Characterization of the pharmacophore properties of novel selective estrogen receptor downregulators (SERDs)

Article abstract of DOI:10.1021/jm100047k

Selective estrogen receptor (ER) down-regulators (SERDs) reduce ERα protein levels as well as block ER activity and therefore are promising therapeutic agents for the treatment of hormone refractory breast cancer. Starting with the triarylethylene acrylic acid SERD 4, we have investigated how alterations in both the ligand core structure and the appended acrylic acid substituent affect SERD activity. The new ligands were based on high affinity, symmetrical cyclofenil or bicyclo[3.3.1]nonane core systems, and in these, the position of the carboxyl group was extended from the ligand core, either retaining the vinylic linkage of the substituent or replacing it with an ether linkage. Although most structural variants showed binding affinities for ERα and ERβ higher than that of 4, only the compounds preserving the acrylic acid side chain retained SERD activity, although they could possess varying core structures. Hence, the acrylic acid moiety of the ligand is crucial for SERD-like blockade of ER activities.

Full text of DOI:10.1021/jm100047k

3320 J. Med. Chem. 2010, 53, 3320–3329
DOI: 10.1021/jm100047k
Characterization of the Pharmacophore Properties of Novel Selective Estrogen Receptor
Downregulators (SERDs)
Karen J. Kieser,†,‡,§ Dong Wook Kim,†, ,§ Kathryn E. Carlson,† Benita S. Katzenellenbogen,‡ and
John A. Katzenellenbogen*,†
†
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, and ‡Department of Molecular and
Integrative Physiology, University of Illinois, 407 South Goodwin Avenue, Urbana, Illinois 61801. §These authors contributed equally to this work.
Current address: Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-712, Korea.
Received January 13, 2010
Selective estrogen receptor (ER) down-regulators (SERDs) reduce ERR protein levels as well as block ER
activity and therefore are promising therapeutic agents for the treatment of hormone refractory breast
cancer. Starting with the triarylethylene acrylic acid SERD 4, we have investigated how alterations in both
the ligand core structure and the appended acrylic acid substituent affect SERD activity. The new ligands
were based on high affinity, symmetrical cyclofenil or bicyclo[3.3.1]nonane core systems, and in these, the
position of the carboxyl group was extended from the ligand core, either retaining the vinylic linkage of the
substituent or replacing it with an ether linkage. Although most structural variants showed binding affinities
for ERR and ERβ higher than that of 4, only the compounds preserving the acrylic acid side chain retained
SERD activity, although they could possess varying core structures. Hence, the acrylic acid moiety of the
ligand is crucial for SERD-like blockade of ER activities.
Introduction
4, Figure 1) is a hydroxylated analogue of compound 1 with
higher ER affinity,11,12 analogous to 4-hydroxytamoxifen,
being a high affinity analogue and metabolite of tamoxifen.14
It has been proposed that the SERD activities of both 4 and
ICI result from their distortion of the conformation of the ER
ligand binding domain (LBD) so that hydrophobic surfaces
become exposed, thereby promoting accelerated degradation of
the ER protein.15,16 The X-ray crystallographic structure of an
ERR-LBD complex with a structural analogue of ICI shows
the ligand bound in an inverted mode relative to estradiol, with
the long 7R-substituent projecting outward from the ligand
binding pocket, displacing helix-12 of the LBD and occupying
the coactivator binding groove.15 A recent structure of 4 with
ERR shows that helix-12 is tilted from the normal SERM posi-
tion through interaction of the carboxylic acid group of the
ligand with the positive end of the helix-12 dipole.16 In both
cases, fluorescent probes of exposed protein hydrophobic re-
gions have revealed that these ER-SERD structures are more
hydrophobic than are complexes with estrogen agonists (estra-
diol, DES) or SERMs (hydroxytamoxifen and raloxifene).16
Although the distortion of helix-12 in the ERR structure
complexed with 4 suggests an important role for the carboxylic
