Synthesis of selenazolopyridine derivatives with capability to induce apoptosis in human breast carcinoma MCF-7 cells through scavenge of intracellular ROS

Article abstract of DOI:10.1016/j.ejmech.2015.03.069

A series of selenazolopyridine derivatives have been synthesized and characterized by X-ray diffraction, high resolution NMR and Mass spectrum. The in vitro anticancer activities of the synthetic compounds were screened against a panel of human cancer cell lines, human breast carcinoma MCF-7 cells, human liver carcinoma HepG2 cells and L02 normal cell line by MTT assay. By analyzing the structure-activity relationship among the synthetic compounds, it was found that 2-(phenylamino) selenazolo [5,4-b] pyridine, (PSeD, 7) had higher growth inhibitory effect on MCF-7 cells. The intracellular mechanism of cell death was evaluated by flow cytometric analysis and ROS assay, which revealed that PSeD could induce MCF-7 cells apoptosis by scavenging intracellular ROS. Taken together, we regard PSeD as an antioxidant which could inhibit cancer cell growth through induction of apoptosis.

Full text of DOI:10.1016/j.ejmech.2015.03.069

European Journal of Medicinal Chemistry 96 (2015) 92e97
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of selenazolopyridine derivatives with capability to induce
apoptosis in human breast carcinoma MCF-7 cells through scavenge of
intracellular ROS
,
,
,
Meiyun Zhou a 1, Shengbin Ji a 1, Zhaojun Wu a, Yiqun Li a, Wenjie Zheng a *, Hua Zhou b,
Tianfeng Chen a
,
*
a Department of Chemistry, Jinan University, Guangzhou 510632, PR China
b Department of Food Science and Engineering, Jinan University, Guangzhou 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of selenazolopyridine derivatives have been synthesized and characterized by X-ray diffraction,
high resolution NMR and Mass spectrum. The in vitro anticancer activities of the synthetic compounds
were screened against a panel of human cancer cell lines, human breast carcinoma MCF-7 cells, human
liver carcinoma HepG2 cells and L02 normal cell line by MTT assay. By analyzing the structureeactivity
relationship among the synthetic compounds, it was found that 2-(phenylamino) selenazolo [5,4-b]
pyridine, (PSeD, 7) had higher growth inhibitory effect on MCF-7 cells. The intracellular mechanism of
cell death was evaluated by flow cytometric analysis and ROS assay, which revealed that PSeD could
induce MCF-7 cells apoptosis by scavenging intracellular ROS. Taken together, we regard PSeD as an
antioxidant which could inhibit cancer cell growth through induction of apoptosis.
Received 24 May 2014
Received in revised form
29 March 2015
Accepted 31 March 2015
Available online 1 April 2015
Keywords:
Selenazolopyridine derivatives
Anticancer activity
Cell apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
ROS
1. Introduction
chemotherapeutic agents has kindled great interest of scientists
from diversified disciplines.
Cancer is a health killer in modern life. According to American
Association for Cancer Research (AACR) Cancer Progress Report
2013, it is projected that 580,350 Americans would die from one of
the more than 200 types of cancer in 2013. Fortunately, definitive
progress has been made against cancer. There have been 1,024,400
lives saved from 1990 to 2012 [1]. Breast carcinoma is one of the
most commonly occurring epithelial malignancies in women, with
an estimated 1 million new cases and over 400,000 deaths annually
worldwide [2]. Many women ultimately develop metastatic breast
carcinoma which is essentially an incurable disease, and the
prognosis has changed little over the past decade. The majority of
patients succumb to their disease within 2 years of diagnosis [3].
Chemotherapy is an effective clinic treatment to resist cancer [4].
The chemotherapeutic drug cisplatin is widely used in clinical
cancer treatment for decades [5,6]. However, their clinical utility
tends to be limited by the frequent development of drug resistance
or other side effects [7]. Therefore, the search for novel
Selenium (Se) is an essential trace element for human, for
example, many kinds of selenoproteins have an important role in
human metabolism [8]. Due to its high anticancer activity and low
toxicity, series of organoselenium compounds have been designed
to be potential anticancer drugs, such as ebselen, selenocyanate,
selenobetaine [9]. Thus, many kinds of organoselenium compounds
showed excellent effect on human breast carcinoma MCF-7 cells.
Selenocystine, a naturally occurring selenoamino acid, was found
that it could induce reactive oxygen species generation and cause
DNA damage, with a result of apoptosis in MCF-7 cells [10]. ASDO, a
synthetic selenadiazole derivative, also could cause cell death
through a process of caspase- and p53-dependent apoptosis in
MCF-7 cells [11].
