Bicyclic core estrogens as full antagonists: Synthesis, biological evaluation and structure-activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides

Article abstract of DOI:10.1039/c2ob26531a

Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERβ. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO₂NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ERβ. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.

Full text of DOI:10.1039/c2ob26531a

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 8692
www.rsc.org/obc
PAPER
Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and
structure–activity relationships of estrogen receptor ligands based on bridged
oxabicyclic core arylsulfonamides†
Manghong Zhu,‡a Chen Zhang,‡a Jerome C. Nwachukwu,b Sathish Srinivasan,b Valerie Cavett,b
Yangfan Zheng,a Kathryn E. Carlson,c Chune Dong,a John A. Katzenellenbogen,*c Kendall W. Nettlesb and
Hai-Bing Zhou*a
Received 2nd August 2012, Accepted 18th September 2012
DOI: 10.1039/c2ob26531a
Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are
useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel
estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo-
[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester
(termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERβ.
Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its
partial antagonist character by stabilizing a novel conformation of the ER that involves a significant
distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core
ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or
tertiary sulfonamides (–SO2NR–), isoelectronic and potentially isostructural molecular replacements.
An array of 16 OBHS sulfonamide analogues were prepared through a Diels–Alder reaction of a
3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary
sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity.
In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower
intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM)
hydroxytamoxifen or OBHS, and they were inverse agonists on ERβ. Thus, the behaviour of these
OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant
(ICI 182 780), and their greater antagonist biocharacter appears to arise from an accentuated distortion
of helix-11.
pharmaceutical targets.1,2 In contrast to the pan-agonist activity
of 17β-estradiol (E2, Fig. 1), Selective Estrogen Receptor
Introduction
The estrogen receptors, ERα and ERβ, regulate a diverse set of
Modulators (SERMs) display agonist activity in certain tissues
but antagonist activity in others,3,4 and some SERMs, such as
tamoxifen and raloxifene, are used for the treatment of breast
cancer or for menopausal hormone replacement.5–7 Because
activity profiles of these SERM are not ideal and resistance to
their effectiveness as antitumor agents can develop with time,
there has been interest in finding new SERMs that might prove
more effective as hormonal or therapeutic agents.6,7 ER ligands
that reduce the level (as well as the activity) of ER are termed
Selective Estrogen Receptor Downregulators (SERDs),8,9 and
because they actually lower ER levels, SERDs are distinct from
SERMs. SERDs such as fulvestrant (Fig. 1, ICI 182 780) are
showing promise in the treatment of metastatic breast cancer,
because they can inhibit the growth of tamoxifen-resistant breast
cancer cells.10 Fulvestrant, however, has a poor oral bioavail-
ability; so, there is also a need for improved SERDs.
physiological and pathological processes and are well established
aLaboratory of Combinatorial Biosynthesis and Drug Discovery
(Wuhan University), Ministry of Education, Wuhan University School of
Pharmaceutical Sciences, Wuhan, 430072, China.
E-mail: zhouhb@whu.edu.cn; Fax: +86-27-68759850;
Tel: +86-27-68759586
bDepartment of Cancer Biology, The Scripps Research Institute-Florida,
130 Scripps Way, Jupiter, FL 33458, USA. E-mail: knettles@scripps.edu;
Fax: +1-561-799-8805; Tel: +1-561-228-3209
cDepartment of Chemistry, University of Illinois, 600 South Mathews
Avenue, Urbana, IL 61801, USA. E-mail: jkatzene@illinois.edu;
Fax: +1-217-333-7325; Tel: +1-217-333-6310
†Electronic supplementary information (ESI) available: Synthetic pro-
cedures and characterization of compounds 7–9, and 1H NMR assign-
ment of exo 11. See DOI: 10.1039/c2ob26531a
‡These two authors contributed equally to this work.
8692 | Org. Biomol. Chem., 2012, 10, 8692–8700
This journal is © The Royal Society of Chemistry 2012

View Online
Fig. 1 The structure of estradiol, 4-OH tamoxifen, fulvestrant (ICI
182 780) and representative three-dimensional ER ligands, OBHS and
title compounds.
In a new approach to develop novel SERMs and SERDs, we
prepared compounds having a more three-dimensional central
hydrophobic core topology than is typically found in steroidal
and non-steroidal ER ligands. This design was inspired by struc-
tural studies of ligand complexes with ER that reveal ample
unoccupied space above and below the mean plane of E2,
particularly near the middle of this molecule.11,12 A number of
structural motifs, such as the bridged bicyclo[3.3.1]nonene core
systems (Fig. 1), have been explored by us13 and others14,15 to
probe this extra space and exploit the flexibility of the ligand-
binding pocket (LBP).
Scheme 1 The synthesis of various dienophiles 7–9. (Yields given are
for the final sulfonation step; yields for the other steps are given in the
ESI†.)
We were intrigued by the role that the phenyl sulfonate moiety
played in engendering the partial antagonist activity of OBHS
ligands,17 and because of our interest in finding ER ligands that
have more complete antagonist, even SERD activity, we have, in
this study, queried the structure of OBHS at this very position by
substituting the sulfonate with secondary and tertiary sulfon-
amide (–SO2NR–) linkages, keeping the remainder of the 7-oxa-
bicyclo[2.2.1]hept-5-ene skeleton intact. While the secondary
phenyl sulphonamides are isostructural to the phenyl sulfonate,
the tertiary sulphonamides introduce an additional substituent
that, as will be seen, is of consequence. We prepared a set of
16 OBHS sulfonamides (11a–q) by an efficient Diels–Alder
approach,16 and we evaluated them for ER binding affinity, for
transcriptional activity in a relevant cell culture assay system,
and by computational modelling for structural analysis. In the
process, we have identified some oxabicyclo[2.2.1]heptane
sulphonamides that are more complete antagonists than even
hydroxytamoxifen, having biological character more like that of
fulvestrant (ICI 182 780). Computational modelling indicates
that this more complete antagonist biocharacter arises from a
more accentuated distortion of helix-11 by the sulphonamides
than was effected by the phenylsulfonate of OBHS.
Recently, we evaluated three-dimensional ER ligands based
on a different bridged oxabicyclo[2.2.1]heptene core (Fig. 1).16
The best compound, exo-5,6-bis-(4-hydroxyphenyl)-7-oxa-
bicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester (OBHS),
exhibited relative binding affinity (RBA) values of 9.3% and
1.7% for ERα and ERβ, respectively (RBA[estradiol] = 100%).
OBHS also profiled as a partial antagonist on both ER subtypes,
even though it was structurally unlike typical ER antagonists.
