Synthesis, characterization, and antioxidant activity of some 2-methoxyphenols derivatives

Article abstract of DOI:10.1515/hc-2020-0112

Oxidative stress is a causative factor in the pathophysiology of numerous diseases, such as diabetes, atherosclerosis, cancer, and neurodegenerative and cardiovascular diseases. Therapeutic antioxidants are promising candidates for preventing and treating conditions in which oxidative stress is a contributing factor. In this study, we report the design, synthesis and antioxidant activity of six compounds containing the 2-methoxyphenol moiety core structure. The synthesized derivatives were characterized using 1H NMR, 13C NMR, Fourier-transform infrared (FT-IR), and elemental analysis spectroscopy. The antioxidant properties of the compounds were evaluated using the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and oxygen radical absorbance capacity (ORAC) assay. New phenolic acid-derived compounds with antioxidant activity were identified.

Full text of DOI:10.1515/hc-2020-0112

Heterocycl. Commun. 2020; 26: 112–122
Research Article
Open Access
Shaikha S. AlNeyadi*, Naheed Amer, Tony G. Thomas, Ruba Al Ajeil, Priya Breitener
aSnyd NnMtuhnaewsaris, Characterization, and Antioxidant
Activity of Some 2-Methoxyphenols derivatives
https://doi.org/10.1515/hc-2020-0112
Received June 17, 2020; accepted July 27, 2020.
in the natural antioxidant defenses. At high concentration
free radicals can damage lipids, proteins, and DNA within
cells and tissues. The human body does have essential
defense pathways to counter free radicals in the form
of enzymes such as superoxide dismutase, catalase,
and glutathione peroxidase. However, the unbalances
between the formation and detoxification of free radical
species results in the development of oxidative stress.
This leads to the development of severe diseases, such
as cancer, atherosclerosis, aging, immunosuppression,
inflammation, ischemic heart disease, diabetes, and
neurodegenerative disorders [2]. Antioxidants are
compounds that can interact with free radicals in a
safe way, terminate the reaction, and convert them to a
harmless molecule by offering an electron. Antioxidants
therefore reduce the oxidative stress and thus protecting
the cells from oxidative damage [3].
Abstract: Oxidative stress is a causative factor in the
pathophysiology of numerous diseases, such as diabetes,
atherosclerosis, cancer, and neurodegenerative and car-
diovascular diseases. Therapeutic antioxidants are pro-
mising candidates for preventing and treating conditions
in which oxidative stress is a contributing factor. In this
study, we report the design, synthesis and antioxidant
activity of six compounds containing the 2-methoxyphenol
moiety core structure. The synthesized derivatives were
1
13
characterized using H NMR, C NMR, Fourier-transform
infrared (FT-IR), and elemental analysis spectroscopy. The
antioxidant properties of the compounds were evaluated
using the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH),
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid
(ABTS), and oxygen radical absorbance capacity (ORAC)
assay. New phenolic acid-derived compounds with anti-
oxidant activity were identified.
Eugenol and isoeugenol are examples of natural
methoxyphenols used in perfumes, detergents, air
fresheners and cosmetics (Figure 1). Eugenol is used also
Keywords: phenol, antioxidant, antiradical, ferulic acid, as a part of zinc-oxide eugenol cement in dentistry, and
curcumin
has demonstrated effective antioxidant activity [4]. In
addition,Eugenolpreventsthemetal-mediatedlowdensity
lipoprotein (LDL) oxidation acting as a physiological
antioxidant in vivo and apocynin is an efficient inhibitor
of the nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase complex [5]. The antioxidant properties
Introduction
Free radicals are atoms, molecules or ions that possess of other natural phenols such as eugenol, creosol,
an unpaired electron in orbit [1]. They are continually apocynin and isoeugenol, have already been reported [6].
formed in the body and can become toxic when obtained
Thedesignofnewantioxidantcompoundshasbecome
in high concentration or in the presence of a deficiency an important therapeutic matter given the wide-ranging
damage to cellular macromolecules caused by reactive
oxygen species (ROS). Therefore, extensive research has
*Corresponding author: Shaikha S. AlNeyadi, Department of
been focused to identify new antioxidants to prevent
radical-induced damage. In this study, we concentrated on
design, synthesis, characterization, and evaluation of the
antioxidant activity of newly methoxyphenol derivatives.
The structures of the various synthesized compounds
were verified based on elemental analysis, along with
infrared (IR) and 1D-NMR spectral data.
Chemistry, College of Science, UAE University Al-Ain, 15551 UAE
E-mail: shaikha.alneyadi@uaeu.ac.ae; Tel.:+971501099154
Tony G. Thomas, Ruba Al Ajeil and N Munawar, Department of
Chemistry, College of Science, UAE University Al-Ain, 15551 UAE
Naheed Amer, Department of Pharmacology, College of Health and
Science, UAE University Al-Ain, 17666 UAE
Priya Breitener, General Requirement Department, Fathima College
of Health Science, Abu Dhabi
Open Access. © 2020 AlNeyadi et al., published by De Gruyter.ꢀ
Attribution alone 4.0 License.
ꢀThis work is licensed under the Creative Commons

