Intermediate for antibody-drug conjugate containing SN38 and method for preparing same

Abstract

The present invention provides an intermediate of an antibody-drug conjugate containing SN38, in which a biologically active water-soluble group PEG is introduced into the linker and the PEG is structurally modified, and a method for preparing the same. This preparation method has a simple procedure, is low cost, and is more environmentally friendly since it does not introduce heavy metal ions during the reaction. Furthermore, the intermediate of the antibody-drug conjugate synthesized by this preparation method has higher stability and efficacy than the intermediate of the antibody-drug conjugate using SN38 as a toxin in the conventional technology.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody-drug conjugates, and in particular to intermediates of antibody-drug conjugates and methods for their preparation. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are novel biological targeting agents that combine the potent targeting effect of monoclonal antibodies with the cytotoxicity of small-molecule drugs, making them one of the fastest-growing fields in tumor targeting therapy. The three components of an ADC—antibody, cytotoxin, and linker—combine to form a targeted drug delivery system. The antibody achieves targeting, the linker ensures the stability of the ADC during blood transport, and the toxin exerts its killing effect on cancer cells after reaching the target site. Currently, there are over 60 ADCs in clinical trials for antitumor therapy. Most toxins are tubulin inhibitors, with a minority being DNA inhibitors. DNA inhibitors have two advantages over tubulin inhibitors: 1) DNA inhibitors are more active than tubulin inhibitors (picomolar IC 50 values), making them more effective in treating antigen-poor tumors; and 2) they can kill non-mitotic cancer cells, making them advantageous for treating solid tumors.

[0003] Camptothecin-based compounds are clinically used topoisomerase 1 (TOP1) inhibitors and have shown good clinical efficacy against slow-growing solid tumors. However, the unique structure of camptothecin leads to poor water and lipid solubility, so water solubility needs to be improved. ADC drugs using camptothecin as a "warhead" offer a novel solution to these drawbacks.

[0004] Currently, two camptothecin derivative-based ADC drugs, Enhertu (trastuzumab deruxtecan) and Trodervy (sacituzumab govitecan), have been approved. These drugs address clinical needs in the treatment of oncology, particularly malignant tumors. Enhertu, developed by AstraZeneca / Daiichi Sankyo, uses a cathepsin B-activated GGF-G tetrapeptide as a linker, introducing a small self-cleaving moiety to release Dxd, an exatecan derivative. Ninety-nine patients treated with Enhertu for HER2-positive metastatic breast cancer achieved an objective remission rate of 54.5% and a disease control rate of 93.9%. Sacituzumab govitecan uses an Mcc-triazole spacer-PEG7-x-lysine-PABC linker, which degrades in intracellular lysosomes (pH approximately 5) to release camptothecin (SN38). Triple-negative breast cancer is clinically considered virtually incurable. However, a Phase II clinical trial of sacituzumab govitecan for triple-negative breast cancer demonstrated a high efficacy rate of 30%, with tumor shrinkage observed in 69.5% of patients. Furthermore, sacituzumab govitecan was able to shrink tumors in 60% of patients with small cell lung cancer who had failed multiple treatments. In non-small cell lung cancer patients who had failed chemotherapy, molecular targeted therapy, and PD-1 therapy, sacituzumab govitecan achieved a high control rate of 43%. However, despite significant therapeutic efficacy in clinical trials, serious safety issues remain due to ADC toxicity, including hematologic, neurotoxicity, pulmonary, cutaneous, hepatotoxic, ocular, metabolic, and cardiac toxicity. Therefore, improving the safety of ADC drugs is a major concern in current drug development and must be addressed.

[0005] In addition, the synthesis of linkers and loadings for some ADCs presents the problem of difficulty in removing the metal ion reaction products. For example, CL2A-SN38, used in sacituzumab govitecan, involves a click chemistry reaction (click reaction) during synthesis (see paragraph

[0273] on page 50 of the specification of Chinese Patent Application Publication No. CN102448494A). 2+ It contains and cannot be easily removed.

[0006] The linker plays a crucial role in the ADC structure, affecting its pharmacokinetic parameters, therapeutic index, and efficacy. The linker can maintain the stability of the ADC conjugate in the bloodstream. Stability must be considered when designing a linker. Because ADC drugs have long half-lives, linker stability is essential to prevent the linker from being degraded in the blood and releasing toxins. If the linker is not stable enough in the blood, the ADC may be degraded before entering tumor cells, resulting in reduced efficacy against tumors and even the accidental killing of other cells. Furthermore, the linker must be able to rapidly release the cytotoxic drug during the ADC's internalization into tumor cells. Thus, the linker plays a crucial role in the safety and efficacy of the developed ADC drug. Different linker structures significantly affect the ease, cost, and environmental friendliness of the ADC preparation process, as well as the quality of the ADC drug, such as its stability, which in turn affects the safety of the ADC drug. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an antibody-drug conjugate intermediate containing SN38 and a method for preparing the same. This preparation method involves simple steps, is inexpensive, does not introduce heavy metal ions during the reaction, and is environmentally friendly. Furthermore, the antibody-drug conjugate intermediate synthesized by this preparation method has good stability, significantly improving the safety of ADC drugs. [Means for solving the problem]

[0008] The present invention relates to an intermediate of an antibody-drug conjugate represented by formula (I). [ka] During the ceremony: X1 is an alkane chain or a PEG chain, X2 is H or -C(O)NR 1 R 2 and R 1 is hydrogen, halogen, hydroxy, substituted or unsubstituted C 1-6 Alkyl groups, substituted or unsubstituted C 1-6 Hydroxyalkyl groups, substituted or unsubstituted C 1-6 Aminoalkyl groups, substituted or unsubstituted C 1-6 Alkoxyl group, substituted or unsubstituted C 1-6 Alkyl acyl groups and substituted or unsubstituted C 1-6 alkylaldehyde groups; R 2 are hydrogen, or are unsubstituted or each independently selected from halogen, hydroxy, amino, carbonyl, carboxyl, 5- to 10-membered heterocyclyl groups (wherein the 5- to 10-membered heterocyclyl groups have 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C 1-6 C optionally substituted with 1 to 5 substituents selected from the group consisting of haloalkyl groups 1-6 It is an alkyl group.

[0009] Preferably, X1 is [ka] wherein m is selected from 1, 2, 3, 4, 5, and 6, and is preferably 5; and p is selected from 1, 2, 3, 4, 5, and 6, and is preferably 2.

[0010] Preferably, X1 is [ka] is selected from.

