Effects of C2-Alkylation, N-Alkylation, and N,N′-Dialkylation on the Stability and Estrogen Receptor Interaction of (4R,5S)/(4S,5R)-4,5-Bis(4-hydroxyphenyl)-2-imidazolines

Article abstract of DOI:10.1021/jm0309809

(4R,5S)/(4S,5R)-4,5-Bis(4-hydroxyphenyl)-2-imidazolines bearing 2,2′-H (3a), 2,2′-Cl (3b), 2,2′,6-Cl (3c), and 2,2′-F (3d) substituents in the aromatic rings were C2-alkylated (5a-i), N-alkylated (7, 7a-c), and N,N′-dialkylated (9a-c). The synthesis started from the diastereomerically pure (1R,2S)/(1S,2R)-1,2-diamino-1,2-bis(4-methoxyphenyl)ethanes 1a-d, which were cyclized to the imidazolines 2a-d and 4a-i with triethylorthoesters or iminoethers. Ether cleavage with BBr₃ yielded the (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines 3a-d and 5a-i. The N-alkylation and N,N′-dialkylation of 2b, employed for obtaining 7a-c and 9a-c, were performed prior to the ether cleavage with alkyl iodine in dry THF. By use of HPLC, the influence of the substitution patterns in the aromatic rings and alkyl chains at the C2- or N-atoms on the hydrolysis rate of the imidazolines was studied under in vitro conditions. It appeared that only imidazolines with C2- or N-alkyl substituents show sufficient stability to interact as heterocycles with the estrogen receptor (ER). The resulting gene activation was monitored in a luciferase assay using ERα-positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE _wtcluc. It is interesting to note that C2-alkylation led to a strong reduction or even a complete loss of activity whereas N-alkylation improved the estrogenic profile. The (4R,5S)/(4S,5R)-N-ethyl-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazoline 7b has proven to be the most active compound in this structure-activity relationship study (EC₅₀ = 0.015 μM).

Full text of DOI:10.1021/jm0309809

J . Med. Chem. 2004, 47, 915-927
915
Effects of C2-Alk yla tion , N-Alk yla tion , a n d N,N′-Dia lk yla tion on th e Sta bility
a n d Estr ogen Recep tor In ter a ction of (4R,5S)/
(4S,5R)-4,5-Bis(4-h yd r oxyp h en yl)-2-im id a zolin es
Moriz von Rauch, Miriam Schlenk, and Ronald Gust*
Institute of Pharmacy, Free University of Berlin, Ko¨nigin-Luise Strasse 2+4, D-14195 Berlin, Germany
Received J uly 31, 2003
(4R,5S)/(4S,5R)-4,5-Bis(4-hydroxyphenyl)-2-imidazolines bearing 2,2′-H (3a ), 2,2′-Cl (3b), 2,2′,6-
Cl (3c), and 2,2′-F (3d ) substituents in the aromatic rings were C2-alkylated (5a -i), N-alkylated
(7, 7a -c), and N,N′-dialkylated (9a -c). The synthesis started from the diastereomerically pure
(1R,2S)/(1S,2R)-1,2-diamino-1,2-bis(4-methoxyphenyl)ethanes 1a -d , which were cyclized to the
imidazolines 2a -d and 4a -i with triethylorthoesters or iminoethers. Ether cleavage with BBr3
yielded the (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines 3a -d and 5a -i. The
N-alkylation and N,N′-dialkylation of 2b, employed for obtaining 7a -c and 9a -c, were
performed prior to the ether cleavage with alkyl iodine in dry THF. By use of HPLC, the
influence of the substitution patterns in the aromatic rings and alkyl chains at the C2- or
N-atoms on the hydrolysis rate of the imidazolines was studied under in vitro conditions. It
appeared that only imidazolines with C2- or N-alkyl substituents show sufficient stability to
interact as heterocycles with the estrogen receptor (ER). The resulting gene activation was
monitored in a luciferase assay using ERR-positive MCF-7-2a breast cancer cells stably
transfected with the plasmid EREwtcluc. It is interesting to note that C2-alkylation led to a
strong reduction or even a complete loss of activity whereas N-alkylation improved the
estrogenic profile. The (4R,5S)/(4S,5R)-N-ethyl-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazoline
7b has proven to be the most active compound in this structure-activity relationship study
(EC50 ) 0.015 µM).
Ch a r t 1
In tr od u ction
The estrogen receptors ERR and ERâ are important
targets in the treatment and prevention of several
diseases. The endogenous ligand estradiol (E2) not only
regulates the growth, differentiation, and function of the
male and female reproductive systems through these
receptors but also influences bone maintenance, the
central nervous system, and the cardiovascular system.1-4
Crystal structures of the ligand binding domain (LBD)
of both ER subtypes cocrystallized with E2 or other
agonists, e.g., diethylstilbestrol (DES) or genistein, as
well as selective estrogen receptor modulators (SERMs),
e.g., raloxifene (RAL) and 4-hydroxytamoxifen (4OHT),
providing a good insight into the binding mode of drugs
at the ERs (for structures, see Chart 1).5-7
Agonists build H-bridges to Glu353, Arg394, and
His524 and van der Waals contacts to aliphatic residues
of amino acids in the LBD of ERR.5,6 A comparable
binding mode was determined for ERâ with H-bridges
to Glu305, Arg346, and His475.7 These bindings lead
to a conformational change of the receptor molecule
followed by a dimerization of ER/drug conjugates and
an interaction with the “estrogen response element”
(ERE) of the DNA.
heterocyclic compounds with an unusual ER binding
mode shows a lot of promise in optimizing hormonal
replacement therapy.
In previous papers, we have described new lead
structures for the design of novel hormonally active
drugs.9-11 It was demonstrated that (4R,5S)/(4S,5R)-
4,5-bis(4-hydroxyphenyl)-2-imidazolines and (2R,3S)/
The varying tissue distribution of the ERs encourages
the search for new steroidal or nonsteroidal compounds
with subtype selectivity.8 Moreover, the development of
* To whom correspondence should be addressed. Phone: (030) 838
53272. Telefax: (030) 838 56906. E-mail: rgust@zedat.fu-berlin.de.
10.1021/jm0309809 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/08/2004

916 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
Sch em e 1
(2S,3R)-2,3-bis(4-hydroxyphenyl)piperazines activated
the gene expression in ERR-containing MCF-7-2a cells
despite a spatial structure that prevents the contact to
His524.9,11 These studies led to a new classification of
estrogens. The essentially planar estrogens, e.g., E2 or
DES, are classified as type I (class 1), whereas angular
estrogens, e.g., 2,3-diarylpiperazines and 4,5-diaryl-2-
imidazolines, are type II (class 2) estrogens.9-12 Know-
ing the fact that imidazolines hydrolyze into formamides
that can then act as type I estrogens, we investigated

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 917
F igu r e 1. 1H NMR (a, b) and 13C spectra (c, d) of 5f and 9b.
the stability of (4R,5S)/(4S,5R)-4,5-bis(4-hydroxy/meth-
oxyphenyl)-2-imidazolines under in vitro conditions and
studied the influence of C2-alkyl, N-alkyl, and N,N′-
dialkyl substituents on both the stability and the ER
interaction.
Resu lts
Syn th esis. The synthesis of the imidazolines was
performed by reaction of the respective 1,2-diamino-1,2-
diarylethane (1a -d ) with an excess of triethyl ortho-

918 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
F igu r e 2. Low-energy conformations of 5f, 7b, and 9b.
formate, triethyl orthoacetate, or triethyl orthopropi-
onate to yield 2a -d , 4a ,b, and 4d -g or with the
iminoethers 13a ,b to yield 4c and 4h ,i (methods A and
B, Scheme 1). 13a ,b were obtained utilizing the method
of Pinner by partial hydrolysis of the respective nitrile
in ethanolic HCl.13,14
The weak acidic character of 2b was employed to
synthesize N-alkylated imidazolines. 2b was treated
successively with butyllithium and alkyl iodine to obtain
the imidazolines 6a -c (method D, Scheme 1). If the
imidazoline anion is reacted with an excess of alkyl
iodine, the N,N′-dialkyl-2-imidazolinium salts 8a -c can
additionally be isolated (method E, Scheme 1). 6 was
not available under these reaction conditions. Therefore,
the 1,2-diamino-1,2-diarylethane 1a had to be alkylated
first (method C; compound 10) followed by ring closure
(method A, Scheme 1). All methyl ethers were cleaved
using BBr3 to yield the hydroxy-substituted compounds
3a -d , 5a -i, 7, 7a -c, and 9a -c (method F, Scheme 1).
F igu r e 3. Energy profile for the rotation of (1R,2S)/(1S,2R)-
1-a m in o-2-form a m ido-1,2-bis(2-chloro-4-hydroxyph en yl)-
ethane 11.
planar structure of the 2-imidazoline ring with pseudo-
axially oriented aromatic rings, as already described for
3a -d .10
The 1-amino-2-formamido-1,2-bis(2-chloro-4-hydroxy-
phenyl)ethane 11 was obtained by hydrolysis of the
imidazoline 3b in 0.01 N NaOH.
The orientation of the substituents at the heterocyclic
ring was confirmed by theoretical calculations on the
examples of 5f, 7b, and 9b. As illustrated in Figure 2,
the N-atoms as well as the C2-atom are partially sp2-
hybridized, with the methylene groups being located in
the heterocyclic plane. The CH3 group is oriented oppo-
site the aromatic rings if the ethyl group is N-standing
(7b and 9b) and oriented toward the phenyl rings in
the C2-ethyl derivative 5f (see Figure 2). However, the
ethyl chains are not hindered in rotation, and the energy
barrier for rotation is less than 2 kcal/mol.
Str u ctu r a l Ch a r a cter iza tion . The 1H NMR spectra
of C2-alkylated and N,N′-dialkylated derivatives with
an identical substitution pattern in both aromatic rings
(5a ,b,d -i, 9a -c) demonstrated only one set of signals
for the aromatic protons and a singlet resonance for the
benzylic protons (for the NMR spectra of 5f and 9b, see
Figure 1). This is only possible in the case of a sym-
metrical heterocyclic ring with a delocalized double bond
between both nitrogen atoms. To verify this, the 13C
NMR spectra of 4,5-bis(2-chloro-4-hydroxyphenyl)-2-
imidazoline 3b and its C2-ethyl (5f) and N,N′-diethyl
(9b) derivative were measured. These spectra confirmed
a symmetric conformation of the molecules. Compared
to 3b (eight resonances: 6 × Ar-H; 1 × C2-H; 1 ×
C4,5-H), there are only two additional signals of the
ethyl chains detected for 5f and 9b (see Figure 1). This
indicates a planar arrangement of the N-CdN moiety
with the CH2 groups of the alkyl chains being located
in the plane of the heterocyclic ring.
2-Imidazolines show temperature- and pH-dependent
hydrolysis under formation of amides.15 The coupling
constant of the benzylic protons determined for the
3
hydrolysis product 11 amounted to J ) 5.6 Hz. This
could be an average of the coupling constants of the
possible conformations during the rotation around the
diarylethane bridge or could result from a preferred
conformation with a dihedral angle between the benzylic
protons of about 60°.
Theoretical investigations indicated three low-energy
conformations of comparable energy in which the aro-
matic rings and the amino groups are synclinally
(synclinal I, 13.72 kcal/mol; synclinal II, 12.04 kcal/mol)
or antiperiplanarly (11.09 kcal/mol) oriented (see Figure
3). A conformational change is possible (activation
energies: 6.73 kcal/mol for antiperiplanar f synclinal
II; 9.44 kcal/mol for antiperiplanar f synclinal I).
Sta bility Stu d ies. The stability of (4R,5S)/(4S,5R)-
4,5-diaryl-2-imidazolines was studied under in vitro
The arrangement of the aromatic rings was examined
by coupling constants analysis. 4-(2-Chloro-4-hydroxy-
phenyl)-5-(2,6-dichloro-4-hydroxyphenyl)-2-methyl-2-
imidazoline 5c and the N-monoalkyl derivatives (7, 7a -
c) represent asymmetric molecules with coupling con-
3
stants for the benzylic protons of J ) 12.0-12.9 Hz.
These values correlate with an arrangement of the
benzylic protons in a dihedral angle of 0-15° and a

