INTRODUCTION:

Oxadiazole is a five member heterocyclic ring which is a versatile lead compound for designing potent bioactive agents. This interesting group of compound has diverse biological activities such as antimicrobial, anti-inflammatory, antitubercular, antihypertensive, analgesic, anticonvulsant, anticancer, anti-HIV, hypoglycemic and genotoxic^1-5 etc.

Cinnoline ring^6-8 is a versatile lead molecule that has been investigated widely used in medicinal chemistry due to its important pharmacological activities. This have been reported to exhibit anti-microbial, anti-tubercular, anti-malarial, anti-hypertensive, anti-convulsant, neurological disorders, anti-depressant, anti-pyretic and analgesic etc. Keeping the above facts in view, we considered it of interest to synthesize some new Series of 3-(5-substituted-1,3,4-oxadiazol-2-yl)-6 fluoro cinnolin-4-ol and have been reported as significantly active antibacterial and antifungal agent.

The Anti-bacterial and anti-fungal activity of all the synthesized compounds were evaluated against various microorganisms using disc diffusion method and MIC values were determined by serial dilution method.

MATERIAL AND METHODS:

All the chemicals were of synthetic grade and commercially procured from sigma Aldrich Chemicals. Melting points were recorded on a Buchi capillary melting point apparatus and are uncorrected. IR spectra were recorded on Fourier Transform (SHIMADZU) infrared spectrophotometer, using KBr disc method. The ¹H-NMR spectra were recorded in DMSO- δ6On Perkin Elmer NMR Spectrophotometer 300MHz using TMS as an internal standard. Thin layer chromatography analyses were performed on pre-coated silica gel plates. The oxadiazole based cinnoline were synthesised as given in the following procedure^9-11 and Scheme of reaction. Fig:1

Synthesis of 4-nitro phenyl hydrazono (cyano) acetamide:

Sodium nitrite (0.02mol) dissolved in 20ml of water was added to a suspension of 4-fluoro aniline (0.02mol) in 1N HCl (100ml). And the mixture was stirred for 1hr at 0-5ºC and filtered to obtain the clear salt solution. This solution was added to the well stirred mixture of cyanoacetamide (0.02mol), ethanol (30ml) and water 400ml at 0ºC. sodium acetate (100g) was then added in small portion to keep the mixture alkaline and stirred the mixture for 3 hours at 0ºC. The precipitate was washed thoroughly with water, air dried and recrystallised from ethanol.

Synthesis of 4-amino-6-fluoro cinnoline-3- carboxamide:

A mixture of Cpd-1 (0.025mol), chlorobenzene (25mmol) and anhydrous aluminium chloride (50mmol) was stirred for 1hr and refluxed under anhydrous conditions, cooled and poured into ice and hydrochloride (25ml) was added while stirring. The residue thus obtained was washed with petroleum ether filtered and collected the filtrate and made alkaline with ammonia. The pure base was filtered and washed with DMF.

Synthesis of 4-hydroxy-6fluoro cinnoline-3- carboxylic acid:

5ml of 10% Sodium hydroxide was added to the Cpd-2 and add 20 ml of ethanol and heated on a water bath for 30min at 85ºC. And this mixture was cooled to room temperature and added to crushed ice. The precipitate obtained was collected and washed thoroughly with water, Air dried and recrystalized from ethanol.

Synthesis of 4-Hydroxy-6-fluoro cinnoline-3- carbohydrazide:

Preparation of ethyl 4-hydroxy-6-fluoro cinnoline-3-carboxylate:

This Compound was synthesized by refluxing Cpd-3 (0.0587 mol) with absolute ethanol (12 ml) in presence of conc. Sulphuric acid (0.5 ml) for 5 h at 40-50°C. Excess of alcohol was distilled off and allowed to cool in ice bath.

Preparation of 4-hydroxy-6-fluoro cinnoline-3- carbohydrazide:

This Compounds were synthesized by refluxing a mixture of compound obtained from the above step (0.0602mol) with hydrazine hydrate (5ml) in absolute ethanol (12 ml) for 8hr at 30-40°C. The reaction mixture was cooled to room temperature and poured in ice with constant stirring.

