Journal of Chemistry

Volume 2013 (2013), Article ID 741953, 9 pages

http://dx.doi.org/10.1155/2013/741953

Research Article

Ultrasonication-Induced Synthesis and Antimicrobial Evaluation of Some Multifluorinated Pyrazolone Derivatives

Anil Gadhave,¹ Shashikant Kuchekar,¹ and Bhausaheb Karale²

¹P. G. Department of Chemistry and Research Centre, Padmashri Vikhe Patil College, Pravaranagar, Ahmednagar 413713, India

²Department of Chemistry, Radhabai Kale Mahila Mahavidyalaya, Ahmednagar 414001, India

Received 6 June 2012; Revised 6 August 2012; Accepted 28 August 2012

Academic Editor: Qing Li

Copyright © 2013 Anil Gadhave et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A series of novel fluorine containing pyrazole-pyrazolone (4a–j) and chromone-pyrazolone (5a–i) was synthesized from multifluorinated pyrazolone by the Knoevenagel condensation reaction. All compounds were synthesized by conventional heating as well as ultrasound irradiation technique. It was found that ultrasonication method was more efficient than conventional heating method. The newly synthesized compounds were subjected for in vitro antimicrobial screening against four bacterial pathogens, namely, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa and three fungal pathogens Candida albicans, Aspergillus niger, and Aspergillus clavatus, using broth microdilution (MIC) method (CLSI guidelines). Among them, some compounds exhibited promising antibacterial activity against the tested strains. All synthesized compounds were characterized by IR, ¹H-NMR, mass, and elemental analysis.

1. Introduction

An alarming increment in pathogenic resistance to existing drugs is a serious problem with antimicrobial therapy, and therefore it is very important to search for new class of antimicrobial agents [1]. The introduction of fluorine in organic molecules may lead to significant influence on the biological and physical properties of compounds due to increase of membrane permeability, hydrophobic binding, and stability against metabolic oxidation [2]. As a consequence of key role of fluorine in medicinal chemistry, the interest to incorporate fluorine to prepare biologically active compounds is increased during the past decades. The compounds containing fluorine or trifluoromethyl substituents such as fluoxetine and Prozac are well-known antidepressant drugs (Figure 1). Fluoxetine as well as the related antidepressants functions as selective serotonin reuptake inhibitors (SSRIs).

Figure 1

Pyrazolone is a key heterocyclic moiety present in numerous organic compounds because they possess antifungal [3], antibacterial [4, 5], antimycobacterial [6, 7], anti-inflammatory [8, 9], antitumor [10], gastric secretion stimulatory [11], antidepressant [12], and antifilarial [13] activities. They inhibit the production of TNF-α and decrease levels of proinflammatory cytokines and thereby reduce inflammation and prevent further tissue destruction in disease as rheumatoid arthritis, osteoarthritis, and crohns’ disease [14–16]. Pyrazolone-containing drugs such as phenazone, propyphenazone, ampyrone, and metamizole are useful antipyretic and analgesic drugs [17], while edaravone (MCI-186) has been used for treating brain [18, 19] and myocardial ischemia [20] (Figure 1).

Pyrazole derivatives are known to possess wide spectrum of biological activities such as antibacterial [21–23], antifungal [24, 25], antidiabetic [26], herbicidal [27, 28], and antianxiety [29], and it is present as an active pharmacophore in celecoxib which is a COX-2 inhibitor [30] and sildenafil citrate as cGMP-specific phosphodiesterase type-5 inhibitors [31]. The chromone is an important oxygen containing heterocycle present in numerous naturally occurring compounds exhibiting interesting biological activities such as antiviral [32], anticancer [33], anti-inflammatory [34, 35], and antioxidant [36, 37] activities.

Knoevenagel condensation reaction [38] is an important tool for construction of new carbon-carbon bonds. It is used for synthesis of different pharmaceutical products [39, 40] by using acidic and basic condition. Ultrasonication has increasingly been used for organic synthesis in the last three decades. It has been demonstrated as an alternative energy source for organic reactions ordinarily accomplished by heating. A large number of organic reactions have been carried out in higher yields, shorter reaction time, and milder conditions under ultrasound [41–44].

