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ORIGINAL ARTICLE
Year : 2013  |  Volume : 5  |  Issue : 2  |  Page : 126-135  

Synthesis, in-vitro antimicrobial and antitubercular screening of Schiff bases of 3-amino-1-phenyl-4- [2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one


1 Department of Pharmaceutical Chemistry, Karpagam University, Coimbatore, Tamil Nadu, India
2 KMCH College of Pharmacy, Coimbatore, Tamil Nadu, India

Date of Submission13-Nov-2012
Date of Decision01-Feb-2013
Date of Acceptance02-Mar-2013
Date of Web Publication14-May-2013

Correspondence Address:
K K Sivakumar
Department of Pharmaceutical Chemistry, Karpagam University, Coimbatore, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.111828

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   Abstract 

Purpose: Synthesis and antimicrobial activity of some Schiff bases of 3-amino-1-phenyl-4- [2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-ones (TZP4a-l) are described. Materials and Methods: Structures of the synthesized compounds were confirmed using infrared, 1 H nuclear magnetic resonance, and mass spectral data. Synthesized compounds were tested in-vitro against four Gram-positive and four Gram-negative bacterial strains, three fungal strains and two mycobacterial strains. Title compounds were screened its in-vitro cytotoxicity (IC 50 ) by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using mouse embryonic fibroblasts cell line (NIH 3T3). Results and Discussion: Compounds TZP4 g and TZP4 h were found to be significant activity against Bacillus substilis (bacteria) and Aspergillus niger (fungi). In-vitro anti-tuberculosis (TB) activity of compound TZP4g showed appreciable antitubercular activity against Mycobacterium tuberculosis H37Rv strain (minimum inhibitory concentration [MIC] =0.6.48 × 10−3 μM/mL) which was 1.69 and 3.91 times more active than the standard drug, pyrazinamide (25.38 × 10−3 μM/mL) and streptomycin (MIC = 11.01 × 10−3 μM/mL), respectively. Their in-vitro cytotoxicity (IC 50 ) was determined to establish a selectivity index (SI) (SI = IC 50 /MIC). Compounds TZP4 c, TZP4 g, and TZP4 h have SI 82.85, 168.88, and 199.07, respectively. Conclusion: All the title compounds had mild toxicity on the mouse embryonic fibroblasts NIH 3T3 cells (IC 50 ≥ 100 μM). In comparison to the results of toxicity and antimycobacterial activity tests, it was observed that the activity of the compounds is not due to general toxicity effect; however, their antimycobacterial activity can be possibly because of their selective antimycobacterial effect. We concluded from our investigations that TZP4 c, TZP4 g, and TZP4 h may be considered promising for the development of new anti-TB agents.

Keywords: Antimicrobial, antimycobacterial, cytotoxicity, pyrazolone, thiazole


How to cite this article:
Sivakumar K K, Rajasekaran A. Synthesis, in-vitro antimicrobial and antitubercular screening of Schiff bases of 3-amino-1-phenyl-4- [2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one. J Pharm Bioall Sci 2013;5:126-35

How to cite this URL:
Sivakumar K K, Rajasekaran A. Synthesis, in-vitro antimicrobial and antitubercular screening of Schiff bases of 3-amino-1-phenyl-4- [2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one. J Pharm Bioall Sci [serial online] 2013 [cited 2019 May 25];5:126-35. Available from: http://www.jpbsonline.org/text.asp?2013/5/2/126/111828

Pyrazolone derivatives have a broad spectrum of biological activities being used as analgesic, [1] antipyretic, [2] anti-inflammatory, [3] antimicrobial, [4] antimycobacterium, [5] anticancer, [6] anti-human immunodeficiency virus (HIV), [7] and antivirus. [8] The antibacterial activity caught our attention because antimicrobial resistance developed by important pathogens with available chemotherapeutic agents has increased to an alarming level. [9],[10] In view of the above mentioned factor and as a continuation of our investigations on pyrazolone derivatives we recently described [11] a series of Schiff bases of 5-amino-4-[2-(4-nitro-1,3-benzothiazol-2-yl) hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-one derivatives to determine the effect of various substituents at the phenyl nucleus on antimicrobial and antimycobacterium activities with considerable selective index. Common structure feature for our previous reported pyrazolone compound derivatives are given in [Figure 1]. Therefore, it seems that various substitutions in the pyrazolone ring can be an effective pharmacophore in several classes of antimicrobial agents. To identify new candidates that may be of value in designing new, potent, selective and less toxic antimicrobial agents particularly as anti-tuberculosis (TB) agents, we report herein the synthesis, in-vitro antimicrobial, antimycobacterial screening of Schiff bases of 3-amino-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one.
Figure 1: Common structure feature for our previous reported pyrazolone compound derivatives

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   Materials and Methods Top


