Fluoro-pegylated chalcones as positron emission tomography probes for in vivo imaging of β-amyloid plaques in Alzheimer's disease

Article abstract of DOI:10.1021/jm901057p

This paper describes the synthesis and biological evaluation of fluoro-pegylated (FPEG) chalcones for the imaging of β-amyloid (Aβ) plaques in patients with Alzheimer's disease (AD). FPEG chalcone derivatives were prepared by the aldol condensation reacti

Full text of DOI:10.1021/jm901057p

6394 J. Med. Chem. 2009, 52, 6394–6401
DOI: 10.1021/jm901057p
Fluoro-pegylated Chalcones as Positron Emission Tomography Probes for in Vivo Imaging of
β-Amyloid Plaques in Alzheimer’s Disease
Masahiro Ono,*,†,‡ Rumi Watanabe,† Hidekazu Kawashima,§ Yan Cheng,‡ Hiroyuki Kimura,‡ Hiroyuki Watanabe,†
Mamoru Haratake,† Hideo Saji,‡ and Morio Nakayama*,†
†Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521,
Japan, ‡Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho,
Sakyo-ku, Kyoto 606-8501, Japan, and §Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine, Kyoto
University, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
Received July 16, 2009
This paper describes the synthesis and biological evaluation of fluoro-pegylated (FPEG) chalcones for
the imaging of β-amyloid (Aβ) plaques in patients with Alzheimer’s disease (AD). FPEG chalcone
derivatives were prepared by the aldol condensation reaction. In binding experiments conducted in vitro
using Aβ(1-42) aggregates, the FPEG chalcone derivatives having a dimethylamino group showed
higher Ki values (20-50 nM) than those having a monomethylamino or a primary amine group. When
the biodistribution of 11C-labeled FPEG chalcone derivatives having a dimethyamino group was
examined in normal mice, all four derivatives were found to display sufficient uptake for imaging Aβ
plaques in the brain. 18F-labeled 7c also showed good uptake by and clearance from the brain, although
a slight difference between the 11C and 18F tracers was observed. When the labeling of Aβ plaques was
carried out using brain sections of AD model mice and an AD patient, the FPEG chalcone derivative 7c
intensely labeled Aβ plaques. Taken together, the results suggest 7c to be a useful candidate PET tracer
for detecting Aβ plaques in the brain of patients with AD.
Introduction
as tracers for imaging Aβ plaques in the diagnosis of AD.
Recent reports suggest that Aβ aggregates possess multiple
ligand-binding sites, the density of which differs.13-15 There-
fore, the development of novel probes that bind Aβ aggregates
may lead to critical findings regarding the pathology of AD.
Recently, in a search for novel Aβ-imaging probes, we
found that radioiodinated flavone,16,17 chalcone,18,19 and
aurone20,21 derivatives, which are categorized as flavonoids,
showed excellent characteristics such as high affinity for Aβ
aggregates and good uptake into and rapid clearance from the
brain. The chalcone structure in particular is considered to be
a useful core in the development of new Aβ-imaging probes
because it can be formed by a one-pot condensation reaction.
In addition, because chalcone derivatives show different
characteristics of binding to Aβ aggregates from Congo Red
and thioflavin T, they are expected to provide new informa-
tion from in vivo imaging in AD brains.
The formation of β-amyloid (Aβa) plaques is a key neuro-
degenerative event in Alzheimer’s disease (AD).1,2 Because the
imaging of Aβ plaques in vivo may lead to the presymptomatic
diagnosis of AD, many radiotracers that bind to Aβ plaques
have been developed.3,4 Preliminary reports of positron emis-
sion tomography (PET) suggested that the uptake and reten-
tion of 2-(40-[11C]methylaminophenyl)-6-hydroxybenzothiazole
([11C]PIB, 1)5,6 and 4-N-[11C]methylamino-40-hydroxystilbene
([11C]SB-13, 2)7,8 differed between the brain of AD patients and
those of controls. However, because 11C is a positron-emitting
isotope with a t1/2 of just 20 min, efforts are being made to
develop comparable agents labeled with the isotope 18F (t1/2
=
110 min). [18F]-2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)-
naphthalene-6-yl)ethylidene)malononitrile ([18F]FDDNP, 3)9,10
and [18F]-4-(N-methylamino)-40-(2-(2-(2-fluoroethoxy)etho-
xy)ethoxy)-stilbene ([18F]BAY94-9172, 4)11,12 should be useful
In the present study, we designed and synthesized fluori-
nated chalcone derivatives for the purpose of developing
18F-labeled probes for PET-based imaging of Aβ plaques.
The formation of bioconjugates based on pegylation-fluor-
ination resulting in fluoro-pegylated (FPEG) molecules is
effective for some core structures of Aβ-imaging probes.22
We have adopted a novel approach, adding a short PEG (n=
1-3) to the chalcone backbone and capping the end of the
ethylene glycol chain with a fluorine atom. Indeed, the most
promising 18F-labeled agent 4 possesses PEG (n=3) in the
stilbene backbone. This tracer showed strong affinity (Ki =
6.7 nM) for Aβ plaques, high uptake (7.77%ID/g at 2 min
postinjection), and rapid clearance from the mouse brain
*To whom correspondence should be addressed. For M.O.: phone,
þ81-75-753-4608; fax, þ81-75-753-4568; E-mail, ono@pharm.kyoto-u.
ac.jp. For M.N.: phone, þ81-95-819-2441; fax, þ81-95-819-2441;
E-mail: morio@nagasaki-u.ac.jp.
a Abbreviations: Aβ, β-amyloid; AD, Alzheimer’s disease; PET, posi-
tron emission tomography; PIB, 2-(40-methyaminophenyl)-6-hydroxy-
benzothiazole; SB-13, 4-N-methylamino-40-hydroxystilbene; FDDNP,
2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)naphthalene-6-yl)ethylidene)-
malononitrile; BAY94-9174, 4-(N-methylamino)-40-(2-(2-(2-fluoroetho-
xy)ethoxy)ethoxy)-stilbene; DMIC, 4-dimethylamino-40-iodo-chalcone;
IMPY, 6-iodo-2-(40-dimethylamino)phenyl-imidazo[1,2-a]pyridine; FPEG,
fluoro-pegylated; DAST, diethylamino sulfur trifluoride; DME, 1,2-di-
methoxyethane; MEK, methyl ethyl ketone; [11C]methyl triflate, [11C]Me-
OTf; DAB, 3,30-diaminobenzidine.
r
pubs.acs.org/jmc
Published on Web 09/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6395
Scheme 1
Scheme 2
Scheme 3a
a (i) EtOH, KOH; (ii) EtOH, SnCl2; (iii) DMSO, MeI, K2CO3.
(1.61%ID/g at 60 min postinjection).12 We adopted the
biological data for 4 as criteria to develop novel Aβ-imaging
agents. In this study, we synthesized 12 fluorinated chalcones
and evaluated their biological potential as Aβ-imaging agents
by testing their affinity for Aβ aggregates and Aβ plaques in
sections of brain tissue from AD model mice and an AD
patient and their uptake by and clearance from the brain in
biodistribution experiments using normal mice.
4-fluoroacetophenone was reacted with 4-dimethylaldehyde
to form 40-hydroxy-4-dimethylamino-chalcone
5
and
40-fluoro-4-dimethylamino-chalcone 13 in yields of 84.0 and
41.6%, respectively. Compounds10(a-c) weresynthesized by
an aldol reaction between FPEG acetophenone 9(a-c) and
4-nitrobenzaldehyde. Fluorination of 6(a-c) and 8(a-c)
to prepare 7(a-c) and 9(a-c) was done using diethylamino
sulfur trifluoride (DAST) after introducing three oligoethy-
lene glycol molecules into the phenolic OH of 5 and 9(a-c).
