Remarkable antioxidant properties of a series of hydroxy-3-arylcoumarins

Article abstract of DOI:10.1016/j.bmc.2013.04.015

In the present work we synthesized a series of hydroxy-3-arylcoumarins (compounds 1-9), some of them previously described as MAO-B selective inhibitors, with the aim of evaluating their antioxidant properties. Theoretical evaluation of ADME properties of all the derivatives was also carried out. From the ORAC-FL, ESR and CV data it was concluded that these derivatives are very good antioxidants, with a very interesting hydroxyl, DPPH and superoxide radicals scavenging profiles. In particular compound 9 is the most active and effective antioxidant of the series (ORAC-FL = 13.5, capacity of scavenging hydroxyl radicals = 100%, capacity of scavenging DPPH radicals = 65.9% and capacity of scavenging superoxide radicals = 71.5%). Kinetics profile for protection fluorescein probe against peroxyl radicals by addition of antioxidant molecule 9 was also performed. Therefore, it can operate as a potential candidate for preventing or minimizing the free radicals overproduction in oxidative-stress related diseases.

Full text of DOI:10.1016/j.bmc.2013.04.015

Bioorganic & Medicinal Chemistry 21 (2013) 3900–3906
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Remarkable antioxidant properties of a series
of hydroxy-3-arylcoumarins q
c
a
a
Maria João Matos a,b, , Fernanda Pérez-Cruz , Saleta Vazquez-Rodriguez , Eugenio Uriarte ,
⇑
Lourdes Santana a, Fernanda Borges b, Claudio Olea-Azar c,
⇑
a Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
b CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal
c Department of Inorganic and Analytical Chemistry, Laboratory of Free Radicals and Antioxidants, Faculty of Chemical and Pharmaceutical Sciences,
University of Chile, Santiago de Chile, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work we synthesized a series of hydroxy-3-arylcoumarins (compounds 1–9), some of them
previously described as MAO-B selective inhibitors, with the aim of evaluating their antioxidant proper-
ties. Theoretical evaluation of ADME properties of all the derivatives was also carried out. From the
ORAC-FL, ESR and CV data it was concluded that these derivatives are very good antioxidants, with a very
interesting hydroxyl, DPPH and superoxide radicals scavenging profiles. In particular compound 9 is the
most active and effective antioxidant of the series (ORAC-FL = 13.5, capacity of scavenging hydroxyl
radicals = 100%, capacity of scavenging DPPH radicals = 65.9% and capacity of scavenging superoxide
radicals = 71.5%). Kinetics profile for protection fluorescein probe against peroxyl radicals by addition
of antioxidant molecule 9 was also performed. Therefore, it can operate as a potential candidate for pre-
venting or minimizing the free radicals overproduction in oxidative-stress related diseases.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Received 13 December 2012
Revised 26 March 2013
Accepted 3 April 2013
Available online 19 April 2013
Keywords:
3-Arylcoumarins
Hydroxyl derivatives
ORAC-FL assays
ESR assays
CV assays
Antioxidant capacity
1. Introduction
neurodegenerative pathologies.2 Therefore, antioxidants are very
important for protecting the organisms from oxidative disorders,
Phenolic compounds are bioactive substances widely distrib-
uted in the vegetable kingdom. Generally, this group of compounds
has one or more aromatic rings in their structure and one or more
hydroxyl groups. They have been described to act as natural anti-
oxidants and their presence contributes to prevent or minimize
several types of oxidative processes.1 Due to their antioxidant
activity their ingestion is correlated with interesting benefits to
health. Therefore, the research and characterization of new bioac-
tive phenolic substances from diet has been intensified in the last
years, either for the development of nutraceutics or medicines.1
Due to their antioxidant properties they can protect cells from
the oxidative damage of the reactive oxygen species (ROS). In fact,
the overproduction of free radicals have been related to cellular
membrane and DNA damage, and indirectly with aging and
oxidative-stress related diseases like cancer, cardiovascular and
in which ROS are also involved.3,4 Antioxidants are capable of
decrease or prevent oxidation processes through different mecha-
nisms, such as scavenging free radicals, inhibition of pro-oxidant
enzymes or chelation of transition metal ions.5
An increasing number of reports suggested the involvement of
oxidative stress in neurodegenerative diseases (ND), where the in-
creased formation of ROS can contribute to neuronal damage and
cell death.3,4
Suggestion has been made that the etiology of Parkinson’s (PD)
and Alzheimer’s (AD) diseases may be closely linked to biochemi-
cal changes resultant from this oxidative stress.6–8 Dopamine (DA)
auto-oxidation naturally produces oxidative species and may
contribute to ND such as PD and ischemia/reperfusion-induced
damage. Monoamine oxidase (MAO) enzyme (particularly
MAO-B) is responsible for metabolizing DA and plays an important
role in oxidative stress through altering the redox state of neuronal
and glial cells, leading to neuronal death.9 Consequences are an
over-production of MAO and non-MAO initiated hydrogen perox-
ide (H2O2) by proliferated reactive microglia and inability of
neurons to dispose of H2O2 and other recative species like peroxyl
radicals.10 H2O2 produces highly toxic ROS, namely hydroxyl
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
⇑
Corresponding authors. Tel.: +34 981594912; fax: +34 981528070.
