Novel 18F-labeled benzofuran derivatives with improved properties for positron emission tomography (PET) imaging of β-amyloid plaques in Alzheimer's brains

Article abstract of DOI:10.1021/jm200057u

In vivo imaging of β-amyloid plaques in the brain may lead to the early diagnosis of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of treatment. In the present study, we report on the development of two potential PET probes,

Full text of DOI:10.1021/jm200057u

ARTICLE
pubs.acs.org/jmc
Novel 18F-Labeled Benzofuran Derivatives with Improved Properties for
Positron Emission Tomography (PET) Imaging of β-Amyloid Plaques in
Alzheimer’s Brains
Masahiro Ono,*,† Yan Cheng,† Hiroyuki Kimura,† Mengchao Cui,† Shinya Kagawa,‡ Ryuichi Nishii,‡ and
Hideo Saji*,†
†Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
‡Shiga Medical Center Research Institute, 5-4-30, Moriyama, Moriyama City, Shiga 524-8524, Japan
S Supporting Information
b
ABSTRACT: In vivo imaging of β-amyloid plaques in the
brain may lead to the early diagnosis of Alzheimer’s disease
(AD) and monitoring of the progression and effectiveness of
treatment. In the present study, we report on the develop-
ment of two potential PET probes, [18F]FPYBF-2 ([18F]10)
and [18F]FPHBF-2 ([18F]21), for imaging of β-amyloid
plaques in AD brain. In experiments in vitro, 10 and 21
displayed high affinity for Aβ(1ꢀ42) aggregates (Ki = 2.41
and 3.85 nM, respectively). In biodistribution experiments
using normal mice, they displayed high uptake in the brain
(7.38 and 8.18% ID/g at 2 min postinjection, respectively),
and the radioactivity washed out from the brain rapidly (3.15
and 3.87% ID/g at 60 min postinjection, respectively), which
is highly desirable for β-amyloid imaging agents. In vivo, they clearly labeled β-amyloid plaques in Tg2576 mice. Furthermore,
the specific labeling of β-amyloid plaques by 10 and 21 was observed in autoradiographs of sections of autopsied AD brain.
These new fluorinated benzofuran derivatives are promising PET probes for imaging cerebral β-amyloid plaques.
’ INTRODUCTION
with 11C (t1/2 = 20 min) on a routine basis have limited its
potential as a diagnostic tool. Since additional tracers labeled
with 18F with longer half-life (t1/2 = 110 min) may be more useful
as PET imaging agents for detection and quantification of Aβ
plaques, recent efforts have focused on the development of
comparable agents labeled with 18F. Previous studies with
[18F]2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)naphthalene-
6-yl)ethylidene)malononitrile (FDDNP)13,14 showed differen-
tial uptake and retention in the brain of AD patients for the first
time. PET imaging studies in humans suggest that FDDNP
shows a higher retention in regions of the brain suspected of
having neurofibrillary tangles and Aβ plaques, indicating that it is
not for selectively measuring Aβ plaques in AD brains. More
recently, a PIB analogue, 2-(3-[18F]fluoro-4-methyaminophe-
nyl)benzothiazol-6-ol (GE-067, flutemetamol),15ꢀ17 a stilbene
derivative, (E)-4-(N-methylamino)-40-(2-(2-(2-[18F]fluoroeth-
oxy)ethoxy)ethoxy)stilbene (BAY94-9172, florbetaben) ,18ꢀ20
Alzheimer’s disease (AD), the most common senile dementia,
is characterized by β-amyloid (Aβ) plaques, vascular amyloid,
neurofibrillary tangles, and progressive neurodegeneration. The
formation of Aβ plaques, composed of Aβ peptides, in the brain
is generally accepted as the initial neurodegenerative event in
AD.1,2 The in vivo detection of Aβ plaques in AD brains by
positron emission tomography (PET) should be useful for early
diagnosis and the discovery of effective therapeutic agents for
AD.3ꢀ5
Initial studies with PET suggested that [11C]4-N-methyl-
amino-40-hydroxystilbene (SB-13),6,7 [11C]2-(40-(methylamino
phenyl)-6-hydroxybenzothiazole (PIB),8,9 [11C]2-(2-[2-di-
methylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxa-
zole (BF-227),10 and [11C]-2-[6-(methylamino)pyridin-3-yl]-
1,3-benzothiazol-6-ol (AZD2184),11,12 differed in their uptake
and retention in the brain between AD patients and controls
(Figure 1). Among them, PIB is the best characterized PET
imaging agent for Aβ plaques in the brain. In the past few years,
successful imaging with PIB in thousands of AD patients has been
reported. The utility of PIB to image Aβ plaques in the brain has
provided considerable impetus for further refinement of this
technique. However, the practical challenges of using PIB labeled
and
a
styrylpyridine derivative, (E)-4-(2-(6-(2-(2-(2-(-
[18F]fluoroethoxy)ethoxy)ethoxy)pyridyn-3-ylvinyl)-N-methyl-
benzenamine (AV-45, florbetapir),21ꢀ27 and have been shown to
Received: January 20, 2011
Published: March 23, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of PET imaging agents targeting β-amyloid plaques in AD patients.
In the present study, we designed and synthesized novel
fluorinated pyridylbenzofuran and phenylbenzofuran derivatives
(FPYBF-2 and FPHBF-2, Figure 2) with a fluoropolyethylene
glycol side chain and a monomethylamino group. We here report
the synthesis and biological evaluation of two new 18F benzofur-
an derivatives with a monomethylamino group in comparison
with the corresponding benzofuran derivatives with
dimethylamino group.
a
Figure 2. Chemical structure of 18F-labeled benzofuran derivatives
reported in the present study.
’ RESULTS AND DISCUSSION
be useful for the imaging of Aβ plaques in living brain tissue in
phase II or III clinical trials (Figure 1).
The synthesis of FPYBF-2 (10) and FPHBF-2 (21) is outlined
in Schemes 1 and 2, respectively. The key step in the formation of
the fluorinated pyridylbenzofuran backbone is accomplished by
Suzuki coupling between 5-methoxybenzofuran-2-boronic acid
and 2-amino-5-iodopyridine.34 Suzuki coupling afforded the
desired compound 1 in a yield of 52%. The key step in the
formation of the phenylbenzofuran backbone was readily
achieved by reacting 2-hydroxy-5-methoxybenzaldehyde with
4-nitrobenzyl bromide to produce 11 in a yield of 64%. The
amino derivative 12 was prepared from 11 by reduction with
SnCl2 in a yield of 49%. Conversion of 1 and 12 to the
monomethylamino derivatives 2 and 13 was achieved by the
monomethylation of the amino group by using a method29
previously reported in yields of 92% and 95%, respectively. A
methoxy group of 2 and 13 was converted to a hydroxyl group
using BBr3/CH2Cl2, which afforded 3 and 14 in yields of 99 and
99%, respectively. To prepare compounds with three ethoxy
groups as the polyethylene glycol linkage, 2-[2-(2-chloroethox-
y)ethoxy]ethanol was coupled with the OH group of 3 and 14 to
obtain 4 and 15. The free OH groups of 4 and 15 were
subsequently protected with tert-butyldimethylsilyl chloride
(TBDMSCl) to give 5 and 16. Compounds 6 and 17 were
obtained by protecting the methylamino groups of 5 and 16.
