A method for synthesizing eptifibatide

Abstract

The present invention discloses a method for synthesizing eptifibatide, which relates to the technical field of polypeptide synthesis. After the starting resin is deprotected, Fmoc-Cys(Trt)-OH is used as the first amino acid to load the resin; the remaining amino acids are successively coupled and condensed by solid-phase synthesis method, then solid-phase oxidation is carried out using iodine solution, and then after cleavage, the crude product of eptifibatide is obtained; this synthesis method has high yield, low cost, mild reaction conditions, little environmental pollution, and is conducive to industrialization.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing eptifibatide. Background Art

[0002] Eptifibatide is an artificially synthesized cyclic peptide containing one mercaptopropionic acid and six amino acid residues. It is a specific platelet glycoprotein GPIIb / IIIa receptor antagonist, which selectively and reversibly inhibits the final common pathway of platelet aggregation (the binding of plasma coagulation factor I to GPIIb / IIIa), and can reverse the ischemic state caused by thrombosis. Clinically, eptifibatide is mainly used to treat unstable angina pectoris and acute myocardial infarction. Its main adverse reaction is bleeding. Most bleeding is mild to moderate bleeding, and the main bleeding site is the vascular puncture site during PCI. Cerebral hemorrhage is rare. Eptifibatide was first developed by CORTherapeuties in the United States and was first launched in the United States in July 1998 under the trade name Integrelin.

[0003] Currently, the preparation methods of eptifibatide mainly include solid-phase synthesis and liquid-phase synthesis. Among them, the liquid-phase synthesis has more steps and a long time, and the solid-phase synthesis is the commonly used method for synthesizing eptifibatide at present. However, the current solid-phase synthesis method has problems such as high cost, long synthesis cycle, large amount of reagents used, and large amount of solvents required for liquid-phase oxidation. The present invention provides a method for preparing eptifibatide, which mainly solves the problems of large volume of liquid-phase oxidation and difficulty in storing crude products existing in the prior art. Summary of the Invention

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

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0006] The present invention discloses a method for synthesizing eptifibatide, including:

[0007] Step 1) Perform deprotection treatment on the starting resin;

[0008] Step 2) Sequentially couple and condense protected amino acids by solid-phase synthesis to obtain a linear polypeptide resin:

[0009] Step 3) Perform iodine solid-phase oxidation cyclization to obtain an oxidized and cyclized polypeptide;

[0010] Step 4) Wash the resin with a mixed solution of vitamin C aqueous solution and DMF until there is no iodine color, then wash with DMF, wash with methanol, and dry to obtain eptifibatide cyclic peptide resin;

[0011] Step 5) Crack the resin, add a precipitant for precipitation and then wash to obtain crude eptifibatide.

[0012] The present invention relates to a method for synthesizing eptifibatide. The technical solution of the present 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 to load the resin; gradually coupling the remaining amino acids by the Fmoc method to obtain a linear polypeptide resin; performing solid-phase oxidation using an iodine solution, and using trifluoroacetic acid, triisopropylsilane and water to crack the resin to obtain crude eptifibatide. This synthesis method has a high yield, low cost, mild reaction conditions, little environmental pollution, and is conducive to industrialization.

[0013] For the present invention, the above-mentioned starting resin is selected from one of Rink Amide-AM, Rink Amide-MBHA resin, Ramage Amide AM resin or Ramage Amide MBHA resin.

[0014] For the present invention, the above-mentioned 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.

[0015] For the present invention, the reaction time of the above-mentioned iodine solid-phase oxidation cyclization is 0.1 - 4 h.

[0016] For the present invention, the above-mentioned precipitant is selected from at least one of methyl tert-butyl ether, ethyl ether and isopropyl ether.

[0017] For the present 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-containing polypeptide 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-containing polypeptide condensing agent / HoBt.

[0018] For the present invention, the dosage ratios of the coupling condensation reagents in each of the above combinations are 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 the sulfonyl group-containing polypeptide condensing agent to HoBt is 1:0.8 - 1.3; the mass ratio of HBTU to DIEA is 1:1.7 - 2.5; the mass ratio of HBTU to NMM is 1:1.7 - 2.5.

