Estrogen pyrazoles: Defining the pyrazole core structure and the orientation of substituents in the ligand binding pocket of the estrogen receptor

Article abstract of DOI:10.1016/S0968-0896(00)00228-5

Previously, we reported that certain tetrasubstituted 1,3,5-triaryl-4-alkyl-pyrazoles bind to the estrogen receptor (ER) with high affinity (Fink, B. E.; Mortenson, D. S.; Stauffer, S. R.; Aron, Z. D.; Katzenellenbogen, J. A. Chem. Biol. 1999, 6, 205-219;

Full text of DOI:10.1016/S0968-0896(00)00228-5

Bioorganic & Medicinal Chemistry 9 (2001) 141±150
Estrogen Pyrazoles: De®ning the Pyrazole Core Structure and the
Orientation of Substituents in the Ligand Binding Pocket of the
Estrogen Receptor
Shaun R. Stau€er, Ying Huang, Christopher J. Coletta, Rosanna Tedesco
and John A. Katzenellenbogen*
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA
Received 16 May 2000; accepted 15 August 2000
AbstractÐPreviously, we reported that certain tetrasubstituted 1,3,5-triaryl-4-alkyl-pyrazoles bind to the estrogen receptor (ER)
with high anity (Fink, B. E.; Mortenson, D. S.; Stau€er, S. R.; Aron, Z. D.; Katzenellenbogen, J. A. Chem. Biol. 1999, 6, 205±219;
Stau€er, S. R.; Katzenellenbogen, J. A. J. Comb. Chem. 2000, 2, 318±329; Stau€er, S. R.; Coletta, C. J.; Sun, J.; Tedesco, R.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A. J. Med. Chem. 2000, submitted). To investigate how cyclic permutation of the two
nitrogen atoms of a pyrazole might a€ect ER binding anity, we prepared a new pyrazole core isomer, namely a 1,3,4-triaryl-5-alkyl-
pyrazole (2), to compare it with our original pyrazole (1). We also prepared several peripherally matched core pyrazole isomer sets to
investigate whether the two pyrazole series share a common binding orientation. Our ecient, regioselective synthetic route to these
pyrazoles relies on the acylation of a hydrazone anion, followed by cyclization, halogenation, and Suzuki coupling. We found that the
ER accommodates 1,3,4-triaryl-pyrazoles of the isomeric series only somewhat less well than the original 1,3,5-triaryl series, and it
appears that both series share a common binding mode. This preferred orientation for the 1,3,5-triaryl-4-alkyl-pyrazoles is sup-
ported by binding anity measurements of analogues in which the phenolic hydroxyl groups were systematically removed from each
of the three aryl groups, and the orientation is consistent, as well, with molecular modeling studies. These studies provide additional
insight into the design of heterocyclic core structures for the development of high anity ER ligands by combinatorial methods.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
various subpockets that make up the ligand cavity of
ER.3 Interestingly, we found that there were large dif-
ferences in binding anity (up to 50-fold) for ligands
that had identical peripheral substitution patterns but
di€erent core structures (e.g. the two diazoles, imidazoles
versus pyrazoles; Figure 1).3 Clearly, our initial thought
that the ligand core structure might be acting as a passive
entityÐsimply to position peripheral substituentsÐwas
not true. In the case shown below, the lower ER binding
anity for the imidazoles compared to the pyrazoles
was attributed, at least in part, to the signi®cantly
greater dipole moment of the imidazoles.3
The estrogen receptor (ER) binds a remarkably wide
range of non-steroidal ligands,1 and the diverse core
structures of these ligands span a wide range of synthetic
accessibility.2 We have been intrigued, in particular, by the
design of non-steroidal ligand cores that might be easily
prepared and thus would be well suited to combinatorial
expansion. As part of this investigation, we identi®ed
pyrazoles as favorable heterocyclic core building blocks,3
and we found that by attaching a sucient number of
appropriate substituents onto this core system, we could
obtain high anity ligands for the ER.3 5
In further consideration, we wondered whether the dis-
tribution of heteroatoms within the same heterocyclic
system would also have a signi®cant e€ect on ER binding
anity. In the two imidazoles shown in Figure 1, the dif-
ferent position of the nitrogen atoms had little e€ect on
their binding anity. However, we were curious whether
the related cyclic permutation of the two nitrogen atoms
of a pyrazole might have a more signi®cant e€ect on ER
binding anity.
An issue which had interested us initially about these
systems was whether the heterocyclic core structure
itself plays an active role in ligand±receptor interaction,
or whether it acts merely as a inert sca€old, simply dis-
playing groups in a geometry appropriate for ®lling the
*Corresponding author. Tel.: +1-217-333-6310; fax: +1-217-333-7325;
e-mail: jkatzene@uiuc.edu
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00228-5

142
S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
sets, in which some of the phenolic hydroxyl groups are
systematically deleted, to establish more de®nitively
which core isomer is most favored and to investigate
whether they share a common binding orientation.
Results and Discussion
Synthesis of 1,3,4-triaryl-5-alkyl-pyrazoles
Our initial attempts to synthesize the pyrazole isomer 2
involved conditions similar to those used in previous
studies (Scheme 1).3 We ®rst prepared diketone 3 in
high yield, starting from the deoxybenzoin and pro-
pionic anhydride. However, our attempts to generate
the pyrazole by condensation of the dione with 4-meth-
oxyphenylhydrazine were unsuccessful and resulted in a
complex mixture that contained the two possible retro-
Claisen condensation products from cleavage of the 1,3-
dione.
Figure 1. E€ect of di€erent core structures of selected ®ve-member
heterocycles (diazoles) on their estrogen receptor relative binding a-
nity (RBA).
Because of these diculties and the fact that this route
was not likely to be regioselective, we investigated an
alternative, potentially regioselective approach that
involved a one-pot acylation and cyclization procedure
starting from an appropriate hydrazone, as illustrated in
Figure 3. Unfortunately, we could not isolate the
hydrazone (4), because it rapidly underwent Fischer
indole cyclization to the 2,3-diaryl-indole product (5).
Even under mild conditions, we obtained only starting
material or indole product, but no hydrazone. Appar-
ently, the 4-methoxyphenyl substituent in the deoxy-
benzoin starting material facilitates the [3,3]-sigmatropic
cyclization of the ene-phenylhydrazole intermediate by
stabilizing the transition state through a stilbene-like
structure (Fig. 3).
