Identification of new triarylethylene oxyalkanoic acid analogues as bone selective estrogen mimetics

Article abstract of DOI:10.1016/S0968-0896(01)00038-4

Previously, the estrogen receptor (ER) ligand 4-[1-(p-hydroxyphenyl)-2-phenylethyl]phenoxyacetic acid (5) was found to have differential bone loss suppressive effects in the ovariectomized (OVX) rat approaching those of selective ER modulators (SERMs) suc

Full text of DOI:10.1016/S0968-0896(01)00038-4

Bioorganic & Medicinal Chemistry 9 (2001) 1579–1587
Identification of New Triarylethylene Oxyalkanoic Acid
Analogues as Bone Selective Estrogen Mimetics
Valeria N. Rubin, Peter C. Ruenitz,* F. Douglas Boudinot and Jason L. Boyd
College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA
Received 10 November 2000; accepted 1 February 2001
Abstract—Previously, the estrogen receptor (ER) ligand 4-[1-(p-hydroxyphenyl)-2-phenylethyl]phenoxyacetic acid (5) was found to
have differential bone loss suppressive effects in the ovariectomized (OVX) rat approaching those of selective ER modulators
(SERMs) such as tamoxifen. In an effort to improve efficacy, analogues of this compound were prepared which incorporated fea-
tures designed to reduce polarity/ionizability. Thus, the acetic acid side chain of 5 was replaced by n-butanoic acid and 1H-tetrazol-
4-ylmethyl moieties, to give 8 and 10, respectively. Also, the phenolic hydroxyl of 5 was replaced, giving deoxy analogue 9. We also
developed new methods for the synthesis of triarylethylene variants of 5 and 9, namely 4-{[1-(p-hydroxyphenyl)-2-phenyl-1-bute-
nyl]phenoxy}-n-butanoic acid (6) and its des-hydroxy counterpart (7), because the former of these had in vitro antiestrogenic effects
characteristic of known SERMs. In the OVX rat, 6 and 7 were as effective as 17b-estradiol in suppressing serum markers of bone
resorption/turnover, namely osteocalcin and deoxypyridinoline, but had only 30% of the uterotrophic efficacy of 17b-estradiol.
This study has thus identified two triarylethylene oxybutyric acids, 6 and 7, that have differential bone/uterus effects like those of
known SERMs. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
mimetic in the skeletal system, thus protecting against
bone loss in postmenopausal women.4 A newer SERM,
raloxifene (2) was recently introduced for the prevention
of postmenopausal osteoporosis. Although 2 has some
advantages over 1 and ERT,5a,6 both 1 and 2 possess
significant therapeutic drawbacks. For example, neither
of these SERMs prevents, and in fact might actually
intensify, a common disquieting symptom of estrogen
deficiency known as hot flashes or hot flushes.7 Thus,
identification of novel SERMs remains a priority (Fig. 1).
Estrogens are the endogenous ligands for the estrogen
receptor (ER). They exert a direct effect on the female
reproductive organs (uterus, ovaries, and breast) as well
as several other ER-containing tissues such as those of
the skeletal and cardiovascular systems. The decreased
ovarian production of estrogen characteristic of meno-
pause leads to numerous health complications in both
menopausal and postmenopausal women. Estrogen
replacement therapy (ERT) is widely used to alleviate
some of the discomforts associated with menopause and
to prevent osteoporosis.1 However, ERT is associated
with an increased risk of breast and endometrial cancer.2
The OVX rat has become a widely used animal model
for determining selective estrogen activity of ER
ligands. Specifically, it is used as a model of osteopenia
associated with estrogen deficiency.8 OVX rats treated
with 17b-estradiol (E2), ethynyl estradiol (EE2), 1 or 2
show a decreased rate of bone turnover and main-
tenance of normal bone mass compared to untreated
animals.5,9,10 Similar results are seen clinically with
postmenopausal patients on ERT11 or taking 14 or 212 for
extended periods of time. The OVX rat also serves as a
model for measuring uterotrophic effects of ER ligands.
As an alternative to ERT, selective estrogen receptor
modulators (SERMs) offer the advantage of acting as
estrogen agonists in extra-reproductive tissue (liver,
bone, and brain) while having no effect or antagonizing
the effects of estrogen in reproductive tissue (uterus,
breast). The first SERM to be used clinically, tamoxifen
(1) is widely used for the prevention and treatment of
breast cancer due to its antiestrogenic effect on ER-
positive breast cancer cells.3 But 1 was a full estrogen
Estrogens and SERMs exert their bone protecting
effects by reducing the rate of bone turnover (formation
and resorption). The degree to which these processes are
occurring can be monitored by determining the levels of
osteocalcin (OC) and deoxypyridinoline (Dpd).13 OC is
*Corresponding author: Tel.: +1-706-542-4376; fax: +1-706-542-
5358; e-mail: pruenitz@rx.uga.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00038-4

1580
V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
a polypeptide produced by osteoblasts during bone for-
mation, and a significant percentage is released into the
blood.14 Dpd is released into the blood during break-
down of bone collagen, which accompanies resorp-
tion.15 In the OVX rat, the degree to which ER ligands
are able to suppress serum OC and Dpd parallels their
ability to suppress bone density loss and maintain other
bone histomorphometric parameters.8,16,17 Thus,
assessment of these serum markers can be used to identify
bone protective ER ligands subsequent to more time-
consuming bone densitometric and microanatomical
studies.
Accordingly, we wanted to determine whether 6, like 5,
was capable of selective bone-protective estrogenicity in
the OVX rat. Also, because 5 had less bone-protective
efficacy in the OVX rat than did E2, we prepared and
evaluated 8 and 10, in which 5’s highly ionizable oxya-
cetic acid side chain was replaced with less acidic oxy-
butyric acid or oxymethyltetrazole moieties. And,
because 3 and 4 were suggested to be ‘active metabo-
lites’ produced by facile 4-hydroxylation of their non-
phenolic precursors in the OVX rat,18b,22 we chose to
prepare and evaluate the bone-protective and utero-
trophic effects of 9 and 7, the putative precursors, in
turn, of 5 and 6.