The amino derivatives 11(a-c) and 15 were readily prepared
from 10(a-c) and 14 by reduction with SnCl2. Conver-
sion of 11(a-c) and 15 to the monomethylamino deriva-
tives 12(a-c) and 16 was achieved by methylation with
CH3I under alkaline conditions. Preparation of 11C-labeled
compounds was done as in Scheme 4. 11C-labeled chalcones
Results and Discussion
The synthesis of the FPEG chalcone derivatives is outlined
in Schemes 1, 2, and 3. The most useful way to prepare
chalcones is the condensation of acetophenones with ben
zaldehydes. Using this process, 4-hydroxyacetophenone or

6396 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Ono et al.
Scheme 4
Scheme 5
Table 1. Chemical Structures and Inhibition Constants of Fluorinated
Chalcone Derivatives
Table 2. Biodistribution of Radioactivity after Injection of [11C]7a,
[
11C]7b, [11C]7c, and [11C]13 in Normal Micea
organ
2 min
10 min
30 min
60 min
[
11C]7a
blood
brain
3.65 ( 0.37
6.01 ( 0.61
2.73 ( 0.28
3.24 ( 0.39
2.12 ( 0.18
2.57 ( 0.26
2.22 ( 0.25
2.26 ( 0.41
[
11C]7b
Ki (nM)a
blood
brain
3.48 ( 0.56
4.73 ( 0.47
2.28 ( 0.84
2.23 ( 0.18
2.54 ( 0.96
1.14 ( 0.12
1.44 ( 0.36
1.00 ( 0.19
compd
R1
R2
7a
7b
FCH2CH2O
F(CH2CH2O)2
F(CH2CH2O)3
FCH2CH2O
F(CH2CH2O)2
F(CH2CH2O)3
FCH2CH2O
F(CH2CH2O)2
F(CH2CH2O)3
F
N(CH3)2
N(CH3)2
N(CH3)2
NH2
45.7 ( 7.1
20.0 ( 2.5
38.9 ( 4.2
[
11C]7c
7c
blood
brain
2.44 ( 0.25
4.31 ( 0.33
1.52 ( 0.42
1.38 ( 0.16
1.01 ( 0.15
0.64 ( 0.07
0.68 ( 0.10
0.35 ( 0.03
11a
11b
11c
12a
12b
12c
13
678.9 ( 21.7
1048.0 ( 114.3
790.0 ( 132.1
197.1 ( 58.8
216.4 ( 13.8
470.9 ( 100.4
49.8 ( 6.2
663.0 ( 88.3
234.2 ( 44.0
13.1 ( 3.0
NH2
NH2
[
11C]13
blood
brain
2.61 ( 0.35
3.68 ( 0.35
1.60 ( 0.25
1.53 ( 0.14
0.39 ( 0.05
1.04 ( 0.15
1.40 ( 0.20
1.04 ( 0.20
NHCH3
NHCH3
NHCH3
N(CH3)2
NH2
a Expressed as % of injected dose per gram. Each value represents the
mean ( SD for 4-5 mice.
15
16
F
Table 3. Biodistribution of Radioactivity after Injection of [18F]7c in
F
NHCH3
N(CH3)2
Normal Micea
DMIC
IMPY
I
28.0 ( 4.1
organ
2 min
10 min
30 min
60 min
a Inhibition constants (Ki, nM) of compounds for the binding of
blood
brain
bone
2.09 ( 0.40
3.48 ( 0.47
1.80 ( 0.31
1.94 ( 0.18
1.52 ( 0.03
1.76 ( 0.15
2.35 ( 0.33
1.08 ( 0.09
2.98 ( 0.49
1.87 ( 0.26
1.07 ( 0.17
3.58 ( 0.41
[
125I]DMIC to Aβ(1-42) aggregates. Values are the mean ( standard
error of the mean for 4-9 independent experiments.
a Expressed as % of injected dose per gram. Each value represents the
mean ( SD for 4-5 mice.
were readily synthesized from their N-normethyl pre-
cursors, 12(a-c) and 16, and [11C]methyl triflate ([11C]-
MeOTf). Radiochemical yields of the final product were
28-35%, decay corrected to end of bombardment. Radio-
chemical purity was >99% with a specific activity of 22-28
GBq/μmol. The identity of [11C]7a, [11C]7b, [11C]7c, and
[11C]13 was confirmed by a comparison of HPLC retention
times with the nonradioactive compounds (7a, 7b, 7c, and 13).
18F labeling of 7c was performed on a tosyl precursor 17
undergoing a nucleophilic displacement reaction with the
fluoride anion (Scheme 5). Radiolabeling with 18F was suc-
cessfullyperformed onthe precursor togenerate[18F]7cwitha
radiochemical yield of 45% and radiochemical purity >99%.
The identity of [18F]7c was verified by a comparison of
retention time with the nonradioactive compound. The spe-
cific activity of [18F]7c was estimated to be 35 GBq/mmol at
the end of synthesis.
Experiments in vitro to evaluate the affinity of the FPEG
chalcones for Aβ aggregates were carried out in solutions of
Aβ aggregates with [125I]4-dimethylamino-40-iodo-chalcone
([125I]DMIC)18 as the ligand (Table 1). The Ki values sug-
gested that the binding to Aβ(1-42) aggregates was affected
by substitution at the amino group at position 4 in the
chalcone structure, not by the length of PEG introduced into
the chalcone backbone. The fluorinated chalcones had bind-
ing affinity for Aβ(1-42) aggregates in the following order:
the dimethyamino derivatives (7a, 7b, 7c, and 13) > the
monomethylamino derivatives (12a, 12b, 12c, and 16) > the
primary amino derivatives (11a, 11b, 11c, and 15). The result
of the binding experiments is consistent with that of previous
reports.16,19 In addition, the affinity of the dimethylamino

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6397
Figure 1. Neuropathological staining of 10 μm sections of a Tg2576 mouse brain (A and B) and aged normal brain (C). Fluorescent staining of
compound 7c in the Tg2576 mouse brain (A). Aβ immunostaining with antibody BC05 in the adjacent section (B). Fluorescent staining of
compound 7c in the age-matched control mouse brain (C).
derivatives was in the same range as that of the known
compound, 6-iodo-2-(40-dimethylamino)phenyl-imidazo[1,2-
a]pyridine (IMPY), which is commonly used for inhibition
assays.22-25 We selected the dimethylamino derivatives
(7a, 7b, 7c, and 13), which showed the greatest affinity, for
additional studies.
sections of mouse brain were carried out with compound 7c
(Figure 1). We used Tg2576 transgenic mice as an animal
model of Aβ plaque deposition, which express human
APP695withthe K670N, M671LSwedishdoublemutation.26
By 11-13 months of age, Tg2576 mice show prominent
Aβ deposition in the cingulated cortex, entorhinal cortex,
dentate gyrus, and CA1 hippocampal subfield and have been
frequently used for the evaluation of specific binding of
Aβ plaques in in vitro and in vivo experiments.12,24,27-31
Many Aβ plaques were clearly stained with 7c, as reflected
by the affinity for the aggregates of synthetic Aβ(1-42) in in
vitro competition assays (Figure 1A). The labeling pattern
was consistent with that observed after immunohistochemical
labeling by BC05, a specific antibody for Aβ (Figure 1B),
while wild-type mouse brain displayed no significant accumu-
lation of 7c (Figure 1C). The results indicated that 7c binds
specifically to Aβ plaques in Tg2576 mice brain. A previous
report suggested the configuration/folding of Aβ plaques in
Tg2576 mice to be different from the tertiary/quaternary
structure of Aβ plaques in AD brains.30,32 In addition, the
studies reported with 1 further indicate that the binding of
1 reflects the amount of Aβ plaques in human AD brain but
not in Tg2576 mouse brain, and the detectability of Aβ
plaques by 1 is dependent on the accumulation of specific
Aβ subtypes.28,29 Therefore, we considered that it should be
essential to evaluate the binding affinity for Aβ plaques in
human AD brains because our goal is to develop clinically
useful probes for in vivo imaging of Aβ plaques in humans.
