Der Pharma Chemica
Journal for Medicinal Chemistry, Pharmaceutical Chemistry, Pharmaceutical Sciences and Computational Chemistry

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Research Article - Der Pharma Chemica ( 2018) Volume 10, Issue 8

Antimicrobial Evaluation of Some New Pyrazolyl Pyridinyl Coumarins

Jigar Patel*

SSR College of Arts, Commerce and Science, Sayli road, Silvassa, Dadra and Nagar Haveli-396230, India

*Corresponding Author:
Jigar Patel
SSR College of Arts, Commerce and Science
Sayli road, Silvassa, Dadra and Nagar Haveli-396230, India

Abstract

The synthesis of various pyrazolyl pyridinyl substituted coumarins 3-{4'-[1''-phenyl -3''- (pyridin-3''' -yl)-1H- pyrazol- 4''-yl]- 6'-aryl -pyridin - 2'-yl} coumarins (4a-f) and 3-{4'-[1''-phenyl-3''-(pyridin-4'''-yl)-1H-pyrazol-4''-yl]-6'-aryl-pyridin-2'-yl}coumarins (5a-f) have been carried out by the reaction of 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)acryloyl)coumarins or 3-(3-(1-phenyl–3- (pyridin-4-yl)-1H–pyrazol-4-yl) acryloyl) coumarins with appropriate aroyl methyl pyridinium salts under Krohnke’s reaction condition. The synthesized compounds were fully characterized by Infrared (IR), Proton Nuclear Magnetic Resonance (1H-NMR), Carbon-13 Nuclear Magnetic Resonance (13C-NMR), DEPT- 135, DEPT-90 and representative Mass spectral data. The synthesized compounds were screened for in vitro antimicrobial activity using Broth micro dilution method.

Keywords

Coumarins, Pyridinylcoumarins, Pyrazole, Kröhnke reaction, Antimicrobial activity.

Introduction

Among five membered nitrogen containing heterocycles, pyrazole is a prominent moiety. A large number of compounds having pyra-zole nucleus in their structure are reported to have wide range of biological activities viz., antioxidant [1], anti-invasive [2], antiviral [3], anti-inflammatory [4,5] and are also used as agrochemicals [6] and dyestuffs [7,8]. Celecoxib [9] was the first to market this class of compounds used as anti-inflammatory and analgesic agents. Phenylbutazone [9] has been used in the treatment of severe arthritis. The pyrazole derivative like Tartrazine is used as food colourant [9]. Soliman [10] and Shatik [11] have reported some pyrazole derivatives, which were found to be useful as anticancer agents. Singh et al. [12] have synthesized series of pyrazole derivatives by the condensation of appropriate 2-hydrazino-4- arylthiazoles with various α-cyanoacetophenones. The compounds exhibited anti-inflammatory and anti-antagonist activities. Wariishi [13,14] has reported some pyrazole derivatives having indole moiety. These compounds were reported to possess properties of photographic dye. Lange et al. [15,16] have synthesized antagonistic active pyrazole derivatives. Armand et al. [17,18] have synthesized analgesic and anti-inflammatory pyrazole derivatives. Some selected medicinally useful pyrazoles are shown in (Chart 1).

derpharmachemica-pyrazoles

Chart 1: Medicinally useful pyrazoles

Moffett tested the synthesized pyridyl coumarin derivatives for various physiological activities. Most of them showed a central nervous system depression property [17,18]. 3-(3-Pyridyl)coumarin inhibited monoamine oxidase (MAO) in vitro and hence was considered as possible stimulant. 6-Bromo-3-(2-pyridyl) and 6-bromo-3-(3-pyridyl) coumarins were found to be antifungal agents in vitro [19,20]. Certain derivatives of these class of compounds were also found to be useful UV screening agents and as optical brightening agents for textile. When certain pyridyl derivatives having –NH2 function in the benzene ring were reacted with fluosilicic acid, they formed amine-fluosilicate salts which were found to be effective as moth-proofing agents [21,22].

Srenivasulu et al. [23] have synthesized some 3-(3-pyridyl)coumarin derivatives. The compounds were synthesized by reacting substituted salicylaldehyde/o-hydroxy acetophenone with 3-pyridine acetic acid or its sodium salt under Perkin reaction conditions. These compounds were reported to have fish toxicity and bactericidal activities. Bragg and Wibberely [24] have synthesized 3-(2-pyridyl) and 3-(4-pyridyl) coumarins using Knoevenagel reaction, in which salicylaldehyde was treated with ethyl-2-pyridyl acetate or ethyl-4-pyridyl acetate in the presence of piperidine. Mohareb et al. [25] have synthesized 3-(2-pyridyl) coumarin with substituents in pyridine nucleus. Some selected biologically active pyridyl substituted coumarins are shown in (Chart 2).

derpharmachemica-active-pyridyl

Chart 2: Selected biologically active pyridyl substituted coumarins

Thus pyridinyl coumarins and pyrazole are very important heterocycles from bioactivity view point, therefore in the present work, some pyrazolyl pyridinyl substituted coumarins 4a-f and 5a-f have been carried out by Krohnke’s reaction [26,27], which are combination of both components i.e. pyrazole as well as pyridinyl coumarin in a single scaffold.

Materials and Methods

All melting points (ºC) are uncorrected. The yields of all compounds reported are of crystallized. All solvents used were distilled and dried. The purity of the compounds was checked by TLC (TLC aluminium sheet silica gel 60 F254, Merck). Column chromatography was performed on silica gel (60-120 mesh). C, H, N analysis were carried out on Parkin-Elmer 2400 C-H-N-S-O Analyzer Series II. Infrared (IR) spectra were recorded in KBr pellets on Shimadzu FT-IR 8400-S spectrometer or Parkin-Elmer Frontier Fourier Transform Infrared (FTIR) /FIR spectrometer. Mass spectra were recorded using a Perkin-Elmer Clarus 500 spectrometer. NMR spectra were recorded using a Bruker Avance 400 spectrometer or Varian 400 spectrometer, operating at 400 MHz for Proton Nuclear Magnetic Resonance (1H-NMR) and 100 MHz for Carbon-13 Nuclear Magnetic Resonance (13C-NMR /DEPT-90/DEPT-135. The chemical shift (δ) is reported in ppm using CDCl3/DMSO as a solvent, and calibrated standard solvent signal.

