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.
