Selection of S18326 as a New Potent and Selective Boronic Acid Direct Thrombin Inhibitor

Summary

Using enzymatic microassays, the potency of a series of new boroarginine tripeptides was determined versus thrombin and a panel of serine-proteases implicated in the coagulation and fibrinolysis pathways. The inhibition of the serine-protease complement factor I was also studied. Factor I regulates the alternate pathway of the complement and its inhibition appears to be responsible for the toxic effects of the orally available thrombin inhibitor Ac-D-Phe-Pro-boro-Arg (DuP-714). The structure of the new boronic acid derivatives tested was modified from that of DuP-714 by replacing the proline in the P2 position by N-cycloalkyl-glycine residues of increasing size (SI8989: cyclopropyl; S18563: cyclobutyl; S18326: cyclopentyl; S18229: cyclohexyl). All compounds were found to be slow-tight binding inhibitors of thrombin versus purified human fibrinogen. Replacement of proline by N-cycloalkyl-glycines did not decrease the anti-thrombin potency of the substances up to the cyclopentyl size and this result was confirmed by classical coagulation assays with human plasma in vitro. In contrast, the inhibitory activities of the four new boronic acids were found to be lower than those of DuP-714 versus plasmin, urokinase (u-PA), plasmatic kallikrein, activated protein C (aPC) and complement factor I. The cyclopentyl derivative SI8326 is a slightly more active inhibitor of thrombin than DuP-714 (initial IC₅₀ values 3.99 ± 0.18 nM versus 4.73 ± 0.27 nM, respectively). Moreover SI8326 was identified as the most selective compound 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-PA>kallikrein>aPC> factor I>plasmin>fXa>u-PA. These results indicate that the size of the thrombin hydrophobic pocket S2 is sufficient to accept larger residues than proline in the P2 position of Ac-D-Phe-X-boroArg derivatives while this is not the case for other important serine-proteases of the fibrinolysis, coagulation and complement pathways. The N-cyclopentyl glycine containing derivative SI8326, which is the most potent and the most selective anti-thrombin compound of the series, currently undergoes major preclinical testing.

• References

• 1 Kettner C, Mersinger L, Knabb R. The selective inhibition of thrombin by peptides of boroarginine. J Biol Chem 1990; 265: 18289-18297
• 2 Hussain MA, Knabb R, Aungst BJ, Kettner C. Anticoagulant activity of a peptide boronic acid thrombin inhibitor by various routes of administration in rats. Peptides 1991; 12: 1153-1154
• 3 Tapparelli C, Mettemich R, Ehrhardt C, Zurini M, Cleason G, Scully MF, Stone SR. In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor. J Biol Chem 1993; 268: 4734-4741
• 4 De NanteuilG, Gloanec P, Lila C, Portevin B, Boudon A, Rupin A, Verbeuren TJ. New tripeptidic thrombin inhibitors; Influence of P2 and 3 residues on activity and selectivity. Bioorg Med Chem 1995; 3: 1019-1024
• 5 Callas D, Fareed J. Comparative pharmacology of site directed antithrombin agents. Implication in drug development. Thromb Haemost 1995; 74: 473-481
• 6 Knabb RM, Luettgen JM, Laemy AW, Barbera FA, Kettner CA, Pangbum MJ, and Thoolen MJ. Acute toxicity of synthetic thrombin inhibitors caused by inhibition of complement factor I. Circulation 1996; 94: 001-696
• 7 Fevig JM, Buriak J, Cacciola J, Alexender RS, Kettner CA, Knabb RM, Pruitt JR, Thoolen MJ, Weber P, Wexler RR. Rational design of boropeptide thrombin inhibitors with greater selectivity over complement factor I. In “Abstracts of the 212^thAmerican Chemical Society National Meeting, Division of Medicinal Chemistry”. 1996; n° 127
• 8 Rupin A, Mennecier P, De Nanteuil G, Laubie M, Verbeuren TJ. A screening procedure to evaluate the anticoagulant activity and the kinetic behaviour of direct thrombin inhibitors. Thromb Res 1995; 78: 217-225
• 9 Isenman DE. Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. J Biol Chem 1983; 258: 4238-4244
• 10 Bieth J. Some kinetic consequences of the tight binding of protein-proteinase-inhibitors to proteolytic enzymes and their application to the determination of dissociation constants. In: Bayer-Symposium V: Proteinase inhibitors. Fritz H. et al eds Springer; Berlin: 1974: 463-469
• 11 Stone SS, Tapparelli C. Thrombin inhibitors as antithrombotic agents: the importance of rapid inhibition. J Enzyme Inhibition 1995; 9: 03-15
• 12 Stubbs MT, Bode W. A player of many parts: the spotlight falls on thrombin’s structure. Thromb Res 1993; 69: 01-58
• 13 Verbeuren TJ, Rupin A, Simonet S, Vallez MO, De Nanteuil G. Antithrombotic properties of S 18326. A new potent orally active tripeptidic boronic acid thrombin inhibitor. Thromb Haemost 1995; 73: 1310
• 14 Lefkovits J, Topol EJ. Direct thrombin inhibitors in cardiovascular medicine. Circulation 1994; 90: 1522-1536
• 15 Verstraete M, Zoldhelyi P. Novel antithrombotic drugs in development. Drugs 1995; 49: 856-884
• 16 Verbeuren TJ, Rupin A, Simonet S, Gloannec P, Lila C, Portevin B, De Nanteuil G. Pharmacological evaluation of two new tripeptidic boronic acid thrombin inhibitors, S17436 and SI8326. In “Abstracts of the Xlllth International Symposium on Medicinal Chemistry“ Sept. 19-23 1994. Paris: pOl;