Synthesis, structural evaluation, and estrogen receptor interaction of 4,5-Bis(4-hydroxyphenyl)imidazoles

Article abstract of DOI:10.1002/ardp.200290000

4,5-Bis(4-hydroxyphenyl)imidazoles with 2,2′-H (1), 2,2′-F (2), 2,2′-Cl (3), and 2,2′6-Cl (4) substituents in the aromatic rings were synthesized by oxidation of the respective methoxy-substituted (R,S)/(S,R)-4,5-diaryl-2-imidazolines with MnO₂

Full text of DOI:10.1002/ardp.200290000

Arch. Pharm. Pharm. Med. Chem. 2002, 10, 463–471
4,5-Bis(4-hydroxyphenyl)imidazoles 463
Ronald Gust,
Roland Keilitz,
Kathrin Schmidt,
Moriz von Rauch
Synthesis, Structural Evaluation, and Estrogen
Receptor Interaction of
4,5-Bis(4-hydroxyphenyl)imidazoles
Institute of Pharmacy,
4,5-Bis(4-hydroxyphenyl)imidazoles with 2,2Ј-H (1), 2,2Ј-F (2), 2,2Ј-Cl (3), and
2,2Ј6-Cl (4) substituents in the aromatic rings were synthesized by oxidation of the
respective methoxy-substituted (R,S)/(S,R)-4,5-diaryl-2-imidazolines with MnO2
and subsequent ether cleavage with BBr3. N-alkylation of 1 and 3 with ethyl iodide
yielded the compounds 5 and 6.The imidazoles were characterized by NMR spec-
troscopy and tested for estrogen receptor binding in a competition experiment with
[3H]estradiol using calf uterine cytosol. Gene activation was verified in a luciferase
assay using estrogen receptor positive MCF-7-2a cells stably transfected with the
plasmid EREwtcluc. All halide substituted imidazoles competed with estradiol for the
binding site at the estrogen receptor. The N-ethyl derivative 6 showed the highest
relative binding affinity of 1.26 %.Treatment of MCF-7-2a cells, however, did not lead
to gene activation.The relative activation of 6 amounted only to 10 % at 1µM com-
pared to E2 (100 %).
Free University of Berlin,
Königin-Luise Str. 2 + 4,
D-14195 Berlin, Germany
Keywords:Estrogenic activity;Luciferase assay;Estrogen receptor binding;4,5-Di-
arylimidazoles
Received: February 5, 2002 [FP672]
4OHT result from the orientation of the basic side chain
in a narrow side pocket. H bonding of the piperidine
Introduction
(RAL) or dimethylamino (4OHT) nitrogen to Asp 351
changes the position of the helix 12, which prevents
gene activation.
Estrogens are hormones that regulate numerous biolog-
ical effects in male and female [1–6].The effects are me-
diated by the estrogen receptor (ER).The attachment of
drugs causes a conformational change of the receptor,
dimerisation of the resulting ER/drug-conjugates, and
subsequent binding to the “Estrogen Response Ele-
ment” (ERE) of the DNA [7].This cascade finally leads to
gene activation.For the design of new selective estrogen
receptor modulators (SERM), a knowledge of the bind-
ing mode is essential. While for decades the binding
mode of estradiol (E2) and related agonists, e.g. diethyl-
stilbestrol (DES), but also of antagonists, e.g. raloxifene
(RAL) or 4-hydroxytamoxifen (4OHT) (formulae,
Chart 1), was speculative, the X-ray structures of the
ligand binding domain (LBD) of the ER co-crystallized
with these drugs are now available [8–10]. It could be
shown that agonists and antagonists are primarily
H-bonded to the carboxylate of Glu 353, the guanidinium
residue of Arg 394, and a water molecule. A second
essential anchor to cause estrogenic activity is His 524.
Due to this interaction, helix 12 as part of the LBD is ori-
ented over the binding cavity, and protects the drug from
the environment. The antagonistic effects of RAL and
In a previously published structure activity study we
identified (R,S)/(S,R)-configurated 2,3-diarylpiper-
Correspondence: Ronald Gust, Institut für Pharmazie der
FU Berlin, Königin Luise Str. 2 + 4, D-14195 Berlin, Germany.
Phone:+49 (0)30 838 53272, fax:+49 (0)30 838 56906, e-mail:
rgust@zedat.fu-berlin.de.
Chart 1.The structural formulae of compounds referred
to in the text.
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/10/0463 $ 17.50+.50/0

464 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
azines and 4,5-diaryl-2-imidazolines as new lead struc-
tures for the design of SERM [11]. We could show that
the molecules must bear two hydroxy groups and hydro-
phobic substituents in the aromatic rings. In this class of
compounds, the essential H-bridge to His 524 can be re-
placed by another H-bridge, presumably to Asp 351.The
pharmacophoric 1,2-diarylethane moiety [12] takes in
(R,S)/(S,R)-2,3-diarylpiperazines a synclinal (dihedral
angle about 40–60°, see 7 in Figure 1) and in (R,S)/
(S,R)-4,5-diaryl-2-imidazolines an eclipsed conforma-
tion (pseudo axially oriented aromatic rings:dihedral an-
gle about 0–10° see 8 in Figure 1) [13, 14].Since the spa-
tial structure of the latter induces significantly higher
gene activation, we investigated the ER binding proper-
ties of 4,5-diarylimidazoles, since at the heterocyclic ring
the pharmacophor is fixed in a stable cis-configuration
comparable to the conformation in imidazolines.The in-
fluence of the substitution pattern in the aromatic rings
on the ER binding and the gene activation in MCF-7-2a
cells is described.
