Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains

Article abstract of DOI:10.1021/jm061035y

2,3-Diarylpyrazolo[1,5-α]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

Full text of DOI:10.1021/jm061035y

J. Med. Chem. 2007, 50, 399-403
399
Structure-Guided Optimization of Estrogen Receptor Binding Affinity and Antagonist Potency
of Pyrazolopyrimidines with Basic Side Chains
Hai-Bing Zhou,† Shubin Sheng,‡ Dennis R. Compton,† Younchang Kim,§ Andrzej Joachimiak,§ Sanjay Sharma,|
Kathryn E. Carlson,† Benita S. Katzenellenbogen,‡ Kendall W. Nettles, Geoffrey L. Greene,| and John A. Katzenellenbogen*,†
Department of Chemistry, UniVersity of Illinois, 600 South Mathews AVenue, Urbana, Illinois 61801, Departments of Molecular and IntegratiVe
Physiology, Cell and DeVelopmental Biology, UniVersity of Illinois, 407 South Goodwin AVenue, Urbana, Illinois 61801, Midwest Center for
Structural Genomics and Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439, Ben May Institute for Cancer
Research, UniVersity of Chicago, Chicago, Illinois 60637, and Department of Biochemistry, The Scripps Research Institute, Jupiter, Florida
33458
ReceiVed August 29, 2006
2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we
have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained
with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group
and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity
and antagonist potency and a loss of residual agonist activity.
Scheme 1. Fused Heterocyclics and Pyrazolo[1,5-R]pyrimidines
Systems and Analogs as ER Ligands
Introduction
The estrogen receptors (ERsa) have emerged as attractive
therapeutic targets for a variety of diseases, including osteoporo-
sis and breast cancer. Estrogens act through two ER subtypes,
ERR and ERâ,1 which have different tissue distributions and
significant differences in their ligand-binding preferences.2
Estrogens can have remarkable tissue-selective effects, and this
has led to the development of compounds termed selective ER
modulators (SERMs), which function principally as estrogen
agonists in some tissues (bone, brain and the cardiovascular
system), but as antagonists in others (uterus and breast).3,4
The ERs can bind a variety of steroidal and nonsteroidal
ligands, and the search for better SERMs has driven efforts to
increase the chemical diversity of these compounds, especially
the nonsteroidal ones. Frequent examples are heterocycles,5-8
including fused bicyclic systems such as benzothiophenes,9
indoles,10 benzofurans,11 indazoles,12 and indolizines13 (Scheme
1), as well as recently reported benzoxazole-,14 benzothiazole,15
and quinoline-based,16 ERâ-selective ligands. The majority of
these fused heterocycles contain only a single heteroatom; so,
to extend the structural scope of ER ligands, we explored fused
bicyclic systems that contain multiple heteroatoms. This led us
to synthesize ER ligands having indazole12 and pyrazolo[1,5-
a]pyrimidine17,18 core structures (Scheme 1).
In preparing the pyrazolo[1,5-a]pyrimidines, our original
intent was to have a hydroxy group on the pyrimidine ring
function as the A-ring mimic of estradiol, as in the 6-hydroxy
analogs 1. These compounds, however, had very low ER binding
affinity (Table 1, entry 1).17 Thus, we took an alternative
approach, substituting the pyrazolo[1,5-a]pyrimidine core with
p-hydroxyphenyl rings at the 2 and 3 positions,18 one of which
we imagined might function as a mimic of the phenolic A-ring
of estradiol. Such a “pendant phenol” orientation is thought to
be adopted by other fused heterocyclic systems19,20 and is
illustrated in certain crystal structures, at least in ERâ.21 We
also changed the substituents at the 5 and 7 positions to vary
the hydrophobicity and size of the system.
