Design, synthesis, and biological evaluation of benzoselenazole-stilbene hybrids as multi-target-directed anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2015.03.030

To identify novel multi-target-directed drug candidates for the treatment of cancer, a series of benzoselenazole-stilbene hybrids were synthesised by combining the pharmacophores of resveratrol and ebselen. The biological assay indicated that all of the h

Full text of DOI:10.1016/j.ejmech.2015.03.030

Accepted Manuscript
Design, synthesis, and biological evaluation of benzoselenazole-stilbene hybrids as
multi-target-directed anti-cancer agents
Jun Yan, Yueyan Guo, Yali Wang, Fei Mao, Ling Huang, Xingshu Li
PII:
S0223-5234(15)00197-X
10.1016/j.ejmech.2015.03.030
EJMECH 7775
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 July 2014
Revised Date: 28 January 2015
Accepted Date: 13 March 2015
Please cite this article as: J. Yan, Y. Guo, Y. Wang, F. Mao, L. Huang, X. Li, Design, synthesis, and
biological evaluation of benzoselenazole-stilbene hybrids as multi-target-directed anti-cancer agents,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.03.030.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis, and Biological Evaluation of benzoselenazole-stilbene hybrids
as multi-target-directed Anti-cancer Agents
Jun Yan †,Yueyan Guo †, Yali Wang, Fei Mao, Ling Huang
, Xingshu Li

Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun
Yat-sen University, Guangzhou 510006, China
These properties highlight the potential of compound 6e as a candidate for further
studies on the development of novel drugs for cancer treatment.

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Design, synthesis, and biological evaluation of benzoselenazole-
stilbene hybrids as multi-target-directed anti-cancer agents
†
†
∗
∗
Jun Yan , Yueyan Guo , Yali Wang, Fei Mao, Ling Huang , Xingshu Li
Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical
Sciences, Sun Yat-sen University, Guangzhou 510006, China
ABSTRACT: To identify novel multi-target-directed drug candidates for the
treatment of cancer, a series of benzoselenazole-stilbene hybrids were synthesised by
combining the pharmacophores of resveratrol and ebselen. The biological assay
indicated that all of the hybrids exhibited antiproliferative activities against four
human cancer cell lines and demonstrated good TrxR inhibitory activities. The
mechanism of cell apoptosis was investigated in G /M cell cycle arrest induced by
2
compound 6e and the apoptosis of the human liver carcinoma Bel-7402 cell line. The
significant increase in intracellular ROS confirmed that compound 6e was capable of
causing oxidative stress-induced apoptosis in cancer cells. Our results support the
potential of compound 6e as a candidate for further studies examining the
development of novel drugs for cancer treatment.
KEYWORDS: benzoselenazole-stilbene hybrids, antiproliferative activity, apoptosis,
G /M cell cycle arrest, TrxR, ROS
2
∗
For L. Huang: Tel.: +086-20-3994-3051; Fax: +086-20-3994-3051; e-mail: huangl72@mail.sysu.edu.cn; For X.
Li.: Tel.: +086-20-3994-3050; Fax: +086-20-3994-3050; e-mail: lixsh@mail.sysu.edu.cn
†
J.Y. and Y.G. contributed equally to this work.
1

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1
. Introduction
Resveratrol (Figure 1) is a well-known polyphenol that is abundant in a wide
variety of plants, such as grapes, peanuts, and mulberries.[1] Extensive research
during the past couple of decades has suggested that resveratrol exhibits anticancer
activities and cardioprotective effects.[2-6] Natural trans-resveratrol targets a wide
variety of intracellular mechanisms in different pathways that lead to malignancy.[7,
8
] Among these, the mechanisms underlying arresting cell cycle progression and
triggering tumour cell death by apoptosis have been well-validated in many previous
studies.[9-12]
Mammalian thioredoxin reductase (TrxR), thioredoxin (Trx) and nicotinamide
adenine dinucleotide phosphate (NADPH) constitutes an essential system for many
cellular pathways.[13, 14] The main biological function of TrxR is to maintain the
cellular antioxidant system at normal physiological conditions, helping cellular
defence against apoptosis by reducing oxidised Trx.[15] TrxR is often overexpressed
in tumour cells,[16] and tumour proliferation appears to be crucially dependent on an
active thioredoxin system to eliminate excessive intracellular reactive oxygen species
(ROS),[17] thus making it a potential target for cancer therapy.[15, 18, 19] In recent
years, there have been several reports of anti-cancer drugs targeting TrxR.[20-23]
Increasing evidence shows that selenium-containing compounds play an
important role in cancer prevention and treatment due to their wide-ranging
physiological functions involved in tumour cell migration and invasion.[24, 25]
Among them, ebselen (Figure 1), 2-phenylbenzo[d] [1,2]selenazol-3(2H)-one, one of
the classic mimics of glutathione peroxidase (GPx), was also extensively studied as an
anticancer agent because of its broad spectrum of biological activities,[26] including
2

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the inhibition of cyclooxygenases, lipoxygenases and thioredoxin reductase, which
play an essential functional role in cancer therapy.[27-29]
Inspired by the strategy of multi-target-directed ligands (MTDLs) for the
treatment of diseases with multifaceted conditions, we designed and synthesised a
series of benzoselenazole-stilbene hybrids by combining the pharmacophores of
resveratrol and ebselen, which are expected to possess antiproliferative activity by
arresting cell cycle progression (pharmacological activity from resveratrol moiety)
and TrxR inhibitory activity (pharmacological activity from ebselen moiety)
simultaneously (Figure 1). Herein, we report the synthesis of these hybrids and the
evaluation of their antiproliferative activity, TrxR inhibitory effect, and ability to
increase the intracellular ROS levels.
We introduce the methoxy group into the A ring of all of the benzoselenazole-
stilbene hybrids (6a-6g) because, as reported in the literature, the methylation of
hydroxyl groups is essential to strengthen the efficiency of resveratrol derivatives for
blocking the cell cycle.[30-32] To evaluate the substituted group effect of the
benzoselenazole moiety in antiproliferative activity, we introduced a series of groups,
including fluorine, chlorine and methoxy, into the C ring. In addition, to evaluate the
steric effect of the stilbene backbone on the cytotoxic activity of the compounds, we
changed the trans configuration of compound 6a to yield 9 (cis configuration).[33]
Moreover, to confirm the “synergistic hybridisation” of those benzoselenazole-
stilbene hybrids, we synthesised compound 13, a carbon-counterpart isoindolinone-
stilbene derivative of compound 6e.
Insert Figure 1 here
Figure 1. Design strategy for multifunctional anti-cancer agents.
3

