Discovery and preclinical characterization of (+)-3-[4-(1-piperidinoethoxy)phenyl]spiro[indene-1,1′-indane]-5, 5′-diol hydrochloride: A promising nonsteroidal estrogen receptor agonist for hot flush

Article abstract of DOI:10.1021/jm034134+

In our studies of the development of a novel class of selective estrogen receptor modulators, (+)-3-[4-(1-piperidinoethoxy)phenyl]spiro[indene-1,1′ -indane]-5,5′-diol hydrochloride (1) was found to be an estrogen receptor ligand with beneficial effects in

Full text of DOI:10.1021/jm034134+

J . Med. Chem. 2003, 46, 3961-3964
3961
for the prevention of breast cancer in high-risk women,
is effective for all stages of hormone-dependent breast
cancer, whereas RAL is prescribed for the prevention
and treatment of osteoporosis in postmenopausal
women.6 While SERMs on the market have beneficial
effects on bone and lipid metabolism antagonizing the
effects of estrogens on the uterus and breasts, no SERM
has been reported to have a beneficial effect on hot
flush.6,7 In our studies of the development of a novel
class of SERMs with beneficial effects on hot flush, we
have found spiro[indene-1,1′-indane]-5,5′-diol as a new
framework for estrogen receptor ligands and assumed
that a series of related compounds might be imparted
with unprecedented biological properties due to their
new structural characteristics.8 Herein, we disclose the
synthesis and unique biological profile of (+)-3-[4-(1-
piperidinoethoxy)phenyl]spiro[indene-1,1′-indane]-5,5′-
diol hydrochloride [1, OS-689; ent-1, 2; Figure 1].9
Discover y a n d P r eclin ica l
Ch a r a cter iza tion of (+)-3-[4-(1-
P ip er id in oeth oxy)p h en yl]sp ir o[in d en e-
1,1′-in d a n e]-5,5′-d iol Hyd r och lor id e: A
P r om isin g Non ster oid a l Estr ogen
Recep tor Agon ist for Hot F lu sh
Nobuhide Watanabe,*,† Akihisa Ikeno,‡
Hisao Minato,‡ Hiroshi Nakagawa,†
Chie Kohayakawa,‡ and J un-ichi Tsuji‡
Chemistry Research Laboratories and
Pharmacology & Microbiology Research Laboratories,
Dainippon Pharmaceutical Co., Ltd., Enoki-cho 33-94,
Suita, Osaka 564-0053, J apan
Received J une 29, 2003
Abstr a ct: In our studies of the development of a novel class
of selective estrogen receptor modulators, (+)-3-[4-(1-piperi-
dinoethoxy)phenyl]spiro[indene-1,1′-indane]-5,5′-diol hydro-
chloride (1) was found to be an estrogen receptor ligand with
beneficial effects in rat models for human hot flush. Moreover,
1 was found to have beneficial effects on lipid and bone
metabolism while maintaining marginal effects on the uterus
and breasts. These findings suggest that 1 would provide a
new treatment for hot flush.
Ch em istr y. We envisaged that 1,1′-spirobiindan-3-
one (8), a key intermediate, could be constructed by
means of a rhodium(II) carboxylate catalyzed intramo-
lecular carbon-hydrogen insertion reaction of R-diazo
â-keto esters.10 As shown in Scheme 1, addition of
Grignard reagent 311 to 5-methoxyindan-1-one (4) fol-
lowed by acid-catalyzed dehydration12 and hydrogenoly-
sis with Pd(OH)2-C gave the benzoic acid 5 in 62%
yield. Conversion of 5 into the corresponding acid
chloride followed by condensation with potassium eno-
late of ethyl acetate and subsequent diazo transfer
reaction gave the R-diazo â-keto ester 6 in 96% yield.
Rh2(OAc)4-catalyzed intramolecular carbon-hydrogen
insertion reaction of 6 successfully proceeded in PhCF3
at 60 °C to afford 7 as a single product in 82% yield.
Deethoxycarbonylation of 7 gave 8 in quantitative yield.
The basic side chain was incorporated by addition of the
aryllithium reagent 9, and subsequent dehydration with
TsOH followed by demethylation13 with Ph2PLi gave a
racemic mixture of 1 and 2 in 42% yield in three steps.