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 919
F igu r e 4. HPLC investigations (RP 18 material; methanol/H2SO4 (0.001 N, 20 mM Na2SO4) ) 50:50; λ ) 254 nm) on the stability
of the 4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazoline 3b. HPLC chromatogram at the beginning (A) and after an incubation
time of 55 h (B). The kinetics of decomposition is shown in part C.
conditions by means of HPLC. In a preliminary study,
we used the 4,5-bis(2-chloro-4-hydroxyphenyl)-2-imida-
zoline 3b as a model. 3b was incubated at 37 °C and
pH 7.4 for 55 h. These conditions matched exactly those
used in the luciferase assay to determine the hormonal
activity.
in the rate-determining step by nucleophilic attack of
OH- at the C2-atom (see Scheme 2). These precondi-
tions enabled a calculation of the rate constant using
KSIM (Table 1). 3b hydrolyzed rapidly with a half-live
of t1/2 ) 5.94 h and a rate constant of kobs ) 0.150 h-1
.
O-methylation (2b) and exchange of the ortho Cl sub-
stituents (3a ) did not significantly influence the hy-
drolysis rate (2b, t1/2 ) 4.58 h, kobs ) 0.198 h-1; 3a , t1/2
) 6.36 h, kobs ) 0.122 h-1). However, an additional
2-chlorine substituent (3c, t1/2 ) 13.41 h, kobs ) 0.052
The start chromatogram exhibited a well-defined peak
at a retention time of tR ) 7.52 min, which was assigned
to 3b (see Figure 4A). The peak area decreased during
an incubation time of 55 h, and the appearance of a
h-1) and an N-ethyl group (7b, t1/2 ) 154.03 h, kobs
)
hydrophilic decomposition product was observed (tR
)
0.004 h-1) enhanced the stability. Interestingly, the C2-
hydroxyethyl derivative 5i and the N,N′-diethyl com-
pound 9b remained stable during the incubation time.
5.06 min, Figure 4B). Isolation from the reaction
mixture and spectroscopical characterization indicated
the formation of the formamide 11.
It should be noted that the asymmetric compounds
3c and 7b can form two hydrolysis products. In the case
of 3c both products were found whereby one was not
formed in favor of the other. For 7b we only detected
The hydrolysis of 3b illustrated in Figure 4C repre-
sents a consecutive reaction with two reversible reaction
steps. In the initial reaction step, the weak imidazoline
base gets protonated and the ring opening takes place

920 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
Ta ble 2. Activation of Luciferase Expression in MCF-7-2a
Cells
Sch em e 2
luciferase expression
in MCF-7-2a cells
relative
activation
R5 [%] at 1 µM
EC50
[µM]
RTPa
[%]
R1 R2 R3
R4
3a
0
67
112
0
3b Cl
3c Cl Cl
3d
5a
0.380
0.065
0.021
0.123
F
CH3
2
5b Cl
CH3
0
5c Cl Cl
CH3
CH3
C2H5
20
12
6
5
0.002
5d
5e
F
Ta ble 1. Half Life of 2-Imidazolines under in Vitro Conditionsa
half-life t1/2 kobs
5f Cl
C2H5
C2H5
C2H4OH
C2H4OH
6
0
0
20
0
78
103
86
54
60
5g
5h
F
R1
R2
R3
R4
R5
R6
[h]
[h-1
]
5i Cl
7
C2H5
CH3
C2H5
C3H7
CH3
7a Cl
7b Cl
7c Cl
9a Cl
9b Cl
9c Cl
E2
0.300
0.015
0.150
0.780
0.500
0.027
0.533
0.053
0.010
0.016
CH3
C2H5
C3H7
C2H5
C3H7
6
100
2b Cl OCH3
3a OH
4.58
6.36
5.94
13.41
ndb
0.198
0.122
0.150
0.052
ndb
0.000 08 100
3b Cl OH
3c Cl OH
5i Cl OH
7b Cl OH
9b Cl OH
Cl
C2H4OH
C2H5
C2H5
154.03
0.004
C2H5
ndb
ndb
a
In vitro conditions: solvent ) PBS; pH 7.4; temperature of
11
12
CHO
H
21
44
3
1.5
0.003
0.005
b
37 °C. No decomposition was observed for 5i and 9b within 55
h.
a
Relative transcriptional potency: % RTP ) [EC50(E2)/EC50
-
one decomposition product in the HPLC chromato-
grams. Theoretically, a secondary and a tertiary amide
could be formed. In accordance with de Savignac et al.,16
we propose the favored formation of the secondary
amide.
(ligand)] × 100.
0.021%) < 7a (N-CH3: EC50 ) 0.300 µM; RTP ) 0.027%)
< 7c (N-C3H7: EC50 ) 0.150 µM; RTP ) 0.053%) < 7b
(N-C2H5: EC50 ) 0.015 µM; RTP ) 0.533%).
Biologica l P r op er ties. The interactions of the com-
pounds 3a -d with the ER were previously described10
and are summarized in Table 2. The relative binding
affinity (RBA) to the ER was determined for all com-
pounds in a competition experiment with [3H]-E2 and
calf uterine cytosol. The RBA values, which are less
than 0.1% (data not shown), indicate that none of the
compounds can displace E2 in large amounts from its
binding site. Nevertheless, we investigated the effects
on the ERR-containing MCF-7-2a cell line in order to
evaluate whether the imidazolines interact indepen-
dently of E2 in the LBD of the ER. In this assay, the
expression of luciferase correlates very well with the
estrogenic potency of drugs.17
Despite their low RBA values, the 2-imidazolines 3b
and 3c activated the luciferase expression. The Cl
substituents in the aromatic rings enhanced the hydro-
phobicity and mediated a relative transcriptional po-
tency of RTP ) 0.021% (EC50 ) 0.380 µM) and 0.123%
(EC50 ) 0.065 µM), respectively (Table 2 and ref 9).
N-monoalkylation of 3b increased the estrogenic
activity further: 3b (N-H: EC50 ) 0.380 µM; RTP )
On the other hand, N,N′-dialkylation reduced the
activity. 9c was completely inactive, while 9b (EC50
)
0.500 µM; RTP ) 0.016%) and 9a (EC50 ) 0.780 µM;
RTP ) 0.010%) showed a luciferase expression slightly
lower than 3b (see also Figure 5).
While N-substituents positively influenced the hor-
monal effects, the alkyl chains at C2 reduced the
activity. Only the C2-methyl derivative 5c showed
marginal effects (EC50 ) 5 µM; RTP ) 0.002%) that are
more than 50 times lower than that of its parent
compound 3c. The hydrolysis product 11 that might be
formed during the incubation of 3b with MCF-7-2a cells
significantly induced the luciferase expression only in
the highest used concentration of 10 µM (Figure 5). A
relative activation of 21% was measured at a concentra-
tion of 1 µM. This effect is even lower than that
determined for the 1,2-diamino-1,2-bis(2-chloro-4-hy-
droxyphenyl)ethane 12 (44% at 1 µM).
Finally, it is noted that none of the 2-imidazolines
were able to antagonize the effect of 1 nM E2.

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 921
F igu r e 5. Luciferase expression in MCF-7-2a cells stably transfected with the reporter plasmid EREwtcluc.
Discu ssion
findings led to their classification as type II (class 2)
estrogens.9,12
The stimulation of the ER by endogenous estrogens
plays an important role in both male and female
physiologies.18 Unfortunately, E2 stimulation is also
implicated in the development of breast cancer.19 Con-
sequently, many ER ligands were designed to prevent
E2-mediated tumor growth. This led to the discovery of
antiestrogens for the treatment of hormone-dependent
tumors.
Throughout the 1970s, research focused mainly on
optimizing the antiestrogenic properties of tamoxifen
(TAM). This potent antibreast cancer agent also ex-
pressed increased estrogenic properties in other tissues,
e.g., the endometrium, which limits its therapeutical
use.20 Such a diversity of pharmacological effects led to
its classification as a selective estrogen receptor modu-
lator.21
This categorization as type II estrogens cannot be
done without restriction because 4,5-diarylimiodazolines
may hydrolyze in aqueous solution into 1-amino-2-
formamido-1,2-diarylethanes.
Under conditions comparable to those used in our in
vitro assay (37 °C, pH 7.4, incubation time of 55 h), 3b
rapidly decomposed into the formamide 11 with a half-
life of t1/2 ) 5.94 h (see Figure 4C). Substituents in the
aromatic rings only marginally influenced the hydrolysis
rate, while alkyl chains located at the heterocycle
stabilized the molecule. All C2- and N-substituted as
well as the N,N′-dialkylated compounds proved to be
stable during the incubation time of 55 h. Only the
N-ethyl-2-imidazoline 7c was marginally hydrolyzed by
elongating the incubation time (t1/2 ) 154 h).
The acyclic compounds can adapt three low-energy
conformations with synclinally and antiperiplanarly
arranged aromatic rings (for 11, see Figure 3). The latter
represents a spatial structure comparable to DES or
HES. Despite this structural analogy, the activity of 11
on the MCF-7-2a cell line was distinctly lower than that
of 3b (see Figure 5 and Table 2) and the 1,2-diamino-
1,2-bis(2-chloro-4-hydroxyphenyl)ethane 12 (see Table
2). Interestingly, N-ethylation and N,N′-diethylation not
only enhanced the stability but also respectively in-
creased (7b) or inhibited (9b) the activity of 3b. This
suggests that the 2-imidazolines interact with the ER
without previous hydrolysis.
TAM acts as a prodrug that is activated by hydroxy-
lation of the para position of the 1-phenyl ring, resulting
in 4OHT. In both cases, the ligand recognition by the
ER is achieved through a combination of specific hy-
drogen bonds and van der Waals interactions. Crystal
structures of the LBDs of ERR and ERâ that cocrystal-
lized with agonists and antagonists indicated that the
binding of 4OHT induces a conformation of the LBD
that differs in both secondary and tertiary structural
organization from those driven by E2 binding.5-7 It was
concluded from these studies that agonists have to be
planar, hydrophobic molecules bearing two OH groups
with an O-O distance of about 12 Å.
While the interactions of type I estrogens in the LBD
of the ERs are well-known, we postulated a novel
binding mode for type II estrogens at ERR based on our
results on the ERR-positive MCF-7-2a cell line.
Initially, type II estrogens of the (4R,5S)/(4S,5R)-4,5-
bis(4-hydroxyphenyl)-2-imidazoline series are attached
to the LBD of the ERR contacting the amino acids
Glu353 and Arg394. We proposed these contacts because
they are documented in all crystal structures of agonists
as well as antagonists. Because of the angular structure
of the molecules, the second anchorage, which is es-
sential for the gene activation, can only be achieved in
the neighboring side pocket. It is very likely that they
Our studies, however, revealed that molecules with
quite different spatial structures also interact with the
ER. Especially RS/SR-configurated 2,3-diarylpipera-
zines and 4,5-diaryl-2-imidazolines activated gene ex-
pression in ERR-containing MCF-7-2a breast cancer
cells.9,10 This effect is quite remarkable because the 1,2-
diarylethane pharmacophor takes a conformation with
synclinally (piperazines) or pseudoaxially oriented aro-
matic rings (imidazolines). Compounds from the most
promising 4,5-bis(4-hydroxyphenyl)-2-imidazoline series
acted as full agonists despite their inability to displace
estradiol in high amounts from its binding site (com-
petition experiment with [3H]-E2: RBA < 0.1%). These

