O primeiro estudo amplo da relação estrutura-atividade do benznidazol: atividades in vitro e in vivo contra Trypanosoma cruzi e na enzima nitroredutase

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a significant public health challenge, particularly due to the limitations of current treatments. Benznidazole (BZN) is currently the only drug used in Brazil for the treatment of the disease and, despite its limitations, remains the best therapeutic alternative, as evidenced by recent clinical studies. Its well-established mechanism of action and consolidated efficacy make it a valuable prototype for the development of new molecules. In this context, this work proposed the synthesis and evaluation of 27 BZN derivatives, aiming to optimize their anti-T. cruzi activity and to carry out a preliminary structure–activity relationship (SAR) study, which has not yet been explored or documented in the literature. The derivatives were obtained through a simple and low-cost synthetic route, consisting of two steps performed using a one-pot system. In silico predictions indicated favorable physicochemical and pharmacokinetic properties, including good aqueous solubility, excellent gastrointestinal absorption potential, and compliance with major drug-likeness criteria. Biological activity was determined through in vitro assays against intracellular forms of the parasite (Tulahuen strain) and bloodstream trypomastigotes (Y strain), as well as cytotoxicity in L929 cells. Nine compounds exhibited greater potency and selectivity than BZN (EC50 = 2.52 ± 0.60 μM) against the intracellular form of the parasite, with three of them standing out as the most potent, showing EC50 values ranging from 1.01 to 2.52 μM. All compounds showed CC50 > 200 μM, indicating a high selectivity index and no cytotoxicity up to the maximum concentration tested, highlighting a favorable safety profile. Against bloodstream trypomastigotes, 8 of the 17 compounds tested showed EC50 values lower than BZN (5.8 μM), ranging from 2.37 to 5.18 μM. The in vitro evaluation on the nitroreductase enzyme (TcNTR) classified nine derivatives as artificial substrates. Enzymatic conversion rates, expressed relative to BZN (100%), were equal to or higher than this value for eight compounds, reinforcing the hypothesis of this pathway's involvement in the mechanism of action. The SAR analysis enabled the identification of structural patterns that enhance anti-T. cruzi activity, suggesting potential directions for the development of new compounds. Five of the most promising derivatives were evaluated in vivo, and the compound N-(2-methylbenzyl)-2- (2-nitro-1H-imidazol-1-yl)acetamide was able to reduce parasitemia by 100% and ensure 100% survival of the treated animals. These findings reinforce the potential of these molecules as promising candidates for the development of new anti-Chagas agents.