Synthesis and characterization of styrylchromone derivatives as β-amyloid imaging agents

Article abstract of DOI:10.1016/j.bmc.2006.09.044

Several promising agents have been synthesized and evaluated for in vivo imaging probes of β-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as

Full text of DOI:10.1016/j.bmc.2006.09.044

Bioorganic & Medicinal Chemistry 15 (2007) 444–450
Synthesis and characterization of styrylchromone derivatives
as b-amyloid imaging agents
Masahiro Ono,* Yoshifumi Maya, Mamoru Haratake and Morio Nakayama
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Received 17 August 2006; revised 19 September 2006; accepted 21 September 2006
Available online 10 October 2006
Abstract—Several promising agents have been synthesized and evaluated for in vivo imaging probes of b-amyloid plaques in Alz-
heimer’s disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchr-
omone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and
evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl.
A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques
were assessed by in vitro binding assay using pre-formed synthetic Ab(1–40) aggregates. The new series of styrylchromone deriva-
tives showed high binding affinity to Ab aggregates at the Kd values of 32.0, 17.5 and 8.7 nM for [125I]6, [125I]9, and [125I]12, respec-
tively. In biodistribution studies using normal mice, [125I]6 and [125I]9 examined in normal mice displayed high brain uptakes with
4.9 and 2.8%ID/g at 2 min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60 min post
injection for [125I]6 and [125I]9, respectively). But [125I]12 did not show marked brain uptake, and the washout rate from the brain
was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30 min post injection, respectively). Although additional
modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchro-
mone backbone may be useful as a backbone structure to develop novel b-amyloid imaging agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Several promising agents based on the backbone struc-
ture of DDNP, thioflavin-T, and Congo Red have been
synthesized and evaluated for in vivo imaging probes of
b-amyloid plaques in AD brain. Clinical trials in AD pa-
tients have been reported with several agents including
[18F]-2-(1-(2-(N-(2-fluoroethyl)-N-methylamino)naphtha-
lene-6-yl)ethylidene)malononitrile ([18F]FDDNP),6,7 [11C]-
2-(4-(methylamino)phenyl)-6-hydroxybenzothiazole ([11C]-
6-OH-BTA-1),8,9 [11C]-4-N-methylamino-40-hydroxystil-
bene ([11C]SB-13),10,11 and [123I]-6-iodo-2-(40-dimethylami-
no)phenyl-imidazo[1,2-a]pyridine ([123I]IMPY),12,13 (Fig. 1),
indicating that detecting amyloid plaques in the living
human brain with amyloid imaging agents is potentially
feasible by both PET and SPECT.
Alzheimer’s disease (AD) is the most common neurode-
generative disorder of the elderly and is characterized
clinically by dementia, cognitive impairment, and mem-
ory loss. The neuropathological hallmarks of AD in-
clude abundant deposits of b-amyloid fibrils in senile
plaques (SPs), accumulation of abnormal tau protein fil-
aments in neurofibrillary tangles (NFTs), and extensive
neuronal degeneration and loss. Although the precise
mechanism of neuronal death in AD still remains un-
known, SPs and NFTs have been regarded as a critical
event for the pathogenesis of AD.1,2 Therefore, quanti-
tative evaluation of SPs and/or NFTs in the brain with
non-invasive techniques such as positron emission
tomography (PET) and single photon emission comput-
ed tomography (SPECT) would lead to presymptomatic
detection of AD patients and evaluation of new anti-am-
yloid therapies currently under development.3–5
Recently, we have reported flavone derivatives, which
possess the basic structure of the 2-phenylchromone,
as useful candidates for b-amyloid imaging agents
(Fig. 2).14 This report also suggested that the backbone
except DDNP, thioflavin-T, and Congo Red can be ap-
plied for the development of more useful b-amyloid
imaging agents. In an attempt to further develop novel
ligands for imaging of amyloid plaques in AD, we
designed a series of 2-styrylchromone derivatives, which
Keywords: Alzheimer’s disease; Amyloid; Imaging.
*
Corresponding author. Tel.: +81 95 819 2443; fax: +81 95 819
2442; e-mail: mono@nagasaki-u.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.044

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
445
NC
CN
N
S
NH
11CH3
18F
HO
N
11C]PIB
[
18F]FDDNP
[
N
HO
N
NH
N
123I
11CH3
11C]SB-13
[
123I]IMPY
[
Figure 1. Chemical structures of amyloid imaging agents previously reported.
