Push-pull benzothiazole derivatives as probes for detecting β-amyloid plaques in Alzheimer's brains

Article abstract of DOI:10.1016/j.bmc.2009.08.032

We synthesized push-pull benzothiazole derivatives and evaluated their potential as β-amyloid imaging probes. In binding experiments in vitro, the benzothiazoles showed excellent affinity for synthetic Aβ(1-42) aggregates. β-Amyloid plaques in the mouse and human brain were clearly visualized with the benzothiazoles, reflecting the results in vitro. These compounds may be a useful scaffold for the development of novel PET/SPECT and fluorescent tracers for detecting β-amyloid in Alzheimer's brains.

Full text of DOI:10.1016/j.bmc.2009.08.032

Bioorganic & Medicinal Chemistry 17 (2009) 7002–7007
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Push–pull benzothiazole derivatives as probes for detecting b-amyloid
plaques in Alzheimer’s brains
Masahiro Ono a, , , Shun Hayashi a, , Hiroyuki Kimura a, Hidekazu Kawashima b, Morio Nakayama c,
*
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
c Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized push–pull benzothiazole derivatives and evaluated their potential as b-amyloid imaging
probes. In binding experiments in vitro, the benzothiazoles showed excellent affinity for synthetic Ab(1-
42) aggregates. b-Amyloid plaques in the mouse and human brain were clearly visualized with the ben-
zothiazoles, reflecting the results in vitro. These compounds may be a useful scaffold for the development
of novel PET/SPECT and fluorescent tracers for detecting b-amyloid in Alzheimer’s brains.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 June 2009
Revised 4 August 2009
Accepted 4 August 2009
Available online 20 August 2009
Keywords:
b-Amyloid
Push–pull dye
Imaging
Alzheimer’s disease
1. Introduction
from the brain, and absorbs and emits within the near-infrared re-
gion (650–900 nm), often called the ‘optical window’ (Fig. 1).17
A
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 for this purpose, including PET/
SPECT and MRI tracers, have been developed.3–12 The PET ligand
series of NIAD derivatives have been designed and synthesized
based on a classical push–pull architecture with terminal donor
(hydroxy or dimethylamino group) and acceptor (dicyanomethyl-
ene group) moieties that are interconnected by a highly polarized
bridge (dithienylethenyl group), because various donor and accep-
tor groups can be used to manipulate the relative energies of
HOMO and LUMO and obtain the desired long wavelength of
absorption/emission bands.17
On the basis of this approach to the molecular design, we
planned to develop novel push–pull dyes for detecting Ab plaques
in the brain. We selected benzothiazole or styrylbenzothiazole as
the highly polarized bridge, a dimethylamino group as the donor,
and a dicyanomethylene group as the acceptor. In the present
study, we designed and synthesized two benzothiazole-derived
push–pull dyes (PP-BTA-1 and PP-BTA-2 in Fig. 2), and evaluated
their biological potential as probes for detecting Ab plaques in
the brain. To our knowledge, this is the first time push–pull benzo-
thiazole derivatives have been proposed as Ab imaging probes for
detecting AD.
[
11C]-2-(4-(methylamino)phenyl)-6-hydroxybenzothiazole (6-OH-
BTA-1 or PIB) with a benzothiazole backbone (Fig. 1) has shown
particular promise in early clinical trials and is currently being
used in a number of human studies.13–15 In addition to PET/SPECT
and MRI probes, much attention has focused on the development
of near-infrared fluorescent (NIRF) probes targeting Ab pla-
ques.16–18 NIRF probes are typically small molecule fluorescent
dyes designed to absorb and emit light in the near-infrared region,
where tissue scattering and absorption is lowest. The simple syn-
thesis, low-cost, and long shelf-life of NIRF probes, together with
the low-cost of optical imaging devices, present an attractive alter-
native to MRI and PET/SPECT techniques.
Among NIRF probes reported, to date, NIAD crosses the blood–
brain barrier, selectively binds Ab with high affinity, clears quickly
2. Results and discussion
* 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).
The target benzothiazole derivatives were prepared as shown in
Schemes 1 and 2. PP-BTA-1 (4) was successfully synthesized in a
yield of 21.4% according to methods reported previously (Scheme
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.032

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
7003
N
NH
S
HO
PIB
CN
CN
CN
CN
HO
N
S
S
S
S
NIAD-4
NIAD-16
Figure 1. Chemical structures of PIB, NIAD-4 and NIAD-16.
ties (absorption/emission wavelengths) of PP-BTA-1 and PP-BTA-2.
