Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4 R)- and (4 S)-4-fluoroproline was rationalized by analyzing inhibitor–enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4 R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2×APTT = 190 nM), excellent selectivity versus the digestive enzyme trypsin ( K i = 3300 nM), and excellent oral bioavailability in dogs with moderate clearance ( F = 100%, CL = 12 mL/min/kg).