Liquid phase synthesis method of linker and its synthetic product

Abstract

The present invention relates to the technical field of polypeptide conjugate synthesis, and in particular to a liquid phase synthesis method of a linker and a synthetic product thereof. The method comprises synthesizing by referring to the following synthesis route: #imgabs0#. The new synthesis route can improve the problems of poor crystal form, difficulty in filtration and difficulty in purification caused by Fmoc-Val-Cit formed by sequentially connecting amino acids in the prior art, greatly improve the product purity, yield and reduce the time cost. At the same time, the conversion rate of Fmoc-Val-OH and Cit-PAB is high, which improves the yield of intermediate C.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide conjugate synthesis, and in particular to a liquid phase synthesis method of a linker and a synthetic product thereof. Background Art

[0002] ADC drugs are composed of three parts: antibodies, linkers, and cytotoxins. Antibodies can specifically target specific antigens, which are overexpressed in tumor cells and lowly expressed or not expressed in normal cells. Linkers are bridges for ADC drugs, connected to antibodies through cleavable and non-cleavable linkers. Linkers are stable under physiological conditions and have the characteristics of being released at specific sites. Cytotoxins have the characteristics of high toxic activity and low immunity, and have active groups connected to linkers and clear mechanisms of action.

[0003] The compound represented by the following formula (1) is a commonly used linker:

[0004] In the existing synthesis process, Fmoc-Val-OH, H-Cit-OH, 4-aminobenzyl alcohol and p-nitrobenzene chloroformate are reacted, and then amino acids are sequentially connected from front to back to form the linker. However, in the synthesis of Fmoc-Val-Cit, the product crystal form is poor, the purification operation is extremely difficult and not easy to operate, resulting in low purity of the intermediate; in the synthesis of Fmoc-Val-Cit-PAB, the reaction rate is slow and the reaction time is as long as 36 hours, the time cost is expensive, and the purity is not high; in the synthesis of Fmoc-Val-Cit-PAB-PNP, a compound with higher purity is obtained by preparing a liquid phase, but the purification cost is high and the yield is low, which is not conducive to industrial production.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a liquid phase synthesis method of a linker and a synthetic product thereof. The embodiment of the present invention provides a new liquid phase synthesis method, which improves the conversion rate of raw materials and can improve the yield and purity of the product.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a liquid phase synthesis method of a linker, which is synthesized according to the following synthesis path:

[0009]

[0010] In an optional embodiment, the step of forming intermediate A comprises: condensing Fmoc-L-Cit and p-aminobenzyl alcohol by a condensation agent;

[0011] Preferably, the step of forming intermediate A comprises: mixing the Fmoc-L-Cit, the p-aminobenzyl alcohol, the condensation agent and the solvent for condensation reaction;

[0012] Alternatively, the step of forming the intermediate A comprises: mixing the Fmoc-L-Cit, the p-aminobenzyl alcohol, the condensing agent, the base and the solvent for condensation reaction;

[0013] Preferably, the method further comprises: performing post-treatment after the condensation reaction is completed, wherein the post-treatment comprises filtering, slurrying with methyl tert-butyl ether and dichloromethane, and then filtering and drying.

[0014] In an optional embodiment, the condensing agent is selected from any one or two or more of N,N"-diisopropylcarbodiimide, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, N,N"-dicyclohexylcarbodiimide, N,N"-carbonyldiimidazole, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably N,N"-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole;

[0015] The organic solvent used is any one of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol, and chloroform, or a mixture of any of them in any proportion, preferably DMF;

[0016] The base used is any one of triethylamine, N,N-diisopropylethylamine, sodium bicarbonate, sodium carbonate or DBU or a mixture of any of them in any proportion, preferably N,N-diisopropylethylamine;

[0017] Preferably, the molar ratio of the Fmoc-L-Cit, the p-aminobenzyl alcohol and the condensing agent is 1:1-1.3:2-2.8;

[0018] Preferably, the molar ratio of the Fmoc-L-Cit, the p-aminobenzyl alcohol, the N,N"-dicyclohexylcarbodiimide and the 1-hydroxybenzotriazole is 1:1-1.3:1-1.5:1-1.3, and the best is 1:1.05:1.2:1.3;

[0019] Preferably, the condensation reaction temperature is -10 to 0°C and the time is 10-30 hours, preferably the temperature is -10°C and the time is 18 hours.

