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

The synthesized benzimidazoles compounds were prepared from the condensation reaction between o-Phenylenediamine and various carbonyl compounds, in the presence of ammonium chloride as a catalyst. Ammonium chloride is a commercial and environmentally benign catalyst. The yield of all benzimidazole derivatives was found to be in the range of 75 – 94%. The purity of the compounds was ascertained by melting point and TLC. The synthesized compounds were characterized by using IR,1H NMR, and MASS spectral data together with elemental analysis. The synthesized benzimidazole compounds were screened for acute and chronic anti-anxiety activity in Wistar rats by using an elevated plus maze model with standard Diazepam. The synthesized compounds ZB, ZE, ZF, ZG, and ZH showed potent anti-anxiety activity when compared to the standard Diazepam. The compound ZH exhibited a higher anti-anxiety activity when compared to other prepared benzimidazoles. The results were subjected to statistical analysis by using one-way ANOVA followed by the Tukey-Kramer test, to calculate the significance.


INTRODUCTION
Benzimidazoles and its derivatives represent one of the most biologically active class of compounds, possessing a wide spectrum of activities and these are well-documented in literature. They show selective neuropeptides YY 1 receptor antagonists, [1] potent inhibitors of TIE-2 and VEGFER-2 tyrosine kinase receptors, [2] antitumor agents, [3] gamma-amino butyric acid (GABA) agonists, and 5-HT 3 antagonists. [4] Substituted benzimidazole derivatives have found commercial application in veterinarian medicine as anthelmintic agents and in diverse human therapeutic Nannapaneni, et al. J Young Pharm. 2010;2(3): [273][274][275][276][277][278][279] compounds and synthesis of 3, 4-dihydropyrimidine-2(1H)-ones. [12] However, there are no reports of the use of ammonium salts as catalysts for the synthesis of benzimidazoles. In continuation, on the synthesis of heterocycles [13,14] and on the development of synthetic methodologies, [15][16][17] we herein report a facile method for the synthesis of benzimidazoles by the condensation of 1, 2-phenylenediamine with carbonyl compounds, in the presence of ammonium salts in very good yields. Anxiety is a psychological and physiological state characterized by cognitive, somatic, emotional, and behavioral components. These components combine to create an unpleasant feeling that is typically associated with uneasiness, fear or worry. Anxiety is a generalized mood or condition that occurs without an identifiable triggering stimulus. As such, it is distinguished from fear, which occurs in the presence of an observed threat. Additionally, fear is related to the specific behaviors of escape and avoidance, whereas, anxiety is the result of threats that are perceived to be uncontrollable or unavoidable. Some reports state that benzimidazoles possess anti-anxiety activity. [18] This observation prompted us to evaluate the synthesized benzimidazole derivatives for anti-anxiety activity

MATERIALS AND METHODS
All the chemicals and reagents used were of analytical grade and were procured from NICE Chemicals. It is known that the reaction of o-phenylenediamine (OPDA) with carbonyl compounds, under strong acidic conditions, gives benzimidazoles, whereas, OPDA in the presence of β-ketoesters under neutral reflux conditions, gives benzodiazepin-2-ones, with the elimination of water and alcohol. Under acidic conditions, initially it forms ethyl β-2-amino aniline crotonate a t room temperature and upon heating it gives 2-methyl-1H-benzo[d]imidazole instead of benzodiazepin-2-ones, with the elimination of ethyl acetate. Therefore, we have made an attempt to react OPDA with carbonyl compounds and b-ketoesters, with different ammonium salts to see the feasibility of the formation of compounds. To select favorable reaction conditions, we first examined the model reaction of 1, 2-phenylenediamine (1 mol) with benzaldehyde (1 mol Figure 1, Table 1]. The results, tabulated in Table  2, indicate the formation of benzimidazoles. The synthesized compounds were analyzed by NMR, Mass, and IR spectroscopy. 1 H NMR spectra were recorded on a Varian Gemini 200-and 300-MHz instrument in CDCl 3 and DMSO-d 6 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 u s i n g a Buchi-510 apparatus and were uncorrected.

Typical Experimental Procedure for the Synthesis of Benzimidazoles
Benzaldehyde (Y, 1 mmol) was added to a stirred   Cl (4 mmol) in CHCl3 (5 ml) for five minutes at room temperature. Stirring was continued for four hours. After completion of the reaction (TLC, eluent Hexane / ethyl acetate 30 / 70), the solvent was removed under reduced pressure and extracted with ethyl acetate (20 ml); the organic layer was washed with water (10 ml). Layers were separated and the organic layer was dried over sodium sulfate. The solvent was removed under reduced pressure and the crude product was subjected to column chromatography using petroleum ether = EtOAc (9:1), which gave 2-phenyl-1H-benzo[d]imidazole (Z A ) as a solid in 94% yield.

Pharmacology
Wistar rats weighing 200 ± 25 g, of either sex, were procured at least two weeks prior to the study. The animals were housed in polycarbated cages under conditions of constant temperature (22 ± 2 o C) and humidity, under a 12-hour light / dark schedule, The animals were allowed free access to a standard diet and water, so they could acclimatize to the new environment.

Acute oral toxicity studies
The aim of this study is to determine the lethal dose. In this study the testing drug was administered in a single dose, using the oral route. The dose was increased in a graded manner. LD 50 in the acute toxicity test was observed at the dose of 400 mg/kg-bw. Therefore, one-tenth of the preceding dose (200 mg/kg-bw) was selected for the study, that is, 20 mg/kg-bw. This 20 mg/kg-bw testing dose did not have any impact on the normal locomotion of the animal, which was tested by administering the same dose to the animals, using the oral route. The locomator activity was assessed in an actophotometer.