At this stage, we investigated the possibility of optical
resolution of 4 or 1 and 2 with a variety of resolving
agents. While all resolution attempts failed, we instead
found that the intermediate 10 could be separated by
preparative chiral HPLC (Scheme 2). The basic side
chain was assembled by removal of the allyl group in
(+)-10 with 5 mol % Pd(OAc)2 in HCO2H followed by
Mitsunobu reaction with piperidineethanol. Subsequent
demethylation gave 1 with up to 99% ee.
In tr od u ction . Hot flush, the most common symptom
of the climacteric, is characterized by the sudden onset
of hot feeling, sweating, palpitation, and anxiety and
affects approximately 75% of naturally menopausal
women. The symptom begins 1-2 years before meno-
pause and lasts for 6 months to 5 years. In women with
surgical or chemotherapy-induced menopause, the symp-
tom generally lasts longer and is more frequent and
severe than that associated with natural menopause.1
Although the physiological mechanism for hot flush
remains unclear, a decline in circulating estrogen levels
has been thought to cause dysfunction of the central
thermoregulatory centers.2a
Indeed, hormone replacement therapy (HRT) allevi-
ates the symptom in 80-90% of women and has been
recognized as the most effective treatment for hot
flush.2b Despite the effectiveness of HRT, its use has
been limited because of side effects such as vaginal
bleeding and breast tenderness and because of concerns
about increased risks of hormone-dependent cancer.3
Very recently, HRT was found to increase the risks of
cardiovascular events in women with coronary heart
disease and breast cancer when undergone for a period
of 5 years or more.4 Given clinical evidence that HRT
has an unfavorable benefit/risk ratio, there is an intense
need for an alternative therapy for hot flush. With the
advent of tamoxifen (TAM) and raloxifene (RAL), selec-
tive estrogen receptor modulators (SERMs) have shown
their high therapeutic potential for estrogen-related
diseases.5 Thus, TAM, the first SERM to be approved
R esu lt s a n d Discu ssion . The binding affinity for
estrogen receptor (ER) was determined by a competitive
radiometric binding assay in human breast cancer
(MCF-7) cytosol, using [3H]-17â-estradiol as tracer.14
While both 1 and 2 showed good affinity for the ER, 1
had higher affinity (Ki ) 9.05 nM) than 2 (Ki ) 78.3
nM).15 Somewhat surprisingly, as will be discussed
below, 1 was more potent in terms of in vivo biological
activity than was predicted from its binding affinity.
Because it is known that two subtypes of the estrogen
receptor, ERR and ERâ, may have different biological
roles16 and that ERR is expressed primarily in the breast
and uterus (only in MCF-7 cells) whereas ERâ is found
mainly in the brain and bone, ERâ may be involved in
* To whom correspondence should be addressed. Phone: 81 66
337 5906. Fax: 81 66 338 7656. E-mail: nobuhide-watanabe@
dainippon-pharm.co.jp.
† Chemistry Research Laboratories.
‡ Pharmacology & Microbiology Research Laboratories.
10.1021/jm034134+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/13/2003

3962 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Letters
F igu r e 2. Effects of 1 and 2 on plasma cholesterol in male
rats. Sprague-Dawley (SD) rats (6- to 8-week old) were treated
daily for 4 days with vehicle control (solid line), 1 (closed
circles), or 2 (oral administration, open triangle; subcutaneous
administration, closed triangle) at the indicated doses. Each
point represents the mean percent decrease ((SEM) relative
to vehicle with a group size of n ) 5. The observed effects of 1
at all doses differ significantly from those of the vehicle control
at P < 0.05.
F igu r e 1. Structures of estradiol and SERMs.
Further explanation for the discrepancy should be
limited.20
Sch em e 1a
At the outset of our in vivo investigation, the choles-
terol-lowering effects of 1 and 2 were briefly compared.
As can been seen from Figure 2, orally administered 1
dose-dependently decreased serum cholesterol in rats
with significant effect at a dose of 0.03 mg/(kg‚day).21
However, 2, given orally even at a dose of 1 mg/(kg‚day),
had no effect on rats cholesterol level. The inefficacy of
orally administered 2 seems to be partly due to its poor
oral bioavailability because subcutaneous administra-
tion of 2 produced a decrease in rats cholesterol levels
similar to that of 1. Hence, we used 1 for further
evaluation.