PP-BTA-1 and PP-BTA-2 exhibited absorption/emission peaks at
540/634 nm and 410/529 nm in EtOH, respectively. The extension
NC
NC
CN
CN
N
S
N
S
of
with longer wavelengths. However, PP-BTA-2 showed a shorter
wavelength than PP-BTA-1 despite a longer -conjugation. On
p-conjugation generally leads to absorption/emission bands
N
N
p
PP-BTA-1
PP-BTA-2
the other hand, because the wavelength of PP-BTA-1 is close to
the near-infrared region, a slight modification should lead to a
wavelength appropriate for imaging in vivo. Furthermore, when
PP-BTA-1 and PP-BTA-2 existed in a solution containing Ab(1-42)
aggregates, the fluorescence intensity of PP-BTA-1 and PP-BTA-2
increased with the concentration of Ab(1-42) aggregates, indicat-
ing affinity for Ab aggregates (Fig. 3).
To quantify the affinity of push–pull benzothiazole derivatives
for Ab plaques, we carried out inhibition assays on the binding to
Ab(1-42) aggregates with thioflavin T as a competing ligand. PP-
BTA-1 and PP-BTA-2 displaced thioflavin T in a dose-dependent
manner, indicating that they have affinity for Ab(1-42) aggregates
(Fig. 4). In addition, this result suggests that PP-BTA-1 and PP-BTA-
2 may occupy a binding site on Ab aggregates similar to that of thi-
oflavin T. The apparent IC50 values for PP-BTA-1, PP-BTA-2 and PIB
Figure 2. Chemical structures of push–pull benzothiazole derivatives reported in
this paper.
1).19 The formation of styrylbenzothiazole in the synthesis of PP-
BTA-2 (7) (Scheme 2) was achieved by a Wadsworth–Emmons
reaction between diethyl (4-cyanobenzyl)phosphonate and 6-dim-
ethylaminobenzothiazole-2-carbaldehyde. The desired (E)-styryl-
benzothiazole derivative was prepared in a yield of 23.0%. The
cyano group was converted to a formyl group by a reaction with
DIBAL-H as reported.20 The target PP-BTA-2 was prepared by the
condensation of carbaldehyde with malononitrile.
NIRF imaging in vivo requires the development of new fluores-
cent compounds with optimal fluorescent properties and high
affinity for Ab plaques. First, we evaluated the fluorescent proper-
were 0.12, 0.11 and 0.67
lM, respectively (Table 1). The IC50 of
(CH2O)n,
H2SO4,
Fe, THF
Fe, HCl,
EtOH/H2O
N
N
S
N
S
N
S
1
O2N
H2N
2
CH2(CN)2,
i-PrOH,
pyridine
n-BuLi,
THF,
NC
N
S
CN
N
S
DMF
CHO
N
N
3
PP-BTA-1(4)
Scheme 1.
MeOH,
NaOMe
N
S
CN
N
+
CN
CHO
(OEt)2(O)P
N
S
5
N
NC
CH2(CN)2,
i-PrOH,
pyridine
CN
DIBAL-H,
THF
CHO
N
S
N
S
N
N
6
PP-BTA-2 (7)
Scheme 2.

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M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
Figure 3. Ab-dependent change in the fluorescence spectra of PP-BTA-1 and PP-BTA-2. Green, blue and red lines show the fluorescence spectrum of 0, 5 and 10
Ab(1-42) aggregates, respectively.
lg/mL of
Figure 4. Inhibition assays of PP-BTA-1 and PP-BTA-2 using thioflavin T as the ligand in Ab(1-42) aggregates. Fluorescence spectral change of thioflavin T (3
addition of 0.0611 (orange line), 0.122 (cyan line), 0.486 (red line), or 2.65 (blue line) M of PP-BTA-1 and PP-BTA-2 to Ab(1-42) aggregates (10 g/mL). A pink line shows the
M) with Ab(1-42) aggregates. A green line shows the fluorescence spectrum of thioflavin T (3 M) alone.
lM) upon
l
l
fluorescence spectrum of thioflavin T (3
l
l
type mice displayed no remarkable accumulation of PP-BTA-1
and PP-BTA-2 in brain sections. These results suggest that PP-
BTA-1 and PP-BTA-2 show affinity for Ab plaques in the mouse
brain in addition to having affinity for synthetic Ab(1-42)
aggregates.
