18F-labeled flavones for in vivo imaging of β-amyloid plaques in Alzheimer's brains

Article abstract of DOI:10.1016/j.bmc.2009.01.025

In vivo imaging of β-amyloid (Aβ) aggregates in the brain may lead to early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop novel ¹⁸F-labeled amyloid-imaging probes based on flavones as a core structure. Fluoropegylated (FPEG) flavone derivatives were designed and synthesized. The affinity of the derivatives for Aβ aggregates varied from 5 to 321 nM. In brain sections of AD model mice, FPEG flavones with the dimethylamino group intensely stained β-amyloid plaques. In biodistrubution experiments using normal mice, they displayed high uptake in the brain ranging from 2.9 to 4.2%ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (1.3-2.0%ID/g at 30 min), which is highly desirable for β-amyloid imaging agents. FPEG flavones may be potential PET imaging agents for β-amyloid plaques in Alzheimer's brains.

Full text of DOI:10.1016/j.bmc.2009.01.025

Bioorganic & Medicinal Chemistry 17 (2009) 2069–2076
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
18F-labeled flavones for in vivo imaging of b-amyloid plaques
in Alzheimer’s brains
a
c
b
a
Masahiro Ono a,b, , Rumi Watanabe , Hidekazu Kawashima , Tomoki Kawai , Hiroyuki Watanabe ,
*
Mamoru Haratake a, Hideo Saji b, Morio Nakayama a,
*
a Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
c Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku, 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:
In vivo imaging of b-amyloid (Ab) aggregates in the brain may lead to 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 novel 18F-labeled amyloid-imaging probes based on flavones as a core structure. Fluorop-
egylated (FPEG) flavone derivatives were designed and synthesized. The affinity of the derivatives for Ab
aggregates varied from 5 to 321 nM. In brain sections of AD model mice, FPEG flavones with the dimeth-
ylamino group intensely stained b-amyloid plaques. In biodistrubution experiments using normal mice,
they displayed high uptake in the brain ranging from 2.9 to 4.2%ID/g at 2 min postinjection. The radioac-
tivity washed out from the brain rapidly (1.3–2.0%ID/g at 30 min), which is highly desirable for b-amyloid
imaging agents. FPEG flavones may be potential PET imaging agents for b-amyloid plaques in Alzheimer’s
brains.
Received 10 December 2008
Revised 6 January 2009
Accepted 7 January 2009
Available online 20 January 2009
Keywords:
Alzheimer’s disease
b-Amyloid plaque
PET
Flavone
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
studies with
[
18F]-2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)-
naphthalen-6-yl)ethylidene)malononitrile ([18F]FDDNP)11,12 showed
differential uptake and retention in the brain of AD patients for the
first time. More recently, a stilbene derivative, [18F]BAY94-9172,
has been shown to be useful for the imaging of b-amyloid plaques
in living human brain tissue in clinical trials.13
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder characterized by cognitive decline, irreversible memory
loss, disorientation, and language impairment. The presence of
b-amyloid (Ab) aggregates in the brain is generally accepted as a
hallmark of AD.1,2 Since the only definitive diagnosis of AD is by
pathological examination of postmortem staining of affected brain
tissues, the development of techniques which enable one to image
b-amyloid plaques in vivo has been strongly desired.3–5
To search for more candidates for 18F-labeled b-amyloid imag-
ing agents for PET, we planned to evaluate a new series of flavone
derivatives previously reported as useful for imaging b-amyloid by
single photon emission computed tomography (SPECT).14 The
derivatives showed good affinity for Ab aggregates in vitro in bind-
ing experiments using synthetic Ab aggregates and neuropatho-
logical staining of AD brain sections, suggesting these classes of
radioiodinated flavones to be potential imaging agents.
Recently, Kung et al. exploited a novel approach by using
fluoro-pegylation (FPEG) of the core structure for 18F labeling of
derivatives.15 Since this approach offers a simple and easy way to
incorporate 18F into the target without an appreciable increase in
lipophilicity, we planned to apply it to the labeling of flavone deriv-
atives. In addition to the structural characteristics of flavone as the
pharmacophore, it has been shown that electron-donating groups
such as amino, methylamino, dimethylamino, methoxy, or hydroxy
groups play a critical role in the binding of Ab aggregates.6,8,16,17
With these considerations, we designed 12 fluorinated flavones with
a fluorine or FPEGylation at position 4 and an electron-donating
group at position 40 (Fig. 1).
Recent success in developing radiolabeled agents targeting Ab
aggregates has provided a window of opportunity to improve the
diagnosis of AD. Preliminary reports of positron emission tomography
(PET) imaging suggested that [11C]4-N-methylamino-40-hydroxy-
stilbene (SB-13),6,7
[
11C] 2-(40-(methylaminophenyl)-6-hydroxy
benzothiazole (PIB),8,9 and [11C]2-(2-[2-dimethylaminothiazol-5-
yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole (BF-227)10 showed
differential uptake and retention in the brain of AD patients as
compared to controls. But 11C is a positron-emitting isotope with
a short t1/2 (20 min), which limits its clinical application. Recent
efforts have focused on the development of comparable agents
labeled with a longer half-life isotope, 18F (t1/2: 110 min). Preliminary
* 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, mono@net.nagasaki-u.ac.jp (M. Ono)
morio@nagasaki-u.ac.jp (M. Nakayama).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.025

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M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
2.1.4. 6-Methoxy-40-aminoflavone (4)
R2
A mixture of 3 (3.0 g, 10.2 mmol), SnCl2 (7.6 g, 39.8 mmol), and
EtOH (30 mL) was stirred under reflux for 40 min. After the mix-
ture had cooled to room temperature, 1 M NaOH (50 mL) was
added until the mixture became alkaline. After extraction with
ethyl acetate, the combined organic layers were washed with
brine, dried over Na2SO4, and filtered. The solvent was removed,
and the residue was purified by silica gel chromatography (hex-
ane/ethyl acetate = 1:2) to give 1.3 g of 4 (65.3% yield). 1H NMR
(300 MHz, CDCl3) d: 3.91 (s, 3H), 4.11 (s, broad, 2H), 6.69 (s, 1H),
6.75 (d, J = 6.6 Hz, 2H), 7.24–7.27 (m, 1H), 7.47 (d, J = 6.6 Hz, 1H),
7.59 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 6.6 Hz, 2H).
