Synthesis and biological evaluation of radioiodinated 2,5-diphenyl-1,3,4-oxadiazoles for detecting β-amyloid plaques in the brain

Article abstract of DOI:10.1016/j.bmc.2009.07.020

This paper describes the synthesis and biological evaluation of a new series of 2,5-diphenyl-1,3,4-oxadiazole (1,3,4-DPOD) derivatives for detecting β-amyloid plaques in Alzheimer's brains. The affinity for β-amyloid plaques was assessed by an in vitro bi

Full text of DOI:10.1016/j.bmc.2009.07.020

Bioorganic & Medicinal Chemistry 17 (2009) 6402–6406
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of radioiodinated 2,5-diphenyl-1,3,4-oxadi-
azoles for detecting b-amyloid plaques in the brain
a
a
b
Hiroyuki Watanabe a, Masahiro Ono a,b, , Ryoichi Ikeoka , Mamoru Haratake , Hideo Saji ,
*
Morio Nakayama a,
*
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 Shimoadachi-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:
This paper describes the synthesis and biological evaluation of a new series of 2,5-diphenyl-1,3,4-oxadi-
azole (1,3,4-DPOD) derivatives for detecting b-amyloid plaques in Alzheimer’s brains. The affinity for b-
amyloid plaques was assessed by an in vitro binding assay using pre-formed synthetic Ab42 aggregates.
The new series of 1,3,4-DPOD derivatives showed affinity for Ab42 aggregates with Ki values ranging from
20 to 349 nM. The 1,3,4-DPOD derivatives clearly stained b-amyloid plaques in an animal model of Alz-
heimer’s disease, reflecting the affinity for Ab42 aggregates in vitro. Compared to 3,5-diphenyl-1,2,4-oxa-
diazole (1,2,4-DPOD) derivatives, they displayed good penetration of and fast washout from the brain in
biodistribution experiments using normal mice. The novel radioiodinated 1,3,4-DPOD derivatives may be
useful probes for detecting b-amyloid plaques in the Alzheimer’s brain.
Received 2 June 2009
Revised 10 July 2009
Accepted 11 July 2009
Available online 17 July 2009
Keywords:
Alzheimer’s disease
b-Amyloid plaques
SPECT imaging
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, we successfully designed and synthesized a new ser-
ies of 3,5-diphenyl-1,2,4-oxadiazole (1,2,4-DPOD) derivatives as
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder pathologically characterized by the deposition of b-amy-
loid (Ab) peptides as senile plaques in the brain.1,2 Since the depo-
sition of Ab plaques is an early event in the development of AD, a
validated biomarker of Ab deposition in the brain would likely
prove useful for identifying and following individuals at risk for
AD and assist in the evaluation of new anti-amyloid therapies cur-
rently under development. Therefore, the quantitative evaluation
of Ab 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
SPECT probes for the in vivo imaging of Ab plaques in the brain.19
The 1,2,4-DPOD derivatives are categorized into Ab-imaging agents
with the three-aromatic-ring linked system.20–22 The derivatives
displayed excellent affinity for Ab aggregates in in vitro binding
experiments. The degree to which the DPOD derivatives penetrated
the brain was also very encouraging. However, nonspecific binding
in vivo reflected by a slow washout from the normal mouse brain
makes them unsuitable for the imaging of Ab 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 structural changes, that is, reducing the
lipophilicity, are necessary to improve the in vivo properties of
the DPOD derivatives.
In the past few years, several groups have reported potential
Ab-imaging probes for the detection of Ab plaques in vivo. Tracers
In an attempt to further develop novel ligands for the imaging
of Ab plaques in AD, we designed a series of 2,5-diphenyl-1,3,4-
oxadiazole (1,3,4-DPOD) derivatives, which are structural isomers
of 1,2,4-DPOD and less lipophilic than 1,2,4-DPOD (Fig. 1). To our
such as
[ [ [ [
11C]PIB,6,7 11C]SB-13,8,9 18F]BAY94-9172,10 11C]BF-
227,11
[
18F]FDDNP,12–14 and [123I]IMPY15–18 have been tested clin-
ically and demonstrated utility. [123I]IMPY is the only tracer for
SPECT, the other five tracers are Ab-imaging probes for PET. Since
SPECT is more valuable than PET in terms of routine diagnostic
use, the development of more useful Ab-imaging agents for SPECT
has been a critical issue.
