Triaryl-substituted schiff bases are high-affinity subtype-selective ligands for the estrogen receptor

Article abstract of DOI:10.1021/jm500268j

We have explored the isoelectronic replacement of the C=C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C=N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C=N for C=C substitution might be more widely considered as a general strategy for preparing drug analogues.

Full text of DOI:10.1021/jm500268j

Article
pubs.acs.org/jmc
Triaryl-Substituted Schiff Bases Are High-Affinity Subtype-Selective
Ligands for the Estrogen Receptor
Zong-Quan Liao,† Chune Dong,† Kathryn E. Carlson,‡ Sathish Srinivasan,∥ Jerome C. Nwachukwu,∥
Robert W. Chesnut,§ Abhishek Sharma,‡ Kendall W. Nettles,∥ John A. Katzenellenbogen,*,‡
and Hai-Bing Zhou*,†
†Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, State Key
Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China
‡Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
§Eastern Illinois University, 600 Lincoln Avenue, Charleston, Illinois 61920, United States
∥Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: We have explored the isoelectronic replacement of the CC double bond found at the core of many nonsteroidal
estrogen ligands with a simple Schiff base (CN). Di- and triaryl-substituted imine derivatives were conveniently prepared by
the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines
demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these
imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function
(AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way:
by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional
coactivators. This work suggests that CN for CC substitution might be more widely considered as a general strategy for
preparing drug analogues.
Many nonsteroidal estrogens are di- or triarylethylenes
having a CC double bond core, and in a recent publication,
we examined the replacement of this CC double bond with
the isoelectronic and isostructural BN bond (Figure 1, right,
middle).13 To achieve hydrolytic stability, the electrophilicity of
the boron center had to be sterically masked with a full array of
flanking ortho methyl groups, and analogues with p-OH groups
on the B-phenyl groups were unstable. Despite these
restrictions, some of the anilino bis(2,6-dimethylphenyl)borane
derivatives that we prepared were very stable and demonstrated
reasonably high binding affinity and good cellular potency,
being ERα agonists and ERβ antagonists.13 Those studies
defined the structural determinants of stability and cellular
bioactivity of a BN for CC substitution and provided a
framework for further exploration of “elemental isomerism” for
diversification of drug-like molecules.13
INTRODUCTION
■
There are many motivations for preparing analogues of
pharmaceuticals (e.g., improving drug properties, reducing
drug liabilities, seeking unclaimed intellectual property space,
and simplifying or improving synthesis), and there are
numerous approaches for the preparation of such analogues
(e.g., substituting heteroatoms and replacing peripheral or
structural core elements with sterically or electronically similar
entities). We have sought to expand the chemical diversity of
ligands for the estrogen receptor (ER) by replacing their
internal scaffolding with various heterocycles and other
structurally related elements, and in the process, we have
obtained structurally novel compounds that are generally easy
to prepare.1−7 Because estrogens, acting through their two
receptors, ERα and ERβ, regulate a wide range of physiological
and pathological processes and because various ER ligands can
demonstrate marked tissue selectivity (based on ER subtype
selectivity8,9 or selective engagement of coregulator proteins,
i.e., SERM selectivity10,11), it is not surprising that in some
cases our structurally novel estrogen analogues showed unusual
patterns of estrogenic activity and selectivity.1,12
Here, we have further explored the chemical diversification of
ER ligands by another, simple isostructural and isoelectronic
Received: February 18, 2014
Published: March 26, 2014
© 2014 American Chemical Society
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Figure 1. Triarylethylene nonsteroidal estrogen as well as BN and CN double bond for CC bond isoelectronic replacements.
replacement, substituting a core CC double bond with a
simple Schiff base (CN). As far as we are aware, there
appears to be only limited and rather distant precedent for use
of a CN bond as a core element for estrogens.14,15
Furthermore, on the basis of a substructure search of the
Merck Index, the CN bond does not appear to be well-
recognized as a surrogate for a CC bond in the construction
of bioactive molecules (except as a sub-element in 2-aryl-
benzimidazoles, benzodiazepines, and other related hetero-
cycles). As was the case earlier,13 di- and triarylethylene
nonsteroidal estrogens offer a convenient CC double bond
core (Figure 1, right, top) for replacement by the isostructural
CN element (Figure 1, bottom, right). These Schiff base or
imine-core ligands are very easy to synthesize, in most cases in
one step (and much easier than their CC double bond
analogues), and to this end, we have prepared a series of triaryl-
substituted (and some diaryl-substituted) Schiff base derivatives
by a simple condensation that proceeds without phenolic
hydroxy protection. Their binding affinities and cellular
biological activities showed distinctive structure−activity
relationships (SARs), and they profiled as potent ERα agonists
and ERβ antagonists.
give desired triphenolic product 2n in 94% yield. We also used
this methodology for the synthesis of the corresponding
phenolic Schiff bases 4 (Scheme 1B). Benzophenone
derivatives 1b and 1c were prepared by a Friedel−Crafts
acylation reaction and subsequent condensation with various
anilines then afforded imines 4a−m in good yield. Product 4n
was also prepared in 92% yield via a two-step procedure similar
to that used for 2n. In all cases (4a−l), the (E) form of the
imines was the only diastereoisomer formed, presumably
because the intramolecular hydrogen bond, which can only
form in the (E) isomer, contributes to the imine stability. These
triaryl systems appeared to be hydrolytically stable indefinitely
under aqueous conditions. By contrast, diaryl imines prepared
from anilines and aldehydes or phenyl alkyl ketones displayed
pronounced hydrolytic lability, undergoing substantial hydrol-
ysis in aqueous MeOH at rt within 1−6 h. However, the
diarylimines having an internal hydrogen bond between the
imine nitrogen and an ortho phenolic OH group were
considerably more stable.
Estrogen Receptor Binding Affinity Assays. The
binding affinity of Schiff base analogues 2 and 4−6 for both
ERα and ERβ was determined by a competitive radiometric
assay, using methods that have been previously described, and
are reported in Table 1.18 The affinities are represented here by
relative binding affinity (RBA) values, where estradiol has an
affinity of 100 (absolute affinities for estradiol: Kd 0.2 nM on
ERα and 0.5 nM on ERβ).
As a global observation, most of the imines are gratifyingly
high-affinity ligands for both ERs, although the number and
position of the hydroxyl group in the phenyl ring of the
benzophenone moiety as well as the disposition and size of
substituents on N-phenyl group have a marked influence on
their binding affinity and selectivity. When assayed on the
individual ER subtypes, ERα and ERβ, most compounds show
only a modest binding-affinity preference for ERβ, at most 5-
fold (compound 2d). Binding-affinity comparisons suggest that
one of the p-hydroxy groups on the benzophenone moiety is
playing the role of the phenolic hydroxyl of estradiol, which is
well-known to be the dominant functional group ensuring high
RESULTS
■
Synthesis. Representative members having an imine-core
structure were prepared in three classes: 4,4′-dihydroxybenzo-
phenone and 2,4,4′-trihydroxybenzophenone derivatives of
various anilines and the corresponding diaryl imines. On the
basis of previous work,16 we developed a HCl(g)-catalyzed
condensation reaction without phenolic hydroxy protection for
the synthesis of bisphenolic Schiff bases 2a−l in chlorobenzene
with heating at 140−145 °C for 24 h (Scheme 1A). Although
all yields were moderate, this improved method was superior to
the phenol-protection strategy.17 We were unable to prepare
triphenolic Schiff base 2n by the reaction of 4,4′-dihydrox-
ybenzophenone 1c with 4-aminophenol or by demethylation of
2l. However, when 4,4′-dihydroxybenzophenone 1a was treated
with 4-aminophenyl benzoate, it gave imine 2m in 74% yield,
and deprotection with KOH proceeded at room temperature to
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a
Scheme 1. Synthesis of Triaryl-Substituted Schiff Base Analogues 2 and 4 with the Improved Method
a
Reagents and conditions: (a) aniline derivatives (3 equiv), HCl(g), PhCl, 140−145 °C, 24 h; (b) KOH, MeOH, rt, 3 h; (c) resorcinol, AlCl3,
sulfolane, 65−70 °C, 8 h.
binding affinity.19 (This was confirmed by X-ray crystallog-
raphy; see below.) Curiously, the presence of a para hydroxyl
group in the distal N-phenyl group, which is essential for the
high-affinity binding of a number of bisphenolic estrogens such
as diethylstilbestrol and hexestrol as well as certain phenyl-
indenes,20 actually has a markedly detrimental effect on
binding, reducing affinity by about 7-fold (Table 1, compound
2a vs 2n and 4b vs 4n). Where studied, the second para
hydroxy group in the second ring of the benzophenone unit has
little effect on binding (Table 1, compound 4a vs 4l), although
the methyl ether of this phenol is a good ERβ ligand (Table 1,
4o). Lastly, placement of a second hydroxyl group at the ortho
position of the phenyl ring of the benzophenone moiety, which
enables potential formation of an intramolecular hydrogen
bond as in the series 4 compounds, caused, in most cases, a
decrease in affinity for both ERα and ERβ; this was somewhat
unexpected but can be rationalized from conformational
changes noted in crystal structures (see Discussion and
below). Parallel changes in the nature of the substituents on
the N-phenyl group were made within the two major series,
2c−n and 4c−n, and in nearly every case, the binding affinities
for both ERα and ERβ changed in a coordinated fashion,
although with some bias for one or the other ER subtype. This
is illustrated graphically in Figure 2.
In both series, the highest affinity analogues were those
having somewhat bulky but nonpolar substituents, and where a
substituent (Me or Cl) was placed at the C-2, -3, and -4
positions, there was a general decline in affinity with that
progression of ring positions, although this was more evident in
the ERα series than in the ERβ series. Among the various
substituents, certain ones were preferred at each of the three
positions, but these differed between the two series and with
the two ER subtypes. Addition of a second methyl group, as in
2,6-dimethyl compound 2b, resulted in a dramatic, ca. 50-fold,
reduction in affinity compared to 2-methyl analogue 2c.
