Evaluation of Δ²-1,3,4-thiadiazolines derived from thiosemicarbazones for antichagasic activity: Insights into structural limitations

In the search for new compounds for the treatment of Chagas disease, the cysteine protease cruzipain remains as one of the most attractive biomolecular targets for drug development. Thiosemicarbazones (TSCs) have been identified as cruzipain inhibitors with notable trypanocidal activity and represent a promising scaffold for rational design strategies, including the application of conformational constraints via heterocycle formation. In this work, we report the synthesis of two series of Δ²-1,3,4-thiadiazolines (TDZs) derived from thiosemicarbazones, a comparative analysis of their predicted binding modes and non-covalent free energies of interaction with cruzain, as well as their in vitro inhibitory activity against cruzipain isolated from Trypanosoma cruzi epimastigotes. Despite favourable interactions predicted for both compound series, the experimental data revealed a marked decrease in cruzipain inhibition by the TDZ derivatives when compared to their thiosemicarbazone precursors. These results suggest that the observed loss of activity may stem from differences in binding orientation and/or chemical reactivity toward the catalytic residue Cys25, rather than from changes in non-covalent binding energy alone. Overall, our findings provide useful insights into the structural requirements for cruzipain inhibition and highlight the limitations of Δ²-1,3,4-thiadiazolines as bioisosteric replacements for thiosemicarbazones in this context.