On the basis of accumulating evidence that the
change in tail temperature of ovariectomized (OVX) rats
reflects human symptoms of skin temperature fluctua-
tion, we chose the OVX rat model as a principal model
for human hot flush.22 As has previously been reported,
tail skin temperature of OVX rats was significantly
higher than that of sham-operated controls, and estra-
diol (E2) attenuated this ovariectomy-induced rise in tail
skin temperature (Figure 3A). Likewise, 1 dose-depen-
dently attenuated the rise in tail temperature of OVX
rats and completely restored tail temperature to the
level of sham-operated controls at doses as low as 0.1
mg/(kg‚day). In addition, 1 did not antagonize the effect
of E2 on the change in tail temperature at any dose
(0.1-10 mg/(kg‚day), Figure 3B). These results suggest
that 1 acted as a full estrogen agonist in the ther-
moregulatory centers. This suggestion is supported by
results from the morphine-dependent rat model.23 Thus,
Merchenthaler23c has reported that E2 suppresses the
rise in tail temperature of morphine-dependent OVX
rats induced by naloxone injection and that RAL has
no effect on the change in tail temperature. In our
experiment with a slightly modified method, although
E2 and RAL gave similar results as those mentioned
above, 1 suppressed the rise in tail temperature with
potency virtually similar to that of E2 (Figure 4). All
results from the two models indicate that 1 may have a
therapeutic efficacy on hot flush.
a
Reagents and conditions: (a) 3, THF; (b) 6 N H2SO4, dioxane,
reflux; (c) H2 (1 atm), 20% Pd(OH)2-C, AcOH; (d) (COCl)2, toluene;
(e) AcOEt, KHMDS, THF; (f) p-acetoamidobenzenesulfonyl azide,
Et3N, MeCN; (g) 0.5 mol % Rh2(OAc)4, PhCF3, 60 °C; (h) 90%
aqueous DMSO, 150 °C; (i) 9, THF, -78 °C; (j) TsOH, toluene,
reflux; (k) Ph2PH, n-BuLi, THF, reflux.
Sch em e 2a
a
Reagents and conditions: (a) HPLC chiral separation with a
CHIRALPAK OJ column; (b) HCO2H, Pd(OAc)2, Ph3P, benzene,
reflux; (c) piperidineethanol, DIAZO, Ph3P, THF; (d) Ph2PH,
n-BuLi, THF, reflux.
this discrepancy.17,18 Besides the possibility of the in-
volvement of ERâ, it has been reported that the binding
affinity does not necessarily correlate with the confor-
mation of the receptor-ligand complex, which affects
the transcriptional or nongenotropic activity, while the
binding affinity is a dominant factor for a ligand
potency.19 Additionally, in the case of oral administra-
tion, pharmacokinetic factors such as absorption, dis-
tribution, and metabolism must be taken into account.
Next, we examined the preventive effects of 1 on
osteoporosis.24 1 prevented the loss of total bone mineral
density of the distal femur with a significant effect at a

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3963
F igu r e 5. Effects of E2, RAL, and 1 on bone mineral density
(BMD) in OVX rats. SD rats (9-week old) were ovariectomized
and orally treated daily for 4 weeks with E2, RAL, or 1 at the
indicated doses. Values represent the mean percent protection
((SEM) relative to OVX controls, with sham control values
defined as 100% (dotted line) and OVX control defined as 0%.
The group size was n ) 8. (//) P < 0.01: vs vehicle group.
F igu r e 3. (A) Effects of E2 and 1 on tail skin temperature of
OVX rats. (B) Effects of 1 on the E2-induced change in tail
skin temperature of OVX rats. Two weeks after ovariectomy,
OVX (SD) rats were orally treated daily for 10 days with E2
alone, 1 alone, or 1 plus E2 at the indicated doses. The OVX
rats were allowed free movement as described,22c and tail
temperature was measured for 90 min with a thermistor probe
(Technol Seven, J apan) placed on the tail approximately 5 cm
from its base. Data were analyzed by the Fluclet software
system (Dainippon Pharmaceutical Co., J apan), and values
represent the mean tail skin temperature ((SEM) relative to
vehicle with a group size of n ) 7. (//) P < 0.01: vs vehicle
group (A). (//) P < 0.01: vs E2 alone group (B).