Electron-withdrawing substituents (halogens and CF3) were
associated with higher affinity than alkyl (methyl) or electron-
donating substituents (OH or OMe). Overall, these results
illustrate that ER ligands having simple imine-core structures
can be readily prepared but that high-affinity binding, as one
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a
Table 1. Relative Binding Affinities (RBAs) of Compounds 2 and 4−6 for Estrogen Receptor α and β
a
Determined by a competitive radiometric binding assay with [3H]estradiol; preparations of purified, full-length human ERα and ERβ (Invitrogen)
were used; see the Experimental Section. Values are reported as the mean the range or SD of two or more independent experiments; the Kd for
estradiol for ERα is 0.2 nM and for ERβ, 0.5 nM. Ki values for the reported compounds can be readily calculated using the formula Ki =
(Kd[estradiol]/RBA)100.
might expect, requires an appropriate distribution of bulk,
polarity, and functionality.
Information Table S1). The highest affinity compounds are
benzophenone imines of alkyl or benzyl amines (9), which also
have some ERβ-binding preference. Even on ERβ, however,
these compounds have RBA values of less than 0.3%, and the
affinities of the other diarylimines we prepared are typically
more than 10-fold lower (see Supporting Information Table
S1). As noted earlier, some of these diaryl systems were also
rather hydrolytically labile, which is not the case for all of the
series 2 and 4 compounds.
Previously, in connection with studies of dimethylgallium
chelates that mimic nonsteroidal estrogens, we prepared a
number of smaller imine-core systems (Figure 3), and although
we published their synthesis and structures,16 we had, until
now, not determined their ER-binding affinities. More recently,
we also prepared a few additional diarylimines. In stark contrast
to the triaryl systems, all of these monoaryl and diaryl imines
have very low binding affinity (the data is given in Supporting
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In general, these imines fully stimulated ER-mediated
transcription in cells transfected with wild-type ERα, indicating
that they are potent and highly efficacious ERα agonists (Table
2 and Figure 4A). In most cases, the number and position of
the hydroxyl groups in the phenyl ring as well as the disposition
and size of substituents on the N-phenyl group had no obvious
effect on ERα-mediated transcription. However, compared to
E2, none of the imines fully stimulated ER-mediated tran-
scription in cells transfected with an ER construct that lacks
AF1 because of the deletion of the N-terminal AB domain
(Figure 4B), indicating that these imines do not fully induce
AF2-mediated ER activity but also rely substantially on the
AF1-mediated activity of ER to drive transcription.
ERβ exhibits negligible AF1-mediated activity compared to
ERα, but the AF2-mediated activities of the ER subtypes are
similar in reporter assays,22 suggesting that despite their similar
binding affinities for the two ER subtypes (Figure 2) these
imines would act as partial ERβ agonists. Consistent with this
supposition, all of the Schiff bases tested failed to stimulate ER-
mediated transcription fully in HepG2 cells transfected with
ERβ (Table 2 and Figure 4C). In fact, many of these
compounds acted as potent and nearly complete ERβ-selective
antagonists, suppressing the E2-induced activity of ERβ in the
low nanomolar range (Table 2) and thereby underscoring the
AF1-dependent ER subtype-selective properties of these
compounds.
The N-phenyl substituents appear to affect the potency of
these compounds as ERβ antagonists; however, whether some
of the obvious differences in IC50 reflect variation in RBA
toward ERβ is unclear. For example, an overview of the
compound 4 series, which in general had lower RBAs (Figure
2), suggests that they were also less potent than the compound
2 series (Table 2); yet, in most cases, they were more
efficacious on ERβ (Figure 4C). Overall, our results suggest
that the imines generally profile as potent, efficacious, subtype-
selective ER ligands that depend to a large extent on AF1 to
induce ER-mediated transcription fully, but they poorly
stimulate the AF2-mediated activity of ERα or ERβ. These
differences underlie their ERα-selective agonist and ERβ-
selective antagonist properties.
Figure 2. Graphical presentation of RBA values for imines 2c−n
(black, H) and 4c−n (gray, OH)
Figure 3. Other mono and diaryl imines (also see Supporting
Information Table S1).
Structural Basis for the ER Subtype-Selective Profile
of Triaryl-Substituted Schiff Base Analogues. We
obtained crystal structures of the ERα ligand-binding domain
complexed with the 2-Cl-substituted analogues, compounds 2f
and 4f, and compared these new structures to previously
reported full agonist- and antagonist-bound ERα struc-
tures.23−25 Full agonists, such as E2, fit into the ERα ligand-
binding pocket in an orientation that facilitates hydrogen
bonding of the phenolic OH to the side chains of helix 3
residue Glu353, helix 6 residue Arg394, and, more variably, the
D-ring 17β-OH to helix 11 residue His524 (Figure 5A). This
binding orientation allows the switch helix, helix 12, to dock
against helices 3 and 11, where it forms one side of the
coactivator binding site on the surface that constitutes the
functional core of AF2, that is, it is a coregulator binding site.23
In contrast, antagonists and SERMs, such as 4-hydroxytamox-
ifen, contain an agonist-like core but have a bulky side group
that protrudes between helices 3 and 11 and directly displaces
helix 12 from its active conformation. As result, helix 12 docks
along helix 3, thereby occluding the AF2 surface.24,25
Transcriptional Activity. We determined the effects of
these imines on ER transcriptional activity using an ER-
responsive luciferase reporter gene. Steroid-deprived HepG2
liver cells were transfected with a widely used 3×ERE-luciferase
reporter and an ERα or ERβ expression plasmid for agonist
activity (% efficacy) and potency (EC50) determinations. These
cells were stimulated with increasing concentrations of 17β-
estradiol (E2) or compounds 2a, 2c−k, 2n, 4b−k, and 4n. For
antagonist mode assays (% efficacy and IC50), cells were
stimulated with a combination of estradiol (10 nM) and an
increasing concentration of the various compounds. The next
day, luciferase activity was measured. HepG2 cells are
particularly useful as a test system to distinguish the activity
of estrogens through the two activation functions of the ERs,
the N-terminal AF1, in the A/B domain, and AF2, which is in
the ligand-binding domain.21 SERMs, such as tamoxifen, show
tissue-selective agonist activity in some tissues, such as the
uterus, and in the HepG2 cells via AF1 activity, but they work
as antagonists in the breast through structural mechanisms that
are not understood.
Within the ligand-binding pocket, compounds 2f and 4f
mimicked the binding orientation of the 4-hydroxytamoxifen
core without a protruding side chain (Figure 5C,D), consistent
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Table 2. Effects of Imines on the Transcriptional Activities of Estrogen Receptor α and β
a
a
agonist mode
antagonist mode
ERα
ERβ
ERα
ERβ
EC50 (nM)
eff (% E2)
EC50 (nM)
eff (% E2)
IC50 (nM)
eff (% E2)
IC50 (nM)
eff (% E2)
2a
2c
2d
2e
2f
3
110
103
109
nd
5
6
3
34 12
126
149
93
4
6
5
13
6
17
27
14
10
30
11
6
3
9
0
2
3
2
2
5
2
3
9
7
8
13
7
3
9
9
9
3
3
2
3
1
8
8
3
2
9
1
2
5
6
8
2
5
41
45
19
16
9
nd
1
nd
104
102
114
89
3
3
4
3
4
2
2
2
4
2
3
4
6
3
4
3
4
3
3
37
10
9
83
9
5
8
6
4
2g
2h
2i
10
2
109
138
104
113
23
2
3
24
7
11
8
2j
114
105
95
10
11
16
25
31
9
2k
2n
4b
4c
4d
4e
4f
1
128 10
111
134 34
6
4
14
7
332
628
2
4
300
6
33
102
111
95
23 12
100
101
5
2
16
51
241
6
29
11
21
33
15
15
16
14
13
4
3
9
6
0
1
2
2
6
14
16
0.1
15
4
103
113
117
114
100
108
113
102
97
10 860
385
20
54
15
19
11
23
24
122 34
136 13
118 23
123 18
4g
4h
4i
67
180
28
139
100
57
10
9
95
124
111
102
108
9
6
5
6
4
4j
4k
4l
12 460
276
17
7
38 13
51 10
20 24
25 10
4n
417
a
In the agonist mode, ERE-luciferase assays were performed with 12-point dose curves of the indicated ligands, whereas in the antagonist mode, this
was done in the presence of 10 nM E2.
Figure 4. Graphical comparison of the agonist-mode efficacies of matched compounds on full-length ERα (A), ERα with the N-terminal AF1 deleted
(B), and ERβ (C)
Figure 5. Imines induce a suboptimal conformation of the ERα ligand-binding domain. (A, B) Active and inactive ERα ligand-binding domain
conformations show a ∼1 Å difference in distance between helices 3 and 11. The crystal structures of 17β-estradiol (E2; PDB ID: 1GWR) and 4-
hydroxytamoxifen (TAM; PDB ID: 3ERT) bound complexes are shown. (C, D) Crystal structures of the ERα ligand-binding domain in complex
with compounds 2f and 4f show a TAM-like binding orientation and increased h3−h11 distance compared to E2. (E, F) Crystal structures of the
imine-bound ERα complexes were superposed. Compared to compound 2f (white), the additional hydroxyl group of compound 4f (coral) leads to a
subtle distortion of the ligand-binding orientation.
with their high binding affinities toward ER subtypes. In
addition, the N-phenyl groups were accommodated between
helices 8 and 11, contacting M421, H524, and L525. The
additional hydroxyl group of compound 4f was also
accommodated easily, as this ring was utilized as the A-ring
mimetic that forms a hydrogen bond with Glu353 (Figure 5E);
however, this hydroxyl substitution also led to a 0.8 Å rotation
of the other phenyl ring that forms a hydrogen bond with
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Figure 6. Structure and relative binding affinities (RBAs, estradiol = 100) of various nonsteroidal and seco-steroidal estrogens as well as torsional
angles of the triarylimine propellane conformation.
