In vitro evaluation of the anti-estrogenic activity of hydroxyl substituted diphenylnaphthyl alkene ligands for the estrogen receptor

Article abstract of DOI:10.1016/j.bmc.2004.11.051

There is still a need for additional scaffolds to further explore tissue selectivity and improving efficacy of selective estrogen receptor modulators (SERMs). A series of hydroxyl substituted diphenylnaphthyl alkene ligands for the two estrogen receptors are described that arose from an initial de novo designed diphenylnaphthyl propylene ligand 1. All compounds gave K_is under 10 nM when assayed in the presence of ERα. Generally these compounds had very high affinity for both ER isotypes. Moving the hydroxyl group on naphthalene from the 6- to the 5-position of the α-naphthalene attached compounds (6b and 6e vs 6c and 6f) had little affect on ER binding nor did altering the position of the naphthalene attachment (α or β) to the alkene moiety. In transfection assays none of the compounds displayed agonistic activity in the absence of E₂. In MCF-7 proliferation assays 6a-d, 6f and 12a-c successfully abrogated E₂ stimulation and resulted in greater than 50% inhibition at 1 μM, a level of efficacy similar to that obtained when the cells were treated with raloxifene. Our results show that this new class of SERMs are good candidates for further study as therapeutic agents for the treatment of breast cancer and osteoporosis.

Full text of DOI:10.1016/j.bmc.2004.11.051

Bioorganic & Medicinal Chemistry 13 (2005) 1819–1828
In vitro evaluation of the anti-estrogenic activity of
hydroxyl substituted diphenylnaphthyl alkene ligands for
the estrogen receptorI
Jonathan M. Schmidt,a, Gilles B. Tremblay,b,à Michael A. Plastina,a,§
a,k
Fupeng Ma,a,– Sanjivanjit Bhal (nee Basra), Miklos Feher,a,
**
´
Robert Dunn-Dufaulta, and Peter R. Reddena,*
aSignalGene Inc., 335 Laird Rd, Unit 2, Guelph, Ontario, Canada N1G 4P7
bSignalGene Inc., 8475 Avenue Christophe-Colomb, bureau 1000, Montreal, Quebec, Canada H2M 2N9
Received 27 August 2004; revised 21 November 2004; accepted 24 November 2004
Available online 22 December 2004
Abstract—There is still a need for additional scaffolds to further explore tissue selectivity and improving efficacy of selective estrogen
receptor modulators (SERMs). A series of hydroxyl substituted diphenylnaphthyl alkene ligands for the two estrogen receptors are
described that arose from an initial de novo designed diphenylnaphthyl propylene ligand 1. All compounds gave Kis under 10 nM
when assayed in the presence of ERa. Generally these compounds had very high affinity for both ER isotypes. Moving the hydroxyl
group on naphthalene from the 6- to the 5-position of the a-naphthalene attached compounds (6b and 6e vs 6c and 6f) had little
affect on ER binding nor did altering the position of the naphthalene attachment (a or b) to the alkene moiety. In transfection assays
none of the compounds displayed agonistic activity in the absence of E2. In MCF-7 proliferation assays 6a–d, 6f and 12a–c success-
fully abrogated E2 stimulation and resulted in greater than 50% inhibition at 1 lM, a level of efficacy similar to that obtained when
the cells were treated with raloxifene. Our results show that this new class of SERMs are good candidates for further study as ther-
apeutic agents for the treatment of breast cancer and osteoporosis.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Keywords: Diphenylnaphthyl alkene ligands; Estrogen receptor;
SERM.
q This work was conducted at SignalGene Inc. and is published with
the permission of the Board of Directors.
Stimulation of the estrogen receptor (ER) by endoge-
nous estrogens plays an important role in both male
and female physiology. Estrogens are involved in the
*
Corresponding author. Address: Protana Inc., Lead Discovery and
Optimization Division, 251 Attwell Dr, Toronto, Ontario, Canada
M9W 7H4. Tel.: +1 416 646 6329; fax: +1 416 644 5111; e-mail:
predden@protana.com
regulation of cholesterol and lipid levels, and the func-
tioning of skeletal, central nervous and reproductive sys-
tems.1,2 Estradiol (E2) stimulation, however, is also
implicated in the development of breast cancer.3 Conse-
quently, many ER ligands are being developed with the
aim of preventing E2-mediated tumour growth. Tamox-
ifen, which was originally developed as an ER antago-
nist, is currently the hormonal treatment of choice for
both pre-and post-menopausal women with breast carci-
noma. It is now known that tamoxifen and other selec-
tive estrogen receptor modulators (SERMs) display a
broad range of agonist and antagonist activity depen-
dent on the tissue and species being evaluated, and that
several factors are thought to contribute to this phenom-
enon.4–7 With the discovery of a new ER subtype desig-
nated ERb,8 experiments based on the tissue
Present address: Department of Environmental Biology, University
of Guelph, Guelph, Ontario, Canada N1G 2W1.
à Present address: Alethia Biotherapeutiques, 8475 Avenue Christophe-
Colomb, bureau 1000, Montreal, Quebec, Canada H2M 2N9.
§ Present address: Brantford Chemicals Inc., Station Main, P.O. Box
1976, Brantford, Ontario, Canada N3T 5W5.
– Present address: NPS Pharmaceuticals, Inc., 6850 Goreway Drive,
Mississauga, Ontario, Canada L4V 1V7.
k Present address: NAEJA Pharmaceutical Inc., 4290-91A Street,
Edmonton, Alberta, Canada T6E 5V2.
** Present address: Neurocrine Biosciences, Inc., 10555 Science Center
Drive, San Diego, CA 92121-1102, USA.
Present address: Thermo Electron Corporation, 5344 John Lucas
Drive, Burlington, Ontario, Canada L7L 6A6.
