Development of a18F-Labeled Radiotracer with Improved Brain Kinetics for Positron Emission Tomography Imaging of Translocator Protein (18 kDa) in Ischemic Brain and Glioma

Article abstract of DOI:10.1021/acs.jmedchem.7b00374

We designed four novel acetamidobenzoxazolone compounds 7a-d as candidates for positron emission tomography (PET) radiotracers for imaging the translocator protein (18 kDa, TSPO) in ischemic brain and glioma. Among these compounds, 2-(5-(6-fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (7d) exhibited high binding affinity (K_i = 13.4 nM) with the TSPO and moderate lipophilicity (log D = 1.92). [¹⁸F]7d was radiosynthesized by [¹⁸F]fluorination of the bromopyridine precursor 7h with [¹⁸F]F^- in 12 ± 5% radiochemical yield (n = 6, decay-corrected). In vitro autoradiography and PET studies of ischemic rat brain revealed higher binding of [¹⁸F]7d with TSPO on the ipsilateral side, as compared to the contralateral side, and improved brain kinetics compared with our previously developed radiotracers. Metabolite study of [¹⁸F]7d showed 93% of unchanged form in the ischemic brain at 30 min after injection. Moreover, PET study with [¹⁸F]7d provided a clear tumor image in a glioma-bearing rat model. We demonstrated that [¹⁸F]7d is a useful PET radiotracer for visualizing not only neuroinflammation but also glioma and will translate this radiotracer to a “first-in-human” study in our facility.

Full text of DOI:10.1021/acs.jmedchem.7b00374

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Article
Development of a 18F-labeled radiotracer with improved
brain kinetics for positron emission tomography imaging of
translocator protein (18 kDa) in ischemic brain and glioma
Masayuki Fujinaga, Rui Luo, Katsushi Kumata, Yiding Zhang, Akiko Hatori, Tomoteru Yamasaki, Lin Xie,
Wakana Mori, Yusuke Kurihara, Masanao Ogawa, Nobuki Nengaki, Feng Wang, and Ming-Rong Zhang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017
Downloaded from http://pubs.acs.org on April 20, 2017
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Development of a F-Labeled Radiotracer with Improved Brain Kinetics
for Positron Emission Tomography Imaging of Translocator Protein (18
kDa) in Ischemic Brain and Glioma
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†
,§
‡,§
†,§
†
†
Masayuki Fujinaga , Rui Luo , Katsushi Kumata , Yiding Zhang , Akiko Hatori ,
†
†
†
†,¶
†,¶
Tomoteru Yamasaki , Lin Xie , Wakana Mori , Yusuke Kurihara , Masanao Ogawa ,
†
,¶
‡,
†,
Nobuki Nengaki , Feng Wang *, and MingꢀRong Zhang *
†
Department of Radiopharmaceuticals Development, National Institute of Radiological
Sciences, National Institutes for Quantum and Radiological Science and Technology, 4ꢀ9ꢀ1
Anagawa, Inageꢀku, Chiba 263ꢀ8555, Japan.
‡
Department of Nuclear Medicine, Nanjing First Hospital, Affiliated to Nanjing Medical
University, 68 Chanle Road, Nanjing 210006, China.
¶
SHI Accelerator Service Co., 1ꢀ17ꢀ6 Osaki, Shinagawaꢀku, Tokyo 141ꢀ0032, Japan.
§
These authors contributed equally to this work.
*
Corresponding authors.
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ABSTRACT
We designed four novel acetamidobenzoxazolone compounds 7a–d as candidates for positron
emission tomography (PET) radiotracers for imaging the translocator protein (18 kDa, TSPO)
in
ischemic
brain
and
glioma.
Among
these
compounds,
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2ꢀ(5ꢀ(6ꢀfluoropyridinꢀ3ꢀyl)ꢀ2ꢀoxobenzo[d]oxazolꢀ3(2H)ꢀyl)ꢀNꢀmethylꢀNꢀphenylacetamide
(7d) exhibited high binding affinity (K
i
= 13.4 nM) with the TSPO and moderate lipophilicity
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(LogD: 1.92). [ F]7d was radiosynthesized by [ F]fluorination of the bromopyridine
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ꢀ
precursor 7h with [ F]F in 12 ± 5% radiochemical yield (n = 6, decayꢀcorrected). In vitro
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autoradiography and PET studies of ischemic rat brain revealed higher binding of [ F]7d
with TSPO on the ipsilateral side as compared to the contralateral side, and improved brain
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kinetics than our previously developed radiotracers. Metabolite study of [ F]7d showed 93%
of unchanged form in the ischemic brain at 30 min after injection. Moreover, PET study with
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[
F]7d provided a clear tumor image in a gliomaꢀbearing rat model. We demonstrated that
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[ F]7d is a useful PET radiotracer for visualizing not only neuroinflammation but also
glioma and will translate this radiotracer to a “firstꢀinꢀhuman” study in our facility.
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INTRODUCTION
Translocator protein (TSPO) is an 18ꢀkDa outer mitochondrial membrane protein that
regulates numerous cellular processes including cholesterol, porphyrin, heme, and anion
transport; steroid hormone synthesis; apoptosis; cell proliferation; mitochondrial function;
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immunomodulation; and inflammation. Under normal physiological conditions, TSPO is
highly expressed in steroidꢀproducing and mitochondriaꢀenriched tissues such as myocardium,
renal tissue, and skeletal muscle, whereas tissues such as brain and liver have relatively low
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expression. Elevated TSPO expression is implicated in many inflammatory diseases and
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neuroinflammation as well as in various malignancies including glioma,
astrocytoma,
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breast cancer, colorectal cancer, and hepatoma . In some TSPOꢀoverexpressing tumor
cells, TSPO mediates cholesterol transport into the nucleus, which has been linked to cell
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proliferation.
A large number of positron emission tomography (PET) studies have visualized TSPO
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in neuroinflammation,
peripheral inflammatory diseases,
and cancer
. Many PET
studies used TPSOꢀspecific radiotracers to monitor activated microglia, which overexpress
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TSPO during neuroinflammation.
In fact, TSPO is associated with pathological
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conditions such as Alzheimer’s and Parkinson’s diseases that involve neuroinflammation.
In addition to neuroinflammation imaging, TSPO expression is also emerging as an important
prognostic biomarker in oncology, suggests the imaging utility of TSPOꢀPET for tumor
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detection and diagnosis.
The first
ꢀ(2ꢀchlorophenyl)ꢀNꢀ[ C]methylꢀNꢀ(1ꢀmethylpropyl)ꢀ3ꢀisoquinolinecarboxamide ([ C]PK
PET
tracer
for
TSPO,
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11195, [ C]1) (Figure 1) was used to detect glioblastoma in a patient. PET studies with
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[ C]1 were widely used to visualize Alzheimer’s disease and other neurodegenerative
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diseases.
However, [ C]1 has several limitations including relatively low brain
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uptake, high nonꢀspecific binding, high lipophilicity, and an unfavourable metabolic profile
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for PET imaging. For instance, quantitation of [ C]1 was made difficult by a low
signalꢀtoꢀnoise ratio, which was likely due to relatively high level of nonꢀspecific binding in
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Several dozen C and Fꢀlabeled tracers have been developed for precise
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visualization of TSPO in inflammatory diseases and tumors by PET.