Thr347 (Figure 5F). Therefore, the different binding
orientations of these imines within the ligand-binding pocket
account for the fact that many of the matched 2 and 4 series
compounds have slightly different binding affinities (Figure 2)
but similar ERα-mediated activity profiles (Figure 4).
pockets; therefore, imines are likely to also distort the active
ERβ conformation through a similar structural mechanism,
which underlies their partial ERβ agonist/antagonist phenotype
(Table 2 and Figure 4C).
DISCUSSION
The structures of the imines suggest that reduced AF2
activity derives from a shift in the distance between helices 3
and 11. The active helix 12 conformer docks across helices 3
and 11; therefore, shifting helices 3 and 11 apart would
undoubtedly destabilize helix 12 and lead to a suboptimal
ligand-binding domain conformation that would likely affect the
binding of coregulators. In the full agonist-bound conformation
of ERα, the distance between helices 3 and 11, measured from
the α-carbon of Thr347 to the α-carbon of Leu525, is a
remarkably consistent 9 Å (Figure 5A).23 ER ligands that are
not full agonists, including SERMs, increase this distance in a
ligand-dependent manner. When bound to 4-hydroxytamox-
ifen, this distance is increased by about 1 Å (Figure 5B).25 In
contrast, compounds 2f and 4f increased this distance by 0.5
and 0.7 Å, respectively (Figure 5C,D), indicating that these
compounds induce a suboptimal conformation of the ERα
ligand-binding domain, which explains their inability to
stimulate the AF2-mediated activity of ERα fully (Figure 4B).
Furthermore, the ER subtypes have similar ligand-binding
■
The estrogen receptors are remarkable in binding and
responding to ligands of great structural diversity,8,26,27 and
this eclectic acceptance of ligands offers an opportunity to
investigate chemically novel structures as potential selective
estrogen receptor modulators (SERMs)10,11 or ER subtype-
selective ligands.8,9 For example, recently, simple acyclic
amide,3 diphenylamine,28 monoaryl- or diaryl-substituted
salicylaldoxime,29−33 and anthranylaldoxime34,35 derivatives
have been reported as ER ligands. Thus, the development of
new ER ligands remains an important issue in medicinal
chemistry because novel functions of estrogen are still being
found.36,37
In this article, we have explored diversification of ligands for
the estrogen receptor by replacing the CC double bond with
a simple Schiff Base or imine (CN) core structure. A series of
triaryl-substituted Schiff base derivatives were conveniently
prepared, generally in one step, by the condensation of
benzophenones with various anilines without the need for
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phenolic hydroxy group protection. Many of these compounds
have very high binding affinities for ERs, rivaling or exceeding
that of estradiol (up to 97% RBA for ERα and 140% for ERβ).
Some of the compounds also show significant affinity selectivity
in favor of ERβ (4- to 5-fold), and in cell-based assays for
transcriptional activity, they profiled as ERα agonists and ERβ
antagonists, a form of ER-subtype selectivity that appears to be
based on their preferential activity through AF1, the N-terminal
activation function of the ERs that is more active in ERα than
ERβ. In addition, an unusual distortion of the ligand-binding
domain, revealed by X-ray analysis, suggests that the function of
AF2 is not fully engaged and highlights the diverse ways in
which ligands can regulate the conformation, and ultimately the
activity, of the ERs.
Beyond our general interest in preparing ER ligands of
unusual structure,1 two other factors motivated our inves-
tigation of imine-core systems for ER ligand design. Some time
ago, in an attempt to prepare gallium chelates that might have
good affinity for ER and could be used in Ga-67- or Ga-68-
labeled form for the imaging of ER in breast cancer using
SPECT or PET, we prepared some similar imines, notably,
those having the 2-hydroxyl group on the carbonyl
component.16 Unfortunately, although we could prepare
dimethylgallium complexes, engaging the oxygen and nitrogen
of the o-hydroxyphenyl imine system as a bidentate chelate, and
could even obtain their crystal structures, the aqueous stability
of these complexes was very low.16 Nevertheless, at the time,
we noted that a few of the imines had some ER binding affinity.
(The complete binding data on these compounds is now given
in Table S1.) We also returned to the imines when we prepared
the BN core compounds as part of our attempt to make the
CC and CN systems that were isoelectronic with the
hydrolytically stable BN compounds; however, the one C
N analogue of these more sterically encumbered systems that
we were able to prepare lacked the critical phenolic hydroxyl
group and had other substituents that precluded high-affinity
binding.13 By contrast, in this investigation, we were gratified by
how easy it was to prepare a large series of triaryl-substituted
imines and how many of them had high binding affinity for
both ERα and ERβ.
Tolerance of Polar Groups in the Ligand Interior.
Because the interior of the ER ligand binding pockets is lined
strictly with hydrophobic residues (except for the phenol−
hydrogen bonding glutamate and arginine residues and one
threonine),24 we expected that compounds in series 2 would
have lower affinity compared those in the series 4: the isolated
lone pair on the imine nitrogen in 2 was expected to exact a
large desolvation energy penalty, whereas in the series 4 imines,
the intramolecular hydrogen bond would internally solvate the
imine nitrogen, thereby muting the impact of moving this polar
function from water into the binding pocket. Relevant to this
issue is the higher affinity of genistein than daidzein for both
ERα and ERβ: the ketone group in genistein is shielded by an
intramolecular hydrogen bond, whereas in daidzein, it is an
isolated function (Figure 6).38 A similar intramolecular
hydrogen-bonded system is present in the resorcylic acid
lactone series and macrolides with high affinity for the ERs
exemplified by zeralanol39 as well as in the salicylaldoximine
and anthranylaldoxime systems developed by Minutolo, where
an intermolecular hydrogen bond completes the formation of a
pseudocycle that is thought to mimic the phenolic ring of
estradiol and is important for high affinity binding (Figure 6).34
(In addition, ER ligands with polar, hydrogen-bonding cores,
such as imidazoles and pyridazines, bind much less well than
their less polar analogues, pyrazoles and pyrazines, respec-
tively.1)
Despite these precedents for the importance of intra-
molecular hydrogen bonding to shield a polar element in the
interior of ER ligands, in nearly every case, the imines of series
2, all of which lack this hydrogen bond, have comparable or
higher affinity than the corresponding members in series 4
(Figure 2). This was particularly evident in their ERβ-binding
affinities, where the average ratio of RBA (series 2)/RBA
(series 4) is 1.88 0.70, whereas it is only 1.16 0.78 for ERα.
A reexamination of the crystal structures for compounds 2f
and 4f shows that at the imine side of the ligands there is ample
space between the ligands and the pocket residues, with no
evidence for interaction of the imine lone pair in compound 2f
with any elements of the protein. Also, the additional hydroxyl
group in compound 4f does not engage in hydrogen bonding
with the protein and is nicely accommodated without any
obvious effects on the shape of the pocket at this side of the
ligands. Perhaps the only factor contributing to the differences
between the two series could be the increased twist angle of the
N-phenyl ring noted in the X-ray structure of the series 4
compound 4f versus the series 2 compound 2f (Figure 5C−F),
which appears to interfere with a productive interaction with
the Thr347 residue. It is notable, as well, that in a number of
ligand series, ERβ appears more tolerant to interior polar
groups.8
Number of Substituents on the CN Core. Although
ligands having CC or BN core elements could be
tetrasubstituted, the imine-core ligands, for valency reasons,
can, at most, be trisubstituted. Nevertheless, triaryl ethylene
ligands with a CC core lacking a fourth substituent often
have very good ER-binding affinity (Figure 6),40 as do our
imines. By contrast, the mono and diaryl imines (Table S1)
have uniformly low affinity, presumably because they are too
small. There are a number of ER ligands in which a CC core
has been replaced by two nitrogens (i.e., an azo or NN
group), and these, for valence reasons, can be only
disubstituted. These azophenol or azoresorcinol systems have
low, but clearly measurable, ER-binding affinity (Figure 6) and
are known to be estrogens, although of very low potency.41,42
In these molecules, as with the imines, there are ortho-
substituted hydroxyl groups that can engage in intramolecular
hydrogen bonds, but again, these hydrogen bonds reduce
binding (Figure 6).
Effect of Phenyl Ring Substituents. In our imines,
addition of a single ortho methyl group in the distal N-phenol
ring improved binding considerably (Table 1, compound 2c vs
2a), whereas addition of a second ortho methyl group caused a
precipitous drop in binding affinity (Table 1, compound 2b vs
2c). We encountered the beneficial effect of single ortho methyl
substitution in A-CD estrogens (B-seco steroids lacking an
intact B-ring), where addition of a methyl group (or other small
substituent, Cl, CF3) ortho to the site of attachment of the
phenol to the C-ring increased affinity (Figure 6).43 In these
seco-estrogens, we interpreted the increase in binding resulting
from this single ortho substitution as the dual effect of
supplying bulk that was lost by deletion of the B-ring as well as
twisting the aryl ring relative to the rest of the ligand, thereby
increasing ligand volume, at least on one side. Similarly, in
other ER ligands that we have explored, a single twist-inducing
ortho substituent generally increased binding affinity.2,44,45
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Article
systems (normal and reversed phase), confirming >95% purity (see the
Supporting Information).
Substituents on the N-phenyl ring in our triarylimines are
directed to different regions of the ER ligand-binding pocket
than those in the B-secosteroids, and their enhancement of
binding is most likely just due to increased hydrophobic bulk.
There is a general trend, with binding affinity decreasing with
the series C-2 > C-3 > C-4, which suggests that the ortho-
disposed (C-2) groups might be causing an increased twist of
the N-phenyl group. Simple MM2 energy minimization,
however, shows that all of these triaryl imines adopt a
propeller-like conformation; even the unsubstituted systems,
2a and 4b, show a coordinated twist of all three rings, giving
dihedral angles of 160, 155, and 172° for τ1, τ2, and τ3,
respectively (Figure 6). Notably, addition of the substituents
shown in Table 1, even those at the C-2 position, changes these
torsions by only a few degrees. Even 2,6-dimethyl substitution
in 2c causes only a 10° change in τ3, suggesting that the marked
drop in affinity results from a steric clash of the second methyl
group in the ligand-binding pocket.
