ARTÍCULO

Novel Compounds containing 1,3,4-oxadiazole and pyrazole-3-one Nuclei – Synthesis and in vitro Antimicrobial Evaluation

Krishna Naik1, Aluru Raghavendra Guru Prasad2*, Yadati Narasimha Spoorthy1, Mokkapati Pariplavi3, Lakshmana Rao Krishna Rao Ravindranath1

1Sri Krishnadevaraya University, Anantapur, A.P., India. 2ICFAI Foundation for Higher Education, Hyderabad, A.P., India.3Osmania Medical College, Osmania University, Hyderabad, A.P., India.

*e-mail: guruprasadar@yahoo.co.in


                                                                                                   Recibido el 16 deseptiembre de 2013 -An. Real Acad. Farm. Vol 80, Nľ 1(2014), pág. 201-213

ABSTRACT

New series of heterocycles containing 1,3,4-oxadiazole and pyrazole-3-one moiety have been synthesized and evaluated in vitro against Staphylococus aureus NCCS 2079, Bacillus cereus NCCS 2106, Escherichia coli NCCS 2065, Pseudomanas aeruginosa NCCS 2200, Aspergillus niger NCCS 1196 and Candida albicans NCCS 2106. The compounds demonstrated moderate antimicrobial activity against selected fungal and bacterial strains.

Keywords: Synthesis; 1,3,4-Oxadiazoles; Pyrazole-3-ones; Antimicrobial activity.

RESUMEN

Nuevas series de compuestos heterocíclicos que contienen 1,3,4-oxadiazol y pyrazol-3-ona han sido sintetizadas y evaluadas in-vitro contra Staphylococus aureus NCCS 2079,  Bacillus cereus NCCS 2106, Escherichia coli NCCS 2065, Pseudomanas aeruginosa NCCS 2200, Aspergillus niger NCCS 1196 y Candida albicans NCCS 2106. Los compuestos demostraron actividad antimicrobiana moderada contra estirpes fúngicas y bacterianas seleccionadas.

Palabras clave: Síntesis; 1,3,4-Oxadiazol; Pyrazol-3-uno; Actividad antimicrobiana.


1. introductIon

Heterocycles bearing nitrogen, oxygen and sulphur atoms in their structure have received remarkable attention because of their biological and pharmacological applications [1-3]. Among the wide variety of heterocycles that were explored for developing pharmaceutically important molecules, compounds containing 1,3,4-oxadiazole nucleus [4-7] and pyrazole nucleus [8-13] constitute the important class of compounds exhibiting diverse and extensive spectrum of biological activities. This scenario led us to synthesize the novel compounds VII and IX containing 1,3,4-oxadiazole and pyrazole nuclei and evaluate their antibacterial and antifungal activities.

2. materialS AND METHODS

All chemicals used were analytical grade obtained from Merck India Limited, India. All the glass ware used were of borosilicate grade. The standard bacterial and fungal stains were procured from National Centre for Cell Science, Pune, India. UV-Visible spectrophotometer manufactured by Shimadzu Corporation, Japan was used for absorption measurements. The IR spectra were recorded on a Perkin-Elmer 983 IR spectrometer. The 1H-NMR spectra were recorded on a Bruker AC 300F (200 MHz) NMR spectrometer using DMSO – d6 as solvent and TMS as an internal standard. Mass spectra of the compounds were recorded on a Jeol JMS-D300 mass spectrometer operating at 70 eV.  

2.1. General Synthetic Procedures

The novel compounds were synthesized by specified procedures and the critical intermediate compounds were characterized by elemental analysis and spectral data.

2.1.1. Synthesis of {4-[3-Methyl-5-oxo-4-(4|-substituted phenyl hydrazono)-4,5-dihydro-pyrazol-1-yl]-phenoxy}-acetic acid hydrazide (V).

a.  Synthesis of substituted phenyl diazoniam chloride (I)

The required primary amine was dissolved in a suitable volume of water containing 2.5–3.0 equivalents of hydrochloric acid (or sulphuric acid). The solution was cooled to 0 oC. To the crystals of amine hydrochloride (or sulphate) so obtained, an aqueous solution of sodium nitrite was added portion wise.  An excess of acid was necessary to stabilize the diazonium chloride. Similar procedure was adopted for the preparation of other substituted phenyl diazonium chlorides.

b. Synthesis of substituted phenyl diazonium ethyl acetoacetic ester (II)

To an ice-cold solution of mixture of sodium acetate (1.0 g) in 100 mL of aqueous alcohol and ethyl acetoacetate (0.1 mol) in 50 mL of ethanol, the corresponding diazonium chloride was added till yellow crystals were separated out.  These crystals were filtered, washed with water and dried.

c. Synthesis of 3-methyl-4-(substituted phenyl hydrazono)-pyrazoline-5-one (III)

3-methyl-4-(4|-substituted phenyl hydrazono)-pyrozoline-5-one (III) was synthesized by thecondensation of 4-substituted phenyl hydrazono acetoacetic ester (II) and hydrazine in the presence of required amounts of dimethylformamide. The mixture was subjected to microwave irradiation at 150W intermittently at 30 sec intervals for 2 minutes.  After complete conversion as indicated by TLC, the reaction mixture was cooled and washed with cold water.  The precipitated III was filtered recrystallized from ethanol.   