To investigate this issue, we have prepared a new pyrazole
core isomer, namely a 1,3,4-triaryl-5-alkyl-pyrazole (2),
to compare it with our original 1,3,5-triaryl-4-alkyl-
pyrazole (1) (Fig. 2). This new compound (2) represents
the only other possible pyrazole system capable of dis-
playing the same relative substitution pattern of aryl,
alkyl, aryl, and phenyl groups as does our original pyra-
zole 1. To extend this investigation further, we synthesized
several other peripherally matched core pyrazole isomer
We wondered whether we could avoid this competing
cyclization by omitting the 4-methoxyphenyl substituent
during the synthesis of the pyrazole (Fig. 4). Of course,
this approach would entail adding an aryl group later to
the completed heterocycle, but this could presumably be
done using an appropriate Pd(0)-mediated coupling reac-
tion. This synthetic strategy was also attractive because it
allows for the introduction of additional structural diver-
sity into these systems at a late stage, just before phenol
deprotection, an attractive feature for combinatorial
library expansion.5
According to this approach, we were able to successfully
synthesize pyrazole 2 as well as analogues 10a±d (Scheme
2). Initial formation of hydrazones 6a±c by reaction of
Figure 2. Original 1,3,5-triaryl-pyrazole 1 (the basis of Series I) and
the new 1,3,4-triaryl-pyrazole isomer 2 (the basis of Series II).
Scheme 1. Attempted synthesis of MeO-protected pyrazole isomer.

S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
143
To introduce the last aromatic substituent, the tri-sub-
stituted pyrazoles 7a±d were iodinated by treatment with
a solution of KI and I2,7 and then subjected to Suzuki
coupling conditions with either phenylboronic acid or p-
methoxyphenylboronic acid. Initial conditions involved
using Pd(PPh3)4 as a Pd(0) catalyst and DME/H2O as
solvent (9c 52%, 72h); however, by using Pd(OAc)2 as a
pre-catalyst and an n-PrOH/H2O solvent mixture, we
were able to obtain the tetra-substituted pyrazoles
(9a,b,d,e) with slightly improved yields (60±71%) and
shorter reaction times (1±14 h).8 The protected pyrazole
isomers were subsequently demethylated using BBr3 to
a€ord the desired phenolic products 10a±d and 2.
Comparison of the estrogen receptor binding anities of
the isomeric 1,3,4-triaryl-pyrazoles (Series II) and the
original 1,3,5-triaryl-pyrazoles (Series I)
The binding anities of the pyrazoles for ER were
assayed in a competitive radiometric binding assay; this
assay has been described previously,9 and anities are
expressed as relative binding anity (RBA) values,
where estradiol has an anity of 100% (Table 1). The
1,3,4-triaryl-pyrazole 2, which is the nitrogen ring iso-
mer of our original 1,3,5-triaryl-pyrazole 1, has a very
signi®cant anity of 5.8%, but is 2.6-fold less than the
original pyrazole 1. To determine whether the 1,3,4-
triaryl-pyrazole binds to ER in the same mode as pyra-
zole 1, we investigated the binding anity of several
other 1,3,4-triaryl-pyrazoles (10a±d) and compared their
anities with those of their corresponding 1,3,5-triaryl-
pyrazole isomers (11a,b4 and 11c,d10).
Figure 3. Attempted synthesis of pryazole isomer using an acylation±
cyclization.
When examining the ®rst three sets of isomeric pyr-
azoles (the other two are discussed below), the pyrazoles
in the new isomeric Series II (1,3,4-triaryl-pyrazoles)
have somewhat lower anity than those in the original
Series I (1,3,5-triaryl-pyrazoles), but only by an average
of 2-fold. Within the original pyrazole series (Series I),
certain changes to the peripheral substituents resulted in
an increase in RBA. For example, when a hydroxyl
group is added to the N(1)-phenyl substituent (i.e. from
pyrazole 1 to 11a), a slight increase in anity occurs. A
similar increase in RBA upon hydroxyl substitution is
observed in the isomeric Series II, going from pyrazole 2
to 10a.
Previously, we also showed that modi®cation of the
alkyl chain at C(4)-position of the initial pyrazole Series
I from an ethyl 1 to a propyl substituent 11b results in a
1.6-fold increase in anity, indicating a more favorable
hydrophobic interaction. However, this favorable inter-
action ended at propyl, as the n-butyl isomer experi-
enced a signi®cant drop in binding anity.4 In Series II,
going from R3=Et (2) to n-propyl (10b) results in a
similar but somewhat larger increase in anity than it
does in Series I (2.7-fold versus 1.6-fold).
Figure 4. Retro-synthetic strategy for Pd(0)-mediated coupling
approach to pyrazoles.
the acetophenone compounds with either the hydro-
chloride salt of 4-methoxyphenylhydrazine or phe-
nylhydrazine occurred in moderate yields, and as we
had hoped, no competing cyclization to the indole
occurred with this less stabilized system. Hydrazones
6a±c were then treated with butyllithium to form the
corresponding dianion, which was acylated with an alkyl
anhydride and then cyclized upon addition of HCl.6 The
yields shown for pyrazole formation are based on the
anhydride and are typical for this reaction.
Because the structure±binding anity patterns observed
for both pyrazole isomers (Series I and II) are quite
similar, we postulated that these distinct core structure
pyrazole isomers would be binding in the same orienta-
tion in the ER binding pocket.

144
S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
Scheme 2. Synthesis of 1,3,4-triaryl-5-alkyl-pyrazoles.
Table 1. Binding anity data for pyrazole isomers and original pyr-
azole series
on binding anity, suggesting that these rings are not
the ones that correspond to the A-ring of estradiol.)
R1 R2 R3 R4 Compound
(I)
RBA
(I)a
Compound
(II)
RBA
(II)a
The route in Scheme 2 was used to prepare both the
N(1) and the C(4) monophenolic pyrazole Series II
analogues 10c and 10d, regioselectively and in good
yield. The RBA of pyrazole isomer 10c is 0.43%,
whereas that of pyrazole isomer 10d is only 0.008%.
When these anities are compared to that of the
bisphenolic pyrazole 2 (5.8%) (Table 1), one can see
that removal of the phenolic hydroxyl from the N(1)
phenyl ring results in a 725-fold decrease in anity,
whereas removal of the hydroxyl from the C(4) phenyl
group results in only a 13-fold decrease. Thus, the 56-
fold greater decrease in anity that results from delet-
ing the phenolic hydroxyl from the N(1) phenyl group
versus from the C(4) phenyl group strongly suggests
that the N(1)-phenolic substituent in the Series II pyr-
azoles is acting as the A-ring mimic (Fig. 5).
