Potent anti-proliferative activities of organochalcogenocyanates towards breast cancer

Article abstract of DOI:10.1039/c8ob01891j

The pharmacological importance, particularly the anti-cancer and chemopreventive potentials, of organochalcogen compounds has attracted wide research attention recently. Herein we describe the synthesis of a series of organochalcogenocyanates that have one or more selenocyanate or thiocyanate units in a single molecule. The anti-proliferative activity of these organochalcogenocyanates in different breast cancer cells shows that selenocyanates exhibit much higher anti-proliferative activities than thiocyanates in general. Our study reveals that the activity of benzyl selenocyanate (1, BSC) could be significantly enhanced by 4-nitro substitution (12), which was more selective towards triple-negative breast cancer cells (MDA-MB-231) over other ER+ breast cancer cells (MCF-7 and T-47D). Furthermore, to the best of our knowledge, this is the first report on the synthesis of compounds having more than two selenocyanate units with promising anti-proliferative activities. Our studies further indicate that the apoptotic activities of selenocyanates are associated with modulation of cellular morphology and cell cycle arrest at S-phase. Selenocyanates also inhibited cellular migration and exhibited weak antioxidant activities. An effective binding interaction of compound 12 with serum albumin indicates its feasible transport in the bloodstream for its enhanced anti-cancer properties. Mechanistic studies by western blot analysis demonstrate that benzylic selenocyanates exhibit anti-proliferative activities by modulating key cellular proteins such as Survivin, Bcl-2 and COX-2; this was further supported by molecular docking studies. The results of this study would be helpful in designing suitable chemotherapeutic and chemopreventive drugs in the future.

Full text of DOI:10.1039/c8ob01891j

Organic &
Biomolecular Chemistry
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PAPER
Potent anti-proliferative activities of
organochalcogenocyanates towards breast
cancer†
Cite this: Org. Biomol. Chem., 2018,
16, 8769
Kaustav Banerjee,a Ganesan Padmavathi,b Debojit Bhattacherjee,a,c
a,c
Suchismita Saha,a Ajaikumar B. Kunnumakkarab and Krishna P. Bhabak
*
The pharmacological importance, particularly the anti-cancer and chemopreventive potentials, of
organochalcogen compounds has attracted wide research attention recently. Herein we describe the syn-
thesis of a series of organochalcogenocyanates that have one or more selenocyanate or thiocyanate units
in a single molecule. The anti-proliferative activity of these organochalcogenocyanates in different breast
cancer cells shows that selenocyanates exhibit much higher anti-proliferative activities than thiocyanates
in general. Our study reveals that the activity of benzyl selenocyanate (1, BSC) could be significantly
enhanced by 4-nitro substitution (12), which was more selective towards triple-negative breast cancer
cells (MDA-MB-231) over other ER+ breast cancer cells (MCF-7 and T-47D). Furthermore, to the best of our
knowledge, this is the first report on the synthesis of compounds having more than two selenocyanate
units with promising anti-proliferative activities. Our studies further indicate that the apoptotic activities of
selenocyanates are associated with modulation of cellular morphology and cell cycle arrest at S-phase.
Selenocyanates also inhibited cellular migration and exhibited weak antioxidant activities. An effective
binding interaction of compound 12 with serum albumin indicates its feasible transport in the blood-
stream for its enhanced anti-cancer properties. Mechanistic studies by western blot analysis demonstrate
that benzylic selenocyanates exhibit anti-proliferative activities by modulating key cellular proteins such as
Survivin, Bcl-2 and COX-2; this was further supported by molecular docking studies. The results of this
study would be helpful in designing suitable chemotherapeutic and chemopreventive drugs in the future.
Received 3rd August 2018,
Accepted 30th October 2018
DOI: 10.1039/c8ob01891j
rsc.li/obc
increased resistance of cancer cells towards the drugs and
(iii) additional toxicities. Particularly, the treatment scope of
Introduction
Cancer, characterized by uncontrolled cell proliferation, is one breast cancer in an advanced stage is very much limited due to
of the major and leading causes of mortality throughout the its high resistance to traditional treatments such as chemo-
world.1 Among different organ-specific cancers, breast cancer therapy.2 While the hormonal treatment of the estrogen recep-
has become a very common disease and is the second leading tor positive (ER+) breast cancer is accompanied by undesired
cause of mortality among women worldwide.1 Although side effects, there is no effective treatment to date for estrogen
chemotherapy is the widely used traditional strategy for pre- receptor negative (ER−) breast cancer, especially the triple-
venting or treating cancer, this treatment becomes inefficient negative sub-types, and thus the patients suffer from very poor
in many cases for one or more reasons such as (i) the lack of prognoses.3 Therefore it is very important and necessary to
selectivity of most of the chemotherapeutic drugs, (ii) identify suitable therapeutic compounds for breast cancer,
which would suppress the growth of ER− breast cancer cells as
well as minimize the undesired side effects.
aDepartment of Chemistry, Indian Institute of Technology Guwahati, Guwahati-
Very recently Giles and co-workers have reported a very
781039, Assam, India. E-mail: kbhabak@iitg.ac.in; Fax: +91-361-258-2349;
simple organoselenium compound, diphenyl diselenide
Tel: +91-361-258-3476
bCancer Biology Laboratory & DBT-AIST International Laboratory for Advanced
Biomedicine (DAILAB), Department of Biosciences and Bioengineering, Indian
Institute of Technology Guwahati, Guwahati-781039, Assam, India
cCentre for the Environment, Indian Institute of Technology Guwahati, Guwahati-
781039, Assam, India
(DPDS), which exhibits significantly higher cytotoxicity
towards triple-negative breast cancer (TNBC) cells over other
breast cancer cells.4 It has been described in several studies
that the treatment of cancer by chemotherapeutic drugs such
as cisplatin, cyclophosphamide, doxorubicin etc. is associated
with significant side-effects and toxicities mainly due to an
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob01891j
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overproduction of reactive oxygen species (ROS) or reactive mor effects of chemotherapeutic drugs.13 Dong and co-workers
nitrogen species (RNS).5 An efficient strategy for minimizing have reported an interesting observation that a second gene-
these toxicities is developed by combination therapy utilizing ration organoselenium compound, methylseleninic acid
the chemopreventive approach in the presence of suitable anti- (MeSeO2H), significantly enhanced the anti-cancer potency of
oxidants or pro-antioxidants. It has been shown that natural paclitaxel towards TNBC cells (MDA-MB-231).14 Although
and/or synthetic organoselenium compounds are suitable can- different organoselenium compounds such as monoselenides,
didates for chemoprevention and would dramatically minimize diselenides, selenoesters, and cyclic selenenyl amides have
chemotherapeutic drug-induced toxicities as well as enhance been reported to exhibit significant anti-proliferative activities
their potencies.6 The chemopreventive activities of inorganic towards different organ-specific cancer cells, reports on the
and organic selenium compounds on multiple mammary anti-proliferative activities of organoselenocyanates and the
tumor model systems such as chemically induced mammary corresponding thiocyanates are relatively rare.15 Very recently,
carcinogenesis in rats/mice as well as spontaneous tumorigen- Sharma and co-workers have reported the anti-cancer activity
esis in mice have been reported earlier.7 These studies clearly of a novel non-steroidal anti-inflammatory drug (NSAID)-based
demonstrated that organoselenium compounds are much selenocyanate (8) towards a series of cancer cells.16 The com-
more potent than the corresponding organosulfur compounds pound was found to be specific towards colorectal cancer
and inorganic selenium species.8 In 1995, El-Bayoumy and co- (CRC) cells and inhibited their growth by arresting the cell
workers showed that a simple organoselenium compound, cycle at G1 and G2/M phases. Attention towards TNBC cells
benzyl selenocyanate (1, BSC), effectively inhibits DMBA- was further enhanced by the recent report on benzimidazole-
induced mammary tumors in rats during the initiation phase based selenadiazole compounds 9–11, which exhibit excellent
of carcinogenesis; however, its sulfur analogue benzyl thio- anti-proliferative activities towards MDA-MB-231 cells over
cyanate (2, BTC) and sodium selenite (Na2SeO3) had no effect MCF-7 cells.17 From various studies it has been elucidated that
(Fig. 1).9 Subsequently, in 1997, Reddy et al. reported the the metabolic pathways of organoselenium compounds fol-
chemopreventive ability of two synthetic organoselenium com- lowed by the relative toxicities of major metabolic products are
pounds, namely 1,4-phenylenebis(methylene) selenocyanate crucial for their chemopreventive activities.18 A possible meta-
(3, p-XSC) and p-methoxybenzyl selenocyanate (6), towards bolic pathway of organoselenocyanates such as p-XSC indicates
colon carcinogenesis in rats that were treated with the carcino- its feasible reaction with intracellular glutathione (GSH) to
gen azoxymethane and fed low- or high-fat diets (Fig. 1).10 generate the corresponding glutathione adduct.19 Therefore,
Dietary administration of these drugs during the initiation before considering organoselenocyanates as chemopreventive
and post-initiation periods inhibited colon tumor incidence in agents in combination or adjuvant therapies, it is very impor-
rats maintained on a high-fat diet. The chemopreventive tant to understand their anti-proliferative activities.