[0011] Preferably, the above R 1 or R 2 is H, methyl group, ethyl group, propyl group, butyl group, pentyl group, heptyl group, Cl, Br, [ka] is.

[0012] Preferably, X2 is selected from the following groups: [ka]

[0013] Preferably, the structure of the intermediate of the antibody-drug conjugate is represented by the following formulas (1)-(16): [ka] [ka] [ka] [ka]

[0014] The present invention also provides methods for preparing intermediates of antibody-drug conjugates.

[0015] Furthermore, the intermediate of the antibody-drug conjugate has the following structure: [ka]

[0016] Above R1 is hydrogen, substituted or unsubstituted C 1-6 Alkyl groups, substituted or unsubstituted C 1-6 Hydroxyalkyl groups, substituted or unsubstituted C 1-6 Aminoalkyl groups, substituted or unsubstituted C 1-6 Alkoxyl group, substituted or unsubstituted C 1-6 Alkyl acyl groups, and substituted or unsubstituted C 1-6 alkylaldehyde groups.

[0017] Above R 2 are hydrogen, or are unsubstituted or each independently selected from halogen, hydroxyl, amino, carbonyl, carboxyl, 5- to 10-membered heterocyclyl groups (wherein the 5- to 10-membered heterocyclyl groups have 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C 1-6 C optionally substituted with 1 to 5 substituents selected from the group consisting of haloalkyl groups 1-6 It is an alkyl group.

[0018] Preferably, R 1 or R 2 is H, methyl group, ethyl group, propyl group, butyl group, pentyl group, heptyl group, methoxyl group, ethoxyl group, Cl, Br, [ka] is.

[0019] Furthermore, the compound of formula (4) can be prepared by the following reaction scheme 1: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added at a low temperature, stirred for an appropriate time, and then stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolve the product obtained in Reaction B in a solvent, add the product obtained in Reaction A and an organic alkali, and stir at room temperature for an appropriate time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction D: Dissolving the product obtained in Reaction C in a solvent, adding an acid, stirring at low temperature for a suitable time, and then rotating the mixture to remove the solvent after the reaction is complete; dissolving the product together with Mc-VC-PAB-PNP in a solvent, stirring at low temperature for a suitable time, adding an organic alkali, and then rotating the mixture to remove the solvent after the reaction is complete, and then purifying the product.

[0020] Reaction 1: [ka]

[0021] Furthermore, the compound of formula (12) can be prepared by the following reaction process 2: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added at a low temperature, stirred for an appropriate time, and then stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolve the product obtained in Reaction B in a solvent, add the product obtained in Reaction A and an organic alkali, and stir at room temperature for an appropriate time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction D: The product obtained in Reaction C is dissolved in a solvent, an acid is added, and the mixture is stirred at low temperature for a suitable period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then the mixture is dissolved in a solvent together with MP2-VC-PAB-PNP, and the mixture is stirred at low temperature for a suitable period of time. An organic alkali is added, and after the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified.

[0022] Reaction Step 2: [ka]

[0023] Furthermore, the compound (I-1) can be prepared by the following reaction scheme 3: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added at a low temperature, and then stirred for an appropriate time, and then stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolve the product obtained in Reaction B in a solvent, add the product obtained in Reaction A and an organic alkali, and stir at room temperature for an appropriate time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction D: Dissolve the product obtained in Reaction C in a solvent, add bis(4-nitrophenyl)carbonate and an organic alkali, and stir at a fixed temperature for a suitable time. After the reaction is completed, remove the solvent by rotary drying, and then purify. Reaction E: The product obtained in Reaction D and an amine compound are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at a low temperature for an appropriate period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction F: The product obtained in Reaction E is dissolved in a solvent, an acid is added, the mixture is stirred at a low temperature for an appropriate period of time, and after the reaction is completed, the mixture is rotary dried to remove the solvent, and then the mixture is dissolved together with Mc-VC-PAB-PNP in a solvent, the mixture is stirred at a low temperature for an appropriate period of time, an organic alkali is added, and after the reaction is completed, the mixture is rotary dried to remove the solvent, and then purification is carried out.

[0024] Reaction Step 3: [ka]

[0025] Furthermore, the compound (I-2) can be prepared by the following reaction scheme 4: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added at a low temperature, and then stirred for an appropriate time, and then stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolve the product obtained in Reaction B in a solvent, add the product obtained in Reaction A and an organic alkali, and stir at room temperature for an appropriate time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction D: Dissolve the product obtained in Reaction C in a solvent, add bis(4-nitrophenyl)carbonate and an organic alkali, and stir at a fixed temperature for a suitable time. After the reaction is completed, remove the solvent by rotary drying, and then purify. Reaction E: The product obtained in Reaction D and an amine compound are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at a low temperature for an appropriate period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. Reaction F: The product obtained in Reaction E is dissolved in a solvent, an acid is added, and the mixture is stirred at a low temperature for a suitable period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then the mixture is dissolved together with MP2-VC-PAB-PNP in a solvent, the mixture is stirred at a low temperature for a suitable period of time, an organic alkali is added, and after the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified.

[0026] Reaction Step 4: [ka]

[0027] Furthermore, the compound (I-1) is [ka] [ka] is selected from. The compound (I-2) is [ka] [ka] is selected from.

[0028] Furthermore, the "low temperature conditions" described in any of the above reaction processes are in an ice-water bath.

[0029] Furthermore, in any of the above reaction processes, solvent are independently polar solvent and / or non-polar solvent The above polarity solvent is one or more of THF, DMF, DMA, and NMP. solvent is one or more species in dichloromethane and carbon tetrachloride.

[0030] Furthermore, the organic alkali in any of the above reaction processes can be independently one or more of N,N-diisopropylethylamine, triethylamine, and pyridine, and preferably one or two of N,N-diisopropylethylamine and pyridine.

[0031] Furthermore, the acid is one or two of hydrochloric acid, trifluoroacetic acid, and citric acid.

[0032] Furthermore, the amine compound is a primary amine or a secondary amine.

[0033] Furthermore, in the above reaction A, extraction is carried out with ethyl acetate, and the above purification is carried out by column chromatography using dichloromethane and methanol as eluents.

[0034] Furthermore, in the above reaction B, trituration is carried out using one or a combination of ethyl acetate, n-hexane and dichloromethane.

[0035] Further purification of the above reaction C is carried out by column chromatography using dichloromethane and methanol as eluents.

[0036] Further purification is carried out by column chromatography using dichloromethane and methanol as eluents in reaction D above.

[0037] Further purification of reaction E above is carried out by column chromatography using dichloromethane and methanol as eluents.

