Synthesis, antioxidant and antichagasic properties of a selected series of hydroxy-3-arylcoumarins

Article abstract of DOI:10.1016/j.bmc.2016.11.033

Oxidative stress is involved in several parasitic diseases such as Chagas. Agents able to selectively modulate biochemical processes involved in the disease represent promising multifunctional agents for the delay or abolishment of the progression of this

Full text of DOI:10.1016/j.bmc.2016.11.033

Accepted Manuscript
Synthesis, antioxidant and antichagasic properties of a selected series of hy-
droxy-3-arylcoumarins
Natalia Robledo-O’Ryan, Maria João Matos, Saleta Vazquez-Rodriguez,
Lourdes Santana, Eugenio Uriarte, Mauricio Moncada-Basualto, Francisco
Mura, Michel Lapier, Juan Diego Maya, Claudio Olea-Azar
PII:
S0968-0896(16)30742-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.033
BMC 13400
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 September 2016
30 October 2016
17 November 2016
Please cite this article as: Robledo-O’Ryan, N., João Matos, M., Vazquez-Rodriguez, S., Santana, L., Uriarte, E.,
Moncada-Basualto, M., Mura, F., Lapier, M., Diego Maya, J., Olea-Azar, C., Synthesis, antioxidant and antichagasic
properties of a selected series of hydroxy-3-arylcoumarins, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.11.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis, antioxidant and antichagasic
properties of a selected series of hydroxy-3-
arylcoumarins
Leave this area blank for abstract info.
Natalia Robledo-O´Ryan,a Maria João Matos,b* Saleta Vazquez-Rodriguez,b Lourdes Santana,b Eugenio Uriarte,b
Mauricio Moncada-Basualto,a,c Francisco Mura,a Michel Lapier,d Juan Diego Maya,d Claudio Olea-Azara*
a Free Radical and Antioxidants Laboratory, Inorganic and Analytical Department, Faculty of Chemical and
Pharmaceutical Sciences, University of Chile, Sergio Livingstone Polhammer 1007, Independencia, Santiago,
Chile.
b Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de
Compostela, Spain.
c Department of Environmental Sciences, Faculty of Chemistry and Biology, University of Santiago of Chile,
Santiago, Chile.
d Department of Molecular Pharmacology and Clinical, Faculty of Medicine, University of Chile, Santiago,
Chile.
OH
HO
O
O

Bioorganic & Medicinal Chemistry
Synthesis, antioxidant and antichagasic properties of a selected series of hydroxy-3-
arylcoumarins
Natalia Robledo-O´Ryan,a Maria João Matos,b Saleta Vazquez-Rodriguez,b Lourdes Santana,b Eugenio
Uriarte,b Mauricio Moncada-Basualto,a,c Francisco Mura,a Michel Lapier,d Juan Diego Maya,d Claudio
Olea-Azara*
a Free Radical and Antioxidants Laboratory, Inorganic and Analytical Department, Faculty of Chemical and Pharmaceutical Sciences, University of Chile,
Sergio Livingstone Polhammer 1007, Independencia, Santiago, Chile.
b Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain.
c Department of Environmental Sciences, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile.
d Department of Molecular Pharmacology and Clinical, Faculty of Medicine, University of Chile, Santiago, Chile.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Oxidative stress is involved in several parasitic diseases such as Chagas. Agents able to
selectively modulate biochemical processes involved in the disease represent promising
multifunctional agents for the delay or abolishment of the progression of this pathology. In the
current work, differently substituted hydroxy-3-arylcoumarins are described, exerting both
antioxidant and trypanocidal activity. Among the compounds synthesized, compound 8 showed
the most interesting profile, presenting a moderate scavenging ability for peroxyl radicals
(ORAC-FL=2.23) and a high degree of selectivity toward epimastigotes stage of the parasite T.
cruzi (IC50 = 1.31 µM), higher than Nifurtimox (drug currently used for treatment of Chagas
disease). Interestingly, the current study revealed that small structural changes in the hydroxy-3-
arylcoumarin core allow modulating both activities, suggesting that this scaffold has desirable
properties for the development of promising classes of antichagasic compounds. 2009 Elsevier
Ltd. All rights reserved.
Available online
Keywords:
Hydroxy-3-arylcoumarins
Antioxidant activity
Trypanosome
Chagas disease
———
 Corresponding author. Tel.: +34 981 528070; e-mail: mariacmatos@gmail.com; colea@uchile.cl

lipid peroxidation inhibition. In addition, the α-pyrone ring in the
coumarin framework proved to improve the inhibitory activity of
oxidative reactions.19 This is in accordance with the studies
developed by our group on hydroxyl substituted 3-
arylcoumarins.18
1. Introduction
Chagas’ disease or American trypanosomiasis is a parasitic
disease produced by a flagellated protozoo called Trypanosoma
cruzi (T. cruzi), that represents a major health problem in South,
Central and North America (Mexico and the south of the U.S.A.).
However, during the last decade this disease has been spread to
some non-endemic areas, driving this situation to a worldwide
public health problem. According to statistics of PAHO (Pan
American Health Organization) and WHO (World Health
Organization), 7.7-10 million people are chronically infected
with T. cruzi, and about 10,000-14,000 deaths per year are caused
by this disease.1,2
It has been shown that hydroxylated coumarins have both
antioxidant and anti-T. cruzi activity.8,20 Both properties might be
considered necessary in the creation of new drugs with potential
trypanocidal activity, since during the lifecycle of T. cruzi and
the infection process ROS can be produced by parasite toxic
secretions. Moreover, one of the mechanisms associated with
trypanocidal activity of coumarins is the inhibition of GAPDH
enzyme, related to oxidative stress generation.21,22
Nifurtimox and benznidazole (Figure 1) were developed over
four decades ago, and they still remain the current treatment for
American trypanosomiasis. These drugs are considered far from
ideal, because they cause multiple side effects and have limited
efficacy, especially in patients with chronic disease.3 This makes
necessary and urgent the development of new, more effective and
less toxic drugs. Several molecules have been studied for their
potential as trypanocidal.4,5,6 Some coumarins have shown anti-T.
cruzi properties,7,8,9,10 and could become an alternative for the
treatment of the disease.
In this work a series of hydroxy-3-arylcoumarins (Figure 2) were
synthesized and anti-T. cruzi activity and its relation to the
antioxidant capacity were studied.
Figure 2. General structure of the studied hydroxy-3-
arylcoumarins.
Figure 1. Chemical structures of the currently used antiparasitic
drugs.
Results and Discussion
Coumarins are members of a huge family of compounds,
structurally consisting in a benzene ring fused to a pyrone ring,
with a carbonyl group at position 2.11 The wide variety of
biological properties of coumarins have drawn the attention of
organic chemists. Both natural and synthetic coumarins own
biological properties that depend on their chemical structure.12 In
particular, several compounds of this family present antioxidant
capacity.13,14,15,16
Reactive oxygen species (ROS) are highly reactive molecules,
which are principally generated by the cellular metabolism.
These species induce oxidative damage in biomolecules, such as
carbohydrates, proteins, lipids and nucleic acids. This deleterious
effect on molecules has been related to ageing acceleration and
other chronic diseases, such as neurodegenerative diseases,
cancer and cardiovascular pathologies.17 Antioxidant compounds,
such as hydroxycoumarins, have been studied due to their ability
to prevent neurodegenerative disorders. The potential
mechanisms are related to the scavenging of free radicals and the
delaying or prevention of biomolecules oxidation.18
Thuong et al. studied some coumarins with a catechol moiety,
which showed interesting free radicals scavenging activity and