In this paper, we report the synthesis of a series of heterocyclic
organoselenium compounds, selenazolopyridine derivatives
(SeDs). The compounds have been synthesized by a three-step
protocol [12e14]. Their structures were established by X-ray
diffraction, high resolution NMR and Mass spectrum. Moreover,
their in vitro anticancer activities were tested and the result showed
that 2-(phenylamino) selenazolo [5,4-b] pyridine (PSeD, 7) can
highly induce apoptosis in human breast carcinoma MCF-7 cells.
* Corresponding authors.
E-mail addresses: tzhwj@jnu.edu.cn (W. Zheng), tchentf@jnu.edu.cn (T. Chen).
Authors contributed equally to the work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.03.069
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M. Zhou et al. / European Journal of Medicinal Chemistry 96 (2015) 92e97
93
Table 1
Further investigation on the intracellular mechanisms demon-
Crystal and experimental data of compound 2 and 3.
strated that PSeD had a capability to scavenge intracellular reactive
oxygen species (ROS) with the result of cell apoptosis.
Compound
2
3
Crystallized from
Empirical formula
Formula weight
Crystal color
CHCl3
CHCl3
C13H11N3Se
288.21
C13H11N3Se
288.21
Yellow
0.2 ꢁ 0.4 ꢁ 0.1
1.5417
153 K
orthorhombic
Pbca
14.7219
7.3429
21.8165
90
2. Result and discussion
Yellow
2.1. Chemistry
Crystal dimension [nm]
0.2 ꢁ 0.3 ꢁ 0.08
1.5417
153 K
monoclinic
P2(1)/c
11.2887
11.4122
10.0395
90
Radiation Cu K
a
Temperature [K]
Crystal system
Space group
a (Å)
b (Å)
c (Å)
The synthetic route for SeDs (1e7) is showed in Scheme 1. In
first step, the treatment of anilines with formic acid in the presence
of ZnO under solvent-free conditions brought about highly and
efficient N-formylation to give the corresponding formamides
(1ae7a) in excellent yields [12]. In the second step, solid tri-
phosgene was taken in the one-pot dehydration of the formamides
in dichloromethane containing triethylamine, followed by the re-
action of the nonisolated isocyanides with an excess of Se powder
[13]. This gave an important reaction intermediate, iso-
selenocyanates (1be7b). The yields of 1ae7a and 1be7b were
listed in Table S1 in the Supplementary data. The third step, the
isoselenocyanates reacted with 3-amino-2-chloropyridine to give
the products (1e7) through cyclization [14]. Among them, com-
pound 1 [15] and compound 7 [14] were reported previously,
compounds 2e6 were novel ones which have not been reported.
The reaction processes were monitored by TLC. All the character-
ization data will be listed below.
a
b
g
(deg)
(deg)
(deg)
90
90
90
90
V(Å3)
2358.40(11)
8
1.630
62.710/99.4%
0.0286
0.0992
1196.02(8)
4
1.601
62.780/99.3%
0.0383
0.1183
Z
D calc (g/cmꢂ3
)
q
range(deg)/completeness (%)
Rb[I > 2
(I)]
wR2 b(all data)
s
treatment [17]. The IC50 values of SeDs against three human cell
lines were listed in Table 4. We can see that there are different kinds
of substituent groups on benzene ring of SeDs. Thus, we firstly tried
to explore the relationship between different substituent groups
and the IC50 values structurally. As shown in Table 4, 2-(phenyl-
amino) selenazolo [5,4-b] pyridine (PSeD, 7) exhibited good growth
inhibitory effects on MCF-7 cells with lower IC50 values than those
of its analogs. We found that no matter electron-repulsing group
(like CH3), electron-attracting group (like Br, Cl), or disubstituted
groups (like 3, 4-diCH3), they both had less inhibitory effects than
PSeD (no substituted group on benzene ring) on MCF-7 cells.
Moreover, PSeD expressed relatively low cytotoxicity to L02 normal
For the chemical synthesis, we obtained the single crystal
structures of two new SeDs, compound 2 and 3. The structures of 2
and
Diffraction intensity data were collected with Crys Alis PRO (single
crystal diffractometer using Cu K radiation at low temperature 153
3 were unequivocally established by X-ray diffraction.
a
K). Crystal data collection and structure refinement data of the two
compounds were listed in Table 1. An ORTEP drawing of them with
the atomic numbering scheme were shown in Fig. 1. Crystallo-
graphic data structures of compound 2 and 3 have deposited at the
Cambridge Crystallographic Data Centre as CCDC 991881e991882.
cell with the IC50 value of 163.51 mM approximately. Furthermore,
Compound
2 and 3 have the same molecular formula
from changes in the morphology of MCF-7 cells, it indicated that
the inhibition of MCF-7 cells growth by PSeD was a dose-
dependent process (Fig. 2A, B).