acid function of the acrylic acid side chain, it is not clear whether
this function is positioned optimally or might be improved by
presentation in an alternative manner, through other linkages,
or at different distances from the ligand core. Similarly, it is not
known whether the triarylethylene core structure is essential or
might be replaced by alternative moieties with higher ER
binding affinities. The hydroxytriarylethylene (TAE) core of
4, in particular, might be subject to cis-trans isomerization in
solution because it shares this structural feature with hydroxy-
tamoxifen and diethylstilbestrol, both of which do undergo this
isomerization in a facile manner.17,18
A novel class of compounds capable of modulating the level
and activity of the estrogen receptor (ERa) are termed selective
estrogen receptor downregulators (SERDs). These com-
pounds are mechanistically distinct from ER ligands such as
4-hydroxytamoxifen, which act as either agonists or antago-
nists, depending upon the target tissue, and are termed selective
estrogen receptor modulators (SERMs).1,2 Although certain
SERMs are clinically useful for menopausal hormone replace-
ment and for the prevention and treatment of breast cancer,3
there is a need for new ER ligands that are capable of over-
coming acquired endocrine resistance in breast cancer. Current
SERDs, such as ICI 182,780 (ICI, Figure 1), have a more
antagonistic profile than SERMs and are capable of inhibiting
the growth of tamoxifen-resistant breast cancer cells.4
SERDs are currently used in the treatment of metastatic
breast cancer,4,5 and their improved clinical activity is thought
to be derived from their ability to down-regulate the ER
protein as well as to block ER action.6-9 ICI, however, has
poor oral bioavailability,10 and a search for alternative drugs
led to the identification of GW-5638 (compound 1, Figure 1),
a SERD that shares the triarylethylene ligand core scaffold of
tamoxifen but has the basic side chain replaced by an acrylic
acid unit. Compound 1 has an agonist profile in bone but acts
as an antagonist in the breast11-13 and can inhibit the growth
of tamoxifen-resistant breast tumors.9 GW-7604 (compound
*To whom correspondence should be addressed. Telephone: (217)
333-6310. Fax: (217) 333-7325. E-mail: jkatzene@illinois.edu.
aAbbreviations: BN, bicyclononane; CF, cyclofenil; DES, diethylstil-
bestrol; E2, estradiol; ER, estrogen receptor; SERD, selective estrogen
receptor downregulators; SERM, selective estrogen receptor modulators;
TAE, triarylethylene.
r
pubs.acs.org/jmc
Published on Web 03/24/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3321
Figure 1. Selective estrogen receptor down-regulators (SERDs) and modulators (SERMs) and symmetrical ligands. The SERD GW-5638 (1)
and its hydroxylated metabolite, GW-7604 (4), can potentially undergo cis-trans isomerization (boxed structures, see text). The GW
compounds, as well as trans-hydroxytamoxifen, share a triaryethylene (TAE) structural core. The cyclic-core ligands, cyclofenil (CF, 2) and
bicyclononane (BN, 3), are symmetrical and thus do not exist as cis-trans isomers. ICI is a known SERD.
Figure 2. Series of SERD compound 4 analogues based on cyclofenil (CF) and bicyclononane (BN) and other analogues.
Therefore, to investigate to what extent the SERD activity
of 4 is dependent on the hydroxytamoxifen-type core, the
vinyl linkage, or the acrylic acid side chain, we prepared and
evaluated the SERD activity of a new series of ER ligands
having acidic side chains of varying length and linkages and
built on different ligand core structures, cyclofenil (compound
2) and bicyclononane (compound3), that are symmetrical and
thereforedo notexistasstereoisomers (Figures 1 and 2). From
our initial series, we selected analogues having high binding
affinity for ERR and ERβ, and we further characterized their
ability to alter the subcellular localization of ERR and to
down-regulate its protein levels and inhibit gene stimulation
by estrogen (SERD activity). The two new compounds having
the most favorable SERD-like activity retained the acrylic
acid side chain, although they had different core structures.
