Dolastatin derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to the field of biomedicine. Specifically, provided are a Dolastatin derivative, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, a preparation method therefor, and the use thereof in the preparation of an anti-tumor drug. The Dolastatin derivative is as represented by formula (I), wherein the definitions of the groups in the formula are described in detail in the description. The Dolastatin derivative has relatively good anti-tumor activity.

Description

Dolastatin derivatives and preparation methods and applications thereof

[0001] This application claims priority to Chinese patent application No. 2024100521163, filed on January 12, 2024, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to dolastatin derivatives and preparation methods and applications thereof. Background Art

[0003] Dolastatin-10 is a natural product isolated from the marine animal Aplysia. It is a linear decapeptide-like natural cytotoxic protein composed of 4 amino acids. It has extremely strong inhibitory activity on cellular microtubule aggregation, thereby inhibiting cell proliferation and tumor growth.

[0004] Due to its extremely limited natural source, various synthetic dolastatin-10 derivatives (also known as auristatins) have been extensively studied. Among them, dolastatin-10 derivatives synthesized from the tubulin-related inhibitors MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F) are widely used as ADC payloads in the research and development of various antibody-drug conjugates. Summary of the Invention

[0005] The purpose of the present invention is to provide a series of dolastatin derivatives and their preparation methods, as well as their applications in the preparation of anti-tumor drugs (ADCs) and antibody-drug conjugates.

[0006] Specifically, the present invention optimizes the N-terminus or C-terminus of the dolastatin structure, including dimethylation, replacement, cyclization, and other modifications to the N-methylvaline residue at the N-terminus of dolastatin, or modification of the phenylethyl backbone at the C-terminus of dolastatin. These compounds exhibit potent inhibitory activity against tubulin aggregation, leading to tumor cell apoptosis, and are therefore promising for the development of antibody-drug conjugates and tumor treatment.

[0007] Studies have shown that the N-terminal N,N-dimethylvaline of dolastatin-10 can be modified or replaced without significantly affecting its activity. In addition, the C-terminal phenylethyl group can also be modified without significantly affecting its biological activity.

[0008] In one aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof:

[0009] Wherein, M is selected from Any of the following;

[0010] R1 and R2 are each independently selected from any one of C1-C6 alkyl, halogen-substituted C1-C6 alkyl or C3-C6 cycloalkyl;

[0011] R3 is selected from any one of hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl;

[0012] R4 is selected from any one of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -C1-C6 alkyl-NR6-O-C1-C6 alkyl or -C1-C6 alkyl-O-NR6-C1-C6 alkyl;

[0013] R5 is selected from any one of hydrogen, hydroxy, C3-C6 cycloalkyl or -O-NR6-C1-C6 alkyl;

[0014] R6 is selected from hydrogen or C1-C6 alkyl;

[0015] X is selected from halogen;

[0016] m1 and m2 are each independently selected from 0, 1 or 2.

[0017] In one embodiment, R1 and R2 are each independently selected from hydrogen, halogen-substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl or isobutyl;

[0018] In one embodiment, R1 and R2 are each independently selected from hydrogen, methyl, trifluoromethyl or trifluoroethyl.

[0019] In one embodiment, R3 is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl or isobutyl.

[0020] In one embodiment, Selected from

[0021] In one embodiment, the halogen is selected from -F, -Cl, -Br or -I.

[0022] In one embodiment, m1 and m2 are each independently selected from 1 or 2.

[0023] In one embodiment, Selected from

[0024] In one embodiment, R4 is selected from C1-C6 alkyl, -C1-C6 alkyl-NH-O-C1-C6 alkyl or -C1-C6 alkyl-O-NH-C1-C6 alkyl.

[0025] In one embodiment, R4 is selected from methyl, -CH2-NH-O-CH3 or -CH2-O-NH-CH3.

[0026] In one embodiment, R5 is selected from hydrogen, hydroxy, or -O-NH-C1-C6 alkyl.

[0027] In one embodiment, R5 is selected from hydrogen, hydroxy or -O-NH-CH3.

[0028] The present invention provides the following compounds or pharmaceutically acceptable salts, stereoisomers or prodrugs thereof:

[0029] The present invention provides an antibody-drug conjugate of the aforementioned compound.

[0030] In one embodiment, the antibody-drug conjugate has the structure shown in the following formula (II): (DL)n-Ab (II)

[0031] Wherein, D is the aforementioned compound;

[0032] Ab is the antibody against tumor-associated antigen;

[0033] L is a linker used to connect D and Ab;

[0034] n = 3-8; for example, 3.2, 3.9, 4.3 or 7.9.

[0035] In one embodiment, the linker is a hydrophilic linker.

[0036] In one embodiment, the linker is selected from the following structures:

[0037] in, The positions shown indicate attachment to antibodies. The position shown indicates connection with D;

[0038] In one embodiment, the antibody conjugate is obtained by conjugating the following compound to an antibody:

[0039] The present invention provides the following antibody-drug conjugates:

[0040] wherein n=3-8; for example, 3.2, 3.9, 4.3 or 7.9.

[0041] In one embodiment, Ab is an antibody or antigen-binding fragment thereof to a tumor-associated antigen.

[0042] In one embodiment, the Ab is a nectin-4 antibody or an antigen-binding fragment thereof.

[0043] In one embodiment, the nectin-4 antibody comprises the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 or any variant thereof, and the antibody light chain amino acid sequence shown in SEQ ID NO: 2 or any variant thereof.

[0044] The present invention provides a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof or the aforementioned antibody-drug conjugate and a pharmaceutically acceptable excipient.

[0045] The present invention provides use of the aforementioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, or the aforementioned antibody-drug conjugate, or the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting tumor cell growth or in the preparation of a drug for treating cancer.

[0046] In one embodiment, the tumor cells include any one of esophageal cancer cells, breast cancer cells, gastric cancer cells, and lung cancer cells.

[0047] In one embodiment, the cancer includes any one of esophageal cancer, breast cancer, gastric cancer, and lung cancer.

[0048] Another aspect of the present invention provides a method for treating cancer, comprising the step of administering a therapeutically effective amount of the aforementioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, or the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition to a patient in need thereof.

[0049] In one embodiment, the cancer is a solid tumor.

[0050] In one embodiment, the cancer includes any one of esophageal cancer, breast cancer, gastric cancer, and lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the LCMS spectrum of the antibody-drug conjugate ADC2; A and B are HPLC results, and C and D are MS results.

[0052] Figure 2 is the LCMS spectrum of the antibody-drug conjugate ADC4; A and B are HPLC results, and C and D are MS results.

[0053] Figure 3 is the LCMS spectrum of the antibody-drug conjugate ADC5; A and B are HPLC results, and C is the MS result.

[0054] Figure 4 is the LCMS spectrum of the antibody-drug conjugate ADC6; A and B are HPLC results, and C is the MS result.

[0055] FIG5 is a graph showing the in vivo inhibition of tumor growth in the NCI-H292 model in Test Example 2 (3 mg / kg).

[0056] FIG6 is a graph showing the in vivo inhibition of tumor growth in the NCI-H292 model in Test Example 2 (10 mg / kg). DETAILED DESCRIPTION

[0057] I. Definition

[0058] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0059] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0060] The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis, trans structures) and optical isomers (such as enantiomers), and are therapeutic substances consisting of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present invention.

[0061] The compounds of the present invention also include tautomeric forms, which result from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.

[0062] As used herein, "pharmaceutically acceptable salts" refer to salts formed between the corresponding amine compounds and inorganic or organic acids, or between the corresponding carboxylic acid compounds and alkali metals or alkaline earth metals, or between the corresponding carboxylic acid compounds and organic amines. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like; organic acids include, but are not limited to, acetic acid, propionic acid, butyric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, tartaric acid, and the like; alkali metal or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0063] As used herein, "precursor" refers to a compound that, after entering the human body via an appropriate administration route, undergoes metabolism or simple chemical changes within the patient's body to transform into the compound of Formula 1 of the present invention and its corresponding salt. Precursors of the compound include, but are not limited to, various carboxylic acid esters, carbonates, phosphates, sulfates, sulfonates, amino acid esters, gluconates, and various amides, acetals, hemiacetals, and carbonate amides.

[0064] Numeric ranges herein refer to each integer in the given range. For example, "C1-C6" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0065] When any variable (e.g., Rn) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 Rn, the group may be optionally substituted with up to 5 Rn, with each occurrence of Rn being an independent choice. Furthermore, combinations of substituents and / or their variants are permitted only if such combinations result in stable compounds.

[0066] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 2,2-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate groups. Methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl are preferred.