d.Synthesis of {4-[3-Methyl-5-oxo-4-(substituted phenyl hydrazono)-4,5-dihydro-pyrazol-1-yl]-phenoxy- acetic acid ethyl ester (IV)

A mixture of III, anhydrous K2CO3 and DMF was stirred at room temperature for 8 hours.  The reaction mixture was diluted with ice cold water. The separated solid was filtered and recrystallized from ethanol.

e. Synthesis of {4-[3-Methyl-5-oxo-4-(4|-aryl-hydrazono)-4,5-dihydro-pyrazol-1-yl]-phenoxy}-acetic acid hydrazides (V)

A mixture of IV and hydrazine hydrate in ethanol was refluxed for five hours. The reaction mixture was cooled to room temperature and poured in ice cold water with continuous stirring. The separated solid was filtered, washed with water and recrystallized from ethanol. Other members of the series Vwere prepared on the same lines. The reaction scheme is depicted in Scheme 1.

2.1.2. Synthesis of 2-(4-acetyl- -5,5-disubstituted 4,5-dihydro-[1,3,4]oxadiazole-2-yl(methyloxyphenyl)-5-methyl-4(aryl hydrazono)- -pyrazol-3-ones (VII)

a. Synthesis of {4-[3- methyl- 5- oxo- 4-(4|-phenyl hydrazono)- 4,5-dihydro-pyrazol-1-yl]-phenoxy}-acetic acid (1-phenyl-ethylidene)-hydrazide (VI)

A mixture of V (0.01 mol) in hot methanol (25 mL), acetophenone (0.01 mol) and a drop of glacial acetic acid were refluxed for 3 hours. The solid separated was filtered, washed with cold methanol and recrystallized from methanol to give VIa. Compounds VI b-h were synthesized on similar lines.

b. Synthesis of 2-(4-acetyl-5,5-disubstituted 4,5-dihydro-[1,3,4]oxadiazole-2-yl(methyloxyphenyl)-5-methyl-4(4|-substituted phenyl hydrazono)-pyrazol-3-ones (VII)

A mixture of VIa (0.01 mol) and an excess of acetic anhydride (10 mL) were refluxed for 2 hours.  The excess acetic anhydride was distilled off and the residue was poured on to crushed ice.  The solid obtained was filtered, washed with water and recrystallized from aqueous methanol to get VIIa. The cyclization reaction was extended to other hydrazones VI b–j and in each case the respective compound was isolated. The reaction scheme is given in Scheme 2.

R=H, CH3, OCH3, OC2H5, Cl, Br.

Scheme 1.-Synthesis of {4-[3-Methyl-5-oxo-4-(4|-substituted phenyl hydrazono)-4,5-dihydro-pyrazol-1-yl]-phenoxy}-acetic acid hydrazide (V).

R = -H, -CH3,  -OCH3, -OC2H5, -Cl, -Br, R1 = -CH3, R2 = -C6H5, p-CH3C6H4,p-ClC6H4, p-OCH3C6H4,p-NO2C6H4.

Scheme 2.- Synthesis of 2-(4-acetyl-5-methyl-5-phenyl-4,5-dihydro-[1,3,4]oxadiazole-2-yl(methyl)-5-methyl-4(4|-phenyl hydrazono)-2,4-dihydrazono-pyrazol-3-one (VII).

2.1.3. Synthesis of Mannich bases containing [1,3,4] oxadiazole and pyrazol-3 one  nuclei (IX)

a.    5-methyl- 4-(4|-substituted phenyl hydrazono)-2-(5-thioxo-[1,3,4] oxadiazole-2-yl-methyloxyphenyl)-2,4-dihydro-pyrazol-3-one (VIII)

A mixture of V (19.9 g, 0.1 mol), KOH (5.5 g, 0.1 mol), ethanol (100 mL) and carbon disulphide (6.02 mL, 0.1 mol) was refluxed in a water bath till the evolution of hydrogen sulphide is ceased.  The excess of alcohol was removed by distillation.  The reaction mixture was cooled to room temperature, poured into ice cold water and neutralized with dilute hydrochloric acid. The precipitate so formed was filtered, washed with water, dried and recrystallized from ethanol-dioxane mixture (1:1) to give VIII.

b.    5-methyl-4-(4|-substituted phenyl hydrazono)-2-[5-thioxo-4-[alkyl/phenyl/heterocyclic amino methyl]-4,5-dihydro-[1,3,4] oxadiazol-2-yl-methyl]-2,4-dihydro-pyrazol-3-ones  (IX)

A mixture of VIII (0.01 mol) in ethanol and dioxane (20 mL) was treated with formaldehyde (40%, 1.5 mL).  Appropriate amine (0.01 mol) in ethanol (10 mL) was added to the reaction mixture and stirred over night.  The precipitated Mannich base was filtered, dried and recrystallized from ethanol-DMF mixture (1:1). The reaction sequence is outlined in Scheme 3.

NHR1R2 = morpholinyl, piperazinyl, N-methylpiprazinyl; R1 = -H, R2 = p-Tolyl, p-anisyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-nitrophenyl; R1 = R2 = ethyl or phenyl.

Scheme 3.Synthesis of Mannich bases containing [1,3,4] oxadiazole and pyrazol-3-one  moiety.


