INTRODUCTION

Pyrazole is five-membered ring having two nitrogen as hetero atoms in the system. It is widely found as the core structure in a large variety of compounds that possess important agrochemical and pharmaceutical activities¹.Classical methods for the synthesis of substituted pyrazoles involve approaches based either on the condensations of hydrazines with 1, 3-dicarbonyl compounds and their 1, 3-dielectrophile equivalents, or on intermolecular [3+2] cycloadditions of 1, 3-dipoles to alkynes^{2, 3}. Over the past few years, more efficient and broadly applicable methodologies have been developed with the aim of increasing the regioselectivity in the preparation of 1, 3, 5-tri- and 1, 3, 4, 5-tetrasubstituted pyrazoles.

Pyrazole derivatives have emerged as a group of compounds possessing a broad spectrum of useful medicinal properties such as analgesic, antipyretic, anti-inflammatory, germicidal and anti-fungal activity^4-7. In addition, these compounds are appropriate precursors for industrial preparation of liquid crystal⁸, dyes⁹, thermally stable polymers¹⁰ and color photographical compounds¹¹. As far as the different pyrazole isomers are concerned, 2-pyrazole derivatives became the most frequently studied. Various methods are used for the preparation of 2-pyrazoles. Treatment of α, β-unsaturated aldehydes and ketones with hydrazines seems to be the most popular procedure for this purpose. This reaction has been conducted under various conditions^12-14. As a hydrazine reagent, hydrazine hydrate or phenylhydrazine were used almost in all cases.

It is well reported that ester linkage shows good anti bacterial and anti-fungal activity. Keeping all in this mind, in this article, we have reported synthesis of pyrazole derivatives with ester linkages to evaluate their microbial activity for E. Coli and S. Aureus as antibacterial activity and A. Niger and A. Orazae as antifungal activity and also reported its cytotoxic activities¹⁵.

MATERIALS AND METHODS:

The requisite starting materials such as 4-Hydroxybenzaldehyde, 4-hydroxy acetophenone, sulphuric acid, ethanol, 2, 4-dinitrophenyl hydrazine, acetic acid, appropriate aromatic acid, thionyl chloride, ethyl acetate, hexane etc. were procured from Sigma / Aldrich /Local Company and used without any further purification. All the solvents were purified and dried by standard method. Microanalysis of the compounds was performed on a Coleman carbon–hydrogen analyzer, and the values obtained are in close agreement with those calculated. FTIR spectra were determined for KBr pellets using a Shimadzu IR-408 spectrophotometer. ¹H NMR spectra were obtained with a Perkin-Elmer R-32 spectrometer using tetramethylsilane (TMS) as internal reference standard. The chemical shifts are quoted in parts per million downfield from the reference; DMSO-d₆ was used as solvent for all the compounds.

General Procedure:

Synthesis of 4, 4’-Dihydroxy chalcone (1)¹⁶

Into a 3-necked round bottom flask equipped with a thermometer, a stirrer and a condenser, 0.1 mole of 4-hydroxyacetophenone, 0.1 mole of 4-hydroxybenzaldehyde and 60 mL of methanol were charged, and after the reactants dissolved, 0.05 mol of a 97% sulfuric acid were added, the temperature was raised to 60°C and then the reaction mixture was maintained at the same temperature for 13 hours. The reaction mixture was poured into water for crystallization. The obtained crystals were filtered and washed 4 times with 100 mL of water, and then dried in vacuo at 80°C for 5 hours. Red colored crystals having a M. P of 164° C. Yield: 75 %

Preparation of 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole (2):

A mixture of 0.5 mole of chalcone, 1.1 mole of 2, 4-dinitrophenyl hydrazine and acetic acid (30 mL) was heated at reflux for 8 h. The reaction mixture was allowed to cool up to room temperature. The obtained solid was filtered off and washed it with water. Solid was dried under vacuum oven at 60°C and crystallized it using mixture of DMF and alcohol to obtain pure 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole. M. P.: 297 ° C Yield: 73 %

