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Synthesis, Characterization and Antibacterial Activities of New Fluorinated Chalcones

  • Kayode L. Amole
  • Isaac A. Bello
  • Adebayo O. OyewaleEmail author
Original Article
  • 97 Downloads

Abstract

A series of five fluorinated chalcones (E)-3-(2′-ethoxyphenyl)-1-(4-Fluoro-2-hydroxyphenyl)prop-2-en-1-one(4),(E)-3-(3′,4′-diethoxyphenyl)-1-(4-fluoro-2-hydroxyl-phenyl)prop-2-en-1-one(5),(E)-3-(2′,3′-dihydrobenzofuran-5-yl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one(6),(E)-3-(3′,5′-Bis[trifluoromethyl]phenyl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one(7) and (E)-3-(4′-diethylaminophenyl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one(8) were synthesized by a modified Claisen-Schmidt condensation of 4-fluoro-2-hydroxyacetophenone with the appropriate aromatic aldehydes. The compounds were characterized by spectroscopic methods and evaluated for their antibacterial activity against Gram-positive (Methicillin-resistant S. aureus, Vancomycin-resistant enterococci, S. aureus, S. pyrogenes, S. faecalis) and Gram-negative (E. coli, S. typhi, C. ulcerans, P. mirabilis, P. aeruginosa) pathogenic bacteria strains using the agar diffusion method. The compounds exhibited broad spectrum activity against eight of these pathogens with compounds (4), (5) and (8) found to be the most potent in the series with zones of inhibition ranging from 23 to 28 mm and MIC between 25 and 50 µg/mL. All the compounds except (7) containing 3-(3′,5′-bis[trifluoromethyl]phenyl) moiety, showed a remarkable activity against MRSA [MIC = 25–50 µg/mL]. Compound (8) exhibited good activity against P. aeruginosa [MIC = 50 µg/mL]. Compounds (4), (5) and (8) were active against C. ulcerans [MIC = 50 µg/mL]. The broad-spectrum standard drug, ciprofloxacin, was inactive against MRSA, P. aeruginosa and C. ulcerans.

Keywords

Flavonoids Chalcones Synthesis Fluorinated Antibacterial 

1 Introduction

The rapid emergence of antibiotic resistance has become a notorious, global problem and fluorinated organic compounds have been considered one of the groups of compounds that are intriguing for the development of pharmaceuticals for this tremendous public health concern. Fluoro-organic compounds have inherent biologically activity and the introduction of fluorine into a biological active compound improves its pharmacological properties. Hence, much effort is being made to develop more general and efficient approaches for introducing fluorine atom(s) or fluoroalkyl group(s) into organic compounds [1]. The pharmacological superiority of fluorinated compounds over their non-fluorinated analogues may be due to C–F bond which is ordinarily stronger than C–H bond and the strength of C–F bond conveys stability to fluorine compounds hence are likely to be recalcitrant in the environment or may be partially metabolised to a more toxic metabolite. Also, the introduction of fluorine confers lipophilicity [1, 2]. These factors are operative singly or sometimes cooperatively to affect the pharmacological properties of the fluorinated compounds [3]. Several studies revealed that fluorinated compounds are a significant class of compounds in medicinal and pharmaceutical therapy, for example, 5-fluorouracil and 5-fluoro-2-deoxyuridine have been used in the treatment of cancer [4, 5] while ciprofloxacin, a mono-fluoro aromatic compound, commonly used as antibiotics, is active against both Gram-positive and Gram-negative bacteria [6].

A number of fluorinated chalcones have been synthesized and evaluated for their biological activities. Fluorine groups, either as aryl fluoro or trifluoromethyl have been previously attached on their respective precursors. For example, some derivatives of 4′-fluoro-4-hydroxychalcone have been shown to possess anti-inflammatory, antioxidant and analgesic property [7, 8]. Aryl fluorinated derivatives of 2,4-dihydroxyl-3′,4′-dimethoxy-chalcone were shown to possess anti-peroxidation, anti-inflammation and anti-hypersensitivity properties [9] and trifluoromethyl methoxy chalcone was evaluated as an excellent antimalarial compound [10]. Several studies have shown that derivatives of polyfluorinated chalcones are more active than their monofluorinated counterparts. For instance, various derivatives of aryl difluoro chalcones have been shown to be more effectives against human pancreatic cancer cells and breast cancer cells than the monosubstituted and hydroxylated derivatives in a pharmacological comparative study [11]. A series of (E)-1-(4-fluorophenyl)chalcones were evaluated and found to possess antibacterial activities against E. coli and P. aeruginosa and, antifungal activity against A. niger [12].

