Halogenated flavanones as potential apoptosis-inducing agents: Synthesis and biological activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.10.043

A series of halogenated flavanones were synthesized from 2-hydroxychalcones and tested for their cytotoxicity against a panel of human cancer cell lines. Among the synthesized compounds, 3′,7-dichloroflavanone (2d) showed the highest activity against MCF-7, LNCaP, PC3, Hep-G2, KB and SK-N-MC cells. However, 3′,6-dichloroflavanone (2g) with IC₅₀ value of 2.9 ± 0.9 μM was the most potent compound against MDA-MB-231 cells, being approximately 12 times more active than etoposide as reference drug. According to the flow-cytometric analysis, compound 2g can induce apoptosis by 66.19 and 21.37% in PC3 and MDA-MB-231 cells, respectively. The results of acridine orange/ethidium bromide staining and TUNEL assay suggested that the cytotoxic activity of this compound in PC3 and MDA-MB-231 cells occurs via apoptosis.

Full text of DOI:10.1016/j.ejmech.2012.10.043

European Journal of Medicinal Chemistry 58 (2012) 573e580
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Halogenated flavanones as potential apoptosis-inducing agents: Synthesis and
biological activity evaluation
Maliheh Safavi a, Nasim Esmati b, Sussan Kabudanian Ardestani a, Saeed Emami c, Soheila Ajdari d,
,e,
Jamshid Davoodi a, Abbas Shafiee e, Alireza Foroumadi b
*
a Institute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384, Tehran, Iran
b Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
c Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
d Department of Immunology, Pasteur Institute of Iran, Tehran, Iran
e Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, 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 halogenated flavanones were synthesized from 2-hydroxychalcones and tested for their
cytotoxicity against a panel of human cancer cell lines. Among the synthesized compounds, 30,7-
dichloroflavanone (2d) showed the highest activity against MCF-7, LNCaP, PC3, Hep-G2, KB and SK-N-
Received 21 June 2012
Received in revised form
20 September 2012
Accepted 25 October 2012
Available online 2 November 2012
MC cells. However, 30,6-dichloroflavanone (2g) with IC50 value of 2.9 ꢀ 0.9
mM was the most potent
compound against MDA-MB-231 cells, being approximately 12 times more active than etoposide as
reference drug. According to the flow-cytometric analysis, compound 2g can induce apoptosis by 66.19
and 21.37% in PC3 and MDA-MB-231 cells, respectively. The results of acridine orange/ethidium bromide
staining and TUNEL assay suggested that the cytotoxic activity of this compound in PC3 and MDA-MB-
231 cells occurs via apoptosis.
Keywords:
Anticancer agents
Cytotoxic activity
Apoptosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Flavanones
1. Introduction
have been identified to inhibit proliferation in many kinds of cultured
human cancer cell lines, whereas less or no toxic to human normal
Despite significant investment in the field of cancer chemo-
therapy, limited improvement in patient survival has been achieved
in many countries. Cancer is the second leading cause of death
worldwide after heart diseases [1,2]. Natural or semi-synthetic
compounds may be used to prevent or treat the development of
invasive cancers [2]. Flavonoids are naturally occurring polyphenolic
compounds that have been reported to possess anticancer or anti-
carcinogenic/antimutagenic activities [3]. These compounds possess
a common phenylbenzopyrone structure (C6eC3eC6), consisting of
two aromatic rings linked by three carbons that are usually in an
oxygenated central pyran ring. They are categorized according to the
saturation level and opening of the central pyran ring, mainly into
flavones, isoflavones, flavonols, flavanonols, flavanols and flavanones
(Fig. 1) [1,4,5]. Several beneficial biological effects of flavonoids
including antioxidant, antitumor, and anti-inflammation properties
have been ascertained in several previous studies [6e8]. Flavonoids
cells [9e11]. The significant anticancer properties of flavonoids may
be via induction of apoptosis [5].
Flavanones have been a potential source in the search for new
lead compounds in the field of cancer chemotherapy. Usman et al.
[12] had reported cytotoxic properties of flavanones isolated from
the tree barks of Cryptocarya costata. A study of eight flavanones on
colorectal carcinoma cells indicated that 20-OH flavanone showed
the most potent cytotoxic effect on these cancer cells, and cell death
induced by 20-OH flavanone was via the occurrence of DNA ladders,
apoptotic bodies, and hypodiploid cells, all characteristics of
apoptosis [13]. In 2007, Hsiao et al. described that flavanone and
20-OH flavanone inhibited cell growth of A549, LLC, AGS, SK-Hepl
and HA22T cancer cells, while other flavanones (40-OH flavanone,
6-OH flavanone, naringin and naringenin) showed little or no
inhibition [14]. The results of another study explored that synthetic
flavanone derivatives have strong anti-proliferative effects on
human breast cancer cells by way of p53-mediated apoptosis and
the induction of cell cycle arrest at the G1 phase [15]. Also, Choi
et al. reported that 40,7-dimethoxyflavanone, exhibits potent anti-
cancer activity and induces cell cycle arrest and apoptosis in human
breast cancer MCF-7 cells [16].
* Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
macy and Pharmaceutical Sciences Research Center, Tehran University of Medical
Sciences, Tehran 14176, Iran. Tel.: þ98 21 66406757; fax: þ98 21 66461178.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.043

574
M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
flavanone derivatives 2aek. The structural characterization of
O
O
O
flavanone compounds 2aek is based on their 1H NMR and IR
spectral data. For example, their 1H NMR spectra showed typical
chemical shifts and coupling pattern of the H-2, H-3ax and H-3eq
protons of chroman ring in flavanone structures (Fig. 2). In the 1H
NMR spectra of flavanones (Table 1), the H-2 signal appears at
approximately at 5.24e5.88 ppm as doublet of doublet. The reso-
nances of the diastereotopic H-3ax and H-3eq protons occurred
approximately at 3.03e3.20 and 2.80e2.90 ppm as two doublets of
doublets.
OH
O
O
O
Flavone
Isoflavone
Flavonol
O
O
O
O
OH
OH
The H-3ax and H-3eq of chroman ring are coupled with
a constant of 16.4e17.6 Hz related to the geminal coupling. The
value of the coupling constant between H-2 and H-3ax is too large
(J2,3ax ¼ 12.4e13.6 Hz) which can only arise from a trans-diaxial
coupling, thus H-2 is axial and the 2-aryl group has equatorial
orientation. Furthermore, the value of J2,3eq which is in the range of
2.8e3.2 Hz confirms the conformation of the flavanone structure
(Fig. 2). FT-IR spectra of flavanone derivatives also revealed a very
strong band about 1642e1697 cmꢁ1 related to C]O stretching.
