INTRODUCTION:

Historically the first benzimidazole was prepared in 1872 by Hoebrecker who obtained 2, 5 or 2, 6- dimethyl benzimidazole by the reduction of 2-nitro-4-methylacetanilide. Several years later Ladenburg obtained the same compound by refluxing 3, 4-diaminotoluene with acetic acid. The benzimidazoles are known also as Benzimidazoles or benzoglyoxalines. Thus, benzimidazole according to this nomenclature would be called methenyl-o-phenylenediamine and 2-methylbenzimidazole.The Benzimidazole ring is an important pharmacophore in modern drug discovery. A large variety of 2-substituted benzimidazoles have been found to possess anti-inflammatory¹,antispasmodic²,antihistaminic³, antimicrobial^4,5,6,antitumour⁷, anticancer⁸and cyclooxygenase inhibitors⁹ activities. In addition benzimidazoles have also been investigated for their analgesic¹⁰ and anti-tubercular activity¹¹.

Scheme

O Phenylene diamine	Carboxylic acids			2-substituted benzimidazole

2-substituted benzimidazole		P-dichloro benzene	1-p(chloro phenyl)2-substituted benzimidazole

MATERIAL AND METHOD:

Experimental work:

All the chemicals used were produced from Aldrich and purity of starting materials used for reactions was confirmed by checking their melting point or boiling point and by thin layer chromatography. All the reactions were monitored using thin layer chromatography. The appropriate mobile phases (solvent systems) as applicable were developed using ‘silica gel G’ as stationary phase. Melting points were determined in open capillary tube and are uncorrected. FT-IR (KBr) spectra were recorded on Jasco FTIR-410 Spectrophotometer. ¹HNMR spectra of synthesized compounds were recorded on Bruker Spectrophotometer at 300 MHz frequency in Deuterated chloride (CDCl₃) as well as dimethyl sulfoxide (DMSO) using tetramethylsilane (TMS) as internal standard (chemical shift δ in ppm). Purity of the compounds was checked on ‘Silica Gel G’ coated on laboratory micro slide prepared by dipping method or pre-coated plates, eluent was the mixture of different polar and non-polar solvents in varying proportions and detections was done either by observing in ultraviolet (UV) light or exposure to iodine vapors as required. The absence of thin layer chromatography (TLC) spots for starting materials and appearance of new TLC spot at different R_f value ensured the completion of reaction. The products of all the reactions were purified initially by different workup processes to remove unreacted starting materials if any and then by recrystallization using suitable solvents.The absences of any impurity of starting material or possible bi-product were ensured by performing qualitative organic analytical tests for various functional groups. All the compounds were prepared by conventional method as outlined in the scheme.

Table No. 1:- Physicochemical properties of compound 1A to 1G.

Comp. No.	R	Molecular formula	MP °C	Practical Yield (gm)	% yield	RF value	Mobile phase	Solvent for recrystalisation.
1A		C₁₉H₁₄N₃cl	160-164	2.2	43	0.87	EA :n-H 7:3	Ethanol
1B		C₂₁H₁₇N₂cl	155-159	3	54.74	0.91	EA :n-H 7:3	Ethanol
1C		C₁₉H₁₃N₂O Cl	210-214	2.3	40	0.65	EA :n-H 7:3	Ethanol
1D		C₁₅H₁₁N₂O Cl	103-107	1.1	23	0.96	EA :n-H 7:3	Ethanol
1E		C₁₉H₁₁N₄O₄Cl	230-234	4	51	0.87	EA :n-H 7:3	Ethanol
1F		C₁₉H₁₂N₂Cl₂	160-164	3.8	65	0.91	EA :n-H 7:3	Ethanol
1G		C₁₉H₁₂N₂Cl₃	180-184	4.4	59	0.65	EA :n-H 7:3	Ethanol

EA= Ethyl Acetate : n-H= n-Hexane.

Synthesis of 2- substituted benzimidazole (I)

The O-phenylenediamine (4g, 0.04 mole) was condensed with substituted carboxylic acids (1A-1G) (0.03 mole) in 50 ml 4N HCl. The reaction mixture was stirred for about 4 hr with magnetic stirrer at 80 °C. The compounds were precipitated by adding concentrated ammonia solution, filtered through suction and washed with cold water. Compounds (1A-1G) were recrystalized from water and ethanol.

Synthesis of 1(p- chloro phenyl) 2-substituted benzimidazole (II)

A mixture of 2-substituted benzimidazoles (0.02 mole) and p-dichlorobenzene (2.92 g, 0.002 mole) dissolved in ethanol (20 ml) in the presence of a little quantity of sodium hydroxide (2g) and tetrahydrofuran (40 ml) stirred for 10-16 hr at 40 °C. The precipitated product was filtered and excess solute was removed by distillation. The crude product was washed with water extracted with ethyl acetate and finally recrystalized from water and ethanol. The respective synthesized 2-substituted benzimidazole has been shown in table 1.

Description: EA= Ethyl Acetate : n-H= n-Hexane.