Starting materials were obtained from Sigma, Aldrich, and Merck Chemical supplied by purani hospital supplies Ltd, Poonmani and co, Coimbatore and was used without further purification. Reaction progress was observed by thin layer chromatography (TLC) making use of commercial silica gel plates (Merck). Melting points (MPs) were determined in open capillary tubes on a Sonar melting point apparatus manufactured by sigma industries Chennai and are uncorrected. 1 H nuclear magnetic resonance ( 1 H NMR) spectra were determined by Bruker 300 MHz Fourier transform (FT)-NMR spectrometer in appropriate deuterated solvents and are expressed in parts per million (δ, ppm) downfield from tetramethylsilane (internal standard). NMR data are given as multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) and number of protons. Elemental analysis (C, H, and N) was undertaken with on Bruker Elemental vario EL III Carlo Erba 1108 analyzer. The infrared (IR) spectra were run as KBr disc on Jasco FTIR (4100) spectrophotometer. Mass spectra of the synthesized compounds were recorded in MS (EI) JEOL GC mate spectrometer. Characterizations of molecules were carried out at Indian Institute of Science, Bangalore and Indian Institute of Technology, Madras.

Chemistry

Synthesis of 4-phenylthiazol-2-amine (TZ) and ethyl cyano [2-(4-phenyl-1,3-thiazol-2-yl) hydrazinylidene] acetate (TZE)

The compounds 4-phenylthiazol-2-amine ( TZ ) and ethyl cyano [2-(4-phenyl-1,3-thiazol-2-yl) hydrazinylidene] acetate (TZE) were synthesized using the reported methods. [12],[13]

Synthesis of 3-amino-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4)

The phenyl hydrazine hydrate (0.1 mol, 12.60 mL) was added to the suspension of the synthesized compound (TZE, 0.01 mol, 3.62 g) and anhydrous sodium acetate (0.01 mol, 0.82 g) in 30 mL of glacial acetic acid and the reaction mixture was heated under reflux for 6 h. Then the reaction mixture was allowed to cool in ice, and the precipitated pyrazolone [14] (TZP4) was filtered, washed with water, dried, and recrystallized from ethanol. The solvent system used for TLC was chloroform: ethanol: water (3:5:2); MP (°C) 225-228; Yield: 68%; IR (KBr pellets) 1/cm: 3390.24 (NH str.), 3122.19 (NH 2 str.) 1629.55, (C = O str., pyrazolone ring), 1576.62 (C = N str.), 768.35 (CH = CH str., aromatic); 1 H NMR (DMSO-d6 ) δ ppm: 9.67 (s, 1H, NH of hydrazine, -N = NH-), 7.13-7.96 (m, 10H, Ar-H), 3.05 (s, 1H, NH of pyrazolone), 2.51 (s, 2H, NH 2 ); exact mass: m/z: 286.06 [M] + ; Anal. Calculated for C 12 H 10 N 6 OS: C, 59.65; H, 3.89; N, 23.19; O, 4.41; S, 8.85; Found: C, 59.34; H, 3.74; N, 23.03.

General procedure for synthesis of Schiff bases of 3-amino-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4a-l)

The mixture of TZP4 ( 0.005 mol, 1.43 g) and corresponding aldehydes (0.005 mol) in 20 mL of ethanol was heated under reflux at 80°C for 2-3 h in the presence of glacial acetic acid. [3] The progress of the reaction was monitored by TLC, the resulting mixture was poured into crushed ice, and the precipitated title compound was filtered, dried, and recrystallized from ethanol.

1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-3-[(phenylmethylidene) amino]-4,5-dihydro-1H-pyrazol-5-one (TZP4a)

IR (KBr pellets) cm -1 : 3212.59 (-NH str., secondary amine), 1642.15, (C = O str., pyrazolone ring), 1609.23 (-CONH- out of plane bending), 1515.19 (C = N), 863.51 (Ar-CH = CH), 689.96 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.86 (s, 1H, -NH-N = of hydrazone), 8.78 (s, H, N = CH-), 7.13-8.15 (m, 15H, Ar-H), 6.37 (s, 1H, CH of thiazole); exact mass: m/z: 450.52 [M] + ; Anal. Calculated for C 25 H 18 N 6 OS: C, 66.65; H, 4.03; N, 18.65; O, 3.55; S, 7.1; Found: C, 66.21; H, 3.86; N, 18.52.