Experimental Section

Preparation of 3-acetyl coumarins (Scheme 1)

derpharmachemica-acetyl-coumarins

Scheme 1: Synthesis of 3-acetyl coumarins

In a 100 ml round bottom flask, a mixture of an appropriate salicylaldehyde (0.01 mol), ethyl acetoacetate (0.01 mol) and 3-4 drops of piperidine was stirred for 10 min at room temperature. It was then heated for 30 min in water bath. On cooling, a yellow solid product was obtained, which was filtered out and washed with cold ether. It was recrystallized from chloroform-hexane.

3-Acetyl coumarin: R=R1=R2=H, Yield: 97%, M. p. 119°C (lit. [28] mp 120°C)

8-Methoxy-3-acetyl coumarin: R=R1= H, R2=OCH3, Yield: 95%, M. p. 171°C (lit. [28] M. p. 174°C)

5,6-Benzo-3-acetyl coumarin: R=R1=benzo, R2=H, Yield: 86%, M. p. 185°C (lit. [26,27] M. p. 186°C)

Preparation of 1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-alde-hyde and 1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-aldehyde (Scheme 2)

derpharmachemica-Synthesis-Preparation

Scheme 2: Synthesis of Preparation of 1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-alde-hyde and 1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-aldehyde

The following general procedure was used.

In a 100 ml round bottom flask, a mixture of an appropriate acetylpyridine (0.01 mol), phenyl hydrazine (0.01 mol) and ethanol (5 ml) containing 1-2 drops of glacial acetic acid was warmed on a steam cone for 15 min. The separated phenyl hydrazone was filtered off, washed with cold ethanol (5 ml) and dried. It was recrystallized from ethanol.

The freshly prepared acetyl pyridine phenyl hydrazone (0.06 mol) was taken in a 250 ml three necked round bottom flask fitted with addition funnel and guard tube. Then anhydrous Dimethyl Formamide (DMF) (0.6 mol) was added and the reaction mixture was cooled to 0°C with stirring. To this reaction mixture, phosphorous oxychloride (POCl3) (0.18 mol) was added dropwise with stirring during one hour at 0°C. The reaction mixture was further stirred at 0°C for 1 h and then heated at 65°C-70°C for two hours. It was then poured into crushed ice (200 g) and left overnight in a refrigerator, during which a solid product was separated out which was filtered off, washed with sodium carbonate (5%, 3 × 30 ml) and water. It was then dried and recrystallized from ethanol.

1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-aldehyde

Molecular Formula C15H11N3O
Yield 82%
M. p. 158°C
Analysis %C %H %N
Found 72.17 4.38 16.77
Calculated 72.28 4.45 16.86
IR (cm-1) Vmax 1681 (C=O stretching of aldehyde), 1599 and 1531 (aromatic C=C and C=N stretchings), 681 and 754 (C-H out of plane bending vibrations of mono substituted benzene ring), 3132 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.42-9.14 (10H, multiplet, aromatic protons), 10.08 (1H, singlet, proton of CHO).

1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-aldehyde

Molecular Formula C15H11N3O
Yield 81%
M. P. 176°C
Analysis %C %H %N
Found 72.18 4.35 16.78
Calculated 72.28 4.45 16.86
IR (cm-1) Vmax 1681 (C=O stretching of aldehyde), 1599 and 1518 (aromatic C=C and C=N stretchings), 684 and 759 (C-H out of plane bending vibrations of mono substituted benzene ring), 3094 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.45-9.56 (10H, multiplet, aromatic protons), 10.06 (1H, singlet, proton of CHO).

Preparation of 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl) -acryloyl)coumarins (1a-c) and 3-(3-(1-phenyl-3-(pyridin-4-yl) -1H-pyrazol-4- yl)acryloyl)coumarins (2a-c) (Scheme 3)

derpharmachemica-Synthesis-compounds

Scheme 3: Synthesis of compounds (2a-c)

The following general procedure was used.

In a 100 ml round bottom flask, an appropriate 3-acetyl coumarin (0.01 mol) and an appropriate pyrazole aldehyde (0.015 mol) were taken in 50 ml of ethanol. Catalytic amount of piperidine (1.0 ml) was added and the reaction mixture was stirred for 10 min at room temperature. The reaction mixture was then refluxed on water bath for 4 h. It was allowed to cool to room temperature. A solid product sep arated out was filtered off, washed with cold ethanol and dried. It was recrystallized from ethanol.

Compound 1a: R=R1=R2=H

Molecular Formula C26H17N3O3
Yield 83%
M. P. 220°C
Analysis %C %H %N
Found 74.29 4.07 9.89
Calculated 74.45 4.09 10.02
IR (cm-1) Vmax 1724 (C=O stretching of d-lactone of coumarin), 1685 (C=O stretching of α,β unsaturated carbonyl group), 1609 and 1539 (aromatic C=C and C=N stretchings), 697 and 755 (C-H out of plane bending vibrations of mono substituted benzene ring), 3046 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.35-9.00 (17H, multiplet, fourteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Compound 1b: R=R1=H, R2=OCH3

Molecular Formula C27H19N3O4
Yield 80%
M. P. 210°C
Analysis %C %H %N
Found 72.08 4.22 9.26
Calculated 72.15 4.26 9.35
IR (cm-1) Vmax 1720 (C=O stretching of d-lactone of coumarin), 1662 (C=O stretching of α,β unsaturated carbonyl group), 1604 and 1534 (aromatic C=C and C=N stretchings), 694 and 761 (C-H out of plane bending vibrations of mono substituted benzene ring), 3037 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 4.03 (3H, singlet, OCH3), 7.38-8.99 (16H, multiplet, thirteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Compound 1c: R=R1=Benzo, R2=H

