Synthesis of (R)- and (S)-[O-methyl-11C]N-[2-[3-(2-cyano- phenoxy) -2-hydroxy-propylamino]-ethyl]-N′-(4-methoxy-phenyl)-urea as candidate high affinity β1-adrenoceptor PET radioligands

Article abstract of DOI:10.1002/jlcr.965

Molecular imaging and quantification of myocardial β₁- adrenoceptor (AR) rather than total β-AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β₁-AR density is reduced in patients with chro

Full text of DOI:10.1002/jlcr.965

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 721–733.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.965
Research Article
Synthesis of (R)- and (S)-[O-methyl-11C]N-[2-[3-(2-
cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-
methoxy-phenyl)-urea as candidate high affinity b1-
adrenoceptor PET radioligands
Stefan Wagnera,*, Marilyn P. Lawa, Burkhard Riemanna, Victor W. Pikeb,
Hans-Jorg Breyholza, Carsten Holtkea, Andreas Fausta, Otmar Schobera,
¨
¨
Michael Schafersa and Klaus Kopkaa
¨
a Department of Nuclear Medicine, Albert-Schweitzer-Str. 33, University Hospital
Munster, 48149 Munster, Germany
¨
¨
b Molecular Imaging Branch, National Institute of Mental Health, National Institutes of
Health, 10 Center Drive, Rm B3 C346A, MSC 1003, Bethesda, MD 20892-1003, USA
Summary
Molecular imaging and quantification of myocardial b1-adrenoceptor (AR) rather
than total b-AR density is of great clinical interest since cardiac biopsy studies suggest
that myocardial b1-AR density is reduced in patients with chronic heart failure
whereas cardiac b2-AR density may vary. Positron emission tomography (PET), with
appropriate radioligands, offers the possibility to assess b-AR density non-invasively
in humans. However, no PET radioligand for the selective imaging of cardiac b1-ARs
is clinically available. Here some derivatives of the well characterized b1-AR selective
antagonist, ICI 89,406, namely the enantiomers of N-[2-[3-(2-cyano-phenoxy)-2-
hydroxy-propylamino]-ethyl]-N0-(4-hydroxy-phenyl)-urea (5a and 5b) were synthe-
sized and evaluated in vitro. The (R)-isomer 5a was more b1-selective but has lower
affinity than its (S)-enantiomer 5b (b1-AR selectivity: 6100 vs 1240; b1-affinity:
K1=0.288 nM vs K1=0.067 nM). Etherification of the analogous desmethyl pre-
cursors, 5e and 5f, respectively, with [11C]iodomethane gave 11C-labelled versions of
5a and 5b, namely 5g and 5h, in 44 ꢀ 5% radiochemical yield (decay-corrected) and
97.4 ꢀ 1.3% radiochemical purity with specific radioactivities of 26.4 ꢀ 9.4 GBq/
mmol within 41.2 ꢀ 3.4 min from the end of bombardment (n=14). 5g and 5h are now
*Correspondence to: S. Wagner, Department of Nuclear Medicine, Albert-Schweitzer-Str. 33, University
Hospital Munster, 48149 Munster, Germany. E-mail: stwagner@uni-muenster.de.
¨
¨
Contract/grant sponsor: DFG; contract/grant number: SFB 556
Contract/grant sponsor: DFG; contract/grant number: SCHA 758/5-1
Contract/grant sponsor: SFB; contract/grant number: 656
Copyright # 2005 John Wiley & Sons, Ltd.
Received 24 February 2005
Revised 12 April 2005
Accepted 2 May 2005

722
S. WAGNER ET AL.
being evaluated as candidate radioligands for myocardial b1-ARs. Copyright # 2005
John Wiley & Sons, Ltd.
