Radiosynthesis and evaluation of 5-methyl-N-(4-[11C]methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine ([11C]ADX88178) as a novel radioligand for imaging of metabotropic glutamate receptor subtype 4 (mGluR4)

Article abstract of DOI:10.1016/j.bmcl.2015.12.008

ADX88178 (1) has been recently developed as a potent positive allosteric modulator for metabotropic glutamate receptor 4 (mGluR4). The aim of this study was to develop [¹¹C]1 as a novel positron emission tomography ligand and to evaluate its bi

Full text of DOI:10.1016/j.bmcl.2015.12.008

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
11
Radiosynthesis and evaluation of 5-methyl-N-(4-[ C]
11
methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine ([ C]
ADX88178) as a novel radioligand for imaging of metabotropic
glutamate receptor subtype 4 (mGluR4)
a,y
a,y
a,b
a,b
a
Masayuki Fujinaga , Tomoteru Yamasaki , Nobuki Nengaki , Masanao Ogawa , Katsushi Kumata ,
a
a
a
a
a
a,
⇑
Yoko Shimoda , Joji Yui , Lin Xie , Yiding Zhang , Kazunori Kawamura , Ming-Rong Zhang
a
Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
SHI Accelerator Service Co. Ltd, 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-0032, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
ADX88178 (1) has been recently developed as a potent positive allosteric modulator for metabotropic
Received 11 August 2015
Revised 19 November 2015
Accepted 4 December 2015
Available online xxxx
11
glutamate receptor 4 (mGluR4). The aim of this study was to develop [ C]1 as a novel positron emission
11
tomography ligand and to evaluate its binding ability for mGluR4. Using stannyl precursor 3, [ C]1 was
11
11
efficiently synthesized by introducing an [ C]methyl group into a pyrimidine ring via C– C coupling and
deprotection reactions, in 16 ± 6% radiochemical yield (n = 10). At the end of synthesis, 0.54–1.10 GBq of
1
1
[
C]1 was acquired with >98% radiochemical purity and 90–120 GBq/
lmol of specific activity.
Keywords:
mGluR4
PET
In vitro autoradiography and ex vivo biodistribution study in rat brains showed specific binding of
1
1
[
C]1 in the cerebellum, striatum, thalamus, cerebral cortex, and medulla oblongata, which showed
dose-dependent decreases by administration with multi-dose of unlabeled 1.
Ó 2015 Elsevier Ltd. All rights reserved.
Autoradiography
11
[
C]ADX88178
Metabotropic glutamate receptors (mGluRs) are G-protein cou-
pled receptors that can activate excitatory neurotransmission via
neuroprotective activity in models of Parkinson’s disease, a degen-
erative disorder of dopaminergic neurons in the basal ganglia.4
The development of radioligands for positron emission tomog-
raphy (PET) imaging of mGluR4 in brain has become of increasing
interest. Kil et al. first developed two PET ligands for mGluR4, such
stimulation of secondary messengers in the central nervous system
1
(
CNS). MGluRs are classified into three groups including eight
subtypes based on sequence homology, intracellular transduction
2
11
18
5
pathways, and pharmacological properties. Of these, mGluR4,
as [ C]ML128 and [ F]KALB001 (Fig. 1). However, the binding of
these radioligands did not correspond to the regional distribution
of mGluR4 in rat and monkey brains, likely because of low affinity
mGluR6, mGluR7, and mGluR8 belonging to group III are identified
primarily on presynaptic terminals of GABAergic and glutamater-
gic neurons. These receptors interact with the Gi protein negatively
coupled with adenylate cyclase to inhibit the cAMP-dependent sig-
naling pathway, which is responsible for regulation of synaptic
transmission via inhibition of voltage-gate calcium flow across
5
(ML128 : EC50 = 240 nM; KALB001: EC50 = 50 nM) for mGluR4.
More recently, 5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyra-
zol-4-yl)thiazol-2-amine (ADX88178, 1) (Fig. 2) was developed as
a potent and selective mGluR4 positive allosteric modulator.6
Compound 1 potentiated glutamate-mediated activation of human
mGluR4 with EC50 values of 4 nM without significant effects on
3
the cell membrane within the basal ganglia circuitry of the brain.
