Synthesis and Anti-Tubercular Activity of Substituted Phenylpyrazole having Benzimidazole Ring

 

Prafulla Sabale¹*, Dhiraj Bhagwat¹, Vidya Sabale³

¹Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University,

Nagpur-440 033 M.S.

²Dadashab Balpande College of Pharmacy, Besa, Nagpur- 440037 M.S.

 

ABSTRACT:

Infectious microbial disease remains a major problem worldwide, because microbes have resisted prophylaxis or require longer therapy. Tuberculosis is most common and often deadly infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis (M. Tb) in humans. Drugs used for tuberculosis are inadequate to address emerging challenge of treatment due to drug resistance towards the mycobacteria.  A number of synthetic azole derivatives have been class of interest now days, which inhibits the synthesis of ergosterol in cell wall. They mainly inhibit 14α-demethylase enzyme, which is also present in the mycobacterium cell wall. The present study involves synthesis of substituted benzimidazole clubbed with pyrazole derivatives as 14α-demethylase inhibitor. The synthesized compounds were confirmed by analytical and spectroscopic techniques like m.p., TLC, IR, NMR and Mass spectroscopy. The compounds with high electronegative substituent show optimal activity against tuberculosis, while compound having less electronegative substituent showed less inhibitory activity. The compound having high molecular weight also showed good anti-tubercular activity. Among all synthesized compounds, 4B showed good anti-tubercular activity.

 

 

 

INTRODUCTION:

Benzimidazole is a heterocyclic aromatic organic compound. This bicyclic compound consists of the fusion of benzene ring with imidazole. The benzimidazoles are also known as benziminazoles or benzoglyoxalines. The most prominent benzimidazole compound in nature is N-ribosyl-dimethyl benzimidazole, which serves as an axial ligand for cobalt in vitamin B₁₂¹. Recently the interest in benzimidazole chemistry has been reviewed somewhat by the discovery, the 5,6-dimethylbenzimidazole moiety which is a part of the chemical structure of vitamin B₁₂.

 

 

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-methyl acetanilide. Several years later Ladenburg obtained the same compound by refluxing 3,4-diaminotoluene with acetic acid². Benzimidazole can be synthesized by condensation of o-phenylenediamine with formic acid, or the equivalent trimethyl orthoformate^{2, 3}.

 

Benzimidazoles possess both acidic and basic characteristics, the –NH group present in benzimidazole nucleus is relatively strongly acidic and also weakly basic. They have capacity to form salts. Benzimidazole with unsubstituted –NH group, exhibits fast prototrophic tautomerism which leads to equilibrium mixtures of asymmetrically substituted compounds. The Benzimidazole scaffold is a useful structural modification for the development of molecule of pharmaceutical or biological interest. Appropriately substituted benzimidazole derivatives have therapeutic applications such as anti-ulcer, anti-hypertensive, anti-viral, anti-fungal, anti-cancer, anti-oxidant, anti-microbial, anthelmintic, anti-inflammatory, and anti-tuberculosis activity⁴.

 

Anti-tubercular activity:

Tuberculosis (TB) is a major serious global health problem all over the world⁵. TB is a common and in some cases deadly infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis (M. Tb) in humans^6,7. TB probably appeared in humans about 8000 years ago. TB is the world’s second common cause of death from infectious diseases, after AIDS. In 1993, after 111 years of Robert Koch’s discovery of M. Tb, the WHO declared TB as “a global emergency”⁸.

 

Despite of advances in TB chemotherapy, DOT’s (directly observed treatment) therapy and BCG vaccine are available. M. Tb is an obligate aerobic bacillus that divides at an extremely slow rate and it is clear that monotherapy with any agent lead to the development of resistance and clinical failure in two or five months. Hence, TB chemotherapy and DOT’s therapy involves the administration of multiple drugs since late 1960s⁹.

 

MATERIAL AND METHODS:

Chemicals were commercially available and used as received without further purification. Moisture sensitive reactions were carried out under a dry nitrogen atmosphere in vacuum oven-dried glassware. Thin-layer chromatography was performed on the precoated silica gel F₂₅₄ plates. Silica gel column chromatography was performed using silica gel 60A. Melting points were determined in open glass capillaries using a VEEGO make microprocessor based melting point apparatus having silicone oil bath and are uncorrected. IR spectra (wave numbers in cm⁻¹) were recorded on a BRUKER ALPHA-T FT-IR spectrophotometer using potassium bromide discs. ¹H NMR spectra were recorded on BRUKER AVANCE II 400 MHz instrument in CDCl₃ with TMS as internal standard. Chemical shift values were mentioned in δ, ppm. Mass spectra were recorded on Shimadzu LC MS 2010 spectrometer.

