Antitrypanosomatid and antiamitochondriate activity of new cyclohexyl-quinoxaline-7-carboxylate-1,4-di-N-oxide derivatives: An in silico and in vitro approach toward the elucidation of their mechanisms of action.

A new series of cyclohexyl-quinoxaline-7-carboxylate-1,4-di-N-oxide derivatives was synthesized and evaluated in vitro against the trypanosomatids Trypanosoma cruzi and Leishmania mexicana, and the amitochondriates Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica. The potential mechanism of action was investigated through molecular docking on T. cruzi and L. mexicana trypanothione reductase (TcTR and LmTR, respectively), and on T. cruzi, G. lamblia, and T. vaginalis triosephosphate isomerase (TcTIM, GlTIM, and TvTIM, respectively), and E. histolytica thioredoxin reductase (EhTrxR). Enzyme inhibition assays were performed on recombinant TcTR, TcTIM, GlTIM, and EhTrxR. Compounds 6, 7, and 10 exhibited the best trypanocidal activity against T. cruzi trypomastigotes (IC50 = 1.9, 8.6, and 0.8 µM, respectively), while compound 4 was the most active derivative against T. cruzi intracellular amastigotes (IC50 = 72.96 µM). Compounds 6, 10, 14, and 15 exhibited uncompetitive inhibition on TcTR (Ki = 26, 50, 30, and 146 µM, respectively). Compounds 7 (IC50 = 4.99 µM) and 14 (IC50 = 65.32 µM) were the most leishmanicidal agents against QEPS and M379 L. mexicana strains, respectively; additionally, 7 was the most selective (SI = 34.37). On the other hand, compounds 8 and 14 had the best giardicidal, trichomonacidal, and amoebicidal activity (IC50 < 0.20 µM); additionally, compound 13 had a potent trichomonacidal and amoebicidal activity (IC50 = 0.12 and 0.18 µM, respectively), while compounds 8 and 14 were the most selective (SI > 800). Finally, compounds 7 and 14 exhibited broad-spectrum activity, as they showed noteworthy activity against trypanosomatids and amitochondriate organisms. These results encourage the development of more potent and selective antiparasitic agents using the quinoxaline 1,4-di-N-oxide scaffold.