In Vitro Inhibition of Human Placental Glutathione S-Transferase by 3-Arylcoumarin Derivatives

Article abstract of DOI:10.1002/ardp.201500151

Glutathione S-transferases (EC: 2.5.1.18, GSTs) are phase II detoxification enzymes that catalyze the conjugation of various electrophilic compounds to glutathione (GSH), thus usually producing less reactive and more water soluble compounds. However, ther

Full text of DOI:10.1002/ardp.201500151

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Arch. Pharm. Chem. Life Sci. 2015, 348, 635–642
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Full Paper
In Vitro Inhibition of Human Placental Glutathione S-Transferase by
3-Arylcoumarin Derivatives
1
2
€
Mustafa Muhlis Alparslan and Ozkan DanısS
1
Department of Medicinal Biochemistry, Faculty of Medicine, CS ukurova University, Adana, Turkey
Department of Chemistry, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey
2
Glutathione S-transferases (EC: 2.5.1.18, GSTs) are phase II detoxification enzymes that catalyze the
conjugation of various electrophilic compounds to glutathione (GSH), thus usually producing less
reactive and more water soluble compounds. However, there is evidence that elevated expression of
GSTs, especially GSTP1, is involved in the resistance of tumor cells against chemotherapeutic agents.
In this study, we synthesized and investigated the inhibitory effects of differently substituted
3-arylcoumarin derivatives on human placental GST, identified as GSTP1-1, using 1-chloro-2,4-dinitroben-
zene as a substrate. A known potent inhibitor of GST, ethacrynic acid was used as a positive control.
Among the tested compounds, 6,7-dihydroxy substituted coumarin derivatives exhibited the highest
inhibitory activity (IC50 ¼ 13.50–20.83mM). These results suggest that 6,7-dihydroxy-3-arylcoumarins may
represent a new promising scaffold to discover potent GST inhibitors.
Keywords: Arylcoumarin / Glutathione S-transferase / GSTP1-1 / Multidrug resistance
Received: May 18, 2015; Revised: June 29, 2015; Accepted: July 1, 2015
DOI 10.1002/ardp.201500151
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
of xenobiotics with GSH does not lead to detoxification but to
the formation of episulfonium ion, which can cause mutations
through covalent binding to the DNA [5]. GSTs have two
Introduction
Glutathione S-transferases (EC 2.5.1.18; GSTs) are a family of
enzymes involved in cellular detoxification by catalyzing the
nucleophilic attack of reduced glutathione (GSH) [1, 2].
Therefore, GSTs play an important role in the protection
against reactive molecules, such as electrophilic xenobiotics
(anticancer drugs, pollutants, or carcinogens) or endogenous
a,b-unsaturated aldehydes, quinones, epoxides, and hydro-
peroxides formed as secondary metabolites during oxidative
stress [3]. On the other hand, increased expression of GSTs has
been reported in various tumors, resulting in the develop-
ment of a resistance to chemotherapeutic agents [4]. Further-
more, in some cases, as in the case of haloalkanes, conjugation
active (catalytic) sites per dimer and each active site consists
of a glutathione-binding site (G-site) and a hydrophobic
electrophilic-substrate-binding site (H-site) [6].
The GSTs have also been involved in modulation of stress-
activated protein kinase activity and protection against lipid
peroxidation [7]. There are two major families of soluble GSTs
in mammals, located in the cytosol (a, m, v, p, s, u, and z) and
mitochondria (k) with multiple subfamilies of each class [8].
The cytosolic GSTs are differentially expressed in various
organs; high levels of GSTp (GSTP1) are found in human
erythrocytes, lungs, esophagus, and placenta, whereas only
low levels of GSTP1 are expressed in liver [9]. Individual GSTs
exhibit different substrate specificity and range of cellular
functions [7]. Overexpression of GSTs has been associated
with diseases, such as neurodegeneration, multiple sclerosis,
chronic renal failure, asthma, and particularly prostate, colon,
and ovarian cancers [10].
