Synthesis method of Val-Cit dipeptide linker

Abstract

The invention discloses a synthesis method of a Val-Cit dipeptide linker for ADCs drugs, (S)-2-((S)-2-((((9H-fluorene-9-yl) methoxyl) carbonyl) amino)-3-methylbutyryl amino) is used as an initial raw material for drug synthesis, all synthesis conditions are mild and controllable, the four-step yield is high, and the synthesis method is a novel synthesis process of the Val-Cit dipeptide linker pioneered at home and abroad. Reaction conditions are optimized, and environment-friendly and efficient trimethyl acetic anhydride is used as a condensing agent, so that environmental pollution caused by synthesis is reduced from the source; the product yield is high, and generated impurities are few; the post-treatment operation is simple, easy to implement and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a method for synthesizing a Val-Cit dipeptide linker. Background Art

[0002] In recent years, tumors remain the main cause of death from human diseases. Treatment methods for malignant tumors have gradually shifted from traditional chemotherapy to targeted therapy, but in general, traditional treatment methods are still the first choice in clinical practice, and chemotherapy drugs still occupy the largest cancer treatment market in terms of product quantity. Small molecule chemotherapy drugs are prone to serious toxic side effects due to their poor targeting and biodistribution. Compared with chemotherapy drugs, antibody drugs have better targeting and have played an increasingly important role in the treatment of malignant tumors; however, they also have shortcomings such as poor tissue penetration, weak biological activity, and easy to develop drug resistance. Based on the above problems, the continuous emergence of new therapies is bound to bring disruptive changes to tumor treatment.

[0003] Antibody-drug conjugates (ADCs) are a class of biological drugs that connect cytotoxic small molecule drugs and antibodies through chemical linkers, and they have great development prospects. Targeted antibodies specifically bind to antigens on the tumor cell membrane to achieve endocytosis, allowing the antibody and the small molecule drug connected to it to enter the cell, and then degrade in the lysosome. The small molecule drug is released into the cell, inducing tumor cell apoptosis, thereby exerting the anti-cancer effect of ADCs drugs. Due to the advantages of high activity, small side effects, and long duration of action, the emergence of ADCs drugs provides a new idea for "precision treatment" of tumors. The ADCs structure consists of three parts: small molecule drugs (cytotoxin), antibodies (antibody), and linkers (Linker). Linkers are used to achieve the organic combination of small molecule drugs and antibodies, and are a crucial part. Valine-citrulline (Val-Cit) coupled with p-aminobenzyl (PAB) spacer as a cleavable dipeptide linker has been widely used in ADCs coupling because of its excellent characteristics such as rapid cleavage by cathepsin B in lysosomes and stability in human plasma.

[0004] Val-Cit dipeptide linkers are used in a large number of articles on anticancer drugs and are well described, but only a few articles provide detailed experimental methods for the synthesis of this specific linker. Val-Cit dipeptide linkers are already available on the market, but they are expensive. Therefore, it is necessary to further develop green and low-cost process routes for the synthesis of Val-Cit dipeptide linkers for the construction and application of various drug linkers.

[0005] Deboprosad Mondal et al. reported that Fmoc-Val-Cit-OH was used as the raw material, and p-aminobenzyl alcohol was used as the condensation agent, methanol and dichloromethane were used as solvents, and amide condensation was carried out; NMP was used as the solvent, diethylamine was used to remove Fmoc; and Mc-OSu was reacted, and then reacted with PNP-chloroformate. The reaction requires four steps of synthesis, and the next step of reaction is carried out after post-treatment. This method uses pyridine as a solvent, and column purification is required for post-treatment, which is not easy to achieve in large-scale production.

[0006] Dian Xiao et al. reported that Fmoc-Val-Cit-OH was used as a raw material, and p-aminobenzyl alcohol was used in the presence of EEDQ as a condensing agent to carry out amide condensation; DMF was used as a solvent, and piperidine was used to remove Fmoc; it reacted with Mc-OSu, and then reacted with dicarbonate. The reaction required four steps of synthesis, and after the reaction, all were post-treated before the next step of reaction. This method uses piperidine as a Fmoc removal agent, and the product is not easy to separate.

