Synthesis and first in vivo evaluation of new selective high affinity β1-adrenoceptor radioligands for SPECT based on ICI 89,406

Article abstract of DOI:10.1016/j.bmc.2004.05.027

The results of cardiac biopsies suggest that myocardial β₁-adrenoceptor (AR) density is reduced in patients with chronic heart failure, while changes in cardiac β₂-ARs vary. A technique for visualization and quantification of β₁-AR populations rather than total β-AR densities in the human heart would be of great clinical interest. Molecular imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET), with appropriate radiopharmaceuticals offer the possibility to assess β-AR density noninvasively in humans, but to date, neither a SPECT nor a PET-radioligand is clinically established for the selective imaging of cardiac β₁-ARs. The aim of this study was to design a high affinity selective β₁-AR radioligand for the noninvasive in vivo imaging of cardiac β₁-AR density in man using SPECT. Based on the well-known selective β₁-AR antagonist, ICI 89,406, both the racemic iodinated target compound 11a and the (S)-enantiomer 15a were synthesized. Competition studies using the nonselective AR ligand, [ ¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP), and ventricular membrane preparations from mice showed that 11a and 15a possess higher β₁-AR affinities (up to 265-fold) and β₁-AR selectivities (up to 245-fold) than ICI 89,406. Encouraged by these results, the radioiodinated counterparts of racemic 11a (11b: ¹²⁵I, 11c: ¹²³I) and (S)-configurated 15a (15b: ¹²⁵I, 15c: ¹²³I) were synthesized. The target compounds were evaluated in rats. Biodistribution and metabolism studies in rats indicated that there is a specific heart uptake of 11b-c and especially 15b-c accompanied by rapid metabolism of the radioligands. Therefore, radioiodinated 11c and 15c appeared to be unpromising SPECT-radioligands for assessing β₁-ARs in vivo in the rat. However, the rat may metabolize β-AR ligands more rapidly than other species as demonstrated for (S)-[¹¹C]CGP 12177, a radioligand structurally related to 11a-c and 15a-c. Therefore further studies in a different animal model will be carried out.

Full text of DOI:10.1016/j.bmc.2004.05.027

Bioorganic & Medicinal Chemistry 12 (2004) 4117–4132
Synthesis and first in vivo evaluation of new selective high affinity
b1-adrenoceptor radioligands for SPECT based on ICI 89,406
Stefan Wagner,a,*,ꢀ Klaus Kopka,a,ꢀ Marilyn P. Law,a Burkhard Riemann,a
b
Victor W. Pike, Otmar Schober and Michael Schafers
a
a
€
aDepartment of Nuclear Medicine, Albert-Schweitzer-Str. 33, University Hospital M€unster, 48149 M€unster, Germany
bMolecular Imaging Branch, National Institute of Mental Health, National Institutes of Health,
10 Center Drive, Rm B3 C346A, MSC 1003, Bethesda, MD 20892-1003, USA
Received 11 November 2003; accepted 21 May 2004
Abstract—The results of cardiac biopsies suggest that myocardial b1-adrenoceptor (AR) density is reduced in patients with chronic
heart failure, while changes in cardiac b2-ARs vary. A technique for visualization and quantification of b1-AR populations rather
than total b-AR densities in the human heart would be of great clinical interest. Molecular imaging techniques, either single photon
emission computed tomography (SPECT) or positron emission tomography (PET), with appropriate radiopharmaceuticals offer the
possibility to assess b-AR density noninvasively in humans, but to date, neither a SPECT nor a PET-radioligand is clinically
established for the selective imaging of cardiac b1-ARs. The aim of this study was to design a high affinity selective b1-AR radi-
oligand for the noninvasive in vivo imaging of cardiac b1-AR density in man using SPECT. Based on the well-known selective b1-
AR antagonist, ICI 89,406, both the racemic iodinated target compound 11a and the (S)-enantiomer 15a were synthesized.
Competition studies using the nonselective AR ligand, [125I]iodocyanopindolol ([125I]ICYP), and ventricular membrane preparations
from mice showed that 11a and 15a possess higher b1-AR affinities (up to 265-fold) and b1-AR selectivities (up to 245-fold) than ICI
89,406. Encouraged by these results, the radioiodinated counterparts of racemic 11a (11b: 125I, 11c: 123I) and (S)-configurated 15a
(15b: 125I, 15c: 123I) were synthesized. The target compounds were evaluated in rats. Biodistribution and metabolism studies in rats
indicated that there is a specific heart uptake of 11b–c and especially 15b–c accompanied by rapid metabolism of the radioligands.
Therefore, radioiodinated 11c and 15c appeared to be unpromising SPECT-radioligands for assessing b1-ARs in vivo in the rat.
However, the rat may metabolize b-AR ligands more rapidly than other species as demonstrated for (S)-[11C]CGP 12177, a radi-
oligand structurally related to 11a–c and 15a–c. Therefore further studies in a different animal model will be carried out.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
putative subtype b4-AR has been identified in cardiac
tissue.5
The heterogeneous family of b-ARs is subdivided into at
least three distinct subtypes, the b1- and b2-AR1 and the
atypical b3-AR.2;3
In the normal heart, mainly b1- and b2-ARs control the
adrenergic functionality of the myocardium. Within the
ventricles of the healthy human heart, the b1=b2-AR
ratio is approximately 80:20.6;7 In heart disease, however
both the b-AR density and the b1=b2-AR ratio may
change. For example, reduced myocardial b-AR density
has been observed in hypertension, heart failure, ische-
mia and both hypertrophic (HCM) and dilated cardio-
myopathies (DCM).8–13 Studies of biopsies or post
mortem material have demonstrated that in a number of
heart diseases downregulation of myocardial b1-AR
density is proportionally greater than that of b2-AR
density.4;14 The failing human heart, however, is often
characterized by a selective reduction in b1-ARs without
change in b2-AR density.6
b1-AR agonists increase the heart rate and cardiac
contractility and b2-selective AR agents mainly stimu-
late bronchodilation and vasodepression.4 The so-called
atypical b3-ARs are involved in lipolysis.2 Recently, a
Keywords: 3-Aryloxy-2-propanolamine; ICI 89,406; Selective b1-adreno-
ceptor ligand; SPECT-radioligand.
* Corresponding author. Tel.: +49-251-8347362; fax: +49-251-83473-
63; e-mail: stwagner@uni-muenster.de
ꢀ
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.027

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S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
The molecular imaging techniques, single photon emis-
sion computed tomography (SPECT) and positron
emission tomography (PET), with suitable radioligands
may be used to assess b-AR density noninvasively in
humans.15;16 To date, several nonselective b-AR radio-
ligands have been used in cardiac imaging. Radio-
iodinated derivatives of the b-AR antagonists carazolol
and CGP 12177 were synthesized for imaging b-ARs
with SPECT.17–19 (S)-[11C]CGP 12177, whose racemate
was described by Delforge et al. in 1991,20 has been used
in PET studies to quantify myocardial b-AR density in
heart disease21–23 and recently (S)-[11C]CGP 12388 has
been presented as a radiochemically more easily acces-
sible radioligand for targeting b-ARs in vivo. [18F]CGP
12388 is also radiochemically available.24;25
particular enantiomer, and the (S)-enantiomer the
multi-step synthesis and radiolabelling procedures were
carried out to yield both the radioiodinated racemic
compounds (11b and 11c) and (S)-enantiomers (15b and
15c). All synthesized nonradioactive ICI 89,406 deriva-
tives (8–11a and 12–15a) were assayed in vitro by
binding studies, to examine the structure–activity rela-
tionships (SAR) between the ligands and b1-ARs. Bio-
distribution and metabolism studies were carried out in
rats to evaluate the potential of the 123I-labelled com-
pounds 11c and 15c as feasible SPECT radioligands.
2. Results
As the b1=b2-AR ratio may change in cardiac disease,
however, a procedure for visualization and quantifica-
tion of b1-AR population rather than total b-AR den-
sities in the human heart would be of great clinical
interest.7 A few b1-AR selective radioligands, such as
2.1. Chemistry
ICI 89,406 (Scheme 1) is known as a high affinity and
selective (75–195 fold) b1-AR ligand.34–37 Therefore, the
development of a selective b1-AR radioligand by slight
modification of the lead structure is a reasonable
approach.
(+/))-[11C]HX-CH 44,26
(S)-[11C]bisoprolol27
or
[11C]CGP 20712A28 and its (S)-enantiomer [11C]CGP-
2650529 were and are in development for PET, but their
clinical use is limited due to high nonspecific binding,
rapid metabolism or a tissue uptake that does not reflect
binding to b-ARs.26–29 In summary, no b1-selective
radioligand suitable for the noninvasive assessment of
cardiac b1-AR is clinically established either for SPECT
or for PET.
Six pairs of compounds possessing the 3-aryloxy-2-
propanolamine core of ICI 89,406 (Schemes 4a and b)
were synthesized. Each pair consists of the racemic and
the (S)-aryloxypropanolamine derivative. Three pairs
are chemically identical. Each of them contains a dif-
ferent iodine isotope at the aryloxy unit. The pair 11a
and 15a represents the nonradioactive version of the
125I-labelled pair 11b and 15b that is useful for biodis-
tribution studies. The 123I-labelled pair 11c and 15c is
also suitable for SPECT and metabolism investigations.
The remaining three pairs include the precursors of the
radioiodinated compounds (10, 14) and the esterified
3-aryloxy-2-propanolamine derivatives occurring in the
synthesis sequence (8, 12 and 9, 13, respectively). The
synthesis of the nonradioactive iodo reference com-
pounds (11a, 15a) and their precursors (10, 14) was
realized via a 5–6 step sequence (Schemes 2–4).
We have chosen ICI 89,406, an early example of a well-
designed selective b1-AR antagonist,30–32 as the lead
compound for the development of high affinity b1-AR
selective ligands suitable for radiolabelling with usual
SPECT- or PET-radionuclides. In our initial study we
assessed the b1-AR affinity and selectivity of a number
of novel derivatives of ICI 89,406.33 Of these one, I-ICI-
H (Scheme 1) showed high affinity and selectivity for
b1-ARs in myocardial membranes in vitro but high
nonspecific binding in vivo.34 In the present study, we
extend these results by inserting a hydrophilic func-
tionality (11a and 15a, Schemes 4a and b), the aim being
to reduce the nonspecific binding. In addition to differ-
entiate between the behaviour of the racemate, that can
be synthesized in a more cost-effective manner than one
The 2-silylated phenol 1a, that was needed as starting
material, was prepared as previously described.38;39 The
2-substituted phenols (1a and 1b) and the 1,2-epoxy-3-
substituted-propane derivatives were converted into the
corresponding 3-aryloxy-1,2-epoxy-propanes 2a–d un-
der basic conditions in moderate to excellent chemical
yields (44–91%). In the synthesis of the racemic com-
pounds 2a and 2b bromide and chloride acted as leaving
groups, respectively. The chiral compounds 2c and 2d
were prepared from (S)-glycidyl-3-nitrobenzene sulfo-
nate ((S)-glycidyl nosylate) containing the chiral infor-
mation and a nosylate leaving group (Scheme 2).
NH
NH
NH
O
O
OH
CN
X
X:
X:
X:
ICI 89,406
2-I-ICI-H
The key intermediate amines 6 and 7 were synthesized in
2–3 steps. The condensation of the Boc-protected ethyl-
enediamine 3 and the phenylisocyanate 4, prepared by a
procedure similar to that published elsewhere,40 yielded
the Boc-urea 5 (yield: 88%). After deprotection of 5
under acidic reaction conditions the hydrochloride 6 was
isolated (yield: 95%), that was transformed into the free
I
125I
2-[125I]I-ICI-H
Scheme 1. Lead structure ICI 89,406 and its iodinated derivatives 2-I-
ICI-H and 2-[125I]I-ICI-H.33;34