Preparation of 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole (2) (By microwave method):

A mixture of 0.5 mole of chalcone, 1.1 mole of 2, 4-dinitrophenyl hydrazine and acetic acid (3 mL) was kept in microwave for 2 min at 760W. The reaction mixture was allowed to cool up to room temperature. The obtained solid was filtered off and washed it with water. Solid was dried under vacuum oven at 60°C and crystallized it using mixture of DMF and alcohol to obtain pure 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole. M. P.: 297 ° C Yield: 93 %

¹H NMR spectrum (DMSO-d₆) (400 MHz): δ 6.85 (d, 1H, Ar-H at C-3” and C-5”), 7.10 (d, 2H, Ar-H at C-3’ and C-5’), 7.18 (d, 1H, Ar-H at C-4), 7.30 (d, 2H, Ar-H at C-2” and C-6”), 7.48 (d, 2H, Ar-H at C-2’ and C-6’), 8.10 (d, 1H, Ar-H at C-f), 8.48 (d, 1H, Ar-H at C-e), 8.85 (s, 1H, Ar-H at C-c), 9.90 (s, 1H, Ar-OH at C-4’), 10.10 (s, 1H, Ar-OH at C-4”).

FTIR spectrum (KBr) ν_max/cm^-1: 3200 (-OH), 1600 (Aromatic -C=C-), 1595 (-C=N- stretching of pyrazole), 1525 (Ar-NO₂), 1420, 1325, 1210 (-C-N- stretching of pyrazole), 1150, 1100, 975, 925, 825 and 780

Elemental Analysis: Calculated for C₂₁H₁₄N₄O₆: C, 60.28; H, 3.34; N, 13.39 %. Found: C, 60.21; H, 3.21; N, 13.37 %.

Aromatic acid chlorides (3):

A mixture of 0.01 mol substituted acid was added to 15 mL of thionyl chloride. It was refluxed on water bath till the evolution of hydrogen chloride gas ceased. Distilled of excess thionyl chloride and used it for next step without further purification.

Preparation of 1-(2, 4-dinitrophenyl)-3, 5-(Bis-4’-substitutedbenzoyloxy benzene)pyrazole (4):

0.01 Mole of 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole (2) was dissolved in 10mL dry pyridine and a cold solution of an appropriate 0.01 mole substituted aromatic acid chloride (3) in dry pyridine was added slowly to it with constant stirring in an ice bath. The mixture was allowed to stand over night at room temperature and then heated on water bath for half an hour. It was acidified with cold 1:1 aqueous hydrochloric acid. The solid obtained was filtered and washed successively with water (2 X 50 mL), saturated solution of sodium bicarbonate (2 X 50 mL) and water (2 X 30 mL). The crude material obtained was chromatographed on silica gel (100-200 mesh) using mixture of ethyl acetate and hexane (35:65) as eluent. Removal of solvent from the eluate afforded a solid material, which was crystallized repeatedly from appropriate solvent (As mentioned in Table 1). The purity of all these compounds was checked by thin layer chromatography (Merck kieselgel 60F254 pre-coated plates).

Preparation of 1-(2, 4-dinitrophenyl)-3, 5-(Bis-4’-substitutedbenzoyloxy benzene)pyrazole (4) (By microwave methods):

