2-Acetyl-4-aminoresorcinol derivatives: synthesis, antioxidant activity and molecular docking studies

Article abstract of DOI:10.1007/s00044-018-2139-3

In this study, we synthesized new phenolic compounds that have the potential to serve as antioxidants and slow the effects of free radicals and oxidizing agents, which is beneficial for human health. Three resorcinol derivatives (new amide compounds) were generated by acetylation, nitration, and reduction. The structures of the products were determined by spectroscopic methods. The antioxidant activities of the new compounds were evaluated in vitro by redox reactions with the free radicals of the diammonium salt of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^+●) and 1,1-diphenyl-2-picrylhydrazyl (DPPH^●). The tested compounds showed antioxidant activities similar to that of resveratrol, a natural product known to affect a wide range of intracellular mediators. These derivatives were also submitted to docking simulations to evaluate their possible interactions with the enzyme myeloperoxidase. This analysis showed potential interactions with pockets “A” and “B” in the enzyme, and the best interaction was detected between succinic-amide (4) and pocket A.

Full text of DOI:10.1007/s00044-018-2139-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2139-3
ORIGINAL RESEARCH
2-Acetyl-4-aminoresorcinol derivatives: synthesis, antioxidant
activity and molecular docking studies
María A. Guerra-Vargas1,2 Martha C. Rosales-Hernández2 Nadhynee Martínez-Fonseca3 Itzia Padilla-Martínez4
●
●
●
●
Yadira Fonseca-Sabater1 Federico Martínez-Ramos1
●
Received: 27 July 2017 / Accepted: 20 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
In this study, we synthesized new phenolic compounds that have the potential to serve as antioxidants and slow the effects of
free radicals and oxidizing agents, which is beneficial for human health. Three resorcinol derivatives (new amide
compounds) were generated by acetylation, nitration, and reduction. The structures of the products were determined by
spectroscopic methods. The antioxidant activities of the new compounds were evaluated in vitro by redox reactions with the
free radicals of the diammonium salt of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+●) and 1,1-diphenyl-2-
picrylhydrazyl (DPPH●). The tested compounds showed antioxidant activities similar to that of resveratrol, a natural product
known to affect a wide range of intracellular mediators. These derivatives were also submitted to docking simulations to
evaluate their possible interactions with the enzyme myeloperoxidase. This analysis showed potential interactions with
pockets “A” and “B” in the enzyme, and the best interaction was detected between succinic-amide (4) and pocket A.
●
●
●
●
●
Keywords Resorcinol Synthesis 2-acetyl-4-aminoresorcinol Myeloperoxidase Docking Antioxidant activity
Introduction
biologically important of the ROS are the superoxide anion
●
radical (O2 ), hydroxyl radical (OH●), hydrogen peroxide
As a result of cellular metabolism, major reactive oxygen
species (ROS) are continually produced, and the most
(H2O2), peroxynitrite (ONOO−), nitric oxide (NO●), and
hypochlorous acid (HClO) (Galcerán and Martínez 2000;
Selemidis et al. 2008). From 1 to 5% of the total oxygen
consumed by the mitochondria is converted into ROS by a
partial reduction of the superoxide radical anion molecule
(O2●−) (Weissman et al. 2007; Lee et al. 2010). The radical
anion is chemically or enzymatically converted into H2O2,
which is then transformed into a highly reactive hydroxyl
radical (OH●) in the presence of reduced transition metals
(Dröge 2002). The ROS concentration is determined by the
balance between the production and the elimination rates.
The imbalance between ROS and antioxidant defense
mechanisms in cells leads to oxidative changes in intra-
cellular compounds, such as polyunsaturated fatty acids,
lipids, proteins, and DNA, and causes oxidative stress to the
cells. Oxidative stress has been implicated in inflammatory
and cardiovascular diseases and aging and has a role in the
development of cancer, neurological disorders, diabetes,
and ischemia/reperfusion (Beckman and Ames 1998; Valko
et al. 2007; Gutteridge and Halliwell 2000). Methods to
counteract the damage caused by these chemical species are
being studied to develop new therapeutic targets, such as
antioxidants (Block 1992; Rice-Evans et al. 1996). A large
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2139-3) contains supplementary
material, which is available to authorized users.
