Development of novel β-amyloid probes based on 3,5-diphenyl-1,2,4-oxadiazole

Article abstract of DOI:10.1016/j.bmc.2008.05.054

In the search for novel probes for the imaging in vivo of β-amyloid plaques in Alzheimer's disease (AD) brain, we have synthesized and evaluated a series of 3,5-diphenyl-1,2,4-oxadiazole (DPOD) derivatives. The affinity for β-amyloid plaques was assessed

Full text of DOI:10.1016/j.bmc.2008.05.054

Bioorganic & Medicinal Chemistry 16 (2008) 6867–6872
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel b-amyloid probes based on 3,5-diphenyl-1,2,4-oxadiazole
a
b
a
Masahiro Ono a,b, , Mamoru Haratake , Hideo Saji , Morio Nakayama
*
a Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Simoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for novel probes for the imaging in vivo of b-amyloid plaques in Alzheimer’s disease (AD)
brain, we have synthesized and evaluated a series of 3,5-diphenyl-1,2,4-oxadiazole (DPOD) derivatives.
The affinity for b-amyloid plaques was assessed by an in vitro-binding assay using pre-formed synthetic
Ab42 aggregates. The new series of DPOD derivatives showed excellent affinity for Ab aggregates with Ki
values ranging from 4 to 47 nM. In biodistribution experiments using normal mice, [125I]12, [125I]13,
Received 1 May 2008
Revised 23 May 2008
Accepted 24 May 2008
Available online 29 May 2008
[
125I]14, and [125I]15 examined displayed sufficient uptake for imaging, ranging from 2.2 to 3.3% ID/g.
Keywords:
Alzheimer’s disease
b-Amyloid
PET
But the washout of the four ligands from the brain was relatively slow. Although additional modifications
are necessary to improve the uptake and rapid clearance of non-specifically bound radiotracers, the DPOD
pharmacophore with high-binding affinity for Ab aggregates may be useful as a backbone structure to
develop novel b-amyloid imaging agents.
SPECT
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, a number of groups have reported new b-amyloid-
binding probes without the basic structures of DDNP, thioflavin-T
Alzheimer’s disease (AD) is the most common neurodegenera-
tive disorder of the elderly and is characterized clinically by
dementia, cognitive impairment, and memory loss. The neuro-
pathological hallmarks of AD include abundant deposits of b-amy-
loid plaques and neurofibrillary tangles. The deposition of b-
amyloid plaques has been regarded as an initial event in the path-
ogenesis of AD.1,2 Therefore, the quantitative evaluation of b-amy-
loid plaques in the brain with non-invasive techniques such as
positron emission tomography (PET) and single photon emission
computed tomography (SPECT) could lead to the presymptomatic
detection of AD and new anti-amyloid therapies.3–5
and Congo Red. Most of these probes have two aromatic rings.
Among them, 1,4-diphenyltriazole and 2,5-diphenylthiophene
derivatives have triazole and thiophene between two benzene
rings, respectively, and it has been shown that they have tolerance
for binding to Ab aggregates.15,16 In an attempt to further develop
novel ligands for the imaging of amyloid plaques in AD, we de-
signed a series of 3,5-diphenyl-1,2,4-oxadiazole (DPOO) deriva-
tives, in which triazole or thiophene of the 1,4-diphenyltriazole
and 2,5-diphenylthiophene backbone was replaced with 1,2,4-oxa-
diazole. To our knowledge, this is the first time the use of DPOD
derivatives in vivo as probes to image b-amyloid plaques in the
AD brain has been proposed. Described herein is the synthesis of
a novel series of DPOD derivatives and the characterization as b-
amyloid imaging agents.
Developing b-amyloid imaging probes is currently an emerging
field of research. The basic requirements for suitable probes in-
clude (i) good penetration of the blood–brain barrier, (ii) selec-
tively binding or labeling b-amyloid plaques, and (iii) displaying
clear and contrasting signals between plaques and non-plaques.
