Synthesis of 1,2,3,4-[ 1-N- Methyl Benzimidazole] 3-Phenyl Substituted Thiazolidine, 4-One and Carbohydrazide Derivatives for Antimicrobial Activity.

Palled M. S.*., Lohar V. R., Kalekar M. C., Patil P. B., Bhat A. R..

Department of Pharmaceutical Chemistry, K. L.E.U’s College of Pharmacy, Belgaum

Corresponding author: maheshpalled@rediffmail.com

ABSTRACT:

Research on benzimidazole and thiazolidine-4-one and their synthetic analogs have been revealed to posses wide range of antimicrobial activity. It was our interest to make novel derivatives of fused benzimidazole, thiazolidine-4-one nuclei and evaluate them for antimicrobial activity. Formic acid was treated with o-phenylenediamine to obtain benzimidazole which was treated with CH₃I for N-methylation. Later this compound was subjected for Vilsmeier-Haack Reaction, followed by formation of Schiff bases on treatment with various aromatic amines. The various Schiff bases were further treated with thioglycolic acid to obtain thaizolidine-4-one derivatives. (Scheme-I). In another (Scheme-II) N-methyl substituted benzimidazole-2-carbohydrazide (Vilsmeier-Haack Reaction product) followed by acidification, esterification and hydrazide formation. Finally hydrazide treated with different aldehydes to obtain carbohydrazide derivatives. The structures of synthesized derivatives were identified and characterized by m.p, TLC, IR, ¹HNMR and Mass Spectroscopy. The synthesized compounds BA-SA, BA-SN, AH-BR and AH-CL have shown significant antibacterial activity against E. coli, E.fecalis, Klebsiella and S. aureus and BA-SA, BA-SN, BA-CL and AH-CL shown significant antifungal activity against C. albicans and A. niger.

KEYWORDS: Benzimidazole, Thiazolidine-4-one, Carbohydrazide, Antimicrobial activity.

INTRODUCTION:

Benzimidazole and its synthetic analogs have been found to exhibit a broad spectrum antimicrobial activity. Research on benzimidazole derivatives also have brought about a wide range of activities including antimicrobial activity. The development of antimicrobial drugs represent one of the most important advances in therapeutics both in the control or cure of the serious infections and in prevention and treatment of infectious complications of other therapeutic modalities such as cancer chemotherapy and surgery. Based on these observations we propose to synthesize various derivatives of fused benzimidazole nuclei with Phenyl substituted Thiazolidine-4-one and Phenyl substituted Carbohydrazide. The determination of anti microbial activity which is followed here is a relative method and not absolute. Responses of an organism to an unknown compound are compared with responses to the standard preparation of known composition and concentration.^1-2

MATERIAL AND METHOD:

Melting points were determined in open capillary method and are uncorrected. Physicochemical properties of synthesized compounds presented in table no.1. IR spectra were recorded on Shimadzu Prestige 21 FT-IR using KBr pellets method. The ¹H NMR spectra were recorded on Bruker AC 400 MHz using DMSO as solvent and tetra methyl silane as internal standard.

Proposed scheme I:

Synthesis of 1H-benzimidazole:

Equimolar amount of o-phenylenediamine and corresponding formic acid in 5N HCl were refluxed for 5-6 hrs. The solution was cooled in an ice bath and neutralized with NaHCO₃ solution. The resulting precipitate was filtered off, washed several times with water and re-crystallization by methanol.³

Synthesis of 1-methyl-1H-benzimidazole:

A mixture of 1H-benzimidazole (0.1 mol), 1.25 moles of methyl iodide in dry acetone of 50ml, then 1.5moles of potassium carbonate and it was refluxed for 4-5 hours. Filtered the content, filtrate was collected and recrystallized to get pure product.

