Structure-activity relationships of bisphenol a analogs at estrogen receptors (ERs): Discovery of an ERα-selective antagonist

Article abstract of DOI:10.1016/j.bmcl.2013.05.067

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. H

Full text of DOI:10.1016/j.bmcl.2013.05.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4031–4036
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of bisphenol A analogs at estrogen
receptors (ERs): Discovery of an ERa-selective antagonist
Keisuke Maruyama a, Masaharu Nakamura a, Shusuke Tomoshige a, Kazuyuki Sugita a,
Makoto Makishima b, Yuichi Hashimoto a, Minoru Ishikawa a,
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures,
has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton
can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that
the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen
receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we syn-
thesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonis-
Received 9 May 2013
Revised 17 May 2013
Accepted 20 May 2013
Available online 30 May 2013
Keywords:
tic/antagonistic activities at ERs and ER
was found to be a potent ER -antagonist with high selectivity over ERb and androgen receptor under
our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic
a/ERb subtype selectivity. Among the compounds examined, 18
Nuclear receptor
Estrogen receptor
Antagonist
a
conformation of ER
cancer.
a
. ER
a
-selective antagonists, such as 18, are candidate agents for treatment of breast
Ó 2013 Elsevier Ltd. All rights reserved.
The multi-template approach to drug design is based on the fact
that the number of three-dimensional spatial structures (fold
structures) of human proteins is much smaller (more than 50 times
smaller) than the number of human proteins (50,000–70,000).1–4
Therefore, ignoring physical/chemical interactions, a template/
scaffold structure which is spatially complementary to one fold
structure might serve as a multi-template for structural develop-
ment of ligands that would interact specifically with more than
50 human proteins. In other words, the structures of ligands that
bind to one member of a particular group of fold structures may
be used for the development of novel lead compounds for other
members of the same fold structure group. Lead compounds ob-
tained in this way may possess polypharmacologic character, and
so structural modification is then necessary to optimize target
selectivity.
A skeleton that corresponds structurally to the steroid skeleton
would be a potential multi-template for structural development of
ligands for a number of important proteins, and we have focused
on diphenylmethane. Indeed, non-secosteroidal vitamin D receptor
(VDR) agonists with a diphenylmethane skeleton have been devel-
oped, such as LG1901785,6 (Fig. 1), and they show improved stabil-
ity and reduced calcium-increasing effects. We have extended this
approach to obtain novel ligands of nuclear receptors (NRs) and
steroid-related enzymes, such as VDR/androgen receptor (AR) dual
ligands (VDR agonists/AR antagonists),7,8 AR-selective antago-
nists,9 farnesoid X receptor (FXR) agonists10 and inhibitors of ste-
roid metabolism-related enzymes, including
5a
-reductase11
(Fig. 1). Those results indicated that the diphenylmethane skeleton
is an effective steroid surrogate.4,12
Estrogen receptors (ERs) are activated by the endogenous hor-
mone 17b-estradiol (Fig. 2). Following endogenous ligand binding,
ERs undergo conformational changes and biochemical modifica-
tions that induce release of inhibitory proteins, receptor dimeriza-
tion, and interaction with DNA. Estrogen action is mediated
through two ER subtypes, ERa and ERb, which have distinct target
tissue distributions and functional activities.13 Estrogens play an
important role in the initiation and progression of breast cancer,
and so ERs are regarded as important drug targets. For example,
classical selective ER modulators (SERMs), tamoxifen14 and raloxif-
ene (Fig. 2), have been used for the treatment of breast cancer.
However, acquired resistance to tamoxifen is a serious prob-
lem.15–17 Although many breast cancer patients benefit from
tamoxifen therapy, resistance to this drug develops in the majority
of cases; moreover, if tamoxifen treatment is not discontinued
after the onset of resistance, the drug can actually promote, rather
than inhibit, tumor growth.
The presence of ERa appears to be associated with an increased
risk of breast cancer.18 In addition, this receptor subtype has been
shown to be a prerequisite for growth stimulation of MCF-7 breast
⇑
Corresponding author. Tel.: +81 3 5841 7853; fax: +81 3 5841 8495.
E-mail address: m-ishikawa@iam.u-tokyo.ac.jp (M. Ishikawa).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.067

4032
K. Maruyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4031–4036
Figure 1. Chemical structures of steroid surrogates bearing a diphenylmethane skeleton.