[0038] Furthermore, in the above reaction F, purification is carried out by preparative liquid chromatography using MeCN and 0.1% HCOOH as mobile phase A and H2O and 0.1% HCOOH as mobile phase B.

[0039] Furthermore, all of the above reactions are carried out under nitrogen gas protection. [Effects of the Invention]

[0040] The method for preparing an antibody-drug conjugate intermediate (specifically, a linker-SN38 covalent conjugate) provided by the present invention not only involves simple steps but also reduces safety issues due to residual heavy metals. Furthermore, antibody-drug conjugates prepared using this antibody-drug conjugate intermediate have higher stability in vivo.

[0041] Furthermore, the inventors have surprisingly found that antibody-drug conjugates prepared by combining the linker of the present invention with SN38 exhibit significant inhibitory effects. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 shows the changes in mouse body weight. [Figure 2] FIG. 2 shows the changes in tumor volume. [Figure 3] Figure 3 shows the relative tumor volumes.

[0043] Unless otherwise specified, all abbreviations used herein have the same meaning as understood by a person skilled in the art. Common abbreviations used herein and their definitions are listed below. [Table 1] [Table 2] [Definition]

[0044] Various terms relating to various aspects of the specification are used throughout the specification and claims. Unless otherwise specified, such terms have their ordinary meaning in the art. Other specifically defined terms should be understood to be consistent with the definitions provided herein.

[0045] As used herein, the terms "a," "an," and "said" are used in accordance with standard convention and mean one or more, unless the context indicates otherwise. Thus, for example, reference to an "antibody-drug conjugate" includes a combination of two or more antibody-drug conjugates, and the like.

[0046] It should be understood that, except for cases described herein using the word "comprising," there are similar cases that are further described using the words "consisting of" and / or "consisting essentially of."

[0047] The term "antibody-drug conjugate" as used herein refers to a compound in which an antibody / antibody functional fragment, a linker, and a drug moiety are chemically bonded. Its structure typically consists of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker connecting the antibody or antibody-like ligand to the drug. Currently, the preparation of antibody-drug conjugates is typically divided into two steps: a first step in which the linker and drug moiety are chemically reacted to form a "linker-drug" conjugate; and a second step in which the linker moiety of the "linker-drug" conjugate is covalently bonded to the antibody / antibody functional fragment via a thiol or amino group. The term "antibody-drug conjugate intermediate" in the present invention refers to the above-described "linker-drug" conjugate. Furthermore, the term "antibody-drug conjugate intermediate" in the present invention generally refers to a covalent conjugate of a "linker" and SN38.

[0048] The "antibody-drug conjugate" used in the present invention can be prepared by a conventional method in the art. For example, the antibody-drug conjugate used in the present invention can be prepared by preparing a 10 mg / mL solution of the antibody in a PBS buffer solution at pH 7.4, adding an appropriate equivalent of TCEP and shaking for 1 hour to mix thoroughly, and then adding 5.0 molar equivalents of a linker-toxin (i.e., a compound represented by Formula 1-16) and shaking for 1 hour to react. After the reaction is complete, the remaining small molecules are removed by ultrafiltration, and the conjugate is loaded onto a hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) to measure the DAR, drug distribution, and antibody ratio.

[0049] The present invention aims to provide intermediates for novel drug conjugates. As an example, several ADCs were prepared using Heceptin to verify the effectiveness of the technology. However, it should be understood that the choice of antibody is not a limitation of the present invention. Specific ADC structures are exemplified below. Here, ADC-1 corresponds to the compound of formula (1) in which a linker and a toxin are used, and is prepared using Heceptin by a general method for preparing an "antibody-drug conjugate" in the art. (Similarly, the linker and toxin in ADC-2 corresponds to the compound of formula (2); the linker and toxin in ADC-3 corresponds to the compound of formula (3); the linker and toxin in ADC-4 corresponds to the compound of formula (4); the linker and toxin in ADC-5 corresponds to the compound of formula (5); the linker and toxin in ADC-6 corresponds to the compound of formula (6); the linker and toxin in ADC-7 corresponds to the compound of formula (7); and the linker and toxin in ADC-8 corresponds to the compound of formula (8). (ADC-9 corresponds to a compound of formula (9); ADC-10 corresponds to a compound of formula (10); ADC-11 corresponds to a compound of formula (11); ADC-12 corresponds to a compound of formula (12); ADC-13 corresponds to a compound of formula (13); ADC-14 corresponds to a compound of formula (14); ADC-15 corresponds to a compound of formula (15); ADC-16 corresponds to a compound of formula (16); ADC-17 corresponds to a compound of formula CL2A-SN38), where q may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. [ka] [ka] [ka] [ka]

[0050] The term "linker" according to the present invention refers to a bi- or multi-functional molecule that can react with the protein / antibody molecule and the SN38 reaction, respectively, and thus acts as a "bridge" to connect the protein / antibody to SN38. The linker according to the present invention particularly refers to a group that contains an acyl group in its structure. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples are generally carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents for which specific sources are not specified are conventional reagents purchased commercially. All percentages, ratios, proportions, or parts are by weight unless otherwise specified.

[0052] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0053] Example 1 Preparation of the compound of formula (12) [ka]

[0054] 1) Method for synthesizing compound c (2-Boc-N-methylethylene glycol)

[0055] 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 hours. The reaction system was placed in an ice-water bath, sodium triacetoxyborohydride was added, and the mixture was stirred in the ice-water bath for 1 hour, then returned to room temperature and stirred overnight. The product was detected by LC-MS, and no UV absorption was observed. The mixture was then dried by rotation. solvent The residue was removed, extracted twice with ethyl acetate, dried over anhydrous NaSO, rotary evaporated, and purified by column chromatography eluting with dichloromethane:methanol=20:1 and washed with water to give 1.0 g of product in 66% yield. LC-MS: (M+H) + =262.6

[0056] 2) Method for synthesizing SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0057] 1.0 g of SN38 (camptothecin) and 1.6 g of DNPC (bis(4-nitrophenyl)carbonate) were dissolved in 100 mL of tetrahydrofuran, 2 mL of triethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the mixture was dried by rotation. solvent The solid was removed, triturated with a mixed solvent of 20 mL of ethyl acetate and 100 mL of n-hexane, and then filtered to obtain a solid. The solid was triturated with 20 mL of dichloromethane, filtered, and the product was collected while monitoring by LC-MS to obtain 700 mg of SN38-PNP solid in a yield of 49.3%. LC-MS: (M+H) + =557.4.