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4119
O
O
O
O
O
O
Br
, K2CO3
X: SiMe3,
SiMe3
2a
O
, NaOH
X: I,
Cl
O
OH
I
2b
O
X
Nos
, K2CO3
X: SiMe3,
1a X: SiMe3
1b X: I
O
O
2c
SiMe3
, KOH
X: I,
Nos
O
I
2d
Scheme 2. Synthesis of the oxiranes 2a–d.
2.2. Radiochemistry
OCN
BocHN
The radioiododesilylation of 10 and 14 was performed
under oxidative conditions (NCS) with [125I]NaI or
[
+
NH2
COOMe
123I]NaI, respectively. After HPLC-purification the
3
4
radiolabelled b1-AR ligands 11b, 11c, 15b and 15c were
obtained. The 125I-labelled compounds 11b and 15b gave
better radiochemical yields than the 123I-labelled 11c and
15c (78 vs 41%, Schemes 4a and b). The radiochemical
purity of the compounds was >98% with theoretical
specific radioactivities of 0.16 GBq/lg (79.6 GBq/lmol)
for 11b and 15b and 17.7 GBq/lg (8.77 TBq/lmol) for
11c and 15c. Figure 1 shows the HPLC chromatograms
of a reaction mixture of 15c (Fig. 1a), of the corre-
sponding quality control (Fig. 1b) and the reference
control of 15c (Fig. 1c). No impurities could be detected
in the HPLC quality control of 15c while the radioio-
dinated compound 15c corresponds to its nonradioac-
tive counterpart 15a in the HPLC reference
chromatogram.
NH
NH
BocHN
HCl
O
COOMe
5
NH
NH
NH
H3N+
Cl-
NaOH
O
COOMe
COOMe
6
NH
H2N
O
7
3. Biology
Scheme 3. Synthesis of the amines 6 and 7.
3.1. In vitro studies
amine 7 with an equimolar amount of NaOH (yield:
79%) (Scheme 3).
The protein dependence of [125I]iodocyanopindolol
([125I]ICYP) binding to mouse ventricular membrane
preparations was investigated in previous work.34 The
specific binding increased linearly with the protein con-
centration for up to 50 lg/200 lL. Therefore, 15 lg
protein per 200 lL were used for the in vitro binding
studies. The time course of [125I]ICYP binding to b-ARs
was linear for approximately 15 min and reached equi-
librium after 60 min of incubation. To determine the
maximum binding, assays were carried out for 60 min.
The binding of the nonselective b-AR antagonist
The ring opening of the epoxides 2a–d with the amine
7 or under basic reaction conditions with the hydro-
chloride 6 gave the 3-aryloxy-2-propanolamine deriva-
tives 8, 9, 12 and 13 (yields: 35–55%). The desired
precursors (10 and 14) and nonradioactive reference
compounds (11a and 15a) were made from these esters
8, 9, 12 and 13 via saponification (yields: 33–77%,
Schemes 4a and b).

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S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
NH
NH
NH
O
(a)
O
OH
X
COOR
Product No. Product X Product R Educts / Reagents
8
SiMe3
Me
Me
H
2a (Scheme 2), 7 (Scheme 3)
9
I
2b (Scheme 2), 6 (Scheme 3), NaOH
8, NaOH
10
11a
11b
11c
SiMe3
I
H
9, NaOH
125I
123I
H
10, [125I]NaI, NCS
10, [123I]NaI, NCS
H
NH
NH
NH
O
O
OH
X
(b)
COOR
Product No. Product X Product R Educts / Reagents
12
2c
7
(Scheme 2), (Scheme 3)
SiMe3
Me
13
14
2d
12
6
I
Me
H
(Scheme 2), (Scheme 3), NaOH
SiMe3
, NaOH
15a
15b
15c
13
14
14
I
H
H
H
, NaOH
125I
123I
, [125I]NaI, NCS
, [123I]NaI, NCS
Scheme 4. (a) Synthesis of the racemic b1-AR ligands 8–11c. (b) Synthesis of the (S)-configurated b1-AR ligands 12–15c.
[
125I]ICYP to ventricular membranes was specific, satu-
To determine the structure–activity relationships (SAR)
between the ligands and b1-ARs, the high- and low-
affinity IC50 values of all synthesized and nonradioac-
tive 3-aryloxy-2-propanolamine derivatives 8–11a and
12–15a for the b1- and b2-ARs, respectively, were cal-
culated by nonlinear regression analysis of the compe-
tition studies using [125I]ICYP and mouse ventricular
membrane preparations.
rable and of high affinity. Scatchard transformation of
the saturation data yielded a linear plot with a correla-
tion coefficient greater than 0.95. The dissociation
constant (KD) and the maximum number of binding
sites (Bmax) determined from three experiments for the
binding of
[
125I]ICYP were 32.3 1.9 pM and
38.6 2.9 fmol mgꢀ1 protein, respectively.41 Competition
studies were carried out to assess the inhibition of the
binding of [125I]ICYP by the b1-selective compounds
8–11a and 12–15a (Fig. 2a and b). Nonlinear regression
analysis using the XMGRACE programme (Linux
software) showed that the data of the ligands 8–11a and
12–15a fitted a two-site model significantly better than a
one-site model (F ¼ 2:15, p < 0:05). These compounds
show a significantly higher affinity to b1 than to b2-ARs
(Table 1).
The IC50 values were converted into the high- and low-
affinity inhibition constants (K1 for the b1-ARs and K2
for the b2-ARs) by the method of Cheng–Prusoff 42 using
the experimentally determined KD value of [125I]ICYP
(32.3 1.9 pM). The ratios of the low- to high-affinity
inhibition constants (K2=K1) yield the b1-selectivities of
the unlabelled derivatives 8–11a and 12–15a (Table 1).
Additionally, the calculated log P and log D values

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4121
150 mV
polar fraction
400 mV
300 mV
200 mV
100 mV
precursor 14
100 mV
50 mV
0 mV
0 mV
12500 cps
10000 cps
7500 cps
5000 cps
2500 cps
0 cps
15c
15c
300 cps
200 cps
100 cps
0 cps
side product
30 40
0
10
20
time (min)
30
40
0
10
20
(a)
(b)
time (min)
1200 mV
1000 mV
800 mV
600 mV
400 mV
200 mV
0 mV
15a
160 cps
120 cps
80 cps
40 cps
0 cps
15c
0
10
20
time (min)
30
40
(c)
Figure 1. Representative HPLC chromatogram for (a) a reaction mixture of 15c, (b) a quality control of 15c and (c) a reference control of 15c with
25 lg of its nonradioactive counterpart 15a.
Figure 2. Competition of (a) racemic ICI 89,406 derivatives (8–10, 11a). (b) (S)-Configurated ICI 89,406 derivatives (12–14, 15a) 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 ligands competing for specific binding of [125I]ICYP. Each point represents the mean SEM (n ¼ 4). See Scheme 4a and b
for details of substituents.
(ACD/LogD Suite) for compounds 8–11a and 12–15a
are listed in Table 1 to imply the changes of the lipo-
philicities caused by the chemical modifications of the
lead compound ICI 89,406.
leads to a higher b1-selectivity of 8 compared to com-
pound 12 (299 vs 180). The same effect can be observed
by changing the bulky TMS-group into an iodine. The
resulting racemic b-AR ligand 9 is nearly threefold more
b1-selective than the (S)-compound 13 (1140 vs 430).
Furthermore, both the racemic 9 and the corresponding
(S)-ligand 13 possess higher b1-selectivities than their
TMS-counterparts 8 and 12. In contrast to the pair
8 and 12, the (S)-configurated ligand 13 (K1 ¼ 15:7
nM, K2 ¼ 6700 nM) exhibits a lower b1- and b2-affinity
The esterified racemic silane 8 possesses significantly
higher K1- and K2-values than its (S)-enantiomer 12.
While the K1-value of 12 is reduced about 4-fold
(K1 ¼ 4:48 nM vs K1 ¼ 1:05 nM) the K2-value of 12
decreases from 1340 to 190 nM. This unequal reduction