2.2. Antimicrobial activity

a. Antibacterial activity

The antibacterial activity of synthesized compounds (250 μg/mL in DMSO) was preliminarily studied by disc diffusion method. The procedure followed for the disc diffusion method is given below.

A suspension of Staphylococcus aureus was added to sterile nutrient agar at 45oC.  The mixture was transferred into sterile petri-dishes to a depth of 3 mm and allowed to solidify.  Sterile discs of 5 mm in diameter (made of Whatmann Filter paper)were immersed in solutions of synthesized compounds. Sterile discs immersed in DMSO were used as control. Both chemical-treated and DMSO-treated discs were laid down onto bacteria mixed agar plates. The plates were allowed to stand for 1 hour at room temperature followed by incubation at 37oC for 24 hours and observed for antibacterial activity. The diameter of the zone of inhibition was measured in each plate. The average zone of inhibition was calculated. A similar procedure was adopted for the antibacterial activity studies against other organisms.

b. Antifungal activity

The procedure described above was followed for antifungal activity against Aspergillus niger NCCS 1196 and Candida albicans. Compounds were treated at several different concentrations using DMSO as a solvent. 

c. Determination of Minimum Inhibitor Concentration

The procedure followed to find out MIC by Broth Dilution Method is given below.

Standardized inoculum (matched to McFarland BaSO4 standard) of suspension of organisms was prepared.  A series of glass tubes containing different concentrations of test compounds dissolved in DMSO and spiller in nutrient broth were incubated with one drop of inoculum and shaken gently to mix the contents.  Two growth control tubes were also prepared by mixing 0.1 mL of control and 0.9 mL of sterile saline and its optical density was determined.  The control contained 1 × 10-5 colony forming units /mL which is equivalent to 20 colonies.

Tubes were incubated for 24 hours at 37oC in air.  The turbidity developed in each tube was recorded by UV-Visible spectrophotometer.  The turbidity produced by the broth (without inoculum) was considered as 100 % transparency.  The minimum inhibitory concentration (MIC) was noted as the concentration of the test sample which completely inhibits the growth of the microorganism i.e. 100 % transparency.


3. results and discussion

3.1. Characterization of synthesized compounds

a. Elemental analysis details of compounds V (-R, M.P., Yield, Molecular formula, Element: Found %, (Calc % ))

Va: H, 152 oC, 65 %, C18H22N6O3, C:58.37(58.25), H:5.94(5.86), N:22.70(22.65), O:12.97(12.85).

Vb: CH3, 153 oC, 60 %, C19H24N6O3, C:59.37(59.26), H:6.25 (6.17), N:21.87(21.76), O:12.23(12.14).

Vc: OCH3, 156 oC, 75%, C19H24N6O3, C:57.00(55.85), H:6.00 (5.68), N:21.00 (19.85), O:16.00 (15.85).

Vd: OC2H5, 168 oC, 80 %, C20H26N6O4, C:57.97(57.85), H:6.28(6.18), N:20.28(20.20), O:15.45(15.35).

Ve: Cl, 174 oC, 75 %, C18H21N6O3Cl, C:53.39(53.29), H:5.19(5.10), N:20.76(20.66), O:11.86(11.76), Cl:8.77(8.66).

Vf: Br, 169 oC, 65 %, C18H21N6O3Br, C:48.11(47.98), H:4.71(4.60), N:18.17(18.07), O:10.89(10.78), Br:17.69(17.58).

b. IR (KBr) Spectral data (νmax in cm-1)

Va: 3445, 3425 (NH2), 3305 (NH), 1665 (C=O), 1620 (C=N).

Vb: 3400, 3420 (NH2), 3285 (NH), 1665 (C=O), 1610 (C=N).

Vc: 3425, 3405 (NH2), 3200 (NH), 1615 (C=N), 1555 (C=O).

Vd: 3435, 3415 (NH2), 3300 (NH), 1615 (C=N), 1660 (C=O).

Ve: 3420, 3400 (NH2), 3275 (NH), 1645 (C=O), 1610 (C=N).

Vf: 3444, 3424 (NH2), 3290 (NH), 1650 (C=O), 1605 (C=N).

c. 1H NMR (DMSO – d6) Spectral data (δ in ppm)

Va: 1.2 (s, 3H, CH3), 2.1 (s, 2H, NH2), 3.85 (s, 2H, O-CH2-CO) 6.8 (s, 1H, Ar-NH) 7.1-7.3 (m, 5H, C66666H5 ), 7.4 (d, 2H, C6H4), 7.7(d, 2H,C6H4), 8.4 (s,1H,NH)

Vb: 0.9 (s, 3H, CH3), 1.16 (s, 3H, CH3), 2.06 (s, 2H, NH2), 3.80(s, 2H, O-CH2-CO) 6.8(s,1H,Ar-NH) 7.1-7.3 (m, 4H, C6H4),7.4 (d, 2H, C6H4), 7.7(d, 2H,C6H4), 8.36 (s, 1H, N, CONH)

Vc: 1.12 (s, 3H, CH3), 2.02 (s, 2H, NH2), 3.24 (s, 3H, OCH3),   3.76 (s, 2H, O-CH2-CO), 6.8 (s, 1H, Ar–NH), 7.1-7.3 (m, 4H, C6H4),  7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 8.32 (s, 1H, NH)