 

 

 

The synthesis of compounds was carried out as outlined in Scheme-I. The starting material was commercially available or can be easily prepared using published procedure. The 1-(substituted-1H-benzo[d]imidazol-2-yl) ethanol (1A, 1B) can be synthesized by condensation of o-phenylenediamine with lactic acid which involves cyclization reaction. In step-2, compound 1A-B was oxidized using solution of potassium dichromate in aqueous acetic acid medium at room temperature which was converted into 1-(substituted-1H-benzo[d]imidazol-2-yl) ethanone (2A, 2B). Further, in step-3 Claisen–Smith condensation reaction gave substituted chalcone derivatives (3A, 3B) by reacting compound 2A, 2B with substituted benzaldehyde in aqueous NaOH solution. Compound 1A, 1B, 2A, 2B and 3A, 3B were characterized by m.p, R_f value and characteristics peaks in IR spectrum were mentioned in Table 1 and 2. In last step chalcone derivatives (3A, 3B) undergoes cyclization with hydrazine hydrate and form final pyrazole moiety with benzimidazole (4A-D). The compounds synthesized were purified and characterized by their physical and spectral data. Among all the synthesized compounds 4B was most active compound compared with standard drug Rifampicin.

 

All derivatives were synthesized as per given procedure. All compounds were confirmed by IR, NMR and Mass spectral data and were screened for anti-tubercular activity.

 

1-(5-Nitro-1H-benzo[d]imidazol-2-yl) ethanol (1A)

4-Nitro-o-Phenylenediamine (0.1mol, 0.5 g.) was mixed with lactic acid (0.1mol, 0.6 g.) in a Round bottom flask and refluxed in water bath for 6-8 hour. The reaction mixture was cooled to room temperature and 10% NaOH was added until basic to litmus paper. The product obtained was thoroughly washed with water until it gets free from the base in the product. The product obtained (1A) was dried over a hot air oven and recrystallized with hot water. Yield: 0.42 gm (79.2 %), m.p: 194-196 ºC, R_f : 0.53 (Hexane: Ethyl Acetate, 6:4)

 

1-(5-Methyl-1H-benzo[d]imidazol-2-yl) ethanol (1B)

4-Methyl-o-Phenylenediamine (0.1mol, 0.5 g.) was mixed with lactic acid (0.1mol, 0.6 g.) in a Round bottom flask and refluxed in water bath for 6-8 hour. The reaction mixture was cooled to room temperature and added with 10% NaOH until basic to litmus paper. The product obtained was thoroughly washed with water until it gets free from the base in the product. The product obtained (1B) was dried over a hot air oven and recrystallized with hot water. Yield: 0.39 gm (78.13 %), m.p: 190-192ºC, R_f : 0.46 (Hexane: Ethyl Acetate, 6:4)

 

1-(5-Nitro-1H-benzo[d]imidazol-2-yl) ethanone (2A)

A solution of 1-(5-Nitro-1H-benzo[d]imidazol-2-yl) ethanol (1A) (10 mmol, 0.42 g.) in aqueous acetic acid (5% v/v, 10 ml) was added at room temperature to a solution of potassium dichromate (10 mmol, 0.54 g.) in aqueous acetic acid (5% v/v, 10 ml) and the mixture was stirred for over a period of 15 min by using a magnetic stirrer. The separated product (2A) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.45gm (67.1%) m.p: 198-200ºC, R_f : 0.45 (Hexane: Ethyl Acetate, 6:4)

 

1-(5-Methyl-1H-benzo[d]imidazol-2-yl) ethanone (2B)

A solution of 1-(5-Methyl-1H-benzo[d]imidazol-2-yl) ethanol (1B) (10 mmol, 0.42 g.) in aqueous acetic acid (5% v/v, 10 ml) was added at room temperature to a solution of potassium dichromate (10 mmol, 0.54 g.) in aqueous acetic acid (5% v/v, 10 ml) and the mixture was stirred over a period of 15 min by using a magnetic stirrer. The separated product (2B) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.37 gm (75.71%), m.p: 196-198⁰C, R_f : 0.45 (Hexane: Ethyl Acetate, 6:4).