E-mail addresses: mariacmatos@gmail.com (M.J. Matos), colea@uchile.cl
(C. Olea-Azar).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.015

M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
3901
radical, by Fenton reaction that is catalyzed by iron and neuro-
2.1.2. General procedure for the preparation of hydroxy-3-
arylcoumarins
melanin.11 Concerning the mechanism of the clinical efficacy of
MAO-B inhibitors in PD, the inhibition of DA degradation (a
symptomatic effect) and also the prevention of the formation of
neurotoxic DA degradation products, that is, ROS and DA derived
aldehydes have been speculated.12 The neuroprotective effect of
rasagiline, a well-known MAO-B inhibitor, might be explained
through multiple mechanisms, possibly due to reduction of DA
catabolism with a subsequent increased activity on dopaminergic
D2 receptors and suppressing the action of ROS as well.13 So, the
possible mechanism of neuroprotection of MAO-B inhibitors may
be related not only to MAO-B inhibition but also to induction and
activation of multiple factors related with oxidative stress and
apoptosis.14
To a solution of a methoxy-3-arylcoumarin (0.50 mmol) in ace-
tic acid (5 mL) and acetic anhydride (5 mL), at 0 °C, hydriodic acid
57% (10 mL) was added dropwise. The mixture was stirred under
reflux, for 3 h. The solvent was evaporated under vacuum and
the dry residue was purified by crystallization (CH3CN).23,28,34
2.1.2.1. 3-(30,40-Dihydroxyphenyl)-6-methylcoumarin (4).
Yield: 92%; mp 199–200 °C; 1H NMR (300 MHz, DMSO-d6): d 2.43
(s, 3H, –CH3), 6.43 (s, 1H, H-20), 7.01 (d, J = 7.1 Hz, 1H, H-50), 7.14
(d, J = 7.0 Hz, 1H, H-60), 7.28–7.32 (m, 2H, H-7, H-8), 7.57 (d,
J = 2.2 Hz, 1H, H-5), 7.83 (s, 1H, H-4), 10.40 (s, 2H, –OH); 13C
NMR (75 MHz, DMSO-d6): d 20.7, 100.1, 110.7, 111.6, 115.7,
119.4, 120.1, 127.3, 127.4, 132.1, 133.7, 138.8, 146.5, 146.8,
151.3, 161.6; EI MS m/z: 269 (13), 268 (M+, 100), 241 (31), 240
(70), 239 (22), 165 (30), 125 (12), 111 (10); Anal. Calcd for
Coumarins are a family of compounds widely distributed in the
nature.15 Due to their structural features, and biological properties,
namely anticancer, anti-inflammatory, antioxidant, antithrom-
botic, vasorelaxant, antiviral and enzymatic inhibition agents, they
have been ascribed as important building blocks in Organic
Chemistry and Medicinal Chemistry.16–23
Recently, it was shown by our group that 3-substituted aryl
coumarins are potent and selective MAO-B inhibitors.24–30 In addi-
tion, it has been found that hydroxycoumarins are antioxidants
scavenging ROS and/or chelating transition metals, exhibiting
tissue-protective properties.6,31–33 The complementarity of these
activities for 3-arylcoumarins was not previously studied and
described. The versatility of the used reactions allowed obtaining
a family of compounds with hydroxyl and/or methyl substituents
in different positions of the molecule. The election of these deriva-
tives has considered the previously MAO-B inhibitory pharmaco-
logical evaluation and the low cost of the commercial reagents to
begin with. Also, the influence of the substituents in the desired
activity was taken into account.
C16H12O4: C, 71.64; H, 4.51. Found: C, 71.60; H, 4.49.
2.1.2.2. 3-(30,40-Dihydroxyphenyl)-8-methylcoumarin (5).