S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity ꢁ
ꢁ113
Figure 1ꢂStructures of some natural phenols
Materials and Methods
General
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
All chemicals and reagents were obtained from Sigma- (brs, 1H, aromatic H), 6.81–6.68 (m, 2 H, aromatic H), 5.53
Aldrich Chemical Company (Sigma Aldrich Chemical (brs, 1H, OH, exchanges with D2O), 3.85 (s, 3H, OCH3), 3.66
Company, street. Louis, MO, USA) and used without (t, J = 4.9 Hz, 1H, CH), 3.42 (d, J = 4.9 Hz, 2H, CH2), 1.72
additional purification. Thin-layer chromatography was (s, 6H, CH3); 13C-NMR [CDCl3, 100 MHz]: (δ, ppm) 165.5,
performed on silica gel coated with fluorescent indicator 147.7, 144.9, 128.8, 120.8, 115.6, 113.0, 105.2, 55.9, 46.3, 31.1,
F254 plates purchase from Fluka Company. Column 26.5; Anal. Calcd for C14H16O6 (280.28): C, 60.00; H, 5.75;
chromatography was implemented on Silica gel S, 0.063- Found: 59.97; H, 5.69.
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 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid (7)
model 470 Fourier-transform spectrophotometer. Nuclear
magnetic resonance (1H and 13C spectra) were recorded on a To a solution of 4-hydroxy-3-methoxybenzaldehyde 1
Varian 400 MHz spectrophotometer. Chemical shifts were (1 mmol) and malonic acid 5 (1.2 mmol) in pyridine
expressed in part per million (δ) with tetramethylsilane (2.5 mL/mmol), piperidine (0.04 mmol) was added at
(TMS) as an internal standard. Elemental analysis was 120 °C, and the reaction mixture was heated under reflux
conducted using a Leco CHN-600 elemental analyzer.
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
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) overnight under H2 atmosphere. After completion,
was dissolved in 2-propanol and acetic acid (0.1 equiv). the reaction mixture was filtered and concentrated to
Then,2,2-dimethyl-1,3-dioxane-4,6-dione1(1.5g,10.5mmol) afford compound 7; yield 95 % and 0.18g; mp, 92 °C; IR
was added. Then the reaction was heated to 75 °C for 2 h. (KBr, cm–1): 3409 (-OH), 2938 (CH-aliphatic), 1708 (C=O),
1
The solvent was removed under reduced pressure. The 1605 (C=C); H-NMR [CDCl3, 400 MHz]: (δ, ppm) 10.50
residue was washed 2-3 times with aqueous NaHSO3 (brs, 1H, CO2H, exchanges with D2O), 6.87 (d, J = 8.0 Hz, 1H),
solution (20 % w/v), water and hexane to obtain 6.73 (brs, 1H), 6.71 (d, J = 8.0 Hz, 1H), 5.60 (brs, 1H, OH,
compound 3 in 70 % and 1.9g. Compound 3 was dissolved exchanges with D2O), 3.88 (s, 3H, OCH3), 2.90 (t, J = 7.8 Hz,
in dichloromethane (100 mL) and the solution was cooled 2H), 2.70 (t, J = 7.8 Hz, 2H); 13C-NMR [CDCl3, 100 MHz]:
in an ice bath, then acetic acid (31.0 equiv) was added. (δ, ppm) 179.2, 146.4, 144.1, 132.0, 120.8, 114.4, 110.9, 55.8,
After 15 min, NaBH4 (8.0 equiv) was added within 1 h. 36.0, 30.3.
The reaction was removed from the ice bath and kept
at room temperature at 25oC for 1 h. After discoloration,
the reaction mixture was diluted with 100 mL water and 3-(4-Hydroxy-3-methoxyphenyl)-1-(piperidin-1-yl)
extracted three times with 50 mL with dichloromethane. propan-1-one (8)
The organic layers were collective and washed two times
withwater. TheorganiclayerwasdriedoverMgSO4, andthe N,N-Carbonyldiimidazole (0.545 g, 3.36 mmol) was added
solvent was evaporated in vacuum, yielding compound 4 to a solution of 3-(4-hydroxy-3-methoxyphenyl) propanoic