Analogues of 1 bearing acidic side-chain substituents
have also been found to have selective estrogenic activ-
ity. Arylacrylic acid (3) was found, like 1 and 2, to pre-
vent against bone loss in OVX rats while displaying
dominant estrogen antagonist activity in the uterus.18
The oxyacetic acid side-chain analogues of 1 (4 and 5)
were found to be estrogen agonists in MCF-7 human
breast cancer cells,19,20 a line of estrogen responsive cells
used to assess potency and efficacy of ER ligands.
Nevertheless, 5 was determined to be a selective estro-
gen in the OVX rat, having bone protecting effects
approaching those of 1 while displaying no uterotrophic
effects.8 Extension of the acidic side-chain-substituent of
5 resulted in oxybutyric acid analogue 6. In MCF-7
cells, 6 was found to be a full estrogen antagonist with
greater potency than 1.21 These findings show how small
structural modifications in these acidic side-chain ana-
logues can have a profound effect on their agonist/
antagonist balance, and they suggest that 6, like 3,
might be a SERM.
Results
Synthesis
Scheme 1 outlines the approach used to prepare com-
pounds 6–10. The triarylethylene backbone of these
compounds was obtained through application of the
McMurry olefination reaction using low-valent tita-
nium23 to cross-couple substituted benzophenones (11a–
c) with propiophenone or benzaldehyde. The desired
intermediates (12a–d) were separated from self-coupling
byproducts by column chromatography when needed.
Introduction of the side chain of 6, 7, and 13–15 was
carried out by O-alkylation of monophenols 12a–d.
Reaction of 15 with sodium azide and ammonium
chloride24 followed by debenzylation/hydrogenation,
afforded tetrazole 10.
Figure 1. Structure of selected ER ligands.

V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
1581
Scheme 1. (a) Ti, THF; (b) Br(CH2)3COOC2H5 or BrCH2COOC2H5, K2CO3, acetone, then NaOH, aqueous dioxane; (c) BrCH2CN, K2CO3, ace-
tone; (d) NaN3, DMF; (e) H2, 10% Pd/C, THF/MeOH.
ER Affinities
Previously, synthesis of 6 was carried out starting with
11b and propiophenone, followed by a debenzylation
step.21 The product so obtained was difficult to purify
and the overall yield (4%) was not satisfactory. Use of a
pivaloyl (Pv) instead of a benzyl (Bz) phenolic protect-
ing group resulted in improvement in overall yields to
28% after crystallization. Removal of the excess 4-bro-
mobutyric acid and pivalic acid from the final product
was accomplished by differential liquid–liquid extrac-
tion of 6, taking advantage of 6’s greater relative lipo-
philicity. The starting benzophenone (11c) was prepared
by a modification of the previously reported alkylation
procedure.25
In the present series of acidic triarylethylenes, com-
pound 6 exhibited the highest ER affinity, considerably
greater than that of its nonphenolic analogue (7) and
about one-tenth that of E2 (Table 1). Replacement of
the oxyacetic acid side chain in 5 with an oxybutyric
acid moiety (8) resulted in about a 70% increase in affi-
nity; 5’s oxymethylenetetrazole analogue 10 had about
25% the ER affinity of 5.
Estrogenic effects
In the OVX rat, 6 and its nonphenolic counterpart 7
exhibited similar bone protective efficacies, which were
equivalent to that of E2 (Table 2). But the uterotrophic
efficacy of either 6 or 7, measured with respect to vehicle
treated controls, was only about 25–30% that of E2 or
EE2. The observed efficacy of 6 in bone and uterus was
independent of its route of administration.
The configuration of final compounds (E/Z isomerism,
when relevant) was determined by the relative chemical
shift of the side chain O-methylene protons in NMR
spectra. For example, the spectrum of 7 contained a
single O-methylene triplet centered at 3.87. This was
assigned to the Z configuration of 7 (as shown in
Scheme 1) based on correlation with O-methylene shifts
of corresponding geometric isomers of 1 as well as other
acidic-side chain analogues.21 Similarly, the spectrum of
6 contained two O-methylene triplets of about equal
intensities, centered at 3.87 and 4.03, corresponding to
the Z and E isomers of 6, respectively.
Compound 10, the ‘tetrazole’ analogue of 5, had only a
weak uterotrophic effect. Unfortunately, it had no effect
on serum OC, and its ability to reduce serum total Dpd
was of only borderline (P<0.10) significance. Neither 8
Differences in ionizability (acidity) of 6–8 and 10, com-
pared to 5, were estimated from the pKas of their
respective phenoxy-substituted variants.26 The pKas of
4-phenoxy-n-butyric acid and 5-phenoxy-1,2,3,4-tetra-
zole were 4.44 and 3.49, respectively, and that of phe-
noxyacetic acid was 3.12. On this basis, 10 was 2.3 times
less acidic than 5; and 6–8 were each calculated to be 20-
fold less acidic than 5. Furthermore, the side chain
acidity of 6–8 was suggested to be comparable to that of
3 whose pKa, extrapolated from that of trans-cinnamic
acid,26a was around 4.40.
Table 1. Comparative affinity of acidic triarylethylene derivatives
and standard ER ligands for human ERa
compd
RBAa
1
2
5
6
7
8
9
10
E2
b
6.7
94
0.91
10.3
0.35
1.57
0.26
100
aThe concentration of E2 (5.9 nM) required to displace 50% of speci-
fically bound [3H]E2, divided by the concentration of test compound
required to do this, times 100. Each RBA value is the average of three
separate determinations in which calculated individual values differ by
<10%.
bAt 10 mM, 9 displaced 54% of specifically bound [3H]E2.

1582
V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
Table 2. Skeletal and reproductive tract effects of acidic triarylethylene derivatives in the OVX rat
Compd
Dosing routea
Uterine wet wt., mg
Serum OC, ng/mL
Serum total Dpd, pmol/mL
6
7
8
sc; po
po
sc
167 (25b)cd; 184 (17)cd
195 (30)c,d
75 (13)
76 (4)c; 62 (8)cd
59 (5)c,d
92 (10)
7.4 (0.8)c; 12.3 (3.9)c
10.7 (1.2)c
12.1 (2.3)
9
10
E2
EE2
vehicle
sc
sc
sc
po
sc; po
72 (8)
123 (14)c
96 (6)
93 (9)
11.8 (1.7)
8.7 (1.5)
355 (73)c
65 (12)c
6.3 (1.4)c
428 (53)c
87 (14); 102 (10)
43 (4)c
93 (9); 84 (7)
11.0(2.3) c
11.3 (1.7); 18.3 (2.9)
aDaily dose of each test compound: 10 mmol/kg of body weight. Those of E2 and EE2 were each 0.35 mmol/kg of body weight.
bStandard deviations are in parentheses.
cP<0.05 with respect to relevant vehicle-treated control.
dP<0.05 with respect to relevant estrogen-treated control.
nor 9, the oxybutyric acid and nonphenolic analogues of
5, exhibited any observable effect on uterine weight or
the serum markers of bone resorption.
to a host of other Phase 1 metabolites that, based on
their ER affinities and accumulation in ER-containing
tissues, might contribute to its observed effects.28 Thus,
unequivocal support for the proposition that 7 is a pro-
drug of 6 would seem to require systematic whole-ani-
mal biotransformation studies of 7.