In an effort to develop potent fluorinated antibiotic compounds to stem the effects of drug resistance, the present study was conducted to synthesize various derivatives of 4-fluoro-2-hydroxychalcones (4-Fluoro-2-hydroxyphenyl)prop-2-en-1-one) and screen them for their antibacterial activities.

2 Experimental

2.1 Synthesis

Five derivatives of 4-fluoro-2-hydroxychalcone (4, 5, 6, 7, 8) were synthesized by refluxing 4-fluoro-2-hydroxyacetophenone (3) with each of 4-(N,N-diethylamino)benzaldehyde, 2-ethoxybenzaldehyde, 3,4-diethoxybenzaldehyde, 2,3-dihydrobenzofuran-5-carbaldehyde and 3,5-Bis[trifluoromethyl]benzaldehyde respectively.

Chromatographic technique using TLC aluminum sheet pre-coated plate from Merck (silica gel 60 F254, layer thickness 0.2 mm, 20% ethyl acetate in hexane) was employed to monitor the progress of the reaction. The plate was also used for analytical TLC and to determine the retardation factor (Rf) values. Spots were viewed at 254 nm and 366 nm (Model UVGL-58).

The compounds were purified using open column chromatography on silica gel 60 from Merck (60–120 mesh). The columns were packed wet with n-hexane and the compounds were dissolved in dichloromethane and dry loaded. n-Hexane:ethyl acetate (9:1) solvent mixture was used to elute the mixture through the column.

NMR spectra of the compounds were measured using a Proton decoupled Bruker 400 MHz spectrometer with internal standard residual resonance signal of deuterated chloroform and dimethylsulphoxide (DMSO), for those insoluble in CDCl3, as specified in the spectra. All spectra measurements were carried out at room temperature.

The mass spectra of the compounds were measured using an Agilent technology GC–MS 7890A coupled with MSD 5975C, with GC ALS as injection source. The recording was carried out under these conditions: Injection volume (2 µL), over maximum temperature of 325 °C and equilibrium time of 0.25 min. Column size 30 m × 320 µm × 0.25 µm, initial temperature of 110 °C and flow rate of 1.6282 ml/min. The samples were derivatized using N-methyl-bis[trifluoroacetamide] in methanol as solvent.

All the solid products were purified by recrystallization from 98% ethanol. The melting point (uncorrected) were recorded using a Stuart SMP40 automated melting point apparatus (ramp rate 5 °C per minute, start temperature 50 °C and automatic stop).

The IR spectra of finely ground compounds were recorded on a FTIR-ATR spectrometer (Cary 630), using transmittance method.

2.2 General procedure for the synthesis of E-1-(4-flouro-2-hydroxyphenyl)-3-(substituted phenyl)-2-propen-1-one

A mixture of the appropriate benzaldehyde derivatives (5 mmol) and 4-fluoro-2-hydroxyacetophenones (1) (5 mmol) in 98% ethanol (20 mL) was stirred in a round bottom flask and 50% NaOH solution (2 mL) was added gradually with constant stirring under reflux for about 5 h. The reaction mixture was allowed to cool to room temperature, then poured over crushed ice, acidified with 10% HCl until the reaction mixture was slightly acidic to wet litmus paper and allowed to stand overnight in an ice bath. The product formed was filtered using a vacuum pump, then washed several times with cold distilled water, dried and recrystallized from absolute ethanol [7, 13].

2.2.1 (E)-3-(2′-ethoxyphenyl)-1-(4-Fluoro-2-hydroxyphenyl)prop-2-en-1-one (4)

The title compound (1.14 g, 80%) was prepared from 4-fluoro-2-hydroxyacetophenone (0.77 g, 5 mmol) and 2-ethoxybenzaldehyde (0.75 g, 5 mmol) as a pale yellow crystalline solid, m.pt 90–92 °C. GCMS m/z = 286.2, 259, 241, 165, 133, 110, 91, 65 (ESM1, Online Resource 1); FTIR-ATR (v cm−1) = 1654.9 (C=O), 3414 (–OH), 3000 (Ar C–H), 2914 (Alip C-H), 1490 (Alip C=C) (ESM2, Online Resource 1); 1H-NMR: (400 MHz, DMSO) δ (ppm) = 1.42 (3H, t, J = 6.91 Hz, CH3), 4.48 (2H, q, J = 6.75 Hz, CH2), 13.02 (1H, s, –OH), 6.85 (2H, m), 7.02 (1H, t, J = 7.52 Hz,), 7.09 (1H, d, J = 8.41 Hz), 7.44 (1H, t, J = 7.79 Hz), 7.97 (2H, m), 8.17 (1H, m), 8.29 (1H, t, J = 7.89 Hz) (ESM3, Online Resource 1); 13C-NMR: (100 MHz, DMSO) δ (ppm) = 15.06 (1C, CH3), 64.30 (1C, CH2), 104.71 (C3, d, J = 23.66 Hz), 107.49 (C5, d, J = 22.32 Hz), 113.10 (C3′), 118.47 (C1, Cq), 1201.06 (C5′), 121.60 (α-C), 123.12 (C1′, Cq), 129.54 (C4′), 133.24 (C6′), 133.91 (C6′, d, J = 11.84 Hz) 140.17 (β-C), 158.31 (C2′, C–O), 164.82 (C2, C–OH), 166.92 (C4, d, J = 253.69 Hz, C-F), 192.97 (C=O) (ESM4, Online Resource 1).

