Effects of 7-O substitutions on estrogenic and anti-estrogenic activities of daidzein analogues in MCF-7 breast cancer cells

Article abstract of DOI:10.1021/jm100610w

Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into anti-estrogenic ligands by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of daidzein analogues have been designed and synthesized. When MCF-7 cells were treated with the analogues, those resulting from hydrogen substitution by isopropyl (3d), isobutyl (3f), cyclopentyl (3g), and pyrano- (2) inhibited cell proliferation, estrogen-induced transcriptional activity, and estrogen receptor (ER) regulated progesterone receptor (PgR) gene expression. However, methyl (3a) and ethyl (3b) substitutions of the hydroxyl proton only led to moderate reduction of the estrogenic activities. These results demonstrated the structural requirements for the transformation of daidzein from an ER agonist to an antagonist. The most effective analogue, 2, was found to reduce in vivo estrogen stimulated MCF-7 cell tumorigenesis using a xenograft mouse model.

Full text of DOI:10.1021/jm100610w

J. Med. Chem. 2010, 53, 6153–6163 6153
DOI: 10.1021/jm100610w
Effects of 7-O Substitutions on Estrogenic and Anti-Estrogenic Activities of Daidzein Analogues in
MCF-7 Breast Cancer Cells
Quan Jiang,#,† Florastina Payton-Stewart,#,†,‡,0,§ Steven Elliott,‡,§ Jennifer Driver,‡ Lyndsay V. Rhodes,‡,0,§ Qiang Zhang,†
Shilong Zheng, Deepak Bhatnagar,z Stephen M. Boue,z Bridgette M. Collins-Burow,‡,0,§ Jayalakshmi Sridhar,†
Cheryl Stevens,† John A. McLachlan,‡,^ Thomas E. Wiese,4 Matthew E. Burow,*,‡,0,§,^ and Guangdi Wang*,†
†Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana 70125, ‡Center for Bioenvironmental Research at
Tulane and Xavier Universities, New Orleans, Louisiana 70112, 0Tulane Cancer Center, Tulane University, New Orleans, Louisiana 70112,
§Department of Medicine, Section of Hematology & Medical Oncology, Tulane University School of Medicine, New Orleans, Louisiana 70112,
^Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112, CombiPhos Catalysts, Inc., P.O. Box 220,
Princeton, New Jersey 08542, zUSDA Southern Regional Research Center, New Orleans, Louisiana 70122, and 4College of Pharmacy,
Xavier University of Louisiana, New Orleans, Louisiana 70125. #These authors contributed equally to this work.
Received May 19, 2010
Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into anti-estrogenic
ligands by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural
modifications on the hormonal activities of the resulting compounds, a series of daidzein analogues have
been designed and synthesized. When MCF-7 cells were treated with the analogues, those resulting from
hydrogen substitution by isopropyl (3d), isobutyl (3f), cyclopentyl (3g), and pyrano- (2) inhibited cell
proliferation, estrogen-induced transcriptional activity, and estrogen receptor (ER) regulated progesterone
receptor (PgR) gene expression. However, methyl (3a) and ethyl (3b) substitutions of the hydroxyl proton
only led to moderate reduction of the estrogenic activities. These results demonstrated the structural
requirements for the transformation of daidzein from an ER agonist to an antagonist. The most effective
analogue, 2, was found to reduce in vivo estrogen stimulated MCF-7 cell tumorigenesis using a xenograft
mouse model.
1. Introduction
for estrogen receptor (ER)a binding.5 Additional studies have
proposed several possible signaling pathways that may be
responsible for these effects, such as binding to the epidermal
growth factor receptor kinase11 and interfering with trans-
forming growth factor signaling.12
Daidzein (1) is one of many known isoflavones that natu-
rally occur in plants such as soybeans and kudzu vines
(Peruraria lobata).1 Like other constitutive isoflavones found
in soy tissues (e.g., genistein),2 daidzein has been shown to act
as a variety of biologically active agents ranging from anti-
oxidant, estrogen at doses lower than 20 μM, and breast
cancer cell growth and proliferation promoter.3-5 The estro-
genic effect, however, can be reversed when daidzein concen-
tration exceeds 20 μM where it has been found to inhibit the
growth of differentcancercells.4,6,7 For example, daidzein was
found to antagonize the inhibitory effects of tamoxifen on
pS2 expression8 and proliferation9 of breast tumor cells. The
diverse functionality of daidzein is also reflected in its glucosyl
derivative, 7-O-glucosyl-40-hydroxyisoflavone (daidzin),
which acts as an antialcohol-addiction agent,10 presumably
through binding to the enzyme aldehyde dehydrogenase.
The exact mechanism underlying the cellular inhibitory
effects of high dose daidzein and its closely related analogue
genistein is not completely understood. Previous studies
suggest their role as weak agonists competing with estradiol
Interestingly, in soy, daidzein is also a key intermediate in the
biosynthesis of the anti-estrogenic glyceollins in response to
bacterial infection.13,14 This naturally occurring transforma-
tion of estrogenic to anti-estrogenic function prompted us to
examine the structural features that distinguish the estrogenic
activity of daidzein from the anti-estrogenic activity of the
glyceollins. As previously published, the biosynthesis of gly-
ceollin I from the daidzein precursor leads to the incorporation
of (1) a 7,8-phenol ring of the chromanone structure where a
six-membered ring is formed via an ether linkage13,14 and (2) a
central moiety where a freely rotating biphenyl bond in daid-
zein becomes a rigid pterocarpan structure in glyceollin I. Both
or one of the structural modifications could be responsible for
the functional reversal of ER activity. Molecular modeling
using the crystal structure of ERR indicates that daidzein can fit
in the binding pocket of ERR when the receptor is optimized
with both 17β-estradiol (E2) and 4-hydroxytamoxifen (4-OH-
TAM), whereas glyceollins can only fit the pocket when
the crystal structure of ERRis optimized with 4-OH-TAM. The
modeling results15 suggest that the structural features in the
7-phenolic position may be the critical one responsible for the
anti-estrogenic effects of glyceollin I.
*Corresponding authors. (G.W.) E-mail: gwang@xula.edu; (M.B.)
E-mail: mburow@tulane.edu
a Abbreviations: E2, 17β-estradiol; ER, estrogen receptor; ERE, estro-
gen responsive element; ICI (ICI182,780), fulvestrant; 4-OH-TAM,
4-hydroxytamoxifen; PgR, progesterone receptor; 3ERT, human estro-
gen receptor alpha ligand-binding domain in complex with 4-hydroxy-
tamoxifen; 3ERD, ERR bound to the agonist diethylstilbestrol; MOPAC,
molecular orbital package.
