Design and structure-activity relationship of thrombin inhibitors with an azaphenylalanine scaffold: potency and selectivity enhancements via P2 optimization

Theoretical and structural studies followed by the directed synthesis and in vitro biological tests lead us to novel noncovalent thrombin pseudopeptide inhibitors. We have incorporated an azapeptide scaffold into the central part of the classical tripeptide D-Phe-Pro-Arg inhibitor structure thus eliminating one stereogenic center from the molecule. A series of compounds has been designed to optimize the occupancy of the S2 pocket of thrombin. Increased hydrophobicity at P2 provides an enhanced fit into this active site S2 pocket. In the present paper, we also report on the structure of these inhibitors in solution and conformational analysis of inhibitors in the active site in order to asses the consequences of the replacement of the central alpha-CH by a nitrogen functionality. In vitro biological testing of the designed inhibitors shows that elimination of R, S stereoisomerism and restriction of conformational freedom influences the binding of inhibitors in a favorable fashion.