Synthetic method of eptifibatide

Abstract

The invention discloses a synthesis method of eptifibatide, and relates to the technical field of polypeptide synthesis. The synthesis method comprises the following steps: carrying out deprotection treatment on initial resin, and loading the resin by taking Fmoc Cys (Trt) OH as first amino acid; sequentially carrying out coupling condensation on the remaining amino acids by adopting a solid-phase synthesis method, then carrying out solid-phase oxidation by using an iodine solution, and then carrying out splitting decomposition to obtain an eptifibatide crude product; the synthesis method is high in yield, low in cost, mild in reaction condition, small in environmental pollution and beneficial to industrialization.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis technology, and specifically relates to a method for synthesizing eptifibatide. Background Technology

[0002] Eptifibatide is a synthetic cyclic peptide containing one mercaptopropionic acid and six amino acid residues. It is a specific antagonist of the platelet glycoprotein GPIIb / IIIa receptor, selectively and reversibly inhibiting the final common pathway of platelet aggregation (the binding of plasma coagulation factor I to GPIIb / IIIa), thus reversing ischemic states caused by thrombosis. Clinically, eptifibatide is mainly used to treat unstable angina and acute myocardial infarction. Its main adverse reaction is bleeding, most of which is mild to moderate, with the most common bleeding site being the vascular puncture site during PCI; cerebral hemorrhage is rare. Eptifibatide was first developed by COR Therapeutics in the United States and was first marketed in July 1998 under the trade name Integrelin.

[0003] Currently, there are two main methods for preparing eptifibatide: solid-phase synthesis and liquid-phase synthesis. Liquid-phase synthesis involves more steps and is more time-consuming, while solid-phase synthesis is the commonly used method. However, current solid-phase synthesis methods suffer from problems such as high cost, long synthesis cycle, large reagent usage, and the need for large amounts of solvent for liquid-phase oxidation. This invention provides a method for preparing eptifibatide, mainly addressing the problems of large volume liquid-phase oxidation and difficulty in storing crude products in existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing epitaphthide, which has high yield, low cost, mild reaction conditions, minimal environmental pollution, and is conducive to industrialization.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention discloses a method for synthesizing epitubatine, comprising: Step 1) Deprotect the starting resin; Step 2) The protected amino acids are sequentially coupled and condensed using a solid-phase synthesis method to obtain a linear polypeptide resin: Step 3) Iodine solid-phase oxidative cyclization to obtain the oxidatively cyclized polypeptide; Step 4) Wash the resin with a mixture of vitamin C aqueous solution and DMF until no iodine color remains, then wash with DMF, then with methanol, and dry to obtain epitubapeptide cyclic peptide resin. Step 5) Crack the resin, add a precipitant to precipitate, and wash to obtain crude epitubatide.

[0006] This invention relates to a method for synthesizing eptifibatide. The technical solution of this invention includes the following steps: using RinkAmide-AM resin, Rink Amide-MBHA resin, Ramage Amide-AM resin, or Ramage Amide-MBHA resin as the starting resin; using Fmoc-Cys(Trt)-OH as the first amino acid and loading the resin; the remaining amino acids are sequentially coupled using the Fmoc method to obtain a linear polypeptide resin; solid-phase oxidation is performed using iodine solution, and crude eptifibatide is obtained using trifluoroacetic acid, triisopropylsilane, and water-pyrolyzed resin. This synthetic method has high yield, low cost, mild reaction conditions, low environmental pollution, and is conducive to industrialization.

[0007] For the purposes of this invention, the starting resin is selected from one of Rink Amide-AM, Rink Amide-MBHA resin, Ramage Amide AM resin, or Ramage Amide MBHA resin.

[0008] For the purposes of this invention, the above-protected amino acids include: Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Har(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp(Boc)-OH, and Mpa(Trt)-OH.

[0009] For the purposes of this invention, the reaction time for the above-mentioned iodine solid-phase oxidative cyclization is 0.1-4 h.

[0010] For the purposes of this invention, the precipitant is selected from at least one of methyl tert-butyl ether, diethyl ether, and isopropyl ether.