[0007] In summary, it is necessary to improve the existing process and develop a green and low-cost process route for synthesizing Val-Cit dipeptide linkers. Summary of the invention

[0008] In view of the above problems, the object of the present invention is to provide a greener and more efficient method for preparing a Val-Cit dipeptide linker, which is simple to operate, has a high yield and high purity of the product, and is suitable for industrial production.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for synthesizing a Val-Cit dipeptide linker comprises the following steps:

[0011] Step 1: Acid amine condensation reaction: Compound I Fmoc-Val-Cit-OH: (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino, p-aminobenzyl alcohol are dissolved in a solvent under a nitrogen atmosphere, a condensing agent is added at 30-70°C, and the reaction is carried out for 12-24 hours. In order to completely react compound I, p-aminobenzyl alcohol and a condensing agent are added again, and the reaction is carried out at the same temperature for 12-24 hours. Recrystallization is performed to obtain compound II Fmoc-Val-Cit-PAB-OH, and the reaction formula is as follows;

[0012]

[0013] Step 2: Reaction of removing Fmoc group: dissolve compound II Fmoc-Val-Cit-PAB-OH in a solvent, add base in batches and react at 25-70°C for 5-14h, and recrystallize to obtain compound III Val-Cit-PAB-OH. The reaction formula is as follows;

[0014]

[0015] Step 3: Amine ester condensation reaction: Dissolve compound III Val-Cit-PAB-OH in a solvent, add raw material 6-(maleimido)hexanoic acid succinimidyl ester, and react at 25-70°C for 5-14h. Recrystallize to obtain compound IV MC-Val-Cit-PAB-OH, the reaction formula is as follows;

[0016]

[0017] Step 4: Ester exchange reaction: Dissolve compound IVMC-Val-Cit-PAB-OH and raw material di(p-nitrophenyl) carbonate in a solvent under a nitrogen atmosphere, add a catalyst, react at 25-70°C for 5-14h, and recrystallize to obtain compound VMC-Val-Cit-PAB-PNP, that is, the Val-Cit dipeptide linker. The reaction formula is as follows.

[0018]

[0019] Further, in the reaction of step 1, the condensing agent used in the reaction of step 1 is any one of trimethylacetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, N,N'-carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide / 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide / 1-hydroxybenzotriazole, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate / N-methylimidazole, preferably trimethylacetic anhydride; in the reaction of step 1, according to In terms of moles, the total usage ratio of Fmoc-Val-Cit-OH to p-aminobenzyl alcohol is 1:2-1:3.6; the total usage ratio of Fmoc-Val-Cit-OH to the condensing agent is 1:2-1:3.6; the reaction temperature is 30-70°C; the solvent used in step 1 is N,N-dimethylformamide, toluene, acetonitrile, ethyl acetate, methanol, 1,2-dichloroethane, N,N-dimethylformamide / acetonitrile, N,N-dimethylformamide / 1,2 dichloroethane, N,N-dimethylformamide / ethyl acetate, preferably N,N-dimethylformamide / acetonitrile.

[0020] Furthermore, in the reaction of step 2, the base used is any one of triethylamine, diethylamine, diisopropylamine, piperidine, tetrabutylammonium fluoride, morpholine, and 1,8-diazabicyclo[5.4.0]undec-7-ene, preferably triethylamine; in the reaction of step 2, the molar ratio of Fmoc-Val-Cit-PAB-OH to the base is 1:1.1-1:1.8; the reaction temperature is 25-70°C; the solvent used in step 2 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylformamide.

[0021] Furthermore, in the reaction of step 3, the molar ratio of Val-Cit-PAB-OH to 6-(maleimido)hexanoic acid succinimide ester is 1:1.1-1:1.8; the reaction temperature is 25-70°C; the solvent used in step 3 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylacetamide.

[0022] Furthermore, in the reaction of step 4, the catalyst used is N,N-diisopropylethylamine; in the reaction of step 4, the molar ratio of MC-Val-Cit-PAB-OH to di(p-nitrobenzene) carbonate is 1:1.2-1:2.1, the molar ratio of MC-Val-Cit-PAB-OH to the catalyst is 1:1.2-1:2.0, the reaction temperature is 25-70°C, and the solvent used in step 4 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylacetamide.

