Synthesis of selected 3- and 4-arylcoumarin derivatives and evaluation as potent antioxidants

Article abstract of DOI:10.1007/s11164-016-2445-7

A series of hydroxyl-, methoxy-, and acetoxy-substituted 3- and 4-arylcoumarins were synthesized. All title compounds were screened for their antioxidant capacity, ability to scavenge the 1,1-diphenyl-1-picrylhydrazyl (DPPH) radical, and ability to chelat

Full text of DOI:10.1007/s11164-016-2445-7

Res Chem Intermed
DOI 10.1007/s11164-016-2445-7
Synthesis of selected 3- and 4-arylcoumarin derivatives
and evaluation as potent antioxidants
Ozkan Danis1 Serap Demir1 Cihan Gunduz1
•
•
•
Mustafa Muhlis Alparslan2 Selcuk Altun1
Basak Yuce-Dursun1
•
•
Received: 13 November 2015 / Accepted: 15 January 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A series of hydroxyl-, methoxy-, and acetoxy-substituted 3- and
4-arylcoumarins were synthesized. All title compounds were screened for their
antioxidant capacity, ability to scavenge the 1,1-diphenyl-1-picrylhydrazyl (DPPH)
radical, and ability to chelate iron ions. Furthermore, all derivatives were assessed
using molecular properties prediction and drug likeness using Molinspiration. It was
found that all studied derivatives were potential candidates for further research, as
they complied with Lipinski’s rule of five for drug likeness. 3- or 4-arylcoumarins
that possess two hydroxyl groups in ortho position, such as 4h, 5b, h, and 6a, had
remarkable half-maximal effective concentration (EC50) for radical scavenging,
with better performance than known antioxidants in DPPH and metal-chelating
assays. In addition, the cupric-reducing antioxidant capacity and ferric-reducing
antioxidant power of the synthesized compounds were investigated for antioxidant
activity. Among them, 5g, h and 6a, b showed significantly better Trolox equivalent
antioxidant capacity (TEAC) than standard compounds. The results demonstrate
that the compounds with dihydroxyl groups at 6- and 7-positions of the benzopyrone
ring of the arylcoumarin structure are the most active of the series as antioxidants.
On the basis of these findings, these new coumarin derivatives are potential ther-
apeutic candidates for pathogenesis of many diseases characterized by free-radical
overproduction.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-016-2445-7)
contains supplementary material, which is available to authorized users.
&
Basak Yuce-Dursun
basak.yuce@marmara.edu.tr
1
2
Department of Chemistry, Faculty of Arts and Sciences, Marmara University, Goztepe Campus,
34722 Kadikoy, Istanbul, Turkey
Department of Medicinal Biochemistry, Faculty of Medicine, Cukurova University, Adana,
Turkey
123

O. Danis et al.
Keywords Antioxidant Á Free radical Á Coumarin Á CUPRAC Á FRAP Á DPPH
Introduction
Various natural products, especially phenolic compounds such as flavonoids and
coumarins, are used in many cultures as medicinal agents and food preservatives
due to their wide spectrum of pharmacological activities and low toxicity.
Isolating new pharmacologically active compounds from natural sources and
synthesizing their more potent analogs is a major field of research [1, 2].
Coumarin (2H-chromen-2-one) derivatives constitute one of the most common
families of green plant secondary metabolites. Since many substitutions are
possible on the core structure, coumarin derivatives have diverse pharmacological
activities, including anticoagulant, anticancer, antiviral, antibacterial, antiinflam-
matory, and cholesterol-lowering properties [3–6]. The interesting biological
properties of coumarins make these compounds very attractive targets for organic
synthesis [7]. To enhance the pharmacological activities and lower the toxicity,
numerous coumarin derivatives have been synthesized and published in literature
[8].
All aerobic organisms produce reactive oxygen species (ROS) as byproducts
of aerobic respiration [9]. Organisms have antioxidant defenses that protect
against oxidative damage and numerous damage removal and repair enzymes to
remove or repair damaged molecules [10]. When the balance between
generation and natural elimination of ROS is broken, oxidative stress occurs
[11]. ROS and oxidative stress play important roles in the pathogenesis of
neurodegenerative diseases, cancer, arteriosclerosis, and diabetes [12]. For
prevention and treatment of these diseases, antioxidants are often considered as
drug candidates [13].
It is known that coumarin derivatives are potent antioxidants and can affect
formation and scavenging of ROS, exhibiting tissue-protective antioxidant proper-
ties. These activities are likely related to their structural similarities to flavonoids
and benzophenones, which interact with Fe(III) and thus inhibit hydroxyl radical
and hydrogen peroxide formation by Fenton reactions [14]. However, there have
been very few systematic studies reported on the relationship between antioxidant
activity and coumarin structure [15–17] and even fewer studies have been published
on arylcoumarins [18].
Focusing on the biological importance of hydroxyl groups for the antioxidant
activity and diverse pharmacological properties of the coumarin nucleus, design
and synthesis of some known and novel arylcoumarin derivatives (4c–e, 5e, f)
were carried out, and their in vitro antioxidant and radical-scavenging effects
investigated. We mainly focused on the effect of the number and position of
substitutions of 3- and 4-arylcoumarins possessing methoxy, hydroxy, and acetoxy
groups.
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Synthesis of selected 3- and 4-arylcoumarin derivatives…
Results and discussion
We synthesized and investigated the antioxidant effects of 29 coumarin derivatives
with special focus on peripheral substituents on the A- and B-rings (Schemes 1, 2).
