International Journal of ChemTech Research CODEN( USA): IJCRGG ISSN : 0974-4290 Vol.2, No.

1, pp 45-49, Jan-Mar 2010

Antiepileptic and Antimicrobial Activities of Novel 1-(unsubstituted/substituted)-3,5-dimethyl-1Hpyrazole Derivatives

School of Pharmacy, Masterskill University College of Health Sciences, Taman Kemacahaya 11, Jalan Kemacahaya, Cheras, Selangor, Malaysia
2

Honorary Associate, School of Pharmacy, La Trobe University, Bendigo, Victoria 3552, Australia

Arulmigu Kalasalingam College of Pharmacy, Krishnankoil-626190, Tamilnadu, India

JSS College of Pharmacy, Rocklands, Ooty, Tamilnadu, India *Corres.Author: anand_arg@yahoo.co.in Phone : 0060129057874

Abstract: A series of 1H-pyrazole derivatives (1a-f) were synthesized by cycloaddition of acetyl acetone with respective hydrazine derivatives in ether in the presence of sodium hydroxide to generate the corresponding pyrazole derivatives. The synthesized compounds were confirmed by melting point and TLC. The structure of synthesized compounds was established by elemental analysis and various analytical techniques such as IR and 1HNMR spectral studies. All the newly synthesized compounds were evaluated for their antiepileptic and antimicrobial activities. Keywords: Pyrazoles, Antiepileptic, Antibacterial, Antifungal Introduction
The pyrazole ring system is a five-membered heterocyclic ring structure composed of two nitrogen atoms and used in the synthesis of pharmaceuticals. The pyrazole moiety is a versatile lead molecule in pharmaceutical development and has a wide range of biological activities. In the past few years, the therapeutic interest of pyrazole derivatives in pharmaceutical and medicinal field has been given a great attention to the medicinal chemist. Literature survey reveals that pyrazole derivatives are well known to have antibacterial1, antifungal2, antitubercular3, anticancer 4, analgesic5, anti5 6 inflammatory , antipyretic , anticonvulsant7, silica gel G as stationary phase and chloroformmethanol (9:1) as eluent. IR spectra in max (cm-1) were antidepressant 8, muscle relaxing9, anti-ulcer10, antiarrhythmic11 and antidiabetic12 activities. In recent years, the extensive studies have been focused on pyrazole derivatives because of their diverse chemical reactivity, accessibility and wide range of biological activities. Thus, in this present work, a series of novel pyrazole derivatives were synthesized and tested for their antiepileptic and antimicrobial activities.

Experimental
Melting points were determined using on veego melting point apparatus and were uncorrected. The purity of the compounds was confirmed by TLC using recorded on Shimazdu 8400 series FT-IR Spectrophotometer using KBr disc technique. 1H NMR

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spectra were obtained on Bruker AMX spectrophotometer at 300 MHz in DMSO-d6. Chemical shifts were reported in units (ppm) relative to an internal standard of tetramethysilane. Elemental analysis determinations for final compounds were performed on Carlo Erba 108 and the analyses. The animal study was approved by the animal ethical committee (AKCP/CPCSEA/509/F(2)/2008). Synthesis of novel pyrazole derivatives The respective hydrazine derivative (0.2 mol) was dissolved in 10% NaOH and cooled in an ice bath at 15 0C. Acetyl acetone (0.2 mol) was added drop wise to the above solution and stirred for 1h. To this mixture water (75 mL) and ether (50 mL) were added and shaken well in separating funnel. The aqueous layer was extracted with four successive quantities of ether (15 mL). Then the collected ether extract was combined and excess of ether was removed by distillation gave the corresponding pyrazole derivatives and recrystallized from ethanol13. The physical data of the compounds were recorded in Table 1 and the spectral data were tabulated in Table 2. Anti-epileptic activity The anti-epileptic activity was carried out by maximal electrical shock induced convulsion method14. Albino mice of either sex (20 30 g) were used for this study. Mice were treated with newly synthesized pyrazole derivatives (25 mg/mL, i.p.), standard drug phenytoin (25 mg/mL, i.p.) and control (2% w/v Tween 80). After 30 min, the animals were subjected to electro

