Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen receptor alpha partial agonist

Article abstract of DOI:10.1016/j.bioorg.2019.102977

Selective estrogen receptor modulators (SERMs)act as either agonist or antagonist of estrogen receptor (ER)in a tissue selective manner and have been used in several diseases such as breast cancer, postmenopausal syndrome, osteoporosis, and cardiovascular diseases. However, current SERMs may also increase the risk of serious side effects and trigger drug resistance. Herein, a screening program, that was designed to search for novel SERMs, resulted in the identification of a series of 2-arylbenzofuran-containing compounds that are ligands for ERα, when applying the Gaussia-luciferase reporter assay. One of these compounds, 10-dehydrooxyglycyuralin E (T9)was chemically synthesized. T9 showed anti-estrogenic/proliferative activity in ERα-positive breast cancer cells. Pretreatment of T9 prevented the mRNA expression of GREB1, which is an estrogen response gene. Furthermore, by an in silico docking simulation study we demonstrated that T9 showed interactions directly to ERα. Taken together, these results demonstrated that T9 is a candidate of SERMs and a useful seed compound for the foundation of the selective activity of SERMs.

Full text of DOI:10.1016/j.bioorg.2019.102977

Bioorganic Chemistry 88 (2019) 102977
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen
receptor alpha partial agonist
a
a
a
b
b
b
Nao Saito , Keiko Kawase , Naoya Yamashita , Yingzhan Tang , Ying Wang , Jian Wang ,
b
b
b,c,⁎
b,⁎
c
a,⁎
Yongxiang Liu , Ning Li , Wei Li
, Mao-Sheng Cheng , Kazuo Koike , Yuichiro Kanno ,
a
Kiyomitsu Nemoto
a
b
c
Department of Molecular Toxicology, Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Selective estrogen receptor modulators (SERMs) act as either agonist or antagonist of estrogen receptor (ER) in a
tissue selective manner and have been used in several diseases such as breast cancer, postmenopausal syndrome,
osteoporosis, and cardiovascular diseases. However, current SERMs may also increase the risk of serious side
effects and trigger drug resistance. Herein, a screening program, that was designed to search for novel SERMs,
resulted in the identification of a series of 2-arylbenzofuran-containing compounds that are ligands for ERα,
when applying the Gaussia-luciferase reporter assay. One of these compounds, 10-dehydrooxyglycyuralin E (T9)
was chemically synthesized. T9 showed anti-estrogenic/proliferative activity in ERα-positive breast cancer cells.
Pretreatment of T9 prevented the mRNA expression of GREB1, which is an estrogen response gene. Furthermore,
by an in silico docking simulation study we demonstrated that T9 showed interactions directly to ERα. Taken
together, these results demonstrated that T9 is a candidate of SERMs and a useful seed compound for the
foundation of the selective activity of SERMs.
Estrogen receptor (ER)
Selective estrogen receptor modulators
(
SERMs)
Partial agonist
Breast cancer
Anti-estrogenic activity
Anti-proliferative activity
1
. Introduction
The estrogen receptor (ER) belongs to the nuclear receptor super-
are used to fight estrogen related diseases [7], including post-
menopausal endometriosis [8], uterine leiomyomas [9], and estrogen-
dependent breast cancer [10]. Especially, ERα has been extensively
studied in breast cancer [11,12].
family of ligand-dependent transcription factors [1,2]. ER is classified
into two subtypes, the ERα (NR3A1) and the ERβ (NR3A2) [3–5]. Al-
though both subtypes are activated by the hormone estrogens (17β-
estradiol), there exists a large differences between ERα and ERβ in their
sequence homology, organs/tissues expression, and targeted gene
function [5]. ER plays crucial roles in diverse biological effects de-
pending on target organs such as the development of female re-
productive system, protection of cardiovascular tissue and maintenance
of the bone mass.
Selective estrogen receptor modulators (SERMs), which are com-
petitive partial agonists of the ER, take much advantage of selective
effects of ER in estrogen target tissues than ER full agonists [13,14].
Currently available SERMs, such as tamoxifen [15,16], raloxifene
[17,18] and bazedoxifene [19,20], have been developed into useful
pharmacological agents for the treatment and prevention of breast
cancer and menopausal osteoporosis. However, it has also been re-
ported that the unremitting use of SERMs may increase the risk of
several serious side effects, such as strokes, deep vein thromboembolic
disorders [21], and drug resistance [22]. Therefore, in clinical research
studies, the tissue-selective estrogen complex (TSEC), conjugation of
Hormone replacement therapy (HRT) is used for the regulation of
estrogen levels to alleviate menopausal symptoms (e.g. hot flashes) and
treat diseases (e.g. osteoporosis) [6]. In contrast, anti-estrogenic agents
Abbreviations: SERMs, selective estrogen receptor modulators; ER, estrogen receptor; ERE, estrogen response elements; T9, 10-dehydrooxyglycyuralin E; GREB1,
growth regulation by estrogen in breast cancer 1; E2, 17β-estradiol; HRT, hormone replacement therapy; TSEC, tissue-selective estrogen complex; SERD, selective
estrogen receptor degradator
⁎
Corresponding authors at: Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan (Y. Kanno, W. Li). Key
Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, PR China (M-S.
Cheng).
E-mail addresses: liwei@phar.toho-u.ac.jp (W. Li), mscheng@syphu.edu.cn (M.-S. Cheng), ykanno@phar.toho-u.ac.jp (Y. Kanno).
https://doi.org/10.1016/j.bioorg.2019.102977
Received 4 February 2019; Received in revised form 3 May 2019; Accepted 7 May 2019
Available online 08 May 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
Fig. 1. Chemical structures.
estrogens with a selective SERM, has been expected to be the new
menopausal therapy that will improve vasomotor symptoms and pro-
tect against osteoporosis without concurrent endometrium and breast
stimulation [23,24].
10 μM, T1, T5, and T6 showed the most potent ERα agonistic activity
comparable to that of estradiol (E2), but no cytotoxic effect was ob-
served. However, these compounds were excluded from further
screening as we were searching for partial agonists for ER, as SERM, but
not full (perfect) antagonists or agonists. The compounds T2, T7, T9,
T10, T12, T13, T15, T17, and T19, which showed partial agonistic
activity, were then selected for secondary screening for their ERα
agonistic activity by the estrogen response elements (ERE)-dual luci-
ferase assay. As shown in Fig. 3, five compounds (T2, T7, T9, T10, and
T12) showed partial ERα agonistic effects compared to E2.
Briefly, discovery of novel SERMs are essential and critical for drug
development of estrogen-dependent diseases. In the present study, we
conducted a screening program searching for novel SERMs, and re-
sulted in the identification of a scaffold of 2-arylbenzofuran-containing
compounds showing ERα agonistic activity (Fig. 1). Among them, a
compound named 10-dehydrooxyglycyuralin E (T9) exhibited partial
agonist activity for ERα and anti-proliferative effect on breast cancer
cells.
