5,6,7,8-Tetrahydroimidazo[1,2-a]pyrazine-based thiosemicarbazones as anti-Trypanosoma cruzi agents

Chagas disease, caused by Trypanosoma cruzi, remains a major neglected tropical disease for which current chemotherapies are limited by toxicity and variable efficacy. In the present work, a new series of 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-based thiosemicarbazones was designed, synthesized, and evaluated as potential antikinetoplastid agents. Then, 24 derivatives were prepared through systematic functionalization at the 2- or 3-position of the heterocyclic core and variation of aromatic substituents on the thiosemicarbazone moiety. Phenotypic in vitro screening against T. cruzi trypomastigotes (from cell culture) and intracellular amastigotes revealed several compounds with sub-micromolar to low-micromolar activity. A pronounced positional effect was observed, with 2-substituted derivatives displaying superior potency compared with their 3-substituted counterparts. Compound 10f, bearing a para-nitrophenyl thiosemicarbazone at the 2-position, emerged as the most promising lead among the series, exhibiting half maximal inhibitory concentration (IC₅₀) values of 2.35 μM against trypomastigotes and 4.06 μM against amastigotes, together with a favorable selectivity index relative to benznidazole. Selected compounds also showed moderate activity against Trypanosoma brucei. Preliminary in vivo toxicity assessment using Galleria mellonella larvae indicated good tolerability for the active compounds. Molecular docking studies suggested cruzain as a potential molecular target, and enzymatic assays confirmed non-covalent, mixed-mode inhibition of cruzain by representative derivatives. Overall, this study identifies 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine thiosemicarbazones, and particularly compound 10f, as a promising chemotype for further medicinal chemistry optimization toward new treatments for Chagas disease.