N
O
N N
N
O
I
R1
I
R2
1,3,4-DPOD
1,2,4-DPOD
* Corresponding authors. Tel.: +81 75 753 4608; fax: +81 75 753 4568 (M.O.); tel./
fax: +81 95 819 2441 (M.N.).
E-mail addresses: ono@pharm.kyoto-u.ac.jp (M. Ono), morio@nagasaki-u.ac.jp
(M. Nakayama).
Figure 1. Chemical structure of 1,2,4-DPOD reported previously and 1,3,4-DPOD
reported in this paper. R1 = NH2, NHCH3, N(CH3)2, OCH3, OH; R2 = N(CH3)2, OCH3,
OH, OCH2CH2OH, (OCH2CH2)2OH, (OCH2CH2)3OH.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.020

H. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 6402–6406
6403
knowledge, this is the first time the use of 1,3,4-DPOD derivatives
in vivo as probes to image Ab plaques in the AD brain has been pro-
posed. Described herein is the synthesis of a novel series of 1,3,4-
DPOD derivatives and the characterization as Ab-imaging probes
in comparison with 1,2,4-DPOD derivatives.
tively, while no marked affinity was observed for 6–9. Compound
displayed almost equal affinity for Ab aggregates as the
1,2,4-DPOD derivative with the dimethylamino group
(4-(3-(4-iodophenyl)-1,2,4-oxadiazole-5-yl)-N,N-dimethylamine;
1,2,4-DPOD-DM, Ki = 15 nM).19
3
To confirm the affinity of 1,3,4-DPOD derivatives for Ab plaques
in the brain, fluorescent staining of sections of mouse brain from
an animal model of AD was carried out with compound 3
(Fig. 2). Many fluorescence spots were observed in the brain sec-
tions of Tg2576 transgenic mice (female, 28-month-old) (Fig. 2A),
while no spots were observed in the brain sections of wild-type
mice (female, 28-month-old) (Fig. 2B). The fluorescent labeling
pattern was consistent with that observed with thioflavin S
(Fig. 2C). These results suggested that 3 shows specific binding to
Ab plaques in the mouse brain. In the fluorescent staining of
Tg2576 mouse brain sections, 4 also showed specific binding to
Ab plaques in the brain (data not shown).
2. Results and discussion
The synthesis of 1,3,4-DPOD derivatives is outlined in Schemes
1 and 2. We used the one-pot synthesis method of producing 2,5-
diphenyl-1,3,4-oxadiazoles.23 The 2,5-diphenyl-1,3,4-oxadiazoles
(3 and 4) were prepared by 4-iodobenzhydrazide with 4-dimethyl-
aminobenzaldehyde and 4-methoxybenzaldehyde in the presence
of ceric ammonium nitrate (CAN). Compound 4 was converted to
6 by demethylation with BBr3 in CH2Cl2 (49% yield). Direct alkyl-
ation of 6 with ethylene chlorohydrin, ethylene glycol mono-2-
chloroethyl ether, or 2-[2-(2-chloroethoxy)ethoxy]ethanol with
potassium carbonate in DMF resulted in 7–9. The tributyltin deriv-
atives (2 and 5) were prepared from corresponding compounds (1
and 4) using a halogen to tributyltin exchange reaction catalyzed
by Pd(0) for yields of 8.2% and 6.5%, respectively. The tributyltin
derivatives were used as the starting materials for radioiodination
in the preparation of [125I]3 and [125I]4. Novel radioiodinated 1,3,4-
DPOD derivatives were obtained by an iododestannylation reaction
using hydrogen peroxide as the oxidant which produced the de-
sired radioiodinated ligands (Scheme 3). It was anticipated 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 the radioiodinated
ligands was verified by co-injection with non-radioiodinated com-
pounds from their HPLC profiles. [125I]3 and [125I]4 were each ob-
tained in a radiochemical yield of >45% with a radiochemical
purity of >95% after purification by HPLC.