* Federico Martínez-Ramos
fmartinezr@ipn.mx
1
Laboratorio de Investigación, Departamento de Química
Inorgánica, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Prolongación de Carpio y Plan de Ayala s/n,
11340 Mexico D. F., Mexico
2
Laboratorio de Biofísica y Biocatálisis, Escuela Superior de
Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz
Mirón s/n, 11340 Mexico D. F., Mexico
3
Departamento de Bioprocesos, Unidad Profesional
Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional,
Av. Acueducto s/n Barrio la Laguna, Ticomán, Mexico D. F.,
Mexico
4
Laboratorio de Investigación en Química Orgánica y
Supramolecular, Unidad Profesional Interdisciplinaria de
Biotecnología, Instituto Politécnico Nacional, Av. Acueducto s/n
Barrio la Laguna, Ticomán, Mexico D. F., Mexico

Medicinal Chemistry Research
number of phenolic compounds, such as derivatives of
hydroxamic acid (caffeic, ferulic, and p-coumaric acids)
have been described in the literature as bioactive sub-
stances. Reports have shown that the latter two compounds
remove thiol groups (free radical) (Nardini et al. 1995).
Synthetic phenolic antioxidants such as butylated hydro-
xyanisole, butylated hydroquinone, and butylated hydro-
xytoluene possess antioxidant capacities, and the structural
feature responsible for their antioxidant activities and their
ability to eliminate free radicals is the phenolic hydroxyl
group (Findik et al. 2011). The electron-withdrawing
groups on the ring systems increase the stability of the
phenolic group, and the π-electron delocalization leads to a
more potent antioxidant molecule. There are three main
mechanisms by which polyphenols eliminate free radicals,
namely, hydrogen atom transfer, single electron transfer
followed by a proton transfer and sequential proton loss and
electrons transfer (Wright et al. 2001). Antioxidants are
natural or synthetic molecules capable of interacting with
free radicals and stopping their chain reactions before
essential molecules are damaged. Several synthetic com-
pounds with antioxidant activities have been studied and
reported, including derivatives of phenylcarbamoylbenzoic
acid (Bayoumi and Elsayed 2012), 4-phenyl hydro-
xycoumarin (Veselinovic et al. 2014), Schiff bases (Chen
et al. 2003), phthalimide derivatives (Shankar et al. 2000),
hydrazides (Gurkok et al. 2009), and pyridoxal iso-
nicotinoyl hydrazone (Maurício et al. 2003) as well as,
natural phenolic compounds isolated from Acacia confusa
(Tung et al. 2007), isoeugenol derivatives (Findik et al.
2011), and resveratrol, which are beneficial to human health
(Gulcin 2010).
docking studies were based on the X-ray structure of the
complexed human MPO. MPO consists of 466 amino acids,
contains predominantly α-helical secondary structures, and
is organized into locally folded domains around a Hem
core. In silico molecular modeling plays an important role
in the design and discovery of drugs predicting the con-
formations of ligands in the active site (Van Antwerpen
et al. 2008; Soubhye et al. 2010), and alkaloids of Peganum
harmala can be modeled (Bensalem et al. 2014).
Materials and methods
Chemistry
The target compound was synthesized by acylation of
resorcinol with ZnCl2 (Lewis acid) and concentrated acetic
acid (Domínguez and Dominguez 1990). The product was
directly and regioselectively nitrated at carbon 4 with con-
centrated HNO3 and CH3COOH (Scheme 1). The nitro
group was reduced with Na2S2O4 in MeOH/H2O at reflux to
afford the aromatic amine (2-acetyl-4-aminoresorcinol). The
amine was reacted with different anhydrides (succinic,
glutaric, and phthalic) in tetrahydrofuran (THF) at reflux
with anhydrous K2CO3 to afford 4, 5, and 6.
