Discovery of a new eugenol-benznidazole hybrid active against different evolutive stages of Trypanosoma cruzi

Chagas disease (CD) is a life-threatening illness caused by the protozoan Trypanosoma cruzi and there are only two drugs currently available for pharmacotherapy of this neglected Infection (benznidazole and nifurtimox). Their limited efficacy in chronic phase of the disease, problems of toxicity and the growing resistance by the protozoan are directly associated to high rates of drug discontinuation by the patients. In the context of the search for new trypanocidal drug candidates, our group has been working with the chemical manipulation of eugenol to obtain new agents active against T. cruzi and promising results have been achieved. In this work we designed molecular hybrids between eugenol and benznidazole (BZN) by performing a bioisosteric replacement between the nitroimidazole ring of BZN by the eugenol or analogues nucleus in nitrated or non-nitrated forms. The obtained compounds were initially evaluated against epimastigote stages of T. cruzi for selecting the most active ones. Three hybrids (8, 10 and 15) showed activity against this evolutive stage and the most promising was eugenol nitro derivative 8 (IC₅₀: 24.7 µM) that showed a similar potential than BZN (IC₅₀: 29.9 µM). Compounds 8, 10 and 15 were then evaluated against infective trypomastigote and intracellular amastigote forms of T. cruzi and the cytotoxicity of them was investigated against healthy Vero and H9c2 cells. The nitro derivative 8 was once again the most promising compound against trypomastigote (IC₅₀: 1.8 µM) and amastigote (IC₅₀: 1.6 µM) forms, with selectivity indices greater than 63 considering both health cells evaluated. This compound was more active than BZN, that showed IC₅₀ values of 6.1 µM (trypomastigotes) and 3.1 µM (amastigotes) and showed the most significant effect in reducing the Infection rate and accumulation of parasites in infected cells. Calculated logP values suggest that the higher lipophilicity of derivative 8 (clogP: 3.25) in relation to BZN (clogP: 0.77) may be correlated to a greater ability of this eugenol derivative to cross the membranes of infected cells. Finally, to try understand the possible mode of action for compound 8, we have evaluated three important targets of the Parasite (T. cruzi nitroreductase type I - TcNRT, trypanothione reductase - TR and mitochondrial membrane depolarization), but none of these metabolic pathways seem to be related to the mechanism of action of this compound. All in vitro assays employed the T. cruzi Y strain which exhibits partial resistance to benznidazole and these results indicate compound 8 as a new promising hit for the development of drug candidates to treat CD.

Chagas disease; Eugenol, benznidazole; Mitochondrial membrane depolarization; Molecular hybridization; T. cruzi nitroreductase type I; Trypanothione reductase.