Synthesis and electrochemical and biological studies of novel coumarin-chalcone hybrid compounds

Article abstract of DOI:10.1021/jm400546y

A series of novel hydroxy-coumarin-chalcone hybrid compounds 2a-i has been synthesized by employing a simple and efficient methodology. An electrochemical characterization using cyclic voltammetry and ESR spectroscopy were carried out to characterize the

Full text of DOI:10.1021/jm400546y

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Article
Synthesis, electrochemical and biological studies
on novel coumarin-chalcone hybrid compounds
Fernanda Pérez-Cruz, Saleta Vazquez-Rodriguez, Maria João Matos, Alejandra Herrera-Morales, Frederick
A. Villamena, Amlan Das, bhavani gopalakrishnan, Claudio Olea-Azar, Lourdes Santana, and Eugenio Uriarte
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400546y • Publication Date (Web): 16 Jul 2013
Downloaded from http://pubs.acs.org on July 20, 2013
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Synthesis, electrochemical and biological
studies on novel coumarinꢀchalcone hybrid
compounds
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Fernanda PérezꢀCruz,a Saleta VazquezꢀRodriguez,b,* Maria João Matos,b Alejandra
HerreraꢀMorales,a Frederick A.Villamena,c Amlan Das,c Bhavani Gopalakrishnan,c
Claudio OleaꢀAzar,a,* Lourdes Santanab and Eugenio Uriarteb
aFree Radical and Antioxidants Laboratory, Inorganic and Analytical Department, Faculty
of Chemical and Pharmaceutical Sciences, University of Chile, Sergio Livingstone
Polhammer 1007, Independencia , Santiago, Chile.
bDepartment of Organic Chemistry, Faculty of Pharmacy, University of Santiago de
Compostela, Campus Vida s/n, 15782, Santiago de Compostela, Spain.
cDepartment of Pharmacology and Davis Heart and Lung Research Institute, College of
Medicine, The Ohio State University, Columbus, Ohio 43210, United States.
EMAIL:
ferperezc@ug.uchile.cl svre77@gmail.com
Frederick.Villamena@osumc.edu
mariacmatos@gmail.com
amlan.das@gmail.com
lourdes.santana@usc.es
aherrera0805@gmail.com
Bhavani.Gopalakrishnan@osumc.edu
eugenio.uriarte@usc.es
colea@uchile.cl
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Key words: coumarin, chalcone, Knoevenagel reaction, electrochemical study, antioxidant
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activity, cytotoxicity, cytoprotection, ADME.
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Abstract
A series of novel hydroxyꢀcoumarinꢀchalcone hybrid compounds 2aꢀi has been synthesized
employing a simple and efficient methodology. An electrochemical characterization using
cyclic voltammetry and ESR spectroscopy were carried out in order to characterize the
oxidation mechanism for the target compounds. The antioxidant capacity and reactivity
were determined by ORAC and ESR assays, respectively. Biological assays were assessed
in order to evaluate the cytotoxicity and cytoprotection capacity against ROS/RNS on
BAEC. The results revealed that all tested compounds present ORAC values much higher
than other wellꢀknown antioxidant compounds such as quercetin and catechin. Compound
2e showed the highest ORAC value (14.1) and also presented low oxidation potential, good
scavenging capacity against hydroxyl radicals, low cytotoxicity and high cytoprotective
activity.
Introduction
Free radicals and reactive oxygen and nitrogen species (ROS and RNS) are atoms,
molecules or ions possessing at least one unpaired electron and therefore, highly reactive
with a wide range of other different molecules. They are constantly generated and
maintained in balance on biological systems through metabolic processes and play
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important roles in a variety of normal biochemical functions, such as cell signaling
apoptosis, gene expression, ion transport and pathological processes.1,2
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It is accepted that ROS/RNS play different roles in vivo. Some are positive and are related
to their involvement in energy production, phagocytosis, regulation of cell growth and
intercellular signaling, and synthesis of biologically important compounds.3 However, the
overproduction of ROS/RNS can have damaging effects on many molecules. ROS/RNS are
capable of oxidizing cellular proteins, nucleic acids and lipids, since they are very small
and highly reactive. For example, lipid peroxidation is a free radicalꢀmediated propagation
of oxidative damage to polyunsaturated fatty acids involving several types of free radicals,
and termination occurs through enzymatic means or by free radical scavenging by
antioxidants.4,5 Therefore, the excess of ROS/RNS causes a disturbance in the prooxidantꢀ
antioxidant balance in favor of the former, leading to potential damage, known as oxidative
stress.1,6,7 Oxidative stress considerably contributes to the pathogenesis of inflammatory
and cardiovascular diseases, cancer, diabetes, Alzheimer’s disease, autism or aging among
others.8ꢀ13
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The consumption of natural antioxidants, such as flavonoids, supports the endogenous
antioxidant defense system. Both dietary and enzymatic antioxidants are components of
interrelated systems that interact with each other to control ROS production, thereby
ensuring adequate defenses against oxidative stress.14,15
In this sense, many studies concerning antioxidant capacity of phenolic compounds of
different types (e.g. flavonoids, coumarins or tanins) have been established and their
physicoꢀchemical properties have been correlated with their antioxidant properties.16 In
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general, free radical scavenging, redox potential and the antioxidant activity of these classes
of compounds mainly depends on the number and position of hydrogenꢀdonating hydroxyl
groups on the aromatic ring of the phenolic molecules, and is also affected by other
factors.17 Therefore, low oxidation potential, number of present hydroxyl groups, existence
of electronꢀdonating groups and/or stabilization of a secondary radical via delocalization
through conjugated systems play a key role in the antioxidant activity.18ꢀ21
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Most of the beneficial health effects of flavonoids are attributed to their antioxidant and
chelating abilities. Due to their ability to inhibit lowꢀdensity lipoprotein (LDL) oxidation,
flavonoids have demonstrated unique cardioprotective effects.22,23 The protective effects of
flavonoids in biological systems are attributed to their capacity for transfer electrons to
radicals, chelate metal catalysts,24 activate antioxidant enzymes,25 reduce alphaꢀtocopherol
radicals,26 and inhibit oxidases.27
Chalcones (αꢀphenylꢀβꢀbenzoylethylene) are prominent secondary metabolites precursors
of flavonoids and isoflavonoids in plants. Chemically, they consist of two aromatic rings
linked by a threeꢀcarbon α,βꢀ unsaturated system that are known to exhibit an impressive
array of biological properties.28ꢀ30 Chalcones attract much research attention because of
their pharmaceutical potential due to the radicalꢀscavenging properties,31,32 antitumour,33,34
antiꢀinflammatory35 or neuroprotective properties.36 Thus, it is reasonable to explore,
among others, the radicalꢀscavenging properties of hydroxyl derivatives of chalcones.
