Synthesis and evaluation of 6-[1-(2-[18F]fluoro-3-pyridyl)-5- methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

Article abstract of DOI:10.1016/j.bmc.2010.11.048

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro- 3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [ ¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3- triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/μmol specific activity using 3300-4000 MBq of [¹⁸F]F^-. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.

Full text of DOI:10.1016/j.bmc.2010.11.048

Bioorganic & Medicinal Chemistry 19 (2011) 102–110
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
18
Synthesis and evaluation of 6-[1-(2-[ F]fluoro-3-pyridyl)-5-methyl-
H-1,2,3-triazol-4-yl]quinoline for positron emission tomography
imaging of the metabotropic glutamate receptor type 1 in brain
1
a
a
a
a
a
Masayuki Fujinaga , Tomoteru Yamasaki , Kazunori Kawamura , Katsushi Kumata , Akiko Hatori ,
a
a
a,b
a,b
a,b
Joji Yui , Kazuhiko Yanamoto , Yuichiro Yoshida , Masanao Ogawa , Nobuki Nengaki
Jun Maeda , Toshimitsu Fukumura , Ming-Rong Zhang
,
a
a
a,
⇑
a
Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
SHI Accelerator Service Co. Ltd, 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
b
a r t i c l e i n f o
a b s t r a c t
The purpose of this study was to synthesize 6-[1-(2-[18F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-
Article history:
Received 27 September 2010
Revised 18 November 2010
Accepted 20 November 2010
Available online 25 November 2010
18
18
yl]quinoline ([ F]FPTQ, [ F]7a) and to evaluate its potential as a positron emission tomography ligand
18
for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [ F]7a was
18
synthesized by [ F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline
8
18
(
7b) with potassium [1 F]fluoride. At the end of synthesis, 1280–1830 MBq (n = 8) of [ F]7a was obtained
with >98% radiochemical purity and 118–237 GBq/
l
mol specific activity using 3300–4000 MBq of
Keywords:
18
À
18
[
F]F . In vitro autoradiography showed that [ F]7a had high specific binding with mGluR1 in the rat
Metabotropic glutamate receptor type 1
18
18
brain. Biodistribution study using a dissection method and small-animal PET showed that [ F]7a had
high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled
[
F]FPTQ
Autoradiography
PET
1
8
7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [ F]7a had in vivo specific binding
1
8
with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [ F]7a may have
a limiting potential for the in vivo imaging of mGluR1 by PET.
Ó 2010 Elsevier Ltd. All rights reserved.
1
. Introduction
in the rodent brain.15 Compound [11C]1 is an analog of
JNJ-16259685 (2, K
i
= 0.34 nM), a potent mGluR1 antagonist.16
1
1
Metabotropic glutamate receptors (mGluRs) are a family of
Compound [ C]1 had high uptake of radioactivity in the rat
cerebellum, which is a region with high density of mGluR1;1
however, further evaluation, including clinical investigation using
7,18
G-protein coupled receptors activated by the neurotransmitter glu-
tamate.
with eight subtypes based on their pharmacology, signal transduc-
tion mechanism, and sequence homology. Group I mGluRs, includ-
ing mGluR1 and mGluR5, modulate intracellular calcium by
1
,2
These receptors have been divided into three groups
1
1
18
[ C]1, has not been reported. Recently, two F-labeled triazole
1
8
18
19
18
analogs
[
F]MK-1312 ([ F]3,
K
i
= 0.40 nM)
and [ F]FTIDC
1
8
20,21
([ F]4, K
i
= 3.9 nM)
for imaging mGluR1 have been presented
3
,4
18
18
stimulating phospholipase C and regulate neuronal excitability.
at two meetings. Compound [ F]3 and [ F]4 were shown to have
high uptake in the brains of monkeys and rats, respectively.
Further characterization demonstrated that [ F]3 had in vivo spe-
19,21
MGluR1 is mainly a postsynaptic receptor and may be related to
diseases such as stroke, epilepsy, pain, cerebellar ataxia, Parkin-
son’s disease, anxiety, and mood disorders.4 To elucidate the
1
8
–7
22
cific binding with mGluR1 in the monkey brain. In addition, a
preliminary study reported that [ C]MMTP ([11C]5, K
1
1
= 7.9 nM)
showed a specific location of radioactivity in the cerebellum of
physiological role of mGluR1 in the brain and the therapeutic po-
i
tential of mGluR1 ligands8
–14
in the pathology and/or etiology of
2
3
central nervous system diseases, radioligands (Scheme 1) labeled
with a positron-emitting radioisotope have been developed to
visualize mGluR1 in living brains using positron emission tomogra-
phy (PET).
the monkey brain.
Recently, we have labeled a mGluR1 antagonist YM-202074 (6,
K = 4.8 nM) with C and evaluated its potential as a PET ligand
i
2
4
11
2
5
for mGluR1. In vitro autoradiographic study demonstrated that
As the first PET ligand for mGluR1, [11C]JNJ-16567083 ([11C]1,
[
11
C]6 had high specific binding with mGluR1 in the rat cerebellum
K
i
= 0.87 nM for mGluR1) was developed for imaging this receptor
and its regional distribution was consistent with the distribution
pattern of mGluR1 in the brain. However, the brain uptake of
1
1
⇑
Corresponding author. Tel.: +81 43 382 3708; fax: +81 43 206 3261.
E-mail address: zhang@nirs.go.jp (M.-R. Zhang).
[
C]6 was very low, which hampered its usefulness for in vivo
imaging.
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.048

M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
103
O
O
H CO
N
H CO
N
O
N
3
11CH
3
3
[11
C]JNJ-16567083 ([11C]1)
JNJ-16259685 (2)
N
N
N
N
18F
N
18F
CH3
N
N
N
N
N
O
O
[18F]MK-1312 ([18F]3)
[18F]FTIDC ([18F]4)
O
N
N
N
N
O11CH3 H CO
3
N
N
N
11CH3
S H C
3
S
O
N
[11C]YM-202074 ([11C]6)
[11C]MMTP ([11C]5)
Scheme 1. Chemical structures of ligands for mGluR1.
To develop a PET ligand with improved properties compared to
C]6 for imaging mGluR1 in the brain, we established 6-[1-(2-flu-
precursor 7b was prepared by coupling of 9b with 10 with a total
yield of 33% from aminobromopyridine.
[11
oro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline
a; Scheme 2) as a target compound in the present study. Com-
pound 7a had a potent in vitro binding affinity (IC50 = 3.6 nM and
(FPTQ,
7
2.2. Radiosynthesis
1
.4 nM for human and mouse mGluR1), but its chemical synthesis
Compound [18F]7a was synthesized by heating the bromo pre-
26,27
18
has not been reported.
evaluated [ F]FPTQ ([ F]7a) as a promising PET ligand for
In this study, we synthesized and
cursor 7b with cyclotron-produced potassium [ F]fluoride
1
8
18
18
([ F]KF) in anhydrous DMSO at 150 °C for 10 min (Scheme 3).