O
Flavanone
Flavanonol
Flavanol
Fig. 1. The main structural categories of flavonoids.
It is now well documented that most cytotoxic anticancer agents
induce apoptosis. Therefore, an attractive method for cancer
chemoprevention or chemotherapy is new therapeutic agents that
induce tumor cells death [17e19]. Because no systemic inflamma-
tory responses have been observed in cells induced to undergo
apoptosis, several studies have suggested that the induction of cell
death via apoptosis reserves a physiological advantage in the
cancer treatment [20,21].
In continuation of our previous work on the synthesis of poten-
tial cytotoxic agents and apoptosis inducers [22e25], and based on
the diverse biological activities of flavanone derivatives, in this
paper we have synthesized and evaluated cytotoxicity and
apoptosis-inducing activity of a series of halogenated flavanones 2
in order to develop novel anticancer agents. These compounds are
small molecules which consist of chromanone ring and aryl ring
attached to the 2 position. Since halogens like chlorine, are very
useful to modulate the electronic and steric characteristics of drugs
and may also influence the hydrophilicehydrophobic balance of the
molecules, thus chlorine substitution on the chromanone ring and
on the C-2 attached phenyl ring was used for structural modification
and modulation of basic pharmacophore of flavanones.
3. Biology
3.1. In vitro cytotoxicity assay
The in vitro cytotoxic activity of the test compounds 2aek
against eight human cancer cell lines include MCF-7, MDA-MB-
231 (human breast cancer), LNCaP, PC3 (human prostate cancer),
Hep-G2 (human liver carcinoma), KB (human nasopharyngeal
epidermoid carcinoma), SK-N-MC (human neuroblastoma) and
K-562 (human erythroleukemic) was assessed using MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colori-
metric assay [26]. The results of cytotoxic assay in comparison with
etoposide were listed in Table 2.
3.2. Acridine orange/ethidium bromide staining method
Apoptosis was determined morphologically after staining PC3
and MDA-MB-231 cells with acridine orange/ethidium bromide
using fluorescence microscopy according to the previously
described method [27]. Acridine orange penetrates into living and
dead cells, emitting green fluorescence as a result of intercalation in
double-stranded DNA. Ethidium bromide emits red fluorescence
after intercalation in DNA of cells with an altered cell membrane.
Ethidium bromide staining due to loss of membrane integrity
identifies the population of late stage of apoptotic cells and necrotic
cells. Analysis of the acridine orange/ethidium bromide staining of
the synthetic compounds 2d and 2g are shown in Fig. 3.
2. Chemistry
A general synthesis of the flavanone derivatives 2aek is shown
in Scheme 1. The condensation of 2-hydroxyacetophenone deriva-
tives with the corresponding aldehyde in a basic media afforded
hydroxychalcones 1aek. The compounds 1aek were cyclized in
refluxing ethanol in the presence of sodium acetate to give
O
O
O
NaOH
H
+
R
R'
R'
R
EtOH
OH
OH
1a-k
O
NaOAc, EtOH
Reflux
R = H, Cl
R' = H, Cl
R
O
R'
2a-k
Scheme 1. Synthesis of flavanone derivatives.

M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
575
2b and 2c followed by 2d and 2e (7-chloroflavanone derivatives
2bee) were the most active compounds being more potent than
etoposide. The activity of compounds 2gei against SK-N-MC was
comparable to that of etoposide. The IC50 values of compounds
against K-562 cell line revealed that flavanones with no substituent
on chroman ring and possessing chlorine on phenyl group
(compounds 2iek) showed good growth inhibitory activity at
Hax
8
7
1
Areq
2
O
6
3
Heq
4
5
concentrations less than 25 m
M. Among them, 30-chloro-analog 2j
was the most potent compound against K-562 cell line.
Hax
O
The comparison of parent compound 2a with chlorophenyl
analogs 2iek demonstrated that the introduction of a chlorine
atom on the 2-phenyl ring of flavanone increases the tumor growth
inhibitory effects of flavanone against all cell lines. The cytotoxic
activity of 7-chloro compound 2b was more than that of parent
compound 2a. Thus, the presence of chloro group on chroman ring
was favorable for cytotoxic activity. Although, chloro substituent on
chroman ring or 2-phenyl group individually could increase the
activity, but the combination pattern of substituents had a different
effect on various cell lines. However, the highest activity was
observed with 30-chloro analogs (2d against MCF-7, LNCaP, PC3,
Hep-G2, KB and SK-N-MC, 2g against MDA-MB-231 and 2j against
K-562).
Fig. 2. Atom numbering and half-chair conformation of flavanone structure in which
2-aryl group exists predominantly in the equatorial orientation.
3.3. Terminal deoxynucleotidyl transferase mediated dUTP nick end
labeling (TUNEL) assay
For detection of early stages of apoptosis in PC3 and MDA-MB-
231 cells, TUNEL (terminal deoxynucleotidyl transferase mediated
dUTP nick end labeling) analysis was used. Generation of free 30-OH
DNA fragments was determined using TUNEL analysis and quan-
tified by flow cytometry. The results are shown in Fig. 4.
Among the halogenated compounds described in this study, the
cytotoxic activity of 40-chloroflavanone (2k) against MCF-7 and
MDA-MB-453 cells has been reported [15]. This compound was
included in our study for comparing its activity with poly-
chlorinated compounds and other regio-isomers. As seen from
data, our compounds for example 30,7-dichloroflavanone (2d) had
a better profile of cytotoxic activity in comparison to that of
40-chloroflavanone (2k).
4. Results and discussion
All the synthesized compounds 2aek were evaluated for their
cytotoxic effects on a panel of eight human tumor cell lines. For
each compound, 50% inhibitory concentration (IC50) was calculated
and presented in Table 2.
Results of the MTT assay showed that flavanones with different
chloro-substituent exhibited differential cytotoxicity in various
human cancer cells lines. Although the unsubstituted flavanone
2a showed marginal or no activity against all cell lines (IC50
The synthetic compounds 2d and 2g were used to identify
apoptotic or necrotic cell death in MDA-MB-231 and PC3 cell lines.
Since there is an essential need to develop drugs for ‘triple-nega-
tive’ breast cancer e a tumor subtype that is categorized as being
aggressive and lacking effective targeted treatments, such as
endocrine therapies and anti-HER2 strategies [28] and because of
potent activity of compound 2g against this kind of tumor cell line
(MDA-MB-231), our laboratory used MDA-MB-231 and androgen-
refractory and chemotherapy-resistant PC3 prostate cancer cell
line, to elucidate the death type induced by synthetic compounds.