Next, [125I]3 and [125I]4 were evaluated for their in vivo biodis-
tribution in normal mice. A biodistribution study provides critical
information on brain penetration. Generally, a freely diffusible
compound with an optimal log P value of 2À3 will have an initial
brain uptake of 2À3% dose/whole brain at 2 min after iv injection.
[
125I]3 and [125I]4 examined in this study displayed optimal lipo-
philicity as reflected by log P values of 2.43 and 2.58, respectively
(Table 2). As expected, these ligands displayed good brain uptake
ranging from 3.8% to 5.9% ID/g brain at 2–10 min postinjection,
indicating a level sufficient for brain imaging probes (Table 3). In
addition, they displayed good clearance from the normal brain
with 1.8% and 0.36% ID/g at 60 min postinjection for [125I]3 and
[
125I]4, respectively. These values were equal to a peak in brain up-
take of 30% and 9.6%, respectively. Additionally, all of the other or-
gans or tissues displayed a good initial uptake and a relatively fast
washout with time. To directly compare the brain uptake and
washout of [125I]1,2,4-DPOD and [125I]1,3,4-DPOD, a combined plot
is presented in Figure 3. It is apparent that [125I]1,2,4-DPOD
showed a lower initial uptake with a longer retention, while
The affinity of 1,3,4-DPOD derivatives (3, 4, 6–9) was evaluated
based on inhibition of the binding of [125I]IMPY to Ab42 aggregates.
As shown in Table 1, all 1,3,4-DPOD derivatives showed inhibitory
activity toward Ab aggregates. The affinity of 1,3,4-DPOD deriva-
tives for Ab aggregates varied from 20 to 349 nM. Compound 3
with the dimethylamino group and 4 with the methoxy group
showed high binding affinity with a Ki of 20 and 46 nM, respec-
[
125I]1,3,4-DPOD displayed a higher initial uptake but with a faster
washout from the brain. At 2 or 10 min after iv injection, the up-
take of [125I]1,3,4-DPOD reached a maximum after which the activ-
ity in the normal brain was washed out. It is important to note that
(Ph3P)4Pd
(Bu3Sn)2
N
N
N
N
O
CAN
+
OHC
N
R
C
O
O
NHNH2
dioxane/Et3N
R
N
Bu3Sn
N
1 : R = Br
3 : R = I
2
R = Br or I
Scheme 1.
(Ph3P)4Pd
(Bu3Sn)2
N
N
N
N
O
C
NHNH2
CAN
+
OHC
OMe
I
O
O
I
OMe dioxane/Et3N
Bu3Sn
OMe
4
5
BBr3
HO
Cl
n
N
N
N
N
O
6
OH
n
O
I
OH
K2CO3
DMF
I
O
7 (n = 1)
8 (n = 2)
9 (n = 3)
Scheme 2.

6404
H. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 6402–6406
N
N
N
N
H2O2
O
Bu3Sn
R
O
125I
R
HCl
125I]NaI
[
[
[
125I]3 (R = NMe2)
125I]4 (R = OMe)
2 (R = NMe2)
5 (R = OMe)
Scheme 3.
Table 1
Table 2
Inhibition constants (Ki) for binding of 1,3,4-DPOD derivatives determined using
[
Partition coefficients for 1,2,4-DPOD and 1,3,4-DPOD derivatives
125I]IMPY as the ligand in Ab42 aggregates
Compound
Log Pa
Compound
Kia (nM)
3
2.43 0.07
2.58 0.06
3.22 0.01
3.37 0.04
3
4
6
7
8
9
20.1 2.5
46.1 12.6
229.6 47.3
282.2 61.4
348.6 51.7
257.7 34.8
4
1,2,4-DPOD-DM
1,2,4-DPOD-OMe
a
Octanol/buffer (0.1 M phosphate-buffered saline, pH 7.4) partition coefficients.
Each value represents the mean SD for 2–3 experiments.
a
Values are the mean standard error for the mean for 4–6 independent
experiments.
