Identification of novel estrogen receptor (ER) agonists that have additional and complementary anti-cancer activities via ER-independent mechanism

Article abstract of DOI:10.1016/j.bmcl.2016.01.089

In this study, a series of bis(4-hydroxy)benzophenone oxime ether derivatives such as 12c, 12e and 12h were identified as novel estrogen receptor (ER) agonists that have additional and complementary anti-proliferative activities via ER-independent mechanism in cancer cells. These compounds are expected to overcome the therapeutic limitation of existing ER agonists such as estradiol and tamoxifen, which have been known to induce the proliferation of cancer cells.

Full text of DOI:10.1016/j.bmcl.2016.01.089

Accepted Manuscript
Identification of novel estrogen receptor (ER) agonists that have additional and
complementary anti-cancer activities via ER-independent mechanism
Taelim Kim, Hye-In Kim, Ji-Young An, Jun Lee, Na-Rae Lee, Jinyuk Heo, Ji-
Eun Kim, Jihyun Yu, Yong Sup Lee, Kyung-Soo Inn, Nam-Jung Kim
PII:
S0960-894X(16)30101-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.089
BMCL 23583
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
14 December 2015
25 January 2016
30 January 2016
Please cite this article as: Kim, T., Kim, H-I., An, J-Y., Lee, J., Lee, N-R., Heo, J., Kim, J-E., Yu, J., Lee, Y.S., Inn,
K-S., Kim, N-J., Identification of novel estrogen receptor (ER) agonists that have additional and complementary
anti-cancer activities via ER-independent mechanism, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.01.089
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Graphical Abstract
Identification of novel estrogen receptor
Leave this area blank for abstract info.
(ER) agonists that have additional and
complementary anti-cancer activities via ER-
independent mechanism
Taelim Kim, Hye-In Kim, Ji-Young An, Jun Lee, Na-Rae Lee, Jinyuk Heo, Ji-Eun Kim, Jihyun Yu, Yong Sup
Lee, Kyung-Soo Inn*, Nam-Jung Kim*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Identification of novel estrogen receptor (ER) agonists that have additional and
complementary anti-cancer activities via ER-independent mechanism
a,†
b,†
a
a
b
a
b
Taelim Kim , Hye-In Kim , Ji-Young An , Jun Lee , Na-Rae Lee , Jinyuk Heo , Ji-Eun Kim , Jihyun
a
Yu , Yong Sup Lee , Kyung-Soo Inn and Nam-Jung Kim
a
b, ∗
a, ∗
a
Department of Pharmacy, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea
Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b
ARTICLE INFO
ABSTRACT
Article history:
Received
In this study, a series of bis(4-hydroxy)benzophenone oxime ether derivatives such as 12c, 12e
and 12h were identified as novel estrogen receptor (ER) agonists that have additional and
complementary anti-proliferative activities via ER-independent mechanism in cancer cells.
These compounds are expected to overcome the therapeutic limitation of existing ER agonists
such as estradiol and tamoxifen, which have been known to induce the proliferation of cancer
cells.
Revised
Accepted
Available online
Keywords:
2
009 Elsevier Ltd. All rights reserved.
Estrogen Receptor (ER) Agonists
Anti-Cancer Activities
Bisphenol
Oxime Ether
Complementary
—
——
∗
Corresponding author. Tel.: +82-2-961-0368; fax: +82-2-966-3885; e-mail: innks@khu.ac.kr (K.-S. Inn)
Corresponding author. Tel.: +82-2-961-0580; fax: +82-2-966-3885; e-mail: kimnj@khu.ac.kr (N.-J. Kim)
∗
†
These authors contributed equally to this work.