Analysis of the acridine orange/ethidium bromide staining
revealed that the synthetic compounds 2d and 2g reduced cell
viability and induced apoptosis in human prostate and breast
cancer cell lines. The appearance of chromatin condensation,
nuclear fragmentation and shift from early to late apoptosis are
evident in Fig. 3. In this figure, viable cells are uniformly green,
early apoptotic cells are green and contained chromatin
condensation and nuclear fragmentation. Late apoptotic cells
incorporate ethidium bromide and therefore stained orange and
in contrast to necrotic cells, they show condense and often
fragmented nuclei.
values > 50
mM) but chloro-substituted compounds exhibited
significant activity against various cell lines with the exception of
K-562 cells.
Among the tested compounds, compound 2d with chlorine on 30
and 7 positions showed the highest cytotoxic activity against MCF-
7, LNCaP, PC3, Hep-G2, KB and SK-N-MC cells. Against MDA-MB-231
cell line, 30,6-dichloroflavanone (2g) was the most potent
compound with IC50 value of 2.9 ꢀ 0.9
mM. This compound was
approximately 12 times more active than etoposide as reference
drug. In the case of human neuroblastoma (SK-N-MC), compounds
Table 1
Selected 1H NMR spectral data of compound 2aek.
O
R
These observations were confirmed by the results obtained in
the TUNEL assay, which detects early stages of apoptosis. We used
TUNEL analysis as a quantitative method of determining apoptosis
(Fig. 4(A) and (B)). Fig. 4(C) and (D) summarizes the data and shows
a highly significant increase in the apoptotic index (as demon-
strated by an increase in the fluorescein isothiocyanate (FITC)-
positive fraction) in PC3 and MDA-MB-231 cells treated with
synthetic compounds compared with negative control. The results
also revealed that the percentage of PC3 cells undergoing apoptosis
after 12 h of exposure to compounds 2d and 2g was higher than
etoposide (49.48% and 66.19% apoptosis in the 2d- and 2g-treated
cells, respectively versus 17.25% in etoposide-treated cells). Expo-
sure of MDA-MB-231 cells to IC50 concentrations of 2d and 2g
induced apoptosis in 44.66% and 21.67% of the cells after 12 h,
O
R'
Compound
R
R0
d
H-2
d
H-3
2J3ax,3eq 3J2ax,3eq 3J2ax,3ax
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
7-Cl
H
H
5.24
5.49
3.2
2.88 16.8
3.2
2.8
2.8
3.0
3.0
2.8
3.0
3.0
2.8
2.8
2.8
13.2
12.8
13.6
12.8
12.9
13.6
12.8
12.8
13.2
12.4
12.8
3.08 2.90 17.2
3.03 2.81 16.8
3.09 2.90 16.8
3.03 2.89 16.8
3.05 2.89 17.6
3.09 2.90 16.8
3.09 2.90 16.8
3.04 2.89 16.8
3.03 2.89 16.4
3.04 2.88 16.8
7-Cl 20-Cl 5.88
7-Cl 30-Cl 5.47
7-Cl 40-Cl 5.47
6-Cl 20-Cl 5.87
6-Cl 30-Cl 5.47
6-Cl 40-Cl 5.47
H
H
H
20-Cl 5.88
30-Cl 5.46
40-Cl 5.47
2j
2k

576
M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
Table 2
Cytotoxic activity (IC50
,
m
M)a of synthetic flavanone against a panel of cancer cell line after 48 h treatment.
O
R
O
R'
Compd
R
R0
MCF-7
MDA-MB-231
LNCaP
PC3
Hep-G2
KB
SK-N-MC
K-562
2a
2b
2c
2d
2e
2f
2g
2h
H
H
H
>100
51.8 ꢀ 2.4
8.2 ꢀ 2.9
7.7 ꢀ 1.8
10.9 ꢀ 2.5
9.4 ꢀ 1.3
36.2 ꢀ 5.9
2.9 ꢀ 0.9
15.3 ꢀ 5.5
20.3 ꢀ 4.3
38.3 ꢀ 5.7
44.3 ꢀ 3.9
34.9 ꢀ 4.2
94.6 ꢀ 12.7
59.5 ꢀ 4.9
>100
55.4 ꢀ 7.1
17.7 ꢀ 7.4
>100
>100
>100
77.5 ꢀ 10.6
10.3 ꢀ 2.3
10.9 ꢀ 1.1
12.8 ꢀ 2.1
16.2 ꢀ 1.6
>100
24.0 ꢀ 0.7
23.1 ꢀ 4.6
24.0 ꢀ 7.4
32.2 ꢀ 2.8
38.2 ꢀ 5.5
23.9 ꢀ 1.8
>100
>100
>100
7-Cl
7-Cl
7-Cl
7-Cl
6-Cl
6-Cl
6-Cl
H
19.3 ꢀ 6.0
20.7 ꢀ 8.2
11.3 ꢀ 0.4
13.0 ꢀ 2.6
>100
25.8 ꢀ 2.1
14.5 ꢀ 2.0
19.3 ꢀ 6.2
23.4 ꢀ 9.7
22.9 ꢀ 3.4
31.3 ꢀ 2.5
9.8 ꢀ 1.5
25.2 ꢀ 4.4
47.3 ꢀ 12.3
14.7 ꢀ 0.2
>100
20-Cl
30-Cl
40-Cl
20-Cl
30-Cl
40-Cl
20-Cl
30-Cl
40-Cl
>100
35.9 ꢀ 6.6
58.1 ꢀ 8.3
53.1 ꢀ 4.1
38.1 ꢀ 4.4
55.7 ꢀ 9.5
57.2 ꢀ 4.2
59.9 ꢀ 10.6
56.0 ꢀ 7.0
16.8 ꢀ 2.1
11.6 ꢀ 4.1
12.4 ꢀ 4.5
27.4 ꢀ 1.4
12.2 ꢀ 4.2
47.9 ꢀ 7.5
43.8 ꢀ 5.1
51.3 ꢀ 8.5
28.3 ꢀ 7.7
36.9 ꢀ 4.7
5.7 ꢀ 0.3
21.9 ꢀ 7.2
>100
20.7 ꢀ 3.8
13.7 ꢀ 0.1
11.0 ꢀ 1.5
13.7 ꢀ 0.1
32.5 ꢀ 0.1
19.2 ꢀ 3.1
78.8 ꢀ 9.5
>100
>100
>100
>100
>100
22.9 ꢀ 0.1
18.6 ꢀ 6.6
21.5 ꢀ 2.7
14.3 ꢀ 2.8
76.9 ꢀ 13.4
>100
2i
2j
>100
H
H
26.9 ꢀ 3.5
32.5 ꢀ 5.0
9.9 ꢀ 2.8
2k
Etoposide
a
Data are mean ꢀ SD of 3 independent experiments (n ¼ 3).
respectively. These data suggested that the cytotoxic activity of 2d
and 2g in PC3 and MDA-MB-231 cells occurs via apoptosis.
flow-cytometric analysis, compound 2g can induce apoptosis by
66.19 and 21.37% in PC3 and MDA-MB-231 cells, respectively. The
results of acridine orange/ethidium bromide staining and TUNEL
assay suggested that the cytotoxic activity of this compound in PC3
and MDA-MB-231 cells occurs via apoptosis.
