Synthesis, in vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-2 H -chromene derivatives as potent anti-breast cancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.09.017

A series of 2-aryl-3-nitro-2H-chromenes 4a-u were designed as hybrid analogs of flavanone, β-nitrostyrene and nitrovinylstilbene scaffolds. They were synthesized from the reaction of appropriate β-nitrostyrenes and salicylaldehydes in good yields. In vitro cytotoxic activities of compounds 4a-u were tested against breast cancer cell lines including MCF-7, T-47D and MDA-MB-231. Most compounds exhibited good cytotoxic activity against selected cell lines, being more potent than standard drug etoposide. Representatively, 8-methoxy-3-nitro-2-(4-chlorophenyl)-2H-chromene (4l) with IC₅₀ Combining double low line 0.2 μ4M against MCF-7 cells, was 36-times more potent than etoposide. Apoptosis as a mechanism of cell death for selected compounds 4h and 4l was confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis, as well as caspase-3 activation assay.

Full text of DOI:10.1016/j.ejmech.2014.09.017

Accepted Manuscript
Synthesis, in vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-2H-
chromene derivatives as potent anti-breast cancer agents
Samira Rahmani-Nezhad, Maliheh Safavi, Mahboobeh Pordeli, Sussan Kabudanian
Ardestani, Leila Khosravani, Yaghoub Pourshojaei, Mohammad Mahdavi, Saeed
Emami, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(14)00835-6
10.1016/j.ejmech.2014.09.017
EJMECH 7323
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2014
Revised Date: 18 August 2014
Accepted Date: 5 September 2014
Please cite this article as: S. Rahmani-Nezhad, M. Safavi, M. Pordeli, S.K. Ardestani, L. Khosravani,
Y. Pourshojaei, M. Mahdavi, S. Emami, A. Foroumadi, A. Shafiee, Synthesis, in vitro cytotoxicity and
apoptosis inducing study of 2-aryl-3-nitro-2H-chromene derivatives as potent anti-breast cancer agents,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.017.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis, in vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-2H-chromene
derivatives as potent anti-breast cancer agents
Samira Rahmani-Nezhad, Maliheh Safavi, Mahboobeh Pordeli, Sussan Kabudanian Ardestani, Leila
Khosravani, Yaghoub Pourshojaei, Saeed Emami, Alireza Foroumadi, Abbas Shafiee *
A series of 2-aryl-3-nitro-2H-chromenes 4a-u were designed and synthesized as hybrid analogs of β-
nitrostyrene, nitrovinylstilbene and flavanone scaffolds with apoptosis-inducing cytotoxic activity against
breast cancer cells.

ACCEPTED MANUSCRIPT
Synthesis, in vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-
2
H-chromene derivatives as potent anti-breast cancer agents
a
b
c
Samira Rahmani-Nezhad, Maliheh Safavi, Mahboobeh Pordeli, Sussan Kabudanian
c
a
a
a
d
Ardestani, Leila Khosravani, Yaghoub Pourshojaei, Mohammad Mahdavi, Saeed Emami,
a
a,
Alireza Foroumadi, Abbas Shafiee *
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development
Research Center, Tehran University of Medical Sciences, Tehran, Iran
b
Department of Biotechnology, Iranian Research Organization for Science and Technology,
Tehran, 33535-111, Iran
c
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran,
Tehran, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A series of 2-aryl-3-nitro-2H-chromenes 4a-u were designed as hybrid analogs of flavanone,
β
-nitrostyrene and nitrovinylstilbene scaffolds. They were synthesized from the reaction of appropriate β-
nitrostyrenes and salicylaldehydes in good yields. In vitro cytotoxic activities of compounds 4a-u were
tested against breast cancer cell lines including MCF-7, T-47D and MDA-MB-231. Most compounds
exhibited good cytotoxic activity against selected cell lines, being more potent than standard drug
etoposide. Representatively, 8-methoxy-3-nitro-2-(4-chlorophenyl)-2H-chromene (4l) with IC50 = 0.2 µM
against MCF-7 cells, was 36-times more potent than etoposide. Apoptosis as a mechanism of cell death
for selected compounds 4h and 4l was confirmed morphologically by acridine orange/ethidium bromide
double staining and TUNEL analysis, as well as caspase-3 activation assay.
Keywords: Anti-cancer agents; Apoptosis; Caspase-3; 2H-Chromene; β-Nitrostyrene
*
Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
1

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1
. Introduction
Cancer is one of the critical public health problems in the world. The corresponding incidence
and mortality statistics shows that it is growing in developing as well as developed countries
[1,2]. Despite significant advances in the diagnostic and therapeutic techniques, cancer is now
the second most frequent cause of death after cardiovascular diseases [1,3].
Considering numerous reports and publications on the synthesis of anti-cancer agents, there is no
drug with 100% efficacy. Therefore, there is still considerable demand for drug discovery
leading to efficient anti-cancer compounds with new scaffold or specific mechanism of action to
overcome the problems associated with current chemotherapeutics in cancer treatment, such as
toxicity and drug resistance [4]. Among different strategies used for chemotherapy, induction of
apoptosis or programmed cell death in cancer cells is one of the credible approaches to drug
discovery and development of new anti-cancer agents [5,6].
Apoptosis plays an important role in the pathogenesis of several diseases including cancer
(cancer initiation, progression and regulation of metastasis) [7,8]. It is well documented that
resistance to apoptosis is one of the most important hallmarks of human cancers [9,10]. Three
main types of cell death have been distinguished [11]; caspase-dependent apoptosis is the major
form of controlled cell death in normal and cancer cells. It involves the activation of a family of
cysteine proteases known as caspase. Caspase-3 is the key enzyme in promoting apoptotic
pathway [12,13].
Several studies demonstrated that the mechanism of numerous anticancer agents occurs through
the activation of apoptotic pathways in cancer cells [14-18]. Steifelder et al. have reported that
the nitrovinyl side chain attached to an aromatic ring is an effective pharmacophore for the
development of new pro-apoptotic agents (structure A, Fig. 1) [19]. On the other hand, structure–
activity relationship study of resveratrol analogues showed that the methoxy groups at positions
3
, 4 and 5 of the stilbene backbone (structure B, Fig. 1), greatly enhanced the pro-apoptotic
effect and anti-cancer activity [20]. Also, Sreedhar et al. designed nitrovinylstilbenes (structure
C, Fig. 1) as hybrid analogs of structures A and B exhibiting significant anti-proliferative activity
with potential apoptosis induction ability in human cancer cells [21].
As a program aiming to develop flavanone and chromene derivatives as apoptosis inducers
[22,23], we recently described a series of flavanones (structure E) derived from 2-
2