(C13H11N3Se) and are both built up from fused selenazolo and
pyridine rings. The major difference is that compound 2 is linked to
o-toluidine group, but compound 3 to m-toluidine group. The
dihedral angle between the mean plane of the fused selenoazolo-
pyridine ring system and the toluidine ring in compound 2 is
119.549(2) compared with 13.686(1) in compound 3. Bond lengths
are in normal ranges [16] (Table 2). In the two crystals, molecules
are linked by N/H … N hydrogen bonds forming zigzag chains
(Table 3).
2.2.2. Effects of PSeD on cell cycle distribution
It has been reported that selenium compounds could inhibit the
development of chemically induced tumorigenesis in several ani-
mal models and induce apoptosis in a variety of cell culture systems
in vitro [9]. Next, we proposed to investigate whether the under-
lying mechanism of PSeD caused cell death through apoptosis
pathway or not. In general, there were three ways that anticancer
agents could inhibit cancer cell proliferation or induce cancer cell
death, such as cell apoptosis or cell cycle arrest or a combination of
these two modes [18]. Therefore, we adopted propidium iodide
(PI)-flow cytometric analysis to determine which of the above-
mentioned modes would occur. Fig. 3 showed all the cell cycle
distribution after exposing MCF-7 cells to different concentrations
2.2. Evaluation of biological activity
2.2.1. In vitro anticancer activity
The in vitro anticancer activities of SeDs (1e7) were firstly
screened against a panel of two human cancer cell lines, including
human breast carcinoma MCF-7, human liver carcinoma HepG2 and
a human normal liver cell line, L02 by MTT assay after 72 h
Scheme 1. Synthetic route of SeDs. Reagent and reaction conditions: (a) HCOOH, 70 ꢀC; (b) CH2Cl2, Et3N, solid triphosgene, Se powder, 40 ꢀC; (c) dry 2-propanol, rt.

94
M. Zhou et al. / European Journal of Medicinal Chemistry 96 (2015) 92e97
Fig. 1. ORTEP plots of the molecular structure of compound 2 (A) and compound 3 (B).
Table 2
of PSeD for 72 h. It is obvious that the proportion of apoptotic cells
expressed a dose-dependent increase as reflected by the Sub-G1
cell population. Moreover, no significant change of the S phase
population indicated that no cycle arrest was happened. As a result,
cell death induced by PSeD was primarily attributed to cancer cells
apoptosis.
Selected Crystal Bond Lengths (Å), Bond angles (ꢀ), and Torsion angles (ꢀ) for com-
pound 2 and 3.
Number
Bond distance and angles
2
3
1
2
3
4
5
6
7
8
C1 … N1
C1 … C2
C3 … C4
C2 … C3
C5 … C4
C5 … Se1
C4 … N2
C6 … N2
1.343(4)
1.372(4)
1.383(3)
1.387(4)
1.401(3)
1.890(2)
1.392(3)
1.298(3)
1.347(3)
1.909(2)
123.9(3)
123.5(2)
109.8(1)
125.4(6)
122.5(7)
112.2(2)
83.7(4)
1.336(4)
1.381(5)
1.394(4)
1.374(5)
1.397(4)
1.886(3)
1.382(4)
1.301(4)
1.340(4)
1.917(2)
123.8(3)
124.8(2)
109.7(2)
124.6(2)
126.8(2)
112.4(2)
83.8(5)
2.2.3. PSeD inhibits intracellular ROS generation by scavenging free
radicals
Reactive oxygen species (ROS) are appreciated to function as
signaling molecules to regulate a wide variety of physiology.
Accumulation of ROS and oxidative damage have been linked to
multiple pathologies, including diabetes, cancer, and premature
aging [19]. It has been reported that ROS is a regulator cell apoptosis
induced by chemotherapeutic drugs [20]. Many studies have
illustrated that overproduction of intracellular ROS tended to attack
DNA and cause DNA strand break, with the result of cell apoptosis
[21]. In this study, we tried to make the role of ROS clear in the
course of PSeD induced-apoptosis. Therefore, the intracellular ROS
generation in MCF-7 cells was marked by measuring the DHE
fluorescence intensity. As shown in Fig. 4A, the overproduction of
ROS did not appear while treatment with PSeD led to a dose-
dependent decrease in DHE fluorescence intensity in a relative
short time for 30 min. Moreover the red fluorescence of DHE faded
gradually with the treatment of PSeD, which confirmed this result
(Fig. 4B). It is also reported that antioxidant protein expression, for
example through Sirt3 and FOXO activation, would decrease the
generation of ROS [19]. Therefore, it was indicated that PSeD ten-
ded to induce apoptosis in MCF-7 cells by scavenging intracellular
ROS as an antioxidant. To confirm this assumption, the antioxidant
activity of PSeD was examined by ABTS and DPPH free radical
scavenging assays [22]. In our expectation, PSeD expressed a high
activity in scavenging the two free radicals, especially in ABTS
assay, in a time-dependent course (Fig. 5).