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2
. Results and Discussion
.1 Chemistry
The synthetic routes for the benzoselenazole-stilbene hybrids are outlined in
2
Schemes 1-3. Commercially available 4-nitrobenzaldehyde (1) was reacted with
sodium borohydride in methanol to provide (4-nitrophenyl)methanol, which was then
reacted with phosphorus tribromide in the presence of pyridine to yield 1-
(bromomethyl)-4-nitrobenzene. Finally, this compound was refluxed in triethyl
phosphite to give the Wittig reagent 2. Compound 2 was reacted with different
substituted benzaldehydes in the presence of sodium methoxide to yield stilbene
derivative 3. The reduction of 3 using stannous chloride provided amine 4, which was
reacted with different 2-(chloroseleno)benzoyl chlorides (5a-5e) in the presence of
sodium hydride to provide the target compounds 6a-j. Compounds 5a-5e were
prepared by reacting the corresponding o-aminobenzoic acid with sodium diselenide
and then sulfoxide chloride according to published procedures.[34] Compound 9, the
cis stereoisomer of compound 6a, was obtained through the Wittig reaction of
phosphonium salt 7 and p-nitrobenzaldehyde, followed by reduction with sodium
dithionite and reaction with 2-(chloroseleno)benzoyl chloride (Scheme 2). The
carbon-counterpart isoindolinone-stilbene derivative of 6e (compound 13) was
synthesised as detailed in Scheme 3. First, the methyl esterification of 2-
formylbenzoic acid (10) yielded compound 11, which was treated with amine 4b for
two steps and then cyclised in the presence of sodium methoxide to provide
isoindolinone-stilbene derivative 13.
Insert Scheme 1 here
Scheme 1. Synthesis of the benzoselenazole-stilbene hybrids 6. Reagents and
4

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conditions: a. NaBH , MeOH, 0°C-rt; b. PBr , pyridine, DCM, 0°C-rt; c. triethyl
4
3
phosphate, 120°C; d. different aldehydes, CH ONa, 0°C, 1 h, rt, 12 h; e. SnCl ·2H O,
3
2
2
AcOEt, 90°C; f. NaH, THF, 0°C-rt.
Insert Scheme 2 here
Scheme 2. Synthesis of compound 9. Reagents and conditions: a. p-
nitrobenzaldehyde, n-BuLi, THF, -78°C; b. Na S O , acetone/H O; c. 5a, NaH, THF,
2
2
4
2
0
°C-rt.
Insert Scheme 3 here
Scheme 3. Synthesis of the compound 13. Reagents and conditions: a: SOCl , MeOH;
2
b: 4b, Na SO , ethanol, rt; c: NaBH , ethanol; d: NaOMe, MeOH, reflux.
2
4
4
2
.2 In vitro antiproliferative activity
The antiproliferative activities of the targeted compounds were evaluated in the
cell lines Bel-7402 (human liver carcinoma cell line), A549 (non-small-cell-lung
cancer line), HeLa (human epithelial cervical cancer line) and MCF-7 (human breast
cancer cell line) using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay with resveratrol and ebselen as the reference compounds.
The IC values listed in Table 1 indicated that all of the targeted compounds
5
0
exhibited more potent antiproliferative activities than the reference compounds.
Among them, compounds 6b, 6c, and 6e showed the best results, with IC values
5
0
ranging from 0.23 to 3.37 ꢀM against the four tested human cancer cell lines, showing
these there are nearly 50-fold more active than resveratrol. Compound 6a (IC = 3.49
5
0
ꢀ
M for Bel-7402), with 3,5-dimethoxy on the A ring, showed better antiproliferative
activities than resveratrol (IC = 50.7 ꢀM for Bel-7402), which is consistent with the
5
0
literature results.[30] When the A ring was substituted with 3,4-dimethoxy, the
antiproliferative activity increased to some extent (6e: IC = 1.01 ꢀM for Bel-7402).
5
0
5

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Increasing the numbers of methoxy groups in the A ring did not show any significant
change in the activity (6f, R1=R2=R3=OCH3, IC50 = 1.11 ꢀM for Bel-7402).
However, when the numbers of methoxy groups in the A ring was decreased, the
activity decreased markedly (6g, R1=OCH3, R2=R3=H, IC = 13.2 ꢀM for Bel-7402;
5
0
6
h, R1=R2=R3=H, IC = 12.4 ꢀM for Bel-7402), which indicated that the methoxy
50
group is crucial for anti-cancer activity. Thus, it can be concluded that the compound
possessing two methoxy groups, particularly at the 3, 4-position on the A ring, is
favourable to the activity. In contrast, the results showed that methoxy groups on the
C ring have a negative effect to the activity (6d: IC50 = 25.4 ꢀM for Bel-7402).
Interestingly, when the substituted group in the C ring was changed to fluorine or
chlorine atoms, the antiproliferative activities increased significantly (6g: IC = 0.99
50
ꢀ
M for Bel-7402; 6h: IC = 0.79 ꢀM for Bel-7402). However, as shown in Table 2,
50
the solubility of these halogen-atom-containing compounds in water was very low,
which limited their further study. Unlike the literature results that show that the cis
form of the stilbene backbone was favourable to cytotoxic activity against the
corresponding trans isomers, compound 9, which is the cis form of 6a, gave poor
activity (IC50 = 36.1 ꢀM for Bel-7402). Compared with 6e, the antiproliferative
activity of compound 13 was significantly decreased (above 10-fold), which strongly
demonstrates that the “synergistic hybridisation" of the benzoselenazole-stilbene
hybrids and/or the introduction of selenium were beneficial for the activity.
Insert Table 1 here
Insert Table 2 here
2
.3 In vitro inhibition of TrxR
TrxR is usually overexpressed in tumour cells compared to normal tissues, and
tumour proliferation appears to be crucially dependent on an active thioredoxin
system.[16-18] Thus, TrxR appears to be a potential target for anti-cancer therapy. To
6