[0011] For the purposes of this invention, the coupling condensation reagent used in the above-mentioned coupling condensation is selected from one of the following combinations: DIC / HoBt, DIC / Oxyma, sulfonyl peptide condensing agent / HoBt, HBTU / DIEA, or HBTU / NMM; preferably, the coupling condensation reagent used in the above-mentioned coupling condensation is selected from one of DIC / HoBt, DIC / Oxyma, or sulfonyl peptide condensing agent / HoBt.

[0012] For the purposes of this invention, the mass ratio of the coupling condensing agents in each of the above combinations is as follows: the mass ratio of DIC to HoBt is 1:0.8-1.3; the mass ratio of DIC to Oxyma is 1:0.8-1.3; the mass ratio of sulfonyl polypeptide condensing agent to HoBt is 1:0.8-1.3; the mass ratio of HBTU to DIEA is 1:1.7-2.5; and the mass ratio of HBTU to NMM is 1:1.7-2.5.

[0013] Specifically, the above-mentioned method for synthesizing epitubatide includes the following steps: Step 1) Add DMF to the starting resin (the mass-volume ratio of starting resin to DMF is 1g:10-15mL), swell for 25-35min, filter, add deprotection reagent, stir for 25-35min, filter again, add DMF to wash for 1-3min, dry, and repeat washing 4-7 times. Step 2) Weigh Fmoc-Cys(Trt)-OH, add coupling condensation reagent and DMF, stir and activate for 8-15 min, add to reactor and stir reaction for 2-2.5 h, wash resin to obtain Fmoc-Cys(Trt)-resin; follow the above method to sequentially connect Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, MPa(Trt)-OH to obtain linear polypeptide resin; Step 3) Add iodine solution with a concentration of 30-150 g / L and stir for 0.1-4 h to obtain the oxidized cyclized polypeptide; Step 4) Wash the resin with a mixture of vitamin C aqueous solution and DMF until no iodine color remains, then wash with methanol 2-5 times, dichloromethane 2-5 times, methanol 2-5 times, and dry to obtain epitubapeptide cyclic peptide resin. Step 5) Add lysis buffer to epitubatide cyclic peptide resin, stir in an ice bath for 100-150 min, filter, add the filtrate to a precipitant at -12 to -8℃, centrifuge, wash 2-4 times with a cleaning agent, and dry for 10-15 h to obtain crude epitubatide.

[0014] For the purposes of this invention, in step 1) above, the mass-to-volume ratio of the starting resin to the deprotecting agent is 1g:12-17mL; the deprotecting agent comprises a mixed solution of piperidine and DMF; and the volume concentration of piperidine in the deprotecting agent is 2-50%.

[0015] For the present invention, in step 2) above, the mass ratio of the starting resin to Fmoc-Cys(Trt)-OH is 1:0.8-1.3; the mass ratio of Fmoc-Cys(Trt)-OH to the coupling condensation reagent is 1:0.4-0.55; and the mass-volume ratio of the coupling condensation reagent to DMF is 1g:5-8mL.

[0016] For the purposes of this invention, in step 3) above, the mass-to-volume ratio of the starting resin to the iodine solution is 1g:10-20mL; the solvent of the iodine solution is selected from one or more of DMF, DMAc and NMP; more preferably, the solvent of the iodine solution is selected from DMF.

[0017] For the purposes of this invention, in step 4) above, the concentration of vitamin C in the vitamin C aqueous solution is 40-70 g / L; the volume ratio of vitamin C aqueous solution to DMF is 1:2-2.5.

[0018] For the present invention, in step 5) above, the mass-to-volume ratio of epitubatide cyclic peptide resin to lysis buffer is 1g:10-15mL; the mass-to-volume ratio of epitubatide cyclic peptide resin to precipitant is 1g:0.1-0.14L; and the mass-to-volume ratio of epitubatide cyclic peptide resin to cleaning agent is 1g:0.03-0.07L.

[0019] For the purposes of this invention, the lysis solution comprises TFA, TIS and water; the volume ratio of TFA, TIS and water is 40-35:1-2:1-2.