[0067] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen, or S(O)m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1-4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms; most preferably, it contains 3 to 8 ring atoms; further preferably, it contains 1-3 nitrogen atoms, 3-8 membered heterocyclyl, optionally substituted by 1-2 oxygen atoms, sulfur atoms, or oxo groups, including nitrogen-containing monocyclic heterocyclyl, nitrogen-containing spiroheterocyclyl, or nitrogen-containing fused heterocyclyl.

[0068] The hydrogen atoms described in the present invention can all be replaced by their isotope deuterium.

[0069] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1-3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0070] Refers to the chemical bond connection.

[0071] Drug or pharmaceutical composition

[0072] The medicaments or pharmaceutical compositions of the present invention can be administered orally, topically, parenterally, or mucosally (e.g., buccally, by inhalation, or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. The oral route is generally desirable. The active agent can be administered orally in the form of capsules, tablets, and the like (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0073] For oral administration in the form of tablets or capsules, the active drug component can be mixed with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (such as acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, etc. For oral administration in liquid form, the drug component can be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils), a preservative (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), etc. Stabilizers such as antioxidants (BHA, BHT, propyl citric acid, sodium ascorbate, citric acid) can also be added to stabilize the dosage form.

[0074] Tablets comprising the active compound can be coated by methods well known in the art. The compositions of the present invention comprising the compound of formula I as the active compound can also be introduced into beads, microspheres or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as dry products reconstituted with water or other suitable adjuvants before use. Preparations for oral administration can be suitably formulated to release the active compound in a controlled or delayed manner.

[0075] The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0076] The term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0077] The term "pharmaceutical composition" means a composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0078] The term "effective amount" or "therapeutically effective amount" refers to a non-toxic but sufficient amount of a drug or medicament that can achieve the desired effect. In embodiments of the present invention, when a patient is treated according to the present invention, the amount of a given drug depends on many factors, such as a specific dosage regimen, the type of disease or condition and its severity, the uniqueness (e.g., body weight) of the patient or host in need of treatment, but, according to specific surrounding circumstances, including, for example, the specific drug, route of administration, the condition to be treated, and the patient or host to be treated, the dosage can be conventionally determined by methods known in the art. Typically, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. The desired dosage can be conveniently expressed as a single dose, or simultaneously administered (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range has been given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0079] The term "antibody-drug conjugate (ADC)" is a drug conjugate that connects a biologically active small molecule drug (toxin) to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into the target cells.

[0080] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens typically have multiple binding sites, also known as epitopes, which are recognized by CDRs (complementary determining regions) on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a target antigen or a portion thereof, including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The term "antibody" is an immunoglobulin molecule that can bind to a specific antigen. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule.

[0081] As used herein, the term "toxin," also referred to as a "cytotoxic drug moiety" or "small molecule drug," refers to a compound that has a tumor cell-killing effect. Examples of the cytotoxic drug moiety include at least one of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, a DNA synthesis inhibitor, an RNA polymerase inhibitor, a splicesome inhibitor, a proteolysis-targeting chimera (PROTAC), and an immunomodulator.

[0082] As used herein, the terms "reduce," "inhibit," "mitigate," or "reduce" are used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0083] The term "MMAE (monomethyl auristatin E)" refers to

[0084] The term "MMAF (monomethyl auristatin F)" refers to

[0085] The term "Dolastatin-10" refers to

[0086] II. Specific Examples

[0087] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0088] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0089] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

[0090] DAR value testing and calculation: Based on the RP-HPLC-MS test results, the DAR value of the ADC was analyzed using a Waters Acquity UPLC I-Class / Xevo G2-XS QTOF instrument.

[0091] RP-HPLC parameters: PLRP-S1000A 5UM column, column temperature 70°C. Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, flow rate 0.2 mL / min. Mobile phase gradient: 20-50% B over 18 minutes; 50-95% B over 5 minutes; 95-20% B over 0.1 minutes; 20-20% B over 6.9 minutes.

[0092] MS parameters were set as follows: capillary voltage 2.50 kV, cone voltage 100 V, mass analysis range m / z 200 to 4000, MSE collision energy 20 to 45 eV, ion source temperature 120°C, nebulizer temperature 500°C, nebulizer flow rate 1000 L / Hr, and internal standard leucine enkephalin. The sample was diluted to 1 mg / mL in sample buffer, TCEP was added to a final concentration of 50 mmol / L, and the sample was incubated at 37°C for 20 min. A 5 μL injection was performed. Light chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Similarly, heavy chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Weighted peak areas for the heavy and light chains were calculated by multiplying the peak area percentages by the corresponding drug loading. The DAR value was calculated as: DAR = 2*(Σlight chain weighted peak area + Σheavy chain weighted peak area) / 100.

[0093] In this embodiment, ADC prepared using but not limited to Nectin-4 antibody.

[0094] The amino acid sequence of the heavy chain of the nectin-4 antibody is as follows (SEQ ID NO: 1):

[0095] The amino acid sequence of the light chain of the nectin-4 antibody is as follows (SEQ ID NO: 2):

[0096] Example 1: Trifluoroethyl dolastatin derivative (D1)

[0097] Synthesis of intermediate D1-1

[0098] To a 1000 mL single-necked flask at room temperature, tert-butyl (3R,4S,5R)-3-methoxy-5-methyl-4-(methylamino)heptanoate (8.0 g, 30.84 mmol), DCM (200 mL), Fmoc-Val-OH (20.93 g, 61.68 mmol), HATU (23.45 g, 61.68 mmol), and DIPEA (15.94 g, 123.37 mmol, 21.49 mL) were added in sequence. The reaction was stirred at room temperature for 2 h. 50 g of silica gel powder was added for sand making. The product was purified by silica gel column chromatography to obtain an off-white solid D1-1 (17.78 g, yield 94.30%, HPLC 95%); LCMS: [M+1] + 581.23 (calculated: 580.77).

[0099] Synthesis of D1-2

[0100] To a 1000 mL single-necked eggplant-shaped flask at room temperature, D1-1 (17.78 g, 30.62 mmol), DCM (200 mL), and TFA (174.54 g, 1.53 mol, 117.14 mL) were added in sequence. The reaction solution was stirred at room temperature for 1 h, concentrated, and DCM (200 mL) was added. The solution was washed with water (100 mL*6) to remove trifluoroacetic acid, dried over anhydrous magnesium sulfate, filtered, and concentrated to give an off-white solid D1-2 (15.5 g, yield 92.64%, HPLC 96%); LCMS: [M+1] + 525.53 (calculated: 524.66).

[0101] Synthesis of D1-4

[0102] At room temperature, D1-2 (10 g, 19.06 mmol), DCM (93.36 mL), (2R, 3R)-N-[(1R, 2S)-2-hydroxy-1-methyl-2-phenylethyl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl] acrylamide (D1-3, Dap-NE) (6.11 g, 19.06 mmol), HATU (10.87 g, 28.59 mmol), and DIPEA (7.39 g, 57.18 mmol, 9.96 mL) were added to a 1000 mL reaction bottle in sequence. The reaction mixture was stirred at room temperature for 1 h. 50 g of silica gel powder was added to the mixture and column chromatography was performed using DCM and MeOH to obtain an off-white solid D1-4 (14.1 g, yield 85.87%, HPLC 96%); LCMS: [M+1] + 828.01 (calculated: 827.08).

[0103] Synthesis of D1-5

[0104] At room temperature, D1-4 (2.84 g, 3.43 mmol), ACN (30 mL), and DBU (522.75 mg, 3.43 mmol, 513.50 μL) were added to a 100 mL single-necked eggplant-shaped flask in sequence. The mixture was stirred at room temperature for 2 h. N-cbz-N-2,2,2-trifluoroethylglycine (1 g, 3.43 mmol) and HATU (1.96 g, 5.15 mmol) were added and the reaction was continued for 1.5 h. The mixture was concentrated, extracted with DCM, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain an off-white solid D1-5 (2.98 g, yield 94.89%, HPLC 96%); LCMS: [M+1] + 879.03 (calculated: 878.04).

