Synthesis of coumarin-chalcone hybrids and evaluation of their antioxidant and trypanocidal properties

Article abstract of DOI:10.1039/c3md00025g

Based on the observed biological activities of coumarins and chalcones, we have synthesized coumarin-chalcone hybrids with the aim of evaluating their antioxidant properties and trypanocidal activity against Trypanosoma cruzi, the parasite responsible for

Full text of DOI:10.1039/c3md00025g

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Synthesis of coumarin–chalcone hybrids and evaluation
of their antioxidant and trypanocidal properties†
Cite this: Med. Chem. Commun., 2013,
4, 993
a
b
a
*
´~
~
Saleta Vazquez-Rodriguez, Roberto Figueroa-Guınez, Maria Joao Matos,
Lourdes Santana,a Eugenio Uriarte,a Michel Lapier,b Juan Diego Mayac
b
*
and Claudio Olea-Azar
Based on the observed biological activities of coumarins and chalcones, we have synthesized coumarin–
chalcone hybrids with the aim of evaluating their antioxidant properties and trypanocidal activity
against Trypanosoma cruzi, the parasite responsible for Chagas disease. All derivatives have been proved
to be good antioxidants in spite of their moderate trypanocidal activity in the epimastigote stage (clone
Dm28c). Based on these results, we can conclude that compounds 4 and 5 are potential candidates for
in vitro studies of their antioxidant activity. These preliminary findings encourage us to the future
structural optimization of these kinds of compounds.
Received 18th January 2013
Accepted 22nd April 2013
DOI: 10.1039/c3md00025g
www.rsc.org/medchemcomm
several countries of Latin America and in the southern United
States. The only two available drugs (nifurtimox and benzni-
1 Introduction
Chalcones (1,3-diaryl-2-propen-1-ones) are one of the major
classes of naturally occurring compounds with widespread
distribution in fruits, vegetables, spices, tea and soy based
foodstuff and they have been subject of great interest for their
remarkable pharmacological activities.1,2 Chemically, they are
open chain precursors of avonoids and isoavonoids, in which
the two aromatic rings are linked by a three-carbon a,b-unsat-
urated carbonyl system. Chalcones have been reported to
possess many useful properties, including antibacterial,3,4
antimalarial,5–7 antifungal,8,9 antiviral10,11 and anti-inamma-
tory12–14 properties.
dazole) are limited and suffer from several drawbacks such as
poor efficacy, toxicity, resistance, and multiple side effects.27,28
During the course of T. cruzi infection and disease development,
ROS can be produced as a consequence of tissue destruction
caused by toxic secretions of the parasite, immune-mediated
cytotoxic reactions, and secondary damage in the myocardium.
Therefore, interventions with antioxidant compounds, that
reduce the generation or the effects of ROS, may exert benecial
effects in preventing or arresting oxidative damage.29,30 There-
fore, there is an urgent need for the discovery of new, more
effective, and safer drugs for human use.
On the other hand, coumarins (2H-1-benzopyran-2-ones) are
another family of naturally occurring compounds widely
distributed in plants. These kinds of molecules of both natural
and synthetic origin, have also attracted considerable interest
due to their pharmacological activities,15 for example, their
enzyme inhibitory16,17 antitumor18,19 and antiviral20,21 activities
as well as their anti-inammatory22,23 and antioxidant22,24–26
properties.
