Novel 18F-labeled benzoxazole derivatives as potential positron emission tomography probes for imaging of cerebral β-amyloid plaques in alzheimers disease

Article abstract of DOI:10.1021/jm300251n

Two radiofluoro-pegylated phenylbenzoxazole derivatives, 4-(5-(2-(2-(2-[¹⁸F]fluoroethoxy)ethoxy)ethoxy)benzo[d]oxazol-2-yl)-N- methylaniline ([¹⁸F]24) and 4-(5-(2-(2-(2-[¹⁸F] fluoroethoxy)ethoxy)ethoxy)benzo[d]oxazol-2-yl)

Full text of DOI:10.1021/jm300251n

Article
pubs.acs.org/jmc
18
Novel F-Labeled Benzoxazole Derivatives as Potential Positron
Emission Tomography Probes for Imaging of Cerebral β-Amyloid
Plaques in Alzheimer’s Disease
Mengchao Cui,†,‡ Masahiro Ono,†,* Hiroyuki Kimura,† Masashi Ueda,†,§ Yuji Nakamoto,§
Kaori Togashi,§ Yoko Okamoto,∥ Masafumi Ihara,∥ Ryosuke Takahashi,∥ Boli Liu,‡ and Hideo Saji†
†Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida
Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
‡Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875,
PR China
§Department of Diagnostic Imaging and Nuclear Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
∥Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507,
Japan
S
* Supporting Information
ABSTRACT: Two radiofluoro-pegylated phenylbenzoxazole derivatives, 4-(5-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)benzo-
[d]oxazol-2-yl)-N-methylaniline ([18F]24) and 4-(5-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)benzo[d]oxazol-2-yl)-N,N-dime-
thylaniline ([18F]32), were synthesized and evaluated as probes for imaging cerebral β-amyloid (Aβ) plaques in living brain tissue
by PET. [18F]24 and [18F]32 displayed high affinity for Aβ1−42 aggregates (Ki = 9.3 and 3.9 nM, respectively). In vitro
autoradiography with sections of post-mortem AD brain and transgenic mouse brain confirmed the affinity of these tracers. Initial
high uptake into and rapid washout from the brain in normal mice were observed. [18F]24 also displayed excellent binding to Aβ
plaques in ex vivo autoradiographic experiments with Tg2576 mice. Furthermore, small-animal PET studies demonstrated
significant differences in the clearance profile after the administration of [18F]24 between Tg2576 and wild-type mice. The results
suggest [18F]24 to be a useful PET agent for detecting Aβ plaques in the living human brain.
these Aβ plaques will provide an important tool for the
noninvasive in vivo diagnosis of AD.4,5 Furthermore, it might
also be used to predict the development of AD before the onset
of dementia and to assess the effect of antiamyloid therapy.6
The search for small, neutral, and moderately lipophilic
imaging agents targeting Aβ plaques in the living human brain
has been extensive (Figure 1). Among PET probes investigated
in the past decade, a thioflavin-T derivative, [11C]-2-(4-
(methylamino)phenyl)-6-hydroxybenzothiazol ([11C]1: PIB),
INTRODUCTION
■
Dementia is the third most common cause of death after cancer
and cardiac vascular disorders, and Alzheimer's disease (AD) is
the most common form of dementia, characterized by
progressive memory impairment, disordered cognitive func-
tions, altered behavior, and a decline in language function. The
pathological hallmarks of AD are extracellular deposits of β-
amyloid (Aβ) plaques and intracellular neurofibrillary tangles
(NFTs).1−3 Currently, the clinical diagnosis of AD is only a
probable diagnosis and a histological diagnosis can only be
obtained after death. According to the amyloid cascade
hypothesis, amyloid deposits constitute a central and initial
event in the pathogenesis of AD. Therefore, a tracer agent for
positron emission tomography (PET) that specifically binds to
Special Issue: Alzheimer's Disease
Received: February 24, 2012
Published: June 12, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structure of PET imaging agents targeting Aβ plaques in AD patients.
is the most suitable and most extensively studied. PET studies
indicate that AD and control cases can be clearly distinguished
by using [11C]1.7−9 Recently, Andersson et. al reported a close
analogue of PIB, [11C]-2-[6-(methylamino)pyridin-3-yl]-1,3-
benzothiazol-6-ol ([11C]2: AZD2184), which has the positive
attributes of [11C]1 but with an apparent lower degree of
nonspecific binding in white matter. Preliminary human PET
studies suggested that [11C]2 displayed favorable brain kinetics
and an excellent contrast for Aβ plaques in the brain of AD
patients.10−12 Beside the core structure of thioflavin-T, the
stilbene scaffold has also been selected for developing Aβ
imaging agents. The first 11C-labeled stilbene derivative was
[11C]-4-N-methylamino-4′-hydroxystilbene ([11C]3: SB-13):
PET imaging in vivo with [11C]3 demonstrated potential
usefulness in detecting Aβ plaques in the human brain.13,14
However, the routine clinical use of 11C-labeled tracers is
limited by the short half-life of 11C (T1/2 = 20 min). Additional
tracers labeled with the radionuclide 18F (T1/2 = 110 min, 511
keV, commonly produced by a cyclotron) would be more
useful for PET.
aggregates (Ki = 0.4−9.0 nM). However, limited penetration
of the brain (<1.5%ID after an iv injection in normal mice)
together with nonspecific binding made them unsuitable for
imaging plaques in vivo.21 With additional structural changes,
we then obtained a 11C-labeled benzofuran derivative ([11C]
13) with improved properties.22 More recently, we have
reported a series of novel fluorinated pyridylbenzofuran and
phenylbenzofuran derivatives ([18F]14−17) with polyethylene
glycol side chains as potential 18F-labeled tracers for the
imaging of Aβ plaques by PET.23,24 These ligands showed high
affinity for Aβ1−42 aggregates in vitro and for Aβ plaques in
sections of autopsied AD brain. Compared with the
dimethylated [18F]14 and [18F]15, [18F]16 and [18F]17 with
a monomethylamino group exhibited greater initial uptake into
the brain. The pyridylbenzofuran derivative [18F]16 provided
the best pharmacokinetics of radioactivity in the brain, the
brain2 min/brain60 min ratio being 2.34. In addition, ex vivo
autoradiograms indicated that [18F]16 showed selective binding
of Aβ plaques with little nonspecific binding in the Tg2576
mouse brain.25 Compared with the radioiodinated benzofuran
derivatives, these fluoro-pegylated benzofuran derivatives had
greatly improved pharmacokinetics.