To test the anti-estrogenic effect of these compounds in ER-positive
breast cancer [10], their proliferative effect was evaluated by the MTS
assay in the MCF-7 cells (Fig. 4). Whereas most compounds showed
estrogenic/proliferative effect, T9 did not enhance cell proliferation
(Fig. 4A), and also prevented estrogen induced cell proliferation
2
. Result and discussion
(
Fig. 4B). These results suggested that T9 was a candidate compound
2
.1. Screening of 2-arylbenzofuran-containing compounds as ERα partial
for SERM, and was selected for further detailed investigation of its ERα
agonistic activity.
agonists
Primary screening of ERα agonistic activity was carried out by a
Gaussia-luciferase reporter assay and a few of 2-arylbenzofuran-con-
taining compounds were identified as potential hits (Figs. 1 and 2).
Compounds T1–T22 were a series of 2-arylbenzofuran-contaning nat-
ural products and their derivates, which were chemically synthetized
by a cascade [3,3]-sigmatropic rearrangement/aromatization strategy
in a previous investigation [25]. A test concentration of 10 μM was set
in primary screening (GAL-RE Gaussia-luciferase reporter assay), so that
the potential hits possess acceptable effectiveness to endure further
evaluation assay for drug development. At a final concentration of
2.2. Chemical synthesis of T9
The chroman derivative T9 was synthesized following our previous
reported procedure using a cascade [3,3]-sigmatropic rearrangement/
aromatization strategy as the key step [25]. The ether precursor 5 was
prepared by Mitsunobu reaction from benzofuran-3-ylmethanol 3 and
phenol 4, which were synthesized by known methods starting from
compound 1. The synthesized substrate 5 was mixed with silica gel and
heated to 140 °C under neat conditions for 4 h leading to the desired
2

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
Fig. 2. Effect of compounds T1–T22 on transacti-
vation of Estrogen receptor alpha using GAL-RE
Gaussia-luciferase reporter assay.
HEK293 cells were transfected with the expression
vector for GAL4/DBD-ERα/LBD and GAL-RE
driven Gaussia-luciferase reporter vector. The cells
were treated with compounds (10 μM) and E2
(
10 nM) for 24 h, and Gaussia-luciferase activity
was measured. The results are the average activity
of two different wells (n = 2).
not changed (Fig. 7B). Thus, T9 acts as antagonist of ERα in MCF-7
cells.
To investigate the effect of T9 on cell viability, the cells were treated
with increasing concentration of T9 (0.1–30 μM) in the absence or
presence of E2 and ICI (Fig. 8). E2-dependent cell proliferation was
suppressed by T9. However, T9 caused ER-independent cell growth in
low concentrations, as this observation was not prevented by the co-
treatment with the selective estrogen receptor degradator (SERD), ICI
(
1 µM) [28].
2.4. Docking simulation
Considering the above experimental results, molecular docking
calculation was conducted to explore the binding pattern of T9 with
ERα. The crystal structure of ERα complexed with ligand was selected
as a receptor model for the docking study. Meanwhile, the binding
mode of ERα with this native ligand provided us with a valuable re-
ference to achieve accuracy in the predicted interactions between T9
and ERα. The docked pose displaying the best binding affinity was
analyzed and compared to that of the co-crystal ligand. The binding
energy of T9 with ERα was −7.13 kcal/mol, while for the native ligand
was −8.88 kcal/mol. Navigating the binding pocket, the hydroxy
moiety in benzofuran of T9 is involved in a hydrogen bound interaction
with Glu353 of ERα, as that was also observed in the co-crystal struc-
ture [29–31]. The hydrophobic interactions between T9 and several
residues such as Leu349, Leu346, Leu391, Leu428, Met343, were also
consistent with the binding modes of ERα and its co-crystal ligand
(Fig. 9). Furthermore, a surface, generated with T9 in the docking
conformation with the lowest binding affinity, fit the acceptor well,
which certified the probability of T9 to exert its bioactivity with a direct
interaction with ERα (Fig. 10).
Fig. 3. Effect of compounds T2, T7, T9, T10, T12, T13, T15, T17, and T19 on
transactivation of ERα using ERE luciferase reporter assay.
HEK293 cells were transfected with the expression vector for ERα, ERE driven
luciferase reporter vector, and pGL4.74, Renilla reporter vector. The cells were
treated with compounds (10 μM) or E2 (10 nM) for 24 h, and luciferase activity
was measured. The results are shown as mean ± SD (n = 3).
product 2 with a yield of 73%. After several functional transformations
including O-methylation, hydrogenation, MOM protection of hydroxyl
groups and bromination, the intermediate 10 was obtained in a mod-
erate yield. Finally, the chroman derivative T9 was generated by a
palladium-catalyzed Suzuki coupling between bromide precursor 10
and prenyl boronic ester 11 and a CSA promoted cascade MOM-de-
protection and cyclization reaction (Fig. 5).
2.3. ERα agonist activity
3
. Conclusions
To examine the subtype selectivity of T9 between ERs, HEK293 cells
were transfected with ERE luciferase reporter and co-transfected with
ERα or ERβ. T9 repressed the transcriptional activity of ERα at a con-
centration of 10 μM (Fig. 6A). In contrast, T9 could not repress tran-
scriptional activity of ERβ at the same concentration (Fig. 6B). These
results indicated that T9 is subtype selective ligand for ERα.
SERMs exhibiting tissue-selective and partial agonistic activities are
useful therapeutic drug candidates for the treatment of various es-
trogen-dependent diseases, such as breast cancer, postmenopausal
syndrome, osteoporosis, and cardiovascular diseases. During
a
screening program, that was designed to search for novel SERMs tar-
geting ERα, a series of 2-arylbenzofuran-containing compounds were
identified as partial ERα agonists. Among them, 10-dehydroox-
yglycyuralin E (T9) showed the most promising anti-estrogenic/pro-
liferative activity in ERα-positive breast cancer cells. Thus, T9 was
chemically synthesized and further investigated its ERα agonistic ac-
tivity. T9 was demonstrated to be ER-subtype selective partial agonist
of ERα but not ERβ. Furthermore, pretreatment of T9 prevented E2-
induced GREB1 mRNA expression, but not affected the protein levels of
Next, we examined the antagonistic effects of T9 on ERα-target gene
regulation in MCF-7 cells. Cells were pretreated with T9 for 30 min and
co-treated with E2 for 6 h. mRNA expression level of Growth Regulation
by Estrogen in Breast cancer 1 (GREB1) [26,27], one of the estrogen
response genes, was increased by E2 treatment (Fig. 7A). Pretreatment
of T9 prevents E2-induced GREB1 mRNA expression. Further, we ex-
amined whether T9 affects the protein levels of ERα, treating the MCF-7
cells with T9 in the absence or presence of E2; ERα protein levels were
3

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
Fig. 4. Effect of compounds T1–T22 on es-
trogen-mediated breast cancer cell pro-
liferation.