2.2.2 (E)-3-(3′,4′-diethoxyphenyl)-1-(4-fluoro-2-hydroxyl-phenyl)prop-2-en-1-one (5)

The title compound (1.29 g, 78%) was prepared from 4-fluoro-2-hydroxyacetophenone (0.77 g, 5 mmol) and 3,4-diethoxybenzaldehyde (0.97 g, 5 mmol) as a yellow crystalline solid, m.pt 128–130 °C. GCMS m/z = 330.2, 301, 273, 245, 217, 192, 179, 164, 136, 110, 77, 55 (ESM1, Online Resource 2); FTIR-ATR (v cm−1) = 1654.9 (C=O), 3414 (–OH), 3000 (Ar C-H), 2914 (Alip C-H), 1513 (Alip C=C) (ESM2, Online Resource 2); 1H-NMR: (400 MHz, CDCl3) δ (ppm) = 1.49 (6H, m, CH3), 4.16 (4H, m, CH2), 13.34 (1H, s, –OH), 6.64 (1H, m), 6.69 (1H, m), 6.90 (1H, m), 7.17 (1H, s), 7.26 (1H, dd, J = 2.21 Hz, 8.32 Hz), 7.40 (1H, m), 7.86 (1H, m), 7.94 (1H, dd, J = 8.55 Hz, 14.80 Hz) (ESM3, Online Resource 2); 13C-NMR: (100 MHz, DMSO) δ (ppm) = 14.69 (1C, CH3), 14.82 (1C, CH3), 64.55 (1C, CH2), 64.87 (1C, CH2), 105.11 (C3, d, J = 23.42 Hz), 106.94 (C5, d, J = 22.74 Hz), 112.73 (C2′), 112.78 (C5′), 117.17 (C1, Cq), 117.30 (α-C), 123.75 (C6′), 127.30 (C1′), 131.81 (C6, d, J = 11.77 Hz), 146.08 (β-C), 148.91 (C3′, C–O), 151.88 (C4′, C–O), 166.08 (C2, C–OH), 167.29 (C4, d, J = 256.26 Hz), 192.45 (C=O) (ESM4, Online Resource 2).

2.2.3 (E)-3-(2′,3′-dihydrobenzofuran-5-yl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one (6)

The title compound (1.19 g, 84%) was prepared from 4-fluoro-2-hydroxyacetophenone (0.77 g, 5 mmol) and 2′,3′-dihydrobenzofuran-5-carbaldehyde (0.74 g, 5 mmol) as a yellow crystalline solid, m.pt 123–124 °C. GCMS m/z = 284.2, 256, 207, 165, 146, 133, 117, 91, 82, 63 (ESM1, Online Resource 3); FTIR-ATR (v cm−1) = 1636.3, (C=O), 3414 (–OH), 3000 (Ar C-H), 2918 (Alip C-H), 1490 (Alip C=C) (ESM2, Online Resource 3); 1H-NMR: (400 MHz, CDCl3) δ (ppm) = 3.25 (2H, t, J = 8.20 Hz, CH2), 4.65 (2H, t, J = 8.73 Hz, CH2), 13.38 (1H, s, –OH), 6.63 (1H, td, J = 2.51 Hz, 8.53 Hz), 6.68 (1H, dd, J = 2.49 Hz, 10.39 Hz), 6.82 (1H, d, J = 8.29 Hz), 7.42 (2H, dd, 11.79 Hz, 19.81 Hz), 7.53 (1H, s), 7.90 (2H, m) (ESM3, Online Resource 3); 13C-NMR: (100 MHz, DMSO) δ (ppm) = 29.18 (1C, CH2), 72.05 (1C, CH2), 105.06 (C3, J = 23.46 Hz), 106.90 (C5, J = 22.70 Hz), 109.97 (C7′), 116.57 (α-C), 117.20 (C1, Cq), 125.26 (C4′), 127.36 (Cq, C-CH2), 128.40 (C5′), 130.64 (C6′), 131.75 (C6, Cq, J = 11.84 Hz), 146.11 (β-C), 163.16 (C–O), 166.06 (C2, C–OH), 167.25 (C4, J = 256.35 Hz), 192.47(C=O) (ESM4, Online Resource 3).