Based on these requirements, several promising agents with the
backbone structure of DDNP, thioflavin-T, and Congo Red have
been synthesized and evaluated for use in vivo as probes to image
b-amyloid plaques in AD brain. Clinical trials in AD patients have
2. Results and discussion
The synthesis of the DPOD derivatives is outlined in Schemes 1
and 2. A wide variety of reaction conditions have been published
for the synthesis of 3,5-diphenyl-1,2,4-oxadiazoles.17 Among them,
we used the reaction of an amidoxime with a carboxylic acid to
form 1,2,4-oxadiazoles. In this process, the 3,5-diphenyl-1,2,4,-
oxadiazoles (1 and 5) were prepared by the condensation of 4-bro-
mobenzamidoxime with 4-nitrobenzoic acid and 4-methoxyben-
zoic acid in the presence of DCC and HOBT. The amino derivative
2 was readily prepared from 1 by reduction with SnCl2 (80.8%
yield). Conversion of 2 to the monomethylamino derivative 3
was achieved by a method reported previously18 (50.3% yield).
been conducted with several agents including
[
18F]FDDNP,6,7
18F]BAY94-9172,12 and
123I]IMPY13,14 (Fig. 1) indicating the imaging of b-amyloid plaques
[
[
11C]6-OH-BTA-1,8,9
[
11C]SB-13,10,11
[
in the living human brain to be useful for the diagnosis of AD.
* Corresponding author. Tel.: +81 75 753 4608; fax: +81 75 753 4568.
E-mail address: ono@pharm.kyoto-u.ac.jp (M. Ono).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.054

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M. Ono et al. / Bioorg. Med. Chem. 16 (2008) 6867–6872
Figure 1. Neuropathological staining of compound 14 on 10-lm AD model mouse sections. (A) Many b-amyloid plaques were clearly stained with 14. (B) The serial sections
were immunostained using an antibody against b-amyloid.
OH
N
N
O
HOOC
a
+
N
NH2
NO2
Br
Br
NO2
Br
1
N O
N
N
O
b
c
N
N
H
Br
NH2
d
3
2
N O
N
Br
N
4
Scheme 1. (a) DCC, HOBT, DMF; (b) SnCl2, EtOH; (c) NaOMe, (CH2O)n, NaBH4, MeOH; (d) (CH2O)n, NaCNBH3, AcOH.
OH
N
O
N O
N
HOOC
a
b
+
N
5
N
6
NH2
OMe
Br
OMe
Br
OH
Br
Scheme 2. (a) DCC, HOBT, DMF; (b) BBr3, CH2Cl2.
Compound 2 was also converted to the dimethylamino derivative 4
by an efficient method19 with paraformaldehyde and acetic acid
(68.4% yield). Compound 5 was converted to 6 by demethylation
with BBr3 in CH2Cl2 (50.6% yield). The tributyltin derivatives (7,
8, 9, 10, and 11) were prepared from the corresponding bromo
compounds (2, 3, 4, 5, and 6) using a bromo to tributyltin exchange
reaction catalyzed by Pd(0) for yields of 16.8%, 15.8%, 20.3%, 22.8%,
and 60.1%, respectively. These tributyltin derivatives were readily
reacted with iodine in chloroform at room temperature to give
the iodo derivatives (12, 13, 14, 15, and 16) for yields of 66.1%,
86.0%, 72.4%, 69.2%, and 81.8%. Furthermore, these tributyltin
derivatives can be also used as the starting materials for radioio-
dination in the preparation of [125I]12, [125I]13, [125I]14, and
iodinated products were obtained in 50–70% radiochemical yields
with a radiochemical purity of >95% after HPLC.
The affinity of DPOD derivatives (compounds 12, 13, 14, 15, and
16) was evaluated based on inhibition of the binding of [125I]IMPY
to Ab(1–42) aggregates. As shown in Table 1, all the derivatives
competed well with [125I]IMPY to bind the aggregates. The Ki val-
ues estimated for 12, 13, 14, 15, and 16 were 14, 14, 15, 4, and
47 nM, respectively. The introduction into the DPOD backbone of
an aminophenyl moiety, such as aminophenyl, methylaminophe-
nyl or dimethylaminophenyl, resulted in good affinity. Further
modification of the aminophenyl moiety with another electron-
donating group, such as methoxyphenyl or hydroxyphenyl, re-
sulted in a difference in binding to Ab aggregates, with the meth-
oxy derivative 15 having 10-fold higher affinity than the hydroxy
derivative 16.