On the other hand, coumarins (2Hꢀchromenꢀ2ꢀone) are a large family of compounds, of
both natural and synthetic origin, important because of the pharmacological activities that
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these kind of compounds display,37 such as antimicrobial,38 monoamine oxidase (MAO)
inhibitors,39ꢀ41 antitumor,42 adenosine receptors43 or antioxidant17 among others.
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Coumarin and chalcones represent, therefore, promising scaffolds in the medicinal
chemistry realm. Taking into account the previously mentioned features,18,19 and with the
aim of finding new chemical structures with antioxidant activity, we have design a new
scaffold in which the chalcone fragment is partially incorporated in the coumarin moiety.
The resulting coumarinꢀchalcone hybrid fixes the transꢀconformation of the chalcone
scaffold by sharing the double bond of the pyrane ring of the coumarin nucleus (Scheme 1).
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O
O
O
coumarin
chalcone
ring
closure
O
O
O
coumarin-chalcone hybrid
Scheme 1. Structural rational design of coumarinꢀchalcone hybrid compounds
Chemistry
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The coumarin derivatives 2aꢀi were efficiently synthesized in two steps40 briefly described
as follows: i) synthesis of methoxyꢀ3ꢀbenzoylcoumarins 1aꢀi and ii) synthesis of the
hydroxyꢀ3ꢀbenzoylcoumarins (2aꢀi). The above steps are outlined in Scheme 2.
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Based on the widely used Knoevenagel condensation reaction for the preparation of
coumarin derivatives,37,44 we used an efficient oneꢀstep synthesis to generate the methoxyꢀ
3ꢀbenzoylcoumarin precursors 1aꢀi. These compounds were prepared in good yields (74ꢀ
98%) using the appropriately substituted salicylaldehyde and the corresponding βꢀketoester
in presence of piperidine in ethanol at reflux for 2ꢀ5 hours, obtaining the desired compound
as a precipitate that was separated by filtration and further purified by recrystallization in
MeOH/DCM. The final compounds 2aꢀi were then synthesized by hydrolysis of the
corresponding methoxy precursors 1aꢀi employing an excess of a Lewis acid, BBr3, in
DCM at 80 ºC in a Schlenk tube for 48 hours followed by treatment of MeOH and
purification by flash chromatography of the crude product (69ꢀ98%).
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O
O
R3
CHO
OH
EtO
R2
+
R4
R1
R3 = H, Br, OMe
R4 = H, OMe, Me
R1 = H, OMe
R2 = H, OMe
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a)
O
O
R3
O
R2
R4
R1
1a R1 = R3 = R4 = H; R2 = OMe
1b R1 = R2 = R4 = H; R3 = OMe
1c R1 = R4 = H; R2 = OMe; R3 = Br
1d R1 = R3 = H; R2 = OMe R4= Me
1e R1 = R4 = H; R2 = R3 = OMe
1f R1 = R2 = H; R3 = Br; R4 =OMe
1g R1 = R2 = OMe; R3 = Br; R4 = H
1h R1 = R2 = R4 = OMe ; R3 = H
1i R1 = R2 = R4 = OMe ; R3 = Br
2a R1 = R3 = R4 = H; R2 = OH
2b R1 = R2 = R4 = H; R3 = OH
2c R1 = R4 = H; R2 = OH; R3 = Br
2d R1 = R3 = H; R2 = OH R4= Me
2e R1 = R4 = H; R2 = R3 = OH
2f R1 = R2 = H; R3 = Br; R4 =OH
2g R1 = R2 = OH; R3 = Br; R4 = H
2h R1 = R2 = R4 = OH ; R3 = H
2i R1 = R2 = R4 = OH ; R3 = Br
b)
Scheme 2. Synthesis of methoxylated (1aꢀi) and hydroxylated (2aꢀi) 3ꢀbenzoylcoumarin
derivatives. Reagents and conditions: a) piperidine, EtOH, reflux, 2ꢀ5 h; b) BBr3, DCM, 80
ºC, 48 h.
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Electrochemical Study
Cyclic Voltammetry
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The electrochemical properties of hydroxyꢀcoumarinꢀchalcone derivatives (2aꢀi) were
studied by cyclic voltammetry, employing an electrochemical cell with three electrodes and
different scan rates between 0.050ꢀ0.75 V*sꢀ1. Under these conditions, all derivatives
exhibited one (compounds 2aꢀg) or two (compounds 2h and 2i) well anodic peaks without
the corresponding cathodic peak. The anodic peak potentials (Epa I and Epa II) obtained at
a 0.75 V* sꢀ1 scan rate are listed in Table 1.
2f
2g
2h
2i
2a
2b
2c
2d
2e
110 ꢀA
120 ꢀA
scan
1.4
Scan
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0.2
0.4
0.6
0.8
1.0
1.2
E vs Ag, AgCl / V
E vs Ag, AgCl / V
Figure 1. Cyclic voltammograms of the hydroxyꢀcoumarinꢀchalcone derivatives 2aꢀi in
phosphate buffer pH 7.4 at 0.75 V*sꢀ1.
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Table 1. Oxidation potential for compounds 2aꢀi in study to 0.75 V*sꢀ1
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Compound
Epa I (V)
Epa II (V)
0.77
0.79
0.72
0.77
0.74
0.92
ꢀ
2a
2b
2c
2d
2e
2f
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0.30
0.27
0.27
2g
2h
2i
0.69
0.77
Characterization of coumarinꢀchalcone radicals by ESR spectroscopy
In order to confirm the formation of semiquinone radical from compounds 2aꢀi, an electron
spin resonance (ESR) spectroscopy study was performed. For compounds 2aꢀd and 2g the
spin trap PBN was used. This nitrone derivative is a diamagnetic compound which in
presence of free radicals produce a spin adduct (paramagnetic species) increasing the half
life of radicals species.45 The experiment was carried out by electrolysis of derivatives to
control potential in situ by using DMSO as solvent and TBAP as supporting electrolyte at
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room temperature. Then PBN spin trap was added. In absence of PBN spin trap, for
derivatives 2e, 2f, 2h and 2i, an ESR spectrum was observed. Compound 2e with two
hydroxyl groups, evidenced a symmetric ESR spectrum with five lines (Figure 2a).
