Microwave-Assisted Syntheses of Benzimidazole-Containing Selenadiazole Derivatives That Induce Cell-Cycle Arrest and Apoptosis in Human Breast Cancer Cells by Activation of the ROS/AKT Pathway

Article abstract of DOI:10.1002/cmdc.201600261

The use of selenium-containing heterocyclic compounds as potent cancer chemopreventive and chemotherapeutic agents has been well documented by a large number of clinical studies. In this study we developed a new approach to synthesize four benzimidazole-containing selenadiazole derivatives (BSeDs). The method uses a combination of peptide coupling reagents and microwave irradiation. This strategy features milder reaction conditions, higher yields, and shorter reaction times. The synthetic BSeDs were identified as potent antiproliferative agents against the human MCF-7 and MDA-MB-231 breast cancer cell lines. Compounds 1 b (5-(6-methyl-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole), 1 c (5-(6-chloro-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole), and 1 d (5-(6-bromo-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole) were found to show greater cytotoxicity against the triple-negative breast cancer cell line MDA-MB-231 than MCF-7, and to exhibit dose-dependent inhibition of cell migration, in which a significant decrease in the zone of cell monolayer wound closure was observed relative to untreated controls. Our results demonstrate that BSeDs can cause cell-cycle arrest and apoptosis in MDA-MB-231 cells by inducing DNA damage, inhibiting protein kinase B (AKT), and activating mitogen-activated protein kinase (MAPK) family members through the overproduction of reactive oxygen species (ROS). Taken together, the results of this study provide a facile microwave-assisted strategy for the synthesis of selenium-containing organic compounds that exhibit a high level of anticancer efficacy.

Full text of DOI:10.1002/cmdc.201600261

DOI: 10.1002/cmdc.201600261
Full Papers
Microwave-Assisted Syntheses of Benzimidazole-
Containing Selenadiazole Derivatives That Induce Cell-
Cycle Arrest and Apoptosis in Human Breast Cancer Cells
by Activation of the ROS/AKT Pathway
Yuanwei Liang, Yangliang Zhou, Shulin Deng, and Tianfeng Chen*[a]
The use of selenium-containing heterocyclic compounds as
potent cancer chemopreventive and chemotherapeutic agents
has been well documented by a large number of clinical stud-
ies. In this study we developed a new approach to synthesize
four benzimidazole-containing selenadiazole derivatives
(BSeDs). The method uses a combination of peptide coupling
reagents and microwave irradiation. This strategy features
milder reaction conditions, higher yields, and shorter reaction
times. The synthetic BSeDs were identified as potent antiproli-
ferative agents against the human MCF-7 and MDA-MB-231
breast cancer cell lines. Compounds 1b (5-(6-methyl-1H-ben-
zo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole), 1c (5-(6-chloro-
1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole), and 1d
(5-(6-bromo-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadia-
zole) were found to show greater cytotoxicity against the
triple-negative breast cancer cell line MDA-MB-231 than MCF-
7, and to exhibit dose-dependent inhibition of cell migration,
in which a significant decrease in the zone of cell monolayer
wound closure was observed relative to untreated controls.
Our results demonstrate that BSeDs can cause cell-cycle arrest
and apoptosis in MDA-MB-231 cells by inducing DNA damage,
inhibiting protein kinase B (AKT), and activating mitogen-acti-
vated protein kinase (MAPK) family members through the
overproduction of reactive oxygen species (ROS). Taken togeth-
er, the results of this study provide a facile microwave-assisted
strategy for the synthesis of selenium-containing organic com-
pounds that exhibit a high level of anticancer efficacy.
Introduction
Breast cancer has become the second leading cause of death
among women worldwide.[1] According to the latest records, it
is estimated that about 246660 women will be diagnosed with
invasive breast cancer in 2016, and among them, an additional
61000 new cases of in situ breast cancer will be diagnosed.
About one in 33 women will receive an in situ breast cancer di-
agnosis in her lifetime.[2,3] Breast cancer is the most frequently
diagnosed cancer in women. Due to its high resistance to che-
motherapy, traditional treatments tend to be inefficient, costly,
and are accompanied with a wide range of side effects.[4] There
is currently still no effective treatment for patients with ad-
vanced stages of breast cancer, especially hormone-independ-
ent cancer.[5]
ERÀ breast cancer patients tend to not benefit from endocrine
therapy, and they face many problems such as poor prognosis,
a high rate of distant metastases, and a high death rate.[7]
Therefore, it is essential to identify novel therapeutic agents
that can suppress the growth of ERÀ breast cancer cells on
the one hand and minimize side effects on the other.
