Design of new beta1-selective adrenoceptor ligands as potential radioligands for in vivo imaging.

Article abstract of DOI:10.1016/S0968-0896(03)00297-9

In general, the failing human heart is characterized by a selective reduction in beta(1)-adrenoceptors (beta(1)-ARs) without change in beta(2)-AR density. Medical imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET) with appropriate radioligands, offer the possibility of assessing beta-adrenoceptor density non-invasively in humans. To date, neither a SPECT nor a PET radioligand is available for the selective imaging of cardiac beta(1)-ARs. The aim of this study was to develop potential high affinity beta(1)-selective AR radioligands for the non-invasive in vivo imaging of the beta(1)-AR density in the human heart using SPECT or PET. A variety of racemic N-aryl-N'-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea derivatives and chain-elongated analogues, related to the established beta(1)-AR antagonist, ICI 89,406 8i, were synthesized. Competition studies using the non-selective AR ligand, [(125)I]iodocyanopindolol ([(125)I]ICYP), and ventricular membrane preparations of wild-type mice revealed nine ligands with higher beta(1)-AR affinities (up to 76-fold) and beta(1)-AR selectivities (up to 139-fold) than 8i. Mostly, these ligands possess a 2-substituted phenoxy group and a 4-substituted phenyl residue in contrast to the lead compound 8i. The non-radioactive counterparts of the desired SPECT- and PET-radiotracers were synthesized as reference compounds [e.g., 8f, 8g, 8h and 8l as the non-radioactive analogues of the radioiodinated SPECT radioligands, 8e and 8h as the non-radioactive compounds of C-11 labelled PET-tracers (C-11 in the methoxy group)]. The established library of high affinity beta(1)-selective AR antagonists was screened for chemical precursors for the radiosynthesis of the mentioned radioligands. Furthermore, the library consists of some comparison compounds that are unsubstituted, allyl- and alkyl-substituted or chain-elongated (e.g., 8a, 8j, 8o and 8r-t). Future steps will include radiolabelling and pharmacokinetic evaluation of the beta(1)-selective target compounds, which could be applied as sympathetic innervation agents for in vivo investigations and diagnostics in patients suffering from cardiac diseases like heart failure and ventricular arrhythmias.

Full text of DOI:10.1016/S0968-0896(03)00297-9

Bioorganic & Medicinal Chemistry 11 (2003) 3513–3527
Design of New ꢀ1-Selective Adrenoceptor Ligands as Potential
Radioligands for In Vivo Imaging
Klaus Kopka,a,*,y Stefan Wagner,a,y Burkhard Riemann,a,b Marilyn P. Law,a
Carsten Puke,a Sajinder K. Luthra,c Victor W. Pike,d Thomas Wichter,e
Wilhelm Schmitz,b Otmar Schobera and Michael Schafersa
aDepartment of Nuclear Medicine, Albert-Schweitzer-Str. 33, University Hospital Munster, 48149 Munster, Germany
¨
¨
bInstitute of Pharmacology and Toxicology, Domagkstr. 12, University Hospital Munster, 48149 Munster, Germany
¨
¨
cImaging Research Solutions Limited, Cyclotron Building, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK
dMolecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, 10 Center Drive,
Rm B3C346A, MSC 1003, Bethesda, MD 20892-1003, USA
eDepartment of Cardiology and Angiology, Albert-Schweitzer Str. 33, University Hospital Munster, 48149 Munster, Germany
¨
¨
Received 13 March 2003; accepted 25 April 2003
Abstract—In general, the failing human heart is characterized by a selective reduction in b1-adrenoceptors (b1-ARs) without change
in b2-AR density. Medical imaging techniques, either single photon emission computed tomography (SPECT) or positron emission
tomography (PET) with appropriate radioligands, offer the possibility of assessing b-adrenoceptor density non-invasively in
humans. To date, neither a SPECT nor a PET radioligand is available for the selective imaging of cardiac b1-ARs. The aim of this
study was to develop potential high affinity b1-selective AR radioligands for the non-invasive in vivo imaging of the b1-AR density
in the human heart using SPECT or PET. A variety of racemic N-aryl-N0-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea deri-
vatives and chain-elongated analogues, related to the established b1-AR antagonist, ICI 89,406 8i, were synthesized. Competition
studies using the non-selective AR ligand, [125I]iodocyanopindolol ([125I]ICYP), and ventricular membrane preparations of wild-
type mice revealed nine ligands with higher b1-AR affinities (up to 76-fold) and b1-AR selectivities (up to 139-fold) than 8i. Mostly,
these ligands possess a 2-substituted phenoxy group and a 4-substituted phenyl residue in contrast to the lead compound 8i. The
non-radioactive counterparts of the desired SPECT- and PET-radiotracers were synthesized as reference compounds [e.g., 8f, 8g, 8h
and 8l as the non-radioactive analogues of the radioiodinated SPECT radioligands, 8e and 8h as the non-radioactive compounds of
C-11 labelled PET-tracers (C-11 in the methoxy group)]. The established library of high affinity b1-selective AR antagonists was
screened for chemical precursors for the radiosynthesis of the mentioned radioligands. Furthermore, the library consists of some
comparison compounds that are unsubstituted, allyl- and alkyl-substituted or chain-elongated (e.g., 8a, 8j, 8o and 8r–t). Future
steps will include radiolabelling and pharmacokinetic evaluation of the b1-selective target compounds, which could be applied as
sympathetic innervation agents for in vivo investigations and diagnostics in patients suffering from cardiac diseases like heart failure
and ventricular arrhythmias.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
AR agents mainly stimulate bronchodilation and vaso-
depression. The atypical b3-ARs are involved in lipo-
lysis. Additionally, a putative subtype has been
identified in cardiac tissue, classified as the b4-AR.10
The b-adrenoceptor (AR) family is heterogeneous
in nature and is subdivided into at least three distinct
subtypes, the b1- and b2-AR7 and the atypical b3-AR.8,9
Within the ventricles of the healthy human heart, the b1/
b2-AR ratio is approximately 3:1.1 In heart disease, both
the b-AR density and the b1/b2-AR ratio may change.
Several conditions, including hypertension, heart failure,
ischemia, hypertrophic and dilated cardiomyopathy
(HCM, DCM) are accompanied by a reduced b-AR
density in the heart.11À16 In addition, the failing human
b1-AR agonists are responsible for the stimulation of
heart rate and cardiac contractility, whereas b2-selective
*Corresponding author. Tel.: +49-251-83-47362; fax: +49-251-83-
47363; e-mail: kopka@uni-muenster.de
yThese authors contributed equally to this work.
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00297-9

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K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
An early example of a designed b1-selective AR
antagonist is the compound ICI 89,406 8i,26 which pro-
duces effective b1-adrenoceptor blockade during exer-
cise in patients with angina pectoris even when applied
as the racemate.27,28 This compound was chosen as lead
structure to develop new radiotracers for application in
nuclear medical imaging.
Scheme 1. Lead structure of ICI 89,406 8i.
The aim of the present study was to develop potential
high affinity b1-selective AR antagonists, based on ICI
89,406 8i,3À6 which could be applied as radioligands
with SPECT and PET. The synthesis of ICI 89,406 8i
was verified and improved by developing a straightfor-
ward convergent synthesis via two major fragments, an
aromatic epoxide and an aromatic urea, respectively. By
inserting different substituents within both aromatic
systems a library of compounds was synthesized. The in
vitro pharmacology of these b1-AR antagonists was
examined.
heart is often characterized by a selective reduction in
b1-adrenoceptors (b1-ARs) without change in b2-AR
density.1
Measuring b1- and b2-AR densities in vivo indepen-
dently therefore may prove to be very valuable. Only
functional imaging techniques like SPECT or PET
allow for the non-invasive investigation of b-AR density
and distribution.2 Selective radiolabelled b-AR antago-
nists suitable for PET or SPECT, respectively, have yet
to be designed to measure b1- and b2-AR densities in the
human heart.
Results
The only clinically established radioligand for visuali-
zation of b-AR density with PET is the non-selective b-
AR radioligand (S)-[11C]CGP 12177 which was first
described as the racemic radioligand in 1991 by Del
Forge and coworkers.17 The recently developed and
radiochemically more easily accessible (S)-[11C]CGP
12388 has been presented as a promising alternative for
non-selective b-AR targeting.18 Despite the fact that
SPECT does not share the unique quantifying cap-
abilities of PET, initial attempts were made in the
mid-1990s to develop suitable ligands, for example
radioiodinated carazolol and CGP 12177 derivatives for
b-AR imaging with SPECT.19À21
Chemistry
ICI 89,406 8i (Scheme 1), which is recognized for its b1-
AR selectivity (75- to 195-fold),29À31 and 20 aromati-
cally functionalized compounds with structural simila-
rities to this (aryloxy)propanolamine lead structure were
synthesized. In order to examine the structure–activity
relationships (SARs) between the potential ligands and
b1-ARs, the prepared compounds were modified in the
pattern of aromatic substituents and in length of the
aliphatic chain. These compounds are ligands that may
be useful for elucidating the SAR of b1-ARs, potential
precursors of the corresponding radiolabelled deriva-
tives, or the non-radioactive references of these radio-
active counterparts. Four of these compounds were
previously described in literature as potential b1-selective
AR antagonists (8a, 8i, 8q, 8t).26,32,33 Most of the sub-
stances are accessible via a convergent five-step synthesis
(Scheme 2), starting with the conversion of the corre-
sponding alcohols 1a–h and 3-chloro-1,2-epoxypropane
Up to now, b1-selective AR antagonists available as
drugs for clinical use have been very rare. In cardiac
studies of b-ARs, a b1-selective radioligand is needed
precisely because changes of this subtype occur mainly
in heart disease. Only few b1-AR selective radioligands
like (Æ)-[11C]HX-CH 44,22 (S)-[11C]bisoprolol,23 or
[11C]CGP 20712A24 are in development.25
Figure 1. (a) Protein dependence of [125I]ICYP binding to mouse ventricular membrane preparations. The specific binding was linear for up to 50 mg
protein/200 mL; (b) time course of [125I]ICYP binding to mouse ventricular membranes. The association rate was linear for 15 min and reached a
steady state after 60 min.