[0105] Synthesis of D1

[0106] To a 1000 mL single-necked eggplant-shaped flask at room temperature were added D1-5 (2.98 g, 3.39 mmol), methanol (30 mL), and Pd / C (carbon content 10%, moistened with about 55% water) (1.08 g, 10.18 mmol) in sequence. The atmosphere was replaced with hydrogen three times. The reaction was stirred at room temperature under a hydrogen atmosphere for 1 h, filtered, and concentrated to give an off-white solid D1 (2.42 g, yield 91.06%, HPLC 95%). 1H NMR(500MHz,DMSO-d6)δ7.98(dd,J=8.8,3.6Hz,1H),7.89(d,J=8.7Hz,1H),7 .63(d,J=8.6Hz,0H),7.34-7.22(m,4H),7.22-7.15(m,1H),5.42(d,J=4.5Hz ,1H),5.35(d,J=4.6Hz,0H),4.74(s,0H),4.67(dd,J=9.0,7.4Hz,1H),4.49( s,0H),4.43(s,0H),4.01(s,2H),3.60(s,0H),3.57(s,0H),3.46(s,1H),3.29 -3.15(m,11H),3.12-3.01(m,2H),2.96(s,1H),2.95(dp,J=20.5,6.8Hz,1H) ,2.70(s,1H),2.41(d,J=15.9Hz,1H),2.28(dt,J=15.8,10.2Hz,1H),2.12(t d,J=10.1,9.6,6.4Hz,1H),1.86-1.68(m,1H),1.57(dd,J=13.3,7.2Hz,1H), 1.50(s,1H),1.30(s,1H),1.08-0.94(m,6H),0.96-0.73(m,12H); LCMS:[M+1] + 744.25 (calculated: 743.91).

[0107] Example 2: Difluoroproline dolastatin derivative (D2)

[0108] Synthesis of D2-1

[0109] D1-4 (3.29 g, 3.98 mmol), ACN (30 mL), and DBU (605.97 mg, 3.98 mmol, 595.26 μL) were added to the reaction flask in sequence and stirred at room temperature for 0.5 h. Then, (2S)-1-tert-butyloxycarbonyl-4,4-difluoropyrrolidine-2-carboxylic acid (1 g, 3.98 mmol) and HATU (1.51 g, 3.98 mmol) were added to the reaction flask and the reaction was continued for 1.5 h. The reaction solution was concentrated, DCM (100 mL) was added, and the solution was washed with water (100 mL*6). The solution was purified by silica gel column chromatography and concentrated by rotary evaporation to obtain an off-white solid D2-1 (3.33 g, yield 94.84%, HPLC 95%); LCMS: [M+1] + 839.04 (calculated: 838.05).

[0110] Synthesis of D2

[0111] D2-1 (3.33 g, 3.97 mmol), DCM (15.10 mL), and TFA (22.65 g, 198.68 mmol, 15.20 mL) were added to the reaction flask in sequence. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated, DCM (200 mL) was added, and the mixture was washed with water (100 mL*6), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain an off-white solid D2 (2.35 g, yield 76.94%, HPLC 96%). 1 H NMR(500MHz,DMSO-d6)δ7.98(d,J=8.9Hz,1H),7.70(d,J=8.5Hz,1H),7.32-7.28(m,4H),7 .19(d,J=3.1Hz,1H),3.26-3.16(m,15H),3.09(s,2H),2.96(d,J=7.1Hz,3H),2.69(s,3H) ,2.28(dtt,J=18.6,12.9,7.5Hz,3H),2.17-1.98(m,3H),1.61-1.45(m,2H),1.34-1.18(m ,4H),1.05(dd,J=6.9,2.2Hz,4H),0.94-0.88(m,13H),0.79(d,J=7.5Hz,3H); LCMS:[M+1] + 738.86 (calculated: 737.93).

[0112] Example 3: N-trifluoroethyl auristatin E (D3)

[0113] D1-4 (600 mg, 0.726 mmol), acetonitrile (10 mL) and DBU (111.1 mg, 0.726 mmol) were added to the reaction flask and stirred at room temperature for 4 hours. The reaction was monitored for completion (LCMS / ESI (m / z): [M+H] + 605.80 (theoretical value 604.83). HOBT (98.16 mg, 0.726 mmol), ATU (276.05 mg, 0.726 mmol), Fmoc-L-valine (246.47 mg, 0.726 mmol), DIEA (112.82 mg, 0.871 mmol) and DMF 5 (mL) were added to the reaction solution in sequence. The mixture was stirred at room temperature for 4 hours. Silica gel was added and the sample was purified by normal phase column chromatography using PE / EA = 8 / 92 as the eluent to obtain the product D3-1 (150 mg, two-step combined yield 22.3%) as a white flaky solid; LCMS / ESI (m / z): [M+H] + 927.40 (theoretical value 926.21).

[0114] D3-1 (150 mg, 0.162 mmol), acetonitrile (5 mL), and DBU (24.87 mg, 0.162 mmol) were added to the reaction flask and stirred at room temperature for 4 hours. The reaction was complete after LCMS monitoring (LCMS / ESI (m / z): [M+H] + 705.40 (theoretical value 703.97). DMF (5 mL) was added to the reaction solution, acetonitrile was removed under reduced pressure, 2,2,2-trifluoroethyl trifluoromethanesulfonate (150 mg, 0.648 mmol) and DIEA (105 mg, 0.81 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase chromatography in the range of ACN% = 45-60% to obtain the solid product D3, N-trifluoroethyl auristatin E (50 mg, two-step yield 39.27%); LCMS / ESI (m / z): [M+H] + 786.80 (theoretical value 785.99); 1 H NMR (500MHz, DMSO-d6) δ8.26(d,J=8.8Hz,1H),8.23(d,J=8.6Hz,1H),7.89(d,J=8.7Hz,1H),7.62(d,J=8.5Hz,1H),7.32-7.30(m,2H),7.28-7.25 (m,2H),7.17(td,J=4.0,1.6Hz,1H),4.79-4.71(m,1H),4.61(t,J=8.8Hz ,1H),4.54(t,J=8.8Hz,1H),4.49(d,J=5.9Hz,1H),4.44(d,J=6.6Hz,1H) ,4.02(ddd,J=13.3,7.6,4.1Hz,2H),3.78(dd,J=9.4,2.4Hz,1H),3.61-3 .46(m,2H),3.26-3.18(m,9H),3.12(s,1H),2.98(s,1H),2.44-2.39(m,1 H),2.30-2.25(m,1H),2.15-2.10(m,1H),2.05-1.99(m,1H),1.06-0.97( m,9H),0.87(ddd,J=13.9,6.8,2.6Hz,19H),0.76(dt,J=9.2,7.4Hz,5H).

[0115] Example 4: N-methoxyamine dolastatin derivative (D4)

[0116] Synthesis of D4-1

[0117] MMAF (3.0 g, 4.09 mmol) was dissolved in DMF (2 mL), and formaldehyde (533.48 mg, 17.19 mmol), acetic acid (4.92 g, 81.86 mmol), and NaBH3CN (514.15 mg, 8.19 mmol) were added in sequence. The mixture was stirred at room temperature for 2 h and purified by silica gel column chromatography to obtain methyl MMAF D4-1 (2.65 g, 86.6% yield) as a pale white powdery solid. 1 H NMR(600MHz, DMSO-d6)δ9.64(s,1H),9.01(dd,J=8.5,4.1Hz,1H),8.57(d,J=8.7Hz,1H),7.27-7.15(m,5H),4.74(dd,J=11.0,4.6Hz,1H),4.6 5(t,J=8.5Hz,1H),4.58(t,J=8.5Hz,1H),4.33-4.24(m,1H),4.18(q,J=7.9Hz,1H),4.00(d,J=12.2Hz,2H),3.55(ddd,J=11.6,7.9,5.4Hz,3H ),3.46(d,J=8.3Hz,5H),3.28-3.15(m,7H),3.10(s,2H),3.00(s,2H), 2.87-2.73(m,7H),2.47(s,3H),2.34-2.21(m,2H),2.16-1.98(m,2H), 1.90-1.65(m,3H),1.60-1.41(m,2H),1.36-1.26(m,2H),1.06(dd,J=17.8,6.7Hz,3H),1.01-0.83(m,16H),0.77(t,J=8.7Hz,3H); LCMS:[M+1] + 745.85 (calculated: 745.50).

[0118] Synthesis of D4-2

[0119] In a 25 mL single-necked bottle, D4-1 (600 mg, 803.22 μmol) and N-methoxy-N-methylamine (86.18 mg, 883.54 μmol) were added and dissolved in DMF (3 mL). HATU (305.41 mg, 803.22 μmol) and DIEA (103.81 mg, 803.22 μmol, 139.91 μL) were added in sequence. The mixture was stirred at room temperature for 2 h, quenched with water, and washed three times with dichloromethane and saturated brine, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated to give a pale white solid D4-2 (432 mg, yield 68.08%); LCMS: [M+1] + 791.37 (calculated: 788.54).

