Sustainable Antiparasitic Agents from an Agro-Industrial Waste: Mitochondria-Targeting Cashew Nutshell Liquid-Derived Phosphonium and Ammonium Salts

Abstract

Innovative, sustainable therapies are urgently needed for neglected vector-borne parasitic diseases. In this study, we leveraged cashew nutshell liquid (CNSL), an agro-industrial byproduct, to develop biobased phosphonium and ammonium salts (5-25) targeting parasite mitochondria. By combining CNSL-derived C8 alkyl chains with lipophilic cations, we synthesized novel compounds exhibiting highly potent in vitro and ex vivo activity against Trypanosoma and Leishmania spp., including veterinary-relevant strains like T. b. evansi and T. b. equiperdum. Compounds 5 and 7 outperformed reference drugs, demonstrating subnanomolar efficacy against Trypanosoma brucei spp., high selectivity indices (>1000), and no cross-resistance with current therapies, underscoring their potential as next-generation antitrypanosomal agents. Reduced activity against T. brucei overexpressing alternative oxidase and against Trypanosoma congolense supports a mitochondrial mechanism. Preliminary bioassays in zebrafish and Daphnia magna indicated ecotoxicity lower than antiparasitic activity. These CNSL-derived agents represent promising, environmentally safer antiparasitic candidates aligned with One Health and Green Chemistry principles.

1

Chemical structures of cashew nutshell liquid (CNSL) constituents (1–4).

2

Design and chemical structures of CNSL-derived phosphonium (5–13) and ammonium (14–25) salts, starting from mesylate precursors 26–28.

1. Synthesis of Final Compound 5–25

3

Growth curves of T. b. brucei s427 bloodstream forms in the presence of 5 (panels A, B) or 7 (panels C, D). Cells were either grown continuously in the presence of a concentration of 2× or 5× EC₅₀ of test compound (i.e., 3.8 nM and 1.56 nM respectively), or without test compound as a control (panels A,C). Alternatively, the cells were washed into fresh medium without test compound at the indicated time (panels B,D), in order to test the reversibility of the action of the compound. Cells were counted under phase contrast microscopy using a hemocytometer, samples from three independent cultures grown in parallel. Symbols show the average and SEM of three independent determinations. The dashed red line indicates the limit of detection by hemocytometer.

4

Propidium iodide assays of cellular permeability in T. b. brucei. (A) T. b. brucei bloodstream forms were incubated with various concentrations of 5, and the arsenical phenylarsine oxide (PAO) as positive control, in 96 well plates at 37 °C and 5% CO₂. The fluorescence of propidium was monitored for 10 h. A.U., artificial units of fluorescence. (B) T. b. brucei bloodstream forms were incubated with various concentrations of 5 in an incubator at 37 °C and 5% CO₂. At the indicated time points after the addition of 5, samples were taken, cell densities were normalized and 100 μL added to a well of a 96-well plate containing propidium iodide. The fluorescence was recorded for 30 min and the no-drug control was taken as 0% whereas the cells lysed with PAO were taken as 100%.

5

Representative EPR spectra of the spin label 5-DSA incorporated into the plasma membranes of L. amazonensis promastigotes and J774.A1 macrophages showing untreated samples and those treated with compounds 5 and 7 at the indicated concentrations. The mean ± SD values of the EPR parameter 2A_// (outer hyperfine splitting) are provided for each EPR spectrum. 2A_// is measured directly from the EPR spectrum and is defined as the magnetic field separation between the first and last inverted peaks, indicated by the vertical red lines. The SD is not shown for uninfected macrophages because the compounds do not cause membrane alteration even at high concentrations. All means were significantly different in the first two columns from those of the control samples (without treatment) at P < 0.05. In the third column, there was no change, and in the fourth column, the means were different from the mean of control. The intensity of the spectra is given in arbitrary units (Y-axis), and the total scan range of the magnetic field in each EPR spectrum was 100 G (X-axis).