Chemistry
Compounds 1–10 were efficiently synthesized according to the
synthetic strategy outlined in Scheme 1. The synthetic
methodology involves a two-step synthetic route. The first step is
a Perkin–Oglialoro condensation of the different commercially
available hydroxybenzaldehydes and arylacetic acids, using
potassium acetate (CH3CO2K) in acetic anhydride (Ac2O), under
reflux, for 16 h, to obtain the precursor acetoxy-3-
arylcoumarins.23 Acetylation of the hydroxyl groups and pyrone
ring closure occur simultaneously. The second step is a
hydrolysis of the obtained acetoxy derivatives, in the presence of
aqueous HCl solution and MeOH, under reflux, for 3 h, to
achieve the final substituted hydroxy-3-arylcoumarins (1–10).
Structures of the synthesized compounds were established based
on their spectral data (see the Experimental section).
OCOCH3
CHO
CH3CO2K, Ac2O
HO
OH
H3COCO
HOOC
reflux, 16 h
OH
O
O
HCl, MeOH
reflux, 3 h
OH
HO
O
O
Scheme 1. Synthetic methodology to obtain the hydroxyl
substituted 3-arylcoumarins.

oxidation mechanism of these compounds could be associated to
a chemical reaction followed by an irreversible electrochemical
reaction.26
Electrochemical study
Oxidation potential is a physicochemical parameter that
determines the energy needed for a compound to give
electrons. This parameter was obtained by means of the
electrochemical technique, Cyclic Voltammetry (CV).
Oxidation potentials have been related to the antioxidant
capacity of polyphenols. This usually means that a low
oxidation potential indicates a higher antioxidant capacity.24,25
In this work, determinations of the oxidation potentials and
the oxidation mechanisms of the hydroxy-3-arylcoumarins
were performed in a polar aprotic medium.
The anodic signal of the resorcinol derivatives is attributed to the
oxidation of the corresponding resorcinol group to a meta-
quinone through a meta-semiquinone radical (Scheme 2). This
radical is not thermodynamically stable, and is rapidly reduced to
the meta-quinone, and therefore no reduction peak is evident. The
reduction potential of the hydroxyl groups in a coumarin ring is
influenced by the position of this group.27 This may justify the
lower reactivity of the resorcinol moiety.
OH
OH
OH
To facilitate the discussion, the hydroxy-3-arylcoumarins
were classified according to the position and number of the
hydroxyl groups presented in the coumarin scaffold as:
monohydroxylated derivatives (1-3), catechol derivatives (4,
7-10) and resorcinol derivatives (5 and 6).
- e-
O
O
O
O
O
O
HO
O
O
- H+
- e-
OH
OH
OH
O
ECirrev
+
Among resorcinol derivatives, the CV of compound 5 shows a
single signal for the oxidation cycle, and no signal in the
reverse scan (reduction), indicating an irreversible oxidation
in the experimental conditions (Figure 3). The same tendency
was observed for compound 6, which presents a similar CV
profile and similar oxidation potential.
O
O
O
O
O
O
O
O
O
OH
O
O
Scheme 2. Proposed electrochemical oxidation mechanism for
compound 5.
120
100
80
60
40
20
0
A)
Monohydroxylated derivatives in the 3-aryl ring (1-3) also
contain a second hydroxyl group in the coumarin core.
Compound 2, for example, has two hydroxyl groups in its
structure: one at position 6 of the coumarin skeleton and another
one at position 2’ of the phenyl group. CV of compound 2
showed two anodic peaks, corresponding to the oxidation of both
hydroxyl groups (Figure 4). The anodic peak at lower oxidation
potential corresponds to the hydroxyl group at position 6 of the
coumarin ring, regarding its relative position to the α-pyrone
ring, thereby facilitating oxidation. The second anodic peak at a
higher potential corresponds to the hydroxyl group present in the
phenyl ring. A proposed oxidation mechanism is outlined in
Scheme 3. Since these oxidation processes are faster than the
scan rate, the process is observed as one wide peak. Due to the
structural similarity between compound 2 and the compounds 1
and 3, similar cyclic voltammograms were obtained. As well as
for resorcinol derivatives, 3-aryl monohydroxylated derivatives
1-3 show irreversible oxidation, since there is no signal in the
reverse scan (Figure 4).
2.5 V/s
2.0 V/s
1.0 V/s
0.50 V/s
0.25 V/s
-20
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
B)
2.6x10-5
2.4x10-5
2.2x10-5
2.0x10-5
1.8x10-5
1.6x10-5
1.4x10-5
0.0
0.5
1.0
1.5
2.0
2.5
v (V/s)
Figure 3. A) Cyclic voltammogram for 1 mmol/L of compound 5
for v= 0.1–2.5 mV/s; B) linear relationship between the ratio of
Ipa /v1/ 2 versus scan speed (v).
According to the reversibility criteria proposed by Nicholson and
Shain and the absence of any cathodic peak (Figure 3A), the

100
80
60
40
20
0
B)
60
40
20
0
2.5 V/s
2.0 V/s
1.0 V/s
0.5 V/s
0.25 V/s
0.10 V/s
-20
-20
-40
0.4
0.6
0.8
1.0
1.2
1.4
E/V vs Ag/AgCl
Figure 4. Cyclic voltammogram for 1 mmol/L of compound 2
(black) and 1 (dotted blue line), for v = 2.5 mV/s.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
E/V vs Ag/AgCl
a)
OH
OH
OH
Figure 5. Cyclic voltammogram at v= 0.1–2.5 mV/s for 1
mmol/L of: A) compound 9 and B) compound 10.
- e-
O
O
HO
- e-
- H+
O
O
O
O
O
O
The anodic signal of the dihydroxycoumarins is attributed to the
oxidation of the catechol group to the corresponding ortho-
radical-semiquinone and then to the corresponding ortho-quinone
(Scheme 4). In these cases, substitution in ortho position is a
structural factor that should contribute to electron density
favouring oxidative process, leading to the decrease in the
oxidation potentials.28
b)
O
O
OH
- e-
- e-
O
O
O
- H+
O
O
O
O
O
O
Scheme 3. Electrochemical oxidation mechanism proposed for
compound 2. a) oxidation mechanism of the hydroxyl group at
position 6 of the coumarin ring; it corresponds to an anodic peak
at lower potential. b) oxidation mechanism of the hydroxyl group
at position 3’ of the aromatic ring; it corresponds to an anodic
peak at higher potential.
Scheme 4. Electrochemical oxidation mechanism proposed for
compound 7.
In the catechol derivatives (4 and 7-10), compound 9 presents a
quasi-reversible peak (Figure 5A). When two catechol groups
were present in the molecule, such as for compound 10, two
anodic peaks were observed (Figure 5B).
From the oxidation potentials point of view (Table 1) the
antioxidant capacity of the compounds can be ranked as: 10 > 3 >
8 > 1 > 2 > 4 = 7 = 9 > 5.
Derivatives with a catechol group in their structure presented an
oxidation potential around 0.80 V, with exception of compound
10, which presented two oxidation potential. From the catechol
series, compound 8 shows the lowest anodic peak potential (Epa
= 0.78 V) when compared to compounds 4, 7 and 9, which have
similar anodic peak potentials (Epa = 0.81 V in all three cases).
Compound 10, containing two catechol groups in its structure in
both the aryl ring and the coumarin core, presents oxidation
potentials at 0.64 and 0.79 V, and therefore showing the most
promising antioxidant properties in the studied series.
A)
50
40
2.5 V/s
2.0 V/s
1.0 V/s
0.5 V/s
0.25 V/s
0.10 V/s
30
20
10
0
-10
-20
-30
-40
0.0
0.2
0.4
0.6
E/V vs Ag/AgCl
0.8
1.0
1.2
Resorcinol derivatives (5-6) show higher anodic oxidation
potentials compare to the catechol derivatives due to the meta
position of the second hydroxyl group of the resorcinol
derivatives, which does not provide electron density directly to
the reaction centre (hydroxyl group at position 7). However, the