As an essential trace mineral, selenium has been recognized
has having an important role in maintaining human health.[8,9]
Epidemiological and clinical studies have supported the role of
Se (especially organic selenium) as a component of potent
cancer chemopreventive agents.[10,11] Supplementation of Se
was found to be effective in decreasing the rates of breast,
lung, and liver cancers.[12,13] There are several proposed mecha-
nisms behind the anticancer activity of Se, such as the induc-
tion of apoptosis and cell-cycle arrest, or a combination there-
of. Both mechanisms may be relevant to the modulation of
redox reactions, the detoxification of carcinogens, and even
the inhibition of angiogenesis.[13,14] However, the therapeutic
window for appropriate Se dosage is rather narrow, which
greatly restricts its clinical application. As a trace element in
the body, Se is still toxic at excessive doses.[15–17] Organic Se
compounds are better than inorganic Se species, as they are
less toxic and more effective in anticancer activities.
Breast cancer cells are classified into two major categories:
estrogen receptor positive (ER+) and estrogen receptor nega-
tive (ERÀ), according to their evolution from distinct cell line-
ages. The clinical utility of ER antagonists for ER+ breast
cancer is often limited by side effects,[6] whereas ERÀ breast
cancer, particularly the triple-negative subtype, has no effective
treatments so far, given the lack of specific targets. Therefore,
[a] Y. Liang, Y. Zhou, S. Deng, Prof. T. Chen
Department of Chemistry, Jinan University, Guangzhou 510632 (P.R. China)
E-mail: tchentf@jnu.edu.cn
Our recent studies identified selenadiazole and benzimida-
zole derivatives as novel anticancer agents that can cause
cancer-cell-cycle arrest or apoptosis by reactive oxygen species
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600261.
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(ROS)-mediated DNA damage, the extracellular signal–regulat-
ed kinase (ERK) and protein kinase B (AKT) pathways,[18] inhibit-
ing thioredoxin reductase (TrxR) to disrupt cellular redox bal-
ance, and overcoming hyperglycemia-induced drug resist-
ance.[19,20] These reports demonstrate their potential in cancer
therapy.[21] Although selenadiazole and benzimidazole are
structurally similar, drug developers seldom introduce the two
groups into one compound, so we were interested to fulfill
such a task. In this study we synthesized four benzimidazole-
containing selenadiazole derivatives (BSeDs). The method we
developed is high yielding, mild, and readily amenable for ex-
traction and separation. We then elucidated the inhibitory ef-
fects of these compounds on the growth of two classic human
breast cancer cell lines: MCF-7 and MDA-MB-231. The results
show that all derivatives exhibit higher cytotoxicity against the
triple-negative breast cancer cell line MDA-MB-231 than MCF-
7. Interestingly, nearly all of them displayed good selectivity
between the two cancer cell lines except 1a. However, they all
exhibited dose-dependent inhibition of cell migration in com-
parison with untreated controls, as evidenced by remarkable
decreases in the wound closure zone of cultured cell monolay-
ers. Apoptosis is an important cell-death mechanism in cancer
treatment, and plays a vital role in the treatment of a variety
of carcinomas.[22] Our results demonstrate that BSeDs can
induce cell-cycle arrest and apoptosis in MDA-MB-231 cells by
causing DNA damage, inactivating AKT and activating MAPK
family members through ROS overproduction. Taken together,
the results of this study provide a facile microwave-assisted
strategy for the synthesis of benzimidazole-containing selena-
diazole derivatives with a high level of anticancer efficacy.
Figure 1. Structures of synthetic BSeDs.
(1:1). The benzimidazole was then obtained by microwave-as-
sisted intramolecular dehydration. The structures are shown in
Figure 1, and the synthetic route is presented in Scheme S1
(Supporting Information).
Cytotoxic effects
The antiproliferative activities of BSeDs were first screened
against MCF-7 and MDA-MB-231 cells by MTT assay. The results
(Figure 2A) show that the BSeDs exhibit growth inhibition
Figure 2. Cytotoxicity of BSeDs against A) cancer cells and B) normal cells.
Treatment time: 72 h. Values are the meanÆSD of experiments performed
in triplicate.
