Synthesis , Pharmacological Evaluation , Molecular Docking and in silico ADMET Prediction of Nitric Oxide Releasing Biphenyls as Anti-Inflammatory Agents

Article history: Received on: 31/05/2017 Accepted on: 06/08/2017 Available online: 30/10/2017 Various 2-(4'-methyl-N-phenyl substituted)-1,1'-biphenyl-2-ylcarboxamido-2-oxoethyl nitrate analogues were synthesized and evaluated for analgesic, anti-inflammatory, ulcerogenic activity and nitric oxide release study. Compounds VM-4, VM-6, VM-9, VM-10, VM-11 and VM-12 exhibited good analgesic and anti-inflammatory activity compared to standard drug diclofenac. The compounds also showed decreased gastro-intestinal ulcerogenicity and gastro-protective activity in histopathological studies resulting in absence of mucosal injury. All the synthesized compounds were found to have significant in vivo nitric oxide releasing activity. The molecular docking was performed to understand the binding mode of these compounds. Results of this study indicated that these NO-NSAIDs may have affinity towards COX-2 active site which can further be explored for selective or non-selective inhibitors.


INTRODUCTION
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used medicines for treatment of various inflammatory conditions such as cancers, diabetes, Alzheimer's and Parkinson's disease.(Husain et al., 2016, Mahdi et al., 2015).The anti-inflammatory effect of NSAIDs arises from their ability to inhibit cyclooxygenase (COX) enzyme.Bis-dioxygenation and subsequent reduction of arachidonic acid (AA) to prostaglandin (PG) G2 and PGH2 formation was catalyzed by COX Enzyme (Rouzer and Marnett, 2003).This process leads to gastrointestinal (GI), renal and hepatic side effects in the patients who undergo chronic treatment.GI irritation, ulceration, dyspepsia and bleeding are common side effects of NSAIDS therapy.The treatment options which can be used for inflammatory diseases are unsatisfactory and complicated due to their compromised efficacy and adverse effect profile (Zayed et al., 2014).As a result Morbidity and Mortality are major consequences of toxicity induced by NSAIDs (Bhandari et al., 2010).Hence, the solution for this problem is to boost and reform safety profile of NSAIDs.One of the approaches is attaching the nitric oxide releasing group to the parent NSAID by a short-chain ester linkage called Nitric oxide (NO)-releasing NSAIDs (Miller et al., 2007).
The advantage of NO-NSAIDs is that the antiinflammatory and antipyretic activity of original NSAIDs will remain same.
Further, nitric oxide release leads increase in gastrointestinal defense mechanism, over production of mucous and bicarbonate release (Bhandari et al., 2009).So, NO-NSAIDS have emerged as reliable option.Among the NO-NSAIDs that have come into clinical trials are NO-naproxen, NO-aspirin, NOketoprofen, etc. Potency aggravated by 10 fold with minimizing the animal writhing in NO-naproxen as compared to naproxen after intra-peritoneal injection of acetic acid.In vivo and in vitro studies reveal that NO-aspirin shows more potent anti-thrombotic action as compare to aspirin.Further, NO-flurbiprofen increases renal function as well as reduces nephrotoxicity.NO-flurbiprofen and NO-aspirin debilitated the brain inflammatory response, in an animal model of chronic neurodegenerative disease.
In this paper we report structure optimization, design, synthesis and pharmacological evaluation of some biphenyl derivatives coupled with NO donor moiety.We have performed docking studies using SYBYL-X 1.3 to establish the binding modes of synthesized compounds to understand mode of antiinflammatory activity.

Chemistry
All the chemicals used were procured from commercial sources such as Sigma-Aldrich, Merck and Loba Chemie and were purified prior to use.The melting points of synthesized compounds were taken on Veego VMP-D digital melting point apparatus by open capillary.Merck pre-coated silica gel F 254 TLC plates was used for monitoring of reaction.
TLC plates were visualized using iodine in a chamber or observed under UV light.Fourier transform infrared (FT-IR) spectra were recorded in anhydrous potassium bromide (KBr) disk on "Jasco FTIR 4100" and are reported in cm -1 .
Proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded in DMSO-D 6 using "BrukerAvance (400 MHz) with tetra methyl silane (TMS) as an internal standard.Elemental analysis of compounds was determined on vario MICRO V2.0.3,Elementar analysis system GmbH.

