Carboxylic acid analogues of tamoxifen: (Z)-2-[p-(1,2-Diphenyl-1- butenyl)phenoxy]-N,N-dimethylethylamine. Estrogen receptor affinity and estrogen antagonist effects in MCF-7 cells

Article abstract of DOI:10.1021/jm990078u

The triarylethylene estrogen mimetic (E,Z)-4-[1-(p-hydroxyphenyl)-2- phenyl-1-butenyl]phenoxyacetic acid (4) represents a novel class of estrogen receptor (ER) ligands which, like tamoxifen (1), can elicit estrogen agonist and antagonist effects, in turn,

Full text of DOI:10.1021/jm990078u

3126
J . Med. Chem. 1999, 42, 3126-3133
Ca r boxylic Acid An a logu es of Ta m oxifen :
(Z)-2-[p-(1,2-Dip h en yl-1-bu ten yl)p h en oxy]-N,N-d im eth yleth yla m in e. Estr ogen
Recep tor Affin ity a n d Estr ogen An ta gon ist Effects in MCF -7 Cells
Kelly S. Kraft, Peter C. Ruenitz,* and Michael G. Bartlett
College of Pharmacy, University of Georgia, Athens, Georgia 30602-2352
Received February 16, 1999
The triarylethylene estrogen mimetic (E,Z)-4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phen-
oxyacetic acid (4) represents a novel class of estrogen receptor (ER) ligands which, like tamoxifen
(1), can elicit estrogen agonist and antagonist effects, in turn, in nonreproductive and
reproductive tissues. Analogues of 4, incorporating structural features shown previously in
triarylethylenes to improve ER affinity and estrogen antagonist properties, were prepared with
the ultimate aim of identifying substances with improved estrogenicity exclusive of reproductive
tissues. Thus, the side chain of 4 was elongated to give oxybutyric acid 13, which was further
altered by (a) repositioning of its p-hydroxyl to the neighboring m-position (12) and (b) ethylenic
bond reduction (14). Also, the p-hydroxyl group and oxyacetic acid groups of 4 were, in turn,
shifted to the neighboring m-positions, affording 8 and 9. Oxybutyric acid analogue 13 had
about 2 times the affinity for human ERR as 4, and its antiproliferative effect in MCF-7 cells
was greater than that of 1. Dihydro analogue 14, which was conformationally similar to cis-
13, had very low ER affinity and antiestrogenicity, and m-hydroxy analogue 12 also had reduced
ER affinity and potency, but its MCF-7 cell antiproliferative efficacy was retained. Modest ER
affinity and antiproliferative potency were seen with 8, in which phenolic and phenyl rings
were trans to one another, but 9, in which these rings were cis, was inactive. Our findings
indicate that two-carbon side-chain elongation and/or m-positioning of the hydroxyl group in
4 affords analogues with dominant estrogen antagonist effects in MCF-7 cells.
In tr od u ction
The triarylethylene antiestrogen tamoxifen (1) has
become widely used in management and prevention of
breast cancer.1 The mechanistic basis for this medical
application is thought primarily to involve interaction
of 1 with estrogen receptors (ER) in breast cancer cells,
resulting in antagonism of the growth-promoting effects
of endogenous estrogens. Interaction of 1 with ER in
organs of the reproductive axis also results in antago-
nism of administered estrogen, but in other tissues such
interactions can result in full estrogenic effects. Thus
1, like 17â-estradiol, was shown to prevent bone density
loss in postmenopausal women.2 These tissue-specific
estrogenic responses to 1 are believed to result from
increased intrinsic activity of 1-liganded ER in regard
to its interaction with DNA estrogen response elements
in bone and other ER-containing tissues, relative to that
in reproductive and ER-positive neoplastic cells.3 Rec-
ognition of 1 as an estrogen mimetic in tissues outside
of the reproductive system has stimulated subsequent
investigations aimed at identification of structurally
related ER ligands as tools for investigating the impact
of chemical modification on the degree of tissue-specific
estrogenicity.
(growth inhibitory) potency and efficacy of ER ligands.4
Selection of ER ligands as potential tissue-selective
estrogens has generally been based on demonstration
of estrogen antagonism in these cell types. Thus,
numerous structural variants of 1 bearing basic side
chains have been shown to inhibit estrogen-stimulated
proliferation of MCF-7 or other estrogen-responsive cell
lines and subsequently demonstrated estrogen antago-
nist effects in uterine tissues and estrogen agonist
effects in bone and liver of ovariectomized laboratory
rats.4
Cultured MCF-7 human breast cancer cells, and other
neoplastic human cells naturally endowed with ER,
have been used extensively in initial characterization
of estrogen agonist (growth stimulatory) and antagonist
Similarly, an acidic side-chain-substituted analogue
(2)antagonized 17â-estradiol in vitroand in a uterotrophic
assay in immature rats but was an estrogen mimetic
on bone maintenance.5 Its phenolic counterpart (3) also
* Correspondence to: Dr. Peter C. Ruenitz. Fax: 706-542-5358.
E-mail: pruenitz@rx.uga.edu.