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S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity
acid 7 (0.6 g, 3.06 mmol) in 10 mL of dry THF. After 30 stirringfor3h.Coldwaterwasaddedtothemixture,filtered
min, piperidine (0.302 mL, 3.06 mmol) was added and and dried in vacuum. The product was recrystallized from
the mixture was stirred overnight. The precipitate formed ethanol to obtain compound 12; yield 70 % and 3.2 g; mp,
then was washed with dichloromethane to obtain 8. White 182 °C; IR (KBr, cm–1): 3368 (-OH), 3045 (C-H aromatic),
solid; yield 60 % and 0.48g; mp 120 °C; IR (KBr, cm–1): 3392 2988 (C-H aliphatic), 1639 (C=O), 1577 (C=C), 1515
1
1
(-OH), 3010 (C-H aromatic), 1749 (C=O); H-NMR [DMSO- (C=C aromatic); H-NMR [CDCl3, 400 MHz]: (δ, ppm) 9.83
d6, 400 MHz]: (δ, ppm) 8.68 (brs, 1H, OH, exchanges with (brs, 2H, OH, exchanges with D2O),7.74 (s, 2H, vinylic H),
D2O), 6.77 (d, J = 1.9 Hz, 1H, aromatic H), 6.64 (d, J = 8.0 Hz, 7.09-6.93 ( m, 6H, aromatic H), 3.92 (s, 6H, OCH3), 2.95 – 2.95
13
1H), 6.59 (dd, J = 8.0, 2.0 Hz, 1H), 3.72 (s, 3H, OCH3), 3.31- (m, 4H, CH2), 1.84-1.78 (m, 2H, CH2); C-NMR [CDCl3, 100
3.35 (m, 4H, CH2 piperidine), 2.67 (m, 2H, CH2), 2.51-2.53 MHz]: (δ, ppm) 190.1, 146.4, 137.0, 134.4, 128.5, 124.4, 114.4,
(m, 2H, CH2), 1.36-1.53 (m, 6H, CH2 piperidine); 13C-NMR 113.2, 55.9, 28.5, 23.0; Anal. Calcd for C22H22O5 (366.41): C,
[DMSO-d6, 100 MHz]: (δ, ppm) 170.1, 147.7, 144.9, 132.6, 72.12; H, 6.05; Found: C, 72.09; H, 5.99.
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)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-
2-one (13)
(E)-1-(4-Chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)
prop-2-en-1-one (10)
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
4-Hydroxy-3-methoxybenzaldehyde 1 (1 g, 6.57 mmol) and and allowed to stand for 48 h. Distilled water (50 mL)
1-(4-chlorophenyl)ethenone 9 (852 µL, 6.57 mmol) were was added. Then, 10% aqueous HCl (15 mL) was added.
mixed and stirred in 10 mL of methanol for 5-10 min; then The yellow precipitate formed was filtered, dried and
H2SO4 (98 %, 3.75 mmol) was added and the reaction was recrystallized from ethanol/water to yield compound 13.
heated to 80 °C for 48 h. After completion, the solution This compound was yellow powder; yield 70 % and 2.7 g;
was neutralized by adding a NaOH solution (20 % w/v). mp 126 °C; IR (KBr, cm–1): 3285 (-OH), 3001 (C-H aromatic),
1
The solution then extracted with ethyl acetate after diluted 2849 (C-H aliphatic), 1675 (C=O), 1638 (C=C); H-NMR
with water. The organic layer was dried over Na2SO4 and [CDCl3, 400 MHz]: (δ, ppm) 7.43-7.47 (d, J = 16.2 Hz, olefinic
the ethyl acetate was evaporated. The crude product H), 7.06-7.11 (m, 2H, aromatic H), 6.93-6.95 (d, J = 8 Hz,
was purified using silica gel flash chromatography to 1H, aromatic H), 6.57-6.61 (d, J = 16.2 Hz, olefinic H), 5.91
yield compound 10; yield, 48 % and 0.14g; mp, 218 °C; (s, 1H, OH, exchanges with D2O), 3.94 (s, 3H, OCH3), 2.37
IR (KBr, cm-1): 3430 (-OH), 3093 (C-H aromatic), 2954 (s, 3H, CH3); 13C-NMR [CDCl3, 100 MHz]: (δ, ppm) 198.5,
(C-Haliphatic), 1651(C=O), 1563(C=C), 1522(C=Caromatic); 148.2, 146.9, 143.8, 127.0, 125.0, 123.5, 114.8, 109.2, 55.9, 27.5;
1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 10.31 (brs, 1H, OH, Anal. Calcd for C11H12O3 (192.21): C, 68.74; H, 6.29; Found:
exchanges with D2O), 7.79 – 7.77 (m, 2H, aromatic H), 7.57 C, 68.71; H, 6.23.
(d,d,1H,COCH=CH,J=15Hz),7.52–7.50(m,2H,aromaticH),
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 (1E,4E)-1-(4-Hydroxy-3-methoxyphenyl)-5-
MHz]: (δ, ppm) 185.4, 151.8, 150.6, 148.1, 145.6, 143.6, 139.1, (4-hydroxyphenyl)penta-1,4-dien-3-one (15)
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; Compound9(0.5g,2.60mmol)and4-hydroxybenzaldehyde
H, 4.48.
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):
(2E,6E)-2,6-bis(4-Hydroxy-3-methoxybenzylidene)
cyclohexanone (12)
4-Hydroxy-3-methoxybenzaldehyde 1 (1.9 g, 12.5 mmol) 3585 (-OH), 3069 (C-H aromatic), 2955 (C-H aliphatic), 1697
and cyclohexanone 11 (0.65 mL, 6.25 mmol) were mixed (C=O), 1593 (C=C), 1513 (C=C aromatic); 1H-NMR [DMSO-d6,
and stirred at room temperature for 5 min. Then, conc. 400MHz]:(δ,ppm)10.03(brs,1H,OH,exchangeswithD2O),
hydrochloric acid (4 mL) was added slowly, followed by 9.68 (brs, 1H, OH, exchanges with D2O), 7.65-7.66 (d, J = 15