[0020] In an optional embodiment, the step of forming intermediate B comprises: removing Fmoc from intermediate A by an organic base;

[0021] Preferably, the step of forming the intermediate B comprises: mixing the intermediate A, the organic base and a solvent for reaction;

[0022] Preferably, the method further comprises: performing post-treatment after the reaction is completed, wherein the post-treatment comprises: concentrating to remove the solvent, adjusting the pH, extracting with ethyl acetate, and then collecting the aqueous phase.

[0023] In an optional embodiment, the organic base includes any one of piperidine, diazabicyclo, diethylamine, ethanolamine, cyclohexylamine and morpholine, preferably diethylamine;

[0024] The solvent includes any one of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform or a mixture of any proportions thereof, preferably DMF;

[0025] Preferably, the mass ratio of the intermediate A to the organic base is 1:0.5-3, preferably 1:2.5;

[0026] The reaction temperature is 0-40°C, preferably 25°C, and the reaction time is 1-8 hours, preferably 6 hours.

[0027] In an optional embodiment, the step of forming intermediate C comprises: condensing Fmoc-L-Val-OH and intermediate B by a condensation agent to perform a condensation reaction;

[0028] Preferably, the step of forming the intermediate C comprises: mixing the Fmoc-L-Val-OH, the intermediate B, the condensation agent, the solvent and the base for condensation reaction;

[0029] Preferably, the method further comprises: performing post-treatment after the condensation reaction is completed, and the post-treatment comprises: cooling the reaction solution to crystallize, and then filtering and drying.

[0030] In an optional embodiment, the condensing agent includes: any one or two or more of N,N"-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N"-dicyclohexylcarbodiimide, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole;

[0031] The solvent includes any one of ethyl acetate, dichloromethane, tetrahydrofuran, water, acetonitrile, DMF, dichloromethane and chloroform or a mixture of any proportions thereof, preferably DMF;

[0032] The base includes any one of triethylamine, N,N-diisopropylethylamine, sodium bicarbonate, sodium carbonate and DBU, preferably triethylamine;

[0033] Preferably, the molar ratio of the Fmoc-L-Val-OH, the intermediate B and the condensing agent is 1:1-1.3:2-2.8;

[0034] Preferably, the molar ratio of the Fmoc-L-Val-OH, the intermediate B, the 1-hydroxybenzotriazole and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-1.3:1-1.3:1-1.5; the best is 1:1.05:1.1:1.2;

[0035] Preferably, the reaction temperature is -10°C-0°C, preferably -5°C; the reaction time is 10-20 hours, preferably 15 hours.

[0036] In an optional embodiment, the method comprises: subjecting the intermediate C to an ester exchange reaction with di(p-nitrobenzene) carbonate under alkaline conditions;

[0037] Preferably, the method comprises: mixing the intermediate C, the di(p-nitrobenzene) carbonate, a base and a solvent to carry out an ester exchange reaction;

[0038] Preferably, the method comprises: after the transesterification reaction, adding the reaction solution into an ester solvent for crystallization, and then filtering and drying.

[0039] In an optional embodiment, the base includes any one of triethylamine, N,N-diisopropylethylamine, pyridine, N-methylmorpholine and DBU; preferably N-methylmorpholine;

[0040] The solvent includes any one of 1,4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform or a mixture of any proportions thereof, preferably 1,4-dioxane;

[0041] Preferably, the molar ratio of intermediate C, di(p-nitrobenzene) carbonate and base is 1:1-4:1-4, and the best is 1:1.5:2.5;

[0042] Preferably, the temperature is -10-30°C, optimally 25°C, and the preferred reaction time is 2-8h, optimally 7h.

