Novel benzofuran derivatives for PET imaging of β-amyloid plaques in Alzheimer's disease brains

Article abstract of DOI:10.1021/jm051176k

A novel series of benzofuran derivatives as potential positron emission tomography (PET) tracers targeting amyloid plaques in Alzheimer's disease (AD) were synthesized and evaluated. The syntheses of benzofurans were successfully achieved by an intramolec

Full text of DOI:10.1021/jm051176k

J. Med. Chem. 2006, 49, 2725-2730
2725
Novel Benzofuran Derivatives for PET Imaging of â-Amyloid Plaques in Alzheimer’s Disease
Brains
Masahiro Ono,*,† Hidekazu Kawashima,‡ Akemi Nonaka,† Tomoki Kawai, Mamoru Haratake,† Hiroshi Mori,| Mei-Ping Kung,§
Hank F. Kung,§ Hideo Saji, and Morio Nakayama†
Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki UniVersity, 1-14 Bunkyo-machi, Nagasaki 852-8521,
Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto UniVersity, Shogoin Kawahara-cho, Sakyo-ku,
Kyoto 606-8507, Department of Neuroscience, Osaka City UniVersity Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585,
Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Yoshida Shimoadachi-cho,
Sakyo-ku, Kyoto 606-8501, Japan, and Department of Radiology, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104
ReceiVed NoVember 21, 2005
A novel series of benzofuran derivatives as potential positron emission tomography (PET) tracers targeting
amyloid plaques in Alzheimer’s disease (AD) were synthesized and evaluated. The syntheses of benzofurans
were successfully achieved by an intramolecular Wittig reaction between triphenylphosphonium salt and
4-nitrobenzoyl chloride. When in vitro binding studies using AD brain gray matter homogenates were carried
out with a series of benzofuran derivatives, all the derivatives examined displayed high binding affinities
with Ki values in the subnanomolar range. Among these benzofuran derivatives, compound 8, 5-hydroxy-
2-(4-methyaminophenyl)benzofuran, showed the lowest Ki value (0.7 nM). In vitro fluorescent labeling of
AD sections with compound 8 intensely stained not only amyloid plaques, but also neurofibrillary tangles.
The 11C labeled compound 8, [11C]8, was prepared by reacting the normethyl precursor, 5-hydroxy-2-(4-
aminophenyl)benzofuran, with [11C]methyl triflate. The [11C]8 displayed moderate lipophilicity (log P )
2.36), very good brain penetration (4.8%ID/g at 2 min after iv injection in mice), and rapid washout from
normal brains (0.4 and 0.2%ID/g at 30 and 60 min, respectively). In addition, this PET tracer showed in
vivo amyloid plaque labeling in APP transgenic mice. Taken together, the data suggest that a relatively
simple benzofuran derivative, [11C]8, may be a useful candidate PET tracer for detecting amyloid plaques
in the brains of patients with Alzheimer’s disease.
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disease
characterized by dementia, cognitive impairment, and memory
loss. Postmortem brains of AD patients reveal neuropathological
features: the presence of senile plaques (SPs) and neurofibrillary
tangles (NFTs), which contain â-amyloid (Aâ) peptides and
highly phosphorylated tau proteins.1,2 Although the precise
mechanism of neuronal death in AD is still unknown, it is widely
accepted that SPs and NFTs play a central role in its develop-
ment. Thus, quantitative evaluation of SPs and/or NFTs in the
brain with noninvasive techniques such as positron emission
Figure 1. Chemical structures of amyloid imaging agents previously
reported.
tomography (PET) and single photon emission computed
tomography (SPECT) would allow presymptomatic identifica-
tion of patients and monitoring of putative neuroprotective
effects of novel treatments that are currently being investigated.3-5
A number of groups have worked to develop radiolabeled
amyloid specific imaging agents, and clinical trials in AD
patients have been reported with several agents including [18F]-
2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)naphthalene-6-yl)eth-
ylidene)malononitrile ([18F]FDDNP),6,7 [11C]-2-(4-(methylamino)-
phenyl)-6-hydroxybenzothiazole ([11C]6-OH-BTA-1),8,9 and
[11C]-4-N-methylamino-4′-hydroxystilbene ([11C]SB-13)10,11 (Fig-
ure 1), indicating that detecting amyloid plaques in the living
human brain with amyloid imaging agents is potentially feasible.