This study expands our current understanding of the optimal
pharmacophore requirements for SERD activity and their
capabilities in regulating the activity of the estrogen receptor.
It also highlighted the importance of the acrylic acid side chain
of 4 for engendering SERD-like activity but revealed that
alternative ligand core structures can be used in place of the
tamoxifen-like triarylethylene core used in the GW series.

3322 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Table 1. Estrogen Receptor Binding Assaye
Kieser et al.
(a) Structure and Binding Affinity of Basic TAE, CF, and BN Systems
(b) ERR and ERβ Binding Affinity of Other Ligands with a Bicyclononane Core
a Relative binding affinity (RBA) values are determined by competitive radiometric binding assays and are expressed as IC50[estradiol]/IC50
ꢀ
[compound]
100 (RBA, estradiol = 100). In these assays the Kd for estradiol is 0.2 nM for ERR and 0.5 nM for ERβ. b For each value, the β/R ratio is calculated such
that the ratio is >1 for compounds having higher affinity on ERβ than on ERR. c Compounds selected for further studies are indicated by a bold
underline. d Compound 4 exists as a mixture of E and Z isomers. See Figure 1. e The ERR and ERβ binding affinities of SERDs with the triarylethylene
(TAE), cyclofenil (CF), and bicyclononane (BN) cores with the acrylic acid substituent are presented in the top part of the table (part a). Some individual
variants with the bicyclononane core are presented in the lower part of the table (part b).
Results
Chemical Synthesis. The structures of the SERD 4 analo-
acrylate-substituted system, having longer chain acids linked
through a styryl function (Figure 2, “styrene acids”), as well
as some individual variants, bis-carboxymethyl and a deoxy
analogue or members with vinylphosphonate or trifluoro-
methanesulfonyloxy groups (Figure 2, bottom).
gue series that we prepared are summarized in Figure 2, and
those studied in greater detail are shown in Tables 1 and 2
(see compound numbers with underline). Some analogues
(42, 43, 44) are based on a well-known, high affinity non-
steroidal estrogen, cyclofenil (2, Figure 1), but the majority
(11, 12, 15, 17, 22-26, 29, 32-36, 46) are based on bicyclo-
nonane (3, Figure 1), a related nonsteroidal estrogen that has
exceptionally high binding affinity for both ERR and ERβ
(Table 1a).19 Notably, neither of these symmetrical ligand
cores have cis-trans isomers, as does the TAE-based com-
pound (compound 4, Figure 1). In both the cyclofenil and
bicyclononane series (designated CF and BN for simplicity),
we prepared the direct analogue of 4 containing the acrylic
acid substituent (17, 42), as well as a series with carboxy-
terminated alkoxy substituents (Figure 2, “alkoxy acids”).
Because members of the bicyclononane series proved to have
higher affinity, we prepared additional homologues of the
The synthetic routes used to prepare these compounds are
shown in Schemes 1-4. McMurry coupling was used to pre-
pare the parent cyclofenil and bicyclononane systems (com-
pounds 7-9) from their appropriate benzophenone precur-
sors (compounds 5 and 6, Scheme 1). Members of the alkoxy
ether series were prepared from the parent cyclofenil and
bicyclononane ligands by Williamson ether synthesis using
various ω-bromo esters, followed by ester hydrolysis (com-
pounds 32-36 (Scheme 2) and compound 43 (Schemes 3
and 4)). Good yields of monosubstitution on the bisphenol
parent ligands could be obtained in all cases except with ethyl
bromoacetate. In this case, the bisphenols were first
protected as the mono-TBDMS ethers (compounds 10, 37);
monoalkylation followed by saponification/deprotection
gave the desired monoalkoxymethyl ethers (compounds 15,

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3323
Table 2. Structure and Binding Affinity of the Extended Series of Styryl and Alkoxy Bicyclononanesd
a Relative binding affinity (RBA) values are determined by competitive radiometric binding assays and are expressed as IC50[estradiol]/IC50
ꢀ
[compound]
100 (RBA, estradiol = 100). In these assays the Kd for estradiol is 0.2 nM for ERR and 0.5 nM for ERβ. b For each value, the β/R ratio is calculated such
that the ratio is >1 for compounds having higher affinity on ERβ than on ERR. c Compounds selected for further studies are indicated by a bold
underline. d The ERR and ERβ binding affinity of both the styryl and alkoxy bicyclononane series was determined and expressed similarly to the
compounds in Table 1.