Next, we investigated the binding affinity of [18F]7c for
Aβ plaques by in vitro autoradiography in a human AD brain
section (Figure 2A). The autoradiographic image of [18F]7c
showed high levels of radioactivity in some specific areas of
the brain section. Furthermore, we confirmed that the hot
spots of [18F]7c in an AD brain section corresponded with
those of in vitro thioflavin-S staining in the same brain section
(Figure 2B). In contrast, no significant accumulation of
[18F]7c was observed in the region without Aβ plaques
(Figure 2C). The results demonstrate the feasibility of using
[18F]7c as a probe for detecting Aβ plaques in the brain of AD
patients with PET.
To evaluate brain uptake of the FPEG chalcones, biodis-
tribution experiments were performed in normal mice with
four 11C-labeled FPEG chalcones ([11C]7a, [11C]7b, [11C]7c,
and [11C]13) (Table 2). Because normal mice were used for the
biodistribution experiments, no Aβ plaques were expected in
the young mice; therefore the washout of probes from the
brain should be rapid to obtain a higher signal-to-noise ratio
earlier in the AD brain. Radioactivity after injection of the
11C-labeled FPEG chalcones penetrated the blood-brain
barrier, showing excellent uptake ranging from 3.7 to 6.0%
ID/g brain at 2 min postinjection, a level sufficient for imaging
Aβ plaques in the brain. In addition, they displayed good
clearance from the normal brain with 2.3, 1.0, 0.35, and 1.0%
ID/g at 60 min postinjection for [11C]7a, [11C]7b, [11C]7c, and
[11C]13, respectively. These values were equal to 37.6, 21.1,
8.1, and 28.3% of the initial uptake peak for [11C]7a, [11C]7b,
[11C]7c, and [11C]13, respectively. Compound 7c with the
fastest washout from the brain was labeled with 18F and
evaluated for its biodistribution in normal mice (Table 3).
[18F]7c displayed high uptake (3.48%ID/g) at 2 min postinjec-
tion, a level sufficient for imaging like [11C]7c, and was cleared
over the subsequent 10, 30, and 60 min. The radioactivity
in the brain at 60 min postinjection was 1.07%ID/g, indicat-
ing that this [18F]7c has favorable pharmacokinetics in the
brain. Although we consider that a slight difference of the
radioactivity pharmacokinetics between [11C]7c and [18F]7c
could be attributable to the different physicochemical char-
acteristics of their radiometabolites produced in the brain, the
reason for this difference has remained unclear. Bone uptake
at 60 min was measurable (3.58%ID/g), suggesting defluori-
nation in vivo. Bone uptake has been observed for other 18F
tracers.12,22-24 However, previous reports suggested that free
fluoride was not taken up by brain tissue; therefore, the
interference from free fluoride may be relatively low for brain
imaging. A previous paper regarding the most promising 18F-
labeled agent 4 reported that it showed high uptake (7.77%
ID/g at 2 min postinjection) and rapid clearance from the
brain (1.61%ID/g at 60 min postinjection) with little accu-
mulation in bone (1.77%ID/g at 60 min postinjection) in
biodistribution experiments using normal mice.12 The phar-
macokinetics of 4 appear superior to that of [18F]7c, but the
good biological results obtained with [18F]7c suggest that
further investigation is warranted.
In conclusion, we reported novel FPEG chalcone deriva-
tives, containing an end-capped fluoropolyethylene glycol as
in vivo PET imaging agents for Aβ plaques in the brain. The
FPEG chalcones with a dimethylamino group displayed
greater affinity for synthetic Aβ aggregates than did the
monomethylamino and primary amino derivatives. In biodis-
tribution experiments using normal mice, 11C-labeled FPEG
chalcones displayed sufficient uptake for the imaging of Aβ
plaques in the brain. [11C]7c showed the fastest clearance from
the brain, probably related to a low nonspecific binding.
[18F]7c also displayed high uptake in and good clearance from
To investigate the ability of the fluorinated chalcones to
bind to Aβ plaques in the AD model, fluorescent staining of

6398 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Ono et al.
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-(hydroxyethoxy)ethoxy)-
phenyl)-2-propen-1-one (6b). The reaction described above to prepare
6a was used, and 6b was obtained from 5 and ethylene glycol mono-
2-chloroethyl ether. 1H NMR (CDCl3) δ: 3.05 (s, 6H), 3.69 (t, J=
4.8 Hz, 2H), 3.78 (s, 2H), 3.91 (t, J = 4.8 Hz, 2H), 4.23
(t, J=4.8 Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 6.99 (d, J=9.0 Hz,
2H), 7.35 (d, J=15.3 Hz, 1H), 7.55 (d, J=8.7 Hz, 2H), 7.79 (d, J=
15.6 Hz, 1H), 8.02 (d, J=9.0 Hz, 2H).
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-((hydroxyethoxy)ethoxy)-
ethoxy)phenyl)-2-propen-1-one (6c). The reaction described
above to prepare 6a was used, and 429 mg of 6c was obtained
in a yield of 82.6% from 5 and 2-[2-(2-chloroethoxy)-
1
ethoxy]ethanol. H NMR (CDCl3) δ: 3.04 (s, 6H), 3.62 (t, J=
5.1 Hz, 2H), 3.73-3.75 (m, 6H), 3.90 (t, J=4.8 Hz, 2H), 4.22
(t, J=4.8 Hz, 2H), 6.70 (d, J=9.0 Hz, 2H), 6.99 (d, J=8.7 Hz,
2H), 7.35 (d, J=15.3 Hz, 1H), 7.55 (d, J=9.0 Hz, 2H), 7.78
(d, J=15.3 Hz, 1H), 8.02 (d, J=9.0 Hz, 2H).
Figure 2. In vitro autoradiography of [18F]7c using the human AD
brain section (A). Aβ plaques were confirmed by in vitro staining of
the same section with thioflavin-S (B and C).
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-fluoroethoxy)phenyl)-
2-propen-1-one (7a). To a solution of 6a (100 mg, 0.32 mmol in
1,2-dimethoxyethane (DME) (5 mL) was added DAST (85 μL, 0.64
mmol) in a dry ice-acetone bath. The reaction mixture was stirred
for 1 h at room temperature and then poured into a saturated
NaHSO3 solution and extracted with chloroform. After the organic
phase was separated, dried over Na2SO4, and filtered, and the
residue was purified by preparative TLC (hexane:ethyl acetate=
3:1) to give 39 mg of 7a (38.9%). 1H NMR (CDCl3) δ: 3.09 (s, 6H),
4.30 (d, t, J1=27.6 Hz, J2=4.2 Hz, 2H), 4.79 (d, t, J1=47.4 Hz, J2=
4.2 Hz, 2H), 6.70 (d, J=8.7 Hz, 2H), 7.00 (d, J=9.0 Hz, 2H), 7.35
(d, J=15.6 Hz, 1H), 7.55 (d, J=9.0 Hz, 2H), 7.79 (d, J=15.3 Hz,
1H), 8.03 (d, J=9.0 Hz, 2H). EI-MS: m/z 313 (Mþ).
the brain, although a slight difference was observed between
the 11C and 18F tracers. When the labeling of plaques in vitro
was carried out using sections of brain tissue from an animal
model of AD and an AD patient, compound 7c intensely
labeled Aβ plaques existing in both brains. Taken together,
the results suggest the novel FPEG chalcone 7c to be poten-
tially useful for imaging Aβ plaques in the brain using PET.