Since by this reaction the asymmetric character of the
molecules is lost, imidazoles could be generated either
from (R,S)/(S,R)- or (R,R)/(S,S)-configurated 2-imidazo-
lines. However, the (R,R)/(S,S)-configurated com-
pounds cannot be oxidized, since the aromatic rings,
located above and below the heterocyclic plane hinder
the contact of the manganese dioxide.
For N-alkylation, the acidic character of the imidazole
ring is utilized. The reaction with bases increases the
nucleophilic character of the imidazole nitrogens and N-
alkylation with ethyl iodide can be achieved.While alkyla-
tion of imidazole 1 a was performed nearly quantitatively
with sodium ethanolate, the same reaction failed with the
4,5-bis(2-chloro-4-methoxyphenyl)imidazole 3 a. Only
the use of sodium hydride to convert 3 a into a defined
sodium salt and the subsequent reaction with ethyl
iodide yielded N-ethyl-4,5-bis(2-chloro-4-methoxyphe-
nyl)imidazole 6 a in satisfactory amounts. The hydroxy
substituted imidazoles 1 to 6 were generated by ether
cleavage with BBr3.
Synthesis
Structural evaluation
4,5-Diarylimidazoles were synthesized by oxidation of
4,5-diaryl-2-imidazolines [14] with MnO2 (Scheme 1)
analogously to the method described by Martin et al.
[15].
The imidazole moiety represents a planar aromatic ring
system, which forces all substituents into one plane. In
Figure 1 a low energy conformation of 4-(2-chloro-4-
hydroxyphenyl)-5-(2,6-dichloro-4-hydroxyphenyl)imid-
Scheme 1. Synthesis of 4,5-diarylimidazoles.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
4,5-Bis(4-hydroxyphenyl)imidazoles 465
azole 4 is shown as an example. The cis-configurated
aromatic rings (dihedral angle: 5.8°) are twisted and ori-
ented to the imidazole ring at angles of 70.0° (2-chloro-4-
hydroxyphenyl ring) and 40.1° (2,6-dichloro-4-hydroxy-
phenyl ring). The O–O distance amounts to 8.9 Å.
The structural characterization of the imidazoles was
performed by NMR spectroscopy.In the 1H NMR spectra
of the 4,5-bis(4-hydroxyphenyl)imidazole 1 taken in
[D6]-DMSO (Figure 2) the NH signal is broad and weak
indicating a proton exchange reaction between the nitro-
Figure 1. Low energy conformations of the 2-chloro-4-hydroxy/2Ј,6Ј-dichloro-4Ј-hydroxy substituted 2,3-di-
arylpiperazine 7, 4,5-diaryl-2-imidazoline 8 and 4,5-diarylimidazole 4.
1
Figure 2. 400 MHz H NMR spectra of the imidazoles 1 and 3 in [D6]-DMSO.

466 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
Figure 3. 300 MHz 1H NMR spectra of the imidazole 3 at room temperature (A), at high temperature (B) and after HT
measurement (C); solvent [D6]-DMSO.
1
Figure 4. 400 MHz H NMR spectra of the imidazole 3 in various solvents.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
4,5-Bis(4-hydroxyphenyl)imidazoles 467
gens [16]. Hereby, the 4- and 5-position of the imidazole
become chemically equivalent and only one set of reso-
nances appears in the spectra.
CF3COOD to the [D6]-DMSO solution fastens the proc-
ess and the splitting collapses (Figure 4). Furthermore,
in [D4]-methanol there is only one set of signals for
H1a + b, H3a + b, and H4a + b, respectively (see Figure
4), which indicates an equivalence of the 4- and 5-posi-
tion in 3.These findings show that a stable NH position or
a very slow proton reaction in DMSO led to an asymmet-
ric molecule structure.
The substituents in the 2-positions of the aromatic rings
double the resonances. In the spectra of the 4,5-bis-
(2-halo-4-hydroxyphenyl)imidazoles 2 and 3 (3 see Fig-
ure 2) two signal groups for the protons 1 a + b, 3 a + b,
and 4 a + b are present. The protons H2a and H2b are
isochronous. This splitting could either be the conse-
quence of a hindered rotation of the aromatic rings or an
asymmetric imidazole moiety due to a stably bound NH.
Biological activity
To gain further information, high temperature measure-
ments (HT) were performed (Figure 3).If rotational isom-
erism exists, the signals should coalesce and during the
cooling down to room temperature the equilibrium be-
tween both isomers should take place once more, or the
most stable isomer should originate.
The evaluation of the relative binding affinity (RBA) was
performed in the competition experiment with
[3H]estradiol ([3H]-E2) [17]. Additionally, a luciferase as-
say was used to obtain information about the agonistic
effects on molecular level [18].For this purpose, ER-pos-
itive MCF-7 mammary carcinoma cells were stably
transfected with the plasmid EREwtcluc. After binding of
hormonally active compounds to the ER, the resulting
ER/drug conjugates interact after dimerization with the
ERE at the plasmid and activate the reporter sequence
which codes for luciferase. The expression of luciferase
correlates very well with the estrogenic potency of the
drug [19].