Synthesis of 3-(5-substituted-1,3,4-oxadiazol-2- yl)-6-fluoro cinnolin-4-ol [OC _1-5]:

An equimolar mixture Cpd-4 (0.0054 mol) with various substituted benzoic acids (0.0054 mol) was refluxed with phosphorus oxychloride (5 ml) for 2-3hr on water bath at 100°C.Reaction mixture was cooled to room temperature and poured in ice. The precipitate obtained was filtered off, washed with water and further purified by recrystallization. The physicochemical properties are given in table:1

6-fluoro-3-(5-(4-nitrophenyl)--1,3,4-oxadiazol-2-yl) cinnolin- 4-ol:

Mol.form-C₁₆H₁₀N₅O₄, Mol. Wt 336.07, Yield - 72%, M.p- 165, Rf- 0.58,UVʎ_max475.0, IR(KBr,cm^-1) 3181.97(OH group), 1398.14(F stretching), 1559.66(C=N stretching), 1290.14 (OH bending), 2921.63(azole moiety), 697.1(mono sub. benzene),1027.87 (N-N stretching). MS (m/z) 337

6-fluoro-3-[5-(p-tolyl)-1,3,4-oxadiazol-2-yl]cinnolin-4-ol:

Mol.form-C₁₆H₁₀N₆O₆, Mol.Wt-382.28, Yield-77.2%, M.p-163, Rf-0.63, UVʎ_max 475.0 IR(KBr,cm^-1) 3389.28(OH group), 1600.63(C=N stretching), 2922.59(azole moiety), 1393.32 (F stretching), 1011.19(N-N stretching), 1208.36(OH bending), 749.20(N-N stretching). ¹HNMR(δppm) 7.25(s,1H in OHgroup), 7.45(m,2H,ArH) 7.4 (m,3H,cinnoline), 7.8(d,2H,ArH). MS (m/z) 380.0

6-fluoro-3-[5- (4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl]cinnolin-4-ol:

Mol.form-C₁₅H₉N₆O₄, Mol.Wt-337.06, Yield-72.5%, M.p-157, Rf-0.67, UVʎ_max 478.0 IR(KBr,cm^-1) 3177.15(OHgroup), 1338.36(NO2 stretching), 1102.95(OH bending), 1646.91(C=N in Ar), 1516.74(C=C stretching), 954.31(p-subs phenyl), 2923.56(Azole moiety) ¹HNMR(δppm) 9.7(s,1H,OHgroup), 7.6(d,2H in phenyl), 7.4(m,3H,cinnoline), 7.85(d,2H in phenyl). MS (m/z) 335.0

6-fluoro-3-[5- (4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]cinnolin-4-ol:

Mol.form-C₁₅H₉N₆O₄, Mol.Wt-337.06, Yield-77% , M.p-155, Rf- 0.65, UVʎ_max477.5, IR(KBr,cm^-1) 3412.42(OH group), 1395.25(NO2 stretching) , 1655.59(C=N in Ar), 1596.7(C=C stretching), 2922.59 (azole moiety), 668.214(sub. Phenyl).

5-(4-hydroxy-6-fluoro-3-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]cinnolin-4-ol:

Mol.form-C₁₀H₆N₅O₄, Mol.Wt-292.01, Yield-65%, M.p-187, Rf-0.58, UVʎ_max 471.0, IR (KBr, cm^-1) 3213.79(NH in amine), 3411.46(OH group), 1650.77(C=C in Ar), 1515.7(C=N stretching), 1399.1(NO2 stretching)

Fig:1. Scheme of reaction

Table no:1: Physicochemical parameters of synthesised compounds

compd	Molecular formula	R	Molecular weight	Percentage yield%	colour	Solubility	M. pt	Rf
Oc1	C₁₆H₈FN₅O₄		353.26	61%	Light yellow	DMSO	147˚C	0.64
Oc2	C₁₇H₁₁FN₄O₂		322.29	64%	Orange	DMSO	148˚C	0.62
Oc3	C₁₆H₉FN₄O₃		324.27	65%	Red	DMSO	153˚C	0.58
Oc4	C₁₈H₁₃FN₄O₄		368.32	67%	Light orange	DMSO	156˚C	0.68
Oc5	C₁₆H₈ClFN₄O₂		342.71	69%	yellow	DMSO	160˚C	0.66

Biological activity^12-15

The antibacterial studies were carried out with all the synthesized oxadiazole based Cinnoline derivatives 10µg/disc against gram positive and gram negative bacteria by disc diffusion method. The MIC values of the synthesized compounds were determined. Table:2&3

Antifungal activity of all the synthesized compounds was evaluated against various fungal organisms. Standard Ciprofloxacin and clotrimazole discs 10µg/disc were used as standard. The MIC values of the synthesized compounds were determined. Table:4&5

RESULT AND DISCUSSION:

In the present study various novel 2,5 Di substituted 1,3,4-Oxadiazole derivatives were synthesized. The structure of the synthesized compounds was confirmed by IR, NMR, Mass spectral data. The IR spectrum of all the synthesized compound show characteristic absorption band at 1609.29cm^-1, 3389.28 cm^-1, 1582.31cm^-1 arising from stretching vibration of bands (C=N,O-H,C=C respectively), 1393.21 cm^-1, 749.20 cm^-1 confirms the chemical structure of the compounds synthesized.