Experimental Section
General. All reagents were obtained commercially and used
without further purification unless otherwise indicated. 1H
NMR spectra were obtained on a Varian Gemini 300 spectro-
meter with TMS as an internal standard. Coupling constants
are reported in hertz. Multiplicity was defined by s (singlet),
d (doublet), t (triplet) and m (multiplet). Mass spectra were
obtained on a JEOL IMS-DX instrument. HPLC analysis was
performed on a Shimadzu HPLC system (a LC-10AT pump
with a SPD-10A UV detector, λ=254 nm) using a Cosmosil C18
column (Nakalai Tesque, 5C18-AR-II, 4.6 mm ꢀ 150 mm) using
acetonitrile/water (50/50) as mobile phase at a flow rate of
1.0 mL/min. All key compounds were proven by this method
to show g95% purity.
Chemistry. (E)-3-(4-(Dimethylamino)phenyl)-1-(4-hydroxy-
phenyl)-2-propen-1-one (5). 4-Hydroxyacetophenone (1.36 g,
10 mmol) and 4-dimethylaminobenzaldehyde (1.86 g, 10.0 mmol
were dissolved in EtOH (15 mL). A 30 mL aliquot of a 10%
aqueous KOH solution was then slowly added dropwise to the
reaction mixture. The mixture was stirred for 24 h at 100 °C and
then extracted with ethyl acetate. After the organic layers were
combined and dried over Na2SO4, evaporation of the solvent
afforded 1.50 g of 5 (84.0%). 1H NMR (CD3OD) δ: 3.04
(s, 6H), 6.76 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 7.50
(d, J=15.3 Hz, 1H), 7.59 (d, J=9.0 Hz, 2H), 7.72 (d, J=15.3 Hz,
1H), 7.98 (d, J=8.7 Hz, 2H). 1H NMR (DMSO-d6) δ: 2.99 (s, 6H),
6.74 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 7.62 (s, 2H), 7.68
(d, J=8.7 Hz, 2H), 8.03 (d, J=8.7 Hz, 2H), 10.30 (s, 1H). EI-MS:
m/z 267 (Mþ).
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-hydroxyethoxy)phenyl)-
2-propen-1-one (6a). To a solution of 5 (500 mg, 1.87 mmol and
ethylene chlorohydrin (125 μL, 1.87 mmol) in DMSO (5 mL) was
added anhydrous K2CO3 (775 mg, 5.61 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=1:1) to give 422 mg of 6a (72.7%). 1H NMR (CDCl3)
δ: 3.04 (s, 6H), 4.00-4.01 (m, 2H), 4.17 (t, J=4.8 Hz, 2H), 6.69 (d,
J = 9.0 Hz, 2H), 6.99 (d, J = 6.9 Hz, 2H), 7.35 (d, J =
15.3 Hz, 1H), 7.55 (d, J=9.0 Hz, 2H), 7.79 (d, J=15.3 Hz, 1H),
8.02 (d, J=9.3 Hz, 2H).
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-(fluoroethoxy)ethoxy)-
phenyl)-2-propen-1-one (7b). The reaction described above to pre-
pare 7a was used, and 28 mg of 7b was obtained in a yield of
1
28.0% from 6b. H NMR (CDCl3) δ: 3.04 (s, 6H), 3.77-3.94
(m, 4H), 4.21-4.24 (m, 3H), 4.61 (d, t, J1=47.4 Hz, J2=4.2 Hz
1H), 6.69 (d, J=9.3 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 7.35 (d, J=
15.3 Hz, 2H), 7.55 (d, J=9.0 Hz, 2H), 7.78 (d, J=15.6 Hz, 2H),
8.02 (d, J=9.0 Hz, 2H). EI-MS: m/z 357 (Mþ).
(E)-3-(4-(Dimethylamino)phenyl)-1-(4-(2-((fluoroethoxy)ethoxy)-
ethoxy)phenyl)-2-propen-1-one (7c). The reaction described
above to prepare 7a was used, and 29 mg of 7c was obtained in a
yield of 14.4% from 6c and 2-[2-(2-chloroethoxy)ethoxy]etha-
nol. 1H NMR (CDCl3) δ: 3.04 (s, 6H), 3.73-3.81 (m, 6H),
3.90 (t, J=5.1 Hz, 2H), 4.21 (t, J=5.1 Hz, 2H), 4.49 (t, J=4.5 Hz,
1H), 4.65 (t, J=4.5 Hz, 1H), 6.70 (d, J=8.7 Hz, 2H), 6.98 (d, J=
9.0 Hz, 2H), 7.35 (d, J=15.3 Hz, 1H), 7.55 (d, J=8.7 Hz, 2H),
7.78 (d, J=15.3 Hz, 1H), 8.02 (d, J=9.0 Hz, 2H). EI-MS: m/z
401 (Mþ).
1-(4-(2-Hydroxyethoxy)phenyl)ethanone (8a). The reaction
described above to prepare 6a was used, and 1.79 g of 8a was
obtained in a yield of 99.4% from 4-hydroxyacetophenone and
ethylene chlorohydrin. 1H NMR (CDCl3) δ: 2.75 (s, 3H), 4.20 (s,
2H), 4.35 (t, J=5.1 Hz, 2H), 7.15 (d, J=9.0 Hz, 2H), 8.13 (d, J=
9.0 Hz, 2H).
1-(4-(2-(2-Hydroxyethoxy)ethoxy)phenyl)ethanone (8b). The
reaction described above to prepare 6b was used, and 8b was
obtained from 4-hydroxyacetophenone and ethylene glycol
1
mono-2-chloroethyl ether. H NMR (CDCl3) δ: 2.56 (s, 3H),
3.68 (t, J=4.8 Hz 2H), 3.75-3.79 (m, 2H) 3.90 (t, J=5.1 Hz,
2H), 4.21 (t, J=4.8 Hz, 2H), 6.96 (d, J=8.7 Hz, 2H), 7.94 (d, J=
8.7 Hz, 2H).
1-(4-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)phenyl)ethanone
(8c). The reaction described above to prepare 6a was used, and
8c was obtained from 4-hydroxyacetophenone and 2-[2-(chloro-
ethoxy)ethoxy]ethanol. 1H NMR (CDCl3) δ: 2.50 (s, 3H),
3.72-3.83 (m, 6H), 3.92 (t, J=4.5 Hz, 2H), 4.22 (t, J=5.1 Hz,
2H), 4.49 (t, J=4.2 Hz, 1H), 4.61 (t, J=4.2 Hz, 1H), 6.86 (d, J=
8.7 Hz, 2H), 7.80 (d, J=8.7 Hz, 2H).
1-(4-(2-Fluoroethoxy)phenyl)ethanone (9a). The reaction des-
cribed above to prepare 7a was used, and 1.02 g of 9a was obtained

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6399
in a yield of 63.3% from 8a and DAST. 1H NMR (CDCl3) δ: 4.24
(d, t, J1=28.2 Hz, J2=4.2 Hz, 2H), 4.75 (d, t, J1=47.1 Hz, J2=3.9
Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 7.89 (d, J=9.3 Hz, 2H).
1-(4-(2-(2-Fluoroethoxy)ethoxy)phenyl)ethanone (9b). The re-
action described above to prepare 7b was used, and 9b was
obtained from 9a and DAST. 1H NMR (CDCl3) δ: 2.56 (s, 3H),
3.78 (t, J=3.3 Hz, 1H), 3.86-3.94 (m, 3H), 4.22 (t, J=5.1 Hz,
2H), 4.51 (t, J=3.0 Hz, 1H), 4.67 (t, J=3.0.Hz, 1H), 6.96 (d, J=
8.7 Hz, 2H), 7.93 (d, J=8.7 Hz, 2H). EI-MS: m/z 226 (Mþ).
1-(4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)phenyl)ethanone
(9c). The reaction described above to prepare 7c was used, and
543 mg of 9c was obtained from 8c and DAST. 1H NMR
(CDCl3) δ: 2.56 (s, 3H), 3.69-3.81 (m, 6H), 3.90 (t, J =
4.5 Hz, 2H), 4.21 (t, J=5.1 Hz, 2H), 4.49 (t, J=4.2 Hz, 1H),
4.65 (t, J=4.2 Hz, 1H), 6.95 (d, J=9.3 Hz, 2H), 7.92 (d, J=9.0 Hz,
2H). EI-MS: m/z 270 (Mþ).
