WO2013057736A2

WO2013057736A2 - Preparation of eptifibatide peptide

Info

Publication number: WO2013057736A2
Application number: PCT/IN2012/000386
Authority: WO
Inventors: Mukund Keshav Gurjar; Chinmoy PRAMANIK; Narendra Kumar TRIPATHY; Anupam Badal ROY; Ashok Tukaram CHAUDHARI; Ajit Sahebrao BORHDE; Ganesh Kaluram KARHADE; Balaji Vasant CHAGHULE; Samit Satish Mehta; Arnab Arun PAL
Original assignee: Emcure Pharmaceuticals Limited
Priority date: 2011-06-07
Filing date: 2012-06-04
Publication date: 2013-04-25
Also published as: WO2013057736A3

Abstract

The present invention related to a process for the preparation of eptifibatide using a novel non-linear liquid phase peptide synthesis scheme. This invention makes use of combing precursor peptides synthesised separately in high purity and yield over presently known processes. The disclosed process has several advances over known processes like ease of scale-up and work-up as the process utilises liquid phase peptide preparation chemistry.

Description

PREPARATION OF EPTIFIBATIDE PEPTIDE

DESCRIPTION

This invention relates to preparation of eptifibatide. Eptifibatide is a cyclic heptapeptide containing 6 amino acids and 1 mercaptopropionyl-(des-amino cysteinyl) residue. An interchain disulfide bridge is formed between the cysteine amide and the mercaptopropionyl moieties. Chemically it is N6- (aminoiminomethyl)-N2-(3-mercapto- 1 -oxopropyl)-L-lysylglycyl-L-a-aspartyl-L- tryptophyl-L-prolyl-L-cysteinamide, cyclic-(l→6)-disulfide [1]. Eptifibatide binds to the platelet receptor glycoprotein (GP) Ilb/IIIa of human platelets and inhibits platelet aggregation. It is a white to light yellow powder, which is readily soluble in water.

pa-Har-Gly-Asp-Trp-Pro-Cys

[1]

The invention disclosed describes the new processes of preparation of eptifibatide. The processes of the disclosure are novel in those new schemes of reactants, reagents and intermediate compounds are used. These schemes are having added advantages over the prior schemes in terms of process parameters, industrial scale-up requirements and impurity profiles. These properties may provide advantage in pharmaceutics and therapy over known methods and products. Accordingly the invention is disclosed in the SCHEME 1 with a reaction sequence for the preparation of intermediate peptide fragments and then condensation of these fragments followed by deprotection and cyclization to get the final cyclic molecule of eptifibatide [1]. The scheme depicts the preparation of two new intermediate peptide fragments [EPT-2 & EPT-3] for convergent synthesis of linear eptifibatide precursor [EPT-5] and then cyclization leading to the formation of eptifibatide [1] molecule using solution phase peptide synthesis methods. To achieve the superior enantiomeric purity of the final product the modified amino acid Cys [EPT-1] is prepared using a tartarate intermediate so that the levo enantiomer of the amino acid is present in at least 99% ee with overall purity of at least 99% by HPLC. This reaction chemistry is disclosed in Examples 1 to 4.

The protecting groups shown are illustrative and any other suitable protecting groups may be used. Similarly, reagents, catalyst used are not specifically depicted as several of these are known in the art and can be innovatively combined to get the required products as described in the following examples.

PART 1

Example 1: preparation off H-L-Cys(Trtt)-OH

To a solution of L-cysteine hydrochloride (200.0 gm, 1.27 mol) in DMF (600 mL), trityl chloride (389 gm, 1.39 mol) was added at room temperature and reaction mixture was heated at 60-65°C for 4 h. the reaction was monitored by TLC. After complete consumption of L-cysteine, the reaction mixture was cooled to room temperature and slowly poured into a 10% aq.NaOAc solution (6 L) under stirring. The solid mass was precipitated. The reaction mixture was filtered and the residue was sequentially washed with DM water (6 L), and acetone (4 L). After filtration the wet cake was dried at 50-55 °C under vacuum to yield 400 g (87 %) of H-L-Cys(Trt)-OH [EPT-1 A] as a white solid.