Table 3
Biodistribution of radioactivity after injection of
[
125I]1,3,4-DPOD derivatives in
normal micea
the ideal Ab-imaging agent should have good brain penetration to
deliver the intended dose into the brain, while maintaining a fast
washout from normal tissues. Because the normal brain has no
Ab plaques to trap the agent, the washout from the brain should
also be fast. Once the high affinity ligand is delivered into the re-
gions containing the Ab plaques, imaging agents such as [125I]3
and [125I]4 are expected to be trapped in this region longer due
to its high binding affinity. The differences between the kinetics
in normal and Ab plaque-containing regions will result in a higher
signal to noise ratio (target to non-target ratio) in the AD brain.
Based on the data presented for [125I]1,3,4-DPOD, it is predicted
that the brain trapping of [125I]1,3,4-DPOD in Ab-containing re-
gions will be much better than that of [125I]1,2,4-DPOD. The log P
values of 1,3,4-DPOD derivatives (log P = 2.43 and 2.58 for 3 and
4, respectively) were lower than those of 1,2,4-DPOD derivatives
(log P = 3.22 and 3.37 for 1,2,4-DPOD-DM and 1,2,4-DPOD-OMe,
respectively). Although many factors such as molecular size, ionic
charge, and lipophilicity affect the uptake of a compound into
the brain, the difference in lipophilicity may be one reason for
the difference in brain uptake and washout between 1,3,4-DPOD
and 1,2,4-DPOD. Further structural modifications, that is, decreas-
ing the lipophilicity by introducing the hydrophilic group, should
improve the in vivo properties of 1,3,4-DPOD derivatives.
Tissue
Time after injection (min)
10 30
2
60
[
125I]3
Blood
Liver
3.28 (0.46)
3.51 (0.29)
19.12 (2.43)
7.80 (0.64)
11.34 (1.61)
4.10 (0.40)
4.39 (2.17)
3.55 (1.86)
4.73 (1.86)
5.93 (0.76)
2.53 (0.28)
2.21 (0.41)
15.87 (3.49)
9.14 (1.60)
2.28 (0.55)
3.56 (0.96)
5.32 (0.98)
3.99 (3.10)
1.40 (0.10)
2.98 (0.53)
12.64 (2.44)
5.71 (1.35)
12.58 (2.35)
2.63 (0.54)
2.50 (0.56)
2.03 (0.30)
4.79 (1.12)
3.16 (0.69)
10.01 (1.64)
3.81 (0.64)
16.22 (2.51)
2.05 (0.41)
2.14 (0.90)
1.54 (0.31)
5.50 (0.51)
1.78 (0.41)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
[
125I]4
Blood
Liver
1.84 (0.30)
9.60 (1.73)
7.14 (1.46)
2.07 (0.36)
2.44 (0.28)
4.74 (0.63)
4.80 (1.33)
0.71 (0.13)
3.75 (0.78)
1.60 (0.30)
12.60 (1.14)
4.85 (0.48)
4.49 (0.60)
1.51 (0.26)
1.98 (0.37)
1.73 (0.27)
1.41 (0.91)
2.74 (0.37)
1.26 (0.26)
8.07 (1.66)
4.36 (1.12)
10.06 (1.81)
0.80 (0.10)
0.96 (0.03)
0.92 (0.25)
3.12 (0.90)
1.04 (0.14)
0.80 (0.20)
4.65 (1.34)
2.48 (0.37)
19.85 (4.71)
0.60 (0.26)
0.57 (0.14)
0.43 (0.11)
2.90 (1.57)
0.36 (0.13)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
a
Expressed as % injection dose per gram. Each value represents the mean (SD) for
4–6 animals.
b
Expressed as % injected dose per organ.
3. Conclusion
1,3,4-DPOD derivatives showed affinity for Ab42 aggregates. The
1,3,4-DPOD derivatives clearly stained Ab plaques in an animal
model of AD, reflecting their affinity for Ab aggregates in vitro.
Compared to 1,2,4-DPOD, they displayed good penetration of and
a fast washout from the brain in biodistribution experiments using
In conclusion, we successfully designed and synthesized a new
series of 1,3,4-DPOD derivatives as probes for the in vivo imaging
of Ab plaques in the brain. In in vitro binding experiments, these
Figure 2. Neuropathological staining of compound 3 on 10-lm AD model mouse sections (A) and wild-type mouse sections (B). Labeled plaques were confirmed by staining
of the adjacent sections with thioflavin S (C).

H. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 6402–6406
6405
4H), 7.97 (d, J = 3.0 Hz, 2H). 13C NMR (400 MHz, CDCl3) d 40.1,
97.8, 110.7, 111.6, 123.9, 128.0, 128.4, 138.2, 152.5, 162.9, 165.5.
HRMS m/z C16H14N3OI found 391.0191/ calcd 391.0182 (M+).
7
6
5
4
3
2
1
0
[
[
[
[
125I]3
125I]4
125I]1,2,4-DPOD-DM
125I]1,2,4-DPOD-OMe
4.1.4. 2-(4-Iodophenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole
(4)
The same reaction as described above to prepare 1 was used,
and 40 mg of 4 was obtained in a 8.8% yield from 4-iodobenzohyd-
razide and 4-methoxybenzaldehyde. 1H NMR (300 MHz, CDCl3) d
3.89 (s, 3H), 7.03 (d, J = 2.9 Hz, 2H), 7.86 (q, J = 7.8 Hz, 4H), 8.03
(d, J = 3.0 Hz, 2H). 13C NMR (400 MHz, CDCl3) d 55.5, 98.2, 114.6,
116.2, 123.6, 128.1, 128.8, 138.3, 162.5, 163.6, 164.7. HRMS m/z
C15H11N2O2I found 377.9877, calcd 377.9865 (M+).
0
10
20
30
40
50
60
Time after injection (min)
4.1.5. 2-(4-(Tributylstannyl)phenyl)-5-(4-methoxyphenyl)-
1,3,4-oxadiazole (5)
The same reaction as described above to prepare 2 was used,
and 6 mg of 5 was obtained in a 6.5% yield from 4. 1H NMR
(300 MHz, CDCl3) d 0.87–1.58 (m, 27H), 3.91 (s, 3H), 7.04 (d,
J = 3.1 Hz, 2H), 7.63 (d, J = 2.6 Hz, 2H), 8.06 (q, J = 6.6 Hz, 4H).
Figure 3. Comparison of brain uptake of [125I]3, [125I]4, [125I]1,2,4-DPOD-DM and
[
[
125I]1,2,4-DPOD-OMe in normal mice. The kinetics of the uptake of [125I]3 and
125I]4 may provide a better pattern for the localization of Ab plaques in the brain.
normal mice. Taken together, the present results suggested that
the novel radioiodinated 1,3,4-DPOD derivatives may be useful
probes for detecting Ab plaques in the AD brain.
4.1.6. 4-(5-(4-Iodophenyl)-1,3,4-oxadiazol-2-yl)phenol (6)
BBr3 (0.6 mL, 1 M solution in CH2Cl2) was added to a solution of
4 (36 mg, 0.1 mmol) in CH2Cl2 (16 mL) dropwise in an ice bath. The
mixture was allowed to warm to room temperature and stirred for
5 days. Water (50 mL) was added while the reaction mixture was
cooled in an ice bath. The mixture was extracted with CHCl3
(30 mL) and the water layer was extracted with ethyl acetate.
The organic phase was dried over Na2SO4 and filtered. The solvent
was removed, and the residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate = 2:1) to give 17 mg of 6 (49.0%). 1H
NMR (300 MHz, CDCl3) d 6.98–7.06 (m, 2H), 7.86–7.91 (m, 4H),
8.02–8.09 (m, 2H). HRMS m/z C14H9N2O2I found 363.9712, calcd
363.9709 (M+).
4. Experimental
4.1. General
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), and m (mul-
tiplet). 13C NMR spectra were obtained on an AL400 JEOL
spectrometer with TMS as an internal standard. Mass spectra were
obtained on a JEOL IMS-DX.