9
C6 … N3
C6 … Se1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
N1 … C1 … C2
N1 … C5 … Se1
C4 … C5 … Se1
N2 … C4 … C3
N2 … C6 … N3
C4 … N2 … C6
C5 … Se1 … C6
C6 … N3 … C7
Se1…C6 … N3
N3 … C7 … C8
C6 … se1 … C5 … N1
C6 … Se1 … C5…C4
C4 … N2 … C6 … Se1
C7 … N3 … C6 … Se1
C5 … Se1 … C6 … Se1
124.7(1)
120.9(4)
119.3(1)
128.(3)
117.4(5)
116.6(2)
ꢂ178.3(9)
ꢂ177.2(8)
0.69(2)
1.72 (8)
ꢂ1.1(2)
ꢂ6.6(3)
0.24(17)
ꢂ179.2(7)
ꢂ170.5(9)
5.68(2)
Table 3
Hydrogen bond interactions for compound 2 and 3 [Å and ꢀ].
Compound
DeH … A
D(DeH)
D(H … A)
D(D … A)
<(DHA)
2
3
N3eH3 … N2#1
N3eH3 … N2#2
0.90(1)
0.97(8)
2.06(1)
2.08(2)
2.96 (4)
3.04(5)
176(7)
167(7)
Symmetry transformations used to generate equivalent atoms: #1 ꢂx
2, ꢂy, ꢂz þ 1/2, #2 ꢂx þ 2, y þ 1/2, ꢂz þ 1/2.
þ
1/
Table 4
3. Conclusion
Growth inhibitory effects of SeDs on various human cancer and normal cells.
SeDs
IC50
(
m
M)a
In summary, a series of selenazolopyridine derivatives have
been synthesized through a three-step protocol. And their in vitro
anticancer activities were evaluated against three human cell lines.
The result showed that PSeD displayed strong growth inhibitory
effect on MCF-7 cells by inducing cancer cell apoptosis. Further-
more, the changes of intracellular ROS generation induced by PSeD
were determined and the result revealed that PSeD could decrease
the generation of ROS. It is also reported that cancer cells utilize
ROS to constitutively activate proliferation pathways to promote
tumor growth [19]. Therefore, we confirm that, as an antioxidant,
PSeD has the capabilities of inducing human breast carcinoma
MCF-7 cells apoptosis by scavenging intracellular ROS.
MCF-7
HepG2
L02b
1
2
3
4
5
6
7
185.31 2.1
52.50 1.5
56.37 3.3
91.54 3.5
43.34 0.7
202.45 4.4
22.51 0.5
74.15 2.3
506.85 3.4
56.68 1.3
234.51 5.5
180.65 4.2
144.54 3.4
57.11 1.3
169.37 3.4
121.44 5.3
54.34 1.2
206.61 3.1
84.62 1.2
182.41 2.1
163.51 2.1
a
Cells were treated with various concentrations of tested compounds for 72 h.
Cell viability was determined by MTT assay and IC50 values were calculated as
described in Experimental section.
b
Normal cells.

M. Zhou et al. / European Journal of Medicinal Chemistry 96 (2015) 92e97
95
Fig. 2. (A) Growth inhibition effects of PSeD on MCF-7 cells. Cells were treated with different concentrations of PSeD for 72 h and cell viability was examined by MTT assay. *,
P < 0.05 versus the control. (B) Changes in the morphology of MCF-7 cells under different treatments of PSeD for 72 h.
Fig. 3. (A) Effects of PSeD on cell apoptosis and cell cycle distribution in MCF-7 cells. (B) Dose-dependent cell apoptosis induced by PSeD by measuring the sub-G1 cell population in
MCF-7 cells. *, P < 0.05 versus the control, **, P < 0.01 versus the control.
Fig. 4. (A) Changes of intracellular ROS generation induced by PSeD in MCF-7 cells. Cells incubated with 10
mM DHE in PBS for 30 min were exposed to different concentration of
PSeD. (B) Changes of intracellular ROS generation marked by DHE fluorescence in 30 min. Cells treated with 80
m
M of PSeD.