Antioxidant screening
ABTS+● radical assay
The potential antioxidant activity of each of the compounds
was evaluated in vitro using resveratrol as a positive con-
trol. Their capacity to react with the ABTS+● radical was
measured. The ABTS+● radical is monitored by the color of
the solution, and a spectrophotometric method has been
widely used to evaluate the antioxidant activity of various
substances. This method can be used for hydrophilic and
lipophilic compounds. Potassium persulfate is used to
generate the cation radical, and the absorbance (734 nm) is
The aim of the present research was to study the anti-
oxidant activity of novel phenolic compounds derived from
resorcinol (1,3-dihydroxybenzene), through the design,
synthesis, and in vitro antioxidant evaluation of the selected
compounds for the formation of amides with 2-acetyl-4-
aminoresorcinol and anhydrides. The antioxidant activities
were evaluated by spectroscopic evaluations of the dis-
coloration of ABTS+● (the diammonium salt of 2,2-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid), (Re et al. 1999;
Rice-Evans et al. 1996), and DPPH● (1,1-diphenyl-2-
picrylhydrazyl) initially purple in color, as described by
Blois (1958).
The computational study was performed with the mye-
loperoxidase (MPO) enzyme, which plays a critical role in
the immune system by producing ROS. However, when
ROS are secreted on the surface of neutrophils, they can
cause oxidative damage to the host tissues, resulting in
diseases such as Parkinson (Choi et al. 2005), Alzheimer’s
(Green et al. 2004), cancer (Henderson et al. 2003; Rojas
et al. 2001), atherosclerosis (Klebanoff 2005), and kidney
damage (Rao et al. 2011). Molecular dynamics (MD) and
Scheme 1 Regioselective synthesis of 2-acetyl-4-aminoresorcinol (3).
Reagents and conditions: (i) AcOH (conc), ZnCl2, 140 °C. (ii) AcOH
(conc), HNO3 (conc), room temperature. (iii) MeOH, H2O, Na2S2O4,
reflux

Medicinal Chemistry Research
measured once the antioxidants have lightened the solution,
diphenyl-2-picrylhydrazyl (DPPH), potassium persulfate
(K2S2O8), dimethylsulfoxide (DMSO), resorcinol, succinic
anhydride, glutaric anhydride, phthalic anhydride, THF,
ethyl acetate (AcOEt), methanol (MeOH), anhydrous
sodium sulfate (Na2SO4), and anhydrous potassium carbo-
nate (K2CO3) were used as received. Nuclear magnetic
resonance spectra (NMR 1H, 13C) were recorded on a
Varian Mercury Model spectrometer at 300 MHz using
tetramethylsilane at room temperature as an internal stan-
dard. The splitting patterns are designated s, singlet; d,
doublet; t, triplet; and m, multiplet. Infrared spectra were
recorded on a Microraman Lab Ram Mod HR 800 Horiba
JobinYvon spectrometer with Fourier transform analysis
using an ATR objective with a diamond tip for reflection
with a magnification of 36×. The same objective compacts
the slide reflection during the preparation of the pellet
sample. The spectra were acquired between 4000 and 400
cm−1 with 32 scans. Mass spectra were recorded on a
Bruker Compass model micrOTOF-QII spectrometer and
gas chromatograph model Finnigan Trace GC Ultra with
mass detector PolarizQ.
which occurs because of their ability to reduce ABTS+●
.
DPPH● radical assay
In the middle of the past century, Blois demonstrated for the
first time that the free DPPH● radical can accept a hydrogen
atom (H●). 1,1-Diphenyl-2-picrylhydrazyl is known to be a
stable free radical due to the delocalization of the unpaired
electron over the entire molecule; this prevents the molecule
from dimerizing. The delocalization of the electron inten-
sifies the typical purple color of the radical, which absorbs
at 517 nm in ethanol. When the DPPH radicals react with an
antioxidant that can donate a H●, the purple color fades. The
color change is spectroscopically measured and used to
determine the antioxidant properties (Raghavendran et al.
2005).
Molecular docking studies
MD and docking studies were based on the X-ray structure
of human MPO, (PDB code: 1DNU) at a1.9 Å resolution
(Blair-Johnson et al. 2001). MD simulations were per-
formed using the Gromac-5s.0.4 software. The two-
dimensional geometry of the ligand was generated with
ISIS Draw 2.4 and optimized using the HYPERCHEM
(Version 6.0, Hypercube, Inc. www.hyper.com) and Avo-
gadro software. The ligand and protein were prepared using
the AutoDock tool 1.5.6 included in the AutoDock 4.2
(www.autodock.scripps.edu) software.