oxidation potentials of the catechol derivatives are lower since
the formation of an ortho-semiquinone radical is stabilized by the
presence of the hydroxyl group at ortho position with electron-
donating properties to the reaction centre, thereby moving
oxidation potentials to less positive values.
For the compounds mentioned above, the electroactive group is
the catechol moiety and therefore the intermediate could be the
ortho-semiquinone radical. This was confirmed by the simulation
of the electrochemical oxidation of the coumarin derivative 10 by
EPR-WinSIM (Figure 7). Compound 10 presented a symmetrical
two-line spectrum, which is interpreted in terms of the formation
of a semiquinone radical whose relocation is through the benzene
ring of the coumarin skeleton.
Characterization of radical species generated through
electron spin resonance (ESR) spectroscopy
Electron spin resonance (ESR) is a spectroscopic technique for
the detection of free radicals in the presence of a magnetic field,
when irradiated with microwaves. For this reason, this technique
has been employed for the detection and characterization of
radicals generated by the oxidation of the tested hydroxylated
coumarin derivatives.
A)
The electrochemical oxidation of the hydroxylated coumarin
derivatives was performed under the same conditions as for CV.
Due to the short half-lives of the generated free radicals, it was
necessary to use the spin-trapping methodology by using N-tert-
butyl-α-phenylnitrone (PBN) as the spin-trap agent. The spin-trap
is a diamagnetic compound that interacts with a free radical
forming a paramagnetic spin-adduct. In this way, the half-life of
a free radical increases and thus it makes possible its detection.
PBN allows the detection of carbon- and oxygen-centred radicals.
In the presence of PBN a six lines hyperfine pattern is obtained
(Figure 6), with coupling constants aN = 13.9 G and aH = 2.3 G,
indicating the trapping of a carbon-cantered free radical for
compounds 1, 2, 5 and 6.29
3390
3400
3410
3420
3430
3440
3450
Magnetic field (G)
B)
3390
3400
3410
3420
3430
3440
3450
Magnetic field (G)
Figure 7. A) ESR experimental spectrum corresponding to the
radical derived from the electrochemical oxidation stabilized
derivative compound 10 Zn+2. B) ESR simulated spectrum of
compound 10 by the EPR-WinSim program.
3390
3400
3410
3420
3430
3440
3450
Magnetic Field (G)
Compound 7 has a hyperfine pattern of two lines with a
hyperfine-coupling constant of 3.14 G, showing the same
behaviour as compound 10.
Figure 6. ESR spectra of compound 5-PBN adduct in DMSO.
According to the analysis of the ESR spectrum of catechol
derivatives 4 and 7-10, it could be possible to confirm that the
oxidation of the catechol moiety in these compounds goes
through the formation of an ortho-semiquinonic radical
intermediate. According to this, we can confirm the proposed
electrochemical oxidation mechanism (Scheme 4) for these
compounds.
The free radical intermediates generated by the electrochemical
oxidation of ortho-dihydroxycoumarin derivatives (catechol
derivatives, compounds 4 and 7-10) produced no signal structure
employing the ESR technique. This is because the lifetimes of
the generated radical species are very short to be measured
experimentally, and the final products of oxidation are silent in
ESR. For this reason, a zinc salt (ZnCl2) was used, because
complexation reaction between zinc (II) and the catechol moiety
in these tested compounds brings stability to the ortho-
Study of the reactivity towards the hydroxyl radical
semiquinone radical. As
a result, the radical can be
experimentally measured at room temperature.30
Reactivity against the hydroxyl radical (OH•) was studied by
means of the spin-trapping methodology, which is based on the
competitive reactions between the coumarin derivative and the

spin-trap (DMPO) for the OH•. The OH• was generated in situ by
UV-photolysis of hydrogen peroxide. A hyperfine pattern of four
lines with intensities in the ratio of 1:2:2:1 was obtained. This
spectrum is characteristic of the spin-adduct [DMPO-OH].31
Scavenging of the OH• was calculated by adding a constant
concentration of the corresponding derivative and measuring the
decrease percentage in the integration of the signals regarding to
the control solution, which do not contain the studied compound.
In Table 1 it is shown that compounds 1, 3, 4 and 6-8 have the
no reactivity against OH•. On the other hand, compounds 2 and 5
proved to be display an ability to completely scavenge radicals.
As a consequence of these results, there is not a direct relation
between the number of hydroxyl groups in the coumarin skeleton
and the antioxidant capacity. Compound 2 (presenting only one
hydroxyl group on the coumarin core at position 6) completely
scavenge the OH•. This could be explained due to the relative
position of the hydroxyl group to the coumarin core, favouring
the radical stabilization.
The possible structural differences between the catechol
derivatives 9 and 10 and the resorcinol derivative 5 are mainly
due to the potential formation of an intramolecular hydrogen
bond between the hydroxyl group at the position 8 and the
oxygen of the pyrone ring. While this interaction may take place
in compounds 9 and 10, that does not occur in the resorcinol
derivatives.

Table 1. Results obtained in antioxidant homogeneous assays (ORAC-FL, oxidation potential and scavenging of OH•).
Compounds
Oxidation potential (V)
OH• Scavenging (%)
ORAC-FL index
Epa1
0.79
0.80
0.74
0.81
1.16
--
0.81
0.78
0.81
0.64
--
Epa2
--
1.17
--
--
--
--
--
--
--
--
100
--
--
100
--
--
--
26.09
24.29
31
5.30 ± 0.29
5.47 ± 0.31
3.12 ± 0.50
4.59 ± 0.02
1.81 ± 0.05
1.95 ± 0.14
1.11 ± 0.04
2.23 ± 0.19
2.95 ± 0.50
5.38 ± 0.50
1.0 ± 0.2
1
2
3
4
5
6
7
8
9
0.79
--
10
Trolox*
-- parameter not observed
* Data obtained from references 32 and 33.
Figure 8. A) ORAC-FL profile for compound 2; B) AUC as a
function of the concentration for compound 2.
Determination of antioxidant capacity by ORAC-FL assay
All the studied compounds showed higher antioxidant capacity
than the standard compound, trolox. Regarding to the ORAC-FL
assay, the compound showing the best ORAC-FL index is
compound 2 (Figure 8). Based on the ORAC-FL index, the order
from the highest to the lowest scavenging activity against the
peroxyl radicals in this series is: 2 > 10 > 1 > 4 > 3 > 9 > 8 > 6 >
5 > 7.
Despite monohydroxylated derivatives 1-3 only present one
hydroxyl group in the coumarin core and another hydroxyl group
in the 3-phenyl ring, compounds 1 and 2 displayed the highest
ORAC-FL indexes of all the studied compounds. This might be
attributed to the presence of the hydroxyl group at position 6,
which is located in the opposite position to the cyclic ester. This
makes possible an easy transfer of a hydrogen atom to the
peroxyl radical.
On the other hand, the lowest ORAC-FL index was presented by
compound 7. A possible explanation is that the hydroxyl group at
position 8 forms a hydrogen bond with the oxygen in the pyrone
ring, thus reducing their ability to transfer to peroxyl radicals.
Compounds 7-9 are structurally similar, as they both contain a
catechol group at 7,8-positions of the coumarin ring, and varies
in the presence and/or position of the hydroxyl substituent on the
3-phenyl ring. In general, the presence of a catechol moiety in the
structure increases the antioxidant properties in a polyphenolic
compound due to the stability of the ortho-semiquinone radical.34
However, these compounds have low ORAC-FL values. This has
been attributed to the formation of an intramolecular hydrogen
bond between the hydroxyl group at position 8 and the oxygen of
the α-pyrone ring affecting the ability to donate the hydrogen
atom. Therefore, their antioxidant capacity is reduced. However,
when comparing the catechol derivatives, we observed that the
presence of the hydroxyl group in the 3-phenyl ring (compounds
4, 8-10) increases the antioxidant capacity in at least double
comparing to compound 7. Position and number of the hydroxyl
groups in the 3-phenyl ring plays an important role in the
antioxidant capacity. Compound 9 (3’-OH) presented a slightly
B)
4000
3500
3000
2500
2000
1500
1000
500
0
-500
0.0
0.5
1.0
1.5
2.0
2.5
[2] / 