Biotransformation of 7
Components with HPLC retention times of 32.7 and
35.2 min, inferred to correspond to the Z and E isomers
of 6, respectively (see Discussion), were observed in
extracts obtained from incubation of 7 with rat liver 9S
fraction. These were observed only when both 7 and the
cofactor (NADPH) for oxidative drug metabolism were
present in incubation mixtures. These retention times
corresponded to those of authentic 6, and the chroma-
tographic peak intensity of each of these extract com-
ponents was increased by dilution with 6. However, the
respective relative peak areas of these extract compo-
nents (70and 30%) differed from those in 6 (47 and
53%).
Although 7 was not subject to significant observable
geometric isomerization, 6 (shown in Scheme 1 as the Z
isomer) was always accompanied by its E isomer. Thus,
hydrolysis of 6’s Z-ester precursor (12c) under alkaline
conditions (described below), or under mildly acidic
conditions with prolonged reaction times at room tem-
perature, gave 6 and its E isomer in ca. 1:1 ratios.
Separate batches of 6, prepared from 12c by several
repetitions of the current procedure, contained slightly
varying amounts of 6’s E isomer, as determined directly
by 1H NMR spectroscopy and confirmed by HPLC
analysis. HPLC data signified, incidentally, that geo-
metric isomerization of 6 in the chromatographic
mobile phase was negligible. Also, ‘metabolic’ 6, pro-
duced enzymatically from 7, was found to have under-
gone isomerization to the extent of about 30%. Because
of this tendency, no attempt was made to resolve 6 from
its geometric isomer prior to biological evaluation.
Configurationally stable geometric isomers of other
triarylethylenes, such as 1 and its E isomer, exhibit
contrasting ER affinities and estrogenic/antiestrogenic
potencies and efficacies.29,30 Thus, 6 might owe its
observed ER affinity and bone protective and uterine
effects in part to its E isomer.
The observed conversion rate of 7 to components chro-
matographically identical to 6 in cofactor-enriched
incubations with 9S liver fractions was 2.5% per 20min.
Discussion
In the OVX rat, compounds 6 and 7 were comparable to
established SERMs 1 and 2 in terms of degree of selec-
tive bone/uterus estrogenic efficacy.10b,17 Thus, like 1
and 2, these triarylethylene oxybutyric acids suppressed
serum markers of bone resorption/turnover (Dpd and
OC) to a degree approaching that of E2 and EE2 (Table 2),
but only had about one-fourth to one-third the utero-
trophic efficacy of these steroidal estrogens. Adminis-
tered orally, 7 was equivalent to 6 regarding the
magnitude of its differential estrogenic effects, despite its
low ER affinity (Table 1). This might be a consequence
of in vivo enzymatic hydroxylation. In the present
study, 7 was suggested to undergo regioselective 4-
hydroxylation to 6 in the presence of liver enzymes from
OVX rats. The rate of 4-hydroxylation of 7 was similar
to that of 1 in the presence of rat liver enzymes.27 And
4-hydroxy 1, a major in vivo metabolite of 1, had an
RBA at least 50times greater than 1 for rat uterine
ER.28 But 1 was also metabolized oxidatively in the rat
We determined the interaction of 6–10 with human ERa
(hERa), rather than with rat ER, because much recent
information about the topography of hERa interactions
with its ligands was available for comparison purposes.
Compounds 7 and especially 9 each exhibited low affi-
nity for hERa, presumably due to lack of an appro-
priately positioned phenolic substituent. Crystal
structures of the hERa ligand binding domain (LBD)
complexed with high affinity ligands E2 (or 2) indicates
the critical role of respective 3-hydroxy (6-hydroxy)
groups in anchoring each of these ligands to ER by
completing a hydrogen bonding network involving Glu
353 and Arg 394 of the hERa LBD.31 Accordingly, affi-
nity of phenolic ligand 6 was greater than that of 7, whose

V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
1583
affinity is presumably supported only by hydrophobic
and van der Waals interaction of its triarylethylene
nucleus with amino acid residues lining the ER binding
pocket that bear aliphatic chains or aromatic rings.
uterotrophic potencies in the OVX rat were similar.6
This appears to be a consequence of the ease with which
2 undergoes inactivation by glucuronide conjugation,35
a process to which 1 is not directly susceptible. Analo-
gously, it is speculated that 8 might be a better substrate
for metabolic conjugation than either 5 or 6. However,
the degrees to which 5, 6, and 8 undergo metabolic
conjugation have not been assessed.
However, the hER RBA of 6 was less than that of high
affinity ligands like 2. Formal replacement of the butyric
acid side chain of 6 with either a hydrogen or an N,N-
dimethylaminoethyl chain gives 1 ‘bisphenol’ and 4-
hydroxy 1. These had respective hER RBAs of 76
(determined as a standard in the current study) and
123.30 The higher ER affinity of 4-hydroxy 1 is due to
the ability of its (protonated) side chain to interact by
ionic bonding with Asp 351 of hERa LBD, in addition
to participation of its 4-hydroxyl group in the hydrogen
bonding triad specified above.31,32 The reduced ER affi-
nity of 1 bisphenol relative to 4-hydroxy 1 would there-
fore seem to be a consequence of the former’s inability
to make contact with Asp 351. The further reduction in
ER affinity seen in 6, considered in light of the above
observations, indicates that the butyrate side chain of 6
not only is incapable of interaction with a com-
plementary amino acid residue in the hERa LBD, but
also interferes sterically with the ligand binding process.