After removal of the TBS-protected groups of 5 and 16 with
tetrabutylammonium fluoride (TBAF) in tetrahydrofuran
(THF), the free OH groups of 7 and 18 were converted into
mesylates by reacting with MsCl in the presence of triethylamine
to give 8 and 19. The fluorinated benzofuran derivatives 10 and
21 were successfully obtained by refluxing 8 and 19 in anhydrous
TBAF/THF followed by stirring with TFA to remove the
butyloxycarbonyl (BOC) protected group.
In the search for PET imaging agents with improved proper-
ties, we have recently reported a series of fluorinated benzofuran
derivatives as potential 18F-labeled tracers for the imaging
of Aβ plaques by PET. First, on the basis of our previous
research regarding radioiodinated and 11C-labeled benzofuran
derivatives,28,29 we developed 4-(5-(2-(2-(2-fluoroethoxy)-
ethoxy)ethoxy)benzofuran-2-yl)-N,N-dimethylbenzenamine
(FPHBF-1, Figure 2) with a fluoropolyethylene glycol side chain
and a dimethyaminophenyl group.30 Although the penetration of
brain tissues by this tracer was encouraging, the slow washout of
this probe from the normal mouse brain made it unsuitable for
imaging in vivo. Therefore, a critical need to fine-tune the kinetics
of the uptake and washout of benzofuran derivatives existed.
Previous results regarding uptake into and clearance from the
brain point to high lipophilicity as one of the reasons for a slow
washout from the brain.8,29,31,32
To further improve the pharmacokinetics of the radioactivity in the
brain, we planned to develop a novel fluorinated pyridylbenzofuran
derivative with less lipophilicity by displacing the phenyl group in
phenylbenzofuran with a pyridyl group. Then we designed and
synthesized 5-(5-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzofuran-
2-yl)-N,N-dimethylpyridin-2-amine (FPYBF-1, Figure 2) with a
fluoropolyethylene glycol side chain and a dimethylaminopyridyl
group.33 This tracer displayed faster clearance from the normal mouse
brain than FPHBF-1, without a decrease in uptake and reduction in
affinity for Aβ plaques. These results suggested that the pharmaco-
kinetics were improved by reducing the lipophilicity of the benzofuran
derivative. While 18F-labeled AV-19 ((E)-2-(2-(2-(2-fluoroethoxy)
ethoxy)ethoxy)-5-(4-dimethylaminostyryl)pyridine) showed pro-
mising results in animal studies, its uptake in humans was lower than
expected possibly because of a rapid metabolism of its dimethylamino
group.27 However, monomethylation of AV-19 led to the formation
of AV-45, which exhibited excellent uptake and washout in the brain
in humans.22,27 Furthermore, all PET imaging probes in phase III
clinical trials (AV-45, BAY94-9172, and GE067, Figure 1) have
a monomethylamino group that is stable in vivo and reduces
the lipophilicity of the compounds compared with a dimethy-
lamino group.
To prepare the desired 18F-labeled benzofuran derivatives, the
N-BOC-protected mesylates 8 and 19 were employed as the
precursors (Scheme 3). Each of the mesylates was reacted with
[18F]fluoride/potassium carbonate and Kryptofix 222 in acet-
onitrile. The mixture was then treated with aqueous HCl to
remove the N-BOC-protected group. After purification of the
crude product by HPLC, the 18F-labeled 10 ([18F]FPYBF-2) and
21 ([18F]FPHBF-2) were prepared with an average radiochemical
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Scheme 1a
a Reagents: (a) (Ph3P)4Pd, Na2CO3(aq)/dioxane; (b) (1) NaOMe, MeOH, (CH2O)n; (2) NaBH4; (c) BBr3, CH2Cl2; (d) 2-[2-(2-chloroethox-
y)ethoxy]ethanol, K2CO3, DMF; (e) TBDMSCl, imidazole, CH2Cl2; (f) (BOC)2O, THF; (g) TBAF, THF; (h) MsCl, Et3N, CH2Cl2; (i) TBAF, THF;
(j) TFA, CH2Cl2.
Scheme 2a
a Reagents: (a) K2CO3, DMF; (b) SnCl2, EtOH; (c) NaOMe, (CH2O)n, MeOH; (d) BBr3, CH2Cl2; (e) 2-[2-(2-chloroethoxy)ethoxy]ethanol, K2CO3,
DMF; (f) TBDMSCl, imidazole, CH2Cl2; (g) (BOC)2O, THF; (h) TBAF, THF; (i) MsCl, Et3N, CH2Cl2; (j) TBAF, THF; (k) TFA, CH2Cl2.
Scheme 3a
a Reagents and conditions: (a) Kryptofix 222/K2CO3, 18Fꢀ, MeCN, 120 °C, 5 min; (b) 10%HCl (aq), 120 °C, 5 min.
yield of 52% and radiochemical purity of >99% and a specific
activity of 242 GBq/μmol. The identity of [18F]10 and [18F]21
was verified by a comparison of the retention time with that of the
nonradioactive compound (see Supporting Information).
that they have excellent affinity for Aβ(1ꢀ42) aggregates (Table 1).
The N-monomethylated derivatives (10 and 21) exhibited slightly
less affinity (Ki = 2.41 and 3.85 nM, respectively) than the N,N-
dimethylated derivatives (FPYBF-1 and FPHBF-1) (0.95 and 2.00
nM, respectively). In addition, 10 and 21 showed higher binding
affinity than PIB and IMPY, well-known Aβ imaging probes (Ki =
9.00 and 10.5 nM, respectively), in the same assay. The result
suggests that removing the methyl group from a dimethylamino
group of FPYBF-1 and FPHBF-1 reduces the affinity for Aβ
aggregates. It also indicates that the electronegativity of the nitrogen
Experiments in vitro to evaluate the affinity of the fluorinated
benzofuran derivatives for Aβ aggregates were carried out
in solutions with [125I]2-(40-dimethylaminophenyl)-6-iodoimid-
azo[1,2-a]pyridine ([125I]IMPY) as the ligand according to conven-
tional methods.35,36 Compounds 10 and 21 inhibited the binding of
[
125I]IMPY with a Ki of 2.41 and 3.85 nM, respectively, indicating
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Table 1. Inhibition Constants of Benzofuran Derivatives on
Binding to Aβ42 Aggregates
compd
Ki (nM)a
ClogPb
10
2.41 ( 0.11
3.85 ( 0.22
0.95 ( 0.21
2.00 ( 0.50
9.00 ( 1.31
10.50 ( 1.05
2.32
2.94
3.11
3.73
3.28
3.79
21
FPYBF-1
FPHBF-1
PIB
IMPY
a Values are the mean ( standard error of the mean for three
independent experiments. b The calculated logarithms of waterꢀoctanol
partition coefficients (ClogP) were obtained using ChemDraw Ultra
10.0 software (CambridgeSoft, Cambridge, MA).46
Figure 3. Comparison of brain uptake of four 18F-labeled benzofuran
derivatives in normal mice.