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hydroxychalcones (structure D, Fig. 1) as potential apoptosis-inducing agents [22]. The
importance of chromene scaffold for antitumor activity [24], and the promising anti-proliferative
and apoptosis induction activities of nitrovinylstilbenes (C) and flavanones (E) prompted us to
hybridize their structural requirements. Thus we report here, synthesis, cytotoxicity and
apoptosis-inducing activity of 2-aryl-3-nitro-2H-chromenes (4a-u, Fig. 1). Indeed, the designed
compounds 4a-u are cyclic rigid analogs of nitrovinylstilbenes (C) and originated from
flavanones (E) by retaining the flavan scaffold in their core structure.
2
. Chemistry
The synthetic pathway to obtain compounds namely 2-aryl-3-nitro-2H-chromene derivatives 4a-
u was outlined in Scheme 1. Initially, β-nitrostyrenes 2a-g were prepared by Henry–
Knoevenagel condensation of different benzaldehydes 1a-g with nitromethane, in the presence of
n-butylamine. The desired β-nitrostyrenes 2a-g were obtained in high yields and with little or no
by-product [25]. The reaction of β-nitrostyrenes 2a-g with appropriate salicylaldehyde 3a-c in
the presence of an organocatalyst base namely 1,4-diazabicyclo[2.2.2]octane (DABCO) afforded
corresponding final compounds 4a-u [26]. The required 2,3,4-trimethoxysalicylaldehyde (3c)
was prepared in two steps from commercially available 2,3,4-trimethoxybenzaldehyde (Scheme
2
). The reaction of 2,3,4-trimethoxybenzaldehyde with hydrogen peroxide in the presence of
H SO in methanol afforded 2,3,4-trimethoxyphenol. The latter compound was converted to
2
4
desired salicylaldehyde by using hexamethylenetetramine in refluxing TFA [27]. The structures
1
13
of synthesized compounds were characterized by H NMR, C NMR, MS and CHN analysis as
described in the experimental section.
3
3
3
. Results and discussion
.1. Biological study
.1.1. MTT assay
The in vitro cytotoxicity of synthesized compounds 4a-u was evaluated against three different
breast cancer cell lines MCF-7, T-47D and MDA-MB-231. The MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] assay was used to determine the reduction in cancer cell
3

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viability induced by cytotoxic agents. The yellow tetrazolium is reduced in mitochondria by the
action of dehydrogenase enzymes to an insoluble purple formazan product which quantified
using spectrophotometric means [28,29]. The IC values of compounds 4a-u in comparison to
5
0
etoposide as standard drug are presented in Table 1. Results of the MTT assay revealed that all
compounds showed potent activity (IC s ≤ 4.7 µM) against MCF-7 cells; their activities were
5
0
superior to that of standard drug etoposide. The most potent compound 4l possessing the IC50
value of 0.2 µM was 36-fold more active than etoposide against MCF-7. All compounds with the
exception of 4i, 4q, 4t and 4u, exhibited higher cytotoxic activity against T-47D cell line respect
to the standard drug. Although compound 4i was as potent as etoposide against MDA-MB-231,
but remaining compounds had superior activity in comparison to etoposide. The most potent
compound (4b) against MDA-MB-231 (IC50 = 0.4 µM), was 32-times more active than
etoposide.
Structurally, the designed compounds are divided to three series: un-substituted chromenes 4a-g,
8
-methoxychromones 4h-n and 6,7,8-trimethoxychromenes 4o-u. In each series, the substituent
on the 2-phenyl ring was altered to optimize the cytotoxic activity against three different breast
cancer cells. In the chromenes 4a-g, all compounds showed same activities against MCF-7 cells
and 3,4,5-trimethoxyphenyl derivative was better compound against T-47D cell line.
Furthermore, 3,4-dimethoxyphenyl substituent showed more favorable cytotoxic activity against
MDA-MB-231. The remaining substituents had same effect on this series, as shown by obtained
IC s against MDA-MB-231.
5
0
Modification of substituents on the 2-phenyl ring in 8-methoxychromone 4h-n series revealed
that the 4-chloro group is preferred substituent for growth inhibitory activity against both MCF-7
and T-47D cell lines. Moreover, in 8-methoxychromone derivatives, the 4-nitro followed by 4-
chloro groups improved the cytotoxicity against MDA-MB-231. The IC50 values of 6,7,8-
trimethoxychromenes 4o-u demonstrated that 3,4-dimethoxyphenyl moiety in compound 4p
offered the highest activity against T-47D cells while para-tolyl moiety was the best candidate
for anti-proliferative activity towards MDA-MB-231 cell line.
The results suggested that the effect of methoxy substitution(s) on the chromene core directly
depends on substituents on 2-phenyl ring. Consequently, the simultaneous presence of methoxy
4

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group at the 8-position of chromene ring and para-chloro on the 2-phenyl ring favored the
antitumor activity.
3
.1.2. Acridine orange/ethidium bromide double staining
Since the main goal of our design was to obtain agents that promote or induce apoptosis,
following the determination of the cytotoxicity by MTT assay, compound 4h and 4l with low
IC s against MCF-7 cells were selected to determine apoptotic or necrotic cell death in MCF-7
5
0
and MDA-MB-231 cells. Apoptotic, necrotic and live cells can be distinguished using acridine
orange/ethidium bromide (AO/EB) staining. Fluorescent DNA binding dyes AO and EB were
used to detect the apoptotic and necrotic cells [30]. AO can penetrate living as well as dead cells
and emits green fluorescence as a result of intercalation into double-stranded DNA while EB can
only penetrate dead cells and emits red fluorescence after intercalation into DNA. The AO/EB
staining which is sensitive to DNA was used to access changes in nuclear morphology. Analysis
of the AO/EB staining of the selected compounds 4h and 4l in MCF-7 and MDA-MB-231 cell
lines using fluorescence microscopy is shown in Fig. 2. In this figure, living cells has a normal
green nucleus, but apoptotic cells show orange-stained nuclei with chromatin condensation or
fragmentation, while necrotic cells appear uniformly orange-stained cell nuclei with no
condensed chromatin. Analysis of the AO/EB staining revealed that the tested compounds 4h
and 4l clearly exhibit late-stage apoptotic events (chromatin condensation and nuclear
fragmentation) and result in reduced cell viability in human breast cancer cell lines.
3
.1.3. TUNEL assay
The TUNEL (TdT-mediated dUTP Nick-End Labeling; Promega) assay, has been designed to
detect apoptotic cells by labeling 3′-OH ends of single- and double-stranded DNA fragments
with fluorescein. This method was used for the detection of early stages of apoptosis (after 12 h
treatment) in MDA-MB-231 cells to compare the ability of the compounds 4h and 4l to induce
apoptosis and was quantified by flow-cytometry. As illustrated in Fig. 3, the apoptotic index of
the test compounds was compared with negative control in MDA-MB-231 cells. The highest
percentage of early apoptotic cells detected in this assay was 28%, resulting from treatment with
the compound 4l after 12 h of incubation. Exposure of MDA-MB-231 cells to IC concentration
5
0
of compound 4h induced apoptosis in 16% of the cells after 12 h. Besides, the corresponding
5

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value obtained after treatment with etoposide was 12% following 12 h of incubation. These data
indicate that the cytotoxic activity of 4h and 4l in MDA-MB-231 cells occurs through apoptosis.
3
.1.4. Caspase-3 activation assay
Caspase-3, one of the main executioner caspases, is activated in the apoptotic cells in both
extrinsic and intrinsic pathways. The activation of caspase-3 is useful in the discovery of many
potential anticancer agents. In an attempt to find out whether the induction of apoptosis by
compound 4h and 4l is dependent on caspase‐3 activity or not, we measured the activation of
caspase-3 in MDA-MB-231 cells. Caspase-3 colorimetric assay was used to determine the
increase in caspase-3 activity in early stage of apoptotic cells. The cleavage of the caspase 3-
specific substrate that is labeled with the chromophore p-nitroaniline (pNA) by the activated
caspase-3 leads to the releases of the chromophore pNA. Generation of free molecule pNA from
apoptotic cells was quantitated spectrophotometrically at 405 nm. A significant 3 to 4-fold
increases was observed in the induction of caspase-3 in MDA-MB-231 cells treated with
compounds 4l and 4h relative to the control (Fig. 4). These results suggested that the cytotoxic
activity of 4h and 4l in MDA-MB-231 cells occurs via caspase-3-dependent apoptosis.
4
. Conclusion
In conclusion, we designed 2-aryl-3-nitro-2H-chromenes 4a-u as hybrid analogs of flavanones
and β-nitrostyrenes which previously have been reported as apoptosis-inducing cytotoxic agents.
The compounds 4a-u were easily synthesized from the reaction of appropriate β-nitrostyrenes
and salicylaldehydes in good yields. Most of the synthesized compounds showed excellent
cytotoxic activity against three breast cancer cell lines, more potent than standard drug etoposide.
Representatively, compound 4l with IC50 value of 0.2 µM was 36 times more potent than
etoposide against MCF-7 cells. The SAR study demonstrated that para-chloro substituent on the
2
-phenyl ring led to optimal effect in 8-methoxychromene derivatives. The most potent
compounds against tested breast-cancer cell lines were belonging to the unsubstituted and 8-
methoxylated chromene series. Meanwhile, the introduction of poly-methoxy substituents on 6,
7
, 8 positions of chromene ring could not improve anti-proliferative activity against tested cell
lines. Further biological assessments including flow-cytometric analysis, AO/EB staining,
6