S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity ꢁ
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Hz, ,2H, olefinic-H), 7.60-7.62 (m, 2H, aromatic H), 7.34-7.35 (Molecular Devices, CA, USA). The ABTS assay measured
(d, J = 15 Hz, 2H, olefinic-H), 7.08-7.19 (m, 3H, aromatic H), the free-radical-scavenging activity of the compounds.
13
6.81-6.82 (m, 2H, aromatic H), 3.82 (s, 3H, OCH3); C-NMR Data are presented as mean SEM and expressed as
[DMSO-d6, 100 MHz]: (δ, ppm) 188.5, 160.3, 149.8, 148.3, micromoles Trolox equivalent (μM TE).
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.
Antioxidant Activity by Oxygen Radical Absorbance
Capacity (ORAC) Assay
(1E,4E)-1-(4-Fluorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (17)
The oxygen radical absorbance capacity of each
test compound was determined using the ORAC
Antioxidant Assay Kit (Zenbio, NC, USA) according to
4-fluorobenzaldehyde 16 (0.23 g, 2.60 mmol) was mixed the manufacturer’s instructions. Trolox standards were
with (E)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one prepared in assay buffer (0–100ꢀμM), along with the
9 (0.5 g, 2.60 mmol) in 10 mL ethanol for 5 min. Then, test compounds (100ꢀμM). Fluorescein working solution
2M NaOH (10 mL) was added and stirred overnight. The (150ꢀμL) was added to the central wells of a 96-well black
resulting mixture was acidified by concentrated HCl, and side, clear bottom plate, and mixed with 25ꢀ μL of each of
the residue was washed with cold water and dried in air. the standards or test compounds in triplicate; the plate
The product was recrystallized from an ethanol/water was incubated at 37ꢀ°C for at least 10ꢀmin. The 2,2′-azobis(2-
mixture to obtain 17; yield, 73 % and 0.57 g; mp, 210 °C; amidinopropane) dihydrochloride [AAPH; sold as
IR (KBr, cm-1 ): 3427 (-OH), 3001 (C-H aromatic), 2926 (C-H 2,2′-azobis(2-methylpropionamidine)
dihydrochloride]
aliphatic), 1674 (C=O), 1618 (C=C), 1577 (C=C aromatic); working solution was added to each well (25ꢀμL) to
1H-NMR [DMSO-d6, 400 MHz]: (δ, ppm) 9.87 (brs, 1H, OH, initiate the reaction. The fluorescence was measured
exchanges with D2O), 7.91-7.95 (d, 2H, J = 15 Hz olefinic H), after 30 min incubation at 37ꢀ°C using a multilabel plate
7.72 (brs, 1H, aromatic H), 7.65 (m, 2H, aromatic H), 7.36 reader (Perkin Elmer Victor3, USA) with excitation/
(d, 2H, J = 15 Hz olefinic H), 7.09 (m, 2H, aromatic H), emissionꢀ=ꢀ485/530ꢀnm. Presented as mean SEM and
6.65 (m, 1H, aromatic H), 6.63 (m, 1H, aromatic H), 3.70 expressed as micromoles Trolox equivalent (μM TE). The
(s, 3H, OCH3); 13C-NMR [DMSO-d6, 100 MHz]: (δ, ppm) 177.2, ORAC values are presented as mean SEM and expressed
150.9, 150.7, 144.5, 143.3, 134.3, 133.7, 121.1 , 120.8, 120.1, as μM TE.
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 by 2,2-diphenyl-1-picryl-hydrazyl-
hydrate (DPPH) Assay
Antioxidant Activity
The free radical scavenging ability of the test compounds
was studied using the DPPH assay [7]. The test compounds
Total Antioxidant Capacity Test Using the 2,2’-azino-
in ethanol were prepared separately and added to the
bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
ethanol solution containing DPPH (0.01 mmol) within the
Kinetic Assay (Free-Radical-Scavenging Activity)
range of 5–50 μL and adjusted to a final volume of 3 mL
using ethanol as solvent. The scavenging ability of the test
The total antioxidant capacity was obtained using the
compounds was monitored spectrophotometrically by
ABTS kinetic assay according to the manufacturer’s
measuring the absorbance at 517 nm after 20 min. The %
instructions (Zenbio, Research Triangle Park, NC, USA).
inhibition was obtained using equation (1).
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