Novel compounds (4c, d, e, 5e, f) were efficiently synthesized according to the
protocol outlined in Scheme 1. The general reaction conditions and characterization
data of the new compounds are described in the ‘‘Experimental’’ section. The
preparation of 3- and 4-arylcoumarin derivatives was performed via Perkin and
Pechmann reactions, respectively, using commercially available and affordable
starting materials, and newly synthesized compounds were characterized and
confirmed by nuclear magnetic resonance (NMR) and mass-spectral studies.
The radical-scavenging activity of the 3- and 4-arylcoumarin derivatives was
determined by investigating the scavenging of the stable free radical 2,20-diphenyl-
1-picrylhydrazyl (DPPH); the results are given in Table 1. The DPPH radical is a
stable free radical due to the extensive delocalization of the unpaired electron.
Tested coumarins were able to reduce stable violet DPPH to yellow DPPH-H, with
EC50 values ranging from 0.049 mM for 6,7-dihydroxy-3-(30,40-dihydroxyphenyl)-
2H-1-chromen-2-one (6a) to 16.411 mM for 5,7-diacetoxy-3-(30,40,50-trimethox-
yphenyl)-2H-1-chromen-2-one (4f). As can be seen from Table 1, compounds 4g, h,
5b–e, g, h, and 6a, b are more potent agents for reduction of DPPH radical (with
EC50 values ranging from 0.049 to 0.075 mM) compared with known antioxidants
Trolox, a-tocopherol, butylated hydroxyanisole (BHA), and butylated hydroxy-
toluene (BHT) (with EC50 values of 0.093, 0.132, and 0.331 mM, respectively).
When 3- and 4-aryl analogs were compared, no discernible differences were found;
however, 3-arylcoumarin derivatives are generally more active than their 4-aryl-
coumarin counterparts. These data show that, in determining the radical-scavenging
activity of the arylcoumarins, the number and location of the hydroxyl groups,
rather than the position of the phenyl group, is important. Similarly, Svinyarov and
Bogdanov showed that the radical-scavenging activity is dependent on the number
and position of the hydroxyl groups [19]. A large number of experimental and
Scheme 1 Synthesis and structure of studied 3-arylcoumarin derivatives. i (CH3CO)2O, CH3COONa,
under N2, 160 °C; ii CH3OH/HCl(aq), reflux; iii BBr3, CH2Cl2, dry ice/(CH3)2CO
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O. Danis et al.
Scheme 2 Synthesis and structure of studied 4-arylcoumarin derivatives. i CF3COOH, heat; ii BBr3,
CH2Cl2, dry ice/(CH3)2CO
Table 1 Radical-scavenging
Compound
DPPHa (mM)
Metal chelatingb (mM)
and metal-chelating data
obtained for tested coumarin
derivatives
3-Arylcoumarins
4a
8.651 0.176
1.694 0.020
1.678 0.006
7.311 0.177
1.313 0.169
16.411 0.467
0.109 0.009
0.056 0.003
0.072 0.003
0.072 0.002
0.066 0.004
0.614 0.025
0.049 0.002
0.062 0.002
n.a.
4b
3.105 0.112
3.626 0.180
n.a.
4c
4d
4e
3.261 0.102
n.a.
4f
5a
1.577 0.032
0.847 0.027
1.021 0.027
1.112 0.099
0.874 0.027
1.214 0.025
0.746 0.031
0.798 0.018
5b
5c
5d
5e
5f
6a
6b
4-Arylcoumarins
4g
0.075 0.005
0.059 0.002
11.514 0.609
0.063 0.002
0.055 0.002
1.077 0.049
0.331 0.027
n.d.
1.012 0.031
0.857 0.012
n.a.
Results given as
mean standard deviation (SD,
n = 3)
4h
4i
n.a. no activity, n.d. not
determined
5g
0.792 0.008
0.764 0.012
0.963 0.018
0.984 0.011
0.185 0.012
1.191 0.017
5h
a
Antioxidant activity measured
using DPPH radical assay and
data expressed as EC50 (mM)
BHT
BHA
EDTA
Trolox
b
Ferrous ion chelating effects
of the compounds expressed as
EC50 (mM)
0.093 0.005
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
theoretical studies have revealed that coumarins containing catechol moiety offer
enhanced radical-scavenging activity for natural antioxidants [20]. However, as is
readily apparent by comparison of the EC50 values in Table 1, the true difference
lies in the presence or absence of two ortho phenolic functions. Compounds with
ortho-dihydroxy substitutions, which are converted upon radical scavenging to o-
quinonoid derivatives, showed the highest DPPH scavenging activity. We found that
6,7-substitution is more effective than 7,8-substitution, and similarly substitutions
on the A-ring are more important than those on the B-ring. When hydroxyl groups
were changed for acetoxy group, the DPPH scavenging activity was drastically
reduced. When acetoxy was on the A-ring, there was low activity, but when it was
present on the B-ring, the activity decreased further. Substitution with methoxy
group further reduced the activity. These observations indicate that the hydroxy and
acetoxy groups must be at ortho positions to each other for maximum activity. It is
worth noting that hydroxyl or acetoxy groups at meta position in the A- or B- ring of
the coumarin nucleus contributed very little to inhibition of the DPPH radical.
Polyphenols such as pyrogallol and gallic acid, which have phenolic OH groups at
ortho position, possess strong radical-scavenging activities. It is known that
coumarin derivatives possess antioxidant activities due to their structural analogy
with benzophenones and flavonoids [21]. Bailly et al. [22] investigated the DPPH
radical-scavenging activity of some 3-hydroxycoumarin derivatives and demon-
strated that 6,7-dihydroxycoumarins are potent scavengers. Kancheva et al. showed
that the antioxidant activity of 4-methylcoumarins depends on the relative positions
of the two phenolic hydroxyls. Similar to our data, they found that various
substitutions on the A-ring lead to decreased antioxidant activity in the following
sequence: 6,7-dihydroxy [ 7,8-dihydroxy [ 5,7-dihydroxy [23].
