Novel chromene-derived selective estrogen receptor modulators useful for alleviating hot flushes and vaginal dryness

Article abstract of DOI:10.1021/jm060353u

A novel SERM (selective estrogen receptor modulators), 1-(R), a chromene-derived bisbenzopyran, was discovered to alleviate hot flushes and effectively increase vaginal fluidity in rats. Moreover, 1-(R) was found to have beneficial effects on plasma chole

Full text of DOI:10.1021/jm060353u

3056
J. Med. Chem. 2006, 49, 3056-3059
Novel Chromene-Derived Selective Estrogen
Receptor Modulators Useful for Alleviating Hot
Flushes and Vaginal Dryness
Nareshkumar Jain,* Ramesh M. Kanojia, Jiayi Xu,
Guo Jian-Zhong, Emmanuel Pacia, Muh-Tsann Lai,
Fuyong Du, Amy Musto, George Allan, DoWon Hahn,
Scott Lundeen, and Zhihua Sui
Figure 1. Structure of estradiol and SERMs.
Johnson & Johnson Pharmaceutical Research & DeVelopment LLC,
1000 Route 202, Raritan, New Jersey 08869
backbone that resulted in what is reported as one of the most
potent SERMs.5c,d We envisioned that SERMs with beneficial
effects on hot flushes and potentially vaginal dryness (i.e.
agonists on these tissues) should have a conformation in the
C/D region similar to that of the natural ligand, 17â-estradiol.
Therefore, structures with flexibility in the C/D region between
7 and 8 might offer distinct pharmacological profile. We
designed such structures by replacing the CdC bond in the C
ring of 8 with a CH2O linker. We believe these bisbenzopyran
analogues (I) closely mimic the backbone of steroidal estrogen
estradiol (E2) as well as 8 and perhaps could possess unique
pharmacological properties.
The general synthetic approach of bisbenzopyran structure
(I) is described in Scheme 1. The construction of the key
tetracyclic chromene began with capping the phenolic hydroxyl
groups of 3a to form the tert-butyldimethylsilyl (TBDMS)
derivative 3b and then reduction with DIBAL-H to cleanly
afford the lactol 4.7,8 Addition of a Grignard or lithium derivative
of the dialkylaminoalkoxyphenyl side chain to lactol 4 opened
the pyran ring B to afford the diol 5(a-f), which recyclized
into chromene structure 6(a-f) under either Mitsunobu protocol
or more conveniently by low-temperature acid treatment (concd
HCl) in a hydrocarbon solvent such as toluene. Removal of the
silyl groups from 6 with tetrabutylammonium fluoride (TBAF)
afforded the target 2(a-f) as racemic mixtures.
The choice of R′ group was significant in influencing affinity
as well as potency in ER cell-based functional assays (Table
1). Compound 2a (R′ ) NMe2) and 2c (R′ ) pyrrolidinyl) had
similar binding affinity to ERs and exhibited antagonistic
potency in endometrium Ishikawa cells as well human breast
cancer cell line MCF-7. Compound 2b with R′ as a morpholinyl
group had good binding affinity, but weak inhibitory activity
in MCF-7 cells. Piperidine was the best choice at this position
as shown with the profile of compound 2d. This compound had
greater binding affinity in ER binding assays and also showed
good potency in both Ishikawa as well as MCF-7 cell-based
functional assays. When R′ was further functionalized as azepine
(2e), both binding affinity and potency were significantly
reduced. It was interesting to note that when OCH2CH2R′ was
absent, the compound (2f, structure not shown) showed agonistic
activity in MCF as well as Ishikawa cells. Introduction of a
p-N,N-dimethyl group to the side chain phenyl ring of 2f (2g,
structure not shown) converted the agonistic activity into
antagonistic activity in Ishikawa cells while maintaining ago-
nistic activity in MCF 7 cells. Raloxifene was used as a standard
throughout the studies. In in vitro studies, our lead compound,
2d-((), was slightly more potent.
ReceiVed March 27, 2006
Abstract: A novel SERM (selective estrogen receptor modulators),
1-(R), a chromene-derived bisbenzopyran, was discovered to alleviate
hot flushes and effectively increase vaginal fluidity in rats. Moreover,
1-(R) was found to have beneficial effects on plasma cholesterol and
bone metabolism while maintaining antiestrogenic activity in the uterus.
The biological profile of its enantiomer 1-(S) was also evaluated.
