Pyrazolyl-Tetrazoles and Imidazolyl-Pyrazoles as Potential Anticoagulants and their Integrated Multiplex Analysis Virtual Screening

( )This article reports a novel virtual screening algorithm seeking the rational identification of novel lead anticoagulants. Seven 5-(3-methyl-1-aryl-1H-pyrazol-4-yl)-1H-tetrazoles and seven novel 1-aryl-4-(4.5-dihydro-1H-imidazol-2-yl)-3-methyl-1H-pyrazoles were obtained in three steps starting from arylhydrazine hydrochlorides as raw materials in good yields: 50-72% and 50-85%, respectively. All compounds were submitted to an in silico target-base pipeline named integrated multiplex analysis virtual screening (IMA-VS), which comprises the evaluation of their (i) fitting physicochemical properties to the chemical environment of the target enzyme; (ii) active-site homing electrostatic potential to the target enzyme; (iii) structural fitting to the target active site through molecular docking; and (iv) overall absorption, distribution, metabolism, excretion and toxicity (ADMET) profile. After the virtual selection of potential anticoagulant hits, all molecules were synthesized and candidates were evaluated in vitro for their anticoagulant and hemolytic profile. The most promising candidate pointed out by IMA-VS was compound 1-(3',4'-dichlorophenyl)-4-(4,5-dihydro-1H-imidazol-2-yl)-3-methyl -1H-pyrazole that shown to display factor Xa (FXa)-specific inhibitory activity in vitro, acting as an uncompetitive inhibitor with an inhibition constant (Ki) = 61.16 +/- 12.96 mu M, in addition to the lowest hemolytic activity of the series. Further experiments revealed the antithrombotic activity of this compound in an in vivo model of arterial thrombosis induced by FeCl3.