Molecular Formula C30H19N3O3
Yield 78%
M. P. 218°C
Analysis %C %H %N
Found 76.64 4.05 8.9
Calculated 76.75 4.08 8.95
IR (cm-1) Vmax 1717 (C=O stretching of d-lactone of coumarin), 1684 (C=O stretching of α,β unsaturated carbonyl group), 1597 and 1577 (aromatic C=C and C=N stretchings), 781 and 691 (C-H out of plane bending vibrations of mono substituted benzene ring), 3050 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.52-9.45 (19H, multiplet, sixteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Compound 2a: R=R1=R2=H

Molecular Formula C26H17N3O3
Yield 83%
M. P. 220°C
Analysis %C %H %N
Found 74.41 3.86 9.88
Calculated 74.45 4.09 10.02
IR (cm-1) Vmax 1717 (C=O stretching of d-lactone of coumarin), 1685 (C=O stretching of α,β unsaturated carbonyl group), 1607 and 1576 (aromatic C=C and C=N stretchings), 688 and 761 (C-H out of plane bending vibrations of mono substituted benzene ring), 3033 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.38-8.78 (17H, multiplet, fourteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Compound 2b: R=R1=H, R2=OCH3

Molecular Formula C27H19N3O4
Yield 85%
M. P. 160°C
Analysis %C %H %N
Found 72.11 4.24 9.33
Calculated 72.15 4.26 9.35
IR (cm-1) Vmax 1717 (C=O stretching of d-lactone of coumarin), 1636 (C=O stretching of α,β unsaturated carbonyl group), 1607 and 1576 (aromatic C=C and C=N stretchings), 688 and 761 (C-H out of plane bending vibrations of mono substituted benzene ring), 3034 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 4.03 (3H, singlet, OCH3), 7.21-8.78 (16H, multiplet, thirteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Compound 2c: R=R1=Benzo, R2=H

Molecular Formula C30H19N3O3
Yield 82%
M. P. 188°C
Analysis %C %H %N
Found 76.73 3.92 8.87
Calculated 76.75 4.08 8.95
IR (cm-1) Vmax 1717 (C=O stretching of d-lactone of coumarin), 1685 (C=O stretching of α,β unsaturated carbonyl group), 1607 and 1576 (aromatic C=C and C=N stretchings), 761 and 663 (C-H out of plane bending vibrations of mono substituted benzene ring), 3033 (aromatic C-H stretching).
1H-NMR CDCl3 (δ, ppm) 7.40-9.31 (19H, multiplet, sixteen aromatic protons+C4 proton of coumarin+two olefinic protons).

Preparation of phenacyl bromide (Scheme 4)

derpharmachemica-Phenacyl-bromide

Scheme 4: Synthesis of Phenacyl bromide

A solution of acetophenone (0.33 mol) in anhydrous ether (25 m; l was placed in a three necked flask equipped with a dropping funnel and gas absorption tube. The flask was placed in an ice bath and was allowed to cool to 0ºC-5ºC. A trace amount of anhydrous AlCl3 (0.1 g) was introduced and bromine (0.33 mol) was added dropwise with stirring during 30 min. The reaction mixture was allowed to come to room temperature and was stirred for further 1 h. Then pet. ether 40-60 (50 ml) was added. The phenacyl bromide was separated out as white solid. It was filtered out and was washed with a mixture of pet. ether 40-60 and water (100 ml, 1: 1). It was dried and crystallized from rectified spirit.

Phenacyl bromide: Yield: 85%, M. p. 47°C (lit. [29] M. p. 49°C)

Preparation of 4-methyl phenacyl bromide (Scheme 5)

derpharmachemica-methyl-phenacyl

Scheme 5: Synthesis of 4-methyl phenacyl bromide

A solution of a p-methyl acetophenone (0.5 mol) in acetic acid (100 ml) was placed in a three necked flask equipped with a dropping funnel and a condenser with gas absorption tube. Bromine (0.5 mol) was added gradually from dropping funnel with stirring at room temperature during 30 min. The reaction mixture was stirred for 2 h at room temperature. It was then poured into ice cold water (500 ml). The product was separated out as a white solid. It was filtered out and was washed with water and dried. The product was crystallized from rectified spirit.

4-Methyl phenacyl bromide: R=CH3; Yield: 86%, M. p. 48°C (lit. [29] mp 45°C-49°C)

Preparation of aroyl methyl pyridinium bromide salts (3a-b) (Scheme 6)

derpharmachemica-Synthesis-compounds

Scheme 6: Synthesis of compounds (3a-b)

The following general procedure was used.

An appropriate phenacyl bromide (0.25 mol) was dissolved in dry toluene (100 ml) in a 250 ml three necked flask equipped with a dropping funnel and magnetic needle at room temperature. Dry pyridine (0.25 mol) was added slowly to the above solution with stirring. The reaction mixture was refluxed at 100ºC for 30 min. It was then allowed to come to room temperature. The aroyl methyl pyridinium bromide salt was separated out as a white solid. It was filtered out and washed with dry toluene. It was then dried at 60°C-70ºC for 4-5 h in vacuum oven.

Compound 3a: R3=H; Yield: 80%, mp 193ºC (lit. [29] M. p. 194°C-197ºC)

Compound 3b: R3=CH3; Yield: 85%, mp 203ºC (lit. [30,31] M. p. 205ºC)

Synthesis of 3-{4'-[1''-phenyl-3''-(pyridin-3'''-yl)-1H-pyrazol-4''-yl]-6'-aryl-pyridin-2'-yl}coumarins 4a-f) and 3-{4'-[1''-phe-nyl-3''-(pyridin- 4'''-yl)-1H-pyrazol-4''-yl]-6'-aryl-pyridin-2'-yl}coumarins(5a-f) (Figure 1)

derpharmachemica-Structures-compounds

Figure 1: Structures of compounds (4a-f) and (5a-f)

The following general procedure was used.