Key Words: 3-aryloxy-2-propanolamine; ICI 89,406; b1-adrenoceptor selective ligand;
PET radioligand
Introduction
b-ARs belong to the rhodopsin/b2-adrenergic receptor-like receptors, that
represent a subfamily of the G protein-coupled-receptors (GPCRs).1 They can
be subdivided into at least three discrete subtypes, the b1-, b2-AR2 and the
atypical b3-AR.3,4 b1-AR agonists cause increased cardiac contractility and
heart rate while b2-selective AR agonists mediate vasodepression and
bronchodilation.5 The so-called atypical b3-ARs are involved in lipolysis.3
In heart disease, both the total b-AR density and the b1/b2-AR ratio may
change. For example, reduced myocardial b-AR density has been found in
hypertension, heart failure, ischaemia and both hypertrophic (HCM) and
dilated cardiomyopathies (DCM).6–11
A selective reduction in b1-ARs without change in b2-AR density is often
observed in the failing human heart.5 Therefore, a means for the visualization
and quantification of the b1-AR density rather than total b-AR density in the
human heart is of great interest in basic research and clinical application.12
The molecular imaging techniques, single photon emission computed
tomography (SPECT) and positron emission tomography (PET), with suitable
radioligands may be ideal tools for assessing b-AR density non-invasively in
humans.13,14 Some subtype-b1-AR selective radioligands have been developed
for PET, for example (+/ꢁ)-[11C]HX-CH 44,15 (S)-[11C]bisoprolol,16
[11C]CGP 20712A17 and its (S)-enantiomer, [11C]CGP-26505.18 The clinical
use of these radioligands, however, is limited because of high non-specific
binding, rapid metabolism or tissue uptake that does not reflect binding to
b-ARs.15–18
ICI 89,406 (Scheme 1) is an example of a designed b1-selective AR
antagonist. Although membrane studies show that the (S)-enantiomer is the
more potent enantiomer even the racemate produces effective b1-AR blockade
during exercise in patients with angina pectoris.19–21 Therefore, we chose this
compound as a lead structure to develop new b1-selective AR radioligands. In
our initial study, the b1-AR affinity and selectivity of a number of novel
derivatives of ICI 89,406 were determined.22 One of these derivatives, racemic
I-ICI-H and its radioiodinated analogue (Scheme 1), showed high affinity and
selectivity for b1-ARs in myocardial membranes in vitro but high non-specific
binding in vivo.23 Another derivative, racemic I-ICI-COOH and its
(S)-enantiomer, were also characterized by high b1-AR affinity and selectivity
in vitro.24 The 125/123I-labelled counterparts (Scheme 1) were evaluated as
Copyright # 2005 John Wiley & Sons, Ltd.
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HIGH AFFINITY b1-ADRENOCEPTOR PET RADIOLIGANDS
723
NH
NH
*
NH
O
O
OH
X
Y
ICI 89,406 X: CN
I-ICI-H
Y: H
Y: H
Y: COOH
racemic
X: I, 125I or 123
I
I
racemic
I-ICI-COOH X: I, 125I or 123
(S) or racemic
Scheme 1. Lead structure ICI 89,406 and its iodinated derivatives23,24
potential SPECT ligands using Sprague Dawley rats but were unsuitable
because of a high degree of deiodination.24 It is apparent that high non-specific
binding and rapid deiodination in vivo preclude the use of such iodinated
ligands for SPECT or PET. Ligands labelled with the positron-emitter,
carbon-11 (t1/2=20.4 min), may be less susceptible to problematic metabolism.
The work presented here describes the synthesis and the in vitro pharmacology
of the non-iodinated ICI 89,406 derivatives, (R)- and (S)-N-[2-[3-(2-cyano-
phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-methoxy-phenyl)-urea (5a and
5b, respectively) and their precursors (5c–5f). The in vitro studies showed that
the (R)-isomer (5a) is more selective but has lower affinity for b1-ARs than the
(S)-isomer (5b). Therefore, 11C-labelled versions of each enantiomer (5g and
5h) were synthesized as potential selective and high affinity b1-AR radioligands
for PET (Scheme 2).
Results and discussion
The syntheses of the ureas 5a–f were achieved through a five- or six-step
sequence (Scheme 2). Treatment of the homochiral glycidyl-3-nitrobenzene
sulphonates 2a and 2b with 2-cyano-phenol 1 under basic conditions yielded
the corresponding oxiranes 3a and 3b in good yields (85–92%). Comparison of
the measured optical rotations with literature values showed that the
stereochemistry was retained; the absolute [a]D values were within errors.25
The ureas 5a–d were prepared from the oxiranes 3a and 3b and the amines 4a
and 4b via nucleophilic ring opening. Compounds 4a and 4b were synthesized
through a three-step sequence as previously described (steps not shown in
Scheme 2).22 The chemical yields ranged from 35 to 45%. Finally, 5c and 5d
were deprotected by hydrogenolysis resulting in the phenol precursors 5e and
5f (51–56%).
The structure–activity relationships (SAR) between the ligands and b1-ARs
were assessed in competition studies using [125I]ICYP and mouse ventricular
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 721–733

724
S. WAGNER ET AL.
O
*
NosO
+
OH
2a
(R )-
1
CN
2b
(S)-
Na2CO3,
butanone,
∆
O
O
*
CN
3a
3b
(R)-
(S)-
NH
NH
H3N+
Cl-
, NaOH, H2O, n-propanol
O
OR
4a
R: CH3
4b
R: CH2Ph
NH
NH
*
NH
O
O
OH
CN
OR
5a
(R)-, R: CH3
(S)-, R: CH3
5b
5c
5d
(R)-, R: CH2Ph
(S)-, R: CH2Ph
H2, Pd/C,
MeOH,
5e (R)-, R: H
∆
11CH3I, NaH,
DMF, 40˚C
5f
(S)-, R: H
(R)-, R: 11CH3
5g
(S)-, R: 11CH3
5h
Scheme 2. Synthesis of the chiral b1-AR ligands 5a–h
membrane preparations. The high- and low-affinity IC50 values for the b1- and
b2-ARs of the non-radioactive 3-aryloxy-2-propanolamine derivatives 5a–f
were calculated by non-linear regression analysis of membrane radioactivity.