1-(2,4-Dihydroxyphenyl)ethanone (1)
The dry Lewis acid (ZnCl2, 1.67 g, 12.2 mmol) and a suf-
ficient amount of concentrated acetic acid were mixed to
cover the solid. The mixture was heated until the complete
dissolution of the solid. Resorcinol (1.1 g, 10 mmol) was
slowly added with constant stirring, and then the tempera-
ture was raised to 140 °C. The reaction was continued at
that temperature for 30 min. The mixture was then poured
onto ice. The formed precipitate was filtered and washed
three times (30 ml) with dilute HCl (1 + 3). After vacuum
drying, 0.9074 g (59.7%) of the compound was obtained
and had a melting point of 144 °C.
The protein-binding visualization was performed with
PyMOL Molecular Visualization (www.pymol.org/).
Statistical analysis
In vitro experiments were performed twice in triplicate. The
concentration to inhibit 50% of the free radical (IC50%) was
calculated by interpolation of the linear regression from the
obtained graph. The experimental results are given as the
mean standard deviation (SD), and the differences are
considered statistically significant at probabilities <5%.
1-(2,4-Dihydroxy-5-nitrophenyl)ethanone (2)
The acetylated resorcinol (0.76 g, 5 mmol) was weighed and
combined with 5 ml of concentrated acetic acid at room
temperature. A mixture of 1 ml of nitric acid and 1 ml of
concentrated acetic acid was added dropwise under constant
stirring; the temperature was monitored to ensure it did not
exceed 30 °C. The reaction continued for 7 h. The product
was poured over crushed ice. The precipitate was filtered,
washed with cold water (three times), and dried under
vacuum. The product was purified by silica gel column
chromatography using hexane-ethyl acetate (8:2) as the
mobile phase. The desired product was obtained in a 53.7%
yield (0.529 g), and the melting point was 140–144 °C
Experimental
Chemistry
Reactant and solvents were purchased from commercial
suppliers (commercial products, Sigma-Aldrich). Nitric
(HNO3), acetic (CH3COOH), and hydrochloric (HCl) acids
as well as sodium hydrosulfite (Na2S2O4, reducing agent),
zinc chloride (ZnCl2), the diammonium salt of 2,2-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-
1
(Scheme 1). H NMR (300 MHz, CDCl3) δ 12.91 (1H, s,
-OH5), 11.06 (1H, s, -OH1), 8.65 (1H, s, Ar-H3), 6.59 (1H,
s, Ar-H6), 2.67 (3H, s, -CH3) p.p.m.

Medicinal Chemistry Research
1-(5-Amino-2,4-dihydroxyphenyl)ethanone (3)
was added, and the mixture was filtered. The solvent was
removed by rotary evaporation under vacuum. The residue
was purified by column chromatography with silica gel
using hexane-ethyl acetate (8:2) as the mobile phase.
(Scheme 2).
The nitrated product (0.985 g, 5 mmol) was weighed, 20 ml
of methanol was added, and the mixture was refluxed until
complete dissolution of the solid. A solution of 1 g of
sodium dithionite (Na2S2O4) in 20 ml of H2O was added
dropwise, and the reaction was maintained at reflux for an
additional 2 h. Additional Na2S2O4 (1 g, solid) was added,
and the heating continued. The progress of the reaction was
followed by thin-layer chromatography. If the reaction was
not complete, another gram of the reducing agent was
added. The methanol was evaporated under vacuum, water
was added, and the pH of the solution was adjusted to 6.5. It
was extracted with 30 ml of ethyl acetate (three times), and
then anhydrous Na2SO4 and activated charcoal were added.