3-[[(2-chlorophenyl)methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4b)

IR (KBr pellets) cm -1 : 3234.25 (-NH str., secondary amine), 1636.23, (C = O str., pyrazolone ring), 1602.24 (-CONH- out of plane bending), 1513.21 (C = N), 850 (Ar-Cl str.), 813.97 (Ar-CH = CH), 675.82 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.95 (s, 1H, -NH-N = of hydrazone), 8.82 (s, H, N = CH-), 7.11-8.25 (m, 14H, Ar-H), 6.23 (s, 1H, CH of thiazole); exact mass: m/z: 484.96 [M] + ; Anal. Calculated for C 25 H 17 ClN 6 OS: C, 61.92; H, 3.53; Cl, 7.31; N, 17.33; O, 3.30; S, 6.61; Found: C, 61.71; H, 3.42; N, 17.12.

3-[[(3-chlorophenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4c)

IR (KBr pellets) cm -1 : 3241.12 (-NH str., secondary amine), 1634.12, (C = O str., pyrazolone ring), 1610.24 (-CONH- out of plane bending), 1518.41 (C = N), 868 (Ar-Cl str.), 826.46 (Ar-CH = CH), 682.75 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.83 (s, 1H, -NH-N = of hydrazone), 8.72 (s, H, N = CH-), 7.12-8.21 (m, 14H, Ar-H), 6.27 (s, 1H, CH of thiazole); exact mass: m/z: 484.96 [M] + ; Anal. Calculated for C 25 H 17 ClN 6 OS: C, 61.92; H, 3.53; Cl, 7.31; N, 17.33; O, 3.30; S, 6.61; Found: C, 61.71; H, 3.42; N, 17.12. 3-[[(4-chlorophenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4d)

IR (KBr pellets) cm -1 : 3243.19 (-NH str., secondary amine), 1631.61 (C = O str., pyrazolone ring), 1612.12 (-CONH- out of plane bending), 1511.85 (C = N), 859 (Ar-Cl str.), 821.55 (Ar-CH = CH), 685.76 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.91 (s, 1H, -NH-N = of hydrazone), 8.72 (s, H, N = H-), 7.11-8.22 (m, 14H, Ar-H), 6.17 (s, 1H, CH of thiazole); exact mass: m/z: 484.96 [M] + ; Anal. Calculated for C 25 H 17 ClN 6 OS: C, 61.92; H, 3.53; Cl, 7.31; N, 17.33; O, 3.30; S, 6.61; Found: C, 61.71; H, 3.42; N, 17.12.

3-[[(4-flurophenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4e)

IR (KBr pellets) cm -1: 3249.15 (-NH str., secondary amine), 1643.67 (C = O str., pyrazolone ring), 1615.10 (-CONH- out of plane bending), 1510.54 (C = N), 1416 (C-F str.), 825.12 (Ar-CH = CH), 686.46 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.97 (s, 1H, -NH-N = of hydrazone), 8.89 (s, H, N = CH-), 7.03-8.32 (m, 14H, Ar-H), 6.17 (s, 1H, CH of thiazole); exact mass: m/z: 468.51 [M] + ; Anal. Calculated for C 25 H 17 FN 6 OS: C, 64.09; H, 3.66; F, 4.06; N, 17.94; O, 3.41; S, 6.84; Found: C, 63.86; H, 3.51; N, 17.74.

3-[[(2-hydroxyphenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4f)

IR (KBr pellets) cm -1 : 3368.83 (OH str.), 3253.59 (-NH str., secondary amine), 1646.32 (C = O str., pyrazolone ring), 1604.14 (-CONH- out of plane bending), 1515.19 (C = N), 823.67 (Ar-CH = CH), 689.96 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 10.23 (s, 1H, OH), 9.94 (s, 1H, -NH-N = of hydrazone), 8.62 (s, H, N = CH-), 7.23-8.27 (m, 14H, Ar-H), 6.37 (s, 1H, CH of thiazole); exact mass: m/z: 466.51 [M] + ; Anal. Calculated for C 25 H 18 N 6 O 2 S: C, 64.36; H, 3.89; N, 18.01; O, 6.86; S, 6.87; Found: C, 64.12; H, 3.61; N, 17.93.

3-[[(2,4-dihydroxyphenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4g)

IR (KBr pellets) cm -1 : 3372.64 (OH str.), 3243.46 (-NH str., secondary amine), 1641.82 (C = O str., pyrazolone ring), 1606.97 (-CONH- out of plane bending), 1509.79 (C = N), 823.67 (Ar-CH = CH), 685.76 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 10.33 (d, 2H, OH), 9.82 (s, 1H, -NH-N = of hydrazone), 8.42 (s, H, N = CH-), 7.12-8.17 (m, 13H, Ar-H), 6.23 (s, 1H, CH of thiazole); exact mass: m/z: 482.51 [M] + ; Anal. Calculated for C 25 H 18 N 6 O 3 S: C, 62.23; H, 3.76; N, 17.42; O, 9.95; S, 6.65; Found: C, 62.09; H, 3.51; N, 17.32.