O
O
R1
Compound
8a
R1
R2
FCH2CH2O
F(CH2CH2O)2
F(CH2CH2O)3
FCH2CH2O
FCH2CH2O
F(CH2CH2O)2
F(CH2CH2O)3
F(CH2CH2O)2
F(CH2CH2O)3
F
N(CH3)2
N(CH3)2
N(CH3)2
NH2
NHCH3
NH2
8b
8c
12
13
15b
15c
17b
17c
21
22
23
NH2
2.1.5. 6-Methoxy-40-dimethylaminoflavone (5)
NHCH3
NHCH3
NH2
NHCH3
N(CH3)2
To a mixture of 4 (401 mg, 1.5 mmol) and paraformaldehyde
(450 mg, 15 mmol) in AcOH (10 mL) was added NaCNBH3
(471 mg, 7.5 mmol) in one portion at room temperature. The
resulting mixture was stirred at room temperature for 1.5 h, and
the addition of 1 M NaOH was followed by extraction with CH3Cl.
The organic phase was dried over Na2SO4. The solvent was re-
moved, and the residue was purified by silica gel chromatography
(CHCl3/MeOH = 20:1) to give 371 mg of 5 (83.7% yield). 1H NMR
(300 MHz, CDCl3) d: 3.07 (s, 6H), 3.91 (s, 3H), 6.70 (s, 1H), 6.75
(d, J = 9.0 Hz, 2H), 7.24–7.26 (m, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.59
(d, J = 2.2 Hz, 1H), 7.81 (d, J = 9.0 Hz, 2H).
F
F
Figure 1. Chemical structure of FPEG-flavone derivatives.
We report here the in vitro and in vivo evaluation of a new ser-
ies of flavone derivatives as agents for imaging b-amyloid with PET.
2. Experimental
2.1.6. 6-Hydroxy-40-dimethylaminoflavone (6)
All reagents were commercial products and used without fur-
ther purification unless otherwise indicated. 1H NMR spectra were
obtained on a Varian Gemini 300 spectrometer with TMS as an
internal standard. Coupling constants are reported in hertz. Multi-
plicity is defined by s (singlet), d (doublet), t (triplet), br (broad),
and m (multiplet). Mass spectra were obtained on a JEOL IMS-DX
instrument.
To a solution of 5 (371 mg, 1.5 mmol) in CH2Cl2 (10 mL) at 10 °C
was added BBr3 (7.5 mL, 1 M solution in CH2Cl2) dropwise in an ice
bath. The mixture was allowed to warm to room temperature and
was stirred for 12 h. Water was added while the reaction mixture
was cooled in an ice bath to keep the reaction temperature at 0 °C.
After extraction with CH2Cl2, the combined organic phase was dried
over Na2SO4. The filtrate was concentrated and the residue was puri-
fied by silica gel chromatography (CHCl3/MeOH = 20:1) to give
303 mg of 6 (71.8% yield). 1H NMR (300 MHz, CDCl3) d: 3.05 (s,
6H), 6.74 (d, J = 9.3 Hz, 2H), 7.26 (d, J = 3.0 Hz, 1H), 7.45 (d,
J = 9.0 Hz, 1H), 7.58 (d, J = 3.0 Hz, 1H), 7.77 (d, J = 9.0 Hz, 2H).
2.1. Chemistry
2.1.1. 4-Nitrobenzoic acid 2-acetyl-4-methoxyphenyl ester (1)
To
a stirring solution of 4-nitrobenzoyl chloride (1.8 g,
10 mmol) in pyridine (20 mL) was added 2-hydroxy-5-methoxy-
acetophenone (1.6 g, 10 mmol). The reaction mixture was stirred
at room temperature for 3 h, and poured into a 1 N aqueous HCl/
ice solution with vigorous stirring. The precipitate that formed
was filtered and washed with water to yield acetophenone 1
(2.9 g, 90.4% yield). 1H NMR (300 MHz, CDCl3) d: 2.54 (s, 3H),
3.89 (s, 3H), 7.15–7.16 (m, 2H), 7.38 (d, J = 2.0 Hz, 1H), 8.37 (s, 4H).
2.1.7. 6-Hydroxyethoxy-40-dimethylaminoflavone (7a)
To a solution of 6 (85 mg, 0.30 mmol) and ethylene chlorohy-
drin (100 lL, 1.50 mmol) in DMSO (3 mL) was added anhydrous
K2CO3 (41 mg, 0.90 mmol). The reaction mixture was stirred at
120 °C for 48 h, then poured into water. After extraction with chlo-
roform, the organic layers were combined and dried over Na2SO4.
Evaporation of the solvent afforded a residue, which was purified
by silica gel chromatography (CHCl3/MeOH = 33:1) to give 30 mg
of 7a (30.5%). 1H NMR (300 MHz, CDCl3) d: 3.06 (s, 6H), 3.98–
4.05 (m, 2H), 4.17–4.23 (m, 2H), 6.69 (s, 1H), 6.76 (d, J = 9.3 Hz,
2H), 7.28 (d, J = 3.0 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.60 (d,
J = 3.0 Hz, 1H), 7.80 (d, J = 9.0 Hz, 2H).
2.1.2. 1-(5-Methoxy-2-hydroxyphenyl)-3-(4-
nitrophenyl)propane-1,3-dione (2)
A solution of acetophenone 1 (2.9 g, 9.0 mmol) and pyridine
(50 mL) was heated to 50 °C, and to it was added pulverized potas-
sium hydroxide (2.5 g, 45.2 mmol). The reaction mixture was stir-
red for 90 min, and when it had cooled, 30 mL of 10% aqueous
acetic acid solution was added. The yellow precipitate that formed
was filtered to yield 2 (2.5 g, 88.1% yield). 1H NMR (300 MHz,
CDCl3) d: 3.85 (s, 3H), 6.84 (s, 1H), 6.99 (d, J = 6.9 Hz, 1H), 7.15–
7.18 (m, 1H), 7.22 (d, J = 2.2 Hz, 1H), 8.10 (d, J = 6.6 Hz, 2H), 8.35
(d, J = 6.6 Hz, 2H), 11.5 (s, 1H).
2.1.8. 6-(2-(2-Hydroxy-ethoxy)-ethoxy)-40-
dimethylaminoflavone (7b)
To a solution of 6 (82 mg, 0.29 mmol) and ethylene glycol
mono-2-chloroethyl ether (37 lL, 0.35 mmol) in DMF (3 mL) was
added anhydrous K2CO3 (120 mg, 0.87 mmol). The reaction mix-
ture was stirred at 120 °C for 11 h, then poured into water. After
extraction with chloroform, the organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a resi-
due, which was purified by silica gel chromatography (hexane/
ethyl acetate = 10:1) to give 107 mg of 7b (99.3%). 1H NMR
(300 MHz, CDCl3) d: 3.07 (s, 6H), 3.69 (t, J = 5.1 Hz, 2H), 3.79 (s,
2H), 3.91 (t, J = 4.5 Hz, 2H), 4.26 (t, J = 4.5 Hz, 2H), 6.69 (s, 1H),
6.75 (d, J = 9.0 Hz, 2H), 7.27–7.30 (m, 1H), 7.47 (d, J = 9.0 Hz, 1H),
7.64 (d, J = 2.2, 1H), 7.81 (d, J = 9.0 Hz, 2H).
