Cationic lipid-conjugated dexamethasone as a selective antitumor agent

Article abstract of DOI:10.1016/j.ejmech.2014.06.051

Dexamethasone (Dex) is one of the highly potent synthetic glucocorticoids. It exhibits prominent anti-inflammatory but moderate anti-proliferative activities. It is widely used along side chemotherapy to alleviate toxic side effects. Additionally, Dex is also a potent inducer of gluconeogenesis. However, its overuse critically desensitizes cells against chemotherapy. Herein, we report on the development of a new class of cationic lipid-Dex conjugates in which the C-8 carbon chain analogue (DX8) exhibited glucocorticoid receptor (GR)-mediated, caspase-3-assisted, cancer cell-selective anti-proliferative activity. Melanoma tumors in DX8-treated mice exhibited significantly reduced tumor aggressiveness with respect to tumors in Dex-treated mice. Tumor lysates prepared from DX8-treated group showed elevated levels of p53. DX8-treated cancer cells showed clear degradation of kinase JAK3/STAT3 protein levels. Additionally, DX8-treatment decreased the level of VEGFR2 in tumor-endothelial cells implying DX8's anti-proliferative roles in both tumor cells and tumor neovascular cells. Collectively, our results demonstrate potent anti-angiogenic, and selective JAK3/STAT3 down-regulating anticancer characteristics of DX8, a new dexamethasone-based antitumor molecule.

Full text of DOI:10.1016/j.ejmech.2014.06.051

European Journal of Medicinal Chemistry 83 (2014) 433e447
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cationic lipid-conjugated dexamethasone as a selective antitumor
agent
, b, *
Samaresh Sau a, Rajkumar Banerjee a
a Biomaterials Group, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Tarnaka, Uppal Road, Hyderabad, Andhra Pradesh 500007, India
b Academy of Scientific and Innovative Research (AcSIR), 2 Rafi Marg, New Delhi 110 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Dexamethasone (Dex) is one of the highly potent synthetic glucocorticoids. It exhibits prominent anti-
inflammatory but moderate anti-proliferative activities. It is widely used along side chemotherapy to
alleviate toxic side effects. Additionally, Dex is also a potent inducer of gluconeogenesis. However, its
overuse critically desensitizes cells against chemotherapy. Herein, we report on the development of a
new class of cationic lipideDex conjugates in which the C-8 carbon chain analogue (DX8) exhibited
glucocorticoid receptor (GR)-mediated, caspase-3-assisted, cancer cell-selective anti-proliferative activ-
ity. Melanoma tumors in DX8-treated mice exhibited significantly reduced tumor aggressiveness with
respect to tumors in Dex-treated mice. Tumor lysates prepared from DX8-treated group showed elevated
levels of p53. DX8-treated cancer cells showed clear degradation of kinase JAK3/STAT3 protein levels.
Additionally, DX8-treatment decreased the level of VEGFR2 in tumor-endothelial cells implying DX8's
anti-proliferative roles in both tumor cells and tumor neovascular cells. Collectively, our results
demonstrate potent anti-angiogenic, and selective JAK3/STAT3 down-regulating anticancer characteris-
tics of DX8, a new dexamethasone-based antitumor molecule.
Received 19 March 2014
Received in revised form
23 June 2014
Accepted 24 June 2014
Available online 25 June 2014
Keywords:
Dexamethasone
Cationic lipid
Glucocorticoid receptor
Apoptosis
Tumor
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
(popularly known as glucocorticoid response elements, GREs), i.e.,
with promoter sequences of target genes, resulting in their tran-
Glucocorticoid receptors (GR) are nuclear hormone receptors
ubiquitously expressed in both cancer and non-cancer cells. GRs
play many crucial roles in various cellular mechanisms. These
include cellular energy metabolism involving non-carbohydrate
precursors for glucose homeostasis, regulation of protein and fat
metabolism and various anti-inflammatory and immunosuppres-
sive responses [1,2]. With structural elucidation of ligand-binding
domain (LBD) of GR an upsurge of studies ensued to develop se-
lective GR-ligands. These studies led to better understanding of the
conformation of ligandereceptor complexes and thereby the fate &
functions of GR [3,4]. These also helped in optimizing ligands' anti-
inflammatory activities with reduced side-effect profile.
scriptional activation [5]. For GR-targeting and transactivation, we
chose to use the synthetic GC ligand Dex instead of natural ligand
cortisol due to its superior activity in transactivating glucocorticoid
responsive genes than cortisol. Additionally, Dex exhibits effective
binding with even mutated LBD [6].
Dexamethasone (Dex) is a widely used synthetic GC for inducing
apoptotic cell deaths in malignant lymphoid and in other cancer
cells [7,8]. In general, these GCs are used in patients during and
post-chemotherapeutic treatment of solid tumors to control
dehydration, nausea, acute toxicity, edema, and pain towards pro-
tecting normal tissues of cancer patients against long-term effects
of genotoxic drugs [9]. Dex also exhibits contrasting pharmaco-
logical properties. In one hand, Dex exhibits anticancer activity [8].
One of the possible anticancer mechanisms elucidated so far in-
volves the regulation of STAT kinases [10]. On the other hand, Dex
induces insensitivity in cancer cells against anticancer drugs [11,12].
So the restricted and carefully regulated use of Dex is needed for
effective anticancer treatments.
Classically, glucocorticoids (GC) exert their function by binding
to the intracellular GR. Upon ligand-binding GR translocates to the
nucleus. Therein, it interacts with specific DNA sequences
* Corresponding author. Biomaterials Group, CSIR-Indian Institute of Chemical
Technology (CSIR-IICT), Tarnaka, Uppal Road, Hyderabad 500007, India.
E-mail addresses: banerjee@iict.res.in, rkbanerjee@yahoo.com, rkbanerjee.iict@
gmail.com (R. Banerjee).
Previously, we showed that a Dex-associated cationic liposomal
gene delivery system could selectively transfect cancerous cells and
tumor in vivo via GR [13]. This selectivity was observed in spite of
http://dx.doi.org/10.1016/j.ejmech.2014.06.051
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
GR's ubiquitous presence in both cancer and non-cancer cells. In
this liposomal system Dex was associated as a co-lipid with other
lipids. We questioned: if, instead of mere association as a co-lipid in
a liposome, Dex is conjugated with a cationic lipid will it be able to
exhibit selective anticancer effect? To this end, we undertook
chemical conjugation of ‘cationic, twin carbon chain-length lipid’
with Dex. Lipids with cationic head-groups have inherent affinity
toward negatively charged cell membranes resulting in enhanced
interaction with trans-membrane proteins [14]. Thus, cationic
lipid-conjugated Dex is expected to have more availability and local
concentration inside the cells than free Dex. We found that this
cationic Dex derivative could maintain uncompromised GR-
transactivation property of Dex.
Recently we reported that similar cationic lipid-conjugation to
estrogen, the natural ligand for estrogen receptor (ER), converted
the estrogen molecule into a very potent anti-breast cancer agent.
The molecule acts through induction of apoptosis and autophagy in
breast cancer cells [15]. The cationic lipid-conjugation also con-
verted haloperidol into a potent anticancer agent [14]. Subse-
quently, others have also shown the pharmacologic benefit of
cationic lipid conjugation to benzamide [16]. These prior studies
clearly exemplify that cationic lipid conjugation (or ‘lipidation’) of
known pharmacophores holds promise toward developing new
class of drug molecules. On the basis of these recent observations
(Scheme 1A), we sought to develop newer cancer targeted mole-
cules through conjugation of cationic lipid to GR ligand, Dex. We
hypothesized that the modified Dex-molecule will resurrect pro-
grammed cell death machinery against aggressive cancer. Herein
we report on the development of a new kind of cationic Dex-
derivatives. We show that the lead molecule DX8 exhibited GR-
mediated, effective tumor growth inhibition presumably through
down-regulation of JAK-STAT pathway with concomitant up-
regulation of p53.
2. Results and discussion
2.1. Chemistry
2.1.1. Syntheses of Dex-derivatives
In the present study, we chemically conjugated cationic twin-
carbon chain lipid with Dex. Since different Dex derivatives
differed by respective lengths of carbon chain linked to quaternary
Nitrogen atom, a general procedure was used to synthesize them
(Scheme 1B). At first the amino group of tert-butyl N-(2-
aminoethyl)carbamate was reacted with alkyl bromides of
different carbon chain lengths to obtain respective tertiary amines
(1aee). This was followed by N-deprotection to give free 10 amines
(2ae2e). Separately, Dex was oxidized using NaIO4 to produce free
carboxylic acid functionality-containing compound 3, which con-
tains one less carbon than the Dex [17]. The compound 3 was
coupled to free 10 amine linked to dialkyl amine moiety (2ae2e)
using EDCI/HOBT/DMAP, to produce amide products (4ae4e). The
resulting tertiary amine upon quaternization with methyl iodide
followed by chloride ion exchange, afforded pure target lipids
(5ae5e) [DXn, where n ¼ 2, 4, 8, 12, and 16 numbers of carbon-
containing long chains] (Scheme 1A, B). All Derivatives were
characterized using mass and NMR spectrometry (S1eS43). The
purities of the respective compounds were determined and
confirmed by HPLC (S44eS48).
2.2. Biology
2.2.1. Screening of Dex-derivatives for selective cytotoxicity
For screening the anticancer property of the newly synthesized
Dex-derivatives we chose cancer cell lines (B16F10, MCF-7, and
A549) and non-cancer cells or cells of primary origin (CHO,
HEK293T and mouse skin fibroblast, FB). Both cancer and non-
Scheme 1. A: Chemical structures of cationic lipid conjugated anticancer agents, dexamethasone (Dex) and newly synthesized cationic Dex-derivatives. B: syntheses of dexa-
methasone derivatives.

