Isoxazole Derivative Induces Apoptosis-like Death and Autophagy through Oxidative Stress in Leishmania amazonensis

Leishmaniasis is one of the most important neglected tropical diseases and a significant global health burden, affecting millions of people worldwide. The limitations of current treatments, including toxicity, high cost, and the emergence of drug resistance, underscore the urgent need for new antileishmanial agents. Isoxazoles are a versatile class of heterocyclic compounds with documented antiparasitic activity, making them attractive scaffolds for drug development. In this study, we synthesized and evaluated a new isoxazole derivative, 4-[((4-fluorophenyl)-amino)-methyl]-5-(4-nitrophenyl)-3-[(2E)-N'-(2-pyridinylmethylene)-hydrazinecarbonyl]-isoxazole (4), against Leishmania amazonensis. Compound 4 exhibited potent activity, with IC₅₀ values of 12.7 μM for promastigote forms and 0.96 μM for intracellular amastigotes, the most clinically relevant form, while displaying low cytotoxicity against J774A.1 macrophages and L929 fibroblasts, indicating a favorable safety profile. We further explored its mechanism of action through detailed analyses in promastigote and amastigote forms. Compound 4 induced oxidative stress, evidenced by elevated reactive oxygen species and nitric oxide content, leading to lipid peroxidation, lipid droplet accumulation, mitochondrial depolarization, and altered ATP levels. These effects were accompanied by loss of cell membrane integrity, reduced cell size, and phosphatidylserine exposure. An increase in autophagic vacuoles and acidic compartments was also observed. Morphological and ultrastructural alterations corroborated the biochemical findings. In silico analysis provided an initial drug-likeness and physicochemical context to support early stage prioritization. Collectively, our results demonstrate that compound 4 induces apoptosis-like cell death and autophagy through oxidative stress and mitochondrial dysfunction, highlighting it as a promising lead for the development of antileishmanial agents.