Discovery and synthesis of [6-hydroxy-3-[4-[2-(1- piperidinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)]benzo[b]thiophene: A novel, highly potent, selective estrogen receptor modulator

Article abstract of DOI:10.1021/jm970167b

Raloxifene,[2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1- piperidinyl)ethoxy]phenyl]-methanone hydrochloride (2), is representative of a class of compounds known as selective estrogen receptor modulators (SERMs) that possess estrogen agonist-like actions on bone tissues and serum lipids while displaying potent estrogen antagonist properties in the breast and uterus. As part of ongoing SAR studies with raloxifene, we found that replacement of the carbonyl group with oxygen ([6-hydroxy-3-[4-[2-(1- piperidinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)]benzo[b]thiophene hydrochloride, 4c) resulted in a substantial (10-fold) increase in estrogen antagonist potency relative to raloxifene in an in vitro estrogen dependent cell proliferation assay (IC₅₀ = 0.05 nM) in which human breast cancer cells (MCF-7) were utilized. In vivo, 4c potently inhibited the uterine proliferative response to exogenous estrogen in immature rats following both sc and oral dosing (ED₅₀ of 0.006 and 0.25 mg/kg, respectively). In ovariectomized aged rats, 4c produced a significant maximal decrease (45%) in total cholesterol at 1.0 mg/kg (po) and showed a protective effect on bone relative to controls with maximal efficacy at 1.0 mg/kg (po). These data identify 4c as a novel SERM with greater potency to antagonize estrogen in uterine tissue and in human mammary cancer cells compared to raloxifene, tamoxifen or ICI-182,780.

Full text of DOI:10.1021/jm970167b

© Copyright 1997 by the American Chemical Society
Volume 40, Number 10
May 9, 1997
Expedited Articles
Discover y a n d Syn th esis of [6-Hyd r oxy-3-[4-[2-(1-p ip er id in yl)eth oxy]p h en oxy]-
2-(4-h yd r oxyp h en yl)]ben zo[b]th iop h en e: A Novel, High ly P oten t, Selective
Estr ogen Recep tor Mod u la tor
Alan D. Palkowitz,* Andrew L. Glasebrook, K. J eff Thrasher, Kenneth L. Hauser, Lorri L. Short,
D. Lynn Phillips, Brian S. Muehl, Masahiko Sato, Pamela K. Shetler, George J . Cullinan, Tina R. Pell, and
Henry U. Bryant
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285
Received March 13, 1997X
Raloxifene,[2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]-
methanone hydrochloride (2), is representative of a class of compounds known as selective
estrogen receptor modulators (SERMs) that possess estrogen agonist-like actions on bone tissues
and serum lipids while displaying potent estrogen antagonist properties in the breast and
uterus. As part of ongoing SAR studies with raloxifene, we found that replacement of the
carbonyl group with oxygen ([6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-hydrox-
yphenyl)]benzo[b]thiophene hydrochloride, 4c) resulted in a substantial (10-fold) increase in
estrogen antagonist potency relative to raloxifene in an in vitro estrogen dependent cell
proliferation assay (IC50 ) 0.05 nM) in which human breast cancer cells (MCF-7) were utilized.
In vivo, 4c potently inhibited the uterine proliferative response to exogenous estrogen in
immature rats following both sc and oral dosing (ED50 of 0.006 and 0.25 mg/kg, respectively).
In ovariectomized aged rats, 4c produced a significant maximal decrease (45%) in total
cholesterol at 1.0 mg/kg (po) and showed a protective effect on bone relative to controls with
maximal efficacy at 1.0 mg/kg (po). These data identify 4c as a novel SERM with greater
potency to antagonize estrogen in uterine tissue and in human mammary cancer cells compared
to raloxifene, tamoxifen or ICI-182,780.
In tr od u ction
(3) is distinct in that it possesses no estrogen agonist
properties.3 A growing understanding of the pharma-
cology of these agents has suggested safe clinical uses
of optimal compounds for the treatment of estrogen
dependent diseases such as osteoporosis, breast cancer,
and gynecological disorders.4 Indeed, raloxifene has
been shown in animal models to be an effective antago-
nist in uterine tissue while potently mimicking the
favorable actions of estrogen on bone and lipids.4a,5 As
an extension of this hypothesis to humans, raloxifene
is currently undergoing clinical evaluation for the
treatment and prevention of osteoporosis as an alterna-
tive to hormone replacement therapy.4b
Over the past few years, it has become apparent that
some compounds previously described as estrogen an-
tagonists also demonstrate estrogen agonist properties
in select tissues.1 This selective estrogen receptor
modulator (or SERM) profile has defined a spectrum of
mixed agonist/antagonist activities for several structural
classes of compounds that include the triphenylethyl-
enes exemplified by tamoxifen (1) and benzothiophene
derivatives represented by raloxifene (2) (Figure 1).2 By
contrast, the steroidal estrogen antagonist ICI-182,780
X Abstract published in Advance ACS Abstracts, April 15, 1997.
S0022-2623(97)00167-2 CCC: $14.00 © 1997 American Chemical Society

1408 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Palkowitz et al.
CF3CO2H in dichloromethane for 2 h at room temper-
ature.11 These conditions provided 7 exclusively in 80%
yield with no observed overoxidation to the sulfone.
Introduction of the basic side chain with construction
of the key heteroatom bond was next accomplished by
reaction of 7 with 8a ,b in DMF in the presence of NaH
to give directly sulfoxides 9a and 9b in 37% and 89%
yield, respectively.12 Sulfoxide reduction (LiAlH4, THF)
and conversion to the hydrochloride salt gave 10a (73%)
and 10b (96%).13 Finally, cleavage of the methyl ethers
(BBr3, CH2Cl2) and conversion to the hydrochloride salt
provided 4b and 4c in 60% and 74% yield, respectively.
Attempts to extend the sulfoxide mediated chemistry
to the synthesis of the N-linked derivative 4d were
unsuccessful. Alternatively, 4d was prepared from the
3-bromobenzothiophene 6 via an Ullman coupling to
form the biarylamine linkage.14 Thus treatment of 6
with 4-(benzyloxy)acetanilide in refluxing collidine and
in the presence of catalytic Cu2O for 72 h provided
coupled derivative 11 in 5% yield following isolation by
column chromatography. No attempt was made at this
point to optimize the yield of the coupling reaction.
Compound 11 was readily transformed to the phenolic
derivative 12 by catalytic hydrogenolysis of the benzyl
ether protecting group (Scheme 2). The resulting phenol
was then alkylated with 2-(chloroethyl)piperidine
(Cs2CO3, DMF). The crude acetanilide derivative was
hydrolyzed with concentrated NaOH to form the di-
amine that was transformed to the dihydrochloride salt
(Et2O‚HCl) 13 in 90% yield from 12. Dimethyl ether
cleavage (BBr3, CH2Cl2), followed by conversion to the
hydrochloride salt, provided 4d in 45% yield from 13.
F igu r e 1. Chemical structures of tamoxifen, ICI-182,780,
raloxifene, and 4a -d .