Results and Discussion
toward both MCF-7 and MDA-MB-231 cell lines. Clearly, the in-
hibitory effects of 1a, 1b, 1c, and 1d on MDA-MB-231 cells
are superior to those toward MCF-7 cells. However, the IC50
values of 1a toward MCF-7 (15.4 mm) and MDA-MB-231 cells
(12.3 mm) are close each other. The IC50 values of 1b, 1c, and
1d against MDA-MB-231 cells (1.0, 2.1, and 1.3 mm, respective-
ly) are 4- to 12-fold more potent than those against MCF-7
cells (4.1, 26.3, and 15.5 mm, respectively), suggesting that
these three compounds have good selectivity between MCF-7
and MDA-MB-231 cells. Moreover, cisplatin was chosen as posi-
tive control, and the results of MTT assays show that cisplatin
decreased MCF-7 and MDA-MB-231 cell proliferation by 50%
at 12.2 and 36.4 mm, respectively, suggesting higher potencies
for BSeDs than cisplatin. We also investigated the cytotoxicity
of BSeDs toward the normal cell lines LO2 and Chem 5. As
shown in Figure 2B, the BSeDs displayed lower toxicity toward
normal cells, especially relative to MDA-MB-231 cells. Future
examinations should involve in vivo anticancer efficacy and
toxicity studies to get a complete evaluation of the application
potentials of these synthetic BSeDs.
Chemistry
Conventional heating for synthesis usually involves the use of
a simple oil bath, which mostly heats the reaction vessel wall
by convection. However, the core of the mixture takes a longer
time to reach the target temperature. In contrast, by acting as
an internal heat source, microwave-irradiated absorption is
able to heat the target agents more uniformly, obviating the
need for heating an entire oil bath, thus saving a significant
amount of time and energy.
Generally, benzimidazole is synthesized by using o-phenyle-
nediamine and aldehyde as reactants.[23,24] However, in most
cases, aldehyde-containing reactants are rather difficult to
obtain because of their chemically active and unstable proper-
ties. Carboxyl group reactants are usually inexpensive and
chemically inactive and stable. However, the use of carboxyl
groups and o-phenylenediamine to synthesize benzimidazoles
is relatively difficult and has many disadvantages such as low
yield and harsher reaction conditions.[25–27] In this study we ex-
plored a new method that not only achieved higher yields
(50–60%), but is relatively mild and convenient. First, 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) was used as a coupling agent to form a peptide
bond from carboxyl selenadiazole 1 and o-phenylenediamine
Lipid–water partition coefficient and cellular uptake
From the perspective of structure–activity relationships, it ap-
pears that electron-accepting groups (Br and Cl) are inferior to
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electron-donating groups (CH3) in terms of antitumor activity
(Figure 2). Based on these results, we next studied the lipid–
water partition coefficient (logP) of the BSeDs (Figure 3A), as
the logP value is often closely linked to cellular uptake, and
the degree of cellular uptake can greatly affect a compound’s
cytotoxicity. The compounds were ranked in the following
order of increasing logP value: 1a < 1b < 1c < 1d (Fig-
ure 3B). However, after investigating cellular uptake, we found
Figure 3. A) Lipid–water partition coefficient and B) cellular uptake of BSeDs
(15 mm) in MDA-MB-231 cells. Values are the meanÆSD of experiments per-
formed in triplicate.
that an electron-donating group (CH3) imparts a higher rate of
absorption than electron-accepting groups (Br and Cl). This in-
dicates that as the logP value increases to a certain extent, the
absorption rate declines, meaning that a higher lipid–water
partition coefficient does not guarantee a higher absorption
rate, which may directly affect cytotoxicity. On the other hand,
compound 1b had a lower logP value but the highest absorp-
tion rate and the greatest cytotoxicity among the four com-
pounds.
Figure 4. Intracellular tracking of BSeDs in MDA-MB-231 cells. Cells were ex-
posed to BSeDs (15 mm, green fluorescence) for A) 0 or B) 4 h, co-stained
with lysotracker (Lyso, red fluorescence) for lysosomes and DAPI (blue fluo-
rescence) for nuclei.
Intracellular localization
Normally, as compounds traverse the cell membrane, the com-
pound concentration rises and the fluorescence strengthens. In
other words, fluorescence strength increases with increasing
cellular uptake. Importantly, the intracellular localization of
compounds can be examined. In this case, two fluorescent
probes, lysotracker (red) and DAPI (blue), were used to stain ly-
sosomes and nuclei, respectively. As shown in Figure 4, time
points at 0 h (Figure 4A) and 4 h (Figure 4B) were recorded.