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3515
Scheme 2. Synthesis of the b1-AR ligands 8a–s. Reagents: (a) 2N NaOH or Na, toluene for compound 3h; (b) CH2Cl2; (c) Et2O or THF; (d) HCl,
MeOH; (e) 10N NaOH, n-PrOH, H2O.
2, which results in the first key compounds, the 1,2-
epoxy-3-substituted-propane derivatives 3a–h. The sec-
ond key structures, the N-(2-amino-ethyl)-urea
derivatives 7a–g, were prepared via a three step
sequence (Scheme 2 continued). At first ethylendiamine
4 is protected by a Boc-group in order to couple it
with the aromatic isocyanates 6a–g. After cleavage of
the Boc-group, ring opening of the 1,2-epoxy-3-sub-
stituted-propanes 3a–h with the N-(2-amino-ethyl)-
urea compounds 7a–g resulted in the desired potential
b1-AR antagonists 8a–s (Scheme 2 continued and
Table 1).
The time course of [125I]ICYP binding to b-ARs is
shown in Figure 1b. The binding reaction was linear for
approximately 15 min and reached equilibrium after
60 min of incubation. To determine the maximum
binding, assays were carried out for 60 min.
Scheme 3. Synthesis of the b1-AR ligand 8t. Reagents: (a) H2, Pd/C,
CH3COOH.
Compound 8t was prepared via a hydrogenolysis from
the benzyloxy substituted precursor 8q (Scheme 3),
while the carbamate 8u was built by the reaction of 3d
and 5 (Scheme 4).
Biology
First, the protein dependence of [125I]ICYP binding to
mouse ventricular membrane preparations was investi-
gated. The specific binding increased linearly with the
protein concentration for up to 50 mg/200 mL (Fig. 1a).
Therefore, 15 mg protein per 200 mL were applied for the
in vitro binding studies.
Scheme 4. Synthesis of the b1-AR ligand 8u. Reagents: (a) n-PrOH.

3516
K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
The binding of the nonselective b-AR antagonist
125I]ICYP to ventricular membranes was specific, satur-
and 8j–u) (Fig. 3a–e). Non-linear regression analysis
using the XMGRACE programme (Linux software)
showed that the data of the compounds 8a–o, 8q and 8u
fitted a two-site model significantly better than a one-
site model (F=3.52, P<0.05). These compounds show
a significantly higher affinity to b1- than to b2-ARs
(Table 1).
[
able and of high affinity (Fig. 2a). Scatchard transfor-
mation of the saturation data yielded a linear plot with
a correlation coefficient greater than 0.95 (Fig. 2b). The
dissociation constant (KD) and the maximum number of
binding sites (Bmax) determined from three experiments
for the binding of [125I]ICYP were 32.3Æ1.9 pM and
38.6Æ2.9 fmol mgÀ1 protein, respectively.
The high- and low-affinity IC50 values of the unlabelled
derivatives 8a–u for the b1- and b2-ARs, respectively,
were calculated by non-linear regression analysis of
competition studies using [125I]ICYP and mouse ven-
tricular membrane preparations.
Competition studies were carried out to assess the inhi-
bition of the binding of [125I]ICYP by the b1-selective
lead compound ICI 89,406 8i and its derivatives (8a–h
Figure 2. (a) Saturation curve for the specific binding of [125I]ICYP to mouse ventricular membrane preparations. Each point represents the mean-
ÆSEM (n=3). (b) Scatchard transformation of the saturation isotherm yielded a dissociation constant (KD) of 32.3Æ1.9 pM and a maximum
density of b-AR (Bmax) of 38.6Æ2.9 fmol mgÀ1 protein. Each point represents the meanÆSEM (n=3).
Table 1. Inhibition constants and calculated b1-selectivities of compounds 8a–u obtained via a radioligand binding assay using mouse ventricular
membrane preparations, plus calculated logP/logD values
Compd
R1
n1
n2
R2
KI1 (nM)a
KI2 (nM)a
b1-selectivityb
logP/logDc
8a
8b
8c
8d
8e
8f
8g
8h
8id
8j
H
H
2-Br
2-Br
2-Br
2-I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
H
4-Br
H
4-Br
4-OMe
H
4-I
4-OMe
H
4-Me
4-Br
4-I
4-OMe
4-OBz
H
H
H
8.1Æ0.6
1.8Æ0.4
940Æ200
260Æ40
120Æ20
74Æ11
113Æ16
143Æ15
75Æ14
1.36/0.07
2.25/0.95
2.24/0.94
3.12/1.83
2.19/0.90
2.49/1.19
2.45/1.15
2.45/1.15
1.73/0.44
1.71/0.41
2.16/0.86
2.41/1.11
1.23/À0.07
2.83/1.54
1.99/0.69
1.33/0.03
2.93/1.63
1.00/À0.29
0.87/À0.55
0.82/À0.94
1.27/À0.03
1.8Æ0.4
0.19Æ0.03
0.04Æ0.016
0.045Æ0.005
0.043Æ0.011
0.55Æ0.11
1.3Æ0.2
403Æ79
5220Æ1890
266Æ28
905Æ194
783Æ362
121Æ14
5340Æ182
16,800Æ4200
398Æ48
82Æ33
140Æ50
12Æ2
2-I
2-I
34Æ5
390Æ160
160Æ40
89Æ13
2-CN
2-CN
2-CN
2-CN
2-CN
2-CN
2-allyl
2-OMe
4-OBz
2-CN
H
0.017Æ0.002
0.017Æ0.007
0.12Æ0.04
1.8Æ0.8
8k
8l
260Æ120
44Æ16
8m
8n
8o
8p
8q
8r
8s
8t
8uf
120Æ60
64Æ9
0.022Æ0.006
0.80Æ0.13
1205Æ72
30Æ4
4020Æ1570
35Æ7
27Æ5
1205Æ72
2700Æ300
61Æ7
1.0e
96Æ19
H
H
H
61Æ7
1.0e
3500Æ820
150Æ30
3500Æ820
150Æ30
16,000Æ8000
1.0e
4-OH
1.0e
35Æ1
489Æ260
aDisplacement of specifically bound non-selective b-AR ligand [125I]ICYP binding at b1- and b2-ARs expressed in mouse ventricular membrane
preparations, noted as meanÆSEM, n=4–6.
bThe ratios of the low- over the high-affinity inhibition constants (KI2/KI1) yield the b1-selectivities of the unlabelled derivatives 8a–u, noted as
meanÆSEM, n=4–6.
clogP and logD values calculated by Pallas 3.0 (logD=logP at physiological pH 7.4).
d8i is the lead compound ICI 89,406.
eIf the two-site model was rejected by the F-ratio test the one site model was applied and the b1-selectivity was set to 1.0.
fThe structure of 8u is shown in Scheme 4.

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3517
The IC50 values were converted into the high- and low-
affinity inhibition constants (KI1 for the b1-ARs and KI2
for the b2-ARs) by the method of Cheng–Prusoff 34
using the experimentally determined KD value of
Substitution of one phenyl hydrogen in 8a by a bromine
atom in 4-position decreases the KI1-value about
4-fold, so that the b1-selectivity of 8b raises by 27%.
The exchange of the phenoxy hydrogen by bromine
in 2-position results in an opposite effect. In fact the
KI1-values of 8b and 8c are identical (KI1=1.8 nM);
but in comparison with 8b the b1-selectivity of 8c is
nearly halved (143 vs 75). The substitutions of 8b and
8c combined in 8d leads to a very potent b1-AR ligand,
that shows a more than 6-fold higher affinity for
b1-AR (KI1=0.19 nM) and a 3-fold higher b1-selec-
tivity (403) in comparison with the lead compound
ICI 89,406 8i. Formally, changing the bromine in
[
125I]ICYP (32.3Æ1.9 pM).
The ratios of the low- to high-affinity inhibition con-
stants (KI2/KI1) yield the b1-selectivities of the unla-
belled derivatives 8a–u (Table 1). Additionally, the
calculated logP and logD values (Pallas 3.0) for com-
pounds 8a–u are also listed in Table 1 to imply the
changes of the lipophilicities caused by the chemical
modifications of the lead compound 8i, ICI 89,406.
Figure 3. Competition of ICI 89,406 (8i) and its derivatives: (a) with a hydrogen substituent replacing the cyano moiety of 8i (8a–b); (b) with a
bromine substituent replacing the cyano moiety of 8i (8c–e); (c) with an iodine substituent replacing the cyano moiety of 8i (8f–h); (d) without a
substutition of the cyano moiety of 8i (8j–n); (e) with different substituents replacing the cyano moiety of 8i (8o–q, 8t); (f) with a change in the chain
length of the carbon skeleton (8r–s) with [125I]ICYP binding to mouse ventricular membranes. Ordinate: specific [125I]ICYP binding expressed as
percentage of maximum binding in each experiment. Abscissa: concentration of antagonists competing for specific binding of [125I]ICYP. Each point
represents the meanÆSEM (n=4–6). See Table 1 for details of substituents.