In view of broad spectrum of biological activities associated with pyrazolone, pyrazole, chromone nucleus, high potential of fluorine-containing compounds, and advantageous use of ultrasonication technique and in continuation to our ongoing efforts for search of biologically important heterocycles [45–47], it was envisaged to construct a system which combines all these pharmacophores in a single molecular framework to explore the additive effects towards their biological activities. Herein we wish to report green synthesis and antimicrobial evaluation of some multifluorinated pyrazolone derivatives containing pyrazole and chromone nucleus. The general route for synthesis of multifluorinated pyrazolone derivatives is outlined in Scheme 1.

Scheme 1: Synthesis of multifluorinated pyrazolone containing pyrazole and chromone.

2. Experimental

2.1. Chemistry

Melting points were taken in open capillaries and are uncorrected. The IR spectra were recorded in KBr on a Shimadzu FTIR 8400 spectrophotometer, and only characteristic peaks are reported in cm⁻¹. The ¹H-NMR spectra were recorded in DMSO- on a Bruker Avance spectrometer using TMS as an internal standard at 400 MHz, and chemical shifts are reported in parts per million (ppm). Mass spectra were scanned on a Finnigan mass spectrometer. Elemental analysis was performed on a Perkin-Elmer analyzer. Thin-layer chromatography (TLC, on aluminium plates coated with silica gel 60F254, 0.25 mm thickness, Merck) was used for monitoring the progress of reactions, purity, and homogeneity of the synthesized compounds. UV radiation and iodine were used as the visualizing agents. Experiment under ultrasound irradiation was carried out in ultrasonic cleaner model EN-20U-S manufactured by Enertech Electronics Pvt. Ltd., Mumbai, India, has maximum output of 100 W and 33 KHz operating frequency.

2.2. General Procedure for Synthesis of Pyrazole-Pyrazolone (4a–j) and Chromone-Pyrazolone (5a–i)

2.2.1. Method (A) Conventional Method

Fluorinated pyrazolone 3 (0.001 mole) and 4-formyl pyrazole or 3-formyl chromone (0.001 mole) were taken in 10 mL glacial acetic acid in 50 mL single neck round bottom flask equipped with condensor. The reaction mixture was heated in an oil bath at 100°C till completion of reaction (checked by TLC). After completion of reaction, contents were allowed to cool and then poured into crushed ice. Solid obtained was separated by filtration and purified by recrystallization from acetic acid to get pure compounds. The formation of compounds was confirmed by spectral techniques. The data of synthesized compounds is given in Tables 1 and 2.

Table 1: Physical data of synthesized pyrazole-pyrazolone (4a–j).

Compound
R
R₁
M.P. (°C)
Conventional method
Ultrasound method

Time (min)
Yield (%)
Time (min)
Yield (%)

4a
Propyl
C₆H₅
151-152
60
75
16
90

4b
Propyl
4-FC₆H₄
179-180
60
71
15
91

4c
Propyl
4-MeC₆H₄
192-193
60
69
18
88

4d
Propyl
4-ClC₆H₄
159-160
60
73
16
85

4e
Propyl
2-Thienyl
149-150
60
66
19
90

4f
CF₃
C₆H₅
237-238
60
78
21
86

4g
CF₃
4-FC₆H₄
235-236
60
74
19
93

4h
CF₃
4-MeC₆H₄
253-254
60
71
18
89

4i
CF₃
4-ClC₆H₄
216-217
60
76
17
94

4j
CF₃
2-Thienyl
215-216
60
69
16
90

Table 2: Physical data of synthesized chromone-pyrazolone (5a–i).

Compound
R
R₁
R₂
R₃
M. P. (°C)
Conventional method
Ultrasound method

Time (min)
Yield (%)
Time (min)
Yield (%)

5a
Propyl
H
H
H
144-145
60
70
14
81

5b
Propyl
H
H
Me
146-147
60
68
16
80

5c
Propyl
Cl
H
Cl
194-195
60
69
13
82

5d
Propyl
H
H
Cl
166-167
60
72
17
78

5e
Propyl
H
Me
Cl
241-242
60
75
12
82

5f
CF₃
H
H
H
222-223
60
71
14
85

5g
CF₃
H
H
Me
238-239
60
66
15
81

5h
CF₃
Cl
H
Cl
212-213
60
69
12
82

5i
CF₃
H
H
Cl
208-209
60
71
16
84

2.2.2. Method (B) Ultrasound Method

Fluorinated pyrazolone 3 (0.001 mole) and 4-formyl pyrazole or 3-formyl chromone (0.001 mole) were taken in 10 mL glacial acetic acid in 50 mL round bottom flask. Contents of flask were subjected for ultrasound irradiation for time as shown in Tables 1 and 2, till completion of reaction (checked by TLC). After completion of reaction, contents were poured into crushed ice. Solid obtained was separated by filtration and purified by recrystallization from acetic acid to get pure compounds. The formation of compounds was confirmed by mp, mixed mp, and spectral techniques. The physical data of synthesized compounds is given in Tables 1 and 2.