Example 2: preparation of Fimoc-L-Cys(Tri)-OH To a mixture of THF (1 L) and water (1 L), H-L-Cys(Trt)-OH (200 gm, 0.55 mol) was added at room temperature. Sodium bicarbonate (92.4 g, 1.101 mol) and Fmoc-OSu (176.4 gm, 0.52 mol) were added portion wise and stirred at RT for 3 h. The reaction was monitored by TLC. After complete consumption of H-L- Cys(Trt)-OH, THF was removed under reduced pressure and aqueous layer was extracted with toluene (1 L X 2). The toluene layer was washed with IN HC1 (1 L) and concentrated under reduced pressure. The crude product was stirred in toluene (400 mL) for 2 h. Cyclohexane (2 L) was added into it and stirred for 4 h. White solid product was precipitated. Solid product was filtered and dried at 55- 60 °C under vacuum to obtain 303 gm (94%) of Fmoc-L-Cys(Trt)-OH [EPT-1B] as a white solid.

Example 3: preparation of Fmoc-L-Cys(Trt)-NH2

To a cold solution (0 to -5 °C) of Fmoc-Cys(Trt)-OH (250 gm, 0.426 mol) in THF (2.5L), CDI (138.4 g, 0.853 mol) was charged under nitrogen atmosphere and the reaction mixture was stirred at 0 to -5 °C for 2 h. Aqueous ammonia (25%) (127.5 mL; 1.707 mol) was added into the reaction mixture and stirred for 30 mins at 0 °C to -5 °C. The reaction was quenched by adding of 2N HC1 (1.5 L). THF was removed under reduced pressure and aqueous layer was extracted with ethyl acetate (750 mL X 3). Combined ethyl acetate layer was dried over anhydrous sodium sulphate (60 g), filtered and concentrated under reduced pressure. The crude product was stirred with methanol (1.25 L) for 16 hrs. Solid precipitate was filtered off. The filtrate was concentrated to afford 225 gm (90%) white solid Fmoc-L-Cys(Trt)-NH₂ [EPT-1C] of chiral purity > 99.5%.

Example 4: preparation of L-H-Cys(Trt)-NH₂.1--tar4rate saM

To a solution of Fmoc-L-Cys(Trt)-NH₂ (100 g, 0.171 mol) in acetonitrile (400 mL), tri ethyl amine (71.4 mL, 0.513 mol) were added and stirred for 12 h at room temperature. The reaction was monitored by TLC. After complete consumption of the Fmoc-L-Cys(Trt)-NH₂, acetonitrile was removed under reduced pressure and diluted with methanol (1 L). L-tartaric acid (28.2 g, 0.188 mol) was added and reaction mixture was heated at 80 °C for 2 h. The reaction mixture was cooled to room temperature and H-butanol (500 mL) was added and stirred for 1 h and filtered. The residue was stirred in toluene (500 mL) for 1 h at 75-80 °C. The reaction mixture was cooled to room temperature. Filtered and dried at 50-55 °C under vacuum to yield 77 g (88 %) H-L-Cys(Trt)-NH₂.L-tartrate salt [EPT-1] .

PART 2

Example 5: preparation of Fmoc-HArg(Pbf)-OAl!

To a solution of Fmoc-HArg(Pbf)-OH (150.0 gm, 0.226 mole) in DMF (600 mL), allyl bromide (54.7 gm, 0.452 mole) and potassium carbonate (40.6 gm, 0.294 mole) were added and reaction mixture was stirred vigorously at 25-30 °C for 4 h. the reaction was monitored by TLC. After complete consumption of Fmoc- HArg(Pbf)-OH, the reaction mixture was filtered and filtrate was added slowly to 0.5 N HCl (6 L) under stirring at 10-15 °C. White solid product was precipitated. The reaction mixture was further stirred for 2 h and filtered. The residue was stirred in water (4 L) for 5 h at 25-30 °C, filtered and dried at 50-55 °C under vacuum to yield 143 gm (90 %) of Fmoc-HArg(Pbf)-OAll [EPT-2A].

