An antibody drug conjugate intermediate comprising sn38 and a method of making the same

Abstract

The application provides an antibody drug conjugate intermediate containing SN38 and a preparation method thereof, wherein a water-soluble group PEG with biological activity is introduced on the linker structure of the antibody drug conjugate intermediate, and structural modification is performed on the PEG, the preparation method has simple steps and saves cost, no heavy metal ions are introduced in the reaction, the preparation method has higher environmental friendliness, and the antibody drug conjugate intermediate synthesized by using the preparation method has higher stability and drug efficacy compared with the antibody drug conjugate intermediate containing the toxicant SN38 in the prior art.

Description

Technical Field

[0001] This invention relates to the field of antibody-drug conjugates, and more specifically to an antibody-drug conjugate intermediate and its preparation method. Background Technology

[0002] Antibody-drug conjugates (ADCs), as a novel type of biological targeted therapy, combine the targeted action of monoclonal antibodies with the cytotoxicity of small molecule drugs, and have become one of the fastest-growing areas in targeted cancer therapy. The three components of an ADC (antibody, cytotoxic agent, and linker) together form a targeted drug delivery system. The antibody enables targeting, the linker ensures the stability of the ADC during blood transport, and upon reaching the target site, the toxin exerts its killing effect on cancer cells. Currently, more than 60 ADC drugs are undergoing clinical trials for anti-tumor therapy, the vast majority of which are microtubule inhibitors, with a smaller number being DNA inhibitors. DNA inhibitors have two advantages over microtubule inhibitors: 1) DNA inhibitors (picomolar IC50 values) have higher activity than microtubule inhibitors (sub-nanomolar IC50 values), playing a better role in treating tumors with fewer antigens; 2) They can kill cancer cells not in the dividing phase, giving them an advantage in the treatment of solid tumors.

[0003] Camptothecin compounds are topoisomerase 1 (TOP1) inhibitors used clinically, showing good efficacy against slow-growing solid tumors. However, the unique structure of camptothecin results in poor water and lipid solubility, necessitating water solubility modification. Antibody-adjuvant (ADC) drugs using camptothecin as the target offer a new solution to overcome these limitations.

[0004] Currently, two ADC drugs using camptothecin derivatives as projectiles have been approved for marketing: Enhertu (trastuzumab deruxtecan) and Trodelvy (Sacituzumab govitecan). These drugs address significant clinical needs in the treatment of tumors, particularly malignant tumors. Enhertu, developed by AstraZeneca / Daiichi Sankyo, uses a cathepsin B-activated GGFG tetrapeptide as a linker and incorporates a short self-cleaving structure, releasing the exatecan derivative Dxd. In HER2-positive metastatic breast cancer, Enhertu achieved an objective response rate of 54.5% and a disease control rate of 93.9% in 99 patients. Sacituzumab govitecan uses the Mcc-triazole spacer-PEG7-x-lysine-PABC linker, which breaks down in lysosomes (at approximately pH 5) to release camptothecin (SN38). Phase II clinical trial results for Sacituzumab govitecan in triple-negative breast cancer showed an efficacy rate of up to 30%, with tumor shrinkage in 69.5% of patients, a condition in which triple-negative breast cancer was previously considered "untreatable." Furthermore, in small cell lung cancer that had failed multiple lines of therapy, Sacituzumab govitecan reduced tumor size in 60% of patients; and in non-small cell lung cancer that had failed chemotherapy, targeted therapy, and PD-1 therapy, Sacituzumab govitecan achieved a control rate of up to 43%. However, despite its significant therapeutic effects, clinical trials also revealed serious safety concerns related to ADC toxicity, including hematologic toxicity, neurotoxicity, pulmonary toxicity, skin toxicity, hepatotoxicity, ocular toxicity, metabolic abnormalities, and cardiotoxicity. Therefore, improving the safety of ADC drugs is a crucial issue that requires significant attention and resolution in current drug development.

[0005] Furthermore, the synthesis of some ADC linkers and loads also presents the problem of difficult removal of metal ion reaction products. For example, the CL2A-SN38 used in Sacituzumab govitecan involves a click reaction during its synthesis (see Chinese Patent Application Publication No. CN102448494A, page 50, paragraph

[0273] ), which leads to the presence of Cu in the reaction product. 2+ And it is not easy to remove.

[0006] Linkers play a crucial role in the structure of ADCs (Advanced Drug Acquisition-Contractant) systems, influencing their pharmacokinetic parameters, therapeutic index, and efficacy. Linkers maintain the stability of the ADC complex in the bloodstream. Linker design must prioritize stability because ADC drugs have relatively long half-lives, requiring stable linker properties to prevent premature decomposition and toxin release in the blood. Instability in the bloodstream can lead to ADC degradation before reaching tumor cells, reducing the drug's effectiveness against tumors and potentially causing the death of other cells. Furthermore, during ADC internalization in tumor cells, the linker should rapidly release the cytotoxic drug. Therefore, linkers significantly determine the safety and efficacy of developed ADC drugs. Different linker structures also greatly influence the complexity, production cost, and environmental friendliness of ADC manufacturing processes, significantly impacting ADC drug quality, such as stability, and consequently, safety. Summary of the Invention

[0007] This invention provides an antibody-drug conjugate intermediate containing SN38 and its preparation method. The preparation method is simple, cost-effective, and does not introduce heavy metal ions during the reaction, thus exhibiting higher environmental friendliness. Furthermore, the antibody-drug conjugate intermediate synthesized using this method has good stability, thereby significantly improving the safety of ADC drugs.