action of compound 3 was found to be more pronounced
Inspired by the enhanced chemopreventive efficacies of
when it was administered along with a low-fat diet. The meta- p-XSC (3) as compared to BSC (1), we attempted to synthesise a
bolic pathway of compound 3 was studied further and it was series of benzylic and mesitylenic selenocyanates that have
found that the selenocyanate moiety reacted with the cellular one or more selenocyanate moieties in a single molecule with
abundant glutathione (GSH) with the replacement of the cyano the intention of exploring their efficacy as potential anti-prolif-
group leading to the formation of the corresponding selenenyl erative agents. To understand the importance of selenium or
sulfide compound 5. The chemopreventive activity of 5 was selenocyanate groups in these compounds, the corresponding
also evaluated and found to be almost equal to that of com- thiocyanates were also synthesized for a detailed structure–
pound 3. Detailed studies by Bhattacharya and co-workers activity correlation study under identical test conditions. As
have established that diphenylmethyl selenocyanate (7) has the the breast cancer expressing estrogen receptor (ER) and pro-
ability to counter the oxidative stress induced by xenobiotics of gesterone receptor (PR) have a better prognosis than triple-
diverse nature in vivo.11 They have also developed a series of negative breast cancers, our test compounds were screened
naphthalimide-based selenocyanates that were found to be first for their anti-proliferative activities in vitro towards the
very effective towards the cyclophosphamide (CP)-induced gen- triple-negative cell line MDA-MB-231. Furthermore, the potent
otoxicity and oxidative stress.12 These compounds protect compounds were subsequently screened in other ER+ breast
against chemotherapeutic drug-induced toxicity in vivo and cancer cells such as MCF-7 and T-47D to gain further compara-
chemoprevention can be initiated without blocking the antitu- tive results on breast cancer cells in general. The most potent
compound was studied further to understand its mode of
action towards anti-proliferative activities (Fig. 2).
Results and discussion
Synthesis of chalcogenocyanates
We present herein the synthesis of benzylic and mesitylenic
poly-substituted selenocyanates and the corresponding thio-
Fig. 1 Chemical structures of some organochalcogen-based chemo-
preventive agents 1–11.
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Fig. 2 Chemical structures of benzylic and mesitylenic poly-selenocya-
nates and the corresponding poly-thiocyanate analogues.
cyanates 12–27. These compounds were conventionally pre-
pared in good yields by nucleophilic substitution reactions
from the corresponding halides. The selenocyanates in the
present study were prepared from the corresponding halides
in the presence of potassium selenocyanate (KSeCN) as a
nucleophile following the literature method with minor modi-
fications.20 The thiocyanate counterparts were synthesized
from the same halides in the presence of potassium thio-
cyanate (KSCN) in dry methanol under reflux conditions
following the earlier reported method with minor modifi-
cations.21 The commercially unavailable precursor halides
were synthesized in the laboratory in one or two steps wherever
applicable (ESI†). The final selenocyanates and thiocyanates
were purified by column chromatography and characterized by
NMR, IR and ESI-MS analyses (ESI†).
Fig. 3 Percentage proliferation of MDA-MB-231 cells by the test com-
pounds in a dose-dependent manner. In addition to control experi-
ments, % proliferation was studied at four different concentrations
(1.0 µM, 5.0 µM, 10.0 µM and 25.0 µM). The % proliferation after 72 h as
shown above was calculated from the resultant OD values at 72 h after
subtracting the OD values of an identical set of cells at 0 h. Therefore,
the negative % proliferation values at some compound concentrations
indicate the presence of a smaller number of live cells at 72 h than that
at 0 h (cytotoxicity).
growth inhibitory activities. Interestingly, most of the organo-
selenocyanates exhibited better efficacy than the standard anti-
cancer drug 5-FU under identical experimental conditions in
MDA-MB-231 cells.
Anti-proliferative activity of chalcogenocyanates in breast
cancer cells
After determining the dose-dependency of the test com-
pounds towards growth inhibition of MDA-MB-231 cells,
inhibitory concentrations towards cell proliferation were deter-
mined for the potent compounds to understand their relative
anti-proliferative activities (Table 1). Standard and previously
reported chemopreventive selenocyanate compounds such as
benzyl selenocyanate (BSC, 1) and p-XSC (3) exhibited good
anti-proliferative activities with an IC50 of 11.15 and 12.13 µM,
respectively. Interestingly, compound 12 having 4-NO2 substi-
As discussed earlier that the treatment scope for triple-negative
breast cancer is limited and not well established, we have
chosen MDA-MB-231 cells to understand the efficacy of our
synthesized compounds. The anti-proliferative activity of these
compounds was evaluated using MTT assay in MDA-MB-231
cells. All the experiments were independently carried out at
least three times and the results were summarized after 72 h
of incubation at 37 °C. To understand the dose-dependency of
these compounds for growth inhibition of MDA-MB-231 cells
over 72 h, the percentage of cell proliferation was estimated at
four different concentrations (1.0 µM, 5.0 µM, 10.0 µM and
25.0 µM) and compared to that in the absence of compounds
(controls) as well as with a well-known anti-cancer drug,
5-fluorouracil (5-FU). As shown in Fig. 3, most of the organo-
selenocyanates were found to be active in the growth inhi-
bition of MDA-MB-231 cells but the corresponding thiocyanate
analogues were found to be almost inactive under identical
experimental conditions. A decreased rate of proliferation was
observed with an increase in concentration up to 10.0 µM and
tution at the phenyl ring exhibited very high potency (IC50
=
0.80 µM), which was almost 14-fold and 15-fold more potent
than standard selenocyanates 1 and 3, respectively. Similar to
p-XSC, other bis-selenocyanates such as 16, 18 and 20 exhibi-
Table 1 IC50 values of some active selenocyanates for their anti-prolif-
erative activities in MDA-MB-231 cells as determined by MTT assay
Compound
IC50 a value (µM)
Compound
IC50 a value (µM)
1
3
12
16
11.15 2.75
12.13 2.19
0.80 0.14
11.73 0.23
18
20
24
13.90 0.12
11.66 2.61
6.43 0.10
a
noticeable cytotoxicity (negative % proliferation) was
detected for almost all the selenocyanates at 25.0 µM concen-
tration. A strong inhibition was observed for compound 12
even at 1.0 µM concentration and cytotoxic behaviour was
detected at concentrations 10.0 and 25.0 µM. This could be
indicative of the very high potency of compound 12 as com-
pared to other derivatives. Whereas moderate inhibition and
no cytotoxicity was observed for compounds 1, 14, 22 and 26
up to 25.0 µM concentration, indicating relatively weaker
a Data represent the mean IC50 values ( SD) of % cell proliferation
determined by the MTT assay from the dose–response curves in tripli-
cate after 72 h of incubation at 37 °C. About 4000 cells per 100 µl per
well were seeded in 96-well culture plates and treated with various con-
centrations of test compounds for 72 h. The % proliferation after 72 h
as shown above are calculated from the resultant OD values at 72 h
after subtracting the OD values of an identical set of cells at 0 h.
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ted comparable IC50 values in the range of 11–14 µM as
shown in Table 1. However, the corresponding ortho-deriva-
tive 14 was found to have weaker anti-proliferative activity and
its IC50 could not be determined in a similar concentration
range. A significant difference in anti-proliferative activity
was observed between compounds 22 and 24 having three
selenocyanate units. While an IC50 value could not be deter-
mined for compound 22 up to a concentration of 25 µM, the
corresponding mesitylenic derivative 24 exhibited a signifi-
cantly improved anti-proliferative activity (IC50 = 6.43 µM).
These results indicate that the presence of the methyl group
in benzylic selenocyanates might play important roles in
their anti-proliferative activities, which is clearly reflected in
the activities of compounds having two (14, 16, 18 and 20)
and three (22 and 24) selenocyanate units. However, to our
surprise, the IC50 value could not be determined in the MTT
assay for compound 26 with six selenocyanate units. A
decrease in cell proliferation was observed in the beginning
with increasing concentration of compound 26; however,
negligible change was observed after 40% inhibition. This is
also reflected in the dose–response data as shown in Fig. 3.
Due to the presence of six selenocyanate units in a single
molecule, compound 26 became very lipophilic in nature and
was sparingly soluble in the buffered medium during cellular
assays. Therefore, accurate inhibitory activity could not be
determined at higher concentrations. As thiocyanates were
almost inactive up to 25 µM concentration, IC50 values were
not determined for these compounds and the inhibitory
responses of some representative thiocyanates are shown in
the ESI.† As observed in Fig. 3 and Table 1, it is evident that
the anti-proliferative activity of benzyl selenocyanate 1 can be
dramatically enhanced by a simple substitution of a nitro
group at the 4-position (compound 12) or by a proper balance
of methyl and selenocyanato groups in a single molecule
(compound 24).
Fig. 4 Percentage proliferation of MCF-7 and T-47D cells in the pres-
ence of some selected test compounds in a dose-dependent manner. In
addition to control experiments, % proliferation was studied at four
different concentrations (1.0 µM, 5.0 µM, 10.0 µM and 25.0 µM). The %
proliferation after 72 h as shown above was calculated from the resultant
OD values at 72 h after subtracting the OD values of an identical set of
cells at 0 h. Therefore, the negative % proliferation values at some com-
pound concentrations indicate the presence of a smaller number of live
cells at 72 h than that at 0 h (cytotoxicity).
cancer cells (MDA-MB-231) over other ER+ breast cancer cells
(MCF-7 as well as T-47D).
