Estrogenic triarylethylene acetic acids: Effect of structural variation on estrogen receptor affinity and estrogenic potency and efficacy in MCF-7 cells

Article abstract of DOI:10.1021/jm960517e

Triarylethylenecarboxylic acids exemplified by (E,Z)-2-{4-[1-(p- hydroxyphenyl)-2-phenyl]-1-butenyl}phenoxyacetic acid (8) are a new class of estrogen receptor (ER) ligands capable of tissue selective estrogen agonist and antagonist effects. We report the syntheses of 8 and of analogues incorporating structural features known or anticipated to facilitate ER affinity in triarylethylenes. These studies revealed that the p- hydroxyphenyl moiety, ethylenic bond, and ether oxygen of 8 were all critical for high ER affinity. Although a 1,1-bisphenolic analogue bearing the p- (oxyacetic acid) moiety on its 2-phenyl ring, 12, had low ER affinity, it exhibited estrogenic potency approaching that of 8 in MCF-7 cells. Unlike 8 which was a partial agonist with weak antagonist potency, 12 was a full agonist. A similar profile of potency/efficacy in MCF-7 cells was seen in 9, an ethylenic bond saturated analogue of 8. Growth-promoting effects of 8, 9, and 12 were fully antagonized by the antiestrogen tamoxifen, suggesting that such effects were mediated solely via ER. Thus, our studies in MCF-7 cells have confirmed the estrogenicity of 8 and have enabled identification of two analogues with favorable estrogenic potency and full estrogen efficacy. On this basis, these three (triarylethylene)acetic acids have been selected for more intensive animal studies of their extrareproductive tract estrogenic effects.

Full text of DOI:10.1021/jm960517e

J . Med. Chem. 1996, 39, 4853-4859
4853
Estr ogen ic Tr ia r yleth ylen e Acetic Acid s: Effect of Str u ctu r a l Va r ia tion on
Estr ogen Recep tor Affin ity a n d Estr ogen ic P oten cy a n d Effica cy in MCF -7 Cells
Peter C. Ruenitz,* Caryl S. Bourne, Kelly J . Sullivan, and Susan A. Moore
College of Pharmacy, University of Georgia, Athens, Georgia 30602-2352
Received J uly 17, 1996X
Triarylethylenecarboxylic acids exemplified by (E,Z)-2-{4-[1-(p-hydroxyphenyl)-2-phenyl]-1-
butenyl}phenoxyacetic acid (8) are a new class of estrogen receptor (ER) ligands capable of
tissue selective estrogen agonist and antagonist effects. We report the syntheses of 8 and of
analogues incorporating structural features known or anticipated to facilitate ER affinity in
triarylethylenes. These studies revealed that the p-hydroxyphenyl moiety, ethylenic bond, and
ether oxygen of 8 were all critical for high ER affinity. Although a 1,1-bisphenolic analogue
bearing the p-(oxyacetic acid) moiety on its 2-phenyl ring, 12, had low ER affinity, it exhibited
estrogenic potency approaching that of 8 in MCF-7 cells. Unlike 8 which was a partial agonist
with weak antagonist potency, 12 was a full agonist. A similar profile of potency/efficacy in
MCF-7 cells was seen in 9, an ethylenic bond saturated analogue of 8. Growth-promoting
effects of 8, 9, and 12 were fully antagonized by the antiestrogen tamoxifen, suggesting that
such effects were mediated solely via ER. Thus, our studies in MCF-7 cells have confirmed
the estrogenicity of 8 and have enabled identification of two analogues with favorable estrogenic
potency and full estrogen efficacy. On this basis, these three (triarylethylene)acetic acids have
been selected for more intensive animal studies of their extrareproductive tract estrogenic
effects.
Effects of estrogens have conventionally been associ-
ated with the female reproductive organs (uterus,
ovary), and are mediated primarily if not solely through
estrogen receptors (ER). But estrogens have direct
effects on other tissues as well. The number of tissues
shown to contain significant levels of ER is growing due
to use of more sensitive detection methods. Thus, ER
is now known to be present in specialized cells of the
skeletal1 and cardiovascular systems.2 The molecular
basis for estrogen replacement therapy involves interac-
tions of ligands with ER in extrareproductive as well
as reproductive tissues.3
cells have also been used to evaluate estrogenic (growth
stimulatory) potency and efficacy of ER ligands.4a,6
Accordingly, 1a,b and estradiol had half-maximal growth
stimulatory potencies in MCF-7 cells of 0.19, 0.06, and
0.02 nM, respectively, and estrogenic efficacies of 1a ,b
were in turn 94% and 62% that of estradiol.4a Other
lines of ER positive cancer cells have been used in
bioassays of ER ligands.7 Estrogenicity in such cells is
due to interaction of liganded ER with composite
estrogen response elements (ERE) in DNA, rather than
with only classical palindromic ERE as appears to be
the case in ER-transfected cell lines.8
Being flexible regarding binding of ligands, ER ac-
commodates not only estradiol and other steroidal
compounds but also a diverse array of aromatic non-
steroidal structural types, exemplified by mono- and
dihydroxylated triarylethylenes 1.4 As examples, 1a -d
had respective ER relative binding affinities (RBA
values) of 38, 55, 95, and 160% (estradiol ) 100%) in
rat uterine cytosol.