The DPPH scavenging reaction progress of the most potent antioxidant
compounds and Trolox at the same conditions (at 0.1 mM) was also investigated
and is shown in Fig. 1. It is apparent from this figure that coumarin derivatives are
faster to react and the rate of DPPH consumption increases in the following
sequence: 6a [ 6b [ 5h [ 5g [ Trolox. The reaction mechanism is based on an
electron transfer (ET) reaction, and as occurs in other ET-based assays, the DPPH
scavenging capacity is strongly influenced by steric accessibility.
The ferrozine method was used to analyze iron chelation. It was observed that,
for iron chelation, two hydroxyl groups at adjacent ortho positions correspond to the
highest iron-chelation potency. Relatively lower potency was found for o-acetoxy-
containing coumarin derivatives. Mladenka et al. [24] found similar results for
4-methylcoumarin derivatives. To determine and compare the antioxidant activity
of 3- and 4-arylcoumarin derivatives, the ferric-reducing antioxidant power (FRAP)
and the cupric-reducing antioxidant power (CUPRAC) methods were employed; the
results are given in Fig. 2. Coumarin compounds with an ortho-hydroxylation
pattern on ring A showed remarkable antioxidant efficiency, far greater than that of
Trolox. Compounds possessing ortho-hydroxyl substitutions on the A-ring were
more effective than their analogs with ortho-hydroxyl substitutions on the B-ring.
Similar observations can be made from the results of the DPPH scavenging study,
but the effect of the o-hydroxy group’s location is more apparent in the CUPRAC
and FRAP assays. This difference can be explained by the increased stability of the
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O. Danis et al.
Fig. 1 DPPH scavenging capacity of selected coumarin derivatives as a function of reaction time (s)
Fig. 2 Cupric-reducing antioxidant capacity (CUPRAC) and total ferric-reducing power (FRAP) of
synthesized compounds and standard antioxidants. Results expressed as Trolox equivalent antioxidant
capacity (TEAC), defined as the concentration (mM) of Trolox solution having antioxidant capacity
equivalent to 1.0 mM solution of each substance. Each value represents mean SD (n = 3)
aryloxyl radical of the compound due to the presence of the carbonyl group in
conjugation with ring A [25]. The dissociation energy of the O–H bond is smaller
due to the conjugation of the oxygen-centered unpaired p-orbital with a pair on the
adjacent oxygen atom, which makes the transient radical more stable. As shown in
Fig. 3, data obtained using the CUPRAC method are comparable to those obtained
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
from the FRAP assay (Spearman r = 0.9547), except for compounds 4b, c, e. All
three of these compounds have diacetoxy substitution on their A-ring, and their
TEACFRAP values are approximately three times lower than their TEACCUPRAC
values. The effects of the methoxy substitutions are the lowest among the tested
coumarin derivatives in vitro. Roussaki et al. also demonstrated that low antiox-
idant efficiency of dimethoxy 3-arylcoumarins could be significantly increased by
demethylation of the molecule [26]. Furthermore, orally administered phenolic
compounds undergo hydroxylation and/or glucuronide and sulfate conjugation
primarily by intestinal microflora and secondarily in the liver and other tissues, and
it is well known that phenolic methoxyl groups of coumarins are demethylated in
the liver [27]. These facts suggest that coumarin derivatives that show low activity
in vitro may be converted into more active compounds during metabolism. As
shown in Table 1 and Fig. 2, 6,7-dihydroxy-3-(30,40-dihydroxyphenylcoumarin)
(6a) showed the strongest antioxidant activity, in both radical-scavenging and
reduction power tests. Our results show that the position and type of substituent
attached at the benzenoid rings of the arylcoumarin molecules had an influence on
the radical-scavenging and reduction power potency. In general, substitution at 6,7-
position had more influence on the properties of coumarin than substitution at 7,8-
position. The effect of the hydroxyl groups in the B-ring is less than their effect in
the A-ring. Bailly et al. [22] demonstrated that 6,7-hydroxycoumarin is a potent
scavenger of DPPH radical. Raj et al. [28] suggested that conversion of
diacetoxycoumarins to dihydroxycoumarins occurs in the presence of initiating
free radicals such as DPPH, peroxy radical, or superoxide radical through formation
of reactive ketene. However, Kancheva et al. [23] found that 7,8-diacetoxy-4-
methylcoumarins, which do not have any free phenolic groups, do not have any
chain-breaking antioxidant activity in vitro or any radical-scavenging activity
towards DPPH radical. The results are consistent with a previous report showing
that resonance structures of the radicals derived from coumarins with substitution at
6-position are especially stable because of the ortho-quinone form [27]. In addition,
Fig. 3 Correlation graph for CUPRACTEAC and FRAPTEAC values of synthesized compounds.
Correlation analysis performed by Spearman rho coefficient, p \ 0.0001. AA ascorbic acid, BHA
butylated hydroxyanisole, BHT butylated hydroxytoluene, GA gallic acid
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O. Danis et al.
the type of substituent has a strong influence on the action of coumarins as
antioxidants. Our results indicate that the hydroxy group has the strongest influence,
followed by the acetoxy group, and the methoxy group with least influence. A
similar correlation was also reported for flavonoids [29].
Yang et al. [18] found that 6,7-dihydroxy-3-(30,40-dihydroxyphenyl)coumarin
(6a) has potent antioxidative effect against 2,2’-azobis(2-methylpropionamidine)
dihydrochloride (AAPH)-induced DNA strand breakage and exhibited specific
antiproliferative activity against HL-60 cells (EC50 = 22.1 lM) [18]. Zhang et al.