Compound 2f showed a hyperfine pattern with two lines (Figure 2b) and was simulated and
characterized by two doublets due to two nonꢀequivalent hydrogen atoms.
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b)
a)
Figure 2. a) Experimental (blue) and simulated (black) ESR spectra for compound
2e and b) for compound 2f. Simulated hyperfine splitting constants (hfscs) were AH=3.57
G, AH=1.34 G for compound 2e; AH=2.35 G, AH=2.35 G (two equivalent hydrogen atom)
and AH=1.75 G for compound 2f.
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ORACꢀFL assay
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The capacity for peroxyl radical scavenging by coumarinꢀchalcone hybrids was assessed
using the oxygen radical absorbance capacity (ORAC) assay. This methodology gives a
relative index to a standard molecule (Trolox), a hydrosoluble analog of vitamin E. The
ORAC values obtained for all derivatives are showed in Table 2. All derivatives presented
an ORAC index higher than the wellꢀknown antioxidants quercetin and catechin.46
Compound 2e presented the highest ORAC value of the studied series (14.1).
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Table 2. ORAC values and percentage of radicals scavenging for compounds 2aꢀi as
measured by ESR spin trapping in competition with DMPO.
Compound
ORAC value
9.2 ± 0.4
% radical scavenging
26
39
2a
2b
2c
2d
2e
2f
11.5 ± 0.4
9.5 ± 0.3
28
10.9 ± 0.3
14.1 ± 0.3
8.3 ± 0.3
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58
65
13.0 ± 0.4
8.1 ± 0.6
100
75
2g
2h
2i
8.8 ± 0.5
100
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1.0 ± 0.2
7.3 ± 0.2*
6.8 ± 0.2*
31
Trolox
Quercetion
Catechin
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* Data obtained from reference 46
Antioxidant reactivity by ESR Assay
The reactivity against mixture of radical species for all the studied coumarin derivatives
was carried out adapting the H2O2/NaOH/DMSO/DMPO system47 a nonꢀcatalytic and
competitive fenton reaction, in which the spin trap DMPO and the antioxidant molecule
competes for hydroxyl, superoxide anion and methyl radicals.
Four wide hyperfine lines were observed in the control measure (namely as blank,
absence of the tested or antioxidant molecule, Figure 3) due to the mixture of DMPOꢀOH,
DMPOꢀCH3 and DMPOꢀO2 ꢀ adducts formation.
ꢀ
Blank
2b
Blank
2i
3450 3480 3510 3540 3570
Magnetic Field / Gauss
3450 3480 3510 3540 3570
Magnetic Field / Gauss
Figure 3. ESR spectra for reactivity assays of compounds 2b and 2i.
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Biology
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Cytotoxicity assays
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Cytotoxicity of compounds 2aꢀi on bovine aortic endothelial cells (BAEC) was assessed
and evaluated over a concentration range of 0ꢀ50 ꢁM employing the 3ꢀ(4,5ꢀ
Dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀ2Hꢀtetrazolium Bromide (MTT) assay.47 Results are
shown on Table 3 for concentrations of 10, 20 and 50 ꢁM for each studied compound.
Table 3. Cytotoxicity of compounds 2aꢀi tested at three different concentrations on BAEC.
Results are expressed on percentage (%) of cell viability (n = 3)
Compound
10 ꢁM
76 ± 7
88 ± 2
95 ± 3
87 ± 7
68 ± 1
96 ± 3
102 ± 4
49 ± 2
64 ± 2
20 ꢁM
60 ± 6
95 ± 2
93 ± 9
82 ± 5
94 ± 5
101 ± 1
100 ± 5
68 ± 2
23 ± 2
50 ꢁM
44 ± 5
86 ± 1
104 ± 6
101 ± 8
93 ± 10
93 ± 4
92 ± 5
63 ± 2
71 ± 6
2a
2b
2c
2d
2e
2f
2g
2h
2i
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Cytoprotection assays: ROS/RNS scavenging activity in cell systems
Hydrogen peroxide
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In order to evaluate the cytoprotection against H2O2, derivatives presenting high cell
viability values (compounds 2bꢀg) were selected. These compounds also presented high
antioxidant capacity in ORAC assays and showed notable hydroxyl radical scavenging
activity.
BAEC were incubated with two different concentrations of each derivative (10 and 20 ꢁM)
for 24 h in a 24 well plate, followed by treatment with 1 mM of H2O2 for 2h. Cell viability
was then quantified by MTT reduction assay (Figure 4a).
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Figure 4. Cytoprotection of compounds 2bꢀg at 10 and 20 ꢁM against (a) 1 mM H2O2 and
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(b) 0.5 mM SINꢀ1 induced cytotoxicity on bovine aortic endothelial cells using MTT assay.
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Peroxynitrite
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Cytoprotective property of the compounds against peroxynitrite in BAEC was investigated
by MTT assay. The peroxynitrite was generated in cell using 3ꢀmorpholinoꢀsydnonimine
(SINꢀ1).48 Figure 4b shows the results for cytoprotection assays against peroxynitrite for
compounds 2bꢀg.
Theoretical evaluation of ADME properties.
To better correlate the drugꢀlike properties of the coumarinꢀchalcone hybrid 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.49 Theoretical prediction of
absorption, distribution, metabolism and excretion (ADME) properties of all compounds is
summarized in table 4.
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Table 4. Theoretical predictiona of the ADME properties of compounds 2aꢀi.
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Molecular
Weight
TPSA
(Å2)
nꢀOH
nꢀOHNH Volume
Compound
logP
acceptors
donors
(Å3)
2.92
2.89
3.70
3.32
2.42
3.89
3.21
2.16
2.92
266.25
266.25
345.15
280.28
282.25
345.15
361.15
298.25
377.15
67.51
67.51
67.51
67.51
87.74
67.51
87.74
107.97
107.97
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1
1
1
1
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3
227.00
227.00
244.88
243.56
235.01
244.88
252.90
243.03
260.92
2a
2b
2c
2d
2e
2f
2g
2h
2i
a TPSA, topological polar surface area; nꢀOH, number of hydrogen acceptors; nꢀOHNH, number of hydrogen
bond donors. The data was determined with Molinspiration calculation software.
Results and Discussion
Taking into account the importance of the hydroxyl substituents in the antioxidant
properties,17,50 we have focused the study in hydroxyꢀcoumarinꢀchalcone hybrids. All
coumarin derivatives described in this report (compounds 1aꢀi and 2aꢀi) were efficiently
synthesized according to the simple and generalized experimental procedure outlined in
Scheme 2. Electrochemical, biological and theoretical studies were carried out for the final
tested compounds 2aꢀi.
