Structure-activity relationship of chalcones and related derivatives as ligands for detecting of β-amyloid plaques in the brain

Article abstract of DOI:10.1016/j.bmc.2007.06.055

A series of novel chalcones and their related derivatives were synthesized and evaluated as β-amyloid imaging probes. In the structure-activity relationship of binding affinities to synthetic Aβ(1-42) aggregates, compound 14 displayed the highest binding

Full text of DOI:10.1016/j.bmc.2007.06.055

Bioorganic & Medicinal Chemistry 15 (2007) 6388–6396
Structure–activity relationship of chalcones and related
derivatives as ligands for detecting of b-amyloid plaques in the brain
Masahiro Ono,a,* Miyuki Hori,a Mamoru Haratake,a Takami Tomiyama,b
Hiroshi Morib and Morio Nakayamaa
aDepartment of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan
bDepartment of Neuroscience, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
Received 10 May 2007; revised 25 June 2007; accepted 27 June 2007
Available online 4 July 2007
Abstract—A series of novel chalcones and their related derivatives were synthesized and evaluated as b-amyloid imaging probes. In
the structure–activity relationship of binding affinities to synthetic Ab(1–42) aggregates, compound 14 displayed the highest binding
affinity in vitro. b-Amyloid plaques in the Alzheimer’s model mouse brain were visualized with 14. In biodistribution studies using
normal mice, [125I]14 showed good brain uptake (2.56% ID/g, 2 min postinjection) and rapid washout from the brain (0.21% ID/g,
60 min postinjection). These results suggest that [125I]14 should be further investigated as a potentially useful b-amyloid imaging
probe.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, in an attempt to search for novel amyloid
imaging probes, we found that radioiodinated flavone17
and chalcone18 derivatives, which are categorized as
flavonoids, showed excellent characteristics as new amy-
loid imaging agents, such as high binding affinity to Ab
aggregates and high uptake into the brain and rapid
clearance from the brain (Fig. 1). Especially, the chal-
cone backbone structure is considered to be a useful core
in the development of new amyloid imaging probes be-
cause it can easily be formed by one-pot condensation
reaction.
Alzheimer’s disease (AD) is a progressive neurodegener-
ative disorder pathologically characterized by deposi-
tion of b-amyloid (Ab) peptides as senile plaques in
the brain.1,2 Since the deposition of b-amyloid plaques
is an early event in the development of AD, a validated
biomarker of b-amyloid deposition in the brain would
likely prove useful to identify and follow individuals at
risk for AD and to assist in the evaluation of new
anti-amyloid therapies currently under development.3–5
A number of groups have reported radiolabeled b-amy-
loid imaging agents for positron emission tomography
(PET) and single photon emission computed tomogra-
phy (SPECT) such as [18F]FDDNP,6–8 [11C]PIB,9,10
In the present study, we designed and synthesized novel
chalcone derivatives and related chalcone-like com-
pounds, and evaluated their structure–activity relation-
ship on the binding affinity to b-amyloid aggregates
and in vivo biodistribution using a compound with high
binding affinity.
[11C]SB-13,11,12
[
123I]IMPY,13–15 and [11C]BF-227.16
Recent reports using these amyloid imaging agents have
indicated that detecting b-amyloid plaques in the living
human brain by PET and SPECT may lead to differen-
tiation between AD patients and healthy human.
R
O
O
125I
125I
R
O
Keywords: Alzheimer’s disease; b-Amyloid plaque; PET; SPECT;
Imaging.
flavove
chalcone
*
Figure 1. Chemical structures of radioiodinated flavones and chal-
cones (R = NH2, NHMe, NMe2).
Corresponding author. Tel.: +81 95 819 2443; fax: +81 95 819
2442; e-mail: mono@nagasaki-u.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.055

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
6389
2. Results and discussion
tuted benzaldehydes or heterocyclic aldehydes. In this
process, the substituted ketones were reacted with the
substituted benzaldehyde or heterocyclic aldehydes in
the presence of 10% aqueous KOH and ethanol at room
temperature to form the target chalcone and chalcone-
like compounds (1 and 11–29). The nitro derivatives
(1, 11, 12, and 15) were converted to amino derivatives
(2, 30, 31, and 32) by reduction with SnCl2. Conversion
2.1. Chemistry
The syntheses of chalcone and chalcone-like compounds
are outlined in Schemes 1–3. Chalcone and chalcone-like
compounds were prepared by base-catalyzed condensa-
tion of appropriately substituted ketones with substi-
O
O
O
OHC
a
b
+
Br
O2N
c
O2N
Br
H2N
Br
1
2
O
O
d
H2N
SnBu3
H2N
I
3
4
O
O
O
O
O
e
d
c
N
H
Br
Br
N
H
SnBu3
SnBu3
N
H
I
I
5
6
7
O
d
f
c
N
N
N
8
9
10
Scheme 1. Reagents: (a) EtOH, KOH; (b) EtOH, SnCl2; (c) dioxane, (Bu3Sn)2, (Ph3P)4Pd, Et3N; (d) CHCl3, I2; (e) CH3I, K2CO3, DMSO; (f) AcOH,
(CH2O)n, NaCNBH3.
O
KOH
EtOH
O
+
R2 CHO
R1
CH3
R1
R2
11: R1
13 R1
15: R1
17: R1
19: R1
21: R1
23: R1
25: R1
27: R1
29: R1
=
, R2
=
, 12: R1
=
, R2
=
=
NO2
N
NO2
N
S
S
S
Br
Br
I
I
=
, R2
=
, 14: R1
, 16: R1
, 18: R1
, 20: R1
, 22: R1
, 24: R1
, 26: R1
, 28: R1
=
, R2
S
=
=
=
=
=
=
=
=
, R2
=
=
=
=
=
=
=
=
, R2
, R2
, R2
, R2
, R2
, R2
, R2
=
=
=
=
=
=
=
NO2
I
I
S
Br
O
S
, R2
=
=
=
I
O
, R2
I
I
I
I
S
HN
S
, R2
I
I
N
N
, R2
=
O
S
N
N
, R2
, R2
, R2
=
=
=
S
O
N
N
N
S
O
O
S
I
I
S
N
O
Br
Br
Br
O
N
Scheme 2.