Indeed, the signals H1a + b at 6.73 and 6.84 ppm,
H3a + b at 6.94 and 7.09 ppm, and H4a + b at 9.79 and
9.97 ppm in the spectra of 3, coalesce during heating to
413 K, respectively. After HT measurement the spectra
show signals at 6.78 ppm (H1a + b), 7.01 ppm (H3a + b),
and 9.89 ppm (H4a + b). These resonances cannot be
assigned to one of the set of resonances in the start
spectra (293 K) but represent the average of the respec-
tive signals. The same effect was detected by tempera-
ture dependent measurement of 13C NMR spectra (data
not shown), so it can be concluded that this effect is not
the result of a restricted rotation of the aromatic rings.
With the exception of the 4,5-bis(4-hydroxyphenyl)imi-
dazole 1 and its alkyl derivative 5 all other compounds
were able to compete with E2 for its binding site (Table
1). Introduction of 2-F or 2-Cl substituents in the 4-hy-
droxyphenyl rings increased the RBA from < 0.02 % (1)
to 0.05 % (2) and 0.11 % (3).Further enhancement of the
binding affinity by introduction of substituents in the aro-
matic ring failed. However, N-alkylation of 3 increased
the RBA value enormously to RBA = 1.26 %.
In the start spectra of 3 the NH resonance is sharp but
shows an increase in width and flatness after HT meas-
urement (see Figure 3). This indicates the presence of
tautomeric and proton exchange processes. Addition of
Table 1. Relative binding affinity and relative activation in the luciferase assay of the imidazoles 1 to 6.
Rel. activation [%]
at 1 µM
–OH
–OCH3
R1
R2
R3
RBA [%]
derivatives
derivatives
1
2
3
4
5
6
H
F
Cl
Cl
H
H
H
H
Cl
H
H
H
H
H
H
ethyl
ethyl
< 0.02
0.05
0.11
0.10
< 0.02
1.26
3
2
8
27
4
10
7
3
–3
6
6
5
Cl

468 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
Although the estrogen receptor binding of imidazoles
was verified, the luciferase expression could not be acti-
vated in high amounts. While the imidazoles 1 (rel. acti-
vation: 3 %), 2 (rel. activation: 2 %), and 3 (rel. activation:
8 %) are completely inactive at 1µM (rel.activation of E2:
100 %), the threefold Cl-substituted compound 4 repre-
sents, with 27 %, the most active compound of this se-
ries. Interestingly, N-alkylation of 3 did not change the
estrogenic activity although the RBA value was more
than 10 times higher. O-Methylation of 4 led to a com-
plete loss of activity and confirmed the relevance of free
hydroxy groups for the receptor binding.
to its low stability [12]. Under the in vitro condition, a
fast conversion into the E-isomer takes place. Howev-
er, if the hydroxy group in the 1-phenyl ring is shifted
into the 3- or 5-position, the Z-isomers are stable
enough for testing. In the competition experiment with
[3H]-E2 the Z-isomers are half as active as the E-iso-
mer and possess the hormonal profile of partial estro-
gens [12]. In estrogenically active 1,2-bis(4-acetoxy-
phenyl)-1-phenylbut-2-enes the Z-1,2-bis(4-hydroxy-
phenyl)ethene structure is also realized. A comparison
of the results obtained with the pure E- and Z-isomers
indicates identical activity in the luciferase assay (acti-
vation about 60 % at 1 µM) [23]. These positive results
prompted us to fix the 1,2-diphenylethane pharma-
cophor in a planar 4,5-diarylimidazole structure. The
aromatic rings are fixed in the heterocyclic plane and
the O–O distance amounts to 8.8 Å–8.9 Å.
Discussion
(R,S)/(S,R)-configurated
1,2-diarylethylenediamines
Compared to the piperazines and 2-imidazolines, the
imidazoles could displace E2 from its binding site. The
RBA value increased with the number of hydrophobic
substituents in the aromatic rings. 4-(2-Chloro-4-
hydroxyphenyl)-5-(2,6-dichloro-4-hydroxyphenyl)imid-
azole 4 displaced E2 from the LBD to 0.1 %. N-Ethyla-
tion of 3 results in the most active compound (6) in
this series. Imidazole 6 showed an RBA value of
1.26 %. O-Methylation led to a complete loss of bind-
ing affinity. These results led to the assumption that
imidazoles are bound at the LBD by a combination of
hydrophobic contacts and H bridges. Interestingly, de-
spite the high RBA = 1.26 %, 6 did not activate luci-
ferase expression in MCF-7-2a cells. The non-alkylat-
ed compound 3 was even more active and achieved in
a concentration of 1 µM a relative activation of 27 %.
derived from the synthetic estrogen DES were used as
carrier ligands for the cytostatic metal complex cisplatin.
Dependent on the substitution pattern, the resulting [1,2-
diarylethylenediamine]platinum(II) complexes possess
high estrogenic potency [20–22]. Interestingly, the spa-
tial structures of the molecules differ strongly from that of
known steroidal or synthetic estrogens.In contrast to the
parent compound DES, the 1,2-diarylethane pharma-
cophor takes on a synclinal conformation in hormonally
active complexes and can not be attached to identical
binding sites at the ER.
To gain a better insight into the binding mode, we synthe-
sized drugs with comparable spatial structure [11]. In a
preliminary study we fixed the 1-(2-chloro-4-hydroxy-
phenyl)-2-(2,6-dichloro-4-hydroxyphenyl)ethane phar-
macophor as 2,3-diarylpiperazine and 4,5-diaryl-2-imid-
azoline [11].
