Synthesis and evaluation of novel radioligands for positron emission tomography imaging of metabotropic glutamate receptor subtype 1 (mGluR1) in rodent brain

Article abstract of DOI:10.1021/jm201590g

We designed three novel positron emission tomography ligands, N-(4-(6-(isopropylamino)pyrimidin-4-yl)-1,3-thiazol-2-yl)-4-[ ¹¹C]methoxy-N-methylbenzamide ([¹¹C]6), 4-[ ¹⁸F]fluoroethoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1, 3-thiazol-2-yl]-N-methylbenzamide ([¹⁸F]7), and 4-[ ¹⁸F]fluoropropoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1, 3-thiazol-2-yl]-N-methylbenzamide ([¹⁸F]8), for imaging metabotropic glutamate receptor type 1 (mGluR1) in rodent brain. Unlabeled compound 6 was synthesized by benzoylation of 4-pyrimidinyl-2-methylaminothiazole 10, followed by reaction with isopropylamine. Removal of the methyl group in 6 gave phenol precursor 12 for radiosynthesis. Two fluoroalkoxy analogues 7 and 8 were prepared by reacting 12 with tosylates 13 and 14. Radioligands [ ¹¹C]6, [¹⁸F]7, and [¹⁸F]8 were synthesized by O-[¹¹C]methylation or [¹⁸F]fluoroalkylation of 12. Compound 6 showed high in vitro binding affinity for mGluR1, whereas 7 and 8 had weak affinity. Autoradiography using rat brain sections showed that [ ¹¹C]6 binding is aligned with the reported distribution of mGluR1 with high specific binding in the cerebellum and thalamus. PET study with [ ¹¹C]6 in rats showed high brain uptake and a similar distribution pattern to that in autoradiography, indicating the usefulness of [ ¹¹C]6 for imaging brain mGluR1.

Full text of DOI:10.1021/jm201590g

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Novel Radioligands for Positron
Emission Tomography Imaging of Metabotropic Glutamate Receptor
Subtype 1 (mGluR1) in Rodent Brain
Masayuki Fujinaga,†,∥ Tomoteru Yamasaki,†,‡,∥ Joji Yui,† Akiko Hatori,† Lin Xie,† Kazunori Kawamura,†
Chiharu Asagawa,† Katsushi Kumata,† Yuichiro Yoshida,†,§ Masanao Ogawa,†,§ Nobuki Nengaki,†,§
Toshimitsu Fukumura,† and Ming-Rong Zhang*,†
†Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
‡Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8574, Japan
§SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
S
* Supporting Information
ABSTRACT: We designed three novel positron emission tomography ligands, N-(4-(6-(isopropylamino)pyrimidin-4-yl)-1,3-
thiazol-2-yl)-4-[11C]methoxy-N-methylbenzamide ([11C]6), 4-[18F]fluoroethoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-
thiazol-2-yl]-N-methylbenzamide ([18F]7), and 4-[18F]fluoropropoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-
N-methylbenzamide ([18F]8), for imaging metabotropic glutamate receptor type 1 (mGluR1) in rodent brain. Unlabeled
compound 6 was synthesized by benzoylation of 4-pyrimidinyl-2-methylaminothiazole 10, followed by reaction with
isopropylamine. Removal of the methyl group in 6 gave phenol precursor 12 for radiosynthesis. Two fluoroalkoxy analogues 7
and 8 were prepared by reacting 12 with tosylates 13 and 14. Radioligands [11C]6, [18F]7, and [18F]8 were synthesized by
O-[11C]methylation or [18F]fluoroalkylation of 12. Compound 6 showed high in vitro binding affinity for mGluR1, whereas 7 and 8
had weak affinity. Autoradiography using rat brain sections showed that [11C]6 binding is aligned with the reported distribution of
mGluR1 with high specific binding in the cerebellum and thalamus. PET study with [11C]6 in rats showed high brain uptake and a
similar distribution pattern to that in autoradiography, indicating the usefulness of [11C]6 for imaging brain mGluR1.
Although many PET probes have been developed for various
targets, such as enzymes, transporters, plaques, ion channels,
and receptors, some important molecular targets, including
mGluR1, still lack useful radioligands for clinical study. PET
with a specific mGluR1 ligand could be an important tool for
understanding the role of this receptor in health and disorders
in which mGluR1 is involved and also for the development of
new drugs targeting this receptor.
The distribution of mGluR1 in the brain has been studied
using in vitro autoradiography and histologic analysis.11,12
Studies showed a wide distribution of mGluR1 in brains with,
for example, a high level in the cerebellum, moderate or low
level in the thalamus, striatum, and cerebral cortex, and a very
low level in the brain stem. Intense efforts have been made over
recent years to develop PET ligands for imaging brain mGluR1.
Examples include [11C]JNJ-16567083 ([11C]1),13 [11C]YM-202074
INTRODUCTION
■
Glutamate is an excitatory neurotransmitter in the central
nervous system (CNS) and is involved in many pathological
conditions of CNS. Glutamate receptors were divided into
metabotropic (mGluRs) and iontropic types based on their
biological functions and molecular structures. MGluRs were
classified into three groups including eight subtypes according
to sequence homology, coupling mechanisms to G-protein,
and pharmacological activity.1,2 Group I mGluRs (mGluR1 and
mGluR5) play important physiological roles in regulating ion
channels and synaptic transmission, and in synaptic plasticity,
which underlies memory and learning.3−5 It has been reported
that mGluR1 may be a drug target for the treatment of diseases
such as stroke, epilepsy, pain, cerebellar ataxia, Parkinson’s
disease, anxiety, and mood disorders.6−10
Positron emission tomography (PET) with radioligands is a
molecular imaging technique that enables study of the living
human brain and in particular of specific proteins involved in
pathophysiology or as targets for therapeutic interventions.
Received: November 25, 2011
Published: February 8, 2012
© 2012 American Chemical Society
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Scheme 1. Chemical Structures of PET Ligands for mGluR1
([11C]2),14 [11C]MMTP ([11C]3),15 [18F]MK-1312 ([18F]4a),16
[18F]FTIDC ([18F]4b),17 [18F]FPTQ ([18F]4c),18 and [18F]FPIT
([18F]4d)19 (Scheme 1). These radioligands had high in vitro
binding affinity for mGluR1, but their in vivo binding for
mGluR1 visualized in the brain with PET was much lower than
the corresponding in vitro binding. Moreover, in vivo binding
for mGluR1 was only seen in the cerebellum.13,15,18 No imaging
study of mGluR1 in the human brain has been performed for
clinical usefulness.
Recently, when searching for a pharmacophore different
from the chemical structures reported, we developed 4-
[18F]fluoro-N-[4-(6-(isopropylamino)pyrimidin-4-yl)-1,3-thiazol-
2-yl]-N-methylbenzamide ([18F]5, Scheme 2) as a novel
radioactivity increased in all brain regions during a PET scan,
which may create complication in quantitative analysis for
measuring mGluR1 density with PET. On the other hand, [18F]
5 was prepared by heating a nitro precursor with [18F]F− at
180 °C, at which the nitro precursor was easily decomposed.20
Separation of [18F]5 with from the nitro precursor and by-
product with high-performance liquid chromatography
(HPLC) required more than 20 min.