[0120] Synthesis of D4-3

[0121] At -78°C, D4-2 (200 mg, 253.14 μmol) was added to a 25 mL single-necked vial and dissolved in THF (3 mL). DIBAL-H (1.0 M in THF) (278 μL, 278.46 μmol) was then added and stirred at -78°C for 2 h. The mixture was quenched by the addition of potassium sodium tartrate. The mixture was washed three times with dichloromethane and saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a light yellow solid D4-3 (150 mg, yield 72.14%, HPLC 95%); MS: [M+1] + 730.42 (calculated: 729.50).

[0122] Synthesis of D4

[0123] In a 25 mL single-necked flask, D4-3 (168.54 mg, 205.20 μmol) was added and dissolved in EtOH (5 mL). O-methylhydroxylamine (17.14 mg, 205.20 μmol) and NaOAc (16.83 mg, 205.20 μmol) were added in sequence. The mixture was stirred at room temperature for 2 h. A mixed solution of acetyl chloride (51.38 mg, 654.59 μmol, 39.71 μL) and EtOH (2 mL) was added. Finally, NaBH3CN (25.79 mg, 410.40 μmol) was added. The mixture was stirred at room temperature for 1 h. A saturated aqueous sodium bicarbonate solution was added, and the mixture was washed three times with dichloromethane. The mixture was dried over anhydrous sodium sulfate and concentrated to give a light yellow solid D4 (35 mg, yield 18.61%, HPLC 97%). 1H NMR(600MHz, DMSO-d6)δ9.57(s,1H),8.92(dd,J=8.5,4.1Hz,1H),7.87(d,J=8.7Hz,1H),7.30-7.08(m,5H),4.74(dd,J=11.0,4.6Hz,1H), 4.65(t,J=8.5Hz,1H),4.58(t,J=8.5Hz,1H),4.33-4.24(m,1H),4.18(q,J=7.9Hz,1H),4.00(d,J=12.2Hz,2H),3.55(ddd,J=11.6,7.9,5. 4Hz,3H),3.46(d,J=8.3Hz,5H),3.28-3.15(m,7H),3.10(s,2H),3.00(s,2H),2.87-2.73(m,7H),2.34-2.21(m,2H),2.16-1.98(m,2H),1. 90-1.65(m,3H),1.60-1.41(m,2H),1.36-1.26(m,2H),1.06(dd,J=17.8,6.7Hz,3H),1.01-0.83(m,16H),0.77(t,J=8.7Hz,3H); MS:(M+1) + 761.46 (calculated: 760.55).

[0124] Example 5: N-2',2',2'-trifluoroethylamine dolastatin derivative (D5)

[0125] In a 50 mL single-necked bottle, tert-butyl (2S)-2-[(1R,2R)-3-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate, D5-1 (0.1 g, 2.60 mmol), DCM (10 mL), (2S)-2-amino-3-phenylpropanol, D5-2 (0.39 g, 2.6 mmol) and diisopropyl ether were added. Amine (0.50 g, 3.90 mmol) was added, the reaction was stirred at room temperature for 2 h, 5 g of silica gel powder was added, the solvent was evaporated to dryness, and the product was purified by silica gel column chromatography to obtain tert-butyl (2S)-2-[(1R,2R)-2-{[(2S)-1-hydroxy-3-phenylpropan-2-yl]carbamoyl}-1-methoxy-2-methylethyl]pyrrolidine-1-carboxylate, D5-3 (1.1 g, yield 96.53%); LCMS [M+1] + 421.54 (theoretical value 420.55); 1H NMR(500MHz,DMSO-d6)δ7.62(d,J=8.3Hz,1H),7.20(q,J=5.4,4.2Hz,4H),7.15-7.06(m,1H ),4.78(s,1H),3.97(d,J=4.4Hz,1H),3.39(dt,J=10.1,4.9Hz,1H),3.35-3.25(m,3H),3.1 2(s,1H),2.98(s,1H),2.90(d,J=10.7Hz,1H),2.58(d,J=9.7Hz,1H),2.11(dq,J=9.5,6.7H z,1H),1.75-1.59(m,2H),1.45(dt,J=15.0,7.5Hz,2H),1.36(s,9H),1.02(d,J=6.1Hz,3H).

[0126] In a 50 mL single-necked bottle, D5-3 (600 mg, 1.43 mmol) was dissolved in DCM (10 mL), and Dess-Martin periodinane (1.21 g, 2.86 mmol) was added. The reaction solution was stirred at room temperature for 4 h, and 10 mL of saturated sodium bicarbonate aqueous solution was added, and the mixture was separated. 10 mL of saturated brine was added, and the mixture was separated. 5 g of anhydrous magnesium sulfate was added to the organic phase, and the mixture was dried, filtered, and the solvent was evaporated to dryness to obtain tert-butyl (2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-{[(2S)-1-oxo-3-phenylpropan-2-yl]carbamoyl}ethyl]pyrrolidine-1-carboxylate, D5-4 (800 mg, yield 80.38%); LCMS [M+1] + 419.47 (theoretical value 418.53).

[0127] In a 50 mL single-necked bottle, D5-4 (0.8 g, 1.15 mmol) was dissolved in methanol (8 mL), and 2,2,2-trifluoroethane-1-amine (0.34 g, 3.45 mmol) and sodium acetate (0.28 g, 3.45 mmol) were added. The reaction was stirred at room temperature for 4 h. Sodium cyanoborohydride (0.22 g, 3.45 mmol) and acetic acid (0.35 g, 5.75 mmol) were added. The reaction solution was stirred at room temperature for 2 h. The solvent was evaporated and 10 mL of saturated carbon was added. The organic phase was added with 10 mL of saturated saline solution, the organic phase was extracted, 5 g of anhydrous magnesium sulfate was added, the mixture was filtered, and the solvent was evaporated to dryness to obtain tert-butyl (2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-{[(2S)-1-phenyl-3-[(2,2,2-trifluoroethyl)amino]propan-2-yl]carbamoyl}ethyl]pyrrolidine-1-carboxylate, D5-5 (0.55 g, yield 47.81%); LCMS [M+1]+ 502.39 (theoretical value 501.59).

[0128] In a 50 mL single-necked bottle, D5-5 (0.5 g, 1.0 mmol) was dissolved in DCM (5 mL), and trifluoroacetic acid (0.23 g, 2 mmol) was added. The reaction was stirred at room temperature for 1 h. The solvent was evaporated to dryness. 10 mL of distilled water and 20 mL of ethyl acetate were added to the crude product, and the extract was separated. The crude product D5-6 was dried to obtain a crude product that was used directly in the next reaction. LCMS [M+1] + 402.50 (theoretical value 401.47).

[0129] In a 50 mL single-necked bottle, sodium bicarbonate (0.42 g, 5 mmol) was added to an aqueous solution of D5-6 (0.4 g, 1.0 mmol), and then (3R, 4R, 5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N, 3-dimethylbutyramido)-3-methoxy-5-methylheptanoate, 2,5-dioxopyrrolidine-1-ester, D5-7 (0.4 g, 1.0 mmol) in 1,4-dioxane (5 mL) was slowly added dropwise to the reaction solution. The reaction was stirred at room temperature for 2 h, 20 mL of ethyl acetate was added, and the organic phase was extracted and separated. The phase was dried over anhydrous magnesium sulfate, filtered, and purified by silica gel column chromatography to give (2S)-2-[(2S)-2-(dimethylamino)-3-methylbutanamido]-N-[(3R,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-2-{[(2S)-1-phenyl-3-[(2,2,2-trifluoroethyl)amino]propan-2-yl]carbamoyl}ethyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptane-4-yl]-N,3-dimethylbutanamide, D5 (0.11 g, yield 12.36%); LCMS [M+1] + 813.33 (theoretical value 812.54); 1H NMR(500MHz,DMSO-d6)δ8.02(d,J=8.3Hz,1H),7.25-7.16(m,4H),7.15-7.07(m,1H),4.78-4.62(m,1H),4.61-4.48(m,1H),4 .21-4.02(m,1H),3.98(s,1H),3.61(s,1H),3.32(s,3H),3.30-3.13(m,9H),3.10(s,1H),3.00(d,J=10.6Hz,2H),2.69-2.60 (m,3H),2.44(d,J=15.3Hz,1H),2.36-2.25(m,1H),2.20(d,J=8.2Hz,6H),2.14-2.04(m,1H),1.92(dt,J=13.9,7.9Hz,2H),1 .80-1.65(m,2H),1.57-1.47(m,1H),1.35-1.18(m,4H),1.05(dd,J=15.4,6.8Hz,3H),0.96-0.80(m,14H),0.79-0.60(m,7H).

