Study of coumarin-resveratrol hybrids as potent antioxidant compounds

Article abstract of DOI:10.3390/molecules20023290

In the present work we synthesized a selected series of hydroxylated 3-phenylcoumarins 5-8, with the aim of evaluating in detail their antioxidant properties. From an in depth study of the antioxidant capacity data (ORAC-FL, ESR, CV and ROS inhibition) it

Full text of DOI:10.3390/molecules20023290

Molecules 2015, 20, 3290-3308; doi:10.3390/molecules20023290
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Study of Coumarin-Resveratrol Hybrids as Potent
Antioxidant Compounds
Maria J. Matos 1,2,†,*, Francisco Mura 3,†, Saleta Vazquez-Rodriguez 1,2,†, Fernanda Borges 1,†,
Lourdes Santana 2,†, Eugenio Uriarte 2,† and Claudio Olea-Azar 3,†,
*
1
CIQUP/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,
4169-007 Porto, Portugal; E-Mails: svre77@gmail.com (S.V.-R.);
mfernandamborges@gmail.com (F.B.)
2
Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain; E-Mails: lourdes.santana@usc.es (L.S.);
eugenio.uriarte@usc.es (E.U.)
3
Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile, Casilla 233 Santiago, Chile; E-Mail: franciscomura@gmail.com
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: mariacmatos@gmail.com (M.J.M.);
colea@uchile.cl (C.O.-A.); Tel.: +34-881-814-936 (M.J.M.); Fax: +34-981-544-912 (M.J.M.).
Academic Editor: Derek J. McPhee
Received: 13 January 2015 / Accepted: 12 February 2015 / Published: 16 February 2015
Abstract: In the present work we synthesized a selected series of hydroxylated
3-phenylcoumarins 5–8, with the aim of evaluating in detail their antioxidant properties.
From an in depth study of the antioxidant capacity data (ORAC-FL, ESR, CV and ROS
inhibition) it was concluded that these derivatives are very good antioxidants, with very
interesting profiles in all the performed assays. The study of the effect of the number and
position of the hydroxyl groups on the antioxidant activity was the principal aim of this
study. In particular, 7-hydroxy-3-(3'-hydroxy)phenylcoumarin (8) proved to be the most
active and effective antioxidant of the selected series in four of the performed assays
(ORAC-FL = 11.8, capacity of scavenging hydroxyl radicals = 54%, Trolox index = 2.33
and AI30 index = 0.18). However, the presence of two hydroxyl groups on this molecule did
not increase greatly the activity profile. Theoretical evaluation of ADME properties of all
the derivatives was also carried out. All the compounds can act as potential candidates for
preventing or minimizing the free radical overproduction in oxidative-stress related diseases.

Molecules 2015, 20
These preliminary findings encourage us to perform a future structural optimization of this
3291
family of compounds.
Keywords: hydroxylated 3-phenylcoumarins; antioxidant assays; electrochemical study;
inhibition of ROS; ESR; ADME properties
1. Introduction
Polyphenolic compounds are one of the major families of plant metabolites. These compounds are
bioactive substances that have one or more aromatic rings in their structure, bearing one or more
hydroxyl groups. This family of compounds acts as antioxidants and thereby protect from degenerative
diseases in which reactive oxygen species (ROS) are involved [1]. In fact, the overproduction of free
radicals have been related to cellular membrane, protein, RNA and DNA damage, and indirectly with
aging and oxidative-stress related diseases like cancer, cardiovascular, inflammatory and neurodegenerative
pathologies [2]. The properties of phenolic compounds are related to their chemical structure, which
confers stability to the secondary free radical formed from the antioxidant reaction product with a free
radical [3]. Therefore, the research and characterization of new bioactive phenolic substances from the
diet has been intensified in the last years, either for the development of nutraceuticals or new drugs [4].
Hydroxycoumarins are phenolic compounds which act as potent metal chelators and free radical
scavengers [5–8]. Hydroxylated 3-arylcoumarins are a family of polyphenolic compounds, which are
known to possess anti-inflammatory, anti-thrombotic, enzyme inhibitory and antioxidant properties [9–12].
In particular, our research group has been studying the potential of differently substituted
3-arylcoumarins as antioxidant agents [13–17]. In these studies it was concluded that the position and
number of different electron donator groups (hydroxyl, methoxy or methyl) in the scaffold are structural
factors contributing to modulate the antioxidant capacity of those compounds. The studied coumarins
exhibited, in general, excellent antioxidant activities, which depended on the position and number of
hydroxyl groups [13–17].
Resveratrol, structurally 3,4',5-trihydroxystilbene, is a natural phenolic component of Vitis vinifera L.
and other spermatophyte species, produced in response to exterior or interior damage [18]. Resveratrol
shows a large number of pharmacological activities, including anti-inflammatory, antioxidant, anticancer
and cardioprotective properties [18]. Some of these properties, such as antioxidant activity, are coincident
with those of the hydroxycoumarins.
Taking into account this background, we proposed to study the antioxidant capacity of a selected
series of hydroxylated 3-phenylcoumarins (coumarin-resveratrol hybrids—Scheme 1), with one hydroxyl
group at positions 6, 7 or 8 (compounds 5, 6 and 7, respectively), or two hydroxyl groups at positions 7
and 3' (compound 8). It is our aim to study the effect of the position and the number of hydroxyl groups
on the antioxidant capacity of 3-arylcoumarins. A detailed antioxidant study of the molecules was carried
out, using different assays and methodologies. Since the formation of ROS is presented in several
biochemical processes and pathologies, these compounds could be valuable as multitarget molecules
against several diseases.