(E)-1-(4-(2-Fluoroethoxy)phenyl)-3-(4-nitrophenyl)-2-propen-
1-one (10a). The reaction described above to prepare 5 was used,
and 856 mg of 10a was obtained in a yield of 56.6% from 9a and
4-nitrobenzaldehyde. 1H NMR (CDCl3) δ: 4.32 (d, t, J1 =
27.6 Hz, J2=4.2 Hz, 2H), 4.81 (d, t, J1=47.4 Hz, J2=4.2 Hz,
2H), 7.04 (d, J=8.7 Hz, 2H), 7.65 (d, J=15.6 Hz, 1H), 7.79
(d, J=8.7 Hz, 2H), 7.82 (d, J=12.6 Hz, 1H), 8.06 (d, J=9.0 Hz,
2H), 8.28 (d, J=8.7 Hz, 2H).
(E)-1-(4-(2-(Fluoroethoxy)ethoxy)phenyl)-3-(4-nitrophenyl)-
2-propen-1-one (10b). The reaction described above to prepare
5 was used, and 128 mg of 10b was obtained from 9b and
4-nitrobenzaldehyde. 1H NMR (CDCl3) δ: 3.79 (t, J=4.2 Hz,
1H), 3.88-4.27 (m, 3H), 4.8 (t, J=4.8 Hz, 2H), 4.53 (t, J=
4.2 Hz, 1H), 4.69 (t, J=4.2 Hz, 1H), 7.03 (d, J=8.7 Hz, 2H),
7.66 (d, J=15.6 Hz, 1H), 7.79 (d, J=9.0 Hz, 2H), 7.81 (d, J=
15.6 Hz, 1H), 8.05 (d, J=8.7 Hz, 2H), 8.28 (d, J=9.0 Hz, 2H).
(E)-1-(4-(2-((Fluoroethoxy)ethoxy)ethoxy)phenyl)-3-(4-nitro-
phenyl)-2-propen-1-one (10c). The reaction described above to
prepare 5 was used, and 649 mg of 10c was obtained from 9c. 1H
NMR (CDCl3) δ: 3.71-3.82 (m, 6H), 3.92 (t, J=4.5 Hz, 2H),
4.24 (t, J=4.8 Hz, 2H), 4.50 (t, J=4.2 Hz, 1H), 4.66 (t, J=4.5 Hz,
1H), 7.03 (d, J=9.3 Hz, 2H), 7.66 (d, J=15.6 Hz, 1H), 7.79
(d, J=9.0 Hz, 2H), 7.81 (d, J=15.6 Hz, 1H), 8.05 (d, J=9.3 Hz,
2H), 8.28 (d, J=8.7 Hz, 2H).
(E)-1-(4-(2-Fluoroethoxy)phenyl)-3-(4-(methylamino)phenyl)-
2-propen-1-one (12a). To a solution of 11a (290 mg, 1.02 mmol)
in DMSO (6 mL) were added CH3I (0.18 mL, 3.05 mmol) and
anhydrous K2CO3 (691 mg, 5.08 mmol). The reaction mixture
was stirred at room temperature for 3 h and poured into water.
The mixture was extracted with ethyl acetate. 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=2:1) to give 90 mg of
12a (29.5%). 1H NMR (CDCl3) δ: 2.89 (s, 3H), 4.23 (d, t, J1=
27.9 Hz, J2=4.2 Hz, 2H), 4.79 (d, t, J1=47.4 Hz, J2=4.2 Hz,
2H), 6.59 (d, J=8.7 Hz, 2H), 6.99 (d, J=9.0 Hz, 2H), 7.34 (d, J=
15.3 Hz, 1H), 7.51 (d, J=8.4 Hz, 2H), 7.78 (d, J=15.3 Hz, 1H),
8.02 (d, J=9.3 Hz, 2H). EI-MS: m/z 299 (Mþ).
(E)-1-(4-(2-(Fluoroethoxy)ethoxy)phenyl)-3-(4-(methylamino)-
phenyl)-2-propen-1-one (12b). The reaction described above
to prepare 12a was used, and 22 mg of 12b was obtained from
11b. 1H NMR(CDCl3) δ: 2.90 (s, 3H), 3.78-3.95 (m, 4H), 3.99 (s,
broad, 1H), 4.23 (t, J=4.5 Hz, 2H), 4.53 (t, J=4.5 Hz, 2H), 4.53
(t, J=4.2 Hz, 1H), 4.69 (t, J=4.2 Hz, 1H), 6.60 (d, J=8.7 Hz, 2H),
6.99 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 15.3 Hz, 1H), 7.51
(d, J = 8.7 Hz, 2H), 7.77 (d, J = 15.3 Hz, 1H), 8.02 (d, J =
8.7 Hz, 2H). EI-MS: m/z 343 (Mþ).
(E)-1-(4-(2-((Fluoroethoxy)ethoxy)ethoxy)phenyl)-3-(4-(methy-
amino)phenyl)-2-propen-1-one (12c). The reaction described above
to prepare 12a was used, and 53 mg of 12c was obtained from 11c.
1H NMR (CDCl3) δ: 2.89 (s, 3H), 3.69-3.83 (m, 6H), 3.90 (t, J=
4.8 Hz, 2H), 4.12 (s, broad, 1H), 4.22 (t, J=5.1 Hz, 2H), 4.49 (t, J=
4.2 Hz, 1H), 4.65 (t, J=4.1 Hz, 1H), 6.60 (d, J=8.7 Hz, 2H), 6.98
(d, J=9.0 Hz, 2H), 7.35 (d, J=15.3 Hz, 1H), 7.51 (d, J=8.7 Hz,
2H), 7.76 (d, J=15.3 Hz, 1H), 8.01 (d, J=8.7 Hz, 2H). EI-MS:
m/z 387 (Mþ).
(E)-3-(4-Dimethylaminophenyl)-1-(4-fluorophenyl)-2-propen-
1-one (13). The reaction described above to prepare 5 was used,
and 209 mg of 13 was obtained from 4-fluoroacetophenone and
4-dimethylbenzaldehyde. 1H NMR (300 MHz, CDCl3) δ: 3.03
(s, 6H), 6.68 (d, J=8.7 Hz, 2H), 7.15 (t, J=8.4 Hz, 2H), 7.30
(d, J=15.3 Hz, 1H), 7.54 (d, J=9.0 Hz, 2H), 7.78 (d, J=15.3 Hz,
1H), 8.02-8.06 (m, 2H). EI-MS: m/z 269 (Mþ).
(E)-1-(4-Fluorophenyl)-3-(4-nitrophenyl)-2-propen-1-one (14).
The reaction described above to prepare 5 was used, and 490 mg
of 14 was obtained from 4-fluoroacetophenone and 4-nitroben-
zaldehyde. 1H NMR (300 MHz, CDCl3) δ: 7.21 (t, J=8.7 Hz,
2H), 7.62 (d, J=15.9 Hz, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.84
(d, J=15.9 Hz, 1H), 8.07-8.12 (m, 2H), 8.29 (d, J=8.7 Hz, 2H).
EI-MS: m/z 271 (Mþ).
(E)-3-(4-Aminophenyl)-1-(4-fluorophenyl)-2-propen-1-one (15).