To understand the selectivity of the most active compound
12 towards a cancer cell over normal cells it was screened
further in a representative normal cell line, L-132 (human lung
embryonic epithelial cells), and the results were compared to
that of MDA-MB-231 cells. As the IC50 of compound 12 in
MDA-MB-231 cells was around 1.0 µM, the anti-proliferative
activity was checked at 1.0 µM and at 5-fold higher concen-
tration (5.0 µM). As expected, the anti-proliferative activity was
found to be much weaker in L-132 cells at both the concen-
trations (Fig. S24, ESI†), indicating significant selectivity of
compound 12 towards triple-negative breast cancer cells over
normal cells.
After screening all the selenocyanates and some representa-
tive thiocyanates in triple-negative breast cancer cells
(MDA-MB-231), we further screened some of the active com-
pounds (1, 12 and 24) along with the standard drug 5-FU in
two additional ER+ breast cancer cells (MCF-7 and T-47D) to
understand the impact of these compounds on breast cancer
cells. As shown in Fig. 4, the compounds were found to be
Dose dependent cellular death study by a PI-flow cytometric
assay
potent in both the cell lines. While the activities of As seen in Fig. 3, some of the selenocyanates exhibited cyto-
these selected compounds in both of these ER+ breast toxic behavior at higher concentrations; this was further con-
cancer cells were almost comparable, the activities were signifi- firmed using a propidium iodide (PI)-flow cytometric assay. PI
cantly different as compared to MDA-MB-231 cells. For is a fluorescent compound that can bind to nucleic acids with
example, although compound 12 (IC50 = 0.80 0.14 µM) was little or no sequence preference. An increased red fluorescence
significantly more active than compound 24 (IC50 = 6.43
is indicative of a damaged cell membrane, which in turn rep-
0.10 µM) in MDA-MB-231 cells, a reversal in potency was resents cell death. Among the test compounds used in this
observed in both MCF-7 and T-47D cells. In these cells, com- study, five selenocyanates, namely, 3, 12, 18, 20 and 24, that
pound 12 [IC50 (MCF-7) = 7.71 0.45 µM; IC50 (T-47D) = 13.17 exhibited cytotoxic behavior were chosen for the PI-flow cyto-
3.6 µM] was found to be less active than compound 24 [IC50 metric assay. Treatment of MDA-MB-231 cells with different
(MCF-7) = 4.64
0.13 µM; IC50 (T-47D) = 3.53
0.74 µM]. concentrations of these test compounds for a duration of 72 h
These results indicate that although compound 24 is moder- resulted in profound cell death, which was found to be dose-
ately active on all three breast cancer cells studied herein, sele- dependent as shown in Fig. 5. At the concentrations of 10 and
nocyanate 12 is highly selective towards triple-negative breast 25 µM of selenocyanates, significant cell death was observed,
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Fig. 5 Cytotoxicity profiles of the test compounds on MDA-MB-231
cells in propidium iodide (PI)-flow cytometric studies. About 5 × 104
cells per 2 mL were seeded in 6-well culture plates and treated with two
different concentrations (10 µM and 25 µM) of test compounds for 72 h.
A concentration of 5.0 and 10.0 µM was used only for compound 12.
The cells were stained with 5.0 µl of 1.0 mg mL−1 of PI and analyzed by
the flow-cytometric method.
which is in agreement with the observation from the anti-pro-
liferative activity data obtained from the MTT method. As the
IC50 of compound 12 was much lower, relatively lower concen-
trations (5 and 10 µM) were used and around 40% cell death
was observed at 10 µM concentration. This study along with
anti-proliferative activity data confirms the inhibition of cell
growth and induction of cell death by the organoselenocya-
nates used in the present study.
Fig. 6 Graphical
representation
of
distribution
patterns
of
Cell cycle progression analysis of MDA-MB-231 cells in the
presence of selenocyanates
MDA-MB-231 cells in different phases of cell cycle in the presence of
selenocyanates 12 and 24. About 1 × 105 cells per 2 mL were seeded in
6-well plates and treated with two different concentrations (1.0 and
5.0 µM) of test compounds for 24 h and the cell cycle distribution was
A proper regulation of the cell cycle is an essential process for
normal cell development and cellular functions. A de-regu- analyzed by flow-cytometric assay after staining with PI/RNase.
lation of it may induce aberrant cell proliferation, leading to
cancer. Therefore, an inhibition of abnormal cell cycle pro-
gression, which would result in reduced cell proliferation and
increased apoptosis, is one of the main strategies for cancer
therapy. In the present study, in order to understand the
mechanism of inhibition of cell proliferation by the test com-
pounds, we have investigated the effects of some active seleno-
cyanates such as 12 and 24 on the cell cycle distribution of
MDA-MB-231 cells using PI-based flow-cytometric experiments.
Cell cycle distribution was observed upon the incubation of
cells with test compounds for 24 h at two different concen-
trations, namely 1.0 µM and 5.0 µM. As shown in Fig. 6, a
noticeable arrest in cell cycle progression was observed in the
MDA-MB-231 cells observed in the present study is in contrast
to the effect of selenadiazole derivatives 9–11 on MDA-MB-231
cells.17
Compounds 9–11 induced an effective increase of cell popu-
lation in Sub-G1 and G2/M phases leading to early and late
apoptosis. However, a similar S-phase arrest has been reported
in MCF-7 cells in the presence of an aromatic organoselenium
compound such as bis(4-aminophenyl) diselenide.22
Inhibition of the migration of cancer cells
presence of compounds 12 and 24. These results show that the In addition to primary cancer cells, metastatic cells mainly
test compounds effectively increase the population at S-phase spread the cancer to other parts of the body. As the metastasis
in a dose-dependent manner with a concomitant decrease in takes place due to cell migration, inhibition of cancer cell
population in G1 and G2 phases. For example, S-phase popu- migration is very important for the therapeutic process.
lation is increased to 38.8% (1.0 µM) and 57.9% (5.0 µM) from Furthermore, a high rate of metastasis is observed for the
33.3% (control) in the presence of compound 12. These results TNBC cells and there are a limited number of drugs known to
indicate that the pronounced anti-proliferative activity of com- have significant anti-migratory properties towards TNBC cells.
pound 12 as reflected in apoptotic assays is probably through We have therefore evaluated some selected selenocyanates for
the induction of cell cycle arrest in S-phase. Similarly, com- their anti-migratory properties towards MDA-MB-231 cells
pound 24 having three selenocyanate units also exhibited using a wound healing assay.23 Regular monitoring of the cells
reasonable cell cycle arrest in S-phase, indicating a similar revealed that the compounds inhibited the migration of the
mode of action towards their anti-proliferative activities. The cells to the wounded area, indicating the anti-invasive nature
influence of selenocyanates on the cell cycle distribution of of test compounds (Fig. 7). For example, significant dose-
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Fig. 8 Change in cellular morphology of MDA-MB-231 cells in the
absence and presence of selenocyanates 12 and 24 at a concentration
of 10.0 µM after incubation for 0 h, 24 h, and 48 h. PI-stained images
after incubation of cells for 72 h in the presence of test compounds rep-
resent dead cells.
cells after 72 h were further stained with the nuclear staining
fluorescent dye PI to detect the dead cell nuclei under an
inverted fluorescence microscope. As PI cannot permeate
through the cell membrane of live cells, it can selectively stain
the nuclei of dead cells, indicating the extent of cell death. A
significant number of dead cells were visualized upon PI-stain-
ing as shown in Fig. 8, supporting the occurrence of cell death.
Fig. 7 Wound healing studies on MDA-MB-231 cells in the presence of
compounds 12 and 24 at a concentration of 1.0 µM and 2.5 µM after
incubation for 0 h, 24 h, 48 h and 72 h.
dependent anti-migratory activities of compounds 12 and 24
were observed as compared to the untreated cells under identi-
cal conditions up to a duration of 72 h. These results indicate
that selenocyanates have the capability to suppress the move-
ment and re-population of cells in the scratched wound region
even up to 72 h. Similar dose-dependent anti-migratory activi-
ties of compounds 9–11 were also reported towards
MDA-MB-231 cells for a duration of 24 h and 48 h.17
Antioxidant activities of organoselenocyanates
A number of organoselenium compounds have been reported
to show good to excellent antioxidant properties towards bio-
logical reactive oxygen species (ROS) such as hydrogen per-
oxide (H2O2), peroxynitrite (ONOO−), hydroxyl radical (OH•)
etc.24 Although the in vitro antioxidant activity of organoseleno-
cyanates has not been reported, we have taken the initiative to
Study of cellular morphology upon selenocyanate treatment
The occurrence of apoptosis is accompanied by notable study their in vitro antioxidant properties.
