Pyrazinyl-Substituted\nAminoazoles as Covalent Inhibitors\nof Thrombin: Synthesis, Structure, and Anticoagulant Properties

This study presents a novel series\nof <i>N</i>-acylated\n1,2,4-triazol-5-amines and 1<i>H</i>-pyrazol-5-amines, featuring\na pyrazin-2-yl moiety, developed as covalent inhibitors of thrombin.\nThese compounds demonstrated potent inhibitory activity, with derivatives <b>13a</b> and <b>13b</b> achieving IC<sub>50</sub> values\nas low as 0.7 and 0.8 nM, respectively. Mass-shift assays confirmed\nthat these inhibitors covalently bind to the catalytic Ser195 of thrombin,\nleading to temporary inhibition of its activity through specific acylation.\nThe anticoagulant efficacy of these compounds was validated in plasma\ncoagulation assays, with selected derivatives extending coagulation\ntimes in both an activated partial thromboplastin time (aPTT) and\nprothrombin time (PT) test. Thrombin generation assays further demonstrated\nthat compounds of this series effectively reduced thrombin generation\nwithout substantially prolonging clotting times, suggesting a lower\nrisk of bleeding. Selected compounds also strongly inhibited cancer\ncell- and thrombin-induced platelet aggregation. These results indicate\nthat acylated aminoazoles hold a promise as new anticoagulants.