AURISTATIN DERIVATIVE AND ANTIBODY-DRUG CONJUGATE BASED THEREON

Abstract

The present invention relates to a cytotoxic compound and an antibody-drug conjugate thereof, and relates in particular to an auristatin derivative, a preparation method for an antibody-drug conjugate thereof, and a use.

Description

Auristatin derivatives and antibody-drug conjugates

[0001] This application claims the Chinese patent application filed on September 26, 2023 with the application number 202311256590.X and the invention name “Auristatin derivatives and antibody-drug conjugates thereof”, the Chinese patent application filed on January 25, 2024 with the application number 202410109076.1 and the invention name “Auristatin derivatives and antibody-drug conjugates thereof”, and the Chinese patent application filed on June 20, 2024 with the application number 202410806735.7 and the invention name “ The present application claims priority to the Chinese patent application No. 202411124033.7 filed with the Patent Office of China on August 15, 2024, with the application number 202411296153.5, entitled “Auristatin derivatives and antibody-drug conjugates thereof”, and the Chinese patent application No. 202411296153.5 filed with the Patent Office of China on September 14, 2024, with the application number 202411296153.5, entitled “Auristatin derivatives and antibody-drug conjugates thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application belongs to the field of medicine, and specifically relates to a preparation method and use of an auristatin derivative and an antibody-drug conjugate thereof. Background Art

[0003] Antibody-drug conjugates (ADCs) are a class of targeted biopharmaceuticals that combine a target-specific monoclonal antibody (Ab) with a highly potent cytotoxic drug (payload) via a specific linker. Using the mAb as a carrier, small-molecule cytotoxic drugs are efficiently delivered to target tumor cells in a targeted manner. ADCs combine the advantages of targeted, highly selective antibodies with the advantages of highly potent cytotoxic drugs. While retaining the tumor-killing properties of small-molecule cytotoxic drugs, they selectively reduce their off-target side effects, effectively improving the benefit-risk ratio of anti-tumor therapy.

[0004] Auristatins are decapeptides isolated from the Indian Ocean sea hare. They inhibit tumor growth primarily by targeting tubulin, with activity approximately 1,000 times greater than that of traditional chemotherapy drugs. MMAE (marigoldenin E) and MMAF (methylmarigoldenin F) are the two most commonly used auristatins in ADC development.

[0005] Although significant progress has been made in the clinical development of ADCs in recent years, their design and development still face many challenges and are associated with a high number of clinical adverse reactions. For example, the most common adverse reactions of trastuzumab deruxtecan (trade name Enhertu) are upper respiratory tract infection, anemia, neutropenia, thrombocytopenia, leukopenia, and interstitial lung disease. Brentuximab vedotin (trade name Adcetris) has adverse reactions such as hematological abnormalities and gastrointestinal diseases. Therefore, there is still a need to develop more safe and effective cytotoxic drugs and their antibody-drug conjugates.

[0006] Summary of the Invention

[0007] The present disclosure provides a cytotoxic drug represented by formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof:

[0008] Wherein, M is selected from C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group; the C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group is optionally further selected from C 1-4 Alkyl or NR m1 R m2 The 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0009] R m1 and R m2 Each independently selected from H or C 1-4 alkyl;

[0010] X is selected from O or -N(R x1 )-;

[0011] R x1 Selected from H, OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-4 Alkenyl, C 2-4 Alkynyl or C 3-6 Cycloalkyl; the C 1-4 Alkyl or C 3- 6 cycloalkyl is optionally further substituted by 1-3 groups selected from halogen, CN, deuterium or C 3-6 substituted by a cycloalkyl substituent;

[0012] R 3 and R 4 are each independently selected from H or OH;

[0013] Or, X and R 3 and the atoms to which they are connected together form a 4-8 membered heterocyclic ring, wherein the 4-8 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2;

[0014] Or, X and R 4 and the atoms to which they are connected together form a 4-8 membered heterocyclic ring, wherein the 4-8 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2;

[0015] R 1 and R 2 Each independently selected from H, OH, C 1-4 Alkyl, C 2-4 Alkynyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group, -COOR 1a or -CONR 1b R 1c ; the C 1-4 Alkyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group may be further substituted by 1-3 groups selected from CN, OH, N3, halogen, -COOR 1a 、-CONR 1b R 1c or C 1-4 substituted by an alkyl substituent, and the 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0016] R 1a 、R 1b and R 1c Each independently selected from H or C 1-4 alkyl;

[0017] Or, R 1 、R 2 Together with the carbon atom it is connected to, it forms C 3-6 Cycloalkyl or 3-8 membered heterocyclic group;

[0018] Z is selected from a chemical bond or -(CH2) n -, n is selected from 1 or 2.

[0019] In some embodiments, the M is selected from C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group; the C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group is optionally further selected from C 1-4 Alkyl or NR m1 Rm2 The 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0020] R m1 and R m2 Each independently selected from H or C 1-4 alkyl;

[0021] X is selected from O or -N(R x1 )-;

[0022] R x1 Selected from H, OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-4 Alkenyl or C 2-4 Alkynyl; the C 1-4 The alkyl group is optionally further substituted with 1-3 substituents selected from halogen or CN;

[0023] R 3 and R 4 are each independently selected from H or OH;

[0024] Or, X and R 3 and the atoms to which they are connected together form a 4-8 membered heterocyclic ring, wherein the 4-8 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2;

[0025] Or, X and R 4 and the atoms to which they are connected together form a 4-8 membered heterocyclic ring, wherein the 4-8 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2;

[0026] R 1 and R 2 Each independently selected from H, OH, C 1-4 Alkyl, C 2-4 Alkynyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group, -COOR 1a or -CONR 1b R 1c ; the C 1-4 Alkyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group may be further substituted by 1-3 groups selected from CN, OH, N3, halogen, -COOR 1a 、-CONR 1b R 1c The 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0027] R 1a 、R 1b and R 1c Each independently selected from H or C 1-4 alkyl;

[0028] Or, R 1 、R 2 Together with the carbon atom it is connected to, it forms C 3-6 Cycloalkyl or 3-8 membered heterocyclic group;

[0029] Z is selected from a chemical bond or -(CH2) n -, n is selected from 1 or 2.

[0030] In some embodiments, the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof:

[0031] Wherein, M is selected from C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group; the C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group is optionally further selected from C 1-4 Alkyl or NR m1 R m2 The 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0032] R m1 and R m2 Each independently selected from H or C 1-4 alkyl;

[0033] X is selected from O or -N(R x1 )-;

[0034] R x1 Selected from H, OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-4 Alkenyl or C 2-4 Alkynyl; the C 1-4 The alkyl group is optionally further substituted with 1-3 substituents selected from halogen or CN;

[0035] R 1 and R 2 Each independently selected from H, OH, C 1-4 Alkyl, C 2-4 Alkynyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group, -COOR 1a or -CONR1b R 1c ; the C 1-4 Alkyl, C 2-4 Alkenyl, C 3-6 Cycloalkyl, 3-8 membered heterocyclic group may be further substituted by 1-3 groups selected from CN, OH, N3, halogen, -COOR 1a 、-CONR 1b R 1c The 3-8 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S;

[0036] R 1a 、R 1b and R 1c Each independently selected from H or C 1-4 alkyl;

[0037] Or, R 1 、R 2 Together with the carbon atom it is connected to, it forms C 3-6 Cycloalkyl or 3-8 membered heterocyclic group;

[0038] R 3 and R 4 are each independently selected from H or OH;

[0039] Z is selected from a chemical bond or -(CH2) n -, n is selected from 1 or 2.

[0040] In some embodiments, the cytotoxic drug represented by formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, wherein M is selected from C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group; the C 1-4 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group is optionally further selected from C 1-4 Alkyl or NR m1 R m2 The 3-6 membered heterocyclic group contains 1-3 heteroatoms selected from N, O or S; R m1 and R m2 Each independently selected from H or C 1-2 alkyl.

[0041] In some embodiments, the M is selected from

[0042] In some embodiments, the R x1 Selected from H, -CH3, OH, -OCH3,

[0043] In some embodiments, X is selected from O, -NH-, -N(CH3)-,

[0044] In some embodiments, X is selected from

[0045] In some embodiments, the R 1 and R 2 Each independently selected from H, -CH3, -CH2OH, -CH2F, -CHF2, -CF3, -CH2Cl, -COOH, -COOCH3, -CONH2, -CONHCH3,

[0046] Or, R 1 、R 2 Together with the carbon atoms to which it is attached,

[0047] In some embodiments, the R 3 and R 4 Each independently selected from H or OH, preferably, R 3 and R 4 Each is independently H.

[0048] In some embodiments, the X and R 3 and the atoms to which they are connected together form a 5-6 membered heterocyclic ring, wherein the 5-6 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2;

[0049] Or, X and R 4 and the atoms to which they are connected together form a 5-6 membered heterocyclic ring, wherein the 5-6 membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S or S(O)2.

[0050] In some embodiments, the X and R 3 Together with its connected atoms, it forms

[0051] Or, X and R 4 Together with its connected atoms, it forms

[0052] In some embodiments, Z is selected from a chemical bond or -CH2-.

[0053] In some embodiments, the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from compounds of formula (Ia), (Ib) or (Ic):

[0054] Wherein, Y is selected from O, S or S(O)2, and the M, R 1 、R 2 、R 3 、R 4 、R x1 and Z are as defined in formula (I).

[0055] In some embodiments, the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from compounds of formula (IIa), (IIb) or (IIc):

[0056] Wherein, Y is selected from O, S or S(O)2, and the R 1 、R 2 、R 3 、R 4 、R x1 As defined in formula (I).

[0057] In some embodiments, the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from the compound of formula (IIIa):

[0058] Among them, the R x1 Selected from OH, C 1-4 Alkyl, C 1-4 Alkoxy or C 3-6 Cycloalkyl, and the C 1-4 Alkyl or C 3-6 The cycloalkyl group is further substituted by 1-3 substituents selected from deuterium, methyl, CN or cyclopropyl; preferably, R x1 Selected from -OCH3,

[0059] In some embodiments, the cytotoxic drug, its stereoisomers, isotopic derivatives, solvates, N-oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from:

[0060] The present disclosure further provides an antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, selected from the structure represented by formula (A):

[0061] Wherein, Ab is an antibody or antigen-binding fragment;

[0062] L is a linker group, the left end of which is connected to the NH on the cytotoxic drug and the right end is connected to Ab;

[0063] a is a value selected from 1 to 10;

[0064] The M and R 1 、R 2 、R 3 、R 4 , X and Z are as defined in formula (I).

[0065] In some embodiments, the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, is selected from the structure represented by formula (A-1):

[0066] Wherein, Ab is a Her2 antibody; preferably, Ab is trastuzumab;

[0067] R x1 Selected from H, -CH3, OH, -OCH3, Preferably, R x1 Selected from -OCH3,

[0068] a is selected from a value of 2-8; preferably, a is selected from a value of 4-8;

[0069] L is as defined in formula (A).

[0070] In some embodiments, the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, wherein L is selected from

[0071] In some embodiments, the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, wherein L is selected from

[0072] In some embodiments, the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, wherein a is selected from a value of 2 to 8; preferably, a is selected from a value of 4 to 8.

[0073] The present disclosure further provides a cytotoxic drug-linker, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts, wherein the cytotoxic drug-linker has a structure shown in formula (B),

[0074] Wherein, the L′ is the structure before Ab in L access formula (A);

[0075] Preferably, L' is selected from

[0076] The M and R 1 、R 2 、R 3 、R 4 , X and Z are as defined in formula (I).

[0077] In some embodiments, preferably, L' is selected from

[0078] In some embodiments, the cytotoxic drug-linker, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, is selected from the structure represented by formula (B-1):

[0079] Among them, the R x1 Selected from H, -CH3, OH, -OCH3, Preferably, R x1 Selected from -OCH3,

[0080] The L' is selected from Preferably, L' is

[0081] In some embodiments, the cytotoxic drug-linker is selected from:

[0082] In some embodiments, the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from:

[0083] The Ab is an antibody, preferably a Her2 antibody, more preferably trastuzumab;

[0084] a is selected from a value of 2-8, preferably a value of 4-8.

[0085] In another aspect, the present disclosure provides an antibody-drug conjugate, wherein the drug has the structure shown in the above compound.

[0086] On the other hand, the present disclosure provides a pharmaceutical composition comprising a cytotoxic drug of formula (I), a stereoisomer, isotopic derivative, solvate, nitrogen oxide, prodrug or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate of formula (A), a stereoisomer, isotopic derivative, solvate, nitrogen oxide, prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0087] In some embodiments of the present invention, in the pharmaceutical composition, the content of the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof is selected from 0.1 mg to 1000 mg.

[0088] In some embodiments of the present invention, the content of the antibody drug conjugate, its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof in the pharmaceutical composition is 1%-95%.

[0089] In some embodiments of the present invention, in the pharmaceutical composition, the pharmaceutically acceptable carrier includes one or more of a filler, a disintegrant, a binder, a glidant, and a lubricant.

[0090] On the other hand, the present disclosure provides the use of the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, the cytotoxic drug-linker of formula (B) or formula (B-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, the antibody drug conjugate of formula (A) or formula (A-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof as drugs (i.e., for use in treatment).

[0091] On the other hand, the present disclosure also provides the use of the cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, the cytotoxic drug-linker of formula (B) or formula (B-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, the antibody drug conjugate of formula (A) or formula (A-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof for preparing drugs for treating tumors.

[0092] On the other hand, the present disclosure also provides a method for preventing or treating tumors, comprising administering to a patient an effective preventive or therapeutic amount of a cytotoxic drug of formula (I), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, a cytotoxic drug-linker of formula (B) or formula (B-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, an antibody drug conjugate of formula (A) or formula (A-1), its stereoisomers, isotopic derivatives, solvates, nitrogen oxides, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof.

[0093] In some embodiments of the present invention, the tumor comprises breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, head and neck cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, skin cancer, thyroid cancer, pancreatic cancer or lymphoma.

[0094] In some embodiments of the present invention, the lung cancer is selected from small cell lung cancer and non-small cell lung cancer; the leukemia is selected from acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia; the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma or relapsed anaplastic large cell lymphoma.

[0095] Explanation of terms

[0096] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered ambiguous or unclear without a specific definition, but should be understood according to its ordinary meaning.

[0097] The term "cytotoxic drug" or "drug" refers to a small molecule compound that has the potential to disrupt the normal growth of tumor cells. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects.

[0098] The term "antibody-drug conjugate" refers to an antibody linked to a biologically active cytotoxic drug via a stable linker.

[0099] The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains or two identical light chains connected by interchain disulfide bonds.

[0100] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.

[0101] The term "linker site" refers to the chemical structure segment or bond that connects the Ab.

[0102] The term "linker" refers to a chemical structure fragment connecting the cytotoxic drug and Ab. In the embodiment of the present invention, the linker is -L-.

[0103] It should be noted that for the structural formula

[0104] As those skilled in the art will understand, the sulfur (S) in L is connected to the sulfhydryl group contained in the Ab (e.g., antibody) itself after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing the disulfide bond with the reducing agent TCEP to generate a sulfhydryl group SH). That is, the S between the right end of L and Ab is not an additional external sulfur atom.

[0105] For example,

[0106] -S- in the middle is not an additional external sulfur atom, but the sulfhydryl group contained in Ab after Ab opens the disulfide bond and is connected to the right end of L, for example Perform the connection to form -S-.

[0107] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0108] The term "pharmaceutically acceptable salt" refers to a derivative of a compound of the present invention prepared with a relatively non-toxic acid or base. These salts can be prepared during compound synthesis, separation, and purification, or by reacting the purified free form of the compound with a suitable acid or base. When the compound contains a relatively acidic functional group (e.g., -COOH, -OH, -SO3H, etc.), it reacts with an appropriate inorganic or organic cation (base) to form a base addition salt, including salts formed with alkali metals or alkaline earth metals, ammonium salts formed with amines or their derivatives, and salts formed with amino acids. When the compound contains a relatively basic functional group (e.g., -NH2, etc.), it reacts with an appropriate inorganic or organic anion (acid) to form an acid addition salt, including salts formed with an inorganic acid or organic acid (e.g., carboxylic acid, etc.).

[0109] The term "pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, particularly mammals, and includes, for example, adjuvants, excipients, or vehicles, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the mode of administration and the nature of the dosage form. Pharmaceutically acceptable carriers are formulated within the purview of those skilled in the art based on a wide range of factors. These include, but are not limited to, the type and nature of the active agent being formulated, the subject to whom the composition containing the agent is to be administered, the intended route of administration of the composition, and the intended therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media, as well as a variety of solid and semisolid dosage forms. In addition to the active agent, such carriers include a variety of different ingredients and additives, and the inclusion of such additional ingredients in a formulation for various reasons (e.g., to stabilize the active agent, binders, etc.) is well known to those skilled in the art.

[0110] The term "effective prophylactic or therapeutic amount" refers to a sufficient amount of the compound of the present disclosure, its pharmaceutically acceptable salt, or its isomer to treat the disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention. However, it should be recognized that the total daily dosage of the compound of Formula I or its pharmaceutically acceptable salt and composition of the present disclosure must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0111] The “isomers” described in the present disclosure include geometric isomers and stereoisomers, such as atropisomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof, all of which fall within the scope of the present disclosure.

[0112] The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a type of functional group isomer that has different hydrogen attachment points due to the displacement of one or more double bonds. For example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers in which the molecules have two or more chiral centers and are not mirror images of each other. The term "cis-trans isomer" refers to different spatial configurations in a molecule where double bonds or single bonds of ring carbon atoms cannot rotate freely. The term "atropisomer" refers to stereoisomers that can be separated because single bond rotation is hindered or rotates very slowly. Stereoisomers of the disclosed compounds can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, one enantiomer of a compound of the disclosed compounds can be prepared by asymmetric catalysis or chiral auxiliary derivatization. Alternatively, a compound with a single stereoconfiguration can be obtained from a mixture by chiral resolution techniques. Alternatively, the compound can be prepared directly using chiral starting materials. The separation of optically pure compounds in the present disclosure is usually accomplished using preparative chromatography, employing a chiral chromatographic column to achieve the purpose of separating chiral compounds.

[0113] The term "isotopic derivative" refers to a compound in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass. Examples of isotopes that can be introduced into the compounds of the present disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, 2 H (deuterium, D), 3 H (tritium, T),11 C. 13 C. 14 C. 15 N. 17 O. 18 O. 32 p、 33 p、 33 S. 34 S. 35 S. 36 S. 18 F. 36 Cl, 82 Br, 123 I. 124 I. 125 I. 129 I and 131 I, etc., preferably deuterium. Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced therapeutic efficacy, and extended drug biological half-life. All isotopic composition changes of the compounds disclosed herein, whether radioactive or not, are included in the scope of this disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom, wherein the deuterium replacement can be partial or complete, and partial deuterium replacement means that at least one hydrogen is replaced by at least one deuterium.

[0114] The term "nitrogen oxide" or "N-oxide" refers to a derivative formed by further oxidation of the nitrogen atom in a nitrogen-containing group. Common N-oxides include N-oxides of tertiary amines or N-oxides of nitrogen atoms in nitrogen-containing heterocycles.

[0115] The term "prodrug" refers to certain derivatives of the compounds of the present invention that have little or no pharmacological activity themselves, which have a cleavable group and decompose into the compounds of the present invention through solvolysis or physiological conditions. Such prodrug types include, but are not limited to, amides, esters, anhydrides, and salts. The "ester" refers to a derivative formed with a suitable alcohol when the compounds of the present invention contain an acidic group (such as a carboxylic acid); or a derivative formed with a suitable acid (including an organic acid or an inorganic acid) when the compounds of the present invention contain a hydroxyl group. Methods for preparing prodrugs are well known to those skilled in the art. The compounds of the present invention can exist in unsolvated and solvated forms, including hydrates. Generally speaking, solvated forms are equivalent to unsolvated forms and are also encompassed by the present invention. The absolute stereoconfiguration of a compound can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction can be used, or the absolute configuration of a compound can be confirmed based on the chiral structure of the starting material and the reaction mechanism of asymmetric synthesis. Alternatively, after resolution, the stereoconfiguration can be determined by comparison with the product with a determined absolute configuration. Compounds labeled as "absolute configuration unknown / undetermined" in this article are usually separated from racemic compounds into single isomers by chiral preparative SFC, and then characterized and tested.

[0116] It is known to those skilled in the art that when a cyclic compound has a coplanar delocalized system and the number of π electrons is 4n+2, the ring is aromatic. The expression of the aromatic structure in the compound can be expressed by using dotted lines to represent electron delocalization or by alternating single and double bonds. For example, the structure of a benzene ring can be drawn as Can also be

[0117] The term "optionally substituted" as used herein refers to two situations in which one or more hydrogen atoms of the substituted group may be "substituted" or "unsubstituted" by one or more substituents.

