Synthesis, Characterization, and Antioxidant Activity of Some 2-Methoxyphenols derivatives

General

All chemicals and reagents were obtained from Sigma-Aldrich Chemical Company (Sigma Aldrich Chemical Company, street. Louis, MO, USA) and used without additional purification. Thin-layer chromatography was performed on silica gel coated with fluorescent indicator F₂₅₄ plates purchase from Fluka Company. Column chromatography was implemented on Silica gel S, 0.063-0.1 mm. Melting points were determined on a Gallenkamp melting point apparatus and were uncorrected. IR spectra were measured using KBr pellets on a Thermo Nicolet model 470 Fourier-transform spectrophotometer. Nuclear magnetic resonance (¹H and ¹³C spectra) were recorded on a Varian 400 MHz spectrophotometer. Chemical shifts were expressed in part per million (δ) with tetramethylsilane (TMS) as an internal standard. Elemental analysis was conducted using a Leco CHN-600 elemental analyzer.

Synthesis of methoxyphenol derivatives

5-(4-Hydroxy-3-methoxybenzyl)-2,2-dimethyl-1,3-dioxane-4,6-dione (4)

4-Hydroxy-3-methoxybenzaldehyde 2 (1.5 g, 10.0 mmol) was dissolved in 2-propanol and acetic acid (0.1 equiv). Then, 2,2-dimethyl-1,3-dioxane-4,6-dione 1 (1.5 g, 10.5 mmol) was added. Then the reaction was heated to 75 °C for 2 h. The solvent was removed under reduced pressure. The residue was washed 2-3 times with aqueous NaHSO₃ solution (20 % w/v), water and hexane to obtain compound 3 in 70 % and 1.9g. Compound 3 was dissolved in dichloromethane (100 mL) and the solution was cooled in an ice bath, then acetic acid (31.0 equiv) was added. After 15 min, NaBH₄ (8.0 equiv) was added within 1 h. The reaction was removed from the ice bath and kept at room temperature at 25^oC for 1 h. After discoloration, the reaction mixture was diluted with 100 mL water and extracted three times with 50 mL with dichloromethane. The organic layers were collective and washed two times with water. The organic layer was dried over MgSO₄, and the solvent was evaporated in vacuum, yielding compound 4 after recrystallization from hot ethyl acetate-hexanes. This compound was white powder; yield, 63 % and 1.76 g; mp, 130 °C; IR (KBr, cm^–1): 3518 (-OH), 2874 (C-H aliphatic), 1793, 1755 (C=O); ¹H-NMR [CDCl₃, 400 MHz]: (δ, ppm) 6.85 (brs, 1H, aromatic H), 6.81–6.68 (m, 2 H, aromatic H), 5.53 (brs, 1H, OH, exchanges with D₂O), 3.85 (s, 3H, OCH₃), 3.66 (t, J = 4.9 Hz, 1H, CH), 3.42 (d, J = 4.9 Hz, 2H, CH₂), 1.72 (s, 6H, CH₃); ¹³C-NMR [CDCl₃, 100 MHz]: (δ, ppm) 165.5, 147.7, 144.9, 128.8, 120.8, 115.6, 113.0, 105.2, 55.9, 46.3, 31.1, 26.5; Anal. Calcd for C₁₄H₁₆O₆ (280.28): C, 60.00; H, 5.75; Found: 59.97; H, 5.69.

3-(4-Hydroxy-3-methoxyphenyl)propanoic acid (7)

To a solution of 4-hydroxy-3-methoxybenzaldehyde 1 (1 mmol) and malonic acid 5 (1.2 mmol) in pyridine (2.5 mL/mmol), piperidine (0.04 mmol) was added at 120 °C, and the reaction mixture was heated under reflux for 4 h. The solvent was removed in vacuo as an azeotrope with toluene (3×70 mL). Water (100 mL) was added, and the aqueous phase was extracted with ethyl acetate (3×100 mL) and ethanol (1×100 mL), dried over MgSO₄, and concentrated to obtain 6 in 74 % and 0.14g. Then, the catalyst, 10% Pd/C (0.014 g), was added to a solution of 6 (0.194 g, 1 mmol) in degassed ethyl acetate (100 mL). The reaction mixture was stirred at room temperature overnight under H₂ atmosphere. After completion, the reaction mixture was filtered and concentrated to afford compound 7; yield 95 % and 0.18g; mp, 92 °C; IR (KBr, cm^–1): 3409 (-OH), 2938 (CH-aliphatic), 1708 (C=O), 1605 (C=C); ¹H-NMR [CDCl₃, 400 MHz]: (δ, ppm) 10.50 (brs, 1H, CO₂H, exchanges with D₂O), 6.87 (d, J = 8.0 Hz, 1H), 6.73 (brs, 1H), 6.71 (d, J = 8.0 Hz, 1H), 5.60 (brs, 1H, OH, exchanges with D₂O), 3.88 (s, 3H, OCH₃), 2.90 (t, J = 7.8 Hz, 2H), 2.70 (t, J = 7.8 Hz, 2H); ¹³C-NMR [CDCl₃, 100 MHz]: (δ, ppm) 179.2, 146.4, 144.1, 132.0, 120.8, 114.4, 110.9, 55.8, 36.0, 30.3.

