Synthesis, structural evaluation, and estrogen receptor interaction of 2,3-diarylpiperazines

Article abstract of DOI:10.1021/jm0208368

To develop novel estrogen receptor (ER) ligands, ring-fused derivatives of the hormonally active (1R,2S)/(1S,2R)-1-(2-chloro-4-hydroxyphenyl)-2- (2,6-dichloro-4-hydroxyphenyl)ethylenediamine 4b were synthesized. (2R,3S)/(2S,3R)-2-(2-Chloro-4-hydroxyphenyl

Full text of DOI:10.1021/jm0208368

J . Med. Chem. 2002, 45, 2325-2337
2325
Syn th esis, Str u ctu r a l Eva lu a tion , a n d Estr ogen Recep tor In ter a ction of
2,3-Dia r ylp ip er a zin es
Ronald Gust,* Roland Keilitz, and Kathrin Schmidt
Institute of Pharmacy, Free University of Berlin, Ko¨nigin-Luise Strasse 2+4, D-14195 Berlin, Germany
Received J anuary 29, 2002
To develop novel estrogen receptor (ER) ligands, ring-fused derivatives of the hormonally active
(1R,2S)/(1S,2R)-1-(2-chloro-4-hydroxyphenyl)-2-(2,6-dichloro-4-hydroxyphenyl)ethylenediamine
4b were synthesized. (2R,3S)/(2S,3R)-2-(2-Chloro-4-hydroxyphenyl)-3-(2,6-dichloro-4-hydroxy-
phenyl)piperazine 4 induced ligand-dependent gene expression in MCF-7-2a cells, stably
transfected with the plasmid EREwtcluc and was therefore used as a lead structure. The
influence of the substitution pattern in the aromatic rings (4-OH (1), 2-F,4-OH (2), 2-Cl,4-OH
(3), 2,6-Cl2,3-OH (5), and 2,6-Cl2,4-OH (6)) and the effect of N-ethyl chains on the ER binding
and activation of gene expression were studied. The synthesis started from the respective
methoxy-substituted (1R,2S)/(1S,2R)-configurated 1,2-diarylethylenediamines 6b to 4b, which
were reacted with dimethyl oxalate in order to get 5,6-diarylpiperazine-2,3-diones. Reduction
with BH3‚tetrahydrofuran and ether cleavage with BBr3 yielded the piperazines 1-6. The
N-alkylation of the piperazines 1a -3a , which was employed for obtaining compounds 7-11,
was succeeded by acetic anhydride followed by reduction and ether cleavage. Nuclear magnetic
resonance (NMR) spectroscopical studies revealed a synclinal conformation of the 1,2-
diarylethane pharmacophore and a preference of the substituents at the heterocyclic ring for
an equatorial position. This spatial structure prevents an interaction with the ER analogously
to that of estradiol (E2). Therefore, the piperazines can displace E2 from its binding site only
to a very small extent. Only the N-ethyl (8) and N,N′-diethyl (11) derivatives of piperazine 3
showed relative binding affinity values > 0.1% (8, 0.42%, and 11, 0.17%). Nevertheless, ER-
mediated gene activation was verified for the piperazines 4 (20%), 6 (73%), 7 (34%), 8 (74%),
and 11 (37%) (concentration, 1 µM; E2, 100% activation) on the MCF-7-2a cell line.
O-methylation led to completely inactive compounds and showed the necessity of H bridges
from the piperazines to the ER for activating gene expression.
In tr od u ction
disease, improvements in cognitive function, and a delay
in the onset of Alzheimer’s disease.
Estr ogen Recep tor (ER). Two forms have been
described (R and â). In the present paper, we refer solely
to ERR, since it is verified that this receptor is mainly
present in MCF-7 mammary carcinoma cells. A member
of the steroid receptor gene family acts as a transcrip-
tional factor, which is involved in the regulation of
diverse arrays of target genes.1 To activate the ER, a
ligand-dependent conformational change of the receptor
molecule is necessary. The expression of target genes
takes place after the dimerization of drug-receptor
complexes and binding to the estrogen response ele-
ments (ERE) at the DNA.2
Despite its high benefit, the long-term treatment with
SERMs is often limited due to intolerable side effects,
for example, benign and malignant lesion of the uterus.
Therefore, the search for new SERMs among both
existing and new drugs has been a challenging task in
recent years.10
Several research groups performed modifications at
the 7R- and 11â-position with the aim to change the
hormonal profile of E2 from estrogen to antiestro-
gen.11,12 Fulvestrant (faslodex) is a 7R-alkylamide of E2
and as pure antiestrogen already used in clinical trials.13
Further investigations focused on the design of drugs
with high selectivity for one of the ER subtypes, using
1,3,5-triaryl-4-alkylpyrazoles14 or 2-amino-4,6-diaryl-
pyridines15 as lead structures.
The drugs show an agonistic, antagonistic, or a tissue
selective mixed agonistic/antagonistic profile depending
on their structure.3 Such compounds, called selective
estrogen receptor modulators (SERMs),4 are of great
interest in cancer chemotherapy and estrogen replace-
ment therapy in postmenopausal women.5,6 SERMs
inhibit bone resorption, can prevent osteoporosis,7 and
cause beneficial effects on the cardiovascular system8
and the central nervous system.9 Furthermore, estrogen
exposure is associated with a decrease of coronary heart
Our investigations show that not only flat but also
puckered molecules interact with the ER and activate
ligand-dependent gene expression in MCF-7-2a breast
cancer cells, stably transfected with the plasmid
EREwtcluc.16 The (2R,3S)/(2S,3R)-2-(2-chloro-4-hydroxy-
phenyl)-3-(2,6-dichloro-4-hydroxyphenyl)piperazine 4, a
ring-fused derivative of the hormonally active (1R,2S)/
(1S,2R)-1-(2-chloro-4-hydroxyphenyl)-2-(2,6-dichloro-4-
hydroxyphenyl)ethylenediamine, activated expression of
* To whom correspondence should be addressed. Tel.: (030)838
53272. Fax: (030)838 56906. E-mail: rgust@zedat.fu-berlin.de.
10.1021/jm0208368 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/25/2002

2326 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
Sch em e 1. Synthesis of the 2,3-Diarylpiperazines
Ch a r t 1. Structural Formulas of Compounds Referred
to in the Text
1a -6a
Sch em e 2. N-Alkylation of the 2,3-Diarylpiperazines
1a -3a
the luciferase gene in MCF-7-2a cells. In contrast, 4 was
not able to displace E2 from its binding site at the ER
in the competition experiment with [3H]E2 using calf
uterine cytosol (relative binding affinity (RBA) < 0.02%).
This is the consequence of a synclinal arrangement of
the 1,2-diarylethane moiety already identified as an
essential pharmacophore.17 This spatial structure is
quite different from those of steroidal or nonsteroidal
estrogens such as diethylstilbestrol (DES) and E2 (see
Chart 1). Therefore, a different binding mode at the ER
is assumed. In this paper, experiments to optimize the
ER-mediated effects of 2,3-diarylpiperazines by varia-
tion of the substitution pattern in the aromatic rings
and by N-alkylation are described.
Str u ctu r a l Eva lu a tion . In solution, the piperazine
ring undergoes a dynamic interconversion between two
limiting structures (see Figure 1). Because it is well-
known that substituents influence the dynamic effects
of five- and six-membered rings,21,22 we studied the
conformeric behavior of the heterocycles 1-11 using 1H
nuclear magnetic resonance (NMR) and molecular
modeling methods.
Resu lts
Syn th esis. The synthesis of the piperazines was
performed in a two step reaction according to Scheme
1. Heating of the respective 1,2-diarylethylenediamines
1b-6b18-20 together with dimethyl oxalate in dioxane
under reflux yielded in the first reaction step the
piperazine-2,3-diones, which were reduced to the 2,3-
diarylpiperazines 1a -6a . For N-alkylation, the 2,3-bis-
(4-methoxyphenyl)piperazines 1a -3a were reacted with
acetic anhydride and subsequently reduced with BH3‚
tetrahydrofuran (THF) (Scheme 2).
In the case of the N,N′-diethyl-2,3-diarylpiperazines
10a and 11a , the reduction led to a partial cleavage of
one of the N-standing ethyl groups, so that the N-ethyl-
2,3-diarylpiperazines 7a and 8a could be isolated, too.
The hydroxy-substituted piperazines 1-11 were finally
generated by ether cleavage with BBr3 (Scheme 3).
The (2R,3S)/(2S,3R) configuration forces the phenyl
rings at the piperazine ring in synclinal position (see
Figure 1). In both limiting structures, one of the phenyl
rings is axially standing and the methine protons (H1a
and H1b) as well as the methylene protons (H2a and
H2b) are nonequivalent. During the interconversion, the
molecule becomes symmetric (presumption: Ar ) Ar′),
1
and in the H NMR spectra, the diastereotopic split of
the resonances is lost.
The spectra of the piperazines 1-3, 5, and 6 show
only a singlet resonance for H1a/H1b and two signal