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From the experimental results obtained for the cyclic voltammetry, we observed that under
these conditions, all derivatives exhibited one (compounds 2aꢀg) or two (compounds 2h
and 2i) characteristic anodic peaks without the corresponding cathodic peak (Figure 1),
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.
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The anodic peak potentials (Epa I and Epa II) obtained at a 0.75 V* sꢀ1 scan rate are listed
in Table 1. Monohydroxyl derivatives 2a, 2c and 2d, which contain a hydroxyl substituent
at the para position of the benzoyl moiety, presented an anodic peak potential in the same
positive potential range (Epa II = 0.77, 0.72 and 0.77 V, respectively). The same positive
potential in derivatives 2a and 2d indicates that the methyl substituent at the position 8 of
the coumarin core in 2d is not relevant in the oxidation process. In contrast, the bromine
group in derivative 2c seems to provoke a slight decrease in the anodic peak potential (Epa
II = 0.72 V). The same effect is observed for compound 2e (Epa II = 0.74 V), which in
addition to the hydroxyl group present in the benzoyl moiety also bears an additional
hydroxyl group in the coumarin core. Compounds 2b and 2f with a hydroxyl group in the
coumarin moiety, presented the oxidation potential shifted to more positive values (0.79 V
and 0.92 V, respectively) disfavoring the oxidation process. This effect is more pronounced
for compound 2f due to the interaction through intramolecular hydrogen bonding between
the OH group in position 8 and the oxygen atom of the pyrone ring, as well as due to the
electronwithdrawing properties of this pyrone system.51
Compounds 2g, 2h and 2i, showed a characteristic peak (Epa I), attributed to catechol
moiety. This oxidation occurred at low potential (0.30 V, 0.27 V and 0.27 V, respectively)
due to the electron donating effect of the hydroxyl group at the 3’ position of the benzoyl
moiety and to the favorable quinone formation as final product in these derivatives. In
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addition to this low potential oxidation peak, compounds 2h and 2i showed an additional
second peak, assigned to the hydroxyl group at position 8 of the coumarin core.
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Data obtained for the characterization of coumarinꢀchalcone radicals by ESR spectroscopy
showed that in the absence of the spin trap Nꢀtertꢀbutylꢀαꢀphenylnitrone (PBN), the
oxidation of compounds 2aꢀd and 2g gave no ESR signal. However, in the presence of
PBN, a hyperfine pattern with six lines was observed corresponding to the formation of a
carbonꢀcentered semiquinone spin adduct (aH ≈ 13.5 and aH ≈ 2.3 G were found for all
compounds).52 For compound 2e with two hydroxyl groups, evidenced a symmetric ESR
spectrum with five lines (Figure 2a) and it was interpreted as a semiquinone radical which
hyperfine pattern corresponds to one triplet assigned to two equivalent hydrogen atoms
with aH = 2.37 G and two doublets corresponding to one non equivalent hydrogen atom
with hyperfine constant aH = 2.35, aH = 1.75 G.
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Compound 2f showed a hyperfine pattern with two lines (Figure 2b) and was simulated and
characterized by two doublets due to two nonꢀequivalent hydrogen atoms with aH= 1.34 and
aH= 3.57 G. For radical species of compound 2h, the ESR spectrum (data not shown) was
simulated by two doublets attributed to two nonꢀequivalent hydrogen atoms with aH = 4.35
and aH = 2.37 G. Finally, for the radical species of compound 2i, the hyperfine pattern was
interpreted and simulated by four doublets due to four nonꢀequivalent hydrogen atoms with
hyperfine coupling constants aH = 2.82, aH = 2.93, aH = 2.06 and aH = 1.83 G and also one
triplet with aH= 3.37 G due to two equivalent hydrogen atoms.
The antioxidant capacity of compounds 2aꢀi was studied by ORACꢀFL assays. From the
data obtained, summarized in Table 2, we observed that the difference between the ORAC
values could be related with the substituents present in these derivatives. Derivatives 2a and
2b with one hydroxyl group at the para position of the benzoyl moiety, and at the 6
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position of the coumarin scaffold, respectively, presented ORAC values of 9.2 and 11.5,
correspondingly. Compound 2c and 2d with a hydroxyl group in the same position as in
compound 2a but with an additional bromine substituent in position 6 of the coumarin or a
methyl substituent at the position 8, respectively, presented ORAC values of 9.5 and 10.9.
Derivative 2e, which combines the substitution pattern of compounds 2a and 2b, resulted to
be the compound with the highest ORAC value (14.1).
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On the other hand, structurally comparing compounds 2c and 2g, we observe that the only
difference is the presence in compound 2g of an additional hydroxyl group at the 3’
position of the benzoyl moiety, forming a catechol function. This modification increases the
antioxidant capacity in compound 2g (ORAC = 13.0). ORAC values around 8 were
observed for compound 2f, 2h and 2i. In addition to the catechol function, compounds 2h
and 2i present an additional hydroxyl group at position 8 of the coumarin ring comparing to
the structurally related compound 2g. This additional hydroxyl group could form a
hydrogen bond with the oxygen atom of the pyrone ring, disfavoring therefore the transfer
of the hydrogen atom.
The antioxidant reactivity of compounds 2aꢀi was studied by ESR assay. With this method
it was observed that the intensity of the spectrum decreased in the presence of the tested
coumarinꢀchalcone hybrid compounds added to the system (red line, Figure 3). This
•
behavior was observed in all derivatives 2aꢀi and the percent of the radicals (CH3 , OH•,
•ꢀ
O2 ) scavenging activity is represented in table 2. The scavenging values obtained could be
related with the number and position of the hydroxyl substituents. Compounds 2g and 2i
showed a different hyperfine pattern, which were attributed to carbonꢀcentered spin adducts
formation (Figure 3), meaning that the secondary semiquinone radical for each compound
was trapped by DMPO spin trap and the radical scavenging percentage were close to 100%
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In general, MTT assay only provides qualitative trends in the cytotoxicity of the
compounds and the cytoprotective properties. Cytotoxicity of compounds 2aꢀi on BAEC
using different concentrations (10, 20 and 50 ꢂM) is shown in Table 3. Derivatives 2bꢀg
showed cell viability between 86ꢀ100% up to the maximum concentration of 50 ꢂM.