In a 100 ml round bottom flask equipped with a condenser, guard tube and magnetic needle, an appropriate aroyl methyl pyridinium bromide salts (3a-b) (0.003 mol) in glacial acetic acid (15 ml) was taken. To this, ammonium acetate (0.03 mol) was added with stirring at room temperature. Then a solution of an appropriate 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)acryloyl)coumarin(1a-c) or 3-(3-(1-phenyl-3- (pyridin-4-yl)-1H-pyrazol-4-yl)acryloyl)coumarin (2a-c) (0.003 mol) in glacial acetic acid (15 ml) was added with stirring at room temperature and the reaction mixture was further stirred for 1 h at room temperature. It was then refluxed for 12 h at 140°C. It was then allowed to come to room temperature and was poured into ice-cold water (75 ml). A crude solid obtained was extracted with chloroform (3 × 30 ml). The organic layer was washed with 5% sodium bicarbonate solution (3 × 20 ml), water (2 × 20 ml) and dried over anhydrous sodium sulfate. The removal of chloroform under reduced pressure gave crude material which was subjected to column chromatography using silica gel and chloroform-ethyl acetate (7: 3) as an eluent to give compounds (4a-f) and (5a-f). The compounds were recrystallized from chloroform-hexane.

Results and Discussion

The condensation of 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)acryloyl)coumarins (1a-c) and 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol- 4-yl)acryloyl)coumarins (2a-c) with aroyl methyl pyridinium salts (3a-b) under Krohnke’s reaction condition proceeded smoothly and gave the expected products (4a-f) and (5a-f) (Scheme 7 and Table 1) in 65%-75% yield. The detailed mechanism for the formation of compounds (4a-f) is shown in Scheme 8. Formation of compounds (5a-f) follows similar mechanism.

derpharmachemica-Formation-compounds

Scheme 7: Formation of the compounds (4a-f) and (5a-f) by the Krohnke’s reaction

R R1 R2 R3 R R1 R2 R3
4a H H H H 5a H H H H
4b H H OCH3 H 5b H H OCH3 H
4c Benzo H H 5c Benzo H H
4d H H H CH3 5d H H H CH3
4e H H OCH3 CH3 5e H H OCH3 CH3
4f Benzo H CH3 5f Benzo H CH3

Table 1: Synthesis data of the compounds (4a-f) and (5a-f)

derpharmachemica-Mechanism

Scheme 8: Mechanism of the formation of compounds (4a-f)

Compound 4a: R=R1=R2=R3=H

Molecular Formula C34H22N4O2
Yield 68%
M. p. 230°C-234°C
Analysis %C %H %N
Found 78.62 4.21 10.73
Calculated 78.75 4.28 10.8

Compound 4b: R=R1=R3=H, R2=OCH3

Molecular Formula C35H24N4O3
Yield 72%
M. p. 218°C-220°C
Analysis %C %H %N
Found 76.58 4.37 10.15
Calculated 76.63 4.41 10.21

Compound 4c: R=R1=Benzo, R2=R3=H

Molecular Formula C38H24N4O2
Yield 69%
M. p. 240ºC
Analysis %C %H %N
Found 80.19 4.22 9.77
Calculated 80.27 4.25 9.85

Compound 4d: R=R1=R2=H, R3=CH3

Molecular Formula C35H24N4O2
Yield 67%
M. p. 194°C-196°C
Analysis %C %H %N
Found 78.87 4.46 10.45
Calculated 78.93 4.54 10.52

Compound 4e: R=R1=H, R2=OCH3, R3=CH3

Molecular Formula C36H26N4O3
Yield 72%
M. p. 223ºC
Analysis %C %H %N
Found 76.80 4.57 9.90
Calculated 76.85 4.66 9.96

Compound 4f: R=R1=Benzo, R2=H, R3=CH3

Molecular Formula C39H26N4O2
Yield 74%
M. p. 235°C-237°C
Analysis %C %H %N
Found 80.31 4.44 9.57
Calculated 80.39 4.5 9.62

Compound 5a: R=R1=R2=R3=H

Molecular Formula C34H22N4O2
Yield 75%
M. p. 225°C-227°C
Analysis %C %H %N
Found 78.67 4.23 10.71
Calculated 78.75 4.28 10.8

Compound 5b: R=R1=R3=H, R2=OCH3

Molecular Formula C35H24N4O3
Yield 73%
M. p. 195°C-197°C
Analysis %C %H %N
Found 76.58 4.36 10.16
Calculated 76.63 4.41 10.21

Compound 5c: R=R1=Benzo, R2=R3=H

Molecular Formula C38H24N4O2
Yield 65%
M. p. 230˚C
Analysis %C %H %N
Found 80.21 4.2 9.77
Calculated 80.27 4.25 9.85

Compound 5d: R=R1=R2=H, R3=CH3

Molecular Formula C35H24N4O2
Yield 66%
M. p. 207°C-209°C
Analysis %C %H %N
Found 78.88 4.48 10.46
Calculated 78.93 4.54 10.52

Compound 5e: R=R1=H, R2=OCH3, R3=CH3

Molecular Formula C36H26N4O3
Yield 72%
M. p. 237°C
Analysis %C %H %N
Found 76.82 4.6 9.91
Calculated 76.85 4.66 9.96

Compound 5e: R=R1=H, R2=OCH3, R3=CH3

Molecular Formula C39H26N4O2
Yield 74%
M. p. 236°C-238°C
Analysis %C %H %N
Found 80.35 4.43 9.58
Calculated 80.39 4.5 9.62

The structures of all the compounds (4a-f) and (5a-f) were confirmed by analytical and spectral data.

Thus, the reaction of 3-(3-(1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)acryloyl)coumarin 1a with phenacyl pyridinium bromide salt 3a in the presence of ammonium acetate in refluxing acetic acid gave a compound 4a as a yellow solid product in 68% yield.

The IR spectrum of 4a showed a strong band at 1729 cm-1 which is due to carbonyl stretching of δ-lactone ring present in coumarin nucleus. The bands observed at 1596 and 1499 cm-1 are due to aromatic C=C and C=N stretching vibrations respectively. The band observed at 3031 cm-1 is due to aromatic C-H stretching vibrations. The sharp bands observed at 691 and 751 cm-1 are due to C-H out of plane bending vibrations for mono substituted benzene ring.