On this basis, we developed a series of 44 pyrazolo[1,5-a]-
pyrimidine core ER ligands, the best of which, the bisphenol
analog 3, still exhibited only modest binding affinity for ERR
and ERâ and no ER subtype selectivity, with the relative binding
affinities (RBA) values (measured on a sample newly synthe-
sized for this study) being 0.77% and 1.0% for ERR and ERâ,
respectively (RBA[estradiol] ) 100%; Table 1, entry 4),
comparable to values determined previously.18 The monophe-
nolic analogs, compounds 2a and 2b, showed even lower
affinities (Table 1, entries 2 and 3).18 These compounds profiled
as ER functional antagonists, with some showing modest
potency preference for ERâ and low, residual ERR agonist
activity (e.g., see compound 3, Figure 2C).18 As was the case
in our prior work with members of the pyrazole triphenols
series,7,22 it was difficult, based on comparisons of the binding
affinities and transcriptional potencies of the monophenols (2a,b)
with the bisphenol (3), to ascertain whether the C-2 phenol or
the C-3 phenol was functioning as the estradiol A-ring mimic.
* To whom correspondence should be addressed: Phone: 217-333-6310.
Fax: 217-333-7325. E-mail: jkatzene@uiuc.edu.
† Department of Chemistry, University of Illinois.
‡ Departments of Molecular and Integrative Physiology, Cell and
Developmental Biology, University of Illinois.
§ Argonne National Laboratory.
| University of Chicago.
The Scripps Research Institute.
a Abbreviations: ER, estrogen receptor; SERMs, selective estrogen
receptor modulators; RBA, relative binding affinity; HEC-1, human
endometrial cancer cells; THF, tetrahydrofuran; TLC, thin-layer chroma-
tography.
10.1021/jm061035y CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

400 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Brief Articles
Table 1. ERR and ERâ Binding Affinities of Pyrazolo[1,5-a]pyrimidine
Derivatives
ligand binding
RBAa (%)
Kib (nM)
R/â
R/â
entry cmpd
ERR
ERâ
ratioc ERR ERâ ratioc
Figure 2. Transcription activation through ERR (shown as solid lines)
and ERâ (shown as dotted lines) of compounds 3, 12a, and 12b. HEC-1
were transfected with expression vectors for ERR or ERâ and the
estrogen responsive gene 2xERE-pS2-Luc and were incubated with the
indicated ligand for 24 h. Agonist assays are the upper curves, and
antagonist assays (done in the presence of 1 nM estradiol, E2) are the
lower lines. Values are the mean ( SD of two or more experiments,
1
2
3
4
5
6
7
8
1
2a
0.006 ( 0.002 0.033 ( 0.001 0.18
3333 1515
0.45
0.06
2.5
0.009 ( 0.001
0.39 ( 0.03
0.77 ( 0.04
2.8 ( 0.44
0.42 ( 0.07
0.075 ( 0.01
0.049 ( 0.004
0.36 ( 0.01
0.41 ( 0.09
1.04 ( 0.09
0.70 ( 0.20
0.28 ( 0.06
0.26 ( 0.01
0.32 ( 0.04
0.025 2222
139
122
48
2b
3
12a
12b
12c
12d
1
0.7
4
1.5
0.29
0.15
51
26
1.8
7
71 10
47
266
408
178
192
156
3.8
0.72
0.38
expressed as a percent of the activity of ERR and ERâ with 10-9
M
a RBA values are IC50estradiol/IC50
× 100. The Kd for estradiol is
compound
E2, which is set at 100%. The following IC50 values are estimated from
the antagonist profiles: compound 3, ERR 6 µM, ERâ 600 nM;
compound 12b, ERR 1 µM, ERâ 250 nM; and compound 12a, ERR
90 nM, ERâ 40 nM.
0.2 nM (ERR) and 0.5 nM (ERâ).27 b Ki values of each compound for
each receptor were obtained from the RBA values by the formula Ki )
(100/RBA) × Kd. c In each case, R/â ratios represent the affinity preference
for ERR vs ERâ; the R/â ratios calculated from the Ki values are 2.5-fold
higher than those calculated from the RBA values, because estradiol binds
to ERR 2.5-fold better than to ERR (see Kd values noted in table footnote
a).