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TUNEL and caspase-3 activation assays, revealed that the selected compounds 4h and 4l led to
the induction of apoptosis through the activation of caspase-3 in breast cancer cell lines.
5
. Experimental
5
.1. Chemistry
Melting points are uncorrected and were determined with a Kofler hot-stage apparatus (Reichert,
1
13
Vienna, Austria). H and C NMR spectra were recorded using Bruker 400 or 500 spectrometers.
Chemical shifts (δ) are reported in ppm relative to TMS as internal standard. Coupling constant
(J) values are presented in Hz and spin multiplicities are given as s (singlet), d (doublet), t
(triplet) and m (multiplet). The IR spectra were obtained on a Nicolet Magna FTIR 550
spectrophotometer (potassium bromide disks). Mass spectra were determined on an Agilent
Technology (HP) mass spectrometer operating at an ionization potential of 70 eV. The elemental
analysis for C, H, N was carried out with an Elementar Analysen system GmbH VarioEL. All
reagents and solvents used in this study were commercially available (from Merck chemical) and
were used without further purification. The intermediate compounds β-nitrostyrenes 2a-g and
2
,3,4-trimethoxy salicylaldehyde (3c) were prepared according to the literature methods [25,27].
5
.1.1. General procedure for the preparation of 2-aryl-3-nitro-2H-chromenes (4a–u).
A mixture of appropriate β-nitrostyrene 2a-g (5 mmol), salicylaldehyde 3a-c (10 mmol), and
DABCO (2.5 mmol) was stirred at 40 ºC for 4 h. Then the mixture was directly purified by
column chromatography on silica gel eluting with n-hexane-EtOAc (7:3). The obtained product
was recrystallized from ethanol to give desired compound 4.
-
5
.1.1.1. 3-Nitro-2-(4-methoxyphenyl)-2H-chromene (4a). Yield 72%; mp 94–96 ºC; IR (KBr, cm
1
1
)
: ν 1644, 1608, 1511, 1454, 1326, 1255, 1168, 1111, 1060, 753; H NMR (500 MHz, CDCl ): δ
3
3
.77 (s, 3H, OCH ), 6.53 (s, 1H, H -chromene), 6.83-6.86 (m, 3H, H -chromene and H -
3
2
8
3,5
phenyl), 7.01 (dt, 1H, J = 7.5 and 1.0 Hz, H -chromene), 7.29-7.31 (m, 2H, H -phenyl), 7.32-
6
2,6
1
3
7
7
4 3
.34 (m, 2H, H5,7-chromene), 8.05 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ 56.39,
3.08, 107.36, 117.31, 117.63, 123.29, 124.12, 127.84, 130.08, 130.81, 134.95, 140.27, 143.52,
7

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1
4
48.37, 153.09. Anal. Calcd for C16
.71; N, 4.75.
4
H13NO : C, 67.84; H, 4.63; N, 4.94. Found: C, 67.95; H,
5
.1.1.2. 3-Nitro-2-(3,4-dimethoxyphenyl)-2H-chromene (4b). Yield 90%; mp 87–89 ºC; IR (KBr,
-1
1
cm ): ν 1646, 1601, 1514, 1449, 1413, 1331, 1253, 1142, 1024, 748; H NMR (500 MHz,
CDCl ): δ 3.77 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 6.46 (s, 1H, H -chromene), 6.48 (d, 1H, J =
3
3
3
2
7
.5 Hz, H -phenyl), 6.84-6.88 (m, 3H, H -chromene and H -phenyl), 7.02 (t, 1H, J = 7.5 Hz,
5 8 2,6
1
3
H
6 4
-chromene), 7.29-7.34 (m, 2H, H5,7-chromene), 8.04 (s, 1H, H -chromene). C NMR (125
MHz, CDCl ): δ 55.23, 56.87, 73.11, 107.39, 117.19, 117.64, 123.09, 123.97, 128.07, 130.52,
3
+
1
30.78, 134.65, 140.12, 143.61, 148.40. MS (m/z, %): 313 (m , 36), 267 (100), 251 (18), 223
(
23), 205 (7), 181 (8), 165 (10), 152 (13). Anal. Calcd for C H NO : C, 65.17; H, 4.83; N, 4.47.
17 15 5
Found: C, 65.35; H, 4.76; N, 4.35.
5
.1.1.3. 3-Nitro-2-(3,4,5-trimethoxyphenyl)-2H-chromene (4c). Yield 81%; mp 124–126 ºC; IR
-1
1
(
KBr, cm ): ν 1644, 1593, 1501, 1454, 1321, 1239, 1122, 1004, 830, 753; H NMR (500 MHz,
CDCl ): δ 3.77 (s, 6H, 2×OCH ), 3.81 (s, 3H, OCH ), 6.53 (s, 1H, H -chromene), 6.58 (s, 2H,
3
3
3
2
8 6
H2,6-phenyl), 6.91 (d, 1H, J = 8.0 Hz, H -chromene), 7.01-7.05 (m, 1H, H -chromene), 7.34-7.38
1
3
(
m, 2H, H , -chromone), 8.07 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ 56.19,
5
7
4
3
5
1
6.25, 56.42, 73.80, 104.18, 107.21, 113.28, 129.61, 131.83, 140.05, 142.11, 142.79, 147.88,
+
48.75, 153.38. MS (m/z, %): 343 (m , 52), 297 (100), 281 (8), 264 (18), 253 (14), 239 (11), 168
(
22), 139 (16). Anal. Calcd for C H NO : C, 62.97; H, 4.99; N, 4.08. Found: C, 63.15; H, 4.71;
18 17 6
N, 4.15.
5
.1.1.4. 3-Nitro-2-(4-methylphenyl)-2H-chromene (4d). Yield 67%; mp 120–122 ºC; IR (KBr,
-1
1
cm ): ν 1650, 1602, 1510, 1455, 1324, 1198, 1116, 1060, 819, 750; H NMR (500 MHz,
CDCl
3
): δ 2.31 (s, 3H, CH
3
), 6.55 (s, 1H, H
2
8
-chromene), 6.86 (d, 1H, J = 7.7 Hz, H -chromene),
7
2
.01 (dt, 1H, J = 7.7 and 1.0 Hz, H -chromene), 7.13 (d, 2H, J = 7.7 Hz, H -phenyl), 7.26 (d,
6 3,5
1
3
H, J = 7.7 Hz, H -phenyl), 7.30-7.34 (m, 2H, H , -chromone), 8.05 (s, 1H, H -chromene). C
2
,6
5 7
4
NMR (125 MHz, CDCl
3
): δ 21.18, 73.13, 107.41, 117.62, 118.55, 129.43, 129.87, 130.12,
+
1
2
34.76, 135.52, 139.68, 141.46, 142.67, 147.93, 148.74. MS (m/z, %): 267 (m , 39), 250 (11),
3
21 (100), 205 (15), 191 (10), 178 (34), 165 (9), 115 (7). Anal. Calcd for C16H13NO : C, 71.90;
H, 4.90; N, 5.24. Found: C, 71.65; H, 4.71; N, 5.33.
8

