Towards the preparation of radiolabeled 1-aryl-3-benzyl ureas: Radiosynthesis of [11C-carbonyl] AR-A014418 by [11C] CO2 fixation

Article abstract of DOI:10.1016/j.bmcl.2011.12.139

The highly selective glycogen synthase kinase-3 (GSK-3) inhibitor N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) was radiolabeled with carbon-11 (¹¹C; half-life = 20.4 min) at the urea moiety via [¹¹C]CO₂

Full text of DOI:10.1016/j.bmcl.2011.12.139

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2099–2101
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Towards the preparation of radiolabeled 1-aryl-3-benzyl ureas:
Radiosynthesis of [11C-carbonyl] AR-A014418 by [11C]CO2 fixation
Justin W. Hicks a, Alan A. Wilson a, Elizabeth A. Rubie b, James R. Woodgett b, Sylvain Houle a,
Neil Vasdev a,
⇑
a PET Centre, Centre for Addiction and Mental Health, Department of Psychiatry, Institute of Medical Science, University of Toronto, 250 College St., Toronto, ON, Canada M5T 1R8
b Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, ON, Canada M5G 1X5
a r t i c l e i n f o
a b s t r a c t
The highly selective glycogen synthase kinase-3 (GSK-3) inhibitor N-(4-methoxybenzyl)-N0-(5-nitro-1,3-
thiazol-2-yl)urea (AR-A014418) was radiolabeled with carbon-11 (11C; half-life = 20.4 min) at the urea
moiety via [11C]CO2 fixation. Reaction of [11C]CO2 with 4-methoxybenzylamine in the presence of a
CO2 fixating base was followed by dehydration with POCl3 and addition of 2-amino-5-nitrothiazole to
prepare [11C-carbonyl] AR-A014418. This reaction resulted in an 8% uncorrected radiochemical yield,
Article history:
Received 1 November 2011
Revised 26 December 2011
Accepted 27 December 2011
Available online 10 January 2012
based on [11C]CO2, with high specific activity (4 Ci/
activity assay revealed that AR-A014418 (Ki = 770 nM) is not as potent as previously claimed. The
11C]CO2 fixation methodology described herein should prove generally applicable to preparing 1-aryl-
3-benzyl-[11C-carbonyl] ureas as radiotracers for positron emission tomography.
lmol) within 30 min. An in vitro GSK-3b enzyme
Keywords:
Glycogen synthase kinase
AR-A014418
Carbon-11
CO2 fixation
In vitro
[
Ó 2012 Elsevier Ltd. All rights reserved.
PET
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine ki-
nase comprising of two closely related isoforms ( and b) which
Carbon-11 labeled ureas have previously been prepared via
[
11C]COCl2.13–15 Although the production of [11C]COCl2 has seen re-
a
has an important role in a number of cellular processes including
glycogen synthesis, neuronal cell survival, and apoptosis.1 Dysreg-
ulation of GSK-3 has been implicated in diseases of the central ner-
vous system (CNS) including major depressive disorder,2 bipolar
disorder,3 and Alzheimer’s disease,4 as well as pain5 and several
cancers.6 Whereas small molecule inhibitors of GSK-3 are avidly
sought as potential therapeutics,7 very few attempts have been
made to develop radiotracers for functional imaging of this kinase
in vivo.8–10
cent technical advancements,16,17 the highly specialized apparatus,
extensive upkeep, technical expertise, and replacement of key
components between production runs have limited its use to a
few laboratories worldwide. Metal catalyzed carbonylation with
[
11C]CO to prepare 11C-ureas is an alternative method,18,19 how-
ever, the need for specialized equipment to trap and utilize the
[
11C]CO has limited the wide-spread application of this method.20
In contrast, [11C]CO2 can be efficiently and routinely prepared in
large quantities in most medical cyclotrons. By using a CO2 fixating
base, [11C]CO2 can be quantitatively captured and reacted in situ to
form 11C-carbamates21–23 and 11C-oxazolidinones24 at room tem-
perature. Our laboratory has recently extended the [11C]CO2 fixa-
tion methodology to prepare symmetrical and unsymmetrical
11C-ureas from aliphatic amines;25 however, all attempts to pre-
pare unsymmetrical ureas using aromatic amines have been
unsuccessful to date with this approach. We here report the radio-
synthesis of [11C-carbonyl] AR-A014418, thereby representing the
first example of an unsymmetrical 11C-urea bearing an aromatic
moiety, prepared by [11C]CO2 fixation.
Our laboratory previously radiolabeled the established GSK-3
inhibitor N-(4-methoxybenzyl)-N0-(5-nitro-1,3-thiazol-2-yl)urea
(AR-A014418)11 with carbon-11
[
(
11C; half-life = 20.4 min) via
11C]CH3I at the methoxy position and carried out ex vivo biodistri-
bution studies8 in conjunction with in vivo positron emission
tomography (PET) imaging in rodent models.12 Using AR-
A014418 as the lead compound for radiosynthesis and derivatiza-
tion, the goal of the present work is to develop a broadly applicable
11C-carbonylation strategy that enables the preparation of struc-
turally diverse 1-aryl-3-benzyl-[11C-carbonyl] ureas.
The radiosynthesis of [11C-carbonyl] AR-A014418 was rapidly
completed in a one-pot automated reaction sequence which re-
quired no heating or cooling (Scheme 1).26 In these reactions,
⇑
Corresponding author at present address: Department of Nuclear Medicine and
[
11C]CO2 in a stream of nitrogen was slowly bubbled through an
Molecular Imaging, Massachusetts General Hospital and Department of Radiology,
Harvard Medical School, 55 Fruit St., Boston, MA, 02114, USA. Tel.: +1 617 643 4736;
fax: +1 617 726 6165.
acetonitrile solution of 4-methoxybenzylamine and the fixating
base BEMP (2-tert-butylimino-2-diethylamino-1,3-dimethylper-
E-mail address: vasdev.neil@mgh.harvard.edu (N. Vasdev).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.139