General procedure of 4'-methylbiphenyl-2-carboxamide derivatives (2a-2q)
A mixture of 4'-methyl biphenyl-2-carbonyl chloride (0.01mol), aromatic or heterocyclic amine (0.01mol) and triethylamine (0.01mol) in dry toluene (20 ml) were refluxed with stirring for 14-16 hr.(Shah et al., 2010).The progress of reaction was monitored by TLC.The reaction mixture was filtered and washed with 25 ml of water to remove traces of triethylamine.The toluene layer was dried over anhydrous sodium sulphate and solvent was removed off on rotary evaporator to obtain the product.The residue was crystallized from methanol.

General procedure of N-(2-chloroacetyl)-4'-methyl-N-phenyl substituted-biphenyl]-2-carboxamide (3a-3q)
Chloroacetyl chloride (0.04 mol) was added to a solution of carboxamide (0.04 mol) in acetone (50 ml) and the reaction mixture heated under reflux on water bath for 4 hr (Bhandari et al., 2010).The solvent was removed under vacuum and residue was purified over column of silica gel and eluted with chloroform.The elute was concentrated and product was crystallized with ethanol to obtain the product.

Pharmacology
The 2-(4'-methyl-N-phenyl substituted)-1,1'-biphenyl-2ylcarboxamido-2-oxoethyl nitrate derivatives (VM 1-17) were tested for anti-inflammatory, analgesic and for acute ulcerogenicity.Diclofenac was taken as a reference standard.Albino rats of Wistar strain of either sex (120-140 g) were used to execute the experiment.Animal were fasted for 24 hr prior to the experiments and water provided ad libitum the relative humidity was maintained at 25 ± 2ºC, 50 ± 5% and 12 hr light/dark cycle.The suspension of test compound in 1% aqueous carboxy methyl cellulose (CMC) solution was administered orally to animals under experiment.

Acute oral toxicity study
As per OECD (Organization for Economic Cooperation and Development) (AOT No. 423) guidelines adult albino mice of either sex were subjected to acute toxicity studies.Doses of 550, 1750, 2000 mg/kg of test compounds were administered orally to mice and control group received normal saline 10 ml/kg, p.o.The mice were kept in polypropylene metabolic cages and observed continuously 2hr.for behavioral, neurological and autonomic profiles and for any lethality during next 48 hr.Use of AOT 423 software was made to obtain higher doses for LD 50 determination as per OECD guidelines.

Acetic acid-induced abdominal writhing
Irritant (acetic acid) was injected intraperitoneally to induce pain in mice (Koster et al., 1959).The animals show writhing i.e. contractions of abdomen, turning of trunk and extension of hind limb, which was observed in treated groups of The animals were pre-treated orally with test compounds and diclofenac, 60 min before administration of acetic acid (0.9%, intra-peritoneal).Period of 15 min was allocated for cumulative counting of the number of abdominal constrictions (full extension of both hind paws).The analgesic activity was as mean number of writhes and percent inhibition, which was calculated by following formula: where, Wc and Wt are mean number of writhes observed in vehicle (control) group and treatment group respectively (Table 1).
The hind paw edema was produced by injecting 0.1 ml of 1% carrageenan in the right hind paw of each rat under the subplantar region.Rats were pre-treated with orally administered test compounds and diclofenac 1hr before carrageenan injection.The rat pedal volume up to the ankle joint was measured using plethysmometer (UgoBasile, Italy) at 1, 3, and 5 hr.after the carrageenan injection (time 0 considered).Increase in the paw edema volume was considered as the difference between 1, 3 and 5 hr and expressed as the mean difference in paw volume (ml).Percent inhibition of edema volume between treated and a control group was calculated as following formula and results were presented in Table 2.
% Inhibition = [Vc Vt /Vc ] *100 Where, Vc and Vt represent mean increase in paw volume in control and treated groups, respectively.

Acute ulcerogenicity studies
Literature procedure (Cioli et al., 1979) was followed to evaluate the acute ulcers in rats.Electron microscope was used for examination of mucosal damage.Scoring system was used to assess mucosal damage.Score description 0.0 Normal (no injury).0.5 Latent injury or widespread bleeding.1.0 Slight injury.2.0 Severe injury.3.0 Very severe injury.4.0 Widespread lined injury or widened injury/erosion.
Severity index of gastric mucosal damage was depicted by the mean score of each treated group minus the mean score of control group.Data are expressed as mean ulcer score.One-way ANOVA by Dunnett's test was used to determine the significance of the difference between the standard group and rats treated with the test compounds.The differences in results were considered significant when P was found to be <0.01.

