Radiosynthesis and preliminary evaluation of 4-[18F]fluoro-N-[4- [6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as a new positron emission tomography ligand for metabotropic glutamate receptor subtype 1

Article abstract of DOI:10.1016/j.bmcl.2011.03.046

The purpose of this study was to develop 4-[¹⁸F]fluoro-N-[4-[6- (isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([ ¹⁸F]FITM, [¹⁸F]4) as a new PET ligand for imaging metabotropic glutamate receptor subty

Full text of DOI:10.1016/j.bmcl.2011.03.046

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2998–3001
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiosynthesis and preliminary evaluation of 4-[18F]fluoro-N-[4-[6-(isopro-
pylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as a new
positron emission tomography ligand for metabotropic glutamate
receptor subtype 1
Tomoteru Yamasaki a,b, , Masayuki Fujinaga a, , Yuichiro Yoshida c, Katsushi Kumata a, Joji Yui a,
Kazunori Kawamura a, Akiko Hatori a, Toshimitsu Fukumura a, Ming-Rong Zhang a,
⇑
a Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8574, Japan
c SHI Accelerator Service Co. Ltd, 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
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 develop 4-[18F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-
thiazol-2-yl]-N-methylbenzamide ([18F]FITM, [18F]4) as a new PET ligand for imaging metabotropic
glutamate receptor subtype 1 (mGluR1). [18F]4 was synthesized by [18F]fluorination of a novel nitro pre-
cursor 3 with [18F]KF in the presence of Kryptofix 222. At the end of synthesis, 429–936 MBq (n = 8) of
Article history:
Received 31 January 2011
Revised 10 March 2011
Accepted 11 March 2011
Available online 17 March 2011
[
18F]4 was obtained with >99% radiochemical purity and 204–559 GBq/
lmol specific activity starting
from 6.7 to 13.0 GBq of [18F]FÀ. The brain distribution of [18F]4 was determined by the in vitro and
ex vivo autoradiography using rat brain sections. The in vitro and in vivo specific binding of [18F]4 to
mGluR1 was detected in the cerebellum, thalamus, hippocampus, and striatum. These results suggest
that [18F]4 is a promising PET ligand for the in vivo evaluation of mGluR1.
Keywords:
mGluR1
PET
Ó 2011 Elsevier Ltd. All rights reserved.
Glutamate (Glu) is a major neurotransmitter in the central ner-
vous system (CNS) in mammals and is involved in over one-half of
synapses. The Glu receptors have distinctive metabotropic and
iontropic types (mGluRs and iGluRs). The mGluRs are G-protein-
coupled receptors and are classified into three groups with eight
subtypes based on their pharmacology, signal transduction mech-
anism, and sequence homology. Among the eight subtypes of
mGluRs, mGluR1 and mGluR5 belonging to group I, are located at
the postsynapse, and regulate acute neuroexcitation with the ef-
flux of Ca2+ from the smooth endoplasmic reticulum. Thus, mGluR1
and mGluR5 are involved in brain development, mechanisms of
learning, and neuroprotection, and have been implicated in the
pathophysiology of several neurological and psychiatric disorders,
such as Parkinson’s disease, multiple sclerosis, motor dysfunction,
epilepsy, and stroke.1–5 The development of mGluRs of group I as a
biomarker should lead to better understanding of the role of these
receptors in pathophysiological and biological processes.
mGluR1 have been developed, such as CPCCOEt,9,10 BAY36-
7620,11 R214127,12 JNJ16259685,13 FTIDC,14 and YM-202074.15
Subsequently, several radiolabeled ligands for PET study were syn-
thesized to investigate the in vivo localization and biological char-
acteristics of mGluR1, such as [11C]JNJ-16567083,16
[
18F]FTIDC,17
[
18F]MK-1312,18 11C]MMTP,19 and [11C]YM-202074 (Scheme 1).20
[
Of these, [18F]MK-1312 and [11C]MMTP have progressed to preclin-
ical evaluation using PET studies involving primates. [18F]MK-1312
demonstrated to have specific binding to mGluR1 in the cerebel-
lum of rhesus monkey by displacement of an mGluR1 allosteric
antagonist MK-5435.21 The uptake of [18F]MK-1312 in the thala-
mus and striatum was low, and included non-specific signals,18
although expression of mGluR1 was detected in not only the cere-
bellum but also the hippocampus, and most of the thalamic nu-
clei.22,23 11C]MMTP showed specific binding to mGluR1 in the
[
cerebellum, hippocampus, frontal cortex, and striatum in the
in vitro autoradiography using human brain sections. However,
in PET studies with baboons, sufficient uptake of [11C]MMTP was
detected in the cerebellum only.19
4-Fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-
2-yl]-N-methylbenzamide (FITM, 4) was developed as a novel allo-
steric antagonist for mGluR1.24 This compound was shown to have
potent antagonistic activity against human mGluR1 with an IC50
value of 5.1 nM. Excellent selectivity over other subtypes was
Positron emission tomography (PET) ligands for mGluR5 have
been developed and successfully applied in the in vivo evaluation
of mGluR5.6–8 At the same time, potent and selective ligands for
⇑
Corresponding author. Tel.: +81 43 206 4041; fax: +81 43 206 3261.
E-mail address: zhang@nirs.go.jp (M.-R. Zhang).
Equal contributions.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.046