Table 2. Biodistribution of Radioactivity after Intravenous
Administration of 18F-Labeled Benzofuran Derivatives in
Micea
time after injection (min)
tissue
2
10
30
60
[
18F]10
blood
4.96 (0.38)
7.38 (0.84)
2.65 (0.35)
16.8 (2.20)
11.9 (0.93)
4.24 (0.51)
2.91 (0.42)
5.68 (0.33)
5.14 (0.48)
5.19 (0.39)
3.73 (0.81)
3.02 (0.12)
4.86 (0.41)
1.63 (0.13)
17.5 (1.81)
6.22 (0.66)
8.39 (2.04)
2.15 (0.14)
3.12 (0.21)
2.68 (0.29)
2.51 (0.28)
7.55 (2.09)
2.66 (0.34)
4.03 (0.61)
1.79 (0.17)
19.2 (2.64)
7.83 (1.18)
24.1 (5.76)
3.14 (0.61)
4.34 (0.77)
4.91 (2.21)
3.59 (0.48)
18.8 (5.69)
2.42 (0.13)
3.15 (0.10)
1.72 (0.27)
8.71 (0.99)
3.70 (1.79)
30.9 (8.00)
2.24 (0.35)
2.88 (0.27)
2.55 (0.21)
2.60 (0.23)
22.5 (2.56)
brain
bone
liver
kidney
intestine
spleen
lung
Figure 4. Ex vivo plaque labeling in brain sections from a Tg2576
mouse (A) and a wild-type mouse (B) with [18F]10. The same sections
were also stained with thioflavin-S (C, D).
pancreas
heart
stomachb
[18F]10 and [18F]21 displayed high uptake (7.38 and 8.18% ID/g)
at 2 min postinjection; these values were comparable to those
reported for BAY94-9172 and AV-45, which are under phase III
clinical trials. The uptake of [18F]10 and [18F]21 was higher than
that of [18F]FPYBF-1 and [18F]FPHBF-1 with a dimethylamino
group. The radioactivity in the brain after the injection of [18F]10
and [18F]21 cleared with time (3.15 and 3.87% ID/g at 60 min
postinjection). Since there are no Aβ plaques in the brain of normal
mice, a high initial uptake and rapid washout in normal mouse brain
are highly desirable properties for Aβ plaque-targeting imaging
agents. To directly compare the uptake and washout of four ligands
tested in this study, a combined plot is presented in Figure 3. The
brain2min/brain60min ratio as an index to compare the washout rate is
used for determining the radioactivity pharmacokinetics in vivo.40
The brain2min/brain60min ratio of [18F]10, [18F]21, [18F]FPYBF-1,
and [18F]FPHBF-1 was 2.34, 2.11, 2.11, and 1.02, respectively,
indicating that [18F]10 provided the best profile of radioactivity in
the brain among the four ligands. Although the brain2min/brain60min
ratio of [18F]10 (2.34) was lower than that of [18F]BAY94-9172
(4.8)18 or [18F]AV-45 (3.8),21 it was improved compared to values
for [18F]FPYBF-1 (2.11) reported previously.30 The favorable
in vivo pharmacokinetics of [18F]10 were achieved by changing
the dimethylaminophenyl group in [18F]FPYBF-1 to a monomethy-
laminophenyl group. Although lipophilicity is just one of the factors
affecting the uptake of a compound into the brain, previous results
suggest the lipophilicity of Aβ imaging agents to play an important
role in uptake and washout. The results observed for [18F]10 and
[
18F]21
blood
brain
bone
4.10 (0.56)
8.18 (0.59)
1.57 (0.62)
4.17 (0.32)
6.43 (0.66)
1.98 (0.16)
5.33 (0.33)
4.26 (0.42)
2.07 (0.16)
7.33 (1.01)
3.87 (0.42)
3.30 (0.25)
[
18F]FPYBF-1
2.13 (0.49)
3.75 (0.64)
1.33 (0.28)
blood
brain
bone
2.83 (0.89)
5.16 (0.30)
1.61 (0.33)
1.76 (0.09)
2.78 (0.22)
1.11 (0.13)
1.98 (0.35)
2.44 (0.36)
1.42 (0.24)
[
18F]FPYBF-1
2.48 (0.18)
5.66 (0.31)
1.76 (0.13)
blood
brain
bone
1.64 (0.07)
2.88 (0.46)
1.19 (0.18)
2.68 (0.26)
3.14 (0.26)
3.63 (1.11)
3.41 (0.56)
2.80 (0.06)
2.74 (0.59)
a Expressed as % injected dose per gram. Each value represents the mean
(SD) for five animals at each interval. b Expressed as % injected dose
per organ.
atom of the aminophenyl group plays an important role in determin-
ing binding affinity. A similar change in affinity for Aβ aggregates
after N-methyl groups were added has been observed for stilbene,37
styrylpyridine,38 flavone,31 chalcone,39 and aurone40 derivatives
containing a p-aminophenyl group.
To evaluate the uptake of [18F]10 and [18F]21 in the brain, a
biodistribution experiment was performed in normal mice (Table 2).
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selective binding of Aβ plaques with little nonspecific binding.
The results suggest that [18F]10 should be further investigated as
a potentially useful PET imaging agent for cerebral Aβ plaques.
[18F]10 is currently in preparation for clinical trials.
’ EXPERIMENTAL SECTION
General Remarks. All chemicals used in syntheses were commercial
products used without further purification. 1H NMR spectra were obtained
at 400 MHz on JEOL JNM-AL400 NMR spectrometers at room tempera-
ture with TMS as an internal standard. Chemical shifts are reported as
δ values relative to the internal TMS. Coupling constants are reported in
hertz. Multiplicity is defined by s (singlet), d (doublet), t (triplet), and m
(multiplet). Mass spectra were acquired with a Shimadzu GC-MS-QP2010
Plus (ESI). HPLC was performed with a Shimadzu system (a LC-10AT
pump with a SPD-10A UV detector, λ = 254 nm) with a Cosmosil C18
column (Nakalai Tesque, 5C18-AR-II, 4.6 mm ꢁ 150 mm) using acetoni-
trile/water (60:40) as the mobile phase at a flow rate of 1.0 mL/min.
Fluorescent observation was performed by microscope (Nikon Eclipse 80i)
with a BV-2A filter set (excitation, 400ꢀ440 nm; diachronic mirror, 455 nm;
long pass filter, 470 nm). All key compounds were proven to show g95%
purity by HPLC.