Based on their physiological backgrounds, the therapeutic
potential of group III mGluRs is rapidly expanding. Of these,
mGluR4 has received particular attention because of the potential
benefits of mGluR4 activation in several CNS disorders. In particu-
lar, several pharmaceuticals for mGluR4 were reported to show
other mGluRs (EC50 > 30 lM). In addition, compound 1 showed
neuroprotection in rodent models of anxiety, obsessive–
6
compulsive disorder, fear, depression, and psychosis.
In present study, we developed [ C]ADX88178 ([11C]1, Fig. 2)
as a novel PET ligand for mGluR4. Compound 1 contains methyl
groups in the pyrimidine and thiazole ring. We labeled the methyl
group at the 4-position in pyrimidine to synthesize [ C]1 by a
3
I, using stannyl precursor 2
or 3 (Fig. 2). Although introduction of a [ C]methyl group into
11
11
⇑
Corresponding author. Tel.: +81 43 382 3079; fax: +81 43 206 3261.
E-mail address: zhang@nirs.go.jp (M.-R. Zhang).
Equal contributions.
1
1
11
C– C coupling reaction with [ C]CH
1
1
y
http://dx.doi.org/10.1016/j.bmcl.2015.12.008
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Fujinaga, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.008

2
M. Fujinaga et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
18F
N
O
O
HN
N
Cl
O
1
[
8
F]KALB001
Figure 1. Chemical structures of primary two radioligands for mGluR4.
Figure 2. Targeted compound and radiolabeling strategy for synthesis of [11C]1.
an electron-deficient heteroaromatic ring such as a pyridine or
for C–11C coupling (entry 2). Based on these data, we considered
that the unprotected pyrazole ring in 2 may hinder the C– C cou-
7
11
pyrimidine has been reported by Suzuki et al. To our knowledge,
no practical PET ligand bearing a [11C]methylated pyrimidine moi-
pling reaction.
11
Thus, the precursor for synthesis of [11C]1 was changed from 2
to 3 in which the pyrazole ring was protected with a p-methoxy-
benzyl (PMB) group (entry 3–6). As expected, use of CsF as a base
ety has been reported. Herein, we introduced a [ C]methyl group
11
into pyrimidine ring to synthesize [ C]1 as a practical PET ligand
1
1
bearing [ C]methylated pyrimidine moiety for the first time and
11
11
11
evaluated the binding ability of [ C]1 to mGluR4 in brain
was effective for C– C coupling, giving [ C]4 in a high yield. How-
1
1
in vitro and in vivo.
ever, the following deletion of the PMB group in [ C]4 did not pro-
8
9
10
Compound 1, precursors 2 and 3 were synthesized as shown
Scheme 1. 4-(1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
methylthiazol-2-amine (5) was prepared as described previously.
Reaction of 5 with 2-bromo-4-methyl or (tributylstannyl)pyrim-
ceed by adding TFA to the reaction mixture in DMF (entry 4). When
1
1
in
DMF was partly removed after C-methylation, the deprotection
1
1
11
11
with TFA produced a mixture of [ C]1 and [ C]4 in an approxi-
mately 1:1 ratio (entry 5). As shown in entry 6, to accomplish effi-
1
2
11
idine derivatives in the presence of Pd(OAc)
2
and Xantphos
cient deprotection of PMB in [ C]4, DMF was completely removed
1
1
afforded 3 and 4 in moderate yields of 39% and 52%, respectively.
Debenzylation of 4 with TFA gave 1 in 38% yield. However, deben-
zylation of 3 with TFA did not give the desired compound 2 and
protodestannylation was mainly observed on the pyrimidine ring
in 3. Other research groups had also reported such protodestanny-
lation of heteroarylstannanes under acidic conditions.13 Thus, to
avoid protodestannylation of 3 in the acid condition, 5 was treated
with TFA and then reacted with 2-bromo-4-(tributylstannyl)
pyrimidine to successfully give precursor 2.
under reduced pressure after the C– C coupling reaction, and TFA
was then added to the reaction mixture. Under a neat condition,
1
1
cleavage of the PMB group in [ C]4 proceeded efficiently at
100 °C for 5 min.
