4,5-DIHYDRO-1H-PYRAZOLE-1-CARBALDEHYDE: SYNTHESIS, ANTI-INFLAMMATORY ACTIVITY AND DOCKING STUDY

A series of 3-phenyl-5-aryl-4,5-dihydro-1H-pyrazole-1-carbaldehyde 4(a-j) was prepared by heating chalcones 3(a-j) and hydrazine hydrate in presence of formic acid. The synthesized compounds were investigated for in-vivo anti-inflammatory activity in Carrageenan induced rat paw edema model. Some of the synthesized derivatives exhibited good anti-inflammatory activity as compared to diclofenac, while some derivatives have shown comparable anti-inflammatory activity to that of diclofenac. All the synthesized derivatives were found to be potent anti-inflammatory agents. Some of the derivatives were evaluated for ulcerogenic potential and their ulcer index was found to be less than the standard drug diclofenac. The molecular docking analysis was performed to understand the binding interactions of these compounds to COX-2 enzyme. The results from the present investigation suggests that 3-phenyl-5-aryl-4, 5-dihydro-1H-pyrazole-1-carbaldehyde as a promising template for the design of new anti-inﬂammatory agents.


INTRODUCTION
Inflammation is defined as the local response of living mammalian tissues to injury due to any agent [1].Non sterodial anti-inflammatory drugs (NSAIDs) are one kind of therapeutics, widely used in the world because of their high efficacy in reducing pain and inhibiting inflammation.NSAIDs such as Celecoxib and Diclofenac can inhibit the enzyme cyclooxygenase (COX-1 and COX-2), which catalyze the biotransformation of arachidonic acid to prostaglandins (PGs) and to Thromboxane A 2 .These are the mediators of pain, inflammation and fever.Hence, the development and discovery of new agents that can inhibit the COX-1 and COX-2 activity will be of importance for the controlling inflammation.
Clinical studies have suggested that selective COX-2 inhibitors could cause typical COXmediated side effects such as gastrointestinal injury, high blood pressure, and hypersensitivity this may be due to the presence of carboxyl group.Furthermore, concerns have been raised about the cardiovascular safety of selective COX-2 inhibitors and some of them, like Vioxx or Bextra, have been withdrawn from the market.In our search for synthesis of novel NSAID with similar or greater efficacy than other NSAIDs and with little or no gastric side effects, we synthesized a series of 3-phenyl-5-aryl-4, 5-dihydro-1H-pyrazole-1-carbaldehyde 4(a-j) and evaluated their ability to inhibit carrageenan induced paw edema in rats.Among the numerous 1,5-diarylpyrazoles studied as possible selective COX-2 inhibitors, only few are substituted at the position 4 of the heterocyclic ring, whereas most have a substituent at the position 3.Therefore, in the present work, novel 1,3, 5-trisubstituted pyrazoline derivatives were designed and synthesized having1-carbaldehyde group, 3-phenyl and 5-aryl group (Fig. 1) with the aim of improving their anti-inflammatory activity and reducing their ulcerogenic properties.The aldehyde group is metabolized in vivo by the NAD (P) dependent aldehyde dehydrogenase (ALDH1) to corresponding carboxylic acids, which are excreted from the body as such or as conjugates [2].The compounds were designed in such a way to get the butterfly structure, an essential feature, required for anti inflammatory activity.The synthesized compounds were investigated for in-vivo anti-inflammatory activity in Carrageenan induced rat paw edema model.Some of the derivatives were evaluated for ulcerogenic potential and their ulcer index was found to be less than the standard drug diclofenac.Molecular docking study was also performed using VLife MDS 4.3 to understand the binding interactions of these compounds.Furthermore pyrazole derivatives have a long history of application in agrochemicals and pharmaceutical industry as herbicides and active pharmaceuticals [3].Pyrazolines have been reported to show a broad spectrum of biological activities including antibacterial [4], antifungal [5], anti-inflammatory [6,7] analgesic [8],

Scheme 1 Scheme of synthesis
The chalcones 3(a-j) were obtained via condensation reaction of acetophenone and substituted benzaldehyde, in presence of aqueous alkali.The synthesized chalcones were refluxed with hydrazine hydrate in presence of formic acid to give the target compounds 4(aj).The purity of the synthesized compounds was checked by TLC and melting points were determined in open capillary tubes and are uncorrected.The physical characterization data of the synthesized compounds are presented in Table 1.The data obtained from IR, 1 H NMR, 13   C NMR Elemental analyses and Mass spectroscopy confirmed the proposed structures.