Figure 5. In vitro autoradiograms of sections of AD brain labeled with
[18F]10. Intensive labeling of β-amyloid plaques in brain tissue from AD
patients is shown (A). The control subject exhibits no labeling by this
tracer (B).
[18F]21 suggest that the introduction of further hydrophilic groups
into the pyridylbenzofuran and phenylbenzofuran scaffold may lead
to the development of more useful pyridylbenzofuran and phenyl-
benzofuran derivatives. No marked uptake of [18F]10 and [18F]21 in
the bone was observed (1.72 and 3.30% ID/g, respectively), suggest-
ing little defluorination in vivo, and so interference with the imaging
is expected to be relatively minor.
To further characterize the potential of these probes in living
brain tissue, we carried out autoradiography ex vivo in Tg2576
mice (36 months, male) and in wild-type mice (36 months, male)
as age-matched controls (Figure 4). Tg2576 transgenic mice
show marked Aβ deposition in the brain by 11ꢀ13 months
of age41 and have been frequently used to evaluate the specific
binding of Aβ plaques in experiments in vitro and in vivo.39,42,43
The autoradiography using [18F]10 showed distinctive labeling
of Aβ plaques in the Tg2576 mouse brain (Figure 4A), while
wild-type mouse brain showed no such labeling (Figure 4B).
Aβ plaques were confirmed to be present by costaining the
sections with thioflavin-S, a dye commonly used to stain
Aβ plaques (Figure 4C). When we carried out ex vivo auto-
radiography in Tg2576 using [18F]21, we obtained almost the
same results as with [18F]10 (data not shown). The results
suggest that these probes penetrated the bloodꢀbrain barrier
and selectively labeled the Aβ plaques in the brain, as reflected by
the biodistribution experiments and the in vitro binding assays.
Furthermore, we also investigated the effectiveness for neu-
ropathological staining of Aβ plaques in human AD brain
sections (Figure 5). A previous report suggested the configura-
tion/folding of Aβ plaques in Tg2576 mice to be different from
the tertiary/quaternary structure of Aβ plaques in AD brains.44
Therefore, it is important to evaluate the affinity for Aβ plaques
in human AD brains. [18F]10 clearly stained many Aβ plaques in
AD brains. In contrast, no apparent staining was observed in
normal adult brain sections. The labeling pattern was consistent
with that observed by immunohistochemical labeling with an
antibody specific to Aβ. The same results were observed in in
vitro autoradiography using [18F]21 (data not shown). These
results indicate that [18F]10 may be applicable for in vivo
imaging of Aβ plaques in AD brains and deserves further
investigation as a potential probe for the diagnosis of AD.
In conclusion, on the basis of previous results, we designed
and synthesized two novel fluorinated pyridylbenzofuran
and phenylbenzofuran ligands 10 and 21 for the imaging of
Aβ plaques in the brain. These ligands showed high affinity for
Aβ aggregates in vitro and for Aβ plaques in sections of autopsied
AD brain. In biodistribution experiments using normal mice,
they displayed good uptake into and fast washout from the brain,
especially [18F]10. In addition, ex vivo autoradiograms of brain
sections from Tg2576 mice after the injection of [18F]10 showed
Chemistry.
5-(5-Methoxy-2-benzofuranyl)-2-pyridina-
mine (1). A solution of 5-methoxybenzofuran-2-boronic acid (576
mg, 3.0 mmol), 2-amino-5-iodopyridine (660 mg, 3.0 mmol), and
(Ph3)4Pd (366 mg, 0.3 mmol) in 2 M Na2CO3/dioxane (150 mL,
1:1) was stirred under reflux overnight. After the mixture had cooled to
room temperature, 1 M NaOH (20 mL) was added and extraction with
ethyl acetate was carried out. 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 = 1:1) to give 374
mg of 1 (52.1%). 1H NMR (400 MHz, CDCl3): δ 3.85 (s, 3H), 4.67 (s,
2H), 6.59 (d, 1H, J = 8.8 Hz), 6.76 (s, 1H), 6.82 (dd, 1H, J1= 8.8 Hz, J2=
2.4 Hz), 7.07 (d, 1H, J = 2.4 Hz), 7.36 (d, 1H, J = 8.8 Hz), 7.86 (dd, 1H,
J1= 8.8 Hz, J2= 2.4 Hz), 8.67 (d, 1H, J = 2.4 Hz). MS: m/z 241
(Mþ þ H).
5-(5-Methoxy-2-benzofuranyl)-N-methyl-2-pyridinamine
(2). Sodium methoxide (275 mg, 5.0 mmol) was added to 1 (240 mg, 1.0
mmol) in methanol (15 mL) followed by paraformaldehyde (101 mg,
4.0 mmol). The solution was heated to reflux for 2 h and cooled to 0 °C
with an ice bath. Sodium borohydride (128 mg, 4.0 mmol) was added.
The reaction mixture was brought to reflux again for 1 h and poured onto
crushed ice. After a standard workup with ethyl acetate, the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 1:1) to
give 234 mg of 2 (92.1%). 1H NMR (400 MHz, CDCl3): δ 2.98 (d, 3H,
J = 5.2 Hz), 3.84 (s, 3H), 4.78 (s, 1H), 6.47 (d, 1H, J = 8.8 Hz), 6.76
(s, 1H), 6.84 (dd, 1H, J1 = 8.8 Hz, J2 = 4.4 Hz), 7.00 (d, 1H, J = 2.4 Hz),
7.36 (d, 1H, J = 8.4 Hz), 7.87 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.59
(d, 1H, J = 2.8 Hz). MS: m/z 255 (Mþ þ H).
2-(6-Methylamino-3-pyridinyl)-5-benzofuranol (3). BBr3
(4.8 mL, 1 M solution in CH2Cl2) was added to a solution of 2 (234
mg, 0.92 mmol) in CH2Cl2 (20 mL) dropwise in an ice bath. The
mixture was allowed to warm to room temperature and stirred for 1 h.
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 silica gel chromatography (hexane/ethyl
1
acetate = 1:1) to give 218 mg of 3 (99.0%). H NMR (400 MHz,
CDCl3): δ 2.98 (d, 3H, J = 5.2 Hz), 4.78 (s, 1H), 6.47 (d, 1H, J = 8.8 Hz),
6.76 (s, 1H), 6.84 (dd, 1H, J1 = 8.8 Hz, J2 = 4.4 Hz), 7.00 (d, 1H, J = 2.4
Hz), 7.36 (d, 1H, J = 8.4 Hz), 7.87 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.59
(d, 1H, J = 2.8 Hz). MS: m/z 241 (Mþ þ H).
