Benzothiophene selective estrogen receptor modulators with modulated oxidative activity and receptor affinity

Article abstract of DOI:10.1021/jm070079j

The regulation of estrogenic and antiestrogenic effects of selective estrogen receptor modulators (SERMs) is thought to underlie their clinical use. Most SERMs are polyaromatic phenols susceptible to oxidative metabolism to quinoids, which are proposed to

Full text of DOI:10.1021/jm070079j

2682
J. Med. Chem. 2007, 50, 2682-2692
Benzothiophene Selective Estrogen Receptor Modulators with Modulated Oxidative Activity and
Receptor Affinity
Zhihui Qin, Irida Kastrati, R. Esala P. Chandrasena, Hong Liu, Ping Yao, Pavel A. Petukhov, Judy L. Bolton, and
Gregory R. J. Thatcher*
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, UniVersity of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612-7231
ReceiVed January 19, 2007
The regulation of estrogenic and antiestrogenic effects of selective estrogen receptor modulators (SERMs)
is thought to underlie their clinical use. Most SERMs are polyaromatic phenols susceptible to oxidative
metabolism to quinoids, which are proposed to be genotoxic. Conversely, the redox reactivity of SERMs
may contribute to antioxidant and chemopreventive mechanisms, providing a new approach to improve the
therapeutic properties of SERMs. An improved synthetic strategy was developed to generate a family of
benzothiophene SERMs. Using computational modeling methods and measurements of antioxidant activity
and estrogen receptor (ER) ligand binding, this SERM family was shown to provide both a range of ERR/
ERâ selectivity from 1.2- to 67-fold and a range of redox activity. Antioxidant activity was successfully
modulated by varying a substituent remote from the OH group; the source of the antioxidant capacity. An
efficient synthetic procedure is reported yielding benzothiophene SERMs wherein redox activity and ER
affinity are modulated.
Introduction
(i.e., chemical carcinogenesis). Chemical carcinogenesis can
contribute to cancer initiation through damage to DNA and other
biomolecules following drug bioactivation to redox-active and
electrophilic quinoid metabolites (o-quinones, quinone methides,
and di-quinone methides).10,11 Human estrogens and equine
estrogens contained in current HRT agents are also proposed
to elicit hormonal and chemical carcinogenesis pathways, the
latter via o-quinone metabolites.12,13 Interestingly, many SERMs
in clinical use and clinical development are also highly
susceptible to oxidative metabolism to electrophilic and redox-
active quinoids simply because they are based on polyaromatic
phenol scaffolds.14 The SERMs, raloxifene, desmethylarzoxifene
(DMA), acolbifene, toremifene, and droloxifene are all oxida-
tively metabolized to quinoids, which have been shown to form
adducts with biomolecules, including glutathione (GSH), pro-
teins, and nucleosides (Scheme 1).14-20
Whereas generation of reactive oxygen species (ROS) and
covalent modification of biomolecules by redox-active quinoids
may contribute to initiation and promotion of carcinogenesis,
induction of oxidative stress and oxidation or covalent modifica-
tion of sensor proteins may trigger cellular stress responses that
are cytoprotective.21,22 This balance between the carcinogenic
and the chemopreventive capacity of a drug is determined by
the reactivity toward oxidative bioactivation and the chemistry
of the reactive metabolite formed and, therefore, can be
controlled by structural modification. The contribution of
oxidative bioactivation to therapeutic activity versus toxicity is
of particular relevance to SERMs, which are designed for
chronic use in healthy women who are peri- or postmenopausal
or who have known risk factors. The continued development
of SERMs based on polyaromatic phenolic scaffolds requires
increased understanding of the influence of oxidative bioacti-
vation.
Tamoxifen is the archetypal selective estrogen receptor
modulator (SERMa). Despite the demonstrated, increased risk
of endometrial cancer, tamoxifen has been the therapy of choice
in the endocrine treatment of all stages of hormone-dependent
breast cancer and in the primary and secondary chemoprevention
of breast cancer.1 Although the introduction of aromatase
inhibitors may change this clinical paradigm, SERMs are likely
to be in clinical use for many years.2 The increased use of
SERMs is anticipated on the basis of favorable clinical trial
results for the benzothiophene SERMs, raloxifene and arzox-
ifene, and because SERMs are hoped to provide an alternative
to current hormone replacement therapy (HRT) that has been
causally linked to breast cancer.3 Raloxifene is in current clinical
use in post-menopausal osteoporosis and is expected to find
use in other postmenopausal indications associated with HRT.4,5
The STAR trial (study of tamoxifen and raloxifene) reported
that raloxifene was as effective as tamoxifen in breast cancer
chemoprevention in postmenopausal women at high risk and
was less likely to cause the potentially dangerous side effects
associated with tamoxifen, such as uterine cancer and blood
clots. The RUTH trial (raloxifene use for the heart) did not show
a significantly increased risk of coronary artery disease, although
there is still debate on the potential beneficial or negative effects
of raloxifene on other cardiovascular events.5-7 Arzoxifene,
designed to improve upon the therapeutic properties of ralox-
ifene, is in late stage clinical trials with the promise of substantial
therapeutic benefits and is likely to find use in cancer chemo-
prevention.8,9
The carcinogenic effects of tamoxifen have been attributed
variously to regulation of gene transcription (i.e., hormonal
carcinogenesis) and to genotoxicity due to oxidative metabolites
Intensive research is currently directed at discovery of the
“ideal SERM”: an agent that is antiestrogenic in breast and
endometrial tissue, but proestrogenic in the vasculature and
brain, which would be of use in cancer chemoprevention and
an attractive alternative to HRT. However, there has been little
* To whom correspondence should be addressed. Tel.: 312-355-5282.
Fax: 312 996 7107. E-mail: thatcher@uic.edu.
a Abbreviations: BDE, bond dissociation energy; DMA, desmethylar-
zoxifene; X-DMA, 4′-X-4′-desmethoxyarzoxifene; DPPH, diphenylpicryl-
hydrazyl radical; ER, estrogen receptor; HRT, hormone replacement therapy;
LBD, ligand binding domain; SERM, selective estrogen receptor modulator.
10.1021/jm070079j CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/10/2007

Benzothiophene SERMs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2683
Scheme 1
Scheme 2 a
a Reagents and conditions: (a) KOH, EtOH, EtOAc; (b) PPA, 130 °C; (c) CH3C(O)NHBr; (d) H2O2, TFA, DCM; (e) NaH, C5H10N(CH2)2OC6H4OH,
DMF; (f) (i) HCl, ether; (ii) TMSCl, Ph3P, THF, reflux; (g) (i) HCl, ether; (ii) BF3SMe2; (h) LiAlH4, THF, reflux; (i) (i) HCl, ether; (ii) BF3SMe2.
Scheme 3 a
Results and Discussion
Arzoxifene (1; Scheme 1) is a structural analogue of ralox-
ifene in which the carbonyl hinge has been replaced by an ether
linkage and the 4′-hydroxy group is methylated. Arzoxifene is
in late stage clinical trials as a next generation SERM with
promise of substantial therapeutic benefits24 that are suggested
to result from (a) increased antiestrogenic potency and (b)
improved bioavailability relative to raloxifene.25 DMA (2;
Scheme 1) is an active metabolite of arzoxifene, which has been
observed with highly variable steady-state plasma concentra-
tions.8 In vitro metabolic studies showed that both DMA and
raloxifene undergo bioactivation to electrophilic diquinone
methides (Figure 1), resulting in potentially cytotoxic actions:
depletion of cellular GSH, irreversible inhibition of P450 3A4,
and liver protein modification.15,16,19,20 The desired chemopre-
ventive actions of SERMs will be compromised by the formation
of covalent adducts between electrophilic quinoid metabolites
and cellular proteins or DNA if these adducts cause genotoxicity
or organ toxicity. In an effort to obtain safer benzothiophene
SERMs that retain efficacy and have attenuated reactivity toward
bioactivation, the arzoxifene analogue, 4′-fluoro-4′-des-
methoxyarzoxifene, has been developed (F-DMA, 3, Scheme
1). F-DMA showed similar antiestrogenic activity to both DMA
and raloxifene, and 4′-fluorination was shown to successfully
block the formation of an electrophilic diquinone methide
(Figure 1) and to suppress phase II metabolism. These properties
are predictive of improved bioavailability compared to DMA
and raloxifene.15,16
a Reagents and conditions: (a) NaOMe, cat. AcOEt, CuI, DMF, MeOH,
reflux; (b) L-proline, NaOH, CH3SO2Na, CuI, DMSO.
attention to structural modifications designed to control oxidative
bioactivation and thereby enhance therapeutic activity and
attenuate toxicity.23 This approach requires a family of SERMs
in which structure is used to modulate both redox reactivity
and activity at the estrogen receptor. To that end, a family of
benzothiophene SERMs related to arzoxifene has been synthe-
sized, requiring development of a new synthetic methodology
for arzoxifene itself.