Structural Mechanisms for Ligand-Dependent Modu-
lation of ER Activity. ER binds a diverse collection of ligands
that modulate its activity through distinct structural mecha-
nisms. Full agonists directly drive helix 12 to adopt an active
conformation where it lies across helices 3 and 11, positioned
to form one side of the AF2 coactivator-binding surface.24 In
contrast, the bulky side chain of SERMs protrudes out of the
pocket and directly displaces helix 12 from this active
conformation.24,25 Yet, several other ligands disrupt the active
conformation of helix 12 indirectly by distorting the C-terminal
end of helix 11,7,44 thereby modulating ER activity. Here, we
show that triaryl imine ligands modulate ER activity through a
new type of indirect mechanism that involves a ligand-
dependent increase in the distance separating helices 3 and
11 (Figure 5). Through this new mechanism, these imine
ligands are able to modulate the AF2-mediated but not AF1-
mediated activity of ER (Figure 4).
Chemical Synthesis. General Procedure of the Improved
Method for the Synthesis of Compounds 2a−m and 4a−m. A
mixture of 1 (1 mmol) and aniline derivatives (3 mmol, 3 equiv) was
dissolved in chlorobenzene (4 mL). Under a N2 atmosphere, the
mixture was briefly exposed to HCl(g) and then heated at 140−145
°C for 24 h. The resulting solution was concentrated under reduced
pressure, and the crude product was purified by silica gel
chromatography (petroleum ether/ethyl acetate: Et3N = 4:1:0.5) to
afford target molecules 2 and 4. Further purification was achieved by
recrystallization (ethyl acetate/petroleum ether).
4,4′-((Phenylimino)methylene)diphenol (2a). According to the
general procedure of the improved method, 2a was obtained as a
yellow solid (60% yield) and was further purified by recrystallization
from ethyl acetate/petroleum ether (mp 273−275 °C). 1H NMR (400
MHz, acetone-d6) δ 8.83 (br s, 2H), 7.66−7.60 (m, 2H), 7.15−7.08
(m, 2H), 7.00−6.94 (m, 2H), 6.92−6.83 (m, 3H), 6.77−6.71 (m, 2H),
6.66 (dt, J = 8.4 Hz, 1.6 Hz, 2H). 13C NMR (101 MHz, acetone-d6) δ
167.97, 160.74, 158.37, 153.24, 132.58, 131.96, 131.94, 129.18, 128.51,
123.11, 121.78, 115.68, 115.49. HRMS (MALDI/DHB) calcd for
C19H17NO2 (M + H)+ m/z, 290.11811; found, 290.11756.
4,4′-(((2,6-Dimethylphenyl)imino)methylene)diphenol (2b).
According to the general procedure of the improved method, 2b was
obtained as a yellow solid (58% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 147−150
1
°C). H NMR (400 MHz, acetone-d6) δ 9.05 (s, 1H), 8.88 (s, 1H),
7.66 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 6.88 (dd, J = 14.6
Hz, 8.1 Hz, 4H), 6.75−6.66 (m, 3H), 1.99 (s, 6H). 13C NMR (101
MHz, acetone-d6) δ 167.02, 160.60, 158.84, 150.56, 132.56, 131.93,
131.06, 129.19, 128.35, 126.53, 122.89, 115.59, 115.28, 18.71. HRMS
(MALDI/DHB) calcd for C21H20NO2(M + H)+ m/z, 318.14941;
found, 318.14886.
4,4′-((2-Tolylimino)methylene)diphenol (2c). According to the
general procedure of the improved method, 2c was obtained as a
yellow solid (60% yield) and was further purified by recrystallization
from ethyl acetate/petroleum ether (mp 126−130 °C). 1H NMR (400
MHz, acetone-d6) δ 8.94 (br s, 1H), 8.70 (br s, 1H), 7.65 (d, J = 8.7
Hz, 2H), 7.05 (d, J = 7.4 Hz, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.93−6.85
(m, 3H), 6.82−6.70 (m, 3H), 6.41 (d, J = 7.7 Hz, 1H), 2.12 (s, 3H).
13C NMR (101 MHz, acetone-d6) δ 167.34, 160.67, 158.45, 151.89,
132.53, 131.92, 131.41, 130.54, 128.83, 128.78, 126.62, 123.23, 120.57,
115.69, 115.45, 18.44. HRMS (MALDI/DHB) calcd for C20H19NO2
(M + H)+ m/z, 304.13376; found, 304.13321.
4,4′-((3-Tolylimino)methylene)diphenol (2d). According to the
general procedure of the improved method, 2d was obtained as a
yellow solid (61% yield) and was further purified by recrystallization
from ethyl acetate/petroleum ether (mp 215−218 °C). 1H NMR (400
MHz, acetone-d6) δ 8.96 (br s, 1H), 8.71 (br s, 1H), 7.61 (d, J = 8.7
Hz, 2H), 6.98 (dd, J = 8.1 Hz, 6.2 Hz, 3H), 6.88 (d, J = 8.7 Hz, 2H),
6.74 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 7.5 Hz, 1H), 6.53 (s, 1H), 6.43
(d, J = 7.9 Hz, 1H), 2.17 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ
167.70, 160.65, 158.31, 153.17, 138.60, 132.66, 131.92, 131.90, 129.01,
128.59, 123.86, 122.50, 118.73, 115.65, 115.45, 21.45. HRMS
(MALDI/DHB) calcd for C20H19NO2 (M + H)+ m/z, 304.13376;
found, 304.13321.
The ease with which these CN imine analogues can be
prepared, in some cases much more readily than their CC
double bond counterparts, and the high binding affinity that
they have for the ERs suggest that this form of analogue
development might be worth exploring more generally in drug
discovery. Furthermore, the marked pattern of ER-subtype
differential cellular efficacy, based on differential utilization of
the two ER activation functions, displayed by the CN
analogues appears to be based on a novel mode of
conformational control of the ER ligand-binding pocket that
affects AF2 function, as revealed by our X-ray structural
analyses. This new ligand design paradigm could also be more
widely investigated for the development of estrogens having a
novel spectrum of activities.
4,4′-((4-Tolylimino)methylene)diphenol (2e). According to the
general procedure of the improved method, 2e was obtained as a
yellow solid (63% yield) and was further purified by recrystallization
from ethyl acetate/petroleum ether (mp 128−131 °C). 1H NMR (400
MHz, acetone-d6) δ 8.85 (br s, 2H), 7.64−7.58 (m, 2H), 6.99−6.90
(m, 4H), 6.87 (d, J = 8.7 Hz, 2H), 6.75 (d, J = 8.5 Hz, 2H), 6.56 (d, J
= 8.2 Hz, 2H), 2.19 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ
167.78, 160.64, 158.31, 150.61, 132.74, 132.24, 131.90, 131.87, 129.74,
128.69, 121.83, 115.63, 115.51, 20.82. HRMS (MALDI/DHB) calcd
for C20H19NO2 (M + H)+ m/z, 304.13376; found, 304.13321.
4,4′-(((2-Chlorophenyl)imino)methylene)diphenol (2f). Ac-
cording to the general procedure of the improved method, 2f was
obtained as a yellow solid (72% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 125−127
EXPERIMENTAL SECTION
■
Analytical Techniques and Instrumentation Used. Melting
points were determined on a X-4 Beijing Tech melting point
apparatus. 1H and 13C NMR spectra were recorded on a Bruker
1
AV400 spectrometer (400 MHz, H NMR; 101 MHz, 13C NMR) at
room temperature. NMR spectra were calibrated to the solvent signals
of CDCl3 (δ 7.26 and 77.00 ppm), acetone-d6 (δ 2.05 and 29.84 ppm,
206.26 ppm), or DMSO-d6 (δ 2.50 and 39.43 ppm). The chemical
shifts are provided in ppm, and the coupling constants, in Hz. The
following abbreviations for multiplicities are used: s, singlet; d,
doublet; dd, double doublet; t, triplet; dt, double triplet; q, quadruplet;
m, multiplet; and br, broad. The purity of all compounds for biological
testing was determined by HPLC analysis in two different solvent
3540
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Journal of Medicinal Chemistry
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1
°C). H NMR (400 MHz, acetone-d6) δ 9.05 (br s, 1H), 8.77 (br s,
3H). 13C NMR (101 MHz, acetone-d6) δ 167.76, 160.61, 158.34,
156.40, 146.14, 133.02, 131.94, 131.82, 128.78, 123.23, 115.76, 115.59,
114.45, 55.51. HRMS (MALDI/DHB) calcd for C20H18NO3 (M +
H)+ m/z, 320.12867; found, 320.12812.
1H), 7.69−7.63 (m, 2H), 7.28 (dd, J = 8.0 Hz, 1.3 Hz, 1H), 7.07−7.00
(m, 3H), 6.93−6.85 (m, 3H), 6.78−6.72 (m, 2H), 6.62 (dd, J = 7.9
Hz, 1.5 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 169.68, 161.14,
158.75, 150.72, 132.29, 131.82, 131.22, 129.96, 128.47, 127.89, 125.61,
124.26, 122.60, 115.77, 115.52. HRMS (MALDI/DHB) calcd for
C19H16NO2Cl (M + H)+ m/z, 324.07914; found, 324.07858.
4,4′-(((4-Hydroxyphenyl)imino)methylene)diphenol (2n).
According to the general procedure of the improved method, 2m
was obtained as a yellow solid (74% yield). Then, 2m was dissolved in
THF and MeOH, a solution of KOH (1 equiv) in MeOH was added
to the stirred mixture for 3h, the mixture was concentrated under
reduced pressure, and the residue was isolated by silica gel
chromatography (petroleum ether/ethyl acetate = 2:1, including
0.5% Et3N) to afford the desired product 2n (94% yield), which was
further purified by recrystallization from ethyl acetate/petroleum ether
(mp 297−301 °C). 1H NMR (400 MHz, acetone-d6) δ 7.46 (d, J = 8.7
Hz, 2H), 6.85−6.79 (m, 2H), 6.73 (d, J = 8.7 Hz, 2H), 6.64 (t, J = 8.1
Hz, 2H), 6.51−6.44 (m, 2H), 6.42−6.36 (m, 2H). 13C NMR (101
MHz, acetone-d6) δ167.29, 160.52, 158.30, 153.94, 145.10, 132.99,
131.95, 131.73, 128.93, 123.34, 116.04, 115.82, 115.59. HRMS
(MALDI/DHB) calcd for C19H17NO3 (M + H)+ m/z, 306.11302;
found, 306.11247.