Figure 2. Structure of benzofuran derivatives. Compounds reported
here include the following: R1 ) OCH3, OH; R2 ) NH2, NHCH3,
N(CH3)2.
In an attempt to develop more useful amyloid specific
imaging agents, we chose to investigate a novel series of
benzofuran derivatives, designed to be isosteric analogues of
thioflavin-T (Figure 2). We reported previously that iodinated
benzofuran derivatives displayed excellent binding affinities for
Aâ(1-40) aggregates (Ki ) 0.4-9.0 nM) and good brain
penetration (>1.1%ID after an iv injection in normal mice).
However, the in vivo nonspecific binding of these probes,
reflected by their slow washout from the normal mouse brain,
made them unsuitable for in vivo plaque imaging.12 These
previous findings suggested that additional structural changes
to these benzofurans may lead to the development of useful
amyloid specific imaging agents.
* To whom correspondence should be addressed: Phone:81-95-819-
2443, Fax: 81-95-819-2442. E-mail: mono@net.nagasaki-u.ac.jp.
† Nagasaki University.
‡ Department of Nuclear Medicine and Diagnostic Imaging, Kyoto
University.
| Osaka City University Medical School.
Department of Patho-Functional Bioanalysis, Kyoto University.
§ University of Pennsylvania.
10.1021/jm051176k CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/04/2006

2726 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Ono et al.
Figure 3. Synthesis of benzofuran derivatives. (a) EtOH, NaBH4; (b) acetonitrile, PPh3‚HBr; (c) toluene, 4-nitrobenzoyl chloride, NEt3; (d) EtOH,
SnCl2; (e) CH2Cl2, BBr3; (f) MeOH, NaOMe, (CH2O)n, NaBH4; (g) CH2Cl2, BBr3; (h) AcOH, (CH2O)n, NaCNBH3; (i) CH2Cl2, BBr3.
Table 1. Inhibition Constants of Benzofuran Derivatives Using
[
125I]IMPY as the Ligand in AD Brain Gray Matter Homogenates
compound
Ki (nM)a
Figure 4. Radiosynthesis of [11C]8.
5
6
7
8
9
10
2.3 ( 0.1
11.5 ( 2.5
1.3 ( 0.2
0.7 ( 0.2
12.0 ( 2.0
2.8 ( 0.5
We report here the in vitro and in vivo evaluation of a novel
series of benzofuran derivatives as â-amyloid imaging agents
for PET.
a Values are means ( standard error of the mean of three independent
experiments.
Results and Discussion
tives competed with [125I]IMPY binding to amyloid plaques with
excellent binding affinities (Table 1). Comparing compounds
5, 7, 9 with compounds 6, 8, 10, substitutions of the methoxy
group at 5 position with the hydroxy group resulted in only
small changes in binding affinity. Compounds 7 and 8 with a
monomethylaminophenyl moiety on the phenylbenzofuran
molecule displayed slightly lower (higher binding affinity) as
compared to compounds 5 and 6 with the aminophenyl moiety
or compounds 9 and 10 with the dimethylaminophenyl moiety.
However, all of the benzofuran derivatives evaluated maintained
good binding affinities in the nanomolar range of Ki values.
The results of the binding study strongly support our previous
report that benzofuran derivatives have considerable tolerance
for structural modification.12 Some reports have shown that
preserving the binding affinity for amyloid plaques and provid-
ing compounds with moderate lipophilicity are prerequisites for
successful amyloid imaging agents. Thus, we selected compound
8, with moderate lipophilicity and the highest binding affinity
for amyloid plaques in AD brain homogenates, for C-11 labeling
and additional studies.