Recently, there has been considerable speculation as
to the mechanism for the tissue selective actions of
SERMs.2a,6 One possibility is that effects of compounds
such as raloxifene are mediated through the local
expression of TGF-â3, a multifunctional regulator of cell
growth and differentiation. It has been shown that
raloxifene upregulates the expression of TGF-â3 mRNA
in bone cells in vitro and in vivo, as well as stimulates
the secretion of TGF-â in human MCF-7 cancer cells
and fetal fibroblasts.7 The distinct tissue actions may
be inherent in that the TGF-â3 promoter contains a
sequence that selectively mediates the actions of ralox-
ifene and other SERMs on TGF-â3 expression, and is
not effectively activated by estrogen.8
Our interest in the unique actions of raloxifene has
prompted an expanded structure-activity relationship
(SAR) evaluation of the series.9 A primary goal of our
efforts was to identify novel compounds with enhanced
estrogen antagonist activity that maintain the SERM
profile. One area of focus for our studies has been
examination of point changes directed at the carbonyl
region of raloxifene (4a -d , Figure 1). In this article we
summarize the results of this investigation that have
led to the discovery of [6-hydroxy-3-[4-[2-(1-piperidinyl)-
ethoxy]phenoxy]-2-(4-hydroxyphenyl)]benzo[b]thio-
phene, 4c. To our knowledge, benzothiophene 4c is the
most potent estrogen antagonist described to date that
maintains estrogen agonist actions on bone and lipids.
Biologica l Resu lts a n d Discu ssion
The testing paradigm employed to guide our SAR
studies and characterize the SERM profile consisted of
in vitro and in vivo assays. In vitro, intrinsic estrogen
antagonist activity (IC50) was determined by measuring
the ability of a compound to inhibit estrogen-induced
proliferation of human MCF-7 breast cancer cells.15 In
vivo, estrogen antagonist activity (ED50) was assessed
following either po or sc administration by measuring
the ability of a compound to block estrogen-stimulated
uterine weight gain in immature rats.9,16 To complete
the evaluation of the SERM profile, agonist actions of
compounds were assessed in an ovariectomized (OVX)
adult rat model to determine effects on bone, uterine,
and lipid parameters.4a,17
Table 1 shows a comparison of intrinsic estrogen
antagonist activity for raloxifene and 4a -d in the
MCF-7 cell proliferation assay. Under the conditions
of this experiment whereby the cells are stimulated with
10 pM 17â-estradiol (E2), the IC50 value for raloxifene
as an inhibitor of proliferation was 0.4 nM. Replace-
ment of the raloxifene carbonyl with S, CH2, or NH
(4a ,b,d ) provided modest (2-5-fold) increases in an-
tagonist potency. This effect was maximized with the
introduction of an oxygen at this site (4c). The biaryl
ether derivative 4c produced an IC50 of 0.05 nM in this
experiment, representing an approximately 10-fold in-
crease in intrinsic estrogen antagonist potency over
raloxifene. For comparison, 4-hydroxytamoxifen (the
most active metabolite of tamoxifen) and ICI-182,780
under similar conditions yielded IC50 values of 1.17 and
0.49 nM, respectively.18
Ch em istr y
Compound 4a was prepared from raloxifene by a two-
step reduction process (LiAlH4 followed by Et3SiH) in
44% overall yield. The syntheses of the thioether and
the oxygen-linked derivatives (4b and 4c, respectively)
were accomplished via a convergent synthesis employ-
ing the known 2-arylbenzothiophene derivative 5 as a
key starting material (Scheme 1).9.10 Bromination of 5
(Br2, CHCl3) provided exclusively the 3-bromo derivative
6 in quantitative yield. This material was activated for
nucleophilic displacement of bromine by oxidation of the
sulfur to the sulfoxide 7. The oxidation was accom-
plished by treatment of 6 with 1.0 equiv of H2O2 in 50%

Highly Potent Selective Estrogen Receptor Modulator
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1409
Sch em e 1a
a
Conditions: (a) Br2, CHCl3, 60 °C; (b) H2O2, CF3CO2H, CH2Cl2, 2 h; (c) 8a or 8b, NaH, DMF, then 7, 1 h; (d) LiAlH4, THF, 0 °C; (e)
Et2O‚HCl, EtOAc; (f) BBr3, CH2Cl2, 0 °C, 1 h.
Sch em e 2a
a
Conditions: (a) 4-benzyloxyacetanilide, Cu2O, collidine, reflux, 72 h; (b) H2, 10% Pd/C, EtOH/EtOAc, 1% concentrated HCl; (c)
2-(chloroethyl)piperidine hydrochloride, Cs2CO3, DMF, 25 °C, 24 h; (d) concentrated NaOH, EtOH, reflux, 18 h; (e) Et2O‚HCl, EtOAc; (f)
BBr3, CH2Cl2, -20 to 25 °C, 1-6 h.
Ta ble 1. Physical Properties for 4a -d and in Vitro and in Vivo Antagonism of Estrogen by 1-3 and 4a -d
in vivo immature rat uterine assay
in vitro MCF-7 cell
compound
mp, °Ca
NA
>200
180-190
158-165
140-150
formulaa,b
analysesa proliferation IC50 (nM) ( SEc ED50 (mg/kg/po)c,d,e ED50 (mg/kg/sc)c,d,e
2 (raloxifene)
NA
NA
0.4 ( 0.3 (n ) 46)
0.19 ( 0.10 (n ) 3)
0.28 ( 0.15 (n ) 4)
0.05 ( 0.02 (n ) 6)
0.10 (n ) 2)
0.49 ( 0.27 (n ) 3)
481 ( 399 (n ) 25)
1.17 ( 0.69 (n ) 5)
0.55 ( 0.15 (n ) 16)
0.47 ( 0.33 (n ) 3)
1.68 (n ) 1)
0.05 (n ) 2)
ND
ND
0.006 (n ) 2)
ND
4a
4b
4c
4d
C
C
C
C
NA
NA
NA
28H29NO3S‚1.0HCl
27H27NO3S2‚2.2HCl
27H27NO4S‚1.0HCl
27H28N2O3S‚1.5HCl
C, H, N
C, H, N
C, H, N
C, H, N
NA
0.25 ( 0.12 (n ) 5)
0.67 (n ) 1)
3 (ICI-182,780) NA
1 (tamoxifen) NA
4-OH tamoxifen NA
0.62 (n ) 1)
>10 (n ) 2)
0.17 (n ) 1)
ND
NA
NA
ND
ND
a
b
NA ) not applicable. All compounds had C, H, and N microanalysis within (0.4% theoretical value. c n ) number of individual
experiments. ND ) not determined. e ED50 values were detemined by regression analysis using the linear portion of each dose response
curve (at least three point dose response) with six animals at each dose. For compounds evaluated in this assay in at least three separate
d
experiments, an average ED50 ( SE was calculated. The ethynylestradiol stimulus used in this experiment was 0.1 mg/kg/day (po).
The marked increase in estrogen antagonist activity
demonstrated by 4c suggested that this molecule should
bind to the estrogen receptor with enhanced affinity
relative to that of raloxifene. This was indeed confirmed
by studying the competitive binding of raloxifene and
4c using an MCF-7 cell lysate preparation. Figure 2
(panel A) demonstrates that under physiologic condi-
tions (37 °C), raloxifene produced a relative binding
affinity (IC50 17â-estradiol/IC50 test compound) of 0.2-
0.3 whereas 4c bound as efficiently as E2 to the estrogen
receptor. Interestingly, the binding of 4c to the estrogen
receptor was temperature dependent. As shown in

1410 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Palkowitz et al.