In this study, using [18F]5 as a lead compound, we designed
three novel radioligands [11C]6, [18F]7, and [18F]8 (Scheme 2)
and evaluated their potentials as PET ligands for mGluR1. As
shown in their chemical structures, a methoxy group was
introduced into [11C]6 instead of a fluorine atom in [18F]5. It is
easy to label 6 using [11C]methyl iodide with two levels of
specific activity (37−185 GBq/μmol22 and 3700−7400 GBq/
μmol23) available in our institute. Further, using the same phe-
nol precursor, we synthesized two [18F]fluoroalkoxy analogues
[18F]7 and [18F]8 and characterized their specific binding for
mGluR1 in the brain.
Scheme 2. Chemical Structure of 5−8 and Their Labeled
Compounds
Here, we report (1) chemical synthesis, (2) radiolabeling, (3)
in vitro binding affinity and autoradiography, and (4) small-
animal PET studies of a small set of PET mGluR1 radioligands.
RESULTS AND DISCUSSION
■
Chemistry. The novel unlabeled compounds 6−8 were
synthesized according to reaction sequences delineated in
Scheme 3. Ethoxyvinylpyrimidine 9 was synthesized by coupl-
ing 4,6-dichloropyrimidine with tributyl(1-ethoxyvinyl)-
stannane in the presence of tetrakis(triphenylphosphine)-
palladium (0) (Pd(PPh3)4). Reaction of 9 with N-bromosucci-
nimide, followed by treatment with N-methylthiourea, gave
4-(6-chloropyrimidin-4-yl)-N-methylthiazol-2-amine (10).24
Benzoylation of 10 with 4-methoxybenzoyl chloride afforded
11, which was heated with isopropylamine to give 6 in 19%
total yield from 4,6-dichloropyrimidine. Removal of the methyl
group in 6 with boron tribromide (BBr3) gave the desmethyl
phenol precursor 12 used for the present radiosyntheses. To
prevent contamination by a trace amount of 6, which would
decrease the specific activity of [11C]6, precursor 12 prepared
by the desmethylation was further purified by semipreparative
HPLC (Supporting Information Figure 1).
PET ligand for imaging brain mGluR1.20,21 PET with [18F]5
showed significant signals in rat and monkey brain regions, not
only an mGluR1-rich cerebellum but also mGluR1-moderate or
low regions, such as the thalamus, striatum, and cerebral
cortex.21 The uptake ratio of radioactivity, an index reflecting
the in vivo specific binding of [18F]5 to mGluR1, between the
cerebellum and mGluR1-negligible pons in the rat brain
reached approximately 10, which was the highest among all
reported PET ligands for this receptor. Currently, [18F]5 is
being prepared for clinical imaging study of brain mGluR1 in
our facility.
The objective of this study was to develop new 11C or 18F-
labeled PET ligands with high in vitro binding affinity, in vitro
and in vivo specific binding for mGluR1, and with improved
kinetics over [18F]5 to quantitatively determine mGluR1 in the
brain. A disadvantage of [18F]5 is slow kinetics in the rat and
monkey brains.21 After injection of [18F]5 into rats, uptake of
Two fluoroalkoxy analogues 7 and 8 were synthesized by
heating precursor 12 with tosylates 13 and 14, which were
obtained by the reaction of fluoroalcohol with 4-methylbenze-
nesulfonyl chloride25 in the presence of K2CO3 at 70 °C for 6 h
in 52% and 60% yields, respectively.
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a
Scheme 3. Syntheses of 6−8 and Precursor 12
a
Reagents and conditions: (a) ethyl 1-(tributylstannyl)vinyl ether, Pd(PPh3)4, DMF, 80 °C, 5 h; (b) THF, H2O, 1-bromopyrrolidine-2,5-dione,
room temperature, 2 h; (c) N-methylthiourea, room temperature, 2 h; (d) 4-methoxybenzoyl chloride, Et3N, toluene, 100 °C, 10 h; (e) isopropylamine,
K2CO3, 1,4-dioxane, 80 °C, 16 h; (f) BBr3, CH2Cl2, −40 °C, then room temperature, overnight; (g) p-toluenesulfonyl chloride, pyridine, CH2Cl2, room
temperature, 24 h; (h) K2CO3, DMF, 70 °C, 6 h.
a
Scheme 4. Radiosyntheses of [11C]6, [18F]7, and [18F]8
a
Reagents and conditions: (a) LiAlH4, THF, −15 °C, 2 min; (b) hydroiodic acid, 180 °C, 2 min; (c) DMF, NaOH, 70 °C, 5 min; (d) I2, 630 °C; (e)
trifluoromethanesulfonic anhydride, 2,6-lutidine, CH2Cl2, 0 °C, 1 h; (f) [18F]KF, Kryptofix 222, 1,2-dichlorobenzene, 130 °C, 2 min; (g) NaOH,
DMF, 90 °C, 10 min; (h) [18F]KF, Kryptofix 222, 1,2-dichlorobenzene, 180 °C, 2 min; (i) NaOH, DMF, 120 °C, 10 min.
Radiosynthesis of [11C]6, [18F]7, and [18F]8 was performed
using a homemade automated synthesis system. This system is
equipped with two units for [11C]methylation and [18F]-
fluoroalkylation routinely used in our facility.26
activity (37−185 GBq/μmol), [11C]MeI for radiolabeling was
prepared by reducing cyclotron-produced [11C]carbon dioxide
([11C]CO2) with lithium aluminum hydride (LiAlH4), followed
by iodination with 57% hydroiodic acid.22 After distillation and
drying, [11C]MeI was transferred into a DMF solution of 12
and NaOH. The [11C]methylation proceeded efficiently for
5 min at 70 °C. Semipreparactive HPLC purification for the
The O-[11C]methyl ligand [11C]6 was prepared by reacting
precursor 12 with [11C]methyl iodide ([11C]MeI) of two levels
of specific activity (Scheme 4). As for the conventional specific
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Table 1. Lipophilicity and In Vitro Binding Affinity
c
in vitro binding affinity (nM)
lipophilicity
mGluRl
mGluR5
a
b
compd
R
cLogD
LogD
IC50
Ki
IC50
6
7
8
5
OCH3
2.64
2.79
3.15
2.35
2.57
2.59
2.80
1.45
32.7 1.2
395.1 1.2
>10000
12.6 1.2
152.7 1.2
>5000
>10000
>10000
>10000
>10000
OCH2CH2F
OCH2CH2CH2F
F
d
13.9 1.2
5.4 1.2
a
b
cLogD values were calculated with Pallas 3.4 software. LogD values were determined in the octanol/phosphate buffer (pH = 7.40) by the shaking
c
flash method (n = 3; maximum range, 5%). Values of in vitro binding affinity are the mean SE measured in duplicate brain homogenates.
d
Value of functional IC50 using CHO cells expressing human mGluRl was 5.1 2.0 nM as reported in ref 24.
reaction mixtures gave [11C]6 with 22
5% (n = 21)
Starting from 4.1−6.3 GBq of [18F]F−, 0.7−1.0 GBq of [18F]7,
and 55 MBq of [18F]8 was obtained with specific activity of
330−410 GBq/μmol at EOS. The [18F]fluoropropylation
efficiency for 12 was much lower than the [18F]fluoroethylation
efficiency, which was due to decrease in the inductive effect of
fluorine atom with the prolonging of methylene chains in the
two labeling agents.