[0008] This invention relates to an antibody-drug conjugate intermediate as shown in formula (I):

[0009]

[0010] in:

[0011] X1 is an alkane chain or a PEG chain.

[0012] X2 is H or -C(O)NR 1 R 2 ,and,

[0013] R 1 Selected from hydrogen, halogen, hydroxyl, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Hydroxyalkyl, substituted or unsubstituted C 1-6 Aminoalkyl, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted C 1-6 Alkyl groups and substituted or unsubstituted C 1-6 Alkyl group;

[0014] R 2 Selected from hydrogen and substituted or unsubstituted C1-6 Alkyl groups; wherein each substituted C 1-6 The alkyl group may be optionally substituted by 1 to 5 independent substituents selected from the following: halogen, hydroxyl, amino, carbonyl, carboxyl, 5-10 membered heterocyclic group and C. 1-6 Halogenated alkyl groups, wherein the 5-10 membered heterocyclic group has 1-3 heteroatoms selected from oxygen, nitrogen and sulfur.

[0015] Preferably, X1 is selected from: Wherein m is selected from 1, 2, 3, 4, 5, 6, preferably m is 5; wherein p is selected from 1, 2, 3, 4, 5, 6, preferably p is 2.

[0016] Preferably, X1 is selected from:

[0017]

[0018] Preferably, the R 1 Or R 2 Selected from: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, Cl, Br,

[0019] Preferably, X2 is:

[0020]

[0021] Preferably, the structure of the antibody-drug conjugate intermediate is shown in formulas (1)-(16):

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] The present invention also provides a method for preparing an antibody-drug conjugate intermediate.

[0028] Furthermore, the structure of the antibody-drug conjugate intermediate is as follows:

[0029]

[0030]

[0031] The R mentioned 1 Selected from hydrogen, substituted or unsubstituted C 1-6Alkyl, substituted or unsubstituted C 1-6 Hydroxyalkyl, substituted or unsubstituted C 1-6 Aminoalkyl, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted C 1-6 Alkyl groups and substituted or unsubstituted C 1-6 Alkyl group;

[0032] The R mentioned 2 Selected from hydrogen and substituted or unsubstituted C 1-6 Alkyl groups; wherein each substituted C 1-6 The alkyl group may be optionally substituted by 1 to 5 independent substituents selected from the following: halogen, hydroxyl, amino, carbonyl, carboxyl, 5-10 membered heterocyclic group and C. 1-6 Halogenated alkyl groups, wherein the 5-10 membered heterocyclic group has 1-3 heteroatoms selected from oxygen, nitrogen and sulfur.

[0033] Preferred, R 1 Or R 2 Selected from: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, methoxy, ethoxy, Cl, Br,

[0034] Furthermore, the preparation method of the compound of formula (4) is as follows:

[0035] Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, and then a reducing agent is added under low temperature conditions. After stirring for an appropriate time, the mixture is stirred at room temperature overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried, and purified.

[0036] Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter.

[0037] Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0038] Reaction D: Dissolve the product of reaction C in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with Mc-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

[0039] Reaction process 1:

[0040]

[0041] Furthermore, the preparation method of the compound of formula (12) is as follows:

[0042] Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, then placed under low temperature conditions, a reducing agent is added and stirred for an appropriate time, then placed at room temperature and stirred overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried and purified.

[0043] Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter.

[0044] Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0045] Reaction D: Dissolve the product of reaction C in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with MP2-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

[0046] Reaction process 2:

[0047]

[0048] Furthermore, the preparation method of the compound (I-1) is as follows:

[0049] Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, and then placed under low temperature conditions. A reducing agent is added and stirred for an appropriate time, and then placed at room temperature and stirred overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried and purified.

[0050] Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter.

[0051] Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0052] Reaction D: Dissolve the product of reaction C in a solvent, add di(p-nitrobenzene) carbonate and organic base, stir at a constant temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0053] Reaction E: Dissolve the product of reaction D and the amine compound in a solvent, add an organic base, stir at low temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0054] Reaction F: Dissolve the product of reaction E in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with Mc-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

[0055] Reaction process 3:

[0056]

[0057] Furthermore, the preparation method of the compound (I-2) is as follows:

[0058] Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, and then placed under low temperature conditions. A reducing agent is added and stirred for an appropriate time, and then placed at room temperature and stirred overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried and purified.

[0059] Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter.

[0060] Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0061] Reaction D: Dissolve the product of reaction C in a solvent, add di(p-nitrobenzene) carbonate and organic base, stir at a constant temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0062] Reaction E: Dissolve the product of reaction D and the amine compound in a solvent, add an organic base, stir at low temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify.

[0063] Reaction F: Dissolve the product of reaction E in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with MP2-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

[0064] Reaction process 4:

[0065]

[0066] Furthermore, the compound (I-1) is selected from:

[0067]

[0068]

[0069]

[0070] The compound (I-2) mentioned above is selected from:

[0071]

[0072]

[0073] Furthermore, the "low temperature conditions" mentioned in any of the above reaction processes refer to an ice-water bath.

[0074] Furthermore, the solvent used in any of the above reaction processes can be independently a polar solvent and / or a non-polar solvent. The polar solvent is one or more of THF, DMF, DMA, and NMP; the non-polar solvent is one or more of dichloromethane and carbon tetrachloride.

[0075] Furthermore, the organic base mentioned in any of the above reaction processes may be one or more of N,N-diisopropylethylamine, triethylamine, and pyridine, preferably one or two of N,N-diisopropylethylamine and pyridine.