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
435
Scheme 1. (continued).
cancer cells were treated continuously for 72 h with varying molar
concentrations of Dex-derivatives and Dex. Clearly, among the five
synthesized Dex-derivatives, DX8 induced maximum toxicity to
tested cancer cells (Table 1). The toxicity profiles of DX2, DX4, DX12
and DX16 were more or less comparable to those of free Dex.
Keeping 15 mM as threshold IC50 for lead screening, we found DX8
to be the most potent anticancer compound among all other de-
rivatives. On the basis of this screening result we undertook
mechanistic studies to decipher the cellular and molecular role of
DX8 and finally assayed its therapeutic efficiency in tumor model.
2.2.2. DX8 is more toxic to cancer cells than normal cells
GRs are ubiquitously expressed in all type of cancerous and non-
cancerous cells. So, it was necessary to assess DX8's selectivity (if
any) in killing cancer cells. We also compared DX8's cellular effect
to that of parent molecule Dex. On the basis of data represented in
Table 1 we further demonstrate the cellular effect of DX8 and Dex
against cancer cells (B16F10, MCF-7, and A549) and non-cancer
cells (CHO, HEK293T and mouse skin fibroblast, FB) (Fig. 1a).
Clearly, DX8 exhibited significantly higher toxicity than free Dex in
cancer cells. But, DX8-induced toxicity was insignificant against
non-cancer cells. Notably, unconjugated cationic C8-lipid compo-
nent (i.e., cationic C8-side chain) exhibited some but significantly
lesser cytotoxicity against various cancer cells than that exhibited
by DX8 (data not shown) [We have also demonstrated this limited
cellular toxicity effect of C8-lipid component independently in
Ref. [14]]. Overall, these findings demonstrate DX8's selective
cytotoxicity in cancer cells thereby validating the need to conjugate
cationic lipid moiety to Dex.
Table 1
Anticancer activities (IC50, mM) of Dex and cationic Dex-derivatives in cancer cells
(B16F10, MCF-7 and A549) and non-cancer cells (CHO, HEK293T, mouse skin
fibroblast cells FB).
Compound IC50
(m
M) B16F10
MCF7
A549
CHO HEK293T FB
Dex
>20
>20
>20
>15
>15
>15
>15
>15
>15
>20 >20
>15 >20
>20 >15
>15
DX2
DX4
DX8
DX12
DX16
>20
>20
>15
>15
>15
6.0 0.68 6.5 0.42 7.2 0.54 >15 >15
>15
>15
13.5 0.46 14.0 0.7 >20 >15
>15 14.8 0.53 >15 >20

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S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
Fig. 1. Cytotoxic and apoptosis inducing effect on cancer and non-cancer cells. (a) Viability studies of B16F10, MCF7, A549 (cancer cells) and CHO, HEK293T, mouse skin fibroblast or
FB (non-cancer cells) continuously treated with Dex and DX8 for 72 h. The asterisks (**) denote p < 0.001 for DX8 with respect Dex. (b) Flow cytometric analysis of apoptosis in
B16F10, MCF-7, CHO and HEK293T cells co-stained with FITC-annexin V/propidium iodide (PI). The horizontal and vertical axes represent FITC-annexin V and PI staining,
respectively. Cells were either untreated or treated with 10 mM Dex, DX8 for continuously 48 h.
2.2.3. DX8 induces apoptosis in cancer cells but not in normal cells
Apoptosis inducing effect of the DX8 was determined by con-
ventional propidium iodide (PI)/annexin V binding assay. The initial
events of apoptosis indicate loss of plasma membrane integrity.
Thereafter, phosphatidylserine (PS) translocates from the inner to
outer membrane leaflets, thereby exposing itself to external
environment. Annexin V with high affinity for PS binds to cells
efficiently with externally exposed PS. Thus, more the positive
staining of fluorescently labeled annexin V more is the loss of cell
membrane polarity. This precedes complete loss of membrane
integrity accompanying the later stage of cell death resulting from
either apoptosis or necrosis. On the contrary, PI can only enter into

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
437
cells after loss of membrane integrity. Dual stain with annexin V
and PI thus allows obvious discrimination among unaffected cells
(annexin V negative, PI negative, lower left quadrant), early
apoptotic cells (annexin V positive, PI negative, lower right quad-
rant), late apoptotic cells (annexin V positive, PI positive, upper
right quadrant), and necrotic cells (annexin V negative, PI positive,
upper left quadrant). As indicated, for early apoptotic cells an
elevated amount of annexin V stained cells is expected in lower
right quadrant. To observe selective induction of cellular apoptosis
(if any), we chose four cell lines B16F10, MCF-7 (all cancer cells) and
CHO, HEK293T (non-cancer cells). Fig.1b shows that DX8 treatment
shown). RU-486 is a known GR-antagonist. Notably, the decrease
in DX8-induced toxicity in GR-depleted cells was significant. This
data clearly suggests that the DX8-induced toxicity was GR-
mediated. In other words, DX8-mediated toxicity in cancer cells
was GR-dependent.
2.2.5. Tumor growth inhibition & anti-angiogenic properties of DX8
DX8 was the lead molecule among all the derivatives. Hence,
we wanted to see its in vivo therapeutic effect in a tumor model.
For an in vivo tumor regression study we used highly aggressive
B16F10-based murine melanoma model in C57BL6/J black mice.
Moreover, in this model, the aggressive tumor develops high levels
of angiogenic blood vessels and hence any treatment-related effect
on angiogenesis can be additionally documented. By flow cyto-
metric analyses we showed that DX8 induces prominent, early
signs of apoptosis in B16F10 cells. Hence, DX8 was administered in
mice to estimate its tumor regression efficacy and its tumor
regression property was compared to that of Dex. After the
completion of the experimentation, tumors from individual
groups were cryosectioned for imaging apoptotic signs (if any)
using green fluorescent TUNEL (Terminal deoxynucleotidyl trans-
ferase dUTP nick end labeling) assay kit and using VE-cadherin
antibody (red fluorescence) as angiogenic blood vessels. Fig. 3a
shows that DX8-treatment significantly inhibited tumor growth,
especially with respect to tumor growths in Dex-treated mice and
in untreated (UT) groups. Fig. 3b exhibits the images of repre-
sentative tumors excised from sacrificed mice from individual
groups at the completion of treatment. Fig. 3c indicates the images
of apoptotic regions (green fluorescence) and blood endothelial
cell-associated sites (red fluorescence) obtained from tumor sec-
tions of different treatment groups. Merged images of sections are
also shown. Here, ‘yellow’ areas (merging of green and red areas)
denote sites where vascular endothelial cells underwent
apoptosis. Evidently, tumor sections from DX8-treated group
contain more apoptotic regions than in tumor sections from the
(10
cells (with annexin V positive and PI negative staining, lower right
quadrant) in only cancer. Contrastingly, Dex (10 M, for 48 h)
mM, for 48 h) increased the percentage of early apoptotic
m
induced such effect neither in cancer nor in non-cancer cells.
Therefore, this is obvious that DX8 at the given condition of treat-
ment selectively induced early signs of apoptosis in cancer cells, but
not in non-cancer cells.
2.2.4. DX8 induces GR-mediated toxicity in cancer cells
For this study, we used siRNA-based silencing strategy to knock
down effective GR concentration in B16F10 and A549 cancer cells.
In this experiment, we pre-transfected cells separately with siRNA
against GR (GR-siRNA), and with control universally scrambled
siRNA (Sc-siRNA). Group, wherein cells were not treated with
siRNA was used as untreated (UT) control. Cells were treated with
DX8 and DX8-induced toxicities in different groups were
compared. Flow cytometric analyses of B16F10 (Fig. 2a) and A549
(Fig. 2c) cells clearly show that there were lesser levels of GR in
GR-siRNA treated cells than those in Sc-siRNA treated or UT cells.
Cellular viability trend reveals that DX8-mediated toxicity in GR-
depleted (i.e., GR-siRNA pre-transfected) B16F10 (Fig. 2b) and
A549 (Fig. 2d) cells were significantly lower than that in Sc-siRNA-
treated or UT cells. Additionally, we also observed reversal of DX8-
mediated toxicity in RU 486-pretreated B16F10 cells (data not
Fig. 2. Effect of GR down-regulation in DX8-mediated cytotoxicity in cancer cells. Flow cytometric analysis of GR down-regulation in (a) B16F10 and (c) A549 cell lines. Black line,
dashed line and striped area represent, GR levels in GR-siRNA treated, negative control siRNA treated, and siRNA untreated cells respectively. Viability test in (b) B16F10 and (d)
A549 cells and correspondingly the dark gray ( ), light gray ( ) and black (-) bars represent siRNA-untreated (UT), scrambled control siRNA-treated, GR-siRNA treated cells. **
denotes p < 0.001 for GR-siRNA treated vs. control siRNA-treated groups.

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S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
Fig. 3. In vivo effect of DX8. (a) Tumor regression curve after subcutaneous implantation of B16F10 cells in C57BL6/J mice followed by intraperitoneal injection of Dex, DX8 or kept
untreated (UT). The days of injections are indicated by black arrows. ** denotes p < 0.05 for DX8 treatment with respect to Dex treatment. (b) Representative tumors from sacrificed
mice on day 24th; (I) tumor from untreated group (UT), (II) tumor from Dex-treated group, (III) tumor from DX8-treated group. (c) Microscopic pictures of 10 mm tumor sections
from UT (upper panel), Dex (middle panel) and DX8 treated group (lower panel). Left column indicates tissue architecture in bright field (BF), next from left column shows apoptotic
regions in TUNEL assay (green fluorescent), second from right column exhibits endothelial region stained with VE-cadherin (red fluorescent) and extreme right panel exhibits
merged green and red fluorescent images of middle columns respectively. All the images are taken at 10ꢀ magnification. (d) Western blot analysis of tumor lysates isolated from UT,
Dex and DX8-treated mice group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Dex or UT groups. Clearly, DX8 could induce significant apoptosis
in cultured cancer cells and also, as depicted here, in highly
aggressive melanoma tumor tissues. Illustratively, tumor sections
of DX8-treated group exhibit considerable amount of apoptosis in
cells prevailing in tumor microenvironment. This apoptotic effect
could have resulted in significant inhibition of tumor growth.
Using VE-cadherin staining for blood vessels in tumor sections, we
found that tumor sections of DX8-treated group exhibit very high
levels of apoptosis in vascular endothelial cells also (when
compared to the yellow areas in merged panels). These data
together indicate that the significant tumor-regression in DX8-
treated group could be due to simultaneous apoptosis in both
vascular endothelial cells as well as in tumor cells of tumor mass.
For DX8-treated group, we have provided two sets of tumor sec-
tion panels representing two different areas with different levels
of vascular cells in tumor sections.
p53 protein is a key regulator of apoptosis mediated cell death,
DNA repair and senescence. The inactivation of p53 increases the
threat of cancer. The mechanism of p53 inactivation is however a
complex process. Typically, inactivation of p53 occurs because of
activation of Mdm2 (i.e., preferable conversion to p-Mdm2), which
negatively regulates p53 stability and regulation [18]. Fig. 3d ex-
hibits the relative expression of Mdm2, p-Mdm2, and p53 proteins
in tumor lysates from different treatment groups. Importantly,
significant up-regulation of p53 and down-regulation of p-Mdm2
in tumor lysates from DX8-treated group were observed. These
indicate that DX8-treatment could have affected inhibition of
Mdm2 phosphorylation thereby impeding sequestration of p53-
activation in aggressive tumor mass.
2.2.6. DX8 down-regulates VEGFR2 expression in tumor-associated
angiogenic cells
Tyrosine kinase VEGFR2 is one of the important factors that
have been implicated in tumor angiogenesis and aggression.
Following VEGF-binding it activates and regulates endothelial
blood vessel formation, survival and activation of downstream
kinases, such as Akt [19]. From Fig. 3aeb it is clear that DX8, in
comparison to Dex, was more potent in inducing tumor growth
inhibition presumably due to enhanced apoptosis in tumor-
associated endothelial cells. We hypothesized that reduction in
tumor aggression was linked to down-regulation of the pro-
angiogenic factor, VEGFR2. To visualize this effect, we co-
stained VEGFR2 (with green fluorescent secondary antibody)
and endothelial cell junction protein, VE-cadherin (with red
fluorescent secondary antibody) in tumor sections. The ratio of
VEGFR2 expression to that of stained endothelial cells was min-
imum in tumor sections of DX8-treated group. The ratio was
comparatively higher in tumor sections of Dex-treated and UT
mice (Fig. 4). This comparative loss of potent receptor VEGFR2 on
angiogenic vessels should be expected to eventually reduce
angiogenesis in tumors of DX8-treated group. The reason for this
incidental reduction in VEGFR2 expression level in DX8-treated
group is currently not known. However, we believe that the