Formal etherification of 1d with a single acetic acid
moiety afforded a 1:1 mixture of isomeric (hydroxytri-
arylethylene)oxyacetic acids which had an ER RBA 20%
that of estradiol and exhibited half-maximal estrogenic
potency at a concentration of 35 nM in MCF-7 cells.9
As a major metabolite of the antiestrogen tamoxifen in
the female rat, this oxyacetic acid was not detected in
reproductive tissues, unlike the parent drug and its
basic metabolites.10 On parenteral administration to
the ovariectomized rat, it exhibited an effect on trabe-
cular bone maintenance that was qualitatively similar
to that of estradiol. However it had no observable
uterotrophic effect, a finding consistent with results of
the biotransformation studies but which could also
signify a differential lack of reproductive tract estroge-
nicity. Agents displaying such selective estrogenicity
are of interest due to their potential for reduced
reproductive tract toxicity compared with conventional
estrogens.11
Cultured human and animal cells naturally endowed
with ER have been used extensively in characterizing
effects of steroidal and nonsteroidal ER ligands. In
particular, MCF-7 cells, a clonally durable line derived
from a human breast cancer, have been well established
with regard to assessment of growth suppressive effects
of steroidal and nonsteroidal antiestrogens.5 But MCF-7
An immediate aim of structural studies was to
compare, in MCF-7 cells, the effects of this oxyacetic
acid derivative of 1d with analogues modified structur-
ally in ways consistent with preserving ER affinity. The
goals were to identify structural features critical for ER
affinity and potency and to provide a basis for selection
X Abstract published in Advance ACS Abstracts, October 15, 1996.
S0022-2623(96)00517-1 CCC: $12.00 © 1996 American Chemical Society

4854 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Ruenitz et al.
Sch em e 1. Synthesis of Hydroxytriarylethylene 8 and Hydroxytriarylethane 9a
a
Reagents: (a) Mg, THF; (b) (C5H4N)2Cr2O7, acetone; (c) C6H5CH2MgCl, Et2O; (d) HCl, EtOH; (e) Ti, THF; (f) BrCH2COOC2H5, K2CO3,
acetone, then NaOH, aqueous dioxane; (g) H2, 10% Pd/C, THF (*MeOH); (h) NaCN, DMF.
Sch em e 2. Synthesis of Bisphenolic Oxyacetic Acid 12a
a
Reagents e, f, and g are specified in Scheme 1.
of compounds for in vivo studies. We also wished to
establish the degree to which effects on MCF-7 cells
were mediated by ER in these structural types.
use of the â-bromoethyl protecting group14 in the reac-
tion sequence leading to 8 (Scheme 1, h).
Deoxy analogue 17 was prepared by Willgerodt-
Kindler rearrangement of protected aryl methyl ketone
16 (Scheme 3, k),15 and this intermediate was in turn
prepared by acetylation of the aryllithium produced by
reaction of 14b tetrahydropyran-2-yl ether with n-
butyllithium.16
Resu lts
Syn th esis. Schemes 1-3 illustrate the approaches
used to prepare the aforementioned oxyacetic acid 8, as
well as 9, 12, 15, and 17.12 A central element in our
synthetic strategy was application of the McMurry
reaction in reductive cross-coupling of substituted ben-
zophenones with propiophenone or hydroxypropiophe-
none.13 The intermediate monophenols 6a ,b, 11, and
14b were readily separated from products of self-
coupling by column chromatography.
Catalytic debenzylation of 7a sometimes proceeded
slowly in tetrahydrofuran. Use of methanol as solvent
increased the reaction rate, but resulted in ethylenic
bond reduction as well, an observation applied to the
preparation of 9 from 7c. These observations prompted
In ter a ction of 8 a n d An a logu es w ith ER. Inter-
action of “target” compounds with cell-free rat ER as
well as intracellular human ER was determined, in
turn, in rat uterine cytosol at 0 °C as previously
described17 and in MCF-7 cells as described below. As
derived from Table 1, ER affinities of analogues of 8
were 0.2-2% that of 8 itself for rat ER and were 0.9-
36% that of 8 for human ER.
Effects of 8 a n d An a logu es on MCF -7 Cell P r o-
lifer a tion . Saturated analogue 9 and bisphenol 12
each exhibited greater proliferative potencies (Table 2)

Estrogenic Triarylethylene Acetic Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4855
Sch em e 3. Synthesis of 15 and 17a
a
Reagents d, e, and f: Scheme 1; (i) BBr3, CHCl3; (j) DHP, p-TsOH; n-BuLi in Et2O-hexanes; CH3CON(CH3)2; (k) S, morpholine, then
H2SO4.
Ta ble 1. Affinity of (Triarylethylene)acetic Acids for Rat and
Human ER
to that of estradiol, presumably due to inability of these
analogues to antagonize estradiol.
IC50, nMa(RBA)
Discu ssion
compd
Rat uterine cytosol
MCF-7 cells
Our studies suggest the ER affinity of 8 is dependent
on several structural characteristics in addition to the
hydroxytriarylethylene nucleus. Removal of the ether
oxygen as in 17, repositioning of the oxyacetic acid
moiety as in 12, or saturation of the ethylenic bond as
in 9 resulted in significant reductions in ER affinity
(Table 1). These findings underscore the contribution
each of these features makes to ER affinity.
8
9
12
9 (20)
900 (0.20)
750 (0.24)
4500 (0.04)
1060 (0.17)
450 (0.40)
1.8 (100)
21 (1.54)
660 (0.05)
2390 (0.02)
320 (0.10)
97 (0.33)
15 (Y ) H)
15
17
estradiol
58 (0.56)
0.32 (100)
a
The concentration required to displace by 50% specifically
bound [3H]estradiol in rat uterine cytosol or MCF-7 cells.