[0076] Furthermore, the acid is one or two of hydrochloric acid, trifluoroacetic acid, and citric acid.

[0077] Furthermore, the amine compound is a primary amine or a secondary amine.

[0078] Furthermore, in reaction A, ethyl acetate is used for extraction, and the purification is performed by column chromatography with dichloromethane and methanol as eluents.

[0079] Furthermore, reaction B uses one or more combinations of ethyl acetate, n-hexane, and dichloromethane for pulping.

[0080] Furthermore, the reaction C is purified by column chromatography, with dichloromethane and methanol as the eluent.

[0081] Furthermore, the reaction D is purified by column chromatography, with dichloromethane and methanol as the eluent.

[0082] Furthermore, the reaction E is purified by column chromatography, with dichloromethane and methanol as the eluent.

[0083] Furthermore, the reaction F is purified using a preparative liquid phase method, with mobile phase A being MeCN and 0.1% HCOOH, and mobile phase B being H2O and 0.1% HCOOH.

[0084] Furthermore, all reactions were carried out under nitrogen protection.

[0085] The method for preparing antibody-drug conjugate intermediates (specifically, linker-SN38 covalent conjugates) provided by this invention is not only simple in preparation steps and reduces safety issues caused by heavy metal residues, but also results in antibody-drug conjugates prepared using this intermediate having higher stability in vivo.

[0086] Meanwhile, the inventors were surprised to find that the antibody-drug conjugate prepared by combining the linker in this invention with SN38 can produce a significant tumor-suppressing effect. Attached Figure Description

[0087] Figure 1 This shows the changes in mouse body weight;

[0088] Figure 2 This describes changes in tumor volume.

[0089] Figure 3 Relative tumor volume. Detailed Implementation

[0090] [abbreviation]

[0091] Unless otherwise stated, all abbreviations used in this invention have the same meaning as understood by one of ordinary skill in the art. Commonly used abbreviations and their definitions as used in this invention are as follows:

[0092]

[0093]

[0094] [definition]

[0095] Various terms relating to various aspects of the specification are used throughout the specification and claims. Unless otherwise specified, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in accordance with the definitions provided herein.

[0096] As used herein, the terms “an,” “a,” and “described” are used in accordance with standard convention and mean one or more unless the context indicates otherwise. Thus, for example, a reference to “an antibody-drug conjugate” includes a combination of two or more antibody-drug conjugates, and so on.

[0097] It should be understood that wherever the term “contains” is used to describe an aspect in this document, similar aspects described as “consisting of” and / or “substantially composed of” are also provided.

[0098] The term "antibody-drug conjugate" as used in this invention refers to a compound in which an antibody / antibody functional fragment, a linker, and a drug moiety are chemically linked together. Its structure typically consists of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker that couples the antibody or antibody-like ligand and the drug. Currently, the preparation of antibody-drug conjugates generally involves two steps: the first step is to chemically react the linker and the drug moiety to form a "linker-drug" conjugate; the second step is to covalently couple the linker moiety in the "linker-drug" conjugate with the antibody / antibody functional fragment via a thiol or amino group. The term "antibody-drug conjugate intermediate" as used in this invention refers to the aforementioned "linker-drug" conjugate. Furthermore, the term "antibody-drug conjugate intermediate" in this invention broadly refers to "linker groups" and SN38 covalent conjugates.

[0099] The "antibody-drug conjugate" used in this invention is prepared using a method commonly used in the art. For example, the preparation method of the antibody-drug conjugate used in this invention is as follows: the antibody is prepared into a solution of 10 mg / mL using PBS buffer at pH 7.4, an appropriate amount of TCEP equivalent is added, and the mixture is shaken and mixed for 1 hour. Then, 5.0 molar equivalents of the linker-toxin (i.e., the compounds shown in Formulas 1-16) are added, the mixture is shaken and mixed, and the reaction is carried out for 1 hour. After the reaction is completed, residual small molecules are removed by ultrafiltration, and the mixture is loaded into hydrophobic chromatography (HIC-HPLC) for DAR, drug distribution, and naked antibody ratio detection.

[0100] It is understood that the present invention aims to provide a new drug conjugate intermediate, and exemplary uses Heceptin to prepare multiple ADCs to verify the technical effect. The choice of antibody does not limit this patent. The structure of a specific ADC is given exemplary. In ADC-1, the linker and toxin correspond to the compound of formula (1) used and Heceptin are prepared by the general preparation method of preparing "antibody-drug conjugates" in the art (the same applies below). The linker and toxin in ADC-2 correspond to the compound of formula (2); the linker and toxin in ADC-3 correspond to the compound of formula (3); the linker and toxin in ADC-4 correspond to the compound of formula (4); the linker and toxin in ADC-5 correspond to the compound of formula (5); the linker and toxin in ADC-6 correspond to the compound of formula (6); and the linker and toxin in ADC-7 correspond to the compound of formula (6). Compound (7); the linker and toxin in ADC-8 correspond to compound (8); the linker and toxin in compound ADC-9 correspond to compound (9); the linker and toxin in compound ADC-10 correspond to compound (10); the linker and toxin in ADC-11 correspond to compound (11); the linker and toxin in ADC-12 correspond to compound (12); the linker and toxin in ADC-13 correspond to compound (13); the linker and toxin in ADC-14 correspond to compound (14); the linker and toxin in ADC-15 correspond to compound (15); the linker and toxin in ADC-16 correspond to compound (16); the linker and toxin in ADC-17 correspond to CL2A-SN38) and: where q can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] In this invention, the term "linking group" refers to a molecule having bifunctional or multifunctional groups that can react with both the protein / antibody molecule and SN38, thus acting as a "bridge" to connect the protein / antibody to SN38. The linking groups involved in this invention specifically refer to those groups containing an acyl group in their structure. [Specific Implementation Examples]

