Adjunctive therapeutic strategy for in-stent thrombosis: in vitro pharmacodynamic evaluation of COX-1 inhibitor 3d combined with catheter-directed thrombolysis

Abstract

Twelve benzohydrazide Schiff base derivatives (3a–3l) with systematic variations in the aromatic aldehyde moiety were designed and synthesized to evaluate their anti-inflammatory, antiplatelet, and in vitro antithrombotic activities. Among them, compound 3d (the condensation product of 4-chlorobenzohydrazide and furan-2-carbaldehyde) was identified as the most promising lead, displaying moderately selective COX-1 inhibition (IC₅₀ = 1.63 µM, selectivity index = 8.87) together with moderate 5-LOX inhibition (IC₅₀ = 10.63 µM). In LPS-stimulated macrophages, 3d concentration-dependently suppressed the release of TNF-α, IL-6, IL-1β, and PGE₂. It inhibited ADP- and collagen-induced platelet aggregation (IC₅₀ = 5.48 µM and 4.52 µM, respectively), reduced the expression of platelet activation markers CD62P and PAC-1, and prolonged clotting time while decreasing clot strength in thromboelastography. Under arterial shear conditions in a parallel-plate flow chamber, 3d significantly attenuated thrombus formation in a concentration-dependent manner. Importantly, in an in vitro clot lysis model, 3d (50 µM) enhanced the thrombolytic rate of low-dose urokinase (100 IU per mL) from 35.0% to 45.2%, highlighting its potential to augment thrombolysis. Preliminary ADME profiling revealed moderate plasma protein binding (89.3%) and a metabolic half-life of 0.75 h in liver microsomes. This study provides the first systematic structure–activity relationship analysis of aromatic aldehyde substituents on benzohydrazide Schiff bases and identifies 3d as a lead compound possessing combined anti-inflammatory, antiplatelet, and thrombolysis-enhancing activities. These findings support the potential of 3d as an adjunctive agent for catheter-directed thrombolysis in in-stent thrombosis, pending further in vivo efficacy and safety investigations.