[0058] 3) Method for synthesizing compound d (Boc-N-methylethylglycol-camptothecin)

[0059] 250 mg of SN38-PNP was dissolved in 30 mL of N,N-dimethylformamide, 235 mg of Boc-DMEDA-PEG and 233 μL of N,N-diisopropylethylamine were added, and the mixture was stirred at room temperature for 16 hours. The completion of the reaction was confirmed by LC-MS detection. After the reaction was completed, the mixture was dried by rotation. solvent The residue was removed, and the mixture was purified by column chromatography using dichloromethane:methanol=20:1 as an eluent, followed by washing with water. The product was collected while monitoring by LC-MS, and 213 mg of Boc-DMEDA-PEG-SN38 solid was obtained in a yield of 70%. LC-MS: (M+H) + =680.5.

[0060] 4) Method for synthesizing compound g (10-(2-N-methylethylene glycol)-camptothecin trifluoroacetate)

[0061] 73.0 mg of compound d was dissolved in 3 mL of dichloromethane and 3 mL of trifluoroacetic acid, placed in an ice-water bath, and stirred at a constant temperature for 1 hour. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the mixture was dried by rotation. solvent After removing the solvent, 2 mL of methylbenzene and 2 mL of dichloromethane were added and the mixture was spun dry twice. The product was collected while monitoring by LC-MS to obtain SN38-DMEDA-TFA, which was used directly in the next reaction. LC-MS: (M+H) + =580.6

[0062] 5) Method for synthesizing the compound of formula (12) (maleimidodiethoxy-L-valyl-L-citrulline-p-aminobenzyl-(2-N-methylethylene glycol)-camptothecin)

[0063] 50 mg of MP2-VC-PAB-PNP was dissolved in 4 mL of N,N-dimethylformamide and placed in an ice-water bath to maintain a constant temperature and protected with nitrogen. 38 mg of compound g and 42 μL of triethylamine were added to the reaction mixture, and the mixture was stirred at constant temperature for 0.5 hours. The mixture was then returned to room temperature and allowed to react for another 0.5 hours. LC-MS analysis confirmed the completion of the reaction. After completion of the reaction, the solvent was removed by rotary evaporation at low temperature. A SunFire® Prep C18 OBD™ 5 μm, 19 × 250 mm column was used as the preparative column, and preparative purification chromatography was performed using MeCN as mobile phase A and HO as mobile phase B. The pure product was collected while monitoring by LC-MS, and 18 mg of the compound of formula (12) was obtained as a solid in 22% yield. LC-MS: (M+H) + =1197.2

[0064] Example 2 Preparation of the compound of formula (4) [ka]

[0065] Compound g (10-(2-N-methylethylene glycol)-camptothecin trifluoroacetate) was prepared according to the synthesis route in Example 1. 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 hour. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the solution was dried by rotation. solvent The residue was removed. 2 mL of dichloromethane was added twice and the mixture was spun dry. The product was collected while monitoring by LC-MS to obtain compound g (70 mg). This was used directly in the next reaction. LC-MS: (M+H) + =580.6.

[0066] Compound (4) (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-(2-N-methylethylene glycol)-camptothecin) was prepared according to the synthetic route described in Example 1. Specifically, Mc-VC-PAB-PNP (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-nitrophenyl carbonate) (58 mg) and compound g (42 mg) were dissolved in 2.5 mL of N,N-dimethylformamide and stirred in an ice-water bath for 30 minutes. DIPEA (42 mg) was added dropwise and stirred in an ice-water bath for 1 hour. LC-MS analysis indicated that the reaction was not complete. Next, triethylamine (10 mg) was added and stirred at room temperature for 1 hour. The reaction mixture turned yellow, and completion of the reaction was confirmed by LC-MS analysis. After the reaction was completed, the solvent was removed by rotary drying at low temperature, and preparative purification chromatography was performed using a SunFire® Prep C18 OBD™ 5um, 19 x 250 mm column as the preparative column, MeCN, 0.1% HCOOH as mobile phase A, and HO, 0.1% HCOOH as mobile phase B. The pure product was collected while monitoring by LC-MS, and 34 mg of the compound of formula (4) was obtained as a solid in a yield of 40.5%. LC-MS: (M+H) + =1179.2

[0067] Example 3 Preparation of the compound of formula (1) [ka]

[0068] 1) Method for synthesizing compound c (2-Boc-N-methylethylene glycol)

[0069] 0.6 g of compound a (aminoethylene glycol) and 1.0 g of compound b (N-Boc-(methylamido)acetaldehyde) were dissolved in 20 mL of methanol and stirred at room temperature for 6 hours. The reaction system was placed in an ice-water bath, sodium triacetoxyborohydride was added, and the mixture was stirred in the ice-water bath for 1 hour, then returned to room temperature and stirred overnight. The product was detected by LC-MS, but no UV absorption was observed. The mixture was then dried by rotation. solventThe residue was removed, extracted twice with ethyl acetate, dried over anhydrous Na2SO4, and the organic phase was poured off and rotovapped. After that, it was purified by column chromatography using dichloromethane:methanol=20:1 as eluent and washed with water to give 1.0 g of product in 66% yield. LC-MS: (M+H) + =262.6

[0070] 2) Method for synthesizing SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0071] 1.0 g of SN38 and 1.6 g of DNPC (bis(4-nitrophenyl)carbonate) were dissolved in 100 mL of tetrahydrofuran, 2 mL of triethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the mixture was dried by rotation. solvent The solid was removed, triturated with a mixed solvent of 20 mL of ethyl acetate and 100 mL of n-hexane, and then filtered to obtain a solid. The solid was triturated with 20 mL of dichloromethane, filtered, and the product was collected while monitoring by LC-MS to obtain 700 mg of SN38-PNP solid in a yield of 49.3%. LC-MS: (M+H) + =557.4

[0072] 3) Method for synthesizing compound d (Boc-N-methylethylglycol-camptothecin)

[0073] 500 mg of SN38-PNP was dissolved in 30 mL of N,N-dimethylformamide, 370 mg of compound c and 233 μL of N,N-diisopropylethylamine were added, and the mixture was stirred at room temperature for 3 hours. The completion of the reaction was confirmed by LC-MS detection. After the reaction was completed, the mixture was dried by rotation. solvent The residue was purified by column chromatography using dichloromethane:methanol = 20:1 as an eluent, and then washed with water. The product was collected while monitoring by LC-MS, and 250 mg of compound d was obtained as a solid in a yield of 45%. LC-MS: (M+H) + =680.5.