To confirm the affinity of DPOD derivatives for b-amyloid pla-
ques in the AD brain, fluorescent staining of sections of mouse
brain from an animal model of AD was carried out with compound
14 (Fig. 1). Many b-amyloid plaques were clearly stained with
compound 14, as reflected by the affinity for Ab aggregates in
in vitro competition assays. The labeling pattern was consistent
with that observed by immunohistochemical labeling with an anti-
body specific for Ab, indicating that DPOD derivatives show specific
binding to b-amyloid plaques. Thus, the results suggest that DPOD
[
125I]15. Novel radioiodinated DPOD derivatives were achieved
by an iododestannylation reaction using hydrogen peroxide as
the oxidant which produced the desired radioiodinated ligands
(Scheme 3). It was anticipated that the no-carrier-added prepara-
tion would result in a final product bearing a theoretical specific
activity similar to that of 125I (2200 Ci/mmol). The radiochemical
identities of the radioiodinated ligands were verified by co-injec-
tion with non-radioactive compounds from their HPLC profiles.
The final radioiodinated compounds, [125I]12, [125I]13, [125I]14,
and [125I]15, showed a single peak of radioactivity at a retention
time of 8.4, 15.7, 25.5, 22.3, and 7.1 min, respectively. Four radio-

M. Ono et al. / Bioorg. Med. Chem. 16 (2008) 6867–6872
6869
N
O
12 (R = NH2)
13 (R = NHMe)
14 (R = NMe2)
15 (R = OMe)
R
16 (R = OH)
N
O
N
O
a
b
N
N
N
Bu3Sn
7 (R = NH2)
8 (R = NHMe)
9 (R = NMe2)
10 (R = OMe)
11 (R = OH)
R
I
Br
R
7-11
2 (R = NH2)
3 (R = NHMe)
4 (R = NMe2)
5 (R = OMe)
6 (R = OH)
[
[
[
[
125I]12 (R = NH2)
125I]13 (R = NHMe)
125I]14 (R = NMe2)
125I]15 (R = OMe)
N
O
c
N
125I
R
Scheme 3. (Bu3Sn)2, Pd(PhP3P), Et3N, dioxane; (b) I2, CHCl3; (c) [125I]NaI, H2O2, HCl.
Table 1
Table 2
Inhibition constants for the binding of DPOD derivatives determined using [125I]IMPY
Biodistribution of radioactivity after intravenous administration of [125I]12, [125I]13,
as the ligand in Ab(1–42) aggregates
[
125I]14, and [125I]15 in micea
Compound
Kia (nM)
Tissue
Time after injection (min)
10 30
12
13
14
15
16
14.2 1.4
14.3 3.6
15.4 1.4
4.3 2.1
2
60
[
[
[
[
125I]12
Blood
Liver
4.55 (0.64)
2.32 (0.63)
17.37 (1.72)
6.94 (0.70)
3.08 (0.47)
8.87 (2.35)
3.88 (0.54)
4.02 (0.48)
1.70 (0.18)
2.48 (0.16)
3.09 (0.22)
2.99 (0.31)
21.36 (3.13)
8.24 (1.26)
1.50 (0.36)
6.13 (1.67)
3.69 (2.30)
9.29 (1.66)
0.92 (0.31)
1.61 (0.23)
14.89 (1.47)
5.86 (0.56)
5.84 (0.75)
6.89 (0.81)
3.36 (0.36)
3.48 (0.24)
4.05 (0.96)
3.32 (0.31)
12.33 (2.19)
5.15 (0.74)
8.33 (1.39)
5.02 (0.55)
2.83 (0.32)
3.51 (0.61)
5.15 (1.32)
3.29 (0.58)
47.1 4.1
Kidney
Intestine
Spleen
Pancreas
Heart
a
Values are
means standard error of the mean for 3–6 independent
experiments.
Stomachb
Brain
derivatives may be applicable to the in vivo imaging of b-amyloid
plaques in the brain.
125I]13
Blood
Liver
3.54 (0.26)
14.62 (1.30)
9.53 (0.83)
1.36 (0.21)
4.58 (0.62)
3.34 (0.38)
9.51 (0.77)
1.08 (0.11)
1.44 (0.12)
1.84 (0.24)
12.37 (2.09)
5.30 (0.94)
2.83 (0.93)
4.70 (0.48)
3.91 (0.60)
3.05 (0.50)
2.32 (0.62)
1.98 (0.36)
1.52 (0.13)
8.53 (0.98)
3.39 (0.66)
5.67 (1.44)
2.84 (0.35)
3.06 (0.58)
1.68 (0.21)
4.89 (0.99)
2.56 (0.44)
1.54 (0.09)
7.30 (0.78)
3.02 (0.58)
8.46 (1.36)
2.14 (0.22)
1.88 (0.25)
1.27 (0.20)
7.38 (0.97)
2.70 (0.33)
To evaluate the uptake of DPOD derivatives in the brain, biodis-
tribution experiments in normal mice were performed with four
radioiodinated DPOD derivatives ([125I]12, [125I]13, [125I]14, and
Kidney
Intestine
Spleen
Pancreas
Heart
[
125I]15), which showed strong affinity for Ab aggregates in the
in vitro-binding assays (Table 2). All four ligands penetrated the
brain well with a delayed peak time at 30 and 60 min for uptakes
(2.3–3.3%ID/g brain) in normal mice. But as a prerequisite for an
imaging agent, they should be washed out from the normal brain,
because there is no trapping mechanism for DPOD derivatives.