changes in the cellular morphology of cancerous cells. There
Hydrogen peroxide scavenging activity. To understand the
are several techniques to understand/detect these changes con- capability of test compounds to catalytically reduce H2O2, an
firming the mode of cell death. Cell shrinkage by a reduction in vitro assay was carried out spectrophotometrically using a
of the volume of the cellular nucleus and cytoplasm is com- glutathione (GSH)–glutathione disulfide (GSSG) coupled
monly associated with apoptotic cell death. The morphological assay.24b,25 To evaluate their relative antioxidant potentials, the
changes upon cell death can be easily visualized under a rates were compared to that of the standard antioxidant organo-
microscope. In the present study, MDA-MB-231 cells were incu- selenium compound ebselen (2-phenyl-1,2-benzoselenazol-3-
bated with active selenocyanates 12 and 24 (10.0 µM) and the one, PZ-51). The activities of most of the selenocyanates were
cellular morphology was visualized under a microscope after found to be much lower than that of ebselen under identical
24 h and 48 h. As shown in Fig. 8, significant visual changes experimental conditions (Fig. S25, ESI†). This indicates that
were observed after 24 h and 48 h in the presence of com- selenocyanates in the present study are relatively weaker anti-
pounds 12 and 24. Visual changes in morphology during cell oxidants for the reduction of oxidants such as H2O2 in the
death are due to the formation of dead cell debris, which loses presence of GSH as a co-substrate. Furthermore, we have
its adherent properties and becomes detached from the surface carried out the reaction of standard selenocyanate 1 with aro-
and floats randomly in the culture media. Significant cell death matic thiol such as thiophenol (PhSH) and the product for-
was found for the test compounds and it was enhanced with mation was monitored using 77Se NMR spectroscopy to under-
the incubation time as compared to the control experiments. In stand its reactivity towards thiols, which is an important step
addition to visualization using a microscope, the incubated towards the antioxidant activity of organoselenium com-
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pounds. Interestingly, the formation of the corresponding sele-
In the present study, the binding interaction of selenocya-
nenyl sulfide (PhCH2Se-SPh) upon the elimination of the nates with BSA was studied following an earlier reported pro-
cyano group from selenocyanate 1 was observed. This was con- cedure.28 The BSA solution exhibits strong fluorescence emis-
firmed by the appearance of a peak at 507 ppm with the dis- sion at 344 nm upon excitation at 280 nm. The fluorescence
appearance of a peak due to compound 1 at 306 ppm in 77Se intensity gradually decreases with an increase in the concen-
NMR spectroscopic experiments (S41–S43, ESI†). A similar tration of the test compounds without any shift in the emis-
reaction of selenocyanate 1 with H2O2 led to the formation of sion λmax, indicating that the compound quenches the intrin-
oxidized species such as selenenic or seleninic acid derivatives. sic fluorescence of BSA in a static manner. The fluorescence
In this reaction, a highly de-shielded peak at 1330 ppm was quenching was performed by a titration method and the
observed, which could be ascribed to benzyl seleninic acid binding parameters such as the quenching constant (Kq) and
(PhCH2SeO2H).
Stern–Volmer constant (KSV) were determined using the Stern–
Peroxynitrite scavenging assay. Peroxynitrite (PN) is a reac- Volmer equation as shown in Table 2. The binding constant
tive nitrogen species (RNS), which is produced in the body and (Ka) and the number of binding sites (n) have been determined
can lead to oxidation and nitration of bio-molecules, amino by using a Scatchard plot as reflected in Table 3. These results
acid residues of proteins and enzymes.26 A number of small show that all the selenocyanate compounds exhibited very
molecules especially organochalcogen compounds have been good binding affinity towards BSA with high KSV and Kq values.
developed as catalytic or stoichiometric scavengers of PN as The values of n for all the compounds are nearly 1.0, indicat-
monitored by the oxidation of dihydrorhodamine-123 ing a single molecular bond with BSA. Particularly, the most
(DHR-123) into the highly fluorescent rhodamine-123.27 The
scavenging potentials of selenocyanates were compared to that
of ebselen, which has been shown to be a catalytic scavenger
of PN.27c While ebselen exhibited strong scavenging activity
Table 2 The binding parameters such as KSV (Stern–Volmer constant)
and Kq (quenching constant) of different test compounds with BSA as
obtained from the Stern–Volmer equationa
(IC50 value: 4.2 µM), the activities of selenocyanates were sig-
nificantly lower (Fig. S25, ESI†). Although a few selenocyanates
such as 18, 20 and 24 exhibited good scavenging activity under
identical conditions, the IC50 values could not be calculated
for some of the selenocyanates such as 1, 3, 16, and 26. These
compounds exhibited some scavenging properties in the
beginning; however, the inhibition remained less than 50%
even up to 150–200 µM concentrations. A similar trend was
observed for all the thiocyanates used in the present study. In
general, the chalcogenocyanates used in the present study
were found to be weaker scavengers of PN as compared to
other organochalcogen compounds studied previously.27
Compound
KSV (×104 M−1
)
Kq (×1012 M−1 S−1
)
R2
1
3
1.94
2.81
8.74
2.75
1.53
4.71
3.54
4.81
5.05
1.94
2.81
8.74
2.75
1.53
4.71
3.54
4.81
5.05
0.9907
0.9824
0.9910
0.9946
0.9958
0.9991
0.9969
0.9905
0.9699
12
16
18
20
22
24
26
a The quenching experiment was performed using a fluorescence
spectrophotometer with excitation and emission wavelengths of
280 nm and 344 nm, respectively. BSA (10.0 µM) in Tris-HCl buffered
saline (100 mM, pH 7.2) was titrated with increasing concentrations of
Binding interaction with bovine serum albumin
As serum albumin is one of the highly abundant proteins in test compounds (0.0–50.0 µM).
blood plasma of living systems and plays crucial roles in the
transportation of several essential small molecules, drugs etc.
by sequestering the latter at one site and specific release of the
Table 3 The binding parameters such as Kb (binding constant) and n
same at some other site, understanding the interaction of
serum albumin with selenocyanates in this context is impor-
tant to gain an insight into the pharmacodynamics of these
compounds.28 Bovine serum albumin (BSA) is very commonly
used as a homology protein for human serum albumin (HSA)
owing to its ready and cost-effective availability. Fluorescence
quenching of BSA upon its interaction with small molecules is
a simple but powerful tool for understanding the affinity of
protein–ligand binding interactions. The fluorescence of BSA
mainly arises due to the presence of tyrosine (Tyr) and two
tryptophan (Trp) units, namely Trp-134 and Trp-212, located
on the surface of sub-domain IB and in the hydrophobic
pocket of sub-domain IIA, respectively. Therefore, the quench-
ing of fluorescence of BSA upon binding with the drug/ligand
indicates that the binding site of the protein may be in these
domains.
(number of molecules per unit protein) of different test compounds with
BSA as determined by using a Scatchard plota
Compound
Kb (×104 M−1
)
n
R2
1
3
0.89
1.10
1.04
0.41
5.08
3.27
3.36
4.84
0.10
0.93
0.92
0.81
0.82
1.11
0.97
0.99
1.00
0.64
0.9913
0.9695
0.9924
0.9993
0.9992
0.9988
0.9910
0.9879
0.9956
12
16
18
20
22
24
26
a The quenching experiment was performed using a fluorescencespec-
trophotometer with excitation and emission wavelengths of 280 nm
and 344 nm, respectively. BSA (10.0 µM) in Tris-HCl buffered saline
(100 mM, pH 7.2) was titrated with increasing concentrations of test
compounds (0.0–50.0 µM).
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active selenocyanate 12 exhibits much higher binding affinities dose-dependent manner and significant down-regulation was
as reflected by its higher KSV and Kq values as compared to observed at higher concentrations such as 2.0 and 2.5 µM.
other analogous compounds. The higher binding affinity of 12 These results indicate that the active selenocyanate 12 prob-
to BSA as compared to other compounds studied in this study ably hase high impact on the expression level of important cel-
is probably due to the presence of the 4-NO2 group in addition lular proteins responsible for cancer progression. In contrast
to the selenocyanate moiety, which can have non-bonding to these observations, the levels of proteins such as Akt,
interactions with the proximal amino acid residues in the p-Akt (S476) and VEGF-A were almost unaltered in the presence
binding site of BSA. The results from our study are in agree- of compound 12 up to a concentration of 2.5 µM.32 These obser-
ment with recent observations on the binding affinities of vations indicate that compound 12 exhibits anti-proliferative
some organoselenium compounds with BSA.
activity via the Akt-independent pathway. Although our results
It should be noted that the interaction of naphthalimide- from western blot analysis on the levels of some key signaling
based selenocyanates with BSA was studied in different binary molecules cannot be considered as conclusive remarks on the
solvents and a significant decrease in the fluorescence emis- mode of action of compound 12, these observations could be a
sion intensity of BSA was observed in the presence of seleno- platform for further studies on the molecular mechanisms of
cyanates.29 A similar static interaction of selenadiazole deriva- selenocyanates towards TNBC cells such as MDA-MB-231. More
tives with BSA with a good binding affinity has recently been detailed mechanistic studies will be carried out with active com-
reported by Chen and co-workers.30 They showed that the pounds in a separate study in the future.
binding of selenadiazole derivatives with serum albumin facili-
tated the drug transport through plasma and significantly
enhanced their anti-tumor activities and cellular uptakes.
Molecular interaction of selenocyanates with proteins
As compound 12 exhibited significant potency for the down-
regulation of some key cancer-cellular proteins such as
Survivin, Bcl-2 and COX-2, we carried out docking studies to
understand the possible molecular interaction of 12 with these
proteins. Additionally, the molecular interaction was also studied
with BSA as these compounds exhibited good BSA-binding inter-
actions. Compound 1 was used as a representative selenocyanate
to obtain comparative interaction profiles. As shown in Table 4,
compound 12 has much higher binding affinity than 1 towards
BSA. This is represented by a higher binding energy and lower
inhibitory constant of 12 as compared to 1. Similar to BSA, the
binding affinity of 12 was found to be significantly higher than
that of 1 towards all the cellular proteins such as Survivin, Bcl-2
and COX-2, which are used in the present study.