[13] found that 7,8-dihydroxy-4-(30,40-dihydroxyphenyl)coumarin (5g) has potent
DPPH, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and nitric
oxide radical-scavenging capacity.
To integrate the patterns of data and reduce the dimension of the dataset,
principal component analysis (PCA) was performed, and the resulting biplot graph,
which contains score and loading plots, is shown in Fig. 4. The first two PCs
accounted for 81.37 and 15.31 % of the data variance. Further components
explained 2.14, and 1.19 % of the variance, respectively. The loading plot was used
to assess the importance among the four antioxidant assays. In PC1, the FRAP and
CUPRAC assays had positive loadings, and in PC2, all assays had positive loadings.
The scores plot was used to gain an overview of the similarities or differences
among the coumarin derivatives.
According to the results, it is possible to separate the tested compounds into five
main groups. The most active group contains the compounds 6a, b and 5g, h with
two o-hydroxyl substitutions on both the A- and B-ring of the arylcoumarin system.
The second active group consists of 4g, h and 5a–e, which have one o-hydroxyl
substitution on either ring A or ring B. Coumarin derivatives belonging to both of
these groups showed considerably higher antioxidant activity than standard
antioxidant compounds Trolox, a-tocopherol, BHA, and BHT.
Fig. 4 Biplot graph obtained from principal component analysis of CUPRAC, FRAP, DPPH scavenging,
and metal-chelating assays. BHA butylated hydroxyanisole, BHT butylated hydroxytoluene
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
Table 2 Drug likeness score for coumarin derivatives
Compounds
Log P
Molecular
weight
TPSA
n OH acceptors
n OHNH donors
Volume
4a
4b
4c
4d
4e
4f
1.997
1.997
2.194
1.382
1.981
2.376
2.62
398.367
398.367
398.367
454.387
428.393
428.393
314.293
314.293
342.347
314.293
314.293
314.293
286.239
344.319
344.319
286.239
286.239
286.239
286.239
101.289
101.289
101.289
135.431
110.523
110.523
89.135
8
8
0
0
0
0
0
0
2
2
0
2
2
2
4
2
2
4
4
4
4
340.145
340.145
340.145
378.112
365.691
365.691
267.122
267.122
302.178
267.122
267.122
267.122
232.066
292.668
292.668
232.066
232.066
232.066
232.066
8
10
9
9
4g
4h
4i
6
2.391
3.006
2.391
2.391
2.62
89.135
6
67.147
6
5a
5b
5c
5d
5e
5f
89.135
6
89.135
6
89.135
6
1.974
2.376
2.574
2.005
1.776
1.776
2.005
111.123
98.369
6
7
98.369
7
5g
5h
6a
6b
111.123
111.123
111.123
111.123
6
6
6
6
Data determined using Molinspiration calculation software
log P octanol/water partition coefficient, TPSA topological polar surface area, n OH number of hydrogen
acceptors, n OHNH number of hydrogen-bond donors
A traditional method to evaluate ‘‘drug likeness’’ is to check compliance with
Lipinski’s rule of five, which covers the number of hydrophilic groups, molecular
weight, and hydrophobicity. From the obtained data, it is also remarkable that all the
coumarin derivatives possess log P values (logarithm of the partition coefficient
between water and 1-octanol) compatible with those required to cross membranes.
Topological polar surface area (TPSA), described as a predictive indicator of
membrane penetration, was also found to be positive (Table 2). In addition, it can be
observed that no violations of Lipinski’s rule (molecular weight, log P, number of
hydrogen donors and acceptors) were found. This is important information
regarding the promising potential of these derivatives [30].
Conclusions
Our results show that the position and type of substituents attached at the aromatic
parts of arylcoumarin derivatives influence the radical scavenging and reduction
power. These dihydroxycoumarins are worth further evaluation for their pharma-
cological properties.
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O. Danis et al.
Experimental
Chemicals and apparatus
Starting chemicals were purchased from Aldrich (St Louis, MO, USA) or Merck
(Darmstadt, Germany), unless otherwise stated. Melting points were obtained on a
Gallenkamp apparatus. Infrared (IR) spectra were taken from KBr pellets with a
Shimadzu FT-IR spectrometer, model 8300. 1H and 13C NMR spectra were obtained
with a Varian Mercury 400 MHz spectrometer. Mass spectra were obtained with a
Q-TOF LC–MS instrument (model 6200 series; Agilent, CA, USA).
Methods
The general methods used for synthesis of the studied compounds are summarized
in Schemes 1 and 2. 3-Phenylcoumarin derivatives (4a–f, 5a–f, 6a, b) were
prepared by reaction of substituted hydroxybenzaldehydes with corresponding
arylacetic acids under traditional Perkin conditions [31], while 4-phenylcoumarin
derivatives (4g–i, 5g, h) were obtained using condensation of selected phenols with
ethyl-3,4-dimethoxybenzoylacetate according to the Pechmann [32] reaction. The
1H NMR, 13C NMR, and mass spectra, and proposed structures of novel compounds
are given in the Electronic Supplementary Material.
Synthesis of 2-hydroxy-4,5-dimethoxybenzaldehyde (2a) Boron tribromide (5.0 g,
20 mmol) was slowly added to a stirring solution of 2,4,5-trimethoxybenzaldehyde
(1) (5.0 g, 25 mmol) in CH2Cl2 in a dry ice/acetone bath. After 1 h, the reaction
mixture was warmed to room temperature and stirred for 3 h, then aqueous HCl
(10 %; 20 mL) solution was added. The mixture was extracted with CH2Cl2
(3 9 100 mL). The organic layers were washed with NaCl solution (2 9 100 mL)
and water (100 mL). The product was dried over Na2SO4 and evaporated to produce
2a (4.3 g, 91 %). M.p.: 103 °C, lit. 104 °C [32].