significant higher activity than compound 8 (2’-OH), and
compound 10 (2’,3’-OH) showed the best ORAC-FL index of the
catechol series.
The activity of the coumarin derivatives were evaluated in
mammalian cells as hosts for parasites in order to assess the
cytotoxicity of these compounds in RAW 264.7 murine cells
(macrophages from blood). A preliminary screening based on the
cell viability was carried out using 100 µM concentrations and
comparing the results to a control (Figure 9).
Compounds 4 and 10, with ORAC-FL values of 4.59 and 5.38,
respectively, presented the highest antioxidant capacity in the
catechol series due to the presence of a catechol moiety in the 3-
phenyl ring. Compound 10 increases the antioxidant capacity in
5-fold comparing to compound 7. Compound 4 has not
significant difference with the ORAC-FL index of the 8-hydroxy-
3-arylcoumarin (ORAC-FL = 5.7) previously described by our
group.18
150
100
50
Resorcinol derivatives 5 and 6 showed low ORAC-FL values due
to the meta position of the hydroxyl groups. This position
performs an electro-withdrawing effect affecting the ability to
donate the hydrogen atom, resulting in low ORAC-FL values.35
0
l
1
2
3
4
5
6
7
8
9
0
o
1
r
t
n
According to the obtained results, there is no clear relationship
between the number of hydroxyl groups and the ORAC-FL
values. However, regarding the results herein analyzed the most
beneficial positions of the hydroxyl groups are on the 3-
arylcoumarin frame.
o
C
Figure 9. Cell viability of hydroxy-3-arylcoumarins on RAW
264.7 cells. The results correspond to the mean ± SD of three
independent experiments.
Determination of the cytotoxic activity against T. cruzi
(Dm28c clone) epimastigotes forms
Based on the screening, the IC50 values were calculated in RAW
264.7 macrophages. Results are summarized in Table 2.
Table 2. In vitro cytotoxicity in murine RAW 264.7 macrophages (IC50) and trypanocidal activity in epimastigote.
Compounds
IC50 (µM)
RAW 264.7
˃ 100 **
Epimastigotes
˃ 100 **
SI*
-
-
1
2
3
4
5
6
7
8
9
˃ 100 **
˃ 100 **
115.30 ± 0.99
˃ 100 **
˃ 100 **
26.10 ± 4.19
68.85 ± 0.65
397.60 ± 29.56
˃ 100 **
˃ 100 **
-
84.28 ± 7.38
6.15 ± 5.01
˃ 100 **
8.90 ± 5.25
1.31 ± 2.03
28.55 ± 5.07
1.37
> 16.26
-
2.93
52.56
13.93
97.48 ± 7.95
263.44
46.99 ± 3.41
17.4 ± 1.3
2.07
10
15.14
Nifurtimox
* Selectivity Index IC50 macrophages RAW 264.7/IC50 epimastigotes clone Dm28c
** 100µM was the highest tested concentration. At higher concentrations, the compounds precipitate and data could not be attained.

The viability of the epimastigotes form of T. cruzi was correlated
to the antioxidant capacity of hydroxy-3-arylcoumarins, in order
to obtain information about the possible mechanism of
trypanocidal action.
Based on the IC50 values, exactly half of the studied derivatives
showed low cytotoxicity on RAW 264.7 macrophages.
Compound 7 showed the highest cytotoxicity against the
evaluated mammalian cells (IC50 = 26.10 ± 4.19 µM).
It can be seen in Figure 11, that the hydroxy-3-arylcoumarins
with greater trypanocidal activity (compounds 8 > 5 > 7) have
lower antioxidant capacity (ORAC-FL index similar between
them) than the others. In contrast, the compounds with lower
trypanocidal activity have better antioxidant capacity. Overall,
mono-hydroxylated coumarins in the 3-phenyl ring proved to
display lower activities on epimastigotes form of the parasite.
The determination of viability of epimastigote of T. cruzi Dm28c,
was performed using concentrations 100 and 10 µM of coumarin
derivatives (Figure 10).
A)
150
100
50
0
100
1
2
6
3
4
80
60
40
20
0
10
1
2
3
4
5
6
7
8
9
0
L
O
R
1
T
N
O
C
9
7
1
8
5
B)
150
100
50
2
3
4
5
6
ORAC-FL index
Figure 11. Correlation between antioxidant capacity and viability
of the epimastigote form of T. cruzi in the presence of hydroxy-3-
arylcoumarins. Red circle – compounds with major trypanocidal
activity and minor antioxidant capacity. Blue circle – compounds
with minor trypanocidal activity and major antioxidant capacity.
0
According on the above, it can be stated that there is no direct
relationship between the properties in compounds in study,
indicating two independent action mechanisms.
1
2
3
4
5
6
7
8
9
0
L
1
O
R
T
N
O
C
In order to obtain information about the possible mechanism of
action via oxidative stress, the ESR spectrum was recorded in
parasitical medium.
Figure 10. Parasite viability of the studied 3-arylcoumarins on
epimastigotes of T. cruzi (clone Dm28c) (A) 100 µM and (B) 10
µM. The results correspond to the mean ± SD of three
independent experiments. The results are normalized in relation
to the control, which represents 100% of viability.
Detection of free radicals generated by coumarin derivatives
in a parasitic medium (epimastigote form of T. cruzi) by
means of the spin-trapping methodology assisted by ESR
In general, it is observed that the catechol moiety at 7,8-positions
of the coumarin skeleton improved the trypanocidal activity in
the epimastigote stage. On the other hand, the derivatives with
one hydroxyl group on the coumarin moiety showed low activity
in the epimastigote stage.
To perform the free radical detection in a microsomal parasitic
medium, an ESR technique was employed. A solution of DMSO
was used as a control, without detecting the generation radical
species.
The IC50 values against the epimastigote form of the T. cruzi
were calculated, as well as the selectivity index (SI, Table 2).
Compounds 5, 8 and 9 presented the best selectivity index
ranging from 13.93 to 52.56, being compound 8 the most
selective of the series (3.5 times more selective than Nifurtimox).
In this assay, the most active compounds were compounds 5, 7
and 8, at a concentration of 5 mM (Figure 12). All these
compounds generated the same spectral pattern after ten minutes
of incubation. The strong intensity of the observed signals is
consistent with the trapping of a carbon-centred radical, which
might be the coumarin derivative radical (aH ~ 20.06 G and aN ~
15.76 G).