Anti-inflammatory activity
All the synthesized compounds were screened for anti-inflammatory activity at a dose of 10 mg/kg intra peritoneally in carrageenan induced rat paw oedema model.Standard drug (Celecoxib and Diclofenac) and test compounds were injected intra peritoneally at dose 10 mg/kg.The activity assessed after 1, 2, 3, 6 h of drug administration.The synthesized derivatives 4b, 4c, 4f and 4i showed excellent anti-inflammatory activity, more than diclofenac but less than celecoxib while the derivatives 4d, 4e, 4h, and 4j showed comparable anti-inflammatory with diclofenac.All synthesized compounds exhibited moderate to good anti-inflammatory activity.The data of percentage inhibition of antiinflammatory activity are presented in Table 2.
Table 2 Results of anti-inflammatory activity of title compounds 4 (a-j) against carrageenan induced rat paw edema model in rats.

Compound Code
Mean paw volume in ml ± SEM (% Inhibition)

Ulcerogenic activity
The major side effect of NSAIDs is gastric ulceration.The ulcerogenic liability was evaluated for 4b, 4c, 4f at dose level of 100mg/kg.
calculating the ulcer index in treated and control animals.Diclofenac was used as standard drug for ulcerogenic potential studies.Results are given in these three compounds cause compared to diclofenac.Hence gastric tolerance to these compounds was better than that of standard drug diclofenac.The results are expressed as mean ± sem (n=5).
followed by dunnett's test.**p < 0.01, *p < 0.05 significant from control Those compounds which exhibited good anti-inflammatory activity were further tested for ulcerogenic activity at dose level of 100 mg/kg.Graphical presentation of results of anti inflammatory activity is shown in Fig. 2.

Fig.2 Graph of anti-inflammatory activity
The major side effect of NSAIDs is gastric ulceration.The ulcerogenic liability was evaluated at dose level of 100mg/kg.The gastric ulcerogenic potential was evaluated by calculating the ulcer index in treated and control animals.Diclofenac was used as standard drug for ulcerogenic potential studies.Results are given in

Graphical presentation of results of anti-
The major side effect of NSAIDs is gastric ulceration.The ulcerogenic liability was evaluated The gastric ulcerogenic potential was evaluated by calculating the ulcer index in treated and control animals.Diclofenac was used as standard which indicates that, less gastric ulceration at the above mentioned oral dose as compared to diclofenac.Hence gastric tolerance to these compounds was better than that of sized compounds in comparison to diclofenac

Ulcer index (mean±SEM)
Data analyzed by one way anova followed by dunnett's test.**p < 0.01, *p < 0.05 significant from control The docking score along with number of hydrophobic, hydrogen bonding and the binding energy of compounds with COX-2 enzyme is presented in Table 4.

General procedure for synthesis of chalcones 3(a-j)
Equimolar quantity of acetophenone 1 (0.01mole) and substituted aromatic aldehydes 2 (0.01 mole) were dissolved in ethanol (30 ml) and ice-cold solution of NaOH (10 ml, 10%) was added in portion keeping the temperature 25ºC with continuous stirring on magnetic stirrer.
The reaction mixture was corked and kept in ice chest overnight.The completion of reaction was monitored by TLC.The product obtained was filtered, washed with cold water until neutral to litmus paper and finally washed with 5 ml absolute ethanol.The dried compounds were recrystallized from absolute ethanol.
Representative spectral data for some compounds

MOLECULAR DOCKING STUDY
To identify potential anti-inflammatory lead compounds among compounds 4(a-j), docking calculations were performed using VLifeMDS 4.3 into the 3D structure of the catalytic site of COX-2 enzyme (PDB code: 6COX).Docking procedure was followed using the standard protocol implemented in VLifeMDS 4.3 [13].The conformers thus obtained,