General procedure for synthesis of o-diacetoxy-3-phenylchromenones (4a–f)
A
mixture of benzaldehyde (2a–d) (20 mmol), phenylacetic acids (3a–c) (20 mmol),
and sodium acetate (50 mmol) was stirred in 40 mL acetic anhydride at 160 °C
under N2 atmosphere for 6 h. On reaction completion, the mixture was poured into
ice-cold water (200 mL). The crude product was filtered and recrystallized from
ethanol.
6,7-Dimethoxy-3-(30,40-diacetoxyphenyl)coumarin (4a) A mixture of 2-hydroxy-
4,5-dimethoxybenzaldehyde (2a) (3.6 g, 20 mmol), 3,4-dihydroxyphenylacetic acid
(3a) (3.4 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride
(40 mL) was treated as described above to yield 4a (7.7 g, 97 %). M.p.: 206 °C, lit.
1
207–208 °C [32]. H NMR (400 MHz, CDCl3, 25 °C): d = 2.29 (s, 6H, COCH3),
3.89 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.85 (s, 1H), 6.93 (s, 1H), 7.22 (d,
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
J = 8.2 Hz, 1H), 7.64 (dd, J = 8.2, 2 Hz, 1H), 7.59 (d, J = 2 Hz, 1H), 7.81 (s, 1H)
ppm.
6,7-Diacetoxy-3-(30,40-dimethoxyphenyl)coumarin (4b) A mixture of 2,4,5-trihy-
droxybenzaldehyde (2b) (3.0 g, 20 mmol), 3,4-dimethoxyphenylacetic acid (3b)
(3.9 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride (40 mL)
was treated as described above to produce 4b (4.8 g, 60 %). M.p.: 220 °C, lit.
220 °C [32]. 1H NMR (400 MHz, CD3OD, 25 °C): d = 2.33 (s, 3H, COCH3), 2.34
(s, 3H, COCH3), 3.92 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.91 (d, J = 8.2 Hz, 1H),
7.25 (dd, J = 8.2, 2.3 Hz, 1H), 7.25 (d, J = 2.3 Hz, 1H), 7.29 (s, 1H), 7.41 (s, 1H),
7.70 (s, 1H) ppm.
7,8-Diacetoxy-3-(30,40-dimethoxyphenyl)coumarin (4c) A mixture of 2,3,4-trihy-
droxybenzaldehyde (2c) (3.0 g, 20 mmol), 3,4-dimethoxyphenylacetic acid (3b)
(3.9 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride (40 mL)
was treated as described above to produce 4c (5.2 g, 65 %). M.p.: 218 °C. 1H NMR
(400 MHz, DMSO, TMS): d 8.25 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.30 (dd,
J = 7.80 and 2.08 Hz, 1H), 7.30 (br s, 1H), 7.29 (d, J = 7.80 Hz, 1H), 7.03 (d,
J = 9 Hz, 1H), 3.80 (s, 6H), 2.40 (s, 3H), 2.30 (s, 3H) ppm. MS (m/z): 398.1 [M]?.
5,7-Diacetoxy-3-(30,40-diacetoxyphenyl)coumarin (4d) A mixture of 2,4,6-trihy-
droxybenzaldehyde (2c) (3.0 g, 20 mmol), 3,4-dihydroxyphenylacetic acid (3a)
(3.4 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride (40 mL)
1
was treated as described above to produce 4d (5.2 g, 65 %). M.p.: 185–187 °C. H
NMR (400 MHz, d6-DMSO, ppm): d = 8.13 (s, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.60
(d, J = 8.2 Hz, 1H), 7.34 (dd, J = 8.8 and 2.1 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H),
7.11 (d, J = 2.1 Hz, 1H), 2.38 (s, 3H, -COCH3), 2.29 (s, 6H, –COCH3), 2.28 (s, 3H,
–COCH3). 13C NMR (400 MHz, d6-DMSO, ppm): d = 169.4, 169.0, 168.7, 168.6,
159.4, 154.1, 152.9, 148.0, 142.8, 142.0, 134.6, 133.2, 127.7, 125.9, 124.4, 123.9,
113.6, 111.7, 108.1, 21.4, 21.3, 20.8, 20.7. MS (m/z): 454.1 [M]?.
6,7-Diacetoxy-3-(30,40,50-trimethoxyphenyl)coumarin (4e) A mixture of 2,4,5-
trihydroxybenzaldehyde (2b) (3.0 g, 20 mmol), 3,4,5-trimethoxyphenylacetic acid
(3c) (3.4 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride
(40 mL) was treated as described above to produce 4e (5.2 g, 65 %). M.p.:
185–188 °C. 1H NMR (400 MHz, d6-DMSO, ppm): d = 8.22 (s, 1H), 7.66 (s, 1H),
7.47 (s, 1H), 7.04 (br s, 2H), 3.80 (s, 6H, OCH3), 3.69 (s, 3H, OCH3), 2.31 (s, 3H,
–COCH3), 2.30 (s, 3H, –COCH3). 13C NMR (400 MHz, d6-DMSO, ppm):
d = 168.8, 168.3, 159.6, 153.0, 150.9, 144.7, 139.6, 139.0, 138.6, 130.2, 127.1,
122.7, 118.1, 111.9, 106.7, 60.5, 56.5, 20.8, 20.7. MS (m/z): 428.1 [M]?.
