Structure-activity relationship study to understand the estrogen receptor-dependent gene activation of aryl- and alkyl-substituted 1H-imidazoles

Article abstract of DOI:10.1021/jm061106t

A series of C5-substituted 1,2,4-triaryl-1H-imidazoles was synthesized. Their gene-activating properties were determined on estrogen receptor alpha positive MCF-7 breast cancer cells, stably transfected with the plasmid ERE _wtcluc (MCF-7-2a cells). The influence of 4-OH and 2-Cl substituents on the phenyl rings as well as the significance of a methyl, ethyl, or phenyl group at C5 on the estrogen receptor binding and the resulting gene activation in MCF-7-2a cells was studied. The alkyl and aryl groups at C5 of 1,2,4-tris(4-hydroxyphenyl)-1H-imidazole 1 increased the transactivation, while chlorine atoms on the phenyl rings diminished this effect. 5-Ethyl-1,2,4-tris(4- hydroxyphenyl)-1H-imidazole 9 was identified as the most active compound. Its excellent transcriptional activity did not only depend on the C5 ethyl group, but also on the three hydroxyl groups of the phenyl rings. Compounds (11-14) with a reduced number of hydroxyl groups displayed distinctly lower gene activation.

Full text of DOI:10.1021/jm061106t

J. Med. Chem. 2007, 50, 1475-1484
1475
Structure-Activity Relationship Study To Understand the Estrogen Receptor-Dependent Gene
Activation of Aryl- and Alkyl-Substituted 1H-Imidazoles
Thomas Wiglenda and Ronald Gust*
Institute of Pharmacy, Free UniVersity of Berlin, Ko¨nigin-Luise Strasse 2+4, D-14195 Berlin, Germany
ReceiVed September 22, 2006
A series of C5-substituted 1,2,4-triaryl-1H-imidazoles was synthesized. Their gene-activating properties
were determined on estrogen receptor alpha positive MCF-7 breast cancer cells, stably transfected with the
plasmid EREwtcluc (MCF-7-2a cells). The influence of 4-OH and 2-Cl substituents on the phenyl rings as
well as the significance of a methyl, ethyl, or phenyl group at C5 on the estrogen receptor binding and the
resulting gene activation in MCF-7-2a cells was studied. The alkyl and aryl groups at C5 of 1,2,4-tris(4-
hydroxyphenyl)-1H-imidazole 1 increased the transactivation, while chlorine atoms on the phenyl rings
diminished this effect. 5-Ethyl-1,2,4-tris(4-hydroxyphenyl)-1H-imidazole 9 was identified as the most active
compound. Its excellent transcriptional activity did not only depend on the C5 ethyl group, but also on the
three hydroxyl groups of the phenyl rings. Compounds (11-14) with a reduced number of hydroxyl groups
displayed distinctly lower gene activation.
Scheme 2
Introduction
The estrogen receptor (ER) is an important transcription factor
and regulates physiological processes, which are involved, for
example, in the development and function of the reproductive
and cardiovascular systems or in the bone density changes.1-3
The regulatory properties become evident through the interaction
with its native ligands such as estradiol (E2).
Nonsteroidal estrogenic (e.g., diethylstilbestrol) and anti-
estrogenic (e.g., raloxifene) ligands have been developed to
regulate these processes or their pathological dysfunction,
including breast cancer, osteoporosis, and infertility. New
possibilities of treatment opened the discovery of selective
estrogen receptor modulators (SERMs) which exert tissue- and
cell-selective activity. It was demonstrated in various structure-
activity relationship (SAR) studies that aromatic4-7 and non-
aromatic6,7 heterocycles as well as cycloalkanes can act as potent
SERMs. Their activity clearly depended on the arrangement of
phenolic substituents at the core molecule. Intensive studies on
4-alkyl-1,3,5-tris(4-hydroxyphenyl)-1H-pyrazoles (e.g., pyrazole
9),4 3-alkyl-2,4,5-tris(4-hydoxyphenyl)furans (e.g., furan 9)5
(formulas see Scheme 1), and on N-alkyl-substituted 4,5-bis-
(4-hydroxyphenyl)-2-imidazolines8 and 2,3-bis(4-hydroxy-
phenyl)piperazines9 demonstrated the significance of the kind
of the heteroatoms as well as the presence of alkyl substituents
on the ER binding affinity and/or ER subtype selectivity.
Scheme 1
This observation induced us to optimize 1H-imidazoles6,10
for a specific receptor-ligand interaction. Because of the
negative results obtained with 4,5-bis(4-hydroxyphenyl)-1H-
imidazoles11 and 2,4,5-tris(4-hydroxyphenyl)imidazoles12 the
arrangement of the substituents at the imidazole core was
changed to a 1,2,4-tris(4-hydroxyphenyl)-1H-imidazole scaf-
fold13 corresponding to the hormonally active pyrazole 9 and
furan 9. Indeed, the lead structure 5-ethyl-1,2,4-tris(4-hydroxy-
phenyl)-1H-imidazole 9 (for structure, see Scheme 1) was
hormonally active but to a lesser extent than pyrazole 9 and
* Corresponding author. Phone: (030) 838 53272; Telefax: (030) 838
56906; E-mail: rgust@zedat.fu-berlin.de.
10.1021/jm061106t CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/13/2007

1476 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Wiglenda and Gust
Scheme 3
furan 9. Interestingly, gene expression induced by 9 in estrogen
receptor alpha (ERR) positive MCF-7-2a cells (routinely used
in our laboratory for the evaluation of hormonally active
compounds) depended on the presence of the C5 ethyl group.
1,2,4-Tris(4-hydroxyphenyl)-1H-imidazole 1 was completely
inactive. In contrast to our investigations on meso-configurated
4,5-diaryl-2-imidazolines,6,11 which can also be interpreted as
2,3-dihydro-1H-imidazoles, chlorine substituents on the phenolic
rings did not increase the transcriptional activity. These results
document the relevance of an alkyl chain at the imidazole core
for estrogenic activity.
In this structure-activity relationship study we investigated
the influence of alkyl chains at C5, the influence of the
substituents on the aromatic rings, and the significance of the
C4 aryl ring on the hormonal profile of 1,2,4-triaryl-1H-
imidazoles.
able, was prepared by reaction of 2-chloro-4-methoxybenzal-
dehyde (3e) with gaseous ammonia and lead(IV) acetate as
oxidant.16 The R-bromoketones 5c, 7c, 10c, and 15c were
produced by Friedel-Crafts acylation of anisoles with R-bromo-
substituted acid chlorides (Scheme 2B and 2C). The subsequent
ring formation between R-bromoketones (3c, 5c, 7c, 10c, 11c,
15c) and N-arylbenzamidines (3b, 7b, 12b, 13b) was carried
out at room temperature in a mixture of CHCl3/H2O (6:1)
utilizing K2CO3 as base (Scheme 3). Ring formation occurred
in each case with high regioselectivity and yield. No regioiso-
mers of 3a, 5a-8a, and 10a-16a could be separated from the
reaction mixture.
For the synthesis of the 1H-imidazoles 17a, 18a, and 20a,
the desoxybenzoins 17c and 18c or butyraldehyde were used.
In the first step the educts were brominated with Br2 in a mixture
of dry CH2Cl2 and dioxane, followed by the well-known
cyclization without prior purification (Scheme 3).
The synthesis of the 1,2-diaryl-1H-imidazole 21a is depicted
in Scheme 4. A ring closing reaction of N1-(4-methoxyphenyl)-
ethane-1,2-diamine hydrochloride 21c with 4-methoxybenz-
imidic acid ethyl ester 12e in glacial acid gave the 1,2-bis(4-
methoxyphenyl)-2-imidazoline (21b), which was oxidized to 21a
with MnO2 in benzene under reflux.
N1-(4-Methoxyphenyl)ethane-1,2-diamine hydrochloride (21c)
was obtained by melting of oxazolidin-2-one and anisidine at a
temperature between 170-190 °C. 4-Methoxybenzimide acid
Results and Discussion
Chemistry. The synthesis of the 1,2-diaryl-, 1,2,4-triaryl-,
and 5-alkyl-1,2,4-triaryl-1H-imidazoles was performed by cy-
clization of N-arylbenzamidines and R-bromoketones as de-
scribed earlier.13 The N-arylbenzamidines (3b, 7b, 12b, 13b)
were obtained by reaction of aryl nitriles (3d, 7d, 12d) with
anisidine or aniline according to Gautier14 or Daoust15 using
sodium amide as reaction agent (Scheme 2A). 2-Chloro-4-
methoxybenzonitrile (3d), which was not commercially avail-

Estrogen Receptor-Dependent Gene ActiVation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1477
Scheme 5
Scheme 4
ethyl ester (12e) was obtained from 4-methoxybenzonitrile (12d)
with SOCl2 in a mixture of ethanol and water at about 0 °C.
The ether cleavage to afford the free phenols 1-18, 20, 21
was performed with boron tribromide (Scheme 5; for 21, see
Scheme 4) at -80 °C. Interestingly, if this reaction was carried
out with 5-ethyl-2,4-bis(4-methoxyphenyl)-1-phenyl-1H-imi-
dazole 13a, a loss of the phenyl group at N1 (w 19, Scheme 5)
was observed.
The structural characterization of the 1H-imidazoles was
performed by double resonance nuclear Overhauser enhance-
ment experiment (NOESY). It was confirmed that all oxo group
standing substituents took position C4.