Scheme 2. Synthesis of Pyrazolo[1,5-a]pyrimidine Derivatives
Containing a Basic Side Chain
Figure 1. Comparison of compound 3 versus estradiol in complex
with ERR, as determined by X-ray crystallography. The estradiol/ERR
ligand binding pocket is colored green and shown superimposed
on the compound 3/ERR structure (atom colors). The red arrow
denotes the interaction of the C-3 phenol with helix 12 Leu540, while
the red asterisk highlights the ligand associated positioning of helix 11
His524.
balance of desired and undesired activities, we introduced two
different types of aminoethoxy moieties into the pyrazolo[1,5-
a]pyrimidine core system, placing these at two positions. Thus,
the compounds we made (12a-d) contained either a 2-dim-
ethylaminoethoxy or a 2-N-piperidinylethoxy group, which
was positioned on either the C-2 or the C-3 phenolic hydroxyl
group (Scheme 2). From the crystal structure, as discussed
below, we expected that the C-3-substituted systems (12a,b)
Our interest in preparing more potent, fully antagonistic
analogs in the pyrazolo[1,5-a]pyrimidine series was piqued when
we obtained a crystal structure for compound 3 complexed to
ERR. Because the nature and spatial orientation of the basic
side chains in SERMs relative to their central core structure
are known to influence their tissue selectivity and to affect the

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 401
would show improved affinity and potency; the other regioi-
someric compounds (12c,d) were prepared to challenge this
expectation.
by the ER and might, in fact, enhance both the affinity and
antagonist character of these novel ligands.
Binding Affinity. The binding affinities of compounds
12a-d for both ERR and ERâ were determined using a
competitive radiometric assay and are reported in Table 1.27
These affinities are discussed here as relative binding affinity
(RBA) values, where estradiol has an affinity of 100% (Ki
values, calculated from RBA values, are also given in Table 1;
the R/â ratios shown for the two methods differ by a factor of
2.5, which represents the 2.5-fold higher affinity that the
standard, estradiol, has for ERR vs ERâ).
The contribution of the basic side chain to the binding affinity
of the pyrazolo[1,5-a]pyrimidines is significant, with both the
position and the nature of the basic side chain proving to be
very important. The compounds having the basic side chain at
the para position of 3-aryl group (12a,b) showed good binding
to ERR (Table 1, entries 5 and 6), with pyrazolo[1,5-a]-
pyrimidine 12a having the highest overall RBA for ERR
and ERâ, 2.8% and 0.70%. The former value represents an
increase in binding affinity for ERR that is about 4-fold
compared to the parent compound 3. By contrast, when the basic
side chain was positioned at the para position of C-2 aryl group
(12c,d), ERR binding affinity was markedly lower (entries 7
and 8).
The preferential binding of the C-3 aryl-substituted compound
12a compared to the C-2 regioisomer 12c is consistent with
predictions derived from X-ray structure of the bisphenol
3-ERR complex, which showed that the C-2 (4-hydroxyphenyl)
group was in the A-ring binding pocket and, thus, was not a
favorable site for substitution with the basic side chain. While
not predicted, it is noteworthy that the nature of the basic side
chain also has a significant effect on the binding affinity of the
ligands. Thus, compared to the ligand 12a, containing a
piperidinylethoxy substituent, the dimethylaminoethoxy analog
12b shows considerably lower binding affinity. Affinities of
the new compounds for ERâ were not improved. This might
arise from the fact that the ligand binding pocket in ERâ is,
overall, smaller than that for ERR;21 so, any increase in steric
bulk (such as from the addition of a side chain), even when
extending in a favorable direction, might reduce binding affinity.
Transcriptional Activity and Molecular Modeling. Com-
pounds 12a and 12b and parent compound 3 were assayed for
their transcriptional activity through ERR and ERâ by reporter
gene cotransfection assays in human endometrial cancer cells
(HEC-1; Figure 2).28 All of the compounds are antagonists on
both ERR and ERâ (IC50 values are given in Figure 2 legend).
Most interesting was the activity of the C-3 substituted
piperidinyl analog 12a, which showed considerably enhanced
antagonist potency compared to parent compound 3 on both
ERs, about 70-fold on ERR and 15-fold on ERâ. The other C-3
substituted compound 12b also showed enhanced ERR antago-
nist potency (ca. 6-fold), but a lesser change in ERâ antagonist
potency (only ca. 2-fold), compared to the parent compound
(3). The most potent compound (12a) also lacked the residual
ERR partial agonist efficacy of the parent compound (3).
