Functionalized 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds: A new class of antimicrobials and antioxidants

2.2.1 Synthesis of 2-acetyl benzofuran (2)

A mixture of salicyaldehyde (1) (2 mmol), chloroacetone (2 mmol) and potassium tert-butoxide (2 mmol) in 10 mL of dichloromethane (DCM) containing molecular sieves was refluxed at 30 °C for 3 h. Progress of the reaction was monitored by TLC using hexane:ethyl acetate (8:2) mixture as mobile phase. After the completion of the reaction, the reaction mixture was washed with 10% HCl solution followed by water. The organics were dried over anhydrous sodium sulfate. The yellow solid was obtained by desolventizing in a rotary evaporator at room temperature affords 2-acetyl benzofuran (2). m.p.: 73–75 °C, Yield-92%, IR (KBr) ν_max (cm⁻¹): 3087 (CH furan), 2900 (CH₃), 1675 (C⚌O), 1558 (C⚌C); ¹H NMR (DMSO-d₆ 400 MHz) δppm: 7.46–7.73 (m, 5H, Ar-H), 2.25 (s, 3H, CH₃). ¹³C NMR (DMSO-d₆ 100 MHz) δppm: 186.0, 160.4, 147.8, 127.6, 124.8, 123.3, 120.9, 111.4, 103.9, 25.5; MS (ESI) m/z: 160.05 (M⁺). Anal. calcd. for C₁₀H₈O₂: C, 74.99; H, 5.03; O, 19.98%; found: C, 74.95; H, 5.07; O, 19.96%.

2.2.2 Synthesis of (E)-1-(benzofuran-2-yl)-3-(4-methoxyphenyl) prop-2-en-1-one (3)

Compound (2) (1 mmol) in ethanol (0.5 mL) was treated with LiOH·H₂O (4 mg 0.1 mmol, 10 mol%) under magnetically stirred condition for 10 min at room temperature (∼25–30 °C) followed by 4-methoxy benzaldehyde (1 mmol). The mixture was stirred magnetically until complete consumption of the compound (2) for 35 min. After the completion of the reaction a dark yellow precipitate was formed and this served as an indicator for monitoring the reaction. Ethanol was removed under reduced pressure. The residue was diluted with water (5 mL) neutralized with 2% aqueous HCl and extracted with ethylacetate (3 × 5 mL). The combined ethylacetate extracts were washed with brine solution (5 mL) dried over anhydrous Na₂SO₄ and concentrated under vacuum at room temperature to afford compound (3). Light yellow solid, m.p. : 118–120 °C, Yield-87%, IR (KBr) ν_max (cm⁻¹): 1624 (C⚌O), 1455 (CH⚌CH); ¹H NMR (DMSO-d₆ 400 MHz) δppm: 7.10–7.60 (m, 9H, Ar-H), 6.75 (s, 2H, CH) 3.81 (m, 3H of OCH₃); ¹³C NMR (DMSO-d₆ 100 MHz) δppm: 177.8, 160.4, 155.2, 149.1, 147.8, 145.0, 127.8, 127.5, 124.7, 123.3, 122.9, 121.2, 120.9, 116.7, 111.9, 111.4, 56.1; MS (ESI) m/z: 278.09 (M⁺). Anal. calcd. for C₁₈H₁₄O₃: C, 73.46; H, 4.79; O, 21.75%; found: C, 73.44; H, 4.78; O, 21.73%.

2.2.3 Synthesis of 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)4, 5-dihydro-1H-pyrazole (4)

To a solution of (E)-1-(benzofuran-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3) (1 mmol) in ethanol as solvent, hydrazine hydrate (2 mmol) was added and the reaction mixture was refluxed for 3 h. After cooling the mixture was poured into crushed ice, the precipitate was obtained, filtered and washed with ethanol (2–5 mL) followed by water, dried in air and residue was collected (4). Light brown solid, m.p.: 195–197 °C, Yield-87%, IR (KBr) ν_max (cm⁻¹): 3311 (N–H) and 1595 (C⚌N); ¹H NMR (DMSO-d₆ 400 MHz) δppm: 7.10 (s, 1H, N–H), 6.65–7.85 (m, 9H, Ar-H), 3.90–3.98 (dd, 2H, CH₂, pyrazoline, J = 4.1 Hz, 5.8 Hz), 3.88 (t, 1H, CH of pyrazoline, J = 7.5 Hz), 3.71 (s, 3H of OCH₃); ¹³C NMR (DMSO-d₆ 100 MHz) δppm: 155.6, 155.0, 147.2, 146.7, 137.0, 134.5, 125.1, 124.7, 123.3, 120.9, 119.4, 115.5, 111.5, 110.2, 104.9, 56.1, 49.0, 40.9; MS (ESI) m/z: 292.12 (M⁺). Anal. calcd. for C₁₈H₁₆N₂O₂: C, 70.12; H, 5.23; N, 9.09; O, 15.57%; found: C, 70.10; H, 5.25; N, 9.11; O, 15.55%. The spectral and analytical data for compounds (2), (3) and (4) are in agreement with that reported in the literature (Rangaswamy et al., 2012; Bakr et al., 2009; Basawaraj et al., 2009).