3-(4-Hydroxy-3-methoxyphenyl)-1-(piperidin-1-yl) propan-1-one (8)

N,N-Carbonyldiimidazole (0.545 g, 3.36 mmol) was added to a solution of 3-(4-hydroxy-3-methoxyphenyl) propanoic acid 7 (0.6 g, 3.06 mmol) in 10 mL of dry THF. After 30 min, piperidine (0.302 mL, 3.06 mmol) was added and the mixture was stirred overnight. The precipitate formed then was washed with dichloromethane to obtain 8. White solid; yield 60 % and 0.48g; mp 120 °C; IR (KBr, cm^–1): 3392 (-OH), 3010 (C-H aromatic), 1749 (C=O); ¹H-NMR [DMSO-d₆, 400 MHz]: (δ, ppm) 8.68 (brs, 1H, OH, exchanges with D₂O), 6.77 (d, J = 1.9 Hz, 1H, aromatic H), 6.64 (d, J = 8.0 Hz, 1H), 6.59 (dd, J = 8.0, 2.0 Hz, 1H), 3.72 (s, 3H, OCH₃), 3.313.35 (m, 4H, CH₂ piperidine), 2.67 (m, 2H, CH₂), 2.51-2.53 (m, 2H, CH₂), 1.36-1.53 (m, 6H, CH₂ piperidine); ¹³C-NMR [DMSO-d₆, 100 MHz]: (δ, ppm) 170.1, 147.7, 144.9, 132.6, 120.8, 115.6, 113.0, 55.9, 46.3, 34.8, 31.1, 26.5, 24.5; Anal. Calcd for C₁₅H₂₁NO₃ (263.34): C, 68.42; H, 8.04; N, 5.32; Found: C, 68.39; H, 7.98; N, 5.27.

(E)-1-(4-Chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl) prop-2-en-1-one (10)

4-Hydroxy-3-methoxybenzaldehyde 1 (1 g, 6.57 mmol) and 1-(4-chlorophenyl)ethenone 9 (852 μL, 6.57 mmol) were mixed and stirred in 10 mL of methanol for 5-10 min; then H₂SO₄ (98 %, 3.75 mmol) was added and the reaction was heated to 80 °C for 48 h. After completion, the solution was neutralized by adding a NaOH solution (20 % w/v). The solution then extracted with ethyl acetate after diluted with water. The organic layer was dried over Na₂SO₄ and the ethyl acetate was evaporated. The crude product was purified using silica gel flash chromatography to yield compound 10; yield, 48 % and 0.14g; mp, 218 °C; IR (KBr, cm-1): 3430 (-OH), 3093 (C-H aromatic), 2954 (C-H aliphatic), 1651 (C=O), 1563 (C=C), 1522 (C=C aromatic); ¹H-NMR [DMSO-d₆, 400 MHz]: (δ, ppm) 10.31 (brs, 1H, OH, exchanges with D2O), 7.79 – 7.77 (m, 2H, aromatic H), 7.57 (d, d, 1H, COCH=CH, J = 15 Hz), 7.52 – 7.50 (m, 2H, aromatic H), 7.11 – 6.97 (m, 3H, aromatic H), 6.14 (d, 1H, COCH=CH, J = 15 Hz), 3.67 (s, 3H, OCH₃); ¹³C-NMR [DMSO-d₆, 100 MHz]: (δ, ppm) 185.4, 151.8, 150.6, 148.1, 145.6, 143.6, 139.1, 135.3, 129.9, 128.7, 118.7, 114.8, 107.6, 55.2; Anal. Calcd for C₁₆H₁₃ClO₃ (288.73): C, 66.56; H, 4.54; Found: C, 66.53; H, 4.48.

