Synthesis and ex vivo evaluation of carbon-11 labelled N-(4-methoxybenzyl)- N′-(5-nitro-1,3-thiazol-2-yl)urea ([11C]AR-A014418): A radiolabelled glycogen synthase kinase-3β specific inhibitor for PET studies

Article abstract of DOI:10.1016/j.bmcl.2005.08.037

N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), a highly selective inhibitor of glycogen synthase kinase-3β (GSK-3β), was radiolabelled with carbon-11 (half-life = 20.4 min) for cerebral positron emission tomography (PET) studies. Reac

Full text of DOI:10.1016/j.bmcl.2005.08.037

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5270–5273
Synthesis and ex vivo evaluation of carbon-11 labelled
N-(4-methoxybenzyl)-N0-(5-nitro-1,3-thiazol-2-yl)urea
([11C]AR-A014418): A radiolabelled glycogen synthase
kinase-3b specific inhibitor for PET studies
a
a
c
Neil Vasdev,a,b, Armando Garcia, Winston T. Stableford, Alex B. Young,
*
Jeffrey H. Meyer,a,b Sylvain Houlea,b and Alan A. Wilsona,b
aPET Centre, Centre for Addiction and Mental Health, 250 College St., Toronto, Ont., Canada M5T-1R8
bDepartment of Psychiatry, University of Toronto, 250 College St., Toronto, Ont., Canada M5T-1R8
cDepartment of Chemistry, University of Toronto, 80 St. George St., Toronto, Ont., Canada M5S-1A1
Received 7 July 2005; revised 15 August 2005; accepted 15 August 2005
Available online 3 October 2005
Abstract—N-(4-Methoxybenzyl)-N0-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), a highly selective inhibitor of glycogen synthase
kinase-3b (GSK-3b), was radiolabelled with carbon-11 (half-life = 20.4 min) for cerebral positron emission tomography (PET) stud-
ies. Reaction of desmethyl AR-A014418 with [11C]CH3I produced [11C]AR-A014418 in 17% decay-corrected radiochemical yield,
based on [11C]CO2, with 3230 mCi/lmol specific activity after a 30 min synthesis time. The desmethyl precursor of AR-A014418
was synthesized in 23% yield by a novel one-pot reaction of 2-amino-5-nitrothiazole with in situ generated TMS-protected 4-hydrox-
ybenzylisocyanate, following deprotection with acid. Ex vivo biodistribution studies were conducted after [11C]AR-A014418 was
administered via tail vein injection into Sprague–Dawley rats. Very low levels of radioactivity were found in all brain regions
(0.08% injected dose/gram of tissue) at 5 and 30 min post-injection, uncorrected for vascular compartment. Considering the extreme-
ly poor brain penetration of [11C]AR-A014418 this compound cannot be used to study GSK-3b in cerebral PET studies. Further-
more, the specific pharmacological mechanism(s) of antidepressant-like activity attributed to AR-A014418 should be investigated.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase 3b (GSK-3b) is a serine/threo-
nine kinase with highest abundance in the brain1 and is in-
volved with signal transduction cascades of multiple
cellular processes. GSK-3b facilitates a variety of com-
plex functions including apoptotic mechanisms, neuronal
plasticity, and gene expression.2 Implications of dysregu-
lated GSK-3b activity in certain psychiatric and neurode-
generative diseases have prompted investigations of
GSK-3b inhibitors as therapeutics. Small molecule inhib-
itors of GSK-3b are currently under development for a
broad range of illnesses including AlzheimerÕs disease,
depression, diabetes, stroke, bipolar disorder and malig-
nancy (for several recent reviews see Refs. 3–10).
tomography (PET). Levels of GSK-3b levels could
potentially be measured in the living animal and/or hu-
man brain by use of PET; however, there is no radio-
tracer available to achieve this as yet. It is the goal of
this work to synthesize and evaluate the first radiophar-
maceutical for imaging GSK-3b with PET. The highly
selective inhibitor of GSK-3b, N-(4-methoxybenzyl)-
N0-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)11 was
chosen for radiolabelling with the positron emitting iso-
tope carbon-11 (11C; half-life = 20.4 min). This particu-
lar compound is purported to be a major advance in
central nervous system (CNS) therapeutics and antide-
pressant development because previously developed
inhibitors lacked selectivity for GSK-3b. Furthermore,
a recent study has demonstrated antidepressant-like ef-
fects in rats exposed to a forced-swim test following
administration of AR-A014418.12
One method for quantitation of enzyme levels in the liv-
ing brain is by in vivo imaging with positron emission
Keywords: AR-A014418; Carbon-11; PET; Curtius rearrangement;
Glycogen synthase kinase.
