Design, Synthesis and Biological Evaluation of Novel 1, 2, 5-Substituted Benzimidazole Derivatives as Gastroprotective Anti-inflammatory and Analgesic Agents

In the present investigation, in order to identify GI-safe anti-inflammatory and analgesic agents, a series of novel 1, 2 and 5-substituted benzimidazole derivatives were synthesized and biologically evaluated. The results demonstrated that the compounds 3b, 4b, and 5b could serve as gastroprotective lead compounds for developing a novel class of potent as well as orally active anti-inflammatory and analgesic agents in the future research.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as a first choice of drug for the treatment of various inflammatory diseases as well as to relieve aches and pain of everyday life [1]. However, long term use of NSAIDs is associated with side effects like hepatotoxicity, platelet dysfunction and bleeding [2][3][4]. But the major and potential side effect with chronic use of NSAIDs is gastrointestinal (GI) ulcerations due to inhibition of cyclooxygenase (COX) in tissues where prostaglandins exert their physiological functions, such as gastric mucosal defense and renal homeostasis [5]. The discovery of two COX isoforms, namely COX-1 constitutively expressed in most tissues such as kidney and GI tract, while COX-2 is induced at the sites of inflammation, led to the development of selective COX-2 inhibitors, with the hope of significantly reducing the GI toxicity associated with acute and chronic use of NSAIDs. However, with the increased knowledge of physiological roles of COX-2 enzyme in a variety of tissues, including stomach and kidney, some selective COX-2 inhibitors are withdrawn from the market because of cardiovascular toxicity, have challenged the benefits of selective COX-2 inhibition [6]. As a consequence, the interest for alternative approaches to reduce GI side effects associated with NSAIDs has re-emerged. Different approaches were pursued in the search for GI-sparing NSAIDs like addition of chemical moieties that release gastro protective mediators such as nitric oxide (NO)-releasing or hydrogen sulphide (H 2 S)-releasing NSAIDs, dual cyclooxygenase/5-lipoxygenase or microsomal prostaglandin E synthase-1 inhibitors [7,8]. But these approaches are associated with some limitations, [9][10][11][12][13][14] so a real need exists to develop new antiinflammatory, analgesic agents with better efficacy, less toxicity and fewer side effects of gastric ulceration. According to reported literature, reactive oxygen species or oxidative stress components are involved in pathophysiology of NSAIDs-induced ulcerations. On synthesizing novel chemical entities or chemical modifications/derivatization of existing molecules could lead to neutral molecules with greatly reduced acidic character and oxidative stress as a useful approach to explore safer and potent anti-inflammatory and analgesic agents [15].
Benzimidazoles and its derivatives substituted at 1, 2, 5 and 6-positions fulfill the minimum structural requirements for antiinflammatory, analgesic and antioxidant activity [20,23]. Encouraged by the aforementioned findings and in continuation of an ongoing program aiming at finding new gastroprotective leads with potential anti-inflammatory, analgesic activities, it was rationalized to explore novel benzimidazole derivatives. In the present research work, a novel series of 1, 2 and 5-substituted benzimidazole derivatives have been synthesized and bio-evaluated for their anti-inflammatory, analgesic, ulcerogenic as well as antioxidant activity. Moreover, the drug-likeness properties of the resulted compounds are also presented.

Materials and methods
All research reagents and solvents used in synthesis were of laboratory grade and were procured from SD Fine Chemicals, Qualigens, Loba Chemie and Sigma-Aldrich, India. All the solvents were dried and distilled before use. Reactions were monitored by thin layer chromatography (TLC) on precoated silica gel plates and compounds were visualized by exposure to iodine vapours. Melting points (m.p.) were determined using open capillary tube method and were uncorrected. Spectral data were obtained as follows: Infra-red (IR) spectra were recorded on Bruker Alpha-FT-IR Spectrophotometer Attenuated total reflection mode from ASBASJSM College of Pharmacy, Bela. The 1 H-NMR spectra were recorded on a Bruker Avance DPX-200 (400 MHz) (Panjab University, Chandigarh, India) using tetramethylsilane as an internal standard and the values are expressed in parts per million (ppm). Mass spectra were obtained using electrospray ionization (ESI) technique by Agilent 1100 series LC-MS instrument. Elemental analyses were performed on Leco CHNS-932 (Leco, St. Joseph, MI, USA).
Anal. Calcd for C 22

Biological evaluation
All the experimental procedures used in this study were approved by the Institutional Animal Ethical Committee, registered under Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. Animals were housed individually in polypropylene cages, maintained under standard conditions of alternating light-and-dark cycles at a constant temperature (25±2°C and 35-60% relative humidity). All the animals were allowed free access to food and water (ad libitum).