Table 1
Apparent inhibition constants (IC50
thioflavin T to Ab(1-42) aggregates
,
l
M) of benzothiazoles for the binding of
a
Compound
IC50
(
lM)
PP-BTA-1 (4)
PP-BTA-2 (7)
PIB
0.12 0.001
0.11 0.001
0.67 0.11
Furthermore, we also investigated the effectiveness of PP-BTA-1
and PP-BTA-2 for neuropathological staining of Ab plaques in hu-
man AD brain sections (Fig. 6). A previous report suggested the
configuration/folding of Ab plaques in Tg2576 mice to be different
from the tertiary/quaternary structure of Ab plaques in AD
brains.21 Therefore, it is important to evaluate the binding affinity
for Ab plaques in human AD brains. PP-BTA-1 and PP-BTA-2 clearly
stained many neuritic plaques in AD brains (Fig. 6A and D). In con-
trast, no apparent staining was observed in adult normal brain sec-
tions (Fig. 6C and F). The labeling pattern was consistent with that
observed by immunohistochemical labeling with an antibody spe-
cific to Ab Fig. 6B and E), indicating that PP-BTA-1 and PP-BTA-2
may be applicable for in vivo imaging of Ab plaques in Alzheimer’s
brains and deserve further investigation as a potential tool for early
diagnosis.
a
Each value represents the mean standard error of the mean for three inde-
pendent experiments.
PP-BTA-1 and PP-BTA-2 was lower than that of PIB, which is
commonly used for clinical research, indicating PP-BTA-1 and PP-
BTA-2 to have greater affinity for Ab(1-42) aggregates. While PP-
BTA-1 does not have the phenyl group in the phenylbenzothiazole
structure that PIB possesses, it showed stronger binding to Ab
aggregates than PIB. Moreover, benzothiazole is a compact mole-
cule advantageous for penetration of the blood–brain barrier after
administration in vivo. These results suggest benzothiazole to be a
useful scaffold for the development of Ab imaging agents in vivo.
Next, the usefulness of PP-BTA-1 and PP-BTA-2 for neuropatho-
logical staining of Ab plaques was investigated in an animal model
of AD, the Tg2576 mouse, specifically engineered to overproduce
Ab plaques in the brain. PP-BTA-1 and PP-BTA-2 clearly stained
the plaques as reflected by the high affinity for Ab aggregates in
in vitro competition assays (Fig. 5). The labeling pattern was
consistent with that observed with thioflavin S. In contrast, wild-
Since PP-BTA-1 and PP-BTA-2 possess a dimethylamino group,
they can be used as probes for PET by labeling one of two methyl
groups with 11CH3. In addition, for the application of push–pull
benzothiazole derivatives to optical imaging in vivo, the fine-tun-
ing of absorption/emission wavelengths to
a desired region
continues by optimizing the combination of donor and acceptor
groups.

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
7005
Figure 5. Neuropathological staining of PP-BTA-1 and PP-BTA-2 in 10
lm sections from a mouse model of AD (A and D) and a wild-type mouse (C and F). Ab plaques labeled
with PP-BTA-1 and PP-BTA-2 were confirmed by staining of the serial sections using thioflavin S (B and E).
Figure 6. Neuropathological staining of 5 lm AD brain sections from the temporal cortex (A, B, D and E) and adult normal temporal brain sections (C and F). Many neuritic
plaques are clearly stained with PP-BTA-1 (A) and PP-BTA-2 (D). Intense fluorescence can be seen in the core of neuritic plaques. Ab immunostaining with anti Ab antibodies
in the serial sections shows an identical staining pattern of plaques (B and E). No apparent staining was observed in adult normal brain sections (C and F).
Germany). Other reagents were of reagent grade and used without
further purification unless otherwise indicated.
3. Conclusion
In conclusion, we successfully designed and synthesized benzo-
thiazole-derived push–pull dyes for imaging Ab plaques in the
brain. In binding experiments in vitro, these benzothiazole com-
pounds showed high affinity for Ab(1-42) aggregates. PP-BTA-1
and PP-BTA-2 clearly stained Ab plaques in both mouse brain
and human brain, reflecting their affinity for Ab aggregates in vitro.
These findings suggest that additional structural changes on the
benzothiazole backbone may be applied to potential Ab probes
for not only optical imaging but also PET and SPECT.