MCF-7 cells were seeded on 96-well plates
4
(
1 × 10 cells/well) with phenol red free
DMEM supplemented with 5% charcoal-
stripped FBS. The cells were treated with
compounds (10 μM) in the absence (A) or
presence (B) of E2 (10 nM). After 72 h, cell
viability was evaluated by the MTS assay.
The results are shown as mean ± SD
(
n = 4).
+
ERα, indicating that T9 is also an antagonist of ERα. The in silico mo-
lecular docking simulation suggested that T9 directly interacts with
ERα. Taken together, these results demonstrated that T9 is a potential
SERM candidate for further drug development targeting ERα-related
diseases, but need further pharmacological investigations.
2790, 1614, 1501, 1486, 1110; HRMS (ESI-TOF) m/z [M + Na] Calcd.
for C19 Na 352.1236; Found 352.1230.
H
20 5
O
(6-(Benzyloxy)benzofuran-3-yl)methanol (3). To
a vigorously
stirred solution of potassium carbonate (50 g, 0.36 mol) in water
(400 mL) 2-(2-acetyl-5-(benzyloxy) phenoxy)acetic acid ethyl ester 2
(
100 g, 0.3 mol) was added and the mixture was gently refluxed for 3 h.
The solution was cooled down to 5 °C and acidified carefully with a
solution of concentrated HCl. The deposited precipitate was filtered off
and washed with cold water. The resulting crude product together with
anhydrous sodium acetate (96 g, 1.2 mol) was dissolved in acetic an-
hydride (390 g, 2.8 mol) and the mixture was heated at 160 °C for 3 h.
After cooling, the mixture was diluted with water (900 mL) and then
extracted with diethyl ether. The combined organic layers were washed
4
. Experimental section
4
.1. Experimental procedures and spectroscopic data of the synthesized
compounds
2
-(2-Acetyl-5-(benzyloxy)phenoxy)acetic acid ethyl ester (2). To a
solution of 2,4-dihydroxyacetophenone 1 (100.0 g, 0.657 mol) in dry
acetonitrile (500 mL) was added anhydrous potassium carbonate
with a saturated aqueous solution of Na
2
CO and brine, dried over
3
anhydrous Na
2
SO , and then evaporated to dryness. The mixture of the
4
(
100.0 g, 0.723 mol), and the mixture was refluxed for 1 h. Benzyl
dry crude product and selenium dioxide (33 g, 0.3 mol) in dry 1,4-di-
oxane (200 mL) was refluxed for 48 h. The resulting black precipitate
was filtered off and the crude product was evaporated to dryness. To
the solution of the crude mixture in methanol (100 mL) under stirring
bromide (76.6 mL, 0.644 mol) dissolved in dry acetonitrile (50 mL) was
added dropwise to the mixture. The reaction was refluxed for 3 h. The
reaction mixture was subjected to a silica gel column, eluted with ethyl
acetate and evaporated to dryness. The crude product, anhydrous po-
tassium carbonate (90 g, 0.657 mol), and ethyl chloroacetate (96.5 g,
were added small quantities of NaBH at room temperature until the
4
entire amount of the aldehyde was converted. Then, the reaction mix-
ture was treated with 5 M HCl (10 mL). The crude product was treated
with a mixture of ethyl acetate and water (1:1, v/v). The organic layers
were separated, washed with water, brined and then dried over anhy-
0
4
.788 mol) were dissolved in dry acetone (400 mL) and refluxed for
8 h. The precipitate was filtered off through silica gel, washed with
acetone and the filtrate was evaporated to dryness, purified by flash
column chromatography (ethyl acetate/petroleum ether = 1:20) to
1
drous Na SO , evaporated to dryness, and purified by flash column
give product 2 (129.3 g) as a white solid with a yield of 60%. H NMR
2
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:5,
(
600 MHz, DMSO-d ) δ 7.66 (d, J = 8.9 Hz, 1H), 7.45 (m, 2H), 7.41 (m,
6
R
f
= 0.1) to give product 3 (48.0 g) as a yellow powder with a yield of
2
1
1
1
7
H), 7.4.0 (m, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.9, 2.4 Hz,
1
6
3%. Mp 126.6–127.1 °C; H NMR (600 MHz, CDCl
3
) δ 7.55 – 7.51 (m,
H), 5.18 (s, 2H), 4.95 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 2.57 (s, 3H),
1
3
2H), 7.46 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.34 (d,
.22 (t, J = 7.1 Hz, 3H); C NMR (150 MHz, CDCl
3
) δ 197.8, 168.1,
J = 7.4 Hz, 1H), 7.09 (d, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.5, 2.2 Hz,
63.5, 159.1, 136.2, 133.1, 128.9, 128.5, 127.7, 122.0, 107.0, 100.1,
1H), 5.11 (s, 2H), 4.80 (s, 2H) ; 13C NMR (150 MHz, CDCl ) δ 157.5,
−
1
0.5, 65.7, 61.7, 32.2, 14.3; IR (thin film, cm ) 3410, 3036, 2933,
3
4

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
Fig. 5. Synthetic route to chroman derivative T9.
1
1
9
1
2
56.7, 141.6, 137.0, 128.8, 128.2, 127.6, 120.5, 120.4, 120.2, 112.7,
product 5 (1.9 g) as a yellow oil with a yield of 45%. H NMR (600 MHz,
−
1
7.6, 70.7, 56.2; IR (thin film, cm ) 3402, 3052, 2928, 2843, 1597,
CDCl ) δ 7.57 (s, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 7.4 Hz, 2H),
3
+
483, 1120; HRMS (ESI-TOF) m/z [M+H] Calcd. for C16
H
15
O
3
7.37–7.43 (m, 4H), 7.39 (t, J = 7.5 Hz, 6H), 7.32–7.35 (m, 3H), 7.09 (d,
J = 2.2 Hz, 1H), 6.99 (dd, J = 8.5, 2.2 Hz, 1H), 6.29 (s, 3H), 5.12 (s, 2H),
55.1021; Found 255.1016.
13
6
-(Benzyloxy)-3-((3,5-bis(benzyloxy)phenoxy)methyl)benzofuran (5).