Compounds 2a, 2h and 2i showed significant toxicity at 50 ꢂM, where compound 2a being
the most toxic exhibiting only 44% viability. The varying degrees in the toxicity of the
compounds can be accounted to several factors such differences in their
compartmentalization properties, nature and concentration of ROS produced, ability to
scavenge radicals, and/or initiate proꢀapoptotic signaling pathways.
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Because compounds 2a, 2i and 2h have significant toxicity, they were not used for
cytoprotection assays. However, compounds 2bꢀg, due to their low toxicity, were then
examined at lower doses of 10 and 20 ꢂM for cytoprotection against hydrogen peroxide
(H2O2) or peroxynitrite (ONOO–) generated from SINꢀ1, and are shown in Figures 4a and
4b, respectively. Compounds 2b and 2d showed the highest cytoprotection against H2O2
compared to all compounds, which presented around 40% cytoprotection at 10 ꢂM.
Compounds 2cꢀ2f at 20 ꢂM were cytoprotective against ONOO– by ~90ꢀ95%, whereas
compound 2b imparted no protection at this concentration. Compound 2g was not
cytoprotective against both H2O2 and ONOOꢀ at any of the concentrations tested. All the
studied compounds showed observable cytoprotection, with remarkable results for
compounds 2bꢀf. In our previous work,53 it was shown that lypophilic spin trap such as the
ꢀ
PBN protect BAEC from H2O2 induced toxicity while the more polar DMPO protects cells
more efficiently from ONOOꢀ. The studied compounds show a better cytoprotective effect
against ONOOꢀ. The studied compounds show a better cytoprotective effect against
ONOO–. However, due to the complexity of the mechanisms that are directly and
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indirectly involved in this assay, it is difficult to find correlation between the cytoprotection
and partition coefficients since these latter values were theoretically calculated. Moreover,
the oxidation potentials may not show direct correlation with cytotoxicity/cytoprotection
data due to other cellular events that are involved such as variations in the ability of the
compounds to redox cycle, subcellular compartmentalization, degradation by cellular
oxidoꢀreductants, or the ability to induce (or suppress) nonꢀradical mediated proꢀapoptotic
pathways. Therefore, although we would like to find direct correlation of cytotoxicity with
the physical data such as partition coefficient and redox potentials, the complexity of the
mechanisms involved in cell viability makes this correlation difficult to realize.19,54,55
From the data obtained for the theoretical evaluation of the ADME properties, one
can notice that all the hybrid compounds 2aꢀi do not break any point of the Lipinski’s rule
of five, making them promising leads for drug candidates.56 TPSA and logP values are
compatible with those described as a predictive indicator of the drug capacity of membrane
penetration.57
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Conclusions
In this paper, we describe the synthesis of coumarinꢀchalcone hybrid compounds
employing the Knoevenagel reaction as a key step for the efficient preparation of a selected
series. The electrochemical properties of the studied compounds resulted to be excellent,
presenting low oxidation potentials, very high ORAC values (all of them higher than the
reference compounds quercetin and catechin) and high percentage of radical scavenging. In
most cases, compounds presented low cytotoxicity for BAEC culture even at the highest
concentration tested and good cytoprotective effects against H2O2 and ONOO– induced cell
death. In addition, the whole series of compounds present good theoretical ADME
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properties. Therefore, based on these results, the synthesized coumarinꢀchalcone hybrid
compounds can be considered as promising scaffolds in the studied area.
It is worth noting that compound 2e presented the highest ORAC value (14.1), good
scavenging capacity, low cytotoxicity and high cytoprotection values, especially against
ONOO– induced cell death (almost 100% of cell viability at 20 ꢀM), and so can be
considered as a potential candidate as lead compound for more thorough studies of its
antioxidant properties.
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Experimental Section
Chemistry
Melting points were determined using a Reichert Kofler thermopan or in capillary tubes on
1
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a Büchi 510 apparatus and are uncorrected. H and C NMR spectra were recorded on a
Bruker AMX spectrometer at 300 and 75.47 MHz, respectively, using TMS as internal
standard (chemical shifts in δ values, J in Hz). Mass spectra were obtained using a Hewlettꢀ
Packard 5988A spectrometer. Elemental analyses were performed using a PerkinꢀElmer
240B microanalyser and were within ± 0.4% of calculated values in all cases. Silica gel
(Merck 60, 230–00 mesh) was used for flash chromatography (FC). Analytical thin layer
chromatography (TLC) was performed on plates precoated with silica gel (Merck 60 F254,
0.25 mm). The purity of compounds 1aꢀi and 2aꢀi was assessed by HPLC and was found to
be higher than 95%. All the chemical reagents employed in the synthetic process were
obtained from Aldrich Chemical Company, Fluka, Across or Merck (analytical reagent
grade). All reactions were carried out under deoxygenated and dry Argon atmosphere
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otherwise indicated. Argon was dried flowing it through CaCl2 columns, NaOH stones and
P2O5.
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General procedure for the synthesis of methoxyꢀ3ꢀbenzoylcoumarins 1aꢀi.40 To a
solution of the appropriate βꢀketoester (1 mmol) and the corresponding salicylaldehyde (1
mmol) in ethanol (3 mL) was added piperidine in catalytic amount. The mixture was
refluxed for 2–5 h and after completion (followed by TLC), the reaction was cooled and the
precipitated was filtered and washed with cold ethanol and ether to afford the desired
compound. Compounds were further recrystallized from MeOH/CH2Cl2.
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6ꢀBromoꢀ3ꢀ(4’ꢀmethoxybenzoyl)coumarin (1c). Yellow solid; yield: 92%; mp 222ꢀ224
ºC. 1H NMR (300 MHz, DMSOꢀd6) δ ppm 8.25 (s, 1H, Hꢀ4), 8.05 (s, 1H, Hꢀ5), 7.92 (d, J =
8.5 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.84 (d, J = 8.7 Hz, 1H, Hꢀ7), 7.44 (d, J = 8.7 Hz, 1H, Hꢀ8), 7.05
(d, J = 8.5 Hz, 2H, Hꢀ3’, Hꢀ5’), 3.85 (s, 3H, OCH3). 13C NMR (75 MHz, DMSOꢀd6) δ ppm
190.1, 164.5, 158.1, 153.5, 143.3, 135.9, 132.7, 131.9, 129.0, 128.3, 120.6, 119.0, 116.7,
114.6, 56.2. MS (EI) m/z (%): 359/361 (M+, 28), 135 (100), 92 (19), 77 (20). Anal. Calcd
for C17H11BrO4: C, 56.85; H, 3.09. Found: C, 56.82; H, 3.07.