[0118] When the substituent appears in the structure Indicates that the atom is a bonding atom, for example Indicates that the C atom on the pyrimidine ring is a bonding atom. A dash "-" in a substituent structure indicates the point of attachment for the substituent, for example, -CH3 is attached through a C atom.

[0119] Indicates the absolute configuration of a stereocenter, i.e., R or S configuration. It represents cis or trans configuration. Double real bonds or double imaginary bonds both represent cis configuration, and one real and one imaginary bond represent trans configuration.

[0120] When a substituent's bond can cross-link to a ring, it means that the substituent can be bonded to any atom on the ring. The substituent R can be substituted at any position on the benzene ring.

[0121] When a substituent is listed without indicating the atom via which the substituent is attached to a given group or a given formula, then the substituent may be attached via any bondable atom thereof.

[0122] When any variable (such as R d ) appears more than once in a compound's composition or structure, its definition is independent in each instance. For example, Indicates that the cyclopentyl group is surrounded by 3 R d is replaced, and each R d There are independent options.

[0123] Unless otherwise specified, the term "halogen" means a fluorine, chlorine, bromine or iodine atom.

[0124] Unless otherwise specified, the term "alkyl" refers to a group derived from a branched or straight chain saturated aliphatic alkane having the specified number of carbon atoms by removing one hydrogen. For example, "C 1-10 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 Alkyl, "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 "alkyl"; specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, sec-butyl, 2-methylbutyl, 1,1-dimethylbutyl, etc.

[0125] Unless otherwise specified, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen atom. 1-6 Alkyl, more preferably halogenated C 1-4 Examples of haloalkyl groups include, but are not limited to, monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, tribromomethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, and the like. Alkyl groups are as defined above.

[0126] Unless otherwise specified, the term "hydroxyalkyl" refers to a group derived from an alkyl group in which one or more hydrogen atoms are replaced by a hydroxy group. The "hydroxyalkyl" described in the present disclosure includes "hydroxy C 1-6 Alkyl", "Hydroxy C 1-4 alkyl"; specific examples include but are not limited to -CH2OH, -CH2CH2OH, -CH(OH)CH3, -CH2CH2CH2OH, wait.

[0127] Unless otherwise specified, the term "alkoxy" refers to an alkyl group as defined herein attached to another group through an oxygen atom, i.e., "alkyl-O-". 1-6 Alkoxy" (structure is C 1-6 Alkyl-O-), "C 1-4 "alkoxy", specific examples include but are not limited to methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, etc.; preferably, the "alkoxy" described in the present disclosure is preferably C 1-4 Alkoxy, more preferably C 1-3 Alkoxy, more preferably C 1-2 Alkoxy.

[0128] Unless otherwise specified, the term "haloalkoxy" refers to a group in which one or more hydrogen atoms in an alkoxy group are replaced by halogen. Preferably, the "haloalkoxy" described in the present disclosure is preferably a "haloC 1-6 Alkoxy", "halogenated C 1-4 Alkoxy". Specific examples of the present disclosure include: fluoromethoxy (including monofluoromethoxy, difluoromethoxy, trifluoromethoxy), -OCH2CF3, -OCHFCH3, etc. Alkoxy is as defined above.

[0129] Unless otherwise specified, the term "alkenyl" refers to a radical derived from a straight-chain or branched alkene (containing at least one carbon-carbon double bond) by removing a hydrogen atom, including "C 2-6 Alkenyl", "C 2-5 Alkenyl", "C 2-4 Alkenyl", "C 2-3 "Alkenyl", specific examples include but are not limited to: -CH=CH2, -CH=CHCH3, -C(CH2)=CH2, -CH2CH=CH2, -CH=CHCH2CH3, -CH2CH=CHCH3, etc.

[0130] Unless otherwise specified, the term "alkynyl" refers to a radical derived from a straight-chain or branched alkyne (containing at least one carbon-carbon triple bond) by removing a hydrogen atom, including "C 2-5 Alkynyl", "C 2-4 Alkynyl", "C 2-3 Specific examples include, but are not limited to: -C≡CH, -C≡CHCH3, -CH2C≡CH, HC≡CC≡C-, etc.

[0131] Unless otherwise specified, the term "ring" refers to saturated, partially saturated or unsaturated monocycles and polycycles, and "polycycles" include spirocycles, condensed rings or bridged rings. The group derived from the ring by removing hydrogen atoms is called a "cyclic group", which includes a monovalent ring, a divalent ring (commonly referred to as a subring), a trivalent ring, a tetravalent ring, etc., and the specific valence depends on the number of substituents connected to the ring. The description of "cyclic group" in this disclosure no longer specifically distinguishes the valence of the ring. Representative "cyclic groups" include substituted or unsubstituted cycloalkyl, heterocyclic group, cycloalkenyl group, heterocycloalkenyl group, cycloalkynyl group, heterocycloalkynyl group, aryl group or heteroaryl group. The term "hetero" refers to substituted or unsubstituted heteroatoms and oxidized forms of heteroatoms (also known as heteroatoms). The heteroatoms are generally selected from N, O, S, and P. Oxidized forms generally include NO, SO, S(O)2, and P(O). The nitrogen atom can be substituted, i.e., NR (R is H or other substituents defined herein). The number of atoms in the ring is generally defined as the number of ring members. For example, "3-6 membered heterocycloalkyl" refers to a ring of 3-6 atoms arranged around, each ring optionally containing 1 to 3 heteroatoms and / or heteroatoms, i.e., N, O, S, NO, SO, S(O)2, P(O), or NR, each ring optionally substituted by an R group, where R is a group defined herein.

[0132] Unless otherwise specified, "cycloalkenyl" means a cycloalkyl group in which one or more of the ring bonds is a double bond and the cycloalkenyl group is not aromatic. The carbon atoms in the cycloalkenyl group may be further oxidized, i.e., to form C(O). The cycloalkenyl group includes "3-8 membered cycloalkenyl", "3-6 membered cycloalkenyl", "3-5 membered cycloalkenyl", and "5-6 membered cycloalkenyl". Specific examples include, but are not limited to,

[0133] Unless otherwise specified, the term "heterocyclyl" refers to a saturated cyclic group derived from a cycloalkyl group in which one or more ring carbon atoms are replaced by heteroatoms and / or heteroatomic groups. The heteroatoms and / or heteroatomic groups are generally selected from N, O, S, NO, SO, S(O)2, P(O), and NR, wherein the carbon atoms in the heterocyclic ring are optionally oxoed, i.e., forming -C(O); preferably, the heteroatoms are independently selected from 1-3 N and / or O. The heterocyclyl group includes "3-8 membered heterocyclyl", "3-6 membered heterocyclyl", "3-5 membered heterocyclyl", "4-6 membered heterocyclyl", and "5-6 membered heterocyclyl". Specific examples include, but are not limited to, oxetanyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, and the like.

[0134] Unless otherwise specified, the term "aryl" refers to an unsaturated, usually aromatic, hydrocarbon radical which may be a single ring or multiple rings fused together. 5-10 Aryl, more preferably C 5-8Aryl, most preferably a monocyclic C 5-6 Aryl; examples of aryl include, but are not limited to, phenyl, naphthyl.

[0135] Unless otherwise specified, the term "fused ring aromatic group" refers to a fused ring group formed by two or more aromatic rings sharing two adjacent carbon atoms, including naphthyl, anthracenyl and phenanthrenyl.

[0136] The "heteroaryl" described in the present disclosure refers to a monocyclic group with aromatic properties in which at least one ring atom is a heteroatom and / or a heteroatom group, wherein the heteroatom and / or heteroatom group is generally selected from N, O, S, P, NO, SO, S(O)2, P(O) and NR, R is H or any substituent group, wherein the carbon atoms in the heterocyclic ring are optionally oxidized, i.e., forming -C(O); preferably, the heteroatoms are independently selected from 1-3 N and / or O. The heteroaryl includes "5-6 membered heteroaryl"; specific examples include but are not limited to pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl, pyrazinyl, pyridazinyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isoxazolyl, pyridinyl, and pyrimidinyl.

[0137] Unless otherwise specified, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group. 3-8 Cycloalkyl, more preferably C 3-6 Cycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0138] The compounds of the present application exist in isomers, such as cis-trans isomers, enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, and all of these mixtures fall within the scope of the present application.

[0139] The term "enantiomer" refers to stereoisomers that are mirror images of one another.

[0140] The term "diastereomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other.

[0141] The term "cis-trans isomers" refers to configurations in which a molecule cannot rotate freely about a double bond or a single bond of a ring-forming carbon atom.

[0142] Unless otherwise specified, use a solid wedge key. and dotted wedge key Indicates the absolute configuration of a stereocenter.

[0143] Stereoisomers of the compounds of this application can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, one enantiomer of a compound of this application can be prepared by asymmetric catalysis or chiral auxiliary derivatization. Alternatively, a compound of a single stereoconfiguration can be obtained from a mixture by chiral resolution techniques. Alternatively, they can be prepared directly using chiral starting materials. The separation of optically pure compounds in this application is typically accomplished using preparative chromatography, employing chiral chromatographic columns to achieve the purpose of separating chiral compounds.

[0144] In particular, combinations of substituents and / or variables described herein are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or structure is one that is sufficiently robust to withstand chemical reactions, to be isolated to a useful degree of purity, and to be formulated into an efficacious therapeutic agent.

[0145] In the examples of this application, the naming of the title compound is converted from the compound structure with the help of Chemdraw. If there is an inconsistency between the compound name and the compound structure, it can be determined by integrating relevant information and reaction routes; if it cannot be confirmed by other means, the given compound structure shall prevail. The preparation method of some compounds in this application quotes the preparation method of the aforementioned similar compounds. Those skilled in the art should know that when using or referring to the preparation method cited, the feed ratio of the reactants, the reaction solvent, the reaction temperature, etc. can be appropriately adjusted according to the different reactants.

[0146] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0147] Summary of experimental instruments:

[0148] The structures of the compounds described herein are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS), or ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using a Bruker Neo 400M or Bruker Ascend 400 NMR instrument, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), deuterated chloroform (CDCl3), and heavy water (D2O) as the internal standard, with tetramethylsilane (TMS) as the internal standard.

[0149] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1260-6125B single quadrupole mass spectrometer with a Welch Biomate column (C18, 2.7 μm, 4.6 × 50 mm) or a Waters H-Class SQD2 with a Welch Ultimate column (XB-C18, 1.8 μm, 2.1 × 50 mm) mass spectrometer (electrospray ionization as the ion source).

[0150] Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using a Waters UPLC H-class SQD mass spectrometer (with electrospray ionization as the ion source).

[0151] HPLC determination was performed using Waters E2695-2998 or Waters ARC and Agilent 1260 or Agilent Poroshell HPH high performance liquid chromatography.

[0152] Preparative HPLC was performed using Waters 2555-2489 (10 μm, ODS 250 cm×5 cm) or GILSON Trilution LC, with a Welch XB-C18 column (5 μm, 21.2×150 mm).

[0153] Thin layer chromatography silica gel plates use GF254 silica gel plates from Yantai Jiangyou Silica Gel Development Co., Ltd. or GF254 silica gel plates from Rushan Shangbang New Materials Co., Ltd. The specifications used for TLC are 0.15mm-0.20mm, and the preparative type is 20×20cm. Column chromatography generally uses 200-300 mesh silica gel from Chenghua as a carrier.

[0154] The starting materials in the examples of this application are known and commercially available, or can be synthesized using or according to methods known in the art. Unless otherwise specified, all reactions in this application were carried out under a dry nitrogen or argon atmosphere with continuous magnetic stirring, with dry solvents, and reaction temperatures are expressed in degrees Celsius or °C.

[0155] The preparation method of the compounds of the present application refers to WO2023122228A1 and WO2023081230A1, and the corresponding reaction substrates are replaced according to their structural differences. Alternatively, the compounds of the present application are prepared using the following routes:

[0156] Route 1:

[0157] Route 2:

[0158] Among them, the route R 1 、R 2 H or C 1-6 Alkyl, or R 1 、R 2 Together with the carbon atom it is connected to, it forms C 3-6 Cycloalkyl;

[0159] R 3 Selected from C 2-4 Alkynyl, C 2-4 Alkenyl, -COOH, -COO-C 1-4 Alkyl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, the C 2-4 Alkenyl, C 1-4 The alkyl group may be optionally further substituted with -N3, OH, CN, halogen, or carboxyl.

[0160] R 4 Selected from H or hydroxyl.

[0161] The inhibitory activity IC of the compounds of the present application on HCC1954 and T47D cells 50 <1μM, preferably IC 50 <100nM, more preferably IC 50 <20nM, further optimized IC 50 <10nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 shows the tumor volume change curves of each group in the NCI-N87 transplanted tumor model at a dose of 1 mpk;

[0163] Figure 2 shows the tumor volume change curves of each group in the NCI-N87 transplanted tumor model at a dose of 4 mpk. DETAILED DESCRIPTION

[0164] Example 1:

[0165] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-(3-aminophenyl)-3-chloropropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0166] Reaction route:

[0167] Steps:

[0168] Step A: Compound 1-1 (6.4 g, 22.27 mmol) and cesium carbonate (14.51 g, 44.54 mmol) were added to a single-necked flask. Tetrahydrofuran (80 mL) and benzyl bromide (4.57 g, 26.72 mmol) were added sequentially. The reaction system was incubated at 50°C under nitrogen for 16 hours. After completion of the reaction, as monitored by LCMS, the reaction mixture was filtered and the filter cake was washed with tetrahydrofuran (30 mL x 3). The filtrate was collected and concentrated to afford crude compound 1-2 (9.2 g, yellow oil), which was used directly without further purification.

[0169] LCMS (ESI) M / Z: 400.2 [M+Na + ].

[0170] Step B: Compound 1-2 (9.2 g, crude) was added to a 250 mL single-necked flask, followed by dichloromethane (50 mL) and a solution of HCl in 1,4-dioxane (30.5 mL, 122 mmol, 4 M). The reaction system was allowed to react at room temperature under nitrogen for 3 hours. LCMS monitoring revealed that after completion of the reaction, the reaction solution was concentrated, and the resulting residue was redissolved in dichloromethane (15 mL). MTBE (150 mL) was added and stirred at 0°C for 0.5 hours, resulting in the precipitation of a white solid. The filter cake was then washed with MTBE:dichloromethane (10:1) (10 mL x 3) to afford compound 1-3 (6.6 g, 86.4% yield).

[0171] LCMS (ESI) M / Z: 278.2 [M+H + ].

[0172] Step C: Compound 1-3 (5 g, 11.64 mmol) was added to a 25 mL single-necked flask, followed by dry N,N-dimethylformamide (15 mL), followed by compound 1-4 (3.94 g, 12.57 mmol), HATU (7.08 g, 18.62 mmol), and DIEA (5.79 mL, 34.92 mmol). The reaction system was reacted at room temperature under nitrogen for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was flash purified (acetonitrile / 0.1% formic acid / water) to afford compound 1-5 (6.2 g, 77.4% yield).

[0173] LCMS (ESI) M / Z: 689.5 [M+H + ].

[0174] Step D: Compound 1-5 (6.2 g, 9.0 mmol) was added to a 500 mL single-necked flask, followed by methanol (240 mL) and Pd / C (1.8 g, 10% Pd). The reaction system was reacted at 70°C under a hydrogen atmosphere for 12 hours. After completion of the reaction, the mixture was filtered and the filtrate was concentrated to afford compound 1-6 (5.3 g, 98.48% yield).

[0175] LCMS (ESI) M / Z: 599.9 [M+H + ].

[0176] 1 H NMR (400MHz, DMSO) δ8.13–7.98(m,1H),4.64(s,1H),4.59–4.45(m,1H),4.06–3.90(m,2H),3.83(d d,J=7.1,3.4Hz,1H),3.68–3.45(m,2H),3.40–3.24(m,4H),3.18(d,J=7.0Hz,3H),3.09–2.94(m,3H) ),2.68–2.61(m,1H),2.48–2.39(m,1H),2.39–2.27(m,2H),2.20(d,J=2.4Hz,6H),2.00–1.85(m,4H ),1.84–1.63(m,3H),1.37–1.24(m,1H),1.21–1.07(m,3H),0.94–0.85(m,12H),0.79–0.68(m,6H).

[0177] Step E: Add tetrahydrofuran (10 mL) to a dry reaction flask, then add compound 1-7 (2.00 g, 6.45 mmol) and stir until uniform. Then add borane tetrahydrofuran complex (2.77 g, 32.25 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by column chromatography (dichloromethane / methanol = 95 / 5-90 / 10) to obtain product 1-8 (900 mg, 71.17% yield).

[0178] LCMS (ESI) M / Z: 197.2 [M+H + ].

[0179] Step F: Add dichloromethane (5 mL) to a dry reaction flask, then add compound 1-8 (40 mg, 0.20 mmol) and N,N-diisopropylethylamine (77.54 mg, 0.60 mmol) and stir until uniform. Then add compound 1-6 (100 mg, 0.17 mmol) and HATU (114.07 mg, 0.30 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete by testing. The reaction solution is concentrated and separated by column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 1-9 (130 mg, 82.07% yield).

[0180] LCMS (ESI) M / Z: 777.5 [M+H + ].

[0181] Step G: Add tetrahydrofuran (5 mL) to a dry reaction flask, add compound 1-9 (60 mg, 0.077 mmol), and stir until uniform. Then add thionyl chloride (45.97 mg, 0.39 mmol). Heat in an oil bath to 80°C and stir under reflux. After 2 hours, take a sample for LCMS analysis, which shows the disappearance of the starting material spot and the formation of the target product ion peak. The reaction is confirmed to be complete, and the reaction solution is concentrated and separated by column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 1-10 (40 mg, 65.12% yield).

[0182] LCMS (ESI) M / Z: 795.4 [M+H + ].

[0183] Step H: Add N,N-dimethylformamide (2 mL) to a dry reaction flask, then add compound 1-10 (20 mg, 0.025 mmol) and stir until uniform. Then add 4,4'-bipyridine (1.95 mg, 0.013 mmol) and stir for 5 minutes before adding diboric acid (11.21 mg, 0.13 mmol). The reaction mixture was stirred at room temperature. After 15 minutes, a sample was taken and sent for LCMS analysis. The results showed that the starting material spot disappeared and the target product ion peak was generated. After confirming the completion of the reaction, the reaction mixture was purified by HPLC to obtain the target compound 1 (3.5 mg, 18.19% yield).

[0184] LCMS (ESI) M / Z: 765.4 [M+H + ].

[0185] 1H NMR(400MHz, DMSO-d6)δ7.04–6.93(m,2H),6.64–6.56(m,2H),6.51(d,J=9.2Hz,4H),4.23–4.14(m,1H),4.10–4.03(m,1H),3.67–3.59 (m,3H),3.57–3.51(m,1H),3.44–3.41(m,1H),3.38–3.35(m,2H),3.34(s,3H),3.14(s,3H),2.96–2.92(m,2H),2.86(d,J=7.9Hz,1H), 2.80(d,J=7.6Hz,1H),2.74–2.65(m,3H),2.53(d,J=4.2Hz,2H),2.42(s,6H),2.12–2.08(m,2H),1.81–1.77(m,2H),1.45–1.39(m,2H) ,1.22–1.19(m,3H),1.17–1.13(m,3H),1.04–1.02(m,3H),1.01–1.01(m,3H),1.00–0.99(m,3H),0.91–0.89(m,3H),0.88–0.87(m,3H).

[0186] Example 2:

[0187] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-(3-aminophenyl)-but-3-yn-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0188] Reaction route:

[0189] Steps:

[0190] Step A: Add tetrahydrofuran (10 mL) to a dry reaction flask, then add compound 1-7 (1.00 g, 3.22 mmol) and stir until uniform. Then add borane tetrahydrofuran complex (830.18 mg, 9.66 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken for analysis by LCMS, which shows the disappearance of the starting material spot. BOC anhydride (763.88 mg, 3.51 mmol) and sodium bicarbonate (294.88 mg, 3.51 mmol) are added to the reaction mixture, and the reaction is stirred overnight. A sample is taken for analysis by LCMS, which shows the formation of the target product ion peak. The reaction is confirmed to be complete, and the reaction mixture is concentrated and separated by column chromatography (dichloromethane / methanol = 95 / 5-90 / 10) to obtain product 2-2 (900 mg, 71.17% yield).

[0191] LCMS (ESI) M / Z: 197.2 [M+H + -Boc].

[0192] Step B: Add dichloromethane (15 mL) to a dry reaction flask, then add compound 2-2 (200 mg, 0.67 mmol) and stir until uniform. Then add Dess-Martin periodinane (568.35 mg, 1.34 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-98 / 2) to obtain product 2-3 (150 mg, 75.51% yield).