5,7-Diacetoxy-3-(30,40,50-trimethoxyphenyl)coumarin (4f) A mixture of 2,4,6-tri-
hydroxybenzaldehyde (2d) (3.0 g, 20 mmol), 3,4,5-trimethoxyphenylacetic acid
(3c) (3.4 g, 20 mmol), and sodium acetate (4.1 g, 50 mmol) in acetic anhydride
(40 mL) was treated as described above to produce 4f (4.9 g, 61 %). M.p.:
123

O. Danis et al.
185–188 °C, lit. 186–187 °C [33]. 1H NMR (400 MHz, d6-DMSO, ppm): d = 8.04
(s, 1H), 7.24 (d, J = 2.1 Hz, 1H), 7.10 (d, J = 2.1 Hz, 1H), 6.96 (br s, 2H), 3.80 (s,
6H, OCH3), 3.68 (s, 3H, OCH3), 2.39 (s, 3H, –COCH3), 2.29 (s, 3H, –COCH3). 13
C
NMR (400 MHz, d6-DMSO, ppm): d = 169.4, 169.0, 159.5, 153.9, 152.9, 152.7,
147.8, 138.7, 134.0, 130.2, 127.3, 113.5, 111.7, 108.0, 107.1, 60.5, 56.5, 21.3, 21.2.
General procedure for synthesis of o-dihydroxy-3-phenylchromenones (5a–f) The
o-diacetoxy-3-phenylchromenone derivatives (4a–f) were refluxed with MeOH/HCl
(aq) for 3 h, and methanol was removed using distillation. The crude product was
obtained after ethanol recrystallization and filtration.
6,7-Dimethoxy-3-(30,40-dihydroxyphenyl)chromenone (5a) Compound 4a (6.4 g,
16 mmol) was treated as described above to yield 5a (3.5 g, 70 %). M.p.: 219 °C,
lit. 220–221 °C [32]. 1H NMR (400 MHz, CD3OD): d = 3.77 (s, 3H, OCH3),
3.83(s, 3H, OCH3), 6.71 (d, J = 8.4 Hz, 1H), 6.79 (s, 1H), 6.88 (dd, J = 8.2,
1.8 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 7.13 (s, 1H), 7.61 (s, 1H) ppm.
6,7-Dihydroxy-3-(30,40-dimethoxyphenyl)chromenone (5b) Compound 4b (4.7 g,
12 mmol) was treated as described above to yield 5b (2.9 g, 77 %). M.p.: 255 °C,
lit. 255–256 °C [32]. 1H NMR (400 MHz, CD3OD, 25 °C): d = 3.79 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 6.79 (s, 1H), 6.94 (d, J = 8.9 Hz, 1H), 7.11 (s, 1H),
7.31 (dd, J = 8.4, 2 Hz, 1H), 7.35 (d, J = 2 Hz, 1H), 7.85 (s, 1H) ppm.
7,8-Dihydroxy-3-(30,40-dimethoxyphenyl)chromenone (5c) Compound 4c (4.7 g,
12 mmol) was treated as described above to yield 5c (2,3 g, 72 %). M.p.:
1
220–221 °C, lit. 220 °C [34]. H NMR (400 MHz, CD3OD, 25 °C): d = 3.75 (s,
3H, OCH3), 3.81 (s, 3H, OCH3), 6.71 (d, J = 8.4 Hz, 1H), 6,87 (d, J = 8.4 Hz, 1H),
6.92 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 8.5 and 2 Hz, 1H), 7.22 (d, J = 2 Hz, 1H),
7.80 (s, 1H) ppm.
5,7-Dihydroxy-3-(30,40-dihydroxyphenyl)chromenone (5d) Compound 4d (4.7 g,
12 mmol) was treated as described above to yield 5d (3,1 g, 79 %). M.p.:[300 °C,
1
lit. 339 °C [35]. H NMR (400 MHz, d6-DMSO, ppm): d = 10.60 (s, 1H, –OH),
10.30 (s, 1H, –OH), 8.98 (br s, 2H, –OH), 7.89 (s, 1H), 7.10 (d, J = 2.1 Hz, 1H),
6.92 (dd, J = 8.2 and 2.1 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.24 (d, J = 2.3 Hz,
1H) 6.18 (d, J = 2.3 Hz, 1H). 13C NMR (400 MHz, d6-DMSO, ppm): d = 161.9,
160.8, 156.2, 155.8, 145.9, 145.2, 134.3, 126.9, 120.2, 119.7, 116.0, 115.8, 102.8,
98.7, 94.0.
6,7-Dihydroxy-3-(30,40,50-trimethoxyphenyl)chromenone (5e) Compound 4e (4.7 g,
12 mmol) was treated as described above to yield 5e (2.9 g, 82 %). M.p.:
220–221 °C. 1H NMR (400 MHz, d6-DMSO, ppm): d = 9.80 (br s, 2H, -OH), 8.08
(s, 1H), 7.02 (s, 1H), 6.99 (br s, 2H), 6.75 (s, 1H), 3.79 (s, 6H, OCH3), 3.67 (s, 3H,
OCH3). 13C NMR (400 MHz, d6-DMSO, ppm): d = 160.7, 152.9, 150.9, 148.4,
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
143.5, 141.3, 137.9, 131.2, 122.4, 112.7, 111.9, 106.4, 102.6, 60.5, 56.4. MS (m/z):
344.1 [M]?.
5,7-Dihydroxy-3-(30,40,50-trimethoxyphenyl)chromenone (5f) Compound 4f (4.7 g,
12 mmol) was treated as described above to yield 5f (2,7 g, 76 %). M.p.:
220–221 °C. 1H NMR (400 MHz, d6-DMSO, ppm): d = 10.68 (s, 1H, –OH), 10.39
(s, 1H, –OH), 8.04 (s, 1H), 6.92 (br s, 2H), 6.27 (d, J = 2.1 Hz, 1H), 6.21 (d,
J = 2.0 Hz, 1H), 3.79 (s, 6H, OCH3), 3.67 (s, 3H, OCH3). 13C NMR (400 MHz, d6-
DMSO, ppm): d = 162.4, 160.4, 156.6, 156.1, 152.9, 137.8, 136.1, 131.4, 120.0,
106.3, 102.7, 98.8, 94.1, 60.5, 56.4. MS (m/z): 344.1 [M]?.