shock through ear electrodes of 150 mA for 0.2 sec by electroconvulsiometer and the duration of time for extensor response was noted. The statistical significance of the difference in the mean values was calculated by studentst test15 and tabulated in Table 3. Test microorganisms Gram positive organisms such as Staphylococcus aureus, Streptococcus pyogenes, Gram negative organisms such as Salmonella typhi, Pseudomonas auregenosa and fungus Candida albicans were used for this study. All the bacterial cultures were procured from microbiology lab, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil, Tamilnadu, India. Antibacterial activity All the synthesized compounds were dissolved in 2 % v/v Tween 80 at a concentration of 100 mcg/mL. The antibacterial activity was performed by cup-plate method16. The respective bacterial culture was spread (swabbed) into the nutrient agar plates for uniform distribution of colonies. Using a sterile cork borer, 8 mm wide well was made on each agar plates. All the synthesized compounds (100 mcg/mL) were poured into each wells using a sterile micropipette and ofloxacin (100 mcg/mL) were used as standard. The plates were incubated for 24 h at 37 oC. After incubation, the zone of inhibition was measured and the values were tabulated in Table 4. All the experiments were done in triplicate.

Scheme 1
O O

+ Acetyl acetone Ether

R NHNH2 Hydrazine derivative

Compounds R 1a

-H -CH3

10% NaOH Stirr 1 h

1b 1c 1d

O2N NO2

H3C

N N

1e 1f

O N N

1a-f 1-(unsubstituted/substituted)-3,5-dimethyl-1H-pyrazole

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Antifungal activity The antifungal activity was tested against Candida albicans by cup plate method16. All the synthesized compounds were dissolved in 2 % v/v Tween 80 at a concentration of 100 mcg/mL. The fungal culture was spread (swabbed) into the sabouraud dextrose agar plates for uniform distribution of colonies. Using a sterile cork borer, 8 mm wide well was made on each agar plates. All the synthesized compounds (100 mcg/mL) were poured into each wells using a sterile micropipette and ketoconazole (100 mcg/mL) were used as standard. The plates were incubated for 48 h at 27 oC. After incubation, the zone of inhibition was measured and the values were tabulated in Table 4. All the experiments were done in triplicate.

Results and Discussion

and R f values of the synthesized compound confirmed the formation of novel compounds. All the spectral data elucidated the structure of the synthesized pyrazole derivatives. Elemental analysis indicated by the symbols of the elements is determined and showed close value within 0.4% of theoretical values. Anti-epileptic activity The reduction in the time of extensor phase was taken for anti-epileptic activity. The compounds 1a, 1b, 1c and 1d showed highly significant anti-epileptic activity where as compounds 1e and 1f showed significant anti-epileptic activity at a dose of 25 mg/mL, i.p. when compared to the standard drug phenytoin 25 mg/mL. Antibacterial and antifungal activities All synthesized novel pyrazoles were tested against Gram positive organisms such as S. aureus, S. pyogenes and Gram negative organisms such as S. typhi, P. auregenosa and fungus C. albicans. All the compounds showed mild to moderate antibacterial and antifungal activities at a concentration of 100 mcg/mL when compared with ofloxacin (100 mcg/mL) and ketoconazole (100 mcg/mL) respectively.