PMR spectra of the synthesized Oxadiazole derivatives shown aromatic protons as a multiplet in 7.0-8.0 ppm, 1 Protons of hydroxyl group appeared as a singlet at 5.0 ppm, 4 protons of aromatic group appeared as a multiplet in 6.68- 6.95 ppm. Thus the proton magnetic spectrum of the compound was in full agreement with its molecular formula, with regard to proton count and the chemical shift. The Mass Spectral analysis of the synthesized compounds performed, and the mass spectrum of the compound was in agreement with its molecular weight.

Evaluation of the results from antimicrobial studies showed the synthesized compounds with the zone of inhibition was found to be in the range of (6-20 mm) due to the presence of azole nucleus with cinnoline, fluoro and chloro group substitutions. The MIC of the synthesized compounds against was determined by serial dilution method and was found to be in the range of 1.2- 12.5μg/ml.

Table 2: Anti-bacterial activity of the synthesized compounds (Disc diffusion method)

Micro, organisms	Zone of Inhibition (in mm) of Compounds (10µg/disc)
Micro, organisms	OC1	OC2	OC3	OC4	OC5	*Std Ciprofloxacin**
Staphylococcus aureus	17	10	10	7	6	19
Bacillus Subtilis	14	12	10	11	10	19
Escherichia coli	14	10	10	13	11	17
Salmonella typhi	11	9	10	11	7	18

Table 3: MIC values of the synthesized compounds (serial dilution method)

Micro organisms	MIC values (µg/ml)
Micro organisms	OC1	OC2	OC3	OC4	OC5	*Std Ciprofloxacin**
Staphylococcus aureus	1.2	2.5	1.2	5	2.5	1.2
Bacillus subtilis	5	5	2.5	2.5	2.5	2.5
Escherichia coli	2.5	1.2	1.2	5	2.5	5
Salmonella typhi	5	2.5	5	2.5	2.5	2.5

Table 4: Anti-fungal activity of the synthesized compounds

Micro organisms	Zone of Inhibition (in mm) of Compounds (10µg/disc)
Micro organisms	OC1	OC2	OC3	OC4	OC5	Std* Fluconazole
Candida albicans	16	22	16	18	17	25
Aspergillus niger	16	15	14	17	18	35
Aspergillus flavus	17	18	20	20	18	17
Saccharomyces cerevisiae	12	14	12	17	11	16
Streptomyces griseus	10	11	11	14	14	7

Table no :5 Antifungal MIC Values of the synthesized compounds (serial dilution method)

Micro organisms	MIC Values (µg/ml)
Micro organisms	OC-1	OC-2	OC-3	OC-4	OC-5
Candida albicans	2.5	1.5	2.5	2.5	2.5
Aspergillus niger	2.5	2.5	2.5	3.5	2.5
Aspergillus flavus	6.25	1.25	6.25	1.25	6.25
Saccharomyces cerevisiae	12.5	3.25	12.5	3.25	12.5
Streptomyces griseus	1.2	2.5	2.5	2.5	2.5

MIC values of *Std-fluconazole-1.5µg/ml for all fungal organisms

CONCLUSION:

In Summary, some novel substituted Oxadiazole derivatives have been synthesized and evaluated for antibacterial and anti-fungal activity. All derivatives showed antibacterial and antifungal activity as moderate to good activity against various microorganisms. Further studies on its possible mechanism and in vivo trials in experimental animals to broaden their Pharmacological assessment, may provide a new analogue that can overcome drug resistance, prolonged treatment, complex drug regimen and side effects involved in the treatment of infectious diseases.

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Received on 08.05.2019 Modified on 05.06.2019

Research J. Pharm. and Tech. 2019; 12(10): 4812-4816.

DOI: 10.5958/0974-360X.2019.00832.1