In this study, we explored the potential anti-estrogenic
properties of daidzein analogues by modifying the 7-hydroxyl
r
2010 American Chemical Society
Published on Web 07/29/2010
pubs.acs.org/jmc

6154 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Jiang et al.
functional group while keeping the daidzein skeleton intact.
We started with an analogue that is structurally identical with
glyceollin I on the 7 and 8 ring substitutions but without the
pterocarpan moiety. Using molecular modeling as a guiding
tool, we designed and synthesized an additional eight daidzein
analogues that vary in the 7-O substitution. We also evaluated
the effect of daidzein and daidzein analogues on estrogen
responsive element (ERE) transcriptional activity, ERR bind-
ing affinity, MCF-7 breast cancer cells colonial survival, PgR
gene expression, and ERR docking. Finally, we conducted in
vivo studies of the anti-estrogenic effects of the most potent
analogue on MCF-7 cell tumorigenesis using a xenograft
mouse model. The structure activity relationship studies on
these daidzein analogues reveal the progressive loss of an ER
agonist activity concurrent to the acquisition of ER anta-
gonistic activity with the substitution of the 7-OH hydrogen
with varying hydrophobic groups.
2. Results and Discussion
2.1. Chemistry. The synthetic route of 3-(4-hydroxy-
phenyl)-8,8-dimethyl-8H-pyrano[2,3-f]chromen-4-one (2)
is diagrammed in Scheme 1. Briefly, following a literature
method,16 a condensation reaction between 1 and an un-
saturated aldehyde masked as its acetal in refluxing p-xylene
in the presence of picoline as base brought about the desired
ring closure in position 8 of 1 to give the target compound 2
as the major product. The minor byproduct formed by
substitution and ring closure on daidzein’s 40 and 50 positions
was negligible and did not present a problem in the isolation
and purification of 2.
Scheme 1. Synthesis of 2 from Daidzein
This strategy, however, did not work as hoped for all other
daidzein analogues from 3a to 3h (Scheme 2). The main
problems appeared to be associated with the much higher
proportion of disubstitution on both hydroxyl groups. The
challenge was overcome by adopting a synthetic strategy17
that used the appropriate alkyl halide in a DMSO suspension
of potassium carbonate. The facile reactions proceeded at
room temperature giving the desired compounds as the main
products with varying amounts of byproducts from substitu-
tion on the other hydroxyl group (40-OH). Notably, in some
cases, it was necessary to carefully control reaction time and
substrate ratio in order to achieve the desired outcome of
majority 7-O substitution. In fact, for several compounds
Scheme 2. Synthesis of Daidzein Analogues 3a-3h
Figure 1. Effects of daidzein and daidzein analogues on ERE transcriptional activity in MCF-7 cells. MCF-7 cells were transiently transfected
with pGL2-ERE2x-TK-luciferase plasmid. After a 6 h transfection, cells were treated with compounds (DMSO, daidzein, and daidzein
analogues) and incubated overnight. Data are represented as relative light units (RLUs) normalized to E2 (100 ( SEM). The values are the
means and the SEM of triplicates from a single experiment and representative for at least three independent experiments. a, significant
difference from daidzein, p<0.05, b, significant difference from daidzein, p<0.01, c, significant difference from daidzein, p<0.001.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6155
Table 1. Summary of Biological Activities of 7-O-Substituted Daidzein Derivativesa
a Data are represented as relative agonistic and antagonists activities for individual compounds with double normalization in which E2 (0.1 nM) was
set at 100% with vehicle (DMSO) set at 0%.
(e.g., 3f and 3g), isolation of the target 7-O-substituted
product from their 40-O-substituted byproducts was rather
difficult, and separation by semipreparative HPLC was
necessary to afford greater than 98% purity of these analogues.
2.2. Biological Activities of Daidzein Analogues. Effects of
Daidzein Analogues on ER Transcriptional Activation in
MCF-7 Cells. We have previously described the estrogenic
and anti-estrogenic activities of individual flavonoids and
phytochemicals using a luciferase-based ERE reporter
assay.2,5,14 These studies have demonstrated the potent
estrogenic activities of certain isoflavones such as genistein
or daidzein, as well as the ability of glyceollins to antagonize
estrogen-stimulated ERE activity.15 To test how structural
modifications on the 7-hydroxyl group of daidzein affect
estrogenic properties, the ER transcriptional activity in-
duced by the daidzein analogues was measured and com-
pared to daidzein using the ERE-based luciferase reporter
gene assay. As shown in Figure 1, the estrogenic activities of
synthetic daidzein analogues were plotted as dose-response
curves when MCF-7 cells were treated with increasing con-
centrations of the test compounds. At 1 μM concentration
where daidzein induced maximal estrogenic effect in MCF-7
cells, the relative estrogenic potencies of the analogues are
seen to decrease in roughly the following order: daidzein >
3a > 3f > 3e > 3b = 3c > 3d > 2 > 3g > 3h. While this
order varied slightly as analogue concentrations were in-
creased further to 25 μM (Table 1), several trends have
emerged in the dose-dependent data. First, the parent com-
pound daidzein showed more potent estrogenic activity than
any of its derivatives when hydrogen was replaced with alkyl
groups with varying chain lengths and configurations. Sec-
ond, shorter alkyl groups appeared to have less effect on the
reduction of ER agonist activity than their longer, bulkier
counterparts. For example, at 25 μM concentration, 3b
(ethyl-), 3a (methyl-), and 3c (n-propyl-) daidzein analogues
induced ERE transcriptional activity at 80.4, 70.9, and
38.4%, respectively, retaining greater estrogenic activities
than other analogues with longer chain or bulkier substi-
tuting groups. Finally, the analogue showing little or no
estrogen activity, 2, resembled the naturally occurring gly-
ceollin I15 in the chromanone moiety, suggesting that the
unique ring substitution on the 7O- and 8-chromanone ring
was responsible for the diminished estrogenic activity of the
modified daidzein.
Antagonist Activities of Daidzein Analogues on ER Trans-
criptional Activity. To evaluate the anti-estrogenic activities
of daidzein analogues, MCF-7 cells were treated with the
synthesized compounds at increasing concentrations in the
presence of 0.1 nM E2. The luciferase activities of the treated
cells were then measured by the ERE reporter gene assay.

6156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Jiang et al.