The clinically
used
TSPO
PET
tracers
(Figure
1)
mainly include:
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Nꢀ(2ꢀ[ C],5ꢀdimethoxybenzyl)ꢀNꢀ(5ꢀfluoroꢀ2ꢀphenoxyphenyl)acetamide
([ C]DAA1106,
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[ C]2a),
Nꢀ(5ꢀfluoroꢀ2ꢀphenoxyphenyl)ꢀNꢀ(2ꢀ[ F]fluoroethoxyꢀ
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ꢀmethoxybenzyl)acetamide ([ F]FEDAA1106, [ F]2b),
Nꢀ(2ꢀ[ C]methoxybenzyl)ꢀ
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Nꢀ(4ꢀphenoxypyridinꢀ3ꢀyl)acetamide
([ C]PBR28,
[ C]2c),
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Nꢀ[ F]fluoroacetylꢀNꢀ(2,5ꢀdimethoxybenzyl)ꢀ2ꢀphenoxyaniline ([ F]PBR06, [ C]2d),
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N,Nꢀdiethylꢀ2ꢀ[2ꢀ(4ꢀ[ C]methoxyphenyl)ꢀ5,7ꢀdimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀyl]acetam
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ide
([ C]DPAꢀ713,
[ C]3a),
N,Nꢀdiethylꢀ2ꢀ[2ꢀ(4ꢀ[ F]fluoroethoxyphenyl)ꢀ
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5,7ꢀdimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀyl]acetamide
([ F]DPAꢀ714,
[ F]3b),
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NꢀbenzylꢀNꢀethylꢀ2ꢀ(7,8ꢀdihydroꢀ7ꢀ[ C]methylꢀ8ꢀoxoꢀ2ꢀphenylꢀ9Hꢀpurinꢀ9ꢀyl)acetamide
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(
[ C]ACꢀ5216,
[ C]4a),
NꢀbenzylꢀNꢀmethylꢀ2ꢀ(7,8ꢀdihydroꢀ7ꢀ[ F]fluoroethylꢀ
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8ꢀoxoꢀ2ꢀphenylꢀ9Hꢀpurinꢀ9ꢀyl)acetamide ([ F]FEDAC, [ F]4b),
2ꢀ(6ꢀchloroꢀ2ꢀ(4ꢀ
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(
(
3ꢀ[ F]fluoropropoxy)phenyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀN,Nꢀdiethylacetamide
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[ F]PBR111,
[ F]5a),
N,Nꢀdiꢀnꢀpropylꢀ2ꢀ[2ꢀ(4ꢀ[ C]methoxyphenyl)ꢀ
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6,8ꢀdichloroimidazol[1,2ꢀa]pyridineꢀ3ꢀyl]acetamide
([ C]CB184,
[ C]5b),
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(
S)ꢀN,Nꢀdiethylꢀ9ꢀ(2ꢀ[ F]fluoroethyl)ꢀ5ꢀmethoxyꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀcarbazoleꢀ
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4ꢀcarboxamide ([ F]GEꢀ180, [ F]5c),
and (R)ꢀNꢀsecꢀbutylꢀ4ꢀ(2ꢀchlorophenyl)ꢀ
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Nꢀ[ C]methylquinazolineꢀ2ꢀcarboxamide ([ C]ER176, [ C]5d)
.
Most of these
radiotracers bind to TSPO with high in vitro affinity and have higher signalꢀtoꢀnoise ratios
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than [ C]1 in small animals; however, they often exhibit low in vivo specific binding and
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slow brain kinetics in human brain. Moreover, PET with certain radiotracers such as [ C]2c
showed variable binding potential among individuals: highꢀaffinity binders, lowꢀaffinity
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binders, and those with mixedꢀaffinities.
On the other hand, in tumor, particularly glioma
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imaging studies, challenging points were observed with some radiotracers such as [ F]3b.
This radiotracer showed a definite level of nonꢀspecific uptake accumulation in normal brain
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due to metabolism, which prevented detection of glioma with modest TSPO expression.
Figure 1
We
recently
reported
the
development
of
a
new
PET
tracer,
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ꢀ[5ꢀ(4ꢀ[ C]methoxyphenyl)ꢀ2ꢀoxoꢀ1,3ꢀbenzoxazolꢀ3(2H)ꢀyl]ꢀNꢀmethylꢀNꢀphenylacetamide
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(
[ C]MBMP, [ C]6a) (Figure 2), and its [ F]fluoroethyl derivative ([ F]FEBMP, [ F]6b)
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for imaging TSPO in the brain.
Both radiotracers, which are acetamidobenzoxazolone
analogs, showed high in vitro and in vivo specific bindings with TSPO in the ischemic rat
brains. Furthermore, in vitro autoradiography on postmortem human brains demonstrated that
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[
F]6b binding sites were unaffected by TSPO rs6971 and exhibited similar response for
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TSPO in “binder” and “nonꢀbinder” brain sections. However, like many TSPO radiotracers,
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[
C]6a and [ F]6b had slow brain kinetics, without significant decreases in radioactivity
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during the PET scan.
This was partly attributed to the high lipophilicity (LogD: 3.4 and
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.4) and potent binding affinity (K : 3.9 nM and 6.6 nM for TSPO) of 6a and 6b. Moreover,
i
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radiolabeled metabolites (> 20% of total radioactivity) of [ C]6a and [ F]6b were detected
in the brain, which limits the applicability of these tracers in humans.
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In this study, we designed four new acetamidobenzoxazolone analogs ([ C]7a–c and
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[ F]7d) using [ C]6a as the lead compound (Figure 2) and investigated their potential as
PET tracers for in vivo imaging of TSPO. We evaluated whether these tracers were superior
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to [ C]1, [ C]6a, and [ F]6b in terms of brain kinetics and in vivo metabolism and
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performed PET studies for visualization of TSPO not only in ischemic brain but also in
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glioma. A pyridine ring was introduced into [ C]7a–c and [ F]7d in place of the benzene
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ring in [ C]6a. This structural change could decrease their lipophilicity, and we therefore
expected these radiotracers to show more rapid brain kinetics and reduced nonꢀspecific
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binding in target tissues. We also predicted that [ F]7d would exhibit improved metabolism
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since the radiolabeling position was different from those in [ C]6a and [ C]7a–c. Moreover,
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the longer halfꢀlife of F (110 min vs. 20 min for C) would allow time for transportation to
distant research and clinical facilities.
Figure 2
Here, we report: 1) chemical synthesis of novel unlabeled compounds 7a–d; 2)
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radiosynthesis of [ C]7a–c and [ F]7d; 3) in vitro binding affinity with TSPO and
lipophilicity; 4) in vitro and in vivo specific binding for TSPO and kinetics in the ischemic rat
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brain when used for autoradiography and PET; 5) metabolite analysis of [ C]7c and [ F]7d
in the plasma and ischemic rat brain; and 6) visualization of glioma in rat brain by PET using
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[
F]7d.
RESULTS AND DISCUSSION
Chemistry. Compounds 7a–d and their corresponding precursors 7e–h for radiolabeling
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were synthesized (Scheme 1). The reaction of bromobenzoxazolone acetic acid 8 with
ꢀmethylaminopyridine, 4ꢀmethylaminopyridine, or Nꢀmethylaniline in the presence of
hydroxybenzotriazole (HOBt) and 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide
2
hydrochloride (WSC) produced acetamidobenzoxazolones 9–11 with 44–72% yield. The
coupling reaction of compound 9 or 10 with phenylboronic acid derivatives in the presence of
palladium catalyst produced 7a, 7b, 7e, or 7f with 11–62% yield. Compound 11 coupled with
pyridinylboronic acid derivatives, giving rise to 7c, 7d, or 7g with 52–70% yield, while
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reaction of 11 with 2ꢀbromopyridineꢀ5ꢀboronic acid generated 7h with 6% yield. Due to the
bromo groups separately presented in 11 and the boron reagent, the coupling reaction
generated a complex mixture, from which a small amount of 7h was separated, which was
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nonetheless sufficient for radiolabeling and evaluation of [ F]7d.
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Scheme 1
11
Radiochemistry. Radiosynthesis of [ C]7a–c was carried out using a homeꢀmade
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automated synthesis system. The labeling agent [ C]methyl iodide ([ C]MeI) was prepared
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by reducing cyclotronꢀproduced [ C]CO
2
with lithium aluminum hydride, followed by
11
iodination with 57% hydroiodic acid. The resultant [ C]MeI was purified by distillation and
trapped in a N,Nꢀdimethylformamide (DMF) solution of the desmethyl phenol precursors
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7
e–g and NaOH (Scheme 2). The [ C]methylation of 7e–g with [ C]MeI proceeded
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efficiently for 5 min at 80 °C. HPLC purification for each reaction mixture produced [ C]7a,
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11
[
C]7b, and [ C]7c with radiochemical yields (decayꢀcorrected) of 36 ± 7% (n = 8), 45 ± 6%
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(
n = 6), and 27 ± 7% (n = 22), respectively, based on [ C]CO . Starting with 13.0–18.5 GBq
2
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2
of [ C]CO , 1.3–3.2 GBq of [ C]7a–c was produced with 25–31 min (n ≥ 6 for each
product) of synthesis times from the end of bombardment (EOB).
