Structure-function relationships of estrogenic triphenylethylenes related to endoxifen and 4-hydroxytamoxifen

Article abstract of DOI:10.1021/jm901907u

Estrogens can potentially be classified into planar (class I) or nonplanar (class II) categories, which might have biological consequences. 1,1,2-Triphenylethylene (TPE) derivatives were synthesized and evaluated against 17β-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer cells. All TPEs were estrogenic and, unlike 4-hydroxytamoxifen (4OHTAM) and Endoxifen, induced cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells with the order of potency as followed: E2 > 1,1-bis(4,4′-hydroxyphenyl)-2-phenylbut-1-ene (15) > 1,1,2-tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1- phenylbut-1-en-2-yl)phenol (7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2- yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl) phenol (12) > 4-OHTAM. Transient transfection of the ER-negative breast cancer cell line T47D:C4:2 with wild-type ER or D351G ER mutant revealed that all of the TPEs increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a class I estrogen and the TPEs as class II estrogens. Using available conformations of the ER liganded with 4OHTAM or diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.

Full text of DOI:10.1021/jm901907u

J. Med. Chem. 2010, 53, 3273–3283 3273
DOI: 10.1021/jm901907u
Structure-Function Relationships of Estrogenic Triphenylethylenes Related to Endoxifen and
4-Hydroxytamoxifen
Philipp Y. Maximov,†,‡ Cynthia B. Myers,† Ramona F. Curpan,§ Joan S. Lewis-Wambi,† and V. Craig Jordan*,
†Fox Chase Cancer Center, Philadelphia, Pennsylvania, ‡Russian State Medical University, Moscow, Russia, §Institute of Chemistry,
Romanian Academy, Timisoara, Romania, and Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road,
NW, Research Building, E501, Washington, DC 20057-1468
Received December 23, 2009
Estrogens can potentially be classified into planar (class I) or nonplanar (class II) categories, which
might have biological consequences. 1,1,2-Triphenylethylene (TPE) derivatives were synthesized
and evaluated against 17β-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer
cells. All TPEs were estrogenic and, unlike 4-hydroxytamoxifen (4OHTAM) and Endoxifen, induced
cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells
with the order of potency as followed: E2 > 1,1-bis(4,40-hydroxyphenyl)-2-phenylbut-1-ene (15) >
1,1,2-tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol
(7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-
hydroxyphenyl)but-1-en-2-yl)phenol (12) > 4-OHTAM. Transient transfection of the ER-negative
breast cancer cell line T47D:C4:2 with wild-type ER or D351G ER mutant revealed that all of the TPEs
increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the
importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a class I estrogen and
the TPEs as class II estrogens. Using available conformations of the ER liganded with 4OHTAM or
diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.
Introduction
TAM is a substituted derivative7,8 of the long-acting estro-
gen triphenylethylene.9 TAM efficacy depends on the forma-
tion of clinically active metabolites 4-hydroxytamoxifen
(4OHTAM)10 and Endoxifen11 (Figure 1), which have a
greater affinity to ERR and a much higher antiestrogenic
potency in breast cancer cells compared to the parent drug.
We are unaware of the subtle molecular changes that occur
when estrogen binds to the ER to produce the ER complex
because the whole complex has not been crystallized. As a
consequence of this gap in our knowledge, the modulation of
ERR can only be deduced by exploring structure-function
relationships. However, the ligand binding domain (LBD) of
ERR has been crystallized12,13 with the estrogens 17β-estra-
diol (E2), diethylstilbestrol (DES), and the SERMs, 4OH-
TAM and raloxifene (Figure 1). The resolution of the
structure of the estrogen: LBD complex by X-ray crystal-
lography demonstrates that the planar estrogens E2 and DES
are sealed within the LBD by helix 12.12,13 This activates
activating function (AF)-2 at the upper surface of helix 12 by
the interaction with coactivators to facilitate full estrogen
action. In contrast, the bulky side chain of 4OHTAM and
raloxifene prevents helix-12 from sealing the LBD and this
produces antiestrogenic action.12,13 However, although AF-2
is deactivated, the 4OHTAM:ERR complex has estrogen-like
activity,14 whereas raloxifene does not.15 This is believed to be
because the side chain of raloxifene shields and neutralizes
asp351 to block estrogen action.16 In contrast, the side chain
of tamoxifen is too short. It appears that when helix 12 is not
positioned correctly the exposed asp351 can interact with AF-
1 to produce estrogen action. This estrogen-likeactivitycan be
Breast cancer is one of the most frequently diagnosed
cancers among women in the United States, with an estimated
192370 new cases of invasive disease and 40170 deaths in
2009.1 Although the exact etiology of breast cancer is not
known, there is strong evidence that estrogen plays a role in its
development and progression.2 The effects of estrogen are
mediated via the estrogen receptors (ERsa), ER-alpha (ERR)
and ER-beta (ERβ), which are present in more than 80% of
breast tumors. With regard to the therapy of breast cancer,
ERR remains the most important target and its presence in
breast tumors is routinely used to predict response to selective
ER modulators (SERMs), such as tamoxifen (TAM).3,4 TAM
(Figure1) is alsothe firstchemotherapeutic drug totarget ER-
positivebreast cancer cells5 andprevent tumorigenesisinhigh-
risk women.6 TAM is available worldwide to treat patients
with ER-positive breast cancers.
*To whom correspondence should be addressed. Phone: (202) 687-
2795. Fax: (202) 687-6402. E-mail: vcj2@georgetown.edu.
a Abbreviations: ER, estrogen receptor; SERM, selective estrogen
receptor modulators; TAM, tamoxifen; 4OHTAM, 4-hydroxytamox-
ifen; LBD, ligand binding domain; E2, 17β-estradiol; DES, diethylstil-
bestrol; AF, activating function; TPE, triphenylethylene; EC50, effective
concentration 50%; ERE, estrogen response element; rmsd, root-mean-
square deviation; THF, tetrahydrofuran; RPMI, Roswell Park Memor-
ial Institute; FBS, fetal bovine serum; SFS, stripped fetal bovine serum;
PBS, phosphate buffered saline; OPTI-MEM, Optimum Eagle’s Mini-
mum Essential Media; RCSB, Research Collaboratory for Structural
Bioinformatics; PDB, Protein Data Bank; OPLS, optimized potential
for liquid simulations; IFD, induced fit.
r
2010 American Chemical Society
Published on Web 03/24/2010
pubs.acs.org/jmc

3274 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Maximov et al.
Table 1. The EC50 Values for E2 and the Tested Triphenylethylenes in
MCF-7:WS8 Cells Proliferation Assays
Results
Chemistry. The general synthetic routes used to prepare
substituted 1,1,2-tribenzyl-but-1-ene compounds are out-
lined in Scheme 1. Desoxyanisoin was treated with potas-
sium t-butoxide followed by reflux with ethyl iodide to give 1
in 74% yield. Intermediate 1 was refluxed with the formed
Grignard reagent of 4-bromoanisole and then treated with
phosphoric acid to yield 2. Removal of the methoxide groups
was accomplished with boron tribromide to give 3. Isomers
6-7 were synthesized from 1 by treatment with the formed
Grignard reagent of bromobenzene followed by reflux in
phosphoric acid to yield isomers 4-5. Removal of the two
methoxides was accomplished with boron tribromide result-
ing in isomers 6-7. Compounds 11-12 were obtained by
reaction of desoxyanisoin with glacial acetic acid and hydro-
iodic acid to give 8 in 90% yield. Dihydroxy 8 was protected
using 3,4-dihydro-2H-pyran and p-toluene sulfonic acid to
form 9. Compound 9 was treated with potassium t-butoxide
followed by reflux with ethyl iodide to yield 10 in 87%.
