Avaliação da atividade in vitro dos derivados sintéticos de N-ciclohexil 3-(3-metilfenil)1,2,4-oxadiazol-5-amina sobre a cepa y de Trypanosoma cruzi e um estudo de toxicidade in vivo

Abstract

Chagas disease remains a significant public health problem in Latin America, primarily treated with Nifurtimox and Benznidazole. Despite initial proactive strategies, these medications have limitations, such as low efficacy in the chronic phase and adverse effects. In this context, heterocyclic compounds, such as 1,2,4-oxadiazole derivatives, have been investigated as potential therapeutic alternatives. This study aimed to evaluate the trypanocidal activity and toxicity of molecules 2a, 2f, and 2i against the Trypanosoma cruzi strain using in vitro, in silico, and in vivo assays. The results showed a concentration-dependent effect on epimastigote forms, with IC50 values of 31.4 µM, 44.4 µM, and 7.0 µM for 2a, 2f, and 2i, respectively. In trypomastigotes, LC50 values of 14.2 µM, 2.4 µM, and 2.6 µM were observed for these same molecules, in addition to a significant reduction in the amastigote load in infected cells. The selectivity index was 15, 5.8, 9.8, and 4.7 for molecules 2a, 2f, 2i, and benznidazole, respectively. Flow cytometry analyses indicated compromised cell integrity, induction of oxidative stress, and mitochondrial dysfunction, altering different targets of action of the molecules under study. Using free radical neutralization assays in solution, through antioxidant activity and acetylcholinesterase inhibition, the molecules under study showed a high capacity for scavenging free radicals. These findings suggest that such compounds may interfere with the redox balance of the parasite, contributing to oxidative stress and, consequently, to the observed antiparasitic effects. Subsequently, scanning electron microscopy revealed membrane alterations, cell rounding, or flagellar absence. Among the compositions evaluated, molecule 2a stood out for presenting a better balance between efficacy and selectivity in vitro, in addition to interactions developed with the cruzipain enzyme in in silico analyses. In in vivo assays with zebrafish, molecule 2a showed dose-dependent embryonic toxicity (LC50~15 µM); similar to bzn at higher concentrations, decreasing a specific cytotoxic potential. Taken together, these findings reinforce the potential of 1,2,4-oxadiazole derivatives as promising candidates for the development of new therapeutic strategies against Chagas disease, highlighting molecule 2a as a priority target for future studies.