Development of dual functional SPECT/fluorescent probes for imaging cerebral β-amyloid plaques

Article abstract of DOI:10.1016/j.bmcl.2010.05.027

The imaging of β-amyloid (Aβ) aggregates in the brain may lead to the early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop dual modality SPECT and fluorescent probes based on boron dipyrromethane (BODIPY) as a core structure. We designed and synthesized an ¹²⁵I-labeled derivative of BODIPY (BODIPY7). BODIPY7 had a K _i value of 108 nM for Aβ(1-42) aggregates and exhibited peaks of absorption/emission at 606/613 nm. It detected Aβ plaques in sections of brain tissue from an animal model of AD and displayed low uptake in the brain and high uptake in the liver in normal mice. Although additional modifications of the BODIPY scaffold are necessary to improve brain uptake, these results should aid the development of dual functional SPECT/fluorescent probes for the imaging of Aβ plaques in the brain.

Full text of DOI:10.1016/j.bmcl.2010.05.027

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3885–3888
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of dual functional SPECT/fluorescent probes for imaging cerebral
b-amyloid plaques
a
a
a
a
Masahiro Ono a, , Manami Ishikawa , Hiroyuki Kimura , Shun Hayashi , Kenji Matsumura ,
*
Hiroyuki Watanabe a, Yoichi Shimizu a, Yan Cheng a, Mengchao Cui a, Hidekazu Kawashima b, Hideo Saji a,
*
a Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The imaging of b-amyloid (Ab) aggregates in the brain may lead to the early detection of Alzheimer’s dis-
ease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study
was to develop dual modality SPECT and fluorescent probes based on boron dipyrromethane (BODIPY)
as a core structure. We designed and synthesized an 125I-labeled derivative of BODIPY (BODIPY7). BOD-
IPY7 had a Ki value of 108 nM for Ab(1–42) aggregates and exhibited peaks of absorption/emission at 606/
613 nm. It detected Ab plaques in sections of brain tissue from an animal model of AD and displayed low
uptake in the brain and high uptake in the liver in normal mice. Although additional modifications of the
BODIPY scaffold are necessary to improve brain uptake, these results should aid the development of dual
functional SPECT/fluorescent probes for the imaging of Ab plaques in the brain.
Received 20 April 2010
Revised 7 May 2010
Accepted 11 May 2010
Available online 16 May 2010
Keywords:
Alzheimer’s disease
b-Amyloid plaque
Positron emission tomography
Single photon emission computed
tomography
Ó 2010 Elsevier Ltd. All rights reserved.
Fluorescence
Dual imaging
The formation of b-amyloid (Ab) plaques is a key neurodegener-
ative event in Alzheimer’s disease (AD).1,2 Since the imaging of
these plaques in vivo may lead to the presymptomatic diagnosis
of AD, many molecular probes, including positron emission tomog-
raphy (PET)3–6 and single photon emission computed tomography
(SPECT)7,8 tracers labeled with a radioisotope (RI), have been devel-
oped. The PET ligand [11C]-2-(4-(methylamino)phenyl)-6-hydrox-
ybenzothiazole (PIB) with a benzothiazole backbone has shown
particular promise in early clinical trials and is currently being
used in a number of human studies.9,10 Nuclear imaging with
PET/SPECT probes is an established clinical modality that offers
good sensitivity deep in tissue, which permits whole body quanti-
tative imaging not only in small animals but also in humans. How-
ever, it is limited by factors such as a time-consuming data
acquisition process, expensive equipment, exposure to radioactiv-
ity, the need for highly skilled personnel, and a relatively poor spa-
tial resolution.11
tively new modality that offers real-time, nonradioactive, and,
depending on the technique, high-resolution imaging. Among NIRF
probes reported to date, NIAD-413 and CRANAD-215 cross the
blood–brain barrier, selectively bind Ab with high affinity, clear
quickly from the brain, and absorb and emit within the near-infra-
red region (650–900 nm), often called the ‘optical window’.16 Opti-
cal imaging techniques are not quantitative, especially when the
object is located deep to the skin because of significant signal
attenuation in tissue, but NIRF imaging has the potential to provide
a rapid, inexpensive, and nonradioactive drug screening system for
AD.
Currently, in vivo imaging of Ab plaques in AD brains is primar-
ily performed using nuclear imaging techniques such as SPECT and
PET. We hypothesize that the development of dual functional nu-
clear/fluorescent imaging probes not only can provide complemen-
tary information that may lead to improve diagnosis and
management of AD patients, but also can facilitate the validation
of optical imaging by standard nuclear imaging techniques.
Although several recent papers demonstrate the usefulness of dual
nuclear and fluorescent imaging probes targeting tumor,17,18 such
dual functional probes for Ab plaques have not been reported.
Here, we propose a design strategy for the development of a dual
SPECT/fluorescent probe for Ab plaques in the brain. In this study,
we selected boron dipyrromethane (BODIPY), one of the most use-
ful fluorophores,19–23 and introduced the radiolabeled moiety
In addition to PET/SPECT probes, much attention has focused on
the development of near-infrared fluorescent (NIRF) probes target-
ing Ab plaques.12–14 Optical imaging with NIRF probes is a rela-
* Corresponding authors. Tel.: +81 75 753 4608; fax: +81 75 753 4568 (M.O.); tel.:
+81 75 753 4556; fax: +81 75 753 4568 (H.S.).
E-mail addresses: ono@pharm.kyoto-u.ac.jp (M. Ono), hsaji@pharm.kyoto-u.ac.jp
(H. Saji).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.027