Scheme 2
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[
F]7d was radiosynthesized using another homeꢀmade automated synthesis system. A
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–
mixture of the 2ꢀbromopyridine precursor 7h and dry [ F]F in dimethyl sulfoxide (DMSO)
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was heated at 130 °C for 10 min. After optimizing the HPLC conditions, [ F]7d was easily
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separated from unreacted 7h and other chemical impurities; 0.60–1.04 GBq of [ F]7d was
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8
ꢀ
then produced from 8.9 GBq of [ F]F over a period of 65 ± 6 min (n = 6) of synthesis times
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from EOB. The radiochemical yield of [ F]7d was 12 ± 5% (n = 6, decayꢀcorrected) based
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on [ F]F .
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The identities of the four radioactive products were confirmed by HPLC by coꢀinjecting
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unlabeled 7a–d. In the final formulated solutions, the radiochemical purity of [ C]7a–c and
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[
F]7d exceeded 98% and their specific activities were 70–160 ( C) and 360–420 ( F)
GBq/ mol, respectively, at the end of synthesis (EOS), as determined by comparing the
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assayed radioactivity with the mass measured from the carrier UV peak in the HPLC
chromatogram. There were no peaks corresponding to precursors 7e–h observed in the HPLC
chromatograms of the final products, which also did not show radiolysis at room temperature
11
18
within 90 min ([ C]7a–c) and 180 min ([ F]7d) after formulation, indicating that they were
radiochemically stable within the time frame of at least one PET scan. These analytical
results were consistent with our inꢀhouse quality specifications of radiopharmaceuticals.
In Vitro Binding Assay. We measured in vitro binding affinity (K
i
) of compounds
7
a–d for TSPO in rat brain by assaying competitive binding to the TSPOꢀspecific radiotracer
11
64
[
C]1 within the concentration range of 0.3 nM to 3 µM. Specific binding was > 80% for
all tested compounds; measured K values are shown in Table 1. Compounds 7a, 7c, and 7d
i
showed high binding affinity (20.1, 15.5, and 13.4 nM, respectively) for TSPO, although
these affinities were weaker than those of 1 (4.4 nM) and 6a (3.9 nM). As previously reported,
a pharmacophore model study showed that TSPO binding requires three hydrophobic groups
6
6
and a hydrogen bond acceptor group. Substituting a lipophilic benzene ring in 6a with a
hydrophilic pyridine ring thus decreased the affinity of 7a–d for TSPO. In particular, a
marked decrease in binding affinity was observed for 7b due to the replacement of the
benzene ring with a 4ꢀpyridinyl ring. This result suggested that the lipophilic property
associated with the 4 position of the 6a benzene ring is critical determinant of TSPO binding
affinity. Although 7a, 7c, and 7d had lower affinity for TSPO than 1 and 6a, we considered
that their affinity was sufficient for the purposes of the present imaging study based on our
67
understanding of and experience with TSPO. Moreover, we expected these radiotracers to
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show improved brain kinetics in vivo by more readily releasing from TSPO in the brain, than
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[
C]1 and [ C]6a. On the other hand, compounds 7a–d did not exhibit appreciable binding
to central benzodiazepine receptor (K > 1 µM).
Table 1
Computation and Measurement of Lipophilicity. The calculated lipophilicity values
i
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55
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60
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at pH = 7.4 (cLogD) for [ C]7a–c and [ F]7d ranged from 2.35 to 3.00 (Table 1). These
48,68
differed from the LogD values (1.92–3.04) measured by the shake flask method.
All the
11
18
measured values for [ C]7a–c and [ F]7d are in the range normally considered as suitable
6
9
PET tracers. The presence of a hydrophilic pyridine ring reduced the lipophilicity, which
may reduce nonꢀspecific binding by accelerating the clearance of radioactivity from
nonꢀtarget sites.
In Vitro Autoradiography of Ischemic Rat Brain. TSPO is expressed at low levels
5
4,70
in normal rat brain; we therefore used a wellꢀcharacterized ischemic rat brain model
to
11
18
evaluate the in vitro and in vivo specific binding of [ C]7a–c and [ F]7d for TSPO. In this
model, neuroinflammation accompanied by microglial activation increases TSPO levels in
70
the ischemic areas, such as the cerebral cortex and striatum. Figure 3 shows the in vitro
11
11
11
autoradiograms and result of the quantitative analysis of [ C]7a (A), [ C]7b (B), [ C]7c (C),
18
11
11
18
and [ F]7d (D) in the ischemic rat brain. In control sections for [ C]7a, [ C]7c, and [ F]7d,
radioactivity was higher on the ipsilateral side (arrow) as compared to the contralateral side,
11
whereas no difference was observed between the two sides for [ C]7b. The observed
differences in radioactivity level between the ipsilateral and contralateral sides for the all
radiotracers were abolished by coꢀincubation with an excess of unlabeled 7a–d or 1.
Figure 3
Quantitative analyses of the autoradiograms revealed that the averaged concentration
11
(
binding) ratios between the ipsilateral and contralateral sides were 12.0 ± 1.0 for [ C]7a, 1.2
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± 0.1 for [ C]7b, 21.6 ± 3.9 for [ C]7c, and 29.8 ± 3.1 for [ F]7d, respectively. These ratios
decreased to approximately 1 in the presence of unlabeled 7a–d or 1. Coꢀincubation with
11
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7
a–d or 1 reduced binding on the ipsilateral side to < 10% of the control for [ C]7a, [ C]7c,
18
11
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and [ F]7d. These results demonstrate that [ C]7a, [ C]7c, and [ F]7d specifically bind to
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TSPO in the ischemic rat brain. The low level of specific binding of [ C]7b to TSPO was
i
due to its low in vitro binding affinity (K = 198 nM; Table 1). Based on these results, we did
11
not further evaluate [ C]7b in our in vivo PET imaging studies.
PET Imaging Studies in Ischemic Rat Brains. Figures 4–6 show representative PET
images and timeꢀactivity curves (TACs) in the ischemic rat brains after the injection of
11
11
18
[ C]7a, [ C]7c, or [ F]7d. Higher uptake of radioactivity was observed on the ipsilateral
1
1
11
sides, allowing clear visualization of the ischemic areas ([ C]7a, Figure 4A; [ C]7c, Figure
18
5
A; [ F]7d, Figure 6A). The TACs revealed that the maximum uptake of radioactivity on the
1
1
ipsilateral sides was 1.72 ± 0.08 standardized uptake value (SUV) for [ C]7a (Figure 4D),
11
18
0.98 ± 0.03 SUV for [ C]7c (Figure 5D), and 1.70 ± 0.16 SUV for [ F]7d (Figure 6D). A
low and constant uptake (0.19–0.40 SUV) was observed on the contralateral sides starting 10
min after each radiotracer injection. At 10–15 min after the injection, uptake ratios between
the ipsilateral and contralateral sides reached the maximum values (2.73, 3.55, and 4.20 for
11
11
18
[
C]7a, [ C]7c, and [ F]7d, respectively) (Table 2).
Figures 4–6
Table 2
The PET quantitative value of radiotracer bound to receptor is presented as the
nonꢀdisplaceable binding potential (BPND), which was nonꢀinvasively obtained by simplified
71
reference tissue model (SRTM) analysis using the contralateral side as the reference region.
11
11
18
The BPND values of [ C]7a, [ C]7c, and [ F]7d for the ipsilateral sides are shown in Table
18
11
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2
. [ F]7d had the highest value (2.33) followed by [ C]7c and [ C]7a in that order.
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We previously carried out a PET analysis with [ C]1 in the ischemic rat brain under the
6
3
11
same experimental conditions as used here. In that study, [ C]1 uptake was higher on the
ipsilateral than that on the contralateral side. Initially, the maximum radioactivity uptake was
6
3
1
.00 ± 0.04 SUV (n = 4) on the ipsilateral side, and the maximum uptake ratio (1.80) of
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uptake between the ipsilateral and contralateral sides was observed at 60 min after the
63
injection, with a BPND value on the ipsilateral side of 1.24 (Table 2).