The mixture was filtered, and the solvent was removed by
4-((5-Acetyl-2,4-dihydroxyphenyl)amino)-4-
oxobutanoic acid (4)
Yield 85.6% with melting point (MP) = 278–280 °C
(uncorrected): 1H NMR (300 MHz, DMSO-d6) δ 12.41 (1H,
s, -COOH), 9.22 (1H, s, NH), 8.28 (1H, s, Ar-H6), 6.37 (1H,
s, Ar-H3), 2.62 (2H, t, CH2), 2.51 (2H, t, -CH2), 2.48 (3H, s,
CH3CO) p.p.m. 13C NMR (300 MHz, DMSO-d6) δ 202.44
(C -COMe), 174 (C, -COOH), 170.61 (C, CONH), 160.37
(C, -C1-OH), 156.68 (C, -C5-OH), 125.11 (C, Ar-C3),
119.38 (C, Ar-C4), 111.75 (C, Ar-C2), 102.44 (C, Ar-C6),
30.8 (C, -CH2COOH), 29.2 (C, -CH2CONH), 26.4 (C,
CH3CO) p.p.m. Mass spectrometry (MS) m/z (% relative
intensity): 267 (13.3%, M+). Infrared (IR) selected bands
(cm−1): 3392 (-OH), 3045 (-NH), 1707 (C=O, -COOH),
1660 (C=O, -NH-C=O), 1633 (C=O, -COCH3), 1406
rotary
evaporation
under
vacuum.
2-Acetyl-4-
aminoresorcinol (3) was isolated as a solid in an 89.1%
yield (0.744 g) (Scheme 1).
General procedure for the synthesis of the amides
derived (4, 5, 6) from 2-acetyl-4-aminoresorcinol (3)
(-CH3), 1192 (-OH) cm−1
.
2-Acetyl-4-aminoresorcinol (3) (1 mmol) and 1 mmol of the
corresponding anhydride (succinic, glutaric, or phthalic)
were dissolved in 5 ml of THF. Anhydrous K2CO3 (138.2
mg, 1 mmol) was then added, and the solution was heated at
reflux for 2 or 3 h. (The progress of the reaction was fol-
lowed by thin-layer chromatography). The THF was eva-
porated, the residue was dissolved in H2O, the pH was
adjusted to 4.3 with dilute HCl, the solution was extracted
with 30 ml of ethyl acetate (three times), anhydrous Na2SO4
5-((5-Acetyl-2,4-dihydroxyphenyl)amino)-5-
oxopentanoic acid (5)
Yield 72.7% with MP = 210–212 °C (uncorrected): 1H
NMR (300 MHz, DMSO-d6) δ 12.42 (1H, s, -COOH), 9.16
(1H, s, NH), 8.21 (1H, s, Ar-H6), 6.36 (1H, s, Ar-H3), 2.5
(3H, s, CH3CO), 2.39 (2H, t, -CH2COOH), 2.28 (2H, t,
-CH2CONH), 1.79 (2H, m, CH2) p.p.m. 13C NMR (300
Scheme 2 Synthesis of amides
succinic (4), glutaric (5), and
phftalic (6). (i), (ii), (iii) THF,
corresponding anhydride,
K2CO3, reflux

Medicinal Chemistry Research
MHz, DMSO-d6) δ: 202.44 (C - COMe), 174.29 (C,
-COOH), 171.28 (C, -CONH), 160.55 (C, Ar-C1-OH),
157.13 (C, Ar-C5-OH), 125.93 (C, Ar-C4), 119.17 (C, Ar-
C3), 111.79 (C, Ar-C2), 102.47 (C, Ar-C6), 34.87 (C, C5),
33.05 (C, C4), 26.63 (C, C2), 20.64 (C, C3). MS m/z (%
relative intensity): 281 (12.8%, M+). IR selected bands (cm
−1): 3396 (-OH), 3063 (-NH), 1704 (C=O, -COOH), 1633
(C=O, -NH-C=O), 1616 (C=O, -COCH3), 1398 (-CH3),
compound solution and 2 ml of DMSO in a second tube
(Aj), and combining 2 ml of DPPH with 2 ml of DMSO in a
third tube (Ac) for each concentration of the test com-
pounds. The tubes were mixed and kept in the dark for 1 h.
The absorbance readings were then taken, and the % inhi-
bition was calculated by the following equation:
% Inhibition ¼ ½1 À ðAi À AjÞ=AcŠ  100
1188 (-OH) cm−1
.
where Ai is the absorbance of the solution of the compound
with DPPH, Aj is the absorbance of the solution of the
compound with DMSO, and Ac is the absorbance of the
DPPH solution with DMSO.