Figure 2. Effects of daidzein and daidzein analogues on E2 stimulation on ERE transcriptional activity in MCF-7 cells. MCF-7 cells were
transiently transfected with pGL2-ERE2x-TK-luciferase plasmid. After a 6 h transfection, cells were treated with (A) compounds (DMSO, E2,
daidzein and daidzein analogues plus E2) or (B) with compounds (DMSO, E2, ICI, ICI þ E2, 4-OH-TAM, 4-OH-TAM þ E2) and incubated
overnight. Data are represented as relative light units (RLUs) normalized to E2 (100 ( SEM). The values are the means and the SEM of
triplicates from a single experiment and representative for at least three independent experiments. a, significant difference from E2, p<0.05, b,
significant difference from E2, p<0.01, c, significant difference from E2 p<0.001, and d, significant difference from DMSO p<0.001.
Figure 2 shows the dose-response curves of MCF-7 cells
treated with E2 plus daidzein and its synthetic analogues
where the luciferase activity is normalized to 100 for cells
treated with 0.1 nM E2 alone. The inhibitory effect of some
analogues on E2 became more pronounced as their concen-
tration was increased to 10 μM. At 25 μM (Table 1), the
relative anti-estrogenic potencies of the analogues were
observed to increase in the following order: daidzein <
3b<3a<3c<3h<3e<3d<3g<3f<2. The analogues
that reduced estradiol-dependent luciferase activity by great-
er than 60% at 25 μM included 2, 3f, 3g, and 3d, of which 2
was the most effective anti-estrogen, completely blocking
ERE activity at 25 μM. In contrast, analogues 3a, 3b, 3c, 3h,
and 3e decreased E2 induced transcriptional activity by less
than 47% at the highest ligand concentration. Compared
with estrogenic activity assay results, the analogues showing
strong anti-estrogenic activity exhibited little or no estrogen
activity. For example, 2, 3f, and 3g all induced negligible
ERE activity, but they behaved as potent estrogen inhibitors
(Figures 1 and 2A). Treatment with fulvestrant (ICI) or
4-OH-TAM alone and in combination with estrogen caused
a decrease in ERE transcriptional activity over 90% and
80%, respectively (Figure 2B). On the other hand, the
strongest estrogenic analogues, 3a, 3b, and 3c, failed to
inhibit 50% ERE activity in the presence of E2 at all
concentrations tested (Figure 2A). Taken together, the alkyl
substituted daidzein analogues can be viewed in two groups
in terms of their ER-mediated transcriptional activities. The
first group (3a, 3b, and 3c) where the 7-OH of daidzein is
alkoxylated by the methyl, ethyl, and n-propyl group, res-
pectively, has retained much of the estrogenic function of
daidzein. The second group, consisting of 2, 3g, 3f, and to a

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6157
modifications on the 7-O position of daidzein reduced more
estrogenic potency of the analogues to various degrees at
lower concentrations (10 μM) than at higher doses (25 μM).
However, the reason for higher binding affinities exhibited
by 3h and 3e which are neither estrogenic nor anti-estrogenic
is unclear. The binding curves suggest that at higher con-
centrations (e.g., 25 μM as used in the luciferase reporter
assay), the degree of binding to ER by several analogues may
rapidly approach that of daidzein. This is also consistent
with the fact that both estrogenic (3a, 3b, and 3c) and anti-
estrogenic (2, 3f, 3g) activities of the analogues reached
maximal levels at 25 μM (Figures 1 and 2). For example,
3a reached 38% ERE activity at 10 μM; at 25 μM, its ERE
activity increased dramatically to 70.9% (Figure 1). The
binding curve for 3a confirmed this rapid increase in estro-
genic activity as its %bound value appears to approach that
of daidzein. Notably, for anti-estrogenic compounds, bind-
ing affinities to ERR did not appear to correlate well with
their ability to inhibit E2-induced ERE activity.
Effect of Daidzein Analogues on Estrogen-Stimulated Clono-
genic Proliferation. To further evaluate the estrogenic or
anti-estrogenic activities of the daidzein analogues deter-
mined from the ERE transcriptional assay, we selected two
analogues, 3a and 2, for further test by a clonogenic assay for
their ability to inhibit the estradiol-induced proliferation of
MCF-7 cells. As discussed earlier, 3a behaved as an estro-
gen with a slightly lower potency than daidzein, whereas 2
acted as an anti-estrogen that was able to inhibit 100%
ER-mediated transcriptional activity. In the colony assay,
MCF-7 cells were treated with increasing concentrations of
daidzein, 2, 3a, and 4-OH-TAM in the presence or absence of
0.1 nM E2. Results are illustrated in Figure 4 where estrogen-
dependent cell growth was normalized to 100 for the control
(DMSO) (Figure 4A) or for E2 alone (Figure 4B). The
addition of 0.1 nM E2 to the culture media resulted in an
approximately 3-fold increase in the clonogenicity of the
MCF-7 cells compared to DMSO-treated controls. At 1 μM,
daidzein also promoted the clonogenicity of MCF-7 cells by
over 3-fold, while 3a increased cell proliferation by 43%,
confirming that 3a is a weaker estrogen than daidzein, a
direct consequence of the replacement of 7-hydroxy group by
7-methoxy group. In contrast, 2 inhibited the clonogenicity
by 15% at the same concentration, demonstrating its anti-
estrogenic property at 1 μM concentration. When the con-
centration was raised to 10 μM, daidzein and 3a continued to
increase the clonogenicity of MCF-7 cells by 2.5 times and
24%, respectively. Analogue 2, however, completely blocked
cell clonogenicity at 10 μM, similar to treatment with 4-OH-
TAM at 100 nM (Figure 4B). Thus, the colony assay results
clearly demonstrated the divergence of hormonal activities
in daidzein analogues by simple modifications of the 7-OH
group of daidzein.
Figure 3. Relative binding affinity of daidzein and daidzein ana-
logues for ERR. Increasing concentrations of daidzein analogues
were added to the ER complex and compared to E2. Data points and
error bars represent the mean ( SEM of at least three experiments
(n = 3) for each concentration tested (p<0.05).
lesser extent 3d, has been transformed into potent anti-
estrogens. This complete reversal from estrogenic to anti-
estrogenic activity in certain daidzein analogues highlights
the importance of the structure of 7-O substitution in deter-
mining the ligand’s interaction with the estrogen receptor. It
is also interesting to note the significant difference in ERE
activity between the i-propyl (3d) and n-propyl (3c) substi-
tuted analogue, suggesting that a branched alkyl group may
provide a better binding mechanism toward the ER.