3886
M. Ono et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3885–3888
NIAD-4, 2-(4-[125I]iodophenyl)-5-thiophene, at the 3-position. De-
scribed herein is the synthesis and characterization of this radioio-
dinated derivative of BODIPY. To our knowledge, this is the first
time dual SPECT/fluorescent probes have been proposed as probes
for imaging Ab plaques in the brain.
The target BODIPY derivative was prepared as shown in
Scheme 1. The compound 1 was synthesized in a yield of 21.4%
by the Suzuki coupling reaction. After reduction of the aldehyde
to an alcohol by NaBH4, the desired Wittig reagent 3 was readily
prepared from 2 and triphenylphosphine. The compound 3 was
produced by a Wittig reaction between 3 and pyrroaldehyde. The
key step in the formation of the BODIPY backbone was accom-
plished by the condensation of pyrrole-2-carboxylaldehyde and 4
at low temperature, followed by the addition of BF3ÁOEt2. The bro-
mo compound (5) was reacted with bis(tributyltin) using Pd(0) as a
catalyst, and the corresponding tributyltin derivative (6) was ob-
tained in a yield of 17.0%. The tributyltin derivative (6) was readily
reacted with iodine in chloroform at room temperature to give the
iodo derivative (7) in a yield of 20.0%. The radioiodination was
achieved by the same iododestannylation reaction using hydrogen
peroxide as the oxidant, which produced the desired radioiodin-
ated ligand, [125I]BODIPY7, in a yield of 25% and with greater than
95% radiochemical purity. It is anticipated that the no-carrier-
added preparation resulted in the final product bearing similar the-
oretical specific activity to 125I.
Figure 1. Curve of BODIPY7’s inhibition of the binding of [125I]IMPY to Ab(1–42)
aggregates.
[
125I]IMPY as a competing radioligand. BODIPY7 inhibited the
binding of [125I]IMPY in a dose-dependent manner, indicating that
it has affinity for Ab(1–42) aggregates (Fig. 1). This result suggests
that the binding sites of BODIPY7 and IMPY partly overlap. BODI-
PY7 had a Ki value of 108 nM, indicating sufficiently high affinity
for Ab aggregates. A recent paper reported that the introduction
of the triazole moiety into the BODIPY scaffold afforded dyes that
allowed for the unambiguous differentiation of an unordered
conformation, which is mostly a benign form, from an ordered
To quantify the affinity of BODIPY7 for Ab plaques, we carried
out inhibition assays on the binding to Ab(1–42) aggregates with
Br
a
S
b
S
(HO)2B
CHO
+
I
Br
CHO
1
Br
Br
S
S
d
c
P+Ph3
OH
2
3
Br
e
f
S
N
F
N
F
N
H
B
S
4
5
Br
N
F
N
F
g
N
F
N
F
B
B
S
S
7
6
(BODIPY7)
SnBu3
I
h
N
F
N
F
B
S
[
125I]7
([125I]BODIPY7)
125I
Scheme 1. Reagents: (a) dioxane, (Ph3P)4Pd, Na2CO3; (b) MeOH, NaBH4; (c) CHCl3, Ph3PÁHBr; (d) MeOH, NaOMe, 2-formylpyrrole; (e) CH2Cl2, 3,5-dimethylpyrrole-2-
carboxaldehyde, POCl3, Et3N, EtOBF3; (f) dioxane, (Bu3Sn)2, (Ph3P)4Pd, Et3N; (g) CHCl3, I2; (h) EtOH, HCl, H2O2, [125I]NaI.