H
OH Et OH
OH OH Et OH
1
15.3Æ3
20.3Æ3
25Æ0.01
0.46
2
5.8Æ0.3
7.8Æ4
11a
11b
11c
11d
10a
10b
10c
10d
H
H
H
OH n-Pr OH
OH Et
Et OH
16.0Æ1
H
0.43Æ0.07
H
0.007
0.008Æ0.005
aCompetitive radiometric binding assays were done using 10 nM
[3H]E2 as tracer and lamb uterine cytosol as receptor source; for
details, see Experimental. Compounds 11a±d were prepared as descri-
bed elsewhere.4,10
Elucidation of the aryl group in the pyrazole that mimics
the A-ring of estradiol: Inferring pyrazole orientation in
the ligand binding pocket from the binding anity of
isomeric monophenols
To further support the hypothesis that both pyrazole
isomers have the same binding mode and to more con-
®dently establish which phenolic group is playing the
role of the A-ring phenol in estradiol, we prepared the
individual monophenolic derivatives of pyrazole 1
(Table 1, Series I, 11c and 11d) and pyrazole 2 (Table 1,
Series II, 10c and 10d). Systematic phenol deletion is a
standard approach that has been used in the past to
determine which phenol in an unsymmetrical bisphenolic
non-steroidal ER ligand mimics the crucial A-ring phenol
of estradiol (E2).11,12 The monophenolic analogue having
the highest anity is then presumed to have preserved
the phenol that is acting as the estradiol A-ring mimic,
because the hydroxyl substituent at this position is known
to be essential for high anity binding to the ER.13 (At
the outset, it is of note that removal of one of the phe-
nols from the R1 position (pyrazole 1 versus 11a in Series
I, and pyrazole 2 versus 10a in Series II) has little e€ect
Initial attempts to prepare the C(3) and C(5) mono-
phenol pyrazole analogues for Series I (11c and 11d)
regioselectively using the acylation±cyclization strategy
were unsuccessful. However, these compounds could
successfully be prepared via the corresponding pyr-
azolines by a new regioselective route (not shown) that
will be described elsewhere.10 The binding anities for
the Series I monophenolic isomers 11d and 11c are
0.007% and 0.46%, respectively. Compared to the cor-
responding bisphenolic pyrazole 1 (15.3%) (Table 1), a
much greater decrease in binding anity results when
the hydroxyl group is removed from C(3) phenol (2185-
fold, 11d) than from the C(5) phenol (33-fold, 11c). The
di€erence in anity loss that results from the alternative
hydroxy group deletion in this series (i.e. 2185/33- or 66-
fold) is comparable to that found in Series II (56-fold).
This result indicates that in Series I the C(3) phenol is

S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
145
Figure 6. A model for pyrazole 10a docked and minimized in the
DES-ERa-LBD. The surface of the ligand is shown in yellow, and the
solvent accessible surface of ER as purple dots.
Figure 5. Preferred orientation and proposed A-ring mimic for Series I
and Series II pyrazoles.
mimicking the A-ring of E2. Thus, because the C(3)
phenol in Series I and the N(1) phenol in Series II are
positioned in a congruent fashion, we believe that the
pyrazoles of both Series I and II share a single common
binding mode, as illustrated in Figure 5. According to
this mode, the other two aryl groups are displayed
within a region of the binding pocket resembling the C/
D region of E2, which is known to tolerate a large
number of substituents.13
In the present study, we have used this approach to
examine the six possible binding orientations of the
ethyl triphenol Series II pyrazole 10a. Again, the orien-
tation in which the N(1) phenol is the mimic of the A-
ring of estradiol (cf. Fig. 5) is well accommodated by the
structure and has a reasonable binding energy, although
an alternative orientation with the C(4) phenol in this
position cannot be ruled out by binding energy con-
siderations. Nevertheless, this latter orientation seems
unlikely, given the much higher experimental binding
anity of monophenol 10c versus 10d (Table 1).
Modeling pyrazole orientation in the ligand binding
pocket
In our original study of pyrazole 1, we used molecular
modeling to show that this compound could bind in an
orientation in which the C(5) phenol mimicked the A-
ring of estradiol;3 this is di€erent from that shown in
Figure 5. However, in this earlier study, we had based
our model on the ERa crystal structure with the
antagonist ligand raloxifene.14 Upon further considera-
tion, we now think that the ERa crystal structure we
used previously was not an appropriate one for model-
ing ligands like pyrazole 1, because unlike raloxifene,
these pyrazoles are ERa agonists, not antagonists.4,15
Thus, on the basis of the experimental determinations of
monophenol binding anities and molecular modeling,
we suggest that the preferred binding modes of the pyr-
azoles in the estrogen receptor correspond to those illu-
strated in Figure 5. A pictorial representation of
pyrazole 10a in this orientation is shown in Figure 6.
Conclusions
To explore the e€ect of core structure on the ability of
pyrazole ligands to bind to ER, we developed an e-
cient, regioselective, and ¯exible route to a new isomeric
series of pyrazoles (1,3,4-triaryl-5-alkyl-pyrazoles), and
we evaluated their binding anity to ER. The synthesis
relies on the acylation of a hydrazone anion, followed,
after cyclization and halogenation, by a Pd(0) Suzuki
coupling strategy. We found that the ER accommodates
1,3,4-triaryl-pyrazoles of the new isomeric series (Series
II) only slightly less well than the original 1,3,5-triaryl
series (Series I), and on the basis of the binding anity
of the corresponding monophenols and molecular
modeling studies, both isomers appear to share a com-
mon binding mode. In this binding mode, the putative
A-ring mimic is the C(3) phenol in Series I and the N(1)
phenol in Series II. Thus, as in the case of the imida-
zoles discussed earlier (Fig. 1), it does appear possible
to permute the position of heteroatoms in the azole
ring without having a major e€ect on the ER binding
We have recently completed a more extensive modeling of
a pyrazole triphenol that corresponds to pyrazole diphe-
nol 11b,4 using as a starting point an X-ray structure of
ERa complexed with the agonist diethylstilbestrol, a non-
steroidal estrogen agonist with an RBA of 98%.16 In
this study, we considered six possible starting orienta-
tions (the three phenols placed in the A-ring pocket and
in each case the two alternative orientations of the
remaining two phenols that come from rotation about
the bond linking the A-ring phenol and the pyrazole
core). From this recent study,4 the orientation in which
the C(3) phenol of the Series I pyrazole mimics the A-
ring of estradiol gave the lowest energy structure and
appeared to be quite reasonable on steric grounds,
although it is dicult to rigorously eliminate some of
the other possible orientations. This is the orientation
shown in Figure 5 for the Series I pyrazole.

146
S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
anity, provided that the peripheral substituents
remain disposed with the same geometry and provided
that one remains in the same azole series (i.e. 1,2-azoles
[pyrazoles] not 1,3-azoles [imidazoles]), so that com-
pounds with equivalent dipole moments and polarities
are being compared. These studies provide additional
insight into the design of heterocyclic core structures for
the development of high anity ER ligands by combi-
natorial methods.
Molecular modeling of pyrazole 10a
The protocol for modeling followed that recently
described for a related pyrazole triphenol.4 The starting
conformation for pyrazole 10a used for receptor dock-
ing studies was generated from a random conformational
search performed using the MMFF94 force ®eld as
implemented in Sybyl 6.6. The resulting lowest energy
conformer was then used for docking studies. Charge
calculations were determined using the MMFF94
method and molecular surface properties displayed
using MOLCAD module in Sybyl 6.6.
Experimental
General
Pyrazole 10a, generated as noted above, was pre-posi-
tioned in the DES-ERa-LBD crystal structure16 using a
least squares multi®tting of select atoms within the DES
ligand. Once pre-positioned, DES was deleted and
ligand 10a was optimally docked in the ERa binding
pocket in six orientations (as speci®ed in the text) using
the Flexidock routine within Sybyl (Tripos). Both
hydrogen-bond donors and acceptors within the pocket
surrounding the ligand (Glu353, Arg394, and His524), the
ligand itself and select torsional bonds were de®ned.