922 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
F igu r e 6. Interaction of DES in the LBD of ERR (left) and the postulated mode of action of the N-ethyl-4,5-bis(2-chloro-4-
hydroxyphenyl)-2-imidazoline 7b (right).
1
Elmer model 580 A; H NMR, Burker ADX 400 spectrometer
at 400 MHz (internal standard, TMS); EI-MS spectra, CH-
7A-Varian MAT (70 eV) and Kratos MS 25 RF (80 eV).
Elemental analyses were carried out at the Microlaboratory
of the Free University of Berlin. Stability studies were made
on an HPLC-system with a Kontron high-pressure gradient
system, HPLC pump 422, HPLC autosampler 465, HPLC-
UV detector 430A, (BiotekKontron, Germany), and thermostat
K 20 (Haake, Germany). As the HPLC column a Nucleosil
100-5 C18 250 mm × 4 mm i.d. with Nucleosil 120-5 C18
precolumn 30 mm × 4 mm i.d. (Macharey & Nagel) was used.
All computational graphics were built using SYBYL 6.7 (Tripos
Inc. 1699 South Hanley Road, St. Louis, MO 63144). Geometry
optimization was carried out utilizing the MM3 force field
within the program running on an INDY workstation. The
liquid scintillation counter was a 1450 Microbeta Plus (Wallac
Finnland), and luminescence measurement was carried out
using a Microlumat LB 96 P (EG & G Berthold, Germany).
Syn t h eses. The (1R,2S)/(1S,2R)-1,2-diamino-1,2-diaryl-
ethanes 1a -d and 12 and the (4R,5S)/(4S,5R)-4,5-diaryl-2-
imidazolines 2a -d and 3a -d were synthesized as described
earlier.9,10
are H-bonded to Asp351 because this amino acid also
mediated the agonistic properties of 4OHT. (The sub-
stitution of glycine for aspartate at position 351
(Asp351Gly ER) changed the activity of the 4OHT/ER
complex from estrogen-like to completely antiestro-
genic.22) Nevertheless, Thr347, as a second crucial
amino acid in the hydrophobic side pocket,23 has to be
taken into consideration as we have recently demon-
strated using theoretical methods.24
The endocrine activity of type II estrogens depends
on the hydrophobicity of the molecule. 2-Imidazolines
and piperazines gain activity by introducing halides into
the aromatic rings and alkyl chains at the nitrogen
atoms. The luciferase expression showed a maximum
with 2-Cl substitution; the respective 2-F derivatives
were by far less active.
Alkyl substituents at the 2-imidazolines not only
increased the stability of the compounds but also
increased the hydrophobicity and consequently the
hormonal activity. The best effect was shown by the
N-ethyl derivative 7b (EC50 ) 0.015 µM). Reduction (N-
methyl (7a ), EC50 ) 0.300 µM) or elongation (N-propyl
(7c), EC50 ) 0.150 µM) of the alkyl chain reduced the
activity. A comparable dependence of the ER binding
on the chain length was determined in the HES and
DES series. Therefore, we assume that the alkyl resi-
dues at the imidazoline core interact with comparable
hydrophobic parts in the LBD (see Figure 6). The decline
of activity after N,N′-dialkylation can be interpreted as
a steric hindrance during ER binding comparable to that
indicated for 7R,11â-disubstituted E2 derivatives.25
Two findings cannot be explained at the moment: the
inactivity of the C2-alkyl derivatives and the activity
of the cationic N,N′-dialkyl derivatives. The positive
charge of the compounds 9a ,b did not disturb the ER
interaction, whereas the small methyl group located at
C2 prevented gene activation. These have to be clarified
in further studies.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yl-4,5-
d ia r yl-2-im id a zolin es w ith Tr ieth ylor th oester s (Meth od
A). An aqueous solution (1.00 mmol) of the appropriate 1,2-
diamino-1,2-diarylethane with 0.25 mL of HClconc and 2.00 mL
of either triethyl orthoacetate or triethyl orthopropionate was
heated to reflux. The reaction mixture was cooled to room
temperature and combined with 20 mL of chloroform. The pH
of the solution was adjusted to 1 with 2 N HCl. The organic
layer was separated, and the water phase was washed twice
with chloroform. The water phase was neutralized with a
saturated NaHCO3 solution, and the products were extracted
three times with chloroform. The combined chloroform phases
were dried over Na2SO4, and the solvent was evaporated.
Purification was performed by column chromatography on
silica gel, recrystallization, or isolation as the hydrochloride
by treatment with etheral HCl.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yl-4,5-
d ia r yl-2-im id a zolin es w it h Im in oet h er s (Met h od B).
Amounts of 1.00 mmol of the appropriate 1,2-diamino-1,2-
diarylethane and 1.30 mmol of the iminoether were dissolved
in 15 mL of dry ethanol and heated to reflux. Subsequently,
the solvent was evaporated and the crude product was purified
by column chromatography or recrystallization.
Exp er im en ta l Section
(4R,5S)/(4S,5R)-4,5-Bis(4-m eth oxyp h en yl)-2-m eth yl-2-
im id a zolin e (4a ). 4a was obtained from (1R,2S)/(1S,2R)-1,2-
diamino-1,2-bis(4-methoxyphenyl)ethane 1a (3.0 mmol, 817
Gen er a l P r oced u r es. The following instruments were used
to collect various spectra: IR spectra (KBr pellets), Perkin-

