A method for the synthesis of a val-cit dipeptide linker

Abstract

The application discloses a synthesis method of a Val-Cit dipeptide linker for ADCs drugs, and the synthesis of the drug is carried out by taking (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino as a starting material, all the synthesis conditions are mild and controllable, the yield of the four steps is high, and the new synthesis process of the Val-Cit dipeptide linker is original at home and abroad; the reaction conditions are optimized, green and efficient trimethyl acetic anhydride is used as a condensing agent, and the pollution of the synthesis to the environment is reduced from the source; the product yield is high, the generated impurities are few; the post-treatment operation is simple and easy to realize, and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug synthesis, and particularly relates to a synthesis method of a Val-Cit dipeptide linker. BACKGROUND

[0002] In recent years, tumors are still the main cause of human disease death, and the treatment method for malignant tumors gradually shifts from traditional chemotherapy to targeted therapy. However, overall, the traditional treatment method is still the first choice for clinical treatment, and the number of chemotherapy drugs accounts for the largest cancer treatment market. Small molecule chemotherapy drugs are prone to serious side effects due to their poor targeting and biological distribution. Compared with chemotherapy drugs, antibody drugs have better targeting and play an increasingly important role in the treatment of malignant tumors. However, they also have the disadvantages of poor tissue penetration, weak biological activity, and easy drug resistance. Based on the above problems, the continuous emergence of new therapies will inevitably bring revolutionary changes to tumor treatment.

[0003] Antibody conjugated drugs (ADCs) are a kind of biological drugs that link cytotoxic small molecule drugs and antibodies through chemical linkers, and have great development prospects. The antibody with targeting specificity binds to the antigen on the tumor cell membrane, realizes endocytosis, and makes the antibody and the small molecule drug connected thereto enter the cell, and then is degraded in the lysosome, and the small molecule drug is released into the cell to induce tumor cell apoptosis, so as to play the anti-cancer effect of ADCs drugs. Because ADCs drugs have the advantages of high activity, small side effects, and long action time, their emergence provides a new idea for "precision treatment" of tumors. The structure of ADCs contains three parts: small molecule drugs (cytotoxin), antibodies (antibody), and linkers (linker). The linker is used to realize the organic combination of the small molecule drug and the antibody, and is a crucial part. Valine-citrulline (Val-Cit) and para-aminobenzyl (PAB) spacer region coupling 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] The Val-Cit dipeptide linker is used in a large part of the articles researching anti-cancer drugs, and is well described, but only a few articles provide detailed experimental methods for synthesizing this specific linker. The Val-Cit dipeptide linker has been marketed, but it is expensive. Therefore, in order to construct various drug linkers, it is necessary to further develop a green and low-cost process route for synthesizing the Val-Cit dipeptide linker.

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

[0006] Dian Xiao et al reported that Fmoc-Val-Cit-OH was used as raw material, and p-aminobenzyl alcohol was used as condensing agent in the presence of EEDQ, methanol and dichloromethane were used as solvents for amide condensation reaction; NMP was used as solvent, diethylamine was used to remove Fmoc; and Mc-OSu was reacted, and then reacted with PNP-chloroformate. The reaction needs four steps of synthesis, and after the reaction, the post-treatment is carried out, and then the next step reaction is carried out. This method uses pyridine as a solvent, and column purification is required for post-treatment, and mass production is not easy to realize.

[0007] In summary, it is necessary to improve the existing process and develop a green and low-cost synthesis process of Val-Cit dipeptide linker. SUMMARY

[0008] In view of the above problems, the purpose of the present application is to provide a more green and efficient preparation method of Val-Cit dipeptide linker, which is simple in operation, high in yield and purity, and suitable for industrial production.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0010] A synthesis method of Val-Cit dipeptide linker, comprising 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-methylbutanoyl)amino, p-aminobenzyl alcohol is dissolved in a solvent under nitrogen atmosphere, a condensing agent is added at 30-70℃, and reacts for 12-24h, in order to make compound I completely react, p-aminobenzyl alcohol and condensing agent are added again, and react at the same temperature for 12-24h, and compound II Fmoc-Val-Cit-PAB-OH is obtained by recrystallization, and the reaction formula is as follows:

[0012]

[0013] Step 2: Fmoc group removal reaction: Compound II Fmoc-Val-Cit-PAB-OH is dissolved in a solvent, and a base is added in batches, and the reaction is carried out at 25-70°C for 5-14h, and compound III Val-Cit-PAB-OH is obtained by recrystallization, and the reaction formula is as follows:

[0014]

[0015] Step 3: Amine ester condensation reaction: Compound III Val-Cit-PAB-OH is dissolved in a solvent, and the raw material 6-(maleimide) hexanoic acid succinimidyl ester is added, and the reaction is carried out at 25-70°C for 5-14h. Recrystallization to obtain compound IV MC-Val-Cit-PAB-OH, and the reaction formula is as follows:

[0016]

[0017] Step 4: Ester exchange reaction: Compound IV MC-Val-Cit-PAB-OH and raw material di(p-nitrophenyl) carbonate are dissolved in a solvent under a nitrogen atmosphere, and a catalyst is added, and the reaction is carried out at 25-70°C for 5-14h, and compound V MC-Val-Cit-PAB-PNP is obtained by recrystallization, that is, the Val-Cit dipeptide linker is obtained, and the reaction formula is as follows.