Relative Affinity of Daidzein Analogues for ERr. In an
attempt to understand the biological activity of the synthe-
sized daidzein analogues, we examined the ability of each
daidzein analogues to bind to ERR using a competitive
binding assay with fluorescent detection (Figure 3). Analysis
of the competitive binding curve yielded IC50 values, repre-
senting the concentration of unlabeled ligand required to
displace 50% of the tracer from the ERR. Unlabeled E2 was
used as a reference and its displacement of 50% tracer E2
bound to ERR occurred at 1.5 nM. Compared to daidzein,
which has an IC50 value of 0.45 μM, none of the analogues
displayed higher binding affinities toward the estrogen recep-
tor. The maximal %bound to ERR for the analogues is
summarized in Table 1. At 10 μM ligand concentration, four
analogues showed over 50% bound ERR, that is, 3a, 3b, 3e,
and 3h, while the rest of analogues appeared to have lower
binding affinities toward the ER. The binding data are
partially consistent with the observation that structural
Regulation of ER-Mediated PgR Gene Expression by
Daidzein Analogues. To further examine the differing hormo-
nal activities of daidzein analogues, we monitored ER-
mediated gene expression of progesterone receptor (PgR)
by quantitative real time RT-PCR in MCF-7 cells treated
with daidzein, 2 (an anti-estrogenic analogue), and 3a (an
estrogenic analogue) (Figure 5). Progesterone receptor (PgR)
is an estrogen-responsive gene, whose expression has been
shown to indicate a responsive estrogen receptor pathway. The
expression of PgR mRNA in MCF-7 cells that were treated with
DMSO (control), daidzein, 2 and 3a either in the presence or
absence of E2 were determined. When treated with ligands only,

6158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Jiang et al.
Figure 4. Effects of daidzein, 2 and 3a on E2 stimulation on colony formation on MCF-7 cells. MCF-7 cells were placed in phenol red-
free DMEM supplemented with 5% dextran-coated charcoal-treated FBS for 48 h before plating. MCF-7 cells (1000) were plated in six well
plates. Forty-eight hours later, cells were treated with DMSO (vehicle), E2, daidzein, daidzein analogues (at 1 μM and 10 μM) and 4-OH-TAM
(at 10 nM and 100 nM) (A) and E2, E2 þ daidzein, E2 þ daidzein analogues (at 1 μM and 10 μM) and E2 þ 4-OH-TAM (at 10 nM and 100 nM)
(B). Colonies of >50 cells were counted as positive. Results are normalized to percentage of clonogenic survival from DMSO control cells. The
values are the means and the SEM of triplicates from a single experiment and representative for at least three independent experiments.
Significant difference from vehicle control (A) and E2 (B), *p<0.05, **p<0.01 and ***p<0.001; Tukey test.
Figure 5. Effects of daidzein, 2 and 3a on PgR expression. Total RNA was isolated from MCF-7 cells, reverse-transcribed into cDNA and
subjected to real-time RT-PCR analysis for quantitation. Treatment on MCF-7 cells was as follows: DMSO (vehicle), daidzein, 2, 3a, E2,
daidzein þ E2, 2 þ E2, 3a þ E2 (10 μM and 10 μM þ E2). Results are expressed as the mean unit ( SEM (***p < 0.05; **p < 0.01; *p < 0.001)
with significant differences from vehicle control.
daidzein and 3a induced a 3-fold increase in PgR expression
compared with control, while a 5-fold increase was induced by
E2. However, 2 caused a decrease in PgR expression compared
with DMSO, demonstrating its non-estrogenic nature. When
MCF-7 cells were treated with the ligands in the presence of
0.1 nM E2, both daidzein and 3a were able to promote the level
of PgR expression by 5- and 4-fold, respectively. In contrast, 2
inhibited the E2 induced PgR expression by reducing its upre-
gulation from 5- to 3-fold. Recently, we have demonstrated the
ability of the known anti-estrogens fulvestrant (100 nM) and
4-OH-TAM (100 nM) to suppress E2-stimulated PgR expres-
sion by ∼75% and ∼64% respectively in our MCF-7 cell sys-
tem.15 In comparison, analogue 2 resulted in a ∼40% suppres-
sion of E2-stimulated PgR expression (Figure 5).
In Vivo Anti-Estrogenic Effects of Analogue 2 on MCF-7
Cell Tumorigenesis. Because the anti-estrogenic effects of
analogue 2 on MCF-7 cells observed in our in vitro biological
assays, this compound was selected for further testing of

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6159
Figure 6. Anti-estrogenic effects of analogue 2 on MCF-7 cell tumorigenesis in vivo. 4-6 week old ovariectomized female Nu/Nu mice were
injected bilaterally in the MFP with 5 ꢀ 106 MCF-7 cells in matrigel (reduced factor). All animals were implanted with a 17β-estradiol pellet
(0.72 mg, 60-day release) subcutaneously in the lateral area of the neck at the time of cell injection. Tumors were allowed to form and at day 15
post cell injection mice were randomized into treatment groups (n = 5). Animals were treated daily with i.p. injections of either vehicle (1:5
DMSO/PBS) or analogue 2 (50 mg/kg/animal). Tamoxifen animals were implanted with a slow release tamoxifen pellet (5mg, 60-day release) in
the lateral area of the neck (opposite side from E2 pellet). ICI animals were given a one-time i.p. injection of ICI 182,780 (5 mg in castor oil).
Tumor size was measured 3 times weekly using digital calipers. Data are represented as mean tumor volume þ SEM.
Figure 7. Molecular modeling of daidzein and its analogues 2 and 3a. Binding modes of daidzein (A), 3a (B) to estrogenic conformer of ERR
and 2 (C) to anti-estrogenic conformer of ERR, wherein, the protein is depicted as ribbon/tube model colored by secondary structure and the
compounds are shown as spacefill models. D, E, and F depict the hydrogen bond interactions made by the compounds daidzein, 3a and 2 with
the protein as a ribbon/tube model colored by secondary structure and the compounds as ball and stick models.
estrogen-stimulated MCF-7 cell tumorigenesis using a xeno-
graft model. Ovariectomized female immunocompromised
mice were injected with MCF-7 cells and supplemented with
exogenous estrogen pellets. Once tumors were palpable animals
were treated with analogue 2, tamoxifen, or ICI and tumor
volume compared to vehicle control animals. As shown in
Figure 6, at day 37 post cell injection, a decrease in tumor volume
was observed in all treatment groups compared to control
(351.17 ( 43.88). Compound 2 treatment resulted in reduced
tumor volumes similar to tamoxifen treated animals (241.53 (
68.29 and 178.12 ( 41.18 mm3, respectively), while treatment
with the pure anti-estrogen ICI 182,780 was most effective in

6160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Jiang et al.
attenuating estrogen-induced tumorigenesis (130.95 ( 23.49).