[0108] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or as recommended by the manufacturer; reagents without a specific source are commercially available, conventional reagents. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0109] Unless otherwise defined, all technical and scientific terms used herein are to have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0110] Example 1: Preparation method of compound (12)

[0111]

[0112] 1) Synthetic method of compound c (2-Boc-N-methylethyl ethylene glycol)

[0113] 0.6 g of compound a (aminoethylene glycol) and 1.0 g of compound b (N-Boc-acetaldehyde) were dissolved in 20 mL of methanol and stirred at room temperature for 6 h. The reaction mixture was then placed in an ice-water bath, sodium triacetoxyborohydride was added, and the mixture was stirred in the ice-water bath for 1 h, then returned to room temperature and stirred overnight. LC-MS analysis showed no UV absorption in the product. The solvent was evaporated to dryness, and the product was extracted twice with ethyl acetate, dried over anhydrous Na₂SO₄, and purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent. After washing, 1.0 g of the product was obtained, with a yield of 66%. LC-MS: (M+H) + 262.6.

[0114] 2) Synthesis of SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0115] 1.0 g of SN38 (camptothecin) and 1.6 g of DNPC (di(p-nitrobenzene) carbonate) were dissolved in 100 mL of tetrahydrofuran, and 2 mL of triethylamine was added. The mixture was stirred at room temperature for 1.5 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was slurried with ethyl acetate:n-hexane = 20 mL:100 mL. The solid was filtered, slurried with 20 mL of dichloromethane, filtered, and the product was collected by LC-MS to obtain 700 mg of SN38-PNP solid, with a yield of 49.3%. LC-MS: (M+H) + 557.4.

[0116] 3) Synthesis method of compound d (Boc-N-methylethyldiol-camptothecin)

[0117] 250 mg of SN38-PNP was dissolved in 30 mL of N,N-dimethylformamide, followed by the addition of 235 mg of Boc-DMEDA-PEG and 233 μL of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 16 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and the product was purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent. The product was collected by LC-MS to yield 213 mg of Boc-DMEDA-PEG-SN38 solid. The yield was 70%. LC-MS: (M+H) + 680.5.

[0118] 4) Synthesis of compound g (10-(2-N-methylethyl ethylene glycol)-camptothecin trifluoroacetate)

[0119] Compound d 73.0 mg was dissolved in 3 mL of dichloromethane and 3 mL of trifluoroacetic acid, and the solution was placed in an ice-water bath and stirred at a constant temperature for 1 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and then 2 mL of toluene and 2 mL of dichloromethane were added. The mixture was evaporated to dryness twice. The product was collected by LC-MS to obtain SN38-DMEDA·TFA, which was directly added to the next reaction step. LC-MS: (M+H) + 580.6.

[0120] 5) Synthetic method of compound (maleimide diethoxy-L-valine-L-citrulline-p-aminobenzyl-(2-N-methylethyl ethylene glycol)-camptothecin) of formula (12)

[0121] 50 mg of MP2-VC-PAB-PNP was dissolved in 4 mL of N,N-dimethylformamide and kept at a constant temperature in an ice-water bath under nitrogen protection. 38 mg of the compound was added, followed by 42 μL of triethylamine. The mixture was stirred at a constant temperature for 0.5 h, then allowed to return to room temperature for another 0.5 h. The reaction was confirmed to be complete by LC-MS. After the reaction was complete, the solvent was evaporated at low temperature, and the solution was purified by preparative liquid chromatography using a Sun column. Prep C18 OBD™ 5µm, 19*250mm Column; Mobile phase A: MeCN, Mobile phase B: H2O. LC-MS was used to collect 18mg of the pure product as a solid of compound (12). Yield 22%. LC-MS: (M+H) + 1197.2.

[0122] Example 2 Preparation method of compound (4)

[0123]

[0124] Compound g (10-(2-N-methylethyl ethylene glycol)-camptothecin trifluoroacetate) was prepared according to the synthetic route of Example 1: Compound d (73.0 mg) was dissolved in 3 mL of dichloromethane and 3 mL of trifluoroacetic acid, and the mixture was placed in an ice-water bath and stirred at a constant temperature for 1 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and then 2 mL of dichloromethane was added, evaporated to dryness twice, and the product was collected by LC-MS to obtain compound g (70 mg), which was directly added to the next reaction step. LC-MS: (M+H) + 580.6.

[0125] Compound (maleimide hexanoic acid-L-valine-L-citrulline-p-aminobenzyl-(2-N-methylethyl ethylene glycol)-camptothecin) was prepared according to the synthetic route of Example 1: Mc-VC-PAB-PNP (maleimide hexanoic acid-L-valine-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) (58 mg) and compound g (42 mg) were dissolved in 2.5 mL of N,N-dimethylformamide, stirred in an ice-water bath for 30 mins, and then DIPEA (42 mg) was added dropwise, and stirred in an ice-water bath for 1 h. The reaction was not complete as detected by LC-MS. Triethylamine (10 mg) was added, and the mixture was stirred at room temperature for 1 h. The reaction solution turned yellow, and the reaction was complete as detected by LC-MS. After the reaction was complete, the solvent was evaporated to dryness at low temperature, and the solution was purified by preparative liquid chromatography. Preparative column: Sun PrepC18OBD™ 5µm, 19*250mm Column; Mobile phase A: MeCN, 0.1% HCOOH; Mobile phase B: H2O, 0.1% HCOOH. LC-MS was used to collect the pure product to obtain 34 mg of solid compound (4), with a yield of 40.5%. LC-MS: (M+H) + 1179.2.