Through our recent structural studies of this compound17 as well
as other members of this series,18 it became evident that the high
ER binding affinity of OBHS relies on its two 4-hydroxyphenyl
substituents, one which mimics the A-ring of estradiol, the other
which projects into a subpocket that lies in the 11β direction
with respect to estradiol in the complex with ERα. Of greater
interest, however, was the fact that the large phenyl sulfonate
moiety, which is too long and extended to fit into the ligand-
binding pocket found in crystal structures of typical steroidal and
non-steroidal estrogens and SERMs, was readily accommodated
by a reorganization of the peptide backbone and residues at the
end of helix-11. These changes provided sufficient volume to
accommodate this large and extended group, without encounter-
ing a marked penalty in ligand binding affinity. This distortion of
helix-11 was presumed to result in the overall partial antagonist
character of OBHS-type ligands by an indirect—rather than a
direct—mechanism through which helix-12 becomes displaced
from its agonist conformation because of the dislocation of the
C-terminus of helix-11, rather than by direct ligand contact with
helix-12.19
Results
Synthesis
The target sulfonamides were prepared by Diels–Alder cyclo-
addition16 of 3,4-bis(4-hydroxyphenyl)furan 10 with various sul-
fonamide dienophiles (7a–d, 8a–h and 9a–d, see ESI† for
details),20,21 readily obtained from anilines (Scheme 1). Dieno-
philes with a secondary sulfonamide –SO2NH– system (7a–d)
were prepared in a single-step by the reaction of 2-chloroethane-
sulfonyl chloride (1.2 equiv.) with the aniline 1 (Scheme 1A);22
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8692–8700 | 8693

View Online
tertiary sulfonamides (N-substituents CH3, CH2CH3, CH2CF3)
were synthesized as shown (Scheme 1B and C). Acylation of
anilines 1 with acetic anhydride or trifluoroacetic anhydride gave
compounds 3a–h,23,24 and compounds 4 were obtained by
borane reduction of compounds 3, under optimized
conditions.25–27 The compounds 6 were prepared from 3 in two
steps.28 In the first step, amide 3 was methylated by methyl
iodide in the presence of sodium hydride in THF to afford 5. In
the second step, deacylation of 5 in 10% HCl (0.25 mL for
1 mmol amide) and glycol (0.75 mL for 1 mmol amide) with
refluxing for 24 h gave the compounds 6. Reaction of com-
pounds 1, 4 and 6 with 2-chloroethanesulfonyl chloride gave
dienophiles 7–9.
phenyl group. The RBA values of the secondary sulfonamide
–SO2NH– compounds (11a–d) are all rather low (entries 1–4),
but all of the tertiary sulfonamides (11e–o) bound much better.
The N-methyl compounds 11e–h, in particular, showed moderate
to high binding affinities (entries 5–8), with compound 11g
being best (7.17% and 1.59% for ERα and ERβ, respectively).
Affinity decreased with increasing alkyl chain length, however,
with the trifluoroethyl compounds (11m–o) having binding
affinities similar to or somewhat lower than those of the ethyl
compounds (11i–l).
Substituents on the pendant phenyl group of the tertiary sulfo-
namides also affected binding affinity and selectivity, with most
As was the case with the sulfonamides, the Diels–Alder cyclo-
addition with furan 10 proceeded well at 95 °C without solvent
or catalysts, giving isolated yields of 60–80% (Scheme 2).16 As
noted in the earlier sulfonate series, the products are almost
exclusively exo diastereomers (see ESI† for 1H NMR assign-
ments of exo 11p); apparently, the high rate and ready reversibil-
ity of this reaction results in the predominant formation of the
product of thermodynamic control. All of the products are
racemates.16
Binding affinities
ER binding affinities, determined radiometrically,29 are
expressed as relative binding affinity (RBA) values, with estra-
diol = 100 (Table 1). The ER binding affinities depend on the
nature of substituents on both the sulfonamide nitrogen and
Scheme 2 Diels–Alder reaction of 3,4-dihydroxyphenyl 10 with di-
enophiles 7–9.
Table 1 Relative binding affinities (RBAs) of the compounds 11a–p for ERα and ERβa
a Relative binding affinity (RBA) values, determined by radiometric assays, are expressed as IC50 estradiol/IC50 compound × 100 the range or
standard deviation (RBA, estradiol = 100%). The Kd for estradiol is 0.2 nM (ERα) and 0.5 nM (ERβ).
8694 | Org. Biomol. Chem., 2012, 10, 8692–8700
This journal is © The Royal Society of Chemistry 2012

View Online
Table 2 Effects of compounds 11a–p on ER-mediated transcription
Agonist modea
Antagonist modeb
ERα
ERα
ERβ
Compounde
EC50 (μM)
Eff (%E2)
IC50 (μM)
Eff (%E2)c
IC50 (μM)
Eff (%E2)c
11a
11c
11d
11e
11g
11i
11j
11k
0.10
0.20
—
78
81
42
31
33
11
3
13
1
9
0
3
24
1
35
57
1
4
3
3
1
1
1
1
1
2
0
3
0
1
3
3
—
—
—
83
89
65
56
41
10
3
20
7
3
5
4
3
2
1
2
1
4
1
1
0
2
2
1
3
0.37
0.10
0.10
0.01
0.12
0.33
0.37
—
0.16
2.14
n.d.
4.08
2.36
0.00058
0.00063
0.16
21
−5
−15
−25
−24
−10
−4
3
−12
0
45
7
6
3
1
3
3
2
4
3
1
2
5
2
2
0
2
5
—
0.74
0.45
0.35
0.72
0.75
0.43
0.19
n.d.
0.93
6.49
0.00033
0.0030
0.014
0.008
—
—
—
—
—
—
—
0.086
—
11l
11m
11nd
11o
−12
3
11p
23
−7
35
31
Fulvestrant
4-OH TAM
OBHS
−23
−20
−2
0.0011
0.028
70 12
a Transcriptional activity in HepG2 cells transfected with 3X-ERE-driven luciferase reporter and ERα or ERβ expression vectors treated in triplicate
with doses of the compounds (up to 10−5 M). Average efficacy (mean s.e.m.) is shown as a percentage of 10−7 M E2. b IC50 and average efficacy
(mean s.e.m.) determined in the presence of 10−8 M E2 on ERα or ERβ. c ERs have considerable basal activity in HepG2 cells; compounds with
inverse agonist activity are given negative efficacy values. d A single dose (10−5 M) of 11n was tested. e Compounds 11b, 11f, and 11h were not
assayed.
ligands showing moderate to good binding affinity; those with
no substitution (11a, 11e, 11i and 11m) were overall the best,
with the exception of the ortho-chloro phenyl analogue 11g. The
para chloro and methoxyl phenyl analogues usually gave lower
binding affinities; nevertheless, the sulfonamide with the bulkiest
substituent (α-naphthyl) still bound well. The tertiary sulfona-
mides showed 2.5–11-fold affinity preferences for ERα. It is
notable that the affinities of the N-methyl sulfonamides (11e–h)
were comparable with those of the corresponding sulfonates, on
which we have recently reported (Table 1, right, data in square
Fig. 2 Luciferase activity was measured in HepG2 cells transfected
with 3X-ERE-driven luciferase reporter and expression vectors encoding
brackets),17 the best sulfonamide (11g) being comparable to the
ERα or ERβ, and treated in triplicate with increasing doses (up to 10−5
M) of A. Hydroxytamoxifen; B. Compound 11j. The average efficacy
(mean s.e.m.) is shown as a percentage of 10−7 M 17β-estradiol (E2).