F igu r e 2. Competitive inhibition of [3H]-17â-estradiol binding to human estrogen receptor. Whole cell lysates containing estrogen
receptor were prepared from human MCF-7 cells, mixed with 3H-17â-estradiol in the absence or presence of 17â-estradiol, 4c, or
raloxifene, and incubated at 37 °C (panel A) or 4 °C (panel B) for 20 h. Receptor bound radioactivity was then determined and
results expressed as a percent of control binding in the absence of competitor. *p < 0.05 vs estrogen control and 4c (panel A), vs
estrogen control (panel B).
panel B of Figure 2, 4c binds with significantly less
affinity at 4 °C than at 37 °C, while the binding of
estrogen was thermally unresponsive under the experi-
mental conditions. Not shown was the binding curve
for raloxifene at 4 °C, which like estrogen was un-
changed by temperature. Studies are currently under-
way to provide insight into this phenomenon.
The increase in functional antagonism coupled with
the higher affinity binding of 4c was quite intriguing
as it represented a profound change in the in vitro
pharmacological profile with only minor structural
modification (i.e. oxygen for carbonyl) of a relatively
large and rigid molecule.19 Studies are currently un-
derway to develop an understanding of the structural
and electronic consequences of replacing a carbonyl with
oxygen in the raloxifene structure that may influence
the interaction of the ligand with the estrogen receptor.
For example, it is possible that the introduced change
could significantly alter the three-dimensional place-
ment of the basic side chain, which is an important
pharmacophore element in conferring antagonist activ-
ity to this class of compounds.
The in vivo antagonist activities of 4a -d and ralox-
ifene were compared in a 3-day uterine immature rat
assay. In young female rats (21-day-old Sprague-
Dawley) the uterus is fully responsive to estrogen;
however the ovaries do not produce 17â-estradiol. Since
the uterus remains unstimulated, this model permits
maximal stimulation by exogenous estrogen (ethynyl
estradiol (EE) 0.1 mg/kg, po in this case), and hence a
ready measure of an antagonist action can be deter-
mined. Test compounds were administered daily either
po or sc following the estrogen stimulus. Twenty-four
hours following the third daily dose, the animals were
euthanized and the uteri removed and weighed. The
ratio of uterine wet weight to body weight served as the
pharmacological end point of this experiment. The
results of these studies are summarized in Table 1. As
an inhibitor of uterine stimulation, raloxifene demon-
strated an oral ED50 of 0.55 mg/kg. Of the new
derivatives, 4c was the most potent with an ED50 of 0.25
mg/kg (po). The in vivo antagonism of 4c compared to
raloxifene was surprisingly low given the large separa-
tion of intrinsic estrogen antagonist activity measured
in vitro. An understanding of this discrepancy was
realized when the compounds were compared by an
alternate route of administration. When dosed sc, 4c
produced an ED50 of 0.006 mg/kg, whereas raloxifene
gave an ED50 of 0.05 mg/kg. The large separation in
activity of oral vs sc suggested poor oral bioavailability
of the compounds in this model, presumably due to
extensive first pass conjugation (glucuronidation) of the
6 and 4′ phenolic groups. However, the difference in
activity between 4c and raloxifene when dosed subcu-
taneously was more consistent with the activity differ-
ence in vitro. Nevertheless, the level of activity ob-
served for 4c in vivo is representative of the most potent
estrogen antagonist activity reported to date. For
comparison, oral and sc administration of ICI-182,780
(which is in development as a parenteral agent) pro-
duced ED50 values of 0.62 and 0.17 mg/kg, respectively.
Interestingly, tamoxifen produced a partial agonist
response in this model. Although tamoxifen did sig-
nificantly antagonize the estrogen response (ED50 esti-
mated at >10.0 mg/kg po), the efficacy was limited to
approximately 40% inhibition of the estrogen stimulus
(Figure 3). All other agents studied produced maximal
inhibition at the higher end of the dose response.
Finally, in order to evaluate the consistency of phar-
macological effects of 4c with the SERM profile estab-
lished by raloxifene, studies were performed in OVX
rats. Of particular interest were effects on bone (where
raloxifene, like estrogen, protects against ovariectomy
induced osteopenia), cholesterol metabolism (where
raloxifene, like estrogen, lowers cholesterol), and the
uterus (where raloxifene produces minimal estrogenic
effects on uterine weight, eosinophilia, and epithelial
cell height).
To measure effects on bone, OVX 6-month-old Spra-
gue-Dawley rats were dosed daily with 4c for 35 days.
As shown in Figure 4, 4c increased bone mineral density
at the proximal tibia as compared to the OVX control
(quantified by computed tomography) with significant
effects being achieved at 1.0 mg/kg. This compares
favorably with raloxifene, which produces comparable
effects at 1.0 mg/kg and EE which provides a maximum
response in this model at 0.1 mg/kg. In the same
experiment, 4c likewise displayed estrogen like actions
on lipid metabolism as measured by a maximum 45%
reduction of total serum cholesterol achieved at 1.0 mg/

Highly Potent Selective Estrogen Receptor Modulator
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1411
F igu r e 3. Representative experiment demonstrating estrogen antagonist effects of 1-3 and 4c on the immature rat uterus.
Each point represents the mean uterine weight/body weight ratio (as a percentage of the EE positive control) ( SEM for six rats.
*p < 0.05 vs estrogen control.
F igu r e 5. Effects of 4c, EE (0.1 mg/kg), and raloxifene (1.0
mg/kg) on serum cholesterol in OVX rats after 35 days of
treatment (*p < 0.05 vs OVX control).
F igu r e 4. Antiosteopenic effect of 4c, EE (0.1 mg/kg), and
raloxifene (1.0 mg/kg) in OVX rats after 35 days of treatment
(*p < 0.05 vs OVX control).
Con clu sion
kg (Figure 5). In addition, no increase in uterine weight
was observed relative to the OVX controls for any dose
of 4c tested (Figure 6).
In summary, as an extension of our SAR studies with
raloxifene, we have identified a novel SERM (4c) that
displays the most potent estrogen antagonist activity
in vitro of any known agent using a human mammary
carcinoma cell line (MCF-7). Furthermore, the superior
estrogen antagonism of 4c in vivo is suggestive from
experiments in a rat model of EE-induced uterine
hypeplasia (po and sc administration). In addition to
the antagonist properties of 4c, this agent retains
beneficial estrogen agonist actions on bone and lipid
metabolism. Studies ongoing to expand the SAR of the
To more rigorously evaluate the effects of 4c on
uterine parameters, a study was conducted in a 4-day
version of the OVX model described above. In this
assay, 4c produced no statistically significant effects on
uterine weight, epithelial cell height, or eosinophil
peroxidase activity relative to control at the doses tested
(0.01-10.0 mg/kg), whereas EE produced a marked
elevation of these parameters at 0.1 mg/kg.