4.1.7. 2-(4-(5-(4-Iodophenyl)-1,3,4-oxadiazol-2-
yl)phenoxy)ethanol (7)
4.1.1. 4-(5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl)-N,N-
dimethylbenzenamine (1)
A
mixture of
6
(22 mg, 0.06 mmol), potassium carbonate
L, 0.06 mmol)
(24.5 mg, 0.18 mmol) and ethylene chlorohydrin (4
l
To a solution of 4-bromobenzhydrazide (215 mg, 1 mmol) and
4-dimethylaminobenzaldehyde (149 mg, 1 mmol) in dry CH2Cl2
(10 mL) was added CAN (548 mg, 1 mmol). The reaction mixture
was stirred under reflux for 24 h. Water was added and following
extraction with CHCl3, 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 12 mg of
1 (3.5%). 1H NMR (300 MHz, CDCl3) d 3.08 (s, 6H), 6.76 (d,
J = 9.0 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.98 (dd, J = 5.4, 4.5 Hz,
4H). MS m/z 362 (M+).
in anhydrous DMF (3 mL) was stirred under reflux for 6.5 h. After
cooling to room temperature, water was added, and the reaction
mixture was extracted with CHCl3. The organic layer was sepa-
rated, dried over Na2SO4 and evaporated. The resulting residue
was purified by silica gel chromatography (hexane/ethyl ace-
tate = 2:3) to give 11 mg of 7 (44.6%). 1H NMR (300 MHz, CDCl3)
d 4.03 (q, J = 5.0 Hz, 2H), 4.18 (d, J = 3.0 Hz, 2H) 7.06 (d, J = 3.0 Hz,
2H), 7.87 (q, J = 8.0 Hz, 4H), 8.07 (d, J = 3.0 Hz, 2H). HRMS m/z
C16H13N2O3I found 407.9983, calcd 407.9971 (M+).
4.1.8. 2-(2-(4-(5-(4-Iodophenyl)-1,3,4-oxadiazol-2-
yl)phenoxy)ethoxy)ethanol (8)
4.1.2. 4-(5-(4-Tributylstannyl)phenyl)-1,3,4-oxadiazol-2-yl)-
N,N-dimethylbenzenamine (2)
The same reaction as described above to prepare 7 was used,
and 9 mg of 8 was obtained in a 25.9% yield from 6 and ethylene
glycol mono-2-chloroethyl ether. 1H NMR (300 MHz, CDCl3) d
3.70 (t, J = 3.1 Hz, 2H), 3.79 (q, J = 4.8 Hz, 2H), 3.92 (t, J = 3.2 Hz,
2H), 4.23 (t, J = 3.1 Hz, 2H), 7.06 (d, J = 3.1 Hz, 2H), 7.87 (q,
J = 7.6 Hz, 4H), 8.70 (d, J = 3.1 Hz, 2H). HRMS m/z C18H17N2O4I
found 452.0244, calcd 452.0233 (M+).
A mixture of 1 (19 mg, 0.06 mmol), bis(tributyltin) (0.04 mL)
and (Ph3P)4Pd (3 mg, 0.002 mmol) in a mixed solvent (6 mL, 1:1
dioxane/Et3N) was stirred under reflux for 4.5 h. The solvent was
removed, and the residue was purified by silica gel chromatogra-
phy (hexane/ethyl acetate = 3:1) to give 2.5 mg of 2 (8.2%). 1H
NMR (300 MHz, CDCl3) d 0.87–1.6 (m, 27H), 3.07 (s, 6H), 6.77 (d,
J = 9.0 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 8.01 (dd, J = 9.0, 8.1 Hz, 4H).
4.1.9. 2-(2-(2-(4-(5-(4-Iodophenyl)-1,3,4-oxadiazol-2-
yl)phenoxy)ethoxy)ethoxy)ethanol (9)
4.1.3. 4-(5-(Iodophenyl)-1,3,4-oxadiazol-2-yl)-N,N-
dimethylbenzenamine (3)
The same reaction as described above to prepare 7 was used,
and 7.8 mg of 9 was obtained in a 44.7% yield from 6 and 2-[2-
(chloroethoxy)ethoxy]ethanol. 1H NMR (300 MHz, CDCl3) d 3.61–
3.77 (m, 8H), 3.91 (t, J = 3.1 Hz), 4.23 (t, J = 3.2 Hz, 2H), 7.06 (d,
The same reaction as described above to prepare 1 was used,
and 14 mg of 3 was obtained in a 1.8% yield from 4-iodobenzohyd-
razide and 4-dimethylaminobenzaldehyde. 1H NMR (300 MHz,
CDCl3) d 3.07 (s, 6H), 6.76 (d, J = 3.0 Hz, 2H), 7.85 (d, J = 12.0 Hz,

6406
H. Watanabe et al. / Bioorg. Med. Chem. 17 (2009) 6402–6406
J = 3.0 Hz, 2H), 7.87 (q, J = 7.8 Hz, 4H), 8.06 (d, J = 2.3 Hz, 2H). HRMS
m/z C20H21N2O5I found 496.0525, calcd 496.0495 (M+).
