Synthesis and mechanistic aspects of 2-anilinonicotinyl-pyrazolo[1,5-a]pyrimidine conjugates that regulate cell proliferation in MCF-7 cells via estrogen signaling

Article abstract of DOI:10.1016/j.bmcl.2016.02.072

A series of anilinonicotinyl linked pyrazolo[1,5-a]pyrimidine conjugates (6a-x) were synthesized and evaluated for their antiproliferative activity. Some of these conjugates exhibited promising cytotoxic effects in the MCF-7 cell line and among these 6a a

Full text of DOI:10.1016/j.bmcl.2016.02.072

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and mechanistic aspects of 2-anilinonicotinyl-pyrazolo
[1,5-a]pyrimidine conjugates that regulate cell proliferation in MCF-7
cells via estrogen signaling
a
Ahmed Kamal a, , Shaikh Faazil , S. M. Ali Hussaini a,y, M. Janaki Ramaiah b, M. Balakrishna a,
⇑
Nibedita Patel b, S. N. C. V. L. Pushpavalli b, Manika Pal-Bhadra b,
⇑
a Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Chemical Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of anilinonicotinyl linked pyrazolo[1,5-a]pyrimidine conjugates (6a–x) were synthesized and
evaluated for their antiproliferative activity. Some of these conjugates exhibited promising cytotoxic
effects in the MCF-7 cell line and among these 6a and 6c exhibited significant effects, apart from G2/M
cell cycle arrest. Interestingly they showed profound effects on cyclin D1, Bcl-2 and survivin proteins that
regulate breast cancer cell proliferation. Moreover, ER alpha protein expression was studied to under-
stand regulatory role of these conjugates on estrogen activity in estrogen positive breast cancer cells like
MCF-7 and compounds 6a and 6c reduced their activity.
Received 30 September 2015
Revised 19 February 2016
Accepted 24 February 2016
Available online xxxx
Keywords:
Estrogen receptors
Cell cycle
Ó 2016 Elsevier Ltd. All rights reserved.
Cyclin D1
Bcl-2
Pyrazolopyrimidine
Breast carcinoma is the most common malignancy in women,
estrogen receptor is an important prognosis marker and is con-
sidered as predictive of response to endocrine therapy in patients
with breast cancer.1 Estrogen receptors ER
and ERb are the mem-
inhibitor for many kinase enzymes, they are also known to func-
tion as CNS depressants,13 neuroleptic14 and tuberculostatic
agents.15 Their cytotoxic activities might be attributed to inhibition
of several enzymes such as glutathione peroxidase,16 tyrosine
kinase,17 mammalian target of rapamycin (mTOR),18 cyclin depen-
dent kinase (CDK)19. Various pyrazolo[1,5-a]pyrimidine scaffolds
and its derivatives have been reported as potential anticancer
agents that include inhibition of cyclin-dependent kinases (1,
2),20 checkpoint kinase 1 (CHK1) inhibitors (3),21 estrogen (ER)
selective ligand (4)22 (Fig. 1). However, opportunities exist to
identify and develop new anticancer agents that may possess
superior biological profile compared to the presently identified
molecules. Hence, there is a considerable challenge that exists in
this area towards the identification of new conjugates comprising
of pharmacophores of known antitumor agents for enhancing
the selectivity as well as anticancer activity. 2-Anilinonicotinyl
moiety is present in agents like E7010 (5) which inhibit tubulin
polymerization and exhibits good in vivo antitumor activity
against several rodent as well as human tumours and is presently
in phase II clinical trials.23
a
a
bers of nuclear hormone receptor family that acts as transcription
factors when bound and activated by their ligand.2 In breast cells
ER
a and ERb are expressed and share 97% identity in their DNA-
binding domains and 55% identity in their ligand binding domains.