Next, compound 8 was investigated for its neuropathological
staining of senile plaques in human AD brain sections (Figure
5). Compound 8 stained neuritic plaques, as well as cerebrovas-
cular amyloids (Figure 5, parts A and B). Since it is commonly
assumed that neuritic plaques are formed even in very mild AD
subjects, and that the density of the neuritic plaques is associated
with the severity of dementia and the number of neurons,15,16
clear staining of amyloid plaques with compound 8 demonstrates
that it is a promising compound and deserves further investiga-
tion as a potential tool for early diagnosis. Furthermore,
compound 8 also displayed high binding affinity for NFTs in
Chemistry. The synthesis of the benzofuran derivatives is
outlined in Figure 3. The key step for the formation of the
benzofuran backbone was achieved by an intramolecular Wittig
reaction between triphenylphosphonium salt and 4-nitrobenzoyl
chloride (Figure 3). The desired Wittig reagent, 3, was readily
prepared from 2-methoxy-5-hydroxybenzyl alcohol, 2, and
triphenylphosphine hydrobromide (yield 84%). Wittig reactions
gave the desired benzofuran backbone, 4, at a yield of 33%.
Conversion of 5 to the monomethylamino derivative, 7, was
achieved by first reducing the nitro group to an amino group
with SnCl2, and the subsequent monomethylation of the amino
group was achieved by using a method previously reported.13
Compound 5 was also converted to a dimethylamino derivative,
9, by an efficient method14 with paraformaldehyde, sodium
cyanoborohydride, and acetic acid (yield 39%). The O-methyl
groups of compounds 5, 7, and 9 were removed by reacting
with BBr3 to give 6, 8, and 10 at yields of 47, 39, and 7%,
respectively.
Preparation of [11C]8 was done as in Figure 4. [11C]8 was
readily synthesized from its N-normethyl precursor 6 and [11C]-
methyl triflate. Radiochemical yields of the final formulated
product were 21%, decay corrected to end of bombardment
(EOB). Radiochemical purities were >99% with specific
activities of 37 GBq/µmol. The identity of the radiolabeled [11C]-
8 was further confirmed by a comparison of its HPLC retention
time with two other possible isomers, O-methylated, 5, and N,O-
dimethylated, 7, which were independently prepared. On the
basis of different retention times, it was concluded that the
desired N-monomethylated, [11C]8, was successfully prepared.
Biological Studies. An in vitro binding assay using AD brain
homogenates demonstrated that substituted benzofuran deriva-

11C-Labeled Benzofuran DeriVatiVes as â-Amyloid Imaging Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2727
Figure 5. Neuropathological staining of compound 8 on 5 µm AD brain sections from the temporal cortex. (A) Amyloid plaques are stained with
compound 8 (× 40 magnification). (B) Many cerebrovascular amyloids are intensely stained with compound 8 (× 40 magnification). (C) Compound
8 also stained neurofibrillary tangles (NFTs) (× 40 magnification).
Table 2. Biodistribution of Radioactivity after Intravenous
washout in normal mice, which prevented this ligand from being
Administration of [11C]8 in Micea
useful for in vivo imaging.12 The unfavorable in vivo pharma-
time (min)
brain
blood
cokinetics of [125I]MABF were improved by changing iodine
to a hydroxyl group at 5 position on its benzofuran molecule.
Although many factors such as molecular size, ionic charge,
and lipophilicity affect the brain clearance of compounds,19-21
the precise mechanisms responsible for the rapid clearance of
[11C]8 from the normal brain still remain unknown.
2
5
15
30
60
4.78 (1.10)
2.80 (0.63)
0.80 (0.31)
0.35 (0.08)
0.19 (0.04)
3.86 (0.36)
4.04 (1.14)
2.49 (0.67)
1.32 (0.41)
1.17 (0.14)
a Expressed as % injected dose per gram. Each value represents the mean
(SD) for four animals at each interval.