R2
R1
O
O
O
I
I
O
Styrylchromone derivatives
Flavone derivatives
Figure 2. Chemical structures of flavone derivatives reported previously and styrylchromone derivatives reported in this report. R1 = NHCH3,
N(CH3)2, OCH3, OH; R2 = NH2, NHCH3, N(CH3)2.
replace the 2-phenyl substituent of flavone backbone
with the 2-styryl. In this paper, we report the synthesis
of a novel series of styrylchromone derivatives and the
characterization as b-amyloid imaging agents.
(71.5% yield). The tributyltin derivatives (5, 8, and 11)
were prepared from the corresponding bromo com-
pounds (4, 7, and 10) using a bromo to tributyltin ex-
change reaction catalyzed by Pd(0) for yields of 56.2%,
42.8%, and 37.4%, respectively. These tributyltin deriva-
tives were readily reacted with iodine in chloroform at
room temperature to give the iodo derivatives (6, 9,
and 12). Furthermore, these tributyltin derivatives can
be also used as the starting materials for redioiodination
in preparation of [125I]6, [125I]9, and [125I]12. Novel radi-
oiodinated styrylchromone derivatives were achieved by
an iododestannylation reaction using hydrogen peroxide
as the oxidant, which produced the desired radioiodinat-
ed ligands (Scheme 2). It was anticipated that the no-
carrier-added preparation would result in a final prod-
uct bearing a theoretical specific activity similar to that
of 125I (2200 Ci/mmol). The radiochemical identities of
the radioiodinated ligands were verified by co-injection
with nonradioactive compounds by their HPLC profiles.
The final radioiodinated compounds [125I]6, [125I]9, and
2. Results and discussion
The synthesis of the styrylchromone derivatives is out-
lined in Scheme 1. Various strategies have been devel-
oped for the synthesis of 2-styrylchromone. Among
them, the most utilized procedure for building the 2-sty-
rylchromone ring system is the Baker-Vankataraman
approach, based on the cyclization of 2-cinnamoyl-O-
hydroxyacetophenones, obtained by the condensation
of 2-hydroxyacetophenone with the cinnamic acid chlo-
ride, followed by rearrangement of the intermediate es-
ter. In this process, the 20-cinnamoyloxyacetophenone
1 has been prepared by O-acylation of the 2-hydroxyace-
tophenone with a cinnamoyl chloride in the presence of
pyridine. The arrangement of ester to give the b-dike-
tone 2 was performed by treatment with KOH in pyri-
dine. The cyclodehydration of 2,4-pentadien-1-one, by
refluxing sulfuric acid and acetic acid, gave the corre-
sponding 2-styrylchromone 3. The amino derivative 4
was readily prepared from 3 by reduction with SnCl2
(63.3% yield). Conversion of 4 to the monomethylamino
derivative 7 was achieved by a method previously
reported15 (96.0% yield). Compound 4 was also convert-
ed to the dimethylamino derivative 10 by an efficient
method16 with paraformaldehyde and acetic acid
[
125I]12 showed a single radioactivity peak at retention
times of 5.3, 7.2, and 11.7 min, respectively (Table 1).
Three radioiodinated products were obtained in 50–
70% radiochemical yields with radiochemical purities
of >95% after purification by HPLC.
The binding studies of [125I]6, [125I]9, and [125I]12 to
aggregates of Ab(1–40) were carried out (Fig. 3). Trans-
formation of the saturation binding of [125I]6, [125I]9,
and [125I]12 to Scatchard plots gave linear plots, indicat-
ing that the styrylchromone derivatives have one bind-

446
M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
NO2
OH
O
O
O2N
a
b
+
O
O
Br
COOCl
Br
1
NO2
NH2
OH
O
NO2
d
c
Br
O
O
O
Br
Br
2
O
O
3
4
NH2
NH2
f
e
O
O
Bu3Sn
I
O
O
6
5
NH
NH
NH
e
g
f
O
O
O
Br
Bu3Sn
I
O
O
O
8
9
7
N
N
N
h
f
e
O
O
O
O
Br
Bu3Sn
I
O
O
10
11
12
Scheme 1. Synthetic route for styrylchromone derivatives. Reagents: (a) pyridine; (b) pyridine, KOH; (c) H2SO4, AcOH; (d) EtOH, SnCl2; (e)
dioxane, (Bu3Sn)2, (Ph3P)4Pd, Et3N; (f) CHCl3, I2; (g) MeOH, NaOMe, (CH2O)n, NaBH4; (h) AcOH, (CH2O)n, NaCNBH3.