5. Conclusion
It has been demonstrated that the modulation of flavanone
structure could increase antitumor activity. Thus, chlorine substi-
tution on the chromanone ring and on the C-2 attached phenyl ring
was used for structural modification and modulation of basic
pharmacophore of flavanones. Therefore, a series of halogenated
flavanones were synthesized from 2-hydroxychalcone derivatives
and tested for their cytotoxicity against a panel of human cancer cell
lines. Among the synthesized compounds, 30,7-dichloroflavanone
(2d) showed the better profile of cytotoxicity. However, 30,6-
6. Experimental protocols
All chemicals and solvents used in this study were purchased
from Merck chemical. Merck silica gel F254 plates were used for
analytical TLC. Melting points were determined on a Kofler hot
stage apparatus and are uncorrected. The IR spectra were obtained
on a Shimadzu 470 spectrophotometer (potassium bromide disks).
The 1H NMR and 13C NMR spectra were recorded using Bruker 400
and Bruker 500 spectrometers, respectively. Chemical shifts are
reported in parts per million (ppm) relative to TMS as internal
standard.
dichloroflavanone (2g) with IC50 value of 2.9 ꢀ 0.9
mM was the
most potent compound against MDA-MB-231 cells, being approxi-
mately 12 times more active than etoposide. According to the
Fig. 3. Acridine orange/ethidium bromide double staining of PC3 (A) and MDA-MB-231 (B) cells with characteristic symptoms of apoptosis: a DMSO 1% as control, b, c and d, cells
treated with IC50 concentrations of etoposide, compounds 2d and 2g, respectively for 12 h. White arrow indicates live cells, dotted arrow shows early phase of apoptosis and dashed
arrow indicates late phase of apoptosis. The images of cells were taken with a fluorescence microscope at 400ꢂ.

M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
577
Fig. 4. Flow-cytometric analysis of PC3 and MDA-MB-231 cells. Generation of free 30-OH DNA fragments was determined using TUNEL analysis and quantitated by flow cytometry.
(A) PC3 cells treated with DMSO 1% (negative control) or with IC50 values of etoposide (positive control) and compounds 2d and 2g for 12 h. (B) MDA-MB-231 cells treated with
DMSO 1% (negative control) or with IC50 values of etoposide (positive control) and 2d and 2g for 12 h. Percentage of FITC-positive events (% apoptotic cells) was calculated (C); and
(D) % apoptotic cells of PC3 and MDA-MB-231 cell lines treated with DMSO 1% (negative control) or with IC50 values of etoposide (positive control), compounds 2d and 2g
respectively for 12 h.
6.1. General procedure for the synthesis of 2-hydroxychalcones 1
vacuo. The residues were purified by column chromatography.
Compounds 1a and 1feh, were prepared according to the previ-
ously reported method [29].
To a well stirred solution of the aldehyde (10 mmol) and the
appropriately substituted acetophenone (10 mmol) in methanol
(30 ml), was added 50% NaOH aqueous solution (1 ml) and the
reaction was stirred overnight at room temperature. The precipi-
tated solid was filtered and recrystallized from hot methanol to
give pure products. After completion of the reaction if the solid
was not precipitated, the solution was neutralized with diluted HCl
solution (1 N) and extracted with chloroform. The combined
organic layers were dried (MgSO4), filtered, and evaporated in
6.1.1. (E)-1-(4-chloro-2-hydroxyphenyl)-3-phenylprop-2-en-1-one
(1b)
Yield 85%; m.p. 118e119 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3300e3440
: 13.01 (s, 1H, OH),
(OH), 1638 (C]O); 1H NMR (400 MHz, CDCl3)
d
7.94 (d, 1H alkene, J ¼ 16 Hz), 7.85 (d, 1H phenyl, J ¼ 8.4 Hz), 7.67e
7.70 (m, 2H phenyl), 7.59 (d, 1H alkene, J ¼ 16 Hz), 7.41e7.46 (m, 3H,
phenyl), 7.06 (s, 1H phenyl), 6.93 (d, 1H phenyl, J ¼ 8.4 Hz).

578
M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
6.1.2. (E)-1-(4-chloro-2-hydroxyphenyl)-3-(2-chlorophenyl)prop-
chroman), 7.37e7.53 (m, 5H, phenyl), 7.02e7.09 (m, 3H, H6, H7 and
H8 chroman), 5.24 (dd,1H, J ¼ 3.2 and 13.2 Hz, H2 chroman), 3.2 (dd,
1H, J ¼ 13.2 and 16.8 Hz, H3 chroman), 2.88 (dd, 1H, J ¼ 3.2 and
2-en-1-one (1c)
Yield 81%; m.p. 134e135 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3300e3440
(OH), 1646 (C]O); 1H NMR (400 MHz, CDCl3)
d
: 12.94 (s, 1H, OH),
16.8 Hz, H3 chroman). 13C NMR (125 MHz, CDCl3)
d: 191.96, 161.53,
8.33 (d, 1H alkene, J ¼ 15.6 Hz), 7.83 (d, 1H phenyl, J ¼ 8.8 Hz), 7.76
(d, 1H phenyl, J ¼ 7.2 Hz), 7.57 (d, 1H alkene, J ¼ 15.6 HZ), 7.48 (d, 1H
phenyl, J ¼ 7.2 Hz), 7.32e7.40 (m, 2H phenyl), 7.06 (d, 1H phenyl,
J ¼ 2.4 Hz), 6.93 (d, 1H phenyl, J ¼ 8.8 Hz).