(NOEs 1, 2, and 3). Furthermore, saturation transfers were
observed between the methylene (NOEs b and d, Figure 1) and
methyl protons (NOEs a and c, Figure 1) of the ethyl chain to
the H-C2′ protons of the N1- and C4-standing phenyl rings.
This is only possible if the ethyl group is located between these
rings at C5 of the imidazole ring.
As an example, Figure 1 presents the section 5.8-8.0 ppm
and 0.7-3.6 ppm of the NOESY spectrum of compound 10
(DMSO-d6). All protons at the C2′ position of the phenyl rings
showed NOEs to their neighboring protons at the C3′ position
Estrogen Receptor Binding and Transcriptional Activity.
The interaction of the 1H-imidazoles 1-21 with ERR was tested
in a competition experiment with [3H]E2 using calf uterine
cytosol as source of ERR and in a luciferase assay17 using ERR
Figure 1. NOESY-spectrum of compound 10 in DMSO-d6 (300 K). The saturation transfer of the methyl (a, c) and methylene (b, d) protons of
the ethyl group to the protons at C(A2′) (a, b) and C(B2′) (c, d) of the phenyl rings A and B.

1478 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Wiglenda and Gust
Figure 2. Activation (%) of the luciferase gene expression in MCF-7-2a cells by 1H-imidazoles 1-21. Values expressed are the means ( SE of
3-fold determination in a single experiment.
positive MCF-7 breast cancer cells stably transfected with the
plasmid EREwtcluc (MCF-7-2a cells). It is well accepted that
the binding of hormonal drugs to the ERR leads to a confor-
mational change of the receptor and subsequently to a dimer-
ization of the ER/drug conjugates. The binding of the dimers
Hydrophobic chlorine substituents on the C2 and/or the C4 aryl
ring abolished the gene activation (compounds 6-8, Figure 2B).
In contrast, enhancement of the hydrophobicity by elongation
of the C5 alkyl chain increased the hormonal potency (Figure
2C). The 1H-imidazole 9 bearing a C5 ethyl residue showed a
distinctly lower EC50 value (0.077 µM) than 5 but a maximum
activation of 140%. Interestingly, the ethyl chain compensated
the negative effect of the chlorine on the aromatic rings.
Compound 10 activated the luciferase expression (EC50 ) 0.48
µM) despite the Cl atom on the C2 aryl ring.
In the next step we studied the significance of the OH groups
on the phenyl rings of 9. Each modification, the exchange of
an OH group by H reduced the transcriptional potency. None
of the compounds 11-14 reached the maximum effect of E2
(100%) within the specified concentration range (Figure 2D).
An EC50 ) 0.061 µM was determined for compound 12.
Nevertheless, a structure-activity relationship can be deduced
from these results. Hydroxyl groups on the N1/C4 aryl rings
(12: rel activation at 10 µM: 90%) were more effective than
on the C2/C4 rings (13: rel activation at 10 µM: 60%).
Compound 11 with OH groups on the N1/C2 rings was
completely inactive. The 5-ethyl-2,4-bis(4-hydroxyphenyl)-1H-
to the estrogen response element (ERE) of the plasmid EREwtc
-
luc in MCF-7-2a cells causes the expression of luciferase, which
correlates well with the estrogenic potency of the drug.18
The respective relative binding affinity (RBA) of the 1,2,4-
triaryl-1H-imidazoles was very low. Only 4 (RBA ) 0.09%),
8 (RBA ) 0.11%), 9 (RBA ) 0.11%), and 10 (RBA ) 0.19%)
possessed RBA values >0.05%. Nevertheless, gene activation
measured strongly depended on the substituents of the aromatic
rings and the C5 alkyl chain.
As depicted in Figure 2A, the 1H-imidazoles 1-4 without
C5 alkyl substituents did not induce gene activation. In the case
of the Cl-substituted compounds 2-4, a significant reduction
of the relative activation (% E2) of the luciferase expression
was observed at the highest concentration.
C5 alkyl chains enhanced the transcriptional activity. The
methyl derivative 5 reached the 100% E2 activation at a
concentration of 10 µM and showed an EC50 ) 0.14 µM.

Estrogen Receptor-Dependent Gene ActiVation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1479
imidazole 19 showed a slightly lower activation of luciferase
expression than 13 (which confirmed the ER binding mainly
by the C2/C4 phenolic rings) but with a clearly higher activation
than compound 14 which had only one OH group.
Exchange of the C4 aryl ring (compound 20) and the C5 ethyl
chain (compound 21) led to inactive compounds (Figure 2E).
The comparison with the results for compound 9 showed that
the C4 phenolic ring is not only essential to achieve H bonds
in the ligand binding domain but also for hydrophobic contacts.
Therefore, in the next step of this SAR study the C5 ethyl
chain in 9 was replaced by an aromatic ring. The resulting 1,2,4-
tris(4-hydroxyphenyl)-5-phenyl-1H-imidazole 15 showed lower
hormonal activity than 9 but reached the 100% relative activation
at a concentration of 10 µM (Figure 2F). The EC50 ) 0.35 µM
was comparable to that of the methyl derivative 5. Chlorine
substituents either on the C2 or the C4 phenolic ring of 15
reduced the luciferase expression. At the highest concentration
of 10 µM, 16 and 18 achieved relative activation levels of 50%
and 40%, respectively (Figure 2F).
Figure 3. Model of the interaction of 5-ethyl-1,2,4-tris(4-hydroxyphen-
yl)-1H-imidazole 9 with the estrogen receptor R binding site. Compound
9, generated by Tripos SYBYL software and energy minimized with
MM3, was inserted in the LBD of the 1ERE crystal structure image
from the pdb data base. The physiological ligand estradiol was removed
and replaced by compound 9. (The ligand was fitted out of the E2
molecule plane. The C2-C1′ bond was aligned in the plane of the E2
C13-Me group.) The C4 phenolic group was oriented to the guanidinium
residue of Arg394 and the carboxylate group of Glu353 as well as an
incorporated water molecule. The phenolic substituent at N1 was
oriented towards His524 to develop a second hydrogen bond. The third
C2 phenolic ring was directed towards Met522. The C5 ethyl residue
was embeded in a lipophilic pocket formed by Phe404, Leu346, and
Ile424.
The introduction of an OH group on the C5 phenyl ring led
to a strong decrease of activity. The 1H-imidazole 17 was nearly
inactive. This might be the consequence of hindered hydropho-
bic contacts by the hydrophilic OH group indicating a well
defined orientation of the 1H-imidazoles in the ligand binding
domain (LBD).
The results of the transcriptional assay depended on the
interaction of the drugs with the ERR, because the MCF-7-2a
cells contain this ER subtype. The binding mode of hormones
at the ERR is well-known from X-ray crystallographic studies
and molecular modeling.19-21 Estradiol is attached to Arg394,
Glu353, and a water molecule by hydrogen bonds from the
3-OH group as well by an H bridge from the 17â-OH group to
His524. This orientation is stabilized by van der Waals contacts
of the steroidal skeleton to hydrophobic amino acids. This
binding mode is realized for all linear hormones (type I or class
I estrogens).6,22,23
The binding model of 9 is depicted in Figure 3. In view of
the fact that a hydroxyl group was more effective on the N1/
C4 rings than on the C2/C4 rings (12 more active than 13) and,
moreover, totally ineffective on the N1/C2 rings (11), we assume
that the C4 phenolic ring is directed to Arg394 and Glu353
and mimics ring A of E2. The OH group of the N1 phenolic
ring is oriented to His524. The third phenolic ring contacts
Met522. The importance of Met522 for the binding of hormon-
ally active compounds was already detected by Danielian et al.
They generated mutant mouse estrogen receptors, which differ
in their sensitivity to estrogens. Mutation of Gly525 and Met522
virtually abolished the ability of the receptor to bind estradiol
and to stimulate transcription.27
The C5-standing residue is oriented toward a hydrophobic
pocket well-known from the X-ray structrure of ERR co-
crystallized with DES.20 This pocket tolerates the ethyl chain
but also a C5 aryl ring. Van der Waals contacts are possible to
Phe404, Leu391, Ile 424, and Leu346. Hydrophilic groups such
as those in 17 (C5 phenolic ring) disturb these contacts, leading
to a reorientation of the drug in the LBD and a reduction of
gene activation.
The decreased efficacy of chlorine-bearing compounds might
be a result of steric repulsion during the ER binding. Only if
the hydrophobic character of the entire molecule is high enough,
can this obstacle be overcome as demonstrated for 3 and 16 as
well as 2 and 18.
Finally some comments are necessary regarding the curve
of 9 and those of the chlorine-substituted compounds 2 and 4
depicted in Figure 2A. Compound 9 exceeded the transactivation
level of E2. This might be the consequence of a distinct mode
of action. The additional contacts of 9 in the LBD might induce
a conformation of the ER, which is different to that of E2. As
a consequence co-activator or co-repressor recruitment might
During the last years a lot of angular hormones were
developed, which cannot bind in the LBD in the same
orientation.6,8,24 In a previous SAR study, we investigated meso-
4,5-bis(4-hydroxyphenyl)-2-imidazolines as new SERMs.6,8,25
The prerequesite for their ER binding were the two OH groups
in a distance of 6.8 Å and o-chloro substituents on the aromatic
rings to intensify the hydrophobic contacts. For this and related
compounds, an interaction between the OH groups and the
guanidinium residue of Arg394 and the carboxylate group of
Glu353 as well as an incorporated water molecule on the one
hand and Asp351 or Thr347 residue in the 11â cannel at ERR
on the other hand was proposed.19
The 1H-imidazoles presented in this paper can be assigned
to type I or type II estrogens, because linear (N1/C4 rings and
C2/C4 rings) and angular pharmacophores (N1/C2 rings) can
be realized. From the data of the luciferase assay, however, a
type II estrogen like orientation can be excluded. Compounds
20 and 21 are completely inactive.