Therefore, the efficient binding of compound 12 with BSA in
our present study could be a good indicator of its enhanced
anti-proliferative activity in breast cancer cells.
Analysis of some cellular proteins in MDA-MB-231 cells using
western blot analysis
To understand the mode or pathway of anti-proliferative activi-
ties of active selenocyanates such as compound 12, western
blot analyses were performed on MDA-MB-231 cells. Some
important cellular protein expression levels were estimated in
the presence of active selenocyanate 12 at different concen-
trations (0.0, 0.25, 0.5, 1.0, 2.0 and 2.5 µM) and incubated for
24 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
was used as a housekeeping gene loading control for this
experiment. As shown in Fig. 9, the expression levels of impor-
tant proteins involved in cell survival, proliferation and angio-
genesis such as COX-2, Survivin, Bcl-2 and VEGF-A, Akt,
p-Akt etc. were studied in the presence of compound 12. It is
well-known that while COX-2 expression is crucial for invasive
The value of the inhibitory constant of compound 1
towards all four proteins was enormously decreased by the
introduction of a simple nitro group at the 4-position of the
phenyl ring of compound 1. This increased affinity is probably
breast cancer and resistance to apoptosis, Survivin and Bcl-2 Table 4 The binding parameters such as binding energy (EB), inhibitory
constant (Ki) and the proximal amino acid residues of proteins involved
in non-bonding interactions with ligands
act as anti-apoptotic protein markers, responsible for the pro-
gression of carcinogenesis.31 It is reflected in Fig. 9 that com-
pound 12 reduces the levels of COX-2, Bcl-2 and Survivin in a
EB
Compound Proteina (kcal mol−1
)
Ki (µM) Interactions
1
BSA
−4.2
829.4
—
Survivin −3.8
Bcl-2
COX-2
1750.0
1310.0
331.8
L6.B, W10.A
Y67.B
R376.B
−3.9
−4.7
12
BSA
−7.1
5.9
K114.A
W10.A, L6.B
K22.A, R26.A, R68.A
R376.B, V538.B
Survivin −5.2
Bcl-2
COX-2
154.6
39.5
24.6
−6.0
−6.3
a The docking study was performed with four proteins such as BSA
(PDB: 4F5S), Survivin (PDB: 1F3H), Bcl-2 (PDB: 2 W3L) and COX-2
(PDB: 5KIR). The crystal structures of these proteins were collected
from the RCSB database.
Fig. 9 Western blot analysis for the estimation of key protein
expression levels in MDA-MB-231 cells in the presence of compound 12
at different concentrations (0.0, 0.2, 0.5, 1.0, 2.0 and 2.5 µM) for 24 h.
Equal protein loading was confirmed by analysis of GAPDH expression.
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due to additional non-bonding interactions with the proximal air and moisture sensitive reactions. All melting points were
amino acid residues of proteins involving the nitro group in measured with a Büchi Melting Point B540 apparatus and the
compound 12. Some representative interaction profiles are values were uncorrected. Thin layer chromatographic (TLC)
shown in the ESI† (Fig. S37–S40). These results support our analyses were carried out on pre-coated silica gel on aluminium
experimental results that serum albumin may help in trans- sheets. The NMR spectra were measured with a Bruker Ascend™
porting these selenocyanates in the bloodstream and signifi- 600 spectrometer (1H NMR at 600 MHz, 13C NMR at 150 MHz
cant binding affinities reflect an effective interaction of com- and 77Se NMR at 114 MHz) or a Varian Mercury Plus 400 MHz
pound 12 with cellular proteins.
NMR spectrometer. Chemical shifts are cited with respect to
Me4Si (1H and 13C) as an internal standard and Me2Se (77Se) as
an external standard. High resolution mass spectra (HR-MS) and
liquid chromatography mass spectra (LC-MS) were obtained
using an Agilent 6520 Accurate-Mass Quadrupole Time-of-Flight
Conclusions
In summary, we describe herein the synthesis of benzylic and (Q-TOF) LC/MS spectrometer. IR spectra were recorded with a
mesitylenic organochalcogenocyanate compounds that have PerkinElmer Spectrum Two FT-IR spectrometer.
one or more selenocyanate or thiocyanate units. To the best of
General procedure 120
our knowledge, this is the first report to describe the synthesis
of compounds having more than two chalcogenocyanate units To a stirred solution of benzylic or mesitylenic halides in dry
in a single molecule. The compounds were evaluated for their acetonitrile was added a solution of potassium selenocyanate
anti-proliferative activities in three different breast cancer cells (KSeCN) in acetonitrile and the mixture was stirred at room
including TNBC cells (MDA-MB-231), for which treatment temperature. The appearance of a white precipitate (inorganic
strategies are limited. This study reveals that selenocyanates salt) soon after the addition of KSeCN indicated the progress
exhibit much higher anti-proliferative activities than thiocya- of the reaction with the formation of selenocyanates. The pro-
nates and the standard anti-cancer drug 5-FU. Furthermore, gress and completion of the reaction was monitored by the
the activity of a simple benzyl selenocyanate could be signifi- thin layer chromatographic (TLC) method.
cantly enhanced either by 4-nitro substitution at the phenyl
ring or by derivatizing to mesitylenic selenocyanate. The
General procedure 221
4-nitro substituted selenocyanate was very much selective To a stirred solution of benzylic or mesitylenic halides in dry
towards TNBC cells over ER+ breast cancer cells and normal methanol was added a solution of potassium thiocyanate
cells. Detailed studies reveal that the apoptotic nature of sele- (KSCN) in methanol and the mixture was stirred under reflux.
nocyanates is associated with modulation of cellular mor- The appearance of a white precipitate (inorganic salt) soon
phology and cell cycle arrest at S-phase. Furthermore, our after the addition of KSCN indicated the progress of the reac-
study also demonstrated the possible anti-invasive activity of tion with the formation of thiocyanates. The progress and
these selenocyanates through the inhibition of cellular completion of the reaction was monitored by the thin layer
migration. However, the active selenocyanates were found to chromatographic (TLC) method.
have weak antioxidant activities. Additionally, an effective
Work-up method 1. The reaction mixture was poured into
binding interaction of active selenocyanate with serum cold distilled water and stirred for 30 minutes. The precipitate
albumin indicates its feasible transport in the bloodstream for was filtered off and washed with water to remove any inorganic
its enhanced anti-cancer properties. Further mechanistic salt. The crude solid was dried and purified by the column
studies with the most active compound demonstrate that sele- chromatographic method.
nocyanates exhibit anti-proliferative activities by down-regulat-
Work-up method 2. The solvent was evaporated under
ing key cellular proteins such as Survivin, Bcl-2 and COX-2, reduced pressure and the residue was dissolved in ethyl
which was further supported by effective protein–ligand inter- acetate and diluted with water. The aqueous layer was
actions as predicted by molecular docking studies. Although extracted with ethyl acetate (3 times). The combined organic
the exact mechanism of action of these selenocyanates could layer was washed with brine and dried over sodium sulfate and
not be highlighted here, results from this study would be filtered off. The solvent was evaporated under reduced
helpful in designing a suitable selenocyanate as an anti-prolif- pressure to afford the crude product. The crude product was
erative and chemopreventive drug candidate in the future.
purified by the column chromatographic method.
Synthesis of compound 1
Prepared following the general method
bromide (0.40 mL, 3.47 mmol) and KSeCN (0.50 g, 3.16 mmol)
in acetonitrile (15 mL). The reaction mixture was stirred at
1 using benzyl
Experimental section
Materials and methods
All commercial reagents were used as delivered, without room temperature for 1 h. Work-up method 1 was used to
further purification. Solvents used for purification (ethyl afford the crude product as an off-white solid. The crude solid
acetate, n-hexane, and petroleum ether 60–80) were freshly dis- was dried and purified by silica gel column chromatography
tilled before use. Dry solvents were used as applicable for the using ethyl acetate and petroleum ether 60–80 as eluents.
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Rf = 0.5 (4% ethyl acetate in petroleum ether 60–80). Yield: Synthesis of compound 12
0.39 g (59%). M.P.: 73–75 °C. 1H NMR (CDCl3, 600 MHz):
Prepared following the general method 1 using 4-nitrobenzyl
bromide (0.42 g, 1.85 mmol) and KSeCN (0.40 g, 2.78 mmol)
in acetonitrile (20 mL). The reaction mixture was stirred at
room temperature for 4 h. Work-up method 1 was used to
afford the crude product as an off-white crystalline solid. The
crude solid was dried and purified by silica gel column chrom-
atography using ethyl acetate and petroleum ether 60–80 as
eluents. Rf = 0.7 (25% ethyl acetate in petroleum ether 60–80).
Yield: 0.39 g (83%). M.P. = 120–123 °C. 1H NMR (CDCl3,
600 MHz): δ (ppm) = 8.25–8.24 (d, J = 6.0 Hz, 2H), 7.55–7.54 (d,
J = 6.0 Hz, 2H), 4.30 (s, 2H); 13C NMR (CDCl3, 150 MHz):
δ (ppm) = 148.1, 143.3, 130.1, 124.6, 100.8, 31.1; 77Se NMR
(CDCl3, 114 MHz): δ (ppm) = 312; IR (KBr pellet, cm−1): 2924
(s), 2146 (m), 1597 (m), 1519 (s), 1342 (s). ESI-MS m/z calcd for
C8H6N2O2Se [M + H]+: 242.9673; obs: 242.2854.