[0043] In a second aspect, the present invention provides a liquid phase synthesis product, which is prepared by the liquid phase synthesis method of the linker described in any one of the aforementioned embodiments.

[0044] The present invention has the following beneficial effects: The embodiment of the present invention provides a new synthesis route, which first synthesizes the intermediate fragment, then docks the front-end amino acid, and finally connects the back-end activated ester, thereby improving the problems of poor crystal form, difficult filtration and difficult purification caused by Fmoc-Val-Cit formed by sequentially connecting amino acids in the prior art, greatly improving the product purity, yield and reducing time cost. At the same time, the conversion rate of Fmoc-Val-OH and Cit-PAB is high, which improves the yield of intermediate C. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0046] Figure 1 A comparison diagram of the dotted plates provided for comparative example 3 of the present invention;

[0047] Figure 2 The HPLC spectrum of the reaction product provided in Comparative Example 4 of the present invention;

[0048] Figure 3 The HPLC spectrum of the reaction product provided in Comparative Example 5 of the present invention;

[0049] Figure 4 A comparison diagram of the dotted plates provided for comparative example 6 of the present invention;

[0050] Figure 5 The HPLC spectrum of the reaction product provided for Comparative Example 7 of the present invention. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0052] An embodiment of the present invention provides a liquid phase synthesis method of a linker, comprising:

[0053] It is synthesized according to the following synthesis route:

[0054]

[0055] Specifically, the operations are as follows:

[0056] (1) Formation of intermediate A:

[0057] Fmoc-L-Cit and p-aminobenzyl alcohol are subjected to condensation reaction by a condensation agent; specifically, the Fmoc-L-Cit, p-aminobenzyl alcohol, the condensation agent and a solvent are mixed for condensation reaction; a base can also be added during the reaction. After the reaction is completed, post-treatment is performed, which includes filtering, pulping with methyl tert-butyl ether and dichloromethane, and then filtering and drying.

[0058] Using a specific solvent for pulping is helpful to improve the purity of the product. If the pulping solvent is changed, the purity of the product may be reduced.

[0059] The condensing agent is one of N,N'-diisopropylcarbodiimide (DIC), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-carbonyldiimidazole (CDI), or a combination of two of N-hydroxysuccinimide (HOSu), 1-hydroxybenzotriazole (HOBT), p-nitrophenol (PN) and pentafluorophenol (PFP); preferably, DCC and 1-hydroxybenzotriazole (HOBT) are used.

[0060] The organic solvent used is any one of ethyl acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, DMF, methanol, and chloroform, or a mixture of any of them in any proportion, preferably DMF.

[0061] The base used is triethylamine (Et3N), N,N-diisopropylethylamine (DIEA), sodium bicarbonate, sodium carbonate, DBU or no base is added, preferably N,N-diisopropylethylamine (DIEA).

[0062] The reaction conditions are as follows: the molar ratio of Fmoc-L-Cit, the p-aminobenzyl alcohol and the condensing agent is 1:1-1.3:2-2.8; specifically, the molar ratio of Fmoc-L-Cit, p-aminobenzyl alcohol (PAB), DCC and HOBT is 1:1-1.3:1-1.5:1-1.3, and the best is 1:1.05:1.2:1.3. The temperature of the condensation reaction is -10 to 0°C, and the best is -10°C. The preferred reaction time is 10-30h, and the best is 18h.

[0063] The above-mentioned synthesis conditions are beneficial for obtaining a product with higher purity and also improve the yield of intermediate A.