PHARMACOLOGICAL SCREENING
The new derivatives obtained by the above mentioned procedure were undertaken for the anti-inflammatory studies by the carrageenan induced rat paw edema model.Celecoxib and diclofenac were used as standard drugs.The standard and test compounds were given intraperitoneally at dose level of 10 mg/kg.Carrageenan induced rat paw edema is a nonspecific inflammation resulting from a complex of diverse mediators.Since edema of this type is highly sensitive to NSAIDs, carrageenan induced rat paw edema has been accepted as a useful model for studying new anti-inflammatory agents.This model reliably predicts the anti-inflammatory efficacy of the NSAIDs.[14] Anti-inflammatory activity The animals were procured under the CPCSEA number CPCSEA/IAEC/Pharm .Chem/14/2011-12/56 approved by Institutional Animal Ethics Committee (IAEC).Swiss Albino rats (150-200 g) were supplied by Wockhardt Ltd Aurangabad.The animals were housed in stainless steel cages, divided into groups of five animals each and deprived of food but not water 24 h before the experiment.The anti-inflammatory activity of the compounds under investigation was studied using carrageenan induced rat paw oedema.A suspension of the test compounds 4 (a-j) and standard drugs (celecoxib and diclofenac) in carboxy methyl cellulose (CMC) solution (0.5% w/v in water) was administered intraperitoneally in a dose level of 10 mg/kg.Control animals were treated similarly with CMC solution (0.5% w/v in water).After 1 h, 0.1 mL of freshly prepared 1% carrageenan solution was injected into the sub plantar region of the left hind paw of rats according to the method of Winter et al [15].
The volume was measured before and after carrageenan treatment at 1,2,3,6 h with the help of digital plethysmometer (Ugo Basil 7140, Italy).Paw edema volume was compared with vehicle control group and percent reduction was calculated by formula (Vc-Vt/Vc) × 100, Where Vc = paw volume of control group, Vt = paw volume of test group

Ulcerogenic activity
Adult albino rats weighing 150-200 g were divided into different groups consisting of five animals in each group.Animals were deprived of food but not water 24 h before the experiment.
Ulcerogenic activity was evaluated after oral administration of a suspension of test compounds 4b, 4c, 4f and standard drug diclofenac in carboxy methyl cellulose solution (0.5% w/v in water) in a dose level of 100 mg/kg.Control animals were treated similarly with CMC solution (0.5% w/v in water).After 5 h the rats were sacrificed by decapitation, the stomachs were removed, collected, opened along the greater curvature, washed with distilled water and cleaned gently by dipping in saline.The mucosal damage of each stomach was examined.The mucosal damage was assessed according to following formula and scoring system [16].

Fig. 1
Fig.1 Structure of Celecoxib, Diclofenac and target compound

Figure 3 .Fig. 3 .
Fig. 3. Docking of compounds 4g, 4h , 4j and celecoxib (Lower right panel).Ligands are shown in red color.Hydrogen bonds are shown in green color.Hydrophobic bonds are shown in sky.
were optimized (MMFF) till they reached a rms gradient energy of 0.001 kcal/mol Å. Docking of the conformers of each molecule, into the COX-2 modeled protein was done by positioning with the active site of cavity 1.The complexes were then minimized using the MMFF method, till they reached an rms gradient of 0.1 kcal/mol/ Å.The binding energy in kcal/mol and the ligand-receptor interaction energy obtained after docking the ligands into the enzyme active site can be defined as: E = InterEq + InterEvdW + IntraEq + IntravdW + IntraEtor Where, InterEq: Intermolecular electrostatic energy of complex, InterEvdW: Intermolecular vdW energy of complex, IntraEq: Intramolecular electrostatic energy of ligand, IntraEvdW: Intramolecular vdW energy of ligand and IntraEtor: Intramolecular torsion energy of ligand.

Table 1
Solvent of recrystallization was ethanol; eluants used in TLC were benzene: methanol (4.5:0.5) for all compounds

Table 3
Ulcerogenic effects of synthe

Table 3 ,
which three compounds caused less gastric ulceration at the above mentioned oral dose as compared to diclofenac.Hence gastric tolerance to these compounds was better than that of Ulcerogenic effects of synthesized compounds in comparison to diclofenac The results are expressed as mean ± sem (n=5).Data analyzed by one way anova followed by dunnett's test.**p < 0.01, *p < 0.05 significant from control inflammatory activity were further tested for