Processes for Preparing Eptifibatide

Abstract

The present invention provides, inter alia, convergent processes for preparing eptifibatide that involve coupling a 2-6 eptifibatide fragment to an activated cysteinamide residue to form a 2-7 eptifibatide fragment, attaching a mercaptopropionic acid residue to the 2-7 eptifibatide fragment through disulfide bond formation, coupling the peptide intramolecularly, and removing the protecting group, to form eptifibatide. The invention further provides products produced by the described processes, novel compounds that can be used as synthetic intermediates for the preparation of eptifibatide, and novel compounds that are structurally similar to eptifibatide.

1 . A process comprising:
 providing a compound of formula H:   
     
       
         
         
             
             
         
       
       wherein
 Har is homoarginyl; 
 Gly is glycyl; 
 Asp is aspartyl; 
 Tip is tryptophanyl; 
 Pro is prolyl; 
 Cys-NH 2  is cysteinamide; 
 Mpa is mercaptopropionic acid; and 
 P 2  is a carboxyl protecting group; and 
 
       coupling the Har and Mpa residues to form a compound of formula III: 
     
     
       
         
         
             
             
         
       
     
   
   
       2 . The process of  claim 1  wherein P 2  is t-butyl. 
   
   
       3 . The process of  claim 1  further comprising removing P 2  from the Asp residue of the compound of formula III to form eptifibatide. 
   
   
       4 . The process of  claim 1  further comprising removing P 1  from the Har residue of a compound of formula I: 
     
       
         
         
             
             
         
       
     
     wherein P 1  is an amino protecting group, thereby forming the compound of formula II. 
   
   
       5 . The process of  claim 4  wherein P 2  is stable under conditions suitable for removal of P 1 . 
   
   
       6 . The process of  claim 4  wherein P 2  is t-butyl. 
   
   
       7 . The process of  claim 4  wherein P 1  is 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl. 
   
   
       8 . The process of  claim 4  further comprising attaching an Mpa residue to a 2-7 eptifibatide fragment of the formula:
   P 1 -Har-Gly-Asp(O—P 2 )-Trp-Pro-ACys-NH 2      
     wherein ACys-NH 2  is an activated cysteinamide residue, through a disulfide linkage between the Mpa residue and the ACys-NH 2  residue of the 2-7 eptifibatide fragment, thereby forming the compound of formula I. 
   
   
       9 . The process of  claim 8  further comprising coupling a 2-6 eptifibatide fragment of the formula:
   P 1 -Har-Gly-Asp(O—P 2 )-Trp-Pro-OH   
     to an activated cysteinamide residue through the Pro residue of the 2-6 eptifibatide fragment, thereby forming the 2-7 eptifibatide fragment. 
   
   
       10 . The process of  claim 9  wherein the activated cysteinamide residue is H-Cys(Npys)-NH 2 . 
   
   
       11 . The process of  claim 9  further comprising coupling a 2-3 eptifibatide fragment of the formula:
   P 1 -Har-Gly-OH   
     and a 4-6 eptifibatide fragment of the formula:
   H-Asp(O—P 2 )-Trp-Pro-OH 
 
     through attachment of the Gly residue of the 2-3 eptifibatide fragment to the Asp residue of the 4-6 eptifibatide fragment, thereby forming the 2-6 eptifibatide fragment. 
   
   
       12 . The process of  claim 11  further comprising coupling an amino-terminal protected Asp residue having a protected carboxyl side chain to a Trp-Pro dipeptide through the Trp residue of the dipeptide and removing the amino-terminal protecting group from the Asp residue to form the 4-6 eptifibatide fragment. 
   
   
       13 . The process of  claim 12  further comprising coupling an amino-terminal protected Har residue and a protected or unprotected Gly residue to form the 2-3 eptifibatide fragment. 
   
   
       14 . A process comprising:
 coupling an amino-terminal protected homoarginine residue and a protected or unprotected glycine residue, thereby forming a 2-3 eptifibatide fragment of the formula:
   P 1 -Har-Gly-OH; 
   coupling an amino-terminal protected aspartic acid residue having a protected carboxyl side chain to a tryptophan-proline dipeptide through the tryptophan residue of the dipeptide, and removing the amino-terminal protecting group from the aspartic acid residue, thereby forming a protected 4-6 eptifibatide fragment of the formula:
   P 3 -Asp(O—P 2 )-Trp-Pro-OH; 
   coupling the 2-3 eptifibatide fragment and the 4-6 eptifibatide fragment through attachment of the Gly residue of the 2-3 eptifibatide fragment to the Asp residue of the 4-6 eptifibatide fragment, thereby forming a 2-6 eptifibatide fragment of the formula:
   P 1 -Har-Gly-Asp(O—P 2 )-Trp-Pro-OH 
   wherein
 Har is homoarginyl; 
 Gly is glycyl; 
 Asp is aspartyl; 
 Trp is tryptophanyl; 
 Pro is prolyl; 
 P 1  and P 3  are amino protecting groups; and 
 P 2  is a carboxyl protecting group; 
   coupling the 2-6 eptifibatide fragment to an activated cysteinamide residue through the Pro residue of the 2-6 eptifibatide fragment, thereby forming a 2-7 eptifibatide fragment of the formula:
   P 1 -Har-Gly-Asp(O—P 2 )-To-ACys-NH 2    
   wherein ACys-NH 2  is an activated cysteinamide;   attaching a mercaptopropionic acid residue to the 2-7 eptifibatide fragment through a disulfide linkage between the mercaptopropionic acid residue and the ACys-NH 2  residue of the 2-7 eptifibatide fragment, thereby forming a compound of formula I:   
     
       
         
         
             
             
         
       
       wherein
 Mpa is mercaptopropionic acid; 
 Cys-NH 2  is cysteinamide; 
 
       removing P 1  from the Har residue of the compound of formula I, thereby forming a compound of formula II: 
     
     
       
         
         
             
             
         
       
       coupling the Har and Mpa residues of the compound of formula II, thereby forming a compound of formula III: 
     
     
       
         
         
             
             
         
       
       removing P 2  from the Asp residue of the compound of formula III to form eptifibatide. 
     