5.10 (s, 2H), 5.01 (s, 4H); C NMR (150 MHz, CDCl ) δ 160.8, 160.6,
3
To the solution of (6-(benzyloxy)benzofuran-3-yl)methanol 3 (2.0 g,
mmol), 3,5-dibenzyloxy phenol 4 (2.9 g, 9.6 mmol) and triphenylpho-
sphine (3.2 g, 12 mmol) in dry THF was added diisopropyl azodiformate
157.6, 156.6, 142.5, 137.0, 136.9, 128.8, 128.7, 128.6, 128.2, 128.1,
8
127.7, 127.6, 127.4, 120.5, 120.3, 116.7, 112.9, 97.6, 95.2, 95.0, 70.7,
−1
70.3, 61.5; IR (thin film, cm ) 3430, 3031, 2873, 1624, 1599, 1454,
+
(
2.4 g, 12 mmol) dropwise at 0 °C for 15 min under nitrogen atmosphere.
1378, 1164, 1134, 1058; HRMS (ESI) m/z [M+H] Calcd. for C36
H O
31 5
The reaction was stirred at room temperature for 4 h. The crude mixture
was evaporated to dryness and purified by flash column chromatography
543.2166, Found 543.2174.
3,5-Bis(benzyloxy)-2-(6-(benzyloxy)-3-methylbenzofuran-2-yl)
on silica gel (ethyl acetate/petroleum ether = 1:50, R
f
= 0.3) to give
phenol (6). In
a
solution of 6-(benzyloxy)-3-((3,5-bis(benzyloxy)
Fig. 6. Effect of T9 on transactivation of ERα and
ERβ using ERE luciferase reporter assay.
HEK293 cells were transfected with the expression
vector for ERα (A), ERβ (B), ERE driven luciferase
reporter vector, and pGL4.74, Renilla reporter
vector. The cells were treated with T9 or E2
(
10 nM) for 24 h, and luciferase activity was mea-
sured. The results are shown as mean ± SD
n = 3), p < 0.01, p < 0.01.
*
*
##
(
5

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
phenoxy)methyl)benzofuran 5 (1.0 g, 1.8 mmol) in DCM (10 mL) silica
gel was added (2.0 g) and DCM was removed under vacuum. The
mixture was heated at 140 °C for 4 h. The crude mixture was purified by
flash column chromatography on silica gel (ethyl acetate/petroleum
ether = 1:10, R = 0.2) to give product 6 (713 mg) as a yellow oil with
f
1
a yield of 73%. H NMR (600 MHz, CDCl
3
) δ 7.47 (dd, J = 7.9, 0.9 Hz,
H), 7.44–7.42 (m, 2H), 7.41–7.38 (m, 5H), 7.36–7.32 (m, 2H),
.29–7.27 (m, 3H), 7.26–7.23 (m, 2H), 7.09 (d, J = 2.1 Hz, 1H), 6.98
2
7
(
dd, J = 8.5, 2.2 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 6.29 (d, J = 2.3 Hz,
1
H), 5.95 (s, 1H), 5.13 (s, 2H), 5.05 (s, 2H), 5.03 (s, 2H), 2.10 (s, 3H);
3
1
C NMR (150 MHz, CDCl ) δ 161.7, 158.6, 157.3, 156.7, 155.6, 143.9,
3
1
1
7
1
5
37.1, 136.8, 136.7, 128.8, 128.7, 128.6, 128.3, 128.1, 127.8, 127.7,
27.6, 127.1, 124.1, 119.7, 115.8, 112.2, 100.4, 97.3, 94.7, 94.2, 70.8,
−1
0.6, 70.3, 9.3; IR (thin film, cm ) 3433, 3032, 2921, 2854, 1629,
Fig. 7. Effect of T9 on ER-mediated gene expression and protein levels of ERα
in MCF-7 cells.
+
452, 1384, 1151; HRMS (ESI) m/z [M + H] Calcd. for C36
H
O
31 5
43.2166, Found 543.2173.
MCF-7 cells were pretreated with T9 for 30 min and co-treated with E2 for 6 h.
The cells were harvested. (A) The mRNA expression of GREB1 was measured by
RT-qPCR. The results were normalized against β-actin, and expressed as the
6-(Benzyloxy)-2-(2,4-bis(benzyloxy)-6-methoxyphenyl)-3-methyl-
benzofuran (7). To a suspension of NaH (60% dispersion in paraffin oil)
(40 mg, 1.0 mmol) in THF (4 mL) was added a solution of 3,5-bis(ben-
zyloxy)-2-(6-(benzyloxy)-3-methylbenzo furan-2-yl)phenol 6 (488 mg,
*
*
average of the fold induction over the solvent control (mean ± S.D., n = 3),
p < 0.01. Results show one representative result of two independent experi-
ments. (B) Whole-cell lysates of MCF-7 cells were resolved by SDS PAGE elec-
trophoresis and proteins were detected by immunoblotting using antibodies
against ERα and Tubulin.
0
.9 mmol) in dry THF (1 mL) at 0 °C with stirring under a nitrogen at-
mosphere. The mixture was stirred for 1 h at room temperature and
iodomethane (255 mg, 1.8 mmol) was added dropwise over 10 min. The
mixture was stirred at room temperature overnight; the reaction mix-
ture was quenched by the addition of a saturated aqueous solution of
ammonium chloride (20 mL) and extracted with ethyl acetate (20 mL).
The combined organic layers were washed with brine, dried over an-
hydrous Na
2
SO , evaporated to dryness, and purified by flash column
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:20,
R
f
= 0.3) to give product 7 (473 mg) as a yellow oil with a yield of 85%.
1
H NMR (600 MHz, CDCl
3
) δ 7.49–7.29 (m, 11H), 7.25–7.18 (m, 5H),
7
.09 (d, J = 2.2 Hz, 1H), 6.94 (dd, J = 8.5, 2.2 Hz, 1H), 6.30 (dd,
J = 12.4, 2.2 Hz, 2H), 5.12 (s, 2H), 5.06 (s, 2H), 5.02 (s, 2H), 3.75 (s,
1
3
3
1
1
9
2
H), 2.07 (s, 3H); C NMR (150 MHz, DMSO‑d ) δ 161.6, 160.4, 159.4,
6
56.7, 155.6, 145.2, 137.4, 137.1, 136.7, 128.8, 128.7, 128.5, 128.0,
27.8, 127.7, 127.6, 126.9, 124.4, 119.3, 114.4, 111.4, 102.7, 97.4,
−1
3.6, 92.4, 70.8, 70.7, 70.4, 56.1, 9.0; IR (thin film, cm ) 3428, 2923,
Fig. 8. Effect of T9 on breast cancer cell proliferation in the absence or presence
+
855, 1630, 1384, 1101; HRMS (ESI) m/z [M+H] Calcd. for
of E2 and ICI.
C
37
H
33
O 557.2323, Found 557.2332.