[0193] LCMS (ESI) M / Z: 195.1 [M+H + -BOC].

[0194] Step C: Methanol (5 mL) was added to a dry reaction flask, followed by compound 2-3 (150 mg, 0.51 mmol) and stirring. Potassium carbonate (211.46 mg, 1.53 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (293.93 mg, 1.53 mmol) were then added. The reaction mixture was stirred at room temperature. After 4 hours, a sample was taken and analyzed by LCMS. The results showed that the starting material spot disappeared and the target product ion peak was generated. The reaction was confirmed to be complete. The reaction solution was concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 2-4 (100 mg, 67.58% yield).

[0195] LCMS (ESI) M / Z: 191.1 [M+H + -Boc].

[0196] Step D: Add dichloromethane (4 mL) to a dry reaction flask, add compound 2-4 (100 mg, 0.34 mmol), and stir until uniform. Then add trifluoroacetic acid (1 mL). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and sent for LCMS analysis. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. Filtration and concentration are performed to obtain crude product 2-5 (60 mg, 91.58% yield), which is used directly in the next step without further purification.

[0197] LCMS (ESI) M / Z: 191.1 [M+H + ].

[0198] Step E: Add dichloromethane (5 mL) to a dry reaction flask, then add compound 2-5 (40 mg, 0.21 mmol) and N,N-diisopropylethylamine (81.42 mg, 0.63 mmol) and stir until uniform. Then add compound 1-6 (100 mg, 0.17 mmol) and HATU (119.77 mg, 0.32 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete by analysis. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 2-6 (40 mg, 24.67% yield).

[0199] LCMS (ESI) M / Z: 771.5 [M+H + ].

[0200] Step F: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add 2-6 (130 mg, 0.17 mmol), and stir evenly. Then add 4,4'-bipyridine (0.013 g, 0.085 mmol) and tetrahydroxydiboron (76.20 mg, 0.85 mmol). The reaction solution is stirred at room temperature. After 4 hours, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction solution is purified by HPLC to obtain the product compound 2 (20 mg, yield 16.01%).

[0201] LCMS (ESI) M / Z: 741.5 [M+H + ].

[0202] 1H NMR (400MHz, DMSO-d6) δ8.49(d,J=5.5Hz,1H),8.29(dd,J=12.9,8.3Hz,1H),8.01(t,J=9.2Hz,2H),6.96–6.84(m,2H),6.42(d,J=5.0Hz,2H),6.39 –6.33(m,3H),4.95–4.89(m,1H),4.67–4.63(m,1H),4.52(t,J=8.8Hz,1H ),4.05–3.94(m,2H),3.83–3.80(m,1H),3.27(s,3H),3.18(s,3H),3.16– 3.15(m,2H),2.99(s,3H),2.66–2.63(m,1H),2.63–2.60(m,1H),2.21(s, 6H),2.00–1.87(m,7H),1.76–1.70(m,2H),1.63–1.57(m,1H),1.49–1.43 (m,1H),1.10–1.04(m,3H),1.01(t,J=6.1Hz,3H),0.91–0.90(m,3H),0.8 9–0.88(m,3H),0.88–0.87(m,3H),0.75–0.73(m,3H),0.72–0.69(m,3H).

[0203] Example 3:

[0204] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-amino-3-(3-aminophenyl)-1-oxopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0205] Reaction route:

[0206] Steps:

[0207] Step A: To a dry reaction flask, add N,N-dimethylformamide (4 mL), compound 1-7 (300 mg, 0.97 mmol), N,N-diisopropylethylamine (376.09 mg, 2.91 mmol), and HATU (553.23 mg, 1.46 mmol) and stir until uniform. Then, add ammonium chloride (103.77 mg, 1.94 mmol). The reaction mixture is stirred at room temperature. After filtration, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction mixture is diluted with dichloromethane (20 mL) and extracted with water (30 mL). The oil layer is dried, concentrated, and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 3-2 (280 mg, 93.63% yield).

[0208] LCMS (ESI) M / Z: 210.1 [M+H + -BOC].

[0209] Step B: Add dichloromethane (4 mL) to a dry reaction flask, add compound 3-2 (280 mg, 0.91 mmol), and stir until uniform. Then add trifluoroacetic acid (1 mL). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and sent for LCMS analysis. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. Filtration and concentration are performed to obtain crude compound 3-3 (200 mg, yield 105.61%), which is used directly in the next step without further purification.

[0210] LCMS (ESI) M / Z: 210.1 [M+H + ].

[0211] Step C: Add dichloromethane (5 mL) to a dry reaction flask, then add compound 3-3 (25 mg, 0.12 mmol) and N,N-diisopropylethylamine (46.53 mg, 0.36 mmol) and stir until uniform. Then add compound 1-6 (70 mg, 0.12 mmol) and HATU (68.44 mg, 0.18 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 3-4 (70 mg, 74.15% yield).

[0212] LCMS (ESI) M / Z: 790.5 [M+H + ].

[0213] Step D: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add 3-4 (35 mg, 0.044 mmol), and stir evenly. Then add 4,4'-bipyridine (3.44 mg, 0.022 mmol) and tetrahydroxydiboron (19.72 mg, 0.22 mmol). The reaction solution is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction solution is purified by HPLC to obtain the product compound 3 (1.5 mg, yield 4.45%).

[0214] LCMS (ESI) M / Z: 760.5 [M+H + ].

[0215] 1 H NMR(400MHz, Methanol-d4)δ7.04–6.94(m,1H),6.66–6.47(m,3H),4.67–4.63(m,1H),4.61–4.55(m,1H),4.19–4.03(m,1H),3.88(d,J =1.8Hz,1H),3.79–3.66(m,1H),3.57–3.47(m,1H),3.45–3.37(m,2H),3.36–3.32(m,6H),3.28–3.25(m,2H),3.20–3.16(m,1H),3.19– 3.05(m,3H),2.82–2.70(m,2H),2.53–2.45(m,2H),2.39–2.34(m,6H),2.15–1.97(m,3H),1.96–1.82(m,2H),1.82–1.69(m,2H),1.68– 1.56(m,1H),1.20(d,J=6.8Hz,1H),1.09(t,J=6.2Hz,3H),1.06–1.01(m,6H),1.00–0.95(m,6H),0.91–0.87(m,3H),0.87–0.82(m,3H).

[0216] Example 4

[0217] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-3-(3-aminophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0218] Reaction route:

[0219] Steps:

[0220] Step A: To a dry reaction flask, add N,N-dimethylformamide (4 mL), compound 1-7 (300 mg, 0.97 mmol), N,N-diisopropylethylamine (376.09 mg, 2.91 mmol), and HATU (553.23 mg, 1.46 mmol) and stir until uniform. Then, add methylamine hydrochloride (327.47 mg, 4.85 mmol). The reaction mixture is stirred at room temperature. After filtration, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction mixture is diluted with dichloromethane (20 mL) and extracted with water (30 mL). The oil layer is dried, concentrated, and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 4-2 (250 mg, 79.97% yield).

[0221] LCMS (ESI) M / Z: 224.1 [M+H + -BOC].

[0222] Step B: Add dichloromethane (4 mL) to a dry reaction flask, add compound 4-2 (250 mg, 0.77 mmol), and stir until uniform. Then add trifluoroacetic acid (1 mL). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and sent for LCMS analysis. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete by testing. Filtration and concentration are performed to obtain crude product 4-3 (230 mg, yield 133.26%), which is used directly in the next step without further purification.

[0223] LCMS (ESI) M / Z: 224.1 [M+H + ].

[0224] Step C: Add dichloromethane (2 mL) to a dry reaction flask, then add compound 4-3 (25 mg, 0.12 mmol) and N,N-diisopropylethylamine (42.65 mg, 0.33 mmol) and stir until uniform. Then add compound 1-6 (70 mg, 0.12 mmol) and HATU (62.74 mg, 0.17 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 4-4 (18 mg, yield 19.99%).

[0225] LCMS (ESI) M / Z: 804.5 [M+H + ].

[0226] Step D: Add acetonitrile (1 mL) to a dry reaction flask, add compound 4-4 (18.49 mg, 0.023 mmol), and stir evenly. Then add 4,4'-bipyridine (1.80 mg, 0.011 mmol) and tetrahydroxydiboron (10.31 mg, 0.11 mmol). The reaction solution is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction solution is purified by HPLC to obtain the product compound 4 (5.1 mg, yield 28.65%).

[0227] LCMS (ESI) M / Z: 774.5 [M+H + ].

[0228] 1H NMR (400MHz, DMSO-d6) δ8.18(d,J=8.7Hz,0H),8.05(t,J=8.2Hz,1H),7.99(d,J=8.4Hz,1H ),7.87–7.74(m,1H),6.83(t,J=7.7Hz,1H),6.44–6.28(m,3H),4.73–4.58(m,2H),4.53(t ,J=8.9Hz,1H),4.39(ddd,J=10.5,8.4,4.4Hz,1H),4.02–3.96(m,1H),3.84(dd,J=9.8,2. 1Hz,1H),3.67–3.62(m,3H),3.37–3.33(m,1H),3.33–3.27(m,2H),3.25(d,J=5.5Hz,3H),3 .19(d,J=2.8Hz,3H),3.10(s,1H),3.02–2.96(m,2H),2.93–2.86(m,1H),2.72–2.61(m,2H ),2.60(dd,J=4.6,1.3Hz,3H),2.47–2.36(m,1H),2.34–2.26(m,1H),2.20(d,J=7.7Hz,6H) ,2.18–2.12(m,1H),2.05–1.84(m,3H),1.78–1.66(m,2H),1.65–1.42(m,2H),1.02(d,J=6 .7Hz,3H),0.95–0.90(m,6H),0.89–0.85(m,6H),0.80–0.74(m,3H),0.72(d,J=6.6Hz,3H).

[0229] Example 5

[0230] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(allyl(3-aminophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0231] Reaction route:

[0232] Steps:

[0233] Step A: Add dichloromethane (25 mL) to a dry reaction flask, then add compound 5-1 (1 g, 5.98 mmol) and stir until uniform. Then add Dess-Martin periodinane (3.80 g, 8.97 mmol). The reaction mixture is stirred at room temperature. After 3 hours, a sample is taken for TLC analysis, which shows the disappearance of the starting material spot and the formation of new spots. The reaction is confirmed to be complete by testing. The reaction mixture is concentrated and separated by preparative column chromatography (petroleum ether / ethyl acetate = 95 / 5-80 / 20) to obtain the product, compound 5-2 (500 mg, 50.61% yield).

[0234] 1 H NMR (400MHz, Chloroform-d) δ9.84(t,J=1.7Hz,1H),8.18(tt,J=5.2,2.4Hz,1H),8.11(t,J=1.6Hz,1H),7.58–7.51(m,2H),3.88(d,J=1.6Hz,2H).

[0235] Step B: Add methanol (5 mL) to a dry reaction flask, then add compound 5-2 (100 mg, 0.61 mmol) and 2-propen-1-amine hydrochloride (100 mg, 1.07 mmol) and stir until uniform. After 0.5 hour, add sodium cyanoborohydride (95.83 mg, 1.52 mmol). The reaction mixture was stirred at room temperature. After 4 hours, a sample was taken and analyzed by LCMS. The results showed that the starting material spot disappeared and the target product ion peak was generated. The reaction was confirmed to be complete. The reaction solution was concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 5-3 (46 mg, 36.84% yield).

[0236] LCMS (ESI) M / Z: 207.1 [M+H + ].

[0237] Step C: Add dichloromethane (3 mL) to a dry reaction flask, then add compound 1-6 (130 mg, 0.22 mmol), N,N-diisopropylethylamine (85.30 mg, 0.66 mmol), and HATU (125.48 mg, 0.33 mmol) and stir until uniform. Then add compound 5-3 (46 mg, 0.22 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete by testing. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 5-4 (54 mg, 30.76% yield).

[0238] LCMS (ESI) M / Z: 787.5 [M+H + ].

[0239] Step D: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add compound 5-4, and stir evenly. Then add tetrahydroxydiboron (30.93 mg, 0.35 mmol) and 4,4'-bipyridine (5.39 mg, 0.035 mmol). The reaction mixture is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction mixture is purified by HPLC to obtain the product compound 5 (7.5 mg, yield 14.44%).

[0240] LCMS (ESI) M / Z: 757.5 [M+H + ].

[0241] 1 H NMR(400MHz, Methanol-d4)δ7.09–6.93(m,1H),6.65–6.49(m,3H),5.88–5.72(m,1H ),5.24–5.17(m,1H),5.17–5.09(m,1H),4.73–4.69(m,1H),4.65(dd,J=8.5,1.9Hz,1 H),4.21–4.01(m,2H),4.00–3.90(m,2H),3.89–3.77(m,1H),3.71–3.60(m,2H),3.5 5(t,J=7.3Hz,1H),3.51–3.45(m,1H),3.41(s,3H),3.39–3.36(m,1H),3.34(s,3H),3 .29(s,3H),3.28–3.21(m,1H),3.16–3.08(m,2H),2.91–2.83(m,1H),2.81–2.70(m, 3H),2.68–2.57(m,1H),2.57–2.48(m,1H),2.44(s,6H),2.21–2.11(m,1H),2.10–1.9 9(m,3H),1.97–1.90(m,1H),1.84–1.72(m,2H),1.48–1.37(m,1H),1.24–1.13(m,3H) ,1.06–1.01(m,6H),1.01–0.97(m,6H),0.91(d,J=6.4Hz,3H),0.87(d,J=6.4Hz,3H).

[0242] Example 6

[0243] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(prop-2-yn-1-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0244] Reaction route:

[0245] Steps:

[0246] Step A: Add methanol (5 mL) to a dry reaction flask, then add compound 5-2 (100 mg, 0.61 mmol) and propargylamine hydrochloride (111.68 mg, 1.22 mmol) and stir until uniform. After 0.5 hour, add sodium cyanoborohydride (95.83 mg, 1.52 mmol). The reaction mixture was stirred at room temperature. After 4 hours, a sample was taken and analyzed by LCMS. The results showed that the starting material spot disappeared and the target product ion peak was generated. The reaction was confirmed to be complete. The reaction solution was concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain the product compound 6-2 (59 mg, 47.71% yield).

[0247] LCMS (ESI) M / Z: 205.1 [M+H + ].

[0248] Step B: Add dichloromethane (3 mL) to a dry reaction flask, then add compound 1-6 (76 mg, 0.13 mmol), N,N-diisopropylethylamine (54.28 mg, 0.42 mmol), and HATU (79.85 mg, 0.21 mmol) and stir until uniform. Then add compound 6-2 (29 mg, 0.14 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 6-3 (48 mg, 43.06% yield).

[0249] LCMS (ESI) M / Z: 785.5 [M+H + ].

[0250] Step C: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add compound 6-3 (54 mg, 0.069 mmol), and stir until uniform. Then add tetrahydroxydiboron (27.34 mg, 0.30 mmol) and 4,4'-bipyridine (4.76 mg, 0.030 mmol). The reaction mixture is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction mixture is purified by HPLC to obtain the product compound 6 (6.04 mg, yield 11.52%).

[0251] LCMS (ESI) M / Z: 755.5 [M+H + ].

[0252] 1 H NMR(400MHz, Methanol-d4)δ7.07–6.97(m,1H),6.64–6.49(m,3H),4.74–4.69(m, 1H),4.66–4.62(m,1H),4.25–4.12(m,1H),4.11–4.04(m,2H),3.92(t,J=8.0Hz,1H ),3.69(s,2H),3.65–3.56(m,1H),3.43–3.39(m,3H),3.37–3.34(m,1H),3.32(s,3 H),3.29(d,J=2.6Hz,3H),3.26(s,1H),3.14–3.09(m,3H),2.96–2.90(m,1H),2.84 –2.81(m,2H),2.80–2.78(m,1H),2.78–2.75(m,1H),2.72–2.61(m,2H),2.51–2.4 5(m,1H),2.38(s,6H),2.14–2.10(m,1H),2.06–2.00(m,3H),1.84–1.71(m,3H),1. 44–1.38(m,1H),1.27–1.14(m,3H),1.02(d,J=2.3Hz,3H),1.01(d,J=1.9Hz,3H),0 .99–0.99(m,3H),0.97(d,J=2.5Hz,3H),0.89–0.87(m,3H),0.86(d,J=1.8Hz,3H).

[0253] Example 7

[0254] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(methoxy)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0255] Reaction route:

[0256] Steps:

[0257] Step A: Add methanol (5 mL) to a dry reaction flask, then add compound 5-2 (100 mg, 0.61 mmol) and methoxyamine hydrochloride (101.89 mg, 1.22 mmol) and stir until uniform. After 0.5 hour, add sodium cyanoborohydride (95.83 mg, 1.52 mmol). The reaction mixture is stirred at room temperature. After 4 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1 to 90 / 10) to obtain product 7-2 (46 mg, 38.72% yield).

[0258] LCMS (ESI) M / Z: 197.1 [M+H + ].

[0259] Step B: Add dichloromethane (3 mL) to a dry reaction flask, then add compound 1-6 (76 mg, 0.13 mmol), N,N-diisopropylethylamine (46.53 mg, 0.36 mmol), and HATU (68.44 mg, 0.18 mmol) and stir until uniform. Then add compound 7-2 (23 mg, 0.12 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 7-3 (12 mg, yield 13.17%).

[0260] LCMS (ESI) M / Z: 777.4 [M+H + ].

[0261] Step C: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add compound 7-3 (12 mg, 0.015 mmol), and stir evenly. Then add tetrahydroxydiboron (6.72 mg, 0.075 mmol) and 4,4'-bipyridine (1.17 mg, 0.0075 mmol). The reaction solution is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction solution is purified by HPLC to obtain the product compound 7 (3.0 mg, yield 26.00%).

[0262] LCMS (ESI) M / Z: 747.5 [M+H + ].

[0263] 1 H NMR(400MHz, DMSO-d6)δ8.04(dd,J=15.6,8.7Hz,1H),6.91(td,J=7.7,3.2Hz,1H),6.43–6.33(m,3H),4.94(s,1H),4.76–4.61(m,1H),4.60–4.48 (m,1H),4.04–3.88(m,2H),3.82(dd,J=14.5,7.4Hz,1H),3.72(dd,J=7.5,3.5Hz,1H),3.62(d,J=11.0Hz,3H),3.26(s,3H),3.18(d,J=6.2Hz,6H) ,3.15–3.13(m,2H),3.00(s,3H),2.68–2.65(m,1H),2.65–2.61(m,2H), 2.34–2.27(m,1H),2.20(d,J=9.2Hz,6H),2.03–1.96(m,2H),1.95–1.84( m,4H),1.78–1.61(m,3H),1.51–1.42(m,1H),1.14–1.06(m,3H),0.92–0 .88(m,6H),0.87(d,J=6.3Hz,6H),0.77–0.74(m,3H),0.73–0.69(m,3H).

[0264] Example 8

[0265] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(hydroxy)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0266] Reaction route:

[0267] Steps:

[0268] Step A: Add methanol (5 mL) to a dry reaction flask, then add compound 5-2 (100 mg, 0.61 mmol) and hydroxylamine hydrochloride (84.78 mg, 1.22 mmol) and stir until uniform. After 0.5 hour, add sodium cyanoborohydride (95.83 mg, 1.52 mmol). The reaction mixture was stirred at room temperature. After 4 hours, a sample was taken and analyzed by LCMS. The results showed that the starting material spot disappeared and the target product ion peak was generated. The reaction was confirmed to be complete. The reaction solution was concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 8-2 (19 mg, yield 17.22%).

[0269] LCMS (ESI) M / Z: 183.1 [M+H + ].

[0270] Step B: Add dichloromethane (3 mL) to a dry reaction flask, then add compound 1-6 (76 mg, 0.13 mmol), N,N-diisopropylethylamine (38.77 mg, 0.30 mmol), and HATU (57.03 mg, 0.15 mmol) and stir until uniform. Then add compound 8-2 (19 mg, 0.10 mmol). The reaction mixture is stirred at room temperature. After 6 hours, a sample is taken and analyzed by LCMS. The results show that the starting material spot disappears and the target product ion peak is generated. The reaction is confirmed to be complete. The reaction solution is concentrated and separated by preparative column chromatography (dichloromethane / methanol = 99 / 1-90 / 10) to obtain product 8-3 (25 mg, 31.42% yield).

[0271] LCMS (ESI) M / Z: 763.4 [M+H + ].

[0272] Step C: Add N,N-dimethylformamide (1 mL) to a dry reaction flask, add compound 8-3 (25 mg, 0.033 mmol), and stir until uniform. Then add tetrahydroxydiboron (14.79 mg, 0.17 mmol) and 4,4'-bipyridine (2.58 mg, 0.017 mmol). The reaction mixture is stirred at room temperature. After 15 minutes, a sample is taken for LCMS analysis, which shows that the raw material spot disappears and the target product ion peak is generated. After confirming the completion of the reaction, the reaction mixture is purified by HPLC to obtain the product compound 8 (1.4 mg, 5.83% yield).