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M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
11
12
15
30
31
32
33
34
35
a
b
O
R1
R2
c
36
30: R1
32: R1
34: R1
36: R1
=
=
=
=
, R2
=
, 31: R1
, 33: R1
=
=
, R2
, R2
NH
=
=
NH2
NH2
NH
S
S
S
S
Br
I
, R2
=
NH2
S
S
Br
Br
, R2
=
, 35: R1
=
, R2 =
NH
S
Br
I
, R2
=
N
Br
Scheme 3. Reagents: (a)EtOH, SnCl2; (b) CH3I, K2CO3, DMSO; (c) AcOH, (CH2O)n, NaCNBH3.
of the amino derivatives (2, 30, 31, and 32) to the
monomethylamino derivatives (5, 33, 34, and 35) was
achieved by a methylation with CH3I under alkaline
conditions. The amino derivatives (2 and 32) were also
converted to the dimethylamino derivatives (8 and 36)
by an efficient method19 with paraformaldehyde, sodium
cyanoborohydride, and acetic acid. The tin compounds
(3, 6, 9, and 37) were prepared from the corresponding
bromo compounds (2, 5, 8, and 13) using a bromo-to-
tributyltin exchange reaction catalyzed by Pd(0). The
tributyltin derivatives (3, 6, and 9) were readily reacted
with iodine in CHCl3 at room temperature to give the
iodo derivatives (4, 7, and 10). The tributyltin derivative
37 was used as the starting materials for radioiodination
in preparation of [125I]14. Novel radioiodinated ligand
was achieved by an iododestannylation reaction using
hydrogen peroxide as the oxidant, which produced the
desired radioiodinated ligand (Scheme 4). It was antici-
pated that the no-carrier-added preparation would
result in a final product bearing a theoretical specific
activity similar to that of 125I (2200 Ci/mmol). The
radiochemical identity of [125I]14 was verified by
co-injection with nonradioactive compound by its HPLC
profiles. The [125I]14 showed a single radioactivity peak
at the retention time of 18.2 min. The radioiodinated
ligand was obtained in 60–70% radiochemical yield with
radiochemical purities of >95% after purification by
HPLC.
4.2 nM).18 Binding affinities of chalcone and chalcone-
like compounds were evaluated with inhibition assays
against [125I]DMIC binding on Ab(1–42) aggregates
(Table 1). These Ki values suggested that the new series
of chalcone and chalcone-like compounds had high
binding affinity for Ab(1–42) aggregates in the order
of N,N-dimethylated derivatives (10 and 14) >
N-monomethylated derivatives (7 and 34) > primary
amino derivatives (4 and 31) when comparing in the sim-
ilar core structure. Compounds 4, 7 and 10 with the
substituted group at 40 position and iodine at 4 position
displayed higher Ki values (lower binding affinities) as
compared to compounds, 4-amino-40-iodo-chalcone
(AIC), 40-iodo-4-methylamino-chalcone (IMC) and
DMIC, which have the substituted group and iodine
at the inverse position against compounds 4, 7, and
10. The Ki values of 32, 35, and 36 with the thienyl
group at R position and phenyl group at R0 position
were higher than those of 31, 34, and 14 with the phenyl
group at R position and the thienyl group at R0 posi-
tion, indicating that the binding affinities depended on
the combination of heterocycles introduced at R and
R0 position, not on the position of the substituted group
or iodine (bromine) group. When comparing the Ki
values of heterocyclic compounds with the same substi-
tuted group, the binding affinities increased in the order
of phenyl > thienyl > furanyl at
R
position and
phenyl = thienyl > furanyl at R0 position. The Ki values
of compounds without the substituted group on the ring
at R0 position (16–22) were varied by altering the kind of
heterocycles. Furthermore, in order to obtain informa-
tion on the binding site of the new chalcone-like com-
pounds, inhibition studies were carried out using
Congo Red (CR) and thioflavin T (ThT), which are well
2.2. Binding studies in vitro
(E)-4-Dimethylamino-40-[125I]iodo-chalcone ([125I]DMIC)
was synthesized and used as the radioligand for compe-
tition binding experiments (Kd value of [125I]DMIC is
O
O
O
b
a
S
S
S
N
N
N
125I
Bu3Sn
Br
[
125I]14
13
37
Scheme 4. Reagents: (a) dioxane, (Bu3Sn)2, (Ph3P)4Pd, Et3N; (b) [125I]NaI, HCl, H2O2.

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
6391
Table 1. Inhibition constants of chalcone and chalcone-like derivatives on ligand binding to Ab(1–42) aggregates
O
R'
R
Compound
R
R0
Kia (nM)
4
4-Aminophenyl
4-Methylaminophenyl
4-Dimethylaminophenyl
5-Iodo-2-thienyl
4-Iodophenyl
4-Iodophenyl
4-Iodophenyl
4-Iodophenyl
248 56
23.9 3.6
13.3 1.9
3.9 0.4
151 16
908 212
125 9.2
102 16
93 11
7
10
14
16
4-Dimethylaminophenyl
Phenyl
2-Furanyl
17
18
4-Iodophenyl
4-Iodophenyl
3-Furanyl
2-Thienyl
19
20
4-Iodophenyl
4-Iodophenyl
3-Thienyl
2-Imidazoyl
2-Thiazoyl
21
22
4-Iodophenyl
4-Iodophenyl
797 316
>10,000
23
24
4-Iodophenyl
4-Iodophenyl
5-Dimethylamino-2-furanyl
5-Dimethylamino-2-thienyl
5-Dimethylamino-2-thienyl
5-Dimethylamino-2-furanyl
4-Dimethylaminophenyl
5-Dimethylamino-2-thienyl
5-Dimethylamino-2-furanyl
4-Aminophenyl
1132 344
113 10
137 3.4
1608 85
126 13
2648 222
>10,000
25
26
5-Iodo-2-thienyl
5-Iodo-2-thienyl
5-Bromo-2-furanyl
5-Bromo-2-furanyl
5-Bromo-2-furanyl
5-Iodo-2-thienyl
4-Aminophenyl
5-Iodo-2-thienyl
4-Methylaminophenyl
4-Dimethylaminophenyl
—
27
28
29
31
121 40
476 48
14.1 0.6
198 49
106 7.1
>10,000
32
34
5-Bromo-2-thienyl
4-Methylaminophenyl
5-Bromo-2-thienyl
5-Bromo-2-thienyl
—
35
36
CR
ThT
AICb
IMCc
DMICd
—
4-Iodophenyl
—
4-Aminophenyl
>10,000
105 12
6.3 1.6
2.9 0.3
4-Iodophenyl
4-Iodophenyl
4-Methylaminophenyl
4-Dimethylaminophenyl
b,c,dData from Ref. 18.