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
439
Fig. 4. Effect of DX8 on VEGFR2. VEGFR2 down-regulation in tumor section either from Dex (middle panel), DX8 (lower panel) treated mice or UT (upper panel). From left first
column respectively indicates the tissue architecture in bright field (BF), second column represents VEGFR2 staining (green fluorescent), third panel represents endothelial region
stained with VE-cadherin (red fluorescent) and extreme right as merged images of middle panels. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
effective reduction in VEGFR2 was eventually linked to signifi-
cant reduction in tumor aggressiveness.
cleavage of caspase 3 to form activated caspase 3 were also
observed in DX8-treated cells. Additionally, selective up-
regulation of caspase 7 was also observed in DX8-treated cells.
These sequential events possibly triggered the pathway of
apoptosis, which led to a) high level of cytotoxicity in B16F10
cells (as observed in Fig. 1a), b) increased population of early
apoptotic cells as shown in Annexin V/PI experiment (Fig. 1b and
c) apoptotic lesions in tumor sections as shown in TUNEL data
(Fig. 3c).
The ‘X-linked inhibitors of apoptosis’ (XIAP) inhibits and helps
proteolytic degradation of effector caspase-3. This process is how-
ever antagonized by Smac [22]. Fig. 5b shows that DX8 released
mitochondria derived activator (Smac/Diablo) and inhibited the
expression of XIAP, which possibly led to stabilization of activated
caspase-3. It seems, DX8-induced apoptosis in cancer cells was
causally linked to caspase-induced apoptotic pathway. This was
sequentially revealed by increase in the ratio of BAX/Bcl-2, which
led to the release of cytochrome c. These we believe prominently
increased fragmented (or activated) caspase 3. The stabilization of
fragmented caspase-3 is also related to down-regulation of anti-
apoptotic factor, XIAP and up-regulation of pro-apoptotic factor,
Smac/Diablo [23]. Thus, the increased levels of pro-apoptotic factor
Smac/Diablo and decreased levels of anti-apoptotic factor XIAP
were possibly responsible for DX8-induced apoptosis in B16F10
cells.
2.2.7. DX8 triggers apoptosis in B16F10 cells
The B16F10 cell-based murine melanoma model was chosen
for in vivo study (Fig. 3). Hence, the mechanism of DX8-mediated
toxicity was determined in B16F10 cells. B16F10 cells were either
treated with DX8, Dex or kept untreated (UT) for 48 h. This was
done to elucidate how DX8 could have affected various cancer-
implicated factors in these cells before inducing prominent cell
killing after 72 h. During apoptosis, mitochondria releases
cytochrome-c in cytosol. Eventually, this activates caspase family
of proteases. Activation of caspase 9 by cytochrome c cleaves
procaspase 3 to release effector caspase i.e., activated caspase 3
[20]. The release of caspase 3 leads to induction of apoptosis in
cells. However, Bcl-2 family of proteins regulate the mitochon-
drial release of cytochrome c [21]. A member of this family, such
as BAX (Bcl-2-associated X protein) causes the release of cyto-
chrome c and thus initiate apoptosis. On the other hand anti-
apoptotic members of this family, such as Bcl-2 bind to the
pro-apoptotic BAX protein and inhibit release of cytochrome c. So
a higher value of BAX/Bcl-2 ratio indicates the onset of apoptosis
in cells. As seen in Fig. 5a, the ratio of BAX/Bcl-2 expression in
DX8-treated cells was higher than in Dex-treated or in UT cells.
An elevated expression of caspase 9 and elevated proteolytic

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S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
Fig. 5. Differential expression of different regulators of apoptosis and proliferation in compound-treated B16F10 cells. Cells were either treated with Dex (10
or kept untreated (UT) for 48 h (a) Western blots of apoptosis related proteins such as, BAX, Bcl2, Active Caspase 3, Caspase 3, cytochrome c, Caspase 9 and 7 and loading control
actin are presented. The densitometric ratio of BAX and Bcl2 is determined using ‘Image J’ software. (b) Similarly, respective levels of expression of XIAP and Smac-Diablo with
respect to loading control -actin were estimated.
mM) and DX8 (10 mM)
b-
b
2.2.8. Role of Akt and activation of p53 through JAK/STAT3 pathway
Akt has leading role in tumorigenesis. It crucially acts among
numerous signaling cascades involved in the regulation of cellular
apoptosis and proliferation [24,25]. Further, Tyrosine-kinases such
as, janus kinases (JAK) are also involved in activation of Akt through
JAK/STAT pathway in cell survival machinery [26]. Pro-apoptotic
factor p53 is a potent inhibitor of cell growth. It regulates cell cy-
cle progression at several check-points and induces apoptosis to
control cell proliferation and cell division [27,28]. In tumors from
DX8-treated group, p53 up-regulation was observed. Here we tried
to understand whether the induction of apoptosis at a cellular level
was due to reactivation of p53. P53-up regulation occurs because of
downregulation of p-Mdm2. Phosphorylation of Akt leads to
nucleo-cytoplasmic localization of vital substrates implicated in
cell cycle and apoptosis. In presence of growth factor, p-Akt phos-
phorylates Mdm2, which then enters nucleus. Nuclear trans-
location of p-Mdm2 leads to the formation of Mdm2ep53
complexes, which allows phosphorylation of p53 and its relocation
to the cytoplasm for subsequent ubiquitination and degradation
[18,28]. The oncoprotein Mdm2 and the tumor suppressor p53 are
part of this auto-regulatory feedback. As we can see from Fig. 6a, in
DX8 treated B16F10 cells JAK3/STAT3 kinases were significantly
down-regulated which naturally resulted in the reduction of p-
STAT3 levels. This DX8-mediated STAT3 down-regulation possibly
antagonized p-Akt formation/stabilization; this eventually led to
Akt's destruction (Fig. 6b). We believe Akt's destruction eventually
inhibited phosphorylation of Mdm2 (shown in tumor mass,
Fig. 3d), resulting in its possibly restricted nuclear interaction with
p53. Hence, level of p53 was up-regulated in DX8-treated cancer
cells (Fig. 6c).
anticancer agent while possibly overshadowing its desensitizing
effects. This study has hence established that cationic lipid modi-
fication of Dex clearly afforded a potent anticancer agent.
3. Conclusion
We have modified Dex with cationic lipid and developed a
highly selective anticancer agent called DX8, which exhibited
therapeutic anticancer potency significantly better than the parent
molecule, Dex. Clearly, DX8 showed its pharmacological property
preferentially through modulation of JAK3/STAT3 pathway. This
data shows that cationic lipid-conjugation to Dex could make a
novel class of anticancer agent. Further studies are in progress to-
ward elucidating any drug sensitizing effect of this molecule to
cancer cells, which we believe will be an added advantage while
developing this new class of anticancer agent. We are also looking
into whether or not DX8 exhibits synergism with inhibitors acting
downstream of JAK/STAT pathway. To conclude, the strategy
adopted herein is expected to find future application in enhancing
pharmacological properties and anticancer activities of existing
glucocorticoids.
4. Experimental section
4.1. Chemicals and general procedures for characterizations
Dexamethasone (Dex), trypsin, EDTA, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazoliumbromide (MTT), propidium iodide (PI),
FITC-labeled annexin V, dimethyl sulfoxide (DMSO), Dulbecco's
Modified Eagle Medium (DMEM) and RPMI 1640 were purchased
from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS)
was purchased from Lonza, Swiss chemicals. RU486 purchased
from Calbiochem, Merck, USA. Si-RNAs were purchased from Santa
Cruz biotechnology, USA. The solvents, salts and common reagents
for synthesis were purchased from either Aldrich (Milwaukee, WI,
USA) or S. D. Fine Chem (Mumbai, India). All other chemicals, re-
agents were purchased either from Sigma (St. Louis, MO, USA) or
from Rankem Ltd. (Mumbai, India). They were used without further
purification. Lipofectamine 2000 was obtained from Invitrogen
Corporation (Carlsbad, CA). All other materials for Western blotting
The data as obtained by the treatment of DX8 to cancer cells and
in tumor model revealed that the lipid-modification enhances or
value-adds the existing, known pharmacologic effects of GR ligand,
Dex. The well-known GR ligand Dex reduces NF-kB-mediated anti-
proliferation or inflammatory syndromes. It is co-treated to cancer
patients during chemotherapy. This is done to alleviate various
inflammatory side effects of drug treatment. However, it is also now
clear that its widespread use leads to desensitization of target cells
during drug-based medical treatment [11,12]. Hence, it was a
challenge to chemically modify Dex to make it more potent