The reaction described above to prepare 11(a-c) was used, and
150 mg of 15 was obtained from 14. 1H NMR (300 MHz,
CDCl3) δ: 4.07 (s, broad, 2H), 6.67 (d, J=8.7 Hz, 2H), 7.15
(t, J=8.7 Hz, 2H), 7.31 (d, J=15.6 Hz, 1H), 7.47 (d, J=8.4 Hz,
2H), 7.75 (d, J=15.6 Hz, 1H), 8.03 (t, J=8.7 Hz, 2H). EI-MS:
m/z 241 (Mþ).
(E)-1-(4-Fluorophenyl)-3-(4-methylaminophenyl)-2-propen-1-
one (16). The reaction described above to prepare 12(a-c) was
used, and 14 mg of 16 was obtained from 15. 1H NMR (300 MHz,
CDCl3) δ: 2.90 (s, 3H), 4.20 (s, broad, 1H), 6.60 (d, J=8.7 Hz,
2H), 7.17 (d, J=8.7 Hz, 2H), 7.30 (d, J=15.6 Hz, 1H), 7.50
(d, J=8.7 Hz, 2H), 7.78 (d, J=15.6 Hz, 1H), 8.04 (d, J=8.7 Hz,
2H). EI-MS: m/z 255 (Mþ).
(E)-2-(2-(2-(4-(3-(4-(Dimethylamino)phenyl)acryloyl)phenoxy)-
ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (17). To a solu-
tion of 6c (108 mg, 0.27 mmol) in pyridine (3 mL) was added
tosyl chloride (343.8 mg, 0.621 mmol). The reaction mixture was
stirred for 3 h at room temperature. After water was added, the
mixture was extracted with ethyl acetate. The organic layer was
dried over Na2SO4, and evaporation of the solvent afforded a
residue, which was purified by preparative TLC (hexane:ethyl
acetate=1:1) to give 44 mg of 17 (29.4%). 1H NMR (300 MHz,
CDCl3) δ: 2.43 (s, 3H), 3.04 (s, 6H), 3.62-3.72 (m, 6H),
(E)-3-(4-Aminophenyl)-1-(4-(2-fluoroethoxy)phenyl)-2-propen-
1-one (11a). A mixture of 10a (856 mg, 2.7 mmol), SnCl2 (2.55 g,
13.5 mmol), and EtOH (10 mL) was stirred at 100 °C for 2 h. After
the mixture had cooled to room temperature, 1 M NaOH (10 mL)
was added. The mixture was then extracted with ethyl acetate
(10 mL). The organic phase was dried over Na2SO4 and filtered.
The solvent was removed, and the residue was purified by silica gel
chromatography using chloroform as a mobile phase to give
1
333 mg of 11a (43.0%). H NMR (CDCl3) δ: 4.02 (s, broad,
2H), 4.30 (d, t, J1=27.6 Hz, J2=4.2 Hz, 2H), 4.79 (d, t, J1=47.4
Hz, J2=4.2 Hz, 2H), 6.68 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz,
2H), 7.36 (d, J=15.3 Hz, 1H), 7.48 (d, J=8.4 Hz, 2H), 7.75 (d, J=
15.3 Hz, 1H), 8.03 (d, J=6.9 Hz, 2H). EI-MS: m/z 285 (Mþ).
(E)-3-(4-Aminophenyl)-1-(4-(2-(fluoroethoxy)ethoxy)phenyl)-
2-propen-1-one (11b). The reaction described above to prepare
1
11a was used, and 85 mg of 11b was obtained from 10b. H
NMR (CDCl3) δ: 3.77-3.94 (m, 4H), 4.00 (s, broad, 2H), 4.23
(t, J=4.5 Hz, 2H), 4.53 (t, J=4.2 Hz, 1H), 4.69 (t, J=4.2 Hz,
1H), 6.68 (d, J=8.4 Hz, 2H), 6.99 (d, J=8.7 Hz, 2H), 7.74 (d, J=
15.6 Hz, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.36 (d, J=15.3 Hz, 1H),
8.01 (d, J=9.0 Hz, 2H). EI-MS: m/z 329 (Mþ).
(E)-3-(4-Aminophenyl)-1-(4-(2-((fluoroethoxy)ethoxy)ethoxy)-
phenyl)-2-propen-1-one (11c). The reaction described above to
prepare 11a was used, and 206 mg of 11c was obtained from 10c.
1H NMR (CDCl3) δ: 3.70-3.83 (m, 6H), 3.89 (t, J=4.5 Hz, 2H),
4.12 (s, broad, 2H), 4.21 (t, J=4.8 Hz, 2H), 4.49 (t, J=4.0 Hz,
1H), 4.65 (t, J=3.9 Hz, 1H), 6.67 (d, J=8.7 Hz, 2H), 6.98 (d, J=
8.7 Hz, 2H), 7.36 (d, J=15.3 Hz, 1H), 7.47 (d, J=8.4 Hz, 2H),
7.74 (d, J=15.9 Hz, 1H), 8.01 (d, J=9.0 Hz, 2H). EI-MS: m/z
373 (Mþ).

6400 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Ono et al.
3.85-3.87 (m, 2H), 4.15-4.18 (m, 4H), 6.70 (d, J=8.7 Hz, 2H),
6.98 (d, J=9.0 Hz, 2H), 7.31-7.35 (m, 2H), 7.37 (d, J=9.0 Hz,
1H), 7.55 (d, J=8.7 Hz, 2H), 7.80 (t, J=8.7 Hz, 3H), 8.02 (d, J=
9.0 Hz, 2H). EI-MS m/z 553 (Mþ)
containing 50 μL of test compound (0.2 pM-400 μM in 10%
EtOH), 50 μL of 0.02 nM [125I]DMIC, 50 μL of Aβ(1-42)
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 4.0, and those for the inhibition con-
stant (Ki) were calculated using the Cheng-Prusoff equation:
Ki = IC50/(1 þ [L]/Kd), where [L] is the concentration of
Radiolabeling. Procedure for Labeling of 7a, 7b, 7c, and 13
with 11C. 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
system for the synthesis of 11C-methyl iodide (CUPID C-100;
Sumitomo Heavy Industry Ltd.) and converted sequentially to
[11C]MeOTf by the previously described method of Jewett.33
[11C]Chalcones were produced by reacting [11C]MeOTf with
the normethyl precursor, 7a, 7b, 7c, and 13, (0.5 mg) in 500 μL
of methyl ethyl ketone (MEK). After the complete transfer of
[11C]MeOTf, 11C-methylation was carried out for 5 min and
the reaction solvent was then 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
(Nakalai Tesque, 5C18-AR-II, 10 mm ꢀ 250 mm) with an
isocratic solvent of acetonitrile/water (55/45) at a flow rate of
6.0 mL/min. The desired fraction was collected in a flask
and evaporated dry. The radiochemical yield, purity, and
specific activity of [11C]chalcones were further confirmed by
analytical reverse phase HPLC on a 5C18-AR-300 column
(Nakalai Tesque, 4.6 mm ꢀ 150 mm, acetonitrile/water (60/
40), 1.0 mL/min).
[
125I]DMIC used in the assay and Kd is the dissociation
constant of DMIC (4.2 nM).19 DMIC and IMPY used as test
compounds for the inhibition assay were synthesized as
reported previously.19,34
Biodistribution in Normal Mice. Experiments with animals
were conducted in accordance with our institutional guidelines
and approved by the Nagasaki University Animal Care Com-
mittee and the Kyoto University Animal Care Committee.
A 100 μL amount of a saline solution containing the radiolabel-
ed agent (3.7 MBq), EtOH (10%), and ascorbic acid (1 mg/mL)
was injected directly into the tail vein of ddY mice (5-week-old,
22-25 g). Groups of five mice were sacrificed at various post-
injection time points. The organs of interest were removed and
weighed, and the radioactivity was measured with an automatic
γ counter (COBRAII, Packard).