2.2.4 (E)-3-(3′,5′-Bis[trifluoromethyl]phenyl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one (7)

The title compound (1.04 g, 55%) was prepared from 4-fluoro-2-hydroxyacetophenone (0.77 g, 5 mmol) and 3,5-Bis[trifluoromethyl]benzaldehyde (1.21 g, 5 mmol) as a pale yellow crystalline solid, m.pt 146 -147 °C. GCMS m/z = 377.1, 360, 309, 270, 240, 227, 165, 139, 111, 88, 74, 55 (ESM1, Online Resource 4); FTIR-ATR (v cm−1) = 1654.9 (C=O), 3436 (–OH), 3000 (Ar C-H), 2997 (Alip C-H), 1490 (Alip C=C) (ESM2, Online Resource 4); 1H-NMR: (400 MHz, DMSO) δ (ppm) = 12.79 (1H, Broad, –OH), 6.88 (2H, ddd, J = 2.53 Hz, 9.59 Hz, 13.18 Hz), 7.97 (1H, d, J = 15.64 Hz, α -H), 8.14 (1H, s), 8.29 (1H, d, J = 15.64 Hz, β-H), 8.44 (1H, dd, J = 6.75 Hz, 8.94 Hz), 8.65 (2H, s) (ESM3, Online Resource 4); 13C-NMR: (100 MHz, DMSO) δ (ppm) = 104.77 (C3, d, J = 23.77 Hz), 107.60 (C5, d, J = 22.60 Hz), 118.37 (C1, Cq), 123.67 (2C, d, J = 273.07, C-F3), 123.89 (α-C), 125.99 (C4′), 130.01 (C2′, C6′), 131.38 (C3′, C5′, q, J = 33.98 Hz, 50.00 Hz, Cq), 134.61 (C6, d, J = 11.90 Hz), 137.70 (C1′, Cq), 141.66 (β-C), 165.03 (C2, J = 14.23 Hz, C–OH), 167.27 (C4, d, J = 254.47 Hz), 192.76 (C=O) (ESM4, Online Resource 4).

2.2.5 (E)-3-(4′-diethylaminophenyl)-1-(4-Fluoro-2-hydroxylphenyl)prop-2-en-1-one (8)

The title compound (1.18 g, 75%) was prepared from 4-fluoro-2-hydroxyacetophenone (0.77 g, 5 mmol) and 4-(diethylamino)benzaldehyde (0.89 g, 5 mmol) as a red crystalline solid, m.pt 129–131 °C. GCMS m/z = 312.2, 298, 252, 219, 183, 160, 139, 105, 83, 55 (ESM1, Online Resource 5); FTIR-ATR (v cm−1) = 1654.9 (C=O), 3417 (–OH), 3000 (Ar C-H), 2914 (Alip C-H), 1520 (Alip C=C) (ESM2, Online Resource 5); 1H-NMR: (400 MHz, DMSO) δ (ppm) = 1.13 (6H, t, J = 7.01 Hz, CH3), 3.44 (4H, q, J = 7.01 Hz, CH2), 13.72 (1H, s, –OH), 6.73 (2H, d, J = 9.00 Hz,), 6.82 (2H, m,), 7.71 (3H, dd, J = 12.02 Hz, 18.76 Hz, α-H), 7.82 (1H, d, J = 15.08 Hz, β-H), 8.38 (1H, dd, J = 6.97 Hz, 8.58 Hz) (ESM3, Online Resource 5); 13C-NMR: (100 MHz, DMSO) δ (ppm) = 12.94 (2C, CH3), 44.35 (2C, CH2), 104.62 (C3, d, J = 23.53 Hz), 107.16 (C5, d, J = 22.19 Hz), 111.67 (C3′, C5′), 114.28 (C1, Cq), 118.15 (α-C), 121.42 (C1′, Cq), 132.43 (C2′, C6′), 133.62 (C6, d, J = 11.68 Hz), 147.40 (β-C), 150.54 (C4′, C–N), 165.25 (C2, C–OH), 166.68 (C4, d, J = 252.93 C-F), 192.48 (C=O) (ESM4, Online Resource 5).

2.3 Antibacterial evaluation

The antibacterial activities of the compounds were evaluated using the following organisms, Gram-positive:Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant enterococci (VRE), Staphylococcus aureus, Streptococcus pyrogenes, Streptococcus feacalis andGram-negative:Escherichia coli, Salmonella typhi, Corynebacterium ulcerans, Proteus mirabilis, Pseudomonas aeruginosa. All were clinical isolates and were obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria.