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.051

1820
J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
OH
N
O
O
N
O
N
O
OH
S
HO
OH
Raloxifene
Tamoxifen
1
Figure 1.
distribution and pharmacology of ERa and ERb suggest
that the tissue selectivity of certain estrogens may be re-
lated, at least in part, to their different effects at the ERa
and ER subtypes.6,9,10 Other factors that may contribute
to selectivity include the tissue-specific presence of co-
repressors and co-activators, and the tissue/species pres-
ence of different DNA-response elements.9,11
CH3I or EtBr to give 2b,c, regioselectively demethylated
using BBr3 to give the monomethyl ethers 3a–c and
alkylated with 1-(2-chloroethyl)piperidine to give the
piperidines 4a–c. Two of these, 4b,c, were alkylated with
a variety of halogenated naphthalenes using halogen–
lithium exchange conditions generating tertiary alcohols
that were dehydrated using HCl in ethanol to give the
methoxy ethers 5a–f. These were demethylated using so-
dium ethyl thiolate to give 6a–f. Unfortunately all at-
tempts to alkylate the hydrogen analogue 4a with
halogenated naphthalenes were unsuccessful.
Since the tissue selective effects of tamoxifen were dis-
covered other SERMs with improved selectivity profiles
have been developed. The most notable is the benzothio-
phene, raloxifene that is marketed for the prevention
and treatment of osteoporosis. Although raloxifene dis-
plays improved selectivity compared with tamoxifen,
optimal tissue selectivity has not yet been demonstrated.
As a result there remains a strong need for additional
diversity and new chemical scaffolds to allow for the
exploration of improved tissue selectivity.
At the same time we were pursuing tamoxifen analogues
whereby the phenyl ring geminal to the ethyl group in
tamoxifen was replaced by our naphthalene moieties
and the ethyl group shortened to hydrogen. As pre-
sented in Scheme 2, we prepared the simple alkene deriv-
atives whereby 4,40-dihydroxybenzophenone was
alkylated with 1-(2-chloroethyl)piperidine to give the
monoalkylated piperidine 8. The hydroxynaphthaldehy-
des were prepared from the corresponding halogenated
naphthalenes 9a–c by halogen–lithium exchange condi-
tions followed by formylation with DMF17 generating
10a–c. Demethylation with NaSEt in DMF afforded
the requisite naphthaldehydes 11a–c. Using McMurry
coupling conditions18–20 8 was treated with 11a–c giving
the diphenylnaphthyl ethylenes 12a–c.
We have previously reported the favourable in vitro
characteristics of a diphenylnaphthyl propylene ligand
1 (Fig. 1) for ERa that arose from our de novo design
technology.12 We subsequently embarked on a program
to further explore analogues around this ligand. Specif-
ically we were interested in substituting an ethyl group
in place of the methyl group on the alkene moiety of
1, since ethyl substitution is found in tamoxifen. In addi-
tion we determined the effect of removing the methyl
group on the alkene moiety. The effect of altering the
position of attachment (b vs a) of the naphthalene moi-
ety to the alkene was also evaluated. Since the antago-
nist potency tends to increase when the
piperidinoethoxy side chain replaces the pyrrolidinoeth-
oxy,13,14 all analogues were prepared with the latter
group.
The double bond configuration of the final compounds
(E/Z isomerization) was determined from the chemical
shift and integral of the piperidine basic side chain O-
methylene protons in the NMR spectra.19,21 The shifts
of these protons for the E-isomers as noted in the liter-
ature19,21 are further downfield than for the Z-isomers
and the isomeric composition is given in Table 1. No
real pattern has emerged regarding the final E/Z
composition of the double bond, whether using metal–
halogen exchange conditions followed by NaSEt
demethylation or McMurray coupling although the E-
isomer was the major isomer formed with the exception
of 6d and 12c.
2. Chemistry
2.1. Synthesis
Since we were interested in determining the effects of
ethyl and hydrogen substitution on the alkene portion
of 1, in addition to methyl substitution, it was more effi-
cient to take advantage of the well known a-alkylation
of desoxyanisoin followed by regioselective demethyla-
tion with a Lewis acid.15,16 Hence as shown in Scheme
1, desoxyanisoin 2a, was alkylated using NaH and
2.2. Computational studies
We had previously conducted relatively detailed model-
ling studies of 1 and out of those studies arose the sug-
gestion that altering the position of the hydroxyl group
on the naphthalene moiety from the 6- to the 5-position

J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
1821
N
O
HO
O
O
3 eq. BBr3
45 min.
Na, EtOH
O
N
O
Cl
R
R
O
O
3a, R = H
2a, R = H, desoxyanisoin
2b, R = CH3
2c, R = CH3CH2
R
3b, R = CH3
O
3c, R = CH3CH2
4a, R = H
4b, R = CH3
4c, R = CH3CH2
nBuLi
11a-c
Cl
OH
OH
O
N
N
O
NaSEt
R2
O
O
R2
1
1
R
R
OH
6a, R1 = CH3, R2 = 6-OH-naphth-2-yl
6b, R1 = CH3, R2 = 6-OH-naphth-1-yl
6c, R1 = CH3, R2 = 5-OH-naphth-1-yl
6d, R1 = CH3CH2, R2 = 6-OH-naphth-2-yl
6e, R1 = CH3CH2, R2 = 6-OH-naphth-1-yl
6f, R1 = CH3CH2, R2 = 5-OH-naphth-1-yl
5a, R1 = CH3, R2 = 6-OCH3-naphth-2-yl
5b, R1 = CH3, R2 = 6-OCH3-naphth-1-yl
5c, R1 = CH3, R2 = 5-OCH3-naphth-1-yl
5d, R1 = CH3CH2, R2 = 6-OCH3-naphth-2-yl
5e, R1 = CH3CH2, R2 = 6-OCH3-naphth-1-yl
5f, R1 = CH3CH2, R2 = 5-OCH3-naphth-1-yl
7
Scheme 1.
could improve binding,12 by enabling the establishment
of an additional potential hydrogen bond with His524 of
ERa. To further evaluate this hypothesis and guide our
synthetic efforts, a variety of virtual structures, v13–v19
and two subsequently synthesized compounds, 6e,f
(Scheme 3) were docked into ERa using the GOLD pack-
age. GOLD is known to reproduce experimental binding
positions well22 and we have confirmed this for different
receptor systems including ERa.23 The base docking
speed (times 1) was applied as it provided the best preci-
sion from all predefined GA settings. A particular prob-
lem in establishing the binding modes of these ligands
arises because of the existence of two tautomeric states
of His524 that are in equilibrium. Since the donor and
R1
O
O
R2
Na, EtOH
4
R
N
OH
O
OH
HO
R3
N
8
Cl
R1 R2 R3 R4
Zn, TiCl4
11a-c
9a
9b
9c
H
I
Br
H
H
H
OCH3
OCH3
H
I
H
OCH3
O
N
nBuLi
DMF
R1 R2 R3 R4
R
10a
H
CHO
H
H
H
OCH3
OCH3
H
10b CHO
10c CHO
H
OCH3
OH
NaSEt
R1 R2 R3 R4
12a, R = 6-OH-naphth-2-yl
12b, R = 6-OH-naphth-1-yl
12c, R = 5-OH-naphth-1-yl
11a
H
CHO
H
H
H
OH
OH
H
11b CHO
11c CHO
H
OH
Scheme 2.