radiochemical yields (decay-corrected) based on [11C]CO2
(Supporting Information Figure 2). Starting from 15−20
GBq of [11C]CO2, 0.9−2.1 GBq of [11C]6 was produced
with 25 3 min (n = 21) of synthesis time from the end of
bombardment (EOB). The specific activity of [11C]6 was 95−
140 GBq/μmol at the end of synthesis (EOS).
As for the high specific activity, [11C]6 was prepared by
reacting 12 with [11C]MeI of 3700−7400 GBq/μmol. In our
facility, several [11C]methylated PET ligands high specific
activity have been synthesized and used to visualize and assess
receptors present at extremely low density or new binding sites
in the brain.23,27,28 For radiolabeling, [11C]MeI was produced
by iodination of [11C]CH4 formed in the target chamber in
situ (the single pass I2 method). After the [11C]methylation
The identity of [11C]6, [18F]7, and [18F]8 was confirmed by
coinjection with the corresponding unlabeled 6−8 on reverse
phased−analytical HPLC. In the final product solutions, their
radiochemical purity was higher than 99% (Supporting
Information Figure 3). No significant peak corresponding to
the unreacted 12 was observed on any HPLC charts for the
final products. Moreover, these radioligands did not show
radiolysis at room temperature for 180 min after formulation,
indicating their radiochemical stability with the time of at least
one PET scan. The analytical results were in compliance with
our in-house quality control/assurance specifications of
radiopharmaceuticals.
Computation and Measurement of Ligand Lipophi-
licities. The computed values of lipophilicities at pH = 7.4
(cLogD) for [11C]6, [18F]7, and [18F]8 were 2.64−3.15 (Table 1).
The values of lipophilicities (LogD) were 2.57−2.80 measured by
the Shake Flask method.17 The lipophilicity values of these
radioligands are higher than that of [18F]5 (LogD, 1.45; cLogD,
2.35) and lie in the range normally considered favorable for a PET
ligand.32
In Vitro Binding Assays. In vitro binding affinities (Ki) of
6−8 for mGluR1 were measured from competition against the
binding of mGluR1-selective [18F]5 using rat brain homoge-
nates. As shown in Table 1, 6 showed high binding affinity
(Ki = 12.6 nM) for mGluR1, although the affinity was weaker
than that of 5 (Ki = 5.4 nM). Higher electron density in the
anisole ring in 6 than that in the fluorobenzene ring in 5 may be
the main cause of the decrease in the binding affinity for
mGluR1. Among these compounds, fluoropropyl 8 displayed
the lowest affinity (Ki > 5 μM). This result suggested that the
bulk-increasing group attached to the 4-position of the benzene
ring may not fit the binding site on the mGluR1 domain,
resulting in a significant decrease in the binding affinity of 8.
On the other hand, the binding affinity of 6−8 for mGluR5 was
measured from competition against the binding of mGluR5-
selective [11C]ABP68833 ([11C]17; Supporting Information
reaction, [11C]6 was obtained with 6
1% (n = 3)
radiochemical yield (decay-corrected) based on [11C]CH4
(Supporting Information Figure 2). Starting from about 37
GBq of [11C]CH4, 0.9−1.1 GBq of [11C]6 was produced with
29 1 min (n = 3) of synthesis time from EOB. The specific
activity of [11C]6 was 4370−7840 GBq/μmol at EOS.
Two [18F]fluoroalkoxy ligands [18F]7 and [18F]8 were syn-
thesized by reacting 12 with 1-bromo-2-[18F]fluoroethane
([18F]FEtBr)29 and 1-bromo-3-[18F]fluoropropane ([18F]-
FPrBr), respectively (Scheme 4). The labeling agent [18F]
FEtBr or [18F]FPrBr was prepared by the [18F]fluorination of
2-bromoethyl trifluoromethanesulfonate (15) or 3-bromoprop-
yl trifluoromethanesulfonate (16) with [18F]F− using a home-
made synthetic unit.30,31 After a solution of 15 or 16 in 1,2-
dichlorobenzene was added to dried [18F]F−, the reaction
mixture was immediately heated to produce [18F]FEtBr or
[18F]FPrBr, which was distilled, dried, and trapped in a solution
of 12 and NaOH in DMF. The purification of [18F]FEtBr
(boiling point: 71.5 °C) or [18F]FPrBr (boiling point: 101 °C)
by distillation was effective,29 because this procedure can
separate both reagents from their reaction mixtures to achieve
radiochemical purity of >95% of [18F]FEtBr or [18F]FPrBr.
After trapping each reagent, the [18F]fluoroalkylation mixture
was heated at 90 or 120 °C for 10 min. HPLC purification of
the reaction mixtures gave [18F]7 and [18F]8 in 24 2% (n =
3) and 2% radiochemical yields based on [18F]F− (Supporting
Information Figure 2), corrected for physical decay in the
synthesis times of 59 5 and 58 min from EOB, respectively.
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Figure 1. Representative in vitro autoradiographic images of rat brains with [11C]6 (9.2 MBq, 0.5 nmol, A,B), [18F]7 (0.4 MBq, 5 pmol, C,D), and
[18F]8 (0.4 MBq, 4 pmol, E,F). (A) [11C]6 only; (B) [11C]6 with 18 (1 μM); (C) [18F]7 only; (D) [18F]7 with 18 (1 μM); (E) [18F]8 only; (F)
[18F]8 with 18 (1 μM).
Table 2. Biodistribution (% ID/g Tissue: mean SE, n = 4) in Mice after Injection of [11C]6
tissue
blood
1 min
5 min
15 min
30 min
60 min
3.79 0.33
4.63 1.19
5.17 0.53
8.16 2.68
1.97 0.44
8.67 1.44
3.65 0.44
2.04 0.07
0.72 0.15
3.10 0.34
1.95 0.03
2.09 0.12
2.90 0.16
8.34 0.61
1.96 0.17
5.64 0.33
4.55 0.32
1.22 0.32
0.83 0.05
3.21 0.13
0.96 0.10
1.19 0.17
1.91 0.14
5.57 0.54
1.98 0.23
3.78 0.29
4.98 0.66
0.95 0.15
0.54 0.06
3.14 0.20
0.67 0.03
0.91 0.05
1.92 0.13
4.68 0.22
2.14 0.59
3.32 0.21
4.07 0.44
0.64 0.08
0.38 0.06
2.38 0.17
0.64 0.03
0.81 0.05
1.57 0.18
3.97 0.26
2.36 0.56
2.92 0.28
3.97 0.33
0.52 0.11
0.33 0.10
1.85 025
heart
lung
liver
spleen
kidney
small intestine
muscle
testis
brain
Scheme 1) using the same homogenates of rat brain. As shown
in Table 1, 6−8 did not show significant inhibitory effects (IC50 >
10 μM) on [11C]17 binding in the homogenate, indicating their
weak binding affinities for mGluR5.
appeared in the blood, heart, lung, liver, kidney, and small
intestine. After the initial phage, the radioactivity levels in most
tissues decreased, while that in the liver continually increased
until 5 min and then decreased slowly. High uptake in the liver,
kidney, and small intestine suggested that hepatobiliary and
urinary excretion, as well as the intestinal reuptake pathway,
might dominate the whole-body distribution of radioactivity
and rapid washout from the body after injection of this
radioligand.