Thus, a [1 1C]1 analogue, [18 F]-2-(3-fluoro-4-
methyaminophenyl)benzothiazol-6-ol ([18F]4: GE-067),15 and
a [11C]2 derivative, [18F]-2-(2-fluoro-6-(methylamino)pyridin-
3-yl)benzofuran-6-ol ([18F]5: AZD4694),16 are currently under
phase II clinical testing in Europe. Preliminary studies in AD
patients suggested that they may potentially be useful for PET
imaging of Aβ plaque burden in living brain tissue. At the same
time, two stilbene derivatives with a short length of
polyethylene glycol units (PEG, n = 3), [18F]-4-(N-methyl-
amino)-4′-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-stilbene
([18F]6: BAY94−9172)4,17 and [18F]-(E)-4-(2-(6-(2-(2-(2-
fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methylani-
line ([18F]7: AV-45)18−20 are under commercial development.
In an attempt to develop more promising amyloid imaging
agents, we chose to investigate benzofuran derivatives designed
to be isosteric analogues of thioflavin-T (Figure 2). Initially, we
prepared a series of radioiodinated benzofuran derivatives
([125I]8−11), which displayed excellent affinity for Aβ1−40
A radioiodinated benzoxazole derivative, [125I]-2-(4′-dime-
thylaminophenyl)-6-iodobenzoxazole ([125I]12: IBOX), which
is another close thioflavin-T analogue (Figure 2), displayed
high affinity for Aβ1−40 aggregates in vitro (Ki = 0.8 nM).26
Similar to that observed with radioiodinated benzofuran
derivatives, [125I]12 displayed low brain uptake and high
nonspecific binding. To improve the pharmacokinetic profile of
[
125I]12 for use as a PET imaging agent, the highly lipid soluble
iodine atom was replaced by a short fluorine end-capped
polyethylene glycol chain (n = 3). In addition, compared with
the structure of benzofuran, the nitrogen atom in the oxazole
ring would reduce the lipophilicity of the probe, thus reducing
nonspecific binding and enhancing the signal-to-noise ratio.
After this modification, the in vivo pharmacokinetic profile of
the benzoxazole derivatives may be better than that of
benzofuran derivatives. Here, we report the synthesis and
biological evaluation of two novel fluoro-pegylated phenyl-
benzoxazole derivatives as PET probes for the detection of
cerebral Aβ plaques in vivo.
RESULTS AND DISCUSSION
■
Chemistry and Radiochemistry. The synthesis of 24 and
32 is outlined in Schemes 1 and 2, respectively. The desired
benzoxazoles (20 and 28) were obtained by a condensation
reaction between 2-amino-4-methoxyphenol (19) and 4-
monomethylaminobenzoic acid or 4-dimethylaminobenzoic
acid catalyzed by polyphosphoric acid in yields of 31% and
25%, respectively. The O-methyl groups of 20 and 28 were
removed by reacting with BBr3 in CH2Cl2 to give 21 and 29 in
Figure 2. Chemical structure of radio-labeled benzofuran and
benzoxazole derivatives reported previously.
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a
Scheme 1
a
Reagents and conditions: (a) Pd/C, MeOH, rt; (b) 4-methylaminobenzoic acid, PPA, 180°C; (c) BBr3 (1 M in CH2Cl2), CH2Cl2, −78 °C to rt;
(d) 2-[2-(2-chloroethoxy)ethoxy]ethanol, K2CO3, DMF, 110°C; (e) TBDMSCl, imidazole, CH2Cl2, rt; (f) DAST, CH2Cl2, −78 °C to rt; (g)
(Boc)2O, THF, reflux; (h) TBAF (1 M in THF), THF, rt; (i) MsCl, Et3N, CH2Cl2, rt; (j) (1) 18F−, K2CO3, Kryptofix-2.2.2, acetonitrile, 120 °C, (2)
HCl (1 M), 120 °C.
a
The radiofluorinated ligands [18F]24 and [18F]32 were
Scheme 2
prepared from the corresponding mesylate or tosylate
precursors (Schemes 1 and 2) with radiochemical yields of
30% and 60%, respectively. After purification by high
performance liquid chromatography (HPLC), the radio-
chemical purity of these radiotracers was greater than 98%,
and their specific activity was estimated as approximately 200
GBq/μmol. The identity of [18F]24 and [18F]32 was verified by
a comparison of the retention time with that of the
nonradioactive compound (see Supporting Information).
Biological Evaluation. In Vitro Binding of 24 and 32 to
a
Synthetic Aβ Aggregates. The affinity of 24 and 32 for Aβ1−42
aggregates was examined in solutions with competition binding
assays using [125I]2-(4-dimethylaminophenyl)-6-iodoimidazo-
[1,2-a]pyridine ([125I]IMPY) as the competing radioligand.27
Well-known Aβ imaging probes, IMPY28,29 and PIB,6,7 were
also screened using the same system for comparison. The data
in Table 1 demonstrate that they compete strongly with
Reagents and conditions: (a) 4-(dimethylamino)benzoic acid, PPA,
180 °C; (b) BBr3 (1 M in CH2Cl2), CH2Cl2, −78 °C to rt; (c) 2-[2-
(2-chloroethoxy)ethoxy]ethanol, K2CO3, DMF, 110 °C; (d) TsCl,
pyridine, rt; (e) TBAF (1 M in THF), THF, reflux; (f) 18F−, K2CO3,
Kryptofix-2.2.2, acetonitrile, 120 °C.
yields of 91% and 88%, respectively. To prepare compounds
with three ethoxy units as the polyethylene glycol linkage, 2-[2-
(2-chloroethoxy)ethoxy]ethanol was coupled with the hydroxy
groups of 21 and 29 to obtain 22 and 30. For the
monomethylated compound, the free hydroxy group in 22
was subsequently protected with tert-butyldimethylsilyl chloride
(TBDMSCl) to give 23. Compound 25 was obtained by
protecting the methylamino group of 23 with a butylox-
ycarbonyl (BOC) group. After removal of the TBS-protected
group of 25, the free hydroxy group of 26 was converted into
mesylates by reacting with methanesulfonyl chloride in the
presence of triethylamine to give 27. The fluorinated
compound 24 was successfully obtained by stirring 22 and
diethylaminosulfur trifluoride (DAST) in CH2Cl2 (Scheme 1).