The docked receptor ligand complexes from Flexidock
then underwent a three step minimization. First, non-
ring torsional bonds of the ligand were minimized in the
context of the receptor using the torsmin command.
This was followed by minimization of the side chain
residues within 8 A of the ligand, while holding the
backbone and residues Glu353 and Arg394 ®xed. A ®nal
third minimization of both the ligand and receptor was
conducted using the Anneal function (hot radius 8 A,
interesting radius 16 A from pyrazole 10a) to a€ord the
®nal model.
Melting points were determined on a Thomas-Hoover
UniMelt capillary apparatus and are uncorrected. All
reagents and solvents were obtained from Aldrich,
Fisher or Mallinckrodt. Tetrahydrofuran was freshly
distilled from sodium/benzophenone. Dimethylforma-
mide was vacuum distilled prior to use, and stored over
4 A molecular sieves. n-Butyllithium and t-butyllithium
were titrated with N-pivaloyl-o-toluidine. Et3N was
stirred with phenylisocyanate, ®ltered, distilled, and
stored over 4 A molecular sieves. All reactions were
performed under a dry N2 atmosphere unless otherwise
speci®ed. Reaction progress was monitored by analy-
tical thin-layer chromatography using GF silica plates
purchased from Analtech. Visualization was achieved
by short wave UV light (254 nm) or potassium perman-
ganate. Flash column chromatography was performed
using Woelm 32±63 mm silica gel packing.17
1H and 13C NMR spectra were recorded on either a
Varian Unity 400 MHz or 500 MHz spectrometer using
CDCl3, MeOD or (CD3)2SO as solvent. Chemical
shifts were reported as parts per million down®eld
from an internal tetramethylsilane standard (d 0.0 for
1H) or from solvent references. NMR coupling con-
stants are reported in Hertz. 13C NMR spectra were
determined using either the Attached Proton Test
(APT) or standard 13C pulse sequence parameters. Low
resolution and high resolution electron impact mass
spectra were obtained on Finnigan MAT CH-5 or 70-
VSE spectrometers. Elemental analyses were performed
by the Microanalytical Service Laboratory of the Uni-
versity of Illinois. Hydrazones 6a±c are prone to
decomposition and therefore could only be stored for 2±
3 days at 0 ꢀC. Once the hydrazone products were con-
Chemical syntheses
5-Ethyl-1,4-bis-(4-methoxyphenyl)-3-phenyl-1H-pyrazole
(2). A stirred solution of 9e (46 mg, 0.16 mmol) in
CH2Cl2 (15 mL) was treated with BBr3 (1.62 mL,
1.62 mmol) according to the general demethylation pro-
cedure. After work up and an SiO2 plug (30% EtOAc/
hexanes) a white solid was isolated (40 mg, 94%): mp
220±225 ꢀC; 1H NMR (MeOD-d4, 400 MHz) d 0.90 (t, 3H,
J=7.5), 2.62 (q, 2H, J=7.5), 6.79 (XX0 of AA0XX0, 2H,
JAX=8.3, JXX=2.3), 6.94 (XX0 of AA0XX0, 2H, JAX
=
8.6, JXX=2.7), 7.04 (AA0 of AA0XX00, 2H, JAX=8.2,
0
0
0
JAA =2.4), 7.20±7.27 (m, 2H), 7.34 (AA of AA XX , 2H,
JAX=8.8, JAA =2.6), 7.37±7.44 (m, 3H); 13C NMR
0
1
®rmed by H NMR they were typically used directly in
the acylation±cyclization step without further char-
acterization.
(MeOD-d4, 100 MHz) d 12.4, 17.2, 114.8, 115.2, 118.5,
124.3, 126.9, 127.4, 127.5, 127.6, 131.0, 131.0, 132.9,
144.1, 148.9, 156.2, 157.8; MS (EI, 70 eV) m/z (relative
intensity, %): 356 (M+, 100); Anal calcd for C23H20
.
N2O2 H2O: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.82;
H, 5.95; N, 7.69.
Biological procedures: relative binding anity assay
Ligand binding anities (RBAs) using lamb uterine
cytosol as a receptor source were determined by a com-
petitive radiometric binding assay using 10 nM
[3H]estradiol as tracer and dextran-coated charcoal as
an adsorbent for free ligand.9 All incubations were done
at 0 ꢀC for 18±24 h. Binding anities are expressed
relative to estradiol (RBA=100%) and are reproducible
with a coecient of variation of 0.3.
40-Methoxyacetophenone-4-methoxyphenylhydrazone (6a).
An EtOH (40 mL) solution of 4-methoxyacetophenone
(1 g, 6.67 mmol), sodium acetate (1.09 g, 13.34 mmol),
and 4-methoxyphenylhydrazine hydrochloride (1.74g,
10.0 mmol) was heated to 80 ꢀC for 3.0 h. The reaction
mixture was cooled to room temperature and concentrated
under reduced pressure. The residue was dissolved in ethyl

S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
147
acetate (30 mL) and washed with water (40 mL) and brine
(2Â40mL). After drying over MgSO4 and solvent con-
centration a red and white heterogeneous solid formed.
The product was collected by ®ltration and rinsed with
cold ethanol (20 mL) to a€ord 6a as a white solid (1.18 g,
66%) which was used directly in the next step.
3.83 (s, 3H) 6.45 (s, 1H), 3.85 (s, 3H), 6.98 (XX0 of
AA0XX0, 2H, JAX=8.9, JXX=2.4), 7.09 (XX0 of AA0
XX0, 2H, JAX=9.0, JXX=2.7), 7.39 0(AA0 of AA0XX0,
0
0
0
2H, JAX=8.9, JAA =2.7), 7.79 (AA of AA XX , 2H,
JAX=8.9, JAA =2.8); 13C NMR (CDCl3, 100 MHz) d
0
13.0, 19.6, 55.1, 55.4, 101.2, 113.7, 114.1, 126.2, 126.8,
126.8, 133.0, 146.5, 150.8, 158.9, 159.1.
Acetophenone-4-methoxyphenylhydrazone (6b). A solution
of acetophenone (250 mg, 2.00 mmol) and 4-methoxy-
phenylhydrazine hydrochloride (349 mg, 2.00 mmol)
was reacted similarly to conditions used for 6a to a€ord
a red and white heterogeneous sold. Cold ethanol
(20 mL) was added and the remaining solid collected via
vacuum ®ltration to a€ord 6b as a white solid (1.41 g,
59%) which was used directly in the next step.
1-(4-Methoxyphenyl)-3-phenyl-5-propyl-1H-pyrazole (7b).
Following the general procedure above hydrazone 6a
(1.37 g, 5.71 mmol) was acylated with butyric anhydride
(452 mg, 0.47 mL, 2.86 mmol) and cyclized with HCl.