δ (ppm) = 7.39–7.33 (m, 5H), 4.31 (s, 2H); 13C NMR (CDCl3,
150 MHz): δ (ppm) = 135.7, 129.4, 129.2, 128.9, 102.2, 33.0;
77Se NMR (CDCl3, 114 MHz): δ (ppm) = 283. IR (KBr pellet,
cm−1): 2146 (s), 1491 (m), 1454 (m), 1217 (m), 1191 (m), 1069
(m). ESI-MS m/z calcd for C8H7NSe [M + Na]+: 219.9641; obs:
220.0526.
Synthesis of compound 2
Prepared following the general method
2 using benzyl
bromide (0.40 g, 2.34 mmol) and KSCN (0.34 g, 3.51 mmol) in
methanol (5 mL) and the mixture was refluxed for 3 h. Work-
up method 2 was used to afford the crude product as yellow
solid. The crude solid was dried and purified using silica gel
column chromatography using ethyl acetate and petroleum
ether 60–80 as eluents. Rf = 0.7 (10% ethyl acetate in petroleum
ether). Yield: 0.2 g (57%). M.P.: 39–41 °C. 1H NMR (CDCl3,
600 MHz): δ (ppm) = 7.41–7.35 (m, 5H), 4.17 (s, 2H); 13C NMR
(CDCl3, 150 MHz): δ (ppm) = 134.3, 129.1, 129.0, 128.9, 112.0,
38.3; IR (KBr pellet, cm−1): 2991 (m), 2147 (s), 1493 (m).
ESI-MS (LC-MS) m/z calcd for C8H7NS [M + Na]+: 172.02; obs:
172.00.
Synthesis of compound 13
Prepared following the general method 2 using 4-nitrobenzyl
bromide (0.45 g, 2.08 mmol) and KSCN (0.30 g, 3.12 mmol) in
methanol (5 mL). The reaction mixture was refluxed at 65 °C
for 4 h. Work-up method 2 was used to afford the crude
product as an off-white crystalline solid. The crude solid was
dried and purified by neutral alumina column chromato-
graphy using ethyl acetate and petroleum ether 60–80 as
eluents. Rf = 0.3 (10% ethyl acetate in petroleum ether 60–80).
Yield: 0.17 g (43%). M.P. = 82–84 °C. 1H NMR (CDCl3,
600 MHz): δ (ppm) = 8.28–8.27 (d, J = 6.0 Hz, 2H), 7.57–7.56 (d,
J = 6.0 Hz, 2H), 4.20 (s, 2H); 13C NMR (CDCl3, 150 MHz):
δ (ppm) = 148.1, 141.6, 130.0, 124.4, 110.8, 37.0; IR (KBr pellet,
cm−1): 3106 (w), 2152 (m), 1515 (s), 1344 (s); ESI-MS m/z calcd
for C8H6N2O2S [M + NH4]+: 212.0494; obs: 212.0220.
Synthesis of compound 3
Prepared following the general method 1 using 1,4-phenylene-
bis(methylene) bromide (0.40 g, 1.52 mmol) and KSeCN (0.5 g,
3.47 mmol) in acetonitrile (20 mL). The reaction was stirred at
room temperature for 2 h. Work-up method 2 was used to
afford the crude product as a white amorphous solid. The
crude solid was dried and purified by silica gel column chrom-
atography using ethyl acetate and petroleum ether. Rf = 0.5
(36% ethyl acetate in petroleum ether); yield: 0.46 g (95%).
Synthesis of compound 14
1
M.P. = 152–155 °C. H NMR (CDCl3, 600 MHz): δ (ppm) = 7.38
Prepared following the general method 1 using 1,2-phenylene-
bis(methylene) bromide (0.40 g, 1.52 mmol), and KSeCN
(0.50 g, 3.47 mmol) in acetonitrile (15 mL). The reaction was
stirred at room temperature for 2 h. Work-up method 1 was
used to afford the crude product as a white crystalline solid.
The crude solid was dried and purified by neutral alumina
column using ethyl acetate and hexane. Rf = 0.5 (50% ethyl
(s, 4H), 4.28 (s, 4H); 13C NMR (CDCl3, 150 MHz): δ (ppm) =
136.4, 130.0, 101.8, 32.3; 77Se NMR (CDCl3, 114 MHz): δ (ppm) =
293. IR (KBr pellet, cm−1): 2924 (s), 2853 (m), 2147 (s), 1426 (m),
1190 (m), 1092 (s). ESI-MS m/z calcd for C10H8N2Se2 [M + H]+:
316.9096; obs: 316.9086.
1
Synthesis of compound 4
acetate in hexane). Yield: 0.41 g (88%). M.P. = 105–107 °C. H
NMR (DMSO-d6, 600 MHz): δ (ppm) = 7.34–7.31 (m, 4H), 4.43
(s, 4H); 13C NMR (DMSO-d6, 150 MHz): δ (ppm) = 136.9, 131.8,
129.1, 105.2, 30.3; 77Se NMR (DMSO-d6, 114 MHz): δ (ppm) =
314; IR (KBr pellet, cm−1): 2923 (m), 2148 (s), 1451 (m), 1194
(m). ESI-MS m/z calcd for C10H8N2Se2 [M + Na]+: 337.8916; obs:
337.4143.
Prepared following the general method 2 using 1,4-phenylene-
bis(methylene) bromide (0.40 g, 1.52 mmol), and KSCN
(0.37 g, 3.79 mmol) in methanol (5 mL). The reaction was
refluxed at 65 °C for 4 h. Work-up method 2 was used to afford
the crude product as a white solid. The crude solid was dried
and purified by silica gel column chromatography using ethyl
acetate and petroleum ether. Rf = 0.3 (10% ethyl acetate in pet-
roleum ether). Yield: 0.26 g (79%). M.P. = 134–136 °C. 1H NMR
Synthesis of compound 15
(CDCl3, 600 MHz): δ (ppm) = 7.40 (s, 4H), 4.15 (s, 4H); 13C Prepared following the general method 2 using 1,2-phenylene-
NMR (CDCl3, 150 MHz): δ (ppm) = 135.3, 129.8, 111.7, 37.7; IR bis(methylene) bromide (0.40 g, 1.52 mmol), and KSCN
(KBr pellet, cm−1): 2924 (w), 2154 (s), 1426 (m), 1245 (m); (0.37 g, 3.79 mmol) in methanol (5 mL). The reaction was
ESI-MS m/z calcd for C10H8N2S2 [M + Na]+: 243.0027; obs: refluxed at 65 °C for 5 h. Work-up method 2 was used to afford
243.0188.
the crude product as a white solid. The crude solid was dried
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and purified by neutral alumina column using ethyl acetate Synthesis of compound 19
and petroleum ether. Rf = 0.6 (20% ethyl acetate in petroleum
Prepared following the general method 2 using 1,3-phenylene-
bis(methylene) bromide (0.40 g, 1.52 mmol), and KSCN
(0.44 g, 4.55 mmol) in methanol (5 mL). The reaction was
refluxed at 65 °C for 5 h. Work-up method 2 was used to afford
the crude product as a yellow solid. The crude solid was dried
and purified by neutral alumina column using ethyl acetate
and petroleum ether. Rf = 0.6 (30% ethyl acetate in petroleum
1
ether). Yield: 0.26 g (78%). M.P. = 77–79 °C. H NMR (CDCl3,
600 MHz): δ (ppm) = 7.45–7.41 (m, 4H), 4.31 (s, 4H); 13C NMR
(CDCl3, 150 MHz): δ (ppm) = 132.7, 131.7, 130.2, 111.0, 35.1;
IR (KBr pellet, cm−1): 3003 (m), 2152 (s), 1427 (m), 1237 (s);
ESI-MS m/z calcd for C10H8N2S2 [M + NH4]+: 238.0473; obs:
238.0462.
1
ether). Yield: 0.26 g (78%). M.P. = 60–62 °C. H NMR (CDCl3,
600 MHz): δ (ppm) = 7.45–7.37 (m, 4H), 4.16 (s, 4H); 13C NMR
Synthesis of compound 16
Prepared following the general method 1 using 1,4-bis(bromo- (CDCl3, 150 MHz): δ (ppm) = 135.5, 130.0, 129.5, 129.4, 111.6,
methyl)-2,5-dimethylbenzene (0.30 g, 1.03 mmol), and KSeCN 37.8; IR (KBr pellet, cm−1): 2992 (m), 2151 (s), 1429 (m), 1240
(0.32 g, 2.26 mmol) in acetonitrile (10 mL). The reaction was (m); ESI-MS m/z calcd for C10H8N2S2 [M + NH4]+: 238.0473;
stirred at room temperature for 2 h. Work-up method 1 was obs: 238.0469.
used to afford the crude product as a yellow amorphous solid.
Synthesis of compound 20
The crude solid was dried and purified by silica gel column
chromatography using ethyl acetate and petroleum ether. Rf =
0.5 (25% ethyl acetate in hexane). Yield: 0.23 g (65%). M.P. =
Prepared following the general method 1 using 1,3-bis(bromo-
methyl)mesitylene (0.50 g, 1.63 mmol), and KSeCN (0.50 g,
3.47 mmol) in acetonitrile (15 mL). The reaction was stirred at
room temperature for 2 h. Work-up method 2 was used to
afford the crude product as a white amorphous solid. The
crude solid was dried and purified by silica gel column chrom-
atography using ethyl acetate and petroleum ether. Rf = 0.5
(26% ethyl acetate in petroleum ether), yield: 0.52 g (89%).
