Design and synthesis of novel biologically active thrombin receptor non-peptide mimetics based on the pharmacophoric cluster Phe/Arg/NH2 of the Ser42-Phe-Leu-Leu-Arg46 motif sequence: platelet aggregation and relaxant activities.

ABSTRACT

The identification of the thrombin receptor has promoted the interest for the development of new therapeutic agents capable of selectively inhibiting unwanted biological effects of thrombin on various cell types. In this study we have designed and synthesized two series of new thrombin receptor antagonists based on the thrombin receptor motif sequence S42FLLR46, one possessing two (Phe/Arg) pharmacophoric groups and the other possessing three (Phe/Arg/NH2). N-(6-Guanidohexanoyl)-N'-(phenylacetyl)piperazine (1), N-(phenylacetyl)-4-(6-guanidohexanoylamidomethyl)piperidine (2), and N-(phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3) (group A) carry the two pharmacophoric side chains of Phe and Arg residues incorporated on three different templates (piperazine, 4-aminomethylpiperidine, and 3-aminopyrrolidine). Compounds with three pharmacophoric groups (group B) were built similarly to group A using the same templates with the addition of an extra methylamino group leading to (S)-N-(6-guanidohexanoyl)-N'-(2-amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-3-phenylpropionyl)-4-(6-guanidohexanoylamidomethyl)piperidine (5), and (S)-N-(2-amino-3-phenylpropionyl)-3-(6-guanidohexanoylamido)pyrrolidine (6). Compounds were able to inhibit thrombin-induced human platelet activation even at low concentrations. In particular, among compounds in group A, compound 3 was found to be the most powerful thrombin receptor activation inhibitor, showing an IC50 of approximately 0.11 mM on platelet aggregation assay. Among compounds in group B, compound 4 was the most powerful to inhibit thrombin-induced platelet aggregation, showing an IC50 of approximately 0.09 mM. All compounds were also found to act as agonists in the rat aorta relaxation assay. Interestingly, the order of potency of these compounds as agonists of the endothelial thrombin receptor was the inverse of the order of potency of the same compounds as antagonists of the platelet thrombin receptor. Such compounds that are causing vasodilation while simultaneously inhibiting platelet aggregation would be very useful in preventing the installation of atherosclerotic lesions and deserve further investigation as potential drugs for treating cardiovascular diseases. The above findings coupled with computational analysis molecular dynamics experiments support also our hypothesis that a cluster of phenyl, guanidino, and amino groups is responsible for thrombin receptor triggering and activation.