Drug Design Rationale. Appropriate structural modifications
designed to minimize drug bioactivation are sometimes incor-

2684 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Qin et al.
Figure 3. (A) Docking poses for the synthetic analogs in ERR/LBD.
(B) Superposition of the rigid (green) vs relaxed (cyan) binding cavity
of ERR/LBD with docked 4′-XDMA (X ) NH(CH2)2Cl).
Figure 1. Bioactivation of raloxifene and DMA is blocked for F-DMA.
Figure 4. Retro synthetic analysis of 4′-substituted arzoxifene
analogues.
I. Redox Activity. Antioxidant capacity can be quantified
by the ArO-H bond dissociation energy (BDE), and the relative
oxidative capacity for phase II enzyme induction has been
correlated with EHOMO.
29 Therefore, calculations were performed
to predict antioxidant capacity at the AM1//B3LYP/6-31+G*
level on a series of 4′-substituted benzothiophenes (Figure 2;
X ) NH2, OMe, OH, NHC(O)CH3, Me, H, F, Br, CHO, CN,
SO2Me, NO2). Linear correlations were observed between
standard Hammett parameters and both EHOMO and BDE (r2 )
0.98 and 0.92, respectively; Figure 2). On this basis, to provide
a range of redox reactivity, the 4′-substituted-4′-desmethoxyarzox-
ifenes (X-DMA series) selected for synthesis were X ) NH2,
OMe, OH, H, F, Br, SO2Me, and NHR (R ) alkyl, acyl). This
family contains, arzoxifene, DMA, and F-DMA, in addition to
interesting compounds such as a methylsulfonyl derivative that
in simile with the non-CNS penetrating SERM, LY2066948,30
are predicted to have reduced brain bioavailability and hence
diminished ovarian stimulation via actions in the hypothala-
mus.31
Figure 2. Plots of EHOMO (squares) and relative BDE (triangles) vs
Hammett parameter for benzothiophene derivatives from AM1//B3LYP/
6-31+G* calculations: open circles represent 4-substituted compounds
selected for synthesis.
porated in the lead optimization stage of drug discovery,23 but
most SERMs retain the polyaromatic phenolic core that is
susceptible to oxidative bioactiviation. Although minimizing
drug bioactivation is often thought to reduce the risks associated
with toxic metabolites, bioactivation to a benign redox-active
metabolite that induces oxidative stress and oxidation or covalent
modification of sensor proteins may trigger cytoprotective
cellular responses that contribute to chemoprevention. Keap1
is a notable example of a sensor protein that responds to redox-
active compounds to mediate induction of phase II enzymes.21,22
An additional potential benefit is antioxidant activity, which
has been proposed to contribute to SERM biological activity.26,27
The potential negative outcomes of oxidative bioactivation
include modification of liver proteins, drug-drug interactions,
and liver dysfunction. Further complicating this picture is the
proposal that quinoid metabolites of estrogenic compounds are
ligand-independent ER modulators.28 To explore the influence
of bioactivation on SERM activity and toxicity and hence
improve SERM design, a homologous family of SERMs was
required with modulated ER binding and redox reactivity. A
benzothiophene core was selected because of the importance
of raloxifene and arzoxifene and on the basis of the promising
preliminary studies with F-DMA. Naturally, some, but not all,
of the benzothiophene SERMs selected for synthesis have been
described in patents but, with the exception of arzoxifene itself,
there is little or no data published on biological activity and
reactivity.
II. ER Binding. To assist in drug design, the coordinates
for the ERR and ERâ ligand binding domains (LBD) were
extracted from the crystal data of the raloxifene-ERR/LBD
complex (PDB code: 1ERR) and tamoxifen-ERâ complex
(PDB code: 2FSZ), respectively. The ERR/LBD structure
indicated that the 4′-OH is involved in a hydrogen-bonding
network that includes His-524, with which it forms a hydrogen
bond.32 The simplistic prediction would be that the loss of this
hydrogen bond in derivatives such as H-DMA (12) would reduce
ER affinity. Because His-524 could potentially act as a
nucleophile toward an electrophilic 4′-substituent with potential
to covalently modify the ERR ligand binding site, for X ) NHR,
R ) (CH2)2Cl (18) and C(O)CH2Cl (16) were selected. A
chloromethyl ketone group is known to alkylate histidine
residues,33 and a nitrogen mustard chloroethylamine group is
known to alkylate via an aziridine intermediate.34

Benzothiophene SERMs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2685
Scheme 4 a
a Reagents and conditions: (a) NaN3, CuI, L-proline, NaOH, DMSO, EtOH; (b) (i) HCl, ether; (ii) BF3SMe2; (c) ClCH2CHO, NaBH3CN, HCl, MeOH;
(d) ClCH2COCl, pyridine, CH2Cl2; (e) (i) HCl, ether; (ii) BF3SMe2, 70 °C.
Raloxifene binding in ERR/LBD is achieved through a
combination of specific hydrogen bonds and complementarity
of the binding cavity with the nonpolar portions of the ligand.
Given the similarity between raloxifene and the benzothiophene
SERMs described in this study, docking to ERâ/LBD was
anticipated to provide good predictive binding data. Whether
FlexX was run on its own or with FlexPharm constraints and/
or more stringent docking criteria, the bulkier 4′-substituents
(X ) SO2CH3, NH(CH2)2Cl, NHC(O)CH2Cl, and in some cases
OCH3) failed to position inside the LBD cavity. In FlexX,
X-DMA derivatives with smaller 4′-substituents positioned
inside the LBD, in simile with the raloxifene-ERR/LBD crystal
structure. (Figure 3A). The prediction from the preliminary
computational studies was that the selected family of X-DMA
derivatives would provide a spectrum of ER affinity and redox
reactivity.
Synthesis. It was necessary to develop a new synthetic
methodology toward arzoxifene and its analogues. The published
synthesis of arzoxifene is multistep and is not readily amenable
to adaptation to generate arzoxifene analogs.35 In addition, the
starting material for the published 11-step synthesis of arzox-
ifene, 1-methanesulfonyloxy-4-bromobenzene, is not com-
mercially available. A new synthesis was designed to provide
a versatile common intermediate for preparation of arzoxifene
and novel arzoxifene analogs. The 4′-bromo derivative, 4,
provides an ideal synthetic intermediate because it is amenable
to copper(I) iodide-catalyzed aryl bromide derivatization
(Figure 4).
