Thiochromene Analogues as Antiparasitic Chemotype: Synthesis, Antiplasmodial and Antitrypanosomal Profiling, Experimental Validation, and Computational Insights into Trypanothione Reductase Inhibition

Abstract The emergence of drug resistance and the limited availability of effective therapeutics continue to hinder the treatment of protozoal diseases, highlighting the need for new chemotypes with broad-spectrum antiparasitic potential. In the present study, a series of nitro and trifluoromethyl-substituted thiochromene carboxamides, previously identified as antileishmanial agents, were further explored for their broad-spectrum antiparasitic potential. Biological screening identified several compounds with promising activity and favorable selectivity against Plasmodium falciparum (D10 and W2 strains) and Trypanosoma brucei (AnTat1.1E strain). As the synthesized analogues exhibited a more favorable antitrypanosomal activity and selectivity profile, representative analogues were subsequently evaluated for trypanothione reductase (TryR) inhibition to investigate their potential mechanism of action, as TryR is a validated and highly conserved drug target across kinetoplastid parasites. Among the synthesized molecules, compound 5p showed the highest antiplasmodial potency (D10 EC50 = 2.1 μM), whereas compounds 5c and 5r were the most potent antitrypanosomal analogues (EC50 = 1.8 μM). Structure–activity relationship analysis demonstrated that piperidine and piperazine-containing carboxamide is a key pharmacophoric feature for broad-spectrum activity, whereas incorporation of a nitro substituent enhanced the activity against T. brucei. To investigate the potential mode of killing, representative compounds were evaluated for inhibition of TryR, identifying compound 5v as the most potent enzyme inhibitor. Enzyme kinetic studies established an uncompetitive mode of inhibition (ki ≈ 29.5 μM), suggesting interaction at an allosteric site rather than the active catalytic site. Consistent with these findings, SiteMap analysis identified a druggable allosteric pocket at the dimer interface of substrate-bound TryR, where the active compounds exhibited favorable binding affinities and interaction profiles. Collectively, these results demonstrate that thiochromene carboxamides constitute a promising scaffold for the development of broad-spectrum antiprotozoal agents and support allosteric inhibition of TryR as a viable strategy for antitrypanosomal drug discovery.