Both these receptors function in antagonistic manner wherein ER
a
increases tumor growth and ERb inhibits tumor growth.3 Estrogen
receptors once activated by estrogen form dimers (both homo as
well as hetero) and regulate the genes.4 It is interesting to note that
more than 70% of breast cancers are estrogen positive.5 Thus there
is an urgent need to discover the new drugs that effectively con-
trols the ERa
-positive cell growth.6
Fused heterocycles, pyrazolopyrimidines have been found to
possess significant pharmacological activities7 such as antibacte-
rial,8 antifungal,9 antitumor,10 and antiinflammatory.11 Owing to
the structural similarity with purines,12 and hence exhibits
promising antitumor activity by acting as ATP competitive
Similarly, it is fascinating to observe that in the last 10 years,
numerous piperazine linked derivatives have been design and
synthesized to exploit their chemotherapeutic potential.24 It is
previously reported that piperazine linked bifunctional agents
proved to be promising anticancer class of compounds with
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
Presently working at: Department of Chemistry, University of Saskatchewan, 110
y
Science Place, Saskatoon, SK S7N 5C9, Canada.
http://dx.doi.org/10.1016/j.bmcl.2016.02.072
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kamal, A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.072

2
A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O-
+N
N
N
NHBn
S
N
HN
N
N
HN
N
N
HN
N
OH
N
HN
N
HO
N
Et
N
OH
N
3
1
OH
HO
2
C6H5
N
O
H
N
O
S
O
O
N
N
N
N
CF3
N
R1
R2
N
NH
HO
O
N
N
N
HN
R
CF3
R3
OH
4
6a-x
5
Figure 1. Chemical structures of pyrazolopyrimidines (1–4), E7010 (5), 2-anilinonicotiyl-pyrazolo[1,5-a]pyrimidine conjugates (6a–x).
remarkable selectivity against different types of cancers.25 As
described here, pyrazolopyrimidines and 2-anilinonicotinyl scaf-
folds are of considerable interest in the development of anticancer
compounds. Modifications on the pyrazolo[1,5-a]pyrimidine
nucleus and 2-anilinonicotic acids have resulted in a large number
of compounds that exhibit significant anticancer activities.
In continuation to our efforts on the development of useful syn-
thetic protocols suitable for applications in the field of combinato-
rial chemistry and chemistry-driven drug discovery.26–28
Additionally, the medicinal importance of these moieties with
impressive anticancer profile and their effect on various molecular
targets as potential cancer chemotherapeutic agents an attempt
has been made in the present study to link the pyrazolo[1,5-a]
pyrimidine moiety to the 2-anilinopyridyl structural component
of E7010. The conformationally restricted cyclic piperazine amide
has been employed as a linker for this purpose resulting in a new
class of compounds. These newly synthesized chemical entities
have been evaluated for their antiproliferative potential and cell
cycle perturbations. Moreover detailed studies have been carried
out to understand their mechanistic aspects of regulating the cell
proliferation in MCF7 cell line via estrogen signaling.
The synthesis of the target conjugates have been performed in a
convenient manner as outlined in Schemes 1 and 2. The synthetic
route employed starts with the synthesis of b-diketoesters (8a, 8b,
8c, 8d and 8e) with some modification to the one reported in the
literature.29 For this purpose, different substituted acetophenones
(7a–e) on oxylation by dimethyl oxalate in the presence of
sodium methoxide provides the b-diketoesters (8a–e). Different
acetophenones with 4-chloro, 4-fluoro, 3-methoxy, 4-methoxy
and 3,4,5-trimethoxy substitutions have been employed. A conve-
nient protocol was employed for the synthesis of fused pyrazolo
[1,5-a]pyrimidines compounds. The b-diketoester on cyclization
with an aza-heterocyclic amine, i.e., 3-amino-5-phenyl pyrazolo
in the presence of catalytic amount of cerric ammonium nitrate
(CAN, 5 mol %) in ethanol at room temperature affords various sub-
The synthesis of these new conjugates (6a–x) has been accom-
plished by the synthetic route depicted in Scheme 2. The ethyl
ester of 2-chloronicotinic acid 11 and different substituted anilines
12a–e are refluxed in ethylene glycol to give the coupled product
of 2-anilino nicotinic acid esters 13a–e. The anilines used are with
different substitutions such as 4-fluoro, 4-methoxy, 3,4-dimethoxy
and 3,4,5-trimethoxysubstitutions with a view to bring diversity in
the conjugates for a better understanding of their SAR. The esters
13a–e on base hydrolysis on treating with 2 N sodium hydroxide
solution in ethanol affords 2-anilinonicotinic acids (14a–e) in
quantitative yields. These acids are converted to amides by cou-
pling them with commercially available Boc-protected piperazine
in presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
and hydroxyl benzotriazole (EDCl/HOBt) in dry dimethylfor-
mamide at 0 °C. To effect the coupling of 2-anilinonicotinyl piper-
azineamide
(15a–e)
and
pyrazolo[1,5-a]pyrimidine
acid
intermediates (10a–e), the Boc-protection on the piperazine moi-
ety of 15a–e is removed by treatment with trifluoroacetic acid in
dichloromethane. This was followed by the crucial coupling
assisted by EDCl/HOBt methodology in dry DMF to yield the
desired 2-anilinonicotinyl linked pyrazolo[1,5-a]pyrimidine conju-
gates (6a–x) as shown in Scheme 2 (Supplementary information).