The variation of the substituents on the phenyl rings showed
that mainly the N1/C4 rings act as pharmacophores and should
be bound in the linear way in the LBD. The insertion of a
hydrophobic alkyl residue or a phenyl ring at C5 in the 1,2,4-
tris(4-hydroxyphenyl)-1H-imidazole 1 enhanced the estrogenic
effect, while the C5 phenolic residue abolished the gene
activation. These findings point to strong hydrophobic interac-
tions and to a well defined orientation of the 1H-imidazoles in
the LBD.26

1480 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Wiglenda and Gust
mmol), and benzonitrile 3d (3.53 g, 21.06 mmol). Water (150 mL)
was added to the crude mixture, and it was repeatedly extracted
with CH2Cl2 (3 × 50 mL). The organic layers were combined, dried
over Na2SO4, and concentrated in vacuum. The crude product was
purified by chromatography with stepwise gradient elution (CH2-
Cl2/methanol 95:5, 90:10) to give 3b (1.08 mg, 18%) as violet
amorphous solid (mp: 79 °C). H NMR (DMSO-d6): δ ) 9.50
(bs, 2H, NH2), 7.72 (bs, 1H, ArH), 7.28 (bs, 3H, ArH), 7.09 (bs,
3H, ArH), 3.85 (bs, 3H, OCH3), 3.73 (bs, 3H, OCH3).
N-(4-Methoxyphenyl)-4-methoxybenzamidine (7b). From ani-
sidine (2.43 g, 19.76 mmol), NaNH2 (924 mg, 23.70 mmol), and
4-methoxybenzonitrile (7d) (2.63 g, 19.75 mmol). The crude
product was separated by vacuum filtration, washed with toluene,
and suspended in H2O (150 mL). After being stirred for 30 min,
the slurry was extracted with CH2Cl2 (3 × 60 mL). The organic
layers were combined and dried over Na2SO4. Evaporation gave
the product 7b (2.70 g, 53%) as a colorless solid (mp: 150 °C).
1H NMR (DMSO-d6): δ ) 7.92 (d, 2H, J ) 8.8, ArH), 6.96 (d,
2H, J ) 8.8, ArH), 6.88 (d, 2H, J ) 8.8, ArH), 6.76 (d, 2H, J )
8.7, ArH), 6.07 (s, 2H, NH2), 3.79 (s, 3H, OCH3), 3.72 (s, 3H,
OCH3).
be different. Other reasons would be the interaction with an
additional binding site, which can be the explanation for the
low RBA value, or an influence on the ER turnover rate.
Investigations on these topics are in the planning stage and will
be performed in the future.
The negative activations at 10 µM displayed by both
compounds 2 and 4 were the results of inhibition of the MCF-
7-2a cell growth. This cytotoxic effect was verified in the
hormone-dependent MCF-7 and the hormone-independent MDA-
MB 231 cell lines.13 Therefore, it was deduced that the ER was
not involved in the cytotoxic activity. Furthermore, the cy-
clooxygenase enzymes (COX) were identified as targets for this
class of drugs. The inhibition of the cell growth correlates very
well with the inhibition of the COX enzymes.13
1
Inhibition of cell growth and reduced COX activity were only
observed with compounds 2 and 4 bearing an o-Cl on the C4
phenolic ring. Compound 3 (Cl substituent on the C2 phenolic
ring) showed neither cytotoxicity nor COX-inhibition (data not
shown).
N-(4-Methoxyphenyl)benzamidine (12b). From anisidine (1.85
g, 15.03 mmol), NaNH2 (586 mg, 15.03 mmol), and benzonitrile
12d (1.55 g, 15.03 mmol). Water (150 mL) was added to the crude
mixture, and it was repeatedly extracted with CH2Cl2 (3 × 50 mL).
The organic layers were combined, dried over Na2SO4, and
concentrated in vacuum. The crude product was purified by
chromatography with stepwise gradient elution (CH2Cl2/methanol
95:5, 90:10) to give 12b (470 mg, 14%) as colorless amorphous
solid (mp: 86 °C). 1H NMR (DMSO-d6): δ ) 7.97-7.93 (m, 2H,
ArH), 7.48-7.41 (m, 2H, ArH), 6.92-6.82 (m, 5H, ArH), 6.46
(bs, 2H, NH2), 3.73 (s, 3H, OCH3).
N-Phenyl-4-methoxybenzamidine (13b). From aniline (3.77 g,
40.48 mmol), NaNH2 (1.58 g, 40.50 mmol), and 4-methoxyben-
zonitrile (7d) (5.40 g, 43.87 mmol). The crude product was
separated by vacuum filtration, washed with toluene, and suspended
in H2O (150 mL). After being stirred for 30 min, the slurry was
extracted with CH2Cl2 (3 × 60 mL). The organic layers were
combined and dried over Na2SO4. Evaporation gave the product
13b (4.32 g, 19.1 mmol, 47%) as a colorless solid (mp: 138 °C).
1H NMR (DMSO-d6): δ ) 7.93 (d, 2H, J ) 8.6, ArH), 7.30 (pt,
2H, J ) 7.6, ArH), 6.96 (pt, 3H, J ) 7.4, ArH), 6.83 (d, 2H, J )
7.6, ArH), 6.12 (s, 2H, NH2), 3.81 (s, 3H, OCH3).
General Procedure of the Preperation of Ketones. The
respective acid chloride was added to a cooled solution of AlCl3
in 1,2-dichlorethane (250 mL). Subsequently, the anisole was added
at a rate that maintained the temperature at 0-5 °C. The mixture
was allowed to warm to room temperature, and stirring was
continued for 1.5 h. Then the reaction mixture was poured onto a
mixture of crushed ice (400 g) and 36% HCl (10 mL), the organic
layer was separated, and the aqueous solution was subsequently
extracted with CH2Cl2 (3 × 50-80 mL). The organic layers were
combined and dried over Na2SO4, and the solvent was removed
under reduced pressure. The products were separated if necessary
by column chromatography on SiO2.
2-Bromo-1-(4-methoxyphenyl)propan-1-one (5c). From 2-bro-
mopropionic acid chloride (3.00 mL, 29.75 mmol), AlCl3 (5.95 g,
44.63 mol), and anisole (3.24 mL, 29.75 mmol). Column chroma-
tography with stepwise gradient elution: petroleum ether/diethyl
ether 5:2, 4:1, 2:1. Yield: 2.30 g (32%) of colorless oil. After
standing at room temperature the oil crystallized (mp ) 57 °C).
1H NMR (DMSO-d6): δ ) 8.03 (d, 2H, J ) 8.9, ArH), 7.05 (d,
2H, J ) 9.0, ArH), 5.79 (q, 1H, J ) 6.5, CHBrCH3), 3.86 (s, 3H,
OCH3), 1.76 (q, 3H, J ) 6.5, CHBrCH3).
To get more information about the structural requirement for
cytotoxicity and COX inhibition, an SAR study was initiated.
The results will be presented in a forthcoming paper.
Experimental Section
General Methods. All reagents and solvents were purchased
from Acros Organics, Fluka Chemie, Lancaster, Merck, or Sigma-
Aldrich. All reactions were monitored by TLC, performed on silica
gel plates 60 F254 (Merck, Darmstadt/Germany). Visualization on
TLC was achieved by UV light. Column chromatography was
performed with Merck silica gel 60H, grain size <0.063 mm, 230
mesh ASTM (Darmstadt/Germany). Melting points: 510 Bu¨chi
(Flawil/Schweiz) capillary melting point apparatus. IR spectra (KBr
pellets): Perkin-Elmer Model 580 A (Rodgau-Ju¨gesheim/Germany).
1H NMR: Avance DPX-400 spectrometer (Bruker, Karlsruhe/
Germany) at 400 MHz, AMX 500 spectrometer (Bruker, Karlsruhe/
Germany) at 500.14 MHz (internal standard: TMS). Elemental
analyses: Microlaboratory of the Free University of Berlin. EI-
MS spectra: CH-7A-Varian MAT, 70 eV (Melbourne/Australia).
FAB spectra: CH5 DF (Varian MAT, Bremen, Germany) modified
with focused FAB-gun (AMD-Intectra). Microlumat: Victor2 1420
Multilabel Counter (Wallac, Perkin-Elmer, Life sciences, Turku/
Finnland). Microplate reader: FLASHscan S12 (analytikjena AG/
Germany).
Synthesis. The 1H-imidazoles 1, 2, 4, and 9 and the benzalde-
hyde 3e were synthesized as previously published.13,28 Compounds
7d and 12d were commercially available.
General Procedure for the Preparation of Benzonitrile.
