COMPARISON OF ANTIHELMINTHIC ACTIVITY BETWEEN BISARYL BENZYL PIPERAZINE AND BENZIMIDAZOLE LINKED PIPERAZINE DERIVATIVES

COMPARISON OF ANTIHELMINTHIC ACTIVITY BETWEEN BISARYL BENZYL PIPERAZINE AND BENZIMIDAZOLE LINKED PIPERAZINE DERIVATIVES

Bijesh Shrestha ¹, Janmajoy Banerjee *¹, Pramod Kumar Yadav ¹, Amit Kumar Gupta¹, Hemanta Khanal ²

Department of Pharmacy ¹, Sunsari Technical College, Dharan, Nepal.

Department of Microbiology ¹, Central Campus of Technology, Hattisar, Dharan, Nepal.

ABSTRACT: Helminthes infections are more common in poor countries where people suffer from poor health hygiene and sanitation, though there are lot of marketed drugs available for these disease but the interesting thing about antihelminthics is that they are either pypirazine or benzimidazole derivatives, so as medicinal chemist the attention will be drawn towards these different moieties which exert same biological action, in this present work  five derivatives  of piperazine were synthesized by conventional methods using two schemes, by scheme 1 three compounds were synthesized which were Bis benzyl derivatives of pierazine  and by scheme 2  already prepared piperazine 2,5 di one was reacted with benzimidazole derivatives to yield  finally two benzimidazole linked piperazine derivatavies and these two different classes of piperazine deiatives were  screened for antihelminthic activity against Pheretima posthuma to compare their potency in context of calculating paralyzing and death time. Result showed that bis-benzyl derivatives possess to some extent better activity than that of benzimidazole linked piperazine derivatives.

Piperazine, Benzimidazole,

Pheretima posthuma,

Antihelminthic activity

INTRODUCTION: Piperazine  is  a  saturated  six member heterocyclic  compound  containing  two  nitrogen at  1 and  4  position  (as  called  1, 4‐hexahydropyrazine). Piperazine was first introduced as an anthelmintic in 1953. A large number of piperazine compounds have anthelmintic action. In comparison with benzimidazole (a imidazole derivative which too possess potent antihelminthic activity and even some marketed drugs are available as antihelminthic) has always drawn the attention of many researchers to compare the potency of these two different nucleus, though both of their mechanism of action is different in contrast to antihelminthic activity ¹.

The anthelminitic activity of piperazine and related compounds is said to be, based on blockage of the response of the worm muscle to acetylcholine, at the myoneural junction. This action is mediated by its agonist effects upon the inhibitory GABA receptor, The selectivity for helminths is because vertebrates only use GABA in the CNS and the helminths' GABA receptor is a different from the vertebrates' as a result causing a flaccid paralysis in the worm, which is dislodged from the intestinal wall by normal peristaltic action and expelled in the feces ^{2, 3, 4}.

The anthelmintic action of the piperazine compounds depends on their capacity to produce a state of narcosis in the worms. ⁵. The addition product of piperazine and phosphoric acid is useful as an anthelmintic agent for poultry, canines, felines, equines, porcines, and humans ⁶ whereas benzimidazole derivatives like albendazole selectively bind to nematode ß-tubulin, inhibiting polymerization, thus preventing the formation of microtubules and preventing cell division. The loss of the cytoplasmic microtubules leads to impaired uptake of glucose by the larval and adult stages of the susceptible parasites, and depletes their glycogen stores thus decreasing ATP formation ultimately leads to death of worms ^{4, 7}.

Various methods are reported for the synthesis of piperizine derivatives, though many piperazine derivatives occur naturally, piperazine can be synthesized by reacting alcoholic ammonia with 1,2- dichloroethane, by the action of sodium and ethylene glycol on ethylene diamine hydrochloride, or by reduction of pyrazine with sodium in ethanol ⁷ various research has been cariied out on synthesis on piperizine and pirazine  and  those respective deivatives were found to be useful in combating various diesase some important derivatives of piperazines which were already proved to be pharmacologically and biologically are I,e 5-methylpyrazine-2-Carbohydrazide possess antibacterial ⁹ and antiinflamatory activity ¹⁰ N-aryl and N-alkyl piperazine derivatives ^{11, 12} posses antibacterial activity and enhanced antibacterial activity is achieved by incorporation of triazine ring in the piperizine ring ¹³ Mannich bases derived from n-methyl piperazine is found to active against cancer ¹⁴.