Figure 2. Chemical structures of ER ligands.
cancer cells by estradiol. An MCF-7 cell line without ER
proliferate in the presence of estradiol, but recovered its ability
to proliferate when ER
was reintroduced.19 However, classical
SERMs have essentially no selectivity for either ER subtype. On
a
did not
We hypothesized that BPA, which has a diphenylmethane skel-
eton, would bind to the ligand-binding domain (LBD) of ERs in a
similar manner to 17b-estradiol, and act as a steroid surrogate.
a
Herein, we report SAR studies of BPA analogs by means of ER
a
the other hand, several ER
reported.
a
-selective antagonists20–24 have been
and ERb reporter gene assays, focusing on agonist/antagonist func-
tional switching and subtype selectivity, in order to evaluate our
hypothesis. One of the BPA analogs examined proved to be a potent
Bisphenol A (BPA, 4,40-(propane-2,2-diyl)diphenol, 1) is used
primarily in the manufacture of polycarbonate plastic and epoxy
resins, and is also used as a non-polymer additive to other plastics
(Fig. 3). Several lines of evidence indicate that BPA has estrogenic
activity. For example, BPA induced the expression of estrogen-
responsive genes and promoted proliferation of MCF-7 cells.25
However, it has also been reported that the activity of BPA depends
on both the ER subtype and the cell type.26 Although bisphenol
AF27 and HPTE28 (Fig. 3) have been reported to be an agonist for
and selective ERa-antagonist.
If BPA binds to the LBD of ERs, as many known NR ligands do, it
should be a good lead compound for development of ER antago-
nists, because it has a simple structure so that synthesis of analogs
should be straightforward. Further, it might be possible to utilize
canonical SAR information available for numerous NR ligands,
including structural requirements for switching from agonistic to
antagonistic activity. Specifically, classical SARs for NRs indicate
that introduction of bulky substituents leads to a switch from ago-
nist to antagonist, because bulky substituents interfere with the
folding of helix 12, which is important for agonistic activity. In
addition, classical SARs for NRs also indicate that hydrophobicity
ERa and an antagonist for ERb, respectively, only a few
structure–activity relationship (SAR) studies of BPA have been
reported,29–32 and most of them did not evaluate ER-subtype
selectivity.
Figure 3. Chemical structures of bisphenols.

K. Maruyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4031–4036
4033
analogs 10–13, 17–20 and 22–27. Some BPA analogs, 1, 9, 14–16
and 21, were commercially available. Tetramethyl analog 28 (Table
2) was synthesized from 7 and 2,6-dimethylphenol.
Synthesis of asymmetrical analogs is illustrated in Scheme 2.
Reaction with methyl 4-hydroxybenzoate (29) and R1MgBr yielded
ternary alcohols 30, which were reacted with o-cresol to give 31
and 32. Aniline analog 33 was obtained from 17.7 Sila-analogs 36
and 37 were synthesized as shown in Scheme 3. 4-Bromo-2-meth-
ylphenol (35) was lithiated with n-BuLi, and then reacted with
dialkyldichlorosilane to obtain 36 and 37.
Scheme 1. Reagents and conditions: (a) MsOH, rt, 7–35%.
is important for strong affinity. Therefore, we designed a variety of
BPA analogs in which these factors were modified.
Symmetrical BPA analogs bearing various alkyl chains were
synthesized as shown in Scheme 1. Symmetrical ketones 7 were
treated with phenols 8 in the presence of MsOH to generate BPA
Agonistic and antagonistic activities of BPA analogs were evalu-
ated by means of reporter gene assay using a Gal4-human ER
a or
ERb reporter system.33 Antagonistic activity was measured in the
Table 1
Structure–activity relationships of bisphenol A analogs
Compound
R1
R2
R3
ER
a
ERb
EC50 (nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
9
H
Me
Et
n-Pr
H
Me
Et
n-Pr
n-Bu
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
n-Pr
n-Pr
n-Pr
910
(35%)a
(38%)a
86
99
79
1900
1100
(28%)b
(43%)b
N.A.
N.A.
N.A.
5500
(19%)b
(15%)b
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
(24%)a
(25%)a
93
2200
180
1 (BPA)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
(33%)b
N.A.
N.A.
N.A.
N.A.