[11C]PIB is currently the most widely utilized for detecting
amyloid plaques in AD patients.9,22,23 More recently, it has been
reported that [11C]SB-13 also showed similar performance to
the more established tracer, [11C]PIB.11 Both tracers displayed
appropriate properties as amyloid imaging agents: high binding
affinity for amyloid plaques at 4.3 and 6.0 nM of Ki values for
PIB and SB-13, respectively, in the in vitro binding assay, and
high brain uptake and rapid clearance from the normal brain in
animal studies. [11C]PIB entered the brain rapidly (7.0%ID/g
at 2 min after iv injection in mice) and cleared rapidly from
normal mice brains (0.6%ID/g at 30 min after iv injection in
mice). Since the ratio of 2-to-30 min mouse brain uptake after
iv injection of [11C]8 was 0.07, which was comparable to that
of [11C]PIB, [11C]8 is also expected to have suitable in vivo
pharmacokinetic properties for amyloid imaging in AD patients,
similar to [11C]PIB.
To confirm the in vivo labeling of amyloid plaques in a living
mouse brain, we evaluated [11C]8 using a model mouse of AD,
Tg2576 mice, which are specifically engineered to overproduce
the amyloid plaques in the brain. Autoradiographic images of
Tg2576 mouse brain at 30 min after injection of [11C]8 showed
high radioactivity accumulation in the cerebral cortex and
hippocampus (Figure 6-A). In contrast, wild-type mouse brain
displayed no remarkable accumulation of [11C]8 in the brain
(Figure 6-A). Furthermore, we confirmed that the hot spots of
[11C]8 in Tg2576 brains corresponded with those of in vitro
thioflavin-S staining in the same brain section (Figure 6, parts
B and C). The specific in vivo labeling for amyloid plaques
demonstrates the feasibility of using it as an in vivo PET
imaging agent for detecting amyloid plaques in the brains of
AD patients.
the AD sections (Figure 5C). A previous study reported that a
marked increase in the amount of NFTs accumulation in the
hippocampus and entorhinal cortex was observed in the pre-
clinical AD stage.17 Because compound 8 intensely stained NFTs
in human AD sections, it could detect increased NFT accumula-
tion in the hippocampus and entorhinal cortex of the AD brain.
These findings from the neuropathological staining of human
AD sections suggest that compound 8 can bind amyloid plaques
and NFTs with almost the same pattern of FDDNP6 or X-3418
previously reported, and quantitative evaluation of radiolabeled
compound 8 in the brain may provide useful information on
Aâ and tau pathology.
To predict the permeability of the blood-brain barrier, a
1-octanol/phosphate buffer (pH 7.4) partition coefficient of [11C]-
8 was examined. The log P of [11C]8 was 2.36 at pH 7.4.
Previous studies suggest that the optimal lipophilicity range for
brain entry is observed for compounds with log P values
between 1 and 3.19 Below that range, passive diffusion through
the BBB is poor, and above that range, binding of any
radiotracers to blood components (e.g., red blood cells and
albumin) is so great as to limit the amount available for brain
entry. Since this ligand displayed moderate lipophilicity for BBB
penetration, it was expected to show adequate brain uptake to
detect amyloid plaques following systemic injection.
A biodistribution study in normal mice after iv injection
showed that [11C]8 exhibited excellent brain uptake (4.8%ID/g
of the brain at 2 min) and rapid washout (0.4 and 0.2%ID/g of
the brain at 30 and 60 min, respectively), while the blood levels
were relatively low at all time points measured (Table 2). When
[11C]8 with high binding affinity to amyloid plaques is delivered
into brain regions containing amyloid plaques, [11C]8 is expected
to be trapped in amyloid plaque regions longer due to its high
binding affinity. A biodistribution study of [11C]8 was carried
out using normal mice. Since the normal brain has no Aâ
plaques to trap [11C]8 in the brain, the radioactivity should wash
out from the brain rapidly. Therefore, the rapid clearance of
[11C]8 from the normal brain (over 90% washout of the
radioactivity from the brain in 30 min or less) is an appropriate
pharmacokinetic property for early detection of amyloid plaques
in the AD brain. Our previous study demonstrated that a
radioiodinated benzofuran derivative, 5-iodo-2-(4-methyl-
aminophenyl)benzofuran, ([125I]MABF) showed slow brain
Conclusion
In summary, we introduced here novel benzofuran derivatives
that have high in vitro binding affinity for amyloid plaques as
candidate PET probes for imaging amyloid plaques. [11C]8, one
of these compounds, showed highly desirable properties: fast
high initial uptake kinetics and rapid washout from the non-Aâ
plaque-containing areas. In addition, ex vivo autoradiograms
of brain sections from Tg2576 after injection of [11C]8 showed
selective labeling of amyloid plaques with little nonspecific
binding. These findings in the study suggest that [11C]8 is a
promising PET probe for in vivo imaging amyloid plaques in
the brain.