The ER affinity of bisphenol 12 was much lower than
expected based on its structural similarity to high-
affinity ligand 1d 4b and to 18, which had a RBA of 173
for rat ER.18 Because the bulky, lipophilic iodine
substituent of 18 clearly did not impede affinity, polarity
of the oxyacetic acid side chain of 12, rather than its
bulk, might account for low affinity. Using TLC Rf
values as a measure of comparative lipophilicity, the Rf
value of 12 was one-fourth that of 8, which had much
higher ER affinity.
Ta ble 2. Stimulatory and Inhibitory Effects of
(Triarylethylene)acetic Acids on MCF-7 Cellsa
compd
EC50, nMb (effc)
IC30, µMd (effe)
8
9
12
5.3 ( 0.3 (79)f
16 ( 10 (102)f
46 ( 15 (100)f
300 ( 50 (67)
350 ( 50 (61)
42 ( 10 (83)f
30 ( 5 (22)g
h
h
15 (Y ) H)
15
17
estradiol
20 ( 5 (22)g
13 ( 5 (26)i
2.5 ( 0.5 (51)i
0.7 ( 0.3 pM (100)
Despite relatively low MCF-7 cell ER affinities, 12
and triarylethane 9 were full estrogens with potencies
approaching that of 8. In general, ER RBA values in
substituted triarylethylenes have been shown to cor-
relate well with MCF-7 cell growth stimulating
potencies.4a Regardless of the underlying mechanism-
(s) accounting for our observations, these results clearly
illustrate the need to examine pharmacodynamic prop-
erties of substances displaying “unpromising” ER af-
finities.
Estrogenic potency of these compounds was clearly
enhanced by the presence of a 4-hydroxy group. Thus
the EC50 values (Table 2) of 15 and its desmethyl
analogue were about one order of magnitude higher
than those of the phenolic analogues.
The dominant estrogen-mimicking effect of (aryloxy)-
acetic acid 8 is in contrast to that of arylacrylic acid 19,
which exhibited lower estrogen efficacy than the partial
agonist tamoxifen in Ishikawa cells and in immature
rat uterus.19 These results infer that the lack of
uterotrophic effects of 8, referred in the introduction,
are not accounted for by low intrinsic activity. Gener-
ally, effects of ER ligands in estrogen responsive cells
and in the immature rat (uterus) are at least qualita-
tively comparable.
a
Values are expressed in terms of effects of compounds on
observed cell growth rates. Each value is the average ( standard
b
deviation for 3-6 separate experiments. The concentration
required for half-maximal growth stimulation. c Maximal growth-
d
stimulatory effect, as a percent of that of estradiol. The concen-
tration required for 30% maximal inhibition of growth seen in the
presence of 10 pM estradiol. e The percent by which the prolifera-
tive effect of 10 pM estradiol was maximally inhibited at 10 µM
of the test compound. f This effect was diminished or eliminated
when 1 µM tamoxifen was present. g This effect was not seen when
h
0.1 µM estradiol was present. No inhibitory effect was seen.
i
This effect was partially prevented when 0.1 µM estradiol was
present.
than expected based on their ER affinities. Further-
more, their proliferative effects were completely pre-
vented in the presence of 1 µM tamoxifen (data not
shown). Although not evident from the data in Table
2, all compounds exhibited concentration-dependent
growth stimulatory effects which were maximal at 1-10
µM concentrations.
Estradiol-stimulated MCF-7 cell proliferation was not
inhibited by 9 or 12, but weak inhibition was seen with
the other compounds. This inhibition was at least
partly reversible when increased concentrations of es-
tradiol were present. Efficacy of 9 and 12 was similar

4856 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Ruenitz et al.
was prepared by stirring a mixture of 1.73 g (10 mmol) of
p-bromophenol with 10 mL of dihydropyran to which was
added a small crystal of p-toluenesulfonic acid. After 1 h, 50
mL of ether was added, and the solution was washed with two
20 mL portions of 5% aqueous NaOH and then 20 mL of water.
After 25 µL of triethylamine was added, workup gave 2.5 g
(97%) of a colorless syrup which crystallized on storage at 8
°C. Dibenzyl ether 10 was prepared by dropwise addition of
2.77 g (22 mmol) of benzyl chloride to an ice-cold stirred
solution of 2.14 g (10 mmol) of 4,4′-dihydroxybenzophenone
in 45 mL of dimethylformamide which initially contained 0.52
g (22 mmol) of NaH. The mixture was heated for 3.5 h at 50
°C, then cooled, and poured into 200 mL of ice-water. The
resulting solid was collected and mixed with 50 mL of 5%
NaOH. The mixture was extracted with four 60 mL portions
of chloroform. Workup of the combined extracts gave 4.25 g
(100%) of 10 as a yellow powder: TLC (solvent 2) one spot, Rf
0.81. p-Methoxy-p′-methylbenzophenone (13a ) was prepared
by addition, in portions, of 2.67 g (20 mmol of aluminum
chloride to an ice-cooled solution of 3.1 g (20 mmol) of p-toluoyl
chloride in 5 mL of anisole over a period of 5-10 min with
vigorous swirling. The orange solution was allowed to warm
to room temperature. After 45 min, 10 mL of ice and water
was added. The mixture was extracted with 20 mL of benzene.