Vd: 0.9 (s, 3H, CH3), 1.11 (t, 3H, CH3), 2.06 (s, 2H, NH2), 3.14 (q, 2H, O–CH2),   3.8 (s, 2H, O-CH2-CO), 6.8 (s, H, Ar–NH), 7.1-7.3 (m, 4H, C6H4), 7.4 (d, 2H,C6H4), 7.7(d,2H,C6H4),   8.36 (s,1H,NH)

Ve: 1.08 (s, 3H, CH3), 2.08 (s, 2H, NH2), 3.82 (s, 2H, O-CH2-CO) 6.8 (s, 1H, Ar–NH), 7.1-7.3 (m, 4H, C6H4), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 8.38 (s, 1H, NH)

Vf: 1.04 (s, 3H, CH3), 2.04 (s, 2H, NH2), 3.78 (s, 2H, O-CH2-CO),  6.8 (s, 1H Ar–NH), 7.1-7.3 (m, 4H, C6H4), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 8.34 (s, 1H, NH)

d. Elemental analysis details of compounds VI (-R, -R1, -R2, M.P., Yield, Molecular formula)

VIa: -H, -H, -C6H5,240 oC, 75 %, C26H28N6O3, C:66.10(65.95), H:5.93(5.83), N:17.93(17.80), O:10.16(10.02).

VIb: -CH3, -CH3, -C6H5, 245 oC, 77 %, C27H30N6O3, C:66.66(66.50), H:6.17(6.05), N:17.28(17.12), O:9.87(9.72).

VIc: OCH3, CH3, C6H5, 35 oC, 72 %, C27H30N6O4, C:64.54(64.42), H:5.97(5.83), N:16.73(16.58), O:12.74(12.62).

VId: OC2H5, CH3, C6H5, 250 oC, 73 %, C28H32N6O4, C:65.11(64.95), H:6.20(6.05), N:16.27(16.12), O:12.40(12.22).

VIe: Cl, CH3, C6H5, 230 oC, 75, C26H27N6O3Cl, C:61.59(61.42), H:5.33(5.23), N:16.58(16.46), O:9.47(9.33), Cl:7.00(6.86).

VIf: Br, CH3, C6H5, 255 oC, 78 %, C27H27N6O3Br, C:56.63(56.52), H:4.90(4.78), N:15.24(15.07), O:8.71(8.58), Br:14.50(14.37).

VIg: H, CH3, CH3C6H4, 260 oC, 72 %, C27H30N6O3, C:66.64(66.48), H:6.17(6.05), N:17.20(17.07), O:9.87(9.70).

VIh: H, CH3, ClC6H4, 265 oC, 76 %, C26H27N6O3Cl, VIh: C:61.59(61.45), H:5.33(5.19), N:16.58(16.45), O:9.47(9.32), Cl:7.00(6.85).

VIi: H, CH3, OCH3C6H4, 270 oC, 70 %, C27H30N6O4.,C:64.54(64.42), H:5.97(5.81), N:16.73(16.59), O:12.74(12.60).

VIj: H, CH3, NO2C6H4, 272 oC, 72 %, C26H27N7O5, C:60.34(60.20), H:5.22(5.07), N:18.95(18.82), O:15.47(15.30).

e. IR (KBr) Spectral data (νmax in cm-1)

VIa: 3185 (NH), 1665 (C=O), 1600 (C=N)

VIb: 3175 (NH), 1670 (C=O), 1602 (C=N)

VIc: 3200 (NH), 1665 (C=O), 1605 (C=N)

VId: 3190 (NH), 1670 (C=O), 1604 (C=N)

VIe: 3210 (NH), 1650 (C=O), 1605 (C=N)

VIf: 3215 (NH), 1660 (C=O), 1602 (C=N)

VIg: 3195 (NH), 1670 (C=O), 1605 (C=N)

VIh: 3190 (NH), 1675 (C=O), 1604 (C=N)

VIi: 3205 (NH), 1660 (C=O), 1605 (C=N)

VIj: 3180 (NH), 1660 (C=O), 1604 (C=N)

f. 1H NMR (DMSO-d6) Spectral data (δ in ppm)

VIa: 1.52 (s, 3H, CH3), 2.35 (s, 3H, N=C-CH3), 6.8 (s, H, Ar-NH), 7.0 (s, 2H, O-CH2-CO), 7.1 –7.3 (m, 10H, Ar–H), 7.4 (d, 2H, C6H4), 7.58 (d, 2H, Ar-H),  7.7 (d, 2H, C6H4), 10.92 (s, H, NH)

VIg: 1.50 (s,3H, CH3), 2.26 (s, 3H, CH3), 2.33 (s, 3H, N=C-CH3), 6.8 (s, H,  Ar-NH), 7.1-7.3(m, 9H, Ar-H), 7.23 (s, 2H, O-CH2-CO),  7.4 (d, 2H,C6H4), 7.7 (d, 2H, Ar,C6H4), 10.90 (s, H, NH),

VIh: 1.58 (s, 3H, CH3), 2.39 (s, 3H, N=C-CH3), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.27 (s, 2H, O-CH2-CO),  7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 10.94 (s, H, NH)

VIi: 1.53 (s, 3H, CH3), 2.37 (s, 3H, N=C-CH3), 6.8 (s, H, Ar-NH),  7.1-7.3 (m, 9H, Ar-H), 7.26 (s, 2H, O-CH2-CO),  7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 10.93 (s, H, NH)

VIj: 1.48 (s, 3H, CH3), 2.30 (s, 3H, N=C-CH3), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.20 (s, 2H, O-CH2-CO), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 10.89 (s, H, NH)

g. Elemental analysis data of compounds VII (-R, -R1=CH3, -R2, M.P., Yield, Molecular formula, Element: Found %, (Calc % ))

VIIa: H, 4|-C6H5, 240 oC, 75 %, C28H30N6O4, C:65.36(65.23), H:5.83(5.70), N:16.34(16.21), O:12.45(12.30).