4. Experimental section
an amine (1 mmol), and then the reaction mixture was heated in an
oil bath at 70 ꢀC and stirred with a magnetic stirrer. The process of
the reaction was monitored by TLC. After the reaction was com-
plete, EtOAc was added to the reaction mixture, and ZnO was
removed by filtration. The organic solvent was then washed with
H2O (10 mL) twice and a saturated solution of NaHCO3 and dried
with anhydrous Na2SO4. After removal of the solvent, the pure
4.1. Chemistry
4.1.1. General procedure for the synthesis of corresponding
formamides (1ae7a)
A mixture of HCOOH (3 mmol) and ZnO (0.5 mmol) was added

96
M. Zhou et al. / European Journal of Medicinal Chemistry 96 (2015) 92e97
3457, 3265, 3080, 3014, 1610, 1458, 1378; Elemental analysis calcd
(%): C, 54.18; H, 3.85; N, 14.58; found (%): C, 54.24; H, 3.81; N, 14.48;
ESI-MS (in CHCl3): m/z 288.2, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d
, ppm): 8.19 (d, J ¼ 4.3 Hz, 1H), 7.61 (t, J ¼ 8.2 Hz, 1H), 7.56 (d,
J ¼ 8.0 Hz, 1H), 7.40e7.26 (m, 4H), 7.25e7.05 (m, 1H), 2.36 (d,
J ¼ 36.9 Hz, 3H); 13C NMR (126 MHz, CDCl3,
d, ppm): 169.6, 158.0,
148.3, 142.6, 138.3, 133.4, 131.6, 127.6, 125.5, 125.0, 121.5, 17.9.
4.1.3.3. 2-[(3-Methylphenyl)amino]selenazolo[5,4-b]pyridine
(3).
Yellow solid; yield: 76%; m.p.: 167.3e170.2 ꢀC; IR (KBr, cmꢂ1) v:
3459, 3265, 3210 3013, 1621, 1462, 1372; Elemental analysis calcd
(%): C, 54.18; H, 3.85; N, 14.58; found (%): C, 54.12; H, 3.83; N, 14.52;
ESI-MS (in CHCl3): m/z 287.7, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d
, ppm): 8.23 (d, J ¼ 6.3 Hz, 1H), 7.69 (d, J ¼ 8.1 Hz, 1H), 7.33 (d,
J ¼ 6.4 Hz, 2H), 7.33e7.26 (m, 2H), 7.25 (dd, J ¼ 8.1, 4.7 Hz, 1H), 7.08
(t, J ¼ 4.3 Hz, 1H), 2.41 (s, 3H); 13C NMR (126 MHz, CDCl3,
d, ppm):
167.0, 158.5, 148.3, 143.1, 139.9, 139.9, 129.6, 126.5, 125.9, 122.2,
121.5, 118.4, 21.5.
4.1.3.4. 2-[(4-Methylphenyl)amino]selenazolo[5,4-b]pyridine
(4).
Yellow solid; yield: 76%; m.p.: 131.7e134.9 ꢀC; IR (KBr, cmꢂ1) v:
3446, 3249, 3188, 3104, 2980, 2852; Elemental analysis calcd (%): C,
54.18; H, 3.85; N, 14.58; found (%): C, 54.36; H, 3.87; N, 14.44; ESI-
MS (in CHCl3): m/z 287.8, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d,
ppm): 8.23 (d, J ¼ 4.8 Hz, 1H), 7.75 (d, J ¼ 8.1 Hz, 1H), 7.38 (d,
J ¼ 8.3 Hz, 2H), 7.29e7.24 (m, 4H), 2.40 (s, 3H); 13C NMR (126 MHz,
CDCl3, d, ppm): 167.1, 158.5, 148.5, 143.0, 137.1, 135.8, 130.4, 126.1,
121.8, 121.5, 76.8, 29.8, 21.0.
Fig. 5. Time-dependent antioxidant activities of PSeD as determined by (A) ABTS and
(B) DPPH assays.
4.1.3.5. 2-[(2-Chlorophenyl)amino]selenazolo[5,4-b]pyridine
(5).
Colorless solid; yield: 65%; m.p.: 177.6e179.2 ꢀC; IR (KBr, cmꢂ1) v:
3459, 3152, 3053, 2849, 1601; Elemental analysis calcd (%): C,
46.70; H, 2.61; N, 13.62; found (%): C, 46.56; H, 2.58; N, 13.29; ESI-
product was obtained.
MS (in CHCl3): m/z 308.2, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d,
4.1.2. General procedure for the synthesis of corresponding
isoselenocyanates (1be7b)
ppm) 8.33 (d, J ¼ 8.2 Hz, 1H), 8.29 (d, J ¼ 6.3 Hz, 1H), 7.88 (d,
A mixture of corresponding formamide (5 mmol), Et3N (5 mL)
and molecular sieve was added into the reagent of CH2Cl2 (10 mL).