[0126] Example 3 Preparation method of compound (1)

[0127]

[0128] 1) Synthetic method of compound c (2-Boc-N-methylethyl ethylene glycol)

[0129] 0.6 g of compound a (aminoethylene glycol) and 1.0 g of compound b (N-Boc-(methylamino)acetaldehyde) were dissolved in 20 mL of methanol and stirred at room temperature for 6 h. The reaction mixture was then placed in an ice-water bath, sodium triacetoxyborohydride was added, and the mixture was stirred in the ice-water bath for 1 h, then returned to room temperature and stirred overnight. LC-MS analysis showed no UV absorption in the product. The solvent was evaporated to dryness, and the product was extracted twice with ethyl acetate. Anhydrous Na₂SO₄ was added to precipitate the product. The organic phase was discarded, dried, and evaporated to dryness. Purification was performed by column chromatography using dichloromethane:methanol = 20:1 as the eluent. After washing, 1.0 g of the product was obtained, with a yield of 66%. LC-MS: (M+H) + 262.6.

[0130] 2) Synthesis of SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0131] 1.0 g of SN38 and 1.6 g of DNPC (di(p-nitrobenzene) carbonate) were dissolved in 100 mL of tetrahydrofuran, and 2 mL of triethylamine were added. The mixture was stirred at room temperature for 1.5 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was slurried with ethyl acetate:n-hexane = 20 mL:100 mL. The solid was filtered, slurried with 20 mL of dichloromethane, filtered, and the product was collected by LC-MS to obtain 700 mg of SN38-PNP solid, with a yield of 49.3%. LC-MS: (M+H) + 557.4.

[0132] 3) Synthesis method of compound d (Boc-N-methylethyldiol-camptothecin)

[0133] 500 mg of SN38-PNP was dissolved in 30 mL of N,N-dimethylformamide, and 370 mg of compound c and 233 μL of N,N-diisopropylethylamine were added. The mixture was stirred at room temperature for 3 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and the product was purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent. The product was collected as a solid of compound d by LC-MS, yielding 250 mg. The yield was 45%. LC-MS: (M+H) + 680.5.

[0134] 4) Synthesis method of compound h (Boc-N-methylethyldiethoxy-p-nitro active ester-camptothecin)

[0135] Compound d (220.0 mg) was dissolved in 5 mL of dichloromethane, and 150 mg of di(p-nitrobenzene) carbonate and 86 mg of DIPEA were added. The mixture was stirred at a constant temperature for 16 h. The reaction was confirmed to be complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and the product was purified by column chromatography using dichloromethane:methanol = 50:1 as the eluent. 220 mg of compound h (68.5%) was collected as a solid and directly added to the next reaction step. LC-MS: (M+H) + 845.6.

[0136] 5) Synthetic method of compound i (Boc-N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin)

[0137] 220 mg of compound h (Boc-N-methylethyldiethoxy-p-nitro active ester-camptothecin) and 50 mg of N,N,N-trimethylethylenediamine were dissolved in 5 mL of N,N-dimethylformamide, followed by dropwise addition of DIPEA (65 mg). The mixture was stirred in an ice-water bath for 1 h. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was purified by column chromatography using dichloromethane:methanol = 20:1 as the eluent. 157 mg of compound i (75% yield) was collected and directly added to the next reaction step. LC-MS: (M+H) + 808.6.

[0138] 6) Synthetic method of compound j (N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin-trifluoroacetate)

[0139] 157.0 mg of compound i (Boc-N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin) was dissolved in 3 mL of dichloromethane and 1.3 mL of trifluoroacetic acid, and the solution was placed in an ice-water bath and stirred for 1 h. The reaction was considered complete by LC-MS. After the reaction was complete, the solvent was evaporated to dryness, and then 2 mL of dichloromethane was added. The solution was evaporated to dryness twice to obtain 120 mg of compound j, which was directly added to the next reaction step. LC-MS: (M+H) + 708.3.

[0140] 7) Synthetic method of compound (maleimide hexanoic acid-L-valine-L-citrulline-p-aminobenzyl-(N-methylethyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin) of formula (1)

[0141] 52 mg of Mc-VC-PAB-PNP (maleimide hexanoic acid-L-valine-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) and 35 mg of compound j were dissolved in 2.5 mL of N,N-dimethylformamide and stirred in an ice-water bath for 30 mins. 129 mg of DIPEA was then added dropwise, and the mixture was stirred in an ice-water bath for 0.5 h. LC-MS analysis showed that the reaction was incomplete. 129 mg of DIPEA was added again, and the mixture was stirred at room temperature for 0.5 h. The reaction solution turned yellow, and LC-MS analysis confirmed that the reaction was complete. After the reaction was complete, the solvent was evaporated to dryness at low temperature, and the solution was purified by preparative liquid chromatography. Preparative column: Sun Prep C18OBD™ 5um, 19*250mm Column; Mobile phase A: MeCN, 0.1% HCOOH, Mobile phase B: H2O, 0.1% HCOOH. 20 mg of the pure product was collected by LC-MS monitoring to obtain compound (1) solid, with a yield of 20.5%. LC-MS: (M+H) + 1307.6.