VIIb: CH3, 4|-C6H5, 245 oC, 77 %, C29H32N6O4, C:65.90(65.72), H:6.06(5.92), N:15.90(15.75), O:12.12(12.00).

VIIc: OCH3, 4|-C6H5, 235 oC, 72 %, C29H32N6O4, C:63.91(63.76), H:5.88(5.74), N:15.44(15.30), O:14.70(14.56).\

VIId: OC2H5, 4|-C6H5, 250 oC, 73 %, C30H34N6O5, C:64.51(64.37), H:6.09(5.94), N:15.05(14.92), O:14.33(14.18).

VIIe: Cl, 4|-C6H5, 230 oC, 75 %, C28H29N6O4Cl, C:61.25(61.10), H:5.28(5.12), N:15.31(15.19), O:11.66(11.44), Cl:6.47(6.33).

VIIf: Br, 4|-C6H5, 255 oC, 78 %, C28H29N6O4Br, C:56.67(56.50), H:4.89(4.66), N:14.16(14.02), O:10.79(10.52), Br:13.47(13.30).

VIIg: H, 4|-CH3C6H4, 260 oC, 72 %, C29H32N6O4, C:65.90(65.72), H:6.06(5.87), N:15.90(15.78), O:12.12(11.95).

VIIh: H, 4|-ClC6H4, 265 oC, 76 %, C28H29N6O4Cl, C:61.36(61.21), H:5.11(5.00), N:15.34(15.20), O:11.68(11.47), Cl:6.17(6.02).

VIIi: H, 4|-OCH3C6H4, 270 oC, 70 %, C27H30N6O4, C:64.34(64.17), H:5.97(5.78), N:16.73(16.58), O:12.74(12.60).

VIIj: H, 4|-NOC6H4, 272 oC, 72 %, C26H27N6O5, C:60.34(60.15), H:5.22(5.08), N:18.95(18.80), O:15.74(15.62).

h. IR (KBr) Spectral data (νmax in cm-1)

VIIa: 3206 (NH), 1685 (C=O), 1620 (C=N)

VIIb: 3195 (NH), 1690 (C=O), 1622 (C=N)

VIIc: 3230 (NH), 1685 (C=O),1625 (C=N)

VIId: 3215 (NH), 1695 (C=O), 1624 (C=N)

VIIe: 3230 (NH), 1675 (C=O), 1630 (C=N)

VIIf: 3210 (NH), 1685 (C=O), 1627 (C=N)

VIIg: 3180 (NH), 1695 (C=O), 1627 (C=N)

VIIh: 3195 (NH), 1700 (C=O), 1629 (C=N)

VIIi: 3245 (NH), 1705 (C=O), 1630 (C=N)

VIIj: 3240 (NH), 1685 (C=O), 1630 (C=N)

i. 1H NMR (DMSO-d6) Spectral data (δ in ppm)

VIIa: 2.22 (s, 3H CH3), 2.40 (s, 3H, CH3), 2.46 (s, 3H, COCH3), 5.26 (s, 2H, OCH2), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4(d, 2H, C6H4), 7.7 (d, 2H,C6H4).

VIIg: 2.20 (s, 3H CH3), 2.26 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.44 (s, 3H, COCH3), 5.24 (s, 2H, OCH2), 6.8 (s, H, Ar - NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7. 7 (d, 2H, C6H4).

VIIh: 2.19 (s, 3H, CH3), 2.37 (s, 3H, COCH3), 2.24 (s, 2H, CH3), 4.92 (s, 2H, OCH2), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4).

VIIi: 2.18 (s, 3H CH3), 2.39 (s, 3 H, CH3), 2.43 (s, 3H, COCH3), 3.89 (s, 3H, OCH3), 5.24 (s, 2H, OCH2), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4).

VIIj: 2.16 (s, 3H CH3), 2.35 (s, 3H, CH3), 2.40 (s, 3H, COCH3), 5.22 (s, 2H, OCH2), 6.8 (s, H, Ar - NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4).

j. Elemental analysis data of compounds VIII (M.P., Yield, Molecular formula, Element: Found %, (Calc % )

VIII: 150 oC, 65 %, C19H20N6O3S, C:55.33(55.19), H:4.85(4.68), N:20.38(20.21), O:16.50(16.35), S:7.76(7.62).

k. IR (KBr) Spectral data (νmax in cm-1)

VIII: 3126 (oxadiazole NH), 3180 (NH), 1603 (C=N), 1670 (C=O), 1134 (C=S).

l. 1H NMR (DMSO-d6) Spectral data (δ in ppm)

VIII: 2.3 (s, 3H CH3), 5.45 (s, 2H, OCH2-), 6.8 (s, H, Ar - NH), 7.1–7.3 (m, 5H, Ar - H),7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 14.7 (s, H, thiol-thione tautomeric proton NH)

m. Elemental analysis data of compounds IX (R1=H, R2, M.W., M.P., Yield, Molecular formula, Element: Found %, (Calc % )

IXa: p-tolyl, 240oC, 75 %, C27H25N7O3S, C:61.48(61.32), H:4.47(4.30), N:18.59(18.45), O:9.10(8.93), S:6.07(5.90).