Flash chromatography (30% EtOAc/hexanes) a€orded
1
the title compound as a yellow oil (710 mg, 42%): H
NMR (CDCl3, 400 MHz) d 0.96 (t, 3H, J=7.5) 1.66
(sext, 2H, J=7.5), 2.61 (t, 2H, J=7.7), 3.85 (s, 3H), 6.54
(s, 1H), 6.99 (XX0 of AA0XX0, 2H, JAX=8.7, JXX=2.7),
7.31 (app tt, 1H, J=7.3, 1.5), 7.38±7.43 (m, 4H), 7.89
Acetophenonephenylhydrazone (6c). A solution of aceto-
phenone (1.8 g, 15 mmol), phenylhydrazine hydrochlo-
ride (2.17 g, 15 mmol), and sodium acetate (1.23 g,
15.0 mmol), in anhydrous ethanol (40 mL), was reacted
similarly to conditions used for 6a to a€ord a yellow
and white heterogeneous solid. Cold ethanol (20 mL)
was added to the solid and the product collected via
vacuum ®ltration to a€ord 6c as a white solid (1.67 g,
53%) which was used directly in the next step.
(AA0 of AA0XX0, 2H, JAX=7.2, JAA =1.3); 13C NMR
0
(CDCl3, 100 MHz) d 13.7, 21.9, 27.1, 55.4, 102.2, 114.1,
125.5, 126.9, 127.5, 128.4, 132.9, 133.4, 145.2, 150.9,
159.1; HRMS (EI, M+) calcd for C19H20N2O: 292.1576.
Found: 292.1569.
5-Ethyl-1-(4-methoxyphenyl)-3-phenylpyrazole (7c). Fol-
lowing the general procedure above hydrazone 6b
(4.50 g, 18.75 mmol) was acylated with propionic anhy-
dride (1.22 g, 1.20 m L, 9.38 mmol) and cyclized with
HCl. Upon solvent removal a crude orange solid was
isolated. The crude solid was recrystallized from 20%
ethyl acetate/hexanes to a€ord the title compound as
pale yellow needles (1.82 g, 35%): mp 70±75 ꢀC; 1H
NMR (CDCl3, 400 MHz) d 1.25 (t, 3H, J=7.5), 2.65 (q,
2H, J=7.5), 6.53 (s, 1H), 3.86 (s, 3H), 6.99 (AA0XX0,
2H, J=8.6, 2.6), 7.28±7.33 (m, 1H), 7.37±7.43 (m, 4H),
7.85±7.90 (m, 2H); 13C NMR (CDCl3, 125 MHz) d 13.0,
19.6, 55.4, 101.6, 114.2, 125.5, 126.8, 127.5, 128.4, 132.9,
133.3, 146.6, 151.0, 159.0; MS (EI, 70 eV) m/z (relative
intensity, %): 278 (M+, 100). Anal. calcd for C18H18
N2O: C, 77.67; H, 6.52; N, 10.06. Found: C, 77.62; H,
6.49; N, 10.05.
General acylation±cyclization procedure (7a±d). To a
stirred solution of hydrazone (3.05 mmol) in THF (10
mL) at 0 ꢀC was added 1.29 M BuLi (4.73 mL,
6.10 mmol) dropwise. The resulting deep red solution
was allowed to stir at 0 ꢀC for 0.25 h, then room tem-
perature for 0.25 h, and re-cooled to 0 ꢀC, whereupon a
THF solution (2.0 mL) of the appropriate alkyl anhy-
dride (1.53 mmol) was added dropwise. This mixture
was stirred at 0 ꢀC for 0.25 h, then treated with 3 M HCl
(6 mL, 18 mmol) and re¯uxed for 1.5 h. The biphasic
mixture was cooled to room temperature and the aqu-
eous layer separated and neutralized with saturated
NaHCO3. The neutralized solution was extracted with
ethyl acetate (3Â20 mL) and the organic layers com-
bined with the THF layer from the reaction mixture.
The ®nal organic layers were washed with satd
NaHCO3 (3Â20 mL), dried over MgSO4, and con-
centrated to a€ord a red oil. The crude oil was puri®ed
by ¯ash chromatography.
1,3-Diphenyl-5-propyl-1H-pyrazole (7d). Following the
general procedure above hydrazone 6c (1.30 g, 6.19mmol)
was acylated with propionic anhydride (403 mg, 0.40
mL, 3.10 mmol) and cyclized with HCl. Flash chroma-
tography (20% diethyl ether/hexanes) a€orded the title
compound as a yellow oil (600 mg, 40%): 1H NMR
(CDCl3, 500 MHz) d 1.28 (t, 3H, J=7.4), 2.72 (q, 2H,
J=7.4), 6.58 (s, 1H), 7.32±7.53 (m, 8H), 7.91 (d, 2H,
J=7.5); 13C NMR (CDCl3, 100 MHz) d 13.2, 19.9,
102.3, 125.7, 127.8, 127.9, 128.8, 129.1, 133.4, 140.0,
146.7, 151.5; HRMS (EI, M+) calcd for C17H16N2:
248.1313. Found: 248.1312.
5-Ethyl-1,3-bis-(4-methoxyphenyl)-1H-pyrazole (7a).
Following the general procedure above hydrazone 6a
(824 mg, 3.05 mmol) was acylated with propionic anhy-
dride (198 mg, 0.20 mL, 1.53 mmol) and cyclized with HCl.
Flash chromatography (25% EtOAc/hexanes) a€orded an
inseparable mixture of 4-methoxyacetophenone and 7a
(474 mg). The phenone was reduced by the dropwise
addition of a solution of NaBH4 (27.3 mg, 0.72) in H2O
(5 mL) to a solution of the product mixture in ethanol
(5 mL). The solution was stirred for 18 h at room tem-
perature and poured over 1 M HCl (20 mL). Upon
product isolation (EtOAc, satd NaHCO3, brine) and
solvent removal under reduced pressure an orange oil
was isolated, which upon addition of 25% EtOAc/hex-
anes resulted in selective crystallization of pyrazole 7a
General iodination procedure (8a±d). To a re¯uxing
solution of pyrazole (0.59 mmol) and sodium acetate
(107 mg, 1.13 mmol) in H2O (4.00 mL) was added a
solution of KI (591 mg, 3.56 mmol) and I2 (301 mg,
1.19 mmol) in H2O (4 mL) dropwise. The resulting dark
brown solution was allowed to re¯ux for 3 h and then
was cooled to room temperature and the product
extracted with diethyl ether (3Â25 mL). The organic
1
as ®ne white crystals (200 mg, 44%): H NMR (CDCl3,
400 MHz) d 1.24 (t, 3H, J=7.6), 2.64 (q, 2H, J=7.5),

148
S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
layers were combined, washed with NaS2O2 (3Â20 mL),
NaHCO3 (2Â20 mL), brine (2Â20 mL) and then dried
over MgSO4 and concentrated under reduced pressure
to a€ord the crude iodo-pyrazoles. The products were
puri®ed by recrystallization from hexanes or by ¯ash
chromatography on silica gel.