The original synthetic route to [11C]AR-A014418
involved the preparation of AR-A01441811 and
subsequent demethylation of AR-A014418 to serve as
*
Corresponding author. Tel.: +1 416 535 8501x4680; fax: +1 416 979
4656; e-mail: neil.vasdev@camhpet.ca
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.037

N. Vasdev et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5270–5273
5271
a precursor for radiolabelling with [11C]iodomethane.
While demethylations of anisoles to phenols are stan-
dard organic transformations,13,14 cleavage of an aryl
ether is difficult in the presence of a nitro-group.15 In
addition to containing a nitro-group, AR-A014418 pre-
sents a further complication as substituted 5-nitrothiaz-
oles are highly susceptible to nucleophilic substitution.16
A variety of demethylation conditions were attempted in
the present work. Not surprisingly, all of them resulted
in very low yields and/or halogenation at the thiazole
moiety. For example, attempted demethylation of AR-
A014418 using 5 M HBr in CH3COOH14 or 1 M BBr3
in CH2Cl214 resulted in the formation of a demethylated
and brominated derivative of AR-A014418 as the major
product, likely N-(4-hydroxybenzyl)-N0-(5-bromo-1,3-
thiazol-2-yl)urea, based on characterization by LC–
MS.17 Other attempts to cleave the ether group of
AR-A014418 during this work included the use of
AlCl3/ethanethiol14 and AlCl3/pyridine/EtOAc15 mix-
tures. While these procedures have been successful for
cleavage of ethers in the presence of nitro-groups, reac-
tion with AR-A014418 did not result in the desired
demethylated product.
no-5-nitrothiazole was added for reaction with the
in situ generated isocyanate. Following deprotection
with HCl and silica gel purification, desmethyl AR-
A014418 was obtained in 23% overall yield.19 This syn-
thetic route is generally applicable for the syntheses of
hydroxybenzyl-ureas and offers an alternative to prob-
lematic dealkylation reactions.
For the radiosynthesis of [11C]AR-A014418, desmethyl
AR-A014418 was dissolved in DMF and deprotonated
with tetrabutylammonium hydroxide (TBAOH) imme-
diately prior to reaction with [11C]CH3I, using the
ÔLoopÕ method20 (Scheme 1). Following methylation,
[11C]AR-A014418 was purified by preparative HPLC
and formulated in saline.21 Full-scale production of
[11C]CO2 resulted in 83 mCi [11C]AR-A014418, ready
for injection, corresponding to 17% decay-corrected
radiochemical yield, based on [11C]CO2, with a specific
activity of 3230 mCi/lmol at the end of synthesis.21
Ex vivo biodistribution studies following administra-
tion of high specific activity [11C]AR-A014418 in male
Sprague–Dawley rats (250 8 g) were conducted as
previously described by our group.22,23 Rats were
injected in the tail vein with a saline solution of
[11C]AR-A014418 (0.7 lg/kg) and sacrificed at 5 and
30 min post-injection (n = 6 per time point) and brain
regions were excised and measured for radioactivity.
Blood (trunk) was also collected for radioactivity mea-
surement. Figure 1 shows the distribution of activity
for [11C]AR-A014418 in eight regions of the rat brain
known to contain glycogen synthase kinase-3b,24 the
remainder of the brain, and whole blood. The average
percent injected dose per gram (% ID/g) of wet tissue
in the brain was 0.08% ID/g for all regions at time
points of 5 and 30 min. Whole blood levels of
[11C]AR-A014418 were 0.84% and 0.46% ID/g at 5
and 30 min, respectively. When corrected for radioac-
tivity in the vascular compartment,25 uptake values of
radioactivity are further reduced from those shown in
Alternatively, a novel one-pot synthesis of desmethyl
AR-A014418 was successfully achieved by the reaction
of in situ generated trimethylsiloxy-protected 4-hydrox-
ybenzylisocyanate (prepared by modification of a
previously described method18) with excess 2-amino-5-
nitrothiazole, followed by acid deprotection (Scheme
1).19 As shown in Scheme 1, reaction of 4-hydroxyphe-
nylacetic acid with chlorotrimethylsilane generated the
bis(silylated) hydroxyl acid, followed by conversion to
4-[(trimethylsilyl)oxy]-benzeneacetyl chloride with thio-
nyl chloride. The acid chloride was then reacted with
azidotrimethylsilane at room temperature to generate
the intermediately formed acyl azide and was subse-
quently converted to the isocyanate upon heating the
reaction mixture to reflux (Curtius rearrangement).