1
8
mGluR1. Here, we report: (1) the chemical synthesis of unlabeled
After the [ F]fluorination reaction, semi-preparative HPLC purifi-
1
8
7
a and a novel bromo precursor 7b for labeling, (2) the radiosyn-
cation of the reaction mixture gave [ F]7a in 69 ± 13% (n = 8)
1
8
18
À
thesis of [ F]7a, and (3) the in vitro and in vivo characterization
of [ F]7a for mGluR1 in the rat brain.
radiochemical yields based on [ F]F , corrected for physical decay
for an averaged synthesis time of 75 min (Fig. 1A). The identity of
1
8
1
8
[
F]7a was confirmed by coinjection with unlabeled 7a on analytic
HPLC. At the end of synthesis (EOS), the radiochemical purity and
2
2
. Results
1
8
specific activity of [ F]7a was higher than 99% (Fig. 1B) and
18–237 GBq/mmol, respectively. No significant peak correspond-
1
.1. Chemical synthesis
ing to unreacted 7b or other chemical impurities was observed on
the HPLC chart of the final product (Fig. 1B). The radiochemical
purity of [ F]7a remained higher than 95% after maintaining the
formulated product for at least 3 h at room temperature.
The two triazole analogs 7a and 7b were synthesized according
1
8
to the reaction sequences lineated in Scheme 3. 3-Azido-2-fluoro-
pyridine (8a) was prepared by reacting 2-fluoropyridine with lith-
ium diisopropylamide (LDA) and then an azide agent at À78 °C for
2
.3. Computation of c Log P and c Log D, and measurement of
1
h. Cyclization of 8a with tributyl(1-propynyl)tin in toluene pro-
Log D
duced 9a with a triazole ring. Coupling of 9a with quinolinyltriflate
0 in the presence of tetrakis(triphenylphosphine)palladium as a
catalyst at 80 °C gave 7a. The overall chemical yield of 7a was
1% from 2-fluoropyridine.
-Amino-2-bromopyridine was first treated with sodium nitrite
1
The computed values of c Log P and c Log D (at pH 7.4) for 7a
were 2.93 and 3.05, respectively. The measured Log D value of
2
1
8
[
F]7a was 2.53 ± 0.02 (n = 3).
3
and tetrafluoroboric acid, followed by substitution with sodium
azide to give azidobromopyridine 8b. As with 8a, 8b was reacted
with the alkynyl regent to afford triazole 9b. The novel bromo
2
.4. In vitro autoradiography
Figure 2 and Table 1 show representative in vitro autoradiograms
1
8
and quantitative results of the [ F]7a binding in the brain sections
1
8
of rats (n = 4). The distribution pattern of [ F]7a was heterogenous
with high radioactivity in the cerebellum, a known mGluR1-rich re-
gion (A). The thalamus displayed a moderate level of radioactivity.
Low radioactivity was seen in the hippocampus, striatum, olfactory
bulb, cerebral cortex, and pons-medulla (Table 1). Incubation with
N
N
N
X
FPTQ (7a): X = F
[
N
18
F]FPTQ ([18F]7a): X = 18
F
N
unlabeled 7a (B) and mGluR1 antagonist 2 (C) at 1 lM markedly
Scheme 2. Chemical structures of unlabeled 7a and [18F]7a for mGluR1 in this
study.
decreased the radioactivity in the brain sections to <3% of total
radioactivity (Table 1), respectively. On the other hand, MPEP

1
04
M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
C12H25Ph-SO N3
2
N
N
a
Bu
Bu Sn
Bu
F
C
d
C
CH3
N
N
Bu Sn
N3
3
N
X
N
N
X
1
) NaNO /HBF4 , b
2
H N
8a: X = F
8b: X =Br
9a: X = F
9b: X =Br
2
2
) NaN , c
3
Br
OTf
N
N
N
10
N
N
X
7
7
a: X = F
b: X =Br
N
N
e
N
N
N
N
18F
K18
F
[18F]7a
7
b
f
Scheme 3. Chemical synthesis and radiosynthesis. Reagents and conditions: (a) lithium diisopropylamide, THF, À78 °C, 1 h; (b) 0 °C, 2 h; (c) 0 °C, 2 h; (d) toluene, reflux, 12 h,
18
À
4
3 4
2% (9a), 64% (9b); (e) Pd(PPh ) , CsF, CuI, DMF, 80 °C, 12 h, 49% (7a), 52% (7b); (f) DMSO, 150 °C, 10 min, 69% decay-corrected based on total [ F]F .
A
800
UV
RI
values in most tissues were the highest at 5 min and thereafter de-
creased quickly. High uptake was found in the liver and small
intestine, while low uptake was determined in the blood and other
peripheral organs, including the heart, lung, spleen, kidney, pan-
creas, and muscle. On the other hand, relatively high radioactivity
was detected in the brain, the target tissue of this study. Among all
brain regions examined, the cerebellum showed the highest up-
take. The radioactivity in the thalamus was lower than that in
the cerebellum, but was slightly higher than that in the hippocam-
pus, striatum, cerebral cortex, olfactory bulb, and pons-medulla.
7
b
700
600
500
400
300
200
100
0
[18
F]7a
0
5
10
15
20
25
min
B
800
2.6. Small-animal PET imaging
7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
Figures 3A–D and 4A show representative PET/MRI-fused brain
images and time–activity curves (TACs) of [ F]7a in the brain re-
gions of rats. The summation images (A and B) between 0 and
0 min after radioligand injection showed the highest uptake of
UV
1
8
RI
3
radioactivity in the cerebellum. A lower but detectable radioactiv-
ity level was seen in the thalamus (C), hippocampus (C), striatum
(D), and cerebral cortex (D). The lowest uptake was found in the
medulla (A and B), a region with low mGluR1 density. As shown
7b
[18
F]7a
1
8
0
5
10
15 min
in the TACs (Fig. 4A), [ F]7a entered the brain rapidly after injec-
tion and radioactivity peaked at 1–3 min in all regions examined.
The maximum uptakes were 2.3 standard uptake value (SUV) in
the cerebellum, 2.1 SUV in the thalamus, and 1.1–1.5 SUV in the
hippocampus, striatum, cerebral cortex, and medulla, respectively.
After the initial uptake, the radioactivity level in the cerebellum at
15 and 30 min decreased to 66% and 37% of the maximum. Com-
pared to the cerebellum, other regions showed a faster decrease
in radioactivity. From 20 min after injection, radioactivity in the re-
gions except the cerebellum remained at a similar and low level.
The uptake ratio of radioactivity in the regions to that in the me-
dulla was used as an index to reflect in vivo specific binding of
Figure 1. Chromatograms from the HPLC separation (A) and analysis (B) used in the
radiosynthesis of [18F]7a. See Section 5 for chromatographic conditions.
(
a mGluR5 antagonist) at 1
the cerebellum, thalamus, hippocampus, olfactory bulb, and stria-
tum (D, Table 1), although MPEP at 10 M decreased the binding
to 30–40% of total binding in all regions examined (E).
lM did not reduce the radioactivity in
l
2
.5. Biodistribution by dissection
1
8
The radioactivity distribution was measured in rats (n = 3) at 5,
5, and 30 min after injection of [18F]7a (Table 2). The radioactivity
[
F]7a in the brain. The uptake ratio of each region/medulla was
3.73 ± 0.50 (n = 4) for the cerebellum, 1.91 ± 0.15 for the thalamus,
1

M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
105
Figure 2. Representative in vitro autoradiograms of [18F]7a in brain sections. (A) [18F]7a alone; (B) [18F]7a with 7a (1
l
M); (C) [18F]7a with 2 (1
l
M); (D) [18F]7a with MPEP
(1 l lM).