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S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
Table 1. Inhibition constants and calculated b1-AR selectivities of the ligands determined by a radioligand binding assay using mouse ventricular
membrane preparations, plus calculated ligand log P and log D values.
Cpd.
K1 (nM)a
K2 (nM)a
b1-Selectivityb
log P=log Dc
8
12
4.48 2.29
1340 700
190 16
171 14
299 53
180 68
5.10/4.10
5.10/4.10
3.59/2.64
3.59/2.64
4.80/2.28
4.80/2.28
3.29/0.79
3.29/0.79
1.57/0.21
3.18/1.80
1.05 0.40
0.15 0.01
15.7 9.0
9
13
1140 210
430 140
71 2
6700 3400
1000 340
1090 300
500 170
140 30
10
14
14.0 4.7
14.4 5.0
76 12
843 268
29700 11500
121 14
11a
15a
ICI 89,406d
2-I-ICI-Hd
0.60 0.24
0.0049 0.0008
1.30 0.20
0.045 0.005
160 40
12
2
266 28
a Displacement of specifically bound nonselective b-AR ligand [125I]ICYP binding at b1- and b2-ARs expressed in mouse ventricular membrane
preparations as mean SEM, n ¼ 4.
b The ratios of the low- over the high-affinity inhibition constants (K2=K1) indicate the b1-selectivities of the nonradioactive b1-AR ligands, noted as
mean SEM, n ¼ 4.
c Log P values of the neutral form and log D values calculated by ACD/LogD Suite (log D ¼ log P at physiological pH 7.4 with consideration of
charged species).
d From Ref. 33.
than the racemic mixture 9. (K1 ¼ 0:15 nM, K2 ¼
b1-affinity and b1-selectivity (compare Table 1 data of
ICI 89,406, 9 and 11a).
171 nM).
The TMS-acids 10 (K1 ¼ 14:0 nM, K2 ¼ 1000 nM) and
14 (K1 ¼ 14:4 nM, K2 ¼ 1090 nM) have comparable b1-
and b2-affinities, resulting in similar b1-selectivities (71
vs 76). The replacement of a methoxycarbonyl group in
8 and 12 with a more polar carboxy moiety in 10 and 14
is associated with a decrease in b1-affinity and selectivity.
The b1-affinity and selectivity of the racemic mixture 10
is reduced threefold in comparison to 8 and when the
(S)-configurated 14 is compared with the methoxycar-
bonyl enantiomer 12 a nearly 14-fold decrease of
b1-affinity and a twofold decrease of b1-selectivity
can be observed (K1 ¼ 14:4 nM, b1-selectivity 76 vs
K1 ¼ 1:05 nM, b1-selectivity 180). The b1-affinity of the
nonradioactive racemic target compound 11a is reduced
4-fold in comparison to the esterified derivative 9
(K1 ¼ 0:60 nM vs K1 ¼ 0:15 nM), similar to the TMS-
compounds 8 and 10. The b2-affinities of these deriva-
tives differ in the same range resulting in comparable
b1-selectivities (11a 843 vs 9 1140). The (S)-target com-
pound 15a shows a different behaviour. It has about
3000-fold more b1-affinity than the (S)-ester 13
(K1 ¼ 0:0049 nM vs K1 ¼ 15:7 nM) while the b2-affinity
is reduced only 50-fold resulting in a huge b1-selectivity
(29700). In summary, the pair of acid derivatives 11a
and 15a possesses completely different properties com-
pared to the methyl esters 9 and 13. The acid pair 11a
and 15a is characterized by a very high b1-affinity and
selectivity of the (S)-enantiomer 15a. In contrast the
racemic mixture 9 of the ester pair 9 and 13 has the
higher b1-affinity and selectivity.
3.2. In vivo studies
The clearance of radioactivity from plasma after intra-
venous injection of 11b or radioiodinated 15b or 15c into
rats was rapid (Fig. 3). Predosing rats with nonradio-
active 11a had little effect (data not shown). The distri-
bution of radioactivity in rat tissues at 20 min after
injection of racemic 11b given alone or 5 min after
established b-AR antagonists is shown in Figure 4.
Uptake of radioactivity in the myocardium and lung
was low but predosing with the nonselective b-AR
antagonist propranolol, nonradioactive 11a and both
doses (1 lg/kg or 100 nmol/kg) of the b1-AR antagonist,
ICI 89,406 reduced uptake. In contrast, predosing with
the selective b2-AR antagonist, ICI 118551, had little
effect. None of the antagonists reduced the radioactivity
in the liver or kidney (Fig. 4), or fat or muscle (data not
Compared to 2-I-ICI-H the substitution of the second
phenyl core with a methyl ester or carboxylic acid
moiety leads to a decrease of b1-affinity but an increase
of b1-selectivity (compare Table 1 data of 2-I-ICI-H, 9
and 11a). Furthermore, the two racemic iodine deriva-
tives 9 and 11a are more potent b1-AR ligands than
the lead compound ICI 89,406 caused by increased
Figure 3. Clearance of radioactivity from plasma after i.v. injection of
radioiodinated 11b (d, 8 rats) or 15b, 15c (s, 11 rats). Up to six blood
samples were taken from each rat.

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4123
Figure 5. Radioactivity in tissues 20 min after i.v. injection of radio-
iodinated 11b given alone (
unlabelled 11a at 1 lmol kgꢀ1
) or 5 min after i.v. injection of
) or radioiodinated 15b, 15c given
(
alone (
1 lmol kgꢀ1
ranges.
) or 5 min after i.v. injection of nonradioactive 15a at
( ). Averages for two rats are shown. Bars indicate
Figure 6 shows the radioactivity in heart, liver, kidney
and thyroid as a function of time after injection of
radioiodinated 11b, 15b or 15c. In the tissues, the max-
imum uptake was observed at the first sampling time,
2 min after injection and there was then a loss in
radioactivity from heart, liver and kidney. In the heart,
but not in the other tissues, uptake after radioiodinated
15b or 15c ((S)-configuration) was greater than that
after 11b (racemate). Radioacitivity in the thyroid,
however, increased with time after injection. Predosing
with nonradioactive 15a decreased uptake in the heart
but not in the other tissues.
Figure 7 shows the HPLC profiles of rat plasma at 2 min
after injection of 15c. The HPLC chromatogram after
25 min after injection had the same profile (data not
shown). No radioactivity was detected at the reference
retention time for this compound for samples obtained
at either time after radioligand administration. The
radioactivity in the plasma corresponded almost quan-
titatively to free iodide eluting in the polar fraction. A
similar profile was obtained when 15c was added to a
sample of blood in vitro but when it was added to a
sample of plasma parent 15c was detected.
Figure 4. Radioactivity in heart, lung, liver or kidney at 20 min after
i.v. injection of 11b given alone or 5 min after i.v. injection of ethanol/
saline, nonradioactive ICI 118551, nonradioactive ICI 89,406, non-
radioactive 11a (I-ICI-COOH) or propranolol (a ¼ 100 nmol mol kgꢀ1
,
b ¼ 1 lmol mol kgꢀ1).
Tissue radioactivity ðcpmÞ=tissue wet weight ðgÞ
Uptake index ¼
:
Radioactivity injected ðcpmÞ=body weight ðgÞ
Each bar represents uptake in a single rat.
4. Discussion
4.1. Chemistry
shown). Uptake in the brain was negligible (uptake in-
dex < 0.02).
Because of the clear clinical research need for a selective
b1-AR radioligand, the high affinity selective b1-AR
ligand, ICI 89,406 (75–195 fold b1-selectivity),33–37 was
chosen as the lead compound for the development of
radioligands with a 3-aryloxy-2-propanolamine subunit.
Tissue radioactivity after the injection of racemic 11b or
(S)-configurated 15b or 15c is illustrated in Figure 5.
Uptake after radioiodinated (S)-ligands 15b or 15c was
greater than that after racemic 11b in the thyroid, lung
and heart. Predosing with unlabelled 11a or 15a de-
creased the uptake of radioactivity in heart, lung and
thyroid but not in other examined tissues.
In comparison to the natural catecholamine agonists,
this typical b-AR antagonist is characterized by an
increased hydrophobicity of the aromatic ring system

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S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
250 mV
200 mV
150 mV
100 mV
50 mV
0 mV
polar fraction
polar fraction
30000 cps
20000 cps
10000 cps
0 cps
0
10
20
30
40
time (min)
Figure 7. HPLC analysis of plasma taken from rats at 2 min after
injection of 15c.
ICI 89,406 were identified. Seven of them serve as
nonradioactive counterparts or precursor compounds of
candidate PET- and SPECT-radioligands. The remain-
ing two compounds are useful to evaluate the (SAR)
between the potential ligands and b1-ARs.33 We selected
the potent racemic ligand, 2-I-ICI-H, that showed a
higher b1-selectivity and affinity (266, 0.045 nM) than
ICI 89,406 (121, 1.3 nM) in the in vitro competition
studies, for further evaluation and synthesized its
radioiodinated counterpart 2-[125I]I-ICI-H (Scheme 1).
Due to its high degree of nonspecific binding in vitro
and almost no specific binding to cardiac b1-ARs in vivo
this radiolabelled compound was not suitable as a
radioligand for studies in vivo.34
The present work continues from these results. To re-
duce the lipophilicity and to avoid a high degree of
nonspecific binding at blood plasma proteins, a polar
moiety in 2-I-ICI-H was introduced (i.e. a carboxylate
moiety). The resulting compounds 11a (racemic) and
15a ((S)-configuration) exhibited an amino-acid-like
structure feature with an amino and a carboxylate
group. Its predominant species at physiological pH (pH:
7.4) possess a polar zwitterionic structure similar to
amino acids (95.6%, calculated by ACD/LogD Suite).
The log D value of 11a and 15a (0.79) is significantly less
than that for the iodo compound 2-I-ICI-H (1.80).
Figure 6. Uptake of radioactivity in heart, liver, kidney and thyroid
after i.v. injection of radioiodinated target-compound into rats. Each
symbol is the datum point for an individual rat. Radioiodinated 11b
was given alone (d, 9 rats) or 5 min after i.v. injection of nonradio-
active 11a at 1 lmol kgꢀ1 (s, 4 rats). Radioiodinated 15b, 15c was
given alone (m, 10 rats) or 5 min after unlabelled 15a at 1 lmol kgꢀ1
(n, 5 rats). Note the differences in the scales for uptake indices.
Usually the (S)-enantiomers of b-blockers show
approximately a 100-fold higher affinity to b-ARs than
the corresponding (R)-enantiomers.44;45 Therefore, to
increase the b1-AR selectivity and affinity, we synthe-
sized the (S)-enantiomers 15a–c as well as the racemic
iodinated compounds 11a–c. The synthesized racemic
and (S)-aryloxypropanolamines 8–11a and 12–15a were
tested in binding studies in vitro to gain further infor-
mation about the SAR of these derivatives.
substituted with a cyano moiety and by an increased
distance between the amino group and this aromatic
subunit, which is realized by a substitution of a phen-
oxymethylene moiety against the phenyl ring of the
agonist.43
In previous work, we synthesized several racemic
derivatives of ICI 89,406, mostly modified in the pattern
of aromatic substituents, and examined them in in vitro
binding studies. Nine compounds that possess an
improved b1-AR selectivity and affinity compared with
b1-ARs are a member of the rhodopsin/b2 adrenergic
receptor-like receptors, that represent a subfamily of the
GPCRs (G protein-coupled receptors).46 The binding
sites of b-AR antagonists enclose the carboxylated side
chain of Asp113 in transmembrane helix 3 (TM 3) of