F igu r e 6. (A) Comparison of the effects of 1, E2, RAL, and
TAM on uterine wet weight in OVX rats. SD rats (9-week old)
were ovariectomized and orally treated daily for 4 weeks with
1 (closed circles), E2 (closed triangles), RAL (open triangles),
and TAM (open squares) at the indicated doses. Uteri were
removed and weighed. Each point represents the mean ((SEM)
with a group size of n ) 8-16. Two sets of controls are
shown: vehicle (solid line) and sham (dotted line). The
observed effects of all test compounds at doses of more than
0.1 mg/(kg‚day) differ significantly from those of OVX control
at P < 0.01. (B) Comparison of the effects of 1, RAL, and TAM
on uterine wet weight in immature rats. SD rats (21-day old)
were orally treated daily for 4 days with 1 (closed circles), RAL
(open triangles), and TAM (open squares) at the indicated
doses. Uteri were removed and weighed. Each point represents
the mean ((SEM) with a group size of n ) 5, and the dotted
line indicates sham controls. The observed effects of test
compounds in all experiments differ significantly from those
of the vehicle control at P < 0.01.
F igu r e 4. Effects of 1 on the change in tail skin temperature
in a morphine-dependent rat model. Ten days after ovariec-
tomy, OVX (SD) rats were orally treated daily at 5:00 p.m. for
8 days with E2, 1, or RAL at the indicated doses. From the
third day to the seventh day of treatment, Kadian (Dainippon
Pharmaceutical Co.), a slow releasing pellet of morphine was
orally administered to the rats at 9:00 a.m. and its dose was
increased by a staircase method from 20 mg/(kg‚day) (mor-
phine base) to 60 mg/(kg‚day). On the eighth day of treatment,
measurement of tail temperature was carried out as described
in Figure 3 under ketamine (80 mg/kg, im) anesthesia. The
baseline was recorded for 15 min, and tail skin temperature
measurement was continued for at most 1 h after naloxone-
induced (1 mg/kg, sc) morphine withdrawal. Values represent
the mean of a maximum reponse of change in tail skin
temperature ((SEM) relative to baseline with a group size of
n ) 7-10. (//) P < 0.01: vs vehicle group.
higher stimulatory activity than that of RAL.24a Finally,
in the MCF-7 proliferation assay,14a,25 1 showed con-
siderably weak stimulatory and inhibitory effects on cell
proliferation.26 Taken together, it seems that the in-
trinsic estrogenic effects of 1 on the reproductive tissues
are only very modest and much less than those of E2.
In conclusion, we synthesized 1, a nonsteroidal es-
trogen receptor ligand with a spiro[indene-1,1′-indane]
core structure, capitalizing on the rhodium(II) carboxy-
late catalyzed carbon-hydrogen insertion reaction. Our
data from rat models show that 1 has beneficial effects
on central thermoregulatory centers and bone and lipid
metabolism while maintaining marginal effects on the
uterus and breasts. These findings suggest that 1 would
meet the criteria as a SERM and provide an alternative
dose of 0.1 mg/(kg‚day) and an efficacy as potent as that
of E2 or RAL (Figure 5).
These encouraging results led us to investigate critical
issues related to adverse effects. In OVX rat model, 1
had minimal stimulatory activity toward the uterus
(Figure 6A).24a On the other hand, in an immature rat
model, 1 at doses of 0.1-10 mg/kg acted as a weak estro-
gen agonist in the uterus (Figure 6B), showing slightly

3964 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Letters
(17) We acknowledge a reviewer for suggesting that the discrepancy
may be explained by the involvement of ERâ.
to HRT. Although the biological mechanism by which 1
exerts its unique pharmacological effects is an open
question, our results would facilitate the development
of a new generation SERMs.