Yield: 85%; mp 205–206 °C; 1H NMR (300 MHz, DMSO-d6): d
2.50 (s, 3H, –CH3), 6.80 (d, J = 8.3 Hz, 1H, H-50), 7.05 (dd,
J = 8.2, J = 2.2 Hz, 1H, H-60), 7.22 (d, J = 2.2 Hz, 1H, H-20), 7.25
(d, J = 7.6 Hz, 1H, H-6), 7.44 (dd, J = 7.4, J = 1.0 Hz, 1H, H-7),
7.57 (dd, J = 7.6, J = 1.1 Hz, 1H, H-5), 8.09 (s, 1H, H-4), 9.09
(s, 1H, –OH), 9.24 (s, 1H, –OH); 13C NMR (75 MHz, DMSO-
d6): d 14.9, 115.4, 116.0, 119.5, 119.9, 124.1, 124.6, 125.7,
126.1, 126.5, 132.2, 138.7, 144.8, 146.2, 150.9, 159.9; EI MS
m/z: 270 (12), 269 (76), 268 (M+, 82), 241 (47), 240 (100)
239 (57), 211 (23), 166 (19), 165 (58), 152 (18), 139 (14),
125 (30), 111 (28), 82 (19); Anal. Calcd for C16H12O4: C,
71.64; H, 4.51. Found: C, 71.63; H, 4.49.
2.1.2.3. 3-(30,50-Dihydroxyphenyl)-8-methylcoumarin (6).
Yield: 90%; mp 180–181 °C; 1H NMR (300 MHz, DMSO-d6): d
2.49 (s, 3H, –CH3), 6.70 (s, 3H, H-20, H-40, H-60), 7.26 (t,
J = 7.6 Hz, 1H, H-6), 7.48 (d, J = 7.8 Hz, 1H, H-7), 7.62 (d,
J = 7.7 Hz, 1H, H-5), 8.11 (s, 1H, H-4), 10.27 (s, 2H, OH); 13C
NMR (75 MHz, DMSO-d6): d 15.3, 103.3, 107.2, 119.6, 124.5,
125.1, 126.8, 127.2, 133.1, 136.6, 140.9, 151.6, 158.5, 160.0;
EI MS m/z: 269 (21), 268 (M+, 100), 241 (12), 240 (63), 239
(26); Anal. Calcd for C16H12O4: C, 71.64; H, 4.51. Found: C,
71.60; H, 4.50.
2. Materials and methods
2.1. Chemistry
Melting points were determined using a Reichert Kofler ther-
mopan or in capillary tubes on a Büchi 510 apparatus and are
uncorrected. 1H and 13C NMR spectra were recorded on a Bruker
AMX spectrometer at 300 and 75.47 MHz, respectively, using
TMS as internal standard (chemical shifts in d values, J in Hz). Mass
spectra were obtained using a Hewlett–Packard 5988A spectrome-
ter. Elemental analyses were performed using a Perkin–Elmer 240B
microanalyser and were within 0.4% of calculated values in all
cases. Silica gel (Merck 60, 230-00 mesh) was used for flash chro-
matography (FC). Analytical thin layer chromatography (TLC) was
performed on plates precoated with silica gel (Merck 60 F254,
0.25 mm).
2.1.2.4. 3-(30,40,50-Trihydroxyphenyl)-8-methylcoumarin (7).
Yield: 82%; mp 189–190 °C. 1H NMR (300 MHz, DMSO-d6): d 2.49
(s, 3H, –CH3), 6.95 (s, 2H, H-20, H-60), 7.20 (t, J = 7.4 Hz, 2H, H-6,
H-7), 7.38 (d, J = 7.4 Hz, 1H, H-5), 7.79 (s, 1H, H-4), 10.55 (s, 2H,
–OH), 10.60 (s, 1H, –OH); 13C NMR (75 MHz, DMSO-d6DMSO-d6):
d 15.6, 106.1, 119.4, 124.2, 125.4, 125.7, 127.7, 130.4, 132.7,
135.6, 139.8, 146.7, 146.9, 160.6; EI MS m/z: 285 (16), 284 (M+,
100), 283 (84), 256 (32), 181 (10) 141 (10); Anal. Calcd for
2.1.1. General procedure for the preparation of methoxy-3-
arylcoumarins
C16H12O5: C, 67.60; H, 4.25. Found: C, 67.61; H, 4.28.
To a solution of the conveniently substituted ortho-hydroxy-
benzaldehyde (7.34 mmol) and the corresponding phenylacetic
acid (9.18 mmol) in dimethyl sulfoxide (15 mL), N,N0-dicyclohexyl-
carbodiimide (11.46 mmol) was added. The mixture was heated at
110 °C for 24 h. Then, ice (100 mL) and acetic acid (10 mL) were
added to the reaction mixture. After keeping it at room tempera-
ture for 2 h, the mixture was extracted with ether (3 ꢀ 25 mL).
The organic layers were combined and washed with sodium bicar-
bonate solution (50 mL, 5%) and water (20 mL). Subsequently, the
solvent was evaporated under vacuum and the dry residue was
purified by flash chromatography (hexane/ethyl acetate 9:1), to
give the desired methoxy-3-arylcoumarins.23,28
2.2. Antioxidant assays
2.2.1. Oxygen radical antioxidant capacity-fluorescein (ORAC-FL)
The ORAC analyses were carried out on a Synergy HT multi
detection microplate reader, from Bio-Tek Instruments, Inc.