2-(2-(2-(2-(6-(Methylamino)-3-pyridinyl)-5-benzofurany-
loxy)ethoxy)ethoxy)ethanol (4). To a solution of 3 (197 mg, 0.82
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mmol) and 2-[2-(2-chloroethoxy)ethoxy]ethanol (180 μL, 1.20 mmol)
in DMF (5 mL) was added anhydrous K2CO3 (414 mg, 3.0 mmol). The
reaction mixture was stirred for 18 h at 100 °C and then poured into
water and extracted with chloroform. The organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a residue,
which was purified by silica gel chromatography (hexane/ethyl acetate =
1:6) to give 224 mg of 4 (60.3%). 1H NMR (400 MHz, CDCl3): δ 2.98
(d, 3H, J = 5.2 Hz), 3.62 (d, 2H, J = 4.4 Hz), 3.70ꢀ3.74 (m, 6H), 3.84 (s,
2H), 4.11 (s, 2H), 5.18 (s, 1H), 6.47 (d, 1H, J = 8.8 Hz), 6.76 (s, 1H),
6.84 (dd, 1H, J1 = 8.8 Hz, J2 = 4.4 Hz), 7.00 (d, 1H, J = 2.4 Hz), 7.36 (d,
1H, J = 8.4 Hz), 7.87 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.59 (d, 1H, J =
2.8 Hz). MS: m/z 373 (Mþ þ H).
M in THF, 0.20 mL) was added to a solution of 8 (27 mg, 0.05 mmol)
in anhydrous THF (10 mL). The mixture was refluxed for 4 h. After-
ward, it was cooled to room temperature. After a standard workup with
ethyl acetate, the residue was purified by silica gel chromatography
(hexane/ethyl acetate = 1:1) to give 22 mg of 9 (94.0%). 1H NMR (400
MHz, CDCl3): δ 1.57 (s, 9H), 3.44 (s, 3H), 3.71ꢀ3.83 (m, 6H),
3.88ꢀ3.91 (m, 2H), 4.22ꢀ4.51 (m, 1H), 4.61ꢀ4.63 (m, 1H), 6.91 (dd,
1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 6.94 (s, 1H), 7.05 (d, 1H, J = 6.0 Hz), 7.38
(d, 1H, J = 8.8 Hz), 7.81 (d, 1H, J = 8.8 Hz), 8.02 (dd, 1H, J1 = 8.8 Hz,
J2 = 2.4 Hz), 8.82 (d, 1H, J = 2.4 Hz). MS: m/z 475 (Mþ þ H).
5-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)benzofuran-2-
yl)-N-methylpyridin-2-amine (10). Trifluoroacetic acid (1.27 mL)
was added slowly to a solution of 9 (22 mg, 0.047 mmol) in dichlor-
omethane (2 mL). The mixture was then stirred at room temperature for
1 h. After a standard workup with ethyl acetate, the residue was purified
by silica gel chromatography (hexane/ethyl acetate = 1:1) to give 9 mg of
10 (50.6%). 1H NMR (400 MHz, CDCl3): δ 2.98 (d, 3H, J = 5.2 Hz),
3.71ꢀ3.80 (m, 6H), 3.79ꢀ3.88 (m, 2H), 4.11ꢀ4.18 (m, 2H),
4.49ꢀ4.51 (m, 1H), 4.61ꢀ4.63 (m, 1H), 4.80 (s, 1H), 6.45 (d, 1H,
J = 8.8 Hz), 6.75 (s, 1H), 6.85 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 7.01 (d,
1H, J = 2.4 Hz), 7.36 (d, 1H, J = 9.2 Hz), 7.86 (dd, 1H, J1 = 8.8 Hz, J2 =
2.4 Hz), 8.59 (d, 1H, J = 2.0 Hz). HRMS (EI: m/z calcd for
C20H23FN2O4 (Mþ) 374.1642, found 374.1650.
5-Methoxy-2-(4-nitrophenyl)benzofuran (11). A solution of
2-hydroxy-5-methoxybenzaldehyde (1.22 g, 8.02 mmol), 4-nitrobenzyl
bromide (1.73 g, 8.02 mmol), and K2CO3 (3.33 g, 24.06 mmol) in DMF
(10 mL) was stirred under reflux overnight. The solvent was removed
and the residue was recrystallized with ethyl acetate to give 1.39 g of 11
(64.0%). 1H NMR (400 MHz, CDCl3): δ 3.85 (s, 3H), 6.96ꢀ7.00 (m,
1H), 7.08 (d, 1H, J = 2.8 Hz), 7.18 (d, 1H, J = 2.8 Hz), 7.45 (d, 1H, J = 2.8
Hz), 7.95 (d, 2H, J = 8.8 Hz), 8.31 (d, 2H, J = 8.8 Hz).
5-Methoxy-2-(4-aminophenyl)benzofuran (12). A mixture
of 11 (1.39 g, 5.16 mmol), SnCl2 (5.82 g, 25.8 mmol), and ethanol
(20 mL) was stirred under reflux for 2 h. After the mixture had cooled to
room temperature, 1 M NaOH (20 mL) was added and extraction with
ethyl acetate was carried out. 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 = 7:3) to give 609
mg of 12 (49.0%). 1H NMR (400 MHz, CDCl3): δ 3.85 (s, 3H), 6.71 (d,
2H, J = 8.8 Hz), 6.75 (s, 1H), 6.82 (dd, 1H, J1 = 8.8 Hz, J2 = 1.6 Hz), 6.97
(d, 1H, J = 2.8 Hz), 7.35 (d, 1H, J = 8.8 Hz), 7.65 (d, 2H, J = 8.8 Hz). MS:
m/z 240 (Mþ þ H).
5-(5-(2-(2-(2-(tert-Butyldimethylsilyloxy)ethoxy)ethoxy)-
ethoxy)benzofuran-2-yl)-N-methylpyridin-2-amine (5). 4 (61
mg, 0.17 mmol) and TBDMSCl (41 mg, 0.27 mmol) were dissolved in
dichloromethane (10 mL) followed by imidazole (24 mg, 0.34 mmol).
The solution was stirred at room temperature for 2 h. After a standard
workup with ethyl acetate, the residue was purified by silica gel chroma-
tography (hexane/ethyl acetate = 1:6) to give 73 mg of 5 (87.6%). 1H
NMR (400 MHz, CDCl3): δ 0.08 (s, 6H), 0.90 (s, 9H), 2.98 (d, 3H, J =
5.2 Hz), 3.62 (d, 2H, J = 4.4 Hz), 3.70ꢀ3.74 (m, 6H), 3.84 (s, 2H), 4.11
(s, 2H), 5.18 (s, 1H), 6.47 (d, 1H, J = 8.8 Hz), 6.76 (s, 1H), 6.84 (dd, 1H,
J1 = 8.8 Hz, J2 = 4.4 Hz), 7.00 (d, 1H, J = 2.4 Hz), 7.36 (d, 1H, J = 8.4 Hz),
7.87 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.59 (d, 1H, J = 2.8 Hz). MS: m/z
487 (Mþ þ H).
