Synthesis, Characterization and Biological Evaluation of Benzimidazole Derivatives as Potential Anxiolytics

 

Sudheer K Dokuparthi*¹, J. Baloji Naik¹ K. Sunil Kumar¹, A. Saidulu²

¹Vijaya College of Pharmacy, Hyderabad - 501511, India.

²Department of Botany, Osmania University, Hyderabad, India.

 

ABSTRACT:

Considerable attention has been focused on the synthesis of benzimidazoles due to their broad spectrum of biological activities such as antimicrobial, anti diabetic, anti cancer, anti hypertensive, anti anxiolytic and inflammatory activities etc. As a part of our research work, a series of benzimidazole derivatives were synthesized in good to high yields by reaction of o-phenylenediamine and different aromatic aldehydes in the presence of ammonium chloride (NH₄Cl), as an efficient catalyst at room temperature. This environmentally safe and practical method offers several advantages, such as high yields, use of an available catalyst, mild reaction conditions and easy work-up process. All synthesized compounds were characterized by detailed spectral analysis by using LC-MS, IR, and NMR spectroscopy. The synthesized benzimidazoles were screened for chronic anti anxiety activity in albino rats by using light and dark box model with standard Buspirone. Behavioral parameters were recorded during the test session of 5 minutes. All benzimidazole derivatives have shown significant anti anxiety activity compared to the standard. However, benzimidazole analog (3e) exhibited highest anxiolytic action.

 

 

INTRODUCTION:

Benzimidazoles are the heterocyclic compounds having benzene and imidazole rings fused together and the benzimidazole derivatives are vastly utilized bioactive intermediates in the preparation of diversified pharmacophores. 5, 6-dimethylbenzimidazole was the first isolated benzimidazole derivative by the acid degradation of Vitamin B₁₂ (1). The benzimidazole ring system is present in numerous anti-inflammatory agents (2), poly (ADP-ribose) phosphorylase inhibitors (3), Histamine H4 receptor binders (4), antiparasitic (5), anticancer (6), cardiotonic (7), antiulcer (8), antimicrobials (9), antihypertensives (10), as alpha-glucosidase inhibitors as anti diabetic (11), anti depressant, analgesic (12) and anti cholesterol (13).

 

Substituted benzimidazoles can be synthesized by two methods. The most general method is a condensation reaction of o-phenylenediamine with carboxylic acids or their derivatives such as nitriles, imidates or ortho esters, under strong acid conditions or in combination with very high temperatures and/or microwave irradiations (14). The other one is a two step process, which includes the condensation of o- diaminoaromatic compounds with different aromatic aldehydes with oxidative cyclodehydrogenation of the intermediates formed in the reaction. A variety of oxidants and catalysts have been used for the preparation of benzimidazoles (15). Though these methods are efficient, they have some limitations such as low yields, use of volatile or toxic organic solvents, the requirement of excess amounts of catalysts or reagents, special apparatus and harsh reaction conditions. Synthesis of benzimidazoles with ease, good yields and the environmentally benign procedure is still a challenge.

 

Due to the important biological activity of benzimidazole and in line with research works in the synthesis of this ring system (16–20) we wish to report a simple procedure for preparation of 2-arylbenzimidazoles through a condensation reaction of o-phenylenediamine and aromatic aldehydes in the presence of NH₄Cl as a catalyst (21)^. We also report the anxiolytic activity of these compounds.

 

MATERIALS AND METHODS:

All the chemicals and reagents (analytical grade) were procured from NICE Chemicals. The synthesized compounds were analyzed by NMR, Mass, and IR spectroscopy. 1H NMR spectra were recorded on a Varian Gemini 200- and 300-MHz instrument in CDCl₃ and DMSO using Tetramethylsilane (TMS) as an internal standard. The mass spectra were measured on a Liquid Chromatography / Mass Spectrometry (LCMS) Agilent mass spectrometer. The IR spectra were recorded on a Nicolet 740 Fourier transform infrared (FTIR) spectrometer. The melting points were measured using a Buchi-510 apparatus and were uncorrected.

 

Benzaldehyde (1 mmol) was added to a stirred solution of 1, 2-phenylenediamine (1 mmol) and NH₄Cl (4 mmol) in CHCl₃ (10 ml) for five minutes at room temperature with continuous stirring for four hours. After completion of the reaction (TLC, eluent Hexane / ethyl acetate 30 / 70), the solvent was removed by downward distillation and the residue was extracted with ethyl acetate (20 ml); the organic layer was washed with water (10 ml). Organic layer was separated and dried over sodium sulfate. The solvent was removed by downward distillation and the crude product was purified by solvent re-crystallization technique.

 

2-(4-dimethylamino phenyl)-1H-benzimidazole (3a):

Brown Solid; Molecular formula: C₁₄H₁₂N₂, Yield-55%; m.p. 274-276°C;  1H NMR (dmso-d₆, 300 MHz, δ ppm) : 8.05 (d, 2H, aromatic), 7.62 ( d , 2 H , aromatic) , 7.35 ( d , 2 H , aromatic) , 7.29 ( m , 2H , aromatic) , 2.38 (s, 3H,aliphatic), ¹³C NMR (dmso-d₆): δ  155.5, 152.9, 142.7, 128.4, 123.6, 115.8, 115.2, 112.7, 43.4.  IR (KBr): 3423(-NH), 2922(-CH3), 1123 (-C-N), 1608 (-C=N) cm-1; Mass (LCMS): m/z 209 (M⁺ + H).