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2327
Ta ble 1. NMR Dataa of 2,3-Diarylpiperazines
methine
H1a,H1b
methylene
H2a,H2b
compd
aromatic H
N-ethyl
OH
1
3.99 (s, 2H)
2.64-2.71 (m, 2H) 6.49 (AA′BB′, 4H)
2.97-3.04 (m, 2H) 7.19 (AA′BB′, 4H)
9.00 (s, 2H)
3
2
3
4
4.35 (s, 2H)
4.57 (s, 2H)
2.66-2.74 (m, 2H) 6.33 (dd, J (H, F) ) 12.5 Hz, 4J ) 1.9 Hz, 2H)
9.51 (s, 2H)
9.55 (s, 2H)
2.97-3.05 (m, 2H) 6.38 (dd, 3J ) 8.6 Hz, 4J ) 1.9 Hz, 2H)
7.67 (dd, 3J ) 8.6 Hz, J (H, F) ) 8.8 Hz, 2H)
4
2.66-2.78 (m, 2H) 6.53 (dd, 3J ) 8.6 Hz, 4J ) 2.1 Hz, 2H)
2.95-3.07 (m, 2H) 6.62 (d, 4J ) 2.1 Hz, 2H)
7.85 (d, 3J ) 8.6 Hz, 2H)
4.82 (d, 3J ) 4.9 Hz, 1H)
5.02 (d, 3J ) 4.9 Hz, 1H)
2.84-2.91 (m, 1H) 6.58 (d, 4J ) 2.6 Hz, 1H)
3.00-3.12 (m, 1H) 6.62 (s, 2H)
3.28-3.42 (m, 1H) 6.74 (dd, 3J ) 8.7 Hz, 4J ) 2.6 Hz, 1H)
3.54-3.66 (m, 1H) 8.10 (d, 3J ) 8.7 Hz, 1H)
5
6
7
5.37 (s, br, 2H)
5.20 (s, 2H)
2.96-3.13 (m, 2H) 6.73 (d, 3J ) 8.8 Hz, 2H)
3.26-3.43 (m, 2H) 7.00 (d, br, 3J ) 8.8 Hz, 2H)
2.96-3.09 (m, 2H) 6.65 (s, 4H)
3.34-3.48 (m, 2H)
4.26 (d, 3J ) 3.8 Hz, 1H)
4.37 (d, 3J ) 3.8 Hz, 1H)
2.47-2.52 (m, 1H) 6.24 (dd, J (H, F) ) 12.3 Hz, 4J ) 2.4 Hz, 1H)
0.95 (t, 3J ) 7.1 Hz, 3H) 9.51 (s, 2H)
2.00-2.09 (m, 1H)
2.19-2.28 (m, 1H)
3
2.65-2.75 (m, 1H) 6.27 (dd, 3J ) 8.6 Hz, 4J ) 2.4 Hz, 1H)
3
2.93-3.00 (m, 1H) 6.37 (dd, J (H, F) ) 12.4 Hz, 4J ) 2.4 Hz, 1H)
3.14-3.21 (m, 1H) 6.94 (dd, 3J ) 8.6 Hz, 4J ) 2.4 Hz, 1H)
4
7.08 (dd, 3J ) 8.6 Hz, J (H, F) ) 8.6 Hz, 1H)
4
8.09 (dd, 3J ) 8.6 Hz, J (H, F) ) 8.7 Hz, 1H)
8
4.46 (d, 3J ) 3.8 Hz, 1H)
4.62 (d, 3J ) 3.8 Hz, 1H)
2.52-2.60 (m, 1H) 6.37 (dd, 3J ) 8.6 Hz, 4J ) 2.3 Hz, 1H)
2.82-2.90 (m, 1H) 6.52 (d, 4J ) 2.3 Hz, 1H, ArH-3)
2.90-3.00 (m, 1H) 6.60 (dd, 3J ) 8.6 Hz, 4J ) 2.3 Hz, 1H)
3.14-3.22 (m, 1H) 6.64 (d, 4J ) 2.3 Hz, 1H)
6.96 (d, 3J ) 8.6 Hz, 1H)
0.94 (t, 3J ) 7.1 Hz, 3H) 9.52 (s, 2H)
2.03-2.12 (m, 1H)
2.28-2.38 (m, 1H)
8.15 (d, 3J ) 8.6 Hz, 1H);
9
3.57 (s, 2H)
4.07 (s, 2H)
4.33 (s, 2H)
2.34-2.50 (m, 2H) 6.47 (AA′BB′, 4H)
0.93 (t, 3J ) 7.1 Hz, 6H) 9.04 (s, 2H)
1.90-2.02 (m, br, 2H)
2.22 (br, 2H)
2.98 (br, 2H)
7.00 (AA′BB′, 2H)
3
10
2.51 (covered by
solvent peak);
3.02 (br, 2H)
6.33 (d, br, J (H, F) ) 12.5 Hz, 2H)
0.96 (t, 3J ) 6.8, 6H)
2.02 (br, 2H)
9.59 (s, 2H)
6.38 (d, br, 3J ) 8.1 Hz, 2H)
7.37 (br, 2H)
2.30 (br, 2H)
11
2.49-2.58 (m, 2H) 6.49 (dd, 3J ) 8.6 Hz, 4J ) 2.2 Hz, 2H)
3.05-3.14 (m, 2H) 6.59 (d, 4J ) 2.2 Hz, 2H)
7.39 (br, 2H)
0.94 (t, 3J ) 7.1 Hz, 6H) 9.57 (s, 2H)
2.07-2.16 (m, 2H)
2.17-2.27 (m, 2H)
a
400 MHz spectra in [D6]DMSO (1-3 and 7-11), [D4]methanol (4-6), with TMS as internal standard; δ(TMS) ) 0. Representation of
the chemical shifting (δ) in ppm.
Sch em e 3. Ether Cleavage of the Piperazines 1a -11a
with BBr3
F igu r e 1. Arrangement of the aryl residues during the
conversion of the piperazine ring.
In the spectra of (2R,3S)/(2S,3R)-2-(2-chloro-4-hydroxy-
phenyl)-3-(2,6-dichloro-4-hydroxyphenyl)piperazine 4,
these signal groups are split due to the asymmetric
3
character of the molecule. The coupling constant J H1a-H1b
) 4.9 Hz follows the Karplus dependence23 and cor-
relates with an angle of 35-55°. This value can only be
realized if the piperazine ring adapts a relatively stable
conformation without interconversion. Analogously to
related [1,2-diarylethylenediamine]platinum(II) com-
plexes,18,24 a conformation is assumed in which the 2,6-
dichloro-4-hydroxyphenyl ring is preferably equatorially
arranged and the 2-chloro-4-hydroxyphenyl ring is
groups for H2a and H2b (Table 1) indicating an inter-
conversion of the piperazine ring (conformations I and
II; see Figure 1).
axially arranged (see Figure 1).
These structures were refined by molecular modeling
methods using Tripos Sybyl 6.6 (Tripos Force Field,