Results and Discussion
Synthesis. The synthesis of these systems is illustrated in
Scheme 2. The main feature of these syntheses is the selective
protection of one of the hydroxyl groups on the para position
of the C-2 or C-3 phenyl group on the pyrazolo[1,5-a]pyrimidine
core systems (9a) with the easily removable isopropyl moiety,
starting from an appropriate precursor aryl acetonitrile 4a or
methyl benzoate 4b. A Claisen-type condensation between an
appropriate pair of these two components (5a + 6a or 5b +
6b) gave the 2,3-diaryl-3-oxopropionitriles 7a and 7b, respec-
tively, in high yield. Further condensation of these R-cyanoke-
tones with hydrazine dihydrochloride went smoothly and
afforded the key 3-aminopyrazole intermediates 8a,b. The
pyrazolo[1,5-a]pyrimidine cores 9a,b were assembled by con-
densing the aminopyrazoles 8a,b with hexafluoro-acetylacetone.
The regioisomeric monophenol derivatives 10a and 10b
were obtained by selective cleavage of the isopropyl ether with
AlCl3 in dichloromethane at room temperature, which left the
aryl methyl ether intact.23 Installation of the two types of basic
side chains in the free phenolic positions in 10a and 10b was
effected by a Mitsunobu reaction, and the remaining methyl
ether group was then selectively cleaved with AlCl3-EtSH,
leaving the basic side chain unaffected. By this approach, we
prepared the analogs containing the two types of basic side
chains at the para position of either the C-2 or C-3 phenyl
groups, 12a-12d.
Crystal Structure. We crystallized compound 3 with the
ERR ligand-binding domain and a leucine-rich peptide from the
nuclear receptor interaction box 2 of the glucocorticoid receptor
interacting protein 1 coactivator, known to interact with ERR
(see data table in Supporting Information). The overall structure
resembles the protein bound to full agonists, such as estradiol,
with the ligand fully enclosed in a hydrophobic pocket, which
is sealed by helix 12 Leu540. This structure (Figure 1)
demonstrates that the C-2 phenol functions as the estradiol
A-ring mimic. One notable feature of this structure is the
relocation of helix 11 His524 by the C-5 substituted CF3 group
(Figure 1A, red asterisk). This positioning of His524 is also
associated with a shift in the last two turns of helix 11, resulting
in an approximately 1 Å displacement of the main chain of
amino acids 528-530, compared to the position seen with full
agonist ligands (not shown). This shift in helix 11 has been
previously demonstrated to induce partial agonist or antagonist
activity by destabilizing the helix 12 portion of the coactivator
binding pocket,24,25 and is consistent with the weak partial
agonist activity of this compound, described below.
The structure also demonstrates that the C-3 phenol directly
contacts helix 12 Leu540 (Figure 1, red arrow), extending
roughly in the direction where SERMs typically project their
basic 2-aminoethoxy-phenyl substituents (compare structures 2,
3, and 12 in the lower line of Scheme 1 with raloxifene in the
upper line). Helix 12 also forms part of the coactivator binding
site, and its ligand-induced positioning is the ultimate determi-
nant of the recruitment of coactivators and the degree of
associated transcriptional activity. SERMs such as tamoxifen
or raloxifene contain an extended side chain that displaces helix
12 from its position as part of the coactivator binding site.24,26
The crystal structure thus suggested that substitution of the C-3
phenol in compound 3 with the types of basic side chains
typically found in SERMs would be sterically well tolerated
The improved binding affinity and antagonist activity of the
C-3-substituted analog 12a can be rationalized by the X-ray
crystal structure of the complex of compound 3 with ERR. The
aromatic ring at the C-3 position is oriented in such a direction
that addition of the basic amine side chain would avoid steric
clashes and would place the amine function in a position to
form the salt bridge with Asp351, the residue with which amine
function in the basic side chains of hydroxytamoxifen and
raloxifene are known to interact.26,29 Such an interaction would

402 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Brief Articles
the site for introducing basic side chains that are characteristic
of SERMs. We evaluated the ERR and ERâ binding affinities
and the transcriptional potency and efficacy of four of these
derivatives, and we observed that when the basic side chain is
properly positioned (on the C-3 phenol, as suggested by the
X-ray structure) and has the appropriate structure (2-N-piperidi-
nyl-ethoxy), it can significantly enhance affinity and potency,
with compound 12a, the best of our series, having about a 4-fold
increase in ERR binding affinity, a loss of residual agonist
activity, and up to a 70-fold increase in antagonist potency
compared to that of the parent compound 3.
