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Trypanosoma cruzi is the causative pathogen of Chagas disease and the main culprit for cardiac-related mortality in Latin-America triggered by an infective agent. Uncapable of synthesizing purines de novo, this parasite depends on acquisition and processing of host-derived purines, making purine (nucleoside) analogues a potential source of antitrypanosomal agents. In this respect, hitherto 7-deazaadenosine (tubercidin) analogues attracted most attention. Here, we investigated analogues with an additional nitrogen (N1) removed. Structure-activity relationship investigation showed that C7 modification afforded analogues with potent antitrypanosomal activity. Halogens and small, linear carbon-based substituents were preferred. Compound 11 proved most potent in vitro, showed full suppression of parasitemia in a mouse model of acute infection and elicited 100 % animal survival after oral dosing at 25 mg/kg b.i.d. for five and fifteen days. Cyclophosphamide-induced immunosuppression led to recrudescence. Washout experiments demonstrated a lack of complete clearance of infected cell cultures, potentially explaining the in vivo results.

Up to now, no vaccines are available for Chagas disease, and the current therapy is largely unsatisfactory. Novel imidazole-based scaffolds of protozoan sterol 14α-demethylase (CYP51) inhibitors have demonstrated potent antiparasitic activity with no acute toxicity. Presently our aim was to investigate the effectiveness of the experimental 14α-demethylase inhibitor VFV in the mouse models of Trypanosoma cruzi infection using a naturally drug-resistant Colombiana strain, under monotherapy and in association with the reference drug, benznidazole (Bz). The treatment with VFV resulted in complete parasitemia suppression and 100% animal survival when administered orally (given in 10% DMSO plus 5% Arabic gum) at 25 mg/kg (bid) for 60 days. However, as parasite relapse was found using VFV alone under this treatment scheme, the coadministration of VFV with Bz was assayed giving simultaneously (for 60 days, bid) by oral route, under two different drug vehicles (10% DMSO plus 5% Gum Arabic with or without 3% Tween 80). All tested mice groups resulted in >99.9% of parasitemia decrease and 100% animal survival. qPCR analysis performed on cyclophosphamide immunosuppressed mice revealed that, although presenting lack of cure, VFV given as monotherapy was 14-fold more active than Bz, and the coadministration of Bz plus VFV (given simultaneously, using 10% DMSO plus 5% Gum Arabic as vehicle) resulted in 106-fold lower blood parasitism as compared to the monotherapy of Bz. Another interesting finding was the parasitological cure in 70% of the animals treated with Bz and VFV when the coadministration was given using the VFV suspension in 10% DMSO + Arabic gum + Tween 80 (a formulation that we have found to provide a better pharmacokinetics), even after immunosuppression using cyclophosphamide cycles, supporting the promising aspect of the drug coadministration in improving the efficacy of therapeutic arsenal against T. cruzi.

Sterol 14α-demethylases (CYP51) are cytochrome P450 enzymes essential for sterol biosynthesis in eukaryotes and therapeutic targets for antifungal azoles. Multiple attempts to repurpose antifungals for treatment of human infections with protozoa (Trypanosomatidae) have been undertaken, yet so far none of them have revealed sufficient efficacy. VNI and its derivative VFV are two potent experimental inhibitors of Trypanosomatidae CYP51, effective in vivo against Chagas disease, visceral leishmaniasis, and sleeping sickness and currently under consideration as antiprotozoal drug candidates. However, VNI is less potent against Leishmania and drug-resistant strains of Trypanosoma cruzi and VFV, while displaying a broader spectrum of antiprotozoal activity, and is metabolically less stable. In this work we have designed, synthesized, and characterized a set of close analogues and identified two new compounds (7 and 9) that exceed VNI/VFV in their spectra of antiprotozoal activity, microsomal stability, and pharmacokinetics (tissue distribution in particular) and, like VNI/VFV, reveal no acute toxicity.

Chagas disease is the leading cause of cardiac-related mortality in Latin American countries where it is endemic. Trypanosoma cruzi, the disease-causing pathogen, is unable to synthesize purines de novo, necessitating salvage of preformed host purines. Therefore, purine and purine-nucleoside analogues might constitute an attractive source for identifying antitrypanosomal hits. In this study, structural elements of two purine-nucleoside analogues (i.e., cordycepin and a recently discovered 7-substituted 7-deazaadenosine) led to the identification of novel nucleoside analogues with potent in vitro activity. The structureactivity relationships of substituents at C-7 were investigated, ultimately leading to the selection of compound 5, with a C-7 para-chlorophenyl group, for in vivo evaluation. This derivative showed complete suppression of T. cruzi Y-strain blood parasitemia when orally administered twice daily for 5 days at 25 mg/kg and was able to protect infected mice from parasite-induced mortality. However, sterile cure by immunosuppression could not be demonstrated.