For the antagonist mode the average efficacy (mean s.e.m.) of the com-
pounds was determined in the presence of 10−8 M E2 and is shown as a
percentage of 10−8 M E2.
original OBHS compound, 7.2% and 1.6% (11g) vs. 9.3% and
1.7% (OBHS), for ERα and ERβ, respectively.16
Transcriptional activity
While SERMs generally profile as antagonists in breast cancer
cells, they display considerable gene activation in other cell lines
that correlates more accurately with their uterotrophic activity
in vivo.30 For this reason, we profiled these compounds in human
hepatocarcinoma (HepG2) cells, using expression plasmids for
full-length human ERα or ERβ and an estrogen-responsive luci-
ferase reporter gene.31 Compounds were assayed alone for direct
activation (agonism) and in the presence of 10 nM E2 for anta-
gonism. Since none of the compounds activated ERβ, we report
only ERβ antagonist data. Agonist and antagonist potencies are
given as EC50 and IC50 values, respectively. The intrinsic
activity, noted as the % efficacy (%Eff) compared to 100 nM E2,
is given in all cases. (Note, when efficacy is low, it is difficult to
determine EC50 values for agonists, and when efficacy is high, it
is difficult to determine IC50 values for antagonists.)
In these cells, the SERD, fulvestrant (ICI 182 780), acts as a
full antagonist on both receptors (Table 2), while 4-OH tamoxi-
fen (an active tamoxifen metabolite) displays the expected ERα-
selective partial-activation profile (Table 2, Fig. 2A). While
OBHS is a full antagonist in HEC-1 cells,16 in HepG2 cells it
profiles with greater agonist efficacy than 4-OH tamoxifen.
Because ERα and especially ERβ have considerable basal
activity in HepG2 cells, compounds can show inverse agonist
activity; those with intrinsic activity less than that of the apo-ER
are reported with negative efficacy values.
With ERα there is a clear trend where agonist activity
decreased with an increase in the N-alkyl chain length (11a >
11e > 11i). Overall, the N-ethyl and trifluoroethyl compounds
have very low efficacy, with those having a 2-chlorophenyl
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8692–8700 | 8695

View Online
substitution (11c, 11g, 11k) showing greater agonist activity than
those with substitutions at the 4-position. Also, the naphthyl
derivative (11p) induced nearly full agonist activity on ERα,
despite the bulky N-ethyl sulfonamide substitution, highlighting
that both substituents on nitrogen are important. Shown in
Fig. 2B is a representative assay curve for a low-efficacy com-
pound (11j), which has an intrinsic activity on both ERα and
ERβ as low as that for fulvestrant (Table 2).
selectivity comparable to those of the parent sulfonate systems,
OBHS and its analogues.
The second substituent, which can be added in the sulphon-
amide—but not the sulfonate—system, not only improves
binding affinity, but also leads to enhanced antagonist activity,
giving compounds that are full antagonists of both ER subtypes,
similar to the drug fulvestrant (ICI 182 780), by a novel
mechanism.
On ERβ, most of the compounds profiled as inverse agonists,
similar to fulvestrant. The inability to fully displace E2 for a few
compounds may reflect their low affinity, as they did not activate
ERβ on their own.
Structural analysis of the origin of the enhanced antagonist
character of OBH-sulfonamides
E2 supports transcriptional activation of ERs by stabilizing helix
12 in a conformation forming one side of a binding groove for
transcriptional coactivator proteins (Fig. 3A), while its high
affinity derives both from its relatively flat shape and hydrogen
bonding on both ends of the ligand: helix-3/E353, R394 and
helix 11/H524 in ERα.11 SERMs and full ER antagonists have
typically been developed by starting with an agonist, and then
adding a bulky side group that physically relocates helix-12 out
of this position by direct displacement (Fig. 3B), thus blocking
the recruitment of transcriptional coactivator proteins, such as
histone acetyl transferases (HATs).12 The residual agonist activity
seen with tamoxifen is due to an allosteric activation of a second
coactivator-binding site on the ER N-terminus, termed AF-1, via
an unknown mechanism.
Previously, we reported crystal structures of much smaller
oxabicyclic core derivatives, such as the diethyl ester ODE
(pdb.2QH618), as well as, more recently, that for OBHS itself.17
These structures revealed a novel mechanism of antagonism
without the use of the bulky side chain traditionally found in
SERMs: ligand-induced repositioning of helix-11 indirectly
modulates helix-12 positioning and receptor activity, a process
we had previously termed “passive antagonism”,19 but might
more appropriately be called “indirect antagonism”.
Discussion
In this work, we have expanded the chemical diversity of
OBHS-type ligands by substitution of the sulfonate moiety with
a sulfonamide (–SO2NR–) linkage, a molecular replacement that
is isoelectronic and potentially isostructural. A small array of
16 OBHS analogues (11a–p) were prepared in moderate to good
yield, through a Diels–Alder reaction of a 3,4-diarylfuran with
various dienophiles of N-aryl vinyl sulfonamides under neat,
mild conditions, without catalysts.
The structure–activity relationships that emerge for these
OBHS sulfonamide analogues from binding and cell-based
activity assays reveal that the affinities depend on the nature of
the substituents on both the nitrogen atom and the phenyl group
of the sulfonamides; the highest affinity being observed in
tertiary sulfonamides having small N-alkyl groups and binding
enhancing groups on the phenyl group.
Even though the secondary sulphonamides are more isostruc-
tural to the sulfonates than are the tertiary sulphonamides, their
lower affinity is not surprising. The N–H group in the secondary
sulphonamides presents a hydrogen bond donor in a region of
the receptor that has no available hydrogen bond acceptor. It is
well known that introduction of strong hydrogen bonding
groups, both donors and acceptors, in the “middle” of ER
ligands generally results in significantly reduced binding
affinities.32,33 The tertiary sulphonamides, however, have
masked this hydrogen bond donor with an alkyl group, and
some of these compounds demonstrated affinities and ERα
The diaryl oxabicyclic core ligands achieve this indirect anta-
gonism in a unique way: one of the phenols mimics the role of
the A-ring phenol of E211 or one of the phenols in diethylstilbes-
trol,12 engaging in strong hydrogen bonds with E353 and R394
and a structured water in ERα. The second phenol of OBHS
makes a distinct hydrogen bonding interaction involving helix-3/
Fig. 3 Modeling of ERα bound to oxabicyclic heptane sulphonamide (OBH-sulfonamide). ERα bound to A. E2 (pdb.1ERE); B. 4-Hydroxytamoxi-
fen; C. ODE (pdb.2QH6),18 which has the same oxabicyclic core as the sulfonamides. D. Model of the sulfonamide 11i based on the ODE structure.
