INTRODUCTION:

The wide occurrence of the heterocycles in bioactive natural products and pharmaceuticals has made them as important synthetic targets. 1,3,4-oxadiazoles are a class of heterocycles which have attracted significant interest in medicinal chemistry. Among the 1,3,4-oxadiazoles 2,5-unsymmetrical di-substituted derivatives have attracted considerable attention because of their biological activity namely anticancer¹, anti-inflammatory², analgesic², antibacterial³ and antifungal³, antitubercular⁴. Literature studies revel that 2,5-disubstituted 1,3,4-oxadiazoles have been synthesized by microwave irradiations of the hydrazide and carboxylic acid mixture⁵ by dehydration of diarylhydrazines⁶. 2,5-disubstituted 1,3,4-oxadiazole have also been synthesized by oxidative cyclization using chloramine-T⁷ as an oxidant, ceric ammonium nitrate⁸, lead tetraacetate^9,10, potassium permanganate¹¹ under microwave conditions.

A large number of azole compounds are used as antimicrobial drugs in clinic for example miconazole, clotrimazole and econazole are administrated topically while ketocoazole , itraconazole and fluconazole are useful in the treatment of systemic infections under the frame work of “Green Chemistry” we have developed and environmentally being solvent free approach for the synthesis of 1,3,4-oxadiazole.

We now reported a typical microwave synthesis of 2-(3-Fluorophenyl)-5-(substituted phenyl)1,3,4-oxadiazole by condensing 3-fluoro benzoic acid hydrazide with an aromatic acid and alumina in presence of POCl₃ the reaction is carried under microwave irradiation in microwave made Roto Synth (Milesone-USA). The reaction is completed in 8 to 12 minute in microwave oven.

Chemistry:

As shown in scheme the title compounds were synthesized by the ring closure reaction of various aroyl hydrazides with aromatic acid in present of POCl₃ in good quality and yield. The aroylhydrazides were synthesized by esterification reaction of 3-fluorobenzoic acid by using sulphuric acid as catalyst. The synthesized 1,3,4-oxadiazoles were characterized by IR, 1H NMR. IR spectra of final compounds showed the absence of amide and carbony frequency in the region 1760-1650 cm^-1, and the NH frequency in the region 3400-3200 cm^-1and showed new peak at 1620-1540 cm^-1 due to C=N.

Antibacterial activity:

All the ten compounds were tested in vitro for their antibacterial activity against two Gram positive bacteria namely staphylococcus aureus (ATCC-25923), Bacillus subtilis (ATCC-6633) and Gram-negative bacteria namely, Escherichia coli (ATCC-28922) and Pseudomonas aeruginosa (ATCC-27853).

Minimum inhibitory concentration (MIC) those compounds were determined which are showing activity in primary screening standard antibiotics namely ciprofloxacin was used for comparison with antibacterial activity shown by compound A₁ –A₁₀. All the compounds of the tested series possessed good antimicrobial activity three of these compounds A₃ ,A₆ and A₁₀ exhibited good antibacterial activity against both Gram positive and Gram negative bacteria (15.62 µg/ml) while minimum activity was found in compound A₄.

Antifungal activity:

All the ten compounds were tested in vitro for their antifungal activity against fungi namely Candida albicans. Standard antibiotics namely fluconazole was used for comparison with antifungal activity shown by compounds A₁ –A₁₀. A careful analysis of MIC revealed that all most all of the newly synthesized compound showed comparable antifungal activity with commercial antibiotics fluconazole as shown in table – 2 maximum activity was found in compound A₃A₅,A₆, A₉, A₁₀ (15.62 µg/ml) . Minimum activity was found in compounds A₈(125 µg/ml).

Table -1 Minimum Inhibitory concentration (MIC) (in µg/ml) of compound A1-A10 by using macrodilution method.