[0019] Specifically, the synthesis method of eptifibatide described above includes the following steps:

[0020] Specifically, the synthesis method of eptifibatide described above includes the following steps:

[0021] Step 1) Add DMF to the starting resin (the mass-volume ratio of the starting resin to DMF is 1 g:10 - 15 mL). After swelling for 25 - 35 min, filter by suction, add the deprotection reagent, stir for 25 - 35 min and then filter by suction. Add DMF to wash for 1 - 3 min and then drain dry. Repeat the washing 4 - 7 times;

[0022] Step 2) Weigh Fmoc-Cys(Trt)-OH, add the coupling condensation reagent and DMF, stir and activate for 8 - 15 min, add it to the reactor and stir and react for 2 - 2.5 h. After washing the resin, Fmoc-Cys(Trt)-resin is obtained; successively connect Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, Mpa(Trt)-OH according to the above method to obtain the linear polypeptide resin;

[0023] Step 3) Add an iodine solution with a concentration of 30 - 150 g / L and stir and react for 0.1 - 4 h to obtain the oxidized and cyclized polypeptide;

[0024] Step 4) Wash the resin with a mixed solution of aqueous vitamin C solution and DMF until there is no iodine color, then wash with methanol 2 - 5 times, wash with dichloromethane 2 - 5 times, wash with methanol 2 - 5 times, and dry to obtain the eptifibatide cyclic peptide resin;

[0025] Step 5) Add the cleavage solution to the eptifibatide cyclic peptide resin, stir in an ice bath for 100 - 150 min and then filter by suction. Add the filtrate to a precipitant at -12 to -8 °C, centrifuge, and wash with a cleaning agent 2 - 4 times, and dry for 10 - 15 h; obtain the crude eptifibatide.

[0026] For the present invention, in the above step 1), the mass-volume ratio of the starting resin to the deprotection reagent is 1 g: 12 - 17 mL; the above deprotection reagent comprises a mixed solution of piperidine and DMF; the volume concentration of piperidine in the above deprotection reagent is 2 - 50%.

[0027] For the present invention, in the above step 2), 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; the mass-volume ratio of the coupling condensation reagent to DMF is 1 g: 5 - 8 mL.

[0028] For the present invention, in the above step 3), the mass-volume ratio of the starting resin to the iodine solution is 1 g: 10 - 20 mL; the solvent of the iodine solution is selected from one or more of DMF, DMAc, and NMP; more preferably, the solvent of the above iodine solution is selected from DMF.

[0029] For the present invention, in the above step 4), the concentration of vitamin C in the aqueous vitamin C solution is 40 - 70 g / L; the volume ratio of the aqueous vitamin C solution to DMF is 1: 2 - 2.5.

[0030] For the present invention, in the above step 5), the mass-volume ratio of the eptifibatide cyclic peptide resin to the cleavage solution is 1 g: 10 - 15 mL; the mass-volume ratio of the eptifibatide cyclic peptide resin to the precipitant is 1 g: 0.1 - 0.14 L; the mass-volume ratio of the eptifibatide cyclic peptide resin to the cleaning agent is 1 g: 0.03 - 0.07 L.

[0031] For the present invention, the above cleavage solution comprises: TFA, TIS, and water; the volume ratio of the above TFA, TIS, and water is: 40 - 35: 1 - 2: 1 - 2.

[0032] For the present invention, the above cleaning agent comprises one or more of methyl tert-butyl ether, diethyl ether, or isopropyl ether.

[0033] The present invention discloses the use of the above vitamin C in the washing of peptide resin.

[0034] The present invention discloses the use of the above mixed solution of aqueous vitamin C solution and DMF in the washing of peptide resin.

[0035] The present invention also provides a preparation method of a sulfonyl-containing polypeptide condensing agent, comprising: performing a Suzuki reaction on 4-bromo-2,6-dimethylaniline and 4-[isopropyl(4-methoxybenzyl)sulfonamido]phenylboronic acid to obtain compound a, synthesizing compound b through a diazotization iodination reaction, then synthesizing compound c through an oxidation reaction, and then obtaining the sulfonyl-containing polypeptide condensing agent through an oxidation reaction under acidic conditions.