4,5-Bis(4-hydroxyphenyl)imidazoles were already syn-
thesized and tested by Fink et al. [24]. In accordance
with our results, the alkyl substituents at the 1-position
increased the RBA. However, this effect depends on
an additional 4-OH-phenyl ring in the 2-position. As
most active compound the 2,4,5-tris(4-hydroxyphenyl)-
1-propylimidazole possesses an RBA value of 0.62 %.
The (R,S)/(S,R)-2,3-diarylpiperazine 7 takes on several
low energy chair and twist conformations, with the 2,6-
dichloro-4-hydroxyphenyl ring being equatorially ar-
ranged and the neighbouring 2-chloro-4-hydroxyphenyl
ring standing axially in each energetically preferred
structure [11, 13]. The dihedral angle between the aro-
matic rings varies from 39.4° to 47.9° with O–O dis-
tances of 6.5 Å–7.1 Å.
As interpreted by Fink et al. [24] the overall relatively
low binding affinity of the imidazoles is due to the high
inherent polarity of the heterocyclic system and the
high dipole moment. These effects can be overcome
by the introduction of hydrophobic Cl substituents in
the ortho positions of the aromatic rings and N-alkyla-
tion as shown in this structure activity study. Therefore,
we investigated the dependence of the relative binding
affinity and gene activation of 2,4,5-tris(4-hydroxyphe-
nyl)imidazole on Cl substituents in the ortho positions.
In the (R,S)/(S,R)-4,5-diaryl-2-imidazoline 8, the hetero-
cyclic ring is planar and the phenyl substituents are
pseudo axially oriented in a dihedral angle of 2.8° [11,
14]. The 1,2-diphenylethane moiety is therefore ar-
ranged in a Z-stilbene-like structure.
In a previously published structure-activity study, we in-
vestigated the binding affinity and the hormonal activity
of Cl/OH-substituted stilbenes. Unfortunately, data from
the Z-configurated 1-(2-chloro-4-hydroxyphenyl)-2-(2,6-
dichloro-4-hydroxyphenyl)ethene are not available due
The results will be published in
paper.
a forthcoming

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
4,5-Bis(4-hydroxyphenyl)imidazoles 469
4-(2-Chloro-4-methoxyphenyl)-5-(2,6-dichloro-4-methoxyphe-
nyl)imidazole (4 a)
Experimental
General procedures
From (4R,5S)/(4S,5R)-4-(2-chloro-4-methoxyphenyl)-5-(2,6-
dichloro-4-methoxyphenyl)-2-imidazoline 4 b: 0.920 mmol
(355 mg). Purification by column chromatography on silica gel
with ethyl acetate.Yield: 0.521 mmol (200 mg), 57 %; colorless
powder, mp 76 –78°C. IR (KBr): ν= 3433 s, br (NH); 2966 w;
2839 w (OCH3); 1668 m; 1608 s; 1551 m; 1504 s; 1465 m; 1362
IR spectra (KBr pellets): Perkin Elmer Model 580 A. 1H NMR:
Bruker ADX 400 spectrometer at 400 MHz (internal standard:
TMS). Elemental analyses: Microlaboratory of the Free Univer-
sity of Berlin.EI-MS spectra:CH-7A-Varian MAT (70 eV) or Kra-
tos MS 25 RF (80eV). All computational graphics were built us-
ing SYBYL 6.6, Tripos Inc., 1699 South Hanley Rd., St. Louis,
Missouri, 63144, USA. Geometry optimization was carried out
using the Tripos force field within the SYBYL program, running
on an INDY workstation. Liquid Scintillation Counter: 1450
MicrobetaTM Plus (Wallac, Finland). Microlumat: LB 96 P
(EG & G Berthold, Germany).
1
w; 1289 s; 1241 s; 1182 m; 1043 s; 840 m; 805 m. H NMR
(CDCl3): δ = 3.78 (s, 3 H, OCH3); 3.79 (s, 3 H, OCH3); 6.68 (dd,
3J = 8.6 Hz, 4J = 2.5 Hz, 1 H, ArH-5); 6.88 (s, 2 H, ArЈH); 6.94 (d,
4J = 2.5 Hz, 1 H, ArH-3); 7.09 (d, 3J = 8.6 Hz, 1 H, ArH-6); 7.80
(s, 1 H, N=CH-N).
N-Ethyl-4,5-bis(4-methoxyphenyl)imidazole (5 a)
A solution of 2.93 mmol (821 mg) 4,5-bis(4-methoxyphenyl)-
imidazole 1 a, 3.22 mmol (502 mg, 260 µL) ethyl iodide and
3.22 mmol (174 mg) sodium ethanolate in 20 mL of dry 1,4-di-
oxane was heated to reflux for 12 h. After cooling, 10 mL of
0.1 N NaOH were added and the product was extracted with
CHCl3. The organic layer was dried over Na2SO4 and the sol-
vent was evaporated.Purification of the crude product was per-
formed by column chromatography on silica gel with diethyl
ether/methanol (9 + 1).Yield: 1.95 mmol (600 mg), 66 %; pale
yellow oil. IR (Film): ν= 2935 m; 2836 m (OCH3); 1613 m; 1576
w; 1502 s; 1344 w; 1267 s; 1177 m; 1104 m; 950 m; 825 s. 1H
Syntheses
(1R,2S)/(1S,2R)-4,5-diaryl-2-imidazolines 1 b to 4 b were syn-
thesized as described earlier [14].