Displacement studies using unlabeled 7a (Figure 4B, E), 7c (Figure 5B, E), 7d (Figure
11
11
18
6
B, E), or 1 (Figure 4C, F; Figure 5C, F; Figure 6C, F) for [ C]7a, [ C]7c, or [ F]7d 10
min after radiotracer injection minimized the difference in signal between the ipsilateral and
contralateral sides in PET images. This displacement first produced a transient increase in
uptake in the contralateral side, which was due to the flow of radioactivity from the
TSPOꢀenriched peripheral organs, such the heart, lung, and kidney. Despite the transient
increase, the radioactivity uptake at 20 min after injection was reduced to a low level. This
result would be consistent with the contralateral side having very little specific binding. From
20 min after injection to the end of the PET scans, there was no difference in radioactivity
uptake between the two sides, indicating that displacement by unlabeled compounds reduced
binding on the ipsilateral side. This provides evidence for high in vivo specific binding of
11
11
18
[
C]7a, [ C]7c, and [ F]7d for TSPO in the ischemic brain.
11
18
[ C]7c and [ F]7d showed higher uptake ratios of the ipsilateral to contralateral sides
11
and higher BPND values than [ C]1 (Table 2). Moreover, the maximum ratios between the
11
18
two sides were achieved at 10–15 min after injection for [ C]7c and [ F]7d, as compared to
11
6
0 min for [ C]1. We presumed that the moderate binding affinity for TSPO and
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18
lipophilicity of [ C]7c and [ F]7d were the main reasons for their improved brain kinetics
1
1
63 11
63 18
65
47,48,53
relative to [ C]1, [ C]6a, [ F]6b, and other TSPO radiotracers
developed by our
group. The improved kinetics is important for quantification of targeted brain receptors by
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PET.
Detection of Radiolabeled Metabolites by HPLC. Given the high BPND values of
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[
C]7c and [ F]7d determined in the ischemic brains, we analyzed their metabolites in
plasma and ischemic rat brain by radioꢀHPLC (Figure 7). Over 95% of radioactivity from the
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samples was recovered in the HPLC analysis. In plasma, the percentage of unchanged [ C]7c
[
retention time (t ): 8.0 min] rapidly decreased. A polar radiolabeled metabolite (t : 2.8 min)
R
R
was detected in plasma; a radiolabeled metabolite was also detected in the ischemic brain,
accounting for 62% of total radioactivity at 30 min after injection. Plasma and brain
metabolites had similar t values. Coꢀinjection of the two radioactive fractions into an
R
analytic HPLC column yielded a single peak, confirming that there was a single metabolite.
This metabolite might enter the brain, making it difficult to quantify TSPO by PET. We
11
therefore did not further evaluate [ C]7c.
Figure 7
18
[ F]7d rapidly degraded in plasma; a polar metabolite (t : 2.4 min) was detected at 10
R
11
18
and 30 min after injection. In contrast to [ C]7c, 95% and 93% of [ F]7d (t
R
: 4.3 min) in the
brain was intact at 10 and 30 min, respectively, indicating that the in vivo metabolic route of
18
11
18
[
F]7d was different from that of [ C]7c. The polar metabolite of [ F]7d in the plasma may
be unable to penetrate the bloodꢀbrainꢀbarrier or, in the affirmative case, may not persist in
the brain due to its hydrophilicity, indicating that the in vivo specific binding observed in the
1
8
ischemic rat brain was attributable to [ F]7d itself. Since only trace amounts of the
radiolabeled metabolites were present in the brain, we did not confirm its identity.
18
Biodistribution. We investigated the distribution of [ F]7d in bone and in the whole
18
body of normal mice (Table 3). Intravenous injection of [ F]7d into mice resulted in a high
uptake of radioactivity (71% to 12% injected dose per gram of wet tissue, % ID/g) in the
lungs, heart, and kidneys, which are the organs with high TSPO expression. Medium and low
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levels of radioactivity were observed in the small intestine, muscle, liver, spleen, and testis,
with uptake values from 0.6% ID/g to 2.9% ID/g. The radioactivity concentration in the
blood decreased 5 min after injection and remained at a constant level (0.5–0.7% ID/g),
possibly due to continuous release of radiolabeled metabolite from the liver or from the
enterohepatic circulation. A relatively high initial uptake (1.8% ID/g) was observed in the
mouse brain, the target tissue in this study, and the radioactivity level decreased to
approximately 15% of the initial peak value 60 min after injection. On the other hand, low
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48
bone uptake is a requirement for most useful Fꢀlabeled radiotracers. During the initial
18
phase after [ F]7d injection, relatively low radioactivity (1.2% ID/g) was detected in the
thighbones; this value increased to 2.3% ID/g at 15 min (Table 3). By comparison, 30 min
1
1
after injection of [ C]7c into mice, a moderate level (2.0% ID/g) of radioactivity was also
18
observed in the bone (Supplement Table 2). These results indicate that [ F]7d does not
induce significant defluorination in vivo.
Table 3
1
8
PET Imaging Studies in Glioma. To further evaluate the applicability of [ F]7d for
18
in vivo visualization of TSPO, we carried out PET using [ F]7d to detect glioma in a rat
24ꢀ26
model, in which C6 glioma cells were implanted into the hemisphere of rat brain
.
Previous studies using this model have reported that TSPO expression was upregulated on the
24ꢀ26
tumor side of the brain bearing glioma.
18
Figure 8A shows PET images of gliomaꢀbearing rat brain after injection of [ F]7d. A
high radioactivity uptake in the brain was detected on the tumor (ipsilateral) side, whereas the
1
8
contralateral hemisphere showed a low signal. The tumorꢀselective uptake of [ F]7d
provided a good imaging contrast of radioactive signals between the ipsilateral and
contralateral sides. The radioactivity accumulated steadily in the tumor and was rapidly
cleared from the contralateral side (Figure 8C). Using this side as a reference, the BPND value
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of [ F]7d on the ipsilateral side was determined to be 2.29 ± 0.79. The maximum uptake
ratio between the ipsilateral and contralateral sides was 4.20 ± 1.09, which was observed at
4
5–50 min after radiotracer injection.
Figure 8
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Blocking experiment with 1 at 1 min before [ F]7d injection minimized the radioactive
signal contrast between the tumor and contralateral sides, as revealed by PET images (Figure
8
B) and TACs (Figure 8D). Thus, treatment with 1 reduced the in vivo specific binding of
18
[
F]7d in the tumor area, indicating that the radioactivity reflected TSPO overexpression in
the glioma.
CONCLUSIONS
In this study, we developed four radiotracers for imaging TSPO in the rat brains. Of these
18
compounds, [ F]7d showed the highest in vitro binding affinity for TSPO. In vitro
18
autoradiography experiments demonstrated that [ F]7d bound to TSPO with high specificity
18
in the ischemic rat brain. PET study with [ F]7d confirmed its distribution and specificity for
18
TSPO in the brain. [ F]7d showed superior in vivo brain kinetics than our TSPO radiotracers
11
previously developed and had higher BPND for TSPO in the ischemic brain than [ C]1. In
1
8
addition, there were no [ F]7d metabolites in the brain and no significant uptake of
1
8
radioactivity in the bone. Moreover, PET with [ F]7d enabled visualization of glioma by
providing sufficient contrast in the radioactive signals between the tumor and contralateral
18
sides. These results demonstrate that [ F]7d is a useful PET tracer not only for imaging of
neuroinflammation but also for glioma detection. We are currently performing PET imaging
1
8
studies with [ F]7d to detect other neuroinflammation, peripheral inflammation, and tumors
1
8
and to determine whether the [ F]7d binding site is affected by TSPO rs6971. We will use
this new radiotracer in human study at our facility.
