A selective estrogen receptor modulator for the treatment of hot flushes

Article abstract of DOI:10.1021/jm0509795

A selective estrogen receptor modulator (SERM) for the potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy) phenyl]-5H-6-oxachrysen-2-ol, LSN2120310, potently binds ERα and ERβ and is an antagonist in MCF-7 b

Full text of DOI:10.1021/jm0509795

J. Med. Chem. 2006, 49, 843-846
843
A Selective Estrogen Receptor Modulator for the
Treatment of Hot Flushes
Owen B. Wallace,*.† Kenneth S. Lauwers,†
Jeffrey A. Dodge,† Scott A. May,† Joel R. Calvin,†
Ronald Hinklin,‡ Henry U. Bryant,† Pamela K. Shetler,†
Mary D. Adrian,† Andrew G. Geiser,† Masahiko Sato,† and
Thomas P. Burris†
Lilly Research Laboratories, Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285, and
Array Biopharma, 2600 Trade Center AVenue,
Longmont, Colorado 80503
ReceiVed September 30, 2005
Abstract: A selective estrogen receptor modulator (SERM) for the
potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-
5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol, LSN2120310,
potently binds ERR and ERâ and is an antagonist in MCF-7 breast
adenocarcinoma and Ishikawa uterine cancer cell lines. The compound
is a potent estrogen antagonist in the rat uterus. In ovariectomized rats,
the compound lowers cholesterol, maintains bone mineral density, and
is efficacious in a morphine dependent rat model of hot flush efficacy.
Figure 1.
Table 1 a
Ishikawa
antagonist
binding
MCF7
agonist
Ki ERR
(nM)
Ki ERâ
(nM)
IC50
(nM)
EC50 efficacy
IC50
(nM)
efficacy
(%)
(nM)
(%)
Hot flushes (flashes) are characterized by a warming sensation
that begins in the chest and moves toward the neck and head
and are often accompanied by sweating, palpitations, and
cutaneous flushing. The episodes generally last from 30 s to 10
min. The majority of postmenopausal women will experience
hot flushes, with a significant percentage of these women
continuing to suffer symptoms for more than five years.1,2 The
hot flush event itself is thought to be centrally mediated,
resulting from a transient lowering of the thermoregulatory set
point in the hypothalamus.3 Regulation of the thermoregulatory
process may involve catecholamines, estrogen, testosterone,
opioids, and serotonin, among others.4 In fact, compounds that
modulate the signaling pathways of each of these hormones/
neurotransmitters have been evaluated clinically for the treat-
ment of hot flushes. Unfortunately, all the investigated therapies
suffer from poor efficacy, are associated with unacceptable side
effects, or are contraindicated for certain patient populations.3
Despite being identified as an ailment of menopause for
hundreds of years, the precise mechanism underlying the
alteration of the thermoregulatory set point is not clear. The
link with declining estrogen levels is widely recognized,
however, and compounds that interact with the estrogen receptor
can have a significant effect on the induction or alleviation of
hot flushes. Tamoxifen (Figure 1) was reported to induce hot
flushes in more than 50% of patients,5 and raloxifene was also
reported to increase the incidence of hot flushes in clinical trials.6
Estrogen or hormone replacement therapy (ERT or HRT) is
currently the treatment of choice and is effective in >80% of
women who initiate treatment. However, estrogen replacement
therapy is not recommended for women with a history of breast
cancer, uterine cancer, ovarian cancer, or venous thrombo-
embolism. Recent data also suggests HRT may not be suitable
for women with coronary artery disease.7 Perhaps because of
these concerns and/or the incidence of undesirable side effects,
the compliance rate for women on ERT or HRT is remarkably
1
2
0.3 ( 0.1 0.5 ( 0.2 1.4 (0.9
NC 3.2 ( 1.0 5.3 ( 1.2 100 ( 7
5.8 ( 0.8 54 ( 7
330 ( 100 NC 3.3 ( 1.6 430 ( 100 68 ( 33
a NC: not calculated.
low.8 The need for an improved treatment is clear. A recent
report detailed a spiro indane derivative that was shown to be
efficacious in preclinical models of hot flushes but had weak
ER binding and weak antagonist properties in a breast cancer
cell line (MCF-7).9 In addition, it was not reported to have
antagonist activity on the uterus. We have identified a compound
(1, Figure 1) that possesses a desirable SERM profile on uterine,
breast, bone, and cardiovascular parameters, while display-
ing potent and efficacious activity in a morphine-dependent
ovariectomized (OVX) rat model of hot flush efficacy. The
compound is related to a series of conformationally restricted
raloxifene analogues reported by Grese et al.10
The synthesis began with naphthalene triflate 3 (Scheme 1).
To facilitate liberation of the A-ring phenol at the end of the
synthesis, a protecting group transposition was undertaken. The
HCl salt of 3 was first made in order to minimize ethoxy-
piperidine side chain cleavage during the subsequent BBr3-
mediated demethylation. Treatment of the HCl salt of triflate 3
with boron tribromide afforded a quantitative yield of phenol 4
which was then treated with MsCl and Hunig’s base to afford
5 in excellent yield (99%). This versatile intermediate was then
converted in situ to the corresponding neopentylglycolato
boronic ester under palladium catalysis. Subsequent addition
of 1-bromo-2,3,5-trifluorobenzene and additional bis(acetato)-
bis-(triphenylphosphine)palladium catalyst afforded the desired
coupled product. This material was then filtered and the mesyl
protecting group was cleaved using KOH in MeOH at room
temperature to yield phenol 6 (64% yield from 5). Asymmetric
reduction of the benzophenone carbonyl was achieved using
the Corey protocol.11 Namely, ketone 6 was treated with
(R)-(+)-R,R-diphenyl-2-pyrrolindinemethanol and borane in
THF, followed by addition of ethanolamine to decomplex the
product, to afford chiral alcohol 7 in excellent yield (98%) and
enantiomeric purity (99.2% ee). The final step in the synthesis
involved a base-promoted intramolecular SNAr reaction whereby
* Corresponding author. Tel: +317 433 1311. Fax: + 1 317 433 0715;
e-mail: owen.wallace@lilly.com.
† Lilly Research Laboratories.
‡ Array Biopharma.
10.1021/jm0509795 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/12/2006

