SYNTHESIS, ANTIBACTERIAL, AND ANTIFUNGAL EVALUATION OF NOVEL MANNICH BASES CONTAINING OXADIAZOLE AND PYRAZOLE MOIETIES
Maliki Reddy Dastagiri Reddy, Aluru Raghavendra Guru Prasad, ...
1. Sri Krishnadevaraya University, Anatapur, A.P., India. 2. ICFAI Foundation for higher Education, Hyderabad, A.P., India.
Keywords: 1,3,4-oxadiazole moiety; Pyrazole-3-one moiety; Antibacterial activity; Antifungal activity; Minimum inhibitory concentration
Abstract

Purpose: To synthesize novel series of compounds containing 1,3,4-oxadiazole and pyrazole-3-one moieties in good yields and to evaluate the antimicrobial activity of title compounds. Methods: Title compounds were characterized by IR, 1H NMR and mass spectra and evaluated for their antimicrobial activities against certain selected gram-positive bacteria, gram-negative bacteria and fungi by disk diffusion method and broth dilution method. Results: Fluoro, chloro, bromo, nitro, morphonilyl, piperizynyl, N-methylpiperizine substituted compounds (VII c, d, e, f, i, j and k) showed more antimicrobial activity against than other compounds of the series. Conclusion: Antimicrobial activity was expressed as the corresponding minimum inhibitory concentration (MIC). The compounds have demonstrated considerable antibacterial and antifungal activities.

Article Information

Identifiers and Pagination:
Year:2013
Volume:5
First Page:76
Last Page:88
Publisher Id:JAppPharm (2013 ). 5. 76-88
Article History:
Received:May 11, 2013
Accepted:June 17, 2013
Collection year:2013
First Published:July 1, 2013

INTRODUCTION

Among the wide variety of heterocycles that have been explored for developing new therapeutic molecules, pyrazole-3-ones and 1,3,4-oxadiazoles attracted substantial attention of the medicinal chemists. The widespread use of 1,3,4-oxadiazoles and pyrazole-3-ones as scaffolds in medicinal chemistry establishes these moieties as an important bio-active class of heterocycles. 1,3,4-oxadiazoles have demonstrated versatile range of medicinal activities like antibacterial [1, 2], antifungal [3, 4], antitumor [5], antitubercular [6], anticonvulsant [7] and HIV-I inhibitory activities [8]. Same scale of medicinal importance is associated with compounds containing pyrazole-3-one moiety [9-15]. The present article is an effort to incorporate both these crucial functionalities in a single entity to exploit their collective medicinal potentials.

 

MATERIALS AND METHODS

Materials and instrumentations

Melting points were determined by using melting point apparatus procured from Bio Technics India Ltd. It consists of aluminum cylindrical block for heating, radial holes for a thermometer and glass capillary. All chemicals used were analytical grade obtained from Merck India Limited, India. The bacterial and fungal strains were procured from National Centre for Cell Science, Pune, India. UV/Visible spectrophotometer model 106 manufactured by Synstronics Instruments Ltd., Ahmadabad, India was used in the studies. The infrared spectra were recorded on Perkin–Elmer KBr spectrometer. ? values were expressed in cm-1. 1HNMR spectra were recorded on JEOL MODEL GSX 270 FT NMR Spectrometer and NMR 200 MHz Supercon machine, using CDCl3 and DMSO–d6 as solvents and TMS as an internal standard. Chemical shifts were expressed as d values (ppm). Mass spectra of the compounds were recorded on a Jeol JMS-D300 mass spectrometer operating at 70 eV. . The amount of halogens present in the compound was determined by the procedure reported in the literature [16].