The goal of this study has been to modify the structure
and polarity of 5 in order to identify putative bone-
protective ER ligands with reduced uterine effects rela-
tive to conventional steroidal estrogens. The current
results indicate that 6 and 7 are capable of such differ-
ential estrogenicity. Evaluation of the potency of these
ER ligands in regard to these and other ER mediated
effects in the OVX rat will be the subject of a subsequent
report.
Experimental
Solvents and chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO), Aldrich Chemical Co.
(Milwaukee, WI) and the University of Georgia Central
Research Stores. Moisture-sensitive and air-sensitive
reactions were carried out in flame or oven dried glass-
ware under dry nitrogen atmosphere. Work up of
organic extracts, filtrates and column fractions was car-
ried out by concentration in vacuo at 40ꢀC. Analytical
thin-layer chromatography (TLC) using 0.25 mm Ana-
ltech silica gel GF254 plates was used to monitor reac-
tion progress and analyze column chromatography
fractions and purity of products. TLC plates were
developed using chloroform/2-propanol/glacial acetic
acid (90:10:0.5, v/v/v) [solvent 1] or toluene/chloroform
(50:50, v/v) [solvent 2], and viewed under UV light at
254 nm wavelength. Chromatographic mobilities are
expressed as Rf values. Melting points were determined
using an Electrothermal 9100 apparatus and are uncor-
rected. 400 MHz proton nuclear magnetic resonance
(1H NMR) spectra were obtained using a Bruker AMX
400 spectrometer. NMR samples were prepared using
acetone-d6 as solvent unless otherwise stated. Chemical
shifts (d) are reported in parts per million and were cal-
culated using tetramethylsilane as standard. Positive ion
liquid secondary ion mass spectra (LSIMS) were
obtained on a Micromass AutoSpec series-M high-
resolution magnetic sector mass spectrometer of EBE
geometry. Sample solutions were prepared using gly-
cerol as the matrix. Elemental analyses were performed
by Atlantic Microlab, Inc., Norcross, GA.
The molecular basis for tissue-selective estrogenic effects
expressed by 1, 2 and other SERMs arises from the dis-
tinct conformation they induce in the LBD of the ER,
compared to that induced by E2.33 Inspection of crystal
structures of SERM-liganded and E2-liganded hERa
LBD indicates the former to have a more ‘open’ con-
formation than the latter, due to an inability of helix 12
of the LBD to fold down completely and enclose the
ligand.31,32 Such complexes are evidently less able to
activate DNA estrogen response elements (EREs) in
uterine tissue, but are nearly as effective as E2-ER
complexes in activation of functionally distinct EREs34
such as those in osteoblasts and osteoclasts, cells
responsible for bone remodelling. In light of the analysis
in the preceding paragraph, 6 interacts differently with
the ER, especially with regard to positioning of its side
chain, than do 1 and 2. However, our results indicating
a similarity of differential bone/uterus estrogenic effi-
cacy of 6 compared to these SERMs suggest that the
conformation of the ER LBD liganded with 6 does not
differ greatly from that arising from interaction with 1
or 2. However, arylacrylic acid 3 inhibited the (partial)
agonist effects of 4-hydroxy 1, and 2, in an estrogen-
responsive line of liver cells transfected with hER.18b
This unique antagonist activity profile suggests a diver-
gence in the way that triarylethylene carboxylic acids
interact with the ER, compared to established SERMs 1
and 2.
Unless indicated otherwise, each unsaturated inter-
mediate and final compound characterized in this study
was composed of approximately equal amounts of its
constituent geometric isomers. Furthermore, no attempt
was made to resolve putative optical isomers of 8–10.
The observed lack of systemic effects of 8 seems to be
counter-intuitive. Its ER affinity was greater than that
of 5, shown previously to have differential bone protec-
tive activity.8 And its polarity (acidity) was estimated to
be comparable to those of 3, 6, and 7, ER ligands shown
previously18 or in the current study to exhibit SERM-like
effects. Efficacy of ER ligands is in part determined by
drug metabolism. For example, although 2 had about six
times greater ER affinity than 1, its bone-protective and
Starting materials
4-Hydroxy-40-benzyloxy-benzophenone (11b) was avail-
able as reported.20 4-Hydroxy-40-(trimethylacetoxy)-

1584
V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
benzophenone (11c) was prepared by a modification of
a previously reported procedure.24 A mixture of 4,40-
dihydroxy-benzophenone (5 g, 23.3 mmol), potassium
carbonate (3.2 g, 23.3 mmol) and trimethylace-
tylchloride (2.8 g, 3.0mL, 23.3 mmol) in dry THF
(40mL) was refluxed for 4 h. After cooling, the reaction
was quenched with water (25 mL) and extracted with
ethyl acetate (3ꢁ50mL). The organic layer was dried
with magnesium sulfate, filtered and concentrated to
give a yellow oil, which solidified upon standing at 8 ꢀC.
This was chromatographed on silica gel (45 g, CH2Cl2/
EtOAc, 19:1). The first 190mL of eluate was discarded.
The next 220mL was collected and concentrated to give
a white solid (0.81 g, 12 %). TLC (solvent 1), one spot,
Rf 0.64; 1H NMR (CDCl3) d 1.38 (s, 9H, Pv), 6.90, 7.17,
7.77, 7.80(d, 8H, ArH); LSIMS m/z calcd for C18H19O4
299.1285 (M+H)+, found 299.1264.
(4.45 g, 3.25 mL, 22.7 mmol). The reaction mixture was
refluxed while stirring for 6 h, and then cooled to room
temperature, filtered and concentrated. The resultant
yellow syrup was dissolved in dioxane (15 mL) and 10%
aqueous NaOH (15 mL) was added. After 1 h of stirring
the reaction was cooled in an ice bath and 10% aq HCl
was added until the mixture was slightly acidic. The
suspension was extracted with ether (3ꢁ20mL). After
work up, the product was crystallized from hot CHCl3-
hexanes at 8 ꢀC and collected as white crystals (1.27 g,
39.0%). TLC (solvent 1), one spot, Rf 0.50; mp 120.1–
125.5 ꢀC; H NMR (CDCl3) d 0.92 (t, 3H, CH2CH3),
1
2.03 (m, 2H, CH2CH2COOH), 2.42–2.52 (m, 4H,
CH2CH3,
CH2COOH),
3.86
(t,
2H,
CH2CH2CH2COOH), 6.51, 6.76 (d, 4H, C6H4OH),
7.08–7.35 (m, 10H, ArH); LSIMS m/z calcd for
C26H26O3 386.1881 (M+), found 386.1863. Anal.