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K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
the phenyl residue into a methoxy group (8e) increases
the b1-selectivity dramatically, because compound 8e
possesses a lower KI1-value (0.04 nM) than 8d. The
bromo derivatives 8b–e are potential precursor
compounds of the corresponding radioiodinated sub-
stances (Br/*I-exchange), while the highly b1-selective
compound 8e can act additionally as the non-
radioactive reference for the PET-tracer [methoxy-
11C]8e, in which the positron emitter C-11 is
introduced within the methyl group of the methoxy
moiety.
Discussion
The human heart contains a heterogenous population of
b1- and b2-ARs, both mediating positive inotropic,
chronotropic, dromotropic and lusitropic effects of
catecholamines.30,35 In the normal human heart, b1-ARs
play the major role in the adrenergic control of myo-
cardial function.36 They account for approximately 60–
80% of cardiac b-Ars.37,38 In heart failure the cardiac
and systemic adrenergic drive is activated in order to
maintain cardiac function. This enhanced adrenergic
stimulation leads to a downregulation of myocardial
b-ARs.39,40 In most studies a selective downregulation
of the b1-ARs is predominant whereas the b2-ARs
appear to be unaffected.36,41,42
The iodinated compounds 8f and 8g exhibit lower affi-
nity-constants KI1 and higher b1-selectivities than the
bromo counterparts 8c and 8d. In contrast to 8c and 8d,
their KI1-values are within the same order of magnitude
(ꢀ0.04 nM). In comparison with the methoxy com-
pound 8e the iodinated counterpart 8h shows a lower
affinity to the b1-AR (0.04 nM vs 0.55 nM). Anyhow,
the b1-selectivity of 8h ranges between 8f and 8g. The
mentioned iodinated substances 8f-h serve as the non-
radioactive references for the future radioiodinated and
C-11-methylated compounds.
With functional imaging procedures like SPECT and
PET it is possible to study the behaviour and fate of
radiolabelled receptor ligands in vivo, for example their
biodistribution, the plasma and tissue kinetics and spe-
cific and non-specific binding. Receptors can be studied
in their natural environment and their distribution and
density can be assessed.
The series of cyano derivatives 8i–n exhibit very differ-
ent b1-affinities and selectivities. Compared with the
other cyano substances, the lead structure ICI 89,406
8i and compound 8m possess both moderate affinity
inhibition constants KI1 and b1-selectivities (1.3 nM,
121 and 1.8 nM, 82, respectively). These parameters
can be improved by introducing a more hydro-
phobic substituent in 4-position at the phenyl moi-
ety (8j–l and 8n). The exchange of hydrogen in 8i for a
methyl, bromo or benzyloxy function (8j, 8k and
8n) leads to a potent increase in the b1-affinities
(ꢁ59-fold in comparison with 8i) and b1-selectivities
(ꢁ33-fold in comparison with 8i), respectively. A
weakening effect has been observed in the case of
the iodo analogue 8l. This potential reference com-
pound for a feasible radioiodinated SPECT-tracer
demonstrates a KI1-value of 0.12 nM and a b1-selectivity
of 398.
Radioligands suitable for non-invasive b-AR imaging
should exhibit high selectivity and affinity (nanomolar
KD) as well as antagonist action for stable receptor-
ligand interactions.25 In addition, stereoselectivity is
an important criterion for b-AR ligands. Similar to
the endogenous (S)-enantiomers of epinephrine and
norepinephrine, the (S)-enantiomers of b-AR antago-
nists show approximately a 100-fold higher affinity to
b-AR than the corresponding (R)-enantiomers.43
Development of (S)-enantiomers of new b-AR radi-
oligands rather than racemates should reduce non-
specific binding. Nevertheless, we decided to synthesize
the racemic forms of 8a–u in order to be able to build up
a library of compounds in a fast and cost-effective
manner. From this library highly selective and affine
candidates could be chosen for enantioselective and
radiochemical resynthesis for detailed evaluation in
vitro and in vivo. A further criterion for cardiac b-AR
radioligands is a low lipophilicity. Radioligands with
high lipophilicity can traverse the sarcolemmal mem-
brane and bind to internalized, that is inactive,
b-ARs. This in turn precludes the exact quantification
of cardiac b-AR densities in vivo with PET. In addi-
tion, high lipophilicity (e.g., logP > >2, see also cal-
culated logD values=logP at physiological pH 7.4,
Table 1) results in a high degree of nonspecific binding,
especially in the lungs. Pulmonary trapping reduces the
myocardial contrast of the radioligand after intravenous
injection.
Replacing the cyano moiety of 8i by an allyl group,
forming compound 8o, leads to a notable decrease of b1-
selectivity (35). A complete loss of b1-selectivity can be
observed with the 2-methoxy substituted derivative 8p.
In addition, a loss of b1-selectivity can also be found if
the phenyl group of 8i is replaced by a benzyl residue
(8r) or if both the phenyl and the phenoxy groups are
replaced by the corresponding benzyl and benzyloxy
residues (8s).
Examples for 4-substituted phenoxy compounds are the
benzyloxy derivative 8q and the hydroxy analogue 8t.
Thereby, 8q is a moderate b1-AR ligand (KI1=30 nM,
b1-selectivity=96), while 8t completely loses its
b1-selectivity.
Furthermore, low metabolism of the putative radi-
oligand, at least in the myocardium itself, is an impor-
tant aspect to simplify kinetic modelling and
quantification of b-AR densities with PET. In addition,
low toxicity and mutagenicity of the radioligand is a
prerequisite for applications in the clinical setting. Last
but not least, the putative tracer has to be amenable to
labelling with gamma emitters for SPECT and positron
emitter for PET.
The tert-butyl-carbamate 8u, containing only one aro-
matic subunit, shows no b2-affinity (KI2=16,000 nM)
but a moderate b1-affinity (KI1=35 nM) resulting in a
b1-selectivity of 489.

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3519
Nonselective radioligands for b-ARs such as [11C]CGP
12177 and [11C]CGP 12388 have been successfully used
in vivo with PET.15,17,44À46 In addition, the b2-selective
radioligand [11C]formoterol may provide an unique
approach to quantify b2-AR density with PET.47 To
date however, no b1-selective radioligand suitable for
the non-invasive assessment of cardiac b1-ARs has been
established clinically either for SPECT or for PET.
form attractive interactions with the aromatic and/or
hydrophobic counterparts of the ligands.48À51 In fact the
disubstituted affine ligands 8d–e, 8g–h, 8j–l and 8n pos-
sess at least one hydrophobic or aromatic substituent in
2- or 4-position of their phenyl cores that obviously
increases the b1-affinities. Thereby the b2-affinities are
not increased in the same manner resulting in high
b1-selectivities.
For the present study ICI 89,406 8i was chosen as the
lead compound for b1-AR radioligand development on
the basis of its high affinity and selectivity for b1-ARs,
moderate lipophilicity and amenability to labelling with
A monosubstitution of the phenoxy ring in 2-position
with a sterical demanding or p-electron-donor moiety
(8c, 8f, 8i, 8o) increases the b1-affinities in comparison to
the unsubstituted compound 8a. Obviously, such sub-
stituents extends the attractive interactions with the
aromatic residue cage of b1-ARs without improving
b1-selectivities significantly. In contrast, a complete loss
of b1-selectivity can be observed in the case of a meth-
oxy group inserted at the 2-position of the phenoxy unit
(8p). Additionally, a benzyloxy or especially a hydroxy
moiety in 4-position (8q, 8t) decreases the b1-affinities
and b1-selectivities in comparison to the unsubstituted
counterpart 8a.
[
123I]iodine and [11C]carbon. Furthermore, this com-
pound produces effective b1-AR blockade even when
applied as racemate.27,28 In order to find suitable pre-
cursor and reference compounds for the development of
potential b1-AR PET and SPECT tracers, a series of
new 2-propanol derivatives have been synthesized by
modifying ICI 89,406 8i. The substituents of the aro-
matic groups have been slightly altered, partly varying
the chain length of the carbon skeleton. A few of the
compounds (e.g., non-precursor compounds 8a, 8j, 8n,
8o and 8q) function as derivatives that help to clarify the
change of b1-affinity and b1-selectivity caused by intro-
duction of small substituents into the phenoxy and
phenyl residues. The prepared substances were tested in
competition studies using [125I]ICYP and mouse ven-
tricular membrane preparations to calculate their high-
and low-affinity inhibition constants KI1 and KI2. Ten
compounds were identified that possess a higher b1-affi-
nity (KI1 <1.3 nM) than the lead compound 8i, and 11
derivatives had a higher b1-selectivity (>121) than 8i.
Nine of the derivatives show higher b1-affinities as well
as higher b1-selectivities.
The carbon skeleton of 8i tolerates neither one phenyl-
to-benzyl substitution (8r) nor a replacement of both
phenyl subunits of 8a by benzyl groups (8s) because
both substitutions lead to a loss of b1-selectivity and
decreased b1-affinity. A similar effect was observed by
Nudelman et. al. comparing classical 3-aryloxy-2-pro-
panolamine b1-AR ligands with their more rigid 4-aryl-
3-butenyl-2-ol-amine analogues. The variation of
increased rigidity also results in decreased b1-affinity.52
Conclusion
In eight of these compounds the improvement of both
the b1-affinity and the b1-selectivity was achieved by
substitution of both aromatic groups, especially by
altering the phenoxy group at the 2-position and the
phenyl group at the 4-position.
A library of derivatives of the known b1-selective
antagonist ICI 89,406 8i was synthesized and their
binding to murine ventricular membranes examined in
vitro in order to identify potent b1-affine and b1-selec-
tive AR ligands as possible radiotracers for assessing
b1-ARs in vivo via SPECT and PET. The next step is to
chose a b1-selective AR compound and prepare its
(S)-enantiomer and optical pure radiolabelled analogue
as a possible b1-AR radioligand for in vivo diagnostics
of the human heart.
The mentioned potent b1-AR ligands show this char-
acteristic substitution pattern with the exception of 8f.
The combination of the 2-bromo residue and the 4-
bromo or 4-methoxy group (8d and 8e), the combi-
nation of the 2-iodo and the 4-iodo or 4-methoxy sub-
unit (8g and 8h), and the assembly of the 2-cyano and
the 4-methyl, 4-bromo, 4-iodo or 4-benzyloxy functions
(8j, 8k, 8l and 8n) lead to very b1-affine and b1-selective
AR compounds that are up to 139-fold more selective
than 8i.
The bromo species 8c, 8d, 8e and 8k could function as
precursor compounds for the radioiodination of the hot
counterpart of 8f, 8g, 8h and 8l via the commonly used
[*I]iodo-debromination-reaction.
The highly b1-affine and b1-selective methoxy deriva-
tives 8e und 8h may represent the non-radioactive ana-
logues of the C-11 labelled PET-tracers, that may be
prepared from the corresponding phenolic compounds
by methylation using [11C]methyliodide.
An electrostatic interaction between a conserved aspar-
tate on transmembrane helix 3 in cationic ligand
GPCRs (G protein-coupled-receptors), which include
the b1-ARs, and the cationic neurotransmitter is sug-
gested. In compounds 8a–u the cationic centre is repre-
sented by the protonated secondary amine moiety. A
second interesting feature of cationic neurotransmitter
GPCRs is the presence of a cluster of conserved aro-
matic residues that encages the ammonium-aspartate
ion pair. This hydrophobic aromatic cluster is able to
Further investigations concerning the radiosynthesis of
the above mentioned compounds as feasible radi-
oligands in b1-AR imaging and the in vivo behaviour of
these 2-propanol derivatives are in progress. SPECT
and PET using these b1-selective radioligands may have