The synthesis of 4 was proceeded by the adaptation of
methods developed by us for F-DMA (Scheme 2).15 3-Meth-
oxybenzenethiol and 2,4′-dibromoacetophenone coupling under
basic conditions gave the â-ketosulfide 5, followed by cycliza-
tion and rearrangement at 130 °C in polyphosphoric acid (PPA)
to yield a mixture of rearranged and isomeric products. The
desired rearranged product 6 was readily separated in good yield
by simple ethyl acetate extraction and filtration. Recovered
unrearranged reactant was subjected to subsequent rearragement
to readily provide increased quantities of 6. Bromination of 6
with bromoacetamide in quantitative yield followed by oxidation
of the 3-bromobenzo[b]thiophene 7 with H2O2 gave the corre-
sponding sulfoxide 8, in which the bromide was activated by
the electron-withdrawing sulfoxide toward SNAr reaction.36 The
subsequent phenol displacement proceeded smoothly, and
compound 9 was obtained in high yield. The desired 4′-bromo
synthetic intermediate, 10, was obtained by reduction with Ph3P/
TMSCl,37 requiring prior protonation of the piperidine nitrogen
by acidification. LiAlH4 reduction at sulfur, employed in the
preparation of DMA25 and 4′-F-DMA,15 led to substitution of
the 4′-bromide with hydride and provided a useful synthetic
route to desmethoxyarzoxifene, 12, from 9. Methyl ether
deprotection of 10 and 11 using BF3·SMe238 gave the 4′-bromo-
4′-desmethoxy and 4′-desmethoxy arzoxifene analogues 4 and
12, respectively (attenuation of nitrogen basicity by acidification
was also required prior to use of the BF3 complex).
Arzoxifene itself (1) was obtained by the single-step metha-
nolysis of 4, effected by copper(I) iodide-catalyzed aryl bromide
substitution with concentrated sodium methoxide solution and
a catalytic amount of ethyl acetate that functions to prevent
precipitation of the copper species during the reaction (Scheme
3).39 This modified arzoxifene synthesis reduces the overall
number of steps compared to the published 11-step synthesis,
uses low-priced commercial starting materials, and is amenable
to scale-up.
A sulfone-substituted arzoxifene analogue was readily pre-
pared from 4 by direct introduction of the sulfone at the 4′-
position by coupling with sodium methanesulfinate under the
catalysis of a copper(I) iodide/L-proline sodium salt, a meth-
odology that has been recently reported (Scheme 3).40 The Cu-
(I) iodide/L-proline catalytic system can also be used in the
synthesis of aryl azides by the coupling reaction of the aryl
halide with sodium azide.41 This is an attractive route to 4′-
amino-4′-desmethoxyarzoxifene, an analogue that is predicted
to possess similar estrogenic binding and oxidative bioactivation
properties to DMA. Under catalysis of Cu(I) iodide/L-proline,
the reaction of compound 10 with sodium azide was achieved
by switching the solvent system from the reported EtOH/H2O
to a mixture of DMSO/ethanol heated at around 110 °C (Scheme
4). Instead of the azido product, the reaction directly gave the
desired 4′-amino compound 14 as a single product in good yield,
which might be due to the instability of the aryl azide at high

2686 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Qin et al.
predictions. The observed pseudo first-order rate constants are
shown in Table 1. DMA (2) and NH2-DMA (5) are not
expected to follow a similar pattern to the monophenolic SERMs
because these are able to form quinoids, a diquinone methide
and quinone imine, respectively, and theoretically to quench 2
equiv of DPPH.
Estrogen Receptor Binding Assays. Arzoxifene and its
analogues were assayed in the standard ER competitive radio-
ligand binding assay, using full length human recombinant ERR
and ERâ and compared to raloxifene.44 As shown in Table 1,
X-DMA ligands bind to ERR with affinities ranging from the
most potent ligand 2 (DMA) to 16, which is a very poor ligand
for ERR. All ligands are nonselective or selective for ERR over
ERâ, owing to the larger ligand binding cavity of ERR, hence
X-DMA ligands 13 and 18 are highly selective for ERR.
Docking Studies. X-DMA ligands with smaller 4′-substitu-
ents docked into the rigid LBD binding sites, using FlexX,
giving good correlations between D-Score and the experimental
pIC50 (exp ∆G; r2 ) 0.912 for ERR and r2 ) 0.744 for ERâ).
Of the scoring methods tested (G_Score, PMF_Score, D_Score,
ChemScore, Total-Score), D_Score consistently showed the best
correlation for FlexX-derived binding poses with both ERR and
ERâ. Ligands with bulkier 4′-substituents (X ) SO2CH3,
NH(CH2)2Cl, and NHC(O)CH2Cl and, in some poses, OCH3)
failed to dock inside the rigid LBD cavity, but three of these
were observed experimentally to be reasonable ligands for ERR.
To account for this observation, docking was achieved using
the DMA binding pose (Figure 3) as a starting point, followed
by force field minimization of the protein-ligand complex.
Using this method to examine the X-DMA ligands, D-Score
again provided the best correlation. This scoring function, drawn
from the molecular docking program DOCK, is a classical force
field energy function, which sums van der Waals and electro-
static interactions in the ligand binding complex.45 This result
is compatible with the dominant contributions to ER/LBD
binding from van der Waals interactions with residues, such as
Leu384 and Met421, and electrostatic interactions with (i)
Arg394/Glu353,42 His524, and (iii) Asp351(303), leading to
displacement of helix12 (Figure 6). Thus, computational docking
was able to account for experimental ligand binding using a
rigid receptor for ligands with smaller 4′-substituents and using
an iteratively relaxed receptor cavity for the more sterically
encumbered ligands.
Figure 5. (A) Time plot of relative absorbance (515 nm) showing
decay of DPPH radical in the presence of selected X-DMA SERMs.
(B) Observed rate constants for quenching of DPPH radical in
Using the Powell method46 for energy minimization with the
Tripos force field and Gasteiger-Huckel charges,47,48 relative
energies were calculated for binding of raloxifene and the
X-DMA ligands to ERR and compared to the experimental
binding energies, giving an excellent correlation for the ligands
binding to the rigid receptor (Figure 7) but not for those that
required relaxation of the receptor residues. This observation
demonstrates the limitations of the crystal structure docking
protocol for prediction of ligand binding and emphasizes that
conformational distortion of the receptor in the region of the
4′-DMA position to accommodate larger ligands does not result
in substantial loss of binding affinity. The crystal structure
reveals that the 4′-substituent interacts with a less-ordered
portion of the LBD. This observation suggests that further
modification at the 4′-position can be explored to optimize ERR
binding while ablating ERâ affinity. Crystal structures show
two distinct conformations of the His-524 imidazole ring.32
Despite the indicated proximity of His-524 in the relaxed
receptor docking and the good ERR affinity of 18, the present
data do not provide any evidence for covalent modification of
ERR. Conversely, the good selectivity of 18 for ERR/ERâ
methanolic solutions by X-DMA derivatives plotted against EHOMO
.
temperature (Scheme 4). The subsequent demethylation gave
the product 15. Selective acylation on the introduced amino
group gave the R-chloroacetylated product 16. The aniline
mustard 18 was prepared by reductive amination reaction of
14,42 and BCl3·SMe2 complex43 was used to avoid the possible
halogen exchange that might occur between BF3 and the chloride
of the mustard.
