Design of Weakly Basic Thrombin Inhibitors Incorporating Novel P1 Binding Functions: Molecular and X-ray Crystallographic Studies.

To prep. weakly basic thrombin inhibitors with modified S1 anchoring groups, two series of compds. were synthesized by reaction of guanidine or aminoguanidine with acyl halides and N,N-disubstituted carbamoyl chlorides. PKa measurements of these acylated guanidines/aminoguanidines showed a reduced basicity, with pKa values in the range of 8.4-8.7. These mols. typically showed inhibition consts. in the range of 150-425 nM against thrombin and 360-965 nM against trypsin, even though some bulky derivs., such as N,N-diphenylcarbamoylguanidine/aminoguanidine and their congeners, showed much stronger thrombin inhibitory activity, with inhibition consts. in the range of 24-42 nM. Unexpectedly, very long incubation times with both proteases revealed that aminoguanidine derivs. behaved as irreversible inhibitors. To assess the mol. basis responsible for the high affinity obsd. for these mols. toward thrombin, the crystal structure of the thrombin-hirugen-N,N-diphenylcarbamoylaminoguanidine complex has been solved at 1.90 .ANG. resoln. The structural anal. of the complex revealed an unexpected interaction mode with the protease, resulting in an N,N-diphenylcarbamoyl intermediate covalently bound to the catalytic serine as a consequence of its hydrolysis together with the release of the aminoguanidine moiety. Surprisingly, in this covalent adduct a Ph group was found in the S1 specificity pocket, which usually recognizes pos. charged residues. These findings provide new insights in the design of low basicity serine protease inhibitors.