For the dimethylated compound, tosylation of the free hydroxyl
group present in 30 afforded the precursor 31, which readily
reacted with anhydrous tetra-n-butylammonium fluoride
(TBAF) at reflux to give the fluorinated compound 32
(Scheme 2).
Table 1. Inhibition Constants for the Binding of [125I]IMPY
to Aβ(1−42) Aggregates
a
compd
Ki (nM)
24
9.3 2.2
3.9 0.7
10.5 1.0
9.0 1.3
32
b
b
IMPY
PIB
a
Values are the means
standard errors of the mean of three
b
independent determinations. Data from ref 25.
[
125I]IMPY for binding to Aβ1−42 aggregates (Ki = 9.3 and 3.9
nM, respectively). In addition, the affinity of these benzoxazole
derivatives was comparable to the value for IMPY and PIB
under the same assay conditions.
Autoradiography of Transgenic Mouse Brain and Post-
Mortem Brain Tissue Sections with [18F]24 or [18F]32. The
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Figure 3. Autoradiography of [18F]24 in vitro in mouse brain sections ((A) Tg mouse, C57BL6, APPswe/PSEN1, 12 months old; (C) wild-type,
C57BL6, 12 months old) and human brain sections ((E) AD, 93 years old, female; (G) normal, 71 years old, male). The presence and distribution of
plaques in the sections were confirmed with thioflavin-S (B, D) and immunohistochemical staining using a monoclonal Aβ antibody (F, H).
Figure 4. Autoradiography of [18F]32 in vitro in mouse brain sections ((A) Tg mouse, C57BL6, APPswe/PSEN1, 12 months old; (C) wild-type,
C57BL6, 12 months old) and human brain sections ((E) AD, 93 years old, female; (G) normal, 71 years old, male). The presence and distribution of
plaques in the sections were confirmed with thioflavin-S (B, D) and immunohistochemical staining using a monoclonal Aβ antibody (F, H).
high binding of the radiolabeled tracers ([18F]24 and [18F]32)
to Aβ plaques was further confirmed by in vitro auto-
radiography in sections of brain tissue from AD patients or
Tg model mice (C57BL6, APPswe/PSEN1, 12 months old). As
shown in Figures 3A and 4A, specific labeling of plaques was
observed in the brain sections of transgenic mice. The
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a
Table 2. Biodistribution in Normal ddY Mice after iv Injection of [18F]24 and the Lipophilicity (log D) of the Ligand
[18F]24 (log D = 3.09 0.05)
organ
blood
2 min
10 min
30 min
60 min
4.76 0.23
8.12 0.51
3.17 0.32
8.57 0.51
20.28 2.18
3.90 0.20
8.18 0.92
12.88 0.56
1.59 0.16
11.33 0.83
3.46 0.26
4.41 0.28
2.32 0.20
4.78 0.53
23.15 2.85
4.22 0.67
5.64 0.89
8.45 1.02
2.36 1.16
25.00 3.96
4.67 0.09
4.07 0.07
3.74 0.30
4.84 0.35
19.31 2.23
3.92 0.32
5.30 0.36
6.84 0.55
1.85 0.41
23.92 4.78
4.87 0.24
3.04 0.16
5.78 0.53
4.32 0.49
13.06 1.41
3.42 0.49
5.22 0.45
5.40 0.74
1.87 0.27
23.91 8.84
brain
bone
heart
liver
spleen
lung
kidney
stomach
b
intestine
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Expressed as % injected dose per organ.
a
Table 3. Biodistribution in Normal ddY Mice after iv Injection of [18F]32 and the Lipophilicity (log D) of the Ligand
[18F]32 (log D = 3.45 0.09)
organ
blood
2 min
10 min
30 min
60 min
3.24 0.25
5.29 0.19
1.18 0.19
5.55 0.53
13.04 1.31
3.60 0.30
5.18 0.23
8.71 0.96
1.10 0.13
4.80 0.79
2.26 0.12
3.37 0.21
1.07 0.31
3.52 0.16
11.36 1.10
2.88 0.51
3.64 0.48
5.18 0.36
1.37 0.52
15.70 1.51
2.84 0.13
2.50 0.20
1.96 0.26
3.14 0.18
9.20 0.99
2.53 0.06
3.28 0.22
4.08 0.35
1.51 0.16
22.59 2.94
3.33 0.41
2.12 0.08
3.45 0.31
3.13 0.31
6.92 0.66
2.25 0.37
3.22 0.36
3.68 0.80
1.41 0.34
15.82 1.67
brain
bone
heart
liver
spleen
lung
kidney
stomach
b
intestine
a
b
Expressed as % injected dose per gram. Average for 5 mice standard deviation. Expressed as % injected dose per organ.
Table 4. Comparison of Inhibition Constants (Ki, nM) and Brain Kinetics between Radiofluorinated Benzoxazole Derivatives
and Other Probes
a
a
tracers
Ki (nM)
2 min
60 min
ratio2 min/60 min
log D
[18F]24
[18F]32
9.5 1.3
3.9 0.2
0.8 0.2
2.87 0.17
1.0 0.2
2.0 0.5
2.4 0.1
3.9 0.2
8.12 0.51
5.29 0.19
3.04 0.16
2.12 0.08
2.67
2.50
1.13
3.90
2.11
1.03
2.34
2.11
3.09
3.45
2.09
2.41
2.32
2.94
3.11
3.73
b
b
[
125I]12
1.43 0.23
1.26 0.10
[18F]7
7.33 1.54
5.16 0.30
2.88 0.46
7.38 0.84
8.18 0.59
1.88 0.14
2.44 0.36
2.80 0.06
3.15 0.10
3.87 0.42
[18F]14
[18F]15
[18F]16
[18F]17
a
b
Expressed as % injected dose per gram. Expressed as % injected dose per organ.
distribution of Aβ plaques was consistent with the results of
fluorescent staining with thioflavin-S, a dye commonly used for
staining the Aβ plaques in human brain sections. (Figures 3B
and 4B). As expected, highly dense labeling of plaques was
observed in the brain sections of AD (Figures 3E and 4E).