Prior to refluxing the reaction mixture, excess 2-ami-
O
O
N
C
OH
N
N
O
i - iv
O2N
NH2
S
O2N
NH
NH
S
OTMS
OTMS
O
OH
N
N
O
v, vi
11CH3I, TBAOH, DMF
"Loop Method"
O2N
NH
NH
O2N
NH
NH
S
S
OH
O11CH3
[
11C]AR-A014418
Scheme 1. Synthesis of desmethyl AR-A014418 and [11C]AR-A014418. Reagents and conditions: (i) TMS-Cl, Et3N, toluene, reflux; (ii) SOCl2,
CH2Cl2, reflux; (iii) TMS-N3, dioxane, rt; (iv) reflux (Curtius rearrangement); (v) 2 N HCl; (vi) silica gel purification.

5272
N. Vasdev et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5270–5273
1.000
3. Gould, T. D.; Zarate, C. A.; Manji, H. K. J. Clin.
Psychiatry 2004, 65, 10.
4. Cohen, P.; Goedert, M. Nat. Rev. Drug Disc. 2004, 3, 479.
5. Martinez, A.; Castro, A.; Dorronsoro, I.; Alonso, M.
Med. Res. Rev. 2002, 22, 373.
6. Cohen, P.; Frame, S. Nat. Rev. Mol. Cell. Biol. 2001, 2,
769.
7. Wagman, A. S.; Johnson, K. W.; Bussiere, D. E. Curr.
Pharm. Design 2004, 10, 1105.
Blood
Cerebellum
0.900
0.800
Hippocampus
Striatum
Cortex
Hypothalamus
Thalamus
Rest
0.700
0.600
0.500
White matter
8. Bhat, R. V.; Budd Haeberlein, S. L.; Avila, J. J.
Neurochem. 2004, 89, 1313.
0.400
0.300
0.200
0.100
0.000
9. Gould, T. D.; Manji, H. K. Neuropsychopharmacology
2005, 1.
10. Meijer, L.; Flajolet, M.; Greengard, P. Trends Pharmacol.
Sci. 2004, 25, 471.
11. Bhat, R.; Xue, Y.; Berg, S.; Hellberg, S.; Ormo¨, M.;
Nilsson, Y.; Radesa¨ter, A.-C.; Jerning, E.; Markgren, P.-
˚
O.; Borgegard, T.; Nylo¨r, M.; Gimenez-Cassina, A.;
Hernandez, F.; Lucas, J. J.; Dıaz-Nido, J.; Avila, J. J.
´
´
´
0
5
10
15
20
25
30
Time (min)
Biol. Chem. 2003, 278, 45937.
12. Gould, T. D.; Einat, H.; Bhat, R.; Manji, H. K.
Int. J. Neuropsychopharmacol. 2004, 7, 387.
13. Greene, T. W.; Wuts, P. G. M. In Protective Groups in
Organic Synthesis, 2nd ed.; John Wiley & Sons: New
York, 1991, p 14.
14. Bhatt, M. V.; Kulkarni, S. U. Synthesis 1983, 249.
15. Learmonth, D. A.; Alves, P. C. Synth. Commun. 2002, 32,
641.
16. Forlani, L.; Todesco, P. E.. In Metzger, J. V., Ed.;
Thiazole and its Derivatives; John Wiley & Sons: New
York, 1979; Vol. 34, pp 565–585.
17. LC–MS was conducted by Electrospray Ionization using a
C-18 Zorbax column (50 mm · 2.1 mm, 3.5 lm) eluted
with CH3CN/H2O (40:60 v/v) + 0.5% formic acid using a
flow rate of 250 lL minꢀ1 in conjunction with a MDS
Sciex QStar mass spectrometer. A desmethyl AR-A014418
derivative with substitution at the thiazole moiety is
predicted based on the parent molecular ion for this
compound which showed a characteristic isotopic pattern
for monobromination, [M+H]: m/z = 328 and 330 (1:1).