[0036] The present invention uses a sulfonyl-containing polypeptide condensing agent prepared from 4-[isopropyl(4-methoxybenzyl)sulfonamido]phenylboronic acid for the synthesis of eptifibatide; this sulfonyl-containing polypeptide condensing agent does not cause side reactions; it has high reaction activity and is easy to recycle and reuse; it can enable the prepared eptifibatide to have a high yield, purity and content.

[0037] Specifically, the preparation method of the above-mentioned sulfonyl-containing polypeptide condensing agent includes the following steps:

[0038] Step 1: Add deionized water to DMAc to prepare a mixed solvent, then add 4-bromo-2,6-dimethylaniline. After dissolution, add 4-[isopropyl(4-methoxybenzyl)sulfonamido]phenylboronic acid, potassium phosphate trihydrate, and palladium acetate, heat up to 98-105 °C and react for 3.5-5 h. After the reaction is completed, 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 chromatograph to obtain compound a;

[0039] Step 2: Add compound a to anhydrous acetonitrile. After dissolution, add a sulfuric acid aqueous solution with a concentration of 50-55 wt%. Then, at -8 to -3 °C, slowly add a sodium nitrite aqueous solution with a concentration of 0.2-0.25 g / mL, stir for 0.8-2 h, add urea, and then slowly add a potassium iodide aqueous solution with a concentration of 0.5-0.55 g / mL. Then, raise the temperature to room temperature at a heating rate of 3-4 °C / h, then adjust the pH to 8-9.5, extract with ethyl acetate 2-5 times, wash with a saturated sodium thiosulfate aqueous solution 2-3 times, wash with saturated brine 2-3 times, dry with anhydrous magnesium sulfate, rotary evaporate, and column chromatograph to obtain compound b;

[0040] Step 3: Add deionized water (the volume ratio of the two is 1:0.8-1.2) to tert-butanol 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, heat under reflux for 20-30 h, filter, wash with deionized water, rotary evaporate, and then adjust the pH to 4.5-6, filter to obtain compound c;

[0041] Step 4: Add concentrated hydrochloric acid with a concentration of 33-37 wt% to compound c. Under light-shielded conditions, at -3 to 3 °C, slowly add a sodium hypochlorite aqueous solution with a concentration of 8-15 wt%. Raise the temperature to room temperature at a heating rate of 2-3 °C / h, filter, wash with deionized water until neutral, and dry to obtain the sulfonyl-containing polypeptide condensing agent.

[0042] For the present invention, in the above step 1, 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)sulfamoyl]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; the molar ratio of 4-bromo-2,6-dimethylaniline to palladium acetate is 1:0.08 - 0.14.

[0043] For the present invention, in the above step 2, 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; the molar ratio of compound a to potassium iodide is 1:2.5 - 2.75.

[0044] For the present invention, in the above step 3, 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.

[0045] For the present invention, in the above step 4, 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.

[0046] The present invention also discloses the use of the sulfonyl group-containing polypeptide condensing agent prepared by the above preparation method in the synthesis of eptifibatide.

[0047] The beneficial effects of the present invention include:

[0048] The present invention provides a synthesis method of eptifibatide. After the starting resin is deprotected, Fmoc-Cys(Trt)-OH is used as the first amino acid for resin loading; the remaining amino acids are sequentially coupled and condensed by solid-phase synthesis method, then solid-phase oxidation cyclization is carried out using iodine solution, and then after washing with a mixed solution of vitamin C aqueous solution and DMF, and then through cleavage and precipitation, the crude eptifibatide is obtained; this synthesis method has high yield, low cost, mild reaction conditions, little environmental pollution, and is conducive to industrialization.

[0049] Therefore, the present invention provides a synthesis method of eptifibatide, which has high yield, low cost, mild reaction conditions, little environmental pollution, and is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1The HPLC chromatogram of the crude eptifibatide prepared in Example 1;

[0051] Figure 2 The HPLC chromatogram of the crude eptifibatide prepared in Comparative Example 1. Detailed implementation manners

[0052] To make the objectives, technical solutions and advantages of the present invention clearer and more explicit, the following further describes the technical solutions of the present invention in detail in conjunction with specific implementation manners:

[0053] The abbreviations used in the present invention have the following meanings:

[0054] DIC: Diisopropylcarbodiimide;