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evaluate the ability of the designed compounds to inhibit TrxR and to determine the
possible relationship between in vitro TrxR inhibition and cell growth inhibition, we
used a 5,5’ dithiol-bis-2-nitrobenzoic acid (DTNB) reduction assay;[35, 36] the
results are summarised in Table 2. As expected, compound 6e exhibited the best
inhibitory activity (IC = 3.10 ꢀM) of the tested compounds, which was also better
5
0
than that of the reference compound ebselen. Compounds 6a and 6f, which exhibited
better cell growth inhibition effects compared to 6g and 6h, also showed better TrxR
inhibitory activities in vitro (6a and 6f, IC = 8.27 and 5.68 ꢀM, respectively; 6g and
5
0
6
6
h, IC = 12.8 ꢀM, and 22.4% of inhibition rate at 10 ꢀM, respectively). Compounds
50
b, 6c, 6i and 6j, which possess halogen atoms on the C ring, exhibited lower TrxR
inhibitory activities (36.6%, 36.4%, 31.2%, and 38.4% of the inhibition rate at 10 ꢀM,
respectively). However, compounds 6d, which demonstrated relatively good TrxR
inhibition in vitro (IC = 7.53), exhibited very poor cell growth inhibition, and the
5
0
cause of this inconsistency requires further study. The reference compound resveratrol
exhibited only 26.1% of inhibition rate at 50 ꢀM, and the reference compound ebselen
showed moderate inhibition (IC = 8.54 ꢀM). These data indicated that the TrxR
5
0
inhibition activity benefited from the introduction of the Se atom. Compound 13,
which is characterised by the absence of a Se atom, exhibited no activity at 10 ꢀM,
further proving the necessity of Se in the compounds. Based on the overall
consideration of the antiproliferative activity, TrxR inhibition activity and solubility of
the targeted compounds in water, compound 6e was chosen as the optimised
compound for further mechanistic study.
Insert Table 3 here
2
.4 Cell cycle analysis
7

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To determine whether the antiproliferative activity induced by the hybrids was
due to cell cycle arrest, flow cytometry analysis was performed. Bel-7402 cells were
treated with compound 6e at different concentrations (0.5, 1, 3, and 5 ꢀM), resveratrol
(50 ꢀM), and ebselen (50 ꢀM) for 48 h, and the harvested cells were analysed by flow
cytometry. Compared with the control, resveratrol and compound 6e exhibited modest
cell cycle arrest, whereas the ebselen group did not lead to obvious cell cycle arrest.
These results demonstrated that the optimised compound 6e possessed antitumor
activity via inhibition of multiple pathways, including cell cycle arrest, which was
consistent with resveratrol. In addition, we also treated the cells with compound 6e at
various concentrations (0.5, 1, 3, and 5 ꢀM) for 48 h, as shown in Figure 2B, and
compound 6e resulted in significant cell cycle arrest at the G /M phase, showing a
2
dose-dependent pattern. At a concentration of 0.5 ꢀM, 22.17% of the cell population
was in the G /M phase of the cell cycle, and this percentage increased to 33.8%,
2
4
2.05%, and 44.23% when the concentration was increased to 1, 3, and 5 ꢀM,
respectively. The percentages were comparatively higher than those obtained in the
vehicle group (0.1% DMSO). These data revealed that the target compounds could
arrest the cell cycle progression at the mitosis phase and further confirm the growth
inhibitory effect of compound 6e on liver carcinoma cells.
Insert Figure 2 here
Figure 2. Effect of compound 6e, resveratrol and ebselen on the cell cycle arrest of
Bel-7402 cells. (A) The cells were treated with compound 6e, resveratrol and ebselen
at the IC values for 48 h. (B) The cells were treated with compound 6e at 0.5, 1, 3
5
0
and 5 ꢀM for 48 h. The DNA contents of each cell phase were analysed by flow
cytometry. The experiments were performed three times, and the results of
representative experiments are shown.
8

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2
.5
Cell apoptosis analysis
To further elucidate the mechanism of cell death caused by the hybrids, Bel-7402
cells were treated with compound 6e (0.5, 1, 3 and 5 ꢀM), resveratrol (50 ꢀM), and
ebselen (50 ꢀM) for 48 h, using DMSO as the control. After incubation for 48 h, the
cells were labelled with two dyes (propidium iodide (PI) and annexin-V-FITC), the
red (PI) and green (FITC) fluorescence was then monitored by flow cytometry. The
results of the cell apoptosis assay, which was determined by flow cytometry (the
results are shown in Figure 3A), indicated that resveratrol and compound 6e showed
significant cell apoptosis, whereas ebselen just induced modest cell apoptosis. These
data also demonstrated that the anti-tumour activity of compound 6e was mostly
benefited from the resveratrol part, which was consistent with the cell cycle arrest
results. Similarly, we also treated the cells with compound 6e at various
concentrations (0.5, 1, 3, and 5 ꢀM) for 48 h, as shown in Figure 3B, and compound
6
e resulted in significant cell apoptosis in a dose-dependent manner. The percentage
of cell apoptosis induced by compound 6e was 0.73% at 0 ꢀM, and this percentage
increased to 6.73%, 13.55%, 36.71%, and 53.40% at 0.5, 1, 3, and 5 ꢀM, respectively.
These data demonstrated that compound 6e effectively induced cell apoptosis in Bel-
7
402 cells in a dose-dependent manner, eventually leading to cell death.
Insert Figure 3 here
Figure 3. Effect of compound 6e, resveratrol and ebselen on the cell cycle arrest of
Bel-7402 cells. (A) The cells were treated with compound 6e, resveratrol and ebselen
at IC values for 48 h. (B) The cells were treated with compound 6e at 0.5, 1, 3 and 5
5
0
ꢀ
M for 48 h. The percentages of cells in each stage of cell apoptosis were quantified
by flow cytometry: (upper left quadrant) detection error cell; (upper right quadrant)
necrotic cells; (bottom left quadrant) live cells; and (bottom right quadrant) apoptotic
9

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cells. The experiments were performed three times, and representative experiments
are shown.
2
.6
Measurement of intracellular ROS generation
Previous studies have revealed that oxidative stress is an influential mechanism
in selenium-induced cytotoxicity.[37] ROS plays an important role in cancer cell
apoptosis induced by anti-cancer drugs through DNA damage in the cell nucleus. To
determine whether the cell apoptosis induced by compound 6e is ROS-dependent, we
evaluated the intracellular ROS generation in Bel-7402 cells. The images in Figures
4
A and 4B demonstrate the results visually. As shown in Figures 4A and 4B, when the
cells were untreated, the green fluorescence, which reflects the level of intracellular
ROS, was difficult to observe in the control group. In contrast, after compound 6e,
resveratrol, and ebselen were added for 4 h, the intensity of the green fluorescence
was brighter than that found for the control group. When the concentration of
compound 6e was increased, the intensity of the green fluorescence became
progressively brighter. As shown in Figure 4C, the generation of intracellular ROS
increased to 109.7%, 140.4%, 177.8% and 193.4% compared with the control cells
(100%; p < 0.05) after the Bel-7402 cells were treated with compound 6e at
concentrations of 0.5, 1, 3, and 5 ꢀM, respectively, for 4 h. Under the same
conditions, resveratrol (50 ꢀM) generated a slight increase in intracellular ROS
(118%), whereas ebselen (50 ꢀM) did not cause much change compared to the control
group. These results indicated that compound 6e induced an increase in intracellular
ROS generation, which may result in cell apoptosis and lead to eventual cell death.
Insert Figure 4 here
Figure 4. Intracellular ROS accumulation induced by compound 6e, resveratrol, and
1
0