Scheme I

Table No. 1: Physicochemical properties of synthesized compounds

Compound	Mol. Formula	Mol. Wt. (gm)	Melting Point ⁰C	Yield %
BA-CL	C₁₇H₁₄ClN₃OS	343.5	155-157	81.63
BA-NI	C₁₇H₁₄N₄O₃S	354	110-112	90.39
BA-SN	C₁₇H₁₆N₄O₃S₂	388	183-185	64.43
BA-SA	C₁₉H₁₈N₄O₄S₂	430	169-171	86.00
AH-CL	C₁₆H₁₄ClN₄O	313.5	128-130	65.85
AH-NI	C₁₆H₁₄N₅O₃	324	117-119	83.33
AH-BR	C₁₆H₁₄BrN₄O	358	135-137	69.98

Synthesis of 1-N-methyl-1-H benzimidazole, 2-carbaldehyde:

N,N-Dimethyl Formamide (3.86 ml,0.05 mol) in RBF in an ice cold condition (0-5⁰C) with constant stirring to this POCl₃ (13.04 ml, 0.014 mol) was added drop wise for period of 30 min. and resultant mixture was stirred for further 1 hrs. The appropriate 1-methyl-1H-benzimidazole(0.1 mol) were added to Vilsmeier reagent and stirred for further 30 min. and reaction mixture was kept in water bath at 100⁰C for stipulated period of time. After reaction completed as inferred through TLC. The reaction mixture was poured onto 500 gm of crushed ice with constant manual stirring. It was kept aside for overnight. After the neutralizing above with 4N NaOH, precipitate obtained was washed with water and extracted with ethyl acetate. The combined organic layers collected and dried over anhydrous Na₂SO_4.⁴

Synthesis of 4-substituted-N-[(E)-(1-methyl-1H-benzimidazol-2-yl) methylidene] -aniline {Schiff Base}

Equimolar aromatic amine (2.5 mol) and 1-N-methyl-1-H benzimidazole, 2-carbaldehyde (25 mmol) in 40ml ethanol, reflux for 3-4 hrs. Then cool the reaction mixture, crystals obtained washed with water, re-crystallize.⁵

Synthesis of 2-(1-methyl-1H-benzimidazol-2-yl)-3-(4-substitutedphenyl)-1, 3-thiazolidin-4-one

4-substituted-N-[(E) - (1-methyl-1H-benzimidazol-2-yl) methylidene] aniline {Schiff Base} 0.01 mol and 0.3 mol of thioglycolic acid in 25-30 ml of Glacial acetic acid, reaction mixture was refluxed for 3-4 hrs. Saturated solution of NaHCO₃was prepared and then, ice (crushed) added to that then reaction mixture was poured into that, keep it for 3-4hrs., at 0-5⁰C. Precipitate filtered off, washed with water and re-crystallize.^6-8

Procedure for proposed scheme II:

Up to Synthesis of 1-N-methyl-1-H benzimidazole, 2-carbaldehyde same procedure as per scheme I .

Synthesis of 1-methyl-1H-benzimidazole-2-carboxylic acid:

1-N-methyl-1-H-benzimidazole, 2-carbaldehyde (0.01mol) was slowly added to solution of KMnO₄ (1.25 eq.) in ter-butyl alcohol (1ml/mmol) with continuous stirring for 2-3 hrs. Temperature of exothermic reaction controlled by external ice cooling, then mixture stirred at room temperature for 30 min. residue treated with Na₂SO₄ solution and extracted with ethyl acetate.⁹

Scheme II

Synthesis of ethyl 1-methyl-1H-benzimidazole-2-carboxylate:

Dissolved (0.01 mol) of 1-methyl-1H-benzimidazole-2-carboxylic acid in sufficient amount of ethanol (30 ml) to that add 3-4 drops of conc. H₂SO₄, reflux the reaction mixture for 6-8 hrs., remove the content from RBF and evaporate ethanol. To this added 5-7% of NaHCO₃ solution, extracted reaction mixture with ethyl acetate, separated organic layer, to that added Na₂SO₄, filtered and ethyl acetate evaporated.¹⁰

Table No. 2: Antimicrobial activity of synthesized compounds

COMPOUND CODE	µg/ml -MIC (Minimum Inhibitory Concentration) against strains of bacteria
	Antibacterial activity				Antifungal activity
	*E. coli*	*Klebsiella*	*E.fecalis*	*S. aureus*		*C. albicans*	*A. niger*
AH-BR	6.25	6.25	3.125	6.25		12.5	12.5
AH-NI	12.5	12.5	6.25	3.125		12.5	12.5
AH-CL	3.125	3.125	3.125	6.25		3.125	3.125
BA-NI	6.25	12.5	6.25	6.25		6.25	12.5
BA-SA	3.125	3.125	3.125	3.125		3.125	3.125
BA-CL	6.25	6.25	6.25	6.25		3.125	3.125
BA-SN	1.6	3.125	3.125	1.6		3.125	3.125
Standards	Ciprofloxacin					Flucanazole