2-((5-Acetyl-2,4-dihydroxyphenyl)carbamoyl)
benzoic acid (6)
Yield 84.2% with MP = 266–268 °C (uncorrected): 1H
NMR (300 MHz, DMSO-d6) δ: 12.59 (1H, s, -COOH),
11.03 (1H, s, NH), 7.93 (2H, m, Ar), 7.98 (3H, m, Ar), 6.48
(1H, s, Ar-H3), 3.36 (3H, s, -CH3). 13C NMR (300 MHz,
DMSO-d6) δ: 202.49 (C, -COMe), 167.37 (C, -COOH),
163.96, 161.26, 134.76, 134.5, 131.84, 123.42, 113.1,
111.65, 103.2, 26.70 (C, -CH3). MS m/z (% relative inten-
sity): 297 (18%, M+-H2O). IR selected bands (cm−1): 3275
(-OH), 3052 (-NH), 1709 (C=O, -COOH), 1638 (C=O,
-NH -C=O), 1624 (C=O, -COCH3), 1404 (-CH3), 1187
Molecular docking
From the PDB of the X-ray complex structure of human
MPO (PDB code: 1DNU), the water and the CN-, Br-, and
SCN- anions were removed and subjected to an MD
simulation for 100 ns (nanoseconds) with a cubic box with
21,958 TIP3 water molecules neutralized with 15 chlorides
Table 1 Results of ABTS antioxidant assay
(-OH) cm−1
.
mM
Resveratrol
Phthalic
Glutaric
Succinic
Antioxidant screening
0.025
0.050
0.075
0.100
0.150
0.200
IC50%
4.409
8.102
8.975
9.249
6.725
9.245
9.454
9.396
ABTS
23.932
32.071
69.899
81.956
0.1275 mM
13.965
25.201
46.031
67.378
0.1281 mM
20.373
26.274
60.911
85.713
0.1314 mM
17.979
32.557
61.071
88.345
0.1605 mM
First, 100 ml of 7 mM ABTS salt was prepared in ethanol,
and 65 mg of K2S2O8 was added. The solution was stirred at
room temperature for 16 h in the dark to form the radical.
The resulting solution was diluted with ethanol until the
absorbance was 0.700 k 0.02 at 734 nm. Subsequently, 2
ml of the formed ABTS+● solution was mixed with each of
the six ethanolic solutions of the compounds prepared at
different concentrations (0.025–0.2 mM). The tubes were
protected from light for 6 min, and then the absorbance of
each was read with a ultraviolet-visible spectrophotometer
at 734 nm. The inhibition percentage was calculated using
the following equation:
Percentage of inhibition of resveratrol, 4, 5, 6 vs IC50%
R2 (0.9604–0.9695)
100
Resveratrol
Phthalic
80
Glutaric
% Inhibition ¼ ½ðAc À AsÞ=Ac  100Š
60
Succinic
where Ac is the absorbance of the control and As is the
absorbance of the tested compounds.
40
20
0
DPPH
A 3 × 10−5 M DPPH solution was prepared in DMSO.
Solutions of the synthesized compounds were prepared at
different concentrations (0.05–1.0 mM) in ethanol. The
assay was performed by combining 2 ml of the compound
solution and 2 ml of DPPH (Ai), combining 2 ml of the
0.025
0.050
0.075
0.100
0.150
0.200
mM
Fig. 1 ABTS. Bar graph of the percentage of inhibition of the tested
compounds

Medicinal Chemistry Research
Table 2 Results of DPPH antioxidant assay
1.0
0.8
0.6
0.4
0.2
0.0
mM
Resveratrol
Phthalic
Glutaric
Succinic
0.05
0.08
0.10
0.15
0.20
1.00
IC50%
23.796
25.022
30.718
40.573
45.772
90.035
0.3733 mM
9.466
4.764
6.774
10.635
11.886
13.552
15.900
31.204
1.8459 mM
7.292
10.830
15.291
16.247
20.296
46.182
1.0823 mM
11.758
13.534
15.464
38.851
1.3216 mM
Percentage of inhibition of the resveratrol, 4, 5, 6 vs IC50%
R2 (0.9560–0.9811)
0
20
40
60
80
100
time ns
100
Resveratrol
Fig. 3 The α-carbon root mean square deviation (RMSD) for MPO
show that the protein reached structural stability at approximately 50
ns
Phthalic
80
Glutaric
60
Succinic
HNO3 (conc.), the electrophilic aromatic substitution can be
40
20
0
directed to the four positions. This was confirmed because
1
the H-3 and H-6 hydrogen signals in the H NMR are
singlets, and therefore, they are not adjacent or coupled to
other protons. Subsequently, the nitrated product (2) was
reduced with Na2S2O4 to the aromatic amine (3), which was
used directly in the following reactions.
