Differential lethal action of octyl and oct-4E-enyl p-nitrobenzoates in Leishmania spp. – a mechanistic study

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania . Current treatments, mainly based on pentavalent antimonials and amphotericin B, are limited by severe side effects, high costs, and the emergence of resistant strains, highlighting the need for new therapeutic scaffolds. Gibbilimbol B, an alkylphenol derivative isolated from the Brazilian plant Piper malacophyllum , has shown antileishmanial potential, prompting the design of analogues to optimize its biological activity. Previous studies have investigated gibbilimbol B–inspired derivatives to enhance potency, selectivity, and ADMET properties. Structural modifications of the phenolic ring and side chain improved potency and drug-like properties, particularly in 4-nitrobenzamide derivatives with C 8 side chains. Therefore, the present study investigates two ester analogues - octyl 4-nitrobenzoate ( 1 ) and oct-4 E -enyl 4-nitrobenzoate ( 2 ), as well as 4-nitrobenzoic acid ( 3 ) - for their in vitro antileishmanial activity against L . infantum , L. amazonensis , and L. braziliensis promastigotes and intracellular amastigotes. Compound 1 , which has a saturated C 8 side chain, exhibited significant activity, with EC 50 values of 5.8 ± 0.2, 13.5 ± 0.2 and 1.0 ± 0.1 μg/mL for promastigotes of L. infantum, L. amazonensis and L. braziliensis , respectively. This compound displayed also reduced cytotoxicity to macrophages in vitro (CC 50 = 201 ± 10 μg/mL) and a high selectivity index (SI > 200 for L. braziliensis ). In contrast, compound 2 , featuring a double bond at the C-4 position of a C 8 side chain, exhibited moderate activity. Meanwhile, compound 3 , the original benzoic acid derivative, was inactive. Mechanistic assays with compound 1 revealed mitochondrial depolarization and reactive oxygen species (ROS) generation without loss of plasma membrane integrity, suggesting an apoptotic-like death pathway. Given its simple structure and promising results, octyl-4-nitrobenzoate ( 1 ) can be considered a promising candidate for further SAR (structure-activity relationship) optimization for the development of new antileishmanial drugs. • Octyl 4-nitrobenzoate ( 1 ) and oct-4 E -enyl 4-nitrobenzoate exhibits selective in vitro activity against Leishmania spp. • Antileihsmanial effect promotes parasite death through mitochondrial dysfunction and ROS generation. • Nitroaromatic ester structure enhances selectivity over host macrophages. • Gibbilimbol-inspired analogues supports development of plant-derived therapies for leishmaniasis.