T. Yamasaki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2998–3001
2999
exhibited: IC50 values were 7000 nM (for human mGluR5),
>10,000 nM (mGluR2), and >10,000 nM (mGluR8).24
presence of K2CO3/Kryptofix 222 at 150 °C for 10 min, resulted in
a radiochemical yield of 69 13% (n = 8).26 However, the reaction
of [18F]FÀ, which was eluted by K2CO3 (66 mM) and Kryptofix
222 according to a routine procedure,26 with 3 at 150 °C for
10 min gave [18F]4 only with a low yield (<1%). Although raising
the reaction temperature from 150 to 180 °C slightly improved
the reaction efficiency, decomposition of 3 took place in the pres-
ence of a large amount of K2CO3. Prolonging the reaction time from
10 to 30 min further accelerated the decomposition. To avoid
decomposition of 3, the amount of K2CO3 was reduced from 66
to 10 mM and [18F]FÀ was dried to remove water completely. Un-
der the optimized reaction condition, [18F]fluorination of 3 pro-
ceeded efficiently at 180 °C for 10 min. Purification of the
reaction mixtures using a semi-preparative HPLC system (Fluofix
120 N C18 column: 10 mm ID Â 250 mm, CH3OH/50 mM
CH3CO2NH4 = 5:5, 5.0 mL/min) gave [18F]4 in 14 3% radiochemi-
cal yield (n = 8 based on [18F]FÀ, corrected for decay). [18F]4 could
be separated from 3 by prolonging their retention times (3:
In this Letter, we developed 18F-labeled FITM (4) ([18F]FITM,
[
18F]4; Scheme 1) as a new PET ligand and performed preliminary
evaluation using in vitro and ex vivo autoradiography on rat brain
sections.
For the radiosynthesis, the novel nitro precursor 3 was prepared
according to reaction sequences delineated in Scheme 2.25 Reaction
of 4-pyrimidinyl-2-methylaminothiazole 1 with 4-nitrobenzoyl
chloride afforded the benzoylated compound 2, which was substi-
tuted with isopropylamine to give 3. The authentic product 4 was
prepared according to procedures reported previously.24
Next, we examined the conditions for [18F]fluorination of 3
using
[
a
home-made automated synthesis system.26 For the
18F]fluorination of 3, the reaction proceeded via a SNAr substitu-
tion mechanism. In our previous report, 6-[1-(2-[18F]fluoro-3-pyr-
idyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline was synthesized by
heating the corresponding bromo precursor with [18F]FÀ in the
O
O
N
O
N
N
N
N
11CH3
N
N
MeO
N
N
N
N
N
18F
18F
18F]FTIDC
[
11C]JNJ-16567083
(Ki = 0.87 nM)
[
18F]MK-1312
(Ki =0.40 nM)
[
(Ki = 2.0 nM)
NH
N
O
N
N
O11CH3
N
N
N
N
H3CO
N
N
N
N
N
11CH3
S
O
S
18F
S
O
[
11C]MMTP
[
11C]YM-202074
(Ki = 4.8 nM)
[
18F]FITM ([18F]4)
(This study)
(Ki = 7.9 nM)
Scheme 1. Chemical structures of PET ligands for mGluR1.
Cl
N
NH
N
Cl
N
N
(a)
(b)
N
N
N
S
N
N
N
N
NH
NO2
NO2
S
S
O
O
1
2
3
NH
N
N
N
(c)
N
18F
S
O
18
4
F]
[
Scheme 2. Chemical synthesis and radiosynthesis. Reagents and conditions: (a) p-nitrobenzoyl chloride, Et3N, toluene, 100 °C, 2 h, 65%; (b) isopropylamine, K2CO3, 1,4-
dioxane, 80 °C, 8 h, 49%; (c) [18F]KF, Kryptofix 222, DMSO, 180 °C, 10 min, 18% (decay-corrected) based on total [18F]FÀ.