10.1021/jm990078u CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/23/1999

Antiestrogenic Triarylethylene Carboxylic Acids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3127
Sch em e 1. Synthesis of Hydroxytriarylethylene
was characterized in vitro as an estrogen antagonist,
but replacement of 3’s acrylic acid side chain with an
oxyacetic acid moiety (4) resulted in dominant estrogen
agonist activity in MCF-7 cells.6 Further structural
variation of 4 revealed the importance of the phenolic
hydroxyl group, ether oxygen, and side-chain location
on ER affinity, and although potency in MCF-7 cells was
also affected by these specific structural changes, all
such variants were estrogen agonists.7 Interestingly the
dihydrodesethyl analogue of 4, despite its MCF-7 cell
estrogen mimetic effect, was a selective estrogen in the
ovariectomized rat: its bone maintenance effects were
equivalent to those of 1, but it had no uterotrophic
effect.8 These findings suggested that oxyalkanoic acids
related to 4 were capable of expressing differential
estrogenicity by a second mechanism distinct from
tissue-specific ER modulation. This mechanism was
speculated to involve ER-independent selective distribu-
tion to tissues outside the reproductive system.
Oxyacetic Acid Isomersa
a
Reagents: (a) BrCH2COOEt, K2CO3, acetone; (b) NaOH, aq
dioxane; (c) BBr3, CH2Cl2; (d) H2, 10% Pd/C, THF.
Accordingly, our attention was directed toward fur-
ther structural alteration of 4 with the aims of retaining
ER affinity and attenuating estrogenic efficacy in MCF-7
cells. This approach was envisioned first to afford
antagonists of estrogen-dependent MCF-7 cell prolifera-
tion and ultimately to result in characterization of
substances expressing more potent ER-mediated agonist
effects exclusive of the reproductive tract, due not just
to uncharacterized selective distribution processes but
also to selective ER modulation.
Several types of structural modifications of 4 ap-
peared to be applicable for maximizing antiestrogenic
potency and/or efficacy, based on relevant structure-
activity studies reported for 1 and its analogues. First,
the maximal antiproliferative and antiuterotrophic ef-
ficacies of 1 (X ) OH) observed in MCF-7 cells and in
the immature rat were exceeded by those of its m-
phenolic analogue.4b,9 Second, reduction of the ethylenic
bond in fused ring analogues of 1 resulted in improve-
ment in ER affinity and estrogen antagonist potency
and efficacy.4d And, homologation of the basic ether
linkage in analogues of 1 from two to four atoms
generally resulted in increased ER affinity and/or
estrogen antagonist potency and efficacy.10 Thus we
report the syntheses of analogues of 4 in which we
evaluated the impact of ethylenic bond reduction,
alternate m-positioning of aryl substituents (R and X),
and elongation of the oxyalkanoic acid side chain on ER
affinity and MCF-7 cell proliferation.
respective benzyl ethers of 8† and 9†. (†The specified
triarylethylene oxyacetic acid (cf. Scheme 1) was ac-
companied by a significant amount of its geometric
isomer.) Catalytic debenzylation followed by alkaline
equilibration (see below) of the second of these furnished
9, but 8† benzyl ether underwent excessive concomitant
ethylenic bond hydrogenation during debenzylation.
Thus, it was debenzylated noncatalytically.
Relative chemical shifts of side-chain (and benzyl
group) O-methylene protons in NMR spectra of 8†, 9†,
and subsequent compounds in this study enabled esti-
mation of geometric isomer ratios and assignment of
configurations.9 Accordingly, the spectrum of 8† featured
O-methylene singlets at 4.56 and 4.78 ppm, with an
intensity ratio of 3:1. These were assigned in turn to 8
and its cis isomer, based on correlation with O-meth-
ylene chemical shifts of corresponding geometric isomers
of 1.9,12 Fisher esterification of 8† with ethanol, followed
by preparative thin-layer chromatography and saponi-
fication of the more polar, major component, afforded
8. Equilibration of 9† in dilute sodium hydroxide re-
sulted in nearly complete conversion to 9. In its proton
NMR spectrum, the ratio of O-methylene singlets at
4.73 and 4.39 ppm, indicative of the respective cis and
trans isomers, was 97:3.
Alkylation of 5 and 7 with ethyl 4-bromobutyrate
afforded respective m- and p-benzyl ethers 10 and 11
(Scheme 2). Attempted isolation of the cis isomers of 10
and 11 was not successful. (Excess residual alkylating
agent, present prior to or after saponification of cis
isomer-enriched mixtures of 10 or 11, could not be
removed by preparative TLC or liquid-liquid extrac-
tion.) Also, attempted preparation of the oxypropionate
homologue of 11 by extension of the specified alkylation
method (Scheme 2) afforded only a small amount of the
putative alkylation product, accompanied by a large
amount of a component which comigrated with starting
material on thin layer chromatograms. Hydrolytic de-
Resu lts
Syn th esis a n d Ch a r a cter iza tion of 8, 9, a n d 12-
14. Reductive cross-coupling of substituted benzophe-
nones with propiophenone,11 followed by silica gel
column chromatographic separation of monophenolic
products from those due to self-coupling reactions,
afforded starting p-hydroxytriarylethylenes 5 and 7 in
high yields. However, the m-hydroxy isomer 6 prepared
in this manner contained one or more major impurities
suggested by proton NMR spectroscopy to be 1,1,2-
triarylethane derivatives.
Preparation of the trans-3-hydroxy and cis-3-oxyacetic
acid analogues of 4 is outlined in Scheme 1. Alkylation7
of 5 and 6 with ethyl bromoacetate followed by saponi-
fication of the resultant oxyacetic acid esters gave the

3128 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Ta ble 1. Affinities of 4 and Selected Analogues for Human ERR
Kraft et al.
1
4
8
9
12
13
14
17â-estradiol
5.9
100
IC50, nMa
RBAb
42
14
500
1.2
1440
>11 µM
<0.05
1700
235
2.5
6000
0.4
0.35
0.1
a
b
The concentration of test compound required to displace specifically bound [3H]17â-estradiol by 50%. The relative binding affinity
(RBA) of each compound was determined by dividing the IC50 of 17â-estradiol by that of the test compound and multiplying the resulting
fraction by 100.