(4Z)-4-[(1,3-Diphenyl-1H-pyrazol-4-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4a). Yellow solid; IR (, cm⁻¹): 1688 (C=O lactum), 1595 (C=C), 1515 (C=N), 1121 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.01–1.04 (t, 3H, J = 12 Hz), 1.71–1.79 (m, 2H), 2.56–2.60 (t, 2H, J = 16 Hz), 7.13–7.18 (m, 1H, Ar–H), 7.35–7.39 (m, 1H, Ar–H), 7.46–7.51 (m, 2H, Ar–H), 7.54–7.60 (m, 4H, Ar–H), 7.66–7.69 (m, 2H, Ar–H), 7.87–7.90 (m, 2H, one proton Ar–H & one pyrazole proton), 10.16 (s, 1H, olefinic proton); MS: m/z 504 (M⁺); Anal. Calcd for C₂₈H₂₀F₄N₄O: C, 66.66; H, 4.00; N, 11.11. Found: C, 66.71; H, 3.92; N, 11.04.

(4Z)-4-{[3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4b). Yellow solid; IR (, cm⁻¹): 1697 (C=O lactum), 1580 (C=C), 1521 (C=N), 1133 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.01–1.05 (t, 3H, J = 16 Hz), 1.73–1.79 (m, 2H), 2.56–2.60 (t, 2H, J = 16 Hz), 7.13–7.18 (m, 1H, Ar–H), 7.25–7.29 (m, 2H, Ar–H), 7.36–7.40 (m, 1H, Ar–H), 7.47–7.52 (m, 3H, Ar–H), 7.64–7.68 (m, 2H, Ar–H), 7.86–7.89 (m, 2H, one proton Ar–H & one pyrazole proton), 10.15 (s, 1H, olefinic proton); MS: m/z 522 (M⁺); Anal. Calcd for C₂₈H₁₉F₅N₄O: C, 64.37; H, 3.67; N, 10.72. Found: C, 64.50; H, 3.61; N, 10.65.

(4Z)-4-{[3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4c). Yellow solid;  IR (, cm⁻¹): 1693 (C=O lactum), 1597 (C=C), 1505 (C=N), 1114 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 0.99–1.03 (t, 3H, J = 16 Hz), 1.72–1.77 (m, 2H), 2.46 (s, 3H, Ar–CH₃), 2.56–2.58 (t, 2H, J = 8 Hz), 7.09–7.17 (m, 1H, Ar–H), 7.35–7.38 (m, 3H, Ar–H), 7.44–7.48 (m, 2H, Ar–H), 7.54–7.56 (d, 2H, J = 8 Hz, Ar–H), 7.59 (s, 1H, pyrazole proton), 7.85–7.87 (d, 2H, J = 8 Hz, Ar–H), 10.13 (s, 1H, olefinic proton); MS: m/z 518 (M⁺); Anal. Calcd for C₂₉H₂₂F₄N₄O: C, 67.18; H, 4.28; N, 10.81. Found: C, 67.11; H, 4.37; N, 10.89.

(4Z)-4-{[3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4d). Yellow solid; IR (, cm⁻¹): 1681 (C=O lactum), 1583 (C=C), 1526 (C=N), 1125 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.02–1.06 (t, 3H, J = 16 Hz), 1.74–1.79 (m, 2H), 2.57–2.60 (t, 2H, J = 12 Hz), 7.13–7.18 (m, 1H, Ar–H), 7.36–7.40 (m, 1H, Ar–H), 7.47–7.52 (m, 3H, Ar–H), 7.54–7.56 (m, 2H, Ar–H), 7.62–7.63 (m, 2H, one proton Ar–H & one pyrazole proton), 7.86–7.88 (m, 2H, Ar–H), 10.16 (s, 1H, olefinic proton); MS: m/z 538 (M⁺); Anal. Calcd for C₂₈H₁₉ClF₄N₄O: C, 62.40; H, 3.55; N, 10.40. Found: C, 62.30; H, 3.61; N, 10.35.