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 923
1
3
mg) and triethyl orthoacetate (6.00 mL) by method A. Reaction
time: 5 h. Purification: recrystallization from chloroform.
Yield: 2.3 mmol (680 mg), 77% colorless powder; mp 121 °C.
IR (KBr, cm-1): νj ) 3600-3300 br, w (NH), 3147 m, 2901 m,
2840 m (OCH3), 1612 s, 1584 m, 1514 s, 1469 m, 1255 s, 1173
s, 1040 s, 855 m. H NMR (DMSO-d6): δ 1.33 (t, J ) 7.5 Hz,
3
3H, CH2CH3), 2.77 (q, J ) 7.5 Hz, 2H, CH2CH3), 3.69 (s, 6H,
OCH3), 5.99 (s, 2H, ArCH), 6.77 (dd, 3J ) 8.8 Hz, 4J ) 2.5 Hz,
2H, ArH-5), 6.88 (d, 4J ) 2.5 Hz, 2H, ArH-3), 7.15 (d, 3J ) 8.8
Hz, ArH-6), 10.75 (s, 2H, NH, exchangeable by D2O).
1
m, 1036 m, 799 m. H NMR (DMSO-d6): δ 2.01 (s, 3H, CH3),
(4R,5S)/(4S,5R)-4,5-Bis(2-flu or o-4-m et h oxyp h en yl)-2-
et h yl-2-im id a zolin e (4g). 4g was obtained from (1R,2S)/
(1S ,2R)-1,2-dia m in o-1,2-bis(2-flu or o-4-m et h oxyph en yl)-
ethane 1d (3.0 mmol, 925 mg) and triethyl orthopropionate
(6.00 mL) by method A. Reaction time: 4 h. Isolation: as
hydrochloride and recrystallization from methanol/ether.
Yield: 2.8 mmol (1062 mg), 92%; mp 236 °C. IR (KBr, cm-1):
νj ) 3600-3300 m, br, (NH), 3065 m, 2940 s, 1625 s, 1510 s,
1445 m, 1315 m, 1290 m, 1270 m, 1160 m, 1115 m, 1030 m,
835 m. 1H NMR (DMSO-d6): δ 1.33 (t, 3J ) 7.6 Hz, 3H,
CH2CH3), 2.74 (q, 3J ) 7.6 Hz, 2H, CH2CH3), 3.66 (s, 6H,
OCH3), 5.84 (s, 2H, ArCH), 6.60-6.66 (m, 4H, ArH-3, ArH-5),
3.60 (s, 6H, OCH3), 5.07 (s, 2H, ArCH), 6.58 (AA′BB′, 3J ) 8.6
Hz, 4H, ArH-3, ArH-5), 6.81 (AA′BB′, 3J ) 8.6 Hz, 4H, ArH-2,
ArH-6).
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-m et h oxyp h en yl)-2-
m eth yl-2-im id a zolin e (4b). 4b was obtained from (1R,2S)/
(1S ,2R)-1,2-dia m in o-1,2-bis(2-ch lor o-4-m et h oxyph en yl)-
ethane 1b (3.0 mmol, 1024 mg) and triethyl orthoacetate (6.00
mL) by method A. Reaction time: 5 h. Isolation: as hydro-
chloride and recrystallization from methanol/ether. Yield: 2.4
mmol (975 mg), 81% colorless powder; mp 231 °C. IR (KBr,
cm-1): νj ) 3600-3300 br, m, (NH), 3060 s, 2930 s, 2840 m
(OCH3), 1605 s, 1500 s, 1440 s, 1290 s, 1245 m, 1040 s, 1025
m, 855 s. 1H NMR (DMSO-d6): δ 2.43 (s, 3H, CH3), 3.69 (s,
6H, OCH3), 5.97 (s, 2H, ArCH), 6.82 (dd, 3J ) 8.7 Hz, 4J ) 2.4
Hz, 2H, ArH-5), 6.87 (d, 4J ) 2.4 Hz, 2H, ArH-3), 7.17 (d, 3J )
8.7 Hz, ArH-6), 10.79 (s, 2H, NH, exchangeable by D2O).
3
4
7.10 (dd, J ) 8.8 Hz, J (H,F) ) 8.6 Hz, 2H, ArH-6), 10.79 (s,
2H, NH, exchangeable by D2O).
(4R,5S)/(4S,5R)-4,5-Bis(4-m et h oxyp h en yl)-2-(2-m et h -
oxyeth yl)-2-im id a zolin e (4h ). 4h was obtained from (1R,2S)/
(1S,2R)-1,2-diamino-1,2-bis(4-methoxyphenyl)ethane 1a (3.00
mmol, 817 mg) and the hydrochloride of 3-methoxypropioni-
midic acid ethyl ester 13b (3.90 mmol, 654 mg) by method B.
Reaction time: 3 h. Purification: column chromatography with
chloroform/methanol (9/1) and isolation as hydrochloride.
Yield: 2.34 mmol (882 mg), 78% yellow powder; mp 65 °C. IR
(KBr, cm-1): νj ) 3600-3300 br, m (NH), 3073 m, 2935 m,
2838 m (OCH3), 1611 s, 1583 m, 1515 s, 1252 s, 1463 w, 1300
w, 1180 m, 1114 w, 1031 m. 1H NMR (DMSO-d6): δ 2.98 (t, 3J
) 5.9 Hz, 2H, CH2CH2OCH3), 3.40 (s, 3H, CH2CH2OCH3), 3.63
(4R,5S)/(4S,5R)-4-(2-Ch lor o-4-m et h oxyp h en yl)-5-(2,6-
d ich lor o-4-m eth oxyp h en yl)-2-m eth yl-2-im id a zolin e (4c).
4c was obtained from (1R,2S)/(1S,2R)-1,2-diamino-1-(2-chloro-
4-methoxyphenyl)-2-(2,6-dichloro-4-methoxyphenyl)ethane 1c
(0.50 mmol, 224 mg) and the hydrochloride of acetimidic acid
ethyl ester 13a (0.65 mmol, 80 mg) by method B. Reaction
time: 72 h. Purification: chromatography on silica gel with
chloroform/methanol (4/1) and isolation as hydrochloride.
Yield: 0.41 mmol (179 mg), 82% ochre powder; mp 227 °C. IR
(KBr, cm-1): νj ) 3600-3300 br, m (NH), 3220 m, 3064 m,
2927 m, 2699 m, 1602 s, 1556 m, 1466 m, 1432 m, 1405 m,
1285 m, 1264 m, 1243 m, 1201 m, 1180 m, 1072 m, 1040 m,
3
(s, 6H, OCH3), 3.81 (t, J ) 5.9 Hz, 2H, CH2CH2OCH3), 5.62
3
(s, 2H, ArCH), 6.70 (AA′BB′, J ) 8.7 Hz, 4H, ArH-3, ArH-5),
6.91 (AA′BB′, 3J ) 8.7 Hz, 4H, ArH-2, ArH-6), 10.93 (s, 2H,
NH, exchangeable by D2O).
1
881 m, 852 m, 816 m, 789 m. H NMR (DMSO-d6): δ 2.36 (s,
3
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-m et h oxyp h en yl)-2-
(2-m eth oxyeth yl)-2-im id a zolin e (4i). 4i was obtained from
(1R,2S)/(1S,2R)-1,2-diamino-1,2-bis(2-chloro-4-methoxyphenyl)-
ethane 1b (3.00 mmol, 1024 mg) and the hydrochloride of
3-methoxypropionimidic acid ethyl ester 13b (3.90 mmol, 654
mg) by method B. Reaction time: 3 h. Purification: column
chromatography with chloroform/methanol (9/1) and isolation
as hydrochloride and recrystallization from methanol/ether.
Yield: 1.41 mmol (630 mg), 47% yellow powder; mp 68 °C. IR
(KBr, cm-1): νj ) 3600-3300 br, w (NH), 3059 m, 2929 m, 2839
m (OCH3), 1606 s, 1571 m, 1500 s, 1293 m, 1247 m, 1041 m,
755 m. H NMR (DMSO-d6): δ 3.02 (t, J ) 5.9 Hz, 2H, CH2-
CH2OCH3), 3.40 (s, 3H, CH2CH2OCH3), 3.69 (s, 6H, OCH3),
3.81 (t, J ) 5.9 Hz, 2H, CH2CH2OCH3), 6.01 (s, 2H, ArCH),
6.79 (dd, J ) 8.8 Hz, J ) 2.5 Hz, 2H, ArH-5), 6.88 (d, J )
2.5 Hz, 2H, ArH-3), 7.09 (d, 3J ) 8.7 Hz, 2H, ArH-6), 10.93 (s,
2H, NH, exchangeable by D2O).
Gen er al P r ocedu r e for th e Syn th esis of (4R,5S)/(4S,5R)-
N-Eth yl-1,2-d ia r yl-2-im id a zolin es (Meth od C). A solution
of the appropriate 1,2-diamino-1,2-diarylethane (4.00 mmol)
in 5 mL of dry ethanol was heated to reflux. An amount of
1.00 mmol of ethyl iodine in 2 mL of dry ethanol was added
slowly to the hot solution. The solvent was evaporated, and
the crude was purified by column chromatography on silica
gel. N-Alkyl-1,2-diamino-1,2-diarylethanes can be converted
into (4R,5S)/(4S,5R)-N-alkyl-1,2-diaryl-2-imidazolines by method
A.
(1R,2S)/(1S,2R)-N-Eth yl-1,2-diam in o-1,2-bis(4-m eth oxy-
p h en yl)eth a n e (10). 10 was obtained from (1R,2S)/(1S,2R)-
1,2-diamino-1,2-bis(4-methoxyphenyl)ethane 1a (4.0 mmol
(1089 mg)). Reaction time: 16 h. Purification: column chro-
matography with chloroform/methanol (9/1) under addition of
1% triethylamine. Yield: 0.6 mmol (187 mg), 60% colorless
powder; mp 77 °C. 1H NMR (DMSO-d6): δ 0.84 (t, 3J ) 7.1
Hz, 3H, CH2CH3), 1.55 (br, 3H, NH, NH2, exchangeable by
3H, CH3), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 5.91 (d, J
) 12.8 Hz, 1H, ArCH), 6.37 (d, 3J ) 12.8 Hz, 1H, ArCH), 6.86-
4
6.89 (m, 3H, ArH-3, ArH-5, Ar′H-5), 7.05 (d, J ) 2.7 Hz, 1H,
3
Ar′H-3), 7.53 (d, J ) 9.4 Hz, 1H, Ar′H-6), 10.30 (s, 2H, NH,
exchangeable by D2O).
(4R,5S)/(4S,5R)-4,5-Bis(2-flu or o-4-m et h oxyp h en yl)-2-
m eth yl-2-im id a zolin e (4d ). 4d was obtained from (1R,2S)/
(1S ,2R)-1,2-dia m in o-1,2-bis(2-flu or o-4-m et h oxyph en yl)-
ethane 1d (3.0 mmol, 925 mg) and triethyl orthoacetate (6.00
mL) by method A. Reaction time: 6 h. Purification: recrys-
tallization from chloroform. Yield: 2.3 mmol (778 mg), 76%
colorless powder; mp 206 °C. IR (KBr, cm-1): νj ) 3600-3300
br, m, (NH), 3080 s, 2940 s, 2840 m (OCH3), 1625 s, 1510 s,
1
3
3
1
1445 m, 1315 m, 1290 s, 1160 m, 1115 s, 1025 m, 835 m. H
3
4
4
NMR (DMSO-d6): δ 2.15 (s, 3H, CH3), 3.63 (s, 6H, OCH3), 5.49
3
(s, 2H, ArCH), 6.52-6.55 (m, 4H, ArH-3, ArH-5), 6.97 (dd, J
4
) 8.4 Hz, J (H,F) ) 8.4 Hz, 2H, ArH-6).
(4R,5S)/(4S,5R)-4,5-Bis(4-m eth oxyp h en yl)-2-eth yl-2-im -
id a zolin e (4e). 4e was obtained from (1R,2S)/(1S,2R)-1,2-
diamino-1,2-bis(4-methoxyphenyl)ethane 1a (1.00 mmol, 272
mg) and triethyl orthopropionate (2.00 mL) by method A.
Reaction time: 5 h. Purification: recrystallization from chlo-
roform. Yield: 0.84 mmol (260 mg), 84% yellow powder; mp
138 °C. IR (KBr, cm-1): νj ) 2960 m, 2835 w (OCH3), 1610 m,
1
1512 m, 1260 s, 1095 s, 1025 s, 800 s. H NMR (DMSO-d6): δ
3
3
1.22 (t, J ) 7.6 Hz, 3H, CH2CH3), 2.34 (q, J ) 7.6 Hz, 2H,
CH2CH3), 3.60 (s, 6H, OCH3), 5.07 (s, 2H, ArCH), 6.58 (AA′BB′,
3J ) 8.7 Hz, 4H, ArH-3, ArH-5), 6.82 (AA′BB′, 3J ) 8.7 Hz,
4H, ArH-2, ArH-6).
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-m et h oxyp h en yl)-2-
et h yl-2-im id a zolin e (4f). 4f was obtained from (1R,2S)/
(1S ,2R)-1,2-dia m in o-1,2-bis(2-ch lor o-4-m et h oxyph en yl)-
ethane 1b (3.0 mmol, 1024 mg) and triethyl orthopropionate
(6.00 mL) by method A: Reaction time: 5 h. Isolation: as
hydrochloride and recrystallization from methanol/ether.
Yield: 1.7 mmol (697 mg), 56% colorless crystals; mp 225 °C.
IR (KBr, cm-1): νj ) 3600-3300 br, m, (NH), 3055 m, 2930 s,
2690 m, 1605 s, 1570 m, 1500 s, 1460 m, 1440 m, 1290 s, 1250
3
D2O), 2.18-2.27 (m, 2H, CH2CH3), 3.58 (d, J ) 6.6 Hz, 1H,
3
ArCH), 3.72 (s, 6H, OCH3), 3.85 (d, J ) 6.6 Hz, 1H, ArCH),
6.81-6.83 (m, 4H, ArH-3, ArH-5, Ar′H-3, Ar′H-5), 7.09-7.13
(m, 4H, ArH-2, ArH-6, Ar′H-2, Ar′H-6).