Synthesis of compounds of interest Synthesis of 2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1 yl]acetohydrazide (V) [17].

a. Synthesis of phenyl diazonium chloride (I)

The required primary amine was dissolved in a suitable volume of water containing 2.5 equivalents of hydrochloric acid (or sulphuric acid). The solution thus obtained was cooled to 0oC to crystallize amine hydrochloride (or sulphate). To this, aqueous solution of sodium nitrite was added portion wise until there was excess of free nitrous acid. 

b. Synthesis of ethyl 3-oxo-2-(phenylhydrazono) butanoate (II)

To an ice cold solution of a mixture of sodium acetate (1.0 g, 0.08 mol), 100 mL of aqueous alcohol (50%) and ethylacetoacetate (0.65 mL, 0.1 mol) in 50 mL of ethanol, the diazonium chloride I (0.72 g, 0.1 mol) was added till yellow crystals were separated out. These crystals were filtered, washed with water and dried under vacuum.

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

A mixture of eqimolar quantities of 2-arylhydrazono acetoacetic ester (II), hydrazine and dimethyl formamide (0.5 mL) was subjected to microwave irradiation at 150 W intermittently at 30 s intervals for 2 min. After completion of the reaction, as indicated by TLC, the reaction mixture was cooled to room temperature and washed with cold water. The precipitated 3-methyl-4-(phenyl hydrazono)-pyrozoline-5-one was filtered and recrystallized from hot ethanol. To improve the recovery for reusing, Al2O3 (0.1 mmol) have been used in the reaction.

d. Synthesis of ethyl [(4E)-3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]acetate (IV)

A mixture of III (0.2 mol), ethylchloloroacetate (0.2 mol), anhydrous K2CO3 (0.03 mol) and dimethylformamide was stirred at room temperature for 8 h. After the completion of reaction, reaction mixture was diluted with cold water. The precipitated solid was filtered and recrystallized from hot ethanol.

 

e. Synthesis of 2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]-acetohydrazide (V)

Equimolar solutions of IV (0.1 mol) and 99% hydrazine hydrate (0.1 mol) in ethanol (20 mL) was refluxed for five h. The reaction mixture was cooled and poured into cold water with continuous stirring. The precipitate so obtained was filtered, washed with water and recrystallized from ethanol to give V. The reaction sequence is shown in the Scheme 1.

Synthesis of certain novel Mannich bases bearing pyrazoline-3-one moiety.

a. Synthesis of 3-methyl-1-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]-1H-pyrazole-4,5-dione 4-(phenylhydrazones)(VI)

A mixture of V (19.9 g, 0.1 mol), KOH (5.5 g, 0.1 mol) ethanol (100 mL) and carbon disulphide (6 mL, 0.1 mol) were refluxed in a round bottom flask for 2-3 h. After the completion of the reaction, excess of alcohol was removed by distillation. The reaction mixture was cooled, poured into cold water and neutralized with dilute hydrochloric acid. The solid precipitated (VI) was filtered, washed thoroughly with water and recrystallized from ethanol-dioxane mixture.

Scheme 1. General proedure for the synthesis of 2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]acetohydrazide (V).

 

b. Synthesis of 3-methyl-1-({4-[(N-substituted and N,N-disubstituted amino)methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}methyl)-1H-pyrazole-4,5-dione-4-(phenylhydrazones) (VII)

A solution of VI (0.01 mol) in ethanol and dioxan mixture (1:1, 20 mL) mixed with formaldehyde (40%, 1.5 mL) was stirred over night with the solution of appropriate amine (0.01 mol) in ethanol (10 mL). The precipitated Mannich base (like VIIa) was collected by filtration, dried and recrystallized from ethanol-DMF mixture. The reaction sequence is depicted in the Scheme 2.

The treatment of VI with p-anisylamine/ p-flurophenylamine/ p-chlorophenylamine/ p-bromophenylamine/ p-nitrophenylamine/ diethyl amine/ diphenyl amine/ piperazine/ morpholine/ N-methyl piperizine in the presence of formaldehyde in ethanol-dioxane mixture leads to the formation of respective Mannich base VIIa–k. The purity of all the compounds was confirmed by TLC.  

 

Scheme 2. Synthesis of Mannich bases containing 1,3,4-oxadiazole and pyrazol-3-one moieties.

