Synthesis and Structure-Activity Relationships of Novel Direct Thrombin Inhibitors based on Pentapeptide FM 19.

Abstract

The serine protease thrombin is the main clotting enzyme in the hemostatic system, in addition to being an effective platelet activator. Modulation of the actions of thrombin can be used as a mechanism to treat myocardial infarctions and other acute coronary syndromes. The bradykinin breakdown product, pentapeptide RPPGF, was the starting point to investigate new inhibitors that bind to the enzyme’s active site. Subsequent SAR studies led to the development of a lead compound, FM 19, with the sequence D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH2. More recently, the x-ray structure of FM 19 in the active site of thrombin has been determined, providing insight into modifications to improve binding. The alterations described here include replacements to the D-Arg residue to maintain a crucial interaction with the catalytic Asp189, but eliminate an unfavorable electrostatic interaction between two positively charged groups, the N-terminal amine and guanidino group on the side chain; simultaneous replacement of the Pro-D-Ala residues which do not make any direct contacts with the enzyme, serving instead as scaffolding; modification of the Phe(p-Me) residue to enhance van der Waals interactions with surrounding Trp 215, Leu 99 and Ile 174 residues of thrombin; and modification of the C-terminus of the peptide to add alkyl and aryl substituents to the amide nitrogen. Alterations to two portions of the molecule investigated led to more potent compounds than FM 19 (replacements to the D-Arg residue and the addition of substituents on the C-terminus). Incorporating the best modifications of both types led to the synthesis of compound 56, with the sequence 5-guanidinopentanoic acid-Oic-Pro-D-Ala-Phe(p-Me)-NHC6H11, which has an IC50 of 530 nM, and shows an over 8-fold increase in potency over FM 19. Compound 56 is a promising new lead in the development of a new direct thrombin inhibitor.