5
4
MCF-7 cells were seeded on 96-well plate (1 × 10 cells/well) with phenol red
4
-(6-Hydroxy-3-methylbenzofuran-2-yl)-5-methoxybenzene-1,3-
free DMEM supplemented with 5% charcoal-stripped FBS. The cells were
diol (8). To the solution of 6-(benzyloxy)-2-(2,4-bis(benzyloxy)-6-
methoxyphenyl)-3-methyl-benzofuran 7 (390 mg, 0.7 mmol) in me-
thanol (10 mL) was added 10% Pd/C (40 mg). The reaction vessel was
then evacuated, and the atmosphere replaced with hydrogen. After
treated with T9 (1–30 μM) in the absence or presence of E2 (10 nM) and ICI
(
1 μM). After 72 h, cell viability was evaluated by the MTS assay. The results are
shown as mean ± SD (n = 3).
Fig. 9. The key interactions between the co-crystal ligand (A) and T9 (B) and ERα were displayed in 2D style.
6

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
Fig. 10. Docking pose of T9 binding at the active pocket of ERα.
The protein is depicted in ribbon and the molecule was displayed as a ball and stick model with carbon atoms painted blue. A surface was generated to describe the
shape of the binding pocket.
vigorous stirring for 8 h, the reaction mixture was filtered through silica
gel and the filtrate was evaporated to dryness and purified by flash
column chromatography on silica gel (ethyl acetate/petroleum
5.09 (s, 2H), 3.58 (s, 3H), 3.56 (s, 3H), 3.52 (s, 3H), 3.38 (s, 3H), 2.12
13
(s, 3H); C NMR (150 MHz, CDCl ) δ 158.2, 156.9, 156.3, 155.4,
3
155.3, 144.5, 124.9, 119.5, 115.0, 112.6, 110.2, 101.1, 99.6, 99.5,
−1
ether = 1:2, R
f
= 0.1) to give product 8 (170 mg) as a colorless oil with
95.3, 95.2, 61.5, 56.8, 56.4, 56.1, 8.8; IR (thin film, cm ) 3422, 2922,
1
+
a yield of 85%. H NMR (600 MHz, CD
3
OD) δ 7.25 (d, J = 8.3 Hz, 1H),
2253, 2126, 1652, 1384, 1051, 1026, 1005; HRMS (ESI) m/z [M+H]
6
.80 (d, J = 2.1 Hz, 1H), 6.70 (dd, J = 8.3, 2.1 Hz, 1H), 6.04 (dd,
Calcd. for C22
H
26
O Br 497.0811, Found 497.0815.
8
1
3
J = 6.7, 2.1 Hz, 2H), 3.68 (s, 3H), 2.00 (s, 3H); C NMR (150 MHz,
2-(2-Methoxy-4,6-bis(methoxymethoxy)-3-(3-methylbut-2-en-1-yl)
phenyl)-6-(methoxymethoxy)-3-methylbenzofuran (12). To a solution
of 2-(3-bromo-2-methoxy-4,6-bis(methoxymethoxy)phenyl)-6-(meth-
oxymethoxy)-3-methylbenzofuran 10 (99 mg, 0.2 mmol) in dry di-
methylformamide (5 mL) was added cesium carbonate (130 mg,
0.4 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium
(II) (7 mg, 0.01 mmol), and 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-
yl)-1,3,2-dioxaborolane 11 (58 mg, 0.3 mmoml) under a nitrogen at-
mosphere. The mixture was stirred at 90 °C for 9 h. The reaction was
monitored by thin layer chromatography (TLC) till the complete con-
sumption of the starting material. The reaction mixture was diluted
with ethyl acetate (10 mL) and washed with water (15 mL). The organic
DMSO‑d
6
) δ 160.5, 159.9, 157.8, 155.6, 154.5, 145.1, 123.2, 118.4,
−1
1
13.4, 110.3, 98.9, 97.0, 95.0, 90.7, 54.6, 7.4; IR (thin film, cm
)
+
3
450, 2967, 2788, 1695, 1314; HRMS (ESI) m/z [M+H] Calcd. for
287.0914, Found 287.0910.
C
16
H
O
16 5
2
-(2-Methoxy-4,6-bis(methoxymethoxy)phenyl)-6-(methox-
ymethoxy)-3-methylbenzofuran (9). To a suspension of NaH (60%
dispersion in paraffin oil) (40 mg, 1.0 mmol) in dry dimethylformamide
(
5 mL) was added a solution of 4-(6-hydroxy-3-methylbenzo furan-2-
yl)-5-methoxybenzene-1,3-diol
8 (286 mg, 1.0 mmol) in dry di-
methylformamide (2 mL) at 0 °C under stirring in a nitrogen atmo-
sphere. The mixture was stirred at room temperature for 1 h and
chloromethyl methyl ether (161 mg, 2.0 mmol) was added dropwise at
layers were washed with brine, dried over anhydrous Na
2
SO , and
4
0
6
°C for over 30 min. The reaction was stirred at room temperature for
evaporated to dryness. The crude material was purified by flash column
h, quenched by a saturated aqueous solution of NH Cl (20 mL) and
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:10,
extracted with ethyl acetate (20 mL). The combined organic layers were
R
f
= 0.1) to obtain product 12 (76 mg) as a colorless oil with a yield of
1
washed with brine, dried over anhydrous Na
2 4
SO , and evaporated to
78%. H NMR (600 MHz, CDCl
3
) δ 7.39 (d, J = 8.4 Hz, 1H), 7.21 (d,
dryness. The crude material was purified by flash column chromato-
J = 2.0 Hz, 1H), 6.97 (dd, J = 8.4, 2.1 Hz, 1H), 6.61 (s, 1H), 5.25 (s,
2H), 5.24 – 5.22 (m, 1H), 5.21 (s, 2H), 4.64 (s, 2H), 3.75 (s, 3H), 3.51
(s, 6H), 3.40 (d, J = 6.7 Hz, 2H), 3.16 (s, 3H), 2.09 (s, 3H), 1.78 (s, 3H),
graphy on silica gel (ethyl acetate/petroleum ether = 1:10, R
f
= 0.1) to
1
give product 9 (347 mg) as a colorless oil with a yield of 83%. H NMR
1
3
(
600 MHz, DMSO‑d
H), 6.95 (dd, J = 8.5, 2.1 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.47 (d,
J = 2.1 Hz, 1H), 5.26 (s, 2H), 5.22 (s, 2H), 5.11 (s, 2H), 3.70 (s, 3H),
6
) δ 7.44 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 2.1 Hz,
1.68 (s, 3H); C NMR (150 MHz, CDCl ) δ 158.1, 157.9, 156.3, 155.4,
3
1
155.1, 146.1, 131.1, 125.2, 123.6, 119.3, 117.0, 114.5, 112.4, 107.3,
99.8, 99.7, 95.4, 94.8, 94.5, 57.3, 56.21, 56.17, 56.1, 25.9, 23.3, 18.0,
1
3
−1
3
.43 (s, 3H), 3.40 (s, 3H), 3.25 (s, 3H), 1.98 (s, 3H); C NMR
8.8; IR (thin film, cm ) 3425, 2921, 2827, 1589, 1469, 1381, 1152,
+
(
150 MHz, DMSO‑d ) δ 159.8, 159.7, 157.3, 154.5, 154.4, 145.2, 124.4,
6
1105; HRMS (ESI) [M+H] m/z Calcd. for C27
H
35
8
O 487.2326, Found
1
5
1
19.2, 113.4, 112.3, 102.4, 98.9, 95.6, 94.5, 94.2, 94.0, 93.8, 55.9,
487.2335.