R1=-H;  R2=p-Tolyl, p-anisyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-nitrophenyl, diethyl, diphenyl, morpholinyl, piperazinyl, N-methylpiprrazinyl.

Antibacterial and antifungal activities

Antibacterial and antifungal activities of the synthesized compounds were studied by disc diffusion method [18]. The activity of the synthesized compounds was expressed as minimum inhibitory concentration (MIC). Broth dilution method [19] was used to determine the minimum inhibitory concentration of an antimicrobial agent. The general procedures are given below.

Disc diffusion method

A suspension of the microorganism was added to sterile nutrient agar at 45oC. The mixture was transferred to sterile petri dishes to give a depth of 3 to 4 mm and allowed to solidify. Precautions were taken to get uniform layer of medium on the plate. Sterile discs 5 mm in diameter (made from Whatmann Filter paper) were immersed in the solutions of synthesized compounds (250 µg/ml). An untreated control sample was always kept for comparison.

After the plates were allowed to stand for 1 h at room temperature to minimize the effects of variations in different time, they incubated at 37oC for 24 h and observed for antimicrobial activity. The diameter of the zone of inhibition was measured in each plate. The average zone of inhibition was calculated.

Similar procedure was adopted for studying the antimicrobial activity against the other organisms.

Broth dilution method

Standardized inoculum (matched to McFarland BaSO4 standard) of suspension of organisms were 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 mixed gently by shaking the rack. Two growth control tubes were also prepared without the addition of test compound and its optical density was determined.

Tubes were incubated for 24 h at 37oC in air. The turbidity produced 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 was noted as the concentration of the test substance, which completely inhibits the growth of the microorganism i.e. 100% transparency. 

 

RESULTS AND DISCUSSION

Compounds of each series (V, VI and VII) were characterized by elemental analysis and respective IR, 1H and NMR spectra. The compounds V and VII were also characterized by mass spectra. The details are given below.

Characterization of compounds V, VI and VII

2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]-acetohydrazide (V)

Yeild 65%; m.p.152-153oC; molecular formula C12H14N6O2; elemental analysis: C 52.63 (52.55), H 5.22 (5.14), N 30.71 (30.64), O 11.74 (11.67); IR (KBr) (?max in cm-1): 3445, 3425 (NH2), 3305 (NH), 1665 (C=O), 1620 (C=N); 1H NMR (DMSO-d6): d 1.20 (s, 3H, CH3), 2.10 (s, 2H, NH2), 3.85 (s, 2H, N-CH2-CO), 7.00 (s, 1H, Ar-NH), 7.30-7.40 (m, 5H, C6H5), 8.40 (s, 1H, NH).

Mass spectral data of V

The fragmentation process revealed by the mass spectrum of 2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]acetohydrazide V is depicted in Scheme 3. The spectrum showed the molecular ion (M+) peak at m/z 274 with an intensity of 14.7%. Decomposition of molecular ion A along path I resulted in the formation of the fragment B at m/z 244 (8.2%), B loses a H radical atom to form cation C at m/z 243 (46.2%). Elimination of CHO molecule from C yielded cation D at m/z 216 (18.5%). Loss of methylene radical from D yielded cation E at m/z 202 (25.2%). Elimination of C2H2N2 molecule from E resulted in the formation of radical cation F at m/z 148 (100%). Loss of C6H5 radical from molecular ion resulted in the formation of cation G at m/z 197 (65.7%).

 

Scheme 3. Mass spectral fragmentation of 2-[3-methyl-5-oxo-4-(phenylhydrazono)-4,5-dihydro-1H-pyrazol-1-yl]acetohydrazide (V).