[0020] For the purposes of this invention, the cleaning agent comprises one or more of methyl tert-butyl ether, diethyl ether, or isopropyl ether.

[0021] This invention discloses the use of the above-mentioned vitamin C in peptide resin washing.

[0022] This invention discloses the use of the above-mentioned vitamin C aqueous solution and DMF mixed solution in peptide resin washing.

[0023] The present invention also provides a method for preparing a sulfonyl-containing polypeptide condensing agent, comprising: reacting 4-bromo-2,6-dimethylaniline with 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid via a Suzuki reaction to obtain compound a; synthesizing compound b via a diazotization-iodination reaction; synthesizing compound c via an oxidation reaction; and then obtaining the sulfonyl-containing polypeptide condensing agent via an oxidation reaction under acidic conditions.

[0024] This invention uses a sulfonyl polypeptide condensing agent prepared from 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid for the synthesis of epitubatide. This sulfonyl polypeptide condensing agent does not trigger side reactions, has high reactivity, and is easy to recycle and reuse. It can enable the prepared epitubatide to have high yield, purity, and content.

[0025] Specifically, the preparation method of the above-mentioned sulfonyl polypeptide condensing agent includes the following steps: Step 1: Add deionized water to DMAc to prepare a mixed solvent, then add 4-bromo-2,6-dimethylaniline and dissolve it. Then add 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid, potassium phosphate trihydrate, and palladium acetate. Heat to 98-105℃ and react for 3.5-5 hours. After the reaction is complete, cool to room temperature, then extract with ethyl acetate 2-5 times, wash with saturated brine 2-3 times, dry with anhydrous magnesium sulfate, rotary evaporate, and column chromatography to obtain compound a. Step 2: Add compound a to anhydrous acetonitrile and dissolve it. Then add a 50-55 wt% sulfuric acid aqueous solution. At -8 to -3℃, slowly add a 0.2-0.25 g / mL sodium nitrite aqueous solution and stir for 0.8-2 h. Add urea and then slowly add a 0.5-0.55 g / mL potassium iodide aqueous solution. Then raise the temperature to room temperature at a rate of 3-4℃ / h. Adjust the pH to 8-9.5, extract with ethyl acetate 2-5 times, wash with saturated sodium thiosulfate aqueous solution 2-3 times, wash with saturated brine 2-3 times, dry with anhydrous magnesium sulfate, rotary evaporate, and column chromatography to obtain compound b. Step 3: Add deionized water to tert-butanol (volume ratio of 1:0.8-1.2) to prepare a mixed solvent. Then add compound b and potassium permanganate (the amount of potassium permanganate added is 45-60 wt% of the total amount of potassium permanganate). Heat under reflux for 20-30 h. Then add the remaining potassium permanganate and heat under reflux for 20-30 h. Filter, wash with deionized water, rotary evaporate, adjust the pH to 4.5-6, filter, and obtain compound c. Step 4: Add concentrated hydrochloric acid (33-37 wt%) to compound c. Under light-protected conditions, slowly add sodium hypochlorite aqueous solution (8-15 wt%) at -3 to 3°C. Heat to room temperature at a rate of 2-3°C / h. Filter, wash with deionized water until neutral, and dry to obtain sulfonyl polypeptide condensing agent.

[0026] For the purposes of this invention, in step one above, the volume ratio of DMAc to deionized water is 1:0.8-1.3; the molar volume ratio of 4-bromo-2,6-dimethylaniline to the mixed solvent is 1 mol:8-12 L; the molar ratio of 4-bromo-2,6-dimethylaniline to 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid is 1:1.3-1.8; the molar ratio of 4-bromo-2,6-dimethylaniline to potassium phosphate trihydrate is 1:1.7-2.2; and the molar ratio of 4-bromo-2,6-dimethylaniline to palladium acetate is 1:0.08-0.14.

[0027] For the purposes of this invention, in step two above, the molar volume ratio of compound a to anhydrous acetonitrile is 1 mol: 0.8-1.3 L; the molar volume ratio of compound a to sulfuric acid aqueous solution is 1 mol: 0.2-0.24 L; the molar ratio of compound a to sodium nitrite is 1:1-1.12; the molar ratio of compound a to urea is 1:0.18-0.24; and the molar ratio of compound a to potassium iodide is 1:2.5-2.75.