2728 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Ono et al.
8.7 Hz, 1H), 7.64 (d, J ) 8.1 Hz, 2H). MS m/z 239 (M+). Anal.
C15H13NO2: C, H, N.
5-Hydroxy-2-(4-aminophenyl)benzofuran (6). BBr3 (2.8 mL,
1 M solution in CH2Cl2) was added to a solution of 5 (132 mg,
0.55 mmol) in CH2Cl2 (10 mL) dropwise in an ice bath. The mixture
was allowed to warm to room temperature and stirred for 30 min.
Water (20 mL) was added while the reaction mixture was cooled
in an ice bath. The mixture was extracted with ethyl acetate, and
the organic phase was dried over Na2SO4 and filtered. The filtrate
was concentrated, and the residue was purified by HPLC on a C18
column with an isocratic solvent of acetonitrile/H2O (1/1) at a flow
1
rate of 7.0 mL/min to give 13 mg of 6 (10.5%). H NMR (300
MHz, CDCl3) δ 5.50 (s, 2H), 6.63 (d, J ) 8.4 Hz, 2H), 6.85 (s,
2H), 7.28 (d, 1H), 7.52 (d, J ) 8.7 Hz, 2H), 9.08 (s, 1H). MS m/z
225 (M+). Anal. C14H11NO2: C, H, N.
5-Methoxy-2-(4-methylaminophenyl)benzofuran (7). A solu-
tion of NaOMe (28 wt % in MeOH, 0.27 mL) was added to a
mixture of 5 (40 mg, 0.17 mmol) and paraformaldehyde (18 mg,
0.69 mmol) in methanol (5 mL) dropwise. The mixture was stirred
under reflux for 1 h. After adding NaBH4 (15 mg, 0.47 mmol), the
solution was heated under reflux for 2 h. 1 M NaOH (30 mL) was
added to the cold mixture and extracted with CHCl3 (30 mL). 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 ) 5:1) to give 20 mg of 7 (47.3%). 1H NMR
(300 MHz, CDCl3) δ 2.87 (s, 3H), 3.84 (s, 3H), 6.64 (d, J ) 8.7
Hz, 2H), 6.72 (s, 1H), 6.83 (m, 1H), 7.35 (d, J ) 9.0 Hz, 1H),
7.67 (d, J ) 8.7 Hz, 2H). MS m/z 253 (M+). Anal. C16H15NO2‚
0.25H2O: C, H, N.
Figure 6. Ex vivo plaque labeling in brain sections from an APP
transgenic mouse (A and B) and a wild-type mouse with [11C]8 (A).
Amyloid plaques were confirmed by in vitro staining of the same section
with thioflavin-S (C). Arrows show amyloid plaques labeled by both
[11C]8 and thioflavin-S.
Experimental Sections
All reagents used in syntheses were commercial products and
were used without further purification unless otherwise indicated.
1H NMR spectra were obtained on a Varian Gemini 300 spectrom-
eter 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 JEOL IMS-DX instrument.