1412 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Palkowitz et al.
piperidinyl)ethoxy]phenyl]methanol hydrochloride: 1H NMR
(DMSO-d6) δ 9.80 (bs, 1H), 9.55 (bs, 1H), 7.55 (d, J ) 3.0 Hz,
1H), 7.40 (d, J ) 3.0 Hz, 2H), 7.27 (d, J ) 3.0 Hz, 2H), 7.25 (s,
1H), 6.9 (m, 5H), 6.0 (m, 2H), 3.9 (t, J ) 4.0 Hz, 2H), 3.50 (m,
4H), 3.05 (t, J ) 4.0 Hz, 2H), 1.85 (m, 4H), 1.40 (m, 2H); FD
MS 476. Anal. (C28H30ClNO4) C, H, N.
A solution of [2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-
3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]methanol (710 mg, 1.50
mmol) in 25 mL of CH2Cl2 was prepared. To this solution was
added 210 mg (1.8 mmol) of Et3SiH, and the solution was
cooled to 0 °C and placed under a nitrogen atmosphere. After
several minutes of stirring, 12 mL of CF3CO2H was added.
The reaction was allowed to proceed for 16 h, while slowly
warming to room temperature. The reaction was quenched
with the addition of 50 mL of H2O. The reaction mixture was
made basic with the addition of saturated NaHCO3 solution.
The reaction mixture was extracted with 50 mL portions of
CH2Cl2 (2×). The organic layer was separated, washed with
water, dried (Na2SO4), and evaporated to 500 mg (72%) of the
amorphous base. This material was dissolved in 50 mL of
EtOAc, and a saturated solution of Et2O‚HCl was added until
no more precipitate formed. The solvents were removed by
evaporation in vacuo. [2-(4-Hydroxyphenyl)-6-hydroxybenzo-
[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]methane hy-
drochloride (4a ) (330 mg, 48%) was isolated as a white
crystalline powder: mp > 200 °C; 1H NMR (DMSO-d6) δ 9.75
(s, 1H), 9.60 (s, 1H), 7.30 (m, 4H), 7.10 (s, 1H), 7.05 (d, J ) 3.0
Hz, 1H), 6.90 (m, 4H), 6.80 (d, J ) 6.8 Hz, 1H), 4.30 (t, J )
4.0 Hz, 2H), 4.10 (s, 2H), 3.40 (m, 4H), 2.95 (m, 2H), 1.75 (m,
4H), 1.40 (m, 2H); FD MS 460. Anal. (C28H29NO3S‚1.0HCl)
C, H, N.
F igu r e 6. Effects of 4c, EE (0.1 mg/kg), and raloxifene (1.0
mg/kg) on uterine weight in OVX rats after 35 days of
treatment (*p < 0.05 vs OVX control).
6-Me t h oxy-2-(4-m e t h oxyp h e n yl)-3-b r om ob e n zo[b]-
th iop h en e (6). To a solution of 6-methoxy-2-(4-methoxyphe-
nyl)benzo[b]thiophene (5) (27.0 g, 100 mmol) in 1.10 L of CHCl3
at 60 °C was added bromine (15.98 g, 100 mmol) dropwise as
a solution in 200 mL of CHCl3. After the addition was
complete, the reaction mixture was cooled to room temperature
and the solvent removed in vacuo to provide 34.2 g (100%) of
6-methoxy-2-(4-methoxyphenyl)-3-bromobenzo[b]thiophene (6)
biaryl ether benzothiophene series represented by 4c,
with particular emphasis on improving upon the oral
bioavailability of this agent, will be reported in due
course.
1
as a white solid: mp 83-85 °C; H NMR (DMSO-d6) δ 7.70-
7.62 (m, 4H), 7.17 (dd, J ) 8.6, 2.0 Hz, 1H), 7.09 (d, J ) 8.4
Hz, 2H); FD MS 349, 350. Anal. (C16H13O2SBr) C,H.
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Thomas-
Hoover melting point apparatus and are uncorrected. 1H NMR
spectra were obtained on a GE QE-300 spectrometer at 300.15
MHz in the solvent indicated. Field desorption (FD) mass
spectra were recorded on a VG Analytical ZAB-3F instrument.
High-resolution (HR) mass spectra were recorded on a VG
Analytical ZAB2-SE instrument. Elemental analyses were
determined by the Physical Chemistry Department at Lilly
Research Laboratories and are within (0.4% of the theoretical
values unless otherwise indicated.
[2-(4-Hyd r oxyp h en yl)-6-h yd r oxyben zo[b]th ien -3-yl][4-
[2-(1-p ip er id in yl)eth oxy]p h en yl]m eth a n e Hyd r och lor id e
(4a ). [2-(4-Hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-
(1-piperidinyl)ethoxy]phenyl]methanone (2, raloxifene free
base) (5.40 g, 11.0 mmol) was dissolved in 250 mL of anhydrous
THF. The solution was cooled to 0 °C under a nitrogen
atmosphere. Over a period of 20 min, 2.0 g (53 mmol) of
LiAlH4 was added to the stirring solution. The reaction was
allowed to proceed for 6 h while slowly warming to ambient
temperature. The solvent was then evaporated in vacuo. The
residue was suspended in 100 mL of EtOAc, and 50 mL of H2O
was carefully added. The EtOAc layer was separated and
extracted with additional H2O (2×). The organic layer was
dried by filtration through anhydrous Na2SO4 and the filtrate
evaporated in vacuo. This provided 4.8 g (91%) of [2-(4-
hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-piperidi-
nyl)ethoxy]phenyl]methanol. This material was characterized
as the hydrochloride salt, prepared by the following procedure.
The alcohol was dissolved in 50 mL of EtOAc, and a saturated
solution of Et2O‚HCl was added until no additional precipitate
formed. The precipitate was triturated twice with Et2O. The
white precipitate was collected by filtration to provide an
amorphous powder that was dried in vacuo to yield 4.5 g (88%)
of [2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-
6-Me t h oxy-2-(4-m e t h oxyp h e n yl)-3-b r om ob e n zo[b]-
th iop h en e S-Oxid e (7). To a solution of 6-methoxy-2-(4-
methoxyphenyl)-3-bromobenzo[b]thiophene (10.0 g, 28.6 mmol)
in 50 mL of anhydrous CH2Cl2 was added 50 mL of trifluoro-
acetic acid. After 5 min of stirring, H2O2 (4.0 mL, 28.6 mmol,
30% aqueous solution) was added. The resulting mixture was
stirred at room temperature for 2 h. Solid sodium bisulfite
(1.25 g) was added to the dark solution followed by 15 mL of
H2O. The mixture was stirred vigorously for 15 min and then
concentrated in vacuo. The residue was partitioned between
CH2Cl2 and saturated NaHCO3 solution (200 mL each). The
layers were separated, and the organic was extracted with
saturated NaHCO3 solution. The organic was then dried (Na2-
SO4) and concentrated in vacuo to a solid that was triturated
from Et2O/EtOAc. Filtration provided 8.20 g (80%) of 6-meth-
oxy-2-(4-methoxyphenyl)-3-bromobenzo[b]thiophene S-oxide
(7) as a yellow solid that can be recrystallized from EtOAc:
1
mp 170-173 °C; H NMR (DMSO-d6) δ 7.24 (d, J ) 2.2 Hz,
1H), 7.68 (d, J ) 8.8 Hz, 2H), 7.54 (d, J ) 8.5 Hz, 1H), 7.26
(dd, J ) 8.5, 2.2 Hz, 1H), 7.10 (d, J ) 8.8 H, 2H), 3.86 (s, 3H),
3.80 (s, 3H). Anal. (C16H13O3SBr) C, H.