2.4 Preparation of culture media and compounds

Mueller–Hinton agar (Oxoid, England) was used as the growth medium for the microbes. The dehydrated bacteriological culture media was weighed (36 g) and dissolved in distilled water (100 mL) according to the manufacturer’s specification. The resultant suspension was dispensed into clean conical flask and sterilized at 121 °C for 15 min in an Adelphi bench autoclave and then poured into previously sterilized petri dishes. The plates were allowed to cool and solidify, initial concentration of each of the compounds (200 µg/mL) was prepared by weighing the compound (0.002 g) and dissolving in DMSO (10 mL) to obtain the stock solution [14].

2.5 Antimicrobial profile (zone of inhibition)

The antibacterial screening was carried out using the agar diffusion method [14]. The prepared medium was seeded with standard inoculums (0.1 mL) of the micro-organism. The inoculums were then spread evenly using a sterilized swab over the surface of the medium, seeded plates were allowed to dry at 37 °C for 30 min inside an incubator (GENLAB WIDNES ENGLAND, model: EIS). A standard cork borer, 6 mm in diameter was used to cut a well at the centre of each seeded medium used and 0.1 mL of the solution of the compounds (200 µg/mL) was then introduced into each hole on the surface of the medium. The plates were then incubated at 37 °C for 24 h, after which the plates were observed for zone of inhibition at growth. The zones of inhibition were measured with a transparent ruler and the results recorded in millimeter. Ciprofloxacin (10 µg/mL) was used as the standard drug.

2.6 Determination of minimum inhibitory concentration (MIC)

MIC was carried out using broth dilution method [15, 16]. Mueller–Hinton broth (Oxoid, England) was prepared and 10 mL was dispensed into test tubes and sterilized at 121 °C for 15 min, the broth was allowed to cool. Mac-Farland’s turbidity standard scale number 0.5 was prepared to give a turbid solution. Normal saline was prepared and 10 ml was dispensed into sterilized test tubes and the test micro-organism was inoculated and incubated at 37 °C for 6 h. After incubation, dilution of the micro-organism in normal saline was done until the turbidity matched that of the Mac-Farland scale by visual comparison, at this point the test micro-organisms have a concentration of about 1.5 × 108 cfu/mL.

Two-fold serial dilution of the compound in the sterilized broth was done to obtain the concentration of 200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, and 12.5 µg/mL respectively. The initial concentration was obtained by dissolving 0.002 g of the compound in 10 mL of the sterile broth. From the suspension of the micro-organism in normal saline, 0.1 mL was inoculated into the different concentrations of the compound in the Mueller–Hinton broth. The broths were then incubated at 37 °C for 24 h, after which the test tubes were observed for turbidity which indicates growth of the microorganism. The test tube with lowest concentration of the compound which showed no turbidity was recorded as the minimum inhibitory concentration (MIC).

2.7 Determination of minimum bactericidal concentration (MBC)

MBC was carried out to check whether the test microbes were killed or only their growth were inhibited. Mueller–Hinton agar (Oxoid, England) was prepared according to manufacturer’s instruction, sterilized at 121 °C for 15 min. It was poured into sterilized petri-dishes. The plates were allowed to cool and solidify. The content of the MIC test tubes in the serial dilution were sub-cultured on to the prepared plates. The plates were then incubated at 37 °C for 24 h, after which the plates were observed for colony growth. The MBC was the plate with lowest concentration of the compound without colony growth [15, 16].

3 Results and discussion

3.1 Synthesis of E-1-(2-hydroxylphenyl) prop -2-en-1-one derivatives

An illustration of the synthetic approach to the prepared compounds is illustrated in Scheme 1. The compounds were yellow in colour except for compound (8) which was red in colour which may be due to increased conjugation and the strong complementary electronic effect of the amino substituent at the para position of the B-ring. Product yields range from 55 to 84% (Table 1). The highest yield was obtained for compound (6) containing 2,3-dihydrobenzofuran moiety while compound (7) gave the least yield. The poor yield of the later might be due to the bulky nature of trifluoromethyl substituents at the meta positions of the ring B that may hinder proper condensation of the reactants [13]. In general, the rate of reaction decreased in the order of 2,3-dihydrobenzofuran > 2-ethoxyphenyl > 3,4-diethoxyphenyl > 4-(diethylamino)phenyl > 3,5-bis[trifluoromethyl]phenyl with regard to substituents on the B-ring.
Scheme 1

Synthetic route to the chalcones

Table 1

Physical data of the 4-fluoro-2-hydroxychalcone derivatives

Compound

B-ring

M.F

M.W (g)

Colour

Rf (H:E)

M.P range (oC)

Yield (%)