These results confirm that the anti-estrogenic properties of
compound 2 observed in vitro extend to in vivo conditions
as well.
efficacy of 2 as an ER antagonist, nude mice xenografted with
E2-dependent MCF-7 cells was treated with the analogue 2,
resulting in significant tumor regression.
By synthesizing and testing additional daidzein analogues
we have further demonstrated the effect of varying 7-O-sub-
stitution groups on estrogenic or anti-estrogenic activities of
the compounds. Short chain alkyl substitutions (3a, 3b, 3c)
weakened the estrogenic potency of daidzein but did not
render the compounds anti-estrogenic; longer chain alkyl
substitutions (3e, 3h) appeared to further diminish the estro-
genic activity. On the other hand, 3d, 3f, and 3g where
branched alkyl or cycloalkyl substitutions were introduced,
exhibited significant anti-estrogenic property. Molecular
modeling study of representative daidzein analogues using
the crystal structure of ERR revealed that daidzein and 3a
fitted well in the ligand binding pocket of ER in its estrogen
bound configuration. The anti-estrogenic2 was found to dock
better in ER in its anti-estrogen-bound configuration. These
results demonstrated the structural requirements for the trans-
formation of daidzein from an estrogen to an anti-estrogen.
The most effective compound, 2 may serve as a useful novel
anti-estrogen for breast cancer treatment and prevention.
Molecular Modeling. The estrogenic compound 3a and
anti-estrogenic compound 2, along with the parent com-
pound daidzein were subjected to docking studies. To under-
stand the nature of their interactions with ERR, two protein
conformations were selected as template coordinates for the
docking studies. It has been well established that the binding
mode of ligand to the estrogen receptor causes structural
perturbations in and around the ligand binding pocket
resulting in two protein conformations that contribute to
the estrogenic or anti-estrogenic effects.18-20 Two such
representative structures, 3ERD (ERR bound to the agonist
diethylstilbestrol) and 3ERT (ERR bound to the antagonist
4-OH-TAM) were taken as protein templates. The com-
pounds were docked to the template protein structures using
the Surflex docking program. The Surflex scoring21 and the
binding postures for the compounds daidzein and 3a were
comparable for the estrogenic and anti-estrogenic protein
conformations with a slight preference to the estrogenic
conformation. The binding modes of daidzein and 3a for the
estrogenic protein conformation are represented in Figure 7A,B.
Daidzein forms hydrogen bonds with GLU353, ARG394,
GLY521, and HIS524 (Figure 7D). 3a forms hydrogen
bonds with LEU387, ARG394, and HIS524 (Figure 7E),
resulting in fewer hydrogen bonding interactions than
daidzein which could account for its weaker estrogenic
activity. Compound 2 docked to the anti-estrogenic confor-
mation of the protein with a significantly better Surflex score
than when docked to the estrogenic conformation of the
protein which implies that it can have anti-estrogenic pro-
perty. The binding posture of 2 (Figure 7C,F) for the anti-
estrogenic protein conformation was similar to that of other
ERR antagonists tamoxifen and raloxifene. These docking
studies further confirm the nature of estrogenic and anti-
estrogenic activity exhibited by the compounds 3a and 2.
4. Experimental Section
4.1. Chemistry. All reagents and solvents were purchased
from either Aldrich Chemical Co. (WI, USA) or Acros organics
(NY, USA) and were used as received. All organic solvents used
were of reagent grade quality and were used without further
purification. Daidzein (I) was synthesized by Tyger Scientific
1
Inc. (NJ, USA). H spectra were recorded at 400 MHz on a
Varian Unity-400 spectrometer (Varian Inc., Palo Alto, CA),
using DMSO-d6 as solvent and as internal standard (δ 2.49).
GC-MS analyses were performed on an Agilent Technologies
5975C inert MSD mass spectrometer. Melting points were
determined with a Mel-temp II point apparatus and are un-
corrected. Crude synthetic products were purified by the follow-
ing methods: chromatography on Silica Gel (60-100 mesh,
Fisher Scientific) column and recrystallization from acetone.
Analytical thin layer chromatography (TLC) was performed on
250 μ fluorescent plates Whatman, Germany) and visualized by
using UV light. For all products, the purity was ascertained to be
greater than 95% by the HPLC method using a Shimadzu
(Columbia, MD) 2010 HPLC-UV/MS system with a C-18
reverse phase column.
4.1.1. 3-(4-Hydroxyphenyl)-8,8-dimethyl-8H-pyrano[2,3-f]-
chromen-4-one (2). According to the literature method,16 to a
solution of 1 (1.9 g, 7.2 mmol) in 360 mL of xylene was added
1,1-diethoxy-3-methyl-2-butene (2.4 g, 14.7 mmol) in 15 mL of
xylene and 3-picoline (3.7 mmol in 43 mL xylene). The reaction
mixture was refluxed at 130 ꢀC for 18 h. The solvent was
evaporated under a vacuum and the residue was chromato-
graphed over silica gel using hexanes as the eluant. Final
product was recrystallized from acetone to give 0.34 g of 2 as
colorless needle crystalline, with an overall yield of 15%. mp:
262-264 ꢀC (Dec.). GC-MS (m/z 320 Mþ, m/z 305, m/z 254, m/z
187, m/z 152, m/z 118). 1H NMR (DMSO-d6, 400 MHz, δ): 1.45
(6H, s), 5.94 (1H, d, J = 10.0 Hz), 6.79 (1H, d, J = 10.0 Hz), 6.80
(2H, d, J = 8.8 Hz), 6.92 (1H, d, J = 8.4 Hz), 7.38 (2H, d, J =
8.8 Hz), 7.88 (1H, d, J = 8.4 Hz), 8.36 (1H, s), 9.57 (1H, s, D2O
exchangeable).
3. Conclusion
Previous studies in our laboratory demonstrated the diverse
estrogenic and anti-estrogenic activity of naturally occurring
flavonoid phytochemicals. Through these studies, we identi-
fied glyceollin I, a naturally occurring phytoalexin in activated
soy, behaved as a potent anti-estrogen.15 However, daidzein,
the precursor in the biological synthesis of glyceollin I was
strongly estrogenic. The contrasting hormonal activity of
daidzein and glyceollin I provided the rationale to design and
synthesize a series of daidzein analogues that have modified
structures on the 7-hydroxyl position of daidzein. In parti-
cular, one such analogue, 2 was designed to answer the central
question: what is the structural requirement that is responsible
for the regulation of the agonistic and antagonistic activities
of isoflavone phytochemcials. 2 has retained the daidzein
skeleton with the addition of a chromanone moiety. Results
from ERE luciferase assay, colony assay, and PgR expression
revealed that 2, in contrast to daidzein, acted predominantly as
an anti-estrogen with limited agonist activity. The in vitro anti-
estrogenic potency of 2 was found comparable to glyceollin I,15
strongly suggesting that substitution on the chromanone struc-
ture, not the presence of pterocarpan skeleton, is responsible for
the loss of estrogenic and acquisition of anti-estrogenic acti-
vity from daidzein to glyceollin I. To further test the in vivo
4.1.2. 3-(4-Hydroxyphenyl)-7-methoxychromen-4-one (3a).