[0273] LCMS (ESI) M / Z: 733.5 [M+H + ].

[0274] 1 H NMR (400MHz, DMSO-d6) δ8.03(t,J=8.6Hz,1H),6.89(t,J=7.7Hz,1H),6.49–6.26(m,3H),4.92(s,2H),4.76–4.62(m,1H),4.63–4.46(m, 2H),4.04–3.92(m,1H),3.84(dd,J=18.9,10.0Hz,1H),3.77–3.68(m,1H),3.30(s,3H),3.28(s,3H),3.20–3.19(m,1H),3.17(s,3H),3.1 4–3.08(m,2H),2.99(s,3H),2.71–2.61(m,3H),2.34–2.26(m,1H),2.20(s,6H),2.04–1.97(m,1H),1.96–1.89(m,2H),1.88–1.81(m,2H ),1.78–1.58(m,3H),1.56–1.36(m,1H),1.13–1.02(m,3H),0.94–0.89(m,6H),0.88–0.85(m,6H),0.78–0.74(m,3H),0.72–0.69(m,3H).

[0275] Example 9

[0276] Preparation of (S)-1-(3-aminophenyl)-3-cyanopropyl-2-yl (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(S)-(dimethylamino)-3-methylbutane)-N,3-dimethylbutane)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate.

[0277] Reaction route:

[0278] Steps:

[0279] Step A: Compound 9-1 (2.0 g, 9.52 mmol) was dissolved in dilute sulfuric acid (1 M, 20 mL), then cooled to 0°C and slowly added dropwise with an aqueous sodium nitrite solution (3.9 g / 12 mL, 57.31 mmol). The addition was complete after half an hour, and the system was allowed to warm to room temperature and stirred overnight. LCMS indicated the reaction was complete. The reaction mixture was extracted with ethyl acetate (100 mL), and the organic phase was washed sequentially with water (50 mL) and saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by flash chromatography (acetonitrile / 0.1% ammonium bicarbonate / water) to afford product 9-2 (1.2 g, 60% yield).

[0280] LCMS (ESI) M / Z: 212.0 [M+H + ].

[0281] Step B: Compound 9-2 (100 mg, 0.47 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), then cooled to 0°C and borane tetrahydrofuran (2.8 mL, 2.8 mmol, 1 M tetrahydrofuran) was added dropwise. The reaction system was stirred at room temperature for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was quenched with methanol, diluted with dichloromethane (20 mL), and washed sequentially with saturated sodium bicarbonate (10 mL) and saturated sodium chloride (10 mL). The mixture was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by reverse phase column chromatography (acetonitrile / 0.1% ammonium bicarbonate / water) to obtain compound 9-3 (60 mg, 64% yield).

[0282] LCMS (ESI) M / Z: 393.0 [M+H + ].

[0283] Step C: Dissolve compound 9-3 (100 mg, 0.51 mmol) in anhydrous dichloromethane (3 mL), then cool to 0°C. Add p-toluenesulfonyl chloride (145 mg, 0.76 mmol) and triethylamine (0.18 mL, 0.13 mmol). Allow the reaction to warm to room temperature and stir overnight. LCMS indicates completion of the reaction. Dilute the reaction solution with dichloromethane (10 mL), then wash with water (10 mL) and saturated sodium chloride (10 mL), dry over anhydrous sodium sulfate, and concentrate to obtain a crude product. The crude product is purified by reverse-phase column chromatography (acetonitrile / 0.1% ammonium bicarbonate / water) to afford compound 9-4 (10 mg, 56% yield).

[0284] LCMS (ESI) M / Z: 369.0 [M+H+ ].

[0285] Step D: Compound 9-4 (80 mg, 0.20 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of potassium iodide (38 mg, 0.20 mmol) and sodium cyanide (40 mg, 0.80 mmol). The reaction system was stirred at 75°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with EA (20 mL), then washed with water (10 mL) and saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by reverse phase column (acetonitrile / 0.1% ammonium bicarbonate / water) to obtain product 9-5 (20 mg, 43% yield).

[0286] LCMS (ESI) M / Z: 411.0 [2M-H] — .

[0287] Step E: Compound 1-6 (200 mg, 0.33 mmol) was dissolved in dry dichloromethane (5 mL), followed by the addition of compound 9-5 (199 mg, 0.96 mmol), N,N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol), and 4-dimethylaminopyridine (4 mg, 0.03 mmol). The reaction system was incubated at room temperature under nitrogen for 16 hours. After completion of the reaction, as monitored by LCMS, the reaction was filtered and the filtrate was purified by Flash chromatography to afford compound 9-6 (140 mg, 53% yield).

[0288] LCMS (ESI) M / Z: 787.2 [M+H + ].

[0289] Step F: Dissolve tetrahydroxydiboron (58 mg, 0.64 mmol) in dry N,N-dimethylformamide (5 mL). 4,4'-bipyridine (78 mg, 0.37 mmol) and compound 9-6 (120 mg, 0.15 mmol) were added sequentially and quickly. The reaction system was incubated at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to quench the reaction, and the filtrate was filtered. The filtrate was purified by HPLC to afford the title compound 9 (60 mg, 52% yield).

[0290] LCMS (ESI) M / Z: 757.5 [M+H + ].

[0291] 1H NMR(400MHz,DMSO)δ8.89(d,J=7.7Hz,1H),7.25(m,1H),7.00–6.82(m,3H),5.34–5.09(m,1H) ,4.72–4.50(m,2H),4.00–3.89(m,2H),3.85(m,1H),3.72(m,1H),3.58–3.40(m,2H),3.37–3.2 1(m,4H),3.17(m,3H),3.03(m,3H),2.97–2.81(m,4H),2.75(m,6H),2.47–2.21(m,4H),2.10– 1.63(m,6H),1.38–1.20(m,1H),1.01(d,J=6.8Hz,3H),0.98–0.82(m,15H),0.81–0.72(m,3H).

[0292] Example 10

[0293] Preparation of ((S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(allyl((S)-1-(3-aminophenyl))-3-cyanopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0294] Reaction route:

[0295] Steps:

[0296] Step A: Compound 10-1 (400 mg, 2 mmol) was dissolved in ultra-dry N,N-dimethylformamide (15 mL). Allyl bromide (223 mg, 3 mmol) and potassium carbonate (260 mg, 3 mmol) were added, and the mixture was stirred at 60°C for 16 hours. LCMS indicated the reaction was complete. The mixture was filtered, and the filtrate was purified by preparative HPLC (0.1% FA in water / acetonitrile) to afford product 10-2 (200 mg, 40.01% yield).

[0297] LCMS (ESI) M / Z: 246.1 [M+H + ].

[0298] Step B: Compound 10-2 (200 mg, 0.9 mmol), compound 1-6 (400 mg, 0.67 mmol), NMI (0.27 ml), and TCFH (470 mg, 1.68 mmol) were added to 2 mL of anhydrous MeCN (5 mL) under nitrogen. The resulting mixture was stirred at 60°C overnight. After LCMS showed completion of the reaction, the reaction solution was concentrated, 3 mL of tetrahydrofuran was added, and the mixture was filtered. The filtrate was purified using a reverse-phase column (0.1% FA in water / acetonitrile) to afford compound 10-3 (40 mg, 20.63% yield).

[0299] LCMS (ESI) M / Z: 826.4 [M+H + ].

[0300] Step C: Add tetrahydroxydiboron (80 mg, 0.1 mmol) to anhydrous N,N-dimethylformamide (1 mL), followed immediately by the addition of 4,4'-bipyridine (15 mg, 0.1 mmol) and compound 10-3 (40 mg, 0.1 mmol). The resulting mixture was stirred at room temperature for 5 minutes. After LCMS indicated completion of the reaction, the mixture was filtered, and the filtrate was purified by HPLC to afford the title compound 10 (7 mg, 30.59% yield).

[0301] LCMS (ESI) M / Z: 796.5 [M+H + ].

[0302] 1 H NMR (400MHz, DMSO) δ8.07–7.96(m,1H),6.95–6.87(m,1H),6.43–6.28(m,3H),5.73–5.59(m,1H),5.35–5.08(m,2H),5.05–4.84(m,2H),4.68–4.4 7(m,2H),4.03–3.92(m,2H),3.87–3.76(m,1H),3.62–3.48(m,2H),3.29 (d,J=5.5Hz,2H),3.19(d,J=6.1Hz,5H),3.10(s,2H),3.00(s,3H),2.89– 2.76(m,2H),2.71–2.55(m,4H),2.45–2.26(m,2H),2.20(d,J=10.0Hz,6H),2 .01–1.63(m,8H),1.37–1.28(m,1H),0.97–0.85(m,15H),0.78–0.66(m,6H).

[0303] Example 11

[0304] Preparation of 1-(3-aminophenyl)propan-2-yl(2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate.

[0305] Reaction route:

[0306] Steps:

[0307] Step A: Dissolve the starting material 11-1 (2.5 g, 1.81 mmol) in acetonitrile (25 mL) and water (2.5 mL). Add isopropyl acetate (1.82 g, 1.81 mmol) and salicylic acid (250 mg, 0.181 mmol). Stir for 5 min, then add tert-butyl nitrite (2.8 g, 2.71 mmol). The resulting mixture is reacted at room temperature under nitrogen for 3 h. TLC indicates the reaction is complete. Ammonium chloride (20 mL) is added to quench the reaction, and the mixture is extracted with dichloromethane (50 mL x 3). The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography (PE:EA = 10:1) to afford the product 11-2 (0.9 g, 30% yield).

[0308] 1 H NMR (400MHz, CDCl3) δ8.32–7.90 (m, 2H), 7.52 (d, J = 5.3Hz, 2H), 3.86 (s, 2H), 2.26 (s, 3H).

[0309] Step B: Dissolve compound 11-2 (600 mg, 2.7 mmol) in anhydrous tetrahydrofuran (20 mL). Add borane tetrahydrofuran solution (5.6 mL, 5.6 mmol, 1 M in THF) dropwise at 0°C. Stir the reaction system at room temperature for 16 hours. After TLC indicates completion of the reaction, quench the reaction with methanol, dilute with dichloromethane (20 mL), and wash sequentially with saturated sodium bicarbonate (10 mL) and saturated sodium chloride (10 mL). Dry over anhydrous sodium sulfate, and concentrate to obtain the crude product. The crude product was purified by column chromatography (PE:EA=9:1) to obtain racemate 11-3, which was then subjected to chiral resolution to obtain single-configuration compound 11-3A (200 mg, yield 34%) and compound 11-3B (200 mg, yield 34%). The resolution conditions were: mobile phase: supercritical carbon dioxide and 0.1% ammonia in isopropanol solution; flow rate: 100 mL / min; gradient: maintaining 0.1% ammonia in isopropanol at 20%; detection wavelength: 214 nm; cycle time: 7.5 min.

[0310] The retention time of compound 11-3A is Rt=5.6 min; the retention time of compound 11-3B is Rt=8.2 min.

[0311] 1 H NMR (400MHz, DMSO) δ8.19–7.94(m,2H),7.75–7.49(m,2H),4.65(d,J=4.9Hz,1H),3.87(m, J=11.4,5.5Hz,1H),2.90–2.64(m,2H),1.08(d,J=6.2Hz,3H).

[0312] Step C: Compound 1-6 (132 mg, 0.22 mmol) was dissolved in dry dichloromethane (5 mL), followed by the addition of compound 11-3A (40 mg, 0.22 mmol), N,N'-dicyclohexylcarbodiimide (91 mg, 0.44 mmol), and 4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction system was reacted at room temperature under nitrogen for 16 hours. After completion of the reaction, as monitored by LCMS, the reaction was filtered, and the filtrate was purified by flash chromatography (acetonitrile / 0.1% ammonium bicarbonate / water) to afford compound 11-4 (120 mg, 53% yield).

[0313] LCMS (ESI) M / Z: 762.4 [M+H + ].

[0314] Step D: Dissolve tetrahydroxydiboron (95 mg, 1.01 mmol) in dry N,N-dimethylformamide (5 mL). 4,4'-bipyridine (20 mg, 0.25 mmol) and compound 11-4 (100 mg, 0.15 mmol) were added sequentially and quickly. The reaction system was incubated at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to quench the reaction. The mixture was filtered and the filtrate was purified by HPLC to afford the title compound 11 (30 mg, 52% yield).

[0315] LCMS (ESI) M / Z: 732.3 [M+H + ].

[0316] 1H NMR (400MHz, DMSO) δ8.09–7.97(m,1H),6.95–6.79(m,1H),6.44–6.27(m,3H),5.15–4.86(m,3H),4.75–4.43(m ,2H),4.05–3.91(m,1H),3.88–3.78(m,1H),3.77–3.68(m,1H),3.64–3.46(m,1H),3.41–3.30(m,2H),3.27–3.1 0(m,6H),3.09–2.95(m,3H),2.72–2.59(m,3H),2.46–2.29(m,3H),2.24–2.13(m,6H),2.04–1.83(m,3H),1.81 –1.53(m,4H),1.36–1.25(m,1H),1.23–1.13(m,3H),1.13–1.04(m,3H),0.95–0.85(m,12H),0.80–0.63(m,6H).

[0317] Example 12

[0318] Preparation of 1-(3-aminophenyl)propan-2-yl(2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate.

[0319] Reaction route:

[0320] Steps:

[0321] Step A: Compound 1-6 (32 mg, 0.22 mmol) was dissolved in dry dichloromethane (5 mL), followed by the addition of compound 11-3B (40 mg, 0.22 mmol), N,N'-dicyclohexylcarbodiimide (91 mg, 0.44 mmol), and 4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction system was reacted at room temperature under nitrogen for 16 hours. After completion of the reaction as monitored by LCMS, the reaction was filtered, and the filtrate was purified by flash chromatography (acetonitrile / 0.1% ammonium bicarbonate / water) to afford compound 12-2 (120 mg, 53% yield).

[0322] LCMS (ESI) M / Z: 762.4 [M+H + ].

[0323] Step B: Dissolve tetrahydroxydiboron (95 mg, 1.01 mmol) in dry N,N-dimethylformamide (5 mL), then quickly add 4,4'-bipyridine (20 mg, 0.25 mmol) and compound 12-2 (100 mg, 0.15 mmol). The reaction system is incubated at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol is added, the mixture is filtered, and the filtrate is purified by preparative HPLC (acetonitrile / 0.1% ammonium bicarbonate / water) to afford compound 12 (30 mg, 52% yield).

[0324] LCMS (ESI) M / Z: 732.3 [M+H + ].

[0325] 1 H NMR (400MHz, DMSO) δ8.09–7.97(m,1H),6.95–6.79(m,1H),6.44–6.27(m,3H),5.15–4.86(m,3H), 4.75–4.43(m,2H),4.05–3.91(m,1H),3.88–3.78(m,1H),3.77–3.68(m,1H),3.64–3.46(m,1H),3. 41–3.30(m,2H),3.27–3.10(m,6H),3.09–2.95(m,3H),2.72–2.59(m,3H),2.46–2.29(m,3H),2.2 4–2.13(m,6H),2.04–1.83(m,3H),1.81–1.53(m,4H),1.36–1.25(m,1H),1.23–1.13(m,3H),1.13– 1.04(m,3H),0.95–0.85(m,12H),0.80–0.63(m,6H).

[0326] The following target compounds were prepared by referring to the synthesis methods of the above examples:

[0327] Example 33

[0328] Preparation of (S)-N-((3R,4S,4S,5S)-1-((S)-2-((1R,2R)-3-(allyl(3-((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide)propionamide)phenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0329] Reaction route:

[0330] Steps:

[0331] Step A: Compound 33-1 (20 g, 89.59 mmol) was added to a 500 mL single-necked flask, followed by N,N-dimethylformamide (150 mL), compound 33-2 (17.9 g, 98.55 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.89 g, 98.55 mmol), and HOBt (12.11 g, 89.59 mmol). The reaction system was incubated at 25°C under nitrogen for 12 hours. After completion of the reaction, monitored by LCMS, the reaction solution was poured into water and extracted with ethyl acetate (800 mL). The organic phase was then washed with saturated sodium chloride solution (300 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified on a silica gel column (PE / EA = 3:1) to afford compound 33-3 (31 g, 98.7% yield).

[0332] LCMS (ESI) M / Z: 373.1 [M+H + ].

[0333] 1 H NMR (400MHz, DMSO) δ8.15(d,J=7.0Hz,1H),7.46–7.24(m,6H),5.01(s,2H),4.08(dt,J=12.1,7.3Hz,2H),1.38(s,9H),1.23(dd,J=12.8,7.2Hz,6H).

[0334] Step B: Compound 33-3 (15 g, 42.81 mmol) was added to a 500 mL single-necked flask. Methanol (300 mL), water (15 mL), and 10% Pd / C (3 g) were added sequentially. The reaction system was incubated at 25°C under hydrogen atmosphere for 2 hours. After completion of the reaction, as monitored by LCMS, the reaction was filtered, and the filtrate was concentrated and lyophilized to provide compound 33-4 (9 g, 97.2% yield).

[0335] LCMS (ESI) M / Z: 217.1 [M+H + ].

[0336] 1 H NMR (400MHz, DMSO) δ8.05 (d, J = 7.0Hz, 1H), 4.12 (p, J = 7.2Hz, 1H), 3.26 (q, J = 6.9Hz, 1H), 1.39 (s, 9H), 1.25 (d, J = 7.3Hz, 3H), 1.12 (d, J = 6.9Hz, 3H).

[0337] Step C: Compound 33-4 (8 g, 36.99 mmol) was added to a 250 mL single-necked flask, followed by dry N,N-dimethylformamide (80 mL), followed by compound 33-5 (11.19 g, 44.39 mmol) and 4-methylmorpholine (7.48 mL, 8.13 mL). The reaction system was incubated at 25°C under nitrogen for 12 hours. After completion of the reaction, as monitored by LCMS, the mixture was filtered and the filtrate was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 33-6 (9 g, 68.8% yield).

[0338] LCMS (ESI) M / Z: 376.1 [M+H + ].

[0339] 1 H NMR (400MHz, DMSO) δ8.39(d,J=7.7Hz,1H),8.24(d,J=6.9Hz,1H),7.09(s,2H),4. 31(p,J=7.0Hz,1H),4.15–3.98(m,3H),1.38(s,9H),1.23(dd,J=11.9,7.2Hz,6H).

[0340] Step D: Compound 33-6 (3 g, 9.0 mmol) was added to a 500 mL single-necked flask, followed by dichloromethane (150 mL) and TFA (30 mL). The reaction system was incubated at 25°C under nitrogen for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure using LCMS. The crude product was purified by column chromatography (acetonitrile / water) to afford intermediate INT-1 (2.3 g, 91.1% yield).

[0341] LCMS (ESI) M / Z: 298.2 [M+H + ].

[0342] 1 H NMR (400MHz, DMSO) δ12.50(s,1H),8.38(d,J=7.7Hz,1H),8.21(d,J=7.3Hz,1H),7.09(s,2H),4.32(p, J=7.1Hz,1H),4.18(p,J=7.3Hz,1H),4.13–3.99(m,2H),1.27(d,J=7.3Hz,3H),1.20(d,J=7.0Hz,3H).

[0343] Step E: Compound 5 (30 mg, 0.04 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (2 mL). Intermediate INT-1 (15 mg, 0.04 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (25 mg, 0.06 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion of the reaction, the reaction solution was purified by column chromatography to afford compound 33 (2.18 mg, 20.51% yield).

[0344] LCMS (ESI) M / Z: 1034.7 [M+H + ].

[0345] 1H NMR (400MHz, DMSO) δ9.90–9.76(m,1H),9.58(s,1H),8.91(s,1H),8.48–8.37(m,1H),8.25–8.10(m,1H),7.54–7.37(m,2H),7.27 –7.17(m,1H),7.09(s,2H),6.98–6.86(m,1H),5.87–5.61(m,1H),5.24–5.03(m,2H),4.78–4.55(m,2H),4.44–4.26(m,2H),4.13– 3.70(m,8H),3.59–3.47(m,3H),3.33–3.28(m,4H),3.22–3.09(m,6H),3.00(s,1H),2.80–2.65(m,9H),2.47–2.41(m,1H),2.35– 2.23(m,2H),2.04–1.63(m,7H),1.32–1.28(m,3H),1.23–1.20(m,3H),1.11–1.05(m,2H),0.98–0.84(m,16H),0.80–0.75(m,3H).

[0346] Example 34

[0347] Preparation of N-(((S)-1-(((S)-1-(3-(2-((2R,3R)-N-allyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide.