Based on different researches mostly it is seen that N-alky/phenyl derivaties or bisaryl/benzyl, (benzyl substitution increase antibacterial activity in comparison to methyl phenyl substitution) ^{10, 11} derivatives of pipearazine are potent molecules, the potency is quantified by the nature of electron donating or electron withdrawing groups substituted on the nucleus.

As mention above that various literatures of piperazine signifies N-phenyl /alkyl or bisaryl/benzyl derivatives are found possess various pharmacological and biological activities here the work is aimed to synthesis some bis benzyl (containing N-methyl group)derivatives of piperazine in one hand and benzimidazole linked piperazine derivatives in other hand and compare their antihelminthic activities.

 Chemistry: Synthesis comprises of two schemes, scheme1 is two step reactions in fist step ethylene diamine reacts with substituted benzoic acid in presence of ammonium purpurate and methanol to give N, N’-Bis-(4-substituted benzyl) -ethane-1,2-diamine derivatives which is then reacted in second step with oxalic acid to give 1,4-Bis-(4-substituted benzyl)-piperazine-2,3-dione. ^] (PZ1, PZ2 PZ3) in  second scheme o-phenylene diamine reacts with substituted benzoic acid to give substituted 2-phenyl -1H-benzo[d] imidazole. Separately glycine is reacted with ethylene glycol to form 2, 5-diketopiperazine. Then substituted 2-phenyl -1H- benzo[d] imidazole and 2, 5 diketopiperazine is refluxed to give substituted 2-phenyl-1-((piperazine-1-yl) methyl)-1H-benzo[d] imidazole (PZ-4, PZ-5).

 MATERIALS AND METHODS:

The chemicals used for the experimental work were commercially purchased  from local source of supplier Rankem (New Delhi), and thionyl chloride was used of Merck (Mumbai), These solvents and reagent were of AR and LR grade and purified before use, The commercially available grades of solvents were distilled. All the compounds were synthesized by conventional methods; melting point was determined by open capillary tube method and synthesized compounds were purified by re-crystallizing using suitable solvents and yield value and percentage purity were determined respectively.

 Experimental portion:

1. General procedure for synthesis of 1,4-Bis-(4-dimethyl amino benzyl)-piperazine-2,3-dione:

In a beaker, the solution of ethylene diamine (2ml) and 4-dimethyl amino benzaldehyde (40Mm, 5 gm) in 50 ml of methanol was added with 40mM of ammonium purpurate was added portion wise with continuous stirring for 3 hours at ice cold temperature and allowed to stand for few minutes. The crystalline deposit was settled down which was re-crystallized from methanol to produce the intermediate compound N, N’-Bis-(4-dimethyl amino benzyl)-ethane-1, 2-diamine. Again 7.5 Mm (2.44gm) of intermediate compound and 7.5 Mm (0.675 gm) of oxalic acid was stirred in ether at ice cold temperature for 1 hour.

The reaction mixture was left over night and the separated solid product was obtained by filtration and dried. The crude drug was re-crystallized from ethanol affording 1, 4-Bis-(4-dimethyl amino benzyl)-piperazine-2,3-dione.

FIG.1: SYNTHESIS OF 1, 4-BIS-(4-DIMETHYL AMINO BENZYL)-PIPERAZINE-2, 3-DIONE

1. General procedure for synthesis of 1, 4-Bis-(4-Chloro benzyl)-piperazine-2, 3-Dione:

FIG.2: SYNTHESIS OF 1,4-BIS-(4-CHLORO BENZYL)-PIPERAZINE-2,3-DIONE.

1. General procedure for synthesis of 1, 4-Bis-(2-hydroxy benzyl)-piperazine-2, 3-Dione:

FIG.3: SYNTHESIS OF 1,4-BIS-(2-HYDROXY BENZYL)-PIPERAZINE-2,3-DIONE.