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
441
Fig. 6. Western blot analysis of different signaling kinases in compound-treated B16F10 cells. Cells were either treated with 10
(a) Western blots of kinases such as, JAK2, JAK3, STAT3, p-STAT3 (Tyr 705), p-STAT3 (ser 727) and
AKT 1 and p-AKT (ser 473) and the ratio of both with respect to -actin are determined. (c) Flow cytometric analysis of p53 up-regulation in B16F10 cell line treated after 48 h of
treatment. Black line, striped area, and gray area represent levels of p53 in cells from UT, Dex and DX8-treated cells respectively.
mM of Dex and DX8 or kept untreated (UT) for 48 h.
b-actin are presented. Ratios of JAK3/b-actin and STAT3/b-actin are mentioned (b)
b
were purchased from local company. The final molecules were
characterized by 1H NMR and ESI-HRMS spectral analyses and the
purity was ascertained by HPLC. 1H NMR spectra were recorded on
Varian FT 200 MHz or Bruker FT 300 and 400 MHz spectrometer.
13C NMR was obtained in Bruker FT 75 or 125 MHz. ESI Mass spectra
were obtained by Micromass Quattro LC mass spectrometer. Purity
of final products was determined in Varian ProStar HPLC at 210 nm
at a flow rate of 1 mL/min in Varian microsorb 100-10 BDS column
(4.6 mm ꢀ 250 mm); Mobile Phase: For DX2 (Methanol), for DX4
(Acetonitrile), DX8 (Methanol), DX12 (95% MethanoleWater sys-
tem), DX16 (95% MethanoleWater system); Flow Rate: 1 mL/min;
Column Pressure: 55e60 kg/cm2; Temperature: 25 ꢁC; Wavelength:
210 nm.
[m, 6H, eN(eCH2eCH3)2, eCH2eCH2eNHeCOeOeC(CH3)3],
3.10e3.17 [m, 2H, eCH2eCH2eNHeCOeOeC(CH3)3].
ESIeMS: m/z 217 (Calculated value for C11H24N2O2 ¼ 216.3).
4.1.1.2. Synthesis of tert-butyl N-[2-(dibutylamino)ethyl]carbamate,
compound 1b. tert-Butyl-N-[2-(dibutylamino)ethyl]carbamate, 1b
was synthesized following a similar procedure as described for 1a
synthesis above. Here 1-bromobutane was used. (Compound 1b:
1.11 g, 65.2% yield, Rf ¼ 0.55 in 30% ethyl acetateehexane, v/v).
1H NMR (300 MHz, CDCl3):
d
/ppm
¼
0.83e0.89 [t,
J ¼ 7.9 Hz, 6H, eN(eCH2eCH2eCH2eCH3)2], 1.17e1.28 [m, 4H,
eN(eCH2eCH2eCH2eCH3)2], 1.37e1.45 [m, 13H, eNH
eCOeOeC(CH3)3, eN(eCH2eCH2eCH2eCH3)2], 2.35e2.42 [t, J ¼
6.9 Hz, 4H,eN(eCH2eCH2eCH2eCH3)2], 2.45e2.51 [t, J ¼ 6.9 Hz,
2H, eCH2eCH2eNHeCOeOeC(CH3)3], 3.03e3.31 [m, 2H,
eCH2eCH2eNHeCOeOeC(CH3)3], 5.03 [s, 1H].
4.1.1. Synthesis of tert-butyl N-[2-dialkylamino)ethyl] carbamate,
compounds 1aee
The general syntheses of compounds 1aee are accomplished by
conventional chemical synthesis procedure as described below.
ESIeMS: m/z 273 (Calculated value for C15H32N2O2 ¼ 272.4).
4.1.1.3. Synthesis of tert-butyl N-[2-(dioctylamino)ethyl]carbamate,
compound 1c. tert-Butyl-N-[2-(dioctylamino)ethyl]carbamate, 1c
was synthesized following a similar procedure as described for 1a
synthesis. Here 1-bromooctane was used. (Compound 1c: 1.70 g,
70.0% yield, Rf ¼ 0.6 in 30% ethyl acetateehexane, v/v).
4.1.1.1. Synthesis of tert-butyl N-[2-(diethylamino)ethyl]carbamate,
compound
1a. tert-Butyl-N-(2-aminoethyl)carbamate
(1
g,
6.25 mmol), potassium carbonate (3.45 g, 25 mmol) and 1-
bromoethane (1.92 g, 15.62 mmol) were added in 15 mL dry ethyl
acetate in a 50 mL round-bottom flask fitted with reflux condenser.
The resulting mixture was refluxed over an oil bath at 70e80 ꢁC for
48 h. Then the mixture was cooled, excess ethyl acetate was
removed by rotary evaporation and 50 mL chloroform was added
into this. The whole mixture was transferred to a 500 mL separating
funnel and mixture was washed with water (3 ꢀ 100 mL) followed
by brine (3 ꢀ 100 mL), dried with anhydrous sodium sulfate
(Na2SO4) and evaporated. The resulting solution was purified by
column chromatography using 60e120 mesh silica gel and 7% ethyl
acetateehexane (v/v) as eluent. This yielded compound 1a as a
yellowish liquid (0.74 g, 55% yield, Rf ¼ 0.5 in 30% ethyl aceta-
teehexane, v/v).
1H NMR (400 MHz, CDCl3):
d/ppm ¼ 0.82e0.92 [t, J ¼ 7.7 Hz,
6H,
eN(eCH2eCH2e(CH2)5eCH3)2], 1.11e1.33 [m, 20H,
eN(eCH2eCH2e(CH2)5eCH3)2], 1.43e1.50 [m, 13H, eNH
eCOeOeC(CH3)3, eN(eCH2eCH2e(CH2)5eCH3)2], 2.34e2.43 [t,
J ¼ 6.9 Hz, 4H, eN(eCH2eCH2e(CH2)5eCH3)2], 2.46e2.52 [t,
J ¼ 5.8 Hz, 2H,eCH2eCH2eNHeCOeOeC(CH3)3], 3.07e3.17 [m, 2H,
eCH2eCH2eNHeCOeOeC(CH3)3], 7.32 [s, 1H, CDCl3].
ESIeMS: m/z 385 (Calculated value for C23H48N2O2 ¼ 384.6).
4.1.1.4. Synthesis of tert-butyl N-[2-(didodecylamino) ethyl]carba-
mate, compound 1d.
tert-Butyl-N-[2-(didodecylamino)ethyl]carbamate, 1d was synthe-
sized following a similar procedure as described for 1a synthesis.
1H NMR (400 MHz, CDCl3):
eN(eCH2eCH3)2], 1.36 [s, 9H, eNHeCOeOeC(CH3)3], 2.46e2.56
d/ppm ¼ 0.94e1.01 [t, J ¼ 7.3 Hz, 6H,