924 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
(4R,5S)/(4S,5R)-N-E t h yl-4,5-b is(4-m et h oxyp h en yl)-2-
im id a zolin e (6). 6 was obtained from 10 (1.00 mmol, 300 mg)
and triethyl formate (2.00 mL) by method A. Reaction time:
6 h. Isolation as hydrochloride and recrystallization from
methanol/ether. Yield: 0.88 mmol (304 mg), 88% orange oil.
IR (KBr, cm-1): νj ) 3600-3300 br (NH), 3002 m, 2961 m, 2939
m, 2839 m (OCH3), 1639 m, 1613 m, 1514 s, 1462 m, 1299 m,
zolin iu m Ch lor id es (Meth od E). Amounts of 1.00 mmol of
2b and 1.20 mmol of NaH (suspended in paraffin) were
dissolved in 20 mL of dry THF and heated to reflux. After the
addition of 2.50 mmol of alkyl iodine in 2 mL of dry THF, the
solution was heated for further 18 h. Subsequently, an amount
of 20 mL of water was added at room temperature and the
water phase was extracted twice with dichloromethane. The
combined organic layers were dried over Na2SO4, and etheral
HCl was added. After evaporation of the solvent, the crude
product was purified by column chromatography on silica gel
or recrystallization.
1
1236 s, 1180 m, 1032 m, 838 m, 755 m. H NMR (DMSO-d6):
3
δ 1.17 (t, J ) 7.2 Hz, 3H, CH2CH3), 2.98-3.03 (m, 1H, CH2-
CH3), 3.50-3.56 (m, 1H, CH2CH3), 3.64 (s, 3H, OCH3), 3.64
3
3
(s, 3H, OCH3), 5.64 (d, J ) 12.1 Hz, 1H, ArCH), 5.75 (d, J )
12.1 Hz, 1H, ArCH), 6.71 (AA′BB′, 3J ) 8.7 Hz, 4H, ArH-3,
ArH-5, Ar′H-3, Ar′H-5), 6.93-6.96 (m, 4H, ArH-2, ArH-6, Ar′H-
2, Ar′H-6), 8.94 (s, 1H, NdCH-N), 10.84 (s, 1H, NH, exchange-
able by D2O).
(4R,5S)/(4S,5R)-N,N′-Dim eth yl-4,5-bis(2-ch lor o-4-m eth -
oxyp h en yl)-2-im id a zolin iu m ch lor id e (8a ). 8a was ob-
tained from 2b (0.29 mmol, 120 mg) and methyl iodine (0.73
mmol, 103 mg, 45 µL). Recrystallization from methanol/ether.
Yield: 0.24 mmol (98 mg), 81% yellow needles; mp 181 °C. IR
(KBr, cm-1): νj ) 3600-3400 br, 2935 m, 2813 w, 1660 s, 1607
s, 1573 m, 1500 s, 1465 m, 1440 m, 1327 m, 1294 s, 1247 s,
Gen er al P r ocedu r e for th e Syn th esis of (4R,5S)/(4S,5R)-
N-Alkyl-1,2-bis(2-ch lor o-4-m eth oxy)-2-im idazolin es (Meth -
od D). An amount of 2.20 mmol of butyllithium (1.6 M in
hexane) was added to a cooled solution (-78 °C) of 2b (1.00
mmol) in 10 mL of dry THF. After the mixture was stirred for
15 min, 1.10 mmol of alkyl iodine in 2 mL of dry ethanol was
added slowly. The stirred solution was allowed to warm to
room temperature overnight. Etheral HCl was added, and the
solvent was evaporated. The crude product was purified by
column chromatography on silica gel.
1
1192 m, 1148 m, 1043 s, 871 m. H NMR (DMSO-d6): δ 2.97
(s, 6H, CH3), 3.70 (s, 6H, OCH3), 6.00 (s, 2H, ArCH), 6.77 (dd,
3J ) 8.7 Hz, 4J ) 2.6 Hz, 2H, ArH-5), 6.92 (d, 4J ) 2.6 Hz, 2H,
ArH-3), 7.06 (d, 3J ) 8.7 Hz, 2H, ArH-6), 8.88 (s, 1H, NdCH-
N).
(4R,5S)/(4S,5R)-N,N′-Diet h yl-4,5-b is(2-ch lor o-4-m et h -
oxyp h en yl)-2-im id a zolin iu m Ch lor id e (8b). 8b was ob-
tained from 2b (0.70 mmol, 271 mg) and ethyl iodine (1.75
mmol, 273 mg, 141 µL). Recrystallization from methanol/ether.
Yield: 0.36 mmol (158.3 mg), 51% yellow needles; mp 190 °C.
IR (KBr, cm-1): νj ) 3600-3500 br, 2976 m, 2839 w (OCH3),
1647 s, 1608 s, 1502 s, 1295 m, 1262 s, 1038 m, 878 m, 849 m.
1H NMR (DMSO-d6): δ 1.19 (t, 3J ) 7.2 Hz, 6H, CH2CH3),
3.01-3.20 (m, 2H, CH2CH3), 3.49-3.58 (m, 2H, CH2CH3), 3.69
(4R,5S)/(4S,5R)-N-Meth yl-4,5-bis(2-ch lor o-4-m eth oxy-
p h en yl)-2-im id a zolin e (6a ). 6a was obtained from 2b (0.75
mmol, 291 mg) and methyl iodine (0.83 mmol, 117 mg, 51 µL).
Purification: column chromatography with chloroform/metha-
nol (9/1) and isolation as hydrochloride. Yield: 0.69 mmol (277
mg), 92% orange powder; mp 93 °C. IR (KBr, cm-1): νj ) 3600-
3300 br, m (NH), 3088 m, 2838 m (OCH3), 1650 s, 1607 s, 1500
s, 1294 m, 1244 m, 1044 s. 1H NMR (DMSO-d6): δ 2.95 (s,
3
4
(s, 6H, OCH3), 6.10 (s, 2H, ArCH), 6.77 (dd, J ) 8.7 Hz, J )
4
3
2.5 Hz, 2H, ArH-5), 6.93 (d, J ) 2.5 Hz, 2H, ArH-3), 7.10 (d,
3H, CH3), 3.69 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 5.97 (d, J
3J ) 8.7 Hz, 2H, ArH-6), 8.98 (s, 1H, NdCH-N).
3
) 12.3 Hz, 1H, ArCH), 6.06 (d, 1H, J ) 12.3 Hz, ArCH), 6.73
3
4
3
(dd, J ) 8.8 Hz, J ) 2.6 Hz, 1H, ArH-5), 6.80 (dd, J ) 8.7
(4R,5S)/(4S,5R)-N,N′-Dip r op yl-4,5-bis(2-ch lor o-4-m eth -
oxyp h en yl)-2-im id a zolin iu m Ch lor id e (8c). 8c was ob-
tained from 2b (0.34 mmol, 140 mg) and propyl iodine (0.85
mmol, 145 mg, 79 µL). Recrystallization from methanol/ether.
Yield: 0.26 mmol (117 mg), 77% yellow needles; mp 231 °C.
IR (KBr, cm-1): νj ) 3600-3400 br, 2964 m, 2935 m, 1648 s,
1632 s, 1608 s, 1572 m, 1498 s, 1263 m, 1441 m, 1292 s, 1243
4
4
Hz, J ) 2.6 Hz, 1H, Ar′H-5), 6.88 (d, J ) 2.6 Hz, 1H, ArH-
4
3
3), 6.93 (d, J ) 2.6 Hz, 1H, Ar′H-3), 7.01 (d, J ) 8.8 Hz, 1H,
ArH-6), 7.18 (d, 3J ) 8.7 Hz, 1H, Ar′H-6), 8.94 (s, 1H, NdCH-
N), 10.89 (s, 1H, NH, exchangeable by D2O).
(4R,5S)/(4S,5R)-N-Eth yl-4,5-bis(2-ch lor o-4-m eth oxyph e-
n yl)-2-im id a zolin e (6b). 6b was obtained from 2b (0.70
mmol, 271 mg) and ethyl iodine (0.77 mmol, 129 mg, 67 µL).
Purification: column chromatography with ether/methanol (6/
1). Yield: 0.53 mmol (200 mg), 76% yellow oil. IR (KBr, cm-1):
νj ) 3019 m, 2840 w (OCH3), 1684 w, 1604 m, 1496 m, 1287
1
3
s, 1043 s, 870 m, 851 m. H NMR (DMSO-d6): δ 0.89 (t, J )
7.3 Hz, 6H, CH2CH2CH3), 1.56 (m, 4H, CH2CH2CH3), 3.01 (m,
2H, CH2CH2CH3), 3.43 (m, 2H, CH2CH2CH3), 3.70 (s, 6H,
OCH3), 6.13 (s, 2H, ArCH), 6.77 (dd, 3J ) 8.7 Hz, 4J ) 2.5 Hz,
2H, ArH-5), 6.92 (d, 4J ) 2.5 Hz, 2H, ArH-3), 7.10 (d, 3J ) 8.7
Hz, 2H, ArH-6), 9.03 (s, 1H, NdCH-N).
1
w, 1216 s, 1044 m, 874 w, 758 s. H NMR (DMSO-d6): δ 1.13
(t, 3J ) 7.2 Hz, 3H, CH2CH3), 2.76-2.87 (m, 1H, CH2CH3),
3.14-3.25 (m, 1H, CH2CH3), 3.69 (s, 3H, OCH3), 3.69 (s, 3H,
OCH3), 5.51 (d, 3J ) 11.0 Hz, 1H, ArCH), 5.85 (d, 3J ) 11.0
Gen er a l P r oced u r e for Eth er Clea va ge w ith BBr 3
(Meth od F ). A solution of the methyl ether (1.00 mmol) in 20
mL of dry CH2Cl2 was cooled to -60 °C. At this temperature,
BBr3 (2.25 mmol/methoxy group) in 5 mL of dry CH2Cl2 was
added under N2 atmosphere. Then the reaction mixture was
allowed to warm to room temperature and was stirred for
further 48 h. After the reaction was cooled with an ice bath,
the surplus of BBr3 was hydrolyzed three times with dry
methanol. The resulting crude product (hydrobromide) was
purified by recrystallization.
3
Hz, 1H, ArCH), 6.45 (dd, J ) 8.7 Hz, 1H, ArH-5), 6.62-6.65
4
3
(m, J ) 2.5 Hz, 2H, ArH-3, ArH-5), 6.68 (d, J ) 8.7 Hz, 1H,
4
3
ArH-6), 6.72 (d, J ) 2.5 Hz, 1H, ArH-3), 7.17 (d, J ) 9.1 Hz,
1H, ArH-6), 7.31 (br, 1H, NdCH-N).
(4R,5S)/(4S,5R)-N-P r op yl-4,5-b is(2-ch lor o-4-m et h oxy-
p h en yl)-2-im id a zolin e (6c). 6c was obtained from 2b (0.70
mmol, 271 mg) and propyl iodine (0.77 mmol, 131 mg, 72 µL).
Purification: column chromatography with chloroform/metha-
nol (9/1), isolation as hydrochloride, and recrystallization from
methanol/ether. Yield: 0.09 mmol (40 mg), 13% yellow needles;
mp 103 °C. IR (KBr, cm-1): νj ) 3600-3300 br (NH), 3082 m,
2941 m, 2839 w (OCH3), 1638 s, 1607 s, 1499 s, 1294 m, 1248
m, 1083 m, 1043 s. 1H NMR (DMSO-d6): δ 0.88 (t, 3J ) 6.6
Hz, 3H, CH2CH2CH3), 1.49-1.58 (m, 2H, CH2CH2CH3), 2.83-
2.90 (m, 1H, CH2CH2CH3), 3.40-3.47 (m, 1H, CH2CH2CH3),
(4R,5S)/(4S,5R)-4,5-Bis(4-h yd r oxyp h en yl)-2-m eth yl-2-
im id a zolin e (5a ). 5a was obtained from 4a (1.00 mmol, 296.4
mg). Recrystallization from methanol. Yield: 0.91 mmol (317.2
mg), 91% yellow powder; mp 289 °C. IR (KBr, cm-1): νj )
3600-2500 br, s (OH), 1615 s, 1591 s, 1516 s, 1447 m, 1427
m, 1356 m, 1260 s, 1201 s, 1048 m, 876 m, 807 m. MS (EI,
100 °C): m/z (%) ) 268 (27) [M+•], 161 (23), 147 (100), 120
3
1
3.69 (s, 6H, OCH3), 6.03 (d, J ) 12.2 Hz, 1H, ArCH), 6.09 (d,
(18), 106 (54). H NMR (DMSO-d6): δ 2.40 (s, 3H, CH3), 5.55
3
3
4
3
1H, J ) 12.2 Hz, ArCH), 6.69 (dd, J ) 8.8 Hz, J ) 2.6 Hz,
(s, 2H, ArCH), 6.51 (AA′BB′, J ) 8.5 Hz, 4H, ArH-3, ArH-5),
3
4
6.79 (AA′BB′, 3J ) 8.5 Hz, 4H, ArH-2, ArH-6), 9.32 (s, 2H, OH,
exchangeable by D2O), 10.42 (s, 2H, NH, exchangeable by
D2O). Anal. (C16H16N2O2‚HBr) C, H, N.
1H, ArH-5), 6.84 (dd, J ) 8.6 Hz, J ) 2.6 Hz, 1H, Ar′H-5),
6.87 (d, 4J ) 2.6 Hz, 1H, ArH-3), 6.94 (d, 4J ) 2.6 Hz, 1H,
Ar′H-3), 6.98 (d, 3J ) 8.8 Hz, 1H, ArH-6), 7.25 (d, 3J ) 8.6 Hz,
1H, Ar′H-6), 8.95 (s, 1H, NdCH-N), 10.95 (s, 1H, NH,
exchangeable by D2O).
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-h yd r oxyp h en yl)-2-
m eth yl-2-im id a zolin e (5b). 5b was obtained from 4b (1.00
mmol, 401.7 mg). Recrystallization from methanol. Yield: 0.96
mmol (408.5 mg), 96% brown powder; mp >300 °C. IR (KBr,
Gen er al P r ocedu r e for th e Syn th esis of (4R,5S)/(4S,5R)-
N,N′-Dia lk yl-1,2-bis(2-ch lor o-4-m eth oxyp h en yl)-2-im id a -