The green fluorescence (BSeDs) and the red fluorescence (lyso-
tracker) were found to coincide well, demonstrating that
BSeDs are located mainly in the lysosomes. It is apparent that
the fluorescence intensity of 1b is slightly higher, supporting
the slightly higher absorption rate observed for 1b (Figure 3B).
Figure 5 shows the representative DNA distribution histograms
of MDA-MB-231 cells exposed to various BSeD concentrations.
The results show that all three test compounds effectively
induce an increase in the sub-G1 cell population, indicating the
induction of apoptosis. Moreover, the G2/M phase increased
significantly in 1b-treated cells (Figure 5A). For instance, the
G2/M cell population increased from 21.6% (control) to 60.9%
(2 mm), implying that G2/M-phase arrest induced by 1b may
contribute to cell death. Moreover, treatment of cells with 1d
significantly enhanced the accumulation of cells in G0/G1 phase
(Figure 5C). Taken together, these results suggest that BSeDs
can inhibit cancer cell growth though the induction of apopto-
sis and cell-cycle arrest.
Cancer cell growth inhibition via apoptosis and cell-cycle
arrest
To further confirm the induction of apoptosis by BSeDs, an
Annexin V FITC/PI assay was performed on MDA-MB-231 cells
with an Annexin V FITC/PI Apoptosis Kit. Cells were exposed to
compounds 1b, 1c, and 1d (each at 1 mm) for 48 h. Flow cyto-
metric analysis was then carried out (Figure 6A); cells stained
by Annexin V alone represent early apoptosis, and cells stained
by both Annexin V and propidium iodide (PI) indicate late
Apoptosis and cell-cycle arrest are two important modes that
inhibit the proliferation of cancer cells.[28] To clarify the mode
of cell death induced by BSeDs 1b, 1c, and 1d, cell-cycle dis-
tribution was monitored by flow cytometric analysis. We did
not investigate 1a because of its relatively low activity.
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apoptosis. The results shown in Figure 6B and 6C indicate that
compound 1b effectively induced early- and late-phase apop-
tosis at rates of 23.82 and 33.36%, respectively. Similarly, 1c in-
duced 17.16 and 21.79% of early- and late-phase apoptosis,
while 1d induced 18.70 and 23.82% of early- and late-phase
apoptosis, respectively. Taken together, these results indicate
that apoptosis could be a major mechanism of action for
BSeDs.
Inhibition of cancer cell migration
In many cases, tumor metastasis cause a higher death rate
than the primary tumor, so the identification of ways to sup-
press or prevent metastasis is particularly important in the
therapeutic process. There is still a limited number of drugs
that can effectively control the metastatic progression of
breast cancer cell lines, particularly the triple-negative subtype,
which features a high rate of metastasis.[29] The development
of new drugs that can suppress tumor metastasis is therefore
immensely important. Thus far, the effects of BSeDs toward
tumor metastases had not yet been documented. We therefore
investigated the effects of BSeDs on the migration of MDA-
MB-231 cells using two concentrations (0.5 and 1 mm) at two
time points (24 and 48 h). The results shown that BSeDs are
able to slow the migration of MDA-MB-231 cells in the wound-
healing migration assay (Figure 7A). The ability of the cells to
move and repopulate a scratched wound region was sup-
pressed by BSeDs in a dose-dependent manner. Specifically,
the inhibitory effects of metastasis for the BSeDs were more
apparent at 48 h than at 24 h at both concentrations. On the
other hand, although at 24 h the inhibitory effects of 1b, 1c,
and 1d at 1 mm are close to each other at only 16, 16, and
15% migrated cells relative to untreated control, respectively
(which are all excellent values), at 0.5 mm, these values increase
to 30% for 1c (Figure 7C) and 31% for 1d (Figure 7D), where-
as only 25% migrated cells (Figure 7B) was observed for 1b.
This means that 1b has a better suppressive effect than 1c
and 1d at lower concentration. Therefore, these synthetic
BSeDs have great potential for decreasing the risk of migration
of MDA-MB-231 cells.
Figure 5. Flow cytometric analysis of MDA-MB-231 cells exposed to A) 1b,
B) 1c, or C) 1d at various concentrations for 72 h.