442
S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
Here 1-bromododecane was used. (Compound 1d: 2.09 g, 67.0%
yield, Rf ¼ 0.7 in 30% Ethyl acetateehexane, v/v).
4.1.2.5. Synthesis of [2-(dihexadecylamino)ethyl]ammonium tri-
fluoroacetate, compound 2e. Compound 2e was synthesized
following a similar procedure as described for 2a synthesis (0.260 g,
92.1% yield, Rf ¼ 0.5 active in ninhydrin charring).
1H NMR (300 MHz, CDCl3):
d
/ppm
¼
0.83e0.89 [t,
J ¼ 7.1 Hz, 6H, eN(eCH2eCH2e(CH2)9eCH3)2], 1.17e1.34 [m, 36H,
eN(eCH2eCH2e(CH2)9eCH3)2], 1.42e1.48 [m, 13H, eNHeCO
eOeC(CH3)3, eN(eCH2eCH2e(CH2)9eCH3)2], 2.39e2.46 [t,
J ¼ 6.4 Hz, 4H, eN(eCH2eCH2e(CH2)9eCH3)2], 2.51e2.55 [t,
J ¼ 6.0 Hz, 2H, eCH2eCH2eNHeCOeOeC(CH3)3], 2.87e2.90 [m,
2H, eCH2eCH2eNHeCOeOeC(CH3)3].
ESIeMS: m/z ¼ 510 (Calculated value for C34H73N2 ¼ 509.9).
4.1.3. Synthesis of compound 3
Dexamethasone (0.47 g, 1.19 mmol) was dissolved with ethanol
(93.5 mL), then water (37 mL) was added, followed by addition of
sodium periodate (0.305 g, 1.42 mmol) and 2 M sulfuric acid
(2.4 mL) in a 250 mL round-bottom flask. The resulting solution was
left stirred at room temperature for 12 h. Then excess ethanol was
removed in vacuo and water (36 mL), brine (19 mL) were added to
the reaction mixture. The pH of the solution was adjusted to 12
with 1 M aqueous sodium hydroxide to dissolve the product in
solution. The mixture was washed with DCM (3 ꢀ 100 mL) and then
pH of the solution was adjusted to 3 with 1 M sodium bisulfate
solution and finally extracted using ethyl acetate (2 ꢀ 100 mL), 1:1
DCM:ethyl acetate (2 ꢀ 100 mL). These organic extracts were
combined and dried with anhydrous sodium sulfate followed by
concentration in vacuo to get white compound 3 [17] (0.4 g, 97.0%
yield, Rf ¼ 0.6 in 10%, methanolechloroform v/v).
ESIeMS: m/z 497 (Calculated value for C31H64N2O2 ¼ 496.8).
4.1.1.5. Synthesis of tert-butyl N-[2-(dihexadecylamino)ethyl]carba-
mate, compound 1e. tert-Butyl-N-[2-(dihexadecylamino) ethyl]
carbamate, 1e was synthesized following a similar procedure as
described for 1a synthesis. Here 1-bromohexadecane was used.
(Compound 1e: 2.31 g, 60.0% yield, Rf ¼ 0.75e0.8 in 30% Ethyl
acetateehexane, v/v).
1H NMR (400 MHz, CDCl3):
6H, eN(eCH2eCH2e(CH2)13eCH3)2], 1.07e1.35 [m,
d
/ppm ¼ 0.81e0.93 [t, J ¼ 6.9 Hz,
52H,
eN(eCH2eCH2e(CH2)13eCH3)2], 1.43e1.47 [m, 13H, eNHeCO
eOeC(CH3)3, eN(eCH2eCH2e(CH2)13eCH3)2], 2.33e2.44 [t,
J ¼ 6.4 Hz, 4H, eN(eCH2eCH2e(CH2)13eCH3)2], 2.46e2.55 [t,
J ¼ 5.8 Hz, 2H, eCH2eCH2eNHeCOeOeC(CH3)3], 3.06e3.22 [m, 2H,
eCH2eCH2eNHeCOeOeC(CH3)3], 7.32 [s, 1H, CDCl3].
1H NMR (300 MHz, CDCl3):
20H], 1.11 [s, 3H, C18H], 1.20e1.30 [m, 2H, C15H], 1.33e1.54 [m, 2H,
C7H], 1.56 [s, 3H, C19H], 1.63e1.93 [m, 2H, C12H], 2.0e2.22 [m, 1H,
14H], 2.26e2.50 [m, 2H, C6H], 2.50e2.76 [m, 1H, C8H], 2.92e3.07
d
/ppm ¼ 0.92e0.97 [d, J ¼ 7.1 Hz, 3H,
C
ESIeMS: m/z 610 (Calculated value for C39H80N2O2 ¼ 609.0).
C
4.1.2. Synthesis of tert[2-(dialkylamino)ethyl]ammonium
trifluoroacetate (2aee)
General procedure for Boc-deprotection was followed as
described below.
[m,1H, C16H], 3.3e3.38 [m,1H, C11H], 6.08 [s,1H, C4H], 6.21e6.33 [d,
10.1 Hz, 1H, C2H], 7.27e7.39 [d, J ¼ 10 Hz, 1H, C1H],
ESIeMS: m/z 379, 401 [MNaþ] (Calculated value for
C21H27O5F ¼ 378.43).
4.1.2.1. Synthesis of tert[2-(diethylamino)ethyl]ammonium tri-
fluoroacetate, compound 2a. Compound 1a (0.124 g, 0.57 mmol)
was dissolved in 4 mL of dry DCM in a 25 mL round-bottom flask
and stirred over an ice bath for 15 min. Then 2 mL of trifluoroacetic
acid (TFA) was added drop-wise to the solution and stirring was
continued for another 4 h over an ice bath. Then the resulting
mixture was diluted with chloroform. Excess TFA was removed by
washing with saturated sodium bicarbonate (NaHCO3) solution
(2 ꢀ 50 mL). The organic layer was dried with anhydrous sodium
sulfate (Na2SO4) and evaporation of the organic layer afforded
compound 2a as a reddish gummy liquid (0.062 g, 93.9% yield,
Rf ¼ 0.1, active in ninhydrin charring). As compound 2a was ob-
tained with 95% purity (revealed by TLC), it was directly used for
the next step.
4.1.4. Syntheses of compounds 4aee
Syntheses of compounds 4aee followed a general chemistry
procedure as described previously [29].
4.1.4.1. Synthesis of compound 4a. Compound 3 (0.2 g, 0.53 mmol)
was dissolved in 10 mL of dry DCM in a 25 mL round-bottomed
flask and stirred over an ice bath for 15 min. To this, a mixture of
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (0.101 g,
0.53 mmol) and Hydroxybenzotriazole (HOBT) (0.080 g,
0.52 mmol) were added and stirring was continued for another
30 min. Then compound 2a (0.067 g, 0.57 mmol) was dissolved in
2 mL of dry DCM and added to the reaction mixture. DIPEA was
added drop-wise to the ice cold reaction mixture (under stirring
condition) until it became slightly basic. The resulting solution was
left stirred for 18 h. The reaction mixture was dissolved in 30 mL
chloroform and washed sequentially with water (3 ꢀ 30 mL), 1 N
HCl (2 ꢀ 30 mL), saturated NaHCO3 solution (3 ꢀ 30 mL) and brine
(3 ꢀ 30 mL). The organic layer was dried with anhydrous sodium
sulfate, filtered and the solvent from the filtrate was removed by
rotary evaporation. Then mixture was purified by column chro-
ESIeMS: m/z ¼ 116 (Calculated value for C6H16N2 ¼ 116.0).
4.1.2.2. Synthesis
of
[2-(dibutylamino)ethyl]ammonium
tri-
fluoroacetate, compound 2b. Compound 2b was synthesized
following a similar procedure as described for 2a synthesis above
(0.144 g, 91.1% yield, Rf ¼ 0.1, active in ninhydrin charring).
ESIeMS: m/z ¼ 173 (Calculated value for C10H25N2 ¼ 173.0).
matography
using
>200
mesh
silica
gel
and
3%
4.1.2.3. Synthesis
of
[2-(dioctylamino)ethyl]ammonium
tri-
methanolechloroform (v/v) as eluent. This yielded compound 4a as
a yellow gummy product (0.09 g, 35.8% yield, Rf ¼ 0.40 in 10%
methanolechloroform (v/v).
fluoroacetate, compound 2c. Compound 2c was synthesized
following a similar procedure as described for 2a synthesis (0.271 g,
73.8% yield, Rf ¼ 0.25, active in ninhydrin charring).
1H NMR (300 MHz, CDCl3):
d/ppm ¼ 0.55e0.67 [m, 9H,
ESIeMS: m/z ¼ 285 (Calculated value for C18H41N2 ¼ 285.0).
eN(eCH2eCH3)2, C20H], 1.02 [s, 3H, C18H], 1.30 [s, 3H, C19H],
1.41e1.78 [m, 4H, C7H, C15H], 1.79e2.44 [m, 6H, C6H, C8H, C12H,
4.1.2.4. Synthesis of [2-(didodecylamino)ethyl]ammonium tri-
fluoroacetate, compound 2d. Compound 2d was synthesized
following a similar procedure as described for 2a synthesis (0.264 g,
91.0% yield, Rf ¼ 0.4 active in ninhydrin charring).
C14H], 2.48e2.64 [m, 4H, eN(eCH2eCH3)2], 2.66e2.82 [m, 2H,
eOCeHNeCH2eCH2], 2.95e3.21[m, 2H, C11H, C16H], 3.28e3.5 [m,
2H, eOCeHNeCH2eCH2], 5.97 [s, 1H, C4H], 6.01e6.18 [d, J ¼ 11 Hz,
1H, C2H], 6.99e7.07 [d, J ¼ 10 Hz, 1H, C1H].
ESIeMS: m/z ¼ 398 (Calculated value for C26H57N2 ¼ 397.7).
ESIeMS: m/z 477 (Calculated value for C27H41N2O4F ¼ 476.6).