Synthesis of 1-methyl-1H-benzimidazole-2-carbohydrazide:

Hydrazine hydrate (4 ml) and related ethyl 1-methyl-1H-benzimidazole-2-carboxylate (1.5 mol) in ethanol (5 ml) were refluxed for 4 hrs. The reaction mixture was cooled and poured into water. The crude product was filtered off and re-crystallized from ethanol.¹¹

Synthesis of 1-methyl-N'-[(E)-(4-substitutedphenyl) methylidene]-1H-benzimidazole-2-carbohydrazide:

0.0025 mol of 1-methyl-1H-benzimidazole-2-carbohydrazide was refluxed with 0.0025 mol of appropriate aldehydes in 50 ml ethanol (60%) for 5 hrs. The solid separated was washed with ethanol (60%).¹²

Analytical data of the synthesized compounds:

BA-CL: IR (KBr) cm^-1: 646 (C-S-C) 752 (C-Cl) 1325 (C-N) 1402 (CH₃) 1610 (C=N) 1681 (C=O) 3250 (Ar C-H)

¹H NMR- 3.62 (-CH₂), 3.73 (-CH₃), 7.32-7.85 (4 H, Ar-H), 7.59-7.85 (3 H, Ar-H), mass- M⁺ - 345

BA-NI: IR (KBr) cm^-1: 682 (C-S-C) 1402 (CH₃) 1450 (Ar-NO₂) 1610 (C=N) 1680 (C=O) 3040 (Ar C-H)

BA-SN: IR (KBr) cm^-1: 646 (C-S-C) 1400 (CH₃) 1600 (C=N) 1680 (C=O) 3200 (Ar C-H) 3360 (-NH-)

BA-SA: IR (KBr) cm^-1: 640 (C-S-C) 1400 (CH₃) 1590 (C=N) 1700 (C=O) 2560 (Ar C-H) 3367 (-NH-)

AH-NI: IR (KBr) cm^-1: 1320 (Ar-NO₂) 1360 (C-N) 1420 (CH₃) 1720 (-C=O) 2860 (C-H) 3160 (-N-H)

mass- M⁺ - 322

AH-CL: IR (KBr) cm^-1: 760 (C-Cl) 1440 (CH₃) 1590 (C=N) 1752 (C=O) 3120 (C-H) 3427 (N-H)

¹H NMR- 3.42 (-CH₃), 7.24-7.31 (4 H, Ar-H), 7.54-8.98 (4 H, Ar-H), 10.35 (-NH)

AH-BR: IR (KBr) cm^-1: 680 (-Br) 1480 (CH₃) 1590 (C=N) 1720 (-C=O) 2880 (C-H) 3040 (-N-H)

Antimicrobiological screening:

Determination of MIC (minimum inhibitory concentration):

The antimicrobial activity of the synthesized phenyl substituted thiazolidine-4-one derivatives and carbohydrazide derivatives were carried out using broth dilution method. The compounds were tested against four strains of bacteria viz. E. coli, E.fecalis, Klebsiella and S. aureus and two strains of fungi viz. C. albicans and A. niger. Ciprofloxacin was used as standard drug for antibacterial activity and Flucanazole was used as standard drug for antifungal activity.

Then dilutions are made as follows:

1. 9 dilutions of each drug have to be done with BHI (brain heart infusion) for MIC.

2. In the initial tube 20microliter of drug was added into the 380microliter of BHI broth.

3. For dilutions 200microliter of BHI broth was added into the next 9 tubes separately.

4. Then from the initial tube 200microliter was transferred to the first tube containing 200microliter of BHI broth. This was considered as 10^-1 dilution.

5. From 10^-1 diluted tube 200microliter was transferred to second tube to make 10^-2 dilution.

6. The serial dilution was repeated up to 10^-9 dilution for each drug.

7. From the maintained stock cultures of required organisms, 5microliter was taken and added into 2ml of BHI broth.

8. In each serially diluted tube 200microliter of above culture suspension was added.

The tubes were incubated for 24 hours and observed for turbidity.

The MIC of the synthesized compounds against strains was presented in table no. 2.

RESULT AND DISCUSSION:

The various title compounds were prepared from phenyl acetic acid by various steps. The structures of compound were confirmed by spectra and analytical studies. All the compounds synthesized matched with spectral data.