(2E,6E)-2,6-bis(4-Hydroxy-3-methoxybenzylidene) cyclohexanone (12)

4-Hydroxy-3-methoxybenzaldehyde 1 (1.9 g, 12.5 mmol) and cyclohexanone 11 (0.65 mL, 6.25 mmol) were mixed and stirred at room temperature for 5 min. Then, conc. hydrochloric acid (4 mL) was added slowly, followed by stirring for 3 h. Cold water was added to the mixture, filtered and dried in vacuum. The product was recrystallized from ethanol to obtain compound 12; yield 70 % and 3.2 g; mp, 182 °C; IR (KBr, cm^–1): 3368 (-OH), 3045 (C-H aromatic), 2988 (C-H aliphatic), 1639 (C=O), 1577 (C=C), 1515 (C=C aromatic); ¹H-NMR [CDCl₃, 400 MHz]: (δ, ppm) 9.83 (brs, 2H, OH, exchanges with D₂O),7.74 (s, 2H, vinylic H), 7.09-6.93 ( m, 6H, aromatic H), 3.92 (s, 6H, OCH₃), 2.95 – 2.95 (m, 4H, CH₂), 1.84-1.78 (m, 2H, CH₂); ¹³C-NMR [CDCl₃, 100 MHz]: (δ, ppm) 190.1, 146.4, 137.0, 134.4, 128.5, 124.4, 114.4, 113.2, 55.9, 28.5, 23.0; Anal. Calcd for C₂₂H₂₂O₅ (366.41): C, 72.12; H, 6.05; Found: C, 72.09; H, 5.99.

(E)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one (13)

4-hydroxy-3-methoxybenzaldehyde 1 (3.04 g, 0.020 mol) was dissolved in 12 mL of acetone under stirring at room temperature. Then, 10% aqueous NaOH (9 mL) was added and allowed to stand for 48 h. Distilled water (50 mL) was added. Then, 10% aqueous HCl (15 mL) was added. The yellow precipitate formed was filtered, dried and recrystallized from ethanol/water to yield compound 13. This compound was yellow powder; yield 70 % and 2.7 g; mp 126 °C; IR (KBr, cm^–1): 3285 (-OH), 3001 (C-H aromatic), 2849 (C-H aliphatic), 1675 (C=O), 1638 (C=C); ¹H-NMR [CDCl₃, 400 MHz]: (δ, ppm) 7.43-7.47 (d, J = 16.2 Hz, olefinic H), 7.06-7.11 (m, 2H, aromatic H), 6.93-6.95 (d, J = 8 Hz, 1H, aromatic H), 6.57-6.61 (d, J = 16.2 Hz, olefinic H), 5.91 (s, 1H, OH, exchanges with D₂O), 3.94 (s, 3H, OCH₃), 2.37 (s, 3H, CH₃); ¹³C-NMR [CDCl₃, 100 MHz]: (δ, ppm) 198.5, 148.2, 146.9, 143.8, 127.0, 125.0, 123.5, 114.8, 109.2, 55.9, 27.5; Anal. Calcd for C₁₁H₁₂O₃ (192.21): C, 68.74; H, 6.29; Found: C, 68.71; H, 6.23.

(1E,4E)-1-(4-Hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)penta-1,4-dien-3-one (15)

Compound 9 (0.5 g, 2.60 mmol) and 4-hydroxybenzaldehyde 14 (0.32 g, 2.60 mmol) were mixed in 10 mL ethanol for 5 min. Then, concentrated HCl (0.5 mL) was added, followed by stirring overnight. The mixture was treated with cold water, filtered and dried in a vacuum. Compound 15 was obtained after recrystallized from an ethanol-H₂O mixture; yield 52 % and 0.4 g; mp, 176 °C; IR, (KBr, cm^–1): 3585 (-OH), 3069 (C-H aromatic), 2955 (C-H aliphatic), 1697 (C=O), 1593 (C=C), 1513 (C=C aromatic); ¹H-NMR [DMSO-d₆, 400 MHz]: (δ, ppm) 10.03 (brs, 1H, OH, exchanges with D₂O ), 9.68 (brs, 1H, OH, exchanges with D₂O), 7.65-7.66 (d, J = 15 Hz, ,2H, olefinic-H), 7.60-7.62 (m, 2H, aromatic H), 7.34-7.35 (d, J = 15 Hz, 2H, olefinic-H), 7.08-7.19 (m, 3H, aromatic H), 6.81-6.82 (m, 2H, aromatic H), 3.82 (s, 3H, OCH₃); ¹³C-NMR [DMSO-d₆, 100 MHz]: (δ, ppm) 188.5, 160.3, 149.8, 148.3, 143.3, 142.8, 130.9, 126.7, 126.3, 123.7, 123.4, 122.9, 116.3, 116.1, 111.7, 56.1; Anal. Calcd for C₁₈H₁₆O₄ (296.32): C, 72.96; H, 5.44; Found: C, 72.93; H, 5.39.