€
Correspondence: Ozkan DanısS, Department of Chemistry, Faculty
of Arts and Sciences, Marmara University, Goztepe Campus, 34722
Kadikoy, Istanbul, Turkey.
Chemotherapy is the treatment of choice for many types of
tumors; however, one of the major problems in the treatment
of human cancer is the development of intrinsic or acquired
E-mail: odanis@marmara.edu.tr
Fax: þ90-216-3478783
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resistance of malignant cells against chemotherapeutic
agents [11]. Development of acquired resistance to anticancer
drugs by an initially drug-responsive tumor is the primary
cause of treatment failure [12]. Multidrug resistance (MDR) is
defined as resistance of tumor cells to the cytotoxic action
of multiple structurally dissimilar and functionally divergent
drugs commonly used in chemotherapy [13]. Mechanisms
of this drug resistance are suggested as alteration in drug
transport resulting in impaired entry or enhanced efflux of
drugs from tumor cells and enhanced GST-mediated conju-
gation of chemotherapeutics due to increased GST expres-
sion [4]. Alkylating anticancer drugs have been found to be
effectively conjugated to GSH, mediated by GST, thereby
decreasing the potency of such drugs [14]. Also, it has been
found that many chemotherapeutic agents induce over-
expression of various GST enzymes, thus protecting tumor
cells from the effect of anticancer agents [15]. Similarly, GSTs
have been implicated in development of resistance of cells
and organisms to pesticides, herbicides, and antibiotics [8].
These facts have brought about the urgent need for the
search and design of GSTP1 inhibitors with low toxicity and
high specificity, in order to reverse drug resistance [16].
Various GST inhibitors have been synthesized and used to
prevent MDR in patients undergoing treatment with chemo-
therapeutic agents [17]. However, most of the existing
inhibitors are either too toxic to be used in vivo or are only
effective in vitro [8]. Ethacrynic acid (EA), an effective
GSTP1 inhibitor, has been synthesized and investigated as
a potential anticancer drug [1, 18]. However, the activity of
EA was attributed to diuresis and it was not to specific for
GST enzymes; thus its clinical use as a chemosensitizer is
limited [14]. Therefore, it is utmost important to find novel
GST inhibitors to reverse MDR in cancer therapy.
diversity of their molecular scaffold and their high bioavail-
abilities [23]. In recent years, considerable research has been
conducted to identify naturally occurring chemopreventive
substances, paying special attention to polyphenolic substan-
ces [16]. The in vitro inhibition of human GSTs by many natural
products, such as quercetin, curcumin, a-tocopherol, ellagic
acid, tannic acid, and caffeic acid has previously been reported
[9, 16, 24, 25] and this inhibition activity was attributed to
the high-antioxidant potency of these compounds [14].
Coumarins are polyphenolic secondary metabolites of
many plants with diverse biological and pharmacological
effects including anticoagulant, anticancer, antiviral, choles-
terol lowering [26–29], generally associated with low
toxicity, and are strong inhibitors of various enzymes [30].
However, there are very few studies reporting the effects of
coumarins on the activity of human GSTs [30–32]. Therefore,
we designed and synthesized some known and novel
(4c,d) hydroxyl-, methoxy-, and/or acetoxy-substituted 3-aryl-
coumarin derivatives possessing high-antioxidant capacity
(data not published) and investigated the effect of these
compounds on the activity of human placental GST enzyme
using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate and
EA as a positive control. The results may guide the design
of improved coumarin analogs with low toxicity and more
inhibitory potential toward human p class GST enzyme.
Results and discussion
In this work, the inhibitory potency of differently substituted
3-arylcoumarin derivatives on the activity of human placental
glutathione S-transferase, which is identified as GSTP1-1, was
determined by evaluating the inhibition of GST-mediated
conjugation of GSH to CDNB.