4-(((4-Methoxyphenyl)imino)(phenyl)methyl)benzene-1,3-
diol (4a). According to the general procedure of the improved
method, 4a was obtained as a yellow solid (76% yield) and was further
purified by recrystallization from ethyl acetate/petroleum ether (mp
253−257 °C). 1H NMR (400 MHz, acetone-d6) δ 15.03 (s, 1H), 9.26
(s, 1H), 7.39 (dd, J = 5.1 Hz, 1.6 Hz, 3H), 7.28−7.19 (m, 2H), 6.82
(d, J = 8.8 Hz, 1H), 6.77−6.67 (m, 4H), 6.43 (d, J = 2.2 Hz, 1H), 6.28
(dd, J = 8.8 Hz, 2.2 Hz, 1H), 3.69 (s, 3H). 13C NMR (101 MHz,
acetone-d6) δ 173.34, 165.44, 162.81, 157.50, 141.22, 134.55, 132.51,
132.15, 129.65, 129.52, 129.09, 124.60, 114.53, 107.53, 104.00, 55.54.
HRMS (MALDI/DHB) calcd for C20H18NO3 (M + H)+ m/z,
320.12867; found, 320.12812.
4-((4-Hydroxyphenyl)(phenylimino)methyl)benzene-1,3-
diol (4b). According to the general procedure of the improved
method, 4c was obtained as a yellow solid (70% yield) and was further
purified by recrystallization from ethyl acetate/petroleum ether (mp
249−252 °C). 1H NMR (400 MHz, acetone-d6) δ 14.82 (s, 1H), 9.15
(s, 1H), 8.81 (s, 1H), 7.18 (t, J = 7.8 Hz, 2H), 7.06 (d, J = 8.5 Hz,
2H), 7.02−6.95 (m, 2H), 6.82 (d, J = 8.4 Hz, 4H), 6.43 (d, J = 2.2 Hz,
1H), 6.31 (dd, J = 8.8 Hz, 2.3 Hz, 1H). 13C NMR (101 MHz, acetone-
d6) δ 174.22, 166.08, 163.10, 158.77, 148.44, 134.86, 131.38, 129.31,
126.42, 124.78, 123.50, 115.77, 114.19, 107.47, 104.02. HRMS
(MALDI/DHB) calcd for C19H17NO3 (M + H)+ m/z, 306.11302;
found, 306.11247.
4,4′-(((3-Chlorophenyl)imino)methylene)diphenol (2g). Ac-
cording to the general procedure of the improved method, 2g was
obtained as a yellow solid (54% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 186−190
1
°C). H NMR (400 MHz, acetone-d6) δ 8.90 (br s, 2H), 7.63 (d, J =
8.7 Hz, 2H), 7.13 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.5 Hz, 2H), 6.90
(dd, J = 5.5 Hz, 3.2 Hz, 3H), 6.78 (d, J = 8.5 Hz, 2H), 6.71 (t, J = 1.9
Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ
169.10, 161.04, 158.62, 154.85, 134.35, 132.18, 132.09, 121.90, 130.63,
128.03, 122.90, 121.66, 120.42, 115.74, 115.62. HRMS (MALDI/
DHB) calcd for C19H16NO2Cl (M + H)+ m/z, 324.07914; found,
324.07858.
4,4′-(((4-Chlorophenyl)imino)methylene)diphenol (2h). Ac-
cording to the general procedure of the improved method, 2h was
obtained as a yellow solid (51% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 123−127
1
°C). H NMR (400 MHz, acetone-d6) δ 9.01 (br s, 1H), 8.75 (br s,
1H), 7.66−7.58 (m, 2H), 7.32−7.25 (m, 2H), 7.01−6.95 (m, 2H),
6.92−6.85 (m, 2H), 6.80−6.74 (m, 2H), 6.66−6.59 (m, 2H). 13C
NMR (101 MHz, acetone-d6) δ 168.90, 161.01, 158.62, 152.12,
132.23, 132.10, 131.93, 129.16, 128.14, 127.86, 123.49, 115.76, 115.66.
HRMS (MALDI/DHB) calcd for C19H16NO2Cl (M + H)+ m/z,
324.07914; found, 324.07858.
4,4′-(((3-(Trifluoromethyl)phenyl)imino)methylene)diphenol
(2i). According to the general procedure of the improved method, 2i
was obtained as a yellow solid (74% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 212−215
1
°C). H NMR (400 MHz, acetone-d6) δ 9.02 (br s, 1H), 8.86 (br s,
1H), 7.67 (t, J = 11.2 Hz, 2H), 7.34 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.7
Hz, 1H), 7.05−6.87 (m, 6H), 6.77 (d, J = 8.4 Hz, 2H). 13C NMR (101
MHz, acetone-d6) δ 169.64, 161.17, 158.67, 153.94, 132.24, 131.98,
131.91, 131.21, 130.90, 130.14, 128.65 (d, J = 270 Hz), 127.88, 125.63,
119.15 (d, J = 100 Hz), 115.79, 115.65. HRMS (MALDI/DHB) calcd
for C20H16NO2F3 (M + H)+ m/z, 358.10549; found, 358.10494.
4,4′-(((4-Fluorophenyl)imino)methylene)diphenol (2j). Ac-
cording to the general procedure of the improved method, 2j was
obtained as a yellow solid (51% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 267−271
4-((4-Hydroxyphenyl)(2-tolylimino)methyl)benzene-1,3-diol
(4c). According to the general procedure of the improved method, 4c
was obtained as a yellow solid (58% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 249−252
1
°C). H NMR (400 MHz, acetone-d6) δ 8.97 (br s, 1H), 8.76 (br s,
1H), 7.65−7.59 (m, 2H), 7.00−6.94 (m, 2H), 6.93−6.85 (m, 4H),
6.80−6.74 (m, 2H), 6.71−6.64 (m, 2H). 13C NMR (101 MHz,
acetone-d6) δ 168.80, 160.86, 158.48, 159.53 (d, J = 237 Hz), 149.52,
132.44, 131.99, 131.93, 128.34, 123.32, (d, J = 8 Hz), 115.70, 115.63,
(d, J = 22 Hz), 115.61. HRMS (MALDI/DHB) calcd for C19H16NO2F
(M + H)+ m/z, 308.10869; found, 308.10813.
1
°C). H NMR (400 MHz, acetone-d6) δ 14.86 (s, 1H), 9.12 (s, 1H),
8.77 (s, 1H), 7.11 (d, J = 7.3 Hz, 1H), 7.04 (d, J = 8.3 Hz, 2H), 6.99
(d, J = 8.8 Hz, 1H), 6.95−6.84 (m, 2H), 6.79 (d, J = 8.5 Hz, 2H), 6.56
(d, J = 7.4 Hz, 1H), 6.43 (s, 1H), 6.31 (d, J = 8.7 Hz, 1H), 2.23 (s,
3H). 13C NMR (101 MHz, acetone-d6) δ 174.09, 166.18, 162.98,
158.74, 147.41, 134.87, 130.92, 130.77, 130.37, 126.72, 126.65, 124.96,
122.98, 115.67, 114.10, 107.39, 104.03, 18.58. HRMS (MALDI/DHB)
calcd for C20H19NO3 (M + H)+ m/z, 320.12867; found, 320.12812.
4-((4-Hydroxyphenyl)(3-tolylimino)methyl)benzene-1,3-diol
(4d). According to the general procedure of the improved method, 4d
was obtained as a yellow solid (64% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 239−243
4,4′-(((4-Bromophenyl)imino)methylene)diphenol (2k). Ac-
cording to the general procedure of the improved method, 2k was
obtained as a yellow solid (55% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 127−130
1
°C). H NMR (400 MHz, acetone-d6) δ 9.03 (br s, 1H), 8.77 (br s,
1H), 7.66−7.58 (m, 2H), 7.17−7.11 (m, 2H), 7.01−6.95 (m, 2H),
6.93−6.85 (m, 2H), 6.80−6.74 (m, 2H), 6.71−6.64 (m, 2H). 13C
NMR (101 MHz, acetone-d6) δ 168.85, 160.95, 158.60, 152.54,
132.23, 132.14, 132.13, 131.95, 128.11, 123.94, 115.75, 115.66, 115.58.
HRMS (MALDI/DHB) calcd for C19H16NO2Br (M + H)+ m/z,
368.02862; found, 368.02807.
4,4′-(((4-Methoxyphenyl)imino)methylene)diphenol (2l). Ac-
cording to the general procedure of the improved method, 2l was
obtained as a yellow solid (79% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 117−119
°C). 1H NMR (400 MHz, acetone-d6) δ 7.60 (d, J = 8.7 Hz, 2H), 6.95
(dd, J = 8.7 Hz, 2.4 Hz, 3H), 6.86 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 8.5
Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.8 Hz, 2H), 3.68 (s,
1
°C). H NMR (400 MHz, acetone-d6) δ 14.88 (s, 1H), 9.15 (s, 1H),
8.82 (s, 1H), 7.07−7.00 (m, 3H), 6.97 (d, J = 8.8 Hz, 1H), 6.80 (t, J =
9.3 Hz, 3H), 6.68 (s, 1H), 6.56 (d, J = 7.9 Hz, 1H), 6.42 (s, 1H), 6.30
(d, J = 8.8 Hz, 1H), 2.20 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ
173.98, 166.17, 163.09, 158.78, 148.23, 138.93, 134.82, 131.33, 129,12,
126.44, 125.55, 124.23, 120.50, 115.74, 114.18, 107.45, 104.02, 21.34.
HRMS (MALDI/DHB) calcd for C20H19NO3 (M + H)+ m/z,
320.12867; found, 320.12812.