Nevertheless, the long retention of these probes in the normal
mouse brain suggested extensive non-specific binding which will
contribute to a high level of background noise in vivo. Previously,
we reported radioiodinated benzofuran derivatives as potential
b-amyloid-binding agents.20 The radioiodinated benzofurans pene-
trated the brain well with a peak uptake at 30–60 min postinjec-
tion, but a slow washout in normal mice prevents these probes
from being used for imaging in vivo. More recently, we developed
11C-labeled benzofuran derivatives, which are less lipophilic by
Stomachb
Brain
125I]14
Blood
Liver
Kidney
Intestine
Spleen
Pancreas
Heart
5.42 (0.85)
13.94 (3.26)
7.83 (3.53)
1.59 (0.28)
6.26 (1.53)
3.17 (0.73)
11.04 (2.43)
1.28 (0.21)
1.07 (0.23)
2.41 (0.22)
8.27 (1.49)
7.67 (1.62)
2.40 (0.36)
4.56 (1.11)
3.80 (0.50)
4.81 (1.07)
3.28 (0.36)
1.45 (0.27)
1.79 (0.24)
5.89 (1.75)
4.81 (1.01)
4.12 (0.99)
3.11 (0.62)
2.86 (1.64)
2.20 (0.54)
4.80 (0.56)
2.23 (0.61)
1.56 (0.27)
5.15 (1.92)
3.31 (0.77)
5.37 (1.01)
2.41 (0.29)
2.47 (0.62)
1.24 (0.68)
5.75 (2.47)
2.32 (0.64)
Stomachb
Brain
125I]15
Blood
1.96 (0.66)
11.94 (1.92)
8.77 (1.17)
1.70 (0.27)
4.41 (0.72)
4.55 (0.77)
11.30 (1.92)
0.62 (0.21)
2.06 (0.45)
1.63 (0.14)
9.35 (1.17)
6.22 (0.87)
3.53 (0.32)
3.62 (0.28)
4.94 (0.26)
4.44 (0.67)
1.64 (0.10)
2.76 (0.23)
1.23 (0.39)
6.13 (0.76)
3.85 (1.01)
6.62 (0.90)
2.13 (0.53)
2.84 (0.48)
1.84 (0.32)
2.46 (0.80)
2.82 (0.34)
1.12 (0.26)
4.44 (0.60)
2.95 (0.66)
8.21 (1.84)
1.34 (0.40)
1.45 (0.28)
1.35 (0.29)
3.16 (1.39)
2.01 (0.33)
Liver
replacing the iodine with a hydroxy group.21 11C]benzofuran
[
Kidney
Intestine
Spleen
Pancreas
Heart
showed a higher and faster peak of brain uptake and a faster wash-
out from the brain in the normal mice. The improved properties
in vivo observed with [11C]benzofuran make it a better candidate
for the imaging of b-amyloid plaques. Indeed, the initial result ob-
tained with [11C]benzofuran in a transgenic2576 mouse showed a
relatively high S/N ratio.21 Similar to [125I]benzofuran, the DPOD
derivatives had unfavorable in vivo pharmacokinetics in normal
mice, despite their good affinity for Ab aggregates. Additional
structural changes, that is, reducing the lipophilicity by introduc-
ing a hydrophilic group, are necessary to improve the in vivo prop-
erties of the DPOD derivatives.
Stomachb
Brain
a
Expressed as % injected dose per gram. Each value represents the mean (s.d.) for
3–5 animals at each interval.
b
Expressed as % injected dose per organ.
gree to which the DPOD derivatives penetrated the brain was also
very encouraging. However, non-specific binding in vivo reflected
by a slow washout from the normal mouse brain makes them
unsuitable for the imaging of b-amyloid plaques. The less than
ideal in vivo biodistribution results in normal mice indicate that
there is a critical need to fine-tune the kinetics of brain uptake
and washout. Additional changes to the DPOD pharmacopore with
3. Conclusion
In conclusion, we successfully designed and synthesized a new
series of DPOD derivatives as probes for the in vivo imaging of b-
amyloid plaques in the brain. The derivatives displayed excellent
affinity for Ab aggregates in in vitro-binding experiments. The de-

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M. Ono et al. / Bioorg. Med. Chem. 16 (2008) 6867–6872
high-binding affinity for Ab aggregates may lead to useful probes
for detecting b-amyloid plaques in the AD brain.