 

3-methyl-1-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]-1H-pyrazole-4,5-dione-4-(phenylhydrazone) VI

Yield 65%; m.p.150-151oC; molecular formula C13H12N6O2S; elemental analysis: C 49.50 (49.36), H 3.98 (3.82), N 26.66 (26.52), O 10.70 (10.12), S 10.32 (10.14); IR (KBr) (?max in cm-1): 3126 (oxadiazole NH), 3180 (NH), 1670 (C=O), 1603 (C=N), 1134 (C=S); 1H NMR (DMSO-d6): d 2.30 (s, 3H CH3), 5.45 (s, 2H, N-CH2-), 6.60–7.20 (m, 5H, Ar-H), 7.90 (s, H, Ar-NH), 14.70 (s, H, thiol–thione tautomeric proton NH)

Mannich bases (VII)

5-Methyl-2-{4-[(methyl-p-tolyl-amino)-methyl]-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIa):

Yeild 75%; m.p. 240-241oC; molecular formula C21H21N7O2S; elemental analysis found % (Calc. %): C 57.82 (57.92), H 4.82 (4.86), N 22.45 (22.51), O 7.30 (7.35), S 7.25 (7.36); IR (KBr) (?max in cm-1): 3250 (Ar-NH), 3140 (NH), 2939  (C-H), 1665 (C=O), 1608 (C=N), 1156 (C=S); 1H NMR (DMSO-d6): d2.28 (s, 3H, CH3), 2.36 (s, 3H CH3), 5.00 (s, 2H, NCH2), 5.64 (s, 2H, N-CH2-N), 6.95 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.80 (s, H, Ar-NH), 11.20 (s, H, Ar-NH).

2-(4-{[(4-Methoxy-phenyl)-methyl-amino]-methyl}-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIb):

Yeild 77%; m.p. 245-246oC; molecular formula C21H21N7O3S; elemental analysis found % (Calc. %): C 55.75 (55.86), H 4.60 (4.69), N 21.65 (21.72), O 10.60 (10.63), S 7.00 (7.10); IR (KBr) (?max in cm-1): 3240 (Ar-NH), 3130 (NH), 2925  (C-H), 1660 (C=O), 1620 (C=N), 1150 (C=S); 1H NMR (DMSO-d6): d2.40 (s, 3H CH3), 3.82 (s, 3H, CH3), 5.06 (s, 2H, N-CH2), 5.62 (s, 2H, N-CH2-N), 6.97 (d, 2H, Ar-H), 7.47 (d, 2H, Ar-H), 7.90 (s, H, Ar - NH), 11.10 (s, H, Ar-NH).

 

2-(4-{[(4-Fluoro-phenyl)-methyl-amino]-methyl}-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIc):

Yeild 72%; m.p. 235-236oC; molecular formula C20H18FN7O2S; elemental analysis found % (Calc. %): C 54.45 (54.66), H 4.10 (4.13), N 22.28 (22.31), O 7.25 (7.28), S 7.07 (7.30), Fl 4.26 (4.32); IR (KBr) (?max in cm-1): 3255 (Ar-NH), 3145 (NH), 2945 (C-H), 1670 (C=O), 1610 (C=N), 1160 (C=S).

2-(4-{[(4-Chloro-phenyl)-methyl-amino]-methyl}-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIId):

Yield 73%; m.p. 250-251oC; molecular formula C20H18ClN7O2S; elemental analysis found % (Calc. %): C 52.33 (52.69), H 4.01 (3.98), N 21.13 (21.51), O 7.11 (7.02), S 6.88 (7.03), Cl 7.98 (7.78); IR (KBr) (?max in cm-1): 3253 (Ar-NH), 3172 (NH), 2940 (C-H), 1663 (C=O), 1608 (C=N), 1158 (C=S).

2-(4-{[(4-Bromo-phenyl)-methyl-amino]-methyl}-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIe):

Yield 75%; m.p. 230-231oC; molecular formula C20H18BrN7O2S; elemental analysis found % (Calc. %): C 47.67 (48.01), H 3.54 (3.63), N 20.01 (19.59), O 6.50 (6.40), S 6.55 (6.41), Br 16.11 (15.97); IR (KBr) (?max in cm-1): 3254 (Ar-NH), 3143 (NH), 2943 (C-H), 1665 (C=O), 1609 (C=N), 1155 (C=S).