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
443
4.1.4.2. Synthesis of compound 4b. Compound 3 (0.2 g, 0.53 mmol)
was dissolved in 8 mL of dry DCM and 2 mL dry DMF in a 25 mL
round-bottom flask and stirred over an ice bath for 15 min. To this, a
mixture of EDCI (0.108 g, 0.63 mmol) and HOBT (0.08 g, 0.52 mmol)
was added and stirring was continued for another 30 min. Then,
compound 2b (0.067 g, 0.57 mmol) was dissolved in 2 mL of dry
DCM and added to the reaction mixture. DIPEA was added drop-
wise to the ice cold reaction mixture (under stirring condition)
until it became slightly basic. The resulting mixture was stirred for
20 h. Then work up was done following a similar procedure as used
for compound 4a. The mixture was purified by column chroma-
tography using 60e120 mesh silica gel and 2.2%
methanolechloroform (v/v) as eluent. This yielded compound 4b as
a yellowish product (0.2 g, 70.0% yield, Rf ¼ 0.5 in 10% meth-
anolechloroform (v/v).
60e120 mesh silica gel and 1% methanolechloroform (v/v) as
eluent. This yielded compound 4d as a yellow gummy product
(0.3 g, 58.8% yield, Rf ¼ 0.6 in 10% methanolechloroform (v/v).
1H NMR (300 MHz, CDCl3):
d
/ppm ¼ 0.75e0.90 [m, 9H,
eN(eCH2eCH2e(CH2)9eCH3)2,
1.11e1.29 [m, 41H, eN(eCH2eCH2e(CH2)9eCH3)2, C7H,
C
20H], 1.02 [s, 3H,
C
C
18H],
19H],
1.37e1.98 [m, 10H, eN(eCH2eCH2e(CH2)9eCH3)2, C8H, C12H, C14H,
15H], 2.07e2.91 [m, 9H, N(eCH2eCH2e(CH2)9eCH3)2,
C
eOCeHNeCH2eCH2, C6H, C16H], 3.20e3.33 [t, J ¼ 6.6 Hz, 2H,
eOCeHNeCH2eCH2], 3.38e3.56 [m, 1H, C11H], 6.00 [s, 1H, C4H],
6.16e6.26 [d, J ¼ 7.9 Hz, 1H, C2H], 7.12e7.33 [d, J ¼ 7.9 Hz, 1H, C1H].
ESIeMS: m/z 757 (Calculated value for C47H81N2O4F ¼ 757.1).
4.1.4.5. Synthesis of compound 4e. Compound 3 (0.2 g, 0.53 mmol)
was dissolved in 10 mL of dry DCM in a 25 mL round-bottomed
flask and mixture was stirred over an ice bath for 15 min. To this,
a mixture of EDCI (0.101 g, 0.52 mmol) and HOBT (0.079 g,
0.52 mmol) was added and stirring was continued for another
30 min. Then compound 2e (0.215 g, 0.42 mmol) was dissolved in
2 mL of dry DCM and added to the reaction mixture. DIPEA was
added drop-wise to the ice cold reaction mixture (under stirring
condition) until it became slightly basic. The resulting mixture was
stirred for 48 h. Then work up was done following a similar pro-
cedure as used for compound 4a. The reaction mixture was evap-
orated and purified by column chromatography using 60e120
mesh silica gel and 0.9% methanolechloroform (v/v) as eluent. This
yielded compound 4e as a gummy product (0.19 g, 41.3% yield,
Rf ¼ 0.65 in 10% methanolechloroform (v/v).
1H NMR (300 MHz, CDCl3):
[m, 9H, eN(eCH2e(CH2)2eCH3)2, C20H], 1.05 [s, 3H, C18H], 1.26-
d/ppm
¼
0.81e1.00
1.46
[m,
9H,
eN(eCH2eCH2eCH2eCH3)2,
C7H,
14H,
C
C
19H],
15H],
1.47e1.92 [m, 7H,eN(eCH2eCH2eCH2eCH3)2,
C
2.05e2.74 [m, 6H, C6H, C8H, C12H, C16H], 2.78e2.90 [t, J ¼ 7.0
Hz, 4H, eN(eCH2eCH2eCH2eCH3)2], 2.94e3.19 [m, 2H,
eOCeHNeCH2eCH2], 3.22e3.42 [m, 2H, eOCeHNeCH2eCH2],
3.82e4.06 [m, 1H, C11H], 6.04 [s, 1H, C4H], 6.24e6.30 [d, J ¼ 10 Hz,
1H, C2H], 7.27 [s, 1H, C1H].
ESIeMS: m/z 533 (Calculated value for C31H49N2O4F ¼ 532.7).
4.1.4.3. Synthesis of compound 4c. Compound 3 (0.3 g, 0.8 mmol)
was dissolved in 12 mL dry dimethyl formamide (DMF) and 5 mL
dimethyl sulfoxide (DMSO) in a 25 mL round-bottom flask and the
turbid like mixture was stirred over an ice bath for 15 min. To this, a
mixture of EDCI (0.271 g, 0.95 mmol) and HOBT (0.151 g,
0.78 mmol) was added, and stirring was continued for another
30 min. Then, compound 2c (0.113 g, 0.390 mmol) was dissolved in
2 mL of dry DCM and added to the reaction mixture. DIPEA was
added drop-wise to the ice cold reaction mixture (under stirring
condition) until it became slightly basic. The resulting mixture was
stirred for overnight at room temperature. The work up was done
following a similar procedure as used for compound 4a. The
mixture was purified by column chromatography using 60e120
mesh silica gel and 2.0% methanolechloroform (v/v) as eluent. This
yielded compound 4c as a whitish product (0.2 g, 39.1% yield,
Rf ¼ 0.55 in 10% methanolechloroform (v/v).
1H NMR (300 MHz, CDCl3):
9H, eN(eCH2eCH2e(CH2)13eCH3)2,
d
/ppm
¼
0.75e0.94 [m,
C20H], 0.99 [s, 3H, C18H],
1.04e1.30 [m, 57H, eN(eCH2eCH2e(CH2)13eCH3)2, C7H, C19H],
1.34e1.83 [m, 10H, eN(eCH2eCH2e(CH2)13eCH3)2, C8H, C12H, C14H,
C15H,], 1.91e2.89 [m, 9H, eN(eCH2eCH2e(CH2)13eCH3)2,
eOCeHNeCH2eCH2,C6H, C16H], 2.9e3.10 [t, J ¼ 6.4 Hz, 2H,
eOCeHNeCH2eCH2], 3.15e3.44 [m, 1H, C11H], 5.99 [s, 1H, C4H],
6.11e6.29 [d, J ¼ 9.9 Hz, 1H, C2H], 7.15e7.37[d, J ¼ 9.9 Hz, 1H, C1H].
ESIeMS: m/z 870 (Calculated value for C56H97N2O4F ¼ 869.3).
4.1.5. Syntheses of compounds 5aee
Syntheses of compounds 5aee followed a general protocol as
exemplified below.
1H NMR (300 MHz, CDCl3):
eN(eCH2eCH2e(CH2)4eCH2eCH3)2,
1.08e1.31 [m, 21H, eN(eCH2eCH2e(CH2)4eCH2eCH3)2, C7H, C19H],
1.33e1.86 [m, 11H, eN(eCH2eCH2e(CH2)4eCH2eCH3)2, 14H,
15H], 1.91e2.76 [m, 10H, eN(eCH2eCH2e(CH2)4eCH2eCH3)2, C6H,
d
/ppm ¼ 0.68e0.93 [m, 9H,
4.1.5.1. Synthesis of compound 5a. Compound 4a (0.1 g, 0.21 mmol)
was dissolved in 8 mL methyl iodide (MeI), then potassium car-
bonate (K2CO3) (0.115 g, 0.82 mmol) was added to the mixture in a
15 mL round-bottomed flask. The reaction mixture was stirred for
12 h at room temperature. Then it was filtered, concentrated and
the residue was purified by column chromatography (using
60e120 mesh silica gel and 6e7% methanolechloroform (v/v) as
eluent), followed by chloride ion exchange chromatography (using
Amberlite IRA-400Cl resin and methanol as eluent). This yielded
compound 5a as a colorless gummy solid (0.32 g, 31.0% yield,
Rf ¼ 0.2 in 10% methanolechloroform, v/v).
C
20H], 1.02 [s, 3H, 18H],
C
C
C
C8H, C12H, C16H], 2.91e3.04 [t, J ¼ 6.9 Hz, 2H, eOCeHNeCH2eCH2],
3.10e3.30 [m, 2H, eOCeHNeCH2eCH2], 3.34e3.59 [m, 1H, C11H],
5.99 [s, 1H, C4H], 6.15e6.23 [d, J ¼ 10.1 Hz, 1H, C2H], 7.18e7.26 [d,
J ¼ 10.1 Hz, 1H, C1H].
ESIeMS: m/z 645 (Calculated value for C39H65N2O4F ¼ 644.9).
4.1.4.4. Synthesis of compound 4d. Compound
3
(0.250 g,
1H NMR (300 MHz, CDCl3):
d
/ppm ¼ 0.90e0.95 [d, J ¼ 7.1 Hz,
0.66 mmol) was dissolved in 10 mL of dry DCM in a 25 mL round-
bottom flask and mixture was stirred over an ice bath for 15 min. To
this, a mixture of EDCI (0.126 g, 0.65 mmol) and HOBT (0.099 g,
0.65 mmol) was added, and stirring was continued for another
30 min. Then compound 2d (0.211 g, 0.53 mmol) was dissolved in
2 mL of dry DCM and added to the reaction mixture. DIPEA was
added drop-wise to the ice-cold reaction mixture (under stirring
condition) until it became slightly basic. The resulting mixture was
stirred for 24 h. Then work up was done following a similar pro-
cedure as used for compound 4a. The reaction mixture was evap-
orated and purified by column chromatography using
3H, C20H], 1.04 [s, 3H, C18H], 1.17e1.29 [m, 2H, C7H], 1.34e1.44
[t, J ¼ 6.6 Hz, 6H, eN(eCH2eCH3)2], 1.48e1.90 [m, 6H, C14H,
C
15H, C19H], 2.06e2.73 [m, 6H, C6H, C8H, C12H, C16H], 3.04
[s, 3H, eOCeHNeCH2eCH2eNeCH3], 3.33e3.48 [m, 4H,
eN(eCH2eCH3)2], 3.56e3.71 [m, 5H, eOCeHNeCH2eCH2, 11H],
C
6.09 [s, 1H, C4H], 6.25e6.33 [d, J ¼ 9.8 Hz, 1H, C2H], 7.34e7.42 [d,
J ¼ 9.8 Hz, 1H, C1H].
13C NMR (75 MHz, CDCl3):
d
/ppm ¼ [187.0 (C3), 174.6 (C21),
167.3(C5), 154.2(C1), 129.3(C2), 124.7(C4), 103.8(C9, doublet),
87.7(C17), 71.0 (C11), 59.4 {eNeCH2eCH3}2, 57.1 (O]
CeNHeCH2eCH2e), 56.8 {e(þ)NeCH3}, 48.2 (C10), 44.1 (C14), 36.2