Antioxidant Activity. The simple 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) radical scavenging assay was run to measure
the relative antioxidant capacity of the X-DMA series com-
pounds and raloxifene. DPPH is a stable nitrogen radical, the
scavenging of which can be measured by its decay at 515 nm
(Figure 5). Although scavenging of stable nitrogen radicals is
not a major function of antioxidants in vivo, the assay is
routinely used to quantify antioxidant capacity and is useful
for comparisons within structural families. As shown in Figure
5 for the monophenolic SERMs, an excellent correlation was
seen between the rate of radical scavenging and the calculated
EHOMO for the X-DMA series, validating the computational

Benzothiophene SERMs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2687
Table 1. ER Binding Data and DPPH Radical Scavenging Rates for X-DMA SERMs Compared to Raloxifenea
ER RBAb
ER IC50 (nM)
selectivity
DPPH scavenging
cmpds (X)
raloxifene
1 (OCH3) arzoxifene
2 (OH) DMA
3 (F) F-DMA
4 (Br)
12 (H)
13 (SO2CH3)
15 (NH2)
ER-R
ER-â
R/â
ER-R
ER-â
103 × kobs, s-1
0.91 ( 0.12
0.94 ( 0.29
2.51 ( 0.61
1.07 ( 0.04
0.70 ( 0.12
1.71 ( 0.23
0.71 ( 0.12
0.96 ( 0.08
0.03 ( 0.01
0.56 ( 0.15
0.03 ( 0.01
0.25 ( 0.01
1.77 ( 0.35
0.68 ( 0.26
0.25 ( 0.03
1.00 ( 0.05
0.009 ( 0.0005
0.34 ( 0.01
0.007
30
3.8
1.4
1.6
2.8
1.7
79
2.8
4.3
>50
20.6 ( 2.7
21.5 ( 6.5
7.8 ( 1.9
17.2 ( 0.6
27.0 ( 4.6
10.9 ( 1.5
27.0 ( 4.8
19.3 ( 1.7
666 ( 219
35.6 ( 10
557 ( 146
66.3 ( 3.1
9.6 ( 1.9
27.9 ( 11
66.7 ( 8.5
16.3 ( 0.8
1800 ( 100
48.6 ( 1.9
2360 ( 18
>1600
4.0 ( 0.6
35 ( 7
9.3 ( 0. 4
6.0 ( 0.9
6.5 ( 0.3
8.9 ( 0.9
3.0 ( 0.5
220 ( 0.4
c
16 (NHC(O)CH2Cl)
18 (NH(CH2)2Cl)
c
<0.01
a Data shown is the mean ( S.D. for at least triplicate measurements. b RBA values calculated relative to IC50 assayed for E2 control (RBA ) 1.0). c Not
measured.
Figure 6. Raloxifene (red) and DMA docked in the ERR/LBD showing key residues. The MOLCAD multichannel surface was generated in
SYBYL. Distances between X-DMA ligands and key ERR/LBD residues, Å, are shown in the table below.
ERâ/LBD (PBD ID: 2FSZ), therefore, it was of interest to
screen the X-DMA ligands for binding.49 Most interactions at
the low affinity binding site are van der Waals contacts provided
by the amino acids within a radius of 5.5 Å from tamoxifen,
with core subpocket residues including Leu306, Met309, Ile310
Val 328, Leu331, Glu332, and Trp335 (using ERâ 2FSZ
reference numbering). The weak, nonspecific ligand association
in this hydrophobic grove permits numerous binding poses for
the X-DMA ligands, which would require probing by long-range
molecular dynamics. The low affinity binding site is not
proposed to inhibit estrogen binding to ER, but rather to
antagonize coregulator binding, thus, binding of bulkier X-DMA
ligands to this site cannot account for the competitive binding
assay data shown in Table 1.
Figure 7. Correlation between experimental pIC50 (exp ∆G) and
calculated ∆G binding (r2 ) 0.93).
Comparisons of X-DMA Activity/Reactivity. Except 16 and
18, the family of benzothiophene SERMs reported can be
subdivided into those that are readily able to form quinoids (2,
15) and the subset that are anticipated to form semiquinones
on oxidation (1, 3, 4, 12, 13). For the latter SERMs, two-electron
oxidation is blocked by the 4′-modification, thus it might be
further supports modification of the 4′ position of ben-
zothiophene SERMs to enhance selectivity.
Recently, a second low-affinity binding site for tamoxifen
has been reported in a crystal structure of 4-hydroxytamoxifen-

2688 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Qin et al.
NMR (acetone-d6, 100 MHz): δ 193.9, 160.9, 137.5, 135.6, 132.7,
131.3, 130.7, 128.6, 122.1, 115.3, 113.0, 55.5, 40.8. APCI-MS: m/z
339.0/337.0 (100/98%) [M + H]+.
anticipated that these will undergo oxidation to o-quinones, an
alternate pathway that is not blocked. In the latter subset, the
redox activity is controlled by the remote 4′-substituent, which
is expected to influence the rate of formation of o-quinones.
For example, 13 is 10-fold less redox active than 1 (Table 1).
The range of EHOMO values calculated for the SERM family is
similar to that for families of compounds that have shown a
wide range in chemopreventive index.29 However, the antici-
pated metabolism of both 1 and 12 to yield DMA (2) would
complicate interpretation in more complex systems. Neverthe-
less, the data presented herein presents some interesting
comparisons. For example, 4 and 13 have similar affinity for
ERR, and both have attenuated redox activity because of
electron-withdrawing 4′-substituents, but 13 is highly selective
for ERR/ERâ, and 2 and 12 are comparably antiestrogenic, but
only 2 can form a diquinone methide. The extrapolation of the
spectrum of antiestrogenic and redox activity, measured in
simple models herein, to complex cellular and in vivo systems
awaits the completion of such studies measuring oxidative
metabolism, Phase II enzyme induction, and estrogenic versus
antiestrogenic endpoints.
6-Methoxy-2-(4-bromophenyl)benzo[b]thiophene (6). PPA (70
g) was added to a 250 mL flask and heated to 80 °C with stirring
(keep the speed of stirring as fast as possible). Compound 5 (10 g,
29.8 mmol) was added portionwise within 30 min, then the
temperature of oil bath was elevated to 130 °C, and the reaction
mixture was heated with stirring for 6 h. The reaction mixture was
poured into 500 mL of rapidly stirring ice water to allow the PPA
to be hydrolyzed. After 2 h, 150 mL of ethyl acetate was added
and stirred for 20 min, and the crude product was collected by
filtration and washed with 15 mL of H2O three times. The obtained
brown solid was air-dried overnight to get the title compound, which
was pure enough for the next step (2.9 g, 30%). The ethyl acetate
solution containing unrearranged reactant was washed with H2O
and NaHCO3 solution, concentrated, passed through a short silica
gel column, and subjected to another rearrangement reaction to gave
more desired product. An analytic sample of 6 was obtained by
recrystallization using ethyl acetate. 1H NMR (DMSO-d6, 400
MHz): δ 7.81 (s, 1H), 7.73 (d, 1H, J ) 8.7 Hz), 7.63-7.68 (m,
4H), 7.56 (d, 1H, J ) 2.0 Hz), 7.01 (dd, 1H, J ) 8.7 Hz, 2.2 Hz),
3.83 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 157.5, 140.3,
138.9, 134.2, 133.0, 132.0, 127.6, 124.6, 120.9, 120.4, 114.9, 105.2,
55.5. APCI-MS: m/z 319.1/321.0 (98 /100%) [M + H]+.
Conclusions
6-Methoxy-2-(4-bromophenyl)-3-bromobenzo[b]thiophene (7).
Compound 6 (3.13 g, 9.87 mmol) was suspended in dried DCM
(70 mL), and N-bromoacetamide (1.45 g, 10.05 mmol) was added
with stirring. The reaction mixture was stirred at room temperature
for 2 h, diluted with another 100 mL of DCM, and washed with
H2O and brine, and the organic phase was separated and concen-
trated. Product was obtained after recrystallization, using ethyl
An ideal SERM, antiestrogenic in breast and endometrial
tissue, but proestrogenic in the vasculature and brain would be
of use in cancer chemoprevention and as an alternative to
hormone replacement therapy. The approaches taken have
focused largely on amplifying antiestrogenic activity; screening
families of SERMs for compounds with increased tissue
selectivity and, more recently, isoform selectivity. There has
been relatively little research directed at exploring the contribu-
tion of the redox reactivity and bioactivation that is common
to many SERMs and to test the hypothesis that structural
modification of SERMs to modulate bioactivation will lead to
an improved SERM. The synthesis presented herein provides
access to a structurally conservative family of SERMs related
to the clinically important benzothiophene SERMs that are
shown to manifest a spectrum of redox reactivity and ER ligand
properties.