The organic phase was washed with 20 mL each of dilute
aqueous HCl and water. Workup gave a colorless oil which
was dissolved in 5 mL of benzene. Addition of 25 mL of
petroleum ether resulted in immediate formation of white
crystals which were collected and washed with cold petroleum
ether: 3.1 g (68%); IR (CCl4) 2870 (O-CH stretch), 1660 cm-1
(CdO). Similarly, p-bromo-p′-methoxybenzophenone was pre-
pared by reaction of p-bromobenzoyl chloride with anisole; the
product (14.2 mmol) was O-demethylated by treatment with
57 mmol of aluminum chloride in 100 mL of refluxing benzene
for 8 h. The reaction mixture was poured into 100 mL of ice/
water, and the product was extracted by addition of 40 mL of
ether and worked up. The phenolic benzophenone 13b crys-
tallized from benzene: TLC (solvent 2) one spot, Rf 0.52; IR
(KBr) 3300 (broad, OH stretch) 1640 cm-1 (CdO).
p-[(Tetr ah ydr opyr an -2-yl)oxy]-p′-(ben zyloxy)ben zoph e-
n on e (4a ). A small (0.5-1 mL) portion of a solution of 5 g
(19.6 mmol) of 3 in 17 mL of tetrahydrofuran was added to
0.54 g (22.5 mmol) of magnesium turnings, to which a few
drops of dibromoethane and an iodine crystal had been added.
After ca. 1 h of intermittent stirring, a mild exothermic
reaction started. The remainder of the solution of 3 was added
dropwise to the stirred mixture at a rate so as to maintain
reaction momentum. Then the mixture was heated at reflux
for 1 h. The clear gray solution was cooled to 0 °C, and a
solution of 3.74 g (17.6 mmol) of 2a in 15 mL of tetrahydro-
furan was added dropwise. The mixture was refluxed for 2 h.
The reaction solution was cooled in ice, and 4.0 mL of 30%
NH4Cl (9.2 mmol) was added slowly. The supernatant was
decanted from precipitated salts and was concentrated in
vacuo to give a yellow syrup which solidified on storage at 8
°C. This was mixed with petroleum ether, filtered, and washed
with this solvent to give 6.91 g (100%) of a light orange
powder: TLC (solvent 1) one spot, Rf 0.14. This intermediate
(17.8 mmol) was dissolved in 70 mL of methylene chloride,
and 5.13 g (13.6 mmol) of pyridinium dichromate was added.
The mixture was stirred for 32.5 h. The mixture was concen-
trated in vacuo, and the residue was extracted with four 30
mL portions of ether. The combined extracts were filtered and
concentrated in vacuo to give 6.81 g (99%) of 4a as a gold oil:
IR (neat, NaCl) 1650 cm-1 (CdO).
The lack of proliferative effects of the phenolic tri-
arylethylenes in the presence of “excess” concentrations
of the nonsteroidal antiestrogen tamoxifen suggests that
these proliferative effects are mediated solely by ER.
Tamoxifen (1 µM) similarly antagonized fully the growth-
promoting effect of 1 pM estradiol.
In conclusion, our studies indicate that incorporation
of a polar (oxy)acetic acid moiety into these structural
types results in ER ligands expressing MCF-7 cell
estrogenicity accompanied by little or no estrogen
antagonism, unlike triarylethylenes substituted with
basic or neutral polar side chains, which tend to exhibit
antiestrogenicity in such cells. Extrapolation of these
results to possible therapeutic applications of (triaryl-
ethylene)acetic acids is not justified at this time, because
effects of compounds on MCF-7 cell growth are not
always consistent with effects seen in animals or
humans. Thus, genistein, a flavone with ER affinity
and MCF-7 cell growth stimulatory potency/efficacy
similar to that of 8 and its analogues, is considered to
be a cancer preventative agent.20 In contrast, N-[(p-
benzylphenoxy)ethyl]diethylamine was an inhibitor of
MCF-7 cell growth, but was a cancer-promoting agent
in the rat.21 Therefore, our results require confirmation
using in vivo models of extrareproductive tract estro-
genicity.
Exp er im en ta l Section
1
Infrared (IR) and 400 MHz H NMR spectra were recorded
in turn on Nicollet 510P FT-IR and Bruker AMX 400 spec-
trometers. NMR chemical shifts (δ) were determined using
tetramethylsilane as standard. Mass spectra were obtained
using a Perkin-Elmer Sciex API 1plus mass spectrometer.
Melting points were determined using an Electrothermal 9100
apparatus. Reaction progress, column chromatographic frac-
tions, and purity of products were analyzed qualitatively by
analytical TLC using 0.25 mm Analtech silica gel GF254
plates. Plates were developed with solvent 1 [benzene-
chloroform (50/50, v/v)], solvent 2 [chloroform-methanol-28%
aqueous ammonia (90/10/0.5, v/v)], or solvent 3 [chloroform-
2-propanol-glacial acetic acid (90/10/0.5, v/v)]. Developed
plates were viewed under light of 254 nm wavelength. Reac-
tions involving air-sensitive reagents were run under dry
nitrogen gas. Chromatographic mobilities of compounds are
expressed as Rf values. Reaction mixture solutions in ether
were generally worked up by removal of excess water with
anhydrous sodium sulfate, followed by filtration and concen-
tration in vacuo. In some cases redissolution of product
residues in benzene followed by reconcentration in vacuo was
carried out to remove residual water.