4.1.5. 3-(4-Bromophenyl)-5-(4-methoxyphenyl)-1,2,4-
oxadiazole (5)
The same reaction as described above to prepare 1 was used,
and 153 mg of 5 was obtained in a 23.1% yield from 4-bro-
mobenzamidoxime and 4-anisic acid. 1H NMR (300 MHz, CDCl3)
d 3.91 (s, 3H), 7.05 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H),
8.04 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 9.0 Hz, 2H). MS m/z 330
(M+).
4. Experimental
4.1. General information
All reagents were commercial products and used without fur-
ther purification unless otherwise indicated. 1H NMR spectra were
obtained on a Varian Gemini 300 spectrometer with TMS as an
internal standard. Coupling constants are reported in Hertz. Multi-
plicity is defined by s (singlet), d (doublet), t (triplet), br (broad),
and m (multiplet). Mass spectra were obtained on a JEOL IMS-DX
instrument.
4.1.6. 4-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)phenol (6)
BBr3 (4.5 mL, 1 M solution in CH2Cl2) was added to a solution of
5 (300 mg, 0.91 mmol) in CH2Cl2 (10 mL) dropwise in an ice bath.
The mixture was allowed to warm to room temperature and stirred
for 42 h. Water (30 mL) was added while the reaction mixture was
cooled in an ice bath. The mixture was extracted with chloroform
(30 mL ꢀ 2) and the organic phase was dried over Na2SO4 and fil-
tered. The solvent was removed and the residue was purified by
silica gel chromatography (hexane/ethyl acetate = 4:1) to give
4.1.1. 3-(4-Bromophenyl)-5-(4-nitrophenyl)-1,2,4-oxadiazole
(1)
To a stirring solution of 4-bromobenzamidoxime (645 mg,
3 mmol) and 4-nitrobenzoic acid (495 mg, 3 mmol) in DMF
(10 mL) was added a solution of DCC (3.6 mmol) and HOBT
(6.0 mmol) in DMF (5 mL). The reaction mixture was stirred at
room temperature for 18 h, and then at 100 °C for 2 h. The solvent
was removed, and the residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate = 9:1) to give 370 mg of 1 (35.6%). 1H
NMR (300 MHz, CDCl3) d 7.70 (d, J = 8.7 Hz, 2H), 8.06 (d, J = 8.7 Hz,
2H), and 8.43 (s, 4H). MS m/z 346 (M+).
146 mg of
6 d 6.99 (d,
(50.6%). 1H NMR (300 MHz, CDCl3)
J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 8.1 Hz, 2H), and
8.12 (d, J = 9.0 Hz, 2H). MS m/z 316 (M+).
4.1.7. 4-(3-(4-(Tributylstannyl)phenyl)-1,2,4-oxadiazol-5-
yl)aniline (7)
A mixture of 2 (100 mg, 0.32 mmol), bis(tributyltin) (0.2 mL)
and (Ph3P)4Pd (16 mg, 0.014 mmol) in a mixed solvent (10 mL,
3:2 dioxane/triethylamine mixture) was stirred for 10 h under re-
flux. The solvent was removed, and the residue was purified by
silica gel chromatography (hexane/ethyl acetate = 3:1) to give
28 mg of 7 (16.8%). 1H NMR (300 MHz, CDCl3) d 0.87–1.61 (m,
27H), 4.13 (s, 2H), 6.76 (d, J = 8.7 Hz, 2H), 7.59 (d,J = 8.1 Hz,
2H), 8.01 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.1 Hz, 2H), and 8.28 (s,
1H).
4.1.2. 4-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)aniline (2)
A mixture of 1 (350 mg, 1 mmol), SnCl2 (948 mg, 5 mmol) and
EtOH (15 mL) was stirred under reflux for 2 h. After the mixture
had cooled to room temperature, 1 M NaOH (100 mL) was added
until the mixture became alkaline. After extraction with ethyl ace-
tate (100 mL ꢀ 2), the combined organic layer was washed with
brine, dried over Na2SO4, and evaporated to give 258 mg of 2
(80.8%). 1H NMR (300 MHz, CDCl3)
d 4.16 (s, 2H), 6.75 (d,
4.1.8. N-Methyl-4-(3-(4-(tributylstannyl)phenyl)-1,2,4-
oxadiazol-5-yl)aniline (8)
J = 8.7 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 9.0 Hz, 2H), and
8.03 (d, J = 6.3 Hz, 2H). MS m/z 316 (M+).