Antioxidants are a class of substances presenting a wide
range of different chemical structures. They are able to decrease
or prevent the oxidation of other sensitive molecules through
different mechanisms like chelation of active metal ions, free
radicals scavenging or inhibition of pro-oxidant enzymes.31 Free
radicals and reactive oxygen species (ROS) are atoms, molecules
or ions possessing unpaired electrons, which are highly reactive
with a wide range of other different molecules. They are
constantly generated and eliminated in the biological system
through metabolic processes and play important roles in a
variety of normal biochemical functions, cell signaling
apoptosis, gene expression, ion transport and abnormal path-
ological processes.32,33 The overproduction of ROS can have
damaging effects on many molecules. For example, ROS
producing oxidative stress may contribute to Chagas disease
progression.34
Chagas disease is caused due to the Trypanosoma cruzi
(T. cruzi) parasite and it represents a major health problem in
a
´
´
Departamento de Quımica Organica, Facultad de Farmacia, Universidad de Santiago
de Compostela, Campus Vida s/n, 15782, Santiago de Compostela, Spain. E-mail:
svre77@gmail.com
b
´
´
´
´
Departamento de Quımica Inorganica y Analıtica, Facultad de Ciencias Quımicas y
´
Farmaceuticas, Universidad de Chile, Casilla 233, Santiago, Chile. E-mail: colea@
uchile.cl
It is also known that the antioxidant properties of chalcones
are quite dependent on the two aryl structures,25,35 that is, the
substituents on the two aryl rings of the chalcone moiety and
their substitution patterns. Especially, the hydroxyl substituent
cDepartamento de Farmacologıa Molecular
Universidad de Chile, Santiago, Chile
y
´
´
Clınica, Facultad de Medicina,
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00025g
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reaction treating the appropriate salicylaldehyde and ethyl 3,4-
dimethoxybenzoylacetate with piperidine in ethanol at reux
for 2–6 h. The resulting methoxy derivatives were treated with
boron tribromide in DCM at 80 ꢀC in a Schlenk tube for 48 h to
give the corresponding hydroxy derivatives 1–5 with 69–94%
yield.
3 Electrochemical study
In order to evaluate the antioxidant behavior of the ve
compounds of this family, cyclic voltammetry, the Oxygen
Radical Absorbance Capacity (ORAC FL-PGR) and the Electron
Spin Resonance (ESR) studies were carried out.
Fig. 1 Structural approach employed for the design of new coumarin–chalcone
hybrid compounds.
is one of the key groups that enhance the antioxidant activity of
chalcone mainly due to its easy conversion to phenoxy radicals
through the hydrogen atom transfer (HAT) mechanism. This
phenoxy radical formation may be essential for the antioxidant
properties, which are assessed primarily as radical scavenging
potential of phenolic chalcones. In fact, the hydroxyl substit-
uent is widespread among chalcones from natural sources.
Due to the potential antioxidant property and trypanocidal
activity of the chalcone and coumarin moieties,36,37 in the
present work a series of coumarin–chalcone hybrids (Fig. 1)
were synthesized and their antioxidant capacity and trypanoci-
dal activity were evaluated. The chalcone moiety in the prepared
molecules is included in the coumarin scaffold so that the
geometry of the chalcone remains as a trans isomer due to the
double bond position of the pyrone ring.
3.1 Cyclic voltammetry
The electrochemical properties of hydroxyl coumarin–chalcone
derivatives were studied by cyclic voltammetry. Fig. 2 shows a
cyclic voltammogram at a glassy carbon electrode (GCE)
immersed in DMSO/75 mmol Lꢁ1 phosphate (pH 7.4) buffer
30/70 media containing 1 mmol Lꢁ1 of compound 3 for several
scan rates (v). Under these experimental conditions, two
oxidation processes (peaks I and II) were observed. In general, it
has been proposed that the charge transfer process at peak I
corresponds to the oxidation of the catechol substituent, while
the process at peak II comprises oxidation reaction involving
the hydroxyl groups present in the coumarinic ring.
Results of the cyclic voltammetry, reported in Table 1 show
that coumarin–chalcone hybrids present low oxidation
potentials.
2 Chemistry
In order to achieve the nal products, the synthesis of the
hydroxy-3-benzoylcoumarins was carried out in two steps,
shown in Scheme 1 and briey described as follows: (a)
synthesis of methoxy-3-benzoylcoumarins and (b) hydrolysis of
the previous compounds to afford the nal hydroxyl-3-ben-
zoylcoumarins (1–5).
The methoxy-3-benzoylcoumarins, with a general structure I
were efficiently synthesized with 78–94% yield by a Knoevenagel
Fig. 2 Cyclic voltammogram for 1 mmol Lꢁ1 compound 3 in DMSO/75 mmol Lꢁ1
phosphate (pH 7.4) buffer 30/70 media at a GCE for v ¼ 100, 250, 500, and
2000 mV sꢁ1
.
Table 1 Oxidation potential peaks obtained by cyclic voltammetry
Compounds
Epaa (mV)
1
2
3
4
5
322
449
312
337
247
Scheme 1 Preparation of coumarin–chalcone hybrids. Reagents and conditions:
(a) piperidine, EtOH, reflux, 2–6 h and (b) BBr3, DCM, 80 ꢀC, 48 h.
a
First oxidation peak potential at a scan rate of 2000 mV sꢁ1
.