2328 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
Ta ble 2. NMR Dataa of COSY, NOE, and NOESY Experiments with Piperazine 7
chemical shift
(ppm)
(COSY) cross-peak
with proton
NOE detected in difference
spectra to proton
(NOESY) cross-peak
assignmentb
with proton
0.95
2.05
2.23
2.50
2.70
2.96
3.17
4.26
4.37
6.24
6.27
6.37
6.44
6.94
8.09
7
6′
6
6, 6′
6, 7
6′, 7
2aa, 2ba
2aa, 2ae, 2be
2ae, 2ba, 2be
2aa, 2ba
1a
1b
4a
5a, 3a
4b
5b, 3b
4a
1b, 2be, 6, 6′
2be, 6, 7
1b, 6′, 7
c (under DMSO peak)
2ae, 2be, 5b, 6′
1a, 2ae, 2be
2aa, 2ba, 2be, 5a
1a, 6, 7
1b, 2aa
c
c
c
1b, 2be, 6, 6′
2ba, 2be, 6, 7
1b, 6′, 7
2aa, 2ae, 2ba, 6′, 7
2ae, 2be, 5b, 6′
1a, 2ae, 2be
2aa, 2ba, 2be
1a, 6, 7
1b, 2aa
d
d
d
2be
2ba
2a a
2a e
1b
1a
3a
4a
3b
4b
5a
5b
c
d
1a, 2ae, 4a, 5b
1b, 2ba, 4b, 5a
4a, 5b
2ba, 4b, 5a
4b
a
b
d
See Table 1. See Figure 2. c Not irradiated. Not detected.
fixed conformation of the heterocyclic ring (Figure 1).
1
To verify this assumption, H NMR experiments with
the (2R,3S)/(2S,3R)-N-ethyl-2,3-bis(2-fluoro-4-hydroxy-
phenyl)piperazine 7 were performed.
An appropriate experiment to determine the spatial
structures of piperazines is the nuclear Overhauser
enhancement experiment (NOE).25 In the NOE as well
as the NOE spectroscopy (NOESY) double-resonance
experiments, a saturation transfer is achieved between
protons that are closer to each other than 3.5 Å. Figure
3 shows the NOE difference spectra of 7. The assign-
ment of the signals is based on correlation spectroscopy
(COSY) and NOE experiments (see Table 2).
H2ae and H2be show NOEs to all other methylene
protons while H2aa and H2ba possess only NOEs to H2ae
and H2be. Beside the NOE between H1a and H1b,
strong effects are observed between H1a and H2aa, H2ba
and H5b as well as H5a and H5b. Because of these
saturation transfers, it can be concluded that the
piperazine 7 takes a conformation in which the aromatic
ring B is preferably arranged in axial position and the
ring A is equatorially oriented (Figure 4). Only in this
conformation, H5b and H2ba as well as H1a and H2aa
came close enough to allow NOEs.
F igu r e 2. Low energy structure of the piperazines 3 (chair
conformation) and 4 (twist conformation), the N-ethylpipera-
zine 7, and the N,N′-diethylpiperazine 11.
Gasteiger-Hu¨ckel charges). In contrast to the pipera-
zines 1-3, which exist in a low energetic chair confor-
mation, for 4 as well as 5 and 6, the twist conformation
is favored. Because of van der Waals contacts between
the Cl substituents and the opposite protons of the
piperazine ring, the twist conformation of 5 (4.7 kJ /mol)
and of 6 (10.0 kJ /mol) is of lower energy than the chair
conformation. In each energetically preferred conforma-
tion, both N-H and one aryl ring are equatorially
arranged (see Figure 2).
The different spatial structures of the piperazine ring
influence the torsion angle between the aryl rings in
1-4 only marginally (47.5-47.9°) so the distances of
the hydroxy groups are nearly the same (O-O dis-
tance: 6.8-7.1 Å). However, a 2,6-Cl2 substitution in
each aromatic ring as realized in 6 reduces both the
torsion angle (39.4°) and the O-O-distance (6.6 Å). The
latter can be distinctly surpassed by the 2,6-Cl2-3-OH
phenyl-substituted piperazine 5 during the rotation of
the aromatic ring.
No effects are observed between H1b and H2be or
H2aa and H2ba. The NOE from H5a to H2ae is only
weak in the NOE difference spectra and cannot be
detected in the NOESY spectra. This is an indication
but no proof that the inversion of the piperazine ring of
7 takes place only in limited degree.
In the NOESY spectra, there are strong cross-peaks
between the aromatic protons H5a and H5b and also
H5b and H2ba. The NOEs from H5a to H1a and H5b to
H1b are very weak although the distance of these
protons amounts to 2.4 and 2.5 Å. This indicates a
conformation in which the F atoms are oriented away
from the piperazine moiety. However, the rotation of the
aromatic rings cannot be excluded.
The NOEs of the protons H6 and H6′ support the
results of Carballeira,21 which predict an equatorial
position of N substituents. In 7, this orientation seems
to be energetically favored, too. In the case of an axially
standing ethyl chain, the NOEs between H7 and H2be
should not exist while NOEs between H6/H6′ and H1a/
H2aa should occur. From the methyl group (H7), NOEs
are observed to H1b and H2be but not to H2ba. This
N-Monoalkylation also limits the dynamic of the ring
inversion. The resonances of the methine protons are
both split with coupling constants of 3J ) 3.8 Hz in the
spectra of 7 and 8. This value correlates with a dihedral
angle of 50-60° for the benzylic protons indicating a

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2329
F igu r e 3. 1H NMR spectra and NOE difference spectra of 7 in [D6]DMSO.
seems to be the consequence of an orientation opposite
the aromatic rings.
The spatial structures of the piperazines 6 and 7 were
also evaluated by theoretical methods. Both compounds
can exist in several low-energy chair and twist confor-
mations. The conformations of 7 possess in the absolute
minimum nearly identical energy, while in the case
of 6 the twist conformation is 3.4 kJ /mol of higher

2330 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
On the basis of these results at room temperature, a
fast interconversion of the piperazine ring with a stable
equatorial position of the ethyl chains can be assumed.
1
Because 10 and 11 show comparable H NMR spectra,
we postulate similar spatial structures in solution.
Theoretical considerations indicate great similarities
to the N-monoethyl and the nonalkyl-substituted 2,3-
diarylpiperazines. We calculated a spatial structure in
the absolute minimum of energy with a twist conforma-
tion of the heterocyclic ring and equatorial orientations
of both ethyl chains and one aromatic ring (the chair
conformation would enhance the energy level of 10
by 3.7 kJ /mol and of 11 by 5.4 kJ /mol). The dihedral
angle between the aryl rings in 10 and 11 amounts to
45.3-43.7° with O-O distances of 6.7 and 6.5 Å,
respectively.
Biologica l P r op er ties. The binding affinity to the
ER (RBA) was determined for all compounds in a
competition experiment using calf uterine cytosol.28 In
this test, the amount of displaced [3H]E2 from the
receptor was measured. Therefore, the RBA value is an
indication to which extent the compound can compete
with E2 for the binding to the ER.
To quantify the ligand-induced gene activation, the
MCF-7-2a cell line was used.29 These ER-containing
mammary carcinoma cells were stably transfected with
the plasmid EREwtcluc. The plasmid contains the “es-
trogen response element” of the DNA as an enhancer
sequence and a reporter sequence, which codes for
luciferase. The binding of ER/drug conjugate dimers
leads to expression of luciferase, which correlates very
well with the estrogenic potency of the drug.30
The nonalkylated piperazines 1-6 showed, with the
exception of 6 (RBA ) 0.05%), no significant displace-
ment of E2 from its binding site (RBA < 0.02%).
N-Monoethylation of 2 and 3 increased the RBA to 0.06
(7) and 0.42% (8), while an additional ethyl group at
the second piperazine nitrogen reduced the RBA values
by half to 0.03 (10) and 0.17% (11). On the other hand,
O methylation led in each case to a complete loss of ER
interaction.
F igu r e 4. Postulated conformation of 7 (left) on the basis of
NOE effects (right).
energy than the chair conformation. In all energetically
preferred structures, the ethyl chain is equatorially
arranged and the neighboring aromatic ring stands
axially. The dihedral angle between the rings in piper-
azine 6 amounts to 45.3° with an O-O distance of
6.9 Å. For piperazine 7, 43.7° and 6.7 Å were calculated.
Contrary to the NMR investigations in the energy
minimum, the methyl group of 7 is oriented upward
(Figure 2). However, the difference to a downward
orientation is only 2.8 kJ /mol. In all energetically
favored conformations of 6 and 7, the protons H1a and
H1b take a dihedral angle in the range of 41-48°, which
correlates very well with the results determined by
coupling constant analyses.
Interestingly, N,N′-diethylation increased the dy-
namic effects as demonstrated for the piperazines 10
and 11. The NMR data listed in Table 1 show no
splitting of the methylene and methine protons and
point out dynamic effects and conversion of the pipera-
zine ring.
The resonances of the aromatic protons H5a and H5b
(δ ) 7.45) in the spectra of 11 are broadened (Figure
5), due to a hindered rotation at room temperature (296
K). Heating to 323 K gives a sharp signal with a defined
coupling pattern. On the other hand, measurements at
low temperature show that the dynamic of the molecule
can be frozen.
The hydroxy- and the methoxy-substituted pipera-
zines were tested in a concentration-dependent manner
(10-10 to 10-6 M) in the luciferase assay on the MCF-
7-2a cell line. The maximum effects were obtained at
the highest concentration used (1 µM). The results listed
in Table 3 show that the halogen/hydroxy substitution
pattern in the aromatic rings is essential for estrogenic
activity. The Cl-substituted compounds (3, 8, and 11)
were more active than the respective F-substituted ones
(2, 7, and 10). Furthermore, the relative activation
correlates with the number of chlorine atoms (relative
activation of 3 < 4 < 6). Interestingly, N-monoethylation
of 3 has the same effects as the introduction of a fourth
Cl substituent (E2, activation 100%; 8, relative activa-
tion 74%; 6, relative activation 73%). The N,N′-dieth-
ylated piperazine 10 was inactive (relative activation
4%), and 11 reached a relative activation of 37%, which
is half of its monoethyl congener.
Below 253 K, all methylene and methine protons
become diastereotopic since the interconversion of the
piperazine ring is restricted. By use of the Eyring
equation
q
k ) (kbT/h) e-∆G /RT
from the coalescence temperature (Tc ) 253 ( 5 K) and
the distance between the methine protons H1a and H1b
(∆ν ) 184 Hz), the free enthalpy of activation (∆Gqc)
can be calculated.26 At the coalescence point, kc amounts
to 2.22 ∆ν and the following equation is given:
kbTc
∆Gqc ) RTc‚ln
)
2.22 h ∆ν
Tc
19.14T 10.32 + log
) 49 ( 1.0 kJ /mol
(
)
c
2.22 ∆ν
Discu ssion
This value is in good agreement with previously pub-
lished data for ring inversions. Harris et al.27 calculated
for the interconversion of the N,N′-dimethylpiperazines
Gq265K ) 52.8 kJ /mol at Tc ) 265 K.
We have synthesized a series of 2,3-diarylpiperazines
and investigated their binding to the ER as well as their
effects on the MCF-7-2a cell line. Because ligand-

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2331
F igu r e 5. 1H NMR spectra (300 MHz) of the piperazine 11 in [D4]methanol at 323, 296, 253, and 203 K.
independent activation of the ER molecule can be
excluded, the luciferase expression is the consequence
of the interaction of the piperazines with the ligand-
binding domain (LBD). In the LBD, however, an an-
chorage different from that of E2 is assumed, since none
of the new compounds can displace E2 effectively from
its binding site as demonstrated in the competition
experiment with [3H]E2.
vitro results. The relevance of H bridges for the ER
binding documents the inactivity of all OCH3 deriva-
tives. Furthermore, the position of the hydroxy groups
plays an important role. The shift of the OH from the
4- into the 3-position reduces the relative activation
from 73 to 12%. The shielding of the hydrophilic N
atoms by ortho-standing halide substituents or by
N-alkylation elevates the luciferase expression indicat-
ing the importance of hydrophobic interactions between
ligand and protein areas for the binding to the ER
The structural prerequisite for the interactions of the
new compounds in the LBD can be deduced from the in