tert-Butyl 5-(5-(2-(2-(2-(tert-Butyldimethylsilyloxy)etho-
xy)ethoxy)ethoxy)-2-benzofuranyl)-2-pyridinyl-
(methyl)carbamate (6). 5 (73 mg, 0.15 mmol) was dissolved in
anhydrous THF (5.0 mL) followed by Boc anhydride (66 mg, 0.30
mmol). The solution was refluxed overnight. After a standard workup
with ethyl acetate, the residue was purified by silica gel chromatography
(hexane/ethyl acetate = 1:1) to give 37 mg of 6 (46.7%). 1H NMR (400
MHz, CDCl3): δ 0.03 (s, 6H), 0.86 (s, 9H), 1.50 (s, 9H), 3.37 (s, 3H),
3.62ꢀ3.68 (m, 6H), 3.82ꢀ3.88 (m, 2H), 4.09ꢀ4.12 (m, 2H), 6.86 (d,
1H, J = 4.4 Hz), 6.88 (s, 1H), 6.98 (d, 1H, J = 2.4 Hz), 7.33 (d, 1H, J = 8.8
Hz), 7.76 (d, 1H, J = 10.0 Hz), 7.95 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz),
8.75 (d, 1H, J = 1.6 Hz). MS: m/z 587 (Mþ þ H).
tert-Butyl 5-(5-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)-2-
benzofuranyl)-2-pyridinyl(methyl)carbamate (7). TBAF (1 M
in THF, 0.30 mL) was added via a syringe to a solution of 6 (37 mg,
0.07 mL) in THF (5 mL). The solution was stirred at room temperature
for 5 h. After a standard workup with ethyl acetate, the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 1:1) to
give 31 mg of 7 (93.0%). 1H NMR (400 MHz, CDCl3): δ 1.57 (s, 9H),
3.45 (s, 3H), 3.62ꢀ3.65 (m, 2H), 3.70ꢀ3.78 (m, 6H), 3.83ꢀ3.93 (m,
2H), 4.07ꢀ4.18 (m, 2H), 6.87(d, 1H, J = 6.8 Hz), 6.94 (s, 1H), 7.06 (d,
1H, J = 2.4 Hz), 7.40 (d, 1H, J = 9.2 Hz), 7.82 (d, 1H, J = 8.8 Hz), 8.02
(dd, 1H, J1 = 8.8 Hz, J2 = 2 Hz), 8.83 (d, 1H, J = 2.8 Hz). MS: m/z 473
(Mþ þ H).
5-Methoxy-2-(4-methylaminophenyl)benzofuran (13). A
solution of NaOMe (5 M in MeOH, 4.0 mL) was added to a mixture
of 12 (609 mg, 2.55 mmol) and paraformaldehyde (279 mg, 10.3 mmol)
in methanol (30 mL) dropwise. The mixture was stirred under reflux for
1 h. After NaBH4 (225 mg, 7.0 mmol) was added, the solution was
heated under reflux for 2 h. Then 1 M NaOH (30 mL) was added to the
cold mixture and extraction with CHCl3 (30 mL) was conducted. 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 = 7:3) to give 616 mg of 13 (95.0%). 1H NMR
(400 MHz, CDCl3): δ 2.84 (s, 3H), 3.85 (s, 3H), 6.71 (d, 2H, J = 8.8
Hz), 6.75 (s, 1H), 6.82 (dd, 1H, J1 = 8.8 Hz, J2 = 1.6 Hz), 6.97 (d, 1H, J =
2.8 Hz), 7.35 (d, 1H, J = 8.8 Hz), 7.65 (d, 2H, J = 8.8 Hz). MS: m/z 254
(Mþ þ H).
2-(2-(2-(2-(6-(tert-Butoxycarbonyl)-3-pyridinyl)-5-benzo-
furanyloxy)ethoxy)ethoxy)ethyl Methanesulfonate (8). 7
(30.5 mg, 0.065 mmol) was dissolved in dichloromethane (5 mL) followed
by triethylamine (35 mg, 0.35 mmol). Methanesulfonyl chloride (25 mg,
0.21 mmol) was then added via a syringe. The solution was stirred at room
temperature for 3 h. After a standard workup with ethyl acetate, the residue
was purified by silica gel chromatography (hexane/ethyl acetate = 1:6) to
give 25 mg of 8(74.5%). 1H NMR (400 MHz, CDCl3):δ1.54(s, 9H), 3.06
(s, 3H), 3.44 (s, 3H), 3.69ꢀ3.72 (m, 6H), 3.79ꢀ3.88 (m, 2H), 4.11ꢀ4.17
(m, 2H), 4.37ꢀ4.39 (m, 2H), 6.90 (dd, 1H, J1 = 9.2 Hz, J2 = 2.8 Hz), 6.95
(s, 1H), 7.05 (d, 1H, J = 2.4 Hz), 7.40 (d, 1H, J = 8.8 Hz), 7.83 (d, 1H, J =
8.0 Hz), 8.02 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 8.83 (d, 1H, J = 0.8 Hz).
HRMS EI: m/z calcd for C26H34N2O9S (Mþ) 550.1985, found 550.1989.
tert-Butyl 5-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-2-
benzofuranyl)-2-pyridinyl(methyl)carbamate (9). TBAF (1
5-Hydroxy-2-(4-methylaminophenyl)benzofuran
(14).
BBr3 (12.4 mL, 1 M solution in CH2Cl2) was added to a solution of
13 (616 mg, 2.43 mmol) in CH2Cl2 (40 mL) dropwise in an ice bath.
The mixture was allowed to warm to room temperature and stirred for 1
h. 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 silica gel chromatography (hexane/ethyl
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1
acetate = 7:3) to give 576 mg of 14 (99.0%). H NMR (400 MHz,
CDCl3): δ 2.92 (s, 3H), 3.92 (s, 1H), 6.64 (d, 2H, J = 8.4 Hz), 6.68 (s,
1H), 6.71 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz), 6.93 (d, 1H, J = 2.4 Hz), 7.30
(d, 1H, J = 8.4 Hz), 7.70 (d, 2H, J = 8.4 Hz). MS: m/z 240 (Mþ þ H).
2-(2-(2-(2-(4-(Methylamino)phenyl)benzofuran-5-ylox-
y)ethoxy)ethoxy)ethanol (15). To a solution of 14 (575 mg, 2.41
mmol) and 2-[2-(2-chloroethoxy)ethoxy]ethanol (810 μL, 8.91 mmol)
in DMF (10 mL) was added anhydrous K2CO3 (1.23 g, 8.91 mmol).
