SYNTHESIS OF 1-BENZOYL-3-PHENYL-1H-PYRAZOLE-4-CARBALDEHYDE AND EVALUATION OF THEIR ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITY

Objective: The main objective of this study is to synthesize a series of 1-Benzoyl-3-phenyl-1H-pyrazole-4-carbaldehyde (4a-e) derivatives and evaluation of the synthesized compounds for their antioxidant and anti-inflammatory activity. Methods: A series of substituted acetophenones are condensed with hydrazides to the corresponding hydrazones which are subsequently cyclized by using vilsmier-Haack reaction to give final series of 1-Benzoyl-3-phenyl-1H-pyrazole-4-carbaldehyde (4a-e) derivatives respectively. All newly synthesized compounds were characterized on the basis of infrared, proton nuclear magnetic resonance and mass spectral data and screened for their antioxidant and anti-inflammatory activities. Results: In view of the significant biological activity profile of Pyrazole, the synthesized compounds (4a-e) were evaluated for their antioxidant potency by DPPH, Nitric oxide, Hydroxyl radical scavenging, and Hydrogen Peroxide method. Compounds 4c and 4e showed potent antioxidant activity then standard. Synthesized compounds were also screened for anti-inflammatory activity. Among all the molecules 4c, 4e, and 4d showed significant activity as compared to standard drug diclofenac sodium. Conclusion: in this study, we synthesized 1-Benzoyl-3-phenyl-1H-pyrazole-4-carbaldehyde (4a-e) derivatives. Further, these derivatives showed significant antioxidant and anti-inflammatory activity. Among them, two molecules 4c and 4e have shown near action to the standard.


INTRODUCTION
Pyrazoles are unique in their chemical behavior not only among heterocyclic compounds in general but also among related azoles. Pyrazoles are also a class of compounds that have the ring C3N2. The drugs containing a pyrazole ring are celecoxib and stanozolol.
The possibility that pyrazole is used directly in the synthesis of the amino acid has been investigated by supplying young melon seedlings with pyrazole alone and in association with various amino acids. The term pyrazole was given to this class of compounds by German Chemist Ludwig Knorr in 1883. In a classical method developed by German chemist Hans von Pechmann in 1898, pyrazole was synthesized from acetylene and diazomethane. In 1959, the first natural pyrazole, 1-pyrazolyl-alanine, was isolated from seeds of watermelons [1]. During the past years, considerable evidences have also accumulated to demonstrate the efficacy of pyrazoles including antimicrobial [2,3], anti-Inflammatory [4], anticonvulsant [5], antitubercular [6,7] antiproliferative, antiangiogenic [8], hypoglycemic agent, antimalarial agent, anti-viral, and analgesic activity [9].
In view of above mentioned pharmacological importance of pyrazoles, we aimed to synthesize series of substituted pyrazole analogs by the Vilsmeier-Haack reaction method, in order to investigate the effect of pyrazole on their in vitro antioxidant and anti-inflammatory activity.

MATERIALS AND METHODS
All chemicals used were of Laboratory Reagents grade. Entire chemicals required for the synthesis and other experimental work were obtained from SD fine chemicals India and Molychem. Melting points of all synthesized compounds were determined in open capillaries on tempo apparatus. Spectroscopic data were recorded by the following instruments: Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded by BRUKER-300 MHz; MASS spectra were recorded in apex mass spectrometer. Infrared (IR) spectra were recorded on BRUKER alpha Fourier transform IR spectrometer. Purity of the samples was checked by thin-layer chromatography (TLC) using silica gel-G precoated plates and spots were detected by Ultraviolet chamber.
General procedure for the synthesis of methyl benzoate (1a-e) Substituted benzoic acid (0.01 mol), in 25 ml of methanol and 0.5 ml of conc H 2 SO 4 was added through sides of the walls. The resulting mixture was allow to reflux for 3 h, the contents were allow to cool on water bath and were neutralized with 5% NaHCO 3 solution. The separated precipitate was the filtered and recrystallized from methanol.