Immunohistochemical staining confirmed the presence of
plaques in these sections (Figures 3F and 4F). In contrast,
no apparent labeling was observed in wild-type mice and
normal adult brain sections.
In Vivo Biodistribution in Normal ddY Mice. One
prerequisite for a successful imaging agent for Aβ plaques in
the brain is the ability to penetrate the BBB. It should also have
fast washout kinetics in normal brain.4 Therefore, the ability of
[18F]24 and [18F]32 to penetrate the blood−brain barrier
(BBB) was evaluated in vivo in normal ddY mice. As shown in
Tables 2 and 3, both [18F]24 and [18F]32 exhibited good
penetration of the BBB. Biodistribution experiments in mice
showed high initial uptake into the brain (8.12% and 5.29% ID/
g at 2 min, respectively) and fast washout kinetics from the
healthy brain (3.04% and 2.12% ID/g at 60 min, respectively),
which is highly desirable for Aβ imaging agents. The brain2 min
/
brain60 min ratio as an index to compare washout rates is used
for determining radioactivity pharmacokinetics in vivo. As
shown in Table 4, the brain2 min/brain60 min ratio of [18F]24 and
[18F]32 was 2.67 and 2.50, respectively, indicating that [18F]24
with a monomethylamino group provided the best profile of
radioactivity in the brain, and the pharmacokinetics in vivo were
improved by changing the dimethylamino group in [18F]32 to a
monomethylamino group. This may be due to the lower
lipophilicity of [18F]24 than [18F]32 (log D = 3.09 and 3.45,
respectively). In addition, no marked uptake of [18F]24 and
[18F]32 in bone was observed (5.78% and 3.45% ID/g at 60
min, respectively), suggesting little defluorination in vivo, and
interference with the imaging is expected to be relatively minor.
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Figure 5. Autoradiography of [18F]24 (left) and [18F]32 (right) ex vivo using Tg2576 mice (C57BL6, APPsw, 18 months old) and wild-type
controls (C57BL6, 18 months old). Plaques were also confirmed by the staining of the same sections with thioflavin-S.
Figure 6. μPET images (coronal views) at 2, 10, 20, 30, and 60 min after injection of [18F]24 in an aged Tg2576 mouse (C57BL6, APPsw, 31
months old) and aged wild-type mouse (C57BL6, 31 months old).
[18F]24 and [18F]32 also distributed to several other organs.
The liver and kidneys showed an initial uptake with washout,
whereas the intestines showed an accumulation of radioactivity
over time. In summary, the initial brain uptake of [18F]24
(8.12% ID/g) was comparable to the value observed for [18F]7
(7.33% ID/g),18 which was just approved by the FDA.
Although the brain2 min/brain60 min ratio of [18F]24 (2.67) was
lower than that of [18F]7 (3.90), it was improved compared to
the values for radiofluoro-pegylated benzofuran and pyridyl-
benzofuran derivatives (1.03−2.34).25 Furthermore, compared
with [125I]12, the fluoro-pegylated [18F]24 had greatly
improved pharmacokinetics (Table 4).
Ex Vivo Autoradiography of Transgenic Mouse Brain and
Wild-Type Mouse Brain. To further characterize the potential
of these benzoxazole probes in living mouse brain tissue, ex
vivo autoradiography was carried out in Tg2576 mice
(C57BL6, APPsw, 18 months old) and wild-type mice
(C57BL6, 18 months old). Autoradiography using [18F]24 or
[18F]32 showed dense labeling of the plaques in the cortical
regions and hippocampus of Tg2576 mice (Figure 5A), while
wild-type mouse brain showed no such labeling (Figure 5B).
Furthermore, the labeling of the plaques with both probes
showed a one-to-one correlation with the fluorescent signals
obtained with thioflavin S (Figure 5C). The results were
comparable to those reported previously for fluorinated
benzofuran derivatives.
Figure 7. μPET mean TACs of [18F]24 from brain VOIs in an aged
Tg2576 mouse (31 months old) and aged wild-type mouse (31
months old).
intravenous injection, [18F]24 penetrated the intact BBB
efficiently. The brain region displayed a good initial uptake in
both Tg2576 and wild-type mice. Because there were no Aβ
plaques in the healthy mouse brain, as expected, the
radioactivity of [18F]24 washed out quickly and did not display
any specific binding or prolonged retention. In Tg2576 mice,
however, the cortices and hippocampus, areas known to contain
high concentrations of Aβ plaques, retained the radioactivity of
[18F]24 to a greater extent during the later time points (Figure
6), and we can conclude that [18F]24 specifically binds to Aβ
plaques in the brain of Tg2576 mice. A comparison of time−
activity curves based on VOI analyses in mouse brain (Figure
7), revealed significant differences in the clearance profile after
the administration of [18F]24 between Tg2576 and wild-type
Small-Animal PET Studies of [18F]24 with Tg-2576 and
Wild-Type Mice. Because of the high initial brain uptake of
[18F]24, this probe with a monomethylamino group was
selected for small-animal PET experiments in Tg2576 and wild-
type mouse brain. The μPET images and brain uptake kinetics
(whole brain) are presented in Figures 6 and 7. After an
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mice. The brain2 min/brain20 min ratio of [18F]24 was lower in
Tg2576 mice (1.21) than wild-type mice (1.77).
and after ice water (50 mL) was added, neutralized by K2CO3. The
mixture was extracted with CHCl3 (100 mL), the organic phase was
separated, dried over Na2SO4, and filtered, and the residue was purified
1
by silica gel chromatography to give 21 (437 mg, 91.1%). H NMR
CONCLUSION
■
(400 MHz, CD3OD) δ 7.93 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz,
1H), 6.99 (d, J = 2.1 Hz, 1H), 6.77 (dd, J = 8.7, 2.4 Hz, 1H), 6.69 (d, J
= 8.8 Hz, 2H), 2.85 (s, 3H). MS(ESI): m/z calcd for C14H12N2O2
240.09; found 241.10 (M + H+).