0.05
0.08
0.10
0.15
0.20
1.00
mM
The derived amides (4, 5, and 6) were synthesized in
refluxing MeOH/H2O in the presence of sufficient reducing
agent. The compounds were purified and characterized. The
1H NMR signals of the amidic N-H protons of the com-
pounds were all singlets (9.22, 9.16, and 11.3 p.p.m. (due to
being adjacent to a carboxyl)), and the singlet signals of the
carboxylic hydrogens were at 12.41, 12.42, and 12.59 p.p.
m. In addition, the IR bands were used to characterize the
functional groups: 3392 (-OH), 3045 (-NH), 1707 (C=O,
-COOH), 1660 (C=O, -NH-C=O), and 1633 cm−1(C=O,
-COCH3) for succinic amide (4); for the derivative of glu-
taric anhydride, the bands were at 3396 (-OH), 3063 (-NH),
1704 (C=O, -COOH), 1633 (-NH-C=O), and 1616 cm−1
(C=O, -COCH3); and for the phthalic anhydride derivative,
the bands were at 3275 (-OH), 3052 (-NH), 1709 (C=O,
-COOH), 1638 (C=O, -NH-C=O), and 1624 cm−1 (C=O,
-COCH3). There are also important signals between 1180
and 1195 cm−1 present in the IR spectra of the three amides
due to the free -OH phenolic groups of the resorcinol.
Fig. 2 DPPH. Bar graph of the percentage of inhibition of the tested
compounds
and Gromos53a6 force field (http://www.gromacs.org/).
Snapshots were obtained every 10 ns prior to the docking
studies. Snapshots of the ligands and the protein were
prepared using the hybrid Lamarckian genetic algorithm
method with an initial population of 100 randomly placed
individuals and a maximum number of energy evaluations
of 1 × 107 using the AutoDock tool 1.5.6 software. The
geometries of the ligands were generated and optimized. All
docking simulations were performed on a grid (126 ×
126 × 126 Å) with grid points separated by 0.375 Å. The
resulting docked orientations were clustered with RMSDs
(root mean square deviation) of ≤0.5 Å.
Results and Discussion
ABTS+● generated by oxidation
The MP of acetylated resorcinol was 144 °C (lit 143–145 °
C); this product was reacted with concentrated HNO3 and
H2SO4 (strong acid). This conventional nitration directs the
nitro group to the aromatic carbons at the four and/or six
positions. However, by decreasing the strength of the acid
using AcOH (a weak acid) and limiting the amount of
This assay is mainly based on the electron transfer and
reduction of the radical ABTS+● cations, which may be
more efficient than that of DPPH (Prior et al. 2005). The
activity was determined from the decrease in absorbance at
734 nm. The color of the ABTS+● solution changed due to

Medicinal Chemistry Research

Medicinal Chemistry Research

Medicinal Chemistry Research
Fig. 4 Myeloperoxidase enzyme showing the pocket “A” and pocket “B” adjacent to the HEM group
the capacity of the antioxidant compounds to reduce the
preformed radical. The inhibition of the superoxide pro-
duction and the antioxidant activity were calculated from
the percentage decrease in the absorbance. The compounds
showed similar antioxidant activities; however, at low
concentrations they are moderately more active than
resveratrol. The results of the antioxidant screening (sca-
venger activity) and IC50% are listed in Table 1; the per-
centage of inhibition is shown in Fig. 1.
when DPPH● was removed by an antioxidant compound via
the donation of a hydrogen to form a stable DPPH radical
molecule. In this assay, at the tested concentrations, the
synthesized compounds showed less antioxidant activity
than resveratrol. The results of the scavenger activity and
IC50% values are listed (Table 2). Percentage of inhibition is
shown (Fig. 2).