Through a stirred solution of the respective benzaldehyde and
lead(IV) acetate in toluene (30 mL) was passed gaseous ammonia
for 3 h while heating to reflux. The solution was then poured onto
a mixture of crushed ice (200 g) and 36% HCl (50 mL) and was
subsequently extracted with CH2Cl2 (3 × 40 mL). The combined
organic layers were dried over Na2SO4 and removed under reduced
pressure to provide the product.
2-Chloro-4-methoxybenzonitrile (3d). From 2-chloro-4-meth-
oxybenzaldehyde 3e (1.00 g, 5.86 mmol) and lead(IV) acetate (5.20
g, 11.72 mmol). Yield: 0.92 g (5.49 mmol, 94%) of colorless
crystals (mp: 75 °C). 1H NMR (DMSO-d6): δ ) 7.89 (d, 1H, J )
8.7 Hz, ArH), 7.35 (d, 1H, J ) 2.5 Hz, ArH), 7.11 (dd, 1H, J )
2.5 Hz, J ) 8.7 Hz, ArH), 3.88 (s, 3H, OCH3).
General Procedure for the Preparation of N-Arylbenz-
amidines. A solution of anisidine or aniline in dry toluene (20-
40 mL) was treated with NaNH2 under N2 atmosphere. The
respective benzonitrile was then added after stirring for 3 h at 120-
140 °C. The mixture was held at this temperature for 3 h and then
cooled overnight to room temperature without stirring.
2-Bromo-1-(2-chloro-4-methoxyphenyl)propan-1-one (7c). From
2-bromopropionic acid chloride (3.00 mL, 29.75 mmol), AlCl3 (5.95
g, 44.63 mol), and 3-chloroanisole (3.66 mL, 29.75 mmol). Column
chromatography with stepwise gradient elution: petroleum ether,
petroleum ether/diethyl ether 5:2, 1:1. Yield: 2.88 g (35%) of
colorless oil. After standing at room temperature, the oil solidified
to gelatinous needles. 1H NMR (DMSO-d6): δ ) 7.85 (d, 1H, J )
N-(4-Methoxyphenyl)-2-chloro-4-methoxybenzamidine (3b).
From anisidine (2.59 g, 21.07 mmol), NaNH2 (986 mg, 25.27

Estrogen Receptor-Dependent Gene ActiVation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1481
8.7, ArH), 7.15 (d, 1H, J ) 2.5, ArH), 7.04 (d, 1H, J ) 2.5, ArH),
5.65 (q, 1H, J ) 6.5, CHBrCH3), 3.86 (s, 3H, OCH3), 1.76 (d, 3H,
J ) 6.5, CHBrCH3).
(dd, 1H, J ) 2.5, J ) 8.6, ArH), 3.79 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 2.20 (s, 3H, CH3).
4-(2-Chloro-4-methoxyphenyl)-1,2-bis(4-methoxyphenyl)-5-
methyl-1H-imidazole (7a). From amidine 7b (104 mg, 0.41 mmol),
K2CO3 (75 mg, 0.54 mmol) and R-bromoketone 7c (100 mg, 0.35
mmol). Method A; reaction time: 80 h; column chromatography
with CH2Cl2/methanol 98:2. Yield: 59 mg (33%) of a colorless
solid (mp: 65-70 °C). 1H NMR (DMSO-d6): δ ) 7.46 (d, 1H, J
) 8.6, ArH), 7.29 (d, 2H, J ) 8.8, ArH), 7.26 (d, 2H, J ) 8.8,
ArH), 7.12 (d, 2H, J ) 2.5, ArH), 7.07 (d, 2H, J ) 8.9, ArH), 7.00
(dd, 1H, J ) 2.5, J ) 8.7, ArH), 6.83 (d, 1H, J ) 8.8, ArH), 3.82
(s, 3H, OCH3), 3.72 (s, 6H, OCH3), 1.92 (s, 3H, CH3).
2-Bromo-1-(4-methoxyphenyl)butan-1-one (10c). 2-Bromobu-
tyric acid (3.21 mL, 29.94 mmol) was heated with SOCl2 (3.26
mL, 44.91 mmol) under reflux for 40 min. To the reaction mixture
was added 1,2-dichlorethane (80 mL), and the solution was cooled
with an ice bath. AlCl3 (5.99 g, 44.91 mmol) was added gradually.
Under ice cooling, anisole (3.24 mL, 29.94 mmol) was added
dropwise (30 min) and the mixture was stirred for 0.75-1.5 h.
Separation by column chromatography with stepwise gradient
elution: petroleum ether, petroleum ether/diethyl ether 4:1, 1:1;
1
Yield: 3.30 g (43%) of colorless oil. H NMR (DMSO-d6): δ )
2,4-Bis(2-chloro-4-methoxyphenyl)-1-(4-methoxyphenyl)-5-
methyl-1H-imidazole (8a). From amidine 3b (344 mg, 0.41 mmol),
K2CO3 (257 mg, 1.86 mmol), and R-bromoketone 7c (301 mg, 0.35
mmol). Method A; reaction time: 72 h; column chromatography
with stepwise gradient elution: CH2Cl2/methanol 98:2, 95:5.
8.04 (d, 2H, J ) 8.9, ArH), 7.08 (d, 2H, J ) 9.0, ArH), 5.62 (pq,
1H, J ) 7.6, CHBrCH2CH3), 3.89 (s, 3H, OCH3), 2.15-2.07 (m,
2H, CHBrCH2CH3), 1.00 (t, 3H, J ) 7.3, CHBrCH2CH3).
1-(2-Chloro-4-methoxyphenyl)-2-phenylethan-1-one (15c). From
phenylacetyl chloride (6.00 g, 38.81 mmol), AlCl3 (7.76 g, 58.20
mol), and 3-chloroanisole (4.77 mL, 38.80 mmol). Column chro-
matography with stepwise gradient elution: petroleum ether/diethyl
1
Yield: 363 mg (66%) of a colorless solid (mp: 74 °C). H NMR
(DMSO-d6): δ ) 8.11 (d, 1H, J ) 8.4, ArH), 7.49 (d, 1H, J )
8.5, ArH), 7.20-7.16 (m, 3H, ArH), 7.06 (dd, 1H, J ) 2.0, J )
8.3, ArH), 7.03 (d, 1H, J ) 2.5, ArH), 6.97 (dd, 1H, J ) 2.5, J )
8.6, ArH), 6.93 (d, 2H, J ) 8.9, ArH), 3.95 (s, 3H, OCH3), 3.79
(s, 3H, OCH3), 3.74 (s, 3H, OCH3), 1.96 (s, 3H, CH3).
1
ether 5:1, 4:1. H NMR (DMSO-d6): δ ) 7.83 (d, 2H, J ) 8.7,
ArH), 7.56 (m, 2H, J ) 8.4, ArH), 7.30 (d, 2H, J ) 8.8, ArH),
7.22 (d, 1H, J ) 1.8, ArH), 7.08 (dd, 1H, J ) 1.9, J ) 8.0, ArH),
4.24 (s, 2H, CH2), 3.93 (s, 3H, OCH3).
2-(2-Chloro-4-methoxyphenyl)-5-ethyl-1,4-bis(4-methoxyphen-
yl)-1H-imidazole (10a). From amidine 3b (513 mg, 1.76 mmol),
K2CO3 (366 mg, 2.65 mmol), and R-bromoketone 10c (454 mg,
1.77 mmol): Method A; reaction time: 144 h; column chroma-
tography with stepwise gradient elution: CH2Cl2/methanol 99:1,
98:2. Yield: 342 mg (43%) of a vitreous brownish solid (mp: 65-
General Procedure for the Preparation of Substituted 1H-
Imidazoles. Method A: K2CO3 and the R-bromoketone were
subsequently added to a stirred solution of the N-arylbenzamidine
in CHCl3 (3.00 mL) and H2O (0.50 mL) at room temperature. The
reaction was kept at room temperature for 18-56 h, quenched with
H2O (50 mL), and stirred for additional 20 min. The solution was
extracted with CH2Cl2 (3 × 20 mL), and the organic layer was
separated, dried over Na2SO4, and evaporated under reduced
pressure, to give a crude product, which was purified by chroma-
tography on silica gel.
Method B: Br2 in dry CH2Cl2 (3.00 mL) was added over a period
of 20-60 min to a cooled solution of the ketone in a mixture of
dry CH2Cl2/dioxane (1:1, 5.00 mL) under nitrogen atmosphere.
After decolorization, N-arylbenzamidine, K2CO3 and H2O (1.00 mL)
were added and the reaction mixture was stirred at room temperatur
for 15-49 h. The reaction was quenched with H2O (50 mL) and
stirred for an additional 20 min. The solution was extracted with
CH2Cl2 (3 × 20 mL), and the organic layers were combined, dried
over Na2SO4, and evaporated under reduced pressure, to give a
crude product, which was purified by chromatography on silica
gel.
1
68 °C). H NMR (DMSO-d6): δ ) 7.63 (d, 2H, J ) 8.8, ArH),
7.38 (d, 1H, J ) 8.6, ArH), 7.23 (d, 2H, J ) 8.9, ArH), 7.00 (d,
2H, J ) 8.9, ArH), 6.97 (d, 1H, J ) 2.6, ArH), 6.95 (d, 2H, J )
8.9, ArH), 6.86 (dd, 1H, J ) 2.5, J ) 8.6, ArH), 3.79 (s, 3H, OCH3),
3.75 (s, 6H, OCH3), 2.61 (q, 2H, J ) 7.4, CH2CH3), 1.43 (t, 3H,
J ) 7.4, CH2CH3).