1. General procedure for synthesis of 1-((2-(Substituted phenyl)-1-H-benzo[d]imidazole-1-yl) methyl) piperazine -2,5-dione. (PZ4, PZ5):

Step 1: Preparation of diketopiperazine:

At first, 15 gm of glycine and 75 ml of ethylene glycol was mixed in a three naked flask fitted with condenser and mechanical stirrer. The mixture was heated in sand bath to 175⁰C, maintaining the temperature, it was continuously stirred for 1hour. The dark brown reaction product was cooled to room temperature and kept at refrigerator for overnight. The next day, the obtained liquid was centrifuged at 3000 rpm for 20 minutes which yielded crude 2,5-diketo piperazine which was yellowish white crystal. Then it was crystallized using water and very few amount of decoloursing carbon to yield pure white crystalline 2, 5-diketo piperazine.

 Step 2: Preparation of intermediate compound:

For the compound INT-4, 0.02 mole of o-phenylene diamine (2.16g) and 0.02 moles of 3, 5-dinitro salicylic acid (4.56 gm) were refluxed in presence of 4N HCl at 100⁰C for 3 hours and 10 minutes. Then completion of reaction was checked by TLC. Then 10% NaOH solution was added to make the solution alkaline. The reaction mixture was cooled and allowed to stand for 5 minutes which yield crude  2-(2-hydroxy-3,5-dinitro phenyl)-1H-benzo[d] imidazole which is then recrystallized from ethanol. For INT-5, 2.76 gm of salicylic acid was taken and procedures were same. The compound formed was 2-(2-hydroxy phenyl)-1H-benzo[d] imidazole.

 Step 3: Preparation of final product (PZ4, PZ5):

The intermediate compound (INT-4, INT-5) (0.01 mole) was dissolved in ethanol (15ml) followed by addition of 2, 5-diketo piperazine (0.01mole) (1.1 gm) and formaldehyde solution (40% w/v) (0.015mole) (1.13 ml) to undergo Mannich reaction. The reactants were refluxed for 6 hrs with continuous stirring at 70-75˚C. The completion of reaction was checked by TLC. After completion, the reaction mixture was kept in a refrigerator overnight. The product precipitated out and was filtered, dried and recrystallised from ethanol to give 1-((2-(2-hydroxy - 3, 5 - dinitrophenyl)-1-H-benzo[d]imidazole-1-yl)methyl) piperazine - 2, 5-dione[PZ4], and 1-((2-(2-hydroxy phenyl)-1-H-benzo[d]imidazole-1-yl)methyl) piperazine - 2, 5-dione and 1-((2-(2-hydroxy phenyl)-1-H-benzo [d]imidazole-1-yl)methyl) piperazine - 2, 5-dione.[PZ5].

Anthelmintic Evaluation:

The synthesized compounds (PZ1-PZ5) were evaluated in vitro for their antihelmintic activities     according to the protocol mentioned below. The earthworms were collected from fish farm, Tararha. Sunsari, Nepal and species identification was done from Biology Department of Central Campus of Technology, Hattisar, Dharan, Nepal.

The anthelmintic activity was evaluated on adult Indian earthworms by Mathew et al method ¹⁵. For preliminary evaluation of anthelmintic activity test samples of synthesized compounds was prepared at the concentration of 1000, 800, 600, 400 µg/ml in DMSO (6%) with normal saline and 6 worms Pheretima posthuma of 8-10cm were placed in petridish containing 25 ml of above test solutions of synthesized compounds. Mebendazole (4mg/ml) was used as positive control and normal saline with DMSO (6%) is used as negative control. All the test solutions and standard solutions were prepared freshly before starting the experiment. Observations are made for the time taken for paralysis when movement was lost or no movement. Worms should not relieve even in normal saline. Time for death of worms were recorded after ascertaining that worms neither moved when shaken vigorously nor when dipped in warm water and fading of color of worms was observed.

Preparation of stock solution and test solution of piperazine derivatives:

It was prepared by dissolving 100mg of synthesized compound in 100 ml of 6%DMSO with normal saline, which served as a stock solution. From stock solution 1000ug/ml, 800ug/ml, 600ug/ml, 400ug/ml, were prepared. Standard drug solution was prepared by dissolving 100mg of mebendazole in 25 ml of 6%DMSO with normal saline. 6 earthworms were placed in each petridish containing 25 ml of solution of different concentration of test and standard and paralyzing and death times are noted.