Because the control solution did not show any signal, it may be
deduced that the DMPO does not break down immediately and
that no free radical is being trapped by the DMPO. Moreover, it
is possible to observe the quartet splitting pattern (aN~aH~14.87
G) due to the DMPO-OH radical adduct. This evidences the in
situ formation of the OH• by the incubation of coumarin
derivatives. Subsequently, this adduct can undergo an oxidative
process, thereby forming a paramagnetic compound (DMPOX,
5,5-dimethyl-2-oxo-pyrrolin-1-oxyl),36 which is observed in the
spectrum as a triplet (aN ~ 15.46 G). These results may indicate
that mechanism of action of these hydroxylated coumarins could
go through the generation of ROS (see Figure 13), i.e. a possible
via would be the oxidative stress. These results are related to
ones described by Aguilera-Venegas et al.,6 in which the
generation of oxidative stress is evident, possibly with a
mechanism associated to the generation of mitochondrial
alterations in the parasite.
A)
+
#
+
#
#
#
#
+
#
*
*
*
*
B)
3440
3460
3480
3500
Magnetic field (G)
Figure 12. Experimental spectrum for spin-adduct generated in
microsomes T. cruzi (strain Dm28c) at room temperature: a)
Spectrum of the control sample (DMSO); b) Spectrum recorded
with the microsomal fraction incubated with compound 5 and
DMPO. It has been marked (*) the corresponding hyperfine
pattern for the spin-adduct DMPO-OH. The hashtag (#) indicates
the adduct between a carbon-centred radical and DMPO, and (+)
indicate the DMPOX adduct.
Figure 13. Mechanism of action proposed of hydroxy-3-arylcoumarins against epimastigotes of T. cruzi, generation of metabolites and
their interaction with the spin trap DMPO. Spectra of ESR of DMPO-CUM (carbon centred, sextet of intensity 1:1:1:1:1:1), DMPO-OH
(quartet of intensity 1:2:2:1) and DMPOX (triplet of intensity 1:1:1).

based on the compound 8 structure, for the design of new
hydroxy-3-arylcoumarins.
Determination of activity gainst trypomastigote form of T.
cruzi (Dm28c clone).
The three most active compounds against epimastigote form of
the parasite, were selected to be studied on the trypomastigote
form.
3. Conclusions
Electrochemical studies indicated that coumarins bearing
resorcinol groups at the 3-phenyl ring and a hydroxyl group in
the coumarin core display an irreversible oxidation process.
The compounds were evaluated at 20 and 100 µM (Figure 14)
against the trypomastigote form. Since only compound 8 showed
a significant activity against the trypomastigote form, its IC50
value was the only one determined (Table 3). Compound 8
Coumarins with catechol groups have
a quasi-reversible
oxidation process. The oxidation potentials of these coumarins
proved to be lower than the compounds with resorcinol groups in
their structures. This was detected and characterized by ESR
assays, confirming the mechanism proposed by CV. For the case
of radical species with a short half-life, it was detected by a spin
trapping technique. The antioxidant reactivity towards OH• was
evaluated by the spin trapping methodology and showed that
there is no correlation between the number of hydroxyl groups in
the molecule and its reactivity. All the studied coumarins were
more active than trolox in the ORAC-FL assay. Coumarins
having a hydroxyl group at position 6 and a catechol group in the
3-aryl ring showed the best antioxidant capacity of the studied
series. Most of the evaluated compounds showed low
cytotoxicity against RAW 264.7 macrophages, particularly those
compounds that showed higher antioxidant capacity. In the in
vitro study of the trypanocidal ability, compound 8 was active
against both epimastigote and trypomastigote forms of the
parasite, and up to 13 times more active than Nifurtimox against
the epimastigote form. The presence of a catechol group at 7,8-
positions of the coumarin core proved to be critical for the
trypanocidal activity, as observed for compounds 8 and 9. The
generation of radical species in the epimastigote form after
incubation with coumarins suggests that the cytotoxicity
mechanisms could occur by generating oxidative stress to the
parasite. Rational design using compound 8 as lead compound
would allow the generation of new series of compounds
displaying important trypanocidal properties.
resulted to be more active against the epimastigote form (IC50
=
1.31 µM) than against the trypomastigote one (IC50 = 39.73 µM).
However, in the epimastigote stage, compound 8 was more than
10 times more active than Nifurtimox. The selectivity of
compound 8 between the macrophages (RAW 246.7 cells) and
the parasites (epimastigote form) was higher (SI = 52.56) than
Nifurtimox (SI = 15.14), but the window between the IC50 in both
cell lines was narrower than for Nifurtimox.
150
100
50
0
100 20 100 20 100 20 CONTROL
5
8
9
Figure 14. Parasite viability of compounds 5, 8 and 9 on T. cruzi
trypomastigote form (clone Dm28c) to the concentration of 20
and 100 µM. The results correspond to the mean ± SD of three
independent experiments. The results were normalized for the
control, representing 100% viability.
4. Experimental section
Synthetic procedures
Table 3. In vitro cytotoxicity of compound
epimastigote and trypomastigote stages of T. cruzi).
8
(against
he starting materials and reagents were o tained from
commercial suppliers ( igma-Aldrich and were used without
further purification. elting points ( p are uncorrected and
were determined using a eichert ofler hermopan or in
Compound 8
Nifurtimox
T. cruzi stage
IC50 (µM)
SI*
IC50 (µM)
SI*
1.31 ± 2.03
52.56
17.40 ± 1.30
15.14
Epimastigote
1
capillary tu es in a chi 510 apparatus. H NMR (250 MHz)
and 13C NMR (75.5 MHz) spectra were recorded using a Bruker
AMX spectrometer using CDCl3 or DMSO-d6 as solvent.
Chemical shifts (δ are expressed in parts per million (ppm using
TMS as an internal standard. Coupling constants J are expressed
in Hertz (Hz). Spin multiplicities are given as s (singlet), d
(doublet), dd (doublet of doublets) and m (multiplet). Mass
spectrometry was carried out using a Hewlett-Packard 5988 A
spectrometer. Elemental analyses were performed by using a
Perkin-Elmer 240B microanalyzer and the results are within
±0.4% of calculated values in all cases. The analytical results
document ≥98% purity for all compounds. Flash chromatography
39.73 ± 2.80
1.73
10.00 ± 0.40
26.34
Trypomastigote
* Selectivity Index = IC50 macrophages RAW 264.7/IC50 T. cruzi
(parasite).
From the results for the trypomastigote stage, we can say that
derivatives with a catechol moiety at 7,8-positions in the
coumarin core showed significant activity. This activity
improved when a hydroxyl group was added to position 2’ of the
3-phenyl ring. These results give some interesting information
about the structural properties and the trypanocidal activity,