On the other hand, high initial uptake was found in the
mouse brain, the target tissue in this study, indicating that [11C]
6 passes across the blood−brain barrier and enters the brain,
which is a prerequisite for a favorable PET ligand used in brain
imaging. The considerable brain uptake was consistent with the
suitable lipophilicity (LogD = 2.57) of [11C]6. At 30 min after
the injection, brain uptake remained at >2% ID/g, suggesting
that [11C]6 has some specific binding in the brain.
In Vitro Autoradiography. Figure 1 shows representative
in vitro autoradiograms of [11C]6, [18F]7, and [18F]8 on sagittal
sections of rat brains. As for [11C]6, the distribution pattern of
radioactivity was heterogeneous, with the highest level in the
cerebellum (Figure 1A). Moderate radioactivity was seen in the
thalamus and a low level was determined in the striatum. The
lowest radioactivity was detected in the brain stem. This
autoradiographic result was consistent with the distribution
pattern of mGluR1 in the rat brain.11 Co-incubation with
mGluR1-selective JNJ-1625968534 (18, Figure 1B; Supporting
Information Scheme 1) reduced radioactivity in the sections to
30% of radioactivity in the control sections. The specific bind-
ing of [11C]6 for mGluR1 accounted for >70% of total binding
in the brain, demonstrating high in vitro specificity of this
radioligand for mGluR1.
Small-Animal PET Studies on Rats. The in vivo uptake,
kinetics, and specific binding in the rat brains were examined
using small-animal PET with [11C]6 with conventional specific
activity.
Autoradiograms of [18F]7 (Figure 1C) and [18F]8 (Figure
1E) showed much lower radioactivity in the brain sections than
that of [11C]6 (Figure 1A). The distribution of radioactivity
was homogeneous among all brain regions. Further, coincuba-
tion with 18 (1D,F) did not affect radioactivity in the sections
significantly. These results indicated that the two [18F]fluo-
roalkoxy ligands had a weak signal of mGluR1 in the brain,
which was consistent with their weak in vitro binding affinity
with mGluR1 (Table 1).
On the basis of these in vitro results, we ceased to further
evaluate [18F]7 and [18F]8, and selected [11C]6 for in vivo
evaluation.
Biodistribution Study. The distribution of radioactivity in
mice was measured at designated experimental time points after
injection of [11C]6 (Table 2). At 1 min after injection, high
uptake (>3% injected dose per gram of wet tissue, % ID/g)
Figures 2A−L show representative PET images of rat brains
after injection of [11C]6. To confirm the corresponding brain
regions, anatomical template MRI images of rat brains were
used as shown in Figures 2M−P. Baseline PET showed high
brain penetration and accumulation of radioactivity in the brain
regions (Figure 2A−D). The highest uptake was seen in the
cerebellum (Figure 2A,B), followed by the thalamus (Figure
2A,C) and striatum (Figure 2A,D). The lowest radioactivity
was determined in the pons (Figure 2B). This distribution
pattern of uptake reflected the distribution of mGluR1 in the
brain, which was similar to the in vitro autoradiograms for [11C]
6. As shown in the time−activity curves (TACs) of brain
regions (Figure 3A), radioactivity in the cerebellum gradually
increased after injection, peaked at 45 min, and decreased to
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Figure 2. Representative PET and MRI images of isoflurane-anesthetized rat brains. PET images (A−L) were generated by summing the whole scan
(0−90 min). Brain regions were confirmed by MRI images of anatomical templates (M−P). (A,E,I,M) sagittal images; left numbers, coronal images.
(A−D) [11C]6 (16 MBq, 0.15 nmol) only; (E−H) [11C]6 (17 MBq, 0.18 nmol) after treatment with 6 (1 mg/kg); (I−L) [11C]6 (17 MBq, 0.18
nmol) after treatment with 18 (1 mg/kg).
Figure 3. Time−activity curves of PET with [11C]6 in the cerebellum (circles), thalamus (buried triangles), striatum (blank triangles), and pons
(buried squares) of rat brains. (A) [11C]6 only; (B) [11C]6 after treatment with 6 (1 mg/kg); (C) [11C]6 after treatment with 18 (1 mg/kg). Data
are the means SE.
90% of the maximum at 90 min. Radioactivity in the thalamus
and striatum peaked at 45 min and declined slowly to about
80% of the maximum in the corresponding regions until the
end of the PET scan. Using the mGluR1-negligible pons as a
reference region, the uptake ratio between each region and
pons was calculated to reflect in vivo specific binding in the
brain. The maximum ratio (n = 4) of each region to the pons
distribution of radioactivity became fairly uniform throughout
the brain. Radioactivity in the cerebellum was reduced to about
15% of the control at 45 min after the injection. The maximum
reduction in uptake exceeded 85% in the thalamus and
striatum. Pretreatment with mGluR1-selctive 18 also showed
significant inhibition of the uptake in regions to a level close to
that in the pons and almost abolished the difference in
radioactivity among all brain regions (Figures 2I−L and 3C).
Such differences in TACs between control (Figure 3A) and 6
or 18-treated (parts B or C of Figure 3) groups imply the
presence of a high proportion of specific binding to mGluR1 in
the brain, demonstrating that PET with [11C]6 could provide
significant signals of mGluR1 in brain regions, such as the
cerebellum, thalamus, and striatum.
was 6.98
1.04 for the cerebellum, 5.05
0.62 for the
thalamus, and 4.32 0.21 for the striatum after injection of
[11C]6.
As shown on the PET images (Figures 2E−H) and TACs
(Figure 3B), pretreatment with unlabeled 6 (1 mg/kg)
markedly reduced the uptake compared to the control. Uptake
was significantly inhibited in all brain regions, and the
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Comparison between Wild-Type and MGluR1 Knock-
out Mice. PET scan with [11C]6 was further performed in
mGluR1 knockout mice. Figure 4 shows PET images of wild
regions, such as the striatum, hippocampus, and cerebral
cortex. This study demonstrated that the present pharmaco-
phore is suitable for the development of prospective mGluR1
PET ligands, of which [11C]6 is promising for evaluation of
primate subjects and has further usefulness for the human brain.
EXPERIMENTAL SECTION
1. Materials and Methods. Melting points were measured using a
■
micro melting point apparatus (MP-500P, Yanaco, Tokyo, Japan) and
1
are uncorrected. H NMR (300 MHz) spectra were recorded on a
JEOL-AL-300 spectrometer (JEOL, Tokyo) with tetramethylsilane as
an internal standard. All chemical shifts (δ) were reported in parts
per million (ppm) downfield relative to the chemical shift of
tetramethylsilane. Signals are quoted as s (singlet), d (doublet), dt
(double triplet), t (triplet), q (quartet), br (broad), or m (multiplet).
Fast atom bombardment mass spectra (FAB−MS) and high-resolution
mass spectra (HRMS) were obtained on a JEOL-AL-300 spectrometer
(JEOL) and recorded on the spectrometer. Column chromatography
was performed using Wako gel C-200 (70−230 mesh). HPLC was
performed using the JASCO HPLC system (JASCO, Tokyo) and
Capcell Pack C18 columns (Shiseido, Tokyo): 4.6 mm ID × 250 mm
for analysis and 10 mm ID × 250 mm for semipreparative purification.