 

1-(5-Nitro-1H-benzo[d]imidazol-2-yl)-3-phenylpropan-1-one (3A)

1-(5-Nitro-1H-benzo[d]imidazol-2-yl) ethanone (0.01 mol, 0.45 g) (2A) was taken in aqueous NaOH (10%,30 ml) and added with the benzaldehyde (0.01 mol, 0.51 ml) at room temperature. The reaction mixture was stirred over a period of 4 hour by using a magnetic stirrer. The separated solid, the chalcone derivative of benzimidazole (3A) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.48 gm (70.3%), m.p: 214-216 ºC, R_f : 0.56 (Benzene : Acetone 9:1).

 

3-(4-Methoxyphenyl)-1-(5-nitro-1H-benzo[d]imidazol-2-yl)propan-1-one (3B)

1-(5-Nitro-1H-benzo[d]imidazol-2-yl) ethanone (0.01 mol, 0.45 g) (2A) was taken in aqueous NaOH (10%,30 ml) and added with the 4-methoxy benzaldehyde (0.01 mol, 0.51 ml) at room temperature. The reaction mixture was stirred over a period of 4 hour by using a magnetic stirrer. The separated solid, the chalcone derivative of benzimidazole (3B) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.32 gm (72.89%), m.p: 196-198 ºC, R_f : 0.44 (Benzene : Acetone 9:1)

 

2-(1,3-Diphenyl-1H-pyrazol-5-yl)-5-nitro-1H-benzo[d]imidazole (4A)

To an equimolar amount of (1-(5-Nitro-1H-benzo[d]imidazol-2-yl)-3-phenylpropan-1-one (0.03 mol, 0.4 g) (3A) and phenyl hydrazine (0.03 mol, 0.6 ml) were mixed in ethanolic sodium acetate (25 ml) and refluxed for 6-7 hour. The reactions were monitored by TLC. The mixture was concentrated on water bath and poured into ice-cold water. The precipitate obtained (4A) was filtered,washed with water,dried and purified by recrystallization from ethanol. Yield: 0.45 gm (67.2 %) m.p : 230- 232 ºC, R_f : 0.49 (Benzene : Acetone 9:1)

 

2-(3-(4-Methoxy phenyl)-1-phenyl-1H-pyrazol-5-yl)-5-nitro-1H-benzo[d] imidazole (4B)

To an equimolar amount of mixture of 3-(4-Methoxyphenyl)-1-(5-nitro-1H-benzo[d]imidazol-2-yl)propan-1-one (0.03 mol, 0.5 g) (3B) and phenyl hydrazine (0.03 mol, 0.63ml) were mixed in ethanolic sodium acetate (25 ml) and refluxed for 6-7 hour. The reactions were monitored by TLC. The mixture was concentrated on water bath and poured into ice-cold water. The precipitate obtained (4B) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.47 gm. (69.3 %), m.p.: 250-252 ºC R_f : 0.51 (Benzene : Acetone 9:1)

 

2-(1,3-Diphenyl-1H-pyrazol-5-yl)5-methyl-1H-benzo[d]imidazole (4C)

To an equimolar amount of mixture of 1-(5-Methyl-1H-benzo[d]imidazol-2-yl)-3-phenylpropan-1-one (0.03 mol, 0.6 g) (3C) and phenyl hydrazine(0.03 mol, 0.72 ml) were mixed in ethanolic sodium acetate (25 ml) and refluxed for 6-7 hour. The reactions were monitored by TLC. The mixture was concentrated on water bath and poured into ice-cold water. The precipitate obtained (4C) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.52 gm. (77.4 %) m.p: 226-228ºC R_f : 0.53 (Benzene: Acetone 9:1).

