Synthesis and Evaluation of Novel Radioligands Based on 3-[5-(Pyridin-2-yl)-2H-tetrazol-2-yl]benzonitrile for Positron Emission Tomography Imaging of Metabotropic Glutamate Receptor Subtype 5

Article abstract of DOI:10.1021/acs.jmedchem.6b00209

We found out 3-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]benzonitrile analogues as the candidate for positron emission tomography (PET) imaging agents of metabotropic glutamate receptor subtype 5 (mGluR5). Among these compounds, 3-methyl-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile (10) exhibited high binding affinity (K_i = 9.4 nM) and moderate lipophilicity (cLogD, 2.4). Subsequently, [¹¹C]10 was radiosynthesized at 25 ± 14% radiochemical yield (n = 11) via C-[¹¹C]methylation of the arylstannyl precursor 15 with [¹¹C]methyl iodide. In vitro autoradiography and PET assessments using [¹¹C]10 showed high specific binding in the striatum and hippocampus, two brain regions enriched with mGluR5. Moreover, test-retest PET studies with [¹¹C]10 indicated high reliability to quantify mGluR5 density, such as the intraclass correlation coefficient (0.90) and Pearson r (0.91) in the striatum of rat brain. We demonstrated that [¹¹C]10 is a useful PET ligand for imaging and quantitative analysis of mGluR5. Furthermore, [¹¹C]10 might be modified using its skeleton as a lead compound.

Full text of DOI:10.1021/acs.jmedchem.6b00209

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Article
Synthesis and Evaluation of Novel Radioligands Based on 3-[5-
(Pyridin-2-yl)-2H-tetrazol-2-yl]benzonitrile for Positron Emission
Tomography Imaging of Metabotropic Glutamate Receptor Subtype 5
Yoko Shimoda, Tomoteru Yamasaki, Masayuki Fujinaga, Masanao Ogawa, Yusuke Kurihara, Nobuki Nengaki,
Katsushi Kumata, Joji Yui, Akiko Hatori, Lin Xie, Yiding Zhang, Kazunori Kawamura, and Ming-Rong Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00209 • Publication Date (Web): 25 Mar 2016
Downloaded from http://pubs.acs.org on April 5, 2016
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Synthesis and Evaluation of Novel Radioligands Based on
3-[5-(Pyridin-2-yl)-2H-tetrazol-2-yl]benzonitrile for Positron Emission
Tomography Imaging of Metabotropic Glutamate Receptor Subtype 5
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Yoko Shimoda,†,§ Tomoteru Yamasaki,†,§ Masayuki Fujinaga,†,§ Masanao Ogawa,†,‡ Yusuke
Kurihara,†,‡ Nobuki Nengaki,†,‡ Katsushi Kumata,† Joji Yui,† Akiko Hatori,† Lin Xie,† Yiding
Zhang,†Kazunori Kawamura,† and MingꢀRong Zhang†,*
†Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological
Sciences, 4ꢀ9ꢀ1 Anagawa, Inageꢀku, Chiba 263ꢀ8555, Japan
‡SHI Accelerator Service Co. Ltd., 1ꢀ17ꢀ6 Osaki, Shinagawaꢀku, Tokyo 141ꢀ0032, Japan
§These authors contributed equally.
*Correspondence to:
MingꢀRong Zhang, PhD
Phone: +81ꢀ43ꢀ382ꢀ3709; Fax: +81ꢀ43ꢀ206ꢀ3261; Eꢀmail: zhang@nirs.go.jp
Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological
Sciences, 4ꢀ9ꢀ1 Anagawa, Inageꢀku, Chiba 263ꢀ8555, Japan
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ABSTRACT
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We designed five 3ꢀ[5ꢀ(pyridinꢀ2ꢀyl)ꢀ2Hꢀtetrazolꢀ2ꢀyl]benzonitrile analogs as candidates for positron
emission tomography (PET) imaging of metabotropic glutamate receptor subtype 5 (mGluR5).
Among these compounds, 3ꢀmethylꢀ5ꢀ(5ꢀ(pyridinꢀ2ꢀyl)ꢀ2Hꢀtetrazolꢀ2ꢀyl)benzonitrile (10) exhibited
high binding affinity (Ki = 9.4 nM) and moderate lipophilicity (cLogD: 2.4). Subsequently, [11C]10
was radiosynthesized at 25 ± 14% radiochemical yield (n = 11) via Cꢀ[11C]methylation of the
arylstannyl precursor 15 with [11C]methyl iodide. In vitro autoradiography and PET assessment using
[11C]10 showed high specific binding in the striatum and hippocampus, two brain regions enriched
with mGluR5. Moreover, testꢀretest PET studies with [11C]10 indicated high reliability to quantify
mGluR5 density, such as the intraclass correlation coefficient (0.90) and Pearson r (0.91) in the
striatum of rat brain. We demonstrated that [11C]10 is a useful PET ligand for imaging and
quantitative analysis of mGluR5. Furthermore, [11C]10 might be modified using its skeleton as a lead
compound.