Compound	*S. aureus*	*Bacillus subtilis*	*E. coli*	*Pseudomonas aeruginosa*
A₁	31.25	15.62	15.62	15.62
A₂	15.62	31.25	31.25	31.25
A₃	15.62	15.62	15.62	15.62
A₄	62.5	31.25	31.25	125
A₅	15.62	31.25	31.25	31.25
A₆	15.62	15.62	15.62	15.62
A₇	31.25	62.5	62.5	31.25
A₈	15.62	15.62	15.62	15.62
A₉	31.25	31.25	31.25	31.25
A₁₀	15.62	15.62	15.62	15.62
Ciprofloxacin	7.8	7.8	7.8	7.8

Reaction Scheme-1

Table -2 Minimum Inhibitory concentration (MIC) (in µg/ml) of compound A1-A10 by using macrodilution method.

Compound	MIC (in µg/ml) *Candida albicans*
A₁	62.5
A₂	31.25
A₃	15.62
A₄	31.25
A₅	15.62
A₆	15.62
A₇	31.25
A₈	125
A₉	15.62
A₁₀	15.62
Fluconazole	15.62

Experimental:

Melting points were determined in open capillaries with electrical melting point apparatus and are uncorrected. Infra Red (IR) spectra were recorded on Simandzu FTIR 84005 spectrophotometer (KBr), ¹HNMR spectra were recorded on VARIAN 400 MHZ instrument using CDCl₃ or DMSO-d₆ as solvent and TMS as internal reference. Chemical shifts were reported in parts per million (PPM). The homogeneity of the synthesized compounds was monitored by ascending thin layer chromatography (TLC) on silica gel G-Plates and visualizing by using iodine vapor. Developing solvent was Chloroform: Methanol (6:1).

General Procedure for the preparation of Methyl ester :

Substituted benzoic acids(0.2moles) were dissolved in methanol(50.0ml) in a round bottom flask equipped with reflux condenser and calcium chloride guard tube. Concentrated sulfuric acid (2.0ml) was added and the reaction mixture subjected to reflux for 8-10 hrs and reaction progress monitored by TLC. After completion of reaction the excess alcohol was removed under reduced pressure and resulting oil was poured into water. The oily layer was separated and the aqueous layer was washed with dilute solution of sodium carbonate(100ml) to remove unreacted acid. The organic layer was dried over anhydrous sodium sulphate. The solvent was removed on a rotary evaporator after filtration.

General procedure for the preparation of Hydrazide:

The respective ester (0.02mole) was dissolved in methanol (100ml) in a Round bottom flask fitted with a reflux condenser and calcium chloride dropping tube. Hydrazine hydrate (80 %, 0.04mole) was added slowly and the reaction progress was monitored by TLC. After completion of the reaction the resulting mixture was concentrated under reduced pressure. The resulting solid was filtered, washed with water and recrystallized from aqueous ethanol.

Preparation of 2-(3- fluorophenyl)-5-(substitutedphenyl) 1,3,4-oxadiazole:

A mixture of 3-fluoro benzoic acid hydrazide (0.01mole) and substituted benzoic acid (0.01mole), alumina (1.50gm) and phosphorous oxychloride (5ml) was added to this mixture. The reaction was carried out under microwave irradiation in microwave made Roto Synth (Milestone-USA), MW 300 Watt at 130^oC. The reaction is completed in 8 to 12 minutes in microwave oven. The contents were then cooled and poured into crushed ice and neutralized with NaHCO₃ solution. The resulting solid was dried and recrystallized from ethanol.

Compound A₁:

2-(3- fluorophenyl)-5-(2-chlorophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 68%. Melting range 80-82^oC. FTIR (KBr)cm^-1 1594(C=N ring),1266 (C-O-C)starching, 1109(C-F),740(C-Cl), ¹H NMR(DMSO-d₆)δ: 7.55-8.19(m,8H,Ar-H)

Compound A₂:

2-(3- fluorophenyl)-5-(3-chlorophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 66%. Melting range 164-165^oC. FTIR (KBr)cm^-1 1600 (C=Nring),1260 (C-O-C) starching, 1120(C-F),745(C-Cl), ¹H NMR(DMSO-d₆) δ:8.75-8.19(m,8H,Ar-H)

Compound A₃:

