Synthesis and Antimicrobial Studies Of Some Novel Benzimidazole Derivatives

Vijaya B Reddy, Rajeev K Singla Varadaraj Bhat G^* and Gautham G Shenoy

Department of Pharmaceutical Chemistry, MCOPS, Manipal University, Manipal-576104, State- Karnataka, India

*Corresponding Author E-mail: varad.g@manipal.edu

ABSTRACT

A series of 2-substituted phenoxymethyl benzimidazoles and 1-alkyl-2-substituted phenoxymethyl benzimidazoles (R1X_1-4- R8X_1-4) were prepared. The structures of the benzimidazole derivatives were confirmed on the basis of spectral data. The compounds were screened for their in vitro antibacterial, antifungal, antituberculars activity using bacterial strains (E. Coli, Pseudomonas aeruginosa and Coagulase positive Staphylococcus aureus {COPS}), fungal strains (Candida albicans and Aspergillus niger) and Mycobacterium tuberculosis H₃₇RV strain. The results of preliminary screening showed that all synthesized 2-(substituted phenoxymethyl) 1-H benzimidazoles and 1-alkyl-2-(substituted phenoxymethyl) benzimidazoles has potential antifungal activity against Candida Albicans, but only few compounds showing activity against Aspergillus niger. Similarly all molecules showed good antibacterial activity against E.coli and Pseudomonas aeruginosa, but very few were resistance to Staphylococcus aureus. And all the synthesized compounds showed good activity against Mycobacterium tuberculosis even at 1 mcg/ml concentration.

KEY WORDS: Benzimidazole, Antibacterial, Antifungal, Antitubercular, Phenoxymethyl.

INTRODUCTION:

In recent years, attention has increasingly been given to the synthesis of benzimidazole derivatives as a source of new antimicrobial agents. The synthesis of novel benzimidazole derivatives remains a main focus of medicinal research. Recent observations suggest that substituted benzimidazoles and heterocycles, which are the structural isosters of nucleotides owing fused heterocyclic nuclei in their structures that allow them to interact easily with the biopolymers, possess potential activity with lower toxicities in the chemotherapeutic approach in man^[1,2] . Moreover, these fused heterocylces were distinctively studied for their antitumor, antiviral and antimicrobial activities as the new nonnucleoside topoisomerase I poisons, human immunodeficiency virus-1 reverse transcriptase inhibitors and /or potent DNA gyrase inhibitors ^[3-5]. In addition, benzimidazole derivatives have played a crucial role in the theoretical development of heterocyclic chemistry and are also used extensively in organic synthesis.   

Encouraged by the diverse biological activities of benzimidazoles and phenolic compounds, it was decided to prepare a new series of 1-alkyl-2-substituted phenoxymethyl benzimidazoles (R1X_1-4- R8X_1-4) thus bringing both types of compounds together in a single molecule, and to screen the newly synthesized compounds for their antimicrobial activities.

The classical synthesis of the title compounds involves the acid-catalyzed condensation of substituted Phenoxy acetic acid and o- Phenylenediamine to give 2-substituted phenoxymethyl benzimidazoles which on reaction with alkyl-iodide in alkaline medium give the corresponding 1-alkyl-2-substituted phenoxymethyl benzimidazoles.

EXPERIMENTAL:

All melting points were determined in an open capillary and are uncorrected. The infrared (IR) spectra were recorded on a FTIR-8300 Shimadzu spectrometer using potassium bromide pellets. The proton nuclear magnetic resonance (1H-NMR) spectra were recorded and studied by IICT, Hyderabad  (at 200 MHz) using tetramethylsilane (TMS) as the internal standard. The Mass spectral study was performed by IICT, Hyderabad. All reagents were of the highest purity commercially available. The chemical shifts are expressed in part per million (ppm) downfield