The 1H-NMR spectrum of compound 4a (in CDCl3) showed twenty two protons in the region 7.26-9.00 δ. The C4 proton of coumarin ring appeared as a singlet in the most down field region at 9.00 δ. This proton appears in the most down field region due to peri effect of nitrogen of the pyridine nucleus present at 3rd position of coumarin. The C2′′′ proton of pyridine nucleus which is attached to pyrazolyl ring appeared as a meta coupled doublet at 8.94 δ (J=2.0 Hz). The C6′′′ proton of this pyridine appeared as doublet of a doublet at 8.64 δ (J=4.6 and 1.6 Hz). The C2′′′ and C6′′′ protons appeared in the down field region due to their attachment to the carbons which are directly attached to N1′′′-atom. The C3′ proton of pyridine ring attached to coumarin appeared as meta coupled doublet at 8.48 δ (J=1.2 Hz). The C5′′ proton of pyrazole ring appeared as a sharp singlet at 8.34. The remaining other seventeen protons appeared as a multiplet between 7.26-7.97 δ.

The 13C-NMR spectrum of compound 4a (in CDCl3) showed signals at 102.33, 110.66, 116.16, 118.16, 118.18, 119.29, 120.96, 124.03, 125.43, 126.26, 127.37, 127.64, 127.99, 128.33, 128.79, 129.24, 129.30, 130.33, 132.91, 133.04, 134.49, 143.62, 145.83, 147.28, 147.55, 147.95, 153.60, 154.95, 156.44 and 161.95 δ. The compound is having thirty types of non-equivalent carbon atoms and hence expected number of signals is observed. The most downfield signal appeared at 161.95 δ can be assigned to the carbonyl carbon of the δ-lactone ring of coumarin. The DEPT-90 spectrum of compound 4a (in CDCl3) showed signals at 110.62, 116.96, 118.11, 118.29, 119.96, 124.03, 125.44, 126.26, 127.37, 127.62, 127.97, 128.34, 129.20, 129.36, 130.53, 145.83, 147.55, and 147.95 δ which are due to eighteen tertiary carbons.

The mass spectrum of compound 4a showed M+ peak at 518(75%) (m/z %) along with some other fragments peaks at 474 (100%), 476 (23%), 248 (52%), 220 (28%), 77 (71%), 44 (25%), etc. The appearance of molecular ion peak at 518 mass units supports the structure of compound 4a.

The IR and NMR data for other compounds (4b-f) and (5a-f) are given below (Tables 2-12).

IR (cm-1) νmax 1719 (C=O stretching of d-lactone of coumarin), 1600 and 1477 (aromatic C=C and C=N stretching), 3057 (aromatic C-H stretching), 758 and 693 (C-H bending vibrations of mono substituted benzene ring), 2935 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 3.87 (3H, singlet, OCH3), 7.17-7.98 (16H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.35 (1H, singlet, C5′′ proton of pyrazole ring), 8.50 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.66 (1H, doublet of the doublet, proton at C6′′′, J=4.6 Hz and 1.6 Hz), 8.95 (1H, meta coupled doublet, proton at C2′′′, J=2.0 Hz), 9.01 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 55.85 (OCH3), 102.34 (C), 110.65 (CH), 113.96 (CH), 118.10 (CH), 119.96 (CH), 120.29 (CH), 123.20 (C), 124.00 (CH), 126.29 (CH), 126.42 (CH), 127.37 (CH), 127.69 (CH), 128.71 (C), 129.20 (CH), 129.37 (CH), 130.50 (CH), 132.96 (C), 133.00 (C), 134.00 (CH), 134.42 (C), 140.65 (C), 143.65 (C), 145.86 (CH), 147.42 (C), 147.50 (CH), 147.91 (CH), 148.59 (C), 154.99 (C), 156.45 (C), 160.95 (CO of coumarin).

Table 2: IR and NMR data for Compound 4b

IR (cm-1) νmax 1727 (C=O stretching of d-lactone of coumarin), 1595 and 1498 (aromatic C=C and C=N stretchings), 3049 (aromatic C-H stretching), 757 and 689 (C-H bending vibrations of mono substituted benzene ring), 2926 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 7.26-8.22 (19H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.34 (1H, singlet, C5′′ proton of pyrazole ring), 8.49 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.84 (1H, doublet of the doublet, proton at C6′′′, J=4.6 Hz and 1.6 Hz), 9.06 (1H, meta coupled doublet, proton at C2′′′, J=2.0 Hz), 9.87 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 102.34 (C), 110.62 (CH), 115.54 (CH), 117.73 (C), 118.18 (CH), 119.97 (CH), 122.23 (CH), 123.58 (CH), 124.02 (CH), 126.30 (CH), 126.84 (CH), 127.36 (CH), 127.64 (CH), 128.49 (CH), 128.74 (C), 128.86 (C), 129.25 (CH), 129.35 (CH), 130.14 (CH), 130.28 (C), 130.47 (CH), 132.95 (C), 133.00 (C), 134.00 (CH), 134.46 (C), 143.64 (C), 145.77 (CH), 147.38 (C), 147.49 (CH), 147.88 (CH), 150.65 (C), 154.96 (C), 156.43 (C), 160.94 (CO of coumarin).

Table 3: IR and NMR data for Compound 4c

IR (cm-1) νmax 1719 (C=O stretching of d-lactone of coumarin), 1600 and 1477 (aromatic C=C and C=N stretchings), 3057 (aromatic C-H stretching), 758 and 693 (C-H bending vibrations of mono substituted benzene ring), 815 (C-H bending vibrations of p-disubstituted benzene ring), 2935 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 2.43 (3H, singlet, CH3), 7.26-8.12 (16H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.32 (1H, singlet, C5′′ proton of pyrazole ring), 8.59 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.75 (1H, doublet of the doublet, proton at C6′′′, J=4.6 Hz and 1.6 Hz), 9.05 (1H, meta coupled doublet, proton at C2′′′, J=2.0 Hz), 9.10 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.34 (CH3), 102.32 (C), 110.62 (CH), 116.13 (CH), 118.18 (CH), 119.91 (CH), 120.98 (CH), 123.31 (CH), 124.00 (CH), 125.41 (CH), 126.23 (CH), 127.92 (CH), 128.31 (C), 128.73 (CH), 129.36 (CH), 129.51 (CH), 130.32 (C), 130.54 (CH), 132.97 (C), 133.09 (C), 134.01 (C), 136.04 (C), 143.61 (C), 145.88 (CH), 147.44 (C), 147.53 (CH), 147.91 (CH), 153.00 (C), 154.92 (C), 156.43 (C), 161.94 (CO of coumarin).