[0064] (2) Formation of intermediate B:

[0065] The intermediate A is subjected to organic base to remove Fmoc; the specific operation is as follows: the intermediate A, the organic base and the solvent are mixed to react; after the reaction is completed, the solvent is concentrated to remove the solvent, and then the pH is adjusted and then extracted with ethyl acetate, and then the aqueous phase is collected.

[0066] In the embodiment of the present invention, the collected aqueous phase can be directly used as a reaction raw material for the next reaction through a specific method without further post-treatment, and the water in the aqueous phase will not affect the subsequent reaction.

[0067] The organic base is one of piperidine, diazabicyclo (DBU), diethylamine, ethanolamine, cyclohexylamine and morpholine, preferably diethylamine.

[0068] The solvent used is any one of ethyl acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, DMF, methanol, and chloroform, or a mixture of any of them in any proportion, preferably DMF.

[0069] The reaction conditions include: the mass ratio of intermediate A to organic base is 1:0.5-3, preferably 1:2.5, the reaction temperature is 0-40°C, preferably 25°C, and the reaction time is 1-8h, preferably 6h.

[0070] (3) Formation of intermediate C;

[0071] Fmoc-L-Val-OH and intermediate B are condensed by a condensing agent to carry out a condensation reaction; specifically, the Fmoc-L-Val-OH, the intermediate B, the condensing agent, a solvent and a base are mixed to carry out a condensation reaction; and after the reaction is completed, the reaction solution is cooled to crystallize, and then filtered and dried.

[0072] The embodiment of the present invention adopts cooling crystallization, which can obtain intermediate C with higher purity compared with concentration to remove the solvent.

[0073] Furthermore, the condensing agent is a combination of two of N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCL), N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (HOSu), 1-hydroxybenzotriazole (HOBT), p-nitrophenol (PN) and pentafluorophenol (PFP); preferably EDC.HCL and HOBT.

[0074] The solvent used is any one of ethyl acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), water, acetonitrile, DMF, dichloromethane, and chloroform, or a mixture of any of them in any proportion, preferably DMF.

[0075] The base used was triethylamine (Et3 N), N, N-diisopropylethylamine (DIEA), sodium bicarbonate, sodium carbonate, DBU, preferably triethylamine (Et 3 N).

[0076] The reaction conditions include: the molar ratio of Fmoc-L-Val-OH, the intermediate B and the condensation agent is 1:1-1.3:2-2.8; preferably, the molar ratio of Fmoc-L-Val-OH, the intermediate B, HOBT and EDC.HCL is 1:1-1.3:1-1.3:1-1.5, and the best is 1:1.05:1.1:1.2; the temperature of the condensation reaction is preferably -10-0°C, and the best is -5°C. The preferred reaction time is 10-20h, and the best is 15h.

[0077] (4) Synthetic linkers;

[0078] The intermediate C and di(p-nitrobenzene) carbonate are subjected to an ester exchange reaction under alkaline conditions; specifically, the intermediate C, the di(p-nitrobenzene) carbonate, a base and a solvent are mixed to carry out an ester exchange reaction. After the reaction is completed, the reaction solution is added to an ester solvent for crystallization, and then filtered and dried.

[0079] Wherein, the base includes any one of triethylamine, N,N-diisopropylethylamine, pyridine, N-methylmorpholine and DBU; preferably N-methylmorpholine;

[0080] The solvent includes any one of 1,4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform or a mixture of any proportions thereof, preferably 1,4-dioxane;

[0081] Preferably, the molar ratio of intermediate C, di(p-nitrobenzene) carbonate and base is 1:1-4:1-4, and the best is 1:1.5:2.5;

[0082] Preferably, the temperature is -10-30°C, optimally 25°C, and the preferred reaction time is 2-8h, optimally 7h.