138.71, 136.18, 128.84, 128.76, 127.03, 126.13, 121.60, 120.91, 118.11,
79.58, 44.65. Anal. Calcd for C15H12O2: C, 80.34; H, 5.39. Found: C,
79.98; H, 5.01.
6.2.2. 7-Chloro-2-phenylchroman-4-one (2b)
6.1.3. (E)-1-(4-chloro-2-hydroxyphenyl)-3-(3-chlorophenyl)prop-
Yield 67%; m.p. 87e88 ꢃC; IR (KBr, cmꢁ1 nmax: 1676 (C]O); 1
) H
2-en-1-one (1d)
NMR (400 MHz, CDCl3)
d
: 7.86 (d,1H, J ¼ 8.6 Hz, H5 chroman), 7.39e
Yield 76%; m.p. 124e125 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3340e3461
7.46 (m, 5H, H phenyl), 7.08 (d, 1H, J ¼ 1.6 Hz, H8 chroman), 7.03
(d, 1H, J ¼ 1.6 and 8.6 Hz, H6 chroman), 5.49 (dd, 1H, J ¼ 2.8 and
12.8 Hz, H2 chroman), 3.08 (dd, 1H, J ¼ 12.8 and 17.2 Hz, H3 chro-
man), 2.90 (dd, 1H, J ¼ 2.8 and 17.2 Hz, H3 chroman). 13C NMR
(OH), 1642 (C]O); 1H NMR (400 MHz, CDCl3)
d: 12.92 (s, 1H, OH),
7.84e7.88 (m, 2H phenyl & alkene), 7.67 (s, 1H phenyl), 7.58 (d, 1H
alkene, J ¼ 15.2 Hz), 7.50e7.54 (m, 2H phenyl), 7.40 (d, 1H phenyl,
J ¼ 7.6 Hz), 7.06 (s, 1H phenyl), 6.95 (d, 1H phenyl, J ¼ 8.8 Hz).
(125 MHz, CDCl3) d: 190.39, 161.57, 142.16, 136.72, 134.79, 129.11,
128.31, 127.47, 122.64, 119.44, 118.27, 79.21, 44.26. Anal. Calcd for
C15H11ClO2: C, 69.64; H, 4.29. Found: C, 69.34; H, 4.31.
6.1.4. (E)-1-(4-chloro-2-hydroxyphenyl)-3-(4-chlorophenyl)prop-
2-en-1-one (1e)
Yield 79%; m.p. 129e130 ꢃC; IR (KBr, cmꢁ1
3421(OH), 1635 (C]O); 1H NMR (400 MHz, CDCl3)
)
d
nmax: 3400e
: 12.90 (s, 1H,
6.2.3. 7-Chloro-2-(2-chlorophenyl)chroman-4-one (2c)
Yield 65%; m.p. 62e64 ꢃC; IR (KBr, cmꢁ1 nmax: 1642 (C]O); 1H
)
OH), 7.88 (d, 1H alkene, J ¼ 16 Hz), 7.83 (d, 2H phenyl, J ¼ 8 Hz), 7.60
(d, 1H phenyl, J ¼ 8.8 Hz), 7.55 (d, 1H alkene, J ¼ 16 Hz), 7.42 (d, 2H
phenyl, J ¼ 8 Hz), 7.05 (d, 1H phenyl, J ¼ 2 Hz), 6.93 (dd, 1H phenyl,
J ¼ 8.8 and 2 Hz).
NMR (400 MHz, CDCl3)
d
: 7.89 (d, 1H, J ¼ 8.2 Hz, H5 chroman), 7.45
0
0
0
(d, 1H, J ¼ 7.6 Hz, H3 ), 7.38e7.43 (m, 2H, H4 and H5 ), 7.33 (d, 1H,
0
J ¼ 7.2 Hz, H6 ), 7.10 (d, 1H, J ¼ 1.6 Hz, H8 chroman), 7.06 (dd, 1H,
J ¼ 1.6 and 8.2 Hz, H6 chroman), 5.88 (dd, 1H, J ¼ 2.8 and 13.6 Hz, H2
chroman), 3.03 (dd, 1H, J ¼ 2.8 and 16.8 Hz, H3 chroman), 2.81
(dd, 1H, J ¼ 13.6 and 16.8 Hz, H3 chroman). Anal. Calcd for
6.1.5. (E)-3-(2-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (1i)
C15H10Cl2O2: C, 61.46; H, 3.44. Found: C, 61.32; H, 3.32.
Yield 83%; m.p. 121e123 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3428 (OH),1637
(C]O); 1H NMR (400 MHz, CDCl3)
d
: 12.73 (s, 1H, OH), 8.31 (d, 1H
6.2.4. 7-Chloro-2-(3-chlorophenyl)chroman-4-one (2d)
Yield 58%; m.p. 93e94 ꢃC; IR (KBr, cmꢁ1 nmax: 1697 (C]O); 1H
: 7.86 (d, 1H, J ¼ 8.8 Hz, H5 chroman), 7.49
alkene, J ¼ 15.6 Hz), 7.91 (d, 1H phenyl, J ¼ 7.6 Hz), 7.77 (d, 1H
phenyl, J ¼ 6.8 Hz), 7.63 (d,1H alkene, J ¼ 15.6 Hz), 7.46e7.54 (m, 2H
phenyl), 7.37e7.40 (m, 2H phenyl),7.05 (d, 1H phenyl, J ¼ 8.8 Hz),
6.93 (t, 1H phenyl, J ¼ 7.6 Hz).
)
NMR (400 MHz, CDCl3)
d
0
0
0
0
(s, 1H, H2 ), 7.37 (m, 1H, H5 ), 7.30e7.36 (m, 2H, H4 and H6 ), 7.10
(d, 1H, J ¼ 2.2 Hz, H8 chroman), 7.05 (dd, 1H, J ¼ 2.2 and 8.8 Hz, H6
chroman), 5.47 (dd, 1H, J ¼ 3 and 12.8 Hz, H2 chroman), 3.09 (dd,
1H, J ¼ 12.8 and 16.8 Hz, H3 chroman), 2.90 (dd, 1H, J ¼ 3 and
6.1.6. (E)-3-(3-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one (1j)
16.8 Hz, H3 chroman). 13C NMR (125 MHz, CDCl3)
d: 190.24, 161.50,
Yield 87%; m.p.101e103 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3438 (OH),1642
142.20, 140.24, 134.91, 130.21, 129.05, 128.33, 126.30, 124.10, 122.71,
119.44, 118.29, 79.14, 44.32. Anal. Calcd for C15H10Cl2O2: C, 61.46; H,
3.44. Found: C, 61.39; H, 3.34.
(C]O); 1H NMR (400 MHz, CDCl3)
d: 12.78 (s, 1H, OH), 7.92 (d, 1H
phenyl, J ¼ 8.4 Hz), 7.85 (d, 1H alkene, J ¼ 16 Hz), 7.63e7.67 (m, 2H
phenyl & alkene), 7.52 (t, 2H phenyl, J ¼ 6.8 Hz), 7.36e7.41 (m, 2H
phenyl), 7.04 (d,1H phenyl, J ¼ 8.4 Hz), 6.96 (t,1H phenyl, J ¼ 7.8 Hz).