Table 4: IR and NMR data for Compound 4d

IR (cm-1) νmax 1726 (C=O stretching of d-lactone of coumarin), 1596 and 1477 (aromatic C=C and C=N stretchings), 3033 (aromatic C-H stretching), 742 and 695 (C-H bending vibrations of mono substituted benzene ring), 827 (C-H bending vibrations of p-disubstituted benzene ring), 2929 (aliphatic C-H stretching),
1H-NMR (d, ppm) (CDCl3) 2.43 (3H, singlet, CH3), 3.88 (3H, singlet, OCH3), 7.14-8.12 (15H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.32 (1H, singlet, C5′′ proton of pyrazole ring), 8.42 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.58 (1H, doublet of the doublet, proton at C6′′′, J=4.6 Hz and 1.6 Hz), 8.87 (1H, meta coupled doublet, proton at C2′′′, J=2.0 Hz), 8.93 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.37 (CH3), 55.84 (OCH3), 102.33 (C), 110.64 (CH), 113.64 (CH), 118.09 (CH), 119.95 (CH), 120.28 (CH), 123.21 (C), 123.36 (CH), 124.00 (CH), 126.30 (CH), 126.43 (CH), 128.72 (CH), 129.36 (C), 129.49 (CH), 130.32 (C), 130.52 (CH), 132.96 (C), 133.00 (C), 134.01 (CH), 136.03 (C), 140.63 (C), 143.64 (C), 145.85 (CH), 147.43 (C), 147.49 (CH), 147.89 (CH), 148.60 (C), 154.90 (C), 156.43 (C), 160.93 (CO of coumarin).

Table 5: IR and NMR data for Compound 4e

IR (cm-1) νmax 1726 (C=O stretching of d-lactone of coumarin), 1599 and 1457 (aromatic C=C and C=N stretchings), 3067 (aromatic C-H stretching), 755 and 689 (C-H bending vibrations of mono substituted benzene ring), 832 (C-H bending vibrations of p-disubstituted benzene ring), 2927 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 2.43 (3H, singlet, CH3), 7.26-8.22 (18H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.35 (1H, singlet, C5′′ proton of pyrazole ring), 8.49 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.83 (1H, doublet of the doublet, proton at C6′′′, J=4.6 Hz and 1.6 Hz), 9.06 (1H, meta coupled doublet, proton at C2′′′, J=2.0 Hz), 9.87 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.21 (CH3), 102.34 (C), 110.63 (CH), 115.48 (CH), 117.72 (C), 118.17 (CH), 119.97 (CH), 122.19 (CH), 123.34 (CH), 123.64 (CH), 124.01 (CH), 126.23 (CH), 126.83 (CH), 128.54 (CH), 128.74 (C), 128.85 (C), 129.36 (CH), 129.55 (CH), 130.29 (CH), 130.36 (C), 130.46 (C), 130.47 (CH), 132.96 (C), 133.00 (C), 134.07 (CH), 136.04 (C), 143.63 (C), 145.86 (CH), 147.39 (C), 147.52 (CH), 147.92 (CH), 150.64 (C), 154.97 (C), 156.43 (C), 160.87 (CO of coumarin).

Table 6: IR and NMR data for Compound 4f

IR (cm-1) νmax 1727 (C=O stretching of d-lactone of coumarin), 1598 and 1499 (aromatic C=C and C=N stretchings), 3066 (aromatic C-H stretching), 756 and 691 (C-H bending vibrations of mono substituted benzene ring), 2928 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 7.26-7.96 (17H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.31 (1H, singlet, C5′′ proton of pyrazole ring), 8.50 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.64 (2H, ortho coupled doublet, protons at C2′′′ and C6′′′, J=4.4 Hz), 9.03 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 102.34 (C), 110.61 (CH), 116.18 (CH), 118.20 (CH), 119.96 (CH), 120.99 (CH), 125.36 (CH), 125.43 (C), 126.28 (CH), 127.33 (CH), 127.68 (CH), 127.98 (CH), 128.28 (CH), 128.70 (C), 129.25 (CH), 129.36 (CH), 130.49 (CH), 132.95 (C), 134.48 (C), 140.34 (C), 145.64 (C), 145.85 (CH), 147.40 (CH), 153.63 (C), 153.79 (C), 154.97 (C), 156.43 (C), 160.92 (CO of coumarin).

Table 7: IR and NMR data for Compound 5a

IR (cm-1) νmax 1718 (C=O stretching of d-lactone of coumarin), 1598 and 1478 (aromatic C=C and C=N stretchings), 3049 (aromatic C-H stretching), 758 and 729 (C-H bending vibrations of mono substituted benzene ring), 2935 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 3.88 (3H, singlet, OCH3), 7.16-7.96 (16H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.31 (1H, singlet, C5′′ proton of pyrazole ring), 8.51 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.65 (2H, ortho coupled doublet, protons at C2′′′ and C6′′′, J=4.4 Hz), 9.01 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 55.78 (OCH3), 102.33 (C), 110.27 (CH), 113.95 (CH), 118.09 (CH), 119.96 (CH), 120.31 (CH), 121.36 (CH), 123.21 (C), 126.20 (CH), 126.43 (CH), 127.36 (CH), 127.69 (CH), 128.72 (C), 129.19 (CH), 129.36 (CH), 130.50 (CH), 132.89 (C), 134.42 (C), 140.37 (C), 140.64 (C), 143.64 (C), 145.79 (CH), 147.43 (C), 148.58 (C), 149.84 (CH), 155.01 (C), 156.43 (C), 161.03 (CO of coumarin).