2100
J. W. Hicks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2099–2101
Scheme 1. Reagents and conditions: (a) BEMP, [11C]CO2, rt, 1 min; (b) POCl3, rt, 1 min; and (c) rt, 1 min.
hydro-1,3,2-diazaphosphorine). After 1 min, a solution of 0.4%
POCl3 was added to dehydrate the in situ generated 11C-carbamic
salt to a 11C-isocyanate prior to the addition of a solution of 2-ami-
no-5-nitrothiazole. Following an additional 1 min, the reaction
mixture was quenched and purified by HPLC. The overall synthesis
time including formulation was 28 min and produced clinically
of GSK-3b (data not shown; vide infra). During our preliminary
validation phase, these assays revealed that AR-A014418 had a Ki
value of 770 nM towards GSK-3b (Fig. 1)28 and is significantly less
potent than initially reported (38 nM).11 Concurrent with our
work, and consistent with our results, a recent report also revealed
that AR-014418 is not as potent (IC50 = 330 nM)29 as previously de-
scribed. Nonetheless, the same study identified several structurally
related 1-aryl-3-benzyl ureas that are significantly more potent to-
wards GSK-3b.29 Further evaluation of the scope of the [11C]CO2
fixation methodology reported herein for development of radiotra-
cers to image GSK-3 expression in vivo is currently underway.
useful quantities of
n = 3), resulting in 8.3 3.9% uncorrected radiochemical yield,
based on starting
11C]CO2, with high specific activity
(4.0 1.1 Ci/ mol) at the end of synthesis. Whereas previous at-
[
11C-carbonyl] AR-A014418 (70 33 mCi;
[
l
tempts to synthesize 11C-labeled unsymmetrical ureas bearing an
aromatic moiety have failed by [11C]CO2 fixation,25 the success of the
present method is attributed to judiciously reacting the aromatic
amine (nucleophile) with the in situ generated 11C-isocyanate
(‘fixated amine’), and the preparation of a mixed 1-aryl-3-benzyl
urea is demonstrated.
Acknowledgments
The Ontario Ministry of Research and Innovation is acknowl-
edged for financial support for this work in the form of an Early Re-
searcher Award (N.V.). The authors thank Armando Garcia,
Winston Stableford, and Min Wong for their expertise with the
radiochemistry.
When compared to the preparation of
[
11C-methoxy] AR-
A014418,8 this [11C]CO2 fixation strategy resulted in similar radio-
chemical yield and specific activity, however, the methodology
presented herein is much more broadly applicable. For instance, li-
braries of 11C-ureas can be prepared via [11C]CO2 fixation reactions
with various commercially available amines, thereby alleviating
cumbersome precursor syntheses and the restriction of array scaf-
folds to 11C-methylation reactions. The demonstrated high effi-
ciency reaction and ease of synthesis are positive indicators that
References and notes
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[
11C]CO2 fixation is a strategy amenable to the synthesis of [11C-
carbonyl]-labeled AR-A014418 derivatives and should be generally
applicable to the preparation of radiotracers based on the 1-aryl-3-
alkyl-11C-urea scaffold. We anticipate that this methodology
should also be applicable to similar [14C]CO2 fixation reactions as
a [14C] phosgene equivalent.27 The scope of these reactions are un-
der investigation in our laboratory.
In an attempt to identify more potent and selective GSK-3
inhibitors and to apply the radiolabelling methodology described
herein, preliminary syntheses of novel 1-aryl-3-benzyl ureas and
1-aryl-3-phenyl derivatives of AR-A014418 were carried out and
evaluated in conjunction with in vitro assays to determine inhibition
11. Bhat, R.; Xue, Y. F.; Berg, S.; Hellberg, S.; Ormo, M.; Nilsson, Y.; Radesater, A. C.;
Jerning, E.; Markgren, P. O.; Borgegard, T.; Nylof, M.; Gimenez-Cassina, A.;
Hernandez, F.; Lucas, J. J.; Diaz-Nido, J.; Avila, J. J. Biol. Chem. 2003, 278, 45937.
12. Hicks, J. W.; VanBrocklin, H. F.; Wilson, A. A.; Houle, S.; Vasdev, N. Molecules
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13. Conway, T.; Diksic, M. Appl. Radiat. Isot. 1991, 42, 441.
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18. Kihlberg, T.; Karimi, F.; Långström, B. J. Org. Chem. 2002, 67, 3687.
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Figure 1. Inhibition of GSK-3b enzymatic activity by: various concentrations of
AR-A014418; no inhibitor present; and the potent, non-selective GSK-3b inhibitor
Alsterpaullone, respectively.