[0142] Example 4: Preparation method of compound (9)

[0143]

[0144] Compound (maleimide diethoxy-L-valine-L-citrulline-p-aminobenzyl-(N-methylethyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin) of formula (9) was prepared according to the synthetic route of Example 3: MP2-VC-PAB-PNP (maleimide diethoxy-L-valine-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) (52 mg) and compound j (34 mg) were dissolved in 2.5 mL of N,N-dimethylformamide, stirred in an ice-water bath for 30 mins, and DIPEA (36 mg) was added dropwise, and stirred in an ice-water bath for 0.5 h. The reaction was not completed by LC-MS. DIPEA (8 mg) was added and stirred at room temperature for 1.5 h. The reaction solution turned yellow, and the reaction was completed by LC-MS. After the reaction was completed, the solvent was evaporated at low temperature and purified by preparative liquid chromatography. Column: Sun Prep C18OBD™ 5µm, 19*250mm Column; Mobile phase A: MeCN, 0.1% HCOOH; Mobile phase B: H2O, 0.1% HCOOH. LC-MS analysis yielded 23 mg of the pure compound (9) as solid, with a yield of 35.2%. LC-MS: (M+H) + 1325.4.

[0145] Example 5: Inhibitory effect of ADC-1 on NCI-N87 cells

[0146] The compound of formula (1) and the CL2A-SN38 intermediate were conjugated with Heceptin using conventional methods in the art (see, for example, Example 12 in Chinese Patent Application Publication No. CN102448494A) to prepare ADC-1 and HER2-CL2A-SN38 (ADC-17) antibody-drug conjugates with an average DAR value of 8. NCI-N87 cells were trypsinized and the cell density was adjusted to 50,000 cells / ml. 100 μL / well was added to cell culture plates and incubated at 37°C in a 5% CO2 incubator for 14–20 h. The sample group and control group (test sample components are shown in Table 1) were serially diluted with basal medium and transferred to cell culture plates containing cells at 100 μL / well; incubated at 37°C in a 5% CO2 incubator for 70–74 h. Dilute CCK-8 10 times with culture medium, remove the old culture medium from the 96-well plate, add 100 μL of diluted CCK-8 solution to each well, develop color under 5% CO2 conditions for 2-4 hours, centrifuge to remove air bubbles, and read the results on a microplate reader at a wavelength of 450 nm / 655 nm. The results are shown in Table 2.

[0147] Table 1. Composition of the test sample

[0148] Serial Number Test sample Average DAR Sample 1 ADC-1 8 Control group 1 ADC-17 8 Control group 2 HER2 naked antibody /

[0149] Table 2 Inhibitory effect on NCI-N87 cells

[0150] Serial Number Test sample IC50 (ng / ml) Sample 1 ADC-1 251 Control group 1 ADC-17 210 Control group 2 HER2 naked antibody NA

[0151] Experimental results showed that the inhibitory effects of the ADC-1 group and the ADC-17 group on NCI-N87 cells were comparable.

[0152] Example 6: Stability Experiment Study

[0153] 1) Accurately weigh compound (1) (2.1 mg) and CL2A-SN38 (2.1 mg) and dissolve them in 700 μL of DMSO respectively;

[0154] 2) Prepare 0.2M PB buffer solutions with pH values ​​of 6, 7, and 8 respectively;

[0155] 3) Take 50 μL of DMSO solution of compound (1) into a centrifuge tube, add 950 μL of PB (0.2 M) buffer solution with pH = 6, and the compound concentration is 60 μg / mL. Place in a constant temperature system at 37℃; Take 50 μL of DMSO solution of CL2A-SN38 into a centrifuge tube, add 950 μL of PB (0.2 M) buffer solution with pH = 6, and the compound concentration is 60 μg / mL. Place in a constant temperature system at 37℃;

[0156] 4) Take 40 μL samples at 0h, 2h, 4h, 6h, 24h and 48h respectively, add 120 μL of acetonitrile, centrifuge, and send the supernatant to LC-MS for quantitative detection.

[0157] 5) Result Processing:

[0158] Using the LC-MS concentration at T=0h as the baseline, the data = T / T0*100%, and the results are shown in Tables 3 and 4, respectively.

[0159] Table 3 shows the compounds in formula (1).

[0160] time pH=6 pH=7 pH=8 0 1 1 1 2 0.988606 0.878866 0.56405 4 0.97612 0.801849 0.284131 6 0.92084 0.821934 0.123407 24 0.86821 0.256709 0.009607 48 0.78862 0.122173 0.012562

[0161] At pH 6, 21.2% of compound (1) decomposed after 48 hours;

[0162] At pH 7, 87.8% of the compound of formula (1) decomposed after 48 hours;

[0163] At pH 8, 98.7% of compound (1) decomposed after 48 hours.

[0164] Table 4 CL2A-SN38

[0165]

[0166]

[0167] At pH 6, 98.0% of CL2A-SN38 was decomposed after 48 hours;

[0168] At pH 7, 98.1% of CL2A-SN38 was decomposed after 48 hours;

[0169] At pH 8, CL2A-SN38 decomposed by 98.1% after 48 hours.

[0170] From the results in Tables 3 and 4, it can be seen that:

[0171] 1) Compound of formula (1) is more stable than CL2A-SN38 under weakly acidic conditions.

[0172] 2) Compound (1) under neutral conditions decomposed 18% in 6 hours and 75% in 24 hours. CL2A-SN38 under neutral conditions decomposed 43% in 6 hours and 98% in 24 hours.