The hydroxybenzyl moiety was seen at m/z = 107 in
addition to pairs of fragments corresponding to bromi-
nated thiazolamine, [M+H]: m/z = 179 and 181 (1:1). A
trace of dibrominated product was also seen, [M+H]:
m/z = 406, 408, and 410 (1:2:1), respectively, with both the
thiazole and benzyl fragments containing a bromine.
Time-course of this reaction revealed that bromination
at the thiazole occurs (<15 min) prior to demethylation
(ca. 1 h) therefore, this synthetic route was no longer
pursued.
Figure 1. Distribution of radioactivity in rat brain regions and whole
blood at 5 and 30 min (n = 6 per time point; means SD shown)
following tail vein injection of [11C]AR-A014418 is shown. Homoge-
neous distribution of low levels of radioactivity was seen in all brain
regions.
Figure 1 to 0.04% and 0.06% ID/g of wet tissue at 5
and 30 min, respectively. Thus, the brain is almost
totally impermeable to this compound (successful
radiotracers for imaging the central nervous system
typically express a % ID/g of tissue P0.5% in rodent
brain26).
Ex vivo measurement of radioactivity in tissue after
injection of [11C]AR-A014418 in rats clearly indicates
that this compound cannot be used to study GSK-3b
in the CNS with PET. The lack of brain penetration
of [11C]AR-A014418 is surprising considering that its
logP value between 1-octanol and 0.02 M phosphate
buffer at pH 7.4 was found to be 2.44, using a previously
described procedure,27 a value which is typical of many
brain penetrating compounds.27,28 The lack of brain
penetration is also surprising in light of reported CNS
effects following peripheral administration of AR-
A014418 into rats.12 Studies to investigate peripheral ef-
fects, metabolism and brain barrier transport of AR-
A014418 are presently underway in our laboratory and
hope to gain further insight into the pharmacological
mechanism(s) by which this compound could exert its
reported antidepressant-like effects.12
18. Schwartz, G.; Alberts, H.; Kricheldorf, H. R. Liebigs Ann.
Chem. 1981, 1257.
19. 4-[(Trimethylsilyl)oxy]-benzeneacetyl chloride was pre-
pared according to the literature procedure,18 however,
intermediates were not isolated; solvents were simply
removed by rotary evaporation proceeding each step and
the entire synthesis was successfully accomplished in one
pot. Azidotrimethylsilane (260 lL, 2.0 mmol) was added
to 4-[(trimethylsilyl)oxy]-benzeneacetyl chloride (400 mg,
1.65 mmol) in 1,4-dioxane (5 mL) and stirred at room
temperature for 2 h. Subsequently, 2-amino-5-nitrothiaz-
ole (440 mg, 3.0 mmol) was added and the mixture was
heated to reflux for 15 h. The resulting brown solution was
evaporated to dryness under a flow of nitrogen. The
residue was dissolved in EtOAc (ca. 100 mL) and washed
with 2 N HCl (3· 50 mL). The organic phase was dried
(Na2SO4), filtered, and concentrated prior to purification
by flash chromatography using silica gel (29% hexane, 69%
ethyl acetate, and 2% glacial acetic acid). Desmethyl
Acknowledgments
The Faculty of Medicine at the University of Toronto is
acknowledged for financial support for this work from
the DeanÕs Fund (N.V.). The authors thank Doug
Hussey, Patrick McCormick, and Hermia Cheung for
their expertise with the ex vivo evaluations.