3000
T. Yamasaki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2998–3001
Figure 1. Representative in vitro autoradiographic images of the rat brain with [18F]4 in the absence (A) or presence of 1
lM 4 (B), 1 lM JNJ16259865 (C), or 1 lM MPEP (D).
Figure 2. Representative ex vivo autoradiographic images of the rat brain with [18F]4 in the absence (A) or presence (B) of co-injection with 1 mg/kg JNJ16259865.
14.2 min; [18F]4: 18.5 min) during the HPLC purification. Starting
with 6.7–13.0 GBq of [18F]FÀ, [18F]4 was reliably obtained as an
injectable solution with 429–936 MBq (n = 8) at the end of synthe-
sis (EOS). The identity of [18F]4 was confirmed by co-injection of
non-radioactive 4 on analytic HPLC (CAPCELL PAK C18 column:
4.6 mm ID Â 250 mm, CH3CN/H2O/Et3N = 6:4:0.1%, 1.0 mL/min,
6.6 min). In the final product solution, no significant peak of 3
was observed in its HPLC chart (see Fig. 1 in Supplementary data).
The radiochemical purity of [18F]4 was higher than 99% and the
To confirm the specificity and selectivity of [18F]4 against
mGluR1, we used JNJ1625968513 (mGluR1 antagonist) and MPEP30
(mGluR5 antagonist). Figure 1 shows representative images of
in vitro autoradiography using [18F]4 co-incubated with vehicle
(A), 1 lM 4 (B), 1 lM JNJ16259685 (C), or 1 lM MPEP (D) (also
see Table 1 in Supplementary data). In the section treated [18F]4
with vehicle, strong signals of radioactivity were found in the cer-
ebellum and thalamus, followed by the hippocampus, striatum,
and cerebral cortex, which are known mGluR1-rich regions31
(Fig. 1A). By co-incubation with 4 or JNJ16259685, radioactivity
through the brain section decreased significantly (Fig. 1B and C)
compared with those in the control section (Fig. 1A). On the other
hand, the signals did not change upon co-incubation with MPEP.
The distribution pattern and concentration of radioactivity were
similar to those of the control section (Fig. 1D). These results indi-
cated excellent properties in that [18F]4 had specific and selective
binding to mGluR1 on the rat brain section in the in vitro
conditions.
specific activity was 204–559 GBq/lmol. The radiochemical purity
remained >95% after being maintained at 25 °C for 180 min. The
radioactivity yield, radiochemical purity and stability, and specific
activity of [18F]4 were sufficient for animal experiments.
The distribution coefficient (Log D) of [18F]4 was determined by
the water/octanol system at pH 7.4 using the shaking method.27
The Log D value was measured to be 1.46 0.01 (n = 3). Although
this value is below the range of suitable values of lipophilicity
(Log D = 2–3) as a PET ligand, this lipophilicity may prevent high
non-specific binding with protein in the plasma, giving a high pen-
etration into brain.
Figure 2 shows representative images of ex vivo autoradiogra-
phy using the brain sections of rat co-injected with [18F]4 only
(A)/plus 1 mg/kg JNJ16259685 (B) (also see Table 2 in Supplemen-
tary data). In the control treated with [18F]4 only, radioactivity was
observed at a high level in the cerebellum and thalamus. A modest
Subsequently, we performed preliminary evaluation of [18F]4 by
means of in vitro and ex vivo autoradiography using rat brain
sections.28,29