(Winooski, USA), using white polystyrene 96-well plates, pur-
chased from Nunc (Denmark). Fluorescence was read from the
top, with an excitation wavelength of 485/20 nm and an emission
filter of 528/20 nm. The plate reader was controlled by Gen 5 soft-
ware. The reaction was carried out in 75 mM sodium phosphate
buffer (pH 7.4), and 200
tration) and hydroxy-3-arylcoumarin solutions in methanol with
lL final volume. FL (70 nM, final concen-

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M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
a range of concentration between 0.3 and 2
l
M were placed in
693VA processor, at room temperature, using a three-electrode
each well of 96-well plate. The mixture was pre-incubated for
15 min at 37 °C, before rapidly adding the AAPH solution
(18 mM, final concentration). The microplate was immediately
placed in the reader and automatically shaken prior to each read-
ing. The fluorescence was recorded every 1 min for 120 min. A
blank with FL and 2,20-azobis(2-methylpropionamidine)dihydro-
chloride (AAPH) using methanol instead of the antioxidant solution
were used in each assay. Five calibration solutions using Trolox
cell. A glassy carbon (GC) electrode presenting an area of
0.03 cm2 was used as the working electrode. The electrode surface
was polished to a mirror finish with alumina powder (0.3 and
0.05 lM) before use and after each measurement. Platinum wire
was used as auxiliary electrode and silver-silver chloride (Ag/AgCl,
3 M KCl) of Metrohm Company with a plastic tip was used as a ref-
erence electrode. The CV experiments were carried out in dimethyl
sulfoxide (DMSO) of analytical grade (Sigma–Aldrich) with 0.1 M of
tetrabutylammonium perchlorate (TBAP) as supporting electrolyte.
Superoxide anion radical was generated in DMSO containing TBAP
0.1 M. The scan rate was kept 30 mV/s and potential window was
ꢁ1.0 to ꢁ0.0 V. The atmospheric solubility of oxygen in DMSO was
(0.5–2.5 lM) as antioxidant were also done. The inhibition capac-
ity was expressed as ORAC values and is quantified by integration
of the area under the curve (AUCNET). All reaction mixtures were
prepared in triplicate and at least three independent assays were
performed for each sample. The area under the fluorescence decay
curve (AUC) was calculated integrating the decay of the fluores-
cence where F0 is the initial fluorescence read at 0 min and F is
the fluorescence read at time. The net AUC corresponding to the
sample was calculated by subtracting the AUC corresponding to
the blank. Data processing was performed using Origin Pro 8 SR2
(Origin Lab Corporation, USA).
2.1 mM.44 The final concentration of each derivative (30
lM) was
achieved by additions of the corresponding aliquot of stock solu-
tion (10 mL final volume). Finally, the antioxidant activity was as-
sessed from the change in the cathodic current of the
voltammograms in absence and present of the derivatives, using
pertinent mathematical formulations.
2.2.5. Data statistical analysis
2.2.2. Hydroxyl radical scavenging assay using electron spin
resonance (ESR)
Statistical analyses were conducted using GraphPad Prism 5
software (GraphPad Software, Inc., San Diego, CA). The data are ex-
pressed as means SD. The experimental data were analyzed by
one-way analysis of variance (ANOVA), and differences between
groups were assessed using Tukey’s post-test. The level of signifi-
cance was set at p <0.05, and all experiments were replicated
3 times.
Reactivity of all the hydroxy-3-arylcoumarin derivatives against
the hydroxyl radical was investigated using the non-catalytic Fen-
ton type method. ESR spectra were recorded in the X band
(9.7 GHz) using a Bruker ECS 106 spectrometer with a rectangular
cavity and 50 kHz field modulation, equipped with a high-sensitiv-
ity resonator at room temperature. Spectrometer conditions were:
microwave frequency 9.81 GHz, microwave power 20 mW, modu-
lation amplitude 0.91 G, receiver gain 59 db, time constant
81.92 ms and conversion time 40.96 ms. The scavenging activity
of each derivative was estimated by comparing the DMPO-OH ad-
duct signals in the antioxidant–radical reaction mixture and the
control reaction at the same reaction time, and is expressed as
scavenging percent of hydroxyl radical.