[0055] HOBT: 1-Hydroxybenzotriazole;

[0056] HBTU: Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate;

[0057] DIEA: N,N-Diisopropylethylamine;

[0058] TIS: Triisopropylsilane;

[0059] DMAc: N,N-Dimethylacetamide;

[0060] DMF: N,N-Dimethylformamide;

[0061] NMP: N-Methylpyrrolidone;

[0062] Pbf: Nα-Fluorenylmethoxycarbonyl-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl);

[0063] TFA: Trifluoroacetic acid;

[0064] Boc: tert-Butyloxycarbonyl;

[0065] Trt: Triphenylmethyl;

[0066] OtBu: tert-Butyl;

[0067] Fmoc: Fluorenylmethoxycarbonyl;

[0068] Trp: Tryptophan;

[0069] Gly: Glycine;

[0070] Asp: Aspartic acid;

[0071] Har: Homoarginine;

[0072] Pro: Proline;

[0073] Mpa: Mercaptopropionic acid.

[0074] Example 1:

[0075] A method for synthesizing eptifibatide, comprising the following steps:

[0076] Step 1) Weigh 32 g of Rink Amide-Am resin (substitution degree: 0.94 mmol / g) into a reactor, add 350 mL of DMF to swell the resin for 30 min, then filter by suction. Add 500 mL 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 30 min, then filter by suction. Add 350 mL of DMF, wash for 1 min, then dry by suction. Repeat the washing process 5 times in sequence;

[0077] Step 2) Weigh 35.14 g of Fmoc-Cys(Trt)-OH and 8.1 g of HOBt, add 100 mL of DMF, dissolve with ultrasonic assistance, add 8.51 g of DIC, stir and activate for 10 min, then add to the reactor and stir for reaction for 2 h. After washing the resin, Fmoc-Cys(Trt)-resin is obtained; in the above manner, Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, and Mpa(Trt)-OH are sequentially connected, and the dosages are shown in Table 1;

[0078] Table 1 Raw material dosages in the sequential coupling and condensation process in Example 1

[0079] Amino acid Charging amount Amount of DIC Amount of HOBt Fmoc-Pro-OH 20.23g 8.50g 8.92g Fmoc-Trp(Boc)-OH 31.64g 8.49g 8.93g Fmoc-Asp(OtBu)-OH 24.70g 8.51g 8.91g Fmoc-Gly-OH 17.86g 8.51g 8.92g Fmoc-Har(Pbf)-OH 39.77g 8.51g 8.91g Mpa(Trt)-OH 20.91g 8.51g 8.91g

[0080] Step 3) Add 400 mL of an iodine DMF solution with a concentration of 60 g / L and stir for reaction for 3 h;

[0081] Step 4) Wash the resin with a mixed solution of aqueous vitamin C solution and DMF (the concentration of vitamin C in the aqueous vitamin C solution is 50 g / L, and the volume ratio of the aqueous vitamin C solution to DMF is 1:2.3) until there is no iodine color, then wash 3 times with methanol, 3 times with dichloromethane, 3 times with methanol, and dry;

[0082] Step 5) Add 800 mL of cleavage solution (760 mL of TFA, 20 mL of TIS, 20 mL of water) to the resin, stir in an ice bath for 120 min, then filter by suction. Add the filtrate to 8 L of pre-cooled ether (-10 °C), centrifuge to remove the supernatant, take the solid precipitate, and wash the solid precipitate 2 times with 4 L of ether; put the solid precipitate into a vacuum drying oven and dry for 12 h to obtain the crude eptifibatide; its HPLC chromatogram is as Figure 1as shown; from Figure 1 It can be seen that the impurity peaks near the main peak of the crude eptifibatide are small.

[0083] Example 2:

[0084] The difference between a synthesis method of eptifibatide and Example 1: A sulfonyl group-containing polypeptide condensing agent is used to replace DIC.