Scheme 1. Synthesis of Cyclofenil and Bicyclononane
Analoguesa
The binding affinities of the parent phenols in the triary-
lethylene (TAE), cyclofenil (CF), and bicyclononane (BN)
systems are presented in Table 1a. All three parent com-
pounds have very high binding affinity for ERR and ERβ,
but the highest affinity is shown by the parent bicyclononane
(3). As with hydroxytamoxifen,14 compound 4 exists as a
cis-trans isomer mixture. After careful recrystallization, we
were able to obtain only the Z isomer in pure form (40). The
lower affinity of the pure Z isomer (Table 1a) suggests that
the E isomer is predominant in binding to the ERs. This is
consistent with the X-ray crystal structures of this com-
pound,16 as well as binding affinity measurements and
X-ray structures for hydroxytamoxifen and diethylstilbes-
trol, where the isomer with the corresponding trans-4-
hydroxystilbene isomer is the active one.14,17,22
a Reagents: (a) TiCl4, Zn, THF, reflux, 10 h.
In the acrylic acid series (Table 1), the affinity of the cyclo-
fenil analogue (42) is considerably lower than that of the GW
compound (4); the bicyclononane analogue (17), however,
binds with comparable affinity. The binding affinities of two
alkoxy analogues prepared in both the cyclofenil and bicy-
clononane series (compounds 44 and 43, and compounds 15
and 34, respectively) provided additional evidence for the
higher affinity of members of the bicyclononane series; there-
fore, further synthetic efforts focused on this series. Also of
note are the binding affinities of certain other bicyclononane
analogues (Table 1, Figure 2, bottom) designed to test other
side chain functional groups. Compounds in which the OH is
missing (46) or substituted with a second alkoxy group (12)
have poor binding affinities. The ester (29) has lower affinity
than the corresponding acid 34; the methyl phosphonate 26
binds less well than the corresponding carboxylate 17, and the
trifluoromethane sulfonate (11) binds only moderately well.
Binding affinities of the extended bicyclononane series of
styryl and alkoxy analogues are given in Table 2. In both
44). The mono-TBDMS ethers were also used to prepare
the monotrifluormethane sulfonates (compounds 11, 38),
which where were used in Suzuki coupling to prepare mem-
bers of the styryl series (compounds 17, 22-25 (Scheme 2)
and compound 42 (Schemes 3 and 4)). A complete des-
cription of the syntheses with experimental details and
spectroscopic characterization is given in the Supporting
Information.
Biological Results. Estrogen Receptor Binding Assays and
Structure-Affinity Relationships. The compounds were as-
sayed for their binding affinity for human ERR and ERβ in a
radiometric competitive binding assay using [3H]estradiol as
tracer and estradiol as the reference standard.20,21 Binding
affinities are expressed as relative binding affinity (RBA)
values, with estradiol set at 100. Results are given in Table 1
(comparison of triarylethylene (TAE), cyclofenil (CF) and
bicyclononane (BN) series) and Table 2 (extended BN styryl
and alkoxy series).

3324 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kieser et al.