M. Ono et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3885–3888
3887
Table 1
Biodistribution of radioactivity after injection of [125I]BODIPY7 in normal micea
Tissue
Time after injection (min)
10 30
3.09 (0.17)
2
60
2.44 (1.25)
Blood
Liver
11.67 (1.70)
35.51 (3.62)
5.39 (0.34)
0.48 (0.07)
16.12 (3.89)
1.19 (0.35)
5.59 (1.74)
1.40 (0.10)
0.40 (0.05)
4.09 (0.25)
46.02 (1.80)
4.79 (0.50)
0.93 (0.16)
26.90 (3.98)
0.74 (0.17)
2.95 (0.50)
4.73 (1.86)
0.19 (0.02)
45.87 (2.60)
4.46 (0.41)
1.91 (0.17)
28.21 (3.79)
0.72 (0.15)
2.31 (0.53)
4.79 (1.12)
0.14 (0.01)
45.78 (3.58)
4.43 (0.41)
3.04 (0.18)
21.52 (8.99)
0.92 (0.28)
2.30 (0.35)
5.50 (0.51)
0.14 (0.01)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
a
Expressed as% injection dose per gram. Each value represents the mean (SD) for
4–6 animals.
b
Expressed as% injected dose per organ.
Figure 2. Neuropathological staining of BODIPY7 (A) in a 10-lm section from a
Tg2576 mouse brain. Labeled plaques were confirmed by staining of the adjacent
section with thioflavin S (B).
conformation, largely a neurotoxic form, of Ab(1–42) soluble olig-
omers.24 Although this Letter did not report the Ki value of the
triazole-containing BODIPY, we consider BODIPY7 to potentially
be suitable for monitoring conformational transitions of amyloid
species in vitro. Also, these results support the validity of using
BODIPY as a scaffold for probes to image Ab plaques in vivo.
Next, the usefulness of BODIPY7 for neuropathological staining
of Ab plaques was investigated in an animal model of AD, the
Tg2576 mouse, specifically engineered to overproduce Ab plaques
in the brain.25 BODIPY7 clearly stained the plaques as reflected by
its high affinity for Ab aggregates in in vitro inhibition assays
(Fig. 2A). The labeling pattern was consistent with that observed
with thioflavin S (Fig. 2B). In contrast, wild-type mice displayed
no remarkable accumulation of BODIPY7 in brain sections (data
not shown). These results suggest that BODIPY7 can function as a
probe for detecting Ab plaques in the brain and deserves further
investigation as a potential dual Ab imaging probe.
Figure 3. Absorption (dotted line) and emission (line) spectra of BODIPY7.
and the clear labeling of Ab plaques in mouse brain sections, the
radioiodinated BODIPY did not have characteristics for the imaging
of Ab plaques. Recently, it was reported that CRANAD-2 can pene-
trate the BBB and bind to Ab plaques in the brain in vivo, though it
possesses a difluoroboron core like BODIPY and a larger molecular
weight than BODIPY7.15 Therefore, additional structural changes
may modify the properties of BODIPY derivatives to improve their
suitability for imaging.
Radioiodinated BODIPY7 was tested in normal mice to assess its
ability to cross the blood–brain barrier (BBB) (Table 1). The uptake
of [125I]BODIPY7 in the brain was 0.4%ID/g at 2 min postinjection,
and most radioactivity had been washed out from the brain by
30 min postinjection (0.14%ID/g). Biodistribution experiments also
demonstrated high and persistent levels of radioactivity in the liver
and spleen, and a decrease in uptake into the lung with time. De-
spite its suitable lipophilicity (log P = 2.2) and reasonable molecu-
lar size (mol wt 363),10 the compound’s uptake in the brain was
relatively low, which may be explained by its rapid trapping in
the liver. An initial brain uptake higher than 0.5%ID/organ at
2 min postinjection is preferred for Ab imaging probes, and this ini-
tial uptake should remain at less than 30% at 30 min in normal
mouse brain because of the absence of Ab plaques. Although its ini-
tial uptakes fell short of these criteria, [125I]BODIPY7 showed a ra-
pid washout (30 min) with a value equal to 35% of the initial brain
uptake. Despite good affinity for synthetic Ab(1–42) aggregates
Although BODIPY7 had a high fluorescent quantum yield
(u = 0.36), it exhibited shorter wavelengths of absorption/emission
at 606/613 nm than are appropriate for optical imaging in vivo
(Fig. 3). Recent papers have reported the development of BODIPY
derivatives with absorption/emission bands in the near-infrared
region.26 By introducing additional structural changes to extend
the near-infrared wavelength simultaneously with modifications
to improve penetration for the BBB, more useful SPECT/fluorescent
probes may be developed in the future.
In conclusion, we designed and synthesized a radioiodinated
BODIPY derivative as a dual SPECT/fluorescent probe for imaging
Ab plaques in the brain. In binding experiments in vitro, the BODIPY
derivative showed high affinity for Ab(1–42) aggregates. BODIPY7
clearly stained Ab plaques in mouse brain, reflecting its affinity for
Ab aggregates in vitro. These findings suggest that additional struc-
tural changes to the BODIPY backbone may be applied to potential
dual SPECT/fluorescent probes for the imaging of Ab plaques.

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M. Ono et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3885–3888
7. Newberg, A. B.; Wintering, N. A.; Clark, C. M.; Plossl, K.; Skovronsky, D.; Seibyl,
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This study was supported by the Program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO), a Health Labour Sciences Research
Grant, and a Grant-in-aid for Young Scientists (A) and Exploratory
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Supplementary data
Supplementary data (procedure for the preparation of new
BODIPY derivatives, in vitro binding assay, in vitro fluorescent
staining using Tg2576 mouse brain sections, and biodistribution
studies) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2010.05.027.
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