INTRODUCTION:

Several pyrazole derivatives are well established in the literatures as important biologically effective heterocyclic compounds. The activity of the pyrazoles covers domains such as antimicrobial [1, 2], antiviral [3], antitumor [4], anticonvulsant [5], antihistaminic [6] and antidepressant [7]. The literature survey reveals an excellent analgesic and anti-inﬂammatory activities with some compounds containing heterocyclic ring as pyrazole [8-17]. Encouraged by these ﬁndings we thought of preparing new derivatives of 1-(4-substitutedphenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 4a-e in order to screen them for analgesic and anti-inﬂammatory activities.

EXPERIMENTAL:

Materials

Synthetic starting material, reagents and solvents were of analytical grade or of the highest quality commercially available. The chemicals were purchased from Aldrich Chemical Co. and Merck Chemical Co., respectively, and were dried whenever necessary.

Instrumentation

The melting points were taken in open capillary tube and are uncorrected. IR spectra were recorded with KBr pellets (ABB Bomem FT-IR spectrometer MB 104 ABB Limited Bangaluru, India). Proton (¹H) NMR spectra (Bruker 400 NMR spectrometer Mumbai, India) were recorded with TMS as internal references. Mass spectral data were recorded with a quadrupol mass spectrometer (Shimadzu GC MS QP 5000, Chennai, India), and microanalyses were performed using a vario EL V300 elemental analyzer (Elemental Analysensysteme GmbH Chennai, India). The purity of the compounds was checked by TLC on pre-coated SiO₂ gel (HF₂₅₄, 200 mesh) aluminium plates (E.Merck) and visualized in UV chamber. IR, ¹H-NMR, mass spectral datas and elemental analyses were consistent with the assigned structures of all the compounds.

General Procedure

The synthetic strategy of the target compounds are illustrated in scheme 1. The acetophenone (0.01mole), substituted phenyl hydrazine (0.01mole) and DMF (0.5 mL) was exposed to microwave at 200 W intermittently at 10 sec intervals. The specified reaction time 0.30 min was observed of compound 3. The reaction mixture was cooled with cold water. The precipitate thus obtained was filtered, washed with water and purified by recrystallization from ethanol to furnish 3. On the completion of the reaction, as monitored by TLC method [eluent: CHCl₃-MeOH (7:2)].

The compound 3 (0.01mole) was added portion wise with vilsmeier-haack reagent (0.03mole), After the addition was complete, the reaction flask was kept at room temperature for 5 min and silica gel 3g was added and properly mixed with the help of a glass rod, till free flowing power was obtained. The power is then irradiated in microwave oven at 400 W intermittently at 30 sec intervals. The specified reaction time 5.0 min was observed of compound 4a-e. The reaction mixture was cooled, treated with cold water. The solid obtained by the neutralization of the filtrate with NaHCO₃ was filtered, washed with water and purified by recrystallization from methanol to afford 4a-e. On the completion of the reaction, as monitored by TLC method [eluent: CHCl₃-MeOH (7:2)].

Scheme 1

1,3-diphenyl-1H-pyrazole-4-carbaldehyde (4a)

Yellow Crystals; Yield: 82%; mp. 189-191°C, IR : 3036 (Ar-CH), 2252 (C=N), 1481 (C=C), 1706 (C=O) cm^-1; ¹H-NMR (CDCl₃): δ 8.79 (s, 1H, -CHO), 6.69-7.19 (m, 11H, Ar-H), 7.39 (s, 1H, -CH);EI-MS (m/z, %): [M]+ 248; (Calcd for C₁₆H₁₂N₂O; 248.28). Anal. Calcd for C₁₆H₁₂N₂O; C, 77.40; H, 4.87; N, 11.28; Found: C, 77.29; H, 4.84; N, 11.16.

1-(4-bromophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (4b)

Pale Yellow Solid; Yield: 78%; mp. 177-179°C, IR : 3039 (Ar-CH), 2261 (C=N), 1712 (C=O), 1489 (C=C), 606 (C-Br) cm^-1; ¹H-NMR (CDCl₃): δ 9.12 (s, 1H, -CHO), 7.12-7.49 (m, 9H, Ar-H), 7.62 (s, 1H, -CH); EI-MS (m/z, %): [M+2] 329; (Calcd for C₁₆H₁₁BrN₂O; 327.18). Anal. Calcd for C₁₆H₁₁BrN₂O; C, 58.74; H, 3.39; N, 8.56; Found: C, 58.71; H, 3.43; N, 8.71.