4

2-ethoxyphenyl

C17H15 FO3

286

Pale yellow

0.40

90–92

80

5

2,3-diethoxyphenyl

C19H19FO4

330

Yellow

0.48

128–130

78

6

2,3-dihydrobenzofuran

C17H13FO3

284

Yellow

0.40

123–124

84

7

3,5-Bis[trifluoromethyl]phenyl

C17H9 F7O2

378

Pale yellow

0.82

146–147

55

8

4-(diethylamino)phenyl

C19H20O2FN

313

Red

0.58

129–131

75

M.F molecular formula, M.W molecular weight (theoretical), Rf retardation factor, M.P melting point range, H hexane, E ethyl acetate (9:1)

The 13C NMR spectra showed highly deshielded carbonyl carbon resonance ranging from 192.43 to 192.97 ppm while the hydroxylated carbon (C-2) signals appeared from 164.82 to 166.08 ppm. The shift between 117.30 and 123.89 ppm was assigned to α-C while between 140.17 and 147.40 ppm to β-C of the conjugated chromophores [13] (Table 2). The delocalization of electrons among the three carbon atoms of the chromophore resulted in the β-carbon resonance being more deshielded than α-carbon.
Table 2

Characteristics 13C NMR chemical shift values of 4-fluoro-2-hydroxychalcone derivatives

Compound

B-ring

C–OH δ (ppm)

C=O δ (ppm)

α-C δ (ppm)

β-C δ (ppm)

4

2-ethoxyphenyl

164.82

192.97

121.60

140.17

5

3,4-diethoxyphenyl

166.08

192.43

117.30

146.08

6

2,3-dihydrobenzofuran

166.06

192.47

116.57

146.11

7

3,5-Bis[trifluoromethyl]phenyl

165.10

192.76

123.89

141.66

8

4-(diethylamino)phenyl

165.25

192.48

118.15

147.40

δ chemical shift value

The use of proton decoupled 13C spectrometer for the analyses of the compounds resulted in the doublet splitting of fluorinated aromatic carbon (C-4) peaks and that of all the carbons at ortho (C-3 and C-5) and meta (C-6) positions relative to the fluorine atom on the ring (Fig. 1) with the exception of hydroxylated carbon (C-2). The presence of hydroxyl group on C-2 might be the reason for non-splitting of its peaks. The fluorinated carbon resonances (C-4) appeared highly deshielded between 166.68 to 167.29 ppm with large coupling constants (J) between 252.93 to 256.35 Hz. The C-3 peaks appeared between 104.71 to 105.11 ppm (J between 23.42 to 23.77 Hz); the C-5 peaks was between 106.90 to 107.60 ppm (J between 22.19 to 22.74 Hz and C-6 peaks between 131.75 to 133.95 ppm (J between 11.68 to 11.84 Hz) (Table 3). This is similar to values obtained in other studies (for Aryl C-F δ = 163.7 ppm, J = 255.3 Hz; C-3 and C-5, δ = 105.0 ppm, J = 22.1 Hz) and C-6, δ = 130.6 ppm, J = 8.8 Hz) [17].
Fig. 1

13CNMR spectrum showing the doublets of C-3 and C-5 peaks

Table 3

13C NMR chemical shift and coupling constant of A-ring of 4-fluoro-2-hydroxychalcone derivatives

Compound

B-ring

C-3

δ (ppm)

C-4

δ (ppm)

C-5

δ (ppm)

C-6

δ (ppm)

4

2-ethoxyphenyl

105.11 (d)

J = 23.42 Hz

167.29 (d)

J = 256.26 Hz

106.94 (d)

J = 22.74 Hz

131.81 (d)

J = 11.77 Hz

5

3,4-diethoxyphenyl

105.06 (d)

J = 23.46 Hz

167.25 (d)

J = 256.35 Hz

106.90 (d)

J = 22.70 Hz

131.75 (d)

J = 11.84 Hz

6

2,3-dihydrobenzofuran

104.77 (d)

J = 23.77 Hz

167.27 (d)

J = 254.47 Hz

107.60 (d)

J = 22.36 Hz

134.61 (d)

J = 11.90 Hz

7

3,5-Bis[trifluoromethyl]phenyl

104.62 (d)

J = 23.53 Hz

166.68 (d)

J = 252.93 Hz

107.16 (d)

J = 22.19 Hz

133.62(d)

J = 11.68 Hz

8

4-(diethylamino)phenyl

104.71 (d)

J = 23.66 Hz

166.92 (d)

J = 253.69 Hz

107.49 (d)

J = 22.32 Hz

133.91 (d)

J = 11.84 Hz

d duplet, J coupling constant, δ chemical shift value

For 1H NMR spectra, the two olefinic trans protons of the chromophores were characterized by the presence of large coupling constants ranged from 15 to 19 Hz, which is expected for trans coupled olefinic protons.