Scheme 2. Synthesis of Acid Substituted Cyclononane Analoguesa
a Reagents: (a) TBDMSCl, imidazole, room temp, THF, 24 h; (b) (i) Tf2O, triethylamine, 0 °C to room temp, 8 h; (ii) TBAF, room temp, 15 min; (c)
dimethyl vinylphosphonate, Pd(PPh3)2Cl2, triethylamine, DMF, 120 °C, 24 h; (d) BrCH2COOCH3, Cs2CO3, CH3CN, room temp, 24 h; (e)
CH2dCH(CH2)nCOOR, Pd(PPh3)2Cl2, triethylamine, DMF, 120 °C, 24 h; (f) ethyl acrylate, Pd(PPh3)2Cl2, triethylamine, DMF, 120 °C, 24 h; (g)
BrCH2(CH2)nCH2COOR, Cs2CO3 or K2CO3, CH3CN, room temp, 24 h; (h) 2 N KOH, MeOH, room temp, 24 h.
Scheme 3. Synthesis of Acid Substituted Cyclofenil Analoguesa
a Reagents: (a) TBDMSCl, imidazole, room temp, THF, 24 h; (b) (i) Tf2O, triethylamine, 0 °C to room temp, 8 h; (ii) TBAF, room temp, 15 min; (c)
ethyl acrylate, Pd(PPh3)2Cl2, triethylamine, DMF, 120 °C, 24 h; (d) ethyl 6-bromohexanoate, Cs2CO3, CH3CN, room temp, 24 h; (e) BrCH2COOCH3,
Cs2CO3, CH3CN, room temp, 24 h; (f) 2 N KOH, MeOH, room temp, 24 h.
series and for both receptors, binding affinities (with but one
exception) increase with increasing chain length, reaching
affinities much greater than that of 4 and even rivaling that of
estradiol. This is shown graphically in Figure 3. None of the
compounds in either Table 1 or Table 2 showed significant
selectivity for either ERR or ERβ, although those in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3325
cyclofenil series (Table 1a) tended to be more ERβ selective,
with the cyclofenil acrylic acid (42) being ∼5-fold in favor
of ERβ.
MCF-7 breast cancer cells were treated with these com-
pounds, and the ERR protein was extracted and analyzed
by SDS-PAGE and Western immunoblotting. Because
SERDs are known not only to lower ER levels in cells but
also to affect the subcellular distribution of ER,6-8 we
monitored ER levels in whole cell extracts (WC) and in
cytoplasmic (C), nuclear (N), matrix (M), and insoluble (I)
fractions after treatment with these various compounds. The
procedures used to obtain these extracts are given in the
Experimental Section, and representative Western immuno-
blots are shown in Figure 4.
The well characterized SERD ICI markedly reduced ERR
protein levels in the cytoplasmic fraction and did not raise
ERR levels in the nuclear fraction; the slight elevation of
ERR in the matrix and insoluble fractions is consistent with
reports that this compound does redistribute ERR into the
less soluble cell fractions (Figure 4A).6-8 The known SERD
4 also reduces cytoplasmic ERR protein, but ERR levels in
the nuclear, matrix, and insoluble fractions are elevated,
consistent with what others have shown.23 The bicyclono-
nane analogue (17) redistributed and down-regulated of ER
in a manner very similar to that of the SERD 4, as did the
cyclofenil analogue (compound 42), though the latter to a
somewhat lesser degree (Figure 4A).
Western Immunoblotting. We selected 10 compounds with
good binding affinity for ER for further analysis of their
SERD character (see underlined compound numbers in
Tables 1 and 2). These compounds were the initial SERD
compound (4), the direct analogues of 4 in both the cyclofenil
(42) and bicyclononane (17) series, the high affinity analogues
in the bicyclononane styryl (24 and 25) and alkoxy (15, 33, 34,
and 36) series, and the BN vinyl methyl phosphonate(26). The
well established SERD ICI was also included for comparison.
Scheme 4. Synthesis of the Deoxy Analogue (46) of Bicyclo-
nonane Compound 34a
Surprisingly, although they have higher ERR binding affi-
nities than the three compounds discussed above, none of
the bicyclononane acrylate homologues (styryl compounds
a Reagents: (a) ethyl 6-bromohexanoate, Cs2CO3, CH3CN, room
temp, 24 h; (b) 2 N KOH, MeOH, room temp, 24 h.