General procedure for the synthesis of 4,5-diarylimidazoles
To
a solution of the respective 4,5-diaryl-2-imidazoline
(1.0 mmol) in 20 mL dry CHCl3 6.00 mmol of MnO2 were add-
ed. After stirring the reaction mixture for 24 h, the manganese
dioxide was filtered off. Removal of the solvent left the crude
product, which was purified either by column chromatography
on silica gel or by recrystallization.
3
NMR (CDCl3): δ = 1.27 (t, J = 7.3 Hz, 3 H, CH2CH3); 3.76 (s,
3
3 H, OCH3); 3.81 (q, J = 7.3 Hz, 2 H, CH2CH3); 3.87 (s, 3 H,
OCH3); 6.76 (AAЈBBЈ, 3J = 8.7 Hz, 2 H, ArH-3, ArH-5); 6.99
3
3
4,5-Bis(4-methoxyphenyl)imidazole (1 a)
(AAЈBBЈ, J = 8.5 Hz, 2 H, ArH-3, ArH-5); 7.25 (AAЈBBЈ, J =
8.5 Hz, 2 H, ArH-2, ArH-6);7.41 (AAЈBBЈ, 3J = 8.7 Hz, 2 H, ArH-
2, ArH-6); 7.59 (s, 1 H, N=CH-N).
From (4R,5S)/(4S,5R)-4,5-bis(4-methoxyphenyl)-2-imidazo-
line 1 b: 1.24 mmol (350 mg). Purification by column chroma-
tography on silica gel with methanol. Yield: 1.21 mmol (338
mg), 97 %;colorless powder, mp 178–180 °C.IR (KBr):ν= 3416
m, br (NH); 2838 w (OCH3); 1615 m; 1524 s; 1508 s; 1293 m;
1251 s; 1171 m; 1107 m; 1038 m; 833 s. 1H NMR (CDCl3): δ =
3.82 (s, 6 H, OCH3);6.86 (AAЈBBЈ, 3J = 8.0 Hz, 4 H, ArH-3, ArH-
5); 7.42 (AAЈBBЈ, 3J = 8.0 Hz, 4 H, ArH-2, ArH-6); 7.59 (s, 1 H,
N=CH-N); 9.67 (br, 1 H, NH, exchangeable by D2O).
N-Ethyl-4,5-bis(2-chloro-4-methoxyphenyl)imidazole (6 a)
To a solution of 0.510 mmol (178 mg) 4,5-bis(2-chloro-4-meth-
oxyphenyl)imidazole 3 a in 20 mL of dry THF 0.550 mmol
(22 mg) sodium hydride (60 % in paraffin) and 0.550 mmol (86
mg, 44 µL) ethyl iodide were added and heated to reflux for 4 h.
Subsequently, the reaction mixture was hydrolysed with 20 mL
of water and the product was extracted with CHCl3.The organic
layer was dried over Na2SO4 and the solvent was distilled off.
Yield: 0.498 mmol (188 mg), 98 %; pale yellow oil. IR (KBr): ν=
2959 m; 2839 w (OCH3); 1610 s; 1570 m; 1509 s; 1478 s; 1441
w;1409 w;1289 s;1260 w;1227 s;1041 s;954 m;870 m;844 w;
4,5-Bis(2-fluoro-4-methoxyphenyl)imidazole (2 a)
From (4R,5S)/(4S,5R)-4,5-bis(2-fluoro-4-methoxyphenyl)-2-
imidazoline 2 b: 2.77 mmol (880 mg). Purification by column
chromatography on silica gel with diethyl ether/methanol
(4 + 1).Yield: 1.59 mmol (503 mg), 58 %; colorless powder, mp
187–189 °C.IR (KBr):ν= 3440 s, br (NH);2840 m (OCH3);1627
s; 1577 m; 1525 s; 1480 w; 1425 w; 1293 s; 1241 m; 1193 m;
1148 s;1102 s;1035 m;946 m;856 m;813 m. 1H NMR (CDCl3):
δ = 3.81 (s, 6 H, OCH3); 6.62–6.67 (m, 4 H, ArH-3, ArH-5);
7.25–7.30 (m, 2 H, ArH-6); 7.72 (s, 1 H, N=CH-N).
3
819 m; 754 m. 1H NMR (CDCl3): δ = 1.31 (t, J = 7.4 Hz, 3 H,
CH2CH3); 3.71–3.91 (m, 2 H, CH2CH3); 3.75 (s, 3 H, OCH3);
3.80 (s, 3 H, OCH3); 6.68 (dd, 3J = 8.5 Hz, 4J = 2.6 Hz, 1 H, ArH-
5); 6.76 (dd, 3J = 8.5 Hz, 4J = 2.6 Hz, 1 H, ArH-5); 6.87 (d, 4J =
2.6 Hz, 1 H, ArH-3); 6.98 (d, 4J = 2.6 Hz, 1 H, ArH-3); 7.10 (d, 3J
= 8.5 Hz, 1 H, ArH-6); 7.18 (d, 3J = 8.5 Hz, 1 H, ArH-6); 7.68 (s,
1 H, N=CH-N).
General procedure for the ether cleavage with BBr3
4,5-Bis(2-chloro-4-methoxyphenyl)imidazole (3 a)
A solution of the methyl ether (1.00 mmol) in 20 mL of dry
CH2Cl2 was cooled to –60 °C. At this temperature BBr3
(4.5 mmol) in 5 mL of dry CH2Cl2 was added under N2 atmos-
phere.Then the reaction mixture was allowed to warm to room
temperature and was stirred for a further 48 h. After cooling the
reaction mixture with an ice bath, the surplus of BBr3 was hy-
drolyzed three times with methanol and the phenolic product
was dissolved in 0.1 N NaOH.The alkaline water phase was fil-
tered and the pH was adjusted to 8 with 2 N HCl. The precipi-
tate was collected by suction filtration and dried over P2O5.Sub-
sequently, the crude product was purified if necessary by col-
umn chromatography or fractional crystallization.
