Developing Safer Anticoagulant Candidates: Structure-Activity Relationship Studies of 5-Amino-Triazole-Based FXIIa Inhibitors

Introduction/Objective: Factor XIIa (FXIIa), a serine protease in the intrinsic coagulation pathway, is primarily involved in thrombosis rather than hemostasis, making it an attractive target for safer anticoagulants with reduced bleeding risk. We previously reported a series of potent and selective triazole-based FXIIa inhibitors. To further advance this class, we evaluated sixteen additional derivatives of the lead inhibitor (1). Methods: FXIIa inhibition was assessed using a chromogenic substrate assay under physiological conditions. The new derivatives were structurally modified in two domains: the N1-benzoyl group and the C3-substituent. Kinetic characterization, selectivity profiling, and clotting assays were also performed. Results: We identified three molecules inhibiting FXIIa with more than 40% at 100 μM. Structure– activity analysis revealed superior activity of a halogenated benzoyl group at the N1- position over a 3-phenylpropanoyl substituent and highlighted the importance of meta-position halogenation. A methyl ester at the 3-position of the triazole ring was required for activity, as analogs lacking this group were inactive. Inhibitor 6 was the most potent compound, exhibiting an IC₂⁽ of 56 nM and ~100% efficacy. It demonstrated a marginal-to-high selectivity of 4– 8,900-fold selectivity for FXIIa over thrombin, FXIa, FXa, FIXa, and plasmin. Kinetic analysis suggested active-site binding with a covalent mode of inhibition. In coagulation assays, inhibitor 6 prolonged both prothrombin time and activated partial thromboplastin time, with a greater effect on aPTT. Discussion: These results define key structural determinants of FXIIa inhibition, including the importance of halogenated benzoyl substitution at N1 and a required methyl ester on the triazole scaffold. The high potency and selectivity of inhibitor 6 indicate effective engagement of the FXIIa active site with minimal off-target serine protease inhibition. Preferential prolongation of aPTT relative to PT is consistent with selective modulation of the intrinsic coagulation pathway. Conclusion: This study successfully identified a potent, selective, and covalent FXIIa inhibitor 6 and defined critical structure-activity relationships for the triazole-based chemotype. The findings validate the feasibility of developing FXIIa-targeted anticoagulants. Future work will focus on the rational design of next-generation inhibitors with enhanced potency and selectivity, further mechanistic studies, and in vivo profiling to advance therapeutic potential.