3520
K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
a great impact on the diagnostic and therapeutic man-
agement of patients with cardiac diseases in the future.
(75.5 MHz, CDCl3): d (ppm): 154.94, 133.41, 128.42,
122.50, 113.97, 112.47, 69.61, 49.98, 44.54.
2-(2-Iodo-phenoxymethyl)-oxirane 3c. Yield: 75%. Bp
(3.0 mbar): 140 ꢂC[lit.: 118–123 ꢂC(0.2 Torr)
55
]
1H
(ppm): 7.84 (dd,
Experimental
NMR (400 MHz, CDCl3):
4
d
Synthetic methods
3J=7.3 Hz, J=1.6 Hz, 1H, HAryl), 7.38–7.34 (m, 1H,
HAryl), 6.91 (dd, 3J=8.4 Hz, 4J=1.2 Hz, 1H, HAryl),
3
4
All chemicals, reagents, and solvents for the synthesis of
the compounds were analytical grade and purchased
from commercial sources.
6.80 (dt, J=7.6 Hz, J=1.5 Hz, 1H, HAryl), 4.35 (dd,
2J=11.2 Hz, 3J=2.8 Hz, 1H,
1 CH2), 4.13 (dd,
2J=11.2 Hz, J=5.2 Hz, 1H, 1 CH2), 3.48–3.44 (m, 1H,
CH), 3.00–2.89 (m, 2H, 2 CH2). 13CNMR (75.5 MHz,
CDCl3): d (ppm): 157.55, 139.98, 129.91, 123.58, 113.22,
87.14, 70.12, 50.47, 45.13.
3
The melting points (uncorrected) were determined on a
Stuart Scientific SMP3 capillary melting point appara-
tus. 1H NMR and 13CNMR spectra were recorded on a
Bruker AC200, ARX 300, and AMX 400 spectrometer,
respectively. Mass spectrometry was performed via a
Varian MAT 212 (EI=70 eV) spectrometer and a Bru-
ker MALDI-TOF-MS Reflex IV (matrix: DHB). GC–
MS analysis was undertaken with a Dani 8521 gas
chromatograph containing a DB 5, 30 m column (tem-
perature program: start 60 ꢂC, gradient 20 ꢂC/min, end
280 ꢂC) combined with an ITD 800 Finnigan MAT
(EI=70 eV) mass spectrometer. Elemental analysis was
realised by a Vario EL III analyser (see Table 2).
2-(2-Cyano-phenoxymethyl)-oxirane 3d. Yield: 71%. Mp
65 ꢂC. H NMR (300 MHz, CDCl3): d (ppm): 7.51–7.42
(m, 2H, HAryl), 6.99–6.93 (m, 2H, HAryl), 4.30 (dd,
1
2J=11.5 Hz, 3J=3.0 Hz, 1H,
1 CH2), 4.05 (dd,
2J=11.3 Hz, J=5.2 Hz, 1H, 1 CH2), 3.34–3.29 (m, 1H,
CH), 2.84 (dd, 2J=5.0 Hz, 3J=4.0 Hz, 1H, 1 CH2), 2.76
(dd, 2J=5.1 Hz, 3J=2.6 Hz, 1H, 1 CH2). 13CNMR
(75.5 MHz, CDCl3): d (ppm): 160.51, 134.75, 133.97,
121.78, 116.57, 113.20, 102.74, 69.71, 50,18, 44.81.
3
2-(2-Allyl-phenoxymethyl)-oxirane 3e. Yield: 66%. Bp.
(2.2 mbar): 130 ꢂC[lit.: 109–114 ꢂC(0.8 Torr) 56]. 1H
NMR (200 MHz, CDCl3): d (ppm): 7.24–7.13 (m, 2H,
HAryl), 6.94–6.81 (m, 2H, HAryl), 6.06–5.89 (m, 1H,
Synthesis of oxirane derivatives 3a–g: general procedure
(Scheme 2)
2
3 Equivalents 2, 1 equivalent phenol compound 1a–g
and 1.4 equivalent 2 N NaOH were heated to 50 ꢂCfor
1.5–2.5 h.
¼CH), 5.11–5.00 (m, 2H, ¼CH2), 4.22 (dd, J=11.1 Hz,
3J=3.1 Hz, 1H,
1
CH2), 3.98 (dd, 2J=11.0 Hz,
3J=5.3 Hz, 1H, 1 CH2), 3.37–3.31 (m, 3H, CH2 and CH),
3
2
2.91 (‘t’, J=4.5 Hz, 1H, 1 CH2), 2.78 (dd, J=5.0 Hz,
3J=2.7 Hz, 1H, 1 CH2). 13C NMR (75.5 MHz, CDCl3):
d (ppm): 156.11, 136.83, 129.90, 129.00, 127.24, 121.11,
115.35, 111.66, 68.74, 50.20, 44.49, 34.26.
At rt, the mixture was extracted three times with CHCl3
or CH2Cl2, the combined organic layers were dried
(MgSO4) and the solvent and volatile compounds were
evaporated. In most cases, the residue was destilled in
vacuo fractionally or in a Kugelrohr to provide the
epoxide derivatives as colorless liquids.
2-(2-Methoxy-phenoxymethyl)-oxirane 3f. Yield: 71%.
Bp (1.2 mbar): 120 ꢂC[lit.: 131 ꢂC(3 Torr) 57]. H NMR
1
(300 MHz, CDCl3): d (ppm): 6.95–6.82 (m, 4H, HAryl),
4.19 (dd, 2J=11.4 Hz, 3J=3.6 Hz, 1H, 1 CH2), 4.00 (dd,
2J=11.4 Hz, 3J=5.4 Hz, 1H, 1 CH2), 3.83 (s, 2H, CH2),
3.36–3.31 (m, 1H, CH), 2.84 (dd, 2J=5.0 Hz, 3J=4.1 Hz,
1H, 1 CH2), 2.69 (dd, 2J=5.0 Hz, 3J=2.6 Hz, 1H, 1
CH2). 13C NMR (75.5 MHz, CDCl3): d (ppm): 149.88,
148.18, 122.06, 120.95, 114.66, 112.25, 70.40, 55.97,
50.25, 44.90.
3d and 3g were purified via crystallization from diethyl-
ether to provide colorless solids.
2-Phenoxymethyl-oxirane 3a. Yield: 74%. Bp
(0.94 mbar): 115ꢂC[lit.: 105 ꢂC(0.08 Torr) 53]. H NMR
1
(300MHz, CDCl3): d (ppm): 7.55–7.50 (m, 2H, HAryl),
7.24–7.15 (m, 3H, HAryl), 4.42 (dd, 2J=11.1 Hz,
3J=3.0 Hz, 1H, 1 CH2), 4.15 (dd, 2J=11.1Hz, 3J=5.7 Hz,
1H, 1 CH2), 3.58–3.52 (m, 1H, CH), 3.08 (dd, 2J=4.8 Hz,
3J=4.2Hz, 1H, 1 CH2), 2.90 (dd, 2J=5.0 Hz, 3J=2.6Hz,
1H, 1 CH2). 13CNMR (75.5 MHz, CDCl 3): d (ppm):
158.98, 129.77, 121.64, 115.27, 69.16, 50.53, 45.04.
2-(4-Benzyloxy-phenoxymethyl)-oxirane 3g. Yield: 71%.
ꢂ
Mp 62 ꢂC(lit.: 63–66 C58). 1H NMR (300MHz, CDCl3):
d (ppm): 7.43–7.30 (m, 5H, HAryl), 6.92–6.82 (m, 4H,
HAryl), 5.01 (s, 2H, 2 CH2), 4.15 (dd, 2J=11.1 Hz,
3J=3.3Hz, 1H,
1
CH2), 3.91 (dd, 2J=11.3 Hz,
2-(2-Bromo-phenoxymethyl)-oxirane 3b. Yield: 71%. Bp
(1.4 mbar): 135 ꢂC[lit.: 131 ꢂC(3 Torr) 54]. 1H NMR
(300 MHz, CDCl3): d (ppm): 7.48 (dd, 3J=7.8 Hz,
4J=1.8 Hz, 1H, HAryl), 7.22–7.16 (m, 1H, HAryl), 6.87
(dd, 3J=8.4 Hz, 4J=1.8 Hz, 1H, HAryl), 6.80 (dt,
3J=7.6 Hz, 4J=1.3 Hz, 1H, HAryl), 4.23 (dd,
3J=5.6 Hz, 1H, 1 CH2), 3.34–3.29 (m, 1H, CH), 2.87
(dd, 2J=5.0 Hz, 3J=4.1 Hz, 1H, 1 CH2), 2.72 (dd,
2J=5.1 Hz, 3J=2.7 Hz, 1H,
1
CH2). 13CNMR
(75.5 MHz, CDCl3): d (ppm): 137.38, 152.88, 137.19,
128.46, 127.80, 127.36, 115.94, 115.71, 70.66, 69.48,
50.16, 44.62.
2J=11.3 Hz, 3J=2.9 Hz, 1H,
1 CH2), 3.99 (dd,
3
2J=11.1 Hz, J=5.1 Hz, 1H, 1 CH2), 3.35–3.30 (m, 1H,
CH), 2.84 (dd, 2J=5.1 Hz, 3J=4.2 Hz, 1H, 1 CH2), 2.78
(dd, 2J=5.1 Hz, 3J=2.7 Hz, 1H, 1 CH2). 13CNMR
2-Benzyloxymethyl-oxirane 3h. This was prepared
according to ref 59, but was purified via Kugelrohr-
destillation. Yield: 3%. Bp (1.2 mbar): 90-100 ꢂC[lit.:

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3521
85–86 ꢂC(2.0 Torr) 60]. H NMR (300 MHz, CDCl3): d
HAryl), 6.74 (t, 3J=5.9 Hz, 1H, NH), 3.32 (q,
1
2
3
(ppm): 7.32–7.28 (m, 5H, HAryl), 4.62 (d, J=12.0 Hz,
2
3J=6.1 Hz, 2H, CH2), 2.87 (q, J=6.2 Hz, 2H, CH2).
1H, 1 CH2), 4.55 (d, J=12.0 Hz, 1H, 1 CH2), 3.76 (dd,
2J=11.6 Hz, 3J=3.2 Hz, 1H,
13CNMR (75.5 MHz, DMSO- d6): d (ppm): 156.11,
140.40, 131.78, 120.13, 112.92, 39.72, 37.61.
1
CH2), 3.45 (dd,
3
2J=11.4 Hz, J=5.7 Hz, 1H, 1 CH2), 3.21–3.16 (m, 1H,
CH), 2.79 (‘t’, 3J=4.5 Hz, 1H, 1 CH2), 2.61 (dd,
N-(2-Amino-ethyl)-N0-(4-iodo-phenyl)-urea hydrochloride
7c. Yield: 78%. Mp 215 ꢂC(decomposition). 1H NMR
(200 MHz, DMSO-d6, TMS intern): d (ppm): 9.12 (s,
1H, NH), 7.90 (s, broad, 3H, NH+3 ), 7.54 (d,
2J=5.0 Hz, 3J=2.6 Hz, 1H,
1
CH2). 13CNMR
(75.5 MHz, CDCl3): d (ppm): 137.98, 128.44, 127.76,
73.37, 70.85, 50.86, 44.30.
3
3J=8.8 Hz, 2H, HAryl), 7.27 (d, J=8.9 Hz, 2H, HAryl),
Synthesis of N-mono(tert-butyloxycarbonyl)ethylenedia-
mine 5 (Scheme 2 continued). 16.35 g (74.9 mmol)
Boc2O, dissolved in 150 mL anhydrous CH2Cl2, were
added slowly (3 h) to a solution of 14.08 g (234 mmol) 4
in 150 mL anhydrous CH2Cl2. The mixture was stirred
21 h at rt and filtered by suction. The solid filter cake
was washed with 50 mL CH2Cl2 three times and the
combined organic layers were evaporated to dryness.
The residue was fractionated in vacuo, using a short
vigreux-column, to give 5 as a pale yellow oil. Yield:
9.01 g (56.2 mmol), 75%. Bp (30 mbar): 102–105 ꢂC[lit.:
5.91 (s, broad, 1H, NH), 3.32–3.27 (m, 2H, CH2), 2.87–
2.80 (m, 2H, CH2). 13CNMR (75.5 MHz, DMSO- d6): d
(ppm): 155.67, 140.41, 137.27, 120.19, 83.86, 37.26.
N-(2-Amino-ethyl)-N0-(4-methoxy-phenyl)-urea
hydro-
chloride 7d. Yield: 83%. Mp 185 ꢂC(decomposition).
1H NMR (200 MHz, DMSO-d6, TMS intern): d (ppm):
8.84 (s, 1H, NH), 8.04 (s, broad, 3H, NH+3 ), 7.33–7.26
(m, 2H, HAryl), 6.84–6.78 (m, 2H, HAryl), 6.15 (s, broad,
3
1H, NH), 3.68 (s, 3H, CH3), 3.31 (t, J=5.7 Hz, 2H,
CH2), 2.86 (q, 3J=5.8 Hz, 2H, CH2). 13CNMR
105 ꢂC(20 Torr) 61]. H NMR (300 MHz, CDCl3, TMS
(75.5 MHz, DMSO-d6): d (ppm):. 156.22, 154.20,
133.68, 119.74, 114.05, 55.29, 39.52, 27.35.
1
intern): d (ppm): 4.90 (broad, s, 1H, NH), 3.16 (q,
3
3J=5.9 Hz, 2H, CH2), 2.81 (t, J=5.9 Hz, 2H, CH2),
1.45 (s, 9H, 3 CH3), 1.21 (s, 2H, NH2). 13CNMR
(75.5 MHz, CDCl3, TMS intern): d (ppm): 156.27,
79.20, 43.56, 41.96, 28.31.
N-(2-Amino-ethyl)-N0-(4-benzyloxy-phenyl)-urea hydro-
chloride 7e. Yield: 98%. Mp 205 ꢂC. 1H NMR
(300 MHz, DMSO-d6): d (ppm): 8.83 (s, 1H, NH), 8.05
(s, broad, 3H, NH3+), 7.44–7.28 (m, 7H, HAryl), 6.91–
3
6.87 (m, 2H, HAryl), 6.59 (t, J=5.9 Hz, 1H, NH), 5.03
Synthesis of urea hydrochloride derivatives 7a–g: general
procedure (Scheme 2 continued)
(s, 2H, CH2), 3.32 (t, 3J=6.1 Hz, 2H, CH2), 2.87 (t,
3J=6.2 Hz, 2H, CH2). 13CNMR (75.5 MHz, DMSO-
d6): d (ppm): 156.38, 153.47, 137.73, 134.10, 128.71,
128.05, 127.95, 119.90, 115.30, 69.84, 39.78, 37.58.
5 was dissolved in anhydrous diethylether or THF
(approximately 1.7 mL per mmol 5). At 0 ꢂCan equi-
molar amount of 6a–g was added within 30 min and the
mixture was stirred for further 30 min at 0 ꢂC.
N-(2-Amino-ethyl)-N0-(4-methyl-phenyl)-urea hydrochlo-
ride 7f. Yield: 88%. Mp 213 ꢂC(decomposition). 1H
NMR (200 MHz, DMSO-d6, TMS intern): d (ppm): 8.98
(s, 1H, NH), 8.08 (s, broad, 3H, NH+3 ), 7.29 (d,
3J=8.1 Hz, 2H, HAryl), 7.03–6.99 (m, 2H, HAryl), 6.69
(s, broad, 1H, NH), 3.37–3.28 (m, 2H, CH2), 2.95–2.83
(m, 2H, CH2), 2.20 (s, 3H, CH3). 13CNMR (75.5 MHz,
DMSO-d6): d (ppm): 156.26, 138.16, 130.26, 129.33,
118.31, 37.54, 20.64.
It was filtered, the filter cake was washed with diethy-
lether and dried in vacuo to provide the raw N-aryl/
benzyl - N0 - [2 - [(tert - butoxycarbonyl)amino]ethyl]-urea
derivatives as colorless solids.
These compounds were dissolved in a concd HCl/MeOH
mixture (1:1 v/v, approximately 720 mL per mmol). The
solvent was evaporated at 45–50 ꢂCin vacuo (99–53mbar)
within 2.5 h. The oily residues were treated with anhy-
drous acetone (approximately 2.2 mL per mmol) and
the solvent was removed in vacuo. This procedure was
repeated four times to provide 7a–g as colorless solids.
N-(2-Amino-ethyl)-N0-benzyl-urea hydrochloride 7g.
ꢂ
1
Yield: 83%. Mp 160 C. H NMR (300 MHz, DMSO-
d6): d (ppm): 8.17 (s, broad, 3H, NH+3 ), 7.75 (s, 1H,
NH), 7.30–7.18 (m, 5H, HAryl), 4.19 (s, 2H, CH2), 3.25
(t, 3J=6.3 Hz, 2H, CH2), 2.80 (q, 3J=6.0 Hz, 2H, CH2).
13CNMR (75.5 MHz, DMSO- d6): d (ppm): 158.79,
140.81, 128.41, 127.25, 126.73, 43.16, 39.75, 37.69.
N-(2-Amino-ethyl)-N0-phenyl-urea hydrochloride 7a.
Yield: 84%. Mp 189 ꢂC(lit.: 190–191 ꢂC62). H NMR
1
(200 MHz, DMSO-d6, TMS intern): d (ppm): 9.09 (s,
1H, NH), 8.06 (s, broad, 3H, NH+3 ), 7.41 (dd,
Synthesis of the N-aryl-N0-[2-[3-aryloxy-2-hydroxy-pro-
pylamino]-ethyl]-urea and chain-elongated derivatives
8a–s: general procedure (Scheme 2 continued)
4
3
3J=8.6 Hz, J=1.0 Hz, 2H, HAryl), 7.21 (t, J=7.9 Hz,
2H, HAryl) 6.92–6.84 (m, 1H, HAryl), 6.73 (t, 3J=5.7 Hz,
3
1H, NH), 3.33 (t, J=6.0 Hz, 2H, CH2), 2.92–2.86 (m,
2H, CH2). 13CNMR (75.5 MHz, DMSO- d6): d (ppm):
156.20, 140.75, 128.92, 121.50, 118.14, 37.49, 33.32.
1.00 mmol 3a–h, an equimolar amount of 7a–g and 1.05
equivalents of 10 N NaOH were heated in approxi-
mately 3.0 mL n-propanol and 0.5 mL water up to 90 ꢂC
for 30 min.
N-(2-Amino-ethyl)-N0-(4-bromo-phenyl)-urea hydrochlo-
ride 7b. Yield: 51%. Mp 234 ꢂC(decomposition). 1H
NMR (300 MHz, DMSO-d6): d (ppm): 9.29 (s, 1H,
NH), 8.02 (s, broad, 3H, NH+3 ), 7.42–7.34 (m, 4H,
As much water was added at rt until the mixture gets a
slight turbidity. The suspension was cooled down to

