Eleven amino-substituted 1,4-naphthoquinones were synthesized via the reaction of 1,4-naphthoquinone with different primary and secondary mono-and diamines in the presence of dichloromethane ethanol (1:2) solvent at room temperature. All compounds were purified by flash column chromatography, characterized by TLC, HPLC, 13 C-NMR, 1 H-NMR, and FT-IR spectral analysis and were evaluated in vitro for antifilarial activity using adult bovine filarial worm Setaria digitata by assessing worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction. Seven of the 11 compounds had macrofilaricidal activity with compounds 9 (2-[(1,3-dimethylbutyl) amino] naphthalene-1,4-dione) and 11 (2-(4-methylpiperazin-1-yl) naphthalene-1,4-dione) having maximum activity (ED 50 values of 0.91 and 1.2 mM, respectively, at 48 h). The effect of different substitutions on antifilarial activity is discussed. Drug Dev Res 71: 188-196, 2010.

Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature.

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 +/- 3.41 mg ml(-1), 0.09 +/- 0.019 micromol min(-1) ml(-1), and 0.009 +/- 0.002 micromol min(-1) mg(-1) protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC(50) values were 19.42, 51.41, and 114.86 microM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 microM, respectively, at 48-h incubation while curcumin was effective at 54.29 microM. In MTT reduction assay, the ED(50) values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 microM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.

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Eleven 1,4-naphthoquinone analogues with different amino substitutions at position 3 of the quinone ring earlier reported for macrofilaricidal activity were selected and screened against purified cytosolic GST isolated from the bovine filarial worm Setaria digitata and IC(50) values were determined. Of the 11 compounds tested, 8 showed good inhibition against S. digitata GST. The IC(50) values of the most effective macrofilaricidal compounds-11 [2-(4-methylpiperazin-1-yl)naphthalene-1,4-dione] and 9 {2-[(1,3-dimethylbutyl)amino]naphthalene -1,4-dione}-were 0.872 and 0.994 mM, respectively. Compounds 9 and 11 were further studied for type of enzyme inhibition and found to exhibit competitive and uncompetitive inhibition kinetics, respectively, with respect to substrate GSH. All 11 compounds were in agreement with Lipinski's rule of five and passed through the FAFDrugs ADME/tox filter. Molecular docking was carried out using the modeled 3D structure of wbGST PDB ID:1SFM as receptor and substituted naphthoquinones as ligands using AutoDock 4.0. The binding energy of nine compounds varied from -9.15 to -6.58 Kcal mol(-1), whereas compounds 8 and 10 did not show any binding to the receptor. Among the compounds studied, compound 7 {2-[3-(diethylamino) propyl]aminonaphthalene-1,4-dione} showed maximum affinity towards wbGST as it exhibited the lowest binding energy, followed by compounds 11 and 9. However compound 7 was not macrofilaricidal while 11 and 9 exhibited macrofilaricidal activity. The results of in silico and in vitro studies with the synthesized 1,4 -naphthoquinone analogues on filarial GST and in vitro macrofilaricidal activity against adult bovine filarial worm S. digitata open up a promising biochemical target for antifilarial drug development.

Preclinical Research Lymphatic filariasis is a disfiguring disease caused by parasitic worms that destroy the human lymphatic system leading to substantial morbidity. The current drug of choice for the treatment of filariasis is diethylcarbamazine and ivermectin with albendazole which are only effective against the microfilaria, leaving the adult worm unaffected, requiring the development of “adulticidal drugs.” Thirty amino substituted 2‐hydroxy/5‐hydroxy/2‐methyl‐1,4‐naphthoquinones were synthesized via the reaction of 2‐hydroxy/5‐hydroxy/2‐methyl‐1,4‐naphthoquinones with different primary and secondary amines. Compounds 1–30 were evaluated for in vitro antifilarial activity against the adult bovine filarial worm Setaria digitata as assessed by worm motility and MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) reduction assays. The mutagenecity, tumerogenecity, irritantancy, reproductive toxicity, drug score, druglike, and cLogP properties were calculated using OSIRIS property predictor. Ten compounds showed macrofilaricidal activity with ED50 values ranging between 0.086 and 7.6 μM. Taking into account the biological effects and the promising drug‐like profiles of these compounds, these represent valid leads for the development of antifilarial agents against adult filarial worm.