The same reaction as described above to prepare 7 was em-
ployed, and 23 mg of 8 was obtained in a 15.8% yield from 3. 1H
NMR (300 MHz, CDCl3) d 0.87–1.63 (m, 27H), 2.92 (s, 3H), 4.27
(s, 1H), 6.66 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 8.03 (d,
J = 8.4 Hz, 2H), and 8.08 (d, J = 7.8 Hz, 2H).
4.1.3. 4-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)-N-
methylaniline (3)
A solution of NaOCH3 (28 wt% in MeOH, 0.4 mL) was added to a
mixture of 2 (185 mg, 0.59 mmol) and paraformaldehyde (176 mg,
0.59 mmol) in methanol (10 mL) dropwise. The mixture was stir-
red under reflux for 30 min. After NaBH4 (225 mg, 5.9 mmol) was
added, the solution was heated under reflux for 1.5 h. 1 M NaOH
(50 mL) was added to the cold mixture and extracted with CHCl3
(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 acetate = 4:1) to give 98 mg of
3 (50.3%). 1H NMR (300 MHz, CDCl3) d 2.93 (s, 3H), 4.30 (s, 1H),
6.66 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 8.7 Hz,
2H), and 8.03 (d, J = 8.7 Hz, 2H). MS m/z 330 (M+).
4.1.9. N,N-Dimethyl-4-(3-(4-(tributylstannyl)phenyl)-1,2,4-
oxadiazol-5-yl)aniline (9)
The same reaction as described above to prepare 7 was em-
ployed, and 45 mg of 9 was obtained in a 20.3% yield from 4. 1H
NMR (300 MHz, CDCl3) d 0.87–1.58 (m, 27H), 3.09 (s, 6H), 6.76
(d, J = 9.6 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H),
and 8.08 (d, J = 8.4 Hz, 2H).
4.1.10. 5-(4-Methoxyphenyl)-3-(4-(tributylstannyl)phenyl)-
1,2,4-oxadiazole (10)
The same reaction as described above to prepare 7 was em-
ployed, and 42 mg of 10 was obtained in a 22.8% yield from 5. 1H
NMR (300 MHz, CDCl3) d 0.87–1.59 (m, 27H), 3.91 (s, 3H), 7.04
(d, J = 8.7 Hz, 2H), 7.61 (d, J = 7.8 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H),
and 8.17 (d, J = 9.0 Hz, 2H).
4.1.4. 4-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)-N,N-
dimethylaniline (4)
To a stirred mixture of 2 (35 mg, 0.10 mmol) and paraformalde-
hyde (36 mg, 1.2 mmol) in AcOH (5 mL) was added NaCNBH3
(50 mg, 0.80 mmol) in one portion at room temperature. The
resulting mixture was stirred at room temperature for 2 h. After
1 M NaOH (30 mL) was added and extraction with CH3Cl (30 mL),
the organic phase was dried over Na2SO4. The solvent was removed
and the residue was purified by silica gel chromatography (hexane/
ethyl acetate = 4:1) to give 24 mg of 4 (68.4%). 1H NMR (300 MHz,
CDCl3) d 3.09 (s, 6H), 6.75 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H),
8.35 (d, J = 8.7 Hz, 2H), and 8.43 (d, J = 8.7 Hz, 2H). MS m/z 344.
4.1.11. 4-(3-(4-(Tributylstannyl)phenyl)-1,2,4-oxadiazol-5-
yl)phenol (11)
The same reaction as described above to prepare 7 was em-
ployed, and 28 mg of 11 was obtained in a 60.1% yield from 6. 1H
NMR (300 MHz, CDCl3) d 0.87–1.58 (m, 27H), 6.99 (d, J = 9.0 Hz,
2H), 7.61 (d, J = 7.8 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H), and 8.12 (d,
J = 8.7 Hz, 2H).