From (4R,5S)/(4S,5R)-4,5-bis(2-chloro-4-methoxyphenyl)-2-
imidazoline 3 b:1.51 mmol (530 mg).Purification by recrystalli-
zation from CHCl3/ligroine.Yield: 0.659 mmol (230 mg), 44 %;
colorless powder, mp 194–196 °C.IR (KBr):ν= 3420 s, br (NH);
2838 m (OCH3); 1610 s; 1564 m; 1515 s; 1498 s; 1466 s; 1439
m; 1289 s; 1226 s; 1046 s; 963 m; 864 m; 815 m. MS (EI,
160 °C): m/z (%) = 348 (100) [M]+·; 333 (8) [M–CH3]+; 235 (14);
197 (19); 170 (79). 1H NMR (CDCl3): δ = 3.79 (s, 6 H, OCH3);
6.72 (dd, 3J = 8.6 Hz, 4J = 2.5 Hz, 2 H, ArH-5); 6.92 (d, 4J = 2.5
Hz, 2 H, ArH-3); 7.13 (d, 3J = 8.6 Hz, 2 H, ArH-6); 7.82 (s, 1 H,
N=CH-N); 9.76 (s, 1 H, NH, exchangeable by D2O).

470 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
N-Ethyl-4,5-bis(4-hydroxyphenyl)imidazole (5)
4,5-Bis(4-hydroxyphenyl)imidazole (1)
From N-ethyl-4,5-bis(4-methoxyphenyl)imidazole 5 a: 1.51
mmol (467 mg). Purification by recrystallization from acetone.
Yield: 1.28 mmol (360 mg), 85 %; colorless powder, mp 251–
253 °C. IR (KBr): ν = 3600–2600 s, br (OH); 2984 w; 1613 s;
1522 s; 1497 s; 1445 m; 1347 m; 1249 s; 1171 m; 1100 m; 961
From 4,5-bis(4-methoxyphenyl)imidazole 1 a:0.892 mmol (250
mg).Yield: 0.674 mmol (170 mg), 76 %; grey powder, mp 307–
310 °C. IR (KBr): ν= 3600–2800 m, br (OH); 3583 s (NH); 3357
m; 3210 m; 1616 m; 1521 m; 1508 m; 1481 m; 1462 m; 1243 s;
1168 m; 1102 w; 969 w; 835 s. 1H NMR ([D6]-DMSO): δ = 6.71
3
m; 838 s. 1H NMR ([D6]-DMSO): δ = 1.12 (t, J = 7.1 Hz, 3 H,
3
3
(AAЈBBЈ, J = 8.4 Hz, 4 H, ArH-3, ArH-5); 7.24 (AAЈBBЈ, J =
8.4 Hz, 4 H, ArH-2, ArH-6); 7.62 (s, 1 H, N=CH-N); 9.41 (s, 2 H,
ArOH, exchangeable by D2O);12.15 (s, 1 H, NH, exchangeable
by D2O). Anal. (C15H12N2O2) 252.27: calc.: C 71.42 H 4.79 N
11.10 found: C 71.40 H 4.90 N 11.06.
CH2CH3);3.74 (q, 3J = 7.1 Hz, 2 H, CH2CH3);6.56 (AAЈBBЈ, 3J =
8.3 Hz, 2 H, ArH-3, ArH-5); 6.85 (AAЈBBЈ, 3J = 8.1 Hz, 2 H, ArH-
3, ArH-5); 7.11 (AAЈBBЈ, 3J = 8.1 Hz, 2 H, ArH-2, ArH-6); 7.19
(AAЈBBЈ, 3J = 8.3 Hz, 2 H, ArH-2, ArH-6);7.70 (s, 1 H, N=CH-N);
9.21 (s, br, 1 H, ArOH, exchangeable by D2O); 9.66 (s, br, 1 H,
ArOH, exchangeable by D2O). Anal. (C17H16N2O2) 280.33:
calc.: C 72.84 H 5.75 N 9.99 found: C 72.84 H 5.85 N 9.89.
4,5-Bis(2-fluoro-4-hydroxyphenyl)imidazole (2)
From 4,5-bis(2-fluoro-4-methoxyphenyl)imidazole 2 a: 0.696
mmol (220 mg).Yield: 0.663 mmol (191 mg), 95 %; colorless
powder, mp 298 °C under decomposition. IR (KBr): ν= 3600–
2400 s, br (OH); 2813 w; 1630 s; 1596 m; 1524 m; 1459 s; 1304
s; 1258 m; 1154 s; 1119 m; 1100 m; 1058 m; 971 s; 848 s. MS
(EI, 230 °C):m/z (%) = 288 (100) [M]+·;268 [M–F]+ (5);239 (12).