Staining of Aβ Plaques in Brain Sections of Tg2576 Transgenic
Mice. The Tg2576 transgenic mice (female, 20-month-old)
and wild-type (female, 20-month-old) mice were used as an
Alzheimer’s model and an age-matched control, respectively.
After the mice were sacrificed by decapitation, the brains were
immediately removed and frozen in powdered dry ice. The
frozen blocks were sliced into serial sections 10 μm thick. Each
slide was incubated with a 50% EtOH solution (100 μM) of
compound 7c for 10 min. The sections were washed with 50%
EtOH for 3 min two times. After drying, the sections were then
examined using a microscope (Nikon, Eclipse 80i) equipped
with a B-2A filter set (excitation, 450-490 nm; diachronic
mirror, 505 nm; long-pass filter, 520 nm). Thereafter, the serial
sections were also immunostained with 3,30-diaminobenzidine
(DAB) as a chromogen using monoclonal antibodies against
Aβ (amyloid β-protein immunohistostain kit, WAKO).
In Vitro Autoradiography Using Human AD Brains. Postmor-
tem brain tissues from an autopsy-confirmed case of AD (73-
year-old male) were obtained from BioChain Institute Inc. The
presence and localization of plaques on the sections were con-
firmed with immunohistochemical staining using a monoclonal
Aβ antibody as described above. The sections were incubated
with [18F]7c (54 μCi/200 μL) for 1 h at room temperature. They
were then washed in 50% EtOH (two 1 min wash), before being
rinsed with water for 30 s. After drying, the 18F-labeled sections
were exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan)
for 6 h. Ex vivo autoradiographic images were obtained using a
BAS5000 scanner system (Fuji Film). After autoradiographic
examination, the same sections were stained by thioflavin-S to
confirm the presence of Aβ plaques. For the staining of thioflavin-
S, sections were immersed in a 0.125% thioflavin-S solution
containing 50% EtOH for 3 min and washed in 50% EtOH. After
drying, the sections were then examined using a microscope
(Nikon, Eclipse 80i) equipped with a B-2A filter set (excitation,
450-490 nm; diachronic mirror, 505 nm; long-pass filter, 520 nm).
Procedure for Labeling 7c with 18F. [18F]Fluoride was pro-
duced by the JSW typeBC3015 cyclotron 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 airflow, and the 18F activity was eluted
with 0.5 mL of a Kryptofix 222/K2CO3 solution (11 mg of
Kryptofix 222 and 2.6 mg of K2CO3 in acetonitrile/water (86/
14)). 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 and dissolved in DMSO (1 mL). A solution of
tosylate precursor 17 (1.0 mg) in DMSO (1 mL) was added to
the reaction vessel containing the 18F activity in DMSO. The
mixture was heated at 160 °C for 5 min. Water (5 mL) was
added, and the mixture was passed through a preconditioned
Oasis HLB cartridge (3 cm3) (Waters). The cartridge was
washed with 10 mL of water, and the labeled compound was
eluted with 2 mL of acetonitrile. The eluted compound
was purified by preparative HPLC [YMC-Pack Pro C18
column (20 mm ꢀ 150 mm), acetonitrile/water (75/25), flow
rate 9.0 mL/min]. The retention time of the major byproduct of
hydrolysis (tR =2.7 min) was well-resolved from the desired
18F-labeled product (tR=10.7 min). The radiochemical purity
and specific activity were determined by analytical HPLC
[YMC-Pack Pro C18 column (4.6 mm ꢀ 150 mm), acetoni-
trile/water (60/40), flow rate 1.0 mL/min], and [18F]7c was
obtained in a radiochemical purity of >99% with the specific
activity of 35 GBq/mmol. Specific activity was estimated by
comparing the UV peak intensity of the purified 18F-labeled
compound with a reference nonradioactive compound of
known concentration.
Binding Assays Using the Aggregated Aβ peptides in Solution.
Aβ(1-42) was purchased from Peptide Institute (Osaka,
Japan). Aggregation was carried out by gently dissolving the
peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was
incubated at 37 °C for 42 h with gentle and constant shaking.
Binding experiments were carried out as described pre-
Acknowledgment. This study was supported by the Program
for Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NIBIO), a Health
Labour Sciences Research Grant, and a Grant-in-Aid for Young
Scientists (A) and Exploratory Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
viously.18 125I]DMIC with 2200 Ci/mmol of specific activity
[
and radiochemical purity greater than 95% was prepared
using the standard iododestannylation reaction. A mixture

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6401
Supporting Information Available: Representative HPLC
(18) Ono, M.; Hori, M.; Haratake, M.; Tomiyama, T.; Mori, H.;
Nakayama, M. Structure-activity relationship of chalcones and
related derivatives as ligands for detecting of β-amyloid plaques in
the brain. Bioorg. Med. Chem. 2007, 15, 6388–6396.
chromatograms of [18F]7c. This material is available free of
charge via the Internet at http://pubs.acs.org.
(19) Ono, M.; Haratake, M.; Mori, H.; Nakayama, M. Novel chalcones
as probes for in vivo imaging of β-amyloid plaques in Alzheimer’s
brains. Bioorg. Med. Chem. 2007, 15, 6802–6809.
(20) Maya, Y.; Ono, M.; Watanabe, H.; Haratake, M.; Saji, H.;
Nakayama, M. Novel radioiodinated aurones as probes for
SPECT imaging of β-amyloid plaques in the brain. Bioconjugate
Chem. 2009, 20, 95–101.
(21) Ono, M.; Maya, Y.; Haratake, M.; Ito, K.; Mori, H.; Nakayama,
M. Aurones serve as probes of β-amyloid plaques in Alzheimer’s
disease. Biochem. Biophys. Res. Commun. 2007, 361, 116–121.
(22) Stephenson, K. A.; Chandra, R.; Zhuang, Z. P.; Hou, C.; Oya, S.;
Kung, M. P.; Kung, H. F. Fluoro-pegylated (FPEG) imaging
agents targeting Aβ aggregates. Bioconjugate Chem. 2007, 18,
238–246.
(23) Qu, W.; Kung, M. P.; Hou, C.; Oya, S.; Kung, H. F. Quick
assembly of 1,4-diphenyltriazoles as probes targeting β-amyloid
aggregates in Alzheimer’s disease. J. Med. Chem. 2007, 50, 3380–
3387.
References
(1) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: the amyloid
cascade hypothesis. Science 1992, 256, 184–185.
(2) Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy.
Physiol. Rev. 2001, 81, 741–766.
(3) Nordberg, A. PET imaging of amyloid in Alzheimer’s disease.
Lancet Neurol. 2004, 3, 519–527.
(4) Mathis, C. A.; Wang, Y.; Klunk, W. E. Imaging β-amyloid plaques
and neurofibrillary tangles in the aging human brain. Curr. Pharm.
Des. 2004, 10, 1469–1492.
(5) Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.;
Holt, D. P.; Bergstrom, M.; Savitcheva, I.; Huang, G. F.; Estrada,
S.; Ausen, B.; Debnath, M. L.; Barletta, J.; Price, J. C.; Sandell, J.;
Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni, G.; Mathis, C. A.;
Langstrom, B. Imaging brain amyloid in Alzheimer’s disease with
Pittsburgh Compound-B. Ann. Neurol. 2004, 55, 306–319.
(6) Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.; Debnath,
M. L.; Klunk, W. E. Synthesis and evaluation of 11C-labeled
6-substituted 2-arylbenzothiazoles as amyloid imaging agents.