The high- and low-affinity inhibition constants (K1 for the b1-ARs and K2
for the b2-ARs) were obtained from the IC50 values by the method of
Copyright # 2005 John Wiley & Sons, Ltd.
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HIGH AFFINITY b1-ADRENOCEPTOR PET RADIOLIGANDS
725
Table 1. Inhibition constants and calculated b1-AR selectivities of the ligands 5a–f
determined by a radioligand binding assay using mouse ventricular membrane
preparations, plus calculated ligand logP and logD values of the ligands
Compound
K1 (nM)a
K2 (nM)a
b1-selectivityb
logPc
logDc
5a
5b
5c
5d
0.288 ꢀ 0.032
0.067 ꢀ 0.016
20.7 ꢀ 4.2
0.085 ꢀ 0.019
0.269 ꢀ 0.067
0.328 ꢀ 0.046
0.28 ꢀ 0.12
1760 ꢀ 300
83 ꢀ 33
3900 ꢀ 150
119 ꢀ 19
569 ꢀ 277
149 ꢀ 27
41 ꢀ 3
6100 ꢀ 1540
1240 ꢀ 280
188 ꢀ 45
1400 ꢀ 160
2120 ꢀ 450
453 ꢀ 106
149 ꢀ 86
1.22
1.22
2.87
2.87
0.83
0.83
1.57
ꢁ0.21
ꢁ0.21
1.55
1.55
5e
5f
ICI 89 406
ꢁ0.55
ꢁ0.55
0.13
a Inhibition constants for non-selective b-AR ligand [125I]ICYP binding at b1- and b2-ARs expressed in
mouse ventricular membrane preparations given as mean ꢀ SEM, n=3.
b The ratios of the low- over the high-affinity inhibition constants (K2/K1) indicate the b1-AR selectivities of
the non-radioactive b1-AR ligands, noted as mean ꢀ SEM, n=3.
c logP values of the neutral form and logD values calculated by ACD/LogD Suite (logD=logP at
physiological pH 7.4 with consideration of charged species).
Cheng–Prusoff26 using the previously experimentally determined KD value of
[
125I]ICYP (32.3 ꢀ 1.9 pM).22 The ratios of the low- to high-affinity inhibition
constants (K2/K1) yield the b1-selectivities of the unlabelled compounds 5a–f
(Table 1). The calculated logP and logD values (ACD/LogD Suite) of
compounds 5a–f are also listed in Table 1 and indicate the change in
lipophilicities ensuing chemical modifications of the lead compound, ICI
89,406. The logP and logD values of 5a and 5b are low and similar to the
values for (S)-CGP 12177 (logP=1.81),13 which when labelled with carbon-11
is an effective radioligand for imaging cell surface b-ARs in human
myocardium with PET. All compounds 5a–f possess high affinity to b1-ARs.
With the exception of 5c (K1=20.7 nM) the ligands showed b1-AR affinities in
the subnanomolar range (K1=0.067–0.328 nM).
Usually the (S)-enantiomer of a b-blocker possesses an up to 100-fold higher
affinity to b-ARs than the corresponding (R)-enantiomer.27,28 This feature was
shown by the ligands 5a and 5b. The (R)-enantiomer 5a has at least four times
less b1-AR affinity than the (S)-isomer 5b (K1=0.288 nM vs K1=0.067 nM).
However, the b1-AR selectivity of 5a, is five times greater than that of 5b (6100
vs 1240). Furthermore, the b1-AR selectivities of the methoxy compounds 5a
and 5b are at least an order of magnitude greater than that of the racemic lead
compound, ICI 89,406 (149). A similar trend is seen in the benzyloxy-pair 5c
and 5d. The (R)-enantiomer 5c has about 240-times less b1-affinity than the
(S)-isomer 5d (K1=20.7 nM vs K1=0.085 nM). In contrast, the (S)-enantiomer
5d exhibits higher b1-AR selectivity than its (R)-enantiomer 5c (1400 vs 188).
Contrary to expectations, both enantiomers of the hydroxy-pair 5e and 5f
display nearly equal b1-AR affinities (K1=0.269 and 0.328 nM). Similar to
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726
S. WAGNER ET AL.
compounds 5a and 5b, the (R)-enantiomer 5e possesses a nearly five-fold
higher b1-AR selectivity than its (S)-enantiomer 5f (2120 vs 453).