6.2.5. 7-Chloro-2-(4-chlorophenyl)chroman-4-one (2e)
Yield 54%; m.p. 85e86 ꢃC; IR (KBr, cmꢁ1 nmax: 1677 (C]O); 1H
)
0
0
6.1.7. (E)-3-(4-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
NMR (400 MHz, CDCl3)
d
: 7.86 (d, 2H, J ¼ 8.4 Hz, H3 and H5 ), 7.30e
one (1k)
7.50 (m, 2H, H5 and H6 chroman), 7.08 (s, 1H, H8 chroman), 7.05
Yield 86%; m.p.148e149 ꢃC; IR (KBr, cmꢁ1
)
nmax: 3432 (OH),1631
(d, 2H, J ¼ 8.4 Hz, H2 and H6 ), 5.47 (dd, 1H, J ¼ 3 and 12.9 Hz, H2
0
0
(C]O); 1H NMR (400 MHz, CDCl3)
d: 12.78 (s, 1H, OH), 7.92 (d, 1H
chroman), 3.03 (dd, 1H, J ¼ 12.9 and 16.8 Hz, H3 chroman), 2.89 (dd,
phenyl, J ¼ 8.4 Hz), 7.85 (d, 1H alkene, J ¼ 16 Hz), 7.63e7.67 (m, 2H
phenyl & alkene), 7.52 (t, 2H phenyl, J ¼ 6.8 Hz), 7.36e7.41 (m, 2H
phenyl), 7.04 (d,1H phenyl, J ¼ 8.4 Hz), 6.96 (t,1H phenyl, J ¼ 7.8 Hz).
1H, J ¼ 3 and 16.8 Hz, H3 chroman). 13C NMR (125 MHz, CDCl3)
d:
190.32, 161.51, 142.09, 136.70, 134.73, 129.07, 128.26, 127.44, 122.58,
119.40, 118.23, 79.16, 44.20. Anal. Calcd for C15H10Cl2O2: C, 61.46; H,
3.44. Found: C, 61.35; H, 3.41.
6.2. General procedure for the synthesis of flavanones 2aek
6.2.6. 6-Chloro-2-(2-chlorophenyl)chroman-4-one (2f)
To a well stirred solution of an appropriate chalcone (1 mmol)
and sodium acetate (500 mg) in ethanol (5 ml), was added 3 drops
of water and the mixture was refluxed overnight. The reaction
mixture was poured into cold water and extracted with ethyl
acetate. The organic phase was washed with saturated NaCl solu-
tion and dried (Na2SO4). After removing the solvent, the residue
was purified by column chromatography using petroleum ether/
ethyl acetate (9:1) as eluent to give compound 2aek.
Yield 57%; m.p. 110e112 ꢃC; IR (KBr, cmꢁ1 nmax: 1696 (C]O); 1H
)
NMR (400 MHz, CDCl3) d: 7.92 (s, 1H, H5 chroman), 7.73 (d, 1H,
J ¼ 8 Hz, H7 chroman), 7.32e7.48 (m, 4H phenyl), 7.04 (d, 1H,
J ¼ 8 Hz, H8 chroman), 5.87 (dd, 1H, J ¼ 2.8 and 13.6 Hz, H2 chro-
man), 3.05 (dd, 1H, J ¼ 2.8 and 17.6 Hz, H3 chroman), 2.89 (dd, 1H,
J ¼ 13.6 and 17.6 Hz, H3 chroman). Anal. Calcd for C15H10Cl2O2: C,
61.46; H, 3.44. Found: C, 61.07; H, 3.46.
6.2.7. 6-Chloro-2-(3-chlorophenyl)chroman-4-one (2g)
6.2.1. 2-Phenylchroman-4-one (2a)
Yield 58%; m.p.129e130 ꢃC; IR (KBr, cmꢁ1 nmax: 1689 (C]O); 1H
)
Yield 89%; m.p. 76e78 ꢃC; IR (KBr, cmꢁ1
)
nmax: 1696 (C]O); 1H
NMR (400 MHz, CDCl3)
d
: 7.86 (d, 1H, J ¼ 2.8 Hz, H5 chroman), 7.49
0
0
0
0
NMR (400 MHz, CDCl3)
d: 7.93 (dd, 1H, J ¼ 1.2 and 8 Hz, H5
(s, 1H, H2 ), 7.37 (m, 1H, H5 ), 7.30e7.36 (m, 2H, H4 and H6 ), 7.10

M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
579
(d, 1H, J ¼ 7.2 Hz, H8 chroman), 7.05 (dd, 1H, J ¼ 2.8 and 7.2 Hz, H7
chroman), 5.47 (dd, 1H, J ¼ 3 and 12.8 Hz, H2 chroman), 3.09 (dd,
1H, J ¼ 12.8 and 16.8 Hz, H3 chroman), 2.90 (dd, 1H, J ¼ 3 and
16.8 Hz, H3 chroman). Anal. Calcd for C15H10Cl2O2: C, 61.46; H, 3.44.
Found: C, 61.32; H, 3.32.
trypsinized and diluted in complete growth medium to give a total
cell count of 5 ꢂ 104 cells/ml. The cell suspension (195
ml) was
seeded into the 96-well plates (Nunc, Denmark). The plates were
incubated overnight in a humidified air atmosphere at 37 ꢃC with
5% CO2. After plating, 5 ml of a serial dilution of each compound was
added per well in triplicate. In each plate, there were three control
wells (cells without test compounds) and three blank wells (the
medium with 1% DMSO) for cell viability. Synthetic compounds
were applied in DMSO, and the solvent reached a concentration not
higher than 1% in all experiments. Etoposide was used as positive
control for cytotoxicity. The plates were incubated for further 48 h.