ABScontrol −ABS sample
% Inhibition =
* 100


ABScontrol




The 50 % decline in absorbance of the DPPH solution
solution were added. The kinetic profile of ABTS+ was derived from the graph with the concentration (μM)
scavenging by antioxidant compounds was monitored in a plotted against the absorbance. These concentration
96-well microplate using an Emax Plus microplate reader values were used to determine the IC50 values inμM.

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S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity
Results and Discussion
Synthesis
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.
Knoevenagel-condensations [9] are used to access
Methoxyphenol derivatives have demonstrated extensive carboxylic acids from a malonic acid and an appropriate
therapeutic effects; most of which depend on its aldehyde. 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid
antioxidant properties [8]. There have been recent efforts 7 was synthesized by condensation of 4-hydroxy-3-
to maximize the therapeutic value of Methoxyphenol methoxybenzaldehyde 1 with malonic acid 5 to generate
by creating structurally modified derivatives with (E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid 6 followed
potentially enhanced physicochemical, antioxidant, and by hydrogenation in the presence of Pd/C to give
therapeutic properties. In this study, we synthesized compound 7 with a yield of 95 % yield. This compound
the six different methoxyphenol derivatives for was synthesized and characterized by the reduction of
antioxidant activity measurements. The first compound olefinic compound 6 and the appearance of two triplet
1
5-(4-hydroxy-3-methoxybenzyl)-2,2-dimethyl-1,3- bands in its HNMR spectrum at δ = 2.90 and 2.70 ppm
dioxane-4,6-dione 4, was obtained by condensation of with the coupling constant J = 7. 8 Hz respectively. In
2,2-dimethyl-1,3-dioxane-4,6-dione(Meldrum’sacid)1with the 13CNMR spectrum, the two new carbons bands,
4-hydroxy-3-methoxybenzaldehyde 2 in an acid-catalyzed, representing two CH2, were detected at δ = 36.0 and 30.3
Knoevenagel-like reaction at 75 °C for 2 h generated ppm. The reaction of compound 7 with one equivalent
compound 3, which was reduced to form compound 4 in of 1,1’carbonyldiimidazole in refluxing dry THF led to
coldDCMusinginsitugeneratedNaHB(OAc)3inanicebath. the formation of the corresponding N-acylpiperdine
The obtained material was recrystallized from hot ethyl compound 8 (Scheme 2), which was characterized by
acetate-hexane to obtain pure compound 4 (Scheme 1). NMR spectroscopy, FTIR, and elemental analysis. The
The resultant product was white solid with a melting point IR spectral data of compound 8 showed sharp bands at
at 130 °C and percentage yield of 63%. The structure of 1749 and 3392 cm‐1, indicating the presence of C=O and
compound 4 was confirmed based on elemental analysis OH groups. The structure of 8 was also confirmed by the
and spectroscopy data. The IR spectrum of 4 indicated 1H-NMR spectral data, which showed a characteristic
the presence of a hydroxyl group stretching vibration at signal that resonated at δ = 3.31–3.35 ppm and 1.36–1.53,
3518 cm–1. In addition, signals appeared at 2874, 1793, attributed to the 10 protons of piperidine. The 13C-NMR
and 1755 cm–1, which were attributed to aliphatic H and spectrum showed new signals resonating at δ = 24.5,
carbonyl groups, respectively. The 1H-NMR spectrum 26.5 and 46.3 ppm, corresponding to the piperidine
of 4 showed a characteristic signal at δ = 1.72 ppm carbons.
corresponding to the methyl protons; another signal,
The synthesis of chalcones is well established and is
resonating at δ = 3.85 ppm, was attributed to the methoxy based on the Claisen condensation of substituted
proton. The aromatic protons resonated as a multiplet benzaldehydes and acetophenones, and is achieved
between δ = 6.68 and 6.85 ppm with an integration value under either basic [10] or acid [11] condition. Confirming
of three protons. The reduction of the ethylenic bond of primary literature research [12], because of the
compound 3 was characterized by the appearance of the presence of the phenolic substituent, the acid catalysis
new bands at δ = 3.42 and 3.66 ppm, corresponding to the showed to be more appreciate in most of the cases and
CH2 and CH protons, respectively. The hydroxyl group thus the procedure was used for the prepared of the
appeared at δ = 5.53 ppm due to the exchange with D2O. 4-hydroxy compound 10 (Scheme 3). The structure of
13
The C-NMR spectrum of compound 4 showed two new the compound 10 was established by spectral analysis.
signals at δ = 31.1 and 46.3 ppm, which were assigned to The FTIR spectrum of compound 10 showed characteristic
the CH and CH2 carbons, while the signals at δ = 146.2, absorption bands at 3430, 1651, and 1563 cm–1,
Scheme 1ꢂThe synthetic route to target compound 4

