Identification of novel pyrazolines and isoxazolines as antileishmanial agents via a potential antifolate mechanism: synthesis, biological evaluation, and in silico studies.
Leishmaniasis remains a major neglected tropical disease affecting nearly one million people annually. Due to toxicity and resistance to current therapies, the folate/pterin salvage pathway represents a rational target for antileishmanial inhibitor design. Novel chalcone-derived pyrazolines and isoxazolines were synthesized and evaluated in vitro against Leishmania major. Six compounds (1b-e, 1g, and 1i) showed higher potency than miltefosine. Specifically, the N-formylated pyrazoline derivatives 1g and 1c exhibited exceptional antileishmanial potencies, with IC50 values of 1.41 μM and 1.54 μM against promastigotes as well as 1.71 μM and 1.94 μM against amastigotes, respectively. This represents a significant potency improvement (up to 5.5-fold) over miltefosine (IC50 = 7.83 and 8.07 μM, respectively). The antileishmanial activity was reversed with folic and folinic acid supplementation, supporting an overall antifolate mechanism. Molecular docking and 100 ns molecular dynamics simulations were conducted within the binding pocket of the essential Leishmania major pteridine reductase 1 enzyme (Lm-PTR1; PDB ID: 7PXX), demonstrating stable ligand-protein complexes for the most active candidates. Notably, molecular docking and 100 ns MD simulations of the S-enantiomer of compound 1g against a validated AlphaFold2 model of Lm-DHFR revealed favorable binding scores and stable cofactor-pocket interactions, suggesting predicted dual-target inhibitory potential against both Lm-PTR1 and Lm-DHFR. Compounds 1g and 1c showed minimal cytotoxicity against Vero cells, with selectivity indices (SI) of 148.7 and 260.9, respectively, compared to miltefosine (SI = 12.6). Based on its superior potency, high selectivity, and stable target binding in silico, the S-enantiomer of compound 1g represents a promising lead for further antileishmanial development.