The reaction mixture was stirred and heated at 40 ꢀC. Then, the
solid triphosgene (2.5 mmol) dissolving in 5 mL CH2Cl2 was add
into the reaction mixture drop by drop in a constant pressure
funnel. The reaction was refluxed for 3.5 h. Then an excess of Se
powder was added to form the corresponding isoselenocyanates
after continuous reflux for 4 h.
J ¼ 8.1 Hz, 1H), 7.48 (d, J ¼ 8.0 Hz, 1H), 7.44e7.30 (m, 3H), 7.14 (t,
J ¼ 8.5 Hz, 1H); 13C NMR (126 MHz, CDCl3,
d, ppm): 163.5, 159.3,
148.4, 1473.8, 136.3, 129.7, 128.1, 127.2, 124.9, 123.7, 121.7, 120.7.
4.1.3.6. 2-[(3,4-dimethylphenyl)amino]selenazolo[5,4-b]pyridine (6).
Red solid; yield: 70%; m.p.: 197.6e198.8 ꢀC; IR (KBr, cmꢂ1) v: 3444,
3243, 3187, 2967, 1591, 1453,1375; Elemental analysis calcd (%): C,
55.64; H, 4.34; N, 13.90; found (%): C, 55.38; H, 4.62; N, 13.92; ESI-
MS (in CHCl3): m/z 301.8, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d,
4.1.3. General procedure for the synthesis of compound 1e7
To a stirred solution of 3-amino-2-chloropyridine in dry 2-
propanol (10 mL) at rt, 1.1 equivalents of freshly prepared corre-
sponding isoselenocyanate were added. The mixture was heated to
reflux for 4 h and the formation of solid was observed. The solid
was filtered after the solution was cooled to room temperature and
stirred in aqueous NaOH solution (5e6%) for 15 min. Then, the solid
was filtered and recrystallized from ethyl acetate.
ppm): 8.23 (d, J ¼ 6.3 Hz, 1H), 7.75 (d, J ¼ 9.4 Hz, 1H), 7.29 (s, 2H),
7.26e7.18 (m, 3H), 2.31 (d, J ¼ 7.3 Hz, 6H); 13C NMR (126 MHz,
CDCl3, d, ppm): 167.5, 158.6, 148.4, 143.0, 138.4, 137.4, 134.6, 130.8,
125.9, 123.3, 121.4, 119.3, 19.9, 19.3.
4.1.3.7. 2-(Phenylamino)selenazolo[5,4-b]pyridine
(7). Colorless
solid; yield: 70%; m.p.: 159.2e160.8 ꢀC; IR (KBr, cmꢂ1) v: 3459,
3257, 3197, 3076, 3023, 1605; Elemental analysis calcd (%): C, 54.57;
H, 3.31; N, 15.33; found (%): C, 54.17; H, 3.41; N, 14.92; ESI-MS (in
CHCl3): m/z 273.7, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d, ppm)
4.1.3.1. 2-[(2-Bromophenyl)amino]selenazolo[5,4-b]pyridine
(1).
Yellow solid; yield: 70%; m.p.: 178.3e179.6 ꢀC; IR (KBr, cmꢂ1) v:
3447, 3198, 3152, 3053, 2850, 1601; Elemental analysis calcd (%): C,
40.82; H, 2.28; N, 11.90; found (%): C, 40.90; H, 2.32; N, 11.75; ESI-
8.24 (dd, J ¼ 4.7, 1.5 Hz, 1H), 7.71 (dd, J ¼ 8.1, 1.5 Hz, 1H), 7.52 (s, 2H),
7.51e7.41 (m, 2H), 7.39e7.14 (m, 3H); 13C NMR (126 MHz, CDCl3,
d,
ppm): 166.6, 158.5, 148.4, 143.3, 139.9, 129.8, 126.1, 125.5, 121.5,
121.4, 121.1.
MS (in CHCl3): m/z 354.3, ([MꢂH]ꢂ); 1H NMR (500 MHz, CDCl3,
d,
ppm): 8.32e8.23 (m, 2H), 7.86 (d, J ¼ 9.6 Hz, 1H), 7.65 (d, J ¼ 8.0 Hz,
1H), 7.43 (t, J ¼ 7.1 Hz,1H), 7.35e7.25 (m, 2H), 7.08 (t, J ¼ 8.4 Hz,1H);
4.2. Biological activity
13C NMR (126 MHz, CDCl3,
d, ppm): 163.9, 159.2, 148.3, 143.8, 137.6,
133.0, 128.8, 127.0, 125.6, 121.6, 121.2, 114.6.
4.2.1. Cell culture and MTT assay
The cell lines used in this study, including human breast carci-
noma MCF-7 cells, human liver cancer HepG2 cells and the normal
human liver L02 cells, were obtained from American Type Culture
4.1.3.2. 2-[(2-Methylphenyl)amino]selenazolo[5,4-b]pyridine
(2).