a Values are means standard error of the mean of 3–6 independent experiments.
b 4-Amino-40-iodo-chalcone.
c 40-Iodo-4-methylamino-chalcone.
d 4-Dimethylamino-40-iodo-chalcone.
known as prototypes of amyloid imaging probes.4,5
While compound 14 competed for [125I]DMIC binding
to Ab(1–42) aggregates, CR and ThT did not exhibit a
dose-dependent decrease in the specific binding of
120
100
80
60
40
20
0
[
125I]DMIC (Fig. 2). This result suggests that the bind-
ing site of a series of chalcone-like compounds on
Ab(1–42) aggregates may be different from that of CR
and ThT.
2.3. Neuropathological staining on AD model mouse brain
sections
In order to confirm the binding affinity to b-amyloid
plaques in the AD brain, fluorescent staining on AD
model mouse brain sections was carried out using the
fluorescence of compound 14 (Fig. 3). Compound 14 in-
tensely stained b-amyloid plaques in the brain sections.
Also, clear staining of cerebrovascular amyloids was
observed. This result suggests that compound 14 should
detect b-amyloid plaques in the AD brains.
0
2
4
6
8
log [inhibitor] (pM)
Figure 2. Competition curves of [125I]DMIC against compound 14
(closed circle), Congo Red (closed square), and thioflavin T (closed
triangle).
2.4. Biodistribution studies
biodistribution study provides important information
on brain uptake. The ideal b-amyloid imaging probe
should have good blood–brain barrier penetration to de-
The radioiodinated compound [125I]14 was evaluated for
its in vivo biodistribution in normal mice (Table 2). A

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M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
Table 2. Biodistribution of radioactivity after intravenous adminis-
tration of [125I]14 in micea
ian Gemini 300 spectrometer with TMS as an internal
standard. Coupling constants are reported in Herz.
The multiplicity is defined by s (singlet), d (doublet), t
(triplet), and m (multiplet). Mass spectra were obtained
on a JEOL IMS-DX instrument.
Time after Injection (min)
2
10
0.75
(0.31)
30
0.31
(0.04)
60
0.21
(0.02)
2.46
(0.30)
4.1.1. (E)-3-(4-Bromophenyl)-1-(4-nitrophenyl)-2-propen-
1-one (1). Equimolar portions of 4-nitroacetophenone
(1.67 g, 10.1 mmol) and 4-bromobenzaldehyde (1.86 g,
10.0 mmol) were dissolved in ethanol (10 mL). A 6 mL
aliquot of 10% aqueous potassium hydroxide solution
was then slowly added dropwise to the reaction mixture.
The mixture was allowed to stir for 30 min at room tem-
perature. A precipitate was collected and washed with
ethyl acetate to give 1.98 g of 1 (59.4%). 1H NMR
(300 MHz, CDCl3) d 7.48 (d, J = 15.6 Hz, 1H), 7.52
(d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.79 (d,
J = 15.6 Hz, 1H), 8.15 (d, J = 9 Hz, 2H), 8.36 (d,
J = 9 Hz, 2H).
a Expressed as % injected dose per gram. Each value represents the
mean (SD) for five mice at each interval.
liver a sufficient dose into the brain while achieving rapid
clearance from the normal regions to result in a higher
signal to noise ratio in the AD brain. Initial brain uptake
of [125I]14 was 2.46% of injected dose/gram at 2 min post
iv injection, whereas the radioactivity accumulated in the
brain was rapidly eliminated (0.21% of injected dose/
gram, 60 min post iv injection), indicating highly desir-
able properties for b-amyloid imaging agents.
3. Conclusion
4.1.2. (E)-1-(4-Aminophenyl)-3-(4-bromophenyl)-2-pro-
pen-1-one (2). A mixture of 1 (372 mg, 1.12 mmol),
SnCl2 (1.05 g, 5.55 mmol), and ethanol (5 mL) was stir-
red under reflux for 1 h. After the mixture was cooled to
room temperature, 1 M NaOH (10 mL) was added and
extracted with ethyl acetate (10 mL). The organic phase
was dried over Na2SO4 and filtered. The filtrate was
concentrated to give 190 mg of 2 (56.1%). 1H NMR
(300 MHz, CDCl3) d 4.19 (s, 2H), 6.70 (d, J = 8.7 Hz,
2H), 7.47–7.55 (m, 5H), 7.71 (d, J = 15.6 Hz, 1H), 7.93
(d, J = 8.7 Hz, 2H).
In conclusion, we successfully designed and synthesized
a series of chalcone and related compounds to evaluate
their structure–activity relationship of the binding affin-
ity to Ab aggregates. In in vitro binding studies with Ab
aggregates, a variety of Ki values were found to be inher-
ent to their structure. Compound 14 with the highest
binding affinity to Ab aggregates clearly stained b-amy-
loid plaques and cerebrovascular amyloids as reflected
in in vitro binding studies. Taken together, the data sug-
gest that the new radioiodinated compound 14 should
be further investigated as a potentially useful b-amyloid
imaging probe.
4.1.3. (E)-1-(4-Aminophenyl)-3-(4-tributylstannylphenyl)-
2-propen-1-one (3). A mixture of 2 (200 mg, 0.66 mmol),
(Bu3Sn)2 (0.4 mL), and (Ph3P)4Pd (35 mg, 0.03 mmol) in
a mixed solvent (16 mL, 5:3 dioxane/triethylamine mix-
ture) was stirred under reflux for 8 h. The solvent was re-
moved, and the residue was purified by preparative TLC
(1:1 hexane/ethyl acetate) to give 165 mg of 3 (48.7%).
1H NMR (300 MHz, CDCl3) d 0.87–1.52 (m, 27H),
4.23 (s,2H), 6.68 (d, J = 8.7 Hz, 2H), 7.50–7.58 (m,
5H), 7.76 (d, 15.3 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H).