Chemical purity of 12 and unlabeled 6−8 was analyzed under the
following conditions: flow rate, 1.0 mL/min; acetonitrile (MeCN)/
H2O/triethylamine (Et3N), 6/4/0.01 (v/v/v). Radiochemical purity of
[11C]6, [18F]7, and [18F]8 was assayed by analytical HPLC with a
detector for monitoring radioactivity according to the following
conditions: 1.0 mL/min, MeCN/H2O/Et3N (6/4/0.01, v/v/v) for
[11C]6 and [18F]7; 1.0 mL/min, MeCN/H2O/Et3N (6.5/3.5/0.01,
v/v/v) for [18F]8. Identity of three radioligands was confirmed with
the corresponding unlabeled 6−8 by HPLC. Under radio-HPLC
purification and analysis, effluent radioactivity was monitored using a
NaI (Tl) scintillation detector system. All chemical reagents and
solvents were purchased from commercial sources (Sigma-Aldrich,
MO; Wako Pure Chemical Industries, Osaka, Japan; Tokyo Chemical
Industries, Tokyo) and used as supplied. Compound 18 (JNJ-
16259685) was purchased from Alexis Biochemicals (San Diego, CA).
Carbon-11 (11C) and fluorine-18 (18F) were produced by 14N (p,α)
11C and 18O (p,n) 18F nuclear reactions using CYPRIS HM-18 cyclo-
tron (Sumitomo Heavy Industry, Tokyo). If not otherwise stated,
radioactivity was measured with an IGC-3R Curiemeter (Aloka,
Tokyo). For in vitro binding assay [18F]5 and [11C]17 were prepared
according to the procedures described previously.20,33
Figure 4. Representative sagittal PET images of wild-type (A) and
mGluR1 knockout (B) mouse brains acquired between 0 and 60 min
after injection of [11C]6 (17 MBq, 0.15 nmol).
type (Figure 4A) and mGluR1 knockout (Figure 4B) mice.
These images showed a significant contrast in brain uptake
and distribution. In the wild-type mouse, high brain penetration
and accumulation of radioactivity was seen in the cerebellum and
thalamus, which was similar to PET images of rat brains and
expected for specific binding to mGluR1. In the mGluR1
knockout mouse, only a very low and uniform distribution of
radioactivity was determined in the brain. As calculated from
the TACs (Supporting Information Figure 4), the values of area
under TACs between 0 and 60 min (AUC0−60 min = SUV × min)
were 104 and 100 for the cerebellum and thalamus of wild-type
mice, whereas the value was 24 for the whole brain of mGluR1
kockout mice. This difference further demonstrated the specificity
for the binding to mGluR1 visualized in the brain after injection
of [11C]6.
The present results indicate that [11C]6 is a promising PET
ligand for the imaging of brain mGluR1. Compared to the
radioligands listed in Scheme 1, PET with [11C]6 showed high
in vivo specific binding for mGluR1, not only in the cerebellum
but also in the thalamus, striatum, etc., of the rat brain. [11C]6
had improved kinetics over [18F]5. After injection of [18F]5
into rats, the uptake of radioactivity increased during the
90 min PET scan,21 while at 45 min after the injection of [11C]
6, uptake peaked and thereafter decreased in the brain slowly.
The difference may be related to the decreased binding affinity
of [11C]6 (Ki = 12.6 nM) with mGluR1, in comparison with
[18F]5 (Ki = 5.4 nM). Further, metabolite analysis of rats
showed that higher than 80% of total radioactivity in the brain
homogenate was due to unchanged [11C]6 at 90 min after the
injection, although its rapid metabolism was determined in the
plasma. This suggested that the radiolabeled metabolite of
[11C]6 in the blood could not enter the brain easily, at least
not to any large extent and, consequently, the radiolabeled
metabolite does not contribute to the nonspecific binding of
[11C]6 in the brain. Detailed comparison of metabolite analysis
will be performed between [11C]6 and [18F]5 and will be
reported elsewhere.
2. Chemistry. 4-Chloro-6-(1-ethoxyvinyl)pyrimidine (9).
A
mixture of 4,6-dichloropyrimidine (1.49 g, 10.0 mmol), ethyl
1-(tributylstannyl)vinyl ether (3.4 mL, 10.0 mmol), Pd(PPh3)4
(348 mg, 0.3 mmol), cesium fluoride (3.04 g, 20 mmol), and
copper(I) iodide (190 mg, 1.0 mmol) in N,N-dimethylforma-
mide (DMF, 30 mL) was stirred at 80 °C for 5 h under N2
atmosphere. The reaction mixture was quenched with CH2Cl2/
H2O (1/1, v/v), and the suspension was filtered through Celite
with CH2Cl2. The organic layer was washed with water, dried
over Na2SO4, and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using
n-hexane/ethyl acetate (15/1, v/v) to give 9 (1.47 g, 63.8%
1
yield) as a colorless solid; mp: 35−37 °C. H NMR (CDCl3, δ):
1.45 (3H, t, J = 7.0 Hz), 3.98 (2H, q, J = 7.0 Hz), 4.58 (1H, d, J =
2.2 Hz), 5.71 (1H, d, J = 2.2 Hz), 7.66 (1H, s), 8.89 (1H, s).
FAB−MS: m/z 185 (M + H).
4-(6-Chloropyrimidin-4-yl)-N-methylthiazol-2-amine (10). To a
solution of 9 (1.47 g, 8.0 mmol) in THF/H2O (25 mL; 1/1, v/v) was
added 1-bromopyrrolidine-2,5-dione (1.56 g, 8.8 mmol) at room
temperature and stirred for 2 h. Methylthiourea (718 mg, 8.0 mmol)
was added to the reaction mixture and the mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with water and
the resulting solid was filtered and recrystallized from hot THF/water
to give 10 (1.11 g, 61.3% yield) as a colorless solid; mp: 190−191 °C.
1H NMR (CDCl3, δ): 3.06 (3H, d, J = 5.1 Hz), 5.20 (1H, br), 7.61
(1H, s), 7.92 (1H, s), 8.89 (1H, s). FAB−MS: m/z 227 (M + H).
SUMMARY
■
To visualize brain mGluR1 with PET, three novel ligands 6−8
were designed and labeled with 11C or 18F. Of them, 6 showed
high in vitro binding affinity for mGluR1 and suitable
lipophilicity. This compound was easily labeled with
[11C]MeI to give [11C]6 of two levels of specific activity in a
short synthesis time. PET with [11C]6 of 4370−7840 GBq/μmol
is expected to be used to elucidate mGluR1 in mGluR1-low
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N-[4-(6-Chloropyrimidin-4-yl)-1,3-thiazol-2-yl]-4-methoxy-N-
methylbenzamide (11). To a solution of 10 (227 mg, 1.0 mmol) and
Et3N (607 mg, 6.0 mmol) in toluene (5 mL) was added 4-metho-
xybenzoyl chloride (256 mg, 1.5 mmol) at room temperature under
N2 atmosphere. The reaction mixture was heated at 100 °C for 10 h.
This mixture was quenched with water and extracted with CH2Cl2.
The organic layer was dried over Na2SO4 and evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2/Et3N (1/0.001, v/v) and then
CH2Cl2/ethyl acetate/Et3N (4/1/0.005, v/v/v) to give 11 (251 mg,
69.6% yield) as a colorless solid; mp: 176−178 °C. 1H NMR (CDCl3,
δ): 3.80 (3H, s), 3.89 (3H, s), 7.02 (2H, d, J = 8.8 Hz), 7.60 (2H, d,
J = 8.8 Hz), 8.06 (1H, s), 8.10 (1H, s), 8.95 (1H, s). FAB−MS: m/z
361 (M + H).