[0023] The technical solution of the present invention has the following beneficial effects:

[0024] 1. The present invention uses compound I Fmoc-Val-Cit-OH: (S)-2-((S)-2-(((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino as the starting material for drug synthesis, and completes the synthesis of Val-Cit dipeptide linker compound VMC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate;

[0025] 2. All synthetic conditions are mild and controllable, and the yield of each step is high. This is the first synthetic process of Val-Cit dipeptide linker compound VMC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate in China and abroad;

[0026] 3. The reaction conditions were optimized, and green and efficient trimethylacetic anhydride was used as a condensation agent, which reduced the environmental pollution caused by the synthesis from the source;

[0027] 4. Compound VMC-Val-Cit-PAB-PNP produced by the method of the present invention: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate, which appears as a light yellow solid.

[0028] Compared with the previous process, the invented new process has the following advantages: 1) the use of trimethylacetic anhydride as a condensation agent for the synthesis of Fmoc-Val-Cit-PAB-OH is environmentally friendly and in line with the concept of green sustainable production; 2) the post-treatment operation is simple and easy to implement, and has the potential for large-scale industrial production. In view of the research on condensation agents, the addition of environmentally friendly condensation agents, and the simplification of post-treatment operations, the new synthesis process of Val-Cit dipeptide linkers has higher value and better application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the hydrogen spectrum of the product of step (1) in the embodiment of the present invention;

[0030] Figure 2 is the hydrogen spectrum of the product of step (2) in the embodiment of the present invention;

[0031] Figure 3 is the hydrogen spectrum of the product of step (3) in the embodiment of the present invention;

[0032] Figure 4 This is the hydrogen spectrum of the product of step (4) in the embodiment of the present invention. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0034] Example 1

[0035] A method for synthesizing a Val-Cit dipeptide linker comprises the following steps:

[0036] (1) Compound II Fmoc-Val-Cit-PAB-OH: Synthesis of [(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamic acid (9H-fluoren-9-yl)methyl ester

[0037] Under nitrogen atmosphere, (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (1 g, 2 mmol) and p-aminobenzyl alcohol (0.3720 g, 3 mmol) were dissolved in 6 mL DMF and 15 mL MeCN (0.4 mL, 1 mL). The mixture was stirred at room temperature for 0.5 h. Trimethylacetic anhydride (0.5626 g, 3 mmol) was added. The mixture was heated to 50 °C for 6 h. The mixture was returned to room temperature. P-aminobenzyl alcohol was added. Benzyl alcohol (0.3720 g, 3 mmol) and trimethylacetic anhydride (0.5626 g, 3 mmol) were heated to 50 °C for 8 h. A white solid-liquid mixture was obtained after the reaction, which was directly filtered and the filter cake was washed with ethyl acetate to obtain a portion of the product. The filtrate was added with 20 mL of acetonitrile for recrystallization, filtered, and the filter cake was washed with ethyl acetate to obtain a portion of the product. The total yield was 89.2%, and the product was a white solid. 1HNMR (400 MHz, DMSO-d6) δ9.99 (s, 1H), 8.11 ( d,J=7.6Hz,1H),7.90(d,J=7.5Hz,2H),7.75(t,J=7.9Hz,2H),7.55(d,J=8.2Hz,2H),7.49-7.38(m,3H), 7.33(t,J=7.4Hz,2H),7.24(d,J=8.2Hz,2H),5.98(t,J=5.8Hz,1H),5.41(s,2H),5.11(t,J=5.7Hz,1H), 4.43 (d, J = 5.6 Hz, 3H), 4.36-4.21 (m, 3H), 3.93 (dd, J = 8.9, 7.0 Hz, 1H), 2.97 (dh, J = 26.4, 6.7 Hz, 2H), 2.00 (q, J = 6.8 Hz, 1H), 1.63 (dd, J = 35.3, 9.2 Hz, 2H), 1.46-1.34 (m, 2H), 0.87 (dd, J = 11.1, 6.7 Hz, 6H). The hydrogen spectrum of the product is shown in the attached figure. Figure 1 shown.