Sch em e 2. Synthesis of Oxybutyric Acid Analogues
12-14a
F igu r e 1. Effect of 13 on the extent of interaction of 17â-
estradiol with human ERR. Concentrations of 13 were 0 (b),
70 (2), and 300 (9) nM. Line slopes and y-axis intercepts of
each data series were calculated by linear regression analysis.
of radical cations has been correlated with relative
a
Reagents: (b) see Scheme 1; (e) BrCH2CH2CH2COOEt, K2CO3,
electron affinities of the sample molecule and liquid
matrix used in the experiment.
acetone; (f) HCl, aq ethanol; (g) H2, 10% Pd/C, 10% aq THF.
Estr ogen Recep tor Affin ities of 8, 9, a n d 12-14.
The degree of equilibrium binding of saturating con-
centrations of [3H]17â-estradiol to human recombinant
ERR was determined in turn in the presence of increas-
ing concentrations of unlabeled 17â-estradiol and test
compounds. Plots of specifically bound [3H]17â-estradiol
as a function of competitor concentration yielded slopes
that did not differ substantially from each other,
enabling the use of IC50 values to calculate affinities of
test compounds relative to that of unlabeled 17â-
estradiol (Table 1). Affinities of 1 and 4 were determined
similarly and are included for comparison.
benzylation of 10 and 11, followed by deesterification,
gave m- and p-hydroxylated oxybutyric acids 12 and 13
as 1:1 mixtures of respective geometric isomers. Resolu-
tion of constituent geometric isomers of 12 and 13, using
the procedure by which 8 was prepared, was not
successful. Reisomerization of the chromatographically
separated isomers of 12 and 13 ethyl esters occurred
during mild alkaline or acidic hydrolysis, as was the
case when preparation of trans-4 from its ethyl ester
was attempted.6 Consequently, biologic evaluation was
carried out using 12 and 13 as the indicated (above)
isomer mixtures.
The oxybutyric acid homologue 13 had an RBA about
2 times that of 4. Repositioning of 13’s phenolic hydroxyl
(12) or saturation of its double bond (14) resulted in
considerable losses of ER affinity. Regarding the two
configurationally defined analogues of 4, trans analogue
8 had an RBA about 1/3 that of 4, but cis analogue 9
was essentially without ER affinity.
The degree to which [3H]17â-estradiol, present at
varying concentrations, was displaced from ERR in the
presence of set concentrations of 13 was assessed. Data
are expressed as a double-reciprocal plot (Figure 1).
An tip r olifer a tive Effects in MCF -7 Cells. The
ability of 4, 8, 9, and 12-14 to inhibit growth of MCF-7
cells cultured in non-estrogen-depleted medium in a
concentration-dependent manner was assessed in com-
parison with 1, and results are shown in Table 2. In
separate experiments, restoration of cell proliferation
to drug-free control values was seen when 1, 8, 12, or
13, at respective concentrations of e1 µM, was co-
incubated with 1 nM concentrations of 17â-estradiol. In
Saponification of 11, followed by catalytic hydro-
genolysis/hydrogenation of the intermediate carboxylic
acid, appeared to proceed without geometric isomeriza-
tion. Thus, the proton NMR spectrum of 14 displayed a
single O-methylene triplet centered at 4.01 ppm. Eth-
ylenic bond isomerization prior to its reduction would
have resulted in “pairing” of these signals, based on
analogy with diastereoisomers of dihydro-1.13
Triarylethylenes 8, 12, and 13 and triarylethane 14
exhibited contrasting novel mass spectral features. The
high-resolution liquid secondary ion (LSI) mass spectra
of 8, 12, and 13 each exhibited a protonated molecular
ion (MH+) accompanied by an intense peak (70-95%
relative intensity) representing the radical cation (M+•).
The LSI mass spectrum of 14 contained only the
expected MH+ ion and a pronounced fragment ion
resulting from the loss of 120 Da (C6H5-CH2-CH2-CH3)
from the protonated molecular ion. Processes leading
to radical cation formation in LSI mass spectra have
been studied systematically.14 In particular, appearance

Antiestrogenic Triarylethylene Carboxylic Acids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3129
Ta ble 2. Estrogen Antagonist Effects of Analogues of 4 in MCF-7 Cells
cell number: percent of control ((SEM)a
concn, nM
1
4
8
9
12
13
14
10
100
1000
97 (6)
72 (5)
31 (3)
24 (5)
99 (8)
82 (4)
78 (2)
52 (3)
89 (7)
91 (5)
50 (4)
19 (5)
74 (4)
25 (5)
20 (3)
18 (5)
99 (7)
102 (4)
68 (6)
96 (5)
102 (4)
NMc
10000
estimated IC50, nMb
100 (5)
NMc
190
1040
720
20
3200
a
Cell numbers are expressed in terms of viable totals with respect to those found in drug-free control incubations run in parallel. Each
value is from at least three separate experiments. In each experiment, the inhibitory effect of 1.0 µM 1 was determined in order to
b
cross-validate results for all compounds. The concentration required for one-half of each compound’s “maximal” growth antagonism
observed at 10 000 nM. In individual experiments, this was determined from inspection of the plot of cell growth as a percent of control
vs log concentration of test compound. c No meaningful growth inhibition was observed.
accord with previous studies carried out using different
experimental protocols,6,15 4 and 9 exhibited no growth
inhibitory effects at respective 10 µM concentrations.