[0083] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0084] Example 1

[0085] This embodiment provides a method for synthesizing a linker, comprising:

[0086] The intermediate A is formed by referring to the following synthetic route:

[0087]

[0088] Dissolve 10.00g Fmoc-L-Cit and 3.25g p-aminobenzyl alcohol in 50ml DMF. Cool to -10°C and add 9.33g EEDQ. After 18 hours of reaction, filter with suction, pour the filtrate into 500mL methyl tert-butyl ether, stir for 30min, filter, beat the filter cake twice with 100mL dichloromethane solution, filter with suction to obtain a white solid, dry at 50 degrees Celsius to obtain 9.8g white solid, yield 77.1% (12.7g), purity is 99.3%; MHz, (C 2 D 6 OS): δ = 1.34-1.50 (m, 2H), 1.56-1.71 (m, 2H), 2.91-3.09 (m, 2H), 4.07-4.26 (m, 4H), 4.41-4.43 (d , 2H), , 5.09-5.12(t, 3H), 5.45-5.49(d, 2H), 5.99-6.05(m, 1H), 7.22-7.85(m, 13H), 9.99(s, 1H).

[0089] Intermediate B was synthesized by following the synthetic route:

[0090]

[0091] 2. Dissolve 5.00 g of Fmoc-L-Cit-PAB in 15 mL of DMF, add 7.5 mL of diethylamine and react at 25 ° C for 6 hours. After the reaction is completed, concentrate at 45 ° C to remove diethylamine, then add 45 mL of water, adjust the pH of the solution to 4 with 1 mol / L hydrochloric acid aqueous solution, and then extract impurities with 15 mL of ethyl acetate for 3 times, collect the aqueous phase, and use it directly in the next reaction.

[0092] Intermediate C was synthesized by referring to the following synthetic route;

[0093]

[0094] 3.18g Fmoc-L-Val-OH and 1.39g HOBT were dissolved in the filtrate 2. When the temperature was lowered to -5°C, 2.15g EDCI and 1.82g DIEA were added and the reaction was continued for 15 hours. After the reaction was completed, the reaction solution was added to water for crystallization, filtered, and dried at 50°C to obtain 3.82g of white solid, with a purity of 98% as determined by HPLC and a yield of 67.8% (5.63g). 1 HNMR (400 MHz, C 2 D 6OS): δ=0.82-0.86(m, 6H), 1.30-1.71(m, 4H), 1.92-2.01(m, 1H), 2.84-3.04(m, 2H), 3.88-3.92(m, 1H), 4.17-4.2 3(m, 2H), 4.37-4.42(m, 3H), 5.06-5.09(t, 1H), 5.39(s, 2H), 5.94-5.97(t, 1H), 7.19-8.09(m, 14H), 9.95(s, 1H).

[0095] The linker was synthesized according to the following synthetic route;

[0096]

[0097] 5.00g Fmoc-Val-Cit-PAB was added to 50mL DMF and stirred to dissolve, then 3.79g di(p-nitrobenzene) carbonate was added, and 1.68g N-methylmorpholine was added when the temperature was cooled to 0°C in an ice-salt bath, and the mixture was returned to room temperature for 7 hours. After the reaction was completed, the reaction solution was added to ethyl acetate for crystallization, filtered, and dried at 40°C to obtain 5.10g of a white solid with an HPLC purity of 98% and a yield of 80.0% (6.37g). 1 HNMR (400 MHz, C 2 D 6 OS): δ=0.79-0.87(m, 6H), 1.32-1.71(m, 4H), 1.96-2.03(m, 1H), 2.88-3.03(m, 2H), 3.89-4 .43(m, 5H), 5.21(s, 2H), 5.40(s, 2H), 5.95-5.98(t, 1H), 7.27-8.28(m, 18H), 10.12(s, 1H).

[0098] Comparative Example 1

[0099] Intermediate C was synthesized by following the synthetic route:

[0100]

[0101] (1) 10.00 g of Fmoc-L-Val-OH and 3.73 g of NHS were dissolved in 50 ml of THF, cooled to below 10°C, and 7.29 g of DCC was added. The reaction was allowed to warm naturally for 10 h. After the reaction was completed, the byproduct DCU was filtered off and the filtrate was collected. After the filtrate was concentrated to an oily state, 4 times of MTBE was added for crystallization, and a white solid was obtained by filtration. The yield was 62% (7.90 g).