Human placental GST (GSTp; GSTP1-1), a homodimeric
protein of about 46 kDa, is often found in solid tumors, and its
overexpression after exposure to antitumor drugs has been
reported [14, 19]; it is therefore a preferential anticancer drug
target among the GST enzymes [1, 20]. GSTP1-1 has been
found to be overexpressed in prostate cancer, squamous cell
carcinoma of the head and neck, gastric carcinoma, acute
lymphoblastic leukemia, and chronic lymphoid leukemia
among others [21]. This overexpression of GSTP1-1 could be
induced in response to treatment with certain chemothera-
peutic agents, such as nimustine hydrochloride and cisplatin
[15, 22]. GSTP1-1 not only contributes to MDR phenomenon
by conjugating anticancer drugs to GSH, but also forms a
complex with c-Jun N-terminal kinase (JNK) in non-stressed
cellsandinhibitsapoptosismediatedbythismitogen-activated
protein kinase (MAPK) [17]. Due to its dual mechanism
contributing to the resistance of tumor cells, GSTP1-1 is a
valuable target to design new molecules to overcome MDR by
either inhibiting the catalytic activity of GST in metabolism of
electrophilic drugs or in disruption of the complex formed
between GST and stress signal kinases [14].
Structures and IC50 values of tested coumarin derivatives
are shown in Table 1. All compounds, except 4a in some
degrees, showed potent to moderate inhibitory activity,
which might be due to the presence of an a,b-unsaturated
carbonyl group. Among the tested compounds, 6a and 6b,
which possessed four hydroxyl groups as substituents showed
the highest inhibitory activity against human placental GST
enzyme (IC50 ¼ 13.50 and 17.47 mM, respectively), whereas 4a
which lacks any hydroxyl substituent, exhibited the lowest
(IC50 ¼ 103.53 mM). 6,7-Dihydroxy coumarin derivatives, 6a
and 5b, showed higher inhibitory activity than their 7,8-
dihydroxy coumarin analogs, 6b and 5c. These data confer
that o-hydroxyl groups at A ring are essential to inhibitory
activity of 3-arylcoumarins and compounds bearing hydroxyl
groups at 6 and 7 positions of the coumarin ring are much
more effective than their 7,8-dihydroxy analogs.
Similar to our data, it has been shown that existence
of hydroxyl groups in the A ring is responsible for the GST
inhibitory activity of flavonoids, such as quercetin and galangin
[33]. Abel et al. demonstrated that different orientations of
the dihydroxyl groups immensely alter the inhibition properties
of the metabolites of estradiol [7]. Effect of having vicinal
Natural products represent a valuable source in medicinal
chemistry for the development of structural analogs, due to
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Inhibition of hGST by 3-Arylcoumarins
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Table 1. Chemical structures and GST inhibition data of coumarin derivatives.
IC50 (mM)a)
0
0
R2
Compound
R1
R2
R3
R4
R1
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
–H
–H
–OAc
–H
–H
–H
–OH
–H
–H
–OCH3
–OAc
–OAc
–OAc
–OCH3
–OH
–OH
–OH
–OH
–OH
–OCH3
–OAc
–H
–H
–H
–H
–OAc
–H
–H
–H
–OH
–H
–H
–OAc
–OCH3
–OCH3
–OAc
–OH
–OCH3
–OCH3
–OH
–OAc
–OCH3
–OCH3
–OAc
–OH
–OCH3
–OCH3
–OH
103.53 ꢀ 1.88
36.33 ꢀ 2.15
27.13 ꢀ 0.65
49.67 ꢀ 2.17
37.80 ꢀ 1.74
20.83 ꢀ 2.21
23.87 ꢀ 0.75
32.43 ꢀ 1.64
13.50 ꢀ 0.79
17.47 ꢀ 0.65
–H
–OCH3
–OH
–H
–H
–OH
–H
–OH
–OH
–OH
–OH
–OH
a) The IC50 value is the mM concentration of inhibitor giving 50% inhibition of the enzyme activity assayed at pH 6.6, 25°C with
1 mM 1-chloro-2,4-dinitrobenzene and 2 mM GSH as substrates. Data shown in each group are the means of triplicate samples.