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Table 2 ORAC-FL and ORAC-PGR values and the scavenging hydroxyl radical (%)
Compound
ORAC-FL
ORAC-PGR
% Scavenging
a
a
a
1
2
3
4
4.37 ꢂ 0.22
4.95 ꢂ 0.14
6.02 ꢂ 0.12
8.32 ꢂ 0.27
8.51 ꢂ 0.32
7.28 ꢂ 0.22c
6.76 ꢂ 0.22c
3.3d
84.6 ꢂ 5.3
82.1 ꢂ 7.1
84.8 ꢂ 7.3
84.2 ꢂ 6.4
90.9 ꢂ 8.2
—
0.52 ꢂ 0.05
5
1.17 ꢂ 0.10
Quercetin
Catechin
—
—
—
DHMcoumarinb
Helichrysetin
4.4e
a
b
c
Inactive compound.
6,7-Dihydroxy-4-methylcoumarin.
Data
d
e
collected from ref. 39. Data collected from ref. 49. Data collected
from ref. 50.
5 in phosphate pH 7.4. Therefore, ORAC-FL values would be
more inuenced by the stoichiometry of the reaction (dened as
the number of radicals that each additive molecule can remove)
than by the reactivity of the additives. Fig. 3b shows that the
area under the curve (AUCNET) of the kinetic proles for
compound 5 was linearly related to the concentration of the
additive.
This behavior was observed for all compounds studied.
However, the consumption of PGR by reactive antioxidants is
inhibited without generating induction times.41,42 This charac-
teristic prole is observed for all the studied compounds, as well
as for Trolox (Fig. 4).
Fig. 3 (a) Kinetic profile of fluorescein consumption, AAPH mediated, in the
presence of compound 5. F0 is the fluorescence in the absence of the compound
and F the fluorescence in the presence of compound 5 and (b) graph of AUCNET
(net area under the curve) versus concentration of compound 5.
3.2 ORAC assays
The absence of induction times in the protective kinetic
proles of PGR would imply that the ORAC-PGR index is more
related to the reactivity of the antioxidants than to stoichio-
metric factors. Taking into account the characteristics of the
ORAC-FL and ORAC-PGR assays, it has been recently proposed
that both methods could be considered as rendering comple-
mentary indexes.42 Results of this study are expressed as ORAC-
FL and ORAC-PGR values and are tabulated in Table 2.
All experiments were carried out in triplicate, and the data
represent the mean values (ꢂS.D.).
The capacity of scavenging peroxyl radicals was studied
employing the ORAC method. In this assay, Trolox and 2,20-azo-
bis(2-amidinopropane) dihydrochloride (AAPH) were used as a
reference antioxidant and a peroxyl radical source, respec-
tively.38 This assay evaluates the capacity of antioxidants (or of
their complex mixtures) to inhibit the bleaching of a target
molecule (probe) induced by peroxyl radicals.
The oxidation occurs due to exposition of the target mole-
cule, uorescein (FL)39 or pyrogallol red (PGR),40 to the peroxyl
radical, leading to decay of uorescence emission or decrease of
PGR absorbance with time.
The consumption of FL is commonly inhibited, even by
antioxidants of low reactivity, throughout kinetic proles
characterized by the presence of induction times. Fig. 3a shows
the results of AAPH mediated uorescein oxidation in the
absence and presence of increasing concentration of compound
3.3 ESR assays
In order to study the antioxidant reactivity of all the hydroxyl-
ated coumarin–chalcone hybrids, we have adopted
a
Fig. 4 Time-course of the consumption of PGR, AAPH mediated, in the presence
Fig. 5 ESR spectra for adduct DMPO–OH without an antioxidant molecule
(black) and in the presence of compound 5 (red).
of increasing concentration of Trolox.
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Table 4 Theoretical structural properties of the coumarin–chalcone hybridsa
non-catalytic and competitive Fenton system.23 Electron spin
resonance (ESR) in combination with spin trapping techniques
was employed to further verify that the tested compounds
possess the ability to scavenge hydroxyl radicals, measuring
thus, the percentage of scavenging of the compounds,
summarized in Table 2. DMPO (5,5-dimethyl-1-pyrroline-N-
oxide) reacts with hydroxyl radicals to generate the spin reso-
nance signal, and ESR can quantify this spin resonance signal.