6-Meth oxy-3-[4-[2-(1-p ip er id in yl)eth oxy]th iop h en oxy]-
2-(4-m eth oxyp h en yl)]ben zo[b]th iop h en e S-Oxid e (9a ). To
a solution of hydroxyphenyl disulfide (3.3 g, 13.0 mmol) in 100
mL of anhydrous DMF were added 1-(2-chloroethyl)piperidine
monohydrochloride (9.7 g, 52.0 mmol) and cesium carbonate
(30 g, 91.0 mmol). The mixture was stirred overnight at room
temperature and then concentrated in vacuo. The resulting
residue was partitioned between chloroform and water and
extracted with CHCl3 (3×). The combined CHCl3 layers were
washed with brine, dried (Na2SO4), filtered, and concentrated
in vacuo. The resulting residue was purified by chromatog-
raphy (SiO2, CH3OH/CHCl3/NH3) to yield 4.6 g (75%) of the
disulfide as a yellow oil: 1H NMR (DMSO-d6) δ 7.37 (d, J ) 9

Highly Potent Selective Estrogen Receptor Modulator
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1413
Hz, 4H), 6.94 (d, J ) 9 Hz, 4H), 4.04 (t, J ) 5.7 Hz, 4H), 2.62
(t, J ) 5.7 Hz, 4H), 2.48 (m, 8H), 1.50 (m, 8H), 1.48 (m, 4H);
FD MS 243.
6-Meth oxy-3-[4-[2-(1-p ip er id in yl)eth oxy]th iop h en oxy]-
2-(4-m eth oxyp h en yl)ben zo[b]th iop h en e (10a ). To a solu-
tion of the sulfoxide (0.14 g, 0.27 mmol) in 10 mL of anhydrous
THF was added LiAlH4 (0.012 g, 0.29 mmol). The resulting
slurry was stirred for 90 min at room temperature and was
then quenched by the careful addition of H2O. The solution
was diluted with H2O and then extracted with EtOAc (3×).
The combined organic was extracted with brine (2×), dried
(Na2SO4), filtered, and concentrated in vacuo to a residue. The
residue was purified by chromatography (SiO2, CH3OH/CHCl3,
gradient) to yield 0.11 g (79%) of the desired product as a
yellow oil. This material was transformed to the HCl salt by
treatment with Et2O‚HCl in EtOAc. Isolation by vacuum
filtration provided 10a in 93% yield as a white solid: mp 198-
To a solution of the disulfide in 20 mL of anhydrous THF
was added LiAlH4 (0.48 g, 1.28 mmol). The resulting slurry
was heated to reflux for 1 h and then quenched with saturated
aqueous sodium bicarbonate and extracted with CHCl3 (2×).
The combined organic was dried (Na2SO4), filtered, and
concentrated in vacuo to give 8a as a crude oil that was not
purified. Crude 8a was then dissolved in 10 mL of anhydrous
DMF, and to this solution was added NaH (0.05 g, 1.32 mmol,
60% dispersion in mineral oil) followed by 7 (0.4 g, 1.1 mmol).
The resulting slurry was heated to 90 °C for 1 h and then
cooled to room temperature. The mixture was concentrated
in vacuo, and the residue was partitioned between EtOAc and
H2O. The layers were separated and the aqueous layer was
extracted with EtOAc (2×). The organic was dried (Na2SO4),
filtered, and concentrated in vacuo. The crude product was
purified by chromatography (SiO2, CH3OH/ CHCl3, gradient)
to provide 0.21 g (37% overall) of 9a product as a yellow oil:
1H NMR (DMSO-d6) δ 7.68 (d, J ) 2.3 Hz, 1H), 7.61 (d, J )
8.7 Hz, 2H), 7.20-7.28 (m, 3H), 7.05-7.15 (m, 3H), 6.87 (d, J
) 8.7 Hz, 2H), 4.00 (t, J ) 5.84 Hz, 2H), 3.84 (s, 3H), 3.82 (s,
3H), 2.60 (t, J ) 5.6 Hz, 2H), 2.35-2.45 (m, 4H), 1.43 (m, 4H),
1.30-1.43 (m, 2H); FD MS 521; HRMS calcd for C29H32NO4S2
522.177, found 522.176. Anal. (C29H31NO4S2) H, N; C: calcd
66.77, found 65.51.
P r ep a r a tion of 4-[2-(1-P ip er id in yl)eth oxy]p h en ol (8b).
To a solution of 4-(benzyloxy)phenol (50.50 g, 0.25 mol) in 350
mL of anhydrous DMF was added 2-(chloroethyl)piperidine
(46.30 g, 0.25 mol). After 10 min of stirring, K2CO3 (52.0 g,
0.375 mol) and Cs2CO3 (85.0 g, 0.25 mol) were added. The
resulting heterogeneous mixture was stirred vigorously at
room temperature for 48 h. The reaction mixture was then
poured into H2O (500 mL) and extracted with CH2Cl2. The
organic layer was then extracted with 1 N NaOH several times
and finally washed with brine. The organic was then dried
(Na2SO4) and concentrated in vacuo to an oil. Chromatogra-
phy (SiO2, 1:1 hexane/EtOAc) provided 60.0 g (77%) of 4-[2-
(1-piperidinyl)ethoxy]phenyl benzyl ether as a colorless oil. 1H
NMR (DMSO-d6) δ 7.40-7.27 (m, 5H), 6.84 (q, J AB ) 11.5 Hz,
4H), 4.98 (s, 2H), 3.93 (t, J ) 6.0 Hz, 2H), 2.56 (t, J ) 6.0 Hz,
2H), 2.35-2.37 (m, 4H), 1.48-1.32 (m, 6H); FD MS 311. Anal.
(C20H25NO2) C, H, N.
1
201 °C; H NMR (DMSO-d6) δ 7.63 (d, J ) 8.6 Hz, 2H), 7.62
(d, J ) 2.0 Hz, 1H), 7.58 (d, J ) 8.2 Hz, 1H), 7.07 (d, J ) 8.6
Hz, 2H), 7.02 (dd, J ) 8.2, 2.0 Hz, 1H), 6.92 (q, J AB ) 9.0 Hz,
4H), 4.24 (bt, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.49-3.39 (m,
4H), 2.93 (m, 2H), 1.82-1.62 (m, 5H), 1.38 (m, 1H). Anal.
(C29H32NO3S2‚1.0HCl) C, H, N.