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5
.1.1.5. 3-Nitro-2-(4-chlorophenyl)-2H-chromene (4e). Yield 80%; mp 118–120 ºC; IR (KBr,
-1
1
cm ): ν 1641, 1598, 1487, 1448, 1325, 1221, 1147, 1063, 1006, 753; H NMR (500 MHz,
CDCl ): δ 6.56 (s, 1H, H -chromene), 6.87 (d, 1H, J = 7.5 Hz, H -chromene), 7.03 (dt, 1H, J =
3
2
8
7
7
7
1
3
6
.5 and 1.05 Hz, H -chromene), 7.28-7.30 (m, 2H, H3,5-phenyl), 7.31-7.33 (m, 2H, H2,6-phenyl),
1
3
.34-7.36 (m, 2H, H -chromene), 8.07 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ
5
,7
4
3
3.09, 107.38, 117.53, 118.36, 128.43, 129.81, 130.03, 134.73, 135.46, 139.68, 141.56, 142.67,
47.93, 148.70. Anal. Calcd for C H ClNO : C, 62.62; H, 3.50; N, 4.87. Found: C, 62.84; H,
1
5
10
3
.71; N, 4.69
-
5
.1.1.6. 3-Nitro-2-(4-nitrophenyl)-2H-chromene (4f). Yield 55%; mp 129–131 ºC; IR (KBr, cm
1
1
)
: ν 1651, 1603, 1525, 1455, 1345, 1194, 1153, 1115, 1066, 753; H NMR (500 MHz, CDCl ): δ
3
6
.67 (s, 1H, H -chromene), 6.92 (d, 1H, J = 8.0 Hz, H -chromene), 7.06 (dt, 1H, J = 8.0 and 1.0
2
8
6
Hz, H -chromene), 7.36-7.40 (m, 2H, H5,7-chromene), 7.58 (d, 2H, J = 8.0 Hz, H2,6-phenyl), 8.11
1
3
(
s, 1H, H -chromene), 8.19 (d, 2H, J = 8.0 Hz, H -phenyl). C NMR (125 MHz, CDCl ): δ
4 3,5 3
7
1
3
3.03, 107.31, 117.25, 117.52, 123.19, 124.09, 127.97, 130.02, 130.77, 134.84, 140.19, 143.53,
48.42, 153.05. Anal. Calcd for C H N O : C, 60.41; H, 3.38; N, 9.39. Found: C, 60.33; H,
1
5
10
2
5
.71; N, 9.38.
5
.1.1.7. 3-Nitro-2-(3,4-methylenedioxyphenyl)-2H-chromene (4g). Yield 85%; mp 97–99 ºC; IR
-1
1
(
KBr, cm ): ν 1646, 1603, 1506, 1447, 1323, 1257, 1222, 1107, 1035, 749; H NMR (500 MHz,
CDCl ): δ 5.94 (s, 2H, OCH O), 6.49 (s,1H, H -chromene), 6.74 (d, 1H, J = 8.0 Hz, H - phenyl),
3
2
2
5
6
7
7
1
.85-6.87 (m, 3H, H
8
-chromene and H2,6-phenyl), 7.02 (t, 1H, J = 8.0 Hz, 1H, H
6
-chromene),
1
3
.32-7.34 (m, 2H, H -chromone), 8.05 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ
5
,7
4
3
3.95, 95.08, 102.13, 107.67, 108.53, 117.09, 121.49, 124.37, 126.64, 129.83, 135.32, 138.97,
+
48.45, 153.27. MS (m/z, %): 297 (m , 31), 251 (100), 221 (10), 193 (17), 165 (42), 146 (8), 139
(
12). Anal. Calcd for C H NO : C, 64.65; H, 3.73; N, 4.71. Found: C, 64.41; H, 3.71; N, 4.93.
16 11 5
5
.1.1.8. 8-Methoxy-3-nitro-2-(4-methoxyphenyl)-2H-chromene (4h). Yield 82%; mp 122–124 ºC;
-
1
1
IR (KBr, cm ): ν 1641, 1605, 1575, 1508, 1477, 1320, 1269, 1172, 1055, 755; H NMR (400
MHz, CDCl ): δ 3.82 (s, 3H, OCH ), 3.83 (s, 3H, OCH ), 6.65 (s, 1H, H -chromene), 6.94-6.98
3
3
3
2
(m, 3H, H5,6,7-chromene), 7.12 (d, 2H, J = 8.0 Hz, H3,5-phenyl), 7.30 (d, 2H, J = 8.0 Hz, H2,6-
1
3
phenyl), 8.05 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ 56.12, 56.31, 73.89, 104.01,
4
3
1
16.47, 118.75, 121.87, 122.59, 129.18, 132.08, 139.01, 141.59, 142.75, 148.58, 153.41. MS
9