Histopathology study
Rats were sacrificed 4 hr after the cold stress and stomach specimens were collected and put into 10% formalin solution for histopathology study (Sánchez-Fidalgo et al., 2004).Greater curvature of stomach which included the ulcer base and both sides of the ulcer margin was taken and fixed in 10% formalin for 24 hr at 4º C and embedded in white solid paraffin.Eosin staining was carried out to analyze morphological changes.Changes in GI epithelial morphology were analyzed and recorded in the form of images.The results are shown in Fig. 3.

In vitro Nitric Oxide release assay
Compound (20 ml) was taken in dimethyl sulfoxide (DMSO) and added to 2 ml of 1:1 v/v mixture of either 50 mM phosphate buffer (pH 7.4) or of an HCl solution (pH 1) with methanol, containing 5 × 10 -4 M L-cysteine.The final concentration of drug was 10 -4 M.After 1 hr at 37 ºC, 1 ml of the reaction mixture was treated with 250 ml of Griess reagent [Sulfanilamide(4 g), N-naphthyl ethylene diaminedihydrochloride (0.2 g), 85% phosphoric acid (10 ml) in distilled water (final volume: 100 ml)].The absorbance was measured at 540 nm.Calibration curve was prepared using Sodium nitrite standard solutions (10-80 mmol/ml).The results were expressed as the percentage of NO released (n = 2) relative to a theoretical maximum release of 1 mol NO/mol of test compound (Abadi et al., 2005)

Statistical analysis
Two-way ANOVA followed by post hoc Dunnett's test was used for data analysis.Data from the acetic acid induced writhing model are expressed as the mean number of writhes ± SEM and were analyzed by one-way ANOVA followed by Dunnett's test.The level of significance was set at p < 0.05.GraphPad Prism 5.2 (USA) statistical software was used to perform statistical calculation.

Docking
Docking was carried out using Sybyl-X 1.3.The crystal structure of Cyclooxygenase enzyme (COX-2) complexed with ibuprofen retrieved from Protein Data Bank (4PH9).Hydrogen was added and energy minimization was carried out using Tripos Force Field.All the compounds were designed using Sketch module in Sybyl and energy minimization was carried out using Tripos Force Field.The amino acid residues in a radius 5.0 Å around Ibuprofen were selected as the active site.Other docking parameters were maintained as default.

Prediction of ADMET properties for the designed derivatives
In-silico prediction of ADMET (absorption, distribution, metabolism, excretion and toxicity) properties is very important for lead identification and optimizations.AdmetSAR is a free online server which is used to predict ADMET properties.The ADMET properties of 2-(4'-methyl-N-phenyl-[1,1'-biphenyl]-2ylcarboxamido)-2-oxoethyl nitrate derivatives were estimated using admetSAR online database (www.lmmd.ecust.edu.cn,accessed on 5 th March 2017).It provides inclusive data for different entities linked with known ADMET profiles.

Acute oral toxicity
The acute toxicity of compounds VM-4, VM-6, VM-9, VM-10 and VM-12 in mice was found to be greater than 2000 mg/kg.No lethal or toxic reactions were reported up to the antiinflammatory dose range.All the compounds were having LD 50 greater than 2000 mg/kg.

Analgesic activity
Analgesic activity was carried out using acetic acid induced writhing model.Writhing test is based on tissue injury increase the sensitivity to pain.Compounds VM-4, VM-6, VM-9, VM-12 were active compounds in the present series.The results of analgesic activity are present in Table 1.The graph of analgesic activity is present in Figure 1.

Group
No. of writhing ± SEM % inhibition % NO release

Anti-inflammatory activity
Carrageenan-induced rat paw edema assay method was used to compare anti-inflammatory activity.The obtained results are given in Table 2.The pharmacological experiments of this study showed that among the tested compounds VM-4, VM-6, VM-9, VM-10, VM-11, VM-12 exhibited significant antiinflammatory activities as compared to standard NSAIDs.For comparing the anti-inflammatory activity diclofenac sodium was used as a standard.The biphasic edema induced by carrageenan mediates its first phase by the release of histamine and 5-hydroxytryptamine followed by kinin release and then prostaglandin in the later phase.In most clinically effective antiinflammatory agents the second phase (3 hour) of edema is reported to be most sensitive.Anti-inflammatory effects of these series in third hour of edema suggest involvement of inhibition of prostaglandin.
Compound VM-11 and VM-12 shows highest antiinflammatory activity in the series.Compound VM-6 shows similar activity as compared to standard drug.Comparison of antiinflammatory activity with standard drug is shown in Figure 2.