5-Methyl-2-(4-{[methyl-(4-nitro-phenyl)-amino]-methyl}-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIf):

Yield 78%; m.p. 255-256oC; molecular formula C20H18N8O4S; elemental analysis found % (Calc. %): C 52.00 (51.50), H 3.78 (3.98), N 23.88 (24.02), O 13.56 (13.72), S 7.01 (6.87); IR (KBr) (?max in cm-1): 3245 (Ar-NH), 3135 (NH), 2930 (C-H), 1655 (C=O), 1605 (C=N), 1145 (C=S); 1H NMR (DMSO-d6): d2.50 (s, 3H, CH3), 4.96 (s, 2H, N-CH2), 5.50 (s, 2H, N–CH2-N), 6.80-7.20 (m, 5H, Ar-H), 7.48 (d, 2H, o-protons of p-nitrophenyl), 7.86 (d, 2H, protons of p-nitrophenyl), 7.89 (s, 1H, Ar-NH), 10.23 (s, 1H, NH).

2-(4-Diethylaminomethyl-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl)-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIg):

Yield 72%; M.p. 260-261oC; molecular formula C18H23N7O2S; elemental analysis found % (Calc. %): C 53.65 (53.85),H 5.67 (5.77), N 24.22 (24.42), O 7.65 (7.97) S 7.55 (7.99); IR (KBr) (?max in cm-1): 3230 (Ar-NH), 3125 (NH), 2925 (C-H), 1645 (C=O), 1590 (C=N), 1135 (C=S).

2-{4-[Diphenylamino)-methyl]-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-5-methyl-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIh):

Yield 76%; m.p. 265-266oC; molecular formula C26H23N7O2S; elemental analysis found % (Calc. %): C 62.70 (62.76), H 4.60 (4.66), N 19.65 (19.71), O 6.35 (6.43), S 6.38 (6.44); IR (KBr) (?max in cm-1): 3250 (Ar-NH), 3150 (NH), 2940 (C-H), 1667 (C=O), 1593 (C=N), 1107 (C=S); 1H NMR (DMSO-d6): d2.67 (s, 3H CH3), 5.26 (s, 2H, N-CH2), 5.50 (s, 2H, N-CH2-N), 6.60-7.20 (m, 5H, Ar-H), 7.26-7.56 (m, 10H, aromatic protons of both phenyl rings), 7.60 (s, H, Ar-NH), 7.80 (s, 1H, Ar-NH).

5-Methyl-2-(4-morpholin-4-ylmethyl-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl)-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIi):

Yield 70%; m.p. 270-271oC; molecular formula C18H21N7O3S; elemental analysis found % (Calc. %): C 52.08 (52.04), H 5.33 (5.09), N 23.75 (23.60), O 11.10 (11.55), S 7.50 (7.72); IR (KBr) (?max in cm-1): 3265 (Ar-NH), 3150 (NH), 2955 (C-H),  1675 (C=O), 1610 (C=N), 1165 (C=S); 1H NMR (DMSO-d6): d2.60 (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, NCH2-N), 6.80-7.20 (m, 5H, Ar-H), 7.60 (s, H, Ar-NH),10.20 (s, 1H, NH).

5-Methyl-4-(phenyl-hydrazono)-2-(4-piperazin-1-ylmethyl-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl)-2,4-dihydro-pyrazol-3-one (VIIj):

Yield 72% m.p. 272-273oC; molecular formula C18H22N8O2S; elemental analysis found % (Calc. %): C 52.20 (52.16), H 5.50 (5.35),n N 27.25 (27.03), O 7.68 (7.72), S 7.58 (7.74); IR (KBr) (?max in cm-1): 3260 (Ar-NH), 3145 (NH), 2945 (C-H), 1670 (C=O), 1610 (C=N), 1160 (C=S); 1H NMR (DMSO-d6): d2.58 (s, 3H, CH3), 2.56 (t, 4H CH2-N-CH2), 4.45 (s, 2H, N-CH2-N), 5.20 (s, 2H, N-CH2), 5.45 (s, 2H, NCH2N), 6.70-7.10 (m, 5H, Ar-H), 7.50 (s, H, Ar-NH), 10.19 (s, H, NH).