(4Z)-4-{[1-Phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]methylene}-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (4e). Yellow solid; IR (, cm⁻¹): 1688 (C=O lactum), 1603 (C=C), 1509 (C=N), 1101 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.05–1.08 (t, 3H, J = 12 Hz), 1.79–1.85 (m, 2H), 2.65–2.68 (t, 2H, J = 12 Hz), 7.13–7.18 (m, 1H, Ar–H), 7.24–7.26 (m, 1H, Ar–H), 7.35–7.38 (m, 1H, Ar–H), 7.45–7.51 (m, 3H, Ar–H), 7.54–7.55 (m, 1H, Ar–H), 7.83 (s, 1H, pyrazole proton), 7.86–7.88 (m, 2H, Ar–H), 10.16 (s, 1H, olefinic proton); MS: m/z 510 (M⁺); Anal. Calcd for C₂₆H₁₈F₄N₄OS: C, 61.17; H, 3.55; N, 10.97. Found: C, 61.29; H, 3.49; N, 10.85.

(4Z)-4-[(1,3-Diphenyl-1H-pyrazol-4-yl)methylene]-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (4f). Yellow solid; IR (, cm⁻¹): 1701 (C=O lactum), 1591 (C=C), 1515 (C=N), 1112 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.20 (s, 1H, Ar–H), 7.41–7.44 (m, 1H, Ar–H), 7.46–7.57 (m, 5H, Ar–H), 7.76 (s, 2H, Ar–H), 7.81–7.92 (m, 2H, Ar–H), 7.99 (s, 1H, pyrazole proton), 10.23 (s, 1H, olefinic proton); MS: 530 (M⁺); Anal. Calcd for C₂₆H₁₃F₇N₄O: C, 58.88; H, 2.47; N, 10.56. Found: C, 58.80; H, 2.51; N, 10.50.

(4Z)-4-{[3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (4g). Yellow solid; IR (, cm⁻¹): 1691 (C=O lactum), 1609 (C=C), 1522 (C=N), 1130 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.27–7.31 (m, 3H, Ar–H), 7.42–7.52 (m, 3H, Ar–H), 7.66 (s, 2H, Ar–H), 7.88–7.91 (m, 3H, two proton Ar–H & one pyrazole proton), 10.22 (s, 1H, olefinic proton); MS: 548 (M⁺); Anal. Calcd for C₂₆H₁₂F₈N₄O: C, 56.94; H, 2.21; N, 10.22. Found: C, 56.90; H, 2.30; N, 10.28.

(4Z)-4-{[3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (4   h). Yellow solid; IR (, cm⁻¹): 1708 (C=O lactum), 1600 (C=C), 1510 (C=N), 1122 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 2.46 (s, 3H, Ar–CH₃), 7.19–7.21 (m, 1H, Ar–H), 7.35–7.40 (m, 3H, Ar–H), 7.47–7.50 (m, 2H, Ar–H), 7.54–7.56 (d, 2H, J = 8 Hz, Ar–H), 7.87–7.89 (d, 2H, J = 8 Hz, Ar–H), 7.99 (s, 1H, pyrazole proton), 10.20 (s, 1H, olefinic proton); MS: m/z 544 (M⁺); Anal. Calcd for C₂₇H₁₅F₇N₄O: C, 59.57; H, 2.78; N, 10.29. Found: C, 59.65; H, 2.70; N, 10.20.

(4Z)-4-{[3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (4i). Yellow solid; IR (, cm⁻¹): 1711 (C=O lactum), 1599 (C=C), 1501 (C=N), 1109 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.26 (s, 1H, Ar–H), 7.42–7.61 (m, 7H, Ar–H), 7.87–7.91 (m, 3H, two proton Ar–H & one pyrazole proton), 10.23 (s, 1H, olefinic proton); MS: m/z 564 (M⁺), Anal. Calcd for C₂₆H₁₂ClF₇N₄O: C, 55.29; H, 2.14; N, 9.92. Found: C, 55.21; H, 2.20; N, 9.81.