1
acetate, as slightly brown solid (3.70 g, 95%). H NMR (DMSO-
d6, 400 MHz): δ 7.66-7.76 (m, 6H), 7.17 (dd, 1H, J ) 8.8 Hz,
2.3 Hz), 3.87 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 158.3,
138.5, 133.6, 132.2, 131.9, 131.6, 131.0, 123.9, 122.3, 115.8, 105.4,
104.3, 55.7. APCI-MS: m/z 398.8 [M + H]+.
6-Methoxy-2-(4-bromophenyl)-3-bromobenzo[b]thiophene S-
oxide (8). Compound 7 (2.6 g, 6.53mmol) was dissolved in
dichloromethane (30 mL), TFA (25 mL) was added dropwise with
stirring, then 1.4 mL of H2O2 (30%) was added, and the reaction
mixture was stirred at room temperature for 2 h. Sodium bissulfide
(500 mg) in 5 mL of H2O was added and stirred vigorously for 15
min to quench the reaction. Most of the solvent was removed under
reduced pressure, and the residue was diluted with dichloromethane
and carefully washed with saturated aqueous NaHCO3 solution.
After concentration, the crude product was purified by column
chromatography, eluting with 6:1 hexanes/ethyl acetate. Product
was obtained as slightly yellow solid (1.75 g, 65%). 1H NMR
(DMSO-d6, 400 MHz): δ 7.67-7.80 (m, 5H), 7.62 (d, 1H, J )
8.5 Hz), 7.31 (d, 1H, J ) 8.5 Hz), 3.91 (s, 3H). 13C NMR (DMSO-
d6, 100 MHz): δ 161.2, 144.4, 143.8, 132.1, 131.0, 129.2, 128.9,
125.6, 123.1, 121.9, 118.7, 112.4, 56.3. APCI-MS: m/z 414.9
(100%) [M + H]+.
Experimental Section
Synthesis. 1H and 13C NMR spectra were obtained with Bruker
Ultrashield 400 or Advance 300 spectrometer. Chemical shifts are
reported as δ values in parts per million (ppm) relative to
tetramethylsilane (TMS) for all recorded NMR spectra. Low-
resolution mass spectra were recorded on a Agilent 1100 series
LC/MSD ion trap instrument, using APCI as ionization method.
High-resolution mass spectra were taken on a Micromass QTOF
mass spectrometer, using ESI as the ionization method. All reagents
and solvents were obtained commercially from Acros, Aldrich, and
Fluka and were used without purification.
6-Methoxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-(4-bro-
mophenyl) Benzo[b]thiophene S-oxide (9). The side chain phenol
compound (1.80 g, 8.13 mmol) was dissolved in anhydrous DMF
(50 mL), NaH (328 mg, 60%, dispersed in oil) was added in three
portions within 20 min, and then bromide 8 (3.3 g, 8.01mmol) was
added. The reaction mixture was stirred at room temperature for 1
h and diluted with 200 mL of ethyl acetate, and the resulting
solution was washed with water. The aqueous phase was back
extracted with 50 mL of DCM, combined with the ethyl acetate
solution, and then dried with anhydrous MgSO4. Solvent was
removed under high vacuum to get a yellow solid that was pure
enough for the next step reaction (4.2 g, 94%). Analytical sample
1-(4-Bromophenyl)-2-(3-methoxylphenylsulfanyl)ethanone (5).
KOH (5.2 g, 87%, 80 mmol) was dissolved in ethanol (200 mL),
and 3-methoxy benzenethiol (9.7 mL, 79 mmol) was added and
stirred for 10 min. Ethyl acetate (80 mL) was added to this solution,
then 2,4′-dibromoacetophenone (20 g, 72 mmol) was added in
portions. Another 60 mL of ethyl acetate was added to dissolve
some precipitate that was produced during the reaction after 3 h.
The reaction mixture was stirred overnight at room temperature.
Most solvent was removed under reduced pressure, the residue was
partitioned between ethyl acetate and brine, and the organic phase
was separated and dried over anhydrous MgSO4. Concentration and
recrystallization using ethyl acetate gave desired compound 5 as a
1
was obtained by PTLC (5:1 DCM/MeOH). H NMR (DMSO-d6,
400 MHz): δ 7.54 (m, 1H), 7.59-7.70 (m, 4H), 7.06-7.11 (m,
3H), 7.01(d, 1H, J ) 8.5 Hz), 6.88 (d, 2H, J ) 8.8 Hz), 3.98 (t,
2H, J ) 5.6 Hz), 3.86 (s, 3H), 2.59 (t, 2H, J ) 5.6 Hz), 2.39 (br
s, 4H), 1.42-1.52 (m, 4H), 1.32-1.42 (m, 2H). 13C NMR (DMSO-
1
yellow solid (21.5 g, 89%). H NMR (acetone-d6, 400 MHz): δ
7.96-7.98 (m, 2H), 7.70-7.73 (m, 2H), 7.19-7.23 (m, 1H), 6.93-
6.95 (m, 2H), 6.76-6.79 (m, 1H), 4.53 (s, 2H), 3.77 (s, 3H). 13C

Benzothiophene SERMs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2689
d6, 75 MHz): 160.8, 155.1, 150.1, 148.4, 144.8, 131.9, 129.8, 129.6,
128.8, 125.3, 123.9, 121.6, 118.1, 118.0, 115.7, 112,8, 66.0, 57.3,
56.1, 54.4, 25.6, 23.9. APCI-MS: m/z 554.2/556.1 (100/98%) [M
+ H]+.
the organic phase was separated and dried by anhydrous MgSO4.
The crude product was purified by column chromatography (12:1
to 7:1 DCM/MeOH), the product was obtained as white solid (95
1
mg, 65%). H NMR (Acetone-d6, 400 MHz): δ 7.75-7.77 (m,
2H), 7.39 (m, 2H), 7.35 (d, 1H, J)1.8 Hz), 7.22-7.32 (m, 2H),
6.85-6.92 (m, 5H), 4.07 (t, 3H, J) 5.8 Hz), 2.77 (t, 3H, J) 5.8
Hz), 2.57 (bs, 4H), 1.54-1.58 (m, 4H), 1.42-1.44 (m,2H); 13C
NMR (Acetone-d6, 75 MHz): δ 157.1, 155.2, 152.5, 141.9, 138.2,
133.4, 129.7, 128.6, 128.0, 127.9, 126.2, 123.2, 117.1, 116.4, 115.7,
108.8, 66.9, 58.5, 55.6, 26.4, 24.7; HRMS calcd. for C27H28NO3S
446.1790 [M+H]+, found 446.1785.
6-Methoxy-3-{4-[2-(1-piperidinyl) ethoxy]phenoxy}-2-(4-bro-
mophenyl) Benzo[b]thiophene (10). Compound 9 (4.0 g, 7.2
mmol) was dissolved in DCM (25 mL), and 1 M HCl/ether solution
(15 mL) was added and stirred for 1 h. All solvent was removed
under reduced pressure. The residue was dissolved in anhydrous
THF (70 mL), TMSCl (9.2 mL, 72 mmol) and Ph3P (7.0 g, 26.7
mmol) were added, the reaction mixture was refluxed for 8h. Most
of the solvent was removed, residue was diluted with 200 mL ethyl
acetate, washed with saturated aqueous NaHCO3 solution, concen-
trated, crude product was purified by column chromatography,
eluting with 30:1 DCM/MeOH containing 3‰ HOAc to remove
the excess of Ph3P and Ph3PO, then eluting with 20:1 DCM/MeOH
6-Hydroxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-(4-meth-
oxyphenyl) benzo[b] thiophene (1, Arzoxifene). Compound 4 (65
mg, 0.12 mmol), CuI (24 mg, 0.12 mmol) were added into an argon-
flushed flask, DMF (0.7 mL), anhydrous MeOH (1.5 mL) and ethyl
acetate (40 µL ,0.4 mmol) were added, after the addition of NaOMe
(530 mg, 9.9 mmol, concentration 4.5 M), the reaction mixture were
heated at 110 °C for 8h. The reaction mixture was diluted with 60
mL ethyl acetate, neutralized with aqueous 5N acetic acid, then
washed with brine, the organic phase was separated, after concen-
tration, the residue was purified by column chromatography (12:1
DCM/MeOH), the product was obtained as white solid (48 mg,
1
to get the product (3.3 g, 85%). H NMR (DMSO-d6, 400 MHz):
δ 7.61-7.68 (m, 5H), 7.21 (d, 1H, J)8.8 Hz), 6.95 (dd, 1H, J)8.8
Hz, 2.3 Hz), 6.86-6.89 (m, 4H), 3.95 (t, 3H, J) 5.9 Hz), 3.83 (s,
3H), 2.59 (t, 3H, J) 5.9 Hz), 2.38 (bs, 4H), 1.43-1.48 (m, 4H),
1.35-1.36 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): δ 158.2 154.0,
150.7 141.0, 136.8 132.1 130.9, 128.7, 126.9, 124.3, 122.0, 121.0,
116.2, 115.6, 115.1, 106.1, 65.9, 57.4, 55.6, 54.4, 25.5, 23.9; HRMS
calcd. for C28H29NO3SBr 538.1052 [M+H]+, found 538.1072.