Estrogens, endogenous agonists of estrogen receptors (ERs)
1
known as members of the nuclear receptor gene family , play
Thus, discovery of novel ER agonists that can not only alleviate
postmenopausal syndrome but also lower the risk of cancers
remains necessary to overcome such the therapeutic limitation of
existing ER agonists such as estradiol (1) and tamoxifen (2).
Considering that ER-mediated signaling intrinsically increases
the risk of cancers by inducing the proliferation of cancer cells in
some tissues such as breast and endometrial tissues, it was
assumed that the ER agonists having additional and
complementary anti-cancer activities via ER-independent
mechanism, neutralizing their ER-associated cancer cell
proliferation, might be desired for efficacious and safe treatment
of postmenopausal syndrome and its related diseases.
crucial roles in diverse physiological processes including
reproductive events, depending on the target organs in the
2
,3
reproductive tract, liver, bone, and cardiovascular system. Lots
of women at postmenopausal stage suffer from postmenopausal
syndrome of which symptoms are hot flashes, depression,
osteoporosis, atherosclerosis, acute myocardial infarction and
other cardiovascular diseases, caused by low estrogen level. To
alleviate the symptoms, ER agonists such as estrogens and
selective ER modulators (SERMs) have been used for past
decades, respectively (Figure 1). Estradiol (1) is one of the
estrogens and has been used as a representative agent for
hormone replacement therapy (HRT) when estrogen deficiency
occurs. Recently, its use has been significantly decreased since
the report from Women’s Health Initiative (WHI) that HRT
might be beneficial in patients with postmenopausal syndrome
but rather possible to increase the risk of breast cancer due to its
It has been well known that many ER agonists including
daidzein (3), and bisphenol A (4) are generally made up of a rigid
center and two phenol moieties, which form hydrogen bondings
with Glu353, Arg394 and His524 in the human ER ligand
binding domain (LBD). This interaction is crucial for the
7
4
activation of ER signaling. Recently, some rigid oxime ether (5),
(
ER agonistic activity in breast tissue.
6) and (7), derived from biaryl ketones have been reported to
display anti-proliferative activities in cancer cells, regardless of
their various substituents on biaryl moieties or alkyl substituents
8
on oxime ethers. Based on the insights gained from those reports,
we envisaged that bis(4-hydroxy)benzophenone oxime ether
derivatives comprised of a rigid oxime ether in a center and two
phenol moieties as terminal aryl groups could be novel scaffolds
which not only have ER agonistic activities but also additional
and complementary anti-cancer activities. Thus, it is
distinguished from existing ER agonists such as estradiol (1) and
tamoxifen (2), which have been known to induce the proliferation
of cancer cells. Herein, we report the identification of novel ER
agonists that have additional and complementary anti-
proliferative activities via ER-independent mechanism in cancer
cells. (Figure 2)
Figure 1. Structures of estradiol (1) and tamoxifen (2)
SERMs including tamoxifen (2) have been also used as
therapeutic agents for the treatment of postmenopausal syndrome.
They can act as agonists in the majority of tissues for relieving
the symptom but antagonists of the ERs in breast tissue for
lowering the risk of hormone-dependent ERα positive breast
5
cancer. However, it has been known that tamoxifen (2),
sometimes used as anti-cancer agents for ER-(+) breast cancer,
might be linked to an increased risk of endometrial cancer in
some women, due to its partial ER agonistic activity in
6
endometrial tissues.
CO2Et
O
OH
O
OH
N
N
N
7
O
OMe
HO
OH
N
O
H3CS
HO
O
N
O
Daidzein (3)
Bisphenol A (4)
5
6
N
H
R
R'
n
n
O
N
O
Center
N
aryl
aryl
HO
OH
HO
OH
Bisphenols with
ER agonistic activities
Novel ER agonists that have
additional and complementary
anti-cancer activities
Oxime ethers with
anti-cancer activities
Figure 2. Design of novel estrogen receptor (ER) agonists that have additional and complementary anti-cancer activities