[0102]

[0103] (2) Dissolve 5 g of Fmoc-L-Val-OSU and 2.21 g of L-Cit-OH in THF and water. When the temperature drops to -5°C, add 2.22 g of DIEA and react at room temperature for 12 hours. After the reaction is completed, extract the product with ethyl acetate, wash the ethyl acetate phase with acid water, wash with water, dry with anhydrous sodium sulfate, filter, and concentrate at 50°C to obtain 5.01 g of white solid, with a yield of 87.8% (5.01 g).

[0104]

[0105] (3) Dissolve 5.00 g of Fmoc-Val-Cit-OH and 2.48 g of p-aminobenzyl alcohol in 50 ml of DMF. Cool to -10°C. Add 4.98 g of EEDQ. After reacting at room temperature for 36 hours in the dark, filter with suction. Pour the filtrate into 500 mL of methyl tert-butyl ether, stir for 30 min, filter with suction to obtain a white solid, and dry at 50°C to obtain 3.15 g of a yellow solid. The purity determined by HPLC is 94%, and the yield is 51.9% (3.15 g).

[0106] It can be seen that when the intermediate C is synthesized from Fmoc-L-Val-OH in this comparative example, the yield of the intermediate C in this comparative example is only 28.2%, while the yield of this embodiment is 67.8%. It can be seen that the yield of the intermediate C formed by the method of the present invention is significantly improved.

[0107] Comparative Example 2

[0108] The linker was synthesized according to the following synthetic route;

[0109]

[0110] The intermediate C3.00g Fmoc-Val-Cit-PAB formed in Comparative Example 1 was added to 50mL DMF and stirred to dissolve, and then 2.27g di(p-nitrobenzene) carbonate was added, and 1.32g DIEA was added when the temperature was cooled to 0°C in an ice-salt bath, and the mixture was returned to room temperature for 7 hours. After the reaction was completed, the reaction solution was added to MTBE for crystallization, filtered, and dried at 40°C to obtain 2.71g of a yellow solid with an HPLC purity of 91% and a yield of 71.0% (2.71g).

[0111] Comparative Example 3

[0112] Intermediate A was prepared by referring to the preparation method provided in Example 1, except that the reaction temperature was room temperature (25°C). After the reaction time was the same for 18 hours, the reaction system for forming intermediate A in Example 1 and the reaction system of this comparative example were plated and then observed.

[0113] Results Figure 1 ,according to Figure 1It can be seen that changing the reaction temperature of intermediate A will lead to an increase in the impurity content.

[0114] Comparative Example 4

[0115] Intermediate A was prepared by referring to the preparation method provided in Example 1, except that ethyl acetate was used after the reaction was completed; that is, 500 mL of methyl tert-butyl ether in Example 1 was stirred for 30 min, filtered, and the filter cake was slurried twice with 100 mL of dichloromethane solution, and then 500 mL of ethyl acetate was stirred for 30 min, filtered, and the filter cake was slurried twice with 100 mL of ethyl acetate solution. The purity of the product of this comparative example was then tested, and the purity was 96.9%, see Figure 2 , HPLC see Figure 2 .

[0116] The purity of the intermediate A in Comparative Example 1 is 99.3%. It can be seen that changing the post-treatment method will lead to a significant decrease in the purity of the intermediate A.

[0117] Comparative Example 5

[0118] Intermediate C was prepared by referring to the preparation method provided in Example 1, except that: after the reaction was completed, the reaction solvent was removed by rotary evaporation, and then dried at 50°C. The purity of the obtained solid was 97.3%. HPLC Figure 3 .