S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity ꢁ
ꢁ117
Scheme 2ꢂThe synthetic route to target compound 8
Scheme 3ꢂThe synthetic route to target compounds 10, 12, 15 and 17
corresponding to the –OH, C=O, and C=C functional hydroxide in a mixture that was stirred at room
1
groups. The H-NMR spectrum showed the presence of temperature for 12 h. The reaction mixture was diluted
a broad singlet at δ = 10.31 ppm attributed to phenolic with ice water and neutralized with HCl to obtain a
OH proton. Multiplet bands at δ = 7.52–7.50 ppm and yellow solid 13 with a 70% yield. Compound 13 was
7.77–7.79 ppm were assigned to aromatic protons; a doublet used to prepare the unsymmetrical diarylpentanoid
(J = 15 Hz) at δ = 6.14 ppm was characteristic for the analogues 15 and 17. The targeted diarylpentanoids were
olefinic proton COCH=CH, and another doublet (J = 15 Hz) synthesized to assess the effect of the substitution on the
at δ = 7.57 ppm was characteristic for the second olefinic aromatic rings and to explore the effect of different aryl
COCH=CH. The methyl protons appeared as a singlet in groups upon antioxidant activity. The characterization
the δ = 3.67 ppm. Compound 12, another mono-carbonyl and spectroscopic date of compounds 15 and 17 were
derivative of curcumin was prepared from available investigated. Compounds 15 and 17 were produced by
4-hydroxy-3-methoxybenzaldehyde 1 and cyclohexanone reacting compound 13 with different aldehydes, having
11 in ethanol via enamine intermediate in the presence electron donating and electron withdrawing moieties.
of acidic medium. Compound 12 was obtained in a 70 % It was completely characterized by NMR. For compound 15,
yield (Scheme 3). The FTIR spectrum of compound 12 one double bond and aromatic moiety was added, and 1H
recognized two absorption bands at 3368 and 1639 cm–1, NMR had two more doublets for -CH=CH- with a coupling
distinctive for OH and C=O groups. The 1H NMR spectrum constant of approximately 15 Hz, which is characteristic
had two signals at δ = 7.74 ppm that coordinated the of an H-H trans-coupling system at δ = 7.65 and 7.34 ppm.
vinylic protons of benzylidenes. The 13C NMR spectrum The aromatic multiplet signals appeared at δ = 7.60–7.62
of compound 12 showed that the carbonyl carbon was ppm, 7.08–7.19 ppm, and 6.81–6.82 ppm, which integrated
specified downfield at δ = 190.1 ppm.
to seven protons.
The unsymmetrical analogs of curcumin, compounds
15 and 17, were characterized by the presence of a short
but entirely conjugated unsaturated ketone chain. Both Antioxidant Activity
compounds were prepared from (E)-4-(4-hydroxy-3-
methoxyphenyl)but-3-en-2-one 13. Compound 13 was Free radical scavenging capabilities were determined for
obtained from vanillin and acetone in aqueous sodium all compounds by the DPPH and ABTS assays. DPPH is