2 2 2 2 2
Scheme 1. Synthesis of compounds 12a-m. Reagents and conditions: (a) alkyl halide, DBU, DMF, rt - 50°C, 59-88%; (b) NH NH ·H O, CH Cl , then 2N HCl in
dioxane, 43-96%; (c) 10a-m, EtOH, 45-100%.
We intended to mainly synthesize a series of bis(4-hydroxy)
benzophenone oxime ethers equipped with linear alkyl chains,
based on the previous reports that the compounds possessing
With synthesized compounds in hands, we performed estrogen
responsive element (ERE)-luciferase assay to assess the effects of
the compounds on their ERE-mediated transcriptional activities
and MTT assay to investigate their anti-cancer activities in breast
cancer cells. Assay result of the compounds was shown in Figure
3. Most of synthesized compounds showed significant ERE-
mediated transcriptional activities, similar to bisphenol A (4,
8
linear alkyl oxime ether chains showed anti-cancer activities. As
outlined in Scheme 1, a variety of compounds were conveniently
synthesized from benzophenone 11 via a concise unified strategy.
Most hydroxylamine HCls were prepared from commercially
available N-hydroxyphthalimide (8), which was transformed into
alkoxyphthalimides using DBU and the corresponding alkyl
1
0
BPA), known as one of ER agonists. However, contrary to our
expectation, many derivatives did not inhibit and even increase
the growth of MCF-7, ER-(+)-breast cancer cells, which was in
consistent with the reports that ER agonists could induce
9
halides. Following imide cleavage and salt formation in the
presence of hydrazine monohydrate and HCl provided various
alkoxyamine HCl intermediates. Other alkoxyamine salts were
available from commercial sources. Concise condensations of the
commercially available bis(4-hydroxy)benzophenone 11 with
alkyloxyamine salts yielded the desired compounds (12a-m).
4
proliferation of ER-(+)-breast cancer cells. Notably, a few
compounds such as 12c, 12e and 12h did not induce cancer cell
proliferation but exhibited moderate anti-proliferative activities
on MCF-7 cells. This result implied that these compounds might
be novel ER agonists that have additional and complementary
anti-cancer activities as we intially designed. On the basis of
these results, we selected compound 12c, 12e and 12h as hit
compounds for further biological evaluation of their ER agonistic
activities and anti-cancer activities.
Table 1. EC50s of 12c, 12e and 12h on ERE-mediated transcriptional
activities
R
O
N
HO
OH
a
Compound
R
EC50 (µM)
1
2c
n-butyl
0.075
1
1
2e
4-pentenyl
0.046
0.077
0.290
2h
i-pentyl
BPA (4)
a
EC50 values of the test compounds were estimated from the sigmoidal
Figure 3. (a) ERE-mediated transcriptional activities of synthesized
compounds at 1 µM. (b) Anti-proliferative effects of synthesized compounds
on MCF-7 breast cancer cells at 10 µM.
dose-response curves using SigmaPlot 10.0 software.

To further evaluate the ERE-mediated transcriptional activities
of compound 12c, 12e and 12h, we tried to determine EC50s of
To confirm ER agonistic activities of the compounds, we also
examined the ERα phosphorylation, a hallmark of ER activation.
As shown in Figure 4(b), treatment with 12c, 12e or 12h induced
significant phosphorylation of ERα, indicating its activation.
Based on the all of above results, it was concluded that 12c, 12e
and 12h would be potent ER agonists.
12c, 12e and 12h. As summarized in Table 1, their EC50 values
obtained from the assay were 75, 46 and 77 nM, indicating that
they have significant ERE-mediated transcriptional activities,
compared to BPA (4). With the result, we tried to confirm
whether the identified compounds could activate ER mediated
signal transduction.
Figure 5. X-ray structure of 4 (Cyan, PDB Code: 3UU7) and molecular
docking model of 12c with ERα (Purple, PDB code: 3UU7), which were
1
1
visualized using Chimera 1.10 (UCSF Chimera)
Figure 4. (a) Effects of 12c, 12e and 12h on ER-mediated transcription level
of GREB1 in ER-(+) MCF-7 cells at 10 µM. Data are presented as means ±
SD. (b) Analysis of ER phosphorylation upon treatment with estradiol (E2,
To investigate the binding modes of bis(4-hydroxy)
benzophenone derivatives which were identified as novel and
potent ER agonists in our study, docking analysis of compound
1
0 nM, 100 nM), 12c (10 µM), 12e (10 µM) or 12h (10 µM).
1
2c within hER LBD active site was performed using the
Using the quantitative polymerase chain reaction (qPCR) assay,
12
Autodock 4.2 program (Molecular Graphic Laboratory). As
illustrated in Figure 5, 12c fitted well into the active site. The
estimated free energy of binding was calculated as -8.19 kcal/mol.
It was plausible that two phenol moieties in our compounds
interact the hER LBD through hydrogen bonding with Glu353
and His 524, respectively, whereas n-butyl group occupies a
lipophilic pocket which was surrounded by Thr 347, Leu346, Leu
384 and Trp 383 and makes van der waals interaction with those
residues. Thus, the binding mode of our compound was
postulated in accordance with that of bisphenol A (4).
we investigated the capabilities of compound 12c, 12e and 12i to
increase the cellular level of Growth Regulation by Estrogen in
Breast cancer 1 (GREB1), one of the representative ER
responsive gene of which expression was increased by ER
activation. As depicted in Figure 4(a), treatment with 10 µM of
the compounds resulted in the increase of mRNA level of
GREB1 similar or more, compared to the treatment with estradiol.
Figure 6. Anti-proliferative activities of 12c, 12e and 12h against breast cancer cells. (a) Dose-dependent anti-proliferative activities of 12c, 12e and 12h against
MCF-7 and MDA-MB-231 breast cancer cell lines as determined by MTT assays. (b) DAPI staining of MCF-7 cells treated with 12c, 12e and 12h at 10 µM.
Scale bar: 10 µm. Arrows indicate condensed nuclei. (c)-(e) Analysis of apoptosis of MCF-7 cells treated with 12c (c), 12e (d) and 12h (e) at 10 µM by Annexin
V staining.