R
R
[
125I]NaI
HCl
H2O2
O
O
O
O
R = NH2 ([125I]6)
NHMe ([125I]9)
NMe2 ([125I]12)
Bu3Sn
125I
Scheme 2. Radioiodination reaction of styrylchromone derivatives.
ing site on Ab aggregates. [125I]6, [125I]9, and [125I]12
showed excellent binding affinity for Ab(1–40) aggre-
gates at the Kd value of 32.0 5.0, 17.5 5.0, and
8.7 2.7 nM, respectively. The binding affinity to
Ab aggregates increased in the order of [125I]12 > [125I]9 >
[
125I]6. The result of the binding study was in agreement

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
447
Table 1. HPLC data and partition coefficient for [125I]6, [125I]9, and
125I]12
Table 2. Biodistribution of radioactivity after intravenous adminis-
tration of [125I]6, [125I]9, and [125I]12 in micea
[
Compound
125I]6
125I]9
125I]12
Retention timea (min)
log Pb
Tissue
Time after injection (min)
10 30
[
5.3
7.2
1.65 (0.08)
2.15 (0.08)
2.65 (0.05)
2
60
[
[
125I]6
Blood
Liver
[
11.7
2.34 (0.16)
2.44 (0.19)
2.56 (0.64)
13.23 (1.65) 14.49 (1.10) 10.27 (1.84)
2.27 (0.47)
a Using a mixture of H2O/acetonitrile (3:7) as the mobile phase.
b Octanol/buffer (0.1 M phosphate-buffered saline, pH 7.4) partition
coefficients. Each value represents the mean (SD) of three
experiments.
8.16 (1.33)
3.76 (0.91)
5.51 (0.55) 11.71 (1.27) 16.08 (2.44)
Kidney
Intestine
Spleen
Pancreas
Heart
11.94 (1.49)
2.84 (0.18)
4.23 (0.62)
6.58 (0.69)
8.40 (0.47)
1.24 (0.43)
4.88 (0.47)
6.99 (0.91)
4.91 (1.07)
3.64 (0.38)
5.18 (0.54)
3.78 (0.35)
1.81 (0.51)
4.40 (0.44)
2.92 (0.73)
3.07 (0.94)
2.43 (0.70)
2.17 (0.40)
2.78 (0.72)
2.16 (0.85)
1.11 (0.93)
1.64 (0.29)
3.29 (0.92)
1.62 (0.40)
Stomachb
with that ofpreviousreports,10,14 indicating thatthe binding
affinity increased in the order of N,N-dimethylated deriva-
tive > N-monomethylated derivative > primary amino
derivative. Comparing compounds 6, 9, and 12 with corre-
sponding radioiodinated flavone derivatives, substitutions
of the phenyl group on flavone backbone14 with the styryl
group resulted in a 1.5-fold increase in binding affinity. In
addition, all of the styrylchromone derivatives evaluated
maintained good binding affinities in the nanomolar range
of Kd values. The results of the binding study suggest that
the styrylchromone derivatives have considerable tolerance
for structural modification.