Colorless solid; yield: 75%; m.p.: 121.2e122.1 ꢀC; IR (KBr, cmꢂ1) v:

M. Zhou et al. / European Journal of Medicinal Chemistry 96 (2015) 92e97
97
J. Dennis, Q. Wang, M.K. Humphreys, C. Luccarini, Genome-wide association
analysis identifies three new breast cancer susceptibility loci, Nat. Genet. 44
(2012) 312e318.
Collection (ATCC, Manassas, VA) and maintained in DMEM medium
supplemented with fetal bovine serum (10%), penicillin
(100 units mLꢂ1), and streptomycin (50 units mLꢂ1) at 37 ꢀC in a
humidified incubator with 5% CO2 atmosphere. Cell viability was
examined by MTT assay [23].
ꢀ
[4] N.J. Robert, V. Dieras, J. Glaspy, A.M. Brufsky, I. Bondarenko, O.N. Lipatov,
E.A. Perez, D.A. Yardley, S.Y. Chan, X. Zhou, RIBBON-1: randomized, double-
blind, placebo-controlled, phase III trial of chemotherapy with or without
bevacizumab for first-line treatment of human epidermal growth factor re-
ceptor 2enegative, locally recurrent or metastatic breast cancer, J. Clin. Oncol.
29 (2011) 1252e1260.
4.2.2. Flow cytometric analysis
[5] B. Rosenberg, L. Van Camp, T. Krigas, Inhibition of cell division in Escherichia
coli by electrolysis products from a platinum electrode, Nature 205 (1965)
698e699.
The cell cycle distribution was monitored by flow cytometric
analysis, which the method was described previously [24].
Apoptotic cells with hypodiploid DNA content were measured by
quantifying the sub-G1 peak in the cell cycle pattern.
[6] A. Sak, S. Grehl, M. Engelhard, A. Wierlemann, H.-P. Kaelberlah, P. Erichsen,
€
C. Pottgen, M. Groneberg, M. Stuschke, Long-term in vivo effects of cisplatin
on g-H2AX foci signaling in peripheral lymphocytes of tumor patients after
irradiation, Clin. Cancer Res. 15 (2009) 2927e2934.
4.2.3. Measurement of ROS generation
[7] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo,
G. Kroemer, Molecular mechanisms of cisplatin resistance, Oncogene 31
(2012) 1869e1883.
[8] K. Brown, J. Arthur, Selenium, selenoproteins and human health: a review,
Public Health Nutr. 4 (2001) 593e599.
[9] R. Sinha, K. El-Bayoumy, Apoptosis is a critical cellular event in cancer che-
moprevention and chemotherapy by selenium compounds, Curr. Cancer Drug
Targets 4 (2004) 13e28.
[10] T. Chen, Y.-S. Wong, Selenocystine induces caspase-independent apoptosis in
MCF-7 human breast carcinoma cells with involvement of p53 phosphoryla-
tion and reactive oxygen species generation, Int. J. Biochem. Cell Biol. 41
(2009) 666e676.
Changes of ROS generation induced by PSeD treated MCF-7 cells
were evaluated by DHE assay [25]. The intracellular ROS level was
examined by detecting fluorescence intensity of DHE conducted by
fluorescence microplate reader (excitation and emission wave-
length set as 300 nm and 610 nm).
As to the fluorescence images reflecting changes of ROS gener-
ation, cells were incubated with DHE (10
m
M) in a 2 cm culture dish
at 37 ꢀC for 30 min. Then cells were treated with PSeD (80
mM) and
immediately examined on a fluorescence microscope to trace the
changes of DHE fluorescence in 30 min.
[11] T. Chen, Y.-S. Wong, W. Zheng, J. Liu, Caspase-and p53-dependent apoptosis in
breast carcinoma cells induced by a synthetic selenadiazole derivative, Chem.
Biol. Interact. 180 (2009) 54e60.
[12] M. Hosseini-Sarvari, H. Sharghi, ZnO as a new catalyst for N-formylation of
amines under solvent-free conditions, J. Org. Chem. 71 (2006) 6652e6654.
[13] J.G. Fernandez-Bolanos, O. Lopez, V.c. Ulgar, I. Maya, J. Fuentes, Synthesis of O-
unprotected glycosyl selenoureas. A new access to bicyclic sugar isoureas,
Tetrahedron Lett. 45 (2004) 4081e4084.
[14] P.K. Atanassov, A. Linden, H. Heimgartner, Selenium-containing heterocycles
from isoselenocyanates: synthesis of 2-arylaminoselenazolo [5, 4-b] pyri-
dines, Heterocycles 61 (2003) 569e579.