H2O (0.5 mL). The heterogeneous solution was heated
to re¯ux for 5 h, cooled to room temperature, and ®l-
tered through Celite (EtOAc 4Â5 mL). The ®ltrate was
concentrated and partitioned in diethyl ether and water
and the aqueous layer separated and extracted twice
more with ether. The combined organic layers were
washed with brine, dried over MgSO4 and concentrated
under reduced pressure. The tetrasubstituted pyrazoles
were subsequently puri®ed by ¯ash chromatography.
5-Ethyl-4-iodo-1,3-bis-(4-methoxyphenyl)-1H-pyrazole
(8a). Pyrazole 7a (153 mg, 0.59 mmol) was iodinated
according to the general procedure above. A crude tan
solid was isolated and recrystallized from hexanes to
a€ord the title compound as a white solid (224 mg,
87%): 1H NMR (CDCl3, 400 MHz) d 1.13 (t, 3H,
J=7.4), 2.73 (q, 2H, J=7.5), 3.84 (s, 3H), 3.85 (s, 3H),
6.94±7.02 (m, 4H), 7.36 (AA0XX0, 2H, J=9.0, 2.7), 7.83
(AA0XX0, 2H, J=8.8, 2.5); 13C NMR (CDCl3,
125 MHz) d 13.1, 20.1, 55.1, 55.4, 60.1, 113.4, 114.2,
125.4, 126.9, 129.4, 132.6, 147.2, 151.4, 159.4, 159.5.
5-Ethyl-1,3,4-tris-(4-methoxyphenyl)-1H-pyrazole (9a).
Iodo-pyrazole 8a (187 mg, 0.43 mmol) was coupled with
4-methoxyphenylboronic acid (68 mg, 0.45 mmol)
according to the general procedure above to a€ord a
crude brown oil. Flash chromatography (40% ethyl
acetate/hexanes) furnished the title compound as a clear
1
oil (124 mg, 70%): H NMR (CDCl3, 400 MHz) d 0.93
(t, 3H, J=7.5), 2.63 (q, 2H, J=7.5), 3.77 (s, 3H), 3.85
(s, 3H), 3.87 (s, 3H), 6.78 (AA0XX0, 2H, J=8.9, 2.5),
6.93 (AA0XX0, 2H, J=8.7, 2.5), 7.01 (AA0XX0, 2H,
J=8.9, 2.6), 7.20 (AA0XX0, 2H, J=8.6, 2.5), 7.43
(AA0XX0, 2H, J=8.8, 2.4), 7.46 (AA0XX0, 2H, J=8.9,
2.6); 13C NMR (CD3OD, 100 MHz) d 13.7, 17.8, 54.9,
55.0, 55.4, 113.3, 113.8, 114.1, 125.9, 126.4, 127.1, 127.4,
128.9, 131.3, 133.1, 143.5, 148.7, 158.3, 158.7, 159.1;
HRMS (EI, M+) calcd for C26H26N2O3: 414.1943.
Found: 414.1937.
4-Iodo-1-(4-methoxyphenyl)-3-phenyl-5-propylpyrazole
(8b). Pyrazole 7b (660 mg, 2.26 mmol) was iodinated
according to the general procedure above. A crude oil
was isolated and puri®ed by ¯ash chromatography
(25% ethyl acetate/hexanes) to a€ord a mixture of
unreacted pyrazole 7b and product 8b as a pale-yellow
oil (yield as determined by NMR: 65%; resonances lis-
1
ted for product only): H NMR (CDCl3, 400 MHz) d
0.90 (t, 3H, J=7.4) 1.56 (sext, 2H, J=7.6), 2.70 (t, 2H,
J=7.8), 3.86 (s, 3H), 6.990(XX0 of0AA00XX0, 2H, JAX
=
8.7, JXX =2.8), 7.90 (AA of AA XX , 2H, JAX=8.3,
1,4-Bis-(4-methoxyphenyl)-3-phenyl-5-propyl-1H-pyra-
zole (9b). Iodo-pyrazole 8b (300 mg, 0.72 mmol) was
coupled with 4-methoxyphenylboronic acid (115 mg,
0.75 mmol) according to the general procedure above.
Flash chromatography (25% EtOAc/hexanes) a€orded
0
JAA =1.6), 7.26±7.46 (m, 5H); 13C NMR (CDCl3, 100
0
MHz) d 13.8, 21.9, 28.4, 55.4, 60.9, 114.2, 127.1, 128.0,
128.0, 128.2, 132.8, 146.2, 151.5, 159.5; HRMS (EI,
M+) calcd for C19H19N2OI: 418.0542. Found: 418.0550.
1
the title compound as a glassy solid (203 mg, 71%): H
NMR (CDCl3, 500 MHz) d 0.70 (t, 3H, J=7.4), 1.31
(sext, 2H, J=7.6), 2.59 (t, 2H, J=7.9), 3.84 (s, 3H), 3.86
(s, 3H), 6.92 (AA0XX0, 2H, J=8.4, 2.4), 7.00 (AA0XX0,
2H, J=8.5, 2.5), 7.16±7.27 (m, 5H), 7.45 (AA0XX0, 2H,
J=8.7, 2.4), 7.48±7.54 (m, 2H); 13C NMR (MeOD-d4,
100 MHz) d 13.8, 22.2, 26.5, 55.2, 55.5, 113.9, 114.3,
114.6, 126.4, 127.2, 127.3, 127.9, 128.1, 131.5, 133.3,
133.4, 142.6, 149.0, 158.5, 159.3; HRMS (EI, M+) calcd
for C26H27N2O2: 398.1994. Found: 398.1991.
5-Ethyl-4-iodo-1-(4-methoxyphenyl)-3-phenyl-1H-pyra-
zole (8c). Pyrazole 7c (500mg, 1.80mmol) was iodinated
according to the general procedure above to a€ord the
title compound as a white solid (603mg, 83%): mp 70±
73 ꢀC; 1H NMR (CDCl3, 500 MHz) d 1.14 (t, 3H,
J=7.5), 2.74 (q, 2H, J=7.5), 3.86 (s, 3H), 6.99 (AA0
XX0, 2H, J=8.6, 2.9), 7.34±7.47 (m, 5H), 7.86±7.92 (m,
2H); MS (EI, 70 eV) m/z (relative intensity, %): 404
(M+, 100). Anal. calcd for C18H17N2OI: C, 53.48; H,
4.24; N, 6.93. Found: C, 53.66; H, 4.24; N, 6.89.
5-Ethyl-1,4-bis-(4-methoxyphenyl)-3-phenyl-1H-pyrazole
(9c). To a degassed solution of 2 M Na2CO3 (0.55 mL,
1.09 mmol; 0.5 mL H2O), and DME (3.0 mL) was added
4-methoxyphenylboronic acid (83 mg, 0.55 mmol) and
pyrazole 8c (200 mg, 0.45 mmol). To this solution
Pd(Ph3P)4 (27 mg, 0.025 mmol) was added and the mix-
ture heated to 80 ꢀC for 72 h. The reaction mixture was
then cooled to room temperature and ®ltered through
Celite. The ®ltrate was transferred to a separatory funnel
and the aqueous layers extracted with Et2O (3Â5 mL).