5-Hydroxy-2-(4-methylaminophenyl)benzofuran (8). The same
reaction as described above to prepare 6 was employed, and 8 was
1
obtained at a 26.8% yield from 7. H NMR (300 MHz, CDCl3) δ
2.87 (s, 3H), 3.84 (s, 3H), 6.64 (d, J ) 8.7 Hz, 2H), 6.72 (s, 1H),
6.83 (m,1H), 7.35 (d, J ) 9.0 Hz, 1H), 7.67 (d, J ) 8.7 Hz, 2H).
MS m/z 239 (M+). Anal. C15H13NO2: C, H, N.
Chemistry. 2-Hydroxy-5-methoxybenzyl Alcohol (2). Sodium
borohydride (250 mg, 6.61 mmol) was added to a stirring solution
of 2-hydroxy-5-methoxybenzaldehyde (2.0 g, 13.1 mmol) in ethanol
(20 mL) in an ice bath. The reaction mixture was stirred at room
temperature for 1 h. After the solvent was removed, 1 N aqueous
HCl solution (40 mL) was added to the residue and extracted with
diethyl ether (40 mL). The organic phase was dried over Na2SO4
and filtered. The filtrate was concentrated to give 2.02 g of 2
(99.7%). 1H NMR (300 MHz, CDCl3) δ 3.72 (s, 3H), 4.72 (s, 2H),
6.59 (s, 1H), 6.73 (m, 2H).
5-Methoxy-2-(4-dimethylaminophenyl)benzofuran (9). To a
stirred mixture of 5 (73 mg, 0.31 mmol) and paraformaldehyde
(97 mg, 0.31 mmol) in AcOH (4 mL) was added in one portion
NaCNBH3 (96 mg, 1.53 mmol) at room temperature. The resulting
mixture was stirred at room temperature for 8 h, and 1 M NaOH
(30 mL) was added and extracted with CH3Cl (30 mL). 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 ) 14:1) to give 32 mg of 9 (39.3%). 1H NMR
(300 MHz, CDCl3) δ 3.08 (s, 6H), 6.70-6.77 (m, 3H), 7.43 (d, J
) 9.0 Hz, 1H), 7.72-7.82 (m, 3H), 8.34 (s, 1H). MS m/z 267 (M+).
Anal. C17H17NO2: C, H, N.
5-Hydroxy-2-(4-dimethylaminophenyl)benzofuran (10). The
same reaction as described above to prepare 6 was employed, and
10 was obtained in 6.6% yield from 9. 1H NMR (300 MHz, CDCl3)
δ 2.97 (s, 6H), 6.66 (m, 1H), 6.79 (d, J ) 8.7 Hz, 2H), 6.85 (s,
1H), 6.95 (s, 1H), 7.32 (d, J ) 9.0 Hz, 1H), 7.67 (d, J ) 8.7 Hz,
2H). 9.11 (s, 1H). MS m/z 253 (M+). Anal. C16H15NO2‚0.25H2O:
C, H, N.
2-Hydroxy-4-methoxybenzyltriphenylphosphonium Bromide
(3). A solution of 2 (2.02 g, 13.1 mmol) and triphenylphosphine
hydrobromide (4.50 g, 13.1 mmol) in acetonitrile (40 mL) was
stirred under reflux for 1 h. The solid that formed was filtered and
1
washed with acetonitrile to give 5.28 g of 3 (84.1%). H NMR
(300 MHz, DMSO-d6) δ 3.38 (s, 3H), 4.87 (d, J ) 14.7 Hz, 2H),
6.33 (s, 1H), 6.65-6.71 (m, 2H), 7.67-7.89 (m, 15H), 9.34 (s,
1H).
5-Methoxy-2-(4-nitrophenyl)benzofuran (4). A mixture of 3
(525 mg, 1.10 mmol) and 4-nitrobenzoyl chloride (206 mg, 1.11
mmol) in a mixed solvent (toluene 20 mL and triethylamine 0.5
mL) was stirred under reflux for 2 h. The precipitate was removed
by filtration. The filtrate was concentrated, and the residue was
Radiolabeling. 11C was produced via a 14N(p,R)11C reaction with
16-MeV protons on a target of nitrogen gas with an ultracompact
cyclotron (CYPRIS model 325R; Sumitomo Heavy Industry Ltd.).