[0119] The purity of the intermediate C in Comparative Example 1 is 98%. It can be seen that changing the post-treatment method will lead to a significant decrease in the purity of the intermediate C.

[0120] Comparative Example 6

[0121] The linker was prepared by referring to the preparation method provided in Example 1, except that the reaction temperature was room temperature, and after the same reaction time of 7 hours, the reaction system for forming the linker in Example 1 and the reaction system of this comparative example were plated and then observed.

[0122] Results Figure 4 ,according to Figure 4 It can be seen that changing the reaction temperature of the linker will lead to an increase in the impurity content.

[0123] Comparative Example 7

[0124] The linker was prepared by referring to the preparation method provided in Example 1, except that: after the reaction was completed, the reaction solution was added to methyl tert-butyl ether for crystallization, and then filtered and dried at 40°C. The purity of the obtained product was 91.9%. HPLC: Figure 5 .

[0125] The purity of the linker in Comparative Example 1 is 98%. It can be seen that changing the post-processing method will lead to a significant decrease in the purity of the linker.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A liquid phase synthesis method for a linker, It is characterized in that It is synthesized according to the following synthesis route: ， The step of forming intermediate A comprises: condensing Fmoc-L-Cit with p-aminobenzyl alcohol by a condensation agent; performing post-treatment after the condensation reaction, the post-treatment comprising filtering, beating with methyl tert-butyl ether and dichloromethane, and then filtering and drying; the condensation reaction temperature is -10 to 0°C, and the time is 10-30 hours; The step of forming intermediate B comprises: removing Fmoc from intermediate A by an organic base; The step of forming intermediate C comprises: condensing Fmoc-L-Val-OH with intermediate B by a condensing agent for condensation reaction; the reaction temperature is -10°C to 0°C, and the reaction time is 10-20 hours; The intermediate C is subjected to an ester exchange reaction with di(p-nitrobenzene) carbonate under alkaline conditions, wherein the reaction temperature is -10-30°C and the reaction time is 2-8h; After the transesterification reaction, the reaction solution is added into an ester solvent for crystallization, and then filtered and dried; the drying temperature is 40°C.

2. The liquid phase synthesis method of the linker according to claim 1, It is characterized in that The step of forming intermediate A comprises: mixing the Fmoc-L-Cit, the p-aminobenzyl alcohol, the condensation agent and the solvent for condensation reaction; Alternatively, the step of forming intermediate A comprises: mixing the Fmoc-L-Cit, the p-aminobenzyl alcohol, the condensing agent, the base and the solvent for condensation reaction.

3. The liquid phase synthesis method of the linker according to claim 2, It is characterized in that The condensing agent is selected from any one or two or more of N,N'-diisopropylcarbodiimide, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, N,N'-dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; The organic solvent used is any one of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol, and chloroform, or a mixture of any proportions thereof. The base used is any one of triethylamine, N,N-diisopropylethylamine, sodium bicarbonate, sodium carbonate or DBU or a mixture of any proportions thereof.

4. The liquid phase synthesis method of the linker according to claim 2, It is characterized in that The condensing agent is N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole; The organic solvent used was DMF; The base used was N,N-diisopropylethylamine.

5. The liquid phase synthesis method of the linker according to claim 2, It is characterized in that The molar ratio of the Fmoc-L-Cit, the p-aminobenzyl alcohol and the condensing agent is 1:1-1.3:2-2.

8.

6. The liquid phase synthesis method of the linker according to claim 4, It is characterized in that The molar ratio of the Fmoc-L-Cit, the p-aminobenzyl alcohol, the N,N'-dicyclohexylcarbodiimide and the 1-hydroxybenzotriazole is 1:1-1.3:1-1.5:1-1.

3.

7. The liquid phase synthesis method of the linker according to claim 1, It is characterized in that The condensation reaction temperature was -10°C and the reaction time was 18 hours.