Following a literature method,17 to a solution of 1 (1.27 g, 5 mmol)
in 10 mL of DMSO was added anhydrous K2CO3 (0.9 g, 6.5 mmol)
and iodomethane (0.71 g, 5.0 mmol). The reaction mixture was
stirred at room temperature for 2 h and then poured into ice
water. The resulting mixture was extracted with ethyl acetate.
The organic phases were combined, washed with brine, dried

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6161
over sodium sulfate, filtered, and concentrated under a vacuum.
The residue was chromatographed over silica gel using hexanes/
ethyl acetate (4:1) as eluant. The product was recrystallized from
acetone to provide 0.36 g of 3a as a white powder in 27% isolated
yield. mp: 210-211. (lit:17 210-211). GC-MS (m/z 268 Mþ, m/z
225, m/z 151, m/z 118). 1HNMR (DMSO-d6, 400 MHz, δ):
3.91 (3H, s), 6.81 (2H, d, J = 8.8 Hz), 7.08 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 7.16 (1H, d, J = 2.4 Hz), 7.40 (2H, d, J = 8.8 Hz),
8.03 (1H, d, J = 8.8 Hz), 8.38 (1H, s), 9.58 (1H, s, D2O
exchangeable).
with 1-bromohexane provided 0.68 g of 3h as a colorless crystal-
line in 40% yield. mp: 147-149 ꢀC (lit:23 145-148 ꢀC). GC-MS
1
(m/z 338 Mþ, m/z 254, m/z 226, m/z 137, m/z 118). HNMR
(DMSO-d6, 400 MHz, δ): 0.87 (3H, t, J = 7.2 Hz), 1.31 (4H, m),
1.42 (2H, quintet, J = 7.2 Hz), 1.75 (2H, quintet, J = 7.2 Hz),
4.11 (2H, t, J = 7.0 Hz), 6.80 (2H, d, J = 8.4 Hz), 7.05 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz), 7.13 (1H, d, J = 2.4 Hz), 7.39 (2H, d, J =
8.4 Hz), 8.00 (1H, d, J = 8.8 Hz), 8.352 (1H, s), 9.57 (1H, s, D2O
exchangeable).
4.2. Biological Assays. Cell Culture. Human cancer cell lines
derived from breast, MCF-7 (ER-positive cells) were cultured
in 75 cm2 culture flasks in Dulbecco’s Modified Eagle’s Medium
(DMEM) (Invitrogen, Co.) supplemented with 10% fetal
bovine serum (FBS) (Life Technologies, Inc., Gaithersburg,
MD), basic minimum MEM essential (50ꢀ, Invitrogen Co.)
and MEM nonessential (100ꢀ, Invitrogen, Co.) amino acids,
sodium pyruvate (100ꢀ, Invitrogen Co.), antimycotic-antibiotic
(10 000 U/mL penicillin G sodium; 10 000 μg/mL streptomycin
sulfate; 25 μg/mL amphotericin B as Fungizone), and human
recombinant insulin (4 mg/mL. Invitrogen Co). The culture
flasks were maintained in a tissue culture incubator in a humi-
dified atmosphere of 5% CO2 and 95% air at 37 ꢀC. For
estrogen studies, cells were washed with PBS 3 times and grown
in phenol red-free DMEM supplemented with 5% dextran-
coated charcoal-treated FBS (5% CS-FBS) for 72 h before
plating for each particular experiment.
4.1.3. 3-(4-Hydroxyphenyl)-7-ethoxychromen-4-one (3b). Fol-
lowing the procedure used to prepare 3a, reaction of 1 with
bromoethane provided 0.44 g of 3b as a pale yellow solid in 31%
yield. mp: 131-132 ꢀC. (lit:22 131-133 ꢀC). GC-MS (m/z 282,
1
Mþ, m/z 253, m/z 225, m/z 165, m/z 137, m/z 118). HNMR
(DMSO-d6, 400 MHz, δ): 1.38 (3H, t, J = 6.8 Hz), 4.19 (2H, q,
J = 6.8 Hz), 6.82 (2H, d, J = 8.8 Hz), 7.06 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 7.14 (1H, d, J = 2.4 Hz), 7.40 (2H, d, J = 8.8 Hz),
8.02 (1H, d, J = 8.8 Hz), 8.37 (1H, s), 9.57 (1H, s, D2O
exchangeable).
4.1.4. 3-(4-Hydroxyphenyl)-7-propoxychromen-4-one (3c).
According to the procedure used to prepare 3a, reaction of 1
with 1-bromopropane provided 0.57 g of 3c in 38% yield as
a white powder. mp: 173-175 ꢀC (Dec.). GC-MS (m/z 296 Mþ,
m/z 253, m/z225, m/z137, m/z118) 1HNMR (DMSO-d6, 400
MHz, δ): 0.99 (3H, t, J = 7.0 Hz), 1.77 (2H, sextet, J = 7.2 Hz),
4.08 (2H, t, J = 7.0 Hz), 6.81 (2H, d, J = 8.4 Hz), 7.06 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz), 7.13 (1H, d, J = 2.4 Hz), 7.39 (2H, d, J =
8.4 Hz), 8.01 (1H, d, J = 8.8 Hz), 8.35 (1H, s), 9.56 (1H, s, D2O
exchangeable).
4.1.5. 3-(4-Hydroxyphenyl)-7-isopropoxychromen-4-one (3d).