5-Methyl-2-[4-(4-methyl-piperazin-1-ylmethyl)-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl]-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIk):

Yield 68%; m.p. 267-268oC; molecular formula C19H24N8O2S; elemental analysis found % (Calc. %): C 53.65 (53.25), H 5.98 (5.65), N 25.81 (26.15), O 7.28 (7.47), S 7.36 (7.48); IR (KBr) (?max in cm-1): 3255 (Ar-NH), 3140(NH), 2935 (C-H), 1655 (C=O), 1605 (C=N), 1145 (C=S); 1H NMR (DMSO-d6): d2.55 (s, 3H, CH3), 2.42 (t, 4H, CH2-N-CH2), 4.20 (s, 3H, N-CH3), 4.52 (s, N-CH2-N), 5.18 (s, 2H, N-CH2), 5.40 (s, 2H, NCH2N), 6.60-7.20 (m, 5H, Ar-NH), 7.40 (s, H, Ar – NH), 10.18 (s, H, NH).

Mass spectral data of VII

The mass spectrum of 5-Methyl-2-{4-[(methyl-p-tolyl-amino)-methyl]-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one VIIa (R1=H, R2=p-CH3C6H4) exploited the molecular ion (M+) peaks at m/z 435.

The fragmentation pattern for VIIa is presented in Scheme 4. The spectrum showed the molecular ion (M+) peak at m/z 435 with intensity 12.7%. The loss of a CH3 radical resulted in the formation of the fragment B at m/z 420 (3.2%). Loss of OH radical from molecular ion A led to the formation of cation C at m/z 418 (49.2%). Loss of C6H5 radical from A yielded the cation D at m/z 358 (17.5%). Elimination of C8H10N molecule from A has resulted in the formation of cation F at m/z 315 (22.2%). Elimination of C7H8N from molecular (M+) ion resulted in the formation of cation E at m/z 329 (100%). Cleavage of molecular ion A led to the formation of cations G and H at m/z 291 (9.6%) and 234 (66.7%) respectively.

Scheme 4. Mass spectral fragmentation of 5-Methyl-2-{4-[(methyl-p-tolyl-amino)-methyl]-5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-ylmethyl}-4-(phenyl-hydrazono)-2,4-dihydro-pyrazol-3-one (VIIa)

 

Antibacterial and antifungal activity

The Mannich bases were evaluated for their antimicrobial activities against certain selected gram-positive bacteria, gram-negative bacteria and fungi. Antimicrobial activity was expressed as the minimum inhibitory concentration. The details are given in Table 1.

Compounds of each series (V, VI and VII) were characterized by elemental analysis, IR, 1H NMR spectral data. The elemental analysis data of each compound synthesized indicated that the reported data almost coincides with the theoretical data. In each case the characteristic signals in the spectra indicative of the compound were identified and reported. The compounds V and VII were also characterized by mass spectra. The details of mass spectral fragmentation pattern presented in the Scheme 3 and Scheme 4 were confirmative of corresponding structures.

 

Antibacterial and antifungal activity

The details of antimicrobial studies are shown in Table 1. The gram-positive bacteria screened were Staphylococcus aureus NCCS 2079 and Bacillus cereus NCCS 2106 and the gram negative bacteria screened were Escherichia coli NCCS 2065 and Pseudomonas aeruginosa NCCS 2200. Antifungal activity tests were also performed against Aspergillus niger NCCS 1196 and Candida albicans NCCS 2106. The preliminary antimicrobial activity of synthesized compounds was studied by disc diffusion method. As shown in Table 1, fluoro, chloro, bromo, nitro, morphonilyl, piperizynyl, N-methylpiperizine substituted compounds (VII c, d, e, f, i, j and k) showed more antibacterial activity against Staphylococus aureu , Bacillus cereus, Escherichia coli and Pseudomonos aeruginosa than other compounds of the series. Similar observations were made against Aspergillus niger and Candida albicans in antifungal studies.