Compound 10 was refluxed with the formed Grignard
reagent of 4-bromophenetole, followed by acid hydrolysis
using phosphoric acid to yield isomers 11-12. Synthesis of
15 proceeded from reaction of anisole with 2-phenylbutyryl
chloride to form monomethoxide 13 in 94% yield. Com-
pound 13 was coupled with 4-methoxyphenyl magnesium
bromide, followed by phosphoric acid to produce 14. The
methoxides of 14 were treated with boron tribromide to give
dihydroxy 15.
Figure 1. The formula of tamoxifen and its hydroxylated metabo-
lites Endoxifen, and 4OHTAM. The related SERM raloxifene is
shown for comparison.
inhibited by substituting asp351 for glycine an uncharged
amino acid.17
Planar or nonplanar compounds are both classified as
estrogens based on their actions to cause growth of the
immature rodent uterus or provoke vaginal cornification in
castrate animals. However, knowledge of the structure of the
4OHTAM:ER LBD complex13 led to the idea that all estro-
gens may not be the same in their interactions with ER.18
Previous studies suggest that nonplanar triphenylethylenes
(TPEs) with a bulky phenyl substituent prevents helix-12from
completely sealing the LBD pocket.18 This physical event
creates a putative “antiestrogen like” configuration within
the complex. However, the complex is not antiestrogenic
because Asp351 is exposed to communicate with AF-1, there-
bycausingestrogen-like action. Thus, there are putativeclassI
(planar) and class II (nonplanar) estrogens.18 A similar clas-
sification and conclusion has been proposed,19 but the biolo-
gical consequences of this classification are unknown.
In this report, we further addressed the hypothesis that the
shape of the ER complex can be controlled by the shape of an
estrogen. We have synthesized a range of hydroxylated TPEs
to establish new tools to investigate the relationship of shape
with estrogenic activity through the exposure of Asp351. For
convenience, the structure of nonsteroidal antiestrogens de-
scribed in the text are illustrated in Figure 1 and the test
compounds in Table 1.
Pharmacology. We compared and contrasted the estrogen-
like properties of the hydroxylated TPEs to promote
proliferation in the ERR-positive human breast cancer cell

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3275
Figure 2. Effects of E2, test TPEs 3, 6, 7, 12, and 15 and antiestrogens 4OHTAM and Endoxifen on the proliferation of MCF-7:WS8 breast
cancer cells. Cells were treated with the indicated compounds for 7 days.
Scheme 1. Synthesis of Substituted 1,1,2-Tribenzyl-but-1-ene Compoundsa
a Reaction conditions: (a) KOtBu, ether, 1 h, then, EtI, reflux 12 h; (b) 4-BrMgC6H4R2, THF, refluxed 12 h, then, H3PO4, refluxed 2 h; (c) BBr3,
CH2Cl2, 4 days; (d) HI, AcOH, 130-140 °C, 4 h; (e) C5H8O, p-CH3C6H4SO3H-H2O, 0 °C 4.5 h; (f) AlCl3, CS2, 20 °C, 22 h.
line MCF-7:WS8. Compounds were compared with the
tamoxifen metabolites 4-OHTAM and Endoxifen, which
have a high affinity for the ER (because of the appropriately
positioned phenolic hydroxyl) but are antiestrogenic because
of the alkylaminoethoxy-side chain. To compare the biolo-
gical activities of the tested TPEs, we employed DNA
proliferation assays which are described in the Materials
and Methods.
Figure 2 shows that our MCF-7:WS8 human breast cancer
cells were exquisitely sensitive to E2, which produced a
concentration-dependent increase in growth with maximal
stimulation at 1 ꢀ 10-11 M. All of the TPE’s were potent
agonists with the ability to stimulate MCF-7:WS8 breast
cancer cell growth, however, their agonist potency was less
compared to E2, which had an effective concentration 50%
(EC50) of 1 ꢀ 10-12 M. The most potent of the phenolic TPEs
was bisphenol (15), with an EC50 of approximately 5 ꢀ 10-11
M. The second potent were the E and Z-isomers of the
diphenolic TPEs, compounds 6 and 7, which both had
an EC50 of approximately 1 ꢀ 10-10 M. The triphenolic
TPE (3) was slightly less active, with an EC50 of approxi-
mately 1.5 ꢀ 10-10 M, whereas the ethoxy TPE (12) was the
least potent, with an EC50 of approximately 4 ꢀ 10-9 M.
The EC50 values for all the tested compounds are outlined

3276 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Maximov et al.
Figure 3. The ability of the tested TPEs 3 and 12 and 4OHTAM and Endoxifen to inhibit estradiol-stimulated MCF-7:WS8 breast cancer cell
growth. Cells were treated with indicated compounds for 7 days.
Figure 4. ERE luciferase assay in MCF-7:WS8 cells transiently transfected with an ERE luciferase construct and treated with E2, test TPEs 3,
6, 7, 12, and 15 and 4OHTAM.
in Table 1. The compound 12 was prepared to replicate a
molecule without the alkyl nitrogen group of 4-OHTAM or
Endoxifen, and this derivative had reduced estrogenic po-
tency comparison to the other TPEs, however, the mole-
cule remained a full estrogen agonist in our proliferation
assays. The metabolites, 4-OHTAM and Endoxifen, had no
significant agonist effect in MCF-7:WS8 cells, however,
these compounds at 1 μM were able to completely inhibit
estradiol-stimulated MCF-7:WS8 breast cancer cell growth
(Figure 3), thus confirming their role as antagonists/anti-
estrogens. Similar experiments performed with compounds 3
and 12 showed an inability to block estradiol-stimulated
growth in MCF-7:WS8 cells at concentrations up to 1 μM
(Figure 3). On the basis of these findings, compounds 3
and 12 were classified as estrogens with a pharmacology,
in this assay, indistinguishable from the natural planar
estrogen E2.
It is interesting to note that compounds 6 and 7, which are
the E- and Z-isomers of the diphenolic TPEs, were equiva-
lent in their agonistic potency, thus suggesting that isomer-
ization occurs in vitro given an equilibrium mixture. This
phenomenon has been noted previously with the E-isomer of
4-OHTAM,20 but the true pharmacology of the separate
isomers was eventually resolved by the synthesis of fixed ring
analogues.20,21 Both the E- and Z-isomers of 4-OHTAM are
antiestrogenic because they block the proliferation of estra-
diol-stimulated growth in MCF-7 breast cancer cells and
they inhibit estradiol-stimulated prolactin gene activation.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3277
structures wereused in thisstudy toestablishwhether a class II
compound could become nonestrogenic with the D351G ER
mutant.
Our series of phenolic TPEs were evaluated in the ER-
negative breast cancer cell line T47D:C42,23 which was
transiently transfected with an ERE luciferase plasmid and
either the wild-type ER or the D351G mutant ER. Figure 5A
shows that in the presence of the wild-type ER all of the
tested TPE compounds were potent agonists with the ability
to significantly enhance ERE luciferase activity. In contrast,
when the D351G mutant ER gene was transfected with the
ERE luciferase reporter, only the planar E2 was estrogenic,
whereas the TPEs did not activate the ERE reporter gene
(Figure 5B). Overall, these results confirm the importance of
Asp351 in ER activation by TPE ligands to trigger estrogen
action.