0.01 Mole of 1-(2, 4-dinitrophenyl)-3, 5-dihydroxyphenylpyrazole (2) was dissolved in 2mL dry pyridine and a cold solution of an appropriate 0.01 mole substituted aromatic acid chloride (3) in dry pyridine was added slowly to it with constant stirring in an ice bath. The mixture was allowed to keep in microwave for 30 seconds at 760 W. It was acidified with cold 1:1 aqueous hydrochloric acid. The solid obtained was filtered and washed successively with water (2 X 50 mL), saturated solution of sodium bicarbonate (2 X 50 mL) and water (2 X 30 mL). The crude material obtained was chromatographed on silica gel (100-200 mesh) using mixture of ethyl acetate and hexane (35:65) as eluent. Removal of solvent from the eluate afforded a solid material, which was crystallized repeatedly from appropriate solvent (As mentioned in Table 1). The purity of all these compounds was checked by thin layer chromatography (Merck kieselgel 60F254 pre-coated plates). The elemental analyses, ¹H NMR and FTIR spectral data of representative compounds were found to be consistent with the proposed structure.

R= -H: ¹H NMR spectrum (DMSO-d₆) (400 MHz): δ 7.39-7.88 (m, 11H, Ar-H at C-4, C-3’, C-5’, C-3”, C-5”, C-h, C-i, C-j, C-m, C-n and C-o), 8.19-8.37 (m, 9H, Ar-H at C-g, C-f, C-k, C-l, C-p, C-2’, C-6’, C-2” and C-6”), 8.59 (d, J=8.9 Hz, 1H, Ar-H at C-e), 8.92 (s, 1H, Ar-H at C-c)

FTIR spectrum (KBr) ν_max/cm^-1: 1695 (-COO-), 1605 (Aromatic -C=C-), 1595 (-C=N- stretching of pyrazole), 1520 (Ar-NO₂), 1425, 1320, 1205 (-C-N- stretching of pyrazole), 1125, 1110, 975, 925, 825 and 780

Elemental Analysis: Calculated for C₃₃H₂₂N₄O₆: C, 69.47; H, 3.85; N, 9.82 %. Found: C, 69.29; H, 3.71; N, 9.77 %.

R= -Cl: ¹H NMR spectrum (DMSO-d₆) (400 MHz): δ 7.32-7.82 (m, 10H, Ar-H at C-4, C-3’, C-5’, C-3”, C-5”, C-h, C-j, C-m, C-n and C-o), 8.10-8.25 (m, 9H, Ar-H at C-f, C-g, C-k, C-l, C-p, C-2’, C-6’, C-2” and C-6”), 8.52 (d, J=9.0 Hz, 1H, Ar-H at C-e), 8.82 (s, 1H, Ar-H at C-c)

FTIR spectrum (KBr) ν_max/cm^-1: 1700 (-COO-), 1600 (Aromatic -C=C-), 1590 (-C=N- stretching of pyrazole), 1520 (Ar-NO₂), 1425, 1320, 1200 (-C-N- stretching of pyrazole), 1125, 1110, 975, 925, 825 and 780

Elemental Analysis: Calculated for C₃₃H₂₀N₄O₆Cl₂: C, 61.97; H, 3.12; N, 8.76 %. Found: C, 61.21; H, 3.04; N, 8.67 %.

R= -CH₃: ¹H NMR spectrum (DMSO-d₆) (400 MHz): δ 2.48 (s, 6H, 2 X Ar-CH₃), 7.41-7.89 (m, 10H, Ar-H at C-4, C-3’, C-5’, C-3”, C-5”, C-h, C-j, C-m, C-n and C-o), 8.10-8.28 (m, 9H, Ar-H at C-f, C-g, C-l, C-k, C-p, C-2’, C-6’, C-2” and C-6”), 8.60 (d, J=8.9 Hz, 1H, Ar-H at C-e), 8.85 (s, 1H, Ar-H at C-c)

FTIR spectrum (KBr) ν_max/cm^-1: 1680 (-COO-), 1600 (Aromatic -C=C-), 1590 (-C=N- stretching of pyrazole), 1525 (Ar-NO₂), 1425, 1320, 1210 (-C-N- stretching of pyrazole), 1125, 1110, 975, 925, 825 and 780

Elemental Analysis: Calculated for C₃₅H₂₆N₄O₆: C, 70.23; H, 4.34; N, 9.36 %. Found: C, 70.19; H, 4.25; N, 9.35 %.