By employing this efficient and convenient synthetic route, we
have synthesized twenty four new 2-anilinonicotinyl linked pyra-
zolo[1,5-a]pyrimidine conjugates that are joined through confor-
mationally restricted piperazineamide spacers.
These conjugates have been evaluated for their biological prop-
erties such as cytotoxic activity, cell viability and effects on pro-
teins such as estrogen receptor, Bcl-2, survivin and cyclin D1 in
MCF breast cancer cell lines in order to understand the mechanism
involved during cell division arrest of cancer cells.
In order to study the possible cytotoxic effect by these series of
conjugates (6a–x), MTT assay was conducted. Here SiHa, HeLa (cer-
vical cancer cell line), MCF-7 (breast cancer), IMR-32 (neuroblas-
toma) were treated at concentration ranging from 0.25 to 8 lM
stituted
2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
of conjugates for 48 h. It was observed that conjugates 6a, 6c, 6j,
6m, 6o, 6q, 6r, 6s, 6t and 6w were effective against all the cells.
Conjugates 6a, 6c were found to be most effective among the series
against the cell lines tested (Fig. 2 and Table1). From cytotoxic data
it is also evident that these molecules are specific to MCF-7, HeLa
esters (9a–e), which upon base hydrolysis with sodium hydroxide
provides the corresponding carboxylic acids (10a–e).Then with
pyrazolo[1,5-a]pyrimidine acid intermediates (10a–e) in hand,
we proceeded to synthesize the target conjugates.
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A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
C6H5
N
O
O
O
N
N
C6H5
R1
R2
N
R1
R2
R1
R2
OCH3
CH3
OCH3
ii
NH
i
+
O
O
NH2
R3
8a-e
R3
R3
9a-e
7a-e
iii
a: R1 = R3 = H; R2 = F
b: R1 = H; R2 = R3 = Cl
c: R1 = H; R2 = OMe ; R3 = H
d: R1 = H; R2 = R3 = OCH3
C6H5
e: R1 = OCH3; R2= OCH3; R3 = OCH3
N
N
N
OH
R1
R2
O
10a-e
R3
Scheme 1. Reagents and conditions: (i) dimethyl oxalate, NaOMe, THF, rt, 8 h; (ii) cat. CAN, ethanol, 0.5 h, rt; (iii) 2 N NaOH, MeOH, reflux, 2 h.
