Polyoxygenated cinnamoylcoumarins as conformationally constrained analogs of cytotoxic diarylpentanoids: Synthesis and biological activity

Article abstract of DOI:10.1016/j.ejmech.2013.07.014

A series of polyoxygenated cinnamoylcoumarins was synthesized as conformationally constrained analogs of cytotoxic diarylpentanoids. The title compounds were tested against the viability of human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (MOLT-4) and human breast adenocarcinoma (MCF-7) cell lines by using MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Among them, all 6- or 7-hydroxylated compounds 6a-h exhibited remarkable cytotoxic activity. Particularly, 7-hydroxycoumarin analog 6h showed good antiproliferative activity against all tested cell lines (IC₅₀ values ≤ 5.5 μM). The preliminary study with selected compounds 6e and 6f showed that reactivity towards mitochondrial thiol compounds cab be considered as cytotoxic mechanism of designed compounds. Furthermore, the antioxidant activity evaluation of synthesized compounds showed that hydroxylated compounds had antioxidative potential at higher concentrations.

Full text of DOI:10.1016/j.ejmech.2013.07.014

European Journal of Medicinal Chemistry 68 (2013) 103e110
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Polyoxygenated cinnamoylcoumarins as conformationally constrained
analogs of cytotoxic diarylpentanoids: Synthesis and biological
activity
Fatemeh Molaverdi a, Mehdi Khoobi b, Saeed Emami c, Masoumeh Alipour a,
Omidreza Firuzi d, Alireza Foroumadi b, Gholamreza Dehghan e, Ramin Miri d,
,
Fatemeh Shaki f, Farnaz Jafarpour a, Abbas Shafiee b
*
a School of Chemistry, College of Science, University of Tehran, Tehran, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran
14176, Iran
c Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari,
Iran
d Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
e Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
f Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of polyoxygenated cinnamoylcoumarins was synthesized as conformationally constrained an-
alogs of cytotoxic diarylpentanoids. The title compounds were tested against the viability of human
chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (MOLT-4) and human
breast adenocarcinoma (MCF-7) cell lines by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. Among them, all 6- or 7-hydroxylated compounds 6aeh exhibi-
ted remarkable cytotoxic activity. Particularly, 7-hydroxycoumarin analog 6h showed good anti-
Received 3 February 2013
Received in revised form
11 June 2013
Accepted 4 July 2013
Available online 8 August 2013
proliferative activity against all tested cell lines (IC50 values ꢀ 5.5
mM). The preliminary study with
Keywords:
selected compounds 6e and 6f showed that reactivity towards mitochondrial thiol compounds cab be
considered as cytotoxic mechanism of designed compounds. Furthermore, the antioxidant activity
evaluation of synthesized compounds showed that hydroxylated compounds had antioxidative potential
at higher concentrations.
Curcuminoids
Diarylpentanoids
Coumarin
Anti-cancer
Cytotoxic agent
Antioxidant activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Among the small molecules introduced to prevent or treat
cancer, there is an important group of compounds structurally
Cancer is going to be the leading cause of death worldwide and
affects about one third of people during their lives. Despite notable
advances have been made in detection, prevention and treatment
of cancer diseases, but one-half of cancer patients do not respond to
therapy or relapse following initial response [1]. However,
chemotherapy is still one of the primary bases for the treatment of
cancer diseases. Due to the undesirable side effects of chemother-
apeutic agents and emergence of drug resistance, there is urgent
need for developing new chemotherapeutic agents with more
potent anti-tumor activity and high therapeutic index [2].
contains a,b-unsaturated carbonyl system [3]. This structural
feature was found in many naturally occurring compounds
including flavonoids, isoflavonoids, chalconoids and curcuminoids.
Curcumin (1) is the principal curcuminoid of turmeric (dry rhi-
zomes of Curcumin longa) which is commonly used as a spice and
food colorant [4]. Curcumin and its derivatives have extensive
biological activities, including antioxidant [5] and anticancer ac-
tivities [6]. Structurally, curcumin (Fig. 1) is
a symmetrical
methoxyphenolic dienone with an active methylene center.
Considering the biological importance of curcumin, many efforts
have been devoted to curcumin analogs. These efforts focused
mainly on omitting the active methylene group and one carbonyl
group of curcumin leading to mono-carbonyl derivatives 2e4
(Fig. 1) namely diaryl-1,4-pentadiene-3-ones (diarylpentanoids)
* Corresponding author. Tel.: þ98 21 66406757; fax: þ98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.014

104
F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
O
O
OMe
OH
MeO
HO
1, Curcumin
A
MeO
O
O
O
O
O
OMe
OMe
MeO
MeO
MeO
OMe
MeO
O
2, GO-035
3, GO-Y030
O
OMe
MeO
O
O
OMe
MeO
OMe
OMe
4, GO-Y031
B
O
O
R2
R1
R2
R1
C
O
O
O
6
5
Fig. 1. The strategy for design of cinnamoylcoumarins 6: (A) deletion of CH2CO from curcumin (1) to generate diarylpentanoids 2e4; (B) conformational restriction of diary-
lpentanoids to chromene derivatives 5; (C) introduction of 2-oxo functionality to yield coumarin-diarylpentanoid hybrids 6.
and changing aryl substitution pattern of the molecule [7,8]. Ohori
et al. have been reported several diarylpentanoids GO-035 (2), GO-
Y030 (3) and GO-Y031 (4), which have a high capacity for growth
suppression in many cancer cell lines. These curcumin-type com-
pounds have a stronger potential to induce down-regulation of
2. Chemistry
The target compounds 6aes were synthesized via the routes
illustrated in Scheme 1. Firstly, commercial hydroxysalicylaldehyde 7
was converted to 6- or 7-hydroxy-3-acetylcoumarins 8 using ethyl
acetoacetate in the presence of catalytic amount of piperidine [15]. In
the next step, 3-acetylcoumarins 8 were condensed with several al-
dehydes in refluxing ethanol and in the presence of piperidine as
catalyst to afford compounds 6aeh. On the other hand, O-benzylation
of hydroxysalicylaldehyde 7 in dry acetonitrile produced benzylox-
ysalicylaldehydes 9. Reaction of 9 with ethyl acetoacetate yielded
corresponding 3-acetylcoumarins 10 which subsequently condensed
with appropriate aldehydes to afford final compounds 6ies.