1-(4-chlorophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (4c)

Pale Brown Solid; Yield: 81%; mp. 170-172°C, IR : 3042 (Ar-CH), 2247 (C=N), 1706 (C=O), 1484 (C=C), 794 (C-Cl) cm^-1; ¹H-NMR (CDCl₃): δ 8.96 (s, 1H, -CHO), 6.72-7.12 (m, 9H, Ar-H), 7.27 (s, 1H, -CH); EI-MS (m/z, %): [M+2] 284; (Calcd for C₁₆H₁₁ClN₂O; 282.72). Anal. Calcd for C₁₆H₁₁ClN₂O; C, 67.97; H, 3.92; N, 9.91; Found: C, 67.82; H, 3.72; N, 9.97.

1-(4-flurophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (4d)

Pale Solid; Yield: 76%; mp. 181-183°C, IR : 3064 (Ar-CH), 2252 (C=N), 1717 (C=O), 1471 (C=C), 1021 (C-F) cm^-1; ¹H-NMR (CDCl₃): δ 8.71 (s, 1H, -CHO), 6.94-7.17 (m, 9H, Ar-H), 7.29 (s, 1H, -CH); EI-MS (m/z, %): [M]+ 266; (Calcd for C₁₆H₁₁FN₂O; 266.27). Anal. Calcd for C₁₆H₁₁FN₂O; C, 72.17; H, 4.16; N, 10.52; Found: C, 72.29; H, 4.17; N, 10.59.

1-(4-acetamidophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (4e)

Pale White Solid; Yield: 82%; mp. 182-184°C, 3064 (Ar-CH), 2252 (C=N), 1717 (C=O), 1471 (C=C), 1326 (N-H bending), 3329 (N-H stretching) cm^-1; ¹H-NMR (CDCl₃): ; ¹H-NMR (CDCl₃): δ 8.94 (s, 1H, -CHO), 7.71 (s, 1H, N-H), 6.82-7.19 (m, 9H, Ar-H), 7.34 (s, 1H, -CH), 2.78 (s, 3H, -CH₃); EI-MS (m/z, %): [M]+ 305; (Calcd for C₂₉H₂₉N₃O₂; 305.33). Anal. Calcd for C₂₉H₂₉N₃O₂; C, 70.81; H, 4.95; N, 13.76; Found: C, 70.74; H, 4.81; N, 13.59.

Pharmacology

The animals were procured from the Srinivas college of pharmacy (Mangalore, India), and were maintained in colony cages at 25 ± 2^oC, relative humidity 45–55%, under a 12 h light and dark cycle; they were fed standard animal feed. All the animals were acclimatized for a week before use. The Institutional Animal Ethics committee has approved the protocol adopted for the experimentation of animals. Adult male albino rats (Harlan Sprague-Dawley), weighing 150–180 g, were fasted for 12 hours and used for determining the anti-inflammatory activity. Adult Swiss Webster mice of both sexes (Harlan Sprague-Dawley), weighing 20–25 g, were fasted for 12–24 hours and used for determining the analgesic activity.

Analgesic activity

The synthesized compounds and the reference drugs flufenamic acid were prepared as a suspension in 2% Tween 80. A sensitivity test [18] was carried out one day before drug administration when the animals were injected (i.p.) with 0.2-0.25 mL of 0.02 % freshly prepared solution of p-benzoquinone in distilled water. Animals showing writhing to p-benzoquinone within 30 minutes were chosen for studying the analgesic activity. On the following day, mice were divided into 13 groups of 6 animals each. The drugs were administered orally according to the following protocol: one group received 2 % Tween 80 (solvent and negative control), the second group received flufenamic acid as reference (20 mg kg^-1), while the other groups received doses of the tested compounds 100, 50, 25 and 5 mg kg^-1. One hour latter, 0.02 % solution of p -benzoquinone was administered (i.p.). The animals were observed for 30 minutes after injection of the irritant, during which time the animals showing writhing were counted (writhing is defined as stretch, torsion to one side, drawing up of hind leg, retraction of the abdomen, so that the belly touches the floor). All writhing is considered as a positive response. The analgesic activity was expressed as the percent protection.

Anti-inflammatory activity

The inhibitory activity of the studied compounds on carrageenean-induced rat’s paw edema was determined according to the method [19]. Groups of adult male albino rats, 8 animals each, were orally dosed with tested compounds at doses of 100, 50, 25 and 5 mg kg^-1one hour before carrageenean challenge. Foot paw edema was induced by subplantar injection of 0.05 mL of 1% suspension of carrageenean in saline into the plantar tissue of one hind paw. An equal volume of saline was injected into the other hind paw and served as a control. The animals were decapitated four hours after drug administration. The average mass of edema was estimated for the treated as well as for the control group and the percentage inhibition of edema was evaluated [20]. Indomethacin (5.0 mg kg^-1), suspended in saline, was employed as a reference drug against the test compounds.