P < 0.05.
hydroxyl groups at the B ring is smaller as compared to the
A ring. When hydroxyl groups are replaced with methoxy,
compound significantly loses its activity. These findings
suggest that spontaneous conversion of o-hydroxyl groups to
o-quinones is responsible for the inhibition of GST reaction. It
has been known that several naturally occurring and synthetic
quinones can inhibit GST activity [34]. It has been shown that
in the presence of tyrosinase, which is abundant in melanoma
cells, caffeic acid phenethyl ester (CAPE) turns into its o-quinone
form and strongly inhibits GSTs by alkylation of a key cysteine
residue on the enzyme [18]. The investigated compounds
might exert their inhibitory activity with covalent modification
of the enzyme and depletion of glutathione by Michael
addition. In an early study on direct and cytochrome P450-
dependent reaction of various a,b-unsaturated carbonyl com-
poundswithglutathione, ithas beenfoundthatcoumarinisnot
a substrate for the direct reaction and only causes depletion of
GSH when incubated in the presence of liver microsomes [35].
Potency of coumarin derivatives is also compared with the
known potent GST inhibitor EA in a dose-dependent manner.
All compounds have weaker inhibitory activities toward
GSTP1-1 than EA (IC50 ¼ 8.72 mM), although compounds 6a
and 6b have comparable activity.
and CDNB as varied substrates (Figs 1 and 2). 6,7-Dihydroxy-
3-(30,40-dihydroxyphenyl)coumarin (6a), the most potent
inhibitor, shows mixed inhibition with respect to GSH and
non-competitive inhibition against CDNB with the Ki values
7.54 and 13.62 mM, respectively.
Naturally occurring plant polyphenols constitute one of
the largest groups of plant metabolites and are known to
interact with human GST enzymes. Inhibitory activity of various
polyphenolic compounds on different GSTs has been investi-
gated and ellagic acid, curcumin, and quercetin were found
to be potent inhibitors of GSTA and GSTM [16]. Interestingly,
quercetin, which contains o-hydroxyphenol structure similar
to our synthesized compounds, did not exhibit GSTP1-1
inhibitory activity at 100 mM concentration. This contradictory
resultmaybeexplainedwiththelackofvicinalhydroxylgroups
in the A ring of quercetin structure. Further studies revealed
that quercetin inhibits GSTP1-1 only when incubated for
2 h at a concentration of 25 mM. HPLC and LC-MS studies
indicate that this inhibition occurs through the formation of
a covalent yet reversible bond with GSTP1-1 cysteine residue
at position 47 [10]. Zanden et al. demonstrated that co-
incubation of quercetin with 1 mM ascorbic acid results in
prevention of inhibition and suggested that this inhibition
is due to preventing the formation of GSTP1-1 inhibitory
o-quinone and p-quinone methides by the antioxidant effect
Kinetic inhibition parameters and type of inhibition of the
most potent inhibitor were determined with respect to GSH
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Figure 1. Inhibition of GST P1-1 by 6,7-dihydroxy-3-(30,40-dihydroxy)coumarin (6a) at varied concentrations of GSH. Enzyme activity
measured with 1 mM CDNB in the absence (&) or presence of 5 mM (~), 10 mM (^), or 20 mM (*) 6a. Double reciprocal plot shows
mixed type inhibition with respect to GSH. Each point represents the average of three different measurements ꢀ SE.
of ascorbic acid [33]. Non-competitive inhibition of human
GSTP1-1 by a-tocopherol has been shown by van Haaften et al.
They suggest that this inhibitory activity occurs through the
conformational changes due to structural modifications of
lipophilic regions of the GST monomers [25]. Gyamfi et al. have
reported the hGST P1-1 inhibitory activity of a potent
antioxidant, thonningianin A, isolated from the African
medicinal herb, Thonningia sanguinea, with an IC50 value of
3.6 mM [8].