M); (E) [18F]7a with MPEP (10
Table 1
Regional distribution of [18F]7a in rat brain sections determined by in vitro autoradiography
Region
Radioactivitya (PSL/mm2)
mGluRl antagonists
M) (1
[18
F]7a
mGluR5 antagonists
M) MPEP (10
7
a (1
l
l
M)
MPEP (1
l
l
M)
*
Cerebellum
Thalamus
Hippocampus
Olfactory bulb
Striatum
Cerebral cortex
Pons-medulla
Whole brain
408.8 ± 48.1
188.9 ± 44.4
112.4 ± 29.4
88.7 ± 38.1
70.7 ± 14,2
28.2 ± 4.7
—b
—b
—
—
—
—
—
397.5 ± 31.6
169.4 ± 39.9
97.4 ± 27.7
76.0 ± 30.7
61.7 ± 16.0
284.7 ± 41.4
115.7 ± 20.7
69.4 ± 13.1
49.0 ± 16.9
*
*
*
*
40.1 ± 6.9
15.7 ± 3.3
*
*
23.6 ± 4.2
*
*
17.5 ± 2.7
15.2 ± 2.5
9.5 ± 1.7
0.6 ± 0.2c
0.5 ± 0.2c
—
b
—
b
a
2
Radioactivity concentrations in regions of interest (ROIs) were expressed as photo-stimulated luminescence values (PSL)/area (mm ). Data are the means ± SD (n = 6–8 in
each group).
b
Not determined.
In the presence of 7a or 2, ROIs were placed on the whole brain.
P <0.05 (Student’s paired t-test, compared with [ F]7a alone).
c
*
18
The radioactivity in the cerebellum was reduced to about 30% of
the control, while that in the thalamus, hippocampus, striatum,
and cerebral cortex was decreased to 50–70% of the control. Similar
to 7a, mGluR1-selective 2 also reduced the uptake and diminished
the difference in radioactivity among all brain regions (Figs. 3I–L
and 4C). On the other hand, pretreatment with MPEP did not
change the distribution of [ F]7a in the brain (Figs. 3M–P and
4D). The difference in the TACs of the same brain regions between
the control and MPEP-treatment groups was analyzed by two-way
repeated-measures ANOVA. This comparison showed that differ-
ence in the cerebellum was not significant (P = 0.86), but difference
in the thalamus, hippocampus, striatum, and cerebral cortex was
statistically significant (P <0.05).
Table 2
Biodistribution of [18F]7a in rats (n = 3) by dissection method
Tissues
5 min
15 min
30 min
Blood
Heart
Lung
Liver
Spleen
Kidney
S. intestine
Pancreas
Muscle
0.38 ± 0.01
0.47 ± 0.03
0.50 ± 0.03
2.30 ± 0.35
0.43 ± 0.03
0.76 ± 0.06
3.06 ± 1.22
0.75 ± 0.03
0.28 ± 0.01
0.22 ± 0.02
0.52 ± 0.01
1.21 ± 0.05
0.58 ± 0.02
0.73 ± 0.05
0.54 ± 0.06
0.52 ± 0.06
0.44 ± 0.00
0.47 ± 0.01
0.26 ± 0.04
0.24 ± 0.05
0.31 ± 0.06
2.04 ± 0.29
0.25 ± 0.05
0.57 ± 0.06
4.64 ± 1.39
0.34 ± 0.12
0.14 ± 0.04
0.15 ± 0.02
0.23 ± 0.04
0.54 ± 0.09
0.22 ± 0.05
0.29 ± 0.02
0.23 ± 0.04
0.24 ± 0.08
0.20 ± 0.04
0.21 ± 0.04
0.15 ± 0.05
0.16 ± 0.07
0.17 ± 0.09
1.93 ± 0.20
0.16 ± 0.10
0.39 ± 0.14
1.74 ± 0.35
0.21 ± 0.14
0.10 ± 0.06
0.22 ± 0.05
0.11 ± 0.03
0.20 ± 0.03
0.09 ± 0.03
0.10 ± 0.03
0.11 ± 0.04
0.10 ± 0.02
0.09 ± 0.03
0.13 ± 0.10
1
8
Bone
Whole brain
Cerebellum
Hippocampus
Thalamus
Striatum
Olfactory bulb
Cerebral cortex
Pons-medulla
2
.7. Metabolite analysis
Figure 5 shows the percentages of parent [18F]7a in the cerebel-
lum, the brain except the cerebellum, and the plasma of mouse
measured by analytical HPLC with a detector for radioactivity.
1
8
The fraction corresponding to parent [ F]7a (retention time:
.8 min) in the plasma rapidly decreased to 57% at 5 min, to 23%
5
1
1
.55 ± 0.22 for the striatum, 1.42 ± 0.11 for the hippocampus, and
.31 ± 0.13 for the cerebral cortex between 10 and 15 min after
at 15 min, and to 4% at 30 min after injection. A major radiolabeled
metabolite (retention time: 2.7 min) with high polarity was ob-
served on the HPLC charts. On the other hand, parent [18F]7a in
the cerebellum homogenate remained at 91% of total radioactivity
at 15 min after injection. The percentages of parent [18F]7a were
67% and 34% of total radioactivity in the cerebellum and brain ex-
cept the cerebellum at 30 min, respectively.
injection.
As shown in PET images (Fig. 3E–H) and TACs (Fig. 4B), pretreat-
ment with unlabeled 7a (1.0 mg/kg) led to a remarked reduction in
the uptake compared to the control group. With this treatment, the
radioactivity distribution became fairly uniform in the whole brain.

1
06
M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
Figure 3. Representative PET/MRI-fused images of [18F]7a in the isoflurane-anesthetized rat brains. PET images were generated by summing the whole scan (0–30 min) and
1
8
18
18
were overlaid on MRI images of anatomical templates. (A–D) [ F]7a alone; (E–H) [ F]7a after treatment with 7a (1 mg/kg); (I–L) [ F]7a after treatment with 2 (1 mg/kg);
(
M–P) [18F]7a after treatment with MPEP (1 mg/kg). (A, E, I, M) sagittal images; left numbers: coronal images.
2
1
0
.5
2.5
2
A: [18F]7a
B: [18F]7a + 7a
Cerebellum
Cerebellum
2
Thalamus
Thalamus
Hippocampus
Striatum
Hippocampus
Striatum
.5
1
1.5
1
Cerebral Cortex
Medulla
Cerebral Cortex
Medulla
.5
0
0.5
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
2
1
0
.5
2
C: [18F]7a + 2
2.5
2
D: [18F]7a + MPEP
Cerebellum
Cerebellum
Thalamus
Thalamus
Hippocampus
Striatum
Hippocampus
Striatum
.5
1
1.5
1
Cerebral Cortex
Medulla
Cerebral Cortex
Medulla
.5
0
0.5
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
Figure 4. Time–activity curves in the rat brain regions after injection of [18F]7a. (A) [18F]7a alone; (B) [18F]7a after treatment with 7a (1 mg/kg); (C) [18F]7a after treatment
with 2 (1 mg/kg); (D) [18F]7a after treatment with MPEP (1 mg/kg). The uptakes of radioactivity were decay-corrected to the injection time and are expressed as the
standardized uptake value (SUV), normalized for injected radioactivity and body weight. Data are the means ± SD (n = 4 in each group).