[0028] For the purposes of this invention, in step three above, the molar volume ratio of compound b to the mixed solvent is 1 mol: 8-13 L; the molar ratio of compound b to the total amount of potassium permanganate is 1: 0.2-0.3.

[0029] For the purposes of this invention, in step four above, the molar volume ratio of compound c to concentrated hydrochloric acid is 1 mol: 8-12 L; the molar volume ratio of compound c to sodium hypochlorite aqueous solution is 1 mol: 17-23 L.

[0030] The present invention also discloses the use of the sulfonyl polypeptide condensing agent prepared by the above preparation method in the synthesis of epitubatide.

[0031] The beneficial effects of this invention include: This invention provides a method for synthesizing epitubatide. The method involves deprotecting the starting resin, using Fmoc-Cys(Trt)-OH as the first amino acid for resin loading, and then sequentially coupling and condensing the remaining amino acids using a solid-phase synthesis method. This is followed by solid-phase oxidative cyclization using iodine solution, and then washing with a mixture of vitamin C aqueous solution and DMF. After cleavage and precipitation, crude epitubatide is obtained. This synthesis method features high yield, low cost, mild reaction conditions, minimal environmental pollution, and is conducive to industrialization.

[0032] Therefore, the present invention provides a method for synthesizing epitaphthide, which has high yield, low cost, mild reaction conditions, low environmental pollution, and is conducive to industrialization. Attached Figure Description

[0033] Figure 1 The HPLC chromatogram of crude epitaphthol obtained in Example 1; Figure 2 The image shows the HPLC chromatogram of crude epitaphthol obtained in Comparative Example 1. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described in detail below with reference to specific embodiments: The abbreviations used in this invention have the following meanings: DIC: Diisopropylcarbodiimide; HOBT: 1-Hydroxybenzotriazole; HBTU: Benzotriazole-N,N,N′,N′-Tetramethylurea hexafluorophosphate; DIEA: N,N-diisopropylethylamine; TIS: Triisopropylsilane; DMAc: N,N-dimethylacetamide; DMF: N,N-dimethylformamide; NMP: N-methylpyrrolidone; Pbf: Nα-fluorenylmethoxycarbonyl-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl); TFA: Trifluoroacetic acid; Boc: tert-butyloxycarbonyl; Trt: Triphenylmethyl; OtBu: Tert-butyl; Fmoc: fluorenemethyloxycarbonyl; Trp: Tryptophan; Gly: Glycine; Asp: Aspartic acid; Har: High in arginine; Pro: Proline; Mpa: Mercaptopropionic acid.

[0035] Example 1: A method for synthesizing eptifibatide includes the following steps: Step 1) Weigh 32g of Rink Amide-Am resin (degree of substitution 0.94mmol / g) into the reactor, add 350mL of DMF to swell the resin for 30min and then filter. Add 500mL of deprotection reagent (the deprotection reagent contains a mixed solution of piperidine and DMF; the volume concentration of piperidine in the deprotection reagent is 20%), stir for 30min and then filter. Add 350mL of DMF, wash for 1min and then dry. Repeat the washing process 5 times. Step 2) Weigh 35.14g of Fmoc-Cys(Trt)-OH and 8.1g of HOBt, add 100mL of DMF, dissolve with ultrasonic assistance, add 8.51g of DIC, stir and activate for 10min, add to the reactor and stir to react for 2h. After washing the resin, obtain Fmoc-Cys(Trt)-resin; in the same manner as above, sequentially connect Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, and MPa(Trt)-OH, with the amounts shown in Table 1. Table 1. Raw material consumption during the sequential coupling condensation process in Example 1