After treatment, the medium was removed and 200 ml phenol
red-free medium containing MTT (1 mg/ml), was added to wells,
followed by 4 h incubation. After incubation, the culture medium
6.2.8. 6-Chloro-2-(4-chlorophenyl)chroman-4-one (2h)
Yield 61%; m.p. 116e117 ꢃC; IR (KBr, cmꢁ1 nmax: 1693 (C]O); 1H
)
NMR (400 MHz, CDCl3)
d
: 7.89 (d, 1H, J ¼ 2.8 Hz, H5 chroman), 7.47
0
(dd, 1H, J ¼ 2.8 and 8.8 Hz, H7 chroman), 7.37 (d, 2H, J ¼ 8.6 Hz, H3
0
and H5 ), 7.10 (d, 1H, J ¼ 2.2 Hz, H8 chroman), 7.05 (d, 2H, J ¼ 8.6 Hz,
0
0
H2 and H6 ), 5.47 (dd, 1H, J ¼ 3 and 12.8 Hz, H2 chroman), 3.09 (dd,
1H, J ¼ 12.8 and 16.8 Hz, H3 chroman), 2.90 (dd, 1H, J ¼ 3 and
16.8 Hz, H3 chroman). 13C NMR (125 MHz, CDCl3)
d: 190.41, 159.94,
136.26, 131.64, 129.78, 127.99, 127.47, 127.13, 126.50, 121.72, 119.81,
76.99, 43.14. Anal. Calcd for C15H10Cl2O2: C, 61.46; H, 3.44. Found: C,
61.11; H, 3.15.
was replaced with 100 ml of DMSO and the absorbance of each well
was measured by using a microplate reader (Gen5, Power wave xs2,
BioTek, America) at 492 nm wavelengths. For each compound, the
concentration causing 50% cell growth inhibition (IC50) compared
with the control was calculated from concentrationeresponse
curves by regression analysis.
6.2.9. 2-(2-Chlorophenyl)chroman-4-one (ꢁ21i)
)
nmax: 1672 (C]O); 1H
Yield 68%; m.p. 81e83 ꢃC; IR (KBr, cm
NMR (400 MHz, CDCl3)
d
: 7.97 (d, 1H, J ¼ 8.4 Hz, H5 chroman), 7.76
0
(d, 1H, J ¼ 7.6 Hz, H3 ), 7.53 (t, 1H, J ¼ 7.2 Hz, H7 chroman), 7.32e7.41
(m, 3H phenyl), 7.07e7.11 (m, 2H, H6,8 chroman), 5.88 (dd, 1H,
J ¼ 2.8 and 13.2 Hz, H2 chroman), 3.04 (dd, 1H, J ¼ 2.8 and 16.8 Hz,
H3 chroman), 2.89 (dd, 1H, J ¼ 13.2 and 16.8 Hz, H3 chroman). Anal.
Calcd for C15H11ClO2: C, 69.64; H, 4.29. Found: C, 69.46; H, 4.13.
6.3.3. Acridine orange/ethidium bromide staining method
Apoptosis was determined morphologically after staining cells
with acridine orange/ethidium bromide using fluorescence
microscopy [27]. PC3 and MDA-MB-231 cells grown in 12-well
plates (50,000 cells/well) were treated with and without IC50
concentration of compounds 2d and 2g for 12 h. After washing
three times with phosphate buffered saline (PBS), the cells were
6.2.10. 2-(3-Chlorophenyl)chroman-4-oneꢁ(21 j)
Yield 61%; m.p. 94e95 ꢃC; IR (KBr, cm
)
nmax: 1664 (C]O); 1H
stained with 100
bromide (1:1, 100
m
m
l of a mixture of acridine orange and ethidium
NMR (400 MHz, CDCl3)
d
: 7.93 (d, 1H, J ¼ 8 Hz, H5 chroman), 7.55
g/ml) solutions. Stained cell suspension (10 l)
m
0
(s, 1H, H2 ), 7.53 (t, 1H, J ¼ 7 Hz, H7 chroman), 7.34e7.38 (m, 3H
phenyl), 7.06e7.10 (m, 2H, H6,8 chroman), 5.46 (dd, 1H, J ¼ 2.8 and
12.4 Hz, H2 chroman), 3.03 (dd, 1H, J ¼ 12.4 and 16.4 Hz, H3 chro-
man), 2.89 (dd, 1H, J ¼ 2.8 and 16.4 Hz, H3 chroman). 13C NMR
were placed on a clean microscope slide and covered with
a coverslip. The cells were immediately analyzed by fluorescence
microscope (Axoscope 2 plus, Zeiss, Germany). All experiments
were repeated three times and the cells with condensed or frag-
mented nuclei were counted as apoptotic cells.
(125 MHz, CDCl3) d: 191.36, 161.23, 140.78, 136.33, 134.83, 130.14,
128.87, 127.10, 126.34, 124.14, 121.87, 120.90, 118.10, 78.76, 44.65.
Anal. Calcd for C15H11ClO2: C, 69.64; H, 4.29. Found: C, 69.28; H,
4.38.
6.3.4. TUNEL assay
Apoptosis-induced nuclear DNA fragmentation was detected
using in situ cell death detection kit (fluorescein, Roche) following
the manufacturer’s protocol. Briefly, 12 h following treating the PC3
and MDA-MB-231 cells with and without potent compounds 2d
and 2g at concentrations of IC50, cells were washed with PBS (pH
7.4) and fixed in 4% paraformaldehyde prepared freshly in PBS. The
cells were again washed with PBS and permeabilised using 0.1%
triton X 100 in 0.1% sodium citrate for 2 min on ice. After washing,
the cells were incubated in the TdT incubation buffer (fluorescein/
dNTP mix, TdT and labeling buffer) for 60 min at 37 ꢃC in
a humidified atmosphere in the dark and then washed and resus-
6.2.11. 2-(4-Chlorophenyl)chroman-4-one (ꢁ21k)
)
nmax: 1678 (C]O); 1H
Yield 62%; m.p.138e139 ꢃC; IR (KBr, cm
NMR (400 MHz, CDCl3)
d
: 7.93 (d, 1H, J ¼ 8 Hz, H5 chroman), 7.52
(t, 1H, J ¼ 8 Hz, H7 chroman), 7.39e7.44 (m, 4H phenyl), 7.04e7.09
(m, 2H, H6,8 chroman), 5.47 (dd, 1H, J ¼ 2.8 and 12.8 Hz, H2 chro-
man), 3.04 (dd, 1H, J ¼ 12.8 and 16.8 Hz, H3 chroman), 2.88 (dd, 1H,
J ¼ 2.8 and 16.8 Hz, H3 chroman). Anal. Calcd for C15H11ClO2: C,
69.64; H, 4.29. Found: C, 69.63; H, 4.37.