The present results showed that [18F]24, a novel radiofluoro-
pegylated phenylbenzoxazole derivative, has several favorable
properties: high affinity for Aβ aggregates (Ki = 9.3 nM); easily
labeled with 18F for imaging (radiochemical yield of 30%, which
is suitable for commercial scale production and distribution;
small (MW < 500), lipophilic (measured log D = 3.09), and
neutral; good initial brain uptake and fast washout (8.12% ID/g
at 2 min and 3.04% ID/g at 60 min after iv injection)); lower
nonspecific binding in white matter as demonstrated by
autoradiography in vitro and ex vivo using post-mortem AD
brain sections and Tg2576 mice. These findings suggest that
[18F]24 should be investigated further as a potential PET tracer
for imaging Aβ plaques in living brain tissue.
2-(2-(2-((2-(4-(Methylamino)phenyl)benzo[d]oxazol-5-yl)oxy)-
ethoxy)ethoxy)ethanol (22). To a solution of 21 (480 mg, 2 mmol)
and 2-[2-(2-chloroethoxy)-ethoxy]ethanol (50 μL, 0.66 mmol) in
DMF (15 mL) was added anhydrous K2CO3 (830 mg, 6 mmol). The
reaction mixture was stirred for 4 h at 105 °C and then poured into
water and extracted with CHCl3. The organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a residue,
which was purified by silica gel chromatography to give 22 (550 mg,
73.9%). 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.3 Hz, 2H), 7.38
(d, J = 8.8 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 6.90 (dd, J = 8.8, 2.5 Hz,
1H), 6.67 (d, J = 8.4 Hz, 2H), 4.24−4.14 (m, 2H), 3.91−3.86 (m,
2H), 3.79−3.68 (m, 6H), 3.67−3.61 (m, 2H), 2.92 (s, 3H). MS(ESI):
m/z calcd for C20H24N2O5 372.17; found 373.15 (M + H+).
N-Methyl-4-(5-((2,2,3,3-tetramethyl-4,7,10-trioxa-3-siladodecan-
12-yl)oxy)benzo[d]oxazol-2-yl)aniline (23). A solution of 22 (372
mg, 1 mmol), TBDMSCl (226 mg, 1.5 mmol), and imidazole (140
mg, 2 mmol) in dichloromethane (10 mL) was stirred at room
temperature for 3 h. A white solid formed and was filtered off. After
the filtrate was evaporated, the residue was purified by silica gel
column chromatography to afford 23 (302 mg, 62.1%). 1H NMR (400
MHz, CDCl3) δ 7.96 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.8 Hz, 1H),
7.14 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.8, 2.4 Hz, 1H), 6.58 (d, J = 8.7
Hz, 2H), 4.22 (s, 1H), 4.13−4.04 (m, 2H), 3.84−3.79 (m, 2H), 3.71
(t, J = 5.4 Hz, 2H), 3.69−3.61 (m, 4H), 3.51 (t, J = 5.4 Hz, 2H), 2.81
(s, 3H), 0.83 (s, 9H). MS(ESI): m/z calcd for C26H38N2O5Si 486.25;
found 487.30 (M + H+).
EXPERIMENTAL SECTION
■
General Remarks. All reagents used for chemical synthesis were
commercial products and were used without further purification. The
1H NMR spectra were obtained at 400 MHz on JEOL JNM-AL400
NMR spectrometers in CDCl3 solutions at room temperature with
TMS as an internal standard. Chemical shifts are reported as δ values
relative to the internal TMS. Coupling constants are reported in hertz.
Multiplicity is defined by s (singlet), d (doublet), t (triplet), and m
(multiplet). Mass spectra were acquired with a Shimadzu GC-MS-
QP2010 Plus (ESI). HPLC was performed with a Shimadzu system (a
LC-20AT pump with a SPD-20A UV detector, λ = 254 nm) using a
column of Cosmosil C18 (Nacalai Tesque, 5C18-AR-II, 4.6 mm × 150
mm or 10 mm × 150 mm) and acetonitrile/water as the mobile phase.
Fluorescence was observed with a Nikon Eclipse 80i microscope
equipped with a BV-2A filter set (excitation, 400−440 nm; diachronic
mirror, 455 nm; long pass filter, 470 nm). Purity of the synthesized
compounds was determined using analytical HPLC and was found to
be more than 95%. ddY Mice (five weeks, male) were used for
biodistribution experiments. Transgenic mice (Tg2576, C57BL6,
APPsw), used as an Alzheimer’s model, were purchased from Taconic
Farms, Inc. All protocols requiring the use of mice were approved by
the animal care committee of Kyoto University. Post-mortem brain
tissues from an autopsy-confirmed case of AD (a 93-year-old, female)
and a control (a 71-year-old, male) were obtained from the Graduate
School of Medicine, Kyoto University, and BioChain Institute Inc.,
respectively. Experiments were performed according to the regulations
of the ethics committee of Kyoto University.
4-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)benzo[d]oxazol-2-yl)-
N-methylaniline (24). To a solution of 22 (74 mg, 0.2 mmol) in
CHCl3 (10 mL) was added DAST (100 mg, 0.6 mmol) in a dry ice−
acetone bath. The reaction mixture was stirred for 2 h at room
temperature and then poured into a saturated NaHSO3 solution and
extracted with CHCl3. The organic phase was separated, dried over
MgSO4, and filtered, and the residue was purified by silica gel
1
chromatography to give 24 (7.1 mg, 9.6%). H NMR (400 MHz,
CDCl3) δ 8.04 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.8 Hz, 1H), 7.21 (d, J
= 2.5 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H),
4.63 (dd, J = 4.5, 3.9 Hz, 1H), 4.51 (dd, J = 6.0, 2.3 Hz, 1H), 4.23−
4.13 (m, 2H), 3.95−3.86 (m, 2H), 3.84−3.79 (m, 2H), 3.78−3.71 (m,
4H), 2.92 (s, 3H). HRMS(EI): m/z calcd for C20H23FN2O4 374.1642;
found 374.1637 (M + H+).
tert-Butyl Methyl(4-(5-((2,2,3,3-tetramethyl-4,7,10-trioxa-3-sila-
dodecan-12-yl)oxy)benzo[d]oxazol-2-yl)phenyl)carbamate (25).