[0173] 3) Compound (1) under alkaline conditions decomposed by 97% in 24 hours, while CL2A-SN38 decomposed by 98% in 24 hours.

[0174] Therefore, the compound of formula (1) prepared by the present invention has higher stability in vivo under weakly acidic or neutral conditions compared with CL2A-SN38, and is expected to significantly improve the safety of ADC drugs.

[0175] Example 7: Evaluation of the inhibitory effect of anti-Her2-drug conjugate on the development of in situ breast cancer

[0176] 1. Experimental Methods

[0177] After resuscitation, BT474 cell lines were passaged 1-2 times. Once cell growth stabilized, the cells were expanded for culture, and tumor cell suspensions were prepared. 0.2 x 10⁻⁶ cells were then inoculated onto the milk fat pads of nude mice. 7 One BT474 cell line was inoculated into 60 female BALB / c nude mice. The average tumor volume reached approximately 70 mm. 3 Forty-two animals were selected and randomly divided into six groups according to tumor volume: the negative control group consisted of control group 1 (saline group), control group 2 (ADC-17), experimental group 1 (ADC-4), experimental group 2 (ADC-12), experimental group 3 (ADC-1), and experimental group 4 (ADC-9), with seven animals in each group. After successful model establishment, control group 1 was given intravenous saline, while the other groups were given intravenous drugs at the following doses: control group 2 5 mg / kg, experimental group 1 5 mg / kg, experimental group 2 5 mg / kg, experimental group 3 5 mg / kg, and experimental group 4 5 mg / kg, once a week for 21 weeks. Tumor volume was weighed and measured three times a week, with the final measurement taken on day 21.

[0178] 2. Experimental Results and Analysis

[0179] 1) Changes in mouse body weight:

[0180] Table 5. Weight of mice

[0181]

[0182] No mice died during the experiment. At the end of the experiment, compared with control group 1, control group 2...

[0183] (ADC-17 group), Experimental group 1 (ADC-4), Experimental group 2 (ADC-12), Experimental group 3

[0184] There was no significant difference in body weight between group (ADC-1) and experimental group 4 (ADC-9), as detailed in Table 5. Figure 1 .

[0185] 2) Tumor volume in mice:

[0186] Table 6. Tumor volume in mice

[0187]

[0188] Note: Compared with the control group (saline group), each group, " * "Indicates P < 0.05;" ** " indicates P < 0.01;

[0189] Compared with control group 2, each group, # " indicates P < 0.05, ## "" indicates that P < 0.01

[0190] Twenty-one days after drug administration, the tumor volume in control group 1 reached 107 ± 19.30 mm. 3 The tumor volumes of the five groups (control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4) were 49.17±13.29, 57.12±20.28, 66.26±21.37, 61.32±12.82, 75.76±46.18, and 75.19±25.99 mm, respectively. 3 (See Table 6) The differences between control group 2, experimental group 1, experimental group 2, and experimental group 3 and control group 1 (saline group) were statistically significant (P < 0.01), and the difference between experimental group 4 and control group 1 was statistically significant (P < 0.05). There were no statistically significant differences in tumor volume between experimental group 1, experimental group 2, and experimental group 3 and control group 2 (P > 0.05), and their relative tumor volumes were also not significantly different (P > 0.05), indicating comparable efficacy. The relative tumor proliferation rates (T / C) of control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4 were 49.30%, 55.00%, 62.58%, 62.48%, 67.19%, and 63.20%, respectively. The data above show that the four antibody-drug conjugates in control group 2, experimental group 1, experimental group 2, and experimental group 3 all significantly inhibited BT474 tumor growth with comparable effects, while experimental group 4 also inhibited BT474 tumor growth. The changes in tumor volume and relative tumor volume in each group during the experiment are shown below. Figure 2 and Figure 3 As shown.

[0191] In summary, the ADCs constructed using the drug conjugate intermediates of this invention showed significant antitumor activity against the BT474 breast cancer cell model. The preferred ADCs-1, ADC-4, and ADC-12 have significant advantages and are expected to significantly improve safety under in vivo conditions while also achieving tumor inhibition effects comparable to those of the ADC-17 group.

[0192] This invention has been illustrated through various specific embodiments. However, those skilled in the art will understand that this invention is not limited to the specific embodiments, and various modifications or variations can be made within the scope of this invention. Furthermore, the various technical features mentioned throughout this specification can be combined with each other without departing from the spirit and scope of this invention. Such modifications and variations are all within the scope of this invention.

Claims

1. An antibody-drug conjugate intermediate as shown in formula (Ⅰ): （Ⅰ） in: X1 is The value of m is 1, 2, 3, 4, 5, or 6. X2 is H or -C(O)NR 1 R 2 ,and, R 1 Selected from substituted or unsubstituted C 1-6 aminoalkyl; R 2 Selected from non-substituted C 1-6 alkyl.

2. The antibody-drug conjugate intermediate according to claim 1, characterized in that, The X1 mentioned is selected from: 。 3. The antibody-drug conjugate intermediate according to claim 1, characterized in that, The R mentioned 1 Selected from R 2 Selected from: methyl, ethyl, propyl, butyl, pentyl.

4. The antibody-drug conjugate intermediate according to any one of claims 1-3, characterized in that, The X2 mentioned is selected from: H or .