4-((4-Hydroxyphenyl)(4-tolylimino)methyl)benzene-1,3-diol
(4e). According to the general procedure of the improved method, 4e
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was obtained as a yellow solid (68% yield) and was further purified by
recrystallization from ethyl acetate/petroleum ether (mp 249−252
4-(((4-Bromophenyl)imino)(4-hydroxyphenyl)methyl)-
benzene-1,3-diol (4k). According to the general procedure of the
improved method, 4k was obtained as a yellow solid (58% yield) and
was further purified by recrystallization from ethyl acetate/petroleum
ether (mp 279−282 °C). 1H NMR (400 MHz, acetone-d6) δ 14.45 (s,
1H), 9.18 (s, 1H), 8.82 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.09−7.03
(m, 2H), 6.99 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 2H), 6.77 (dd, J
= 8.7 Hz, 2.2 Hz, 2H), 6.43 (d, J = 2.2 Hz, 1H), 6.32 (dd, J = 8.8 Hz,
2.3 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 174.83, 165.89,
163.26, 158.89, 147.97, 135.07, 132.28, 131.40, 126.21, 125.60, 117.45,
115.88, 114.13, 107.67, 104.00. HRMS (MALDI/DHB) calcd for
C19H16NO3Br (M + H)+ m/z, 384.02354; found, 384.02298.
4-((4-Hydroxyphenyl)((4-methoxyphenyl)imino)methyl)-
benzene-1,3-diol (4l). According to the general procedure of the
improved method, 4l was obtained as a yellow solid (75% yield) and
was further purified by recrystallization from ethyl acetate/petroleum
ether (mp 259−263 °C). 1H NMR (400 MHz, acetone-d6) δ 15.30 (s,
1H), 9.16 (s, 1H), 8.89 (s, 1H), 7.08−7.03 (m, 2H), 6.96 (d, J = 8.8
Hz, 1H), 6.87−6.83 (m, 2H), 6.74 (s, 4H), 6.41 (d, J = 2.4 Hz, 1H),
6.28 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 3.70 (s, 3H). 13C NMR (101 MHz,
acetone-d6) δ 173.49, 162.86, 158.73, 157.47, 141.06, 134.67, 131.37,
126.63, 124.78, 115.90, 114.56, 114.33, 110.90, 107.31, 104.00, 55.55.
HRMS (MALDI/DHB) calcd for C20H19NO4 (M + H)+ m/z,
336.12359; found, 336.12304.
4-((4-Hydroxyphenyl)((4-hydroxyphenyl)imino)methyl)-
benzene-1,3-diol (4n). According to the general procedure of the
improved method, 4m was obtained as a yellow solid (65% yield).
Then, 4m was dissolved in THF and MeOH, a solution of KOH (1
equiv) in MeOH was added to the stirred mixture for 3 h, and the
mixture was concentrated under reduced pressure, and the residue was
isolated by silica gel chromatography (petroleum ether/ethyl acetate =
2:1, including 0.5% Et3N) to afford desired product 4n (92% yield),
which was further purified by recrystallization from ethyl acetate/
petroleum ether (mp 269−271 °C). 1H NMR (400 MHz, acetone-d6)
δ 15.50 (s, 1H), 8.94 (s, 1H), 8.40 (s, 1H), 7.05 (d, J = 8.4 Hz, 2H),
6.96 (d, J = 8.8 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.7 Hz,
4H), 6.41 (d, J = 2.3 Hz, 1H), 6.27 (dd, J = 8.8 Hz, 2.3 Hz, 1H). 13C
NMR (101 MHz, acetone-d6) δ 173.11, 162.84, 158.74, 144.22,
134.58, 132.02, 131.39, 129.65, 126.59, 124.95, 116.00, 115.93, 114.27,
107.23, 104.09. HRMS (MALDI/DHB) calcd for C19H17NO4 (M +
H)+ m/z, 322.10794; found, 322.10739.
Estrogen Receptor Binding Affinity Assays. Relative binding
affinities were determined by a competitive radiometric binding assay,
as previously described,46,47 using 2 nM [3H]estradiol as tracer
([2,4,6,7-3H]-estra-1,3,5(10)-triene-3,17β-diol, 70−115 Ci/mmol, Per-
kinElmer, Waltham, MA) and purified full-length human ERα and
ERβ, which were purchased from PanVera/Invitrogen (Carlsbad, CA).
Incubations were for 18−24 h at 0 °C. Hydroxyapatite (Bio-Rad,
Hercules, CA) was used to absorb the receptor−ligand complexes, and
free 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 to three
independent determinations. Estradiol binds to ERα with a Kd of 0.2
nM and to ERβ with a Kd of 0.5 nM; these values were determined by
Scatchard analysis using the binding assay protocol described
previously.46
1
°C). H NMR (400 MHz, acetone-d6) δ 15.03 (s, 1H), 9.13 (s, 1H),
8.83 (s, 1H), 7.05 (d, J = 7.9 Hz, 2H), 6.97 (t, J = 8.0 Hz, 3H), 6.82 (d,
J = 8.4 Hz, 2H), 6.70 (d, J = 7.7 Hz, 2H), 6.42 (s, 1H), 6.29 (d, J = 8.7
Hz, 1H), 2.20 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ 173.88,
166.41, 163.02, 158.76, 145.50, 134.77, 134.27, 131.36, 129.90, 126.90,
123.49, 115.83, 114.23, 107.40, 104.05, 20.83. HRMS (MALDI/DHB)
calcd for C20H19NO3 (M + H)+ m/z, 320.12867; found, 320.12812.
4-((4-Hydroxyphenyl)(2-chloroimino)methyl)benzene-1,3-
diol (4f). According to the general procedure of the improved
method, 4f was obtained as a yellow solid (52% yield) and was further
purified by recrystallization from ethyl acetate/petroleum ether (mp
226−230 °C). 1H NMR (400 MHz, acetone-d6) δ 14.22 (s, 1H), 9.00
(br s, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.12−6.95 (m, 5H), 6.79 (dd, J =
16.4 Hz, 8.2 Hz, 3H), 6.47−6.43 (m, 1H), 6.37−6.30 (m, 1H). 13C
NMR (101 MHz, acetone-d6) δ 175.65, 165.83, 163.47, 158.98,
146.45, 135.27, 130.72, 130.04, 127.95, 127.23, 126.50, 126.07, 124.81,
115.76, 113.88, 107.76, 104.02. HRMS (MALDI/DHB) calcd for
C19H16NO3Cl (M + H)+ m/z, 340.07405; found, 340.07350.
4-((4-Hydroxyphenyl)(3-chloroimino)methyl)benzene-1,3-
diol (4g). According to the general procedure of the improved
method, 4g was obtained as a yellow solid (55% yield) and was further
purified by recrystallization from ethyl acetate/petroleum ether (mp
209−211 °C). 1H NMR (400 MHz, acetone-d6) δ 14.74 (s, 1H), 9.20
(s, 1H), 8.86 (s, 1H), 7.05 (d, J = 8.3 Hz, 2H), 6.96 (dd, J = 18.6 Hz,
8.9 Hz, 3H), 6.87−6.79 (m, 4H), 6.43 (d, J = 2.0 Hz, 1H), 6.31 (dd, J
= 8.8 Hz, 1.9 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 174.82,
166.03, 163.12, 161.53, 158.82, 144.83, 134.96, 131.40, 126.30, 125.16,
125.09, 116.00, 115.87, 115.78, 114.16, 107.56, 103.99. HRMS
(MALDI/DHB) calcd for C19H16NO3Cl (M + H)+ m/z, 340.07405;
found, 340.07350.
4-((4-Hydroxyphenyl)(4-chloroimino)methyl)benzene-1,3-
diol (4h). According to the general procedure of the improved
method, 4h was obtained as a yellow solid (60% yield) and was further
purified by recrystallization from ethyl acetate/petroleum ether (mp
255−257 °C). 1H NMR (400 MHz, acetone-d6) δ 14.50 (s, 1H), 9.25
(s, 1H), 8.90 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.3 Hz,
2H), 6.99 (d, J = 8.8 Hz, 1H), 6.83 (dd, J = 8.5 Hz, 2.1 Hz, 4H), 6.43
(d, J = 2.0 Hz, 1H), 6.32 (dd, J = 8.8 Hz, 2.1 Hz, 1H). 13C NMR (101
MHz, acetone-d6) δ 174.91, 165.87, 163.20, 158.85, 147.55, 135.05,
131.40, 129.63, 129.30, 126.19, 125.19, 115.87, 114.12, 107.58, 103.95.
HRMS (MALDI/DHB) calcd for C19H16NO3Cl (M + H)+ m/z,
340.07405; found, 340.07350.
4-((4-Hydroxyphenyl)((3-(trifluoromethyl)phenyl)imino)-
methyl)benzene-1,3-diol (4i). According to the general procedure
of the improved method, 4i was obtained as a yellow solid (61% yield)
and was further purified by recrystallization from ethyl acetate/
petroleum ether (mp 238−242 °C). 1H NMR (400 MHz, acetone-d6)
δ 14.28 (s, 1H), 9.26 (s, 1H), 8.86 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H),
7.31 (d, J = 7.6 Hz, 1H), 7.17−7.05 (m, 4H), 7.00 (t, J = 8.4 Hz, 1H),
6.83 (d, J = 8.2 Hz, 2H), 6.45 (s, 1H), 6.34 (d, J = 8.6 Hz, 1H). 13C
NMR (101 MHz, acetone-d6) δ 175.56, 165.78, 165.25, 163.38,
158.87, 135.30, 135.26, 131.41, 130.28, 127.38, 127.31, 126.38 (d, J =
269 Hz), 120.75 (d, J = 98 Hz), 115.88, 115.83, 107.78, 107.75,
103.96. HRMS (MALDI/DHB) calcd for C20H16NO3F3 (M + H)+ m/
z, 374.10041; found, 374.09985.