The ESR spectrum shows four hyperne lines due to the DMPO–
OH adduct formation. The coumarin–chalcone hybrids
compete with DMPO for hydroxyl radicals. Thus, the ESR signal
will diminish as presented in Fig. 5 for compound 5.
n-OH
acceptors
n-OHNH
donors
2
3
˚
˚
Compd
log P
TPSA (A )
Volume (A )
1
2
3
4
5
2.43
2.85
1.93
1.63
2.92
87.74
87.74
107.97
128.20
107.97
5
5
6
7
6
2
2
3
4
3
235.01
251.58
243.03
251.05
260.92
a
TPSA, topological polar surface area; n-OH, number of hydrogen
acceptors and n-OHNH, number of hydrogen bond donors. The data
were determined with Molinspiration calculation soware.
4 Pharmacology
4.1 Trypanocidal activity
6 Results and discussion
Taking into account the importance of the hydroxyl substitu-
ents in the antioxidant properties,26,45 we have focused the study
on hydroxylated derivatives of the coumarin–chalcone hybrid
compounds, which contain a catechol fragment in the benzoyl
ring at the 3 position of the coumarin.
Studies on trypanocidal activitywere carried out in order to
investigate a possible relationship between the antioxidant
capacity of the compounds and their antiparasitic activity.
Trypanocidal activity was evaluated against the T. cruzi epi-
mastigote stage (clone Dm28c). It was measured through
the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay,43 widely used for in vitro measurement of the
metabolic activity or viability of cell cultures. Aer incubation
time, we determined the number of viable parasites by
measuring absorbance at 570 nm in a multiwell reader (Asys
Expert Plusª, Austria). Untreated parasites were used as
controls (100% of viability). Results are reported as the
percentage of non-viable epimastigotes regarding the control.
Results of two different concentrations of the synthesized
compounds (10 and 100 mmol Lꢁ1) are reported in Table 3.
According to the data obtained in the cyclic voltammetry, all
derivatives exhibited two anodic peaks without the corre-
sponding cathodic peak, indicating an irreversible oxidation
process as a result of the oxidation of one hydroxyl group in any
of the aromatic rings, forming the semiquinone radical. We
have thus rstly investigated the inuence of the scan rate (v) in
order to access in more detail the characteristics of electro-
chemical and chemical reactions that take place at the elec-
trode/solution interface. The current ratio Ipc/Ipa progressively
increases as a function of v, corroborating an EC-type mecha-
nism46 associated with peak I. It is known that the antioxidant
capacity is conceivably related to the electrochemical behavior,
being indicative that a low oxidation potential corresponds to a
high antioxidant power.47 In our case, this fact could be
explained as mainly due to the strong electron donating effect of
the catechol group, present in all the synthesized derivatives,
which can form stable radicals in which the remaining o-OH
group stabilizes the radical as a result of the intramolecular
hydrogen bonding.48
Considering the ORAC-FL assays, we observed that the
highest ORAC-FL values were found for compounds 4 and 5
(8.32 and 8.51 respectively). The results obtained are better than
for quercetin (7.28) and catechin (6.76), avonoids currently
used as reference compounds,39 as well as other simple
coumarins, such as the 6,7-dihydroxy-4-methylcoumarin
(DHMcoumarin) studied by our group49 or chalcones collected
from bibliographic sources such as helichrystein.50 It is also
worth mentioning the activity of compound 3 (6.02), compa-
rable to catechin.
5 Theoretical evaluation of ADME
properties
To better correlate the drug-like properties of the coumarin–
chalcone hybrid compounds the lipophilicity, expressed as the
octanol/water partition coefficient and herein called log P, as
well as other theoretical calculations such as the TPSA, the
number of hydrogen acceptors and the number of hydrogen
bond donors were calculated using the Molinspiration property
program. The ADME properties of the studied compounds were
calculated using the Molinspiration property program, and
results are summarized in Table 4.44
Table 3 Trypanocidal activity results for the coumarin–chalcone hybrids and
nifurtimox (Nfx)
% Trypanocidal
% Trypanocidal
Cmpd
activity at 10 mmol Lꢁ1
activity at 100 mmol Lꢁ1
The different ORAC-FL values can be related to the substit-
uents existing in the coumarin moiety and their pattern of
substitution. In addition, the presence of a benzoyl substituent
improves the electron delocalization with respect to the
coumarin skeleton, inuencing ORAC values.