Table 8: IR and NMR data for Compound 5b

IR (cm-1) νmax 1735 (C=O stretching of d-lactone of coumarin), 1598 and 1406 (aromatic C=C and C=N stretchings), 3074 (aromatic C-H stretching), 756 and 690 (C-H bending vibrations of mono substituted benzene ring), 2927 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 7.26-7.86 (19H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.30 (1H, singlet, C5′′ proton of pyrazole ring), 8.49 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.62 (2H, ortho coupled doublet, protons at C2′′′ and C6′′′, J=4.4 Hz), 9.81 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 102.34 (C), 110.58 (CH), 115.48 (CH), 117.74 (C), 118.27 (CH), 119.98 (CH), 121.34 (CH), 122.23 (CH), 123.60 (CH), 126.23 (CH), 126.83 (CH), 127.34 (CH), 127.66 (CH), 128.54 (CH), 128.74 (C), 128.78 (C), 129.25 (CH), 129.36 (CH), 130.12 (C), 130.30 (CH), 130.50 (CH), 132.96 (C), 134.48 (C), 140.30 (C), 143.64 (C), 145.86 (CH), 147.41 (C), 149.87 (CH), 150.55 (C), 154.96 (C), 156.44 (C), 160.89 (CO of coumarin).

Table 9: IR and NMR data for Compound 5c

IR (cm-1) νmax 1726 (C=O stretching of d-lactone of coumarin), 1599 and 1490 (aromatic C=C and C=N stretchings), 3067 (aromatic C-H stretching), 755 and 689 (C-H bending vibrations of mono substituted benzene ring), 832 (C-H bending vibrations of p-disubstituted benzene ring), 2927 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 2.43 (3H, singlet, CH3), 7.26-7.96 (16H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.32 (1H, singlet, C5′′ proton of pyrazole ring), 8.51 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.65 (2H, ortho coupled doublet, protons at C2′′′ and C6′′′, J=4.8 Hz), 9.03 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.29 (CH3), 102.29 (C), 110.56 (CH), 116.20 (CH), 118.20 (CH), 119.94 (CH), 120.99 (C), 121.33 (CH), 123.32 (CH), 125.42 (CH), 126.31 (CH), 127.96 (CH), 128.28 (CH), 128.73 (C), 129.34 (CH), 129.57 (CH), 130.38 (C), 130.46 (CH), 132.49 (C), 136.00 (C), 140.34 (C), 143.65 (C), 145.85 (CH), 147.44 (C), 149.78 (CH), 153.63 (C), 154.99 (C), 156.45 (C), 160.94 (CO of coumarin).

Table 10: IR and NMR data for Compound 5d

IR (cm-1) νmax 1726 (C=O stretching of d-lactone of coumarin), 1594 and 1477 (aromatic C=C and C=N stretchings), 3057 (aromatic C-H stretching), 776 and 695 (C-H bending vibrations of mono substituted benzene ring), 827 (C-H bending vibrations of p-disubstituted benzene ring), 2935 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 2.44 (3H, singlet, CH3), 3.88 (3H, singlet, OCH3), 7.12-7.95 (15H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.30 (1H, singlet, C5′′ proton of pyrazole ring), 8.49 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.63 (2H, ortho coupled doublet, protons at C2′′′ and C6′′′, J= 4.8 Hz), 8.99 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.34 (CH3), 55.85 (OCH3), 102.33 (C), 110.58 (CH), 113.97 (CH), 118.09 (CH), 119.88 (CH), 120.34 (CH), 121.36 (CH), 123.26 (CH), 123.40 (C), 126.19 (CH), 126.44 (CH), 128.72 (C), 129.24 (C), 129.28 (CH), 129.56 (CH), 130.40 (CH), 132.87 (C), 136.05 (C), 140.35 (C), 140.64 (C), 143.65 (C), 145.87 (CH), 147.38 (C), 148.58 (C), 149.85 (CH), 154.98 (C), 156.44 (C), 160.97 (CO of coumarin).

Table 11: IR and NMR data for Compound 5e

IR (cm-1) νmax 1735 (C=O stretching of d-lactone of coumarin), 1508 and 1406 (aromatic C=C and C=N stretchings), 3074 (aromatic C-H stretching), 756 and 690 (C-H bending vibrations of mono substituted benzene ring), 824 (C-H bending vibrations of p-disubstituted benzene ring), 2927 (aliphatic C-H stretching).
1H-NMR (d, ppm) (CDCl3) 2.44 (3H, singlet, CH3), 7.26-7.9.81 (18H, multiplet, aromatic protons except protons at C4, C3′, C5′′, C2′′′ and C6′′′), 8.31 (1H, singlet, C5′′ proton of pyrazole ring), 8.48 (1H, meta coupled doublet, proton at C3′, J=1.2 Hz), 8.63 (2H, ortho coupled doublet, proton at C2′′′ and C6′′′, J=5.6 Hz), 9.81 (1H, singlet, C4 proton of coumarin ring).
13C-NMR (d, ppm) (CDCl3) 21.32 (CH3), 102.34 (C), 110.61 (CH), 115.54 (CH), 117.76 (C), 118.27 (CH), 119.96 (CH), 121.47 (CH), 122.34 (CH), 123.30 (CH), 123.61 (CH), 126.30 (CH), 126.83 (CH), 128.49 (CH), 128.78 (C), 128.89 (C), 129.35 (CH), 129.56 (CH), 129.87 (C), 130.17 (CH), 130.30 (C), 130.35 (CH), 130.47 (C), 132.89 (C), 136.03 (C), 143.31 (C), 145.85 (CH), 147.39 (C), 149.88 (CH), 150.54 (C), 154.96 (C), 156.45 (C), 160.95 (CO of coumarin).