The rapid, structure-guided optimization of antagonist char-
acter and potency that we experienced here in the pyrazolo-
[1,5-a]pyrimidine series can be contrasted with our experiences
in an earlier study in which we endeavored to convert an ERR
agonist ligand of the pyrazole-triphenol class (propylpyrazole
triol) into an ERR-selective antagonist by adding a basic side
chain.31 Although ultimately successful, without the guidance
of an X-ray structure, we had to prepare all four possible
regioisomers and several analogs31-33 to find the one (methyl-
piperidinopyrazole) that showed good binding and effective ERR
antagonism.32,33 By contrast, the results in this study illustrate
the value of X-ray structures of ER-ligand complexes in
guiding an efficient optimization of the pharmacological
characteristics of an ER ligand.
Figure 3. Model of compound 12a ERR (atom colors) compared to
raloxifene ERR (green).
enforce the antagonist conformation of ER and, thereby, enhance
ligand antagonist character, as appears to be the case with
compound 12a. This compound bears the piperidinylethoxyl side
chain on the C-3 phenol, which leaves the C-2 phenol free to
function as the estradiol A-ring mimic. As expected, the C-2/
C-3 regioisomer, compound 12c, is unable to reverse the roles
of the two phenols and, hence, suffers from very low binding
affinity.
To understand why the piperidinylethoxyl side group provides
a significant increase in affinity and antagonist potency com-
pared to the dimethylamino group (compounds 12a,b), we
generated molecular models of these compounds based on the
X-ray structure we determined for compound 3. A comparison
of the previously published tamoxifen26 and raloxifene-bound
ER structures29 shows that they differ in that the piperidine group
of raloxifene forms direct, van der Waals contacts with helix
12, whereas the dimethylamino group in tamoxifen does not
contact helix 12. Thus, these SERMs are thought to differ in
how they control the dynamics of helix 12 and the associated
recruitment of transcriptional coregulator proteins.30
Experimental Section
General Procedure for the Installation of Basic Side Chain
(BSC) by a Mitsunobu Reaction (11). To a solution of corre-
sponding monophenols 10, triphenylphosphine (5 equiv), and
corresponding amino alcohols (5 equiv) in dry THF was added
diisopropyl diazodicarboxylate (5 equiv). The resulting solution was
stirred at room temperature under N2 for 2 days and concentrated
under reduced pressure. The residue was chromatographed over a
silica gel column eluted with 30-50% EtOAc/hexane to afford the
product.
In our structure, the C-3 phenyl group of compound 3 is
oriented in a slightly different manner than the corresponding
group in the raloxifene-ER structure (Figure 3). We generated
a model of compound 3 bound to ER in the antagonist
conformation by superimposing our structure with the raloxifene/
ER complex. We then introduced by molecular modeling the
piperidine or dimethylamino groups onto the C-3 phenol in
compound 3 by superimposing the tamoxifen or raloxifene
compounds, using the respective phenols that correspond to the
C-3 phenol, thereby generating models of compounds 12a and
12b. These models showed that the basic side chains are
optimally positioned to form an electrostatic interaction with
Asp351, but are not able to directly contact helix 12. We then
asked whether a rotation of the core molecule in these models
might allow the basic side chains in compounds 12a and 12b
to contact helix 12. The rotation of the molecule is limited by
the C-7 CF3 group, which lies in a pocket between Leu428 and
Met421. In this position, the CF3 group is located 3.3 Å from
Leu426 and 3.8 Å from Met421, which allows it to rotate the
10-15 degrees necessary for the piperidine group to form
stabilizing contacts with helix 12. These models suggest that
the higher affinity of compound 12a relative to 12b is associated
with a direct interaction that only the larger piperidine group
can make with helix 12.