4.5. Determination of partition coefficient determination
Partition coefficients were measured by mixing [125I]3 and
[
125I]4 with 1.5 mL each of 1-octanol and buffer (0.1 M phosphate,
4.2. Iododestannylation reaction
pH 7.4) in a test tube. The test tube was vortexed for 20 s three
times. Two weighed samples (1 mL each) from the 1-octanol and
buffer layers were measured for radioactivity with a gamma coun-
ter. The partition coefficient was determined by calculating the ra-
tio of cpm/1 mL of 1-octanol to that of the buffer.
The radioiodinated forms of compounds 3 and 4 were prepared
from the corresponding tributyltin derivatives by iododestannyla-
tion. Briefly, to initiate the reaction, 50
to mixture of tributyltin derivative (50
125I]NaI (0.1–0.2 mCi, specific activity 2200 Ci/mmol), and 50
lL
lL of H2O2 (3%) was added
a
a
l
g/50 EtOH),
lL
[
4.6. In vivo biodistribution in normal mice
of 1 N HCl in a sealed vial. The reaction was allowed to proceed
at room temperature for 3 min and terminated by addition of NaH-
SO3. After neutralization with sodium biocarbonate and extraction
with ethyl acetate, the extract was dried by passing 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
(4:6) at a flow rate of 1.0 mL/min.
A saline solution (100
lL) of radiolabeled agents (0.2–0.4 lCi)
containing ethanol (10 L) was injected intravenously directly into
l
the tail of ddY mice (5-week-old, 22–25 g). The mice were sacri-
ficed at various time points postinjection. The organs of interest
were removed and weighed, and radioactivity was measured with
an automatic gamma counter (Aloka, ARC-380).
Acknowledgments
4.3. Binding assays using the aggregated Ab peptide in solution
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 a Health Labour Sciences Re-
search Grant.
A solid form of Ab42 was purchased from Peptide Institute
(Osaka, Japan). Aggregation was carried out by gently dissolving
the peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was incu-
bated at 37 °C for 42 h with gentle and constant shaking. Binding
References and notes
assays were carried out as described previously.24
[
125I]IMPY
1. Klunk, W. E. Neurobiol. Aging 1998, 19, 145.
2. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
(6-iodo-2-(4’-dimethylamino)phenyl-imidazo[1,2]pyridine) with
2200 Ci/mmol specific activity and greater than 95% radiochemical
purity was prepared using the standard iododestannylation reac-
tion as described previously.15 Binding assays were carried out in
3. Mathis, C. A.; Lopresti, B. J.; Klunk, W. E. Nucl. Med. Biol. 2007, 34, 809.
4. Mathis, C. A.; Wang, Y.; Klunk, W. E. Curr. Pharm. Des. 2004, 10, 1469.
5. Nordberg, A. Lancet Neurol. 2004, 3, 519.
6. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.; Debnath, M. L.; Klunk, W. E. J.
Med. Chem. 2003, 46, 2740.
7. 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.