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4125
b-ARs, that form a salt bridge between the positively
charged amine group of the antagonists and the recep-
tor.47–49 This cationic centre is represented by the pro-
tonated secondary amine moiety of compounds 8–15a.
The phenoxyoxygen atom of the synthesized target
compounds 8–15a may be involved in a hydrogen bond
with Asp312 in TM 7, that has been identified as a
critical interaction point,46;50 and the aryloxycore of the
ligands 8–15a may be stabilized by p-stacking with
Phe289 and Phe290 of TM 6 similar to the proposed
interactions of the agonist catechol ring and these aro-
matic amino acid residues.48;51;52 In general the antago-
nist binding site of b-ARs is a nonsolvent exposed
pocket, that is buried in the transmembrane core and
differs from the binding site of the neurotransmitter
agonists.53–55
resynthesized radiochemically to evaluate the resulting
radioligands 11b–c and 15b–c in studies in vivo.
4.2. Biodistribution studies
The first biodistribution study of 11b in rats showed that
at 20 min after intravenous injection of the radioligand,
uptake of radioactivity in heart and lung was low
compared with that in liver and kidney (Fig. 4). Pre-
dosing the rats with the nonselective b-AR antagonist
propranolol, unlabelled 11a and two doses (1 lg/kg or
100 nmol/kg) of the b1-AR antagonist, ICI 89,406, re-
duced uptake. In contrast, predosing with the selective
b2-AR antagonist, ICI 118551, had little effect. None of
the antagonists reduced the radioactivity in the liver or
kidney (Fig. 4), or fat or muscle (data not shown). This
result suggests that 11b binds selectively to b1-ARs in
the heart and lung and that nonspecific binding is low in
these tissues.
The b1-AR ligand pair 8 and 12 is characterized by
stereospecific binding properties, that are known for the
classical aryloxyalkylamine antagonists. The silylated
(S)-compound 12 possesses a higher b1-affinity than the
racemic mixture 8 (K1 ¼ 1:05 nM vs K1 ¼ 4:48 nM).
However, on changing the bulky TMS group into a iodo
atom the affinity behaviour of the resulting b1-AR
ligands 9 and 13 is reversed. The enantiomeric pure (S)-
compound 13 (K1 ¼ 15:7 nM) is about 100-fold less
potent than the racemate 9 (K1 ¼ 0:15 nM). This unex-
pected fact may be based on a different binding orien-
tation of 12 and 13 caused by the steric demand of the
bulky TMS group on the one hand and a negatively
polarized halogen atom on the other.
Uptake of radioactivity after injection of the radio-
iodinated (S)-enantiomer 15b or 15c was greater than
that of the racemic compound 11b in the heart, lung and
thyroid but not in the other studied tissues (Fig. 5).
Predosing with nonradioactive 11a or 15b reduced the
tissue radioactivity in the heart and lung to similar
values. This is consistent with published results that
demonstrate that the (S)-enantiomers of b-blockers
show higher affinity to b-AR than the corresponding
racemates or (R)-enantiomers. In contrast although
predosing did reduce the radioactivity in the thyroid,
uptake after the (S)-enantiomer with predosing was
greater than that after the racemate without predosing
(Fig. 5).
The TMS acid pair 10 and 14 looses b1-affinity and
selectivity compared to their esterified counterparts 8
and 12 (affinity: 3–13-fold decrease, selectivity: 2–4-fold
decrease). Probably the negatively charged carboxylate
moiety in combination with the TMS residue decreases
the potency of the b1-AR ligands 10 and 14 in
comparison to 8 and 12 due to more repulsion forces
between receptor and ligand. Both acid derivatives 10
and 14 possess similar b1-affinities and selectivities
(Table 1).
Although the biodistribution at 20 min suggests that
radioiodinated target-compounds bind selectively to
b1-AR in the heart and lung, the time–activity curves
indicate that radioactivity was lost from all studied tis-
sues with the exception of the thyroid (Fig. 6). In the
heart (Fig. 6) clearance of the (S)-enatiomers 15b–c was
greater than that after the racemic compound 11b and
predosing with nonradioactive 11a or 15a reduced the
maximal uptake but the time profile was not optimal for
functional imaging. The data for the thyroid showed
considerable scatter but there was a gradual increase in
radioactivity following injection of both 11b and 15b–c
with or without predosing. This increase in thyroid
radioactivity suggested that free iodide ion was present,
presumably due to metabolism of the radioligand. The
HPLC analysis of plasma taken from rats at 2 and
25 min after injection of 15c did not detect parent 15c
(Fig. 7). Radioactivity in the plasma eluted at the same
time as free iodide ion. Rapid breakdown occurred when
15c was added to rat blood in vitro although when it was
added to plasma parent 15c was detected. Therefore
radioiodinated 11c and 15c appear to be unpromising
radioligands for assessing b1-AR in vivo in the rat. The
rat, however, may metabolize b-AR-ligands more rap-
idly than other species. For example, comparative
studies of metabolism of (S)-[11C]CGP 12177 in rat, dog
and man demonstrated significant metabolism in rat but
The target pair 11a and 15a shows different binding
features. The iodinated (S)-acid 15a is a selective high
affinity b1-AR ligand. This compound shows a 122-fold
increased b1-affinity (K1 ¼ 0:0049 nM vs K1 ¼ 0:60 nM)
and a 35-fold higher selectivity (29700 vs 843) than the
racemic mixture 11a. In contrast to the TMS acids 10
and 14, that loose b1-affinity and selectivity in compar-
ison to their TMS esters 8 and 12, the iodinated acids
11a and 15a possess partially increased b1-affinities and
selectivities compared to their ester counterparts 9 and
13. The stereospecific binding properties of 11a and 15a
are similar to those of the known aryloxyalkylamine
antagonists for which the (S)-enantiomers have a higher
affinity than their corresponding racemates. It appears
that the substitution of the methyl ester group with a
polar carboxylate group leads to a stereospecific binding
orientation of ligand and b1-AR that preferentially
binds the (S)-enantiomer. In summary, the in vitro
binding studies identified the acids 11a and especially
15a as promising and potent b1-AR ligands. Both were

