INTRODUCTION:

The synthesis of heterocyclic compounds has always drawn attention of organic chemist over the years mainly because of their important biological properties. The role of nitrogen and oxygen containing hetero compound which are endowed with unique structure and potent antibacterial activity need to be over emphasized ^[1]

Literature survey reveals that majority of the pharmacologically active agents are heterocyclic compounds. These compounds are reported to possess a wide spectrum of therapeutic properties. Numerous reports have highlighted their chemistry and use. Diverse biological activities, such as anti-tubercular, anti-inflammatory, analgesic, antipyretic and anticonvulsant have been found to be associated with oxadiazole derivatives. For this reason our aim was to synthesize various 1, 3, 4-oxadiazole-2-thiol derivatives to make notable contributions to this class of heterocyclic compounds.

The 2, 5-disubstituted 1, 3, 4-oxadiazole derivatives were prepared in this study as potential antibacterial and antifungal drugs. The idea came from previous interest in these compounds, in addition to the data that were published in the literature about the promising effects of some related compounds. The importance of developing new antibacterial active compounds needs not to be emphasized, especially if one considers the problem of resistance and multi-resistance properties arising continuously among pathogenic bacteria.^[2]

There is already evidence that antibacterial resistance is associated with an increase in mortality. In view of the increased resistance of microorganism to currently available antimicrobial agents, there is an urgent need for synthesis of new antimicrobial agents. These observations and the research efforts are directed towards the synthesis of novel 2, 5-disubstituted 1, 3, 4-oxadiazole derivatives that are as effective as antimicrobial agents.

An attempt is made to synthesize, characterize novel 2, 5-disubstituted 1, 3, 4-oxadiazole derivatives and evaluate them for their antibacterial and antifungal activity.^[3]

MATERIALS AND METHODS:

All research chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Lancaster Co. (Ward Hill, MA, USA) and were used as such for the reactions. Solvents except for those with laboratory reagent grade were dried and purified according to the literature, when necessary. Reactions were monitored by thin layer chromatography (TLC) on pre-coated silica gel plates from E. Merck and Co. (Darmstadt, Germany).

Melting points of the synthesized compounds were determined in a Thermonik (Mumbai, India) melting point apparatus and are uncorrected. IR spectra were recorded using KBr disk on a Thermo Nicolet 200 FT-IR Spectrometer (Nicolet, Madison WI, USA). The ¹H-NMR spectra were recorded on Bruker AVANCE II 400 MHz (Bruker, Rheinstetten/Karlsruhe, Germany) using DMSO-d₆ as solvent. Chemical shifts are reported in δ [ppm] units with respect to TMS as internal standard. The purity of compounds was examined by TLC on silica gel plate using n-Hexane and ethyl acetate (6:4) as a mobile phase and iodine vapor as visualizing agent.

General procedure for the preparation of methyl 3-[(substituted phenyl) amino] propanoate (Ia-i)^[4-5]:

A mixture of substituted aniline (0.5 mol), methyl acrylate (0.5 mol) and glacial acetic acid (15 ml) was refluxed on an oil bath at 120°C for 15 h. The reaction mixture was poured onto a crushed ice. The precipitated solid was filtered, washed with cold water, dried and recrystallised from appropriate solvent.

General procedure for the preparation of 3-[(substituted phenyl) amino] propanehydrazide (IIa-i) ^[^6]:

A mixture of methyl 3-[(substituted phenyl) amino] propanoate (Ia-i) (0.01 mol) and hydrazine hydrate (0.01 mol, 99%) were dissolved in absolute ethanol under heating, the reaction mixture was refluxed on steam bath for 12 h. Excess of ethanol was distilled off under reduced pressure and residue was precipitated with ice cold water. The precipitated solid was filtered, dried and recrystallised from absolute ethanol.