The FTIR spectra showed broad intense peaks mostly at 3414 cm−1 for hydrogen bonded hydroxyl groups. The bands ranging from 1636 to 1654 cm−1 was observed for C=Ostr, 3000 cm−1 for aromatic C–Hstr, 2914 and 2996 cm−1 aliphatic C-Hstr and 1490 and 1520 cm−1 for Cα = Cβ stretching of the chromophores. The m/z values of the compounds were equivalent to their respective theoretical molecular weights which confirmed the products.

4 Antibacterial evaluation

All the compounds showed varying degrees of antibacterial activities against both Gram-positive and Gram-negative species based on the observed zones of inhibition which ranged from 20 to 28 mm (Table 4). The compounds that showed positive activity were further tested to determine their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (Table 5). The activities of the compounds compared favourably with that of the standard drug (ciprofloxacin) and in some cases where the microbes were resistant to the standard drug, they were sensitive to some of the prepared compounds. For instance, all the compounds, except compound (6) containing the 3′,5′-Bis[trifluoromethyl]phenyl moiety, have a broad-spectrum activity against MRSA with zone of inhibition ranging from 23 to 28 mm while the standard drug exhibited no potency against this microbe. Compounds (4), (5) and (8) containing the 4′-(diethylamino), 2′-ethoxyl and 3′,4′-diethoxyl substituents respectively on the B-ring showed remarkable activity against C. ulcerans whereas standard drug was inactive against this microbe. P. aeruginosa was resistant to standard drug as well as all the compounds with the exception of compound (8) containing the 4′-(diethylamino)phenyl moiety. The activity of this compound against P. aeruginosa made it a potential drug against this multi-drug resistance species.
Table 4

Zone of inhibition (mm, m ± s) of prepared 4-fluoro-2-hydroxychalcone derivatives

Compound/Microbes

4

5

6

7

8

CIP

MRSA

25.3 ± 1.25

28.0 ± 0.82

26.3 ± 1.25

0

23.0 ± 1.63

0

VRE

24.0 ± 1.63

25.7 ± 1.25

20.0 ± 1.63

23.0 ± 1.63

20.0 ± 1.63

28.0 ± 0.82

S. aureus

27.0 ± 0.82

25.7 ± 1.25

21.0 ± 1.63

25.3 ± 1.25

26.3 ± 1.25

29.0 ± 0.82

S. pyrogenes

26.0 ± 0.82

24.3 ± 1.25

23.0 ± 1.63

28.0 ± 0.82

27.3 ± 1.25

27.0 ± 0.82

S. feacalis

0

0

20.0 ± 1.63

0

24.7 ± 1.25

30.0 ± 0.82

C. ulcerans

23.0 ± 1.63

26.0 ± 0.82

0

0

26.3 ± 1.25

0

E. coli

28.0 ± 0.82

27.3 ± 1.25

0

0

0

31.0 ± 0.82

P. mirabilis

0

0

0

0

0

27.0 ± 0.82

P. aeruginosa

0

0

0

0

26.3 ± 1.25

0

S. typhi

24.0 ± 1.63

0

25.3 ± 1.25

28.0 ± 0.82

27.3 ± 1.25

38.0 ± 0.82

CIP ciprofloxacin, m mean zone of inhibition, s standard deviation, MRSA methicillin-resistant Staphylococcus aureus, VRE vancomycin-resistant enterococci

Table 5

MIC & MBC (µg/mL) regimes of prepared 2-hydroxylchalcone derivatives

Compounds

4

5

6

7

8

Microbe

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MRSA

50

100

25

50

50

100

50

100

VRE

50

100

50

100

50

200

50

100

50

100

S. aureus

25

100

50

100

50

200

50

100

50

100

S. pyogenes

50

100

50

100

50

100

50

50

25

50

S. feacalis

50

200

50

100

C. ulcerans

50

200

50

100

50

100

E. coli

25

50

25

P. mirabilis

P. aeruginosa

50

100

S. typhi

50

100

50

100

25

50

50

100

MIC minimum inhibition concentration, MBC minimum bactericidal concentration

The MIC of all the compounds were between 25 and 50 µg/mL while MBC ranged from 50 to 200 µg/mL. The activities of the compounds against the tested microbes were observed to decrease in order of compound containing 4′-(diethylamino)phenyl > 2′-ethoxyphenyl > 3′,4′-diethoxyphenyl > 2,3-dihydrobenzofuran > 3,5-Bis[trifluoromethyl]phenyl moieties. This observation might be due to electronic effect of substituents on the B-ring with compound (8) containing the 4′-(diethylamino) substituent being a strong electron releasing group and at the para position of the ring. This is the most active compound against the bacteria strains. The combined effects of the fluorine atom on the A-ring and the electron releasing group on the B-ring in (8) which also has the added advantage of forming an ammonium ion in physiological pH conferring a positive charge on the compound could enable it to easily penetrate the negatively charged bacterial cell wall especially of hardy Gram-negative bacteria. The negative inductive effect of the trifluoromethyl substituent in compound (6) might be responsible for its lower activity.