Zanden et al. have also reported the GST inhibitory activity
of antioxidants galangin, kaempferol, and quercetin against
human GSTP1-1 in MCF7 cells and found that these
Figure 2. Inhibition of GST P1-1 by 6,7-dihydroxy-3-(30,40-dihydroxy)coumarin (6a) at varied concentrations of CDNB. Enzyme activity
measured with 2 mM GSH in the absence (&) or presence of 5 mM (~), 10 mM (^), or 20 mM (*) 6a. Double reciprocal plot shows
noncompetitive inhibition with respect to CDNB. Each point represents the average of three different measurements ꢀ SE.
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Inhibition of hGST by 3-Arylcoumarins
Archiv der Pharmazie
compounds effectively inhibit GSTP1-1 activity (IC50 ¼ 14.4,
23.1, and 25.9, respectively) [36].
without further purification. 3-Arylcoumarin derivatives were
prepared by the reaction of substituted hydroxybenzalde-
hydes with the corresponding arylacetic acids under the
traditional Perkin conditions [38–40]. Scheme of synthesis of
the 3-arylcoumarin derivatives has been shown in Scheme 1.
Melting points were obtained on a Gallenkamp apparatus
Research on inhibitory activities of coumarin derivatives on
human GST is relatively scarce. In a study to evaluate some
novel natural products as inhibitors of GSTP1-1, Mukanga-
nyama et al. have found that 5,7-dihydroxy-6-(1-butanoyl)-4-
phenylcoumarin inhibits human GSTP1-1 enzyme with an IC50
value of 100 mM [31]. Relatively low activity of this arylcou-
marin derivative than our compounds could be explained by
the lack of vicinal hydroxyl groups. There are several reports in
the literature about inhibition of plant GSTs by coumarins.
Hossain and Fujita investigated the inhibitory effect of various
coumarin derivatives on pumpkin GSTs (cmGSTU3) and found
that although 6,7-dihydroxycoumarin (esculetin) is highly
effective, 6-methoxy-7-hydroxycoumarin (scopoletin) shows
very low activity (% inhibition at 25 mM concentration ¼ 52
and 3%, respectively) [30]. Esculetin was also found to be a
potent inhibitor of onion GSTs, whereas its parent compound
coumarin or 7-hydroxycoumarin shows very low activity [32].
These data suggest that o-dihydroxy groups are important for
GST inhibitory activity of coumarin derivatives which are in
agreement with our results.
Besides their activities as enzyme inhibitors, coumarin
derivatives are also known for their anticarcinogenic activi-
ties [23, 27]. It has been shown that 6,7-dihydroxycoumarin,
which is structurally similar to 6a and 5b, exhibits an
antiproliferative effect on HL-60 cells by inducing apoptosis
that is associated with cytochrome c translocation and caspase
activation [37]. Considering the low IC50 values, compound 6a
and 6b might be an effective inhibitor of GST in vivo and may
act as a chemopreventive agent by preventing the formation
of carcinogens, scavenging of activated carcinogens, and
inhibiting the DNA/carcinogen complex. Furthermore, it
might also act as an antiproliferative agent by inducing
apoptosis.
1
(MDP 360, UK). H and 13C NMR spectra were recorded on a
400 MHz High Performance Digital NMR spectrometer (Bruker
Daltonics DPX-400, Billerica, MA, USA) instrument.
Chemistry
Synthesis of 3-arylcoumarin derivatives
A mixture of variously substituted benzaldehydes (2a–d)
(20 mmol), phenylacetic acids (3a–c) (20 mmol), and sodium
acetate (50 mmol) was stirred in acetic anhydride (40 mL) at
160°C under N2 atmosphere for 6 h. The reaction mixture was
cooled, poured into ice-cold water (200 mL), and then filtered.
The crude products were purified via crystallization from
ethanol [39, 40]. The NMR spectra and proposed structures of
novel compounds are given in the supplementary data.
6,7-Dimethoxy-3-(30,40-diacetoxyphenyl)coumarin (4a)
The mixture of 2-hydroxy-4,5-dimethoxybenzaldehyde (2a)
(3.6 g, 20 mmol) and 3,4-dihydroxyphenylacetic acid (3a)
(3.4 g, 20 mmol) was treated as described above to yield 4a
(7.1 g, 89%). Mp 206°C (207–208°C in the literature [39]).