N.A.
(23%)b
(31%)b
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
n-Bu
R1,R2 = –(CH2)4–
170
520
270
560
R1,R2 = –(CH2)5–
H
Me
Et
n-Pr
H
Me
Et
n-Pr
n-Bu
(26%)a
1200
25
N.A.
(25%)a
260
4.9
14
84
230
230
490
2900
(17%)a
(38%)a
(15%)a
140
150
770
790
1300
2600
4500
(35%)a
(23%)a
(14%)a
n-Bu
R1,R2 = –(CH2)4–
R1,R2 = –(CH2)5–
Et
Et
n-Pr
n-Bu
Et
n-Pr
n-Bu
n-Pr
n-Bu
Et
n-Pr
n-Bu
N.A.
a
% Inhibition at 3 lM.
% To maximal activation of 17b-estradiol at 10 lM.
b
Table 2
Structure–activity relationships of bisphenol A analogs
Compound
X
R1
R2
R3
R4
R5
ER
a
ERb
EC50 (nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
1 (BPA)
31
16
10
32
17
28
33
36
C
C
C
C
C
C
C
C
Si
C
Si
Me
Me
Me
Et
Et
Et
Et
Et
Et
n-Bu
n-Bu
H
H
H
Me
H
H
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
Me
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
NH2
OH
OH
OH
(33%)a
(26%)a
(31%)a
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
(38%)b
(48%)b
1200
86
110
25
74
98
72
1100
(28%)a
(19%)a
(28%)a
(19%)a
(15%)a
N.A.
N.A.
N.A.
N.A.
N.A.
(25%)b
(26%)b
(25%)b
93
380
260
300
43
800
150
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
19
37
14
N.A.
(30%)b
N.A.
a
% To maximal activation of 17b-estradiol at 10 lM.
% Inhibition at 3 lM.
b

4034
K. Maruyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4031–4036
30-positions. Indeed, the length of the alkyl chains at these posi-
tions was important for ERs-antagonistic activities, that is, the or-
der of potency of antagonistic activities was: Me (15–21) > H (9–
14) > Et (22–24) > n-Pr (25–27). Asymmetric mono-methyl analogs
31 and 32 showed lower activity than the corresponding dimethyl
analogs 16 and 17, respectively. Tetramethyl analog 28 also
showed lower activities than the dimethyl analog 17. These results
indicated that dimethyl analogs are the most suitable to obtain po-
tent antagonistic activity. Aniline 33 was distinctive among the
compounds reported here, that is, it showed twofold ERb-selective
antagonistic activity.
As mentioned above, central alkyl chains were critical for po-
tent ERs antagonistic activities, indicating that the hydrophobicity
at the central moiety is important for strong antagonistic activity.
On the other hand, sila-substitution (C/Si exchange) of existing
drugs is an attractive approach to find new drug candidates with
a clear intellectual property position: for example, silicon-contain-
ing analogues are more lipophilic and larger in molecular size than
their carbon analogues.34–37 Therefore, we designed sila-analogs
36 and 37 with the aim of increasing the hydrophobicity or molec-
ular size. However, both sila-analogs 36 and 37 showed decreased
activities compared with carbon derivatives 17 and 19,
respectively.
Scheme 2. Reagents and conditions: (a) R1MgBr, THF, 0 °C to rt, 66–84%; (b) o-
cresol, H2SO4, 0 °C to rt, 16–18%; (c) o-toluidine–HCl, neat, 180 °C, 58%.
Overall, we identified several important SARs, and discovered
the n-Pr analog 18, which possessed the most potent ERa-antago-
nistic activity (IC50 4.9 nM) and the greatest selectivity for ER
a
over ERb (28-fold) among the compounds examined.38
X-Ray crystal structures of two complexes, that is, a complex
consisting of ER LBD mutant (Y537S) and BPA, and a complex
consisting of wild-type ER LBD and bisphenol C (BPC), have re-
a
a
cently been reported.39 BPC is reported to act as an ER antagonist.39
Both BPA and BPC bind to the LBD, as 17b-estradiol does (Fig. 4). A
common feature of BPA and BPC is the hydrogen-bonding interac-
tion between their phenol moieties and amino acid residues E353
and R394. However, there are differences in the binding modes of
the two complexes, that is, (1) one of the phenol rings of BPC is
positioned in an alternative pocket, different from that into which
the corresponding ring of BPA binds, and (2) the chlorine atoms in
BPC are directed toward M421, which forms a hydrogen bond with
the phenol moiety in the case of BPA. As a result, the structures of
Scheme 3. Reagents and conditions: (a) NBS, CH3CN, 0 °C to rt, 63%; (b) n-BuLi,
(R1)2SiCl2, THF, ꢀ78 °C to rt, 2–39%.