844 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Letters
Scheme 1a
a a. i. HCl ii. BBr3. b. MsCl, i-Pr2NEt. c. i. bis(neopentyl glycolato)diboron, Pd(OAc)2(PPh3)2, CsF, ACN, 75 °C. ii. 1-bromo-2,3,5-trifluorobenzene. iii.
KOH, MeOH. d. i. (R)-(+)-R,R-diphenyl-2-pyrrolidinemethanol, BH3-THF, 45 °C. ii. ethanolamine e. i. KOtBu, THF, ii. HCl.
Figure 2. Three week old Sprague Dawley female rats were orally
treated with estradiol (0.1 mg/kg) and 1.0, 0.1, and 0.01 mg/kg SERM
for 3 days, 6 rats per group. Test compounds were dissolved in 20%
â-hydroxy-propylcyclodextrin and administered orally 15 min prior to
E2. Animals were weighed and then euthanized (by carbon dioxide
asphyxiation), and the uteri were collected and weighed. The percentage
inhibition of the estrogen-induced response was calculated as: 100 ×
(UWRestrogen - UWRtest compound/UWRestrogen - UWRcontrol) where UWR
) uterine weight/body weight ratio. *Significant decrease from estradiol
alone for each dose, p < 0.05.
Figure 3. 75 day old SD rats were ovariectomized and were orally
treated with ethinyl estradiol (EE2, 0.1 mg/kg) or compound 1 once
daily starting 14 days post-ovariectomy. After 4 days of treatment,
eosinophil peroxidase (EPO) activity was measured. *All groups
significantly different from control, p < 0.05.
240-fold more potent than its antipode, a trend that is consistent
with the decreased binding affinity of 2. Prior to testing the
compounds in vivo for their ability to act as agonists or
antagonists on the uterus, the compounds were profiled in an
Ishikawa human uterine cell line. Agonist activity was deter-
mined by quantitating alkaline phosphatase induction by the
compound alone, and antagonist activity was determined by
inhibition of alkaline phosphatase induction by the compound
in the presence of 1 nM 17â-estradiol.14 Compound 1 was thus
determined to be a highly potent (IC50 ) 5.3 ( 1.2 nM) and
efficacious (100 ( 7% efficacy) antagonist, while displaying
weak and variable agonist activity of approximately 3.2%
relative to 17â-estradiol.
In vivo uterine effects of the compound were assessed in an
immature rat model of estrogen antagonist activity and in an
ovariectomized rat model of estrogen agonist activity on uterine
stimulation. In the immature (3 week old) rats, benzopyran 1
was capable of inhibiting the effects of administered estrogen
at oral doses of 0.01-1 mg/kg/day for 3 days (Figure 2) and was
remarkably potent in this assay (ED50 ) 0.03 mg/kg). Converse-
ly, compound 2 displayed no dose dependent antagonism of
estradiol stimulation (data not shown), which again is consistent
with the weak binding and functional profile of 2 in vitro.
In a 4-day OVX model, we measured eosinophil peroxidase
(EPO) activity which is a very sensitive gauge of estrogenic
activity in the uterus.15 Activity in this model was compared to
8 (LY117018, Figure 1), an analogue of raloxifene that has been
characterized extensively in vivo.16 Compound 1 displayed
alcohol 7 was heated with K-OBu-t in THF to afford (R)-(+)-
7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxach-
rysen-2-ol, 1, in 97% yield.12 The (S) enantiomer 2 was isolated
by chiral chromatography of a racemic mixture resulting from
a nonasymmetric reduction of benzophenone 6 followed by
cyclization.
Benzopyran 1 was initially examined for its ability to bind
to the estrogen receptor using a cell free competition binding
assay where displacement of 3H-17â-estradiol by the compound
was measured (Table 1).13 The compound was tested a total of
seven times and found to possess high affinity to both ERR (Ki
) 0.3 ( 0.1 nM) and ERâ (Ki ) 0.5 ( 0.2 nM) and was
considerably more potent than its enantiomer 2 (Ki ERR ) 5.8
( 0.8 nM; Ki ERâ ) 54 ( 7 nM).
Since the breast is highly estrogen responsive, and HRT has
been implicated with an increase in the risk of breast cancer,
we considered it important that desirable compounds were
estrogen antagonists in mammary cells. In addition, the estrogen
receptor ligand previously reported to be efficacious as a hot
flush agonist possessed only very weak activity in a breast
cancer proliferation assay.9 Profiling was therefore undertaken
to determine the antagonist effects of our compounds on breast
adenocarcinoma-derived MCF-7 cells stimulated by 10 pM 17â-
estradiol.13 Compound 1 was a potent inhibitor of cell prolifera-
tion in this assay (IC50 ) 1.4 ( 0.9 nM) and was approximately