[0348] Reaction route:

[0349] Steps:

[0350] Step A: Compound 34-1 (1.5 g, 5.60 mmol) was dissolved in dry N,N-dimethylformamide (20 mL). HATU (2.34 g, 6.15 mmol), compound 34-2 (1.66 g, 5.60 mmol), and N,N-diisopropylethylamine (1.52 g, 11.74 mmol) were added sequentially. The reaction system was reacted at room temperature under nitrogen for 2 hours. After completion of the reaction, as monitored by LCMS, the reaction solution was purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to provide compound 34-3 (2.4 g, 92.01% yield).

[0351] LCMS (ESI) M / Z: 489.0 [M+H + ].

[0352] Step B: Compound 34-3 (500 mg, 1.07 mmol) was dissolved in dichloromethane (50 mL), and TFA (15 mL) was added dropwise. The reaction system was stirred under argon for 3 hours. After completion of the reaction as monitored by LCMS, the reaction solution was concentrated and the crude product was slurried with diethyl ether to obtain intermediate INT-2 (400 mg, 90.94% yield).

[0353] LCMS (ESI) M / Z: 411.1 [M+H + ].

[0354] 1 H NMR (400MHz, DMSO) δ9.12(s,2H),8.14(d,J=7.3Hz,1H),8.03(d,J=7.7Hz,1H),4.46–4.26(m,1H),4.18(t,J=7.3Hz,1H),3 .41(s,3H),2.56(t,J=7.1Hz,2H),2.30(t,J=7.3Hz,2H),1.86–1.73(m,2H),1.27(d,J=7.3Hz,3H),1.19(d,J=7.1Hz,3H).

[0355] Step C: Compound 5 (200 mg, 0.26 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (15 mL), followed by the addition of intermediate INT-2 (113.12 mg, 0.28 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (60 mg, 0.34 mmol). The reaction system was reacted at room temperature under nitrogen for 20 minutes. After completion of the reaction as monitored by LCMS, the solvent was evaporated under reduced pressure at low temperature, and the resulting residue was purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution) to afford compound 34 (30 mg, 32.25% yield).

[0356] LCMS (ESI) M / Z: 1149.8 [M+H + ].

[0357] 1 H NMR(400MHz,DMSO)δ9.91–9.65(m,1H),9.57(s,1H),9.15–9.04(m,2H),8 .97–8.83(m,1H),8.39–8.07(m,2H),7.58–7.40(m,2H),7.27–7.14(m,1H ),6.97–6.86(m,1H),5.86–5.64(m,1H),5.21–5.03(m,2H),4.78–4.54(m ,2H),4.42–4.23(m,2H),4.04–3.91(m,3H),3.90–3.81(m,3H),3.79–3.75 (m,2H),3.56–3.45(m,3H),3.41(s,3H),3.35–3.25(m,4H),3.21–3.15( m,4H),3.11–2.99(m,2H),2.82–2.70(m,8H),2.59–2.55(m,2H),2.38–2. 22(m,4H),2.05–1.98(m,1H),1.93–1.76(m,5H),1.73–1.59(m,2H),1.32 –1.28(m,3H),1.23–1.18(m,3H),1.09–1.04(m,2H),0.99–0.74(m,21H).

[0358] Example 36

[0359] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(allyl(3-(S)-2-[(S)]-(S)-2-(3-((2-(methylsulfonyl)pyrimidinyl)-5-sulfonamido)propionamido)propionamido)phenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-methoxy-5-oxoheptan-4-yl)-2-(S)2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0360] Reaction route:

[0361] Steps:

[0362] Step A: Compound 5 (78 mg, 0.10 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (2 mL). Compound 36-1 (30 mg, 0.12 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (31 mg, 0.13 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion of the reaction, the reaction mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to afford compound 36-2 (100 mg, 97% yield).

[0363] LCMS (ESI) M / Z: 999.7 [M+H + ].

[0364] Step B: Compound 36-2 (60 mg, 0.06 mmol) was added to a 25 mL single-necked flask, followed by dichloromethane (5 mL) and trifluoroacetic acid (2.5 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the pH of the reaction solution was adjusted to approximately 8 with ammonium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate and spin-dried to afford compound 36-3 (50 mg, 92.63% yield).

[0365] LCMS (ESI) M / Z: 899.5 [M+H + ].

[0366] Step C: Compound 36-3 (50 mg, 0.067 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (5 mL). Compound 36-4 (37 mg, 0.134 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (25 mg, 0.131 mmol), and N,N-diisopropylethylamine (25 mg, 0.194 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 1 hour. After completion of the reaction, the reaction solution was purified by column chromatography (dichloromethane / methanol = 10:1) to afford compound 36-5 (40 mg, 62.10% yield).

[0367] LCMS (ESI) M / Z: 1158.5 [M+H + ].

[0368] Step D: Compound 36-5 (15 mg, 0.013 mmol) was added to a 25 mL single-necked vial. Dry 2-methyltetrahydrofuran (2 mL) and water (0.5 mL) were then added, followed by potassium peroxymonosulfonate (11 mg, 0.032 mmol). The reaction system was incubated at room temperature under nitrogen for 1 hour. After completion of the reaction, the reaction mixture was purified by column chromatography (dichloromethane / methanol = 10:1) to afford compound 36 (7 mg, 62.10% yield).

[0369] LCMS (ESI) M / Z: 1188.5 [M+H + ].

[0370] 1 H NMR (400MHz, DMSO) δ9.92–9.72(m,1H),9.53(s,1H),9.39(s,2H),8.90(s,1H),8.34(s,1H),8.30–8.05(m,2H),7.61–7.35(m,2H),7.26– 7.16(m,1H),7.00–6.83(m,1H),5.94–5.59(m,1H),5.20–5.00(m,2H),4.76–4.55(m,2H),4.42–4.20(m,3H),4.05–3.92(m,3H),3.89–3. 77(m,3H),3.75–3.68(m,2H),3.65–3.51(m,7H),3.20–3.10(m,6H),3.00(s,2H),2.84–2.67(m,8H),2.40–2.21(m,4H),2.05–1.83(m,5H ),1.72–1.60(m,2H),1.51–1.43(m,1H),1.35–1.27(m,5H),1.21–1.16(m,3H),1.12–1.03(m,3H),0.97–0.83(m,15H),0.81–0.74(m,3H).

[0371] Example 37

[0372] Preparation of (S)-2-(((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-(S)-2--(1R,2R)-3-(3-(((S)-2-(S)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)phenethyl)(prop-2-yn-1-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-dimethylbutoxy.

[0373] Reaction route:

[0374] Steps:

[0375] Step A: Compound 6 (1 g, 1.32 mmol) was dissolved in dry N,N-dimethylformamide (30 mL), followed by the addition of intermediate INT-1 (394 mg, 1.32 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (489 mg, 1.98 mmol). The reaction system was reacted at room temperature under nitrogen for 2 hours. After completion of the reaction as monitored by LCMS, the reaction solution was directly purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to provide compound 37 (210 mg, 15.4% yield).

[0376] LCMS (ESI) M / Z: 1032.6 [M+H + ].

[0377] 1 H NMR (400MHz, DMSO) δ10.01–9.77(m,1H),9.56(s,1H),8.90(d,J=5.1Hz,1H),8.44(d,J=6.3Hz,1H),8.17(d,J=6 .9Hz,1H),7.65–7.35(m,2H),7.32–7.16(m,1H),7.11(s,2H),7.03–6.86(m,1H),4.77–4.48(m,4H),4.41–4.23( m,4H),4.19–3.83(m,12H),3.73–3.69(m,2H),3.22–3.09(m,5H),3.04–2.92(m,2H),2.89–2.69(m,8H),2.36–2 .25(m,1H),2.16–1.53(m,6H),1.31(d,J=6.5Hz,3H),1.26–1.07(m,5H),1.00–0.82(m,18H),0.80–0.74(m,3H).

[0378] Example 38

[0379] Preparation of N-(((S)-1-(((S)-1-(3-(2-((2R,3R)-N-propargyl-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-carboxamide.

[0380] Reaction route:

[0381] Steps:

[0382] Step A: Compound 6 (600 mg, 0.794 mmol) was dissolved in dry dichloromethane (30 mL), followed by the addition of intermediate INT-2 (113.12 mg, 0.794 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (294 mg, 0.1.19 mmol). The reaction system was reacted at room temperature for 30 min under nitrogen protection. After completion of the reaction as monitored by LCMS, the solvent was evaporated under reduced pressure at low temperature, and the residue was purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to afford compound 38 (230 mg, 25.40% yield).

[0383] LCMS (ESI) M / Z: 1147.5 [M+H + ].

[0384] 1 H NMR (400MHz, DMSO) δ9.90–9.80 (m, 1H), 9.60 (s, 1H), 9.11 (s, 2H), 8.91 (d, J = 7. 6Hz,1H),8.11(d,J=5.1Hz,2H),7.60–7.40(m,2H),7.28–7.16(m,1H),7.01–6. 86(m,1H),4.77–4.62(m,1H),4.61–4.53(m,1H),4.43–4.36(m,1H),4.34–4.27 (m,1H),4.23–4.08(m,3H),4.06–3.88(m,4H),3.84–3.67(m,3H),3.64–3.45(m, 3H),3.41(s,3H),3.37–3.33(m,1H),3.30–3.25(m,1H),3.20–3.10(m,5H),3.0 0(s,2H),2.86–2.70(m,8H),2.61–2.53(m,2H),2.47–2.41(m,1H),2.37–2.23(m ,4H),2.05–1.90(m,2H),1.88–1.79(m,3H),1.75–1.58(m,2H),1.30(d,J=6.9H z,3H),1.22(d,J=7.0Hz,3H),1.11(dd,J=12.9,6.6Hz,1H),1.04–0.73(m,22H).

[0385] Example 40

[0386] Preparation of (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-(S)-2--(1R,2R)-3-(3-((S,2-(S)-2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)phenethyl)(methoxy)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide.

[0387] Reaction route:

[0388] Steps:

[0389] Step A: Compound 7 (500 mg, 67 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (10 mL), followed by the addition of intermediate INT-1 (211.11 mg, 0.71 mmol), HATU (331.18 mg, 0.87 mmol), and 2-6-lutidine (215.37 mg, 2.01 mmol). The reaction system was incubated at room temperature under nitrogen for 15 minutes. After completion of the reaction as monitored by LCMS, the reaction solution was purified by column chromatography (tetrahydrofuran / 0.1% trifluoroacetic acid aqueous solution) to afford compound 40 (470 mg, 68.41% yield).

[0390] LCMS (ESI) M / Z: 1026.7 [M+H + ].

[0391] 1H NMR (400MHz, DMSO) δ9.81 (s, 1H), 9.67 (s, 1H), 8.93 (d, J = 7.8Hz, 1H), 8.45 (d, J = 7.2 Hz,1H),8.22–8.12(m,1H),7.49(s,1H),7.41(t,J=6.8Hz,1H),7.19(t,J=7.8Hz,1H ),7.09(s,2H),6.92(d,J=7.3Hz,1H),4.76–4.54(m,2H),4.43–4.27(m,2H),4.15–4 .04(m,2H),3.99(dd,J=2.2,1.6Hz,1H),3.94–3.82(m,2H),3.77–3.68(m,2H),3.66– 3.59(m,4H),3.53–3.45(m,1H),3.29(d,J=6.2Hz,1H),3.24(s,2H),3.19(d,J=2.3H z,3H),3.13(s,1H),3.01(s,2H),2.78(s,9H),2.49–2.42(m,1H),2.39–2.20(m,2H) ,2.06–1.96(m,1H),1.95–1.79(m,3H),1.79–1.55(m,3H),1.30(dd,J=6.9,3.9Hz,4 H),1.22(d,J=7.0Hz,3H),1.14–1.04(m,3H),0.98–0.84(m,16H),0.81–0.74(m,3H).

[0392] Example 41

[0393] Preparation of N-(((S)-1-(((S)-1-(3-(2-((2R,3R)-3-(S)-1-((3R,4S,5S)-4-((S,2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-N,-3-dimethoxy-2-methylpropanamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)5-hexanamide.

[0394] Reaction route:

[0395] Steps:

[0396] Step A: Compound 7 (600 mg, 0.80 mmol) was added to a 25 mL single-necked flask, followed by dry N,N-dimethylformamide (12 mL). Intermediate INT-2 (350 mg, 0.82 mmol), HATU (395.44 mg, 1.04 mmol), and 2,6-lutidine (257.16 mg, 2.4 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 15 minutes. After completion of the reaction, as monitored by LCMS, the product was purified by column chromatography (tetrahydrofuran / 0.1% trifluoroacetic acid in water) to afford compound 41 (780 mg, 85.23% yield).

[0397] LCMS (ESI) M / Z: 1139.8 [M+H + ].

[0398] 1 H NMR (400MHz, DMSO) δ9.82 (s, 1H), 9.66 (s, 1H), 9.14–9.07 (m, 2H), 8.93 (d, J = 7.8Hz ,1H),8.12(t,J=5.6Hz,2H),7.46(dd,J=19.7,11.3Hz,2H),7.19(t,J=7.8Hz,1H), 6.91(d,J=7.4Hz,1H),4.76–4.52(m,2H),4.44–4.24(m,2H),4.06–3.94(m,1H),3. 90–3.84(m,1H),3.80–3.68(m,2H),3.60(dd,J=22.7,9.5Hz,4H),3.53–3.43(m,1H) ,3.41(s,3H),3.37–3.27(m,2H),3.24(s,2H),3.21–3.05(m,5H),3.01(s,2H),2.7 9(t,J=13.8Hz,9H),2.57(t,J=7.1Hz,2H),2.46(d,J=8.2Hz,1H),2.39–2.21(m,4H) ,2.09–1.96(m,1H),1.92–1.57(m,7H),1.30(dd,J=7.0,4.2Hz,4H),1.22(dd,J=7. 1,2.3Hz,3H),1.07(dd,J=11.7,6.6Hz,3H),0.99–0.83(m,16H),0.81–0.74(m,3H).

[0399] Example 42

[0400] Preparation of (S)-1-cyano-3-(3-(S)-2-(S)-2-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propylamine)propylamine)phenyl)propyl-2-yl(2R,3R)-3-((S)-1-((S)-(S)-2-(dimethylamino)-3-methylbutane)-N,3-dimethylbutane)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methoxy-2-methylpropanoate.

[0401] Reaction route:

[0402] Step A: Compound 9 (40 mg, 0.04 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (2.5 mL). Intermediate INT-1 (12 mg, 0.04 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (12.35 mg, 0.05 mmol) were then added sequentially. The reaction system was incubated under nitrogen at room temperature for 15 minutes. After completion of the reaction, as monitored by LCMS, the reaction solution was purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to afford compound 42 (9 mg, 16.4% yield).

[0403] LCMS (ESI) M / Z: 1034.5 [M+H + ].

[0404] 1 H NMR (400MHz, DMSO) δ9.98–9.80(m,1H),8.48–8.33(m,1H),8.27–8.18(m,1H),8.09–7.95(m,1H),7.59–7.37(m,2H),7.29–7. 14(m,1H),7.08(s,2H),6.91(d,J=7.1Hz,1H),5.27–5.07(m,1H),4.71–4.55(m,1H),4.51(t,J=8.6Hz,1H),4.43–4.24(m,2H) ,4.15–3.79(m,7H),3.29–3.22(m,3H),3.19–3.15(m,3H),3.05–2.78(m,8H),2.70–2.56(m,2H),2.46–2.28(m,3H),2.21–2.1 4(m,6H),2.00–1.66(m,7H),1.31–1.26(m,3H),1.24–1.17(m,3H),1.05–0.96(m,3H),0.94–0.85(m,12H),0.79–0.67(m,6H).

[0405] Example 43

[0406] Preparation of (R)-1-(3-((S)-2-(2-(2,5-dioxo-2,5-dihydro-2,5-pyridin-1-ylacetamido)propionamide-propionamidopropyl)propionamide-phenyl-2-propane-2-ol-2-propane-2-propane-2-propane-3-methylisoheptenone-ethylisoheptenone)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate.

[0407] Reaction route:

[0408] Steps:

[0409] Step A:

[0410] Compound 11 (20 mg, 0.03 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (2 mL), followed by intermediate INT-1 (15 mg, 0.04 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (25 mg, 0.06 mmol). The reaction system was reacted at room temperature under nitrogen for 2 hours. After completion of the reaction as monitored by LCMS, the reaction solution was purified by column chromatography (tetrahydrofuran / 0.1% trifluoroacetic acid aqueous solution) to afford compound 43 (2.12 mg, 35.31% yield).

[0411] LCMS (ESI) M / Z: 1011.5 [M+H + ].

[0412] 1H NMR (400MHz, DMSO) δ9.81(s,1H),9.53(s,1H),8.92(s,1H),8.43(d,J=7.2Hz,1H),8.16(d,J=7.2H z,1H),7.51–7.39(m,2H),7.22–7.15(m,1H),7.09(s,2H),6.91(d,J=7.4Hz,1H),5.08–4.93(m,1H) ,4.68–4.54(m,2H),4.41–4.28(m,2H),4.15–3.90(m,4H),3.78–3.67(m,3H),3.52–3.48(m,1H),3. 23–3.17(m,3H),3.14–3.13(m,2H),3.07–2.99(m,3H),2.81–2.73(m,8H),2.45–2.26(m,5H),2.17– 1.88(m,2H),1.88–1.49(m,6H),1.31–1.28(m,3H),1.23–1.18(m,6H),1.0 7–1.01(m,3H),0.96–0.88(m,12H),0.86–0.82(m,3H),0.79–0.74(m,3H).

[0413] Example 44

[0414] Preparation of (S)-1-(3-((S)-2-(2-(2,5-dioxo-2,5-dihydro-2,5-pyridin-1-ylacetamido)-propionamide-propionamidopropyl)-phenyl-2-propane-2-ol, 2-propane-2-propane-3-methylisoheptenone-ethylisohepten)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate.

[0415] Reaction route:

[0416] Steps:

[0417] Step A:

[0418] Compound 12 (20 mg, 0.03 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (2 mL), followed by intermediate INT-1 (15 mg, 0.04 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (25 mg, 0.06 mmol). The reaction system was reacted at room temperature under nitrogen for 2 hours. After completion of the reaction as monitored by LCMS, the reaction solution was purified by column chromatography (tetrahydrofuran / 0.1% trifluoroacetic acid aqueous solution) to afford compound 44 (2.39 mg, 35.37% yield).

[0419] LCMS (ESI) M / Z: 1011.5 [M+H + ].

[0420] 1 H NMR (400MHz, DMSO) δ9.83(s,1H),8.43(d,J=7.3Hz,1H),8.18(d,J=6.8Hz,1H),8.03(d,J=8.1Hz,1H),7.56–7.37(m,2H),7.20(t,J=7.9Hz,1H),7 .10(s,2H),6.91(d,J=7.4Hz,1H),5.08–4.93(m,1H),4.69–4.49(m,2H), 4.41–4.30(m,2H),4.09(d,J=4.1Hz,2H),4.02–3.75(m,4H),3.54–3.47( m,2H),3.25(s,3H),3.19(s,2H),3.03(d,J=19.8Hz,3H),2.84–2.75(m,2 H),2.64(d,J=10.0Hz,1H),2.47–2.29(m,4H),2.20(d,J=6.1Hz,6H),1.9 8–1.67(m,8H),1.31(d,J=7.0Hz,3H),1.22(d,J=7.0Hz,3H),1.15(d,J=6 .1Hz,3H),1.03(d,J=6.8Hz,3H),0.93–0.86(m,12H),0.78–0.69(m,6H).

[0421] Example 45

[0422] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((3-aminophenethyl)(cyanomethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(S)2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0423] Reaction route:

[0424] Steps:

[0425] Step A: Dissolve compound 45-1 (500 mg, 2.48 mmol) and N,N-diisopropylethylamine (960 mg, 7.44 mmol) in acetonitrile (10 mL). Stir at room temperature for 10 minutes. Then slowly add bromoacetonitrile (320 mg, 2.69 mmol) dropwise. Stirring at room temperature continues for 1 hour. LCMS indicates the reaction is complete. The reaction solution is concentrated, and the crude product is purified by reverse-phase column chromatography to afford compound 45-2 (310 mg, 61.29% yield).

[0426] LCMS (ESI) M / Z: 206.2 [M+H] + .