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S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
no detectable metabolism during the duration of a PET
scan in dog and man.56;57 Therefore the target com-
pounds 11c and 15c should be evaluated in different
animal models.
6.2. Synthesis of the oxirane compounds 2a–d (Scheme 2)
6.2.1. 2-(2-Trimethylsilyl-phenoxymethyl)-oxirane 2a.
5.80 g (34.9 mmol) 2-(Trimethylsilyl)-phenol 1a, pre-
pared as previously described,38;39 19.11 g (139 mmol)
bromomethyl-oxirane and 17.29 g (125 mmol) anhy-
drous K2CO3 were refluxed in 40 mL dry 2-butanone for
6 h under Ar atmosphere. The hot mixture was filtered
and the filter cake was washed with 2-butanone. The
combined filtrates were evaporated and the crude reac-
tion product was purified by silica gel chromatography
(hexane/diethyl ether 49:1) to give 2a (Rf : 0.16) as a
colourless liquid. Yield: 6.52 g (29.3 mmol), 84%. 1H
NMR (300 MHz, CDCl3, TMS intern): d [ppm]: 7.40–
7.37 (m, 1H, Haryl), 7.34–7.28 (m, 1H, Haryl), 6.96 (dt,
3J ¼ 7:3 Hz, 4J ¼ 1:0 Hz, 1H, Haryl), 6.79 (t, 3J ¼ 8:4 Hz,
1H, Haryl), 4.18 (dd, 2J ¼ 10:8 Hz, 3J ¼ 3:3 Hz, 1H,
5. Conclusion
The newly synthesized nonradioactive compounds 11a
(racemate) and especially 15a ((S)-enantiomer) possess
excellent b1-affinities and b1-selectivities. In contrast to
their esterified precursors 9 and 13, where the racemate 9
has more b1-AR affinity and selectivity than the
(S)-isomer 13, the pair 11a and 15a showed stereo-
selective binding behaviour to b1-AR, that is charac-
teristic for b1-AR antagonists with such a 3-aryloxy-2-
propanolamine structure. The (S)-enantiomer 15a fea-
tured higher b1-AR affinity (122-fold) and b1-AR
selectivity (35-fold) than 11a. The radiochemically syn-
thesized counterparts 11b–c (racemic) and 15b–c ((S)-
enantiomer) were evaluated in vivo in the rat to test their
suitability as SPECT-radioligands. Although the results
argue against the suitability of 11c and 15c as promising
SPECT-radioligands for assessing b1-ARs in vivo in the
rat because of rapid radioligand metabolism, the rat
seems to be an inappropriate animal model. b-AR-
ligands may be metabolized more rapidly in rats than in
other species (mouse, dog, man) as demonstrated for
(S)-[11C]CGP 12177, which is structurally related to
11a–c and 15a–c. Therefore further studies in different
animal models (e.g. nonhuman primate, mice) will be
carried out with the aim to use these b1-AR selective
radioligands in the study of patients with cardiac dis-
eases in the future.
2
3
1CH2), 3.97 (dd, J ¼ 11:1 Hz, J ¼ 5:7 Hz, 1H, 1CH2),
3.37–3.33 (m, 1H, CH), 2.89 (dd, 2J ¼ 5:0 Hz,
3J ¼ 4:1 Hz, 1H, 1CH2), 2.72 (dd, 2J ¼ 5:1 Hz,
3J ¼ 2:7 Hz, 1H, 1CH2), 0.29 (s, 9H, Si(CH3)3). 13C
NMR (75.5 MHz, CDCl3, TMS intern): d [ppm]: 163.96,
136.02, 131.57, 129.06, 121.83, 111.09, 69.53, 51.02,
45.56, )0.87. GC–MS (EI): m=z (intensity %): 222 (Mꢂþ,
13), 177 (100), 161 (43), 151 (45), 135 (31), 91 (46). Anal.
Calcd for C12H18O2Si: C, 64.82; H, 8.16. Found: C,
64.49; H, 8.31.
6.2.2. 2-(2-Iodo-phenoxymethyl)-oxirane 2b. 7.19 g
(77.7 mmol) chloromethyl-oxirane, 4.80 g (21.8 mmol) 2-
iodo-phenol 1b and 15 mL 2 N NaOH were heated to
50 ꢁC for 2.5 h. Then the mixture was extracted three
times with CH2Cl2, the combined organic layers were
dried (MgSO4) and the solvent and volatile compounds
were evaporated. The residue was distilled in vacuo
fractionally to provide 2b as a colourless liquid. Yield:
4.50 g (16.3 mmol), 75% (lit.58: 44%). bp (3.0 mbar):
140 ꢁC (lit.58: 118–123 ꢁC (0.2 Torr)). 1H NMR
(400 MHz, CDCl3): d [ppm]: 7.84 (dd, 3J ¼ 7:3 Hz,
4J ¼ 1:6 Hz, 1H, Haryl), 7.38–7.34 (m, 1H, Haryl), 6.91
(dd, 3J ¼ 8:4 Hz, 4J ¼ 1:2 Hz, 1H, Haryl), 6.80 (dt,
3J ¼ 7:6 Hz, 4J ¼ 1:5 Hz, 1H, Haryl), 4.35 (dd, 2J ¼
11:2 Hz, 3J ¼ 2:8 Hz, 1H, 1CH2), 4.13 (dd, 2J ¼ 11:2 Hz,
3J ¼ 5:2 Hz, 1H, 1CH2), 3.48–3.44 (m, 1H, CH), 3.00–
2.89 (m, 2H, 2CH2). 13C NMR (75.5 MHz, CDCl3): d
[ppm]: 157.55, 139.98, 129.91, 123.58, 113.22, 87.14,
70.12, 50.47, 45.13.
6. Experimental section
6.1. Synthetic methods
All chemicals, reagents and solvents for the synthesis of
the compounds were analytical grade and purchased
from commercial sources.
The melting points (uncorrected) were determined on a
Stuart Scientific SMP3 capillary melting point appara-
tus. 1H NMR and 13C NMR spectra were recorded on a
Bruker ARX 300 or AMX 400 spectrometer, respec-
tively. Mass spectrometry was performed via a Varian
MAT 212 (EI ¼ 70 eV) spectrometer and a Bruker
MALDI-TOF-MS Reflex IV (matrix: DHB). ½aꢁ values
6.2.3. (S)-2-(2-Trimethylsilyl-phenoxymethyl)-oxirane 2c.
Compound 2c was prepared from 1.78 g (10.7 mmol)
2-(trimethylsilyl)-phenol 1a, 5.55 g (21.5 mmol) (S)-
glycidyl-3-nitrobenzene sulfonate and 5.28 g (38.2 mmol)
anhydrous K2CO3 in 20 mL dry 2-butanone similar to
the procedure described for 2a. Yield: 1.82 g (8.2 mmol),
76%. 1H NMR (300 MHz, CDCl3, TMS intern): d
[ppm]: 7.39 (dd, 3J ¼ 7:2 Hz, 4J ¼ 1:8 Hz, 1H, Haryl),
7.35–7.29 (m, 1H, Haryl), 6.96 (dt, 3J ¼ 7:2 Hz,
4J ¼ 0:6 Hz, 1H, Haryl), 6.79 (dd, 3J ¼ 8:1 Hz,
4J ¼ 0:3 Hz, 1H, Haryl), 4.18 (dd, 2J ¼ 10:8 Hz,
3J ¼ 3:6 Hz, 1H, 1CH2), 3.97 (dd, 2J ¼ 10:8 Hz,
3J ¼ 5:4 Hz, 1H, 1CH2), 3.37–3.32 (m, 1H, CH), 2.90–
D
were determined on a Perkin–Elmer polarimeter 341.
Elemental analysis was realized by a Vario EL III ana-
lyzer. Separation of the radiosynthesized compounds,
analyses of the radiochemical yields and the radio-
chemical purity were performed by gradient radio-
HPLC using a Knauer K-500 and a Latek P 402 pump,
a Knauer K-2000 UV-detector (wavelength 254 nm) and
a Crismatec Na(Tl) Scintibloc 51 SP51 c-detector.
Sample injection was carried out using a Rheodyne
injector block (type 7125 incl. 200 lL loop). The
recorded data were processed by the NINA radio-HPLC
software (GE Functional Imaging GmbH).