(4Z)-4-{[1-Phenyl-3-(2-thienyl)-1H-pyrazol-4-yl]methylene}-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (4j). Yellow solid; IR (, cm⁻¹): 1701 (C=O lactum), 1605 (C=C), 1505 (C=N), 1116 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.22 (s, 1H, Ar–H), 7.41–7.56 (m, 6H, Ar–H), 7.85–7.89 (m, 2H, Ar–H), 8.22 (s, 1H, pyrazole proton), 10.20 (s, 1H, olefinic proton); MS: m/z 536 (M⁺); Anal. Calcd for C₂₄H₁₁F₇N₄OS: C, 53.74; H, 2.07; N, 10.44. Found: C, 53.69; H, 2.10; N, 10.55.

(4Z)-4-[(4-Oxo-4H-chromen-3-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (5a). Yellow solid; IR (, cm⁻¹): 1700 (C=O lactum), 1659 (C=O chromone), 1623 (C=C), 1599 (C=N), 1129 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 0.99–1.02 (t, 3H, J = 12 Hz), 1.69–1.75 (m, 2H), 2.71–2.74 (t, 2H, J = 12 Hz), 7.59–7.63 (m, 1H, Ar–H), 7.79–7.81 (d, 1H, J = 8 Hz, Ar–H), 7.90–7.94 (m, 1H, Ar–H), 8.05 (s, 1H, Ar–H), 8.11–8.19 (m, 2H, Ar–H), 10.35 (s, 1H, olefinic proton); MS: m/z 430 (M⁺); Anal. Calcd for C₂₂H₁₄F₄N₂O₃: C, 61.40; H, 3.28; N, 6.51. Found: C, 61.31; H, 3.22; N, 6.61.

(4Z)-4-[(6-Methyl-4-oxo-4H-chromen-3-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (5b). Yellow solid; IR (, cm⁻¹): 1694 (C=O lactum), 1669 (C=O chromone), 1627 (C=C), 1588 (C=N), 1140 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 0.97–1.01 (t, 3H, J = 16 Hz), 1.68–1.73 (m, 2H), 2.45 (s, 3H, Ar–CH₃), 2.69–2.72 (t, 2H, J = 12 Hz), 7.66–7.72 (m, 2H, Ar–H), 7.95 (s, 1H, Ar–H), 8.03 (s, 1H, Ar–H), 8.07–8.11 (m, 1H, Ar–H), 10.35 (s, 1H, olefinic proton); MS: m/z 444 (M⁺); Anal. Calcd for C₂₃H₁₆F₄N₂O₃: C, 62.16; H, 3.63; N, 6.30. Found: C, 62.24; H, 3.59; N, 6.23.

(4Z)-4-[(6,8-Dichloro-4-oxo-4H-chromen-3-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (5c). Yellow solid; IR (, cm⁻¹): 1707 (C=O lactum), 1659 (C=O chromone), 1624 (C=C), 1577 (C=N), 1126 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.03–1.07 (t, 3H, J = 12 Hz), 1.77–1.82 (m, 2H), 2.68–2.71 (t, 2H, J = 12 Hz), 7.10–7.16 (m, 1H, Ar–H), 7.77 (s, 1H, Ar–H), 7.99 (s, 1H, Ar–H), 8.13 (s, 1H, Ar–H), 10.66 (s, 1H, olefinic proton); MS: m/z 449 (M⁺); Anal. Calcd for C₂₂H₁₂Cl₂F₄N₂O₃: C, 52.93; H, 2.42; N, 5.61. Found: C, 52.86; H, 2.47; N, 5.70.

(4Z)-4-[(6-Chloro-4-oxo-4H-chromen-3-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (5d). Yellow solid; IR (, cm⁻¹): 1695 (C=O lactum), 1655 (C=O chromone), 1605 (C=C), 1580 (C=N), 1119 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.06–1.09 (t, 3H, J = 12 Hz), 1.79–1.85 (m, 2H), 2.71–2.74 (t, 2H, J = 12 Hz), 7.14–7.19 (m, 1H, Ar–H), 7.52–7.54 (d, 1H, J = 8 Hz, Ar–H), 7.69–7.72 (m, 1H, Ar–H), 8.08 (s, 1H, Ar–H), 8.25–8.26 (d, 1H, J = 4 Hz, Ar–H), 10.65 (s, 1H, olefinic proton); MS: m/z 464 (M⁺); Anal. Calcd for C₂₂H₁₃ClF₄N₂O₃: C, 56.85; H, 2.82; N, 6.03. Found: C, 56.78; H, 2.87; N, 6.11.