Compound 1 is the structurally simplest compound, pre-
senting a catechol group in the benzoyl ring, with no substitu-
ents on the coumarin skeleton. This feature could explain why
1
2
3
4
23.8 ꢂ 0.4
27.9 ꢂ 0.3
38.7 ꢂ 2.1
47.9 ꢂ 1.0
18.9 ꢂ 0.4
46.4 ꢂ 0.8
a
a
5
Nfx
9.5 ꢂ 0.2
24.1 ꢂ 0.7
100 ꢂ 3.2
52.5 ꢂ 2.2
a
Inactive at concentration tested.
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compound 1 presents the lowest ORAC value (4.37) of all the NMR (300 MHz) and 13C NMR (75.4 MHz) spectra were recorded
studied compounds, but still being 4 times more active than the with a Bruker AMX spectrometer using DMSO-d6 or CDCl3 as
employed reference compound Trolox. If we introduce a methyl solvent. Chemical shis (d) are expressed in parts per million
group at position 6 (compound 2), we observe a slight increase (ppm) using TMS as an internal standard. Coupling constants
in the ORAC-FL index (4.95) compared to compound 1. When J are expressed in hertz (Hz). Spin multiplicities are given as s
analyzed the obtained ORAC-FL values of compounds 1–3 based (singlet), d (doublet), dd (doublet of doublets) and m (multiplet).
on the chemical structure and pattern of substitution, we Mass spectrometry was carried out with a Kratos MS-50 or
observed that, among other features, the higher the electron a Varian MAT-711 spectrometer. Elemental analyses were
donating effect of the substituent, the more active the performed by a Perkin-Elmer 240B microanalyzer and were
compound. Compound 3 containing a hydroxyl at position 6, within ꢂ0.4% of calculated values in all cases. The analytical
increases its ORAC value to 6.02 compared to the simplest results were $95% purity for all compounds. Flash Chromatog-
synthesized compound 1.
raphy (FC) was performed on silica gel (Merck 60, 230–400 mesh)
Compounds with the highest activities (3–5), with ORAC and analytical TLC was performed on precoated silica gel plates
values 6.02, 8.51 and 8.32 respectively, all present, in addition to (Merck 60 F254). Organic solutions were dried over anhydrous
the catechol group in the benzoyl ring, at least one hydroxyl sodium sulfate. Concentration and evaporation of the solvent
group that increases the electronic density, favoring the aer reaction or extraction was carried out on a rotary evaporator
¨
hydrogen atom transfer (HAT) mechanism. Compound 4, with (Buchi Rotavapor) operating at reduced pressure.
two hydroxyl groups in the coumarin moiety, showed a
considerable increase in the ORAC-FL value (8.32) compared to 7.2 Synthesis
compound 3, in which only one hydroxyl substituent is present
in the coumarin ring. In any case, the highest ORAC-FL value
was found for compound 5, in which one hydroxyl group is
7.2.1 General procedure for the synthesis of hydroxy-3-
benzoylcoumarins (1–5). To a solution of the appropriate
b-ketoester (1 mmol) and the corresponding salicylaldehyde
present at position 8 and a bromide atom is present at position
(1 mmol) in ethanol (5 mL) was added piperidine in a catalytic
6 of the coumarin. Therefore, it has been found that the pres-
amount. The reaction mixture was reuxed for 2–6 h and aer
completion (followed by TLC), the reaction mixture was cooled
ence, the number and the position of substituents with
different electronic effects on the coumarin skeleton are
and the precipitate was ltered and washed with cold ethanol
important structural factors that contribute to increase the
and ether to afford the corresponding methoxy-3-benzoylcou-
antioxidant capacity of these coumarin–chalcone hybrids.
marin compounds with a general structure I. The appropriate
Regarding the ESR assays, whose results are reported in
methoxy derivative compound (1 mmol) was dissolved in DCM
Table 2 we observed accuracy with the obtained ORAC values
(1 mL) and BBr3 in DCM (20 mmol, 1 M) was added in a Schlenk
and the radical scavenging activity, as high as 80% for all tested
tube. The tube was sealed, and the reaction mixture was heated
compounds.
at 80 ꢀC for 48 h. The resulting crude product was treated with
On the other hand, results reported in Table 3 have shown
MeOH and rotated to dryness. The obtained precipitate was
that all compounds present moderate trypanocidal activity with
recrystallized in MeOH or puried by ash chromatography
exception of compound 4 that resulted to be inactive at the
using hexane–ethyl acetate mixtures as an eluent, to afford the
concentration tested. However, these compounds have less
desired hydroxy derivatives (Scheme 1).
activity than the positive control nifurtimox, the drug currently
used against T. cruzi in the treatment of Chagas disease.