The reaction mixture was stirred for 18 h at 100 °C and then poured into
water and extracted with chloroform. The organic layers were combined
and dried over Na2SO4. Evaporation of the solvent afforded a residue,
which was purified by silica gel chromatography (hexane/ethyl acetate =
1:6) to give 654 mg of 15 (73.2%). 1H NMR (400 MHz, CDCl3): δ 2.84
(s, 3H), 3.49ꢀ3.52 (m, 2H), 3.58ꢀ3.64 (m, 6H), 3.71ꢀ3.82 (m, 2H),
4.08ꢀ4.11 (m, 2H), 6.59 (d, 2H, J = 8.8 Hz), 6.65 (s, 1H), 6.77 (dd, 1H,
J1 = 8.8 Hz, J2 = 2.8 Hz), 6.94 (d, 1H, J = 2.4 Hz), 7.26 (d, 1H, J = 8.8 Hz),
7.60 (d, 2H, J = 8.8 Hz). MS: m/z 486 (Mþ þ H).
tert-Butyl 4-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ben-
zofuran-2-yl)phenyl(methyl)carbamate (20). TBAF (1 M in
THF, 0.50 mL) was added to a solution of 20 (55.3 mg, 0.10 mmol) in
anhydrous THF (5 mL). The mixture was refluxed for 2 h and cooled to
room temperature. After a standard workup with ethyl acetate, the
residue was purified by silica gel chromatography (hexane/ethyl acetate
= 1:1) to give 46 mg of 20 (97.2%). 1H NMR (400 MHz, CDCl3): δ 1.47
(s, 9H), 3.29 (s, 3H), 3.70ꢀ3.73 (m, 6H), 3.78ꢀ3.80 (m, 2H),
4.15ꢀ4.18 (m, 2H), 4.48ꢀ4.62 (m, 2H), 6.89ꢀ6.92 (m, 2H), 7.04 (d,
1H, J = 2.8 Hz), 7.30 (d, 2H, J = 8.8 Hz), 7.37 (d, 1H, J = 5.2 Hz), 7.79 (d,
2H, J = 8.8 Hz). MS: m/z 474 (Mþ þ H).
4-(5-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)benzofuran-2-
yl)-N-methylbenzenamine (21). Trifluoroacetic acid (2.70 mL)
was added slowly to a solution of 20 (46.0 mg, 0.10 mmol) in
dichloromethane (3 mL). The mixture was then stirred at room
temperature for 1 h. After a standard workup with ethyl acetate, the
residue was purified by silica gel chromatography (hexane/ethyl acetate
= 1:1) to give 16 mg of 21 (43.4%). 1H NMR (400 MHz, CDCl3): δ 2.88
(s, 3H), 3.72ꢀ3.75 (m, 6H), 3.87ꢀ3.89 (m, 2H), 4.05ꢀ4.11 (m, 2H),
4.49ꢀ4.63 (m, 2H), 6.65 (d, 2H, J = 8.8 Hz), 6.71 (s, 1H), 6.82 (dd, 1H,
J1 = 8.8 Hz, J2 = 2.8 Hz), 7.00 (d, 1H, J = 2.4 Hz), 7.33 (d, 1H, J = 8.8 Hz),
7.66 (d, 2H, J = 8.8 Hz). HRMS EI: m/z calcd for C21H24FNO4 (Mþ)
373.4180, found 373.1683.
4-(5-(2-(2-(2-(tert-Butyldimethylsilyloxy)ethoxy)ethoxy)-
ethoxy)benzofuran-2-yl)-N-methylbenzenamine (16). 15
(383 mg, 1.03 mmol) and TBDMSCl (243 mg, 1.61 mmol) were
dissolved in dichloromethane (10 mL) followed by imidazole (142 mg,
2.06 mmol). The solution was stirred at room temperature for 4 h. After
a standard workup with ethyl acetate, the residue was purified by silica
gel chromatography (hexane/ethyl acetate = 1:1) to give 453 mg of 16
Binding Assays Using the Aggregated Aβ Peptides in
Solution. Aβ(1ꢀ42) 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 incubated at 37 °C for 42
h with gentle and constant shaking. A mixture containing 50 μL of test
compounds (0.008 pM to 400 μM in 10% EtOH), 50 μL of 0.02 nM
1
(90.6%). H NMR (400 MHz, CDCl3): δ 0.02 (s, 6H), 0.83 (s, 9H),
2.84 (s, 3H), 3.49ꢀ3.52 (m, 2H), 3.58ꢀ3.64 (m, 6H), 3.71ꢀ3.82 (m,
2H), 4.08ꢀ4.11 (m, 2H), 6.59 (d, 2H, J = 8.8 Hz), 6.65 (s, 1H), 6.77 (dd,
1H, J1 = 8.8 Hz, J2 = 2.8 Hz), 6.94 (d, 1H, J = 2.4 Hz), 7.26 (d, 1H, J = 8.8
Hz), 7.60 (d, 2H, J = 8.8 Hz). MS: m/z 486 (Mþ þ H).
tert-Butyl 4-(5-(2-(2-(2-(tert-Butyldimethylsilyloxy)etho-
xy)ethoxy)ethoxy)benzofuran-2-yl)phenyl-
[
125I]IMPY, 50 μL of Aβ(1ꢀ42) aggregates, and 850 μL of 10% EtOH
was incubated 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 of the filters containing the bound 125I
ligand was measured in a γ counter. Values for the half maximal
inhibitory concentration (IC50) were determined from displacement
curves of three independent experiments using GraphPad Prism 5.0, and
those for the inhibition constant (Ki) were calculated using the
ChengꢀPrusoff equation:45 Ki = IC50/(1 þ [L]/Kd), where [L] is the
concentration of [125I]IMPY used in the assay and Kd is the dissociation
constant of IMPY (4.2 nM).
(methyl)carbamate (17). Compound 16 (452 mg, 0.93 mmol) was
dissolved in anhydrous THF (20 mL) followed by Boc anhydride (601
mg, 1.79 mmol). The solution was refluxed overnight. After standard
workup with ethyl acetate, the residue was purified by silica gel
chromatography (hexane/ethyl acetate = 7:3) to give 412 mg of 17
1
(75.6%). H NMR (400 MHz, CDCl3): δ 0.02 (s, 6H), 0.83 (s, 9H),
3.20 (s, 3H), 3.47ꢀ3.52 (m, 2H), 3.59ꢀ3.67 (m, 6H), 3.71ꢀ3.82 (m,
2H), 4.08ꢀ4.11 (m, 2H), 6.82 (d, 2H, J = 8.4 Hz), 6.95 (d, 1H, J = 3.6
Hz), 7.23 (d, 2H, J = 8.8 Hz), 7.29 (d, 1H, J = 8.4 Hz), 7.70 (d, 2H, J = 8.8
Hz). MS: m/z 586 (Mþ þ H).