(4Z)-4-[(6-Chloro-7-methyl-4-oxo-4H-chromen-3-yl)methylene]-5-propyl-2-(2,3,5,6-tetrafluorophenyl)-2,4-dihydro-3H-pyrazol-3-one (5e). Yellow solid; IR (, cm⁻¹): 1711 (C=O lactum), 1649 (C=O chromone), 1630 (C=C), 1588 (C=N), 1135 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 1.05–1.09 (t, 3H, J = 16 Hz), 1.79–1.84 (m, 2H), 2.53 (s, 3H, Ar–CH₃), 2.70–2.74 (t, 2H, J = 16 Hz), 7.14–7.18 (m, 1H, Ar–H), 7.45 (s, 1H, Ar–H), 8.09 (s, 1H, Ar–H), 8.24 (s, 1H, Ar–H), 10.63 (s, 1H, olefinic proton); MS: m/z 478 (M⁺); Anal. Calcd for C₂₃H₁₅ClF₄N₂O₃: C, 57.69; H, 3.16; N, 5.85. Found: C, 57.60; H, 3.29; N, 5.89.

(4Z)-4-[(4-Oxo-4H-chromen-3-yl)methylene]-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (5f). Yellow solid; IR (, cm⁻¹): 1699 (C=O lactum), 1655 (C=O chromone), 1625 (C=C), 1596 (C=N), 1133 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.52–7.60 (m, 3H, Ar–H), 7.77–7.82 (m, 1H, Ar–H), 8.30–8.33 (d, 1H, J = 8 Hz, Ar–H), 8.53 (s, 1H, Ar–H), 10.69 (s, 1H, olefinic proton); MS: m/z 456 (M⁺); Anal. Calcd for C₂₀H₇F₇N₂O₃: C, 52.65; H, 1.55; N, 6.14. Found: C, 52.53; H, 1.46; N, 6.08.

(4Z)-4-[(6-Methyl-4-oxo-4H-chromen-3-yl)methylene]-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (5g). Yellow solid; IR (, cm⁻¹): 1705 (C=O lactum), 1662 (C=O chromone), 1620 (C=C), 1593 (C=N), 1141 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 2.48 (s, 3H, Ar–CH₃), 7.20–7.23 (m, 2H, Ar–H), 7.44–7.46 (d, 1H, J = 8 Hz, Ar–H), 8.06 (s, 1H, Ar–H), 8.51 (s, 1H, Ar–H), 10.64 (s, 1H, olefinic proton); MS: m/z 470 (M⁺); Anal. Calcd for C₂₁H₉F₇N₂O₃: C, 53.63; H, 1.93; N, 5.96. Found: C, 53.53; H, 1.89; N, 5.90.

(4Z)-4-[(6,8-Dichloro-4-oxo-4H-chromen-3-yl)methylene]-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (5h). Yellow solid; IR (, cm⁻¹): 1711 (C=O lactum), 1677 (C=O chromone), 1622 (C=C), 1599 (C=N), 1135 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-): 7.52–7.57 (m, 1H, Ar–H), 7.69–7.71 (m, 1H, Ar–H), 8.01 (s, 1H, Ar–H), 8.40 (s, 1H, Ar–H), 10.59 (s, 1H, olefinic proton); MS: m/z 525 (M⁺); Anal. Calcd for C₂₀H₅Cl₂F₇N₂O₃: C, 45.74; H, 0.96; N, 5.33. Found: C, 45.61; H, 0.88; N, 5.41.

(4Z)-4-[(6-Chloro-4-oxo-4H-chromen-3-yl)methylene]-2-(2,3,5,6-tetrafluorophenyl)-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one (5i). Yellow solid;IR (, cm⁻¹): 1695 (C=O lactum), 1671 (C=O chromone), 1611 (C=C), 1580 (C=N), 1109 (Ar–F); ¹H-NMR δ (400 MHz, DMSO-d₆): 7.54–7.56 (m, 2H, Ar–H), 7.72–7.74 (d, 1H, J = 8 Hz, Ar–H), 8.26 (s, 1H, Ar–H), 8.46 (s, 1H, Ar–H), 10.66 (s, 1H, olefinic proton); MS: m/z 490 (M⁺); Anal. Calcd for C₂₀H₆ClF₇N₂O₃: C, 48.95; H, 1.23; N, 5.71. Found: C, 48.90; H, 1.12; N, 5.64.