Analysis of the Induced Fit Models for Tested TPEs. Data
analysis was performed on top ranked poses for each of the
tested TPEs and for comparison reasons on 4OHTAM
(Figure 6A). The top ranked structure from induced fit for
4OHTAM has a ligand root-mean-square deviation (rmsd)
˚
of 0.55 A compared with the experimental structure. In
addition to the low ligand rmsd, there is a good similarity
between the 3ert crystal structure and the top-ranked struc-
tures from docking (Figure 6B), the conformations of D351,
E353, R394, T347, H524, and the rest of amino acids which
line the binding site are nearly superimposable in both
structures. Also, the well-known network of H-bonds is
formed between 4OHTAM and E353, R394, and water
molecules. The most significant difference is that in the top
docked pose of 4OHTAM, the antiestrogenic chain is moved
closer to D351 to form the interaction between the amino
group of 4OHTAM and carboxylate of D351. Induced fit
docking of the TPE derivatives: 3, 6, 7, 12, 15, and Endoxifen
in the ligand binding domain of ERR (3ert) has yielded
ligand poses which display a binding mode (Figure 6B) very
similar with that of 4OHTAM in the ER binding site
(Figure 6A). Thus, the superimposition of the top ranked
poses of each ligand onto the 4OHTAM cocrystallized with
ERR (binding cavity filled with water) shows the ligands
binding to the receptor in a similar mode with 4OHTAM,
having the propensity to form the same hydrophobic con-
tacts with the amino acids lining the binding cavity. Further-
more, the complex H-bond network is formed with E353,
R394, H524, and a highly ordered water molecule positioned
between E353 and R394 (Figure 6B). Interestingly, a H-bond
has been noticed between the hydroxyl group of 15, 3, 7, and
the side chain of T347 is stabilized by an additional interac-
tion with a water molecule from close proximity and pre-
cludes the interaction of the ligands with D351. The situation
is different when water is removed from the binding site. In
this case, the OH is shifted so that the H interacts with the
carboxylate group of D351 and the HO group of T347 is
shifted to form a H-bond with the oxygen. (data not shown).
The molecular docking results have shown that most of the
compounds form the H-bond network encountered in the
case of agonists (E353, R394, H524, water) and display
hydrophobic interactions with the amino acids lining the
binding site. An interesting interaction is the hydrogen bond
with T347 which seems to be stabilized by a water molecule
and it was observed in different docking simulations (flexible
and rigid). However, analysis of other ER crystal structures
has not revealed additional data to confirm this interaction.
Additional work has to be done to verify the hypothesis
Figure 5. Luciferase assay in ER-negative T47D:C4:2 cells, tran-
siently transfected with ERE luciferase and wild type (A) and
D351G (B) mutant ER constructs, respectively, and treated with
E2, tested TPEs 3, 6, 7, 12, and 15 and 4OHTAM. Results
demonstrate that substitution of Asp351 to Gly in ER abrogates
the agonistic activity on all tested TPEs (class II estrogens), except
planar E2 (class I estrogen).
The E-isomer is however approximately 1/100 the potency of
the Z-isomer.
To determine the ability of the test TPEs to activate the
ER, MCF-7:WS8 cells were transiently transfected with an
estrogen response element (ERE)-luciferase reporter gene
encoding the firefly reporter gene with five consecutive EREs
under the control of a TATA promoter. The binding of
ligand-activated ER complex at the EREs in the promoter of
the luciferase gene activates transcription. The measurement
of the luciferase expression levels permits a determination of
agonist activity of the TPE:ER complex. Figure 4 shows that
all the phenolic TPEs were estrogenic, but E2 was 100 times
more potent than the most potent TPE bisphenol (15). The
order of potency was as follows: E2 > 15 > 3 > 7 > 6 >
12 > 4-OHTAM. None of the tested TPEs were antiestro-
genic in this assay.
Our goal was to confirm and advance the hypothesis that
the shape of the estrogen ER complex was different for
planar and nonplanar (TPE -like) estrogens. This hypothesis
has been advanced independently by ourselves18,22 and
Gust’s group.19 Through a series of studies using mutant
ER expression in an ER negative breast cancer cell line, we
found that the mutant D351G ER completely suppressed
estrogen-like properties of 4-OHTAM at an endogenous
TGFR target gene.17 Use of this assay led us to classify
planar estrogens (DES or E2) as class I and nonplanar
estrogens (TPE-type) as class II. A broad group of compound

3278 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Maximov et al.
˚
weak (3.8-4 A), and it was mostly noticed when the simula-
tions were run with the receptor without water in the binding
site. This would mean that D351 is exposed and not shielded
so it could communicate intrinsic estrogenic properties of the
complex to AF-1.
The best poses of the tested TPEs 3, 6, 7, 12, 15, and
Endoxifen, obtained from docking simulations ran against
the antagonist conformation of the ER, were superimposed
on the experimental agonist conformation of the ER (ER
cocrystallized with estradiol, PDB code 1GWR) (Figure 7A).
This has shown that these ligands are unlikely to be accom-
modated in the agonist conformation of the ER due to the
sterical clashes between “Leu crown”, mostly Leu525 and
Leu540, helix 12, and ligands as depicted in Figure 7B,
indicating, that these ligands most likely bind to ER’s
conformation more closely related with the antagonist form.
Discussion
The aim of this structure function relationship study was to
evaluate the pharmacological properties of synthetic TPEs as
estrogens in MCF-7 human breast cancer cells using the DNA
proliferation assay and ERE luciferase assays. Our results
show that all of the synthesized TPEs possess potent estrogen-
like properties in our MCF-7 human breast cancer cells. These
TPEs markedly increased cell growth and enhanced ERE
luciferase activity. In contrast, the tamoxifen metabolites
4OHTAM and Endoxifen, which possess an alkylami-
noethoxy side chain in their structure, failed to induce growth
or increase ERE luciferase activity, thus confirming their role
as antiestrogens.
X-ray crystallography of ER-4OHTAM and ER-Raloxi-
fene complexes demonstrate that the presence of the alkya-
minoethoxy side chain of 4OHTAM is crucial for the ER to
gain an antagonistic conformation by displacing the H12 of
the receptor by 4OHTAM’s bulky side chain, thus preventing
the binding of the coactivators.13 On the basis of the results of
our proliferation assays and the luciferase assays, it is clear
that repositioning of the hydroxyl groups changed the biolo-
gical potencies of the tested TPE compounds, which lowered
their estrogenic potency compared to thatof E2. However, the
fact that these TPEs were able to significantly induce growth
and ERE activation in MCF-7:WS8 cells demonstrated that
they are still full agonists. The absence of the alkyami-
noethoxy side chain on the tested TPEs does not allow these
compounds to act as antiestrogens, like 4-OHTAM or En-
doxifen, which possesses the alkyaminoethoxy side chain.13
However, despite the changes in biological potencies of the
tested TPEs, due to repositioning of the hydroxyl groups and
addition of the ethoxy group, these compounds also main-
tainedtheir abilityto activatethe ERE as was demonstrated in
our ERE luciferase assays.
