Anthelmintic Evaluation of Some Novel Synthesized 1 , 2 , 4-Triazole Moiety Clubbed with Benzimidazole Ring

A series of N-[3-{(1H-benzo[d]imidazol-2-ylthio) methyl}-5-mercapto-4H-1, 2, 4-triazol-4yl]-2-substituted phenoxy acetamide 6(a-g) were synthesized by the mixture of the compound of potassium 2-(2-(1H-benzo[d]imidazol-2-ylthio) acetyl) hydrazinecarbodithioate (4) and aryloxy acid hydrazide (5) in presence of hydrochloric acid. The predicted structures of the synthesized compounds were confirmed by different spectral analysis studies. The title compounds 6(a-g) were screened for anthelmintic activity against Pheretima posthumous. The entire compounds were exhibited good anthelmintic activity when compared with standard drugs such as Albendazole and Piperazine.


INTRODUCTION
There are two important types of worm infections, those in which the worms live in the alimentary canal of the host and worms live in other tissues of the host body.Common examples of worms 1 in the alimentary canal are the tapeworm, intestinal round worm, trematodes or flukes, tissue round worm and diseases caused by helminthes are ascariasis, trichirians, hook worm and tape worm infection, thread worm infection, schistosomiasis and giardial infection 2 .During the last decades the compounds bearing heterocyclic nuclei have received much more attention due to their medicinal value in the development of novel anthelmintics drugs.Benzimidazole derivatives have been a wide range of biological activities, such as analgesic 3 , antiinflammatory 4 , antiparasitic 5 , antitubercular 6 , antiviral 7 , anticonvulsant 8 and antimicrobial activities 9- 10 .Literature survey indicated that the compounds consists of 1, 2, 4-triazole ring have been reported to possess different biological activities such as antimicrobial 11 , antifungal 12 , anti-inflammatory 13 , antioxidant [14][15] , antiviral 16 , anticancer 17 and anticonvulsant activity 18 depending on the substituent in the ring system.In view of such biological reports, the present work is aimed to design and develop the synthesis of some new potential substituted aryloxy triazolyl Benz[b] imidazole moiety and explores the evaluation of their in-vitro anthelmintic activity.

EXPERIMENTAL
The chemicals and solvents used for the experimental work were procured from E. Merck, India, and Qualigens, India.Silica gel G used for analytical chromatography (TLC) was obtained from E. Merck.Melting points were determined in an open glass capillary using a Kjeldahl flask containing paraffin and are uncorrected.The proton magnetic resonance spectra ( 1 H NMR) were recorded on a Bruker 300 MHz instrument (Bruker, Germany) in dimethylsulfoxide-d 6 /CDCl 3 using tetramethylsilane as internal standard.Chemical shifts (δ) are expressed in ppm.The infrared spectra of compounds were recorded in KBr on infrared spectrophotometer Jasco, Japan.Purity of the compounds was checked by TLC using silica gel G.

Preparation of 2-mercapto Benzimidazole (1)
A mixture of o-phenylenediamine 32.4gm (0.3mole) potassium ethyl xanthate 52.8gm (0.33mole), 95% ethanol 300ml and 45ml of water in a 1-1, flask was heated under reflux for 3 hours.Noritz (12gm) was then added cautiously and the mixture was heated at the reflux temperature for 10 minutes.Then Noritz was removed by filtration, the filtrate was heated to 60-70 °C, 300ml of warm tap water (60-70 °C) was added followed by 25ml of acetic acid in 50ml of water with stirring.The product separated out as glistening white crystal and the mixture was placed in a refrigerator for 3 hours to complete the crystallization.The product is collected on a Buchner funnel dried overnight at 40°C.Yield 86%, m.p 303-304°C.

Preparation of Ethyl 2(Benzimidazolylthio) acetate (2)
A mixture of 2-mercaptobenzimidazole (0.1mole), ethylchloroacetate (0.1mole) and potassium carbonate (0.1mole) and acetone (distilled) was taken in a round bottom flask and refluxed for 6-7 hrs.After the completion of reaction, the mixture was cooled at room temperature and then it was poured into the ice cold water.The solid obtained was filtered.Yield 63%, m.p .208°C.

Synthesis of Substituted Aryloxy hydrazide 5(a-g)
The substituted acid (0.1mole) and ethanol (50 ml) were taken with a few drops of concentrated sulfuric acid and it was refluxed for 6 hours.The reaction mixture was concentrated by distilling of the excess of ethanol under reduced pressure and treated with a saturated solution of sodium bicarbonate.The ester obtained used for the preparation of hydrazide directly.The ester (0.1mole) was dissolved in appropriate quantity of ethanol and to this hydrazine hydrate (0.1mole) was added.The reaction mixture was taken in a round bottomed flask and refluxed for a period of 12-18 hours.Excess of ethanol was distilled off under reduced pressure.It was then poured into ice cold water and the solid obtained was filtered.It was crystallized from ethanol.

Anthelmintic activity
The newly synthesized compounds were tested for anthelmintic activity 19 against Pheretima posthumous (earth worms collected from Ekahabaor Botanical Garden, Near to Regional plant Resource Centre, Bhubaneswar, Odisha, India) of nearly equal size (6 ± 1 cm) were selected randomly for the experimental study.The worms were acclimatized to laboratory conditions before experimentation.The earthworms were divided into four groups of six earth worms each.Albendazole and Piperazine citrate diluted with normal saline solution to obtain 0.1, 0.2, 0.5 and 1 mg/mL act as standard and were poured into Petri dishes.The synthesized compounds were dissolved in water and add minimal quantity of Tween 80 and make to prepare four concentrations of 0.1, 0.2, 0.5 and 1mg/mL of each compound in form suspension. Normal saline served as a control.The time taken for complete paralysis and death was noted.The mean paralysis time and mean lethal time were calculated for each compound (each reading was taken in triplicate).Paralysis was considered when no movement was observed even after shaken vigorously.Death was considered when the worm loss their motility followed by change in body color.Statistically analysis was performed using ANOVA to find the significance of the test.

CONCLUSION
Present research work involves synthesis of some benzimidazole derivatives clubbed with triazolyl moiety and to explore their anthelminthetic activity.The compounds (6d, 6e) have been exhibited good anhelminthetic activity against earth worm due to presence triazolyl moiety with phenyl substituted electron withdrawing groups and conjugated with pharmacophore group such as benzimidazole ring in the same molecular structural frame.