1822
J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
Table 1. ERa and ERb binding, receptor activity and MCF-7 inhibition of diphenylnaphthyl alkenes
R1
O
OH
N
N
R2
O
R2
OH
1
R
B
A
Compound E/Z Ratioa Scaffold
R1
R2
ER binding (Ki, nM)
ERa ERb
6.02 1.97e 37.9 13.8
Receptor activityb MCF-7 growth inhibitionc Agonismd
1
6a
9:1
2:1
++
++
++
++
++
++
+
No
No
No
No
No
No
No
No
No
No
No
Ndh
A
A
A
A
A
A
CH3
CH3
CH3
6-OH 1.06 0.49 0.99 0.49
6-OH 4.61 1.66 59.4 18.3
5-OH 4.33 1.89 20.8 8.50
+++
+++
++
6b
6c
7:1
4:1
6d
6e
1:2
CH3CH2 6-OH 0.82 0.47 0.45 0.56
CH3CH2 6-OH 4.11 1.08 39.7 16.3
CH3CH2 5-OH 4.45 1.46 9.01 3.51
10.1 5.36 48.9 28.1
+++
+++
+++
+++
+++
+++
+++
+++
8.5:1
8.5:1
6f
7f
++
—
12a
12b
12c
Ralg
19:1
1:1
B
B
B
H
H
H
6-OH 1.16 0.51 2.43 1.42
6-OH 0.9 0.38 1.37 0.82
++
++
++
++
1:1.8
5-OH 1.57 0.82 2.66 0.81
0.57 0.20 2.41 1.14
a E/Z Ratio determined from the integral of the respective OCH2 triplets in the NMR spectra.
b Cos-1 cells transfected with human ERa and an ERE-bLuc reporter in the presence of 10 nM estradiol and increasing SERM (Ô+++Õ,
EC50 > 50 nM; Ô++Õ, EC50 > 200 nM; Ô+Õ, EC50 > 200 nM).
c MCF-7 cells stimulated with 10 nM estradiol for 72 h in the presence of 1 lM SERM (Ô++Õ, >50% inhibition; Ô+Õ >50% inhibition; Ô—Õ, no
inhibition).
d HeLa cells transfected with human ERa and an ERE-bLuc reporter in the presence of increasing SERM (ÔnoÕ, no induction of reporter gene; ÔyesÕ,
measurable reporter gene activity).
e 3.7 nM.12
f Chlorine in place of pyrrolidine.
g Raloxifene.
h Not determined.
acceptor positions of His are switched in the two states,
this can strongly affect ligand orientations. Therefore in
order to account for this effect, the ligands were docked
to two forms of the receptor, each containing one of the
His tautomers.
OH
For the virtual analogues of 1, in addition to moving the
hydroxyl group on the naphthalene from the 6- to the 5-
position (v13) we also docked compounds with the hy-
droxyl group at the 7- (v14) and 4-positions (v15) as well
as the 4,5-dihydroxynaphthalene (v16). The correspond-
ing analogues containing the ethyl group and with piper-
idine in the basic side chain as opposed to the
pyrrolidine group, v17–v19, 6e,f were also docked.
O
N
(
)n
R1
R2
R5
R4
R3
From the results of these GOLD docking studies two
compounds, the 5-OH naphthalene v14 (corroborating
our previous modelling studies12) and the 4,5-dihydr-
oxynaphthalene v16 were predicted to establish hydrogen
bonds with His524. Interestingly, the corresponding
ethyl analogues (including the two ultimately synthe-
sized, 6e,f) did not appear to establish interactions with
His524.
n
R1
R2
R3
R4
R5
1
1
1
1
1
1
2
2
2
2
2
CH3
CH3
CH 3
CH3
CH3
CH3CH2
H
OH
H
H
H
OH
H
H
H
H
OH
H
H
H
H
H
H
OH
H
OH
H
H
OH
H
H
H
H
OH
OH
H
H
H
v13
v14
v15
v16
6e
v17
6f
H
CH3CH2 OH
CH3CH2
CH3CH2
CH3CH2
H
H
H
3. Biology
v18
v19
OH
OH
OH
For the compounds in this study the questions we
wished to answer pertained to their relative affinities
Scheme 3.

J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
1823
for both ER isotypes, the ability of these molecules to
act as anti-estrogens in cellular transfection assays, their
efficacy in inhibiting the E2-stimulated growth of the
human breast cancer cell line, MCF-7, and assessment
as to whether these molecules behaved as partial ago-
nist/antagonists (SERMs) or as pure anti-estrogens.
using a reporter under the control of a synthetic ERE,
a natural E2-dependent promoter, pS2, also produced
similar results (data not shown). We also observed anti-
estrogen activity in the presence of ligand-stimulated
ERb, albeit with reduced efficacy as predicted by the
overall reduction in affinity of these compounds for this
ER isotype.
Several different assays were used to address these ques-
tions. These included ER binding studies using in vitro
transcribed–translated ERs, receptor transcriptional
activity via reporter genes under the control of estrogen
response elements (EREs), and inhibition of estrogen
stimulated MCF-7 cells, with all results presented in
Table 1.
Other experiments were carried out to gauge the SERM
potential of these compounds. For example, although
these compounds were shown to be very good inhibitors
of ER activity in the presence of E2, we wished to deter-
mine if any of these molecules exhibited inherent estro-
genic activity. We measured this activity in a similar
fashion to the assay described above but utilized a basal
luciferase reporter plasmid with one copy of an ERE to
minimize background transcription. As shown in Table
1, none of the compounds displayed agonistic activity in
the absence of E2.