For the compounds 5a–f, with their specific substitution pattern of a
2-cyanophenyloxy core and a 4-hydroxyphenyl or an 4-alkoxyphenyl unit, two
SAR-conclusions can be drawn. Firstly, a more sterically demanding residue
and/or a more lipophilic aromatic substituent at the oxygen of the phenyl-4-
position (5c and 5d R:Bn) leads to an increased difference between the b1-AR
affinities of the (R)- and (S)-enantiomers compared to enantiomer-pairs with
a smaller substituent at this position (5a and 5b R:Me; 5e and 5f R:H). The
(R)-enantiomer 5c has 244-fold less b1-affinity than the (S)-enantiomer 5d.
The tendency is reduced in the methoxy-pair 5a and 5b (four-fold) and the
hydroxy-pair 5e and 5f (0.8-fold). Secondly, an (S)-enantiomer with a benzyl-
substituent at the phenolic oxygen is more b1-selective than its (R)-enantiomer,
whereas smaller substituents shift the increased b1-AR selectivity to the (R)-
enantiomer ((R)-enantiomer to (S)-enantiomer ratios of b1-AR selectivities: 5c
and 5d R:Bn: 0.13; 5a and 5b R:Me: 4.9; 5e and 5f R:H:4.7).
As a result of these binding affinity determinations, the potent methoxy
derivatives 5a and 5b were chosen for conversion into corresponding potential
high affinity and selective b1-AR radioligands for PET, namely 5g and 5h,
respectively. Thus, the desmethyl precursors 5e and 5f were treated with
sodium hydride and [11C]iodomethane in DMF at 408C for 5 min to yield
11C-labelled 5g and 5h, respectively (Scheme 2), which were then purified with
HPLC. The activities of 5g and 5h ranged from 1.2 to 2.7 GBq (EOS),
representing 44.1 ꢀ 5.0% decay-corrected radiochemical yields at
41.2 ꢀ 3.4 min from the end of radionuclide production. Radiochemical
purities of 97.4 ꢀ 1.3% and specific activities of 26.4 ꢀ 9.4 GBq/mmol (13.3–
47.4 GBq/mmol) were achieved (n=14), as determined by analytical radio-
HPLC. The new radioligands 5g and 5h are now being evaluated in rodents
with PET.
Experimental
General 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
1
SMP3 capillary melting point apparatus. H-NMR and 13C-NMR spectra
were recorded on a Bruker ARX 300 spectrometer and an AMX 400
spectrometer, respectively. Mass spectrometry was performed using a Varian
MAT 212 (EI=70 eV) spectrometer and a Bruker MALDI-TOF-MS Reflex
IV (matrix: DHB). [a]D values were determined on a Perkin-Elmer 341
polarimeter. Elemental analysis was realised by a Vario EL III analyser.
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HIGH AFFINITY b1-ADRENOCEPTOR PET RADIOLIGANDS
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Radiosyntheses were carried out using an automated PET tracer synthesizer
(TRACERLab Fxc; GE Functional Imaging GmbH). Separation of the
radiosynthesized compounds, and determinations of the radiochemical yields
were performed with radio-HPLC using a Syknm S1021 pump, a Knauer
K-2001 UV-detector (wavelength 254 nm),
a Raytest Ramona-90/92
g-detector, a Nucleosil 100-10 C18 precolumn (20 ꢂ 8 mm2) and a Nucleosil
100-10 C18 column (250 ꢂ 8 mm2). Sample injection was carried out using a
VICI injector block (type C6W incl. 1000 ml loop). The recorded data were
processed by the TRACERLab C software (GE Functional Imaging GmbH).
The radiochemical purities and the specific activities were acquired with a
radio-HPLC system composed of a Syknm S1021 pump, a Knauer K-2501
UV-detector (wavelength 254 nm), a Crismatec Na(Tl) Scintibloc 51 SP51
g-detector, a Nucleosil 100-3 C18 column (200 ꢂ 3 mm2), a VICI injector block
(type C1 incl. 20 ml loop) and the NINA version 4.8, Rev. 4 software (GE
Functional Imaging GmbH).
Synthetic methods
Synthesis of oxiranes (3a–b)
General procedure. 2-Cyano-phenol 1 (2 eq.), (R)-glycidyl-3-nitrobenzene
sulphonate 2a or (S)-glycidyl-3-nitrobenzene sulphonate 2b (1 eq.) and
anhydrous K2CO3 (6 eq.) were refluxed in dry 2-butanone (3.8 ml/mmol
sulphonate) for 6–7 h and then stirred at RT for 16–72 h under an argon
atmosphere. The mixture was filtered and the filter cake washed with 2-
butanone. Solvent from the combined filtrates was evaporated off. The residue
was dissolved in water and CH2Cl2. The pH of the aqueous layer was adjusted
to be within the range 11–13 and the aqueous layer was extracted. The layers
were separated. The aqueous layer was extracted with CH2Cl2 twice, the
combined organic layers dried (Na2SO4) and the solvent evaporated off. The
residue was recrystallized from diisopropyl ether–CHCl3 (ꢃ3:2 v/v) at ꢁ308C
to provide 3a–b as colourless solids.