The latter aldol products 6 were only in E-configuration as
thermodynamically favored structures. The coupling constant of
15.3e16 Hz between the olefinic protons clearly showed that the
compound 6 possess the E-configuration.
oncoproteins, including
ras, than curcumin [9].
b-catenin, ErbB-2, c-Myc, cyclin D1, and Ki-
Recently, we have described some potential cytotoxic agents
namely 1-(2H-chromen-3-yl)-3-phenylprop-2-en-1-ones (5) as
chromene-chalcone hybrids (Fig. 1) [10]. Indeed, these compounds
are conformationally restricted analogs of diarylpentanoids.
On the other hand, coumarins belong to the flavonoid class of
plant secondary metabolite, which have been found to exhibit a
variety of biological activities, including antioxidant [11], and
cytotoxic effects [12,13].
Encouraged by the aforementioned findings and in continuation
of our research program to find novel anticancer agents [10,14], it
was of our interest to design and synthesize cinnamoylcoumarins 6.
Indeed, these compounds could be considered as coumarinedia-
rylpentanoid hybrids, in which ester bond of the coumarin nucleus
partially restricts the conformation of the diarylpentanoid system.
Thus, we describe here, the synthesis and biological evaluation of
polyoxygenated cinnamoylcoumarins as new antiproliferative and
antioxidant agents.
3. Biology
3.1. Cytotoxic assay
The cytotoxic activity of synthesized compounds 6aes against
K562 (humanchronicmyelogenousleukemia), MOLT-4 (humanacute

F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
105
R1
O
O
O
a
H
H
HO
OH
OH
Cl
R1
9
7
b
b
R1
O
O
O
O
HO
O
8
O
O
10
c
c
R1
O
O
R2
O
R2
HO
O
O
O
O
6a-h
6i-s
Scheme 1. Synthesis of substituted cinnamoylcoumarins 6aes. Reagents and conditions: (a) NaHCO3, KI, dry CH3CN; (b) ethyl acetoacetate, EtOH, piperidine; (c) appropriate
benzaldehyde, piperidine, EtOH.
lymphoblastic leukemia) and MCF-7 (human breast adenocarci-
noma) cell lines was evaluated using MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay [16]. The percent inhi-
bition of viability for 3e4 concentrations of the compounds was
calculated with respect to the control. The IC50 values of compounds
were estimated with the software CurveExpert version 1.34 for
Windows. Each experiment was repeated 4e5 times. The IC50 values
of tested compounds in comparison with cisplatin as standard drug
compounds 6aeh (with exception of 6g) exhibited remarkable
cytotoxic activity possessing IC50 values between 1.3 and 17.3 M.
Among the benzyloxy derivatives 6ies, compound 6o bearing a 2,6-
dichloro substituent on cinnamoyl moiety showed significant ac-
m
tivity (IC50 values ꢀ 15.6
mM) against all cell lines while the rest of
compounds exhibited weak or no activity.
The comparison of IC50 values against K562 cells demonstrated
that carboxy methyl ester derivative 6h had the most potent
are presented in Table 1 as mean ꢁ SD (in
m
M).
inhibitory activity against this cell line (IC50 value ¼ 4.4
However, 3,4,5-trimethoxycinnamoyl analogs 6b and 6f with IC50
M showed potent anti-proliferative activity against
K562 cells. Interestingly, carboxy methyl ester derivatives 6d and
6h were the most potent compounds in the term of cytotoxic ac-
mM).
values ꢀ8.8
m
3.2. Antioxidant activity evaluation
Antioxidant activity can be considered as an index of therapeutic
usefulness of new compounds [17]. Numerous methods are avail-
able for the assessment of antioxidant capacity of compounds. In
this study, two commonly used methods, the DPPH (1,1-diphenyl-
2-picrylhydrzyl) radical scavenging and ferric reducing antioxidant
power (FRAP) assays were used to determine the antioxidant po-
tential of the target compounds 6 in comparison with quercetin.
The results are summarized in Table 2.
tivity against MCF-7 cells. The 6-hydroxy analog 6d (IC50 ¼ 1.3
showed better inhibition respect to the 7-hydroxy derivative 6h
M) against the latter cell line. In the case of human
mM)
(IC50 ¼ 5.5
m
acute lymphoblastic leukemia cells (MOLT-4), 3,4-methylenedioxy
derivative of 6-hydroxycomarin (compound 6c) with IC50 value of
2.0 mM exhibited the highest activity. However, 3,4,5-trimethoxy
derivatives (compounds 6b and 6f) as well as carboxy methyl
ester analog (compound 6h) were amongst the more potent com-
pounds against MOLT-4 (IC50 values ¼ 3.7e3.9
mM).
3.3. Mitochondrial total thiol assay
In overall, the 7-hydroxycoumarin analog 6h containing carboxy
methyl ester group on the cinnamoyl moiety showed good anti-
proliferative activity against all tested cell lines.
The effect of selected compounds 6e and 6f on total mito-
chondrial thiol content was determined using 5,50-dithiobis-2-
nitrobenzoic acid (DTNB) as indicator, and the absorbance was
measured at 412 nm by spectrophotometry. Corrected absorbance
values were used to calculate mitochondrial total thiols using the
glutathione (GSH) standard curve [18].
4.2. Reactivity towards mitochondrial thiols
A number of studies have demonstrated that conjugated enones
such as diarylpentanoids act as Michael acceptor for intracellular
thiol compounds [19e21]. The Michael addition of intracellular
thiol compounds such as glutathione (GSH) to the olefinic double
bond of enones results in the formation of corresponding conju-
gated adducts. In general, an initial reaction of cytotoxic agents
with cellular thiols sensitizes the malignant cells to oxidative stress
and modulates processes such as DNA synthesis, enzyme
4. Results and discussion
4.1. Cytotoxic activity
The IC50 values of target compounds 6 against K562, MCF-7, and
MOLT-4 cells in Table 1 revealed that all 6- or 7-hydroxylated

106
F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
Table 1
The IC50 values (mM) of compounds 6aes against different cell lines.