[0130] Example 6: Preparation of BrAc-PEG2-GVA-ha-D1 (A1)

[0131] Synthesis of A1-1

[0132] In a 100 mL single-necked flask, D1 (1.2 g, 1.56 mmol) and (2R)-N-(chloromethyl)-2-(1,3-dioxoisoindolin-2-yl)propionamide (Int101) (497.07 mg, 1.72 mmol) were dissolved in DCM (15 mL). DIPEA (201.62 mg, 1.56 mmol, 271.72 μL) was added and stirred at room temperature for 2 h. 5 g of silica gel powder was added to the reaction solution, the solvent was evaporated, and A1-1 (1.3 g, 83.58% yield) was isolated by silica gel column chromatography. 1H NMR(500MHz, CDCl3)δ7.81(td,J=8.0,6.7,3.1Hz,2H),7.73-7.65(m,2H),7.40-7.30(m,4H),7.26(q,J=6.9Hz,2H),4.98-4.81(m ,2H),4.74-4.58(m,2H),4.38-4.17(m,2H),4.15-3.98(m,2H),3.89-3.78(m,1H),3.65-3.15(m,15H),3.01(d,J=50.1Hz,2H),2.6 3(s,1H),2.51-2.33(m,2H),2.16-2.01(m,2H),1.90-1.77(m,3H),1.75-1.62(m,4H),1.35(d,J=18.9Hz,1H),1.25(dd,J=10.9,4 .8Hz,4H),1.12-1.05(m,1H),0.98(td,J=20.4,19.2,6.8Hz,7H),0.86(dt,J=23.3,8.9Hz,6H),0.70(d,J=6.5Hz,2H); LCMS:[M+1] + 974.62 (calculated 973.51).

[0133] Synthesis of A1-2

[0134] In a 100 mL single-necked bottle, A1-1 (0.8 g, 802.32 μmol) was dissolved in ACN (10 mL), and 80% hydrazine hydrate (602.47 mg, 9.63 mmol) was added. The mixture was stirred at room temperature for 12 h. 20 mL of water was added to the reaction solution, and the mixture was extracted three times with DCM. The mixture was washed with saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and concentrated to afford A1-2 (750 mg, yield 99.68%, HPLC 93%); LCMS: [M+1] + 844.21 (calculated value 843.51).

[0135] Synthesis of A1-3

[0136] In a 100 mL single-necked bottle, A1-2 (750 mg, 778.54 μmol) was dissolved in DCM (10 mL), and FmocPEG2-GV-OSu (526.05 mg, 778.54 μmol) was added. The mixture was stirred at room temperature for 2 h. 5 g of silica gel powder was added, and the solvent was evaporated. A1-3 (1 g, yield 91.45%) was obtained by silica gel column chromatography. 1HNMR(500MHz, CDCl3)δ7.76(d,J=7.2Hz,2H),7.60(d,J=7.2Hz,3H),7.47-7.36(m,4H),7.33(dd, J=13.2,5.7Hz,5H),4.95(s,3H),4.39(s,3H),4.31-3.98(m,7H),3.85(d,J=8.1Hz,4H),3.66-3. 20(m,24H),2.58-2.26(m,4H),1.86(s,13H),1.35(dd,J=21.0,7.0Hz,4H),1.25(d,J=6.8Hz,4H) ,1.09(d,J=6.5Hz,2H),0.96(ddd,J=32.7,17.0,7.4Hz,17H),0.83(t,J=7.3Hz,3H); LCMS:[M+1] + 1381.72 (calculated value 1380.76).

[0137] Synthesis of A1

[0138] In a 25 mL single-necked bottle, A1-3 (300 mg, 213.58 μmol) was dissolved in DCM (3 mL), and DBU (32.52 mg, 213.58 μmol, 31.94 μL) was added. The mixture was stirred at 25 °C for 0.5 h to obtain A1-4 (LCMS: [M+1] + 1159.39; calculated value: 1158.69), DIC (32.34 mg, 256.30 μmol, 39.69 μL) and 2-bromoacetic acid (32.64 mg, 234.94 μmol) were added directly without treatment, and the mixture was stirred at 25°C for 1 h. 3 g of silica gel powder was added, and the solvent was evaporated. A1 was separated by silica gel column chromatography to obtain 150 mg, two-step yield 53.89%; 1H NMR(500MHz,DMSO-d6)δ8.17-8.10(m,1H),7.82(dt,J=16.6,7.7Hz,1H),7.29(dt,J=14.8,7.6Hz,3H),7.22-7.13(m,1H),4.78-4.5 4(m,2H),4.46(dd,J=31.9,6.4Hz,1H),4.28-4.11(m,3H),4.08-3.93(m,3H),3.86(s,1H),3.80-3.73(m,3H),3.71-3.56(m,10H),3 .45-3.30(m,15H),3.24(dt,J=11.7,7.0Hz,8H),3.18(d,J=4.7Hz,1H),3.07(d,J=19.2Hz,2H),2.95(s,1H),2.38(t,J=6.4Hz,2H), 2.29(d,J=8.5Hz,1H),2.16-1.89(m,3H),1.87-1.45(m,5H),1.39-1.19(m,6H),1.07-0.97(m,5H),0.96-0.69(m,16H); LCMS:[M+1] + 1281.75 (calculated 1280.33).

[0139] Example 7: Preparation of BrAc-PEG2-GVA-ha-D2 (A2)

[0140] Synthesis of A2-1

[0141] To a 10 mL reaction flask were added D2 (1.85 g, 2.51 mmol), DMF (10 mL), (2R)-N-(chloromethyl)-2-(1,3-dioxoisoindolin-2-yl)propionamide (668.58 mg, 2.51 mmol), and DIPEA (324.02 mg, 2.51 mmol, 436.68 μL) in sequence at room temperature. The reaction was stirred at room temperature for 0.5 h. 5 g of silica gel powder was added to the reaction solution, and column chromatography was performed to obtain A2-1 (2.11 g, yield 83.46%, HPLC 96%); LCMS: [M+1] + 968.07 (calculated: 967.52).

[0142] Synthesis of A2-2

[0143] At room temperature, A2-1 (1 g, 1.03 mmol), methanol (10 mL), and hydrazine hydrate (620.50 mg, 12.39 mmol) were added to the reaction flask in sequence. The reactants were stirred at room temperature for 5 h, extracted with DCM (100 mL), washed with water, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a light yellow solid A2-2 (740 mg, yield 76.94%, HPLC 94%). 1 H NMR (500MHz, DMSO-d6) δ8.42(s,1H),8.07(dd,J=9.1,3.1Hz,1H),7.88(d,J=8.7Hz,1H),7.63(d,J=8.5Hz,0H),7.34-7.26 (m,4H),7.28-7.20(m,0H),7.19(td,J=6.9,2.2Hz,1H),5.42(d,J=5.0Hz,1H),5.35(d,J=5.0Hz,0H),4.74(s,0H),4.68(d d,J=9.1,6.9Hz,0H),4.62-4.55(m,0H),4.50(t,J=5.4Hz,0H),4.43(t,J=5.8Hz,1H),4.14(td,J=16.1,15.2,4.5Hz,1H), 4.08-4.01(m,1H),4.02(s,1H),4.00(s,1H),3.95(dd,J=14.4,7.1Hz,0H),3.84-3.74(m,0H),3.70-3.55(m,2H),3.25(s, 3H),3.23(d,J=10.0Hz,3H),3.19(s,1H),3.10(s,2H),3.04(dt,J=11.6,9.2Hz,0H),2.96(s,1H),2.90(s,0H),2.56(dd,J =15.5,9.7Hz,1H),2.42(dd,J=15.8,8.5Hz,1H),2.30(t,J=10.2Hz,1H),2.26(s,1H),2.18-2.05(m,2H),2.00(dt,J=13.0 ,6.7Hz,1H),1.83(d,J=8.3Hz,1H),1.81-1.67(m,1H),1.55(s,1H),1.49(q,J=8.2Hz,1H),1.30(s,2H),1.25(d,J=2.7Hz, 1H),1.20-1.09(m,4H),1.04(dd,J=11.9,5.9Hz,4H),1.01(s,1H),0.99(d,J=6.6Hz,2H),0.97-0.72(m,12H).LCMS:[M+1] +839.05 (calculated: 837.52).

[0144] Synthesis of A2-3

[0145] A2-2 (740 mg, 883.02 μmol), DMF (5 mL), and Fmoc-PEG2-GV-OSu (576.34 mg, 883.02 μmol) were added to the reaction flask in sequence at room temperature. The reaction was stirred at room temperature for 0.5 h. 5 g of silica gel powder was added to make sand. The product was purified by silica gel column chromatography to obtain a light yellow solid A2-3 (814 mg, 92% yield); LCMS: [M+1] + 1376.32 (calculated: 1375.66).

