Pyrazinyl-Substituted Aminoazoles as Covalent Inhibitors of Thrombin: Synthesis, Structure, and Anticoagulant Properties.

Abstract

This study presents a novel series of N-acylated 1,2,4-triazol-5-amines and 1H-pyrazol-5-amines, featuring a pyrazin-2-yl moiety, developed as covalent inhibitors of thrombin. These compounds demonstrated potent inhibitory activity, with derivatives 13a and 13b achieving IC₅₀ values as low as 0.7 and 0.8 nM, respectively. Mass-shift assays confirmed that these inhibitors covalently bind to the catalytic Ser195 of thrombin, leading to temporary inhibition of its activity through specific acylation. The anticoagulant efficacy of these compounds was validated in plasma coagulation assays, with selected derivatives extending coagulation times in both an activated partial thromboplastin time (aPTT) and prothrombin time (PT) test. Thrombin generation assays further demonstrated that compounds of this series effectively reduced thrombin generation without substantially prolonging clotting times, suggesting a lower risk of bleeding. Selected compounds also strongly inhibited cancer cell- and thrombin-induced platelet aggregation. These results indicate that acylated aminoazoles hold a promise as new anticoagulants.