2332 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
Ta ble 3. ER Affinity and Transcription Efficacy in MCF-7-2a, 1-11, (1a -11a )
relative activationb
(% of E2) of luciferase
expression at 10-6
M
-OCH3
derivatives
R1
R2
R3
R4
RBAa
1
2
3
4
5
6
7
8
9
4-OH
4-OH
H
H
H
H
H
H
ethyl
ethyl
ethyl
ethyl
ethyl
H
H
H
H
H
H
H
H
<0.02
<0.02
<0.02
<0.02
<0.02
0.05
0.06
0.42
<0.02
0.03
-1
4
7
20
12
73
34
74
8
5
4
1
3
8
5
3
4
4
7
0
2-F, 4-OH
2-Cl, 4-OH
2,6-Cl2, 4-OH
2,6-Cl2, 3-OH
2,6-Cl2, 4-OH
2-F, 4-OH
2-Cl, 4-OH
4-OH
2-F, 4-OH
2-Cl, 4-OH
2-Cl, 4-OH
2,6-Cl2, 3-OH
2,6-Cl2, 4-OH
2-F, 4-OH
2-Cl, 4-OH
4-OH
ethyl
ethyl
ethyl
10
11
2-F, 4-OH
2-Cl, 4-OH
2-F, 4-OH
2-Cl, 4-OH
4
37
0.17
a
Relative binding affinity (RBA), % ) [E2]/[I] × 100; [E2] and [I] are the molar concentrations of nonradioactive E2 and inhibitor
b
required to decrease the bound radioactivity by 50%; E2 ) 17â-estradiol. Mean value of three determinations. Values are means of three
independent experiments.
F igu r e 6. Interactions of estradiol (left), raloxifene (right), and 4-hydroxytamoxifen (middle) in the LBD of the ER according to
Brzozowski30 and Shiau.32
(relative activation: 6, 73%; 7, 34%; 8, 74%). Intro-
duction of a second ethyl chain in 7 and 8 decreases
the expression of luciferase (relative activation: 10, 4%;
11, 37%), which leads to the suggestion that probably
steric effects abolish the hydrophobic contacts to the
ER.
Because of their different geometry, the estrogenically
active 2,3-diarylpiperazines were assigned to a second
class of estrogens (type II estrogens).16 An interaction
in the LBD comparable to that of E2 is impossible.
Therefore, a speculative binding model was derived from
the crystal structure of the LBD cocrystallized with E2,
raloxifene (RAL),31 and 4-hydroxytamoxifen (4OHT)32
(see Chart 1 and Figure 6).
During the past decades, many structure-activity
relationship studies (SAR) on hormonally active com-
pounds were performed to characterize the LBD. Mean-
while, the LBD/E2 conjugate could be crystallized and
analyzed by X-ray analysis31,32 allowing an insight into
the binding mode of E2 and related compounds.
The selective hormone recognition by the ER occurs
by a combination of H bonding and hydrophobic con-
tacts. The phenolic OH group of E2 is bound by
hydrogen bridges to the γ-carboxylate of Glu 353, the
guanidinium residue of Arg 394 and a water molecule.
The 17â-OH group is linked to His 524. Furthermore,
hydrophobic interactions with aliphatic residues of
amino acids above and below the steroidal skeleton
(Figure 6) are essential.
Structure-activity studies together with available
X-ray data of LBD/drug conjugates revealed that drugs
with high RBA values possess comparable van der
Waals volumes and activate the ER by attachment to
identical binding sites. These drugs (type I estrogens)
must have a planar structure having two OH groups in
a distance comparable to E2 (10.9 Å).
RAL is attached to the LBD comparable to E2 by
hydrophilic and hydrophobic interactions.31 Beside the
H bridges known from E2 (see Figure 6), an additional
H bond is built in a narrow side pocket from the
piperidine nitrogen of the side chain of RAL to Asp 351.
4OHT is also attached to Glu 353, Arg 394, a water
molecule (phenolic OH), and to Asp 351 (dimethylamino
group of the side chain). The missing H bridge to His
524 seems to be compensated by van der Waals contacts
of the hydrophobic unsubstituted 2-phenyl ring.33
Further information about ligand-ER interactions is
given by structure-activity studies in the class of
estrogenically and antiestrogenically active 7R- and
11â-substituted E2 derivatives.12,34-38 Because of the
steroidal skeleton, these compounds should bind in the
same region of the LBD as E2. Large side chains at C-7
or C-11 can be positioned in analogy to a “one pocket”
model.39,40 In the case of a C-7 substitution, the steroid
has to rotate around a virtual C2 symmetry axis between
C-3 and C-17 (Figure 7). Hereby, the 7R- and the 11â-

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2333
F igu r e 7. Orientation of 7R- and 11â-substituted E2 deriva-
tives at the ER.
F igu r e 9. Postulated interactions of piperazine 4 in the LBD
of the ER.
active position can be excluded, since 4 causes an
activation of the luciferase gene but not an inhibition
of the agonistic effects of E2 in our test model.
N-Monoethylated piperazines exist as a mixture of
enantiomers, since the ethyl residue can be located
either at N-1 or at N-4 of the piperazine ring as well.
According to Figure 8, the ethyl group imitates the
hydrophobic 13â-methyl group of E2 or an alkyl chain
at C-7. The hydrophobic interaction is strengthened in
this region of the LBD, and the binding affinity in-
creases. Reduced activity after N,N′-diethyl substitution
could be interpreted as a steric hindrance during ER
binding comparable to 7R,11â-disubstituted E2 deriva-
tives.38,40
F igu r e 8. Superposition of low energy structures of raloxifene,
4-hydroxytamoxifen, and RU 39411 (upper left), 11â-(4-hy-
droxyphenyl)estradiol and piperazine 4 (twist conformation)
(upper right), and the enantiomers of 8 and 11â-(4-hydroxy-
phenyl)estradiol.
positions become nearly equivalent for the binding at
the ER.
Substitution with hydrophobic groups at either the
7R- or the 11â-position leads to compounds with es-
sentially the same binding affinities.39 This suggests
that these compounds may interact with the same
hydrophobic region in the LBD. Polar groups at the 7R-
and 11â-positions decrease the RBA value depending
on the distance from the E2 core.38
If both the 7R- and the 11â-position in E2 are
substituted, the residue at C-7 has to be relatively small.
Otherwise, the orientation of a large moiety analogous
to the basic side chain of RAL in the side pocket appears
to be prevented.40 The (4-dimethylaminoethoxyphenyl)
residue is well-tolerated in both the 7R- (RBA ) 87%)
and the 11â-position (RBA ) 130%). The binding affinity
of 11â-(4-dimethylaminoethoxyphenyl)-E2 (RU 39411)
surpasses even that of E2.
RU 39411 (see Chart 1) shows a good structural
analogy to RAL, 4OHT, and the 2,3-diarylpiperazine 4
(Figure 8). A superposition of the 11â-(4-hydroxy-
phenyl)-E2 moiety with 4 indicates an orientation of
the 2-chloro-4-hydroxyphenyl ring in the side pocket.
Although the amino acid Asp 351 was identified as an
essential anchor for antiestrogens (the binding of the
basic side chains of antiestrogens leads to a displace-
ment of the helix 12 away from the side pocket), the
spatial structure of 2,3-diarylpiperazines (type II estro-
gens) also allows de facto the contact to Asp 351.
However, a displacement of helix 12 from its normal,
Figure 9 presents a theoretical binding mode of 2,3-
diarylpiperazines in the LBD. The molecule is positioned
in such a way that H bridges to Glu 353, Arg 394, and
H2O are possible. The OH group of the other aromatic
ring interacts with Asp 351. Hydrophobic parts of the
molecule, in particular the ortho-Cl substituents or the
N-ethyl chain, are in van der Waals contacts with
hydrophobic residues of amino acids in the LBD.
The significance of Asp 351 for estrogenic as well as
antiestrogenic properties of RAL, 4OHT, and related
compounds has been verified by the group of J ordan.41,42
They hypothesized that alterations in the charge of
the amino acid 351 and changes in the interaction with
the side chain of antiestrogens are critical for the
estrogenicity of the ER/drug complex. This is confirmed
by the effects of RAL and 4OHT in an assay using the
induction of mRNA for the TGFR gene in MDA-MB-231
breast cancer cells stably transfected with the cDNA for
the wild-type Asp351 ER and the mutant Asp351Tyr
ER (Tyr 351 instead of Asp 351). In this test, the
Asp351Tyr ER can enhance the estrogenic properties
of 4OHT and change the pharmacology of RAL by
converting it from antiestrogen to estrogen.
On the other hand, substitution of glycine for aspar-
tate at position 351 (Asp351Gly) changed the activity
of the ER/4OHT complex from estrogen-like to com-
pletely antiestrogenic. In the same assay, 4OHTP P , a
nonisomerizable derivative of tamoxifen without a basic