M. Ono et al. / Bioorg. Med. Chem. 16 (2008) 6867–6872
6871
4.1.12. 4-(3-(4-Iodophenyl)-1,2,4-oxadiazol-5-yl)aniline (12)
To a solution of 7 (27 mg, 0.05 mmol) in CHCl3 (5 mL) was
added a solution of iodine in CHCl3 (1 mL, 50 mg/mL) at room tem-
perature. The mixture was stirred at room temperature for 10 min
and a saturated NaHSO3 solution (25 mL) was added. The mixture
was stirred for 5 min and the organic phase was separated. The
aqueous phase was extracted with CH3Cl (25 mL ꢀ 2), and the
combined organic phase was dried over Na2SO4 and filtered. The
solvent was removed and the residue was washed with hexane
to give 12 mg of 12 (66.1%). 1H NMR (300 MHz, CDCl3) d 4.15 (s,
2H), 6.76 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.89 (d,
J = 8.4 Hz, 2H), and 8.01 (d, J = 8.7 Hz, 2H). HRMS m/z C14H10N3OI
found 362.9855/ calcd 362.9869 (M+).
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 solu-
tions were incubated at 37 °C for 42 h with gentle and constant
shaking. Binding experiments were carried out as described previ-
ously.22
[
125I]IMPY
(6-iodo-2-(40-dimethylamino)phenyl-imi-
dazo[1,2]pyridine) with 2200 Ci/mmol specific activity and
greater than 95% radiochemical purity was prepared using the
standard iododestannylation reaction as described previously.13
Binding assays were carried out in 12 ꢀ 75 mm borosilicate glass
tubes. The reaction mixture contained 50
l
L of Ab(1-42) aggre-
L of [125I]IMPY
L of inhibitor (8 pM-12.5
lL of 10% EtOH. Non-specific binding
4.1.13. 4-(3-(4-Iodophenyl)-1,2,4-oxadiazol-5-yl)-N-
methylaniline (13)
gates (29 nM in the final assay mixture), 50
(0.02 nM diluted in 10% EtOH), 50
diluted in 10% EtOH), and 850
l
l
lM
The same reaction as described above to prepare 12 was em-
ployed and 20 mg of 13 was obtained in a 86.0% yield from 8. 1H
NMR (300 MHz, CDCl3) d 2.93 (s, 3H), 4.29 (s, 1H), 6.66 (d,
J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), and
8.02 (d, J = 8.4 Hz, 2H). HRMS m/z C15H12N3OI found 377.0022/
calcd 377.0025 (M+).
was defined in the presence of 400 nM IMPY in the same assay
tubes. The mixture was incubated at 37 °C for 3 h and the bound
and the free radioactivities were separated by vacuum filtration
through Whatman GF/B filters using a Brandel M-24R cell har-
vester followed by 2 ꢀ 3 mL washes with 10% EtOH at room tem-
perature. Filters containing the bound I-125 ligand were placed
in a gamma counter (Aloka, ARC-380) for measuring radioactivity.
Under the assay conditions, the specifically bound fraction ac-
counted for less than 15% of the total radioactivity. Values for the
half-maximal inhibitory concentration (IC50) were determined
from displacement curves using GraphPad Prism 4.0 (GraphPad
Software, San Diego, CA), and those for the inhibition constant
(Ki) were calculated using the Cheng-Prusoff equation.23
4.1.14. 4-(3-(4-Iodophenyl)-1,2,4-oxadiazol-5-yl)-N,N-
dimethylaniline (14)
The same reaction as described above to prepare 12 was em-
ployed and 34 mg of 14 was obtained in a 72.4% yield from 9.
1H NMR (300 MHz, CDCl3) d 3.09 (s, 6H), 6.75 (d, J = 9.3 Hz,
2H), 7.83 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 8.7 Hz, 2H), and 8.04
(d, J = 9.3 Hz, 2H). HRMS m/z C16H14N3OI found 391.0192/ calcd
391.0182 (M+).
4.4. Neuropathological staining of model mouse brain sections
4.1.15. 3-(4-Iodophenyl)-5-(4-methoxyphenyl)-1,2,4-
oxadiazole (15)
The Tg2576 transgenic mice (female, 23-month-old) were used as
Alzheimer’s model mice. After the mice were sacrificed by decapita-
tion, the brains were immediately removed and frozen in powdered
The same reaction as described above to prepare 12 was em-
ployed and 17 mg of 15 was obtained in a 69.2% yield from 10.