Table 1. Physical data of novel pyrazole derivatives Comp. Molecular M.W. m.p.( Rf 0 value code formula C) 1a 1b 1c 1d 1e 1f C5H8N2 C6H10N2 C11H12N2 C11H10N4O4 C12H12N2O C13H12N4 96.13 110.16 172.23 262.22 200.24 224.26 88 96 92 106 122 116 0.951 0872 0.910 0.841 0.896 0.864

% yield 73.35 62.46 64.50 61.23 56.98 58.24 C 62.47 (62.42) 65.42 (65.40) 76.71 (76.74) 50.38 (50.32) 71.98 (71.96) 69.62 (69.60)

Elemental analysis % calculated (%found) H N 8.39 29.14 (8.42) (29.12) 9.15 25.43 (9.16) (25.40) 7.02 16.27 (7.06) (16.24) 3.84 21.37 (3.88) (21.34) 6.04 13.99 (6.08) (13.96) 5.39 24.98 (5.36) (24.96)

O 24.41 (24.44) 7.99 (7.96) -

Comp. code Compound code, M.W. Molecular weight, m.p. - Melting point

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Table 2. Spectral data of novel pyrazole derivatives Compound IR (KBr disc) max (cm-1) code 3384 (NH), 1620.45 (C=N), 1512.22 (N-N), 1a 1476 (CH3) 1618.22 (C=N), 1515.82 (N-N), 1472.44 1b (CH3) 1615.75 (C=N), 1505.82 (N-N), 1468 (CH3) 1c 1d 1e 1f 1611.90 (C=N), 1520 (N-N), 1462.38 (CH3), 1611.90 (Ar-NO2) 1624 (C=N), 1522.40 (N-N), 1470 (CH3), 1664 (C=O), 1618.52 (C=N), 1512.22 (N-N), 1476 (CH3)

H NMR (DMSO-d6) (ppm)

2.34 (s, CH3), 2.72 (s, CH3), 5.86 (s, CH pyrazole), 13.4 (s, NH) 2.86 (s, CH3), 3.82 (s, NCH3), 5.74 (s, CH pyrazole) 2.62 (s, CH3), 5.96 (s, CH pyrazole), 7.437.82 (m, ArH) 2.78 (s, CH3), 6.06 (s, CH pyrazole), 7.688.26 (m, ArH) 2.92 (s, CH3), 5.96 (s, CH pyrazole), 7.989.16 (m, ArH) 2.88 (s, CH3), 5.66 (s, CH pyrazole), 7.849.42 (m, ArH)

Table 3. Anticonvulsant activity of novel pyrazole derivatives Compound code Duration of time (Mean SEM, sec) % Protection Extensor Clonus Stupor

Control 16.22 0.12 22.64 0.44 44.62 5.42 Standard drug 3.12 0.026** 11.32 0.32** 14.54 4.26** 80.76 1a 62.27 6.12 0.24** 16.52 0.12** 19.6 4.74** 1b 5.94 0.18** 17.98 0.70** 22.2 3.44** 63.38 1c 70.41 4.80 0.16** 14.50 0.36** 17.4 2.16** 1d 54.01 7.46 0.09** 15.12 0.44** 18.5 2.64** 1e 10.94 0.22* 18.34 0.56* 32.0 2.82* 32.55 1f 11.67 0.46* 17.45 0.78* 38.18 6.86* 28.05 Values are Mean SEM, *P < 0.05 and **P < 0.01 statistically significant from control group (n=6) Table 4. Anti-bacterial and Antifungal activities of novel pyrazole derivatives Diameter of Zone of inhibition (mm) Microorganisms Standard S. aureus S. pyogenes S. typhi P. aurgenosa C. albicans n=3 20 21 18 22 19 1a 15 15 14 16 17 1b 10 12 09 12 14 1c 13 11 10 14 13 1d 15 09 06 15 16 1e 12 08 12 11 10 1f 13 11 14 10 10

Acknowledgements
The authors are thankful to the management, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil, Tamilnadu, India and for providing facilities and technical assistance. Also the authors are grateful to Dato Prof. Dr. Ishak Bin Tambi Kechik, Vice-

Chancellor, Dato Prof. Dr. P.Promwichit, Deputy Vice-Chancellor and Dato Edmund Santhara, CEO, Masterskill University College of Health Sciences, Malaysia, for their continuous encouragement and support.

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