IXb: p-anisyl, 245oC, 77 %, C27H25N7O4S, C:59.66(59.48), H:4.60(4.56), N:18.04(17.85), O:11.78(11.62), S:5.89(5.69).

IXc: p-flurophenyl, 235oC, 78 %, C26H22N7O3SFl, C:58.76(58.65), H:4.14(3.98), N:18.45(18.33), O:9.04(8.86), S:6.02(5.80), Fl:3.55 (3.38).

IXd: p-chlorophenyl, 250oC, 73 %, C26H22N7O3SCl, C:56.98(56.75), H:4.01(3.83), N:17.88(17.69), O:8.76(8.61), S:5.84(5.69), Cl:6.48(6.31).

IXe: p-bromophenyl, 230oC, 80 %, C26H23N7O3Br, C:52.71(52.58), H:3.71(3.59), N:16.55(16.38), O:8.10(7.92), S:5.40(5.23), Br:13.49(13.30).

IXf: p-nitrophenyl, 255oC, 83 %, C26H22N8O5S, C:55.91(55.74), H:3.94(3.78), N:20.07(19.87), O:14.33(14.21), S:5.73(5.57).

IXg: diethyl, 260oC, 72 %, C28H37N8O3S, C:59.46(59.28), H:6.54(6.39), N:19.82(19.70), O:8.49(8.32), S:5.66(5.44).

IXh: diphenyl, 266 oC, 70 %, C32H27N7O3S, C:65.29(65.18), H:4.52(4.62), N:17.01(16.63), O:8.03(8.14), S:5.27(5.44).

IXi: morpholinyl, 270oC, 70 %, C20H18N7O4S, C:53.09(52.89), H:3.98(3.76), N:21.68(21.52), O:14.15(13.98), S:7.07(6.84).

IXj: piperazinyl, 272oC, 72 %, C22H20N7O3S, C:57.14(56.98), H:4.32(4.17), N:21.21(21.07), O:10.38(10.23), S:6.92(6.76).

IXk: N-methyl piperizinyl, 267oC, 68 %, C22H21N7O3S, C:57.01 (56.63), H:4.53 (4.38), N:21.16 (21.02), O:10.36 (10.12), S:6.91 (6.83).

n. IR (KBr) Spectral data (νmax in cm-1)

IXa: 1608 (C=N), 1665 (C=O), 1156 (C=S), 2939 (C-HStr), 3140 (NH), 3250 (Ar – NH).

IXb: 1620 (C=N), 1660 (C=O), 1150 (C=S), 2925 (C-HStr), 3130 (NH), 3240 (Ar – NH).

IXc: 1610 (C=N), 1670 (C=O), 1160 (C=S), 2945 (C-HStr), 3145 (NH), 3255 (Ar – NH).

IXd: 1608 (C=N), 1663 (C=O), 1158 (C=S), 2940 (C-HStr), 3172 (NH), 3253 (Ar – NH),

IXe: 1609 (C=N), 1665 (C=O), 1155 (C=S), 2943 (C-HStr), 3143 (NH), 3254 (Ar – NH).

IXf: 1605 (C=N), 1655 (C=O), 1145 (C=S), 2930 (C-HStr), 3135 (NH), 3245 (Ar – NH).

IXg: 1590 (C=N), 1645 (C=O), 1135 (C=S), 2925 (C-HStr), 3125 (NH), 3230 (Ar – NH).

IXh: 1593 (C=N), 1667 (C=O), 1107 (C=S), 2940 (C-HStr), 3150 (NH), 3250 (Ar – NH).

IXi: 1610(C=N), 1675 (C=O), 1165 (C=S), 2955 (C-HStr), 3150 (NH), 3265 (Ar – NH).

IXj: 1610 (C=N), 1670 (C=O), 1160 (C=S), 2945 (C-HStr), 3145 (NH), 3260 (Ar – NH).

IXk: 1605 (C=N), 1655 (C=O), 1145 (C=S), 2935 (C-HStr), 3140 (NH), 3255 (Ar – NH).

o. 1H NMR (DMSO-d6) Spectral data (δ in ppm)

IXa: 2.28 (s, 3H, CH3),  2.36 (s, 3H, CH3), 5.0 (s, 2H, NCH2),  5.64 (s, 2H, N-CH2-N),  6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 11.2 (s, H, Ar-NH).

IXb: 2.40 (s, 3H, CH3), 3.82 (s, 3H, CH3), 5.06 (s, 2H, N-CH2),  5.62 (s, 2H, N-CH2-N), 6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 11.1 (s, H, Ar-NH).