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ebselen. (A) Bel-7402 cells were treated with compound 6e, resveratrol and ebselen at
IC values for 4 h. (B) Bel-7402 cells were treated with compound 6e (0.5, 1, 3, and
5
0
5
ꢀM) for 4 h, and the intracellular ROS was measured using the ROS indicator
DCFH-DA, as described in the Experimental Section. Representative fluorescent
images of the intracellular ROS level obtained using an Arrayscan VTI High Content
Analysis System (magnification, 200×). The nuclei were stained with Hoechst 33342
as indicated by blue fluorescence, and the accumulated ROS was indicated by green
fluorescence. (C) The fluorescence intensity, which corresponded to the intracellular
ROS level, was measured using a microplate reader with the excitation and emission
wavelengths set at 488 and 525 nm, respectively. The data are shown as the means ±
SD (n = 3); *p < 0.05 and ***p < 0.001 compared with the control (0.1% DMSO).
3
. Conclusions
In summary, we have designed, synthesised, and evaluated a series of novel
benzoselenazole-stilbene hybrids as anti-cancer agents. The results demonstrated that
all of the designed compounds possessed potential antiproliferative activities against
four human cancer cell lines. In this study, we describe the mechanism through which
compound 6e induces G /M cell cycle arrest and apoptosis of the human liver
2
carcinoma Bel-7402 cell line. The TrxR inhibition assay revealed a possible
relationship between the in vitro TrxR inhibition and cell growth inhibition, and
compound 6e, which showed good antiproliferative activities against four human
cancer cell lines, also exhibited the best TrxR inhibitory activity. Considering the
important role of TrxR in oxidation-reduction systems, we also studied intracellular
ROS generation in Bel-7402 cells after treatment with compound 6e. The significant
increase in the intracellular ROS levels confirmed that compound 6e could cause
oxidative stress-induced apoptosis in cancer cells. The multi-target-directed anti-
11

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cancer properties of compound 6e support its role as a candidate for further studies
directed toward the development of novel drugs for cancer treatment.
4
4
. Experimental Section
.1 Chemistry
1
13
The H NMR and C NMR spectra were acquired on a Bruker Avance III
spectrometer using TMS as the internal standard. High-resolution mass spectra (HR-
MS) were recorded on an Agilent LC-MS 6120 instrument with an ESI mass selective
detector in the positive ion mode. The melting points were determined using an SRS-
OptiMelt automated melting point instrument. The purity of the synthesised
compounds was determined by high-performance liquid chromatography (HPLC)
with a TC-C18 column (4.6 × 250 mm, 5 ꢀm), an acetonitrile/water or methanol/water
mobile phase, and a flow rate of 1.00 mL/min.
4
.1.1 General procedure for the preparation of 3
To a solution of CH ONa (1.350 g, 7.5 mmol) in DMF (10 mL), 2 (1.296 g, 5
3
mmol) was added. After the mixture was stirred at 0°C under nitrogen for 1 h, a
substituted benzaldehyde (7.5 mmol) in DMF (5 mL) was added dropwise to the
solution. The reaction mixture was slowly warmed to room temperature and stirred
overnight. Ice water was added, and the mixture was filtered to yield the crude
product. Purification of the crude product by recrystallisation from ethyl acetate
provided the target product.
(E)-1, 3-dimethoxy-5-(4-nitrostyryl) benzene (3a)
1
Yellow solid, yield 74%. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H),
3
7
.63 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 16.3 Hz, 1H), 7.11 (d, J = 16.3 Hz, 1H), 6.70 (d,
+
J = 2.1 Hz, 2H), 6.46 (t, J = 2.1 Hz, 1H), 3.84 (s, 6H). LC-MS(ESI): 286.0ꢀM+Hꢁ .
1
2

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(E)-1, 2-dimethoxy-4-(4-nitrostyryl) benzene (3b)
1
Yellow solid, yield 63.8%. H NMR (400 MHz, CDCl ) δ 8.21 (d, J = 8.4 Hz,
3
2
H), 7.60 (d, J = 8.5 Hz, 2H), 7.29-7.16 (m, 1H), 7.11 (d, J = 8.0 Hz, 2H), 7.00 (d, J =
1
6.2 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H).LC-MS(ESI): 286.0
+
ꢀ
M+Hꢁ .
(E)-1, 2, 3-trimethoxy-5-(4-nitrostyryl) benzene (3c)
1
Yellow solid, yield 57%. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 7.7 Hz, 2H),
3
7
.63 (s, 2H), 7.39
-7.14 (m, 1H), 7.05 (dd, J = 16.5, 4.7 Hz, 1H), 6.78 (d, J = 5.0 Hz,
2
H), 3.94 (d, J = 5.0 Hz, 6H), 3.90 (d, J = 5.1 Hz, 3H). LC-MS(ESI): 316.0ꢀM+Hꢁ
+
.
(E)-1-methoxy-3-(4-nitrostyryl) benzene (3d)
1
Yellow solid, yield53%. H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 8.8 Hz, 2H),
7
.59 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 16.3 Hz, 1H), 7.00 (d, J
+
=
16.3 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H). LC-MS(ESI): 256.0ꢀM+Hꢁ .
(E)-1-nitro-4-styrylbenzene (3e)
1
Yellow solid, yield 60%. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H),
3
7
.64 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 7.1 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.34 (d, J =
7
.2 Hz, 1H), 7.27 (t, J = 8.1 Hz, 1H), 7.15 (d, J = 16.3 Hz, 1H). LC-MS(ESI): 226.1
+
ꢀ
M+Hꢁ .
4
.1.2 General procedure for the synthesis of compound 4
To a solution of compound 3 (1 eq.) in ethyl acetate, stannous chloride (5 eq.)
was added. After refluxing for 6 h, the mixture was cooled to room temperature, and
saturated sodium bicarbonate solution was added until the solution was basic. Ethyl
acetate was added, and the organic layer was separated. Solvent removal yielded the
crude product, which was purified by silica gel column chromatography
(AcOEt/petroleum ether as the eluent) to provide the target product.
1
3