Under a nitrogen atmosphere, 23 (300 mg, 0.62 mmol) was dissolved
in anhydrous tetrahydrofuran (THF) (20 mL) followed by Boc-
anhydride (1.35 g, 6.2 mmol). The solution was refluxed for 48 h.
After the reaction was complete, the solvent was removed and the
residue was purified by silica gel column chromatography to afford 25
(273 mg, 74.7%). 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz,
2H), 7.37 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 2.5
Hz, 1H), 6.91 (dd, J = 8.8, 2.5 Hz, 1H), 4.19−4.09 (m, 2H), 3.88−
3.80 (m, 2H), 3.71 (t, J = 5.4 Hz, 2H), 3.69−3.61 (m, 4H), 3.51 (dd, J
= 5.6, 5.1 Hz, 2H), 3.26 (s, 3H), 1.42 (s, 9H), 0.83 (s, 9H). MS(ESI):
m/z calcd for C31H46N2O7Si 586.31; found 587.40 (M + H+).
tert-Butyl (4-(5-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)benzo[d]-
oxazol-2-yl)phenyl)(methyl)carbamate (26). To a solution of 25
(270 mg, 0.46 mmol) in dry THF (10 mL) was added anhydrous
TBAF (1.0 mL, 1 M in THF). The solution was stirred at room
temperature for 2 h. After removal of the THF, the residue was
Chemistry. 2-Amino-4-methoxyphenol (19). A mixture of 18 (5.1
g, 40 mmol) and Pd/C (10%, 0.5 g) in absolute methanol (200 mL)
under a hydrogen atmosphere (balloon) was strongly stirred for 24 h
at room temperature. The mixture was filtered, and the filtrate washed
with methanol (20 mL) and concentrated in vacuo to give 19 (3.72 g,
89.2%).
4-(5-Methoxybenzo[d]oxazol-2-yl)-N-methylaniline (20). To a
mixture of 19 (1.39 g, 10 mmol) and 4-methylaminobenzoic acid
(1.51 g, 10 mmol) was added polyphosphoric acid (10 g). The mixture
was stirred at 180 °C for 30 min, and after ice water (400 mL) was
added, neutralized by K2CO3. The mixture was extracted with ethyl
acetate (200 mL), and the organic phase was separated and dried over
Na2SO4. After the solvent was removed, the residue was purified by
1
silica gel chromatography to afford 20 (783 mg, 30.8%). H NMR
(400 MHz, CD3OD) δ 7.94 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 8.7 Hz,
1H), 7.12 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.9, 2.6 Hz, 1H), 6.69 (d, J
= 8.9 Hz, 2H), 3.84 (s, 3H), 2.85 (s, 3H). MS(ESI): m/z calcd for
C15H14N2O2 254.11; found 255.10 (M + H+).
2-(4-(Methylamino)phenyl)benzo[d]oxazol-5-ol (21). To a sol-
ution of 20 (510 mg, 2 mmol) was added BBr3 (1 M in CH2Cl2, 10
mL) dropwise in a dry ice−acetone bath. The reaction mixture was
stirred for 1 h at −78 °C and an additional 12 h at room temperature,
1
purified by silica gel chromatography to give 26 (281 mg, 75.7%). H
NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.8 Hz, 2H), 7.39−7.30 (m,
3H), 7.22 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 4.11 (dd, J = 6.2, 2.9 Hz,
2H), 3.88−3.75 (m, 2H), 3.70−3.60 (m, 6H), 3.58−3.51 (m, 2H),
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3.24 (s, 3H), 1.41 (s, 9H). MS(ESI): m/z calcd for C25H32N2O7
472.22; found 473.30 (M + H+).
10 mL of dry tetrahydrofuran (THF) was added anhydrous TBAF
(300 μL, 1 M in THF). The reaction mixture was refluxed for 3 h.
After removal of the THF, the residue was purified by silica gel
2-(2-(2-((2-(4-((tert-Butoxycarbonyl)(methyl)amino)phenyl)-
benzo[d]oxazol-5-yl)oxy)ethoxy)ethoxy)ethyl methanesulfonate
(27). To a solution of 26 (281 mg, 0.6 mmol) in dichloromethane
(10 mL) was added triethylamine (121 mg, 1.2 mmol). Methane-
sulfonyl chloride (137 mg, 1.2 mmol) was then added via a syringe.
The solution was stirred at room temperature for 2 h. Next, 20 mL of
water was added and the solution was extracted with CHCl3. The
organic layer was dried over MgSO4. After the solvent was removed,
the residue was purified by silica gel chromatography to afford 27 (173
mg, 52.4%). 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.7 Hz, 2H),
7.45 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 2.5 Hz,
1H), 6.97 (dd, J = 8.9, 2.5 Hz, 1H), 4.42−4.35 (m, 2H), 4.21−4.14
(m, 2H), 3.88 (dd, J = 5.3, 4.0 Hz, 2H), 3.81−3.77 (m, 2H), 3.76−
3.69 (m, 4H), 3.33 (s, 3H), 3.06 (s, 3H), 1.49 (s, 9H). MS(ESI): m/z
calcd for C26H34N2O9S 550.20; found 551.25 (M + H+).