The model was constructed by substitution of the ODE ester functions with the N-ethyl phenyl sulfonamide; the strong hydrogen bonding patterns of
the phenols maintained the position of the ligand core. Accommodation of the aryl sulfonamide would require a shift in helix 11, predicted to disrupt
the helix 11–helix 12 interface and block agonist activity by an indirect antagonism mechanism.19
8696 | Org. Biomol. Chem., 2012, 10, 8692–8700
This journal is © The Royal Society of Chemistry 2012

View Online
T347. This latter interaction is energetically favourable, because
deletion of the second phenolic OH or its etherification greatly
reduces ER binding affinity.16 While this second phenol points
in the E2 11β direction, as do the third aryl groups in the
SERMs, hydroxytamoxifen and raloxifene, it is not long enough
to interact directly with helix-12 and displace it, as do these
SERMs. Consequently, OBHS does not have a hydrogen
bonding interaction to constrain helix 11 by interaction with
H524 (Fig. 3C). The crystal structures show that the large, non-
polar phenyl sulfonate group in OBHS,17 and to a lesser extent
the ethyl carboxylate group in the smaller analogues,18 make
strong steric clashes with helix-11, displacing H524 and reposi-
tioning helix-11 in a manner that indirectly modulates helix-12
positioning and reduces receptor agonist activity, which is the
essence of indirect antagonism.19
Molecular modelling indicates that the aryl sulfonamide sub-
stituents could be accommodated in a manner similar to that of
aryl sulfonate groups in the OBHS analogues (Fig. 3D). The
additional bulk of the tertiary sulphonamides, however, would be
expected to accentuate this clash with helix-11, which would
result in a greater reduction in agonistic efficacy than that shown
by the sulfonates. This is illustrated with the N-ethyl sulfonamide
11i, which is a low efficacy compound that is an analogue of
both ODE and OBHS.
Thus, it appears that the enhanced indirect antagonism of the
OBH-sulfonamides compared to the OBH-sulfonates, can
account for their ability to achieve full antagonism, without the
need for a bulky side chain that disrupts helix-12 directly. While
it is possible that the scaffold flips to allow the sulfonamide to
exit towards helix 12, this would require accommodation of the
L-shaped configuration of the two phenols within the pocket,
which up to now has never been seen with an ER ligand, as well
as the loss of the key hydrogen bonding interaction with T347.
Thus, the oxabicyclic heptane sulfonamides may represent a
novel binding epitope to generate full antagonists on both ERα
and ERβ.
Bruker Biospin AV400 (400 MHz) instrument. The chemical
shifts are reported in ppm and are referenced to either tetra-
methylsilane or the solvent. Mass spectra were recorded under
electron impact conditions at 70 eV. Melting points were
obtained on SGW X-4 melting point apparatus and are uncor-
rected. Flash chromatography was performed with silica gel
(0.040–0.063 mm) packing.
General procedure for the synthesis of dienophiles 7–9
The synthesis of dienophiles 7. 2-Chloroethanesulfonyl chloride
(1.2 equiv.) was added slowly to a solution of aniline
(0.5 equiv.) in acetone at 0 °C. The mixture was stirred overnight
at 0–10 °C, then evaporated in vacuo. The residue was dissolved
into a mixture of CH2Cl2 (25 mL) and water (25 mL), then
extracted with CH2Cl2 (3 × 30 mL). The combined organic layer
was washed with saturated NaCl, dried over Na2SO4, filtered and
evaporated in vacuo. The product 7 was purified by column
chromatography.
The synthesis of dienophiles 8 and 9
A solution of N-substituted anilines (4 or 6, 1.0 equiv.) in
methylene chloride (10 mL) and water (10 mL) was stirred at
0 °C, and 25% NaOH (2 mL for 1 mmol 2-chloroethanesulfonyl
chloride) and 2-chloroethanesulfonyl chloride (1.2 equiv.) were
added simultaneously and slowly under 0 °C, keeping the pH
between 8.5 and 9.5. After 12 h, the mixture was extracted with
CH2Cl2 (3 × 30 mL). The combined organic phase was washed
with saturated NaCl and dried with Na2SO4, filtered, evaporated
in vacuo, and purified by flash chromatography on silica gel to
give the dienophiles 8 and 9.
General procedure for the Diels–Alder reaction of dienophiles
7–9 and 10
3,4-Diphenol furans 10 (1.0 equiv.) and dienophiles 7–9
(1.2 equiv.) were placed in a round flask, and the mixture was
stirred under Ar2 atmosphere at 95 °C for 24 h. The crude
product was purified by flash chromatography (EtOAc–petro-
leum ether = 1 : 3).
Conclusion
Oxabicyclic heptane sulfonamides appear to represent a novel
binding epitope that can generate full ERα and ERβ antagonists
with intrinsic activity as low as that of fulvestrant (and possibly
also SERD activity). This is an issue that we are exploring
further.
5,6-Bis(4-hydroxyphenyl)-N-phenyl-7-oxabicyclo[2.2.1]hept-5-
ene-2-sulfonamide (11a). Yellow solid (69% yield; mp
94–96 °C); 1H NMR (400 MHz, acetone-d6) δ 8.82 (s, 1H), 8.68
(s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.34–7.27 (m, 2H), 7.20–7.08
(m, 3H), 7.02 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.7 Hz, 4H), 6.73
(d, J = 8.7 Hz, 2H), 5.48 (d, J = 1.0 Hz, 1H), 5.31 (dd, J = 4.4,
1.1 Hz, 1H), 3.49 (dd, J = 8.4, 4.5 Hz, 1H), 2.30 (dt, J = 12.0,
4.5 Hz, 1H), 1.98 (dd, J = 12.4, 8.0 Hz, 1H); 13C NMR
(101 MHz, acetone-d6) δ 158.26, 158.17, 139.34, 138.27,
130.18, 129.66, 129.46, 125.26, 125.08, 124.53, 121.56, 121.49,
116.43, 116.37, 85.03, 83.62, 68.10, 26.18; HRMS (ESI) calcd
for C24H21NO5SNa, 458.1031 (M + Na+); found, 458.10327.
Experimental section
Materials and methods
Unless otherwise noted, reagents and materials were obtained
from commercial suppliers and were used without further purifi-
cation. Tetrahydrofuran and toluene were dried over Na and dis-
tilled prior to use. Dichloromethane was dried over CaH2 and
distilled prior to use. Glassware was oven-dried, assembled
while hot, and cooled under an inert atmosphere. Unless other-
wise noted, all reactions were conducted in an inert atmosphere.