3-[[(3,4-dihydroxyphenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4h)

IR (KBr pellets) cm -1 : 3373.53 (OH str.), 3223.79 (-NH str., secondary amine), 1647.12 (C = O str., pyrazolone ring), 1608.12 (-CONH- out of plane bending), 1512.67 (C = N), 831.47 (Ar-CH = CH), 685.53 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 10.24 (s, 2H, OH), 9.75 (s, 1H, -NH-N = of hydrazone), 8.33 (s, H, N = CH-), 7.09-8.27 (m, 13H, Ar-H), 6. 31 (s, 1H, CH of thiazole); exact mass: m/z: 482.51 [M] + ; Anal. Calculated for C 25 H 18 N 6 O 3 S: C, 62.23; H, 3.76; N, 17.42; O, 9.95; S, 6.65; Found: C, 62.14; H, 3.62; N, 17.23.

3-[[(3-methoxyphenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4i)

IR (KBr pellets) cm -1: 3249.71 (-NH str., secondary amine), 1645.92 (C = O str., pyrazolone ring), 1614.42 (-CONH- out of plane bending), 1516.21 (C = N), 1072.25 (C-O-C str.), 829.61 (Ar-CH = CH), 699.17 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.75 (s, 1H, -NH-N = of hydrazone), 8.48 (s, H, N = CH-), 7.05-8.26 (m, 14H, Ar-H), 6. 28 (s, 1H, CH of thiazole), 3.36 (s, 3H, OCH3); exact mass: m/z: 480.54 [M] + ; Anal. Calculated for C 26 H 20 N 6 O 2 S: C, 64.98; H, 4.20; N, 17.49; O, 6.66; S, 6.67; Found: C, 63.54; H, 4.02; N, 17.24.

3-[[(4-hydroxy-3-methoxyphenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4j)

IR (KBr pellets) cm -1 : 3369.26 (OH str.), 3253.71 (-NH str., secondary amine), 1644.32 (C = O str., pyrazolone ring), 1614.47 (-CONH- out of plane bending), 1511.59 (C = N), 1086.31 (C-O-C str.), 829.27 (Ar-CH = CH), 681.36 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 10.34 (s, 1H, OH), 9.75 (s, 1H, -NH-N = of hydrazone), 8.58 (s, H, N = CH-), 7.11-8.36 (m, 13H, Ar-H), 6. 27 (s, 1H, CH of thiazole), 3.36 (s, 3H, OCH3); exact mass: m/z: 496.54 [M] + ; Anal. Calculated for C 26 H 20 N 6 O 3 S: C, 62.89; H, 4.06; N, 16.93; O, 9.67; S, 6.46; Found: C, 62.45; H, 3.92; N, 16.32.

3-[{[4-(dimethylamino) phenyl] methylidene} amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4k)

IR (KBr pellets) cm 1 : 3245.19 (-NH str., secondary amine), 1639.84 (C = O str., pyrazolone ring), 1613.64 (-CONH- out of plane bending), 1509.96 (C = N), 1319.16 (N (CH 3 ) 2 ), 823.27 (Ar-CH = CH), 676.14 (C-S-C str.); 1 H NMR (DMSO-d 6 ) δ ppm: 9.89 (s, 1H, -NH-N = of hydrazone), 8.78 (s, H, N = CH-), 6.96-8.12 (m, 15H, Ar-H), 6.32 (s, 1H, CH of thiazole), 3.14 (s, 6H, N (CH 3 ) 2 ); exact mass: m/z: 493.58 [M] + ; Anal. Calculated for C 27 H 23 N 7 OS: C, 65.70; H, 4.70; N, 19.86; O, 3.24; S, 6.50; Found: C, 65.32; H, 4.55; N, 19.28.

3-[[(4-nitrophenyl) methylidene] amino]-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-one (TZP4l)

IR (KBr pellets) cm -1: 3243.34 (-NH str., secondary amine), 1645.61 (C = O str., pyrazolone ring), 1612.37 (-CONH- out of plane bending), 1512.59 (C = N), 1341.54 (NO 2 ), 821.77 (Ar-CH = CH), 683.58 (C-S-C str.); 1 H NMR (DMSO-d6 ) δ ppm: 9.89 (s, 1H, -NH-N = of hydrazone), 8.78 (s, H, N = CH-), 7.23-8.32 (m, 15H, Ar-H), 6.31 (s, 1H, CH of thiazole); exact mass: m/z: 495.51 [M] + ; Anal. Calculated for C 25 H 17 N 7 O 3 S: C, 65.70; H, 4.70; N, 19.86; O, 3.24; S, 6.50; Found: C, 65.41; H, 4.49; N, 19.47.[Table 1]
Table 1: Physicochemical properties of synthesized compounds (TZP4a-l)


