Integrated <i>In Vitro</i> and <i>In Silico</i> Profiling of Piperazinyl Thiosemicarbazone Derivatives Against <i>Trypanosoma cruzi</i>: Stage-Specific Activity and Enzyme Inhibition

<b>Background:</b> <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease, remains a major public health concern, and there is a continued need for new antitrypanosomal agents. Thiosemicarbazone (TSC) derivatives have emerged as a promising class of compounds with potential antiparasitic activity. <b>Objectives:</b> This study aimed to report the synthesis, characterization, and biological profiling of a novel series of thiosemicarbazone derivatives as antitrypanosomal agents against <i>Trypanosoma cruzi</i>. <b>Methods:</b> Fourteen new compounds and six previously described analogues were prepared and characterized by ¹H/¹³C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As a preliminary <i>in vitro</i> screen, activity was assessed by direct parasite counting in epimastigote and bloodstream trypomastigote forms, as tractable models of replicative and infective stages sharing core metabolic targets with intracellular amastigotes. Epimastigote potency was quantified as half-maximal effective concentrations (EC₅₀) derived from dose–response curves, whereas trypomastigote response was evaluated as percent viability after treatment at a fixed concentration of 20 µM. Mechanistic profiling included inhibition assays against the cysteine protease cruzipain (CZP) and selected redox defense enzymes, complemented by <i>in silico</i> similarity clustering and binding-pose affinity scoring. <b>Results:</b> A nitro-methoxy-substituted TSC showed potent CZP inhibition but limited trypomastigote efficacy, whereas brominated analogues displayed dual-stage activity independent of CZP inhibition. Tanimoto similarity analysis identified distinct structure–activity clusters, linking nitro-methoxy substitution to epimastigote selectivity and brominated scaffolds to broader antiparasitic profiles, with hydrophobicity and steric complementarity as key determinants. Enzymatic assays revealed no significant inhibition of cytosolic tryparedoxin peroxidase (cTXNPx) or glutathione peroxidase type I (TcGPx-I), suggesting redox disruption is not a primary mode of action. <i>In vitro</i> and <i>in silico</i> analyses showed low or no non-specific cytotoxicity under the tested conditions, supporting further optimization of these derivatives as antitrypanosomal preliminary hits. Key hits included derivative <b>3e</b> (epimastigote EC₅₀ = 0.36 ± 0.02 µM) and brominated analogues <b>2c</b> and <b>2e</b> (epimastigote EC₅₀ = 3.92 ± 0.13 and 4.36 ± 0.10 µM, respectively), while docking supported favorable binding-pose affinity (e.g., ΔG<i>_S</i>_-pose = −20.78 ± 2.47 kcal/mol for <b>3e</b>). <b>Conclusions</b>: These results support further optimization of the identified thiosemicarbazone derivatives as preliminary antitrypanosomal hits and provide insight into structure–activity relationships and potential mechanisms of action.