Figure 3. Dependence of ligand relative binding affinity on acid chain length. N represents the number of intervening atoms between the
phenyl and the carboxyl moieties, irrespective of whether they are carbon or oxygen.
Figure 4. Subcellular distribution and down-regulation of ERR protein by ICI and compounds 36, 25, 4, 42, 17 (A) and 33, 34, 24, 15, and 26
(B). MCF-7 cells were treated with 1 μM compounds for 16 h, and the level of cellular ERR in the indicated subcellular fraction and in whole cell
extracts was measured by Western immunoblotting. Sp1 was monitored as an internal reference for gel loading.

3326 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kieser et al.
Discussion
In this study, we have examined a range of compounds
structurally related to the selective estrogen receptor down-
regulator (SERD), compound 4. In all of these analogues, the
isomerization-prone hydroxyltriarylethylene ligand core of 4
was replaced by two symmetrical cyclic systems, cyclofenil and
bicyclononane, that are known to engender high ligand binding
affinity for both estrogen receptors, ERR and ERβ. In addi-
tion, homologues were prepared in which the carboxylic acid
group on the acrylic acid side chain was extended (keeping the
styrene linkage) or analogues in which it was replaced by an
ether-linked carboxyalkyl substituent. While in both analogue
series, extension of the acid function away from the ligand core
increased ER binding affinity substantially, it did not result in
compounds having SERD activity. In fact, the only com-
pounds capable of reducing levels of ERR protein, bicyclono-
nane 17 and cyclofenil 42, were ones that retained the acrylic
acid group present in 4. Of the two new SERDs, 17 and 42, the
higher affinity bicyclononane compound (17) proved to be the
better, and in most respects it closely replicated the activity of 4.
The marked dampening of ER action shown by compounds 4
and 17 indicates an obliteration of transactivation from both
AF-1 and AF-2, a known effect of pure antagonist/SERD
binding to the ER.28-30
Structural studies have suggested that the acrylic acid side
chain of 4 down-regulates ERR protein levels by interactions
with specific amino acids, namely, D351 in the ligand binding
domain of the receptor.31 This charge-charge repulsion blocks
the proper positioning of the critical helix 12,31,32 preventing it
from adopting either the folded-back orientation found in ago-
nist structures or the extended conformation found in SERM
structures, where helix 12 occludes the hydrophobic groove of
the AF-2 function, blocking interaction with coregulators.16
With helix 12 thus mal-disposed, hydrophobic patches are
exposed, and the receptor is thought to become a target for
ubiquitination and processing by the ubiquitin-proteasome
pathway.33 A related mechanism is proposed for the activity of
ICI-type SERDs,16 although in this case, helix-12 appears to be
completely disordered,34 with even greater exposure of hydro-
phobic regions for targeting ubiquitination.16
Figure 5. Western immunoblot analysis of of ERR protein level
and subcellular distribution with compound and estradiol treat-
ment. MCF-7 cells were treated with 1 μM compound for 16 h and
then (1 nM estradiol for 4 h prior to cell fractionation and
monitoring of ERR by Western immunoblots. Leftmost two lanes
show vehicle or E2 treatment for 20 h. Sp1 was monitored as an
internal reference for gel loading.