6-Met h oxy-3-[4-[2-(1-p ip er id in yl)et h oxy]p h en oxy]-2-
(4-m et h oxyp h en yl)b en zo[b]t h iop h en e H yd r och lor id e
(10b). To a solution of 6-methoxy-3-[4-[2-(1-piperidinyl)-
ethoxy]phenoxy]-2-(4-methoxyphenyl)benzo[b]thiophene S-ox-
ide (3.00 g, 5.94 mmol) in 200 mL of anhydrous THF under
N2
at 0 °C was added LiAlH4 (0.34 g, 8.91 mmol) in small
portions. After 30 min of stirring, the reaction was quenched
by the careful addition of 5.0 mL of 2.0 N NaOH. The mixture
was stirred vigorously for 30 min, and additional 2.0 N NaOH
was added to dissolve salts. The mixture was then distributed
between H2O and 10% EtOH/EtOAc. The layers were sepa-
rated, and the aqueous layer was extracted several times with
10% EtOH/EtOAc. The organic was dried (Na2SO4) and
concentrated in vacuo to an oil. The crude product was
dissolved in 50 mL of 1:1 EtOAc/Et2O and treated with excess
Et2O‚HCl. The resulting precipitate was collected and dried
to provide 2.98 g (96%) of 6-methoxy-3-[4-[2-(1-piperidinyl)-
ethoxy]phenoxy]-2-(4-methoxyphenyl)benzo[b]thiophene hy-
1
drochloride (10b) as a white solid: mp 216-220 °C; H NMR
(DMSO-d6) δ 10.20 (bs, 1H), 7.64 (d, J ) 8.7 Hz, 2H), 7.59 (d,
J ) 1.5 Hz, 1H), 7.18 (d, J ) 9.0 Hz, 1H), 7.00 (d, J ) 8.7 Hz,
1H), 6.96 (dd, J ) 9.0, 1.5 Hz, 1H), 6.92 (q, J AB ) 9.0 Hz, 4H),
4.31 (m, 2H), 3.83 (s, 3H), 3.77 (s, 3H), 3.43 (m, 4H), 2.97 (m,
2H), 1.77 (m, 5H), 1.37 (m, 1H); FD MS 489. Anal. (C29H31
NO4S‚1.0HCl) C, H, N.
-
4-[2-(1-Piperidinyl)ethoxy]phenyl benzyl ether (21.40 g,
68.81 mmol) was dissolved in 200 mL of 1:1 EtOH/EtOAc
containing 1% concentrated HCl. The solution was transferred
to a Parr apparatus, and 5% Pd/C (3.4 g) was added. The
mixture was hydrogenated at 40 psi for 2 h. The mixture was
then passed through a plug of Celite to remove catalyst. The
filtrate was concentrated in vacuo to a solid that was slurried
in Et2O and filtered to provide 12.10 g (83%) of 4-[2-(1-
piperidinyl)ethoxy]phenol (8b): mp 148-150 °C; 1H NMR
(DMSO-d6) δ 8.40 (s, 1H), 6.70 (q, J AB ) 11.5 Hz, 4H), 3.93 (t,
J ) 6.0 Hz, 2H), 2.59 (t, J ) 6.0 Hz, 2H), 2.42-2.38 (m, 4H),
1.52-1.32 (m, 6H); FD MS 221. Anal. (C13H19NO2) C, H, N.
6-Met h oxy-3-[4-[2-(1-p ip er id in yl)et h oxy]p h en oxy]-2-
(4-m eth oxyp h en yl)ben zo[b]th iop h en e S-oxid e (9b). To
a solution of 4-[2-(1-piperidinyl)ethoxy]phenol (0.32 g, 1.43
mmol) in 5 mL of anhydrous DMF at room temperature was
added NaH (0.57 g, 1.43 mmol, 60% dispersion in mineral oil).
After 15 min of stirring, 6-methoxy-2-(4-methoxyphenyl)-3-
bromobenzo[b]thiophene S-oxide (0.50 g, 1.37 mmol) was added
in small portions. After 1 h of stirring, the reaction was judged
complete by TLC analysis. The solvent was removed in vacuo,
and the residue was distributed between H2O and 10% EtOH/
EtOAc. The organic was washed several times with H2O and
then dried (Na2SO4). Concentration in vacuo gave an oil that
was triturated from EtOAc/hexane to provide 0.62 g (89%) of
6-methoxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-methox-
yphenyl)benzo[b]thiophene S-oxide as a light yellow solid: mp
6-Hyd r oxy-3-[4-[2-(1-p ip er id in yl)eth oxy]th iop h en oxy-
2-(4-h yd r oxyp h en yl)ben zo[b]th iop h en e (4b). To a solu-
tion of 6-methoxy-3-[4-[2-(1-piperidinyl)ethoxy]thiophenoxy]-
2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride (160 mg,
0.29 mmol) in 15 mL of anhydrous CH2
Cl2 at 0 °C under N2
was added BBr3 (0.15 mL). The resulting dark solution was
stirred for 1 h at 0 °C and then immediately poured into a
stirred solution of EtOAc/saturated NaHCO3 solution (50 mL
each). The layers were separated, and the aqueous layer was
washed with EtOAc (3 × 30 mL). The organic was dried (Na2-
SO4) and concentrated in vacuo to a white solid. Chromatog-
raphy (SiO2, 0-5% CH3OH/CHCl3) provided of 6-hydroxy-3-
[4-[2-(1-piper idin yl)et h oxy]t h ioph en oxy]-2-(4-h ydr oxy-
phenyl)benzo[b]thiophene that was converted to the hydro-
chloride salt as described above for 10a . Isolation provided
95 mg (60%) of 4b as a white solid: mp 180-190 °C; 1H NMR
(DMSO-d6) δ 9.86 (s, 1H), 9.79 (s, 1H), 7.46 (d, J ) 8.5 Hz,
2H), 7.42 (d, J ) 8.7 Hz, 1H), 7.29 (d, J ) 2.0 Hz, 1H), 6.96 (d,
J ) 8.7 Hz, 2H), 6.86-6.81 (m, 5H), 4.27 (m, 2H), 3.41-3.37
(m, 4H), 2.96-2.84 (m, 2H), 1.77-1.60 (m, 5H), 1.35-1.28 (m,
1H); FD MS 477. Anal. (C27H27NO3S2‚2.2HCl) C, H, N.
6-H yd r oxy-3-[4-[2-(1-p ip er id in yl)et h oxy]p h en oxy]-2-
(4-h yd r oxyp h en yl)ben zo[b]th iop h en e (4c). 6-Methoxy-3-
[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-methoxyphenyl)benzo-
[b]thiophene hydrochloride (10b) (10.0 g, 19.1 mmol) was
dissolved in 500 mL of anhydrous CH2Cl2 and cooled to 8 °C.
To this solution was added BBr3 (7.20 mL, 76.2 mmol). The
resultant mixture was stirred at 8 °C for 2.5 h. The reaction
was quenched by pouring into a stirring solution of saturated
NaHCO3 (1 L) and cooled to 0 °C. The CH2Cl2 layer was
separated, and the remaining solids were dissolved in CH3-
1
97-100 °C; H NMR (DMSO-d6) δ 7.68 (d, J ) 2.1 Hz, 1H),
7.62 (d, J ) 8.8 Hz, 2H), 7.06-6.92 (m, 6H), 6.85 (d, J ) 8.8
Hz, 2H), 3.94 (t, J ) 6.0 Hz, 2H), 3.81 (s, 3H), 3.72 (s, 3H),
2.56 (t, J ) 6.0 Hz, 2H), 2.39-2.32 (m, 4H), 1.47-1.32 (m, 6H).
Anal. (C29H31NO5S) C, H, N.

1414 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Palkowitz et al.
OH/EtOAc. The aqueous layer was then extracted with 5%
CH3OH/EtOAc (3 × 500 mL). All of the organic extracts
(EtOAc and CH2Cl2) were combined and dried (Na2SO4).