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2. Results and Discussion
2.1. Chemistry
In order to obtain the desired final products, coumarin derivatives 5–8 were efficiently synthesized
according to the protocol outlined in Scheme 1 [15,19–24]. The synthetic methodology was carried out
in two steps, and is briefly described as follows: (a) synthesis of acetoxy-3-phenylcoumarins 1–4 and (b)
hydrolysis of the previous compounds to afford the final hydroxy-3-phenylcoumarins 5–8. The general
reaction conditions of the synthesized compounds are described in the Experimental Section. The
acetoxy-3-phenylcoumarins were efficiently synthesized in 85%–92% yield by a Perkin-Oglialoro
condensation [25], treating the appropriate ortho-hydroxybenzaldehyde with the adequate phenylacetic
acid, in the presence of potassium acetate and acetic anhydride. Hydroxyl derivatives were obtained in
with 90%–94% yield from the abovementioned acetoxy-substituted precursors by acidic hydrolysis,
using hydrochloric acid in the presence of methanol.
O
H
HOOC
R'
R
OH
a)
3'
6'
2'
1'
4'
5'
R'
5
8
4
3
6
7
R
2
O
O
1: R = 6-OCOCH3 ; R' = H
2: R = 7-OCOCH3 ; R' = H
3: R = 8-OCOCH3 ; R' = H
5: R = 6-OH ; R' = H
6: R = 7-OH ; R' = H
7: R = 8-OH ; R' = H
8: R = 7-OH ; R' = 3'-OH
b)
4: R = 7-OCOCH3 ; R' = 3'-OCOCH3
Reagents and conditions: (a) CH3CO2K, Ac2O, reflux, 16 h; (b) HCl 2N, MeOH, reflux, 4 h.
Scheme 1. Synthetic methodology to obtained hydroxy-3-phenylcoumarins 5–8.
2.2. Antioxidant Capacity Assays
The main objective of this study was to assess in detail the antioxidant capacity of the
hydroxy-3-phenylcoumarins 5–8 in order to compare the profiles of these compounds as antioxidant
agents. To achieve this goal, the oxidation potentials of compounds 5–8 were determined by CV [26].
This parameter gave information not only for evaluating the antioxidant potentials of the compounds, but
also for understanding their reaction mechanisms [26]. The evaluation of the antioxidant activity of the
studied compounds towards different types of ROS: peroxyl, hydroxyl, alkoxyl and superoxide radicals,
was also performed [27]. ORAC, ESR and CV were the techniques used to obtain the desired results.

Molecules 2015, 20
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2.2.1. Cyclic Voltammetry (CV)
The first assay performed to better understand the antioxidant potential of the studied compounds was
the evaluation of oxidative potentials by CV. Anodic peak potential (Epa) values for compounds 5–8 are
reported in Table 1, and demonstrated that the coumarin derivatives present a variety of oxidation
potentials depending on their substitution pattern.
Table 1. Oxidant potentials recorded for the studied compounds.
Compounds
Epa (V) a
0.762
5
6
7
8
0.640
0.652
0.720
a
Epa: anodic peak potential. First oxidation peak potential at scan rate of 2000 mV/s. All experiments were
carried out in triplicate. The data are expressed as means ± SD.
In the experimental conditions, a single oxidation process (peak I) was observed. In general, it has
been proposed that the charge transfer process at peak I corresponded to the oxidation of the hydroxyl
substituent. The compounds with lowest oxidation potential were compounds 6 (Epa = 0.640 V) and 7
(Epa = 0.652 V). It is known that the antioxidant capacity is conceivably related to the electrochemical
behaviour, being indicative that a low oxidation potential corresponds to a high antioxidant power.
1.6
5.0 V/s
4.0 V/s
3.0 V/s
120
1.4
100
80
60
40
20
0
2.0 V/s
1.0 V/s
0.5 V/s
1.2
1.0
0.8
0.6
0.4
0.2
-20
-40
0.8
1.2
1.6
2.0
2.4
0,0
0.0
0,2
0.2
0,4
0.4
0,6
0.6
0,8
0.8
1,0
1.0
1,2
1.2
1/2
-1 1/2
)
E vs Ag/AgCl (V)
v
/ (mV s
(A)
(B)
Figure 1. (A) Cyclic voltammogram for 1 mmol/L of compound 6, in DMSO/75 mmol/L
phosphate (pH 7.4) buffer 40/60 media at a GCE, for v = 0.5–5 mV/s. (B) Graphic of the
anodic peak current versus the root of the scanning rate.
However, this does not present a linear relationship. The low oxidation potential values are only
indicative of a good antioxidant profile. Therefore, compound 6 proved to be the molecule with best
profile of the series. As an example, the cyclic voltammogram of compound 6 is illustrated in Figure 1.
It was recorded at a GCE, in a pH 7.4, containing 1 mmol/L of compound 6, for several scan rates (v).
Analysing the results, it was observed by CV that all the studied coumarin derivatives showed the same
oxidation pattern; a single oxidation signal, near 0.7 V, corresponding to an irreversible type of reaction