2.1.3. 6-Methoxy-40-nitroflavone (3)
A mixture of the diketone 2 (2.5 g, 8.0 mmol), concentrated sul-
furic acid (2 mL), and glacial acetic acid (40 mL) was heated at
100 °C for 2 h and cooled to room temperature. The mixture was
poured onto crushed ice, and the resulting precipitate was filtered
to yield 3 (1.5 g, 63.5%). 1H NMR (300 MHz, CDCl3) d: 3.93 (s, 3H),
6.92 (s, 1H), 7.31–7.37 (m, 1H), 7.54–7.61 (m, 2H), 8.11 (d,
J = 9.3 Hz, 2H), 8.39 (d, J = 9.3 Hz, 2H).

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
2071
2.1.9. 6-(2-(2-(2-Hydroxy-ethoxy)-ethoxy)ethoxy)-40-
dimethylaminoflavone (7c)
NMR (300 MHz, CDCl3) d: 3.88–4.02 (m, 2H), 4.13–4.20 (m, 2H),
6.89 (s, 1H), 7.38–7.41 (m, 1H), 7.59 (d, J = 9.3 Hz, 1H), 7.67 (d,
J = 3.3 Hz, 1H), 8.12 (d, J = 9.0 Hz, 2H), 8.46 (d, J = 9.0 Hz, 2H).
To a solution of 6 (100 mg, 0.36 mmol) and 2-[2-(2-chloroeth-
oxy)ethoxy]ethanol (62 lL, 0.43 mmol) in DMF (3 mL) was added
anhydrous K2CO3 (148 mg, 1.07 mmol). The reaction mixture was
stirred at 120 °C for 17 h, then poured into water. After extraction
with chloroform, the organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which was
purified by preparative TLC (CHCl3/MeOH = 20:1) to give 76 mg
of 7c (51.1%). 1H NMR (300 MHz, CDCl3) d: 3.07 (s, 6H), 3.62–
3.76 (m, 8H), 3.91 (t, J = 4.5 Hz, 2H), 4.26 (t, J = 4.5 Hz, 2H), 6.69
(s, 1H), 6.77 (d, J = 9.3 Hz, 2H), 7.28–7.33 (m, 1H), 7.47 (d,
J = 9.0 Hz, 1H), 7.60 (d, J = 2.2, 1H), 7.81 (d, J = 9.0 Hz, 2H).
2.1.15. 6-(2-(2-Hydroxy-ethoxy)-ethoxy)-40-nitroflavone (10b)
The same reaction as described above to prepare 7b was used,
and 830 mg of 10b was obtained from 9. 1H NMR (300 MHz, CDCl3)
d: 3.70 (t, J = 5.1 Hz, 2H), 3.79 (s, 2H), 3.93 (t, J = 5.0 Hz, 2H), 4.28 (t,
J = 4.8 Hz, 2H), 6.90 (s, 1H), 7.37–7.41 (m, 1H), 7.56 (d, J = 9.3 Hz,
1H), 7.66 (d, J = 3.3 Hz, 1H), 8.10 (d, J = 9.0 Hz, 2H), 8.40 (d,
J = 9.0 Hz, 2H).
2.1.16. 6-(2-(2-(2-Hydroxy-ethoxy)-ethoxy)ethoxy)-40-
nitroflavone (10c)
2.1.10. 6-Fluoroethoxy-40-dimethylaminoflavone (8a)
To a solution of 7a (30 mg, 0.09 mmol) in ethylene glycol di-
methyl ether (3 mL) was added dimethylamino sulfur trifluoride
The same reaction as described above to prepare 7c was used,
and 10c was obtained from 9 in a yield of 83.1%. 1H NMR
(300 MHz, CDCl3) d: 3.64 (t, J = 4.5 Hz, 2H), 3.71–3.78 (m, 6H),
3.93 (t, J = 4.8 Hz, 2H), 4.27 (t, J = 4.5 Hz, 2H), 6.90 (s, 1H), 7.38–
7.42 (m, 1H), 7.55 (d, J = 9.0 Hz, 1H), 7.61 (d, J = 3.1 Hz, 1H), 8.10
(d, J = 8.7 Hz, 2H), 8.39 (d, J = 8.7 Hz, 2H). EI-MS m/z 415 (M+).
(DAST) (30 lL, 0.23 mmol) in a dry ice-acetone bath. The reaction
mixture was stirred for 6 h at room temperature. The mixture
was then poured into a saturated NaHSO3 solution and after
extraction with chloroform, the organic phase was separated, dried
over Na2SO4, and filtered. The residue was purified by silica gel
chromatography (hexane/ethyl acetate = 2:1) to give 16 mg of 8a
(53.0%). 1H NMR (300 MHz, CDCl3) d: 2.93 (s, 6H), 4.26–4.40 (m,
2H), 4.70–4.92 (m, 2H), 6.71 (s, 1H), 6.76 (d, J = 9.0 Hz, 2H), 7.29–
7.35 (m, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 3.3 Hz, 1H), 7.82
(d, J = 9.3 Hz, 2H). EI-MS m/z 327 (M+).
2.1.17. 6-(2-Fluoro-ethoxy)-40-nitroflavone (11)
The same reaction as described above to prepare 8a was used,
and 24 mg of 11 was obtained from 10a in a yield of 41.6%. 1H
NMR (300 MHz, CDCl3) 4.26–4.41 (m, 2H), 4.71–4.92 (m, 2H),
6.91 (s, 1H), 7.42–7.44 (m, 1H), 7.56–7.61 (m, 2H), 8.11 (d,
J = 9.0 Hz, 2H), 8.39 (d, J = 9.3 Hz, 2H).
2.1.11. 6-(2-(2-Fluoro-ethoxy)-ethoxy)-40-
dimethylaminoflavone (8b)
To a solution of 7b (29 mg, 0.08 mmol) in 1,2-dimethoxyethane
(DME) (5 mL) was added DAST (21 lL, 0.16 mmol) in a dry ice-ace-
tone bath. The reaction mixture was stirred for 1.5 h at room tem-
perature. The mixture was then poured into a saturated NaHSO3
solution and after extraction with chloroform, after the organic
phase was separated, dried over Na2SO4, and filtered. The residue
was purified by preparative TLC (CHCl3/MeOH = 20:1) to give
15 mg of 8b (51.5%). 1H NMR (300 MHz, CDCl3) d: 3.07 (s, 6H),
3.79 (t, J = 4.2 Hz, 1H), 3.89–3.96 (m, 3H), 4.26 (t, J = 4.8 Hz, 2H),
4.54 (t, J = 4.2 Hz), 4.69 (t, J = 4.2 Hz), 6.70 (s, 1H), 6.76 (d,
J = 9.0 Hz, 2H), 7.27–7.33 (m, 1H), 7.49 (d, J = 9.3 Hz, 1H), 7.59 (d,
J = 3.0, 1H), 7.81 (d, J = 9.0 Hz, 2H). EI-MS m/z 371 (M+).