Another interesting aspect in our study is the importance of
Asp351 in activation of the ER thereby acting as a molecular
test for the presumed structure of the TPE:ER complex. On
the basis of the X-ray crystallography of the ER in complex
with 4OHTAM13 and Raloxifene,12 it was determined thatthe
basic side chains of these antiestrogens are in proximity of
Asp351 in the ER. It was hypothesized that this interaction
with Raloxifene actually neutralizes and shields Asp351,
preventing it from interacting with ligand-independent acti-
vating function 1 (AF-1). In contrast, 4OHTAM possesses
some estrogenic activity because the side chain is too short.13
Substitution of Asp351 with glycine leads to loss of estrogenic
Figure 6. (A) Cartoon representation of the human ERR ligand
binding domain complexed with 4-hydroxy tamoxifen, the antagonist
conformation of the receptor. Helix 12 is depicted in blue, the amino
acids involved in the H-bond network with the ligand are displayed as
sticks. and the ligand is colored in purple. (B) Molecular docking of
TPE derivatives into the binding site of ERR. For comparison
reasons, the top ranked ligand-protein complex is superimposed on
the crystal structure of the receptor cocrystallized with 4-OHT; the
amino acids lining the biding sites of both complexes are shown and
the complex H-bond network between ligand and the binding site is
displayed. The induced fit docking poses of the ligands are colored as
follow: 15 in cyan, 3 in blue, 6 in orange, 7 in pink, 12 in green,
Endoxifen in yellow, while the crystal structure is depicted in purple.
(docking, binding energy calculations through semiempircal
and/or ab initio methods, etc.). The interaction with D351 is

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3279
activity of the ER bound with 4OHTAM.17,24 Results from
ERE luciferase assays in T47:C4:2 cells transiently transfected
with wild type and D351G mutant ER expression plasmids
demonstrated that wild type ER was activated by all of the
tested TPEs, however substitution of Asp351 by Gly pre-
vented the increase of ERE luciferase activity by all TPEs and
only planar E2, which does not interact with Asp351 at all, or
exposes it on the surface of the complex, was able to activate
ERE in D351G ER transfected cells. This confirms and
expandsthe classification of estrogens, whereplanar estrogens
such as E2 are classified as class I and all TPE-related
estrogens are classified as class II estrogens based on the
mechanism of activation of the ER.18
It is important to note that all of the tested TPEs were
agonists in our wild type ER assay systems, however, exten-
sive studies of the structure-function relationship of phenolic
TPEsby Gust and co-workers,25,26 demonstrated thatsomeof
these compounds were potent antagonists in their MCF-7:2A
cells stably transfected with an ERE luciferase plasmid.
Specifically, these investigators found that compounds 1,1,2-
tris(4-hydroxyphenyl)but-1-ene and 1,1-bis(4,40-hydroxy-
phenyl)-2-phenylbut-1-ene, which correspond to compounds
3 and 15 in this study, were able to completely inhibit
estradiol-stimulated ERE luciferase activity at 100 nM. The
antagonistic potency of these compounds, however, did not
correlate with results from the cytotoxicity assays perfor-
med in their wild-type MCF-7 cells.25,26 Both compounds
1,1,2-tris(4-hydroxyphenyl)but-1-ene (designated as 3 in this
study) and 1,1-bis(4,40-hydroxyphenyl)-2-phenylbut-1-
ene (designated as 15 in this study) produced weak cytotoxic
effects only at concentrations above 5 uM, which were well
beyond the concentration range used in our study. Thus it is
possible that the variation in findings between our laboratory
and that of Gust and co-workers25,26 might be due to differ-
ences in our in vitro model systems and our experimental
design.
Conclusions
Wehave confirmed and advanced the hypothesis18,19,22 that
estrogens can be classified into planar class I compounds (E2)
and nonplanar class II compounds (TPEs). Armed with these
new tools, we are now poised to examine the biological
consequences of estrogen classification based on the shape
of the resulting ER complex.
Figure 7. (A) View of ERR binding cavity. The X-ray crystal
structure of ERR complexed with estradiol (PDB code 1GWR),
the agonist conformation of the receptor. The amino acids lining
the binding site are depicted as sticks colored by element. The color
code is blue for carbon, red for oxygen, gray for nitrogen, and
yellow for sulfur. The ligand is represented as sticks having the
same colored code like the receptor and the ligand’s surface is
colored in gray. (B) View of ERR binding cavity. The best poses of
BisPhen, TriOHTPE, EDiOHTPE, ZDiOHTPE, Z4EthoxDiO-
HTPE, Endox obtained from docking simulations ran against
the antagonist conformation of the receptor are superimposed on
the agonist conformation of the receptor, ERR cocrystallized
with estradiol (PDB code 1GWR). The amino acids involved in
steric clashes with the ligands, Leu525 and Leu540, are depicted
as molecular surfaces colored in blue while the rest of amino acids
lining the binding site are depicted as sticks colored by element, the
color code is blue for carbon, red for oxygen, gray for nitrogen,
and yellow for sulfur. The ligands are represented in sticks with the
associated molecular surfaces. They respect the same coloring
code with the exception of carbons which are colored as follow:
BisPhen in cyan, TriOHTPE in blue, EDiOHTPE in orange,
ZDiOHTPE in pink, ZEthoxDiOHTPE in green, Endoxifen in
yellow. For clarity, waters and hydrogen atoms were omitted from
the binding site.
Materials and Methods
Chemistry. 1,1,2-Tris(4-hydroxyphenyl)but-1-ene (3). 1,1,2-
Tris(4-hydroxyphenyl)but-1-ene (3) was synthesized according
to the method of Lubczyk, Bachmann, and Gust.26
1,2-Bis(4-methoxyphenyl)butanone (1). Potassium tert-butox-
ide (1.35 g, 12 mmol) was added to a solution of desoxyanisoin
(2.55 g, 10 mmol) in anhydrous ether under a nitrogen atmo-
sphere, and the mixture was stirred for 1 h. At which time,
iodoethane (0.8 mL, 10 mmol) was added dropwise and the
mixture was refluxed for 12 h. Water (40 mL) was added, and the
product was extracted with ether. The ether extracts were
combined, dried over sodium sulfate, and evaporated under
reduced pressure. The crude product was dissolved in carbon
tetrachloride (10 mL), and petroleum ether was added to crystal-
lize unreacted desoxyanisoin. Dexosyanisoin was filtered off,
and the filtrate was evaporated in vacuo to yield 1 as colorless oil
(2.11 g, 74%). 1H NMR (CDCl3): δ = 0.88 (t, 3H, J = 7.5 Hz),
1.81 (m, 1H), 2.15 (m, 1H), 3.75 (s, 3H, OCH3), 3.82 (s, 3H,
CH3), 4.34 (t, 1H, J = 7.5 Hz), 6.84 (d, 2H, J = 8.7 Hz), 6.88

3280 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Maximov et al.
(d, 2H, J = 9.0 Hz), 7.26 (d, 2H, J = 9.0 Hz), 7.97 (d, 2H, J =
9.0 Hz).
atmosphere. Then, 3,4-dihydro-2H-pyran (6 mL, 65.6 mmol)
was added, followed by p-toluenesulfonic acid monohydrate
(53 mg, 0.28 mmol), and the solution was stirred at 0 °C for 4.5 h.