Synthesis of 6,7-dihydroxy-3-(30,40-dihydroxyphenyl)chromenone (6a) As described
previously, boron tribromide (3.0 g, 12 mmol) was slowly added to a stirring
solution of 5a (2.9 g, 10 mmol) in CH2Cl2 in a dry ice/acetone bath. After 1 h, the
reaction mixture was warmed to room temperature and stirred for 3 h, then aqueous
HCl (10 %; 20 mL) solution was added. The mixture was extracted with CH2Cl2
(3 9 100 mL). The organic layers were washed with NaCl solution (2 9 100 mL)
and water (100 mL). The product was dried over Na2SO4 and evaporated to produce
1
6a (2.1 g, 72.4 %). M.p.: 298 °C, lit. [300 °C [36]. H NMR (400 MHz, DMSO,
TMS): d 7.89 (s, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.01 (s, 1H), 6.95 (dd, J = 8.2 and
2.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H).
Synthesis of 7,8-dihydroxy-3-(30,40-dihydroxyphenyl)chromenone (6b) Compound
5c (2.9 g, 10 mmol) was treated as described above to yield 6b (2,3 g, 79,2 %).
M.p.: 294 °C, lit. 295 °C [37]. 1H NMR (400 MHz, DMSO): d = 6,69 (d,
J = 8.4 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.95 (dd, J = 8.4 Hz and 2.0 Hz, 1H),
7.17 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 2 Hz, 1H) 7.91 (s, 1H), 9.07 (s, 1H), 9.13
(1H), 9.36 (s, 1H), 9.91 (s, 1H) ppm.
General procedure for synthesis of o-methoxy-4-phenylchromenones (4g–i) Using
a typical procedure for cyclization reaction, a mixture of phenols (2e–g) (47 mmol),
ethyl-3,4-dimethoxybenzoylacetate (3d) (47 mmol), and CF3COOH (15 mL) was
refluxed for 6 h, cooled, collected by filtration, and washed with water, and solvent
was evaporated under vacuum.
7,8-Dihydroxy-4-(30,40-dimethoxyphenyl)chromenone (4g) A mixture of 1,2,3-
benzenetriol (2e; 5.99 g, 47 mmol), ethyl-3,4-dimethoxybenzoylacetate (3d;
11.97 g, 47 mmol), and CF3COOH (15 mL) was refluxed for 6 h, cooled, collected
by filtration, washed with water, and dried under vacuum to yield 4g (7.89 g, 52 %).
1
M.p.: 271–272 °C, lit. 274.1–274.4 °C [38]. H NMR (400 MHz, CD3OD, 25 °C):
d = 3.78 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.10 (s, 1H), 6.72 (d, J = 2.0 Hz, 1H),
6.85 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.92 (dd, J = 8.5, 1.6 Hz, 1H).
6,7-Dihydroxy-4-(30,40-dimethoxyphenyl)chromenone (4h) A mixture of 1,2,4-
benzenetriol (2f; 5.99 g, 47 mmol), ethyl-3,4-dimethoxybenzoylacetate (3d;
123

O. Danis et al.
11.97 g, 47 mmol), and CF3COOH (15 mL) was treated as described above to
produce 4h (7.28 g, 48 %). M.p.:[300 °C, lit.[300 °C [31]. 1H NMR (400 MHz,
CD3OD, 25 °C): d = 3.79 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.01 (s, 1H), 6.70 (d,
J = 2.0 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.86 (s, 1H), 7.0 (dd, J = 8.5, 1.6 Hz, 1H).
6,7-Dimethoxy-4-(30,40-dimethoxyphenyl)chromenone (4i)
A mixture of 3,4-
dimethoxyphenol (2g; 7.2 g, 47 mmol), ethyl-3,4-dimethoxybenzoylacetate (3d;
11.97 g, 47 mmol), and CF3COOH (15 mL) was treated as described above to
produce 4i (8.24 g, 42.9 %). M.p.: 221 °C, lit. 222–223 °C [39]. 1H NMR (CDCl3):
d = 7.07 (dd, J = 8.3, 1.9 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.98 (d, 1H,
J = 1.9 Hz), 6.97 (s, 1H), 6.92 (s, 1H), 6.25 (s, 1H), 3.98 (s, 6H), 3.92 (s, 3H), 3.79
(s, 3H).
Synthesis of 7,8-dihydroxy-4-(30,40-dihydroxyphenyl)chromenone (5g) As described
in 5.1.1, boron tribromide (4.5 g, 18 mmol) was slowly added to a stirring solution
of 4g (4.35 g, 15 mmol) in CH2Cl2 in a dry ice/acetone bath. After 1 h, the reaction
mixture was warmed to room temperature and stirred for 3 h, then aqueous HCl
(10 %; 30 mL) solution was added. The mixture was extracted with CH2Cl2
(3 9 100 mL). The organic layers were washed with NaCl solution (2 9 100 mL)
and water (100 mL). The product was dried over Na2SO4 and evaporated to produce
1
5g (3.6 g, 86 %). M.p.: 301 °C, lit. [300 °C [38]. H NMR (500 MHz, DMSO,
TMS): d = 6.91 (br d, J = 8.75 Hz, 1H), 6.86–6.84 (m, 2H),6.77 (s, 1H), 6.76 (dd,
J = 8.90 and 1.50 Hz, 1H), 6.50 (br s, 1H), 6.00 (d, J = 1.5 Hz, 1H).