Sta r tin g Ma ter ia ls. Benzyl ether 2a , propiophenone, and
4-hydroxypropiophenone were obtained from Aldrich Chemical
Co. â-Bromoethyl ether 2b was prepared by refluxing a stirred
mixture of 5 g (41 mmol) of p-hydroxybenzaldehyde, 50 mL of
10% aqueous NaOH, 1 g (2.9 mmol) of tetra-n-butylammonium
hydrogen sulfate, and 8.5 mL (18.5 g, 98 mmol) of 1,2-
dibromoethane for 2 h. After cooling, the mixture was
extracted with two 60 mL portions of ether. The combined
extracts were washed with 50 mL of water and worked up to
give 3.1 g (40%) of 2b. Tetrahydropyran-2-yl (THP) ether 3
For conversion to p-(benzyloxy)-p′-hydroxybenzophenone
(5a ), 4a was dissolved in 30 mL of acetone. The solution was
cooled in ice, and 3 mL of 10% HCl was added dropwise. After
1 h at room temperature, the reaction solution was concen-
trated in vacuo and the residue was shaken with 100 mL of
ether and 50 mL of 1% aqueous HCl. The organic layer was
washed with 50 mL of water and worked up routinely after
addition of 10 mL of benzene. The residue was stirred for 2 h
with 25 mL of 10% ethyl acetate in hexanes. The resulting
orange solid was filtered and washed with this solvent to give
4.03 g (75%) of an orange powder: TLC (solvent 2) one spot

Estrogenic Triarylethylene Acetic Acids
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4857
Rf 0.60. A small sample was crystallized from ethyl acetate-
hexanes as shiny yellow plates: mp 136-137.5 °C. Anal.
(C20H16O3) C, H.
O-Dem eth yla tion of 14a . A stirred solution of 1.5 g (4.5
mmol) of 14a in 30 mL of methylene chloride was cooled in
ice-water, and a solution of 3.4 g (13.6 mmol) of boron
tribromide in 3.4 mL of methylene chloride was added drop-
wise. The mixture was allowed to warm to room temperature,
and after 3.5 h, a mixture of 5 mL of 28% aqueous ammonia
and 5 g of ice was added. The mixture was shaken with 35
mL of ether, and the aqueous layer was discarded. The organic
extract was washed with two 15 mL portions of water and
worked up. The residue was crystallized from petroleum ether
to give two crops of white crystals, 1.03 g (73%): IR (KBr) 3610
cm-1 (s, OH).
Gen er a l Meth od for Con ver sion of (Tr ia r yleth ylen e)-
m on op h en ols to Oxya cetic Acid s. The synthesis of 12
dibenzyl ether is typical. To a solution of 1.14 g (2.22 mmol)
of 11 in 25 mL of acetone was added 1.86 g (1.25 mL, 11.13
mmol) of ethyl bromoacetate and 0.78 g (5.56 mmol) of
potassium carbonate. The mixture was stirred and refluxed
for 6 h, after which time TLC (solvent 1) indicated a major
component, Rf 0.56, and the absence of 11. The cooled mixture
was filtered and concentrated. The resulting yellow syrup was
dissolved in 19 mL of dioxane, and 10 mL of 5% NaOH was
added. After 0.5 h, the solution was cooled in ice and 7 mL of
10% HCl was added. The resulting suspension was extracted
with three 40 mL portions of ether. Workup left a white solid.
In order to remove residual bromoacetic acid, this was mixed
with 10 mL of acetone and then 20 mL of water was added.
The mixture was filtered and washed with cold acetone-water
(10/20, v/v). Drying (60 °C, 0.05 mmHg, 4 h) gave 1.03 g (81%)
of halogen-free white powder: TLC (solvent 3) one spot, Rf
0.51.
Application of these methods gave benzophenone 5b, mp
135-139 °C (lit.14 mp 139-142 °C) in 81% yield.
Gen er a l Meth od for Olefin a tion of 5, 10, a n d 13. The
synthesis of 1-[(4-(benzyloxy)phenyl]-1-(4-hydroxyphenyl)-2-
phenylbut-1-ene (6a ) is typical. To a cold (-15 °C), well-stirred
suspension of 5.07 g (79 mmol) of zinc powder in 60 mL of
tetrahydrofuran was added slowly by syringe 4.4 mL (39 mmol)
of titanium tetrachloride. The mixture was heated at reflux
for 2 h and then cooled to ca. 40 °C. A solution of 4 g (13.2
mmol) of 5a and 1.77 g (13.2 mmol) of propiophenone in 24
mL of tetrahydrofuran was added dropwise via syringe to the
stirred suspension. The mixture was refluxed for 4 h, then
cooled, and poured into a solution of 7 g of potassium carbonate
in 70 mL of water. After standing overnight, the mixture was
filtered and the collected precipitate was washed with tet-
rahydrofuran. The combined filtrate and washings were
concentrated in vacuo to give a mixture of oil and water. This
was shaken with 50 mL of benzene. The organic layer was
dried and concentrated in vacuo to give 8.05 g of a gold oil.
This was chromatographed on 50 g of silica gel using benzene
as eluting solvent. The first 170 mL of eluate was discarded.
The next 240 mL was collected and concentrated to give 4.4 g
(82%) of a gold oil which solidified on standing at 8 °C.
A
portion of this was crystallized from petroleum ether, giving
1
6a : mp 107.5-111 °C; H NMR (acetone-d6) δ 0.91 (t, J ) 7
Hz, 3, CH3), 2.48 (q, J ) 7 Hz, 2, CH2), 4.97 and 5.14 (s, 2,
OCH2Ar), 6.48-7.04 (m, 18, ArH). Anal. (C29H26O2) C, H.
By this procedure the following triarylethylenes were pre-
pared. â-Bromoethyl ether 6b (40%): TLC (solvent 1) one spot,
Rf 0.30; 1H NMR (acetone-d6) δ 0.92 (t, J ) 7 Hz, 3, CH3), 2.51
(q, J ) 7 Hz, 2, CH2), 3.78 and 3.80 (t, J ) 6 Hz, 2, CH2Br),
4.22 and 4.41 (t, J ) 6 Hz, 2, OCH2), 6.50-7.22 (m, 13, ArH).