12 Â 75 mm borosilicate glass tubes. A mixture containing 50
of test compound (0.2 pM–400 in 10% EtOH), 50
125I]IMPY (0.02 nM diluted in 10%EtOH), 50
L of Ab42 aggregates,
L of 10% ethanol was incubated at room temperature for
lL
lM
lL of
[
l
and 850
l
3 h. The mixture was then filtered through Whatman GF/B filters
using a Brandel M-24 cell harvester, and the filters containing
the bound 125I ligand were placed in a gamma counter (Aloka,
ARC-380). Values for the half-maximal inhibitory concentration
(IC50) were determined from displacement curves of three inde-
pendent experiments using GraphPad Prism 4.0, and those for
the inhibition constant (Ki) were calculated using the Cheng–Prus-
off equation.25
8. Ono, M.; Wilson, A.; Norbrega, J.; Westaway, D.; Verhoeff, P.; Zhuang, Z. P.;
Kung, M. P.; Kung, H. F. Nucl. Med. Biol. 2003, 30, 565.
9. 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.
10. Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan, R.; Pike, K. L.; O’Keefe, G.;
Tochon-Danguy, H.; Chan, G.; Berlangieri, S. U.; Jones, G.; Dickinson-Rowe, K.
L.; Kung, H. P.; Zhang, W.; Kung, M. P.; Skovronsky, D.; Dyrks, T.; Holl, G.;
Krause, S.; Friebe, M.; Lehman, L.; Lindemann, S.; Dinkelborg, L. M.; Masters, C.
L.; Villemagne, V. L. Lancet Neurol. 2008, 7, 129.
11. Kudo, Y.; Okamura, N.; Furumoto, S.; Tashiro, M.; Furukawa, K.; Maruyama, M.;
Itoh, M.; Iwata, R.; Yanai, K.; Arai, H. J. Nucl. Med. 2007, 48, 553.
12. Agdeppa, E. D.; Kepe, V.; Liu, J.; Flores-Torres, S.; Satyamurthy, N.; Petric, A.;
Cole, G. M.; Small, C. W.; Huang, S. C.; Barrio, J. R. J. Neurosci. 2001, 21, RC189.
13. 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.
14. 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.
15. 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.
16. Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plossl, K.; Hou, C.; Holtzman,
D. M.; Kung, H. F. J. Med. Chem. 2003, 46, 237.
17. 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.
18. 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, 78.
19. Ono, M.; Haratake, M.; Saji, H.; Nakayama, M. Bioorg. Med. Chem. 2008, 16, 6867.
20. Neaterov, E. E.; Skoch, J.; Hyman, B. T.; Klunk, W. E.; Bacskai, B. J.; Swager, T. M.
Angew. Chem. Int. Ed. 2005, 44, 5452.
21. Chabdra, R.; Kung, M. P.; Kung, H. F. Bioorg. Med. Chem. Lett. 2006, 16, 1350.
22. Qu, W.; Kung, M. P.; Hou, C.; Oya, S.; Kung, H. F. J. Med. Chem. 2007, 50, 3380.
23. Dabiti, M.; Salehi, P.; Baghbanzadeh, M.; Bahramnejad, M. Tetrahedron Lett.
2006, 47, 6983.
4.4. Neuropathological staining of model mouse brain sections
The experiments with animals were conducted in accordance
with our institutional guidelines and were approved by Nagasaki
University Animal Care Committee. The Tg2576 transgenic mice
(female, 28-month-old) and wild-type mice (female, 28-month-
old) were used as the Alzheimer’s model and control mice, respec-
tively. After the mice were sacrificed by decapitation, the brains
were immediately removed and frozen in powdered dry ice. The
frozen blocks were sliced into serial sections, 10
lm thick. Each
slide was incubated with a 50% EtOH solution (100
l
M) of com-
pounds 3 and 4 for 30 min. The sections were washed in 50% EtOH
for 1 min two times, and examined using a microscope (Nikon
Eclipse 80i) equipped with a UV-1A filter set (excitation, 365–
375 nm; diachronic mirror, 400 nm; longpass filter, 400 nm).
Thereafter, the serial sections were also stained with thioflavin S,
a pathological dye commonly used for staining Ab plaques in the
brain, and examined using a microscope (Nikon Eclipse 80i)
equipped with a B-2A filter set (excitation, 450–480 nm; dia-
chronic mirror, 505 nm; longpass filter, 520 nm).
24. Kung, M. P.; Hou, C.; Zhuang, Z. P.; Skovronsky, D.; Kung, H. F. Brain Res. 2004,
1025, 98.
25. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.