[15] Z. Bo, H. Du Shu, L. Wei, Z. Mei Yun, N-(2-bromo-phen-yl)-1,3-selenazolo[5,4-
b]pyridin-2-amine, Acta Crystallogr. Sect. E Struct. Rep. Online 69 (2013)
o1537.
4.2.4. ABTS, DPPH free radical scavenging assay
ꢀ
ꢀ
~
ꢀ
ABTS, DPPH free radical scavenging activity of PSeD was
measured according to the method as previously described [11].
Briefly, 20 mL of test samples at different concentrations was mixed
with 180 mL of ABTS (or DPPH) solution for 30 min in the dark.
Then, the change in absorbance at 734 nm for ABTS and 517 nm for
DPPH was measured on a microplate reader. DMSO was used as a
negative control.
[16] P.K. Atanassov, A. Linden, H. Heimgartner, Synthesis of 4-(phenylamino)qui-
nazoline-2(1H)-selones and diselenides from isoselenocyanates: dimroth
rearrangement of an intermediate, Helv. Chim. Acta 87 (2004) 1873e1887.
[17] T. Chen, Y. Wong, Selenocystine induces apoptosis of A375 human melanoma
cells by activating ROS-mediated mitochondrial pathway and p53 phos-
phorylation, Cell. Mol. Life Sci. 65 (2008) 2763e2775.
[18] D. Chan, W. Van Dyke, M. Bahls, S. Connell, P. Critser, J. Kelleher, M. Kramer,
S. Pearce, S. Sharma, C. Neu, Mechanostasis in apoptosis and medicine, Prog.
Biophys. Mol. Biol. 106 (2011) 517e524.
[19] L.A. Sena, N.S. Chandel, Physiological roles of mitochondrial reactive oxygen
species, Mol. Cell 48 (2012) 158e167.
[20] T. Chen, W. Zheng, Y.-S. Wong, F. Yang, Mitochondria-mediated apoptosis in
human breast carcinoma MCF-7 cells induced by a novel selenadiazole de-
rivative, Biomed. Pharmacother. 62 (2008) 77e84.
[21] A. Sallmyr, J. Fan, F.V. Rassool, Genomic instability in myeloid malignancies:
increased reactive oxygen species (ROS), DNA double strand breaks (DSBs)
and error-prone repair, Cancer Lett. 270 (2008) 1e9.
[22] K. Thaipong, U. Boonprakob, K. Crosby, L. Cisneros-Zevallos, D. Hawkins Byrne,
Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant
activity from guava fruit extracts, J. Food Compos. Anal. 19 (2006) 669e675.
[23] W. Liu, X. Li, Y.-S. Wong, W. Zheng, Y. Zhang, W. Cao, T. Chen, Selenium
nanoparticles as a carrier of 5-fluorouracil to achieve anticancer synergism,
ACS Nano 6 (2012) 6578e6591.
[24] S. Liu, W. Cao, L. Yu, W. Zheng, L. Li, C. Fan, T. Chen, Zinc (II) complexes
containing bis-benzimidazole derivatives as a new class of apoptosis inducers
that trigger DNA damage-mediated p53 phosphorylation in cancer cells,
Dalton Trans. 42 (2013) 5932e5940.
[25] Y. Huang, L. He, W. Liu, C. Fan, W. Zheng, Y.-S. Wong, T. Chen, Selective cellular
uptake and induction of apoptosis of cancer-targeted selenium nanoparticles,
Biomaterials 34 (2013) 7106e7116.
Acknowledges
This work was supported by 863 National High Technology
Research and Development Program of China (SS2014AA020538),
Guangdong Natural Science Funds for Distinguished Young Scholar
(S2013050014667), the National Natural Science Foundation of
China (NO. 31000816, NO. 31101323) and Research Fund for the
Doctoral Program of Higher Education of China (20114401110004).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.069.
References
[1] C.L. Sawyers, C. Abate-Shen, K.C. Anderson, A. Barker, J. Baselga, N.A. Berger,
M. Foti, A. Jemal, T.S. Lawrence, C.I. Li, AACR cancer progress report 2013, Clin.
Cancer Res. 19 (2013) S1eS98.
[2] V. Ribrag, J. Dupuis, H. Tilly, F. Morschhauser, F. Laine, R. Houot, C. Haioun,
C. Copie, A. Varga, J. Lambert, A dose-escalation study of SAR3419, an anti-
CD19 antibody maytansinoid conjugate, administered by intravenous infu-
sion once weekly in patients with relapsed/refractory B-cell non-hodgkin
lymphoma, Clin. Cancer Res. 20 (2014) 213e220.
[3] M. Ghoussaini, O. Fletcher, K. Michailidou, C. Turnbull, M.K. Schmidt, E. Dicks,