Dibenzyl ether 11 (42%): mp 146-148 °C; TLC (solvent 1)
one spot, Rf 0.21; 1H NMR (CDCl3) δ 0.95 (t, J ) 7 Hz, 3, CH3),
2.42 (q, J ) 7 Hz, 2, CH2), 4.63 (s, 1, OH), 4.94 and 5.09 (s, 2
each, CH2Ar), 6.59-7.44 (m, 22, ArH). Anal. (C36H32O3) C,
H. [Note: addition of the starting ketones as a solution
required 50 mL of warm (50 °C) tetrahydrofuran.]
Methyl ether 14a crystallized on standing, and was recrys-
tallized from ethanol in 55% yield, thus obviating chroma-
tography: TLC (solvent 1) one spot, Rf 0.67; mp 94.5-95.5 °C
(sublimed at 90 °C, 0.05 mmHg); 1H NMR (CDCl3) δ 0.92 (t, J
) 7.6 Hz, 3, CH2CH3), 2.37 (s, 3, ArCH3), 2.47 (q, J ) 7.6 Hz,
2, CH2CH3), 3.67 (s, 3, OCH3), 6.54 (d, J ) 8.7 Hz, 2, ArH ortho
to -O-), 6.78 (d, J ) 8.7 Hz, 2, ArH meta to -O-), 7.09-
7.16 (m, 9, remaining ArH). Anal. (C24H24O) H; C: calcd,
87.76; found, 86.92.
By this procedure the following (triarylethylene)oxyacetic
acids were prepared. Benzyl ether 7a crystallized from
chloroform-hexanes in 48% yield: mp 115-117 °C; TLC
(solvent 3) one spot, Rf 0.42; 1H NMR (acetone-d6) δ 0.90 (t, J
) 7.3 Hz, 3, CCH3), 2.48 (q, J ) 7.3 Hz, 2, CH2), 4.55 and 4.72
(s, 1.06 and 0.94, OCH2CdO), 4.96 and 5.13 (s, 0.96 and 1.04,
OCH2Ar), 6.60-7.40 (m, 18, ArH). Its desethyl counterpart
7c was obtained as white crystals from chloroform (25%): mp
143-147 °C; TLC (solvent 3) two components of equal inten-
1
sity, Rf 0.38 and 0.44; H NMR (acetone-d6) δ 4.57 and 4.73
(s, ca. 1 each, OCH2CdO), 4.93 and 5.13 (s, ca. 1 each, OCH2-
Ar), 6.55-7.38 (m, 18, ArH). â-Bromoethyl ether 7b (75%):
TLC (solvent 3) one spot, Rf 0.48. p-Methyl analogue 15 was
prepared from O-desmethyl 14a in 94% yield after crystal-
lization from acetone-water: mp 150-151 °C; 1H NMR (acetone-
d6) δ 0.93 (t, J ) 7.3 Hz, 3, CCH3), 2.15 and 2.34 (s, 1.5 each,
ArCH3), 2.44 (q, J ) 7.3 Hz, 2, CH2), 4.58 and 4.64 (s, 1 each,
OCH2CdO), 6.98-7.71 (m, 13, ArH). Anal. (C25H24O3‚0.25H2O)
C, H.
Ca ta lytic Hyd r ogen olysis of 7a ,c a n d 11-Ben zyl Eth er .
To a solution of 1.15 g (2.48 mmol) of 7a in 20 mL of
tetrahydrofuran was added 155 mg of 10% palladium on
carbon. The mixture was shaken for 11.5 h under 44 psi of
H2. TLC (solvent 3) suggested the reaction to contain 90-
95% 8 (Rf 0.16) accompanied by 5-10% of 7a . The mixture
was filtered, and the collected catalyst was washed well with
tetrahydrofuran. The combined filtrate and washings were
concentrated in vacuo to give 1.14 g of a light yellow oil which
solidified on storage at 8 °C. This was stirred with 10 mL of
dry CHCl3-hexanes (50/50) at room temperature for 1 h. The
suspended product was filtered and washed with two 5 mL
portions of the above solvent to give 676 mg (73%) of 8 as a
white powder, after drying at 60 °C (0.05 mmHg) for 7 h: TLC
(solvent 3) one spot, Rf 0.16; mp 215-219 °C; 1H NMR
(acetone-d6) δ 0.92 (t, J ) 7 Hz, 3, CCH3), 2.49 (q, J ) 7 Hz, 2,
CH2), 4.47 and 4.74 (s, ca. 1 each, OCH2CdO), 6.51-7.11 (m,
13, ArH); EIMS m/ z (rel intensity) 374 (M, 52), 315 (14, M -
CH2COOH), 44 (100). Anal. (C24H22O4‚1.5H2O) C, H.
By this same procedure was prepared bisphenol oxyacetic
acid 12 from 1.03 g (1.81 mmol) of its dibenzyl ether, using 1
mL of acetic acid in 80 mL of tetrahydrofuran as solvent and
590 mg of 10% palladium on carbon. The crude product, from
which residual solvent was removed by lyophilization, was
crystallized from chloroform: 440 mg (63%) of beige crystals:
mp 82-85 °C; TLC (solvent 3) one spot, Rf 0.04; 1H NMR
Bromophenol 14b crystallized from ethanol in 70% yield
after chromatography: TLC (solvent 1) one spot, Rf 0.24; H
1
NMR (CDCl3) δ 0.91 and 0.93 (t, J ) 7.4 Hz, 3, CCH3), 2.45
and 2.49 (q, J ) 7.4 Hz, 2, CH2), 4.59 and 4.82 (s, 1, OH), 6.47
and 6.70 (d, J ) 8.5 Hz, ca. 2, ortho and meta ArH to OH in
E-isomer), 6.73 and 6.81 (d, J ) 8.4 Hz, ca. 2, ArH ortho and
meta to OH in Z-isomer), 7.47 (d, J ) 8.2 Hz, 2, ArH ortho to
Br in E-isomer), 7.07-7.18 (m, 7, remaining ArH).