When an antioxidant is added to the radical solution, the
color of the solution will change, which is related to the
formation of the radical DPPH● and ABTS+● cations:
DPPH● radical
DPPHꢀðABTSþꢀÞ þ AH ! DPPH À HðABTSþÞ þ Aꢀ
Polyphenolic compounds can act as antioxidants, and
their properties depend on the redox potentials of their
hydroxyl–phenolic groups and the potential for electronic
delocalization through their chemical structures. These
methods are efficient, the reagents are not expensive, and
common instruments found in research laboratories are used
for the analyses.
This assay is based on electron transfer and hydrogen atom
abstraction, so the reductive capacity of the antioxidant
compounds toward DPPH is measured according to a pre-
viously reported procedure (Soong and Barlow 2004).
DPPH● is considered a valid and easy method for evaluat-
ing the antioxidant radical scavenging activity of the com-
pounds since the radical compound (DPPH●) is stable and
does not have to be freshly generated like in other radical
scavenging assays. The synthesized compounds have
chromophores that interfere with measuring the absorbance
of the DPPH solution at 517 nm, which is the wavelength
typically used in DPPH assays. The absorbance decreased
MPO docking
Because antioxidants derived from phenols have been
designed as inhibitors of MPO, the compounds obtained in

Medicinal Chemistry Research
Fig. 5 Pocked “A”. Amino acid
interactions with resveratrol
(A1), glutaric (A2), succinic
(A3), and phthalic (A4)
derivatives
this study were subjected to molecular modeling studies to
assess their possible interactions with the active pockets of
MPO.
According to the RMSD values (Fig. 3), the system
reaches convergence after 50 ns, and the conformers were
analyzed with the Pymol program. Therefore, the system
was explored at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and
100 ns (Table 3), and the chains derived from the molecular
dynamics studies of MPO were present in pocket “A” and
pocket “B” (Fig. 4).
For all the compounds synthesized in the snapshots, the
ΔG values of their pocket “A” sites are lower than those of
their pocket “B” sites. The interactions are with the amino
acids Asn555, Phe552, Arg490, Ser482, Leu384, Trp477,
and Gly335 (Table 3). The resveratrol forms hydrogen
bonds between the -OH and -C=O peptide of Phe552,
-C=O peptide of Arg490, -NH peptide of Gly335, and
-COO of Glu483. Ligands also interact via bridging
hydrogen bonds from the carboxyl of the guanidine group
of Arg490 and -NH peptide of Gly335, the -C=OCH3
(acetyl) group with -NH (indole) of Trp477 and the -C=O
peptide of Ser482, and the aromatic -OH group with the
-OH of Ser482 and the -NH peptide of Arg333 (Fig. 5).
Pocket “B”: The values of ΔG (free energy) of the syn-
thesized products are generally lower than those of resver-
atrol (Table 3). Additionally, the ligands are adjacent to the
pyrrole D of the Hem group with residues Arg239, Phe366,
and Phe407 (Van Antwerpen et al. 2008). The resveratrol
forms bridging hydrogen bonds between its -OH group and
the guanidine group of Arg239, the Arg239 peptide -C=O,
and the peptide -C=O of Glu404. The three synthesized
compounds are characterized by hydrogen bonds to the
-C=OCH3 (acetyl) group and the -C=O (carboxyl) group
with the guanidine part of Arg239 and Arg405 and the
aromatic peptide -NH linkers of residues Phe407 and
Phe147 (Fig. 6).
Conclusion
It is worth mentioning that the three synthesized compounds
are good antioxidants at low concentrations (0.025 and
0.050 mM), and at higher concentrations, they are similar to
the reference (resveratrol, ABTS method). The succinic-
amide (4) showed the best antioxidant capacity with an
IC50% very similar to that of the positive control. However,

Medicinal Chemistry Research
Fig. 6 Pocked “B”. Amino acid
interactions with resveratrol
(B1), glutaric (B2), succinic
(B3), and phthalic (B4)
derivatives
Benabdesselam F, Bedjou F, Van Antwerpen P, Duez P (2014)
Inhibition ofmyeloperoxidase activity by the alkaloids of Pega-
in the DPPH assay, the synthesized compounds showed
approximately 50% lower efficiencies than the control.
The results of the docking study show that strong affi-
nities are possible in two pockets: “B” adjacent to the HEM
group and “A”, which is the pocket with lower ΔG values in
almost all the MPO conformers analyzed (Table 3).
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L.
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Conflict of interest The authors declare that they have no conflict of
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