After optimization of conditions for C–11C coupling and depro-
11
tection reactions, radiosynthesis of [ C]1 was achieved according
to entry 6 using a home-made automated synthesis system.16 Rev-
11
ersed-phase HPLC purification of the reaction mixtures gave [ C]1
in 16 ± 6% (n = 10) radiochemical yield (n = 10, based on [11C]CO
,
,
2
Radiosynthesis conditions of [ C]1 via C–[11C]methylation
1
4
11
corrected for decay). Starting from 22.2 to 24.0 GBq [ C]CO
11
2
11
11
and results are summarized in Table 1. [ C]CH
3
I was obtained
with LiAlH , fol-
I was dis-
tilled from the reaction mixture and trapped in a DMF solution
containing precursor 2 or 3, Pd (dba) , P(o-tol) , CuCl, and base.
When K CO was used as a base, only trace [ C]1 was produced
and unreacted [ C]CH
the base from K CO to CsF did not improve the reaction efficiency
0.54–1.10 GBq [ C]1 was produced within 45 min (n = 10) of aver-
11
by the reduction of cyclotron-produced [ C]CO
2
4
aged synthesis time from the end of bombardment. In the final pro-
lowed by treatment with 48% HI.15 The resulting [ C]CH
11
duct solution, identity of [ C]1 was confirmed by co-injection with
11
3
1
4
11
unlabeled 1 onto analytic HPLC. The radiochemical purity of [ C]
1 was greater than 98% and the specific activity was 90–120 GBq/
2
3
3
11
2
3
lmol at the end of synthesis. No significant peaks corresponding
1
1
3
I was mainly observed (entry 1). Changing
to chemical impurities were observed on HPLC charts for the final
1
1
2
3
product solutions. Moreover, [ C]1 did not show radiolysis at room
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M. Fujinaga et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
R
N
S
N
N
N
S
N
2
H N
R
N
a
Br
b
HN
1
N
(R = CH3)
N
N
N
PMB
5
3; R = SnBu3 PMB
; R = CH3
4
TFA
R = SnBu3)
c
(
N
S
Bu Sn
N
3
H N
Bu Sn
N
Br
2
3
N
S
N
N
HN
N
d
N
H
N
N
H
6
2
Scheme 1. Chemical syntheses of compound 1–3. Reagents and conditions: (a) Pd(OAc)
2 2 3
, Xantphos, Cs CO , 1,4-dioxane, 100 °C, 10–17 h, for 3: 39%, for 4: 52%; (b) TFA, 90 °C,
3
h, 38%; (c) TFA, 90 °C, 4 h, 88%; (d) Pd(OAc) , Xantphos, Cs CO , 1,4-dioxane, 100 °C, 6 h, 22%.
2
2
3
temperature for 90 min after formulation. The analytical results
were in compliance with our in-house quality control/assurance
specifications of radiopharmaceuticals.
Table 1
Effect of the reaction conditions for synthesis of [11C]1
a
11
To determine the binding ability of [ C]1 for mGluR4, we per-
17
formed in vitro autoradiography using rat brain sections. Figure 3
3
Bu Sn
N
H311C
shows representative in vitro autoradiograms with [ C]1 containing
11
N
N
S
N
[11
N
S
N
C]CH3I
vehicle (control, A) or 10 lM unlabeled 1 (blockade, B). In the control
HN
HN
section, strong radioactivity signals were detected in the cerebellum,
striatum, thalamus, midbrain, cerebral cortex, and medulla oblon-
gata, while low radioactivity signals were found in the frontal cortex,
hypothalamus, and pons. The radioactivity was significantly
decreased by treatment with unlabeled 1; a decrease of 12.3% for
the cerebellum, 13.8% for the thalamus, 12.5% for the medulla oblon-
gata, and 15.8% for the striatum.