2-(4-Methoxyphenyl)-3-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-
5,7-bis(trifluoromethyl)pyrazole-[1,5-a]pyrimidine (11a). Re-
crystallized from ethyl acetate/hexane to give a yellow solid (75%
1
yield; mp 149-150 °C). H NMR (500 MHz, CDCl3) δ1.43 (m,
2H), 1.59 (m, 4H), 2.46 (m, 4H), 2.81 (t, 2H, J ) 6.0 Hz), 3.82 (s,
3H), 4.14 (t, 2H, J ) 6.0 Hz), 6.90 (d, 2H, J ) 8.5 Hz), 6.96 (d,
2H, J ) 8.5 Hz), 7.39 (s, 1H), 7.41 (d, 2H, J ) 8.5 Hz), 7.60 (d,
2H, J ) 8.5 Hz); 13C NMR (125 MHz, CDCl3) δ 24.42, 26.14,
55.28, 55.52, 58.18, 66.17, 102.15, 112.18, 114.29, 115.03, 119.91
(q, J ) 274.8 Hz), 121.31 (q, J ) 274.8 Hz), 122.42, 124.36,
130.75, 131.39, 135.31 (q, J ) 37.5 Hz), 145.21 (q, J ) 37.5 Hz),
146.59, 156.57, 158.79. 160.82; LRMS m/z 564.2; HRMS (EI) calcd
for C28H26F6N4O2, 564.1960 (M+); found, 564.1964.
General Procedure for the Synthesis of Compounds 12. The
pyrazolo[1,5-a]pyrimidine 11 (0.5 mmol) was dissolved in 10 mL
of dichloromethane, and to the resulting solution was added AlCl3
(10 equiv) and EtSH (5 equiv). The solution was stirred overnight
and was quenched by careful addition of MeOH, and 10 mL of
water was added. The solution was then extracted (3 × 50 mL)
with ethyl acetate, and the organic layers were combined. The
combined organic layers were washed (2 × 25 mL) with a saturated
NaCl solution and dried with Na2SO4. The ethyl acetate was
removed by rotary evaporation, and the crude product was purified
by flash chromatography (40-60% EtOAc/hexanes), and further
purification was effected using preparative TLC.
2-(4-Hydroxyphenyl)-3-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-
5,7-bis(trifluoromethyl)pyrazole-[1,5-a]pyrimidine (12a). Purified
by preparative TLC (50% EtOAc/hexane) to give a yellow solid
(82% yield) that was recrystallized from ethyl acetate (mp 147-
Conclusions
In this study, we show how the X-ray structure of an ER-
ligand complex can serve as a useful guide that enabled us to
enhance the affinity, potency, and antagonist character of
SERMs of the pyrazolo[1,5-a]pyrimidine core class by directing
1
149 °C). H NMR (400 MHz, CDCl3) δ 1.50 (m, 2H), 1.70 (m,
4H), 2.66 (m, 4H), 2.90 (t, 2H, J ) 5.5 Hz), 4.18 (t, 2H, J ) 5.5
Hz), 4.59 (br s, 1H, OH), 6.77 (d, 2H, J ) 8.8 Hz), 6.82 (d, 2H, J

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 403
(15) Barlaam, B.; Bernstein, P.; Dantzman, C.; Warwick, P. Preparation
of benzoxazoles and benzothiazoles as selective ligands for human
b-estrogen receptor. PCT Int. Appl. (Astrazeneca AB, Sweden) WO,
2002; p 71.
(16) Vu, A. T.; Cohn, S. T.; Manas, E. S.; Harris, H. A.; Mewshaw, R.
E. ERâ ligands. Part 4: Synthesis and structure-activity relationships
of a series of 2-phenylquinoline derivatives. Bioorg. Med. Chem. Lett.
2005, 15, 4520-4525.
) 8.8 Hz), 7.35 (s, 1H), 7.39 (d, 2H, J ) 8.8 Hz), 7.53 (d, 2H, J
) 8.8 Hz); 13C NMR (100 MHz, CDCl3) δ 24.07, 25.17, 54.95,
58.15, 64.40, 101.64, 114.70, 116.27, 117.33 (q, J ) 274.9 Hz),
119.36 (q, J ) 274.9 Hz), 121.52, 123.00, 128.73, 130.92, 131.19,
132.27, 135.42 (q, J ) 37.8 Hz), 144.49 (q, J ) 37.8 Hz), 149.47,
158.33, 158.56; HRMS (ESI) calcd for C27H24F6N4O2H, 551.1882
(M + H+); found, 551.1882.