.
(C26H26O3 0.25 H2O) C, H.
General method for olefination of 11a–c. 1-Phenyl-1-(p-
hydroxyphenyl)-2-phenylbut-1-ene (12a) was prepared
by a procedure previously reported23 with slight mod-
ifications. To a cold (ꢂ15 ꢀC), stirred suspension of zinc
powder (4.9 g, 75.5 mmol) in dry tetrahydrofuran
(35 mL) was added slowly titanium tetrachloride (5.7 g,
3.3 mL, 30.2 mmol). The reaction mixture was refluxed
for 2 h and then cooled to 40 ꢀC at which point a solu-
tion of 4-hydroxybenzophenone (11a) (3.0g, 15.1 mmol)
and propiophenone (2.5 g, 2.5 mL, 18.2 mmol) in THF
(15 mL) was added dropwise to the stirred suspension.
The mixture was refluxed for another 4 h, then cooled
and poured into an aqueous solution of 10% potassium
carbonate (300 mL). After standing overnight, the mix-
ture was filtered and the filtrate was concentrated to
give 5.06 g (>100%) of yellow oil. This was used in the
next step without further purification. TLC (solvent 2),
one major spot (>95%), Rf 0.35.
By this same procedure the following compounds were
prepared. Z-4-{[1-(p-benzyloxyphenyl)-2-phenyl-1-ethe-
nyl]phenoxy}-n-butanoic acid (13) crystallized from
CHCl3-hexanes at 8 ꢀC (52%): TLC (solvent 1), one
spot, Rf 0.75; 1H NMR (CDCl3) d 2.13 (m, 2H,
CH2CH2COOH), 2.60(t, 2H, CH2COOH), 4.00 (t, 2H,
CH2CH2CH2COOH), 5.06 (s, 2H, CH2Ph), 6.81–7.46
(m, 18H, ArH); nominal mass calcd for C31H28O4 464,
LSIMS m/z found 464 (M+ꢃ).
4-{[1-(p-Hydroxyphenyl)-2-phenyl-1-butenyl]phenoxy}-n-
butanoic acid (6). A solution of 12c (0.93 g, 2.33 mmol)
in acetone (15 mL) was alkylated with ethyl-4-bromo-
butyrate (2.27 g, 1.67 mL, 11.63 mmol). Upon saponifi-
cation with 10% aqueous NaOH (20 mL) in dioxane
(20mL), and work up, the crude extract was shaken
with 25 mL of ether and 3ꢁ20 mL 0.05 M potassium
phosphate buffer, pH 7.04. The ether layer was worked
up and the residue was crystallized from ether-petro-
leum ether upon standing at 8 ꢀC for several days. Three
batches of white crystals (0.37 g, 40%) were collected:
TLC (solvent 1), one spot, Rf 0.51; mp 141.6–147.6 ꢀC;
1H NMR (CDCl3) d 0.92 (t, 3H, CH2CH3), 2.02, 2.13
(q, 2H, CH2CH2COOH), 2.44–2.62 (m, 4H, CH2CH3,
CH2COOH), 3.87, 4.03 (t, 2H, CH2CH2CH2COOH),
6.45, 6.51, 6.71, 6.73, 6.75, 6.79, 6.85 (d, 7H, 1.75 O-
C6H4), 7.08–7.25 (m, 6H, C6H5, 0.25 O-C6H4); LSIMS
m/z calcd for C26H26O4 402.1831 (M+ꢃ), found
By this same procedure the following compounds were
prepared. Crude 1-[(p-(benzyloxyphenyl)-1-(4-hydro-
xyphenyl)-2-phenyl]ethene (12b) was obtained as a
golden oil after workup. This was chromatographed on
silica gel (49 g, CHCl3/toluene, 50:50). The product
solidified from CHCl3-hexanes as a white solid (34%):
1
TLC (solvent 2), one spot, Rf 0.34; H NMR (CDCl3) d
5.06 (s, 2H, CH2Ph), 6.76–7.42 (m, 13H, ArH).
1-(4-Trimethylacetoxyphenyl)-1-(p-hydroxyphenyl)-2-
phenylbut-1-ene (12c). Yield 69%. TLC (solvent 2), one
spot, Rf 0.20. 1H NMR d 0.89 (t, 3H, CH2CH3), 1.35 (s,
9H, Pv), 2.46 (q, 2H, CH2CH3), 6.49–6.70(d, 4H,
C6H4OH), 7.10–7.29 (m, 9H, ArH); LSIMS m/z calcd
for C27H28O3 400.2038 (M+ꢃ), found 400.2032.
.
402.1828. Anal. (C26H26O4 0.5 H2O) C, H.
4-(1,2-Diphenyl-1-ethenyl)phenoxyacetic acid (14). Soli-
dified from CHCl3-hexanes at 8 ꢀC (56%). TLC (solvent
1
1), one spot, Rf 0.55; H NMR (CDCl3) d 4.70(s, 2H,
CH2COOH), 6.85–7.32 (m, 14H, ArH).
1,2-Diphenyl-1-(p-hydroxyphenyl)ethene (12d). Yield
30%. TLC (solvent 2), one spot, Rf 0.29; 1H NMR
(CDCl3) d 1.59 (br s, 1H, OH), 6.77–7.65 (m, 14 H,
ArH).
Z-4-[1-(p-Benzyloxyphenyl)-2-phenyl-1-ethenyl]phenox-
yacetonitrile (15). Synthesized by a similar procedure as
described above. A solution of 12b (1.03 g, 2.72 mmol)
in acetone (25 mL) was stirred with K2CO3 (1.09 g,
2.9 mmol) and bromoacetonitrile (1.45 g, 0.84 mL,
12.1 mmol). The mixture was refluxed for 2 h after
which it was filtered. The filtrate was then concentrated
to give a yellow oil (1.8 g), which solidified upon stand-
ing. This was dissolved in toluene and the solution was
General method for alkylation of phenols 12a–d. The
synthesis of Z-4-[p-(1,2-diphenyl-1-butenyl)phenoxy]-n-
butanoic acid (7) is typical. To a solution of 12a (2.53 g,
8.45 mmol) in acetone (20mL) was added potassium
carbonate (1.4 g, 10.1 mmol) and ethyl-4-bromobutyrate

V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
1585
filtered through a silica gel column (36 g, toluene) to
Animal studies
remove impurities. A total of 250mL of eluate were
collected, combined and concentrated to give white
crystals (1.0g, 88%). TLC (solvent 1), one spot, Rf 0.83;
1H NMR (CDCl3) d 4.77 (s, 2H, CH2CN), 5.07 (s, 2H,
CH2Ph), 6.81–7.44 (m, 13H, ArH).