[0018]

[0019] Further, in the step 1 reaction, the condensing agent used in the step 1 reaction is any one of trimethylacetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, N,N'-carbonyldiimidazole, N,N'-dicyclohexyl carbodiimide / 4-dimethylaminopyridine, N,N'-dicyclohexyl carbodiimide / 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / 1-hydroxybenzotriazole, N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate / N-methylimidazole, preferably trimethylacetic anhydride; the total amount ratio of Fmoc-Val-Cit-OH to p-aminobenzyl alcohol in step 1 reaction is 1:2-1:3.6 by mole; the total amount ratio of Fmoc-Val-Cit-OH to 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] Further, in the step 2 reaction, the base used is any one of triethylamine, diethylamine, diisopropylamine, piperidine, tetrabutylammonium fluoride, morpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene, preferably triethylamine; the ratio of Fmoc-Val-Cit-PAB-OH to the base used in step 2 reaction is 1:1.1-1:1.8 by mole; the reaction temperature is 25-70 DEG C; the solvent used in step 2 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, 1,2-dichloroethane, preferably N,N-dimethylformamide.

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

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

[0023] The technical scheme of the present application has the following beneficial effects:

[0024] 1. The present application uses compound I Fmoc-Val-Cit-OH: (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoyl)amino as a starting material for drug synthesis, and completes the synthesis of Val-Cit dipeptide linker compound V MC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutanoylamino)-5-ureidopentanoylamino)benzyl(4-nitrophenyl) carbonate;

[0025] 2. All synthesis conditions are mild and controllable, and each step has a high yield, which is the first 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)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate synthesis process at home and abroad;

[0026] 3. The reaction conditions are optimized, and green and efficient trimethyl acetic anhydride is used as a condensing agent, which reduces environmental pollution from the source;

[0027] 4. The compound VMC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate produced by the method has a light yellow solid appearance.

[0028] Compared with the previous process, the new process has the following advantages: 1) using trimethyl acetic anhydride as a condensing agent for synthesizing Fmoc-Val-Cit-PAB-OH has environmental friendliness, which meets the green and sustainable production concept; 2) the post-treatment operation is simple and easy to realize, and has a large potential for industrial production. In view of the research on the condensing agent, the addition of the environmentally friendly condensing agent, and the simplification of the post-treatment operation, the new process for synthesizing the Val-Cit dipeptide linker has higher value and better application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The hydrogen spectrum of the product of step (1) in the embodiment of the application;

[0030] Figure 2 The hydrogen spectrum of the product of step (2) in the embodiment of the application;

[0031] Figure 3 The hydrogen spectrum of the product of step (3) in the embodiment of the application;

[0032] Figure 4 The hydrogen spectrum of the product of step (4) in the embodiment of the application. DETAILED DESCRIPTION

[0033] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present application.

[0034] Embodiment 1

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

[0036] (1) Synthesis of compound II Fmoc-Val-Cit-PAB-OH: [(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] (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutyrylamino)(l g, 2 mmol), p-aminobenzyl alcohol (0.3720 g, 3 mmol) were dissolved in 6 mL of DMF and 15 mL of MeCN (0.4 mL, 1 mL), stirred at room temperature for 0.5 h, trimethylacetic anhydride (0.5626 g, 3 mmol) was added, heated to 50 °C for 6 h, returned to room temperature, and p-aminobenzyl alcohol (0.3720 g, 3 mmol) and trimethylacetic anhydride (0.5626 g, 3 mmol) were added, heated to 50 °C for 8 h. After the reaction, a white solid-liquid mixture was obtained, which was directly filtered, and the filter cake was washed with ethyl acetate to obtain a part of the product, and the filtrate was recrystallized with 20 mL of acetonitrile, filtered, and the filter cake was washed with ethyl acetate to obtain a part of the product, and the total yield was 89.2%, and the product was a white solid.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.9 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.49 - 7.38 (m, 3H), 7.33 (t, J = 7.4 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 5.98 (t, J = 5.8 Hz, 1H), 5.41 (s, 2H), 5.11 (t, J = 5.7 Hz, 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). The1H NMR spectrum of the product is shown in FIG. 1. Figure 1

[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-fluoren-9-yl)methyl ester (0.9 g, 1.5 mmol) was dissolved in 10 mL of DMF, triethylamine (3 g, 30 mmol) was added slowly, heated to 50 °C for 8 h. After that, the reaction was allowed to cool to room temperature, the reaction was added dropwise into 30 mL of ethyl acetate, stirred for 2 h, a white solid-liquid mixture was obtained, filtered, the filter cake was washed with ethyl acetate. (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4- (hydroxymethyl)phenyl)-5-ureidopentanamide was obtained with a yield of 94.7%, the product was a white solid.1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 6.05 (q, J = 7.4, 5.9 Hz, 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 = 6.8 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H). The hydrogen spectrum of the product is shown in Figure 1. Figure 2