TABLE I

CODE	Mol. Formula	% Yield	M.P. (^oC)	Rf	λmax(nm)	IR(cm^-1)
R1X1	C14H12N2O	70	180	0.81	281.6	3456,3045,2916,2869,1442,1213,1072,746
R1X2	C15H14N2O	78	105	0.84	281.3	3053,2923,2858,1494,1242,1070,750
R1X3	C16H16N2O	70	124	0.92	281.1	3050,2916,2869,1442,1218,1072,746
R1X4	C17H18N2O	65	140	0.95	281.3	3049,2912,2860,1438,1222,1060,746
R2X1	C14H11N3O3	65	170	0.71	281.5	3357,3114.8,2848,1508,1340 ,1255,1110,850,745
R2X2	C15H13N3O3	76	120	0.83	281.4	3084,2923,2852,1591,1504,1340,1255,1110,880,752
R2X3	C16H15N3O3	68	140	0.87	281.7	3085,2920,2842,1591,1506,1340,1253,1109,850,752
R2X4	C17H17N3O3	50	160	0.86	281.1	3085,2923,2852,1591,1438,1340,1253,1109,848,752
R3X1	C14H11N3O3	55	250	0.66	281.4	3448,2715,1527,1461,1346,1064,856,736
R3X2	C15H13N3O3	75	195	0.69	281.4	3182,3031,2927,1523,1450,1346,1245,1068,817,736
R3X3	C16H15N3O3	62	230	0.72	281.4	3057,2904,2709,1529,1485,1348,1247,1062,862,734
R3X4	C17H17N3O3	48	240	0.70	281.4	3057,2906,2840,1529,1485,1384,1247,1062,862,734
R4X1	C14H11ClN2O	70	220	0.83	281.3	3340,2930,2870,1732,1485,1230,1076,829,736,644
R4X2	C15H13ClN2O	80	195	0.87	281.5	3082,2914,2815,1490,1244,1064,831,740,655
R4X3	C16H15ClN2O	68	210	0.77	281.6	3020,2916,2840,1490,1244,1066,831,740,655
R4X4	C17H17ClN2O	56	215	0.77	281.6	3018,2930,2855,1488,1234,1066,831,740,655
R5X1	C14H11BrN2O	67	260	0.77	281.6	3317,3050,2931,2837,1485,1433,1218,1049.809,596
R5X2	C15H13BrN2O	76	140	0.81	281.6	3030,1483,1448,1232,1058,823,748,561
R5X3	C16H15BrN2O	61	160	0.79	281.4	3042,2921,2852,1485,1434,1220,1051,817,738,596
R5X4	C17H17BrN2O	52	180	0.79	281.6	3073,2942,2858,1431,1222,1045,813,601
R6X1	C15H14N2O	68	170	0.79	281.6	3375,3043,2920,2854,1616,1438,1238,1072,825,748
R6X2	C16H16N2O	72	110	0.84	281.7	3085,2943,1508,1438,1234,1068,752
R6X3	C17H18N2O	64	115	0.83	281.7	3093,2927,2895,1510,1452,1234,1068,792,750
R6X4	C18H20N2O	55	150	0.83	281.6	3043,2920,2858,1512,1238,1072,825,748
R7X1	C15H14N2O	52	194	0.77	281.3	3409,3035,2934,2842,1660,1446,1265,1072,744
R7X2	C16H16N2O	73	140	0.81	281.2	3042,2923,2823,1587,1446,1263,1074,775,738,690
R7X3	C17H18N2O	63	155	0.83	281.3	3051,2922,2821,1587,1444,1263,1074,775,738,690
R7X4	C18H20N2O	50	180	0.79	281.3	3029,2923,2821,1587,1444,1263,1074,775,738,688
R8X1	C15H13ClN2O	51	198	0.79	281.5	3446,3025,2920,2804,1585,1245,1041,918,867,642
R8X2	C16H15ClN2O	79	110	0.84	281.4	3186,2927,1573,1469,1411,1249,1099,914,759,642
R8X3	C17H17ClN2O	67	140	0.84	281.2	3055,2923,1581,1461,1249,1041,929,821,752,642
R8X4	C18H19ClN2O	54	160	0.88	281.3	3027,2922,1568,1475,1242,1034,912,864,748,642

from the internal standard; the coupling constants are in Hz, and signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). High Performance Thin layer chromatography(HPTLC) analytical separation were conducted with silica gel 60 F-254 (Merck) plates of 0.25 mm thickness using Shimadzu wincats software eluted with ethyl acetate: n-Hexane(35:65) and were visualized with UV (254 nm) or iodine to check the purity of the compounds.