Induction of intracellular ROS generation
Reactive oxygen species (ROS) are chemically reactive oxygen-
containing compounds such as peroxides, superoxide, and hy-
droxyl radicals. They are formed naturally as byproducts of the
normal metabolism of oxygen and play vital roles in cell signal-
ing and homeostasis. However, under environmental stress
(e.g., drug exposure), ROS levels can increase markedly. This is
likely to result in significant damage to cell structures.[30] There
is much evidence to suggest that in the biological context,
ROS act as critical mediators in apoptosis or cell-cycle arrest. In
our previous studies, most of the Se-based compounds were
observed to cause an upregulation of ROS levels to varying de-
grees. Thus, we continued to investigate the generation of in-
tracellular ROS, which was measured by the fluorescent probe
dicholorofluorescein (DCF). As shown in Figure 8, after incuba-
Figure 6. Apoptosis induced by BSeDs (1 mm) as analyzed by flow cytometry.
A) Annexin V/PI double-labeled assay performed on MDA-MB-231 cells;
B) early and C) late apoptosis rates. Values are the meanÆSD of experiments
performed in triplicate.
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Figure 7. Inhibition of metastasis of MDA-MB-231 cells by BSeDs. A) 1b, 1c, and 1d significantly suppressed MDA-MB-231 cell migration at different concen-
trations (0.5 and 1 mm) for 0, 24, and 48 h. Quantitative analysis of cell migration after exposure to B) 1b, C) 1c, or D) 1d at 24 h by manual counting. Values
are the meanÆSD of experiments performed in triplicate.
tion with BSeDs 1b, 1c, and 1d, MDA-MB-231 cells showed
a dose-dependent increase in intracellular ROS. Basically, the
curvilinear trend rises dramatically in the first 30 min and then
peaks between 30 and 40 min. These results demonstrate the
important role of superoxide anions in BSeD-induced apopto-
sis.
Induction of DNA-damage-mediated p53 phosphorylation
Activation of the tumor suppressor p53 can occur in response
to a variety of cellular stresses such as DNA damage, histanox-
ia, and oxidative damage. Several forms of DNA damage have
been shown to activate p53, including ionizing radiation, radio-
mimetic drugs, and chemicals. Owing to the fairly short half-
life of this polypeptide, p53 levels are maintained at a rather
low state under normal circumstances. Activation of p53 in re-
Figure 8. Intracellular ROS generation in MDA-MB-231 cells induced by
sponse to DNA damage involves a rapid increase in p53 ex-
pression levels and with an increased capacity for p53 to bind
DNA. Evidence supports that ROS-mediated DNA damage in-
duced by chemotherapeutic or chemopreventive drugs can
result in cell death by triggering cell-cycle arrest or apoptosis
via various signaling pathways, such as AKT, MAPKs, ataxia te-
langiectasia mutated (ATM), and p53.[31]
A) 1b, B) 1c, or C) 1d at various concentrations. ROS levels were measured
by DCF fluorescence. Values are the meanÆSD of experiments performed in
triplicate.
of BSeDs. We first investigated the expression levels of phos-
phorylated histone (P-histone), as P-histone is closely related
to DNA damage, especially DNA double-strand breakage. Fig-
ure 9A shows that histone phosphorylation is significantly trig-
In this study, western blot assays were performed to investi-
gate the underlying mechanisms behind the anticancer action
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ation related proteins. ERK is the best-characterized MAPK in
multiple physiological processes, whereas the JNK signal trans-
duction pathway is relatively complicated. There are three
genes that encode JNK, with 12 possible isoforms. The p38
MAPK appears to play a major role in apoptosis, cell-cycle
arrest, inflammation, and other stress responses.[36] To deter-
mine the possible role of MAPKs and AKT, we determined the
protein expression levels of MAPKs and AKT in BSeD-treated
cells. As shown in Figure 10, 1b, 1c, and 1d exhibited similar
effects on the phosphorylation of p38, JNK, ERK, and AKT. The
phosphorylation of pro-apoptotic kinases p38 and JNK dis-
played a trend of upregulation. In contrast, the phosphoryla-
tion of anti-apoptotic kinases AKT and ERK were effectively
suppressed by these three compounds. These results therefore
illustrate that the regulation of MAPK and AKT signaling path-
ways are involved in apoptosis induced by BSeDs.
Figure 9. Induction of DNA damage and p53-mediated apoptotic response
by BSeDs. MDA-MB-231 cells were treated with 1b (1.0 mm), 1c (2.1 mm), or
1d (1.3 mm) for 72 h. Equal protein loading was confirmed by analysis of b-
actin protein expression.
gered by BSeDs. The expression levels of phosphorylated ATM
(P-ATM) were enhanced as well, and P-ATM subsequently pro-
moted the activation of P-BRCA1, which can then result in the
upregulation of phosphorylated p53. Our data indicates that
exposing cells to BSeDs results in ROS-mediated DNA damage.