2.3. Antimicrobial Activity

The antimicrobial activity of all newly synthesized compounds was evaluated by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI 2005, formerly known as NCCLS) [48]. The results were determined using minimum inhibitory concentration (MIC) values in μg/mL. Antibacterial activity was screened against two Gram-positive (Staphylococcus aureus MTCC 96 and Streptococcus pyogenes MTCC 443) and two Gram-negative (Escherichia coli MTCC 442 and Pseudomonas aeruginosa MTCC 441) bacterial strains using ampicillin as a standard antibacterial agent. Antifungal activity was screened against three fungal species (Candida albicans MTCC 227, Aspergillus niger MTCC 282, and Aspergillus clavatus MTCC 1323) where griseofulvin was used as a standard antifungal agent. All MTCC cultures were collected from Institute of Microbial Technology, Chandigarh.

Sterile 96-well microtiter plates were used in this assay. All tests were performed in Mueller Hinton broth for bacterial strains and Sabouraud’s dextrose broth for fungal strains. Overnight broth cultures of each strain were prepared, and the final inoculum concentration in each well was adjusted to 2 × 10⁶ CFU mL⁻¹ for the bacterial strains and 2 × 10⁷ CFU mL⁻¹ for fungal strains. The inoculum concentration was adjusted by diluting broth culture supernatant in sterile distilled water to a density of 0.5 McFarland standards using a nephelometer. Stock solutions of all compounds at 2000 μg/mL were prepared by dissolving them in DMSO. Serial dilutions were prepared in primary and secondary screening. In primary screening, 500, 250, and 200 μg/mL concentrations of the synthesized compounds were taken. The compounds found active in primary screening were further tested in a second set of dilutions against all microorganisms. The compounds found active in primary screening were similarly diluted to obtain 100, 62.5, 50, and 25 μg/mL concentrations. In the tests, triphenyltetrazolium chloride (TTC) was also added to culture medium as growth indicator. The final concentration of TTC after inoculation was 0.05%. The microbial growth was determined after 24 h incubation at 37°C for the bacteria and at 25°C after 48 h for the fungi. The MIC is defined as the lowest concentration of a compound at which the microorganism does not demonstrate visible growth. All determinations were performed in duplicate to check the accuracy of results. The antimicrobial activity results (MIC) are presented in Table 3.

Table 3: Antibacterial and antifungal data of newly synthesized pyrazole-pyrazolone (4a–j) and chromone-pyrazolone (5a–i) indicated by MIC values (g/mL).

Compound
Minimum inhibitory concentration (MIC, g/mL)