Example 6: preparation of H-HArg(Pbf)-OAll

To a solution of Fmoc-HArg(Pbf)-OAll (140 gm, 0.199 mol) in DMF (560 mL) triethyl amine (80.6 gm, 0.798 mole) was added and reaction mixture was stirred at 25-30 °C for 16 h. The reaction was monitored by TLC. After complete consumption of Fmoc-HArg(Pbf)-OAll, the reaction mixture was poured into 0.5 N HCl (2.8 L) and precipitated solid was filtered off. The filtrate was washed with MTBE (150 mL x 2), diluted with water (2.8 L) and basified by solid sodium bicarbonate (45 gm). The resulting solution was extracted with ethyl acetate (300 mL x 3), combine organic layer was dried over sodium sulphate, filtered and concentrated. 78.5 gm (82%) of H-HArg(Pbf)-OAll [EPT-2B]. was obtained as a light yellow viscous liquid.

Example 7: preparation of MPA(Trt)-HArg(Pbf)-OAH To a solution of H-HArg(Pbf)-OAll (74.3gm, 0.155 mol) in DMF ( 370 mL) at 5- 10 °C , MPA(Trt)-OH (45 gm, 0.129 mol), HOBt (23.7 gm, 0.155 mol), EDC.HCl (32.0 gm, 0.168 mol) and DIPEA (11.2 mL, 0.065 mol) were added slowly with stirring at 5-10 °C under nitrogen atmosphere. The reaction mixture was allowed to warm at 25-30 °C and stirred for 2 h. the reaction was monitored by TLC. After complete consumption of MPA(Trt)-OH, the reaction mixture was added drop wise to 0.2 N HC1 (3.7 L). White solid product was precipitated. The reaction mixture was further stirred for 2 h and filtered. The residue was stirred in water (3 L) for 4 h at 25-30 °C and filtered. The residue was dried at 50-55 °C under vacuum to afford 96 gm (91%) of 3-Mpa(Trt)-HArg(Pbf)-OAll [EPT-2C] as a white solid.

Example 8: preparation of MPA(Trt)-HArg(Pbf)-OH

To a solution of MPA(Trt)-HArg(Pbf)-OAll (95 gm, 0.117 mol) in DMSO (380 mL), Pd(PPh₃)₄ (2.03 gm, 0.00176 mol) and morpholine (40.8 gm, 0.468 mol) were added at 25- 30 °C under N₂ atmosphere and stirred for 2 h. the reaction was monitored by TLC. After complete consumption of MPA(Trt)-HArg(Pbf)-OAll, reaction mixture was filtered through celite bed and the filtrate was added slowly into 0.5 N HC1 (3.8 L). A light yellow solid was precipitated. The reaction mixture was further stirred for 3 h and filtered. The residue was stirred in water (2 L) for 2 h at 25-30 °C and filtered. The residue was dried at 50-55 °C under vacuum. 81 gm (89 %) of 3-Mpa(Trt)-HArg(Pbf)-OH [EPT-2B] was obtained as a light yellow solid. Example 9: preparatiom of MPA(Trt)-HArg(PIbf)-GIy-OAil

To a solution of HCl.H-Gly-OAll (18.9 gm, 0.125 mol) in DMF (800 ml) at 5- 10 °C, 3-Mpa(Trt)-HArg(Pbf)-OH (80.0 gm, 0.104 mol), HOBt (19.1 gm, 0.125 mol) and EDC.HCl (25.8 gm, 0.135 mol) were added under nitrogen atmosphere. DIPEA (30.7 mL, 0.126 mol) was added slowly over a period of 15-20 min. The reaction mixture was allowed to warm to 25-30 °C and stirred for 2 h. the reaction was monitored by TLC. After complete consumption of 3-Mpa(Trt)-HArg(Pbf)- OH, the reaction mixture was added drop wise to 0.2 N HC1 (8.0 L). Light yellow solid was precipitated. The reaction mixture was further stirred for 2 h and filtered. The residue was stirred in water (3 L) for 2 H at 25-30 °C and filtered. The residue was dried at 50-55 °C under vacuum. 82 gm (91 %) of 3-Mpa(Trt)-H- Arg(Pbf)-Gly-OAll [EPT-2E]. was obtained as a light yellow solid.