Acute ulcerogenicity studies
The compounds VM-4, VM-6, VM-9, VM-10 and VM-12 which possessed promising analgesic activity and antiinflammatory activity were further screened for ulcerogenicity activity of stomach.Compounds VM-6, VM-9, VM-10 and VM-12 showed less signs of gastric ulceration compared to standard drug.The ulcer index is shown in Table 3.A less incidence of gastric erosion was observed for the highest active compound VM-12.

Histopathology study
The protective mucous layer of stomach specimen showed complete disruption along with severe ulceration in diclofenac treated rat (Fig. 3 specimen b).The gastric epithelial layer showed complete disruption by proliferation and migration of some epithelial cells from ulcer margin into ulcer crater in the tissue of diclofenac treated rat.
Electron microscope was used for scanning of stomach specimen which unfolded that the rat treated with 2-(4'-methyl-Nphenyl-[1,1'-biphenyl]-2-ylcarboxamido)-2-oxoethyl nitrate derivatives (Fig. 3 specimen c and d) showed less gastric mucosal injury compared with diclofenac treated group, while disruption of gastric epithelial layer was observed in Fig. 3 specimen e but less than diclofenac.

Molecular Docking
To investigate interaction between Cyclooxygenase enzyme and synthesized derivatives molecular docking was carried out.
Active compounds VM-4 binds with ARG amino acid.While VM-6 binds with ARG and ASN amino acid with docking score of 4.5 and 4.9 respectively.

CONCLUSION
A novel series of biphenyl derivatives possessing a variety of hetero aryl amines attached to nitric oxide releasing group were synthesized.Structure of all final compounds were characterized by IR, H-NMR, mass and elemental analysis and were subjected for analgesic and anti-inflammatory activity by in vivo models and were subjected to nitric oxide releasing studies.The compound VM-12 was found as the most active.The synthesized compounds VM-4, VM-6, VM-9, VM-10 VM-11 exhibited significant analgesic and anti-inflammatory activity compared to standard drug.Furthermore, these compounds showed little ulcerogenic activity as that of diclofenac.Histopathological studies showed that synthesized derivatives not just maintained but showed aggravated anti-inflammatory activity and are without gastro intestinal toxicities and also exhibit good nitric oxide releasing property.Structure activity relationship study indicates that VM-11 having fluorine atom in the structure increases the activity.VM-12 having fluorine and chlorine atoms and VM-6 having trifluoro group results in good activity.VM-9 having NO 2 in the structure also increases the activity.The synthesized compounds along with their anti-inflammatory activity were further subjected for docking and ADMET studies.Molecular docking study revealed that these compounds fit into the cavity of COX-2 receptor via interaction with ARG amino acid.Also, the pharmacokinetic parameters for the entire 17 synthesized compounds were obtained within the acceptable range defined for human use revealing their potential as possible druglike compounds.Hence these compounds can serve as good leads for further modification and optimization to obtain better compounds.
for their support.The authors are thankful to Dr. S.S. Kadam, Vice Chancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Pune for their help.
Financial support and sponsorship: Nil.

Conflict of Interests:
There are no conflicts of interest.

Fig. 1 :
Fig. 1: Analgesic activity of VM 1-17 compared with standard drug determined by acetic acid induced writhing in mice.

Fig. 3 :
Fig. 3: Haematoxylin and eosin immunohistochemical staining of gastric ulcers after ulcer induction in rats.As illustrated in figure specimen: (a) showed intact mucous membrane in control treated rat showing granular tissues composed of macrophages, fibroblasts and endothelial cells forming microvessels.(b) Congestion of mucosal blood vessels was observed in diclofenac treated group.(c) Less gastric damage was observed to mucosa of rat treated with test compound, VM 16.(d) Disruption of gastric epithelial layer was observed for VM 6 specimen but less than diclofenac.(e) Disruption of gastric epithelial layer was observed for VM 4 specimen but less than diclofenac.

Table 1 :
Effect of VM1-16 series on writhing induced by acetic acid in mice and in vitro release of nitric oxide.

Table 3 :
Ulcer index of most active compounds.