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INTRODUCTION
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Glutamate is the major excitatory neurotransmitter in the brain and its receptors are classified into
two types, ionotropic and metabotropic. Metabotropic glutamate receptors (mGluRs) are G
proteinꢀcoupled receptors categorized into three groups including eight subtypes (mGluR1–8) based
on their structure, signal transduction pathways, and pharmacological properties.1,2 Of these groups,
group I mGluRs (mGluR1 and mGluR5) are localized postsynaptically in glutamatergic neurons,
activate phospholipase C after Gq protein coupling, and subsequently form inositol 1,4,5ꢀtriphosphate
and triacylglycerol. These second messengers promote intracellular calcium release, finally
activating protein kinase C.3,4 Although mGluR1 and mGluR5 have a high degree of homology, each
receptor has a distinct regional distribution in the brain.5,6 The mGluR5 is mainly expressed in the
striatum and can trigger the activities of the NꢀmethylꢀDꢀaspartate receptor and dopamine transporter,
and modulate neurotransmission in the central nervous system (CNS).7,8 Based on the physiological
properties, a number of modulators targeting mGluR5 have been developed for treatment of several
CNS disorders such as anxiety, depression, fragile X syndrome, and dyskinesia in Parkinson’s
disease.9–11 Subsequently, several positron emission tomography (PET) ligands for mGluR5 imaging
were developed with chemical structures based on 2ꢀmethylꢀ6ꢀ(phenylethynyl)pyridine (MPEP, 1)12
and 3ꢀ[(2ꢀmethylꢀ1,3ꢀthiazolꢀ4ꢀyl)ethynyl]pyridine (MTEP, 2), two positive allosteric mGluR5
modulators.13 These PET ligands labeled with carbonꢀ11 (11C; halfꢀlife: 20.4 min) or fluorineꢀ18 (18F;
halfꢀlife: 109.8 min) show specific accumulation of radioactivity in the mGluR5ꢀenriched striata of
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rodent
(E)ꢀ3ꢀ((6ꢀmethylpyridinꢀ2ꢀyl)ethynyl)cyclohexꢀ2ꢀenoneꢀOꢀ[11C]methyloxime
[11C]3),
and
primate
brains.14,15
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them,
((E)ꢀ[11C]ABP688,
(E)ꢀ3ꢀ(pyridinꢀ2ꢀylethynyl)cyclohexꢀ2ꢀenoneꢀOꢀ(3ꢀ(2ꢀ[18F]ꢀfluoroethoxy)propyl)oxime
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([18F]PSS232, [18F]4), 3ꢀ[18F]fluoroꢀ5ꢀ(pyridinꢀ2ꢀylethynyl)benzonitrile ([18F]FPEB, [18F]5),
3ꢀfluoroꢀ5ꢀ((2ꢀ([18F]fluoromethyl)thiazolꢀ4ꢀyl)ethynyl)benzonitrile ([18F]SP203, [18F]6), and
3ꢀfluoroꢀ5ꢀ(3ꢀ(5ꢀfluoropyridinꢀ2ꢀyl)ꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl)benzoꢀ[11C]nitrile ([11C]AZD9272, [11C]7)
have progressed to clinical studies (Figure 1).16–19
Figure 1
The most widely used PET ligand for mGluR5 imaging is [11C]3.20–22 However, it has been
reported that the (Z) geometrical isomer is also formed while radiosynthesizing [11C]3 as an (E)
isomer,23,24 and the (Z) isomer has much poorer efficacy for mGluR5 imaging than [11C]3.25
Ametamey et al. reported approximately 10% (Z) isomer during synthesis of [11C]3 at their PET
center.23 Additionally, we have previously found that conventional radiosynthesis of [11C]3 at our
laboratory includes 20% (Z) isomer,25 and (Z) isomer contamination leads to ambiguous results in
testꢀretest PET studies with [11C]3.26,27 Furthermore, average percentage differences between test and
retest PET studies with [11C]3 to determine the nondisplaceable binding potential (BPND), a
quantitative value for evaluation of binding to mGluR5, have been reported as 28% for humans and
12% for baboons in all regions of interest (ROIs). Similarly, testꢀretest PET studies with radiolabeled
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derivatives of 3 also demonstrate relatively low reliability.28,29 On the other hand, a previous study
demonstrated that mGluR5 modulators containing an alkyne moiety formed glutathione conjugates at
their alkyne moiety.30 This result strongly suggests that the presence of an alkyne moiety may
produce individual difference of brain uptake of the ligand via catalysis by glutathione Sꢀtransferase.
Accordingly, development of novel PET ligands lacking the alkyne moiety is desired for in vivo
imaging of mGluR5 in the brain.
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In the field of medicinal chemistry, tetrazole is known as a bioisoster for alkyne.
3ꢀ[5ꢀ(Pyridinꢀ2ꢀyl)ꢀ2Hꢀtetrazolꢀ2ꢀyl]benzonitrile (8) has been reported as a new potent antagonist of
mGluR5.31,32 Different from 3, 4, 5, and 6 containing alkyne moieties, compound 8 contains a
tetrazole ring. Despite this change, 8 and its derivatives retain relatively high affinities for mGluR5.31
Additionally, it was expected that 8 have higher affinity for mGluR5 than a previous nonꢀalkyne type
ligand 7.32
In this study, to develop a novel PET ligand for mGluR5 imaging, we designed five ligands,
9–13 (Figure 2), using 8 as a lead compound. Compounds 9 and 13 are easily labeled with
[11C]methyl iodide ([11C]MeI) or 1ꢀbromoꢀ2ꢀ[18F]fluoroethane by [11C]/[18F]alkylation of desmethyl
precursor 14. Compound 10 can be labeled with [11C]MeI by a Cꢀ[11C] coupling reaction. Since the
[11C]CH3 directly attached to the benzene ring, [11C]10 may be stable in vivo compared with 9 and
13. Compounds 11 or 12 bearing fluorine or methyl groups can be labeled with [18F]KF or [11C]MeI
at the 6ꢀposition in the pyridine ring, which enables investigation of the substituent effect of the
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pyridine ring on the in vivo potential. Here, we report the synthesis of PET ligand candidates 9–13 as
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well as in vitro and in vivo evaluations of their potential for imaging mGluR5 in the brain.
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Figure 2
RESULTS AND DISCUSSION
Chemistry. It has been found that some 1,3ꢀdisubstituted aryl compounds derived from 3 and 8 show
similar or improved binding affinities compared with their corresponding parent compounds.31 Here,
we synthesized 1,3ꢀdisubstituted analogs 9–13 of 8 (Scheme 1). To label 9 and 10, we synthesized
precursors 14 and 15.
To construct a tetrazole ring, a 1,3ꢀdipolar cycloaddition reaction was performed between tosyl
hydrazone 20 and aryldiazonium salt 24. Compound 20 was synthesized by reacting
2ꢀpyridinecarboxaldehydes 17–19 with pꢀtoluenesulfonyl hydrazide, which was used for the next
reaction without purification. Diazonium compound 24 was prepared by reacting anilines 21–23 with
NaNO2 under acidic conditions in situ. The reaction of 20 and 24 under basic conditions produced
tetrazole compounds 9–12 and 16 with 23–50% chemical yields. Demethylation of 9 using BBr3 was
conducted at −40 °C to produce 14 that was used for the subsequent fluoroethylation to yield 13.
Stannylation of 16 using bis(tributyltin) in the presence of Pd(PPh3)4 did not produce the
tributylstannyl compound. In place of bis(tributyltin), reacting 16 with bis(trimethyltin), a more
reactive reagent for stannylation, produced the trimethylstannyl compound 15 with a 24% chemical
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yield.
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Scheme 1
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In Vitro Profiles of PET Ligand Candidates. The binding affinities of PET ligand candidates 9–13
for mGluR5 were measured using a binding assay with the mGluR5ꢀselective radioligand [11C]3 in
rat brain homogenates. As shown in Figure 3 and Table 1, these compounds exhibited a wide range
of binding affinities for mGluR5 with Ki values of 9.4–2280 nM. The Ki values of compounds 9 and
10 in the assay with [11C]3 were 10.8 and 9.4 nM, respectively. This result was similar to previous
data in which the Ki values of 9 and 10 were 6 and 7 nM for mGluR5 in an assay with competition to
3ꢀ[3H]methoxyꢀ5ꢀ(pyridinꢀ2ꢀylethynyl)pyridine.31 The change of the methoxy group in 9 to a
relatively large fluoroethoxy group in 13 significantly decreased the binding affinity for mGluR5.