Molecules 2015, 20
3294
(Figure 1A). From the analysis of the anodic peak current (Ipa) versus v1/2 (Figure 1B), a straight line
with r2 > 0.99 was obtained, indicating that the electrode process was controlled by diffusion.
2.2.2. ORAC-Fluorescein (ORAC-FL)
The ORAC-fluorescein (ORAC-FL) assay, that can assess the scavenging ability of coumarins against
peroxyl radicals in competition with a fluorescent sensor, was performed and the results expressed as
ORAC-FL index are presented in Table 2 [28,29]. In this study, AAPH was used as the peroxyl radical
source [30].
Table 2. ORAC-FL values calculated for the studied and reference compounds.
Compounds
ORAC-FL Index a
11.0 ± 2.3
9.6 ± 1.3
5.7 ± 0.6
11.8 ± 1.4
1
5
6
7
8
Trolox
Quercetin
7.28 b
6,7-Dihydroxy-4-methylcoumarin
3.3 c
a ORAC-FL studies were carried out in 75 mmol/L sodium phosphate buffer (pH 7.4) with coumarin solutions
in methanol (range of concentration between 0.3 and 2 μM); b Reference [26]; c Reference [27]. All experiments
were carried out in triplicate. The data are expressed as means ± SD.
Analyzing the ORAC-FL results, compounds 5 and 8 proved to be the best candidates of the series
(ORAC-FL = 11.0 and 11.8, respectively). Nevertheless, compound 6 is also considered a good candidate,
presenting an ORAC-FL index of 9.6, significantly higher than quercetin, used as reference compound.
The results obtained are better than the ORAC-FL value of simple coumarins, such as the 6,7-dihydroxy-
4-methylcoumarin, studied by our group (ORAC-FL = 3.3). An enhanced activity of compound 8, comparing
to compound 6, was found against this radical, proving that the presence of two hydroxyl groups in the
molecules could slightly improve the antioxidant capacity. It was observed that the derivative with the
lowest rate is the one bearing the hydroxyl group at position 8 (compound 7). This may be due to the
formation of an intramolecular hydrogen bond with the oxygen of the lactone, depleting the availability
of the hydrogen.
The consumption of FL is commonly inhibited, even by antioxidants of low reactivity, throughout
kinetic profiles. Figure 2 illustrates the results of AAPH-mediated FL oxidation in the absence and
presence of an increasing concentration of compounds 5–8, and demonstrates that the AUCNET of the
kinetic profiles for these compounds were linearly related to the concentration of the additive. Of the
studied series, compounds 6 and 8 presented an induction time, indicating that these coumarins display
better activity to protect the sensor of the peroxyl radical than compounds 5 and 7, which showed no
induction time. In addition, the presence of an induction time observed in the case of compound 6 is
accompanied by the fact that the slopes are similar for all concentrations. This indicates that the
antioxidant was completely consumed before the fluorescence decay, due to the attack of free radicals
remaining on the sensor.