RESULT AND DISCUSSION:

 TABLE 1: PHYSICAL PARAMETER OF SYNTHESIZED FINAL PRODUCTS

All the ccompounds were soluble in water, while none was soluble in diethyl ether and PZ-2, PZ-3 and PZ-5 were soluble in benzene while others were insoluble in benzene.

Melting Points: The melting points of synthesized organic compound were determined by open capillary tube method in a heavy liquid paraffin bath.

Melting point is valuable criterion of purity for an organic compound as pure crystal is having definite and sharp melting point.
TABLE 2: MELTING POINT OF SYNTHESIZED COMPOUND

 Anti helminthes screening:

 TABLE 3: DATA FOR ANTIHELMINTHIC ACTIVITY

*standard = mebendazole for antihelmintics activity, the antihelmintics activity were reported as mean paralyzing time and mean death time.

DISCUSSION: Various piperazine derivatives were prepared by two different schemes viz. scheme 1 and scheme 2. In scheme 1, ethylene diamine was reacted with substituted benzaldehyde in presence of ammonium purpurate (reductive alkylation reaction) followed by reaction with oxallic acid (cyclization process) to afford 1, 4-Bis-(4-substituted benzyl)-piperazine-2,3-dione. All the compounds were purified through recrystallization by ethanol. All the synthesized compound have been characterised by physico chemical datas such as melting point, colour, %yield, solubility etc.compunds PZ-2 and PZ-3 were synthesized earlier and were found to be active against   Enterobious vermicularis and Fasciola hepatica ³.

PZ-1 was synthesized for the first time by substituting p-Cl, o-OH by N-(CH3)2 in the benzyl ring of Bis phenyl piperazines derivatives. The synthesized derivatives(PZ1-3) posess 2,3 dione moiety as common, as it has been reported earlier 2,3-dione moiety in the structure of piperazine was considered as promising candidates as an antihelminthic agent ^{3, 16}. In scheme 2, the targeted compounds were synthesized fusing benzimidazole and piperazine through Mannich reaction. The synthesized compounds were subjected to antihelminthic activity as per referred protocol using mebendazole as standard drug against   Pheretima posthuma. 

The anti helmenthic activity data focus that PZ-1(mean paralyzing time 28.25 minute and mean death time 67.5 minute respectively) and PZ-3(mean paralyzing time 26.25 minute and mean death time 53.5 minute respectively)showed better activity among all the synthesized compounds and even in comparison to benzimidazole linked piperazine derivatives i,e PZ-4(mean paralyzing time 52.75 minute and mean death time 108 minute respectively) and PZ-5(mean paralyzing time 32.75 minute and mean death time 59.5 minute respectively) as it was anticipated that benzimidazole ring if incorporated in pierazine ring may exert better antihelminthic  activity than other piperazine derivatives, but here the result does not comply with this above justification. Bis benzylderivatives were found to have better antihelminthic activity than bezimidazole derivatives.

CONCLUSION: Compared to two different schemes, the compound produced by scheme:  PZ-1 got more yield value. Among them PZ-2 yield percentage was high, 80.8%. Scheme 2 had good yield value but took comparatively longer time of reaction as it produced complex or higher molecular weight piperizine derivatives. It proves that ethylene diamine is the good precursor for synthesizing piperazine and its derivatives.

The synthesized compounds were screened for antihelmintic and antioxidant activity. From scheme 1: PZ1, PZ3 were found to have very good anti helminthic activity. It indicates that the presence of 4-dimethyl amino group and 2-hydroxy group at para position of benzyl moiety attached to the piperazine ring enhanced the activity compared to other groups like 4-chloro derivatives at the same position. Similarly from scheme 2: PZ-5 was potent which had 2-hydroxy benzyl group attached to benzimidazole. It shows that incorporation of 2-hydroxy benzyl group in drug structure was found to be active against helminthes (earthworms).

ACKNOWLEDGEMENT: The authors want to acknowledge Sunsari Technical College Dharan, Nepal for assisting necessary facilities required for the research work.

CONFLICT OF INTEREST: Poster presentation was done in 3^rd International Conference in Medicinal chemistry and drug Design, conducted by Med Chem. India, Hyderbad September (10-11) 2015.

