Anti-inflammatory, analgesic evaluation and molecular docking studies of o-benzoyl benzoic acid based 1,3,4-oxadiazole analogues

A novel series of o-benzoyl benzoic acid based 1,3,4-oxadiazoles were prepared and subjected to anti-inflammatory, analgesic activity as well as molecular docking studies to target cyclooxygenase-2 enzyme.1,3,4-Oxadiazole derivatives were screened for anti-inflammatory activity in carrageenan-induced rat paw edema and analgesic activity by tail immersion method. In synthesized compounds, the free carboxylic group, which is responsible for gastric side effects, was derivatized by heterocyclic 1,3,4-oxadiazole bioactive core, which showed good interaction with COX-2 receptor with good docking score. Among all thesynthesized compound having m-methoxy-p-hydroxy have emerged out as potential COX-2 inhibitor.


INTRODUCTION
• Non-steroidal anti-inflammatory drugs (NSAIDs) form an imperative class of commonly used therapeutic agents due to their antiinflammatory, analgesic and antipyretic effects.
• The biological activity of NSAIDs is associated to suppression of prostaglandin biosynthesis by inhibiting the enzyme cyclooxygenase (COX). COX is an endogenous enzyme which catalyzes the conversion of arachidonic acid into prostaglandins.
• The isoforms (COX-1 and COX-2) catalyze the same biochemical transformation but are subject to a dissimilar expression regulation. COX-1 is responsible for the physiological function of prostaglandins (PGs) like maintenance of the integrity of the gastric mucosa and provides adequate vascular homeostasis whereas COX-2 is expressed only after an inflammatory stimulus.

Introduction continued
• 1,3,4-oxadiazole nucleus possess a diversity of useful biological effects such as anti-edema and anti-inflammatory activities.
• 1,3,4-Oxadiazoles have anti-inflammatory activity by virtue of dual mechanism i.e., inhibiting both COX/LOs to reduce gastric ulceration [2]. The reported literature confirms that gastrointestinal side effects of NSAIDs such as irritation, GI bleeding etc. are due to the presence of a free carboxylic group in drug.
• Our studies and studies of other researchers [6] have shown that derivatization of the carboxylate function of some NSAIDs resulted in an increased antiinflammatory activity with a reduced ulcerogenic effect.
• Hence, by including the oxadiazolyl moiety, expecting to get better antiinflammatory compounds.

Material and Methods • Preparation of target compounds
Target compounds were prepared previously in our laboratory ( Figure 1). The aromatic esters were prepared by Fischer esterification. Further these esters were subjected to formation of aryl hydrazide in presence of hydrazine hydrate and cyclized in presence of bromine, acetic acid and sodium acetate by stirring for 2-3hours. Similarly compounds (13b-e) were prepared. The physical properties and spectral characterization were already discussed.

 Chemicals and instruments
• All chemicals and reagents used in the estimation were of analytical grade.
• Carrageenan was purchased from sigma chemicals.
• Molecular docking studies were carried out on Molecular Virtual Docker 5.0.

Acute toxicity studies
It has been fond from literature survey that 1,3,4-oxadiazole derivatives in a dose of 1000 mg/kg produces mortality (LD 50 ) in rats.
So one tenth of the LD 50 i.e., 100 mg/kg (ED 50 ) was selected as a dose for anti-inflammatory and analgesic activity.
• In vivo anti-inflammatory activity In carrageenan model albino rats of all groups were treated with subcutaneous injection of 0.1 ml of 1% w/v solution of carrageenan into the sub plantar region of the right hind paw. The paw was marked with permanent marker at the planter region where paw volume was to be measured.
• The diclofenac sodium (10mg/kg) and test compounds (100mg/kg) were suspended in 0.3% sodium carboxy methyl cellulose. The test compounds, vehicle (control) were administered p.o with the help of gastric cannula half an hour after the injection of carrageenan in sub planter region of right paw.
• Mean normal paw volume was measured 30 min. prior to carrageenan injection by using plethysmometer. Mean increase in the paw volume for control group (after carrageenan injection) and test group was measured at 1hr, 2hr and 3hr [9]. Percent inhibition of inflammation after test/standard was calculated using the formula 1.

% inhibition = V c -V t /V c X 100 …………… (1)
Where V t is the of paw volume (ml) of test/standard compound at corresponding time and V c is paw volume (ml) of control.

• Analgesic activity
Tail immersion method is based on the observation that morphine like drugs selectively prolongs the reaction time of the typical tail withdrawal reflex in mice.
• Albino rats were divided in twenty six groups each containing six animals. The tail of mice was immersed (1-2 cm) in warm water kept constant at 55ºC.
• The reaction time was recorded through stop watch (the reaction time is the time taken by the rat to flick their tails).
• The latent period of the tail flick response will determine before and 15, 30, 60 and 120 min. after drug administration.
• Statistical analysis • The results were expressed as mean±SEM for six animals in each group for anti-inflammatory and analgesic activity. All the grouped data was statistically evaluated. • Hypothesis testing method included one way analysis of variance (ANOVA) followed by Dunnett's comparison test. P-values of less than 0.05 were considered to indicate statistical significance.

 Molecular docking studies
Comparative docking of set of ligands with specific proteins involves methodology with Easy user interface and their respective scoring function provided by Molegro Virtual Docker. Molecular docking studies were performed to target COX-2. The compound which have shown potent antiinflammatory and analgesic activity (13e) were subjected to molecular docking to target COX-2 (Pdb-1CX2) .

Steps in methodology:
1) Importing a protein file and ligand file and preparation of ligands.
2) Protein preparation and detecting cavities of protein molecules.
3) Executing a docking set up through docking wizard panel.

4) Poses of protein-ligand complex obtained after docking process with
their specific mol dock scores displayed in output file.

 Results
In vivo anti-inflammatory activity 1,3,4-Oxadiazole of o-benzoyl benzoic acid series compounds were evaluated for anti-inflammatory activity in carrageenan induced rat paw edema. Among all the compounds, 13e have shown maximum activity at 100mg/kg (Table 1).    Figure 2 and 3).