MS m/z 513 (MH+).
4. Experimental
4.1. General information
All reagents used in syntheses were commercial products
and were used without further purification unless other-
wise indicated. H NMR spectra were obtained on Var-
1
Figure 3. Neuropathological fluorescent staining of compound 14 on AD model mouse brain sections. (a) Compound 14 intensely stained b-amyloid
plaques. (b) Clear staining of cerebrovascular amyloids was also observed.

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
6393
4.1.4. (E)-1-(4-Aminophenyl)-3-(4-iodophenyl)-2-propen-
1-one (4). To a solution of 3 (90 mg, 0.18 mmol) in
CHCl3 (5 mL) was added a solution of iodine in CHCl3
(2 mL, 0.25 M) at room temperature. The mixture was
stirred at room temperature for 20 min, and saturated
NaHSO3 solution was added. After the organic phase
was separated, dried over Na2SO4, and filtered, the sol-
vent was removed, and the residue was purified by pre-
parative TLC (1:1 hexane/ethyl acetate) to give 39 mg of
1H), 7.69 (d, J = 15.9 Hz, 1H), 7.74 (d, J = 8.4 Hz,
2H), 8.00 (d, J = 9 Hz, 2H). MS m/z 377 (M+).
4.1.9. (E)-1-(4-Dimethylaminophenyl)-3-(4-tributylstan-
nylphenyl)-2-propen-1-one (9). The same reaction as
described above to prepare 3 was used, and 36 mg of 9
was obtained in a 31.4% yield from 8. 1H NMR
(300 MHz, CDCl3) d 0.87–1.66 (m, 27H), 3.09 (s, 6H),
6.71 (d, J = 8.7 Hz, 2 H), 7.34–7.62 (m, 5H), 7.77(d,
J = 15.9 Hz, 1H), 8.01(d, J = 9 Hz, 2H). MS m/z 541
(MH+).
1
4 (63.6%). H NMR (300 MHz, CDCl3) d 4.17 (s, 2H),
6.70 (d, 8.4 Hz, 2H), 7,36 (d, J = 8.7 Hz, 2H), 7.54 (d,
J = 15.6 Hz, 1H), 7.69 (d, J = 15.6 Hz, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H). MS m/z 349
(M+).
4.1.10. (E)-1-(4-Dimethylaminophenyl)-3-(4-iodophenyl)-
2-propen-1-one (10). The same reaction as described
above to prepare 4 was used, and 11 mg of 10 was
1
4.1.5. (E)-3-(4-Bromophenyl)-1-(4-methylaminophenyl)-
2-propen-1-one (5). To a solution of 2 (230 mg,
0.76 mmol) in DMSO (5 mL) were added methyl iodide
(0.2 mL) and anhydrous K2CO3 (526 mg, 3.81 mmol).
The reaction mixture was stirred at room temperature
for 5 h. After it was poured into water (50 mL), the mix-
ture was extracted with ethyl acetate (50 mL). The or-
ganic layers were combined and dried over Na2SO4.
Evaporation of the solvent afforded a residue, which
was purified by silica gel chromatography (hexane/ethyl
acetate = 3:1) to give 78 mg of 5 (32.4%). 1H NMR
(300 MHz, CDCl3) d 2.89–2.94 (m, 3H), 4.36 (s, 1H),
6.61 (d, J = 8.7 Hz, 2H), 7.50–7.57 (m, 3H), 7.70 (d,
J = 15.6 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.96 (d,
J = 8.7 Hz, 2H).
obtained in a 38.2% yield from 9. H NMR (300 MHz,
CDCl3) d 3.09 (s, 6H), 6.71 (d, J = 9 Hz, 2H), 7.36 (d,
J = 8.4 Hz, 2H), 7.56 (d, J = 15.6 Hz, 1H), 7.68 (d,
J = 16.2 Hz, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.96 (d,
J = 9.0 Hz, 2H). MS m/z 363 (M+).
4.1.11.
(E)-1-(5-Bromo-2-thienyl)-3-(4-nitrophenyl)-2-
propen-1-one (11). The same reaction as described above
to prepare 1 was used, and 686 mg of 11 was obtained in
a 66.8% yield from 2-acetyl-5-bromothiophene and
4-nitrobenzaldehyde. 1H NMR (300 MHz, CDCl3) d
7.19 (d, J = 3.9 Hz, 1H), 7.48 (d, J = 15.6 Hz, 1H),
7.63 (d, J = 3.9 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.85
(d, J = 15.6 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H).
4.1.12. (E)-1-(5-Iodo-2-thienyl)-3-(4-nitrophenyl)-2-pro-
pen-1-one (12). The same reaction as described above
to prepare 1 was used, and 625 mg of 12 was obtained
in a 84.1% yield from 2-acetyl-5-iodothiophene and 4-
nitrobenzaldehyde. 1H NMR (300 MHz, CDCl3) d
7.38–7.43 (m, 2H), 7.51 (d, J = 4.2 Hz, 1H), 7.78 (d,
J = 8.4 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 8.29 (d,
J = 9 Hz, 2H).
4.1.6. (E)-1-(4-Methylaminophenyl)-3-(4-tributylstannyl-
phenyl)-2-propen-1-one (6). The same reaction as de-
scribed above to prepare 3 was used, and 70 mg of 6
was obtained in a 38.2% yield from 5. 1H NMR
(300 MHz, CDCl3) d 0.87–1.59 (m, 27H), 2.93 (d,
J = 5.1 Hz, 3H), 4.31 (s, 1H), 6.61 (d, J = 8.7 Hz, 2H),
7.50–7.60 (m, 5H), 7.77 (d, J = 15.6 Hz, 1H), 7.97 (d,
J = 9 Hz, 2H). MS m/z 527 (MH+).
4.1.13.
(E)-1-(5-Bromo-2-thienyl)-3-(4-dimethylamino-
4.1.7. (E)-3-(4-Iodophenyl)-1-(4-methylaminophenyl)-2-
propen-1-one (7). The same reaction as described above
to prepare 4 was used, and 16 mg of 7 was obtained in
phenyl)-2-propen-1-one (13). The same reaction as
described above to prepare 1 was used, and 565 mg of
13 was obtained in a 82.8% yield from 2-acetyl-5-bromo-
thiophene and 4-dimethylaminobenzaldehyde. 1H NMR
(300 MHz, CDCl3) d 3.05 (s, 6H), 6.69 (d, J = 8.7 Hz,
2H), 7.12 (d, J = 15.3 Hz, 1H), 7.13 (d, J = 3.9 Hz,
1H), 7.55–7.57 (m, 3H), 7.18 (d, J = 15.3 Hz, 1H). MS
m/z 337 (MH+).