Reaction progress was monitored using analytical thin-layer
chromatography (TLC). Visualization was achieved by UV light
(254 nm). 1H NMR and 13C NMR spectra were obtain on
5,6-Bis(4-hydroxyphenyl)-N-(4-methoxyphenyl)-7-oxabicyclo-
[2.2.1]hept-5-ene-2-sulfonamide (11b). Yellow solid (73% yield;
1
mp 96–98 °C); H NMR (400 MHz, acetone-d6) δ 8.70 (s, 1H),
8.65 (s, 1H), 7.25 (d, J = 8.9 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H),
Org. Biomol. Chem., 2012, 10, 8692–8700 | 8697
This journal is © The Royal Society of Chemistry 2012

View Online
7.07 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 6.77 (dd,
J = 8.5, 1.7 Hz, 4H), 5.48 (s, 1H), 5.30 (d, J = 4.3 Hz, 1H),
3.75 (s, 3H), 3.39 (dd, J = 8.4, 4.5 Hz, 1H), 2.28 (dt, J = 12.1,
4.4 Hz, 1H), 1.69 (dd, J = 11.5, 4.8 Hz, 1H); 13C NMR
(101 MHz, acetone-d6) δ 158.24, 158.21, 141.84, 138.34,
131.58, 129.77, 129.50, 125.21, 125.15, 124.87, 124.78, 116.49,
116.39, 115.22, 85.10, 83.59, 61.80, 55.74, 20.93; HRMS (ESI)
124.64, 116.58, 116.41, 114.94, 85.31, 83.63, 60.67, 55.79,
32.66, 20.93; HRMS (ESI) calcd for C26H25NO6SNa, 502.1282
(M + Na+); found, 502.12948.
N-(2-Chlorophenyl)-5,6-bis(4-hydroxyphenyl)-N-methyl-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11g). Yellow solid
1
(77% yield; mp 97–99 °C); H NMR (400 MHz, acetone-d6) δ
8.70 (s, 1H), 8.67 (s, 1H), 7.52 (ddd, J = 7.2, 6.3, 4.3 Hz, 2H),
7.36 (ddd, J = 6.4, 3.4, 2.2 Hz, 2H), 7.22 (dd, J = 8.7, 3.3 Hz,
4H), 6.81 (dd, J = 8.6, 7.2 Hz, 4H), 5.56 (s, br, 1H), 5.36 (dd,
J = 4.4, 1.1 Hz, 1H), 3.68 (dd, J = 8.3, 4.4 Hz, 1H), 3.29 (s,
3H), 2.36 (dt, J = 11.1, 4.1 Hz, 1H), 2.20 (dd, J = 11.9, 8.3 Hz,
1H); 13C NMR (101 MHz, acetone-d6) δ 158.29, 158.21,
140.11, 138.46, 134.96, 132.74, 131.33, 130.64, 130.39, 129.91,
129.49, 128.88, 125.26, 124.61, 116.52, 116.36, 85.42, 83.68,
60.62, 39.33, 18.85; HRMS (ESI) calcd for C25H22NO5SClNa,
506.0787 (M + Na+); found, 506.07994.
calcd for C25H23NO6SNa, 488.1135 (M
488.11383.
+
Na+); found,
N-(2-Chlorophenyl)-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo-
[2.2.1]hept-5-ene-2-sulfonamide (11c). Yellow solid (65% yield;
1
mp 93–95 °C); H NMR (400 MHz, acetone-d6) δ 8.70 (s, 1H),
8.38 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.45 (d, J = 9.5 Hz, 1H),
7.36–7.29 (m, 1H), 7.19 (d, J = 6.6 Hz, 3H), 7.09 (d, J = 6.7
Hz, 2H), 6.77 (dd, J = 8.7, 7.1 Hz, 4H), 5.49 (s, br, 1H), 5.34
(dd, J = 4.4, 1.1 Hz, 1H), 3.57 (dd, J = 8.0, 4.1 Hz, 1H), 2.36
(dt, J = 12.0, 4.4 Hz, 1H), 1.98–1.96 (m, 1H); 13C NMR
(101 MHz, acetone-d6) δ 158.29, 158.18, 142.12, 138.26,
135.54, 135.46, 130.71, 129.66, 129.57, 128.77, 127.26, 125.39,
125.31, 124.47, 116.45, 116.37, 85.15, 83.65, 63.80, 18.85;
HRMS (ESI) calcd for C24H20NO5SClNa, 492.0656 (M + Na+);
found, 492.06429.
N-(4-Chlorophenyl)-5,6-bis(4-hydroxyphenyl)-N-methyl-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11h). Yellow solid
(71% yield; mp 86–88 °C); 1H NMR (400 MHz, acetone-d6) δ =
8.70 (s, 1H), 8.65 (s, 1H), 7.46 (d, J = 8.8, 2H), 7.36 (d, J = 8.7,
2H), 7.17 (t, J = 8.6, 4H), 6.80 (dd, J = 12.7, 8.6, 4H), 5.46 (s,
1H), 5.30 (d, J = 4.2, 1H), 3.60 (dd, J = 8.3, 4.5, 1H), 3.39 (s,
3H), 2.42–2.31 (m, 1H), 2.27 (dd, J = 8.6, 4.7, 1H); 13C NMR
(101 MHz, acetone-d6) δ 158.32, 158.26, 142.00, 138.14,
132.70, 130.43, 130.31, 129.81, 129.52, 128.83, 125.10, 124.54,
116.55, 116.39, 85.24, 83.67, 60.56, 39.15, 20.94; HRMS (ESI)
calcd for C25H22NO5SClNa, 506.0793 (M + Na+); found,
506.07994.
N-(4-Chlorophenyl)-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo-
[2.2.1]hept-5-ene-2-sulfonamide (11d). Yellow solid (72% yield;
1
mp 121–123 °C); H NMR (400 MHz, acetone-d6) δ 8.96 (s,
1H), 8.72 (s, 1H), 7.40–7.37 (m, 2H), 7.34–7.31 (m, 2H), 7.17
(d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.76 (dd, J = 8.7,
6.0 Hz, 4H), 5.47 (d, J = 1.0 Hz, 1H), 5.31 (dd, J = 4.4, 1.1 Hz,
1H), 3.52 (dd, J = 8.4, 4.4 Hz, 1H), 2.28 (dt, J = 12.0, 4.5 Hz,
1H), 2.02 (dd, J = 8.7, 3.3 Hz, 1H); 13C NMR (101 MHz,
acetone-d6) δ 158.27, 158.17, 141.95, 138.18, 130.10, 130.01,
129.64, 129.51, 125.04, 124.50, 123.09, 123.02, 116.43, 116.36,
85.03, 83.62, 62.65, 18.85; HRMS calcd for C24H20NO5SClNa,
492.0643 (M + Na+); found, 492.06429.
N-Ethyl-5,6-bis(4-hydroxyphenyl)-N-phenyl-7-oxabicyclo-
[2.2.1]hept-5-ene-2-sulfonamide (11i). Yellow solid (75% yield;
mp 102–104 °C); 1H NMR (400 MHz, acetone-d6) δ 8.72
(s, 1H), 8.67 (s, 1H), 7.37–7.33 (m, 4H), 7.33–7.30 (m, 1H),
7.19 (dd, J = 8.6, 1.7 Hz, 4H), 6.80 (dd, J = 14.2, 8.6 Hz, 4H),
5.47 (d, J = 1.1 Hz, 1H), 5.31 (dd, J = 4.4, 1.1 Hz, 1H), 3.84
(q, J = 7.1 Hz, 2H), 3.51 (dd, J = 8.3, 4.5 Hz, 1H), 2.17 (dt, J =
11.9, 4.5 Hz, 1H), 2.05–2.00 (m, 1H), 1.03 (t, J = 7.1 Hz, 3H);
13C NMR (101 MHz, acetone-d6) δ 158.35, 158.24, 140.32,
138.33, 130.40, 130.10, 129.91, 129.89, 129.44, 128.40, 125.21,
124.57, 116.56, 116.37, 85.30, 83.63, 62.59, 47.10, 18.86,
14.94; HRMS (ESI) calcd for C26H25NO5SNa, 486.1334
(M + Na+); found, 486.13457.
