The novel ligand, 1-(phthalazin-1(2H)-one)[(pyridin-2-yl)ethylidene]hydrazone,(APN), derived from the antihypertensive drug, hydralazine hydrochloride, was synthesized and characterized by spectroscopic methods (IR, 1H NMR). The X-ray crystallographic data indicates that APN has an exocyclic C=N bond on the hydralazine moiety. APN revealed significant anti-onchocercal activity with IC50 values of 0.3125 μg/mL on microfilaria and 10 μg/mL on adult worms compared to the standard drug, ivermectin.
Hydralazine, a 1-substituted phthalazine, commonly called apresoline, is a powerful areterial vasodilator that has been effectively used for the treatment of hypertensive disorders particularly in pregnant patients and also in the treatment of patients with congestive heart failure. Schiff bases of hydralazine (hydrazones) have shown important medicinal properties including antifungal activities [
Human onchocerciasis or subcutaneous filariasis, commonly known as river blindness, is one of the 17 neglected tropical diseases caused by the filarial worm Onchocerca volvulus and transmitted by the black fly, Simulium damnosum. By World Health Organization (WHO) estimates about 123 million people are at risk of becoming infected with river blindness and 25 million people worldwide are already infected with Onchocera volvulus, 300,000 of whom are blind and 800,000 are suffering from some kind of visual impairment [
The drug of choice for the treatment of onchocerciasis is ivermectin which is very effective against microfilariae with no discernible effect on macrofilariae. A yearly dose of prescribed ivermectin for 15 - 18 years may eradicate microfilariae and break the chain of transmission. This long period of treatment may lead to patients’ noncompliance and drug resistance in the parasite. The emergence of ivermectin resistance in parasitic nematodes in veterinary medicine [
These challenges coupled with adverse effects of ivermectin to humans have prompted an urgent search for other sources of new and more potent drugs for the treatment of river blindness. Our objective in this study is to synthesize a new hydralazine-derived Schiff base and to test it against Onchocerca ochengi, a close relative of Onchocerca volvulus, the causative agent of human onchocerciasis.
Hydralazine hydrochloride, 2-acetylpyridine and other solvents were used as purchased from commercial sources without further purification. Elemental analysis was performed on a VARIO EL (Heraeus) analyzer. IR spectra were obtained from a Perkin-Elmer System 2000 FT-IR spectrophotomer using KBr pellets. The mass spectra (ESI) were recorded with an FT-IR (APEX II) mass spectrometer from Bruker Daltonics and the 1H NMR spectra were run in MeOD on a 400-MHz spectrometer
A mixture of equimolar amounts of hydralazine hydrochloride (500 mg, 2.54 mmol) and 2-acetylpyrideine (307 mg, 2.54 mmol) and sodium acetate (buffering agent) (350 mg, 2.56 mmol) in 50 mL of ethanol was refluxed for 4 hours while stirring (Scheme 1). The mixture was left to cool overnight. The resultant solid was vacuum-fil- tered, washed several times with distilled water and ethanol to give a yellow precipitate on air-drying.
Yield 79%. Anal.Calcd for C15H13N5: C 58.40%; H 5.60%; N 22.70%. Experimental: C 58.40%; H 4.40%; N 33.43%. IR (KBr, cm−1) 3438.6, 3317.1, 3045, 3006, 2922.1, 1605.6, 1591.5, 1569.1, 1532.6.1468.1, 1432.8, 1250.1, 1146.8, 1023, 780.5. ESI [m/z (%)]: 286.1 (100%) [M + Na]+; 264.2 (35%) [M]+. 1H NMR (δ ppm): 2.64 (s, 3H at position 8’), 7.74 (m, H at positions 6,7), 8.05 (d, H at position 5), 8.12 (d,H at position 8), 8.56 (s, H at position 4), 7.79, 7.74 (m, Hs at positions 4’ and 5’), 7.96 (d, H at position 3’), 8.71 (d, proton at position 6’) (
The crystallographic data of APN were collected on a Gemini diffractometer (Agilent Technologies) using Mo-Kα radiation (λ = 71.073 pm), ω-scan rotation. CrysAlis Pro [
Scheme 1. Synthesis of APN.
SCALE3 ABSPACK [
Both O. ochengi microfilariae and macrofilariae were isolated and cultured by the known methods [
All assays were conducted for 120 hours after adding compound. The FDA-approved compound, auranofin which has been reported to be active on adult worms at 10 µM, was used as positive control for the adult worm assays [
The condensation reaction between hydralazine hydrochloride and 2-acetylpyridine gave APN in good yield (Scheme 1).
Elemental analysis of the ligand was in good agreement with literatures values [
The IR spectra of the APN (
and 3317 cm−1 corresponding to the stretching vibrations of the O-H bond of residual water molecules and N-H stretching vibrations respectively. The bands that cluster around 3000 cm−1 are the stretching vibrations of =CH and the sharp bands at 2992 cm−1 can be assigned to the methyl group bonds. The strong bands in the range 1532 - 1606 cm−1 are due to the C=C vibrations respectively [
The proton NMR spectra of APN (
A single x-ray analysis performed on APN (
APN was screened on the juvenile form of the O. ochengi beginning at the highest compound concentration of 20 µg/mL. It inhibited microfilariae motility completely at 1.25 µg/mL (
APN compound was also seen to be most cytotoxic on LLCMK2 cells. APN killed the cells completely at 1.25 µg/mL and had a selectivity index of 2 (
drugs which are macro-/microfilaricidal is still highly solicited.
The long duration of treatment of onchocerciasis with ivermectin is because the drug is only microfilaricidal. APN has shown both micro- and macrofilaricidal properties raising hope for an effective anti-onchocercal drug.
Compound code | Concentration of compound (µg/mL) | Percent inhibition of mfs motility | Cytotoxicity on LLCMK2 cells | IC50 of parasite (µg/mL) | CC50 of LLCMK2 cell (µg/mL) | Selectivity Index |
---|---|---|---|---|---|---|
APN | 20 | 100 | 100 | 0.3125 | 0.625 | 2 |
10 | 100 | 100 | ||||
5 | 100 | 100 | ||||
2.5 | 100 | 100 | ||||
1.25 | 100 | 100 | ||||
0.625 | 50 | 50 | ||||
0.3125 | 50 | 0 | ||||
0 | 0 | 0 | ||||
Ivermectin | 10 | 100 |
APN derived from hydralazine hydrochloride was synthesized and characterized by spectroscopic methods. The X-ray crystallographic data show that APN has an exocyclic carbon-nitrogen double bond on the hydralazine moiety indicating the stable form of the compound in the solid state. APN showed significant anti-onchocercal activity with IC50 value of 0.3125 µg/mL on microfilariae and 10 µg/mL on adult worms. The anti-onchocercal activities of this compound are reported for the first time and the results indicate that this novel ligand can be exploited as an anti-onchocercal agent.
We wish to thank the Department of Chemistry at the University of Buea for providing the necessary support to perform the experiments, Professor Rhett Kempe of the Institute of Inorganic Chemistry, University of Bayreuth, Germany, for providing the chemicals and Professor Dr. Evamarie Hey-Hawkins of the Institute of Inorganic Chemistry, University of Leipzig, Germany, for doing the X-ray analysis.
Joseph N. Yong,Felicite Majoumo-Mbe,Moses Samje,Emmanuel N. Nfor, (2016) Synthesis, Molecular Structure and Anti-Onchocercal Studies of 1-(Phthalazin-1(2H)-one)[(Pyridin-2-yl)ethylidene]hydrazone. International Journal of Organic Chemistry,06,77-84. doi: 10.4236/ijoc.2016.61008