2.2.4 General procedure for the synthesis of functionalized 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds (4a-q)

3-(Benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (1.2 mmol) was suspended in dry THF (5 mL) in an inert atmosphere (N₂). To this suspension, at room temperature triethylamine (1.5 mmol) and different benzoyl chlorides (RCOCl) (1 mmol in 3 mL of THF) were added and reaction mixture was stirred for 3–4 h. The progress of reaction mixture was monitored by TLC using hexane:ethylacetate (6:4). The reaction mixture was then desolventized in a rotary evaporator and the compound was extracted in ethylacetate. The ethylacetate layer was washed with water and dried over anhydrous sodium sulfate. The products were obtained by further desolventation in a rotary evaporator at 50 °C. The respective products were purified through column chromatography using hexane:ethylacetate (6:4).

2.3.1 Antibacterial studies

The antibacterial activities of newly synthesized compounds (4a-q) were determined by the well plate method in Mueller–Hinton Agar (Arthington-Skaggs et al., 2000). The antibacterial activity was carried out against 24 h old cultures of bacterial strains. In this work, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were used to investigate the activity. The test compounds were dissolved in dimethyl sulfoxide (DMSO) at concentration of 1 and 0.5 mg/mL. 20 mL of sterilized agar media was poured into each pre-sterilized petri dish. Excess of suspension was decanted and plates were dried by placing in an incubator at 37 °C for an hour. About 60 mL of 24 h old culture suspension was poured and neatly swabbed with the pre-sterilized cotton swabs. Six millimeter diameter well was then punched carefully using a sterile cork borer and 30 mL of test solutions of different concentrations was added into each labeled well. The plates were incubated for 24 h at 37 °C. The inhibition zone that appeared after 24 h, around the well in each plate was measured as zone of inhibition in mm. Experiments were triplicates and standard deviation was calculated.

2.3.2 Antifungal studies

Antifungal studies of newly synthesized 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds (4a-q) were carried out against Aspergillus flavus, Chrysosporium keratinophilum, Candida albicans. Sabourands agar media were prepared by dissolving peptone (10 g), d-glucose (40 g) and agar (20 g) in distilled water (1000 mL) and adjusting the pH to 5.7. Normal saline was used to make a suspension of spore of fungal strains for lawning. A loopful of particular fungal strain was transferred to 3 mL saline to get a suspension of corresponding species (Mac Lowry et al., 1970). 20 mL of agar media was poured into each petri dish. Excess of suspension was decanted and plates were dried by placing in an incubator at 37 °C for 1 h. Using sterile cork borer punched carefully, wells were made on these seeded agar plates and different concentrations of the test compounds in DMSO were added into each labeled well. A control was also prepared for the plates in the same way using solvent DMSO. The petri dishes were prepared in triplicate and maintained at 25 °C for 72 h. Antifungal activity was determined by measuring the diameter of inhibition zone. Activity of each compound was compared with fluconazole as standard and zones of inhibition were determined for all the synthesized compounds (4a-q).

2.4.1 DPPH radical scavenging assay

The evaluation of antioxidant activity of newly synthesized compounds was done by DPPH radical scavenging activity (Blois, 1958). Internal standard BHA and the synthesized compounds of different concentrations were prepared in distilled ethanol, 1 mL of each compound solution having different concentrations (10, 25, 50, 100, 200 and 500 μM) was taken in different test tubes, 4 mL of 0.1 mM ethanol solution of DPPH was added and shaken vigorously. The tubes were then incubated in the dark room at RT for 20 min. A DPPH blank was prepared without compound and ethanol was used for the baseline correction. Changes (decrease) in the absorbance at 517 nm were measured using a UV–visible spectrophotometer and the remaining DPPH was calculated. The percent decrease in the absorbance was recorded for each concentration and percent quenching of DPPH was calculated on the basis of the observed decrease in absorbance of the radical. The radical scavenging activity was expressed as the inhibition percentage and was calculated using the formula: $$\text{Radical scavenging activity}(\%)=[(A_0-A_1)/A_0\times 100]$$ where A₀ is the absorbance of the control (blank, without compound) and A₁ is the absorbance of the compound.