6-Hydroxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-(4-bro-
mophenyl) benzo[b] thio phene (4). Compound 10 (300 mg, 0.56
mmol) was dissolved in DCM (3 mL), 1N HCl in diethyl ether (3
mL) was added, the mixture was stirred at room temperature for
30 min. All solvents was removed and the obtained slightly yellow
foam was redissolved in 15 mL DCM, the flask was filled with
argon, BF3 dimethyl sulfide complex (2.2 mL) was added, the
resulting mixture was stirred at room temperature for 5h. The
reaction was diluted with 30 mL ethyl acetate, and washed with
saturated NaHCO3 aqueous solution, organic phase was separated
and dried by anhydrous MgSO4. The crude product was purified
by column chromatography (15:1 DCM/MeOH), the product was
obtained as slight yellow solid (240 mg, 82%). 1H NMR (DMSO-
d6, 400 MHz): δ 9.92 (s, 1H), 7.58-7.67 (m, 4H), 7.30 (d, 1H,
J)1.9 Hz), 7.14 (d, 1H, J)8.7 Hz), 6.85 (bs, 4H), 6.82 (dd, 1H,
J)8.7 Hz, 1.9 Hz), 3.97 (t, 2H, J) 5.9 Hz), 2.59 (t, 2H, J) 5.8
Hz), 2.38(m, 4H), 1.43-1.48 (m, 4H), 1.32-1.38 (m, 2H) ; 13C
NMR (DMSO-d6, 100 MHz): δ 156.4, 154.0, 150.7, 141.2, 136.7,
132.0 , 131.1 , 128.6, 125.9, 123.1, 122.2, 120.8, 116.2, 115.6,
115.2, 108.0, 65.8, 57.3, 54.3, 25.4, 23.8 ; HRMS calcd. for C27H27-
NO3SBr 524.0895 [M+H]+, found 524.0887.
6-Methoxy-3-{4-[2-(1-piperidinyl) ethoxy]phenoxy}-2-phenyl
benzo[b] thiophene (11). Compound 9 (270 mg, 0.47 mmol) was
dissolved in anhydrous THF (5 mL), LiAlH4 (55 mg, 1.45 mmol)
was added, the reaction mixture was refluxed for 6h. The reaction
was quenched by adding 2N aqueous NaOH solution (0.5 mL),
then diluted with 30 mL ethyl acetate, more NaOH was added until
most of the white precipitate dissolved. The organic phase was
separated and concentrated, the residue was purified by column
chromatography (50:3 DCM/MeOH), product was obtained as white
solid (180 mg, 83%). 1H NMR (CDCl3, 400 MHz): δ 7.76 (d, 2H,
J) 7.4 Hz), 7.25-7.37 (m, 5H), 6.86-6.91 (m, 3H), 6.79-6.81
(m, 2H), 4.05 (t, 2H, J) 6.0 Hz), 3.87 (s, 3H), 2.76 (t, 2H, J) 6.0
Hz), 2.52 (bs, 4H), 1.59-1.65 (m, 4H), 1.42-1.48 (m, 2H); 13C
NMR (CDCl3, 75 MHz): δ158.2, 154.2, 151.9, 141.0, 137.4, 132.6,
128.9, 128.2, 127.8, 127.6, 126.7, 122.7, 116.6, 115.7, 114.6, 105.5,
66.5, 58.1, 55.8, 55.2, 26.0, 24.8; HRMS calcd. for C28H30NO3S
460.1946 [M+H]+, found 460.1935.
1
85%). H NMR (Acetone-d6, 400 MHz): δ 7.67-7.69 (m, 2H),
7.32 (d, 1H, J) 2.0 Hz), 7.21 (d, 1H, J)8.6 Hz), 6.94-6.96 (m,
2H), 6.86-6.88 (m, 5H), 4.14 (t, 2H, J) 5.8 Hz), 3.80 (s, 3H),
2.92 (t, 2H, J)5.8 Hz), 2.73 (m, 4H), 1.56-1.66 (m, 4H), 1.43-
1.45 (m, 2H); 13C NMR (Acetone-d6, 75 MHz): δ 160.3, 156.8,
154.9, 152.7, 140.7, 137.7, 129.4, 128.1, 126.3, 125.8, 122.8, 117.1,
116.5, 115.6, 115.1, 108.9, 66.3, 58.2, 55.6, 55.4, 25.9, 24.3; HRMS
calcd. for C28H30NO4S 476.1896 [M+H]+, found 476.1893.
6-Hydroxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-[(4-meth-
anesulfonyl)phenyl] benzo[b] thiophene (13). Compound 4 (50
mg, 0.095mmol), CH3SO2Na (78 mg, 0.76 mmol), L-proline (8.7
mg, 0.076 mmol), NaOH (6 mg, 0.15 mmol), CuI (14.5 mg, 0.076
mmol) were added to a 5 mL flask, then filled with argon,
anhydrous DMSO (1.5 mL) was added, the resulting reaction
mixture was stirred at 110 °C for 13 h. The reaction mixture was
partitioned between ethyl acetate (30 mL) and water (5 mL), the
undissolved solid was filtered off, solid was washed with another
10 mL ethyl acetate. The organic phase was combined, separated
and concentrated, crude product was purified by column chroma-
tography (DCM/MeOH 20:1), product was obtained as slightly
1
green foam (45 mg, 90%). H NMR (Acetone-d6, 400 MHz): δ
7.93-8.01 (m, 4H), 7.38 (d, 1H, J)1.9 Hz), 7.27 (d, 1H, J)8.7
Hz), 6.80-6.93(m, 5H), 4.03 (t, 2H, J) 5.9 Hz), 3.12 (s, 3H), 2.68
(t, 2H, J)5.9 Hz), 2.48 (m, 4H), 1.50-1.54 (m, 4H), 1.39-1.41
(m, 2H) 13C NMR (Acetone-d6, 75 MHz): δ 157.8, 155.6, 152.1,
144.1, 140.7, 138.9, 138.4, 128.8, 128.3, 127.6, 124.0, 123.9, 117.3,
116.5, 116.2, 108.9, 67.2, 58.6, 55.6, 44.3, 26.7, 24.9. HRMS calcd.