P r ep a r a tion of 6c. Reaction of 4.62 g (12 mmol) of 4a with
a 1.5 molar excess of benzylmagnesium chloride was carried
out by a standard procedure.5c The resulting carbinol was
dissolved in 15 mL of ethanol, and 2.5 mL of 5% HCl was
added. After 1.5 h, 20 mL of ether and 3 mL of 10% aqueous
sodium carbonate was added, and the mixture was worked up,
affording 5.45 g of a brown oil. This was chromatographed
on 38 g of 60-200 mesh silica gel, with benzene as eluent.
The first 125 mL of eluate was discarded. The next 170 mL
was collected and concentrated in vacuo to give 5.04 g (112%)
of 6c as a light yellow oil which solidified on storage at 8 °C.
A 200 mg sample of this was further purified by washing with
two 5 mL portions of hexanes, followed by trituration with 5
mL of methanol. The precipitate was collected and washed
with cold methanol to give 57 mg of 6c as a white powder:
TLC (serial development two times with solvent 1) one spot,
Rf 0.39; mp 129-132 °C. Anal. (C27H22O2) C, H.

4858 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Ruenitz et al.
(acetone-d6) δ 0.90 (t, J ) 7 Hz, 3, CCH3), 2.42 (q, J ) 7 Hz, 2,
CCH2), 4.62 (s, 2, OCH2), 6.49 (d, J ) 8 Hz, 2, ArH ortho to
one phenolic OH), 6.6-6.9 (m, 8, unassigned ArH), 7.07 (d, J
) 7.5 Hz, 2, meta ArH in Ar-O-C); EIMS m/ z (rel intensity)
390 (M, 100), 331 (7, M - CH2COOH), 44 (41). Anal.
(C24H22O5‚0.25H2O) C, H.
organic phase was washed with 15 mL of water and then
shaken with 20 mL of 10% aqueous sodium carbonate. The
aqueous extract was washed with 20 mL of ether, acidified
with 10% HCl, and extracted twice with 25 mL portions of
ether. Workup of the combined extracts gave 95 mg (19%) of
a brown gum. Storage over benzene for several weeks resulted
in crystallization of a tan solid, which was filtered and washed
with cold benzene to give 28 mg of tan crystals: mp 189.5-
192 °C (uncharacterized transition at 160 °C); TLC (solvent
3) one component, Rf 0.40; 1H NMR (acetone-d6) δ 0.91 (t, J )
7 Hz, 3, CH3), 2.46 and 2.48 (q, J ) 7 Hz, 2, allylic CH2), 3.46
and 3.66 (s, 1 each, ArCH2-CO-), 6.50-7.35 (13, ArH); EIMS
m/ z (rel intensity) 358 (M, 14), 299 (4, M - CH2COOH), 44
(100). Anal.(C24H22O3‚0.25 H2O) C, H.
Hyd r ogen olysis/Hyd r ogen a tion of 7c. A solution of 1.46
g (3.34 mmol) of 7c in 100 mL of methanol was shaken with
0.16 g of 10% palladium on carbon for 2 h under 44 psi of H2.
The mixture was filtered. The filtrate was concentrated in
vacuo, and the residue was dissolved in 10 mL of dioxane. The
solution was diluted with 4 mL of 5% NaOH. After 0.5 h, the
mixture was cooled in ice, acidified with 4 mL of 10% HCl,
and extracted with 50 mL of ether. The extract was washed
with 30 mL of water. Addition of 10 mL of benzene, followed
by workup, gave 1.83 g of a gold oil. This was dissolved in 1
mL of dry chloroform, and the solution was diluted with 25
mL of hexanes. The resulting precipitate solidified on storage
at 8 °C. This was crystallized from 3 mL of alcohol-free
chloroform to give a total of 0.78 g (67%) of 9 as beige crystals
after drying at room temperature for 16 h (0.05 mmHg): mp
Estr ogen Recep tor Bin d in g Affin ity. Cell-free competi-
tive assays were conducted using aliquots (0.2 mL) of cytosol
prepared from immature rat uterus. Incubations were run in
triplicate at 0 °C for 4 h and contained 5 nM [3H]estradiol (56
Ci/mmol) and concentrations of test compound ranging from
3
1 nM to 10 µM. The extent of specific H bound was plotted
as a function of test compound concentration.17
1
143-148 °C (sublimed); TLC (solvent 3) one spot, Rf 0.23; H
Competitive binding in MCF-7 cells was determined as
previously described,22 with the following modifications. In-
cubation wells contained ca. 2 × 105 attached cells. To each
well was added 3 mL of serum-free growth medium which
contained 0.35 nM (56 nCi) of [3H]estradiol. Medium also
contained test compounds in varying concentrations, from 1
nM to 10 µM. Incubations were run in triplicate at 37 °C for
60 min. Medium and unbound ligands were removed by
aspiration. Suspensions of cell nuclei were prepared by
adaptation of methods to be described elsewhere.23 Radioac-
tivity in aliquots of lysed cells with suspended cell nuclei was
determined in Ecolite liquid scintillation fluid. Specific 3H
bound was calculated as a function of test compound concen-
tration by subtracting nonspecific binding found in incubations
containing 10 µM estradiol from total 3H bound at each test
compound concentration.