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1,0
5
1.0
1.0
1,0
6
CONTROL
0,8
0.8
0.8
0,8
0.3 μM
0.7 μM
1.0 μM
1.5 μM
2.0 μM
CONTROL
0.3 μM
0.7 μM
1.0 μM
1.5 μM
0.6
0,6
0.6
0,6
0.4
0,4
0.4
0,4
0.2
0.2
0,2
0,2
0.0
0.0
0,0
0,0
0
2000 4000 6000 8000 10000 12000
0
0
2000 4000 6000 8000 10000 12000
2000 4000 6000 8000 10,000 12,000
0
2000
4000
6000
8000 10,000 12,000
Time / s
Time / s
1.0
1,0
1.0
1,0
7
8
0.8
0,8
0.8
0,8
Control
0.6 μM
1.2 μM
1.5 μM
Control
0.6
0,6
0.3 μM
0.7 μM
1.0 μM
1.5 μM
2.0 μM
0.6
0,6
0,4
0.4
0.4
0,4
0.2
0,2
0,2
0.2
0.0
0,0
0,0
0.0
0
0
2000 4000 6000 8000 10000 12000 14000
2000 4000 6000 8000 10,000 12,000 14,000
0
0
2000 4000 6000 8000 10000 12000 14000
2000 4000 6000 8000 10,000 12,000 14,000
Time / s
Time / s
Figure 2. Kinetic profiles of FL consumption AAPH-mediated in presence of compounds
5–8. F0 is the fluorescence in absence of the compound and F the fluorescence in presence
of the compound. Last graphic: AUCNET versus concentration of compound 6.
2.2.3. Electron Spin Resonance (ESR)
In order to test the ability of the selected compounds to scavenge hydroxyl radicals a non-catalytic
and competitive-type Fenton system was examined [31]. The percentage of scavenging of hydroxyl
radicals by the studied compounds was measured and is summarized in Table 3. Antioxidant capacity
against alkoxyl radicals, generated by photolysis of AAPH in aqueous medium, was also determined by
spin trapping methodology, and the results expressed as Trolox indexes are summarized in Table 3.

Molecules 2015, 20
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Table 3. Percentage of scavenging of hydroxyl radicals and Trolox index calculated for
compounds 5–8.
Compounds % Scavenging of Hydroxyl Radicals a Trolox Index
5
6
7
8
24.7 ± 6.3
44.7 ± 1.2
10.9 ± 1.1
51.4 ± 4.5
-
2.28 ± 0.05
2.32 ± 0.05
1.23 ± 0.01
2.33 ± 0.05
1
Trolox
a
Scavenging activity of hydroxyl 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. All experiments were carried out in triplicate. The data are expressed as means ± SD.
To study the antioxidant capacity of all the synthesized coumarins towards hydroxyl radicals in depth,
a non-catalytic and competitive type Fenton system was set up, employing DMPO as a spin trap. The
percentage of scavenging of the studied compounds was measured and is summarized in Table 3. In this
assay, DMPO reacts with hydroxyl radicals to generate the spin resonance signal, which was quantified
by ESR [32]. The ESR spectrum illustrates four hyperfine lines due to the DMPO-OH adduct formation.
In particular, ESR spectrum obtained to the control (DMPO + N,N-dimethylformamide + NaOH + H2O2)
presents four hyperfine lines illustrated in magenta (Figure 3). Coumarin derivatives competed with
DMPO for hydroxyl radicals, diminishing the ESR signal, as represented in Figure 3 for compounds 5–8.
C ontrol
5
6
7
8
3400
3425
3450
3475
3500
3525
3550
Magnetic field / G
Mag netic field / G
Figure 3. ESR spectra of the control and 3-phenylcoumarins 5–8.
As expected, the intensity of the spectra decreased when the coumarin derivatives were added to the
system. This type of response was observed for all derivatives, reflecting different percentages of
hydroxyl radical scavenging activity (Table 3). Compound 8 proved to be the best compound of the
studied series, presenting the highest hydroxyl radical scavenging capacity (51.4%). This value could be
due to the presence of two hydroxyl groups in its structure. Compounds 6 and 8, bearing a hydroxyl
group at position 7, proved to be the molecules with highest capacity to scavenge hydroxyl radicals. An
enhanced activity of compound 8, compared to compound 6, was found against this radical, proving that the
presence of two hydroxyl groups in the molecules could slightly improve the antioxidant capacity.

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2.2.4. Antioxidant Capacity against Alkoxyl Radicals by ESR
Antioxidant capacity against alkoxyl radicals generated by photolysis of AAPH in aqueous medium
was monitored by spin trapping methodology [33–36]. The antioxidant capacity was considered
proportional to the fall of the signal height compared to the control, in absence of antioxidant
(Figure 4). All the results were contrasted with Trolox, being the Trolox index proportional to the
antioxidant capacity against alkoxyl radicals (Table 3).
C ontrol
5
6
7
8
3400
3450
3500
3550
Mag netic field / G
Figure 4. Spectrogram of the adduct formed between AAPH-derived radical by photolysis
and DMPO. G-value: 2.0023. Microwave frequency: 9.81 GHz, Modulation amplitude: 0.95
G, Time constant: 81.92 ms, Conversion time: 40.96 ms. Simple LW = 0.598.
The results described in Table 3 showed the same tendency observed to the ORAC-FL, in which
compounds 5, 6 and 8 presented a similar antioxidant capacity, bigger than compound 7. The second
hydroxyl group in the 3-phenyl moiety (compound 8) seems to have not significantly improve the
antioxidant capacity against alkoxyl radicals.
2.2.5. Antioxidant Activity against Superoxide Radicals by CV
In order to investigate the antioxidant activity of the coumarins against the superoxide anion radical
−
(O2 ) generated electrochemically [37,38], a stock solution of the coumarins was added gradually to the
−
solution, leading to a decrease of O2 Ipa, while the intensity of O2 cathodic current was not significantly
modified, as shown in Figure 5. The oxidation potential peaks of the studied coumarins did not interfere
with the superoxide radical couple signal, which appeared at more negative potentials. For each antioxidant
compound, a series of Ipa values was determined from the CVs recorded for increasing antioxidant
concentrations. All antioxidant substrates exhibited a similar effect upon the O2 reduction. At higher
concentrations of some antioxidant substrates, a not-well defined reduction peak was observed at a potential
more positive than the oxygen reduction peak.
By analogy, with the inhibiting concentration of antioxidant (IC), the antioxidant index values
expressed by the substrate concentration needed to consume 30% (AI30 index) by the concentration in