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 845
Figure 5. 60 day old SD rats were ovariectomized and were orally
treated with either ethinylestradiol (EE2, 0.3 mg/kg) or test compound
once daily starting on day 14 postovariectomy. On days 15 and 17, a
single 75 mg morphine pellet was surgically implanted. On day 21,
animals were anesthetized with ketamine (80 mg/kg, im) 2 h after the
final dose of compound. Temperature-sensitive probes were applied
to the dorsal side of the tail base, and temperature measurements were
recorded every 15 s for a 1 h period. 45 min after ketamine
administration, animals were treated with naloxone (1 mg/kg, sc), and
temperature changes (AUC) were recorded for 45 min. *Significant
decrease from control, p < 0.05. (a) Data for compound 1. (b) Data
for compound 8.
eral density (BMD) and cholesterol changes were measured.
As can be seen in Figure 4a, compound 1 significantly
maintained BMD relative to the loss seen in OVX control rats
at doses as low as 0.1 mg/kg. The ability to block ovariectomy-
induced bone loss was comparable to compound 8. Compound
1 also displayed potent lowering of cholesterol in this model
where just 0.01 mg/kg of compound reduced serum cholesterol
by approximately 50% (Figure 4b). We also measured uterine
growth response to see if long-term dosing would cause an
agonist response that was not seen with the short-term dosing
described above. As shown in Figure 4c, there was minimal
activity on uterine wet weight, less than that seen with 8. Thus,
benzopyran 1 possesses the desired agonist profile on the skeletal
and cardiovascular systems, while maintaining potent antagonist
activity in reproductive tissues.
The ability of the compound to act as a potential therapy for
the treatment of hot flushes was assessed using a slight
modification of the morphine dependent rat model. In this assay,
estrogen, or compounds that act as estrogen agonists in the
thermoregulatory centers, attenuate the increase in tail skin
temperature observed on treatment of morphine dependent rats
with an opioid antagonist such as naloxone. This model, or
modifications thereof, has been widely used to evaluate the
effect of SERMs and estrogens on vasomotor activity.17
Compound 1 exhibited a dose-dependent response and had a
significant effect on temperature at doses of 1, 5, and 10 mg/
kg (Figure 5a). Conversely, raloxifene analogue 8 in an identical
assay showed no effect on temperature relative to the morphine
control at doses of 0.1 to 30 mg/kg (Figure 5b).
Figure 4. Virgin 6 month old SD rats were ovariectomized and were
orally treated with compound once daily starting 3 days postovariec-
tomy. After 42 days of treatment, animals were sacrificed. (a)
Volumetric bone mineral density (vBMD) of the mid-transverse section
of lumbar vertebra L-4 was measured using quantitative computed
tomography. *Significant increase from OVX control, p < 0.05. (b)
Serum cholesterol levels were determined by measurement of choles-
terol esterase/cholesterol oxidase activity utilizing a Roche/Hitachi 917
automated chemistry analyzer. *Significant decrease from OVX control,
p < 0.05. (c) Uteri were extracted and their wet weight was measured.
*Significant increase from OVX control, p < 0.05.
minimal agonist effects in this assay, with slight effects on EPO
activity observed and comparable to benzothiophene 8 (Figure
3). The data from the immature rat model and the 4-day OVX
model is consistent with the in vitro Ishikawa data, which
suggested that 1 is a potent antagonist and weak agonist in the
uterus.
Bone and cardiovascular effects as well as long-term effects
on the uterus were assessed in a 42 day OVX rat model of
estrogen deficiency-induced osteopenia, where both bone min-
In summary, we have developed an asymmetric synthesis of
(R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-