 

2-(3-(4-Methoxy phenyl)-1-phenyl-1H-pyrazol-5-yl)-5-methyl-1H-benzo[d]imidazole (4D)

To an equimolar amount of mixture of 3-(4-Methoxy phenyl)-1-(5-methyl-1H-benzo[d]imidazol-2-yl) propan-1-one (0.03 mol, 0.48 g) (3D) and phenyl hydrazine (0.03 mol, 0.6 ml) were mixed in ethanolic sodium acetate (25 ml) and refluxed for 6-7 hour. The reactions were monitored by TLC. The mixture was concentrated on water bath and poured into ice-cold water. The precipitate obtained (4D) was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 0.45 gm. (67.12 %) m.p: 244-246 ºC R_f : 0.55 (Benzene : Acetone, 9:1)

 

RESULTS AND DISCUSSION:

All the synthesized compounds are characterized for physicochemical properties as shown in Table 1.

 

 

 

 

Table: 1 Physiochemical data of all synthesized compounds

Compounds	Molecular Formula	Mol. Wt. gm.	M.P °C	Yield %	Rf	Solvent System
1A	C9H9N3O3	207.19	194-196	79.2	0.53	A
1B	C10H12N2O	176.21	190-192	78.13	0.46	A
2A	C9H7N3O3	205.18	198-200	67.1	0.45	A
2B	C10H10N2O	174.2	196-198	75.71	0.45	A
3A	C18H19N3O3	325.37	214-216	70.3	0.56	B
3B	C19H22N2O	294.39	196-198	72.89	0.44	B
4A	C22H15N5O2	381.39	230-232	67.2	0.49	B
4B	C23H17N5O3	411.41	250-252	69.3	0.51	B
4C	C23H18N4	350.42	226-228	77.4	0.53	B
4D	C24H20N4O	380.45	244-246	67.12	0.55	B

Hexane: Ethyl acetate (6:4), B. Benzene: Acetone (9:1)

 

 

Formation of substituted benzimidazole with pyrazole derivatives were confirmed by characteristic peaks in IR as shown in Table 2.

 

Table 2: FT-IR Spectroscopic Data of Compounds (2A-C, 3A-C and 4A-C)

Comp. No.	IR (cm-1)
1A	3570 (-OH str.), 3108 (Ar -C-H str), 3340 (-NH str), 1510 (C=N), 1350(-NO2), 1175 (C-N)
1B	3440 (-OH str.), 3034 (Ar -C-H str), 3320 (-NH str), 1505 (C=N), 1150 (C-N)
2A	3114 (Ar -C-H str), 3384 (-NH str), 1505 (C=N), 1590 (-C=O str), 1365(-NO2), 1168 (C-N)
2B	3080 (Ar -C-H str), 3335 (-NH str), 1513 (C=N), 1599 (-C=O str), 1154 (C-N)
3A	3015 (Ar -C-H str), 3324 (-NH str), 1513 (C=N), 1642 (-C=O str), 1154 (C-N) 862(-CH= CH).
3B	3090 (Ar -C-H str), 3330 (-NH str), 1513 (C=N), 1624 (-C=O str), 1144 (C-N) 890(-CH= CH).

 

Synthesized compounds were confirmed from IR, ¹HNMR and Mass spectral studies.

 

2-(1,3-diphenyl-1H-pyrazol-5-yl)-5-nitro-1H-benzo[d]imidazole (4A):

The compound 4A which was confirmed by characteristic peak at 1569 (-C=N str.), 3323 (-NH₂ str.), 883 (-CH=CH str.) in IR spectrum and, M⁺ peak at 381.39 in mass spectrum. The ¹H NMR of compound gave signal at 8.63 (d, 1H, CH of Ar), 7.46-8.19 (m, 2H, CH of Ar), 7.2- 7.34 (m, 5H, CH of Ar), 7.32-7.48 (m, 5H, CH of Ar), 6.7(s, 1H of Pyrazole), 5.0 (m, 1H of Benzimidazole).

 

2-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-5-yl)-5-nitro-1H-benzo[d]-imidazole (4B):

The compound 4B which was confirmed by characteristic peak at 3319 (-NH₂ str.), 1597 (-C=N str.), 2922 (-OCH₃ str.) in IR spectrum and, M⁺ peak at 411.41 in mass spectrum. The ¹H NMR of compound gave signal at 8.54 (d, 1H, CH of Ar), 7.42-8.12 (m, 2H, CH of Ar), 7.2- 7.33 (m, 5H, CH of Ar), 7.31-7.32 (m, 4H, CH of Ar), 6.5(s, 1H of Pyrazole), 5.1 (m, 1H of Benzimidazole), 3.70 (s, 3H of OCH₃).