The organic layers were combined and concentrated
under reduced pressure. Flash chromatography (25%
EtOAc/hexanes) a€orded 9c as a white solid (100 mg,
53%): 1H NMR (CDCl3, 400 MHz) d 0.93 (t, 3H,
J=7.5), 2.64 (q, 2H, J=7.6), 3.85 (s, 3H), 3.86 (s, 3H),
6.93 (AA0XX0, 2H, J=8.7, 2.4), 7.00 (AA0XX0, 2H,
J=9.2, 2.7), 7.18±7.27 (m, 5H), 7.46 (AA0XX0, 2H,
J=9.0, 2.7), 7.49±7.53 (m, 2H); HRMS (EI, M+) calcd
for C25H24N2O3: 384.1834. Found: 384.1838.
5-Ethyl-4-iodo-1,3-diphenyl-1H-pyrazole (8d). Pyrazole
7d (248 mg, 1.13 mmol) was iodinated according to the
general procedure above to a€ord a crude orange solid.
Recrystallization from hexanes a€orded 8d as small
white crystals (312 mg, 74%): mp 82±85 ꢀC; H NMR
1
(CDCl3, 500 MHz) d 1.61 (t, 3H, J=7.5), 2.78 (q, 2H,
J=7.5), 7.35±7.55 (m, 8H), 7.86±7.91 (m, 2H); 13C
NMR (CDCl3, 125 MHz) d 13.3, 20.3, 61.1, 125.6,
128.2, 128.3, 128.4, 128.6, 129.3, 132.9, 139.9, 147.3,
152.1; HRMS (EI, M+) calcd for C17H15N2I: 374.0280.
Found: 374.0277.
General procedure for Suzuki coupling (9a,b,d,e). To a
stirred solution of iodo-pyrazole (0.43 mmol) in n-propa-
nol (2mL) were added the appropriate boronic acid
(0.45 mmol), Pd(OAc)2 (3 mg, 0.013 mmol), PPh3 (10 mg,
0.039 mmol), 2 M Na2CO3 (0.47 mL, 0.95 mmol), and

S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
149
5-Ethyl-1-(4-methoxyphenyl)-3,4-diphenyl-1H-pyrazole
(9d). Iodo-pyrazole 8c (400 mg, 0.99 mmol) was cou-
pled with phenylboronic acid (144 mg, 1.04 mmol)
according to the general procedure above to a€ord a
crude brown oil. Flash chromatography (15% THF/
hexanes) furnished the product and a mixture of the
product and unreacted starting material. The mixture
was treated to a second column under the same condi-
tions to a€ord additional 9d (50 mg combined, 14%
isolated yield; 217 mg for remaining mixture, 60% total
J=8.4), 6.93 (d, 2H, J=8.8), 7.03 (d, 2H, J=8.8), 7.21
(d, 2H, J=8.4), 7.32 (d, 2H, J=8.8); 13C NMR
(MeOD-d4, 100 MHz) d 12.4, 17.2, 114.3, 114.8, 115.1,
118.0, 124.2, 124.6, 127.4, 128.9, 131.0, 131.2, 143.9,
149.1, 156.0, 156.6, 157.7; HRMS (EI, M+) calcd for
C23H20N2O3: 372.1474. Found: 372.1468.
1,4-Bis-(4-hydroxyphenyl)-3-phenyl-5-propyl-1H-pyrazole
(10b). A stirred solution of 9b (100 mg, 0.25 mmol) in
CH2Cl2 (10 mL) was treated with BBr3 (2.50 mL,
2.50 mmol) according to the general demethylation pro-
cedure. After work up a red solid was isolated. The solid
was recrystallized from 10% CH3OH/CHCl3 to a€ord
10b as small white crystals (75 mg, 81%): mp 233±
1
yield as determined by NMR): H NMR (MeOD-d4,
500 MHz) d 0.94 (t, 3H, J=7.4), 2.66 (q, 2H, J=7.5),
7.02 (AA0XX0, 2H, J=8.9, 2.7), 7.17±7.53 (m, 12H); 13
C
NMR (MeOD-d4, 100 MHz) d 13.7, 17.8, 55.4, 114.1,
118.7, 126.6, 127.2, 127.9, 127.9, 128.3, 130.2, 132.8,
133.1, 133.9, 143.7, 148.8, 159.3.
236 ꢀC; H NMR (MeOD-d4, 400 MHz) d 0.67 (t, 3H,
1
J=7.3), 1.29 (sext, 2H, J=7.5), 2.59 (t, 2H, J=7.9),
6.79 (AA0XX0, 2H, J=8.4, 2.2), 6.94 (AA0XX0, 2H,
J=8.6, 2.5), 7.03 (AA0XX0, 2H, J=8.3, 2.1), 7.20±7.26
(m, 3H), 7.31±7.43 (m, 4H); 13C NMR (MeOD-d4,
100 MHz) d 12.4, 21.5, 25.9, 114.9, 115.2, 119.1, 124.4,
127.0, 127.4, 127.5, 127.6, 131.1, 131.2, 132.9, 142.8,
148.9, 156.2, 157.8; HRMS (EI, M+) calcd for
C24H22N2O2: 370.1681. Found: 370.1685.
5-Ethyl-4-(4-methoxyphenyl)-1,3-diphenyl-1H-pyrazole
(9e). Iodo-pyrazole 8d (244mg, 0.65mmol) was coupled
with 4-methoxyphenylboronic acid (105mg, 0.69mmol)
according to the general procedure above to a€ord a yel-
low oil. Flash chromatography (15% EtOAc/hexanes)
was performed to a€ord a pale yellow oil which was
then recrystallized from hexanes to a€ord the title com-
pound as white crystals (160 mg, 70%): 1H NMR
(MeOD-d4, 500 MHz) d 0.94 (t, 3H, J=7.5), 2.70 (q,
2H, J=7.5), 3.85 (s, 3H), 6.94 (AA0XX0, 2H, J=8.8,
2.6), 7.19±7.28 (m, 5H), 7.43 (app tt, 1H, J=7.4, 1.7),
7.48±7.55 (m, 4H), 7.55±7.60 (m, 2H); 13C NMR
(MeOD-d4, 125 MHz) d 13.7, 17.9, 55.1, 113.8, 118.8,
125.7, 126.1, 127.2, 127.8, 127.9, 129.0, 131.3, 133.2,
140.0, 143.6, 149.3, 158.4.