2.1.18. 6-(2-Fluoro-ethoxy)-40-aminoflavone (12)
The same reaction as described above to prepare 4 was used,
and 22 mg of 12 was obtained from 11 in a yield of 41.6%. 1H
NMR (300 MHz, CDCl3) 4.10 (s, 2H), 4.27–4.39 (m, 2H), 4.71–4.88
(m, 2H), 6.70 (s, 1H), 6.76 (d, J = 9.0 Hz, 2H), 7.29–7.35 (m, 1H),
7.49 (d, J = 9.3 Hz, 1H), 7.58 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H). EI-MS
m/z 299 (M+).
2.1.19. 6-(2-Fluoro-ethoxy)-40-methylaminoflavone (13)
To a solution of 12 (22 mg, 0.07 mmol) in DMSO (2 mL) were
added methyl iodide (0.14 mL) and K2CO3 (50.8 mg, 0.37 mmol).
The reaction mixture was stirred at room temperature for 5 h,
and poured into water (30 mL). After extraction with ethyl acetate
(2 ꢀ 30 mL), the organic layers were combined and dried over
Na2SO4. Evaporation of the solvent afforded a residue, which was
purified by reversed phase HPLC (acetonitrile/H2O = 3:2) to give
10 mg of 13 (43.4% yield). 1H NMR (300 MHz, CDCl3) 2.93 (s, 3H),
4.22 (s, 1H), 4.26–4.40 (m, 2H), 4.70–4.91 (m, 2H), 6.71 (s, 1H),
6.76 (d, J = 9.0 Hz, 2H), 7.29–7.35 (m, 1H), 7.50 (d, J = 9.3 Hz, 1H),
7.58 (s, 1H), 7.78 (d, J = 8.7 Hz, 2H). EI-MS m/z 313 (M+).
2.1.12. 6-(2-(2-(2-Fluoro-ethoxy)-ethoxy)ethoxy)-40-dimethyla-
minoflavone (8c)
To a solution of 7c (141 mg, 0.34 mmol) in DME (5 mL) was
added DAST (90 lL, 0.68 mmol) in a dry ice-acetone bath. The reac-
tion mixture was stirred for 1 h at room temperature. The mixture
was then poured into saturated NaHSO3 solution and extracted
with chloroform. After the organic phase was separated, dried over
Na2SO4 and filtered, the residue was purified by preparative TLC
(hexane/ethyl acetate = 1:5) to give 21 mg of 8c (14.9%). 1H NMR
(CDCl3) d: 3.08 (s, 6H), 3.69–3.81 (m, 6H), 3.91 (t, J = 4.8 Hz, 2H),
4.24 (t, J = 4.8 Hz, 2H), 4.49 (t, J = 4.5 Hz, 1H), 4.65 (t, J = 4.5 Hz,
1H), 6.69 (s, 1H), 6.76 (d, J = 9.0 Hz, 2H), 7.27–7.33 (m, 1H), 7.48
(d, J = 9.0 Hz, 1H), 7.59 (d, J = 2.2, 1H), 7.81 (d, J = 9.0 Hz, 2H). EI-
MS m/z 415 (M+).
2.1.20. 6-(2-(2-Hydroxy-ethoxy)-ethoxy)-40-aminoflavone (14b)
The same reaction as described above to prepare 4 was used,
and 251 mg of 14b was obtained from 10b in a yield of 37.9%. 1H
NMR (CDCl3) d: 3.69 (t, J = 5.1 Hz, 2H), 3.79 (s, 2H), 3.91 (t,
J = 4.5 Hz, 2H), 4.09 (s, 2H), 4.27 (t, J = 4.2 Hz, 2H), 6.69 (s, 1H),
6.76 (d, J = 8.7 Hz, 2H), 7.27–7.32 (m, 1H), 7.48 (d, J = 9.3 Hz, 1H),
7.65 (d, J = 3.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H). EI-MS m/z 387 (M+).
2.1.21. 6-(2-(2-(2-Hydroxy-ethoxy)-ethoxy)ethoxy)-40-amino-
flavone (14c)
2.1.13. 6-Hydroxy-40-nitroflavone (9)
The same reaction as described above to prepare 6 was used,
and 560 mg of 9 was obtained from 3 and BBr3. EI-MS m/z 283
(M+).
The same reaction as described above to prepare 4 was used,
and 553 mg of 14c was obtained from 10c in a yield of 58.8%. 1H
NMR (300 MHz, CDCl3) d: 3.62–3.65 (m, 2H), 3.71–3.78 (m, 6H),
3.91 (t, J = 4.8 Hz, 2H), 4.11 (s, 2H), 4.25 (t, J = 4.5 Hz, 2H), 6.68 (s,
1H), 6.75 (d, J = 8.7 Hz, 2H), 7.27–7.32 (m, 1H), 7.50 (d, J = 9.0 Hz,
1H), 7.59 (d, J = 2.2 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H). EI-MS m/z
387 (M+).
2.1.14. 6-(2-Hydroxy-ethoxy)-40-nitroflavone (10a)
The same reaction as described above to prepare 7a was used,
and 40 mg of 10a was obtained from 9 in a yield of 9.9%. 1H

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M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
2.1.22. 6-(2-(2-Fluoro-ethoxy)-ethoxy)-40-aminoflavone (15b)
The same reaction as described above to prepare 8b was used,
and 10 mg of 15b was obtained from 14b in a yield of 9.1%. 1H
NMR (CDCl3) d: 3.79 (t, J = 4.2 Hz, 1H), 3.86–3.95 (m, 3H), 4.11 (s,
2H), 4.25 (t, J = 4.5 Hz, 2H), 4.53 (t, J = 4.2 Hz, 1H), 4.70 (t,
J = 4.2 Hz, 1H), 6.68 (s, 1H), 6.75 (d, J = 9.0 Hz, 2H), 7.28–7.33 (m,
1H), 7.47 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 3.0 Hz, 1H), 7.74 (d,
J = 8.4 Hz, 2H). EI-MS m/z 343 (M+).