4.1. Chemistry
4.1.1. 1,3-Benzothiazol-6-amine (1)
To a mixture of 6-nitrobenzothiazole (2.5 g, 13.9 mmol) and
concentrated HCl (1.93 mL, 22.7 mmol) in 80% EtOH (63 mL)
was added powdered iron (3.7 g, 55.6 mmol). The reaction
mixture was stirred for 1 h under reflux, and then cooled to
room temperature. The precipitate of iron oxides and hydroxy
salts was removed by filtration. The solvent was removed and
the solid residue was extracted into a heterogeneous mixture
of EtOAc (50 mL Â 2) and a 10% aqueous solution of Na2CO3
(50 mL). The EtOAc extract was dried (Na2SO4) and the solvent
was removed under vacuum to yield 1 (1.91 g, 91.7%). 1H NMR
(400 MHz, CDCl3) d 8.70 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.17
(d, J = 2.4 Hz, 1H), 6.87 (dd, J = 8.8, 2.4 Hz, 1H), 3.85 (br s, 2H).
MS m/z 151 [MH+].
4. Experimental
1H NMR spectra were obtained on a JEOL JNM-LM400 with TMS
as an internal standard. Coupling constants are reported in hertz.
Multiplicity was defined by s (singlet), d (doublet), t (triplet), br
(broad) and m (multiplet). Mass spectra were obtained on a SHI-
MADZU LCMS-2010 EV. PIB was purchased from ABX (Radeberg,

7006
M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
4.1.2. N,N-Dimethyl-1,3-benzothiazol-6-amine (2)
2H), 7.85 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.50 (d,
J = 16.4 Hz, 1H), 7.38 (d, J = 16.4 Hz, 1H), 7.07 (d, J = 2.4 Hz, 1H),
6.96 (dd, J = 8.8, 2.4 Hz, 1H), 3.06 (s, 6H). MS m/z 309 [MH+].
A solution of 1 (1.47 g, 9.8 mmol) in THF (40 mL) was slowly
added to
a stirred mixture of 40% aqueous formaldehyde
(7.24 mL, 98 mmol) and 4 M H2SO4 (7.95 mL, 29.4 mL). Powdered
iron (4.36 g, 78.4 mL) was then added and the mixture was vigor-
ously stirred for 3 h. The precipitate of iron salts was removed by
filtration and washed with EtOAc (20 mL Â 2). The combined or-
ganic solutions were made strongly basic with 1 N NaOH (50 mL)
and extracted with EtOAc (50 mL Â 2). The combined EtOAc ex-
tracts were dried (Na2SO4) and the solvent was removed on a ro-
tary vacuum evaporator. The oily residue was purified by silica
4.1.7. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzylidene)malononitrile (PP-BTA-2, 7)
The same reaction as described above to prepare 5 was used,
and 45 mg of 7 was obtained in a 63.5% yield from 6. 1H NMR
(400 MHz, CDCl3) d 7.94 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.8 Hz, 1H),
7.73 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 16.4 Hz, 1H), 7.35
(d, J = 16.4 Hz, 1H), 7.08 (s, 1H), 6.97 (d, J = 10.0 Hz , 1H), 3.08 (s,
6H). MS m/z 357 [MH+]. Anal. Calcd for C21H16N4S: C, 70.76; H,
4.52; N, 15.72; S, 9.00. Found: C, 70.48; H, 4.57; N, 15.43; S, 8.99.
gel chromatography (hexane/EtOAc = 4:1) to give
2 (460 mg,
26.3%). 1H NMR (400 MHz, CDCl3)
d
8.67 (s, 1H), 7.95 (d,
J = 8.8 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz,
1H), 3.04 (s, 6H). MS m/z 179 [MH+].
4.2. Fluorescence experiments
4.1.3. 6-(Dimethylamino)-1,3-benzothiazole-2-carbaldehyde (3)
To a vigorously stirred solution of n-BuLi (0.5 mL, 2.6 M in hex-
ane, 1.3 mmol) in THF (5.8 mL) at À78 °C under N2 was added
slowly a solution of 2 (220 mg, 1.23 mmol). The reaction mixture
was stirred, warmed to À50 °C and after 1 h cooled to À78 °C. To
the resulting solution of aryllithium salt was added slowly anhy-
drous DMF (0.38 mL). The solution was stirred for 2 h, poured into
H2O (9 mL), neutralized with an aqueous saturated solution of
NH4Cl and subsequently extracted with EtOAc (20 mL Â 2). The
combined extracts were dried over Na2SO4 and the solvent was re-
PP-BTA-1 and PP-BTA-2 were dissolved in 5% EtOH at 10 lM.