[0085] The preparation method of the sulfonyl group-containing polypeptide condensing agent includes the following steps:

[0086] Step 1: Add deionized water to DMAc to obtain a mixed solvent, then add 4-bromo-2,6-dimethylaniline. After dissolution, add 4-[isopropyl(4-methoxybenzyl)sulfonamido]phenylboronic acid (CAS No.: 913835-96-8), potassium phosphate trihydrate, and palladium acetate. Heat to 98 °C and react for 5 h. 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 perform column chromatography to obtain compound a;

[0087] The hydrogen spectrum of compound a is: 1 H NMR (400 MHz, CDCl3): 1 H NMR (400 MHz, 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, N-C H ), 1.04 (6H, CH-C H 3), 4.47 (2H, N-C H 2), 6.85 - 7.13 (4H, O-Ar- H ), 3.81 (3H, O-C H 3). HRMS(ESI): C 25 H 30 N2O3S, m / z [M+H] + , 438.20.

[0088] Step 2: Add compound a to anhydrous acetonitrile. After dissolution, add a sulfuric acid aqueous solution with a concentration of 50 wt%, then slowly add a sodium nitrite aqueous solution with a concentration of 0.2 g / mL at -8 °C, stir for 0.8 h, add urea, and then slowly add a potassium iodide aqueous solution with a concentration of 0.5 g / mL. Then, raise the temperature to room temperature at a rate of 3 °C / h, adjust the pH to 8, extract twice with ethyl acetate, wash twice with a saturated sodium thiosulfate aqueous solution, wash twice with a saturated brine solution, dry with anhydrous magnesium sulfate, rotary evaporate, and perform column chromatography to obtain compound b;

[0089] The hydrogen spectrum of compound b is as follows: 1 H NMR (400 MHz, CDCl3): 1 H NMR (400 MHz, CDCl3): 2.35 (6H, Ar-C H 3), 7.42 (2H, CH3-Ar- H ), 7.85 (4H, S-Ar- H ), 2.86 (1H, N-C H ), 1.03 (6H, CH-C H 3), 4.44 (2H, N-C H 2), 6.85 - 7.13 (4H, O-Ar- H ), 3.80 (3H, O-C H 3). HRMS(ESI): C 25 H 28 INO3S, m / z [M + H] + , 549.08.

[0090] Step 3: Add deionized water to tert-butanol (the volume ratio of the two is 1:0.8) to obtain a mixed solvent, then add compound b and potassium permanganate (the amount of potassium permanganate added is 45 wt% of the total amount of potassium permanganate), heat under reflux for 20 h, then add the remaining potassium permanganate, heat under reflux for 20 h, filter by suction, wash with deionized water, rotary evaporate, and then adjust the pH to 5, filter to obtain compound c;

[0091] The hydrogen spectrum of compound c is as follows: 1 H NMR (400 MHz, CDCl3): 1 H NMR (400 MHz, CDCl3): 12.68 (2H, O=C-O H ), 8.85 (2H, O=C-Ar- H ), 7.85 (4H, S-Ar- H ), 2.85 (1H, N-C H ), 1.04 (6H, CH-C H 3), 4.46 (2H, N-CH 2), 6.85 - 7.13 (4H, O - Ar - H ), 3.81 (3H, O - C H 3). HRMS(ESI): C 25 H 24 INO7S, m / z [M + H] + , 609.03.

[0092] Step 4: Add concentrated hydrochloric acid with a concentration of 33 wt% to compound c. Under dark conditions, slowly add an aqueous sodium hypochlorite solution with a concentration of 8 wt% at -3°C, raise the temperature to room temperature at a rate of 2°C / h, filter, wash with deionized water until neutral, and dry to obtain a sulfonyl - containing polypeptide condensing agent;

[0093] Among them, in Step 1, 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)sulfonamido]phenylboronic acid is 1:1.3; the molar ratio of 4 - bromo - 2,6 - dimethylaniline to potassium phosphate trihydrate is 1:1.7; the molar ratio of 4 - bromo - 2,6 - dimethylaniline to palladium acetate is 1:0.08;

[0094] In Step 2, 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; the molar ratio of compound a to potassium iodide is 1:2.5;

[0095] In Step 3, 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;

[0096] In Step 4, the molar - volume ratio of compound c to concentrated hydrochloric acid is 1 mol:8 L; the molar - volume ratio of compound c to the aqueous sodium hypochlorite solution is 1 mol:17 L.