2334 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
(m, 2H, NCH2C); 3.20-3.27 (m, 2H, NCH2C); 3.71 (s, 6H,
side chain (see Chart 1), shows no estrogenic properties
although it is an estrogen by use of Asp351 ER. In
contrast to E2 and DES, the triphenylethylene deriva-
tives activate gene expression and coactivator binding
not through the activation function AF2 but through
AF2b, a novel binding site for coactivators.42
On the basis of these data, J ordan et al. proposed a
classification of estrogens.43 Class I estrogens are planar
and related to E2 and DES (type I estrogens) and can
easily be sealed within the hydrophobic pocket by helix
12. They will use the AF2 coactivator binding site, which
will synergize AF1 to produce optimal estrogen-like
action. 4OHTP P and related angular triphenylethyl-
enes are class II estrogens (type II estrogens) and use
AF2b, which includes acidic surface amino acids on a
repositioned helix 12, an exposed aspartate at position
351 and AF1.43 This classification of estrogens correlates
very well with our investigations, wherein we assume
a comparable mode of action for our hormonally active
piperazines, imidazoline, or imidazoles.16
3
4
OCH3); 4.66 (s, 2H, ArCH); 6.46 (dd, J (H, F) ) 12.4 Hz, J )
2.5 Hz, 2H, ArH-3); 6.51 (dd, 3J ) 8.7 Hz, 4J ) 2.5 Hz, 2H,
3
4
ArH-5); 7.80 (dd, J ) 8.7 Hz, J (H, F) ) 8.7 Hz, 2H, ArH-6).
(2R,3S)/(2S,3R)-2,3-Bis(2-ch lor o-4-m eth oxyp h en yl)p ip -
er a zin e (3a ). (1R,2S)/(1S,2R)-1,2-Bis(2-chloro-4-methoxy-
phenyl)ethylenediamine 3b: 5.84 mmol (2.00 g). Reaction
time: 48 h. Purification: recrystalization from CHCl3/diethyl
ether. Yield: 3.72 mmol (1.20 g), 56%, colorless powder, mp
159-160 °C. IR (KBr): νj ) 3438 s, br (NH); 2837 w (OCH3);
1606 s; 1493 s; 1458 m; 1285 m; 1235 m; 1125 m; 1040 s; 862
m; 826 m. 1H NMR (CDCl3): δ 1.70 (s, 2H, NH); 2.92-3.00
(m, 2H, NCH2C); 3.19-3.26 (m, 2H, NCH2C); 3.71 (s, 6H,
OCH3); 4.87 (s, 2H, ArCH); 6.64 (dd, 3J ) 8.8 Hz, 4J ) 2.7 Hz,
4
3
2H, ArCH-5); 6.78 (d, J ) 2.7 Hz, 2H, ArH-3); 7.99 (d, J )
8.8 Hz, 2H, ArH-6).
(2R,3S)/(2S,3R)-2-(2-Ch lor o-4-m et h oxyp h en yl)-3-(2,6-
dich lor o-4-m eth oxyph en yl)piper azin e (4a). (1R,2S)/(1S,2R)-
1-(2-Chloro-4-methoxyphenyl)-2-(2,6-dichloro-4-methoxyphenyl)-
ethylenediamine 4b: 1.06 mmol (400 mg). Reaction time: 72
h. Purification: chromatography on silica gel with diethyl
ether/methanol (4 + 1). Yield: 0.199 mmol (80 mg), 19%, yellow
1
oil. H NMR (CDCl3): δ 2.30 (s, 2H, NH); 2.75-3.70 (m, 4H,
3
NCH2C); 3.75 (s, 3H, OCH3); 3.80 (s, 3H, OCH3); 4.90 (d, J )
5 Hz, 1H, ArCH); 5.10 (d, 3J ) 5 Hz, 1H, ArCH); 6.70-7.00
Ma ter ia ls a n d Meth od s
3
(m, 4H, ArH-3, ArH-5, Ar′H); 8.20 (d, J ) 8 Hz, 1H, ArH-6).
(2R,3S)/(2S,3R)-2,3-Bis(2,6-d ich lor o-3-m eth oxyp h en yl)-
p ip er a zin e (5a ). (1R,2S)/(1S,2R)-1,2-Bis(2,6-dichloro-3-meth-
oxyphenyl)ethylenediamine 5b: 1.46 mmol (600 mg). Reaction
time: 5 days. Purification: chromatography on silicagel with
CH2Cl2/methanol (29 + 1). Yield: 0.275 mmol (120 mg), 19%,
yellow crystals, mp 79-81 °C. IR (KBr): νj ) 3435 s, br (NH);
Gen er a l P r oced u r es. IR spectra (KBr pellets): Perkin-
Elmer model 580 A. H NMR: Bruker ADX 400 spectrometer
1
at 400 MHz (internal standard, tetramethylsilane (TMS)).
Elemental analyses: Microlaboratory of Free University of
Berlin. EI-MS spectra: CH-7A-Varian MAT (70 eV) or Kratos
MS 25 RF (80 eV). All computational graphics were built using
SYBYL 6.6, Tripos Inc., 1699 South Hanley Rd., St. Louis, MO,
63144. Geometry optimization was carried out using the Tripos
force field within the SYBYL program, running on an INDY
workstation. Liquid Scintillation Counter: 1450 Microbeta
Plus (Wallac, Finland). Microlumat: LB 96 P (EG & G
Berthold, Germany).
1
1630 s; 1459 m; 1436 m; 1282 s; 1110 m; 1070 m; 804 m. H
NMR (CDCl3): δ 2.45 (s, 2H, NH); 3.13-3.25 (m, 2H, NCH2C);
3.31-3.43 (m, 2H, NCH2C); 3.79 (s, 6H, OCH3); 5.41 (s, 2H,
3
3
ArCH); 6.68 (d, J ) 8.9 Hz, 2H, ArH-4); 7.07 (d, J ) 8.9 Hz,
2H, ArH-5).
(2R,3S)/(2S,3R)-2,3-Bis(2,6-d ich lor o-4-m eth oxyp h en yl)-
p ip er a zin e (6a ). (1R,2S)/(1S,2R)-1,2-Bis(2,6-dichloro-4-meth-
oxyphenyl)ethylenediamine 6b: 1.46 mmol (600 mg). Reaction
time: 5 days. Purification: chromatography on silica gel with
CH2Cl2/methanol (29 + 1). Yield: 0.410 mmol (179 mg), 28%,
yellow powder, mp 78-79 °C. IR (KBr): νj ) 3432 m, br (NH);
3411 m, br (NH); 2836 w (OCH3); 1601 s; 1554 s; 1465 m; 1431
Syn th eses. (1R,2S)/(1S,2R)-1,2-Diarylethylenediamines 1b-
6b were synthesized as described previously.18-20
Gen er a l P r oced u r e for th e Syn th esis of 2,3-Dia r ylp ip -
er a zin es (Meth od A). To a solution of the respective 1,2-
diarylethylenediamine 1b-6b in dry 1,4-dioxane, dimethyl
oxalate was added and heated to reflux. Subsequently, the
solvent was evaporated and the crude product was dried. To
the 5,6-diarylpiperazine-2,3-dione, suspended in 20 mL of dry
THF, was added under nitrogen atmosphere 5 mL of a 1 M
boran‚THF complex. After the mixture was heated to reflux
for 12 h, it was hydrolyzed with 10 mL of water and 10 mL of
3 N HCl. The THF was distilled off, and the acidic solution
was heated for an additional hour. After it was cooled to room
temperature, the aqueous solution was shaken with diethyl
ether and alkalized with NaOH (20%), and the product was
extracted with CH2Cl2. The organic layer was dried over Na2-
SO4 and evaporated to dryness.
1
m; 1270 m; 1172 w; 1112 w; 1041 s; 906 w; 839 m; 793 m. H
NMR (CDCl3): δ 2.54 (s, 2H, NH); 3.09-3.23 (m, 2H, NCH2C);
3.28-3.43 (m, 2H, NCH2C); 3.73 (s, 6H, OCH3); 5.24 (s, 2H,
ArCH); 6.69 (s, 4H, ArH).
Gen er a l P r oced u r e for th e Syn th esis of N,N′-Dia cetyl-
2,3-d ia r ylp ip er a zin es (Meth od B). Under nitrogen atmo-
sphere, acetic anhydride (5 mmol) in 15 mL of dry THF was
added at -10 °C to a solution of the respective piperazine 1a -
3a dissolved in 5 mL of dry THF. After the mixture was heated
to reflux, the reaction mixture was evaporated to dryness.
(2R,3S)/(2S,3R)-N,N′-Diacetyl-2,3-bis(4-m eth oxyph en yl)-
p ip er a zin e (1c). (2R,3S)/(2S,3R)-2,3-Bis(4-methoxyphenyl)-
piperazine 1a : 1 mmol (298 mg). Reaction time: 1 h. Purifi-
cation: chromatography on silica gel with diethyl ether/
methanol 4 + 1. Yield: 0.662 mmol (253 mg), 66%, colorless
powder, mp 74-76 °C. 1H NMR ([D4]methanol): δ 1.99 (br,
6H, CH3CO); 3.73 (s, 6H, OCH3); 3.88 (br, 2H, NCH2C); 4.23
(br, 2H, NCH2C); 5.63 (br, 2H, ArCH); 6.74 (AA′BB′, br, 4H,
ArH).
(2R,3S)/(2S,3R)-2,3-Bis(4-m et h oxyp h en yl)p ip er a zin e
(1a ). (1R,2S)/(1S,2R)-1,2-Bis(4-methoxyphenyl)ethylenedi-
amine 1b: 12.0 mmol (3.27 g). Reaction time: 12 h. Purifica-
tion: chromatography on silica gel with methanol. Yield: 6.51
mmol (1.94 g), 54%, colorless oil. IR (KBr): νj ) 3436 s, br (NH);
2833 w (OCH3); 1611 s; 1511 m; 1458 w; 1249 s; 1176 m; 1031
m; 831 m; 803 w. 1H NMR (CDCl3): δ 1.87 (s, 2H, NH,); 2.88-
2.98 (m, 2H, NCH2C); 3.24-3.35 (m, 2H, NCH2C); 3.70 (s, 6H,
OCH3); 4.21 (s, 2H, ArCH); 6.69 (AA′BB′, 3J ) 8.8 Hz, 4H, ArH-
(2R,3S)/(2S,3R)-N,N′-Dia cetyl-2,3-bis(2-flu or o-4-m eth -
oxyp h en yl)p ip er a zin e (2c). (2R,3S)/(2S,3R)-2,3-Bis(2-fluoro-
4-methoxyphenyl)piperazine 2a : 1.18 mmol (396 mg). Reaction
time: 16 h. Purification: chromatography with diethyl ether/
methanol 2 + 1. Yield: 0.956 mmol (400 mg), 81%, colorless
3
3, ArH-5); 7.33 (AA′BB′, J ) 8.8 Hz, 4H, ArH-2, ArH-6).
(2R,3S)/(2S,3R)-2,3-Bis(2-flu or o-4-m eth oxyp h en yl)p ip -
er a zin e (2a ). (1R,2S)/(1S,2R)-1,2-Bis(2-fluoro-4-methoxy-
phenyl)ethylenediamine 2b: 7.14 mmol (2.20 g). Reaction
time: 48 h. Purification: chromatography on silica gel with
diethyl ether/methanol 4 + 1. Yield: 1.98 mmol (661 mg), 28%,
colorless crystals, mp 53-55 °C. IR (KBr): νj ) 3440 m, br
(NH); 3415 m, br (NH); 2837 m (OCH3); 1624 s; 1582 m; 1507
s; 1444 m; 1288 s; 1192 m; 1154 s; 1133 m; 1100 s; 1032 s; 950
m; 838 m. 1H NMR (CDCl3): δ 1.75 (s, 2H, NH); 2.91-3.03
1
powder, mp 78-81 °C. H NMR (CDCl3): δ 1.99 (s, 6H, CH3-
CO); 3.73 (s, 6H, OCH3); 3.78-3.93 (m, 2H, NCH2C); 4.32 (br,
2H, NCH2C); 5.86 (br, 2H, ArCH); 6.38 (dd, 3J (H, F) ) 12.3
4
3
4
Hz, J ) 2.3 Hz, 2H, ArH-3); 6.51 (dd, J ) 8.7 Hz, J ) 2.3
3
4
Hz, 2H, ArH-5); 6.86 (dd, J ) 8.7 Hz, J (H, F) ) 8.6 Hz, 2H,
ArH-6).