1
chromatography to give 32 (22 mg, 56.7%). H NMR (400 MHz,
CDCl3) δ 8.08 (d, J = 9.0 Hz, 2H), 7.38 (d, J = 8.8 Hz, 1H), 7.21 (d, J
= 2.5 Hz, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.77 (d, J = 9.1 Hz, 2H),
4.67−4.59 (m, 1H), 4.55−4.45 (m, 1H), 4.24−4.14 (m, 2H), 3.93−
3.85 (m, 2H), 3.84−3.65 (m, 6H), 3.07 (s, 6H). HRMS(EI): m/z
calcd for C21H25FN2O4 388.1798; found 388.1805 (M + H+).
Radiosynthesis. [18F]Fluoride trapped on an anion exchange
cartridge was eluted with a K2CO3 solution (33 mM). Kryptofix222 (6−
8 mg) was dissolved in the solution of [18F]fluoride in water. The
solvent was removed at 120 °C under a stream of nitrogen gas. The
residue was azeotropically dried with 0.3 mL of anhydrous acetonitrile
twice at 120 °C under a stream of nitrogen gas.
For [18F]24, a solution of the mesylate precursor 27 (1.0 mg) in
CH3CN (0.2 mL) was added to the reaction vessel containing the 18F−
activity. The mixture was heated at 120 °C for 6 min. After the
solution had cooled to room temperature, HCl (1 M aqueous solution,
0.2 mL) was added and the mixture was heated at 120 °C again for 5
min. An aqueous solution of K2CO3 was added to adjust the pH to
basic (pH 8−9). Water (5 mL) was added, and the mixture was passed
through a preconditioned Sep-Pak Plus PS-2 cartridge (Waters). The
cartridge was washed with 20 mL of water, and the labeled compound
was eluted with 3 mL of acetonitrile. After the solvent was removed,
the residue was dissolved in CH3CN and subjected to HPLC for
purification (Nacalai Tesque, 5C18-AR-II, 4.6 mm × 150 mm,
CH3CN/water = 1/1; flow rate = 1 mL/min). The retention time of
[18F]24 was 8.22 min in this HPLC system. The preparation took 60
min, and the radiochemical yield was 30% (decay corrected).
For [18F]32, a solution of the tosylate precursor 31 (1.0 mg) in
CH3CN (0.2 mL) was added to the reaction vessel containing the 18F−
activity. The mixture was heated at 120 °C for 6 min. Water (5 mL)
was added, and the mixture was passed through a preconditioned Sep-
Pak Plus PS-2 cartridge (Waters). The cartridge was washed with 20
mL of water, and the labeled compound was eluted with 3 mL of
acetonitrile. The eluted compound was purified by HPLC (Nacalai
Tesque, 5C18-AR-II, 4.6 mm × 150 mm). The retention time of [18F]
32 was 7.55 min in this HPLC system (CH3CN/water = 6/4; flow rate
= 1 mL/min). The preparation took 40 min and the radiochemical
yield was 60% (decay corrected). The radiochemical purity of both
tracers was greater than 98%.
In Vitro Binding Studies of 24 and 32. Inhibition experiments
were carried out in 12 mm × 75 mm borosilicate glass tubes according
to procedures described previously with some modifications.25,30
Briefly, 100 μL of aggregated Aβ fibrils (60 nM in the final assay
mixture) was added to a mixture containing 100 μL of radioligands
([125I]IMPY) at an appropriate concentration, 10 μL of inhibitors
(10−5−10−10 M in ethanol), and 790 μL of PBS (0.2 M, pH = 7.4) in a
final volume of 1 mL. Nonspecific binding was defined in the presence
of 1 μM IMPY. The mixture was incubated for 2 h at 37 °C with
constant shaking, and then the bound and free radioactive fractions
were separated by vacuum filtration through borosilicate glass fiber
filters (Whatman GF/B) using a M-24 cell harvester (Brandel,
Gaithersburg, MD). The radioactivity from filters containing the
bound 125I ligand was measured in a γ-counter (WALLAC/Wizard
1470, USA) with 70% efficiency. Under the assay conditions, the
specifically bound fraction accounted for about 10% of total
radioactivity. The half-maximal inhibitory concentration (IC50) was
determined using GraphPad Prism 4.0, and the inhibition constant
(Ki) was calculated using the Cheng−Prusoff equation: Ki = IC50/(1 +
[L]/Kd).31
4-(5-Methoxybenzo[d]oxazol-2-yl)-N,N-dimethylaniline (28). To
a mixture of 19 (1.39 g, 10 mmol) and 4-(dimethylamino)benzoic acid
(1.65 g, 10 mmol) was added polyphosphoric acid (30 g). The mixture
was stirred at 180 °C for 30 min, and after ice water (400 mL) was
added, neutralized by K2CO3. The mixture was extracted with ethyl
acetate (200 mL), and the organic phase was separated and dried over
Na2SO4. After the solvent was removed, the residue was purified by
1
silica gel chromatography to afford 28 (672 mg, 25.1%). H NMR
(400 MHz, CDCl3) δ 8.08 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.8 Hz,
1H), 7.21 (d, J = 2.5 Hz, 1H), 6.86 (dd, J = 8.8, 2.5 Hz, 1H), 6.77 (d, J
= 8.9 Hz, 2H), 3.86 (s, 3H), 3.07 (s, 3H). MS(ESI): m/z calcd for
C16H16N2O2 268.12; found 269.15 (M + H+).
2-(4-(Dimethylamino)phenyl)benzo[d]oxazol-5-ol (29). To a
solution of 28 (536 mg, 2 mmol) was added BBr3 (1 M in CH2Cl2,
10 mL) dropwise in a dry ice−acetone bath. The reaction mixture was
stirred for 1 h at −78 °C and an additional 12 h at room temperature,
and after ice water (50 mL) was added, neutralized by K2CO3. The
mixture was extracted with CHCl3 (100 mL), the organic phase was
separated, dried over Na2SO4, and filtered, and the residue was purified
1
by silica gel chromatography to give 29 (447 mg, 88.0%). H NMR
(400 MHz, CD3OD) δ 7.99 (d, J = 8.9 Hz, 2H), 7.38 (d, J = 8.7 Hz,
1H), 7.00 (d, J = 2.4 Hz, 1H), 6.84 (d, J = 8.9 Hz, 2H), 6.78 (ddd, J =
8.8, 2.4, 0.8 Hz, 1H), 3.07 (s, 6H). MS(ESI): m/z calcd for
C15H14N2O2 254.11; found 255.10 (M + H+).