[0036] Step 3) Add 400 mL of 60 g / L iodine DMF solution and stir for 3 h; Step 4) Wash the resin with a mixed solution of vitamin C aqueous solution and DMF (the concentration of vitamin C in the vitamin C aqueous solution is 50 g / L, and the volume ratio of vitamin C aqueous solution to DMF is 1:2.3) until no iodine color remains. Then wash it three times with methanol, three times with dichloromethane, and three times with methanol. Then dry it. Step 5) Add 800 mL of lysis buffer (760 mL TFA, 20 mL TIS, 20 mL water) to the resin, stir in an ice bath for 120 min, then filter. Add the filtrate to 8 L of pre-cooled diethyl ether (-10 °C), centrifuge to remove the supernatant and collect the solid precipitate. Wash the solid precipitate twice with 4 L of diethyl ether. Place the solid precipitate in a vacuum drying oven and dry for 12 h to obtain crude epitubatide. Its HPLC chromatogram is shown below. Figure 1 As shown; by Figure 1 It can be seen that the impurity peaks near the main peak of crude epitaphtide are relatively small.

[0037] Example 2: The difference between the synthesis method of epitaphthide and Example 1 is that a sulfonyl polypeptide condenser is used instead of DIC.

[0038] A method for preparing a sulfonyl polypeptide condensing agent includes the following steps: Step 1: Add deionized water to DMAc to prepare a mixed solvent, then add 4-bromo-2,6-dimethylaniline and dissolve it. Then add 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid (CAS No.: 913835-96-8), potassium phosphate trihydrate, and palladium acetate. Heat to 98℃ and react for 5 hours. After the reaction is completed, cool to room temperature, then extract twice with ethyl acetate, wash twice with saturated brine, dry with anhydrous magnesium sulfate, rotary evaporate, and column chromatography to obtain compound a. The proton spectrum of compound a is as follows: 1 H NMR (400MHz, CDCl3): 1 H NMR (400MHz, CDCl3): 5.29 (2H, Ar-N H 2), 2.11 (6H, Ar-C H 3), 7.53 (2H, CH3-Ar- H ), 7.83 (4H, S-Ar- H ), 2.86 (1H, NC) H ), 1.04 (6H, CH-C) H 3), 4.47 (2H, NC) H 2), 6.85-7.13 (4H, O-Ar- H ), 3.81 (3H, OC) H 3). HRMS (ESI): C 25 H 30 N₂O₃S, m / z [M+H] + ,438.20.

[0039] Step 2: Compound a was added to anhydrous acetonitrile and dissolved. Then, a 50 wt% sulfuric acid aqueous solution was added. At -8°C, a 0.2 g / mL sodium nitrite aqueous solution was slowly added and stirred for 0.8 h. Urea was added, followed by a 0.5 g / mL potassium iodide aqueous solution. The temperature was then raised to room temperature at a rate of 3°C / h. The pH was adjusted to 8. The mixture was extracted twice with ethyl acetate, washed twice with saturated sodium thiosulfate aqueous solution, and washed twice with saturated brine. The mixture was dried over anhydrous magnesium sulfate, rotary evaporated, and subjected to column chromatography to obtain compound b. The proton NMR spectrum of compound b is as follows: 1 H NMR (400MHz, CDCl3): 1 H NMR (400MHz, CDCl3): 2.35 (6H, Ar-C H 3), 7.42 (2H, CH3-Ar- H ), 7.85 (4H, S-Ar- H ), 2.86 (1H, NC) H ), 1.03 (6H, CH-C) H 3), 4.44 (2H, NC) H 2), 6.85-7.13 (4H, O-Ar- H ), 3.80 (3H, OC) H 3). HRMS (ESI): C 25 H 28 INO3S, m / z [M+H] + , 549.08.

[0040] Step 3: Add deionized water (volume ratio of 1:0.8) to tert-butanol to prepare a mixed solvent. Then add compound b and potassium permanganate (the amount of potassium permanganate added is 45wt% of the total amount of potassium permanganate). Heat under reflux for 20 h. Then add the remaining potassium permanganate and heat under reflux for 20 h. Filter, wash with deionized water, rotary evaporate, adjust the pH to 5, filter, and obtain compound c. The proton spectrum of compound c is as follows: 1 H NMR (400MHz, CDCl3): 1 H NMR (400MHz, CDCl3): 12.68 (2H, O=CO H ), 8.85 (2H, O=C-Ar- H ), 7.85 (4H, S-Ar- H ), 2.85 (1H, NC) H ), 1.04 (6H, CH-C) H 3), 4.46 (2H, NC) H2), 6.85-7.13 (4H, O-Ar- H ), 3.81 (3H, OC) H 3). HRMS (ESI): C 25 H 24 INO7S, m / z [M+H] + ,609.03.