Molecules 2015, 20
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millimoles of antioxidant substrate needed to consume superoxide radicals was determined as revealed
s
by a current decrease to 30% of the initial anodic current (Ipa°) (AI30 = (Ipa° − Ipa )/Ipa° = 0.30). With this
antioxidant characterization, the lower the AI30 values are, the more antioxidant capacity the substrate
has against superoxide [39,40]. Recorded data is listed in Table 4.
6
I
pa
4
2
0
0.00 mM
-2
-4
0.04 mM
0.08 mM
0.12 mM
0.16 mM
0.20 mM
0.24 mM
0.28 mM
0.32 mM
0.36 mM
Scan
-6
-8
I
pc
-10
-1,0
-1.0
-0,8
-0.8
-0,6
-0.6
-0,4
-0.4
-0,2
0,0
0.0
-0.2
E vs Ag/AgCl / V
Figure 5. Cyclic voltammograms of superoxide radical at different concentrations of
compound 6 in DMSO + TBAP 0.1 M, on GC (working electrode) versus Ag/AgCl, at room
temperature, with scan rate of 30 mV/s.
Table 4. AI30 index calculated for compounds 5–8.
Compounds
AI30/mM
0.19 ± 0.02
0.23 ± 0.02
0.23 ± 0.04
0.18 ± 0.04
0.24 ± 0.02
5
6
7
8
Trolox
All experiments were carried out in triplicate. The data are expressed as means ± SD.
It was observed that the best superoxide scavenger of this series was once again compound 8, which
had the lowest AI30. However, as in some of the above-described assays, none of the studied compounds
presented significant differences in their AI30 values (Figure 6).

Molecules 2015, 20
3299
0,5
0.5
7
8
0,4
0.4
0,3
0.3
0.2
0,2
0.1
0,1
AI
30
0.0
0,0
0,00
0.00
0,05
0.05
0,10
0.10
0,15
0.15
0,20
0.20
0,25
0.25
0,30
0.30
0,35
0.35
0,40
0.40
C onc entration / mM
Figure 6. Graphs of decreasing anodic peak current between the control (Ipa°) and the
s
substrate (Ipa ) against increasing concentrations of substrate, in this case, compounds 7 and 8;
AI30 is the concentration of inhibition at 30% of superoxide radical.
2.2.6. Inhibition of ROS
An assay on cell models (macrophages from the strain RAW 264.7) was performed to better understand
the antioxidant potential of the studied compounds in a biological environment [41]. This assay allowed
determining the amount of ROS formed after an oxidation process. This process is particularly sensitive
to the detection of H2O2 and peroxyl radicals. The percentage of disappearance of fluorescence in presence
of menadione (50 μM) and different concentrations of the 3-phenylcoumarins (1, 10 and 100 μM) was
evaluated, and the results of this study are presented in Table 5 (1 μM) and Figure 7 (10 μM).
Table 5. Percentage of disappearance of fluorescence in presence of menadione (50 μM)
and 3-phenylcoumarins 5–8 (1 μM), (p < 0.05).
Compounds % Disappearance of Fluorescence
5
92.57 ± 0.09
92.52 ± 0.03
42.88 ± 0.30
88.69 ± 0.08
1.48 ± 0.01
6
7
8
Trolox
All experiments were carried out in triplicate. The data are expressed as means ± SD.
The quenching capacity was biologically studied using radicals generated by stimulation of oxidative
stress due to the inclusion of menadione in a cellular model. Menadione is capable of generating a wide
concentration responsible for the occurrence of free radical fluorescence due to oxidation of the sensor.