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S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity
a very stable free radical, which is reduced by accepting lowest antioxidant activity (40.6 µM). The difference in
hydrogen from a hydrogen donor compound. Reduction free radical scavenging activity among the six compounds
of DPPH results in the disappearance of its violet color, could be related to the presence of different characteristic
indicating the presence of free radical scavenging groups at the end of their carbon side chain. The
compounds in the reaction mixture. Similarly, the ABTS compounds having the least polar groups attached with
radical cation can be produced in aqueous solution. The the 2-methoxyphenol moiety of derived compounds,
blue color of the ABTS radical absorbs light at 734 nm and (compound 4 and 8), had a higher free radical scavenging
can be used to monitor the radical scavenging capacity activity (13.3 μM and 15.1 μM, respectively) in the DPPH
of a substance relative to the antioxidant potential of this assay, compared with the compounds having more
substance.
hydrophilic end groups (compounds 12 and 15; IC50
The DPPH and ABTS reducing capabilities of all 40.1 µM and 40.6 µM, respectively) (Table 1). High
synthetic compounds were evaluated by determining their antioxidant activity by ferulic acid derivatives bearing
IC50 values. The antioxidant activity analysis of the six non-polar groups in the side carbon chain has been
synthetic compounds indicated a strong reducing activity. reported already by Nenadis et al. [13].
The IC50 values of all compounds obtained in the DPPH,
An interesting structural modification with a
ABTS, and ORAC assays are presented in Table 1. The IC50 positive effect on the reducing properties of our synthetic
value corresponds to the concentration of a sample, which compounds was the introduction of halogen atoms, Cl
has ability to scavenge 50% of the free radicals present and F, at the end of the side chain of the compounds.
in the reaction mixture. Low IC50 values indicate a high The substitution of Cl and F doubled the free radical
antioxidant activity of the sample compound.
scavenging activity of compounds 10 (18.1 µM) and 17
As shown in the Table 1 and Figure 2, all compounds (19.3 µM), compared to compounds 12 (40.1 µM) and
had free radical scavenging activity in the DPPH 15 (40.6 µM) with an -OH group at the end of the side
assay. The IC50 values in the DPPH assays were in the chain. Our results confirm the finding by Puskullu
range of 13.3–40.6 µM DPPH. Compound 4, which has et al. [14]. Their study also indicated that according to the
one -OH group, showed highest antioxidant activity structure activity relationship, halogenated derivatives
(13.3 µM). Compound 15 with two -OH groups had the have a better antioxidant activity. The IC50 of ferulic acid
Table 1ꢂAntioxidant 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
12
15
17
18.14 1.6
40.18 2.4
40.6 2.8
19.3 1.2
165.9 24.4
231.1 10.4
159.8 34.5
156.7 28.5
299.8 4.4
280.7 6.3
302.3 3.0
310.3 4.5

S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity ꢁ
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350
300
250
200
150
100
50
0
4
8
10
12
15
17
IC50 (µM) DPPH
ABTS (µM TE)
ORAC (µM TE)
Figure 2ꢀComparison of antioxidant activity of phenolic acid derivatives using the DPPH, ABTS, and ORAC assays
has been reported by Aleksandra et al.[15]; the DPPH fluorescent molecule from degradation. Out of the six
value is 44.6 µmol/L. Therefore, all compounds had test compounds, the maximum antioxidant activity was
a higher antioxidant activity than ferulic acid in the observed with compound 4 (IC50 242 µM TE), followed
DPPH assay. Moreover, the comparison of antioxidant by compound 8 (IC50 276.2 µM TE). A similar observation
activities of all compounds with the standard antioxidant was made with the other two assays. However, instead of
molecules like ascorbic acid and Trolox revealed that compound 12 and 15, which showed the lowest activity
all compounds have lower IC50 vales than IC50 values in the DPPH and ABTS assays, compound 17 showed the
of ascorbic acid (41.25μg/ml) and Trolox (63.69 μg/mL) lowest antioxidant activity in the ORAC assay. The varying
in DPPH assay, which have been reported by Matuszewska trends in the antioxidant activity by different assays could
et al. [16].
be explained with the effect of the reaction medium used
The measurements of the ABTS and the ORAC assay in the different assays. Different compounds could have
showed lower antioxidant activities of compounds, different hydrogen abstraction mechanisms depending on
compared to DPPH assay results. The IC50 values by the the reaction medium.
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 Mechanism Suggested for Hydrogen Abstraction from
the Antioxidants
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. Free radicals (ROO•) react with phenols (ArOH) via the
Therefore, the compounds, which are less soluble in following prominent mechanisms
water have less activity in the ABTS assay than in the
DPPH assay. As shown in Table 1, the DPPH assay results (i) Hydrogen atom transfer (HAT) is the abstraction of
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
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

