A dual fluorinated and iodinated radiotracer for PET and SPECT imaging of β-amyloid plaques in the brain

Article abstract of DOI:10.1016/j.bmcl.2011.08.063

We report a fluorinated and iodinated radiotracer as a probe for PET/SPECT to detect of β-amyloid (Aβ) plaques in the brain of patients with Alzheimer's disease (AD). We successfully designed and synthesized the fluorinated and iodinated aurone derivative

Full text of DOI:10.1016/j.bmcl.2011.08.063

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6519–6522
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Bioorganic & Medicinal Chemistry Letters
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A dual fluorinated and iodinated radiotracer for PET and SPECT imaging
of b-amyloid plaques in the brain
Hiroyuki Watanabe a,b, Masahiro Ono a,b, , Hiroyuki Kimura a, Shinya Kagawa c, Ryuichi Nishii c,
⇑
a
Takeshi Fuchigami b, Mamoru Haratake b, Morio Nakayama b, , Hideo Saji
⇑
a Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
c Shiga Medical Center Research Institute, 5-4-30 Moriyama, Moriyama City, Shiga 524-8524, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a fluorinated and iodinated radiotracer as a probe for PET/SPECT to detect of b-amyloid (Ab)
plaques in the brain of patients with Alzheimer’s disease (AD). We successfully designed and synthesized
the fluorinated and iodinated aurone derivative (3) and its radiolabels ([125I]3 and [18F]3). In binding
experiments in vitro, 3 showed high affinity for Ab aggregates (Ki = 6.81 nM). In brain sections of AD
model mice, 3 intensely stained Ab plaques. Furthermore, a specific plaque labeling signal was observed
on the autoradiography of postmortem AD brain sections using [125I]3. In biodistribution experiments
using normal mice, [125I]3 and [18F]3 displayed good uptake into and a rapid washout from the brain,
properties highly desirable for Ab imaging agents. These results suggest that 3 may function as a PET/
SPECT dual imaging agent for detecting Ab plaques in AD brains.
Received 31 May 2011
Revised 12 August 2011
Accepted 13 August 2011
Available online 24 August 2011
Keywords:
Alzheimer’s disease
b-Amyloid peptide
PET
Ó 2011 Elsevier Ltd. All rights reserved.
SPECT
Dual imaging
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder characterized by cognitive decline, irreversible memory
loss, disorientation, and language impairment. Senile plaques con-
taining b-amyloid (Ab) peptides and neurofibrillary tangles in post-
mortem brain are two pathological hallmarks of AD.1,2 Excessive
production of Ab via various normal or abnormal mechanisms is
considered to be the initial neurodegenerative event in AD. Cur-
rently, it is difficult for clinicians to differentiate between the cog-
nitive decline associated with normal aging and the cognitive
decline associated with AD. There is no simple and definitive diag-
nostic method to detect Ab plaques in the brain without postmor-
tem pathological staining of brain tissue. Thus, the development of
imaging agents for positron emission tomography (PET) or single
photon emission computed tomography (SPECT), which can detect
Ab plaques in vivo may assist with the early diagnosis of AD.3–5
The nuclear imaging modality of choice in the clinic is often
PET rather than SPECT in part because of superior sensitivity,
resolution, and quantitative ability. Therefore, in the past few
years, several groups’ efforts have focused on the development
of potential PET probes for the detection of Ab plaques
[
[
18F]GE-068,8 [18F]BAY94-9172,9,10
[ [
11C]AZD2184,11,12 11C]PIB,13,14
11C]SB-13,15,16 and [18F]FDDNP17,18 have been tested clinically and
demonstrated potential utility for the diagnosis of AD. Conversely,
more SPECT scanners have been installed for routine clinical imag-
ing than PET imaging devices, which provides a certain advantage
to the use of SPECT imaging agents. Therefore, SPECT is thought to
be more valuable than PET in terms of routine diagnostic use.
While the development of more useful Ab imaging probes for
SPECT has been a critical issue, [123I]IMPY is the only SPECT probe
to be tested in humans.19–21 The preliminary clinical data for
[
123I]IMPY showed a poor signal-to-noise ratio making it difficult
to distinguish AD patients, possibly due to high lipophilicity and
low stability.
Although many PET or SPECT imaging probes for cerebral Ab
plaques have been reported, there has been no report on the devel-
opment of dual imaging probes. To develop novel radiotracers
offering both PET (18F) and SPECT (123I) imaging using a single
compound, we designed a new candidate compound which has
both fluoride and iodide in a single chemical structure. We have re-
cently reported the use of radioiodinated aurone derivatives as a
new backbone structure in the development of Ab imaging probes
for SPECT.22,23 The derivatives showed strong binding to Ab aggre-
gates, good penetration of the brain, and a fast washout from the
brain despite their substituted groups. Among them, AR-1 (Fig. 1)
showed the most promising characteristics as a SPECT probe in
terms of affinity for Ab aggregates and radioactivity pharmacoki-
netics in the brain.
in vivo. As
a result, PET probes such as [
18F]AV-45,6,7
⇑
Corresponding authors. Tel.: +81 75 753 4608; fax: +81 75 753 4568 (M.O.); tel./
fax: +81 95 819 2441 (M.N.).
E-mail addresses: ono@pharm.kyoto-u.ac.jp (M. Ono), morio@nagasaki-u.ac.jp
(M. Nakayama).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.063