N
N
N
N
R
R
2
; R = H
[11C]1; R = H
C]4; R = PMB
TFA
3
; R = PMB
11
[
Entry Precursor Base
TFA
L)
RCY (%) of [11C]
4
RCY (%) of [11C]
d
d
(
l
1
The distribution pattern of radioactive accumulation in the con-
trol section was similar to the reported biological distribution of
1
2
3
4
5
6
2
2
3
3
3
3
K
2
CO
CsF
CO
3
3
—
—
—
—
—
35
91
45
Trace
Trace
Trace
—
0
52
1
8
mGluR4 in the rat brain. Because of high selectivity of 1 for
1
1
K
2
mGluR4 over other mGluRs, we assumed that [ C]1 could have
some in vitro specific binding with mGluR4 in the rat brain. How-
ever, radioactive accumulation was identified in some negligible
regions, such as the cerebral cortex and midbrain, suggesting that
CsF
CsF
CsF
300
500
b
500c
97
a
11
Reagents and conditions: from 2; Pd
2 3 3
(dba) , CuCl, P(o-tol) , base, DMF, 80 °C,
specific binding of [ C]1 may contain binding to other receptors.
5
5
min. from 3; (i) Pd
min.
2
(dba)
3
, CuCl, P(o-tol)
3
, base, DMF, 80 °C, 5 min, (ii) TFA, 100 °C,
11
We further verified specific binding of [ C]1 in vivo by biodis-
b
tribution studies using dose–response assessments with unlabeled
Before addition of TFA, DMF was partly removed.
Before addition of TFA, DMF was completely removed under reduced pressure.
The radiochemical yield was determined by analyzing the reaction mixture
1
9
11
c
1. Figure 4A shows time–activity curves of [ C]1 in the blood
and brain regions (cerebellum, cerebral cortex, basal ganglia con-
taining striatum and thalamus, and whole brain). All radioactivity
d
using radio-HPLC.
Figure 3. Representative in vitro autoradiograms of rat sagittal brain sections with [11C]1 in presence of vehicle (A) or 10
lM unlabeled 1 (B).
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4
M. Fujinaga et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 4. Ex vivo distribution study of [11C]1 in the brain (n = 3, in each time point). (A) Time-course of radioactivity (%ID/g) in the blood, cerebellum, cerebral cortex, basal
ganglia, and whole brain. (B) Time-course of tissue-to-blood ratio in the cerebellum, cerebral cortex, basal ganglia, and whole brain.
Acknowledgment
We thank the staff of the National Institute of Radiological
Sciences (Chiba, Japan) for their support with the cyclotron opera-
tion, radionuclide production and animal experiments.
References and notes
1.
2.
3.
Gladding, C. M.; Fitzjohn, S. M.; Molnar, E. Pharmacol. Rev. 2009, 61, 395.
Kew, J. N.; Kemp, J. A. Psychopharmacology (Berl) 2005, 179, 4.
(a) Pinheiro, P. S.; Mulle, C. Nat. Rev. Neurosci. 2008, 9, 423; (b) Trombley, P. Q.;
Westbrook, G. L. J. Neurosci. 1992, 12, 2043.
4
5
6
.
.
.
(a) Maj, M.; Bruno, V.; Dragic, Z.; Yamamoto, R.; Battaglia, G.; Inderbitzin, W.;
Stoehr, N.; Stein, T.; Gasparini, F.; Vranesic, I.; Kuhn, R.; Nicoletti, F.; Flor, P. J.
Neuropharmacology 2003, 45, 895; (b) Marino, M. J.; Williams, D. L., Jr.; O’Brien,
J. A.; Valenti, O.; McDonald, T. P.; Clements, M. K.; Wang, R.; DiLella, A. G.; Hess,
J. F.; Kinney, G. G.; Conn, P. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13668.
(a) Kil, K. E.; Poutiainen, P.; Zhang, Z.; Zhu, A.; Choi, J. K.; Jokivarsi, K.; Brownell,
A. L. J. Med. Chem. 2014, 57, 9130; (b) Kil, K. E.; Zhang, Z.; Jokivarsi, K.; Gong, C.;
Choi, J. K.; Kura, S.; Brownell, A. L. Bioorg. Med. Chem. 2013, 21, 5955; (c) Engers,
D. W.; Niswender, C. M.; Weaver, C. D.; Jadhav, S.; Menon, U. N.; Zamorano, R.;
Conn, P. J.; Lindsley, C. W.; Hopkins, C. R. J. Med. Chem. 2009, 52, 4115.