R1
O
O
O
β
4
5
R2
O
6
7
α
R2
HO
O
O
8
1
O
6a-h
6i-s
Compound
6- or 7-substitution
R1
R2
K562
MCF-7
MOLT-4
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6
6
6
6
7
7
7
7
6
6
6
7
7
7
7
7
7
7
7
e
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
4-(COOMe)
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
4-(COOMe)
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
H
4-Me
2,4-Cl2
2,6-Cl2
4-MeO
2,3-(MeO)2
2,5-(MeO)2
3,4-(OCH2O)
17.3 ꢁ 4.8
8.8 ꢁ 4.6
14.6 ꢁ 3.2
15.8 ꢁ 5.9
14.6 ꢁ 1.6
7.3 ꢁ 3.2
31.7 ꢁ 6.3
4.4 ꢁ 0.75
48.9 ꢁ 23.5
66.4 ꢁ 8.0
>50
8.8 ꢁ 2.9
10.9 ꢁ 6.5
9.7 ꢁ 2.3
1.3 ꢁ 0.7
16.6 ꢁ 6.8
8.8 ꢁ 2.8
38.9 ꢁ 6.3
5.5 ꢁ 0.7
21.3 ꢁ 10.6
26.5 ꢁ 3.9
>50
6.7 ꢁ 1.0
3.9 ꢁ 0.7
2.0 ꢁ 0.4
7.1 ꢁ 1.2
7.0 ꢁ 0.6
3.8 ꢁ 0.9
26.2 ꢁ 10.5
3.7 ꢁ 0.4
23.6 ꢁ 10.5
10.3 ꢁ 4.1
>50
e
e
e
e
e
e
e
H
H
H
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
>100
33.7 ꢁ 9.8
>100
>50
ND
>50
>50
NDa
ND
15.6 ꢁ 7.5
10.1 ꢁ 4.3
12.5 ꢁ 2.0
>100
>100
>100
45.5 ꢁ 7.0
>100
>100
35.1 ꢁ 8.7
>100
>100
22.8 ꢁ 8.9
13.8 ꢁ 0.4
>100
6s
Cisplatin
12.2 ꢁ 2.0
10.9 ꢁ 3.7
3.1 ꢁ 0.4
a
Not determined.
activation, selective gene expression, and regulation of the cell
cycle [22]. Physiological cell death (apoptosis) can be initiated from
mitochondria via cytochrome c release following mitochondrial
permeability transition (MPT) pores opening. It has been shown
GSH have an essential role in MPT pores and oxidation of thiol
groups in the inner mitochondrial membrane cause conformational
changes in the pore complex that leading to MPT pores opening and
imitation of apoptosis signaling [23,24].
Accordingly, we investigated the effect of selected compounds
6e and 6f on the total thiol content of mitochondria after 1 h
exposure. As shown in Fig. 2, the tested compounds 6e and 6f
decreased the mitochondrial thiol content in a concentration
dependent manner. Therefore, thiol alkylation and induction of
mitochondrial pathway of apoptosis cab be considered as cytotoxic
mechanism of designed compounds. Although, we have shown that
cinnamoylcoumarins such as 6e and 6f prototypes act as Michael
acceptor for mitochondrial thiol compounds, but we cannot rebut
other mechanisms such as tubulin polymerization inhibition which
are responsible for cytotoxic activity.
that 2,3-dimethoxy analogs of 6- or 7-hydroxycoumarins (com-
pounds 6a and 6e) had superior antioxidant power in FRAP assay.
The relative capability of these compounds to reduce the ferric ions
respect to quercetin was about 0.3. Among the benzyloxycoumar-
ins, compound 6p exhibited the highest antioxidant power to
reducing ferric ions.
In general, the 2,3-dimethoxycinnamoyl-hydroxycoumarins 6a
and 6e exhibited the most potent antioxidant activity in both DPPH
and FRAP methods. Although compounds 6a and 6e were not as
potent as reference drug quercetin, but their antioxidant activity
could be considered as attendant property of the title compounds.
5. Conclusion
We described a series of polyoxygenated cinnamoylcoumarins
as conformationally constrained analogs of cytotoxic diary-
lpentanoids. Two set of compounds bearing hydroxyl or benzyloxy
moiety on coumarin ring were synthesized and tested against the
viability of three different cell lines. Among them, all 6- or 7-
hydroxylated compounds 6aeh exhibited remarkable cytotoxic
activity. Particularly, 7-hydroxycoumarin analog 6h containing
carboxy methyl ester group on cinnamoyl moiety showed good
antiproliferative activity against all tested cell lines (IC50
4.3. Antioxidant activity
As shown in Table 2, all hydroxycoumarin derivatives 6aeh
exhibited better DPPH radical scavenging activity with IC50 values
less than 0.55 mM. The 2,3-dimethoxy analogs 6a and 6e were the
most potent compounds in the scavenging of DPPH radicals
(IC50 z 0.15 mM). Furthermore, compounds 6b and 6f bearing
3,4,5-trimetoxycinnamoyl showed significant radical scavenging
activity with 50% inhibitory concentrations of about 0.18 mM.
The FRAP assay shows the capability of compound to reduce the
ferric 2,4,6-tripyridyl-s-triazine complex to the colored ferrous
complex [25]. The results of FRAP assay in Table 2 demonstrated
values
ꢀ
5.5
mM). Besides carboxy methyl ester, 3,4,5-
trimethoxyphenyl scaffold was found to be a good functionality
for exhibiting the cytotoxic potential of cinnamoylcoumarins. The
effect of selected compounds 6e and 6f on total mitochondrial
thiol content demonstrated that cinnamoylcoumarins act as
Michael acceptor for mitochondrial thiol compounds. Further-
more, the antioxidant activity evaluation of synthesized com-
pounds showed that hydroxylated compounds had antioxidative
potential at higher concentrations.

F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
107
Table 2
Antioxidant properties of compounds 6 in comparison with quercetin.
