Process for preparing eptifibatide
View Patent ↗The present invention provides processes for preparation of eptifibatide that involve coupling of amino acids in a (2+5), (4+3) and (3+4) sequence method. The invention further provides products produced by the described processes, novel compounds that can be used as synthetic intermediates for the preparation of eptifibatide.
1. A process for preparation of eptifibatide of Formula I (SEQ ID NO:1):
wherein Mpr is mercaptopropionic acid, Har is homoarginyl, Gly is glycyl, Asp is aspartyl, Trp is tryptophanyl, Pro is prolyl, and Cys-NH 2 is cysteinamide, the process comprising the steps of:
a) coupling Cys(P 5 )—NH 2 and Trp(P 4 )-Pro-OH to obtain a peptide fragment of formula Trp(P 4 )-Pro-Cys(P 5 )—NH 2 ;
b) coupling the peptide fragment of formula Trp(P 4 )-Pro-Cys(P 5 )—NH 2 and Asp(P 3 )—OH to obtain a peptide fragment of formula Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:5);
c) coupling the peptide fragment of formula Asp(P 3 )-Trp(P 4 )—Pro-Cys(P 5 )—NH 2 (SEQ ID NO:5) and Gly-OH to obtain a peptide fragment of formula Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:6);
d) purifying the peptide fragment of formula Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:6) produced in step c) to less than 0.1% Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )(D)-NH 2 (SEQ ID NO:7), Gly-Asp(P 3 )(D)-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:8), Gly-Asp(P 3 )-Trp(P 4 )(D)-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:9), or Gly-Asp(P 3 )-Trp(P 4 )-Pro(D)-Cys(P 5 )—NH 2 (SEQ ID NO:10) by HPLC;
e) coupling the peptide fragment of formula Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ ID NO:6) and a 1-2 peptide fragment of formula Mpr(P 1 )-Har(P 2 )—OH to obtain protected linear peptide of formula Mpr(P 1 )-Har(P 2 )-Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ NO:3);
f) optionally purifying Mpr(P 1 )-Har(P 2 )-Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ NO:3);
g) deprotection of the Mpr(P 1 )-Har(P 2 )-Gly-Asp(P 3 )-Trp(P 4 )-Pro-Cys(P 5 )—NH 2 (SEQ NO:3) to obtain linear peptide of formula Mpr-Har-Gly-Asp-Trp-Pro-Cys-NH 2 (SEQ ID NO:4); and
h) cyclizing the two sulphur atoms of the Mpr-Har-Gly-Asp-Trp-Pro-Cys-NH 2 (SEQ ID NO:4) to obtain eptifibatide of Formula I (SEQ ID NO:1),
wherein each one of P 1 and P 5 is one of hydrogen and a sulphur protecting group, each one of P 2 and P 4 is one of hydrogen and an amino protecting group, and P 3 is one of hydrogen and a carboxyl protecting group.
2. The process of claim 1 , wherein the coupling is carried out with a suitable coupling agent selected from the group consisting of HOBT, HOAT, DCC, EDC, CDI, ethyl 2-cyano-2-(hydroxyimino)acetate, and mixtures thereof.
3. The process of claim 1 , wherein the step g) is carried out with a suitable deprotecting agent selected from the group consisting of trifluoroacetic acid, hydrobromic acid, acetic acid, and hydrogen fluoride; and
wherein the step h) is carried out in the presence of atmospheric air, oxygen gas or hydrogen peroxide.
4. The process of claim 1 further comprising the step of subjecting the eptifibatide obtained from step h) to a flash chromatography system using an eluent to obtain pure eptifibatide.
5. The method of claim 4 , wherein the eluent is selected from the group consisting of alcohols, nitrile solvents, water, acids, and mixtures thereof.
6. The method of claim 4 , wherein the eluent is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, propionitrile, acetic acid, trifluoro acetic acid, water, and mixtures thereof.
7. The method of claim 4 , wherein the pure eptifibatide has a purity of at least 99%.