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(E)-4-(3, 5-dimethoxystyryl) aniline (4a)
1
White solid, yield: 70%. H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.3 Hz, 2H),
7
6
.00 (d, J = 16.2 Hz, 1H), 6.85 (d, J = 16.2 Hz, 1H), 6.66 (dd, J = 14.4, 5.0 Hz, 4H),
+
.36 (s, 1H), 3.83 (s, 6H). LC-MS(ESI): 256.1ꢀM+Hꢁ .
(E)-4-(3, 4-dimethoxystyryl) aniline (4b)
1
Yellow solid, yield 83.6%. H NMR (400 MHz, CDCl ) δ 7.31 (d, J = 8.4 Hz,
3
2
H), 7.07 – 6.96 (m, 2H), 6.86 (dd, J = 14.7, 5.1 Hz, 3H), 6.67 (d, J = 8.4 Hz, 2H),
+
3
.92 (d, J = 10.5 Hz, 3H), 3.87 (d, J = 11.8 Hz, 3H). LC-MS(ESI): 256.1ꢀM+Hꢁ .
(E)-4-(3, 4, 5-trimethoxystyryl) aniline (4c)
1
Yellow solid, yield 55%. H NMR (400 MHz, CDCl ) δ 7.32 (d, J = 8.4 Hz, 2H),
3
6
.93 (d, J = 16.2 Hz, 1H), 6.84 (d, J = 16.2 Hz, 1H), 6.77-6.62 (m, 4H), 3.90 (d, J =
+
2
.3 Hz, 6H), 3.85 (d, J = 2.4 Hz, 3H), 3.74 (s, 2H). LC-MS(ESI): 286.1ꢀM+Hꢁ .
(E)-4-(3-methoxystyryl) aniline (4d)
1
Yellow solid, yield 86%. H NMR (400 MHz, CDCl ) δ 7.33 (d, J = 8.4 Hz, 2H),
3
7
.25 (dd, J = 9.5, 6.3 Hz, 1H), 7.10 – 6.97 (m, 3H), 6.89 (d, J = 16.3 Hz, 1H), 6.77
(dd, J = 8.1, 2.3 Hz, 1H), 6.67 (d, J = 8.4 Hz, 2H), 3.84 (s, 3H), 3.74 (s, 2H). LC-
+
MS(ESI): 226.1ꢀM+Hꢁ .
(E)-4-styrylaniline (4e)
1
White solid, yield 70.2%. H NMR (400 MHz, CDCl ) δ 7.47 (d, J = 7.4 Hz, 2H),
3
7
.33 (t, J = 7.8 Hz, 4H), 7.21 (d, J = 7.4 Hz, 1H), 7.02 (d, J = 16.3 Hz, 1H), 6.92 (d, J
+
=
16.3 Hz, 1H), 6.72-6.62 (m, 2H). LC-MS(ESI): 196.0ꢀM+Hꢁ .
4
.1.3 General procedure for the synthesis of compounds 6
Compound 4 (1 mmol) was dissolved in THF and stirred at 0 °C, and NaH (2.5
mmol) was then added to the solution. After the reaction mixture was stirred at 0 °C
for 0.5 h under nitrogen, compounds 5a-5e (1.2 mmol) were added, and the mixture
was slowly warmed to room temperature and stirred for 3 h. The solvents were
1
4

ACCEPTED MANUSCRIPT
evaporated under vacuum, and ice water was added to the residue. The crude product
was obtained by filtration and was purified by recrystallisation from tetrahydrofuran
to provide the target compound.
(E)-2-(4-(3, 5-dimethoxystyryl) phenyl)benzo[d][1,2]selenazol-3(2H)-one (6a)
1
White solid, yield 55.0%, m.pꢂ238.8-239.7 ꢃ. H NMR (400 MHz, DMSO-d )
6
δ 8.10 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 7.4 Hz, 1H), 7.69 (s, 5H), 7.50 (t, J = 7.4 Hz,
1
H), 7.31 (d, J = 16.3 Hz, 1H), 7.20 (d, J = 16.4 Hz, 1H), 6.81 (s, 2H), 6.44 (s, 1H),
1
3
3
.80 (s, 6H). C NMR (101 MHz, DMSO-d ) δ 165.03, 160.64, 139.19, 139.01,
6
1
38.71, 134.35, 132.28, 128.51, 128.49, 128.09, 127.16, 126.27, 125.78, 124.48,
+
1
04.46, 99.96, 55.19. HRMS (ESI) (m /z) [M+H] calcd for C H NO Se, 438.0603;
23
19
3
found,.438.0809 Purity: 96.5% (by HPLC).
(
E)-2-(4-(3,5-dimethoxystyryl)phenyl)-6-fluorobenzo[d][1,2]selenazol-3(2H)-one
6b)
(
1
Yellow solid, yield 48.4%, m.pꢂ257.0-258.7 ꢃ. H NMR (400 MHz, DMSO-d )
6
δ 8.04 (d, J = 8.9 Hz, 1H), 7.92 (dd, J = 8.2, 5.6 Hz, 1H), 7.66 (s, 4H), 7.30 (d, J =
1
3
1
5.6 Hz, 2H), 7.18 (d, J = 16.4 Hz, 1H), 6.80 (s, 2H), 6.42 (s, 1H), 3.79 (s, 6H). C
NMR (101 MHz, DMSO-d ) δ 165.66, 164.10, 163.17, 160.64, 141.02 (d, J = 10.8
6
Hz), 139.06 (d, J = 9.4 Hz), 134.35, 130.11 (d, J = 9.7 Hz), 128.48, 128.09, 127.17,
1
25.43, 124.44, 114.48 (d, J = 23.9 Hz), 112.50 (d, J = 26.9 Hz), 104.45, 99.94, 55.18.
+
HRMS (ESI) (m /z) [M+H] calcd for C H FNO Se, 456.0509; found,.456.0517.
2
3
18
3
Purity: 98.6% (by HPLC).
(E)-6-chloro-2-(4-(3,5-dimethoxystyryl)phenyl)benzo[d][1,2]selenazol-3(2H)-one
(6c)
1
Green solid, yield 65.4%, m.p 236.3-239.2ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
8
.31 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.68 (s, 4H), 7.53 (d, J = 8.2 Hz, 1H), 7.32 (d, J
1
3
=
16.3 Hz, 1H), 7.20 (d, J = 16.3 Hz, 1H), 6.81 (s, 2H), 6.44 (s, 1H), 3.80 (s, 6H). C
1
5