3. Results
3.1. Chemical synthesis
Coumarin derivatives 1–9 were efficiently synthesized accord-
ing to the protocol outlined in Scheme 1. The general reaction con-
ditions and the characterization data of the new compounds were
described in the experimental section. Perkin condensation of dif-
ferent ortho-hydroxybenzaldehydes with the adequate arylacetic
acid, using N,N0-dicyclohexylcarbodiimide (DCC) as dehydrating
agent,28 afforded the methoxy-3-arylcoumarins. Hydroxyl deriva-
tives were obtained from the above-mentioned methoxy substi-
tuted precursors by acidic hydrolysis, using hydriodic acid 57% in
the presence of acetic acid and acetic anhydride.28
To prepare the samples, 150
and 50 L of NaOH (3 mM) were mixed, followed by the addition of
50 L of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap (30 mM
final concentration) and finally 50 L of hydrogen peroxide 30%. The
lL of N,N-dimethylformamide (DMF)
l
l
l
mixture was put in an ESR cell and the spectrum was recorded after
five minutes of reaction. All the compounds were studied to 4 mM
final concentration (300 lL final volume).
2.2.3. DPPH radical scavenging assay using ESR
3.2. Antioxidant capacity assays
The 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging
capacity35–38 of the hydroxy-3-arylcoumarin derivatives was
determined by an ESR spectrometry method.39–43 Each hydroxy-
3-arylcoumarin solution was mixed with DPPH stock solution to
initiate the antioxidant–radical reaction. All the reaction mixtures
contained 1.0 mM of DPPH and 1.0 mM of the studied compound.
The control solution was prepared in absence of the studied com-
pounds. Both DPPH and compounds solutions were prepared in
acetonitrile. ESR signals were recorded after five minutes of reac-
tion. Spectrometer conditions were: microwave frequency
9.81 GHz, microwave power 20 mW, modulation amplitude 0.95
G, receiver gain 59 db, time constant 81.92 ms and conversion time
40.96 ms. The scavenging activity of each compound was esti-
mated by comparing the DPPH signals in the antioxidant–radical
reaction mixture and the control reaction at the same reaction
time, and was expressed as scavenging percent of DPPH.
The evaluation of the antioxidant activity of the studied com-
pounds was performed towards different types of reactive oxygen
or nitrogen species—peroxyl, hydroxyl, superoxide and DPPH rad-
icals. ORAC-FL, ESR and CV assays were the techniques used to ob-
tain the desired results.
The peroxyl radical scavenging activity of the synthesized
hydroxy-3-arylcoumarins was evaluated by the oxygen radical
absorbance capacity (ORAC) method45 This assay use a fluores-
cence-based technology (ORAC-FL) and allow obtaining a relative
antioxidant index by using as reference trolox, a hydrosoluble vita-
min E derivative. The exposition of the fluorophore, in this case
fluorescein (FL) to the peroxyl radical lead to an oxidation process
reflected as a decay of fluorescence emission through time. In
ORAC assays, the loss of fluorescence of FL generally corresponds
to an induction time and is reliant on antioxidant capacity of a
compound. In fact, it refers to the time in which the FL is protected
against the oxidative damage of peroxyl radicals and this behavior
is associated to a competitive reaction between the radical and the
antioxidant.46,47 ORAC data take into account the induction time,
2.2.4. Superoxide antioxidant assay using cyclic voltammetry
(CV)
Cyclic voltammetry (CV) measurements were performed in a
Metrohm 693VA instrument with a 694VA stand convertor and a

M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
3903
R1
CHO
OH
COOH
i
R
R1
R
O
O
R or R1 = OCH3
ii
1: R = 6-CH3 ; R1 = 4'-OH
2:
3:
R = 6-CH3 ; R1 = 3'-OH
R = 6-CH3 ; R1 = 2'-OH
R1
4: R = 6-CH3 ; R1 = 3',4'-OH
R
5:
6:
R = 8-CH3 ; R1 = 3',4'-OH
R = 8-CH3 ; R1 = 3',5'-OH
O
O
7: R = 8-CH3 ; R1 = 3',4',5'-OH
8:
9:
R = 8-OH ; R1 = 4'-CH3
R = 8-OH ; R1 = 4'-OH
Scheme 1. Reagents and conditions: (i) DCC, DMSO, 110 °C, 24 h; (ii) HI 57%, AcOH, Ac2O, reflux, 3 h.
Table 1
ORAC-FL index and hydroxyl, DPPH and superoxide radical scavenging data obtained
for compounds 1-9
Compd
ORAC-
FL
index
% Scavenging
hydroxyl
% Scavenging
% Scavenging
superoxide
radicalsb
DPPH radicalsa
radicalsa
1
2
3
4
5
6
7
8
6.1
6.7
8.4
5.3
5.7
5.5
5.3
6.3
100
5.2
100
6.05
100
75
100
16
100
—
27.7
3.4
17.4
17.5
25
28.9
77.3
22.6
76.5
17.4
71.5
—
10.6
28.3
66.7
11.2
100
28.6
65.9
—
9
13.5
1.0
Trolox
Quercetin
Catechin
7.28c
6.76c
—
—
20.0d
44.5d
—
—
Figure 1. ORAC-FL profile (kinetic profile for protection FL probe against peroxyl
radicals) for compound 9.
a
The scavenging activity of hydroxyl and DPPH radicals effect was calculated as
follows: [(A0 ꢁ Ax)/A0] ꢀ 100, where Ax and A0 are the double-integral ESR for the
first line of samples in the presence and absence of test compounds, respectively.