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4127
2
3
2.87 (m, 1H, 1CH2), 2.72 (dd, J ¼ 5:0 Hz, J ¼ 2:6 Hz,
C16H23N3O5: C, 56.96; H, 6.87; N, 12.46. Found: C,
56.58; H, 6.80; N, 12.54.
1H, 1CH2), 0.30 (s, 9H, Si(CH3)3). 13C NMR
(75.5 MHz, CDCl3, TMS intern): d [ppm]: 163.04,
135.10, 130.65, 128.13, 120.91, 110.16, 68.63, 50.10,
20
D
44.63, )0.91. ½aꢁ +17.2 (c 1.0, C2H5OH). Anal. Calcd
6.3.2. N-(2-Amino-ethyl)-N0-(4-methoxycarbonyl-phenyl)-
urea hydrochloride 6. 3.37 g (10.0 mmol) N-[2-[(tert-
Butoxycarbonyl)amino]ethyl]-N0-(4-methoxycarbonyl-
phenyl)-urea 5 were dissolved in 7 mL of a conc. HCl/
MeOH mixture (1:1, v/v). The solvents were evaporated
at 50 ꢁC in vacuo (600 fi 30 mbar) within 1.5 h. Then
30 mL dry acetone were added and the solvent was re-
moved in vacuo. This procedure was repeated two times.
Thereafter the product was suspended in 25 mL acetone,
stirred for 30 min, filtered and dried in vacuo to provide
6 as a colourless solid. Yield: 2.61 g (9.5 mmol), 95%.
mp: 215 ꢁC, decomposition. 1H NMR (300 MHz,
DMSO-d6, TMS intern): d [ppm]: 9.68 (s, 1H, NH), 8.05
(broad, s, 3H, NH3þ), 7.83–7.80 (m, 2H, Haryl), 7.56–7.52
for C12H18O2Si: C, 64.82; H, 8.16. Found: C, 64.43; H,
7.99.
6.2.4. (S)-2-(2-Iodo-phenoxymethyl)-oxirane 2d. To a
solution of 5.00 g (22.7 mmol) 2-iodo-phenol 1b in
568 mL dry toluene, 17.1 mL 1.3 M KOH (22.2 mmol)
were added. After stirring for 30 min at RT, the mixture
was evaporated to dryness. Acetonitrile was added and
evaporated to a volume of approximately 190 mL. After
heating to reflux, a solution of 5.88 g (22.7 mmol)
(S)-glycidyl-3-nitrobenzene sulfonate in 170 mL dry
acetonitrile was added dropwise. Thirty minutes after
complete addition, the reaction mixture was cooled to
RT and stirred for 2 h. The mixture was evaporated to
dryness, 200 mL water and 200 mL CH2Cl2 were added
and the organic layer was separated. The mixture was
extracted two times with CH2Cl2, the combined organic
layers were dried (Na2SO4) and the solvent and volatile
compounds were evaporated. The residue was distilled
in vacuo in a Kugelrohr and the high boiling fraction
(bp (2.1 mbar): 150–210 ꢁC) was purified by silica gel
chromatography (PE/diethyl ether 9:1) to give 2d (Rf :
0.14) as a colourless liquid. Yield: 5.69 g (20.6 mmol),
91%. 1H NMR (300 MHz, CDCl3, TMS intern): d
[ppm]: 7.76 (dd, 3J ¼ 7:5 Hz, 4J ¼ 1:5 Hz, 1H, Haryl),
7.30–7.24 (m, 1H, Haryl), 6.83 (dd, 3J ¼ 8:3 Hz,
4J ¼ 1:4 Hz, 1H, Haryl), 6.74–6.69 (m, 1H, Haryl), 4.26
(dd, 2J ¼ 11:4 Hz, 3J ¼ 3:0 Hz, 1H, 1CH2), 4.04 (dd,
2J ¼ 11:1 Hz, 3J ¼ 5:1 Hz, 1H, 1CH2), 3.40–3.35 (m,
1H, CH), 2.90–2.87 (m, 2H, 2 CH2). 13C NMR
3
(m, 2H, Haryl), 6.93 (t, J ¼ 6:0 Hz, 1H, NH), 3.77 (s,
3
3H, CH3), 3.33 (q, J ¼ 6:1 Hz, 2H, CH2), 2.88 (broad,
s, 2H, CH2). 13C NMR (75.5 MHz, DMSO-d6): d [ppm]:
166.13, 155.54, 145.26, 130.42, 121.91, 116.93, 51.82,
51.77, 37.26. Anal. Calcd for C11H16CIN3O3: C, 48.27;
H, 5.89; N, 15.35. Found: C, 48.08; H, 5.91; N, 15.28.
6.3.3. N-(2-Amino-ethyl)-N0-(4-methoxycarbonyl-phenyl)-
urea 7. 1.20 g (4.38 mmol) N-(2-Amino-ethyl)-N0-(4-
methoxycarbonyl-phenyl)-urea hydrochloride 6 were
dissolved in 24 mL water and 438 lL 10 N NaOH. The
aqueous solution was refluxed with 50 mL CHCl3 for
20 min and the layers were separated. This procedure
was repeated four times. The combined organic layers
were dried (Na2SO4) and the solvent was evaporated to
give 7 as a colourless solid. Yield: 825 mg (3.48 mmol),
1
79%. mp: 190 ꢁC, decomposition. H NMR (300 MHz,
DMSO-d6): d [ppm]: 8.97 (broad, s, 1H, NH), 7.81 (d,
3
(75.5 MHz, CDCl3): d [ppm]: 157.05, 139.51, 129.44,
3J ¼ 8:7 Hz, 2H, Haryl), 7.50 (d, J ¼ 8:7 Hz, 2H, Haryl),
20
D
123.10, 112.70, 86.66, 69.43, 50.04, 44.67. ½aꢁ +6.9 (c
6.35 (t, 3J ¼ 5:6 Hz, 1H, NH), 3.78 (s, 3H, CH3), 3.08 (q,
3
1.0, C2H5OH). Anal. Calcd for C9H9IO2: C, 39.16; H,
3.29. Found: C, 38.75; H, 3.33.
3J ¼ 5:9 Hz, 2H, CH2), 2.62 (t, J ¼ 6:2 Hz, 2H, CH2).
13C NMR (75.5 MHz, DMSO-d6): d [ppm]: 166.13,
155.01, 145.44, 130.42, 121.67, 116.78, 51.72, 42.56,
41.73. Anal. Calcd for C11H15N3O3: C, 55.69; H, 6.37;
N, 17.71. Found: C, 55.88; H, 6.32; N, 17.46.
6.3. Synthesis of 2-aminoethyl-ureas 5-7 (Scheme 3)
6.3.1. N-[2-[(tert-Butoxycarbonyl)amino]ethyl]-N0-(4-
methoxycarbonyl-phenyl)-urea 5. 2.14 g (13.4 mmol) N-
6.4. Synthesis of the 3-aryloxy-2-propanolamine deriva-
tives 8–15 (Scheme 4a and b)
Mono(tert-butyloxycarbonyl)ethylenediamine
3 were
dissolved in 9 mL dry ether. At 0 ꢁC 2.37 g (13.4 mmol)
4-methoxycarbonyl-phenylisocyanate 4, prepared using
a procedure similar to that published elsewhere,59 in
15 mL dry ether were added within 30 min. The mixture
was left at +4 ꢁC for 13 h and filtered. The filter cake was
washed with 50 mL ether and dried in vacuo to provide 5
as a colourless solid. Yield: 3.95 g (11.7 mmol), 88%. mp:
6.4.1.
( )-N-(4-Methoxycarbonyl-phenyl)-N0-[2-[3-(2-
trimethylsilyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-
urea 8 and its (S)-enantiomer 12. N-(2-Amino-ethyl)-N0-
(4-methoxycarbonyl-phenyl)-urea 7 was dissolved in hot
anhydrous acetonitrile (2.6 mL/mmol) under Ar atmo-
sphere. An equimolar amount of 2-(2-trimethylsilyl-
phenoxymethyl)-oxirane 2a or its (S)-enantiomer 2c was
added, and the mixture was stirred under reflux for
2.5 h, at RT for 12 h, and again under reflux for 7.5 h.
The reaction mixture was cooled to RT and filtered, the
filter cake was washed with acetonitrile and the com-
bined evaporated filtrates were purified by silica gel
chromatography (ethyl acetate/MeOH 9:1) to give 8 as a
colourless hygroscopic oil that solidified after some
1
172 ꢁC. H NMR (300 MHz, DMSO-d6): d [ppm]: 8.96
3
(s, 1H, NH), 7.82 (d, J ¼ 8:4 Hz, 2H, Haryl), 7.50 (d,
3J ¼ 8:4 Hz, 2H, Haryl), 6.79 (broad, s, 1H, NH), 6.31 (t,
3J ¼ 4:8 Hz, 1H, NH), 3.79 (s, 3H, CH3), 3.14 (q,
3
3J ¼ 6:0 Hz, 2H, CH2), 3.01 (t, J ¼ 5:8 Hz, 2H, CH2),
1.36 (s, 9H, 3CH3). 13C NMR (75.5 MHz, DMSO-d6): d
[ppm]: 187.63, 166.14, 154.95, 145.30, 130.42, 121.81,
116.84, 77.79, 51.75, 40.41, 39.27, 28.35. Anal. Calcd for

4128
S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
weeks at 4 ꢁC, and its (S)-enantiomer 12 (Rf : 0.15) as a
colourless hygroscopic solid, respectively.
Haryl), 7.74 (dd, 3J ¼ 7:7 Hz, 4J ¼ 1:5 Hz, 1H, Haryl),
7.50 (d, 3J ¼ 8:7 Hz, 2H, Haryl), 7.34–7.28 (m, 1H, Haryl),
3
4
6.99 (dd, J ¼ 8:4 Hz, J ¼ 0:9 Hz, 1H, Haryl), 6.72 (dt,
3J ¼ 7:5 Hz, 4J ¼ 1:2 Hz, 1H, Haryl), 6.34 (t, 3J ¼ 5:1 Hz,
1H, NH), 4.85 (broad, s, 1H, OH), 4.00–3.90 (m, 3H,
6.4.2.
( )-N-(4-Methoxycarbonyl-phenyl)-N0-[2-[3-(2-
trimethylsilyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-
3
CH2CH), 3.79 (s, 3H, CH3), 3.19 (q, J ¼ 5:7 Hz, 2H,
1
urea 8. Yield: 55%. mp: 50–52 ꢁC. H NMR (300 MHz,
CH2), 2.85–2.64 (m, 4H, 2CH2). 13C NMR (75.5 MHz,
DMSO-d6): d [ppm]: 166.09, 157.25, 154.94, 145.40,
139.02, 130.43, 129.82, 122.73, 121.69, 116.77, 112.91,
86.74, 71.62, 68.08, 52.39, 51.75, 49.21, 32.51. MS
(MALDI-TOF): m=z: 514 (M+H)þ. Anal. Calcd for
C20H24IN3O5: C, 46.80; H, 4.71; N, 8.19. Found: C,
46.48; H, 4.32; N, 7.93.
CDCl3, TMS intern): d [ppm]: 8.25 (broad, s, 1H, NH),
7.87 (d, 3J ¼ 8:7 Hz, 2H, Haryl), 7.41–7.25 (m, 4H, Haryl),
3
4
6.95 (dt, J ¼ 7:3 Hz, J ¼ 0:7 Hz, 1H, Haryl), 6.75 (d,
3
3J ¼ 8:4 Hz, 1H, Haryl), 6.08 (t, J ¼ 5:3 Hz, 1H, NH),
4.16–3.87 (m, 3H, CH2CH), 3.84 (s, 3H, CH3), 3.38–3.33
(m, 2H, CH2), 3.08 (broad, s, 2H, OH and NH), 2.94–
2.77 (m, 4H, 2CH2), 0.25 (s, 9H, Si(CH3)3). 13C NMR
(75.5 MHz, CDCl3, TMS intern): d [ppm]: 166.93,
162.98, 156.26, 143.85, 135.26, 130.88, 127.66, 123.86,
121.06, 118.08, 110.26, 77.19, 69.96, 68.64, 52.02, 49.63,
39.98, )0.60. MS (MALDI-TOF): m=z: 460 (M+H)þ,
482 (M+Na)þ. Anal. Calcd for C23H33N3O5SiÆ0.5H2O:
C, 58.95; H, 7.53; N, 8.97. Found: C, 58.85; H, 7.16; N,
8.51.
6.4.6.
(S)-N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propyl-
13.
amino]-ethyl]-N0-(4-methoxycarbonyl-phenyl)-urea
Yield: 37%. mp: 134–135 ꢁC. 1H NMR (300 MHz,
DMSO-d6): d [ppm]: 9.02 (s, 1H, NH), 7.81 (d,
3J ¼ 8:7 Hz, 2H, Haryl), 7.74 (dd, 3J ¼ 7:8 Hz,
4J ¼ 1:5 Hz, 1H, Haryl), 7.50 (d, J ¼ 8:7 Hz, 2H, Haryl),
3
7.34–7.28 (m, 1H, Haryl), 6.99 (dd, 3J ¼ 8:3 Hz,
4J ¼ 1:4 Hz, 1H, Haryl), 6.72 (dt, 3J ¼ 7:4 Hz,
6.4.3. (S)-N-(4-Methoxycarbonyl-phenyl)-N0-[2-[3-(2-tri-
methylsilyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-urea
12. Yield: 35%. mp: 75–76 ꢁC. 1H NMR (300 MHz,
CDCl3, TMS intern): d [ppm]: 8.05 (broad, s, 1H, NH),
7.87 (d, 3J ¼ 9:0 Hz, 2H, Haryl), 7.39–7.25 (m, 4H, Haryl),
4J ¼ 1:2 Hz, 1H, Haryl), 6.36 (t, J ¼ 5:4 Hz, 1H, NH),
3
4.93 (broad, s, 1H, OH), 4.03–3.90 (m, 3H, CH2CH),
3.78 (s, 3H, CH3), 3.19 (q, 3J ¼ 5:9 Hz, 2H, CH2), 2.85–
2.64 (m, 4H, 2CH2). 13C NMR (75.5 MHz, DMSO-d6): d
[ppm]: 166.13, 157.24, 154.98, 145.42, 139.04, 130.44,
3
4
6.94 (dt, J ¼ 7:3 Hz, J ¼ 0:8 Hz, 1H, Haryl), 6.74 (d,
129.84, 122.76, 121.71, 116.78, 112.90, 86.74, 71.63,
20
D
3
3J ¼ 8:4 Hz, 1H, Haryl), 5.48 (t, J ¼ 5:3 Hz, 1H, NH),
68.08, 52.40, 51.77, 49.22, 32.95. ½aꢁ )12.3 (c 1.0,
4.12–3.90 (m, 3H, CH2CH), 3.84 (s, 3H, CH3), 3.33 (q,
C2H5OH). Anal. Calcd for C20H24IN3O5: C, 46.80; H,
4.71; N, 8.19. Found: C, 46.78; H, 4.55; N, 8.39.
3J ¼ 5:4 Hz, 2H, CH2), 2.89–2.73 (m, 4H, 2CH2), 2.44
(broad, s, 2H, OH and NH), 0.24 (s, 9H, Si(CH3)3). 13
C
NMR (75.5 MHz, CDCl3, TMS intern): d [ppm]: 166.87,
162.96, 156.00, 143.77, 135.21, 130.86, 127.63, 123.86,
120.97, 118.05, 110.17, 77.14, 69.97, 68.87, 51.96, 49.57,
6.4.7.
( )-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-trimeth-
ylsilyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-urea 10
and its (S)-enantiomer 14. 1 Equiv ( )-N-(4-methoxy-
carbonyl-phenyl)-N0-[2-[3-(2-trimethylsilyl-phenoxy)-2-
hydroxy-propylamino]-ethyl]-urea 8 or its (S)-enantio-
mer 12 and 11.1 equiv 1 N NaOH were stirred at RT for
66–93 h. The pH value was adjusted to 6–7 with 1 N
HCl, the product was filtered off, washed with water and
stirred under reflux in a ethyl acetate/acetonitrile mix-
ture (1:1). The mixture was cooled down to )20–
()30) ꢁC for several hours, the filtered compounds were
washed with ethyl acetate/acetonitrile (1:1) and dried in
vacuo to provide 10 and its (S)-enantiomer 14 as col-
ourless solids, respectively.
40.23, )0.65. MS (MALDI-TOF): m=z: 460 (M+H)þ,
20
482 (M+Na)þ. ½aꢁ )12.5 (c 1.0, C2H5OH). Anal. Calcd
D
for C23H33N3O5SiÆ0.5H2O: C, 58.95; H, 7.53; N, 8.97.
Found: C, 59.11; H, 7.23; N, 8.72.
6.4.4.
( )-N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propyl-
amino]-ethyl]-N0-(4-methoxycarbonyl-phenyl)-urea 9 and
its (S)-enantiomer 13. 1.00 mmol N-(2-Amino-ethyl)-N0-
(4-methoxycarbonyl-phenyl)-urea hydrochloride 6, an
equimolar amount of 2-(2-iodo-phenoxymethyl)-oxirane
2b or its (S)-enantiomer 2d and 1.05 equiv of 10 N
NaOH were heated in approximately 3.0 mL n-propanol
and 0.5 mL water up to 90 ꢁC for 30–60 min. Water was
added at RT until the mixture gets a slight turbidity. The
suspension was cooled down to +4 ꢁC for about 20 h, the
product was filtered off, washed with water, after that
with ethyl acetate, and finally dried in vacuo. The raw
products were recrystallized two times from a ethyl
acetate/acetonitrile mixture (8:1 and 10:1) to provide 9
and 13 as colourless solids, respectively.
6.4.8.
silyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-urea
( )-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-trimethyl-
10.
Yield: 77%. mp: 175–177 ꢁC. 1H NMR (300 MHz,
CDCl3, TMS intern): d [ppm]: 9.39 (broad, s, 1H, NH),
7.80 (d, 3J ¼ 8:7 Hz, 2H, Haryl), 7.47 (d, 3J ¼ 8:4 Hz, 2H,
Haryl), 7.29 (d, 3J ¼ 7:2 Hz, 2H, Haryl), 6.92–6.89 (m, 3H,
NH, Haryl), 5.29 (broad, s, 3H, NH, OH, COOH), 4.04–
3
3.92 (m, 3H, CH2CH), 3.27 (d, J ¼ 5:4 Hz, 2H, CH2),
2.93–2.74 (m, 4H, 2CH2), 0.24 (s, 9H, Si(CH3)3). 13C
NMR (75.5 MHz, CDCl3, TMS intern): d [ppm]: 168.56,
163.33, 155.39, 144.40, 134.72, 131.04, 130.41, 126.79,
125.07, 120.41, 116.57, 110.54, 70.22, 67.55, 52.02, 48.94,
38.46, )0.57. MS (MALDI-TOF): m=z: 446 (M+H)þ,
6.4.5.
amino]-ethyl]-N0-(4-methoxycarbonyl-phenyl)-urea
( )-N-[2-[3-(2-Iodo-phenoxy)-2-hydroxy-propyl-
9.
1
Yield: 44%. mp. 137 ꢁC. H NMR (300 MHz, DMSO-
d6): d [ppm]: 9.00 (s, 1H, NH), 7.81 (d, 3J ¼ 8:4 Hz, 2H,