More than 50% of tumor cell types show various degrees of
mutant p53, and MDA-MB-231 cells are no exception. Al-
though the extent of p53 mutation varies with the tumor type,
it is now widely recognized that p53 mutations are the most
common genetic event in human cancer.[32] It stands to reason
that the normal function of p53 is compromised in most
human tumors. Because at least half of tumors exhibit p53 mu-
tations, the activity of those that retain wild-type p53 can be
attenuated by other mechanisms.[33] Moreover, it has been re-
ported that the mutation of p53 is not equivalent to simply
losing wild-type p53 function.[34] In other words, it does not
necessarily lead to a loss of p53 protein response.
Figure 10. BSeDs activate MAPK and inhibit AKT activity. MDA-MB-231 cells
were treated with 1b (1.0 mm), 1c (2.1 mm), or 1d (1.3 mm) for 72 h. Shown
are the effects of BSeDs on the expression levels of total (T) and phosphory-
lated (P) AKT (panel A) and p38, JNK, and ERK (panel B). Equal protein load-
ing was confirmed by analysis of b-actin expression.
To determine whether BSeD-induced apoptosis is still in-
duced via a p53-related pathway, we performed western blot
assays on p53 and apoptosis-related genes. The results
showed that BSeDs can downregulate the expression of Bcl-2
and Bcl-xl. Furthermore, the p53 target genes Noxa (a member
of the pro-apoptotic BH3-only proteins in the Bcl-2 family) and
Puma were investigated as well. Interestingly, 1b, 1c, and 1d
all induced an upregulation of Puma and Noxa expression
levels. We also investigated the expression level of mouse
double minute 2 (MDM2), which is the predominant negative
regulator of p53; it can bind to and inactivate p53. It was
found that the expression level of MDM2 in MBA-MD-231 was
downregulated to a certain degree by BSeDs. The results sug-
gest that Noxa, Puma, and MDM2 may contribute to BSeD-in-
duced apoptosis as mediated by p53.
Conclusions
The use of carboxyl-group reactants and o-phenylenediamine
to synthesize benzimidazole derivatives is conventionally diffi-
cult, requiring high temperature and long reaction times for
rather low yields. In this study we used HBTU as coupling
agent under alkaline conditions to form peptide bonds; prod-
ucts were then obtained via intramolecular dehydration by mi-
crowave irradiation. This novel method has advantages such as
short reaction time, milder reaction conditions, and higher
yield.
Breast cancer is the second most common type of cancer
after lung cancer (both sexes included) and by far the most
common cancer among women worldwide, with an incidence
rate more than twice that of colorectal cancer and cervical
cancer. Surgery and radiotherapy are both common therapeu-
tic approaches. However, surgery has the disadvantages of the
risk of bleeding, infections, damage to healthy tissues, among
others. Radiotherapy, in turn, may cause a series of side effects
including fatigue, sore skin, and hair loss. There still remains
only a limited number of drugs available for the treatment of
early-stage breast cancer. Therefore, there is still a significant
need to develop new agents that inhibit the proliferation of
breast cancer cells. The BSeDs synthesized in this study exhibit
AKT inhibition and MAPK activation
The MAPK and AKT pathways involve in a series of protein
kinase cascades, playing a pivotal role in the regulation of cell
proliferation and cell-cycle progression via transmitting extra-
cellular signals from the cell membrane to the nucleus.[35]
There are three primary MAPK family members that have been
characterized: p38, Jun kinase (JNK), and extracellular signal-
regulated kinase (ERK). These kinases are involved in the regu-
lation of cell signaling pathways, along with other cell prolifer-
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5-(6-chloro-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadia-
zole (1c): Yellow solid, yield: 53%, mp: 270–2728C; 1H NMR
([D6]DMSO, 500 MHz): d=13.37 (s, 1H, NH), 8.63 (s, 1H, Ar-H), 8.39
(d, 1H, Ar-H), 8.02 (d, 1H, Ar-H), 7.69 (m, 2H, Ar-H), 7.30 ppm (d,
1H, Ar-H); 13C NMR ([D6]DMSO, 125 MHz): d=160.43, 160.05,
130.62, 128.29, 124.19, 120.87 ppm; IR (KBr): n˜max =3446, 3130,
1683, 1539, 1399 cmÀ1; Anal. calcd for C13H7ClN4Se: C 46.80, H 2.11,
N 16.79%, found: C 46.82, H 2.14, N 16.75%; ESI-MS m/z (%): 333
[MÀH]À; HRMS calcd for C13H7ClN4Se [M+1]: 334.9524, found:
334.9597.