6.3. Biology
pended in 500 ml PBS. In humid chamber terminal deoxynucleotidyl
transferase (TdT) catalyzes polymerization of fluorescein-labeled
deoxynucleotides to free 30-OH DNA ends in a template-indepen-
dent manner. Free 30-OH DNA in apoptotic cells was detected and
quantified based on green fluorescence by flow cytometer (FACS-
can, LYSIS II, Becton Dickinson).
6.3.1. Cell lines and cell culture
Eight human cancer cell lines include MCF-7, MDA-MB-231
(human breast cancer), LNCaP, PC3 (human prostate cancer), Hep-
G2 (human liver carcinoma), KB (human nasopharyngeal epider-
moid carcinoma), SK-N-MC (human neuroblastoma) and K-562
(human erythroleukemic) were purchased from National Cell Bank
of Iran (NCBI). The cells were maintained in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum (from
GibcoeBRL, UK) and 100 mg/ml streptomycin and 100 U/ml peni-
cillin at 37 ꢃC in a humidified atmosphere with 5% CO2 in air.
Acknowledgments
This work was supported by grants from the research council of
Tehran University of Medical Sciences and Iran National Science
Foundation.
6.3.2. In vitro cytotoxicity assay
References
The in vitro cytotoxic activity of the test compounds 2aek was
assessed in comparison with etoposide using MTT colorimetric
assay [26]. Briefly, cultures in the exponential growth phase were
[1] W.Y. Huang, Y.Z. Cai, Nutr. Cancer 62 (2010) 1e20.
[2] L. Reddy, B. Odhav, K.D. Bhoola, Pharmacol. Ther. 99 (2003) 1e13.

580
M. Safavi et al. / European Journal of Medicinal Chemistry 58 (2012) 573e580
[3] V.P. Androutsopoulos, A. Papakyriakou, D. Vourloumis, A.M. Tsatsakis,
D.A. Spandidos, Pharmacol. Ther. 126 (2010) 9e20.
[18] C. Fabris, G. Valduga, G. Miotto, L. Borsetto, G. Jori, S. Garbisa, E. Reddi, Cancer
Res. 61 (2001) 7495e7500.
[4] Y. Cai, Q. Luo, M. Sun, H. Corke, Life Sci. 74 (2004) 2157e2184.
[5] W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Med. Res. Rev. 23 (2003) 519e534.
[6] C.C. Chen, T.H. Hung, Y.H. Wang, C.W. Lin, P.Y. Wang, C.Y. Lee, S.F. Chen, PLoS
One 7 (2012) e30294.
[7] Y.C. Chen, S.C. Shen, W.R. Lee, H.Y. Lin, C.H. Ko, C.M. Shih, L.L. Yang, Arch.
Toxicol. 76 (2002) 351e359.
[8] H.Y. Lin, S.H. Juan, S.C. Shen, F.L. Hsu, Y.C. Chen, Biochem. Pharmacol. 66
(2003) 1821e1832.
[9] S. Kuntz, U. Wenzel, H. Daniel, Eur. J. Nutr. 38 (1999) 133e142.
[10] C. Pouget, F. Lauthier, A. Simon, C. Fagnere, J.P. Basly, C. Delage, A.J. Chulia,
Bioorg. Med. Chem. Lett. 11 (2001) 3095e3097.
[11] U. Wenzel, S. Kuntz, M.D. Brendel, H. Daniel, Cancer Res. 60 (2000) 3823e3831.
[12] H. Usman, E.H. Hakim, T. Harlim, M.N. Jalaluddin, Y.M. Syah, S.A. Achmad,
H. Takayama, Z. Naturforsch. C 61 (2006) 184e188.
[13] S.C. Shen, C.H. Ko, S.W. Tseng, S.H. Tsai, Y.C. Chen, Toxicol. Appl. Pharmacol.
197 (2004) 84e95.
[19] S.W. Lowe, A.W. Lin, Carcinogenesis 21 (2000) 485e495.
[20] P.G. Satchell, J.L. Gutmann, D.E. Witherspoon, Int. Endod. J. 36 (2003)
237e245.
[21] A.W. Tolcher, Hematol. Oncol. Clin. North Am. 16 (2002) 1255e1267.
[22] M. Mahmoodi, A. Aliabadi, S. Emami, M. Safavi, S. Rajabalian,
M.A. Mohagheghi, A. Khoshzaban, A. Samzadeh-Kermani, N. Lamei, A. Shafiee,
A. Foroumadi, Arch. Pharm. (Weinheim) 343 (2010) 411e416.
[23] M. Khoobi, A. Foroumadi, S. Emami, M. Safavi, G. Dehghan, B.H. Alizadeh,
A. Ramazani, S.K. Ardestani, A. Shafiee, Chem. Biol. Drug Des. 78 (2011)
580e586.
[24] B.H. Alizadeh, A. Foroumadi, S. Emami, M. Khoobi, F. Panah, S.K. Ardestani,
A. Shafiee, Eur. J. Med. Chem. 45 (2010) 5979e5984.
[25] M. Nakhjiri, M. Safavi, E. Alipour, S. Emami, A.F. Atash, M. Jafari-Zavareh,
S.K. Ardestani, M. Khoshneviszadeh, A. Foroumadi, A. Shafiee, Eur. J. Med.
Chem. 50 (2012) 113e123.
[26] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
[27] D. Ribble, N.B. Goldstein, D.A. Norris, Y.G. Shellman, B.M.C. Biotechnol. 5
(2005) 12e19.
[14] Y.C. Hsiao, Y.S. Hsieh, W.H. Kuo, H.L. Chiou, S.F. Yang, W.L. Chiang, S.C. Chu,
J. Biomed. Sci. 14 (2007) 107e119.
[15] E.J. Choi, J.I. Lee, G.H. Kim, Int. J. Mol. Med. 25 (2010) 293e298.
[16] E.J. Choi, J.I. Lee, G.-H. Kim, Arch. Pharm. Res. 34 (2011) 2125e2130.
[17] Z. Zhang, M. Li, E.R. Rayburn, D.L. Hill, R. Zhang, H. Wang, Am. J. Pharmaco-
genomics 5 (2005) 397e407.
[28] C.S. Di, J. Baselga, Eur. J. Cancer 44 (2008) 2781e2790.
[29] H. Aryapour, Gh. Riazi, A. Foroumadi, S. Ahmadian, A. Shafiee, O. Karima,
M. Mahdavi, S. Emami, M. Sorkhi, S. Khodadadi, Med. Chem. Res. 20 (2011)
503e510.