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EXPERIMENTAL SECTION
All chemicals were purchased from commercial sources. Melting points (mp) were
determined using a Yanaco MPꢀ500P micromelting point apparatus (Yanaco New Science,
10
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1
Kyoto, Japan). H NMR (300 MHz) spectra were recorded using a JEOLꢀALꢀ300 NMR
spectrometer (JEOL, Tokyo, Japan) with tetramethylsilane 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). Highꢀresolution fast
atom bombardment mass spectra (HRMS) were acquired using a JEOL NMSꢀSX102 102A
18
11
spectrometer. Radioisotopes F and C were produced using a CYPRIS HMꢀ18 cyclotron
(
Sumitomo Heavy Industry, Tokyo, Japan). Silica gel column chromatography was performed
with a Cꢀ200 gel (70–230 mesh; Wako Pure Chemical Industries, Osaka, Japan). High
performance liquid chromatography (HPLC) separation and analysis were performed using a
JASCO HPLC system (JASCO, Tokyo, Japan). HPLC analysis was performed with a Capcell
Pak C18 column (4.6 mm i.d. × 250 mm, Shiseido, Tokyo) or Fluofix 120N column (4.6 mm
i.d. × 250 mm, Wako, Tokyo). The chemical purity of compounds 7a–h was ≥ 95% by HPLC
analysis. The analytic conditions were as follows: 7a–c, 7f, and 7g: 1.0 mL/min,
MeCN/H O/Et N (50/50/0.1); 7d and 7h: 1.0 mL/min, MeCN/H O/Et N (60/40/0.1); 7e: 1.0
2
3
2
3
11
18
mL/min, MeCN/H
2 3
O/Et N (55/45/0.1). Radiochemical purity of [ C]7a–c and [ F]7d was
analyzed by HPLC with a detector to monitor radioactivity under the following conditions:
11
11
11
[
C]7a: 1.0 mL/min, MeCN/H
2 3
O/Et N (55/45/0.1); [ C]7b and [ C]7c: 1.0 mL/min,
18
MeCN/H O/Et N (50/50/0.1); and [ F]7d: 1.0 mL/min, MeOH/H O (50/50). Effluent
2
3
2
radioactivity in radioꢀHPLC was monitored with a NaI (Tl) scintillation detector system.
Unless otherwise stated, radioactivity was measured with an IGCꢀ3R Curiemeter (Aloka,
11
11
Tokyo, Japan). For the in vitro binding assay, [ C]PK 11195 ([ C]1) was synthesized by
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reacting a desmethyl precursor with [ C]MeI according to a previously reported method.
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The radiochemical purity and specific activity of [ C]1 were ≥ 98% and in the range of
0−100 GBq/µmol, respectively, at EOS.
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Chemical Synthesis
2-(5-(4-Methoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-2-yl)aceta
mide (7a). A mixture of 9 (362 mg, 1.0 mmol), 4ꢀmethoxyphenylboronic acid (182 mg, 1.2
mmol), Pd(PPh
3 4 2 3
) (80 mg, 0.07 mmol), and 1 M aqueous K CO (3 mL, 3.0 mmol) in
1
,4ꢀdioxane (25 mL) was heated at 100 °C for 9 h under a N atmosphere. The mixture was
2
poured into brine and extracted with AcOEt. The organic layer was washed with brine, dried
over Na SO , and evaporated under reduced pressure. Column chromatographic separation of
2
4
the residue on silica gel using hexane/AcOEt (4/1) followed by hexane/AcOEt (2/1) yielded
1
7
a (238 mg, 61.1%) as a white powder. mp. 135–136 °C. H NMR (CDCl
3
): δ 3.43 (3H, s),
3
.85 (3H, s), 4.82 (2H, s), 6.94–6.99 (2H, m), 7.11 (1H, d, J = 1.5 Hz), 7.20–7.33 (4H, m),
7.45–7.50 (2H, m), 7.83 (1H, td, J = 1.7, 7.9 Hz), 8.50 (1H, dt, J = 1.0, 4.0 Hz). HRMS m/z:
3
20 3 4
90.1475 (calculated for C22H N O : 390.1454).
2
-(5-(4-Methoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-4-yl)aceta
mide (7b). A mixture of 10 (362 mg, 1.0 mmol), 4ꢀmethoxyphenylboronic acid (182 mg,
1.2 mmol), Pd(PPh (80 mg, 0.07 mmol), and 1 M aqueous K CO (3 mL, 3.0 mmol) in
,4ꢀdioxane (25 mL) was heated at 100 °C for 6 h under a N atmosphere. The miture was
3
)
4
2
3
1
2
poured into brine and extracted with AcOEt. The organic layer was washed with brine, dried
over Na SO , and evaporated under reduced pressure. Column chromatographic separation of
2
4
the residue on silica gel using hexane/AcOEt (3/1) followed by hexane/AcOEt (1/1) yielded
1
7
b (125 mg, 32.1%) as a white powder. mp. 197–198 °C. H NMR (CDCl
3
): δ 3.37 (3H, s),
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3.84 (3H, s), 4.49 (2H, s), 6.94–7.01 (3H, m), 7.23–7.32 (4H, m), 7.44 (2H, d, J = 8.8 Hz),
20 3 4
8.73 (2H, d, J = 5.9 Hz). HRMS m/z: 390.1451 (calculated for C22H N O : 390.1454).
2
-(5-(5-Methoxypyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetami
(7c). mixture of 11 (72 mg, 0.2 mmol),
ꢀmethoxyꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridine (47 mg, 0.2 mmol),
PdCl (dppf) (10 mg, 0.012 mmol), and K CO (42 mg, 0.3 mmol) in dimethoxyethane
DME)/H O (5/1, 6 mL) was heated at 80 °C for 7 h under a N atmosphere. The mixture was
poured into brine and extracted with AcOEt. The organic layer was washed with brine, dried
over Na SO , and evaporated under reduced pressure. Column chromatographic separation of
the residue on silica gel using CH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
de
A
3
2
2
3
(
2
2
2
4
2
Cl
2
followed by CH
Cl
2 2
/MeOH (97/3) yielded 7c (41 mg,
1
5
2%) as a pale brown powder. mp. 207–208 °C (decomposition). H NMR (CDCl
3
): δ 3.33
(
3H, s), 3.94 (3H, s), 4.73 (2H, s), 7.02 (1H, s), 7.28 (2H, d, J = 1.1 Hz), 7.31–7.47 (3H, m),
7.50–7.61 (3H, m), 8.31 (1H, d, J = 2.9 Hz), 8.40 (1H, d, J = 1.8 Hz). HRMS m/z: 390.1489
(calculated for C22 : 390.1454).
20 3 4
H N O
2
-(5-(6-Fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamid
e (7d)
A mixture of 11 (180 mg, 0.5 mmol), 2ꢀfluoropyridineꢀ5ꢀboronic acid (85 mg, 0.6
mmol), Pd(PPh (58 mg, 0.05 mmol), and K CO (104 mg, 0.75 mmol) in DME/H O (3/1,
2 mL) was heated at 100 °C for 4 h under a N atmosphere. The mixture was poured into
4
brine and extracted with AcOEt. The organic layer was washed with brine, dried over Na SO ,
3
)
4
2
3
2
1
2
2
and evaporated under reduced pressure. Column chromatographic separation of the residue
on silica gel using hexane/AcOEt (1/1) yielded 7d (132 mg, 70%) as a white powder. mp.
1
2
20–222 °C. H NMR (CDCl
3
): δ 3.33 (3H, s), 4.36 (2H, s), 7.00 (1H, s), 7.01 (1H, dd, J =
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3.3, 8.1 Hz), 7.21–7.29 (2H, m), 7.35 (2H, d, J = 7.3 Hz), 7.43–7.55 (3H, m), 7.92 (1H, dt, J
=
2.9, 8.4 Hz), 8.37 (1H, d, J = 1.8 Hz). HRMS m/z: 378.1295 (calculated for C21
17 3 3
H O N F:
378.1254).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-(5-(4-Hydroxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-2-yl)aceta
mide
ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenol (365 mg, 1.66 mmol), PdCl
mg, 0.024 mmol), and K CO (480 mg, 3.45 mmol) in DME/H
at 80 °C for 8 h under a N atmosphere. The mixture was poured into brine and extracted with
(7e).
A
mixture
of
9
(500
mg,
1.38
mmol),
4
2
(dppf) (20
2
3
2
O (10/1, 11 mL) was heated
2
2 4
AcOEt. The organic layer was washed with brine, dried over Na SO , and evaporated under
reduced pressure. Column chromatographic separation of the residue using silica gel under
CH
2
Cl
2
/MeOH (100/1) followed by CH
2
Cl
2
/MeOH (97/3) yielded 7e (320 mg, 61.8%) as a
): δ 3.41 (3H, s), 4.95 (2H, s), 6.85
1
pale brown powder. mp. 209–210 °C. H NMR (DMSOꢀd
6
(2H, d, J = 8.8 Hz), 7.30–7.38 (3H, m), 7.46–7.52 (3H, m), 7.64 (1H, d, J = 8.1 Hz), 7.93 (1H,
t, J = 7.0 Hz), 8.52 (1H, d, J = 3.3 Hz), 9.54 (1H, s). HRMS m/z: 376.1304 (calculated for
21 18 3 4
C H N O : 376.1297).