Preparation of 2-[(4-Chloro-3-methylphenoxy) methyl]-1H-benzimidazole (R8X1):

To a mixture of 0.01M of the 4-Chloro-3-methylphenol and 3.5ml of 33% sodium hydroxide solution, added 0.01M of 50% chloroacetic acid solution. The mixture was heated on a water bath for 1 hour. After cooling, the mixture was acidified with dilute Hydrochloric acid and extracted by shaking with 25ml of 5% Sodium carbonate solution. The extract was acidified with dilute Hydrochloric acid. The 4-chloro-3-methylphenoxyacetic acid separates out and recrystallized from aqueous ethanol. A mixture of  4-Chloro-3-methylphenoxyacetic acid (0.01 M)  and o-Phenylenediamine(0.01 M) was  heated in hydrochloric acid at 100oC for 4Hrs, cooled and poured into ice cold 10% aqueous sodium carbonate. The solid product, which separated out, was collected, washed with water and recrystallized from methanol. M.P.198 °C. Yield 51%. EIMS: 272 m/z value.  IR (KBr): 3446(NH str.), 3020, 3110(Ar. Ring str.), 2992(CH3 str.), 2796(CH2 str.), 1568(C=N str.), 1475, 1423(Ar. C=C str.), 1242.1039(C-O-C str.), 864,692(Ar. meta substitution), 642(C-Cl str.), 748(Ar. bending).  1HNMR (DMSO); 2.3(d-CH2), 2.6(s-CH3), 4.1(s-NH), 6.6(m-1Ar-H), 6.8(s-1ArH), 6.9(d-1ArH), 7.2(m-1ArH), 7.3(d-1ArH) confirms the compound R8X1.

Preparation of 2-[(3-Nitrophenoxy) methyl]-1methyl-benzimidazole (R3X2):

3-Nitrophenoxy acetic acid was synthesized from 3-Nitrophenol as explained above. A mixture of 3-Nitrophenoxy acetic acid (0.01 Ml) and o-Phenylenediamine (0.01 M) was heated in hydrochloric acid at 100oC for 4hrs, cooled and poured into ice cold 10% aqueous sodium carbonate. The solid product, which separated out, was collected and washed with water and recrystallized with methanol. Dissolved Took 0.01mol of 2-[(3-Nitrophenoxy) methyl]-1H-benzimidazole in a dry N, N-Dimethylformamide and dry methanol. After 10 minutes of stirring at room temperature methyl iodide (0.01 M) was added in 2-3 portions and the resultant suspension was then

TABLE II. Antibacterial activity of the novel compounds Zone of inhibition in mm

CODE	E. coli			P. aeruginosa			COPS
	1µg/ml	10µg/ml	100µg/ml	1µg/ml	10µg/ml	100µg/ml	1µg/ml	10µg/ml	100µg/ml
R1X1	16	17	17	14	14	16	R	R	R
R1X2	15	19	21	13	14	17	R	R	R
R1X3	14	17	18	13	14	17	14	R	R
R1X4	14	15	22	16	19	21	14	R	R
R2X1	16	19	23	12	13	14	14	R	R
R2X2	13	17	21	11	14	17	12	R	R
R2X3	R	14	16	14	15	18	R	R	R
R2X4	11	14	15	12	17	23	R	R	R
R3X1	11	14	16	14	16	19	14	R	R
R3X2	R	16	14	16	16	21	21	R	R
R3X3	R	14	14	12	14	16	R	R	R
R3X4	R	14	15	12	16	16	R	R	R
R4X1	16	19	20	13	14	16	R	R	18
R4X2	14	16	20	11	12	21	R	11	16
R4X3	17	13	21	12	14	14	R	12	24
R4X4	13	18	19	12	12	16	R	R	11
R5X1	15	15	16	13	14	15	R	R	11
R5X2	14	15	23	13	16	19	R	R	12
R5X3	14	17	19	12	13	15	R	13	14
R5X4	14	17	19	11	13	23	R	R	14
R6X1	20	20	21	11	14	26	R	R	R
R6X2	12	22	24	13	19	19	R	13	R
R6X3	12	20	22	14	16	17	R	R	R
R6X4	15	14	24	14	14	14	R	13	24
R7X1	14	15	28	12	14	14	R	R	15
R7X2	14	18	16	13	13	18	R	R	12
R7X3	15	15	21	11	15	17	R	18	12
R7X4	15	17	19	13	16	19	R	14	31
R8X1	14	16	18	12	12	14	R	16	18
R8X2	15	19	19	11	12	13	R	R	12
R8X3	16	18	20	13	14	17	R	R	12
R8X4	17	19	22	12	13	22	R	R	14