3.1. ER binding
The compounds were assayed as mixtures of the E/Z iso-
mers. With the exception of 6a, 6d, 12b and 12c all com-
pounds contained the E-isomer at a minimum of 80%.
All of the compounds tested, except for 712 gave Kis
under 10 nM when assayed in the presence of ERa.
When assayed with ERb, six compounds (1, 6b,c and
7) had calculated Kis greater than 10 nM. Of the com-
pounds that displayed better binding to ERb (6a and
6d) neither had greater than a two-fold preference for
this ER isotype. These binding results suggest that some
of these compounds had very high affinity for both ER
isotypes and there was a modest preference for ERa.
3.3. Inhibition of MCF-7 cells
In vitro profiling demonstrated that these molecules
have a very high affinity for ER, with a modest prefer-
ence for the ERa isotype, and results from the transfec-
tion assays indicated several behaved as potent anti-
estrogens. We therefore wished to determine if these
molecules could effectively inhibit the proliferation of
ER+ human breast cancer MCF-7 cells. We routinely
obtained approximately a twofold stimulation in prolif-
eration in the presence of E2 over the untreated cells
indicating that the ER+ MCF-7 cells were responding
to hormone. Treatment of these stimulated cells with
the test compounds resulted in a wide range of re-
sponses. Compound 7, which lacks a basic side chain
was unable to inhibit the proliferation of MCF-7 cells.
In contrast compounds 1, 6a–d, 6f and 12a–c, which
all contain the basic side chain, successfully abrogated
E2 stimulation and resulted in greater than 50% inhibi-
tion at 1 lM, a level of efficacy similar to that obtained
when the cells were treated with raloxifene. This con-
firms the correlation of effective inhibition of breast can-
cer cell proliferation with the presence of a basic side
chain in the molecule, presumably as a result of interac-
tion with Asp351.26 Overall, these results demonstrate
that several of the compounds were quite effective in this
assay and could inhibit proliferation to a level that was
comparable to raloxifene.
Moving the hydroxyl group on naphthalene from the
6- to the 5-position of the a-naphthalene attached com-
pounds (6b and 6e vs 6c and 6f) had little affect on bind-
ing. The hydroxyl group is positioned a number of
different ways in the various crystal structures of ERa
with diethylstilbesterol, raloxifene and is even absent
with 4-hydroxytamoxifen.24 There are indications that
a variety of lipophilic substituents (e.g., CH3, F) can
be substituted for the OH group of raloxifene25 that
can interact with His524, so perhaps we were too opti-
mistic in our additional binding expectations from the
modelling studies. Unfortunately we were unable to pre-
pare the hydrogen-substituted compounds, which may
have provided additional insight.
3.2. Receptor activity
These assays were designed to measure receptor tran-
scriptional activity via reporter genes under the control
of estrogen response elements (EREs) in order to deter-
mine the mechanistic influences of the compounds on
receptor activity.
The biological profiling of compounds of this nature
(mixtures of E/Z isomers) have been reported previ-
ously.19,21,27 It has been suggested, however, that it is
the Z-isomer of tamoxifen that is anti-estrogenic27 whilst
the E-isomer acts as an estrogen agonist. Moreover it is
known that facile isomerization with these types of com-
pounds frequently occurs and it proves difficult to sepa-
rate the isomers chromatographically.20 Therefore,
although the compounds in this study were tested as
E/Z mixtures and have thus far generated appropriate
in vitro SERM activity, due to the possibility of the
two isomers displaying different activity further work
would be required to determine the biological effects of
the individual isomers. Nonetheless in terms of potency
There are many permutations of the transfection assay
that can be used to address the stimulation or repression
of receptor activity. For example, under conditions
where the receptor is stimulated by E2, we observed that
all compounds efficiently abrogated ERa transcriptional
activity with EC50 values that correlated, for the most
part, with receptor affinity. Data presented in Table 1
was obtained using Cos-1 cells, but we observed very
similar results in HeLa and HepG2 cells (Data not
shown). In addition, although this data was obtained

1824
J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
the data indicates that at least one of the two E/Z
isomers is at least as potent as the mixture. It is also
unlikely that either isomer has substantial estrogenic
activity (although the higher affinity compound may
mask this).
was separated and the aqueous layer washed with
CH2Cl2. The combined organic layers were washed with
water then brine, dried and concentrated to give a pur-
ple oil that was purified by flash column chromatogra-
phy to give 2.6 g (95%) of 3c as white crystals.
1
400 MHz H NMR (CDCl3) d 7.94–7.96 (d, 2H, ArH);
7.13–7.16 (d, 2H, ArH); 6.85–6.87 (d, 2H, ArH); 6.75–
6.77 (d, 2H, ArH); 5.70 (br s, 1H, OH); 4.34 (t, 1H,
CH); 3.81 (s, 3H, OCH3); 2.13 (m, 1H, CH3CH2); 1.81
(m, 1H, CH3CH2); 0.88 (t, 3H, CH3CH2).
4. Conclusion
In conclusion, we have carried out the in vitro character-
ization of several members of the novel hydroxyl substi-
tuted diphenylnaphthyl alkene family of nonsteroidal
potential SERMs containing the piperidine basic side
chain. Minor structural modifications/changes such as
extending the alkyl portion on the alkene moiety, alter-
ing the position of attachment of the naphthalene moi-
ety (a to b) to the alkene or moving the hydroxyl
group on the naphthalene moiety produced minor effects
in terms of binding affinity and functional effects. Our
results clearly show that these compounds (as E/Z mix-
tures) have very high affinity for the a and b ERs and
display effective SERM-like activity in cell-based assays.
Most abrogate transcriptional activity of E2-stimulated
ERs and can efficiently prevent the proliferation of hu-
man MCF-7 breast cancer cells. At the same time cell
transfection assays in the presence of the TGFb pro-
moter in HeLa cells indicate that our compounds may
behave like raloxifene, a known SERM, and not like
the pure anti-estrogen ICI182,780.28 Our results show
that these new naphthalene SERMs as a class are good
candidates for further study as therapeutic agents for the
treatment of breast cancer and osteoporosis, or perhaps
in hormone replacement therapy.
5.1.3.