In order to study the antioxidant reactivity of all the synthe-
sized hydroxy-3-arylcoumarin derivatives towards hydroxyl radi-
cals, a non-catalytic and competitive type Fenton system in
which the DMPO spin trap was performed.48 The ESR spin-trapping
spectrum obtained in the control assay (DMPO+N,N-dimethylform-
amide + NaOH + H2O2) presents four hyperfine lines, due to the
DMPO-OH adduct formation, as it is shown in Figure 2 (red line).
For each putative antioxidant coumarin compounds ESR spectra
were also acquired to check their capacity of scavenging hydroxyl
radicals. The data obtained with compound 2 is depicted in Figure 2
(black line).
The intensity of the spectra decreases when the hydroxy-3-aryl-
coumarin derivatives were added into the system. For compound 9,
100% of scavenging of hydroxyl radicals was obtained (Fig. 3—black
line). This type of response was observed for all derivatives, reflect-
ing different percentage of the hydroxyl radical scavenging activity
(Table 1).
The stable free radical DPPH assay has been used for detecting
the antioxidant activity in several chemical analyses.35–38 Cur-
rently, DPPH assay is considered an easy and accurate method,
appropriate for measuring the antioxidant capacity of fruits, vege-
tables, juices or extracts.39 This is due to the electronic properties
shared by DPPH and peroxyl radicals (the unpaired electron is
delocalized through the pair of nitrogen or oxygen atoms, respec-
b
The scavenging activity of superoxide radical effect was calculated as follows:
[(Ipc blank ꢁ Ipc aox)/Ipc blank] ꢀ 100, where Ipc blank is cathodic current in the
absence of the studied compounds and Ipc aox is the cathodic current in the
presence of the studied compounds.
c
Data collected from Ref. 45.
Data collected from Ref. 49.
d
initial rate and the range of total antioxidant inhibition in one
value.46
Results expressed as ORAC-FL values are presented in Table 1.
All the obtained ESR results for the% scavenging of the hydroxyl
and DPPH radicals are also illustrated in Table 1. In addition, % of
superoxide radical scavenging, performed by CV, are also repre-
sented in Table 1.
The ORAC-FL profile, intensity of fluorescence at 528 nm versus
the incubation time was obtained for all derivatives. Compounds 3
and 9 (8.4 and 13.5, respectively) display the highest ORAC-FL in-
dexes, comparing with the flavonoids quercetin and catechin that
are very well known natural antioxidant compounds. Figure 1
shows the kinetic profile for protection of FL probe against peroxyl
radicals obtained in presence of increasing concentrations of com-
pound 9. It was obtained a profile of fluorescence measure at
528 nm versus the incubation time at different concentration, for
all derivatives.

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M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
Figure 2. ESR spectra obtained for the control (adduct DMPO-OH without
antioxidant molecule—red line) and for adduct DMPO-OH in the presence of
compound 2 (black line).
Figure 5. Cyclic voltammograms of superoxide radical in absence (blank) and
presence of compounds 5, and in DMSO + TBAP 0.1 M, on GC (working
electrode) versus. Ag/AgCl, at room temperature, with scan rate of 30 mV/s.
7
9
Superoxide anion radical was generated by one electron reduc-
tion of the atmospheric molecular oxygen dissolved in DMSO at
room temperature (25 °C). Then, the voltammetric performance
of the nine compounds was studied, one by one, in DMSO and TBAP
0.1 M. The resultant CV responses for derivative 5, 7 and 9, and in
absence of any derivative (blank) are represented in Figure 5.
3.3. Theoretical evaluation of ADME properties
In order to better understand the overall properties of the de-
scribed compounds, the lipophilicity (expressed as the octanol/
water partition coefficient and herein called logP), was calculated
using the Molinspiration property calculation program.50 The the-
oretical prediction of ADME properties (molecular weight, logP,
number of hydrogen donors and acceptors) of all the compounds
was carried out and is presented in Table 2.51,52
Figure 3. ESR spectra obtained for the control (adduct DMPO-OH without
antioxidant molecule—red line) and for adduct DMPO-OH in the presence of
compound 9 (black line).