ACCEPTED MANUSCRIPT
+
(
m/z, %): 313 (M , 4), 297 (42), 267 (26), 251 (100), 236 (15), 208 (30), 165 (38). Anal. Calcd
for C H NO : C, 65.17; H, 4.83; N, 4.47. Found: C, 65.39; H, 4.71; N, 4.36.
1
7
15
5
5
.1.1.9. 8-Methoxy-3-nitro-2-(3,4-dimethoxyphenyl)-2H-chromene (4i). Yield 89%; mp127–129
-1
1
ºC; IR (KBr, cm ): ν 1649, 1599, 1575, 1509, 1474, 1325, 1260, 1140, 1025, 750; H NMR (400
MHz, CDCl ): δ 3.83 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 6.63 (s, 1H, H -
3
3
3
3
2
chromene), 6.75 (d, 1H, J = 8.0 Hz, H -chromene), 6.87 (dd, 1H, J = 8.0 and 2.0 Hz, H -
7
6
chromene), 6.95-6.99 (m, 3H, H2,5,6-phenyl), 7.04 (d, 1H, J = 8.0 Hz, H
5
-chromene), 8.06 (s, 1H,
1
3
H -chromene), C NMR (100 MHz, CDCl ): δ 56.06, 56.26, 60.77, 74.05, 103.98, 116.59,
4
3
1
18.86, 121.98, 122.63, 129.22, 132.03, 138.78, 141.53, 142.70, 148.66, 153.34. Anal. Calcd for
C H NO : C, 62.97; H, 4.99; N, 4.08. Found: C, 62.78; H, 4.81; N, 4.14.
18
17
6
5
.1.1.10. 8-Methoxy-3-nitro-2-(3,4,5-trimethoxyphenyl)-2H-chromene (4j). Yield 70%; mp 131–
-1
1
1
33 ºC; IR (KBr, cm ): ν 1651, 1602, 1509, 1450, 1339, 1268, 1126, 1048, 860, 786; H NMR
): δ 3.78 (s, 6H, 2×OCH ), 3.81 (s, 3H, OCH ), 3.87 (s, 3H, OCH ), 6.63 (s,
(400 MHz, CDCl
3
3
3
3
1
H, H -chromene), 6.64 (s, 2H, H -phenyl), 6.95-6.99 (m, 3H, H5,6,7-chromene), 8.06 (s, 1H,
2
2,6
1
3
H
4
3
-chromene). C NMR (100 MHz, CDCl ): δ 56.08, 56.22, 60.73, 61.67, 74.03, 103.87,
1
15.99, 118.82, 122.08, 122.67, 129.31, 131.95, 138.75, 141.48, 142.70, 148.59, 153.29. Anal.
Calcd for C H NO : C, 61.12; H, 5.13; N, 3.75. Found: C, 61.35; H, 5.38; N, 3.82.
1
9
19
7
5
.1.1.11. 8-Methoxy-3-nitro-2-(4-methylphenyl)-2H-chromene (4k). Yield 74%; mp 125–127 ºC;
-
1
1
IR (KBr, cm ): ν 1645, 1608, 1574, 1512, 1481, 1323, 1268, 1212, 1055, 750; H NMR (500
MHz, CDCl ): δ 2.31 (s, 3H, CH ), 3.81 (s, 3H, OCH ), 6.64 (s, 1H, H -chromene), 6.93-6.97
3
3
3
2
(m, 3H, H5,6,7-chromene), 7.12 (d, 2H, J = 7.7 Hz, H3,5-phenyl), 7.30 (d, 2H, J = 7.7 Hz, H2,6-
1
3
phenyl), 8.04 (s, 1H, H -chromene). C NMR (100 MHz, CDCl ): δ 21.28, 60.68, 74.03, 103.85,
4
3
1
16.60, 118.92, 121.84, 122.63, 129.25, 132.07, 139.18, 141.53, 142.66, 148.73, 149.57. Anal.
Calcd for C17
H15NO
4
: C, 68.68; H, 5.09; N, 4.71. Found: C, 68.95; H, 5.34; N, 4.63.
5
.1.1.12. 8-Methoxy-3-nitro-2-(4-chlorophenyl)-2H-chromene (4l). Yield 92%; mp 130–132 ºC;
-1
1
IR (KBr, cm ): ν 1651, 1598, 1577, 1512, 1478, 1317, 1265, 1187, 1082, 783; H NMR (400
3 3 2
MHz, CDCl ): δ 3.84 (s, 3H, OCH ), 6.64 (s, 1H, H -chromene), 6.96-7.00 (m, 3H, H5,6,7-
chromene), 7.29-7.31 (m, 2H, H -phenyl), 7.34-7.36 (m, 2H, H -phenyl), 8.06 (s, 1H, H -
3
,5
2,6
4
1
3
chromene). C NMR (100 MHz, CDCl ): δ 56.42, 74.03, 104.05, 116.60, 118.74, 121.87,
3
+
1
22.63, 129.43, 132.19, 139.22, 141.73, 142.56, 148.67, 149.83. MS (m/z, %): 319 ([M+2] , 20),
1
0

ACCEPTED MANUSCRIPT
+
3
17 (M , 48), 271 (100), 256 (12), 228 (22), 178 (18), 165 (50). Anal. Calcd for C16
4
H12ClNO :
C, 60.48; H, 3.81; N, 4.41. Found: C, 60.35; H, 3.77; N, 4.56.
5
.1.1.13. 8-Methoxy-3-nitro-2-(4-nitrophenyl)-2H-chromene (4m). Yield 60%; mp 138–140 ºC;
-
1
1
IR (KBr, cm ): ν 1654, 1608, 1574, 1512, 1516, 1478, 1348, 1268, 1058, 857; H NMR (400
MHz, CDCl ): δ 3.84 (s, 3H, OCH ), 6.67 (s, 1H, H -chromene), 6.96-6.99 (m, 3H, H5,6,7-
3
3
2
chromene), 7.30 (d, 2H, J = 8.4 Hz, H -phenyl), 8.02 (s, 1H, H -chromene), 8.19 (d, 2H, J = 8.4
2
,6
4
1
3
Hz, H3,5-phenyl). C NMR (125 MHz, CDCl
3
): δ 56.29, 73.09, 104.01, 116.95, 117.72, 123.32,
+
1
3
24.16, 128.07, 130.09, 131.23, 134.64, 140.53, 143.76, 148.62, 153.07. MS (m/z, %): 328 (m ,
8), 282 (100), 236 (46), 193 (45), 165 (73), 152 (42), 139 (42), 63 (46). Anal. Calcd for
C H N O : C, 58.54; H, 3.68; N, 8.53. Found: C, 58.35; H, 3.71; N, 8.32.
16
12
2
6
5
.1.1.14. 8-Methoxy-3-nitro-2-(3,4-methylenedioxyphenyl)-2H-chromene (4n). Yield 65%; mp
-1
1
1
43–136 ºC; IR (KBr, cm ): ν 1649, 1602, 1574, 1496, 1448, 1330, 1266, 1101, 1034, 971; H
NMR (400 MHz, CDCl ): δ 3.83 (s, 3H, OCH ), 5.94 (s, 2H, OCH O), 6.64 (s, 1H, H -
3
3
2
2
chromene), 6.76 (d, 1H, J = 8 Hz, H -chromene), 6.87 (dd, 1H, J = 8.0 and 2 Hz, H -chromene),
7
6
6
.96-7.01 (m, 3H, H2,5,6-phenyl), 7.04 (d, 1H, J = 8.0 Hz, H
5
4
-chromene), 8.04 (s, 1H, H -
1
3
chromene). C NMR (100 MHz, CDCl ): δ 56.42, 73.85, 95.05, 102.06, 107.53, 108.43, 121.34,
3
+
1
2
4
24.23, 126.64, 129.59, 135.40, 139.17, 148.13, 153.27. MS (m/z, %): 327 (m , 18), 281 (100),
66 (9), 251 (5), 238 (13), 208 (6), 152 (22). Anal. Calcd for C H NO : C, 62.39; H, 4.00; N,
1
7
13
6
.28. Found: C, 62.45; H, 4.18; N, 4.51.
5
.1.1.15. 6,7,8-Trimethoxy-3-nitro-2-(4-methoxyphenyl)-2H-chromene (4o). Yield 65%; mp
-1
1
1
30–132 ºC; IR (KBr, cm ): ν 1642, 1608, 1507, 1461, 1422, 1379, 1309, 1247, 1027, 750; H
NMR (500 MHz, CDCl ): δ 3.63 (s, 3H, OCH ), 3.77 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 3.94
3
3
3
3
(
s, 3H, OCH ), 6.57 (s, 1H, H -chromene), 6.61 (s, 1H, H -chromene), 6.83 (d, 2H, J = 8.5 Hz,
3 2 5
1
3
H -phenyl), 7.32 (d, 2H, J = 8.5 Hz, H -phenyl), 8.02 (s, 1H, H -chromene). C NMR (125
3
,5
2,6
4
MHz, CDCl
3
): δ 56.18, 56.29, 56.41, 60.85, 73.82, 104.18, 107.24, 113.28, 129.49, 131.65,
+
1
38.81, 140.12, 141.99, 142.53, 147.76, 148.59, 153.41. MS (m/z, %): 373 (m , 14), 327 (100),
3
11 (22), 297 (20), 283 (21), 266 (8), 155 (20), 139 (12), 127 (16). Anal. Calcd for C H NO :
19 19 7
C, 61.12; H, 5.13; N, 3.75. Found: C, 61.35; H, 5.41; N, 3.58.
1
1