1-(4-Methoxyphenyl)-2-[4-(2-piperidin-1-yl-eth-
oxy)phenyl]ethan-1-one, 4a. To a solution of 3a16
(0.7 g, 2.9 mmol) in absolute ethanol (35 mL) that was
refluxing vigorously was added Na (0.13 g, 5.8 mmol).
After further refluxing for 30 min 1-(2-chloro-
ethyl)piperidine hydrochloride (0.59 g, 3.2 mmol) was
added. After refluxing for 1 h the reaction was filtered,
washed with acetone and concentrated. The residue
was purified by silica gel flash chromatography using a
gradient of 35% ethyl acetate in hexanes to 40–50% ace-
tone in hexanes to give 0.16 g (26%) of 4a. 400 MHz 1H
NMR (CDCl3) d 7.98–8.00 (d, 2H, ArH); 7.15–7.17 (d,
2H, ArH); 6.91–6.93 (d, 2H, ArH); 6.85–6.87 (d, 2H,
ArH); 4.16 (2, 2H, CH2); 4.07 (t, 2H, OCH2); 3.86 (s,
3H, OCH3); 2.75 (t, 2H, NCH2); 2.49 (br s, 4H,
CH2NCH2); 1.60 (m, 4H, CH2CH2NCH2CH2); 1.44
(m, 2H, NCH2CH2CH2).
5.1.4.
1-(4-Methoxyphenyl)-2-[4-(2-piperidin-1-yl-eth-
oxy)phenyl]propan-1-one, 4b. Prepared following the
method for 4a using 3b. Yield 0.88 g (45%). 200 MHz
1H NMR (CDCl3) d 7.92–7.96 (d, 2H, ArH); 7.15–7.20
(d, 2H, ArH); 6.81–6.88 (d, 4H, ArH); 4.60 (q, 1H,
CH); 4.06 (t, 2H, OCH2); 3.82 (s, 3H, OCH3); 2.76 (t,
2H, NCH2); 2.51 (br s, 4H, CH2NCH2); 1.60 (m, 4H,
CH2CH2NCH2CH2); 1.48 (d, 3H, CH3); 1.44 (m, 2H,
NCH2CH2CH2).
5. Experimental
5.1. General
All solvents were reagent grade and dried appropriately
prior to their use. NMR spectra were obtained using a
Gemini 200 or Bruker 400 MHz spectrometer using
CDCl3, DMSO-d6, acetone-d6 or methanol-d4 as sol-
vents. Silica gel (200–430 mesh) was used for flash col-
umn chromatography.
5.1.5.
1-(4-Methoxyphenyl)-2-[4-(2-piperidin-1-yl-eth-
oxy)phenyl]butan-1-one, 4c. Prepared following the
method for 4a using 3c. Yield 2.9 g (53%). 200 MHz
1H NMR (CDCl3) d 7.93–7.97 (d, 2H, ArH); 7.18–7.21
(d, 2H, ArH); 6.79–6.88 (d, 4H, ArH); 4.33 (q, 1H,
CH); 4.04 (t, 2H, OCH2); 3.81 (s, 3H, OCH3); 2.73 (t,
2H, NCH2); 2.48 (br s, 4H, CH2NCH2); 2.14 (m, 1H,
CH2); 1.83 (m, 1H, CH2); 1.58 (m, 4H,
CH2CH2NCH2CH2); 1.43 (m, 2H, NCH2CH2CH2);
0.87 (t, 3H, CH3).
5.1.1. 2-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)propan-
1-one, 3b. Prepared from 2b15 following the method
given for 3c. Yield (88%). 400 MHz H NMR (CDCl3)
1
d 7.93–7.95 (d, 2H, ArH); 7.12–7.14 (d, 2H, ArH);
6.85–6.87 (d, 2H, ArH); 6.75–6.77 (d, 2H, ArH);
4.59 (q, 1H, CH); 3.82 (s, 3H, OCH3); 1.48 (d, 3H,
CH3CH).
5.1.6. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(6-methoxynaphthalen-2-yl)propene, 5a.
2-Bromo-6-methoxynaphthalene (406 mg, 1.71 mmol)
was dissolved in anhydrous THF (20 mL) and cooled
to ꢀ78 °C. n-BuLi (2 M in cyclohexane, 0.86 mL,
1.71 mmol) was added and after 5 min 4b (210 mg,
0.57 mmol) dissolved in THF was added. The reaction
was removed from the cooling bath and allowed to come
to rt. After 1 h the reaction was quenched with methanol
and concentrated. The residue was dissolved in 10%
CH3OH in CHCl3 and the tertiary alcohol was partially
purified using silica gel chromatography using 10%
CH3OH in CHCl3. After concentrating the partially
5.1.2. 2-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)butan-1-
one, 3c. Compound 2c15 (2.85 g, 0.01 mol) was dissolved
in CH2Cl2 (50 mL) and placed in a CO2/acetone bath for
15 min. BBr3 (1 M in CH2Cl2, 30 mL, 0.03 mol) was
slowly added and the reaction left in the CO2/acetone
bath for 15 min. The reaction was allowed to come to
room temperature and after an additional 30 min TLC
indicated the reaction was complete. The reaction was
added dropwise to satd NaHCO3. The organic layer

J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
1825
purified tertiary alcohol was dissolved in abs ethanol
(15 mL). Concd HCl (1.5 mL) was added and the reac-
tion refluxed for 1 h. After evaporating the solvent the
residue was dissolved in CH2Cl2, water was added, the
mixture adjusted to pH 8 with satd NaHCO3 and ex-
tracted with CH2Cl2. The CH2Cl2 layer was washed with
1 N NaOH, water, brine, dried, concentrated and puri-
fied by silica gel flash chromatography using a gradient
of CHCl3–hexanes (1/1) to CHCl3 to 2% CH3OH in
2.78 (t, 2H, NCH2); 2.53 (m, 4H, CH2NCH2); 2.18 (q,
2H, CH2); 1.61 (m, 4H, CH2CH2NCH2CH2); 1.46 (m,
2H, NCH2CH2CH2); 0.76 (t, 3H, CH3).