[0427] Step B: Dissolve compound 1-6 (883 mg, 1.48 mmol) in anhydrous dichloromethane (10 mL). Slowly add oxalyl chloride (0.16 mL) and N,N-dimethylformamide (1 drop) dropwise at 0°C. The resulting mixture is stirred at 0°C for 1 hour. The reaction system is then spin-dried at 0°C, and the residue is dissolved in 2 mL of dichloromethane for later use. Dissolve compound 45-2 (250 mg, 1.21 mmol) in dichloromethane (10 mL), add triethylamine (0.37 mL), and slowly add the dichloromethane solution of the acyl chloride dropwise at 0°C. Stir at room temperature for 1 hour. LCMS indicates the reaction is complete. The reaction solution is concentrated, and the crude product is purified by reverse-phase column chromatography to obtain compound 45-3 (408 mg, 42.77% yield).

[0428] LCMS (ESI) M / Z: 786.8 [M+H] + .

[0429] Step C: Dissolve tetrahydroxydiboron (158 mg, 1.78 mmol) in anhydrous N,N-dimethylformamide (5 mL). Under nitrogen, add 4,4-bipyridine (35 mg, 0.22 mmol) and compound 45-3 (400 mg, 0.51 mmol). Stir the resulting mixture at room temperature for 5 minutes. After LCMS showed completion of the reaction, the reaction solution was purified by reverse-phase column chromatography to obtain the target compound 45 (350 mg, 85.76% yield).

[0430] LCMS (ESI) M / Z: 756.4 [M+H] + .

[0431] 1H NMR (400MHz, DMSO) δ8.18–8.00(m,1H),6.99–6.88(m,1H),6.52(s,1H),6.46–6.34(m,3H),5.01( s,1H),4.73–4.25(m,4H),4.00–3.91(m,1H),3.80–3.41(m,5H),3.28–3.24(m,2H),3.21–3.09(m, 5H),2.99(s,2H),2.79–2.60(m,4H),2.46–2.40(m,1H),2.33–2.19(m,6H),2.01–1.56(m,8H),1. 35–1.28(m,1H),1.18–1.07(m,1H),1.00(d,J=6.6Hz,1H),0.95–0.84(m,15H),0.79–0.70(m,6H).

[0432] Example 46

[0433] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(cyanomethyl)(3-(S)-2-[(S)-2](2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)phenethylamino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(S)2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0434] Reaction route:

[0435] Steps:

[0436] Step A: Compound 45 (800 mg, 1.05 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (20 mL). Intermediate INT-1 (350 mg, 1.1 mmol), HATU (500 mg, 1.3 mmol), and 2,6-lutidine (0.45 mL) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 20 minutes. After completion of the reaction, the reaction solution was concentrated under reduced pressure at low temperature, and the residue was purified by column chromatography to afford compound 46 (900 mg, 85.67% yield).

[0437] LCMS (ESI) M / Z: 1035.8 [M+H] + .

[0438] 1H NMR (400MHz, DMSO) δ9.93–9.79(m,1H),9.60(brs,1H),8.89(brs,1H),8.43(d,J=7.3Hz,1H),8.17(d,J=7.1Hz,1H),7.57–7.36(m, 2H),7.29–7.18(m,1H),7.09(s,2H),7.03–6.88(m,1H),4.80–4.50(m,3H),4.49–4.25(m,4H),4.17–3.92(m,4H),3.79–3.68(m,2H) ,3.59–3.49(m,2H),3.31–3.25(m,3H),3.22–3.10(m,5H),2.99(s,2H),2.90–2.70(m,9H),2.36–2.19(m,2H),2.06–1.84(m,4H),1. 81–1.60(m,3H),1.30(d,J=6.7Hz,3H),1.21(d,J=7.0Hz,3H),1.11(dd,J=12.6,6.2Hz,1H),1.04–0.83(m,19H),0.81–0.74(m,3H).

[0439] Example 47

[0440] Preparation of N-((S)-1-(((S)-1-((3-(2-((2R,3R)-N-(cyanomethyl)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)ethyl)phenyl)-1-oxopropan-2-amino.

[0441] Reaction route:

[0442] Steps:

[0443] Step A: Compound 45 (300 mg, 0.33 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (10 mL). Intermediate INT-2 (150 mg, 0.35 mmol), HATU (195 mg, 0.42 mmol), and 2,6-lutidine (0.15 mL) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 20 minutes. After completion of the reaction, the solvent was evaporated under reduced pressure at low temperature. The residue was dissolved in N,N-dimethylformamide and purified by column chromatography to afford compound 47 (300 mg, 76.75% yield).

[0444] LCMS (ESI) M / Z: 1148.6 [M+H + ].

[0445] 1 H NMR (400MHz, DMSO) δ9.90–9.82(m,1H),9.12–9.09(m,2H),8.17–7.97(m,4H),7. 56–7.44(m,2H),7.29–7.20(m,1H),7.03–6.91(m,1H),4.75–4.55(m,2H),4.54– 4.47(m,1H),4.46–4.28(m,4H),4.07–3.87(m,2H),3.78–3.75(m,1H),3.69–3.5 9(m,1H),3.59–3.44(m,3H),3.34–3.30(m,4H),3.27(s,2H),3.20(s,1H),3.17– 3.15(m,3H),3.12(s,1H),2.99(s,2H),2.90–2.61(m,5H),2.59–2.55(m,2H),2. 48–2.40(m,1H),2.34–2.30(m,2H),2.22–2.20(m,2H),2.19(s,3H),1.96–1.77( m,7H),1.74–1.64(m,2H),1.32–1.29(m,3H),1.21(d,J=7.1Hz,3H),1.12(dd,J= 13.8, 6.6Hz, 1H), 1.01 (d, J=6.7Hz, 1H), 0.94–0.84 (m, 15H), 0.76–0.69 (m, 6H).

[0446] Example 48

[0447] Preparation of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((R)-2-(3-aminophenyl)morpholinyl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0448] Reaction route:

[0449] Steps:

[0450] Step A: Compound 48-1 (10 g, 41.15 mmol) was added to a 500 mL single-necked flask and dissolved in ethanol (200 mL). Sodium borohydride (1.51 g, 39.92 mmol) was then added and stirred at room temperature for 1 hour. Potassium hydroxide (2.3 g, 41.07 mmol) was then added and stirred at room temperature for another 2 hours. After the reaction was complete, the ethanol was removed under reduced pressure. The residue was added with water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 40:1) to afford compound 48-2 (5 g, 73.5% yield).

[0451] 1 H NMR (400MHz, CDCl3) δ8.19–8.02(m,2H),7.61(d,J=7.7Hz,1H),7.52(t,J=8.0Hz,1H),4.05–3.77(m,1H),3.36–3.09(m,1H),2.98–2.57(m,1H).

[0452] Step B: Compound 48-2 (5.54 g, 33.55 mmol), TosNH2 (11.49 g, 67.1 mmol), TEBA (760 mg, 3.35 mmol), and potassium carbonate (460 mg, 3.35 mmol) were sequentially added to a 250 mL single-necked flask. 1,4-dioxane (70 mL) was added to dissolve the mixture, and the mixture was heated to 90°C for 4 hours. After completion of the reaction, as monitored by LCMS, 1,4-dioxane was removed by distillation under reduced pressure, and the residue was purified by column chromatography (acetonitrile / 0.1% aqueous ammonium bicarbonate solution) to afford compound 48-3 (6.93 g, 61.42% yield).

[0453] LCMS (ESI) M / Z: 359.1 [M+H + ].

[0454] Step C: Compound 48-3 (7.18 g, 5.95 mmol) was added to a single-necked flask and dissolved in anhydrous dichloromethane (270 mL). Sodium hydroxide (2.56 g, 64.05 mmol) was added under ice-cooling and stirred at this temperature for half an hour. (2-bromoethyl)diphenylsulfonium trifluoromethanesulfonate (14.20 g, 32.03 mmol) was then added and the mixture was allowed to warm to room temperature for 2 hours. After completion of the reaction, as monitored by LCMS, water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by Flash chromatography (acetonitrile / 0.1% aqueous formic acid) to afford compound 48-4 (1.86 g, 20.04% yield).

[0455] LCMS (ESI) M / Z: 385.1 [M+H+ ].

[0456] Step D: Compound 48-4 (2.6 g, 7.17 mmol) was added to a single-necked flask, followed by phenol (4.04 g, 43.03 mmol) and HBr (29 mL, 33% in AcOH). The mixture was reacted at 75°C for 2 hours. After completion of the reaction, the reaction solution was purified by column chromatography (acetonitrile / 0.1% aqueous formic acid) using LCMS to afford compound 48-8 (1.2 g, 80% yield). Chiral separation of the product afforded 48-5-P1 (550 mg) and 48-5-P2 (610 mg). Separation conditions: Mobile phase: supercritical carbon dioxide and 0.1% ammonia in ethanol; Flow rate: 50 mL / min; Gradient: 0.1% ammonia in isopropanol to 35%; Detection wavelength: 214 nm; Cycle time: 4.91 min.

[0457] The retention time of compound 48-5-P1 is Rt=6.27 min; the retention time of compound 48-5-P2 is Rt=8.84 min.

[0458] LCMS (ESI) M / Z: 209.1 [M+H + ].

[0459] Step E: Compound 48-5-P1 (500 mg, 2.40 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (20 mL). Compound 1-6 (1.44 g, 2.4 mmol), HATU (1.37 g, 3.6 mmol), and N,N-diisopropylethylamine (1.19 mL, 7.2 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 48-6 (1.05 g, 55.42% yield).

[0460] LCMS (ESI) M / Z: 789.5 [M+H + ].

[0461] Step F: Tetrahydroxydiboron (408.8 mg, 4.56 mmol) was added to a 25 mL single-necked flask, followed by dry N,N-dimethylformamide (12 mL). 4,4'-Bipyridine (178.05 mg, 1.14 mmol) and compound 48-6 (900 mg, 1.14 mmol) were then added rapidly, sequentially. The reaction system was incubated at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to aid solubilization. The product was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 48 (650 mg, 75.07% yield).

[0462] LCMS (ESI) M / Z: 759.6 [M+H + ].

[0463] 1 H NMR (400MHz, DMSO) δ8.07–7.93(m,1H),7.04–6.92(m,1H),6.67–6.42(m,3H),5.12–4.96(m,2H),4.78–4.43(m,2H) ,4.36–4.12(m,2H),4.09–3.70(m,5H),3.65–3.40(m,3H),3.35(s,1H),3.30–3.27(m,1H),3.20(d,J=12.0Hz,2H), 3.16–3.04(m,3H),3.03–2.86(m,3H),2.83–2.68(m,1H),2.67–2.55(m,2H),2.47–2.39(m,1H),2.35–2.13(m,7H), 1.98–1.63(m,7H),1.34–1.26(m,1H),1.20–1.09(m,2H),1.08–1.03(m,1H),0.94–0.85(m,11H),0.81–0.67(m,8H).

[0464] Example 49

[0465] Preparation of (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-1-(S)-2--(1R,2R)-3-((R)-2-(3-(S)-2-(S)-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)phenyl)morpholinyl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide.

[0466] Reaction route:

[0467] Steps:

[0468] Step A: Compound 48 (300 mg, 0.40 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (15 mL). Intermediate INT-1 (126.04 mg, 0.42 mmol), HATU (197.72 mg, 0.52 mmol), and 2,6-lutidine (128.58 mg, 1.20 mmol) were then added sequentially. The reaction system was reacted at room temperature under nitrogen for 20 minutes. After completion of the reaction, the mixture was evaporated under reduced pressure at low temperature. The residue was dissolved in N,N-dimethylformamide and purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution) to afford compound 49 (195 mg, 47.52% yield).

[0469] LCMS (ESI) M / Z: 1038.7 [M+H + ].

[0470] 1 H NMR (400MHz, DMSO) δ10.00–9.82(m,1H),9.59(s,1H),8.88(dd,J=30.5,7.7Hz, 1H),8.44(dd,J=7.0,3.9Hz,1H),8.16(dd,J=10.8,7.3Hz,1H),7.76–7.63(m,1 H),7.52(d,J=8.1Hz,1H),7.32–7.20(m,1H),7.18–6.99(m,3H),4.78–4.50(m, 2H),4.46–4.28(m,4H),4.14–3.97(m,5H),3.91–3.82(m,1H),3.62–3.46(m,4H) ,3.38–3.27(m,4H),3.20(d,J=10.7Hz,2H),3.16–3.04(m,3H),2.99(d,J=6.7H z,2H),2.78(t,J=14.9Hz,7H),2.62(dd,J=28.1,11.8Hz,1H),2.44–2.16(m,2H ),2.14–1.56(m,7H),1.30(t,J=6.3Hz,3H),1.20(dd,J=13.2,6.8Hz,4H),1.12 (d,J=6.6Hz,1H),1.05(t,J=6.5Hz,1H),0.99–0.82(m,15H),0.81–0.68(m,5H).

[0471] Example 50

[0472] Preparation of N-((S)-1-(S)-1-[(S)-1,[(3-(R)-4-(2R,3R,3R)-3-(S)-1[(3R,4S,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-ethoxy-2-methylpropanoyl)morpholin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide.

[0473] Reaction route:

[0474] Steps:

[0475] Step A: Compound 48 (200 mg, 0.26 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (15 mL). Intermediate INT-2 (113.12 mg, 0.28 mmol), HATU (128.52 mg, 0.34 mmol), and 2,6-lutidine (83.58 mg, 0.78 mmol) were then added sequentially. The reaction system was reacted at room temperature under nitrogen for 20 minutes. After completion of the reaction as monitored by LCMS, the product was concentrated under reduced pressure at low temperature. The residue was dissolved in N,N-dimethylformamide and purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to afford compound 50 (280 mg, 92.25% yield).

[0476] LCMS (ESI) M / Z: 1151.8 [M+H + ].

[0477] 1H NMR (400MHz, DMSO) δ9.97–9.86 (m, 1H), 9.60 (s, 1H), 9.10 (d, J = 8.1Hz, 2H), 8.9 7–8.81(m,1H),8.11(t,J=7.6Hz,2H),7.70(d,J=17.5Hz,1H),7.56(d,J=5.7Hz ,1H),7.34–7.20(m,1H),7.18–6.99(m,1H),4.69–4.53(m,2H),4.41–4.26(m,4 H),4.10–3.88(m,4H),3.84–3.67(m,2H),3.61–3.47(m,2H),3.41(s,3H),3.38 –3.27(m,4H),3.26–3.15(m,3H),3.12(t,J=6.2Hz,2H),3.08–2.88(m,3H),2.8 5–2.69(m,7H),2.57(t,J=7.1Hz,2H),2.43(d,J=21.3Hz,1H),2.36–2.25(m,3H ),2.14–1.57(m,9H),1.30(t,J=6.4Hz,3H),1.20(dd,J=15.2,6.8Hz,4H),1.12 (d,J=6.7Hz,1H),1.05(t,J=6.5Hz,1H),0.99–0.82(m,15H),0.81–0.68(m,5H).

[0478] Example 51

[0479] Preparation of ((S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-2-(3-aminophenyl)morpholinyl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide.

[0480] Reaction route:

[0481] Steps:

[0482] Step A: Compound 48-5-P2 (550 mg, 2.62 mmol) was added to a 50 mL single-necked flask, followed by dry N,N-dimethylformamide (20 mL). Compound 1-6 (1.60 g, 2.62 mmol), HATU (1.50 g, 3.93 mmol), and N,N-diisopropylethylamine (1.32 mL, 7.92 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion, the reaction was filtered, and the filtrate was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 51-2 (1.20 g, 56.52% yield).

[0483] LCMS (ESI) M / Z: 789.3 [M+H + ].

[0484] Step B: Tetrahydroxydiboron (408.8 mg, 4.56 mmol) was added to a 25 mL single-necked flask, followed by dry N,N-dimethylformamide (12 mL). 4,4'-bipyridine (178.05 mg, 1.14 mmol) and compound 51-2 (900 mg, 1.14 mmol) were then added rapidly, sequentially. The reaction system was incubated at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to aid solubilization. The product was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 51 (800 mg, 90.12% yield).

[0485] LCMS (ESI) M / Z: 759.6 [M+H + ].

[0486] 1 H NMR(400MHz, DMSO)δ8.02(d,J=8.6Hz,1H),7.05–6.92(m,1H),6.67–6.40(m,3H),5.05(s,2H),4.78–4.47(m, 2H),4.41–4.10(m,2H),4.07–3.89(m,4H),3.86–3.71(m,1H),3.64–3.40(m,3H),3.31–3.29(m,2H),3.22–3.1 1(m,5H),3.08–2.96(m,2H),2.93–2.71(m,2H),2.69–2.55(m,2H),2.38–2.11(m,7H),2.03–1.57(m,8H),1.21 (d,J=6.7Hz,1H),1.12(dd,J=14.5,6.6Hz,2H),1.05(d,J=6.6Hz,1H),0.96–0.82(m,13H),0.78–0.68(m,6H).

[0487] Example 52

[0488] Preparation of (S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N-((3R,4S,5S)-1-(S)-2--(1R,2R)-3-((S)-2-(3-(S)-2-(S)-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propionamido)phenyl)morpholinyl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide.

[0489] Reaction route:

[0490] Steps:

[0491] Step A: Compound 51 (300 mg, 0.40 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (15 mL). Intermediate INT-1 (126.04 mg, 0.42 mmol), HATU (197.72 mg, 0.52 mmol), and 2,6-lutidine (128.58 mg, 1.20 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 20 minutes. After completion of the reaction, the mixture was concentrated under reduced pressure at low temperature, and the residue was purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid in water) to afford compound 52 (255 mg, 55.47% yield).

[0492] LCMS (ESI) M / Z: 1038.7 [M+H + ].

[0493] 1H NMR (400MHz, DMSO) δ9.91(s,1H),9.67(brs,1H),9.00–8.87(m,1H),8.44(d,J=7.2Hz,1H),8.17(d,J=6.6Hz,1H),7.69( d,J=7.1Hz,1H),7.59–7.46(m,1H),7.34–7.21(m,1H),7.19–6.95(m,3H),4.78–4.45(m,2H),4.41–4.27(m,4H),4.09–3 .86(m,6H),3.80–3.70(m,1H),3.67–3.40(m,3H),3.33(dd,J=15.1,7.8Hz,4H),3.24–3.12(m,6H),3.01(d,J=11.7Hz,2 H),2.95–2.84(m,1H),2.79–2.56(m,8H),2.39–2.19(m,1H),2.08–1.58(m,6H),1.32–1.05(m,11H),0.94–0.73(m,18H).

[0494] Example 53

[0495] Preparation of N-((S)-1-((S)-1-((3-(S)-4-((2R,3R)-3-(S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamine)-4-((S)-2-(S)-2-(dimethylamino)-3-methylbutanamine)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-N,3-dimethylbutanamine)-1-oxopropyl-2-yl)amino-1-oxopropyl-2-yl)amino-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexazol-5-ylamide.

[0496] Reaction route:

[0497] Steps:

[0498] Step A: Compound 51 (250 mg, 0.33 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (10 mL). Intermediate INT-2 (142 mg, 0.35 mmol), HATU (160 mg, 0.42 mmol), and 2,6-lutidine (105 mg, 0.98 mmol) were then added sequentially. The reaction system was reacted at room temperature under nitrogen for 20 minutes. After completion of the reaction, the solvent was evaporated under reduced pressure at low temperature. The residue was dissolved in N,N-dimethylformamide and purified by column chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution) to afford compound 53 (320 mg, 76.78% yield).

[0499] LCMS (ESI) M / Z: 1151.7 [M+H + ].

[0500] 1 H NMR (400MHz, DMSO) δ9.97–9.87(m,1H),9.62(s,1H),9.11(s,2H),8.97–8. 84(m,1H),8.12(d,J=6.9Hz,2H),7.70(d,J=9.6Hz,1H),7.61–7.52(m,1H), 7.35–7.22(m,1H),7.14–6.96(m,1H),468–4.55(m,2H),4.41–4.28(m,4H), 4.09–3.87(m,4H),3.83–3.65(m,2H),3.62–3.51(m,2H),3.41(s,3H),3.37 –3.29(m,3H),3.24–3.10(m,6H),3.06–2.96(m,2H),2.94–2.87(m,1H),2. 82–2.70(m,7H),2.59–2.55(m,2H),2.48–2.43(m,1H),2.36–2.26(m,3H),2 .07–1.56(m,9H),1.31(d,J=7.0Hz,4H),1.22(d,J=7.0Hz,4H),1.16(d,J=6 .6Hz,1H),1.11(d,J=6.5Hz,1H),1.06(d,J=6.4z,1H),0.99–0.74(m,20H).

[0501] The following target compounds were prepared by referring to the synthesis methods of the above examples:

[0502] Example 60

[0503] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((R)-2-(3-aminophenyl)thiomorpholinyl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohexyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide (Compound 60-P1 or Compound 60-P2) and ( Preparation of S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(S)-2-(3-aminophenyl)thiomorpholino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (Compound 60-P2 or Compound 60-P1).