Additionally 4, in contrast to 12-14, was shown to
reverse the growth suppressive effect of 1 µM 1 in a
concentration-dependent manner, with maximal efficacy
equal to that of 17â-estradiol. However 8, the 3-hydroxy
regioisomer of 4, antagonized proliferation with modest
potency and efficacy relative to 1.
precursor (11), featured a large (12 Hz) benzhydryl-
benzyl proton coupling constant in its NMR spectrum.
This suggested an antiperiplanar orientation of these
protons, thus implicating a gauche relationship between
the phenolic and phenyl rings. The NMR spectrum of
14 also exhibited an O-methylene triplet above 4 ppm,
typical of triarylethylenes in which the aryl-O-methyl-
ene and phenyl rings are on opposite sides of the
ethylenic bond.
Oxybutyric acid analogue 13 compared favorably to
1 in terms of estrogen antagonist potency and maximal
effectiveness. Repositioning of its phenolic hydroxyl (12)
resulted in no change in efficacy, but reduction in
potency; hydrogenation of its ethylenic bond (14) caused
reduction in both.
Although the ERR affinity of 13 was 1/5 that of 1, it
exhibited about a 10-fold greater antiestrogenic potency
than 1 (Table 2). This discrepancy is unlikely to be a
consequence of differential interaction of either ligand
with other ER isoforms besides ERR in MCF-7 cells.
Although ER heterogeneity, in particular the presence
of ERâ and ERâ2, has been demonstrated in reproduc-
tive and/or nonreproductive tissues,19 MCF-7 cells ap-
pear to contain only ERR.19a,20 The overall ER affinities
of 1 and 13 (Table 1) do not reveal the specific amino
acid residues in the ERR hormone binding domain with
which these ligands interact. In particular, the amino
acid residue(s) in ERR which interact with the cationic
side chain of 1 would presumably differ from those that
accommodate the anionic side chain of 13. This differ-
ence in specific amino acid bonding in the ERR hormone
binding domain might affect differentially, by one or
more of several mechanisms,21 the ability of respective
liganded ERR complexes to interact effectively with
DNA estrogen response elements, resulting in greater
antagonist potency of 13.
Another possibility is that differences in permeability
of these compounds into MCF-7 cells might account for
the observed discrepancy between ER affinity and
antagonistic potencies of these ligands. The concentra-
tion of 13 available for interaction with nuclear ER
might be greater than that of 1 due to more efficient
diffusion through cell membranes, a process not in-
volved in interaction of 1 and 13 with isolated ERR.
Isolation of 13’s geometric isomers was impeded by
facile reisomerization of its cis- and trans-ethyl ester
precursors during or after aqueous hydrolysis (see
Results). Similar results were obtained with the corre-
sponding ethyl esters of 12. Consequently, the degree
to which the constituent geometric isomers of these
compounds contribute to their observed ER affinities
and estrogen antagonist effects is not known.
Discu ssion
Two-carbon homologation of the acidic side chain of
the estrogen mimetic 4 resulted in improvement of ER
affinity and reduction in estrogenicity. Thus, 13 was a
potent, effective antiestrogen in MCF-7 cells (Table 2).
This finding indicates that greater ER affinity and
antiestrogenic effectiveness can be achieved by side-
chain elongation of triarylethylene carboxylic acids, just
as was the case in analogous triarylethylenes with basic
side chains.10
Previously, binding of 1 and 17â-estradiol to rat
uterine ER was shown to be competitive.16 It has
generally been assumed that interactions of newer
analogues of 1 with ER are also competitive with 17â-
estradiol, but an uncompetitive interaction of a non-
steroidal substance with ER has been reported.17 The
data in Figure 1 indicate that like 1, interaction of 13
with human ERR was competitive with respect to 17â-
estradiol. Together, these results indicate that 1, 13, and
17â-estradiol interact with a common site within the
hormone binding domain of ER.
In the presence of ERR, 8 exhibited modest affinity
but its isomer (9) was ineffective. This difference is
suggested to be due primarily to the established prefer-
ence by ER for triarylethylenes endowed with trans-
hydroxystilbene moieties, such as in 8, over their cis
counterparts, such as 9.9,18 (However, variance in the
position of side-chain attachment (p vs m) in 8 and 9
might also contribute to observed affinity differences.)
Similarly, a cis-like orientation of phenolic and phenyl
rings in 14 could account for its low ER affinity. The
analogous dihydro-4-hydroxytamoxifen produced by hy-
drogenation of 1 (X ) OH) was suggested to resemble
its opposite geometric isomer, cis-1 (X ) OH), confor-
mationally.13 Compound 14, prepared from its trans
Nevertheless, our studies extend earlier findings
suggesting that antiestrogenic activity can be found in
hydroxytriarylethylene carboxylic acids endowed with
side chains of sufficient length5 or in which the p-
phenolic hydroxyl is shifted to the m-position in the ring

3130 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Kraft et al.
geminal to the one bearing the side chain.4b The
antiestrogenic potency and efficacy of 13 were compa-
rable to those of 1 and 2, which have been shown
experimentally to be full estrogen mimetics in bone and/
or liver.5,22 Future efforts will investigate the degree to
which 13 is capable of similar tissue-selective estroge-
nicity.
boron tribromide. The orange solution was stirred for 24 h. A
cold solution of 5% aqueous sodium bicarbonate was added in
portions to the reaction solution until the mixture was pH 8-9
to litmus. The reaction mixture was concentrated in vacuo,
and the remaining basic aqueous layer was washed with three
20-mL portions of ether. The aqueous layer was then acidified
with 10% aqueous HCl (until pH 1-2 to litmus) and extracted
with 35 mL of ether. The organic layer was washed with two
20-mL portions of water, dried, and concentrated, giving a
yellow solid. Crystallization of this from chloroform and
hexanes (1:1) gave 49 mg (60%) of a 1:1 mixture of 8 and its
cis isomer: TLC (solvent 2) one spot, Rf 0.39; mp 137.2-140.0
°C; 1H NMR 7.7-7.0, m, 13H (ArH); {4.74 and 4.58, s}, 2H
(OCH2COOH); 2.47, m, 2H (CH2CH3); 0.91, m, 3H (CH2CH3).