Exannp!e 10: preparation of MPA(Trt)-HArg(Pbf)-Gly-OH:

To a solution of MP A(Trt)-HArg(Pbf)-Gly-OAll (80.0 gm, 0.092 mol) in DMSO

(320 mL), Pd(PPh₃)₄ (1.6 gm, 1.4 mmol) and morpholine (32.1 gm, 0.368 mol) were added at 25- 30 °C under nitrogen atmosphere and stirred for 2 h. The reaction was monitored by TLC. After complete consumption of starting material, reaction mixture was filtered through celite bed and filtrate was added slowly to 0.5 N HC1 (3.2 L). A light yellow solid was precipitated. The reaction mixture was stirred for 2 h and filtered. The residue was stirred in water (3 L) for 2 h at 25-30 °C and filtered. The residue was dried at 50-55 °C under vacuum. 69 gm (90 %) of 3-Mpa(Trt)-H-Arg(Pbf)-Gly-OH [EPT-2] was obtained as a light yellow solid.

PART 3

Example 11: preparation, of Boc-Trp-OH

To a solution of H-Trp-OH (100.0 gm, 0.490 mole) in THF (300 mL) and water (300 mL), sodium bicarbonate (82.3 g, 0.979 mole) was added portion wise and reaction mixture was cooled to 10-15°C. Boc anhydride (123.8 g, 0.539 mol) was added to the reaction mixture slowly at 10-15 °C and stirred for 15 hrs. Reaction was monitored by TLC. After complete reaction, THF was removed under reduced pressure and aqueous layer was washed with MTBE (500 mL). Aqueous layer was added slowly to 1.ON HC1 (2 L). White solid was precipitated. Reaction mixture was stirred for 3 hrs and filtered. The residue was stirred in water (1 L) for 2 hrs at 25-30 °C and filtered. The residue was dried at 50-55 °C under ' vacuum. 140 gm (94%) of Boc-Trp-OH was obtained as a white solid. Example 12: preparation of Boc-Pro-OH

To a solution of H-Pro-OH (200.0 gm, 1.73 mole) in acetonitrile (400 mL) and water (400 mL), triethylamine (289 mL, 2.07 mol) and Boc anhydride (436 mL, 1.9 mol) were added at 5-10°C and stirred for 15 h at RT. Reaction was monitored by TLC. After complete consumption of starting material acetonitrile was removed under reduced pressure and aqueous layer was washed with toluene (1 L). Aqueous layer was acidified by adding 1.0N HC1 (1 L). White solid was precipitated. Reaction mixture was stirred for 1 h and filtered. The residue was dried at 50-55 °C under vacuum. 325 gm (86%) of Boc-Pro-OH was obtained as a white solid.

Example 13: preparation of Boc-Pro-OAll

To a solution of Boc-Pro-OH (150.0 gm, 0.698 mol) in DMF (750 mL), potassium carbonate (154g, 1.11 mol) and allyl bromide (85 g, 0.977 mol) were added and stirred for 15 hrs at RT. Reaction was monitored by TLC. After complete reaction, reaction mixture was poured in to DM water (7.5 lit) and reaction mixture was extracted with ethyl acetate (750 mL X 2). Combined organic layer was dried on sodium sulfate and solvent was removed under reduced pressure at below 50 °C. to obtain Boc-Pro-OAll as a viscous liquid.