potential anticancer activities against the two classic breast
cancer cell lines, MCF-7 and MDA-MB-231. Compounds 1b, 1c,
and 1d were found to show greater cytotoxicity against the
triple-negative breast cancer cell line MDA-MB-231 than MCF-7
cells, and to exhibit dose-dependent inhibition of cell migra-
tion, in which a significant decrease in the zone of wound clo-
sure was observed relative to untreated controls. Our results
demonstrate that BSeDs can induce cell-cycle arrest and apop-
tosis in MDA-MB-231 cells by inducing DNA damage, inactivat-
ing AKT and activating MAPK family members through ROS
overproduction. The results of this study provide a facile micro-
wave-assisted strategy for the synthesis of organic selenium-
containing compounds that exhibiting high anticancer efficacy.
5-(6-bromo-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadia-
zole (1d): Yellow–brown solid, yield: 54%, mp: 277–2798C;
1H NMR ([D6]DMSO, 500 MHz): d=8.63 (s, 1H, Ar-H), 8.39 (d, H, Ar-
H), 8.01 (d, 1H, Ar-H), 7.85 (s, H, Ar-H), 7.61 (d, 1H, Ar-H), 7.41 ppm
(d, 1H, Ar-H); 13C NMR ([D6]DMSO, 125 MHz): d=160.46, 160.03,
130.57, 128.28, 124.19, 120.90 ppm; IR (KBr): n˜max =3446, 3131,
1683, 1540, 1399 cmÀ1; Anal. calcd for: C 41.30, H 1.87, N 14.82%,
found: C 41.31, H 1.91, N 14.85%; ESI-MS m/z (%): 377 [MÀH]À;
HRMS calcd for C13H7BrN4Se [M+1]: 378.9019, found: 378.9093.
Experimental Section
Synthesis and characterization
The synthesis of compounds 1, 1a, 1b, 1c, and 1d is shown in
Biological methods
Scheme S1 (Supporting Information).
Synthesis of 1: Commercially available 3,4-diaminobenzoic acid
was used as starting material. A solution of 3,4-diaminobenzoic
acid (1.54 g, 10 mmol) and selenium dioxide (1.11 g, 10 mmol) in
0.2n HCl (150 mL) was stirred for 2 h at room temperature. The
product was filtered, vacuum dried, and washed with water and
ethanol (twice each) and vacuum dried to obtain compound 1;
Yield: 93%.
Cell culture and MTT assay: MCF-7 and MDA-MB-231 breast
cancer cell lines were obtained from the American Type Culture
Collection. Cells were routinely grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), penicillin (100 UmLÀ1), and streptomycin (50 UmLÀ1
)
at 378C in a humidified incubator with 5% CO2 (in air). Cell viability
was determined by measuring the capacity of cells to transform 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
into the purple formazan dye, as previously described.[37]
Synthesis of 1a, 1b, 1c, and 1d: A mixture of compound
1 (228 mg, 1 mmol), HBTU (417 mg, 1.1 mmol), and N,N-diisopropy-
lethylamine (DIPEA; 260 mg, 2 mmol) in DMF (16 mL) was stirred at
room temperature for 2 h. Variously substituted 1,2-phenylenedia-
mine derivatives (1 mmol) dissolved in DMF (8 mL) were added,
and the mixture was stirred for another 12 h. The mixture was
transferred to a glass reaction pot (30 mL). The reaction was carried
out on a Discover SP Microwave Synthesizer (CEM Corporation)
under stirring at 1508C for 30 min. After cooling to room tempera-
ture, the reaction solution was poured into 100 mL water, filtered,
washed with water twice and vacuum dried. The resulting solid
was monitored by TLC and separated by silica gel column chroma-
tography (CH2Cl2/CH3OH 60:1) to afford 1a, 1b, 1c, and 1d.
Wound-healing migration assay: According to
a published
method[38] with minor modification, 1ꢁ105 MDA-MB-231 cells were
plated into 60 mm dishes for 48 h to achieve 80% confluency. A
wound on the confluent monolayer was created by scratching the
monolayer with a yellow Gilson d200 pipette tip. Cell debris was
removed by rinsing the scratched monolayer with PBS. Cells were
then incubated in DMEM containing 5% FBS in the presence or ab-
sence of various BSeD concentrations. The relative rate of cell mi-
gration for each sample was determined by differences in unclosed
wound area measured at specific time points (0, 24, and 48 h) rela-
tive to those of untreated control. Migrated cells were photograph-
ed by a digital camera (Olympus inverted microscope); micro-
graphs were then enlarged to a certain degree, and migrated cells
were quantified by manual counting in three independent experi-
ments.