N-[4-[6-(Isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-
methoxy-N-methylbenzamide (6). To a solution of 11 (250 mg,
0.7 mmol) and K2CO3 (193 mg, 1.4 mmol) in 1,4-dioxane (7 mL) was
added isopropylamine (1 mL, 11.7 mmol) at room temperature. The
reaction mixture was heated at 80 °C for 16 h. This mixture was quenched
with water and extracted with ethyl acetate. The organic layer was washed
with brine, dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using n-hexane/
ethyl acetate/Et3N (1/4/0.005, v/v/v) to give 6 (191 mg, 71.2% yield) as
a colorless solid; mp: 186−188 °C. 1H NMR (CDCl3, δ): 1.29 (6H, d, J =
6.2 Hz), 3.78 (3H, s), 3.88 (3H, s), 4.12 (1H, br), 4.85 (1H, br), 7.00
(2H, d, J = 8.8 Hz), 7.06 (1H, s), 7.59 (2H, d, J = 8.8 Hz), 7.90 (1H, s),
8.57 (1H, s). HRMS (FAB) calcd for C19H22O2N5S, 384.1494; found,
384.1451.
for 6 h at 70 °C. This mixture was quenched with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography using n-hexane/ethyl
acetate/Et3N (1/2/0.003, v/v/v) to give 7 (58 mg, 52.0% yield) as a
colorless solid; mp: 172−174 °C. 1H NMR (CDCl3, δ): 1.29 (6H, d, J =
6.2 Hz), 3.77 (3H, s), 4.11 (1H, br), 4.30 (2H, dt, J = 4.2, 27.9 Hz), 4.80
(2H, dt, J = 4.0, 47.3 Hz), 4.82 (1H, br), 7.03 (2H, d, J = 8.8 Hz), 7.06
(1H, s), 7.59 (2H, d, J = 8.8 Hz), 7.90 (1H, s), 8.57 (1H, s). HRMS
(FAB) calcd for C20H23O2N5FS, 416.1557; found, 416.1540.
4-Fluoropropoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-
thiazol-2-yl]-N-methylbenzamide (8). To a solution of 12 (100 mg,
0.27 mmol) and K2CO3 (57 mg, 0.41 mmol) in anhydrous DMF (5 mL)
was added 14 (70 mg, 0.30 mmol). The reaction mixture was stirred for
6 h at 70 °C. This mixture was quenched with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried
over Na2SO4, and evaporated under reduced pressure. The residue
was purified by silica gel column chromatography using n-hexane/
ethyl acetate/Et3N (1/1/0.002, v/v/v) to give 8 (70 mg, 60.4%
1
yield) as a colorless solid; mp: 162−164 °C. H NMR (CDCl3, δ):
1.29 (6H, d, J = 6.2 Hz), 2.14−2.30 (2H, m), 3.78 (3H, s), 4.14
(1H, br), 4.18 (2H, t, J = 6.0 Hz), 4.68 (2H, dt, J = 5.7, 47.3 Hz),
4.85 (1H, br), 7.00 (2H, d, J = 6.6 Hz), 7.06 (1H, s), 7.58 (2H, d,
J = 6.6 Hz), 7.90 (1H, s), 8.57 (1H, s). HRMS (FAB) calcd for
C21H25O2N5FS, 430.1713; found, 430.1679.
3. Radiochemistry. N-(4-(6-(Isopropylamino)pyrimidin-4-yl)-
1,3-thiazol-2-yl)-4-[11C]methoxy-N-methylbenzamide ([11C]6).
For Conventional Specific Activity. [11C]MeI with conventional
specific activity was synthesized from cyclotron-produced
[11C]CO2 as described previously.22 Briefly, [11C]CO2 was
bubbled into 0.04 M LiAlH4 in anhydrous tetrahydrofuran
(THF, 300 μL). After evaporation of THF, the remaining
complex was treated with 57% hydroiodic acid (300 μL) to give
[11C]MeI, which was distilled at 180 °C and transferred by
helium gas into a solution of 12 (1.0 mg) and NaOH (5 μL,
0.5 M) in anhydrous DMF (300 μL) at −15 to −20 °C. After
radioactivity reached a plateau, this reaction mixture was heated
at 70 °C for 5 min. The reaction mixture was applied to the
semipreparative HPLC system. HPLC purification was
completed using the mobile phase of MeCN/H2O/Et3N
(6/4/0.01, v/v/v) at a flow rate of 5.0 mL/min. The radioactive
fraction corresponding to the desired product was collected in a
sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL
of sterile normal saline, and passed through a 0.22 μm Millipore
filter to give 2.1 GBq of [11C]6. The tR of [11C]6 was 7.1 min
for purification and 6.2 min for analysis on HPLC. Synthesis
time from EOB, 25 min; radiochemical yield (decay-corrected),
25% based on [11C]CO2; radiochemical purity, > 99%; specific
activity at EOS, 140 GBq/μmol.
4-Hydroxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-
2-yl]-N-methylbenzamide (12). BBr3 in dry CH2Cl2 (8.3 mL, 1.0 M;
8.3 mmol) was added dropwise to a solution of 6 (640 mg, 1.66 mmol) in
dry CH2Cl2 (20 mL) at −40 °C under N2 atmosphere. The reaction
mixture was stirred at −40 °C for 1 h and then at room temperature
overnight. This mixture was quenched with 2% aqueous Na2CO3 solution
and extracted with CH2Cl2. The organic layer was washed with brine,
dried over Na2SO4, and evaporated under reduced pressure. The residue
was purified by silica gel column chromatography using n-hexane/ethyl
acetate/Et3N (1/2/0.003, v/v/v) and then ethyl acetate/methanol/Et3N
(4/1/0.005, v/v/v) to give 12 (185 mg, 30.2% yield) as a colorless solid;
1
mp: 227−229 °C. H NMR (DMSO-d6, δ): 1.16 (6H, d, J = 6.2 Hz),
3.68 (3H, s), 4.12 (1H, br), 6.89 (2H, d, J = 8.1 Hz), 7.10 (1H, s), 7.45
(1H, d, J = 7.0 Hz), 7.57 (2H, d, J = 8.4 Hz), 7.92 (1H, s), 8.41 (1H, s),
10.21 (1H, br). HRMS (FAB) calcd for C18H20O2N5S, 370.1338; found,
370.1379.
2-Fluoroethyl 4-Methylbenzenesulfonate (13). To a solution of 2-
fluoroethanol (192 mg, 3.0 mmol) and pyridine (480 μL, 6.0 mmol)
in dry CH2Cl2 (5 mL) was added 4-methylbenzenesulfonyl chloride
(629 mg, 3.3 mmol) at 0 °C. The reaction mixture was stirred for 24 h
at room temperature. This mixture was quenched with water and
extracted with CH2Cl2. The organic layer was washed with brine, dried
over Na2SO4, and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography using n-hexane/ethyl
acetate (3/1, v/v) to give 13 (428 mg, 65.4% yield) as a colorless oil.
1H NMR (CDCl3, δ): 2.46 (3H, s), 4.27 (2H, dt, J = 4.0, 27.1 Hz),
4.58 (2H, dt, J = 4.1, 47.1 Hz), 7.36 (2H, d, J = 8.4 Hz), 7.82 (2H, d,
J = 8.4 Hz).