T. Yamasaki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2998–3001
3001
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2009, 19, 5464.
radioactive signal was also observed in the hippocampus, striatum,
and cerebral cortex (Fig. 2A). This distribution pattern was similar
to that in the in vitro conditions. Moreover, co-injection with
JNJ16259685 decreased the uptake of radioactivity significantly
in the brain section, indicating that [18F]4 was bound to mGluR1
specifically in the in vivo conditions (Fig. 2B). The signals of radio-
activity in the brain sections of rat co-injected with JNJ16259685
decreased by 86–91% in each mGluR1-rich region compared with
those of the control.
In summary, we succeeded in the development of [18F]4 as a
new PET ligand for mGluR1, and praliminary demonstrated the
specific binding for mGluR1 in the in vitro and ex vivo autoradiog-
raphy using rat brain sections. Thus, [18F]4 is a promising PET li-
gand to evaluate pathophysiological and biological processes
mediated by mGluR1. In further experiments, the determination
of specific uptake for mGluR1 in PET study using rodents and pri-
mates will be performed to confirm the usefulness of [18F]4.
25. Synthesis of nitro precursor 3: 4-nitro-N-[4-(6-(isopropylamino)pyrimidin-4-
yl)-1,3-thiazol-2-yl]-N-methylbenzamide: To a stirred solution of 2 (188 mg,
0.5 mmol) and K2CO3 (207 mg, 1.5 mmol) in dioxane (5 mL) was added excess
isopropylamine (1 mL) at room temperature. The reaction mixture was stirred
at 80 °C for 8 h until the full conversion of 2 (TLC monitoring). The reaction
mixture was quenched with water and extracted with AcOEt. The organic layer
was washed with water and saturated NaCl, and dried over Na2SO4. The solvent
was removed to give a residue. Column chromatography of the residue on silica
gel under hexane/AcOEt (1:1) with Et3N (1%) gave the title compound (98 mg,
49%) as a white powder, mp: 208–210 °C. The chemical purity of 3 was higher
than 98% (Fluofix 120 N C18 column: 4.6 mm ID Â 250 mm, CH3OH/
50 mMCH3CO2NH4 = 6:4, 0.8 mL/min, 8.5 min).
Acknowledgments
We thank Nobuki Nengaki and Masanao Ogawa (SHI Accelera-
tor Service Co. Ltd) for their help with radiosynthesis. We are also
grateful to the staff of the National Institute of Radiological Sci-
ences for their support with the cyclotron operation and radionu-
clide production.
1H NMR (300 MHz, CDCl3) d: 1.29 (d, J = 6.6 Hz, 6H), 3.72 (s, 3H), 4.10 (br, 1H),
4.83 (br, 1H), 7.02 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 8.39 (d, J = 8.4 Hz,
2H), 8.57 (s, 1H); GCMS (EI), m/z: 399.
Supplementary data
26. Fujinaga, M.; Yamasaki, T.; Kawamura, K.; Kumata, K.; Hatori, A.; Yui, J.;
Yanamoto, K.; Yoshida, Y.; Ogawa, M.; Nengaki, N.; Maeda, J.; Fukumura, T.;
Zhang, MR. Bioorg. Med. Chem. 2011, 19, 102.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.046.
27. Distribution coefficient values were measured by mixing [18F]4 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 3500 rpm for 5 min. An aliquot of 1 mL PBS and 1 mL n-
octanol was removed, weighed, and counted. 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 the
count per minute (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 performed in
triplicate.
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room temperature in fresh buffer with [18F]4 (1 MBq/mL in saline with 5%
ethanol and Tween80) in the presence or absence of
1 lM 4, 1 lM
JNJ16259685 (ENZO, USA), or 1 M MPEP (Sigma, Japan). After incubation,
l
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analyzer (BAS 5000; Fuji Film, Japan).
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radioactivity, rats were sacrificed by cervical dislocation, and their brains were
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