Exp er im en ta l Section
Solvents, chemicals, and biochemicals were purchased from
Aldrich Chemical Co. (Milwaukee, WI), Sigma Chemical Co.
(St. Louis, MO), or the University of Georgia Central Research
Stores. Hydroxytriarylethylene 4 was available from previous
studies.7 The tetrahydrofuran (THF) used in moisture-sensi-
tive reactions was distilled from lithium aluminum hydride
immediately prior to use. All air-sensitive reactions were
performed in dry glassware under a nitrogen atmosphere.
Workup of organic extracts of reaction products was carried
out by removal of water by addition of magnesium sulfate,
followed by filtration and concentration in vacuo. Analytical
thin-layer chromatography (TLC), using 5- × 20-cm glass-
backed 0.25-mm silica gel GF254 plates (Analtech, Inc., Newark,
DE) was used to monitor the course of reactions, the progress
of column chromatography, and determine the purity of final
products. Plates were developed in either solvent 1 [benzene/
chloroform (50/50 v/v)] or solvent 2 [chloroform/ 2-propanol/
glacial acetic acid (90/10/0.5 v/v/v)] and viewed under light of
254-nm wavelength. Preparative TLC was carried out as
described above except using 20- × 20-cm plates coated with
1-mm layers of silica gel GF254, with developing solvents as
specified below. Melting points were determined on an Elec-
trothermal 9100 apparatus and are uncorrected. Proton nuclear
magnetic resonance (NMR) spectra were obtained using a
Bruker AMX 400 spectrometer. The solvent for NMR analyses
was acetone-d6 unless otherwise stated. Chemical shifts (δ)
are reported as downfield from the internal standard, tetra-
methylsilane. Positive ion liquid secondary ion mass spectra
The above geometric isomer mixture was dissolved in 10
mL of absolute ethanol, and 0.25 mL of concentrated HCl was
added. The mixture was stirred for 1 h at room temperature.
A solution of 10% aqueous sodium bicarbonate was added, and
solvent was removed under a stream of nitrogen. The residual
oil was dissolved in 0.25 mL of acetone and subjected to
preparative TLC (1.0-mm silica gel GF plate; Analtech,
Newark, DE). The plate was developed in benzene-triethy-
lamine (90-10, v/v). Two zones, Rf 0.51 and 0.54, approximate
intensity ratio 3:1, were present. The lower of these was
removed from the plate and stirred with 5 mL of ethanol for
15 min. The mixture was filtered, and the filtrate was
concentrated. The residue was dissolved in 5 mL of 1,4-
dioxane, and 1 mL of 10% aqueous NaOH was added. After
0.5 h at room temperature, the solution was acidified with 2
mL of 10% aqueous HCl and extracted with three 10-mL
portions of ether. The combined extracts were dried and
concentrated to yield a yellow residue, which was dissolved
in 5 mL of chloroform, followed by dilution with 3 mL of
hexanes. Storage at 8 °C afforded 15.3 mg of 8 as a pale yellow
solid: mp 134.4-136.6 °C; 1H NMR {7.25-7.05 and 6.80-6.70,
m}, 9H (C6H5 and HO-C6H4); {6.82 and 6.61, d, J ) 8 Hz},
4H (CH2C6H4); 4.58, s, 2H (OCH2COOH); 2.47, q, J ) 7 Hz,
2H (CH2CH3); 0.89, t, J ) 7 Hz, 3H (CH2CH3); HRMS (LSIMS)
m/z calcd for C24H23O4 375.1596 (MH+), found 375.1614, rel
ion intensity M+•/MH+ ) 0.95. Anal. (C24H22O4‚2.25H2O) C,
H.
P r ep a r a t ion of 3-[1-(4-H yd r oxyp h en yl)-2-p h en yl-1-
bu ten yl]p h en oxya cetic Acid (9). Compound 6 (1.03 g, 2.5
mmol) was converted to 9 benzyl ether as described for 8. The
product, a yellow oil, was stored at 8 °C over hexanes. The
resulting white powder was filtered and washed with cold
hexanes: 481 mg (41%). To a solution of 450 mg of this in 15
mL of tetrahydrofuran was added 50 mg of 10% Pd on carbon.
The mixture was shaken under 47 psi of H2 for 24 h. Filtration
and concentration gave 132 mg of light brown oil. This was
dissolved in 5 mL of methanol containing HCl gas. After 24 h,
solvent was evaporated and the residue was dissolved in 0.6
mL of methanol. The solution was subjected to preparative
TLC using benzene-triethylamine (90-10, v/v) as mobile
phase. Bands with Rf 0.27-0.31 were cleanly separated from
those with Rf 0.20-0.23, characterized separately by proton
NMR spectrometry as dihydro-9 methyl ester. The upper
bands were scraped off the plates, combined, and eluted with
25 mL of methanol. The mixture was filtered and concentrated
in vacuo. The residue was dissolved in 2 mL of dioxane, and 1
mL of 5% NaOH was added. After 15 min of stirring, the
mixture was extracted with 2 mL of ether, acidified with 10%
aqueous HCl, and extracted with two 3-mL portions of ether.