R1
O
O
O
β
4
5
R2
O
6
7
α
R2
HO
O
O
8
1
O
6a-h
6i-s
Compound
6- or 7-substitution
R1
R2
DPPH radical scavenging activity (IC50, mM)
FRAP value (mM Fe2þ) (100
mg)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6
6
6
6
7
7
7
7
6
6
6
7
7
7
7
7
7
7
7
e
e
e
e
e
e
e
e
H
H
H
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
4-(COOMe)
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
4-(COOMe)
2,3-(MeO)2
3,4,5-(MeO)3
3,4-(OCH2O)
H
4-Me
2,4-Cl2
2,6-Cl2
4-MeO
2,3-(MeO)2
2,5-(MeO)2
3,4-(OCH2O)
0.16 ꢁ 0.01
0.18 ꢁ 0.01
0.48 ꢁ 0.01
ND
0.15 ꢁ 0.01
0.19 ꢁ 0.01
0.54 ꢁ 0.01
ND
1.27 ꢁ 0.02
1.15 ꢁ 0.03
0.57 ꢁ 0.01
0.89 ꢁ 0.01
2.63 ꢁ 0.04
0.48 ꢁ 0.01
0.94 ꢁ 0.02
0.93 ꢁ 0.02
0.72 ꢁ 0.01
0.75 ꢁ 0.01
0.46 ꢁ 0.01
8.12 ꢁ 0.13a
25.7 ꢁ 0.24
9.6 ꢁ 0.08
15.3 ꢁ 0.07
ND
25.2 ꢁ 0.10
8.7 ꢁ 0.10
13.7 ꢁ 0.26
ND
4.4 ꢁ 0.01
8.1 ꢁ 0.07
12.6 ꢁ 0.11
10.4 ꢁ 0.09
6.6 ꢁ 0.02
7.1 ꢁ 0.09
5.3 ꢁ 0.02
14.3 ꢁ 0.10
7.6 ꢁ 0.11
9.5 ꢁ 0.07
7.5 ꢁ 0.06
87.1 ꢁ 1.4
6s
Quercetin
a
IC50 in mM.
6. Experimental
points were measured on a Kofler hot stage apparatus and are
uncorrected. The IR spectra were taken using Nicolet FT-IR Magna
550 spectrometer (KBr disks). The 1H NMR spectra were recorded
6.1. Chemistry
on a NMR instrument Bruker 500 MHz. The chemical shifts (d) and
6.1.1. General methods
All starting material and reagents were purchased from Acros
Organics and Merck, and used without further purification. Melting
coupling constants (J) are expressed in parts per million (ppm) and
Hertz (Hz), respectively. An HP (Agilent technologies) 5937 Mass
Selective Detector was used to obtain Mass spectra of the com-
pounds. Elemental analyses were carried out with a PerkineElmer
model 240-C apparatus. The results of elemental analyses (C, H, N)
were within ꢁ0.4% of the calculated values.
6.1.2. General procedure for the synthesis of
benzyloxysalicylaldehyde derivatives 9
A suspension of hydroxysalicylaldehyde (1.0 mmol), NaHCO3
(1.5 mmol), and catalytic amounts of KI in dry acetonitrile (3.0 mL)
was stirred at room temperature for 5 min. Then, appropriate
benzyl halide (1.2 mmol) was added to the mixture and the mixture
was allowed to warm to 90 ꢂC under argon atmosphere. After
completion of the reaction, the solvent was evaporated under
reduced pressure. The residue was mixed with ethyl acetate (5 mL)
and the insoluble solids was filtered and washed with ethyl acetate
(3 ꢃ 5 mL). The organic phase was concentrated under reduced
pressure and the residue was purified by silica gel column chro-
matography using petroleum ethereethyl acetate (8:2) as eluent to
give the pure product 9.
6.1.3. General procedure for the synthesis of 3-acetylcoumarin
derivatives 8 or 10
A cold mixture of salicylaldehyde derivative 7 or 9 (0.2 mol)
and ethyl acetoacetate (0.2 mol) in absolute ethanol (25 mL) was
treated with piperidine (0.2 mL). The mixture was allowed to
warm to 50 ꢂC. After completion of the reaction, the precipitated
Fig. 2. Effect of compounds 6e and 6f on mitochondrial total thiol content. Isolated
liver mitochondria (1 mg/mL) were incubated for 1 h with 6f (25 and 50
(50 and 100 M). Then, total thiol content was measured using DTNB method. Values
represented as mean ꢁ SD (n ¼ 3). *P < 0.05 compared with control group.
mM) and 6e
m

108
F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
solid was filtered off and subsequently washed with ethanol and
recrystallized from watereethanol (30:70) to give pure product.
CDCl3) d: 10.56 (s, 1H, OH), 8.56 (s, 1H, H4 coumarin), 7.88 (d,
J ¼ 15.4 Hz, 1H, Hb), 7.74 (d, J ¼ 15.4 Hz, 1H, Ha), 7.53 (d, J ¼ 7.8 Hz,
1H, H5 coumarin), 7.45 (s, 1H, H8 coumarin), 6.90e6.84 (m, 3H, H6
coumarin and H2,6 phenyl), 3.92 (s, 6H, 2 ꢃ OCH3), 3.88 (s, 3H,
OCH3). Anal. Calcd for C21H18O7 (382.36): C, 65.96; H, 4.74. Found:
C, 66.01; H, 4.59.
6.1.4. General procedure for the synthesis of cinnamoylcoumarins 6
To a mixture of 3-acetylcoumarin derivative 8 or 10 (1 mmol)
and appropriate aldehyde (1 mmol) in absolute ethanol (3 mL)
were added catalytic amount of piperidine (3 drops). The mixture
was heated under reflux for 12e24 h. After completion of the re-
action, water was added and the mixture was extracted with ethyl
acetate. The solvent was evaporated under reduced pressure and
the residue was purified by silica gel column chromatography using
petroleum ethereethyl acetate (8:2) as eluent to give the pure
product 6.
6.1.4.7. (E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-7-hydroxy-2H-
chromen-2-one (6g). Yellow solid; yield 88%; mp: >250 ꢂC; IR (KBr,
cmꢄ1): 3330 (OH), 1695 (C]O), 1615 (C]O). 1H NMR (500 MHz,
CDCl3)
d
: 8.43 (s, 1H, H4 coumarin), 7.72 (d, J ¼ 15.7 Hz, 1H, Hb), 7.62
(d, J ¼ 15.7 Hz, 1H, Ha), 7.37 (d, J ¼ 8.5 Hz, 1H, H5 coumarin), 7.08 (s,
1H, H2 phenyl), 7.02 (d, J ¼ 8.0 Hz,1H, H6 phenyl), 6.75e6.70 (m, 3H,
H6,8 coumarin and H5 phenyl), 5.91 (s, 2H, OCH2O). Anal. Calcd for
6.1.4.1. (E)-3-(3-(2,3-Dimethoxyphenyl)acryloyl)-6-hydroxy-2H-
chromen-2-one (6a). Yellow solid; yield 85%; mp: >250 ꢂC; IR (KBr,
cmꢄ1): 3262 (OH), 1684 (C]O), 1589 (C]O). 1H NMR (500 MHz,
C19H12O6 (336.29): C, 67.86; H, 3.60. Found: C, 65.61; H, 3.32.