Brain
[
125I]9
Blood
Liver
3.21 (0.38)
2.44 (0.08) 2.19 (0.29)
14.55 (2.64) 14.41 (2.52) 11.13 (1.63)
1.79 (0.23)
7.82 (0.94)
3.52 (1.36)
Kidney
Intestine
Spleen
Pancreas
Heart
10.41 (1.33)
2.59 (0.38)
3.88 (0.74)
4.92 (0.54)
6.92 (1.41)
1.00 (0.23)
2.84 (0.35)
7.05 (0.56)
5.48 (0.97)
8.55 (1.21) 16.72 (2.58) 19.16 (2.63)
4.42 (1.05)
3.56 (0.96)
3.31 (0.69)
1.89 (0.33)
2.96 (0.25)
2.66 (0.47)
2.25 (0.63)
2.09 (0.39)
2.01 (0.53)
1.86 (0.30)
1.51 (0.19)
1.32 (0.33)
1.47 (0.27)
1.90 (0.29)
0.99 (0.09)
Stomachb
Brain
[
125I]12
Blood
Liver
2.62 (0.42) 1.44 (0.57)
11.17 (2.23) 10.15 (1.43)
1.13 (0.37) n.d.c
7.72 (1.83)
3.69 (0.19)
One of the important prerequisites for an in vivo imag-
ing agent of the brain is to penetrate the blood–brain
barrier after an iv injection. The logP values of [125I]6,
Kidney
Intestine
Spleen
Pancreas
Heart
6.70 (0.92)
1.45 (0.31)
2.43 (0.75)
3.02 (0.57)
6.25 (1.27)
0.70 (0.16)
1.14 (0.20)
4.51 (0.62)
4.95 (0.96) 12.68 (2.31)
[
125I]9, and [125I]12 were 1.65, 2.15, and 2.65, respective-
2.01 (0.39)
2.73 (0.54)
1.75 (0.73)
1.14 (0.62)
1.76 (0.24)
1.38 (0.34)
1.34 (0.39)
1.27 (0.24)
1.23 (0.52)
1.37 (0.36)
ly (measured by a partition between 1-octanol and pH
7.4 phosphate buffer) (Table 1). Previous studies suggest
that the optimal lipophilicity range for brain entry is ob-
served for compounds with logP values between 1 and
3.17–19 Since these ligands displayed moderate lipophilic-
ity for BBB penetration, it was expected to show ade-
quate brain uptake to detect amyloid plaques
following systemic injection. To test the brain uptake
and washout from the brain, three styrylchromone
derivatives were injected into normal mice (Table 2).
Generally, the ideal amyloid imaging agents should have
a high initial brain uptake and a fast washout from the
normal brain area. Since the normal brain has no amy-
loid plaques to trap the agent, the radioactivity washout
should be fast from the normal brain to obtain a higher
signal to noise ratio in the AD brain. In the biodistribu-
tion studies, initial brain uptake of [125I]6 and [125I]9 at
2 min after iv injection was relatively high (4.88 and
2.84%ID/g, respectively), whereas the brain retention
at later time points of [125I]6 and [125I]9 was low (1.62
and 0.99%ID/g at 60 min post iv injection, respectively).
Stomachb
Brain
a Expressed as % injected dose per gram. Each value represents the
mean (SD) for 3–5 animals at each interval.
b Expressed as % injected dose per organ.
c Not determined.
These in vivo properties observed with [125I]6 and [125I]9
(a high initial brain uptake and fast washout from the
normal mice brain) suggest that these radioiodinated
styrylchromones may be novel candidates of amyloid
imaging agents. Compared to the radioiodinated flav-
ones previously reported,14 125I]6 and [125I]9 showed
[
very similar in vivo pharmacokinetics in normal mice.
On the other hand, [125I]12 did not show marked initial
brain uptake in the normal mice after iv injection
(1.14%ID/g at 2 min postinjection), whereas the radioac-
tivity level of blood was similar to that of [125I]6 and
Figure 3. Scatchard plots of [125I]6, [125I]9, and [125I]12 binding to Ab(1–40) aggregates. Three ligands showed one-site binding. High binding
affinities with a Kd value in a nanomolar range were obtained (Kd = 32.0 5.0, 17.5 2.5, and 8.7 2.7 nM for [125I]6, [125I]9, and [125I]12,
respectively).

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M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
[
125I]9. The brain uptake of [125I]12 peaked at 10 min
the development of new amyloid imaging agents, appro-
priate structural changes on the styrylchromone back-
bone may lead to useful amyloid imaging agents.
with a maximum brain uptake of 1.76%ID/g, and the
washout rate from the brain was relatively slow
throughout the time course that was evaluated
(1.37%ID/g at 30 min postinjection), which is unsuitable
for in vivo imaging. Compound [125I]12 with the highest
logP displayed the lowest brain uptake among three
radioiodinated styrylchromones, and the brain uptake
of three ligands increased in order of their decreasing
logP value. Although many factors such as molecular
size, ionic charge, and lipophilicity affect the brain up-
take of a compound, the high lipophilicity of [125I]12
may be one reason for the low brain uptake of
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
ian Gemini 300 spectrometer with TMS as an internal
standard. Coupling constants are reported in Hertz.
The multiplicity is defined by s (singlet), d (doublet), t
(triplet), br (broad), and m (multiplet). Mass spectra
were obtained on a JEOL IMS-DX instrument.