presence of 0.5 nM 17b-estradiol. Under these assay conditions,
BPA (1) showed weak agonistic and antagonistic activities towards
ERa at 3 lM, indicating that it is a weak ERa partial agonist under
our assay conditions. With ERb, BPA (1) showed agonistic activity
with the EC50 value of 1100 nM. First, we investigated SARs regard-
ing the central propyl group, as shown in Table 1. Removal of two
methyl groups (9) increased the ER
910 nM), with retention of the ERb-agonistic activity, so 9 was an
ER and ERb dual agonist. Introduction of longer linear alkyl chains
at the central carbon (10–12) decreased the agonistic activities for
both ER and ERb, but increased the antagonistic activities for both
ER and ERb, compared with 1. For example, compound 10 pos-
sessing two ethyl groups showed decreased agonistic activity for
both ERs, but exhibited both ER - and ERb-antagonistic activities
a
-agonistic activity (EC50
ERa bound with BPA and 17b-estradiol display the canonical active
conformation with helix 12 capping the ligand-binding pocket and
the steroid receptor coactivator-1 (SRC-1) peptide bound to the
transcriptional activation function (AF-1) surface, whereas the
a
a
structure of ERa with BPC displays an antagonist conformation
a
similar to that observed in the hydroxytamoxifen-bound structure,
with helix 12 occupying the coactivator binding groove. To exam-
ine the binding mode of the most potent and selective antagonist
a
with the IC50 values of 86 and 93 nM, respectively. These results
were consistent with expectation, that is, increase of bulkiness
by introduction of longer alkyl chains at the central carbon led to
a switch from agonist to antagonist. Cyclic alkyl analogs 13 and
18, this molecule was computationally docked into ER
3UUC) using AutoDock 4.2. The docking model of the complex of
18 with the active site of ER shows the hydrogen-bonding inter-
a (PDB ID:
a
action with the phenol and E353, as seen in the X-ray crystal struc-
tures of BPA and BPC. The most noteworthy result of the docking
simulation was that the bulky n-Pr chains at the central carbon
are directed toward M421, like the chlorine atoms of BPC. These
X-ray crystal data and the docking simulation are consistent with
our hypothesis that the diphenylmethane skeleton of BPA acts as
a steroid surrogate, and introduction of bulky substituents causes
a switch from agonist to antagonist. The precise molecular mecha-
14 also showed ERa/ERb dual antagonistic activities.
Next, we investigated the effect of introduction of two methyl
groups at the 3- and 30-positions because all representative steroid
surrogates with a diphenylmethane skeleton (Fig. 1) possess two
methyl groups at the 3- and 30-positions. The dimethyl analog of
BPA 16 showed an increase of ERa-antagonistic activity and a de-
crease of ERb-agonistic activity. In particular, dimethyl analogs
with longer alkyl chains at R1 and R2 (17–19) showed increased
nism of the selectivity of 18 for ER
there is a a difference in amino acid sequences between ER
ERb in the region of the ligand-binding pocket, that is, M421 in
ER corresponds to I328 in ERb. Because M421 is a key amino acid
for the interaction with ER ligands, this difference in amino acid
sequence between ER and ERb might result in different interac-
tions with 18, possibly via changes in the size or electrostatic char-
a
over ERb is unclear. However,
ER
tent ER
a
-antagonistic activity. Among them, n-Pr analog 18 showed po-
-antagonistic activity with an IC50 value of 4.9 nM, afford-
a
and
a
ing 28-fold selectivity over ERb. Cyclic alkyl analogs 20 and 21
exhibited weaker antagonistic activity than the linear alkyl analogs
17–19. These SARs regarding dimethyl analogs prompted us to
synthesize alkyl analogs bearing Et or n-Pr groups at the 3- and
a
a
a

K. Maruyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4031–4036
4035
(a)
(b)
L391
F404
M421
R394
R394
H524
H524
E353
E353
A350
T347
(c)
(e)
(d)
M421
M421
R394
E353
E353
Figure 4. (a) X-ray crystal structure of ER
a
with 17b-estradiol (PDB ID: 3UUD); (b) X-ray crystal structure of ERa with BPA (PDB ID: 3UU7); (c) X-ray crystal structure of ERa
with BPC (PDB ID: 3UUC); (d) docking simulation of ER
a
LBD and 18; (e) chemical structure of BPC.