(E)-3-(4-fluorophenyl)chalcones have been reported to show antitubercular and antibacterial activities against S. aureus and antifungal activity against C. albicans. The research further revealed that fluorinated chalcones with fluoro-substitution in position 2 and or position 5 of its B ring showed higher antibacterial potency against S. aureus, S. pyogenes, E. feacalis, E. coli, P. aeruginosa and antifungal activity against C. albicans, C. glabrata and C. parapsilosis. However, the presence of hydroxyl group on the A ring decreases the antitubercular activity of theses fluorinated chalcones [18]. Also, fluorinated chalcones-1,2,3-triazoles conjugates have been reported to show significant activity against some bacterial and fungal strains [19]. Several other reports on biological evaluation of fluorinated chalcones revealed some of its pharmacological importance. For instance, (E)-1-(2-hydroxyphenyl)-3-(4-fluorophenyl)chalcones was shown to possess antibacterial activity against E. coli, B. pumilis and B subtilis and, antifungal activity against A. niger [20]. These findings on the potency, against various pathogens, of the synthesized chalcones are similar to the observations reported in the cited literatures,

Apart from the reported effect of fluoro groups on biological activitiy, other reports suggested that the α,β-unsaturated carbonyl group in chalcones are responsible for their antimicrobial activity [21] and the presence of more than one electron withdrawing group on B-ring of the chalcone skeleton might reduce this observed antimicrobial activity [22].

5 Conclusion

This work reports the synthesis, characterization and antibacterial activities of five derivatives of (E)-1-(4-fluoro-2-hydroxylphenyl)chalcone with 4′-(diethylamino)phenyl, 2′-ethoxyphenyl, 3′,4′-diethoxyphenyl, 2′,3′-dihydrobenzofuran and 3′,5′-Bis[trifluoromethyl]phenyl moieties as the B-ring. These compounds were synthesized in good yield by a modified Claisen-Schmidt condensation and the structures characterized using NMR and GCMS spectral analysis. The fluorinated chalcones were then evaluated for antibacterial activities against MRSA, VRE, Staphylococcus aureus, Streptococcus pyrogenes, Streptococcus feacalis (Gram-positive) and Escherichia coli, Salmonella typhi, Corynebacterium ulcerans, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative). The antibacterial activity of the compounds compared favourably with that of ciprofloxacin and appeared to have broad spectrum activity. Compounds containing the electron releasing moieties of 4′-(diethylamino)phenyl, 2′-ethoxyphenyl and 3′,4′-diethoxyphenyl were found to be the most potent in that order while the compound containing the strong electron withdrawing moiety of 3′,5′-Bis[trifluoromethyl]phenyl was found to be the least potent especially against Gram-negative bacterial strain. The results suggest that the synthesized compounds, especially the compound with the 4′-(diethylamino) substituent, were better, and could be used, after in vivo and clinical tests and establishing their safety profiles, to supplement or even replace current drug therapies. Especially for drugs which pathogenic microorganisms have or are developing resistance, for example MRSA and P. aeruginosa which are responsible for life-threatening, and other nosocomial infections such as life-threatening diseases for example pneumonia, meningitis, toxic shock syndrome, sepsis, urinary tract infections (UTIs), and bacteremia.

Notes

Acknowledgments

The authors are thankful to Prof. John Igoli of Federal University of Makurdi, Nigeria and Mr. Bashir of Multi-User Laboratory, Ahmadu Bello University, Nigeria for spectroscopic analyses.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

42250_2019_43_MOESM1_ESM.pdf (187 kb)
Supplementary material 1 (PDF 187 kb)
42250_2019_43_MOESM2_ESM.pdf (160 kb)
Supplementary material 2 (PDF 159 kb)
42250_2019_43_MOESM3_ESM.pdf (174 kb)
Supplementary material 3 (PDF 174 kb)
42250_2019_43_MOESM4_ESM.pdf (158 kb)
Supplementary material 4 (PDF 158 kb)
42250_2019_43_MOESM5_ESM.pdf (172 kb)
Supplementary material 5 (PDF 171 kb)

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Copyright information

© The Tunisian Chemical Society and Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Faculty of Physical Sciences, Department of ChemistryAhmadu Bello UniversityZariaNigeria

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