Example 14: preparation of Boc-Trp-Pro-OAM

The solution of Boc-Pro-OAll (101 g, 0.394 mol) in 9-11% EtOAc.HCl (600 ml) was stirred at 25-30 °C for 4 hrs. Reaction was monitored by TLC. After complete consumption of Boc-Pro-OAll, volatilities were distilled out at below 45 °C under reduced pressure and co evaporated with toluene (150 mL). The residue was dissolved in DMF (500 mL) under nitrogen atmosphere and cool to 10-15 °C. DIPEA (102.7 mL, 0.59 mol), Boc-Trp-OH (100.0 g, 0.328 mol), HOBt (60.2 g, 0.394 mol) and EDC.HC1 (81.7 g, 0.427 mole) were added. Reaction mixture was allowed to warm at 25-30 °C and stirred for 1.5 hrs. Reaction was monitored by TLC. After complete consumption of Boc-Trp-OH reaction mixture was added into 0.2 HC1 (2.5 L) and stirred for 15 min. The solution was extracted with ethyl acetate (1.0 L x 2). Combine ethyl acetate layer was washed with 10 % sodium bicarbonate solution (1.0 L) and water (2.5 L). Ethyl acetate layer was dried over anhydrous sodium sulfate (50 g), filtered and concentrated under reduced pressure at below 50 °C. 140 gm (96%) of Boc-Trp-Pro-OAU [EPT-3A] was obtained as a white solid.

Example 15: preparation of Fmoc-Asp(OtBra)-Trp-Pro-OAll

The solution of Boc-Trp-Pro-OAll (120 g, 0272 mol) in 9 -11% EtOAc.HCl (1.2

L) was stirred at 25-30 °C for 4 hrs. Reaction was monitored by TLC. After complete consumption of Boc-Trp-Pro-OAll, volatilities were distilled out at below 45 °C under reduced pressure. The residue was stirred in ethyl acetate (600 mL) for 30 min at 25-30 °C and filtered. The residue was dried under vacuum at 25-30 °C. The solid obtained was dissolved in DMF (600 mL) and cooled to 10- 15 °C under nitrogen atmosphere. Fmoc-Asp(OtBu)-OH (81.2 g, 0.197 mol), HOBt (36.23 g, 0.236 mol), EDC.HC1 (49.5 g, 0.258 mol) and DIPEA (64.0 mL, 0.367 mol) were added into the reaction mixture slowly under stirring at 10-15 °C. Reaction mixture was allowed to warm at 25-30 °C and stirred for 2 h. Reaction was monitored by TLC. After complete consumption of Fmoc-Asp(OtBu)-OH reaction mixture was added slowly into 0.2 N HC1 (6.0 L) under vigorous stirring and stirred for 2 hrs at 25-30 °C. Solid was filtered and again stirred in water (3.0L) for 2 hrs at 25-30 °C. Solid was filtered, washed with water (300 mL) and dried under vacuum at 50-55 °C. 140 gm (97%) of Fmoc-Asp(QfBu)-Trp-Pro- OA11 [EPT-3C] was obtained as a yellowish solid. Example 16: preparation of Fmoc-Asp(OtBii)-Trp-Pro-OH

To a solution of Fmoc-Asp(OtBu)-Tip-Pro-QAll (125 g, 0.170 mol) in MDC (625 mL), Pd(PPh₃)₄ (5.9 g, 5.1 mmol), triphenylphosphine (6.7g, 0.025 mol) and sodium-2- ethylhexanoate (30.7 g, 0.187 mol) were added under nitrogen atmosphere at 25-30 °C and stirred for 2 h. Reaction was monitored by TLC. After complete consumption of Fmoc-Asp(OtBu)-Trp-Pro-OAll, solvent was removed completely and the residue was dissolved in DMF (250 mL) and water (500 ml). The solution was washed with MTBE (625 mL x 3). The aqueous layer was acidified by adding 1 N HC1 (1 L) and extracted with ethyl acetate (625 mL x 2). Combine ethyl acetate layer was evaporated and the residue was dissolved in DMF (310 mL). The solution was added slowly to water (5 L) under stirring at 25-30 °C. Solid product was precipitated. Stir the reaction mixture for 3 h and filtered. The solid was stirred again in D M water (2.5 lit) for 3 h at 25-30 °C, filtered and dried at 50-55 °C under vacuum. 102 gm (86%) of Fmoc-Asp(OtBu)- Trp-Pro-OH [EPT-3B] was obtained as a yellowish solid. Example 17: preparation of Fmoc-Asp(OtBii)-Trp-Pro-Cys(Trt)-NH2