[0074] 4) Method for synthesizing compound h (Boc-N-methylethylenedioxy p-nitro active ester-camptothecin)

[0075] 20.0 mg of compound d was dissolved in 5 mL of dichloromethane, 150 mg of bis(4-nitrophenyl)carbonate and 86 mg of DIPEA were added, and the mixture was stirred at a constant temperature for 16 hours. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the mixture was dried by rotation. solvent The residue was removed and purified by column chromatography using dichloromethane:methanol=50:1 as an eluent. The product was collected to give 220 mg of compound h as a solid in 68.5% yield. This was used directly in the next reaction. LC-MS: (M+H) + =845.6

[0076] 5) Method for synthesizing compound i (Boc-N-methyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin)

[0077] 220 mg of compound h (Boc-N-methyldiethoxy p-nitro active ester-camptothecin) and 50 mg of N,N,N-trimethylethylenediamine were dissolved in 5 mL of N,N-dimethylformamide, and then DIPEA (65 mg) was added dropwise and stirred in an ice-water bath for 1 hour. The completion of the reaction was detected by LC-MS, and the mixture was then dried by rotation. solvent The residue was removed and purified by column chromatography using dichloromethane:methanol=20:1 as eluent. The product was collected to give compound i as a solid (157 mg) in 75% yield, which was used directly in the next reaction. LC-MS: (M+H) + =808.6

[0078] 6) Method for synthesizing compound j (N-methyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin-trifluoroacetate)

[0079] 157.0 mg of compound i (Boc-N-methyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin) was dissolved in 3 mL of dichloromethane and 1.3 mL of trifluoroacetic acid, placed in an ice-water bath, and stirred at constant temperature for 1 hour. The completion of the reaction was confirmed by detection using LC-MS. After the reaction was completed, the mixture was dried by rotation. solvent The residue was removed. 2 mL of dichloromethane was added and the mixture was dried by rotation twice. 120 mg of compound j was obtained. This was used directly in the next reaction. LC-MS: (M+H) + =708.3.

[0080] 7) Method for synthesizing the compound of formula (1) (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-(N-methyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin)

[0081] 52 mg of Mc-VC-PAB-PNP (maleimidohexanoic acid-L-valyl-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 minutes. 129 mg of DIPEA was added dropwise and stirred in an ice-water bath for 0.5 hours. LC-MS analysis indicated that the reaction was not complete. Next, 129 mg of DIPEA was added and stirred at room temperature for 0.5 hours. The reaction mixture turned yellow, and completion of the reaction was confirmed by LC-MS. After completion of the reaction, the solvent was removed by rotary evaporation at low temperature. A SunFire® Prep C18 OBD™ 5 μm, 19 × 250 mm column was used as a preparative column. The mixture was purified by preparative chromatography using MeCN, 0.1% HCOOH as mobile phase A and HO, 0.1% HCOOH as mobile phase B. The pure product was collected while monitoring by LC-MS to obtain 20 mg of the compound of formula (1) as a solid in a yield of 20.5%. LC-MS: (M+H) + =1307.6.

[0082] Example 4 Preparation of the compound of formula (9) [ka]

[0083] Compound (9) (maleimidethoxy-L-valyl-L-citrulline-p-aminobenzyl-(N-methyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin) was prepared according to the synthetic route described in Example 3. Specifically, MP2-VC-PAB-PNP (maleimidediethoxy-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) (52 mg) and compound j (34 mg) were dissolved in 2.5 mL of N,N-dimethylformamide and stirred in an ice-water bath for 30 minutes. DIPEA (36 mg) was added dropwise and stirred in an ice-water bath for 0.5 hours. LC-MS analysis indicated that the reaction was not complete. Next, DIPEA (8 mg) was added and stirred at room temperature for 1.5 hours. The reaction mixture turned yellow, and completion of the reaction was confirmed by LC-MS. After the reaction was completed, the solvent was removed by rotary drying at low temperature, and preparative purification chromatography was performed using a SunFire® Prep C18 OBD™ 5 μm, 19 x 250 mm column as the preparative column, MeCN, 0.1% HCOOH as mobile phase A, and HO, 0.1% HCOOH as mobile phase B. The pure product was collected while monitoring by LC-MS, and 23 mg of the compound of formula (9) was obtained as a solid in a yield of 35.2%. LC-MS: (M+H) + =1325.4.

[0084] Example 5 Inhibitory effect of ADC-1 on NCI-N87 cells

[0085] The compound of formula (1) and the CL2A-SN38 intermediate were each sulfhydryl-conjugated to HER2-CL2A-SN38 (ADC-17) antibody-drug conjugates with an average DAR of 8 were prepared using conventional methods (see, for example, Example 12 of Chinese Patent Application Publication No. CN102448494A). NCI-N87 cells were digested with trypsin and adjusted to a cell density of 50,000 cells / mL. 100 μL / well of the conjugates were added to cell culture plates and cultured at 37°C in a 5% CO2 incubator for 14-20 hours. The sample and control groups (see Table 1 for the composition of the test samples) were diluted with basal medium and transferred to the plated cells at 100 μL / well. The cells were then cultured at 37°C in a 5% CO2 incubator for 70-74 hours. CCK-8 was diluted 10-fold with culture medium, the old medium was discarded from the 96-well plate, and 100 μL of the diluted CCK-8 solution was added to each well. The plate was incubated for 2-4 hours in 5% CO2. After centrifugation to remove air bubbles, the plate was read using a microplate reader at a detection wavelength of 450 nm / 655 nm. The results are shown in Table 2.

[0086] [Table 3]

[0087] [Table 4]

[0088] The test results showed that the ADC-1 and ADC-17 groups exhibited comparable inhibitory effects on NCI-N87 cells.

[0089] Example 6 Experimental Study on Stability

[0090] 1) The compound of formula (1) (2.1 mg) and CL2A-SN38 (2.1 mg) were precisely weighed and dissolved in 700 μL of DMSO.

[0091] 2) PB (0.2 M) buffer solutions with pH values ​​of 6, 7, and 8 were prepared.

[0092] 3) 50 μL of a DMSO solution of the compound of formula (1) was taken and added to a centrifuge tube, and 950 μL of PB (0.2 M) buffer solution (pH 6) was added to adjust the compound concentration to 60 μg / mL. The tube was then placed in an incubator at 37°C. 50 μL of a DMSO solution of CL2A-SN38 was taken and added to a centrifuge tube, and 950 μL of PB (0.2 M) buffer solution (pH 6) was added to adjust the compound concentration to 60 μg / mL. The tube was then placed in an incubator at 37°C.

[0093] 4) 40 μL samples were collected at 0 h, 2 h, 4 h, 6 h, 24 h, and 48 h, and 120 μL of acetonitrile was added and centrifuged. The supernatant was subjected to LC-MS quantification.