24 and 25) or the carboxyalkoxy analogues (compounds 15,
33, 34, and 36) that we tested demonstrated substantial
SERD-like activity (Figure 4). While cytoplasmic ERR levels
were somewhat lower, nuclear ER, and in some cases also
matrix and insoluble fractions, showed substantially in-
creased ERR levels. These compounds all lack the acrylic
acid side chain moiety which is present in compounds 4, 17,
and 42.24
Western immunoblot analysis of MCF-7 cells treated with
ICI and compounds 4, 42, and 17, alone or with E2, are
shown in Figure 5. In the presence of E2, all of the SERDs
tested were capable of lowering ERR protein levels. This
down-regulation was seen particularly in the whole cell lysate
and the cytoplasmic and the nuclear fractions. The cyclofenil
acrylic acid (42) showed lower activity (i.e., less ER protein
reduction), especially in the cytoplasmic fraction. The se-
questration of ERR protein in the insoluble fraction in-
creased upon concomitant E2 treatment. This may repre-
sent ligand-dependent turnover of the receptor,25 as it is well-
known that E2 enhances ER turnover.26
Remarkably, the SERD activity of the compounds in this
series appears to be uniquely associated with the acrylic acid
unit that is common to compound 4, cyclofenil 42, and
bicyclononane 17. Even replacing it with a carboxymethyl
ether, which enables the carboxyl group to access the same
space as it occupies in the acrylic acid, did not suffice to
engender SERD activity. This suggests that the rigidity of the
acrylate unit, in fact, might be essential for maintaining the
critical charge-charge repulsion with D351. Thus, the higher
binding affinity found when the carboxylic acid group is
spaced further from the aromatic ring might indicate relief
of this ligand carboxylate-ERR D351 Coulombic repulsion,
and it suggests that the critical interaction needed to distort
the position of helix 12 and induce ER degradation (the very
essence ofSERD activity) comeswitha cost in binding energy.
Relevant to this point, others have found that maintaining the
double bond but replacing the carboxylic acid group with a
carboxamide or methyl ketone, which would maintain rigidity
but remove the charge, also results in loss of SERD activity.24
While activation of the ubiquitin-proteasome pathway is a
plausible explanation for the down-regulation of ERR pro-
tein, Wijayaratne and McDonnell did not find a large increase
in ERR ubiquitination upon treatment with compound 4.35
Analysis of ERr Mediated Transcription Regulation. Tran-
scriptional analysis of established ERR target genes was
carried out on MCF-7 cells treated with the best three
compounds in the absence or presence of E2. The RNA
levels of three E2-upregulated genes (pS2, progesterone
receptor, carbonic anhydrase 12) were in most cases reduced
by the SERDs 4, 17, and 42 (Figure 6A). Compounds 4 and
17 significantly blocked the E2-mediated stimulation of the
pS2 and PR genes, but compound 42 was ineffective, con-
sistent with its less effective down-regulation of the ER
protein. However, 42 did block the E2-mediated activation
of the CA12 gene even more so than did compound 17.
Compound 4 was the most effective in blocking estrogen
action on all three of these E2-stimulated genes.
The three SERDs were also effective in reversing estrogen
action on two genes that are repressed by E2, namely, BLNK
and ENC1 (Figure 6B).27 Compounds 4 and 17 did, in fact,
reduce the E2-mediated repression of BLNK and ENC1 gene
expression (Figure 6B), increasing RNA levels for these
genes relative to the E2-repressed state, but the repression
was not fully reversed to the control, basal level of expres-
sion.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3327
Figure 6. Impact of different compounds on ERR mediated transcriptional activity. MCF-7 cells were treated with 1 μM compounds for 16 h
and then with vehicle (0.1% ethanol) or 1 nM estradiol for 4 h. Real-time PCR analysis was performed for E2-stimulated ERR-target genes (A)
or E2-repressed ERR-target genes (B) as a measure of compound ability to antagonize estradiol action.
There are, however, other reports implicating the protea-
some-ubiquitin pathway in ER turnover. Duong et al.
described a ternary complex between ERR, p53, and the
oncogenic ubiquitin-ligase Mdm2 in MCF-7 cells.36 Also,
receptor down-regulation is blocked by proteasome-inhibitor
MG132 treatment.37-39 It has been suggested that the 26S
proteasome is necessary for the cycling of ERR protein
needed to maintain transcription of target genes.25 These
studies all suggest the involvement of the ubiquitin-protea-
some pathway in the down-regulation of ERR protein.