Gram-positive bacteria
Gram-negative bacteria
Fungi

S. aureus
S. pyogenes
E. coli
P. aeruginosa
C. albicans
A. niger
A. clavatus

4a
250
200
200
100
>1000
>1000
>1000

4b
125
100
62.5
100
500
1000
1000

4c
200
250
100
125
1000
1000
1000

4d
100
100
62.5
200
1000
>1000
>1000

4e
125
62.5
200
250
>1000
>1000
>1000

4f
200
250
250
250
1000
500
500

4g
250
200
100
200
>1000
250
500

4h
500
250
250
125
>1000
500
500

4i
500
500
200
200
500
>1000
>1000

4j
250
500
125
250
500
1000
1000

5a
200
250
250
125
500
1000
1000

5b
100
62.5
200
100
1000
>1000
>1000

5c
125
100
250
200
1000
>1000
>1000

5d
200
200
250
200
>1000
500
500

5e
100
100
500
200
250
1000
>1000

5f
62.5
200
500
500
250
>1000
>1000

5g
100
250
200
200
500
>1000
>1000

5h
250
200
100
250
1000
>1000
>1000

5i
250
200
125
62.5
1000
500
1000

Ampicillin
250
100
100
100
—
—
—

Griseofulvin
—
—
—
—
500
100
100

3. Result and Discussion

3.1. Chemistry

In the present investigation, our aim was to do the synthesis of multifluorinated pyrazole-pyrazolone (4) and chromone-pyrazolone (5) by using green technique such as ultrasonication and investigate their antibacterial and antifungal activities (Scheme 1). The starting material multifluorinated pyrazolone (3) was prepared by literature known procedure [49] using β-ketoester (1) and 2,3,5,6-tetra-fluoro phenyl hydrazine (2). The pyrazolone (3) was treated with 3-formyl chromone or 4-formyl pyrazole in acetic acid under conventional heating as well as ultrasound irradiation to give pyrazole-pyrazolone (4) and chromone-pyrazolone (5). The structures of all compounds were confirmed on the basis of spectral analysis. The IR spectra of compounds 4 and 5 showed a peak in the range 1680–1711 cm⁻¹ for C=O stretching of five membered lactum ring. They also exhibited a peak in the range 1500–1600 cm⁻¹ for C=N stretching. The ¹H-NMR spectra of compounds 4a–e and 5a–e clearly showed peaks for CH₃–CH₂–CH₂–(propyl group) protons, while such peaks were absent in compounds 4f–j and 5f–i as they have no propyl group. The exocyclic methylene group proton appeared as a singlet in the range of 10.11–10.66 δ. The aromatic protons present in all compounds were also confirmed by ¹H-NMR spectra. Further the structures of all compounds were also confirmed by molecular ion peak in their mass spectra. For all the synthesized compounds, elemental analysis values are in good agreement with theoretical data.

It was observed that ultrasound irradiation method was practically more superior than conventional heating method in terms of higher yields, rapid, and environmentally benign process which makes this protocol more useful.

3.2. Antimicrobial Activity

Antibacterial activity was screened against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa bacterial strains using ampicillin as a standard antibacterial agent. Antifungal activity was screened against fungal species Candida albicans, Aspergillus niger, and Aspergillus clavatus where griseofulvin was used as a standard antifungal agent.

Reviewing the antibacterial activity data (Table 1) of all newly synthesized pyrazole-pyrazolone (4a–j) and chromone-pyrazolone (5a–i), it was revealed that the compounds (4b, 4d), (5i), (5f), and (4e, 5b) showed highest activity, that is, 62.5 μg/mL against E. coli, P. aeruginosa, S. aureus, and S. pyogenes, respectively. Among pyrazole-pyrazolone, the compounds 4b, 4c, 4d, 4e containing propyl group and compound 4f containing trifluoromethyl group, similarly among chromone-pyrazolone, the compounds 5a, 5b, 5c, 5d, 5e containing propyl group and compounds 5f, 5g containing trifluoromethyl group are found to possess high potency against Gram-Positive S. aureus bacterial strain as compared with standard bactericidal ampicillin. So it is concluded that compounds with propyl group rather than CF₃ group exhibited high potency against gram positive S. aureus bacterial strain. It is also observed that compounds (4c, 4g, 5h), (4a, 4b, 5b), (4a, 4g, 4j, 5h, 5i), and (4b, 4d, 5c, 5e) showed promising activity against E. coli, P. aeruginosa, S. aureus, and S. pyogenes, respectively, as compared with standard ampicillin drug.

Antifungal activity study of synthesized compounds reveals that compounds 5e and 5f exhibited more potency while compounds 4b, 4i, 4j, 5a, and 5g showed good antifungal activity against C. albicans as compared with standard fungicidal griseofulvin. All the synthesized compounds showed moderate-to-poor antifungal activity profile against A. niger and A. clavatus fungal strains.

4. Conclusion

Present study describes synthesis of a series of novel multifluorinated pyrazolone containing pyrazole and chromone nucleus by conventional heating as well as ultrasonication technique. The ultrasonication method was more efficient in terms of short reaction time, high yields, and clean reaction process. The antimicrobial activity result reveals that some of the synthesized compounds exhibited promising activity against the tested bacterial and fungal strains. The insights gained in this study will be useful for further studies on potential anti-infective agents. A study regarding structure activity relationship is in progress.

Acknowledgments

The authors are thankful to Dr. S. R. Walunj (Principal, PVP, College), Dr. T. N. Gholap (Principal, RKMM, Ahmednagar), and Management, PRES, for providing necessary facilities and constant encouragement. The authors also thank SAIF, Chandigarh, for spectral analysis.

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