[0097] The hydrogen spectrum of the sulfonyl - containing polypeptide condensing agent is as follows: 1 H NMR (400 MHz, CDCl3): 8.82 (2H, O = C - Ar - H ), 7.85 (4H, S - Ar - H ), 2.85 (1H, N - C H ), 1.04 (6H, CH - C H 3), 4.45 (2H, N - C H 2), 6.85 - 7.13 (4H, O - Ar -H ), 3.80 (3H, O-C H 3). HRMS(ESI): C 25 H 22 INO7S, m / z [M+H] + , 607.02.

[0098] Comparative Example 1:

[0099] A method for synthesizing eptifibatide, comprising the following steps:

[0100] Weigh 30 g of Rink Amide-AM resin (substitution degree 0.92 mmol / g) into a reactor, add 350 mL of DMF to swell the resin for 30 min, then filter by suction. Add 500 mL 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 30 min, then filter by suction. Add 350 mL of DMF, wash for 1 min and then drain. Repeat the washing process 5 times in sequence;

[0101] Weigh 37 g of Fmoc-Cys(Trt)-OH and 8.90 g of HOBt, add 8.51 g of DIC, stir and activate for 10 min, add 100 mL of DMF, assist in dissolving by ultrasonic wave, and let stand and activate for 10 min. Add it to the reactor and stir for 2 h. After washing the resin, Fmoc-Cys(Trt)-resin is obtained; according to the above deprotection and then condensation method, connect Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Har(Pbf)-OH, Mpa(Trt)-OH respectively, and the dosages are shown in Table 2;

[0102] Table 2 Raw material dosages in the sequential coupling and condensation process in Comparative Example 1

[0103] Amino acid Charging amount Amount of DIC Amount of HOBt Fmoc-Pro-OH 20.25g 8.51g 8.91g Fmoc-Trp(Boc)-OH 31.62g 8.51g 8.91g Fmoc-Asp(OtBu)-OH 24.69g 8.51g 8.91g Fmoc-Gly-OH 17.84g 8.51g 8.91g Fmoc-Har(Pbf)-OH 39.77g 8.51g 8.91g Mpa(Trt)-OH 20.91g 8.51g 8.91g

[0104] The condensed heptapeptide resin is washed 3 times with methanol, 3 times with dichloromethane, 3 times with methanol, and after drying, add 800 mL of cleavage solution (700 mL of TFA, 20 mL of TIS, 20 mL of water, 60 mL of EDT) to the resin, stir in an ice bath for 120 min and then filter by suction;

[0105] Add the filtrate to 8 L of pre-cooled methyl tert-butyl ether (-11 °C), centrifuge to remove the supernatant and take the solid precipitate, and wash the solid precipitate twice with 4 L of methyl tert-butyl ether; put the solid precipitate into a vacuum drying oven and dry for 12 h;

[0106] The dried solid precipitate was added to an acetonitrile aqueous solution (volume ratio of acetonitrile to water was 1:2) for dissolution to prepare a 1 mg / mL solution, and then 3 wt% hydrogen peroxide (mass-volume ratio of solid precipitate to hydrogen peroxide was 1 g:4 mL) was added for oxidation at 25 °C for 2 h, followed by concentration under reduced pressure to obtain the crude eptifibatide; its HPLC chromatogram was as shown in Figure 2 shown; compared with Figure 1 , there were relatively large impurities near the main peak of the crude eptifibatide.

[0107] Comparative Example 2:

[0108] The difference between a method for synthesizing eptifibatide and Example 1 was as follows: the washing method in step 4) was different: the resin was washed with acetic acid until the iodine color disappeared.

[0109] Test Example 1:

[0110] Product purity and yield test

[0111] The prepared eptifibatide sample was tested for purity and yield.

[0112] Table 3 Test results of purity and yield of eptifibatide samples

[0113] Experimental grouping Yield / wt% Purity / % Content / % Comparative example 1 101.01 77.01 65.01 Comparative example 2 99.05 82.38 73.74 Example 1 98.97 85.1 75.02 Example 2 99.23 89.4 80.65

[0114] The above tests were carried out on Examples 1-2 and Comparative Examples 1-2, and the results were as shown in Table 3. It can be seen from Table 3 that compared with Comparative Example 1, the purity and content of the eptifibatide sample in Example 1 were both improved, indicating that compared with the prior art, the eptifibatide sample prepared by the synthesis method provided by the present invention had good purity and content; compared with Example 1, the yield, purity and content of the eptifibatide sample in Example 2 were all improved, indicating that the eptifibatide sample prepared using a sulfonyl group-containing polypeptide condensing agent also had 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 washing the peptide resin with vitamin C made the prepared eptifibatide sample have good purity and content.