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Antimicrobial evaluation

Determination of zone of inhibition

All newly synthesized compounds (TZP4a-l) were screened for their preliminary antibacterial activity against four Gram-positive strains: Micrococcus luteus, Staphylococcus aureus, Bacillus. substilis, and Coryne bacterium; four Gram-negative strains: Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, and Klebsiella pneumonia; and three fungal strains: Candida albicans, Aspergillus niger, and Aspergillus parasites by disc diffusion method. [15] A standard inoculum (1-2 × 10 7 cfu/mL 0.5 McFarland standards) was introduced on to the surface of sterile agar plates, and a sterile cotton swab was used for even distribution of the inoculum. The discs measuring 6.25 mm in diameter were prepared from Whatman No. 1 filter paper and sterilized by dry heat at 140°C for 1 h. The sterile discs previously soaked in a known concentration (100 μg/mL) of the test compounds were placed in nutrient agar medium. The plates were inverted and incubated for 24 h at 37 ± 1°C for bacteria and 72-96 h at 27 ± 1°C for fungi. After the incubation zone of inhibition was measured. The media used was nutrient agar medium and Sabouraud dextrose medium for antibacterial and antifungal activity, respectively. Ciprofloxacin (5 μg/disc) and clotrimazole (5 μg/disc) were used as standard drugs for antibacterial and antifungal activity, respectively. Triplicate was maintained for all tested strains. Activity was determined by measuring the diameter of the zone showing complete inhibition. The average mean results of the antibacterial and antifungal studies are listed in [Table 2], [Table 3], [Table 4], respectively.
Table 2: Antibacterial screening of titled compounds (TZP4a-l)e against Gram-positive bacteria


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Table 3: Antibacterial screening of titled compounds (TZP4a-l)e against Gram-negative bacteria


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Table 4: Antifungal screening of titled compounds (TZP4a-l)e


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Determination of minimum inhibitory concentration

The minimum inhibitory concentration [16] (MIC) in μg/mL of the titled compounds was carried out by two-fold serial dilution method. The synthesized compounds (TZP4a-l) were dissolved in dimethyl sulfoxide (DMSO) to obtain 1 mg/mL stock solution. Seeded broth (broth containing microbial spores) was prepared in nutrient broth from 24 h old bacterial cultures on nutrient agar at 37 ± 1°C while fungal spores from 1 to 7 days old Sabouraud agar slant cultures were suspended in Sabouraud dextrose broth. The colony forming units (cfu) of the seeded broth were determined by plating technique and adjusted in the range of 104-105 cfu/mL. The final inoculums size was 105 cfu/mL for antibacterial assay and 1.1-1.5 cfu/mL × 102 cfu/mL for antifungal assay. Testing is performed at pH 7.4 ± 0.2 for bacteria and at a pH 5.6 for fungi. Exactly 0.4 mL of the solution of test compound was added to 1.6 mL of seeded broth to form the first dilution. One mL of this was diluted with a further 1 mL of seeded broth to give the second dilution and so on till six of such dilutions are obtained. A set of assay tubes containing only seeded broth was kept as control. The tubes were incubated in BOD incubators at 37 ± 1°C for bacteria and 28 ± 1°C for fungi. The MICs were recorded through visual observations after 24 h (for bacteria) and 72-96 h (for fungi) of incubation. Ciprofloxacin was used as standard for bacterial studies and clotrimazole was used as standard for fungal studies. The lowest concentration at which there was no visible growth was taken as MIC. The results of the MIC study are listed in [Table 2], [Table 3], [Table 4].

Determination of minimum bactericidal/fungicidal concentration

The minimum bactericidal concentration [17] (MBC) and fungicidal concentration (MFC) were determined by sub-culturing 100 μl of culture from each tube that remained clear in the MIC determination into fresh medium. MBC and MFC values represent the lowest concentration of compound that produces a 99.9% end point reduction.

Evaluation of antimycobacterial activity

A preliminary antimycobacterial activity [18] of titled compounds TZP4a-l at 100-1.56 μg mL against Mycobacterium smegmatis (MTCC99) in Middlebrook 7H9broth medium by the two fold serial dilution method was carried out in Department of Microbiology, KMCH College of Pharmacy, Coimbatore. Compounds demonstrating at least 90% inhibition in the preliminary evaluation were screened at 100-1.56 μg/mL concentration in a broth micro-dilution assay with Alamar Blue, called as microplate Alamar Blue assay, against Mycobacterium tuberculosis H37Rv (MTB) to determine MIC. These results in comparison with Pyrazinamide and Streptomycin as the reference drugs and the results are listed in [Table 5]. To prepare inoculum, microorganism was grown in middle brook 7H9 broth medium until a bacterial density corresponding to 0.5 McFarland turbidity standards. 200 μl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate to minimize evaporation of medium in the test wells during incubation. The 96 wells plate received 100 μl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on the plate. The final drug concentrations tested were 1.56-100.0 μg/mL. Plates were covered and sealed with parafilm and incubated at 37°C for 5 days. After this time, 25 μl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 h. A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth. The MIC was defined as lowest drug concentration which prevented the color change from blue to pink.