[0041] Step 4: Add 33wt% concentrated hydrochloric acid to compound c. Under light-protected conditions, slowly add 8wt% sodium hypochlorite aqueous solution at -3℃. Raise the temperature to room temperature at a rate of 2℃ / h. Filter, wash with deionized water until neutral, and dry to obtain sulfonyl polypeptide condensing agent. In step one, the volume ratio of DMAc to deionized water is 1:0.8; the molar volume ratio of 4-bromo-2,6-dimethylaniline to the mixed solvent is 1 mol:8 L; the molar ratio of 4-bromo-2,6-dimethylaniline to 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid is 1:1.3; the molar ratio of 4-bromo-2,6-dimethylaniline to potassium phosphate trihydrate is 1:1.7; and the molar ratio of 4-bromo-2,6-dimethylaniline to palladium acetate is 1:0.08. In step two, the molar volume ratio of compound a to anhydrous acetonitrile is 1 mol: 0.8 L; the molar volume ratio of compound a to sulfuric acid aqueous solution is 1 mol: 0.2 L; the molar ratio of compound a to sodium nitrite is 1:1; the molar ratio of compound a to urea is 1:0.18; and the molar ratio of compound a to potassium iodide is 1:2.5. In step three, the molar volume ratio of compound b to the mixed solvent is 1 mol: 8 L; the molar ratio of compound b to the total amount of potassium permanganate is 1: 0.2. In step four, the molar volume ratio of compound c to concentrated hydrochloric acid is 1 mol: 8 L; the molar volume ratio of compound c to sodium hypochlorite aqueous solution is 1 mol: 17 L.

[0042] The 1H NMR spectrum of the sulfonyl-containing polypeptide condensing agent is as follows: 1 H NMR (400MHz, CDCl3): 8.82 (2H, O=C-Ar- H ), 7.85 (4H, S-Ar- H ), 2.85 (1H, NC) H ), 1.04 (6H, CH-C) H 3), 4.45 (2H, NC) H 2), 6.85-7.13 (4H, O-Ar- H ), 3.80 (3H, OC) H 3). HRMS (ESI): C 25H 22 INO7S, m / z [M+H] + ,607.02.

[0043] Comparative Example 1: A method for synthesizing eptifibatide includes the following steps: Weigh 30g of Rink Amide-AM resin (degree of substitution 0.92mmol / g) into the reactor, add 350mL of DMF to swell the resin for 30min, then filter. Add 500mL of deprotection reagent (the deprotection reagent contains a mixed solution of piperidine and DMF; the volume concentration of piperidine in the deprotection reagent is 20%), stir for 30min, then filter. Add 350mL of DMF, wash for 1min, then dry. Repeat the washing process 5 times. Weigh 37g of Fmoc-Cys(Trt)-OH and 8.90g of HOBt, add 8.51g of DIC, stir and activate for 10min, add 100mL of DMF, sonicate to dissolve, and let stand to activate for 10min. Add to the reactor and stir for 2h. After washing the resin, obtain Fmoc-Cys(Trt)-resin; according to the above deprotection and condensation method, connect Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, and MPa(Trt)-OH respectively, with the amounts shown in Table 2; Table 2. Raw material consumption during the sequential coupling condensation process in Comparative Example 1