6.1.4.8. Methyl (E)-4-(3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-en-1-yl)benzoate (6h). Yellowish solid; yield 60%; mp:
243e245 ꢂC; IR (KBr, cmꢄ1): 3442 (OH), 1725 (C]O), 1701 (C]O).
CDCl3) d: 10.45 (br s, 1H, OH), 8.25 (s, 1H, H4 coumarin), 7.49 (d, 1H,
J ¼ 15.4 Hz, Hb), 7.41 (d, 1H, J ¼ 15.4 Hz, Ha), 7.24 (d, 1H, J ¼ 8.2 Hz, H7
coumarin), 6.89 (s, 1H, H5 coumarin), 6.84 (d, J ¼ 8.2 Hz, 1H, H8
coumarin), 6.55e6.52 (m, 3H phenyl), 3.01 (s, 6H, 2 ꢃ OCH3). Anal.
Calcd for C20H16O6 (352.33): C, 68.18; H, 4.58. Found: C, 68.36; H, 4.49.
1H NMR (500 MHz, DMSO-d6)
d: 8.65 (s, 1H, H4 coumarin), 8.00 (d,
J ¼ 8 Hz, 2H, H3,5 phenyl), 7.88 (d, J ¼ 16.0 Hz, 1H, Hb), 7.84 (d,
J ¼ 8.0 Hz, 2H, H2,6 phenyl), 7.77 (d, J ¼ 8.5 Hz, 1H, H5 coumarin),
7.72 (d, J ¼ 16.0 Hz, 1H, Ha), 6.84 (d, J ¼ 8.5, 1H, H6 coumarin), 6.74
(s, 1H, H8 coumarin), 3.85 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3)
6.1.4.2. (E)-6-Hydroxy-3-(3-(3,4,5-trimethoxyphenyl)acryloyl)-2H-
chromen-2-one (6b). Yellow solid; yield 82%; mp: >250 ꢂC; IR (KBr,
cmꢄ1): 3337 (OH), 1731 (C]O), 1648 (C]O). 1H NMR (500 MHz,
d
: 186.2, 165.7, 164.6, 159.0, 157.3, 148.5, 141.0, 139.1, 132.5, 130.7,
129.7, 128.6, 127.1, 119.4, 114.4, 110.9, 101.9, 52.27. Anal. Calcd for
C20H14O6 (350.08): C, 68.57; H, 4.03. Found: C, 68.32; H, 3.95.
CDCl3)
d
: 8.42 (s, 1H, H4 coumarin), 7.74 (d, 1H, J ¼ 15.7 Hz, Hb), 7.59
(d, 1H, J ¼ 15.7 Hz, Ha), 7.38 (d, 1H, J ¼ 8.4 Hz, H7 coumarin), 6.75e
6.70 (m, 4H, H5,8 coumarin and H2,6 phenyl), 3.77 (s, 6H, 2 ꢃ OCH3),
3.74 (s, 3H, OCH3). Anal. Calcd for C21H18O7 (382.36): C, 65.96; H,
4.74. Found: C, 65.76; H, 4.49.
6.1.4.9. (E)-6-(Benzyloxy)-3-(3-(2,3-dimethoxyphenyl)acryloyl)-2H-
chromen-2-one (6i). Yellow solid; yield 93%; mp: 172e174 ꢂC; IR
(KBr, cmꢄ1): 1728 (C]O), 1662 (C]O). 1H NMR (500 MHz, CDCl3)
d:
8.50 (s, 1H, H4 coumarin), 8.20 (d, J ¼ 15.9 Hz, 1H, Hb), 7.96 (d,
J ¼ 15.9 Hz, 1H, Ha), 7.46e7.31 (m, 7H, H7,8 coumarin, H6 phenyl and
5H benzyloxy), 7.14 (d, 1H, J ¼ 2.3 Hz, 1H, H5 coumarin), 7.10 (t,
J ¼ 8.0 Hz, 1H, H5 phenyl), 6.99 (d, J ¼ 8.0 Hz, 1H, H4 phenyl), 5.14 (s,
2H, CH2 benzyl), 3.92 (s, 3H, OCH3), 3.90 (s, 3H, OCH3). Anal. Calcd
for C27H22O6 (442.46): C, 73.29; H, 5.01. Found: C, 73.56; H, 5.36.
6.1.4.3. (E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-6-hydroxy-2H-
chromen-2-one (6c). Yellow solid; yield 90%; mp: >250 ꢂC; IR (KBr,
cmꢄ1): 3337 (OH), 1607 (C]O), 1731 (C]O). 1H NMR (500 MHz,
CDCl3)
d: 9.49 (br s, 1H, OH), 8.47 (s, 1H, H4 coumarin), 7.75 (m, 2H,
Ha b), 7.52 (s, 1H, H5 coumarin), 7.24e7.11 (m, 4H, H7,8 coumarin and
,
H
2,6 phenyl), 6.88 (d, J ¼ 7.6 Hz, H5 phenyl), 6.07 (s, 2H, OCH2O). Anal.
Calcd for C19H12O6 (336.29): C, 67.86; H, 3.60. Found: C, 67.70; H, 3.36.
6.1.4.10. (E)-6-(Benzyloxy)-3-(3-(3,4,5-trimethoxyphenyl)acryloyl)-
2H-chromen-2-one (6j). Yellow solid; yield 93%; mp: 178e180 ꢂC; IR
6.1.4.4. Methyl (E)-4-(3-(6-hydroxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-en-1-yl)benzoate (6d). Yellowish solid; yield 60%; mp:
243e245 ꢂC; IR (KBr, cmꢄ1): 3433 (OH), 1724 (C]O), 1685 (C]O).