2.4.2 Inhibition of microsomal lipid peroxidation assay

Liver excised from adult male Wister rats, were homogenized with a polytron (speed setting 7–8) in 10 mL of ice cold Tris–HCl buffer (20 mM, pH 7.4) by the method of Liu and Ng, 2000. The homogenate was centrifuged at 14,000 rpm for 15 min. The supernatants (1 mL) were incubated with different concentrations of compounds (10, 25, 50, 100, 200 and 500 μM) in the presence of 10 μM FeSO₄ and 0.1 mM ascorbic acid at 37 °C for 1 h. The reaction was terminated by the addition of 1.0 mL of trichloroacetic acid (TCA; 28%) and 1.5 mL of thiobarbituric acid (TBA 1%). The solution was heated at 100 °C for 15 min, cooled to room temperature, and centrifuged at 2500 rpm for 15 min, and the color of the MDA–TBA complex in the supernatant was read at 532 nm using a spectrophotometer. BHA was used as a positive control. The inhibition ratio (%) was calculated using the following formula: $\text{inhibition ratio}(\%)=(A-A_1)/A\times 100$ , where A is the absorbance of the control and A₁ is the absorbance of the test sample.

2.4.3 ABTS⁺ free radical scavenging assay

The ability of the test sample to scavenge ABTS⁺ radical cation was determined according to the literature method (Re et al., 1999). The ABTS⁺ radical cation was pregenerated by mixing 7 mM ABTS⁺ stock solution with 2.45 mM potassium persulfate (final concentration) and incubating for 12–16 h in the dark at room temperature until the reaction was complete and the absorbance was stable. The absorbance of the ABTS⁺ solution was equilibrated to 0.70 (±0.02) by diluting with water at room temperature, then 1 mL was mixed with different concentrations of the test sample (10, 25, 50, 100, 200 and 500 μM) and the absorbance was measured at 734 nm after 6 min. The scavenging capability of ABTS⁺ radical was calculated using the following equation: $${\text{ABTS}}^{+}\text{scavenging effect}(\%)=[(A_{\text{c}}-A_{\text{s}})/A_{\text{c}}]\times 100$$ where, A_c is the initial concentration of the ABTS⁺ and A_s is the absorbance of the remaining concentration of ABTS⁺ in the presence of compounds.

3.2.1 Antibacterial activity

The antibacterial activity of newly synthesized functionalized 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds (4a-q) was determined by the well plate method. In this study, E. coli ATCC 25922 (Gram-negative), S. aureus ATCC 25923 (Gram-positive) and P. aeruginosa ATCC 27853 (Gram-negative) were selected due to their infectious nature. The test compounds were dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 and 0.5 mg/mL. (Table 1)

Table 1 Chemical structures and yields of synthesized compounds (4a-q).

Compound number	R	Yield %	Compound number	R	Yield %
4a		87.53	4j		71.86
4b		75.26	4k		71.60
4c		68.30	4l		80.28
4d		77.00	4m		74.35
4e		65.50	4n		67.77
4f		75.06	4o		79.80
4g		82.00	4p		76.82
4h		72.80	4q		78.35
4i		69.11

The antibacterial screening revealed that some of the tested compounds showed good inhibition against various tested microbial strains (Table 2). The results indicated that among the tested compounds 4h (p-fluro substituted) and 4j (p-chloro substituted) on the 1-substituted phenyl ring showed excellent activity against P. aeruginosa and E. coli at the concentrations of 1 and 0.5 mg/mL compared to standard drug streptomycin. Compounds 4i (p-bromo substituted) and 4k (m-chloro substituted) showed similar activity as that of standard, against E. coli. Whereas, compounds 4b (p-nitro substituted) and 4c (3,5-dinitro substituted) revealed moderately good activity against E. coli at 1 mg/mL concentrations. The remaining compounds showed very least activity against all of the three tested bacterial strains. From these results it could be concluded that, the chloro, fluoro, bromo, and nitro functionalized derivatives showed better activity and the others showed less activity against all bacterial strains.

3.2.2 Antifungal activity

Newly synthesized compounds (4a-q) were also screened for their antifungal activity against A. flavus MTCC 3306, C. keratinophilum MTCC 2827 and C. albicans MTCC 3017, because of their infectious nature. The compounds were dissolved in DMSO and antifungal activity was determined by the well plate method at concentrations of 1 and 0.5 mg/mL.