OVX Sprague–Dawley rats (10–12 weeks old) were
obtained from Harlan, Inc., Indianapolis, IN. Animals
were housed and fed as described.8 Test compounds (6
and 8–10) for subcutaneous (sc) dosing were adminis-
tered in 5% benzyl alcohol–corn oil. For oral (po) dos-
ing, compounds 6 and 7 were each dissolved in 80%
ethanol containing an equimolar amount of trometha-
mine. At the time of use, such stock solutions were
diluted to 1/10the original compound concentration by
addition of 0.11% aqueous methylcellulose. Daily dos-
ing was carried out five days per week for three weeks.
Each treatment group had seven animals, and each
experiment included groups treated in turn with vehi-
cle or estrogen, in addition to groups receiving test
compounds.
Z-1-(p-Benzyloxyphenyl)-1-[4-(1H-tetrazol-2-ylmethoxy)
phenyl]-2-phenyl-ethene (16). A solution of 15 (1.0g,
2.4 mmol) and NH4Cl (0.38 g, 7.2 mmol) in DMF
(25 mL) was stirred and NaN3 (0.31 g, 4.8 mmol) was
carefully added. The reaction was refluxed for 48 h, then
filtered and concentrated to give a brown oil. The oil
was dissolved in DMF (20mL) and 10% aq HCl
(10mL) was added. The mixture was extracted with
ether (3ꢁ15 mL). The combined ether extracts were
shaken with 10% aq NaOH (30 mL). The aqueous
extract was acidified to pH 4 by addition of 10% HCl.
A white solid separated. This was filtered: (0.9g, 82 %).
TLC (solvent 1), one spot, Rf 0.56; 1H NMR d 5.14 (s, 2H,
CH2Ph), 5.56 (s, 2H, CH2-Ar), 6.91–7.27 (m, 13H, ArH).
At the end of the study period, each animal was eutha-
nized under carbon dioxide. Blood was aspirated by
syringe from the abdominal aorta and allowed to coa-
gulate in a Vacutainer tube at room temperature for 2 h.
Serum was obtained by centrifugation for 10min at
3000 rpm, and samples were stored at ꢂ80 ꢀC until
analyzed for OC or Dpd. Uterine tissue was removed,
dissected free of fat and connective tissue, and weight
was recorded.
Catalytic hydrogenation/hydrogenolysis of 13, 14, and
16. To a solution of 13 (0.7 g, 1.5 mmol) in ethanol
(40mL) and THF (10mL) was added 10% palladium
on activated carbon (70mg). The mixture was shaken
under ꢄ45 psi of H2 for 1.5 h. The mixture was filtered
after addition of methylene chloride (20mL). The fil-
trate was concentrated in vacuo to give a yellow oil.
This was chromatographed on silica gel (25 g, 15%
EtOAc in hexanes) to give 4-{4-[1-(p-hydroxyphenyl)-2-
phenylethyl]phenoxy}-n-butanoic acid (8) (0.34 g,
59.6%), which crystallized from CHCl3-hexanes. TLC
For drug metabolism studies, livers from four of the
aqueous vehicle-treated animals were dissected, com-
bined, minced, and homogenized in three volumes of
1.15% ice-cold aq KCl using a tissue homogenizer. The
homogenate was centrifuged at 9000ꢁg for 25 min at
4 ꢀC. Aliquots (5 mL) of the supernatant were lyophilized
and stored at ꢂ80 ꢀC prior to use.
(solvent 1), one spot, Rf 0.72; mp 124.7–128.8 ꢀC; H
1
NMR d 2.47 (t, 2H, CH2COOH), 3.29 (d, 2H), 3.97 (t,
2H, CH2CH2CH2COOH), 4.17 (t, 1H), 6.70, 6.79 (d,
4H, C6H4OH), 7.00–7.17 (m, 9H, ArH); nominal mass
Assays for OC and Dpd. Serum samples were thawed by
placing containers on ice for 2 h. Properly diluted sam-
ples were assayed for OC using an enzyme immu-
noassay kit (Biomedical Technologies, Inc., Stoughton,
MA). The procedure was carried out in a 96-well poly-
styrene plate in which a monoclonal antibody to the N-
terminal region of rat OC was bound to each well sur-
face. After overnight incubation with the diluted serum
sample, wells were washed and incubated with a second
antibody (goat polyclonal), which interacted with the C-
terminal region of the immobilized OC. Subsequent
incubation with horseradish peroxidase (HRP) con-
jugated donkey-anti goat IgG, and then a solution of
HRP substrate, 3,30,5,50-tetramethylbenzidine, was car-
ried out. Absorbance at 405 nm, which accompanied
substrate oxidation, was determined using a plate
reader. The amount of OC in the sample was calculated
by comparing its absorbance with those of standards,
which contained known amounts of rat OC. Absor-
bance intensity was directly proportional to the amount
of OC present in the sample.
calcd for C24H24O4 376, LSIMS m/z found 377
+
.
(M+H) . Anal.(C24H24O4 H2O) C, H.
By this same procedure the following compounds were
prepared. 4-(1,2-diphenylethyl)phenoxyacetic acid (9)
(28%): TLC (solvent 1), one spot, Rf 0.57; mp 161.0–
167.0 ꢀC; H NMR d 3.36 (d, 2H), 4.31 (t, 1H), 4.64 (s,
1
2H, CH2COOH), 6.82 (d, 2H, 0.5 O-C6H4), 7.07–7.32
.
(m, 12H, 0.5 O-C6H4, ArH). Anal. (C22H20O3 H2O) C,
H.