M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
107
1
00
vitro study using
a
selective radioligand [3H]R21412718 for
mGluR1 in the rat brain showed the highest density of mGluR1
in the cerebellum, moderate density in the thalamus, hippocam-
pus, and olfactory bulb, and low density in the cerebral cortex,
5
min
15 min
0 min
80
60
40
20
0
18
3
striatum, and pons-medulla. The present distribution of [ F]7a
in the rat brain was similar to that of [ H]R214127 or [11C]6.
3
25
Although the radioactivity level was different among the brain re-
1
8
gions examined, the specific binding of [ F]7a accounted for
higher than 97% of total binding, as determined by incubating
with unlabeled 7a or mGluR1-selective 2. On the other hand,
mGluR5-selective MPEP (IC50: 10 nM for mGluR5 and >10 mM
1
0
for mGluR1) at 10 mM decreased the binding to 30–40% of the
1
8
control, suggesting that [ F]7a may have a very weak affinity
with mGluR5 (IC50 >10 M) based on this in vitro autoradiogra-
phy. However, MPEP at 1 M did not inhibit the binding in most
l
l
Cerebellum
Brain except
cerebellum
Plasma
regions with statistical significance. This autoradiography demon-
18
strated that [ F]7a has high in vitro binding for mGluR1 in the rat
Figure 5. Metabolite analysis of [18F]7a in the cerebellum, brain except the
cerebellum, and plasma of mice (n = 3 in each group).
cerebellum.
Dissection method and small-animal PET were used to deter-
1
8
mine the in vivo distribution of [ F]7a in the rat. Compound
1
8
[
F]7a had high uptake in the liver and small intestine, suggest-
2
.8. Protein binding
ing that the hepatability and urinary excretion, as well as intesti-
nal reuptake pathway dominated the whole-body distribution of
The binding of [18F]7a with protein in the plasma was deter-
mined by ultra-filtration. The free (not protein-bound) fraction
was 8.5–9.9%.
18
[
F]7a and a rapid washout of radioactivity from the body (also
see TACs of peripheral organs described in Supplementary data).
18
The brain uptake of [ F]7a was relatively high, indicating that
this radioligand passes across the BBB and enters the brain, which
is a prerequisite for a favorable PET ligand used in brain imaging.
This uptake was probably related to the suitable lipophilicity
3
. Discussion
In this study, we synthesized [18F]7a and determined its in vitro
18
(Log D: 2.53) of [ F]7a and relatively high percentage (8.5–
and in vivo specific binding with mGluR1 in the rat brain using
autoradiography and small-animal PET.
9.9%) of free fraction in the plasma. Regional distribution of
1
8
[
F]7a in the brain was heterogeneous and the highest radioac-
We described the synthetic route of unlabeled 7a, which was
adapted from the patent literature26 and is the first full report of
its reaction, purification, and characterization data. We prepared
tivity was detected in the cerebellum among all brain regions
(Table 2).
PET imaging showed that the distribution and uptake of [18F]7a
in the brain was consistent with the results obtained by dissection
7
a by three-step reactions using commercially available materials
18
(Scheme 3). By treating fluoropyridine with LDA and then an azide
as well as by in vitro autoradiography. The uptake of [ F]7a in the
1
1
25
reagent, the azide group was successfully introduced to the pyri-
dine ring to give 8a. However, this introducing route is not suit-
able for the synthesis of bromo 8b, which is a key intermediate
for preparing the novel bromo precursor 7b. Because LDA is a
strong base and would have caused the lithiodebromination of
cerebellum was fivefold higher than that of [ C]6 in the rat cer-
1
8
19
ebellum, and was similar to that of [ F]3 in the monkey cerebel-
11
15
lum, although the level was approximately 1/3 that of [ C]1.
Pretreatment with 7a (Figs. 3E–H and 4B) and 2 (Figs. 3I–L and
4C) significantly reduced the uptake and completely diminished
the difference in uptakes among all brain regions. The obvious
blockade by 7a and 2 showed that the binding in the receptor-rich
cerebellum was mGluR1 mediated. On the other hand, the uptake
was not changed in the cerebellum by treatment with mGluR5-
selective MPEP, but slightly decreased in the thalamus with statis-
tical significance, suggesting the low specific binding in the thala-
mus may also be related to mGluR5.
2
-bromopyridine, we determined another approach instead of
using LDA. After aminobromopyridine was diazonized, treatment
of the reaction mixture with sodium azide afforded 8b. Unstable
8
a or 8b was not purified and was directly used for cyclization.
Subsequent coupling of 9a or 9b with triflate 10 produced the de-
sired 7a or 7b.
Since 7a structurally has a fluorine atom attached to the 2-po-
sion of the pyridine ring, this compound is easily labeled with
Presence of a radiometabolite in plasma may reduce the specific
binding of a parent PET ligand if the radiometabolite enters the
brain and is retained/bound at the target sites. Although in vivo
metabolite analysis showed the presence of a radiometabolite in
the plasma, the main radioactive component in the cerebellum
1
8
F by nucleophilic [18F]fluorination. This labeling did not change
the chemical structure and pharmacological profile of 7a.
1
8
[
F]Fluorination of the bromo precursor 7b proceeded efficiently
1
8
to give [ F]7a, which could be separated from the unreacted pre-
1
8
18
cursor by prolonging their retention times (7b: 19.6 min; [ F]7a:
3.7 min) during the HPLC purification (Fig. 1). At EOS, the radioac-
tivity amount, radiochemical purity, stability, and specific activity
and other regions was parent [ F]7a at 15 min after injection
2
(Fig. 5). On the other hand, although defluorination of the
1
8
[
F]ligand is a possible metabolite route that could hamper brain
1
8
29
of [ F]7a were sufficient for animal experiments.
imaging and quantitative analysis of receptors in the brain, no
considerable radioactivity was visualized in the skull, as shown
in the present PET images (Fig. 3). Dissection also demonstrated
no obvious increase of radioactivity in the bone between 5 and
30 min after injection (Table 2). Thus, despite the presence of a
small amount of radiometabolite in the brain, we assumed that
the specific binding to mGluR1 determined in the brain until
The measured Log D value of [18F]7a was found to be 2.53 and
was appreciably different from the computed value (3.05). The
measured value lies in the range normally considered favorable
for good penetration of the blood–brain barrier (BBB) in the ab-
sence of any effect of efflux transporters.28
In vitro autoradiographic results (Fig. 2, Table 1) showed that
the distribution pattern of [18F]7a in the brain regions was consis-
15 min after radioligand injection was mainly due to parent
1
7,18
18
tent with the distribution of mGluR1 reported previously.
In
[ F]7a.