These results suggested that the presence of only small substituents, such as methyl and methoxy
groups, at the 3ꢀposition of the benzene ring provided a better fit for the binding site of the mGluR5
domain, although the affinities of 9 and 10 were slightly weaker than that of 3 (Ki = 5.5 nM). On the
other hand, compounds 11 and 12 with a fluorine atom and methyl group at the 6ꢀposition of the
pyridine ring showed low affinities for mGluR5. This result was different from the high affinities of
compounds 1 and 3 for mGluR5, which included 6ꢀmethylpyridine moieties, and the reason remains
unclear.
The values of calculated (cLogD) and measured lipophilicity (LogD) are shown in Table 1. The
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cLogD values for compounds 9–13 were 2.4–2.9, which were within the range generally considered
to be suitable for PET ligands in brain imaging.33 Following radiolabeling with 11C, the LogD values
were measured for [11C]9 and [11C]10 (radiochemical results are described in the following section).
The LogD values of [11C]9 and [11C]10 were 2.4 and 2.8, which were similar and higher than that of
[11C]3 (LogD = 2.4), respectively.
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Figure 3
Table 1
Radiochemistry. Because of their potent binding affinities for mGluR5, we selected compounds 9
and 10 for radiolabeling with [11C]MeI. [11C]MeI was produced by reducing cyclotronꢀproduced
[11C]CO2 with LiAlH4, followed by iodination with 57% HI (Scheme 2). To yield [11C]9,
Oꢀ[11C]methylation of desmethyl precursor 14 with [11C]MeI in the presence of NaOH proceeded
easily at 70 °C for 5 min. For [11C]10, arylstannyl precursor 15 was added to a mixture containing
[11C]MeI and a Pd catalyst, which was prepared in a reaction vial containing Pd2(dba)3, CuCl, and
P(oꢀtol)3 in DMF. This reaction mixture was heated at 80 °C for 5 min. After termination of the Oꢀ
and Cꢀ[11C]methylation reactions, separation of these mixtures was performed by reverse phase
HPLC to obtain two formulated products, [11C]9 and [11C]10, respectively.
Starting from 14.8–22.2 GBq [11C]CO2, 0.58–0.91 GBq [11C]9 was produced with 14 ± 4% (n =
3) radiochemical yield at 32 min after the end of bombardment (EOB). Starting from 22.2–28.1 GBq
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[11C]CO2, 0.86–2.99 GBq [11C]10 was obtained with 25 ± 14% (n = 11) radiochemical yield at 32
min after EOB. In the final product solution, the specific activities of [11C]9 and [11C]10 were 57–71
and 52–190 GBq/µmol, respectively. The radiochemical purity of both products was higher than 98%
at the end of synthesis (EOS).
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The two products did not show radiolysis at room temperature for 90 min after formulation,
indicating their radiochemical stability for the duration of at least one PET scan. The analytical
results of [11C]9 and [11C]10 were in compliance with our inꢀhouse quality control/assurance
specifications for radiopharmaceuticals.
Scheme 2
In Vitro Autoradiography. To confirm the specificity and selectivity of [11C]9 and [11C]10 binding
to mGluR5, in vitro autoradiography was performed using rat brain sections (Figure 4). In the control
sections for both radioligands (A and D), the distribution pattern of radioactivity was heterogeneous
with the highest level in the striatum. Moderate radioactivity was seen in the hippocampus and
cerebral cortex, and a low level was found in the thalamus. The lowest radioactivity was seen in the
cerebellum. This distribution pattern was consistent with the biological distribution of mGluR5 in the
rat brain.34 Coꢀincubation with mGluR5ꢀselective compound 1 (1 ꢁM) dramatically diminished the
radioactivity in the brain sections of both groups (B and E). Furthermore, coꢀincubation with
mGluR1ꢀselective
(3,4ꢀdihydroꢀ2Hꢀpyrano[2,3]b
quinolinꢀ7ꢀyl)
(cisꢀ4ꢀmethoxycyclohexyl)
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methanone (JNJ16259685, 25)35 (1 ꢁM) had almost no effect on the binding of [11C]9 and [11C]10 (C
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Figure 4
Table 2 shows the ratios of radioactive binding in interesting regions to the cerebellum, a region
of negligible mGluR5 expression, as determined from autoradiograms of [11C]9 and [11C]10. In the
control sections, the ratios of the mGluR5ꢀrichest striatum to cerebellum were 14.9 for [11C]9 and
15.8 for [11C]10. By coꢀincubation with compound 1, these ratios decreased to 2.7 for [11C]9 and 2.1
for [11C]10. These results indicated that [11C]10 had higher specific binding for mGluR5 and lower
nonspecific binding than [11C]9.
Table 2
Because of the high level of in vitroꢀspecific binding to mGluR5 in the rat brain, we pursed
[11C]10 for in vivo evaluation using PET to assess its potential for mGluR5 imaging in the brain.
PET Assessment. To evaluate the in vivo availability of [11C]10, PET assessments were performed
using rats.
Figure 5 shows timeꢀactivity curves (TACs) of [11C]10 in the striatum, hippocampus, frontal
cortex, thalamus, and cerebellum of the baseline (A)ꢀ and compound 1 (1 mg/kg) (B)ꢀtreated rats. In
baseline rats (n = 7), radioactivities in all ROIs immediately reached maximum levels after injection
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of [11C]10 and then decreased quickly. The uptake ratio [Ctissue(t)/Ccerebellum(t)] of each region in the
cerebellum at the equilibrium state [Ctissue(t)dt/Ccerebellum(t)dt = 0] of specific binding was 3.0 ± 0.1 in
the striatum, 2.4 ± 0.1 in the hippocampus, 2.3 ± 0.1 in the frontal cortex, and 2.0 ± 0.1 in the
thalamus (Supporting Information Figure 4A). In blocking experiments (n = 4), the ratios were
reduced by 1.3–1.6 in each brain region (Supporting Information Figure 4B). These results suggested
that [11C]10 showed significant specific binding to mGluR5. The BPND value of [11C]10 was
estimated by kinetic analysis with the simplified reference tissue model (SRTM)36 to quantitatively
evaluate the binding to target molecules in vivo.37 The BPND of [11C]10 in each region was 1.30 ±
0.10 in the striatum, 0.94 ± 0.09 in the hippocampus, 0.82 ± 0.09 in the frontal cortex, and 0.72 ±
0.09 in the thalamus. As shown in Figure 6, parametric PET images of [11C]10 with the BPND scale
clearly visualized regional differences with the highest signal in the striatum. Moderate signals were
detected in the hippocampus and cerebral cortex. Low radioactivity was found in the pons and
cerebellum. These specific signals were homogenously diminished by pretreatment with 1 (1 mg/kg),
demonstrating that PET using [11C]10 provided significant specific visualization of mGluR5 in brain
regions.