Anti-tubercular screening:

Anti-tubercular activity was evaluated against Mycobacterium tuberculosis H37 RV using Microplates Alamar blue assay (MABA) method^12,13. Anti-tubercular susceptibility test was performed in black, clear-bottomed, 96-well microplates in order to minimize background fluorescence. Initial drug dilutions were prepared in dimethyl sulfoxide and subsequent two-fold dilutions were performed in 0.1 ml of 7H9GC media in the microplates. An aliquot (100 μl) of 2000CFU/ml of M. tuberculosis H37 RV were added to each well of 96-well micro liter plate containing test compounds. Three control well plates containing drug and medium, bacteria and medium, and medium only were also prepared. All micro liter plates were incubated at 37 °C for seven days. At day 7 of incubation, Alamar Blue dye solution (20 μl Alamar Blue solution and 12.5 ml of 20 % Tween 80) was added to all the wells and the plates re-incubated at 37 °C for 24 hr.

M. tuberculosis H37RV was considered as resistant if there were colour changes (pink or deep red to violet) in the test samples bottle in question greater than in the 10 % diluted growth control on the same day. The bottles that did not showed any colour change and remain the same (no development of pink colour), were considered as M. tuberculosis H37RV sensitive to the test sample.

Those test samples were further incubated for 10 days and 14 days for confirmation of results. On the 10th day and 14th day again 0.5ml of a mixture of three reagents (25 µl of concentrated HCl, 50 µl of 2% sulphanilamide and 50 µl of 1% n-1-napthyl-ethylenediamine dihydrochloride) was added in all these bottles. If bottles did not show any colour change and remain the same then it was confirmed that M. tuberculosis H37RV was sensitive to that test samples.

CHARACTERIZATION OF SYNTHESIZED COMPOUNDS:-

1A = of1 (2-amino phenyl, 4- chloro phenyl)-benzimidazole. IR(KBr) 1689(Ar-CH),3288-3476 (NHstr.), 2985 (C-H) 1292 (C-N). 1H NMR ( DMSO d6) 2.4 (2H, R-NH2), 7.1 to 8. 3H, (Ar-H)10.5 (1H, -CHO).m/z175

1B = 1-(2- cinnamyl phenyl,4- chloro)- benzimidazole, IR(KBr) 1688 (Ar-CH), 3345 (NHstr.), 2924 (C-H) 1256 (C-N), 583 (C-Brstr.) 1H NMR ( DMSO d6) 1.1, 2.5(1H, CH-CO)4.5 (2H, -CH=CH-), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z263.

1C = 1-(2- hydroxyl phenyl, 4- chloro) - benzimidazole. IR(KBr) 1266(Ter. Amine C-N) 1689 (Ar-CH), 3345 (NHstr.), 2924 (C-H) 1256 (C-N), 1H NMR ( DMSO d6) 1.1, 2.5 (1H, CH-CO) 4.5 (2H, -CH=CH-) 3.1 to 3.6 (6H, N-R2 ), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z 343.

1D= 1-(2- carboxyl phenyl,4- chloro)- benzimidazole, IR(KBr) 3366(O-Hstr) 1689 (Ar-CH), 2960 (C-H) 1286 (C-N), 1H NMR ( DMSO d6) 1.1, 2.5 (1H, CH-CO) 4.5 (2H, -CH=CH-) 3.1 to 3.6 (6H, N-R2 ), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z243.

1E = 1-(3, 5 dinitro phenyl, 4- chloro) - benzimidazole, IR(KBr) 3366 (O-Hstr) 1689 (Ar-CH), 2960 (C-H) 1286 (C-N), 1H NMR ( DMSO d6) 1.1, 2.5 (1H, CH-CO) 4.5 (2H, -CH=CH-) 3.1 to 3.6 (6H, N-R2 ), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z 343.

1F = 1-(2, 4 –Dichloro phenyl, 4-chlorophenyl)-benzimidazole, IR(KBr) 3366 (O-Hstr) 1689 (Ar-CH), 2965 (C-H) 1186 (C-N), 1H NMR ( DMSO d6) 1.1, 2.5 (1H, CH-CO) 4.5 (2H, -CH=CH-) 3.6 (6H, N-R2 ), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z 234.

1G = 1-(2-bromo phenyl, 4- chloro phenyl)-Benzimidazole, IR(KBr) 3366 (O-Hstr) 1689 (Ar-CH), 2965 (C-H) 1186 (C-N), 1H NMR ( DMSO d6) 1.1, 2.5 (1H, CH-CO), 3.6 (6H, N-R2 ), 7.1 to 8. 3H, (Ar-H) 10.5 (1H, -CHO).m/z534.

RESULT AND DISCUSSION:

Anti-tubercular activity

The compound 1A, 1G (200µ/ml) did not show development of pink color after addition of 0.5ml of a mixture of three reagents (25 µl of concentrated HCl, 50 µl of 2% sulphanilamide and 50 µl of 1% n-1-napthyl-ethylenediamine dihydrochloride). The compound 1A, 1G was found to possess significant anti tubercular activity as compared with Rifampicin.

CONCLUSION:

An efficient synthesis of different novel N-substituted and 2-substituted derivatives by the Phillips Condensation by condensing the o-phenylenediamine and carboxylic acid derivatives in 4N HCl. N-substituted derivatives have been synthesized by reaction with alkyl/aryl halide in presence of base sodium hydroxide. The compound 1A and 1F showed significant anti tubercular activity as compared with Rifampicin.

ACKNOWLEDGEMENT:

We are also thankful to the Principal Prof Dr. C S Magdum, Vice-Principle Dr. S K Mohite and Management of Rajarambapu College of pharmacy, Kasegaon for providing the necessary facilities to carry out this work.

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Received on 05.11.2013 Modified on 15.12.2013

Asian J. Research Chem. 7(2): February 2014; Page 137-140