5,6-Bis(4-hydroxyphenyl)-N-methyl-N-phenyl-7-oxabicyclo-
[2.2.1]hept-5-ene-2-sulfonamide (11e). Yellow solid (71% yield;
1
mp 86–88 °C); H NMR (400 MHz, acetone-d6) δ 8.71 (s, 1H),
8.67 (s, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.35 (dd, J = 8.0, 3.0 Hz,
2H), 7.27 (d, J = 7.3 Hz, 1H), 7.20–7.13 (m, 4H), 6.79 (dd, J =
10.9, 8.6 Hz, 4H), 5.45 (s, 1H), 5.29 (d, J = 4.3 Hz, 1H), 3.57
(dd, J = 8.1, 4.0 Hz, 1H), 3.39 (s, 3H), 2.43–2.27 (m, 1H), 1.97
(dd, J = 8.2, 3.7 Hz, 1H); 13C NMR (101 MHz, acetone-d6)
δ 158.31, 158.25, 141.88, 138.27, 130.40, 129.80, 129.77,
129.49, 127.66, 127.37, 125.17, 124.59, 116.50, 116.34, 85.24,
83.61, 61.71, 39.18, 18.84; HRMS (ESI) calcd for C25H23-
NO5SNa, 472.1191 (M + Na+); found, 472.11892.
N-Ethyl-5,6-bis(4-hydroxyphenyl)-N-(4-methoxyphenyl)-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11j). Yellow solid
(71% yield; mp 92–93 °C); 1H NMR (400 MHz, acetone-d6)
δ 8.85 (s, 1H), 8.79 (s, 1H), 7.25 (d, J = 7.5 Hz, 2H), 7.19
(d, J = 8.6 Hz, 4H), 6.89–6.76 (m, 6H), 5.46 (s, 1H), 5.31
(d, J = 4.1 Hz, 1H), 3.79 (s, 3H), 3.76 (q, J = 7.1 Hz, 1H), 3.47
(dd, J = 7.7, 4.1 Hz, 1H), 2.20 (dt, J = 8.9, 3.8 Hz, 1H), 1.72
(dd, J = 13.8, 7.0 Hz, 1H), 1.03 (t, J = 7.1 Hz, 3H); 13C NMR
(101 MHz, acetone-d6) δ 159.90, 158.22, 141.83, 138.43,
132.65, 131.99, 131.51, 129.97, 129.39, 125.28, 124.64, 116.56,
116.36, 114.95, 85.34, 83.59, 62.29, 55.74, 47.29, 31.29, 14.90;
HRMS (ESI) calcd for C27H27NO6SNa, 516.1450 (M + Na+);
found, 516.14513.
5,6-Bis(4-hydroxyphenyl)-N-(4-methoxyphenyl)-N-methyl-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11f). Yellow solid
1
(73% yield; 89–91 °C); H NMR (400 MHz, acetone-d6) δ 8.70
(s, 1H), 8.64 (s, 1H), 7.32 (d, J = 8.9 Hz, 2H), 7.17 (dd, J = 8.6,
3.4 Hz, 4H), 6.86 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H),
6.78 (d, J = 8.6 Hz, 2H), 5.45 (s, br, 1H), 5.30 (d, J = 3.9 Hz,
1H), 3.78 (s, 3H), 3.53 (dd, J = 8.3, 4.4 Hz, 1H), 3.33 (s, 3H),
2.16 (dt, J = 11.8, 4.4 Hz, 1H), 2.01 (dd, J = 8.8, 3.1 Hz, 1H);
13C NMR (101 MHz, acetone-d6) δ 159.49, 158.31, 158.22,
141.85, 138.30, 135.61, 129.89, 129.48, 129.20, 125.22,
N-(2-Chlorophenyl)-N-ethyl-5,6-bis(4-hydroxyphenyl)-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11k). Yellow solid
8698 | Org. Biomol. Chem., 2012, 10, 8692–8700
This journal is © The Royal Society of Chemistry 2012

View Online
(68% yield; mp 92–94 °C); 1H NMR (400 MHz, acetone-d6)
δ 8.78 (s, 1H), 8.75 (s, 1H), 7.51 (dd, J = 7.5, 1.7 Hz, 2H),
7.38 (dd, J = 9.1, 4.1 Hz, 2H), 7.21 (d, J = 6.4 Hz, 4H), 6.80 (t,
J = 8.3 Hz, 4H), 5.53 (s, 1H), 5.34 (d, J = 4.1 Hz, 1H), 3.77 (dd,
J = 22.3, 14.8 Hz, 2H), 3.62 (dd, J = 8.1, 4.4 Hz, 1H),
2.30–2.24 (m, 1H), 2.20 (d, J = 9.2 Hz, 1H), 1.06 (t, J = 7.1 Hz,
3H); 13C NMR (101 MHz, acetone-d6) δ 158.39, 158.29,
142.09, 139.71, 138.50, 137.38, 134.30, 131.36, 130.69, 129.93,
129.42, 128.56, 125.26, 124.59, 116.55, 116.38, 85.50,
83.65, 64.10, 47.05, 32.66, 14.580; HRMS (ESI) calcd for
C26H24NO5SClNa, 520.0960 (M + Na+); found, 520.09559.
1H), 4.51 (q, J = 8.7 Hz, 2H), 3.56–3.53 (m, 2H), 2.50–2.41 (m,
1H), 2.17 (dd, J = 8.2, 3.8 Hz, 1H); 13C NMR (101 MHz,
acetone-d6) δ 158.41, 158.37, 142.14, 139.40, 137.98, 134.43,
131.46, 130.24, 130.02, 129.37, 124.95, 124.35, 116.56, 116.37,
85.22, 83.61, 63.38, 60.59, 31.37; HRMS (ESI) calcd for
C26H21NO5SF3ClNa, 574.0665 (M + Na+); found, 574.06733.