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Figure 5
Figure 6
Table 3 shows the reliability of testꢀretest PET studies with [11C]10. A scatter plot comparing the
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individual outcomes is shown in Figure 7. In the striatum, which is the mGluR5ꢀrichest region in the
brain, the percentage of variability, intraclass correlation coefficient (ICC), and Pearson’s correlation
coefficient (r) were 3.0, 0.90, and 0.91, respectively. Additionally, correlation (Pearson’s r) between
test and retest outcomes in all ROIs was 0.96, indicating high reliability of PET with [11C]10.
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Table 3
Figure 7
Ex Vivo Metabolite Analysis. To investigate metabolism of [11C]10 in the plasma and brain,
metabolite analysis was performed using rats (Figure 8). The fraction corresponding to unchanged
[11C]10 (tR = 5.2–5.6 min) in the plasma gradually decreased to 57 ± 19% at 5 min, 18 ± 4% at 30
min, and 11 ± 3% at 60 min after injection. A polar radiolabeled metabolite (tR = 2.1–2.4 min) was
observed in the HPLC charts of plasma and brain samples (Supporting Information Figure 5). On the
other hand, unchanged [11C]10 in the brain remained at 93% of total radioactivity at 30 min and
decreased to 84% at 60 min. Considering that the treated brain homogenate was slightly
contaminated with blood from sampling and brain capillaries (3−5% of whole rat brain38) the actual
percentages of unchanged [11C]10 in the brain tissue might be larger than the experimentally
determined values. Because the radioactive uptake of [11C]10 in the brain was very low from 30 min
after injection, metabolite correction for quantitative analysis was unnecessary.
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Figure 8
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Comparison of [11C]3 and [11C]10. To confirm the utility of [11C]10 for PET, we compared [11C]10
with [11C]3, the most widely used PET ligand for mGluR5 imaging.
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The in vitro binding affinities of both unlabeled compounds for mGluR5 were measured under
the same experimental conditions. Although the binding affinity of compound 10 (Ki = 9.4 nM) for
mGluR5 was slightly weaker than that of 3 (Ki = 5.5 nM), [11C]10 showed significant in
vitroꢀspecific binding on the autoradiograms of rat brain sections (Figure 4).
The average BPND value of [11C]10 in PET assessments using SpragueꢀDawley (SD) rats was
half of that of [11C]3: 1.30 vs. 2.49 ([11C]10 vs. [11C]3) in the striatal BPND.39 However, as an
important index in quantitative PET analysis, the testꢀretest reliability of SRTM analysis with
[11C]10 in the striatum of the rat brain was superior to that with [11C]3: 3.0 vs. 4.8 ([11C]10 vs.
[11C]3) for the percentage of variability; 0.90 vs. 0.88 for ICC, and 0.91 vs. 0.88 for Pearson’s r.
Moreover, the in vivo stability of [11C]10 in rat plasma was higher than that of [11C]3. At 5 min after
injection, the percentage of unchanged radioligand was 57% for [11C]10 and <40% for [11C]3,40
which might be caused by different metabolizing routes of the radioligands.
Taken together, although the in vitroꢀ and in vivoꢀbinding affinities of [11C]10 for mGluR5 were
slightly weaker than those of [11C]3, the high testꢀretest reliability of PET and in vivo stability of
[11C]10 provide some advantages, especially in improving incoherence, which has been found in
quantitative PET analysis with [11C]3 for mGluR5 imaging in animal and human studies.26–29
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Biodistribution Analysis. The distribution of radioactivity in mice was measured at five time points
(1, 5, 15, 30, and 60 min) after injection of [11C]10 (Figure 9). At 1 min, high uptake (>5% injected
dose per gram of wet tissue, %ID/g) appeared in the lung (6.3 ± 0.8), liver (7.0 ± 1.6), kidney (6.0 ±
0.6), small intestines (7.0 ± 2.5), and brain (5.2 ± 0.6 %ID/g). After the initial increments, the
radioactivity in many tissues decreased dramatically, while that in the liver and small intestines
increased continually until reaching maximum levels (liver: 13.6 ± 1.0 %ID/g at 5 min; small
intestines: 47.7 ± 14.1 %ID/g at 15 min) and then decreased quickly. Accumulation of radioactivity
in the liver and small intestines suggested that hepatobiliary excretion as well as the intestinal
reuptake pathway might dominate the whole body distribution of radioactivity and rapid washout
from the body after injection.
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Figure 9
SUMMARY
In the current study, we demonstrated that compound 10 is a promising PET ligand candidate for
mGluR5 imaging by in vitro binding assays, and subsequently synthesized [11C]10 via
Cꢀ[11C]methylation of the trimethylstannyl precursor 15 with [11C]MeI in a short synthesis time with
reliable radiochemical yield. PET with [11C]10 in rat brains visualized the regional distribution of
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mGluR5 and demonstrated high reliability for quantification of the mGluR5 density in testꢀretest
PET studies. Hence, [11C]10 is a potential PET ligand for imaging and quantification of mGluR5 in
the brain, which may warrant evaluation in humans. This radioligand is also a useful lead compound
for development of new PET ligands with higher affinity and more specific binding to mGluR5.
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EXPERIMENTAL SECTION
Melting points were measured using a micro melting point apparatus (MPꢀ500P, Yanaco, Tokyo,
Japan) and were uncorrected. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on
a JEOLꢀALꢀ300 spectrometer (JEOL, Tokyo, Japan) using TMS as an internal standard. All chemical
shifts (δ) are reported as ppm downfield relative to the TMS signal. Signals are quoted as s (singlet),
d (doublet), t (triplet), br (broad), or m (multiplet). FABꢀMS and HRMS spectra were obtained on a
NMSꢀSX102 spectrometer (JEOL) and recorded on the spectrometer. Silica gel column
chromatography was performed using Wako gel Cꢀ200 (70–230 mesh, Wako Pure Chemical
Industries, Osaka, Japan). HPLC analysis was performed using the JASCO HPLC system (JASCO,
Tokyo, Japan) and a Capcell Pak C18 column (4.6 mm i.d. × 250 mm, Shiseido, Tokyo). Compounds
9–15 indicated ≥ 95% purity by HPLC analysis. The chemical purities of compounds 9–15 were
analyzed under the following conditions: 9, 13, and 14: 1.0 mL/min, MeCN/H2O/Et3N (50/50/0.1,
v/v/v); 10 and 15: 1.5 mL/min, MeCN/H2O/Et3N (55/45/0.1, v/v/v); 11 and 12: 1.0 mL/min,
MeCN/H2O/Et3N (60/40/0.1, v/v/v). Radiochemical purity of [11C]9 and [11C]10 was analyzed by
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HPLC with a detector to monitor radioactivity under the following conditions: [11C]9: 1.0 mL/min,
MeCN/H2O/Et3N (50/50/0.1, v/v/v); [11C]10: 1.5 mL/min, MeCN/H2O/Et3N (55/45/0.1, v/v/v). For
radioꢀHPLC separation and analysis, effluent radioactivity was monitored using a NaI (Tl)
scintillation detector system. All chemical reagents and solvents were purchased from commercial
sources (SigmaꢀAldrich, St. Louis, MO; Wako Pure Chemical Industries; Tokyo Chemical Industries,
Tokyo, Japan) and used as supplied. Compounds 1 hydrochloride and 25 were purchased from Enzo
Life Sciences (Farmingdale, NY).