1
a 38.2% yield from 6. H NMR (300 MHz, CDCl3) d
2.93 (d, J = 4.5 Hz, 3H), 4.35 (s, 1H), 6.61 (d,
J = 9 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.56 (d,
J = 15.6 Hz, 1H), 7.68 (d, J = 16.2 Hz, 1H), 7.74 (d,
J = 8.1 Hz, 2H), 7.96 (d, J = 9 Hz, 2H). MS m/z 363
(M+).
4.1.14. (E)-1-(5-Iodo-2-thienyl)-3-(4-dimethylaminophe-
nyl)-2-propen-1-one (14). The same reaction as described
above to prepare 1 was used, and 274 mg of 14 was
obtained in a 69.3% yield from 2-acetyl-5-iodothiophene
4.1.8. (E)-3-(4-Bromophenyl)-1-(4-dimethylaminophenyl)-
2-propen-1-one (8). To a stirred mixture of 2 (300 mg,
0.99 mmol) and paraformaldehyde (315 mg, 10.5 mmol)
in AcOH (15 mL) was added in one portion NaCNBH3
(300 mg, 4.77 mmol) at room temperature. The resulting
mixture was stirred at room temperature for 4 h, 1 M
NaOH (50 mL) was added, and extracted with CH3Cl
(50 mL). The organic phase was dried over Na2SO4
and filtered. The solvent was removed, and the residue
was purified by silica gel chromatography (hexane/ethyl
and
4-dimethylaminobenzaldehyde.
1H
NMR
(300 MHz, CDCl3) d 3.05 (s, 6H), 6.79 (d, J = 8.7 Hz,
2H), 7.11 (d, J = 15.3 Hz, 1H), 7.32 (d, J = 3.9 Hz,
1H), 7.45 (d, J = 3.9 Hz, 1H), 7.54 (d, J = 8.7 Hz, 2H),
7.81 (d, J = 15 Hz, 1H). MS m/z 383 (M+).
4.1.15. (E)-3-(5-Bromo-2-thienyl)-1-(4-nitrophenyl)-2-pro-
pen-1-one (15). The same reaction as described above to
prepare 1 was used, and 422 mg of 15 was obtained in a
41.2% yield from 4-nitroacetophenone and 5-bromothi-
1
acetate = 4:1) to give 150 mg of 8 (45.7%). H NMR
(300 MHz, CDCl3) d 3.09 (s, 6H), 6.71 (d, J = 9 Hz,
2H), 7.36 (d, J = 8.4 Hz 2H), 7.59 (d, J = 15.3 Hz,

6394
M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
ophene-2-carboxaldehyde. 1H NMR (300 MHz, CDCl3) d
7.09 (d, J = 3.9 Hz, 1H), 7.14–7.20 (m, 2H), 7.86 (d,
J = 15.6 Hz, 1H), 8.12 (d, J = 8.7 Hz, 2H), 8.35 (d,
J = 8.7 Hz, 2H).
61.6% yield from 4-iodoacetophenone and 2-thiazole-
carboxaldehyde. 1H NMR (300 MHz, CDCl3) d 7.52
(d, J = 3.0 Hz, 1H), 7.75–7.79 (m, 3H), 7.85–7.92 (m,
3H), 7.99 (d, J = 3.6 Hz, 1H). MS m/z 341 (M+).
4.1.16. (E)-40-Iodochalcone (16). The same reaction as
described above to prepare 1 was used, 293 mg of 16
was obtained in a 87.4% yield from 40-iodoacetophe-
4.1.23. (E)-1-(4-Iodophenyl)-3-(5-dimethylamino-2-fura-
nyl)-2-propen-1-one (23). The same reaction as described
above to prepare 1 was used, and after purification by
silica gel chromatography (2:1 hexane/ethyl acetate),
25 mg of 23 was obtained in a 6.8% yield from 4-iodo-
acetophenone and 5-dimethylamino-2-furaldehyde.20
1H NMR (300 MHz, CDCl3) d 3.04 (s, 6H), 5.22 (d,
J = 3.9 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 6.89 (d,
J = 15.0 Hz, 1H), 7.46 (d, J = 14.7 Hz, 1H), 7.70 (d,
J = 8.1 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H). MS m/z 367
(M+).
1
none and benzaldehyde. H NMR (300 MHz, CDCl3)
d 7.42–7.44 (m, 3H), 7.47 (d, J = 15.6 Hz, 1H), 7.63–
7.66 (m, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.82 (d,
J = 15.9 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H). MS m/z 334
(M+).
4.1.17.
(E)-3-(2-Furanyl)-1-(4-iodophenyl)-2-propen-1-
one (17). The same reaction described above to prepare
1 was used, and 212 mg of 17 was obtained in a 65.4%
yield from 4-iodoacetophene and 2-furaldehyde. 1H
NMR (300 MHz, CDCl3) d 6.52–6.54 (m, 1H), 6.74 (d,
J = 3.6 Hz, 1H), 7.39 (d, J = 15.0 Hz, 1H), 7.54 (d,
J = 1.8 Hz, 1H), 7.60 (d, J = 15.6 Hz, 1H), 7.74 (d,
J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H). MS m/z 324
(M+).
4.1.24. (E)-3-(5-Dimethylamino-2-thienyl)-1-(4-iodophe-
nyl)-2-propen-1-one (24). The same reaction as described
above to prepare 1 was used, and after purification by
silica gel chromatography (4:1 hexane/ethyl acetate),
18 mg of 24 was obtained in a 4.7% yield from 4-iodo-
acetophenone and 5-dimethylamino-2-thiophenecarbox-
aldehyde.20 1H NMR (300 MHz, CDCl3) d 3.04 (s, 6 H),
5.22 (d, J = 3.9 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 6.89
(d, J = 15.0 Hz, 1H), 7.46 (d, J = 14.7 Hz, 1H), 7.70
(d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H). MS m/z
383 (M+).