(1E,4E)-1-(4-Fluorophenyl)-5-(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (17)

4-fluorobenzaldehyde 16 (0.23 g, 2.60 mmol) was mixed with (E)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one 9 (0.5 g, 2.60 mmol) in 10 mL ethanol for 5 min. Then, 2M NaOH (10 mL) was added and stirred overnight. The resulting mixture was acidified by concentrated HCl, and the residue was washed with cold water and dried in air. The product was recrystallized from an ethanol/water mixture to obtain 17; yield, 73 % and 0.57 g; mp, 210 °C; IR (KBr, cm-1 ): 3427 (-OH), 3001 (C-H aromatic), 2926 (C-H aliphatic), 1674 (C=O), 1618 (C=C), 1577 (C=C aromatic); ¹H-NMR [DMSO-d₆, 400 MHz]: (δ, ppm) 9.87 (brs, 1H, OH, exchanges with D₂O), 7.91-7.95 (d, 2H, J = 15 Hz olefinic H), 7.72 (brs, 1H, aromatic H), 7.65 (m, 2H, aromatic H), 7.36 (d, 2H, J = 15 Hz olefinic H), 7.09 (m, 2H, aromatic H), 6.65 (m, 1H, aromatic H), 6.63 (m, 1H, aromatic H), 3.70 (s, 3H, OCH₃); ¹³C-NMR [DMSO-d₆, 100 MHz]: (δ, ppm) 177.2, 150.9, 150.7, 144.5, 143.3, 134.3, 133.7, 121.1 , 120.8, 120.1, 120.2, 119.5, 112.4, 112.3, 111.1, 65.1; Anal. Calcd for C₁₈H₁₅FO₃ (298.31): C, 72.47; H, 5.07; Found: C, 72.44; H, 5.01.

Antioxidant Activity

Total Antioxidant Capacity Test Using the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) Kinetic Assay (Free-Radical-Scavenging Activity)

The total antioxidant capacity was obtained using the ABTS kinetic assay according to the manufacturer’s instructions (Zenbio, Research Triangle Park, NC, USA). After diluting the myoglobin working solution with a dilution buffer, 10 μL of 100 μM of test compounds or Trolox standard or assay buffer were dispensed into a 96-well microplate. To start the reaction, 20 μL of the myoglobin working solution and 100 μL of the ABTS solution were added. The kinetic profile of ABTS+ scavenging by antioxidant compounds was monitored in a 96-well microplate using an Emax Plus microplate reader (Molecular Devices, CA, USA). The ABTS assay measured the free-radical-scavenging activity of the compounds. Data are presented as mean±SEM and expressed as micromoles Trolox equivalent (μM TE).

Antioxidant Activity by Oxygen Radical Absorbance Capacity (ORAC) Assay

The oxygen radical absorbance capacity of each test compound was determined using the ORAC Antioxidant Assay Kit (Zenbio, NC, USA) according to the manufacturer’s instructions. Trolox standards were prepared in assay buffer (0–100 μM), along with the test compounds (100 μM). Fluorescein working solution (150 μL) was added to the central wells of a 96-well black side, clear bottom plate, and mixed with 25 μL of each of the standards or test compounds in triplicate; the plate was incubated at 37 °C for at least 10 min. The 2,2′-azobis(2-amidinopropane) dihydrochloride [AAPH; sold as 2,2′-azobis(2-methylpropionamidine) dihydrochloride] working solution was added to each well (25 μL) to initiate the reaction. The fluorescence was measured after 30 min incubation at 37 °C using a multilabel plate reader (Perkin Elmer Victor3, USA) with excitation/ emission = 485/530 nm. Presented as mean±SEM and expressed as micromoles Trolox equivalent (μM TE). The ORAC values are presented as mean±SEM and expressed as μM TE.

Antioxidant Activity by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) Assay

The free radical scavenging ability of the test compounds was studied using the DPPH assay [7]. The test compounds in ethanol were prepared separately and added to the ethanol solution containing DPPH (0.01 mmol) within the range of 5–50 μL and adjusted to a final volume of 3 mL using ethanol as solvent. The scavenging ability of the test compounds was monitored spectrophotometrically by measuring the absorbance at 517 nm after 20 min. The % inhibition was obtained using equation (1).

The 50 % decline in absorbance of the DPPH solution was derived from the graph with the concentration (μM) plotted against the absorbance. These concentration values were used to determine the IC₅₀ values in μM.