R= -NO₂: ¹H NMR spectrum (DMSO-d₆) (400 MHz): δ 7.42-7.90 (m, 10H, Ar-H at C-4, C-3’, C-5’, C-3”, C-5”, C-h, C-j, C-m, C-n and C-o), 8.16-8.31 (m, 9H, Ar-H at C-f, C-g,C-k, C-l, C-p, C-2’, C-6’, C-2” and C-6”), 8.58 (d, J=9 Hz, 1H, Ar-H at C-e), 8.90 (s, 1H, Ar-H at C-c)

FTIR spectrum (KBr) ν_max/cm^-1: 1700 (-COO-), 1605 (Aromatic -C=C-), 1595 (-C=N- stretching of pyrazole), 1530 (Ar-NO₂), 1425, 1320, 1215 (-C-N- stretching of pyrazole), 1125, 1110, 975, 925, 825 and 780

Elemental Analysis: Calculated for C₃₃H₂₀N₆O₁₀: C, 60.00; H, 3.03; N, 12.72 %. Found: C, 59.81; H, 2.95; N, 12.57 %.

ANTIMICROBIAL ACTIVITY:

The antimicrobial activity was assayed by using cup-plate diffusion method by measuring the inhibition zones in mm. Compounds were screened in vitro for their antimicrobial activity against a variety of bacterial strains such as Staphylo-coccus aureus and Escherichia coli and fungal strains such as Aspergillus niger and Aspergillus oryzae at 1 mg/mL and compared with known antibiotics like Furacin and Griseofulvin as standard.

Antibacterial Activity¹⁷

For anti bacterial activity, we had taken 20 gms of Luria-Bertani broth (Hi media M-575) and 25 gms of agar-agar in 1000 mL distilled water, plugged with cotton and wrapped with paper and heated till dissolved. Then, the six petri dishes having flat bottom, pipette, above prepared nutrient agar flask mixture were sterilized by autoclave at 15 lbs pressure and 121°C for 15 minutes. Here, agar-agar was used to solidify the solution. Overlay the plate with 4 mL soft agar-agar containing 0.1 mL test culture. Bored four well on each plate with 8 mm diameter cork bore aseptically. All the compounds were evaluated by using 1 mg/mL solution in DMF and added 0.1 mL of testing solution into each well. This solution was allowed to diffuse at 4°C. After 20 minutes diffusion, the plate was incubated at 37°C for over night. After incubation, the zone of inhibition was measured with standard furacin and Grieseofulvin at same concentration.

Antifungal Activity¹⁷

Aspergillus niger and Aspergillus oryzae were employed for testing fungicidal activity using cup plate method. The cultures were maintained on Sabouraud's agar slants. Sterilized Sabouraud's agar medium was inoculated with 72 hours old suspension of fungal spores in a separate flask. About 25 mL of the inoculated medium was evenly spread in a sterilized petri dish and allowed to settle down for 2 hours. The cups (10 mm in diameter) were punched in petri dish and loaded with 0.1 mL sample solution in DMF. The plates were incubated at room temperature (37°C) for 48 hours. After the completion of the incubation period, the zones of inhibition of growth of compounds in the form of diameter in mm were measured. Along the test solution in each petri dish, one cup was filled with solvent which acted as control. The antifungal activity of compound was compared with known standard drugs mentioned above, which are recorded in table.

RESULTS AND DISCUSSION:

The FTIR spectrum of compound 2, shows broad hydroxyl group (-OH) absorption peak at 3200 cm^-1, while –C=N- and –C-N- stretching absorption band of pyrazole was observed at 1595 and 1210 cm^-1. The FTIR spectrums, for all the representative members showed absorption bands in the region of ν_max/ cm^-1 1680 to 1700 for (-COO-) stretching, 1590-1595 for (-C=N-) stretching of pyrazole ring, 1520-1525 for (Ar-NO₂), 1200-1215 for (-C-N-) stretching of pyrazole ring.