444
S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
(C16), 35.2 (O]CeNHeCH2eCH2e), 34.3 (C12), 33.1(C13), 31.9 (C8),
31.1(C15), 29.6 (C6), 27.9 (C7), 22.6 (C19), 15.8(C18), 14.1(C20),
8.0(eNeCH2eCH3)].
[m, 41H, eN(eCH2eCH2e(CH2)9eCH3)2, C7H, C19H], 1.57e1.90 [m,
10H, eN(eCH2eCH2e(CH2)9eCH3)2, C8H, 12H, 14H, 15H],
1.97e2.50 [m, 7H, eN(eCH2eCH2e(CH2)9eCH3)2, C6H, C16H], 3.05
[s, 3H, eOCeHNeCH2eCH2eNeCH3], 3.11e3.28 [t, 2H, J ¼ 6.7 Hz,
eOCeHNeCH2eCH2], 3.44e3.74 [m, 3H,eOCeHNeCH2eCH2,
C
C
C
ESIeMS: m/z 491.
ESIeHRMS
¼
491.3298
(calculated
mass
for
C
28H44N2O4F ¼ 491.3285).
C
11H], 6.04 [s, 1H, C4H], 6.27e6.32 [d, J ¼ 10.3 Hz, 1H, C2H], [s, 1H,
C1H].
13C NMR (125 MHz, CDCl3):
(C5), 153.9 (C1), 129.2 (C2), 124.6 (C4), 103.8 (C9, doublet), 87.5 (C17),
71.1(C11), 62.0 {eNeCH2e(CH2)10eCH3}2, 61.6 (O]
HPLC purity ¼ 99.53%.
d/ppm ¼ [186.7 (C3), 174.1(C21), 167.0
4.1.5.2. Synthesis of compound 5b. Compound 5b was synthesized
following a similar procedure as described for 5a synthesis above
(26.5% yield, Rf ¼ 0.48 in 10% methanolechloroform, v/v).
CeNHeCH2eCH2e), 60.9{e(þ)NeCH3}, 48.7 (C10), 44.0 (C14), 36.0
(C16), 35.1 (O]CeNHeCH2eCH2e), 34.4 (C13), 34.1(C8), 32.8 (C15),
32.5{eNe(CH2)9eCH2eCH2eCH3}2, 31.7 (C6), 31.0{eNe(CH2)7
1H NMR (300 MHz, CDCl3):
d
/ppm ¼ 0.87e0.92 [d, J ¼ 7.7 Hz, 3H,
C
C
20H], 1.01e1.08 [t, J ¼ 7.2 Hz, 9H,eN(eCH2eCH2eCH2eCH3)2,
18H], 1.17e1.51 [m, 6H, eN(eCH2eCH2eCH2eCH3)2, C7H],
eCH2e(CH2)3eCH3}2,
29.29{eNe(CH2)5eCH2e(CH2)5eCH3}2, 29.2 {eNe(CH2)3eCH2
e(CH2)7eCH3}2, 29.0 (C7), 27.7{eNe(CH2)4eCH2
29.3
{eNe(CH2)6eCH2e(CH2)4eCH3}2,
1.55e1.91 [m, 9H, C8H, C12H, C14H, C15H, C19H], 2.12e2.74 [m, 7H,
eN(eCH2eCH2eCH2eCH3)2, C6H, C16H], 3.06 [s, 3H, eOCeHNeCH2
eCH2eNeCH3],
3.24e3.44
[m,
7H,eN(eCH2eCH2
e(CH2)6eCH3}2, 26.2{eNe(CH2)2eCH2e(CH2)8eCH3}2, 23.0 (C19),
22.5{eNe(CH2)10eCH2eCH3}2, 22.1{eNeCH2eCH2e(CH2)9eCH3}2,
16.9 (C18), 15.6 (C20), 13.9{eNe(CH2)11eCH3}].
ESIeMS: m/z 771.
eCH2eCH3)2,eOCeHNeCH2eCH2, C11H], 3.56e3.62 [d, J ¼ 6.7 Hz,
2H,eOCeHNeCH2eCH2], 6.08 [s, 1H, C4H], 6.25e6.33 [d, J ¼ 11 Hz,
1H, C2H], 7.38e7.43 [d, J ¼ 11.5 Hz, 1H, C1H].
13C NMR (75 MHz, CDCl3):
d/ppm ¼ [186.7 (C3), 173.9 (C21), 167.0
ESIeHRMS
¼
771.6446
(calculated
mass
for
(C5), 153.8 (C1), 129.1 (C2), 124.6 (C4), 101.2 (C9, doublet), 87.9 (C17),
70.1 (C11), 62.1{eNeCH2e(CH2)2eCH3}2, 61.8 (O]
C
48H84N2O4F ¼ 771.6415).
HPLC purity ¼ 95.85%.
CeNHeCH2eCH2e), 60.5{e(þ)NeCH3}, 48.0 (C10), 44.0 (C14),
36.1(C16), 35.2 (O]CeNHeCH2eCH2e), 34.0 (C13), 32.8 (C8), 32.3
(C15), 30.9 (C6), 29.5 (C7), 24.1{eNeCH2eCH2eCH2eCH3}2, 19.5
(C19), 17.6 {eNeCH2eCH2eCH2eCH3}2, 16.0 (C18), 14.0(C20), 13.8
{eNe(CH2)3eCH3}].
4.1.5.5. Synthesis of 5e. Compound 5e was synthesized following
the similar procedure as described for compound 5a (36.8% yield,
Rf ¼ 0.8 in 10% methanolechloroform, v/v).
1H NMR (300 MHz, CDCl3):
J ¼ 6.9 Hz, eN(eCH2eCH2e(CH2)13eCH3)2, C20H], 0.98 [s, 3H, C18H],
1.07e1.36 [m, 57H, eN(eCH2eCH2e(CH2)13eCH3)2, C7H, C19H],
1.41e1.81 [m, 10H, eN(eCH2eCH2e(CH2)13eCH3)2, C8H, C12H, C14H,
d
/ppm ¼ 0.70e0.87 [m, 9H,
ESIeMS: m/z 547.
ESIeHRMS
¼
547.3936
(calculated
mass
for
C
32H52N2O4F ¼ 547.3911).
HPLC purity ¼ 97.46%.
C
15H], 1.84e2.80 [m, 7H, eN(eCH2eCH2e(CH2)9eCH3)2, C6H, C16H],
3.01 [s, 3H, eOCeHNeCH2eCH2eNeCH3], 3.11e3.24 [t, 2H,
6.7 Hz, eOCeHNeCH2eCH2], 3.29e3.56 [m,
4.1.5.3. Synthesis of compound 5c. Compound 5c was synthesized
following a similar procedure as described for compound 5a (20.5%
yield, Rf ¼ 0.55 in 10% methanolechloroform, v/v).
J
¼
3H,eOCeHNeCH2eCH2, C11H], 6.02 [s, 1H, C4H], 6.20e6.28 [d,
J ¼ 9.4 Hz, 1H, C2H], 7.30e7.38 [d, J ¼ 9.9 Hz, 1H, C1H], 13C NMR
1H NMR (300 MHz, CDCl3):
d
/ppm ¼ 0.81e0.97 [m, 9H,
(125 MHz, CDCl3):
d
/ppm ¼ [186.9 (C3), 174.4 (C21), 167.0 (C5), 153.9
J ¼ 6.7 Hz, eN(eCH2eCH2e(CH2)5eCH3)2, C20H], 1.02 [s, 3H, C18H],
1.16e1.42 [m, 25H, eN(eCH2eCH2e(CH2)5eCH3)2, C7H, C19H],
1.45e1.90 [m, 10H,eN(eCH2eCH2e(CH2)5eCH3)2, C8H, C12H, C14H,
(C1), 129.3 (C2), 124.7 (C4), 103.6 (C9, doublet), 87.5 (C17), 71.4 (C11),
62.0 {eNeCH2e(CH2)14eCH3}2, 61.7 (O]CeNHeCH2eCH2e), 61.1
{eN(þ)eCH3}, 48.4 (C10),44 (C14), 37.3 (C16), 37.0 (O]
CeNHeCH2eCH2e), 35.9(C13), 35.2 (C8), 32.6(C15), 32.9
{eNe(CH2)13-CH2eCH2eCH3}2, 31.9 (C6), 31.3 {-N-(CH2)11-CH2-
(CH2)3eCH3}2, 31.1 {-N-(CH2)10-CH2-(CH2)4eCH3}2, 30.1 {-N-
(CH2)9-CH2-(CH2)5eCH3}2, 30.0 {-N-(CH2)8-CH2-(CH2)6eCH3}2,
C
C
J
15H,], 2.05e2.75 [m, 7H, eN(eCH2eCH2e(CH2)5eCH3)2, C6H,
16H], 3.06 [s, 3H, eOCeHNeCH2eCH2eNeCH3], 3.19e3.30 [t, 2H,
¼
6.7
Hz,
eOCeHNeCH2eCH2],
3.35e3.59
[m,
3H,eOCeHNeCH2eCH2, C11H], 6.07 [s, 1H, C4H], 6.27e6.33 [d,
J ¼ 10.1 Hz, 1H, C2H], 7.35e7.40 [d, J ¼ 10.1 Hz, 1H, C1H].
29.6
(CH2)8eCH3}2, 24.3 {-N-(CH2)5-CH2-(CH2)9eCH3}2, 29.0 {-N-
(CH2)3-CH2-(CH2)11eCH3}2, 26.3(C7), 22.9 {-N-(CH2)4-CH2-
{-N-(CH2)7-CH2-(CH2)7eCH3}2,
29.4
{-N-(CH2)6-CH2-
13C NMR (125 MHz, CDCl3):
d
/ppm ¼ [186.9 (C3), 174.3(C21),
167.1(C5), 154.2 (C1), 129.3 (C2), 124.7 (C4), 103.8 (C9, doublet), 87.8
(C17), 70.6 (C11), 62.3 {eNeCH2e(CH2)6eCH3}2, 61.7 (O]
CeNHeCH2eCH2e), 61.4 {e(þ)NeCH3}, 48.2 (C10), 44.2 (C14), 36.7
(C16), 35.1 (O]CeNHeCH2eCH2e), 35.2 (C13), 34.4 (C8), 33.0 (C15),
32.7{eNe(CH2)5eCH2eCH2 eCH3}2, 31.5 (C6), 31.2{eNe(CH2)3
eCH2e(CH2)3eCH3}2, 29.6 (C7), 27.9 {eNe(CH2)4eCH2e(CH2)2
eCH3}2, 26.3 {eNe(CH2)2eCH2e(CH2)4eCH3}2, 23.0 (C19), 22.5
{eNe(CH2)6eCH2eCH3}2, 22.3{eNeCH2eCH2e(CH2)5eCH3}2, 17.0
(C18), 16.0 (C20), 14.0{eNe(CH2)7eCH3}].
(CH2)10eCH3}2, 22.6 {-N-(CH2)2-CH2-(CH2)12eCH3}2, 22.2 (C19),19.6
{-N-CH2-(CH2)13eCH2eCH3}2, 17.0 {-N-CH2-CH2-(CH2)13eCH3}2,
15.4 (C18), 14.4 (C20), 14.1{-N-(CH2)15-CH3}].
ESIeMS: m/z 884.
ESIeHRMS
¼
883.7663
(calculated
mass
for
C
56H100N2O4F ¼ 883.7667).
HPLC purity ¼ 97.41%.
ESIeMS: m/z 659.
4.2. Biological experiments
4.2.1. Antibodies
Caspase 3 (ab44976), Smac/Diablo (ab 32023), Mdm2 (ab
38618), XIAP were purchased from Abcam, UK; BAX (PA5-17806),
Bcl-2 (MA5-14937), Akt-1 (MA5-14898), p-Akt (ser 473,PA1-
14032), Caspase 9 (PA5-16358), Active Caspase 3 (PA1-29157),
STAT3 (PA5-16187), p-STAT3 (Tyr705, MA5-1118), p-STAT3 (Ser727,
PA1-14393), JAK2 (OPA1-03052A), JAK3, Cytochrome C (PA1-9586),
ESIeHRMS
¼
659.5155
(calculated
mass
for
C
40H68N2O4F ¼ 659.5157).
HPLC purity ¼ 98.41%.
4.1.5.4. Synthesis of compound 5d. Compound 5d was synthesized
following a similar procedure as described for compound 5a (66.6%
yield, Rf ¼ 0.68 in 10% methanolechloroform, v/v).
1H NMR (300 MHz, CDCl3):
d/ppm ¼ 0.87e0.93 [t, 9H, J ¼ 6.9,
eN(eCH2eCH2e(CH2)9eCH3)2, C20H], 1.02 [s, 3H, C18H], 1.21e1.39
Caspase 7, b-Actin (PA5-16914) and goat anti rabbit IgG alkaline