Carrageenan-induced paw edema model:
The newly synthesized test compounds were subjected to in vivo anti-inflammatory studies using carrageenan-induced rat paw edema model by employing 1% carrageenan solution as the phlogistic agent [34]. The test compounds were administered p.o. as suspensions in 2% sodium carboxy For the synthesis of benzimidazole derivatives, different substituted aryl amines were added to the solution of compound 2a-c (2g, 0.006 mol), potassium iodide (2 g, 0.012 mol) and potassium hydroxide (1 g, 0.017 mol) in dry ethanol in a round bottom flask. The mixture was refluxed for 24 h and then poured into ice-cold water. The precipitates formed were filtered at the pump, washed, dried and crystallized by using hot water. methylcellulose (CMC), 30 min before the injection of the phlogistic agent, at a dose level of 100 mg/kg body weight. Indomethacin was used as a standard at a dose level of 50 mg/kg body weight. Groups of six SD rats of either sex were used in each experiment. The 2% sodium CMC was served as a control. The paw edema volume was measured with the help of plethysmograph by mercury displacement method at 0 h and 3 h (immediately after injection and 3h post injection of carrageenan). The % edema is shown in Table 1. The percent anti-inflammatory activity was calculated according to the formula as given below: Vt and Vc is the edema volume in drug treated and control groups, respectively.
Acetic acid-induced writhing assay: The most active compounds (3b, 4b and 5b) emerged in the anti-inflammatory study were further tested for their analgesic potential by employing acetic acid-induced writhing assay [35]. Albino mice of either sex (20-25 g, body weight) were divided into control, standard and different test groups of six mice each. The control group was administered p.o., with 2% sodium CMC, whereas standard and test compound groups were administered with acetyl salicylic acid and different test compounds, respectively at a dose level of 100 mg/kg suspended in 2% sodium CMC, 30 min before the i.p. injection of the acetic acid solution (0.6% v/v in distilled water) at a dose level of 1 ml/kg. The number of writhes per animal was recorded for 15 min. The analgesic activity was expressed as percentage of protection and the results are presented in Table 2.

% Protection=100-[(Vt/Vc) × 100]
Ulcerogenic assay: SD rats of either sex weighing 150-200 g were divided into vehicle control, standard (indomethacin) and different test compound groups (n=6). The test compounds and indomethacin were administered orally in 2% sodium CMC orally at a dose of 100 and 50 mg/kg, respectively. After 6 h later, the rats were sacrificed, and their stomach was removed. Formal-saline (2% v/v) was then injected into the totally ligated stomach for storage overnight. The next day, the stomach were opened along the greater curvature, washed in warm water, and examined under 3-fold magnifier. The lengths of the longest diameters of the lesions were measured and summated to give a total lesion score (in mm) for each animal, the mean count for each group being calculated [36] and results are depicted in Table 3.
Statistical analysis: Statistical analysis of the biological activity of synthesized compounds on animals was performed by one-way variance (ANOVA) followed by Tukey's test; as "p" value of less than 0.05 was considered as statistically significant. All values were expressed as mean ± SEM. The SIGMASTAT, version 2.0 by Jandel Corporation was used for statistical analysis.
In vitro Antioxidant activity: The in vitro antioxidant activity for all the newly synthesized compounds was performed by FRAP assay based on the reduction of a colorless Fe 3+ -tripyridyltriazine complex into a blue-colored Fe 2+ -tripyridyltriazine complex [37]. The working FRAP reagent was prepared by mixing 10 ml of sodium acetate buffer (pH=3.6) with 1 ml of TPTZ (2,4,6-tri(2-pyridyl)-1,3,5-triazine) solution and 1 ml FeCl 3 .6H 2 O in a volume ratio of 10:1:1, respectively. The FRAP reagent was warmed to 37°C before being used and the assay was started by adding 228 μl of FRAP reagent into 96-well microtiterplate. The 12 μl of methanol as blank, test samples dissolved in methanol and ascorbic acid as standard dissolved in water, were taken into the wells. The reaction was allowed to run for 30 min and absorbance was read at 593 nm. The experiments were performed in triplicate and their mean was calculated for each compound and the absorbance change was translated into FRAP value (in μM) by following formula:

Synthesis of compounds
A novel series of compounds are designed by taking cue from the structures of indomethacin and sulindac with the absence of acidic moiety while retaining the functional groups that are required to bind with cyclooxygenase enzyme. Most of the COX-2 inhibitors contain sulfone moiety and the oxygen of the sulfone group interacts Values are mean ± standard error of mean (SEM) (n=6) a Statistically significant compared to control group (p ≤ 0.05) Data was analyzed by unpaired one-way ANOVA test  Values are mean ± SEM (n=6). a Statistically significant compared to Indomethacin (p ≤ 0.05).  [25]. Sulfonylation of compounds 1a, 1b and 1c was done in the presence of dry pyridine and benzenesulfonyl chloride to give intermediate compound 2a, 2b and 2c [26], which were further reacted with aryl amines of choice to get target compounds 3a-c, 4a-c and 5a-c. The with Hist90, Arg513, and Gln192 and forms hydrogen bonds with the enzyme pocket. Moreover, benzimidazole is recently emerged as pharmacophore of choice for anti-inflammatory analgesic action [24]. Inspired from abovementioned findings and with a new hope to develop gastroprotective NSAIDs, we designed novel 1, 2, 5-substitited benzimidazole derivatives (Figure 1). The synthetic strategy employed for the construction of novel benzimidazole derivatives is given in purity of the compounds was achieved by column chromatographic separation using silica gel as stationary phase and chloroform: methanol as mobile phase.

Biological assays
The entire series of newly synthesized benzimidazole derivatives were biologically tested in order to evaluate their pharmacological activity. The anti-inflammatory activity of all the test compounds was carried out in rats by the carrageenan-induced rat paw edema model test with interesting activity ranging from 20.90-46.27%. It is observed that the tested compounds 3b, 4b, and 5b with electron donating substituents exhibited significant reduction in edema, 3h post dosing of test compounds, i.e. 40.30, 46.27 and 31.34% comparable to standard drug indomethacin, which exhibited reduction in edema as 47.76% ( Table 1). The test compounds 3b, 4b, and 5b that exhibited potent antiinflammatory activity were further selected to evaluate their analgesic activity. The compounds 3b, 4b, and 5b exhibited percentage protection as 56.87, 61.37 and 48.10% comparable to acetyl salicylic acid which was found to 63.51% against acetic acid-induced writhing test. The ulcerogenic potential of 3b, 4b and 5b was also studied in rats, which was appreciably lower than indomethacin. The mean lesion scores for the test compounds were 16.33, 14.67 and 19.00 mm when compared to indomethacin which was found to 30.67 mm. These findings suggest that the compounds 3b, 4b and 5b can produce less gastric ulceration and may be considered as safer for treating inflammatory conditions. The analgesic and ulcerogenic activity results are illustrated in Table 2 and Table 3, respectively.
It has been well known that local generation of various reactive oxygen species plays a significant role in the formation of gastric ulceration associated with NSAIDs therapy [27,28]. These observations indicate that NSAIDs with antioxidant activity could be a useful approach to prevent gastric ulcers. In present investigation, the antioxidant potential of all the newly synthesized compounds was accessed in vitro by Ferric reducing antioxidant power FRAP assay. The increase in absorbance is proportional to the antioxidant content. From the results, it is observed the compounds 3b, 4b and 5b were the most efficient compounds in FRAP assay with strongest ferric reductants FRAP values as 272.4, 325.6 and 230.6 μM (Table 4).

Drug-likeness properties
The present study also attempts the calculation of drug-likeness properties of all the newly synthesised derivatives. Lipinski's Rule of five is a rule to predict high probability of success or failure due to druglikeness for molecules complying with 3 or more of the following [29]:


High lipophilicity (expressed as logP less than 5)  Molecular weight less than 500 g/mol The molecule's hydrophilicity can be reliably evaluated using its logP value (logarithm partition coefficient). Molecules with logP<5 and molecular weight (MW) of 500 or less are considered to the best candidates for better oral absorption. The newly synthesized derivatives have logP in the range of 3.2-4.7 and MW in the range of 393.4-453.4 g/mol indicates good bioavailability with less than 10 freely rotatable bonds [30,31]. The polar surface area (PSA) of all the derivatives is in the range of 72.3-164.0 shows their ability to permeate cell membranes [32]. Similarly, all the derivatives exhibited fewer than 6 hydrogen bond donors (HBD) are viable drug candidates with no violation of drug-likeness [33]. Drug-likeness properties of all newly synthesized compounds were assessed by using ACD/Labs software and the results are disclosed in Table 5.

Conclusion
In our attempt to discover potent and safer anti-inflammatory, analgesic agents with lower gastric ulceration, a series of 1, 2 and 5-substituted benzimidazole derivatives were synthesized and biologically evaluated. Further, the results suggested that the compounds 3b, 4b and 5b were found to possess encouraging antiinflammatory and analgesic activity with significant reduction in ulcerogenic side effect. The absence of gastric damage may be attributed to their antioxidant activity. Hopefully, in the future, the compounds 3b, 4b and 5b could serve as lead compounds for developing a novel class of potent as well as orally active GI-safer anti-inflammatory analgesic agents.