1H NMR (400 MHz, CDCl3, 25°C): d ¼ 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, J ¼ 8.2 Hz, 1H), 7.64 (dd, J ¼ 8.2, 2 Hz, 1H), 7.59 (d, J ¼ 2 Hz,
1H), and 7.81 (s, 1H) ppm.
6,7-Diacetoxy-3-(30,40-dimethoxyphenyl)coumarin (4b)
2,4,5-Trihydroxybenzaldehyde (2b) (3.0 g, 20 mmol) and 3,4-
dimethoxyphenylacetic acid (3b) (3.9 g, 20 mmol) was treated
as described above to produce 4b (5.5 g, 69%). Mp 220°C
(220°C in the literature [39]). 1H NMR (400 MHz, CD3OD, 25°C):
d ¼ 2.33 (s, 3H, COCH3), 2.34 (s, 3H, COCH3), 3.92 (s, 3H, OCH3),
3.96 (s, 3H, OCH3), 6.91 (d, J ¼ 8.2 Hz, 1H), 7.25 (dd, J ¼ 8.2,
2.3 Hz, 1H), 7.25 (d, J ¼ 2.3 Hz, 1H), 7.29 (s, 1H), 7.41 (s, 1H), and
7.70 (s, 1H) ppm.
Conclusion
From this literature, it may be concluded that because of their
high-inhibitory activities and probable low toxicities, 6,7-
dihydroxy-3-arylcoumarin derivatives are promising and
potential scaffolds for designing novel GST inhibitors for
use as adjuvants in cancer treatment to overcome MDR where
it is related to overexpression of GST.
7,8-Diacetoxy-3-(30,40-dimethoxyphenyl)coumarin (4c)
2,3,4-Trihydroxybenzaldehyde (2c) (3.0 g, 20 mmol) and 3,4-
dimethoxyphenylacetic acid (3b) (3.9 g, 20mmol) was treated
as described above to produce novel coumarin compound 4c
(5.2 g, 65%). Mp 218°C. 1H NMR (400 MHz, DMSO, TMS):
d ¼ 8.25 (s, 1H), 7.70 (d, J ¼ 8.7 Hz, 1H), 7.30 (dd, J ¼ 7.80 and
2.08 Hz, 1H), 7.30 (br s, 1H), 7.29 (d, J ¼ 7.80 Hz, 1H), 7.03 (d,
J ¼ 9 Hz, 1H), 3.80 (s, 6H), 2.40 (s, 3H), and 2.30 (s, 3H).
Experimental
General
GST assay kit and starting materials for the synthesis of
coumarin derivatives were purchased from Sigma–Aldrich
(St. Louis, MO, USA). GST was purchased from Sigma–Aldrich
(Cat. no. G8642) and the enzyme is identified as hGSTP1-1
with 26 U/mg solid (55 U/mg protein) by Kudugunti et al. [18].
All other chemicals were of analytical grade and were used
5,7-Diacetoxy-3-(30,40-diacetoxyphenyl)coumarin (4d)
2,4,6-Trihydroxybenzaldehyde (2d) (3.0 g, 20 mmol) and 3,4-
dihydroxyphenylacetic acid (3a) (3.4 g, 20 mmol) was treated
as described above to produce novel coumarin compound 4d
(5.2 g, 65%). Mp 185–187°C. 1H NMR (400 MHz, d6-DMSO,
ppm): d ¼ 8.13 (s, 1H), 7.61 (d, J ¼ 2.1 Hz, 1H), 7.60 (d, J ¼ 8.2 Hz,
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M. M. Alparslan and O. DanısS
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Scheme 1. Synthesis and structures of the studied 3-arylcoumarin derivatives; (i) acetic anhydride 160°C, under N2 atmosphere,
(ii) methanol, HCl, N2, reflux, and (iii) BBr3, CH2Cl2, dry ice, acetone, and HCl.