3522
K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
+4 ꢂCfor about 20 h, the product was filtered off,
washed with water, thereafter with diethylether and
dried in vacuo. Compounds 8a–s were isolated as col-
orless solids, in some cases as hemihydrates (8e, 8h, 8l
and 8n).
3J=5.4 Hz, 1H, NH), 4.83 (s, broad, 1H, OH), 4.05–
3.88 (m, 3H, CH2CH), 3.68 (s, 3H, CH3), 3.14 (q,
3J=5.9 Hz, 2H, CH2), 2.80–2.55 (m, 4H, 2 CH2). 13C
NMR (75.5 MHz, DMSO-d6): d (ppm): 155.92, 155.28,
154.24, 134.15, 133.27, 129.30, 122.32, 119.71, 114.30,
111.51, 71.96, 68.39, 55.52, 52.63, 49.76. MS (EI): m/z
(intensity%): 439 (Mꢃ+, 2), 437 (Mꢃ+, 2), 260 (88), 258
(91), 149 (41), 123 (100), 108 (69). Anal.
N-[2-(3-Phenoxy - 2 - hydroxy - propylamino) - ethyl]-N0-
phenyl-urea 8a. Yield: 30%. Mp 136 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d (ppm): 8.51 (s, 1H, NH), 7.37
.
(C19H24BrN3O4 0.5H2O) C, H, N.
3
4
(dd, J=8.6 Hz, J=1.0 Hz, 2H, HAryl), 7.27–7.17 (m,
4H, HAryl), 6.93–6.84 (m, 4H, HAryl), 6.14 (t, 3J=5.4 Hz,
1H, NH), 4.93 (s, 1H, OH), 3.96–3.14 (m, 5H, CH2CH
and CH2), 2.71–2.57 (m, 4H, 2 CH2), 1.85 (s, broad, 1H,
NH). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):
157.05, 153.65, 138.96, 127.77, 126.94, 118.77, 119.23,
115.92, 112.85, 69.02, 66.55, 53.97, 50.64. Anal.
(C18H23N3O3) C , H, N.
N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propylamino]-ethyl]-
N0-phenyl-urea 8f. Yield: 62%. Mp 158–159 ꢂC. 1H
NMR (400 MHz, DMSO-d6): d (ppm): 8.51 (s, 1H,
NH), 7.74 (dd, 3J=8.2 Hz, 4J=1.8 Hz, 1H, HAryl), 7.38–
7.36 (m, 2H, HAryl), 7.34–7.29 (m, 1H, HAryl), 7.19 (t,
3J=8.0 Hz, 2H, HAryl), 7.00 (dd, 3J=8.4 Hz,
3
4J=1.0 Hz, 1H, HAryl), 6.86 (t, J=7.4 Hz, 1H, HAryl),
6.72 (dt, 3J=7.5 Hz, 4J=1.0 Hz, 1H, HAryl), 6.14 (t,
3J=5.6 Hz, 1H, NH), 4.92 (s, 1H, OH), 4.00–3.15 (m,
5H, CH2CHCH2), 2.83–2.63 (m, 4H, CH2CH2), 2.10 (s,
1H, NH). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):
156.45, 154.61, 139.93, 138.20, 129.01, 127.91, 120.20,
116.96, 111.99, 85.91, 70.82, 67.29, 51.65, 48.62, 38.60.
Anal. (C18H22IN3O3) C , H, N.
N-(4-Bromo-phenyl)-N0-[2-(3-phenoxy-2-hydroxy-propy-
lamino)-ethyl]-urea 8b. Yield: 42%. Mp 157 ꢂC. 1H
NMR (400 MHz, DMSO-d6): d (ppm): 8.68 (s, 1H,
NH), 7.36–7.23 (m, 6H, HAryl), 6.93–6.88 (m, 3H,
3
HAryl), 6.19 (t, J=5.4 Hz, 1H, NH), 4.93 (s, 1H, OH),
3.97–3.13 (m, 5H, CH2CH and CH2), 2.71–2.56 (m, 4H,
2
C
H), 1.81 (s, broad, 1H, NH). 13CNMR
2
(100.6 MHz, DMSO-d6): d (ppm): 160.48, 156.88,
141.84, 133.10, 131.14, 122.22, 121.26, 116.28, 113.93,
72.45, 69.99, 54.06, 51.02, 40.73. Anal. (C18H22BrN3O3)
C , H, N.
N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propylamino]-ethyl]-
N0-(4-iodo-phenyl)-urea 8g. Yield: 61%. Mp 163 ꢂC. H
1
NMR (400 MHz, DMSO-d6): d (ppm): 8.66 (s, 1H,
NH), 7.74 (dd, 3J=7.6 Hz, 4J=1.6 Hz, 1H, HAryl), 7.52–
3
4
7.21 (m, 5H, HAryl), 6.99 (dd, J=8.4 Hz, J=1.2 Hz,
1H, HAryl), 6.74–6.69 (m, 1H, HAryl), 6.19 (t, 3J=5.4 Hz,
1H, NH), 4.93 (s, 1H, OH), 4.00–3.14 (m, 5H, CH2CH
and CH2), 2.82–2.64 (m, 4H, 2 CH2), 2.00 (s, broad, 1H,
NH). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):
158.56, 156.48, 141.97, 140.34, 138.56, 131.15, 124.05,
121.32, 114.01, 88.07, 84.77, 72.92, 69.40, 53.74, 50.63,
40.72. Anal. (C18H21I2N3O3) C , H, N.
N-[2-[3- (2-Bromo- phenoxy) -2-hydroxy -propylamino]-
ethyl]-N0-phenyl-urea 8c. Yield: 41%. Mp 158 ꢂC. 1H
NMR (400 MHz, DMSO-d6): d (ppm): 8.51 (s, 1H,
NH), 7.54 (dd, 3J=7.8 Hz, 4J=1.8 Hz, 1H, HAryl), 7.38–
7.28 (m, 3H, HAryl), 7.19 (t, 3J=8.0 Hz, 2H, HAryl), 7.10
3
4
(dd, J=8.2 Hz, J=1.4 Hz, 1H, HAryl), 6.88–6.84 (m,
3
2H, HAryl), 6.15 (t, J=5.4 Hz, 1H, NH), 4.95 (s, 1H,
OH), 4.01–3.14 (m, 5H, CH2CH and CH2), 2.79–2.62
(m, 4H, 2 CH2), 1.73 (s, broad, 1H, NH). 13CNMR
(100.6 MHz, DMSO-d6): d (ppm): 156.03, 155.62,
141.35, 133.64, 129.67, 129.32, 122.69, 122.53, 121.61,
118.43, 111.85, 72.29, 68.74, 52.98, 50.03, 40.00. Anal.
(C18H22BrN3O3) C , H, N.
N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propylamino]-ethyl]-
N0-(4-methoxy-phenyl)-urea 8h. Yield: 53%. Mp 148 ꢂC.
1H NMR (300 MHz, DMSO-d6): d (ppm): 8.29 (s, 1H,
NH), 7.69 (dd, 3J=7.7 Hz, 4J=1.7 Hz, 1H, HAryl), 7.65–
7.59 (m, 1H, HAryl), 7.30–7.24 (m, 3H, HAryl), 7.07 (dt,
4
3J=7.4 Hz, J=0.7 Hz, 1H, HAryl), 6.82–6.77 (m, 2H,
3
N-[2-[3-(2-Bromo - phenoxy) - 2 - hydroxy - propylamino] -
ethyl]-N0 -(4-bromo-phenyl)-urea 8d. Yield: 30%. Mp
HAryl), 6.03 (t, J=5.4 Hz, 1H, NH), 5.01 (s, 1H, OH),
4.19–3.92 (m, 3H, CH2CH), 3.68 (s, 3H, CH3), 3.18–
3.11 (m, 2H, CH2), 2.76–2.55 (m, 4H, 2 CH2), 1.92 (s,
broad, 1H, NH). 13CNMR (75.5 MHz, DMSO- d6): d
(ppm): 157.51, 155.94, 154.24, 139.27, 134.15, 130.08,
122.87, 119.72, 114.25, 113.16, 87.00, 71.90, 68.35,
55.53, 52.70, 49.77. MS (EI): m/z (intensity%): 306 (84),
159 ꢂC. H NMR (400 MHz, DMSO-d6): d (ppm): 8.68
1
(s, 1H, NH), 7.54 (dd, 3J=7.8 Hz, 4J=1.8 Hz, 1H,
3
HAryl), 7.35–7.28 (m, 5H, HAryl), 7.10 (dd, J=8.2 Hz,
4J=1.4 Hz, 1H, HAryl), 6.88–6.86 (m, 1H, HAryl), 6.19 (t,
3J=5.4 Hz, 1H, NH), 4.94 (s, 1H, OH), 4.00–3.15 (m,
5H, CH2CH and CH2), 2.75–2.62 (m, 4H, 2 CH2), 1.86
(s, broad, 1H, NH). 13CNMR (100.6 MHz, DMSO- d6):
d (ppm): 155.45, 155.25, 140.41, 133.27, 131.67, 129.30,
122.32, 119.83, 114.27, 112.50, 111.48, 71.91, 68.37,
52.59, 49.56, 39.64. Anal. (C18H21Br2N3O3) C , H, N.
149
(98),
123
(100),
(C19H24IN3O4 0.5H2O) C, H, N.
108
(86).
Anal.
.
N-[2-[3-(2-Cyano - phenoxy) - 2 - hydroxy - propylamino] -
ethyl] - N0 - phenyl-urea 8i. Yield: 41%. Mp 154 ꢂC. H
1
NMR (200 MHz, DMSO-d6): d (ppm): 8.52 (s, 1H,
NH), 7.67 (dd, 3J=7.7 Hz, 4J=1.7 Hz, 1H, HAryl), 7.62–
7.57 (m, 1H, HAryl), 7.39–7.15 (m, 5H, HAryl), 7.06 (t,
N-[2-[3-(2-Bromo-phenoxy) - 2 - hydroxy - propylamino] -
ethyl]-N0 -(4-methoxy-phenyl)-urea 8e. Yield: 43%. Mp
140 ꢂC. H NMR (300 MHz, DMSO-d6): d (ppm): 8.31
3J=7.2 Hz, 1H, HAryl), 6.86 (t, J=7.2 Hz, 1H, HAryl),
1
3
3
(s, 1H, NH), 7.69 (dd, 3J=7.7 Hz, 4J=1.7 Hz, 1H,
6.16 (t, J=5.5 Hz, 1H, NH), 5.05 (s, 1H, OH), 4.15–
3.11 (m, 5H, CH2CH and CH2), 2.78–2.59 (m, 4H, 2
3
HAryl), 7.34–7.25 (m, 3H, HAryl), 7.11 (dd, J=8.3 Hz,
4J=1.4 Hz, 1H, HAryl), 6.90–6.78 (m, 3H, HAryl), 6.05 (t,
CH2). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3523
160.27, 155.21, 140.52, 134.91, 133.55, 128.49, 120.89,
117.47, 116.35, 113.10, 100.56, 71.50, 67.81, 51.86,
49.08. Anal. (C19H22N4O3) C , H, N.
121.35, 119.87, 116.79, 114.24, 113.57, 101.05, 71.99,
68.32, 55.52, 52.34, 49.74. MS (EI): m/z (intensity%):
205 (45), 149 (45), 123 (100) 108 (100). Anal.
(C20H24N4O4) C , H, N.
N-[2-[3-(2-Cyano-phenoxy) - 2 - hydroxy - propylamino] -
ethyl] - N0 - (4 - methyl -phenyl)-urea 8j. Yield: 63%. Mp
N-(4-Benzyloxy-phenyl)-N0 -[2-[3-(2-cyano-phenoxy)-2-
hydroxy-propylamino]-ethyl]-urea 8n. Yield: 73%. Mp
149 ꢂC. H NMR (300 MHz, DMSO-d6): d (ppm): 8.35
1
(s, 1H, NH), 7.69 (dd, 3J=7.7 Hz, 4J=1.4 Hz, 1H,
105 ꢂC. H NMR (300 MHz, DMSO-d6): d (ppm): 8.32
1
3
HAryl), 7.64–7.58 (m, 1H, HAryl), 7.24 (d, J=8.4 Hz,
3
(s, 1H, NH), 7.70 (dd, 3J=7.7 Hz, 4J=1.6 Hz, 1H,
HAryl), 7.64–7.59 (m, 1H, HAryl), 7.43–7.24 (m, 8H,
HAryl), 7.06 (t, 3J=7.4 Hz, 1H, HAryl), 6.87 (d,
4
3H, HAryl), 7.06 (dt, J=7.6 Hz, J=0.8 Hz, 1H, HAryl),
3
3
6.99 (d, J=8.1 Hz, 2H, HAryl), 6.06 (t, J=5.4 Hz, 1H,
3
3
NH), 4.99 (d, J=4.8 Hz, 1H, OH), 4.16–3.89 (m, 3H,
3
3J=9.4 Hz, 2H, HAryl), 6.04 (t, J=5.4 Hz, 1H, NH),
CH2CH), 3.14 (q, J=5.8 Hz, 2H, CH2), 2.75–2.60 (m,
5.05–5.02 (m, 3H, OH and CH2), 4.16–3.12 (m, 5H,
CH2CH and CH2), 2.75–2.61 (m, 4H, 2 CH2). 13C
NMR (75.5 MHz, DMSO-d6): d (ppm): 160.74, 155.90,
153.28, 137.77, 135.35, 134.37, 133.99, 128.71, 128.04,
127.94, 121.35, 119.67, 116.80, 115.33, 113.58, 101.05,
4H, 2 CH2), 2.19 (s, 3H, CH3), 1.80 (s, broad, 1H, NH).
13CNMR (75.5 MHz, DMSO- d6): d (ppm): 160.74,
155.75, 138.41, 135.33, 133.99, 129.96, 129.33, 121.33,
118.21, 116.78, 113.58, 112.50, 101.07, 71.98, 68.32,
52.32, 49.56. Anal. (C20H24N4O3) C , H, N.
71.99,
(C26H28N4O4 0.5H2O) C, H, N.
69.84,
.
68.32,
52.34,
49.73.
Anal.
N-(4-Bromo - phenyl) - N0 - [2 - [3 - (2 - cyano - phenoxy) - 2 -
hydroxy-propylamino]-ethyl]-urea 8k. Yield: 58%. Mp
N-[2-[3-(2-Allyl-phenoxy)-2-hydroxy-propylamino]-ethyl]
-N0 -phenyl-urea 8o. Yield: 42%. Mp 143 ꢂC. H NMR
148 ꢂC. H NMR (300 MHz, DMSO-d6): d (ppm): 8.65
1
1
(s, 1H, NH), 7.69 (dd, 3J=7.7 Hz, 4J=2.0 Hz, 1H,
HAryl), 7.64–7.58 (m, 1H, HAryl), 7.38–7.35 (m, 4H,
HAryl), 7.24 (d, 3J=8.4 Hz, 1H, HAryl), 7.06 (dt,
(400 MHz, DMSO-d6): d (ppm): 8.51 (s, 1H, NH), 7.37
(dd, J=8.6 Hz, J=1.0 Hz, 2H, HAryl), 7.21–7.08 (m,
3
4
3
4H, HAryl), 6.93 (d, J=8.4 Hz, 1H, HAryl), 6.88–6.83
3
4
3
3J=7.6 Hz, J=1.0 Hz, 1H, HAryl), 6.17 (t, J=5.4 Hz,
(m, 2H, HAryl), 6.14 (t, J=5.5 Hz, 1H, NH), 5.98–5.92
3
1H, NH), 4.99 (d, J=4.8 Hz, 1H, OH), 4.16–3.89 (m,
3
(m, 1H, ¼CH), 5.05–4.97 (m, 2H, ¼CH2), 4.90 (s, 1H,
OH), 3.94–3.14 (m, 7H, CH2CH and 2 CH2), 2.72–2.48
(m, 4H, 2 CH2), 2.00 (s, broad, 1H, NH). 13CNMR
(100.6 MHz, DMSO-d6): d (ppm): 156.15, 155.22,
140.54, 137.01, 129.38, 128.51, 127.97, 127.30, 120.80,
120.24, 117.49, 115.31, 111.56, 70.59, 68.16, 52.25,
49.25, 33.86. Anal. (C21H27N3O3) C , H, N.
3H, CH2CH), 3.15 (q, J=5.9 Hz, 2H, CH2), 2.75–2.60
(m, 4H, 2 CH2), 1.84 (s, broad, 1H, NH). 13CNMR
(75.5 MHz, DMSO-d6): d (ppm): 160.73, 155.47, 140.42,
135.32, 131.65, 121.33, 119.85, 116.78, 113.56, 112.50,
101.06, 138.41, 135.33, 133.99, 129.96, 121.33, 118.21,
116.78, 113.58, 101.07, 72.00, 68.33, 52.36, 49.71. Anal.
(C19H21BrN4O3) C , H, N.
N-[2-[3-(2-Methoxy-phenoxy)-2-hydroxy-propylamino]-
0
ꢂ
1
N-[2-[3-(2-Cyano - phenoxy) - 2 - hydroxy - propylamino] -
ethyl] - N0 - (4 - iodo - phenyl)-urea 8l. Yield: 90%. Mp
150 ꢂC(decomposition). 1H NMR (300 MHz, DMSO-
ethyl]-N -phenyl-urea 8p. Yield: 48%. Mp 143 C. H
NMR (400 MHz, DMSO-d6): d (ppm): 8.51 (s, 1H,
NH), 7.37 (d, 3J=7.6 Hz, 2H, HAryl), 7.20 (t,
3J=8.0 Hz, 2H, HAryl), 6.88–6.73 (m, 5H, HAryl), 6.14 (t,
3J=5.6 Hz, 1H, NH), 4.90 (s, 1H, OH), 3.92–3.78 (m,
3
d6): d (ppm): 8.64 (s, 1H, NH), 7.69 (dd, J=7.7 Hz,
4J=1.4 Hz, 1H, HAryl), 7.65–7.59 (m, 1H, HAryl), 7.52–
3
7.48 (m, 2H, HAryl), 7.25–7.21 (m, 2H, HAryl), 7.06 (dt,
3H, CH2CH), 3.67 (s, 3H, CH3), 3.16 (q, J=6.0 Hz,
4
3
3J=7.6 Hz, J=0.8 Hz, 1H, HAryl), 6.17 (t, J=5.4 Hz,
1H, NH), 5.00 (s, broad, 1H, OH), 4.16–3.91 (m, 3H,
CH2CH), 3.22–3.08 (m, 2H, CH2), 2.78–2.60 (m, 4H, 2
CH2), 2.15 (s, broad, 1H, NH). 13CNMR (75.5 MHz,
DMSO-d6): d (ppm): 160.54, 155.24, 140.71, 137.30,
135.14, 133.80, 121.15, 120.08, 116.60, 113.38, 100.87,
83.50, 71.79, 68.13, 52.13, 49.37. MS (EI): m/z (inten-
sity%): 245 (100), 219 (73), 205 (16) 99 (30). Anal.
2H, CH2), 2.72–2.48 (m, 4H, 2 CH2), 2.02 (s, broad, 1H,
NH). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):
154.54, 152.54, 151.99, 139.80, 127.83, 120.12, 116.81,
114.65, 113.81, 70.60, 67.49, 54.58, 51.55, 48.56. Anal.
(C19H25N3O4) C , H, N.
N-[2-[3-(4-Benzyloxy-phenoxy)-2-hydroxy-propylamino]-
ethyl]-N0-phenyl-urea 8q. Yield: 48%. Mp 140 ꢂC. 1H
NMR (400 MHz, DMSO-d6): d (ppm): 8.52 (s, 1H,
.
(C19H21IN4O3 0.5H2O) C, H, N.
3
NH), 7.43–7.30 (m, 7H, HAryl), 7.20 (t, J=8.0 Hz, 2H,
N-[2-[3-(2-Cyano-phenoxy) - 2 - hydroxy - propylamino] -
ethyl]-N0-(4-methoxy-phenyl)-urea 8m. Yield: 65%. Mp
HAryl), 6.91–6.81 (m, 5H, HAryl), 6.15 (t, 3J=5.4 Hz, 1H,
NH), 5.01 (s, 2H, CH2), 4.91 (s, 1H, OH), 3.89–3.14 (m,
5H, CH2CH and CH2), 2.69–2.55 (m, 4H, 2 CH2), 1.67
(s, broad, 1H, NH). 13CNMR (100.6 MHz, DMSO- d6):
d (ppm): 154.57, 152.20, 151.61, 139.89, 136.63, 127.86,
127.62, 126.95, 126.83, 120.14, 116.83, 114.96, 114.65,
70.59, 68.95, 67.50 51.57, 48.58, 38.53. Anal.
(C25H29N3O4) N, H, C: calcd, 68.95; found, 68.43.
137 ꢂC. H NMR (300 MHz, DMSO-d6): d (ppm): 8.31
1
(s, 1H, NH), 7.75 (dd, 3J=7.7 Hz, 4J=1.7 Hz, 1H,
3
HAryl), 7.35–7.24 (m, 3H, HAryl), 7.00 (dd, J=8.3 Hz,
4J=1.4 Hz, 1H, HAryl), 6.79 (dd, 3J=6.8 Hz,
4J=2.3 Hz, 2H, HAryl), 6.73 (dd, 3J=7.6 Hz,
3
4J=1.1 Hz, 1H, HAryl), 6.04 (t, J=5.4 Hz, 1H, NH),
4.91 (s, broad, 1H, OH), 4.01–3.90 (m, 3H, CH2CH),
3.68 (s, 3H, CH3), 3.16 (q, J=5.9 Hz, 2H, CH2), 2.84–
2.62 (m, 4H, 2 CH2). 13CNMR (75.5 MHz, DMSO- d6):
d (ppm): 160.74, 155.94, 154.26, 135.35, 134.10, 133.99,
N-Benzyl-N0-[2-[3-(2-cyano-phenoxy)-2-hyꢂdroxy-propyl-
3
1
amino]-ethyl]-urea 8r. Yield: 32%. Mp 144 C. H NMR
(300 MHz, DMSO-d6): d (ppm): 7.70–7.59 (m, 2H,