2-(5-(4-Hydroxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-4-yl)aceta
mide
ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenol (365 mg, 1.66 mmol), PdCl
mg, 0.024 mmol), and K CO (480 mg, 3.45 mmol) in DME/H
at 80 °C for 10 h under a N atmosphere. The mixture was poured into water and extracted
with AcOEt. The organic layer was washed with brine, dried over Na SO , and evaporated
(7f).
A
mixture
of
10
(500
mg,
1.38
mmol),
4
2
(dppf) (20
2
3
2
O (10/1, 11 mL) was heated
2
2
4
under reduced pressure. Column chromatographic separation of the residue on silica gel using
CH
2
Cl
2
/MeOH (100/1) followed by CH
2 2
Cl /MeOH (97/3) gave 7f (57.1 mg, 11.0%) as a pale
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7
8
9
1
brown solid. mp. 260–261 °C. H NMR (DMSOꢀd
6
): δ 3.40 (3H, s), 4.86 (2H, s), 6.86 (2H, d,
J = 8.4 Hz), 7.30–7.39 (2H, m), 7.47–7.57 (5H, m), 8.63 (2H, d, J = 5.9 Hz), 9.55 (1H, s).
18 3 4
HRMS m/z: 376.1293 (calculated for C21H N O : 376.1297).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-(5-(5-Hydroxypyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetami
de (7g). mixture of 11 (72 mg, 0.2 mmol),
ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ3ꢀpyridinol (44 mg, 0.2 mmol), PdCl (dppf)
10 mg, 0.012 mmol), and K CO (61 mg, 0.44 mmol) in DME/H O (5/1, 6 mL) was heated
at 80 °C for 8 h under a N atmosphere. The reaction mixture was poured into brine and
A
5
2
(
2
3
2
2
extracted with AcOEt. The organic layer was washed with brine, dried over Na
2 4
SO , and
evaporated under reduced pressure. Column chromatographic separation of the residue on
silica gel using CH
2
Cl
2
followed by CH
2
Cl
2
/MeOH (97/3) yielded 7g (46 mg, 61%) as a pale
1
brown powder. mp. 222–223 °C (decomposition). H NMR (DMSOꢀd
6
): δ 3.21 (3H, s), 4.40
(2H, s), 7.39–7.61 (9H, m), 8.14 (1H, s), 8.35 (1H, s), 10.07 (1H, s). HRMS m/z: 376.1342
(calculated for C21 : 376.1297).
18 3 4
H N O
2
-(5-(6-Bromopyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamid
e (7h). A mixture of 11 (864 mg, 2.4 mmol), 2ꢀbromopyridineꢀ5ꢀboronic acid (585 mg, 2.9
mmol), PdCl (dppf) (120 mg, 0.14 mmol), and K CO (504 mg, 3.6 mmol) in DME/H O (5/1,
7 mL) was heated at 80 °C for 5 h under a N atmosphere. The mixture was then poured into
brine and extracted with CH Cl . The organic layer was washed with brine, dried over
Na SO , and evaporated under reduced pressure. Column chromatographic separation of the
2
2
3
2
2
2
2
2
2
4
residue on silica gel using hexane/AcOEt (2/1) yielded 7h (28 mg, 6%) as a white powder.
1
mp. 239–241 °C. H NMR (CDCl
3
): δ 3.33 (3H, s), 4.37 (2H, s), 6.99 (1H, d, J = 1.5 Hz),
7.23–7.37 (4H, m), 7.46–7.57 (4H, m), 7.70 (1H, dd, J = 2.7, 8.3 Hz), 8.54 (1H, d, J = 1.8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
17 3 3
Hz). HRMS m/z: 438.0450 (calculated for C21H O N Br: 438.0453).
2-(5-Bromo-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-2-yl)acetamide (9). A
66
mixture of 5ꢀbromoꢀ2ꢀoxobenzo[d]oxazole (8, 3.27g, 12.0 mmol), HOBt (1.62 g, 12.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mmol), 2ꢀmethylaminopyridine (1.56 g, 14.4 mmol), and WSC (3.45 g, 18.0 mmol) in DMF
(
50 mL) was stirred at room temperature for 3 h. After removing DMF under reduced
pressure, the residue was combined with saturated NaHCO solution and then extracted with
AcOEt. The organic layer was washed with brine, dried over MgSO , and evaporated under
reduced pressure. The residue was purified by column chromatography using CH Cl ,
3
4
2
2
1
yielding 9 (1.94 g, 44.5%) as a colorless solid. mp 180–182 °C. H NMR (CDCl
s), 4.78 (2H, s), 7.05–7.13 (2H, m), 7.21–7.29 (3H, m), 7.84 (1H, dt, J = 2.0, 7.8 Hz), 8.49
1H, d, J = 4.8 Hz). HRMS m/z: 362.0117 (calculated for C15 Br: 362.0140).
3
): δ 3.43 (3H,
(
13 3 3
H N O
2-(5-Bromo-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-4-yl)acetamide (10). A
mixture of 8 (3.27g, 12.0 mmol), HOBt (1.62 g, 12.0 mmol), 4ꢀmethylaminopyridine (1.56 g,
1
4.4 mmol), and WSC (3.45 g, 18.0 mmol) in DMF (50 mL) was stirred at room temperature
for 18 h. After removing DMF under reduced pressure, the residue was combined with
saturated NaHCO solution and then extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO , and evaporated under reduced pressure. The residue was
3
4
2 2
purified by column chromatography using CH Cl , yielding 10 (3.00 g, 69.1%) as a colorless
1
solid. mp. 166–167 °C. H NMR (CDCl ): δ 3.38 (3H, s), 4.44 (2H, s), 7.04–7.09 (2H, m),
3
7.20–7.23 (1H, m), 7.32 (2H, d, J = 6.2 Hz), 8.76 (2H, d, J = 6.2 Hz). HRMS m/z: 362.0148
(calculated for C H N O Br: 362.0140).
1
5
13
3
3
2-(5-Bromo-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (11). A mixture
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5
6
7
8
9
of 8 (3.48g, 12.6 mmol), HOBt (2.04 g, 15.1 mmol), Nꢀmethylaniline (1.63 ml, 15.1 mmol),
and WSC (3.62 g, 18.9 mmol) in DMF (50 mL) was stirred at room temperature for 18 h.
After removal of DMF under reduced pressure, the residue was added to saturated NaHCO
solution and then extracted with AcOEt. The organic layer was washed with brine, dried over
MgSO , and evaporated under reduced pressure. The residue was purified by column
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
2 2
chromatography using CH Cl , yielding 11 (3.28 g, 72.1%) as a colorless solid. mp.
1
1
27–128 °C. H NMR (CDCl ): δ 3.32 (3H, s), 3.86 (3H, s), 4.35 (2H, s), 6.96–7.00 (3H, m),
3
7.19–7.23 (2H, m), 7.34 (2H, d, J = 7.7 Hz), 7.41–7.54 (5H, m). HRMS m/z: 361.0209
(calculated for C H N O Br: 361.0188).
1
6
14
2
3
Radiosynthesis
11
2
-(5-(4-[ C]Methoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-2-yl)a
11
11
cetamide ([ C]7a). After irradiation, the cyclotronꢀproduced [ C]CO was bubbled into
2
0.4 M lithium aluminium hydride in anhydrous tetrahydrofuran (THF, 0.3 mL). After
evaporating the THF, the remaining complex was treated with 57% hydroiodic acid (0.3 mL)
11
to obtain [ C]MeI, which was distilled and immediately transferred under N
2
gas flow into a
solution of 7e (1.0 mg, 2.6 ꢀmol) and 0.5 M aqueous NaOH (7 ꢀL) in DMF (300 ꢀL) at –15
to –20 °C. When the radioactivity reached a plateau, the reaction mixture was heated at 80 °C
for 5 min. After completion of the reaction, 1.0 mL of the preparative HPLC mobile phase
was added into the reaction vial, and then the reaction solution was applied to the HPLC
system. HPLC separation was completed using a Capcell Pack C18 column (10 mm i.d. × 250
mm) with MeCN/H
2
O/Et
3
N (50/50/0.1) at 5.0 mL/min. The radioactive fraction
11
corresponding to [ C]7a (t : 9.0 min) was collected in a sterile flask containing polysorbate
R
80 (100 ꢀL) and 25% ascorbic acid (100 ꢀL), evaporated to dryness under vacuum,
redissolved in 3 mL of sterile normal saline, and passed through a 0.22 ꢀm Millipore filter
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
11
R
(Waters, Milford, MA, USA), yielding 3.2 GBq of [ C]7a. The identity of [ C]7a (t : 8.1
min) was confirmed by analytical HPLC with 7a. The synthesis time was 28 min from EOB;
11
radiochemical yield (decayꢀcorrected) was 43% based on [ C]CO
2
; radiochemical purity was
>
99%; and specific activity at EOS was 130 GBq/ꢀmol.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11
2
-(5-(4-[ C]Methoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-(pyridin-4-yl)a
11
11
cetamide ([ C]7b). [ C]MeI was transferred into a solution of 7f (1.0 mg, 2.7 ꢀmol) and
.5 M aqueous NaOH (7 ꢀL) in DMF (300 ꢀL) at –15 to –20 °C. The reaction mixture was
heated at 80 °C for 5 min. HPLC separation was performed using a Capcell Pack C column
0
18
(10 mm i.d. × 250 mm) with MeCN/H
2
O/Et
: 9.1 min) was collected and treated to obtain 3.1 GBq of
: 7.0 min) was confirmed by analytical HPLC with 7b.