2-(3- fluorophenyl)-5-(4-chlorophenyl) 1,3,4-oxadiazole

Recrystallized form ethanol to yield 69%. Melting range 180-182^oC. FTIR (KBr)cm^-1 1592 (C=N ring), 1270 (C-O-C) starching, 1110(C-F),740(C-Cl), ¹H NMR (DMSO-d₆) δ:7.55-8.19(m,8H,Ar-H)

Compound A₄:

2-(3- fluorophenyl)-5-(2,3-dichlorophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 72%. Melting range 170-172^oC. FTIR (KBr)cm^-1 1596 (C=N ring),1262 (C-O-C) starching, 1090(C-F),742(C-Cl), ¹H NMR (DMSO-d₆) δ:7.5-8.2 (m,7H,Ar-H)

Compound A₅:

2-(3- fluorophenyl)-5-(2,4-dichlorophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 76%. Melting range 126-127^oC. FTIR (KBr)cm^-1 1594 (C=N ring), 1266 (C-O-C)starching, 1109 (C-F), 740 (C-Cl), ¹H NMR (DMSO-d₆) δ:7.5-8.2(m,7H,Ar-H)

Compound A₆:

2-(3- fluorophenyl)-5-(3-nitrophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 72%. Melting range 136-137^oC. FTIR (KBr) cm^-1 1592 (C=N ring),1268 (C-O-C) starching, 1089(C-F), 1196 (C-N)starching,1530 and 1350(C-NO₂), ¹H NMR (DMSO-d₆) δ:7.5-8.23 (m,8H,Ar-H)

Compound A₇:

2-(3- fluorophenyl)-5-(4-nitrophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 78%. Melting range 138-140^oC. FTIR (KBr)cm^-1 1598 (C=N ring), 1260 (C-O-C)starching, 1102(C-F), 1198(C-N) starching,1530 and 1354(C-NO₂), ¹H NMR (DMSO-d₆) δ:7.5-8.23(m,8H,Ar-H)

Compound A₈:

2-(3- fluorophenyl)-5-(3,5-dinitrophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 66%. Melting range 204-206^oC. FTIR (KBr)cm^-1 1596 (C=N ring), 1270 (C-O-C) starching, 1104 (C-F), 1200(C-N) starching, 1534 and 1348 (C-NO₂), ¹H NMR (DMSO-d₆) δ:7.5-8.23 (m,7H,Ar-H)

Compound A₉ :

2-(3-fluorophenyl)-5-(2-Chloro,4-nitrophenyl)1,3,4-oxadiazole Recrystallized form ethanol to yield 78%. Melting range 126-127^oC. FTIR (KBr)cm^-1 1592 (C=N ring), 1268 (C-O-C) starching, 1100 (C-F), 1194 (C-N) starching, 1540 and 1340 (C-NO₂), ¹H NMR (DMSO-d₆) δ:7.5-8.2(m,7H,Ar-H)

Compound A₁₀:

2-(3- fluorophenyl)-5-(3- fluorophenyl)1,3,4-oxadiazole

Recrystallized form ethanol to yield 82%. Melting range 106-108^oC. FTIR (KBr)cm^-1 1596(C=N ring),1270 (C-O-C) starching, 1110 (C-F) ¹H NMR (DMSO-d₆) δ:7.5-8.2 (m,8H,Ar-H)

CONCLUSION:

We have described here in efficient and convenient synthesis of unsymmetrical 2,5disubstituted 1,3,4-oxadiazoles by the ring closure reactions of 3-fluorobenzohydrazide with aromatic acid in presence of POCl₃ under microwave irradiation. The antibacterial and antifungal activities of compounds have proved them potential antibacterial and antifungal agents.

ACKNOWLEDGMENT:

The authors were thankful to Dr. Bhavin Bhatt, Sterling Hospital, Bhavnagar for his assistance in antibacterial and antifungal studies and Dr. Vishnu Sutarai, Shree Dhanvantari Institute of Pharmaceutical for FT.I.R, analysis.

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Received on 16.04.2013 Modified on 30.04.2013

Asian J. Research Chem. 6(5): May 2013; Page 486-489