Concentration in vacuo provided a tan solid that was chro-
matographed (SiO2, 1-7% CH3OH/CHCl3) to provide 7.13 g
(81%) of 6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]-2-(4-
hydroxyphenyl)benzo[b]thiophene as a white solid. This mate-
rial was converted to the hydrochloride salt (4c) by treatment
with Et2O‚HCl in EtOAc and isolated in 91% yield as a white
solid: mp 158-165 °C; 1H NMR (DMSO-d6) δ 9.79 (s, 1H), 9.74
(s, 1H), 7.40 (d, J ) 8.6 Hz, 2H), 7.23 (d, J ) 2.0 Hz, 1H), 7.04
(d, J ) 8.6 Hz, 1H), 6.86 (q, J AB ) 9.3 Hz, 4H), 6.76 (dd, J )
8.6, 2.0 Hz, 1), 6.74 (d, J ) 8.6 Hz, 2H), 4.26 (bt, 2H), 3.37 (m,
4H), 2.91 (m, 2H), 1.72 (m, 5 H), 1.25 (m, 1H); FD MS 461.
Anal. (C27H27NO4S‚1.0HCl) C, H, N.
isolated oil was converted to the hydrochloride salt by treat-
ment with Et2O‚HCl in EtOAc. Isolation by filtration provided
1.37 g (100%) of 6-methoxy-2-(4-methoxyphenyl)-3-[4-[[2-(1-
piperidinyl))ethoxy]amino]phenyl]benzo[b]thiophene hydro-
chloride (13) as a white solid: mp 126-130 °C; 1H NMR
(DMSO-d6) δ 7.54 (d, J ) 8.8 Hz, 2H), 7.48 (d, J ) 2.2 Hz,
1H), 7.29 (d, J ) 8.9 Hz, 1H), 6.92 (d, J ) 8.8 Hz, 2H), 6.91
(dd, J ) 8.9, 2.2 Hz, 1H), 6.73 (d, J ) 8.9 Hz, 2H), 6.50 (d, J
) 8.9 Hz, 2H), 4.21 (t, J ) 4.7 Hz, 2H), 3.78 (s, 3H), 3.71 (s,
3H), 3.43-3.31 (m, 4H), 2.97-2.90 (m, 2H), 1.80-1.61 (m, 5H),
1.35 (m, 1H); FD MS 488. Anal. (C29H34N2O3S‚2.0HCl) C, H,
N.
6-Hydr oxy-2-(4-h ydr oxyph en yl)-3-[4-[[2-(1-piper idin yl)-
et h oxy]a m in o]p h en yl]b en zo[b]t h iop h en e H yd r och lo-
r id e (4d ). To a solution of 6-methoxy-2-(4-methoxyphenyl)-
3-[4-[[2-(1-piper idin yl)et h oxy]a m in o]ph en yl]ben zo[b]-
thiophene hydrochloride (0.70 g, 1.25 mmol) in 40 mL of
anhydrous CH2Cl2 under N2 10 °C was added BBr3 (0.36 mL,
3.80 mmol). The solution was allowed to gradually warm to
room temperature and stirred for a total of 6 h. The reaction
was quenched by pouring the mixture into an excess of
saturated NaHCO3 solution. The aqueous layer was then
extracted several times with 5% EtOH/EtOAc. The organic
layer was combined and dried (Na2SO4) and then concentrated
in vacuo to a foam. The crude product was chromatographed
(1-6% CH3OH/CHCl3) to provide 325 mg (49%) of 6-hydroxy-
2-(4-hydroxyphenyl)-3-[4-[[2-(1-piperidinyl)ethoxy]amino]phe-
nyl]benzo[b]thiophene as an oil. This material was converted
to the hydrochloride salt (4d ) by treatment with Et2O‚HCl in
EtOAc and isolated as a white solid in 92% yield by filtration:
mp 140-150 °C; 1H NMR (DMSO-d6) δ 9.57 (s, 2H), 7.40 (d, J
) 8.6 Hz, 2H), 7.19 (d, J ) 10.1 Hz, 1H), 7.16 (d, J ) 2.0 Hz,
1H), 6.75 (dd, J ) 10.1, 2.0 Hz, 1H), 6.70 (d, J ) 8.6 Hz, 2H),
6.64 (d, J ) 8.8 Hz, 2H), 6.46 (d, J ) 8.8 Hz, 2H), 4.21 (m,
2H), 3.50-3.30 (m, 4H), 3.01-2.95 (m, 2H), 1.81-1.63 (m, 5H),
1.38 (m, 1H); FD MS 461. Anal. (C27H28N2O3S‚1.5HCl) C, H,
N.
6-Meth oxy-2-(4-m eth oxyp h en yl)-3-[4-(ben zyloxy)-N-a c-
eta m id op h en yl]ben zo[b]th iop h en e (11). To a solution of
6-methoxy-2-(4-methoxyphenyl)-3-bromobenzo[b]thiophene
(34.20 g, 0.098 mol) and 4-(benzyloxy)acetanilide (23.7 g, 0.98
mol) in 100 mL of 2,4,6-collidine under N2 was added Cu2O
(14.0 g, 0.98 mol). The resulting mixture was heated to reflux
for 72 h. Upon cooling, the reaction mixture was diluted with
CH2Cl2 and the precipitated solids were removed by filtration.
The filtrate was concentrated in vacuo, and the residue was
dissolved in EtOAc. The organic was then extracted several
times with 5.0 N HCl. The organic was dried (Na2SO4) and
concentrated in vacuo to a solid. Chromatography (SiO2,
CHCl3) provided 2.43 g (5%) of [6-methoxy-2-(4-methoxyphe-
nyl)-3-[4-(benzyloxy)-N-acetamidophenyl]benzo[b]thiophene (11)
as an amber foam: 1H NMR (DMSO-d6) δ (doubling due to
amide rotamers) 7.70-6.80 (m, 16H), 4.90 (bs, 2H), 3.78-3.75
(m, 6H), 2.11 and 1.78 (s, 3H); FD MS 509. Anal. (C31H27
NO4S) C, H, N.
-
6-Meth oxy-2-(4-m eth oxyp h en yl)-3-(4-h yd r oxy-N-a ceta -
m id op h en yl)b en zo[b]t h iop h en e (12). 6-Methoxy-2-(4-
methoxyphenyl)-3-[4-(benzyloxy)-N-acetamidophenyl]benzo-
[b]thiophene (2.43 g, 4.77 mmol) was dissolved in 50 mL of
1:1 EtOH/EtOAc containing 5% concentrated HCl. To this
solution was added 1.0 g of 5% Pd/C. The resulting mixture
was hydrogenated at 40 psi for 4 h using a Parr apparatus.
The mixture was then filtered through Celite to remove the
catalyst. The filtrate was concentrated in vacuo to a semisolid.
Chromatography (SiO2, CHl3) provided 1.32 g (83%) of 6-meth-
oxy-2-(4-methoxyphenyl)-3-(4-hydroxy-N-acetamidophenyl)-
benzo[b]thiophene (12) as an amber foam: 1H NMR (DMSO-
d6) δ (doubling due to amide rotamers) 9.53 and 9.36 (s, 1H),
7.62-6.51 (m, 11 H), 3.78 and 3.58 (s, 6H), 2.01 and 1.76 (s,
3H); FD MS 419. Anal. (C24H21NO4S‚0.19CHCl3) C, H, N.