[
125I]12. Furthermore, this result suggests that the opti-
mal logP value for brain uptake of styrylchromone
derivatives may be approximately 1.7 or less. Despite
the highest binding affinity for Ab aggregates, [125I]12
cannot serve as in vivo amyloid imaging due to this
unfavorable in vivo pharmacokinetics. In addition to
in vivo pharmacokinetics, in vivo stability of a com-
pound is one of important prerequisites for b-amyloid
imaging agents. Although the radiometabolites derived
from radioiodinated styrylchromone derivatives in the
brain and blood have not been investigated in this study,
little radioactivity was observed in the stomach and
blood in biodistribution studies, indicating that at least
these styrylchromone derivatives possess high stabilities
against in vivo deiodination in mice.
4.1.1. (E)-2-Acetyl-bromophenyl 3-(4-nitrophenyl)acry-
late (1). To a stirring solution of trans-4-nitrocinnamoyl
chloride (545 mg, 2.58 mmol) in pyridine (10 mL) in an
ice bath was added 50-bromo-20-hydroxyacetophenone
(500 mg, 2.58 mmol). The reaction mixture was stirred
at room temperature for 60 min and poured into 1 N
aqueous HCl solution with vigorous stirring. The pre-
cipitate which formed was filtered and washed with
water to yield acetophenone (1) (1.07 g, 99.2% yield).
1H NMR (300 MHz, CDCl3) d 6.80 (d, J = 16.2 Hz,
1H), 7.10 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 2.4 Hz, 1H),
7.62–7.77 (m, 4H), 7.95 (d, J = 2.4 Hz, 1H), 8.29 (d,
J = 9.0 Hz, 1H).
[
123I]IMPY is currently the most promising radioiodin-
ated tracer for detecting amyloid plaques in AD pa-
tients.12,13,20,21 This tracer displays appropriate
properties as amyloid imaging agents: high binding
affinity for amyloid plaques at 15 nM of Ki value in
the in vitro binding assay, and high brain uptake and ra-
pid clearance from the normal brain in animal studies.
4.1.2. 1-(5-Bromo-2-hydroxyphenyl)-3-(4-nitrophenyl)pent-
4-ene-1,3-dione (2). A solution of acetophenone 1
(500 mg, 6.34 mmol) and pyridine (12 mL) was heated
to 50 ꢁC and to it was added pulverized potassium
hydroxide (220 mg, 3.84 mmol). The reaction mixture
was stirred for 30 min, and upon cooling, 20 mL of
10% aqueous acetic acid solution was added. The pale
yellow precipitate which formed was filtered to yield 2
[
123I]IMPY entered the brain rapidly (2.88%ID/organ
at 2 min after iv injection in mice) and cleared rapidly
from normal mice brains (0.26%ID/organ at 30 min
after iv injection in mice). The in vivo pharmacokinetics
of the styrylchromone derivatives reported in this study
appears inferior to that of IMPY, although their high
binding affinities for amyloid plaques are sufficient for
in vivo imaging of b-amyloid plaques. Therefore, addi-
tional structural changes are essential to further improve
the in vivo properties of the styrylchromone derivatives
to be suitable for imaging.
1
(410 mg, 82.0% yield). H NMR (300 MHz, CDCl3) d
6.34 (s, 1H), 6.77 (d, J = 16.2 Hz, 1H), 6.98 (s, 1H),
7.12 (d, J = 5.7 Hz, 2H), 7.71 (m, 3H), 8.27 (d,
J = 9.0 Hz, 2H).
4.1.3. 6-Bromo-2-(40-nitrostyryl)chromone (3). A mixture
of the diketone 2 (860 mg, 2.20 mmol), concentrated sul-
furic acid (1.2 mL), and glacial acetic acid (15 mL) was
heated at reflux for 1 h and cooled to room temperature.
The mixture was poured onto crushed ice, and the
resulting precipitate was filtered to yield 3 (790 mg,
94.0% yield).
3. Conclusion
In conclusion, we reported the synthesis of a new series of
styrylchromone derivatives as amyloid imaging agents.