[0146] Synthesis of A2-4

[0147] At room temperature, A2-3 (400 mg, 290.77 μmol), DMF (1.2 mL), and DBU (44.27 mg, 290.77 μmol, 43.48 μL) were added sequentially in a 10 mL single-necked bottle. The mixture was stirred at room temperature for 1 h. 10 mL of methyl tert-butyl ether was added and the mixture was centrifuged (10000 r / min) for 5 min to give a light yellow solid A2-4 (300 mg, yield 83.19%, HPLC 93%); LCMS: [M+1] + 1154.26 (calculated value: 1153.42).

[0148] Synthesis of A2

[0149] At room temperature, A2-4 (300 mg, 260.10 μmol), DMF (3 mL), and (2-bromoacetyl) 2-bromoacetate (67.60 mg, 260.10 μmol) were added sequentially to a 10 mL single-necked bottle. The mixture was stirred at room temperature for 1 h. 5 g of silica gel powder was added to the reaction solution, and the mixture was purified by silica gel column chromatography to obtain an off-white solid A2 (250 mg, yield 72.41%, HPLC 96%). 1H NMR (500MHz, DMSO-d6) δ8.41 (dt, J=11.1, 6.2Hz, 1H), 8.14 (dq, J=15.6, 6.0, 4.7Hz, 2H), 7.98 (dd, J=8. 3,4.0Hz,1H),7.81(t,J=8.3Hz,1H),7.34-7.24(m,4H),7.22-7.15(m,1H),4.65-4.54(m,1H),4.49(d, J=6.3Hz,1H),4.18(dtt,J=26.7,13.1,5.9Hz,3H),4.08-4.00(m,1H),4.00(s,2H),3.98-3.90(m,1H), 3.87(s,1H),3.81-3.73(m,3H),3.64-3.55(m,3H),3.50(s,4H),3.27-3.20(m,8H),3.19(s,1H),3.09(s ,2H),2.95(s,1H),2.71-2.58(m,3H),2.39(t,J=6.6Hz,3H),2.31-2.23(m,2H),2.12(dq,J=11.9,6.5H z,1H),2.05-1.88(m,3H),1.83(s,1H),1.74-1.64(m,1H),1.63(dq,J=9.1,4.9,4.5Hz,1H),1.55(d,J= 7.0Hz,1H),1.48(s,1H),1.36(p,J=10.4,9.6Hz,1H),1.32-1.22(m,10H),1.25-1.19(m,1H),1.07-0.9 5(m,6H),0.93(s,1H),0.97-0.86(m,4H),0.84(dt,J=20.6,6.3Hz,12H),0.81-0.73(m,2H); LCMS:[M+1] + 1275.22 (calculated: 1274.35).

[0150] Example 8: Preparation of mc-PEG2-GVA-ha-D4 (A4)

[0151] Synthesis of A4-1

[0152] D4 (200 mg, 257.72 μmol) was dissolved in DCM (2.89 mL) at room temperature. TEA (78.24 mg, 773.16 μmol, 107.76 μL) and intermediate Int1 (68.99 mg, 257.72 μmol) were added sequentially. The mixture was stirred at room temperature for 2 h, dried by spin drying, and purified by reverse phase column chromatography to give a light yellow solid A4-1 (259.33 mg, 95% yield). 1 H NMR (500MHz, DMSO-d6) δ8.62(dt,J=22.0,6.1Hz,1H),8.05(dd,J=13.2,8.6Hz,1H),7.90(p,J=5.9,5.3Hz,5H),7.31-7.12(m,6H),4.79(pd,J=9.0,8 .1,4.3Hz,2H),4.64(t,J=8.8Hz,1H),4.56(t,J=8.8Hz,1H),4.41-4.26(m ,2H),4.14(ddd,J=29.2,13.3,5.8Hz,2H),4.02(dd,J=9.3,5.1Hz,1H),3. 84(dd,J=9.3,2.4Hz,1H),3.68(t,J=6.7Hz,1H),3.60(ddd,J=12.5,7.9,5 .4Hz,1H),3.52(s,3H),3.46(s,2H),2.65(dh,J=20.2,4.3Hz,5H),2.23(s ,8H),2.15(d,J=3.1Hz,1H),1.62(d,J=7.3Hz,3H),1.42-1.15(m,9H),1.1 5-1.01(m,7H),1.00-0.85(m,16H),0.78(tt,J=13.7,6.6Hz,9H);MS:[M+1] + 991.24 (calculated: 990.62).

[0153] Synthesis of A4-2

[0154] A4-1 (177.50 mg, 178.88 μmol) was dissolved in DCM (8 mL) at room temperature, and hydrazine hydrate (35.82 mg, 715.53 μmol) was added. The mixture was stirred at 30°C for 26 h, dried, and purified by reverse phase column chromatography to give a light yellow solid A4-2 (119.5 mg, 72.83% yield). 11H NMR (500 MHz, DMSO-d6) δ 8.30 (dt, J = 24.8, 6.4 Hz, 1H), 8.08 (t, J = 7.6 Hz, 1H), 7.91 (d, J = 8.9 Hz, 0H), 7.31 - 7.20 (m, 4H), 7.22 - 7.15 (m, 1H), 4.79 (dd, J = 11.0, 4.9 Hz, 1H), 4.67 (q, J = 9.2 Hz, 1H), 4.58 (t, J = 8.8 Hz, 0H), 4.34 (tt, J = 16.8, 7.0 Hz, 1H), 4.22 (ddd, J = 26.3, 13.2, 6.0 Hz, 1H), 4.04 (dd, J = 9.4, 5.1 Hz, 1H), 3.62 (ddd, J = 12.4, 7.8, 5.2 Hz, 1H), 3.55 (s, 2H), 3.49 (d, J = 9.2 Hz, 1H), 3.43 (s, 0H), 3.30 (d, J = 9.0 Hz, 3H), 3.24 (d, J = 11.8 Hz, 3H), 3.18 (s, 1H), 3.10 (dt, J = 11.7, 7.1 Hz, 0H), 3.05 (s, 1H), 3.05 - 2.98 (m, 1H), 2.95 (s, 1H), 2.79 (s, 1H), 2.71 (q, J = 9.1 Hz, 1H), 2.67 (s, 0H), 2.63 (d, J = 8.2 Hz, 2H), 2.48 (t, J = 15.9 Hz, 1H), 2.37 - 2.31 (m, 1H), 2.26 (d, J = 8.1 Hz, 6H), 2.21 - 2.15 (m, 1H), 2.18 - 2.10 (m, 1H), 2.07 - 1.96 (m, 1H), 1.98 - 1.91 (m, 0H), 1.88 (d, J = 7.8 Hz, 1H), 1.86 (s, 2H), 1.79 (ddt, J = 22.4, 10.9, 5.5 Hz, 1H), 1.59 (dq, J = 12.4, 7.3 Hz, 1H), 1.53 (s, 1H), 1.46 - 1.30 (m, 3H), 1.29 (s, 1H), 1.18 (d, J = 6.9 Hz, 3H), 1.10 (dd, J = 16.3, 6.7 Hz, 3H), 1.04 - 0.86 (m, 13H), 0.80 (dt, J = 10.0, 5.6 Hz, 5H), 0.76 (s, 1H); MS: [M+1] + 862.24 (calculated: 861.18).

[0155] Synthesis of A4-3

[0156] At room temperature, Fmoc-PEG2-GV-OSu (148.99 mg, 227.92 μmol) was added to DCM (2 mL), and a solution of A4-2 (200 mg, 228.27 μmol) in DCM (6 mL) was added. The mixture was stirred at room temperature for 1 h, dried by spin drying, and purified by reverse phase column chromatography to obtain a light yellow solid A4-3 (185 mg, 57.90% yield); 1 HNMR(500MHz,DMSO-d6)δ8.29(dt,J=27.4,6.1Hz,1H),8.11(td,J=7.6,4.7Hz,2H),8 .03(s,1H),7.89(d,J=7.6Hz,2H),7.84-7.73(m,2H),7.69(d,J=7.5Hz,2H),7.42(t, J=7.4Hz,2H),7.33(t,J=7.2Hz,3H),7.25-7.09(m,6H),4.32(s,3H),4.36-4.27(m,3 H),4.25-4.09(m,3H),3.78-3.72(m,2H),3.59(q,J=9.5,8.0Hz,3H),3.49(d,J=6.0Hz ,6H),3.41(d,J=15.0Hz,2H),3.28-3.19(m,6H),3.16(d,J=19.4Hz,3H),3.13(s,3H) ,2.69-2.52(m,7H),2.47-2.35(m,4H),2.21(d,J=6.2Hz,7H),2.03-1.90(m,2H),1.31 (s,3H),1.28-1.17(m,4H),1.05(dd,J=16.3,6.6Hz,3H),0.91(t,J=7.2Hz,6H),0.91 -0.84(m,14H),0.84-0.80(m,3H),0.83-0.72(m,6H),0.72(d,J=9.5Hz,2H);MS:[M+1] + 1399.00 (calculated value: 1397.86).