The endogenous steroid estrogen is a critical mediator of
many physiological functions related to development, growth,
and maintenance of a large number of tissues in both females
and males. Selective estrogen receptor modulators (SERMa) are
compounds that act as estrogen agonists on selected targets while
being estrogen antagonists on others.1 Hot flushes and vaginal
dryness are the most common symptoms associated with
menopause in women. A hot flush is characterized by the sudden
onset of a hot feeling, sweating, palpitation, and anxiety and
affects approximately 75% of women as they enter into
menopause.2 More than 50% postmenopausal women experience
lack of vaginal lubrication that leads to several genital com-
plaints associated with a diminished frequency of all forms of
sexual behavior.3 Indeed, hormone therapy (HT) alleviates both
of these symptoms in 80-90% of women and has been
recognized as the most effective treatment for hot flushes and
vaginal dryness.4 Despite the effectiveness of HT on these
symptoms as well as the beneficial effects on bone and plasma
cholesterol levels, its use has been limited because of side effects
such as vaginal bleeding, breast tenderness, concerns about
increased risks of breast cancer, and more recently, the concerns
about cardiovascular safety in about 25% of patients. Given the
clinical evidence that HT has an unfavorable benefit/risk ratio,
there is an intense need for an alternative therapy for hot flushes
and vaginal dryness. Both tamoxifen (TAM) and raloxifene
(RAL) have shown their high therapeutic potential for estrogen-
related diseases. Thus, TAM, the first SERM approved for breast
cancer, is effective for all stages of hormone-dependent breast
cancer, whereas RAL is indicated for the prevention and
treatment of osteoporosis in postmenopausal women.5 These
SERMs have beneficial effects on bone and lipid metabolism,
while antagonizing the effects of estrogens on the uterus and
breasts. However, there are no SERMs reported to have a clinical
beneficial effect on hot flushes and vaginal dryness.4b,6
Upon examining several recently reported potent SERM-
pharmacophores, we noted in the tricyclic benzopyran scaffold
of 7 (EM-652), the pendant phenolic ring D is orthogonal to
the benzopyran ring (Figure 1). In contrast, in 8 (LY-357489),
C and D rings were tied up into an almost planar tetracyclic
To evaluate in vitro as well as in vivo pharmacology of 2d-
(R) and 2d-(S), several attempts were made for chiral separation
of racemic 2d. During these efforts we realized free phenolic
groups in the bisbenzopyrans I appeared to be sensitive to the
a SERM, selective estrogen receptor modulators; HT, hormone therapy;
RAL, raloxifene; TAM, tamoxifen; ovex, ovariectomized.
10.1021/jm060353u CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/11/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3057
Scheme 1a
Table 2. In Vitro Profiles of 1-(R) and 1-(S) and Its Active Metabolites
2d-(R) and 2d-(S)a
human
human
breast
endometrial
ERR binding ERâ binding Ishikawa cellsb MCF-7 cellsb
compd
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
ethinyl
estradiol
raloxifene
1-(R)
2d-(R)
1-(S)
2.0
8.1
NA
NA
1.8
>10000
51
>1000
0.61
8.2
>10000
20.3
>10000
1.3
24.6
175c
43.3
99c
222
510c
599
670c
416
2d-(S)
17.1
a All compounds were tested more than three times in these assays with
high reproducibility. The average is shown here. b No agonist activity was
found at the 10 µM concentration. c 1-(R) and 1-(S) may be getting
hydrolyzed to 2d-(R) and 2d-(S), respectively, in these cell-based functional
assay.17
metabolites 2-(R) and 2-(S) showed activity in both functional
assays, with similar potency to raloxifene. These compounds,
1-(R) and 1-(S) or 2d-(R) and 2d-(S), were devoid of any
stimulatory activity in human MCF-7 breast cancer cells or
Ishikawa endometrial cell assay, indicative of antiestrogenic
activity in these cell lines. Table 2 summarizes the in vitro
profiles for 1-(R), 1-(S), 2d-(R), and 2d-(S).
a Reagents and conditions: a, TBSCl/Et3N/DCM/ rt; b, DIBAL-H/
toluene/DCM/-78 °C; c, M-Ar, where M ) MgI or Li; d, diethyl
azidodicarboxylate, DEAD/THF/PPh3 /rt or concd HCl/toluene/0-10 °C;
e, TBAF/THF/ rt;f, pivaloyl chloride/Et3N/DCM/rt. g, ChiralPak A/(5 cm
× 50 cm, 20 m), 20% MeOH in IPA as mobile phase; h, Et2NH (excess),
MeOH, 60 °C in sealed tub.
