Chagas disease, caused by Trypanosoma cruzi parasite, was described thousands of years ago. Currently, it affects millions of people, mostly in Latin America, and there are not suitable drugs for treating it. As an attempt to find appropriate drugs to deal with this problem, we report here on the design, synthesis, and characterization of 82 new compounds. Trypanosomicidal behavior in vitro showed more than 20 outstanding derivatives with anti-Trypanosoma cruzi activity. Furthermore, we studied the nonspecific toxicity against mammalian cells determining their selectivity and also performed mutagenicity studies. Proof of concept, in vivo studies, was conducted with two of the most promising derivatives (77 and 80). They were identified as candidates because they have (i) very simple and cost-effective syntheses; (ii) activity against different stages and strains of the parasite showing excellent in vivo behavior during the acute phase of Chagas disease; and (iii) neither nonspecific toxicity nor mutagenic activity.

Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.

Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection.

For a fourth approach of quinoxaline N,N′-dioxides as anti-trypanosomatid agents against T. cruzi and Leishmania, we found extremely active derivatives. The present study allows us to state the correct requirements for obtaining optimal in vitro anti-T. cruzi activity. Derivatives possessing electron-withdrawing substituents in the 2-, 3-, 6-, and 7-positions were the most active compounds. With regard to these features and taking into account their mammal cytotoxicity, some trifluoromethylquinoxaline N,N′-dioxides have been proposed as candidates for further clinical studies. Consequently, mutagenicity and in vivo analyses were performed with the most promising derivatives. In addition, with regard to the mechanism of action studies, it was demonstrated that mitochondrial dehydrogenases are involved in the anti-T. cruzi activity of the most active derivatives.

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