(17) Compton, D. R.; Carlson, K. E.; Katzenellenbogen, J. A. Pyrazolo-
[1,5-a]pyrimidines as estrogen receptor ligands: defining the orienta-
tion of a novel heterocyclic core. Bioorg. Med. Chem. Lett. 2004,
14, 5681-5684.
(18) Compton, D. R.; Sheng, S.; Carlson, K. E.; Rebacz, N. A.; Lee, I.
Y.; et al. Pyrazolo[1,5-a]pyrimidines: estrogen receptor ligands
possessing estrogen receptor beta antagonist activity. J. Med. Chem.
2004, 47, 5872-5893.
(19) Kohno, H.; Bocchinfuso, W. P.; Gandini, O.; Curtis, S. W.; Korach,
K. S. Mutational analysis of the estrogen receptor ligand-binding
domain: influence of ligand structure and stereochemistry on
transactivation. J. Mol. Endocrinol. 1996, 16, 277-285.
(20) Anstead, G. M.; Peterson, C. S.; Katzenellenbogen, J. A. Hydroxy-
lated 2,3-diarylindenes: synthesis, estrogen receptor binding affinity,
and binding orientation considerations. J. Steroid Biochem. 1989,
33, 877-887.
Acknowledgment. Supported by grants from the NIH
(DK15556 to J.A.K., CA18119 to B.S.K., and CA89489 to
G.L.G.) and USAMRMC W81XWH-04-1-0791 to G.L.G.
Funding for NMR and MS instrumentation is from the Keck
Foundation, NIH, and NSF.
Supporting Information Available: Experimental description
for ER binding affinity assay, gene transcriptional activity assay,
protein purification and X-ray crystallography, molecular modeling,
and characterization of compounds 10a,b, 11b-d, and 12b-d;
HPLC results and chromatograms for compounds 12a-d. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(21) Pike, A. C.; Brzozowski, A. M.; Hubbard, R. E.; Bonn, T.; Thorsell,
A. G.; et al. Structure of the ligand-binding domain of oestrogen
receptor beta in the presence of a partial agonist and a full antagonist.
EMBO J. 1999, 18, 4608-4618.
(22) Stauffer, S. R.; Coletta, C. J.; Tedesco, R.; Nishiguchi, G.; Carlson,
K.; et al. Pyrazole ligands: structure-affinity/activity relationships
and estrogen receptor-alpha-selective agonists. J. Med. Chem. 2000,
43, 4934-4947.
(23) Banwell, M. G.; Flynn, B. L.; Stewart, S. G. Selective cleavage of
isopropyl aryl ethers by aluminum trichloride. J. Org. Chem. 1998,
63, 9139-9144.
(24) Nettles, K. W.; Greene, G. L. Ligand control of coregulator
recruitment to nuclear receptors. Annu. ReV. Physiol. 2005, 67, 309-
333.
(25) Shiau, A. K.; Barstad, D.; Radek, J. T.; Meyers, M. J.; Nettles, K.
W.; et al. Structural characterization of a subtype-selective ligand
reveals a novel mode of estrogen receptor antagonism. Nat. Struct.
Biol. 2002, 9, 359-364.
(26) Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; et
al. The structural basis of estrogen receptor/coactivator recognition
and the antagonism of this interaction by tamoxifen. Cell 1998, 95,
927-937.
(27) Carlson, K. E.; Choi, I.; Gee, A.; Katzenellenbogen, B. S.; Katzenel-
lenbogen, 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.
(28) Sun, J.; Meyers, M. J.; Fink, B. E.; Rajendran, R.; Katzenellenbogen,
J. A.; et al. Novel ligands that function as selective estrogens or
antiestrogens for estrogen receptor-alpha or estrogen receptor-beta.
Endocrinology 1999, 140, 800-804.
(29) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn,
T.; et al. Molecular basis of agonism and antagonism in the oestrogen
receptor. Nature 1997, 389, 753-758.
(30) Webb, P.; Nguyen, P.; Kushner, P. J. Differential SERM effects on
corepressor binding dictate ERR activity in vivo. J. Biol. Chem. 2003,
278, 6912-6920.
(31) Stauffer, S. R.; Huang, Y. R.; Aron, Z. D.; Coletta, C. J.; Sun, J.; et
al. Triarylpyrazoles with basic side chains: development of pyrazole-
based estrogen receptor antagonists. Bioorg. Med. Chem. 2001, 9,
151-161.