acter of the pocket occupied by the n-propyl groups, leading in turn
to increased selectivity for ERb.
our previous studies on generation of steroid surrogates bearing
a diphenylmethane skeleton, we speculated that the diphenylme-
thane derivative BPA would bind to the ligand binding domain of
ERs like 17b-estradiol, acting as a steroid surrogate. Clear SARs
for BPA was obtained, that is, introduction of alkyl chains at the
central carbon atom led to a switch from agonist to antagonist,
and introduction of two methyl groups at the 3- and 30-positions
Next, 16 and 17 were also docked into ER
a (PDB ID: 3UUC) to
explain the SARs of liner alkyl groups at the central carbon. A com-
mon feature of 16 and 17 was the direction of the liner alkyl
groups, that is, the methyl groups or ethyl groups at the central
carbon were directed toward M421, like the n-Pr groups of 18.
However, lowest binding energy derived from these docking stud-
ies was different, and it is noteworthy that the rank order of lowest
binding energy (18 < 17 < 16) was corresponded to the order of
increased the antagonistic activity and selectivity for ER
ERb. These structural developments of BPA resulted in the discov-
ery of 18 as a potent and selective ER -antagonist under our assay
conditions. Although BPA is also reported to possess anti-androgen
activity, 18 showed also showed high selectivity for ER over AR,
as compared with BPA. Docking studies suggested that 18 would
bind to the antagonistic conformation of ER . These results further
confirm the versatility of the multi-template approach and the util-
ity of the diphenylmethane skeleton. Based on its ER -selective
a over
a
ER
the SARs, that is, the length of the liner alkyl groups at the central
carbon was very critical for ER -antagonistic activity.
a IC50 (18 < 17 < 16). Therefore, this docking study supported
a
a
In 2003, BPA was reported to possess anti-androgen activity as
well.40 Therefore, we checked the AR-antagonistic activity of repre-
sentative compounds by means of AR reporter gene assay using the
pSG5-human AR reporter system.37 Antagonistic activity was mea-
sured in the presence of 0.5 nM dihydrotestosterone, an AR ago-
nist. BPA showed anti-androgen activity under our assay
conditions with an IC50 value of 5900 nM (Table 3). Under this con-
dition, 18 showed weaker AR-antagonistic activity (IC50 value of
25,000 nM) than BPA. Thus, 18 showed 28-fold and 5000-fold
a
a
antagonistic activity, 18 is considered to be a candidate agent for
treatment of breast cancer.
Table 3
AR-antagonistic activity of bisphenol A analogs
selectivity as an antagonist for ERa over ERb and AR, respectively.
These results indicated that lead compounds obtained via the mul-
ti-template approach can indeed be structurally modified to gener-
ate target-selective compounds. Other representative compounds
17, 33, 36 and 37 showed weak AR-antagonistic activity.
We believe that the multi-template approach for drug discov-
ery, focusing on proteins with similar fold structures, is effective
for generating lead compounds for multiple targets. Inspired by
Compound
AR IC50 (nM)
1 (BPA)
18
17
33
36
5900
25,000
4500
5400
1900
8200
37

4036
K. Maruyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4031–4036
18. Fowler, A. M.; Solodin, N.; Preisler-Mashek, M. T.; Zhang, P.; Lee, A. V.; Alarid, E.
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Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science.
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38. 1H NMR (500 MHz, CDCl3): d 6.90 (s, 2H), 6.85 (dd, J = 8.4, 2.0 Hz, 2H), 6.64 (d,
J = 8.4 Hz, 2H), 2.20 (s, 6H), 1.95–1.91 (m, 4H), 0.99–0.92 (m, 4H), 0.84 (t,
J = 7.3 Hz, 6H).; mp: 172–174 °C.; HRMS (FAB, m/z, M+) calcd for C21H28O2,
312.2089; found 312.2123.
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