4.1.18.
(E)-3-(3-Furanyl)-1-(4-iodophenyl)-2-propen-1-
one (18). The same reaction as described above to pre-
pare 1 was used, and 279 mg of 18 was obtained in a
86.1% yield from 4-iodoacetophenone and 3-furalde-
hyde. 1H NMR (300 MHz, CDCl3)
d 6.70 (d,
J = 1.8 Hz, 1H), 7.17 (d, J = 15.3 Hz, 1H), 7.48 (s,
1H), 7.69–7.75 (m, 4H), 7.86 (d, J = 9.0 Hz, 2H). MS
m/z 324 (M+).
4.1.25.
(E)-3-(5-Dimethylamino-2-thienyl)-1-(5-iodo-2-
thienyl)-2-propen-1-one (25). The same reaction as
described above to prepare 1 was used, and after purifi-
cation by silica gel chromatography (4:1 hexane/ethyl
acetate), 15 mg of 25 was obtained in 7.9% yield from
2-acetyl-5-iodothiophene and 5-dimethylamino-2-thio-
4.1.19. (E)-1-(4-Iodophenyl)-3-(2-thienyl)-2-propen-1-one
(19). The same reaction as described above to prepare 1
was used, and 546 mg of 19 was obtained in a 79.1%
yield from 4-iodoacetophenone and 2-thiophenecarbox-
1
phenecarboxaldehyde. H NMR (300 MHz, CDCl3) d
3.07 (s, 6H), 5.85 (d, J = 4.2 Hz, 1H), 6.63 (d,
J = 14.7 Hz, 1H), 7.15 (d, J = 4.5 Hz, 1H), 7.29 (d,
J = 3.9 Hz, 1H), 7.38 (d, J = 4.2 Hz, 1H), 7.87 (d,
J = 14.7 Hz, 1H). MS m/z 389 (M+).
1
aldehyde. H NMR (300 MHz, CDCl3) d 7.09–7.12 (m,
1H), 7.26 (d, J = 15.0 Hz, 1H), 7.38 (d, J = 3.6 Hz, 1H),
7.44 (d, J = 5.4 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 7.87
(d, J = 8.4 Hz, 2H), 7.95 (d, J = 15.3 Hz, 1H). MS m/z
340 (M+).
4.1.26. (E)-3-(5-Dimethylamino-2-furyl)-1-(5-iodo-2-thie-
nyl)-2-propen-1-one (26). The same reaction as described
above to prepare 1 was used, and after purification by
silica gel chromatography (3:1 hexane/ethylacetate),
21 mg of 26 was obtained in a 11.5% yield from 2-acet-
yl-5-iodothiophene and 5-dimethylamino-2-furalde-
4.1.20. (E)-1-(4-Iodophenyl)-3-(3-thienyl)-2-propen-1-one
(20). The same reaction as described above to prepare 1
was used, and 295 mg of 20 was obtained in a 86.7%
yield from 4-iodoacetophenone and thiophene-3-car-
1
1
boxaldehyde. H NMR (300 MHz, CDCl3) d 7.26 (d,
hyde. H NMR (300 MHz, CDCl3) d 3.04 (s, 6H), 5.22
J = 15.6 Hz, 1H), 7.38–7.43 (m, 2H), 7.62–7.65 (m,
1H), 7.71 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 15.6 Hz,
1H), 7.87 (d, J = 8.4 Hz, 1H). MS m/z 340 (M+).
(d, J = 3.6 Hz, 1H), 6.73 (d, J = 14.4 Hz, 1H), 6.79 (d,
J = 3.9 Hz, 1H), 7.29 (d, J = 3.9 Hz, 1H), 7.50 (d,
J = 3.9 Hz, 1H), 7.45 (d, J = 14.7 Hz, 1H). MS m/z 373
(M+).
4.1.21. (E)-1-(4-Iodophenyl)-3-(1H-imidazol-2-yl)-2-pro-
pen-1-one (21). The same reaction as described above
to prepare 1 was used, and after purification by silica
gel chromatography (1:1 hexane/ethyl acetate), 142 mg
of 21 was obtained in a 43.8% yield from 4-iodoacetoph-
enone and 2-imidazolecarboxaldehyde. MS m/z 324
(M+).
4.1.27. (E)-1-(5-Bromo-2-furanyl)-3-(4-dimethylamino-
phenyl)-2-propen-1-one (27). The same reaction as de-
scribed above to prepare 1 was used, and 470 mg of
27 was obtained in a 55.4% yield from 2-acetyl-5-bro-
mofuran21 and 4-dimethylaminobenzaldehyde. 1H
NMR (300 MHz, CDCl3) d 3.04 (s, 6H), 5.22 (d,
J = 3.9 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 6.89 (d,
J = 15.0 Hz, 1H), 7.46 (d, J = 14.7 Hz, 1H), 7.70 (d,
J = 8.1 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H). MS m/z 321
(MH+).
4.1.22. (E)-1-(4-Iodophenyl)-3-(thiazol-2-yl)-2-propen-1-
one (22). The same reaction as described above to pre-
pare 1 was used, and 210 mg of 22 was obtained in a

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
6395
4.1.28.
(E)-1-(5-Bromo-2-furyl)-3-(5-dimethylamino-2-
J = 8.7 Hz, 2H), 7.80 (d, J = 15.3 Hz, 1H). MS m/z 369
thienyl)-2-propen-1-one (28). The same reaction as
described above to prepare 1 was used, and after purifi-
cation by silica gel chromatography (7:2 hexane/ethyl
acetate), 18 mg of 28 was obtained in a 11.4% yield from
2-acetyl-5-bromofuran and 5-dimethyl-2-thiophenecar-
(M+).
4.1.35. (E)-3-(5-Bromo-2-thienyl)-1-(4-methylaminophe-
nyl)-2-propen-1-one (35). The same reaction as described
above to prepare 5 was used, and after purification by
silica gel chromatography (4:1 hexane/ethyl acetate),
228 mg of 35 was obtained in a 25.4% yield from 32.