The 11CO2 produced was transported to an automated synthesis
system of 11C-methyl iodide (CUPID C-100; Sumitomo Heavy
Industry Ltd.), and converted sequentially to [11C]methyl triflate
([11C]CH3OTf) by the previously described method of Jewett.24
[11C]8 was produced by reacting [11C]CH3OTf with the normethyl
precursor, 5-hydroxy-2-(4-aminophenyl)benzofuran, 6 (0.4 mg), in
250 µL of methyl ethyl ketone (MEK). After complete transfer of
the [11C]CH3OTf, the reaction solvent was dried with a stream of
nitrogen gas. The residue taken up in 200 µL of acetonitrile was
purified by a reverse phase HPLC system (a Shimadzu LC-6A
isocratic pump, a Shimadzu SPD-6A UV detector and a Aloka
NDW-351D scintillation detector) on a Cosmosil C18 column
(Nacalai Tesque, 5C18-AR-300, 10 × 250 mm) with an isocratic
solvent of 0.1 M AcONH4/acetonitrile (45/55) at a flow rate of 6.0
1
recrystalized with ethyl acetate to give 97 mg of 4 (32.9%). H
NMR (300 MHz, CDCl3) δ 3.87 (s, 3H), 6.96-7.00 (m, 1H), 7.08
(d, 1H), 7.18 (d, 1H), 7.45 (d, J ) 8.7 Hz, 1H), 7.96-7.99 (m,
2H), 8.31 (d, J ) 9.3 Hz, 2H).
5-Methoxy-2-(4-aminophenyl)benzofuran (5). A mixture of 4
(170 mg, 0.63 mmol), SnCl2 (1.50 g, 7.91 mmol) and ethanol (20
mL) was stirred under reflux for 2 h. After the mixture cooled to
room temperature, 1 M NaOH (20 mL) was added and extracted
with ethyl acetate. The organic phase was dried over Na2SO4 and
filtered. The filtrate was concentrated and the residue was purified
by silica gel chromatography (hexane:ethyl acetate ) 3:1) to give
140 mg of 5 (92.9%). 1H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H),
6.73 (d, J ) 8.7 Hz, 2H), 6.84 (m, 2H), 6.99 (s, 1H), 7.36 (d, J )

11C-Labeled Benzofuran DeriVatiVes as â-Amyloid Imaging Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2729
mL/min. The desired fraction (Rt 7.6 min) was collected in a flask
and evaporated to dryness. Radiochemical yields, purities and
specific activity of [11C]8 were further confirmed by analytical
reverse phase HPLC on 5C18-AR-300 column (Nacalai Tesque,
4.6 × 150 mm, 0.1 M AcONH4/acetonitrile (50/50), 1.0 mL/min,
Rt-precursor 3.0 min; Rt-product 4.4 min).
Ex Vivo Plaque Labeling with [11C]8 in Transgenic Mice.
The ex vivo evaluation was performed using Tg2576 transgenic
(female, 22-month-old) and wild type (female, 22-month-old) mice
which were kindly provided by Dr. Tooyama (Shiga University of
Medical Science, Otsu, Japan). A saline solution (200 µL) contain-
ing ascorbic acid (1 mg/mL) of the labeled agent (7.4-9.3 MBq)
was injected directly into the tail vein. The mice were sacrificed
by decapitation at 30 min after intravenous injection. The brains
were immediately removed and frozen in powdered dry ice. Sections
of 30 µm were cut and exposed to a BAS imaging plate (Fuji Film,
Tokyo, Japan) for 3 h. Ex vivo autoradiographic images were
obtained using a BAS2500 scanner system (Fuji Film). After
autoradiographic examination, the same sections were stained with
thioflavin-S to confirm the presence of amyloid plaques. For the
staining with thioflavin-S, sections were immersed in 0.125%
thioflavin-S solution containing 50% ethanol for 3 min and washed
in 50% ethanol. After drying, the sections were then examined using
a TE-300 Fluoromicroscope with a COOLPIX910 CCD camera
(Nikon, Tokyo) equipped with an EPI-FL filter block.
