Synthesis, characterization, analgesic and anti-inflammatory activity of new pyrazole derivatives

The synthesis of nine new pyrazole derivatives was achieved from β-hydroxy enones and hydrazines by using a trace of piperidine in methanol at room temperature. The use of piperidine provides more yield and purity of products in lesser time when compared to the other reagents. All synthetic compounds were characterized by their physical properties, NMR and LC mass spectral data. The pyrazole derivatives were screened for their analgesic and anti-inflammatory activities in vivo. The derivatives exhibited moderate to significant activities in comparison to control, and most of the pyrazoles were competent with standard drugs (Pentazocine and Indomethacin).


Introduction
Pyrazoles are important heteroaromatic compounds with two nitrogen atoms in the five-membered ring system. They play a vital role in the pharmaceutical 1 and agrochemical industry. 2,3 They are classed as azoles. 4 Azoles occupy a domain of interest in natural and synthetic chemistry. Pyrazoles resemble benzene, and most of their properties were analyzed in comparison with the derivatives of benzene. 5 Knorr synthesized triaryl pyrazoles for the first time, by condensation of hydrazines with β-diketones. 6 Varma and co-workers developed a room temperature protocol for pyrazole synthesis using water as the solvent. 7 The binding of heterocyclic compounds with an acyclic sugar moiety, forming thus acyclic-nucleosides, has commanded worldwide attention of many research groups because of their high potential to exhibit chemotherapeutic activity. 8,9 Substituted pyrazole and its analogs have been used as precursors for the synthesis of various biologically active molecules.

Results and Discussion
The importance and biological activities of pyrazoles led us to prepare new pyrazoles in three steps followed by a study of their analgesic and anti-inflammatory activities in vivo. The synthetic process initially involved condensation of cinnamic acids 33 with o-hydroxy-4'-acetoxyacetophenones in pyridine using POCl3 as a condensing agent, to obtain 2'-cinnamoyloxyacetophenones (5)(6)(7). These were treated with powdered KOH in pyridine to give β-hydroxy-enones, 34,35 and then converted into pyrazoles by treating with alkaline hydrazine. The procedure for the preparation of the desired pyrazoles 11-19 is given in Scheme 1.
The optimization of the reaction conditions was carried out with β-hydroxy-enones as model substrate using different bases and solvents, at room temperature ( Table 1). The best results were attained when the reaction was run at room temperature with methanol as a solvent in the presence of piperidine in a catalytic amount, providing the new pyrazoles (11)(12)(13)(14)(15)(16)(17)(18)(19). Piperidine proved to be very effective in increasing the yields as well as the purity of the products within considerably less time than the other reagents. The completion of the reaction and purity of the products were monitored by thin-layer chromatography. Pinto et.al 36 has reported some pyrazoles which were prepared from 2-styrylchromones under reflux conditions in methanol.
All the final products were confirmed from their spectral data, viz. 1 H and 13 C-NMR, LC-Mass. Signals in PMR at δ 6.54 -7.15 (1H, s) indicate H(4) in pyrazole ring. The δ value at 7.27-7.90 was due to shielding of αprotons in the styryl group, whereas β-protons resonate at relatively higher δ values because of polarization of π-electrons, in the range 7.40-7.93 ppm. The trans configuration of Hα and Hβ of styryl group was established from the corresponding coupling constant, JHα-Hβ ≈ 16 Hz. The aromatic protons gave signals at various δ values depending on the nature and position of the substituent. Phenolic protons have appeared at δ 11.30-12.48 ppm. The methoxy protons gave a strong signal at 3.80 and methyl protons of the ester group at δ 2.08 ppm.
The 13 C-NMR values obtained at δ 107, 136 and 147 ppm, confirmed the presence of the pyrazole group. The high δ values at 160, 154, etc. confirmed that the aromatic carbons substituted with -OH, -OCH3 groups, the values at δ 167-168 ppm reveals the presence of carbonyl group in methyl ester.
All the synthesized final products showed strong peaks in their LC-Mass spectra at respective mass values in positive ion and negative ion modes.   (17) 0.340 g. Biological testing data All pyrazole derivatives except 12 exhibited thermal pain response much more effectively than Pentazocine in the same dose ( Figure 1). This could be due to the pyrazole ring structure with the nature and position of substituents in the styryl group and at N(1) of pyrazole. All the compounds tested by Eddy's hot plate method showed better activity at the dosage of 30 mg/kg. The significant activity of all pyrazoles was due to the -Cl, -OMe at position 4 of the styryl group. In the absence of these groups, the compounds showed lower or nonsignificant activity, especially compound 12 has much lesser activity at 60 minutes, while 11, 13, and 15 have lesser activity at 30 minutes. The synthesized pyrazoles exhibited significant anti-inflammatory activity when compared to the control group ( Figure 2). The anti-inflammatory activities of compounds 11-19 were studied in the carrageenaninduced edema model of inflammation in mice. All the pyrazole derivatives showed significant antiinflammatory activity. The compound 16, 17, showed better activity might be due to the presence of methoxyl and chloro substituents at 4-position in the styryl ring of pyrazole. The compounds 11 and 12 exhibited good activity might well be due to the presence of the nitro group in the styryl ring. Hence, the anti-inflammatory activity of pyrazole derivatives was increased with the styryl group substituted by an electron releasing group at position 4.
Tables S1 and S2 (Supplementary file) give full details of the analgesic and anti-inflammatory testing data of the individual compounds reported here. The results are summarized in Figures 1 and 2 below.

Conclusions
We have synthesized new pyrazole derivatives containing styryl groups at one position of pyrazole ring. The yield and purity of the final products were enhanced by using a trace amount of piperidine at the third stage of preparation. All reactions gave their target products in good to excellent yield. The reactions were rapid, facile and accomplished at room temperature. Almost all the synthesized compounds exhibited significant analgesic and anti-inflammatory activity against Pentazocine and Indomethacin in vivo of albino mice and rats respectively

Experimental Section
General: Melting points were measured by a Stuart Scientific melting point apparatus in open capillaries. NMR spectra were recorded on a Bruker spectrometer operating at 400 MHz for proton & 100 MHz for 13 C-spectra, using DMSO-d6 as a solvent with TMS as an internal standard. Mass spectra were recorded on an Agilent-1100 period LC-MSD. Elemental analysis was performed using a EURO EA 3000 instrument. Acme silica gel-G and Merck silica gel (100 to 200, 60 to 120 meshes) were used for analytical TLC and column chromatography respectively. All other analytical grade chemicals and solvents were obtained from commercial sources and used as received.