−
1
5.8, 55.6, 55.5, 8.4; IR (thin film, cm ) 3422, 2922, 2853, 1804,
6-(6-Hydroxy-3-methylbenzofuran-2-yl)-5-methoxy-2,2-di-
methylchroman-7-ol (T9). To a solution of 2-(2-methoxy-4,6-bis
(methoxymethoxy)-3-(3-methylbut-2-en-1-yl)phenyl)-6-(methox-
ymethoxy)-3-methylbenzofuran 12 (71 mg, 0.2 mmol) in methanol
(5 mL) was added CSA (23 mg, 0.1 mmol) and the resulting mixture
was stirred at 50 °C for 12 h. The reaction was monitored by TLC till
the complete consumption of the starting material. The reaction
mixture was quenched by the addition of a saturated aqueous solu-
+
697, 1492, 1454, 1153, 1025; HRMS (ESI) [M+H] m/z Calcd. for
C
22
H
27
O 419.1700, Found 419.1709.
8
2
-(3-Bromo-2-methoxy-4,6-bis(methoxymethoxy)phenyl)-6-(meth-
oxymethoxy)-3-methylbenzofuran (10). To a solution of 2-(2-methoxy-
4
,6-bis(methoxymethoxy)phenyl)-6-(methoxymethoxy)-3-methylben-
zofuran 9 (418 mg, 1.0 mmol) in DCM (10 mL) was added a solution of
N-bromosuccinimide (178 mg, 1.0 mmol) in DCM (5 mL) dropwise
under stirring in a nitrogen atmosphere at 0 °C. The mixture was stirred
at room temperature for 1 h. The reaction mixture was evaporated to
dryness. The crude material was purified by flash column chromato-
tion of Na
2
CO (10 mL) and extracted with ethyl acetate (10 mL). The
3
organic layers were washed with brine, dried over anhydrous Na
2
SO
4
and evaporated to dryness. The crude material was purified by flash
column chromatography on silica gel (ethyl acetate/petroleum
graphy on silica gel (ethyl acetate/petroleum ether = 1:10, R
f
= 0.1) to
1
give product 10 (174 g) as a colorless oil with a yield of 35%. H NMR
ether = 1:2, R = 0.2) to give product T9 (39 mg) as a colorless oil
f
(
600 MHz, CDCl
3
) δ 7.42 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H),
with a yield of 75%. The spectroscopic data of T9 was identical with
7
.00 (dd, J = 8.4, 2.1 Hz, 1H), 6.90 (s, 1H), 5.30 (s, 2H), 5.22 (s, 2H),
previous reported [25].
7

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
4
.2. Cell culture
(D8H8) antibody (1: 1000 dilution; Cell Signaling Technology, Danvers,
MA) and anti-Tubulin (1: 10,000 dilution; MBL, Nagoya, Japan) as
primary antibodies and horseradish peroxidase (HRP)-conjugated anti-
rabbit immunoglobulin G (IgG) (1: 4000 dilution; Cell Signaling
Technology) as a secondary antibody. Band intensity was detected
using a Luminograph Cheminluminescent Imaging System (ATTO,
Tokyo, Japan) and analyzed by ImageJ software (National Institutes of
Health).
Cells of the human embryonic kidney cell line HEK293 were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka,
Japan) containing 10% fetal bovine serum (FBS) and penicillin-strep-
tomycin in a humidified atmosphere containing 5% CO
2
at 37 °C.
Human breast cancer cell line MCF-7 was cultured in DMEM containing
5
% FBS and penicillin-streptomycin in a humidified 5% CO at 37 °C.
2
4.3. Plasmid construction
4.8. Reverse transcription quantitative real-time PCR (RT-qPCR)
Full-length cDNA encoding human ERα and ERβ were inserted into
Total RNA was isolated using the ISOGEN II (Nippon Gene, Tokyo,
Japan) according to the manufacturer’s protocol. cDNA was synthesized
pcDNA5 vector (Invitrogen/Thermo Fisher Scientific, Waltham, MA).
The construction of expression vector encoding a GAL4-DBD fused ERα-
LBD (GAL4/DBD-ERα/LBD) was cloned into the pcDNA5 vector
using
a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan).
Quantitative real-time PCR (qPCR) was conducted using SYBR qPCR
Mix (Toyobo) according to the manufacturer’s protocol and using the
7500 fast system SDS software 2.0.6 (Applied Biosystems, Foster City,
CA). The following pairs of primer were used GREB1: forward 5′-CAC
(
Invitrogen). The GAL-responsible elements (GALREs)/TATA-box were
inserted into pMCS-Gaussia Luc vector (Thermo Fisher Scientific) to
insert the GAL-RE driven Gaussia-luciferase reporter plasmid. ERE
driven luciferase reporter vector was previously constructed [32].
ATCTATCCTAGACATTTA-3′,
reverse
5′-CGCGGACTTTTTTTCTTA
GGA-3′ and β-actin: forward 5′-TCCTCCTGAGCGCAAGTACTC-3′, re-
4
.4. Gaussia-luciferase reporter assay
verse 5′-CTGCTTGCTGATCCACATCTG-3′.
HEK293 cells maintained in phenol-red-free DMEM, containing 5%
4.9. Docking
charcoal-stripped FBS (csFBS) were transfected with the expression
vector for GAL4/DBD-ERα/LBD, GAL-RE driven Gaussia-luciferase re-
porter vector, using PEI Max Reagent (Polysciences Inc., Warrington,
PA). After overnight incubation, the cells were treated with compounds
T1–T22 (10 µM) or Estradiol (10 nM) for 24 h, and the Gaussia-luci-
ferase activity was measured using the BioLux Gaussia Luciferase Assay
Kit (BioRad, Hercules, CA).
