SELECTION OF S18326 AS A NEW POTENT AND SELECTIVE BORONIC ACID DIRECTTHROMBIN INHIBITOR

Using enzymatic microassays, the potency of a series of new boroargini ne tripeptides was determined versus thrombin and a panel of serine-pr oteases implicated in the coagulation and fibrinolysis pathways. The i nhibition of the serine-protease complement factor I was also studied. Factor I regulates the alternate pathway of the complement and its in hibition appears to be responsible for the toxic effects of the orally available thrombin inhibitor Ac-D-Phe-Pro-boroArg (DuP-714). The stru cture of the new boronic acid derivatives tested was modified from tha t of DuP-714 by replacing the proline in the P2 position by N-cycloalk yl-glycine residues of increasing size (S18989: cyclopropyl; S18563: c yclobutyl; S18326: cyclopentyl; S18229: cyclohexyl). All compounds wer e found to be slow-tight binding inhibitors of thrombin versus purifie d human fibrinogen. Replacement of proline by N-cycloalkyl-glycines di d not decrease the anti-thrombin potency of the substances up to the c yclopentyl size and this result was confirmed by classical coagulation assays with human plasma in vitro. In contrast, the inhibitory activi ties of the four new boronic acids were found to be lower than those o f DuP-714 versus plasmin, urokinase (u-PA), plasmatic kallikrein, acti vated protein C (aPC) and complement factor I. The cyclopentyl derivat ive S18326 is a slightly more active inhibitor of thrombin than DuP-71 4 (initial IC50 values 3.99 +/- 0.18 nM versus 4.73 +/- 0.27 nM, respe ctively). Moreover S18326 was identified as the most selective compoun d of the series with relative potencies being 2 to 29 fold higher than that of DuP-714 versus the panel of serine-proteases tested; the rank order of potency versus the other serine-proteases for S18326 was t-P A>kallikrein>aPC>factor I>plasmin>fXa>u-PA. These results indicate tha t the size of the thrombin hydrophobic pocket S2 is sufficient to acce pt larger residues than proline in the P2 position of Ac-D-Phe-X-boroA rg derivatives while this is not the case for other important serine-p roteases of the fibrinolysis, coagulation and complement pathways. The N-cyclopentyl glycine containing derivative S18326, which is the most potent and the most selective anti-thrombin compound of the series, c urrently undergoes major preclinical testing.