[0504] Reaction route:

[0505] Steps:

[0506] Step A: Compound 60-1 (5.0 g, 33.1 mmol) was placed in a 100 mL single-necked flask, followed by dry dichloromethane (30 mL). Trimethylsilyl cyanide (4.6 mL, 37.1 mmol) was added at 0°C and stirred at room temperature for 6 hours. Zinc iodide (0.21 g, 0.66 mmol) was then added and refluxed at 50°C for 30 minutes. 2M hydrochloric acid (100 mL) and diethyl ether (150 mL) were then added and stirred for 16 hours. After completion of the reaction by LCMS, the reaction mixture was extracted with diethyl ether. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a residue. The residue was purified by normal phase purification (petroleum ether:ethyl acetate = 1:1) to afford compound 60-2 (5.26 g, 89.24% yield).

[0507] 1 H NMR (400MHz, CDCl3) δ8.35 (t, J=1.9Hz, 1H), 8.15 (ddd, J=8.2, 2.2, 0.9Hz, 1H), 7.84 (d, J=7.8 Hz,1H),7.51(t,J=8.0Hz,1H),5.34(s,1H),3.76(s,3H).

[0508] Step B: Compound 60-2 (5.0 g, 28.08 mmol) was placed in a 100 mL single-necked flask, followed by addition of methanolic hydrochloric acid (30 mL, 6 M in MeOH) and reflux with stirring for 3 hours. After completion of the reaction by LCMS, the reaction solution was extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain a residue. The residue was purified by normal phase purification (petroleum ether:ethyl acetate = 1:1) to afford compound 60-3 (5.35 g, 90.26% yield).

[0509] 1 H NMR (400MHz, CDCl3) δ8.34(t,J=1.7Hz,1H),8.23–8.14(m,1H),7.82(d,J=7.7Hz,1H ),7.56(t,J=8.0Hz,1H),5.33(d,J=5.0Hz,1H),3.84(d,J=5.0Hz,1H),3.81(s,3H).

[0510] Step C: Compound 60-3 (5.0 g, 23.68 mmol) was added to a 100 mL single-necked flask, followed by dry dichloromethane (5 mL). Carbon tetrabromide (11.70 g, 35.28 mmol) and triphenylphosphine (9.30 g, 35.46 mmol) were then added sequentially at 0°C. The reaction system was incubated at room temperature under nitrogen for 2 hours. After LCMS indicated completion of the reaction, the reaction solution was concentrated and purified by normal phase purification (petroleum ether:ethyl acetate = 10:1) to afford compound 60-4 (5.56 g, 85.98% yield).

[0511] 1 H NMR (400MHz, CDCl3) δ8.35(t,J=1.9Hz,1H),8.15(ddd,J=8.2,2.2,0.9Hz,1H),7.84(d,J=7.8Hz,1H),7.51(t,J=8.0Hz,1H),5.34(s,1H),3.76(s,3H).

[0512] Step D: Compound 60-4 (5.06 g, 18.46 mmol) was added to a 500 mL single-necked flask, followed by ethanol (150 mL), followed by mercaptoethylamine hydrochloride (2.11 g, 18.57 mmol) and potassium carbonate (2.56 g, 18.55 mmol). The reaction system was incubated at 85°C under nitrogen for 10 hours. After LCMS analysis, the reaction mixture was concentrated, slurried with petroleum ether, and filtered to obtain compound 60-5 (2.06 g, 46.83% yield).

[0513] LCMS (ESI) M / Z: 239.1 [M+H+ ].

[0514] Step E: Compound 60-5 (0.5 g, 2.10 mmol) was added to a 100 mL single-necked flask, followed by dry tetrahydrofuran (10 mL). Borane dimethyl sulfide complex (6.2 mL, 1 M in THF) was added at 0°C. The reaction system was incubated at 70°C under nitrogen for 2 hours. After completion of the reaction, p-toluenesulfonic acid monohydrate (0.40 g, 2.10 mmol) was added and stirring continued for 1 hour. LCMS indicated that the reaction was complete. Methanol was slowly added at 0°C to quench the reaction. The reaction solution was then concentrated and the crude product was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 60-6 (0.319 g, 67.78% yield). Chiral separation afforded 60-6-P1 (119 mg) and 60-6-P2 (129 mg). Separation conditions: mobile phase: supercritical carbon dioxide and 0.1% ammonia in methanol; flow rate: 140 mL / min; gradient: maintaining 0.1% ammonia in methanol at 30%; detection wavelength: 214 nm; cycle time: 8 min.

[0515] The retention time of compound 60-6-P1 is Rt=6.5-9 min; the retention time of compound 60-6-P2 is Rt=9.8-14 min.

[0516] LCMS (ESI) M / Z: 225.1 [M+H + ].

[0517] Step F: Compound 60-6-P1 (50 mg, 0.22 mmol) was added to a 25 mL single-necked flask, followed by dry dichloromethane (5 mL). Compound 1-6 (200 mg, 0.33 mmol), HATU (170 mg, 0.447 mmol), and N,N-diisopropylethylamine (86 mg, 0.67 mmol) were then added sequentially. The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion, the reaction was filtered, and the filtrate was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 60-7-P1 (150 mg, 83.57% yield).

[0518] Similarly, compound 60-6-P2 (20 mg, 0.089 mmol) was prepared under the same conditions to give compound 60-7-P2 (75 mg, yield 86.57%).

[0519] LCMS (ESI) M / Z: 805.9 [M+H + ].

[0520] Step G: Compound 60-7-P1 (150 mg, 0.19 mmol) and tetrahydroxydiboron (89 mg, 0.99 mmol) were added to a 25 mL single-necked vial. Dry N,N-dimethylformamide (5 mL) was then added, followed by 4,4'-bipyridine (19 mg, 0.12 mmol). The reaction system was allowed to react at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to aid solubilization. The product was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 60-P1 (73 mg, 50.55% yield).

[0521] LCMS (ESI) M / Z: 775.5 [M+H + ].

[0522] 1 H NMR (400MHz, DMSO) δ8.97–8.79(m,1H),7.19–7.03(m,1H),6.83–6.59(m,3H),4.79–4.53(m,3H),4.44–4.24(m,2H ),3.93–3.79(m,6H),3.74–3.63(m,4H),3.62–3.50(m,3H),3.36–3.28(m,4H),3.22–3.15(m,4H),3.12–3.07(m,1 H),3.04–2.97(m,2H),2.78–2.76(m,3H),2.74(s,3H),2.36–2.20(m,2H),2.08–1.98(m,1H),1.96–1.84(m,3H),1 .80–1.64(m,3H),1.35–1.16(m,1H),1.14–1.05(m,2H),0.97–0.93(m,6H),0.90–0.81(m,8H),0.79–0.74(m,4H).

[0523] Similarly, compound 60-7-P2 (75 mg, 0.09 mmol) was reacted with the same conditions to give compound 60-P2 (35 mg, yield 52.75%).

[0524] LCMS (ESI) M / Z: 775.6 [M+H + ].

[0525] 1H NMR (400MHz, DMSO) δ8.11–7.87(m,1H),7.07–6.88(m,1H),6.63–6.41(m,3H),5.20–5.01(m,2H) ,4.79–4.42(m,3H),4.33–4.14(m,1H),4.06–3.91(m,2H),3.80–3.54(m,5H),3.27–3.19(m,5H), 3.15–3.07(m,3H),3.00(s,2H),2.92–2.76(m,2H),2.71–2.59(m,3H),2.46–2.39(m,1H),2.20(s ,6H),1.98–1.74(m,6H),1.24(s,1H),1.22–1.03(m,3H),0.94–0.83(m,12H),0.79–0.66(m,8H).

[0526] Example 61

[0527] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(R)-2-(3-aminophenyl)-1,1-dioxidethiomorpholino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide (Compound 61-P1 or Compound 61-P2) and ( Preparation of S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(S)-2-(3-aminophenyl)-1,1-dioxidethiomorpholino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-(S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide (Compound 61-P2 or Compound 61-P1).

[0528] Reaction route:

[0529] Steps:

[0530] Step A: Compound 60-7-P1 (50 mg, 0.22 mmol) was added to a 25 mL single-necked vial. 2-Methyltetrahydrofuran / water (2:1 = 2 mL / 0.5 mL) was added, followed by potassium peroxymonosulfate (273 mg, 0.446 mmol). The reaction system was incubated at room temperature under nitrogen for 2 hours. After completion, the reaction was filtered, and the filtrate was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 61-2-P1 (40 mg, 80.26% yield).

[0531] Similarly, compound 60-7-P2 (50 mg, 0.22 mmol) was reacted with compound 61-2-P2 (40 mg, yield 80.26%) under the same conditions.

[0532] LCMS (ESI) M / Z: 837.3 [M+H + ].

[0533] Step B: Compound 61-2-P1 (40 mg, 0.062 mmol) and tetrahydroxydiboron (25 mg, 0.227 mmol) were added to a 25 mL single-necked flask. Dry N,N-dimethylformamide (2 mL) was added, followed by 4,4'-bipyridine (8 mg, 0.40 mmol). The reaction system was reacted at room temperature under nitrogen for 5 minutes. After completion of the reaction, as monitored by LCMS, a small amount of methanol was added to aid solubilization. The product was purified by column chromatography (acetonitrile / 0.1% formic acid in water) to afford compound 61-P1 (25 mg, 60.31% yield).

[0534] LCMS (ESI) M / Z: 807.6 [M+H + ].

[0535] 1H NMR (400MHz, DMSO-d6) δ8.98–8.75(m,1H),7.19–7.04(m,1H),6.80–6.57(m,3H),4.85–4.66(m,2H),4.60– 4.47(m,2H),4.45–4.30(m,2H),4.27–4.18(m,2H),4.06–4.03(m,1H),3.98–3.93(m,2H),3.64–3.59(m,2H ),3.34–3.30(m,3H),3.28–3.24(m,2H),3.21–3.16(m,3H),3.02–2.96(m,3H),2.78–2.68(m,6H),2.36–2. 12(m,3H),2.05–1.63(m,8H),1.31–1.22(m,1H),1.18–1.05(m,3H),0.97–0.84(m,12H),0.81–0.68(m,8H).

[0536] Similarly, compound 61-2-P2 (40 mg, 0.062 mmol) was reacted with the same conditions to give compound 61-P2 (25 mg, yield 60.31%).

[0537] LCMS (ESI) M / Z: 807.6 [M+H + ].

[0538] 1 H NMR(400MHz,DMSO-d6)δ8.97–8.77(m,1H),7.17–7.02(m,1H),6.76–6.56(m,3H),4.87–4.52 (m,4H),4.43–4.20(m,4H),4.07–3.94(m,5H),3.35–3.30(m,3H),3.24–3.20(m,3H),3.16–3 .11(m,2H),3.06–2.99(m,3H),2.79–2.73(m,5H),2.46–2.40(m,1H),2.35–2.17(m,3H),2.0 7–1.66(m,8H),1.23–1.18(m,1H),1.14–1.04(m,3H),1.00–0.83(m,16H),0.80–0.73(m,4H).

[0539] Trastuzumab was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. The antibody sequence is as follows:

[0540] The amino acid sequence of the light chain is shown in SEQ ID NO: 1:

[0541] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 2:

[0542] The method for coupling the payload-linker conjugate with the trastuzumab antibody of the present invention is as follows:

[0543] Coupling Example 1: ADC-1

[0544] Trastuzumab was exchanged into 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration. Antibody concentration was determined using UV light. A 10 mM TCEP solution (3.2 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 8°C for 3.5 hours. Subsequently, a 10 mM mc-vc-PAB-MMAE (MCE, Catalog No. HY-15575) solution in dimethyl sulfoxide (6.5 equivalents) was added, mixed, and reacted at 8°C for 2.0 hours. After the coupling reaction was complete, 100 mM N-acetyl-cysteine ​​(Merck, Catalog No. 1009005) solution was added at a quenching ratio of 20:1 (quencher:antibody), shaken, and quenched at room temperature for 30 minutes to terminate the coupling reaction. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.02 M His-HAc solution at pH 5.5), concentrated by ultrafiltration, and filtered through a 0.2 μm filter to obtain ADC-1 in PBS buffer (12.13 mg / mL, 2.2 mL), which was stored frozen at 4°C. The average value of toxin-bound amount per antibody molecule (DAR) calculated by RP-HPLC was α = 4.14, and the purity by SEC was 98.8%.

[0545] Coupling Example 2: ADC-2

[0546] The reference linker-payload was synthesized by referring to the method of compound 28 of patent WO2023081230A1.

[0547] Trastuzumab was exchanged into 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration. The antibody concentration was determined using UV light. A 10 mM TCEP solution (10 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 37°C for 3 hours. Subsequently, a 10 mM dimethyl sulfoxide solution (20 equivalents) of the reference linker-loaded substance was added, mixed, and reacted at 25°C for 2.5 hours. After the coupling reaction, the ADC-2 was purified by desalting using a Sephadex G25 gel column (eluting phase: 0.02 M His-HAc solution at pH 5.5). After ultrafiltration and concentration, the ADC-2 was filtered through a 0.2 μm filter to obtain ADC-2 in PBS buffer (11.62 mg / mL, 2.5 mL) and stored frozen at 4°C. The average value of the toxin-bound amount per antibody molecule (DAR) was calculated by RP-HPLC: a = 7.78, and the purity by SEC was 99.3%.

[0548] Coupling Example 3: ADC-3

[0549] Trastuzumab was exchanged into 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration. The antibody concentration was determined using UV light. A 10 mM TCEP solution (10 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 37°C for 3 hours. A 10 mM dimethyl sulfoxide solution (20 equivalents) of compound 46 was then added, mixed, and reacted at 25°C for 2.5 hours. After the coupling reaction, the ADC-3 was purified by desalting using a Sephadex G25 gel column (eluting phase: 0.02 M His-HAc solution at pH 5.5). After ultrafiltration and concentration, the ADC-3 was filtered through a 0.2 μm filter to obtain PBS buffer (15.3 mg / mL, 1.7 mL) and stored frozen at 4°C. The average value of the toxin-bound amount per antibody molecule (DAR) was calculated by RP-HPLC: a = 7.82, and the purity by SEC was 99.0%.

[0550] Coupling Example 4: ADC-4

[0551] Trastuzumab was exchanged into 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration. The antibody concentration was determined using UV light. A 10 mM TCEP solution (10 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 37°C for 3 hours. Subsequently, a 10 mM dimethyl sulfoxide solution (20 equivalents) of compound 37 was added, mixed, and reacted at 25°C for 2.5 hours. After the coupling reaction, the ADC-4 was purified by desalting using a Sephadex G25 gel column (eluting phase: 0.02 M His-HAc solution at pH 5.5). After ultrafiltration and concentration, the ADC-4 was filtered through a 0.2 μm filter to obtain ADC-4 in PBS buffer (4.67 mg / mL, 6.2 mL) and stored frozen at 4°C. The average DAR (number of toxins attached per antibody molecule) was calculated by RP-HPLC: a = 7.78, and the purity by SEC was 99.2%.

[0552] Coupling Example 5: ADC-5

[0553] Trastuzumab was exchanged into 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration. The antibody concentration was determined using UV light. A 10 mM TCEP solution (10 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 37°C for 3 hours. Subsequently, a 10 mM dimethyl sulfoxide solution (20 equivalents) of compound 40 was added, mixed, and reacted at 25°C for 2.5 hours. After the coupling reaction, the ADC-5 was purified by desalting using a Sephadex G25 gel column (eluting phase: 0.02 M His-HAc solution at pH 5.5). After ultrafiltration and concentration, the ADC-5 was filtered through a 0.2 μm filter to obtain ADC-5 in PBS buffer (12.67 mg / mL, 2.2 mL) and stored frozen at 4°C. The average DAR (amount of toxin attached per antibody molecule) was calculated by RP-HPLC: a = 7.65, and the SEC purity was 98.8%.

[0554] Coupling Example 6: ADC-6

[0555] Trastuzumab was exchanged into 10 mM phosphate buffer at pH 7.0 by desalting chromatography or ultrafiltration. The antibody concentration was determined using UV light. 10 mM TCEP solution (3.0 equivalents) was added to the antibody solution (10 mg / mL, 3 mL), mixed, and reduced at 8°C for 3.5 hours. A 10 mM dimethyl sulfoxide solution (6 equivalents) of compound 46 was then added, mixed, and reacted at 8°C for 2 hours. After the coupling reaction, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluting phase: 0.02 M His-HAc solution at pH 5.5). ADC-6 was filtered through a 0.2 μm filter to obtain ADC-6 in PBS buffer (4.52 mg / mL, 5.6 mL) and stored frozen at 4°C. The average value of the toxin-bound amount per antibody molecule (DAR) was calculated by RP-HPLC: a = 4.34, and the purity by SEC was 98.8%.

[0556] Biological test example 1: Cytotoxicity test of compounds

[0557] 96-well transparent flat-bottom white plate (Corning, #3610)

[0558] RPMI1640 culture medium (Gibco, #A10491-01)

[0559] Fetal Bovine Serum (Sigma-Aldrich, #F8687-500ML)

[0560] Luminescent Cell Viability Assay (Promega, #G7572 / G7571)

[0561] Experimental methods:

[0562] Human gastric cancer cells NCI-N87 (ATCC, catalog number: CRL-5822), human breast cancer cells MDA-MB-468 (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, catalog number: TCHu136), and human breast cancer cells KPL-4 (Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60379) were washed with PBS and trypsinized with 0.25% Trypsin-EDTA for approximately 3-10 minutes. Digestion was terminated with complete cell culture medium, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the desired cell density was adjusted. 90.0 μL of cell suspension was added to each well, at a density of 3000 cells / well. The cell plates were incubated overnight in a 37°C, 5% CO2 cell culture incubator.

[0563] In the experiment, a DMSO control group and a test sample group were set up respectively. The cell plate was taken out of the incubator to observe the cell adhesion status. After the cells adhered, 10.00 μL of sample (starting with a final concentration of 10 μM, 5-fold dilution, 9 concentrations) was added to each well, and the plate was gently shaken and placed in the incubator for incubation.

[0564] After 3 days of incubation, 100.0 μL / well CellTiter-Glo TM (Promega, Cat. No. G7572 / G7571) working solution was added and shaken on a thermostatic shaker to lyse the cells. The plate was read on a microplate reader after 10 min.

[0565] The calculation formula of cell proliferation inhibition rate is: cell proliferation inhibition rate = (1-RLU 供试品孔 / RLU DMSO对照孔 )×100%.

[0566] Data analysis: GraphPad Prism 8.0 software was used to plot the sample concentration log value as the horizontal axis and the inhibition percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC of each test sample. 50 value.

[0567] Table 1. Inhibitory activity of the compounds of the present invention on cell proliferation

[0568] Note: NT stands for Not Tested.

[0569] Conclusion: The disclosed compounds have a strong inhibitory effect on the proliferation of KPL-4, NCI-87 and MDA-MD-468 cells.

[0570] Biological Test Example 2: Hematotoxicity of Compounds

[0571] Experimental purpose: To investigate the effect of cytotoxic drugs on CD34 + The effect of drug on the differentiation and development of hematopoietic stem / progenitor cells into megakaryocytes to evaluate their potential hematologic toxicity causing thrombocytopenia.

[0572] Experimental Design: After bone marrow-mobilized peripheral blood-derived HSCs were revived, they were seeded at 2000 cells / well / 180 μL in ultra-low-adsorption 96-well round-bottom plates and stabilized for 4 hours. Five-fold serial dilutions were performed with a maximum dose of 1 μM, and drug treatment was performed at a total of 9 concentration points for 10 days. Cell viability was detected using CTG reagent (CellTiter-Glo luminescent cell viability assay kit, manufacturer: Promega, cat. no.: G7571).

[0573] Table 2 Hematotoxicity test results of cytotoxic drugs

[0574] The structure of the control compound is as follows:

[0575] Conclusion: Compared with compound 1 of WO2023081230A1, the compound disclosed herein has a better effect on CD34 + The cell inhibition effect was weak, indicating that its potential hematotoxicity was low.

[0576] Biological Test Example 3: In vitro plasma stability

[0577] Experimental purpose: To detect the stability of the HER2-ADC drug disclosed herein in various plasma species in vitro.

[0578] Experimental Methods: In a sterile environment, ADC samples, healthy human plasma (donated by colleagues from the Qilu Pharmaceutical Employee Hospital), SD rat plasma (Shandong Xinbo Pharmaceutical Research Co., Ltd.), and CD1 mouse plasma (Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd.) were sterilized by filtration using a 0.22 μm filter. The drug stock solution was diluted to 100 μg / mL using plasma from different species and incubated in a 37°C cell culture incubator. The day of incubation was designated as day 0, and samples were removed on day 21 for free toxin detection.