Synthesis

Methoxyphenol derivatives have demonstrated extensive therapeutic effects; most of which depend on its antioxidant properties [8]. There have been recent efforts to maximize the therapeutic value of Methoxyphenol by creating structurally modified derivatives with potentially enhanced physicochemical, antioxidant, and therapeutic properties. In this study, we synthesized the six different methoxyphenol derivatives for antioxidant activity measurements. The first compound 5-(4-hydroxy-3-methoxybenzyl)-2,2-dimethyl-1,3-dioxane-4,6-dione 4, was obtained by condensation of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid) 1 with 4-hydroxy-3-methoxybenzaldehyde 2 in an acid-catalyzed, Knoevenagel-like reaction at 75 °C for 2 h generated compound 3, which was reduced to form compound 4 in cold DCM using in situ generated NaHB(OAc)₃ in an ice bath. The obtained material was recrystallized from hot ethyl acetate-hexane to obtain pure compound 4 (Scheme 1). The resultant product was white solid with a melting point at 130 °C and percentage yield of 63%. The structure of compound 4 was confirmed based on elemental analysis and spectroscopy data. The IR spectrum of 4 indicated the presence of a hydroxyl group stretching vibration at 3518 cm^–1. In addition, signals appeared at 2874, 1793, and 1755 cm^–1, which were attributed to aliphatic H and carbonyl groups, respectively. The ¹H-NMR spectrum of 4 showed a characteristic signal at δ = 1.72 ppm corresponding to the methyl protons; another signal, resonating at δ = 3.85 ppm, was attributed to the methoxy proton. The aromatic protons resonated as a multiplet between δ = 6.68 and 6.85 ppm with an integration value of three protons. The reduction of the ethylenic bond of compound 3 was characterized by the appearance of the new bands at δ = 3.42 and 3.66 ppm, corresponding to the CH₂ and CH protons, respectively. The hydroxyl group appeared at δ = 5.53 ppm due to the exchange with D₂O. The ¹³C-NMR spectrum of compound 4 showed two new signals at δ = 31.1 and 46.3 ppm, which were assigned to the CH and CH₂ carbons, while the signals at δ = 146.2, 144.7, 128.8, 122.5, 114.3, and 112.6 ppm were caused by the aromatic carbons. The signal at δ = 165.5 ppm was assigned to the two carbonyl carbons.

Scheme 1

The synthetic route to target compound 4

Knoevenagel-condensations [9] are used to access carboxylic acids from a malonic acid and an appropriate aldehyde. 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid 7 was synthesized by condensation of 4-hydroxy-3-methoxybenzaldehyde 1 with malonic acid 5 to generate (E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid 6 followed by hydrogenation in the presence of Pd/C to give compound 7 with a yield of 95 % yield. This compound was synthesized and characterized by the reduction of olefinic compound 6 and the appearance of two triplet bands in its ¹HNMR spectrum at δ = 2.90 and 2.70 ppm with the coupling constant J = 7.8 Hz respectively. In the ¹³CNMR spectrum, the two new carbons bands, representing two CH₂, were detected at δ = 36.0 and 30.3 ppm. The reaction of compound 7 with one equivalent of 1,1’carbonyldiimidazole in refluxing dry THF led to the formation of the corresponding N-acylpiperdine compound 8 (Scheme 2), which was characterized by NMR spectroscopy, FTIR, and elemental analysis. The IR spectral data of compound 8 showed sharp bands at 1749 and 3392 cm^‐1, indicating the presence of C=O and OH groups. The structure of 8 was also confirmed by the ¹H-NMR spectral data, which showed a characteristic signal that resonated at δ = 3.31–3.35 ppm and 1.36–1.53, attributed to the 10 protons of piperidine. The ¹³C-NMR spectrum showed new signals resonating at δ = 24.5, 26.5 and 46.3 ppm, corresponding to the piperidine carbons.