STANDARD: GENTAMYCIN 100µg/ml (E. coli:21)(P. aeruginosa: 19)(COPS:21)

Figure 1: Reaction Scheme

stirred for 8Hr.  The reaction mixture was poured into an ice bath (15 ml) and left over night. The product was precipitated, which was filtered and washed with cold water. The product was recrystallized from methanol. M.P: 195°C. Yield 75%. IR (KBr): 3181, 3031(Ar. Ring str.), 2927(CH3 str.), 1523, 1346(C=N str.), 1450(Ar. C=C str.), 1245, 1068(C-O-C str.), 817.8(Ar. Meta substitution), 736(Ar. bending). 1HNMR (DMSO): 2.3(s-CH3), 5.7(d-

TABLE III. Antifungal activity of the novel compounds Zone of inhibition in mm

CODE	Candida albicans			Aspergillus niger
	1 µg/ml	10 µg/ml	100 µg/ml	1 µg/ml	10µg/ml		100µg/ml
R1X1	12	12	24	R	R		R
R1X2	12	13	16	R	R		R
R1X3	13	14	15	R	R		10
R1X4	11	13	14	R	11		15
R2X1	11	13	13	R	R		R
R2X2	13	13	15	R	R		R
R2X3	12	16	16	R	R		R
R2X4	13	15	17	R	R		R
R3X1	13	16	19	R	R		R
R3X2	12	13	15	R	R		R
R3X3	15	17	18	R	R		R
R3X4	11	16	17	R	R		R
R4X1	12	14	14	11	27		27
R4X2	11	15	17	13	13		14
R4X3	12	16	17	R	20		20
R4X4	11	14	16	R	14		19
R5X1	11	13	14	R	R		R
R5X2	11	11	13	R	R		R
R5X3	13	13	15	R	R		R
R5X4	14	14	16	R	R		R
R6X1	13	14	16	R		R	R
R6X2	13	14	15	R		R	R
R6X3	13	15	15	R		11	12
R6X4	13	14	16	11		12	13
R7X1	11	11	15	R		R	R
R7X2	11	13	17	R		R	R
R7X3	11	14	20	R		R	15
R7X4	11	15	17	11		17	R
R8X1	11	14	14	11		22	27
R8X2	12	14	14	12		29	29
R8X3	11	13	15	11		13	13
R8X4	13	14	19	12		15	15

STANDARD: Fluconazole 100µg/ml (C. albicans: 20);(A. niger:19)

CH3), 7.4(d-2ArH), 7.6(m-2ArH), 7.7(d-2ArH), 7.9(d-2ArH) confirms the presence of compound R3X2.

All the 32 compounds were synthesized by the same procedure and their formulas, melting points, yields and analytical data are shown in Table I.