[0094] 5) Processing the results:

[0095] The LC-MS concentration at T=0 h is used as the standard, and T / T0 × The calculation was based on 100%. The results are shown in Tables 3 and 4, respectively.

[0096] [Table 5]

[0097] At a pH of 6, the compound of formula (1) was decomposed by 21.2% after 48 hours.

[0098] When the pH was 7, the compound of formula (1) was decomposed by 87.8% after 48 hours.

[0099] At a pH of 8, the compound of formula (1) was decomposed by 98.7% after 48 hours.

[0100] [Table 6]

[0101] At a pH of 6, CL2A-SN38 was degraded by 98.0% after 48 hours.

[0102] At a pH of 7, CL2A-SN38 was degraded by 98.1% after 48 hours.

[0103] At a pH of 8, CL2A-SN38 was degraded by 98.1% after 48 hours.

[0104] The results in Tables 3 and 4 reveal the following.

[0105] 1) Under weakly acidic conditions, the compound of formula (1) was more stable than CL2A-SN38.

[0106] 2) The compound of formula (1) was decomposed by 18% in 6 hours and by 75% in 24 hours under neutral conditions, while CL2A-SN38 was decomposed by 43% in 6 hours and by 98% in 24 hours under neutral conditions.

[0107] 3) Under alkaline conditions, the compound of formula (1) was decomposed by 97% in 24 h, and CL2A-SN38 was decomposed by 98% in 24 h.

[0108] Therefore, compared with CL2A-SN38, the compound of formula (1) prepared in the present invention is more stable under weakly acidic or neutral conditions in vivo, and is expected to significantly improve the safety of ADC pharmaceuticals.

[0109] Example 7 Evaluation of the inhibitory effect of anti-Her2 drug conjugates on the development of orthotopic breast cancer

[0110] 1. Test Method

[0111] The BT474 cell line was regenerated for 1-2 generations. After the cell proliferation state stabilized, expansion culture was performed to prepare a tumor cell suspension. BT474 cells were then applied to nude mice cream pads at a concentration of 0.2 × 10 7 A total of 60 female BALB / c nude mice were inoculated with the IgG1 gene. The average tumor volume was approximately 70 mm. 3At this point, 42 mice were selected and randomly divided into six groups of seven mice per group according to tumor volume: Control Group 1 (normal saline group), which was the negative control 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). After successful model establishment, control group 1 was intravenously administered normal saline, while the remaining groups were intravenously administered the drugs at doses of 5 mg / kg (control group 2), 5 mg / kg (experimental group 1), 5 mg / kg (experimental group 2), 5 mg / kg (experimental group 3), and 5 mg / kg (experimental group 4) once weekly for 21 weeks. Mouse weights and tumor volumes were measured three times weekly, and on day 21, the weights and tumor volumes of the mice were measured.

[0112] 2. Test results and analysis

[0113] 1) Changes in mouse weight:

[0114] [Table 7]

[0115] No mice died during the study. At the end of the study, there were no significant differences in body weight between control group 1 and control group 2 (ADC-17 group), experimental group 1 (ADC-4), experimental group 2 (ADC-12), experimental group 3 (ADC-1), and experimental group 4 (ADC-9). See Table 5 and Figure 1 for details.

[0116] 2) Mouse tumor volume:

[0117] [Table 8] Note: When comparing each group with the control group, the saline group, * " indicates P<0.05, and " ** " indicates P<0.01. When each group was compared with the control group 2, # " indicates P<0.05, and " ## " indicates P<0.01.

[0118] The tumor volume in control group 1 was 107±19.30 mm on the 21st day after administration. 3 The tumor volumes in 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 four groups (control group 2, experimental group 1, experimental group 2, and experimental group 3) showed highly statistically significant differences (P<0.01) compared with control group 1 (saline group), and experimental group 4 showed a statistically significant difference (P<0.05) compared with control group 1. The tumor volumes in experimental groups 1, 2, and 3 were not statistically significantly different (P>0.05) compared with control group 2, and the relative tumor volumes were also not significantly different (P>0.05), indicating comparable efficacy. Furthermore, the relative tumor growth rates (T / C) in the five groups (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. These data indicate that the four antibody-drug conjugates tested in this study, corresponding to control group 2, experimental group 1, experimental group 2, and experimental group 3, were all able to highly significantly inhibit BT474 tumor growth with comparable efficacy, and experimental group 4 was also able to inhibit BT474 tumor growth. The changes in tumor volume and relative tumor volume for each group during the study are shown in Figures 2 and 3, respectively.

[0119] In summary, the ADCs constructed using the intermediate drug conjugates of this invention showed significant antitumor activity in a breast cancer BT474 cell model. The preferred ADCs, ADC-1, ADC-4, and ADC-12, have clear advantages, significantly improved safety under in vivo conditions, and are expected to provide tumor-suppressing effects comparable to those of the ADC-17 group.

[0120] Although the present invention has been described using various specific examples, the present invention is not limited to these specific examples, and a person skilled in the art can make various changes or modifications within the scope of the present invention. Furthermore, it will be understood by a person skilled in the art that the technical features mentioned in this specification can be combined with each other without departing from the spirit and scope of the present invention, and that such changes and modifications are also within the scope of the present invention. Furthermore, the present invention includes the following aspects. [Aspect 1] An intermediate of an antibody-drug conjugate represented by the following formula (I): [ka] (In the formula, X 1 is an alkane chain or a PEG chain, X 2 is H or -C(O)NR 1 R 2 and R 1 is hydrogen, halogen, hydroxy, substituted or unsubstituted C 1-6 Alkyl groups, substituted or unsubstituted C 1-6 Hydroxyalkyl groups, substituted or unsubstituted C 1-6 Aminoalkyl groups, substituted or unsubstituted C 1-6 Alkoxyl group, substituted or unsubstituted C 1-6 Alkyl acyl groups, and substituted or unsubstituted C 1-6 alkylaldehyde groups; R 2 are hydrogen or unsubstituted or each independently a halogen, a hydroxy group, an amino group, a carbonyl group, a carboxyl group, a 5-10 membered heterocyclyl group (wherein the 5-10 membered heterocyclyl group has 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C 1-6 C optionally substituted with 1 to 5 substituents selected from the group consisting of haloalkyl groups 1-6 is an alkyl group. [Aspect 2] X 1 teeth, [ka] wherein m is 1, 2, 3, 4, 5, or 6, preferably m is 5, and p is 1, 2, 3, 4, 5, or 6, preferably p is 2; Preferably, the X 1 teeth, [ka] 2. The intermediate of an antibody-drug conjugate according to embodiment 1, wherein the intermediate is selected from the group consisting of: [Aspect 3] R 1 or R 2 are independently H, methyl, ethyl, propyl, butyl, pentyl, heptyl, methoxyl, ethoxyl, Cl, Br,