N-Ethyl-5,6-bis(4-hydroxyphenyl)-N-(naphthalen-2-yl)-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11p). Yellow solid
1
(71% yield; mp 165–167 °C); H NMR (400 MHz, acetone-d6)
δ 8.80 (d, J = 8.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.19 (d, J =
8.5 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.1 Hz, 1H),
7.15 (d, J = 8.7 Hz, 2H), 6.82–6.68 (m, 6H), 6.58 (d, J =
8.7 Hz, 2H), 5.32 (d, J = 5.2 Hz, 1H), 5.27 (s, br, 1H), 3.66 (dd,
J = 8.3, 4.6 Hz, 1H), 3.47 (q, J = 7.2 Hz, 2H), 2.41 (dt, J = 11.8,
4.5 Hz, 1H), 1.89 (dd, J = 11.8, 8.4 Hz, 1H), 1.39 (t, J = 7.2 Hz,
3H); 13C NMR (101 MHz, acetone-d6) δ 158.28, 157.92,
150.59, 141.99, 138.45, 134.76, 131.66, 129.88, 129.02, 128.84,
125.95, 125.53, 125.11, 124.59, 123.95, 122.89, 120.67, 116.41,
116.31, 116.22, 101.72, 85.01, 83.83, 65.83, 38.91, 14.28;
HRMS (ESI) calcd for C30H27NO5SNa, 536.1513 (M + Na+);
found, 536.15022.
N-(4-Chlorophenyl)-N-ethyl-5,6-bis(4-hydroxyphenyl)-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11l). Yellow solid
1
(69% yield; mp 107–109 °C); H NMR (400 MHz, acetone-d6)
δ 8.70 (s, 1H), 8.65 (s, 1H), 7.38 (d, J = 5.5 Hz, 4H), 7.19 (dd,
J = 8.6, 1.7 Hz, 4H), 6.82 (d, J = 8.6 Hz, 2H), 6.78 (d, J =
8.7 Hz, 2H), 5.48 (d, J = 1.1 Hz, 1H), 5.32 (dd, J = 4.3, 1.0 Hz,
1H), 3.84 (q, J = 7.1 Hz, 2H), 3.53 (dd, J = 8.3, 4.5 Hz, 1H),
2.27 (dd, J = 13.0, 7.1 Hz, 1H), 2.15 (dt, J = 11.9, 4.4 Hz, 1H),
1.04 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, acetone-d6)
δ 158.34, 158.24, 141.87, 139.21, 138.24, 133.54, 131.59,
129.92, 129.90, 129.47, 125.17, 124.57, 116.57, 116.39,
85.29, 83.65, 62.90, 47.06, 20.94, 14.85; HRMS (ESI) calcd for
C26H24NO5SClNa, 520.0953 (M + Na+); found, 520.09559.
Estrogen receptor binding assays
Relative binding affinities were determined by a competitive
radiometric binding assay, as previously described,29 using 2 nM
[3H]estradiol as tracer (Perkin Elmer, Waltham, MA) and
purified full-length human ERα and ERβ (PanVera/InVitrogen,
Carlsbad, CA). Incubations were for 18–24 h at 0 °C. Then the
receptor–ligand complexes were absorbed onto hydroxyapatite
(BioRad, Hercules, CA), and unbound ligand was washed away.
The binding affinities are expressed as relative binding affinity
(RBA) values, with the RBA of estradiol set to 100. The values
given are the average range or SD of two or more independent
determinations. Estradiol binds to ERα with a Kd of 0.2 nM and
to ERβ with a Kd of 0.5 nM.
5,6-Bis(4-hydroxyphenyl)-N-phenyl-N-(2,2,2-trifluoroethyl)-7-
oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11m). Yellow solid
(59% yield; mp 91–93 °C); H NMR (400 MHz, acetone-d6) δ
1
8.76 (s, 1H), 8.70 (s, 1H), 7.48–7.45 (m, 2H), 7.36 (dd, J = 5.2,
2.0 Hz, 3H), 7.19 (d, J = 3.5 Hz, 2H), 7.17 (d, J = 3.5 Hz, 2H),
6.82 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 5.53 (d, J =
1.0 Hz, 1H), 5.33 (dd, J = 4.3, 1.0 Hz, 1H), 4.59 (q, J = 8.7 Hz,
2H), 3.58 (dd, J = 8.3, 4.5 Hz, 1H), 2.50–2.40 (m, 1H),
2.14 (dd, J = 8.2, 3.8 Hz, 1H); 13C NMR (101 MHz, acetone-d6)
δ 158.47, 158.32, 142.10, 140.54, 138.07, 130.19, 130.04,
129.85, 129.35, 129.18, 125.03, 124.36, 116.56, 116.36, 85.20,
83.59, 62.99, 60.59, 30.62; HRMS (ESI) calcd for C26H22NO5-
SF3Na, 540.1076 (M + Na+); found, 540.10630.
5,6-Bis(4-hydroxyphenyl)-N-(4-methoxyphenyl)-N-(2,2,2-trifluoro-
ethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11n). Yellow
solid (80% yield; 117–119 °C); 1H NMR (400 MHz, acetone-d6)
δ 8.80 (s, 1H), 8.73 (s, 1H), 7.35–7.32 (m, 2H), 7.19 (dd,
J = 8.6, 6.6 Hz, 4H), 6.85 (t, J = 9.1 Hz, 4H), 6.78 (d, J =
8.5 Hz, 2H), 5.52 (d, J = 1.1 Hz, 1H), 5.33 (dd, J = 4.3, 0.9 Hz,
1H), 4.51 (q, J = 8.7 Hz, 2H), 3.79 (s, 3H), 3.55–3.53 (m, 1H),
2.50–2.41 (m, 1H), 2.17 (dd, J = 8.2, 3.8 Hz, 1H); 13C NMR
(101 MHz, acetone-d6) δ 160.37, 158.45, 158.27, 142.10,
138.13, 132.78, 131.29, 130.16, 129.33, 125.11, 124.45, 116.59,
116.37, 115.21, 85.25, 83.57, 62.62, 60.59, 55.79, 31.37;
HRMS (ESI) calcd for C27H24NO6SF3Na, 570.1173 (M + Na+);
found, 570.11687.
Luciferase reporter gene assays
HepG2 cells cultured in Dulbecco’s minimum essential medium
(DMEM) (Cellgro by Mediatech, Inc., Manassas, VA) sup-
plemented with 10% fetal bovine serum (FBS) (Hyclone by
Thermo Scientific, South Logan, UT), and 1% non-essential
amino acids (Cellgro), Penicillin–Streptomycin–Neomycin anti-
biotic mixture and Glutamax (Gibco by Invitrogen Corp., Carls-
bad, CA), were maintained at 37 °C and 5% CO2. Cells were
transfected with 10 μg of 3X ERE-luciferase reporter plus 1.6 μg
of ERα or ERβ expression vector per 10 cm dish using
FugeneHD reagent (Roche Applied Sciences, Indianapolis, IN).
The next day, the cells were transferred to phenol red-free
growth media supplemented with 10% charcoal-dextran sulfate-
stripped FBS at a density of 20 000 cells per well, incubated
in 384-well plates overnight at 37 °C and 5% CO2, and stimu-
lated with various concentrations of compounds in triplicate.
Luciferase activity was measured after 24 h using BriteLite
reagent (Perkin-Elmer Inc., Shelton, CT) according to manufac-
turer’s protocol.