5-(1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadiazole
(1a):
Yellow solid, yield: 58%, mp: 273–2758C; 1H NMR ([D6]DMSO,
500 MHz): d=13.22 (s, 1H, NH), 8.63 (s, 1H, Ar-H), 8.43 (d, 1H, Ar-
H), 7.98 (d, 1H, Ar-H), 7.61(m, 2H, Ar-H), 7.28 ppm (s, 1H, Ar-H);
13C NMR ([D6]DMSO, 125 MHz): d=160.41, 160.17, 150.40, 131.11,
128.50, 124.10, 120.40 ppm; IR (KBr): n˜max =3446, 3123, 1684, 1541,
1398 cmÀ1; Anal. calcd for C13H8N4Se: C 52.19, H 2.70, N 18.73%,
found: C 52.15, H 2.74, N 18.75%; ESI-MS m/z (%): 301 [M+H]+;
HRMS calcd for C13H8N4Se [M+1]: 300.9914, found: 300.9984.
Flow cytometric analysis: As previously reported,[38] cell-cycle dis-
tribution was determined by flow cytometric analysis (MultiCycle
software, version: 6-16-03-F32). MDA-MB-231 cells were treated
with the indicated BSeD concentrations. Treated cells were then
stained with propidium iodide (PI) for the process of cell-cycle
analysis. The proportions of cells in G0/G1, S, and G2/M phases were
plotted as DNA histograms. Apoptotic cells with hypodiploid DNA
content were measured by quantifying the sub-G1 peak in the cell-
cycle pattern. For each experiment, 10000 events per sample were
recorded.
5-(6-methyl-1H-benzo[d]imidazol-2-yl)benzo[c][1,2,5]selenadia-
zole (1b): Yellow solid, yield: 51%, mp: 266–2688C; 1H NMR
([D6]DMSO, 500 MHz): d=13.06 (s, 1H, NH), 8.60 (s, 1H, Ar-H), 8.41
(d, 1H, Ar-H), 8.00 (d, 1H, Ar-H), 7.54 (d, H, Ar-H), 7.44 (s, 1H, Ar-H),
7.11 ppm (d, 1H, Ar-H); 13C NMR ([D6]DMSO, 125 MHz): d=160.37,
160.20, 131.19, 128.54, 124.02, 120.06, 21.84 ppm; IR (KBr): n˜max
=
Annexin V (FITC)/PI apoptosis detection: Annexin V (FITC)/PI
apoptosis detection was carried out with a BD 556547 Annexin V
FITC/PI Apoptosis Kit. MDA-MB-231 cells were treated with indicat-
ed concentrations of BSeDs, then washed three times with PBS.
After dissociation, cells were then resuspended in PBS, and the PBS
3446, 3122, 1683, 1540, 1398 cmÀ1; Anal. calcd for C14H10N4Se: C
53.68, H 3.22, N 17.89%, found: C 53.65, H 3.26, N 17.92%; ESI-MS
m/z (%): 313 [MÀH]À; HRMS calcd for C14H10N4Se [M+1]: 315.0071,
found: 315.0145.
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Keywords: apoptosis
irradiation · reactive oxygen species · selenadiazoles
·
benzimidazoles
·
microwave
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Received: May 22, 2016
Revised: August 3, 2016
Published online on && &&, 0000
&
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FULL PAPERS
Here’s a better way: We developed
a new way to synthesize benzimidazole-
containing selenadiazole derivatives
(BSeDs) by combining peptide coupling
reagents and microwave irradiation.
These derivatives were found to have
high efficacy against the triple-negative
breast cancer cell line MDA-MB-231 and
to exhibit dose-dependent inhibition of
cell migration. BSeDs cause MDA-MB-
231 cell-cycle arrest and apoptosis by
inducing DNA damage, inactivating AKT,
and activating MAPK family members
by the overproduction of reactive
oxygen species.
Y. Liang, Y. Zhou, S. Deng, T. Chen*
&& – &&
Microwave-Assisted Syntheses of
Benzimidazole-Containing
Selenadiazole Derivatives That Induce
Cell-Cycle Arrest and Apoptosis in
Human Breast Cancer Cells by
Activation of the ROS/AKT Pathway
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