To a suspension of H-Cys (Trt)-NH₂.tartrate salt (89.0 g, 172 mmol) in DM water (900mL) and ethyl acetate (900mL), aq ammonia (54.0 mL) was added and stirred for 30 min. at 25-30 °C Reaction mixture was filtered through cellite bed. From filtrate layers were separated, upper ethyl acetate layer contained the product. Aqueous layer was again extracted with ethyl acetate (2 x 450 mL). Combined ethyl acetate layer was dried over anhydrous sodium sulphate, filtered and concentrated at below 45 °C. To this residue DMF was added at 25-30 °C followed by Fmoc-Asp (OtBu)-Trp-Pro-OH (100.0 g, 0.144 mole), HOBt (26.5 g, 0.173 mole) , EDC.HC1 (35.9 g, 0.187 mole) and DIPEA (12.5 ml 0.072mole) were added under nitrogen atmosphere at 10-15 °C. Reaction mixture was allowed to warm to 25-30 °C and stirred for 1.5 h. Reaction was monitored by TLC. After complete consumption of Fmoc-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ reaction mixture was added slowly to 0.2 N HC1 (5.6 L), stirred for another 2 h at 25-30 °C and filtered. The solid was again stirred in water (4 L) at 25-30 °C for 2 h, filtered and dried at 50-55 °C under vacuum. 144 gm (96%) of Fmoc-Asp (OtBu)-Trp- Pro-Cys (Trt)-NH₂ [EPT-3E] was obtained as an off white solid.

PART 4

Example 18: preparation off Mpa(Tri)-HArg(Pbf)-Gly-Asp(OiBia)-Trp-Pro-

To a solution of Fmoc-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ (100.0 g, 0.096 mol) in DMF (400 mL), triethylamine (40.0 mL, 0.288 mol) was added and reaction mixture was stirred at 25-30 °C for 15 h. Reaction was monitored by TLC. After complete consumption of Fmoc-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ reaction mixture was cooled to 10-15 °C and 1.5 N HCl (400 mL) was added. The reaction mixture was washed with 20% ethyl acetate in MTBE (1.0 L x 2). The aqueous layer was diluted with water (2.0 L) and basified with sodium bicarbonate (45 g). The solution was extracted with ethyl acetate (500 mL x 2), combine ethyl acetate layer was dried over anhydrous sodium sulfate (75 g), filtered and concentrate. The residue was dissolved in DMF (440 mL) at 10-15 °C under nitrogen atmosphere with constant stirring. Mpa(Trt)-HArg(Pbf)-Gly-OH (66.0 g, 0.797 mole), HOBt (14.7 g, 0.096 mole), EDC.HC1 (20.0 g, 0.105 mole) and DIPEA (10.4 mL, 0.06 mole) were added into the reaction mixture under stirring at 10-15 °C . Reaction mixture was allowed to warm to 25-30 °C and stirred for 1.5 h. Reaction was monitored by TLC. After complete consumption of Mpa(Trt)- HArg(Pbf)-Gly-OH reaction mixture was added slowly to 0.2 N HCl (4.5 L). Solid product was precipitated. The reaction mixture was stirred for another 2 hrs and filtered. The residue was again stirred in water (2.2 L) at 25-30 °C for 1 h, filtered and dried under vacuum at 50-55 °C. 120 gm (92%) of Mpa(Trt)- HArg(Pbf)-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ [EPT-4]was obtained as a yellowish solid.