Binding Studies. Binding studies were carried out according to
the method described previously.25
[
125I]IMPY (6-iodo-2-(4′-
dimethylamino)phenyl-imidazo[1,2]pyridine) with 81.4 TBq/mmol
specific activity and greater than 95% radiochemical purity was
prepared using the standard iododestannylation reaction, and
purified by a simplified C-4 mini column as described previously.26
Binding assays were carried out in 12 × 75 mm borosilicate glass
tubes. The reaction mixture contained 50 µL of AD brain homo-
genates (20-50 µg), 50 µL of [125I]IMPY (0.04-0.06 nM diluted
in PBS), and 50 µL of inhibitors (10-5-10-10 M diluted serially
in PBS containing 0.1% bovine serum albumin) in a final volume
of 1 mL. Nonspecific binding was defined in the presence of 600
nM IMPY in the same assay tubes. The mixture was incubated at
37 °C for 2 h, and the bound and free radioactivity were separated
by vacuum filtration through Whatman GF/B filters using a Brandel
M-24R cell harvester followed by 2 × 3 mL washes with PBS at
room temperature. Filters containing the bound I-125 ligand were
counted in a gamma counter (Packard 5000) with 70% counting
efficiency. Under the assay conditions, the specifically bound
fraction was less than 15% of the total radioactivity. The results of
inhibition experiments were subjected to nonlinear regression
analysis using EBDA,27 from which Ki values were calculated.
Partition Coefficient Determination. The determination of
partition coefficient of [11C]8 was performed according to the
procedure previously reported with some modifications.28 1-Octanol
(3 mL) and 0.1 M phosphate buffer (pH 7.4, 3 mL) were pipetted
into a test tube with 2 µL of [11C]8 (370 kBq). The test tube was
vortexed for 3 min at room temperature, followed by centrifugation
for 5 min. Two weighed samples (500 µL each) from the 1-octanol
and buffer layers were counted in a well counter. The partition
coefficient was determined by calculating the ratio of cpm/500 µL
of 1-octanol to that of buffer. Samples from 1-octanol layer were
repartitioned until consistent partitions of coefficient values were
obtained. The measurement was done in triplicate and repeated four
times.
Staining of Senile Plaques in Human AD Brain Sections.
Postmortem brain tissues from AD cases (74 and 79 years old)
were confirmed by conventional neuropathology, including amyloid
and tau staining, in addition to silver staining. Experiments were
performed under the regulations of the ethics committee of Osaka
City University Medical School. Five-micrometer-thick serial
sections of paraffin-embedded blocks from the temporal cortex were
used for staining. Paraffin sections were first taken through five
10-min incubations in xylene, five 10-min incubations in 100%
ethanol to completely deparaffinize them, followed by two 5-min
washes in water and then PBS. Tissue sections were immersed in
the compound solution at various concentrations. At more than 0.5
mM concentration, we used 50% ethanol in PBS to ensure the full
solubilization of the compound. Finally, the sections were washed
in PBS for 15 min. Thereafter, the sections were incubated in
ethanol and xylene and embedded in Entellan Neu (Merck,
Darmstadt, Germany). Fluorescent sections were viewed using a
BX50 Fluoromicroscope with an M-3204C CCD camera (Olympus,
Tokyo) equipped with a G-filter. The sections were also immuno-
stained with DAB as a chromogen using antibodies against Aâ29
and tau30 protein, as previously described.
Acknowledgment. Financial support was provided in part
by a Grant-in-Aid for Young Scientists (B) (Grant No.
16790734) and Scientific Research (B) (Grant No. 15390014)
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan. Tg2576 transgenic mice were kindly
provided by Dr. Ikuo Tooyama (Shiga University of Medical
Science, Otsu, Japan).
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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