(R)-2-(2-cyano-phenoxymethyl)-oxirane (3a). Yield: 85%. Mp.: 85–868C.
1H-NMR (300 MHz, CDCl3): d [ppm]: 7.58–7.50 (m, 2H, HAr), 7.09–7.01 (m,
2H, HAr), 4.37 (dd, 2J=11.6 Hz, 3J=3.2 Hz, 1H, 1CH2), 4.12 (dd,
2J=11.6 Hz, 3J=5.3 Hz, 1H, 1CH2), 3.40–3.37 (m, 1H, CH), 2.93 (dd,
3
2
3
2J=4.8 Hz, J=4.2 Hz, 1H, 1CH2), 2.84 (dd, J=5.0 Hz, J=2.4 Hz, 1H,
1CH2). 13C-NMR (75.5 MHz, CDCl3): d [ppm]: 160.01, 134.35, 133.87, 121.44,
116.26, 112.79, 102.50, 69.49, 49.85, 44.55. [a]2D0=ꢁ15.88 (c=1.0, EtOH); Lit.:
[a]2D5=ꢁ16.08 (c=1.0, EtOH).25 Analytically calculated for C10H9NO2: C
68.56, H 5.12, N 8.00. Found: C 68.93, H 5.07, N 8.16.
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S. WAGNER ET AL.
(S)-2-(2-cyano-phenoxymethyl)-oxirane (3b). Yield: 92%. Mp.: 868C; Lit.:
88–898C.25 1H-NMR (300 MHz, CDCl3): d [ppm]: 7.58–7.49 (m, 2H, HAr),
2
3
7.10–7.01 (m, 2H, HAr), 4.37 (dd, J=11.4 Hz, J=3.0 Hz, 1H, 1CH2), 4.12
(dd, 2J=11.4 Hz, 3J=5.4 Hz, 1H, 1CH2), 3.41–3.37 (m, 1H, CH), 2.93
2
3
(‘t’, J=4.5 Hz, 1H, 1CH2), 2.84 (dd, J=5.0 Hz, J=2.6 Hz, 1H, 1CH2).
13C-NMR (75.5 MHz, CDCl3): d [ppm]: 159.99, 134.35, 133.84, 121.42,
116.26, 112.80, 102.45, 69.47, 49.85, 44.54. [a]2D0=+18.18 (c=1.0, EtOH); Lit.:
[a]2D5=+17.698 (c=1.0, EtOH).25 Analytically calculated for C10H9NO2:
C 68.56, H 5.12, N 8.00. Found: C 68.80, H 4.96, N 8.38.
Synthesis of the N-aryl-N0-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea
derivatives (5a–d)
General procedure. 3a or 3b (1 eq.), N-(2-amino-ethyl)-N0-(4-methoxy-phenyl)-
urea hydrochloride 4a (1 eq.) or N-(2-amino-ethyl)-N0-(4-benzyloxy-phenyl)-
urea hydrochloride 4b (1 eq.), prepared as previously described,22 and 1 N
NaOH (1.05 eq.) were heated in n-propanol (1.8–2.9 ml/mmol oxirane) and
water (1.0–2.4 ml/mmol oxirane) up to 908C for 2–3 h.
Purification procedure A (5a and 5c): The mixture was evaporated to
dryness. Water was added and stirred. The product was filtered off and dried
in vacuo. The crude product was refluxed in methanol, the hot suspension was
filtered and the filtrate was left for crystallization at +4 to ꢁ308C. Additional
amounts of product were obtained by crystallization from the mother liquor.
Purification procedure B (5b and 5d): Water was added. The product was
filtered off and washed with water–diethyl ether. The crude product was
purified by silica gel chromatography (ethyl acetate–MeOH 4:1 v/v). The
product fraction was evaporated and recrystallized from methanol.
After the purification procedures A or B, the urea derivatives 5a–d were
obtained as colourless solids.