6-Meth oxy-2-(4-m eth oxyph en yl)-3-[4-[[2-(1-piper idin yl)-
et h oxy]a m in o]p h en yl]b en zo[b]t h iop h en e H yd r och lo-
r id e (13). To a solution of 6-methoxy-2-(4-methoxyphenyl)-
3-(4-hydroxy-N-acetamidophenyl)benzo[b]thiophene (1.31 g,
3.13 mmol) and Cs2CO3 (4.10 g, 12.50 mmol) in 15 mL of
anhydrous DMF was added 2-(chloroethyl)piperidine hydro-
chloride (1.15 g, 6.26 mmol). The resulting mixture was stirred
vigorously at room temperature for 5 h. The reaction mixture
was diluted with 200 mL of H2O and then extracted several
times with EtOAc. The combined organic was then washed
with H2O several times and dried (Na2SO4). Concentration
in vacuo provided and oil. Chromatography (1% CH3OH/
CHCl3, SiO2) provided 1.49 g (90%) of 6-methoxy-2-(4-meth-
oxyphenyl)-3-[4-[2-(1-piperidinyl)ethoxy]-N-acetamidophenyl]-
benzo[b]thiophene as an amber oil: 1H NMR (DMSO-d6) δ
(doubling due to amide rotamers) 7.66-6.70 (m, 11 H), 3.93
(m, 2H) 3.78 and 3.58 (s, 6H), 2.8-2.30 (m, 6H), 2.01 and 1.78
(s, 3H), 1.52-1.11 (m, 6H); FD MS 530. Anal. (C31H34N2O4S)
C, H, N.
P h a r m a cologica l Meth od s: Estr ogen Recep tor Bin d -
in g. Serial dilution of test compounds or 17â-estradiol were
mixed with 0.5 nM of [3H]-17â-estradiol, along with 0.5 mg/
mL of protein from MCF-7 cell lysates, to a total volume of
0.14 mL. Binding took place for 1 h at 37 °C, followed by
addition of 0.07 mL of dextran/charcoal and centrifugation to
remove nonbound radioligand. Aliquots of supernatant con-
taining bound radioligand were mixed with scintillant and
counted. Relative binding affinity (RBA) was calculated as
(IC50 17â-estradiol/IC50 test compound) × 100.
MCF -7 Br ea st Ca n cer Cell P r olifer a tion . MCF-7 breast
adenocarcinoma cells (ATCC HTB 22) were maintained in
MEM (Minimal Essential Medium, minus phenol red, Sigma,
St. Louis, MO) supplemented with 10 % fetal bovine serum
(FBS) (V/V), L-glutamine (2 µM), sodium pyruvate (1 µM),
HEPES [N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic
acid), 10 µM], nonessential amino acids, and bovine insulin (1
mg/mL). Ten days prior to assay, MCF-7 cells were switched
to maintenance medium supplemented with 10% dextran-
coated FBS in place of 10% FBS to deplete internal stores of
steroids. MCF-7 cells were removed from maintenance flasks
using cell dissociation medium (Ca2+/Mg2+ free HBSS (phenol
red-free) supplemented with 10 µM HEPES and 2 nM EDTA).
Cells were washed twice with assay medium and adjusted to
80 000 cells/mL. Approximately 100 µL (8000 cells) was added
to flat-bottomed microculture wells (Costar 3596) and incu-
bated at 37 °C in a 5% CO2 humidified incubator for 48 h to
allow for cell adherence and equilibration transfer. Serial
dilutions of test compounds or DMSO as a diluent control were
prepared in assay medium and 50 µL transferred to triplicate
microcultures followed by 50 µL assay medium for a final
volume of 200 µL. After an additional 48 h incubation,
microcultures were pulsed with 1 µCi [3H]thymidine (specific
activity 6.7 Ci/mmol; DuPont NEN) for the last 4-6 h of
culture and the assay terminated by freezing at -70 °C.
Microcultures were then thawed and harvested using a Ska-
tron semiautomatic cell harvester. Samples were counted by
liquid scintilation using a Wallace BetaPlate â counter, and
To a solution of 6-methoxy-2-(4-methoxyphenyl)-3-[4-[2-(1-
piperidinyl))ethoxy)-N-acetamidophenyl)benzo[b]thiophene (1.49
g, 2.80 mmol) in 10 mL of absolute EtOH was added 10 mL of
50% NaOH solution. The resulting mixture was heated to
reflux for 18 h. Upon cooling, the mixture was diluted with
200 mL of H2O. The aqueous layer was then extracted with
EtOAc (3 × 100 mL). The organic layer was combined, washed
with brine, and then dried (Na2SO4). Concentration in vacuo
provided a foam that was chromatographed (SiO2, CHCl3). The

Highly Potent Selective Estrogen Receptor Modulator
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1415
plotting of dpm versus compound concentration was used to
determine the mean IC50 ( standar error (SE).
Ack n ow led gm en t. The authors thank J . Dodge, J .
Sluka, K. Fahey, M. Martin, S. J ones, R. Murff, and K.
Marron for the preparation of ICI-182,780 and 4-hy-
droxytamoxifen.
3-Da y in Vivo An tiestr ogen Assa y. In vivo antiestrogen
activity was determined in 21-day-old Sprague-Dawley rats
(Charles River Laboratories, Portage, MI). Animals were
grouped based on a 12 h light:12 h dark cycle with room
temperature set at 22 °C. The animals had ad libitum access
to both food and tap water. Ethynylestradiol (Sigma Chemical,
St. Louis, MO) at a dose of 0.1 mg/kg/d (po) was used as the
estrogenic stimulus to increase uterine weight in these ani-
mals. Test compounds were administered by oral gavage in a
volume of 0.2 mL, 15 min prior to the ethynylestradiol gavage.
Dosing with the test compounds was continued for 3 consecu-
tive days. The animals were fasted over the night following
the final dose. On the following morning, the animals were
weighed and then euthanized (by CO2 asphyxiation), and uteri
were rapidly collected and weighed. All test compounds and
ethynylestradiol were dissolved in 20% â-hydroxycyclodextrin
(SAF Bulk Chemical). Nonestrogenic controls were given the
vehicle (po) only.
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Uterine weight/body weight ratios (UWR) were calculated
for each animal. The percent inhibition of the estrogen-
induced response was then calculated by the following for-
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Dawley rats (75-day-old) were obtained from Charles River
and maintained under similar conditions as described above.
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dextrin) was initiated 14 days after ovariectomy by oral gavage
and continued daily for 4 consecutive days. The animals were
fasted the evening following the final dose. On the following
morning the animals were weighed and then anesthetized
(CO2), and a blood sample was collected by cardiac puncture.
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the uteri were collected and weighed. One horn of the uterus
was weighed separately and transferred into Tris buffer for
analysis of uterine eosinophil peroxidase activity. Uteri were
homogenized on ice in 50 mM Tris buffer (pH ) 8.0; 50 µL/mg
tissue) containing Triton X-100 (0.05%). Samples were cen-
trifuged at 3000 rpm for 10 min at 4 °C. The resulting
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velocity was determined by monitoring the oxidation of o-
phenylenediamine at 490 nm on a kinetic microplate reader.
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and stored at -70 °C for subsequent analytical procedures.
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assay.
5-Week OVX Ra t Bon e Assa y. Virgin, OVX Sprague-
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and maintained under similar conditions as described above.
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J M970167B