Molecules 2015, 20
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1,5
1.5
C ontrol
1,4
1.4
5
6
7
8
1.3
1,3
1.2
1,2
1.1
1,1
1.0
1,0
0
10
20
30
40
T im e / m in
Figure 7. Graphic of ROS inhibition (control and compounds 5–8 at 10 μM).
Subsequently, with the addition of coumarin at concentrations of 1, 10 and 100 μM, a decrease in
fluorescence was observed, indicating that the coumarin effectively entered the cell and protected it from
oxidation (Figure 7). In this assay, the smaller is the slope, and the less is the fluorescence, better
antioxidant capacity the compounds present. At 10 μM, the effect of all the studied coumarins was less
than 10% of appearance of fluorescence (Figure 7). At lower concentrations (1 μM), three of the four
studied compounds (compounds 5, 6 and 8) still presented less than 10% of appearance of fluorescence.
However, compound 7 presented 57% of appearance of fluorescence.
2.3. ADME Theoretical Properties Calculation
To better correlate the drug-like properties of the studied compounds the lipophilicity, expressed as
the octanol/water partition coefficient and herein called logP, as well as other theoretical calculations
such as the topological polar surface area (TPSA), the number of hydrogen acceptors and the number of
hydrogen bond donors were calculated using the Molinspiration property program [42]. TPSA is a commonly
used medicinal chemistry metric for the optimization of a drug’s ability to permeate cells and achieve
the desired target. The obtained in silico results are summarized in Table 6 [42].
Table 6. Theoretical structural properties of the 3-phenylcoumarins 5–8. a
Compd. logP TPSA (Å2) n-OH Acceptors n-OHNH Donors Volume (Å3)
5
6
7
8
3.23
3.23
3.23
2.73
50.44
50.44
50.44
70.67
3
3
3
4
1
1
1
2
208.01
208.01
208.01
216.03
a 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 [42].
From the data obtained for the theoretical evaluation of the ADME properties, it can be noticed that
none of the compounds 5–8 break any of the Lipinski rule of five, making them promising leads for drug

Molecules 2015, 20
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candidates. TPSA and logP values are compatible with those described as a predictive indicator of the
drug capacity of membrane penetration [42,43].
3. Experimental Section
3.1. Synthesis
General procedure for the synthesis of acetoxy-3-phenylcoumarins 1–8. Compounds 1–4 were
synthesized under anhydrous conditions, using materials previously dried at 60 °C for at least 12 h and
at 300 °C during few minutes immediately before use. A solution containing anhydrous potassium
acetate (CH3CO2K, 2.94 mmol), the conveniently substituted phenylacetic acid derivative (1.67 mmol)
and the corresponding 2-hydroxybenzaldehyde (1.67 mmol) in acetic anhydride (Ac2O, 1.2 mL) was
refluxed (138 °C) for 16 h. The reaction mixture was cooled, neutralized with 10% aqueous sodium
bicarbonate (NaHCO3), and extracted (3 × 30 mL) with ethyl acetate (EtOAc). The organic layers were
combined, washed with distilled water, dried with anhydrous sodium sulfate (Na2SO4), and evaporated
under reduced pressure. The product was purified by recrystallization in ethanol (EtOH) and dried, to
afford the desired compound. Hydroxylated coumarins 5–8 were obtained by hydrolysis of their
acetoxylated counterparts 1–4. In general, the appropriate acetoxylated coumarin was mixed with 2 N
aqueous hydrochloric acid (HCl) and methanol (MeOH) and refluxed (100 °C) with stirring during 4 h.
The resulting reaction mixtures were cooled in an ice-bath and the reaction products, obtained as solids,
were filtered, washed with cold distilled water, and dried under vacuum, to afford the desired compound.
Characterization of the compounds was previously reported [14,19–24].
3.2. Antioxidant Assays
3.2.1. Oxidation Potential Determination by Cyclic Voltammetry (CV)
Cyclic voltammetry (CV) was carried out using a Metrohm instrument (Riverview, FL, USA) with a
797 VA stand convertor and a 797 VA processor, in DMSO/75 mM phosphate (pH 7.4) buffer 40/60
DMSO/(~1.0 Å, ~10.3 M) at room temperature with KCl (~0.1 M), using a three-electrode cell. A glassy
carbon electrode (GCE) was used as the working electrode, a platinum wire as the auxiliary electrode,
and saturated calomel (Ag/AgCl) as the reference electrode. The assays were performed containing 1
mmol/L of each studied compound, for several scan rates (v = 0.5–5 mV/s).
3.2.2. Oxygen Radical Antioxidant Capacity-Fluorescein (ORAC-FL)
The oxygen radical absorbance capacity (ORAC-FL) studies were carried out on a Synergy™ HT
multidetection microplate reader from Bio-Tek Instruments, Inc. (Winooski, VT, USA), using white
polystyrene 96-well plates, purchased from Nunc (Roskilde, Denmark) [27,28,30,44–46]. 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 software. The reaction was carried out in 75 mM sodium
phosphate buffer (pH 7.4), and 200 μL final volume. Fluorescence (FL; 40 nM, final concentration) and
coumarin solutions in methanol (range of concentration between 0.3 and 2 μM) were placed in each well
of the 96-well plate. The mixture was pre-incubated for 15 min at 37 °C, before rapidly adding the