(18) (a) Kuiper, G. G. J . M.; Carlsson, B.; Grandien, K.; Enmark, E.;
Ha¨ggblad, J .; Nilsson, S.; Gustafsson, J .-A° . Comparison of the
Ligand Binding Specificity and Transcript Tissue Distribution
of Estrogen Receptors R and â. Endocrinology 1997, 138, 863-
869. (b) Watanabe, T.; Inoue, S.; Ogawa, S.; Ishii, Y.; Hiroi, H.;
Ikeda, K.; Orimo, A.; Muramatsu, M. Agonistic Effect of Tamox-
ifen Is Dependent on Cell Type, ERE-Promoter Context, and
Estrogen Receptor Subtype: Functional Difference between
Estrogen Receptors R and â. Biochem. Biophys. Res. Commun.
1997, 236, 140-145.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the synthesis of 1, and physical and spectral data
for key compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(19) For detailed descriptions of the current understanding of tissue-
specific steroid hormone activities, see the following. (a) Katzenel-
lenbogen, J . A.; O’Malley, B. W.; Katzenellenbogen, B. S.
Tripartite Steroid Hormone Receptor Pharmacology: Interaction
Refer en ces
(1) For a recent review, see the following. Stearns, V.; Ullmer, L.;
Lo´pez, J . F.; Smith, Y.; Isaacs, C.; Hayes, D. F. Hot Flushes.
Lancet 2002, 360, 1851-1861.
with Multiple Effector Sites as
a Basis for the Cell- and
(2) For recent reviews, see the following. (a) Freedman, R. R.
Physiology of Hot Flashes. Am. J . Hum. Biol. 2001, 13, 454-
464. (b) Shanafelt, T. D.; Barton, D. L.; Adjei, A. A.; Loprinzi,
C. L. Pathphysiology and Treatment of Hot Flashes. Mayo Clin.
Proc. 2002, 77, 1207-1218.
Promoter-Specific Action of These Hormones. Mol. Endocrinol.
1996, 10, 119-131. (b) Kousteni, S.; Bellido, T.; Plotkin, L. I.;
O’Brien, C. A.; Bodenner, D. L.; Han, L.; Han, K.; DiGregorio,
G. B.; Katzenellenbogen, J . A.; Katzenellenbogen, B. S.; Rober-
son, P. K.; Weinstein, R. S.; J ilka, R. L.; Manolagas, S. C.
Nongenotropic, Sex-Nonspecific Signaling through the Estrogen
or Androgen Receptors: Dissociation from Transcriptional Activ-
ity. Cell 2001, 104, 719-730.
(3) Seibel, M. M. Treating Hot Flushes without Hormone Replace-
ment Therapy. J . Fam. Pract. 2003, 52, 291-296.
(4) (a) Hulley, S.; Grady, D.; Bush, T.; et al. Randomized Trial of
Estrogen plus Progestin for Secondary Prevention of Coronary
Heart Disease in Postmenopausal Women. J . Am. Med. Assoc.
1998, 280, 605-613. (b) Collaborative Group on Hormonal
Factors in Breast Cancer. Breast Cancer and Hormone Replace-
ment Therapy. Lancet 1997, 350, 1047-1059.
(20) Similar discrepancies have been reported. See the following. (a)
Grese, T. A.; Cho, S.; Finley, D. R.; Godfrey, A. G.; J ones, C. D.;
Lugar, C. W., III; Martin, M. J .; Matsumoto, K.; Pennington, L.
D.; Winter, M. A.; Adrian, M. D.; Cole, H. W.; Magee, D. E.;
Phillips, D. L.; Rowley, E. R.; Short, L. L.; Glasebrook, A. L.;
Bryant, H. U. Structure-Activity Relationships of Selective
Estrogen Receptor Modulators: Modifications to the 2-Arylben-
zothiophene Core of Raloxifene. J . Med. Chem. 1997, 40, 146-
167. (b) Labaree, D. C.; Reynolds, T. Y.; Hochberg, R. B.
Estradiol-16R-Carboxylic Acid Esters as Locally Active Estro-
gens. J . Med. Chem. 2001, 44, 1802-1814.
(5) For recent reviews, see the following. (a) J ordan, V. C. Anties-
trogens and Selective Estrogen Receptor Modulators as Multi-
functional Medicines. 1. Receptor Interactions. J . Med. Chem.