1H NMR (300 MHz, CDCl3) d 2.92–2.94 (m, 3H), 4.33
(s, 1H), 6.61 (d, J = 8.7 Hz, 2H), 7.02–7.06 (m, 2H),
7.26 (d, J = 15.3 Hz, 1H), 7.78 (d, J = 15.6 Hz, 1H),
7.93 (d, J = 9 Hz, 2H). MS m/z 323 (MH+).
1
boxaldehyde. H NMR (300 MHz, CDCl3) d 3.08 (s,
6H), 5.85 (d, J = 3.9 Hz, 1H), 6.48 (d, J = 3.6 Hz, 1H),
6.72 (d, J = 15 Hz, 1H), 7.14–7.16 (m, 2H), 7.91 (d,
J = 15.0 Hz, 1H). MS m/z 327 (MH+).
4.1.29.
(E)-1-(5-Bromo-2-furyl)-3-(5-dimethylamino-2-
furyl)-2-propen-1-one (29). The same reaction as
described above to prepare 1 was used, and after purifi-
cation by silica gel chromatography (4:1 hexane/ethyl
acetate), 29 mg of 29 was obtained in a 17.7% yield from
2-acetyl-5-bromofuran and 5-dimethylamino-2-furalde-
4.1.36.
(E)-3-(5-Bromo-2-thienyl)-1-(4-dimethylamino-
phenyl)-2-propen-1-one (36). The same reaction as
described above to prepare 8 was used, and after purifi-
cation by silica gel chromatography (4:1 hexane/ethyl
acetate), 53 mg of 36 was obtained in a 5.7% yield from
32. 1H NMR (300 MHz, CDCl3) d 3.09 (s, 6H), 6.70 (d,
J = 9 Hz, 2H), 7.02–7.06 (m, 2H), 7.28 (d, J = 15.3 Hz,
1H), 7.78 (d, J = 15 Hz, 1H), 7.96 (d, J = 9 Hz, 2H).
MS m/z 337 (MH+).
1
hyde. H NMR (300 MHz, CDCl3) d 3.05 (s, 6H), 5.25
(d, J = 3.6 Hz, 1H), 6.48 (d, J = 3.3 Hz, 1H), 6.78–6.83
(m, 2 H), 7.14 (d, J = 3.3 Hz, 1H), 7.49 (d,
J = 14.7 Hz, 1H). MS m/z 311 (MH+).
4.1.30. (E)-1-(5-Bromo-2-thienyl)-3-(4-aminophenyl)-2-
propen-1-one (30). The same reaction as described above
to prepare 2 was used, 626 mg of 30 was obtained in a
4.1.37. (E)-1-(5-Tributylstannyl-2-thienyl)-3-(4-dimethyl-
aminophenyl)-2-propen-1- one (37). The same reaction as
described above to prepare 3 was used, and 10 mg of 37
was obtained in a 15.1% yield from 13. 1H NMR
(300 MHz, CDCl3) d 0.88–1.63 (m, 27H), 3.04 (s, 6H),
6.70 (d, J = 9 Hz, 2H), 7.21–7.27 (m, 2H), 7.55 (d,
J = 9 Hz, 2H), 7.81 (d, J = 15.3 Hz, 1H), 7.92 (d,
J = 3.6 Hz, 1H). MS m/z 547 (MH+).
1
43.6% yield from 11. H NMR (300 MHz, CDCl3) d
4.03 (s, 2H), 6.68 (d, J = 9 Hz, 2H), 7.11–7.16 (m,
2 H), 7.47 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 3.9 Hz,
1H), 7.78 (d, J = 15.3 Hz, 1H). MS m/z 309 (MH+).
4.1.31. (E)-3-(4-Aminophenyl)-1-(5-iodo-2-thienyl)-2-pro-
pen-1-one (31). The same reaction as described above to
prepare 2 was used, and 586 mg of 31 was obtained from
12. Compound 31 was used in the next reaction without
further purification. 1H NMR (300 MHz, CDCl3) d 4.03
(s, 2H), 6.68 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 15.3 Hz,
1H), 7.33 (d, J = 3.9 Hz, 1H), 7.44–7.49 (m, 3H), 7.78
(d, J = 15.3 Hz, 2H). MS m/z 355 (M+).
4.2. Iododestannylation reaction
The radioiodinated form of compound 14 was prepared
from corresponding tributyltin derivatives by an iodo-
destannylation. Briefly, 50 lL of H2O2 (3%) was added
to a mixture of a tributyltin derivative (100 lg/50 lL
in EtOH), [125I]NaI (3.7–7.4 MBq, specific activity
2200 Ci/mmol), and 100 lL of 1 N HCl in a sealed glass
vial. The reaction was allowed to proceed at room tem-
perature for 2 min and terminated by addition of 100 lL
of a saturated aqueous NaHSO3. After addition of
100 lL of a saturated aqueous NaHCO3, the reaction
mixture was extracted with ethyl acetate (1 mL). The
extract was dried by passing through an anhydrous
Na2SO4 column and was then blown to dryness with a
stream of nitrogen gas. The radioiodinated ligand was
purified by HPLC on a Cosmosil C18 column with an
isocratic solvent of H2O/acetonitrile (2/3) at a flow rate
of 1.0 mL/min.
4.1.32. (E)-3-(5-Bromo-2-thienyl)-1-(4-aminophenyl)-2-
propen-1-one (32). The same reaction as described above
to prepare 2 was used, and 453 mg of 32 was obtained in
1
a 23.5% yield from 15. H NMR (300 MHz, CDCl3) d
4.16 (s, 2H), 6.80 (d, J = 8.4 Hz, 2H), 7.03–7.07 (m,
2 H), 7.23 (d, J = 15.6 Hz, 1H), 7.78 (d, J = 15 Hz,
1H), 7.89 (d, J = 8.7 Hz, 2H). MS m/z 309 (MH+).
4.1.33. (E)-1-(5-Bromo-2-thienyl)-3-(4-methylaminophe-
nyl)-2-propen-1-one (33). The same reaction as described
above to prepare 5 was used, 40 mg of 33 was obtained
in a 18.0% yield from 30. 1H NMR (300 MHz, CDCl3) d
2.91 (s, 3H), 4.19 (s, 1H), 6.60 (d, J = 8.7 Hz, 2H), 7.09–
7.14 (m, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.56 (d,
J = 4.2 Hz, 1H), 7.80 (d, J = 15.6 Hz, 1H). MS m/z 323
(MH+).