O
O
O
OCH2CH3
NH2
R'
(i)
OH
OCH2CH3
(ii)
+
N
Cl
11
N
NH
R'
N
NH
R'
12a-e
13a-e
14a-e
a:R'= 4-Fluorophenyl
b:R'= 4-Chlorophenyl
c:R' = 4-Methoxyphenyl
d:R' = 2,4-Dimethoxylphenyl
e:R'= 3,4,5-Trimethoxyphenyl
(iii)
R1
R2
R3
O
O
O
N
O
N
(iv)
N
N
N
N
O
N
NH
R'
N
N
NH
R'
Ph
6a-x
15a-e
: R' = phenyl; R1 = R3 = H; R2 = F
6a
6b: R' = phenyl; R1 = H; R2 = R3 = 3, 4-dichloro
6c: R' = phenyl; R1 = H; R2 = OCH3; R3 = H
6d: R' = phenyl; R1 = H; R2 = R3 = 3, 4-dimethoxy
6e: R' = phenyl; R1 = R2 = R3 = 3, 4, 5-trimethoxy
6f: R' = 4-f luorophenyl; R1 = R3 = H; R2 = F
R' = 4-methoxyphenyl; R1 = H; R2 = R3 = 3, 4-dimethoxy
: R' = 4-methoxyphenyl; R1 = R2 = R3 = 3, 4, 5-trimethoxy
6n:
6o
6p:
R' = 3,4-dimethoxyphenyl; R1 = R3 = H; R2 = F
6q: R' = 3,4-dimethoxyphenyl; R1 = H; R2 = R3 = 3, 4-dichloro
6r: R' = 3,4-dimethoxyphenyl; R1 = H; R2 = OCH3; R3 = H
6g: R' = 4-f luorophenyl; R1 = H; R2 = R3 = 3, 4-dichloro
6h: R' = 4-fluorophenyl; R1 = H; R2 = OCH3; R3 = H
6s: R' = 3,4-dimethoxyphenyl; R1 = H; R2 = R3 = 3, 4-dimethoxy
6t: R' = 3,4-dimethoxyphenyl; R1 = R2 = R3 = 3, 4, 5-trimethoxy
6u: R' = 3,4,5-trimethoxyphenyl; R1 = R3 = H; R2 = F
R' = 4-f luorophenyl; R1 = H; R2 = R3 = 3, 4-dimethoxy
R' = 4-fluorophenyl; R1 = R2 = R3 = 3, 4, 5-trimethoxy
6i:
6j:
6k:
6v: R' = 3,4,5-trimethoxyphenyl; R1 = H; R2 = R3 = 3, 4-dichloro
R' = 4-methoxyphenyl; R1 = R3 = H; R2 = F
6w: R' = 3,4,5-trimethoxyphenyl; R1 = H; R2 = OCH3; R3 = H
6l: R' = 4-methoxyphenyl; R1 = H; R2 = R3 = 3, 4-dichloro
6m:R' = 4-methoxyphenyl; R1 = H; R2 = OCH3; R3 = H
R' = 3,4,5-trimethoxyphenyl; R1 = H; R2 = R3 = 3, 4-dimethoxy
6x:
Scheme 2. Reagents and conditions: (i) ethylene glycol, 160 °C, 6 h; (ii) 2 N NaOH, ethanol, reflux 2 h; (iii) Boc-piperazine, EDCI/HOBt, dry DMF 0 °C-rt, 8 h; (iv) (a) TFA, DCM,
rt, 2 h; (b) substituted 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic acid, EDCI/HOBt, dry DMF 0 °C-rt, 8 h.
and SiHa cells. Conjugates 6r, 6s and 6v have shown specific effects
in SiHa; conjugates 6q, 6r and 6s have shown specific effects in
MCF-7; conjugates 6a, 6c, 6l and 6m have shown specific effects
in HeLa cells and are found to cause strong cytotoxic effects. How-
ever, IMR-32 cells did not respond significantly to these
conjugates.
With the help of cell viability studies discussed above, structure
activity relationships have been determined. Electron donating
substituents on the phenyl ring of pyrazolopyrimidine scaffold as
in 6c, 6j, 6m, 6o, 6r, 6t, 6s and 6w were found to be fruitful for
activity. These compounds exhibited good activity against one or
more of the tested cell lines irrespective of the substituents present
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A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
MTT assay in SiHa cells
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
(a)
MTT Assay in MCF-7 cells
1.2
1
0.8
0.6
0.4
0.2
0
(b)
MTT assay in HeLa cells
2.5
2
1.5
1
(c)
0.5
0
MTT assay in IMR-32 cells
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
(d)
Figure 2. (a–d) SiHa, HeLa (cervical cancer), MCF-7 (breast cancer), IMR-32 (neuroblastoma) were studied at 4 lM of conjugates for 24 h using MTT assay.
on 2-anilinonicotinoyl group. Similarly, 6a and 6q containing weak
electron withdrawing substituents such as fluoro and chloro on
pyrazolopyrimidine scaffold displayed appreciable activity only
in presence of certain substituents on 2-anilinonicotinoyl group.