2003, 46, 883-908. (b) J ordan, V. C. Antiestrogens and Selective
Estrogen Receptor Modulators as Multifunctional Medicines. 2.
Clinical Considerations and New Agents. J . Med. Chem. 2003,
46, 1081-1111. (c) Riggs, B. L.; Hartmann, L. C. Selective
Estrogen-Receptor ModulatorssMechanisms of Action and Ap-
plication to Clinical Practice. N. Engl. J . Med. 2003, 348, 618-
629.
(6) Plouffe, L., J r.; Facoq, C. M. Selective Estrogen Receptor
Modulators (SERMs) in Clinical Practice. J . Soc. Gynecol. Invest.
2000, 7, S38-S46.
(7) (a) Davies, G. C.; Huster, M. W. J .; Lu, Y.; Plouffe, L., J r.;
Lakshmanan, M. Adverse Events Reported by Postmenopausal
Women in Controlled Trials with Raloxifene. Obstet. Gynecol.
1999, 93, 558-565. (b) Loprinzi, C. L.; Zahasky, K. M.; Slaoan,
J . A.; Novotny, P. J .; Quella, S. K. Tamoxifen-Induced Hot
Flashes. Clin. Breast Cancer 2000, 1, 52-56.
(8) Estrogen receptor ligands possessing spiroindenes have been
reported. See the following. Blizzard, T. A.; Morgan, J . D., II;
Mosley, R. T.; Birzin, E. T.; Frisch, K.; Rohrer, S. P.; Hammond,
M. L. 2-Phenylspiroindenes: A Novel Class of Selective Estrogen
Receptor Modulators (SERMs). Bioorg. Med. Chem. Lett. 2003,
13, 479-483.
(9) The absolute configuration of 1 has not been determined.
(10) For recent reviews, see the following. (a) Ye, T.; McKervey, M.
A. Organic Synthesis with R-Diazocarbonyl Compounds. Chem.
Rev. 1994, 94, 1091-1160. (b) Doyle, M. P.; McKervey, M. A.
Recent Advances in Stereoselective Synthesis Involving Diazo-
carbonyl Intermediates. J . Chem. Soc., Chem. Commun. 1997,
983-989.
(11) Marxer, A.; Rodriguez, H. R.; McKenna, J . M.; Tsai, H. M. Spiro
Piperidines. I. Synthesis of Spiro[isobenzofuran-1(3H),4′-pip-
eridines] and Spiro[isobenzofuran-1(3H),3′-piperidines]. J . Org.
Chem. 1975, 40, 1427-1433.
(12) The reaction exclusively gave the spirolactone. See Supporting
Information.
(13) Ireland, R. E.; Walba, D. M. Demethylation of Methyl Aryl
Ethers: 4-Ethoxy-3-Hydroxybenzaldehyde. Org. Synth. 1988, VI,
567-570 (Collective).
(14) (a) Rufbin, V. N.; Ruenitz, P. C.; Boyd, J . L.; Boudinot, F. D.;
Wiese, T. E. Characterization of Selective Estrogen Receptor
Modulator (SERM) Activity in Two Triarylethylene Oxybutyric
Acids. Biochem. Pharmacol. 2002, 63, 1517-1525. (b) Wang, T.
T.; Sathyamoorthy, N.; Phang, J . M. Molecular Effects of
Genistein on Estrogen Receptor Mediated Pathways. Carcino-
genesis 1996, 17, 271-275.
(15) The apparent inhibition constant (Ki) values were calculated
according to the following equation: Ki ) IC50/(1 + S/K), where
S is the concentration of labeled E2 and K is the Kd value of E2
(0.027 nM) for the estrogen receptor.
(16) (a) Couse, J . F.; Korach, K. S. Estrogen Receptor Null Mice:
What Have We Learned and Where Will They Lead Us? Endocr.
Rev. 1999, 20, 358-417. (b) Hewitt, S. C.; Couse, J . F.; Korach,
K. S. Estrogen Receptor Transcription and Transactivation.
Estrogen Receptor Knockout Mice: What Their Phenotypes
Reveal about Mechanisms of Estrogen Action. Breast Cancer Res.
2000, 2, 345-352.