8. The liquid phase synthesis method of the linker according to claim 1, It is characterized in that The step of forming the intermediate B comprises: mixing the intermediate A, the organic base and a solvent for reaction.

9. The liquid phase synthesis method of the linker according to claim 1, It is characterized in that Also includes: After the reaction is completed, post-treatment is performed, which includes: concentrating to remove the solvent, then adjusting the pH and then extracting with ethyl acetate, and then collecting the aqueous phase.

10. The liquid phase synthesis method of the linker according to claim 8, It is characterized in that The organic base includes any one of piperidine, diazabicyclo, diethylamine, ethanolamine, cyclohexylamine and morpholine, The solvent includes any one of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform, or a mixture of any proportions of the solvent.

11. The liquid phase synthesis method of the linker according to claim 8, It is characterized in that The organic base is diethylamine; and the solvent is DMF.

12. The liquid phase synthesis method of the linker according to claim 8, It is characterized in that The mass ratio of the intermediate A to the organic base is 1:0.5-3, the reaction temperature is 0-40°C, and the reaction time is 1-8 hours.

13. The liquid phase synthesis method of the linker according to claim 8, It is characterized in that The mass ratio of the intermediate A to the organic base is 1:2.5; The reaction temperature is 25°C and the reaction time is 6 hours.

14. The liquid phase synthesis method of the linker according to claim 1, It is characterized in that The step of forming the intermediate C comprises: mixing the Fmoc-L-Val-OH, the intermediate B, the condensation agent, the solvent and the base to carry out a condensation reaction.

15. The liquid phase synthesis method of the linker according to claim 14, It is characterized in that Also includes: After the condensation reaction is completed, post-treatment is performed, which includes: cooling the reaction solution to crystallize, and then filtering and drying.

16. The liquid phase synthesis method of the linker according to claim 14, It is characterized in that The condensing agent includes: any one or two or more of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; The solvent includes any one of ethyl acetate, dichloromethane, tetrahydrofuran, water, acetonitrile, DMF, dichloromethane and chloroform or a mixture of any proportions thereof. The base includes any one of triethylamine, N,N-diisopropylethylamine, sodium bicarbonate, sodium carbonate and DBU.

17. The liquid phase synthesis method of the linker according to claim 14, It is characterized in that The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole; The solvent is DMF; The base is triethylamine.

18. The liquid phase synthesis method of the linker according to claim 16, It is characterized in that The molar ratio of the Fmoc-L-Val-OH, the intermediate B and the condensing agent is 1:1-1.3:2-2.

8.

19. The liquid phase synthesis method of the linker according to claim 17, It is characterized in that The molar ratio of the Fmoc-L-Val-OH, the intermediate B, the 1-hydroxybenzotriazole and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-1.3:1-1.3:1-1.5; The reaction temperature for forming intermediate c is -5°C and the reaction time is 15 hours.

20. The liquid phase synthesis method of a linker according to claim 1, characterized in that: include: The intermediate C, the di(p-nitrobenzene) carbonate, a base and a solvent are mixed to carry out an ester exchange reaction.

21. The liquid phase synthesis method of the linker according to claim 20, It is characterized in that The base includes any one of triethylamine, N,N-diisopropylethylamine, pyridine, N-methylmorpholine and DBU; the solvent includes any one of 1,4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform or a mixture of several of them in any proportion.

22. The liquid phase synthesis method of the linker according to claim 20, It is characterized in that The base is N-methylmorpholine; The solvent is 1,4-dioxane.

23. The liquid phase synthesis method of the linker according to claim 20, It is characterized in that The molar ratio of intermediate C, di(p-nitrobenzene) carbonate and base is 1:1-4:1-4.

24. The liquid phase synthesis method of the linker according to claim 20, It is characterized in that The molar ratio of intermediate C, di(p-nitrobenzene) carbonate and base is 1:1.5:2.5; The temperature was 25°C and the reaction time was 7h.

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