Both 1-(R) and 1-(S) were evaluated in several in vivo models
for estrogen action. These models were used to assess the tissue
selective activity of the compound. In these studies, 1-(R) and
1-S) were orally administered to animals in an aqueous
homogeneous suspension (in 0.5% Methocel) daily in all studies
except the hot flush assay. In the latter, sesame oil was used as
the vehicle. The active drug components 2-(R) and 2-(S) were
not evaluated for the in vivo activity because of their long-
term stability concerns.12
Compounds 1-(R) and 1-(S) were tested for their in vivo
effects on uterine weight in immature rats (uterotopic assay).
This assay is used to demonstrate in vivo activity.18a In the
agonist mode, 1-(R) and 1-(S) were administered orally once
daily (1.4 mg/kg) for 3 days and the effect on uterine wet weight
was compared with vehicle, estrone, tamoxifen, and raloxifene.18b
In these uterotopic assays, when 1-(R), 1-(S), or raloxifene were
administered alone, they showed no significant agonistic activity
to stimulate uterine growth, while estrone and tamoxifen
significantly increased the uterine wet weight. In the antagonist
mode, the daily oral administration (3 days) of 1-(R) and 1-(S)
were studied to evaluate the ability of the compounds to block
the increase in uterine wet weight induced by estrone. Oral
administration (3 days) of both 1-(R) and 1-(S) led to a decrease
in uterine wet weight in a dose dependent manner to 27%, 55%,
and 75% inhibition of estrone-stimulated uterine weight increase
at 0.14, 0.42, and 1.4 mg/kg doses, respectively while 1-(S) led
to 55%, 88%, and 100% inhibition of estrone-stimulated uterine
weight increase at respective doses of 0.14, 0.42, and 1.4 mg/
kg. The effects of 1-(R) and 1-(S) on estrone-induced uterine
weight in immature rats were similar to raloxifene.20
Table 1. SAR of Chromene-Derived Bisbenzopyransa
ERR binding ERâ binding Ishikawa cells,b MCF-7 cells,c
compd
RBA, %
RBA, %
IC50 (nM)
IC50 (nM)
2a
2b
2c
61
31
35
40
90
47
180
410
80
220
1400
150
2d
2e
2f
2g
340
55
13
6.4
64
170
50
110
42
12
13
110
660
(24)d
3800
23
(94)d
(71)d
222
raloxifene
58
a All compounds were tested more than three times in these assays with
high reproducibility. The average is shown here. b No agonist activity were
found at 10 µM concentration except compound 2f. c No agonist activity
were found at 10 µM concentration except compound 2f and 2g. d Agonists,
EC50 shown in parentheses; showed 13-22% inhibition at 10 µM
concentration in antagonist format.
ambient conditions. It slowly decomposed upon long standing.
Decomposition was also observed during the chiral separations
using commercial available preparative chiral columns such as
chiralPak-AD or chiralPak-OD. We, therefore, decided to use
a pro-drug approach, where both phenolic groups were protected
as the pivaloyl esters. The pivaloylation of 2d was carried out
using pivaloyl chloride and Et3N and resulted in a white stable
solid 1-((). Chromene 1-(() was resolved using preparative
chiral HPLC (Chiralpak-AD column and IPA as mobile phase)
to yield the title compounds 1-(S) which was first to elute and
1-(R) which was second to elute during chiral separation.9a
Heating 1-(R) and 1-(S) in sealed tube at 55 °C with 1:1 mixture
of diethylamine and MeOH afforded 2d-(R) and 2d-(S),
respectively. 9b
Both 1-(R) and 1-(S) were evaluated in in vitro assays for
their estrogenic as well antiestrogenic activities and were
compared with raloxifene and ethinyl estradiol.10 In binding
assays, neither 1-(R) nor 1-(S) binds to the purified estrogen
receptors ERR or ERâ. The active metabolites 2d-(R) and 2d-
(S) of 1-(R) and 1-(S) bind to both ERR and ERâ with affinities
similar to raloxifene and ethinyl estradiol. Both 1-(R) and 1-(S)
demonstrated antiestrogen activity in the two cell-based assays,
MCF-7 breast cancer cell proliferation assay and Ishikawa
endometrial cell alkaline phosphatase assay.11 The active
Next we examined the effects of 1-(R) and 1-(S) on adult
ovariectomized (ovex) rats to see the estrogenic effects on
multiple tissues, including uterus, vagina, bone, and liver
(plasma lipids). For these studies, the rats were ovariectomized
and then treated, orally, with the compounds 1-(R), 1-(S), and
raloxifene for 6 weeks. Neither 1-(R) nor 1-(S) significantly
increased uterine wet weight when administered alone to the
ovex rats. Similar to the data on uterine weight, 1-(R) and 1-(S)
had minimal effect on epithelial cell height, similar to ralox-
ifene.14 To assess the skeletal effect of 1-(R) and 1-(S) in these
animals, total and trabecular bone mineral densities of the tibia

3058 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Letters
Figure 3. Effect of 1-(R) on the change in tail skin temperature in a
morphine-dependent rat model. ∆T represents the mean of maximum
change in tail skin temperature relative to baseline; po, n ) 24 vs vehicle
Figure 2. Effect of 2 weeks of treatment with 1-(R) on vaginal fluidity
levels in comparison to ethynylestradiol, raloxifene, and 1-(S) in
ovariectomized rats. *Significantly different from OVX control (p <
0.05).