J. W. Hicks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2099–2101
2101
26. A Scanditronix MC 17 cyclotron was used for [11C]CO2 production via the
27. Dean, D. C.; Wallace, M. A.; Marks, T. M.; Melillo, D. G. Tetrahedron Lett. 1997,
38, 919.
14N(p, 11C nuclear reaction using a 40
a) lA proton beam current to irradiate
14N2 containing 0.5% O2. Carbon-11 labeled CO2 was dispensed in a stream of
nitrogen (10 mL/min) into a 1 mL conical vial containing the phosphazene base
BEMP (170 mM) and 4-methoxybenzylamine (7.5 mM) in anhydrous CH3CN
28. Active, purified GSK-3b (10 ng; Millipore) in water (5
of the substrate phospho-glycogen Synthase peptide-2 (2.5
8 mM Mops and 0.2 mM EDTA buffer (5 L, pH = 7.0) with the presence of a
range of concentrations (0, 10, 20, 30, 60 and 100 M in 2.5 L) of AR-A014418
and 1 mCi/mL
32P]-ATP (3000 Ci/mmol) in 5 mM magnesium chloride
(10 L) amounting to a final volume of 25 L. After 10 min at 30 °C, 20
l
L) was added to 20
lM
lL; Millipore) in
l
(100
lL). After 1 min, a 0.4% (v/v) solution of POCl3 in CH3CN (100
lL) was
l
l
added, via Labview (National Instruments) controlled solenoid-valves,
followed 1 min later by a solution of 2-amino-5-nitrothiazole (34 mM) in
[c-
l
l
lL
DMSO (100
after 1 min and purified by HPLC. Reverse-phase HPLC [Phenomenex Prodigy
C18, 250 ꢀ 10 mm, 10 m] was performed using a mobile phase consisting of
CH3CN/H2O (40:60 v/v) + 0.1 N ammonium formate at a flow rate of 8 mL/min
to separate
11C-carbonyl] AR-A014418 (tR = 12.2 min) from the reaction
mixture. The fraction eluting at 12.2 min was collected, evaporated, and
formulated in saline (10 mL) and 8.4% sodium bicarbonate solution (1 mL).
Confirmation of identity was performed by analytical HPLC as previously
described.8
l
L). The reaction was quenched with 10:90 CH3CN/H2O (800
l
L)
was spotted onto P81 phosphocellulose paper (2 ꢀ 4 cm), dried, and then
washed 8 times (5 min per wash) in a solution of 1% phosphoric acid. The dried
l
filter papers were counted in the presence of scintillation fluid using a
Beckman LS 6500 liquid scintillation counter. Radioactive phosphate
incorporation was corrected for non-specific background (<1%) by
subtracting counts obtained in the absence of added GSK-3b.
[
29. Monte, F. L.; Kramer, T.; Boländer, A.; Plotkin, B.; Eldar-Finkelman, H.; Fuertes,
A.; Dominguez, J.; Schmidt, B. Bioorg. Med. Chem. Lett. 2011, 21, 5610.