The synthesis time was 30 min from EOB; radiochemical yield (decayꢀcorrected) was 50%
3
N (55/45/0.1) at 5.0 mL/min. The radioactive
11
fraction corresponding to [ C]7b (t
R
11
11
[
C]7b. The identity of [ C]7b (t
R
11
based on [ C]CO ; radiochemical purity was > 99%; and specific activity at EOS was 160
2
GBq/ꢀmol.
11
2
-(5-(5-[ C]Methoxypyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylac
11
11
etamide ([ C]7c). [ C]MeI was transferred into a solution of 7g (1 mg, 2.7 ꢀmol) and 0.5
M aqueous NaOH (7 µL) in DMF (300 µL) at –15 to –20 °C. The reaction mixture was
heated at 80 °C for 5 min. HPLC separation was carried out using a Capcell Pack C18 column
(
10 mm i.d. × 250 mm) with MeCN/H
2
O/Et
3
N (50/50/0.1) at 5.0 mL/min. The radioactive
11
fraction corresponding to [ C]7c (t
R
: 8.1 min) was collected and treated to obtain 2.4 GBq of
11
11
[ C]7c. The identity of [ C]7c (t : 7.6 min) was confirmed by analytical HPLC with 7c. The
R
synthesis time was 27 min from EOB; radiochemical yield (decayꢀcorrected) was 33% based
11
on [ C]CO
2
; radiochemical purity was > 99%; specific activity at EOS was 150 GBq/ꢀmol.
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5
6
7
8
9
1
8
2
-(5-(6-[ F]Fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylaceta
18
18
18
ꢀ
18
mide ([ F]7d). [ F]Fluorine anion ([ F]F ) was produced using a cyclotron by the O(p,
1
8
18
2
n) F reaction on 98 atom % H O (Rotem Industries, Arava, Israel), and was separated from
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
H
2
O using the SepꢀPak Accell Plus QMA Plus Light cartridge (Waters). The produced
18
[
F]HF was eluted from the cartridge with a mixture of aqueous K
2 3
CO (0.4 mg/0.2 mL) and
a solution of 4,7,13,16,21,24ꢀhexaoxaꢀ1,10ꢀdiazabicyclo[8,8,8]hexacosane (7.5 mg) in MeCN
18
ꢀ
(
0.2 mL), and transferred into a reaction vessel in a hot cell. The [ F]F solution was dried at
1
20 °C for 30 min to remove H O and MeCN. A solution of 7h (1.5 mg, 3.4 µmol) in DMSO
2
18
ꢀ
(300 µL) was combined with [ F]F and heated at 130 °C for 10 min. After completion of the
18
[
F]fluorination reaction, MeOH/50 mM MeCO
reaction mixture, which was separated by HPLC using an Atlantis T3 column (10 mm i.d. ×
50 mm, Waters) with MeOH/50 mM MeCO NH (6/4) at 5.0 mL/min. The radioactive
2 4
NH (6/4, 500 µL) was added into the
1
2
4
18
fraction corresponding to [ F]7d (t : 9.3 min) was collected in a sterile flask containing
R
polysorbate 80 (100 ꢀL) and 25% ascorbic acid (100 ꢀL), evaporated to dryness under
vacuum, reꢀdissolved in 3 mL sterile saline, and passed through a 0.22 ꢀm Millipore filter to
18
obtain the final product. The identity of [ F]7d (t
R
: 7.7 min) was confirmed by analytical
HPLC with 7d. The synthesis time was 65 min from EOB; radiochemical yield
1
8
ꢀ
(decayꢀcorrected) was 17% based on [ F]F ; radiochemical purity was > 99%; and specific
activity at EOS was 420 GBq/ꢀmol.
11
Computation and Measurement of Lipophilicity. The cLogD values of [ C]7a–c and
18
[ F]7d were determined computationally using ChemDraw software (ChemBioDraw Ultra
12.0; PerkinꢀElmer, Waltham, MA, USA). The LogD value was measured by mixing
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[
C]7a–c or [ F]7d [radiochemical purity: > 99%; about 200,000 counts per minute (cpm)]
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with nꢀoctanol (3.0 g) and phosphate buffered saline (PBS; 3.0 g, 0.1 M, pH 7.4) in a test
tube, which was vortexed for 3 min at room temperature, followed by centrifugation at 3500
g for 5 min. An aliquot of 0.65 mL PBS and 0.65 mL nꢀoctanol was removed and weighed
and its radioactivity was counted with a 1480 Wizard autogamma counter (PerkinꢀElmer),
respectively. Each sample from the remaining organic layer was removed and repartitioned
until a consistent LogD value was obtained. The LogD value was calculated by comparing
the ratio of cpm/g of nꢀoctanol to that of PBS and is expressed as LogD = Log[cpm/g
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(nꢀoctanol)/cpm/g (PBS)]. All measurements were performed in triplicate.
Animals. Animals were maintained and handled in accordance with the recommendations
of the National Institute of Health and institutional guidelines of the National Institute of
Radiological Sciences (NIRS). All experiments conducted at NIRS were approved by the
Animal Ethics Committee. Male ddY mice (7 weeks old, 32−35 g) and SpragueꢀDawley (SD)
rats (male, 7 weeks old, 220−240 g) were purchased from Japan SLC (Shizuoka, Japan), and
housed under a 12/12ꢀh dark/light cycle under optimal conditions.
In Vitro Assays of TSPO Binding. The TSPO binding assay was performed as previously
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4
described. Briefly, four rats were sacrificed by cervical dislocation under 5% (v/v)
isoflurane anaesthesia. The brains were quickly removed and homogenized in iceꢀcold
TrisꢀHCl buffer (50 mM, pH 7.4) containing 120 µM NaCl. The homogenate was centrifuged
at 40,000 g for 15 min at 4 °C. The supernatant was discarded and then the pellet was
resuspended, homogenized, and centrifuged under the same conditions. This procedure was
repeated twice. The pellet was resuspended in 50 mM TrisꢀHCl buffer (pH 7.4) at a
concentration of 100 mg original wet tissue per mL and used for binding assays.
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Crude mitochondrial preparations (100 µL) were incubated with [ C]1 (final
concentration: 2.5 nM) and various concentrations (0.3 nM–3 µM) of compounds 7a−d and 1
in a total volume of 1 mL TrisꢀHCl buffer at room temperature for 30 min, and the reaction
was terminated by rapid filtration through a Whatman GF/C glass fiber filter pretreated with
0.3% polyethylenimine using an Mꢀ24 cell harvester (Brandel; Neuroscience, Tokyo, Japan).
The filters were washed three times with 5 mL of iceꢀcold TrisꢀHCl buffer (50 mM), and the
filterꢀbound radioactivity was counted using the autogamma counter. Nonspecific binding
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was examined using a saturating concentration (10 µM) of 1. The K
i
of each compound for
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TSPO was determined according to following equation:
IC50
K =
i
[
L]
1+
Kd
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where [L] is the concentration of [ C]1 (nM). The dissociation constant (K
d
) of [ C]1 for
TSPO was obtained by Scatchard plot analysis.
Ischemic Rat Brain Model. Mild focal ischemia was induced by intraluminal occlusion of
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the middle cerebral artery for 30 min based on the intraluminal thread model.