1-(p-Hydroxyphenyl)-1-[4-(1H-tetrazol-4-ylmethoxy)phe-
nyl]-2-phenyl-ethane (10). Crystallized as white crystals
from acetone-H2O at 8 ꢀC (72%): TLC (solvent 1), one
ꢀ
1
spot, Rf 0.40; mp 108.2–109.5 C; H NMR d 3.33 (d,
2H), 4.24 (t, 1H), 5.49 (s, 2H, CH2-Ar), 6.73, 6.95 (d,
4H, C6H4OH), 7.12–7.17 (m, 7H, C6H5, 0.5 O-C6H4),
7.26 (d, 2H, 0.5 O-C6H4); LSIMS m/z calcd for
C22H21N4O2 373.1664 (M+H)+, found 373.1643. Anal.
.
(C22H20N4O2 2H2O) C, H, N.
Estrogen receptor affinity. The ability of 6–10, and stan-
dard ER ligands 1, 2, and 1-‘bisphenol’, to displace
specifically bound [3H]17b-estradiol from human
recombinant ERa was determined as described pre-
viously.21
Alternatively, thawed serum samples were analyzed for
total Dpd, using a hydrolysis/competitive enzyme
immunoassay procedure (Metra Biosystems, Inc.,
Mountain View, CA). Each serum sample was mixed
with 6 N HCl plus solubilizing agent. Precipitated

1586
V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
protein was separated by centrifugation at 10,000ꢁg for
10min. An aliquot of the supernatant was heated at
99 ꢀC for 18 h to hydrolyze that portion of serum Dpd
linked to polypeptides. This was neutralized by addition
of 10N NaOH, and an aliquot was transferred to the 96
well assay plate, each well containing monoclonal anti-
Dpd antibody. Then a fixed amount of Dpd-alkaline
phosphatase conjugate was added. After a 2 h incuba-
tion, wells were washed and a solution of p-nitrophenyl
phosphate was added. After a second 2 h incubation,
alkaline stop solution was added and absorbance of
formed p-nitrophenoxide was determined at 405 nm
using a plate reader. The amount of Dpd in the sample
was calculated by comparing absorbance of the sample
with that of standards, run in parallel, which contained
known amounts of Dpd. Absorbance intensity was
inversely proportional to the amount of Dpd originally
present in the sample.
3. (a) Magarian, R. A.; Overacre, L. B.; Singh, S.; Meyer,
K. L. Curr. Med. Chem. 1994, 1, 61. (b) Jordan, V. C. Annu.
Rev. Pharmacol. 1995, 35, 195.
4. (a) Love, R. R.; Mazers, R. B.; Tormey, D. C.; Barden,
H. S.; Newcomb, P. A.; Jordan, V. C. Breast Cancer Res.
Treat 1988, 12, 297. (b) Fornander, T.; Rutquist, L. E.; Sjo-
berg, H. E.; Blomquist, L.; Mattsson, A.; Glas, U. J. Clin.
Oncol. 1990, 8, 1019.
5. (a) Black, L. J.; Sato, M.; Rowley, E. R.; Magee, D. E.;
Bekele, A.; Williams, D. C.; Cullinan, G. J.; Bendele, R.;
Kauffmann, R. F.; Bensch, W. R.; Frolik, C. A.; Termine,
J. D.; Bryant, H. U. J. Clin. Invest. 1994, 93, 63. (b) Turner,
C. H.; Sato, M.; Bryant, H. U. Endocrinology 1994, 135, 2001.
(c) Riggs, B. L.; Melton, L. J. III. N. Engl. J. Med. 1992, 327,
620.
6. Sato, M.; Rippy, M. K.; Bryant, H. U. FASEB J. 1996, 10,
905.
7. (a) Dhingra, K. Invest. New Drugs 1999, 17, 285. (b)
Merchenthaler, I.; Funkhouser, J. M.; Carver, J. M.; Lundeen,
S. G.; Ghosh, K.; Winneker, R. C. Maturitas 1998, 30, 307. (c)
Khovidhunkit, W.; Shoback, D. M. Ann. Int. Med. 1999, 130,
431.
Biotransformation of 7. Metabolism of 7 with 9000ꢁg in
supernatant (9S) fraction prepared from pooled livers of
vehicle-treated OVX rats was carried out as follows.
Triplicate incubations were run in 12ꢁ75 mm poly-
propylene tubes. The standard incubation mixture
(1.0mL) contained 20mM potassium phosphate buffer,
pH 7.05, 90 mM potasssium chloride, 5 mM magnesium
chloride, 0.4 mM NADP, 6.5 mM glucose 6-phosphate,
and 9S fraction equivalent to 50mg of wet liver. Each
incubation was started by addition of 7 in 20 mL of
DMF to give a final concentration of 0.1 mM (38 mg/
mL). In control incubations, either the cofactor mixture
(NADP and glucose-6-phosphate), or 7, wasꢀomitted.
Incubations were shaken at 70cycles/min at 37 C for 20
min, and then to each was added 0.1 mL of 50 mM
EDTA disodium salt. Each mixture was vortexed and
poured into 3 mL of methanol. The mixture was shaken
for 5 min and then centrifuged for 10min at 450 ꢁg. The
supernatant was concentrated at 40 ꢀC to low volume
under a stream of compressed nitrogen gas, and the
aqueous concentrate was lyophilized. The residue was
dissolved in 1 mL of water and the mixture was shaken
for 5 min with 3 mL of ether. The mixture was cen-
trifuged for 10min at 450 ꢁg. The ether layer (2.0mL)
was concentrated as before. The residue was recon-
stituted in 100 mL of HPLC mobile phase and subjected
to high performance liquid chromatography (HPLC).
Column: 4.6ꢁ250mm stainless steel, packed with 10 mm
Whatman1Partisil1 ODS-3 (Mitchell modification);
mobile phase: MeOH ꢂ40mM sodium phosphate buf-
fer, pH 2.45 (67/33, v/v), 1.0mL/min; UV detection at
277 nm; 20 mL flushed loop injection. Retention times
(relative % area) for the geometric isomers of 6 were
32.7 min (47%) and 35.2 min (53%).
8. Ruenitz, P. C.; Shen, Y.; Li, M.; Whitehead, R. D., Jr.;
Pun, S.; Wronski, T. J. Bone 1998, 23, 537.