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[11C]CO2 was produced by a CYPRIS HM18 cyclotron (Sumitomo Heavy Industry, Tokyo,
Japan). [11C]MeI was synthesized from cyclotronꢀproduced [11C]CO2 as described previously.41
[11C]3 for the in vitro binding assay was synthesized according to a previous report.25 At EOS (n
= 3), 2.3–3.5 GBq [11C]3 was obtained with >99% radiochemical purity and 113–239 GBq/µmol SA.
Chemistry
3-Methoxy-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile
(9).
A
mixture
of
3ꢀaminoꢀ5ꢀmethoxybenzonitrile (296 mg, 2 mmol), ethanol (6 mL), and 42% aqueous
tetrafluoroboric acid (1.4 mL) was stirred at −5 °C in an iceꢀsalt bath. A solution of NaNO2 (152 mg,
2.2 mmol) in water (1 mL) was added dropwise to this mixture to produce a solution of diazonium
salt. In another flask, a mixture of 2ꢀpyridinecarboxaldehyde (214 mg, 2 mmol) and
pꢀtoluenesulfonyl hydrazide (372 mg, 2 mmol) in ethanol (5 mL) was stirred at room temperature for
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15 min. Following removal of ethanol under reduced pressure, the residue was dissolved in pyridine
(2 mL) and cooled to −10 °C. The solution of diazonium salt was added dropwise to this mixture,
followed by stirring at room temperature for 3.5 h. The reaction mixture was poured into water and
extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Purification of the residue by silica gel column
chromatography (ethyl acetate/nꢀhexane, 1/2 to 1/1, v/v) yielded 9 (269 mg, 48%) as slightly orange
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crystals (mp: 176–177 °C). H NMR (CDCl3): δ 3.98 (3H, s), 7.30 (1H, s), 7.46–7.50 (1H, m),
7.91–7.96 (1H, m), 8.06 (1H, dd, J = 1.8, 1.8 Hz), 8.20 (1H, s), 8.37 (1H, d, J = 8.0 Hz), 8.86 (1H, d,
J = 4.8 Hz). 13C NMR (CDCl3): δ 56.3, 109.8, 114.7, 115.4, 117.2, 119.1, 123.0, 125.4, 137.3, 138.1,
146.0, 150.6, 160.8, 165.2. HRMS m/z: 279.1046 (calculated for C14H11N6O: 279.0994).
3-Methyl-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile
(10).
A
mixture
of
3ꢀaminoꢀ5ꢀmethylbenzonitrile (90 mg, 0.7 mmol), ethanol (2 mL), and 42% aqueous tetrafluoroboric
acid (555 µL) was stirred at −5 °C in an iceꢀsalt bath. A solution of NaNO2 (52 mg, 0.8 mmol) in
water (1 mL) was added dropwise to this mixture to produce a solution of diazonium salt. In another
flask, a mixture of 2ꢀpyridinecarboxaldehyde (65 µL, 0.7 mmol) and pꢀtoluenesulfonyl hydrazide
(128 mg, 0.7 mmol) in ethanol (2 mL) was stirred at room temperature for 20 min. After removal of
ethanol under reduced pressure, the residue was dissolved in pyridine (2.0 mL) and cooled to −5 °C.
The solution of diazonium salt was added dropwise to this solution, followed by stirring at room
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temperature for 2 h. The reaction mixture was poured into water and extracted with CH2Cl2. The
organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced
pressure. Purification of the residue by silica gel column chromatography (CH2Cl2 to ethyl
acetate/CH2Cl2, 1/9, v/v) yielded 10 (86 mg, 48%) as slightly orange crystals (mp: 139 °C). 1H NMR
(CDCl3): δ 2.55 (3H, s), 7.45–7.50 (1H, m), 7.62 (1H, s), 7.90–7.96 (1H, m), 8.34–8.41 (3H, m),
8.84–8.86 (1H, m). 13C NMR (CDCl3): δ 21.2, 100.5, 113.9, 117.4, 120.5, 123.0, 124.6, 125.4, 133.6,
136.9, 137.3, 141.8, 146.1, 150.6. HRMS m/z: 263.0988 (calculated for C14H11N6: 263.1045).
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3-(5-(6-Fluoropyridin-2-yl)-2H-tetrazol-2-yl)-5-methoxybenzonitrile (11).
A
mixture of
3ꢀaminoꢀ5ꢀmethoxybenzonitrile (148 mg, 1 mmol), ethanol/water (4 mL, 1/1, v/v), and conc. HCl
(250 µL) was stirred at −5 °C in an iceꢀsalt bath. A solution of NaNO2 (76 mg, 1.1 mmol) in water (1
mL) was added dropwise to this mixture to produce a solution of diazonium salt. In another flask, a
mixture of 6ꢀfluoropyridineꢀ2ꢀcarboxaldehyde (182 mg, 1.4 mmol) and pꢀtoluenesulfonyl hydrazide
(182 mg, 1.0 mmol) in ethanol (5.0 mL) was stirred at room temperature for 1 h, followed by
addition of 1 M NaOH (5.0 mL). After the solution was cooled to −10 °C, the solution of diazonium
salt was added dropwise, followed by stirring at room temperature for 2 h. This reaction mixture was
poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried
over anhydrous Na2SO4, and concentrated under reduced pressure. Purification of the residue by
silica gel column chromatography (CH2Cl2 to ethyl acetate/CH2Cl2, 1/9, v/v) yielded 11 (67 mg,
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23%) as slightly yellow crystals (mp: 172–174 °C). 1H NMR (CDCl3): δ 3.98 (3H, s), 7.14 (1H, dd, J
= 2.9, 8.0 Hz), 7.30–7.32 (1H, m), 8.03–8.08 (2H, m), 8.18 (1H, dd, J = 1.5, 1.5 Hz), 8.21 (1H, dd, J
= 2.2, 7.5 Hz). 13C NMR (CDCl3): δ 56.4, 109.8, 111.8 (d, JCꢀF = 36.4 Hz), 114.8, 115.4, 117.1, 119.2,
120.4 (d, JCꢀF = 3.2 Hz), 138.0, 142.4 (d, JCꢀF = 7.4 Hz), 144.4 (d, JCꢀF = 14.2 Hz), 160.8, 164.0,
163.6 (d, JCꢀF = 241.1 Hz). HRMS m/z: 297.0896 (calculated for C14H10ON6F: 297.0900).