1H NMR (300 MHz, CDCl3) d 3.89 (s, 3H), 6.97 (d, J = 9.3 Hz, 2H),
7.48 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 9.3 Hz, 2H), and 7.85 (d,
J = 9.0 Hz, 2H). HRMS m/z C15H11N2O2I found 377.9865/ calcd
377.9872 (M+).
dry ice. The frozen blocks were sliced into serial sections, 10-
thick. Each slide was incubated with a 50% ethanol solution
(100 M) of compound 14. The sections were washed in 50% ethanol
lm
l
for 3 min two times. Fluorescent observation was performed with the
Nikon system. Thereafter, the serial sections were also immuno-
stained with DAB as a chromogen using monoclonal antibodies
against b-amyloid (Amyloid b-Protein Immunohistostain kit, WAKO).
4.1.16. 4-(3-(4-Iodophenyl)-1,2,4-oxadiazol-5-yl)phenol (16)
The same reaction as described above to prepare 12 was
employed and 14 mg of 16 was obtained in
a 81.8% yield
from 11. 1H NMR (300 MHz, CDCl3) d 6.99 (d, J = 8.7 Hz, 2H),
7.87 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.1 Hz, 2H), and 8.12 (d,
J = 8.1 Hz, 2H). HRMS m/z C14H9N2O2I found 363.9704/ calcd
363.9709 (M+).
4.5. In vivo biodistribution in normal mice
The experiments with animals were conducted in accordance
with our institutional guidelines and were approved by Nagasaki
University Animal Care Committee. A saline solution (100
lL) con-
taining ethanol (10 L) of radiolabeled agents (0.5
l
lCi) was
4.2. Iododestannylation reaction
injected intravenously 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 interest were removed and
weighed, and the radioactivity was measured with an automatic
gamma counter (Aloka, ARC-380).
The radioiodinated forms of compounds 12, 13, 14, and 15 were
prepared from the corresponding tributyltin derivatives by iodo-
destannylation. Briefly, to initiate the reaction, 50
was added to a mixture of a tributyltin derivative (100
EtOH), [125I]NaI (0.1–0.2 mCi, specific activity 2200 Ci/mmol), and
100 L of 1 N HCl in a sealed vial. The reaction was allowed to pro-
lL of H2O2 (3%)
lg/50
lL
Acknowledgments
l
ceed at room temperature for 10 min and terminated by addition
of NaHSO3. After neutralization with sodium bicarbonate and
extraction with ethyl acetate, the extract was dried by passing it
through an anhydrous Na2SO4 column and then blown dry 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 (3/7) at a flow rate of 1.0 mL/min. The purified li-
gand was stored at ꢁ20 °C for the in vitro binding and biodistribu-
tion experiments.
This study was supported by the Program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO) and Health Labour Sciences Re-
search Grant.
References and notes
1. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
2. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.

6872
M. Ono et al. / Bioorg. Med. Chem. 16 (2008) 6867–6872
Krause, S.; Friebe, M.; Lehman, L.; Lindemann, S.; Dinkelborg, L. M.; Masters, C.
3. Selkoe, D. J. Nat. Biotechnol. 2000, 18, 823.
4. Nordberg, A. Lancet Neurol. 2004, 3, 519.
L.; Villemagne, V. L. Lancet Neurol. 2008, 7, 129.
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.;
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.
13. Kung, M.; 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,
78.
15. Qu, W.; Kung, M. P.; Hou, C.; Oya, S.; Kung, H. F. J. Med. Chem. 2007, 50,
3380.
16. Chandra, R.; Kung, M. P.; Kung, H. F. Bioorg. Med. Chem. Lett. 2006, 16, 1350.
17. Santagada, V.; Frecentese, F.; Perissutti, E.; Cirillo, D.; Terracciano, S.; Caliendo,
G. Bioorg. Med. Chem. Lett. 2004, 14, 4491.
18. Barluenga, A. M.; Bayron, G.; Asensio, A. J. Chem. Soc., Chem. Commun. 1984,
1334.
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.
19. Gribble, G. W.; Nutaitis, C. F. Synthesis 1987, 709.
20. Ono, M.; Kung, M. P.; Hou, C.; Kung, H. F. Nucl. Med. Biol. 2002, 29, 633.
21. Ono, M.; Kawashima, H.; Nonaka, A.; Kawai, T.; Haratake, M.; Mori, H.; Kung,
M.-P.; Kung, H. F.; Saji, H.; Nakayama, M. J. Med. Chem. 2006, 49, 2725.
22. Kung, M. P.; Hou, C.; Zhuang, Z. P.; Skovronsky, D.; Kung, H. F. Brain Res. 2004,
1025, 98.
11. 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.
12. 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.;
23. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.