6520
H. Watanabe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6519–6522
sired radioiodinated ligand (Scheme 1). It was anticipated that
the no-carrier-added preparation would result in a final product
bearing a theoretical specific activity similar to that of 125I
(2200 Ci/mmol). The free OH groups of 5 were converted into tosy-
lates by reacting with TsCl in the presence of pyridine to give 6. To
make the desired 18F-labeled aurone, [18F]3, the tosylate 6 was em-
ployed as the precursor. A solution of 6 in acetonitrile (200 lL) was
added to a reaction vessel containing 18F and the mixture was
heated at 100 °C for 10 min (Scheme 2). The radiochemical identity
of [125I]3 and [18F]3 was verified by a comparison of retention
times with the nonradioactive compound. These products were ob-
tained in >23% radiochemical yields with a radiochemical purity of
>95% after purification by HPLC.
In vitro binding experiments to evaluate the affinity of the aur-
one derivative 3 for Ab aggregates were carried out in solution with
[
[
125I]IMPY as the ligand. The compound inhibited the binding of
Figure 1. Flow chart of the development of PET/SPECT dual imaging probes for Ab
plaques.
125I]IMPY to Ab(1–42) aggregates in a dose-dependent manner,
indicating an affinity for Ab aggregates (Fig. 2). The Ki value esti-
mated for 3 was 6.81 nM. This value suggested that 3 had binding
affinity sufficient for the in vivo imaging of Ab(1–42) aggregates in
the brain. The affinity was in the same range as that of aurone
derivatives reported previously,22,23 indicating no decrease in
binding even though the iodide and the fluoroethoxy group were
both introduced into the aurone scaffold.
In the present study, we designed and synthesized a new fluo-
rinated and iodinated aurone derivative (FIAR) with a 2-fluoroeth-
oxy group at the 40 position of AR-1 (Fig. 1) and evaluated its
biological potential as a PET/SPECT dual imaging probe for Ab by
testing its affinity for Ab aggregates in vitro and its uptake by
and clearance from the brain in biodistribution experiments using
normal mice.
The target aurone derivatives were prepared as shown in
Schemes 1 and 2. The synthesis of the aurone backbone was
achieved by an Aldol reaction of benzofuranone with benzaldehyde
using Al2O3.24 In this process, 5-iodobenzofuran-3(2H)-one was re-
acted with 4-(2-fluoroethoxy)benzaldehyde 2 or 4-(2-hydroxyeth-
oxy)benzaldehyde 1 in the presence of Al2O3 in chloroform at room
temperature to form 3 and 5 in yields of 27% and 48%, respectively.
The tributyltin derivative 4 was prepared from the corresponding
compound 3 using a halogen to tributyltin exchange reaction cat-
alyzed by Pd(0) for yields of 32.2%. The tributyltin derivative was
used as the starting material for radioiodination in the preparation
of [125I]3. [125I]3 was achieved by an iododestannylation reaction
using hydrogen peroxide as the oxidant, which produced the de-
To confirm the affinity of aurone derivatives for Ab plaques in
the mouse brain, neuropathological staining with 3 was carried
out using brain sections from transgenic mouse (Tg2576)
(Fig. 3). Many Ab plaques were clearly stained with 3, as reflected
by the high affinity for Ab aggregates in inhibition assays in vitro
(Fig. 3A). The labeling pattern of 3 was consistent with that of
thioflavin-S, a dye commonly used for the detection of Ab pla-
ques (Fig. 3B), indicating that 3 has affinity for Ab plaques in
the mouse brain in addition to having binding affinity for syn-
thetic Ab42 aggregates. Conversely, no apparent staining of 3
was observed in age-matched control mouse brain sections
(Fig. 3C).
Furthermore, we investigated the affinity of [125I]3 for Ab pla-
ques by in vitro autoradiography in human AD brain sections
(Fig. 4). The autoradiographic images of [125I]3 showed high levels
Scheme 1. Synthesis of [125I]3.