Scheme 2

The synthetic route to target compound 8

The synthesis of chalcones is well established and is based on the Claisen condensation of substituted benzaldehydes and acetophenones, and is achieved under either basic [10] or acid [11] condition. Confirming primary literature research [12], because of the presence of the phenolic substituent, the acid catalysis showed to be more appreciate in most of the cases and thus the procedure was used for the prepared of the 4-hydroxy compound 10 (Scheme 3). The structure of the compound 10 was established by spectral analysis. The FTIR spectrum of compound 10 showed characteristic absorption bands at 3430, 1651, and 1563 cm^–1, corresponding to the –OH, C=O, and C=C functional groups. The ¹H-NMR spectrum showed the presence of a broad singlet at δ = 10.31 ppm attributed to phenolic OH proton. Multiplet bands at δ = 7.52–7.50 ppm and 7.77–7.79 ppm were assigned to aromatic protons; a doublet (J = 15 Hz) at δ = 6.14 ppm was characteristic for the olefinic proton COCH=CH, and another doublet (J = 15 Hz) at δ = 7.57 ppm was characteristic for the second olefinic COCH=CH. The methyl protons appeared as a singlet in the δ = 3.67 ppm. Compound 12, another mono-carbonyl derivative of curcumin was prepared from available 4-hydroxy-3-methoxybenzaldehyde 1 and cyclohexanone 11 in ethanol via enamine intermediate in the presence of acidic medium. Compound 12 was obtained in a 70 % yield (Scheme 3). The FTIR spectrum of compound 12 recognized two absorption bands at 3368 and 1639 cm^–1, distinctive for OH and C=O groups. The ¹H NMR spectrum had two signals at δ = 7.74 ppm that coordinated the vinylic protons of benzylidenes. The ¹³C NMR spectrum of compound 12 showed that the carbonyl carbon was specified downfield at δ = 190.1 ppm.

Scheme 3

The synthetic route to target compounds 10, 12, 15 and 17

The unsymmetrical analogs of curcumin, compounds 15 and 17, were characterized by the presence of a short but entirely conjugated unsaturated ketone chain. Both compounds were prepared from (E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one 13. Compound 13 was obtained from vanillin and acetone in aqueous sodium hydroxide in a mixture that was stirred at room temperature for 12 h. The reaction mixture was diluted with ice water and neutralized with HCl to obtain a yellow solid 13 with a 70% yield. Compound 13 was used to prepare the unsymmetrical diarylpentanoid analogues 15 and 17. The targeted diarylpentanoids were synthesized to assess the effect of the substitution on the aromatic rings and to explore the effect of different aryl groups upon antioxidant activity. The characterization and spectroscopic date of compounds 15 and 17 were investigated. Compounds 15 and 17 were produced by reacting compound 13 with different aldehydes, having electron donating and electron withdrawing moieties. It was completely characterized by NMR. For compound 15, one double bond and aromatic moiety was added, and ¹H NMR had two more doublets for -CH=CH- with a coupling constant of approximately 15 Hz, which is characteristic of an H-H trans-coupling system at δ = 7.65 and 7.34 ppm. The aromatic multiplet signals appeared at δ = 7.60–7.62 ppm, 7.08–7.19 ppm, and 6.81–6.82 ppm, which integrated to seven protons.

Antioxidant Activity

Free radical scavenging capabilities were determined for all compounds by the DPPH and ABTS assays. DPPH is a very stable free radical, which is reduced by accepting hydrogen from a hydrogen donor compound. Reduction of DPPH results in the disappearance of its violet color, indicating the presence of free radical scavenging compounds in the reaction mixture. Similarly, the ABTS radical cation can be produced in aqueous solution. The blue color of the ABTS radical absorbs light at 734 nm and can be used to monitor the radical scavenging capacity of a substance relative to the antioxidant potential of this substance.

The DPPH and ABTS reducing capabilities of all synthetic compounds were evaluated by determining their IC₅₀ values. The antioxidant activity analysis of the six synthetic compounds indicated a strong reducing activity. The IC₅₀ values of all compounds obtained in the DPPH, ABTS, and ORAC assays are presented in Table 1. The IC₅₀ value corresponds to the concentration of a sample, which has ability to scavenge 50% of the free radicals present in the reaction mixture. Low IC₅₀ values indicate a high antioxidant activity of the sample compound.

Table 1

Antioxidant activity of phenolic acid derivatives using the DPPH, ABTS, and ORAC assays

Compound	Structure	IC50 (μM) DPPH	ABTS (μM TE)	ORAC (μM TE)
4		13.31 ± 1.5	183.3 ± 7.7	242.0 ±1.9
8		15.11± 1.7	141.8 ± 32.3	276.2 ±15.9
10		18.14± 1.6	165.9 ± 24.4	299.8 ± 4.4
12		40.18± 2.4	231.1 ± 10.4	280.7 ±6.3
15		40.6± 2.8	159.8 ± 34.5	302.3 ±3.0
17		19.3± 1.2	156.7 ± 28.5	310.3 ± 4.5

As shown in the Table 1 and Figure 2, all compounds had free radical scavenging activity in the DPPH assay. The IC₅₀ values in the DPPH assays were in the range of 13.3–40.6 μM DPPH. Compound 4, which has one -OH group, showed highest antioxidant activity (13.3 μM). Compound 15 with two -OH groups had the lowest antioxidant activity (40.6 μM). The difference in free radical scavenging activity among the six compounds could be related to the presence of different characteristic groups at the end of their carbon side chain. The compounds having the least polar groups attached with the 2-methoxyphenol moiety of derived compounds, (compound 4 and 8), had a higher free radical scavenging activity (13.3 μM and 15.1 μM, respectively) in the DPPH assay, compared with the compounds having more hydrophilic end groups (compounds 12 and 15; IC₅₀ 40.1 μM and 40.6 μM, respectively) (Table 1). High antioxidant activity by ferulic acid derivatives bearing non-polar groups in the side carbon chain has been reported already by Nenadis et al. [13].