These last ether extracts were worked up to give a colorless
oil. This was dissolved in 0.5 mL of dry chloroform, and the
solution was diluted with 0.4 mL of hexanes. Storage at 8 °C
gave white crystals, which were filtered and washed with cold
chloroform-hexanes (50-50) to give 23 mg of 9: mp 159.5-
165.2 °C (partial), 180.1-181.5 °C (complete); 1H NMR 7.30-
7.10, m, 5H (C6H5); {6.84 and 6.55, d, J ) 8.5 Hz}, 4H
(C6H4OH); 6.80-6.60, m, 4H (C6H4O-CH2); 4.73, s, 2H (CH2-
OAr); 2.45 q, J ) 7 Hz, 2H (CH2CH3); 0.97, t, J ) 7 Hz, 3H,
(CH2CH3). Anal. (C24H22O4‚0.5H2O) C, H.
were recorded on
a Micromass AutoSpec series-M high-
resolution magnetic sector mass spectrometer of EBE geom-
etry. The instrument used a cesium ion gun operated at 27
keV at an accelerating voltage of 8 kV. The scan speed was
1.5/decade, and 10 scans were averaged for each mass spec-
trum. Concentrations of the sample solutions for the neat
compounds were about 1 µg/µL using glycerol as the matrix.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA.
Sta r tin g Ma ter ia ls. Compound 7 was available as re-
ported;7 5 and 6 were prepared in a similar manner. Thus 5
was isolated in 71% yield: TLC (solvent 1) one spot, Rf 0.34;
mp 107.6-109.5 °C (initial softening at 87.3-88.7 °C); 1H NMR
7.7-7.0, m, 18H (ArH); {5.5, s and 4.71, s}, 2H (OCH2Ar); 2.1,
m, 2H (CH2CH3); 0.9, m, 3H, (CH2CH3). Crude 6 solidified on
standing (61% yield): 1H NMR 7.5-6.5, m, 18H (ArH); {5.12,
s and 4.95, s}, 2H (OCH2Ar); 2.47, q, J ) 7 Hz, 2H (CH2CH3);
0.97, t, J ) 7 Hz, 3H, (CH2CH3).
P r ep a r a t ion of 4-[1-(3-H yd r oxyp h en yl)-2-p h en yl-1-
bu ten yl]p h en oxya cetic Acid (8). Ethyl bromoacetate (2.55
mL, 22.9 mmol) and potassium carbonate (0.91 g, 6.6 mmol)
were added to a solution of 5 (0.80 g, 1.9 mmol) in 25 mL of
acetone. The mixture was refluxed for 11 h. The solution was
cooled, filtered, and concentrated. The resulting oil was
dissolved in 20 mL of dioxane, and 14 mL of 5% aqueous NaOH
was added. After stirring for 0.5 h, the reaction mixture was
cooled to 0 °C and acidified with 10 mL of 10% aqueous HCl.
The product was extracted with 50 mL of ether, washed with
two 20-mL portions of water, then dried, and concentrated.
Crystallization from acetone and water (2:1) afforded 0.52 g
(60%) of 8 benzyl ether as white crystals: TLC (solvent 2) one
1
spot, Rf 0.57; mp 148.4-152.0 °C; H NMR 7.7-7.0, m, 18H
(ArH); {5.11, s and 4.78, s}, 2H (OCH2Ar); {4.74 and 4.55, s},
2H (OCH2COOH); 2.42, m, 2H (CH2CH3); 0.85, m, 3H (CH2CH3).
To a solution of 100 mg (0.22 mmol) of 8 benzyl ether in 30
mL of methylene chloride was added 125 mg (0.50 mmol) of
P r epar ation of tr a n s-4-{[1-(4-Ben zyloxyph en yl)-2-ph en -

Antiestrogenic Triarylethylene Carboxylic Acids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3131
yl-1-bu ten yl]p h en oxy}-n -bu ta n oic Acid Eth yl Ester (11).
Ethyl 4-bromobutyrate (4.4 mL, 32.8 mmol) and potassium
carbonate (3.22 g, 23.3 mmol) were added to a solution of 7
(1.76 g, 4.33 mmol) in 50 mL of acetone. The mixture was
refluxed for 10 h. The solution was cooled, filtered, and
concentrated. Hot hexanes was added, and the solution cooled
to 4 °C. The resulting white crystals were collected and washed
with cold hexanes, affording 0.98 g (44%) of 11 ethyl ester:
TLC (solvent 1) one spot, Rf 0.60; 1H NMR (CDCl3) 7.45-7.15,
m, 10H (C6H5); {7.09 and 6.95, d, J ) 8H}, 4H (Bz-O-C6H4);
{6.76 and, 6.52, J ) 8 Hz}, 4H (R-O-C6H4); 5.07, s, 2H (OCH2-
Ar); 4.11, q, J ) 8 Hz, 2H (COOCH2CH3); 3.86, t, J ) 6 Hz,
2H (ArOCH2CH2); 2.55-2.43, m, 4H (CH2COOR and CH2CH3);
2.02, p, J ) 6 Hz, 2H (CH2CH2CH2); 1.23, t, J ) 8 Hz, 3H
(COOCH2CH3); 0.93, t, J ) 7 Hz, 3H (CH2CH3).
obtained as crystals from water-acetone (1:2): TLC (solvent
2) one spot, Rf 0.51; mp >400 °C. Anal. (C26H25O4Na‚2.25H2O)
C, H.