N-Ethyl-4,5-bis(2-chloro-4-hydroxyphenyl)imidazole (6)
From N-ethyl-4,5-bis(2-chloro-4-methoxyphenyl)imidazole 6 a:
0.477 mmol (180 mg). Purification by column chromatography
on silica gel with acetone.Yield: 0.395 mmol (138 mg), 83 %,
yellow powder, mp 100–103 °C. IR (KBr): ν= 3600–2500 s, br
(OH);2968 w;2927 m;2760 w;2651 w;1700 m;1613s;1572 m;
1512 s; 1444 s; 1345 m; 1288 s; 1224 s; 1129 m; 1069 m; 1045
m; 901 s; 857 m; 823 s. MS (EI, 190 °C): m/z (%) = 348 (100)
[M]+·; 319 (5) [M–C2H5]+; 313 (17) [M–Cl]+; 284 (17) [M–Cl–
C2H5]+; 278 (10) [M–2Cl]+; 249 (6) [M–2Cl–C2H5]+; 139 (27). 1H
NMR ([D6]-acetone): δ = 1.27 (t, 3J = 7.3 Hz, 3 H, CH2CH3);
3.77–3.87 (m, 1 H, CH2CH3);3.89–3.99 (m, 1 H, CH2CH3);6.67
(dd, 3J = 8.4 Hz, 4J = 2.5 Hz, 1 H, ArH-5); 6.80 (d, 4J = 2.5 Hz,
1 H, ArH-3); 6.81 (dd, 3J = 8.4 Hz, 4J = 2.5 Hz, 1 H, ArH-5); 6.96
1H NMR ([D6]-DMSO): δ = 6.46 (dd, J(H, F) = 12.1 Hz, J =
1.7 Hz, 1 H, ArH-3); 6.55–6.63 (m, 3 H, ArH-3, 2 x ArH-5); 7.03
(dd, 3J = 8.8 Hz, 4J(H, F) = 8.8 Hz, 1 H, ArH-6);7.26 (dd, 3J = 8.7
Hz, 4J(H, F) = 8.7 Hz, 1 H, ArH-6); 7.76 (s, 1 H, N=CH-N); 9.79
(s, 1 H, ArOH, exchangeable by D2O); 9.99 (s, 1 H, ArOH, ex-
changeable by D2O);12.30 (s, 1 H, NH, exchangeable by D2O).
Anal. (C15H10F2N2O2) 288.25: calc.: C 62.50 H 3.50 N 9.72
found: C 62.54 H 3.45 N 9.66.
3
4
4
3
(d, J = 2.5 Hz, 1 H, ArH-3); 7.12 (d, J = 8.4 Hz, 1 H, ArH-6);
7.13 (d, 3J = 8.4 Hz, 1 H, ArH-6);7.76 (s, 1 H, N=CH-N);8.86 (br,
1 H, ArOH, exchangeable by D2O); 9.21 (br, 1 H, ArOH, ex-
changeable by D2O). Anal. (C17H14Cl2N2O2) 349.22: calc.: C
58.42 H 4.01 N 8.02 found: C 58.24 H 4.22 N 7.91.
4,5-Bis(2-chloro-4-hydroxyphenyl)imidazole (3)
From 4,5-bis(2-chloro-4-methoxyphenyl)imidazole 3 a: 0.458
mmol (160 mg). Purification by stirring in hot acetone. Yield:
0.316 mmol (114 mg), 77 %;colorless powder, mp 295–297 °C.
IR (KBr): ν= 3600–2300 s, br (OH); 3407 w; 3258 w; 2767 w;
2654 w;1611 s;1513 m;1470 s;1445 s;1363 w;1280 s;1222 s;
1051 m;1028 m;900 s;861 m;821 s.MS (EI, 250 °C):m/z (%) =
320 (100) [M]+᭹; 284 (17) [M–Cl]+; 250 (29) [M–2Cl]+; 221 (15);
Biological methods
Biochemicals, chemicals and materials
1
3
139 (14); 125 (14). H NMR ([D6]-DMSO): δ = 6.66 (dd, J =
Dextran, 17β-estradiol, L-glutamine (L-glutamine solution:
29.2 mg/mL phosphate buffered saline (PBS)), and Minimum
Essential Medium Eagle (EMEM) were purchased from Sigma
(Munich, Germany); Dulbecco’s Modified Eagle Medium with-
out phenol red (DMEM) was from Gibco (Eggenstein, Germa-
ny); bovine calf serum (BCS) was from Bio Whittaker (Verviers,
Belgium);N-hexamethylpararosaniline (crystal violet) and gen-
tamicin sulfate were from Fluka (Deisenhofen, Germany); glu-
tardialdehyde (25 %) was from Merck (Darmstadt, Germany);
trypsin (0.05 %) in ethylenediaminetetraacetic acid (0.02 %)
(trypsin/EDTA) was from Boehringer (Mannheim, Germany);
penicillin-streptomycin gold standard (10 000 IE penicillin/mL,
10 mg streptomycin/mL) and geneticin disulfate (geneticin so-
lution: 35.71 mg/mL PBS) were from ICN Biomedicals GmbH
(Eschwege, Germany);norit A (charcoal) was from Serva (Hei-
delberg, Germany); cell culture lysis reagent (5×) (diluted 1 : 5
with purified water before use) and the luciferase assay reagent
were from Promega (Heidelberg, Germany); optiphase
HiSafe3 liquid szintillator was from Wallac (Turku, Finland);
NET-317-estradiol[2,4,6,7-3H(N)] (17β-[3H]estradiol) was from
Du Pont NEN (Boston, Maryland); CDCl3, [D6]-acetone, [D4]-
methanol and [D6]-DMSO were from Aldrich (Steinheim, Ger-
many); PBS was prepared by dissolving 8.0 g of NaCl, 0.2 g of
KCl, 1.44 g of Na2HPO4 ·2 H2O, and 0.2 g of KH2PO4 (all pur-
chased from Merck or Fluka) in 1000 mL of purified water.