These styrylchromone derivatives displayed excellent
binding affinities for in vitro binding assay using synthetic
Ab(1–40) aggregates. In biodistribution studies using
normal mice, [125I]6 and [125I]9 showed a good initial
brain penetration and fast washout from the brain, desir-
able properties for an amyloid plaque-specific imaging
agent. However, the slow brain washout observed with
4.1.4. 6-Bromo-2-(40-aminostyryl)chromone (4). A mix-
ture of
3 (361 mg, 0.97 mmol), SnCl2 (1.28 g,
4.74 mmol), and EtOH (30 mL) was stirred under reflux
for 2 h. After the mixture was cooled to room tempera-
ture, 1 M NaOH (100 mL) was added until the mixture
became alkaline. After extraction with ethyl acetate
(100 mL), the combined organic layers was washed with
brine, dried over Na2SO4, and evaporated to gave
[
125I]12 in normal mice made it unsuitable for in vivo pla-
1
que imaging. Since styrylchromone backbone with high
binding affinity for amyloid plaques is very useful for
210 mg of 4 (63.3%). H NMR (300 MHz, CDCl3) d
3.99 (s, 2H), 6.25 (s, 1H), 6.55 (d, J = 15.9 Hz, 1H),

M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
449
6.68 (d, J = 8.4 Hz, 2H), 7.38–7.47 (m, 4H), 7.71 (d,
J = 8.1 Hz, 1H), 8.30 (d, J = 2.4 Hz, 1H).
(m, 3H), 7.94 (d, J = 15.6 Hz, 1H), 8.50 (d, J = 2.1 Hz,
1H). MS m/z 403 (M+).
4.1.5. 6-Tributylstannyl-2-(40-aminostyryl)chromone (5).
A mixture of 4 (242 mg, 0.71 mmol), bis(tributyltin)
(0.55 mL), and (Ph3P)4Pd (40 mg, 0.034 mmol) in a
mixed solvent (20 mL, 3:2 dioxane/triethylamine mix-
ture) was stirred at 90ꢁC for 6 h. The solvent was re-
moved, and the residue was purified by silica gel
chromatography (3:2 hexane/ethyl acetate) to give
4.1.10.
6-Bromo-2-(40-dimethylaminostyryl)chromone
(10). To a stirred mixture of 4 (194 mg, 0.57 mmol)
and paraformaldehyde (170 mg, 5.67 mmol) in AcOH
(10 mL) was added NaCNBH3 (214 mg, 3.40 mmol) in
one portion at room temperature. The resulting mixture
was stirred at room temperature for 3 h, 1 M NaOH
(50 mL) was added followed by extraction with CH3Cl
(50 mL). The organic phase was dried over Na2SO4.
The solvent was removed, and the residue was purified
by silica gel chromatography using chloroform to give
1
220 mg of 5 (56.2%). H NMR (300 MHz, CDCl3) d
0.86–1.54 (m, 27H), 4.07 (s, 2H), 6.27 (s, 1H), 6.57 (d,
J = 15.9 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H), 7.42 (d,
J = 9.0 Hz, 1H), 7.48–7.54 (m, 3H), 7.74 (d,
J = 8.1 Hz, 1H), 8.28 (s, 1H). MS m/z 496 (M+).
1
150 mg of 10 (71.5%). H NMR (300 MHz, CDCl3) d
3.04 (s, 6H), 6.24 (s, 1H), 6.57 (d, J = 15.9 Hz, 1H),
6.72 (d, J = 2.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 1H),
7.46–7.56 (m, 3H), 7.71 (d, J = 7.8 Hz, 1H), 8.31 (d,
J = 2.4 Hz, 1H). MS m/z 371 (MH+).
4.1.6. 6-Iodo-2-(40-aminostyryl)chromone (6). To a solu-
tion of 5 (220 mg, 0.40 mmol) in CHCl3 (5 mL) was add-
ed a solution of iodine in CHCl3 (3 mL, 0.25 M) at room
temperature. The mixture was stirred at room tempera-
ture for 10 min, and NaHSO3 solution (15 mL) was add-
ed. The mixture was stirred for 5 min, and the organic
phase was separated. The aqueous phase was extracted
with CH3Cl, and the combined organic phase was dried
over Na2SO4 and filtered. The solvent was removed, and
the residue was purified by silica gel chromatography
(2:1 hexane/ethyl acetate) to give 60 mg of 6 (38.7%).
1H NMR (300 MHz, CDCl3) d 3.99 (s, 2H), 6.56 (d,
J = 15.9 Hz, 1H), 6.69 (d, J = 8.4 Hz, 2H), 7.27 (d,
J = 8.7 Hz, 1H), 7.39–7.53 (m, 3H), 7.89 (m, 1H), 8.50
(d, J = 9.0 Hz, 1H). MS m/z 389 (M+).