Evaluation of in-vitro cytotoxicity activity

The NIH 3T3 mouse embryonic fibroblasts line (NIH 3T3) was obtained from National Center for Cell Science, Pune, and grown in Dulbeccos Modified Eagles Medium containing 10% fetal bovine serum (FBS). All the cells were maintained at 37°C, 5% CO 2 , 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. The monolayer cells were detached with trypsin-ethylene diamine tetra acetic acid to make single cell suspension, and viable cells were counted using a hemocytometer and diluted using medium with 5% FBS to give final density of 1 × 10 5 cells/mL. One hundred microlitres per well of cell suspension was seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37°C, 5% CO 2 , 95% air, and 100% relative humidity. After 24 h, the cells were treated with serial concentrations of the synthesized compounds TZP4a-l. They were initially dissolved in neat DMSO and further diluted in serum free medium to produce various concentrations. One hundred microlitres per well of each concentration were added to plates to obtain final concentrations of 1000, 500, 250, 125, and 63 μM. The final volume in each well was 200 μl and the plates were incubated at 37°C, 5% CO 2 , 95% air, and 100% relative humidity for 48 h. The medium without samples served as control. Triplicate was maintained for all concentrations. After 48 h, the cells in each well were quantified by MTT assay. [19],[20] Briefly, 15 mL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37°C for 4 h. The medium with MTT was then flicked off, and the formed formazan crystals were solubilized in 100 mL of DMSO and then measured the absorbance at 570 nm using a micro-plate reader. The % cell inhibition was determined using the following formula:

% Cell inhibition = 100 − Abs (sample)/Abs (control) ×100.

Non-linear regression graph was plotted between % cell inhibition and Log 10 concentration and IC 50 was determined using Graph Pad Prism software.

Selectivity index

Selectivity index [21] (SI) was calculated for all the titled compounds taking into account the MIC against M. tuberculosis H37Rv and the IC 50 on mouse embryonic fibroblasts cell line (NIH 3T3) (SI = IC 50 /MIC) by the MTT assay.


   Results and Discussion Top


Chemistry

The synthetic procedures adopted to obtain the target compounds are depicted in [Figure 2]. The starting material, 4-phenylthiazol-2-amine (TZ) was prepared by heating acetophenone with thiourea in the presence of iodine following a procedure described earlier by Pandeya, et al.[12] Diazonium salt of 4-phenylthiazol-2-amine coupled with ethyl cyano acetate in ethanol to afford product (TZE) that was identified as ethyl cyano [2-(4-phenyl-1,3-thiazol-2-yl) hydrazinylidene] acetate. Diazotization is carried out in concentrated acid is used for the diazo component, since hydrolysis of the diazonium salt occurs in dilute acid. The temperature 0-5°C is necessary to avoid decomposition of diazonium salt solution. To complete the coupling with ethylcyanoacetate, particularly for reactions using nitrosyl hydrochloric acid in the diazotization, the pH of the reaction mixture was adjusted to approximately 4-5. Thus, an appropriate amount of 10% sodium acetate solution was slowly added below 5°C. The key intermediate 4-(2-(4-phenylthiazol-2-yl) hydrazono)-3-amino-1-phenyl-1H-pyrazol-5 (4H)-one (TZP4) was prepared by cyclization reaction of TZE with phenyl hydrazine hydrate in ethanol to afford compound TZP4. The compound (TZP4) containing amino group at third position in the pyrazolone ring is appreciably labile to participate in the Schiff reaction. These primary amines in pyrazolone moiety were subjected to Schiff reaction with various aldehydes to yield title compounds (TZP4a-l). To reduced reaction time and increase percentage yield, the Schiff reaction is catalyzed by few drops of glacial acetic refluxing for 2-3 h. The structures of compounds (TZP4a-l) were assigned by IR, 1 H NMR and mass spectral and elemental analysis data, which were consistent with the proposed molecular structures. Structures of key intermediates (TZP4) were confirmed by IR spectra, which showed that the disappearance of the characteristic bands of the cyano group and carboxylic acid ester at 2362 and 1754 cm -1 respectively and the appearance of a band at 3312-3318 and 1605-1638 cm -1 attributed to the stretching vibration of the NH 2 group, pyrazolone ring C = O groups. The IR spectra of the Schiff base (TZP4a-l) confirmed the presence of -NH-N = C, HN-C = O, -CH-S-C and fused ring system present in the synthesized compounds by the presence of IR stretching bands at 3284-3269, 1630-1652, 1248-1045, and 1602-1623 cm -1 , respectively. Singlet signals derived from-NH-N = C protons at 9.5-10.30 δ ppm indicates the hydrazone formation. The aromatic protons appeared as multiplets ranging from 6.58 to 8.43 δ ppm. In case of Schiff bases (TZP4a-l) singlet signals appeared within the range 6.21-6.40 δ ppm due to -N = CH- proton which confirmed the formation of Schiff bases. Further, the formations of title compounds were confirmed by recording their mass spectrums which were in full agreement with their molecular weights and results of elemental analysis were ± 0.4% of the theoretical values.
Figure 2: Scheme for synthesis of Schiff base of pyrazol-5-one moiety containing 3-(hydrazinyl)-4-phenyl-1,3 thiazole

