Antileishmanial activities of three chalcone derivatives and their association with plasma membrane rigidity as assessed by EPR spectroscopy.

• LQFM332 causes cell membrane rigidity, as detected by EPR spectroscopy. • Membrane rigidity was associated with antileishmanial activity. • EPR spectra suggest that chalcone derivatives cause oxidation in membrane proteins. Novel chalcone-like compounds, LQFM332 (4), LQFM333 (5), and LQFM341 (6), were synthesized and their activity against Leishmania (L.) amazonensis was evaluated and compared. Compound 4 was synthesized from the molecular hybridization of vanillin (9) and butylated hydroxytoluene (BHT), forming a chalcone scaffold. BHT was replaced by another apocynin to give compound 5, while for compound 6 the Michael acceptor double bond from compound 4 was reduced. The IC 50 values for compounds 4, 5, and 6 against the Leishmania parasite were 7, 20, and 16 µM for the promastigote form, and 19, 11, and 1 µM for the amastigote form inside infected macrophages, respectively. Electron paramagnetic resonance (EPR) spectroscopy of a lipid spin label incorporated into the parasite plasma membrane demonstrated that treatment with these compounds in culture medium causes membrane rigidity in a concentration-dependent manner, owing to oxidative processes. From the IC 50 values with different cell concentrations, the membrane-water partition coefficient (K M/W) of the compounds could be estimated, as well as their concentrations in the membrane (c m50) and in the aqueous culture medium (c w50) that reduce antiproliferative activity by 50%. Values for these biophysical parameters were similar to those reported for the antileishmanial drug, miltefosine. K M/W values indicated that the compounds have high affinity for the parasite membrane, with the compound containing the BHT group having the highest affinity. Compound 5, which contained a two 4-OH-3-methoxybenzene scaffold, showed the lowest values of c w50 and c m50. Compound 6 showed that the reduction of the double bond decreased the membrane affinity and the c w50 value, but maintained the c m50 , demonstrating the same activity as compound 4 for assays with higher concentration of cells. As cytotoxic activities were modulated by effects on the cell membrane, our results suggest that the primary action of the compounds is on the parasite membrane. [ABSTRACT FROM AUTHOR]