From the data obtained for the theoretical evaluation of the
ADME properties, one can notice that all the hybrid compounds
1–5 do not break any point of the Lipinski's rule of ve, making
them promising leads for drug candidates.51 Topological polar
7.3 Cyclic voltammetry
Cyclic voltammetry was carried out using a potentiostat/galva-
nostat VersaSTAT 3 provided with a V3-Studio electrochemistry
soware package, in 50 mM sodium phosphate buffer (pH 7.4),
at room temperature, using a three electrode cell, a glassy
surface area (TPSA) and log P values are compatible with those
carbon electrode was used as a working electrode, a platinum
described as a predictive indicator of the drug capacity of
wire as an auxiliary electrode and Ag, AgCl/KCl (ca. 3.5 M) as a
reference electrode. All coumarin–chalcone hybrids were
studied to 1 mM in 20% methanol as nal concentration.
membrane penetration.52
It is noteworthy that compounds with the higher trypanoci-
dal activities were those with lower ORAC values. These data can
be taken into account for future studies on the mechanism of
action of this kind of drug against T. cruzi.
7.4 Determination of ORAC
The ORAC analyses were carried out on a Synergy HT multi
detection microplate reader, from Bio-Tek Instruments, Inc.
(Winooski, USA), using white polystyrene 96-well plates,
purchased from Nunc (Denmark). The consumption of the
7 Experimental
7.1 General methods
Starting materials and reagents were obtained from commercial probe molecules, FL or PGR, associated with its incubation in
suppliers and were used without purication. Melting points the presence of AAPH, was estimated from uorescence (F) and
(mp) are uncorrected and were determined with a Reichert Koer absorbance (A) measurements, respectively. PGR consumption
1
thermopan or in capillary tubes in a Buchi 510 apparatus. H was evaluated from its absorption intensity (A) decrease at
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540 nm, while FL consumption was evaluated from its decrease incubated at 28 ꢀC for 4 h and then solubilized with sodium
in the uorescence intensity (F, excitation: 485/20 nm; emis- dodecyl sulfate (10%) in HCl (0.1 mmol Lꢁ1) and incubated
sion: 528/20 nm). The plate reader was controlled by Gen 5 overnight. Aer incubation time, the number of viable parasites
soware. The reaction was carried out in 75 mM sodium was determined by measuring the absorbance at 570 nm in a
phosphate buffer (pH 7.4), and a nal volume of 200 mL. FL multiwell reader (Asys Expert Plusª, Austria). Untreated parasites
(40 nM, nal concentration) or PGR (70 mM, nal concentration) were used as controls (100% of viability). Results are reported as
and coumarin–chalcone hybrid solutions in methanol with a the percentage of non-viable epimastigotes regarding the control.
range of concentrations between 0.3 mM and 2 mM were placed Statistical analysis was performed using GraphPad Prism 5 so-
in each well of 96-well plate. The mixture was preincubated for ware (GraphPad Soware, Inc. San Diego, CA). The data are
15 min at 37 ꢀC, before rapidly adding the AAPH solution expressed as mean ꢂ S.D. The experimental data were analyzed
(18 mM and 0.1 M for FL and PGR nal concentration, by one-way analysis of variance (ANOVA), and differences
respectively). The microplate was immediately placed in the between groups were assessed using the Tukey's post-test. The
reader and automatically shaken prior to each reading. The level of signicance was set at p < 0.05, and all experiments were
uorescence and absorbance were recorded every 1 min for 120 replicated three times.
min. A blank with FL or PGR and AAPH using methanol instead
of the antioxidant solution and ve calibration solutions using
Trolox (0.5 mM to 2.5 mM for Fl and 50 mM to 250 mM for PGR) as
7.7 Theoretical evaluation of ADME properties
The ADME properties of the studied compounds were calcu-
lated using the Molinspiration property program.44 log P is
calculated by the methodology developed by Molinspiration as a
sum of fragment-based contributions and correction factors.