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3-Methoxy-5-(5-(6-methylpyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile (12).
A
mixture of
3ꢀaminoꢀ5ꢀmethoxybenzonitrile (297 mg, 2 mmol), ethanol (6 mL), and 42% aqueous
tetrafluoroboric acid (1.4 mL) was stirred at −5 °C in an iceꢀsalt bath. A solution of NaNO2 (158 mg,
2.3 mmol) in water (1 mL) was added dropwise to produce a solution of diazonium salt. In another
flask, a mixture of 6ꢀmethylꢀ2ꢀpyridinecarboxaldehyde (250 mg, 2.1 mmol) and pꢀtoluenesulfonyl
hydrazide (382 mg, 2.1 mmol) in ethanol (6.0 mL) was stirred at room temperature for 15 min. After
removal of ethanol under reduced pressure, the residue was dissolved in pyridine (2.0 mL). After the
solution was cooled to −10 °C, the solution of diazonium salt was added dropwise, followed by
stirring at room temperature for 3.5 h. The reaction mixture was poured into CH2Cl2, washed with
brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Purification of the
residue by silica gel column chromatography (CH2Cl2) and then recrystallization from
1
CH2Cl2/nꢀhexane yielded 12 (296 mg, 50%) as colorless crystals (mp, 178–180 °C). H NMR
(CDCl3): δ 2.73 (3H, s), 3.98 (3H, s), 7.29 (1H, dd, J = 1.5, 2.6 Hz), 7.34 (1H, d, J = 7.7 Hz), 7.81
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(1H, dd, J = 7.7, 7.7 Hz), 8.05 (1H, dd, J = 2.2, 2.2 Hz), 8.13 (1H, s), 8.16 (1H, dd, J = 1.5, 1.5 Hz).
13C NMR (CDCl3): δ 24.6, 56.3, 109.8, 114.5, 115.3, 117.2, 119.0, 120.2, 125.2, 137.4, 138.0, 145.3,
159.6, 160.7, 165.3. HRMS m/z: 293.1108 (calculated for C15H13N6O: 293.1151).
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3-(2-Fluoroethoxy)-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile (13). A mixture of 14 (100
mg, 0.4 mmol), K2CO3 (78 mg, 0.6 mmol), and 2ꢀfluoroethylꢀ4ꢀmethylbenzenesulfonate (92 mg, 0.4
mmol) in DMF (3 mL) was stirred at 70 °C for 5 h. The reaction mixture was poured into saturated
aqueous K2CO3 and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Purification of the residue by silica gel column
chromatography (ethyl acetate/nꢀhexane, 2/3, v/v) yielded 13 (40 mg, 34%) as colorless crystals (mp:
1
171–173 °C). H NMR (CDCl3): δ 4.39 (2H, dt, J = 4.0, 27.4 Hz), 4.82 (2H, dt, J = 4.0, 50.9 Hz),
7.34 (1H, s), 7.48 (1H, dd, J = 1.8, 7.7 Hz), 7.94 (1H, ddd, J = 1.8, 7.7, 7.7 Hz), 8.10 (1H, dd, J = 1.8,
1.8 Hz), 8.23 (1H, s), 8.36 (1H, d, J = 7.7 Hz), 8.86 (1H, d, J = 4.4 Hz). 13C NMR (CDCl3): δ 68.3 (d,
JCꢀF = 20.3 Hz), 81.2 (d, JCꢀF = 171.4 Hz), 110.2, 114.9, 115.9, 117.0, 119.8, 123.1, 125.4, 137.3,
138.1, 146.0, 150.6, 159.7, 165.3. HRMS m/z: 311.1089 (calculated for C15H12FN6O: 311.1057).
3-Hydroxy-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile (14). BBr3 (1 M) in CH2Cl2 (5 mL)
was added dropwise to a solution of 9 (278 mg, 1 mmol) in CH2Cl2 (20 mL) at −40 °C. The reaction
mixture was warmed to room temperature slowly and stirred overnight. Then, the reaction mixture
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was adjusted to pH 7 with saturated aqueous Na2CO3 and extracted with ethyl acetate. The organic
layer was washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure.
Purification of the residue by silica gel column chromatography (ethyl acetate/nꢀhexane, 1/1, v/v)
10
11
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yielded 14 (251 mg, 95%) as slightly orange crystals (mp: 211–212 °C). H NMR (DMSOꢀd6): δ
7.42 (1H, s), 7.60–7.65 (1H, m), 7.87 (1H, s), 8.03–8.10 (2H, m), 8.27 (1H, d, J = 7.7 Hz), 8.80–8.82
(1H, m), 11.2 (1H, br s). 13C NMR (DMSOꢀd6): δ 111.5, 113.8, 117.5, 120.5, 122.9, 125.8, 137.6,
137.8, 145.5, 150.4, 159.1, 164.5. HRMS m/z: 265.0833 (calculated for C13H9N6O: 265.0838).
3-(5-(Pyridin-2-yl)-2H-tetrazol-2-yl)-5-(trimethylstannyl)benzonitrile (15). A mixture of 16 (197
mg, 0.6 mmol), hexamethylditin (237 mg, 0.7 mmol), and Pd(PPh3)4 (36 mg, 5 mol%) in 1,4ꢀdioxane
(5 mL) was stirred at 110 °C for 16 h under N2 gas. The reaction mixture was poured into water and
extracted with CH2Cl2. The organic layer was washed with brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Purification of the residue by silica gel column
chromatography (ethyl acetate/nꢀhexane, 1/2, v/v) yielded 15 (60 mg, 24%) as colorless crystals (mp:
1
156–157 °C). H NMR (CDCl3): δ 0.44 (9H, s), 7.45–7.50 (1H, m), 7.88–7.96 (2H, m), 8.38 (1H, d,
J = 7.7 Hz), 8.50–8.52 (1H, m), 8.60–8.61 (1H, m), 8.85–8.88 (1H, m). 13C NMR (CDCl3): δ −9.03,
113.5, 117.7, 123.0, 125.3, 130.5, 136.2, 137.3, 140.1, 146.2, 147.8, 150.6, 165.2. HRMS m/z:
413.0587 (calculated for C16H17N6Sn: 413.0539).
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3-Bromo-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile
(16).