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4129
468 (M+Na)þ. Anal. Calcd for C22H31N3O5Si: C, 59.30;
H, 7.01; N, 9.43. Found: C, 59.51; H, 6.65; N, 9.12.
Haryl), 7.74 (dd, 3J ¼ 7:6 Hz, 4J ¼ 1:5 Hz, 1H, Haryl),
7.46 (d, 3J ¼ 8:4 Hz, 2H, Haryl), 7.34–7.29 (m, 1H, Haryl),
7.00 (d, 3J ¼ 7:5 Hz 1H, Haryl), 6.80 (broad, s, 1H, NH),
6.75–6.70 (m, 1H, Haryl), 4.33 (broad, s, 3H, NH, OH,
COOH), 4.03–3.97 (m, 3H, CH2CH), 3.27–2.77 (m, 6H,
3CH2). 13C NMR (75.5 MHz, DMSO-d6): d [ppm]:
168.33, 157.19, 155.33, 144.40, 139.06, 130.45, 129.88,
6.4.9.
silyl-phenoxy)-2-hydroxy-propylamino]-ethyl]-urea
(S)-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-trimethyl-
14.
Yield: 69%. mp: 185–186 ꢁC. 1H NMR (300 MHz,
CDCl3, TMS intern): d [ppm]: 9.42 (broad, s, 1H, NH),
7.80 (d, 3J ¼ 8:4 Hz, 2H, Haryl), 7.47 (d, 3J ¼ 8:7 Hz, 2H,
Haryl), 7.29 (d, 3J ¼ 7:2 Hz, 2H, Haryl), 6.97–6.88 (m, 3H,
NH, Haryl), 6.11 (broad, s, 3H, NH, OH, COOH), 3.99–
3.90 (m, 3H, CH2CH), 3.25–2.69 (m, 6H, 3CH2), 0.24 (s,
9H, Si(CH3)3). 13C NMR (75.5 MHz, CDCl3, TMS in-
tern): d [ppm]: 168.55, 163.39, 155.39, 144.30, 134.40,
131.04, 130.37, 126.79, 125.53, 120.36, 116.50,
110.50, 70.29, 67.88, 52.28, 49.11, 38.81, )0.59. MS
122.87, 116.63, 112.97, 86.76, 71.49, 67.35, 51.90, 48.97,
20
D
33.04. MS (MALDI-TOF): m=z: 500 (M+H)þ. ½aꢁ )4.5
(c 0.067, H2O/C2H5OH 2/1). Anal. Calcd for
C19H22IN3O5: C, 45.71; H, 4.44; N, 8.42. Found: C,
45.47; H, 4.25; N, 8.29.
6.4.13. ( )-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-[125I]iodo-
phenoxy)-2-hydroxy-propylamino]-ethyl]-urea 11b, the
(MALDI-TOF): m=z: 446 (M+H)þ, 468 (M+Na)þ.
[
123I]iodo-derivative 11c and its (S)-enantiomers 15b–c.
20
D
½aꢁ )13.5 (c 0.067, H2O/C2H5OH 2/1). Anal. Calcd for
A suspension of 3.3 mg (24.7 lmol) N-chlorosuccinimide
(NCS) in 20 lL water for injection were added to a
solution of 0.3 mg (0.67 lmol) ( )-N-(4-carboxy-phen-
yl)-N0-[2-[3-(2-trimethylsilyl-phenoxy)-2-hydroxy-propyl-
amino]-ethyl]-urea 10 or its (S)-enantiomer 14 in 12 lL
acetic acid (HOAc) and 12 lL 8% NaOAcÆ3H2O in
HOAc. The radiosynthesis was started by adding this
mixture to 6–49 MBq n.c.a. [125I]NaI in 5–14 lL 0.05 N
NaOH and 75–316 MBq n.c.a. [123I]NaI in 5–31 lL
0.05 N NaOH, respectively. The reaction mixture was
vortexed and allowed to stand 2 min at RT. The reaction
was terminated by the addition of 100 lL eluent B (see
below).
C22H31N3O5Si: C, 59.30; H, 7.01; N, 9.43. Found: C,
59.38; H, 6.64; N, 9.09.
6.4.10. ( )-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-iodo-phen-
oxy)-2-hydroxy-propylamino]-ethyl]-urea 11a and its
(S)-enantiomer 15a. 1 Equiv ( )-N-[2-[3-(2-iodo-phen-
oxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-methoxycarb-
onyl-phenyl)-urea 9 or its (S)-enantiomer 13 and
11.1 equiv 1 N NaOH were stirred under reflux for 1–2 h
and then at RT for 2–14 h. The pH value was adjusted to
6.2–6.8 with 1 N HCl, the product was filtered off and
stirred under reflux in an ethyl acetate/methanol mixture
(10:1). The mixture was cooled down to )20–()30) ꢁC
for several hours, the filtered compounds were washed
with ethyl acetate and dried in vacuo. The products were
suspended in an ethyl acetate/methanol mixture (5:1),
the mixture was heated under reflux for 2 h, cooled
down to )30 ꢁC for 17 h and filtered. The products were
washed with methanol to provide 11a and its (S)-enan-
tiomer 15a as colourless solids, respectively.
The diluted reaction mixture was injected onto a
gradient RP-HPLC-chromatograph with a Nucleosil
100 column (C-18.5 l250 · 4.6 mm) with a correspond-
ing precolumn (20 · 4.6 mm) and combined c-/UV-
detectors to isolate the radiolabelled compounds 11b,
11c, 15b and 15c, respectively. Radiochemical yield for
11b and 15b: 78%. Radiochemical yield for 11c and 15c:
41%.
HPLC
conditions:
Eluent A:
Eluent B:
CH3CN/H2O/TFA
950/50/1
CH3CN/H2O/TFA
50/950/1
Eluent B from 92% to
50% within 45 min and
then from 50% to 92%
within 10 min
1.5 mL/min
254 nm
6.4.11. ( )-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-iodo-phen-
oxy)-2-hydroxy-propylamino]-ethyl]-urea 11a. Yield:
1
48%. mp: 185–186 ꢁC. H NMR (300 MHz, DMSO-d6):
3
d [ppm]: 9.17 (s, 1H, NH), 7.80 (d, J ¼ 8:7 Hz, 2H,
Haryl), 7.74 (dd, 3J ¼ 7:8 Hz, 4J ¼ 1:5 Hz, 1H, Haryl),
7.47 (d, 3J ¼ 8:7 Hz, 2H, Haryl), 7.35–7.29 (m, 1H, Haryl),
Time
program:
3
4
7.00 (dd, J ¼ 8:1 Hz, J ¼ 0:9 Hz, 1H, Haryl), 6.73 (dt,
3J ¼ 7:5 Hz, 4J ¼ 1:1 Hz, 1H, Haryl), 6.69 (t, 3J ¼ 3:8 Hz,
1H, NH), 6.37 (broad, s, 3H, NH, OH, COOH), 4.03–
Flow:
k:
3
3.97 (m, 3H, CH2CH), 3.24 (q, J ¼ 5:7 Hz, 2H, CH2),
2.93–2.73 (m, 4H, 2CH2). 13C NMR (75.5 MHz,
DMSO-d6): d [ppm]: 168.00, 157.18, 155.20, 144.51,
139.01, 130.43, 129.80, 122.79, 116.59, 112.93, 86.73,
71.52, 67.54, 52.02, 49.03, 33.50. MS (MALDI-TOF):
m=z: 500 (M+H)þ, 522 (M+Na)þ. Anal. Calcd for
C19H22IN3O5: C, 45.71; H, 4.44; N, 8.42. Found: C,
45.52; H, 4.24; N, 8.20.
Rt (product 27.0–29.0 min (maximum
fraction): 27.6 min, average).
6.5. Quality control
6.4.12. (S)-N-(4-Carboxy-phenyl)-N0-[2-[3-(2-iodo-phen-
oxy)-2-hydroxy-propylamino]-ethyl]-urea 15a. Yield:
The product fraction (50 lL) was re-injected into the
HPLC column. The quality control did not show any
impurities within the c-range. Only the injection peak
was detectable within the UV range.
1
33%. mp: 193–194 ꢁC. H NMR (300 MHz, DMSO-d6):
3
d [ppm]: 9.24 (s, 1H, NH), 7.80 (d, J ¼ 8:7 Hz, 2H,