J. Med. Chem. 2003, 46, 2740–2754.
(7) Verhoeff, N. P.; Wilson, A. A.; Takeshita, S.; Trop, L.; Hussey, D.;
Singh, K.; Kung, H. F.; Kung, M. P.; Houle, S. In vivo imaging of
Alzheimer disease β-amyloid with [11C]SB-13 PET. Am. J. Geriatr.
Psychiatry 2004, 12, 584–595.
(24) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung, H.
F. F-18 stilbenes as PET imaging agents for detecting β-amyloid
plaques in the brain. J. Med. Chem. 2005, 48, 5980–5988.
(25) Kung, M. P.; Hou, C.; Zhuang, Z. P.; Zhang, B.; Skovronsky, D.;
Trojanowski, J. Q.; Lee, V. M.; Kung, H. F. IMPY: an improved
thioflavin-T derivative for in vivo labeling of β-amyloid plaques.
Brain Res. 2002, 956, 202–210.
(8) Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.;
Zhuang, Z. P.; Kung, M. P.; Kung, H. F. 11C-Labeled stilbene
derivatives as Abeta-aggregate-specific PET imaging agents for
Alzheimer’s disease. Nucl. Med. Biol. 2003, 30, 565–571.
(9) Small, G. W.; Kepe, V.; Ercoli, L. M.; Siddarth, P.; Bookheimer,
S. Y.; Miller, K. J.; Lavretsky, H.; Burggren, A. C.; Cole, G. M.;
Vinters, H. V.; Thompson, P. M.; Huang, S. C.; Satyamurthy, N.;
Phelps, M. E.; Barrio, J. R. PET of brain amyloid and tau in mild
cognitive impairment. N. Engl. J. Med. 2006, 355, 2652–2663.
(10) Shoghi-Jadid, K.; Small, G. W.; Agdeppa, E. D.; Kepe, V.; Ercoli,
L. M.; Siddarth, P.; Read, S.; Satyamurthy, N.; Petric, A.; Huang,
S. C.; Barrio, J. R. Localization of neurofibrillary tangles and
β-amyloid plaques in the brains of living patients with Alzheimer
disease. Am. J. Geriatr. Psychiatry 2002, 10, 24–35.
(11) Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan, R.; Pike, K. L.;
O’Keefe, G.; Tochon-Danguy, H.; Chan, G.; Berlangieri, S. U.;
Jones, G.; Dickinson-Rowe, K. L.; Kung, H. P.; Zhang, W.; Kung,
M. P.; Skovronsky, D.; Dyrks, T.; Holl, G.; Krause, S.; Friebe, M.;
Lehman, L.; Lindemann, S.; Dinkelborg, L. M.; Masters, C. L.;
Villemagne, V. L. Imaging of amyloid β in Alzheimer’s disease with
18F-BAY94-9172, a novel PET tracer: proof of mechanism. Lancet
Neurol. 2008, 7, 129–135.
(12) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung,
H. F. F-18 polyethyleneglycol stilbenes as PET imaging agents
targeting Aβ aggregates in the brain. Nucl. Med. Biol. 2005, 32,
799–809.
(13) Lockhart, A. Imaging Alzheimer’s disease pathology: one target,
many ligands. Drug Discovery Today 2006, 11, 1093–1099.
(14) Ye, L.; Morgenstern, J. L.; Gee, A. D.; Hong, G.; Brown, J.;
Lockhart, A. Delineation of positron emission tomography ima-
ging agent binding sites on β-amyloid peptide fibrils. J. Biol. Chem.
2005, 280, 23599–235604.
(26) Hsiao, K.; Chapman, P.; Nilsen, S.; Eckman, C.; Harigaya, Y.;
Younkin, S.; Yang, F.; Cole, G. Correlative memory deficits,
Aβ elevation, and amyloid plaques in transgenic mice. Science
1996, 274, 99–102.
(27) Kuntner, C.; Kesner, A. L.; Bauer, M.; Kremslehner, R.; Wanek,
T.; Mandler, M.; Karch, R.; Stanek, J.; Wolf, T.; Muller, M.;
Langer, O. Limitations of small animal PET imaging with
[
18F]FDDNP and FDG for quantitative studies in a transgenic
mouse model of Alzheimer’s disease. Mol. Imaging Biol. 2009, 11,
236–240.
(28) Klunk, W. E.; Lopresti, B. J.; Ikonomovic, M. D.; Lefterov, I. M.;
Koldamova, R. P.; Abrahamson, E. E.; Debnath, M. L.; Holt,
D. P.; Huang, G. F.; Shao, L.; DeKosky, S. T.; Price, J. C.; Mathis,
C. A. Binding of the positron emission tomography tracer Pitts-
burgh compound-B reflects the amount of amyloid-β in Alzhei-
mer’s disease brain but not in transgenic mouse brain. J. Neurosci.
2005, 25, 10598–10606.
(29) Maeda, J.; Ji, B.; Irie, T.; Tomiyama, T.; Maruyama, M.; Okauchi,
T.; Staufenbiel, M.; Iwata, N.; Ono, M.; Saido, T. C.; Suzuki, K.;
Mori, H.; Higuchi, M.; Suhara, T. Longitudinal, quantitative
assessment of amyloid, neuroinflammation, and anti-amyloid
treatment in a living mouse model of Alzheimer’s disease enabled
by positron emission tomography. J. Neurosci. 2007, 27, 10957–
10968.
(30) Toyama, H.; Ye, D.; Ichise, M.; Liow, J. S.; Cai, L.; Jacobowitz, D.;
Musachio, J. L.; Hong, J.; Crescenzo, M.; Tipre, D.; Lu, J. Q.;
Zoghbi, S.; Vines, D. C.; Seidel, J.; Katada, K.; Green, M. V.; Pike,
V. W.; Cohen, R. M.; Innis, R. B. PET imaging of brain with the
β-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model
of Alzheimer’s disease. Eur. J. Nucl. Med. Mol. Imaging 2005, 32,
593–600.
(31) Skovronsky, D. M.; Zhang, B.; Kung, M. P.; Kung, H. F.;
Trojanowski, J. Q.; Lee, V. M. In vivo detection of amyloid plaques
in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci.
U.S.A 2000, 97, 7609–7614.
(15) Lockhart, A.; Ye, L.; Judd, D. B.; Merritt, A. T.; Lowe, P. N.;
Morgenstern, J. L.; Hong, G.; Gee, A. D.; Brown, J. Evidence for
the presence of three distinct binding sites for the thioflavin T class
of Alzheimer’s disease PET imaging agents on β-amyloid peptide
fibrils. J. Biol. Chem. 2005, 280, 7677–7684.
(16) Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.; Arano, Y.;
Mori, H.; Nakayama, M. Radioiodinated flavones for in vivo
imaging of β-amyloid plaques in the brain. J. Med. Chem. 2005,
48, 7253–7260.
(32) Saido, T. C.; Iwatsubo, T.; Mann, D. M.; Shimada, H.; Ihara, Y.;
Kawashima, S. Dominant and differential deposition of distinct
β-amyloid peptide species, A β N3(pE), in senile plaques. Neuron
1995, 14, 457–466.
(33) Jewett, D. M. A simple synthesis of [11C]methyl triflate. Int. J.
Radiat. Appl. Instrum. A 1992, 43, 1383–1385.
(17) Ono, M.; Watanabe, R.; Kawashima, H.; Kawai, T.; Watanabe,
H.; Haratake, M.; Saji, H.; Nakayama, M. 18F-Labeled flavones
for in vivo imaging of β-amyloid plaques in Alzheimer’s brains.
Bioorg. Med. Chem. 2009, 17, 2069–2076.
(34) Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plossl, K.;
Hou, C.; Holtzman, D. M.; Kung, H. F. Structure-activity
relationship of imidazo[1,2-a]pyridines as ligands for detecting
β-amyloid plaques in the brain. J. Med. Chem. 2003, 46, 237–243.