 

2-(4-chloro phenyl)-1H-benzimidazole (3b):

Grayish white Solid; Molecular formula: C₁₃H₉N₂Cl, Yield-58%, m.p. 230-233°C; 1H NMR (dmso-d₆, 300 MHz, δ ppm): 8.23 (d, 2H, aromatic), 7.59 ( d , 2 H , aromatic) , 7.55 (d, 2H, aromatic), 7.24 (m, 2H,aromatic) is tabulated in fig no:6.3, ¹³C NMR (dmso-d₆): δ  152.9, 141.7, 134.2, 129.4, 128.9, 123.8, 115.2. IR (KBr): 3380(-NH), 3052(Ar-CH), 1596 (-C = N), 743 (-C-Cl) cm-1; Mass (LCMS): m/z 229 (M+ + H)

 

2-(3, 4-dihydroxy phenyl)-1H-benzimidazole (3c):

Brownish white  solid ; Molecular formula: C₁₃H₁₀N₂O₂, Yield-74%, m.p. 236-237°C; 1H NMR (dmso-d₆, 300 MHz, δ ppm) : 10.18 (bs, 2H, OH), 8.05(s, 2 H, NH, OH) , 7.59 ( d , 2 H , aromatic) , 7.28 (m, 2H, aromatic), 6.98 (d, 1H,aromatic), 6.97 (d, 1H,aromatic), ¹³C NMR (dmso-d₆): δ 152.9, 147.4, 145.8, 142.7, 124.4, 123.6, 123.3, 116.3, 115.8, 114.2. IR (KBr):3379(-NH), 3020 (-OH), 2912 (-Ar-CH), 1513 (C = N), 1271 (-C-O) cm-1; Mass (LCMS): m/z 227 (M+ + H).

 

2-(3, 4-dimethoxy phenyl)-1H-benzimidazole (3d):

Brownish red solid; Molecular formula: C₁₅H₁₄N₂O₂, Yield-85% ; m.p. 232-234°C; 1H NMR (dmso-d₆, 300 MHz, δ ppm): 8.16 (s, 1H, aromatic), 8.12 ( d , 1 H , aromatic), 7.59 ( m , 2 H , aromatic) , 7.42 ( d ,  2H , aromatic), 7.25 (s, 1H, aromatic), 7.22 (bs, 1H, NH); ¹³C NMR (dmso-d₆): δ 152.9, 150.3, 149.6, 141.3, 123.6, 122.6, 115.8, 112.9, 111.7, 56.1    IR (KBr): 3423(-NH), 2922 (Ar-CH), 1123 (-OCH3), 1608(-C=N), 615 (-C-O) cm-1; Mass (LCMS): m/z 255 (M+ + H).

 

2-(4-nitro phenyl)-1H-benzimidazole (3e):

Grey  Solid; Molecular formula: C₁₃H₉N₃O₂, Yield- 63%; m.p. 298-299°C;  1H NMR (dmso-d₆, 300 MHz, δ ppm): 8.38 (d, 2H, aromatic), 8.35 ( d , 2 H , aromatic) , 7.59 ( d , 2 H , aromatic) , 7.15 ( m , 2H , aromatic). ¹³C NMR (dmso-d₆): δ 152.9, 147.9, 135.6, 127.4, 124.6, 123.0, 115.8, 115.2. IR (KBr): 3400(-NH), 2920(Ar-CH), 1601 (-NO2), 1514 (-C=N), 1341(-N-O) cm-1; Mass (LCMS): m/z 240 (M⁺ + H).

 

Pharmacology:

Albino rats (150-250 g) were used in the experiments. They were procured from Sainath agencies, musheerabad (Hyderabad). After randomization into various groups and before initiation of the experiment, the rats were acclimatized for a period of 10 days. Animals were housed in polypropylene cages and maintained under standard environmental conditions such as temperature (26 ± 2ºc), relative humidity (45-55%) and 12hr dark/light cycle. The animals were fed with rodent pellet diet (Golden Mohur Lipton India Ltd.) and water ad libitum. The study protocol was approved by the institutional animal ethics committee (IAEC) before the commencement of experiment (1292/ac/09/CPCSEA).

 

Determination of acute toxicity (OECD guidelines 423):

The benzimidazole analoges were screened according to the OECD guidelines (22) for acute toxicity at a dose of 100 mg/kg, 200mg/kg, 400 mg/kg, 800 mg/kg and 2000 mg/kg p.o in albino rats (6 rats in each group). All the rats were observed for toxicity signs of compounds for 24hrs followed by 14 days. On 8th-day &14th-day, the body weight of rats was recorded. The rats were devoid of any toxic signs at all the given doses of the compounds and there is no change in body weight of rats after 14 days. Hence 2000 mg/kg (highest dose) was selected as safe dose and 1/10 of 2000mg/kg i.e., 200mg/kg of benzimidazole compounds were selected for the in-vivo studies.