(300 MHz, CDCl3) d: 6.81 (s, 2H), 7.02 (d, J = 9.0 Hz, 1H), 7.45 (d,
J = 9.0 Hz, 1H), 7.68 (s, 1H), 8.11 (d, J = 8.7 Hz, 2H), 8.36 (d,
J = 8.7 Hz, 2H), 11.7 (s, 1H).
2.1.30. 6-Fluoro-40-nitroflavone (20)
The same reaction as described above to prepare 3 was used,
and 2.0 g of 20 was obtained from 19 in a yield of 85.3%. EI-MS
m/z 285 (M+).
2.1.23. 6-(2-(2-(2-Fluoro-ethoxy)-ethoxy)ethoxy)-40-amino-
flavone (15c)
2.1.31. 6-Fluoro-40-aminoflavone (21)
The same reaction as described above to prepare 4 was used,
and 944 mg of 21 was obtained from 20 in a yield of 67.4%. 1H
NMR (300 MHz, CDCl3) d: 4.13 (s, broad, 2H), 6.74 (s, 1H), 6.76
(d, J = 9.0 Hz, 2H), 7.35–7.42 (m, 1H), 7.51–7.56 (m, 1H), 7.75 (d,
J = 8.7 Hz, 2H), 7.82–7.85 (m, 1H).
The same reaction as described above to prepare 8 was used,
and 85 mg of 15c was obtained from 14 in a yield of 81.3%. 1H
NMR (CDCl3) d: 3.62–3.65 (m, 2H), 3.70–3.78 (m, 7H), 3.82 (t,
J = 3.9 Hz, 1H), 3.90 (t, J = 4.5 Hz, 2H), 4.22 (t, J = 4.5 Hz, 2H), 4.49
(t, J = 4.2 Hz, 1H), 4.66 (t, J = 4.2 Hz, 1H), 6.68 (s, 1H), 6.75 (d,
J = 8.7 Hz, 2H), 7.27–7.32 (m, 1H), 7.46 (d, J = 9.3 Hz, 1H), 7.57 (d,
J = 2.2 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H).
2.1.32. 6-Fluoro-40-methylaminoflavone (22)
To a mixture of 21 (300 mg, 1.2 mmol) and paraformaldehyde
(179 mg, 5.9 mmol) in MeOH (15 mL) was added a solution of
NaOMe (0.34 mL, 28 wt % in MeOH) dropwise at 0 °C. The mixture
was stirred under reflux for 1 h. After addition of NaBH4 (246 mg,
6.5 mmol), the solution was heated under reflux for 45 min. To
the cold mixture, 1 M NaOH was added followed by extraction
with CHCl3. The organic phase was dried over Na2SO4 and filtered.
The solvent was removed, and the residue was purified by silica gel
chromatography (hexane/ethyl acetate = 5:3) to give 314 mg of 22
(99.2%). 1H NMR (300 MHz, CDCl3) d: 2.91 (s, 3H), 4.37 (s, broad,
1H), 6.63 (s, 1H), 6.66 (s, 2H), 7.32–7.39 (m, 1H), 7.49–7.53 (m,
1H), 7.74 (d, J = 8.7 Hz, 2H), 7.82–7.85 (m, 1H).
2.1.24. 6-(2-(2-Hydroxy-ethoxy)-ethoxy)-40-
methylaminoflavone (16b)
The same reaction as described above to prepare 13 was used,
and 41 mg of 16b was obtained from 14b in a yield of 37.9%. 1H
NMR (CDCl3) d: 3.49 (s, 3H), 3.69 (t, J = 3.6 Hz, 2H), 3.77–3.79 (m,
2H), 3.91 (t, J = 4.8 Hz, 2H), 4.27 (t, J = 4.0 Hz, 2H), 6.65 (s, 1H),
6.68–6.69 (m, 2H), 7.29–7.32 (m, 1H), 7.47 (d, J = 9.0 Hz, 1H),
7.65 (d, J = 3.0 Hz, 1H), 7.78 (d, J = 9.0 Hz, 2H). EI-MS m/z 355 (M+).
2.1.25. 6-(2-(2-(2-Hydroxy-ethoxy)-ethoxy)ethoxy)-40-
methylaminoflavone (16c)
The same reaction as described above to prepare 13 was used,
and 145 mg of 16c was obtained from 14c in a yield of 64.8%. 1H
NMR (CDCl3) d: 2.92 (d, J = 3.0 Hz, 3H), 3.63 (t, J = 5.4 Hz, 2H)
3.72–3.76 (m, 6H), 3.91 (t, J = 5.1 Hz, 2H), 4.25 (t, J = 4.8 Hz, 3H),
6.65 (s, 1H), 6.68 (s, 2H), 7.28–7.32 (m, 1H), 7.46 (d, J = 9.3 Hz,
1H), 7.59 (d, J = 2.2 Hz, 1H), 7.77 (d, J = 8.7 Hz, 2H).
2.1.33. 6-Fluoro-40-dimethylaminoflavone (23)
The same reaction as described above to prepare 5 was used,
and 203 mg of 23 was obtained from 21 in a yield of 61.0%. 1H
NMR (300 MHz, CDCl3) d: 3.08 (s, 6H), 6.69 (s, 1H), 6.76 (d,
J = 9.3 Hz, 2H), 7.35–7.41 (m, 1H), 7.51–7.56 (m, 1H), 7.81 (d,
J = 9.0 Hz, 2H), 7.83–7.86 (m, 1H).
2.1.26. 6-(2-(2-Fluoro-ethoxy)-ethoxy)-40-methylaminoflavone
(17b)
2.1.34. 6-(2-Tosyloxyethoxy)-40-dimethylaminoflavone (24a)
To a solution of 8a (136 mg, 0.28 mmol) in pyridine (4 mL) was
added tosyl chloride (122 mg, 0.65 mmol) in an ice bath. The reaction
mixture was stirred for 32 h at room temperature following the reac-
tion in an ice bath for 1 h. The organic phase was dried over Na2SO4
and filtered. The solvent was removed, and the residue was purified
by silica gel chromatography (chloroform/MeOH = 20:1) to give
50 mg of 24a (36.8%). 1H NMR (300 MHz, CDCl3) d: 2.45 (s, 3H),
3.07 (s, 6H), 4.23 (t, 2H, J = 4.5 Hz), 4.41 (t, J = 5.1 Hz, 2H), 6.68 (s,
1H), 6.75 (d, J = 9.0 Hz, 2H), 7.12–7.18 (m, 1H), 7.35 (d, J = 8.1 Hz,
2H), 7.43–7.56 (m, 2H), 7.82 (t, J = 9.0 Hz, 4H). EI-MS: m/z 479 [M+].