ACCEPTED MANUSCRIPT
5
.1.1.16. 6,7,8-Trimethoxy-3-nitro-2-(3,4-dimethoxyphenyl)-2H-chromene (4p). Yield 60%; mp
-1
1
1
40–142 ºC; IR (KBr, cm ): ν 1654, 1580, 1521, 1453, 1425, 1265, 1113, 1030, 848,756; H
NMR (500 MHz, CDCl ): δ 3.65 (s, 3H, OCH ), 3.83 (s, 3H, OCH ), 3.85 (s, 6H, 2×OCH ), 3.94
3
3
3
3
(
s, 3H, OCH
3
), 6.57 (s, 1H, H
2
5
-chromene), 6.61 (s,1H, H -chromene), 6.75 (d, 1H, J = 8.3 Hz,
H -phenyl), 6.86 (d, 1H, J = 8.3 Hz, H -phenyl), 7.01 (s, 1H, H -phenyl), 8.02 (s, 1H, H -
5
6
2
4
1
3
chromene). C NMR (125 MHz, CDCl
3
): δ 55.83, 55.92, 56.41, 61.42, 61.52, 73.80, 107.19,
10.54, 110.80, 113.43, 119.25, 128.49, 129.50, 140.23, 141.84, 142.80, 147.70, 148.57, 149.14,
49.93. Anal. Calcd for C H NO : C, 59.55; H, 5.25; N, 3.47. Found: C, 59.35; H, 5.51; N,
1
1
3
20
21
8
.35.
5
.1.1.17. 6,7,8-Trimethoxy-3-nitro-2-(3,4,5-trimethoxyphenyl)-2H-chromene (4q). Yield 54%;
-1
mp 147–149 ºC; IR (KBr, cm ): ν 1642, 1592, 1508, 1458, 1421, 1278, 1129, 1108, 1064, 968;
1
H NMR (500 MHz, CDCl
3 3 3 3
): δ 3.72 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 3.81 (s, 3H, OCH ),
3
.85 (s, 6H, 2×OCH ), 3.95 (s, 3H, OCH ), 6.58 (s, 1H, H -chromene), 6.61 (s, 3H, H -
3
3
2
5
1
3
chromene, H -phenyl), 8.03 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ 56.14,
2
,6
4
3
5
1
6.27, 56.42, 60.79, 61.42, 61.56, 73.80, 104.10, 107.21, 113.24, 129.57, 131.73, 138.87, 140.09,
+
42.01, 142.71, 147.82, 148.68, 153.36. MS (m/z, %): 433 (M , 23), 387 (100), 357 (26), 343
9
(9), 329 (7), 311 (10), 157 (12). Anal. Calcd for C21H23NO : C, 58.20; H, 5.35; N, 3.23. Found:
C, 58.35; H, 5.41; N, 3.22.
5
.1.1.18. 6,7,8-Trimethoxy-3-nitro-2-(4-methylphenyl)-2H-chromene (4r). Yield 90%; mp 134–
-1
1
1
36 ºC; IR (KBr, cm ): ν 1642, 1608, 1564, 1483, 1423, 1378, 1308, 1271, 1115, 992; H NMR
(
500 MHz, CDCl ): δ 2.31 (s, 3H, CH ), 3.65 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 3.94 (s, 3H,
3 3 3 3
OCH ), 6.59 (s, 1H, H -chromene), 6.61 (s, 1H, H -chromene), 7.12 (d, 2H, J = 7.8 Hz, H -
3
2
5
3,5
1
3
phenyl), 7.28 (d, 2H, J = 7.8 Hz, H2,6-phenyl), 8.02 (s, 1H, H
4
-chromene). C NMR (125 MHz,
CDCl ): δ 21.19, 56.39, 61.40, 61.44, 73.85, 107.26, 113.39, 114.10, 127.01, 128.56, 129.46,
3
1
6
29.54, 133.12, 139.48, 140.21, 141.89, 142.74, 147.66, 148.51. Anal. Calcd for C19
3.86; H, 5.36; N, 3.92. Found: C, 63.65; H, 5.51; N, 3.73.
6
H19NO : C,
5
.1.1.19. 6,7,8-Trimethoxy-3-nitro-2-(4-chlorophenyl)-2H-chromene (4s). Yield 87%; mp 139–
-1
1
1
41 ºC; IR (KBr, cm ): ν 1638, 1602, 1564, 1486, 1458, 1423, 1378, 1270, 1116, 829; H NMR
): δ 3.68 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 3.95 (s, 3H, OCH ), 6.59 (s, 1H,
H -chromene), 6.61 (s, 1H, H -chromene), 7.30 (d, 2H, J = 8.1 Hz, H -phenyl), 7.34 (d, 2H, J =
(500 MHz, CDCl
3
3
3
3
2
5
3,5
1
2

ACCEPTED MANUSCRIPT
1
3
8
6
1
4
4 3
.1 Hz, H2,6-phenyl), 8.03 (s, 1H, H -chromene). C NMR (125 MHz, CDCl ): δ 56.39, 61.39,
1.47, 73.16, 107.30, 113.03, 128.38, 129.01, 129.80, 134.70, 135.41, 139.68, 141.63, 142.67,
47.88, 148.71. Anal. Calcd for C H ClNO : C, 57.23; H, 4.27; N, 3.71. Found: C, 57.39; H,
1
8
16
6
.33; N, 3.77.
5
.1.1.20. 6,7,8-Trimethoxy-3-nitro-2-(4-nitrophenyl)-2H-chromene (4t). Yield 50%; mp 147–149
-1
1
ºC; IR (KBr, cm ): ν 1645, 1576, 1481, 1451, 1339, 1254, 1228, 1145, 1039, 753; H NMR (500
MHz, CDCl ): δ 3.68 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 6.58 (s, 1H, H
chromene), 6.61 (s, 1H, H -chromene), 7.57 (d, 2H, J = 8.5 Hz, H -phenyl), 8.02 (s, 1H, H -
3
3
3
3
2
-
5
2,6
4
1
3
chromene), 8.19 (d, 2H, J = 8.5 Hz, H -phenyl). C NMR (125 MHz, CDCl ): δ 56.42, 61.34,
3
,5
3
6
1
1.55, 73.22, 107.35, 113.13, 128.44, 128.93, 129.79, 134.68, 135.40, 139.63, 141.56, 142.65,
+
48.03, 148.61, 153.08. MS (m/z, %): 388 (m , 4), 342 (10), 212 (100), 197 (89), 169 (80), 154
16 2 8
(27), 126 (41), 83 (21). Anal. Calcd for C18H N O : C, 55.67; H, 4.15; N, 7.21. Found: C, 55.58;
H, 4.41; N, 7.23.
5
.1.1.21. 6,7,8-Trimethoxy-3-nitro-2-(3,4-methylenedioxyphenyl)-2H-chromene (4u). Yield 75%;
-1
mp 150–152 ºC; IR (KBr, cm ): ν 1648, 1561, 1485, 1457, 1319, 1270, 1139, 1036, 1039, 987;
1
H NMR (500 MHz, CDCl ): δ 3.66 (s, 3H, OCH ), 3.86 (s, 3H, OCH ), 3.94 (s, 3H, OCH ),
3
3
3
3
5
.88 (s, 2H, OCH O), 6.56 (s, 1H, H -chromene), 6.62 (s, 1H, H -chromene), 6.76 (d, 1H, J = 8.3
2
2
5
Hz, H
5
-phenyl), 6.85 (d, 1H, J = 8.3 Hz, H
6
2 4
-phenyl), 7.01 (s, 1H, H -phenyl), 8.05 (s, 1H, H -
1
3
chromene). C NMR (100 MHz, CDCl ): δ 55.78, 55.91, 60.25, 73.83, 96.23, 102.35, 107.59,
3
1
08.63, 113.75, 121.44, 124.30, 126.64, 129.59, 135.40, 139.35, 148.17, 153.38. Anal. Calcd for
C H NO : C, 58.92; H, 4.42; N, 3.62. Found: C, 58.78; H, 4.71; N, 3.35.
19
17
8
5
5
.2. Biology
.2.1. Cell lines and cell culture
Three different breast cancer cell lines including MCF-7, T-47D and MDA-MB-231 were
purchased from National Cell Bank of Iran (NCBI). The cells were routinely maintained in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (from
GibcoeBRL, UK) and 100 µg/mL streptomycin and 100 U/mL penicillin at 37 ºC in a humidified
incubator with 5% CO
2
.
5
.2.2. In vitro cytotoxicity assay
1
3