(KBr, cmꢄ1): 1714 (C]O), 1638 (C]O). 1H NMR (500 MHz, CDCl3)
d:
8.51 (s, 1H, H4 coumarin), 7.85 (d, J ¼ 15.7 Hz, 1H, Hb), 7.79 (d,
J ¼ 15.7 Hz, 1H, Ha), 7.46e7.33 (m, 7H, H7,8 coumarin and 5H benzy-
loxy), 7.14 (d, J ¼ 2.3 Hz, 1H, H5 coumarin), 6.91 (s, 2H, H2,6 phenyl),
5.14 (s, 2H, CH2 benzyl), 3.93(s, 6H, 2ꢃ OCH3), 3.91(s, 3H, OCH3). Anal.
Calcd for C28H24O7 (472.48): C, 71.38; H, 5.12. Found: C, 71.44; H, 5.36.
1H NMR (500 MHz, DMSO-d6)
d: 9.93 (s, 1H, OH), 8.60 (s, 1H, H4
coumarin), 8.01 (d, J ¼ 8.0 Hz, 2H, H3,5 phenyl), 7.86 (d, J ¼ 8.0 Hz,
2H, H2,6 phenyl), 7.76 (br s, 2H, Ha b), 7.33 (d, J ¼ 9.0 Hz, 1H, H8
,
coumarin), 7.24 (d, J ¼ 2.5 Hz, 1H, H5 coumarin), 7.16 (dd, J ¼ 9.0 and
2.5 Hz, 1H, H7 coumarin), 3.86 (s, 3H, OCH3). 13C NMR (125 MHz,
6.1.4.11. (E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-6-(benzyloxy)-
2H-chromen-2-one (6k). Yellow solid; yield 94%; mp: 198e200 ꢂC;
IR (KBr, cmꢄ1): 1719 (C]O), 1661 (C]O). 1H NMR (500 MHz, CDCl3)
CDCl3) d: 187.3, 165.7, 158.6, 154.0, 147.9, 147.2, 142.0, 138.9, 130.9,
129.7, 128.8, 127.1, 125.2, 122.6, 118.8, 117.1, 113.9, 52.3. Anal. Calcd
for C20H14O6 (350.08): C, 68.57; H, 4.03. Found: C, 68.28; H, 4.33.
d: 8.51 (s, 1H, H4 coumarin), 7.80 (br s, 2H, Ha,b), 7.46e7.30 (m, 7H,
H7,8 coumarin and 5H benzyloxy), 7.13 (d, J ¼ 2.5 Hz, 1H, H5
coumarin), 7.17 (dd, J ¼ 8.0 and 2.1 Hz, 1H, H6 phenyl), 7.22 (d,
J ¼ 2.1 Hz, 1H, H2 phenyl), 6.85 (d, J ¼ 8.1 Hz, 1H, H5 phenyl), 6.04 (s,
2H, OCH2O), 5.14 (s, 2H, CH2 benzyl). Anal. Calcd for C26H18O6
(426.41): C, 73.23; H, 4.25. Found: C, 73.44; H, 4.36.
6.1.4.5. (E)-7-Hydroxy-3-(3-(2,3-dimethoxyphenyl)acryloyl)-2H-
chromen-2-one (6e). Yellow solid; yield 89%; mp: 181e183 ꢂC; IR
(KBr, cmꢄ1): 3416 (OH), 1703 (C]O), 1661 (C]O). 1H NMR
(500 MHz, CDCl3) d: 11.15 (s, 1H, OH), 8.67 (s, 1H, H4 coumarin),
7.89e7.82 (m, 3H, H5 coumarin and H4,6 phenyl), 7.33 (t, J ¼ 7.5 Hz,
1H, H5 phenyl), 7.16e7.14 (m, 2H, Ha,b), 6.86 (d, J ¼ 7.5 Hz, 1H, H6
coumarin), 6.78 (s, 1H, H8 coumarin), 3.84 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3). Anal. Calcd for C20H16O6 (352.33): C, 68.18; H, 4.58. Found: C,
68.36; H, 4.39.
6.1.4.12. 3-Cinnamoyl-7-((3-methoxybenzyl)oxy)-2H-chromen-2-
one (6l). Yellow solid; yield 92%; mp: 131e133 ꢂC; IR (KBr, cmꢄ1):
1714 (C]O), 1650 (C]O). 1H NMR (500 MHz, CDCl3)
d: 8.59 (s, 1H,
H4 coumarin), 8.02 (d, J ¼ 15.7 Hz, 1H, Hb), 7.87 (d, J ¼ 15.7 Hz, 1H,
Ha), 7.70e7.68 (m, 2H, H2,6 phenyl), 7.58 (d, J ¼ 8.7 Hz, 1H, H5
coumarin), 7.42e7.41 (m, 3H, H3,4,5 phenyl), 7.34 (t, 1H, J ¼ 7.8 Hz,
H5 benzyloxy), 7.03e6.98 (m, 3H, H4,6 benzyloxy and H6 coumarin),
6.92e6.90 (m, 2H, H2 benzyloxy and H8 coumarin), 5.16 (s, 2H, CH2
6.1.4.6. (E)-7-Hydroxy-3-(3-(3,4,5-trimethoxyphenyl)acryloyl)-2H-
chromen-2-one (6f). Yellow solid; yield 91%; mp: >250 ꢂC; IR (KBr,
cmꢄ1): 3337 (OH), 1730 (C]O), 1646 (C]O). 1H NMR (500 MHz,

F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
109
benzyl), 3.84 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3)
d: 186.3,
benzyloxy and 3H phenyl), 5.25 (s, 2H, CH2 benzyl), 3.84 (s, 3H,
OCH3), 3.79 (s, 3H, OCH3), 3.76 (s, 3H, OCH3). 13C NMR (125 MHz,
CDCl3) d: 186.3, 163.7, 159.3, 158.8, 156.8, 152.8, 148.1, 137.4, 137.1,
132.0,129.6,128.0,125.6,124.4,119.9,119.0,115.0,114.0,113.5,113.4,
112.2, 101.1, 70.0, 60.8, 55.7, 55.0. Anal. Calcd for C28H24O7 (472.48):
C, 71.18; H, 5.12. Found: C, 71.39; H, 5.25.
164.1, 159.9, 159.7, 157.5, 148.4, 144.5, 136.8, 134.9, 131.3, 130.6,
129.9, 128.8, 124.1, 121.4, 119.6, 114.4, 113.8, 113.0, 112.5, 101.3, 70.6,
55.2. Anal. Calcd for C26H20O5 (412.43): C, 75.72; H, 4.89. Found: C,
75.90; H, 4.99.