3524
K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3
2
3
HAryl), 7.31–7.19 (m, 6H, HAryl), 7.06 (dd, J=7.5 Hz,
3
(s, 1H, OH), 4.11 (dd, J=10.0 Hz, J=6.0 Hz, 1H, 1
3
4J=0.9 Hz, 1H, HAryl), 6.36 (t, J=6.0 Hz, 1H, NH),
CH2), 4.05 (dd, J=10.0 Hz, J=4.6 Hz, 1H, 1 CH2),
2
2
5.91 (t, 3J=5.6Hz, 1H, NH), 4.97 (s, 1H, OH), 4.20–3.90
(m, 5H, CH2CH and CH2), 3.10 (q, 3J=6.0Hz, 2H, CH2),
2.74–2.56 (m, 4H, 2 CH2) 1.86 (s, broad, 1H, NH). 13C
NMR (75.5 MHz, DMSO-d6): d (ppm): 160.77, 158.55,
141.31, 135.33, 133.99, 128.52, 127.37, 125.84, 121.34,
116.77, 113.61, 101.10, 72.03, 68.35, 52.40, 50.04, 43.33.
Anal. (C20H24N4O3) C , H, N: calcd, 15.21; found, 14.66.
3.89–3.86 (m, 1H, CH), 2.99 (q, J=5.9 Hz, 2H, CH2),
2.69–2.54 (m, 4H, 2 CH2), 1.75 (s, broad, 1H, NH), 1.36
(s, 9H, C(CH3)3). 13CNMR (100.6 MHz, DMSO- d6): d
(ppm): 159.24, 154.48, 133.83, 132.49, 119.81, 115.26,
112.04, 99.52, 76.29, 70.44, 66.84, 50.77, 47.98, 38.97,
27.09. Anal. (C17H25N3O4) C , H, N.
Table 2. Elemental analytical data of the target compounds 8a–u
N-Benzyl-N0 -[2-(3-benzyloxy-2-hydroxy-propylamino)-
ethyl]-urea 8s. Yield: 19%. Mp 116 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d (ppm): 7.36–7.20 (m, 10H,
HAryl), 6.39 (t, 3J=6.0 Hz, 1H, NH), 5.91 (t, 3J=5.6 Hz,
1H, NH), 4.67 (s, 1H, OH), 4.47 (s, 2H, CH2) 4.19 (d,
3J=6.0 Hz, 2H, CH2), 3.71–3.35 (m, 5H, CH2CH and
CH2), 2.61–2.45 (m, 4H, 2 CH2) 1.80 (s, broad, 1H,
NH). 13CNMR (100.6 MHz, DMSO- d6): d (ppm):
157.88, 140.68, 138.35, 127.90, 127.88, 127.15, 127.02,
126.71, 126.20, 72.90, 71.98, 68.39, 52.36, 49.37, 42.64,
39.37. Anal. (C20H27N3O3) C , H, N.
No. Formula
Calcd
Found
N
C
H
N
C
H
8a C18H23N3O3
8b C18H22BrN3O3
65.63 7.04 12.76 65.34 6.95 12.66
52.95 5.43 10.29 52.79 5.46 10.12
52.95 5.43 10.29 52.88 5.48 10.25
44.38 4.34 8.63 44.22 4.39 8.48
8c
C18H22BrN3O3
8d C18H21Br2N3O3
.
8e
8f
C19H24BrN3O4 0.5H2O 51.02 5.63 9.39 51.08 5.41 9.28
C18H22IN3O3
47.48 4.87 9.23 47.41 4.93 8.88
37.20 3.64 7.23 37.43 3.77 7.05
8h C19H24IN3O4 0.5H2O 46.17 5.10 8.50 46.31 4.88 8.40
8g C18H21I2N3O3
.
8i
8j
C19H22N4O3
C20H24N4O3
64.39 6.26 15.81 64.02 6.30 15.52
65.20 6.57 15.21 64.80 6.55 15.17
52.67 4.88 12.93 52.49 4.84 12.96
8k C19H21BrN4O3
.
Synthesis of N-[2-[3-(4-hydroxy-phenoxy)-2-hydroxy-
propylamino]-ethyl]-N0-phenyl-urea 8t (Scheme 3).
850 mg (1.95 mmol) 8q were dissolved in 20 mL acetic
acid and 92 mg Pd/C(10%) were added. The mixture
was stirred under a H2-atmosphere for 69 h. The cata-
lyst was filtered off and the filtrate was evaporated to
dryness. 20 mL acetonitrile were added. After stirring
for 20 h at rt 10 mL acetonitrile were added. Then the
mixture was refluxed and decanted from the residual
oily brown solid. This procedure was repeated two
times. The solution was cooled down to À15 ꢂCfor 3 h
and the precipitate was filtered off to provide the acetate
of 8t as a colorless solid. Yield: 172 mg (0.42 mmol),
8l
8m C20H24N4O4
C19H21IN4O3 0.5H2O 46.64 4.53 11.45 46.92 4.48 11.62
62.49 6.29 14.57 62.19 6.34 14.58
66.50 6.23 11.93 66.24 6.06 11.92
68.27 7.37 11.37 67.99 7.37 11.05
63.49 7.01 11.69 63.09 6.96 11.58
68.95 6.71 9.65 68.43 6.64 9.43
65.20 6.57 15.21 64.87 6.48 14.66
67.20 7.61 11.76 67.03 7.54 12.04
59.25 6.71 10.36 59.07 6.81 10.35
60.88 7.51 12.53 60.90 7.66 12.92
.
8n C26H28N4O4 0.5H2O
8o C21H27N3O3
8p C19H25N3O4
8q C25H29N3O4
8r
8s
8t
C20H24N4O3
C20H27N3O3
C20H27N3O6
8u C17H25N3O4
Pharmacological methods
ꢂ
1
22%. Mp 134 C. H NMR (300 MHz, DMSO-d6): d
(ppm): 8.57 (s, 1H, NH), 7.35 (d, 3J=7.8 Hz, 2H, HAryl),
The chemicals were reagent grade.
3
3
7.17 (t, J=8.0 Hz, 2H, HAryl), 6.84 (t, J=7.5 Hz, 1H,
HAryl), 6.71 (d, 3J=9.0 Hz, 2H, HAryl), 6.62 (d,
Tissue preparation
3J=9.0 Hz, 2H, HAryl), 6.24 (t, J=5.1 Hz, 1H, NH),
The biological profile of the compounds at the b1- and
b2-ARs listed in Table 1 was assessed on ventricular
membrane preparations of wild-type DAB mice.
Microsomes were prepared by homogenizing ventricles
at 4 ꢂCfor 90 s in 1 mL of a buffer A containing 10 mM
EDTA, 10 mM HEPES, 0.1 mM benzamidine (pH 7.4),
using a Polytron PT 3000 (Kinematica, Lucerne, Swit-
zerland). Homogenates were centrifuged at 45,000Âgmax
for 15 min at 4 ꢂC. The pellets were again resuspended in
1 mL of a buffer B containing 1 mM EDTA, 10 mM
HEPES, 0.1 mM benzamidine (pH 7.4) and recen-
trifuged at 45,000Âgmax for 15 min at 4 ꢂC. The pellets
were resuspended in 1 mL of buffer B and centrifuged at
10,000Âgmax for 10 min at 4 ꢂC. The supernatants were
recentrifuged at 45,000Âgmax for 15 min at 4 ꢂC. The
pellets, partially enriched membranes, were resuspended
in 50 mM Tris–HCl, 5 mM MgCl2 (pH 7.4), and stored
frozen at À80 ꢂC. Membranes were used for the
measurement of b-AR density.
3
5.00 (s, broad, 3H, OH and NH+2 ), 3.82–3.12 (m, 5H,
CH2CH and CH2), 2.68–2.54 (m, 4H, 2 CH2), 1.86 (s,
3H, CH3). 13CNMR (75.5 MHz, DMSO- d6): d (ppm):
172.63, 155.73, 151.89, 151.55, 141.01, 128.94, 121.23,
117.97, 116.06, 115.85, 71.72, 68.37, 52.54, 49.56, 21.73.
MALDI-TOF: 345 (M+H)+, 368 (M+Na)+, 384
(M+K)+. Anal. (C20H27N3O6) C , H, N.
Synthesis of N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-pro-
pylamino]-ethyl]-carbamic acid tert-butyl ester 8u
(Scheme 4). 187 mg (1.07 mmol) 3d and 171 mg
(1.07 mmol) 5 were heated in 1.5 mL n-propanol over
10 min at 100 ꢂC. The solvent was removed in vacuo and
the residue became completely solid after 1 day at rt. It
was extracted with 10 mL hot diethylether, the organic
layer was cooled down to +4 ꢂC(1 h), the resulting
solid was filtered off, washed with n-pentane and dried.
The isolated 8u was a colorless solid. Yield: 147 mg
(0.44 mmol), 41%. Mp 55 ꢂC. 1H NMR (400 MHz,
3
4
DMSO-d6): d (ppm): 7.69 (dd, J=7.6 Hz, J=1.6 Hz,
1H, HAryl), 7.63 (ddd, 3J=8.6 Hz, 3J=7.3 Hz,
[
125I]ICYP binding experiments
4J=1.5 Hz, 1H, HAryl), 7.24 (d, J=8.4 Hz, 1H, HAryl),
7.09–7.05 (m, 1H, HAryl), 6.67 (s, broad, 1H, NH), 4.98
The b-AR antagonist [125I]ICYP represents the most
useful ligand for labelling b-ARs on cell membrane
3