2-(2-(2-((2-(4-(Dimethylamino)phenyl)benzo[d]oxazol-5-yl)oxy)-
ethoxy)ethoxy)ethanol (30). To a solution of 29 (380 mg, 1.5 mmol)
and 2-[2-(2-chloroethoxy)-ethoxy]ethanol (510 mg, 3 mmol) in DMF
(10 mL) was added anhydrous K2CO3 (280 mg, 2 mmol). The
reaction mixture was stirred for 3 h at 110 °C and then poured into
water and extracted with CHCl3. The organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a residue,
which was purified by silica gel chromatography to give 30 (250 mg,
43.1%). 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 7.9 Hz, 2H), 7.37
(d, J = 8.8 Hz, 1H), 7.24 (d, J = 2.3 Hz, 1H), 6.88 (dd, J = 8.8, 2.5 Hz,
1H), 6.72 (d, J = 8.2 Hz, 2H), 4.17 (dd, J = 4.9, 3.8 Hz, 2H), 3.86 (dd,
J = 4.9, 3.8 Hz, 2H), 3.78−3.42 (m, 8H), 3.02 (s, 6H). MS(ESI): m/z
calcd for C21H26N2O5 386.18; found 387.27 (M + H+).
2-(2-(2-((2-(4-(Dimethylamino)phenyl)benzo[d]oxazol-5-yl)oxy)-
ethoxy)ethoxy)ethyl 4-Methylbenzenesulfonate (31). To a solution
of 30 (250 mg, 0.65 mmol) in pyridine (10 mL) was added tosyl
chloride (248 mg, 1.30 mmol). The reaction mixture was stirred for 3
h at room temperature, 50 mL of water was added, and the mixture
was extracted with CHCl3. The organic layer was dried over Na2SO4,
and evaporation of the solvent afforded a residue, which was purified
1
by silica gel chromatography to give 31 (150 mg, 42.7%). H NMR
Measurement of Partition Coefficient. [18F]24 or [18F]32 (370
kBq) was added to a premixed suspension containing 3 g of n-octanol
and 3 g of PBS (0.05 M, pH = 7.4) in a test tube. The test tube was
vortexed for 3 min at room temperature and centrifuged for 5 min at
3000 rpm. Two weighed samples from the n-octanol (100 μL) and
buffer (500 μL) layers were measured. The partition coefficient was
expressed as the logarithm of the ratio of the count per gram from n-
octanol versus PBS. Samples from the n-octanol layer were
(400 MHz, CDCl3) δ 8.06 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 8.3 Hz,
2H), 7.37 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 2.5
Hz, 1H), 6.87 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 9.1 Hz, 2H),
4.20−4.11 (m, 4H), 3.88−3.81 (m, 2H), 3.74−3.56 (m, 6H), 3.04 (s,
6H), 2.40 (s, 3H). MS(ESI): m/z calcd for C28H32N2O7S 540.19;
found 541.29 (M + H+).
4-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)benzo[d]oxazol-2-yl)-
N,N-dimethylaniline (32). To a solution of 31 (54 mg, 0.1 mmol) in
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Journal of Medicinal Chemistry
Article
repartitioned until consistent partition coefficient values were
obtained. The measurement was done in triplicate and repeated
three times.
ACKNOWLEDGMENTS
■
This study was supported by the Funding Program for Next
Generation World-Leading Researchers (NEXT Program)
from the Japan Society for the Promotion of Science (JSPS)
and the China Scholarship Council (CSC).
In Vitro Autoradiography of Transgenic Mouse Brain
Sections and AD Brain Sections. Paraffin-embedded mouse brain
sections (Tg mouse, C57BL6, APPswe/PSEN1, 12 months old; wild-
type, C57BL6, 12 months old) and human brain sections (AD, 93
years old, female; normal, 71 years old, male) were deparaffinized with
2 × 20 min washes in xylene, 2 × 5 min washes in 100% ethanol, a 5
min wash in 90% ethanol/H2O, a 5 min wash in 80% ethanol/H2O, a
5 min wash in 60% ethanol/H2O, and a 10 min wash in running tap
water and then incubated in PBS (0.2 M, pH = 7.4) for 30 min. The
sections were incubated with [18F]24 or [18F]32 (370 kBq/100 μL)
for 1 h at room temperature, washed with 50% ethanol for 1 min, and
rinsed with water for 1 min. After drying, the labeled sections were
exposed to a Fuji Film imaging plate overnight. The in vitro
autoradiographic images were obtained using a BAS5000 scanner
system (Fuji Film). The presence and location of plaques in the
sections were confirmed with fluorescent staining using thioflavin S or
immunohistochemical staining using a monoclonal Aβ1−42 antibody,
BC05 (Wako Pure Chemical Inc., Osaka, Japan).
In Vivo Biodistribution in Normal ddY Mice. Normal male ddY
mice (5 weeks, 20−22 g, n = 5) were injected with [18F]24 or [18F]32
(370 kBq/100 μL saline containing 5% EtOH) directly into a tail vein.
The mice were sacrificed at various time points postinjection. The
organs of interest were removed and weighed, and the radioactivity
was measured with an automatic γ-counter (WALLAC/Wizard 1470,
USA). The percent dose per gram of wet tissue was calculated by a
comparison of the tissue counts to suitably diluted aliquots of the
injected material.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, β-amyloid; NFTs, neurofibrillary
tangles; PET, positron emission tomography; PEG, poly-
ethylene glycol; BBB, blood−brain barrier; TBDMSCl, tert-
butyldimethylsilyl chloride; BOC, butyloxycarbonyl; HPLC,
high performance liquid chromatography; THF, tetrahydrofur-
an; TBAF, tetra-n-butylammonium fluoride; DAST, diethyla-
minosulfur trifluoride; Tg, transgenic
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ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC chromatograms of 24, 32, [18F]24, and [18F]32. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-75-753-4608. Fax: +81-75-753-4568. E-mail:
ono@pharm.kyoto-u.ac.jp.
Notes
The authors declare no competing financial interest.
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