H. Watanabe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6519–6522
6521
Scheme 2. Synthesis of [18F]3.
immunohistochemical staining (Fig. 4B). In contrast, normal hu-
man brain displayed no remarkable accumulation of
125I]3
(Fig. 4C). These results demonstrate the feasibility of using 3 as a
probe for detecting Ab plaques in the brains of AD patients by
PET/SPECT.
110
90
[
70
The biodistribution of [125I]3 and [18F]3 in vivo was tested in
normal mice (Table 1). A biodistribution study provides important
information on brain uptake. The ideal Ab imaging probe should
have good blood–brain barrier penetration to deliver a sufficient
dose into the brain while achieving rapid clearance from normal
regions to result in a higher signal-to-noise ratio in the AD brain.
The initial brain uptake of [125I]3 was 2.34% of injected dose/gram
at 2 min postinjection, whereas the radioactivity accumulated in
the brain was rapidly eliminated (0.19% of injected dose/gram at
60 min postinjection), properties highly desirable for Ab imaging
probes. [18F]3 displayed high uptake (3.66% of injected dose/gram
at 2 min postinjection) into and rapid clearance (1.75% of injected
50
30
10
-10
0
2
4
6
8
log[inhibitor] (pM)
Figure 2. Competition curve for 3 against [125I]IMPY.
of radioactivity in the brain sections (Fig. 4A). We confirmed that
the hot spots of [125I]3 corresponded with those of in vitro
Figure 3. Fluorescent staining of 3 in 10-lm Tg2576 mouse brain sections (A). Labeled plaques were confirmed by staining of the adjacent sections with thioflavin-S (B). No
apparent staining of 3 was observed in the age-matched control mouse brain section (C).
Figure 4. In vitro ARG of [125I]3 revealed the distinct labeling of Ab plaques in AD brain sections (A). The presence and localization of plaques in the sections were confirmed
with immunohistochemical staining using a monoclonal b-amyloid antibody (B). Conversely, there was very little labeling of [125I]3 in a control brain section (C).

6522
H. Watanabe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6519–6522
Table 1
in-aid for Young Scientists (A) and Exploratory Research from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan.
Biodistribution of radioactivity after injection of [125I]3 and [18F]3 in normal micea
Tissue
Time after injection (min)
10 30
2
60
Supplementary data
[
125I]3 (log P = 2.45 0.04)b
Blood
Liver
6.70 (2.97)
3.13 (0.58)
16.50 (1.65)
7.53 (0.85)
6.53 (1.44)
4.33 (0.87)
1.33 (0.33)
1.95 (0.26)
1.46 (1.85)
0.84 (0.23)
1.94 (0.21)
1.29 (0.14)
17.06 (1.48)
6.96 (0.76)
2.02 (0.48)
3.74 (0.48)
3.44 (0.58)
4.90 (0.86)
0.61 (0.07)
2.34 (0.36)
12.44 (0.72)
5.49 (1.21)
12.35 (1.21)
3.61 (0.85)
0.80 (0.16)
1.54 (0.22)
0.79 (0.51)
0.19 (0.03)
10.96 (2.06)
5.22 (1.95)
15.74 (1.12)
2.37 (0.59)
1.35 (0.09)
1.19 (0.53)
0.89 (0.35)
0.12 (0.06)
Supplementary data (procedure for the preparation of FIAR,
in vitro binding assay, in vitro autoradiography using AD brain sec-
tions, and biodistribution studies) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2011.08.063.
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachc
Brain
References and notes
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observed between the 125I and 18F tracers. A specific plaque label-
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Acknowledgments
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The study was supported by the Funding Program for Next Gen-
eration World-Leading Researchers (NEXT Program), and a Grant-