Radiolabeling with 18F. [18F]Fluoride was produced by cyclotron
(CYPRIS HM-18, Sumitomo Heavy Industries, Tokyo) via an 18O (p,
n)18F reaction and passed through a Sep-Pak Light QMA cartridge
(Waters) as an aqueous solution in 18O-enriched water. The cartridge
was dried by N2, and the 18F activity was eluted with 1.0 mL of a
Kryptofix 222/K2CO3 solution (9.5 mg of Kryptofix 222 and 1.7 mg of
K2CO3 in acetonitrile/water (96/4)). The solvent was removed at
120 °C under a stream of argon gas. The residue was azeotropically dried
with 1 mL of anhydrous acetonitrile twice at 120 °C under a stream of
nitrogen gas. A solution of the mesylate precursors 8 and 19 (1.0 mg) in
acetonitrile (200 μL) was added to the reaction vessel containing the 18F
activity. The mixture was heated at 120 °C for 5 min and was cooled for 1
min. HCl (10% aqueous solution, 450 μL) was then added, and the
mixture was heated at 120 °C again for 5 min. An aqueous solution of
NaOH was added to adjust the pH to 8ꢀ9. The mixture was extracted
with ethyl acetate (1 mL ꢁ 2), and the solvent was removed under
nitrogen gas. The residue was purified by preparative HPLC [YMC-Pack
Pro C18 column (20 mm ꢁ 150 mm), acetonitrile/water (70/30), flow
rate of 4.0 mL/min]. The retention time of the desired 18F-labeled
product is 13.3 min. The radiochemical purity and specific activity were
determined by analytical HPLC [YMC-Pack Pro C18 column (4.6 mm
ꢁ 150 mm), acetonitrile/water (50/50), flow rate of 1.0 mL/min], and
[18F]10 and [18F]21 were obtained in a radiochemical purity of >99%
tert-Butyl 4-(5-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)-
benzofuran-2-yl)phenyl(methyl)carbamate (18). TBAF (1 M
in THF, 1.83 mL) was added via a syringe to a solution of 17 (412 mg,
0.70 mL) in THF (10 mL). The solution was stirred at room temperature
for 2 h. After a standard workup withethyl acetate, theresiduewas purified
by silica gel chromatography (hexane/ethyl acetate = 1:1) to give 324 mg
of18 (97.9%). 1H NMR (400 MHz, CDCl3): δ 1.40 (s, 9H), 3.18 (s, 3H),
3.49ꢀ3.53 (m, 2H), 3.58ꢀ3.66 (m, 6H), 3.74ꢀ3.79 (m, 2H), 6.82 (d,
2H, J = 8.4 Hz), 6.95 (d, 1H, J = 3.6 Hz), 7.22 (d, 2H, J = 8.8 Hz), 7.29 (d,
1H, J = 8.4 Hz), 7.70 (d, 2H, J = 8.8 Hz). MS: m/z 472 (Mþ þ H).
2-(2-(2-(2-(4-(tert-Butoxycarbonyl)phenyl)benzofuran-5-
yloxy)ethoxy)ethoxy)ethyl Methanesulfonate (19). 18 (324
mg, 0.688 mmol) was dissolved in dichloromethane (20 mL) followed
by triethylamine (344 mg, 3.44 mmol). Methanesulfonyl chloride (237
mg, 2.06 mmol) was then added via a syringe. The solution was stirred at
room temperature for 2 h. After a standard workup with ethyl acetate,
the residue was purified by silica gel chromatography (hexane/ethyl
1
acetate = 1:1) to give 374 mg of 19 (98.8%). H NMR (400 MHz,
CDCl3): δ 1.47 (s, 9H), 3.02 (s, 3H), 3.26 (s, 3H), 3.63ꢀ3.72 (m, 6H),
3.79ꢀ3.82 (m, 2H), 4.32ꢀ4.35 (m, 2H), 6.86 (dd, 1H, J1 = 8.8 Hz, J2 =
2.4 Hz), 6.90 (s, 1H), 7.02 (d, 1H, J = 2.8 Hz), 7.30 (d, 2H, J = 8.8 Hz),
7.36 (d, 1H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.8 Hz). HRMS EI: m/z calcd
for C27H35NO9S (Mþ) 549.6331, found 549.2025.
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ARTICLE
with a specific activity of 242 GBq/μmol. Specific activity was estimated
by comparing the UV peak intensity of the purified 18F-labeled
compound with a reference nonradioactive compound of known
concentration.
’ ABBREVIATIONS USED
AD, Alzheimer’s disease; Aβ, β-amyloid; NFT, neurofibrillary
tangle; PET, positron emission tomography; SB-13, 4-N-meth-
ylamino-40-hydroxystilbene; PIB, 2-(40-(methylaminophenyl)-6-
hydroxybenzothiazole; FDDNP, 2-(1-(2-(N-(2-fluoroethyl)-N-
methylamino)naphthalene-6-yl)ethylidene)malononitrile; FPHBF-
1, 4-(5-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)-
N,N-dimethylbenzenamine; FPYBF-1, 5-(5-(2-(2-(2-fluoroetho-
xy)ethoxy)ethoxy)benzofuran-2-yl)-N,N-dimethylpyridin-2-amine;
TBAF, tetra-n-butylammonium fluoride; THF, tetrahydrofuran;
BOC, tert-butyloxycarbonyl; TBDMSCl, tert-butyldimethylsilyl chlor-
ide; IMPY, 2-(40-dimethylaminophenyl)-6-iodoimidazo[1,2-
a]pyridine
Biodistribution in Normal Mice. Experiments with animals were
conducted in accordance with our institutional guidelines and approved
by the Kyoto University Animal Care Committee. While under anesthesia
with isoflurane, ddY mice (22ꢀ25 g, male) were injected directly into the
tail vein with 100 μL of a 0.1% BSA solution containing [18F]10 and
[18F]21 (185ꢀ370 kBq). The mice (n = 5 for each time point) were
sacrificed at 2, 10, 30, and 60 min postinjection. The organs of interest
were removed and weighed, and radioactivity was measured with an
automaticγcounter(COBRAII, Packard). Percentage doseper organwas
calculated by a comparison of the tissue counts to suitably diluted aliquots
of the injected material. The % dose/g of samples was calculated by
comparing the sample counts with the count of the diluted initial dose.
In Vitro Autoradiography Using Brain Sections from AD
Patients. Post-mortem brain sections (5 μm, temporal lobe) of an AD
patient and a control subject (5 μm, temporal lobe) were obtained from
BioChain Institute Inc. They were incubated with [18F]10 and [18F]21
(444 kBq/50 μL) for 1 h at room temperature. The sections were then
dipped in saturated Li2CO3 in 40% EtOH (two 2-min washes), washed
with 40% EtOH (one 2-min wash), and rinsed with water for 30 s. After
drying, the 18F-labeled sections were exposed to a BAS imaging plate
(Fuji Film, Tokyo, Japan) overnight. Autoradiographic images were
obtained using a BAS5000 scanner system (Fuji Film).
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Supporting Information. Preparation for 18F-labeling of
S
b
10 and 21. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For M.O.: phone, þ81-75-753-4608; fax, þ81-75-753-4568;
e-mail, ono@pharm.kyoto-u.ac.jp. For H.S.: phone, þ81-75-
753-4556; fax, þ81-75-753-4568, e-mail, hsaji@pharm.kyoto-u.
ac.jp.
’ ACKNOWLEDGMENT
The study was supported by the Funding Program for Next
Generation World-Leading Researchers and a Grant-in-Aid for
Young Scientists (A) and Exploratory Research from the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
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Journal of Medicinal Chemistry
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