TRIS-buffer (pH = 7.5) was prepared by dissolving 1.211 g of
trishydroxymethylaminomethane, 0.37224 g of Titriplex III and
0.19503 g of sodium azide (all from Merck or Fluka) in 1 L of pu-
8.4 Hz, 4J = 2.4 Hz, 2 H, ArH-5); 6.73 (d, 4J = 2.4 Hz, 1 H, ArH-
3); 6.84 (d, 4J = 2.4 Hz, 1 H, ArH-3); 6.94 (d, J = 8.4 Hz, 1 H,
3
ArH-6);7.09 (d, 3J = 8.4 Hz, 1 H, ArH-6);7.70 (s, 1 H, N=CH-N);
9.79 (s, br, 1 H, ArOH, exchangeable by D2O); 9.97 (s, br, 1 H,
ArOH, exchangeable by D2O);12.26 (s, 1 H, NH, exchangeable
by D2O). Anal. (C15H10Cl2N2O2) 321.16: calc.: C 56.10 H 3.14 N
8.72 found: C 56.07 H 3.09 N 8.64.
4-(2-Chloro-4-hydroxyphenyl)-5-(2,6-dichloro-4-hydroxyphe-
nyl)imidazole (4)
From 4-(2-chloro-4-methoxyphenyl)-5-(2,6-dichloro-4-meth-
oxyphenyl)imidazole 4 a: 0.430 mmol (165 mg). The crude
product was prepurified by column chromatography on silica
gel with diethyl ether/acetone (6 + 1) and subsequent recrystal-
lization from CHCl3 and acetone (6 + 1). Yield: 0.225 mmol
(80 mg), 52 %;pale yellow powder, mp 220 °C under decompo-
sition.IR (KBr):ν= 3600–2700 s, br (OH);2752 w;2643 w;1610
s; 1570 m; 1510 m; 1436 m; 1357 w; 1284 m; 1227 m; 1105 w;
1061 m; 1029 w; 946 m; 899 m; 854 m; 808 m. MS (EI, 230 °C):
m/z (%) = 354 (94) [M]+·; 319 (25) [M–Cl]+; 284 (71) [M–2Cl]+;
255 (24); 190 (17); 142 (26); 139 (17). 1H NMR ([D4]-methanol):
δ = 6.60 (dd, 3J = 8.4 Hz, 4J = 2.3 Hz, 1 H, ArH-5); 6.80 (s, 2 H,
ArЈH-3, ArЈH-5); 6.83 (d, 4J = 2.3 Hz, 1 H, ArH-3); 7.01 (d, 3J =
8.4 Hz, 1 H, ArH-6); 7.78 (s, 1 H, N=CH-N). Anal.
(C15H9Cl3N2O2) 355.61: calc.: C 50.66 H 2.55 N 7.88 found: C
50.73 H 2.48 N 7.67.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 463–471
4,5-Bis(4-hydroxyphenyl)imidazoles 471
rified water. Deionized water was produced by means of a Milli-
pore Milli-Q Water System, resistivity > 18 MΩ.T-75 flasks, re-
action tubes, 96-well plates, and 6-well plates were purchased
from Renner GmbH (Dannstadt, Germany).
References
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The applied method was already described by Hartmann et al.
[17] and was used with some modifications. The relative bind-
ing affinity (RBA) of the test compounds to the estrogen recep-
tor was determined by the displacement of 17β-[3H]estradiol
from its binding site.For this purpose, the test compounds were
dissolved in ethanol and diluted with TRIS-buffer to 6–8 appro-
priate concentrations (300 µL). They were incubated shaking
with calf uterine cytosol (100 µL) and 17β-[3H]estradiol
(0.723 pmol in TRIS-buffer (100 µL); activity: 2249.4 Bq/tube)
at 4 °C overnight.To stop the reaction 500 µL of a dextran-char-
coal suspension in TRIS-buffer was added to each tube. After
shaking for 90 min at 4 °C and centrifugation 500 µL HiSafe3
was mixed with 100 µL supernatant of each sample and the re-
activity was determined by liquid scintillation spectroscopy.The
same procedure was used to quantify the binding of 17β-
[3H]estradiol (0.723 pmol – control). Non-specific binding was
calculated using 2 nmol of 17β-estradiol as the competing lig-
and. On a semilog plot the percentage of maximum bound la-
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[3H]estradiol vs. concentration of the competitor (log-axis) is
plotted. At least six concentrations of each compound were
chosen to estimate its binding affinity. From this plot those mo-
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50 %.
3
c[
at 50 % inhibition
H]–Estradiol
RBA =
· 100 %
csample at 50 % inhibition
Luciferase assay
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(0.5 mL cell suspension and 1.5 mL medium per well) at grow-
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50 h. Before harvesting, the cells were washed twice with PBS
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10–8 M Estradiol control (100 %).
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Acknowledgements
The authors are grateful to Prof. E. von Angerer for pro-
viding the MCF-7-2a cell line.The technical assistance of
S. Bergemann and I. Schnautz is acknowledged. The
presented work was supported by Grant Gu285/3-1 from
the Deutsche Forschungsgemeinschaft.
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