5.1.12. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(6-hydroxynaphthalen-2-yl)propene,
6a. To 5a (208 mg, 0.41 mmol) dissolved in
DMF (10 mL) was added NaSEt (80%, 0.86 g,
10.2 mmol) and refluxed for 1.5 h. Water was added,
the mixture adjusted to pH 9 with satd NaHCO3, ex-
tracted with EtOAc, washed with water, brine, dried
and concentrated. The residue was purified by silica
gel flash chromatography using a gradient of CHCl3–
hexanes (1/1) to CHCl3 to 3–8% CH3OH in CHCl3 to
1
CHCl3 to give 0.21 g (72%) of 5a. 200 MHz H NMR
(CDCl3) d 6.62–7.80 (m, 14H, ArH); 4.00–4.17 (t, 2H,
OCH2); 3.73–3.95 (s, 6H, OCH3); 2.70–2.86 (t, 2H,
NCH2); 2.54 (m, 4H, CH2NCH2); 2.19–2.22 (s, 3H,
CH3); 1.64 (m, 4H, CH2CH2NCH2CH2); 1.49 (m, 2H,
NCH2CH2CH2).
1
give 0.144 g (73%) of 6a. 400 MHz H NMR (DMSO-
d6) d 9.11–9.63 (s, 2H, OH); 6.37–7.66 (m, 14H, ArH);
3.83–3.93 (t, 2H, OCH2); 2.40–2.60 (t, 2H, NCH2);
2.32 (m, 4H, CH2NCH2); 1.96–1.99 (s, 3H, CH3);
1.40 (m, 4H, CH2CH2NCH2CH2); 1.28 (m, 2H,
NCH2CH2CH2).
5.1.7. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(6-methoxynaphthalen-1-yl)propene, 5b.
Prepared following the method for 5a using 4b and 1-
iodo-6-methoxynaphthalene.12 Yield 0.185 g (67%).
200 MHz 1H NMR (CDCl3) d 6.47–8.00 (m, 14H,
ArH); 4.09 (t, 2H, OCH2); 3.67–3.93 (s, 6H, OCH3);
2.77 (t, 2H, NCH2); 2.53 (m, 4H, CH2NCH2); 1.84 (s,
3H, CH3); 1.63 (m, 4H, CH2CH2NCH2CH2); 1.48 (m,
2H, NCH2CH2CH2).
5.1.13. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(6-hydroxynaphthalen-1-yl)propene,
6b. Prepared following the method for 6a. Yield 0.03 g
(51%). 200 MHz 1H NMR (acetone-d6) d 6.46–8.00
(m, 14H, ArH); 3.85–4.09 (t, 2H, OCH2); 2.72 (t, 2H,
NCH2); 2.50 (m, 4H, CH2NCH2); 1.82–2.21 (s, 3H,
CH3); 1.53 (m, 4H, CH2CH2NCH2CH2); 1.44 (m, 2H,
NCH2CH2CH2).
5.1.8. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(5-methoxynaphthalen-1-yl)propene, 5c.
Prepared following the method for 5a using 4b and 1-
iodo-5-methoxynaphthalene (see method for 1-iodo-6-
methoxynaphthalene12). Yield 0.142 g (52%). 200 MHz
1H NMR (CDCl3) d 6.50–8.27 (m, 14H, ArH); 4.11 (t,
2H, OCH2); 3.67–4.02 (s, 6H, OCH3); 2.80 (t, 2H,
NCH2); 2.56 (m, 4H, CH2NCH2); 1.85 (s, 3H, CH3);
1.64 (m, 4H, CH2CH2NCH2CH2); 1.49 (m, 2H,
NCH2CH2CH2).
5.1.14. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(5-hydroxynaphthalen-1-yl)propene,
6c. Prepared following the method for 6a. Yield 0.06 g
(45%). 400 MHz 1H NMR (acetone-d6) d 6.38–8.07
(m, 14H, ArH); 3.75–4.01 (t, 2H, OCH2); 2.50–2.62 (t,
2H, NCH2); 2.41 (m, 4H, CH2NCH2); 1.73 (s, 3H,
CH3); 1.47 (m, 4H, CH2CH2NCH2CH2); 1.34 (m, 2H,
NCH2CH2CH2).
5.1.9. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(6-methoxynaphthalen-2-yl)butene, 5d.
Prepared following the method for 5a using 4c and 2-
bromo-6-methoxynaphthalene. Yield 0.263 g (91%).
200 MHz 1H NMR (CDCl3) d 6.52–7.76 (m, 14H,
ArH); 3.95–4.13 (t, 2H, OCH2); 3.68–3.93 (s, 6H,
OCH3); 2.65–2.80 (t, 2H, NCH2); 2.47 (m, 6H,
CH2NCH2 + CH2); 1.60 (m, 4H, CH2CH2NCH2CH2);
1.45 (m, 2H, NCH2CH2CH2); 0.97 (t, 3H, CH3).
5.1.15. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(6-hydroxynaphthalen-2-yl)butene, 6d.
Prepared following the method for 6a. Yield 0.124 g
(51%). 400 MHz 1H NMR (methanol-d4) d 6.41–7.65
(m, 14H, ArH); 3.97–4.05 (t, 2H, OCH2); 2.65–2.75 (t,
2H, NCH2); 2.40–2.58 (m, 4H, CH2NCH2 + CH2);
1.55 (m, 4H, CH2CH2NCH2CH2); 1.43 (m, 2H,
NCH2CH2CH2); 0.90 (m, 3H, CH3).
5.1.10. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(6-methoxynaphthalen-1-yl)butene, 5e.
Prepared following the method for 5a using 4c and
1-iodo-6-methoxynaphthalene. Yield 0.185 g (85%).
200 MHz 1H NMR (CDCl3) d 6.50–7.99 (m, 14H,
ArH); 4.10 (t, 2H, OCH2); 3.66–3.92 (s, 6H, OCH3);
2.79 (t, 2H, NCH2); 2.53 (m, 4H, CH2NCH2); 2.20 (q,
2H, CH2); 1.62 (m, 4H, CH2CH2NCH2CH2); 1.46 (m,
2H, NCH2CH2CH2); 0.77 (t, 3H, CH3).
5.1.16. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(6-hydroxynaphthalen-1-yl)butene, 6e.