IXf: 2.50 (s, 3H, CH3), 4.96 (s, 2H, N-CH2),   5.50 (s, 2H, N-CH2-N),   6.8 (s, 1H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 7.4 (d, 2H, C6H4), 7.7 (d, 2H, C6H4), 10.23 (s, 1H, NH).

IXi: 2.60 (s, 3H, CH3), 2.62 (t, 4H CH2-N-CH2), 3.70 (t, 4H, CH2-O-CH2),  4.50 (s,  2H, N-CH2-N), 5.24 (s, 2H, N-CH2),   5.48 (s, 2H, N-CH2-N),  6.8 (s, H, Ar-NH), 7.1-7.3 (m, 9H, Ar-H), 6.8 (s, H, Ar-NH), 7.4 (d,2H,C6H4), 7.7(d, 2H, C6H4), 10.20 (s, 1H, NH).

IXj: 2.56 (t, 4H CH2-N-CH2), 2.58 (s, 3H, CH3), 4. 45 (s, 2H, N-CH2-N),    5.20 (s, 2H, N–CH2),  5.45 (s, 2H, N-CH2-N),  4.45 (s, 2H, N-CH2-N), 6.8 (s, H, Ar-NH), 7.1-7.3 (m,9H, Ar-H), 7.4(d,2H,C6H4), 7.7 (d, 2H,C6H4), 10.19 (s, H, NH).

3.2. Antimicrobial activity

All synthesized compounds were subjected to preliminary antibacterial screening by disc diffusion method against Staphylococus aureus NCCS 2079, Bacillus cereus NCCS 2106, Escherichia coli NCCS 2065 and Pseudomanas aeruginosa NCCS 2200. As shown in Table 1, VII series showed moderate activity against tested organism. In this series chloro, bromo and nitro substituted compounds showed more activity than other compounds. Whereas among the compounds of IX series, fluoro, chloro, bromo, nitro, morphonilyl, piperizynyl, N-methylpiperizine showed more activity than other compounds.

All synthesized compounds were subjected to preliminary antifungal screening by disc diffusion method against Aspergillus niger NCCS 1196 and Candida albicans NCCS 2106. Among the compounds of VII series, Chloro, bromo and nitro substituted compounds showed more activity against Aspergillus niger NCCS 1196 and Candida albicans NCCS 2106. The compounds of IX series have demonstrated good antifungal activity particularly fluoro, chloro, bromo, nitro, morphonilyl, piperizynyl and N-methyl piperizine showed good antifungal activity. The details are given in Table 1 and 2.

Table 1.-  Antifungal activity of novel compounds synthesized. 

Compound

-R

-R1

-R2

Zone of inhibition in mm

(MIC in μg/mL)

Aspergillus niger

NCCS 1196

Candida albicans

NCCS 2106

VIIa

H

CH3

C6H5

2.25 (55)

1.75(52.5)

VIIb

CH3

CH3

C6H5

1.75(45)

1.25(52.5)

VIIc

OCH3

CH3

C6H5

1.5(52.5)

1.5(52.5)

VIId

OC2H5

CH3

C6H5

1.5(47.5)

1.25(50)

VIIe

Cl

CH3

C6H5

1.75(40)

2(120)

VIIf

Br

CH3

C6H5

1.75(40)

1.75(42.5)

VIIg

H

CH3

CH6C6H5

1.25(52.5)

1.25(50)

VIIh

H

CH3

ClC6H4

2(37.5)

1.75(33.75)

VIIi

H

CH3

OCH3C6H4

1.5(195)

1.5(>50)

VIIj

H

CH3

NO2C6H4

1.75(41.25)

1.75(41.25)

IXa

H

H

p-tolyl

2.5(45)

2.25(45)

IXb

H

H

p-anisyl

2.25(47.5)

2(45)

IXc

H

H

p-fluoro phenyl

3.5(35)

3(37.5)

IXd

H

H

p-chloro phenyl

3.25(37.5)

3.5(36.25)

IXe

H

H

p-bromo phenyl

3.25(37.5)

3(37.5)

IXf

H

H

p-nitro phenyl

3.75(32.5)

3.25(36.25)

IXg

H

H

diethyl

2(47.5)

1.75(47.5)

IXh

H

H

di phenyl

2(47.5)

2(47.5)

IXi

H

H

morphonilyl

3.5(35)

3.25(35)

IXj

H

H

Piperizyinyl

3.25(37.5)

3(35)

IXk

H

H

N-methyl piperizine

3.25(35)

3.25(32.5)

Table 2.-  Antibacterial activity of novel compounds synthesized.