1H), 7.34 (dd, J ¼ 8.8 and 2.1 Hz, 1H), 7.26 (d, J ¼ 2.1 Hz, 1H),
7.11 (d, J ¼ 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 ¼ 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, and 20.7.
Synthesis of 7,8-dihydroxy-3-(30,40-dimethoxyphenyl)-
coumarin (5c)
Compound 4c (4.7 g, 12 mmol) was treated as described
above to yield 5c (2.3 g, 72%). Mp 220–221°C (220°C in
the literature [40]). 1H NMR (400 MHz, CD3OD, 25°C): d ¼ 3.75
(s, 3H, OCH3), 3.81 (s, 3H, OCH3), 6.71 (d, J ¼ 8.4 Hz, 1H), 6,87
(d, J ¼ 8.4 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz, 1H), 7.15 (dd, J ¼ 8.5
and 2 Hz, 1H), 7.22 (d, J ¼ 2 Hz, 1H), and 7.80 (s, 1H) ppm.
Synthesis of 6,7-dimethoxy-3-(30,40-dihydroxyphenyl)-
coumarin (5a)
Compound 4a (6.4 g, 16 mmol) was refluxed with MeOH/
HCl(aq) for 3 h, and the precipitate was filtrated and purified
by crystallization from ethanol to yield 5a (3.5 g, 70%).
Mp 219°C (220–221°C in the literature [39]). 1H NMR (400 MHz,
CD3OD): d ¼ 3.77 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.71
(d, J ¼ 8.4 Hz, 1H), 6.79 (s, 1H), 6.88 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.10
(d, J ¼ 1.8 Hz, 1H), 7.13 (s,1H), and 7.61 (s, 1H) ppm.
Synthesis of 5,7-dihydroxy-3-(30,40-dihydroxyphenyl)-
coumarin (5d)
Compound 4d (4.7 g, 12 mmol) was treated as described above
to yield 5d (3.1 g, 79%), (2.3 g, 79.2%). Mp >300°C (339°C
in the literature [41]). 1H NMR (400 MHz, d6-DMSO, ppm):
d ¼ 10.60 (s, 1H, –OH), 10.30 (s, 1H, –OH), 8.98 (br s, 2H, –OH),
7.89 (s, 1H), 7.10 (d, J ¼ 2.1 Hz, 1H), 6.92 (dd, J ¼ 8.2 and 2.1 Hz,
1H), 6.72 (d, J ¼ 8.2 Hz, 1H), 6.24 (d, J ¼ 2.3 Hz, 1H), 6.18 (d,
J ¼ 2.3 Hz, 1H). 13C NMR (400 MHz, d6-DMSO, ppm): d ¼ 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, and 94.0.
Synthesis of 6,7-dihydroxy-3-(30,40-dimethoxyphenyl)-
coumarin (5b)
Compound 4b (4.7 g, 12 mmol) was treated as described
above to yield 5b (2.9 g, 77%). Mp 255°C (255–256°C in the
literature [39]). 1H NMR (400 MHz, CD3OD, 25°C): d ¼ 3.79 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 6.79 (s, 1H), 6.94 (d, J ¼ 8.9 Hz,
1H), 7.11 (s, 1H), 7.31 (dd, J ¼ 8.4, 2 Hz, 1H), 7.35 (d, J ¼ 2 Hz,
1H), and 7.85 (s, 1H) ppm.
Synthesis of 6,7-dihydroxy-3-(30,40-dihydroxyphenyl)-
coumarin (6a)
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
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Inhibition of hGST by 3-Arylcoumarins
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dry ice/acetone bath for 1 h. After reaction mixture was
warmed to room temperature and stirred for another 4 h, an
aqueous solution of HCl (10%, 20 mL) was added. The mixture
was extracted with CH2Cl2. The product was evaporated to
produce 6a (1.9 g, 65%). Mp 298°C (>300°C in the litera-
ture [42]). 1H NMR (400 MHz, DMSO, TMS): d ¼ 7.89 (s, 1H), 7.14
(d, J ¼ 2.2 Hz, 1H), 7.01 (s, 1H), 6.95 (dd, J ¼ 8.2 and 2.2 Hz, 1H),
6.75 (d, J ¼ 8.2 Hz, 1H), and 6.73 (s, 1H).