(21) In this experiment, coadministration (3 mg/kg, sc) of ICI-182780,
a pure antiestrogen, reduced the cholesterol-lowering effect of
1 (0.2 mg/kg, po) by 64%, which is evidence that the in vivo
activity of 1 was mediated through the ER.
(22) (a) J elinek, J .; Kappen, A.; Scho¨nbaun, E.; Lomax, P. A Primate
Model of Human Postmenopausal Hot Flushes. J . Clin. Endo-
crinol. Metab. 1984, 59, 1224-1228. (b) Kobayashi, T.; Tamura,
M.; Hayashi, M.; Katsuura, Y.; Tanabe, H.; Ohta, T.; Komoriya,
K. Elevation of Tail Skin Temperature in Ovariectomized Rats
in Relation to Menopausal Hot Flushes. Am. J . Physiol.: Regul.,
Integr. Comp. Physiol. 2000, 278, R863-R869. (c) Hosono, T.;
Chen, X.-M.; Miyatsuji, A.; Yoda, T.; Yoshida, K.; Yanase-
fujiwara, M.; Kanosue, K. Effects of Estrogen on Thermoregu-
latory Tail Vasomotion and Heat-Escape Behavior in Freely
Moving Female Rats. Am. J . Physiol.: Regul., Integr. Comp.
Physiol. 2001, 280, R1341-R1347. (d) Berendsen, H. H. G.;
Weekers, A. H. J .; Kloosterboer, H. J . Effect of Tibolone and
Raloxifene on the Tail Temperature of Oestrogen-Deficient Rats.
Eur. J . Pharmacol. 2001, 419, 47-54.
(23) (a) Simpkins, J . W.; Katovich, M. J .; Song, I.-C. Similarities
between Morphine Withdrawal in the Rat and the Menopausal
Hot Flush. Life Sci. 1983, 32, 1957-1966. (b) Katovich, M. J .;
Simpkins, J . W.; Berglund, L. A.; O’Meara, J . Regional Skin
Temperature Changes in a Rat Model for the Menopausal Hot
Flush. Maturitas 1986, 8, 67-76. (c) Merchenthaler, I.; Funk-
houser, J . M.; Carver, J . M.; Lundeen, S. G.; Ghosh, K.;
Winneker, R. C. The Effect of Estrogens and Antiestrogens in a
Rat Model for Hot Flush. Maturitas 1998, 30, 307-316.
(24) (a) Qu, Q.; Zheng, H.; Dahllund, J .; Laine, A.; Cockcroft, N.; Peng,
Z.; Koskinen, M.; Hemminki, K.; Kangas, L.; Va¨a¨na¨nen, K.;
Ha¨rko¨nen, P. Selective Estrogenic Effects of a Novel Triphenyl-
ethylene Compound, FC1271a, on Bone, Cholesterol Level, and
Reproductive Tissues in Intact and Ovariectomized Rats. En-
docrinology 2000, 141, 809-820. (b) Sato, M.; Turner, C. H.;
Wang, T.; Adrian, M. D.; Rowley, E.; Bryant, H. U.
LY353381.HCl: A Novel Raloxifene Analog with Improved
SERM Potency and Efficacy in Vivo. J . Pharmacol. Exp. Ther.
1998, 287, 1-7.
(25) Kanemura, Y.; Mori, H.; Kobayashi, S.; Islam, O.; Kodama, E.;
Yamamoto, A.; Nakanishi, Y.; Arita, N.; Yamasaki, M.; Okano,
H.; Hara, M.; Miyake, J . Evaluation of in Vitro Proliferative
Activity of Human Fetal Neural Stem/Progenitor Cells Using
Indirect Measurements of Viable Cells Based on Cellular
Metabolic Activity. J . Neurosci. Res. 2002, 69, 869-879.
(26) The stimulatory activities of the compounds were estimated from
their percent maximal increase relative to a maximal response
of E2 (E2 ) 100% at 0.1 nM): 1, 27% (100 nM); RAL, 26% (0.1
nM); TAM, 34% (0.1 nM). The inhibitory activities were esti-
mated from IC30 values in the presence of 0.1 nM of E2: 1, 1200
nM; RAL, 5.1 nM; TAM, 8.2 nM.
J M034134+