References and notes
1. Woodgett, J. R. EMBO J. 1990, 9, 2431.
2. Grimes, C. A.; Jope, R. S. Prog. Neurobiol. 2001, 65, 391.

N. Vasdev et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5270–5273
5273
AR-A014418 was obtained as a yellow solid in 23% overall
yield. 1H NMR (Me2SO-d6, 499.8 MHz) d (ppm, relative to
TMS): 11.9 (br s, Dm1/2 = 119 Hz, 1H,), 9.34 (br s, Dm1/
2 = 2.5 Hz, 1H), 8.50 (s, 1H), 7.19 (br t, Dm1/2 = 13.9 Hz,
J = ca. 5.5 Hz, 1H; confirmed to be NH–CH2Ph by COSY),
7.11 (AA0 XX0, JA = 3.5 Hz, JX = 1.4 Hz, J = 8.3 Hz,
(40 mg mLꢀ1 in H2O) and heated to dryness at 70 ꢁC,
under vacuum. The product was dissolved in 10 mL saline
and transferred into a vial containing 1 mL of 8.4%
sodium bicarbonate. The pH of the final solution was
between 7 and 8. Analytical HPLC was performed using a
C-18 column (Phenomenex Prodigy ODS-Prep,
250 mm · 4.6 mm, 10 lm) eluted with CH3CN/H2O
J0 = ꢀ0.3 Hz, 2H), 6.72 (AA0 XX0, JA = 3.5 Hz, JX
=
1.4 Hz, J = 8.3 Hz, J0 = ꢀ0.3 Hz, 2H), 4.24 (d, J = 5.8 Hz,
2H). 13C NMR (Me2SO-d6, 125.7 MHz) d (ppm, relative to
TMS): 64.3, 156.5, 153.3, 143.3,140.8, 128.9, 128.7, 115.1,
42.7. HRMS (ESI) calcd for C11H11N4O4S, 295.0495;
found: 295.0471 (M++H).
(40:60 v/v) + 0.1 N AF using a flow rate of 4 mL minꢀ1
.
Authentic AR-A014418 (Calbiochem) co-eluted with the
11C-labelled
product under these conditions
(tR = 5.2 min). In addition, three other HPLC conditions
were used to confirm the co-elution of authentic AR-
A014418 and [11C]AR-A014418: (1) analytical C-18 Prod-
igy column eluted with THF/H2O (50:50 v/v) + 0.1 N AF
using a flow rate of 3.5 mL minꢀ1; tR = 2.1 min; (2)
analytical C-18 Prodigy column eluted with CH3CN/
H2O (40:60 v/v) + 0.1 N AF + 0.5% formic acid using a
flow rate of 4 mL minꢀ1, tR = 4.7 min; (3) Econosil C-8
column (Alltech; 250 mm · 4.6 mm,10 lm) eluted with
CH3CN/H2O (40:60 v/v) + 0.1 N AF using a flow rate of
4 mL minꢀ1, tR = 3.3 min.
20. Wilson, A. A.; Garcia, A.; Jin, L.; Houle, S. Nucl. Med.
Biol. 2000, 27, 529.
21. Radiosynthesis was shown to be reliable and efficient (30
min) for the production of mCi quantities of chemically
and radiochemically pure [11C]AR-A014418 (>98%;
n = 5). In a full-scale production of [11C]CO2, 1% O2 in
N2 was bombarded in a 17 MeV cyclotron with 45 lA
proton beam current for 20 min. Desmethyl AR-A014418
(1.0 mg, 3.4 lmol) was dissolved in 75 lL DMF and
6.5 lL of
a
1.0 M solution of tetrabutylammonium
22. All animal experiments were conducted under humane
conditions, with approval from the Animal Care Com-
mittee at CAMH and in accordance with guidelines set
forth by the Canadian Council of Animal Care.
23. Wilson, A. A.; DaSilva, J. N.; Houle, S. Nucl. Med. Biol.
1996, 23, 141.
24. Leroy, K.; Brion, J.-P. J. Chem. Neuroanat. 1999, 16,
279.
25. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle,
S. Life Sci. 2001, 68, 1223.
26. Wong, D. F.; Pomper, M. G. Mol. Imaging Biol. 2003, 5,
350.
27. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle,
S. Appl. Radiat. Isot. 2001, 54, 203.
28. Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376.
hydroxide (TBAOH) in methanol was added prior to a
5 min at room temperature reaction with [11C]iodome-
thane using the ÔLoopÕ method.20 Reverse-phase semi-
preparative HPLC (Phenomenex Prodigy ODS-Prep, 250·
10 mm, 10 lm) was performed using a mobile phase
consisting of CH3CN/H2O (50:50 v/v) + 0.1 N ammonium
formate (AF) at a flow rate of 7 mL minꢀ1 to separate
[11C]AR-A014418 (tR = 5.9 min) from unreacted desmeth-
yl AR-A014418 (tR = 3.1 min) and undesired byproducts.
The reaction mixture was monitored during purification
and analysis using an in-line UV (k = 254 nm) detector in
series with a NaI crystal radioactivity detector. The
product eluting at 5.9 min was led into a rotary evapora-
tion flask containing 250 lL ascorbic acid solution