On the other hand, substituents on 2-anilinonicotinoyl moiety dis-
played random effects on the activity.
Cell cycle analysis has been performed to explore the basis of
anti-proliferative properties of these effective conjugates (6a and
6c), wherein doxorubicin was used as the positive control. MCF-7
cells were treated with doxorubicin (Doxo), 6a, 6c at 4–32 lM con-
centration for 48 h. Control cells have shown only 25% of cells in
G2/M phase of cell cycle whereas cells treated by compounds lead
to huge accumulation of cells in G2/M phase.
Here doxorubicin caused 48%, 50%, 49%, 46% of G2/M phase cells
at 4, 8, 16 and 32
treated cells have shown accumulation in the range of 25–39% G2/
M phase cells. Surprisingly concentration of 4 M and above has
given almost the similar percentage of G2/M phase cells as it
lM respectively. Whereas compounds 6a and 6c
l
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A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 1
Cytotoxicity of the compounds 6a–x expressed in lM
R1
R2
R3
O
O
N
N
N
N
N
N
NH
Ph
R'
6a-x
Compound
R’
R1
R2
R3
SiHa
MCF-7
HeLa
IMR-32
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
H
H
H
H
H
4-F
4-F
4-F
H
H
H
H
OCH3
H
H
H
H
OCH3
H
H
H
H
OCH3
H
H
H
H
F
Cl
OCH3
OCH3
OCH3
F
H
Cl
H
OCH3
OCH3
H
Cl
H
OCH3
OCH3
H
Cl
H
OCH3
OCH3
H
Cl
H
OCH3
OCH3
H
2.65
5.93
3.33
5.93
5.10
7.21
5.27
5.93
5.64
5.25
4.56
3.92
4.36
5.10
3.87
6.62
3.73
3.73
5.93
3.73
4.07
3.65
6.08
7.65
3.93
1.79
5.30
2.16
6.12
6.93
3.34
4.73
4.97
7.02
6.28
8.08
6.12
2.93
2.85
3.02
2.69
5.95
2.53
2.61
4.24
7.59
5.38
3.83
7.34
0.473
2.29
2.57
2.43
2.88
2.99
3.86
4.27
3.79
3.16
2.57
6.60
2.64
3.72
4.62
2.85
6.33
5.11
5.87
5.70
6.08
4.31
4.52
4.24
4.24
1.25
4.65
7.87
4.75
6.5
7.25
5.62
5.87
5.87
5.87
5.75
5.37
5
6.37
5.87
5.87
5.25
7.15
5.12
5.25
5.75
5.0
5.62
6.62
4.88
4.5
Cl
OCH3
OCH3
OCH3
F
4-F
4-F
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4-diOCH3
3,4,5-triOCH3
3,4,5-triOCH3
3,4,5-triOCH3
3,4,5-triOCH3
—
Cl
OCH3
OCH3
OCH3
F
Cl
OCH3
OCH3
OCH3
F
OCH3
H
H
H
H
Cl
Cl
H
OCH3
—
OCH3
OCH3
—
Doxorubicin (Doxo)
—
may be the saturation concentration for inducing cell cycle effects.
In addition the increased concentration resulted in increased per-
centage of cells that undergo apoptosis as indicated by cells accu-
mulated in G0 phase (Fig. 3).
Since breast cancer cells are highly resistant to anticancer drugs
due to multidrug resistance (MDR), we were interested to examine
the molecular mechanism of action of these molecules in ER posi-
inhibits apoptosis induced by anticancer drugs, radiation, and
other DNA-damaging agents.32 Moreover 17-b-estradiol induces
bcl-2 gene transcription in human breast cancer cell line MCF-7
via two estrogen response element (ERE’s)33 and also BCl-2 and
survivin proteins are responsible for rapid cell proliferation by
functioning as anti-apoptotic proteins (IAPs). It was considered of
interest to examine the protein levels in the compound treatment
scenario. To our surprise these molecules inhibits the expression of
Bcl-2 and survivin. This can function by inhibiting the interaction
between ERE and ER alpha at Bcl-2 and effect the downstream sur-
viving protein. Further we have also examined the possibility of
effect of compounds on cyclin D1, the protooncogene that is func-
tions at G1-S phase transition, no significant changes were
observed in the level of this protein (Fig. 6).
tive MCF-7 cells. In breast cells estrogen receptors such as ER
a, ERb
are expressed and are activated by estrogen hormone.30 High ER
a
expression is associated with tumor growth in breast and high
levels ERb inhibit tumor growth.3
It is well established fact that ligands such as 17-b-estradiol and
genistein bind to estrogen receptor and cause breast cancer.