1
207–209 °C. H NMR (DMSO-d6, 600 MHz): δ (ppm) = 7.13 (s,
2H), 4.32 (s, 4H), 2.29 (s, 6H); 13C NMR (DMSO-d6, 150 MHz):
δ (ppm) = 136.1, 134.6, 133.0, 105.2, 31.1, 19.1; 77Se NMR
(DMSO-d6, 114 MHz): δ (ppm) = 292; IR (KBr pellet, cm−1):
2953 (m), 2922 (s), 2854 (m), 2147 (s), 1447 (m). ESI-MS m/z
calcd for C12H12N2Se2 [M + NH4]+: 361.9675; obs: 361.9665.
1
M.P. = 154–157 °C; H NMR (CDCl3, 600 MHz): δ (ppm) = 6.95
(s, 1H), 4.48 (s, 4H), 2.47 (s, 3H), 2.40 (s, 6H); 13C NMR (CHCl3,
150 MHz): δ (ppm) = 138.6, 137.2, 131.6, 129.9, 101.7, 29.0,
20.3, 16.1; 77Se NMR (DMSO-d6, 114 MHz): δ (ppm) = 252; IR
(KBr pellet, cm−1): 2916 (w), 2148 (s), 1458 (m), 1190 (s);
ESI-MS m/z calcd for C13H14N2Se2 [M + NH4]+: 375.9831; obs:
375.9830.
Synthesis of compound 17
Prepared following the general method 2 using 1,4-bis(bromo-
methyl)-2,5-dimethylbenzene (0.40 g, 1.37 mmol), and KSCN
(0.33 g, 3.42 mmol) in methanol (5 mL). The reaction was
refluxed at 65 °C for 5 h. Work-up method 2 was used to afford
the crude product as a white solid. The crude solid was dried
and purified by silica gel column chromatography using ethyl
acetate and petroleum ether. Rf = 0.5 (20% ethyl acetate in pet-
roleum ether). Yield: 0.17 g (49%). M.P. = 194–196 °C. 1H NMR
(CDCl3, 400 MHz): δ (ppm) = 7.14 (s, 2H), 4.16 (s, 4H), 2.38 (s,
6H); 13C NMR (DMSO-d6, 150 MHz): δ (ppm) = 134.3, 133.9,
132.4, 112.6, 34.8, 18.1, IR (KBr pellet, cm−1): 2923 (m), 2151
(s), 1432 (w); ESI-MS m/z calcd for C12H12N2S2 [M + NH4]+:
266.0786; obs: 266.0838.
Synthesis of compound 21
Prepared following the general method 2 using 1,3-bis(bromo-
methyl)mesitylene (0.40 g, 1.31 mmol), and KSCN (0.38 g,
3.92 mmol) in methanol (5 mL). The reaction was refluxed at
65 °C for 5 h. Work-up method 2 was used to afford the crude
product as a white solid. The crude solid was dried and puri-
fied by silica gel column chromatography using ethyl acetate
and petroleum ether. Rf = 0.8 (30% ethyl acetate in petroleum
ether). Yield: 0.11 g (32%). M.P. = 107–109 °C. 1H NMR (CDCl3,
600 MHz): δ (ppm) = 6.98 (s, 1H), 4.37 (s, 4H), 2.48 (s, 3H),
2.41 (s, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) = 138.8,
137.4, 131.4, 129.0, 111.8, 33.6, 20.0, 15.8; IR (KBr pellet,
cm−1): 2926 (w), 2147 (s), 1380 (w); ESI-MS m/z calcd for
C13H14N2S2 [M + Na]+: 285.0496; obs: 285.0485.
Synthesis of compound 18
Prepared following the general method 1 using 1,3-phenylene-
bis(methylene) bromide (0.40 g, 1.52 mmol), and KSeCN
(0.50 g, 3.47 mmol) in acetonitrile (20 mL). The reaction was
stirred at room temperature for 2 h. Work-up method 2 was
used to afford the crude product as a pale yellow solid. The
crude solid was dried and purified by silica gel column chrom-
atography using ethyl acetate and petroleum ether. Rf = 0.4
Synthesis of compound 22
(32% ethyl acetate in petroleum ether). Yield: 0.46 g (97%). Prepared following the general method 1 using 1,3,5-tris
M.P. = 108–110 °C. 1H NMR (CDCl3, 600 MHz): δ (ppm) = (bromomethyl) benzene (0.2 g, 0.56 mmol), and KSeCN (0.26 g,
7.39–7.33 (m, 4H), 4.27 (s, 4H); 13C NMR (CDCl3, 150 MHz): 1.8 mmol) in acetonitrile (7 mL). The reaction was stirred at
δ (ppm) = 136.9, 130.2, 129.6, 129.5, 101.9, 32.3; 77Se NMR room temperature for 2 h. Work-up method 2 was used to
(CDCl3, 114 MHz): δ (ppm) = 295; IR (KBr pellet, cm−1): 2923 afford the crude product as an off-white crystalline solid. The
(m), 2150 (s), 1200 (m). ESI-MS m/z calcd for C10H8N2Se2 crude solid was dried and purified by silica gel column chrom-
[M + Na]+: 337.0937; obs: 337.1142.
atography using ethyl acetate and hexane. Rf = 0.6 (50% ethyl
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1
acetate in hexane). Yield: 0.13 g (53%). M.P. = 158–160 °C. H KSeCN (0.15 g, 0.97 mmol) in acetonitrile (10 mL). The reac-
NMR (DMSO-d6, 600 MHz): δ (ppm) = 7.30 (s, 3H), 4.29 (s, 6H); tion was stirred at room temperature for 7 h. Work-up method
13C NMR (DMSO-d6, 150 MHz): δ (ppm) = 139.7, 129.4, 105.5, 1 was used to afford the crude product as a white solid, which
32.9; 77Se NMR (DMSO-d6, 114 MHz): δ (ppm) = 315; IR (KBr was insoluble in water and non-polar organic solvents such as
pellet, cm−1): 2925 (m), 2151 (s), 1455 (m), 1430 (m), 1196 (m). dichloromethane, ethyl acetate, methanol and acetonitrile.
ESI-MS m/z calcd for C12H9N3Se3 [M + Na]+: 455.0897; obs: The product could not be eluted on a silica gel TLC plate.
455.2540.
Yield: 0.47 g (93%). M.P. = 224–227 °C. 1H NMR (DMSO-d6,
600 MHz): δ (ppm) = 4.69 (s, 12H); 13C NMR (DMSO-d6,
150 MHz): δ (ppm) = 137.6, 104.4, 27.9; 77Se NMR (DMSO-d6,
Synthesis of compound 23
Prepared following the general method 2 using 1,3,5-tris 114 MHz): δ (ppm) = 305; IR (KBr pellet, cm−1): 2924 (m), 2162
(bromomethyl)benzene (0.20 g, 0.70 mmol), and KSCN (0.22 g, (s), 2148 (s), 1493 (m), 1169 (s); ESI-MS m/z calcd for
2.8 mmol) in methanol (3 mL). The reaction was refluxed at C18H12N6Se6 [M + H]+: 792.6193; obs: 793.3620.
65 °C for 5 h. Work-up method 2 was used to afford the crude
Synthesis of compound 27
product as a white solid. The crude solid was dried and puri-
fied by silica gel column chromatography using ethyl acetate To
a stirred solution of 1,2,3,4,5,6-hexakis(bromomethyl)
and petroleum ether. Rf = 0.4 (30% ethyl acetate in petroleum benzene (0.15 g, 0.24 mmol) in dry MeOH was added a solu-
ether). Yield: 71 mg (44%). M.P. = 112–114 °C. 1H NMR tion of KSCN (0.15 g, 1.5 mmol) in dry MeOH (6 mL) and the
(CDCl3, 400 MHz): δ (ppm) = 7.39 (s, 3H), 4.17 (s, 6H); 13C solution was refluxed for 7 h at 65 °C. The progress of the reac-
NMR (CDCl3, 150 MHz): δ (ppm) = 136.8, 129.9, 111.3, 37.4; IR tion was monitored by the thin layer chromatographic (TLC)
(KBr pellet, cm−1): 2989 (w), 2156 (s), 1431 (m); ESI-MS m/z method. After completion, the solvent was evaporated under
calcd for C12H9N3S3 [M + NH4]+: 309.0302; obs: 309.3262.
reduced pressure. The residue was stirred with water to dis-
solve the inorganic salts, followed by filtration and subsequent
drying under reduced pressure to yield the crude product. Rf =
Synthesis of compound 24
Prepared following the general method 1 using 1,3,5-tris 0.6 (65% ethyl acetate in petroleum ether). Yield: 70 mg (59%).
1
(bromomethyl)mesitylene (0.40 g, 1.00 mmol), and KSeCN M.P. = 190–192 °C. H NMR (DMSO-d6, 600 MHz): δ (ppm) =
(0.58 g, 4.03 mmol) in acetonitrile (15 mL). The reaction was 4.81 (s, 12H); 13C NMR (DMSO-d6, 150 MHz): δ (ppm) = 137.1,
stirred at room temperature for 2 h. Work-up method 2 was 112.4, 31.7; IR (KBr pellet, cm−1): 2924 (w), 2161 (s), 2147 (s),
used to afford the crude product as a white amorphous solid. 1190 (m); ESI-MS m/z calcd for C18H12N6S6 [M + NH4]+:
The crude solid was dried and purified by silica gel column 521.9791; obs: 521.9907.
chromatography using ethyl acetate and petroleum ether. Rf =
Cell culture
0.5 (40% ethyl acetate in petroleum ether). Yield: 0.42 g (88%).