3-Fluoropropyl 4-Methylbenzenesulfonate (14). To a solution of
3-fluoropropanol (781 mg, 10.0 mmol) and pyridine (1.6 mL, 20.0 mmol)
in dry CH2Cl2 (15 mL) was added 4-methylbenzenesulfonyl chloride
(2.1 g, 11 mmol) at 0 °C. The reaction mixture was stirred for
24 h at room temperature. This mixture was treated as described for
13. Purification by silica gel column chromatography using n-hexane/
ethyl acetate (5/1, v/v) gave 14 (1.67 g, 72.0% yield) as a colorless oil.
1H NMR (CDCl3, δ): 1.96−2.12 (2H, m), 2.46 (3H, s), 4.16 (2H, t,
For High Specific Activity. [11C]MeI with high specific activity was
synthesized from cyclotron-produced [11C]CH4 as described pre-
viously.23 Briefly, prior to real production, target gas was irradiated at
20 μA for 5 min and then purged. After the irradiation had been repeated
three times, fresh target gas was irradiated at 20 μA for 30 min. The
resulting [11C]CH4 was recovered from the target, concentrated on a
Porapak Q trap, passed through a quartz tube kept at 50 °C (I2 part) and
630 °C (empty part) by helium gas (50 mL/min), and converted to
[11C]MeI. Passed through an Ascarite and phosphorus pentoxide column,
[11C]MeI was purified and collected in a solution of 12 (1.0 mg) and
NaOH (5 μL, 0.5 M) in anhydrous DMF (300 μL) at −15 to −20 °C.
After radioactivity reached a plateau, this mixture was warmed to 70 °C
and maintained for 5 min. This reaction mixture was treated as described
for conventional specific activity to give 1.1 GBq of [11C]6. Synthesis time
from EOB, 29 min; radiochemical yield (decay-corrected), 6% based on
[11C]CH4; radiochemical purity, >99%; specific activity at EOS, 7840
GBq/μmol.
J = 6.2 Hz), 4.49 (2H, dt, J = 5.7, 46.9 Hz), 7.36 (2H, d, J = 8.1 Hz),
7.80 (2H, d, J = 8.1 Hz).
4-Fluoroethoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-
thiazol-2-yl]-N-methylbenzamide (7). To a solution of 12 (100 mg,
0.27 mmol) and K2CO3 (57 mg, 0.41 mmol) in anhydrous DMF (5 mL)
was added 13 (65 mg, 0.30 mmol). The reaction mixture was stirred
4-[18F]Fluoroethoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-
thiazol-2-yl]-N-methylbenzamide ([18F]7). H218O (95 atom%) was
used for irradiation. [18F]HF was recovered from the target, separated
from [18O]H2O, and concentrated on a QMA short column. After
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elution from the column with 400 μL of a solution containing aqueous
K2CO3 (110 mg/8 mL), Kryptofix (330 mg), and MeCN (8 mL), the
aqueous [18F]KF was transferred into a reaction vial and evaporated to
remove H2O and MeCN at 110 °C for 15 min. After 15 (7 μL) in 1,2-
dichlorobenzene (150 μL) was added to the vial, the reaction mixture
was heated at 130 °C to give [18F]FEtBr, which was distilled at once
by helium gas (10 mL/min) for 2 min. With the helium flow,
[18F]FEtBr was trapped in a solution of 12 (1.0 mg) and NaOH (5 μL,
0.5 M) in anhydrous DMF (300 μL) at −15 to −20 °C. After radio-
activity reached plateau, the reaction mixture was heated at 90 °C for
10 min. HPLC purification was completed using a mobile phase of
MeCN/H2O/Et3N (5.5/4.5/0.01, v/v/v) at a flow rate of 5.0 mL/min.
The radioactive fraction corresponding to the desired product was
collected in a sterile flask, evaporated to dryness in vacuo, redissolved
in 3 mL of sterile normal saline, and passed through a 0.22 μm
Millipore filter to give 1.0 GBq of [18F]7. The tR of [18F]7 was 8.2 min
for purification and 6.0 min for analysis on HPLC. Synthesis time,
59 min from EOB; radiochemical yield (decay corrected), 25% based
on [18F]F−; radiochemical purity, >99%; specific activity at EOS,
400 GBq/μmol.
incubated with [18F]5 (3 nM in PBS) and 100 μL of tested com-
pounds 5−8 (10−6 to 10−10 M in 0.1% dimethylsulfoxide) in a final
volume of 1.0 mL buffer, respectively. These mixtures were incubated
for 1 h at room temperature. The bound and free radioactivity were
separated by vacuum filtration through 0.3% polyethylenimine-
pretreated Whatman GF/C glass fiber filters using a cell harvester
(M-24, Brandel, Gaithersburg, MD), followed by washing with
prechilled buffer three times. The filters containing the bound [18F]
5 were assayed for radioactivity by a 1480 Wizard autogamma
scintillation counter (Perkin-Elmer, Waltham, MA). In the present
study, the dissociation constant (Kd) of [18F]5 for mGluR1 in the
brain homogenate was determined by using the Scatchard analysis.
Subsequently, we performed the same assay with [11C]17, an
antagonistic radioligand for mGluR5, as described above. The results
of inhibitory experiments were subjected to nonlinear regression
analysis using Prism 5 (GraphPad Software, La Jolla, CA) by which
IC50 and the inhibition constant (Ki) values were calculated.
7. In Vitro Autoradiography. Rat brain sections (20 μm) were
preincubated for 20 min in 50 mM Tris-HCl buffer (pH 7.4)
containing 1.2 mM MgCl2 and 2 mM CaCl2 at room temperature.
After preincubation, these sections were incubated for 30 min at room
temperature in the fresh buffer containing [11C]6 (9.2 MBq, 0.5 nM),
[18F]7 (0.4 MBq, 5 pM), or [18F]8 (0.4 MBq, 4 pM). Compound 18
(1 μM) were used to determine the specific binding of these
radioligands for mGluR1. After incubation, these brain sections were
washed (3 × 5 min) with cold buffer, dipped in cold distilled water,
and dried with cold air. These sections were placed on contact with
imaging plates (BAS-MS2025, FUJIFILM, Tokyo). Autoradiograms
were obtained and photostimulated luminescence values (PSL) in the
regions of interest were measured using a Bio-Imaging Analyzer
System (BAS5000, FUJIFILM).
4-[18F]Fluoropropoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-
1,3-thiazol-2-yl]-N-methylbenzamide ([18F]8). To a vial containing
dried [18F]KF was added 16 (10 μL) in 1,2-dichlorobenzene (150 μL).
The reaction mixture was heated at 180 °C for 2 min to produce
[18F]FPrBr, which was distilled by helium gas and trapped into a
solution of 12 (1.0 mg) and NaOH (5 μL, 0.5 M) in anhydrous DMF
(300 μL) at −15 to −20 °C. After trapping was complete, the reaction
mixture was heated at 120 °C for 10 min. HPLC purification was
completed using a mobile phase of MeCN/H2O/Et3N (5.5/4.5/0.01,
v/v/v) at a flow rate of 5.0 mL/min to give 55 MBq of [18F]8. The tR
of [18F]8 was 12.1 min for purification and 6.2 min for analysis on
HPLC. Synthesis time from EOB, 58 min; radiochemical yield (decay-
corrected), 2% based on total [18F]F−; radiochemical purity, >99%;
specific activity at EOS, 410 GBq/μmol.