5-Ethyl-1,2-bis(4-methoxyphenyl)-4-phenyl-1H-imidazole (11a).
From amidine 7b (677 mg, 2.64 mmol), K2CO3 (1.09 g, 7.92 mmol),
and R-bromoketone 11c (600 mg, 2.64 mmol). Method A; reaction
time: 60 h; column chromatography with stepwise gradient
elution: CH2Cl2/methanol 98:2, 95:5. Yield: 321 mg (32%) of a
colorless solid (mp: 150 °C). 1H NMR (DMSO-d6): δ ) 7.65 (d,
2H, J ) 8.8, ArH), 7.57-7.54 (m, 3H, ArH), 7.41-7.39 (m, 2H,
ArH), 7.23 (d, 2H, J ) 8.9, ArH), 7.01 (d, 2H, J ) 8.8, ArH), 6.80
(d, 2H, J ) 8.9, ArH), 3.80 (s, 3H, OCH3), 3.71 (s, 3H, OCH3),
2.58 (q, 2H, J ) 7.4, CH2CH3), 0.92 (t, 3H, J ) 7.4, CH2CH3).
5-Ethyl-1,4-bis(4-methoxyphenyl)-2-phenyl-1H-imidazole (12a).
From amidine 12b (457 mg, 2.02 mmol), K2CO3 (838 mg, 6.06
mmol), and R-bromoketone 10c (727 mg, 2.83 mmol). Method A;
reaction time: 48 h; column chromatography with CH2Cl2/methanol
98:2. Yield: 234 mg (30%) of a colorless solid (mp: 103 °C). 1H
NMR (DMSO-d6): δ ) 7.66 (d, 2H, J ) 7.8, ArH), 7.34 (d, 4H,
J ) 7.0, ArH), 7.25 (bs, 3H, ArH), 7.07 (d, 2H, J ) 7.7, ArH),
7.02 (d, 2H, J ) 7.7, ArH), 3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
2.60 (q, 2H, J ) 7.1, CH2CH3), 0.95 (t, 3H, J ) 7.2, CH2CH3).
5-Ethyl-2,4-bis(4-methoxyphenyl)-1-phenyl-1H-imidazole (13a).
From amidine 13b (500 mg, 2.21 mmol), K2CO3 (916 mg, 6.62
mmol), and R-bromoketone 10c (795 mg, 3.09 mmol). Method A;
reaction time: 72 h; column chromatography with stepwise gradient
elution: CH2Cl2/methanol 98:2, 95:5. Yield: 84 mg (10%) of a
colorless solid (mp: 121 °C). 1H NMR (DMSO-d6): δ ) 7.65 (d,
2H, J ) 8.8, ArH), 7.57-7.54 (m, 3H, ArH), 7.42-7.39 (m, 2H,
ArH), 7.23 (d, 2H, J ) 8.9, ArH), 7.01 (d, 2H, J ) 8.8, ArH), 6.80
(d, 2H, J ) 8.8, ArH), 3.80 (s, 3H, OCH3), 3.71 (s, 3H, OCH3),
2.60 (q, 2H, J ) 7.4, CH2CH3), 0.92 (t, 3H, J ) 7.4, CH2CH3).
5-Ethyl-2-(4-methoxyphenyl)-1,4-diphenyl-1H-imidazole (14a).
From amidine 13b (1.00 g, 4.41 mmol), K2CO3 (1.83 g, 13.26
mmol) and R-bromoketone 11c (1.59 g, 7.00 mmol). Method A
(CHCl3 (5.00 mL) and H2O (1.00 mL)); reaction time: 72 h; column
chromatography with stepwise gradient elution: CH2Cl2/methanol
98:2, 95:5. Yield: 302 mg (19%) of a colorless solid (mp: 114-
2-(2-Chloro-4-methoxyphenyl)-1,4-bis(4-methoxyphenyl)-1H-
imidazole (3a). From amidine 3b (200 mg, 0.69 mmol), K2CO3
(143 mg, 1.03 mmol), and R-bromoketone 3c (237 mg, 1.04 mmol).
Method A; reaction time: 20 h; column chromatography with CH2-
Cl2/methanol 98:2. Yield: 250 mg (86%) of a colorless solid (mp:
76 °C). 1H NMR (DMSO-d6): δ ) 7.88 (s, 1H, 5-H), 7.78 (d, 2H,
J ) 8.7, ArH), 7.48 (d, 1H, J ) 8.5, ArH), 7.16 (d, 2H, J ) 8.8,
ArH), 7.02 (d, 1H, J ) 2.5, ArH), 6.98-6.92 (m, 5H, ArH), 3.79
(s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.74 (s, 3H, OCH3).
1,2,4-Tris(4-methoxyphenyl)-5-methyl-1H-imidazole (5a). From
amidine 7b (691 mg, 2.70 mmol), K2CO3 (495 mg, 3.58 mmol),
and R-bromoketone 5c (580 mg, 2.39 mmol). Method A; reaction
time: 48 h; column chromatography with CH2Cl2/methanol 98:2.
1
Yield: 310 mg (29%) of a colorless solid (mp: 78 °C). H NMR
(DMSO-d6): δ ) 7.49 (d, 2H, J ) 8.9, ArH), 7.28 (d, 2H, J )
8.8, ArH), 7.07-7.03 (m, 4H, ArH), 6.88 (d, 2H, J ) 8.9, ArH),
6.82 (d, 2H, J ) 8.8, ArH), 3.82 (s, 3H, OCH3), 3.71 (s, 6H, OCH3),
1.90 (s, 3H, CH3).
2-(2-Chloro-4-methoxyphenyl)-1,4-bis(4-methoxyphenyl)-5-
methyl-1H-imidazole (6a). From amidine 3b (202 mg, 0.69 mmol),
K2CO3 (128 mg, 0.93 mmol), and R-bromoketone 5c (150 mg, 0.62
mmol). Method A; reaction time: 72 h; column chromatography
with CH2Cl2/methanol 98:2. Yield: 292 mg (97%) of a colorless
solid (mp: 196-198 °C). 1H NMR (DMSO-d6): δ ) 7.64 (d, 2H,
J ) 8.7, ArH), 7.39 (d, 1H, J ) 8.6, ArH), 7.19 (d, 2H, J ) 8.9,
ArH), 7.01-6.98 (m, 3H, ArH), 6.95 (d, 2H, J ) 8.9, ArH), 6.89
1
116 °C). H NMR (DMSO-d6): δ ) 7.74 (d, 2H, J ) 7.3, ArH),

1482 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Wiglenda and Gust
7.57-7.55 (m, 3H, ArH), 7.46-7.41 (m, 4H, ArH), 7.28 (t, 1H, J
) 8.0, ArH), 7.24 (d, 2H, J ) 8.9, ArH), 6.81 (d, 2H, J ) 8.8,
ArH), 3.71 (s, 3H, OCH3), 2.64 (q, 2H, J ) 7.5, CH2CH3), 0.94 (t,
3H, J ) 7.4, CH2CH3).
N1-(4-Methoxyphenyl)ethane-1,2-diamine Hydrochloride (21c).
Oxazolidin-2-one (6.75 g, 77.52 mmol) and anisidine hydrochloride
(3.68 g, 23.13 mmol) were heated to 170-190 °C for 16 h. The
melt was cooled to room temperature and dissolved in a mixture
of methanol/diethyl ether (3:2). After being refluxed for 30 min,
the product was allowed to crystallize at 7 °C. The precipitate was
separated by vacuum filtration and washed with methanol/diethyl
ether (3:2). Yield: 2.75 g (59%) of a gray amorphous powder
1,2,4-Tris(4-methoxyphenyl)-5-phenyl-1H-imidazole (15a). From
amidine 7b (300 mg, 1.17 mmol), K2CO3 (214 mg, 1.55 mmol),
and R-bromoketone 15c (470 mg, 1.54 mmol). Method A; reaction
time: 67 h; column chromatography with petroleum ether/diethyl
ether 1:1. Yield: 206 mg (38%) of a colorless solid (mp: 176-
1
(mp: 185 °C). H NMR (DMSO-d6): δ ) 7.44 (d, 2H, J ) 9.0,
1
ArH), 6.88 (d, 2H, J ) 9.0, ArH), 6.77 (s, 1H, NH), 3.79 (t, 2H,
J ) 7.8, CH2CH2), 3.71 (s, 3H, OCH3), 3.37 (t, 2H, J ) 7.9,
CH2CH2), 3.31 (s, 3H, NH3).
180 °C). H NMR (DMSO-d6): δ ) 7.38 (d, 2H, J ) 8.9, ArH),
7.33-7.28 (m, 5H, ArH), 7.23-7.20 (m, 2H, ArH), 7.16 (d, 2H, J
) 8.8, ArH), 6.86 (d, 4H, J ) 8.9, ArH), 6.81 (d, 2H, J ) 8.9,
ArH), 3.73 (s, 3H, OCH3), 3.71 (s, 6H, OCH3).