 

Table 1. Antibacterial and antifungal activities of Mannich bases

Compound

Zone of inhibition in mm and MIC in µg/mL*

Antibacterial activity

Antifungal activity

Staphylococus aureus

NCCS 2079

Bacillus cereus

NCCS 2106

Escherichia coli

NCCS2065

Pseudomonos aeruginosa

 NCCS 2200

Aspergillus niger

NCCS 1196

Candida albicans

NCCS 2106

VIIa

1.50 (39.0)

1.75 (35.0)

1.25 (40.0)

1.50 (40.0)

2.50 (38.0)

2.25 (28.0)

VIIb

1.75 (40.0)

1.50 (38.0)

1.50 (40.0)

1.25 (38.0)

2.25 (35.0)

2.00 (28.0)

VIIc

2.50 (33.0)

2.75 (30.7)

2.25 (28.0)

2.50 (30.7)

3.75 (25.0)

3.00 (20.0)

VIId

3.00 (33.0)

2.50 (30.8)

2.25 (25.0)

2.75 (30.8)

3.50 (25.0)

3.75 (25.0)

VIIe

2.50 (30.8)

2.75 (30.7)

2.00 (28.0)

2.25 (33.0)

3.50 (22.3)

3.00 (22.3)

VIIf

2.75 (33.0)

2.50 (33.0)

2.50 (35.0)

2.50 (35.0)

15.00 (20.0)

3.50 (20.0)

VIIg

1.75 (50.0)

2.00 (55.7)

1.50 (60.0)

1.25 (60.0)

8.00 (45.5)

1.75 (40.0)

VIIh

1.75 (45.5)

1.75 (40.0)

1.25 (40.0)

1.50 (47.5)

2.00 (40.0)

2.00 (35.3)

VIIi

2.50 (30.7)

3.00 (30.8)

2.50 (35.3)

2.75 (30.8)

3.75 (22.2)

3.50 (20.0)

VIIj

2.75(20.0)

2.50 (25.0)

2.25 (35.23)

2.50 (20.0)

3.50 (18.5)

3.00 (18.5)

VIIk

2.50 (22.2)

2.75 (27.2)

2.25 (35.3)

2.75 (27.3)

3.50 (20.0)

3.50 (20.0)

*Average of three determinations. Zone of inhibition in mm and MIC (in side the brackets) in µg/mL

 

As is evident from the literature, extensive reports pertaining to the antimicrobial screening of compounds containing 1,3,4-oxadiazole moiety [1-8] or pyrazole-3-one moiety are available [9-15]. The pharmacological applications of Mannich bases containing different moieties are also reported in the literature [20-29], but pharmacological applications of Mannich bases incorporating 1,3,4-oxadiazole or pyrazole-3-one moieties have not beenexplored. Moreover the reported information witnesses disadvantages such as requirement of high concentration of the drug [20, 24, 26, 27, 29] or low zone of inhibition [25] or the compounds are active against bacteria only [20, 27, 29] or active against gram positive bacteria only [21] or active against fungi only [22, 23, 28]. The present article reports the antibacterial and antifungal activities of Mannich bases containing pharmacologically potential 1,3,4-oxadiazole and pyrazole-3-one moieties. The discern is clearly evident from the fact that the Mannich bases (VIIa-VIIk) demonstrated low magnitudes of MIC, higher magnitudes of zone inhibition and wide spectrum of activity against bacteria and fungi.

 

CONCLUSION

Series of new compounds containing 1,3,4 oxadiazole and pyrazole-3-one moiety were synthesized. The compounds were characterized by elemental analysis, IR, 1H NMR and mass spectral data. All compounds were screened for antibacterial and antifungal activities. The activities of the compounds were assessed by the disc diffusion method and the minimum inhibitory concentration was established by broth dilution method. The results reveal that all the compounds exhibited promising antimicrobial activities.

 

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Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

Bibliography

Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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Abbreviation: J App Pharm
doi: http://dx.doi.org/10.21065/19204159
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