General procedure for synthesis of 5-{2-[(substituted phenyl) amino] ethyl}-1, 3, 4-oxadiazole-2-thiol (IIIa-i)^7-8:

A mixture of 3-[(substituted phenyl) amino] propanehydrazide (IIa-i) (0.01 mol) and carbon disulphide (0.01 mol) in alcoholic KOH was refluxed for 6-8 h until the evolution of hydrogen sulphide gas ceased. The mixture was allowed to cool to room temperature and poured on to the crushed ice, neutralized with acetic acid. The resulting solid was filtered, washed with cold water, dried and recrystallised from ethanol.

RESULTS AND DISCUSSION:

The synthesis of 5-{2-[(substituted phenyl) amino] ethyl}-1, 3, 4-oxadiazole-2-thiol (IIIa-i) was achieved through the versatile and efficient synthetic route outlined in Scheme. Various substituted anilines were condensed with methyl acrylate in presence of glacial acetic acid in order to produce the methyl 3-[(substituted phenyl) amino] propionate (Ia-i). Condensation of methyl-3-[(substituted phenyl) amino] propanoate (Ia-i) with hydrazine hydrate (99%) in the presence of absolute ethanol yielded 3-[(substituted phenyl) amino] propanehydrazide (IIa-i). The titled compounds (IIIa-i) were synthesized conveniently by refluxing acid hydrazides (IIa-i) with carbon disulphide in presence of alcoholic KOH. The results of the Physical and Spectral data of the series of compounds (IIIa-i) are summarized in Table 1 and 2, respectively.

R R

a H f 3-Cl

b 4-Cl g 3-NO₂

c 4-CH₃ h 3-CH₃

d 4-NO₂ i 3,4-(Cl)₂

e 4-OCH₃

Synthesis of 5-{2-[(substituted phenyl) amino] ethyl}-1, 3, 4-oxadiazole-2-thiol

Biological evaluation:

All the newly synthesized compounds (IIIa-i) were evaluated for their in-vitro antibacterial activity against two Gram-positive bacterial strains namely, Staphylococcus aureus (NCIM 2602) and Bacillus subtilis (NCIM 2613) and two Gram-negative bacterial strains namely, Escherichia coli (NCIM 2666) and pseudomonas aeruginosa using the conventional agar-dilution method. Inoculum was added to 30 ml of sterile nutrient agar medium and was poured into sterile Petri dishes for solidifying. After diffusion, the Petri dishes were incubated at 37 ⁰C for 24 h. Ciprofloxacin was used as the reference standard.^[9-11]

Table 1: Physical data of the synthesized compounds

Compound

Yield (%)

M.P. (⁰C)

Rf^§

Mol. Formula

Mol. Weight

IIIa

IIIb

IIIc

IIId

IIIe

IIIf

IIIg

IIIh

IIIi

4-Cl

4-CH₃

4-NO₂

OCH₃

3-Cl

3-NO₂

CH₃

3,4-(Cl)₂

181

115

175

190

185

152

141

152

161

0.57

0.49

0.61

0.45

0.81

0.70

0.51

0.75

0.55

C₁₀H₁₁SN₃O

C₁₀H₁₀SN₃ClO

C₁₁H₁₃SN₃O

C₁₀H₁₀SN₄O₃

C₁₁H₁₃SN₃O₂

C₁₀H₁₀SN₃ClO

C₁₀H₁₀SN₄O₃

C₁₁H₁₃SN₃O

C₁₀H₉SN₃Cl₂O

221

254

235

266

251

254

266

235

287

All synthesized compounds were purified by column chromatography using n-hexane and ethyl acetate (6:4) as a mobile phase and iodine vapors as visualizing agent.