(R)-N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-meth-
1
oxy-phenyl)-urea (5a). Yield: 35%. Mp.: 126–1278C. H-NMR (400 MHz,
3
4
DMSO-d6): d [ppm]: 8.29 (s, 1H, NH), 7.69 (dd, J=7.7 Hz, J=1.7 Hz, 1H,
HAr), 7.61 (ddd, J=8.7 Hz, J=7.4 Hz, J=1.4 Hz, 1H, HAr), 7.27–7.23 (m,
3
3
4
3
3H, HAr), 7.06 (dt, J=7.5 Hz, J=0.9 Hz, 1H, HAr), 6.78 (dd, J=6.8 Hz,
4
3
3
4J=2.3 Hz, 2H, HAr), 6.02 (t, J=5.1 Hz, 1H, NH), 5.01 (s, br, 1H, OH),
4.16–3.92 (m, 3H, CH2CH), 3.67 (s, 3H, CH3), 3.14 (q, 3J=5.9 Hz, 2H, CH2),
2.75–2.60 (m, 4H, 2CH2). 13C-NMR (100.6 MHz, DMSO-d6): d [ppm]: 160.52,
155.71, 154.03, 135.14, 133.89, 133.76, 121.13, 119.53, 116.58, 114.02, 113.35,
100.83, 71.79, 68.12, 55.30, 52.13, 49.53, 39.89. Analytically calculated for
C20H24N4O4: C 62.49, H 6.29, N 14.57. Found: C 62.25, H 6.24, N 14.62.
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HIGH AFFINITY b1-ADRENOCEPTOR PET RADIOLIGANDS
729
(S)-N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-methoxy-
phenyl)-urea (5b). Yield: 45%. Mp.: 128–1298C. 1H-NMR (400 MHz,
3
DMSO-d6): d [ppm]: 8.30 (s, 1H, NH), 7.69 (d, J=7.8 Hz, 1H, HAr), 7.62
(‘t’, J=7.4 Hz, 1H, HAr), 7.27–7.23 (m, 3H, HAr), 7.06 (t, J=7.5 Hz, 1H,
3
3
3
3
HAr), 6.78 (d, J=8.7 Hz, 2H, HAr), 6.03 (t, J=5.3 Hz, 1H, NH), 5.02 (s, br,
3
1H, OH), 4.16–3.90 (m, 3H, CH2CH), 3.68 (s, 3H, CH3), 3.15 (q, J=5.7 Hz,
2H, CH2), 2.76–2.61 (m, 4H, 2CH2). 13C-NMR (100.6 MHz, DMSO-d6): d
[ppm]: 160.50, 155.68, 154.01, 135.13, 133.89, 133.77, 121.13, 119.48, 116.54,
114.01, 113.35, 100.82, 71.76, 68.05, 55.29, 52.09, 49.49, 39.91. Analytically
calculated for C20H24N4O4: C 62.49, H 6.29, N 14.57. Found: C 62.11, H 6.12,
N 14.17.
(R)-N-(4-benzyloxy-phenyl)-N0-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propyla-
1
mino]-ethyl]-urea (5c). Yield: 41%. Mp.: 117–1188C. H-NMR (300 MHz,
3
DMSO-d6): d [ppm]: 8.31 (s, 1H, NH), 7.68 (d, J=7.5 Hz, 1H, HAr), 7.63–
7.58 (m, 1H, HAr), 7.43–7.23 (m, 8 H, HAr), 7.06 (t, 3J=7.4 Hz, 1H, HAr), 6.87
3
3
(d, J=9.0 Hz, 2H, HAr), 6.04 (t, J=5.6 Hz, 1H, NH), 5.02 (s, 3H, OH and
3
CH2), 4.16–3.90 (m, 3H, CH2CH), 3.15 (q, J=5.1 Hz, 2H, CH2), 2.76–2.58
(m, 4H, 2CH2), 1.85 (s, br, 1H, NH). 13C-NMR (75.5 MHz, DMSO-d6): d
[ppm]: 160.48, 155.63, 153.03, 137.52, 135.08, 134.13, 133.74, 128.46, 127.78,
127.69, 121.08, 119.39, 116.54, 115.06, 113.32, 100.81, 71.74, 69.59, 68.08,
52.12, 49.49. Analytically calculated for C26H28N4O4: C 67.81, H 6.13, N
12.17. Found: C 67.56, H 6.04, N 12.11.
(S)-N-(4-benzyloxy-phenyl)-N0-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propyla-
1
mino]-ethyl]-urea (5d). Yield: 36%. Mp.: 113–1148C. H-NMR (300 MHz,
3
DMSO-d6): d [ppm]: 8.31 (s, 1H, NH), 7.68 (d, J=7.5 Hz, 1H, HAr), 7.63–
7.58 (m, 1H, HAr), 7.43–7.23 (m, 8 H, HAr), 7.06 (t, 3J=7.5 Hz, 1H, HAr), 6.87
3
3
(d, J=9.0 Hz, 2H, HAr), 6.04 (t, J=5.1 Hz, 1H, NH), 5.02 (s, 3H, OH and
3
CH2), 4.16–3.90 (m, 3H, CH2CH), 3.15 (q, J=5.4 Hz, 2H, CH2), 2.76–2.61
(m, 4H, 2CH2), 1.96 (s, br, 1H, NH). 13C-NMR (75.5 MHz, DMSO-d6): d
[ppm]: 160.50, 155.64, 153.02, 137.53, 135.10, 134.15, 133.75, 128.45, 127.78,
127.70, 121.09, 119.40, 116.55, 115.08, 113.32, 100.81, 71.74, 69.59, 68.09,
52.12, 49.49, 39.84. Analytically calculated for C26H28N4O4: C 67.81, H 6.13,
N 12.17. Found: C 62.48, H 5.97, N 12.16.