4. Measurement and Computation of Lipophilicity. The
LogD values were measured by mixing [11C]6, [18F]7, or [18F]8
(radiochemical purity: 100%; about 200000 cpm) with n-octanol
(3.0 g) and sodium phosphate buffer (PBS; 3.0 g, 0.1 M, pH 7.4) in a test
tube. The tube was vortexed for 3 min at room temperature, followed
by centrifugation at 2330g for 5 min. An aliquot of 1 mL of PBS and
1 mL of n-octanol was removed, weighted, and counted, respectively.
Each samples from the remaining organic layer was removed and
repartitioned until consistent LogD value was obtained. The LogD
value was calculated by comparing the ratio of cpm/g of n-octanol to
that of PBS and expressed as LogD = Log[cpm/g (n-octanol)/cpm/g
(PBS)]. All measurements were performed in triplicate. Meanwhile,
the values of cLogD of three radioligands were computed using Pallas
3.4 software (CompuDrug, Sedona, AZ).
8. Biodistribution Study in Mice. A saline solution of [11C]6
(4.8 MBq/140 μL, 40 pmol) was injected into mice via the tail vein.
Four mice were sacrificed at 1, 5, 15, 30, and 60 min after the injection
by cervical dislocation, respectively. The whole brain, heart, liver, lung,
spleen, testis, kidney, small intestine (including contents), muscle, and
blood samples were quickly removed. Radioactivity present in these
tissues was measured with an autogamma counter (1480 Wizard) and
expressed as the percentage of injected dose per gram of wet tissue
(% ID/g). All radioactivity measurements were decay-corrected.
9. Small-Animal PET Studies. For Normal Rats. Prior to PET
scans, anatomical template images of rat brain were generated
by a high-resolution magnetic resonance imaging (MRI) system.35
Briefly, a rat was anesthetized with sodium pentobarbital
(50 mg/kg, ip), and scanned with a 400 mm bore, 7 T horizontal
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: repetition time = 8000 ms, effective echo
time = 15 ms, field of view = 35 mm ×35 mm, and slice thick-
ness = 0.6 mm.
A rat was placed in a small-animal PET scanner (Siemens Medical
Solutions, Knoxville, TN). Body temperature was maintained with a
40 °C water circulation system (T/Pump TP401, Gaymar Industries,
Orchard Park, NY). The rat was kept under anesthesia with 1.5%
(v/v) isoflurane during the scan. To inject [11C]6, a 24-gauge needle
with catheter (Terumo Medical Products, Tokyo) was placed into the
rat tail vein. A dynamic emission scan in 3D list mode was performed
for 90 min (1 min ×4 scans, 2 min ×8 scans, 5 min ×14 scans). For
inhibitory study, unlabeled 6 (1 mg/kg) or 18 (3 mg/kg) was injected
via the tail vein catheter just before a bolus injection of [11C]6 (37−55
MBq/100 μL, 0.3−0.5 nmol). PET dynamic images were
reconstructed with filtered back-projection using a Hanning’s filter, a
Nyquist cutoff of 0.5 cycle/pixel.
5. Animal. DdY mice (male; 8 weeks old; 34−36 g), c57BL/6j
mouse (male; 11 weeks old; 24 g), and Sprague−Dawley rats (male;
7−8 weeks old; 210−280 g) were purchased from Japan SLC
(Shizuoka, Japan). MGluR1 knockout mouse (male; 12 weeks old;
34 g) was supplied by RIKEN Bioresource Center (Tsukuba, Japan).
These animals were housed under a 12 h dark−light cycle and were
allowed free access to food pellets and water. The animal experiments
were approved by the Animal Ethics Committee of the National
Institute of Radiological Sciences.
6. In Vitro Binding Assays. Rats (n = 5) were sacrificed by
decapitation under ether anesthesia. The whole brains were rapidly
removed and homogenized in 10 volumes of 50 mM Tris-HCl (pH =
7.4) containing 120 mM NaCl with a SilentCrusher S homogenizer
(Heidolph Instruments, Schwabach, Germany). The homogenate was
centrifuged in polypropylene tube at 40000g for 15 min at 4 °C using
Optima-TLX (Beckman Coulter, Brea, CA). After the supernatant was
discarded, the pellet was resuspended, homogenized, and centrifuged
in the same buffer. This procedure was repeated twice to obtain a final
pellet of brain homogenate stored at −80 °C before use.
The brain homogenate was diluted to 100 mg/mL in 50 mM Tris-
HCl buffer containing 120 mM NaCl, 2 mM KCl, 1 mM MgCl2, and
1 mM CaCl2. Each preparation of 100 μL of homogenate was
For Mice. A wild-type or mGluR1 knockout mouse was secured in a
custom-designed chamber, placed in the Inveon PET scanner, and
prepared as described above. A 29-gauge needle with 12−15 cm of
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polyethylene 10 tube prepared in-house was placed into the tail vein of
mouse to inject [11C]6 (5 MBq/100 μL, 45 pmol). A dynamic
emission scan in 3D list mode was performed for 60 min (1 min ×4
scans, 2 min ×8 scans, 5 min ×8 scans).
PBS, phosphate buffer solution; Pd(PPh3)4, tetrakis-
(triphenylphosphine)palladium(0); PET, positron emission
tomography; SUV, standardized uptake value; TAC, time−
activity curve; tR, retention time
Image Acquisition and Data Analysis. The PET images were
reconstructed using ASIPro VM software (Analysis Tools and System
Setup/Diagnostics Tool, Siemens Medical Solutions) with each PET
image by individual experience. Volumes of interest were placed on the
striatum, thalamus, pons, and cerebellum using ASIPro VM (Siemens
Medical Solutions) with reference to the MRI template. Brain uptake
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 radioactivity) × gram body
weight.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 81-43-382-3708. Fax: 81-43-206-3261. E-mail:
zhang@nirs.go.jp. Address: Department of Molecular Probes,
Molecular Imaging Center, National Institute of Radiological
Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
■
Author Contributions
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We thank the staff of National Institute of Radiological Sciences
for support with the cyclotron operation, radioisotope pro-
duction, radiosynthesis, and animal experiments. We are also
grateful to Dr. Tatsumi Hirata (National Institute of Genetics,
Shizuoka, Japan) for development of mGluR1 knockout mouse.
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ABBREVIATIONS USED
■
AUC, area under time−activity curve; BBr3, boron tribromide;
[11C]CH4, [11C]methane; CNS, central nervous system; [11C]
CO2, [11C]carbon dioxide; DMF, N,N-dimethyl formamide;
EOB, end of bombardment; EOS, end of synthesis; Et3N,
triethylamine; FAB-MS, fast atom bombardment mass spectra;
[18F]FEtBr, 1-bromo-2-[18F]fluoroethane; [18F]FPrBr, 1-bromo-
3-[18F]fluoropropane; HPLC, high-performance liquid chroma-
tography; HRMS, high-resolution mass spectra; %ID/g,
percentage of the injected dose per gram of wet tissue;
Kryptofix 222, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-
[8,8,8]hexacosane; LiAlH4, lithium aluminum hydride; MeCN,
acetonitrile; [11C]MeI, [11C]methyl iodide; mGluR1, metabo-
tropic glutamate receptor type 1; mGluR5, metabotropic
glutamate receptor type 5; MRI, magnetic resonance imaging;
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