S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
445
Phosphatase-conjugated (31340) were purchased from Pierce
Biotechnology) (diluted to 50
complexed with 2.5
15e20 min. An amount of 900
free DMEM was added to the six well plates and kept for 4 h. Then,
10% FBS containing DMEM was freshly added into the same well
and left the plates as such for 36 h. After that cells were washed
with PBS and harvested, counted, and again seeded at a density of
5000 cells/well in 96-well plates for MTT assay. However, after 12 h
cells in 96-well plate cells were either kept untreated or treated
m
L with serum free DMEM) were
Protein Research, USA; p53 (#2524), GAPDH (#5174),were pur-
chased from Cell Signaling Technology; Anti-KDR/Flk-1/VEGFR2
(07-158) was purchased from Millipore; VE-cadherin (sc-9989),
and mouse mouse-anti-rabbit IgG-FITC (sc-2359), goat anti-rabbit
IgG-FITC (sc-3839), Anti-GR (sc-8992), were purchased from
Santa Cruz Biotechnology; Goat anti-mouse IgG Alkaline Phos-
phatase (DC05L) were purchased from Calbiochem, Germany, IL-6
assay kit was purchased from e-Bioscience, USA.
m
L of Lipofectamine 2000 (Invitrogen) for
mL siRNA-complex containing serum
with 10 mM DX8 for another 36 h in B16F10 and A549 experiment.
4.2.2. Samples preparation and treatment
For this study viability assay was performed after 36 h of compound
treatment. After completion of treatment the cells were washed
assayed for viability using MTT as described earlier.
These Synthesized compounds were dissolved in cell culture
grade DMSO to get a primary stock. Then stocks were progressively
diluted with DMSO to make secondary stock and finally working
concentrations of those compounds were obtained by adding the
secondary DMSO stocks in 10% fetal bovine serum (FBS) containing
cell culture medium. The concentration of DMSO in 10% FBS con-
taining working medium never exceeded more than 1%. For the cell
GR siRNA(m) [cat no. sc-35506]
1060
1971
2103
CCACUAUGACAUGAAUAC
GCAUGUAUGACCAAUGUA
GCCAAGAGUUAUUUGAUG
viability studies an amount of 100
mL of cell culture solutions
GR siRNA(H) [cat no.sc-35505]
2096
2178
containing respective concentrations of derivatives were added to
each well of 96-well plates and few wells were kept untreated. For
quantification of apoptosis studies an amount of 1.5 mL of culture
medium containing respective concentrations of compounds were
added to each well of 6-well plates.
GGCUUCAGGUAUCUUAUG
CCAAGAGCUAUUUGAUGA
CUCCAGUAUUCUUGUCAA
3202
Control siRNA [Qiagen Inc.]
UUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATT
4.2.3. Cell culture
MCF-7 (human breast adenocarcinoma), B16F10 (murine mel-
anoma), CHO (Chinese hamster ovary), human lung carcinoma
(A549), Human embryo kidney (HEK293T) cell lines were pur-
chased from the American Type Cell Culture (ATCC, USA) and Na-
tional Center for Cell Sciences (Pune, India). Freshly isolated mouse
skin fibroblast (FB) cell was obtained from Center for Cellular and
Molecular Biology (Hyderabad, India) and was grown in myco-
plasma free condition. Cells were cultured in DMEM supplemented
4.2.6. Flow cytometric analysis
The remaining cells after seeding from the above experiment
were used to evaluate the extent of down-regulation of GR by flow
cytometry. They were re-plated, and after 48 h they were har-
vested, washed, fixed with 2% formaldehyde in PBS for 10e15 min,
and then permeabilized with 0.01% triton-x in PBS for 5e10 min at
room temperature [14]. Then they were collected by centrifugation,
washed, and treated with rabbit raised full-length glucocorticoid
receptor primary antibody obtained from Santa Cruz Biotechnology
for A549 cells and from Cell Signaling Technology for B16F10 (at
1:100 dilutions, in PBS containing 2% fetal bovine serum) at 4 ꢁC for
2 h. After incubation, cells were collected by centrifugation,
washed, and treated with FITC-conjugated goat-anti rabbit sec-
ondary antibody (Santa Cruz Biotechnology) at 1:500 dilution (in
PBS containing 2% fetal bovine serum) and incubated at 4 ꢁC for 1 h
in the dark. Finally cells were washed and analyzed using a flow
cytometer (FACS Canto II, Becton Dickinson, San Jose, CA, U.S.) and
data were analyzed with FCS Express V3 software. Minimum of
10,000 events were gated per sample.
with 10% US origin FBS (Lonza, USA), 50
mg/mL penicillin, 50 mg/mL
streptomycin and 100
m
g/mL kanamycin at 37 ꢁC in a humidified
condition of 5% CO2 in air. Cultured healthy cells of 75e85% con-
fluency were used for experiments. Cells were trypsinized, counted
and seeded, in 6-well plates for quantification of apoptosis studies,
96-well plates for cell viability studies and in 25 cm2 or 75 cm2
tissue culture flasks for Western blot studies. The cells were kept to
adhere overnight before they were used for experiments.
4.2.4. Cytotoxicity studies
Cytotoxicity assay of the compounds were determined by the
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT). Briefly, cells were plated well in 96-well plates
with density of 5000 cells/well usually 12e18 h before experi-
ments. Solutions containing respective concentrations of following
compounds were added in at least triplicate wells or more. In the
screening experiments dexamethasone derivatives (DXn) were
continuously treated to the cells for 72 h. Following termination of
treatment, cells were washed with Phosphate buffer saline (PBS)
and assayed for viability using MTT. Results were represented as
percentage of viability ¼ {[A550 (treated cells) ꢂ background]/
[A550 (untreated cells) ꢂ background]} ꢀ 100.
For p53 level detection in B16F10 cells, cells were treated with
10 mM DX8 and Dex for 48 h. Following which cells were harvested,
washed, fixed with 2% formaldehyde in PBS for 10e15 min, and
then permeabilized with 0.01% triton-x in PBS for 5e10 min at room
temperature. As described above, cells were collected and treated
with mouse monoclonal p53 antibody (Cell Signaling Technology,
USA) at 1: 100 dilution (in PBS containing 2% fetal bovine serum)
and incubated at 4 ꢁC for 2 h in the dark. The cells were washed
with PBS and incubated with PE-conjugated goat-anti-mouse sec-
ondary antibody for 1 h at 4 ꢁC. Cells were finally washed and
analyzed using a flow cytometer (FACS Canto II, Becton Dickinson,
San Jose, CA, U.S.) and data were analyzed with FCS Express V3
software. Minimum of 10,000 events were gated per sample.
4.2.5. Glucocorticoid receptor (GR) down-regulation by siRNA
delivery
GR down-regulation siRNA was based on following a similar
procedure as described earlier [14]. Briefly, B16F10 and A549 cells
were plated with a density of 2.5 ꢀ 105 cells per well in a 6-well
plate usually between 12 and 18 h before transfection. Then
4.2.7. Quantification of apoptosis studies
The annexin V-FITC-labeled apoptosis detection kit (Sigma) was
used to detect and quantify apoptosis by flow cytometry as per
manufacturer's instructions. In brief, cells (2 ꢀ 105 cells/well) were
seeded in six-well plates and cultured overnight in 10% serum
25 pmol of negative control siRNA (Qiagen) (diluted to 50
mL with
serum free DMEM) or 25 pmol of GR-siRNA (Santa Cruz

446
S. Sau, R. Banerjee / European Journal of Medicinal Chemistry 83 (2014) 433e447
containing complete medium. After 16e18 h cells were kept un-
treated (UT) or treated with 10 M Dex or 10 M DX8 for 48 h. Then
0.15 M NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1%
SDS, which was premixed with 1ꢀ protease inhibitor cocktail and
2 mM sodium vanadate. For tumor cell lysates preparation: after
completion of in vivo experiment mice were sacrificed and small
pieces of tumors were lysed in same buffer as described above.
Respective cell lysates were run in different % of SDS-PAGE gradient
gel according to antigen's molecular weight, and then the bands
were transferred to Nitrocellulose membrane. BCIP/NBT was used
for detection of protein bands by colorimetric method.
m
m
the cells were harvested and collected by centrifugation for 5 min at
1000 g. Cells were then re-suspended at a density of 1 ꢀ 106 cells/
mL in 1ꢀ binding buffer and stained simultaneously with FITC-
labeled annexin V (25 ng/mL) and propidium iodide (50 ng/mL).
Cells were analyzed using a flow cytometer (FACS Canto II), and
data were analyzed with FCS Express V3 software. Minimum of
10,000 events were gated per sample.
4.2.8. Preclinical studies using C57BL6/J mice
4.3. Statistical analysis
Female C57BL6/J mice were obtained from National Institute of
Nutrition (NIN, Hyderabad, India). The protocols for animal exper-
imentation were approved through Institutional Animal Ethical
Committee. Female C57BL6/J mice, 8e10 weeks old, were subcu-
taneously inoculated with 2.2 ꢀ 105 B16F10 cells in the lower right
abdomen. Fourteen days following cancer cell implantation, mice
were grouped as per treatment. The groups were as follows: (i)
group kept untreated, UT (four mice) (ii) group treated with Dex
(four mice) and (iii) group treated with DX8 (four mice). For DX8
treatment group an amount of 20 mg/kg compound in 5% glucose
having DMSO concentration 5% was injected and for Dex treatment
group equivalent amount of DX8 equal to an amount of 12 mg/kg in
5% glucose having DMSO concentration 5% was injected. Five
intraperitoneal injections with every alternate day were adminis-
tered. Tumors were measured every alternate day and tumor sizes
were expressed in volume (mm3) and calculated using the formula
(0.5 ab2), where ‘a’ is the longest dimension and ‘b’ is the shortest
dimension of the tumors. Animals were humanely euthanized
when the average tumor volume of the untreated (UT) group
reached~2000 mm3.
Data were expressed as the mean (SD) and statistically analyzed
by the two-tailed, unpaired, Student's t-test using Microsoft Excel
(Seattle, WA). For animal studies, scores were considered signifi-
cant when p < 0.05. For in vitro studies, scores were considered
significant when p < 0.01.
4.4. Software
Chemical structures were drawn with the help of ChemDraw
software. For graph plotting Microsoft excel software was used.
Images were prepared in Photoshop CS2.
Acknowledgment
SS acknowledges Council of Scientific & Industrial Research
(CSIR), Govt. of India for his doctoral fellowship; RB acknowledges
DST, Govt. of India [SR/S1/OC-64/2008] and CSIR Network Project
‘ADD’ [CSC 0302] for research support. Authors thankfully
acknowledge Dr. Arabinda Chaudhuri, CSIR-IICT, India for his crit-
ical reading of the manuscript and providing valuable corrections
and suggestions for the manuscript.
4.2.9. TUNEL assay
After completion of the above experiment one mouse from each
group was sacrificed for detection of apoptosis in tumor tissues. The
tumors were frozen in Jung tissue freeze medium (Leica Micro-
Abbreviations used
system Germany) followed by cryo-sectioning of 10 mm thin sec-
Dex
dexamethasone
tions using Leica CM1850 Cryostat (Germany). The sections were
fixed with 4% formalin for 15 min and TUNEL assayed using Dead-
End fluorometric TUNEL assay kit (Promega, Madison, WI) as per
manufacturer's protocol. The same cryosections used for TUNEL
assay were again considered for blood vessel staining by VE-
cadherin. The tissue sections were washed and incubated for 2 h
at 4 ꢁC with VE-cadherin mouse monoclonal antibody (Santa Cruz)
followed by one hour incubation with Texas Red conjugated goat-
anti-mouse secondary antibody. Images were acquired in Nikon
TE 2000E microscope at 10ꢀ magnification. Duplicate panel in
TUNEL assay of DX8 treated tumor section is to represent signature
of apoptosis in both tumor and tumor associated endothelial cells in
two different areas of tumor section.
TFA
trifluoroacetic acid
sodium periodate
potassium carbonate
dichloromethane
glucocorticoid receptor
glucocorticoid responsive element
NaIO4
K2CO3
DCM
GR
GRE
Mdm2 mouse double minute 2
STAT3
DMEM DMEM Dulbecco's modified eagle medium
VEGFR2 vascular endothelial growth factor receptor 2
Bcl2
signal transducer and activator of transcription 3
B-cell lymphoma 2
Bcl-2-associated X protein
X-linked inhibitor of apoptosis protein
1-hydroxybenzotriazole
diisopropylethyl amine
BAX
XIAP
HOBT
DIPEA
EDCI
4.2.10. VEGFR2-downregulation analysis
VEGFR2 down-regulation was based on a similar procedure as
described earlier [30]. Thin 10 mm tumor sections were fixed and
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.
incubated with VEGFR2-rabbit monoclonal antibody for 2 h at 4 ꢁC.
This was followed by washing with PBS (3 ꢀ 10 min) and then kept
in goat anti-rabbit FITC conjugated secondary antibody for another
45 min. The sections were visualized using Nikon TE 2000E mi-
croscope at 10ꢀ magnification.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.051.
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