ACCEPTED MANUSCRIPT
NMR (101 MHz, DMSO-d ) δ 164.08, 160.63, 140.73, 139.18, 139.02, 136.99,
6
1
34.28, 129.23, 128.42, 128.11, 127.98, 127.15, 126.53, 125.60, 124.35, 104.44,
+
9
9.94, 55.18. HRMS (ESI) (m /z) [M+H] calcd for C H ClNO Se, 472.0213;
2
3
18
3
found,.472.0225. Purity: 96.3% (by HPLC).
(
E)-2-(4-(3,5-dimethoxystyryl)phenyl)-5,6-dimethoxybenzo[d][1,2]selenazol-
(2H)-one (6d)
Red solid, yield 42.3%, m.p 222.8-223.8 ꢃ. H NMR (400 MHz, DMSO-d ) H
3
1
1
6
NMR (400 MHz, DMSO-D6) δ 7.69 (t, J = 7.8 Hz, 5H), 7.41 (s, 1H), 7.32 (d, J =
1
3
1
1
6.3 Hz, 1H), 7.21 (d, J = 16.4 Hz, 1H), 6.84 (d, J = 2.1 Hz, 2H), 6.55 – 6.40 (m, 1H),
1
3
.94 (s, 3H), 3.92 (s, 3H), 3.85 (s, 6H). C NMR (101 MHz, DMSO-d ) δ 165.00,
6
60.65, 153.05, 148.53, 139.58, 139.04, 133.99, 131.24, 128.30, 128.13, 127.14,
24.12, 120.58, 109.00, 107.34, 104.44, 99.92, 55.80, 55.65, 55.18. HRMS (ESI) (m
+
/
z) [M+H] calcd for C H NO Se, 498.0814; found,.498.0825 Purity: 98.0% (by
2
5
23
5
HPLC).
(E)-2-(4-(3,4-dimethoxystyryl)phenyl)benzo[d][1,2]selenazol-3(2H)-one (6e)
1
Yellow solid, yield 64%, m.p 226.5-228.3 ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
8
.12 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.73-7.62 (m, 5H), 7.50 (t, J = 7.4
Hz, 1H), 7.27 (d, J = 1.3 Hz, 1H), 7.19 (d, J = 2.6 Hz, 2H), 7.12 (dd, J = 8.3, 1.5 Hz,
1
3
1
H), 6.96 (d, J = 8.3 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H). C NMR (101 MHz, DMSO-
d6) δ 165.00, 148.94, 148.78, 138.76, 138.71, 134.88, 132.21, 129.93, 128.58, 128.49,
27.89, 126.74, 126.24, 125.82, 125.42, 124.53, 119.99, 111.79, 109.21, 55.49.
1
HRMS (ESI) (m /z) [M+H]+ calcd for C23H19NO3Se, 438.0603; found,.438.0620.
Purity: 98.8% (by HPLC).
(E)-2-(4-(3, 4, 5-trimethoxystyryl) phenyl) benzo[d] [1, 2] selenazol-3(2H)-one (6f)
1
Red solid, yield 55.7%, m.p 207.2-209.2 ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
7
.95 (s, 1H), 7.79 (d, J = 6.5 Hz, 2H), 7.41 (d, J = 7.7 Hz, 2H), 7.21 (d, J = 16.3 Hz,
1
6

ACCEPTED MANUSCRIPT
1
3
1
H), 7.13 (d, J = 16.2 Hz, 1H), 6.92 (s, 2H), 3.83 (s, 6H), 3.67 (s, 3H). C NMR (101
MHz, DMSO-d6) δ 165.02, 153.02, 138.90, 138.71, 137.31, 134.61, 132.68, 132.30,
28.62, 128.47, 127.92, 126.93, 126.29, 125.76, 124.57, 103.87, 60.05, 55.85. HRMS
ESI) (m /z) [M+H]+ calcd for C24H21NO4Se, 468.0709; found,.468.0719. Purity:
7.5% (by HPLC).
1
(
9
(E)-2-(4-(3-methoxystyryl)phenyl)benzo[d][1,2]selenazol-3(2H)-one (6g)
1
Yellow solid, yield 46.5%, m.p 196.5-197.8 ꢃ. H NMR (400 MHz, DMSO-d )δ
6
8
1
=
.14-8.05 (m, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 2.3 Hz, 5H), 7.52-7.45 (m,
H), 7.25 (dd, J = 10.2, 4.9 Hz, 3H), 7.18 (s, 2H), 6.83 (d, J = 8.0 Hz, 1H), 3.78 (d, J
13
2.3 Hz, 3H). C NMR (101 MHz, DMSO-d δ 165.01, 159.56, 139.12, 138.70,
6
1
38.41, 134.40, 132.30, 129.68, 128.50, 128.37, 127.93, 127.84, 127.15, 126.29,
+
1
25.77, 124.49, 119.07, 113.51, 111.42, 55.03. HRMS (ESI) (m /z) [M+H] calcd for
C H NO Se, 408.0497; found,.408.0509. Purity: 99.2% (by HPLC).
22
17
2
(E)-2-(4-styrylphenyl) benzo[d][1,2]selenazol-3(2H)-one (6h)
1
Yellow solid, yield 82%, m.p 232.6-233.4 ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
8
2
.12 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.69 (s, 5H), 7.63 (d, J = 7.4 Hz,
1
3
H), 7.51 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.6 Hz, 2H), 7.30 (d, J = 9.1 Hz, 3H). C
NMR (101 MHz, DMSO-d ) δ 165.05, 139.11, 138.76, 136.93, 134.46, 132.27,
6
1
28.69, 128.53, 128.44, 127.92, 127.65, 127.55, 127.13, 126.44, 126.27, 125.79,
1
24.50. HPLC purity: 97.4%. LC-MS(ESI): 378.0ꢀM+Hꢁ+. HRMS (ESI) (m /z)
[
M+H]+ calcd for C H NOSe, 378.0392; found, 378.0401. Purity: 98.6% (by
21 15
HPLC).
(
E)-5-chloro-2-(4-(3,5-dimethoxystyryl)phenyl)benzo[d][1,2]selenazol-3(2H)-one
6i)
(
1
Yellow solid, yield 57%, m.p 222.1-224.0 ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
8
.19 (dd, J = 8.4, 4.3 Hz, 1H), 7.87 (d, J = 2.1 Hz, 1H), 7.82-7.61 (m, 5H), 7.26 (dt, J
1
7

