Design and Evaluation of Novel Multi-Target, Stage-Specific Piperazinyl Thiosemicarbazones Against Trypanosoma Cruzi

Abstract

This study explores the rational design, synthesis, and biological evaluation of a series of thiosemicarbazone (TSC) derivatives as potential antitrypanosomal agents, with emphasis on substituent effects, molecular conformation, and steric interactions. In vitro screening against Trypanosoma cruzi (T. cruzi) and molecular docking studies were employed to elucidate structure-activity relationships (SAR) across different parasite life stages. A nitro-methoxy-substituted derivative exhibited the most potent cruzipain (CZP) inhibition, achieving near-complete suppression of epimastigote proliferation (EC50 = 0.36 μM), attributed to its favorable S-pose binding to the CZP active site. However, this compound displayed limited efficacy against the clinically relevant trypomastigote form. In contrast, brominated analogues demonstrated dual-stage activity, effectively targeting both trypomastigotes and epimastigotes through a CZP-independent mechanism. Tanimoto similarity clustering corroborated these findings, grouping nitro-methoxy compounds with epimastigote-selective profiles and distinguishing brominated scaffolds with broader antitrypanosomal potency. Steric complementarity and hydrophobicity emerged as key determinants of activity, with notable outliers such as an unsubstituted phenyl derivative and a chlorothiophene analogue displaying balanced stereoelectronic features. Importantly, assays on antioxidant defence enzymes, cytosolic tryparedoxin peroxidase (cTXNPx) and glutathione peroxidase type I (TcGPx-I), indicated no significant inhibition, excluding redox disruption as a major cytotoxic mechanism. These results highlight the potential of tailored TSC scaffolds for stage-specific chemotherapy and support a multi-targeted approach in the development of new treatments for Chagas disease.