(a) Le Poul, E.; Bolea, C.; Girard, F.; Poli, S.; Charvin, D.; Campo, B.; Bortoli, J.;
Bessif, A.; Luo, B.; Koser, A. J.; Hodge, L. M.; Smith, K. M.; DiLella, A. G.; Liverton,
N.; Hess, F.; Browne, S. E.; Reynolds, I. J. J. Pharmacol. Exp. Ther. 2012, 343, 167;
Figure 5. Dose–response of unlabeled 1 (0, 0.01, and 0.1 mg/kg) in the brain uptake
of [ C]1. Specific radioactive uptakes in the tissue were estimated using ratio to
blood under the equilibrium state (5 min after the injection).
11
(
% of injection dose per gram tissue, %ID/g) in brain regions peaked
(
b) Kalinichev, M.; Rouillier, M.; Girard, F.; Royer-Urios, I.; Bournique, B.; Finn,
11
at 1 min after the injection of [ C]1 and then showed a quick
decrease. To estimate equilibrium state for binding of [11C]1, the
ratio of tissue to blood in brain regions was calculated. Figure 4B
shows time-course of tissue-to-blood ratio in brain regions. The
T.; Charvin, D.; Campo, B.; Le Poul, E.; Mutel, V.; Poli, S.; Neale, S. A.; Salt, T. E.;
Lutjens, R. J. Pharmacol. Exp. Ther. 2013, 344, 624.
Suzuki, M.; Doi, H.; Koyama, H. WO 2010/074272.
7
8
.
.
Compound
1 (5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-
2
-amine, ADX88178): The title compound was prepared as previously
estimated equilibrium state for binding of [11C]1 was heteroge-
11
1
reported. colorless powder; mp: 256–258 °C; H NMR (300 MHz, DMSO-d
d: 2.41 (3H, s), 2.43 (3H, s), 6.87 (1H, d, J = 5.1 Hz), 7.87 (2H, br s), 8.43 (1H, d,
J = 4.8 Hz), 11.38 (1H, s), 12.93 (1H, br s). HRMS (FAB) calcd for C12 S,
73.0922; found, 273.0952.
9. Compound (5-methyl-4-(1H-pyrazol-4-yl)-N-(4-(tributylstannyl)pyrimidin-2-
yl)-thiazol-2-amine): colorless solid; mp: 204–206 °C;
DMSO-d ) d: 0.85 (9H, t, J = 7.3 Hz), 1.06–1.21 (6H, m), 1.25–1.37 (6H, m),
.49–1.70 (6H, m), 2.40 (3H, s), 7.09 (1H, d, J = 4.8 Hz), 7.80 (1H, br s), 7.94 (1H,
br s), 8.34 (1H, d, J = 4.4 Hz), 11.34 (1H, s), 12.94 (1H, s). HRMS (FAB) calcd for
SSn, 549.1822; found, 549.1840.
6
)
neously determined at 5 min after the injection of [11C]1, for exam-
13 6
H N
ple, 1.27 for the cerebellum, 1.13 for the cerebral cortex, 1.23 for
the basal ganglia, and 1.19 for the whole brain, respectively. By
administration with multi-doses of unlabeled 1, these ratios
decreased with dose-dependency (Fig. 5). Compared with the base-
line rat, percentages of decreased radioactivity in the brain regions
of rats administrated with 0.1 mg/kg unlabeled 1 were 22–26%.
Previously, in vivo blocking study in PET with [18F]KALB001, the
most promising PET ligand for mGluR4 until now, showed
2
2
1
H
NMR (300 MHz,
6
1
23 37 6
C H N
10.