[0157] Synthesis of A4-4

[0158] At room temperature, A4-3 (185 mg, 133.50 μmol) was added to DCM (6 mL), and then DBU (10.16 mg, 66.75 μmol, 9.98 μL) was added. The mixture was stirred at room temperature for 1 h, dried by spin drying, and purified by reverse-phase column chromatography to obtain a light yellow solid A4-4 (145 mg, 88.68% yield). 1H NMR(500MHz,DMSO-d6)δ9.74(s,1H),8.93(t,J=6.8Hz,1H),8.28(q,J=6.8Hz,1H),8.22-8.09(m,2H),7.88-7 .82(m,2H),7.81(dd,J=8.9,3.0Hz,1H),7.29(dd,J=17.2,7.5Hz,1H),7.16(ddt,J=32.2,14.8,7.5Hz,5H),4 .65(q,J=7.2,5.9Hz,1H),4.31(td,J=7.2,2.9Hz,2H),4.16(ddt,J=37.0,13.6,7.4Hz,3H),4.08-3.97(m,1H ),3.75(t,J=9.3Hz,3H),3.65-3.58(m,4H),3.55(dd,J=15.1,10.2Hz,7H),3.43(s,3H),3.33(dd,J=19.1,10. 6Hz,3H),3.27-3.19(m,5H),3.14(d,J=29.6Hz,3H),3.08-2.90(m,5H),2.78(d,J=8.0Hz,6H),2.58(ddq,J=1 8.3,11.8,7.5,6.4Hz,4H),2.49-2.36(m,3H),2.29(tq,J=14.9,6.8Hz,2H),2.03(dddt,J=53.0,18.9,12.8,6 .5Hz,4H),1.85(d,J=8.4Hz,1H),1.77(p,J=6.3Hz,1H),1.73-1.66(m,1H),1.53(dd,J=13.2,6.7Hz,1H),1.3 2(dq,J=17.9,9.7,8.0Hz,2H),1.26-1.18(m,3H),1.05(dd,J=17.3,6.7Hz,3H),1.00-0.74(m,26H);MS:[M+1] + 1177.48 (calculated value: 1176.55).

[0159] Synthesis of A4

[0160] At room temperature, succinimidyl (6-(maleimido)hexanoate or mc-Osu (31.80 mg, 102.80 μmol) was dissolved in DCM (20 mL), and A4-4 (118.56 mg, 103.14 μmol) was added. The mixture was stirred at room temperature for 0.5 h, and then directly dried by rotary evaporation. The mixture was purified by reverse phase chromatography to give a light yellow solid A4 (106 mg, 72.77% yield). 1H NMR (500MHz, DMSO-d6) δ8.33(t,J=5.6Hz,2H),8.28(t,J=6.3Hz,2H),8.13-8.05(m,4H),7.65(d,J=8.8Hz,2H),7.52(s,4H),7.25(s,2H) ,6.30(s,4H),5.43(d,J=2.3Hz,4H),5.28(s,4H),4.24(dt,J=33.4,13.1,6.2Hz,7H),4.13(dd,J=8.9,6.7Hz,2H),3.86(s,4H),3.74(d,J =6.3Hz,4H),3.60(t,J=6.4Hz,5H),3.56(s,7H),3.49(s,12H),3.31(t,J=8.0Hz,5H),2.89(dq,J=22.7,8.2,7.3Hz,4H),2.38(t,J=6.4Hz ,5H),1.86(dh,J=21.4,7.2Hz,5H),1.76(h,J=6.8Hz,3H),1.17(d,J=7.1Hz,6H),0.88(t,J=7.3Hz,7H),0.67(d,J=6.7Hz,11H); MS:[M+1] + 1370.47 (calculated value: 1369.76).

[0161] Example 9: Preparation of dBPA-PEG2-GVA-ha-D4 (A5)

[0162] 2,6-Dibromomethylpyridinium 4-oxyacetic acid (27.82 mg, 81.82 μmol) dissolved in DCM (3 mL) was added to a reaction flask at room temperature. DIC (8.60 mg, 68.18 μmol, 10.56 μL) was added and stirred for 5 minutes. A solution of A4-4 (78.38 mg, 68.18 μmol) in DCM (3 mL) was then added and stirred at room temperature for 0.5 hours. The reaction solution was concentrated and purified by reverse phase column chromatography to give a light yellow solid A5 (35.4 mg, 33.54% yield). 1H NMR(500MHz,DMSO-d6)δ9.57(s,1H),8.15(dt,J=22.9,6.0Hz,1H),7.88-7.74(m ,1H),7.38-7.26(m,2H),7.26-7.19(m,2H),7.19-7.11(m,2H),4.92(s,1H),4.76 (s,3H),4.35-4.25(m,2H),4.17(dt,J=23.5,15.5,7.4Hz,3H),3.76(t,J=6.3Hz ,6H),3.64(s,14H),3.63-3.58(m,17H),3.49-3.41(m,5H),3.40-3.10(m,12H),3 .01(d,J=3.8Hz,1H),2.79(q,J=11.5,8.2Hz,5H),2.58(dd,J=18.6,9.5Hz,2H), 2.39(dq,J=6.4,4.0,2.7Hz,2H),2.30(dq,J=15.8,8.4,7.8Hz,2H),1.98(tt,J=1 6.9,6.8Hz,2H),1.49(dt,J=21.9,6.8Hz,1H),1.30(t,J=13.0Hz,2H),1.26-1.17 (m,4H),1.11-1.01(m,2H),0.87(ddd,J=48.9,28.3,15.6,7.5Hz,19H);MS:[M+1] + 1496.72 (calculated value: 1497.52).

[0163] Example 10: Preparation of Nectin-4-Ac-PEG2-GVA-ha-D2 (DAR4) (ADC2)

[0164] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.02 mL), and react at 10°C for 3 h.

[0165] Compound A2 (0.58 mg, 0.45 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed, and reacted at 10°C for 4 h. After the reaction was completed, small molecules were removed using a NAP-5 gel column (Cytiva) and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH = 6.2, to obtain antibody-drug conjugate ADC2 (3.0 mg / mL, 2 mL).

[0166] RP-MS Analysis: RP-HPLC-MS analysis of ADC DAR values ​​was performed using a Waters Acquity UPLC I-Class / Xevo G2-XS QTOF instrument. RP-HPLC parameters included a PLRP-S1000A5UM column, a column temperature of 70°C, mobile phase A consisting of 0.1% formic acid in water, and mobile phase B consisting of 0.1% formic acid in acetonitrile, at a flow rate of 0.2 mL / min. The mobile phase gradient was 20-50% B over 18 minutes; 50-95% B over 5 minutes; 95-20% B over 0.1 minutes; and 20-20% B over 6.9 minutes. MS parameters were set as follows: capillary voltage 2.50 kV, cone voltage 100 V, mass analysis range m / z 200 to 4000, MSE collision energy 20 to 45 eV, ion source temperature 120°C, nebulizer temperature 500°C, nebulizer flow rate 1000 L / Hr, and internal standard leucine enkephalin. The sample was diluted to 1 mg / mL in sample buffer, TCEP was added to a final concentration of 50 mmol / L, and the sample was incubated at 37°C for 20 min. A 5 μL injection was performed. Light chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Similarly, heavy chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Weighted peak areas for the heavy and light chains were calculated by multiplying the peak area percentages by the corresponding drug loading. The DAR value was calculated as: DAR = 2*(Σlight chain weighted peak area + Σheavy chain weighted peak area) / 100.

[0167] The average value calculated by RP-MS was n=3.2; the MS results showed that the light chain and heavy chain (HL) were connected to one and two linker-payloads ( FIG1 ).

[0168] Example 11: Preparation of Nectin-4-mc-PEG2-GVA-ha-D4(DAR8) (ADC4)

[0169] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0170] Compound A4 (1.27 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, small molecules were removed using a NAP-5 gel column (Cytiva) and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH = 6.2, to obtain antibody-drug conjugate ADC4 (3.0 mg / mL, 2 mL).