[0038] (2) Synthesis of Compound III Val-Cit-PAB-OH: (S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

[0039] [(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamic acid (9H-fluorene-9-yl)methyl ester (0.9g, 1.5mmol) was dissolved in 10mL DMF, triethylamine (3g, 30mmol) was slowly added, and the reaction was heated to 50°C for 8h. After that, the reaction solution was brought to room temperature, and the reaction solution was added dropwise to 30mL ethyl acetate, stirred for 2h, and a white solid-liquid mixture was obtained, which was filtered and the filter cake was washed with ethyl acetate. (S)-2-((S)-2-amino-3-methylbutyramide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide was obtained with a yield of 94.7% and a product as a white solid. 1H NMR (400MHz, DMSO-d6) δ10.08 (s, 1H), 8.16 (d, J = 8.1Hz, 1H), 7.56 (d, J = 8.3Hz, 2H), 7.24 (d, J = 8.3Hz, 2H), 6.05 (q, J = 7.4, 5.9Hz, 1H), 5.44 (s, 2H), 5.13 (s, 1H), 4.43 (s, 3H), 3.15-2.87 (m, 4H), 2.00-1.30 (m, 6H), 0.89 (d, J = 6.8Hz, 3H), 0.79 (d, J = 6.8Hz, 3H). The hydrogen spectrum of the product is shown in the attached figure. Figure 2 shown.

[0040] (3) Synthesis of Compound IV MC-Val-Cit-PAB-OH: 6-(2,5-dioxo-2,5-dihydro-1-pyrrolyl)-N-[(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureido-2-pentyl]amino]-3-methyl-1-oxo-2-butyl]hexanamide

[0041] (S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanoyl (0.4 g, 1.05 mmol) was dissolved in 5 mL DMAc, 6-(maleimido)hexanoic acid succinimidyl ester (0.3887 g, 1.26 mmol) was added, and the mixture was heated to 50°C for 7 h. After that, the reaction solution was cooled to room temperature, and the reaction solution was added dropwise to 20 mL ethyl acetate and stirred for 2 h to obtain a white solid-liquid mixture, which was filtered and the filter cake was washed with ethyl acetate. 6-(2,5-dioxo-2,5-dihydro-1-pyrrolyl)-N-[(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureido-2-pentyl]amino]-3-methyl-1-oxo-2-butyl]hexanamide was obtained with a yield of 89.6% and the product was a white solid. 1H NMR(400MHz, DMSO-d6)δ9.91(s,1H),8.07(d,J=7.5Hz,1H),7.82(d,J=8.7Hz,1H),7.58-7.49(m,2H),7.23(d,J=8.3 Hz,2H),7.01(s,2H),5.98(t,J=5.8Hz,1H),5.41(s,2H),5.10(t,J=5.7Hz,1H),4.43(d,J=5.7Hz,2H),4.19(dd,J=8 .7,6.8Hz,1H),2.99(ddd,J=26.6,13.2,6.7Hz,2H),2.90(s,2H),2.74(s,2H),2.15(ddq,J=21.3,14.2,7.3Hz,2H),2.02-1.88(m,2H),1.77-1.56(m,2H),1.48(p,J=7.1Hz,4H),1.25-1.12(m,2H),0.84(dd,J=12.9,6.7Hz,6H). The hydrogen spectrum of the product is shown in the attached figure. Figure 3 shown.

[0042] (4) Compound V: Synthesis of MC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate

[0043] Under nitrogen atmosphere, 6-(2,5-dioxo-2,5-dihydro-1-pyrrolyl)-N-[(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureido-2-pentyl]amino]-3-methyl-1-oxo-2-butyl]hexanamide (0.3 g, 0.52 mmol) and di(p-nitrobenzene) carbonate (0.2867 g, 0.94 mmol) were dissolved in 4 mL DMAc and stirred at room temperature for 0.5 h. N,N-diisopropylethylamine (0.1083 g, 0.84 mmol) was added and heated to 50 ° C for 7 h. After that, the reaction solution was cooled to room temperature and added dropwise to 15 mL ethyl acetate and stirred for 2 h to obtain a solid-liquid mixture. The mixture was filtered and the filter cake was washed with ethyl acetate. The yield was 78.7% and the product was light yellow solid particles. 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.35-8.28(m,2H),8.12(d,J=7.4Hz,1H),7.82(d,J=8.6Hz,1H) ,7.69-7.60(m,2H),7.61-7.54(m,2H),7.41(d,J=8.6Hz,2H),7.01(s,2H),5.99(t,J=5.9Hz,1H),5.4 3 (s, 2H), 5.25 (s, 2H), 4.42-4.37 (m, 2H), 4.20 (dd, J = 8.6, 6.8 Hz, 1H), 2.99 (dp, J = 26.8, 6.6 Hz, 3H), 2.15 (ddq, J = 21.4, 14.3, 7.4 Hz, 3H), 1.18 (td, J = 7.3, 2.5 Hz, 4H), 0.84 (dd, J = 13.5, 6.7 Hz, 6H). The hydrogen spectrum of the product is shown in the attached figure. Figure 4 shown.