A
mixture
of
5
6
7
8
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3ꢀaminoꢀ5ꢀbromobenzonitrile (591 mg, 3.0 mmol), ethanol (4.0 mL), and 42% aqueous
tetrafluoroboric acid (2.4 mL) was stirred at −5 °C in an iceꢀsalt bath. A solution of NaNO2 (380 mg,
5.5 mmol) in water (2 mL) was added dropwise to this mixture to produce a solution of diazonium
salt. In another flask, a mixture of 2ꢀpyridinecarboxaldehyde (284 µL, 3 mmol) and
pꢀtoluenesulfonyl hydrazide (563 mg, 3 mmol) in CH2Cl2 (4 mL) was stirred at room temperature for
15 min. After removal of CH2Cl2 under reduced pressure, the residue was dissolved in pyridine (4
mL). The solution of diazonium salt was added dropwise to this solution at −5 °C, followed by
stirring at room temperature for 3.5 h. The reaction mixture was poured into water and extracted with
ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Purification of the residue by silica gel column
chromatography (ethyl acetate/CH2Cl2, 1/9, v/v) yielded 16 (464 mg, 47%) as colorless crystals( mp:
164–166 °C). 1H NMR (CDCl3): δ 7.47–7.52 (1H, m), 7.92–7.97 (2H, m), 8.37 (1H, ddd, J = 1.1, 1.1,
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8.0 Hz), 8.57 (1H, dd, J = 1.8, 1.8 Hz), 8.76 (1H, dd, J = 1.8, 1.8 Hz), 8.85–8.88 (1H, m). C NMR
(CDCl3): δ 115.6, 115.8, 121.8, 123.1, 124.2, 125.6, 127.0, 135.6, 137.3, 137.6, 145.8, 150.6, 165.5.
HRMS m/z: 326.9983 (calculated for C13H8N6Br: 326.9994).
Radiochemistry
3-[11C]Methoxy-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile ([11C]9). After irradiation, the
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cyclotronꢀproduced [11C]CO2 was bubbled into 0.4 M LiAlH4 in anhydrous THF (0.3 mL). After
evaporation of THF, the remaining complex was treated with 57% HI (0.3 mL) to produce [11C]MeI
that was distilled and transferred under N2 gas flow into a solution of 14 (1.3 mg, 4.9 µmol) and
NaOH (0.5 M, 7 µL) in anhydrous DMF (0.3 mL) at −15 to −20 °C. When the radioactivity reached
a plateau, the reaction mixture was heated at 70 °C for 5 min. The [11C]methylation was competed by
addition of MeCN/H2O/Et3N (50/50/0.1, 1 mL), and the reaction mixture was applied to a
semiꢀpreparative HPLC system. HPLC separation was performed on a Capcell Pak C18 column (10
mm i.d. × 250 mm) using MeCN/H2O/Et3N (50/50/0.1, v/v/v) at 5 mL/min. The radioactive fraction
corresponding to [11C]9 (tR = 9.5 min) was collected in a sterile flask, evaporated to dryness under
reduced pressure, redissolved in 3 mL sterile saline, and passed through a 0.22ꢀµm Millipore filter to
obtain 0.8 GBq [11C]9. The identity of [11C]9 (tR = 8.6 min) was confirmed by analytical HPLC with
unlabeled 9. The specific activity was calculated by comparing the assayed radioactivity to the mass
measured at UV 254 nm.
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3-[11C]Methyl-5-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile ([11C]10). Copper(I) chloride (1.5
mg, 15 µmol), CsF (2.8 mg, 19 µmol), and Pd2(dba)3 (1.4 mg, 1.6 µmol) were added to a 1ꢀmL
minivial containing a septum and stirring bar. The vial was purged with nitrogen gas, and a solution
of P(oꢀtol)3 (1.8 mg, 5.9 µmol) in DMF (0.2 mL) was added. This mixture was purged with N2 gas
and stirred for 3 min at room temperature. The prepared solution was transferred into a reaction vial
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using a syringe equipped with an automated synthetic unit. [11C]MeI was transferred under N2 gas
flow into the reaction vial at −15 to −20 °C. When the radioactivity of [11C]MeI reached a plateau, a
solution of precursor 15 in DMF (0.2 mL) was added, and the reaction mixture was heated at 80 °C
for 5 min. After 1 mL of the preparative HPLC mobile phase was added, the reaction mixture was
filtered through a glass fiber prefilter (GF53, 30 µm; Agilent Technologies, Santa Clara, CA) and
applied to the HPLC system. HPLC separation was performed using a Capcell Pak C18 column (10
mm i.d. × 250 mm) and MeCN/H2O/Et3N (55/45/0.1, v/v/v) at 5.0 mL/min. The radioactive fraction
corresponding to [11C]10 (tR = 7.7 min) was collected in a sterile flask, evaporated to dryness in
vacuo, redissolved in 3 mL sterile normal saline, and passed through a 0.22ꢀµm Millipore filter to
yield 2.1 GBq [11C]10. The identity of [11C]10 (tR = 4.6 min) was confirmed by analytical HPLC
with unlabeled 10. The specific activity was calculated by comparing the assayed radioactivity to the
mass measured at UV 254 nm.
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Evaluation of Calculated and Measured Lipophilicities. Measurement of the lipophilicities
(LogD) of [11C]9 and [11C]10 was performed by mixing with nꢀoctanol (3 g) and phosphateꢀbuffered
saline (PBS; 3 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 3,500 g for 5 min. Aliquots of 0.65 mL PBS and 0.65 mL nꢀoctanol
were sampled, weighed, and counted. The LogD value was calculated by comparing the ratio of
cpm/g of nꢀoctanol to that of PBS and expressed as LogD = Log[cpm/g (nꢀoctanol)/cpm/g (PBS)].
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All measurements were performed in triplicate. The calculated lipophilicities (cLogD) for each
ligand were estimated by simulations using ADMET Predictor™ (Simulations Plus, Lancaster, CA).
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Animals. The ddY mice (male, 8 weeks old) and SD rats (male, 8–9 weeks old) were purchased
from Japan SLC (Shizuoka, Japan), maintained in a temperatureꢀcontrolled environment with a 12 h
lightꢀdark cycle, and fed a standard diet (MBꢀ1; Funabashi Farm, Chiba, Japan).
Ethics Statement. Animals were treated and handled according to the Recommendations for
Handling of Laboratory Animals for Biomedical Research compiled by the Committee on the Safety
and Ethical Handling Regulations for Laboratory Animal Experiments, National Institute of
Radiological Sciences, and this study was approved by the committee.
In Vitro Binding Assay
Three rats were sacrificed by decapitation under anesthesia (3% isoflurane in air). The whole brains
except the cerebellum were rapidly removed and homogenized in five volumes of 50 mM TrisꢀHCl
(pH 7.4) containing 120 mM NaCl with a Silent Crusher S homogenizer (Heidolph Instruments,
Schwabach, Germany). The homogenate was centrifuged in a polypropylene tube at 40,000 g for 15
min at 4 °C using an OptimaꢀTLX (Beckman Coulter, Brea, CA). After discarding the supernatant,
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the pellet was resuspended, homogenized, and centrifuged in the same buffer. This procedure was
repeated twice to obtain the final pellet of brain homogenate, which was stored at −80 °C before use.