Figure 2

Comparison of antioxidant activity of phenolic acid derivatives using the DPPH, ABTS, and ORAC assays

An interesting structural modification with a positive effect on the reducing properties of our synthetic compounds was the introduction of halogen atoms, Cl and F, at the end of the side chain of the compounds. The substitution of Cl and F doubled the free radical scavenging activity of compounds 10 (18.1 μM) and 17 (19.3 μM), compared to compounds 12 (40.1 μM) and 15 (40.6 μM) with an -OH group at the end of the side chain. Our results confirm the finding by Puskullu et al. [14]. Their study also indicated that according to the structure activity relationship, halogenated derivatives have a better antioxidant activity. The IC₅₀ of ferulic acid has been reported by Aleksandra et al.[15]; the DPPH value is 44.6 μmol/L. Therefore, all compounds had a higher antioxidant activity than ferulic acid in the DPPH assay. Moreover, the comparison of antioxidant activities of all compounds with the standard antioxidant molecules like ascorbic acid and Trolox revealed that all compounds have lower IC₅₀ vales than IC₅₀ values of ascorbic acid (41.25μg/ml) and Trolox (63.69 μg/mL) in DPPH assay, which have been reported by Matuszewska et al. [16].

The measurements of the ABTS and the ORAC assay showed lower antioxidant activities of compounds, compared to DPPH assay results. The IC₅₀ values by the ABTS method ranged from 134.5–231.1 μM TE (Table 1). This difference between DPPH and ABTS radical scavenging activities can be attributed to the solubility of a compound in a specific reaction medium. The DPPH assays are normally conducted in 50 % ethanol medium, and the ABTS assays are conducted in aqueous solution. Therefore, the compounds, which are less soluble in water have less activity in the ABTS assay than in the DPPH assay. As shown in Table 1, the DPPH assay results showed that compound 12 had the lowest antioxidant activity (231.1 μM TE) in the ABTS assay. Overall, despite the solubility issues in the DPPH and ABTS assay reaction mixtures, the antioxidant activities of the compounds were similar in these two assays, suggesting that the synthesized compounds possessed reliable, significant antioxidant properties.

In addition to the DPPH and ABTS free radical scavenging activities, the antioxidant activities of all six compounds were tested with the ORAC assay. The assay indicates the oxidative degradation of a fluorescent compound after being mixed with a free radical producer. The presence of the antioxidant protects the fluorescent molecule from degradation. Out of the six test compounds, the maximum antioxidant activity was observed with compound 4 (IC₅₀ 242 μM TE), followed by compound 8 (IC₅₀ 276.2 μM TE). A similar observation was made with the other two assays. However, instead of compound 12 and 15, which showed the lowest activity in the DPPH and ABTS assays, compound 17 showed the lowest antioxidant activity in the ORAC assay. The varying trends in the antioxidant activity by different assays could be explained with the effect of the reaction medium used in the different assays. Different compounds could have different hydrogen abstraction mechanisms depending on the reaction medium.

Mechanism Suggested for Hydrogen Abstraction from the Antioxidants

Free radicals (ROO^•) react with phenols (ArOH) via the following prominent mechanisms

1. Hydrogen atom transfer (HAT) is the abstraction of the hydrogen atom of ArOH by free radicals where the bond dissociation energy of O-H bond controls the rate of reaction. The HAT is dominant in a polar solvents and non-protic polar solvents [17]. These solvents do not support ionisation of ArOH thus the release of hydrogen as free radical from ArOH is slow therefore decreasing the rate of ArOH-free radical reactions. Hydrogen atom transfer (HAT) being very slow requires a higher concentration of the antioxidant to get 50% inhibition of free radicals, which would result in higher IC₅₀ value. Antioxidant action reduced with an increase in proportion of water. The consequence can elucidate in terms of Bond Dissociation Energy (BDE) of phenol [18]. Solvents such as alcohols and water are able to both accept and donate hydrogen bonds and therefore could either improve or reduce the Bond Dissociation Energy (BDE) of phenol. Water is a better donor but a poorer acceptor of hydrogen bonds than alcohols and hence should be able of better solvating the phenoxyl radical and solvating the parent phenol less or ethanol solvate curcuminoids rather than water. By increasing the percentage of water, the degree of solvation decreases and the phenoxide ion production decreases and this will affect the antioxidant activity. The buffer used as medium in both methods ABTS and ORAC, thereby decreasing the concentration of phenoxide ion formed from reacting antioxidants. Therefore, quenching of free radical produced requires a higher concentration of antioxidant samples. IC_5o values in ABTS and ORAC method are higher owing to the reduction of phenoxide ion in the lack of medium as shown in Scheme 4.