ACCEPTED MANUSCRIPT
1
3
=
16.3, 12.5 Hz, 2H), 6.82 (s, 2H), 6.44 (s, 1H), 3.80 (d, J = 4.2 Hz, 6H). C NMR
(
101 MHz, DMSO-d ) δ 163.74, 160.64, 138.99, 137.57, 134.51, 131.95, 131.34,
6
1
30.46, 128.55, 128.06, 127.96, 127.17, 126.91, 124.45, 104.46, 99.97, 55.18. HRMS
ESI) (m /z) [M+H]+ calcd for C23H18ClNO3Se, 472.0213; found,.472.0225. Purity:
6.2% (by HPLC).
(
9
(E)-5-chloro-2-(4-(3,4-dimethoxystyryl)phenyl)benzo[d][1,2]selenazol-3(2H)-one
(6j)
1
Yellow solid, yield 57%, m.p 223.0-224.0 ꢃ. H NMR (400 MHz, DMSO-d ) δ
6
8
.10 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.74 (dd, J = 8.6, 2.3 Hz, 1H), 7.65
(s, 4H), 7.26 (d, J = 1.6 Hz, 1H), 7.18 (d, J = 3.6 Hz, 2H), 7.11 (dd, J = 8.3, 1.7 Hz,
1
3
1
H), 6.96 (d, J = 8.4 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 3H). C NMR (101 MHz, DMSO-
d6) δ 164.22, 149.39, 149.26, 138.79, 137.93, 135.69, 132.61, 131.92, 130.76, 130.35,
29.15, 128.30, 127.50, 127.27, 125.82, 125.10, 120.56, 112.16, 109.55, 55.94.
1
HRMS (ESI) (m /z) [M+H]+ calcd for C23H18ClNO3Se, 472.0213; found,.472.0220.
Purity: 99.5% (by HPLC).
(Z)-1,3-dimethoxy-5-(4-nitrostyryl)benzene (8)
Compound 8 was synthesised following a previously reported procedure.[30]
Briefly, to a solution of phosphonium bromide salt 7 (4.934 g, 10 mmol) in anhydrous
tetrahydrofuran at -78°C, n-butyllithium (2.5 M in hexanes, 6 mL) was added. After
stirring for 1 h, a solution of p-nitrobenzaldehyde in THF was added dropwise over a
period of 30 min. The reaction mixture was slowly warmed to room temperature and
stirred overnight. The resulting suspension was poured into water and extracted with
DCM. Compound 8 was obtained by flash chromatography. Yellow solid, yield 68%.
1
H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.18
(s, 1H), 7.13 (s, 1H), 6.70 (d, J = 2.1 Hz, 2H), 6.46 (s, 1H), 3.84 (s, 6H). LC-MS(ESI):
+
2
86.0ꢀM+Hꢁ .
1
8

ACCEPTED MANUSCRIPT
(Z)-2-(4-(3, 5-dimethoxystyryl) phenyl) benzo[d] [1, 2] selenazol-3(2H)-one (9)
Compound 8 was reduced using a reported method.[30] The method uses sodium
dithionite (5.0 equiv) to reduce nitro derivative 8 (1.0 equiv) in acetone/water (10:5
mL) at 50°C for 8 h. The crude products could be used in the next reaction without
purification. The next step was similar to the method used for the synthesis of
1
compound 6. Colourless solid, yield 45%, m.p. 96.7-97.9°C. H NMR (400 MHz,
DMSO-d6) δ 8.08 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.69 (dd, J = 11.1, 4.1
Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H),
1
3
6
.62 (dd, J = 25.1, 12.3 Hz, 2H), 6.40 (dd, J = 11.9, 2.1 Hz, 3H), 3.64 (s, 6H). C
NMR (101 MHz, DMSO-d6) δ 160.27, 138.78, 138.67, 138.51, 134.15, 132.27,
1
9
30.17, 129.60, 129.34, 128.48, 127.90, 126.26, 125.76, 124.85, 124.04, 106.44,
+
9.49, 54.96. HRMS (ESI) (m /z) [M+H] calcd for C23H19NO3Se, 438.0603;
found,.438.0623 Purity: 96.5% (by HPLC).
Methyl 2-formylbenzoate (11)
To the solution of compound 10 (750 mg, 5 mmol) in 5 mL SOCl , 5 mL of
2
MeOH was slowly added. The mixture was stirred at room temperature for 30 min.
Then, solvent removal yielded the crude product, which was purified by silica gel
column chromatography (petroleum ether/AcOEt=10:1) to provide 11. Colourless oil,
1
yield: 80%. H NMR (400 MHz, CDCl3) δ 10.57 (s, 1H), 7.98 – 7.81 (m, 2H), 7.72 –
7
.56 (m, 2H), 3.94 (s, 3H). LC-MS(ESI): 165.0ꢀM+Hꢁ+.
(E)-methyl 2-((4-(3,4-dimethoxystyryl)phenylamino)methyl)benzoate (12)
Compound 4b (2 mmol) and compound 11(2 mmol) were dissolved in ethanol,
and the resulting mixture was stirred at room temperature for 3 h. NaBH was then
4
added at 0°C. After the mixture was stirred for 1 h, water (10 mL) was added, and the
mixture was extracted with ethyl acetate. The solvents were removed under reduced
pressure to obtain the crude product, which was purified by recrystallisation or flash
1
9

ACCEPTED MANUSCRIPT
1
chromatography on silica gel to provide 12. Yellow solid, yield 65%. H NMR (400
MHz, DMSO-d ) δ 7.84 (d, J = 7.4 Hz, 1H), 7.51 (dd, J = 6.5, 1.2 Hz, 2H), 7.35 (s,
6
1
H), 7.24 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 1.8 Hz, 1H), 6.99-6.91 (m, 1H), 6.91-6.84
(m, 2H), 6.79 (d, J = 16.3 Hz, 1H), 6.51 (d, J = 8.5 Hz, 2H), 4.59 (d, J = 4.8 Hz, 2H),
+
3
.83 (s, 3H), 3.77 (s, 3H), 3.72 (s, 3H). LC-MS(ESI): 404.0ꢀM+Hꢁ .
(E)-2-(4-(3,4-dimethoxystyryl)phenyl)isoindolin-1-one (13)
CH ONa (16 mg, 0.3 mmol) was added to the solution of compound 12 (40 mg, 0.1
3
mmol) in MeOH, and the mixture was then refluxed for 3 h under nitrogen protection.
Water was poured into the resulting mixture, and the mixture was extracted with ethyl
acetate. The solvents were removed under reduced pressure to obtain the crude
product, which was purified by recrystallisation to provide 13. Yellow solid, yield
1
7
5%, m.p. 211.6-213.8°C. H NMR (400 MHz, DMSO-d ) δ 7.90 (d, J = 8.7 Hz, 2H),
6
7
.77 (d, J = 7.6 Hz, 1H), 7.64 (dd, J = 15.8, 7.4 Hz, 4H), 7.58-7.49 (m, 1H), 7.22 (d, J
=
1.5 Hz, 1H), 7.13 (d, J = 2.0 Hz, 2H), 7.08 (dd, J = 8.3, 1.6 Hz, 1H), 6.93 (d, J = 8.4
1
3
Hz, 1H), 5.02 (s, 2H), 3.81 (s, 3H), 3.75 (s, 3H). C NMR (101 MHz, DMSO-d ) δ
6
1
1
68.47, 150.83, 150.54, 142.83, 140.32, 135.13, 134.26, 134.12, 131.98, 130.06,
29.58, 128.55, 127.51, 125.16, 125.05, 121.68, 121.18, 113.72, 111.05, 57.38, 52.27.
+
HRMS (ESI) (m/z) [M+H] calcd for C H NO , 372.1594; found, 372.1598. Purity:
24
21
3
9
8.9% (by HPLC).
4
4
.2 Biological assays
.2.1 Cell lines and culture
The four normal cell lines used in this study were obtained from the Laboratory
Animal Centre of Sun Yat-sen University. The Bel-7402 (human liver carcinoma),
A549 (non-small-cell-lung cancer), HeLa (human epithelial cervical cancer) and
2
0