 

Effect of Benzimidazole derivatives in Light and Dark box Test induced anxiety in rats:

The experiment was performed on albino rats (150-250 gm) of either sex procured from Sainath agencies, Musheerabad (Hyderabad). The animals were housed in colony cages at an ambient temperature of 26 ± 2°C and relative humidity (45-55%), with a 12h/12h light dark cycle and access to food and water ad libitum. The food was restricted during experiments. Stock solutions of Buspirone (1 mg/kg) and benzimidazole derivatives were prepared in 2% acacia suspension.

 

Weigh and mark the animals. Divide the animals into three groups control(C), test (T), and standard(S) each consisting of three rats. All the animals were subjected to light and dark box for assessment of anxiety levels. The light and dark box apparatus consists of two compartments  chamber (40x60x20cm) comprising of a brightly illuminated area (40x40cm)  and dark area (40x20cm) separated by a wall hole (7cm diameter). All groups of rats were given treatment for a period of 10 days, wherein control rats were treated with distilled water (P.O), test rats with novel benzimidazole compounds (200 mg/kg, P.O) and standard rats with Buspirone (1 mg/kg, P.O). On the 11^th day, test session was performed, in which control rats were placed individually in the illuminated part of the cage and following parameter were recorded during the test session of 5minutes.

1)    Time spent in the light box.

2)    Time spent in dark box

3)    Total number of entries in entries and dark box during a 5min session.

 

The same procedure was repeated for test and standard groups, after 1hr of administration of (benzimidazole derivatives 200mg/kg) and Buspirone (1mg/kg) and the above parameters were recorded on the 11^th day of the test session.

 

Statistical Analysis:

The values are represented as mean ± S.E.M, and statistical significance between treated and control groups was analyzed using One way ANOVA, Followed by Dunnett’s test where P<0.001, P<0.01 and P<0.05 was considered statistically significant.

 

RESULTS AND DISCUSSIONS:

 

Scheme 1: Synthetic strategy for synthesis of benzimidazole derivatives:

Though there are diversified schemes were reported for the synthesis of benzimidazole analogues, here we are reporting a new method for the preparation of benzimidazole derivatives (scheme 1) by using ammonium chloride as a catalyst, which was inexpensive and decreased the reaction time, with moderate to good yields. This method could be easily practiced in laboratories within the stipulated time.

 

The synthesized benzimidazole derivatives (Table 1) exhibited significant in-vivo anxiolytic potency in light and dark box transition test. All the compounds exhibited anxiolytic activities. But the compound 3e was found to possess more anxiolytic activity by allowing the rats to spend more time in the light box (244.33 ± 1.96 seconds) and by increasing the number of entries in the light box (5 ± 1.46) when compared to control rats (0.66 ± 0.12, 19 ± 1.84). Table II, III and IV represent the anxiolytic activity by using light and dark box apparatus.

 

Table 1: Summary of synthesized compounds

S.No.	2(a-e)	Time (hrs)	3(a-e)	Yield (%)
1		8		55
2		6		58
3		10		74
4		4		85
5		5		63

 

Table II:  Control (Distilled water p.o)

S.No	Body weight(g)	No. of entries in Light box	No .of  entries in the dark box	Time spent in  the dark box(sec)	Time spent in the light box(sec)
1	240	2	3	257	24
2	185	0	2	274	18
3	220	0	1	291	15
	AVERAGE	0.66 ± 0.12	2	274	19 ± 1.84

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

 

 

Table III: Test (Benzimidazole analogues)

Analogue I (3a):

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

Analogue II (3b):

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

ANALOGUE III (3c):

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

Analogue IV (3d):

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

Analogue V (3e):

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

Table III: Standard Group :( Buspirone 1mg/kg)

P<0.001***, P<0.01** and P<0.05* was considered statistically significant.

 

 

 

 

CONCLUSION:

A series of benzimidazole derivatives were synthesized in good to high yields by reaction of o-phenylenediamine and different aromatic aldehydes in the presence of ammonium chloride (NH₄Cl) as an efficient catalyst at room temperature. This environmentally benign and practical method offers several advantages, such as high yields, use of an available catalyst, mild reaction conditions and easy workup procedure. The structures of all synthesized compounds were characterized by using LC-MS, IR and NMR spectroscopy.  All compounds exhibited significant anxiolytic potency when screened by using light and dark box apparatus.  Though, the exact mechanism behind the anxiolytic potential is not clear. But, it can be hypothesized that these benzimidazole compounds may enhance GABA transmission in the brain that may result in anti-anxiety. Further, neuro-chemical studies are required to know exact mechanism behind the anxiolytic potential of these benzimidazole derivatives.

 

ACKNOWLEDGEMENT:

Acknowledgement is given to all the members in the research group of Pharmacognosy, Pharmacology and the management of Vijaya College of Pharmacy for providing the required facilities.

 

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Received on 09.08.2017         Modified on 28.08.2017

Accepted on 13.09.2017      © RJPT All right reserved