The same reaction as described above to prepare 8 was used,
and 9 mg of 17b was obtained from 16b in a yield of 21.9%. 1H
NMR (CDCl3) d: 2.93 (d, J = 5.1 Hz, 3H), 3.79 (t, J = 4.2 Hz, 1H),
3.85–3.95 (m, 3H), 4.26 (t, J = 4.8 Hz, 3H), 4.53 (t, J = 4.2 Hz, 1H),
4.70 (t, J = 4.5 Hz, 1H), 6.65 (s, 1H), 6.68 (s, 2H), 7.28–7.32 (m,
1H), 7.47 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 3.0 Hz, 1H), 7.78 (d,
J = 9.0 Hz, 2H). EI-MS m/z 357 (M+).
2.1.27. 6-(2-(2-(2-Fluoro-ethoxy)-ethoxy)ethoxy)-40-
methylaminoflavone (17c)
The same reaction as described above to prepare 8 was used,
and 20 mg of 17c was obtained from 16c in a yield of 13.8%. 1H
NMR (CDCl3) d: 2.92 (d, J = 4.8 Hz, 3H), 3.69–3.76 (m, 5H), 3.82 (t,
J = 4.5 Hz, 1H), 3.91 (t, J = 4.8 Hz, 2H), 4.25 (t, J = 4.2 Hz, 3H), 4.50
(t, J = 4.2 Hz, 1H), 4.66 (t, J = 4.5 Hz, 1H), 6.65 (s, 1H), 6.68 (s, 2H),
7.28–7.31 (m, 1H), 7.46 (d, J = 9.3 Hz, 1H), 7.59 (d, J = 3.0 Hz, 1H),
7.77 (d, J = 8.7 Hz, 2H). EI-MS m/z 401 (M+).
2.1.35. 6-(2-(2-Tosyloxyethoxy)ethoxy)-40-
dimethylaminoflavone (24b)
The same reaction as described above to prepare 24a was used,
and 111 mg of 24b was obtained from 8b in a yield of 34.1%. 1H
NMR (300 MHz, CDCl3) d: 2.41 (s, 3H), 3.08 (s, 6H), 3.76–3.85 (m,
4H), 4.12 (t, J = 5.1 Hz, 2H), 4.22 (t, J = 5.1 Hz, 2H), 6.70 (s, 1H),
6.76 (d, J = 9.0 Hz, 2H), 7.25–7.33 (m, 3H), 7.47 (d, J = 9.0 Hz, 1H),
7.55 (d, J = 3.0 Hz, 1H), 7.79–7.83 (m, 4H). EI-MS m/z 523 (M+).
2.1.28. 4-Nitrobenzoic acid 2-acetyl-4-fluorophenyl ester (18)
The same reaction as described above to prepare 1 was used, and
2.5 g of 18 was obtained from 2-hydroxy-5-fluoroacetophenone and
4-nitrobenzoyl chloride in a yield of 85.6%. 1H NMR (300 MHz, CDCl3)
d: 2.56 (s, 3H), 7.23–7.34 (m, 2H), 7.56–7.60 (m, 1H), 8.37 (s, 4H).
2.1.36. 6-(2-(2-(2-Tosyloxyethoxy)ethoxy)ethoxy)-40-
dimethylaminoflavone (24c)
The same reaction as described above to prepare 24a was used,
and 35 mg of 24c was obtained from 8c in a yield of 39.9%. 1H NMR
(300 MHz, CDCl3) d: 2.43 (s, 3H), 3.08 (s, 6H), 3.62–3.73 (m, 6H),
3.87 (t, J = 4.8 Hz, 2H), 4.16–4.21 (m, 4H), 6.70 (s, 1H), 6.76 (d,
J = 9.0 Hz, 2H), 7.28–7.33 (m, 3H), 7.47 (d, J = 9.0 Hz, 1H), 7.60 (d,
J = 2.2 Hz, 1H), 7.79–7.83 (m, 4H). EI-MS m/z 567 (M+).
2.1.29. 1-(5-Fluoro-2-hydroxyphenyl)-3-(4-
nitrophenyl)propane-1,3-dione (19)
The same reaction as described above to prepare 2 was used,
and 2.5 g of 19 was obtained from 18 in a yield of 96.3%. 1H NMR

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
2073
2.2. Radiolabeling
scitillation detector) on
a
YMC Hydrosphere C18 column
(20 ꢀ 150 mm) with acetonitrile/water (70:30) at a flow rate of
9.0 mL/min to obtain [18F]8(a–c). The radiochemical purity and
specific activity were determined by analytical HPLC on a YMC
Pack Pro C18 column (4.6 ꢀ 150 mm, acetonitrile/water (60:40),
1.0 mL/min).
[
18F]Fluoride produced by an ultracompact cyclotron (CYPRIS
model 325R; Sumitomo Heavy Industry Ltd) via an 18O(p,n)18
reaction was adsorbed to a strong-base anion exchange resin
(Bio-Rad), and was eluted with 500 L of K2CO3 solution (33 mM)
F
l
into 1 mL of acetonitrile containing Kryptofix 222 (K222)
(20 mg). The solvent was removed azeotropically with anhydrous
acetonitrile at 120 °C under a nitrogen stream. A solution of tosyl-
2.3. Binding assays using the aggregated Ab peptide in solution
ate precursor 24(a–c) (0.2 mg) in 400
l
L of DMSO was added to
A solid form of Ab(1-42) was purchased from Peptide Institute
(Osaka, Japan). Aggregation of peptides 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 solu-
tions were incubated at 37 °C for 42 h with gentle and constant
the reaction vessel containing [18F]Fluoride. The mixture was
heated at 160 °C for 5 min. The reaction mixture was purified by
the reversed phase HPLC system (a Shimadzu LC-6A isocratic
pump, a Shimadzu SPD-6A UV detector and an Aloka NDW-351D
O
O
NO2
OH
O
NO2
OH
a
b
MeO
O
MeO
MeO
c
NO2
O
O
O
Cl
1
2
NH2
N
NO2
O
O
O
e
O
d
MeO
MeO
MeO
O
O
5
4
3
f
f
NO2
NH2
N
O
O
O
O
F
HO
O
O
HO
n
O
O
6
15(b,c)
9
g
g
h
NO2
NH2
N
O
O
O
d
HO
HO
HO
O
O
n
n
n
O
O
O
7(a-c)
10(a-c)
14(b,c)
h
h
i
H
N
NO2
N
O
O
O
HO
F
F
F
O
O
O
O
n
n
O
O
11
O
8(a-c)
16(b,c)
d
h
H
N
NH2
O
O
a: n = 1
b: n = 2
c: n = 3
F
O
n
O
O
17(b,c)
12
i
H
N
O
F
O
O
13
O
Scheme 1. Reagents: (a) pyridine; (b) KOH, pyridine; (c) H2SO4, AcOH; (d) EtOH, SnCl2; (e) (CH2O)n, NaCNBH3, AcOH; (f) CH2Cl2, BBr3; (g)
(h) DAST, DME; (i) DMSO, CH3I, K2CO3.