According to the procedure used to prepare 3a, reaction of 1
with 2-bromopropane provided 0.26 g of 3d in 18% yield as a
white powder. mp: 179-180 ꢀC (Dec.). GC-MS (m/z 296, Mþ,
m/z 253, m/z 225, m/z 137, m/z 118). 1HNMR (DMSO-d6, 400
MHz, δ): 1.32 (6H, d, J = 6.4 Hz), 4.82 (sept, 1H, J = 6.0 Hz),
6.796 (2H, d, J = 8.4 Hz), 7.03 (1H, dd, J = 8.8 Hz, J = 2.4 Hz),
7.13 (1H, d, J = 2.4 Hz), 7.38 (d, 2H, J = 8.4 Hz), 8.00 (1H, d, J =
8.8 Hz), 8.35 (1H, s), 9.56 (1H, s, D2O exchangeable).
4.1.6. 7-Butoxy-3-(4-hydroxyphenyl) Chromen-4-one (3e). Using
the procedure used to prepare 3a, reaction of 1 with 1-bromo-
butane provided 0.66 g of 3e as a white powder in 43% yield. mp:
162-164 ꢀC. GC-MS (m/z 310, Mþ, m/z254, m/z253, m/z226, m/z
225, m/z 197, m/z 137, m/z 118). 1HNMR (DMSO-d6, 400 MHz,
δ): 0.94 (3H, t, J = 7.2 Hz), 1.45 (2H, sextet, J = 7.2 Hz), 1.737
(2H, quint, J = 7.2 Hz), 4.12 (2H, t, J = 7.0 Hz), 6.80 (d, 2H, J =
8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, J = 2.4 Hz), 7.13 (1H, d, J =
2.4 Hz), 7.39 (2H, d, J = 8.4 Hz), 8.00 (1H, d, J = 8.8 Hz), 8.35
(1H, s), 9.57 (1H, s, D2O exchangeable).
4.1.7. 3-(4-Hydroxyphenyl)-7-isobutoxychromen-4-one (3f).
By the same procedure used to prepare 3a, reaction of 1 with
2-bromobutane provided 0.39 g of 3f in 25% yield as a white
powder. mp: 176-178 ꢀC. GC-MS (m/z 310 Mþ, m/z 253, m/z
137, m/z 118). 1HNMR (DMSO-d6, 400 MHz, δ): 0.990 (6H, d,
J = 6.4 Hz), 2.05 (1H, nonet, J = 6.40 Hz), 3.89 (2H, d, J =
6.4 Hz), 6.792 (2H, d, J = 8.8 Hz), 7.061 (1H, dd, J = 8.8 Hz,
J = 2.4 Hz), 7.119 (1H, d, J = 2.4 Hz), 7.381 (2H, d, J = 8.8 Hz),
8.001(1H, d, J = 8.8 Hz), 8.350 (1H, s), 9.57 (1H, s, D2O
exchangeable).
ERE-Luciferase Assay. As previously described,15,24 MCF-7
cells, grown for 2 days in 5%-CS-FBS containing phenol-red
free DMEM, were plated in 24-well plates at a density of 5 ꢀ
105 cells/well in the same media and allowed to attach overnight.
After 18 h, cells were transfected with 300 μg of pGL2-ERE2X-
TK-luciferase plasmid (Panomics) for 6 h according to the
manufacturer’s protocol using Effectene (Qiagen) and treated
with vehicle DMSO, E2, various concentrations of daidzein
analogues (all with and without estrogen), ICI and 4-OH-TAM
overnight. Media was removed and cells were lysed with reporter
lysis buffer. Relative light units (RLUs) were measured in an
Opticomp II luminometer (MGM Laboratories) using luciferase
reagent (Promega).
ERr Binding Assays. Receptor binding determinations of
daidzein analogues were achieved using the method of Bolger25
et al. as was applied by Burow14 et al. In this method, recombi-
nant ER is in equilibrium with a fluorescent ligand (ES2) and a
concentration of the competitor (daidzein analogues). The relative
displacement of the ES2 is measured as a change in polarization
anisotropy. Serial dilutions of competitors (daidzein analogues
and estradiol) were prepared from DMSO stock solutions in
screening buffer at the desired concentrations. The ER and ES2
were combined with each competitor aliquot to a final concentra-
tion of 2 nM ER and 3 nM ES2, respectively. In addition, both a no
binding control (ER þ ES2, equivalent to 0% competitor in-
hibition) and a 100% binding control (only free ES2, no ER,
equivalent to 100% competitor inhibition) were prepared. All
competitor and controls were prepared in duplicate within a
binding experiment. After 2-h incubation at room temperature,
the anisotropy values for each sample and control were measured
using the Beacon 2000. Anisotropy values were converted to
percent inhibition using the following formula: I% = (A0 -A)/
(A0 - A100) ꢀ 100, where I% is the percent inhibition, A0 is 0%
inhibition, A100 is 100% inhibition, and A represents the observed
value. This conversion to percent inhibition makes the data more
intuitive and normalizes the experiment-to-experiment differences
in the range of anisotropy values. The percent inhibition versus
competitor concentration curves were analyzed by nonlinear
least-squares curve fitting (Prism 5.0a, GraphPad Software, San
Diego, California USA, www.graphpad.com) to yield IC50 values
(the concentration of competitor needed to displace half of the
bound ligand). To compare binding affinities of the test compounds
to those reported in the literature, IC50 values were converted to
4.1.8. 7-Cyclopentyloxy-3-(4-hydroxyphenyl)chromen-4-one (3g).
According to the procedure used to prepare 3a, reaction of 1 with
bromocyclopentane provided 0.35 g of 3g as a colorless needle
crystalline in 22% yield. mp: 204-206 ꢀC. GC-MS (m/z 322 Mþ,
1
m/z 254, m/z 225, m/z 137, m/z 118). HNMR (DMSO-d6, 400
MHz, δ): 1.61 (2H, m), 1.74 (4H, m), 1.99 (2H, m), 5.01 (1H, m),
6.81 (2H, d, J = 8.4 Hz), 7.02 (1H, d, J = 8.8 Hz, J = 2.0 Hz), 7.09
(1H, d, J = 2.0 Hz), 7.39 (2H, d, J = 8.4 Hz), 8.01 (1H, d, J = 8.8
Hz), 8.35 (1H, s), 9.57 (1H, s, D2O exchangeable).
4.1.9. 7-Hexyloxy-3-(4-hydroxyphenyl)chromen-4-one (3h).
According to the procedure used to prepare 3a, reaction of 1

6162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Jiang et al.
relative binding affinities (RBA) using E2 as a standard. The E2
RBA was set equal to 100 RBA = (IC50/IC50 of E2) ꢀ 100.
Tumor size was measured 3 times weekly using digital calipers.
The volume of the tumor was calculated using the following
formula: 4/3 πLS∧2 (L = larger radius; S = shorter radius). All
procedures involving these animals were conducted in compli-
ance with State and Federal laws, standards of the U.S. Depart-
ment of Health and Human Services, and guidelines established
by Tulane University Animal Care and Use Committee. The
facilities and laboratory animals program of Tulane University
are accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care.