Compound 3 (4-(1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-methyl-N-(4-(tributyl-
1
stannyl)pyrimidin-2-yl)thiazol-2-amine): yellow solid; mp: 75–77 °C; H NMR
(300 MHz, DMSO-d ) d: 0.85 (9H, t, J = 7.3 Hz), 1.06–1.20 (6H, m), 1.25–1.37
6
1
0–20% decline compared with baseline rats.5 Hence, the present
(6H, m), 1.46–1.67 (6H, m), 2.39 (3H, s), 3.73 (3H, s), 5.29 (2H, s), 6.92 (2H, d,
11
J = 8.1 Hz), 7.09 (1H, d, J = 4.0 Hz), 7.26 (2H, d, J = 8.1 Hz), 7.74 (1H, s), 8.01 (1H,
study suggested that in vivo specific binding of [ C]1 for mGluR4
13
3
s), 8.34 (1H, d, J = 5.1 Hz), 11.33 (1H, s). C NMR (75 MHz, CDCl ) d: 10.1, 11.7,
might be higher than that of [18F]KALB001.
1
3.7, 27.3, 29.0, 55.3, 55.6, 114.1, 117.9, 118.5, 122.9, 127.5, 128.4, 129.3,
137.8, 138.0, 154.2, 155.6, 156.2, 159.4, 186.4. HRMS (FAB) calcd for
45ON SSn, 669.2398; found, 669.2416.
In summary, we successfully synthesized [11C]1 as a novel radi-
oligand for mGluR4 by introducing a [11C]methyl group into the
C
31
H
6
1
1
1. Borea, C. WO 2010/079239.
pyrimidine ring. This labeling technique enables synthesis of PET
2. 2-Bromo-4-(tributylstannyl)pyrimidine was synthesized by trapping with
tributylstannyl chloride via magnesiation at the 4-position in 2-
bromopyrimidine using TMPMgClꢀLiCl, see reference: Marc, M.; Paul, K. Org.
Lett. 2008, 10, 2497.
1
1
ligands containing electron-deficient
[ C]methylpyrimidine.
1
1
Although [ C]1 showed low specific binding for mGluR4 in vitro
and in vivo, development of new candidates with higher binding
affinity to mGluR4 than 1 is required.
13. (a) Getmanenko, Y. A.; Twieg, R. J. J. Org. Chem. 2008, 73, 830; (b) Mathieu, J.;
Gros, P.; Fort, Y. Chem. Commun. 2000, 951.
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M. Fujinaga et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
1
4. Radiosynthesis of [11C]1 from precursor 3: CsF (2.3 mg, 15
chloride (1.5 mg, 15 mol), and Pd (dba) (1.3 mg, 1.4 mol) were added to a
minivial (1 mL) equipped with a septum and stirring bar. The vial was purged
with N gas, and a solution of P(o-tol) (1.7 mg, 5.6 mol) in DMF (0.15 mL)
was added to the vial. After the reaction mixture was stirred for 4 min at room
temperature, a solution of 3 (2.0 mg, 3 mol) in DMF (0.1 mL) was added to the
reaction mixture. This mixture was purged with N gas and stirred for 1 min.
The prepared solution was transferred with a syringe into a reaction vial
lmol), copper(I)
temperature-controlled environment with a 12-h light-dark cycle, and fed a
standard diet (MB-1; Funabashi Farm, Chiba, Japan). Animal experiments were
performed according to the recommendations of the Committee for the Care
and Use of Laboratory Animals, National Institute of Radiological Sciences. (b)
In vitro autoradiography: Three rats anesthetized with 1.5% (v/v) isoflurane
were sacrificed by the cervical dislocation and their brains were quickly
removed and frozen. The sagittal brain sections (20 lm) were prepared from
frozen rat brains using a cryostat (HM560, Carl Zeiss, Oberkochen, Germany).
Brain sections were pre-incubated for 10 min in Tris-buffer (50 mM Tris–HCl,
l
2
3
l
2
3
l
l
2
1
1
equipped with the automated synthetic unit. [ C]CH
gas flow into the prepared solution at ꢁ15 to ꢁ20 °C. After radioactivity of [ C]
CH I reached at a plateau, the reaction mixture was heated at 80 °C for 5 min.