The brain homogenate was diluted to 100 mg/mL in 50 mM TrisꢀHCl buffer containing 120 mM
NaCl, 2 mM KCl, 1 mM MgCl2, and 1 mM CaCl2. Each preparation of 0.1 mL homogenate was
incubated with [11C]3 (1 nM in buffer) and 0.1 mL test compounds 9–13 (10−2 to 10−7 M in 0.1–1%
DMSO) in a final volume of 1 mL buffer. These mixtures were incubated for 30 min at room
temperature. The bound and free radioactivities were separated by vacuum filtration through 0.3%
polyethylenimineꢀpretreated Whatman GF/C glass fiber filters using a cell harvester (Mꢀ24, Brandel,
Gaithersburg, MD), followed by three washes with prechilled buffer. The radioactivity of filters
containing the bound [11C]3 was counted with a 1480 Wizard autogamma scintillation counter
(PerkinꢀElmer, Waltham, MA). In the present study, the radioligand affinity (1/Kd) of [11C]3 for
mGluR5 in the brain homogenate was determined using GraphPad Prism 5 (GraphPad Software, La
Jolla, CA). The results of inhibitory experiments were subjected to nonlinear regression analysis
using GraphPad Prism 5, in which IC50 and inhibition constant (Ki) values were calculated.
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In Vitro Autoradiography. Rats (n = 3) were killed by cervical dislocation under anesthesia (3%
isoflurane in air). Their brains were quickly removed and frozen in crushed dry ice. Sagittal brain
sections (20 µm) were prepared with a cryostat (HM560; Carl Zeiss, Oberkochen, Germany). Rat
brain sections were preincubated for 10 min in 50 mM TrisꢀHCl buffer (pH 7.4) containing 1.2 mM
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MgCl2 and 2 mM CaCl2 at room temperature. After preincubation, the sections were incubated for 30
min at room temperature in fresh buffer containing [11C]9 (7.6 MBq, 0.1 nM). For blocking
experiments, compounds 1 (1 ꢁM) and 25 (1 ꢁM) were used to determine the specificity and
selectivity for mGluR5. After incubation, brain sections were washed (3 × 5 min) with cold buffer,
immersed in cold distilled water, and then dried with cold air. The sections were placed in contact
with imaging plates (BASꢀMS2025, FUJIFILM, Tokyo, Japan). Autoradiograms were obtained and
photoꢀstimulated luminescence (PSL) values in the ROIs were measured using a BioꢀImaging
Analyzer System (BAS5000, FUJIFILM).
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PET Assessment. A rat was secured in a customꢀdesigned chamber and placed in a small animal
PET scanner (Inveon, Siemens Medical Solutions, Knoxville, TN). Body temperature was
maintained using a 40 °C water circulation system (T/Pump TP401, Gaymar Industries, Orchard
Park, NY). A 24 G intravenous catheter (Terumo Medical Products, Tokyo, Japan) was placed in the
tail vein of the rat for a bolus injection. A bolus of [11C]10 (1 mL, 37–49 MBq, 0.1–0.7 nmol) was
injected at flow rate of 0.5 mL/min via the catheter inserted into the tail vein. For blocking
experiments, compound 1 (n = 4, 1 mg/kg) was administrated prior to a bolus injection of [11C]10.
Dynamic emission scans in threeꢀdimensional list mode were performed for 60 min (10 s × 12
frames, 20 s × three frames, 30 s × three frames, 60 s × three frames, 150 s × three frames, and 300 s
× nine frames). Acquired PET dynamic images were reconstructed by filtered back projection using
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Hanning’s filter with a Nyquist cutoff of 0.5 cycle/pixel, which were summed using analysis
software (ASIPro VM™, Siemens Medical Solutions). The TACs of [11C]10 were acquired from
volumes of interest in the frontal cortex, striatum (caudate/putamen), hippocampus, thalamus, and
cerebellum referring to a rat brain MRI template42 using PMOD software (version 3.4; PMOD
technology, Zurich, Switzerland). The radioactivity was decayꢀcorrected to the injection time and is
expressed as the standardized uptake value (SUV) that was normalized to the injected radioactivity
and body weight. SUVs were calculated according to the following formula: SUV = (radioactivity
per milliliter tissue per injected radioactivity) × gram of body weight. To obtain BPND, kinetic
analysis with SRTM was performed using PMOD software. A reference region for mGluR5 was
chosen in the cerebellum.40 Representative parametric images scaled with BPND were reconstructed
by PMOD software using TACs in mGluR5ꢀenriched and reference regions. For testꢀretest PET
studies, seven male SD rats were used twice within 7–10 days (247 ± 45 g at first and 303 ± 44 g at
the second scan), and the reliability of the data was assessed by the ICC. The parameters were
calculated as follows:
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a. Relative difference (%) = (scan 2 – scan 1)/scan 1 × 100
b. Testꢀretest variability (%) = |scan 2 – scan 1|/[(scan 2 + scan 1)/2] × 100
c. Coefficient of variation (%CV) = standard deviation (s.d.)/mean × 100
d. ICC with BSMSS as “mean sum of squares between subjects” and WSMSS as “mean sum of
squares within subjects”: ICC = (BSMSS – WSMSS)/(BSMSS + WSMSS). An ICC value of −1
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denotes no reliability, whereas 1 indicates maximum reliability.39
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Ex Vivo Metabolite Analysis. Following intravenous injection of [11C]10 (60–108 MBq, 0.2–0.4
mL, 1–2 nmol), the rats (n = 3 for each time point) were sacrificed by cervical dislocation at 5, 30,
and 60 min. Blood and whole brain samples were obtained quickly. The blood sample was
centrifuged at 15,000 g for 2 min at 4 °C to separate the plasma. The supernatant (0.5 mL) was
collected in a test tube containing MeCN (0.5 mL), and the resulting mixture was vortexed for 15 s
and then centrifuged at 15,000 g for 2 min for deproteinization. The resulting supernatant was
collected. The rat brain was homogenized with a Silent Crusher S homogenizer in an iceꢀcooled
MeCN/H2O (1/1, 2 mL) solution. The resulting homogenate was centrifuged at 15,000 g for 2 min at
4 °C. The supernatant (0.5 mL) was collected, resuspended with MeCN (0.5 mL), and then
centrifuged at 15,000 g for 2 min for deproteinization. An aliquot of the supernatant (0.1–1.0 mL)
obtained from the plasma or brain homogenate was injected into the HPLC system with a
radioactivity detector42 and analyzed using a Capcell Pak C18 column (4.6 mm i.d. × 250 mm) with a
mobile phase (MeCN/H2O/Et3N, (55/45/0.01, v/v/v)) at a flow rate of 1.5 mL/min. The percentage
ratio of [11C]10 (tR = 5.1–5.4 min) to total radioactivity (corrected for decay) on the HPLC
chromatogram was calculated as % = (peak area for [11C]ligand/total peak area) × 100.
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Biodistribution Analysis. Each mouse (34–38 g) was injected a bolus of [11C]10 (4.8 MBq/0.1 mL,
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