4.1.11. 6-Tributylstannyl-2-(40-dimethylaminostyryl)chro-
mone (11). The same reaction as described above to pre-
pare 5 was employed, and 204 mg of 11 was obtained in
1
a 37.4% yield from 10. H NMR (300 MHz, CDCl3) d
0.86–1.74 (m, 27H), 3.04 (s, 6H), 6.27 (s, 1H), 6.54 (d,
J = 15.9 Hz, 1H), 6.69 (d, J = 8.7 Hz, 2H), 7.42 (m,
1H), 7.47–7.59 (m, 3H), 7.74 (d, J = 8.1 Hz, 1H), 8.23
(s, 1H). MS m/z 580 (M+).
4.1.12. 6-Iodo-2-(40-dimethyaminostyryl)chromone (12).
The same reaction as described above to prepare 6 was
used, and 18 mg of 12 was obtained in a 71.8% yield from
1
11. H NMR (300 MHz, CDCl3) d 3.04 (s, 6H), 6.25 (s,
4.1.7. 6-Bromo-2-(40-methylaminostyryl)chromone (7).
To a mixture of 4 (710 mg, 2.07 mmol) and paraformal-
dehyde (231 mg, 7.70 mmol) in MeOH (15 mL) was add-
ed a solution of NaOMe (28 wt% in MeOH, 0.45 mL)
dropwise. The mixture was stirred under reflux for 1 h.
After adding NaBH4 (270 mg, 7.13 mmol), the solution
was heated under reflux for 2 h. To the cold mixture
1 M NaOH (30 mL) was added followed by extraction
with CHCl2 (2· 30 mL). The organic phase was dried
over Na2SO4 and filtered. The solvent was removed,
and the residue was purified by silica gel chromatogra-
phy using chloroform to give 690 mg of 7 (96.0%).
1H NMR (300 MHz, CDCl3) d 2.89 (s, 3H), 4.18 (s,
1H), 6.25 (s, 1H), 6.56–6.61 (m, 3H), 7.29–7.45 (m,
4H), 7.71 (m,1H), 8.30 (d, J = 2.4 Hz, 1H). MS m/z
355 (M+).
1H), 6.56 (d, J = 15.9 Hz, 1H), 6.65 (d, J = 8.7 Hz, 2H),
7.23 (d, J = 8.1 Hz, 1H), 7.44–7.59 (m, 3H), 7.94 (d,
J = 8.7 Hz, 1H), 8.45 (s, 1H). MS m/z 417 (MH+).
4.2. Iododestannylation reaction
The radioiodinated forms of compounds 6, 9, and 12 were
prepared from the corresponding tributyltin derivatives
by an iododestannylation. Briefly, to initiate the reaction
50 lL H2O2 (3%) was added to a mixture of a tributyltin
derivative (50 lg/50 lL EtOH), [125I]NaI (0.1–0.2 mCi,
specific activity 2200 Ci/mmol), and 100 lL of 1 N HCl
in a sealed vial. The reaction was allowed to proceed at
room temperature for 10 min and terminated by addition
of NaHSO3. The reaction, after neutralization with sodi-
um bicarbonate, was extracted with ethyl acetate. The ex-
tract 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 sol-
vent of H2O/acetonitrile (3:7) at a flow rate of 1.0 mL/
min. The purified ligand was stored at ꢀ20 ꢁC for in vitro
binding and biodistribution studies.
4.1.8.
6-Tributylstannyl-2-(40-methylaminostyryl)chro-
mone (8). The same reaction as described above to pre-
pare 5 was employed, and 204 mg of 8 was obtained in
1
a 42.8% yield from 7. H NMR (300 MHz, CDCl3) d
0.86–1.52 (m, 27H), 2.89 (s, 3H), 4.11 (s, 1H), 6.26 (s,
1H), 6.57–6.62 (m, 3H), 7.43–7.54 (m, 4H), 7.74 (d,
J = 8.1 Hz, 1H), 8.28 (s, 1H). MS m/z 567 (MH+).
4.3. Binding assays using the aggregated Ab peptide in
solution
4.1.9. 6-Iodo-2-(40-methyaminostyryl)chromone (9). The
same reaction as described above to prepare 6 was used,
and 32 mg of 9 was obtained in a 22.4% yield from 8. 1H
NMR (300 MHz, CDCl3) d 2.94 (s, 3H), 4.21 (s, 1H),
6.25 (s, 1H), 6.54 (d, J = 15.6 Hz, 1H), 6.61 (d,
J = 8.7 Hz, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.42–7.59
A solid form of Ab(1–40) was purchased from Peptide
Institute (Osaka, Japan). Aggregation of peptides was
carried out by gently dissolving the peptide (0.5 mg/
mL) in a buffer solution (pH 7.4) containing 10 mM

450
M. Ono et al. / Bioorg. Med. Chem. 15 (2007) 444–450
sodium phosphate and 1 mM EDTA. The solutions
were incubated at 37 ꢁC for 42 h with gentle and con-
stant shaking. Binding studies were carried out in
12 · 75 mm borosilicate glass tubes according to the
procedure described before22 with some modification.