1
08
M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
4
. Conclusions
toluene under reduced pressure gave a residue, which was purified
by chromatography on a silica gel column under hexane/AcOEt
(3:1) with Et
N (1%) to give 9a (489 mg, 42%) as a brown oil. 1
NMR (300 MHz, CDCl ) d: 0.90 (9H, t, J = 7.1 Hz), 1.12–1.42 (12H,
This study described the synthesis and characterization of a PET
3
H
1
8
18
ligand [ F]7a for imaging mGluR1 in the rat brain. [ F]7a was
3
1
8
synthesized by [ F]fluorination of the bromo precursor 7b with
m), 1.54–1.62 (6H, m), 2.29 (3H, d, J = 2.2 Hz), 7.40–7.45 (1H, m),
1
1
8
8
À
[
[
F]F at high and reproducible radiochemical yields. Compound
7.96–8.02 (1H, m), 8.37–8.39 (1H, m). HRMS (FAB) calcd for
+
F]7a exhibited in vitro and in vivo binding with mGluR1 in the
C H
20 34
N
4
FSn: 469.1789; found: 469.1761 (M ).
brain regions such as the cerebellum, suggesting its usefulness
for visualizing mGluR1 in the cerebellum. However, the rapid dis-
5.1.2. 1-(2-Bromo-3-pyridyl)-5-methyl-4-tri-n-butylstannyl-1H-
1,2,3-triazole (9b)
1
8
sociation of [ F]7a in the brain may show limited specificity for
mGluR1 and its brain kinetics may be easily influenced by blood
flow. Moreover, the presence of a low amount of radiolabeled
metabolite in the brain may disturb determination for the specific
To
a
suspension of 3-amino-2-bromopyridine (1.04 g,
6.0 mmol) in water (8.0 mL) was added dropwise a solution of so-
dium nitrite (500 mg, 7.2 mmol) and tetrafluoroboric acid (10 mL,
42%) at 0 °C for 2 h. Sodium azide (19 mg, 0.05 mmol) was added
with caution and the mixture was stirred at 0 °C for 2 h. The reac-
18
binding of this radioligand. Thus, [ F]7a may be of limited applica-
tion for in vivo investigation of mGluR1. This problem would
be solved by the further development of more potent mGluR1-
selective PET ligands with improved pharmacological profiles, such
as binding affinity and in vivo metabolism.
tion mixture was quenched with saturated Na
with AcOEt. The organic layer was washed with water and satu-
rated NaCl, and dried over Na SO . The solvent was removed to
2 3
CO and extracted
2
4
give crude 3-azido-2-bromopyridine (8b).
Tributyl(1-propynyl)tin (1.16 mL, 2.31 mmol) was successively
added to a solution of the above 8b in dry toluene (10 mL) and
the reaction mixture was heated at reflux for 12 h. Removal of tol-
uene under reduced pressure gave a residue, which was chromato-
5
. Experimental section
Melting points were uncorrected. 1H NMR spectra were re-
corded on a JNM-AL-300 spectrometer (JEOL; Tokyo, Japan) with
tetramethylsilane as an internal standard. All chemical shifts (d)
were reported in parts per million (ppm) downfield from the
standard. High resolution (HR) FAB-MS was obtained on a JEOL
NMS-SX102 spectrometer (JEOL). Column chromatography was
performed on Wakogel C-200 (Wako Pure Chem Ind; Osaka, Japan).
HPLC was performed using a JASCO HPLC system (JASCO; Tokyo,
Japan): effluent radioactivity was monitored using a NaI (Tl)
scintillation detector system. Compounds 2, 6, and MPEP were pur-
chased from ALEXIS BIOCHEMICALS (San Diego, CA). All chemical
reagents of the highest grade commercially available were pur-
chased from Aldrich Chem (Milwaukee, WI) or Wako. Fluorine-18
graphed on silica gel under hexane/AcOEt (3:1) with Et
3
N (1%) to
afford 9b (2.03 g, 64%) as a yellowish oil. H NMR (CDCl ) d: 0.90
9H, t, J = 7.1 Hz), 1.12–1.39 (12H, m), 1.55–1.63 (6H, m), 2.22
3H, s), 7.47–7.51 (1H, m), 7.73–7.77 (1H, m), 8.55–8.57 (1H, m).
BrSn: 529.0989; found: 529.1019
1
3
(
(
34 4
HRMS (FAB) calcd for C20H N
+
(
M ).
5
.1.3. Quinoline-6-yl trifluoromethanesulfonate (10)
Trifluoromethane sulfonic anhydride (1.40 mL, 8.5 mmol) was
added dropwise to a solution of 6-quinolinol (1.01 g, 7.0 mmol)
and pyridine (1.1 mL, 14.0 mmol) in CH Cl (35 mL) at 0 °C under
atmosphere. The reaction mixture was stirred at room temper-
ature overnight. Removal of CH Cl under reduced pressure gave a
residue, which was purified by chromatography on a silica gel col-
umn using CH Cl with Et N (1%) to give 10 (1.40 g, 72%) as a yel-
low solid; mp: 33–34 °C. H NMR (300 MHz, CDCl
2
2
N
2
1
8
(
F) was produced using a CYPRIS HM-18 cyclotron (Sumitomo
2
2
Heavy Industry; Tokyo, Japan). If not otherwise stated, radioactiv-
ity was measured with an IGC-3R Curiemeter (Aloka; Tokyo,
Japan). All animal experiments were performed according to the
recommendations of the Committee for the Care and Use of Labo-
ratory Animals, National Institute of Radiological Sciences (Chiba,
Japan). Animals were maintained and handled in accordance with
the recommendations of National Institute of Health and the insti-
tutional guidelines of National Institute of Radiological Sciences.
Sprague–Dawley male rats (220–240 g, 7 weeks) were provided
by Japan SLC (Shizuoka, Japan).
2
2
3
1
3
) d: 7.50–7.54
m, 1H), 7.62 (1H, dd, J = 2.7, 9.2 Hz), 7.77 (1H, d, J = 2.6 Hz), 8.22
2H, d, J = 9.2 Hz), 9.00–9.02 (1H, m). HRMS (FAB) calcd for
(
(
+
C H
10 7
O
3
3
NF S: 278.0099; found: 278.0083 (M ).
5
.1.4. 6-[1-(2-Fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-
yl]quinoline (7a)
A mixture of 9a (481 mg, 1.0 mmol), 10 (277 mg, 1.0 mmol), tet-
rakis(triphenylphosphine)palladium (0) (58 mg, 0.05 mmol), ce-
sium fluoride (304 mg, 2.0 mmol), and copper (I) iodide (19 mg,
5
.1. Chemical synthesis
0
.1 mmol) in DMF (5 mL) was heated at 80 °C for 12 h under N
2
5
1
.1.1. 1-(2-Fluoro-3-pyridyl)-5-methyl-4-tri-n-butylstannyl-1H-
,2,3-triazole (9a)
Normal butyllithium in hexane (2.4 mL, 1.6 M; 3.8 mmol) was
atmosphere. The reaction mixture was quenched with AcOEt and
the suspension was filtered through Celite with AcOEt. After the or-
ganic layer was washed with water and dried over Na SO , the sol-
2
4
added dropwise to a solution of diisopropylamine (0.53 mL,
.8 mmol) in THF (5 mL) at À78 °C under N atmosphere. The reac-
tion mixture was stirred at À78 °C for 5 min and then at 0 °C for
vent was removed to give a residue. Column chromatography of
3
2
the residue on silica gel under hexane/AcOEt (1:3) with Et N (1%)
3
1
gave 7a (150 mg, 49%) as a white powder; mp: 176–178 °C.