Synthesis of the N-aryl-N0-[2-[3-aryloxy-2-hydroxy-propylamino]-ethyl]-urea
derivatives (5e–f)
General procedure. 5c or 5d (1 eq.), methanol (46 ml/mmol) and Pd/C (10%,
47 mg/mmol) were stirred in a H2-atmosphere at RT for 15–16 h. Then the
mixture was refluxed for 6–8.5 h. The hot reaction mixture was filtered and the
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S. WAGNER ET AL.
filtrate was evaporated to dryness. The residue was purified by silica gel
chromatography (ethyl acetate–MeOH 4:1 v/v) to provide the urea derivative
5e or 5f (Rf: 0.15) as a colourless solid.
(R)-N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-hydro-
1
xy-phenyl)-urea (5e). Yield: 51%. Mp.: 120–1228C. H-NMR (300 MHz,
DMSO-d6): d [ppm]: 8.87 (s, br, 1H, OH), 8.14 (s, 1H, NH), 7.69 (dd,
3J=7.7 Hz, 4J=1.4 Hz, 1H, HAr), 7.61 (ddd, 3J=8.4 Hz, 3J=7.7 Hz,
4J=1.1 Hz, 1H, HAr), 7.24 (d, 3J=8.7 Hz, 1H, HAr), 7.14–7.03 (m, 3H,
3
4
3
HAr), 6.78 (dd, J=6.8 Hz, J=2.1 Hz, 2H, HAr), 5.98 (t, J=5.4 Hz, 1H,
3
NH), 4.98 (s, br, 1H, OH), 4.16–3.88 (m, 3H, CH2CH), 3.13 (q, J=5.8 Hz,
2H, CH2), 2.75–2.59 (m, 4H, 2CH2). 13C-NMR (75.5 MHz, DMSO-d6):
d [ppm]: 160.50, 155.77, 152.01, 135.11, 133.76, 132.27, 121.10, 119.93, 116.54,
115.19, 113.34, 100.81, 71.76, 68.07, 52.12, 49.55. Analytically calculated for
C19H22N4O4: C 61.61, H 5.99, N 15.13. Found: C 61.24, H 5.98, N 14.92.
(S)-N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-hydro-
xy-phenyl)-urea (5f). Yield: 56%. Mp.: 118–1198C. 1H-NMR (300 MHz,
DMSO-d6): d [ppm]: 8.87 (s, br, 1H, OH), 8.14 (s, 1H, NH), 7.69
(dd, 3J=7.7 Hz, 4J=1.4 Hz, 1H, HAr), 7.61 (ddd, 3J=8.7 Hz, 3J=
7.4 Hz, 4J=1.5 Hz, 1H, HAr), 7.24 (d, 3J=8.7 Hz, 1H, HAr), 7.14–7.03
3
4
3
(m, 3H, HAr), 6.78 (dd, J=6.8 Hz, J=2.0 Hz, 2H, HAr), 5.97 (t, J=5.4 Hz,
1H, NH), 5.01 (s, br, 1H, OH), 4.14–3.89 (m, 3H, CH2CH), 3.13
3
(q, J=6.0 Hz, 2H, CH2), 2.75–2.60 (m, 4H, 2CH2). 13C-NMR (75.5 MHz,
DMSO-d6): d [ppm]: 160.49, 155.81, 152.02, 135.12, 133.78, 132.27, 121.13,
119.96, 116.57, 115.18, 113.33, 100.82, 71.77, 68.08, 52.11, 49.56. Analytically
calculated for C19H22N4O4: C 61.61, H 5.99, N 15.13. Found: C 61.14, H 5.94,
N 14.88.
Radiosynthesis of (R)- and (S)-[O-methyl-11C]N-[2-[3-(2-cyano-phenoxy)-
2-hydroxy-propylamino]-ethyl]-N0-(4-methoxy-phenyl)-urea (5g and 5h).