for C28H30NO5S2 524.1565 [M+H]+, found 524.1575
6-Methoxy-3-{4-[2-(1-piperidinyl) ethoxy]phenoxy}-2-(4-ami-
nophenyl) benzo[b] thiophene (14). Compound 10 (150 mg, 0.278
mmol), NaN3 (217 mg, 3.36 mmol), L-proline (28 mg, 0.25 mmol),
NaOH (11.1 mg, 0.27 mmol), CuI (47 mg, 0.25 mmol) were added
to a 10 mL flask, then filled with argon, a mixture of 4 mL DMSO/2
mL EtOH was added by syringe, the resulting reaction mixture was
stirred at 110 °C for 10h. The reaction was partitioned between
ethyl acetate and water, the undissolved solid was filtered off. The
organic phase was separated and concentrated, crude product was
purified by column chromatography (DCM/MeOH), product was
obtained as slightly green syrup (98 mg, 74%). 1H NMR (DMSO-
d6, 400 MHz): δ 8.31 (s,1H), 7.51 (d, 1H, J)2.0 Hz), 7.37 (d, 2H,
J)8.4 Hz), 7.12 (d, 1H, J)8.7 Hz), 6.81-6.91 (m, 5H), 6.55 (d,
2H, J)8.4 Hz), 5.40 (s, 2H), 3.95 (t, 2H, J) 5.7 Hz), 3.80 (s, 3H),
2.59 (t, 2H, J)5.6 Hz), 2.39 (bs, 4H), 1.45-1.50 (m, 4H), 1.35-
1.40 (m, 2H); 13C NMR (DMSO-d6, 100 MHz): δ 157.3, 153.7,
151.0, 148.9, 137.7, 135.4, 127.9, 127.7, 127.4, 120.9, 118.8, 115.9,
115.5, 114.3, 113.8, 106.0, 65.8, 57.4, 55.5, 54.3, 25.5, 23.9; HRMS
calcd. for C28H31N2O3S 475.2055 [M+H]+, found 475.2045.
6-Hydroxy-3-{4-[2-(1-piperidinyl) ethoxy]phenoxy}-2-(4-ami-
nophenyl) benzo[b] thiophene (15). Compound 14 (250 mg, 0.53
6-Hydroxy-3-{4-[2-(1-piperidinyl) ethoxy]phenoxy}-2-phenyl
benzo[b] thiophene (12). Compound 11 (150 mg 0.33 mmol) was
dissolved in DCM (5 mL), 1N HCl in diethyl ether (1 mL) was
added, the mixture was stirred at room temperature for 30 min.
All solvents was removed and the residue was redissolved in 8
mL DCM, the flask was filled with argon, BF3 dimethyl sulfide
complex (1.7 mL) was added, the resulting mixture was stirred at
room temperature for 5h. The reaction was diluted with 30 mL
ethyl acetate, and washed with saturated NaHCO3 aqueous solution,

2690 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Qin et al.
mmol) was dissolved in DCM (10 mL), 1M HCl in ether (3 mL)
was added and the mixture was stirred at room temperature for 30
min. All solvents was removed and the residue was redissolved in
DCM (12 mL), the flask was filled with argon, BF3 dimethyl sulfide
complex (2.5 mL) was added, the resulting mixture was stirred at
room temperature for 5h. The reaction was diluted with 30 mL
ethyl acetate, and washed with saturated NaHCO3 aqueous solution,
the organic phase was separated and dried by anhydrous MgSO4.
The crude product was purified by column chromatography (10:1
DCM/MeOH), the product was obtained as slightly yellow solid
(175 mg, 72%). 1H NMR (Acetone-d6, 400 MHz): δ 7.47 (d, 2H,
J)8.68 Hz), 7.28 (d, 1H, J)1.86 Hz), 7.16 (d, 1H, J)8.62 Hz),
6.38-6.91 (m, 5H), 6.66 (d, 2H, J ) 6.89 Hz), 4.89 (bs, 2H), 4.21
(t, 2H, J) 5.45 Hz), 3.19 (t, 2H, J) 5.36 Hz), 3.00 (m, 4H), 1.71-
1.76 (m, 4H), 1.52-1.56 (m, 2H); 13C NMR (Acetone-d6, 100
MHz): δ 156.3, 155.1, 152.6, 139.5, 137.1, 129.0, 128.4, 127.7,
122.4, 121.5, 116.9, 116.3, 115.2, 115.1, 108.7, 67.3, 58.7, 55.7,
26.7, 24.9; HRMS calcd. for C27H29N2O3S 461.1899 [M+H]+,
found 461.1901.
6-Hydroxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-[4-(2-
chloroacetamide)phenyl] benzo[b] thiophene (16). Compound 15
(46 mg, 0.1 mmol), pyridine (80 µL) were dissolved in anhydrous
CH2Cl2 (1.5 mL) and cooled in ice bath, chloroacetyl chloride (6
µL) was added by a syringe. The reaction mixture was gradually
warmed up to room temperature and stirred overnight. Reaction
mixture was diluted with CH2Cl2 (25 mL), washed with saturated
aqueous NaHCO3 solution. The organic phase was separated and
concentrated, crude product was purified by flash column chro-
matography (AcOEt/CH2Cl2/MeOH 30:25:8). The product was
obtained as slightly yellow foam (31 mg, 57%). 1H NMR (Acetone-
d6, 400 MHz): δ 9.56 (s, 1H), 7.74-7.69 (m, 4H), 7.33 (d, 1H,
J) 2.0 Hz), 7.22 (d, 1H, J) 8.65 Hz), 6.91-6.83 (m, 5H), 4.24
(s,1H), 4.01 (t, 2H, J) 5.96 Hz), 2.66 (t, 2H, J)5.96 Hz), 2.46
(m, 4H), 1.53-1.49 (m, 4H), 1.40-1.35 (m, 2H); 13C NMR
(Acetone-d6, 75 MHz): δ 165.5, 157.1, 155.4, 152.5, 141.7, 139.1,
138.0, 129.2, 128.6, 128.1, 125.9, 123.2, 120.7, 117.2, 116.5, 115.8,
108.9, 67.4, 58.8, 55.8, 44.2, 26.8, 25.1; HRMS calcd. for
C29H30N2O4SCl 537.1615 [M+H]+, found 537.1595.
5.9 Hz), 4.10 (t, 2H, J) 5.8 Hz), 3.73 (t, 2H, J)6.1 Hz), 3.51-
3.56 (m, 2H), 2.79 (t, 2H, J)5.7 Hz), 2.60 (m, 4H), 1.56-1.62
(m, 4H), 1.40-1.46 (m, 2H); 13C NMR (Acetone-d6, 75 MHz): δ
156.5, 155.1, 152.8, 148.8, 139.7, 137.3, 129.3, 128.5, 127.5, 122.5,
121.9, 117.1, 116.5, 115.4, 113.4, 108.9, 66.9, 58.5, 55.5, 45.9,
43.9, 26.3, 24.7; HRMS calcd. for C29H32N2O3SCl 523.1822
[M+H]+, found 523.1809.
Estrogen Receptor Binding Assays. ER competitive binding
assay with [3H]-estradiol. The assay was slightly modified from
the original protocol.49 Twenty-four hours before the assay, 50%
v/v hydroxyapatite (HAP) slurry was prepared using 10 g hydroxy-
lapatite/60 mL of TE buffer (50 mM Tris-Cl, 1 mM EDTA, pH
7.4) and stored at 4 °C. ER binding buffer (10 mM Tris, 10%
glycerol, 2 mM dithiothrietol, 1 mg/mL bovine serum albumin, pH
7.5), ERR (40 mM Tris, 100 mM KCl, pH 7.5) and ERâ (40 mM
Tris, pH 7.5) wash buffers were prepared subsequently. The reaction
mixture consisted of 5µL of test samples in DMSO, 5µL of pure
human recombinant diluted ERR or ERâ (0.5pmol) in ER binding
buffer, 5µL of “Hot Mix” (400nM, prepared fresh using 3.2µL of
3
25µM, 83Ci/mM H-estradiol, 98.4µL of ER binding buffer), and
85µL ER binding buffer. The incubations were performed at room
temperature for 2 h or at 4 °C overnight, then 100µL of 50% HAP
slurry was added and the tubes were incubated on ice for 15 min
with votexing every 5 min. The appropriate ER wash buffer was
added (1 mL), and the tubes were vortexed and then centrifuged at
2000 g for 5 min. The supernatant was discarded and this wash
step was repeated three times. The HAP pellet, containing the
ligand-receptor complex, was then resuspended in 200 µL of
ethanol and transferred to scintillation vials. Cytoscint (4 mL/vial)
was added, and the samples were counted using a Beckman
(Schaumburg, IL) LS 5801 liquid scintillation counter. The percent
inhibition of [3H] estradiol binding to each ER was determined as
follows: [1 - (dpmsample - dpmblank)/(dpmDMSO - dpmblank)] × 100.