4. Discussion
In previous works, 3-arylcoumarin derivatives were described
as potent and selective MAO-B inhibitors.28 This family of com-
pounds and their remarkable data on the selective inhibition of
MAO-B isoenzyme and putative application for ND therapy were
the inspiration for this work. In particular, compounds 1–3 were
previously described as potent and selective MAO-B inhibitors,
with IC50 values between 120 and 650 nM.28 On the other hand
the recent medicinal chemistry paradigms in the drug design,
namely the rational discovery of multi-target drugs, as a promising
strategy to combat this type of multifactorial diseases prompted us
to look for other properties for this type of coumarins. Based on
this data, a new family of derivatives sharing the same scaffold
and type of substituents was designed and synthesized.
tively), in such way that the reaction rate between DPPH and sev-
eral antioxidants provides a good approximation for scavenging
activities with lipid peroxyl radicals.40,41 ESR spectra of DPPH in
absence and presence of compounds 4, 7 and 9 are showed in
Figure 4.
The evaluation of the antioxidant activity of the hydroxy-3-
arylcoumarin compounds was performed towards different types
of reactive oxygen or nitrogen species—peroxyl, hydroxyl, superox-
ide and DPPH radicals. ORAC-FL, ESR reactivity and CV assays were
the techniques used to achieve the goals. From the obtained data, it
was concluded that the antioxidant scavenging activity is related
with the type of substituents presented in the 3-arylcoumarin
skeleton.
Compound 9 was found to be the most interesting coumarin of
the series. This compound has two hydroxyl groups in its structure,
one at position 8 and another at position 40 of the 3-arylcoumarin
scaffold. The other compounds have structural combinations of
two types of substituents (methyl and one, two or three hydroxyl
groups). Compounds 1, 3, 5 and 7 have ORAC-FL values between
Figure 4. ESR signal from DPPH radical in absence (blank) and presence of
compounds 4, 7 and 9.

M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
3905
Table 2
Theoretical structural properties of the hydroxy-3-arylcoumarins derivatives 1–9a
Compd
logP
Molecular weight
TPSA
n-OH acceptors
n-OHNH donors
Volume
1
2
3
4
5
6
7
8
9
3.68
3.66
3.89
3.19
3.17
3.11
2.88
3.92
2.99
252.27
252.27
252.27
268.27
268.27
268.27
284.27
252.27
254.24
50.44
50.44
50.44
70.67
70.67
70.67
90.90
50.44
70.67
3
3
3
4
4
4
5
3
4
1
1
1
2
2
2
3
1
2
224.57
224.57
224.57
232.59
232.59
232.59
240.61
224.57
216.03
a
logP—octanol/water partition coefficient; TPSA–topological polar surface area; n-OH—number of hydrogen acceptors; n-OHNH—number of hydrogen bond donors. The
data was determined with Molinspiration calculation software.50
5.3 and 8.4 and the higher scavenging activity towards hydroxyl
radicals (100% ꢁ total scavenging). From these derivatives, com-
pound 7 presented the best DPPH and superoxide radicals scaveng-
ing (100 and 76.5%, respectively). Compounds 1 and 3 have on their
structure a methyl group at position 6 of the coumarin moiety and
a hydroxyl group in the 3-aryl ring. The ORAC-FL indexes are 6.1
and 8.4, respectively, but the position of the hydroxyl group in
the exocyclic aromatic ring seems to have no influence on the hy-
droxyl radical scavenging capacity (100%, in both cases). The DPPH
radical scavenging values of these compounds are, respectively,
27.7% and 10.6% and superoxide radical scavenging values are,
respectively, 17.4% and 25%. Their profiles are, therefore, very sim-
ilar. Compounds 5 and 7 have a methyl group at position 8 of the
coumarin moiety, and two or three hydroxyl groups, respectively,
in the 3-aryl ring. Both ORAC-FL values (5.7 and 5.3, respectively),
hydroxyl radical scavenging capacity (100%) and superoxide radi-
cal scavenging capacity (77.3% and 76.5%, respectively) are similar,
proving that the presence of the extra hydroxyl group does not
seem to significantly affect the peroxyl, hydroxyl and superoxide
radicals scavenging activities. One the other hand, DPPH radical
scavenging capacity is 66.7% for compound 5 and 100% for com-
pound 7. Compounds 2, 4 and 8, with ORAC-FL values between
5.3 and 6.7, presented the lowest hydroxyl scavenging capacity
(between 5.2% and 16%). Compound 2 presented also the lowest
DPPH radical scavenging capacity (3.4%) and compounds 1 and 8
the lowest superoxide radical scavenging capacities (17.4%). Com-
paring the data obtained for compound 4 (30,40-dihydroxy substi-
tuted) and for compound 1 (40-hydroxy substituted), it can be
concluded that the presence of two hydroxyl groups led to the
decline in the ORAC-FL index (6.1–5.3) and the hydroxyl radical
scavenging capacity (100%–6.05%). Comparing compound 4 with
2 (30-hydroxy substituted), it is also noted a decrease in the
ORAC-FL index (from 6.7 to 5.3), caused by the presence of two hy-
droxyl groups in the molecule. However, the hydroxyl radical scav-
enging capacity is almost the same (6.05% and 5.2%, respectively).