[0579] Free toxin detection method: Add 300 μL of acetonitrile solution containing internal standard (internal standard: 0.1 ng / mL aprepitant) to the prepared biological sample or biological sample of unknown concentration (50 μL), vortex mix for 5 minutes, centrifuge at 4000 rpm (4°C) for 10 minutes, take 100 μL of the supernatant and add 100 μL of aqueous solution containing 0.2% formic acid, vortex for 3 minutes, and transfer to a 96-well sample plate for LC-MS / MS analysis (Shimadzu Exion LC-AB Triple The release rates of the Her2-ADC compounds of the disclosed embodiments in plasma are shown in Table 3.

[0580] DAR value detection method: Human plasma samples were captured using human her2 protein beads, added to PBST (EZ-Buffers E10X PBST Buffer, manufacturer: Sangon, catalog number: C520004-0500), and incubated at 1000 rpm for 1 hour. The beads were then washed with 200 μL of PBST, PBS (pH 7.4, manufacturer: Yuanpei, catalog number: B320KJ), ultrapure water, and 10% acetonitrile, followed by elution with 100 μL of 20% acetonitrile (containing 0.5% formic acid) for 5 minutes. The supernatant was removed and 20 μL of 100 mM TCEP (manufacturer: Sigma, catalog number: C4706-2G) was added. The beads were shaken at 1000 rpm at room temperature for 1 hour before being loaded for analysis (QTOF-MS). The results of the in vitro DAR value loss rate test for human plasma are shown in Table 4.

[0581] Table 3 In vitro plasma stability test results of ADC

[0582] As can be seen from Table 3, the in vitro plasma stability of the ADC disclosed herein is superior to that of the control group ADC-1, and the free toxin shedding rates of various species are all lower than 2%.

[0583] Table 4 DAR loss rate of ADC in human plasma in vitro

[0584] As can be seen from Table 4, the DAR value of ADC-3 of the present disclosure did not decrease in human plasma, indicating that the stability of its cytotoxic drug-linker is better than that of other ADCs.

[0585] Biological Test Example 4: Hematotoxicity of ADC

[0586] Experimental purpose: To investigate the effect of ADC molecules on CD34 + The effect of drug on the differentiation and development of hematopoietic stem / progenitor cells into megakaryocytes to evaluate their potential hematologic toxicity causing thrombocytopenia.

[0587] Experimental Design: After bone marrow-mobilized peripheral blood-derived HSCs were revived, they were seeded at 2000 cells / well / 180 μL in ultra-low-adsorption 96-well round-bottom plates and stabilized for 4 hours. Three-fold serial dilutions were performed with a maximum dose of 1 μM, and the drug was added at a total of 9 concentration points for 10 days. Cell viability was detected using CTG reagent. The experimental results are shown in Table 5.

[0588] Table 5 ADC blood toxicity test results

[0589] Conclusion: The ADC disclosed herein has a significant effect on CD 34+ The inhibitory effect on cells is weak, its blood toxicity is low, and it has the potential beneficial effect of reducing adverse blood reactions.

[0590] Biological Test Example 5: In vitro Neurotoxicity Assessment

[0591] Experimental purpose: To investigate the inhibitory effect of ADC on mouse dorsal root ganglion cells;

[0592] Experimental design: After mouse dorsal root ganglia (Punosai, Wuhan) were revived, approximately 7,500 cells / well were inoculated in a 96-well plate at a dose of 200 nM for 5 days. Cell viability was detected using CTG reagent. The test results are shown in Table 6.

[0593] Table 6 Inhibitory rate of ADC on mouse dorsal root ganglion cells

[0594] Conclusion: At the same concentration, the inhibitory effect of the disclosed ADC on mouse ganglia is significantly weaker than that of the ADC-1 control group, and is safer.

[0595] Biological Test Example 6: In Vitro Bystander Effect

[0596] The bystander effect refers to the ability of an ADC to kill cells with low or no antigen expression in the presence of antigen-expressing cells. This experiment aims to evaluate the in vitro bystander effect of the ADC drug of the present invention.

[0597] The method used in this experiment is the medium transfer method.

[0598] Her2-positive cell plate: SKBR3 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 1-2 mL of complete medium (complete medium DMEM + 10% FBS). The cell suspension was mixed with an equal volume of trypan blue dye and the cells were counted using a Countstar counter. The cell density was adjusted to 5 × 10 cells / mL using complete medium. 4According to the "Sample Layout," add the adjusted cells to the corresponding 96-well plate at 100.0 μL / well and culture overnight. Seal the plate edges with PBS at 200 μL / well.

[0599] Incubate with Her2-positive cells for 6 days: Dilute the test article to a 2x working solution in complete culture medium, starting at 100 nM, and make a 3-fold dilution gradient over 10 concentrations. Add 100.0 μl / well of the prepared test article to the corresponding 96-well plate. After administration, gently tap the plate to mix. Incubate the 96-well plate in an incubator for 6 days.

[0600] Her2-negative cell plate: MDA-MB-468 cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 1-2 ml of complete medium (complete medium: RPMI1640 + 10% FBS). The cell suspension was mixed with an equal volume of trypan blue dye and the cells were counted using a Countstar counter. The cell density was adjusted to 2 × 10 cells / mL using complete medium. 4 Cells were added to the corresponding 96-well plates at 100 μl / well according to the "Sample Layout" and cultured overnight. An MDA-MB-468 dosing plate (100 μl of SKBR3 cell supernatant) was set up. The plates were sealed with PBS at 200 μl / well.

[0601] Media transfer method: Transfer supernatant from the Her2-positive cell dosing plate to an MDA-MB-468 dosing plate, 100 μl per well. Incubate in an incubator for 6 days. After incubation, remove the 96-well cell culture plate and let it rest at room temperature for 30 minutes. Add 50.0 μl of CellTiter-Glo luminescent dye to each well and shake at 200 rpm in a microplate shaker at room temperature for 10 minutes. All procedures must be performed in the dark.

[0602] Data processing: The data of this experiment were processed using the data processing software GraphPad prism 8.0.

[0603] Table 7 Bystander effect of ADC

[0604] Conclusion: As can be seen from Table 7, the bystander effect of ADC-5 is stronger than that of the control ADC.

[0605] Biological Test Example 7: In vivo efficacy evaluation of the test substance in the NCI-N87 human breast cancer model

[0606] BALB / c nude mice were used as experimental animals to evaluate the in vivo efficacy of the ADC of the present invention and the control group ADC against NCI-N87 xenograft tumors.

[0607] Female BALB / c nude mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and were adapted to live in an SPF animal room for 7 days before being used in subsequent studies. NCI-N87 cells were cultured at 5×10 6 0.1 mL was inoculated subcutaneously in the right axilla of nude mice. When the average tumor volume reached 150 mm 3 When the tumor size was about 10 days, 66 mice with moderate tumor volume were selected and divided into 11 groups, with 6 mice in each group. The grouping day was the 10th day after cell inoculation, and intravenous administration was started, with a single dose of 0.9% sodium chloride injection by vehicle. Tumor size was measured and weighed twice a week. The long and short diameters of the tumor were measured using a vernier caliper, and the tumor volume (mm 3 ) = 0.5 × major diameter × minor diameter. Tumor inhibition rate (%) = (Crtv - Trtv) / Crtv (%), where Crtv and Trtv are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

[0608] The test results are shown in Table 8 and Figures 1-2. Compared to the vehicle, each test molecule significantly inhibited NCI-N87 tumor growth. At the low-dose level of 1 mpk, ADC-3 of the present invention showed slightly superior efficacy to the ADC-1 control group, while ADC-4 and ADC-5 were essentially equivalent. At the high-dose level of 4 mpk, the anti-tumor activity of ADC-3 and ADC-5 of the present invention was slightly superior to that of the ADC-1 control group. Mice in all treatment groups showed an increased body weight, demonstrating good tolerance.

[0609] In summary, the drug described in the present invention has good efficacy and safety, and has great research and application value.

[0610] Table 8 Evaluation indexes of antitumor efficacy of the test substances

[0611] Note: a, mean ± standard error;

[0612] b, tumor inhibition rate (%) = [1-(T 38 -T 10 ) / (V 38 -V 10 )]×100%;

[0613] Biological Test Example 8: Rat Tolerance Experiment

[0614] Experimental plan: Tail vein injection, once every 3 weeks, three male rats in a group (SD rats, SPF grade, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were administered at doses of 10 mpk, 20 mpk, 30 mpk, 40 mpk, and 60 mpk. The body weight of the rats was observed, and blood routine and biochemical indicators were tested.

[0615] Random inspection and observation: observe weight indicators and death phenomena, and record abnormal indicators.

[0616] Table 9 Changes in physical signs of rats in each dose group after administration

[0617] Conclusion: As shown in Table 9, in the SD rat model, the maximum tolerated dose of the ADC-1 control group was 30 mpk, while the tolerated doses of the other groups exceeded 60 mpk. Compared to the control groups ADC-1 and ADC-2, the ADC-3 and ADC-4 of the present invention showed no adverse effects at all doses, demonstrating good safety. However, the control groups ADC-1 and ADC-2 exhibited reproductive toxicity, weight loss, or death at medium and high doses.

[0618] Table 10 Blood routine data of healthy rats and rats in each treatment group at a dose of 10 mpk on the 4th day after administration

[0619] Conclusion: At a dose of 10 mpk, the ADC-1 control group showed obvious leukopenia, neutrophil count, lymphocyte count, monocyte count and reticulocyte count on the fourth day after administration, while the ADC disclosed in the present invention showed no abnormalities in routine blood indicators. White blood cells are divided into lymphocytes, neutrophils, eosinophils, basophils, monocytes, etc., with lymphocytes and neutrophils being the most important. Leukopenia indicates that the drug has a bone marrow suppressive effect and is prone to secondary serious infection. Reticulocyte count is an important indicator reflecting bone marrow hematopoietic function. Reticulocyte decrease indicates decreased bone marrow hematopoietic function, which is common in aplastic anemia and myeloid anemia. The above data show that under the condition of 10 mpk, the ADC-1 control group showed obvious blood toxicity, while the ADC disclosed in the present invention showed no obvious blood toxicity.

[0620] Table 11 Liver function of healthy rats and rats in each treatment group at 40 mpk dose on the 4th day after administration

[0621] Conclusion: At a dose of 40 mpk, the ADC-1 control group showed elevated alanine aminotransferase and aspartate aminotransferase levels, abnormal liver function, and potential hepatotoxicity; no abnormalities in these two enzyme indicators were observed in the other groups.

Claims

1. A cytotoxic drug represented by formula (I), or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof: where M is chosen from C 1-6 alkyl, C 3-6 cycloalkyl and 3-8-membered heterocyclyl; wherein C 1-6 alkyl, C 3-6 cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with a substituent selected from C 1-4 alkyl and NR m1 R m2 , and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R m1 and R m2 independently selected from H and C 1-4 alkyl; X is chosen from O and -N(R x1 )-; R x1 selected from H, OH, C 1-4 alkyl, C 1-4 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl and C 3-6 cycloalkyl; wherein C 1-4 alkyl or C 3-6cycloalkyl is optionally further substituted with 1-3 substituents selected from halogen, CN, deuterium and C 3-6 cycloalkyl; each of R 3 and R 4 independently selected from H and OH; or X and R 3 together with the atoms to which they are attached, form a 4- to 8-membered heterocyclic ring, and the 4- to 8-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S, and S(O)2; or X and R 4 together with the atoms to which they are attached, form a 4- to 8-membered heterocyclic ring, and the 4- to 8-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S, and S(O)2; each of R 1 and R 2 independently selected from H, OH, C 1-4 alkyl, C 2-4 alkynyl, C 2-4 alkenyl, C 3-6 cycloalkyl, 3-8-membered heterocyclyl, -COOR 1a and -CONR 1b R 1c ; in this case, C 1-4 alkyl, C 2-4 alkenyl, C3-6 cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with 1-3 substituents selected from CN, OH, N3, halogen, -COOR 1a , -CONR 1b R 1c and C 1-4 alkyl, and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R 1a , R 1b and R 1c independently selected from H and C 1-4 alkyl; or R 1 and R 2 together with the carbon atom to which they are attached, they form C 3-6 cycloalkyl or 3-8-membered heterocyclyl; Z is selected from the chemical bond and -(CH2) n -, and n is chosen from 1 and 2.

2. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to claim 1, where M is chosen from C 1-6 alkyl, C 3-6 cycloalkyl and 3-8-membered heterocyclyl; wherein C 1-6 alkyl, C 3-6cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with a substituent selected from C 1-4 alkyl and NR m1 R m2 , and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S: each of R m1 and R m2 independently selected from H and C 1-4 alkyl; X is chosen from O and -N(R x1 )-; R x1 selected from H, OH, C 1-4 alkyl, C 1-4 alkoxy, C 2-4 alkenyl and C 2-4 alkynyl; wherein C 1-4 alkyl is optionally further substituted with 1-3 substituents selected from halogen and CN; each of R 3 and R 4 independently selected from H and OH; or X and R 3 together with the atoms to which they are attached, form a 4- to 8-membered heterocyclic ring, and the 4- to 8-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S, and S(O)2; or X and R 4together with the atoms to which they are attached, form a 4- to 8-membered heterocyclic ring, and the 4- to 8-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S, and S(O)2; each of R 1 and R 2 independently selected from H, OH, C 1-4 alkyl, C 2-4 alkynyl, C 2-4 alkenyl, C 3-6 cycloalkyl, 3-8-membered heterocyclyl, -COOR 1a and -CONR 1b R 1c ; while C 1-4 alkyl, C 2-4 alkenyl, C 3-6 cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with 1-3 substituents selected from CN, OH, N3, halogen, -COOR 1a and -CONR 1b R 1c , and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R 1a , R 1b and R 1c independently selected from H and C 1-4 alkyl; or R 1 and R 2together with the carbon atom to which they are attached, they form C 3-6 cycloalkyl or 3-8-membered heterocyclyl; Z is selected from the chemical bond and -(CH2) n -, and n is chosen from 1 and 2.

3. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to claim 1 or 2, where M is chosen from C 1-6 alkyl, C 3-6 cycloalkyl and 3-8-membered heterocyclyl; wherein C 1-6 alkyl, C 3-6 cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with a substituent selected from C 1-4 alkyl and NR m1 R m2 , and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R m1 and R m2 independently selected from H and C 1-4 alkyl; X is chosen from O and -N(R x1 )-; R x1 selected from H, OH, C 1-4 alkyl, C 1-4alkoxy, C 2-4 alkenyl and C 2-4 alkynyl; wherein C 1-4 alkyl is optionally further substituted with 1-3 substituents selected from halogen and CN; each of R 1 and R 2 independently selected from H, OH, C 1-4 alkyl, C 2-4 alkynyl, C 2-4 alkenyl, C 3-6 cycloalkyl, 3-8-membered heterocyclyl, -COOR 1a and -CONR 1b R 1c ; in this case, C 1-4 alkyl, C 2-4 alkenyl, C 3-6 cycloalkyl or 3-8-membered heterocyclyl is optionally further substituted with 1-3 substituents selected from CN, OH, N3, halogen, -COOR 1a and -CONR 1b R 1c , and 3-8-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R 1a , R 1b and R 1c independently selected from H and C 1-4 alkyl; or R 1 and R 2 together with the carbon atom to which they are attached, they form C 3-6cycloalkyl or 3-8-membered heterocyclyl; each of R 3 and R 4 independently selected from H and OH; Z is selected from the chemical bond and -(CH2) n -, and n is chosen from 1 and 2.

4. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein M is selected from C 1-4 alkyl, C 3-6 cycloalkyl and 3-6-membered heterocyclyl; wherein C 1-4 alkyl, C 3-6 cycloalkyl or 3-6-membered heterocyclyl is optionally further substituted with a substituent selected from C 1-4 alkyl and NR m1 R m2 , and 3-6-membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; each of R m1 and R m2 independently selected from H and C 1-2 alkyl.

5. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein M is selected from 6. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R x1 selected from H, -CH3, OH, -OCH3, 7. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein X is selected from O, -NH-, -N(CH3)-, 8. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-7, wherein each of R 1and R 2 independently selected from H, -CH3, -CH2OH, -CH2F, -CHF2, -CF3, -CH2Cl, -COOH, -COOCH3, -CONH2, -CONHCH3, or R 1 and R 2 together with the carbon atom to which they are attached, they form 9. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-8, wherein each of R 3 and R 4 independently selected from H and OH; preferably each of R 3 and R 4 independently represents N.

10. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-9, where X and R 3together with the atoms to which they are attached, form a 5-6-membered heterocyclic ring, and the 5-6-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S and S(O)2; or X and R 4 together with the atoms to which they are attached, form a 5-6-membered heterocyclic ring, and the 5-6-membered heterocyclic ring contains 1-3 heteroatoms or groups selected from N, O, S and S(O)2.

11. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of paragraphs 1-10, where X and R 3 together with the atoms to which they are attached, they form or or X and R 4 together with the atoms to which they are attached, they form 12. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein Z is selected from a chemical bond and -CH2-.

13. A cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to any one of claims 1 to 12, wherein the cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt is selected from a compound of general formula (Ia), (Ib) or (Ic): where Y is selected from O, S and S(O)2, and M, R 1 , R 2 , R 3 , R 4 , R x1 and Z have the meanings defined in any of paragraphs 1-12.

14. A cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to any one of claims 1 to 13, wherein the cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt is selected from a compound of general formula (IIa), (IIb) or (IIc): where Y is selected from O, S and S(O)2, and R 1 , R 2 , R 3 , R 4 and R x1 have the meanings defined in any of paragraphs 1-13.

15. A cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to any one of claims 1 to 14, wherein the cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt is selected from a compound of general formula (IIIa): where R x1 selected from OH, C 1-4 alkyl, C 1-4 alkoxy and C 3-6 cycloalkyl, and at the same time C 1-4 alkyl or C 3-6 cycloalkyl is further substituted with 1-3 substituents selected from deuterium, methyl, CN and cyclopropyl; preferably R x1 selected from -ОСН3, 16. A cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to claim 1, wherein the cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof is selected from: And 17. An antibody-drug conjugate or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof, wherein said antibody-drug conjugate or stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof is selected from a structure represented by formula (A): where Ab is an antibody or antigen-binding fragment; L is a linking group whose left end is linked to NH of the cytotoxic drug and whose right end is linked to Ab; and is selected from a numerical value from 1 to 10; M, R 1 , R 2 , R 3 , R 4 , X and Z have the meanings defined in any of paragraphs 1-16.

18. The antibody-drug conjugate or stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to claim 17, wherein the antibody-drug conjugate or stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof is selected from the structure represented by formula (A-1): wherein Ab is an antibody to Her2; preferably Ab is trastuzumab; R x1selected from H, -CH3, OH, -OCH3, preferably R x1 selected from -ОСН3, a is selected from a numerical value from 2 to 8; preferably a is selected from a numerical value from 4 to 8; L is as defined in paragraph 17.

19. An antibody-drug conjugate or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to claim 17 or 18, wherein L is selected from:

20. An antibody-drug conjugate or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of claims 17-19, wherein a is selected from a numerical value of 2 to 8; preferably, a is selected from a numerical value of 4 to 8.

21. A cytotoxic drug linker or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof, wherein said cytotoxic drug linker has a structure represented by formula (B), where L' is the structure of L before binding to Ab according to any one of claims 17-20; preferably L' is selected from M, R 1 , R 2 , R 3 , R 4 , X and Z have the meanings defined in any of paragraphs 1-16.

22. The cytotoxic drug linker or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to claim 21, wherein the cytotoxic drug linker or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt is selected from the structure represented by formula (B-1): where R x1 selected from H, -CH3, OH, -OCH3, preferably R x1 selected from - OSN3, L' is chosen from And preferably L' is 23. A cytotoxic drug linker or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to claim 21 or 22, wherein the cytotoxic drug linker is selected from:

24. The antibody-drug conjugate or stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof according to any one of claims 17-20, wherein the antibody-drug conjugate or stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug, or pharmaceutically acceptable salt thereof is selected from: where Ab is an antibody, preferably an antibody to Her2 and more preferably trastuzumab: and is selected from a numerical value from 2 to 8, preferably a numerical value from 4 to 8.

25. A pharmaceutical composition comprising a cytotoxic drug or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to any one of claims 1-16 or an antibody-drug conjugate or its stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt according to any one of claims 17-20 or 24, and a pharmaceutically acceptable carrier.

26. The use of a cytotoxic drug or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1-16, an antibody-drug conjugate or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 17-20 or 24, a cytotoxic drug-linker or a stereoisomer, isotopic derivative, solvate, nitric oxide, prodrug or pharmaceutically acceptable salt thereof according to any one of claims 21-23 or a pharmaceutical composition according to claim 25 for the preparation of a medicament for the treatment of a tumor.

27. The use according to claim 26, wherein the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, neuroglioma, neuroblastoma, sarcoma, lung cancer, head and neck cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

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