1,2-Bis(4-methoxyphenyl)-2-imidazoline (21b). Ethane-1,2-
diamine hydrochloride 21c (1.00 g, 4.98 mmol) was stirred with
the iminoethyl ether 12e (900 mg, 2.25 mmol) in glacial acetic
acid (15 mL) for 1 h and then heated to reflux for 17 h. The reaction
mixture was added to a 15% aqueous NaHCO3 solution (200 mL),
stirred for 15 min, and extracted with CH2Cl2 (3 × 50 mL). The
organic layers were combined, dried over Na2SO4, and evaporated
under reduced pressure to give a crude product which was purified
by column chromatography using stepwise gradient elution with
CH2Cl2/methanol 98:2, 95:5, and 90:10. Compound 21b (631 mg,
45%) was isolated from the main fraction as colorless vitreous solid
2-(2-Chloro-4-methoxyphenyl)-1,4-bis(4-methoxyphenyl)-5-
phenyl-1H-imidazole (16a). From amidine 3b (675 mg, 2.32
mmol), K2CO3 (285 mg, 2.06 mmol), and R-bromoketone 15c (514
mg, 1.68 mmol). Method A; reaction time: 17 h; column chroma-
tography with stepwise gradient elution: petroleum ether/diethyl
ether 1:1, 1:2. Yield: 479 mg (42%) of a colorless solid (mp: 153
1
°C). H NMR (DMSO-d6): δ ) 7.46 (d, 2H, J ) 8.5, ArH), 7.37
(d, 2H, J ) 9.0, ArH), 7.32-7.30 (m, 3H, ArH), 7.22-7.19 (m,
2H, ArH), 7.05-7.01 (m, 2H, ArH), 6.90 (dd, 1H, J ) 2.7, J )
8.6, ArH), 6.81 (d, 2H, J ) 9.0, ArH), 6.74 (d, 2H, J ) 9.0, ArH),
3.77 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.65 (s, 3H, OCH3).
1
(mp: 97-102 °C). H NMR (DMSO-d6): δ ) 7.35 (d, 2H, J )
8.8, ArH), 6.98 (d, 2H, J ) 8.9, ArH), 6.90 (d, 2H, J ) 8.9, ArH),
6.85 (d, 2H, J ) 9.0, ArH), 4.03 (t, 2H, J ) 9.0, CH2CH2), 3.90 (t,
2H, J ) 9.2, CH2CH2), 3.75 (s, 3H, OCH3), 3.71 (s, 3H, OCH3).
1,2-Bis(4-methoxyphenyl)-1H-imidazole (21a). The 2-imida-
zoline 21b (282 mg, 1.01 mmol) was dissolved in dry benzene (30
mL) and heated to reflux for 3 h to remove water using a Dean-
Stark trap. MnO2 (508 mg, 5.84 mmol) was added to this solution
and was heated for additional 18 h. The solution was filtered, the
residue was washed with toluene, and the organic layers were
combined. 1H-Imidazole 21a (0.28 g, 98%) was isolated from the
1,2,4,5-Tetrakis(4-methoxyphenyl)-1H-imidazole (17a). From
desoxybenzoine 17c (250 mg, 0.80 mmol) with Br2 (128 mg. 0.80
mmol; 60 min). Method B (amidine 7b (259 mg, 1.01 mmol), K2-
CO3 (124 mg, 0.90 mmol)); reaction time: 15 h; column chroma-
tography with stepwise gradient elution: petroleum ether/diethyl
ether 1:1, 1:2. Yield: 498 mg (30%) of a colorless solid (mp: 209
°C). H NMR (DMSO-d6): δ ) 7.40 (d, 2H, J ) 8.9, ArH), 7.30
(d, 2H, J ) 8.9, ArH), 7.15 (pt, 4H, J ) 8.3, ArH), 6.88-6.81 (m,
8H, ArH), 3.73 (s, 9H, OCH3), 3.71 (s, 3H, OCH3).
4-(2-Chloro-4-methoxyphenyl)-1,2-bis(4-methoxyphenyl)-5-
phenyl-1H-imidazole (18a). From ketone 18c (900 mg, 3.45 mmol)
with Br2 (180 mg, 1.13 mmol; 60 min). Method B (amidine 7b
(870 mg, 3.39 mmol), K2CO3 (480 mg, 4.84 mmol)); reaction
time: 48 h; column chromatography with CH2Cl2/methanol 98:2.
Yield: 448 mg (27%) of a colorless solid (mp: 139 °C). 1H NMR
(DMSO-d6): δ ) 7.35 (d, 1H, J ) 8.6, ArH), 7.28 (d, 2H, J )
8.8, ArH), 7.19-7.14 (m, 5H, ArH), 6.99 (d, 1H, J ) 2.6, ArH),
6.95-6.90 (m, 5H, ArH) 6.85 (d, 2H, J ) 8.9, ArH), 3.77 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 3.73 (s, 3H, OCH3).
1
1
solution as colorless solid. H NMR (DMSO-d6): δ ) 7.34 (d,
1H, J ) 1.0, CHCH), 7.24 (d, 2H, J ) 8.9, ArH), 7.21 (d, 2H, J
) 8.9, ArH), 7.10 (d, 1H, J ) 1.2, CHCH), 7.00 (d, 2H, J ) 8.9,
ArH), 6.86 (d, 2H, J ) 8.9, ArH), 3.79 (s, 3H, OCH3), 3.72 (s, 3H,
OCH3).
General Procedure for the Ether Cleavage with BBr3. A
solution of the methyl ether (1.00 mmol) in 15 mL of dry CH2Cl2
was cooled to 0 °C. BBr3 (4.5 mmol) in 5 mL of dry CH2Cl2 was
added at this temperature under N2 atmosphere. Then the reaction
mixture was allowed to warm up to room temperature and was
stirred for further 18 h. After the reaction mixture was cooled with
an ice bath, the excess BBr3 was hydrolyzed with three portions of
a mixture of methanol and Cl2CH2 and then methanol. The phenolic
product was dissolved in 10% aqueous NaHCO3 (50 mL), and the
solution was extracted with ethyl acetate (3 × 20 mL). The organic
layers were combined and dried over Na2SO4. The solvent was
removed under reduced pressure, and the resulting crude product
was purified by chromatography on silica gel.
5-Ethyl-1,2-bis(4-methoxyphenyl)-1H-imidazole (20a). From
butanal (500 µL, 5.55 mmol) and Br2 (887 mg, 5.55 mmol, 20 min).
Method B (amidine 7b (500 mg, 1.95 mmol), K2CO3 (0.77 g, 11.94
mmol)); reaction time: 48 h; column chromatography with CH2-
Cl2/methanol 95:5. Yield: 373 mg (58%) of brownish high viscous
1
oil. H NMR (DMSO-d6): δ ) 7.23 (d, 2H, J ) 8.9, ArH), 7.20
(d, 2H, J ) 9.0, ArH), 7.04 (d, 2H, J ) 8.9, ArH), 6.86 (s, 1H,
4-H), 6.81 (d, 2H, J ) 8.9, ArH), 3.81 (s, 3H, OCH3), 3.71 (s, 3H,
OCH3), 2.32 (q, 2H, J ) 7.5, CH2CH3), 1.05 (t, 3H, J ) 7.5,
CH2CH3).
2-(2-Chloro-4-hydroxyphenyl)-1,4-bis(4-hydroxyphenyl)-1H-
imidazole (3). From 3a (296 mg, 0.70 mmol) and BBr3 (266 µL,
2.81 mmol). Column chromatography with CH2Cl2/methanol 9:1.
Yield: 250 mg (94%) of a colorless solid (mp: 262 °C). 1H NMR
(DMSO-d6): δ ) 10.16 (s, 1H, OH), 9.68 (s, 1H, OH), 9.35 (s,
1H, OH), 7.72 (s, 1H, 5-H), 7.63 (d, 2H, J ) 8.6, ArH), 7.30 (d,
1H, J ) 8.1, ArH), 7.00 (d, 2H, J ) 8.7, ArH), 6.78-6.74 (m, 4H,
ArH), 6.71 (d, 2H, J ) 8.7, ArH). Anal. (C21H15ClN2O3) C, H, N.
1,2,4-Tris(4-hydroxyphenyl)-5-methyl-1H-imidazole (5). From
5a (687 mg, 1.72 mmol) and BBr3 (162 µL, 1.71 mmol). Column
chromatography with stepwise gradient elution: CH2Cl2/methanol
99:1, 95:5, 90:10. Yield: 223 mg (36%) of a colorless solid (mp:
Preparation of 1,2-Bis(4-methoxyphenyl)-1H-imidazole 21a.
4-Methoxybenzimidic Acid Ethyl Ester (12e). SOCl2 (5.14 mL,
70.59 mmol) was added over a period of 30 min to a stirred solution
of 4-methoxybenzonitrile (12d) (10 g, 75.11 mmol), ethanol (4.37
mL, 75.11 mmol), diethyl ether (4.40 mL), and water (1.10 mL) at
0-5 °C. The reaction mixture was stirred for 4 h at room
temperature and then stored in a refrigerator overnight and cooled
to -30 °C. The solution was kept at this temperature to provide a
colorless amorphous solid. The crude product was separated by
vacuum filtration, washed with diethyl ether, and treated for a short
time with 15% aqueous NaHCO3 solution (200 mL). The resulting
emulsion was extracted with CH2Cl2 (3 × 40 mL). The organic
layers were combined, dried over Na2SO4, and concentrated in
vacuum to obtain a colorless oil (6.53 g, 40%). 1H NMR (DMSO-
d6): δ ) 8.66 (s, 1H, CdNH), 7.78 (d, 2H, J ) 9.0, ArH), 6.97
(d, 2H, J ) 8.8, ArH), 4.22 (q, 2H, J ) 7.1, OCH2CH3), 3.80 (s,
3H, OCH3), 1.31 (t, 3H, J ) 7.1, OCH2CH3).