P r ep a r a tion of 4-{[1-(3-Hyd r oxyp h en yl)-2-p h en yl-1-
bu ten yl]p h en oxy}-n -bu ta n oic Acid (12). Alkylation of 1.88
g (4.62 mmol) of 5 with ethyl 4-bromobutyrate was carried out
as described above for synthesis of 11. Crystallization of the
crude product from hexanes afforded 600 mg (25%) of 10 as a
white solid: TLC (solvent 2) one spot, Rf 0.80; mp 108.2-109.6
°C; NMR (CDCl3) 7.40-6.50, m, 18H ArH; 5.04, s, 2H (OCH2-
Ar); 4.11, q, J ) 8 Hz, 2H (COOCH2); 3.86, t, J ) 6 Hz, 2H
(OCH2R); 2.47-2.43, m, 4H (CH2COOR and CH2CH3); 2.02,
p, J ) 6 Hz, 2H (CH2CH2CH2); 1.23, t, J ) 8 Hz, 3H
(COOCH2CH3); 0.89, t, J ) 7 Hz, 3H (CH2CH3). Hydrolytic
debenzylation/saponification of 175 mg (0.34 mmol) of 10 was
carried out as described in step B above. There was obtained
58 mg (43%) of 12 as an amorphous yellow solid. The sodium
salt of 12 obtained in an analogous manner to that described
for 13: TLC (solvent 2) one spot, Rf 0.50; 1H NMR (methanol-
d4) 7.20-6.30, m, 13H (ArH); {4.01, t, 3.84, t, J ) 6 Hz}, 2H
(OCH2R); 2.47, m, 2H (CH2CH3); {2.35 and 2.26, t, J ) 6 Hz},
2H (CH2COOH); {2.06 and 1.96, p, J ) 6 Hz}, 2H (CH2CH2-
CH2); 0.90, m, 3H (CH2CH3); HRMS (LSIMS) m/z calcd for
Rea ctivity of 11. A. Sa p on ifica tion a n d Hyd r ogen a -
tion /Hyd r ogen olysis: P r ep a r a tion of 4-{[1-(4-Hyd r oxy-
p h en yl)-2-p h en yl-1-b u t a n yl]p h en oxy}-n -b u t a n oic Acid
(14). To a solution of 0.98 g of 11 in 20 mL of dioxane was
added 10 mL of 10% aqueous NaOH. After stirring at room
temperature for 1 h, the solution was cooled to 0 °C, acidified
with 10% aqueous HCl, and extracted with three 30-mL
portions of ether. The organic extracts were combined, dried,
and concentrated to give a pale yellow oil. Crystallization from
chloroform-hexanes (1:2) afforded 0.42 g (65%) of 11 free
acid: TLC (solvent 2) one spot, Rf 0.57; 1H NMR spectrum was
identical to that of 11 except signals at 4.11 and 1.23 ppm were
absent. To a solution of 262 mg of this (0.53 mmol) in 20 mL
of THF were added 2 mL of water and 0.63 g of 10% palladium
on carbon. The suspension was shaken under 44 psi of
hydrogen gas for 24 h. The reaction mixture was filtered and
concentrated to yield a milky white liquid. This was extracted
with three 50-mL portions of ether, and the combined extracts
were dried and concentrated to afford a yellow oil which
solidified on standing. Crystallization from chloroform-hex-
anes (1:1) yielded 90 mg (42% yield) of 14 as white crystals:
TLC (solvent 2) one spot, Rf 0.43; mp 157.2-158.8 °C; 1H NMR
{7.39 and 7.24, d, J ) 8 Hz}, 4H (R-O-C6H4); 7.15-6.95, m,
5H (C6H5); {6.88 and 6.50, d, J ) 8 Hz}, 4H (HO-C6H4); 4.15,
d, J ) 12 Hz, 1H (Ar2CH); 4.01, t, J ) 6 Hz, 2H (OCH2); 3.39,
ddd, J 1 ) J 2 ) 12 Hz, J 3 ) 2.7 Hz, 1H (PhCHEt); 2.49, t, J )
6 Hz, 2H (CH2COOH); 2.03, p, J ) 6 Hz, 2H (CH2CH2CH2);
{1.62 and 1.42, m}, 2H (CH2CH3); 0.60, t, J ) 8 Hz, 3H
(CH2CH3); HRMS (LSIMS) m/z calcd for C26H29O4 405.2065
C
26H27O4 403.1909 (MH+), found 403.1894, rel ion intensity
M+•/MH+ ) 0.70. Anal. (C26H25O4Na‚2H2O) C, H.