REFERENCES:

1. Jain VK, Jain B, Sharma UK, Saha D. Synthesis, characterization and antimicrobial screening of some 4-substituted-1-(4-substituted phenyl) piperazine derivatives. Int J Curr Pharm Res.3:66-70.
2. Debell JT. A long look at neuromuscular junctions in nematodes. Quarterly Review of Biology. 1965;40(3):233-51.
3. Hussein MA, Diab AK. Synthesis of some new 1, 4-distributed Piperazine-2, 3-dione derivatives of potential antihelminthic activity. Bulletin of Pharmaceutical Science. 2005;28(1):34-7.
4. Manzeera AS, Sireesha PN, Aswini M, Rao PM, Sree KN, Mallikharjunarao KLN. Synthesis and anti-inflamatory activity of pyrazine derivatives. Asian Journal of Research in Pharmaceutical Sciences and Biotechnology. 2013; 1(1):10-5.
5. Miniyar P, Makhija S. Synthesis and antibacterial activity of 5-methylpyrazine-2-carbohydrazide derivatives. Int J Drug Dev & Res. 2009;1(1):19-26
6. Jain VK, Jain B, Sharma UK, Saha D. Synthesis, characterization and antimicrobial screening of some 4-substituted-1-(4-substituted phenyl) piperazine derivatives. Int J Curr Pharm Res. 2011; 3(1):66-70.
7. Kaminski ZJ, Kolesinska B, Kolesinska J, Sabatino G, Chelli M, Rovero P, et al. N-Triazinylammonium tetrafluoroborates. A new generation of efficient coupling reagents useful for peptide synthesis. Journal of the American Chemical Society. 2005; 127(48):16912-20.
8. Thriveni KS, Basavaraj Padmashali, Siddesh MB And Chidananda.Synthesis and antimicrobial screening of napthofuran- 1,3,4-oxadiazole linked piperazines. BN UJP 2013; 02 (06): 43-6.
9. Yeap CW, Bian CK, Abdullah AFL. A review on benzylpiperazine and trifluoromethylphenypiperazine: origins, effects, prevalence and legal status. Health Environ J. 2010;1(2):38-50.
10. Ahmadi A, Khalili M, Chavrogh S, Nahri-Niknafs B. Synthesis and Anti-inflammatory Performance of Newly Cyclizine Derivatives on Adult Male Wistar Rats. Iranian Journal of Pharmaceutical Research. 2012; 11(4):1027.
11. Preeti Chaudhary, Rupesh Kumar, Akhilesh K. Verma, Devender Singh, Vibha Yadav, Anil K. Chhillar, G. L. Sharmab and Ramesh Chandra, Synthesis and antimicrobial activity of N-alkyl and N-aryl piperazine derivatives, Bioorganic & Medicinal Chemistry, 2006; 14:1819–26.
12. Singh KK, Joshi SC, Mathela CS. Synthesis and in vitro antibacterial activity of N-alkyl and N-aryl piperazine derivatives. Indian Journal of Chemistry-Part B Organic Including Medicinal. 2011; 50(2):196.
13. Desai SD, Mehta AG. Design, Synthesis, Characterization and Biological Evaluation of Various Nsubstituted Piperazine Annulated s-Triazine Derivatives. Research Journal of Chemical Sciences.2014; 4(5):14-19.
14. Kumbhare RM, Kumar KV, Ramaiah MJ, Dadmal T, Pushpavalli S, Mukhopadhyay D, et al. Synthesis and biological evaluation of novel Mannich bases of 2-arylimidazo [2, 1-b] benzothiazoles as potential anti-cancer agents. European journal of medicinal chemistry. 46(9):4258-66.
15. Mathew AS, Patel KW, Shah BK, Investigation on antifeedant and antihelminthic potential of Adatoda vasica Nees. Indian J Nat Prod, 1995; 14(1): 11.
16. Narasimhan B, Sharma D, Kumar P. Benzimidazole: a medicinally important heterocyclic moiety. Medicinal Chemistry Research. 2012; 21(3):269-83
    

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How to cite this article:

Shrestha B, Banerjee J, Yadav PK, Gupta AK and Khanal H: Comparison of Antihelminthic Activity between Bisaryl Benzyl Piperazine and Benzimidazole linked Piperazine Derivatives. Int J Pharm Sci Res 2016; 7(4): 1547-55.doi: 10.13040/IJPSR.0975-8232.7(4).1547-55.

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