H
5
min. After the mixture was cooled again to À78 °C, a solution
NMR (300 MHz, CDCl ) d: 2.56 (3H, d, J = 1.8 Hz), 7.44–7.53 (m,
3
of 2-fluoropyridine (0.33 mL, 3.8 mmol) in THF (2 mL) was added
and the mixture was stirred for 10 min. To this mixture was added
a solution of n-dodecylbenzenesulfonyl azide (0.89 g, 2.5 mmol) in
THF (3 mL), and the reaction mixture was stirred for 1 h at À78 °C.
This mixture was quenched with water and extracted with AcOEt.
2H), 8.11 (1H, dt, J = 1.6, 8.3 Hz), 8.17–8.25 (4H, m), 8.47 (1H, d,
J = 4.8 Hz), 8.96 (1H, dd, J = 1.5, 4.0 Hz). HRMS (FAB) calcd for
+
C17H13N F: 306.1155; found: 306.1189 (M ).
5
5.1.5. 6-[1-(2-Bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-
yl]quinoline (7b)
2 4
The organic layer was dried over Na SO and the solvent was re-
moved to give crude 3-azido-2-fluoropyridine (8a).
A mixture of 9b (1.11 g, 2.1 mmol), 10 (554 mg, 2.0 mmol) and
tetrakis(triphenylphosphine)palladium (0) (266 mg, 0.1 mmol), ce-
sium fluoride (608 mg, 4.0 mmol), and copper (I) iodide (38 mg,
0.2 mmol) in DMF (5 mL) was heated at 80 °C for 12 h under N2
Tributyl(1-propynyl)tin (0.91 mL, 3.0 mmol) was successively
added to a solution of the above 8a in dry toluene (20 mL) and
the reaction mixture was heated at reflux for 12 h. Removal of

M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
109
atmosphere. The reaction mixture was quenched with AcOEt and
the suspension was filtered through Celite with AcOEt. After the or-
ganic layer was washed with water and dried over Na SO , the sol-
vent was removed to give a residue. Column chromatography of
the residue on silica gel under hexane/AcOEt (1:3) with Et N (1%)
gave 7b (380 mg, 52%) as a white powder; mp: 163–165 °C.
NMR (300 MHz, CDCl ) d: 2.52 (3H, s), 7.47 (1H, m), 7.57 (1H, m),
.86 (1H, dd, J = 1.8, 7.7 Hz), 8.23–8.29 (4H, m), 8.65 (1H, dd,
J = 1.8, 4.8 Hz), 8.96 (1H, dd, J = 1.5, 4.4 Hz). HRMS (FAB) calcd for
5.4. In vitro autoradiography
2
4
Four rat brains were quickly removed and frozen on powdered
dry ice. Brain sagittal sections (20 mm) were cut on a cryostat
microtome (HM560; Carl Zeiss, Germany) and thaw-mounted on
glass slides, which were then dried and stored at À80 °C until used
3
1
H
´
for experiments. Brain sections were preincubated (3 5 min) in
3
7
Tris–HCl (50 mM, pH 7.4, containing 1.2 mM MgCl
CaCl ) at room temperature. After preincubation, these sections
were incubated for 30 min at room temperature in fresh buffer
2
and 2 mM
2
+
17 13 5
C H N Br: 366.0354; found: 366.0363 (M ).
1
8
with [ F]7a (0.023 nM, 0.3 MBq/200 mL). Unlabeled 7a or 2
(
1 mM) was used to determine the specific binding of [18F]7a for
5
.2. Radiosynthesis
mGluR1. Subgroup selectivity was investigated by MPEP (1 and
10 mM). After incubation, the sections were washed (3 Â 5 min)
with cold buffer, dipped in cold distilled water, and dried with cold
air. These sections were placed in contact with imaging plates
(BAS-MS2025; FUJIFILM, Tokyo, Japan). Autoradiograms were ob-
tained and photo-stimulated luminescence values (PSL) in the re-
gions of interest (ROIs) were measured using a Bio-Imaging
Analyzer System (BAS5000, FUJIFLIM). Radioactivity concentra-
.2.1. 6-[1-(2-[18F]Fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-
5
4
1
8
18
-yl]quinoline ([ F]FPTQ, [ F]7a)
18
All synthetic sequences of [ F]7a were carried out using a
3
0
18
À
home-made automated synthesis system.
[
F]F was produced
1
8
18
18
by the O (p, n) F reaction on 95 atom% H
2
O using 18 MeV pro-
tons (14.2 MeV on target) from the cyclotron and separated from
1
8
18
À
[
O]H
was eluted from the resin with aqueous K
into a vial containing a solution of 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8,8,8]hexacosane (Kryptofix 222, 25 mg) in CH CN
1.5 mL) and transferred into another reaction vessel in the hot cell.
2
O using Dowex 1-X8 anion exchange resin. The [ F]F
2
tions in ROIs were expressed as PSL/area (mm ) and calculated
2
CO (3.3 mg/300 L)
3
l
2
by subtracting the background (1.5 PSL/mm ).
3
5
.5. Biodistribution by dissection
saline solution of [18F]7a (average of 17 MBq/200
lL,
(
1
8
À
2 3
The [ F]F solution was dried to remove H O and CH CN at
A
1
20 °C for 15 min. After the dryness, a solution of 7b (1 mg) in
L) was heated with [18F]F at 150 °C for
0 min. The [ F]fluorinating reaction was terminated by adding
À
0.11 nmol) was injected into rats through the tail vein. Three rats
for each time point were sacrificed by cervical dislocation at 5,
anhydrous DMSO (300
1
l
1
8
1
5, and 30 min after injection. Whole brain, lung, heart, liver, kid-
CH OH/50 mM CH CO NH (4:6, 500 L) and this mixture was ap-
3
3
2
4
l
ney, spleen, pancreas, muscle, small intestine, bone and blood sam-
ples were quickly removed and weighed. From the brain
hemispheres, cerebellum, olfactory bulb, striatum, hippocampus,
thalamus, pons-medulla, and cerebral cortex were further dis-
sected and weighed. The radioactivity in these tissues was mea-
sured with a 1480 Wizard autogamma counter (Perkin–Elmer;
Tokyo, Japan) and expressed as a percentage of the injected dose
per gram of wet tissue (% ID/g). All radioactivity measurement
was corrected for decay.
plied to a semi-preparative HPLC system. HPLC purification was
completed on a Fluofix 120 N C18 column (10 mm  250 mm;
Wako Pure Chem Ind, Osaka, Japan) using a mobile phase of
CH
3 3 2 4
OH/50 mM CH CO NH (4:6) at a flow rate of 5.0 mL/min.
18
The retention time for [ F]7a was 23.5 min, whereas that for 7b
1
8
was 19.6 min. The radioactive fraction corresponding to [ F]7a
was collected in a sterile flask containing polysorbate 80 (100 L)
and 25% ascorbic acid (100 L), evaporated to dryness under vac-
uum, re-dissolved in 3 mL sterile saline, and passed through a
l
l
0
1
.22
lm Millipore filter to obtain the final product. At EOS,
18
5.6. Small-animal PET imaging
280–1830 MBq of [ F]7a was obtained as an intravenously
injectable solution at a beam current of 15 mA and 10 min proton
18
À
Anatomical template images of rat brain were generated by a
high-resolution magnetic resonance imaging (MRI) system.25
bombardment from 3300–4000 MBq of [ F]F .