Sudha et al.
The reaction mixture was left overnight at room temperature and the solid separated was collected by filtration. It was washed thoroughly with water. The product was purified by recrystallization from methanol.
General procedure for the synthesis of N 1 -(1-phenyl ethylidene) benzohydrazide (3a-e) Mixture of acid hydrazides (2a-e) (0.01 mol) and substituted acetophenone (0.01 mol) in methanol (30 ml) and 3-4 drops of glacial acetic acid was heated under refluxed for 2 h. The reaction mixture was cool to room temperature. The separated precipitate was the filtered and washes thoroughly with water, dried, and recrystallized with methanol.
General procedure for the synthesis of 1-Benzoyl-3-phenyl-1Hpyrazole-4-carbaldehyde w(4a-e) N 1 -(1-phenyl ethylidene) benzohydrazide (3a-e) (0.004 mol) and mixture of Vilsmeier-Hacck reagent (prepared from 10 ml of DMF and 1.1 ml [0.012 mole] POCl 3 at °C) was added in small aliquots at a time and the reaction mixture was stirred at 60-65°C for 4 h. Completion of the reaction was judged by TLC. The reaction mixture was slowly quenched into crushed ice with stirring and neutralized it with solid NaHCO 3 . The precipitate was filtered off, dried, and purified by recrystallization from methanol.

In vitro antioxidant activity 2,2-Diphenylpicrylhydrazyl (DPPH) free radical -scavenging activity
The DPPH free radical scavenging capability was performed as the method described by Altarejos et al. [11]. Methanolic solution of DPPH (1.0 mL, 0.1 mM) was mixed with 3.0 mL of sample solution of different concentrations ranging from 10 to 320 µg/ml. The reaction mixture was incubated in dark at room temperature for 30 min and the absorbance was recorded at 517 nm against a blank. The assay was carried out in triplicate for each sample. The radical scavenging activity of ascorbic acid was also determined as standard. IC 50 values (concentration required to scavenge 50% of free radicals) of both ascorbic acid and test samples were determined. The activity was expressed by the inhibition percentage (I %) of DPPH radical, following the equation (1).
Where, Ac and As are the absorbance of the control and of the test/ standard sample respectively.

Nitric oxide scavenging activity
Nitric oxide radical scavenging activity was determined according to the method reported by Marcocci et al. [12]. In this assay, sample solution (4 ml) at different concentration (10-320 µg/ml) was mixed with 1.0 mL of 25 mM sodium nitroprusside solution in a test tube, and incubated for 2 h at 37°C. Incubated solution (2 mL) was mixed with 1.2 mL Griess reagent (1% sulfanilamide in 5% H 3 PO 4 and 0.1% naphthyl ethylenediamine dihydrochloride). which results in diazotization of the nitrite with sulfanilamide and subsequent coupling with naphthyl ethylenediamine dihydrochloride to form a chromophore. The absorbance of chromophore was measured immediately at 570 nm. Control experiment was also carried out in similar manner taking same volume of distilled water in the place of sample solution. The experiment was performed in triplicate, ascorbic acid was used as positive control and percentage scavenging activity was calculated using the equation (1).

Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of sample was assayed by using the 2-deoxyribose oxidation method Sakanaka et al. [13]. To the reaction mixture containing 0.2 ml KH 2 PO 4 -KOH (100 mM), 0.2 ml deoxyribose (15 mM), 0.2 ml FeCl 3 (500 mM), 0.1 ml EDTA (1 mM), 0.1 ml ascorbic acid (1 mM), and 0.1 ml H 2 O 2 (10 mM) were mixed with 0.1 ml sample. The mixture was incubated at 37°C for 1 h. After incubation time, 1.0 ml of TBA (1% w/v) was added to mixture followed by the addition of 1.0 ml of TCA (2.8% w/v). The resultant mixture was heated on a water bath at 80°C for 20 min, so that pink color developed. The absorbance of the solution was measured at 532 nm. Ascorbic acid was used as the positive control. The scavenging activity of test sample or standard (I %) was calculated using the equation (1).

Hydrogen peroxide scavenging activity
This assay was carried out according to the method of Bozin et al. [14].  (1).

Anti-inflammatory activity Acute toxicity studies
The acute toxicity study was determined in rats. Rats fasted for 12 h were randomly divided into different groups of 3 rats per group. All the animals were then allowed free access to food and water and observed over a period of 48 h for signs of acute toxicity. The number of deaths within this period was recorded. Based on the results of the preliminary toxicity test, the doses of 50 mg/kg, 100 mg/kg body weight (according to OECD Guideline 423) of were chosen for further experiments.