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2335
(2R,3S)/(2S,3R)-N,N′-Dia cetyl-2,3-bis(2-ch lor o-4-m eth -
oxyp h en yl)p ip er a zin e (3c). (2R,3S)/(2S,3R)-2,3-Bis(2-chloro-
4-methoxyphenyl)piperazine 3a : 1.63 mmol (600 mg). Reaction
time: 16 h. Purification: chromatography on silica gel with
diethyl ether/methanol 10 + 1. Yield: 1.55 mmol (698 mg),
Gen er a l P r oced u r e for th e Eth er Clea va ge w ith BBr 3
(Meth od C). 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
atmosphere. The reaction mixture was allowed to warm and
was stirred for further 48 h. After the reaction mixture was
cooled with an ice bath, surplus of BBr3 was hydrolyzed three
times with methanol and the phenolic product was dissolved
in 0.1 N NaOH. The alkaline water phase was filtered, and
the pH was adjusted to 8 with 2 N HCl. The precipitate was
collected by suction filtration and dried over P2O5. Subse-
quently, the crude product was purified by column chroma-
tography or fractional crystallization.
1
95%, colorless powder, mp 165-168 °C. H NMR (CDCl3): δ
1.94 (s, 6H, CH3CO); 3.75 (s, 6H, OCH3); 3.85-3.97 (m, 2H,
NCH2C); 4.34 (br, 2H, NCH2C); 5.99 (br, 2H, ArCH); 6.62 (dd,
3J ) 8.8 Hz, 4J ) 2.6 Hz, 2H, ArH-5); 6.71 (d, 4J ) 2.6 Hz, 2H,
3
ArH-3); 6.87 (d, J ) 8.8 Hz, 2H, ArH-6).
Red u ction of th e Am id e Str u ctu r e of th e (2R,3S)/
(2S,3R)-N,N′-Dia cet yl-2,3-d ia r ylp ip er a zin es Accor d in g
t o Met h od A. (2R,3S)/(2S,3R)-N,N′-Diet h yl-2,3-b is(4-
m et h oxyp h en yl)p ip er a zin e (9a ). (2R,3S)/(2S,3R)-N,N′-
Diacetyl-2,3-bis(4-methoxyphenyl)piperazine 1c: 0.662 mmol
(253 mg). Purification: chromatography on silica gel with meth-
anol. Yield: 0.522 mmol (185 mg), 79%, yellow crystals, mp
(2R,3S)/(2S,3R)-2,3-Bis(4-h ydr oxyph en yl)piper azin e (1).
(2R,3S)/(2S,3R)-2,3-Bis(4-methoxyphenyl)piperazine 1a : 0.757
mmol (226 mg). Reaction time: 24 h at room temperature.
Purification: recrystallization from methanol. Yield: 0.610
mmol (165 mg), 81%, colorless powder, mp 189-190 °C. IR
(KBr): νj ) 3600-3000 m, br (OH); 1611 m; 1511 s; 1377 m;
1
3
69-71 °C. H NMR (CDCl3): δ 1.01 (t, J ) 7.1 Hz, 6H, CH3-
CH2); 2.03-2.12 (m, 2H, CH3CH2); 2.25-2.45 (m, 2H, CH3CH2);
2.55-2.60 (m, 2H, NCH2C); 3.10-3.15 (m, 2H, NCH2C); 3.72
1
1317 m; 1253 s; 1177 m; 891 m; 826 s. H NMR (see Table 1).
3
(s, 6H, OCH3); 3.75 (s, 2H, ArCH); 6.64 (AA′BB′, J ) 8.5 Hz,
Anal. (C16H18N2O2) C H N.
4H, ArH-3, ArH-5); 7.15 (AA′BB′, br, 4H, ArH-2, ArH-6).
(2R,3S)/(2S,3R)-N-E t h yl-2,3-b is(2-flu or o-4-m e t h oxy-
p h en yl)p ip er a zin e (7a ) a n d (2R,3S)/(2S,3R)-N,N′-Dieth yl-
2,3-bis(2-flu or o-4-m eth oxyph en yl)piper azin e (10a). (2R,3S)/
(2S,3R)-N,N′-Diacetyl-2,3-bis(2-fluoro-4-methoxyphenyl)pipera-
zine 2c: 0.956 mmol (400 mg). Separation: chromatography
on silica gel with diethyl ether.
(2R,3S)/(2S,3R)-2,3-Bis(2-flu or o-4-h ydr oxyph en yl)piper -
a zin e (2). (2R,3S)/(2S,3R)-2,3-Bis(2-fluoro-4-methoxyphenyl)-
piperazine 2a : 0.598 mmol (200 mg). Reaction time: 24 h at
room temperature. Purification: recrystallization from metha-
nol. Yield: 0.434 mmol (133 mg), 73%, colorless powder, mp
at 210 °C under decomposition. IR (KBr): νj ) 3600-2600 m,
br (OH); 1624 s; 1509 m; 1472 s; 1357 w; 1300 s; 1252 m; 1154
m; 1134 w; 1092 s; 966 s; 846 s; 817 m. MS (EI, 150 °C): m/z
(%) ) 306 (30) [M]+•; 276 (12); 167 (23); 153 (27); 138 (42); 112
(2R,3S)/(2S,3R)-N-E t h yl-2,3-b is(2-flu or o-4-m e t h oxy-
p h en yl)p ip er a zin (7a ). Yield: 0.259 mmol (94 mg), 27%,
colorless oil. 1H NMR (CDCl3): δ 1.05 (t, 3J ) 7.2 Hz, 3H, CH3-
CH2); 2.16-2.25 (m, 1H, CH3CH2); 2.36-2.45 (m, 1H, CH3CH2);
2.63-2.69 (m, 1H, NCH2C); 2.88-2.95 (m, 1H, NCH2C); 3.19-
3.26 (m, 1H, NCH2C); 3.31-3.37 (m, 1H, NCH2C); 3.69 (s, 3H,
1
(24); 30 (100). H NMR (see Table 1). Anal. (C16H16F2N2O2) C
H N.
(2R,3S)/(2S,3R)-2,3-Bis(2-ch lor o-4-h yd r oxyp h en yl)p ip -
er a zin e (3). (2R,3S)/(2S,3R)-2,3-Bis(2-chloro-4-methoxyphen-
yl)piperazine 3a : 0.871 mmol (320 mg). Reaction time: 36 h
at room temperature. Purification: recrystallization from
methanol. Yield: 0.598 mmol (203 mg), 69%, colorless powder,
mp at 175 °C under decomposition. IR (KBr): νj ) 3600-2700
m, br (OH); 2955 w; 1606 s; 1496 s; 1453 s; 1363 m; 1268 s;
1239 s; 1125 m; 1038 m; 900 m; 853 m. MS (EI, 180 °C): m/z