The solution changed from purple to pink to clear. It was poured
into saturated sodium bicarbonate solution and extracted with
ethyl acetate. The combined ethyl acetate layers were washed
with water, dried over magnesium sulfate, and evaporated in
vacuo to a yellow solid. The residue was triturated with carbon
tetrachloride to remove unreacted pyran. The white solid
(548 mg) was collected by filtration, and the filtrate was purified
by column chromatography over silica (2.7 ꢀ 4 on 2.7 ꢀ 22). The
product was eluted with 100 mL of pet ether, 200 mL of 10%
ether 90% pet ether, 200 mL of 20% ether 80% pet ether,
200 mL of 30% ether 70% pet ether, and 200 mL of 40% ether
60% pet ether. Fractions 33-40 were combined and evaporated
in vacuo to give 243 mg of additional product (791 mg, 65%
E/Z-4,40-(1-Phenylbut-1-ene-1,2-diyl)bis(methoxybenzene) (4)
and (5). Bromobenzene (1.12 mL, 1.664 g, 10.6 mmol) was added
dropwise over 30 min to a stirred solution of magnesium turn-
ings (0.26 g, 10.6 mmol) in dry tetrahydrofuran (THF) (10 mL)
under a nitrogen atmosphere. Once the Grignard reagent
formed and went into solution, 1 (2.03 g, 7.0 mmol) in THF
(10 mL) was added dropwise over 60 min. The reaction was
refluxed for 12 h then quenched with water (10 mL) and the THF
removed under reduced pressure. The aqueous layer was ex-
tracted with ether (3 ꢀ 50 mL). The ether extracts were washed
with saturated sodium bicarbonate and water and dried over
sodium sulfate. The crude carbinol was refluxed with 85%
phosphoric acid (10 mL) in dry THF (20 mL) for 2 h. The
reaction mixture was diluted with water (30 mL) and extracted
with of dichloromethane (3 ꢀ 50 mL). The dichloromethane
layers were washed with sodium bicarbonate and water and
dried over sodium sulfate. It was filtered and the solvent
removed under reduced pressure, yielding a brown oil. Purifica-
tion by flash chromatography over silica (3.0 ꢀ 30 cm) and
elution with 200 mL of petroleum ether, 300 mL of 5% ether
95% pet ether, and 500 mL of 10% ether 90% petroleum ether
yielded two isomers. Isomer E 4 (0.322 g; 15% yield) was
collected in fractions 17 to 19 while Z-isomer 5 was collected
1
yield). H NMR (CDCl3): δ = 1.59-2.00 (m, 12H), 3.60 (m,
2H), 3.86 (m, 2H), 4.16 (s, 2H), 5.38 (t, 1H, J = 3.0 Hz), 5.50
(t, 1H, J = 3.0 Hz), 7.00 (d, 2H, J = 8.7 Hz), 7.07 (d, 2H, J =
8.7 Hz), 7.17 (d, 2H, J = 8.7 Hz), 7.97 (d, 2H, J = 9.0 Hz).
1,2-Bis(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)butan-1-one (10).
Potassium tert-butoxide 9 (229 mg, 2.04 mmol) was added to
1,2-bis(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)ethanone (9)
(672 mg, 1.69 mmol) dissolved in anhydrous THF (25 mL)
under a nitrogen atmosphere with stirring. The mixture was
stirred at room temperature for 1 h. Next, iodoethane (0.136 mL
1.70 mmol) was added dropwise, and the reaction mixture was
refluxed for 6 h. After cooling, the THF was removed under
reduced pressure. Water (30 mL) was added, and the product
was extracted with ether. The ether extracts were combined,
dried over sodium sulfate, and evaporated under reduced pres-
sure to 10 (623 mg, 87% yield). 1H NMR (CDCl3): δ = 0.88 (t,
3H, J = 7.2 Hz), 1.60-1.97 (m, 12H), 2.13 (m, 2H, J = 7.2 Hz),
3.58 (m, 2H), 3.864 (m, 2H), 4.34 (t, 1H, J = 7.2 Hz), 5.34 (t, 1H,
J = 3.0 Hz), 5.46 (t, 1H, J = 3.0 Hz), 6.95 (d, 2H, J = 8.4 Hz),
7.01 (d, 2H, J = 8.4 Hz), 7.20 (d, 2H, J = 8.7 Hz), 7.93 (d, 2H,
J = 8.7 Hz).
1
in fractions 20 to 28 (0.746 g, 31% yield). H NMR E-isomer
(CDCl3): δ = 0.96 (t, 3H, J = 7.5 Hz), 2.49 (q, 2H, J = 7.5 Hz),
3.76 (s, 3H), 3.84 (s, 3H), 6.71 (d, 2H, 8.7 Hz), 6.89 (dd, 4H, J =
1
8.7 and 2.1 Hz), 6.88-7.05 (m, 5H), 7.15 (d, 2H, J = 8.7). H
NMR Z-isomer (CDCl3): δ = 0.93 (t, 3H, J = 7.5 Hz), 2.44 (q,
2H, J = 7.5 Hz), 3.71 (s, 3H), 3.78 (s, 3H), 6.57 (d, 2H, 8.7 Hz),
6.72 (d, 2H, J = 8.7 Hz), 6.89 (d, 2H, J = 8.7 Hz), 7.05 (d, 2H,
J = 8.7), 7.22-7.37 (m, 5H).
E/Z-4-(1-(4-Hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6)
and (7). Boron tribromide (1.23 mL; 3.25 g; 0.0129 mols) in
dichloromethane (5 mL) was added dropwise over 60 min to
4,40-(1-phenylbut-1-ene-1,2-diyl)bis(methoxybenzene) 4 or 5
(0.746, 2.17 mmol) in dry dichloromethane (20 mL) cooled in
a dry ice/ethanol bath while stirring under a nitrogen atmo-
sphere. The solution turned dark immediately and was allowed
to warm to room temperature after the addition was complete.
The reaction mixture was stirred for a total of 4 days at room
temperature. Excess boron tribromide was removed using a
nitrogen stream then anhydrous methanol (3 ꢀ 25 mL) was
added and it was evaporated in vacuo three times. It was
recrystallized from benzene and purified further by preparative
HPLC using 70% methanol 30% water. Fractions were col-
lected as follows: E-isomer 6 (28-39 min, 1.814 abs; 40 mg); Z-
isomer 7 (41-58 min, 2.007 Abs; 78 mg). 1H NMR E-isomer 6
(MeOD): δ = 0.90 (t, 3H, J = 7.5 Hz), 2.47 (q, 2H, J = 7.5 Hz),
6.39 (d, 2H, J = 8.4 Hz), 6.65 (d, 2H, J = 8.7), 6.76 (d, 2H, J =
8.4 Hz), 7.02 (d, 2H, J = 8.7), 7.07-7.12 (m, 5H). 1H NMR Z-
isomer 7 (MeOD): δ = 0.90 (t, 3H, J = 7.5 Hz), 2.38 (q, 2H, J =
7.5 Hz), 6.43 (d, 2H, 8.4 Hz), 6.59 (d, 2H, J = 8.4 Hz), 6.66 (d,
2H, J = 8.4 Hz), 6.93 (d, 2H, J = 8.4 Hz), 7.16-7.32 (m, 5H).
MS m/z calcd for C22H20O2 315.14 (M - H)-; found 315 for
both samples.