ACCEPTED MANUSCRIPT
MCF-7 (human breast cancer) cells were cultivated in Dulbecco’s modified Eagle
medium (DMEM, GIBCO) containing 10% (v/v) heat-inactivated foetal bovine serum
(FBS, GIBCO), 100 IU/mL penicillin, and 100 ꢀg/mL streptomycin (GIBCO). The
cells were incubated at 37 °C under a 5% CO atmosphere.
2
4
.2.2 In vitro antiproliferative activity assays
The antiproliferative activity of the designed compounds in four human cancer
cell lines (Bel-7402, A549, HeLa and MCF-7) was examined by MTT assay. When
the cells were in the logarithmic phase, the cells were harvested and plated into each
3
well of 96-well plates (5×10 cells/well) for 24 h. The cells were then exposed to
different concentrations of the test compounds for 48 h. Subsequently, 20 ꢀL of MTT
(5 mg/mL, Sigma) was added, and the cells were incubated for 4 h. The suspension
was then discarded, and 150 ꢀL of DMSO was added to each well. After shaking the
plates to dissolve the dark blue crystals (formazan) for 10 min, the absorbance at 570
nm was measured using a multifunction microplate reader (Molecular Devices, Flex
Station 3). All of the experiments were repeated at least three times. The data were
calculated using GraphPad Prism version 5.0. The IC values were fitted using a
5
0
nonlinear regression model with a sigmoidal dose response.
4
.2.3 In vitro inhibition of TrxR
The DTNB assay was used to determine the TrxR inhibitory activity of the target
compounds.[35, 36] The method was performed according to the manufacturer’s
instructions (Sigma product information sheet T9698) with moderate modifications.
All of the assays were conducted at room temperature in 384-well plates with a total
volume of 80 ꢀL. First, 0.6 ꢀL of rat liver TrxR (Sigma) was added to 71 ꢀL of assay
solution (containing 100 mM potassium phosphate, 10 mM EDTA, 0.24 mM NADPH
2
1

ACCEPTED MANUSCRIPT
(Sigma), and 0.2 mg/mL bovine serum albumin (BSA), pH 7.4). Then, 2 ꢀL of the
compound at various concentrations was added to each well, and this step was
followed by a 5-min pre-incubation period. The same amount of DMSO was added to
the control experiments. The reaction was initiated by the addition of 6.4 ꢀL of DTNB
(Sigma), and TrxR (Sigma) activity was measured by the increase in absorbance at
4
12 nm over the initial 300 s using a multifunction microplate reader (Molecular
Devices, Flex Station 3). All of the experiments were repeated at least three times.
The data were calculated using GraphPad Prism version 5.0. The IC values were
5
0
fitted using a nonlinear regression model with a sigmoidal dose response.
4
.2.4 Flow cytometric analysis of the cell cycle distribution
For the flow cytometric analysis of the DNA content, Bel-7402 cells were plated
5
in six-well plates (3×10 cells/well) and incubated in the presence or absence of
compound 6e at various concentrations (0.5, 1, 3, and 5 ꢀM), resveratrol (50 ꢀM) and
ebselen (50 ꢀM) for 48 h. The cells were harvested with 0.25% trypsin and fixed with
ice-cold 70% ethanol overnight. Ethanol was removed by centrifugation, and the cells
were washed with cold 10% phosphate buffer solution (PBS). The cells were then
treated with RNAse A (100 ꢀg/mL, Beyotime) at 37 °C for 30 min and DNA staining
solution (PI, Beyotime) at 4 °C for 15 min. The DNA contents of 10,000 events were
measured by a flow cytometer (Beckman Coulter, Epics XL) at 488 nm. The data
regarding the number of cells in different phases of the cell cycle were analysed by
the EXPO32 ADC Analysis software.
4
.2.5 Analysis of cellular apoptosis
Bel-7402 cells were plated in six-well plates (3×10 cells/well) and incubated in
the presence or absence of compound 6e at various concentrations (0.5, 1, 3, and 5
5
2
2

ACCEPTED MANUSCRIPT
ꢀ
M), resveratrol (50 ꢀM) and ebselen (50 ꢀM) for 48 h to induce cell apoptosis; 0.1%
DMSO was used as a vehicle control. After the cells were induced for 48 h, both
treated and untreated cells were harvested and washed once with PBS. The
percentages of apoptotic cells were assessed by staining with Annexin-V-FITC and PI
(Beyotime Annexin-V-FITC Apoptosis Detection Kit) according to the manufacturer’s
instructions with moderate modifications. Almost 10,000 events were collected for
each sample and analysed using a flow cytometer (Beckman Coulter, Epics XL). The
percentage of cells undergoing apoptosis was calculated using the EXPO32 ADC
Analysis software.
4
.2.6 Measurement of intracellular ROS accumulation
The intracellular ROS accumulation was detected by fluorescence microscopy
using the cell-permeable fluorogenic probe 2’,7’-dihydrofluorescein diacetate
5
(
DCFH-DA, Sigma). Bel-7402 cells were cultured in 12-well plates (1×10 cells/well)
for 24 h. After adding compound 6e (0.5, 1, 3, and 5 ꢀM), resveratrol (50 ꢀM) and
ebselen (50 ꢀM) for 4 h, the culture supernatants were removed, and the cells were
then incubated with 10 ꢀM DCFH-DA in fresh medium at 37 °C for 30 min. After
incubation, the culture medium was removed, and the cells were washed with PBS
buffer three times. The production of ROS could be measured by changes in the
fluorescence intensity due to the intracellular accumulation of dichlorofluorescein
(DCF) caused by the oxidation of DCFH. The intracellular ROS level, as indicated by
the DCF fluorescence, was determined by measuring the fluorescence intensity on a
multifunctional monochromator-based microplate reader with the excitation and
emission wavelengths set at 488 and 525 nm, respectively. To observe the intracellular
ROS level visually, we used the Arrayscan VTI High Content Analysis System to
acquire cell images after the cell nuclei were stained with Hoechst 33342 (Sigma) for
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0 min in dark conditions.
SUPPORTING INFORMATION
HPLC chromatograms and NMR spectra of target compounds.
ACKNOWLEDGMENT
We thank the Natural Science Foundation of China (No. 21302235, 20972198), the
Ph.D. Programs Foundation of the Ministry of Education of China (20120171120045)
and Distinguished Young Talents in Higher Education of Guangdong
(2012LYM_003)for financial support of this study.
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