(FC) was performed on silica gel (Merck 60, 230–400 mesh);
analytical TLC was performed on precoated silica gel plates
(Merck 60 F254). Organic solutions were dried over anhydrous
sodium sulfate. Concentration and e aporation of the sol ent
after reaction or extraction were carried out on a rotary
e aporator ( chi ota apor) operating under reduced pressure.
100 %). Ana. Elem. Calc. for C15H10O5: C, 66.67; H, 3.73;.
Found: C, 66.65; H, 3.70.
7,8-Dihydroxy-3-(2’-hydroxyphenyl)coumarin (8). Mp. 215-
216 oC. Yield 93 %. 1H NMR (250 MHz, DMSO-d6) δ: 6.84-6.90
(m, 2H, H-3’, H-5’ , 7.01-7.06 (m, 1H, H-4’ , 7.12-7.34 (m, 3H,
H-5, H-6, H-6’ , 7.91 (s, 1H, H-4), 9.45 (s, 1H, OH), 10.13 (s,
1H, OH), 10.36 (s, 1H, OH). 13C NMR (75.5 MHz, DMSO-d6) δ:
112.7, 115.7, 119.0, 121.2, 122.8, 123.8, 129.7, 131.0, 132.0,
133.2, 136.5, 143.3, 149.2, 155.1, 160.1. MS m/z: 270 ([M+1]+,
100 %). Ana. Elem. Calc. for C15H10O5: C, 66.67; H, 3.73;.
Found: C, 66.67; H, 3.71.
General procedure for the synthesis of acetoxy-3-
arylcoumarins. The compounds were synthesized under
anhydrous conditions, using a material previously dried at 60 °C
for at least 12 h and at 300 °C for a few minutes immediately
before use. A solution containing anhydrous CH3CO2K (2.94
mmol), the corresponding arylacetic acid (1.67 mmol) and the
corresponding hydroxysalicylaldehyde (1.67 mmol), in Ac2O (1.2
mL), was refluxed for 16 h. The reaction mixture was cooled,
neutralized with 10% aqueous NaHCO3, and extracted with
EtOAc (3 x 30 mL). The organic layers were combined, washed
with water, dried over anhydrous Na2SO4 and the solvent was
evaporated under reduced pressure. The product was purified by
recrystallization in EtOH and dried in vacuum to afford the
desired compound.
7,8-Dihydroxy-3-(3’-hydroxyphenyl)coumarin (9). Mp. 240-
241 oC. Yield 88 %. 1H NMR (250 MHz, DMSO-d6) δ: 6.74-6.83
(m, 2H, H-4’, H-6’ , 7.07-7.13 (m, 3H, H-5, H-6, H-2’ , 7.18-
7.24 (t, 1H, H-5’ , 8.06 (s, 1H, H-4), 9.51 (s, 1H, OH), 9.93 (s,
1H, OH), 10.28 (s, 1H, OH). 13C NMR (75.5 MHz, DMSO-d6) δ:
112.9, 115.2, 115.4, 119.1, 119.4, 122.1, 129.3, 131.9, 136.5,
141.6, 149.7, 157.2, 160.1, 171.4, 180.9. MS m/z: 270 ([M+1]+,
100 %). Ana. Elem. Calc. for C15H10O5: C, 66.67; H, 3.73;.
Found: C, 66.69; H, 3.75.
General procedure for the synthesis of hydroxy-3-
arylcoumarins (1–10). Compounds 1–10 were obtained by
hydrolysis of their acetoxylated counterparts, respectively. The
appropriate acetoxylated coumarin, mixed with 2N aqueous HCl
and MeOH, was refluxed for 3 h. The resulting reaction mixture
was cooled in an ice-bath and the reaction product and the
obtained solid was filtered, washed with cold distilled water, and
dried under vacuum, to afford the desired compounds (1–10).
Evaluation of ORAC-FL activity
The assays were carried out in a Synergy HT Multi-Detection
Microplate Reader, from BioTek Instruments, Inc. (Winooski,
USA), using polystyrene 96-well plate, purchased from Nunc,
Denmark. Fluorescence was measured from the top, at an
excitation wavelength of 485/20 nm and an emission at 528/20
nm. The plate reader was controlled by Gen5 software. The
reaction was performed at 37 °C in 75 mM of phosphate buffer
solution (pH = 7.4), to obtain a final volume of 200 µL.
Fluorescein (final concentration, 40 nM) and stocks solutions of
compounds were prepared in methanol because of their low
solubility in buffer at high concentrations. A set of compounds
solutions, in a range of final concentrations between 0.5 and 2.5
µM, was placed in each well of a 96-well plate. These
concentrations allowed the separation of the fluorescence decay
curves. The mixture was pre-incubated for 15 min at 37°C before
6-Hydroxy-3-(2’-hydroxyphenyl)coumarin (1). Mp. 226-227
oC. Yield 87 %. 1H NMR (250 MHz, DMSO-d6) δ: 6.84-6.90 (m,
2H, H-3’, H-5’ , 7.01-7.06 (m, 2H, H-7, H-4’ , 7.24-7.43 (m, 3H,
H-5, H-8, H-6’ , 7.91 (s, 1H, H-4), 9.54 (1H, OH), 9.70 (1H,
OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 115.6, 118.1, 118.8,
119.3, 120.7, 124.7, 125.0, 126.8, 129.4, 136.1, 141.0, 141.7,
144.4, 157.2, 171.8. MS m/z: 254 ([M+1]+, 100 %). Ana. Elem.
Calc. for C15H10O4: C, 70.86; H, 3.96. Found: C, 70.84; H, 3.93.
8-Hydroxy-3-(3’-hydroxyphenyl)coumarin (3). Mp. 245-246
oC. Yield 89 %. 1H NMR (250 MHz, DMSO-d6) δ: 6.83-6.89 (m,
1H, H-4’ , 7.12-7.29 (m, 3H, H-2’, H-5’, H-6’ , 7.14-7.25 (m,
3H, H-5, H-6, H-7), 7.91 (s, 1H, H-4), 9.63 (1H, OH), 9.89 (1H,
OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 113.9, 117.8, 118.2,
119.7, 120.1, 123.6, 125.3, 127.2, 129.7, 135.4, 140.7, 141.9,
145.9, 158.1, 169.3. MS m/z: 254 ([M+1]+, 100 %). Ana. Elem.
Calc. for C15H10O4: C, 70.86; H, 3.96. Found: C, 70.87; H, 3.95.
the
addition
of
the
2,2′-azobis(2-
methylpropionamidine)dihydrochloride (AAPH) solution (final
concentration, 18 mM). The microplate was immediately placed
in the reader and automatically shaked prior to each reading. The
fluorescence was recorded every 1 min for 120 min. A control
assay with fluorescein, AAPH, and methanol (instead of the
antioxidant solution) was performed for each assay.37 Trolox was
employed as standard antioxidant, in a final concentration range
of 0.5–2.5 µM. The inhibition capacity was expressed as ORAC-
FL 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 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.
5,7-Dihydroxy-3-(3’-hydroxyphenyl)coumarin (6). Mp. 276-
277 oC. Yield 93 %. 1H NMR (250 MHz, DMSO-d6) δ: 6.21-6.27
(m, 2H, H-6, H-8), 6.71-6.79 (m, 1H, H-4’ , 7.02-7.23 (m, 3H,
H-2’, H-5’, H-6’ , 8.00 (s, 1H, H-4), 9.51 (s, 1H, OH), 10.38 (s,
1H, OH), 10.75 (s, 1H, OH). 13C NMR (75.5 MHz, DMSO-d6) δ:
93.8, 102.4, 113.2, 115.2, 119.8, 120.3, 120.9, 129.4, 131.2,
136.7, 138.0, 156.3, 157.2, 162.2, 172.1. MS m/z: 270 ([M+1]+,