   
   
       15 . The process of  claim 14  wherein P 2  is stable under conditions suitable for removal of P 1 . 
   
   
       16 . The process of  claim 14  wherein P 2  is t-butyl. 
   
   
       17 . The process of  claim 14  wherein P 1  is 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl. 
   
   
       18 . The process of  claim 14  wherein ACys-NH 2  is H-Cys(Npys)-NH 2 . 
   
   
       19 . A compound having formula IV: 
     
       
         
         
             
             
         
       
     
     wherein
 R 1  is hydrogen or P 1 ; 
 P 1  is an amino protecting group; and 
 P 2  is a carboxyl protecting group. 
 
   
   
       20 . The compound of  claim 19  wherein P 2  is t-butyl. 
   
   
       21 . The compound of  claim 19  wherein R 1  is hydrogen. 
   
   
       22 . The compound of  claim 21  wherein P 2  is t-butyl. 
   
   
       23 . The compound of  claim 19  wherein R 1  is P 1 . 
   
   
       24 . The compound of  claim 23  wherein P 1  is 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl. 
   
   
       25 . The compound of  claim 24  wherein P 2  is t-butyl. 
   
   
       26 . A compound selected from the group consisting of:
 Fmoc-Har-Gly-OH;   Fmoc-Har-Gly-O—P 4 ; and   Fmoc-Har-Gly-Asp(O—P 5 )-Trp-Pro-OH   
     wherein
 Fmoc is 9-fluorenylmethoxycarbonyl; 
 Har is homoarginine; 
 Gly is glycine; 
 Asp is aspartic acid; 
 Tip is tryptophan; 
 Pro is proline; 
 P 4  and P 5  are carboxyl protecting groups. 
 
   
   
       27 . The compound of  claim 26  wherein P 4  is pentafluororphenol. 
   
   
       28 . The compound of  claim 26  wherein P 5  is t-butyl. 
   
   
       29 . 3-nitro-2-pyridinesulfenyl-cysteinamide (H-Cys(Npys)-NH 2 ). 
   
   
       30 . A compound having the following formula: 
     
       
         
         
             
             
         
       
     
   
   
       31 . A composition comprising eptifibatide and Gly-eptifibatide. 
   
   
       32 . The composition of  claim 31  comprising at least 99% eptifibatide and Gly-eptifibatide in the range of about 0.01% to about 1%. 
   
   
       33 . The composition of  claim 32  comprising at least 99% eptifibatide and Gly-eptifibatide in the range of about 0.01% to about 0.1%. 
   
   
       34 . The product produced by a process according to  claim 1 . 
   
   
       35 . The product produced by a process according to  claim 4 . 
   
   
       36 . The product produced by a process according to  claim 8 . 
   
   
       37 . The product produced by a process according to  claim 9 . 
   
   
       38 . The product produced by a process according to  claim 11 . 
   
   
       39 . The product produced by a process according to  claim 12 . 
   
   
       40 . The product produced by a process according to  claim 13 . 
   
   
       41 . A process for purifying eptifibatide comprising:
 contacting an eptifibatide solution with a stationary phase comprising octadecyl carbon chains attached to silica;   washing the stationary phase contacted with the eptifibatide solution with a trifluoroacetic acid/acetonitrile solution;   optionally washing the stationary phase contacted with the eptifibatide solution with an acetic acid/acetonitrile solution; and   washing stationary phase contacted with the eptifibatide solution with an ammonium acid/acetonitrile solution.   
   
   
       42 . The process of  claim 41  wherein the trifluoroacetic acid/acetonitrile solution is a solution comprising 95% of a 0.1% aqueous trifluoroacetic acid solution and 5% of an acetonitrile solution. 
   
   
       43 . The process of  claim 42  further comprising washing the stationary phase contacted with the eptifibatide solution with a trifluoroacetic acid/acetonitrile solution comprising 50% of a 0.1% aqueous trifluoroacetic acid solution and 50% of an acetonitrile solution. 
   
   
       44 . The process of  claim 41  wherein the acetic acid/acetonitrile solution is a solution comprising 95% of a 0.5% aqueous solution of acetic acid and 5% of an acetonitrile solution. 
   
   
       45 . The process of  claim 44  further comprising washing the stationary phase contacted with the eptifibatide solution with an acetic acid/acetonitrile solution comprising 50% of a 0.5% aqueous solution of acetic acid and 50% of an acetonitrile solution. 
   
   
       46 . The process of  claim 41  wherein the ammonium acid/acetonitrile solution is a solution comprising 95% of an aqueous 100 mM ammonium acid solution and 5% of an acetonitrile solution.