1
8
Radioactive [ F]7a was analyzed by radio-HPLC coupled with a
Briefly,
a rat was anesthetized with sodium pentobarbital
UV detector to determine the radiochemical purity and specific
(
50 mg/kg, ip), and scanned with a 400 mm bore, 7 T horizontal
activity (Fluofix 120 N
C
18
,
4.6 mm  250 mm; CH
3 2
CN/H O/
magnet (NIRS/KOBELCO, Kobe; Japan/Bruker BioSpin, Ettlingen,
Germany) equipped with 120 mm diameter gradients (Bruker
BioSpin). A 72 mm diameter coil was used for radiofrequency
transmission, and signals were received by a 4-channel surface
coil. Coronal T2-weighted MRI images were obtained by a fast
spin-echo sequence with the following imaging parameters: repe-
tition time = 8000 ms, effective echo time = 15 ms, field of view
(FOV) = 35 mm´ 35 mm, and slice thickness = 0.6 mm.
Et N = 1:1:0.05%, 0.8 mL/min, 11.2 min). The specific activity of
3
8
1
[
F]7a was calculated by comparing the assayed radioactivity to
the mass associated with the carrier UV peak at 254 nm.
5
.3. Measurement of partition coefficients (Log D)
Partition coefficient values were measured by mixing [18F]7a
(
(
radiochemical purity: 100%; about 150,000 cpm) with n-octanol
3.0 g) and sodium phosphate buffer (PBS, 3.0 g; 0.1 M, pH 7.40)
PET scans were performed using a small-animal Inveon PET
scanner (Siemens Medical Solutions USA, Knoxville, TN), which
provides 159 transaxial slices 0.796 mm (center-to-center) apart,
a 10 cm transaxial FOV, and a 12.7 cm axial FOV. Before scans,
the rats were anesthetized with 5% (v/v) isoflurane, and main-
tained thereafter by 1–2% (v/v) isoflurane. Emission scans were ac-
quired for 60 min in 3-dimensional list-mode with an energy
window of 350–750 keV, immediately after intravenous injection
in a test tube. The tube was vortexed for 3 min at room tempera-
ture, followed by centrifugation at 3500 rpm for 5 min. An aliquot
of 1 mL PBS and 1 mL n-octanol was removed, weighted, and
counted, respectively. Samples from the remaining organic layer
were removed and re-partitioned until consistent Log D values
were obtained. The Log D value was calculated by comparing
the ratio of cpm/g of n-octanol to that of PBS and expressed as
Log D = Log[cpm/g (n-octanol)/cpm/g(PBS)]. All assays were per-
formed in triplicate. Meanwhile, the values of c Log D and c Log P
1
8
of [ F]7a (16–18 MBq/200 mL, 0.07–0.11 nmol). To determine
in vivo specific binding, unlabeled 7a, 2, or MPEP (1 mg/kg dis-
solved in 300 mL saline containing 10% ethanol and 5% polysorbate
1
8
18
of [ F]7a were computed using Pallas 3.4 software (CompuDrug;
80) was injected at 1 min before injection of [ F]7a. Four rats were
Sedona, AZ).
used for each experiment. All list-mode acquisition data were

1
10
M. Fujinaga et al. / Bioorg. Med. Chem. 19 (2011) 102–110
sorted into 3-dimensional sinograms, which were then Fourier
Acknowledgments
rebinned into 2-dimensional sinograms (frames  min: 4  1,
8
 2, 8  5). Dynamic images were reconstructed with filtered
We thank the crew of the Cyclotron Operation Section and
Molecular Imaging Center of National Institute of Radiological
Sciences for support in operation of the cyclotron, production of
radioisotope, and animal experiments.
back-projection using a Hanning’s filter, a Nyquist cutoff of 0.5 cy-
cle/pixel. ROIs were placed on the striatum, hippocampus, cerebral
cortex, thalamus, medulla, and cerebellum using ASIPro VM™
(
Analysis Tools and System Setup/Diagnostics Tool, Siemens Med-
ical Solutions USA) with reference to the MRI template. Brain up-
take of radioactivity was decay-corrected to the injection time
and was expressed as the standardized uptake value (SUV), which
was normalized to the injected radioactivity and body weight.
SUV = (radioactivity per milliliter tissue/injected radioactiv-
ity) Â gram body weight.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.048.
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.7. Protein binding
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.
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Swanson, C. J.; Bures, M.; Johnson, M. P.; Linden, A. M.; Monn, J. A.; Schoepp, D.
D. Nat. Rev. Drug Discovery 2005, 4, 131.
method using a Centrifree YM-30 filter unit (Millipore; Carrigtwo-
hill, Ireland) according to a previously described method.31 A blood
sample (1.0 mL) was obtained from rats (n = 3) and was main-
7. Vanejevs, M.; Jatzke, C.; Renner, S.; Müller, S.; Hechenberger, M.; Bauer, T.;
Klochkova, A.; Pyatkin, I.; Kazyulkin, D.; Aksenova, E.; Shulepin, S.; Timonina,
O.; Haasis, A.; Gutcaits, A.; Parsons, C. G.; Kauss, V.; Weil, T. J. Med. Chem. 2008,
18
tained at 37 °C in a centrifuge tube. Then, [ F]7a (5 mL, approxi-
mately 10 Bq) was added and mixed in the blood sample. An
aliquot (20 mL) of blood sample was taken to measure radioactiv-
ity in the sample using the autogamma counter. The remainder of
blood sample was centrifuged at 3900 rpm for 5 min at 4 °C to
obtain plasma. The plasma sample (40 mL) was loaded into the
filter unit and centrifuged at 2000 rpm for 15 min at 25 °C. The
remainder of plasma sample (20 mL) was used for measurement.
An aliquot (100–150 mL) was taken from the ultrafiltrate and
radioactivity was measured. The amount of nonspecific binding
5
1, 634.
8. Schkeryantz, J. M.; Kingston, A. E.; Johnson, M. P. J. Med. Chem. 2007, 31, 2563.
9.
Litschig, S.; Gasparini, F.; Rueegg, D.; Stoehr, N.; Flor, P. J.; Vranesic, I.; Prézeau,
L.; Pin, J. P.; Thomsen, C.; Kuhn, R. Mol. Pharmacol. 1999, 55, 453.
1
0. Carroll, F. Y.; Stolle, A.; Beart, P. M.; Voerste, A.; Brabet, I.; Mauler, F.; Joly, C.;
Antonicek, H.; Bockaert, J.; Müller, T.; Pin, J. P.; Prézeau, L. Mol. Pharmacol. 2001,
59, 965.
11. Lavreysen, H.; Janssen, C.; Bischoff, F.; Langlois, X.; Leysen, J. E.; Lesage, A. S.
Mol. Pharmacol. 2003, 63, 1082.
12. Kohara, A.; Toya, T.; Tamura, S.; Watabiki, T.; Nagakura, Y.; Shitaka, Y.;
Hayashibe, S.; Kawabata, S.; Okada, M. J. Pharmacol. Exp. Ther. 2005, 315, 163.
of [ F]7a to the filter was also determined by adding [18F]7a to
1
8
13. Wu, W. L.; Burnett, D. A.; Domalski, M.; Greenlee, W. J.; Li, C.; Bertorelli, R.;
Fredduzzi, S.; Lozza, G.; Veltri, A.; Reggiani, A. J. Med. Chem. 2007, 50, 5550.
saline and following the same procedure as for the plasma sample.