The fluorescence of PP-BTA-1 and PP-BTA-2 was measured with a
spectrophotometer (RF-1500, Shimadzu, Japan). For some mea-
surements, the spectra of PP-BTA-1 and PP-BTA-2 were determined
with or without Ab(1-42) aggregates (0, 5 and 10 lM).
4.3. Binding experiments using Ab(1-42) aggregates
A solid form of Ab(1-42) was purchased from Peptide Institute
(Osaka, Japan). Aggregation was carried out by gently dissolving
the peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was incu-
bated at 37 °C for 42 h with gentle and constant shaking. Thiofla-
vin-T was used as the tracer for the competition binding
experiments. A mixture (3.6 mL of 10% EtOH) containing PP-BTA-
moved under vacuum to give
3
(255 mg, 97.3%). 1H NMR
(400 MHz, CDCl3) d 10.06 (s, 1H), 8.03 (d, J = 10.0 Hz, 1H), 7.07–
7.04 (m, 2H), 3.12 (s, 6H). MS m/z 207 [MH+].
4.1.4. ((6-(Dimethylamino)-1,3-benzothiazol-2-yl)methylene)-
malononitrile (PP-BTA-1, 4)
1, PP-BTA-2 and PIB (final concn 61.1 nMÀ5.48
l
M), thioflavin-T
A solution of 3 (124 mg, 0.6 mmol), malononitrile (60 mg,
0.9 mmol) and pyridine (0.12 mL) in 2-propanol (7.2 mL) was stir-
red and refluxed for 30 min. The mixture was poured into H2O
(20 mL) and extracted with CHCl3 (20 mL Â 3). The combined ex-
tracts were dried over Na2SO4 and the solvent was removed under
vacuum to give 4 (152 mg, 91.7%). 1H NMR (400 MHz, CDCl3) d 7.99
(s, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.08 (dd, J = 9.2, 2.4 Hz, 1H), 7.02 (d,
J = 2.4 Hz, 1H), 3.16 (s, 6H). MS m/z 255 [MH+]. Anal. Calcd for
C13H10N4S: C, 61.40; H, 3.96; N, 22.03; S, 12.61. Found: C, 61.34;
H, 3.84; N, 21.82; S, 12.64.
(final concn 3 M), and Ab(1-42) aggregates (final concn 10 l
l
g/
mL) was incubated at room temperature for 10 min. Fluorescence
intensity at an excitation wavelength of 445 nm was plotted, and
values for the apparent half-maximal inhibitory concentration
(IC50) were determined from a calibration curve of fluorescence
intensity at 478 nm in three independent experiments.
4.4. Staining of Ab plaques in Tg2576 mouse brain sections
Theexperimentswithanimalswereconductedinaccordancewith
our institutional guidelines and approved by the Kyoto University
Animal Care Committee. The Tg2576 transgenic mice (female, 27-
month-old) and wild-type mice (female, 27-month-old) were used
as the Alzheimer’s model and control mice, respectively. After the
miceweresacrificedbydecapitation, thebrainswereimmediatelyre-
moved and frozen in powdered dry ice. The frozen blocks were sliced
4.1.5. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzonitrile (5)
To
a solution of (4-cyanobenzyl)phosphonate (403.6 mg,
1.6 mmol) in MeOH (12.8 mL) was added NaOMe (0.632 mL). The
mixture was cooled in an ice bath, and stirred under reflux for
3 h after the addition of 3 (330 mg, 1.6 mmol). The solid that
formed in the reaction mixture was filtered to give 5 (385 mg,
78.8%). 1H NMR (400 MHz, CDCl3) d 7.84 (d, J = 9.6 Hz, 1H), 7.64
(dd, J = 21.2, 8.0 Hz, 4H), 7.45 (d, J = 16.4 Hz, 1H), 7.32 (d,
J = 16.4 Hz, 1H), 7.06 (d, J = 2.8 Hz, 1H), 6.95 (dd, J = 9.6, 2.8 Hz,
1H), 3.06 (s, 6H). MS m/z 306 [MH+].
into serial sections, 10
lm thick. Each slide was incubated with a 50%
EtOH solution (100 M) of PP-BTA-1 and PP-BTA-2 for 10 min. The
l
sectionswerewashedin50%EtOHfor1 mintwotimes,andexamined
using a microscope(Nikon Eclipse 80i) equippedwith a G-2Afilter set
(excitation, 510–560 nm; diachronic mirror, 575 nm; longpass filter,
470 nm) for PP-BTA-1, and a B-2A filter set (excitation, 450–480 nm;
diachronic mirror, 505 nm; longpass filter, 520 nm) for PP-BTA-2.