[11C]Carbon dioxide was produced by the 14N(p,a)11C nuclear reaction on
research grade nitrogen containing 2.5% oxygen, using 11 MeV proton beams
at currents of 40 mA from a CTI-RDS-111 cyclotron. The [11C]carbon dioxide
was trapped from the target gas in a stainless steel loop cooled with liquid
nitrogen to ꢁ1508C. [11C]Iodomethane was prepared from [11C]carbon dioxide
as previously described.29 Phenol precursor 5e or 5f (1.0 mg; 2.70 mmol) was
treated with NaH (60% oil suspension; 1.5 mg; 37.5 mmol) in DMF (200 ml)
and [11C]CH3I at 408C for 5 min. After heating the reaction mixture to 508C,
water for injection (200 ml) was added and the crude mixture was loaded onto a
semi-preparative HPLC-column (see Synthetic methods). Product 5g or 5h
was eluted with 20 mM Na2HPO4-buffer/EtOH 4/1 at a flow of 4 ml/min at
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HIGH AFFINITY b1-ADRENOCEPTOR PET RADIOLIGANDS
731
11.9–14.5 min. The product solution was filtered through a sterile filter REF
F60 (0.2 mm, pvb Medizintechnik GmbH). The time of synthesis and
purification was 41.2 ꢀ 3.4 min from the EOB. The radiochemical yield
averaged 44.1 ꢀ 5.0% (decay-corrected) and the radiochemical purity,
determined via radio-HPLC (see Synthetic methods, eluent: H2O/MeCN/
TFA 700/300/1, flow: 0.3 ml/min, retention time: 11.1 min), was 97.4 ꢀ 1.3%
with a specific activity of 26.4 ꢀ 9.4 GBq/mmol (13.3–47.4 GBq/mmol) at EOS
(n=14).
Ligand binding assay
Microsomes were prepared by homogenizing ventricles from DBA mice at 48C
for 90 s in buffer A (1 ml) containing 10 mM EDTA, 10 mM HEPES and
0.1 mM benzamidine (pH 7.4), using a Polytron PT 3000 (Kinematica,
Lucerne, Switzerland). Homogenates were centrifuged at 45 000 ꢂ gmax for
15 min at 48C. The pellets were resuspended again in buffer B (1 ml) containing
1 mM EDTA, 10 mM HEPES and 0.1 mM benzamidine (pH 7.4) and
recentrifuged at 45 000 ꢂ gmax for 15 min at 48C. The pellets were resuspended
in buffer B (1 ml) and centrifuged at 10 000 ꢂ gmax for 10 min at 48C. The
supernatants were recentrifuged at 45 000 ꢂ gmax for 15 min at 48C. The pellets,
partially enriched membranes, were resuspended in buffer C (50 mM
Tris ꢄ HCl, 5 mM MgCl2 (pH 7.4)), and stored frozen at ꢁ808C. For
competition binding studies, the prepared membranes were resuspended in
buffer D (10 mM TrisHCl, 154 mM NaCl, 0.1 mM ascorbic acid, pH 7.4).
15 mg of membranes were incubated with a constant concentration of
[
125I]ICYP (80 pM) and with varying concentrations (1 pM–100 mM) of
compounds 5a–f (Scheme 2). Reactions were conducted at 378C for 60 min.
Reactions were stopped by filtering onto Whatman GF/B filters and washed
with water for injection. The membrane bound radioactivity was determined
in a g-scintillation counter. Competition binding curves were analysed by non-
linear regression analysis as previously described30–32 using the XMGRACE
program (Linux software). The data of the ligands 5a–f fitted a two-site model
significantly better than a one-site model (F=2.15, p50.05). The resulting
high- and low-affinity IC50 values of all synthesized and non-radioactive 3-
aryloxy-2-propanolamine derivatives 5a–f 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–Prusoff26 using the experimentally determined KD value
of [125I]ICYP (32.3 ꢀ 1.9 pM).22 The ratios of the low- to high-affinity
inhibition constants (K2/K1) yield the b1-selectivities of the unlabelled
derivatives 5a–f (Table 1). These compounds show a significantly higher
affinity to b1- than to b2-ARs. Additionally, the calculated logP and logD
values (ACD/LogD Suite) for compounds 5a–f are listed in Table 1 to indicate
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S. WAGNER ET AL.
the changes of the lipophilicities caused by the chemical modifications of the
lead compound, ICI 89,406.
Conclusion
The syntheses, radiosyntheses and in vitro pharmacology of (R)- and (S)-N-[2-
[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N0-(4-methoxy-phenyl)-
urea (5a and 5b, respectively), and their 11C-labelled versions (5g and 5h,
respectively), potential new high affinity b1-AR selective PET radioligands, are
reported. The non-radioactive counterparts 5a and 5b of the radiolabelled
target compounds 5g and 5h showed high affinity to b1-ARs in murine
myocardial membranes and moderate lipophilicity. The radiosyntheses of 5g
and 5h were achieved with good radiochemical and acceptable specific
activities. The formulation of the preparations is suitable for preliminary
in vivo studies using small animal PET.
Acknowledgements
This work was supported by the SFB 556, 656 and SCHA 758/5-1 of the
Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany. The authors
gratefully thank Ms Monika Trub and Ms Sandra Schroer for technical
¨
assistance.
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