A convergent strategy for Factor Xa inhibition: synthesis, bioinformatics, and biological analysis of 3,5-disubstituted isoxazoles

Thrombosis affects millions of individuals worldwide and is characterized by the formation of a blood clot within veins or arteries, leading to obstruction of normal blood flow. Approximately one in four deaths globally is associated with thrombotic events. Direct Factor Xa (fXa) inhibitors are widely used to prevent and treat this condition. Factor Xa is a well-established pharmacological target for the design of oral anticoagulants due to its amplifying role in the coagulation process. In this study, a series of novel aryl-lactam compounds based on 3,5-disubstituted isoxazoles was synthesized and evaluated for their fXa inhibitory activity. Among them, compounds 10j, 10m, 10p, 10q, and 10r were identified as the most active, exhibiting IC₅₀ values between 83.7 and 160.5 µM, consistent with moderate inhibitory activity. Moreover, these compounds prolonged PT and aPTT at concentrations of 200–400 μM, while not significantly affecting thrombin-induced clot retraction. Also, by slowing the coagulation process and fXa generation through dilution of PT and aPTT reagents, these compounds prolonged clotting times at 100 μM. Additionally, their in silico ADMET profiles were predicted, showing a balanced pharmacokinetic profile. In vitro cytotoxicity assays of these compounds in HEK-294, EA.hy926, HaCat, and HCT-116 cell lines revealed negligible to low cytotoxic effects. Finally, molecular docking and molecular dynamics simulations were conducted to rationalize the observed activity and to identify structural aspects that may support future optimization.