(%) ) 338 (24) [M]+•; 303 (2) [M-Cl]+; 210 (12); 183 (42); 170
(54); 154 (100); 141 (54); 128 (22). 1H NMR (see Table 1). Anal.
(C16H16Cl2N2O2) C H N.
3
OCH3); 3.71 (s, 3H, OCH3); 4.51 (d, J ) 3.7 Hz, 1H, ArCH);
4.68 (d, 3J ) 3.7 Hz, 1H, ArCH); 6.33-6.39 (m, 2H, ArH-3,
ArH-5); 6.47 (dd, 3J (H, F) ) 12.3 Hz, 4J ) 2.5 Hz, 1H, ArH-3);
3
4
3
6.56 (dd, J ) 8.7 Hz, J ) 2.5 Hz, 1H, ArH-5); 7.08 (dd, J )
4
3
8.6 Hz, J (H, F) ) 8.6 Hz, 1H, ArH-6); 8.16 (dd, J ) 8.5 Hz,
4J (H, F) ) 8.5 Hz, 1H, ArH-6).
(2R,3S)/(2S,3R)-N,N′-Dieth yl-2,3-bis(2-flu or o-4-m eth oxy-
p h en yl)p ip er a zin e (10a ). Yield: 0.538 mmol (210 mg), 56%,
colorless oil. 1H NMR (CDCl3): δ 1.05 (t, 3J ) 7.2 Hz, 6H, CH3-
CH2); 2.13-2.22 (m, 2H, CH3CH2); 2.33-2.43 (m, 2H, CH3CH2);
2.60-2.65 (m, 2H, NCH2C); 3.09-3.15 (m, 2H, NCH2C); 3.73
(s, 6H, OCH3); 4.32 (s, 2H, ArCH); 6.43 (dd, 3J (H, F) ) 12.2
(2R,3S)/(2S,3R)-2-(2-Ch lor o-4-h yd r oxyp h en yl)-3-(2,6-
d ich lor o-4-h yd r oxyp h en yl)p ip er a zin e (4). (2R,3S)/(2S,3R)-
2-(2-Chloro-4-methoxyphenyl)-3-(2,6-dichloro-4-methoxyphenyl)-
piperazine 4a : 0.212 mmol (85 mg). Reaction time: 48 h under
reflux. Purification: chromatography on silica gel with diethyl
ether/methanol 4 + 1. Yield: 0.099 mmol (37 mg), 47%,
colorless powder, mp 148-150 °C. IR (KBr): ν 3600-2900 s,
br (OH); 2943 w; 1604 s; 1493 w; 1439 s; 1379 w; 1272 s; 1234
4
3
4
Hz, J ) 2.4 Hz, 2H, ArH-3); 6.48 (dd, J ) 8.7 Hz, J ) 2.4
Hz, 2H, ArH-5); 7.56 (br, 2H, ArH-6).
(2R,3S)/(2S,3R)-N-E t h yl-2,3-b is(2-ch lor o-4-m et h oxy-
p h en yl)p ip er a zin e (8a ) a n d (2R,3S)/(2S,3R)-N,N′-Di-
eth yl-2,3-bis(2-ch lor o-4-m eth oxyph en yl)piper azin e (11a).
(2R,3S)/(2S,3R)-N,N′-Diacetyl-2,3-bis(2-chloro-4-methoxy-
phenyl)piperazine 3c: 1.55 mmol (698 mg). Separation: chroma-
tography on silica gel with diethyl ether.
1
m; 1101 m; 1039 m; 953 m; 901m; 854 m. H NMR (see Table
1). Anal. (C16H15Cl3N2O2) C H N.
(2R,3S)/(2S,3R)-2,3-Bis(2,6-d ich lor o-3-h yd r oxyp h en yl)-
p ip er a zin e (5). (2R,3S)/(2S,3R)-2,3-Bis(2,6-dichloro-3-meth-
oxyphenyl)piperazine 5a : 0.229 mmol (100 mg). Reaction
time: 96 h under reflux. Purification: chromatography on
silica gel with diethyl ether/methanol 10 + 1. Yield: 0.098
mmol (40 mg), 43%, yellow powder, mp 147-149 °C. IR
(KBr): νj ) 3600-2900 s, br (OH); 2938 w; 1631 m; 1569 m;
1448 s; 1293 s; 1202 w; 1103 w; 815 m. 1H NMR (see Table 1).
Anal. (C16H14Cl4N2O2) C H N.
(2R,3S)/(2S,3R)-2,3-Bis(2,6-d ich lor o-4-h yd r oxyp h en yl)-
p ip er a zin e (6). (2R,3S)/(2S,3R)-2,3-Bis(2,6-dichloro-4-meth-
oxyphenyl)piperazine 6a : 0.195 mmol (85 mg). Reaction
time: 5 days under reflux. Purification: chromatography on
silica gel with diethyl ether/methanol 10 + 1. Yield: 0.066
mmol (27 mg), 34%, yellow powder, mp 168-170 °C. IR
(KBr): νj ) 3600-2700 s, br (OH); 2957 w; 1603 s; 1571 s; 1437
s; 1272 s; 1173 m; 1093 m; 1059 m; 952 m; 855 s; 793 s. 1H
NMR (see Table 1). Anal. (C16H14Cl4N2O2) C H N.
(2R,3S)/(2S,3R)-N-E t h yl-2,3-b is(2-ch lor o-4-m et h oxy-
p h en yl)p ip er a zin e (8a ). Yield: 0.354 mmol (140 mg), 23%,
colorless oil. 1H NMR (CDCl3): δ 1.05 (t, 3J ) 7.2 Hz, 3H, CH3-
CH2); 2.23-2.32 (m, 1H, CH3CH2); 2.41-2.50 (m, 1H, CH3CH2);
2.69-2.73 (m, 1H, NCH2C); 3.05-3.13 (m, 1H, NCH2C); 3.19-
3.26 (m, 1H, NCH2C); 3.32-3.39 (m, 1H, NCH2C); 3.70 (s, 3H,
3
OCH3); 3.72 (s, 3H, OCH3); 4.78 (d, J ) 3.6 Hz, 1H, ArCH);
3
3
4
4.83 (d, J ) 3.6 Hz, 1H, ArCH); 6.47 (dd, J ) 8.7 Hz, J )
2.6 Hz, 1H, ArH-5); 6.66 (d, 4J ) 2.6 Hz, 1H, ArH-3); 6.69 (dd,
3J ) 8.7 Hz, 4J ) 2.6 Hz, 1H, ArH-5); 6.78 (d, 4J ) 2.6 Hz, 1H,
3
3
ArH-3); 7.13 (d, J ) 8.7 Hz, 1H, ArH-6); 8.18 (d, J ) 8.7 Hz,
1H, ArH-6).
(2R,3S)/(2S,3R)-N,N′-Diet h yl-2,3-b is(2-ch lor o-4-m et h -
oxyp h en yl)p ip er a zin e (11a ). Yield: 1.11 mmol (470 mg),
72%, colorless powder, mp 62-65 °C. 1H NMR (CDCl3): δ 1.02
(t, 3J ) 7.1 Hz, 6H, CH3CH2); 2.18-2.27 (m, 2H, CH3CH2);
2.30-2.41 (m, 2H, CH3CH2); 2.49-2.70 (m, 2H, NCH2C); 3.14-
3.22 (m, 2H, NCH2C); 3.71 (s, 6H, OCH3); 4.55 (s, 2H, ArCH);
(2R ,3S )/(2S ,3R )-N -E t h yl-2,3-b is(2-flu or o-4-h yd r oxy-
p h en yl)p ip er a zin e (7). (2R,3S)/(2S,3R)-N-Ethyl-2,3-bis(2-
fluoro-4-methoxyphenyl)piperazine 7a : 0.828 mmol (300 mg).
3
4
4
6.55 (dd, J ) 8.8 Hz, J ) 2.6 Hz, 2H, ArH-5); 6.70 (d, J )
2.6 Hz, 2H, ArH-3); 7.26 (br, 2H, ArH-6).