[0044] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for synthesizing a Val-Cit dipeptide linker, characterized in that: The steps include: Step 1: Acid amine condensation reaction: Compound I Fmoc-Val-Cit-OH and p-aminobenzyl alcohol are dissolved in a solvent under a nitrogen atmosphere, a condensing agent is added at 30-70°C, and the reaction is carried out for 12-24 hours. P-aminobenzyl alcohol and the condensing agent are added again, and the reaction is carried out at the same temperature for 12-24 hours. Compound II Fmoc-Val-Cit-PAB-OH is obtained by recrystallization. The reaction formula is as follows; Step 2: Reaction of removing Fmoc group: dissolve compound II Fmoc-Val-Cit-PAB-OH in a solvent, add base in batches and react at 25-70°C for 5-14h, and recrystallize to obtain compound III Val-Cit-PAB-OH. The reaction formula is as follows; Step 3: Amine ester condensation reaction: Compound III Val-Cit-PAB-OH is dissolved in a solvent, and raw material 6-(maleimido)hexanoic acid succinimidyl ester is added, reacted at 25-70° C. for 5-14 hours, and recrystallized to obtain compound IV MC-Val-Cit-PAB-OH, and the reaction formula is as follows; Step 4: Transesterification reaction: Compound IV MC-Val-Cit-PAB-OH and raw material di(p-nitrophenyl) carbonate are dissolved in a solvent under a nitrogen atmosphere, a catalyst is added, and the reaction is carried out at 25-70° C. for 5-14 hours, and the compound V MC-Val-Cit-PAB-PNP is obtained by recrystallization, that is, the Val-Cit dipeptide linker is obtained. The reaction formula is as follows; 2. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 1, the condensing agent used is any one of trimethylacetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, N,N'-carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide / 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide / 1-hydroxybenzotriazole, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate / N-methylimidazole, preferably trimethylacetic anhydride.

3. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 1, the total amount ratio of Fmoc-Val-Cit-OH to p-aminobenzyl alcohol is 1:2-1:3.6 by mole; the total amount ratio of Fmoc-Val-Cit-OH to the condensing agent is 1:2-1:3.6; the reaction temperature is 30-70°C; the solvent used in step 1 is one of N,N-dimethylformamide, toluene, acetonitrile, ethyl acetate, methanol, 1,2-dichloroethane, N,N-dimethylformamide / acetonitrile, N,N-dimethylformamide / 1,2 dichloroethane, and N,N-dimethylformamide / ethyl acetate, preferably N,N-dimethylformamide / acetonitrile.

4. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 2, the base used is any one of triethylamine, diethylamine, diisopropylamine, piperidine, tetrabutylammonium fluoride, morpholine, and 1,8-diazabicyclo[5.4.0]undec-7-ene, preferably triethylamine.

5. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 2, the ratio of Fmoc-Val-Cit-PAB-OH to the base is 1:1.1-1:1.8 by mole; the reaction temperature is 25-70° C.; the solvent used in step 2 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylformamide.

6. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 3, the molar ratio of Val-Cit-PAB-OH to 6-(maleimido)hexanoic acid succinimide ester is 1:1.1-1:1.8; the reaction temperature is 25-70°C; the solvent used in step 3 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylacetamide.

7. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 4, the catalyst used is N,N-diisopropylethylamine.

8. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that: In the reaction of step 4, the molar ratio of MC-Val-Cit-PAB-OH to di(p-nitrobenzene) carbonate is 1:1.2-1:2.1, the molar ratio of MC-Val-Cit-PAB-OH to the catalyst is 1:1.2-1:2.0, the reaction temperature is 25-70°C, and the solvent used in step 4 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane, preferably N,N-dimethylacetamide.

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