(n = 1–3) K2CO3, DMF;
Cl
n
H

2074
M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
shaking. Binding experiments were carried out as described previ-
ously.14 125I]DMFV ([125I]6-iodo-40-dimethylaminoflavone) with
505 nm; longpass filter, 520 nm). Thereafter, the serial sections
were also immunostained with DAB as a chromogen using mono-
clonal antibodies against b-amyloid (Amyloid b-Protein Immuno-
histostain kit, WAKO).
[
81.4 TBq/mmol specific activity and greater than 95% radiochemi-
cal purity was prepared using the standard iododestannylation
reaction.14
(0.2 pM-400
A
l
mixture containing 50
M in 10% EtOH), 50
L of 0.02 nM [125I]DMFV,
50 lL of Ab(1-42) aggregates and 850 lL of 10% EtOH was incu-
lL of test compounds
l
2.5. In vivo biodistribution in normal mice
bated at room temperature for 3 h. The mixture was then filtered
through Whatman GF/B filters using a Brandel M-24 cell harvester,
and the radioactivity on the filters containing the bound 125I ligand
was measured in a gamma counter (Aloka, ARC-380). Values for
the half-maximal inhibitory concentration (IC50) were determined
from displacement curves of three independent experiments using
GraphPad Prism 4.0, and those for the inhibition constant (Ki) were
calculated using the Cheng-Prusoff equation:18 Ki = IC50/(1 + [L]/
Kd), where [L] is the concentration of [125I]DMFV used in the assay,
and Kd is the dissociation constant of DMFV (12.3 nM).14
A saline solution (100 lL) containing ethanol (5 lL) of radiola-
beled agents (18.5 kBq) was injected directly into a tail vein of
ddY mice (5-week-old, 22–25 g). While under isoflurane anesthe-
sia, the mice were sacrificed at various time points postinjection.
The organs of interest were removed and weighed, and radioactiv-
ity was measured with an automatic gamma counter (Packard Co-
bra Auto-Gamma Counter 5003).
3. Results and discussion
The target FPEG flavone derivatives were prepared as shown in
Scheme 1. The most common method of synthesizing flavones is
known as the Baker-Venkataraman transformation.19 In this pro-
cess, a hydroxyacetophenone is first converted into a benzoyl ester
1, and this species is then treated with a base, forming a 1,3-diketone
2. Treatment of this diketone with acid leads to generation of the de-
sired flavone 3. In the route for the synthesis of dimethylamino
derivatives, the free amino derivative 4 was readily prepared from
3 by reduction with SnCl2. Compound 5 was converted to 6 by
demethylation with BBr3 in CH2Cl2. To prepare compounds with 1-
3ethoxygroupsasthePEGlinkage, commerciallyavailablechlorides
were coupled with the OH group of 6 to obtain 7(a–c), respectively.
The fluorinated flavones, 8(a–c), were successfully obtained by
reacting 7(a–c) with DAST in DME or ethylene glycol dimethyl ether.
In the route for the synthesis of monomethylated derivatives and the
primary amino derivatives, the demethylation of 3 with BBr3 and the
introduction of 1-3 ethoxy groups into 9 gave 10(a–c). To prepare
2.4. Staining of amyloid plaques in transgenic mouse brain
sections
Animal studies were conducted in accordance with institu-
tional guidelines and approved by the Kyoto University Animal
Care Committee. Tg2576 transgenic mice (female, 20-month-
old) were used as an Alzheimer’s model. While under isoflurane
anesthesia, the mice were sacrificed by decapitation, and the
brains were immediately removed and frozen in powdered dry
ice. The frozen blocks were sliced into serial sections 10
using a cryostat (Leica Instruments, CM1900). Each slide was
incubated with a 50% ethanol solution (100 M) of compound
lm thick
l
8a, 8b, or 8c, which have the characteristics to emit fluorescence.
The sections were washed in 50% ethanol for 3 min two times,
and examined using a microscope (Nikon, Eclipse 80i) equipped
with a B-2A filter set (excitation, 450–490 nm; dichronic mirror,
O
O
NO2
OH
O
OH
O
NO2
a
b
O
F
F
F
NO2
NO2
O
O
Cl
18
19
NH2
c
d
O
O
O
F
F
O
20
21
e
f
H
N
N
O
O
O
F
F
O
23
22
Scheme 2. Reagents: (a) pyridine; (b) KOH, pyridine; (c) H2SO4, AcOH; (d) EtOH, SnCl2; (e) (CH2O)n, NaOMe, NaBH4; (f) (CH2O)n, NaCNBH3, AcOH.
N
N
N
O
O
O
a
b
TsO
18F
HO
O
O
O
n
n
n
O
O
O
[
18F]8(a-c)
8(a-c)
24(a-c)
Scheme 3. Reagents: (a) tosyl chloride, pyridine; (b) K2CO3, [18F]Fꢁ, kryptofix[222], DMSO/acetonitrile.

M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
2075
the FPEG flavone with one ethoxy group (n = 1) (12 and 13), the fluo-
rination of 10a with DAST, the reduction of 11 with SnCl2 and the
methylation of 12 were performed. The primary amino derivatives
of FPEG flavones (n = 2 and 3) (15b and 15c) were synthesized by
the fluorination of 14b and 14c with DAST following the reduction
of the nitro group in 10b and 10c. The monomethylated FPEG flav-
ones (n = 2 and 3) (17b and 17c) were synthesized by the methyla-
tion of 16b and 16c following the fluorination of 14b and 14c with
DAST. We successfully synthesized the flavone derivatives (21, 22,
and 23) with fluorine directly bound to the phenyl group according
to a procedure reported previously (Scheme 2). To make the desired
18F-labeled FPEG flavones, [18F]8(a–c), the tosylates 24(a–c) were
employed as the precursors. The free OH groups of 8(a–c) were con-
vertedintotosylatesbyreactingwithTsClinthepresenceofpyridine
to give 24(a–c) (Scheme 3). Each of the tosylates, 24(a–c), was mixed
with [18F]fluoride/potassium carbonate and Kryptofix 222 in DMSO
and heated at 160 °C for 5 min. The crude product was purified by
HPLC (radiochemical purity >99%, radiochemical yield 5–13%, decay
corrected). The total synthesis time was 70 min, and the specific
activity was estimated to be 33.3–55.5 GBq/mmol at the end of
synthesis.