846 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Letters
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6-oxachrysen-2-ol (LSN2120310), 1, in high yield and excellent
ee from naphthalene triflate 3. Benzopyran 1 exhibits a typical
SERM profile in vitro, in that it potently binds ERR and ERâ
and is an antagonist in breast (MCF-7) and uterine (Ishikawa)
cancer cell lines. In vivo, compound 1 is a potent antagonist of
estrogen action on the uterus in an immature rat model. In a
longer term in vivo assay in ovariectomized rats, the compound
increases bone mineral density, lowers serum cholesterol, and
exhibits minimal uterine agonist activity. Compound 1 also
displays dose-dependent activity in an ovariectomized rat model
of hot flush efficacy and may have utility for the treatment of
a variety of menopausal symptoms.
(12) The absolute configuration was assigned based on an X-ray crystal
structure of 1 bound to ERR.
(13) For a detailed description of the ER binding and MCF-7 assay
protocols, see: Wallace, O. B.; Lauwers, K. S.; Jones, S. A.; Dodge,
J. A. Tetrahydroquinoline-based selective estrogen receptor modula-
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Wallace, O. B.; Bryant, H. U.; Shetler, P. K.; Adrian, M. D.; Geiser,
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Bioorg. Med. Chem. Lett. 2004, 14, 5103-5106.
Acknowledgment. We thank members of the Lead Opti-
mization Biology group for conducting ER binding, and MCF-7
and Ishikawa assays, and Ms. Julie Ruff for her assistance with
the preparation of this manuscript.
Supporting Information Available: Detailed experimental
procedures for the synthesis and characterization of 1. This material
is available free of charge via the Internet at http://pubs.acs.org.
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