5. The antibody-drug conjugate intermediate according to claim 1, characterized in that: The structure of the antibody-drug conjugate intermediate is shown in formula (1) or (4): （1）， （4）。 6. A method for preparing an antibody-drug conjugate intermediate, wherein the structure of the antibody-drug conjugate intermediate is shown in formula (4) or (I-1): (4), or （Ⅰ-1）， The R mentioned 1 Selected from substituted or unsubstituted C 1-6 aminoalkyl; The R mentioned 2 Selected from non-substituted C 1-6 alkyl; The preparation method is selected from the following reaction processes: Reaction process 1: ,or Reaction process 3: 。 7. The preparation method according to claim 6, characterized in that, The R mentioned 1 for The R mentioned above 2 Selected from: methyl, ethyl, propyl, butyl, pentyl.

8. The preparation method according to claim 6, characterized in that, The structure of the aforementioned formula (Ⅰ-1) is shown in formula (1): （1）。 9. The preparation method according to claim 6, characterized in that, The reaction process 1 includes the following conditions: Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, and then a reducing agent is added under low temperature conditions. After stirring for an appropriate time, the mixture is stirred at room temperature overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried, and purified. Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter. Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify. Reaction D: Dissolve the product of reaction C in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with Mc-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

10. The preparation method according to claim 6, characterized in that, The reaction process 3 includes the following operating conditions: Reaction A: Compounds a and b are dissolved in a solvent, stirred at room temperature for an appropriate time, and then placed under low temperature conditions. A reducing agent is added and stirred for an appropriate time, and then placed at room temperature and stirred overnight. After the reaction is completed, the solvent is evaporated and the mixture is extracted, dried and purified. Reaction B: Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and then pulp and filter. Reaction C: Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify. Reaction D: Dissolve the product of reaction C in a solvent, add di(p-nitrobenzene) carbonate and an organic base, stir at a constant temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify. Reaction E: Dissolve the product of reaction D and the amine compound in a solvent, add an organic base, stir at low temperature for an appropriate time, and after the reaction is complete, evaporate the solvent and purify. Reaction F: Dissolve the product of reaction E in a solvent, add acid, stir at low temperature for an appropriate time, after the reaction is complete, evaporate the solvent and dissolve it together with Mc-VC-PAB-PNP in a solvent, stir at low temperature for an appropriate time, add organic base, after the reaction is complete, evaporate the solvent and purify.

11. The preparation method according to any one of claims 9-10, wherein the low temperature condition is an ice-water bath.

12. The preparation method according to any one of claims 9-10, characterized in that... The solvent is a polar solvent and / or a non-polar solvent. The polar solvent is one or more of THF, DMF, DMA, and NMP; the non-polar solvent is one or more of dichloromethane and carbon tetrachloride.

13. The preparation method according to any one of claims 9-10, characterized in that... The organic base is one or more of N,N-diisopropylethylamine, triethylamine, and pyridine.

14. The preparation method according to claim 13, characterized in that, The organic base is selected from one or two of N,N-diisopropylethylamine and pyridine.

15. The preparation method according to any one of claims 9-10, characterized in that... The acid mentioned is one or two of hydrochloric acid, trifluoroacetic acid, and citric acid.

16. The preparation method according to claim 10, characterized in that... The amine compounds mentioned are primary or secondary amines.

17. The preparation method according to any one of claims 9-10, characterized in that... In reaction A, ethyl acetate was used for extraction, and the purification was performed by column chromatography with dichloromethane and methanol as eluents.

18. The preparation method according to any one of claims 9-10, characterized in that... In reaction B, one or more of ethyl acetate, n-hexane, and dichloromethane are used for pulping.

19. The preparation method according to any one of claims 9-10, characterized in that... The reaction C described herein is purified by column chromatography, with dichloromethane and methanol as the eluents.

20. The preparation method according to any one of claims 9-10, characterized in that... The reaction D was purified by column chromatography, with dichloromethane and methanol as the eluents.

21. The preparation method according to claim 10, characterized in that... The reaction E was purified by column chromatography, with dichloromethane and methanol as the eluents.

22. The preparation method according to claim 10, characterized in that... The reaction F was purified using a preparative liquid phase method. Mobile phase A was MeCN, 0.1% HCOOH, and mobile phase B was H2O, 0.1% HCOOH.

23. The preparation method according to any one of claims 6-10, characterized in that, All reactions were carried out under nitrogen protection.

24. An antibody-drug conjugate, the structure of which is shown below: in: X1 is The value of m is 1, 2, 3, 4, 5, or 6. X2 is H or -C(O)NR 1 R 2 ,and, R 1 Selected from substituted or unsubstituted C 1-6 aminoalkyl; R 2 Selected from non-substituted C 1-6 alkyl; Ab represents antibodies; q is selected from 1, 2, 3, 4, 5, 6, 7, 8.

25. The antibody-drug conjugate according to claim 24, characterized in that, The X1 mentioned is selected from: 。 26. The antibody-drug conjugate according to claim 25, characterized in that, The R mentioned 1 for ;R 2 Selected from: methyl, ethyl, propyl, butyl, pentyl.

27. The antibody-drug conjugate according to any one of claims 24-26, characterized in that, The X2 mentioned is selected from: H or .

28. The antibody-drug conjugate according to claim 26, characterized in that, The structure of the antibody-drug conjugate is shown in formula ADC-1 or ADC-4: ADC-1, ADC-4。 29. Use of the antibody-drug conjugate intermediate according to any one of claims 1-5 in the preparation of antibody-drug conjugates.

30. Use of the antibody-drug conjugate according to any one of claims 27-28 in the preparation of a drug for treating tumors.

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