Colony Formation Assay. Similar to our previous studies,15,24
cells were cultured in 5% FBS-DMEM and media was changed
to phenol red-free 5% CS-DMEM for 48 h prior to assay. MCF-
7 cells were seeded at a density of 1 ꢀ 103 cells/well in a six-well
plate containing phenol red-free 5% CS-DMEM. The cells were
allowed to attach overnight and treated on the following day with
DMSO vehicle, 1 nM E2, the individual daidzein analogues (with
and without estrogen) and 4-OH-TAM. Cells were incubated at
37 ꢀC, 5% CO2 in a humidified incubator. Media was replaced
every 7 days and treated with appropriate drug for 3 weeks. After
3 weeks, the media were removed and the cells were fixed with
formaldehyde and dried overnight. The cells were then washed and
stained with crystal violet and dried. The colonies were counted.
Real Time RT-PCR. Total cellular RNA was extracted using
the RNeasy mini column (Qiagen), following the manufac-
turer’s instructions. Briefly, a maximum of 1 ꢀ 107 cells are
disrupted in buffer containing guanidine isothiocyanate and
homogenized. Ethanol is added to the lysate, creating condi-
tions that promote selective binding of RNA to the RNeasy
silica-gel membrane. The sample is then applied to the RNeasy
mini-column. Total RNA binds to the membrane, and high-
quality RNA is then eluted in RNase-free water. All binding,
wash, and elution steps were performed by centrifugation in a
microcentrifuge. The concentration of RNA was determined
using an ultraviolet spectrophotometer. Reverse transcription
(RT) was carried out using the SuperScript First-Strand Synth-
esis System for RT-PCR (Invitrogen). One microgram of total
RNA was reverse transcribed to cDNA following the manufac-
turer’s instructions. For each RT, a blank was prepared using all
the reagents except the RNA sample (for which an equivalent
volume of DEPC treated water was substituted). This blank was
used as a nontemplate control in the real-time PCR experiments.
The level of PgR transcripts was determined using a real-time
quantitative PCR. Primers for PCR were designed to span
intron/exon junctions to minimize amplification of residual
genomic DNA. The primer sequence for PgR is (50-TACC-
CGCCCTATCTCAACTACC-30; 50-TGCTTCATCCCCACAG-
ATTAAACA-30). PCR mix contained optimal concentrations
of primers, cDNA and SYBR Green PCR Master Mix (Bio-
Rad). Quantification and relative gene expression were calcu-
lated with internal controls. The ratio between these values
obtained provided the relative gene expression levels.
Molecular Modeling. The coordinates for the estrogen recep-
tor alpha (ERR) bound to the agonist diethylstilbestrol (3ERD.
pdb)18 and bound to the antagonist 4-OH-TAM (3ERT.pdb)21
were taken from the Protein Data Bank (http://www.rcsb.org).
Oxygen atoms representing water were removed and hydrogen
atoms were added to the template proteins using Amber99 force
field. The representative compounds daidzein, 3a and 2 were
utilized for the docking studies. The 3D structures of the mole-
cules were built using the Tripos SYBYL 8.1 Program (Tripos,
St. Louis, MO). Initial geometric optimizations of the ligands
were carried out using the standard MMFF94 force field, with a
0.001 kcal/mol energy gradient convergence criterion and a
distance-dependent dielectric constant employing Gasteiger
and Marsili charges. Additional geometric optimizations were
performed using the semiempirical method molecular orbital
package (MOPAC). The compounds were docked into the
binding pockets of 3ERD and 3ERT using the Surflex docking
program. Surflex21 is a fully automatic flexible molecular dock-
ing algorithm that combines the scoring function from the
Hammerhead docking system26 with a search engine that relies
on a surface-based molecular similarity method as a means to
rapidly generate suitable putative poses for molecular fragments.
A protomol27 representing the binding site was first generated
using the position of the native ligand of the crystal structures.
This protomol consists of the preferred locations of various
molecular probes (CH4, CdO, N-H), which are then used by
the docking engine to search for the best three-dimensional
morphological similarity between the protomol and the ligand
to dock. A proto-thresh value of 0.5 and a proto-bloat value of
0 were used to generate a compact protomol to which the com-
pounds were docked. The Surflex scores and visual inspection of
docking results wereconsideredfor obtaining the optimalbinding
posture. The resulting protein-ligand complexes were then sub-
jected to staged minimization using 200 steps of Steepest descent
and 1000 steps of Conjugate gradient methods (with Amber99
force fields).
In Vivo Assay of Anti-Estrogenic Activities of Analogue 2.
Xenograft experiments were conducted using previously pub-
lished protocols (Salvo et al 2006, Rhodes et al 2010). Briefly,
4-6 week old female ovariectomized NU/Nu mice were ob-
tained from Charles River Laboratories (Wilmington, MA).
The animals were allowed a period of adaptation in a sterile and
pathogen-free environment with phytoestrogen-free food and
water ad libitum. MCF-7 cells were harvested in the exponential
growth phase using a PBS/EDTA solution and washed. Mice
were injected bilaterally in the mammary fat pad (MFP) with 5 ꢀ
106 viable cells suspended in 50 μL sterile PBS mixed with 100 μL
Matrigel (reduced factor; BD Biosciences, Bedford, MA). 17β-
Estradiol pellets (0.72 mg, 60-day release; Innovative Research
of America, Sarasota, FL) were implanted subcutaneously in
the lateral area of the neck using a precision trochar (10 gauge)
at the time of cell injection. All procedures in animals were
carried out under anesthesia using a mix of isofluorane and
oxygen delivered by mask. Tumors were allowed to form and at
day 15 post cell injection mice were randomized into groups of
5 mice each. Mice were treated daily with intraperitoneal injec-
tions of either vehicle (1:5 DMSO/PBS) or analog 2 (50 mg/kg/
animal) for 21 days. Tamoxifen animals were implanted with a
slow release tamoxifen pellet (5mg, 60-day release) in the lateral
area of the neck on the opposite side from the estrogen pellet.
ICI animals were given a onetime intraperitoneal injection of
ICI 182,780 (5 mg in castor oil) on day 15 post cell injection.
Statistical Analysis. Data were summarized as the mean (
standard error of the mean (SEM) using the Graph Pad Prism
V.4 software program. Analysis of variance models were em-
ployed to compare relative ERE transcriptional reporter assays
between controls versus treatment. A Tukey post-test was
performed to compare differences between groups where a
p value < 0.05 was considered significant.
Acknowledgment. This study was supported by a coopera-
tive agreement with the U.S. Department of Agriculture
Grant 58-6435-7-019; Department of Defense Grant
W81XWH-04-1-0557; the Louisiana Cancer Research Con-
sortium (LCRC); Office of Naval Research Grant N00014-
99-1-0763; and National Center for Research Resources
RCMI program through Grant 1G12RR026260-01.
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