6.1.4.13. (E)-7-((3-Methoxybenzyl)oxy)-3-(3-(p-tolyl)acryloyl)-2H-
chromen-2-one (6m). Yellow solid; yield 91%; mp: 174e176 ꢂC; IR
6.1.4.18. (E)-3-(3-(2,5-Dimethoxyphenyl)acryloyl)-7-((3-
methoxybenzyl)oxy)-2H-chromen-2-one (6r). Yellow solid; yield
89%; mp: 147e149 ꢂC; IR (KBr, cmꢄ1): 1726 (C]O), 1655 (C]O). 1H
(KBr, cmꢄ1): 1717 (C]O), 1652 (C]O). 1H NMR (500 MHz, CDCl3)
d:
8.58 (s, 1H, H4 coumarin), 7.98 (d, J ¼ 15.7 Hz, 1H, Hb), 7.85 (d,
J ¼ 15.7 Hz, 1H, Ha), 7.59e7.57 (m, 3H, H2,6 phenyl and H5
coumarin), 7.34 (t, J ¼ 7.8 Hz, 1H, H5 benzyloxy), 7.22 (d, J ¼ 7.9 Hz,
2H, H3,5 phenyl), 7.03e6.98 (m, 3H, H4,6 benzyloxy and H6
coumarin), 6.92e6.90 (m, 2H, H2 benzyloxy and H8 coumarin), 5.16
(s, 2H, CH2 benzyl), 3.84 (s, 3H, OCH3), 2.40 (s, 3H, CH3). 13C NMR
NMR (500 MHz, CDCl3)
d: 8.56 (s, 1H, H4 coumarin), 8.19 (d,
J ¼ 15.8 Hz, 1H, Hb), 8.01 (d, J ¼ 15.8 Hz, 1H, Ha), 7.58 (d, J ¼ 8.7 Hz,
1H, H5 coumarin), 7.34 (t, J ¼ 7.9 Hz, 1H, H5 benzyloxy), 7.23 (s, 1H,
H2 benzyloxy), 7.03e6.86 (m, 7H, H6,8 coumarin, H4,6 benzyloxy and
3H phenyl), 5.16 (s, 2H, CH2 benzyl), 3.88 (s, 3H, OCH3), 3.84 (s, 3H,
(125 MHz, CDCl3)
d
: 186.4, 164.0, 159.9, 159.7, 157.4, 148.3, 144.7,
OCH3), 3.83 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3)
d: 186.7, 163.9,
141.2, 136.8,132.2,131.3, 129.9,129.6,128.9,123.1, 121.6, 119.6, 114.3,
113.8, 113.0, 112.5, 101.3, 70.6, 55.2, 21.5. Anal. Calcd for C27H22O5
(426.46): C, 76.04; H, 5.20. Found: C, 76.37; H, 5.44.
159.9, 159.7, 157.4, 153.4, 148.1, 139.6, 136.8, 131.2, 129.9, 124.6,
124.5, 121.8, 119.6, 117.9, 114.3, 113.8, 113.2, 113.0, 112.5, 112.4, 101.3,
70.6, 56.1, 55.8, 55.2. Anal. Calcd for C28H24O7 (472.48): C, 71.18; H,
5.12. Found: C, 71.07; H, 5.34.
6.1.4.14. (E)-3-(3-(2,4-Dichlorophenyl)acryloyl)-7-((3-
methoxybenzyl)oxy)-2H-chromen-2-one (6n). Yellow solid; yield
85%; mp: 177e179 ꢂC; IR (KBr, cmꢄ1): 1723 (C]O), 1655 (C]O). 1H
6.1.4.19. (E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-7-((3-
methoxybenzyl)oxy)-2H-chromen-2-one (6s). Yellow solid; yield
95%; mp: 169e171 ꢂC; IR (KBr, cmꢄ1): 1725 (C]O), 1694 (C]O). 1H
NMR (500 MHz, CDCl3)
d: 8.62 (s, 1H, H4 coumarin), 8.18 (d,
J ¼ 15.8 Hz, 1H, Hb), 8.00 (d, J ¼ 15.8 Hz, 1H, Ha), 7.78 (d, J ¼ 8.5 Hz,
1H, H5 coumarin), 7.60 (d, J ¼ 8.8 Hz, 1H, H6 phenyl), 7.46 (s, 1H, H3
phenyl), 7.34 (t, J ¼ 7.2 Hz, 1H, H5 benzyloxy), 7.30 (d, J ¼ 8.8 Hz, 1H,
H5 phenyl), 7.01e6.90 (m, 5H, H6,8 coumarin and H2,4,6 benzyloxy),
5.17 (s, 2H, CH2 benzyl), 3.84 (s, 3H, OCH3). 13C NMR (125 MHz,
NMR (500 MHz, CDCl3) d: 8.56 (s, 1H, H4 coumarin), 8.19 (d,
J ¼ 15.8 Hz, 1H, Hb), 8.01 (d, J ¼ 15.8 Hz, 1H, Ha), 7.58 (d, J ¼ 8.7 Hz,
1H, H5 coumarin), 7.34 (t, J ¼ 7.9 Hz, 1H, H5 benzyloxy), 7.23 (s, 1H,
H2 benzyloxy), 7.16 (d, J ¼ 8.1 Hz, 1H, H6 phenyl), 7.03e6.84 (m, 6H,
H
2,5 phenyl, H6,8 coumarin and H4,6 benzyloxy), 6.03 (s, 2H, OCH2O),
CDCl3)
d: 185.8, 173.3, 164.3, 159.8, 157.6, 148.9, 138.5, 136.7, 136.5,
5.16 (s, 2H, CH2 benzyl), 3.84 (s, 3H, OCH3). 13C NMR (125 MHz,
CDCl3) : 186.1, 164.0, 159.8, 159.7, 157.4, 148.3, 144.5, 136.8, 131.3,
131.8, 131.5, 129.9, 128.8, 127.5, 126.7, 120.9, 119.6, 114.5, 113.8, 113.0,
112.5, 101.3, 70.6, 55.2. Anal. Calcd for C26H18Cl2O5 (481.32): C,
64.88; H, 3.77. Found: C, 65.01; H, 3.58.
d
129.9,129.5,125.6,122.2,121.5,119.6,114.3,113.8,113.0,112.5,108.5,
107.1, 101.6, 101.3, 70.6, 55.2. Anal. Calcd for C27H20O7 (456.44): C,
71.05; H, 4.42. Found: C, 71.07; H, 4.38.