ACCEPTED MANUSCRIPT
The synthesized compounds 4a-u were tested against three human breast cancer cell lines using
MTT colorimetric assay [28,29]. Briefly, cells in the log-phase of growth were harvested by
4
trypsinization, and diluted in complete growth medium to give a total cell count of 5 ×10
cells/mL. The cell suspension (195 µL) from different cell lines was seeded in 96-well plates
(Nunc, Denmark) and the plates were incubated overnight in a humidified air atmosphere at 37
ºC with 5% CO . After plating, 5 µL of the media containing various concentrations of the
2
compounds 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) as negative
controls for cell viability. Synthetic compounds were initially dissolved in DMSO, and the final
concentration of DMSO was less than 1% in all experiments. Etoposide was used as positive
control for cytotoxicity. After 48 h further incubation, the medium was removed and 200 µL
phenol red-free medium containing MTT (1 mg/mL, final concentration), was added to wells,
followed by 4 h incubation. Then, the culture medium was replaced with 100 µL of DMSO and
the absorbance values were determined at 492 nm using a multi-well plate reader (Gen5, Power
wave xs2, BioTek, America). The IC50 values compared with the control were calculated by
nonlinear regression analysis and expressed in mean ±SD.
5
.2.3. AO/EB staining method
Apoptosis in treated cancer cells was determined morphologically after staining with acridine
orange/ethidium bromide (AO/EB) using fluorescence microscopy [30]. MDA-MB-231 cell
grown in 12-well plates (50,000 cells/well) were treated with and without IC50 concentrations of
compounds 4h and 4l for 48 h. After washing three times with phosphate buffered saline (PBS),
9
µL of cell suspension were stained with 1 µL of dye mixture (100 mg/mL AO and 100 mg/mL
EB in PBS). Stained cell suspension (10 µL) were placed on a clean microscope slide and
covered with a coverslip. The suspension was immediately examined by fluorescence
microscope (Axoscope 2 plus, Zeiss, Germany). All experiments were repeated three times.
5
.2.4. TUNEL assay
Apoptosis-induced nuclear DNA fragmentation in early stage of apoptosis was detected using in
situ cell death detection kit (fluorescein, Roche) following the manufacturer’s protocol. Briefly,
after 12 h treating the MDA-MB-231 cell with and without IC concentrations of compounds 4l
5
0
and 4h, cells were washed with PBS (pH 7.4) and fixed in 4% paraformaldehyde prepared
1
4

ACCEPTED MANUSCRIPT
freshly in PBS. The cells were again washed with PBS and permeabilized using 0.1% triton X
1
00 in sodium citrate 0.1% for 2 min on ice. Following 2 times washes in PBS buffer, the cells
were incubated in the TdT incubation buffer (fluorescein/ dNTP mix, TdT and labeling buffer)
for 60 min at 37 ºC in a humidified atmosphere in the dark and then washed and resuspended in
5
00 mL PBS. In humid chamber, terminal deoxynucleotidyltransferase (TdT) catalyzes
polymerization of fluorescein-labeled deoxynucleotides to free 3′-OH DNA ends in the absence
of a template. Free 3΄-OH DNA in apoptotic cells was detected and quantified based on green
fluorescence by flow cytometer (FACScan, LYSIS II, Becton Dickinson).
5
.2.5. Caspase-3 activation assay
The activity of caspase-3 was determined in early stage of apoptosis (after 12 h) using caspase-3
5
colorimetric assay [31]. Briefly, MDA-MB-231 cells (5×10 cells/well) in 6-well plate were
treated with IC concentrations of compounds 4l and 4h for 12 h. The control and treated cells
5
0
were harvested, washed once with PBS and were lysed on ice for 10 min in lysis buffer
containing 20 mM PIPES, 10 mM KCl, 2 mM MgCl , 4 mM DTT, 2 mM EDTA and 1 mM
2
EGTA freshly supplemented with protease inhibitors. After centrifuging for 15 min (10,000 ×g)
cytosol fraction was transferred to fresh tubes and put on ice for performing the assays. Protein
concentrations were determined by the Bradford method [32] using bovine serum albumin (BSA)
as the standard. One hundred micrograms protein was used for each assay in the presence of 200
µM colorimetric caspase substrate (Ac-DEVD-pNA, Sigma) in 100 µl reaction buffer (100 mM
NaCl, 50 mM HEPES, 10 mM DTT, 0.1% CHAPS, 0.1 mM EDTA, 10% Glycerol). The
absorbance was measured kinetically every 60 seconds for 120 minutes at 405 nm in an ELISA
reader. Caspase-3 activity was expressed as the change of the activity in treated cancer cells
compared to the untreated controls.
Acknowledgments
This research has been supported by a grant from the Research Council of Tehran University of
Medical Sciences and Iran National Science Foundation (INSF).
1
5

ACCEPTED MANUSCRIPT
References
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[
[
[
[
[
[
[
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Captions:
Fig. 1. Structural derivatization of some reported compounds (structures A–E) as pro-apoptotic
anti-cancer agents. Compounds 4a–u namely 2-aryl-3-nitro-2H-chromenes were designed in this
work, as new pro-apoptotic anti-cancer agents.
Fig. 2. Acridine orange/ethidium bromide double staining of MCF-7 (A) and MDA-MB-231 (B)
cells with characteristic symptoms of apoptosis: a) DMSO 1% as control, b) cells after exposure
to etoposide for 48 h, c) cells treated with compound 4l for 48 h, d) cells treated with compound
4
h for 48 h. White arrow indicates live cells, dashed arrow shows apoptosis. The images of cells
were taken with a fluorescence microscope at 400×.
Fig. 3. Flow-cytometric analysis of MDA-MB-231 cell line. DNA fragmentation and generation
of free 3′-OH DNA fragments was determined by TUNEL assay. MDA-MB-231 cells treated
with DMSO 1% (negative control) or with IC50 values of etoposide (positive control) and
compounds 4h and 4l, for 12 h. The presence of apoptotic cells is demonstrated by histogram
analys is indicating increase in fluorescence intensity (RN1gate) in treated cells with synthetic
compounds.
Fig. 4. Effect of etoposide (positive control), compounds 4h and 4l on caspase-3 activity in
MDA-MB-231 cell line compared to the negative control (untreated cells) after 12 h. Caspase-3
activities were determined by mixing the cell lysates with DEVD-pNA and monitoring
colorimetric substrate hydrolysis at 405 nm using absorbance plate reader. The values are shown
as a fold-increase compared to the control.
Scheme 1. Synthesis of compounds 4a-u. Reagents and conditions: (a) nitromethane, n-
butylamine, glacial acetic acid, reflux, 4 h; (b) DABCO, 40 ºC, 2 h.
Scheme 2. Synthesis of intermediate 3c. Reagents and conditions: (a) H O , H SO , MeOH, 0
2
2
2
4
ºC, 1h; (b) HMTA, TFA, reflux, 2 h.
1
8