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 925
3
3
cm-1): νj ) 3600-2500 br, s (OH), 1603 s, 1578 s, 1499 s, 1432
s, 1287 s, 1258 s, 1212 s, 1050 m, 1030 m, 896 m, 857 m, 820
m. MS (EI, 100 °C): m/z (%) ) 336 (31) [M+•], 181 (100), 156
(25), 140 (42), 105 (24). 1H NMR (DMSO-d6): δ 2.40 (s, 3H,
CH3), 5.91 (s, 2H, ArCH), 6.57 (dd, J ) 8.6 Hz, J ) 2.1 Hz,
2H, ArH-5), 6.63 (d, 4J ) 2.1 Hz, 2H, ArH-3), 7.02 (d, 3J ) 8.6
Hz, 2H, ArH-6), 9.94 (s, 2H, OH, exchangeable by D2O), 10.57
(s, 2H, NH, exchangeable by D2O). Anal. (C16H14Cl2N2O2‚HBr‚
0.5H2O) C, H, N.
(4R,5S)/(4S,5R)-4-(2-Ch lor o-4-h yd r oxyp h en yl)-5-(2,6-
d ich lor o-4-h yd r oxyp h en yl)-2-m eth yl-2-im id a zolin e (5c).
5c was obtained from 4c (0.25 mmol, 109.1 mg). Recrystalli-
zation from methanol. Yield: 0.20 mmol (90.4 mg), 80% ochre
powder; mp >300 °C. IR (KBr, cm-1): νj ) 3600-2500 br, s
(OH), 1602 s, 1567 m, 1501 m, 1460 w, 1429 m, 1259 m, 1212
m, 1074 m, 954 m, 898 w, 861 w, 788 w. MS (EI, 400 °C): m/z
(%) ) 370 (31) [M+•], 215 (41), 181 (100), 156 (20), 140 (45),
ArH-3), 6.45 (dd, J ) 8.3 Hz, 2H, ArH-5), 6.95 (dd, J ) 8.3
Hz, 2H, ArH-6), 9.90 (s, 2H, OH, exchangeable by D2O), 10.56
(s, 2H, NH, exchangeable by D2O). Anal. (C17H16F2N2O2‚HBr‚
0.5H2O) C, H, N.
3
4
(4R,5S)/(4S,5R)-4,5-Bis(4-h ydr oxyph en yl)-2-(2-h ydr oxy-
eth yl)-2-im id a zolin e (5h ). 5h was obtained from 4h (0.90
mmol, 340.4 mg). Recrystallization from methanol/ether.
Yield: 0.55 mmol (217.8 mg), 61% gray powder; mp 231 °C.
IR (KBr, cm-1): νj ) 3600-2500 br, s (OH), 1610 s, 1517 s,
1447 m, 1340 w, 1263 m, 1210 m, 1176 m, 1064 m. MS (EI,
380 °C): m/z (%) ) 147 (20), 106 (14), 94 (100). 1H NMR
3
(DMSO-d6): δ 2.84 (t, J ) 5.8 Hz, 2H, CH2CH2OH), 3.88 (t,
3J ) 5.8 Hz, 2H, CH2CH2OH), 5.31 (br, 1H, CH2CH2OH,
3
exchangeable by D2O), 5.56 (s, 2H, ArCH), 6.50 (AA′BB′, J )
8.5 Hz, 4H, ArH-3, ArH-5), 6.81 (AA′BB′, 3J ) 8.5 Hz, 4H, ArH-
2, ArH-6), 9.32 (s, 2H, OH, exchangeable by D2O), 10.45 (s,
2H, NH, exchangeable by D2O). Anal. (C17H18N2O3‚HBr‚H2O)
C, H, N.
1
3
105 (20). H NMR (DMSO-d6): δ 2.35 (s, 3H, CH3), 5.89 (d, J
3
) 12.9 Hz, 1H, ArCH), 6.32 (d, J ) 12.9 Hz, 1H, ArCH), 6.61
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-h yd r oxyp h en yl)-2-
(2-h yd r oxyeth yl)-2-im id a zolin e (5i). 5i was obtained from
4i (1.00 mmol, 445.8 mg). Recrystallization from methanol/
ether. Yield: 0.82 mmol (402.1 mg), 82% ochre powder; mp
232 °C. IR (KBr, cm-1): νj ) 3600-2500 br, s (OH), 1603 s,
1578 m, 1500 s, 1434 m, 1332 w, 1289 m, 1253 m, 1216 m,
1059 m, 1040 m, 911 w, 862 m, 826 w. MS (EI, 170 °C): m/z
4
4
(d, J ) 2.4 Hz, 1H, ArH-3), 6.64 (d, J ) 2.4 Hz, 1H, Ar′H-3),
3
4
4
6.67 (dd, J ) 8.6 Hz, J ) 2.4 Hz, 1H, Ar′H-5), 6.79 (d, J )
2.4 Hz, 1H, ArH-5), 7.39 (d, 3J ) 8.6 Hz, 1H, Ar′H-6), 10.12
(s, 1H, OH, exchangeable by D2O), 10.54 (s, 1H, OH, exchange-
able by D2O), 10.63 (s, 1H, NH, exchangeable by D2O), 10.81
(s, 1H, NH, exchangeable by D2O). Anal. (C16H13Cl3N2O2‚HBr‚
H2O) C, H, N.
1
(%) ) 208 (17), 128 (100), 100 (11), 65 (95). H NMR (DMSO-
3
3
(4R,5S)/(4S,5R)-4,5-Bis(2-flu or o-4-h yd r oxyp h en yl)-2-
m eth yl-2-im id a zolin e (5d ). 5d was obtained from 4d (0.50
mmol, 169.6 mg). Recrystallization from methanol. Yield: 0.39
mmol (165.6 mg), 78% brown powder; mp >300 °C. IR (KBr,
cm-1): νj ) 3600-2500 br, m (OH), 1631 s, 1606 s, 1501 m,
1460 m, 1295 m, 1253 m, 1202 m, 1152 w, 1104 m, 1042 m,
967 w, 845 w. MS (EI, 120 °C): m/z (%) ) 304 (31) [M+•], 165
(100), 124 (55), 96 (17). 1H NMR (DMSO-d6): δ 2.39 (s, 3H,
CH3), 5.76 (s, 2H, ArCH), 6.32 (dd, 3J (H,F) ) 12.1 Hz, 4J )
2.2 Hz, 2H, ArH-3), 6.43 (dd, 3J ) 8.5 Hz, 4J ) 2.2 Hz, 2H,
d6): δ 2.85 (t, J ) 5.9 Hz, 2H, CH2CH2OH), 3.87 (t, J ) 5.9
Hz, 2H, CH2CH2OH), 5.02 (br, 1H, CH2CH2OH, exchangeable
by D2O), 5.93 (s, 2H, ArCH), 6.64 (dd, 3J ) 8.6 Hz, 4J ) 2.4
Hz, 2H, ArH-5), 6.64 (d, 4J ) 2.4 Hz, 2H, ArH-3), 7.01 (d, 3J )
8.6 Hz, 2H, ArH-6), 9.94 (s, 2H, OH, exchangeable by D2O),
10.61 (s, 2H, NH, exchangeable by D2O). Anal. (C17H16Cl2N2O3‚
HBr‚2H2O) C, H, N.
(4R,5S)/(4S,5R)-N-E t h yl-4,5-b is(4-h yd r oxyp h en yl)-2-
im id a zolin e (7). 7 was obtained from 6 (0.26 mmol, 90 mg).
Recrystallization from methanol/ether. Yield 0.24 mmol (104.0
mg), 94% brown powder; mp 206 °C. IR (KBr, cm-1): νj )
3600-2500 br, s (OH), 1640 s, 1612 m, 1518 s, 1443 m, 1368
m, 1338 m, 1268 m, 1224 m, 1172 m, 1107 m, 1042 w, 878 w,
809 m. MS (EI, 135 °C): m/z (%) ) 282 (30) [M+•], 150 (100),
107 (22). 1H NMR (DMSO-d6): δ 1.17 (t, 3J ) 7.2 Hz, 3H,
CH2CH3), 2.98-3.07 (m, 1H, CH2CH3), 3.47-3.56 (m, 1H, CH2-
3
4
ArH-5), 6.94 (dd, J ) 8.5 Hz, J (H,F) ) 8.5 Hz, 2H, ArH-6),
9.90 (s, 2H, OH, exchangeable by D2O), 10.56 (s, 2H, NH,
exchangeable by D2O). Anal. (C16H14F2N2O2‚HBr‚2H2O) C, H,
N.
(4R,5S)/(4S,5R)-4,5-Bis(4-h yd r oxyp h en yl)-2-eth yl-2-im -
id a zolin e (5e). 5e was obtained from 4e (0.50 mmol, 155.2
mg). Recrystallization from methanol. Yield: 0.45 mmol (172.8
mg), 90% brown powder; mp 281 °C. IR (KBr, cm-1): νj )
3600-2500 br, s (OH), 1604 m, 1517 s, 1445 m, 1271 s, 1215
s, 1176 m, 1104 m, 1085 m, 807 m. MS (EI, 150 °C): m/z (%)
) 282 (35) [M+•], 161 (100), 146 (12), 106 (43). 1H NMR (DMSO-
3
3
CH3), 5.55 (d, J ) 12.0 Hz, 1H, ArCH), 5.65 (d, J ) 12.0 Hz,
1H, ArCH), 6.53 (AA′BB′, 3J ) 8.2 Hz, 4H, ArH-3, ArH-5, Ar′H-
3, Ar′H-5), 6.77-6.81 (m, 4H, ArH-2, ArH-6, Ar′H-2, Ar′H-6),
8.89 (s, 1H, NdCH-N), 9.33 (s, 1H, OH, exchangeable by D2O),
9.41 (s, 1H, OH, exchangeable by D2O), 10.63 (s, 1H, NH,
exchangeable by D2O). Anal. (C17H18N2O2‚HBr‚3H2O) C, H, N.
3
3
d6): δ 1.33 (t, J ) 7.6 Hz, 3H, CH2CH3), 2.75 (q, J ) 7.6 Hz,
3
2H, CH2CH3), 5.56 (s, 2H, ArCH), 6.52 (AA′BB′, J ) 8.5 Hz,
(4R,5S)/(4S,5R)-N-Met h yl-4,5-b is(2-ch lor o-4-h yd r oxy-
p h en yl)-2-im id a zolin e (7a ). 7a was obtained from 6a (0.15
mmol, 60.2 mg). Recrystallization from methanol/ether.
Yield: 0.05 mmol (21.9 mg), 32% brown powder; mp 292 °C.
IR (KBr, cm-1): νj ) 3600-2500 br, s (OH), 1657 s, 1607 s,
1577 m, 1501 s, 1437 m, 1290 m, 1265 m, 1226 m, 1044 m,
903 m. MS (EI, 180 °C): m/z (%) ) 338 (19) [M+•], 184 (13),
4H, ArH-3, ArH-5), 6.79 (AA′BB′, 3J ) 8.5 Hz, 4H, ArH-2, ArH-
6), 9.33 (s, 2H, OH, exchangeable by D2O), 10.43 (s, 2H, NH,
exchangeable by D2O). Anal. (C17H18N2O2‚HBr‚H2O) C, H, N.
(4R,5S)/(4S,5R)-4,5-Bis(2-ch lor o-4-h yd r oxyp h en yl)-2-
eth yl-2-im id a zolin e (5f). 5f was obtained from 4f (0.50 mmol,
193.8 mg). Recrystallization from methanol. Yield: 0.44 mmol
(192.7 mg), 87% beige powder; mp 308 °C. IR (KBr, cm-1): νj
) 3500-2600 br, m (OH), 1688 s, 1602 s, 1578 s, 1500 m, 1428
m, 1303 m, 1263 s, 1169 m, 1027 m. MS (EI, 290 °C): m/z (%)
) 350 (27) [M+•], 195 (100), 140 (40), 105 (17). 1H NMR (DMSO-
1
170 (100). H NMR (DMSO-d6): δ 2.95 (s, 3H, CH3), 5.88 (d,
3
3J ) 12.0 Hz, 1H, ArCH), 5.98 (d, 1H, J ) 12.0 Hz, ArCH),
3
4
3
6.54 (dd, J ) 8.6 Hz, J ) 2.3 Hz, 1H, ArH-5), 6.60 (dd, J )
8.5 Hz, 4J ) 2.3 Hz, 1H, Ar′H-5), 6.63 (d, 4J ) 2.3 Hz, 1H,
ArH-3), 6.68 (d, 4J ) 2.6 Hz, 1H, Ar′H-3), 6.86 (d, 3J ) 8.6 Hz,
1H, ArH-6), 7.02 (d, 3J ) 8.5 Hz, 1H, Ar′H-6), 8.91 (s, 1H, Nd
CH-N), 9.95 (s, 1H, OH), 10.02 (s, 1H, OH), 10.89 (br, 1H,
NH). Anal. (C16H14Cl2N2O2‚HBr‚2H2O) C, H, N.
3
3
d6): δ 1.31 (t, J ) 7.6 Hz, 3H, CH2CH3), 2.74 (q, J ) 7.6 Hz,
3
4
2H, CH2CH3), 5.92 (s, 2H, ArCH), 6.57 (dd, J ) 8.6 Hz, J )
4
2.4 Hz, 2H, ArH-5), 6.64 (d, J ) 2.4 Hz, 2H, ArH-3), 7.00 (d,
3J ) 8.6 Hz, 2H, ArH-6), 9.55 (s, 2H, OH, exchangeable by
D2O), 10.55 (s, 2H, NH, exchangeable by D2O). Anal. (C17H16
Cl2N2O2‚HBr‚H2O) C, H, N.
-
(4R,5S)/(4S,5R)-N-Eth yl-4,5-bis(2-ch lor o-4-h yd r oxyp h e-
n yl)-2-im id a zolin e (7b). 7b was obtained from 6b (0.15
mmol, 60.2 mg). Recrystallization from methanol/ether.
Yield: 0.50 mmol (189.6 mg), 64% colorless powder; mp 262
°C. IR (KBr, cm-1): νj ) 3600-2500 br, s (OH), 1643 s, 1608 s,
1500 s, 1437 m, 1261 m, 1218 m, 1044 m, 903 m, 861 w. MS
(4R,5S)/(4S,5R)-4,5-Bis(2-flu or o-4-h yd r oxyp h en yl)-2-
eth yl-2-im id a zolin e (5g). 5g was obtained from 4g (1.00
mmol, 382.8 mg). Recrystallization from methanol. Yield: 0.95
mmol (390.2 mg), 95% red-brown powder; mp 196 °C. IR (KBr,
cm-1): νj ) 3600-2500 br, s (OH), 1628 m, 1609 m, 1513 w,
1461 w, 1299 w, 1100 w. MS (EI, 210 °C): m/z (%) ) 318 (28)
[M+•], 179 (100), 124 (44), 96 (15). 1H NMR (DMSO-d6): δ 1.31
1
(EI, 200 °C): m/z (%) ) 350 (16) [M+•], 184 (100), 141 (13). H
3
NMR (DMSO-d6): δ 1.16 (t, J ) 7.2 Hz, 3H, CH2CH3), 2.92-
3.01 (m, 1H, CH2CH3), 3.47-3.57 (m, 1H, CH2CH3), 5.96-6.02
3
3
3
4
(t, J ) 7.6 Hz, 3H, CH2CH3), 2.71 (q, J ) 7.6 Hz, 2H, CH2-
CH3), 5.77 (s, 2H, ArCH), 6.33 (dd, 3J (H,F) ) 12.1 Hz, 2H,
(br, 2H, ArCH), 6.51 (dd, J ) 8.7 Hz, J ) 2.5 Hz, 1H, ArH-
5), 6.61-6.63 (m, 2H, ArH-3, Ar′H-5), 6.69 (d, 4J ) 2.4 Hz,