Topological Polar Surface Area (TPSA) is calculated based on the
methodology published by Ertl et al. as a sum of fragment
contributions.52 Oxygen and nitrogen centered polar fragments
are considered. PSA has been shown to be a very good descriptor
characterizing drug absorption, including intestinal absorp-
tion, bioavailability, Caco-2 permeability and blood–brain
barrier penetration. The method for calculation of molecular
volume developed by Molinspiration is based on group contri-
butions. These have been obtained by tting the sum of frag-
ment contributions to “real” 3D volume for a training set of
about twelve thousand, mostly drug-like molecules. 3D molec-
ular geometries for a training set were fully optimized by the
semiempirical AM1 method.
antioxidant were also used in each assay. The inhibition
capacity was expressed as ORAC-FL and ORAC-PGR values, and
is quantied by integration of the area under the curve (AUC-
NET). All reaction mixtures were prepared in triplicate and at
least three independent assays were performed for each sample.
The area under the uorescence decay curve (AUC) was calcu-
lated integrating the decay of the uorescence where F0 is the
initial uorescence read at 0 min and F is the uorescence read
at time. The net AUC corresponding to the sample was calcu-
lated by subtracting the AUC corresponding to the blank. The
ORAC-PGR values were calculated like we indicated previously.
Data processing was performed using Origin Pro 8 SR2 (Origin
Lab Corporation, USA).
7.5 ESR spectroscopy
To a solution of 100 mL of NaOH (50 mL, 4 mM), 5,5-dimethyl-1-
pyrroline-N-oxide (DMPO) (50 mL, 30 mM) and hydrogen
peroxide (50 mL, 30%) was added the antioxidant compound
(50 mL, 3 mM in DMF). The resulting mixture was placed in an
EPR cell, at room temperature (25 ꢀC) and the spectrum was
recorded aer ve minutes of reaction in the X band (9.68 GHz)
using a Bruker ECS 106 spectrometer with a rectangular cavity
and 50 kHz eld modulation.
8 Conclusions
In conclusion, we have conrmed the good antioxidant activity
of the hydroxyl coumarin–chalcone hybrids 1–5, two of them
(compounds 4 and 5) presenting better ORAC-FL values than
the reference compounds quercetin and catechin. The use of a
catechol group in the benzoyl ring and the presence of hydroxyl
and bromine groups in the coumarin moiety make a big
7.6 Determination of trypanocidal activity
Trypanocidal activity was evaluated against the T. cruzi epi- improvement in the antioxidant activity, compound 5 being the
mastigote stage (clone Dm28c) and it was measured through the most active of the series under study. The results of the anti-
MTT assay,43 using 0.22 mg mLꢁ1 phenazine metosulfate (as oxidant assay using ESR showed signicant reactivity against
electron carrier). The T. cruzi epimastigotes Dm28c strain, from the hydroxyl radical for all tested compounds. A very interesting
´
´
our own collection (Programa de Farmacologıa Molecular y Clınica, nding is that compounds 4 and 5 resulted to be very reactive
Facultad de Medicina, Universidad de Chile) were grown at 28 ꢀC in and present good antioxidant capacity and scavenging
the Diamond's monophasic medium as reported earlier53 but percentage against hydroxyl and peroxyl radicals, as well as low
replacing blood by 4 mM hemin and adding fetal calf serum (5%). oxidation potentials. Based on these results, we can conclude
The coumarin–chalcone hybrids (10 and 100 mM in DMSO) were that compounds 4 and 5 are potential candidates for deeper
added to 3 ꢃ 106 parasites per mL in the RPMI 1640 culture studies of antioxidant activity. On the other hand, it is worth
ꢀ
medium (5% bovine fetal serum) for 24 h at 28 C. The DMSO mentioning that molecules with higher trypanocidal activities
nal concentration was less than 0.1% v/v. The same procedure showed a lower ORAC-FL index, which can be a key in the
was followed with nifurtimox as the positive control. The tetra- understanding of the mechanism of action of the compounds
zolium salt was added at a nal concentration of 0.5 mg mLꢁ1
,
on the T. cruzi parasite.
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Acknowledgements
This work was partially supported by the Ministerio de Sanidad
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1090078) and Anillo ACT112 (Chile). SVR thanks Ministerio de
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Educacion y Ciencia for her PhD grant FPU (AP2008-04263).
RFG gratefully acknowledges CONICYT-Chile for his PhD grant
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no. 24121574. MJM thanks Fundaçao para a Ciencia e Tecno-
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