ACCEPTED MANUSCRIPT
Table 1
Structures and in vitro cytotoxic activities of synthesized choromenes 4a-u against breast cancer cell lines after 48 h
treatment.
R1
NO2
R2
O
R4
R3
a
1
2
3
4
Cytotoxicity (IC50 µM)
Compounds
R
R
R
R
MCF-7
T-47D
MDA-MB-231
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
4-OCH
3,4-(OCH
3,4,5-(OCH )
3 3
3
3.3 ± 0.9
3.7 ± 0.2
4.3 ± 0.6
3.7 ± 0.5
3.7 ± 0.2
3.4 ± 0.3
3.8 ± 0.4
1.6 ± 0.2
3.1 ± 0.6
4.6 ± 0.5
3.9 ± 0.1
0.2 ± 0
3.9 ± 2.4
3.2 ± 2.3
2.1 ± 0.9
3.3 ± 2.5
4.5 ± 1.2
4.5 ± 1.6
5.6 ± 0.4
3.9 ± 1.2
9.1 ± 1.06
6.2 ± 0.5
4.6 ± 0.3
3.6 ± 2.1
7.5 ± 0.3
6.3 ± 0.4
4.7 ± 0.7
3.0 ± 0.2
12.7 ± 1.2
5.4 ± 1.3
4.5 ± 0.4
10.3 ± 0.2
9.5 ± 1.3
7.7 ± 0.7
4.1 ± 0.1
0.4 ± 0.2
4.9 ± 1.0
4.6 ± 0.5
4.1 ± 0.1
4.2 ± 0.1
4.5 ± 0.4
4.7 ± 0.6
12.8 ± 2.3
4.9 ± 0.1
3.3 ± 0.8
2.0 ± 0.2
0.5 ± 0.2
4.7 ± 0.4
4.8 ± 0.6
4.6 ± 0.1
6.8 ± 0.1
3.6 ± 0.3
5.2 ± 0.1
6.2 ± 0.7
6.1 ± 0.4
12.8 ± 1.0
H
H
)
3 2
H
H
H
H
4-CH
4-Cl
3
H
H
H
H
4-NO
3,4-methylenedioxy
4-OCH
3,4-(OCH
3,4,5-(OCH
2
g
h
i
H
H
H
H
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
H
H
3 2
)
j
H
H
3 3
)
k
l
H
H
4-CH
4-Cl
3
H
H
m
n
o
p
q
r
s
H
H
4-NO
3,4-methylenedioxy
4-OCH
3,4-(OCH
3,4,5-(OCH
2
2.3 ± 0.5
3.5 ± 0.07
3.8 ± 0.2
4.2 ± 0.1
3.9 ± 0.3
3.1 ± 0.7
3.5 ± 0.1
3.0 ± 1.4
4.7 ± 0.8
7.2 ± 0.8
H
H
OCH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
3
OCH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
3
3
)
3 2
)
3 3
4-CH
4-Cl
3
t
4-NO
2
u
3,4-methylenedioxy
Etoposide
a
Values are the mean ± SD. All experiments were performed at least three times.

ACCEPTED MANUSCRIPT
O
O
O
O
NO2
R
OH
R
R'
O
Nitrostyrenes (A)
Poly-methoxylated
Chalcones (D)
stilbene (B)
O
O
NO2
O
O
R
O
R'
R
Nitrovinylstilbenes (C)
Flavanones (E)
R1
R2
NO2
O
R4
R3
Designed compounds
4a-u)
(
Fig. 1. Structural derivatization of some reported compounds (structures A–E) as pro-apoptotic
anti-cancer agents. Compounds 4a–u namely 2-aryl-3-nitro-2H-chromenes were designed in this
work, as new pro-apoptotic anti-cancer agents.

ACCEPTED MANUSCRIPT
A
B
Fig. 2. Acridine orange/ethidium bromide double staining of MCF-7 (A) and MDA-MB-231 (B)
cells with characteristic symptoms of apoptosis: a) DMSO 1% as control, b) cells after exposure
to etoposide for 48 h, c) cells treated with compound 4l for 48 h, d) cells treated with compound
4
h for 48 h. White arrow indicates live cells, dashed arrow shows apoptosis. The images of cells
were taken with a fluorescence microscope at 400×.

ACCEPTED MANUSCRIPT
Etoposide
4h
Control
4l
Fig. 3. Flow-cytometric analysis of MDA-MB-231 cell line. DNA fragmentation and generation
of free 3′-OH DNA fragments was determined by TUNEL assay. MDA-MB-231 cells treated
with DMSO 1% (negative control) or with IC50 values of etoposide (positive control) and
compounds 4h and 4l, for 12 h. The presence of apoptotic cells is demonstrated by histogram
analys is indicating increase in fluorescence intensity (RN1gate) in treated cells with synthetic
compounds.

ACCEPTED MANUSCRIPT
Fig. 4. Effect of etoposide (positive control), compounds 4h and 4l on caspase-3 activity in
MDA-MB-231 cell line compared to the negative control (untreated cells) after 12 h. Caspase-3
activities were determined by mixing the cell lysates with DEVD-pNA and monitoring
colorimetric substrate hydrolysis at 405 nm using absorbance plate reader. The values are shown
as a fold-increase compared to the control.

ACCEPTED MANUSCRIPT
O
O N
2
a
H
R4
R4
R1
1a-g
2a-g
NO2
b
R2
O
R4
O
R3
R1
R2
H
4a-u
OH
R3
a-c
3
Scheme 1. Synthesis of compounds 4a-u. Reagents and conditions: (a) nitromethane, n-
butylamine, glacial acetic acid, reflux, 4 h; (b) DABCO, 40 ºC, 2 h.

ACCEPTED MANUSCRIPT
O
O
O
O
O
O
a
H
H
b
O
OH
O
OH
O
O
O
3
c
Scheme 2. Synthesis of intermediate 3c. Reagents and conditions: (a) H O , H SO , MeOH, 0
2
2
2
4
ºC, 1h; (b) HMTA, TFA, reflux, 2 h.

ACCEPTED MANUSCRIPT
Highlights
•
•
Compounds 4a-u were designed as new cytotoxic and apoptosis inducing agents.
Compounds 4a-u were synthesized from β-nitrostyrenes and salicylaldehydes.
•
•
•
The cytotoxicity of compounds 4a-u was tested against breast cancer cell lines.
Most of the tested compounds were more potent than standard drug etoposide.
Selected compounds 4h and 4l induce apoptosis via the activation of caspase-3.

ACCEPTED MANUSCRIPT
Supporting information
Synthesis, in vitro cytotoxicity and apoptosis inducing study of 2-aryl-3-nitro-
2
H-chromene derivatives as potent anti-breast cancer agents
Samira Rahmani Nezhad, Maliheh Safavi, Mahboobeh Pordeli, Sussan Kabudanian Ardestani,
Leila Khosravani, Yaghoub Pourshojaei, Mohammad Mahdavi, Saeed Emami, Alireza
Foroumadi, Abbas Shafiee*
1

ACCEPTED MANUSCRIPT
2