926 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
von Rauch et al.
3
1H, Ar′H-3), 6.84 (d, J ) 8.7 Hz, 1H, ArH-6), 7.07-7.09 (m,
(EI, 80 °C): m/z (%) ) 322 (11), 194 (51), 167 (12), 156 (60),
1
1H, Ar′H-6), 8.95 (s, 1H, NdCH-N), 9.95 (s, 1H, OH, ex-
changeable by D2O), 10.03 (s, 1H, OH, exchangeable by D2O),
10.76 (br, 1H, NH, exchangeable by D2O). Anal. (C17H16
Cl2N2O2‚HBr) C, H, N.
140 (10), 128 (100), 105 (10), 65 (29). H NMR (DMSO-d6): δ
1.61-1.96 (br, 2H, NH2, exchangeable by D2O), 4.43 (d,3J )
5.6 Hz, 1H, ArCHNH2), 5.54 (dd,3J ) 5.6 Hz, 3J ) 9.0 Hz, 1H,
-
3
4
ArCHNHCHO), 6.56 (dd, J ) 8.6 Hz, J ) 2.4 Hz, 1H, Ar′H-
4
5), 6.61 (d, J ) 2.4 Hz, 1H, Ar′H-3), 6.66-6.75 (m, 2H, ArH-
(4R,5S)/(4S,5R)-N-P r opyl-4,5-bis(2-ch lor o-4-h ydr oxyph e-
n yl)-2-im id a zolin e (7c). 7c was obtained from 6c (0.07 mmol,
30.0 mg). Recrystallization from methanol/ether. Yield: 0.06
mmol (26.8 mg), 82% brown powder; mp 245 °C. IR (KBr,
cm-1): νj ) 3600-2500 br, m (OH), 2962 m, 2925 m, 2853 m,
1602 m, 1497 m, 1457 m, 1440 m, 1360 w, 1261 s, 1212 m,
1180 m, 1096 m, 1038 m, 902 w, 857 w, 803 m, 690 w. MS (EI,
250 °C): m/z (%) ) 364 (22) [M+•], 236 (37), 198 (99), 167 (13),
3
3
3, ArH-5), 6.87 (d, J ) 8.6 Hz, 1H, Ar′H-6), 7.20 (d, J ) 8.6
3
Hz, 1H, ArH-6), 7.94 (s, 1H, CHO), 8.43 (d, J ) 9.0 Hz, 1H,
NHCHO, exchangeable by D2O), 9.66 (br, 1H, OH, exchange-
able by D2O), 9.74 (br, 1H, OH, exchangeable by D2O). Anal.
(C15H14Cl2N2O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Im in oeth er s.
An amount of 100 mmol of the appropriate nitrile was
dissolved in a mixture of 110 mmol of dry ethanol and 100
mL of dry ether and cooled with an ice bath. Dry HCl was
added until a precipitate formed. The flask was sealed and
stored in a refrigerator for 24 h. The precipitate was collected
by suction filtration and dried over P2O5.
1
156 (17), 128 (100), 105 (14). H NMR (DMSO-d6): δ 0.87 (t,
3J ) 7.4 Hz, 3H, CH2CH2CH3), 1.50-1.56 (m, 2H, CH2CH2-
CH3), 2.86-2.93 (m, 1H, CH2CH2CH3), 3.41-3.50 (m, 1H, CH2-
3
3
CH2CH3), 5.97 (d, J ) 12.1 Hz, 1H, ArCH), 6.03 (d, J ) 12.1
3
4
Hz, 1H, ArCH), 6.50 (dd, J ) 8.6 Hz, J ) 2.4 Hz, 1H, ArH-
5), 6.62-6.64 (m, 2H, ArH-3, Ar′H-5), 6.69 (d, 4J ) 2.4 Hz,
Acetim id ic Acid Eth yl Ester (13a ). 13a was obtained
from acetonitrile (50.0 mmol, 2.05 g, 2.59 mL). Recrystalliza-
tion from dry ether. Yield: 42.0 mmol (5.63 g), 84% white
powder; mp 112 °C. IR (KBr, cm-1): νj ) 2980 br (NH2), 1710
s, 1640 s, 1600 s, 1480 s, 1455 s, 1395 s, 1345 s, 1145 s, 1015
s, 835 s. 1H NMR (DMSO-d6): δ 1.35 (t, 3J ) 7.0 Hz, 3H,
3
1H, Ar′H-3), 6.83 (d, J ) 8.6 Hz, 1H, ArH-6), 7.09-7.11 (m,
1H, Ar′H-6), 8.97 (s, 1H, NdCH-N), 9.96 (s, 1H, OH, ex-
changeable by D2O), 10.05 (s, 1H, OH, exchangeable by D2O),
10.81 (br, 1H, NH, exchangeable by D2O). Anal. (C18H18
Cl2N2O2‚HBr) C, H, N.
-
(4R,5S)/(4S,5R)-N,N′-Dim et h yl-4,5-b is(2-ch lor o-4-h y-
d r oxyp h en yl)-2-im id a zolin iu m br om id e (9a ). 9a was ob-
tained from 8a (0.24 mmol, 100 mg). Yield: 0.22 mmol (96
mg), 85% yellow powder; mp 184 °C. IR (KBr, cm-1): νj )
3500-3000 br, m (OH), 2934 m, 1662 s, 1608 s, 1577 m, 1500
s, 1437 m, 1291 s, 1261 s, 1222 m, 1044 m, 902 m, 861 m. MS
(EI): m/z (%) ) 133 (23), 111 (26), 97 (48), 83 (50), 74 (93), 59
3
OCH2CH3), 2.36 (s, 3H, CH3), 4.40 (q, J ) 7.0 Hz, 2H, OCH2-
CH3), 10.86 (br, 1H, NH, exchangeable by D2O), 11.75 (br, 1H,
NH, exchangeable by D2O).
3-Meth oxyp r op ion im id ic Acid Eth yl Ester (13b). 13b
was obtained from 3-methoxypropionitrile (50.0 mmol, 4.26 g,
4.53 mL). Recrystallization from dry ether. Yield: 38.0 mmol
(6.37 g), 76% white powder; mp 95 °C. IR (KBr, cm-1): νj )
2890 br (NH2), 1640 s, 1600 s, 1440 s, 1390 s, 1130 s, 1090 m,
995 m. 1H NMR (DMSO-d6): δ 1.18 (t, 3J ) 7.1 Hz, 3H,
1
(100), 43 (52). H NMR (DMSO-d6): δ 2.97 (s, 6H, CH3), 5.92
3
4
(s, 2H, ArCH), 6.57 (dd, J ) 8.6 Hz, J ) 2.4 Hz, 2H, ArH-5),
6.67 (d, 4J ) 2.4 Hz, 2H, ArH-3), 6.89 (d, 3J ) 8.6 Hz, 2H,
ArH-6), 8.89 (s, 1H, NdCH-N), 10.02 (s, 2H, OH, exchange-
able by D2O). Anal. (C17H17BrCl2N2O2‚H2O) C, H, N.
3
OCH2CH3), 2.50 (q, J ) 6.1 Hz, 2H, OCH2CH2), 3.22 (s, 3H,
3
3
OCH3), 3.53 (q, J ) 6.1 Hz, 2H, OCH2CH2), 4.05 (q, J ) 7.1
Hz, 2H, OCH2CH3), 11.26 (br, 1H, NH, exchangeable by D2O),
12.03 (br, 1H, NH, exchangeable by D2O).
(4R,5S)/(4S,5R)-N,N′-Dieth yl-4,5-bis(2-ch lor o-4-h ydr oxy-
p h en yl)-2-im id a zolin iu m br om id e (9b). 9b was obtained
from 8b (0.23 mmol, 100.0 mg). Recrystallization from methanol/
ether. Yield: 0.20 mmol (90.7 mg), 88% yellow powder; mp
>300 °C. IR (KBr, cm-1): νj ) 3600-2500 br, m (OH), 1649 s,
1608 s, 1499 m, 1438 w, 1259 m, 1221 m, 1044 w, 901 w. MS
(EI, 50 °C): m/z (%) ) 350 (21), 251 (12), 222 (22), 184 (100),
Sta bility Stu d ies. The mobile phase was methanol/H2SO4
(0.001 N, 20 mM Na2SO4) in the range of 50/50 to 30/70. The
probes were prepared as aqueous solutions (1.0 mM in MilliQ
water). Prior to analysis, they were diluted with PBS to 0.1
mM, placed in the autosampler (tempered to 37 °C) and
immediately injected. Stability was monitored for 55 h. Detec-
tion of the eluted probes was made with UV at 254 nm.
1
3
141 (17), 128 (24), 105 (10). H NMR (DMSO-d6): δ 1.18 (t, J
) 7.2 Hz, 6H, CH2CH3), 3.02-3.10 (m, 2H, CH2CH3), 3.48-
3
Biologica l Meth od s. 1. Bioch em ica ls, Ch em ica ls, a n d
Ma ter ia ls. The following is a list of materials and the
companies from which they were purchased: dextran, 17â-
estradiol, L-glutamine (L-glutamine solution, 29.2 mg/mL PBS),
and minimum essential medium Eagle (EMEM) from Sigma
(Munich, Germany); Dulbecco’s modified Eagle medium with-
out phenol red (DMEM) from Gibco (Eggenstein, Germany);
fetal calf serum (FCS) from Bio whittaker (Verviers, Belgium);
gentamycin sulfate from Fluka (Deisenhofen, Germany); trypsin
(0.05%) in ethylenediaminetetraacetic acid (0.02%) (trypsin/
EDTA) from Boehringer (Mannheim, Germany); penicillin-
streptomycin gold standard (10 000 IE penicillin/mL, 10 mg
of streptomycin/mL) and geneticin disulfate (geneticin solution,
35.71 mg/mL PBS) from ICN Biomedicals GmbH (Eschwege,
Germany); norit A (charcoal) from Serva (Heidelberg, Ger-
many); cell culture lysis reagent (5×) (diluted 1:5 with purified
water before use) and luciferase assay reagent from Promega
(Heidelberg, Germany); Optiphase HiSafe3 scintillation liquid
from Wallac (Turku, Finland); NET-317-estradiol[2,4,6,7-3H-
(N)] (17â-[3H]estradiol) from Du Pont NEN (Boston, Mary-
land).
3.57 (m, 2H, CH2CH3), 6.01 (s, 2H, ArCH), 6.57 (dd, J ) 8.6
Hz, 4J ) 2.4 Hz, 2H, ArH-5), 6.68 (d, 4J ) 2.4 Hz, 2H, ArH-3),
6.94 (d, 3J ) 8.6 Hz, 2H, ArH-6), 8.97 (s, 1H, NdCH-N), 10.07
(s, 2H, OH, exchangeable by D2O). Anal. (C19H21BrCl2N2O2‚
H2O) C, H, N.
(4R,5S)/(4S,5R)-N,N′-Dipropyl-4,5-bis(2-chloro-4-hydroxy-
p h en yl)-2-im id a zolin iu m Br om id e (9c). 9c was obtained
from 8c (0.23 mmol, 100 mg). Yield: 0.16 mmol (78 mg), 69%
yellow powder; mp >300 °C. IR (KBr, cm-1): νj ) 3600-3000
br, m (OH), 1643 s, 1609 s, 1499 m, 1436 m, 1264 m, 1217 m,
1044 m, 904 m, 861 m. H NMR (DMSO-d6): δ 0.89 (t, J )
7.3 Hz, 6H, CH2CH2CH3), 1.56 (m, 4H, CH2CH2CH3), 3.02 (m,
2H, CH2CH2CH3), 3.41 (m, 2H, CH2CH2CH3), 6.04 (s, 2H,
ArCH), 6.56 (dd, 3J ) 8.5 Hz, 4J ) 2.3 Hz, 2H, ArH-5), 6.66
1
3
4
3
(d, J ) 2.3 Hz, 2H, ArH-3), 6.94 (d, J ) 8.5 Hz, 2H, ArH-6),
9.02 (s, 1H, NdCH-N), 10.02 (s, 2H, OH, exchangeable by
D2O) Anal. (C21H25BrCl2N2O2‚H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of th e (1R,2S)/
(1S,2R)-1-Am in o-2-for m am ido-1,2-bis(2-ch lor o-4-h ydr oxy-
p h en yl)eth a n e (11). An amount of 0.50 mmol (202.0 mg) of
2b was dissolved in 100 mL of 0.01 N NaOH and stirred for
12 h at room temperature. The solution was extracted three
times with ethyl acetate. The combined organic layers were
then dried over Na2SO4. After the evaporation of the solvent,
the crude product was purified by recrystallization from CH2-
Cl2. Yield: 0.38 mmol (128.1 mg), 75% yellow powder; mp 75
°C. IR (KBr, cm-1): νj ) 3600-2500 br, m (OH), 1668 m (Cd
O), 1606 s, 1541 m (NH), 1499 s, 1445 m, 1385 m, 1260 m,
1238 m, 1095 w, 1040 m, 904 m, 856 m, 815 m, 691 w. MS
Phosphate buffered saline (PBS) was prepared by dissolving
8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4‚2H2O, 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, 0.3722
g of Titriplex III, and 0.195 g of sodium azide (all from Merck
or Fluka) in 1000 mL of purified water. Deionized water was
produced by means of a Millipore Milli-Q water system,