As others have seen, SERDs such as ICI not only reduce
ERR protein levels overall but also redistribute it from the
most soluble cellular compartments, the cytoplasm and
nucleus, to less soluble compartments, the matrix and in-
soluble fractions. Presumably, at these locations it cannot
function in its role as a ligand-modulated transcription
factor. In fact, the movement of ER into these compart-
ments might be a consequence of its ubiquitination, as others
have noted.40-42 Our observations of such down-regulations
and alterations in intracellular distribution of ER after
treatment with these new SERDs are consistent with their
prevention of estrogen-regulated gene expression and high-
light the importance of the acrylic acid side chain in enabling
SERD activity.
Conclusion
Compounds capable of suppressing estrogen action by
reducing estrogen receptor levels appear to have particular
promise for novel endocrine therapies in breast cancer. It is

3328 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kieser et al.
hoped that they might not succumb to the development of
acquired resistance that typically follows treatment with
SERMs, and this appears to be the case in model systems.43
Our study has demonstrated that other ligand core structures,
in addition to the triarylethylene ligand core in traditional
SERMs like tamoxifen and in the original GW SERD (4), can
be used to produce SERD compounds (namely, the cyclofenil
compound 42 and the bicyclononane compound 17) that are
still very effective at moderating the activity of ER. Our
biological analyses of these novel SERDs underscore the
importance of the acrylic side chain in effecting the down-
regulation of ERR and the suppression of ER-regulated gene
expression.
GGCCTCCCCCTCAGTCTCT; ENC1 reverse (r), GCACT-
CACTACTGCGGCGT. The primers used for pS2, progester-
one receptor, CA12, and BLNK were those previously des-
cribed.27,44,46,47 Statistical analysis was performed with repeated
measures one-way ANOVA using the Tukey’s t test.
Estrogen Receptor Binding Affinity Assays. Relative binding
affinities were determined by a competitive radiometric binding
assay, as previously described,20,21 using 10 nM [3H]estradiol as
tracer (GE Healthcare, Piscataway, NJ) and purified full-length
human ERR and ERβ (PanVera/InVitrogen, Carlsbad, CA).
Incubations were for 18-24 h at 0 °C. Then the receptor-ligand
complexes were absorbed onto hydroxyapatite (BioRad,
Hercules, CA) and unbound ligand was washed away. The
binding affinities are expressed as relative binding affinity
(RBA) values, with the RBA of estradiol set to 100. The values
given are the average ( range or SD of two or more independent
determinations. Estradiol binds to ERR with a Kd of 0.2 nM and
to ERβ with a Kd of 0.5 nM.
Experimental Section
Synthesis Materials and Methods. See Supporting Informa-
tion for details on synthesis, spectroscopic characterizations,
and purity determination by normal and reversed phase HPLC
analysis. All compounds assayed were >95% pure in both
HPLC systems.
Biological Materials and Methods. Compounds. E2 and ICI
182,780 (ICI) were purchased from Sigma Chemical Co. (St.
Louis, MO).
Acknowledgment. We thank Zeynep Madak-Erdogan for
helpful experimental advice. This work was supported by
grants from the National Institutes of Health [NIH Grant
CA 018119 (B.S.K.), NIH Grant DK 015556 (J.A.K.)] and a
Breast Cancer Research Foundation grant (B.S.K.).
Cell Culture. The human breast cancer MCF-7 cell line was
maintained in culture as previously described.44 For Western
analysis, cells were steroid depleted in phenol red-free media
supplemented with 5% charcoal-dextran-treated calf serum for
5 days prior to the indicated ligand treatment. Media were
changed on day 2 of culture, and cells were then treated with
ligands. For real-time PCR gene expression analysis, cells were
steroid depleted in phenol red-free media supplemented with 5%
charcoal-dextran-treated calf serum for 7 days prior to the
indicated ligand treatment. Media were changed on days 2 and 4
of culture, and cells were then treated with ligands.
Supporting Information Available: Synthesis and spectro-
scopic characterization (9-12, 14-46), 1H and 13C NMR
spectra (9-12, 14-46), and normal and reversed phase HPLC
chromatograms (11, 15, 17, 22-26, 29, 31-36, 42-44, 46). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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