*
group. Significantly different from ovex control (p < 0.05).
were measured with pQCT. Both 1-(R) and 1-(S) effectively
prevented bone loss in a dose-dependent manner, and to a similar
level as raloxifene.20 Following 6 weeks of treatment, blood
was collected and total cholesterol levels were determined in
the serum. 1-(R) and 1-(S) both reduced the circulating serum
total cholesterol levels at all doses tested. The maximum
reduction was to approximately 33-35% that of the control
vehicle treated animals. These responses for bone density and
cholesterol were similar to that seen in raloxifene-treated animals
and reported previously.14, 20
Figure 4. Effect of 1-(R) in rat hot flush model illustrated by AUC.
*Significantly different from vehicle control (p < 0.05, n ) 24).
Importantly, in these same ovex animals there was a key
differentiation from raloxifene, that is, the impact on the vagina.
Raloxifene has no beneficial effect on vaginal dryness, a critical
cause for genital complaints in postmenopausal women.3 We
studied effect of 1-(R), 1-(S), and raloxefene on the production
of vaginal fluid in ovex rats. The fluid in the vagina was
collected using a cotton swab and the absolute weight of the
fluid was measured for each animal after 2 weeks of treatment
(Figure 2). In addition, the cytology of the vaginal smear from
each animal was evaluated. Cytological observation showed that
ovex rats treated with vehicle, 1-(S), 1-(R), and raloxifene were
all in diestrus, while the rats treated with ethinyl estradiol were
in estrus. Ethinyl estradiol showed an increase in vaginal fluid,
while raloxifene showed no effect. Compound 1-(R) increased
the vaginal fluid in ovex rats at the 0.14 mg/kg/day and 1.4
mg/kg/day doses but not at lower doses and which are
comparable to ethinyl estradiol, while the enantiomer 1-(S)
decreased the vaginal fluid in ovex rats.
On the basis of literature evidence, a change in tail temper-
ature of morphine dependent rats, following morphine with-
drawal, reflects the human symptoms of a hot flush.16b This
hot flush model has been used to characterize several
SERMs.5b,15,16 We also evaluated 1-(R) and 1-(S) in the hot flush
model. Our data demonstrated that 1-(R) effectively suppresses
the increase of tail skin temperature ∆T ) 1.51 at 1.4 mg /kg.day
while raloxifene had no effect in suppressing the tail skin
temperature (Figure 3). In a separate experiment, the values of
AUC (area under curve) from the first 15 min following
morphine withdrawal were also measured (Figure 4). In a
separate study (data not shown), 1-(S) had no effect on
suppressing the tail skin temperature. The result from this hot
flush model indicates that 1-(R) may have therapeutic efficacy
for hot flushes.
(antagonist on vagina). Such drastic changes in pharmacology
could be attributed to certain changes in conformation of the
ligand-bound receptor complex and its interaction with the
coactivators and corepressors.19
In conclusion, we discovered a series of nonsteroidal estrogen
receptor modulators with chromene-derived bisbenzopyran as
the core structure. Our data from in vivo studies demonstrated
that 1-(R) possesses ideal SERM profile in animal studies. It
not only exhibits estrogen agonistic effects on bone and lipid,
and antagonistic effects on mammary glands and uterus, but
also alleviates hot flushes and increases the amount of vaginal
fluid. This unique pharmacological profile made compound
1-(R) an ideal clinical candidate for treatment of hot flushes
and vaginal dryness in postmenopausal women, without the risk
of uterine or breast cell stimulation. The biology and pharma-
cology associated with the unique profile of 1-(R) as well as
results from detailed preclinical and clinical studies will be
reported in due course.
Acknowledgment. We are grateful to Drs. Rehka Shah,
Yong Zhou, and Brigitte Segmuller for stereochemistry discus-
sions and Dr. Mark J. Macielag for his guidance.
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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(20) See Supporting Information for details.
(12) Possible path way for auto oxidation is described in ref 8b.
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