Anti-anxiety activity
In this activity, the elevated plus maze model was used. For this model, Wistar rats were divided into 10 groups of six animals each. Group-I (control) animals were administered the vehicle, Group-II, Group-III, Group-IV, Group-V, Group-VI, Group-VII, Group-VIII, and Group-IX animals were administered the benzimidazole derivatives of Z A -Z H, respectively, with a dose of 20 mg/kg-bw (p.o), and Group-X was administered diazepam 2 mg/kgbw (p.o), for 1 day in case of acute study and for 10 days in case of chronic study. The number of entries and time spent in the open and closed arms of the elevated plus maze, using rats, was observed in acute and chronic studies for 1day and 10 days, respectively. Diazepam was used as a reference standard. The experiment was conducted in a sound attenuated room. In an acute study, the animals of all groups were treated with the respective drugs 30 minutes prior to the experiment. In a chronic study, animals of all groups were treated with the respective drugs for 10 days, and on the tenth day, treatment was given 30 minutes prior to the experiment. In both acute and chronic studies, each rat was placed in the center of the maze facing one of the enclosed arms, and during a ten-minute session the following parameters were noted; Number of entries into the open arm, number of entries into the closed arm, time spent in the open arm, time spent in the closed arm, and total number of entries into the open and closed arms. [23,24]

Statistical Analysis
The values were expressed as mean ± SEM for six animals. The results were subjected to statistical analysis by using one-way ANOVA followed by Tukey-Kramer test, to calculate the significant difference, if any, among the groups. P < 0.05 was considered as significant.

RESULTS
The compounds (Z A -Z H ) were obtained by the reaction between aldehyde and o-phenylediamine in the presence of ammonium salts according to Figure 1. The synthesized compounds were confirmed by thin layer chromatography (TLC), Melting Point (mp), IR, 1 H NMR, and mass spectroscopy (MS) spectral analysis. The yields and melting points for all the synthesized compounds are listed in Table 2. The titled compounds were confirmed by IR spectral data showing characteristic bands at 1384 -3200 cm -1 , indicating the presence of -NO 2 and -OH stretching; and sharp bands, ranging between 1680 -1750 cm -1 , indicating the presence of C = N. Compounds Z A -Z H were confirmed by stretching at 3500 cm -1 , due to the presence of -NH. Compounds Z A -Z H were confirmed by 1 H NMR spectral analysis. The NMR proton peak at 6.00 -6.18 ppm revealed the presence of -NH. Further appearance of the molecular ion peak at 225 (m + 1) and 209 (m + 1) confirmed the structure of Z D and Z E . The synthesized compounds Z B , Z E , Z F , Z G , and Z H were found to have potent anti-anxiety activity. Compound Z H exhibited more activity when compared to other prepared benzimidazoles.

Anti-anxiety activity
In the acute and chronic studies of elevated plus maze models [Tables 3 and 4], the number of entries into the open and closed arms and the time spent in the open arm were increased; and time spent in the closed arm was decreased in synthetic preparation-treated animals when compared with the control animals, which was comparable with that of the reference standard, diazepam.

DISCUSSION
Several researchers reported a synthesis of benzimidazole derivatives, but in our present study we synthesized the benzimidazole derivatives by using ammonium salts as catalysts, which were inexpensive and decreased the reaction time, with very good yields. This method could be easily practiced in laboratories within the stipulated time.
In the evaluation of anti-anxiety activity, the experimental model used in our study was an elevated plus maze. This was based on the assumption that unfamiliar, nonprotective, and brightly lit environmental stress provoked inhibition of normal behavior. This normal behavioral inhibition was further augmented in the presence of fear or an anxiety-like state. [24] The elevated plus maze test was a well-established animal model for testing anxiolytic drugs. A known anxiolytic drug, diazepam, was used as the standard, which is one of the well-recognized anxiolytic drugs. In this model, the changes that occurred due to anxiety were, decreased time spent in the open arm, increased time spent in the closed arm, and decreased number of entries between the arms, than that which was observed in the control animals.
After treatment with the synthetic preparation, at a dose of 20 mg/kg-bw, the compounds Z B , Z E , Z F , Z G , Z H , and the standard, significantly increased the time spent in the open arm, decreased the time spent in the closed arm, and increased the number of entries between the arms, in the acute study [ Table 3]. All these may be due to decreased fear, increased exploratory behavior, and the behavioral disinhibitory effect [24] of the standard (Diazepam) and synthetic preparation. Similar findings were observed in the chronic study also [ Table 4]. This may be due to the same mechanism, as stated earlier in the acute study.

CONCLUSION
The present study describes a simple, inexpensive, and easy method for synthesis of benzimidazole derivatives  Significance: All values are given in mean ± SEM, n ═ 6. One way ANOVA followed by Tukey's multiple comparison tests, ***P < 0.001, **P < 0.01, *P < 0.05 vs. control in a stipulated time, without using any drastic conditions. The yield of all benzimidazole derivatives were found to be in the range of 75 -94%. The purity of the compounds were ascertained by a melting point and TLC. The assigned structure was further established by IR, 1 HNMR, and MS spectral studies.
The acute and chronic studies for anti-anxiety activity of the synthesized compounds were screened using elevated plus maze method in Wistar rats. Diazepam was used as the reference drug. In the prepared benzimidazole derivatives, it seemed that the compounds Z From the present study, it can be concluded that the benzimidazole derivatives can potentially be developed into useful anti-anxiety agents, which can prompt future researchers to synthesize a series of benzimidazole derivatives containing a wide variety of substituent's, with the aim of producing a novel heterocyclic system, with enhanced activity.