Estr ogen Recep tor Bin d in g Affin ity. [3H]Estradiol (51
Ci/mmol) was obtained from Amersham Pharmacia Biotech,
Inc., Piscataway, NJ . Human estrogen receptor alpha (ERR)
from recombinant baculovirus-infected insect cells was ob-
tained from PanVera Corp., Madison, WI. Aliquots (20 µL) of
the thawed preparation were stored at -80 °C until use. For
preparation of incubation mixtures, an aliquot was warmed
to 4 °C and diluted with 16 mL of assay buffer: 10 mM Tris,
pH 7.50, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol,
0.1% bovine serum albumin. Incubations were run in triplicate
for 20 h at 4 °C in 12- × 75-mm polypropylene tubes and
contained 200 µL (0.34 pmol) of diluted ERR, 5.7 nM [3H]-
estradiol added in 10 µL of ethanol, and increasing concentra-
tions (1 nM to 10 µM) of test compounds added as solutions in
10 µL of N,N-dimethylacetamide. Control incubations con-
tained 10 µL of N,N-dimethylacetamide alone, and nonspecific
binding was determined in incubations to which 10 µM
estradiol had been added. Unbound [3H]estradiol was removed
by addition of 400 µL of a suspension of dextran-coated
charcoal (prepared by stirring 100 mg of dextran and 1 g of
Norit A in 100 mL of water at 4 °C for 16 h, followed by
centrifugation of the mixture at 450g for 10 min and resus-
pension of the pellet in assay buffer), and incubation was
continued for 15 min. Tubes were centrifuged at 600g for 15
min, and a 400-µL aliquot of each supernatant was shaken
for 10 min with 4 mL of scintillation fluid. Specific binding
was determined using a single label percent of reference
program, with respect to nonspecific and total bound radio-
activity (counts/min).
(MH+), found 405.2063, rel ion intensity MH+ - 120/MH+
0.11. Anal. (C26H28O4‚0.25H2O) C, H.
)
B. Hyd r olytic Deben zyla tion : P r ep a r a tion of 4-{[1-(4-
Hydr oxyph en yl)-2-ph en yl-1-bu ten yl]ph en oxy}-n -bu tan o-
ic Acid (13). To a solution of 360 mg (0.69 mmol) of 11 in 50
mL of ethanol was added 40 mL of concentrated aqueous HCl.
The mixture was stirred and refluxed under nitrogen gas for
10 h. The mixture was concentrated to about 1/3 its original
volume in vacuo. The supernatant was discarded, and the
gummy residue was washed with 20 mL of water and dissolved
in 10 mL of dioxane. Then 3 mL each of water and 10% NaOH
were added, and the mixture was stirred for 1 h. The mixture
was extracted with two 20-mL portions of ether. The aqueous
phase was cooled in ice, acidified with 10% HCl, and extracted
with two 25-mL portions of ether. These last extracts were
combined and worked up to give a gold oil, which solidified on
storage at 8 °C yielding 204 mg (73%) of 13, as a light yellow
semisolid after drying for 24 h at 60 °C (0.05 mmHg): TLC
(solvent 2) one spot, Rf 0.51; 1H NMR 7.20-7.10, m, 5H + 1H
(C6H5 + 0.25 O-C6H4); {7.07, 6.95, 6.85, 6.78, 6.70, 6.59, 6.49,
d, J ) 8 Hz}, 7 H (1.75 O-C6H4); {4.08 and 3.91, t, J ) 6 Hz},
2 H (OCH2R); 2.52-2.41, m, 4H (CH2COOH and CH2CH3);
2.06-1.95, m, 2H (CH2CH2CH2); 0.91, t, J ) 7 Hz, 3H
(CH2CH3); HRMS (LSIMS) m/z calcd for C26H27O4 403.1909
(MH+), found 403.1929, rel ion intensity M+•/MH+ ) 0.70.
Attempted crystallization of this product was not successful.
A 40-mg portion of 13 was dissolved in 2 mL of methanol, and
the solution was passaged through a 5-g column of 100-200
mesh Amberlite CG-50 (Na+). The methanol eluent was
concentrated in vacuo, and the sodium salt of 13 (35 mg) was
Effects on MCF -7 Cell Gr ow th . Cells (1 × 105) were
conditioned at 37 °C in a humidified atmosphere containing
5% carbon dioxide, in 75-cm2 flasks containing 10 mL of growth
medium (phenol red-free Dulbecco’s modified Eagle’s medium-
Ham’s nutrient mixture, 50-50) containing 15 mM HEPES
buffer, 5 mM L-glutamine, 1.2 g/L sodium bicarbonate, 5 mg/L
gentamycin, 1 mg/L insulin, and 10% heat-inactivated new-
born calf serum,15 and then passaged into 25-cm2 flasks with
5 mL of growth medium. Test compounds, in final concentra-
tions ranging from 10 to 10 000 nM, were added as solutions
in dimethyl sulfoxide, without or with similar addition of 17â-
estradiol or 1 at respective final concentrations of 1 nM and 1
µM. Medium and test compounds were changed every 3-4
days. After 7 days of incubation, growth medium was aspirated
from flasks, and cell numbers/flask were determined as
described previously.15 In each experiment, MCF-7 cell uni-
formity was validated by assessment of the growth suppressive
effect of 1 µM 1 in parallel incubations.
Ack n ow led gm en t. This research was supported by
Grant AR 42069 from the National Institutes of Health.

3132 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Kraft et al.
(7) Ruenitz, P. C.; Bourne, C. S.; Sullivan, K. J .; Moore, S. A.
Estrogenic Triarylethylene Acetic Acids: Effect of Structural
Variation on Estrogen Receptor Affinity and Estrogenic Potency
and Efficacy in MCF-7 Cells. J . Med. Chem. 1996, 39, 4853-
4859.
Dr. Kraft is the recipient of a predoctoral fellowship
from the American Foundation for Pharmaceutical
Education.
(8) Ruenitz, P. C.; Shen, Y.; Li, M.; Liang, H.; Whitehead, J r., R.
D.; Pun, S.; Wronski, T. J . Specific Bone-Protective Effects of
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Antiestrogenic Activity of Monophenolic Analogues of Tamoxifen,
(Z)-2-[p-(1,2-Diphenyl-1-butenyl)phenoxy]-N,N-dimethylethyl-
amine. J . Med. Chem. 1982, 25, 1056-1060.
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