Thereafter, the serial sections were also stained with thioflavin S, a
pathological dye commonly used for staining Ab plaques in the brain,
andexaminedusingamicroscope(NikonEclipse80i)equippedwitha
BV-2A filter set (excitation, 400–440 nm; diachronic mirror, 455 nm;
longpass filter, 470 nm).
4.1.6. 4-((E)-2-(6-(Dimethylamino)-1,3-benzothiazol-2-yl)
vinyl)benzaldehyde (6)
To a solution of 5 (61 mg, 0.2 mmol) in THF (3.3 mL) was added
DIBAL-H (1 M in hexane, 0.5 mL) at À78 °C. The reaction mixture
was stirred at room temperature overnight. Thereafter, 10% acetic
acid (15 mL) was added and the mixture was extracted with CHCl3
(20 mL Â 2). After the organic layer was washed with saline, the
combined extracts were dried over Na2SO4. The residue was purified
by silica gel chromatography (hexane/EtOAc = 2:1) to give 6 (28 mg,
45.4%). 1H NMR (400 MHz, CDCl3) d 10.02 (s, 1H), 7.90 (d, J = 8.4 Hz,
4.5. Staining of Ab plaques in human AD brain sections
Postmortem brain tissues from an autopsy-confirmed case of AD
(73-year-old male) and a control subject (36-year-old male) were

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 7002–7007
7007
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temperature. The sections were washed in 50% EtOH for 1 min two
times, and examined using a microscope (Nikon Eclipse 80i)
equipped with a G-2A filter set (excitation, 510–560 nm; diachronic
mirror, 575 nm; longpassfilter, 470 nm) for PP-BTA-1, and a B-2Afil-
ter set (excitation, 450–480 nm; diachronic mirror, 505 nm; long-
pass filter, 520 nm) for PP-BTA-2. The presence and localization of
plaques on the same sections were confirmed with immunohisto-
chemical staining using a monoclonal Ab antibody, BC05 (Wako).
Acknowledgements
11. Higuchi, M.; Iwata, N.; Matsuba, Y.; Sato, K.; Sasamoto, K.; Saido, T. C. Nat.
Neurosci. 2005, 8, 527.
12. Poduslo, J. F.; Curran, G. L.; Peterson, J. A.; McCormick, D. J.; Fauq, A. H.; Khan,
M. A.; Wengenack, T. M. Biochemistry 2004, 43, 6064.
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
Research from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.
13. Bacskai, B. J.; Frosch, M. P.; Freeman, S. H.; Raymond, S. B.; Augustinack, J. C.;
Johnson, K. A.; Irizarry, M. C.; Klunk, W. E.; Mathis, C. A.; Dekosky, S. T.;
Greenberg, S. M.; Hyman, B. T.; Growdon, J. H. Arch. Neurol. 2007, 64, 431.
14. Johnson, K. A.; Gregas, M.; Becker, J. A.; Kinnecom, C.; Salat, D. H.; Moran, E. K.;
Smith, E. E.; Rosand, J.; Rentz, D. M.; Klunk, W. E.; Mathis, C. A.; Price, J. C.;
Dekosky, S. T.; Fischman, A. J.; Greenberg, S. M. Ann. Neurol. 2007, 62, 229.
15. Pike, K. E.; Savage, G.; Villemagne, V. L.; Ng, S.; Moss, S. A.; Maruff, P.; Mathis, C.
A.; Klunk, W. E.; Masters, C. L.; Rowe, C. C. Brain 2007, 130, 2837.
16. Hintersteiner, M.; Enz, A.; Frey, P.; Jaton, A. L.; Kinzy, W.; Kneuer, R.; Neumann,
U.; Rudin, M.; Staufenbiel, M.; Stoeckli, M.; Wiederhold, K. H.; Gremlich, H. U.
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