RESULTS AND DISCUSSION:

Chemistry

The synthesized series of heterocycles, 1-(4-substitutedphenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 4a-e by the reaction of acetophenone with substituted phenyl hydrazine in the presence of vilsmeier-haack reagent. The IR, ¹H-NMR, mass spectroscopy and elemental analysis for the new compound is in accordance with the assigned structures. The ¹H NMR spectra of compounds 4a-e showed -CHO signal of pyrazole-4-carbaldehyde appear at 8.79 (s), 9.12 (s), 8.96 (s), 8.71 (s), 8.94 (s) ppm respectively. The position and presence of CHO signal in the ¹H NMR spectra of final compounds conforms with the pyrazole moiety. This clearly envisages that intermediate 3 involve in 1-(4-substitutedphenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 4a-e formation. All these observed facts clearly demonstrate that the intermediate 3 is converted into 1-(4-substitutedphenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 4a-e as indicated in scheme 1 and conforms with the proposed structure Fig 1.

Pharmacology

The synthesized compounds 4a-e compounds showed significant analgesic activity at doses of 50 and 100 mg kg^-1. None of the tested components showed analgesic activity at doses of 25 and 5 mg kg^-1, Compounds 4b, 4c and 4d showed higher activity ranging from 68 to 98 % at a dose of 100 mg kg^-1than the reference drug flufenamic acid at a dose of 20 mg kg^-1. The protection of these compounds ranged from 30 to 62 % at a dose of 50 mg kg^-1. The results of the tested compounds are given in Table 1.

The newly synthesized compounds 4a-e were evaluated for their anti-inflammatory activity against carrageenean induced rat’s paw edema by administration of 100, 50, 25 and 5mg kg^-1(p.o.) using indomethacin as a reference drug 5 mg kg^-1. None of the tested compounds showed anti-inflammatory activity at doses of 3 and 5 mg kg^-1, whereas compounds 4b, 4c, 4d exhibited remarkable anti-inflammatory activity ranging from 35 to 69 % at a dose of 50 mg kg^-1and from 54 to 78 % at a dose of 100 mg kg^-1. The results of the tested compounds are given in Table 2. Higher anti-inflammatory activity of the mentioned compounds 4b, 4c, 4d compared to the others may be due to the presence of the halo atoms in their skeletons. The most potent compound was 4c with anti-inflammatory activity of 78 % at a dose of 100 mg kg^-1and 69 % at a dose of 50 mg kg^-1, respectively.

Structure-activity relationship studies

Structure-activity relationship studies revealed that different substitutions on the pyrazole nucleus exerted remarkable biological activity. The electronic nature of the substituent groups at 4' positions in phenyl ring showed signiﬁcant analgesic and anti-inflammatory activity. Among the series compounds substituted by electron-withdrawing (-Br, -Cl and -F) groups are enhanced biological activity.

Fig 1

Table 1 Analgesic activity of synthesized compounds ^a (4a-e)

Compd	Dose mg kg^-1	Inhibition (%)
4a	100	71
4a	50	30
4b	100	77
4b	50	44
4c	100	98
4c	50	62
4d	100	84
4d	50	51
4e	100	68
4e	50	47
Negative control ^b	0	0
Flufenamic acid	20	69

^ap-Benzoquinone was used for inducing writhing in mice.

^bNegative control- 2%Tween 80; also solvent test compounds and reference drugs.

Table 2 Anti-inflammatory activity of synthesized compounds (4a-e)

Compd^a	Dose mg kg^-1	Inhibition (%)
4a	100	61
4a	50	35
4b	100	74
4b	50	64
4c	100	78
4c	50	69
4d	100	71
4d	50	60
4e	100	54
4e	50	52
Negative control	0	0
Indomethacin	5	62

^aNegative control- saline, also solvent for test compounds and indomethacin.

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Received on 13.11.2011 Accepted on 12.12.2011

Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 126-129

T. Panneer Selvam*¹, G. Saravanan², C. R. Prakash³, P. Dinesh Kumar⁴

¹Department Pharmaceutical Chemistry, Srinivas College of Pharmacy, Mangalore -574142, Karnataka, India

T. Panneer Selvam*1, G. Saravanan2, C. R. Prakash3, P. Dinesh Kumar4

1Department Pharmaceutical Chemistry, Srinivas College of Pharmacy, Mangalore -574142, Karnataka, India

T. Panneer Selvam*¹, G. Saravanan², C. R. Prakash³, P. Dinesh Kumar⁴

¹Department Pharmaceutical Chemistry, Srinivas College of Pharmacy, Mangalore -574142, Karnataka, India