IC50 values were calculated from binding of the sample expressed
as a percentage relative to E2 (50 nM, 100%). Relative binding
affinity (RBA; relative to E2) was calculated from IC50(E2)/IC50-
(sample). The samples were assayed in triplicate at at least five
concentrations.
Antioxidant Activities of SERMs. Antioxidant activities of
SERMs were monitored using DPPH assay. The 0.2 mM solution
of DPPH in methanol and 1.2 mM of SERMs in DMSO were mixed
in a 1 mL spectrophotometer cell. The maximum volume of DMSO
used in experiments was less than 4% compare to methanol. Kinetic
traces were obtained at 515 nm using a HP8452A diode array
spectrometer. Observed rate constants (kobs) were obtained from
first-order fitting of the corresponding kinetic traces. Excellent
pseudo first-order kinetics were observed for DPPH degradation
in the presence of monophenolic SERMs (Figure 5), although for
2 and 15, the curve shape suggested more complex behavior.
Computational Methods. DFT molecular orbital calculations
were performed using Spartan 4.0 for Windows (Wavefunction Inc.,
CA) using minimum energy conformations obtained at the semiem-
pirical AM1 level. All molecular modeling studies were performed
on an SGI computer with the Sybyl 7.2 software packages. The
coordinates for the estrogen receptor alpha (ERR) LBD were
extracted from the cocrystal structure data of the complex between
ERR LBD and raloxifene (PDB code: 1ERR). The coordinates for
the estrogen receptor beta (ERâ) LBD were extracted from the
cocrystal structure data of the complex between ERâ and tamoxifen
(PDB code: 2FSZ). The active site was designated to consist of
the amino acid residues within a radius of 4.5 Å from the original
ligand raloxifene or tamoxifen. According to ERR (ERâ in
parenthesis) reference numbering, Asp351(303), Glu353(305),
Arg394(346), and His524(475) were set as a core subpocket. The
following FlexX-Pharm settings were used to restrict the binding
of the ligands to the raloxifene/tamoxifen binding site: Asp351-
(303) is an optional hydrogen-bond acceptor, and a spatial constraint
of r ) 3 Å is set around carboxylate oxygens of Asp351(303) and
the ligand’s nitrogen.
6-Methoxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-{4-[(2-
chloroethyl) amino] phenyl} benzo[b] thiophene (17). Compound
14 (60 mg 0.12 mmol) was dissolved in 2 mL methanol, chloro-
acetaldehyde (30 µL 45% aqueous solution, 0.16 mmol), NaBH3-
CN (15 mg, 0.17 mmol), 6N HCl in methanol (30 µL) were added,
the reaction mixture was stirred at room temperature for 3 days,
solvent was removed under reduced pressure, residue was purified
by column chromatography (DCM/MeOH 20:1 containing 1‰
NH3‚H2O) to get the product (25 mg, 37.3%). 1H NMR (Acetone-
d6, 400 MHz): δ 7.55(d, 2H, J)8.7 Hz), 7.43(d, 1H, J)2.1 Hz),
7.21 (d, 1H, J)8.7 Hz), 6.84-6.9 3 (m, 5H), 6.69 (d, 2H, J)8.7
Hz), 5.50 (t, 1H, J) 5.96 Hz), 4.01 (t, 2H, J) 5.9 Hz), 3.86 (s,
3H), 3.73 (t, 2H, J)6.2 Hz), 3.52-3.55 (m, 2H), 2.66 (t, 2H, J)6.0
Hz), 2.46 (m, 4H), 1.49-1.55 (m, 4H), 1.38-1.41 (m, 2H); 13C
NMR (Acetone-d6, 75 MHz): δ 158.8, 155.2, 152.6, 148.8, 139.6,
137.2, 129.2, 129.2, 128.3, 122.3, 121.7, 116.9, 116.4, 115.1, 113.4,
106.5, 67.3, 58.7, 55.9, 55.7, 45.8, 43.8, 26.7, 24.9; HRMS calcd.
for C30H34N2O3SCl 537.1979 [M+H]+, found 537.1983.
6-Hydroxy-3-{4-[2-(1-piperidinyl)ethoxy]phenoxy}-2-{4-[(2-
chloroethyl) amino] phenyl} benzo[b] thiophene (18). Compound
17 (20 mg, 0.03 7mmol) was dissolved in DCM (1.5 mL), 1N HCl
in diethyl ether (0.3 mL) was added, the mixture was stirred at
room temperature for 30 min. All solvents was removed and the
obtained brown foam was redissolved in DCM (1.5 mL), the flask
was filled with argon, BCl3 dimethyl sulfide complex (0.3 mL)
was added, the resulting mixture was stirred at 70 °C for 10 h. The
reaction was diluted with 10 mL DCM, and washed with saturated
NaHCO3 aqueous solution, the organic phase was separated and
dried by anhydrous MgSO4. Crude product was purified by column
chromatography (15:1 DCM/MeOH). Product was obtained as slight
1
yellow solid (15 mg, 77%). H NMR (Acetone-d6, 400 MHz): δ
7.54 (d, 2H, J)8.7 Hz), 7.29 (d, 1H, J)1.9 Hz), 7.16 (d, 1H, J)8.6
Hz), 6.83-6.89 (m, 5H), 6.69 (d, 2H, J)8.7 Hz), 5.49 (t, 1H, J)
Arzoxifene and its 4′-position modified analogs were screened.
After ligand docking was performed by the FlexX and Flex-Pharm

Benzothiophene SERMs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2691
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modules in Sybyl, the 30 best poses were selected for each ligand
and saved for analysis by CScore. We used a combination of several
functions and the criterion of consensus g4 to select the best pose
for each ligand. The binding modes of the docked ligands were
found to be consistent with those expected for compounds that are
structurally related to raloxifene and tamoxifen. The selection of
the binding poses was based on FlexX score. Scoring and ranking
of the poses using the CScore scoring function performed reason-
ably well for the ligands able to dock within the LBD cavity. Bulkier
4′-substituents failed to dock inside the cavity. For these ligands,
manual docking was designed based on the top scoring DMA pose.
Furthermore, the most meaningful pose or manually docked pose
for each ligand was co-minimized with the receptor LBD by
employing the Powell method with initial Simplex optimization
(20 steps) using the Tripos force field, Gasteiger-Huckel charges,
dielectric constant ) 1, and nonbonding cutoff of 8 Å, until a
convergence of 0.05 kcal/mol·Å was reached. The minimized poses
were re-evaluated with CScore functions.
To examine the second, low-affinity reported binding site for
tamoxifen in ERâ, a second screening for arzoxifene analogs was
devised based on PBD ID: 2FSZ. Because most of the interaction
of the low affinity binding site are based on van der Waals contacts,
the second active site was designated to consist of amino acids
with a radius of 5.5 Å from tamoxifen, with core subpocked residues
such as Leu306, Met309, Ile310 Val 328, Leu331, Glu332, and
Trp335 according to ERâ 2FSZ reference numbering. The binding
resulted in weak, nonspecific association of hydrophobic 4′-
substituents, with the hydrophobic groove of the coactivator
recognition surface.
Energy Evaluation. To compare the energetic interactions of
the arzoxifene series with the estrogen receptor ERR vs raloxifene
interaction with ERR, the following equation was used
∆Gbind ) (GER-analog complex - Ganalog) -
(GER-raloxifene complex - GER-raloxifene
)
Solute energy was evaluated for the lowest energy conformer in
the same method as mentioned above for the ERR-analog complex.
Acknowledgment. This work was supported by NIH Grants
CA102590 and CA79870.
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