Antibacterial activity:

Synthesized compounds from scheme-I, 3-(4-substitutedphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)-1, 3-thiazolidin-4-one and synthesized compounds from scheme-II,1-methyl-N'-[(E)-(4-nitrophenyl)methylidene]-1H-benzimidazole-2-carbohydrazide From scheme-I subjected compounds like BA-CL, BA-NI, BA-SN, BA-SA and from scheme-II subjected compounds like AH-CL, AH-NI and AH-BR were tested for antibacterial activity.

The tested compounds BA-SA, BA-SN, AH-BR and AH-CL have shown significant antibacterial activity at concentration (6.25µg/ml) and (3.12 µg/ml) against E. coli, E.fecalis, Klebsiella and S. aureus. While other compounds shown moderate activity. Ciprofloxacin was used as standard drug.

Antifungal activity:

Synthesized compounds from scheme-I 3-(4-substitutedphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)-1, 3-thiazolidin-4-one and synthesized compounds from scheme-II,1-methyl-N'-[(E)-(4-nitrophenyl)methylidene]-1H-benzimidazole-2-carbohydrazide From scheme-I subjected compounds like BA-CL, BA-NI, BA-SN, BA-SA and from scheme-II subjected compounds like AH-CL, AH-NI and AH-BR were tested for antifungal activity.

The tested compounds BA-SA, BA-SN, BA-CL and AH-CL have shown significant antifungal activity (12.5 µg/ml), (6.25 µg/ml) and (3.12 µg/ml) against C. albicans and A. niger. While other compounds shown moderate activity. Flucanazole was used as standard drug.

ACKNOLEDGEMENT:

Author thanks to Dr. A. D. Taranalli Principal, K.L.E.U’s college of pharmacy, Belgaum, for his kind help and encouragement during our research work.

REFERENCES:

1. Valdez-Padilla David , Rodriguez-Morales Sergio , Hernandez-Campos Alicia , Hernandez-Luis Francisco, Yepez-Mulia Lilian , Tapia-Contreras Amparo , Castillo Rafael, Synthesis and Antiprotozoal Activity of Novel 1-MethylBenzimidazole derivatives, Bioorganic and Medicinal Chem., 17, 1^st Jan 2009; 1724-1730.

2. Verma Amit, Saraf Shailendra K., 4-Thiazolidinone - A biologically active scaffold, European Journal of Medicinal Chemistry, 43 (2008): 897-905.

3. Agarwal RK, Sharma S., A new synthesis of substituted benzimidazole-2-ones, Ind. J. Chem., 1982: 21 (b): 967-968.

4. Nandha Kumar R, Suresh T and Mohan P S, Vilsmeier-Haack reaction on quinaldines, Indian Journal of Chemistry, Vol.42-B, September, 2003; 2069-2073.

5. Gadaginamath G.S. , Kavali R. R., Shyadligeri A.S. and Oddamani H.P, Synthesis and antimicrobial activity of novel 3-thiazolyl /imidazo(2,1-b)-1,3,4-thiadiazolyl/ anilinoacetyl / phenoxyacetyl indole derivatives, Indian Journal of Heterocyclic Chemistry , Vol.9,July- September, 1999; 33-38.

6. Shingare M., Synthesis of new thiazolidinones from pyrimidine schiff bases and their biological activity, Ind. J. Chem, 1983; 22(B): 714-715.

7. Xin –ping Hui, thang –Hu-chu and Zi Zhang, Indian J. Chem; 2002; 41B:2176-2179.

8. Organic analysis; ELBS and Logman Group Ltd., 1971, (3) , 580.

9. Tony K, Preparation of carboxylic acid from aldehyde oxidation using potassium permanganate. J. Med. Chem, 1994, 37, 1696.

10. L Ziemnik Schlater, Fischer D., Esterification of various aromatic ring substituted benzoic acids catalyzed by microwave energy, Chemistry Research, 1999, 169.

11. Ansari KF, Lal C, Synthesis, physicochemical properties and antimicrobial activity of some new benzimidazole derivatives, Eu. J. Med. Chem, 2009, 44: 4028-33.

12. Afaf H., El-masry, Fahmy H. H. and Ali Abdelwahed S. H., Synthesis and antimicrobial activity of some new benzimidazole derivatives. Molecules 2000:5; 1429–38.

Received on 24.06.2011 Modified on 30.06.2011

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 249-252