Discovery of Tetra‐Substituted Imidazole Inhibitors of Plasmin

ABSTRACT Plasmin mediates fibrinolysis and contributes to pathological conditions such as hyperfibrinolysis, hemorrhagic disorders, and tumor progression. Direct small‐molecule plasmin inhibitors remain limited. We synthesized a library of 19 imidazole‐based molecules, including tetra‐ and tri‐substituted analogs with 1,2,3‐triazole linkers and benzene‐sulfonamide moieties, to identify potential plasmin inhibitor leads. Screening using a chromogenic substrate hydrolysis assay at 100 µM identified tetra‐substituted molecules 7 , 12 , 14 , and 15 , and tri‐substituted inhibitor 8 as inhibitors of plasmin with efficacy of ≥40%. Dose–response analysis revealed inhibitors 7 , 8 , 12 , 14 , and 15 with IC 50 values of 42.4 ± 4.0, 48.4 ± 4.5, 121.3 ± 5.9, 73.6 ± 5.2, and 102.1 ± 5.9 µM, respectively, and efficacy ranging from 54.0% ± 2.5% to 97.0% ± 2.1%. Enzyme kinetic studies confirmed competitive inhibition of plasmin by inhibitor 7 . Selectivity assays demonstrated ≥11.8‐fold selectivity over thrombin and negligible inhibition of factors Xa and XIIa at 100 µM. Molecular docking provided a working hypothesis for the binding interactions, suggesting key interactions with the catalytic triad and S1–S4 subsites that may contribute to the observed potency. Overall, we put forward inhibitor 7 as a lead for further development of plasmin inhibitors.