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2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH) solution (18 mM, final concentration). The
microplate was immediately placed in the reader and automatically shaken prior to each reading. The
fluorescence was recorded every 1 min for 120 min. A blank with FL and AAPH using methanol instead
of the antioxidant solution was used in each assay. Five calibration solutions using Trolox® (0.5–2.5 μM)
as the antioxidant were also used in each assay. The inhibition capacity was expressed as ORAC-FL
values and it was quantified by integration of the area under the fluorescence decay curve (AUC). All
reaction mixtures were prepared in triplicate and at least three independent assays were performed for
each sample. The AUC was calculated integrating the decay of the fluorescence, 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, Northampton, MA, USA).
3.2.3. Hydroxyl Radical Scavenging Assay Using Electron Spin Resonance (ESR)
Reactivity of all the hydroxy-3-phenylcoumarin derivatives against the hydroxyl radical was investigated
using the non-catalytic Fenton type method. Electron spin resonance (ESR) spectra were recorded in the
X band (9.7 GHz) using an ECS 106 spectrometer (Bruker, Coventry, UK) with a rectangular cavity and
50 kHz field modulation, equipped with a high-sensitivity resonator at room temperature. Spectrometer
conditions were: microwave frequency 9.81 GHz, microwave power 20 mW, modulation amplitude 0.91
G, receiver gain 59 db, time constant 81.92 ms and conversion time 40.96 ms [46]. The scavenging
activity of each derivative was estimated by comparing the 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO-OH) adduct signals in the antioxidant-radical reaction mixture and the control reaction at the
same reaction time, and was expressed as scavenging percent of hydroxyl radical. To prepare the
samples, 150 μL of N,N-dimethylformamide (DMF) and 50 μL of NaOH (3 mM) were mixed, followed
by the addition of 50 μL of DMPO spin trap (30 mM final concentration) and finally 50 μL of hydrogen
peroxide 30%. The mixture was put in an ESR cell and the spectrum was recorded after five minutes of
reaction. All the compounds were studied at 4 mM final concentration (300 μL final volume).
3.2.4. Determination of Alcoxyl Radicals Generated by Photolysis of AAPH Assay Using Electron
Spin Resonance (ESR)
Antioxidant capacity against alcoxyl radicals generated by photolysis of AAPH in aqueous
medium was monitored by spin trapping methodology (ORAC-like assay). The most popular spin trap
for oxygen-centered free radicals is the previously described DMPO. The photolysis of AAPH, at
392 nm, had been identified to generate alcoxyl radicals, observing a splitting pattern of 4 lines with
hyperfine coupling constant of aN = 14.27 G and aH = 14.67 G, for the adduct DMPO/RO• [33–35].
Moreover, it was not found the appearance of methyl radical/DMPO adduct, product of the reaction of
hydroxyl radical and DMSO-d6 [34]. This was an evidence that pattern of lines is due to DMPO/RO•,
not DMPO/HO•. To achieve this goal, coumarins were prepared in DMSO at concentration of 0.1 mM,
DMPO was prepared in 0.1 M phosphate buffer pH 7.4 at concentration of 200 mM, and AAPH was
prepared in buffer medium at concentration of 20 mM. The antioxidant capacity was considered proportional
to the decrease of signal height compared to the control, in absence of antioxidant.