5-Ethyl-1-(4-hydroxyphenyl)-3,4-diphenyl-1H-pyrazole
(10c). A stirred solution of 9c (37 mg, 0.10 mmol) in
CH2Cl2 (5 mL) was treated with BBr3 (1.00 mL, 1.00
mmol) according to the general demethylation proce-
dure. After work up a brown oil was a€orded. The
crude oil was puri®ed by ¯ash chromatography (5%
CH3OH/CH2Cl2) to a€ord 10c as a white solid (30 mg,
86%): mp 210±220 ꢀC; H NMR (MeOD-d4, 400 MHz)
1
d 0.90 (t, 3H, J=7.60 Hz), 2.65 (q, 2H, J=7.4), 6.95
(AA0XX0, 2H, J=9.1, 2.7), 7.19±7.26 (m, 5H), 7.31±7.42
(m, 7H); 13C NMR (MeOD-d4, 100 MHz) d 13.8, 17.8,
116.5, 118.8, 126.8, 127.5, 127.6, 128.1, 128.1, 128.5,
130.3, 132.1, 132.9, 133.9, 144.2, 149.1, 156.5; HRMS
(EI, M+) calcd for C23H20N2O: 340.1576. Found:
340.1576.
General procedure for demethylation using BBr3 (2, 10a±d).
To a stirred solution of pyrazole (9a±e, 0.28 mmol) in
CH2Cl2 (10 mL) at 78 ꢀC was added BBr3 (2.82 mL,
2.82 mmol) dropwise as a 1 M solution in CH2Cl2. The
mixture was allowed to warm to room temperature and
stirred for 3 h. The reaction was quenched at 0 ꢀC by the
addition of H2O (10 mL). The resulting solid was solu-
bilized by the addition of ethyl acetate and the resulting
biphasic solution transferred to a separatory funnel and
the aqueous layer isolated. The aqueous layer was acid-
i®ed with 3 M HCl (3 mL) and extracted with ethyl
acetate (2Â15 mL). The combined organic layers were
washed with brine and dried over MgSO4. The solvent
was removed under reduced pressure and the phenolic
products puri®ed by ¯ash chromatography and/or crys-
tallization.
5-Ethyl-4-(4-hydroxyphenyl)-1,3-diphenyl-1H-pyrazole
(10d). A stirred solution of 9e (100 mg, 0.28 mmol) in
CH2Cl2 (10 mL) was treated with BBr3 (2.82 mL,
2.82 mmol) according to the general demethylation pro-
cedure. After work up a crude brown solid was isolated.
The solid was puri®ed by ¯ash chromatography (5%
CH3OH/CH2Cl2) to a€ord 10d as a white solid (89 mg,
1
93%): H NMR (MeOD-d4, 500 MHz) d 0.93 (t, 3H,
J=7.5), 2.68 (q, 2H, J=7.6), 6.78 (XX00 of XX0AA0, 2H,
0
0
0
JAX=8.4, JXX =2.5), 7.10 (AA of AA XX , 2H, JAX
=
0
5-Ethyl-1,3,4-tris-(4-hydroxyphenyl)-1H-pyrazole (10a).
A stirred solution of 9a (46 mg, 0.11 mmol) in CH2Cl2
(5 mL) was treated with BBr3 (1.10 mL, 1.10 mmol)
according to the general demethylation procedure. After
work up a reddish brown oil isolated. The oil was dis-
solved in methanol and concentrated under reduced
pressure. No e€ort was made to remove trace amounts
of methanol remaining in the sample. The residue was
triturated by adding CH2Cl2 dropwise until a precipitate
formed. The precipitate was isolated by vacuum ®ltra-
8.4, JAA =2.5), 7.21±7.28 (m, 3H), 7.40±7.52 (m, 6H),
7.53±7.58 (m, 2H); 13C NMR (MeOD-d4, 100 MHz) d
13.7, 17.9, 115.6, 119.1, 125.8, 125.9, 127.4, 128.0, 128.1,
128.2, 129.2, 131.5, 133.0, 139.8, 143.9, 149.5, 154.9;
HRMS (EI, M+) calcd for C23H20N2O: 340.1576.
Found: 340.1571.
Acknowledgements
1
tion to a€ord 10a as a white solid (40 mg, 93%): H
NMR (MeOD-d4, 400 MHz) d 0.89 (t, 3H, J=7.6) 2.59
(q, 2H, J=7.5), 6.65 (d, 2H, J=8.4), 6.79 (d, 2H,
We are grateful for support of this research through
grants from the US Army Breast Cancer Research Pro-
gram (DAMD17-97-7076) and the National Institutes of

150
S. R. Stau€er et al. / Bioorg. Med. Chem. 9 (2001) 141±150
Health (PHS 5R37 DK15556 and T32 CA 09067
(Training Grant for Y. H.)). We thank Kathryn E.
Carlson for performing binding assays and for helpful
comments. NMR spectra were obtained in the Varian
Oxford Instrument Center for Excellence in NMR
Laboratory. Funding for this instrumentation was pro-
vided in part from the W. M. Keck Foundation and the
National Science Foundation (NSF CHE 96-10502).
Mass spectra were obtained on instruments supported
by grants from the National Institute of General Medi-
cal Sciences (GM 27029), the National Institutes of
Health (RR 01575), and the National Science Founda-
tion (PCM 8121494).
5. Stau€er, S. R.; Katzenellenbogen, J. A. J. Comb. Chem.
2000, 2, 318.
6. Foote, R. S.; Beam, C. F.; Hauser, C. R. Heterocycle
Chem. 1970, 7, 589.
7. Holzer, W.; Gruber, H. Heterocycle Chem. 1995, 32, 1351.
8. Hu€, B. E.; Koenig, T. M.; Staszak, M. A. Org. Synth.
1998, 75, 53.
9. Katzenellenbogen, J. A.; Johnson, H. J. Jr; Myers, H. N.
Biochemistry 1973, 12, 4085.
10. Huang, Y.; Katzenellenbogen, J. A. Org. Lett. 2000, 2,
2833.
11. Anstead, G. M.; Wilson, S. R.; Katzenellenbogen, J. A. J.
Med. Chem. 1989, 32, 2163.
12. Anstead, G. M.; Peterson, C. S.; Katzenellenbogen, J. A.
J. Steroid Biochem. 1989, 33, 877.
13. Anstead, G. M.; Carlson, K. E.; Katzenellenbogen, J. A.
Steroids 1997, 62, 268.
References and Notes
14. Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R.
E.; Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G.L.; Gus-
tafsson, J.-A.; Carlquist, M. Nature 1997, 389, 753.
15. Sun, J.; Meyers, M. J.; Fink, B. E.; Rajendran, R.; Kat-
zenellenbogen, J. A.; Katzenellenbogen, B. S. Endocrinology
1999, 140, 800.
16. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.;
Kushner, P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95, 927.
17. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923.
1. Gao, H.; Katzenellenbogen, J. A.; Garg, R.; Hansch, C.
Chem. Rev. 1999, 99, 723.
2. Magarian, R. A.; Overacre, L. B.; Singh, S.; Meyer, K. L.
Curr. Med. Chem. 1994, 1, 61.
3. Fink, B. E.; Mortensen, D. S.; Stau€er, S. R.; Aron, Z. D.;
Katzenellenbogen, J. A. Chem. Biol. 1999, 6, 205.
4. Stau€er, S. R.; Coletta, C. J.; Sun, J.; Tedesco, R.; Katze-
nellenbogen, B. S.; Katzenellenbogen, J. A. J. Med. Chem.
2000, submitted.