8ꢀMethylꢀ3ꢀ(4’ꢀmethoxybenzoyl)coumarin (1d). White solid; yield 93%; mp 250ꢀ252 ºC.
1HꢀNMR (300 MHz, DMSOꢀd6) δ ppm 8.34 (s, 1H, Hꢀ4), 7.91 (d, J= 8.7 Hz, 2H, Hꢀ2’, Hꢀ
4’), 7.73ꢀ7.24 m, 3H, Hꢀ5, Hꢀ6, Hꢀ7), 7.06 (d, J = 8.7 Hz, 2H, Hꢀ3’, Hꢀ5’), 3.86 (s, 3H,
OCH3), 2.41 (s, 3H, CH3). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 190.2, 164.4, 158.4,
152.9, 152.8, 144.7, 134.7, 132.3, 129.6, 127.7, 125.8, 124.7, 118.5, 114.6, 56.1, 15.0. MS
(EI) m/z (%): 295 ([M+1]+, 19), 294 (M+, 100), 135 (88), 77 (31). Anal. Calcd for
C18H14O4: C, 73.46; H, 4.79; Found: C, 73.39; H, 4.57.
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6ꢀMethoxyꢀ3ꢀ(4’ꢀmethoxybenzoyl)coumarin (1e). Yellow solid; yield 80%; mp 182ꢀ184
ºC. 1HꢀNMR (300 MHz, DMSOꢀd6) δ ppm 7.84 (s, 1H, Hꢀ4), 7.71 (d, J = 8.9 Hz, 2H, Hꢀ2’,
Hꢀ6’), 7.13ꢀ6.94 (m, 3H, Hꢀ5, Hꢀ7, Hꢀ8) 6.77 (d, J = 8.9 Hz, 2H, Hꢀ3’, Hꢀ5’), 3.83 (s, 3H,
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OCH3), 3.71 (s, 3H, OCH3). C NMR (75 MHz, DMSOꢀd6) δ ppm 190.2, 164.0, 158.3,
155.9, 148.6, 144.2, 132.3, 128.9, 127.3, 121.1, 118.9, 117.6, 114.2, 111.6, 55.9, 55.8. MS
(EI) m/z (%): 311 ([M+1]+, 29), 310 (M+, 92), 135 (100), 92 (22), 77 (27). Anal. Calcd for
C18H14O5: C, 69.67; H, 4.55. Found: C, 69.45; H, 4.33.
6ꢀBromoꢀ3ꢀ(3’,4’ꢀdimethoxybenzoyl)coumarin (1g). Yellow solid; yield 98%; mp 223ꢀ
1
224 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 8.23 (s, 1H, Hꢀ4), 8.05 (d, J = 2.4 Hz, 1H,
Hꢀ5), 7.84 (dd, J = 8.8, 2.4 Hz, 1H, Hꢀ7), 7.56 (dd, J = 8.4, 2.0 Hz, 1H, Hꢀ6’), 7.52ꢀ7.38
(m, 2H, Hꢀ8, Hꢀ2’), 7.05 (d, J = 8.4 Hz, 1H, Hꢀ5’), 3.86 (s, 3H, 4’ꢀOCH3), 3.81 (s, 3H, 3’ꢀ
OCH3). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 189.8, 157.7, 154.3, 153.2, 149.0, 142.8,
135.6, 131.6, 128.7, 127.9, 125.8, 120.4, 118.8, 116.4, 111.3, 111.1, 56.1, 55.9. MS (EI)
m/z (%): 389/391 (M+, 59), 165 (100), 71 (31). Anal. Calcd for C18H13BrO5: C, 55.55; H,
3.37. Found: C, 55.54; H, 3.39.
8ꢀMethoxyꢀ3ꢀ(3’,4’ꢀdimethoxybenzoyl)coumarin (1h). Pale yellow solid; yield: 95%; mp
238ꢀ240 ºC. 1H NMR (300 MHz, DMSOꢀd6) δ ppm 8.28 (s, 1H, Hꢀ4), 7.54 (dd, J = 8.2, 2.1
Hz, 1H, Hꢀ6’), 7.46 (d, J = 2.1 Hz, 1H, Hꢀ2’), 7.43ꢀ7.27 (m, 3H, Hꢀ5, Hꢀ6, Hꢀ7), 7.04 (d, J
= 8.2 Hz, 1H, Hꢀ5’), 3.93 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.81 (s, 3H, OCH3). 13C
NMR (75 MHz, DMSOꢀd6) δ ppm 190.2, 157.9, 154.1, 148.9, 146.6, 144.6, 143.5, 128.8,
127.1, 125.7, 124.9, 120.7, 119.0, 115.5, 111.1, 111.0, 56.3, 56.0, 55.8. MS (EI) m/z (%):
341 ([M+1]+, 25), 340 (M+, 97), 165 (100), 137 (9), 77 (10). Anal. Calcd for C19H16O6: C,
67.05; H, 4.74. Found: C, 67.06; H, 4.74.
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6ꢀBromoꢀ8ꢀmethoxyꢀ3ꢀ(3’,4’ꢀdimethoxybenzoyl)coumarin (1i). Pale yellow solid; yield
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90%; mp 276ꢀ277 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 8.21 (s, 1H, Hꢀ4), 7.67ꢀ7.49
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(m, 3H, Hꢀ5, Hꢀ7, Hꢀ6’), 7.46 (s, 1H, Hꢀ2’), 7.04 (d, J = 8.5 Hz, 1H, Hꢀ5’), 3.96 (s, 3H, 4’ꢀ
OCH3), 3.85 (s, 3H, 8ꢀOCH3), 3.80 (s, 3H, 3’ꢀOCH3). 13C NMR (75 MHz, DMSOꢀd6) δ
ppm 195.3, 155.1, 149.9, 149.8, 148.2, 143.6, 138.1, 129.4, 128.9, 128.7, 126.0, 123.4,
119.0, 116.8, 112.6, 112.0, 59.0, 57.6, 56.7. MS (EI) m/z (%): 418/420 (M+, 98), 165 (100),
77 (18). Anal. Calcd for C19H15BrO6: C, 54.43; H, 3.61. Found: C, 54.43; H, 3.62.
General procedure to the synthesis of hydroxyꢀ3ꢀbenzoylcoumarins (2aꢀi).40 To the
corresponding methoxyꢀ3ꢀbenzoylcoumarin (1 mmol), BBr3 in DCM (20 mmol, 1M) was
added in a Schlenk tube. Tube was sealed, and the reaction mixture was heated at 80 ºC for
48 h. The resulted crude product was treated with MeOH and rotated to dryness. The
obtained precipitate was recrystallized from MeOH or purified by flash chromatography,
using hexane/ethyl acetate mixtures as eluent, to afford the desired hydroxy derivative.