With the result that 12c, 12e and 12h showed potent ER
agonistic activities, we turned our attention to further study of
their additional and complementary anti-cancer activities,
neutralizing ER-mediated cell proliferation in cancer cells.
Through the MTT assay results (Figure 3), 12c, 12e and 12h
were found to have anti-cancer activities in MCF-7, breast cancer
cells. To investigate whether their anti-cancer activities are
associated with ER dependent mechanism, we performed MTT
assay with increasing concentrations at ER-(+) or ER-(-) cancer
cells. These compounds inhibited the growth of MCF7, ER-(+)
breast cancer cells in dose dependent manners. Notably, the
compounds also showed dose dependent anti-proliferative
activities toward MDA-MB231 cells, ER-(-) breast cancer cell
lines, indicating that anti-cancer activities of these compounds
might not be resulted from their ER-mediated signaling but from
ER-independent signaling (Figure 6(a)). Next, we have examined
whether anti-cancer activities of these compounds were caused
by the induction of apoptosis. As shown in Figure 6(b), DAPI
staining of MCF-7 cells treated with 10 µM of the compounds
showed the morphological changes including membrane blebbing,
or condensed nuclei, which have been known as typical features
of apoptosis in cancerous cells. To further evaluate the apoptotic
cell death by the compounds, additional analysis of MCF7 cells
treated with 12c, 12e and 12h was performed. As shown in
Figure 6(c)-(e), the cell death of cancer cell, caused by the
compounds, was associated with apoptosis as determined by
AnnexinV staining, followed by flow cytometry analysis.
Moreover, increment of subG1 population in 12c, 12e and 12h
treated cells were observed in cell cycle analysis by PI staining.
All of these results indicated that the compounds have anti-
proliferative activities by inducing ER-independent apoptosis of
(NRF), which was funded by the Ministry of Science, ICT &
Future Planning (NRF-2013R1A1A1062292).
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Figure 7. Cell cycle analysis of MCF-7 cells treated with 12c, 12e and 12h
at 10 µM by PI staining
1
3. Compound 12h also showed the anti-cancer effects on endometrial
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Oncol. 2015, 47, 280.
In summary, we identified
a
series of bis(4-
hydroxy)benzophenone oxime ether derivatives as ER agonists
that have additional and complementary anti-proliferative
activities via ER-independent mechanism in cancer cells, based
on rational design, convenient synthetic approaches and
biological evaluation. In particular, the compounds such as 12c,
Supplementary Material
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
appropriate editorial office.
12e and 12h showed significant estrogenic activities but inhibit
the growth of cancer cells through ER-independent mechanism,
which is distinguished from existing ER agonists such as
estradiol and tamoxifen, which have been known to induce the
proliferation of cancer cells. Further work for the development of
therapeutically useful novel ER modulators based on our current
study is in progress.
Acknowledgments
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea

Graphical Abstract
Identification of novel estrogen receptor
Leave this area blank for abstract info.
(ER) agonists that have additional and
complementary anti-cancer activities via ER-
independent mechanism
Taelim Kim, Hye-In Kim, Ji-Young An, Jun Lee, Na-Rae Lee, Jinyuk Heo, Ji-Eun Kim, Jihyun Yu, Yong Sup
Lee, Kyung-Soo Inn*, Nam-Jung Kim*
In this study, we identified a series of bis(4-hydroxy)benzophenone oxime ether derivatives as novel ER agonists that
have additional and complementary anti-proliferative activities via ER-independent mechanism in cancer cells, based
on rational design, convenient synthetic approaches and biological evaluation. In particular, the compounds such as 12c,
1
2e and 12h showed significant estrogenic activities but inhibit the growth of cancer cells through ER-independent
mechanism, which is distinguished from existing ER agonists such as estradiol and tamoxifen, which have been
known to induce the proliferation of cancer cells.