Table 2: Spectral data of the synthesized compounds

Compound	R	IR spectra (KBr cm^-1)	¹H NMR spectra (δ, ppm)
III a	H	3283 (NH str), 1624 (C=N str), 1599 (C=C str).	6.80-7.27 (m, 5H, Ar-H),5.26 (s, 1H, NH), 3.60 (t, 2H, CH₂), 3.03 (t, 2H, -CH₂), 3.06 (s, 1H, SH).
III b	4-Cl	3278 (NH str), 1619 (C=N str), 1523 (C=C str).	6.53-7.16 (m, 4H, Ar-H), 5.26 (s, 1H, NH), 3.51 (t, 2H, CH₂), 2.96 (t, 2H, -CH₂), 3.06 (s, 1H, SH).
III c	4-Me	3310 (NH str), 1660 (C=N str), 1593 (C=C str).	6.57-7.16 (m, 4H, Ar-H), 5.58 (s, 1H, NH), 3.58 (t, 2H, CH₂), 2.98 (t, 2H, -CH₂), 2.26 (s, 3H, CH₃), 3.06 (s, 1H, SH).
III d	4-NO₂	3236 (NH str), 1600 (C=N str), 1544 (C=C str).
III e	4-OMe	3364 (NH str), 1619 (C=N str), 1522 (C=C str).	6.57-6.77 (m, 4H, Ar-H), 5.54 (s, 1H, NH), 3.45 (t, 2H, CH₂), 2.89 (t, 2H, -CH₂), 3.86 (s, 3H, OCH₃), 3.00 (s, 1H, SH).
III f	3-Cl	3211 (NH str), 1601 (C=N str), 1546 (C=C str).
III g	3-NO₂	3250 (NH str), 1607 (C=N str), 1552 (C=C str).	6.64-7.24 (m, 4H, Ar-H), 5.21 (s, 1H, NH), 3.64 (t, 2H, CH₂), 3.03 (t, 2H, -CH₂), 3.06 (s, 1H, SH).
III h	3-Me	3234 (NH str), 1597 (C=N str), 1548 (C=C str).
III i	3,4(Cl)₂	3394 (NH str), 1617 (C=N str), 1534 (C=C str).	6.93 (s, 1H, Ar-H), 5.55 (s, 1H, NH), 3.45 (t, 2H, CH₂), 2.78 (t, 2H, -CH₂), 6.45-7.34 (d, 2H, Ar-H ), 3.06 (s, 1H, SH).

Table 3: Antibacterial and antifungal activity of synthesized compounds

Compound	R	Zone of Inhibition in mm
		Antibacterial activity				Antifungal activity
		S. aureus	B. *subtilis*	E. *coli*	P. *aeruginosa*	*C. albicans*	*A. niger*
III a	H	19	14	15	13	10	12
III b	4-Cl	24	17	18	16	21	17
III c	4-Me	10	15	9	7	15	16
III d	4-NO₂	18	13	11	9	11	9
III e	4-OMe	13	7	8	7	10	9
III f	3-Cl	18	16	13	17	17	15
III g	3-NO₂	10	8	9	10	11	10
III h	3-Me	12	16	10	11	14	15
III i	3,4 (Cl)₂	25	18	18	21	20	19
DMSO	-	-	-	-	-	-	-
Ciprofloxacin	-	30	24	32	28	-	-
Gresiofulvin	-	-	-	-	-	28	30

Standard cultures of Candida albicans and Aspergillus niger (NCIM 813) were used for the antifungal study. The fungi were maintained by subculturing and used at regular intervals. An inoculum was prepared by suspending a single isolated colony in normal saline. Gresiofulvin was taken as standard reference.^[12] The results of the antibacterial and antifungal activity of the series of compounds (IIIa-i) are summarized in Table 3.

CONCLUSION:

The compounds investigated in the present study showed antibacterial and antifungal activities against the strains used in this investigation. Among the series tested, two compounds (IIIb and IIIi) exhibited excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. Compound III b which showed a very strong activity against Candida albicans is a very promising antifungal drug to be modified. The same applies to compound III i concerning its activity against Aspergillus niger.

The result of antimicrobial activity indicates that the presence of chloro group at para position enhances the activity. Suitable molecular modification of these compounds may generate potent antimicrobial agents in future.

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Received on 02.02.2011 Modified on 25.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 766-769