Data processing was performed using Origin Pro 8 SR2 (Origin
Lab Corporation, USA).). The ORAC-FL indexes were
calculated according to the following equation (1):
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 msec;
and conversion time 40.96 msec. The results are given as a
percentage of scavenging of the OH• in comparison to the control
solution (which does not contain any antioxidant).
ORAC-FL index=
[(AUCAH–AUCcontrol)/(AUCtrolox–AUCcontrol) x [Trolox]/[AH]
Evaluation of redox properties by CV
Evaluation of the cytotoxic and trypanocidal activity
Cytotoxicity assay
CV measurements were performed in a Metrohm 693VA
instrument with a 694VA stand convertor and a 693VA
processor, at room temperature, using a three-electrode cell. A
glassy carbon electrode presenting an area of 0.03 cm2 was used
as the working electrode. The electrode surface was polished
with alumina powder (particle sizes, 0.3 and 0.05 mm) before use
and after each measurement. Platinum wire was the auxiliary
electrode and silver/silver chloride (Ag/AgCl, 3 M KCl) of
Metrohm Company was used as a reference electrode. The CV
experiments were carried out in DMSO, with 0.1 M of
tetrabutylammonium perchlorate (TBAP) as supporting
electrolyte and 1 mM of each coumarin derivative. Potential
sweeps were executed between 0.0 and + 1.4 V, and 0.1–2.5 V/s.
The effect of drug treatments on RAW 264.7 cells was evaluated
through the tetrazolium dye (MTT; 3[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) assay as a viability test.38
riefly, 10 μl of 5 mg/mL plus 0.22 mg/mL phenazine
metosulfate (electron carrier), were added to each well containing
AW 264.7 cell culture in 100 μL P I 1640 without phenol
red. Compounds under study, dissolved in DMSO, were added to
the culture media at the concentrations demonstrated in the
figures and tables. DMSO final concentration was less than
0.25% v/v. After incubation for 4 h at 37 °C, the generated water-
insoluble formazan dye was dissol ed y the addition of 100 μL
of 10% w/v sodium dodecyl sulphate (SDS) in 0.01 M HCl. The
plates were further incubated overnight at 37 °C, and optical den-
sity of the wells was determined using a microplate reader (Asys
Expert Plus©, Asys Hitach, Austria) at 570 nm. Under these
conditions, the optical density is directly proportional to the
viable cell number in each well. All experiments were performed
at least three times and data reported as means and their standard
deviations from triplicate cultures. Results are reported as the
percentage of non-viable RAW 264.7 cells regarding the control
(cells in culture medium).
Monitoring of electrochemically generated radicals by ESR
ESR spectra were recorded in the X band (9.85 GHz) using a
Bruker ECS106 spectrometer with a rectangular cavity and 50-
kHz field modulation. The hyperfine splitting constants were
estimated to be accurate within 0.05 G. The phenoxyl radicals
were generated by an in situ electrolytic oxidation process under
the same experimental conditions as those of CV, in DMSO with
0.1 M of TBAP. The oxidation potentials were acquired from
CV. Every spectrum was obtained after 50 scans. For catechol
systems, radicals were stabilized by adding 0.01 M of zinc
chloride. For monohydroxylated and resorcinol systems, radicals
were trapped by adding 200 mM of N-tert-butyl-α-phenylnitrone
(PBN). ESR spectra were simulated using the program EPR-
WinSIM Version 0.98.
Determination of trypanocidal activity
Epimastigote stage viability study
Trypanocidal activity was evaluated against the T. cruzi
epimastigote stage (clone Dm28c). It was measured through the
MTT assay using 0.22 mg mL-1 phenazine metosulfate (as an
electron carrier).39 T. cruzi epimastigote (Dm28c strain), from the
authors own collection (Programa de Farmacología Molecular y
Clínica, Facultad de Medicina, Universidad de Chile, Santiago,
Chile) were grown at 28 °C in Diamond’s monophasic medium,
as reported earlier, ut replacing lood with 4 μ hemin.40 Fetal
calf serum was added to a final concentration of 5%. In this
colorimetric assay for testing the trypanocidal activity, the
coumarin derivatives were dissolved in DMSO and were added
to 3 × 106 parasites mL-1 at 10 μ final concentrations in P I
1640 culture medium for 24 h at 28 °C.
Reactivity against the hydroxyl radical (OH•)
The spin trapping technique involves short half-life free radicals
with a diamagnetic molecule (spin trap), driving to the formation
of a more stable free radical (spin-adduct) than the original
radical, this new species can be detected by ESR technique.
In this case the OH• was studied and the spin trap DMPO (5,5-
dimethyl-1-pyrroline) was employed. The OH• was generated by
means of photolysis with ultraviolet light. This solution was
prepared with 100 µL of 30% H2O2, 50 µL of DMPO solution
(200 mM) and 50 µL of a 1 mM antioxidant solution. This
solution was placed in a quartz EPR cell and after 2 minutes of
UV-radiation the spectrum was taken.
DMSO final concentration was less than 0.1% v/v. Likewise,
Nifurtimox was added as a positive control. Tetrazolium salt was
added at a final concentration of 0.5 mg mL-1, incubated at 28°C
for 4 h and then solubilized with 10% SDS/0.1 mM HCl and
incubated overnight. After incubation, the number of viable
ESR spectra were recorded in the X band (9.7 GHz) using a
Bruker ECS 106 spectrometer with a rectangular cavity and 50-