2. The sequential proton loss electro transfer mechanism (SPLET) [17] operates predominantly in polar protic solvents like methanol and ethanol.

Scheme 4

HAT in a polar and non-protic solvents

The kind of solvent and polarity might affect the process of single electron transfer and the hydrogen atom transfer, which are main features in the measurement of antioxidant activity. In DPPH assay method ethanol was used as a solvent, the antioxidants showed the maximum reducing ability. This behavior was attributed to polarity of ethanol establishing intermolecular hydrogen bonding amongst the solvent and thus hindering ArOH (substrate) and S (solvent) interaction as shown in Scheme 5.

Scheme 5

Proton Loss Electron Transfer (SPLET) in protic solvents

Therefore, substrate to solvent equilibrium do not occur in ethanol, allowing the ionisation of substrate (ArOH) depending on the properties of the solvent such as its relative permittivity (dielectric constant, ε_r), molecular property, its relative capability to solvate stabilising anions (ArO^–), as measured by Swain et al’s A value [17]. Ethanol as a solvent having high dielectric constant equal to 25 supports ionisation, ArOH loses proton to form stable phenoxide ion at fast rate. Therefore, quenching of DPPH occurs at low concentration of ArOH, obtaining a very low IC₅₀ value. Hence resolved that the ionisation affinity of solvent medium support the SPLET or relief of proton as Scheme 6.

Scheme 6

Anomalous behavior of phenolic antioxidants in ethanol

Structure activity relationship in DPPH

In DPPH reaction the structure activity relationships with respect to the solvent ethanol is analysed. The influence and effect of ethanol on antioxidant capacity is well defined as studied by various solvent effect kinetics studies [17, 18]. As for the ABTS and ORAC assay the reacting medium is a buffer solution and hence difficult to interpret the quenching ability of antioxidants synthesized in terms of two reactants. Compounds of which has half curcumin structure is having an IC₅₀ value comparable to curcumin of 17.88μM [19]. In Group 1 the compounds 4 and 8 exhibited excellent antioxidant activity using DPPH method with IC₅₀ values 13.31 ± 1.5 μM and 15.11± 1.7 μM respectively as compared with curcumin (17.88μM), which could be attributed to the heterocyclic ring centers and phenolic ring in the structures which enhances the antioxidant activity [20]. In Group 2 compounds 10 (18.14± 1.6 μM) and 17 (19.3± 1.2 μM) members have comparable IC₅₀ values, contributing part will be half curcumin and the halogen substituted in the second benzene ring[14]. Finally, Group 3 represented by compounds 12 and 15 showed the lower activities with IC₅₀ values 40.18± 2.4 μM and 40.6± 2.8 μM and this may be attributed to the steric effects offered by the extended ring structure (Table 2 and Figure 3). A brief investigation of the structure-activity relationship (SAR) revealed that halogen substitution and the presence of heteroring contributed to better antioxidant activity.

Figure 3

Free radicals centers of the synthesized compounds

Table 2

IC₅₀ value of DPPH (μM) of the synthesized compounds

Group.no	Compound	IC50 value of DPPH (μM)
1		4: 13.31 ± 1.5 8: 15.11 ± 1.7
2		10: 18.14 ± 1.6 17: 19.3 ± 1.2
3		12: 40.18 ± 2.4 15: 40.6 ± 2.8
	Curcumin	17.88

In curcumin, the phenolic group is accepted as the major reaction site where the electron capture takes place either as ArO^· or as ArO^-; the single electron moves towards the di-ketone moiety, giving free radical i, ii, and iii (Figure 4). The product analysis study also confirms that diketo moiety contribution is very less towards the antioxidant property [21]. However, experimental evidence indicates the relative importance of different mechanisms in different solvents and contributing centers, accounting for the antioxidant activity of curcumin and other antioxidants. The solvent attributes for the difference in IC₅₀ values got by DPPH, ABTS, and ORAC method.

Figure 4

Free radical centers of curcumin