Animals
Albino (Wistar Strain) rats (150-250 g) were used in the study of anti-inflammatory activity and international principle and local regulations concerning the care and use of laboratory animals were taken into account [15]. Animal ethics committee approval was obtained from the institutional ethical committee (Registration number: 1305/ ac/09/ CPCSEA). The animals were maintained under environmental condition and had free access to standard diet and freshwater ad libitum. They were housed in animal cages at room temperature (30±2°C) and 60-65% relative humidity.

Anti-inflammatory activity (carrageenan-induced rat hind paw edema model)
The anti-inflammatory activity of test compounds was carried out by carrageenan-induced rat hind paw edema method by Winter et al. [16].
Carboxy methylcellulose (0.5% w/v solution) was selected as vehicle to suspend the standard drugs and the test compounds. The albino rats weighing between 150 and 200 g were starved for 18 h prior to the experiment. The animals were weighed, marked for identification, and divided into 12 groups each group containing 6 animals. Edema was induced in the left hind paw of all rats by subcutaneously to the sub plantar injection of 0.1 ml of 1% carrageenan in normal saline into their footpads. The first group served as control received 0.5 ml of carboxymethyl chitosan (CMC) (0.5% w/v solution). The second group (standard) was treated with diclofenac sodium at a dose of 10 mg/kg and test compounds were administered p.o. as suspension in 0.5% CMC, 1 h prior the injection of the test compounds at dose levels of 50 mg/ kg and 100 mg/kg as suspension in 0.5% CMC. The paw volume was measure immediately (0 h) and after 30-180 min, respectively, using digital plethysmometer. Percentage inhibition in the paw edema was calculated according to the following equation (2).
Where, A is the mean paw volume after administration of drug (ml), X is the mean paw volume before administration of drug (ml), B is the mean paw volume of treated control rats, Y is the mean paw volume of treated control rats before administration of carrageenan. Percentage inhibition shown by tested compounds is recorded in Table 1 and Fig. 2.

RESULTS AND DISCUSSION
In the present work, five derivatives of pyrazoles were synthesized. The structures of synthesized compounds were established by their spectral data. According to IR spectroscopic data compounds (4a-4e) showed absorption bands at 2987, 2790, 1756, 1640, 1605, and 1340 cm −1 indicating the presence of Ar C-H, C=O, aldehyde C-H,C=O, and pyrazole C=N, C-N groups, respectively. 1H NMR spectra of the synthesized compounds were confirmed by the appearance of -CHO proton at δ 9.4 and CH proton of pyrazole ring at δ 8.2 as singlet. Peaks due to two phenyl groups appeared at δ 7.2-7.8 as multiplet. Further, LC mass spectrum showed molecular ion peak at m/z 306 (100%) in agreement with their molecular formula.
The synthesized compounds were screened for their in vitro antioxidant activity by DPPH, hydrogen peroxide, Nitric Oxide, and hydroxyl radical scavenging method and the results are reported in Figs. 3-6, respectively. The data reveal that synthesized compounds showed moderate to good inhibition activity compared to the standard. Out of all the synthesized compounds, 4e (4-OH) and 4c (H) derivatives showed good antioxidant activity in all the four methods. The synthesized compounds were subjected to anti-inflammatory activity by the carrageenan-induced rat hind paw edema method at the low and high dose of 50 mg/kg and 100 mg/kg and the results are summarized in Table 1 and Fig. 2, respectively. The entire compounds have a good response for anti-inflammatory activity. Among all these 4-hydroxy and unsubstituted derivatives showed good reduction of edema at high dose (100 mg/ kg). Similarly, compound 4d (4-methoxy) showed good protection effect at low dose (50 mg/kg). Compounds 4a, 4b, and 4c have relatively moderate anti-inflammatory activity. It was further concluded that presence of electron-donating groups at para position of phenyl nucleus showed maximum antioxidant and anti-inflammatory activity.

CONCLUSION
In this work, a series of 1-Benzoyl-3-phenyl-1H-pyrazole-4carbaldehyde derivatives is prepared. The synthesized compounds were purified and well-characterized by TLC, IR, 1 HNMR, and GC-MS data. The antioxidant and anti-inflammatory data revealed that the compound with electron-donating group at para position on the ring exhibit significant action it makes the ring with prompt aromatic to exhibit action and therefore might serve as a lead molecule to obtain more clinically useful, novel entities in the future.