parasites was determined by absorbance measures at 570 nm in a
multiwell plate reader (Asys Expert Plus). Untreated parasites
were used as controls (100% of viability). Results are reported as
the percentage of non-viable epimastigotes regarding the control
(parasite in culture medium).
7
8
9
Figueroa-Guiñez, R.; Matos, M. J.; Vazquez-Rodriguez, S.;
Santana, L.; Uriarte, E.; Borges, F.; Olea-Azar, C.; Maya, J. D.
Curr. Top. Med. Chem. 2015, 15, 850.
Pérez-Cruz, F.; Serra, S.; Delogu, G.; Lapier, M.; Maya, J. D.;
Olea-Azar, C.; Santana, L.; Uriarte, E. Bioorg. Med. Chem. Lett.
2012, 22, 5569.
Hamdi, N.; Puerta, M. C.; Valerga, P. Eur. J. Med. Chem. 2008,
43, 2541.
Trypomastigote stage viability study
10 Vazquez-Rodriguez, S.; Figueroa-Guiñez, R.; Matos, M. J.; Olea-
Azar, C.; Maya, J. D.; Uriarte, E.; Santana, L. Med. Chem. 2016,
In press.
11 Iaroshenko, V.O.; Erben, F.; Mkrtchyan, S.; Hakobyan, A.;
Vilches-Herrera, M.; Dudkin, S.; Bunescu, A.; Villinger, A.;
Sosnovskikh, V. Y.; Langer, P. Tetrahedron 2011, 67, 7946.
12 Matos, M.J.; Viña, D.; Picciau, C.; Orallo, F.; Santana, L.; Uriarte,
E. Bioorg. Med. Chem. Lett. 2009, 19, 5053.
13 Beillerot, A.; Domínguez, J. C.; Kirsch, G.; Bagrel, D. Bioorg.
Med. Chem. Lett. 2008, 18, 1102.
14 Lin, H. C.; Tsai, S. H.; Chen, C. S.; Chang, Y. C.; Lee, C. M.; Lai,
Z. Y.; Lin, C. M.; Biochem. Pharmacol. 2008, 75, 1416.
15 Ća ar, .; o ač, F.; aksimo ić, . Food Chem. 2012, 133,
930.
Vero cells were infected with Dm28c trypomastigotes at a 1:3
(cell:parasite) ratio. T. cruzi trypomastigotes were initially
obtained from primary cultures of peritoneal macrophage from
chagasic mice. Vero cells were cultured in 5% fetal bovine serum
supplemented RPMI 1640 medium in humidified air with 5%
CO2 at 37 °C. Vero cell cultures were then infected with
trypomastigotes and incubated at 37 °C in humidified air and 5%
CO2 for 5–7 days. After that time, the culture medium was
collected, centrifuged at 500 × g for
5 min, and the
16 Zhang, H. Y.; Wang, L. F. J. Mol. Struc. THEOCHEM. 2004, 673,
199.
17 Castro, L.; Freeman, B. A. Nutrition 2001, 17, 161.
18 Matos, M. J.; Pérez-Cruz, F.; Vazquez-Rodriguez, S.; Uriarte, E.;
Santana, L.; Borges, F.; Olea-Azar, C. Bioorg. Med. Chem. 2013,
21, 3900.
19 Thuong, P. T.; Hung, T. M.; Ngoc, T. M.; Ha do, T.; Min, B. S.;
Kwack, S. J.; Kang, T. S.; Choi, J. S.; Bae, K. Phytother. Res.
2010, 24, 101.
20 Figueroa, R.; Matos, M.; Vazquez-Rodriguez, S.; Santana, L.;
Uriarte, E.; Olea-Azar, C.; Maya, J. D. Synthesis and evaluation of
antioxidant and trypanocidal properties of a selected series of
coumarin derivatives. Fut. Med. Chem. 2013, 5, 1911.
21 Freitas, R. F.; Prokopczyk, I. M.; Zottis, A.; Oliva, G.;
Andricopulo, A. D.; Trevisan, M. T. S.; Vilegas, W.; Silva, M. G.
V.; Montanari, C. A. Bioorg. Med. Chem. 2009, 17, 2476.
22 de Alcantara, F. C.; Lozano, V. F.; Vale Velosa, A. S.; dos Santos,
M. R. M.; Pereira, R. M. S. Polyhedron 2015, 101, 165.
23 Kabeya, L.; de Marchi, A. A.; Kanashiro, A.; Lopes, N. P.; da
Silva, C. H.; Pupo, M. T.; Lucisano-Valim, Y. M. Bioorg. Med.
Chem. 2007, 15, 1516.
trypomastigote-containing pellet was re-suspended in free-serum
RPMI 1640 and penicillin–streptomycin at a final density of 1 ×
107 parasites/mL. Trypomastigote viability assays were
performed using the MTT reduction method as described
previously. 1 × 107 parasites/ mL were incubated in free-serum
RPMI 1640 culture medium at 37 °C, over 24 h with or without
the studied compounds. An aliquot of the parasite suspension was
extracted and incubated in a 96-well flat-bottom plate and MTT
was added at a final concentration of 0.5 mg/mL using 0.22 mg
mL-1 phenazine metosulfate (as an electron carrier), incubated at
28 °C during 4 h and then made soluble with 10% SDS–0.1 mM
HCl and incubated overnight. Formazan formation was measured
at 570 nm, with a reference wavelength at 690 nm, in a multiwell
plate reader (Asys Expert Plus). Untreated parasites were used as
controls (100% viability). Results are reported as the percentage
of non-viable parasites regarding the control (parasite in culture
medium).
24 imić, A.; anojlo ić, D.; Šegan, D.; odoro ić, . Molecules
2007, 12, 2327.
25 Chevion, S.; Roberts, M. A.; Chevion, M. Free Radic. Biol. Med.
2000, 28, 860.
Acknowledgments
26 Rodríguez, J.; Olea-Azar, C.; Barriga, G.; Folch, C.; Gerpe, A.;
Cerecetto, H.; González, M. Spectrochim. Acta. A Mol. Biomol.
Spectrosc. 2008, 70, 557.
The authors thank University of Santiago de Compostela. M.J.M.
thanks to the Xunta de Galicia for the postdoctoral grant ED481B
2014/086-0. S.V.R. thanks to the Xunta de Galicia for the
postdoctoral grant ED481B 2014/027-0. The authors thank
FONDECYT 1150175 and M.L. thanks to FONDECYT
postdoctoral 3160022.
27 Nasr, B.; Abdellatif, G.; Cañizares, P.; Sáez, C.; Lobato, J.;
Rodrigo, M. A. Environ. Sci. Technol. 2005, 39, 7234.
28 Bubnov, M. P.; Teplova, I. A.; Cherkasov, V. K.; Abakumov, G.
A. Eur. J. Inorg. Chem. 2003, 2003, 2519.
29 Kubow, S.; Janzen, E. G.; Bray, T.M. J. Biol. Chem. 1984, 259,
4447.
30 Le Nest, G.; Caille, O.; Woudstra, M.; Roche, S.; Burlat, B.; Belle,
V.; Guigliarelli, B.; Lexa, D. Inorg. Chim. Acta 2004, 357, 2027.
31 Laurie, V. F.; Zúñiga, M. C.; Carrasco-Sánchez, V.; Santos, L. S.;
Cañete, A.; Olea-Azar, C.; Ugliano, M.; Agosin, E. Food Chem.
2012, 131, 1510.
References and notes
32 Vazquez-Rodriguez, S.; Figueroa-Guiñez, R.; Matos, M. J.;
Santana, L.; Uriarte, E.; Lapier, M.; Maya, J. D.; Olea-Azar, C.
Med. Chem. Commun. 2013, 4, 993.
33 Pérez-Cruz, F.; Vazquez-Rodriguez, S.; Matos, M. J.; Herrera-
Morales, A.; Villamena, F.; Das, A.; Gopalakrishnan, B.; Olea-
Azar, C.; Santana, L.; Uriarte, E. J. Med. Chem. 2013, 56, 6136.
34 Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. J. Nutr. Biochem.
2002, 13, 572.
35 Díaz-Urrutia, C.A.; Olea-Azar, C.; Zapata, G.A.; Lapier, M.;
Mura, F.; Aguilera-Venegas, B.; Arán, V.J.; López-Múñoz, R.A.;
Maya, J.D. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2012, 95,
670.
1
2
3
4
Rassi, A. Jr.; Rassi, A.; Marcondes de Rezende, J. Infect. Dis.
Clin. North Am.2012, 26, 275.
Nunes, M. C.; Dones, W.; Morillo, C. A.; Encina, J. J.; Ribeiro, A.
L. J. Am. Coll. Cardiol. 2013, 62, 767.
Muschietti, L.V.; Sülsen, V. P.; Martino, V. S. Studies in Natural
Products Chemistry, Elsevier, 2013, Chapter 9.
Castro, D.; Boiani, L.; Benitez, D.; Hernández, P.; Merlino, A.;
Gil, C.; Olea-Azar, C.; González, M.; Cerecetto, H.; Porcal, W.
Eur. J. Med. Chem. 2009, 44, 5055.
5 Pagano, M.; Demoro, B.; Toloza, J.; Boiani, L.; González, M.;
Cerecetto, H.; Olea-Azar, C.; Norambuena, E.; Gambino, D.;
Otero, L. Eur. J. Med. Chem. 2009, 44, 4937.
6 Aguilera-Venegas, B.; Olea-Azar, C.; Aran, V. J.; Speisky, H. Fut.
Med. Chem. 2013, 5, 1843.
36 Ća ar, .; o ač, F.; aksimo ić, . Food Chem. 2009, 117,
135.
37 Matos, M. J.; Mura, F.; Vazquez-Rodriguez, S.; Borges, F.;
Santana, L.; Uriarte, E.; Olea-Azar, C. Molecules 2015, 20, 3290.
38 Mosmann, T. Methods 1983, 65, 55.

39 Bisby, R.H.; Brooke, R.; Navaratnam, S. Food Chem. 2008, 108,
1002.
40 Vieites, M.; Otero, L.; Santos, D.; Figueroa-Guiñez, R.;
Norambuena, E.; Olea-Azar, C.; Aguirre, G.; Cerecetto, H.;
González, M.; Morello, A.; Maya, J.D.; Garat, B.; Gambino, D. J.
Inorg. Biochem. 2008, 102, 1033.