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S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity
(BDE) of phenol [18]. Solvents such as alcohols and Therefore, substrate to solvent equilibrium do not occur
water are able to both accept and donate hydrogen in ethanol, allowing the ionisation of substrate (ArOH)
bonds and therefore could either improve or reduce depending on the properties of the solvent such as its
the Bond Dissociation Energy (BDE) of phenol. relative permittivity (dielectric constant, εr), molecular
Water is a better donor but a poorer acceptor of property, its relative capability to solvate stabilising
hydrogen bonds than alcohols and hence should anions (ArO–), as measured by Swain et al’s A value [17].
be able of better solvating the phenoxyl radical and Ethanol as a solvent having high dielectric constant equal
solvating the parent phenol less or ethanol solvate to 25 supports ionisation, ArOH loses proton to form stable
curcuminoids rather than water. By increasing the phenoxide ion at fast rate. Therefore, quenching of DPPH
percentage of water, the degree of solvation decreases occurs at low concentration of ArOH, obtaining a very low
and the phenoxide ion production decreases and IC50 value. Hence resolved that the ionisation affinity of
this will affect the antioxidant activity. The buffer solvent medium support the SPLET or relief of proton as
used as medium in both methods ABTS and ORAC, Scheme 6.
thereby decreasing the concentration of phenoxide
ion formed from reacting antioxidants. Therefore,
quenching of free radical produced requires a higher Structure activity relationship in DPPH
concentration of antioxidant samples. IC5o values
in ABTS and ORAC method are higher owing to the In DPPH reaction the structure activity relationships
reduction of phenoxide ion in the lack of medium as with respect to the solvent ethanol is analysed. The
shown in Scheme 4.
influence and effect of ethanol on antioxidant capacity
(ii) The sequential proton loss electro transfer mechanism is well defined as studied by various solvent effect kine-
(SPLET) [17] operates predominantly in polar protic tics studies [17, 18]. As for the ABTS and ORAC assay the
solvents like methanol and ethanol.
reacting medium is a buffer solution and hence difficult
to interpret the quenching ability of antioxidants synthe-
The kind of solvent and polarity might affect the process sized in terms of two reactants. Compounds of which has
of single electron transfer and the hydrogen atom half curcumin structure is having an IC50 value compara-
transfer, which are main features in the measurement of ble to curcumin of 17.88µM [19]. In Group 1 the compounds
antioxidant activity. In DPPH assay method ethanol was 4 and 8 exhibited excellent antioxidant activity using
used as a solvent, the antioxidants showed the maximum DPPH method with IC50 values 13.31 1.5 µM and 15.11
reducing ability. This behavior was attributed to polarity 1.7 µM respectively as compared with curcumin (17.88µM),
of ethanol establishing intermolecular hydrogen bonding which could be attributed to the heterocyclic ring centers
amongst the solvent and thus hindering ArOH (substrate) and phenolic ring in the structures which enhances the
and S (solvent) interaction as shown in Scheme 5. 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 antioxi-
dant activity.
KHAT
ArO + ROOH
ArOH + ROO
HAT
Scheme 4ꢂHAT in a polar and non-protic solvents
ArO-
+ H +
ArOH
ArO-
ArO + ROO-
ROOH
+ ROO
ROO-
H+
+
In curcumin, the phenolic group is accepted as the
major reaction site where the electron capture takes place
Scheme 5ꢂProton Loss Electron Transfer (SPLET) in protic solvents
ArO-
DPPH
H+
- S
fast
ArOH-
ArO
ArOH + S
+
+ DPPHH
Scheme 6ꢂAnomalous behavior of phenolic antioxidants in ethanol

S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity ꢁ
ꢁ121
·
either as ArO or as ArO-; the single electron moves towards Conclusions
the di-ketone moiety, giving free radical i, ii, and iii
(Figure 4). The product analysis study also confirms In summary, a series of compounds containing the
that diketo moiety contribution is very less towards 2-methoxyphenol moiety were successfully synthesized
the antioxidant property [21]. However, experimental with a good yield of 48–95 %. The characterisation of
evidence indicates the relative importance of different compounds were identified using different spectral
mechanisms in different solvents and contributing studies. The synthesized compounds exhibited a wide-
centers, accounting for the antioxidant activity of ranging of possibly promising antioxidant activities.
curcumin and other antioxidants. The solvent attributes All compounds displayed antioxidant activities using
for the difference in IC50 values got by DPPH, ABTS, and the DPPH, ABTS, and ORAC method. Compounds
ORAC method.
8 and 13 showed the highest antioxidant activity with
Table 2ꢂIC50 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
Figure 3ꢂFree radicals centers of the synthesized compounds
Figure 4ꢀFree radical centers of curcumin

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122ꢁ
S. S. AlNeyadi et al.: Synthesis, Characterization, and Antioxidant Activity
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Compounds 10 and 17 showed antioxidant activity using
[8] Koleva L, Angelova S, Dettori M, Fabbri D, Delogu G, Kancheva V.
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Antioxidant activity of selected o-methoxyphenols and
compounds could generate free radical centers, which
were stabilized by the hyper-conjugative effect and the
electron-withdrawing groups present in the structures.
The mechanism of oxidation of phenolic compounds in
a reaction with DPPH is considered as sequential proton-
loss electron transfer (SPLET) based mechanism in protic
solvent like EtOH. All test phenolic compounds showed
a higher antioxidant activity by DPPH assay because this
assay was done in EtOH as compared to other assays, ABTS
and ORAC, which were performed in aqueous solution.
Both, ABTS and ORAC, are HAT mechanism-based assays
involve donation of H-atom in quenching of free radicals.
The major factor contributes in scavenging of free radical
in HAT mechanism is bond dissociation energy (BDE) of
O-H bond that is related to the stability of the phenoxyl
radical (ArO.).
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Conflicts of Interest: The authors declare no competing
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