BackgroundChagas’ disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people, mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas’ disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl) quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro.Methodology/Principal FindingsQuinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and more effective against the proliferative forms. The cytotoxicity against LLCMK2 cells provided selective indices above one for all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction against LLCMK2 cells. Quinoxaline 4 induced several ultrastructural alterations, including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane integrity of treated-parasites.Conclusion/SignificanceOur results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further development of a combination therapy as an alternative more effective in Chagas’ disease treatment.

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Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis. The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (⌬⌿m), mitochondrial superoxide anion (O 2 · ؊ ) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ⌬⌿m, an increase in the O 2 · ؊ concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of action of LSPN329 and LSPN331 and the decrease in lesion thickness in vivo brought about by LSPN329 and LSPN331 make them potential candidates for new drug development for the treatment of leishmaniasis.