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3.2.5. Superoxide Antioxidant Assay Using Cyclic Voltammetry (CV)
Superoxide anion radical was generated one electron reduction of the atmospheric molecular oxygen in
DMSO of analytical grade (Sigma-Aldrich, St. Louis, MO, USA), with 0.1 M tetrabutylammonium
perchlorate (TBAP) as supporting electrolyte. Then, the voltammetric performance of the compounds
was studied. Coumarins were added incrementally to the in situ generated radical and the resultant
behavior was recorded. The concentration of the coumarins in the electrochemical cell was in the range
of 0.0 to 0.4 mM. The scan rate was kept at 50 mV/s and the potential window was −1.0–0.0 V [47,48].
The atmospheric solubility of oxygen in DMSO was 2.1 mM. The antioxidant activity was assessed from
the change in the cathodic current of the voltammograms in absence and present of the derivatives, using
pertinent mathematical formulations. The relative decrease in the intensity signal was expressed as
s
(Ipa° − Ipa )/Ipa°; where Ipa° is the current peak in the oxidative scan in absence of substrate, and Ipas is the
current peak in the oxidative scan in presence of substrate. CV measurements were performed in a
Metrohm instrument with a 694VA stand convertor and a 693VA processor, at room temperature, using
a three-electrode cell. A 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 reference electrode.
3.3. Statistical Data Analysis
Statistical analyses were conducted using GraphPad Prism 5 software (GraphPad Software, Inc.,
San Diego, CA, USA). The data are expressed 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 significance was set at p < 0.05, and all experiments were replicated 3 times.
3.4. Inhibition of Radical Oxygen Species (ROS)
A variety of reductive enzymes such as NADPH-cytochrome P450 reductase and NADH
microsomal-ubiquinone oxidoreductase (complex I) are able to metabolize quinones by reductive reactions
via electron [41,49]. The resulting semiquinone radical can enter a redox cycle, if oxygen is present,
regenerating the starting quinone and produce ROS. In the present assay, the used quinone source was
menadione (2-methylnaftoquinone) 50 µM/well, and the fluorescent sensor was
dichlorodihydrofluoscein diacetate (DCFH2-DA) 20 µM/well, which is a non-fluorescent compound that
penetrated by diffusion into the cell, and was hydrolysed to 2',7'-dichlorodihydrofluorecein (DCFH2)
inside the cell, making it susceptible to be oxidized by ROS, giving the fluorescent compound
2',7'-dichlorofluorescein (DCF) [41,49]. Measuring the fluorescence emitted from the DCF at 530 nm,
after being excited at 495 nm, allowed determining the amount of ROS formed after the oxidation
process. This process is particularly sensitive to detection of H2O2 and peroxyl radicals. The cell models
used in this study were macrophages from the strain RAW 264.7. The medium used for the assay was a
phosphate buffer solution 0.01 M (0.138 M NaCl; 2.7 mM KCl; pH 7.4). The studied coumarins were
prepared in DMSO/buffer (no more tan 1% DMSO) to final concentrations of 1, 10 and 100 µM. The
macrophage cells were diluted in a phosphate buffer solution until concentrations of 50.000 cell/mL.

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3.5. Theoretical Evaluation of ADME Properties
The ADME properties of the studied compounds were calculated using the Molinspiration
property program. LogP was calculated by the methodology developed by Molinspiration as a sum of
fragment-based contributions and correction factors [42]. Topological Polar Surface Area (TPSA) was
calculated based on the methodology published by Ertl et al. as a sum of fragment contributions. Oxygen
and nitrogen centered polar fragments were considered. PSA had been shown to be a very good descriptor
characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and
blood-brain barrier penetration. Method for calculation of molecule volume developed at Molinspiration
was based on group contributions. These had been obtained by fitting sum of fragment contributions to
“real” 3D volume for a training set of about twelve thousand, mostly drug-like molecules. 3D molecular
geometries for a training set were fully optimized by the semiempirical AM1 method.
4. Conclusions
Oxidation potentials of all the studied hydroxylated 3-phenylcoumarins were determined, with
compounds 6 and 7 giving the lowest oxidation potentials, associated to a higher antioxidant activity
within the series. Through the ORAC-FL test, derivative 8, bearing two hydroxyl groups on the scaffold,
presented the highest value (11.8), similar to compound 5 (11.0). In the spin-trapping assays, the highest
percentage of scavenging of hydroxyl radicals (51.4%) and alkoxyl radicals (trolox index = 2.33) also
corresponded to compound 8. However, these values are similar to those of compound 6 (scavenging of
hydroxyl radicals = 44.7% and Trolox index = 2.32). An enhanced activity of compound 8 (compared
to compound 6) was found, particularly against peroxyl and hydroxyl radicals. The AI30 index of
compound 8 was also the best one in the series (0.18). Finally, in the biological assay, it was observed
that all coumarins decreased ROS generation in an oxidative stress situation due to the metabolism of
menadione. As main conclusion, the position of the hydroxyl group on the benzene ring of the coumarin
scaffold is in general critical for the antioxidant activity, even though all the derivatives presented a good
profile. Also, in general, the increase of a hydroxyl group in the 3-phenyl ring did not improve
significantly the antioxidant profile of the derivatives. Therefore these preliminary findings have
encouraged us to perform a future structural optimization of this kind of compounds.
Acknowledgments
This project was partially supported by the FONDECYT (projects 1110029 and 1090078), PhD
fellowship CONICYT, fellowship for operational expenses (N°21120376), Spanish researchers personal
founds, University of Santiago de Compostela and Fundação para a Ciência e Tecnologia (FCT) for the
Pest/C-QUI/UI0081/2013. MJ Matos was supported by the fellowship from Fundação para a Ciência e
Tecnologia (FCT), POPH (Programa Operacional Potencial Humano) and QREN (Quadro de Referência
Estratégica Nacional) (SFRH/BPD/95345/2013). S Vazquez-Rodriguez was supported by the Universidade
de Porto postdoctoral grant NORTE-07-0124-FEDER-000065.

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Author Contributions
MJM, FB, LS, EU and COA designed research; MJM and SVR performed research and analyzed the
data related to the synthetic methodologies; FM performed research and analyzed the data related to the
electrochemical studies; MJM and FM wrote the paper. FB, LS, EU and COA contributed to the SVR
study and revised the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).