N-(4-Chlorophenyl)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoro-
ethyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonamide (11o). Yellow
1
solid (69% yield; mp 93–95 °C); H NMR (400 MHz, acetone-
d6) δ 8.80 (s, 1H), 8.73 (s, 1H), 7.33 (d, J = 9.0 Hz, 2H), 7.19
(dd, J = 8.6, 6.6 Hz, 4H), 6.85 (t, J = 9.1 Hz, 4H), 6.78 (d, J =
8.5 Hz, 2H), 5.52 (d, J = 1.1 Hz, 1H), 5.33 (dd, J = 4.3, 0.9 Hz,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8692–8700 | 8699

View Online
15 R. Sibley, H. Hatoum-Mokdad, R. Schoenleber, L. Musza, W. Stirtan,
D. Marrero, W. Carley, H. Xiao and J. Dumas, Bioorg. Med. Chem. Lett.,
2003, 13, 1919–1922.
16 H. B. Zhou, J. S. Comninos, F. Stossi, B. S. Katzenellenbogen and
J. A. Katzenellenbogen, J. Med. Chem., 2005, 48, 7261–7274.
17 Y. Zheng, M. Zhu, S. Srinivasan, J. C. Nwachukwu, V. Cavett, J. Min,
K. E. Carlson, P. Wang, C. Dong, J. A. Katzenellenbogen, K. W. Nettles
and H. B. Zhou, ChemMedChem, 2012, 7, 1094–1100.
18 K. W. Nettles, J. B. Bruning, G. Gil, J. Nowak, S. K. Sharma,
J. B. Hahm, K. Kulp, R. B. Hochberg, H. Zhou, J. A. Katzenellenbogen,
B. S. Katzenellenbogen, Y. Kim, A. Joachmiak and G. L. Greene,
Nat. Chem. Biol., 2008, 4, 241–247.
19 A. K. Shiau, D. Barstad, J. T. Radek, M. J. Meyers, K. W. Nettles,
B. S. Katzenellenbogen, J. A. Katzenellenbogen, D. A. Agard and
G. L. Greene, Nat. Struct. Biol., 2002, 9, 359–364.
Acknowledgements
We are grateful to the NSFC (91017005, 20972121, 81172935),
the Program for New Century Excellent Talents in University
(NCET-10-0625), the National Mega Project on Major Drug
Development (2009ZX09301-014-1), and the Research Fund
for the Doctoral Program of Higher Education of China
(20100141110021) for support of this research. Research support
from the National Institutes of Health (PHS 5R37 DK015556 to
J.A.K. and R01 DK077085 to K.W.N.) is gratefully acknowl-
edged. We are grateful to Teresa Martin to help in the binding
assays.
20 P. Forgione, P. D. Wilson and A. G. Fallis, Tetrahedron Lett., 2000, 41,
17–20.
21 S. J. Liu, B. Zhou, H. Y. Yang, Y. T. He, Z. X. Jiang, S. Kumar, L. Wu
and Z. Y. Zhang, J. Am. Chem. Soc., 2008, 130, 8251–8260.
22 J. Morris and D. G. Wishka, J. Org. Chem., 1991, 56, 3549–3556.
23 J. Ohtaka, T. Sakamoto and Y. Kikugawa, Tetrahedron Lett., 2009, 50,
1681–1683.
24 J. Salazar, S. E. Lepez and O. Rebollo, J. Fluorine Chem., 2003, 124, 111–113.
25 G. La Regina, R. Silvestri, V. Gatti, A. Lavecchia, E. Novellino,
O. Befani, P. Turini and E. Agostinelli, Bioorg. Med. Chem., 2008, 16,
9729–9740.
Notes and references
1 V. C. Jordan, Sci. Am., 1998, 60–67.
2 B. S. Katzenellenbogen and J. A. Katzenellenbogen, Breast Cancer Res.,
2000, 2, 335–344.
3 T. A. Grese and J. A. Dodge, Curr. Pharm. Des., 1998, 4, 71–92.
4 Y. F. Shang and M. Brown, Science, 2002, 295, 2465–2468.
5 L. J. Black, M. Sato, E. Rowley, D. Magee, A. Bekele, D. Williams,
G. Cullinan, R. Bendele, R. Kauffman and W. Bensch, J. Clin. Invest.,
1994, 93, 63.
26 S. Sato, H. Watanabe and M. Asami, Tetrahedron: Asymmetry, 2000, 11,
4329–4340.
6 V. C. Jordan, J. Med. Chem., 2003, 46, 883–908.
7 V. C. Jordan, J. Med. Chem., 2003, 46, 1081–1111.
8 D. P. McDonnell and S. E. Wardell, Curr. Opin. Pharmacol., 2010, 10,
620–628.
9 S. Dauvois, P. S. Danielian, R. White and M. G. Parker, Proc. Natl. Acad.
Sci. U. S. A., 1992, 89, 4037–4041.
10 K. Smolnikar, Drugs Today (Barc.), 2001, 37, 783–789.
11 A. M. Brzozowski, A. C. Pike, Z. Dauter, R. E. Hubbard and T. Bonn,
et al., Nature, 1997, 389, 753–758.
12 A. K. Shiau, D. Barstad, P. M. Loria, L. Cheng, P. J. Kushner,
D. A. Agard and G. L. Greene, Cell, 1998, 95, 927–937.
13 `R. S. Muthyala, S. B. Sheng, K. E. Carlson, B. S. Katzenellenbogen and
J. A. Katzenellenbogen, J. Med. Chem., 2003, 46, 1589–1602.
14 L. G. Hamann, J. H. Meyer, D. A. Ruppar, K. B. Marschke, F. J. Lopez,
E. A. Allegretto and D. S. Karanewsky, Bioorg. Med. Chem. Lett., 2005,
15, 1463–1466.
27 H. B. Zhou, K. W. Nettles, J. B. Bruning, Y. Kim, A. Joachimiak,
S. Sharma, K. E. Carlson, F. Stossi, B. S. Katzenellenbogen, G. L. Greene
and J. A. Katzenellenbogen, Chem. Biol., 2007, 14, 659–669.
28 Y. Y. Peng, H. L. Liu, M. Tang, L. S. Cai and V. Pike, Chin. J. Chem.,
2009, 27, 1339–1344.
29 K. E. Carlson, I. Choi, A. Gee, B. S. Katzenellenbogen and
J. A. Katzenellenbogen, Biochemistry (Mosc.), 1997, 36, 14897–14905.
30 D. P. McDonnell, C. E. Connor, A. Wijayaratne, C. Y. Chang and
J. D. Norris, Recent Prog. Horm. Res., 2002, 57, 295–316.
31 E. M. McInerney, M. J. Tsai, B. W. O’Malley and B. S. Katzenellenbogen,
Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 10069–10073.
32 G. M. Anstead, K. E. Carlson and J. A. Katzenellenbogen, Steroids,
1997, 62, 268–303.
33 A. C. Pike, A. M. Brzozowski, R. E. Hubbard, T. Bonn, A. G. Thorsell,
O. Engstrom, J. Ljunggren, J. A. Gustafsson and M. Carlquist, Embo J.,
1999, 18, 4608–4618.
8700 | Org. Biomol. Chem., 2012, 10, 8692–8700
This journal is © The Royal Society of Chemistry 2012