[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-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (0.4 g, 1.05 mmol) was dissolved in 5 mL of DMAc, 6-(maleimidyl)hexanoic acid succinimidyl ester (0.3887 g, 1.26 mmol) was added, heated to 50 °C for 7 h. After that, the reaction was allowed to cool to room temperature, the reaction was added dropwise into 20 mL of ethyl acetate, stirred for 2 h, a white solid-liquid mixture was obtained, filtered, 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%, the product was a white solid. 1 ​H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.58 - 7.49 (m, 2H), 7.23 (d, J = 8.3 Hz, 2H), 7.01 (s, 2H), 5.98 (t, J = 5.8 Hz, 1H), 5.41 (s, 2H), 5.10 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.7 Hz, 2H), 4.19 (dd, J = 8.7, 6.8 Hz, 1H), 2.99 (ddd, J = 26.6, 13.2, 6.7 Hz, 2H), 2.90 (s, 2H), 2.74 (s, 2H), 2.15 (ddq, J = 21.3, 14.2, 7.3 Hz, 2H), 2.02 - 1.88 (m, 2H), 1.77 - 1.56 (m, 2H), 1.48 (p, J = 7.1 Hz, 4H), 1.25 - 1.12 (m, 2H), 0.84 (dd, J = 12.9, 6.7 Hz, 6H). The product hydrogen spectrum is shown in Figure 1 1. Figure 3

[0042] (4) Synthesis of Compound V MC-Val-Cit-PAB-PNP: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-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-ureidopentyl]amino]-3-methyl-1-oxo-2- butyl]hexanamide (0.3 g, 0.52 mmol), bis(p-nitrophenyl)carbonate (0.2867 g, 0.94 mmol) were dissolved in 4 mL of DMAc, stirred at room temperature for 0.5 h, added N,N- diisopropylethylamine (0.1083 g, 0.84 mmol), heated to 50 °C for 7 h, after which the reaction was allowed to come to room temperature, the reaction was added dropwise to 15 mL of ethyl acetate, stirred for 2 h, to obtain a solid-liquid mixture, filtered, the filter cake was washed with ethyl acetate. The yield was 78.7%, the product was a light yellow solid particle.1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.35 - 8.28 (m, 2H), 8.12 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.69 - 7.60 (m, 2H), 7.61 - 7.54 (m, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.01 (s, 2H), 5.99 (t, J = 5.9 Hz, 1H), 5.43 (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 product1H NMR spectrum is shown in Figure Figure 4

[0044] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which shall be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.​

Claims

1. A method for synthesizing a Val-Cit dipeptide linker, characterized in that, Includes the following steps: Step 1: Acid-amine condensation reaction: Compound I, Fmoc-Val-Cit-OH, and p-aminobenzyl alcohol were dissolved in a solvent under a nitrogen atmosphere. A condensing agent was added, and the reaction was carried out at 30-70℃ for 12-24 hours. Then, p-aminobenzyl alcohol and the condensing agent were added again, and the reaction was carried out at the same temperature for 12-24 hours. Recrystallization yielded compound II, Fmoc-Val-Cit-PAB-OH, as shown in the following reaction formula: ； Step 2: Removal of Fmoc group: Compound II Fmoc-Val-Cit-PAB-OH was dissolved in a solvent, and alkali was added in portions. The reaction was carried out at 25-70℃ for 5-14 hours, and recrystallized 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 was dissolved in a solvent, and the starting material 6-(maleimide)hexanoic acid succinimide ester was added. The reaction was carried out at 25-70℃ for 5-14 h, and recrystallization yielded compound IV MC-Val-Cit-PAB-OH. The reaction formula is as follows: ； Step 4: Transesterification reaction: Compound IV MC-Val-Cit-PAB-OH and the starting material di(p-nitrophenyl) carbonate were dissolved in a solvent under a nitrogen atmosphere. A catalyst was added, and the reaction was carried out at 25-70℃ for 5-14 hours. Recrystallization yielded compound V MC-Val-Cit-PAB-PNP, which is the Val-Cit dipeptide linker described above. The reaction formula is as follows: ； The condensing agent is trimethylacetic anhydride; the catalyst is N,N-diisopropylethylamine.

2. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that, In step 1, the total ratio of Fmoc-Val-Cit-OH to p-aminobenzyl alcohol is 1:2 to 1:3.6 (based on molar ratio); the total ratio of Fmoc-Val-Cit-OH to condensing agent is 1:2 to 1:3.6; the reaction temperature is 30-70℃; and the solvent used in step 1 is any 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.

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

4. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that, In step 2, the ratio of Fmoc-Val-Cit-PAB-OH to alkali is 1:1.1-1:1.8 (molar). The reaction temperature is 25-70℃. The solvent used in step 2 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane.

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

6. The method for synthesizing a Val-Cit dipeptide linker according to claim 1, characterized in that, In 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 catalyst is 1:1.2-1:2.0, the reaction temperature is 25-70℃, and the solvent used in step 4 is any one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, acetonitrile, tetrahydrofuran, and 1,2-dichloroethane.

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