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Claims

1. An intermediate of an antibody-drug conjugate represented by formula (I): 【Chemical 1】 (In the formula, X １ is an alkylene chain or a PEG chain, X ２ is H or —C(O)NR １ R ２ and R １ is hydrogen, halogen, hydroxy, substituted or unsubstituted C １－６ alkyl group, substituted or unsubstituted C １－６ Hydroxyalkyl groups, substituted or unsubstituted C １－６ Aminoalkyl groups, substituted or unsubstituted C １－６ Alkoxyl group, substituted or unsubstituted C １－６ Alkyl acyl groups, and substituted or unsubstituted C １－６ alkylaldehyde groups; R ２ are selected from the group consisting of hydrogen and substituted or unsubstituted C 1-6 alkyl groups, each of which is independently selected from the group consisting of halogen, hydroxy, amino, carboxyl, 5- to 10-membered heterocyclyl groups (wherein the 5- to 10-membered heterocyclyl groups have 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C １－６ and substituted with 1 to 5 substituents selected from the group consisting of haloalkyl groups.

2. The X １ but, 【Chemistry 2】 2. The intermediate of antibody-drug conjugate according to claim 1, characterized in that: (wherein m is selected from 1, 2, 3, 4, 5 and 6, and p is selected from 1, 2, 3, 4, 5 and 6).

3. An intermediate of the antibody-drug conjugate described in claim 2, characterized in that m is 5 and p is 2.

4. X 1 is 【Chemistry 3】 The intermediate of the antibody-drug conjugate according to claim 2, characterized in that it is selected from the group consisting of:

5. The R １ or R ２ are independently H, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a heptyl group, a methoxyl group, an ethoxyl group, Cl, Br, 【Chemistry 4】 The intermediate of the antibody-drug conjugate according to claim 1, characterized in that it is selected from the group consisting of:

6. The X ２ but, 【Chemistry 5】 The intermediate of the antibody-drug conjugate according to any one of claims 1 to 5, characterized in that it is selected from the group consisting of:

7. Formulas (1) to (16): 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 An intermediate of an antibody-drug conjugate, which is one selected from the group consisting of: 【Request 8】 【Chemical 10】 Formula (Wherein, the R １ is hydrogen, substituted or unsubstituted C １－６ alkyl group, substituted or unsubstituted C １－６ Hydroxyalkyl groups, substituted or unsubstituted C １－６ Aminoalkyl groups, substituted or unsubstituted C １－６ Alkoxyl group, substituted or unsubstituted C １－６ Alkyl acyl groups and substituted or unsubstituted C １－６ alkylaldehyde groups; The R ２ are selected from the group consisting of hydrogen and substituted or unsubstituted C 1-6 alkyl groups, each of which independently is selected from the group consisting of halogen, hydroxy, amino, carboxyl, 5- to 10-membered heterocyclyl groups (wherein the 5- to 10-membered heterocyclyl groups have 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C １－６ and substituted with 1 to 5 substituents selected from the group consisting of haloalkyl groups. A method for preparing an intermediate of an antibody-drug conjugate represented by the formula: The preparation method includes the following reaction steps 1 to 4: Reaction Step 1: 【Chemistry 11】 or Reaction Step 2: 【Chemistry 12】 or Reaction Step 3: 【Chemistry 13】 or Reaction Step 4: 【Chemistry 14】 A method for preparing an intermediate of an antibody-drug conjugate selected from the group consisting of:

9. R 1 and R 2 are H, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a heptyl group, a methoxyl group, an ethoxyl group, Cl, Br, 【Chemistry 15】 9. The method of claim 8, wherein the compound is selected from the group consisting of:

10. The compound (I-1) is 【Chemistry 16】 【Chemistry 17】 or the compound (I-2) is selected from the group consisting of: 【Chemistry 18】 【Chemistry 19】 9. The method according to claim 8, characterized in that the compound is selected from the group consisting of:

11. The reaction process 1 is performed under the following conditions: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added under low temperature conditions, stirred for an appropriate time, and then stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate period of time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolving the product obtained in Reaction B in a solvent, adding the product obtained in Reaction A and an organic alkali, stirring at room temperature for an appropriate time, and after the reaction is completed, rotating and drying the mixture to remove the solvent, and then purifying the mixture. The preparation method of claim 8, characterized in that it includes: Reaction D: dissolving the product obtained in Reaction C in a solvent, adding an acid, stirring at low temperature for a suitable time, and after the reaction is completed, rotating and drying to remove the solvent; dissolving Mc-VC-PAB-PNP in a solvent, stirring at low temperature for a suitable time, adding an organic alkali, and after the reaction is completed, rotating and drying to remove the solvent; and purifying.

12. The reaction process 2 is performed under the following conditions: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added under low temperature conditions, stirred for an appropriate time, and stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate period of time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolving the product obtained in Reaction B in a solvent, adding the product obtained in Reaction A and an organic alkali, stirring at room temperature for an appropriate time, and after the reaction is completed, rotating and drying the mixture to remove the solvent, and then purifying the mixture. The preparation method of claim 8, characterized in that it comprises: Reaction D: dissolving the product obtained in Reaction C in a solvent, adding an acid, stirring at low temperature for a suitable time, and after the reaction, rotating and drying to remove the solvent, and then dissolving MP2-VC-PAB-PNP in a solvent, stirring at low temperature for a suitable time, adding an organic alkali, and after the reaction, rotating and drying to remove the solvent, and then purifying.

13. The reaction process 3 is carried out under the following operating conditions: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added under low temperature conditions, and then stirred for an appropriate time, and stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate period of time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolving the product obtained in Reaction B in a solvent, adding the product obtained in Reaction A and an organic alkali, stirring at room temperature for an appropriate time, and after the reaction is completed, rotating and drying the mixture to remove the solvent, and then purifying the mixture. Reaction D: Dissolve the product obtained in Reaction C in a solvent, add bis(4-nitrophenyl)carbonate and an organic alkali, and stir at a fixed temperature for a suitable time. After the reaction is completed, the solution is dried by rotary drying to remove the solvent, and then purified. Reaction E: The product obtained in Reaction D and an amine compound are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at a low temperature for an appropriate period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. The preparation method according to claim 8, characterized in that it comprises: Reaction F: dissolving the product obtained in Reaction E in a solvent, adding an acid, stirring at low temperature for a suitable time, and after the reaction is completed, rotating and drying to remove the solvent, and then dissolving it together with Mc-VC-PAB-PNP in a solvent, stirring at low temperature for a suitable time, adding an organic alkali, and after the reaction is completed, rotating and drying to remove the solvent, and then purifying.