Stability and Interaction of Imidazolines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 927
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well plates were purchased from Renner GmbH (Dannstadt,
Germany).
2. Tr a n scr ip tion a l Bin d in g Assa y. Lu cifer a se Assa y.
The pertinent in vitro assay was described earlier by Hafner
et al.17 One week before starting the experiment, MCF-7-2a
cells were cultivated in DMEM supplemented with L-glutamine,
antibiotics, and dextran/charcoal-treated FCS (ct-FCS, 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 of cell suspension and 2 mL of medium per well) under
growing conditions (see above). After 24 h, an amount of 25
µL of a stock solution of the test compounds was added to
achieve concentrations ranging from 10-5 to 10-10 M and the
plates were incubated for 50 h. Before harvesting, the cells
were washed twice with PBS, and then an amount of 200 µL
of cell culture lysis reagent was added into each well. After
20 min of lysis at room temperature, cells were transferred
into reaction tubes and centrifuged. Luciferase was assayed
using the Promega luciferase assay reagent. An amount of 50
µL 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 Bradford26) of each sample with the mass of
luciferase. Estrogenic activity was expressed as percent activa-
tion of a 10-8 M estradiol control (100%).
Ack n ow led gm en t . The technical assistance of S.
Bergemann and I. Schnautz is acknowledged. The study
presented was supported by Grants Gu285/3-1 and
Gu285/3-2 from the Deutsche Forschungsgemeinschaft.
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