In vitro binding experiments to evaluate the affinity of the FPEG
flavones for Ab aggregates were carried out in solutions with
[
125I]DMFV as the ligand. The affinity of flavone derivatives for
Ab aggregates varied from 5 to 321 nM (Table 1). The flavone deriv-
atives had affinity for Ab(1-42) aggregates in the following order:
the dimethyamino derivatives (8a, 8b, 8c, and 23) > the monom-
ethylamino derivatives (13, 17b, 17c, and 22) > the primary amino
derivatives (12, 15b, 15c, and 21). The results of the binding exper-
iments are consistent with those of previous reports.14,20,21 The Ki
values indicated that the affinity for Ab(1-42) aggregates was af-
fected by the substituted group at position 40 in the flavone struc-
ture, not by the length of the PEG introduced into the flavone
backbone. We selected the dimethylamino derivatives (8a, 8b,
and 8c), which showed greater binding affinity than the monom-
ethylamino derivatives and the primary amino derivatives, for
additional study.
Table 1
Inhibition constants (Ki, nM) of compounds for the binding of [125I]DMFV to Ab(1-42)
aggregatesa
Compound
Ki (nM)
5.3 0.8
14.4 2.5
19.3 4.0
234.3 63.5
99.0 11.8
321.1 74.4
Compound
Ki (nM)
8a
8b
8c
12
13
15b
15c
17b
17c
21
234.0 60.6
54.5 10.3
45.1 5.8
260.5 43.3
110.0 47.4
73.9 5.3
22
23
a
Values are the mean standard error of the mean for 4–9 experiments.
Three 18F FPEG flavones ([18F]8a, [18F]8b, and [18F]8c) were
examined for their biodistribution in normal mice (Table 2). All
three ligands displayed high uptake from the brain 2.89–4.17%ID/
g, at 2 min postinjection, indicating a level sufficient for imaging.
In addition, they displayed good clearance from the normal brain
with 1.89, 2.00, and 1.31%ID/g at 30 min postinjection for [18F]8a,
Table 2
Biodistribution of 18F-labeled flavones in normal micea
Organ
18F]8a
2 min
10 min
30 min
60 min
[
[
18F]8b, and [18F]8c, respectively. These values were equal to
45.3%, 56.5%, and 45.3% of the initial uptake peak for [18F]8a,
18F]8b, and [18F]8c, respectively. A rapid initial uptake in normal
Blood
Brain
Bone
2.80 0.41
4.17 0.77
2.02 0.53
2.71 0.13
3.62 0.21
2.83 0.23
2.53 0.17
1.89 0.13
4.51 0.55
3.25 0.31
2.19 0.18
6.21 0.84
[
[
18F]8b
brain coupled with a fast washout are highly desirable properties
for b-amyloid-imaging probes, as they lead to a high signal to back-
ground ratio. [18F]8(a–c) showed the bone uptake (3.74–6.21%ID/
g) at 60 min postinjection, suggesting there may be in vivo defluo-
rination. However, the free fluorine was not taken up by brain tis-
sue; therefore, the interference from this free fluoride is expected
to be relatively low for brain imaging.22
Blood
Brain
Bone
2.09 0.35
3.54 0.54
1.13 0.22
2.30 0.07
2.75 0.21
1.65 0.10
2.50 0.21
2.00 0.20
2.42 0.38
2.94 0.27
2.13 0.10
3.74 0.30
[
18F]8c
Blood
Brain
Bone
2.35 0.54
2.89 0.74
1.53 0.52
1.50 0.26
2.23 0.36
2.38 0.39
1.40 0.04
1.31 0.14
4.06 0.49
1.88 0.08
1.37 0.11
5.21 0.98
a Expressed as % of injected dose per gram. Each value represents the mean SD for
4–5 mice at each interval.
To confirm the affinity of FPEG chalcone derivatives for b-amy-
loid plaques in the brain, neuropathological fluorescent staining
Figure 2. Neuropathological staining of flavone derivatives 8a (A), 8b (B), and 8c (C) in 10-
against b-amyloid (D, E, and F) in the adjacent sections of A, B, and C, respectively.
lm brain sections of Tg2576 mice. Immunohistological staining with an antibody

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M. Ono et al. / Bioorg. Med. Chem. 17 (2009) 2069–2076
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with 8a, 8b, and 8c was carried out using the Alzheimer’s model
(Fig. 2A–C). Many fluorescence spots were observed in the brain
sections of Tg2576 transgenic (female, 20-month-old) mice, while
no spots were observed in the brain sections of wild-type (female,
22-month-old) mice (data not shown). The fluorescent labeling
pattern was consistent with that obtained by immunohistochemi-
cal labeling with an antibody specific for Ab (Fig. 2D–F), indicating
that FPEG flavones show specific binding to b-amyloid plaques in
the mouse brain.
In conclusion, we successfully designed and synthesized novel
18F labeled flavones with the FPEG strategy for PET imaging of b-
amyloid in the brain. The affinity of the derivatives for Ab aggre-
gates varied from 5 to 321 nM. When in vitro plaque labeling
was carried out using sections of brain from Tg2576 mice, FPEG
flavones intensely stained b-amyloid plaques. In addition, they dis-
played good uptake into and a rapid washout from the brain after
injection in normal mice. The combination of high binding affinity
for b-amyloid plaques, high brain uptake, and good clearance in
mice of the FPEG-flavone derivatives may provide a series of prom-
ising in vivo amyloid imaging agents for PET.
14. Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.; Arano, Y.; Mori, H.; Nakayama,
M. J. Med. Chem. 2005, 48, 7253.
15. Stephenson, K. A.; Chandra, R.; Zhuang, Z. P.; Hou, C.; Oya, S.; Kung, M. P.; Kung,
H. F. Bioconjugate Chem. 2007, 18, 238.
Acknowledgments
16. Ono, M.; Kung, M. P.; Hou, C.; Kung, H. F. Nucl. Med. Biol. 2002, 29, 633.
17. Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plossl, K.; Hou, C.; Holtzman,
D. M.; Kung, H. F. J. Med. Chem. 2003, 46, 237.
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Ems, B. L.; Kakodkar, S. V.; Kelm, G. R.; Kershaw, W. C., et al J. Med. Chem. 1995,
38, 4937.
This study was supported by the Industrial Technology
Research Grant Program from the New Energy and Industrial Tech-
nology Development Organization (NEDO) of Japan, and the
Program for Promotion of Fundamental Studies in Health Sciences
of the National Institute of Biomedical Innovation (NIBIO).
20. Ono, M.; Haratake, M.; Mori, H.; Nakayama, M. Bioorg. Med. Chem. 2007, 15,
6802.
21. Ono, M.; Hori, M.; Haratake, M.; Tomiyama, T.; Mori, H.; Nakayama, M. Bioorg.
Med. Chem. 2007, 15, 6388.
References and notes
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