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Biological evaluation

In-vitro antimicrobial activity

Agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Ciprofloxacin and clotrimazole were used as reference drugs. The results were recorded for each tested compound in triplicate as the average diameter of inhibition zones (IZs) of bacterial or fungal growth around the disks in mm. The MIC measurement was determined for compounds showed growth IZs (>8 mm) using twofold serial dilution method. The IZ diameters and MIC [(μM/mL × 10−3 )] values are recorded in [Table 2], [Table 3], [Table 4]. The results are described in [Table 2], [Table 3], [Table 4] revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative bacterial strains as well as against fungal strains. In general, most of the synthesized compounds demonstrated better activity against Gram-positive bacteria with the % of zone of inhibition range between 38 and 92 as well as fungal strains with the % of zone of inhibition range between 56 and 100. In case of S. aureus, all the title compounds displayed antibacterial activity. Compounds TZP4 d, TZP4e, TZP4f, and TZP4g were found to be most potent against M. luteus with 72%, 72%, 92%, and 80% inhibition, respectively, whereas compounds TZP4d, TZP4e, TZP4 g, and TZP4 h exhibited most potent antibacterial activity against B. substilis having 78%, 78%, 83%, and 83% inhibition and compound TZP4 g showed most significant antibacterial activity against S. aureus (% inhibition = 88). On the other hand, synthesized compounds demonstrated moderate activity against Gram-negative bacteria.

The antifungal activity results [Table 4] indicated that compound TZP4 g, TZP4 h, and TZP4k was found equipotent antifungal agent against A. niger having 100% growth inhibition comparable to standard. In case of C. albicans compound TZP4g, TZP4h, TZP4j, TZP4k, and TZP4l exhibited maximum 90% growth inhibition. Compounds TZP4 g and TZP4 h exhibited remarkable MIC activity (MIC = 12.95 × 10−3 μM/mL), TZP4j marvelous MIC activity (MIC = 06.29 × 10−3 μM/mL) against B. substilis which was 1.45 and 2.99 times more active than the standard drugs Ciprofloxacin. On the other hand, compounds TZP4d, TZP4g, TZP4h, TZP4i, TZP4j, and TZP4k demonstrated more MIC activity against A. niger compared with the standard clotrimazole. All the newly synthesized compounds are bactericidal and fungicidal in nature.

In-vitro antimycobacterial activity

In-vitro
antimycobacterial potential of synthesized Schiff bases was determined against M. smegmatis (MTCC99) and M. tuberculosis (H37Rv). In this study, M. smegmatis is used as model microorganism for the research analysis of other Mycobacteria species in laboratory experiments due to fast growing and non-pathogenic nature. [22] Results of antimycobacterial activity are given in [Table 5] indicated that varying degree of antimycobacterial activity was observed and also antimycobacterial activity of the synthesized compounds against M. smegmatis was well interconnected with the M. tuberculosis activity. However, this slight dissimilarity observed in the MIC results may be due to a different method of assay procedure adopted or difference in strain. Pyrazinamide and streptomycin was used as the standard in the tests. All the 12 compounds showed good in-vitro activity against M. tuberculosis with MIC ranging from 06.48 to 53.59 μM. The effect of various substituents on the phenyl ring of pyrazolones (TZP4a-l) producing antitubercular activity in the descending order was found to be: m, p-diOH (TZP4h); >m-Cl (TZP4c); >o, p-diOH (TZP4g); >p-N (CH 3 ) 2 (TZP4k); >H (TZP4a); >m-OCH 3 , p-OH (TZP4j); >m-OCH 3 (TZP4i); >p-NO 2 (TZP4l); >m-Cl (TZP4b); and p-Cl (TZP4d); >o-Cl (TZP4e); >p-OH (TZP4f). The results revealed that compound TZP4h exhibited tremendous antitubercular activity (MIC = 06.48 × 10−3 μM/mL) which was 3.9 and 1.69 times more active than the standard drugs Pyrazinamide and streptomycin (MIC = 25.38, 11.01 × 10−3 μM/mL), respectively. In general, the synthesized compounds with electron donating group on the phenyl ring (TZP4 g, TZP4 h, TZP4i, TZP4j, and TZP4k) were showed appreciable antitubercular activity against M. tuberculosis (MIC = 12.95, 12.96, 25.17, and 26.01 × 10−3 μM/mL) which was equipotent to Pyrazinamide but less potent than Streptomycin. Synthesized compounds to inhibition of M. tuberculosis growth might be confirmed by evaluation its cytotoxicity.
Table 5: Screening of antimycobacterium, cytotoxicity, and selectivity index of the titled compounds (TZP4a-l)