1
185 °C). H NMR (DMSO-d6): δ ) 9.90 (s, 1H, OH), 9.58 (s,
1H, OH), 9.36 (s, 1H, OH), 7.53 (d, 2H, J ) 8.4, ArH), 7.28 (d,
2H, J ) 8.7, ArH), 7.13 (d, 4H, J ) 8.9, ArH), 6.88 (d, 2H, J )
8.4, ArH), 6.83 (d, 2H, J ) 8.7, ArH), 2.12 (s, 3H, CH3). Anal.
(C22H18N2O3) C, H, N.
2-(2-Chloro-4-hydroxyphenyl)-1,4-bis(4-hydroxyphenyl)-5-
methyl-1H-imidazole (6). From 6a (220 mg, 0.51 mmol) and BBr3

Estrogen Receptor-Dependent Gene ActiVation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1483
(215 µL, 2.27 mmol). Column chromatography with CH2Cl2/
solid (mp: 119-125 °C). 1H NMR (DMSO-d6): δ ) 9.58 (s, 1H,
OH), 7.73 (d, 2H, J ) 7.6, ArH), 7.57-7.54 (m, 3H, ArH), 7.45
(m, 4H, ArH), 7.27 (t, 1H, J ) 7.3, ArH), 7.12 (d, 2H, J ) 8.6,
ArH), 6.61 (d, 2H, J ) 8.6, ArH), 2.63 (q, 2H, J ) 7.5, CH2CH3),
0.93 (t, 3H, J ) 7.4, CH2CH3). Anal. (C23H20N2O) C, H, N.
1,2,4-Tris(4-hydroxyphenyl)-5-phenyl-1H-imidazole (15). From
15a (377 mg, 0.82 mmol) and BBr3 (346 µL, 3.66 mmol). Column
chromatography with stepwise gradient elution: CH2Cl2/methanol
98:2, 95:5, 90:10. Yield: 255 mg (75%) of a colorless solid (mp:
> 300 °C). 1H NMR (DMSO-d6): δ ) 9.71 (s, 1H, OH), 9.63 (s,
1H, OH), 9.31 (s, 1H, OH), 7.29-7.25 (m, 5H, ArH), 7.20 (d, 2H,
J ) 8.7, ArH), 7.18-7.15 (m, 2H, ArH), 6.98 (d, 2H, J ) 8.7,
ArH), 6.66-6.61 (m, 6H, ArH). Anal. (C27H20N2O3) C, H, N.
methanol 9:1. Yield: 132 mg (67%) of a colorless solid (mp: 260
1
°C). H NMR (DMSO-d6): δ ) 10.13 (s, 1H, OH), 9.78 (s, 1H,
OH), 9.40 (s, 1H, OH), 7.50 (d, 2H, J ) 8.6, ArH), 7.22 (d, 1H, J
) 8.4, ArH), 7.02 (d, 2H, J ) 8.7, ArH), 6.82 (d, 2H, J ) 8.7,
ArH), 6.74 (d, 3H, J ) 8.7, ArH), 6.68 (dd, 1H, J ) 2.4, J ) 8.5,
ArH), 2.17 (s, 3H, CH3). Anal. (C22H17ClN2O3) C, H, N.
4-(2-Chloro-4-hydroxyphenyl)-1,2-bis(4-hydroxyphenyl)-5-
methyl-1H-imidazole (7). From 7a (246 mg, 0.57 mmol) and BBr3
(241 µL, 2.55 mmol). Reaction time after reaching room temper-
ature: 48 h. Column chromatography with stepwise gradient
elution: CH2Cl2/methanol 98:2, 95:5, 90:10. Yield: 182 mg (85%)
of a colorless solid (mp: 263 °C). 1H NMR (DMSO-d6): δ ) 9.92
(s, 2H, OH), 9.60 (s, 1H, OH), 7.36 (bs, 1H, ArH), 7.17 (bs, 4H,
ArH), 6.95-6.82 (m, 4H, ArH), 6.66 (bs, 2H, ArH), 1.91 (s, 3H,
CH3). Anal. (C22H17ClN2O3) C, H, N.
2-(2-Chloro-4-hydroxyphenyl)-1,4-bis(4-hydroxyphenyl)-5-
phenyl-1H-imidazole (16). From 16a (398 mg, 0.80 mmol) and
BBr3 (340 µL, 3.61 mmol). The solution was stirred for a period
of 48 h after reaching room temperature. Column chromatography
with CH2Cl2/methanol 9:1. Yield: 243 mg (67%) of a colorless
2,4-Bis(2-chloro-4-hydroxyphenyl)-1-(4-hydroxyphenyl)-5-
methyl-1H-imid azole (8). From 8a (281 mg, 0.60 mmol) and BBr3
(255 µL, 2.69 mmol). Column chromatography with CH2Cl2/
methanol 9:1. Yield: 200 mg (78%) of a colorless solid (mp: 254
1
solid (mp: 200-203 °C). H NMR (DMSO-d6): δ ) 10.11 (s,
1H, OH), 9.58 (s, 1H, OH), 9.31 (s, 1H, OH), 7.28-7.23 (m, 6H,
ArH), 7.19-7.15 (m, 2H, ArH), 6.86 (d, 2H, J ) 8.7, ArH), 6.80
(d, 1H, J ) 2.4, ArH), 6.69 (dd, 1H, J ) 2.37, J ) 8.4, ArH), 6.61
(d, 2H, J ) 8.7, ArH), 6.53 (d, 2H, J ) 8.6, ArH). Anal. (C27H19-
ClN2O3) C, H, N.
1
°C). H NMR (DMSO-d6): δ ) 10.04 (s, 1H, OH), 9.94 (s, 1H,
OH), 9.76 (s, 1H, OH), 7.31 (d, 1H, J ) 8.4, ArH), 7.19 (d, 1H, J
) 8.4, ArH), 7.02 (d, 2H, J ) 8.4, ArH), 6.91 (d, 1H, J ) 1.8,
ArH), 6.81 (dd, 1H, J ) 1.8, J ) 8.0, ArH), 6.74 (pd, 3H, J ) 9.0,
ArH), 6.67 (d, 1H, J ) 8.4, ArH), 1.96 (s, 3H, CH3). Anal. (C22H16-
Cl2N2O3) C, H, N.
1,2,4,5-Tetrakis(4-hydroxyphenyl)-1H-imidazole (17). From
17a (400 mg, 0.81 mmol) and BBr3 (460 µL, 4.87 mmol). Column
chromatography with stepwise gradient elution: CH2Cl2/methanol
9:1, 8:2. Yield: 218 mg (62%) of a colorless solid (mp: 200-203
2-(2-Chloro-4-hydroxyphenyl)-5-ethyl-1,4-bis(4-hydroxyphen-
yl-1H-imidazole (10). From 10a (239 mg, 0.53 mmol) and BBr3
(227 µL, 2.39 mmol). Column chromatography with CH2Cl2/
methanol 9:1. Yield: 205 mg (95%) of a colorless solid (mp: 140-
1
°C). H NMR (DMSO-d6): δ ) 9.69 (s, 1H, OH), 9.60 (s, 1H,
OH), 9.52 (s, 1H, OH), 9.27 (s, 1H, OH), 7.29 (d, 2H, J ) 8.6,
ArH), 7.18 (d, 2H, J ) 8.6, ArH), 6.97-6.93 (m, 4H, ArH), 6.66-
6.61 (m, 8H, ArH). Anal. (C27H20N2O4) C, H, N.
1
145 °C). H NMR (DMSO-d6): δ ) 10.05 (s, 1H, OH), 9.75 (s,
1H, OH), 9.35 (s, 1H, OH), 7.49 (d, 2H, J ) 8.6, ArH), 7.20 (d,
1H, J ) 8.4, ArH), 7.05 (d, 2H, J ) 8.7, ArH), 6.80 (d, 2H, J )
8.6, ArH), 6.74-6.72 (m, 3H, ArH), 6.65 (dd, 1H, J ) 2.3, J )
8.4, ArH), 2.60 (q, 2H, J ) 7.4, CH2CH3), 0.93 (t, 3H, J ) 7.4,
CH2CH3). Anal. (C23H19ClN2O3) C, H, N.
4-(2-Chloro-4-hydroxyphenyl)-1,2-bis(4-hydroxyphenyl)-5-
phenyl-1H-imidazole (18). From 18a (140 mg, 0.28 mmol) and
BBr3 (96 µL, 1.02 mmol). Addition of BBr3 at -80 °C over 1 h,
reaction time 20 h (-80 °C f RT). Column chromatography with
stepwise gradient elution: CH2Cl2/methanol 98:2, 95:5, 90:10.
Yield: 126 mg (98%) of a colorless solid (mp: >300 °C). 1H NMR
(DMSO-d6): δ ) 9.86 (s, 1H, OH), 9.77 (s, 1H, OH), 9.63 (s, 1H,
OH), 7.22 (d, 1H, J ) 8.3, ArH), 7.18-7.13 (m, 5H, ArH), 7.00
(d, 2H, J ) 8.3, ArH), 6.92-6.89 (m, 2H, ArH), 6.78 (d, 1H, J )
2.0, ArH), 6.72-6.68 (m, 3H, ArH), 6.64 (d, 2H, J ) 8.6, ArH).