[0115] The conventional techniques in the above examples were the prior art well-known to those skilled in the art, so they will not be elaborated in detail here.

[0116] The above is only the specific implementation manner 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, and all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A method for synthesizing eptifibatide, comprising: Step 1) Deprotecting the starting resin; Step 2) Sequentially coupling and condensing protected amino acids by solid-phase synthesis to obtain a linear polypeptide resin: Step 3) Performing iodine solid-phase oxidation cyclization to obtain the oxidized and cyclized polypeptide; Step 4) Washing the resin with a mixed solution of aqueous vitamin C solution and DMF until the iodine color disappears, then washing with DMF, washing with methanol, and drying to obtain the eptifibatide cyclic peptide resin; Step 5) Cleaving the resin, then adding a precipitant to precipitate and washing to obtain the crude eptifibatide; The coupling and condensation reagent used in the coupling and condensation is selected from sulfonyl-containing polypeptide condensing agents / HoBt; The preparation method of the sulfonyl-containing polypeptide condensing agent includes: Step 1: Add deionized water to DMAc to prepare a mixed solvent, then add 4-bromo-2,6-dimethylaniline. After dissolution, add 4-[isopropyl(4-methoxybenzyl)sulfonamido]phenylboronic acid, tripotassium phosphate trihydrate, and palladium acetate. Heat to 98-105 °C and react for 3.5-5 h. After the reaction is completed, 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 perform column chromatography to obtain compound a; Step 2: Add compound a to anhydrous acetonitrile. After dissolution, add a sulfuric acid aqueous solution with a concentration of 50-55 wt%. Then, at -8 to -3 °C, slowly add a sodium nitrite aqueous solution with a concentration of 0.2-0.25 g / mL, stir for 0.8-2 h, add urea, then slowly add a potassium iodide aqueous solution with a concentration of 0.5-0.55 g / mL, and then raise the temperature to room temperature at a heating rate of 3-4 °C / h. Then 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 perform column chromatography to obtain compound b; Step 3: Add deionized water to tert-butanol, with a volume ratio of 1:0.8-1.2 between the two, to prepare a mixed solvent. Then add compound b and the first portion of potassium permanganate, heat to reflux for 20-30 h, then add the remaining potassium permanganate, heat to reflux for 20-30 h, filter, wash with deionized water, rotary evaporate, and then adjust the pH to 4.5-6, filter to obtain compound c; The addition amount of the first portion of potassium permanganate is 45-60 wt% of the total amount of potassium permanganate; Step 4: Add concentrated hydrochloric acid with a concentration of 33-37 wt% to compound c. Under light-shielded conditions, at -3 to 3 °C, slowly add a sodium hypochlorite aqueous solution with a concentration of 8-15 wt%, raise the temperature to room temperature at a heating rate of 2-3 °C / h, filter, wash with deionized water until neutral, and dry to obtain the sulfonyl-containing polypeptide condensing agent; The hydrogen spectrum of the polypeptide condensing agent containing sulfonyl group is as follows: 1 H NMR (400 MHz, CDCl3): 8.82 (2H, O=C-Ar- H ), 7.85 (4H, S-Ar- H ), 2.85 (1H, N-C H ), 1.04 (6H, CH-C H 3), 4.45 (2H, N-C H 2), 6.85 - 7.13 (4H, O-Ar- H ), 3.80 (3H, O-C H 3); HRMS(ESI): C 25 H 22 INO7S, m / z [M+H] + , 607.

02.

2. The synthesis method of 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. A method for synthesizing eptifibatide according to claim 1, wherein: 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. A method for synthesizing eptifibatide according to claim 1, wherein: The reaction time for the iodine solid-phase oxidation cyclization is 0.1 - 4 h.

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

6. Use of the sulfonyl group-containing polypeptide condensing agent described in claim 1 in the synthesis of eptifibatide.

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