1,2-Bis(4-hydroxyphenyl)ethanone (8). Desoxyanisoin (1.0 g;
3.90 mmol) was dissolved in glacial acetic acid (1 mL) with
stirring. Next, hydroiodic acid (5 mL, 36.5 mmol) was added
and the solution was heated to 130-140 °C for 4 h. The reaction
mixture was poured into water (50 mL) and the blue-gray
colored solid was filtered and washed with water. It was dried in
vacuo to yield 9 (0.80 g; 90%). Melting point 205-208 °C. 1H
NMR (MeOD): δ = 4.13 (s, 2H), 6.71 (d, 2H, J = 8.7 Hz), 6.83
(d, 2H, J = 8.7 Hz), 7.06 (d, 2H, J = 8.7 Hz), 7.93 (d, 2H, J =
8.7 Hz).
E/Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)-
phenol (11) and (12). 4-Bromophenetole (0.160 mL, 1.11 mmol)
in dry THF (10 mL) was added dropwise over 30 min to
magnesium turnings (27 mg, 1.11 mmol) with stirring under a
nitrogen atmosphere. An iodine crystal was added to initiate the
reaction, and it was refluxed until the magnesium turnings dis-
solved. Next, 1,2-bis(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)-
butan-1-one (10) (0.311 g; 0.735 mmol) was added and the
reaction was refluxed for 12 h. After cooling, the reaction
mixture was evaporated in vacuo to an orange residue. Water
(20 mL), dichloromethane (20 mL), and acetic acid (1 drop) were
added to the orange residue, and the aqueous layer was ex-
tracted with dichloromethane (3 ꢀ 25 mL). The organic extracts
were washed with saturated sodium bicarbonate and water and
dried over sodium sulfate. Filtration and evaporation provide
the crude carbinol, which was hydrolyzed by refluxing for 2 h
with 85% phosphoric acid (1 mL) in dry THF (10 mL). The
reaction mixture was diluted with water (30 mL) and extracted
with dichloromethane (3 ꢀ 50 mL). The dichloromethane was
washed with sodium bicarbonate and water and dried over
sodium sulfate. It was filtered, and the solvent was removed
under reduced pressure to a yellow oil. It was purified by prep
HPLC over C-18 Delta Pak column eluting with 40% H2O and
60% MeOH. Flow rate was 100 mL/min. The elution was
monitored by UV set to 254. Two fractions were collected: E-
isomer 11 (54-64 m, 14 mg) and Z-isomer 12 (92-100 m, 16
mg). 1H NMR 11 (CDCl3): δ = 0.92 (t, 3H, J = 7.5 Hz), 1.34 (t,
3H, J = 6.9 Hz), 2.44 (q, 2H, J = 7.5 Hz), 3.90 (q, 2H, J = 6.9
Hz), 6.55 (d, 2H, J = 8.7 Hz), 6.63 (d, 2H, J = 8.4 Hz), 6.76 (d,
2H, J = 8.7 Hz), 6.80 (d, 2H, J = 8.7 Hz), 6.97 (d, 2H, J =
8.7 Hz), 7.12 (d, 2H, J = 8.4 Hz). 1H NMR 12 (CDCl3): δ = 0.92
(t, 3H, J = 7.5 Hz), 1.40 (t, 3H, J = 6.9 Hz), 2.45 (q, 2H, J =
7.5 Hz), 4.02 (q, 2H, J = 6.9 Hz), 6.49 (d, 2H, J = 8.7 Hz), 6.63
1,2-Bis(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)ethanone (9).
1,2-Bis(4-hydroxyphenyl)ethanone (8) (700 mg, 3.07 mmol) was
suspended in benzene (35 mL) with stirring under nitrogen

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3281
(d, 2H, J = 8.4 Hz), 6.73 (d, 2H, J = 7.8 Hz), 6.86 (d, 2H, J =
8.4 Hz), 6.97 (d, 2H, J = 8.4 Hz), 7.12 (d, 2H, J = 8.7 Hz). MS
m/z calcd for C24H24O3 360; (M - H)- found 359; (M þ H)þ
found 361.
were maintained in phenol-red RPMI 1640 medium containing
10% fetal bovine serum (FBS), 2 mM glutamine, penicillin at
100 units/mL, streptomycin at 100 μg/mL, 1ꢀ nonessential
amino acids (all from Invitrogen, Carlsbad, CA), and bovine
insulin at 6 ng/mL (Sigma-Aldrich, St. Louis, MO). The hor-
mone-independent T47D:C4:2 cells were subcloned from T47D:
C4 clones of T47D cells that were originally obtained from the
ATCC (Rockville, MD). The T47D:C4:2 cells are ER-negative
hormone-independent cells and they do not re-express ERR
following growth in estrogen-containing media.23 T47D:C4:2
cells were maintained in estrogen-free RPMI 1640 medium,
containing 10% dextran charcoal-stripped fetal bovine serum
(SFS). All cells were cultured in T185 culture flasks (Nalge Nunc
International, Rochester, NY) and passaged twice a week in 1:4
ratio. All cultures were grown in 5%CO2, 37 °C.
Cell Proliferation Assays. MCF-7:WS8 cells were cultured in
estrogen-free medium (phenol red-free RPMI 1640 media sup-
plemented with 10% charcoal-stripped FBS) for 4 days before
beginning the proliferation assay. On day 0 of the experiment,
MCF-7:WS8 cells were seeded in estrogen-free RPMI media
containing 10% SFS at a density of 20000 cells per well
respectively in NunclonΔ Surface 24-well plates (Nalge Nunc
International, Rochester, NY). After 24 h, cells were treated
with various concentrations of the tested compounds, prepared
via serial dilutions. All concentration points were performed in
triplicate. The compound-containing medium was changed on
days 3 and 5, and the experiment was stopped on day 7. Cells
were washed with cold PBS (Invitrogen, Carlsbad, CA) at least
twice and analyzed with Fluorescent DNA quantification kit
(Bio-Rad, Hercules, CA) according to manufacturers instruc-
tions, and samples were read on Mithras LB540 fluorometer/
luminometer (Berthold Technologies, Oak Ridge, TN) in black
wall 96-well plates (Nalge Nunc International, Rochester, NY) .
DNA Plasmids. Estrogen Response Element activity was
determined via Luciferase assays with pERE(5X)TA-ffLuc
and pTA-srLuc reporter plasmids. These plasmids contained
the TATA-box basal promoter firefly and the Renilla luciferase
reporter genes, respectively, and were constructed by insertion
via HindIII linkers of the nucleotides -31 and þ31 region of the
herpes simplex virus thymidine kinase promoter into pGL3-
Basic and phRG-B (Promega, Madison, WI).27 For transient
expression of wild-type ERR and 351 aspartate-to-glycine-
substituted mutant ERR, pSG5HEGO and pSG5D351-
GER plasmids were used, respectively. pSG5HEGO was ori-
ginally provided by Professor Pierre Chambon, University of
Strasbourg, France, and pSG5D351GER was generated using
QuichChange Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA) and pSG5HEGO as a template.17 All plasmids for
this study were purified using HiSpeed Plasmid Maxi Kit
(Qiagen, Valencia, CA) and were grown via OneShot TOP10
Chemically Competent Escherichia coli cells (Invitrogen,
Carlsbad, CA).