Prepared following the method for 6a. Yield 0.124 g
(51%). 400 MHz 1H NMR (methanol-d4) d 6.31–7.83
(m, 14H, ArH); 3.99 (t, 2H, OCH2); 2.69 (t, 2H,
NCH2); 2.47 (m, 4H, CH2NCH2); 2.07 (m, 2H, CH2);
1.54 (m, 4H, CH2CH2NCH2CH2); 1.40 (m, 2H,
NCH2CH2CH2); 0.66 (m, 3H, CH3).
5.1.11. (E/Z)-1-(4-Methoxyphenyl)-2-(4-(2-piperidin-1-yl)-
ethoxy)phenyl-1-(5-methoxynaphthalen-2-yl)butene, 5f.
Prepared following the method for 5a using 4c and
1-iodo-5-methoxynaphthalene. Yield 0.238 g (83%).
200 MHz 1H NMR (CDCl3) d 6.48–8.25 (m, 14H,
ArH); 4.09 (t, 2H, OCH2); 3.64–4.00 (s, 6H, OCH3);
5.1.17. (E/Z)-1-(4-Hydroxyphenyl)-2-(4-(2-piperidin-1-
yl)ethoxy)phenyl-1-(5-hydroxynaphthalen-1-yl)butene, 6f.
Prepared following the method for 6a. Yield 0.111 g
(69%). 400 MHz H NMR (DMSO-d6) d 10.04 (s, 1H,
OH); 9.08 (s, 1H, OH); 6.28–8.23 (m, 14H, ArH); 3.94
(t, 2H, OCH2); 2.57 (t, 2H, NCH2); 2.35 (m, 4H,
1

1826
J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
CH2NCH2); 2.00 (m, 2H, CH2); 1.41 (m, 4H,
CH2CH2NCH2CH2); 1.30 (m, 2H, NCH2CH2CH2)
0.60 (m, 3H, CH3).
5.1.23. 6-Hydroxy-1-naphthaldehyde, 11b. Prepared fol-
lowing 11a using 10b. Yield 0.085 g (39%). 400 MHz
1H NMR (acetone-d6) d 10.35 (s, 1H, CHO); 9.16 (d,
1H, ArH); 8.93 (br s, 1H, OH); 8.05 (d, 1H, ArH);
7.94 (d, 1H, ArH); 7.65 (dd, 1H, ArH); 7.35 (m, 2H,
ArH).
5.1.18. 4-(2-Piperidin-1-yl)ethoxy-40-hydroxybenzophe-
none, 8. Na (0.23 g, 10 mmol) was added to refluxing
abs ethanol (150 mL). After 5 min at reflux, 4,40-dihydr-
oxybenzophenone (1 g, 4.7 mmol) was added and the
reaction refluxed for 0.5 h. 1-(2-Chloroethyl)piperidine
hydrochloride (0.95 g, 5.1 mmol) was added as the free
base by treatment with K2CO3 (1.4 g, 10.2 mmol) in
abs ethanol and continued refluxing for 1 h. The solvent
was evaporated and the residue dissolved in acetone, fil-
tered, concentrated and purified by silica gel flash chro-
matography using a gradient of 3–4.5% CH3OH in
5.1.24. 5-Hydroxy-1-naphthaldehyde, 11c. Prepared fol-
lowing 11a using 10c. Yield 0.06 g (35%). 400 MHz H
1
NMR (acetone-d6) d 10.43 (s, 1H, CHO); 9.40 (br s,
1H, OH); 8.75 (d, 1H, ArH); 8.62 (d, 1H, ArH); 8.15
(d, 1H, ArH); 7.70 (dd, 1H, ArH); 7.53 (dd, 1H,
ArH); 7.07 (d, 1H, ArH).
5.1.25. (E/Z)-1-(4-Hydroxyphenyl)-1-(4-(2-piperidin-1-
yl)ethoxy)phenyl-2-(6-hydroxynaphthalen-2-yl)ethene, 12a.
To a suspension of Zn (0.1 g, 1.5 mmol) in anhydrous
THF (4 mL) under Ar at ꢀ10 °C was added TiCl4
(0.068 mL, 0.62 mmol) slowly. The cooling bath was re-
moved and the reaction gently refluxed for 2 h. The heat
was removed and a solution of 8 (0.1 g, 0.31 mmol) and
11a (0.08 g, 0.46 mmol) in anhydrous THF (22 mL) was
added. The reaction mixture was then refluxed for a fur-
ther 2.5 h. Additional 17a (0.06 g, 0.35 mmol) was added
in anhydrous THF (7 mL) and refluxed for a further
40 min. The reaction mixture was cooled, poured into
10% K2CO3 and EtOAc and stirred overnight. After
separating, the EtOAc layer was washed with brine,
dried, concentrated and purified by silica gel flash chro-
matography using a gradient of EtOAc–hexanes (1/1) to
4% CH3OH/36% hexanes in EtOAc to give 0.05 g (35%).
1
CHCl3 to give 0.73 g (48%) of 8. 400 MHz H NMR
(DMSO-d6) d 6.80–7.75 (m, 8H, ArH); 4.18 (m, 2H,
OCH2); 2.70 (m, 2H, NCH2); 2.44 (m, 4H, CH2NCH2);
1.50 (m, 4H, CH2CH2NCH2CH2); 1.41 (m, 2H,
NCH2CH2CH2).
5.1.19. 6-Methoxy-2-naphthaledhyde, 10a. To a solution
of 2-bromo-6-methoxynaphthaldehyde (0.5 g, 2.1 mmol)
in anhydrous THF (20 mL) cooled to ꢀ78 °C under Ar
was added n-BuLi (2 M in cyclohexane, 2.6 mL,
5.3 mmol) and stirred for 15 min. Anhydrous DMF
(1.0 mL, 12.7 mmol) was added and the reaction stirred
for a further 15 min at ꢀ78 °C. The reaction was poured
into 3 N HCl (15 mL), extracted with ether, washed with
water, satd NaHCO3, brine, dried and concentrated.
The residue was purified by silica gel flash chromatogra-
phy using 5% acetone in hexanes to give 0.29 g (74%).
400 MHz 1H NMR (CDCl3) d 10.10 (s, 1H, CHO);
7.80–7.94 (m, 3H, ArH); 7.16–7.25 (m, 2H, ArH); 3.94
(s, 3H, OCH3).