4130
S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
6.6. Reference control
11a or 15a (1 lmol kgꢀ1), the selective b1-AR antagonist
ICI 89,406 (1 lmol kgꢀ1 or 100 nmol kgꢀ1), the selective
b2-AR antagonist, ICI 118551 (1 lmol kgꢀ1 or
100 nmol kgꢀ1) or the nonselective b-AR antagonist,
propranolol (as HCl salt; 1 lmol kgꢀ1). Six sequential
arterial blood samples (ca. 100 lL) were taken from each
animal. An aliquot of whole blood was taken and the
remainder centrifuged to separate the plasma. Animals
were sacrificed by intravenous injection of sodium pen-
tobarbitone (Euthatal) at 200 mg (kg body weight)ꢀ1 at
selected times after injection of radioligand and tissues
were rapidly removed.
The radioiodinated products 11b, 11c, 15b or 15c were
verified by concentrating 25 lL of the isolated c-fraction
with 25 lg of the nonradioactive compounds 11a and
15a, each dissolved in 25 lL eluent B. The concentrated
50 lL mixtures were again injected into the HPLC col-
umn. Both the radiolabelled product and the nonradio-
active reference standard corresponded to each other.
7. Biology
7.1. Tissue preparation
In some animals, heads were removed using large scis-
sors, the brain removed from the cranium and brain
regions dissected. In all animals, the thorax was opened
by an incision on each side of the sternum. The heart
and lungs were removed together. The heart was dis-
sected into five regions, left and right atrial walls, left
and right ventricular walls and interventricular septum,
excluding the heart valves. The individual lobes of the
lung were separated and samples (ca. 50 mg) taken from
each lobe were combined. Samples (100–200 mg) from
liver, kidney and striated muscle were also removed and
a sample of urine taken from the bladder. Tissue sam-
ples were blotted and transferred to weighed vials for
reweighing and measurement of radioactivity using an
automated c-counter (Wallac Wizard 300 , Perkin–Elmer
Life Sciences, Boston, USA). Radioactivity was ex-
Microsomes were prepared by homogenizing ventricles
from DBA mice at 4 ꢁC for 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 (Ki-
nematica, Lucerne, Switzerland). Homogenates were
centrifuged at 45,000 · gmax for 15 min at 4 ꢁC. The pel-
lets were resuspended again in 1 mL of a buffer B con-
taining 1 mM EDTA, 10 mM HEPES, 0.1 mM
benzamidine (pH 7.4) and recentrifuged at 45,000 · gmax
for 15 min at 4 ꢁC. The pellets were resuspended in 1 mL
of buffer B and centrifuged at 10,000 · gmax for 10 min at
4 ꢁC. The supernatants were recentrifuged at
45,000 · gmax for 15 min at 4 ꢁC. The pellets, partially
enriched membranes, were resuspended in buffer C
(50 mM TrisÆHCl, 5 mM MgCl2 (pH 7.4)), and stored
frozen at )80 ꢁC.
pressed as cpm (g wet tissue)ꢀ1
.
In a previous study the tissue uptake of radioactivity
20 min after injection of 2-[125I]I-ICI-H (Scheme 1)
increased linearly with injected dose and predosing with
2-I-ICI-H 1b had no effect. It was assumed that radio-
labelled acids (11b–c and 15b–c) would be comparable.
Therefore, to correct for differences in animal body
weight and injected dose, results were expressed as an
uptake index, defined as
For competition binding studies, the prepared mem-
branes were resuspended in buffer D (10 mM Tris HCl,
154 mM NaCl, 0.1 mM ascorbic acid, pH 7.4). 15 lg of
membranes were incubated with a constant concentra-
tion of [125I]ICYP (80 pM) and with varying concentra-
tions (1 pM–100 lM) of compounds 8–11a and 12–15a
(Scheme 4). Reactions were conducted at 37 ꢁC for
60 min. Reactions were stopped by filtering onto
Whatman GF/B filters and washed with 0.9% NaCl. The
membrane bound radioactivity was determined in a c-
scintillation counter. Competition binding curves were
analyzed by nonlinear regression analysis as previously
described.60–62
Uptake index
Tissue radioactivity ðcpmÞ=tissue wet weight ðgÞ
¼
:
Radioactivity injected ðcpmÞ=body weight ðgÞ
Radioactive metabolites in plasma were assessed by
gradient RP-HPLC as described above. Rats (250–320g)
were anaesthetized for insertion of catheters into the
ventral tail artery and one lateral tail vein and were al-
lowed to recover from the anaesthesia for ꢃ2–3 h before
injection of 37 MBq 15c via the tail vein catheters. Blood
(1 mL) was collected from the tail artery catheter at 2 or
25 min after injection of radioligand. It was spiked with
45 lg nonradioactive 15a. Cell-free plasma was prepared
by centrifuging each blood sample at 2000g for 2 min.
Ice-cold acetonitrile (0.7 mL) was added to the plasma
(0.5 mL) and the precipitated proteins were removed by
centrifugation. 200 lL of the resulting spiked superna-
tant was injected on to the HPLC column (HPLC-con-
ditions: see above). To evaluate the method 1.4 MBq 15c
were added to 1 mL blood in vitro and processed as
described above.
7.2. Animals
Adult male Sprague Dawley rats (250–320 g) were
anaesthetized by isoflurane/N2O/O2 and catheters (o.d.
1 mM) were inserted into the ventral tail artery and one
lateral tail vein of each rat. Animals were allowed to
recover from the anaesthesia for 2–3 h and during the
studies they were conscious but under light restraint.
Each radioiodinated compound (11b, 15b or 15c) at
2–7 MBq kgꢀ1 body weight) was injected as a bolus
(1 lL gꢀ1 body weight) via the tail vein. Aliquots of each
injectate were diluted in ethanol/saline and measured to
determine the radioactivity injected into each animal. In
some animals, nonradioactive antagonists were injected
as a bolus (1 lL gꢀ1 body weight) via the tail vein 5 min
before injection of the radioligand. Ligands used were

S. Wagner et al. / Bioorg. Med. Chem. 12 (2004) 4117–4132
4131
€
21. Schafers, M.; Dutka, D.; Rhodes, C. G.; Lammertsma, A.
A.; Hermansen, F.; Schober, O.; Camici, P. G. Circ. Res.
1998, 82, 57–62.
Acknowledgements
This work was supported by the SFB 556 (grants C1 and
C4) ‘Heart Failure and Arrhythmias’ of the Deutsche
Forschungsgemeinschaft (DFG), Bonn, Germany. The
authors gratefully thank Mrs. Monika Trub and Mrs.
€
22. Schafers, M.; Lerch, H.; Wichter, T.; Rhodes, C. G.;
Lammertsma, A. A.; Borggrefe, M.; Hermansen, F.;
Schober, O.; Breithardt, G.; Camici, P. G. J. Am. Coll.
Cardiol. 1998, 32, 181–186.
€
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