4-(((4-Fluorophenyl)imino)(4-hydroxyphenyl)methyl)-
benzene-1,3-diol (4j). According to the general procedure of the
improved method, 4j was obtained as a yellow solid (57% yield) and
was further purified by recrystallization from ethyl acetate/petroleum
ether (mp 241−245 °C). 1H NMR (400 MHz, acetone-d6) δ 14.75 (s,
1H), 9.19 (s, 1H), 8.85 (s, 1H), 7.05 (d, J = 8.6 Hz, 2H), 7.02−6.90
(m, 3H), 6.87−6.79 (m, 4H), 6.43 (d, J = 2.3 Hz, 1H), 6.31 (dd, J =
8.8 Hz, 2.4 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 175.14,
165.23, 164.53 (d, J = 253 Hz), 158.85, 150.31, 135.21, 134.40, 131.38,
130.73, 124.61, 123.47, 122.15 (d, J = 8 Hz), 116.83 (d, J = 20 Hz),
107.72, 103.95. HRMS (MALDI/DHB) calcd for C19H16NO3F (M +
H)+ m/z, 324.10360; found, 324.10305.
Gene Transcriptional Activity. Assays were performed as
previously described.44 HepG2 cells cultured in Dulbecco’s minimum
essential medium (DMEM) (Cellgro by Mediatech, Inc. Manassas,
VA) supplemented with 10% fetal bovine serum (FBS) (Hyclone by
Thermo Scientific, South Logan, UT), 1% nonessential amino acids
(Cellgro), penicillin−streptomycin−neomycin antibiotic mixture, and
Glutamax (Gibco by Invitrogen Corp. Carlsbad, CA) were maintained
at 37 °C and 5% CO2. HepG2 cells were transfected with 10 μg of
3×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/well,
incubated in 384-well plates overnight at 37 °C with 5% CO2, and
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assayed in dose curves ranging from 10 μM to 100 pM for ERE
luciferase assays in HepG2 cells. Luciferase activity was measured after
24 h using BriteLite reagent (PerkinElmer Inc., Shelton, CT)
according to the manufacturer’s protocol. Raw data measured as
relative light units were normalized for each plate using the average of
DMSO-treated samples as 0% and the average of the top of the E2
curve as 100%.
(3) Stauffer, S. R.; Sun, J.; Katzenellenbogen, B. S.; Katzenellenbogen,
J. A. Acyclic amides as estrogen receptor ligands: Synthesis, binding,
activity and receptor interaction. Bioorg. Med. Chem. 2000, 8, 1293−
1316.
(4) Fink, B. E.; Mortensen, D. S.; Stauffer, S. R.; Aron, Z. D.;
Katzenellenbogen, J. A. Novel structural templates for estrogen-
receptor ligands and prospects for combinatorial synthesis of
estrogens. Chem. Biol. 1999, 6, 205−219.
(5) Ghosh, U.; Ganessunker, D.; Sattigeri, V. J.; Carlson, K. E.;
Mortensen, D. J.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A.
Estrogenic diazenes: Heterocyclic non-steroidal estrogens of unusual
structure with selectivity for estrogen receptor subtypes. Bioorg. Med.
Chem. 2003, 11, 629−657.
(6) Mortensen, D. S.; Rodriguez, A. L.; Carlson, K. E.; Sun, J.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Synthesis and
biological evaluation of a novel series of furans: Ligands selective for
estrogen receptor alpha. J. Med. Chem. 2001, 44, 3838−3848.
(7) Zhu, M.; Zhang, C.; Nwachukwu, J. C.; Srinivasan, S.; Cavett, V.;
Zheng, Y.; Carlson, K. E.; Dong, C.; Katzenellenbogen, J. A.; Nettles,
K. W.; Zhou, H. B. Bicyclic core estrogens as full antagonists:
Synthesis, biological evaluation and structure-activity relationships of
estrogen receptor ligands based on bridged oxabicyclic core
arylsulfonamides. Org. Biomol. Chem. 2012, 10, 8692−8700.
(8) Minutolo, F.; Macchia, M.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Estrogen receptor beta ligands: Recent
advances and biomedical applications. Med. Res. Rev. 2011, 31, 364−
442.
(9) Nilsson, S.; Koehler, K. F.; Gustafsson, J. A. Development of
subtype-selective oestrogen receptor-based therapeutics. Nat. Rev.
Drug Discovery 2011, 10, 778−792.
(10) McDonnell, D. P.; Wardell, S. E. The molecular mechanisms
underlying the pharmacological actions of ER modulators: Implica-
tions for new drug discovery in breast cancer. Curr. Opin. Pharmacol.
2010, 10, 620−628.
(11) Bryant, H. U. Selective estrogen receptor modulators. Rev.
Endocr. Metab. Disord. 2002, 3, 231−241.
(12) Saijo, K.; Collier, J. G.; Li, A. C.; Katzenellenbogen, J. A.; Glass,
C. K. An ADIOL-ERbeta-CtBP transrepression pathway negatively
regulates microglia-mediated inflammation. Cell 2011, 145, 584−595.
(13) Zhou, H. B.; Nettles, K. W.; Bruning, J. B.; Kim, Y.; Joachimiak,
A.; Sharma, S.; Carlson, K. E.; Stossi, F.; Katzenellenbogen, B. S.;
Greene, G. L.; Katzenellenbogen, J. A. Elemental isomerism: A boron-
nitrogen surrogate for a carbon-carbon double bond increases the
chemical diversity of estrogen receptor ligands. Chem. Biol. 2007, 14,
659−669.
(14) Nomura, Y. Molecular structure and estrogenic activity of
methylenimine derivatives. II. Nippon Kagaku Zasshi 1956, 77, 1072−
1074.
(15) Nomura, Y. Molecular structure and estrogenic activity of
methylenimine derivatives. III. Nippon Kagaku Zasshi 1956, 77, 1074−
1075.
X-ray Crystallography. As previously described,48 human ERα-
Y537S ligand-binding domain was expressed in BL21(DE3)
Escherichia coli cells, purified, mixed with SRC2 peptide, and
crystallized at room temperature by the hanging-drop vapor-diffusion
method. The ERα crystals obtained were then soaked in the different
imine ligands. The X-ray diffraction data was scaled using HKL-2000
software.49 The crystal structures were solved via molecular
replacement using the PHENIX software suite,50,51 with the protein
components of PDB 2B1V as a starting model.52 The new structures
were then completed upon ligand docking and extensive combinatorial
refinement.53
ASSOCIATED CONTENT
* Supporting Information
■
S
ER-binding affinities of compounds 5−9, analytical method-
ology, summary of purities, and HPLC verification of the purity
of the final compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(H.-B.Z.) Tel.: 86 27 68759586; E-mail: zhouhb@whu.edu.cn.
*(J.A.K.) Tel.: 217 333 6310; E-mail: jkatzene@illinois.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the NSFC (91017005,
81172935, and 81373255), the Key Project of the Ministry of
Education (313040), the Scientific and Technological In-
novative Research Team of Wuhan (2013070204020048),
Hubei Province’s Outstanding Medical Academic Leader
Program, and the Fundamental Research Funds for the Central
Universities (201130602020001) as well as research support
from the National Institutes of Health (PHS 5R37 DK015556
to J.A.K. and 5R01 DK077085 to K.W.N.). S.S. is supported by
Frenchman’s Creek Women for Cancer Research. X-ray data
reported in this study was collected at beamline 11-1, Stanford
Synchrotron Radiation Lightsource, SLAC National Accelerator
Laboratory.
(16) Chesnut, R. W.; Cesati, R. R.; Cutler, C. S.; Pluth, S. L.;
Katzenellenbogen, J. A. Four-coordinate dimethylgallium compounds
vary in stability toward hydrolysis. Organometallics 1998, 17, 4889−
4896.
(17) Takanashi, K.; Yamamoto, K. Divergent approach for synthesis
and terminal modifications of dendritic polyphenylazomethines. Org.
Lett. 2007, 9, 5151−5154.
(18) Carlson, K. E.; Choi, I.; Gee, A.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Altered ligand binding properties and
enhanced stability of a constitutively active estrogen receptor:
Evidence that an open pocket conformation is required for ligand
interaction. Biochemistry 1997, 36, 14897−14905.
(19) Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J. A. The
estradiol pharmacophore: Ligand structure-estrogen receptor binding
affinity relationships and a model for the receptor binding site. Steroids
1997, 62, 268−303.
(20) Anstead, G. M.; Wilson, S. R.; Katzenellenbogen, J. A. 2-
Arylindenes and 2-arylindenones: Molecular structures and consid-
ABBREVIATIONS USED
■
E2, estradiol; ER, estrogen receptor; AF1, transcriptional
activation function 1; AF2, transcriptional activation function
2; SERMs, selective estrogen receptor modulators; RBA,
relative binding affinity; HepG2, hepatoma-derived cancer
cells; ERE, estrogen response element; DMEM, Dulbecco’s
minimum essential medium; FBS, fetal bovine serum
REFERENCES
■
(1) Katzenellenbogen, J. A. The 2010 Philip S. Portoghese Medicinal
Chemistry Lectureship: Addressing the “core issue” in the design of
estrogen receptor ligands. J. Med. Chem. 2011, 54, 5271−5282.
(2) Min, J.; Wang, P.; Srinivasan, S.; Nwachukwu, J. C.; Guo, P.;
Huang, M.; Carlson, K. E.; Katzenellenbogen, J. A.; Nettles, K. W.;
Zhou, H. B. Thiophene-core estrogen receptor ligands having
superagonist activity. J. Med. Chem. 2013, 56, 3346−3366.
3543
dx.doi.org/10.1021/jm500268j | J. Med. Chem. 2014, 57, 3532−3545

Journal of Medicinal Chemistry
Article
erations in the binding orientation of unsymmetrical nonsteroidal
ligands to the estrogen receptor. J. Med. Chem. 1989, 32, 2163−2171.
(21) Gould, J. C.; Leonard, L. S.; Maness, S. C.; Wagner, B. L.;
Conner, K.; Zacharewski, T.; Safe, S.; McDonnell, D. P.; Gaido, K. W.
Bisphenol A interacts with the estrogen receptor alpha in a distinct
manner from estradiol. Mol. Cell. Endocrinol. 1998, 142, 203−214.
(22) Cowley, S. M.; Parker, M. G. A comparison of transcriptional
activation by ER alpha and ER beta. J. Steroid Biochem. Mol. Biol. 1999,
69, 165−175.