1
400 MHz H NMR (acetone-d6) d 9.71 (br s, 1H, OH);
9.55 (br s, 1H, OH); 6.72–7.51 (m, 14H, ArH); 4.04
(m, 2H, OCH2); 2.66 (m, 2H, NCH2); 2.44 (br s, 4H,
CH2NCH2); 1.49 (br s, 4H, CH2CH2NCH2CH2); 1.38
(br s, 2H, NCH2CH2CH2).
5.1.20. 6-Methoxy-1-naphthaledhyde, 10b. Prepared fol-
lowing the method for 10a from 1-iodo-6-methoxynaph-
thalene.12 Yield 0.26 g (76%). 200 MHz 1H NMR
(CDCl3) d 10.34 (s, 1H, CHO); 9.18 (d, 1H, ArH);
8.00 (d, 1H, ArH); 7.84 (m, 1H, ArH); 7.60 (dd, 1H,
ArH); 7.35 (dd, 1H, ArH); 7.22 (dd, 1H, ArH); 3.94
(s, 3H, OCH3).
5.1.26. (E/Z)-1-(4-Hydroxyphenyl)-1-(4-(2-piperidin-1-
yl)ethoxy)phenyl-2-(6-hydroxynaphthalen-1-yl)ethene, 12b.
Prepared following the method for 12a using 6-hydroxy-
1-naphthaldehyde. Yield 0.025 g (66%). 400 MHz 1H
NMR (DMSO-d6) d 9.71 (br s, 1H, OH); 9.58 (br s,
1H, OH); 6.56–7.94 (m, 14H, ArH); 4.09 (m, 2H,
OCH2); 2.68 (m, 2H, NCH2); 2.42 (br s, 4H,
CH2NCH2); 1.50 (br s, 4H, CH2CH2NCH2CH2); 1.38
(br s, 2H, NCH2CH2CH2).
5.1.21. 5-Methoxy-1-naphthaledhyde, 10c. Prepared fol-
lowing the method for 10a from 1-iodo-5-methoxynaph-
thalene. Yield 0.18 g (69%). 200 MHz 1H NMR (CDCl3)
d 10.42 (s, 1H, CHO); 8.80 (d, 1H, ArH); 8.62 (d, 1H,
ArH); 8.02 (m, 1H, ArH); 7.61 (m, 2H, ArH); 6.95 (d,
1H, ArH); 3.97 (s, 3H, OCH3).
5.1.27. (E/Z)-1-(4-Hydroxyphenyl)-1-(4-(2-piperidin-1-
yl)ethoxy)phenyl-2-(5-hydroxynaphthalen-1-yl)ethene, 12c.
Prepared following the method for 12a using 5-hydroxy-
1-naphthaldehyde. Yield 0.025 g (60%). 400 MHz 1H
NMR (DMSO-d6) d 10.12 (br s, 1H, OH); 9.59 (br s,
1H, OH); 6.55–8.00 (m, 14H, ArH); 4.09 (m, 2H,
OCH2); 2.68 (m, 2H, NCH2); 2.42 (br s, 4H,
CH2NCH2); 1.50 (br s, 4H, CH2CH2NCH2CH2); 1.38
(br s, 2H, NCH2CH2CH2).
5.1.22. 6-Hydroxy-2-naphthaldehyde, 11a. To 10a
(0.25 g, 1.3 mmol) and NaSEt (80%, 0.45 g, 5.4 mmol)
was added anhydrous DMF (15 mL) and refluxed for
20 min. After cooling the reaction was poured into
3 N HCl (50 mL), extracted with EtOAc, washed with
water, brine, dried and concentrated. The residue was
purified by silica gel flash chromatography using a gra-
dient of hexanes to 50% ether in hexanes to give 0.13 g
5.2. ER binding
1
(59%). 200 MHz H NMR (DMSO-d6) d 10.35 (br s,
Human cDNAs encoding ERa and ERb were used as
templates to express receptor proteins in vitro. The pro-
teins were produced with rabbit reticulocyte lysates as
supplied by Promega (TNT kit) that couples transcrip-
1H, OH); 10.06 (s, 1H, CHO); 8.42 (d, 1H, ArH);
8.03 (d, 1H, ArH); 8.69 (m, 2H, ArH); 7.20 (m, 2H,
ArH).

J. M. Schmidt et al. / Bioorg. Med. Chem. 13 (2005) 1819–1828
1827
tion and translation in a single reaction. The amount
of template used in each reaction was determined
empirically and expression was monitored in parallel
reactions where [35S]methionine was incorporated into
the receptor followed by gel electrophoresis and expo-
sure to film.
using MCF-7 cells (ATCC, Manassas, VA). These cells
contain ERa and have been shown to be responsive to
treatment with E2. Briefly, MCF-7 cells were seeded in
48-well plates in IscoveÕs phenol red-free medium con-
taining 10% FBS and 10 lg/mL insulin. The next day,
the cells were starved for 72 h in medium containing
10% dextran-coated charcoal treated serum in the ab-
sence of insulin. The cells were treated with 10 nM E2
in the absence or presence of increasing concentration
of raloxifene or test compound. Cell number was deter-
mined after 96 h using the MTS assay as described by
the manufacturer (Promega Corp.).
Binding reactions were carried out in 100 lL final vol-
umes in TEG buffer (10 mM tris, pH 7.5, 1.5 mM
EDTA, 10% glycerol). Five microlitres of in vitro tran-
scribed–translated receptor was used in each binding
reaction in the presence of 0.5 nM [3H]estradiol (E2).
All compounds were routinely tested from 10ꢀ11 to
10ꢀ6 M and diluted in ethanol. The reactions were incu-
bated at 4 °C overnight and bound E2 was quantified by
adding 200 lL dextran-coated charcoal. After a 15 min
rotation at 4 °C, the tubes were centrifuged for 10 min
and 150 lL of the supernatant was added to 5 mL scin-
tillation cocktail for determination of cpms by liquid
scintillation counting.
References and notes
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´
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Typically cells are seeded in 12-well plates and allowed
to grow until 60–80% confluence. Transfection of
DNA into cells is carried out using a liposome-based
delivery system from Qiagen, Polfect reagent. During
transfection experiments, the cells were maintained in
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was set to 100%.
5.4. Inhibition of MCF-7 cells
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