14. The reaction process 4 is carried out under the following operating conditions: Reaction A: Compound a and compound b are dissolved in a solvent, stirred at room temperature for an appropriate time, then a reducing agent is added under low temperature conditions, and then stirred for an appropriate time, and stirred at room temperature overnight. After the reaction is completed, the solvent is removed by rotary drying, and then extraction, drying, and purification are carried out. Reaction B: SN38 and DNPC are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at room temperature for an appropriate period of time. After the reaction is completed, the mixture is dried by rotation to remove the solvent, and then triturated and filtered. Reaction C: Dissolving the product obtained in Reaction B in a solvent, adding the product obtained in Reaction A and an organic alkali, stirring at room temperature for an appropriate time, and after the reaction is completed, rotating and drying the mixture to remove the solvent, and then purifying the mixture. Reaction D: Dissolve the product obtained in Reaction C in a solvent, add bis(4-nitrophenyl)carbonate and an organic alkali, and stir at a fixed temperature for a suitable time. After the reaction is completed, the solution is dried by rotary drying to remove the solvent, and then purified. Reaction E: The product obtained in Reaction D and an amine compound are dissolved in a solvent, an organic alkali is added, and the mixture is stirred at a low temperature for an appropriate period of time. After the reaction is completed, the mixture is rotary dried to remove the solvent, and then purified. The preparation method of claim 8, characterized in that it includes: Reaction F: dissolving the product obtained by Reaction E in a solvent, adding an acid, stirring at low temperature for a suitable time, and after the reaction, rotating and drying to remove the solvent, and then dissolving it together with MP2-VC-PAB-PNP in a solvent, stirring at low temperature for a suitable time, adding an organic alkali, and after the reaction, rotating and drying to remove the solvent, and then purifying.

15. The preparation method according to any one of claims 11 to 14, wherein the "low temperature conditions" refer to an ice-water bath.

16. The method according to any one of claims 11 to 14, wherein the solvent is a polar solvent selected from the group consisting of THF, DMF, DMA, NMP, and combinations thereof, and / or a non-polar solvent selected from the group consisting of dichloromethane, carbon tetrachloride, and combinations thereof.

17. The preparation method according to any one of claims 8 to 14, characterized in that the organic alkali is selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and combinations thereof.

18. The method of claim 17, wherein the organic alkali is one or both of N,N-diisopropylethylamine and pyridine.

19. The preparation method according to any one of claims 8 to 14, characterized in that the acid is one or two of hydrochloric acid, trifluoroacetic acid, and citric acid.

20. The preparation method according to any one of claims 8 to 14, characterized in that the amine-based compound is a primary amine or a secondary amine.

21. The preparation method according to any one of claims 11 to 14, characterized in that in Reaction A, the extraction is carried out using ethyl acetate, and the purification is carried out by column chromatography using dichloromethane and methanol as eluents.

22. The preparation method according to any one of claims 11 to 14, characterized in that in the reaction B, the trituration is carried out using one or a combination of two or more of ethyl acetate, n-hexane, and dichloromethane.

23. The preparation method according to any one of claims 11 to 14, characterized in that in reaction C, the purification is carried out by column chromatography using dichloromethane and methanol as eluents.

24. The preparation method according to any one of claims 13 to 14, characterized in that in reaction D, the purification is carried out by column chromatography using dichloromethane and methanol as eluents.

25. The preparation method according to any one of claims 13 to 14, characterized in that in reaction E, the purification is carried out by column chromatography using dichloromethane and methanol as eluents.

26. In the reaction F, the purification was carried out using MeCN and 0.1% HCOOH as mobile phase A and H as mobile phase B. ２ 15. The method according to any one of claims 13 to 14, characterized in that it is carried out by preparative liquid chromatography using O and 0.1% HCOOH.

27. The preparation method according to any one of claims 11 to 14, characterized in that all of the reactions are carried out under nitrogen gas protection.

28. Formula: 【Chemistry 20】 (In the formula, X 1 is an alkylene chain or a PEG chain; X 2 is H or —C(O)NR 1 R 2 , and R 1 is selected from the group consisting of hydrogen, halogen, hydroxy, a substituted or unsubstituted C 1-6 alkyl group, a substituted or unsubstituted C 1-6 hydroxyalkyl group, a substituted or unsubstituted C 1-6 aminoalkyl group, a substituted or unsubstituted C 1-6 alkoxyl group, a substituted or unsubstituted C 1-6 alkylacyl group, and a substituted or unsubstituted C 1-6 alkylaldehyde group; R 2 is selected from the group consisting of hydrogen and substituted or unsubstituted C 1-6 alkyl groups, each of which is independently substituted with 1 to 5 substituents selected from the group consisting of halogen, hydroxyl, amino, carboxyl, 5- to 10-membered heterocyclyl groups (wherein the 5- to 10-membered heterocyclyl groups have 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur), and C 1-6 haloalkyl groups; Ab is an antibody; q is selected from 1, 2, 3, 4, 5, 6, 7 and 8. An antibody-drug conjugate represented by the formula:

29. X 1 is 【Chemical 21】 (where, m is selected from 1, 2, 3, 4, 5 and 6; p is selected from 1, 2, 3, 4, 5 and 6 The antibody-drug conjugate of claim 28, wherein the antibody-drug conjugate is selected from the group consisting of:

30. The antibody-drug conjugate of claim 29, wherein m is 5 and p is 2.

31. X １ but, 【Chemical 22】 30. The antibody-drug conjugate of claim 29, wherein the antibody-drug conjugate is selected from the group consisting of:

32. R 1 or R 2 is independently H, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a heptyl group, a methoxyl group, an ethoxyl group, Cl, Br, 【Chemical 23】 The antibody-drug conjugate of claim 28, wherein the antibody-drug conjugate is selected from the group consisting of:

33. Formulas (ADC-1) to (ADC-16): 【Chemistry 24】 【Chemistry 25】 【Chemical 26】 【Chemical 27】 The antibody-drug conjugate is one selected from the group consisting of:

34. Use of an antibody-drug conjugate intermediate described in any one of claims 1 to 5 in the preparation of an antibody-drug conjugate.

35. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 28 to 33 for use in treating a tumor.

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