3
I transferred under N
2
11
2 2
1.2 mM MgCl , and 2 mM CaCl , pH 7.4) at room temperature. After pre-
3
incubation, these sections were incubated for 30 min at room temperature in
After removal of DMF under reduced pressure, TFA (0.5 mL) was added to the
fresh buffer containing [11C]1 (37 MBq/mL in saline) in the absence (vehicle,
residue and heated at 100 °C for 5 min. The reaction mixture was neutralized
0.1% DMSO) or presence of 10 lM unlabeled 1 (dissolved in DMSO, final
with a solution of 2 mol/L CH
3
CO
2
Na (0.7 mL) in MeOH (0.3 mL), filtered with a
concentration: 0.1%). After incubation, these sections were washed three times
for 5 min each time with cold buffer, dipped in cold distilled water, and dried
in air. These sections were placed in contact with an imaging plate (BAS-
MS2025; Fujifilm, Tokyo, Japan). Autoradiograms were acquired using a Bio-
Imaging Analyzer System (BAS5000; Fujifilm).
glass fiber prefilter (GF53, 30 mm; Agilent Technologies, Santa Clara, CA), and
applied to the HPLC system. HPLC purification was performed using a JASCO
HPLC system (JASCO, Tokyo, Japan) under the following conditions. Column:
Capcell Pak C18 (Shiseido, Tokyo, Japan), 10 mm ꢂ 250 mm; detector: UV
2
54 nm; eluent: MeOH/H
2
O/TFA = 5/5/0.01 (v/v/v); flow rate: 5.0 mL/min. The
18. Testa, C. M.; Standaert, D. G.; Young, A. B.; Penney, J. B., Jr. J. Neurosci. 1994, 14,
3005.
1
1
radioactive fraction corresponding to [ C]1 [retention time (t
collected in a sterile flask containing polysorbate (80) (75
150
normal saline, and passed through a 0.22-
for analysis and the animal experiments. HPLC analysis was performed under
the following conditions. Column: Capcell Pak (Shiseido)
.6 mm ꢂ 250 mm; detector: UV 254 nm; eluent: MeOH/H O/TFA = 5/5/0.01
v/v/v); flow rate: 1.0 mL/min; 8.3 min. The identity of
R
): 7.9 min] was
l
L) and ethanol
19. Respective mouse (n = 3 in each time point, 34–41 g) was injected a bolus of
11
(
l
L), evaporated to dryness under vacuum, re-dissolved in 3 mL of sterile
[
C]1 (8.6 MBq/0.1 mL, 79 pmol) via its tail vein. Three mice were killed at
l
m Millipore filter to obtain [11C]1
each experimental time point (1, 5, 15, 30, and 60 min) after the injection of
11
[
C]1 by cervical dislocation, respectively. For the dose–response assessment,
C
18
mice (n = 3 in each group, 36–41 g) were administrated with multi-doses (0,
4
(
2
0.01, and 0.1 mg/kg) of unlabeled 1 prior to the injection of [11C]1 (5.2 MBq/
11
11
t
R
:
[
C]1 with
0.1 mL, 49 pmol). Three mice were killed at 5 min after the injection of [ C]1.
unlabeled 1 was confirmed by analytical HPLC under the same condition.
5. Zhang, M.-R.; Kida, T.; Noguchi, J.; Furutsuka, K.; Maeda, J.; Suhara, T.; Suzuki,
K. Nucl. Med. Biol. 2003, 30, 513.
6. Suzuki, K.; Inoue, O.; Hashimoto, K.; Yamasaki, T.; Kuchiki, M.; Tamate, K. Int. J.
Appl. Radiat. Isot. 1985, 36, 971.
The brain and blood samples were quickly removed. Subsequently, removed
brains were separated into the cerebellum, cerebral cortex, basal ganglia, and
residue. The radioactivity in these tissues was measured by an autogamma
scintillation counter (1480 Wizard, Perkin-Elmer, Waltham, MA) and
expressed as the percentage of injected dose per gram of wet tissue (%ID/g).
All radioactivity measurements were decay-corrected.
1
1
1
7. (a) Animals: Male Sprague-Dawley rats (8 weeks old) and ddY mice
(
8–9 weeks old) were purchased from Japan SLC (Shizuoka, Japan), kept in a
Please cite this article in press as: Fujinaga, M.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.008