4.3. Binding assays using the aggregated Ab peptide in
solution
A solid form of Ab(1–42) was purchased from Peptide
Institute (Osaka, Japan). Aggregation of peptides was
carried out by gently dissolving the peptide (0.25 mg/
mL) in a buffer solution (pH 7.4) containing 10 mM
sodium phosphate and 1 mM EDTA. The solutions
were incubated at 37 ꢁC for 42 h with gentle and
constant shaking. Binding studies were carried out in
12 · 75 mm borosilicate glass tubes according to the
4.1.34.
(E)-1-(5-Iodo-2-thienyl)-3-(4-methylaminophe-
nyl)-2-propen-1-one (34). The same reaction as described
above to prepare 5 was used, and 45 mg of 34 was ob-
tained in a 22.8% yield from 31. H NMR (300 MHz,
CDCl3)
J = 8.4 Hz, 2H), 7.11 (d, J = 15.3 Hz, 1H), 7.32 (d,
1
d 2.90 (s, 3H), 4.22 (s, 1H), 6.59 (d,
J = 4.2 Hz, 1H), 7.44 (d, J = 3.9 Hz, 1H), 7.50 (d,

6396
M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 6388–6396
procedure described previously.17 A mixture containing
50 lL of test compounds (8 pM–12.5 lM in 10% etha-
nol), 50 lL of 0.02 nM [125I]DMIC, 50 lL of Ab(1–42)
aggregates, and 850 lL of 10% ethanol was incubated
at room temperature for 3 h. The mixture was then fil-
tered through Whatman GF/B filters using a Brandel
M-24 cell harvester, and the filters containing the bound
125I ligand were counted in a gamma counter. Values for
the half-maximal inhibitory concentration (IC50) were
determined from displacement curves of three indepen-
dent experiments using GraphPad Prism 4.0, and those
for the inhibition constant (Ki) were calculated using
the Cheng-Prusoff equation22: Ki = IC50/(1 + [L]/Kd),
where [L] is the concentration of [125I]DMIC used in
the assay, and Kd is the dissociation constant of DMIC
(4.2 nM).18
Industrial Technology Development Organization
(NEDO) of Japan.
References and notes
1. Selkoe, D. J. J. Neuropathol. Exp. Neurol. 1994, 53, 438.
2. Selkoe, D. J. Physiol Rev 2001, 81, 741.
3. Selkoe, D. J. Nat Biotechnol. 2000, 18, 823.
4. Mathis, C. A.; Wang, Y.; Klunk, W. E. Curr. Pharm. Des.
2004, 10, 1469.
5. Nordberg, A. Lancet Neurol. 2004, 3, 519.
6. Agdeppa, E. D.; Kepe, V.; Liu, J.; Flores-Torres, S.;
Satyamurthy, N.; Petric, A.; Cole, G. M.; Small, G. W.;
Huang, S. C.; Barrio, J. R. J. Neurosci. 2001, 21, RC189.
7. 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. Am. J. Geriatr.
Psychiatry 2002, 10, 24.
8. 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. N. Engl. J. Med. 2006, 355, 2652.
9. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.;
Debnath, M. L.; Klunk, W. E. J. Med. Chem. 2003, 46,
2740.
10. 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.
Ann. Neurol. 2004, 55, 306.
11. Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.;
Verhoeff, P.; Zhuang, Z. P.; Kung, M. P.; Kung, H. F.
Nucl. Med. Biol. 2003, 30, 565.
12. Verhoeff, N. P.; Wilson, A. A.; Takeshita, S.; Trop, L.;
Hussey, D.; Singh, K.; Kung, H. F.; Kung, M. P.; Houle,
S. Am. J. Geriatr. Psychiatry 2004, 12, 584.
13. Kung, M. P.; Hou, C.; Zhuang, Z. P.; Zhang, B.;
Skovronsky, D.; Trojanowski, J. Q.; Lee, V. M.; Kung,
H. F. Brain Res. 2002, 956, 202.
14. Newberg, A. B.; Wintering, N. A.; Plossl, K.; Hochold, J.;
Stabin, M. G.; Watson, M.; Skovronsky, D.; Clark, C. M.;
Kung, M. P.; Kung, H. F. J. Nucl. Med. 2006, 47, 748.
15. Newberg, A. B.; Wintering, N. A.; Clark, C. M.; Plossl,
K.; Skovronsky, D.; Seibyl, J. P.; Kung, M. P.; Kung, H.
F. J. Nucl. Med. 2006, 47, 78P.
4.4. Staining of amyloid plaques in double transgenic
mouse brain sections
Double transgenic mice (6 months of age) produced by
Tg2576 crossed with mutated PS1 (A260V) mice were
used as Alzheimer’s model mice. Brain tissues were
obtained followed by fixation with 10% formaldehyde.
Dehydrated tissues with ethanol and xylene were paraff-
inized and the resultant wax blocks were sliced into seri-
al sections of 5 lm thickness. The tissue slides were
deparaffinized with xylene, ethanol, and distilled water.
After incubation with PBS for 30 min, each slide was
incubated with 50% ethanol solution (100 lM) of com-
pound 14. Finally, the sections were washed in PBS
for 15 min. Thereafter, the sections were incubated in
ethanol and xylene, and embedded in Entellan Neu
(Merck, Darmstadt, Germany). Fluorescent observation
was performed by the Leica TCS SP2 system with
DMIRE2 fluorescence microscope. Staining with com-
pound 14 was detected using filter set with 458 nM exci-
tation and 540–580 nM emission. The sections were also
immunostained with DAB as a chromogen using mono-
clonal antibodies against b-amyloid as previously
reported.23
4.5. In vivo biodistribution in normal mice
Animal studies were conducted in accordance with our
institutional guidelines and were approved by Nagasaki
University Animal Care Committee. A saline solution
(100 lL) containing [125I]14 (4.2–6.3 kBq) and 10% eth-
anol was injected directly into the tail vein of ddY mice
(5-week-old, average weight 23–25 g). The mice were
sacrificed at various time points postinjection. The or-
gans of interest were removed and weighed, and the
radioactivity was counted with an automatic gamma
counter (Aloka, ARC-380).
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Acknowledgment
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This study was supported by Industrial Technology Re-
search Grant Program in 2005 from New Energy and
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