AutoDock 4 [33] was applied to investigate the possible binding
mode of a certain molecule with a certain protein. The crystal structure
of human Estrogen Receptor (PDB code: 1X7E [31]) was used in
docking calculation. A grid box was generated with a grid spacing of
0.375 Å and its center was defined with the coordination of the co-
crystal ligand. Structure of T9 was built and optimized in SYBYL 6.9.1
software package (Tripos Inc.) with Tripos force field and Gastei-
ger–Hückel charges. The Lamarckian genetic algorithm was used during
the molecular docking, the optimized parameters were set as follows:
the maximum number of energy evaluations per run was set to
25 000 000, the iterations of Solis & Wets local search were set to 3000,
the number of the individuals was up to 300, and the number of gen-
erations was set to 100. The poses generated with a positional root
mean square deviation (RMSD) less than 2 Å were divided into one
cluster, and the conformation with the lowest binding energy and the
highest percentage of frequency was selected as a representative result.
All other parameters were kept as default.
4.5. Dual-luciferase reporter assay
HEK293 cells maintained in phenol-red-free DMEM containing 5%
csFBS were transfected with estrogen receptor expression vector, ERE
driven luciferase reporter vector, and pGL4.74 (hRluc/TK; Promega,
Madison, WI); as an internal standard, we used PEI Max Reagent
(
Polysciences Inc.). After overnight incubation, the cells were treated
with compounds (T2, T7, T9, T10, T12, T13, T15, T17, and T19) or E2
for 24 h and the luciferase activity was measured using the Dual-
Luciferase Reporter Assay System (Promega). The activities of firefly
luciferase were normalized to those of Renilla luciferase.
Acknowledgements
4.6. Cell proliferation assay
Funding: This work was supported by the Japan Society for the
Promotion of Science, Grant-in-Aid for Scientific Research (KAKENHI)
[Grants 23790104 and 25460175], and the National Natural Science
Foundation of China [Grant No. 81628012 and 81673323].
MCF-7 cells were seeded at 1 × 104 cells/ml in phenol-red-free
DMEM containing 5% csFBS in 96-well plates. The cells were treated
with compounds in the absence or presence of E2 (10 nM) and a se-
lective estrogen receptor degradator (SERD; ICI 182780; Sigma Aldrich,
St Louis, MO) (1 µM) [28] as indicated. After 72 h of incubation, the
viability of MCF-7 cells was evaluated using the CellTiter 96 Aqueous
One Solution Cell Proliferation Assay Kit (Promega) according to the
manufacturer’s instructions. The number of living cells was directly
proportional to the absorbance at 490 nm of a formazan product re-
duced from living cells by MTS. The absorbance at 490 nm was mea-
sured in a multimode detector (BECKMAN COUNTER, CA, USA).
References
[1] S. Green, P. Walter, V. Kumar, A. Krust, J.M. Bornert, P. Argos, P. Chambon, Human
oestrogen receptor cDNA: sequence, expression and homology to v-erb-A, Nature
3
20 (1986) 134–139, https://doi.org/10.1038/320134a0.
[
2] D.J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schütz, K. Umesono,
B. Blumberg, P. Kastner, M. Mark, P. Chambon, R.M. Evans, The nuclear receptor
superfamily: the second decade, Cell 83 (1995) 835–839 http://www.ncbi.nlm.nih.
gov/pubmed/8521507.
[
[
3] G.G. Kuiper, E. Enmark, M. Pelto-Huikko, S. Nilsson, J.A. Gustafsson, Cloning of a
novel receptor expressed in rat prostate and ovary, Proc. Natl. Acad. Sci. USA 93
4.7. Western blotting
(
1996) 5925–5930 http://www.ncbi.nlm.nih.gov/pubmed/8650195.
4] S. Mosselman, J. Polman, R. Dijkema, ER beta: identification and characterization
of a novel human estrogen receptor, FEBS Lett. 392 (1996) 49–53 http://www.ncbi.
nlm.nih.gov/pubmed/8769313.
The cells were seeded in 24-well plates in phenol red-free DMEM
containing 5% csFBS. The next day, the cells were treated with T9
[
[
[
5] S. Nilsson, J.-å Gustafsson, Estrogen receptors: therapies targeted to receptor sub-
types., Clin. Pharmacol. Ther. 89 (2011) 44–55. doi: 10.1038/clpt.2010.226.
6] A.M. Kaunitz, J.E. Manson, Management of menopausal symptoms, Obstet.
Gynecol. 126 (2015) 859–876, https://doi.org/10.1097/AOG.0000000000001058.
7] K.A. Burns, K.S. Korach, Estrogen receptors and human disease: an update, Arch.
Toxicol. 86 (2012) 1491–1504, https://doi.org/10.1007/s00204-012-0868-5.
(
10 µM) or E2 (10 nM) for 6 h. Total protein was extracted with sodium
dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCI pH 6.8, 4% SDS,
2
0% glycerol, 10% dithiothreitol, and 0.01% bromophenol blue]. The
protein samples were separated by SDS-polyacrylamide gel electro-
phoresis (PAGE). Immunoblotting was performed with rabbit anti-ERα
8

N. Saito, et al.
Bioorganic Chemistry 88 (2019) 102977
[
[
8] T. Harada, Endometriosis: pathogenesis and treatment, Endometr. Pathog. Treat. 10
(1998) 1609–1618, https://doi.org/10.1056/NEJM199811263392207.
(
2014) 1–477, https://doi.org/10.1007/978-4-431-54421-0.
[23] R. Lindsay, J.C. Gallagher, R. Kagan, J.H. Pickar, G. Constantine, Efficacy of tissue-
selective estrogen complex of bazedoxifene/conjugated estrogens for osteoporosis
prevention in at-risk postmenopausal women, Fertil. Steril. 92 (2009) 1045–1052,
https://doi.org/10.1016/j.fertnstert.2009.02.093.
9] G.P. Flake, J. Andersen, D. Dixon, Etiology and pathogenesis of uterine leiomyomas:
a review, Environ. Health Perspect. 111 (2003) 1037–1054, https://doi.org/10.
1
289/ehp.5787.
[
[
10] M. Clemons, P. Goss, Estrogen and the risk of breast cancer, N. Engl. J. Med. 344
[24] A. Andersson, A.I. Bernardi, M. Nurkkala-Karlsson, A. Stubelius, L. Grahnemo,
C. Ohlsson, H. Carlsten, U. Islander, Suppression of experimental arthritis and as-
sociated bone loss by a tissue-selective estrogen complex, Endocrinology 157
(2016) 1013–1020, https://doi.org/10.1210/en.2015-1820.
(
2001) 276–285, https://doi.org/10.1056/NEJM200101253440407.
11] B. Manavathi, O. Dey, V.N.R. Gajulapalli, R.S. Bhatia, S. Bugide, R. Kumar, Derailed
estrogen signaling and breast cancer: an authentic couple, Endocr. Rev. 34 (2013)
1
–32, https://doi.org/10.1210/er.2011-1057.