The activated ERa regulate gene expression by binding to con-
sensus and non-consensus ERE (50-GGTCAnnnTGACC-30) on target
promoters and thus acts as transcription factor that regulate the
breast cancer cell proliferation.31 Thus we have examined the
Model proposed: Conjugates inhibited the expression of ER alpha
and down regulated the proteins that are vital for breast cancer cell
proliferation such as Bcl-2, cyclin D and survivin that finally result
in cell cycle perturbations and apoptosis (see Fig. 7)
effect of these conjugates on ERE and ERa protein expression by
conducting luciferase based studies by treating MCF-7 cells with
the effective compounds (6a, 6c, 6j, 6m, 6o, 6q, 6r, 6s, 6t, 6w)
obtained from MTT assay at 1 lM concentration for 24 h, wherein
etoposide (ETO) and doxorubicin (Doxo) are used as standard
To summarise, in this work new chemical entities based on the
conjugation of potent moieties pyrazolo[1,5-a]pyrimidine and 2-
anilinonicotinic acid linked by conformationally restricted piper-
azineamide spacers were synthesized by a convenient synthetic
route. These chemical entities are tested for their anticancer prop-
erties followed by detailed biological studies to understand their
mechanism of action. Some of the conjugates like 6a, 6c, 6r and
6s are found to be promising antiproliferative agents by regulating
antiapoptotic proteins and proto-oncogene. Thus it could be con-
drugs as shown in Figures 4 and 5.
Small molecules that target ERE play a crucial role in controlling
breast cancer cell proliferation. Bcl-2 is the major anti-apoptotic
protein that confers resistance to apoptosis by reducing the effec-
tiveness of chemotherapy and inhibits the mitochondrial apoptosis
pathway. Further studies also indicate that over expression of Bcl-2
cluded that conjugates 6a and 6c that effects ERE–ERa interaction
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A. Kamal et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 3. The cell cycle analysis was conducted in MCF-7 cells treated with 6a, 6c at concentrations of 4, 8, 16, 32 lM for 48 h. Here doxorubicin (Doxo), the anthracycline
antibiotic as well as one of most effective anticancer agent used to treat against breast cancer was used as positive control. Conjugates induced G2/M cell cycle arrest.
Increased concentration of drug caused apoptosis as indicated by percentage of cells in G0 phase.
Effect on ERE
12000
ERα expression
12000
10000
8000
6000
4000
2000
0
10000
8000
6000
4000
2000
0
Figure 4. Effect on ERE: the effect compound on ERE was studied in MCF-7 cells at
Figure 5. Effect on ER alpha: the effect of compounds on ER alpha was studied in
1
l
M conc of 6a, 6c, 6j, 6m, 6o, 6q, 6r, 6s, 6t and 6w for 24 h. Here ETO: etoposide.
MCF-7 cells at 4 lM conc of 6a, 6c, 6f, 6m, 6n, 6o, 6p, 6r, 6s for 24 h. Here ETO:
Doxo: doxorubicin. Initially the transfection with ERE-Luc is carried out for 48 h
etoposide. Doxo: doxorubicin. Initially the transfection with ERa are carried out for
followed by compound treatment for 24 h.
48 h followed by compound treatment for 24 h.
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7
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.02.
072.
References and notes
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as well as 6r and 6s that affects ERa could be considered as effec-
tive molecules and have the potential to be taken up for preclinical
studies as promising therapeutics for the treatment of breast
cancer.
Acknowledgments
F.S., M.B. and S.M.A.H. thank UGC and CSIR, New Delhi, India for
the award of research fellowships and the financial support
received under the 12th Five Year plan project ‘‘Affordable Cancer
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Please cite this article in press as: Kamal, A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.072