Compound

Zone of inhibition in mm

(MIC in μg/mL)

Staphylococus aureus

NCCS 2079

Bacillus Cereus

NCCS 2106

Escherichia coli

NCCS2065

Pseudomanas aeruginos

NCCS 2200

VIIa

1.5(50)

1.25(52.5)

1.25(52.5)

1.75(45)

VIIb

1.5(50)

1.25(52.5)

1(55)

1.5(50)

VIIc

1(50)

1.25(50)

1(55)

1.25(52.5)

VIId

2(42.5)

1.5(47.5)

1(50)

1.75(47.5)

VIIe

2(31.25)

2.25(35)

1.5(47.5)

2(32.5)

VIIf

1.75(35)

2(35)

1.5(45)

1.25(47.5)

VIIg

1(55)

1.25(55)

1(60)

1.25(60)

VIIh

2(31.25)

2(36.25)

1.5(40)

1.75(32.5)

VIIi

1(50)

1.5(47.5)

1.5(45)

1.25(50)

VIIj

1.75(33.75)

2(35)

1.5(38.75)

1.75(41.25)

IXa

1.5(52.5)

1.75(57.5)

1.25(57.5)

1.5(60)

IXb

1.75(50)

1.5(52.5)

1.5(55)

1.25(57.5)

IXc

2.5(36.25)

2.75(38.75)

2.25(38.75)

2.5(38.75)

IXd

3(35)

2.5(38.75)

2.25(38.75)

2.75(36.25)

IXe

2.5(37.5)

2.75(38.75)

2(40)

2.25(37.5)

IXf

2.75(36.25)

2.5(38.75)

2.5(35)

2.5(35)

IXg

1.75(50)

2(52.5)

1.5(57.5)

5(57.5)

IXh

1.75(50)

1.75(50)

5(60)

1.5(55)

IXi

2.5(37.5)

3(35)

2.5(37.5)

2.75(35)

Compound

Zone of inhibition in mm

(MIC in μg/mL)

Staphylococus aureus

NCCS 2079

Bacillus Cereus

NCCS 2106

Escherichia coli

NCCS2065

Pseudomanas aeruginos

NCCS 2200

IXj

2.75(32.5)

2.5(37.5)

2.25(40)

2.5(37.5)

IXk

2.5(35)

2.75(35)

2.25(37.5)

2.75(35)

4. CONCLUSSION

All the novel compounds have demonstrated moderate antimicrobial activity against selected of fungal and bacterial stains.

5. REFERENCeS

1.   Ericsson, J.M.; Sherris, J.C.; Antibiotic sensitivity testing. Report of an International Collaborative Study. Acta Pathol Microbiol Scand., 1971;217, 1-90

2.   Brown, B.A.; Wallace Jr., R.J.;  Onyi, G.O. Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. Antimicrob. Agents Chemother., 1992; 36, 1987-1990

3.   El-Kashef, H.S.;  Abd-Alla, M.A.;  Bayoumi, B E.;  El-Timawy, A. A. M.; Synthesis and antibacterial activity of some new pyrazolone dyes. J. Chem. Technol. Biot.,1983; 33, 294-298

4.   Fan, X.; Zhang, X.; Zhou, L.;  Keith, K.A.;  Kernb, E. R.;  Torrencea, P. F.;   A pyrimidine-pyrazolone nucleoside chimera with potent in vitro anti-orthopoxvirus activity. Bioorg. Med. Chem. Lett.,2006;16, 3224-3228

5.   Guckian, K.;  Carter, M.B.;  Lin, E.Y.;  Choi, M.;  Sun, L.;  Boriack-Sjodin, P. A.;  Chuaqui, C.;  Lane B.;  Cheung, K.;  Ling, L.;  Lee, W. C.   Pyrazolone based TGFbetaR1 kinase inhibitors. Bioorg. Med. Chem. Lett.,2010; 20, 326-329

6.   Jignesh, P.R.; Arpita, B.S.; Nilesh, H.P.;  Hemul, V.P.;  Pradip, S.P.; Kashyap, K. B.; Kishor, R.D. Synthesis and anti-tubercular activity of novel pyrazol-5(H)-one derivatives. Eur. J. Chem., 2011;2, 238-242

7.   Manojkumar, P.;  Ravi, T.K.; Gopalakrishnan, S. Antioxidant and antibacterial studies of arylazopyrazoles and arylhydrazonopyrazolones containing coumarin moiety. Eur. J. Med. Chem.,2009; 44, 4690-4694

8.   Chandrakantha, B.; Shetty, P.; Nambiyar, V.; Isloor, N.; Isloor, A.M. Synthesis, characterization and biological activity of some new 1,3,4-oxadiazole bearing 2-flouro-4-methoxy phenyl moiety. Eur. J. Med. Chem.,2010; 45, 1206-1210

9.   Farshori, N.N.;  Banday, M.R.;  Ahmad, A.;  Khan, A U.;  Rauf, A. Synthesis, characterization, and in vitro antimicrobial activities of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles. Bioorg. Med. Chem.Lett.,2010; 15, 1933-1938

10. Hussain, A.; Ajmal, M. Synthesis of novel 1,3,4-oxadiazole derivatives and their biological properties.  Acta Pharm., 2009; 59, 223-233

11. Rakesh, S.; Awani, K.R.; Kesari, A.N.; Yar, M.S.  Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole. Asian J. Res. Chem., 2009; 2, 34-42

12. Rakesh, C.; Anshu, A.; Manoj Kumar, P.; Chander Sharma, P.; Sukumar, M.;  Thengungal Ravi, K.Synthesis of novel 1,3,4-oxadiazole derivatives as potential antimicrobial agents. Acta Pol. Pharm., 2010; 67, 247-253

13. Ravitas, D.; Jha, A.K.; Alok Singh, T.; Dhansay, D. Synthesis and antibacterial activity of  some 1,3,4-oxadiazole derivatives and their thione analogues.Intl. J. Biomed. Pharma.Sci., 2011; 2, 215-219.