K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
3525
preparations because it is characterized by very high
B ¼ Bmax1=ð1 þ I=IC1Þ þ Bmax2=ð1 þ I=IC2Þ
affinity, which results in low non-specific binding at the
dissociation constant (KD) concentration range, and by
radioiodination, requiring only small tissue amounts to
be added to the assay.63 Furthermore, [125I]ICYP is
considered as nonselective on the basis of linear
Scatchard plots derived from saturation binding experi-
ments on tissues containing a mixed population of b-
ARs.64,65
where B is the amount of radioligand bound, Bmax1 and
Bmax2 are the total numbers of binding sites labelled by
this concentration of radioligand, I is the free concen-
tration of the competing ligand, and IC1 and IC2 are the
concentrations of the competing ligand that inhibit 50%
of the binding of each subtype. Analysis of the compe-
tition curves by nonlinear regression analysis with the
XMGRACE program (Linux Software) yielded the
high- and low-affinity IC50 values of ICI 89,406 8i and
its derivatives.67 In addition, all inhibition data were
fitted to the equation describing the binding of a non-
selective b-AR antagonist with a single binding site:
B ¼ Bmax=ð1 þ I=ICÞ. The F-test was performed in order
to test for a significant improvement of the two-site fit
as compared to the one-site fit. If the two-site model was
rejected the one-site model was applied and the b1-
selectivity was set to 1.0.
First, the protein dependence of [125I]ICYP binding to
mouse ventricular membrane preparations was investi-
gated. [125I]ICYP binding was linear up to a protein
amount of 50 mg in a total volume of 200 mL. Non-
specific binding was simultaneously determined in a
parallel experiment using the same membrane and
ligand conditions in the presence of 20 mM alprenolol. A
protein amount of 15 mg in 200 mL was subsequently
chosen for kinetic studies.
Subsequently, association kinetics of
[
125I]ICYP
binding to b-ARs were investigated. The association
kinetics were linear for approximately 15 min and an
equilibrium was reached after 60 min. Therefore, assays
were carried out for 60 min to calculate kinetic para-
meters.
The IC50 values were converted into the high- and
low-affinity inhibition constants (KI1 and KI2) by
the method of Cheng–Prusoff34 using the equation
Ki ¼ IC50=ð1 þ S=KdÞ, where S is the concentration
of [125I]ICYP, and KD is the dissociation constant
of b-ARs for [125I]ICYP determined independently
by Scatchard analysis.66 The ratios between KI2 and
KI1 represent the b1-selectivities of the compounds
tested.
Total b-AR density (Bmax) and the dissociation constant
(KD) of b-ARs were determined by incubating 15 mg of
membrane protein with increasing concentrations of
125
.
[
I]ICYP (1–300 pM) in a buffer of 10 mM Tris HCl,
154 mM NaCl, 0.1 mM ascorbic acid (pH 7.4). The
nonspecific binding was measured in the presence of
20 mM alprenolol (Fig. 2a). Binding reactions (200 mL)
were incubated at 37 ꢂCfor 1 h prior to vacuum fil-
tration onto Whatman GF/B filters. Subsequently,
membrane bound [125I]ICYP was determined in a
gamma-counter. The maximum number of binding sites
(Bmax) and the dissociation constants (KD) were calcu-
lated from plots according to the method of Scatchard
(Fig. 2b).66
Statistics
The experimental data given in the text, Figures 2 and 3,
and Table 1 are meansÆSEM. Statistical analysis was
performed using the Student’s t-test. P<0.05 was con-
sidered as denoting statistical significance. The dis-
sociation constants (KD) and the maximum number of
binding sites (Bmax) were calculated from plots accord-
ing to Scatchard.66 Analysis of the inhibition of
[
125I]ICYP binding by ICI 89,406 8i and its derivatives
was performed by nonlinear regression analysis, as
described by Engel et al.64 Statistical analysis of the
competition experiments was performed using the F-
ratio test to measure the goodness of fit of the competi-
tion curves for either one or two binding sites. The IC50
values were converted to KI values according to the
method of Cheng and Prusoff.34
For competition binding studies, 15 mg of membranes
were incubated with [125I]ICYP (c=80 pM) and with 24
concentrations of ICI 89,406 8i and its derivatives
(1 pM–100 mM), respectively. Reactions were conducted
at 37 ꢂCfor 60 min. Samples were filtered onto Whatman
GF/B filters, washed three times, and the membrane
bound activity was determined in a gamma-scintillation
counter. This screening method was applied for the
indirect determination of the half maximum inhibitory
concentrations (IC50) of the selective b1-AR antagonists
as compared to a nonselective b-AR radioligand. Sub-
sequently, the obtained IC50 values were converted into
the high- and low-affinity inhibition constants KI1 and
KI2, respectively.
Acknowledgements
This work was supported by the Jung-Stiftung fur Wis-
senschaft und Forschung, Hamburg, Germany and The
Royal Society, London, UK as well as by the SFB 556
(grants C1 and C4) ‘Heart Failure and Arrhythmias’ of
the Deutsche Forschungsgemeinschaft (DFG), Bonn,
Germany. The authors express their gratitude to Mr. G.
Goder from the Institute of Medical Informatics and
Biometrics, University of Munster for support with the
statistical analysis. Additionally the authors gratefully
thank Mrs Monika Trub and Mrs Sandra Schroer for
technical assistance.
Calculation of Ki values
Each competition binding curve was analyzed accord-
ing to the following equation describing the interaction
of a selective competitor with two classes of binding
sites:

3526
K. Kopka et al. / Bioorg. Med. Chem. 11 (2003) 3513–3527
27. Majid, P. A.; Schreuder, J. E.; de Feyter, P. J.; Roos, J. P.
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