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In-vitro cytotoxicity

The compounds (TZP4f, TZP4g, and TZP4h) showed highest IC 50 at 1310, 2187, and 1290 μM, compounds (TZP4b, TZP4c, TZP4d, and TZP4l) showed IC 50 at 989.90, 1068, 1153, and 1025 μM, respectively, thus suggesting that their anti-TB activity was not due to some general cytotoxicity. The SI was calculated by dividing IC 50 by the MIC of TB activity values. SI is defined as the ratio of the measured IC 50 in mouse embryonic fibroblasts cell line to the MIC of TB activity described above. Furthermore, compounds TZP4c, TZP4 g, and TZP4 h showed excellent SI of 82.85, 168.88, and 199.07. On the other hand, the compounds TZP4d, TZP4f, and TZP4l showed better SI of 22.37, 24.44 and 20.32. If the SI is ≥10, the compound is then investigated further. Compounds TZP4c, TZP4d, and TZP4l have the potential to be selected as a lead compound for the development of antitubercular agents. Structural requirements for antimicrobial and antitubercular activity of synthesized compounds is given in [Figure 3].
Figure 3: Structural requirements for antimicrobial and antitubercular activity of synthesized compounds

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From the results of the in-vitro antitubercular and antimicrobial activity of the synthesized substituted pyrazolone derivatives, the following structure activity relationship (SAR) can be derived.

SAR

Compounds with electron donating OH groups at para-/meta- or ortho-position (TZP4 h and TZP4 g) on the phenyl ring displayed potent activity against tested mycobacterium strains. Among the di-substituents electron donating groups on the phenyl ring, the highest antitubercular activity was obtained with substituent having lowest lipophilicity, polarizability, and electron donating power (TZP4g-h and TZP4j). Title compounds reduced general cytotoxicity and improved specific antitubercular activity compared with our previous reported compounds (Schiff bases of 5-amino-4-[2-(4-nitro-1,3-benzothiazol-2yl) hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-one) by replacement of hydrogen, 4-nitro-1,3-benzothiazole moiety at first, fourth position on the pyrazolone by phenyl and 4-phenyl-1,3-thiazole moiety. On the other hand, presence of phenyl and 4-phenyl-1,3-thiazole moiety on the pyrazolone reduced Gram-negative bacteria activity compared with previously reported compounds. Presence of hydroxy group on the phenyl ring reduced cytotoxicity among the title compounds.


   Conclusion Top


In conclusion, a series of Schiff bases (TZP4a-l) of 3-amino-1-phenyl-4-[2-(4-phenyl-1,3-thiazol-2-yl) hydrazin-1-ylidene]-4,5-dihydro-1H-pyrazol-5-ones have been synthesized and screened for antimicrobial activity against Gram-positive, Gram-negative bacterial as well fungal strains. Results of antimicrobial study indicated that the synthesized were significant against fungi strains. Compound TZP4 g, TZP4h, and TZP4k (% inhibition = 100) exhibited equal potent antibacterial activity compared with standard drug clotrimazole against A. niger. In-vitro anti-TB activity of compound TZP4 g was showed appreciable antitubercular activity against M. tuberculosis H37Rv strain (MIC = 06.48 × 10 −3 μM/mL), which was 1.69 and 3.91 times more active than the standard drug, Pyrazinamide (25.38 × 10−3 μM/mL) and streptomycin (MIC = 11.01 × 10 −3 μM/mL), respectively. In comparison to the results of toxicity and antimycobacterial activity tests, it was observed that the activity of the compounds is not due to general toxicity effect; however, their antimycobacterial activity can be possibly because of their selective antimycobacterial effect. We concluded from our investigations that TZP4c, TZP4 g, and TZP4 h may be considered promising for the development of new anti-TB agents.


   Acknowledgments Top


The authors express their sincere gratitude to Dr. Thavamani D. Palaniswami, Managing Trustee, Kovai Medical Research and Education Trust, Coimbatore and Karpagam University, Coimbatore for providing facilities to carry out this research work. The authors are grateful to Indian Institute of Science, Bangalore and Indian Institute of Technology, Madras for providing NMR and Mass spectral data. Authors also thank Dr. Kishore G Bhat, Maratha mandals NGH Institute of Dental Sciences and Research Centre, Karnataka for evaluation of antimycobacterial activity.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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