All assays were carried out in triplicates, and data were
expressed as the mean ꢀ SD. Coumarin derivatives were
assessed by one-way ANOVA in order to determine whether
there is a significant difference between the IC50 values. In
addition, a binary comparison of coumarin derivatives was
performed by two-tailed t-test. The criterion for statistical
significance was P < 0.05.
Coumarin derivatives were dissolved in DMSO and diluted
by DPBS. Thus, the reaction contained 0.1% DMSO due to
inhibitor delivery. In combination with the ethanol used to
deliver CDNB, the reaction contained a maximum of 1.1%
solvent (0.1% DMSO and 1% ethanol). Solvents used at this
concentration have no effect on activity of the GSTs [13].
Synthesis of 7,8-dihydroxy-3-(30,40-dihydroxyphenyl)-
coumarin (6b)
Compound 5c (2.9 g, 10 mmol) was treated as described above
to yield 6b (2.3 g, 79.2%). Mp 294°C (295°C in the litera-
ture [38]). 1H NMR (400 MHz, DMSO): d ¼ 6.69 (d, J ¼ 8.4 Hz,
1H), 6.85 (d, J ¼ 8.4 Hz, 1H), 6.95 (dd, J ¼ 8.4 and 2.0 Hz, 1H),
7.17 (d, J ¼ 8.4 Hz, 1H), 7.25 (d, J ¼ 2Hz, 1H), 7.91 (s, 1H), 9.07
(s, 1H), 9.13 (1H), 9.36 (s, 1H), and 9.91 (s,1H) ppm.
This work was supported by Marmara University, Commission
of Scientific Research Project under grant FEN-C-YLP-071211-
0308. The authors gratefully acknowledge the suggestions
and advice of Drs. Umit Salan and Cihan Gunduz, Department
of Chemistry, Marmara University, Istanbul, concerning the
synthesis of coumarin derivatives and evaluation of structural
analysis results.
Enzyme assay and inhibition studies
GST enzyme activity was determined spectrophotometrically
by measuring the initial rate of absorbance change at 340 nm
using GST assay kit according to the manufacturer’s instruc-
tions. IC50 values of the coumarin derivatives were deter-
mined with CDNB as electrophilic substrate on an Epoch UV
Plate Reader (BioTek, Winooski, VT, USA) using a series of
inhibitor concentrations. Briefly, various concentrations of
inhibitor compounds (final concentration 5–50 mM, 20 mL)
were incubated with the GST enzyme from human placenta
(0.65 U/mL, 20 mL) and Dulbecco’s phosphate buffered saline
(DPBS) (pH 6.6, 60 mL) in a 96-well microplate for 30 min at
room temperature and a substrate solution containing 2 mM
GSH and 1 mM CDNB in DPBS (100 mL) was added to start the
reaction [18, 43, 44]. Reaction mixture was mixed well by
gentle shaking for a few seconds and absorbance in the
Epoch UV Plate Reader (BioTek) was measured at 340 nm
after immediately preparing the reaction tests for 6 min
(the extinction coefficient; e ¼ 5.3/M/cm). Correction for the
spontaneous nonenzymatic reaction was obtained by sub-
tracting the reaction rate of GSH with CDNB in the absence of
enzyme from the rate in the presence of enzyme. The IC50
values are defined as the inhibitor concentration that gives
50% inhibition of the enzymatic activity and determined by
using GraphPad Prism 6.0 software. IC50 values were obtained
from nonlinear regression analysis by fitting the relationship
between the log10 of at least six different inhibitor concen-
trations versus the percentage of inhibition and are given in
Table 1.
The authors have declared no financial or other relationship
that might lead to a conflict of interest.
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