All the aromatic protons were observed in the range of δ 7.85-8.48 ppm, while ydroxyl protons (-OH) was observed at δ 9.90 and 10.10 ppm for intermediate compound (2). The formation of ester by condensation of acid chloride and hydroxyl derivative has been confirmed on the basis of ¹H NMR. Here, the characteristic of two hydroxyl protons peak of intermediate compound (2) was not observed in final compounds. Formation of ester bond was confirmed by disappearance of hydroxyl protons in final ¹H NMR of the representative compounds. All aromatic protons were observed in the range of δ 7.32-8.92 ppm. Six methyl protons of Ar-CH₃ were obtained as double at δ 2.48 ppm.

The microbial activity data was recorded in table 1. All the synthesized pyrazole derivatives were screened for the microbial activity. In the present study, all the compounds showed moderate microbial activity. For series I, the nitro substituted derivatives showed better anti-bacterial activity compared to other derivatives. While all the synthesized compounds showed moderate anti-fungal activity. All the synthesized compounds were tested for cytotoxic activity by the BLST bioassay method. Among them compounds Ia and IC showed a dose dependent cytotoxic activity at concentration (Ia) 25.27 μg/mL, (Ic) 32.10 μg/mL respectively. The other compounds are less activity. The degree of lethality is directily proportional to the concentration of synthesized compounds. All synthesized compounds having moderate cytotoxic properties.

Where Ar = Phenyl, 4-Chlorophenyl, 4-Methylphenyl, 4-Nitrophenyl, Nicotin, Isonicotin, 4-Chloro-3-nitrophenyl.

Reagents and conditions: (a) Methanol, H₂SO₄, 60°C (b) 2,4-Dinitrophenylhydrazine, Glacial acetic acid, Reflux or In microwave (c) Thionyl chloride, Reflux (d) Dry Pyridine, Cold aqueous HCl (1:1); Reflux or In microwave

Scheme 1: The synthetic route to series I compound

Table 1:Microbial activity data for Series I Compounds

Sr. No.	Ar	Anti Bacterial Blank 12 mm		Anti Fungal Blank 12 mm
Sr. No.	Ar	*E. Coli*	*S. Aureus*	*A. Niger*	A. *Orazae*
1	Phenyl	12.25	12.00	12.00	12.00
2	4-Chlorophenyl	12.25	12.50	12.50	12.00
3	4-Methylphenyl	12.25	12.25	12.25	12.75
4	4-Nitrophenyl	13.50	13.00	12.25	13.25
5	Nicotin	12.50	12.25	12.50	13.25
6	Isonicotin	12.25	12.50	12.25	14.50
7	4-Chloro-3-nitro phenyl	13.50	13.75	13.25	12.50
8	Furacin (Standard)	17.50	17.50	-	-
9	Grieseofulvin (Standard)	-	-	-	15.00

Note: All the compounds were crystallized by DMF-Ethanol mixture or by DMF.

Table 2: Cytotoxic activity of data for synthesized series I

Ar	Solubility	ED₅₀ μg/mL
Phenyl	DMSO	25.27
4-Chlorophenyl	DMSO	41.23
4-Methylphenyl	DMSO	32.10
4-Nitrophenyl	DMSO	40.23
Nicotin	DMSO	41.34
Isonicotin	DMSO	42.36
4-Chloro-3-nitrophenyl	DMSO	39.18
Podophyllotoxin	DMSO	3.88

CONCLUSION:

All the newly synthesized pyrazole derivatives with ester linkage have good anti-bacterial and anti-fungal activity. Therefore, they can be used for the development of new drugs for treatment of bacterial and fungal diseases.

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Received on 13.08.2013 Modified on 15.09.2013

Asian J. Research Chem. 6(11): November 2013; Page 1060-1064