[0171] The average value calculated by RP-MS was: n=7.9; MS results showed that the light chain (L) was connected to one linker-payload, and the heavy chain (H) was connected to three linker-payloads (Figure 2).

[0172] Example 12: Preparation of Nectin-4-mc-PEG2-GVA-ha-D4 (DAR4) (ADC5)

[0173] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.02 mL), and react at 10°C for 3 h.

[0174] Compound A4 (0.63 mg, 0.45 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed, and reacted at 10°C for 4 h. After the reaction was completed, small molecules were removed using a NAP-5 gel column (Cytiva) and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH = 6.2, to obtain antibody-drug conjugate ADC5 (3.0 mg / mL, 2 mL).

[0175] The average value calculated by RP-MS was: n=3.9; MS results showed that the light chain and heavy chain (HL) were connected to two linker-payloads (Figure 3).

[0176] Example 13: Preparation of Nectin-4-PA-PEG2-GVA-ha-D4 (DAR4) (ADC6)

[0177] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0178] Compound A5 (1.11 mg, 0.74 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, small molecules were removed using a NAP-5 gel column (Cytiva) and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH = 6.2, to obtain the antibody-drug conjugate ACN100546-CL60-D4 (3.0 mg / mL, 2 mL).

[0179] The average value calculated by RP-MS was n=4.3; the MS results showed that the light and heavy chains (HL) were connected to two linker-payloads ( FIG4 ).

[0180] Test Example 1: Inhibition of tumor cell growth activity

[0181] Human esophageal cancer cells OE33, human breast cancer cells SK-BR-3, human breast cancer cells MDA-MB-231, human gastric cancer cells NCI-N87, human lung cancer cells NCI-H1975, and human esophageal cancer cells TE12 were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax). Tumor cells in the exponential growth phase were diluted with culture medium to 1×10 5 cells / mL, 100 μL was added to each well of a 96-well cell culture plate and returned to a 37°C, 5% CO2 incubator for overnight incubation. The next day, the compound was diluted to 10,000 nM, 2,000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.13 nM using culture medium, and 2 μL of the diluted compound was added to each well of the 96-well cell culture plate. Three replicates were set for each concentration. For the negative control and blank control groups without compound addition, 2 μL of the dilution was added to each well. After addition, the plates were returned to a 37°C, 5% CO2 incubator and incubated for an additional 72 hours. After incubation, the cell culture plates were removed, the culture medium was aspirated, and 100 μL of culture medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 hours. After incubation, remove the culture plate, protect from light, and place it in an ELISA plate. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance value, use the four-parameter regression in GraphPad to calculate the IC 50 Values ​​(Table 1).

[0182] For IC 50 Values, where “++++” means 10nM>IC 50 ; “+++” means 100nM>IC 50 ≥10nM; “++” indicates 500nM>IC 50≥100nM; “+” indicates 1000nM>IC 50 ≥500μM.

[0183] Table 1 IC of compounds for inhibiting tumor cell growth 50 (nM) value

[0184] Note: “-” means not tested.

[0185] "Dxd" is a positive control drug, and its structural formula is

[0186] The compounds provided in the examples of the present invention all have a good inhibitory effect on the growth of tumor cells. 50 The values ​​were all lower than 500nM, even lower than 0.5nM, showing significant anticancer activity.

[0187] Test Example 2: In vivo tumor growth inhibition activity of ADC

[0188] ADC in vitro inhibitory activity test method: NCI-H292 lung cancer cells were cultured in monolayer in vitro. When the cell saturation reached 80%-90%, they were digested with trypsin-EDTA, centrifuged and the supernatant was discarded. The cells were resuspended in PBS and the cell suspension was adjusted to an appropriate concentration. NCI-H292 cells (2-10×10 6 cells / 0.1 mL) were subcutaneously inoculated into BALB / c nude mice, and the animals and transplanted tumor growth were regularly observed. 3 Animals were randomly divided into groups of 6 per group based on tumor volume and body weight. The drug was administered intravenously once a day and monitored until day 22. The experimental groups are shown in Table 2 below. Tumor long diameter (a) (mm), short diameter (b) (mm) (mm) and mouse body weight were measured twice weekly with a vernier caliper. Tumor volume (V) was calculated according to the following formula: V = 1 / 2 × a × b 2 (mm 3 ), where a and b represent tumor length and width, respectively. Growth curves were plotted, and tumors were removed and weighed. Statistical analysis was performed using GraphPad Prism software based on the data of tumor volume and weight of tumor-bearing mice at the end of the experiment to generate tumor inhibition results (Figures 5 and 6).

[0189] Table 2 Dosage regimen

[0190] Among them, DXd-Nectin-4 is It is obtained by coupling with the Nectin-4 antibody described in the examples of this application.

[0191] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof: wherein, M is selected from any one of the following; R1 and R2 are each independently selected from any one of C1-C6 alkyl, halogen-substituted C1-C6 alkyl, or C3-C6 cycloalkyl; R3 is selected from any one of hydrogen, C1-C6 alkyl, or C3-C6 cycloalkyl; R4 is selected from any one of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, -C1-C6 alkyl-NR6-O-C1-C6 alkyl, or -C1-C6 alkyl-O-NR6-C1-C6 alkyl; R5 is selected from any one of hydrogen, hydroxy, C3-C6 cycloalkyl, or -O-NR6-C1-C6 alkyl; R6 is selected from hydrogen or C1-C6 alkyl; X is selected from halogen; m1 and m2 are each independently selected from 0, 1, or 2.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein, R1 and R2 are each independently selected from hydrogen, halogen-substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl, or isobutyl; Preferably, R1 and R2 are each independently selected from hydrogen, methyl, trifluoromethyl, or trifluoroethyl; Preferably, R3 is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or isobutyl.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein, selected from 4. A compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof according to any one of claims 1-3, wherein, The halogen is selected from -F, -Cl, -Br, or -I; Preferably, m1 and m2 are each independently selected from 1 or 2; Preferably, Selected from 5. A compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof according to any one of claims 1-4, wherein, R4 is selected from C1-C6 alkyl, -C1-C6 alkyl-NH-O-C1-C6 alkyl, or -C1-C6 alkyl-O-NH-C1-C6 alkyl; Preferably, R4 is selected from methyl, -CH2-NH-O-CH3, or -CH2-O-NH-CH3; Preferably, R5 is selected from hydrogen, hydroxy, or -O-NH-C1-C6 alkyl; Preferably, R5 is selected from hydrogen, hydroxy, or -O-NH-CH3.

6. The compound or its pharmaceutically acceptable salt, stereoisomer or prodrug according to any one of claims 1-5, wherein the compound of formula (I) includes the following compounds:

7. An antibody-drug conjugate comprising the compound according to any one of claims 1-6; Preferably, the antibody-drug conjugate has the structure shown in the following formula (II): (D-L)n-Ab (II) Among them, D is the compound according to any one of claims 1-6; Ab is an antibody against a tumor-associated antigen; L is a linker for connecting D and Ab; n＝3-8； Preferably, the linker is a hydrophilic linker; Preferably, the linker is selected from the following structures: Among them, The position shown indicates attachment to the antibody, The indicated position represents connection to D; Preferably, the antibody conjugate is obtained by conjugating the following compound with an antibody:

8. The following antibody-drug conjugates: Among them, n＝3-8； Preferably, Ab is an antibody against a tumor-associated antigen or its antigen-binding fragment; preferably an anti-Nectin-4 antibody or its antigen-binding fragment; Preferably, the anti-Nectin-4 antibody comprises the heavy-chain amino acid sequence of the antibody shown in SEQ ID NO: 1 or any variant thereof, and the light-chain amino acid sequence of the antibody shown in SEQ ID NO: 2 or any variant thereof.

9. A pharmaceutical composition comprising the compound according to any one of claims 1-6 or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof, or the antibody-drug conjugate according to claim 7 or 8 and a pharmaceutically acceptable excipient.

10. Use of the compound according to any one of claims 1-6 or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof, or the antibody-drug conjugate according to claim 7 or 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug for inhibiting the growth of tumor cells; Preferably, the tumor cells include any one of esophageal cancer cells, breast cancer cells, gastric cancer cells, and lung cancer cells.

11. A method for treating cancer, which comprises the step of administering a therapeutically effective amount of the compound or its pharmaceutically acceptable salt, stereoisomer or prodrug according to any one of claims 1-6, or the antibody-drug conjugate according to claim 7 or 8, or the pharmaceutical composition according to claim 9 to a patient in need; Preferably, the cancer is a solid tumor; Preferably, the cancer includes any one of esophageal cancer, breast cancer, gastric cancer, and lung cancer.

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