Briefly,
healthy SD rats were anesthetized with 4% (v/v) isoflurane and maintained under anaesthesia
with 1.8% isoflurane. A 4.0ꢀmonofilament nylon suture coated with silicon was inserted into
the internal carotid artery up to the level of the middle cerebral artery branches
(
approximately 16−18 mm from the internal carotid artery), and the neck incision was closed
with a silk suture; 30 min after rat had regained consciousness from anaesthesia, they were
again anesthetized and the filament was carefully removed for reperfusion. Body temperature
was monitored and maintained at optimal levels throughout the surgery. Rats were then used
for in vitro autoradiography, PET imaging, and metabolite analysis at 7 days after ischemic
surgery.
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Glioma Rat Model. Fourteen days before PET imaging, healthy male SD rats were
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stereotactically inoculated in the right hemisphere with 1.0 × 10 C6 glioma cells (American
Type Tissue Collection, Manassas, VA, USA) according to a previously described
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protocol.
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In Vitro Autoradiography of Ischemic Rat Brain. Coronal sections (20 µm) were
prepared from frozen ischemic rat brains using a cryostat (HM560; Carl Zeiss, Jena,
Germany). Brain sections were preincubated for 20 min in 50 mM TrisꢀHCl buffer (pH 7.4),
and the sections were then incubated for 30 min at room temperature in fresh buffer
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containing [ C]7a−c (16−18 MBq, 1.1−1.3 nM) or [ F]7d (1.0 MBq, 0.015 nM). For the
1
1
inhibition studies, unlabeled 7a−d (10 µM) or 1 (10 µM) was incubated with [ C]7a–c or
18
[
F]7d; brain sections were then washed (3 × 2 min) with cold buffer, dipped in cold distilled
water, and dried with cold air. The sections were placed on imaging plates (BASꢀMS2025,
Fujifilm, Tokyo, Japan), and autoradiograms were analyzed using a BAS5000 bioꢀimaging
system (Fujifilm). The concentrations of radioactivity determined in brain sections are
2
expressed as photoꢀstimulated luminescence (PSL) per mm .
Radiolabeled Metabolite Analysis in Ischemic Rats. Ischemic rats were intravenously
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injected via the tail vein with [ C]7c (52 MBq, 0.7 nmol) or [ F]7d (20 MBq, 0.054 nmol)
solutions, then sacrificed by cervical dislocation 10 or 30 min later (n = 4 for each point).
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 resultant mixture was vortexed for 15 s and
then deproteinized by centrifugation at 15,000 g for 2 min. Rat brain was homogenized in
iceꢀcooled MeCN/H
2
O (1/1, 2 mL); the homogenate was centrifuged at 15,000 g for 2 min at
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4 °C and the supernatant (0.5 mL) was collected, resuspended in MeCN (0.5 mL), and
deproteinized by centrifugation at 15,000 g for 2 min. An aliquot of the supernatant (0.2–1.0
mL) obtained from the plasma or brain homogenate was injected into an HPLC system with a
radioactivity detector and analyzed using a Capcell Pak C column (4.6 mm i.d. × 250 mm)
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1
1
2 3
with MeCN/H O/Et N (50/50/0.1) at a flow rate of 1.0 mL/min for [ C]7c, and
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MeCN/H
2
O/Et
3
N (60/40/0.1) at a flow rate of 1.5 mL/min for [ F]7d. The percentage ratio
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of [ C]7c (t
R
= 7.1–8.0 min) or [ F]7d (t
R
= 4.2–4.7 min) to total radioactivity (corrected for
decay) on the HPLC chromatogram was calculated as % = (peak area for radiotracer/total
peak area) × 100.
PET Study and Image Analysis.
Ischemic rat model. PET scan was carried out using a smallꢀanimal PET scanner (Siemens
Medical Solutions USA, Knoxville, TN, USA), which provides 159 transaxial slices 0.796
mm (centerꢀtoꢀcenter) apart, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial
7
0
FOV for imaging. Ischemic rats were anesthetized with 1.5% (v/v) isoflurane during the
scan, and their body temperatures were maintained using a 40°C water circulation system
(
T/Pump TP401; Gaymar Industries, Orchard Park, NY, USA). Rats were intravenously
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injected via the tail vein with [ C]7a (38.7 ± 1.5 MBq/0.52 ± 0.02 nmol), [ C]7c (40.0 ± 1.6
1
8
MBq/0.54 ± 0.02 nmol), or [ F]7d (18.3 ± 1.0 MBq/0.05 ± 0.003 nmol), and a 60ꢀmin
listꢀmode emission scan was conducted immediately. The time frame reconstruction was as
follows: 1 min × 4 frames, 2 min × 8 frames, and 5 min × 8 frames. For the displacement
experiments, unlabeled compounds 7a, 7c, 7d (1 mg/kg), or 1 (3 mg/kg), dissolved in 500 µL
of saline containing 10% ethanol and 5% Tween 80, was injected 10 min after PET scans
were initiated. Four independent experiments were performed for each group.
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PET scan data modeling was performed using threeꢀdimensional sinograms, which were
transformed into twoꢀdimensional sinograms by Fourier rebinning. Dynamic image
reconstruction was achieved by filtered backꢀprojection using Hanning’s filter with a Nyquist
cutꢀoff frequency of 0.5 cycles/pixel. PET images were analyzed using ASIPro VM™
(Analysis Tools and System Setup/Diagnostics Tool; Siemens Medical Solutions). The
regions of interest were manually placed on ipsilateral and contralateral sides of the striatum
with the same area with reference to the template images of nuclear magnetic resonance.
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The BPND on the ipsilateral side of the striatum was calculated with SRTM using the
contralateral side as the reference region. The model analysis was performed using PMOD
version 3.4 image analysis software (PMOD Technologies, Zurich, Switzerland).
Glioma rat model. PET scan was carried out using the same smallꢀanimal PET scanner as
mentioned above. Gliomaꢀbearing rats were intravenously injected via the tail vein with
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[
F]7d (19.0 ± 0.8 MBq/0.05 ± 0.002 nmol), and a 60ꢀmin listꢀmode emission scan was
immediately performed. The time frame reconstruction was as follows: 1 min × 4 frames, 2
min × 8 frames, and 5 min × 8 frames. For the displacement experiments, unlabeled 1 (3
mg/kg), dissolved in 500 ꢀL of saline containing 10% ethanol and 5% Tween 80, was
injected via the tail vein 1 min before PET scans were initiated. Four to five independent
experiments were performed for each group. PET scan data were modelled as described
above. The BPND on the tumor (ipsilateral side) was calculated with the SRTM using the
same area on the contralateral side as the reference region.
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Biodistribution Study. Mice were injected via the tail vein with [ F]7d (1.0 MBq, 2.7
pmol) and were sacrificed at five time intervals (1, 5, 15, 30, and 60 min). Blood samples
were collected from each group (n = 4) and the brain, heart, kidneys, liver, lungs, spleen,
small intestine, testis, muscles, and bones (thighbone) were quickly removed and weighed.
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The radioactivity level in each tissue was measured with the autogamma counter and is
expressed as percentage of the injected dose per gram of wet tissue (% ID/g). Decay
correction was taken into account during the radioactivity measurements.
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Statistics. All quantitative data are expressed as the mean ± standard deviation (SD). The
relationship between the regional outcome parameters under test and retest conditions were
estimated using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).
ASSOCIATED CONTENT
Supporting Information
Purity of compounds 7a–h determined by HPLC; HPLC analytical charts for 7a–h; HPLC
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separation and analytic charts for [ C]7a–c and [ F]7d; and biodistribution of [ C]7c in
mice
AUTHOR INFORMATION
Corresponding Authors
Feng Wang, PhD., Department of Nuclear Medicine, Nanjing First Hospital, Affiliated to
Nanjing Medical University, 68 Chanle Road, Nanjing 210006, China. Phone:
86ꢀ25ꢀ52271456; eꢀmail: fengwangcn@hotmail.com.
MingꢀRong Zhang, PhD., Department of Radiopharmaceuticals Development, National
Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science
and Technology, 4ꢀ9ꢀ1 Anagawa, Inageꢀku, Chiba 263ꢀ8555, Japan. Phone: 81ꢀ43ꢀ382ꢀ3709;
fax: 81ꢀ43ꢀ206ꢀ3261; eꢀmail: zhang.mingꢀrong@qst.go.jp.
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