3ꢀ(4’ꢀHydroxybenzoyl)coumarin (2a). White solid; yield: 78%; mp 242ꢀ243 ºC. (mp
literature 239 ºC)58 1H NMR (300 MHz, DMSOꢀd6) δ ppm 10.58 (bs, 1H, OH), 8.30 (s, 1H,
Hꢀ4), 7.89ꢀ7.75 (m, 3H, Hꢀ5, Hꢀ2’, Hꢀ6’), 7.78ꢀ7.64 (m, 1H, Hꢀ7), 7.51ꢀ7.37 (m, 2H, Hꢀ6,
Hꢀ8), 6.86 (d, J = 8.8, 2H, Hꢀ3’,Hꢀ5’). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 190.1, 163.4,
158.5, 154.3, 144.2, 133.6, 132.9, 129.9, 127.8, 127.5, 125.3, 118.7, 116.7, 115.8. MS (EI)
m/z (%): 267 ([M+1]+, 20), 266 (M+, 93), 237 (25), 121 (100), 93 (22), 65 (25). Anal Calcd
for C16H10O4: C, 72.18; H, 3.79. Found: C, 71.83; H, 3.53.
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6ꢀBromoꢀ3ꢀ(4’ꢀhydroxybenzoyl)coumarin (2c). Pale yellow solid; yield: 73%; mp 295ꢀ
298 ºC. 1H NMR (300 MHz, DMSOꢀd6) δ ppm 10.61 (bs, 1H, OH), 8.22 (s, 1H, Hꢀ4), 8.05
(d, J = 2.4 Hz, 1H, Hꢀ5), 7.93ꢀ7.73 (m, 3H, Hꢀ7, Hꢀ2’, Hꢀ6’), 7.45 (d, J = 8.8 Hz, 1H, Hꢀ8),
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6.86 (d, J = 8.7 Hz, 2H, Hꢀ3’, Hꢀ5’). C NMR (75 MHz, DMSOꢀd6) δ ppm 190.0, 163.6,
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158.1, 153.4, 142.8, 136.2, 133.0, 131.8, 128.5, 127.6, 120.7, 119.0, 116.7, 115.9. MS (EI)
m/z (%): 343/345 (M+, 49), 121 (100). Anal. Calcd for C16H9BrO4: C, 55.68; H, 2.63.
Found: C, 55.30; H, 2.45.
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3ꢀ(4’ꢀHydroxybenzoyl)ꢀ8ꢀmethylcoumarin (2d). White solid; yield: 56%; mp 252ꢀ253
ºC. 1H NMR (300 MHz DMSOꢀd6) δ ppm 10.56 (bs, 1H, OH), 8.27 (s, 1H, Hꢀ4), 7.80 (d, J
= 8.4 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.63 (d, J = 7.5 Hz, 1H, Hꢀ5), 7.57 (d, J = 7.5 Hz, 1H, Hꢀ7), 7.30
(t, J = 7.5 Hz, 1H, Hꢀ6), 6.86 (d, J = 8.4 Hz, 2H, Hꢀ3’, Hꢀ5’), 2.40 (s, 3H, CH3). 13C NMR
(75 MHz, DMSOꢀd6) δ ppm 190.2, 163.4, 158.6, 152.6, 144.6, 134.6, 132.9, 127.8, 127.7,
127.2, 125.6, 124.8, 118.5, 115.9, 15.3. MS (EI) m/z (%): 281 ([M+1]+, 54), 280 (M+, 96),
252 (46), 251 (51), 120 (100), 93 (56), 65 (67). Anal. Calcd for C17H12O4: C, 72.85; H,
4.32. Found: C, 72.67; H, 4.12.
6ꢀHydroxyꢀ3ꢀ(4’ꢀhydroxybenzoyl)coumarin (2e). Brown crystals; yield: 65%; mp 301ꢀ
1
303 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 10.58 (bs, 1H, 4’ꢀOH), 9.86 (bs, 1H, 8ꢀ
OH,), 8.21 (s, 1H, Hꢀ4), 7.79 (d, J = 8.8 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.37ꢀ7.24 (m, 1H, Hꢀ5), 7.19ꢀ
7.01 (m, 2H, Hꢀ7, Hꢀ8), 6.86 (d, J = 8.8 Hz, 2H, Hꢀ3’, Hꢀ5’). 13C NMR (75 MHz, DMSOꢀ
d6) δ ppm 190.3, 163.4, 158.8, 154.4, 147.7, 144.0, 132.9, 127.8, 127.6, 121.6, 119.2,
117.6, 115.9, 113.7. MS (EI) m/z (%): 283 ([M+1]+, 9), 282 (M+, 51), 121 (100), 93 (15),
65 (18). Anal. Calcd for C16H10O5: C, 68.09; H, 3.57. Found: C, 68.23; H, 3.33.
3ꢀBenzoylꢀ6ꢀbromoꢀ8ꢀhydroxycoumarin (2f). Pale yellow solid; yield: 98%; mp 293ꢀ295
ºC. 1H NMR (300 MHz, DMSOꢀd6) δ ppm 10.92 (s, 1H, OH), 8.30 (s, 1H, Hꢀ4), 7.93 (dd, J
= 8.1, 1.4 Hz, 2H, Hꢀ2’, Hꢀ6’), 7.79ꢀ7.63 (m, 1H, Hꢀ5), 7.63ꢀ7.44 (m, 3H, Hꢀ3’, Hꢀ4’, Hꢀ
5’), 7.34ꢀ7.24 (m, 1H, Hꢀ8). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 191.9, 157.9, 146.3,
144.8, 142.7, 136.3, 134.5, 130.0, 129.2, 127.8, 121.9, 121.8, 120.9, 116.2. MS (EI) m/z
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(%): 344/346 (M+, 46), 105 (100), 77 (59). Anal. Calcd for C16H9BrO5: C, 53.21; H, 2.51.
Found: C, 52.97; H, 2.41.