Synthesis of 6,7-dihydroxy-4-(30,40-dihydroxyphenyl)chromenone (5h) Compound
4h (4.35 g, 15 mmol) was treated as described above to yield 5h (3.4 g, 81 %).
1
M.p.: 304 °C, lit. [300 °C [40]. H NMR (500 MHz, DMSO, TMS): d = 6.94 (s,
1H), 6.87 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.77 (s, 1H), 6.76 (dd,
J = 8.1 and 2.0 Hz, 1H), 5.96 (s, 1H).
Antioxidant assays
DPPH radical-scavenging activity
The scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH)
using the method described by Blois [41] with slight modifications. In summary,
1 mL DPPH (in methanol, 0.1 mM) and 250 lL coumarin derivative (in methanol
containing 5 lL DMSO, conc. 10–100 lg/mL) were shaken vigorously and allowed
to stand at room temperature in the dark for 30 min. The absorbance was measured
at 517 nm. The capacity for scavenging free radicals was calculated from the
following formula:
% Inhibition¼ðAcÀAs=AcÞ Â 100;
where Ac is the absorbance of the control and As is the absorbance of the test
compound. The radical-scavenging activity of the samples is expressed in terms of
123

Synthesis of selected 3- and 4-arylcoumarin derivatives…
EC50 (concentration required for a 50 % decrease in absorbance of DPPH radical).
EC50 values were calculated from the graph of % scavenging activity against the
various concentrations of the samples plotted using GraphPad Prism 5.0 (GraphPad
Software, San Diego, CA. USA). A lower EC50 (mM) value indicates greater
antioxidant activity. To investigate the reaction progress of the DPPH scavenging,
250 lL of compounds 6a–g, 5g, h, and Trolox (0.1 mM) and 1 mL DPPH (in
methanol, 0.1 mM) were shaken briefly and the absorbance was measured at
517 nm at 2-s intervals for 60 s.
Ferric-reducing antioxidant power (FRAP) assay
Ferric ion (Fe3?) reducing power was evaluated using the method developed by
Oyaizu with slight modifications [42]. Sample (100 lL, dissolved in phosphate
buffer 50 lM, pH 6.6, containing 5 lL DMSO) was mixed with phosphate buffer
(200 lL, 50 lM, pH 6.6) and K3Fe(CN)6 (200 lL, 1 %). The mixture was
incubated at 50 °C for 20 min. A portion (250 lL) of trichloroacetic acid (10 %)
was added to the mixture, which was then centrifuged at 10,000g for 10 min. The
upper layer of the solution (700 lL) was mixed with FeCl3 (150 lL, 0.1 %), and the
absorbance was measured at 700 nm. BHT, BHA, and Trolox were used as positive
controls. Results are expressed as Trolox equivalent antioxidant capacity (TEAC),
defined as the concentration (mM) of Trolox solution having antioxidant capacity
equivalent to 1.0 mM solution of the substance under investigation. A higher TEAC
value indicates greater reducing power ability.
Cupric-reducing antioxidant capacity (CUPRAC) assay
Cupric-reducing antioxidant capacity (CUPRAC) was determined as described by
Apak et al., with slight changes [43]. Briefly, 800 lL of coumarin derivatives was
dissolved in NH4Ac buffer pH 7, containing 5 lL DMSO, and mixed with 160 lL
NH4Ac buffer pH 7, 160 lL CuCl2 (10 mM), 160 lL neocuproine (7.5 mM). After
30 min, absorbance was measured at 450 nm. Results are expressed as TEAC
values. A higher TEAC value indicates greater reducing power ability.
Metal chelating
Ferrous ion (Fe2?) chelating activity was determined by inhibiting ferrous–ferrozine
complex formation after treatment of test material with ferrous ion (Fe2?).
Compounds at concentrations of 20–100 lg/mL in 500 lL methanol were added to
a solution of 2 mM FeCl2 (500 lL). The reaction was initiated by addition of 5 mM
ferrozine (200 lL) in methanol. Then, the mixture was shaken vigorously at room
temperature for 10 min. Absorbance of the solution was then measured spectropho-
tometrically at 562 nm [44]. The percentage inhibition of ferrozine–Fe2? complex
formation was calculated as
123

O. Danis et al.
½ðA0 À AsÞ=AsŠ  100;
where A0 is the absorbance of the control and As is the absorbance of the
extract/standard. Na2EDTA was used as positive control [45]. The ferrous ion
chelating effects of the compounds are expressed in terms of EC50 (concentration
required for chelating 50 % of ferrous ions). A lower EC50 (mM) value indicates
greater antioxidant activity.
Theoretical evaluation of ADME properties
The most widely used absorption, distribution, metabolism, and excretion (ADME)
model is Lipinski’s rule of five [46], which is based on four simple rules related to
molecular properties. To better understand the overall properties of the described
compounds, theoretical prediction of ADME properties [molecular weight,
lipophilicity (expressed as log P), number of hydrogen donors and acceptors] of
all the compounds was carried out and is presented in Table 2 [47].
Statistical analysis
All experiments were performed in triplicate, and data obtained from experiments
were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA,
USA). Results are expressed as mean SD. The level of significance was defined
as p \ 0.05. The results from the different antioxidant assays were subjected to
principal component analysis using OriginPro 9.1 (OriginLab Corporation,
Northampton, MA).
Acknowledgments This work was supported by Marmara University, Commission of Scientific
Research Project, FEN-A-110908-0223.
Compliance with ethical standards
Conflict of interest Authors declare that there is no conflict of interest.
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