Anal. (C27H19ClN2O3) C, H, N.
5-Ethyl-1,2-bis(4-hydroxyphenyl)-4-phenyl-1H-imidazole (11).
From 11a (256 mg, 0.67 mmol) and BBr3 (188 µL, 2.00 mmol).
Column chromatography with stepwise gradient elution: CH2Cl2/
methanol 98:2, 95:5. Yield: 219 mg (96%) of a colorless solid
(mp: 250-255 °C). 1H NMR (DMSO-d6): δ ) 9.93 (s, 1H, OH),
9.60 (s, 1H, OH), 7.72 (d, 2H, J ) 7.3, ArH), 7.42 (pt, 2H, J )
7.7, ArH), 7.26 (t, 1H, J ) 7.3, ArH), 7.17 (d, 4H, J ) 8.7, ArH),
6.88 (d, 2H, J ) 8.7, ArH), 6.63 (d, 2H, J ) 8.7, ArH), 2.60 (q,
2H, J ) 7.4, CH2CH3), 0.96 (t, 3H, J ) 7.4, CH2CH3). Anal.
(C23H20N2O2) C, H, N.
5-Ethyl-1,4-bis(4-hydroxyphenyl)-2-phenyl-1H-imidazole (12).
From 12a (215 mg, 0.56 mmol) and BBr3 (159 µL, 1.68 mmol).
Column chromatography with stepwise gradient elution: CH2Cl2/
methanol 98:2, 95:5. Yield: 128 mg (67%) of a colorless solid
(mp: 285 °C). 1H NMR (DMSO-d6): δ ) 9.94 (s, 1H, OH), 9.40
(s, 1H, OH), 7.53 (d, 2H, J ) 8.6, ArH), 7.36-7.33 (m, 2H, ArH),
7.28-7.23 (m, 2H, ArH), 7.18 (d, 3H, J ) 8.6, ArH), 6.88 (d, 2H,
J ) 8.6, ArH), 6.83 (d, 2H, J ) 8.6, ArH), 2.57 (q, 2H, J ) 7.4,
CH2CH3), 0.94 (t, 3H, J ) 7.4, CH2CH3). Anal. (C23H20N2O2) C,
H, N.
5-Ethyl-2,4-bis(4-hydroxyphenyl)-1-phenyl-1H-imidazole (13).
From 13a (169 mg, 0.44 mmol) and BBr3 (104 µL, 1.10 mmol).
Addition of BBr3 at -80 °C over 1 h, reaction time 18 h (-80 °C
f RT). Column chromatography with stepwise gradient elution:
CH2Cl2/methanol 98:2, 95:5, 90:10. Yield: 148 mg (94%) of a
colorless solid (mp: 149 °C). 1H NMR (DMSO-d6): δ ) 9.55 (s,
1H, OH), 9.36 (s, 1H, OH), 7.55-7.51 (m, 5H, ArH), 7.37-7.35
(m, 2H, ArH), 7.10 (d, 2H, J ) 8.7, ArH), 6.82 (d, 2H, J ) 8.6,
ArH), 6.59 (d, 2H, J ) 8.7, ArH), 2.57 (q, 2H, J ) 7.5, CH2CH3),
0.90 (t, 3H, J ) 7.4, CH2CH3). Anal. (C23H20N2O2) C, H, N.
5-Ethyl-1,4-diphenyl-2-(4-hydroxyphenyl)-1H-imidazole (14).
From 14a (100 mg, 0.28 mmol) and BBr3 (98 µL, 1.04 mmol).
Addition of BBr3 at -80 °C over 1 h, reaction time 16 h (-80 °C
f RT). Column chromatography with stepwise gradient elution:
CH2Cl2/methanol 98:2, 95:5. Yield: 57 mg (60%) of a colorless
5-Ethyl-2,4-bis(4-hydroxyphenyl)-1H-imidazole (19). From
13a (140 mg, 0.36 mmol) and BBr3 (104 µL, 1.10 mmol). Column
chromatography with stepwise gradient elution: CH2Cl2/methanol
98:2, 95:5, 90:10, 70:30. Yield: 44 mg (43%) of a colorless solid
1
(mp: 180 °C). H NMR (DMSO-d6): δ ) 12.40 (bs, 1H, NH),
9.83 (s, 1H, OH), 9.53 (s, 1H, OH), 7.81 (d, 2H, J ) 8.6, ArH),
7.40 (d, 2H, J ) 8.5, ArH), 6.87-6.83 (m, 4H, ArH), 2.70 (q, 2H,
J ) 7.5, CH2CH3), 1.23 (t, 3H, J ) 7.5, CH2CH3). Anal.
(C17H16N2O2) C, H, N.
5-Ethyl-1,2-bis(4-hydroxyphenyl)-1H-imidazole (20). From
20a (373 mg, 1.21 mmol) and BBr3 (349 µL, 3.68 mmol). Column
chromatography with stepwise gradient elution: CH2Cl2/methanol
98:2, 95:5, 90:10. Yield: 181 mg (53%); colorless solid (mp: 235
1
°C). H NMR (DMSO-d6): δ ) 9.84 (s, 1H, OH), 9.53 (s, 1H,
OH), 7.09 (d, 2H, J ) 8.7, ArH), 7.05 (d, 2H, J ) 8.6, ArH), 6.83
(d, 2H, J ) 8.6, ArH), 6.79 (s, 1H, 4-H), 6.60 (d, 2H, J ) 8.7,
ArH), 4.61 (q, 2H, J ) 7.4, CH2CH3), 1.04 (t, 3H, J ) 7.5,
CH2CH3). Anal. (C17H16N2O2) C, H, N.
1,2-Bis(4-hydroxyphenyl)-1H-imidazole (21). From 21a (200
mg, 0.71 mmol) and BBr3 (337 µL, 3.57 mmol). Column chroma-
tography with stepwise gradient elution: CH2Cl2/methanol 98:2,
95:5, 90:10. Yield: 60 mg (33%) of a colorless solid (mp: 279
1
°C). H NMR (DMSO-d6): δ ) 9.78 (s, 1H, OH), 9.61 (s, 1H,
OH), 7.26 (d, 1H, J ) 1.3, CH)CH), 7.13 (d, 2H, J ) 8.7, ArH),
7.06 (d, 2H, J ) 8.9, ArH), 7.04 (d, 1H, J ) 1.5, CH)CH), 6.80
(d, 2H, J ) 8.64, ArH), 6.65 (d, 2H, J ) 8.61, ArH). Anal.
(C15H12N2O2) C, H, N.

1484 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Wiglenda and Gust
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Biological Methods. Transcriptional Binding Assay. The
transactivation on the estrogen receptor was tested by using MCF-
7-2a cells, stably transfected with the plasmid EREwtcluc. The cells
were cultivated in DMEM supplemented with L-glutamine, antibiot-
ics, and dextran/charcoal-treated BCS (ct-BCS, 50 mL/L) one week
before starting the experiment. Cells from an almost confluent
monolayer were removed by trypsinization and suspended to
approximately 2.2 × 105 cells/mL in the growth medium mentioned
above. The cell suspension was then cultured in six-well flat-
bottomed plates (0.5 mL cell suspension and 1.5 mL medium per
well) at growing conditions (see above). After 24 h, 20 µL of a
stock solution of the test compounds were added to achieve
concentrations ranging between 10-5 to 10-10 M, and the plates
were incubated for 50 h. Before harvesting, the cells were washed
twice with PBS and 200 µL of cell lysis reagent was added to each
well. After 20 min lysis at room temperature, cells were transferred
into reaction tubes and centrifuged. Luciferase was assayed using
the Promega luciferase assay reagent. A 50 µL amount of each
supernatant was mixed with 50 µL of substrate reagent. Lumines-
cence (in relative light units, RLU) was measured for 10 s using a
microlumat. Measurements were corrected by correlating the
quantity of protein (quantified according to Bradford29) in each
sample with the mass of luciferase. Estrogenic activity was
expressed as % activation of a 10-8 M estradiol control (100%).
Estrogen Receptor Binding Assay. The relative binding affinity
of the test compounds was determined by displacement of 17â-
[3H]estradiol. At 4 °C the test compounds were shaken with calf
uterine cytosol and 17â-[3H]estradiol for 16 h. To stop the
incubation, dextran-coated charcoal was added, and after centrifuga-
tion, the radioactivity of a 200 µL supernatant aliquot was counted.
On a semilog plot the percentage of bound labeled steroid vs
concentration of the competitor was plotted. Six concentrations of
each compound were chosen to get a linear graph. From the plot,
the molar concentration of unlabeled estradiol and of the competitor
that reduced the binding of the radioligand by 50% was determined.
Acknowledgment. The technical assistance of S. Bergemann
and I. Schnautz is acknowledged. The presented study was
supported by Grants Gu285/3-1 and Gu285/3-2 from the
Deutsche Forschungsgemeinschaft.
Supporting Information Available: Elemental analyses of the
target compounds 3 and 5-21 and additional analytical data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(23) Jordan, V. C.; MacGregor Schafer, J. I.; Levenson, A. S.; Liu, H.;
Pease, K. M.; Simons, L. A.; Zapf, J. W. Molecular classification of
estrogens. Cancer Res. 2001, 61, 6619-6623.
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