9. (a) Moon, L. Y.; Wakley, G. K.; Turner, R. T. Endocri-
nology 1991, 129, 1568. (b) Turner, R. T.; Wakley, G. K.;
Hannan, K. S.; Bell, N. H. Endocrinology 1988, 122, 1146.
10. (a) Wronski, T. J.; Cintron, M.; Doherty, A. L.; Dann,
L. M. Endocrinology 1988, 123, 681. (b) Williams, D. C.; Paul,
D. C.; Black, L. J. Bone Miner. 1991, 14, 205.
11. (a) Quigley, M. E. T.; Martin, P. L.; Curnier, A. M.;
Brooks, P. Am. J. Obstet. Gynecol. 1987, 156, 1516. (b) Cau-
ley, J. A.; Seeley, D. G.; Ensrud, K.; Ettinger, B.; Black, D.;
Cummings, S. R. Ann. Intern. Med. 1995, 122, 9.
12. Delmas, P. D.; Bjarnason, N. H.; Mitlak, B. H.; Ravoux,
A. C.; Shah, A. S.; Huster, W. J.; Draper, M.; Christiansen,
C. N. Engl. J. Med. 1997, 337, 1641.
13. Delmas, P. D. J. Bone Miner. Res. 8, S549. 1993.
14. (a) Tarallo, P.; Henny, J.; Fournier, B.; Sniest, G. Scand.
J. Clin. Lab. Invest. 1990, 50, 649. (b) Brown, J. P.; Delmas,
P. D.; Malaval, L.; Edouard, C.; Chapuy, M. C.; Meunier,
P. J. Lancet 1984, 1, 1091.
15. Black, D.; Farquharson, C.; Robins, S. P. Calcif. Tissue
Int. 1989, 44, 343.
16. Delmas, D. D.; Malaval, L.; Artol, M. E.; Meunier, P. J.
Bone 1985, 6, 339.
17. Frolik, C. A.; Bryant, H. V.; Black, E. C.; Magee, D. E.;
Chandrosekhar, S. Bone 1996, 18, 621.
18. (a) Willson, T. M.; Henke, B. R.; Momtahen, T. M.;
Charifson, P. S.; Batchelor, K. W.; Lubahn, D. B.; Moore,
L. B.; Oliver, B. B.; Sauls, H. R.; Triantafillou, J. A.; Wolfe,
S. G.; Baer, P. G. J. Med. Chem. 1994, 37, 1550. (b) Willson,
T. M.; Norris, J. D.; Wagner, B. L.; Asplin, I.; Baer, P.;
Brown, H. R.; Jones, S. A.; Henke, B.; Sauls, H.; Wolfe, S.;
Morris, D. C.; Macdonnell, D. P. Endocrinology 1997, 138,
3901.
19. Wilson, S.; Ruenitz, P. C.; Ruzicka, J. A. J. Steroid Bio-
chem. Mol. Biol. 1992, 42, 613.
20. Ruenitz, P. C.; Bourne, C. S.; Sullivan, K. J.; Moore, S. A.
J. Med. Chem. 1996, 39, 4853.
21. Kraft, K. S.; Ruenitz, P. C.; Bartlett, M. G. J. Med. Chem.
1999, 42, 3126.
References
22. Ruenitz, P. C.; Bai, X. Drug Metab. Dispos. 1995, 23, 993.
23. Coe, P. L.; Scriven, C. E. J. Chem. Soc., Perkin Trans. 1
1986, 475.
24. Finnegan, W. G.; Henry, R. A.; Lofquist, R. J. Amer.
Chem. Soc. 1958, 80, 3908.
1. Griffing, G. T.; Allen, S. H. Postgrad. Med. 1994, 96, 131.
2. (a) Colditz, G. A.; Hankinson, S. E.; Hunter, D. J.; Willett,
W. C.; Manson, J. E.; Stampfer, M. J.; Hennekens, C.; Ros-
ner, B.; Speizer, F. E. N. Engl. J. Med. 1995, 332, 1589. (b)
Grady, D.; Gebretsadik, T.; Kerlikowski, K.; Ernster, V.;
Petitti, D. Obstet. Gynecol. 1995, 85, 304.
25. Gauthier, S.; Mailhot, J.; Labrie, F. J. Org. Chem. 1996,
61, 3890.

V. N. Rubin et al. / Bioorg. Med. Chem. 9 (2001) 1579–1587
1587
26. (a) Brown, H. C.; McDaniel, D. H.; Hafliger, O. In
Determination of Organic Structures by Physical Methods;
Braude, E. A.; Nachod, F. C., Eds.; Academic: New York,
1955; Vol. 1, pp 567–662. (b) Lange’s Handbook of Chemistry;
Dean, J. A., Ed.; McGraw-Hill, Inc.: New York, 1999; Vol.
15, pp 8. 24–8, 79.
30. Bignon, E.; Pons, M.; Crastes de Paulet, A.; Dore, J.-C.;
Gilbert, J.; Abecassis, J.; Miquel, J.-F.; Ojasoo, T.; Raynaud,
J.-P. J. Med. Chem. 1989, 32, 2092.
31. Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard,
R.E., Bonn, T., Engstrom, O., Ohman, L., Greene, G.L.,
˚
Gustafsson, J-A., Carlquist, M. Nature 1997, 389, 753.
27. Ruenitz, P. C.; Bagley, J. R.; Pape, C. W. Drug Metab.
Dispos. 1984, 12, 478.
28. (a) Robertson, D. W.; Katzenellenbogen, J. A.; Long,
D. J.; Rorke, E. A.; Katzenellenbogen, B. S. J. Ster. Biochem.
1982, 16, 1. (b) Ruenitz, P. C.; Bagley, J. R. Drug Metab.
Dispos. 1985, 13, 582.
32. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.;
Kushner, P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95, 927.
33. Levenson, A. S.; Jordan, V. C. Eur. J. Cancer 1999, 35,
1628.
34. DiPippo, V. A.; Powers, C. A. J. Pharmacol. Exp. Ther.
1997, 281, 142.
29. (a) Jordan, V. C.; Haldemann, B.; Allen, K. E. Endocri-
nology (Baltimore) 1981, 108, 1353. (b) Jordan, V. C. Phar-
macol. Rev. 1984, 36, 245.
35. Lindstrom, T. D.; Whitaker, N. G.; Whitaker, G. W.
Xenobiotica 1984, 14, 841.