1-(4-Methoxyphenyl)-2-phenylbutan-1-one (13). Anisole (5.861 g,
54.2 mmol) and 2-phenylbutyryl chloride (9.90 g, 54.2 mmol) were
dissolved in 20 mL of carbon disulfide with stirring under a nitrogen
atmosphere. The reaction mixture was cooled in an ice bath while
AlCl3 (7.6 g, 57.0 mmol) was added, keeping the temperature
o
between 10 and 20 C. It was stirred at room temperature for
20 h. The dark-red reaction mixture was poured into ice, and the
aqueous layer was extracted with ether (3 ꢀ 75 mL). The combined
ether layers were washed with 10% KOH, 10% HCl, and saturated
sodium bicarbonate solutions. The ether layer was then dried over
MgSO4 and evaporated under reduced pressure to a yellow solid 18
(13.01 g; 94% yield); mp 41-42.5 o C. TLC: (CHCl3) Rf = 0.57.
NMR (CDCl3): δ = 0.90 (t, 3H, J = 7.2 Hz), 1.84 (m, 1H, J = 7.2
Hz), 2.19 (m, 1H, J= 7.2 Hz), 3.82 (s, 3H), 4.40 (t, 1H, J=7.2Hz),
6.86 (d, 2H, J = 9 Hz), 7.16-7.41 (m, 5H), 7.96 (d, 2H, J = 9 Hz).
1,1-Bis(4,40-methoxyphenyl)-2-phenylbut-1-ene (14). Com-
pound 18 (1.0 g, 3.93 mmol) in 10 mL of THF was added
dropwise over 15 min to a 1 M THF solution of 4-methoxyphe-
nyl magnesium bromide (5.8 mL, 5.8 mmol) cooled in an ice
bath under a nitrogen atmosphere with stirring. The solution
was refluxed for 17.5 h. The reaction mixture was poured into
20 mL of water and 8 mL of 6N acetic acid. The aqueous layer
was extracted with ether (3 ꢀ 50 mL). The ether extracts were
washed with saturated sodium bicarbonate and brine, dried over
sodium sulfate, and evaporated under reduced pressure to an oil.
The crude carbinol was refluxed with 85% phosphoric acid
(10 mL) in dry THF (20 mL) for 2 h. The reaction mixture was
diluted with water (60 mL) and extracted with dichloromethane
(3 ꢀ 50 mL). The dichloromethane layers were washed with
sodium bicarbonate and water then dried over sodium sulfate.
The solvent was removed under reduced pressure, yielding a
cream solid. Purification by flash chromatography over silica
(2.7 ꢀ 37 cm) and elution with 500 mL of hexanes gave pure 19 in
fractions 7-18, which were combined and evaporated in vacuo;
yield 1.30 g (96%); mp 116-118 o C. TLC: (CHCl3) Rf = 0.68.
NMR (CDCl3): δ = 0.93 (t, 3H, J = 7.5 Hz), 2.48 (q, 2H, J =
7.5 Hz), 3.68 (s, 3H), 3.83 (s, 3H), 6.54 (d, 2H, J = 9 Hz), 6.77 (d,
2H, J = 9 Hz), 6.88 (d, 2H, J = 9 Hz), 7.07-7.19 (m, 7H).
1,1-Bis(4,40-hydroxyphenyl)-2-phenylbut-1-ene (15). Compound
19 (1.9 g, 3.79 mmol) in 18 mL dry dichloromethane was cooled to
o
-55 C under a nitrogen atmosphere with stirring. Then, BBr3
(2.17 mL, 22.95 mmol) in 10 mL of dichloromethane was added
over 30 min while the temperature was kept at -55 o°C. The
reaction mixture was stirred at room temperature for 90 h. The
reaction was quenched by addition of methanol. The methanol was
evaporated, and this was performed three more times, which
resulted in a green residue. The crude product was purified by
flash chromatography on a silica column (4 cm ꢀ 26 cm) equili-
brated with hexane. It was eluted with chloroform:methanol
(85:15), and 20 mL fractions were collected. Fractions 18-27 were
combined and evaporated under reduced pressure. The resulting
solid was recrystallized from chloroform but contained a small
impurity by HPLC. It was purified by prep HPLC over C-18 Delta
Pak column eluting with 20% H2O and 80% MeOH. Flow rate
was 30 mL/min. The UV detector was set to 254λ. The product was
collect from 20 to 24 min, and the solvent was evaporated in vacuo
to give 20 (75 mg, 5% yield); mp 206-206.5 o C. TLC: (CHCl3 9:
methanol 1) Rf = 0.50. NMR (CDCl3): δ = 0.92 (t, 3H, J = 7.5
Hz), 2.47 (q, 2H, J= 7.5 Hz), 4.48 (s, 1H), 4.71 (s. 1H), 6.46 (d, 2H,
J = 8.4 Hz),; 6.73 (d, 2H, J = 8.4 Hz),; 6.81 (d, 2H, J = 8.4 Hz),
7.10-7.16 (m, 7H).
Transient Transfections and Luciferase Activity Assays. MCF-
7:WS8 cells were cultured in estrogen-free RPMI media for 24 h
prior to transfection. On the day of the experiment, cells were
seeded in estrogen-free media at a density of 100000 cells per well
in 24-well plates. After 24 h, MCF-7:WS8 cells were transfected
with 28.8 μg of pERE(5X)TA-ffLuc and 9.6 μg of pTA-srLuc
reporter plasmids, using 3 μL of TransIT-LT1 transfection
reagent (Mirus Biolabs, Madison, WI) per 1 μg of plasmid
DNA in 52.5 mL of OPTI-MEM serum-free media (Invitro-
gen, Carlsbad, CA). Transfection mix containing transfection
complexes of the transfection reagent and plasmid DNA in
OPTI-MEM media was added to cell in growth media to a final
concentration of 0.3 μg pERE(5X)TA-ffLuc and 0.1 μg of pTA-
srLuc reporter plasmids per well. After 18 h, transfection
reagents were removed and fresh media was added. Cells were
then treated with the various test compounds for 24 h. At the
indicated time point, cells were washed once with cold PBS
(Invitrogen, Carlsbad, CA), lysed, and ERE luciferase activity
Cell Culture. The ER-positive human breast cancer cell line
MCF-7:WS8 and the ER-negative breast cancer cell line T47D:
C4:2 were used in our study. MCF-7:WS8 cells were cloned from
wild type MCF-7 cells that were originally obtained from Dr.
Dean Edwards (University of Texas, San Antonio, TX) and

3282 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Maximov et al.
was determined using the Dual-Luciferase Reporter Assay System
(Promega, Madison, WI) according to manufacturers recommen-
dations. Samples were then read on a Mithras MB540 fluorom-
eter/luminometer (Berthold Technologies, Oak Ridge, TN).
T47D:C4:2 cells were seeded in estrogen-free RPMI 1640
media at a density of 200000 cells per well in 24-well plates.
