.31, approximately 5.32, approximately 5.33, approximately 5.34, approximately 5.35, approximately 5.36, approximately 5.37, approximately 5.38, approximately 5.39, approximately 5.4, approximately 5.41, approximately 5.42, approximately 5.43, approximately 5.44, approximately 5.45, approximately 5.46, approximately 5.47, approximately 5.48 Approximately 5.49, approximately 5.5, approximately 5.51, approximately 5.52, approximately 5.53, approximately 5.54, approximately 5.55, approximately 5.56, approximately 5.57, approximately 5.58, approximately 5.59, approximately 5.6, approximately 5.61, approximately 5.62, approximately 5.63, approximately 5.64, approximately 5.65, approximately 5.66, approximately 5.67, approximately 5.68, approximately 5.

69. Approximately 5.7, approximately 5.71, approximately 5.72, approximately 5.73, approximately 5.74, approximately 5.75, approximately 5.76, approximately 5.77, approximately 5.78, approximately 5.79, approximately 5.8, approximately 5.81, approximately 5.82, approximately 5.83, approximately 5.84, approximately 5.85, approximately 5.86, approximately 5.87, approximately 5.88, approximately 5.89, approximately 5.9, approximately 5 .91, about 5.92, about 5.93, about 5.94, about 5.95, about 5.96, about 5.97, about 5.98, about 5.99, about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6. 33, approximately 6.34, approximately 6.35, approximately 6.36, approximately 6.37, approximately 6.38, approximately 6.39, approximately 6.4, approximately 6.41, approximately 6.42, approximately 6.43, approximately 6.44, approximately 6.45, approximately 6.46, approximately 6.47, approximately 6.48, approximately 6.49, approximately 6.5, approximately 6.51, approximately 6.52, approximately 6.53, approximately 6.5 4. Approximately 6.55, 6.56, 6.57, 6.58, 6.59, 6.6, 6.61, 6.62, 6.63, 6.64, 6.65, 6.66, 6.67, 6.68, 6.69, 6.7, 6.71, 6.72, 6.73, 6.74, 6.7 5. Approximately 6.76, 6.77, 6.78, 6.79, 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, 6.9, 6.91, 6.92, 6.93, 6.94, 6.95, 6.96 Approximately 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.1, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17 Approximately 7.18, 7.19, 7.2, 7.21, 7.22, 7.23, 7.24, 7.25, 7.26, 7.27, 7.28, 7.29, 7.3, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38 Approximately 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0, approximately 8.01, approximately 8.02, approximately 8.03, approximately 8.04, approximately 8.05, approximately 8.06, approximately 8.07, approximately 8.08, approximately 8.09, approximately 8.1, approximately 8 .11, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.23, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33, about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, approximately 8.42, approximately 8.43, approximately 8.44, approximately 8.45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, approximately 8.55, approximately 8.56, approximately 8.57, approximately 8.58, approximately 8.59, approximately 8.6, approximately 8.61, approximately 8.62, approximately 8.63, approximately 8.64, approximately 8.65, approximately 8.66, approximately 8.67, approximately 8.68, approximately 8.69, approximately 8.7 Approximately 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77, 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.

0.

23. A pharmaceutical composition comprising a compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug thereof; a pharmaceutical conjugate of any one of claims 18-21 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug thereof; the composition of claim 22; and one or more pharmaceutically acceptable carriers.

24. A medicine box, which contains: a) at least one compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug as a first therapeutic agent; a drug conjugate of any one of claims 18-21 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug as a first therapeutic agent; a composition of claim 22; or a pharmaceutical composition of claim 23. b) At least one other therapeutic agent optionally present as a second therapeutic agent, or a pharmaceutical composition comprising another therapeutic agent as a second pharmaceutical composition; and c) Optional packaging and / or instructions.

25. The use of the compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug of any one of claims 18-21 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug of claim 22, the composition of claim 23, or the cassette of claim 24 in the preparation of a medicament for treating diseases of abnormal cell proliferation; Preferably, the disease is a tumor, such as an advanced solid tumor; Preferably, the tumor is selected from brain tumors, lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

26. The compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug of any one of claims 18-21 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope-labeled compound, metabolite, or prodrug of any one of claims 18-21, the composition of claim 22, the pharmaceutical composition of claim 23, or the kit of claim 24, for the treatment of diseases, particularly diseases of abnormal cell proliferation; Preferably, the disease is a tumor, such as an advanced solid tumor; Preferably, the tumor is selected from brain tumors, lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

27. A method for treating a disease of abnormal cell proliferation, comprising the steps of: administering a therapeutically effective amount of the compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug of any one of claims 18-21 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite, or prodrug of claim 22, the composition of claim 23, or the cassette of claim 24 to an individual in need of it; Preferably, the disease is a tumor, such as an advanced solid tumor; Preferably, the tumor is selected from brain tumors, lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

28. Use of the compound of any one of claims 1-17 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitride, isotope label, metabolite or prodrug thereof for the preparation of a drug conjugate (e.g., an antibody-drug conjugate).

29. The following are examples of compounds or their salts, esters, stereoisomers, polymorphs, solvates, nitrides, or isotopically labeled compounds: Where R 1 R 2 Lg, X and X' are as defined in any one of claims 1-21; PG 1 Each group is independently an H or amino protecting group. The amino protecting group can be an alkoxycarbonyl group, such as benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); an acyl group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), tert-pentanoyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; or an alkyl group, such as triphenylmethyl (Trt), C 1-6 Alkyl-substituted triphenylmethyl, p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn), or (trimethylsilyl)ethoxymethyl (SEM); PG 2 Each is independently an H or hydroxyl protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, triphenylmethyl (Trt), methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, trifluoromethanesulfonyl (Tf), or p-nitrobenzoyl. PG 3 Each is an independent H or carboxyl protecting group, and the carboxyl protecting groups are, for example, each independently selected from C. 1-6 Alkyl, allyl, benzyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, methoxyethoxymethyl, pentafluorophenyl, 4-p-methylbenzyloxybenzyl; Preferably, the compound is selected from:

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