Example 19: preparations off Eptifibat e crade

To the solution of Mpa(Trt)-HArg(Pbf)-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH₂ (15 g) in MDC (60 mL) at 5-10 °C, DTT (6.0 g), triethylsilane (15 mL) and TFA (45 mL) were added under stirring. Reaction mixture was allowed to warm to 25- 30 °C. After 45 min, volatilities were distilled out under reduced pressure at below 32°C. The residue was cooled to 10- 20 °C and MTBE (225 mL) was added slowly with vigorous stirring. Yellow solid was precipitated; it was stirred for 30 min. Solid was filtered under nitrogen atmosphere and washed with MTBE (50 mL). Solid was stirred in MTBE (150 mL) for 30 min at 25-30 °C, filtered and dried under vacuum at 25-30 °C. The solid obtained was dissolved in a mixture of D M water (365 mL) and acetonitrile (365 mL) and added into the mixture of D M water (7.1 lit) and acetic acid (15.5 mL) at 25-30 °C. The solution of I₂ (3.51 gm) in methanol (77.0 ml) was added slowly into the reaction mixture under stirring at 25-30 °C. The reaction mixture was stirred for 30 min; ascorbic acid (2.6 g) was added to the reaction mixture at 25-30 °C and stirred for 15 min. The reaction mixture was filtered through a bed of Hyflow supercel (350.0 gm) followed by 0.4 micron filter paper. The filtrate was purified by preparative HPLC to obtain pure eptifibatide (>99.0% purity).

Example 20: purification of Eptifibatide by preparative HPLC method:

Purification Step 1

1. The primary purification of eptifibatide involved a trifluroacetic acid/ acetonitrile/ water based purification.

2. The HPLC purity of the crude sample was >70.0 %. Daiso gel CI 8, 10μ, was used as a stationary phase with column of 50mm diameter & 300mm length. Flow rate was 50 ml/min and detection wavelength was 220 nm.

3. The composition of mobile phase were as follows :- Phase A) 0.1 % TF A in water (v/v)

Phase B) 0.1 % TFA in CH₃CN (v/v)

4) The column was equilibrated with 100% mobile phase A for 20 min. The purification gradient was as follows: started elution of 12% mobile phase B over 60 min; then 12% mobile phase B to 20 % mobile phase over B 65 min; and eluted with 100% mobile phase B over final 20 min.

5) The fractions collected at the time of above purification were monitored by analytical HPLC.

6) Main fraction (Mf) - >94% purity & individual impurity < 1.3 %. Side fraction (Sf)- > 84 % purity. The above fractions were stored at 2° C to 8° C.

ff MIcatiom Step 2 1) The secondary purification involved an acetic acid / water / acetonitrile based purification. Here the residual TFA counter ions were replaced by the acetate ions.

2) Daiso gel CI 8, 10μ, was used as a stationary phase with 50 mm diameter & 300 mm length. Flow rate was 50 ml/min and detection wavelength was 220 nm.

3) The composition of mobile phase were as follows:-

Phase A) 0.1 % AcOH in water (v/v).

Phase B) 0.1% AcOH acid in CH₃CN (v/v).

4) The main fraction (Mf) from Purification Step 1 was loaded on the preparative column followed by desalting using 3% ammonium acetate in water (pH4, adjusted by AcOH) and 1% AcOH in water respectively.

5) The column was equilibrated by eluting with the mobile phase A over 20 min. The purification gradient was as follows:- started elution of 5% mobile phase B; gradient rise 5% mobile phase B to 15 % mobile phase B over 150 min; then elution of 100 % mobile phase B over 20 min.

6) The fractions collected at the time of above purification were monitored by analytical HPLC.

8) Main fraction (Mf) - >99% purity & individual impurity < 0.5 %.

Side fraction (Sf)- > 84% purity.

9) Then all the main fractions were mixed & concentrate to remove the solvent.

10) The above solution was stored at 2° C to 8° C.

11 ) The above solution was lyophilized.

Accordingly the invention is disclosed in an illustrative SCHEME 1 with a reaction sequence for the preparation of intermediate peptide fragments and then condensation of these fragments followed by deprotection and cyclization to get the final cyclic molecule of eptifibatide [1] using a set of reactants and reagents. The set of reactants and reagents used in the scheme are illustrative and any other suitable reactants and reagents may be replaced in these reactions.