1
M.P. = 160–163 °C. H NMR (DMSO-d6, 600 MHz): δ (ppm) = The TNBC cell line MDA-MB-231 was a kind gift from Dr Anil
4.52 (s, 6H), 2.41 (s, 9H); 13C NMR (DMSO-d6, 150 MHz): Mukund Limaye, Associate Professor, Department of
δ (ppm) = 138.0, 132.6, 104.3, 29.9, 17.4; 77Se NMR (DMSO-d6, Biosciences and Bioengineering, Indian Institute of
114 MHz): δ (ppm) = 251; IR (KBr pellet, cm−1): 2923 (s), 2853 Technology Guwahati (IIT Guwahati), Assam, India. RPMI
(m), 2148 (m), 1730 (m), 1461 (m), 1191 (m); ESI-MS m/z calcd 1640 medium (Gibco) added with 10% fetal bovine serum
for C15H15N3Se3 [M + Na]+: 497.1695; obs: 497.8654.
(FBS) (Gibco) and 1% Pen-Strep (Gibco) was used as a nutrient
supplement for the MDA-MB-231 cells. T-47D cells were pro-
cured from Dr VGM Naidu, Associate Professor, Department of
Synthesis of compound 25
Prepared following the general method 2 using 1,3,5-tris Pharmacology
&
Toxicology,
National
Institute
of
(bromomethyl)mesitylene (0.50 g, 1.25 mmol), and KSCN Pharmaceutical Education and Research (NIPER) Guwahati.
(0.49 g, 5.01 mmol) in methanol (6 mL). The reaction was MCF-7 and L-132 cells were purchased from National Centre
refluxed at 65 °C for 4 h. Work-up method 2 was used to afford for Cell Science (NCCS), Pune, India. The T-47D, MCF-7 and
the crude product as a white solid. The crude solid was dried L-132 cells were cultured in DMEM (Gibco) supplemented with
and purified by silica gel column chromatography using ethyl 10% fetal bovine serum (FBS) (Gibco) and 1% Pen-Strep (Cell
acetate and petroleum ether. Rf = 0.4 (20% ethyl acetate in pet- Clone). All the cells were maintained in a CO2-regulated
roleum ether). Yield: 0.36 g (86%). M.P. = 146–148 °C. 1H NMR humidified (95% air/5% CO2 atmosphere) incubator at 37 °C.
(CDCl3, 400 MHz): δ (ppm) = 4.40 (s, 6H), 2.53 (s, 9H); 13C
MTT assay
NMR (CDCl3, 150 MHz): δ (ppm) = 138.7, 130.2, 111.3, 50.9,
33.9, 16.7; IR (KBr pellet, cm−1): 2957 (w), 2154 (s), 2141 (s), The cells were plated in 96-well culture plates at a density of
1443 (m), 1232 (s); ESI-MS m/z calcd for C15H15N3S3 [M + Na]+: 2 × 103 cells per 100 µl per well for L-132 cells and 4 × 103 cells
356.0326; obs: 356.0300.
per 100 µl per well for all other cells and treated with 0.0, 1.0,
5.0, 10.0 and 25.0 µM of test compounds for 0 h (Set 1) and
72 h (Set 2). At the end of the treatment period, 10.0 µL of
Synthesis of compound 26
Prepared following the general method 1 using 1,2,3,4,5,6- 5.0 mg mL−1 of MTT was added to the plate (Set 1) and incu-
hexakis(bromomethyl)benzene (0.10 g, 0.16 mmol), and bated for 2 h. Following the 2 h incubation, the culture
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Paper
medium from the plate was removed and the purple formazan Western blot analysis
crystals were dissolved using 100 µl of DMSO (Merck Life
MDA-MB-231 cells were treated with 0.0, 0.2, 0.5, 1.0, 2.0 and
2.5 µM of compound 12 for 24 h and the whole cell protein
lysate was made using lysis buffer (20 µM HEPES buffer, 1 M
NaCl, 0.5 M EDTA, 1 mg mL−1 leupeptin, 100 mM PMSF,
5 mg mL−1 aprotinin, 1 M DTT and 0.1% (v/v) Triton-X100).
Total protein concentration was estimated with the Bradford
protein assay; 20 µg of protein lysate was loaded and separated on
a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel by electro-
phoresis and transferred to a nitrocellulose membrane. Protein
transfer was confirmed by Ponceau-S (HiMedia) staining. The blots
were blocked with 5% non-fat dry milk or BSA in 1X TBST buffer
for 2 h at room temperature and incubated with an appropriate
dilution of the primary antibodies specific for COX-2, Survivin, Bcl-
2, VEGF-A, Akt, p-Akt and GAPDH (Cell Signaling Technology) over-
night at 4 °C. Following the primary antibody incubation, the blots
were washed and incubated with HRP-conjugated secondary anti-
bodies (Abcam) for 2 h and developed with an Optiblot ECL Detect
Kit (Abcam) and ChemiDoc™ XRS System (Bio-Rad). The house-
keeping gene GAPDH was used as a loading control.
Science Pvt Ltd) and the absorbance at 570 nm was measured
using a microplate reader (TECAN Infinite 200 PRO multimode
reader). In Set 2, a similar MTT treatment protocol was followed
only after 72 h. The mean OD values of a 0 h plate (Set 1) were
subtracted from the mean OD values of identical wells at a 72 h
plate (Set 2) ΔOD and the inhibition of proliferation was calcu-
lated keeping the ΔOD of the untreated control as 100%.
Propidium iodide (PI) flow cytometric assay
5 × 104 cells per 2 mL per well were plated in 6-well plates and
treated with 0.0, 10.0 and 25.0 µM of selected selenocyanates
(1, 3, 18, 20 and 24) and 0.0, 5.0 and 10.0 µM of compound 12
for 72 h. At the end of 72 h, the cells were harvested and
washed with PBS (1X), stained with 5.0 µl of 1.0 mg mL−1 pro-
pidium iodide (PI) (Sigma-Aldrich), a nucleic acid intercalating
fluorescent dye, and analysed using a BD FACSCalibur™
instrument (BD Biosciences).
Cell cycle analysis
1 × 105 cells per 2 mL per well were seeded in 6-well plates and
treated with 0.0, 1.0 and 5.0 µM of compounds 12 and 24 and
incubated for 24 h. Following the 24 h drug treatment, the
cells were collected; washed with PBS (1X); fixed with ice-cold
70% ethanol at −20 °C for 30 min; again washed with PBS (1X)
and stained with PI/RNase staining buffer (BD Biosciences) for
10 min at room temperature. The prepared samples were then
Docking studies
AutoDock 4.2 MGL Tools and Lamarckian Generic Algorithm
(LGA) were used for protein-fixed ligand-flexible docking calcu-
lations.33 Auto grid version 4.2 was used to calculate the grid para-
meters and the grid size was set at 40 × 40 × 40 points with a grid
spacing of 0.375 Å, 0.519 Å, 0.375 Å, and 0.5916 Å for Bcl-2, BSA,
COX-2 and Survivin, respectively. Twenty search attempts (i.e.,
ga_run parameter) were performed for each ligand. The maximum
number of energy evaluations before the termination of the LGA
run was 250 000 and the maximum number of generations of LGA
run before termination was 27 000. Other docking parameters
were set to the default values of AutoDock 4.2 program.
analysed using
Biosciences).
a
BD FACSCalibur™ instrument (BD
Wound healing (scratch) assay
MDA-MB-231 cells were seeded in a 24-well plate at a concen-
tration of 1.5 × 105 cells per 2 mL per well and incubated in a
CO2 incubator at 37 °C to form a monolayer of cells. After the
monolayer formation, the cells were pre-incubated in serum
free medium for 6 h. Using a 100 µl tip, a scratch was made on
the monolayer and the debris was removed by washing with
PBS (1X). Then, the cells were re-incubated with serum free
medium and treated with 0.0, 1.0 and 2.5 µM of active seleno-
cyanates 12 and 24. Images of the scratch were captured at
regular time intervals (0, 24, 48 and 72 h) using a Nikon
inverted microscope and a camera. The extent of wound
healing denotes the anti-migratory effect of the compounds
tested.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This project was supported by the Science and Engineering
Research Board (SERB), India. KPB acknowledges SERB for the
award of a DST-INSPIRE Faculty Fellowship (Ref.: IFA12-CH-68)
and IIT Guwahati for a Start-Up Research Grant. We thank the
Central Instruments Facility (CIF), IIT Guwahati for NMR and
the DST-FIST for ESI-MS facilities. KB, GP, DB and SS thank
IIT Guwahati for their research fellowships.
Assessment of cellular morphology and apoptotic bodies
5 × 104 cells per 1 mL per well were plated in 24-well plates
and treated with 0.0 and 10.0 µM of compounds 12 and 24.
The cells were observed for any morphological changes and
the change in morphology was captured at a regular time inter-
vals (0, 24 and 48 h) using a Nikon inverted microscope and a
camera. After 72 h, the formation of apoptotic nuclei was ana-
lysed by staining the cells with 5 µl of 1 mg mL−1 PI and
images were taken using an Eclipse Ti-S inverted fluorescence
microscope.
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