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6ꢀBromoꢀ3ꢀ(3’,4’ꢀdihydroxybenzoyl)coumarin (2g). Yellow solid; yield: 72%; mp 264ꢀ
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266 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 10.14 (s, 1H, OH), 9.47 (s, 1H, OH), 8.19
(s, 1H, Hꢀ4), 8.05 (d, J = 2.1 Hz, 1H, Hꢀ5), 7.84 (dd, J = 8.8, 2.2 Hz, 1H, Hꢀ7), 7.45 (d, J =
8.8 Hz, 1H, Hꢀ8), 7.39ꢀ7.20 (m, 2H, Hꢀ2’, Hꢀ6’), 6.82 (d, J = 8.1 Hz, 1H, Hꢀ5’). 13C NMR
(75 MHz, DMSOꢀd6) δ ppm 189.6, 157.9, 153.3, 152.5, 145.9, 142.3, 135.7, 131.7, 128.4,
127.8, 124.5, 120.5, 119.0, 116.7, 116.3, 115.7. MS (EI) m/z (%): 360/362 (M+, 87), 253
(47), 251 (45), 227 (14), 225 (18), 137 (100), 109 (44). Anal. Calcd for C16H9BrO5 : C,
53.21; H, 2.51. Found: C, 53.23; H, 2.52.
8ꢀHydroxyꢀ3ꢀ(3’,4’ꢀdihydroxybenzoyl)coumarin (2h). Bright yellow solid; yield: 81%;
1
mp 277ꢀ279 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 10.35 (s, 1H, OH), 10.10 (s, 1H,
OH), 9.47 (s, 1H, OH), 8.20 (s, 1H, Hꢀ4), 7.36ꢀ7.06 (m, 5H, Hꢀ5, Hꢀ6, Hꢀ7, Hꢀ2’, Hꢀ6’),
6.82 (d, J = 8.2 Hz, 1H, Hꢀ5’). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 190.2, 158.4, 152.3,
145.8, 145.0, 144.2, 142.8, 128.0, 127.4, 125.3, 124.2, 119.7, 119.5, 116.4, 115.7. MS (EI)
m/z (%): 299 ([M+1]+, 8), 298 (M+, 41), 155 (16), 137 (100), 91 (31). Anal. Calcd for
C16H10O6: C, 64.43; H, 3.38. Found: 64.43; H, 3.39.
6ꢀBromoꢀ8ꢀhydroxyꢀ3ꢀ(3’,4’ꢀdihydroxybenzoyl)coumarin (2i). Pale yellow solid; yield
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69%; mp 300ꢀ302 ºC. H NMR (300 MHz, DMSOꢀd6) δ ppm 10.89 (s, 1H, OH), 10.14 (s,
1H, OH), 9.48 (s, 1H, OH), 8.14 (s, 1H, Hꢀ4), 7.45 (d, J = 2.2 Hz, 1H, Hꢀ5), 7.38ꢀ7.12 (m,
3H, Hꢀ7, Hꢀ2’, Hꢀ5’), 6.81 (dd, J = 8.1, 3.0 Hz, 1H, Hꢀ6’). 13C NMR (75 MHz, DMSOꢀd6)
δ ppm 189.3, 157.4, 152.0, 145.8, 145.5, 142.3, 141.9, 128.0, 127.4, 124.0, 121.1, 121.0,
120.5, 115.9, 115.8, 115.2. MS (EI) m/z (%): 376/378 (M+, 100), 269 (30), 267 (24), 137
(80), 109 (74). Anal. Calcd for C16H9BrO6: C, 50.95; H, 2.41. Found: C, 50.92; H, 2. 43.
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Cyclic voltammetry
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Cyclic Voltammetry was carried out using a potentiostat/galvanostat VersaSTAT 3
provided with a V3ꢀStudio electrochemistry software package, in 50 mM sodium phosphate
buffer (pH 7.4), room temperature, using a three electrode cell, a glassy carbon electrode
was used as working electrode, a platinum wire as auxiliary electrode and Ag, AgCl/KCl
(ca. 3.5 M) as reference electrode. All coumarinꢀchalcone hybrids were studied to 1 mM in
20% methanol as final concentration.
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Characterization of coumarinꢀchalcone radicals by ESR spectroscopy. ESR
measurements were carried out on an ESR spectrometer equipped with a highꢀsensitivity
resonator at room temperature. Unless otherwise indicated, the instrument settings used for
general spectral acquisition are microwave power 10 mW, modulation amplitude 1 G,
receiver gain 1.0 × 104 or 1.0 × 105, scan time 21.5 s, time constant 40.0 s, and sweep width
120 G. The oxidation process was carried out by using 30% H2O2 and NaOH (25 mM) in
presence of chalconeꢀcoumarins hybrid. Scans were integrated using the Bruker WINEPR
v.2.11b software. Sample cells used were 50 ꢁL glass capillary tubes. An automatic fitting
program carried out the spectrum simulation. The hyperfine splitting constants were
estimated to be accurate within 0.05 G.
Radical generating system to test coumarinꢀchalcone hybrids To the corresponding
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coumarinꢀchalcone hybrid compound (1.2 mmol) in dimethyl sulfoxide (20 ꢂL), a
hydrogen peroxide solution (50 ꢂL, 30%) and a sodium hydroxide solution (50 ꢂL, 25 mM)
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were added. The total volume of the resulting solution (300 ꢂL) was completed with
DMSO in order to achieve a final 4 mM solution of the tested compounds.
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ORACꢀFL assay. The ORACꢀFL assays were carried out on a Synergy HT multidetection
microplate reader, from BioꢀTek Instruments Inc. (Winooski, USA), using white
polystyrene 96ꢀwell plates. 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 oxygen radical absorbance capacity was determined as
previously described46 with slight modifications. The reaction was carried out in 75 mM
sodium phosphate buffer (pH 7.4), and 200 ꢁL final volume, FL (70 nM, final
concentration) and coumarinꢀchalcone solutions in methanol with a range of concentration
between 0.3 ꢁM to 2 ꢁM and 0.5 ꢁM to 7 ꢁM for monohydroxy and dihydroxy derivatives,
respectively, were placed in each well of 96ꢀwell plate. This range of concentration was
selected empirically in order to obtain a good separation between the fluorescence curves.
This separation was crucial for a more accurate data treatment.59,60 The mixture was preꢀ
incubated for 15 min at 37 °C, before rapidly adding the AAPH solution (18 mM, final
concentration). The microplate was immediately placed in the reader and automatically
shaken prior to each reading. The fluorescence was recorded every 1 min for 120 min. A
blank with FL and AAPH using methanol instead of the antioxidant solution and five
calibration solutions using Trolox (0.5 ꢁM to 2.5 ꢁM) as standard molecule were also used
in each assay. The inhibition capacity was expressed as ORAC values, and it was quantified
employing the equation 1. All reaction mixtures were prepared in triplicate and at least
three independent assays were performed for each sample.
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