RESULTS AND DISCUSSION:

The structures of compounds R1X1-4- R8X1-4 were confirmed on the basis of spectral analysis. As seen by the UV spectral analysis, the benzimidazoles showing a characteristic peak at 281 nm. The IR spectrum of R₁X_1-4- R₈X_1-4 exhibited a band due to C =N str. (1438–1568 cm^–1), C-O-C str. (1242,1039 cm^–1) showing the formation of phenoxymethyl benzimidazole derivatives, further  N–H Str.(3300-3450 cm^–1 ) on in R_1-8X₁ showing the substitution at 1N-benzimidazole in rest of the derivatives. Further, in their 1H-NMR (DMSO) spectrum, the appearance of a signal at 2.6 (s- CH3) in R8X₁ showing the presence of methyl substitution at aromatic ring and 4.1 (s-NH) in R8X₁ showing the presence of unsubstituted Nitrogen in aromatic ring

which were absent in case of R₃X₂ ,  further confirms by the peak at 5.7(d-CH3) in R3X2 which was methyl substitution on 1N-benzimidazole ring.

ANTIMICROBIAL STUDY:

Antibacterial activity:

The target molecules were tested for antibacterial activity against the variety of test organisms Escherichia coli, Pseudomonas aeruginosa (gram-negative bacteria) and Coagulase positive Staphylococcus aureus (COPS) (gram-positive bacteria) by the punch well and Disc diffusion methods on the Muller Hinton agar medium using Gentamycin(100µg/ml) as the standard drug. The antibacterial screening was carried out with three different concentration of the synthesized novel molecules i.e. 1µg/ml, 10µg/ml, 100µg/ml using 50% Dimethylformamide as solvent. The screening results indicate that all compounds show promising activity  against E. coli and P. aeruginosa and fewer compounds against COPS at all concentration levels (SEE TABLE II). The compounds showed activity which is comparable with Gentamycin against E. coli in increasing order of R2X1>R6X1>R4X1>R8X1>R5X1>R1X1. This shows that para position may be important to exhibit significant activity against E. coli. It is quite evident from the above sequence that the compound R2X1 which contain nitro group (strong electron withdrawing group) at para position is highly active. The activity decreasing as the electron withdrawing ability of the substituent decreased. The role of substituent at position 1 didn’t had any effect on the activity against E. coli for all derivatives except the compounds R2X1,R2X2,R2X3,R2X4, which showed decrease in activity as the chain length of the X increased.

The derivatives R1X4, R2X4, R3X1,R3X2, R4X2, R5X4, R6X1, R7X4 and R8X4 exhibited good activity in comparison to the Gentamycin against P. aeruginosa. R6X1 showed highest activity than the standard.

The compounds R4X3, R6X4, R7X4 showed higher activity than the standard against Gram positive COPS. Some of the compounds exhibited a non-linearity between the concentration used and the zone of inhibition.

Antifungal studies:

The target molecules were tested for antifungal activity against the variety of test organisms Candida albicans and Aspergillus niger by the punch well and Disc diffusion methods on the Sabourads Dextrose Agar Broth using Fluconazole(100 µg/ml) as the standard drug. The antifungal screening was carried out with three different concentration of the synthesized novel molecules i.e. 1µg/ml, 10µg/ml,100µg/ml using 50% Dimethylformamide as solvent. The screening results indicate that all compounds show promising activity against Candida albicans, of which only R1X1, R3X1, R3X3, R7X3 and R8X4 showed comparable activity with the standard Fluconazole. (SEE TABLE III).

Fewer compounds exhibited activity against Aspergillus niger, R1X3, R4X1, R8X1 and R8X2 showed higher activity than the standard Fluconazole.

Some of the compounds exhibited a non-linearity between the concentration used and the zone of inhibition.

Antituberculars study:

The target molecules were tested for antifungal activity against the Mycobacterium tuberculosis H37RV strain using Lowenstein-Jensen Medium and Isoniazid and Rifampicin as standard drug. The media was prepared as per the procedure recommended by the international union against tuberculosis.  All the compounds exhibited very good anti tubercular activity even at 1µg/ml against M. tuberculosis strain H37RV compared to the standards viz., Rifampicin and Isoniazid. (SEE TABLE IV)

CONCLUSION:

Most of the synthesized compounds exhibited good activity against the studied set of microorganisms. Since a fewer species have been used in this study, it is warranted to screen these compounds with varied species and resistant strains. All the compounds showed very good antituberculars activity even at less concentration. Hence, it is evident that the 2-Phenoxymethylbenzimidazoles are potent candidates for extensive Antitubercular studies.