³ methanol, 80 °C,

EPT-1C 2h EPT-1.Tartarate

EPT-1

O^OH Allyl bromide _NHpbf Triethyl amine

NHPbf O.

DMF _, DMF NHPbf

HN N NHFmoc HN N 'NHFmoc

HN N NH, H RT, 4h H

RT, 16h H

EPT-2A EPT-2B

EPT-2E EPT-2 PART 3

Eptifibatide

Claims

A process of the preparation of eptifibatide acetate by non-linear liquid phase peptide synthesis, comprising the steps of:

a) coupling of C-terminus protected L-homoarginine(Pbf) with S- trityl-3-mercaptopropionic acid, in the presence of a coupling reagent, leading to the formation of protected dipeptide ester of sequence MPA[Trt]-L-homoArg(Pbf)-0-All;

b) deprotecting the dipeptide ester of step a) and coupling of said deprotected dipeptide with H-Gly-OAllyl leading to the formation of protected tripeptide of sequence MPA[Trt]-L- homoArg(Pbf)-Gly-OAllyl and deprotecting it leading to the formation of tripetide of sequence MPA[Trt]-L-homoArg(Pbf)- Gly-OH;

c) coupling of N-terminus protected Boc-Trp-OH with H-L-Pro- OAllyl, in the presence of a coupling reagent, leading to the formation of protected dipeptide of sequence Boc-Trp-Pro- OAllyl and deprotecting it to H-Trp-Pro-OAll;

d) coupling of said deprotected dipeptide of step c) with Fmoc- Asp(OtBu)-OH and deprotecting leading to the formation of tripeptide of sequence Fmoc-Asp(OtBu)-T -Pro-OH; e) coupling of said deprotected tripeptide of step d) with H-Cys- (Trt)-NH₂ and selectively deprotecting it leading to the formation of tetrapeptide of sequence H-Asp(OtBu)-Trp-Pro- ( Cys(Trt)-NH₂;

f) reacting tripeptide of step b) with the tetrapetide of step e) leading to the formation of helptapeptide of sequence MPA[Trt]-L-homoArg(Pbf)-Gly-Asp(OtBu)^-Pro-Cys[Trt]- NH₂;

g) deprotecting heptapeptide of step f) in the presence of

trifluoroacetic acid and DTT leading to the formation of deprotected heptapeptide of sequence MPA-L-homoArg-Gly- Asp-Trp-Pro-Cys-NH_2.TFA salt; and

h) oxidizing the heptapeptide of step g) with iodine in a protic solvent leading to the formation of cyclic eptifibatide salt of sequence MPA-L-homoArg-Gly-Asp-Trp-Pro-Cys-NH₂ having a disulphide bond (1-7) between MPA and Cys residues.

2. A process of Claim 1, wherein the tripeptide obtained in step b) is isolated prior to used in step f); the tetrapeptide obtained in step e) is isolated prior to use in step f); and the heptapeptide obtained in step g) is isolated to high purity.

3. A process of Claim 2, wherein the isolation is by precipitation,

crystallization, extraction, or chromatography.

4. A process according to Claim 1 or 3, further comprising the steps:

a) purifying the eptifibatide by HPLC chromatography; and b) drying the solution of the peptide acetate so obtained.

5. A process of according to Claim 4, wherein the drying is by lyophilizing or spray drying.

6. A process of the preparation of modified amino acid H-L-Cys[Trt]-NH₂ using L-tartarate as a resolving intermediated having enantiomeric purity of at least 99% ee with overall at least 99% purity by an HPLC metjod.

7. Eptifibatide obtained according to Claim 1 having a purity of at least about 98.0% as determined by HPLC method.

8. Eptifibatide obtained according to Claims 1 and 4 contains no

more than about 1% impurity of any other peptide and no more

than 2% total impurities.

9. A novel tripeptide of sequence MPA[Trt]-L-homoArg(Pbf)- Gly-OH according to Claim 1.

10. A novel tetrapeptide of sequence H-Asp(OtBu)-Trp-Pro- Cys(Trt)-NH₂ according to Claim 1.