Table 12: IR and NMR data for Compound 5f

The newly synthesized target compounds 4a-f and 5a-f were evaluated for their in vitro antibacterial activity against two Gram positive bacteria Staphylococcus aureus (MTCC 96) and Bacillus subtilis (MTCC 441) and two Gram negative bacteria Escherichia coli (MTCC 443) and Salmonella typhi (MTCC 98). They were also evaluated for their in vitro antifungal activity against Candida albicans (MTCC 227) and Aspergillus niger (MTCC 282) as fungal strains. Broth dilution method was used for the determination of the antibacterial and antifungal activity as recommended by NCCLS [18]. Ampicillin, Chloramphenicol and Norfloxacin were used as standard antibacterial drugs, whereas Griseofulvin and Nystatin were used as standard antifungal drugs. All MTCC cultures were collected from Institute of Microbial Technology, Chandigarh and tested against above mentioned known drugs. Mueller-Hinton broth was used as the nutrient medium for the test bacteria and Sabouraud Dextrose broth was used for the test fungi. Inoculum size for the test strains was adjusted to 108 CFU (Colony Forming Unit per milliliter) per milliliter by comparing the turbidity. Each synthesized compound was diluted with DMSO so as to have the stock solution of 2000 μg/ml concentration as a stock solution. The results were recorded in the form of primary and secondary screening. The synthesized compounds (5a-r) were screened for their antibacterial and antifungal activity at the concentration of 1000, 500 and 250 μg/ml for the primary screening. The synthesized compound showing activity against microbes in the primary screening were further screened in a second set of dilution at concentrations of 200, 100, 62.5, 50 and 25 μg/ml. The suspension of 10 μl from each were further well incubated and growth was noted at 37°C after 24 h for bacteria and 48 hour for fungi. The lowest concentration which showed no visible growth (turbidity) after spot subculture was considered as the minimum inhibitory concentration (MIC) for each compound.

The investigation of the data summarized in (Table 13) reveals that many compounds were found to be active against Gram-positive bacteria while some of the compounds were found to be active against Gram-negative bacterial and fungal species as compared to that of the standard antimicrobial drugs.

Compound Minimum Inhibitory Concentration (MIC, μgml-1)
Gram +ve bacteria Gram –ve bacteria Fungi
Bacillus subtillus Staphylocossus aureus Escherichia coli Salmonella typhii Asperigillus niger Candida albicans
4a 250 250 250 200 >1000 250
4b 100 100 62.5 100 >1000 1000
4c 200 250 100 125 >1000 1000
4d 250 200 250 200 >1000 >1000
4e 100 100 200 125 500 500
4f 125 100 250 200 250 >1000
5a 125 200 250 250 500 >1000
5b 62.5 200 100 100 >1000 250
5c 250 250 125 200 1000 1000
5d 200 125 200 250 >1000 500
5e 200 200 100 100 >1000 1000
5f 125 125 250 500 >1000 500
Ampicillin 250 250 100 100 - -
Chloramphenicol 50 50 50 50 - -
Ciprofloxacin 50 50 25 25 - -
Norfloxacin 100 10 10 10 - -
Gentamycin 0.5 0.25 0.05 1 - -
Griseofulvin - - - - 100 500
Nystatin - - - - 100 100

Table 13: Estimation of the minimum inhibitory concentration (MIC)

The assessment of antimicr.obial screening data reveals that almost all the compounds 4a-f and 5a–f, exerted significant inhibitory activity against gram positive bacteria compared to Ampicillin (MIC=250 μg/ml) against Bacillus subtilis. Compounds 5b (MIC = 62.5 μg/ml), 4b and 4e (MIC=100 μg/ml) exhibited excellent activity compared to Ampicillin (MIC=250 μg/ml) and comparable activity to Norfloxacin (MIC=100 μg/ml) against Bacillus subtilis. Compounds 4f, 5a, and 5f (MIC=125 μg/ml) displayed better inhibition against B. subtilis compared to Ampicillin (MIC=250 μg/ml). Compounds 4c, 5d, and 5e (MIC=200 μg/ml) displayed comparable activity to Ampicillin (MIC=250 μg/ml). Compounds 4a and 4d (MIC=250μg/ml) were found to have equipotent activity compared to Ampicillin (MIC=250 μg/ml).

Compounds 4b, 4e and 4f (MIC=100 μg/ml), 5d and 5f (MIC=125 μg/ml) were found to be more potent against S. aureus compared to Ampicillin (MIC=250 μg/ml). Compounds 5d and 5f (MIC=200 μg/ml) were found to be more potent against gram positive bacteria S. aureus compared to Ampicillin (MIC=250 μg/ml). Compounds 4a, 4c and 5c (MIC=250 μg/ml) were found to have equipotent activity compared to Ampicillin (MIC=250 μg/ml).

Compounds 4b (MIC=62.5 μg/ml) exerted excellent inhibition against gram negative bacteria E. coli compared to Ampicillin (MIC=100 μg/ml) whereas compounds 4c and 5b (MIC=100 μg/ml) were found to be equipotent against E. coli compared to Ampicillin (MIC=100 μg/ml).

Compounds 4b, 5b and 5e (MIC=100 μg/ml) showed equal inhibition to Ampicillin against gram negative bacteria S. typhi.

Compounds 4a and 5b (MIC=250 μg/ml) were found to be more active than Griseofulvin (MIC=500 μg/ml) whereas, Compounds 4e, 5d and 5f (MIC=500 μg/ml) were found to be equipotent to Griseofulvin (MIC=500 μg/ml) against C. albicans. None of the tested compounds showed better activity than standard drugs against A. niger.

Among the compounds 4a-f and 5a-f compounds 4b and 5b exhibited excellent inhibitory activity than other derivatives. The enhanced activity of the above compounds can be attributed to the presence of -OCH3 group in coumarin ring. All the compounds exhibited excellent activity against B. subtilis and S. aureus compared to the standard drugs. Examining the antimicrobial data, it has been observed that the derivatization of the parent molecule moderately changed the antimicrobial potency of the synthesized analogs. Thus compounds 4b and 5b were found to be the most efficient members of the series.

Conclusion

From present study, we summarized that employed synthetic strategy provide efficient route for the synthesis of 3-{4'-[1''-phenyl-3''-(pyridin-3''' -yl)-1H-pyrazol-4''-yl]-6'-aryl-pyridin-2'-yl} coumarins (4a-f) and 3-{4'-[1''-phenyl-3''-(pyridin-4'''-yl)-1H-pyrazol-4''-yl]-6'-aryl-pyridin-2'-yl}coumarins (5a-f) by Krohnke’s protocol in good yield. Moreover the starting precursors were also easy to prepare from synthesis point of view. Antimicrobial study on target compounds concluded that the all the compounds exerted promising activity against gram positive bacteria and gram negative. The target compounds showed feeble activity against fungal pathogens. Compounds 4b and 5b were found to be the most efficient members of the series.

Acknowledgement

We sincerely thank Principal and management of SSR College of Arts, Commerce and Science and HOD Chemistry Department for providing laboratory facilities.

References

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