[0044] The condensed heptapeptide resin was washed three times with methanol, three times with dichloromethane, and three times with methanol. After drying, 800 mL of lysis buffer (700 mL TFA, 20 mL TIS, 20 mL water, and 60 mL EDT) was added to the resin. The mixture was stirred in an ice bath for 120 min and then filtered. Add the filtrate to 8L of pre-cooled methyl tert-butyl ether (-11℃), centrifuge to remove the supernatant and collect the solid precipitate, and wash the solid precipitate twice with 4L of methyl tert-butyl ether; place the solid precipitate in a vacuum drying oven and dry for 12h. The dried solid precipitate was dissolved in an acetonitrile aqueous solution (acetonitrile to water volume ratio of 1:2) to prepare a 1 mg / mL solution. Then, 3 wt% hydrogen peroxide (solid precipitate to hydrogen peroxide mass-to-volume ratio of 1 g:4 mL) was added for oxidation at 25°C for 2 hours. The solution was then concentrated under reduced pressure to obtain crude epitubatide. Its HPLC chromatogram is shown below. Figure 2 As shown; with Figure 1 In comparison, the crude eptifibatide has larger impurities near the main peak.

[0045] Comparative Example 2: The difference between the synthesis method of eptifibatide and that in Example 1 is that the washing method in step 4) is different: the resin is washed with acetic acid until no iodine color remains.

[0046] Experimental Example 1: Product purity and yield testing The purity and yield of the prepared epitubatide sample were tested.

[0047] Table 3. Purity and yield test results of eptifibatide samples

[0048] The above tests were performed on Examples 1-2 and Comparative Examples 1-2, and the results are shown in Table 3. Table 3 shows that, compared with Comparative Example 1, the purity and content of the eptifibatide sample in Example 1 were improved, indicating that the eptifibatide sample prepared by the synthesis method provided by this invention has good purity and content compared with the prior art. Compared with Example 1, the yield, purity, and content of the eptifibatide sample in Example 2 were improved, indicating that the eptifibatide sample prepared using a sulfonyl peptide condensing agent also has good yield, purity, and content. Compared with Comparative Example 2, the purity and content of the eptifibatide sample in Example 1 were also improved, indicating that the use of vitamin C to wash the peptide resin resulted in a eptifibatide sample with good purity and content.

[0049] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0050] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for synthesizing eptifibatide, comprising: Step 1) Deprotect the starting resin; Step 2) The protected amino acids are sequentially coupled and condensed using a solid-phase synthesis method to obtain a linear polypeptide resin: Step 3) Iodine solid-phase oxidative cyclization to obtain the oxidatively cyclized polypeptide; Step 4) Wash the resin with a mixture of vitamin C aqueous solution and DMF until no iodine color remains, then wash with DMF, then with methanol, and dry to obtain epitubapeptide cyclic peptide resin. Step 5) Crack the resin, then add a precipitant to precipitate and wash to obtain crude epitubatide.

2. The method for synthesizing eptifibatide according to claim 1, characterized in that: The starting resin is selected from one of Rink Amide-AM, Rink Amide-MBHA resin, Ramage Amide AM resin, or Ramage Amide MBHA resin.

3. The method for synthesizing eptifibatide according to claim 1, characterized in that: The protected amino acids include: Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Har(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp(Boc)-OH, and Mpa(Trt)-OH.

4. The method for synthesizing eptifibatide according to claim 1, characterized in that: The reaction time for the iodine solid-phase oxidative cyclization is 0.1-4 h.

5. The method for synthesizing eptifibatide according to claim 1, characterized in that: The precipitant is selected from at least one of methyl tert-butyl ether, diethyl ether, and isopropyl ether.

6. The method for synthesizing eptifibatide according to claim 1, characterized in that: The coupling condensing agent used in the coupling condensation is selected from one of the following combinations: DIC / HoBt, DIC / Oxyma, sulfonyl polypeptide condenser / HoBt, HBTU / DIEA, or HBTU / NMM.

7. The method for preparing the sulfonyl polypeptide condensing agent according to claim 6, comprising: Compound a was prepared by reacting 4-bromo-2,6-dimethylaniline with 4-[isopropyl(4-methoxybenzyl)sulfonamide]phenylboronic acid via the Suzuki reaction. Compound b was then synthesized via a diazotization-iodization reaction, followed by an oxidation reaction to synthesize compound c. Finally, under acidic conditions, an oxidation reaction was carried out to obtain a sulfonyl-containing polypeptide condensing agent.

8. The use of the sulfonyl polypeptide condensing agent prepared by the method of claim 7 in the synthesis of eptifibatide.

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