For saturation studies, a solution of [125I]6, [125I]9, and
Industrial Technology Development Organization
(NEDO) of Japan.
References and notes
[
125I]12 (final concentration, 1–125 nM) was prepared
1. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
2. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
3. Selkoe, D. J. Nat. Biotechnol. 2000, 18, 823.
4. Nordberg, A. Lancet Neurol. 2004, 3, 519.
5. Mathis, C. A.; Wang, Y.; Klunk, W. E. Curr. Pharm. Des.
2004, 10, 1469.
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. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.;
Debnath, M. L.; Klunk, W. E. J. Med. Chem. 2003, 46, 2740.
9. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.;
Blomqvist, G.; Holt, D. P.; Bergstorm, M.; Savitcheva, I.;
Huang, G. F.; Estrada, S.; Ausen, B.; Debnath, M. L.;
Barietta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall,
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Ann. Neurol. 2004, 55, 306.
10. 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.
11. Verhoeff, N. P.; Wilson, A. A.; Takeshita, S.; Trop, L.;
Hussey, D.; Singh, K.; Kung, M. P.; Kung, H. F. Am. J.
Geriatr. Psychiatry 2004, 12, 584.
12. Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.;
Plossl, K.; Hou, C.; Holtzman, D. M.; Kung, H. F. J.
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by mixing nonradioactive 6, 9, and12. Nonspecific bind-
ing was defined in the presence of 1 lM nonradioac-
tive12. The binding assay was performed by mixing
50 lL Ab(1–40) aggregates (57 nM in the final assay
mixture), an appropriate concentration of 50 lL of
[
125I]6, [125I]9, and [125I]12, and 900 lL of 10% ethanol.
After incubation for 3 h at room temperature, the bind-
ing mixture was filtered through GF/B filters (Whatman,
Kent, UK) using a M-24 cell harvester (Brandel, Gai-
thersburg, MD). Filters containing the bound 125I ligand
were counted in a gamma camera counter. The dissoci-
ation constant (Kd) of compounds 6, 9, and 12 was
determined by Scatchard analysis using GraphPad
Prism 4.0 (GraphPad Software, San Diego, CA).
4.4. Partition coefficient determination
Partition coefficients were measured by mixing the radi-
oiodinated tracers with 3 mL each of 1-octanol and buff-
er (0.1 M phosphate, pH 7.4) in a test tube. The test tube
was vortexed for 3 min at room temperature, followed
by centrifugation for 5 min. Two weighed samples
(0.5 mL each) from the 1-octanol and buffer layers were
counted in a well counter. The partition coefficient was
determined by calculating the ratio of cpm/0.5 mL of
the 1-octanol to that of buffer. A sample from the octa-
nol layer was re-partitioned with the same volume of
buffer until consistent partition coefficient values were
obtained. The measurement was done in triplicate and
repeated three times.
14. Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.;
Arano, Y.; Mori, H.; Nakayama, M. J. Med. Chem. 2005,
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4.5. In vivo biodistribution in normal mice
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Skovronsky, D.; Trojanowski, J. Q.; Lee, V. M.; Kung,
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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 ethanol (10 lL) of radiolabeled
agents (0.5 lCi) was injected directly into the tail vein of
ddY mice (5-week-old, 22–25 g). The mice were sacrificed
at various time points post injection. The organs of inter-
est were removed and weighed, and the radioactivity was
counted with an automatic gamma counter (Aloka 3000).
21. Kung, M. P.; Hou, C.; Zhuang, Z. P.; Cross, A. J.; Maier,
D. L.; Kung, H. F. Eur. J. Nucl. Med. Mol. Imaging 2004,
31, 1136.
Acknowledgment
22. Zhuang, Z. P.; Kung, M. P.; Hou, C.; Skovronsky, D. M.;
Gur, T. L.; Plossl, K.; Trojanowski, J. Q.; Lee, V. M.;
Kung, H. F. J. Med. Chem. 2001, 44, 1905.
This study was supported by Industrial Technology Re-
search Grant Program in 2005 from New Energy and