6.1.4.15. (E)-3-(3-(2,6-Dichlorophenyl)acryloyl)-7-((3-
methoxybenzyl)oxy)-2H-chromen-2-one (6o). Yellow solid; yield
87%; mp: 162e164 ꢂC; IR (KBr, cmꢄ1): 1715 (C]O), 1663 (C]O). 1H
6.2. Biological assays
NMR (500 MHz, CDCl3)
d: 8.60 (s, 1H, H4 coumarin), 8.12 (d,
6.2.1. Cell viability assay
J ¼ 16.0 Hz, 1H, Hb), 7.94 (d, J ¼ 16.0 Hz, 1H, Ha), 7.60 (d, J ¼ 8.5 Hz,1H,
H5 coumarin), 7.38 (d, J ¼ 8.0 Hz, 2H, H3,5 phenyl), 7.34 (t, J ¼ 8.0 Hz,
1H, H4 phenyl),7.20 (t, J ¼ 7.8 Hz,1H, H5 benzyloxy), 7.02e6.91 (m, 5H,
The human cancer cell lines including K562 (human chronic
myelogenous leukemia), MOLT-4 (human acute lymphoblastic
leukemia) and MCF-7 (human breast adenocarcinoma) cells were
obtained from the National Cell Bank of Iran, Pasteur Institute,
Tehran, Iran. Cells were maintained at 37 ꢂC in humidified air
containing 5% CO2. All cell lines were maintained in RPMI 1640
supplemented with 10% FBS, and 100 units/mL penicillin-G and
H
6,8 coumarin and H2,4,6 benzyloxy), 5.16 (s, 2H, CH2 benzyl), 3.84 (s,
3H, OCH3). 13C NMR (125 MHz, CDCl3)
d: 186.3, 164.2, 159.9, 159.5,
157.6, 148.8, 137.3, 136.7, 135.4, 132.5, 132.0, 131.4, 129.9, 128.7, 127.7,
121.1, 119.6, 114.4, 113.8, 113.0, 112.4, 101.3, 70.6, 55.2. Anal. Calcd for
C26H18Cl2O5 (481.32): C, 64.88; H, 3.77. Found: C, 64.96; H, 3.90.
100 mg/mL streptomycin. MCF-7 cells were grown in monolayer
cultures, while K562 and MOLT-4 cells were grown in suspension.
Compounds were all first dissolved in DMSO and then diluted
with the growth medium. The final concentration of DMSO in the
wells did not exceed 0.5%. Cell viability following exposure to
compounds 6aes side by side to the reference drug cisplatin was
determined by using the MTT reduction assay [14e,16]. The MCF-7,
K562 and MOLT-4 cells were plated in 96-well microplates at
densities of 30,000, 40,000 and 40,000 cells/ml, respectively
6.1.4.16. (E)-7-((3-Methoxybenzyl)oxy)-3-(3-(4-methoxyphenyl)
acryloyl)-2H-chromen-2-one (6p). Yellow solid; yield 92%; mp:
167e169 ꢂC; IR (KBr, cmꢄ1): 1718 (C]O), 1655 (C]O). 1H NMR
(500 MHz, CDCl3)
d
: 8.58 (s, 1H, H4 coumarin), 7.91 (d, J ¼ 15.5 Hz,
1H, Hb), 7.85 (d, J ¼ 15.5 Hz, 1H, Ha), 7.65 (d, J ¼ 8.2 Hz, 2H, H2,6
phenyl), 7.57 (d, J ¼ 8.7 Hz, 1H, H5 coumarin), 7.34 (t, J ¼ 7.9 Hz, 1H,
H5 benzyloxy), 7.03e6.92 (m, 7H, H3,5 phenyl, H6,8 coumarin and
H2,4,6 benzyloxy), 5.16 (s, 2H, CH2 benzyl), 3.86 (s, 3H, OCH3), 3.84
(100 m
L/well). After overnight incubation at 37 ꢂC, different con-
(s, 3H, OCH3). 13C NMR (125 MHz, CDCl3)
d: 186.2,163.9,161.7,159.9,
centrations of test compounds were added to the wells and the cells
were further incubated for 72 h. Then, the medium was replaced
with fresh medium containing 0.5 mg/mL of MTT. Plates were
incubated for another 4 h at 37 ꢂC, the media was removed and
159.7, 157.4, 148.2, 144.5, 136.8, 131.2, 130.7, 129.9, 127.7, 121.8, 121.6,
119.6, 114.3, 113.8, 113.0, 112.6, 101.3, 70.6, 55.3, 55.2. Anal. Calcd for
C
27H22O6 (442.46): C, 73.29; H, 5.01. Found: C, 73.47; H, 5.34.
formazan crystals formed in the cells were dissolved in 200 mL of
6.1.4.17. (E)-3-(3-(2,3-Dimethoxyphenyl)acryloyl)-7-((3-
methoxybenzyl)oxy)-2H-chromen-2-one (6q). Yellow solid; yield
87%; mp: 142e144 ꢂC; IR (KBr, cmꢄ1): 1719 (C]O), 1654 (C]O). 1H
DMSO. Optical density was measured at 570 nm with background
correction at 655 nm using a Bio-Rad microplate reader (Model
680). Blank wells of all compounds, which contained the same
concentrations of test compounds but did not contain MTT were
run in parallel.
NMR (500 MHz, CDCl3)
d: 8.71 (s, 1H, H4 coumarin), 7.89e7.81 (m,
3H, Ha and H5 coumarin), 7.33e6.93 (m, 9H, H6,8 coumarin, 4H
,b

110
F. Molaverdi et al. / European Journal of Medicinal Chemistry 68 (2013) 103e110
6.2.2. DPPH radical scavenging assay
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This work was supported by grants from the research council of
Tehran University of Medical Sciences, Iran National Elite Founda-
tion (INEF) and Iran National Science Foundation (INSF). The au-
thors also wish to thank the financial support of Shiraz University of
Medical Sciences, Vice Chancellor for Research (Grant 5712).
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.014.