The antifungal result data (Table 3) indicated that, the synthesized compounds showed a variable degree of inhibition against the tested fungi. As a result, the presence of chloro substituent on the 1-substuted phenyl ring in compound (4j) has exhibited a highly potent activity against C. albicans compared with standard, fluconazole. Compounds (4h), (4i) and (4k) incorporating with fluoro, bromo, chloro moieties on the 1-substituted phenyl ring at different positions displayed considerable activity against C. keratinophilum. The other analogues of 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole showed less activity against all the tested microorganisms compared to standard. It can be concluded that some of the prepared compounds were superior to standard against C. albicans tested microbial strains. From these results it could be generalized that, the presence of halogen substituents on the phenyl ring showed better activity compared to other analogues.

3.3.1 DPPH radical scavenging assay

Free radical scavenging is one of the best known mechanisms by which antioxidants inhibit the oxidation and offer a rapid technique for screening the radical scavenging activity of specific compounds. All tested 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds (4a-q) exhibited as certain degree of radical scavenging activity. Initially, benzofuran scaffold (4) showed considerable activity, this could be due to the presence of the electron donating methoxy group on the phenyl ring and also the presence of the N–H functional group in the pyrazole ring (Mei and Fang, 2004). Further, introducing benzoyl chlorides having different functional sites gave the significant enhancement in activity. The inhibition results of the derivatives appeared to be related to the nature of the substituent groups on the 1-susbtituted phenyl ring. Compounds (4g) and (4q) exhibited better radical scavenging potential (Table 4), this may be due to the presence of more than one hydroxyl group on the 1-substituted phenyl ring at different positions. The conjugation of these two benzoyl chlorides (3,4,5-tri-hydroxybenzoyl chloride and 3,5-di-hydroxybenzoyl chloride) to compound (4) have exhibited thirty folds more activity compared to 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (4) and even more higher activity than that of standard, butylated hydroxyanisole. The presence of electron donating substituents, such as hydroxy, methoxy and methyl on the phenyl ring in compounds (4d-f, 4l-n and 4p) showed good radical scavenging activity but slightly less than the standard, whereas, compounds (4b-c and 4h-k) possessing electron withdrawing (nitro, halogens) substituents exhibited less activity and compound (4a) and (4o) without any substituents and acetyl substituents showed least DPPH radical scavenging potential.

3.3.2 Inhibition of microsomal lipid peroxidation assay

A well-recognized result of oxidant injury is peroxidation of membrane lipids to organic peroxyl radicals, which initiates a chain reaction that may explain many membrane-mediated effects of reactive oxygen species (ROS). The concentration-dependent inhibitory effects of 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds (4a-q) on lipid peroxidation (LP) in liposomes were estimated by the amount of thiobarbituric acid reactive substances (TBARS). BHA was used as a reference antioxidant (Table 4). All tested compounds exhibited some degree of inhibitory effect on the Fe³⁺/ascorbic acid-induced LP whereas, two compounds showed higher than that of standard. Based on the IC₅₀ values (effective concentration at which TBARS were reduced to 50% with respect to controls), the compounds (4g) and (4q) possessing 3,4,5-tri-hydroxy and 3,5-di-hydroxy substituents on the 1-substituted phenyl ring accounted for significant LPO inhibitors than the other 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds. The compounds (4d-f), (4l), (4m) and (4n), have good inhibitory effect, this may be due to the presence of hydroxyl and methoxy functions on the 1-substituted ring respectively. Whereas, compounds having halogens (4h-k), nitro (4b-c), acetyl (4o), methyl (4p) and non-substituted (4a) showed lowest inhibitory effect compared with the standard as well as other compounds.

3.3.3 ABTS radical scavenging assay

The ABTS method is based on the ability of hydrogen or electron-donating antioxidants to decolorize the performed radical monocation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) generated due to oxidation of ABTS with potassium persulfate. The radical scavenging abilities showed by 3-(benzofuran-2-yl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole scaffolds toward this assay typically revealed that, compounds (4g) and (4q) exhibit significantly better activity than standard BHA. Whereas para, meta, ortho-free hydroxy substituted (4d-f) and methoxy substituted (4l-n) derivatives permit the moderate radical scavenging ability, respectively. The presence of electron-withdrawing groups on the 1-substituted phenyl ring at 3–5 positions might not favor the activity. Thus, compounds (4b-c) and (4h-k) which contain NO₂ and halogen groups on the phenyl ring at different positions showed low ABTS radical activity than the other compounds (Table 4). The data presented in Table 2 reflect that compounds (4g) and (4q) exhibit effective antioxidant potential compared to the standard BHA.