ACKNOWLEDGEMENT:

Our deepest appreciation and heartfelt thanks to IICT, Hyderabad and Dr. Sugandhi Rao, Head of Department of Microbiology for rendering their help in spectral studies and screening respectively.

TABLE IV: Antitubercular study of novel compounds

CODE	Mycobacterium tuberculosis H37RV
	1µg/ml	10µg/ml	100µg/ml
R1X1	+	+	+
R1X2	+	+	+
R1X3	+	+	+
R1X4	+	+	+
R2X1	+	+	+
R2X2	+	+	+
R2X3	+	+	+
R2X4	+	+	+
R3X1	+	+	+
R3X2	+	+	+
R3X3	+	+	+
R3X4	+	+	+
R4X1	+	+	+
R4X2	+	+	+
R4X3	+	+	+
R4X4	+	+	+
R5X1	+	+	+
R5X2	+	+	+
R5X3	+	+	+
R5X4	+	+	+
R6X1	+	+	+
R6X2	+	+	+
R6X3	+	+	+
R6X4	+	+	+
R7X1	+	+	+
R7X2	+	+	+
R7X3	+	+	+
R7X4	+	+	+
R8X1	+	+	+
R8X2	+	+	+
R8X3	+	+	+
R8X4	+	+	+
Rifampicin	+	+	+
Isoniazid	+	+	+

+ indicates no growth or very little growth of M. tuberculosis organism i.e. antimycobacterial activity.

REFERENCES:

1. Haugwitz RD. et al. Antiparasitic agents Synthesis and antihelmintic activities of novel 2- substituted isothiocyanatobenzoxazoles and benzimidazole. J. Med. Chem. 25, 1982, 969-974.

2. Hisano T. et al. Synthesis of benzoxazloes, benzothiazoles and benzimidazoles and evaluation of their antifungal, insecticidal and herbicidal activities. Chem. Pharm. Bull. 30, 1982, 2996-3004.

3. Hubschwerlen, C. et al., Pyrimido [1,6-a]benzimidazoles: New class of DNA gyrase inhibitors, J. Med. Chem. 35, 1992, page no. 1385.

4. Kim, J.S. et al., Structure activity relationships of benzimidazoles and related heterocylces as topoisomerase I poison. Borg. Med. Chem., 4, 1996, 621-630.

5. Shi, D.F. et al., Synthesis of 2-(4-aminophenyl) benzothiazoles and evaluation of their activities against breast cancer cell lines in vitro and in vivo. J. Med. Chem. 39, 1996, 3375-3384.

6. S. Wirsching et al., Journal of bacteriology. 182(2000) page no.400-404.

7. Ilkay oren et al., “ Synthesis and antimicrobial activity of some novel2,5- and/or 6- substituted benzoxazole and benzimidazole derivatives”, European journal of pharmaceutical sciences,7,1998,153-160

8. Vera Klimesova et al., “Synthesis and preliminary evaluation of benzimidazole derivatives as antimicrobial agents”, Eur. J. Med. Chem, 37, 2002, 409-418.

9. Pawar.N.S et al.,” Studies of antimicrobial activity of N-alkyl and N-acyl 2-(4-thiazolyl)-1H-benzimidazoles”, Eur. J. Pharmaceutical Sciences, 21, 2004, 115-118.

10. Gulgan Ayhan-Kilergil et al.,”Synthesis and antimicrobial activities of some new benzimidazole derivatives” IL Farmaco, 2003, 58, 1345-1350.

11. Hakan Goker et al., “Synthesis of some new benzimidazolcarboximides and evaluation of their antimicrobial activity”, IL farmaco, 1998, 415-420.

12. Asutosh Bhatt.k et al., “Synthesis of benzimidazole derivatives and their antimicrobial and antifungal activities”. Indian Journal of Heterocycli Chemistry, 13, Oct-Dec 2003, 187-188.  

Received on 30.01.2009        Modified on 20.03.2009

Accepted on 08.05.2009        © AJRC All right reserved

Asian J. Research Chem.  2(2): April.-June, 2009 page 162-167