(32) Huang, Y. R.; Katzenellenbogen, J. A. Regioselective synthesis of
1,3,5-triaryl-4-alkylpyrazoles: novel ligands for the estrogen receptor.
Org. Lett. 2000, 2, 2833-2836.
References
(1) Kuiper, G. G.; Enmark, E.; Pelto-Huikko, M.; Nilsson, S.; Gustafsson,
J. A. Cloning of a novel receptor expressed in rat prostate and ovary.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5925-5930.
(2) Pettersson, K.; Gustafsson, J. A. Role of estrogen receptor beta in
estrogen action. Annu. ReV. Physiol. 2001, 63, 165-192.
(3) Park, W. C.; Jordan, V. C. Selective estrogen receptor modulators
(SERMS) and their roles in breast cancer prevention. Trends Mol.
Med. 2002, 8, 82-88.
(4) Sato, M.; Grese, T. A.; Dodge, J. A.; Bryant, H. U.; Turner, C. H.
Emerging therapies for the prevention or treatment of postmenopausal
osteoporosis. J. Med. Chem. 1999, 42, 1-24.
(5) 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.
(6) Mortensen, D. S.; Rodriguez, A. L.; Carlson, K. E.; Sun, J.;
Katzenellenbogen, B. S.; et al. Synthesis and biological evaluation
of a novel series of furans: ligands selective for estrogen receptor
alpha. J. Med. Chem. 2001, 44, 3838-3848.
(7) Stauffer, S. R.; Huang, Y.; Coletta, C. J.; Tedesco, R.; Katzenellen-
bogen, J. A. Estrogen pyrazoles: defining the pyrazole core structure
and the orientation of substituents in the ligand binding pocket of
the estrogen receptor. Bioorg. Med. Chem. 2001, 9, 141-150.
(8) Zhou, H. B.; Comninos, J. S.; Stossi, F.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Synthesis and evaluation of estrogen receptor
ligands with bridged oxabicyclic cores containing a diarylethylene
motif: estrogen antagonists of unusual structure. J. Med. Chem. 2005,
48, 7261-7274.
(9) Palkowitz, A. D.; Glasebrook, A. L.; Thrasher, K. J.; Hauser, K. L.;
Short, L. L.; et al. Discovery and synthesis of [6-hydroxy-3-[4-[2-
(1-piperidinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)]benzo[b]thiophene:
a novel, highly potent, selective estrogen receptor modulator. J.
Med. Chem. 1997, 40, 1407-1416.
(10) Gruber, C.; Gruber, D. Bazedoxifene (Wyeth). Curr. Opin. InVest.
Drugs 2004, 5, 1086-1093.
(11) Teo, C. C.; Kon, O. L.; Sim, K. Y.; Ng, S. C. Synthesis of 2-(p-
chlorobenzyl)-3-aryl-6-methoxybenzofurans as selective ligands for
antiestrogen-binding sites. Effects on cell proliferation and cholesterol
synthesis. J. Med. Chem. 1992, 35, 1330-1339.
(12) De Angelis, M.; Stossi, F.; Carlson, K. A.; Katzenellenbogen, B. S.;
Katzenellenbogen, J. A. Indazole estrogens: highly selective ligands
for the estrogen receptor beta. J. Med. Chem. 2005, 48, 1132-1144.
(13) Jorgensen, A. S.; Jacobsen, P.; Christiansen, L. B.; Bury, P. S.;
Kanstrup, A.; et al. Synthesis and estrogen receptor binding affinities
of novel pyrrolo[2,1,5-cd]indolizine derivatives. Bioorg. Med. Chem.
Lett. 2000, 10, 2383-2386.
(33) Sun, J.; Huang, Y. R.; Harrington, W. R.; Sheng, S.; Katzenellen-
bogen, J. A.; et al. Antagonists selective for estrogen receptor alpha.
Endocrinology 2002, 143, 941-947.
(14) Harris, H. A.; Albert, L. M.; Leathurby, Y.; Malamas, M. S.;
Mewshaw, R. E.; et al. Evaluation of an estrogen receptor-beta agonist
in animal models of human disease. Endocrinology 2003, 144, 4241-
4249.
JM061035Y