NMR (acetone-d6) δ 3.30 (d, J ) 8 Hz, 2, CH2Ph), 4.21 (t, J )
8 Hz, 1, CHAr2), 4.62 (s, 2, OCH2), 6.75 (“t”, J ) 9 Hz, 4, C6H4-
OH), 7.05-7.25 (m, 9, remaining ArH), 8.30 (s, 1, ArOH).
Anal. (C22H20O4‚0.5 H2O) C, H.
P r ep a r a tion of 8 via Cya n olysis of 7b. To a solution of
1.0 g (2.09 mmol) of 7b in 60 mL of N,N-dimethylformamide
was added 2.4 g (49 mmol) of powdered NaCN. The mixture
was stirred for 48 h. The mixture was concentrated in vacuo.
The residue was shaken with 70 mL each of ether and 5%
aqueous NaHCO3. The organic phase was discarded, and the
aqueous phase was washed with two 50 mL portions of ether.
Then it was acidified to pH <2 by addition of 10% aqueous
H2SO4. The resulting suspension was extracted with two 60
mL portions of methylene chloride. Workup gave an oily
residue which crystallized from chloroform-hexanes: 0.51 g
1
(64%). Its TLC Rf value, mp, and H NMR and mass spectra
Effects on MCF -7 Cell Gr ow th . Cells (0.5-5 × 105) were
conditioned at 37 °C in a humidified atmosphere containing
5% carbon dioxide, in 75 cm2 flasks containing 5 mL of
complete growth medium (phenol red free Dulbecco’s modified
Eagle’s medium-Ham’s nutrient mixture, 50/50) containing 5%
newborn calf serum, then passaged into 25 cm2 flasks using
low-estrogen medium.23 Then, varying concentrations (1-
10000 nM) of test compound without or with specified con-
centrations of estradiol or tamoxifen (see Table 2) were added.
[Up to nine flasks were prepared this way for each compound
concentration.] This was repeated on days 4, 7, and 9. At each
of these time intervals, cells in two or three flasks were
trypsinized and lysed to release cell nuclei. Aliquots of
suspended nuclei were counted as described.24 For sets of
identically prepared flasks, ranges of cell numbers were within
5% of averages. Data were plotted as the log of the number
of cell nuclei per flask as a function of incubation time. From
such plots, observed generation (doubling) times (g) were
found. These were used to calculate first-order growth rates
(µ) by the formula µ ) 0.693/g. Calculated growth rates were
plotted as a function of test compound concentration in order
to determine potencies (EC50, IC30) and efficacies.
were nearly identical to that of the product of debenzylation
of 7a .
Con ver sion of 14b to 16 THP eth er . The THP ether of
14b was prepared from 2.31 g (6.09 mmol) of 14b as described
for 3. This was dissolved in 25 mL of ether. The solution was
cooled to -13 °C, and to the stirred suspension was added
slowly by syringe 7.6 mL of a 1.6 M solution of n-BuLi in
hexane (12.18 mmol). After 0.5 h under these conditions, a
solution of 0.65 mL (6.5 mmol) of neat N,N-dimethylacetamide
(stored over KOH pellets) was added dropwise via syringe to
the orange solution. After being warmed to room temperature,
the light yellow reaction suspension was cooled in ice and 1.1
mL of 30% aqueous NH4Cl was added slowly. The mixture
was separated, and the upper layer was washed with water
and worked up to give 3.71 g of yellow syrup. Chromatography
on 30 g of 60-200 mesh silica gel (Baker) using benzene as
eluent gave fractions containing a single component, Rf 0.23
(solvent 1), which eluted after an unwanted component, Rf
0.46. Evaporation of solvent left 2.08 g (80%) of 16 THP ether.
Preparative TLC of 160 mg of this on two Analtech silica gel
GF254 TaperPlates gave 45 mg (28%) of 16 THP ether as a
glassy white solid: IR (CCl4) 1685 cm-1 Ar-CdO; 1H NMR
(CDCl3) δ 0.93 (t, J ) 7.4 Hz, 3, CH2CH3), 1.50-2.00 (m, 6,
aliph ring CH2), 2.44 (q, J ) 7.4 Hz, 2, CH2CH3), 2.61 (s, 3,
CH3CdO), 3.54 and 3.84 (m, 1 each, OCH2), 5.26 (t, J ) 3.2
Hz, 1, O-CH-O), 6.70 (dd, J 1 ) 6.5 Hz, J 2 ) 2.7 Hz, 2, arom
H ortho to -O-), 6.74 (dd, J 1 ) 6.5 Hz, J 2 ) 2.7 Hz, 2, arom
H meta to -O-), 7.09-7.18 (m, 5, Ph), 7.35 (d, J ) 8 Hz, 2,
arom meta to CdO), 7.95 (d, J ) 8 Hz, 2, arom ortho to CdO).
Syn th esis of 17. A mixture of 570 mg (1.33 mmol) of 16
THP ether, 64 mg (2 mmol) of sulfur, and 7 mL of morpholine
was stirred at 120 °C for 15 h. The brown mixture was mixed
with 20 mL of ether and 10 mL of water. The ether was
washed with 10 mL of 1% HCl and concentrated to give 0.54
g of yellow foam. This was dissolved in 7 mL of acetic acid,
and 0.8 mL of water and 0.64 mL of sulfuric acid were added.
The solution was heated at 120 °C for 5 h. The mixture was
shaken with 30 mL of ether and 10-15 mL of water. The
Ack n ow led gm en t. This research was supported by
Grant AR 42069 from the National Institutes of Health.
Mass spectral analyses were carried out by Dr. Dennis
R. Phillips of our Mass Spectrometry Facility. Ms.
Sullivan was the recipient of a predoctoral fellowship
from the American Foundation for Pharmaceutical
Education.
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