[25] Y. Tang, C. Jiang, X. Zhang, C. Liu, J. Lin, Y. Wang, C. Du, X. Peng, W. Li, Y. Liu,
M. Cheng, Collective syntheses of 2-(3-methylbenzofuran-2-yl)phenol-derived nat-
ural products by a cascade [3,3]-sigmatropic rearrangement/aromatization
strategy, J. Org. Chem. 82 (2017) 11102–11109, https://doi.org/10.1021/acs.joc.
7b02066.
[
[
[
12] M. Dalvai, K. Bystricky, Cell cycle and anti-estrogen effects synergize to regulate
cell proliferation and ER target gene expression, PLoS One 5 (2010) e11011. doi:
1
0.1371/journal.pone.0011011.
13] B.L. Riggs, L.C. Hartmann, Selective estrogen-receptor modulators – mechanisms of
action and application to clinical practice, N. Engl. J. Med. 348 (2003) 618–629,
https://doi.org/10.1056/NEJMra022219.
[26] M.G. Ghosh, D.A. Thompson, R.J. Weigel, PDZK1 and GREB1 are estrogen-regu-
lated genes expressed in hormone-responsive breast cancer, Can. Res. 60 (2000)
6367–6375 http://www.ncbi.nlm.nih.gov/pubmed/11103799.
14] P.Y. Maximov, T.M. Lee, V.C. Jordan, The discovery and development of selective
estrogen receptor modulators (SERMs) for clinical practice, Curr. Clin. Pharmacol. 8
[27] H. Mohammed, C. D’Santos, A.A. Serandour, H.R. Ali, G.D. Brown, A. Atkins,
O.M. Rueda, K.A. Holmes, V. Theodorou, J.L.L. Robinson, W. Zwart, A. Saadi,
C.S. Ross-Innes, S. Chin, S. Menon, J. Stingl, C. Palmieri, C. Caldas, J.S. Carroll,
Endogenous purification reveals GREB1 as a key estrogen receptor regulatory
factor, Cell Rep. 3 (2013) 342–349, https://doi.org/10.1016/j.celrep.2013.01.010.
[28] A.E. Wakeling, M. Dukes, J. Bowler, A potent specific pure antiestrogen with
clinical potential, Cancer Res. 51 (1991) 3867–3873, https://doi.org/10.1158/
0008-5472.CAN-10-3145.
(
2013) 135–155, https://doi.org/10.1016/j.mce.2014.11.015.
[
[
15] V.C. Jordan, Tamoxifen: a most unlikely pioneering medicine, Nat. Rev. Drug
Discov. 2 (2003) 205–213, https://doi.org/10.1038/nrd1031.
16] J. Cuzick, J. Forbes, R. Edwards, M. Baum, S. Cawthorn, A. Coates, A. Hamed,
A. Howell, T. Powles, IBIS investigators, first results from the International Breast
Cancer Intervention Study (IBIS-I): a randomised prevention trial, Lancet (London,
England) 360 (2002) 817–824, https://doi.org/10.1016/S0140-6736(13)60140-3.
17] C. Jochems, U. Islander, A. Kallkopf, M. Lagerquist, C. Ohlsson, H. Carlsten, Role of
raloxifene as a potent inhibitor of experimental postmenopausal polyarthritis and
osteoporosis, Arthritis Rheum. 56 (2007) 3261–3270, https://doi.org/10.1002/art.
[
[
[29] V. Delfosse, M. Grimaldi, J.-L. Pons, A. Boulahtouf, A. le Maire, V. Cavailles,
G. Labesse, W. Bourguet, P. Balaguer, Structural and mechanistic insights into bi-
sphenols action provide guidelines for risk assessment and discovery of bisphenol A
substitutes, Proc. Natl. Acad. Sci. 109 (2012) 14930–14935, https://doi.org/10.
1073/pnas.1203574109.
2
2873.
18] P.D. Delmas, N.H. Bjarnason, B.H. Mitlak, A.C. Ravoux, A.S. Shah, W.J. Huster,
M. Draper, C. Christiansen, Effects of raloxifene on bone mineral density, serum
cholesterol concentrations, and uterine endometrium in postmenopausal women, N.
Engl. J. Med. 337 (1997) 1641–1647, https://doi.org/10.1056/
[30] R.W. Hsieh, S.S. Rajan, S.K. Sharma, G.L. Greene, Molecular characterization of a B-
ring unsaturated estrogen: implications for conjugated equine estrogen components
of premarin, Steroids 73 (2008) 59–68, https://doi.org/10.1016/j.steroids.2007.
08.014.
NEJM199712043372301.
[
[
19] R.A. Lobo, J.V. Pinkerton, M.L.S. Gass, M.H. Dorin, S. Ronkin, J.H. Pickar,
G. Constantine, Evaluation of bazedoxifene/conjugated estrogens for the treatment
of menopausal symptoms and effects on metabolic parameters and overall safety
profile, Fertil. Steril. 92 (2009) 1025–1038, https://doi.org/10.1016/j.fertnstert.
[31] E.S. Manas, R.J. Unwalla, Z.B. Xu, M.S. Malamas, C.P. Miller, H.A. Harris, C. Hsiao,
T. Akopian, W.-T. Hum, K. Malakian, S. Wolfrom, A. Bapat, R.A. Bhat, M.L. Stahl,
W.S. Somers, J.C. Alvarez, Structure-based design of estrogen receptor-beta selec-
tive ligands, J. Am. Chem. Soc. 126 (2004) 15106–15119, https://doi.org/10.1021/
ja047633o.
2
009.03.113.
20] B.S. Komm, Y.P. Kharode, P.V.N. Bodine, H.A. Harris, C.P. Miller, C.R. Lyttle,
Bazedoxifene acetate: a selective estrogen receptor modulator with improved se-
lectivity, Endocrinology 146 (2005) 3999–4008, https://doi.org/10.1210/en.2005-
[32] Y. Kanno, Y. Takane, Y. Takizawa, Y. Inouye, Suppressive effect of aryl hydrocarbon
receptor repressor on transcriptional activity of estrogen receptor alpha by pro-
tein–protein interaction in stably and transiently expressing cell lines, Mol. Cell.
Endocrinol. 291 (2008) 87–94, https://doi.org/10.1016/j.mce.2008.05.004.
[33] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, https://doi.org/10.
1002/jcc.21256.
0
030.
[
[
21] H.B. Nichols, L.A. DeRoo, D.R. Scharf, D.P. Sandler, Risk-benefit profiles of women
using tamoxifen for chemoprevention, J. Natl. Cancer Inst. 107 (2015) 354, https://
doi.org/10.1093/jnci/dju354.
22] C.K. Osborne, Tamoxifen in the treatment of breast cancer, N. Engl. J. Med. 339
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