(23) Warnmark, A.; Treuter, E.; Gustafsson, J. A.; Hubbard, R. E.;
Brzozowski, A. M.; Pike, A. C. Interaction of transcriptional
intermediary factor 2 nuclear receptor box peptides with the
coactivator binding site of estrogen receptor alpha. J. Biol. Chem.
2002, 277, 21862−21868.
(24) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.;
Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.;
Carlquist, M. Molecular basis of agonism and antagonism in the
oestrogen receptor. Nature 1997, 389, 753−758.
(25) Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.;
Agard, D. A.; Greene, G. L. The structural basis of estrogen receptor/
coactivator recognition and the antagonism of this interaction by
tamoxifen. Cell 1998, 95, 927−937.
(26) Katzenellenbogen, J. A. The structural pervasiveness of
estrogenic activity. Environ. Health Perspect. 1995, 103, 99−101.
(27) Katzenellenbogen, J. A.; Muthyala, R. S. Interactions of
exogenous endocrine active substances with nuclear receptors. Pure
Appl. Chem. 2003, 75, 1797−1817.
(28) Ohta, K.; Chiba, Y.; Ogawa, T.; Endo, Y. Promising core
structure for nuclear receptor ligands: Design and synthesis of novel
estrogen receptor ligands based on diphenylamine skeleton. Bioorg.
Med. Chem. Lett. 2008, 18, 5050−5053.
(29) Minutolo, F.; Bertini, S.; Papi, C.; Carlson, K. E.;
Katzenellenbogen, J. A.; Macchia, M. Salicylaldoxime moiety as a
phenolic “A-ring” substitute in estrogen receptor ligands. J. Med. Chem.
2001, 44, 4288−4291.
(30) Minutolo, F.; Antonello, M.; Bertini, S.; Rapposelli, S.; Rossello,
A.; Sheng, S. B.; Carlson, K. E.; Katzenellenbogen, J. A.; Macchia, M.
Synthesis, binding affinity, and transcriptional activity of hydroxy- and
methoxy-substituted 3,4-diarylsalicylaldoximes on estrogen receptors
alpha and beta. Bioorg. Med. Chem. 2003, 11, 1247−1257.
(31) Minutolo, F.; Bellini, R.; Bertini, S.; Carboni, I.; Lapucci, A.;
Pistolesi, L.; Prota, G.; Rapposelli, S.; Solati, F.; Tuccinardi, T.;
Martinelli, A.; Stossi, F.; Carlson, K. E.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A.; Macchia, M. Monoaryl-substituted salicylal-
doximes as ligands for estrogen receptor β. J. Med. Chem. 2008, 51,
1344−1351.
(32) Minutolo, F.; Bertini, S.; Granchi, C.; Marchitiello, T.; Prota, G.;
Rapposelli, S.; Tuccinardi, T.; Martinelli, A.; Gunther, J. R.; Carlson, K.
E.; Katzenellenbogen, J. A.; Macchia, M. Structural evolutions of
salicylaldoximes as selective agonists for estrogen receptor β. J. Med.
Chem. 2009, 52, 858−867.
(36) van der Windt, D. J.; Kok, N. F.; Ijzermans, J. Estrogen and
tumors: For better or for worse? Science 2007, 318, 1239−1240 ;
author reply pp 1239−1240.
(37) Prins, G. S.; Korach, K. S. The role of estrogens and estrogen
receptors in normal prostate growth and disease. Steroids 2008, 73,
233−244.
(38) Muthyala, R. S.; Ju, Y. H.; Sheng, S.; Williams, L. D.; Doerge, D.
R.; Katzenellenbogen, B. S.; Helferich, W. G.; Katzenellenbogen, J. A.
Equol, a natural estrogenic metabolite from soy isoflavones:
Convenient preparation and resolution of R- and S-equols and their
differing binding and biological activity through estrogen receptors
alpha and beta. Bioorg. Med. Chem. 2004, 12, 1559−1567.
(39) Katzenellenbogen, B. S.; Katzenellenbogen, J. A.; Mordecai, D.
Zearalenones: Characterization of the estrogenic potencies and
receptor interactions of a series of fungal beta-resorcylic acid lactones.
Endocrinology 1979, 105, 33−40.
(40) Lubczyk, V.; Bachmann, H.; Gust, R. Investigations on estrogen
receptor binding. The estrogenic, antiestrogenic, and cytotoxic
properties of C2-alkyl-substituted 1,1-bis(4-hydroxyphenyl)-2-phenyl-
ethenes. J. Med. Chem. 2002, 45, 5358−5364.
(41) Takahashi, T. The estrogenic azo compounds. Nippon Kagaku
Kaishi 1953, 74, 673−675.
(42) Urushibara, Y.; Takahashi, T. 2,2′,4,4′-Tetrahydroxyazobenzene
a synthetic estrogen of a new type. Bull. Chem. Soc. Jpn. 1953, 26, 62−
63.
(43) Wright, J. S.; Shadnia, H.; Anderson, J. M.; Durst, T.; Asim, M.;
El-Salfiti, M.; Choueiri, C.; Pratt, M. A.; Ruddy, S. C.; Lau, R.; Carlson,
K. E.; Katzenellenbogen, J. A.; O’Brien, P. J.; Wan, L. A-CD estrogens.
I. Substituent effects, hormone potency, and receptor subtype
selectivity in a new family of flexible estrogenic compounds. J. Med.
Chem. 2011, 54, 433−448.
(44) Zheng, Y.; Zhu, M.; Srinivasan, S.; Nwachukwu, J. C.; Cavett, V.;
Min, J.; Carlson, K. E.; Wang, P.; Dong, C.; Katzenellenbogen, J. A.;
Nettles, K. W.; Zhou, H. B. Development of selective estrogen
receptor modulator (SERM)-like activity through an indirect
mechanism of estrogen receptor antagonism: defining the binding
mode of 7-oxabicyclo[2.2.1]hept-5-ene scaffold core ligands. Chem-
MedChem 2012, 7, 1094−1100.
(45) Wang, P.; Min, J.; Nwachukwu, J. C.; Cavett, V.; Carlson, K. E.;
Guo, P.; Zhu, M.; Zheng, Y.; Dong, C.; Katzenellenbogen, J. A.;
Nettles, K. W.; Zhou, H. B. Identification and structure-activity
relationships of a novel series of estrogen receptor ligands based on 7-
thiabicyclo[2.2.1]hept-2-ene-7-oxide. J. Med. Chem. 2012, 55, 2324−
2341.
(46) Carlson, K. E.; Choi, I.; Gee, A.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Altered ligand binding properties and
enhanced stability of a constitutively active estrogen receptor:
Evidence that an open pocket conformation is required for ligand
interaction. Biochemistry 1997, 36, 14897−14905.
(47) Katzenellenbogen, J. A.; Johnson, H. J., Jr.; Myers, H. N.
Photoaffinity labels for estrogen binding proteins of rat uterus.
Biochemistry 1973, 12, 4085−4092.
(33) Bertini, S.; De Cupertinis, A.; Granchi, C.; Bargagli, B.;
Tuccinardi, T.; Martinelli, A.; Macchia, M.; Gunther, J. R.; Carlson, K.
E.; Katzenellenbogen, J. A.; Minutolo, F. Selective and potent agonists
for estrogen receptor beta derived from molecular refinements of
salicylaldoximes. Eur. J. Med. Chem. 2011, 46, 2453−2462.
(34) Minutolo, F.; Antonello, M.; Bertini, S.; Ortore, G.; Placanica,
G.; Rapposelli, S.; Sheng, S.; Carlson, K. E.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A.; Macchia, M. Novel estrogen receptor ligands
based on an anthranylaldoxime structure: Role of the phenol-type
pseudocycle in the binding process. J. Med. Chem. 2003, 46, 4032−
4042.
(35) Tuccinardi, T.; Bertini, S.; Martinelli, A.; Minutolo, F.; Ortore,
G.; Placanica, G.; Prota, G.; Rapposelli, S.; Carlson, K. E.;
Katzenellenbogen, J. A.; Macchia, M. Synthesis of anthranylaldoxime
derivatives as estrogen receptor ligands and computational prediction
of binding modes. J. Med. Chem. 2006, 49, 5001−5012.
(48) Nettles, K. W.; Bruning, J. B.; Gil, G.; Nowak, J.; Sharma, S. K.;
Hahm, J. B.; Kulp, K.; Hochberg, R. B.; Zhou, H.; Katzenellenbogen, J.
A.; Katzenellenbogen, B. S.; Kim, Y.; Joachmiak, A.; Greene, G. L.
NFkappaB selectivity of estrogen receptor ligands revealed by
comparative crystallographic analyses. Nat. Chem. Biol. 2008, 4,
241−247.
(49) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode; Academic Press: New York, 1997; pp
307−326.
(50) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis,
I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-
Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.
J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.
PHENIX: A comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66,
213−221.
3544
dx.doi.org/10.1021/jm500268j | J. Med. Chem. 2014, 57, 3532−3545

Journal of Medicinal Chemistry
Article
(51) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Echols,
N.; Headd, J. J.; Hung, L. W.; Jain, S.; Kapral, G. J.; Grosse Kunstleve,
R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R. D.; Read, R. J.;
Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H.
The Phenix software for automated determination of macromolecular
structures. Methods 2011, 55, 94−106.
(52) Hsieh, R. W.; Rajan, S. S.; Sharma, S. K.; Guo, Y.; DeSombre, E.
R.; Mrksich, M.; Greene, G. L. Identification of ligands with bicyclic
scaffolds provides insights into mechanisms of estrogen receptor
subtype selectivity. J. Biol. Chem. 2006, 281, 17909−17919.
(53) Nwachukwu, J. C.; Southern, M. R.; Kiefer, J. R.; Afonine, P. V.;
Adams, P. D.; Terwilliger, T. C.; Nettles, K. W. Improved
crystallographic structures using extensive combinatorial refinement.
Structure 2013, 21, 1923−1930.
3545
dx.doi.org/10.1021/jm500268j | J. Med. Chem. 2014, 57, 3532−3545