 

2-(1,3-Diphenyl-1H-pyrazol-5-yl)-5-methyl-1H-benzo[d]imidazole (4C):

The compound 4C which was confirmed by characteristic peak at 3345 (-NH₂ str.), 1607 (-C=N str.), 2920 (-CH str.), in IR spectrum and, M⁺ peak at 350.42 in mass spectrum. The ¹H NMR of compound gave signal at 8.52 (d, 1H, CH of Ar), 7.41-8.12 (m, 2H, CH of Ar), 7.1- 7.32 (m, 5H, CH of Ar), 7.30-7.32 (m, 5H, CH of Ar), 6.4 (s, 1H of Pyrazole), 5.1 (m, 1H of Benzimidazole), 2.35 (s, 3H of CH₃).

2-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-5-yl)-5-methyl-1H-benzo[d]-imidazole (4D):

The compound 4D which was confirmed by characteristic peak at 3328 (-NH₂ str.), 1597 (-C=N str.), 2925 (-OCH₃ str.) in IR spectrum and, M⁺ peak at 380.44 in mass spectrum. The ¹H NMR of compound gave signal at 8.51 (d, 1H, CH of Ar), 7.52-8.12 (m, 7H, CH of Ar), 7.34-7.37 (m, 4H, CH of Ar), 6.4 (s, 1H of Pyrazole), 5.1 (m, 1H of Benzimidazole), 2.35 (s, 3H of CH₃) 3.74 (m, 3H of OCH₃).

 

Biological activity:

The in vitro screening of test compounds was done by different techniques. Different media were available for cultivating mycobacteria. Because there is no general agreement that, which media is the best for the routine isolation of acid fast organisms, culture media usually selected on the basis of personal preference and laboratory tradition. Anti-tubercular screening had been carried out by Agar based media method. The test compounds were subjected to screening by Lowenstein Jensen Method using H37Rv strain of M. Tb. (L.J. method).

 

The drug susceptibility test was carried out by primary isolation or a sub-culture on L.J. medium. The test compounds (0.5 ml) were homogenized with 5 ml of media in a sterile screw capped bottle (e.g. 14 ml McCartney bottle) containing 1 ml of sterile distilled water. The mixture was homogenized in a vortex mixer for a minute and necessary opacity was adjusted by adding sterile distilled water. Screw-capped Tubes containing 5 ml of medium were inspissated at 85 ºC for 40-45 minutes. This inspissation was repeated 3 times. During this step, media was solidified. Then culture inoculation was carried out. For culture inoculation, dilution of 1 μg/ml of the standard suspension was prepared and 0.2 ml of the inoculums was incorporated to each tube. After inoculation the tubes were incubated at 37 ºC in a slanted position with the screw cap slightly loosened to allow evaporation of the inoculum. After 24-48 h, screw caps were tightened and the tubes were further incubated.

 

The reading of result was carried out at the 21^th day after inoculation. The reading of the results is based on counting of growth on the different slants and calculation of the proportion of bacilli by comparing counts on drug free (control) and drug containing L.J. media¹⁰. Table 3 represents the growth results of anti-tubercular activity.

Table: 3 Anti tubercular activity of the synthesized compounds

Sr No.	Compound Name	Concentration of Compound (100 μg/ml)
1	4A	No growth	Active
2	4B	No growth	Active
3	4C	Growth detected	Not active
4	4D	No growth	Active

CONCLUSION:

From the above results, it is concluded that Benzimidazole substituted with pyrazole has vital need for treatment of tuberculosis. 4B showed potent anti-tubercular activity at concentration (100 μg/ml). Among four compounds, three compounds 4A, 4B and 4D were active against M. Tb due to high lipophilicity and electronegativity of the attached substituents (4-OCH₃, 4-NO₂) and they are high molecular weight compounds. The 4C did not show activity because of its low lipophilicity and less electronegativity.

 

AKNOWLEDGEMENT:

All the authors are thankful to the director, SAIF, Punjab University, Chandigarh and Oxygen healthcare private ltd. Ahmedabad for providing spectroscopic analysis of the compounds. The authors are also thankful to All India Council for Technical Education, New Delhi for the financial assistance in the form of fellowship. We would like to thank University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for providing necessary infrastructure and facility.

 

We would also like to thanks Late Mr. Karan Parwe, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for his equal contribution in research and compilation of work.

 

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Accepted on 03.05.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3599-3608.