2336 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Gust et al.
Reaction time: 36 h at room temperature. Purification: chroma-
tography on silica gel with acetone. Yield: 0.508 mmol (170
mg), 61%, colorless powder, mp 152-153 °C. IR (KBr): νj )
3600-2800 s, br (OH); 1625 s; 1508 s; 1458 s; 1385 m; 1299 s;
1239 m; 1150 s; 1093 s; 967 s; 846 s. MS (EI, 150 °C): m/z (%)
) 334 (6) [M]+•; 305 (10) [M-C2H5]+; 182 (100); 168 (20); 152
Safe3 liquid scintillator was from Wallac (Turku, Finland);
NET-317-estradiol[2,4,6,7-3H(N)] (17â-[3H]estradiol) was from
Du Pont NEN (Boston, MA); CDCl3, [D6]acetone, [D6]DMSO,
and [D4]methanol were from Aldrich (Steinheim, Germany);
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
purchased from Merck or Fluka) in 1000 mL of purified water.
TRIS- buffer (pH ) 7.5) was prepared by dissolving 1.211 g of
trishydroxymethylaminomethane, 0.372 24 g of Titriplex III,
and 0.190 53 g of sodium azide (all from Merck or Fluka) in
1 L of purified water. Deionized water was produced by means
of a Millipore Milli-Q Water System, resistivity >18 MΩ.
T-75 flasks, reaction tubes, 96 well plates, and 6 well plates
were purchased from Renner GmbH (Dannstadt, Germany).
ER Bin d in g Assa y. The applied method was described
already by Hartmann et al.28 and used with some modifica-
tions. The RBA of the test compounds to the ER was deter-
mined by the displacement of 17â-[3H]estradiol from its
binding site. For this purpose, the test compounds were
dissolved in ethanol and diluted with TRIS-buffer to 6-8
appropriate concentrations (300 µL). They were incubated
shaking with calf uterine cytosol (100 µL) and 17â-[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-
charcoal suspension in TRIS-buffer was added to each tube.
After it was shaken for 90 min at 4 °C and centrifuged, 500
µL of HiSafe3 was mixed with 100 µL of supernatant of each
sample and the reactivity was determined by liquid scintilla-
tion spectroscopy. The same procedure was used to quantify
the binding of 17â-[3H]estradiol (0.723 pmol control). Nonspe-
cific binding was calculated using 2 nmol of 17â-estradiol as
the competing ligand. On a semilog plot, the percentage of
maximum-bound labeled steroid corrected by the nonspecifi-
cally bound 17â-[3H]estradiol vs concentration of the competi-
tor (log-axis) is plotted. At least six concentrations of each
compound were chosen to estimate its binding affinity. From
this plot, those molar concentrations of unlabeled estradiol and
of the competitors were determined, which reduced the binding
of the radioligand by 50%.
1
(21); 138 (21); 125 (42); 58 (88). H NMR (see Table 1). Anal.
(C18H20F2N2O2) C H N.
(2R,3S)/(2S,3R)-N-E t h yl-2,3-b is(2-ch lor o-4-h yd r oxy-
p h en yl)p ip er a zin e (8). (2R,3S)/(2S,3R)-N-Ethyl-2,3-bis(2-
chloro-4-methoxyphenyl)piperazine 8a : 0.354 mmol (140 mg).
Reaction time: 36 h at room temperature. Purification: re-
crystallization from methanol. Yield: 0.150 mmol (55 mg),
42%, colorless powder, mp 178-180 °C. IR (KBr): νj ) 3600-
2600 s, br (OH); 3316 m (NH); 2938 w, 2851 w; 1606 s; 1494
s; 1474 s; 1457 s; 1382 w; 1355 w; 1273 s; 1248 s; 1139 m;
1036 m; 901 s; 856 m. MS (EI, 190 °C): m/z (%) ) 366 (5)
[M]+•; 337 (8) [M-C2H5]+; 210 (11); 198 (100); 184 (25); 168
1
(17); 155 (23); 141 (42). H NMR (see Table 1). Anal. (C18H20
Cl2N2O2) C H N.
-
(2R,3S)/(2S,3R)-N,N′-Dieth yl-2,3-bis(4-h yd r oxyp h en yl)-
p ip er a zin e (9). (2R,3S)/(2S,3R)-N,N′-Diethyl-2,3-bis(4-meth-
oxyphenyl)piperazine 9a : 1.75 mmol (620 mg). Reaction
time: 24 h at room temperature. Purification: chromatography
on silica gel with methanol. Yield: 1.23 mmol (400 mg), 70%,
colorless powder, mp 162-164 °C. IR (KBr): νj ) 3600-2600
s, br (OH); 2973 m; 2814 m; 1610 s; 1512 s; 1453 m; 1374 w;
1249 s; 1174 m; 1147 m; 1103 m; 836 s. MS (EI, 150 °C): m/z
(%) ) 326 (47) [M]+•; 297 (36) [M-C2H5]+; 176 (100); 162 (28);
1
148 (41); 134 (32); 120 (14); 107 (29). H NMR (see Table 1).
Anal. (C20H26N2O2) C H N.
(2R,3S)/(2S,3R)-N,N′-Dieth yl-2,3-bis(2-flu or o-4-h ydr oxy-
ph en yl)piper azin e (10). (2R,3S)/(2S,3R)-N,N′-Diethyl-2,3-bis-
(2-fluoro-4-methoxyphenyl)piperazine 10a : 0.435 mmol (170
mg). Reaction time: 36 h at room temperature. Purification:
recrystallization from CH2Cl2/ligroin. Yield: 0.268 mmol (97
mg), 62%, colorless powder, mp 118-121 °C. IR (KBr): νj )
3600-2600 s, br(OH); 2974 m; 2819 w; 1624 s; 1507 s; 1463 s;
1390m; 1298 s; 1242 m; 1150 s; 1090 s; 966 s; 845 s. MS (EI,
180 °C): m/z (%) ) 362 (44) [M]+•; 333 (65) [M-C2H5]+; 248
(22); 194 (100); 180 (44); 166 (72); 152 (76); 138 (25); 125 (48);
c[3H]-estradiol at 50% inhibition
1
RBA )
× 100%
115 (82). H NMR (see Table 1). Anal. (C20H24F2N2O2) C H N.
csample at 50% inhibition
(2R,3S)/(2S,3R)-N,N′-Dieth yl-2,3-bis(2-ch lor o-4-h ydr oxy-
ph en yl)piper azin e (11). (2R,3S)/(2S,3R)-N,N′-Diethyl-2,3-bis-
(2-chloro-4-methoxyphenyl)piperazine 11a : 0.723 mmol (306
mg). Reaction time: 36 h at room temperature. Purification:
chromatography on silica gel with acetone. Yield: 0.493 mmol
(195 mg), 68%, colorless powder, mp 171-173 °C. IR (KBr): νj
) 3600-2700 s, br (OH); 2973 m; 2828 w; 1725 m; 1606 s;
1494 s; 1450 s; 1378 m; 1354 m; 1282 s; 1229 s; 1144 m; 1038
m; 979 w; 900 s; 853 m; 830 m. MS (EI, 180 °C): m/z (%) )
394 (25) [M]+•; 366 (35) [M-C2H5]+; 211 (100); 197 (34); 185
Lu cifer a se Assa y. The pertinent in vitro assay was
described earlier by Hafner et al.29 One week before starting
the experiment, MCF-7-2a cells were cultivated in DMEM
supplemented with L-glutamine, antibiotics, and dextran/
charcoal-treated BCS (ct-BCS, 50 mL/L). 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
cultivated in six well flat-bottomed plates (0.5 mL cell suspen-
sion and 2.0 mL medium per well) at growing conditions (see
above). After 24 h, 25 µL of a stock solution of the test
compounds was added to achieve concentrations ranging from
10-5-10-10 M and the plates were incubated for 50 h. Before
they were harvested, the cells were washed twice with PBS
and then 200 µL of cell culture lysis reagent was added into
each well. After a 20 min lysation at room temperature, cells
were transferred into reaction tubes and centrifugated. Lu-
ciferase was assayed using the Promega luciferase assay
reagent. Fifty microliters of each supernatant was mixed with
50 µL of substrate reagent. Luminescence (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 Bradford44) of each sample
with the mass of luciferase. Estrogenic activity was expressed
as percent activation of a 10-8 M estradiol control (100%).
1
(29); 177 (77); 168 (51); 154 (20); 141 (45); 115 (76). H NMR
(see Table 1). Anal. (C20H24Cl2N2O2) C H N.
Biologica l Met h od s. Bioch em ica ls, Ch em ica ls, a n d
Ma ter ia ls. Dextran, 17â-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 without phenol red (DMEM) was from Gibco
(Eggenstein, Germany); bovine calf serum (BCS) was from
Bio whittaker (Verviers, Belgium); N-hexamethylpararos-
aniline (crystal violet) and gentamicin sulfate were from Fluka
(Deisenhofen, Germany); glutardialdehyde (25%) was from
Merck (Darmstadt, Germany); trypsin (0.05%) in ethylene-
diaminetetraacetic acid (0.02%) (trypsin/EDTA) was from
Boehringer (Mannheim, Germany); penicillin-streptomycin
gold standard (10 000 IE penicillin/mL, 10 mg strepto-
mycin/mL) and geneticin disulfate (geneticin solution: 35.71
mg/mL PBS) were from ICN Biomedicals GmbH (Eschwege,
Germany); Norit A (charcoal) was from Serva (Heidelberg,
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 Hi-
Ack n ow led gm en t . We are grateful to Prof. E.
von Angerer for providing the MCF-7-2a cell line. The
technical assistance of S. Bergemann and I. Schnautz
is acknowledged. The presented work was supported

2,3-Diarylpiperazines
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2337
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