The free fraction in plasma was calculated as the ratio of radioac-
tivity in ultrafiltrate to radioactivity in plasma corrected for a stick
factor. The stick factor is radioactivity in a known volume of ultra-
filtered saline divided by radioactivity in the same volume of
saline.
1
4. Satoh, A.; Nagatomi, Y.; Hirata, Y.; Ito, S.; Suzuki, G.; Kimura, T.; Maehara, S.;
Hikichi, H.; Satow, A.; Hata, M.; Ohta, H.; Kawamoto, H. Bioorg. Med. Chem. Lett.
2009, 19, 5464.
1
5. Huang, Y.; Narendran, R.; Bischoff, F.; Guo, N.; Zhu, Z.; Bae, S. A.; Lesage, A. S.;
Laruelle, M. J. Med. Chem. 2005, 48, 5096.
16. Lavreysen, H.;Wouters, R.;Bischoff, F.;Nóbrega Pereira, S.; Langlois, X.;Blokland,
S.; Somers, M.; Dillen, L.; Lesage, A. S. Neuropharmacology 2004, 47, 961.
1
1
1
7. Fotuhi, M.; Sharp, A. H.; Glatt, C. E.; Hwang, P. M.; von Krosigk, M.; Snyder, S.
H.; Dawson, T. M. J. Neurosci. 1993, 13, 2001.
8. Lavreysen, H.; Pereira, S. N.; Leysen, J. E.; Langlois, X.; Lesage, A. S.
Neuropharmacology 2004, 46, 609.
9. Eng, W.; Kawamoto, H.; O’Malley, S.; Krause, S.; Patel, S.; Ryan, C.; Riffel, K.; Ito,
S.; Suzuki, G.; Cook, J.; Ozaki, S.; Ohta, H.; Burns, D.; Hargreaves, R.; Hostetler, E.
J. Labelled Compd. Radiopharm. 2009, 52, S84.
5
.8. Metabolite assay for mouse plasma and brain tissue
After intravenous injection of [18F]7a (3.7 MBq/100
L) into ddy
l
mice (n = 3), these mice were sacrificed by cervical dislocation at 5,
5, and 30 min. Blood (500 mL) and whole brain samples were re-
moved quickly. The blood sample was centrifuged at 15,000 rpm
for 2 min at 4 °C to separate plasma, which (250 L) was collected
in a test tube containing CH CN (500 L) and a solution of unla-
beled 7a (0.8 mg/5.0 mL of CH CN, 10 L). After the tube was vor-
2
0. Suzuki, G.; Kimura, T.; Satow, A.; Kaneko, N.; Fukuda, J.; Hikichi, H.; Sakai, N.;
Maehara, S.; Kawagoe-Takaki, H.; Hata, M.; Azuma, T.; Ito, S.; Kawamoto, H.;
Ohta, H. J. Pharmacol. Exp. Ther. 2007, 321, 1144.
1
l
21. Ohgami, M.; Haradahira, T.; Takai, N.; Zhang, M.-R.; Kawamura, K.; Yamasaki,
K.; Yanamoto, K. Eur. J. Nucl. Med. Mol. Imag. 2009, 36, S310.
3
l
2
2. Hostetler, E.; Eng, W.; Joshi, A.; Sanabria-Bohórquez, S.; Kawamoto, H.; Ito, S.;
O’Malley, S.; Krause, S.; Ryan, C.; Patel, S.; Williams, M.; Riffel, K.; Suzuki, G.;
Ozaki, S.; Ohta, H.; Cook, J.; Burns, H. D.; Hargreaves, R. Synapse 2010.
doi:10.1002/syn.20826.
3
l
texed for 15 s and centrifuged at 15,000 rpm for 2 min for
deproteinization, the supernatant was collected. The extraction
2
3. Prabhakaran, J.; Majo, V. J.; Milak, M. S.; Kassir, S. A.; Palner, M.; Savenkova, L.;
Mali, P.; Arango, V.; Mann, J. J.; Parsey, R. V.; Kumar, J. S. Bioorg. Med. Chem. Lett.
2010, 20, 3499.
3
efficiency of radioactivity into the CH CN supernatant ranged from
7
0% to 92% of the total radioactivity in the plasma. On the other
2
2
4. Kohara, A.; Takahashi, M.; Yatsugi, S.; Tamura, S.; Shitaka, Y.; Hayashibe, S.;
Kawabata, S.; Okada, M. Brain Res. 2008, 1191, 168.
5. Yanamoto, K.; Konno, F.; Odawara, C.; Yamasaki, T.; Kawamura, K.; Hatori, A.;
Yui, J.; Wakizaka, K.; Nengaki, N.; Takei, M.; Zhang, M.-R. Nucl. Med. Biol. 2010,
hand, the cerebellum and brain except the cerebellum were dis-
sected from the mouse brain and homogenized together in an
ice-cooled CH CN/H O (1:1, 1.0 mL) solution, respectively. The
3 2
homogenate was centrifuged at 15,000 rpm for 2 min at 4 °C and
supernatant was collected. The recovery of radioactivity into the
supernatant was 68–87% based on the total radioactivity in the
brain homogenate.
3
7, 615.
26. Kawamoto, H.; Ito, S.; Satoh, A.; Nagatomi, Y.; Hirada, Y.; Kimura, T.; Suzuki, G.;
Sato, A.; Ohta, H. WO 2005/085214 A1, 2005.
27. Suzuki, G.; Kawagoe-Takaki, H.; Inoue, T.; Kimura, T.; Hikichi, H.; Murai, T.;
Satow, A.; Hata, M.; Maehara, S.; Ito, S.; Kawamoto, H.; Ozaki, S.; Ohta, H. J.
Pharmacol. Sci. 2009, 110, 315.
An aliquot of the supernatant (100–500 lL) obtained from the
plasma or brain homogenate was injected into the radio-HPLC sys-
28. Pike, V. W. Trends Pharmacol. Sci. 2009, 30, 431.
29. Zhang, M.-R.; Maeda, J.; Ogawa, M.; Noguchi, J.; Ito, T.; Yoshida, Y.; Okauchi, T.;
tem, and analyzed under the same analytical conditions described
Obayashi, S.; Suhara, T.; Suzuki, K. J. Med. Chem. 2004, 47, 2228.
0. Fukumura, T.; Suzuki, H.; Mukai, K.; Zhang, M.-R.; Yoshida, Y.; Nemoto, K.;
Suzuki, K. J. Labelled Compd. Radiopharm. 2007, 50, S202.
1. Kawamura, K.; Naganawa, M.; Konno, F.; Yui, J.; Wakizaka, H.; Yamasaki, T.;
Yanamoto, K.; Hatori, A.; Takei, M.; Yoshida, Y.; Sakaguchi, K.; Fukumura, T.;
Kimura, Y.; Zhang, M.-R. Nucl. Med. Biol. 2010, 37, 625.
3
3 2
above except that the mobile phase was CH CN/H O (4:6) and flow
18
rate was 1.5 mL/min. The percentage rate of [ F]7a to total radio-
3
activity (corrected for decay) on the HPLC chromatogram was cal-
culated as% = (peak area for [18F]7a/total peak area) Â 100.