T47D:C4:2 cells are ER-negative cells, therefore these cells were
transiently transfected with wild-type ERR (pSG5HEGO) or
D351G mutant ERR (pSG5D351GER), along with pERE-
(5X)TA-ffLuc and pTA-srLuc reporter plasmids. Transfection
mix contained 7.2 μg of pSG5HEGO or 7.2 μg of pSG5D351GER,
7.2 μg of pERE(5X)TA-ffLuc, and 2.4 μg of pTA-srLuc reporter
plasmids, and 3 μL of FuGene HD transfection reagent (Roche
Diagnostics, Indianapolis, IN) per 1 μg of plasmid DNA, and
13 mL of OPTI-MEM serum-free media (Invitrogen, Carlsbad,
CA) for 1 ꢀ 24-well plate. Transfection complexes of the reagent
and plasmid DNA were added to cells in growth media to a final
concentration of 0.3 μg of pSG5HEGO or 0.3 μg of pSG5D-
351GER, 0.3 μg of pERE(5X)TA-ffLuc, and 0.1 μg of pTA-
srLuc per well. After 18 h, transfection reagents were removed
and fresh media was added. Cell were then treated with the
various test compounds for 24 h, and ERE luciferase activity
was determined as described above.
Data Bank (PDB),28 entry 3ert, was selected for further model-
ing with ER in antagonist conformation and 1GWR was
selected for modeling of the ER in the agonist conformation.
The protein was prepared for the docking experiments using the
Protein Preparation Workflow (Protein Preparation Wizard,
€
Schrodinger, LLC, Portland, OR) implemented in Schrodinger
suite and accessible from within the Maestro program (Maestro
€
€
8.5, Schrodinger, LLC, Portland, OR). Briefly, the hydrogen
atoms were added, water molecules beyond 5 A from the ligand
˚
were deleted, and the orientation of hydroxyl groups, Asn, Gln,
and the protonation state of His were optimized to maximize
hydrogen bonding. All Asp, Glu, Arg, and Lys residues were left
in their charged state. Finally, the ligand-protein complex was
refined with a restrained minimization performed by Impref
utility, which is based on the Impact molecular mechanics
€
engine (Impact 4.5, Schrodinger, LLC, Portland, OR) and the
OPLS2001 force field, setting a max rmsd of 0.30.
Ligands preparation for docking was performed with LigPrep
€
(LigPrep 2.1, Schrodinger, LLC, Portland, OR) application
which consists of a series of steps that perform conversions,
apply corrections to the structure, generate ionization states and
tautomers, and optimize the geometries.
Molecular docking was performed using Glide 4.5 (Glide 4.5,
€
Schrodinger, LLC, Portland, OR) followed by the Induced Fit
Reagents and Supplies. Estradiol (E2), 4-hydroxy-tamoxifen
(4OHTAM), and bovine insulin, was obtained from Sigma, St.
Louis, MO. Endoxifen (Z-isomer) was a kind gift from Dr.
James Ingle (Mayo Clinic). Fetal bovine serum (FBS), 2 mM
glutamine, penicillin at 100 units/mL, streptomycin at 100 μg/
mL, 1ꢀ nonessential amino acids, RPMI 1640 with phenol-red
media, PBS buffer, RPMI 1640 phenol-red-free media, and
OPTI-MEM serum-free media were all obtained from Invitro-
gen, Carlsbad, CA. Fluorescent DNA quantification kit ob-
tained from Bio-Rad, Hercules, CA. HiSpeed Plasmid Maxi Kit
was obtained from Qiagen, Valencia, CA. TransIT-LT1 trans-
fection reagent was obtained from Mirus Biolabs, Madison, WI.
FuGene HD transfection reagent was obtained from Roche
Diagnostics, Indianapolis, IN. Dual-Luciferase Reporter Assay
System was obtained from Promega, Madison, WI. Anhydrous
ether was purchased from Fisher. Potassium tert-butoxide,
desoxyanisoin, magnesium turnings, bromobenzene, boron tri-
bromide, anhydrous methylene chloride, 3,4-dihydro-2H-pyr-
an, oxalyl chloride, N,O-dimethylhydroxylamine, aluminum
chloride, and cyclohexene were purchased from Acros. Ethyl
iodide, hydroiodic acid (55% ACS unstabilized), p-toluene
sulfonic acid monohydrate, 2-fluoro-3-trifluoromethylbenzoic
acid, 2,4-dimethoxymagnesium bromide (0.5 M solution in
THF), anisole, 2-phenylbutyryl chloride, phenyl magnesium
bromide (1 M solution in THF), sodium hydride (60% in
mineral oil), and allylbromide was purchased from Aldrich.
Carbon disulfide was purchased from Baker. THF was distilled
and stored over calcium hydride. Benzene was distilled from
calcium hydride and stored over molecular sieves. Flash chro-
matography was run using Whatman 230-400 mesh silica gel
60. Preparative chromatography was run on a Waters Delta
Prep 3000 HPLC system using a prep pak C-18 delta-pak
column (47 nn ꢀ 300 mm). UV detection was set at 254λ. Flow
rate was 50 mL per min. 1H NMR was performed on a Bruker
WB Advance 300 MHz instrument. MS analysis performed by
HT Laboratories (San Diego, CA) using electrospray ioniza-
tion. LC-MS was performed using a Waters 2545 binary gra-
dient module and a 2487 dual wavelength detector set to 254 and
365, a 2424 ELS detector, and a 3100 MS detector. The gradient
was linear 5% MeOH 95% H2O to 95% MeOH 5% H2O over
20 min. The column was a Waters Delta Pak C-18 15μ 100A 3.9
mm ꢀ 300 mm (catalogue number 11797) run at a flow rate of
0.8 mL per min. The purity of all compounds was determined by
LC-MS to be 95% or greater.
€
protocol (Induced Fit protocol, Schrodinger, LLC, Portland,
OR), which is intended to circumvent the inflexible binding site
and accounts for the side chain or backbone movements, or both,
upon ligand binding.29 In the first stage of the IFD protocol,
softened-potential docking step, 20 poses per ligand were re-
tained. In the second step, for each docking pose, a full cycle of
protein refinement was performed, with Prime 1.6 (Prime 1.6,
€
Schrodinger, LLC, Portland, OR) on all residues having at least
one atom within 8 A of an atom in any of the 20 ligand poses. The
˚
Prime refinement starts with a conformational search and mini-
mization of the side chains of the selected residues and after
convergence to a low-energy solution, an additional minimiza-
tion of all selected residues (side chain and backbone) is per-
formed with the truncated-Newton algorithm using the OPLS
parameter set and a surface Generalized Born implicit solvent
model. The obtained complexes are ranked according to Prime
calculated energy (molecular mechanics and solvation), and
those within 30 kcal/mol of the minimum energy structure are
used in the last step of the process, redocking with Glide 4.5
(extended precision), and scoring. In the final round, the ligands
used in the first docking step is redocked into each of the receptor
structures retained from the refinement step. The final ranking of
the complexes is done by a composite score which accounts for
the receptor-ligand interaction energy (GlideScore) and recep-
tor strain and solvation energies (Prime energy).29
Acknowledgment. We thank Dr. Richard Schneider from
the Organic Synthesis Core Facility at Fox Chase Cancer
Center for his help in synthesis of the tested compounds. We
particularly thank Helen Kim, Laboratory Manager at the
Lombardi Comprehensive Cancer Center for her dedicated
work assembling this manuscript for publication. This work
was supported by the Department of Defense Breast Program
under award no. BC050277 (V.C.J.), Fox Chase Cancer
Center Core Grant no. NIH P30 CA006927-47, NIH Career
Development Grant K01CA120051-01A2 (J.S.L.W.), American
Cancer Society Grant IRG-92-027-14 (J.S.L.W.), and the
Hollenbach Family Fund (J.S.L.W.).
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