Superoxide radical scavenging capacity is also similar (28.9 and
17.5, respectively). Compounds 4 and 5 have the same substitution
pattern, changing only the position of the methyl group from 6 to
8. This modification strongly affects the hydroxyl radical scaveng-
ing capacity (from 6.05% to 100%), the DPPH radical scavenging
capacity (from 28.3% to 66.7%) and superoxide radical scavenging
capacity (from 28.9% to 77.3%). Compound 6, with a methyl group
at position 8 of the coumarin moiety and two hydroxyl groups at
positions 30 and 50 of the 3-aryl ring, in spite of presenting an
ORAC-FL value of 5.5 and low DPPH and superoxide radicals scav-
enging (11.2 and 22.6, respectively), evidence a significant ten-
dency to scavenge hydroxyl radicals (75%).
paring with trolox (ORAC-FL = 1.0), the interesting ORAC-FL values
of compounds 3 and 9 make them promising antioxidant mole-
cules. It is important to notice that compound 5, 7 and 9 presented
good trends against all the studied radicals.
From the obtained data, it is also remarkable that all the couma-
rin derivatives possess logP values compatible with those required
to cross membranes. TPSA, described to be a predictive indicator of
membrane penetration, is also found to be positive. In addition, it
can be observed that no violations of Lipinski’s rule (molecular
weight, logP, number of hydrogen donors and acceptors) were
found. This is important information about the promising potential
of these derivatives.
All the synthesized compounds, in spite of presenting different
chemical substituents, disclose interesting ORAC-FL values, in most
cases accompanied by a remarkable ability to scavenge hydroxyl,
DPPH and superoxide radicals. Therefore, the data acquired so far
are relevant allowing proposing hydroxy-3-arylcoumarins as a va-
lid scaffold for the design of novel antioxidants.
5. Concluding remarks
In conclusion, in the current work coumarins presenting very
promising antioxidant profiles were described. Compound
9
proved to be the most interesting molecule of the whole series,
with an ORAC-FL of 13.5, 100% of scavenging of hydroxyl radicals,
65.9% of scavenging of DPPH radicals and 71.5% of scavenging of
superoxide radicals. This derivative has presented good antioxi-
dant capacity towards different types of reactive oxygen or nitro-
gen species—peroxyl, hydroxyl, superoxide and DPPH radicals.
Compound 3, previously describe as very good selective MAO-B
inhibitor, presented also a very interesting antioxidative profile.
It is important to notice that compound 5 and 7 also presented
good trends against all the studied radicals. In addition, it can be
observed that no theoretical violations of Lipinski’s rule were ob-
served for all the studied derivatives. Therefore, the described
compounds seem to present desirable ADME properties. Based on
these results, it can be concluded that especially compounds 3
and 9 are potential candidates for a further optimization process
and could be successfully employed in the prevention or minimiza-
tion of the oxidative damage caused by overproduction of oxygen
free radicals.
Acknowledgments
The current work was supported by Mecesup (Project
UCH-0601), CONICYT-Chile (Project N 24110059), Fundação para
a Ciência e Tecnologia (Project PTDC/QUI-QUI/113687/2009) and
personal funds from the researchers. M.J. Matos thanks Fundação
para a Ciência e Tecnologia (SFRH/BD/61262/2009) Ph.D. Grant, F.
Pérez-Cruz thanks CONICYT-Chile PhD. grant, Becas-Chile and
As said before, the highest ORAC-FL values were found for com-
pounds 3 and 9 (8.4 and 13.5, respectively). The results are very
interesting comparing with the ORAC-FL values of catechin (6.76)
and quercetin (7.28), very well known natural antioxidants. Com-

3906
M. J. Matos et al. / Bioorg. Med. Chem. 21 (2013) 3900–3906
24. Matos, M. J.; Viña, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.;
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Fulbright doctoral stay fellowships and S. Vazquez-Rodriguez
thanks Ministerio de Educación y Ciencia (AP2008-04263) PhD.
Grant.
26. Matos, M. J.; Viña, D.; Janeiro, P.; Borges, F.; Santana, L.; Uriarte, E. Bioorg. Med.
Chem. Lett. 2010, 20, 5157.
27. Matos, M. J.; Vazquez-Rodriguez, S.; Uriarte, E.; Santana, L.; Viña, D. Bioorg.
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