Diphenylpropynone derivatives as probes for imaging β-amyloid plaques in Alzheimer's brains

Article abstract of DOI:10.1016/j.bmcl.2010.11.058

A new series of diphenylpropynone (DPP) derivatives for use in vivo to image β-amyloid (Aβ) plaques in the brain of patients with Alzheimer's disease (AD) were synthesized and characterized. Binding experiments in vitro revealed high affinity for Aβ (1-42

Full text of DOI:10.1016/j.bmcl.2010.11.058

Bioorganic & Medicinal Chemistry Letters 21 (2011) 117–120
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diphenylpropynone derivatives as probes for imaging b-amyloid plaques
in Alzheimer’s brains
Masahiro Ono a,b, , Hiroyuki Watanabe a,b, Rumi Watanabe b, Mamoru Haratake b, Morio Nakayama b,
⇑
⇑
,
Hideo Saji a
a Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2010
Revised 9 November 2010
Accepted 13 November 2010
Available online 23 November 2010
A new series of diphenylpropynone (DPP) derivatives for use in vivo to image b-amyloid (Ab) plaques in
the brain of patients with Alzheimer’s disease (AD) were synthesized and characterized. Binding exper-
iments in vitro revealed high affinity for Ab (1–42) aggregates at a Ki value ranging from 6 to 326 nM.
Furthermore, specific labeling of plaques was observed in sections of brain tissue from Tg2576 transgenic
mice stained using one of the compounds, 1. In biodistribution experiments with normal mice, [125I]1 dis-
played moderate uptake (1.55% ID/g at 2 min) and clearance from the brain with time (0.76 ID/g at
60 min). Taken together, DPP can serve as a new molecular scaffold for developing novel Ab imaging
agents by introducing appropriate substituted groups.
Keywords:
Alzheimer’s disease
b-Amyloid plaque
Positron emission tomography (PET)
Single photon emission computed
tomography (SPECT)
Ó 2010 Elsevier Ltd. All rights reserved.
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 (Ab) plaques and neurofibrillary tangles. The deposition of Ab
plaques has been regarded as an initial event in the pathogenesis of
AD.1,2 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 tomogra-
phy (SPECT) could lead to the presymptomatic detection of AD and
new anti-amyloid therapies.3–5
[
18F]AZD469420 indicating the imaging of Ab plaques in the living hu-
man brain to be useful for the diagnosis of AD.
Recently, to develop more useful PET/SPECT probes, a number of
groups have reported new Ab-binding probes without the basic
structure of DDNP, thioflavin-T and Congo Red. Kung et al. reported
several diphenylacetylenes as PET/SPECT probes for Ab plaques,
which are a simplified version of stilbene derivatives; the double
bond in the stilbene derivative is replaced by a triple bond
(Fig. 1).21–23 Two reasons prompted them to investigate this class
of compound. The first reason was that the compounds can be
synthesized by a Sonogashira reaction, which is tolerant of a wide
variety of functional groups. The second and more important reason
Developing Ab imaging probes is currently an emerging field of
research. The basic requirements for suitable probes include: (i) good
penetration of the blood–brain barrier, (ii) selectively binding or
labeling of Ab plaques, and (iii) 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 Ab plaques in AD brain. Clinical trials in AD
patients have been conducted with several agents including
R
R
R'
R'
Stilbene deriv.
O
Diphenylacetylene deriv.
O
[
18F]FDDNP,6,7
[
11C]6-OH-BTA-1,8,9
[
11C]SB-13,10,11
[
18F]BAY94-
9172,12,13
[
123I]IMPY,14,15 18F]AV-45,16–18
[
[
11C]AZD2184,19 and
R
R
R'
R'
⇑
Corresponding authors. Tel.: +81 75 753 4608; fax: +81 75 753 4568 (M.O.);
tel./fax: +81 95 819 2441 (M.N.).
Chalcone deriv.
Diphenylpropynone deriv.
E-mail addresses: ono@pharm.kyoto-u.ac.jp (M. Ono), morio@nagasaki-u.ac.jp
(M. Nakayama).
Figure 1. Chemical structure of the stilbene, diphenylacetylene, chalcone, and
diphenylpropynone derivatives.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.058

118
M. Ono et al. / Bioorg. Med. Chem. Lett. 21 (2011) 117–120
O
O
Pd(OAc)2
N
Cl
+
Et3N
R
R
R = I, F
N
1 : R = I
2 : R = F
Scheme 1.
O
O
Pd(OAc)2
OMe
Cl
+
I
Et3N
I
I
OMe
OH
3
O
BBr3
4
Scheme 2.
O
O
5
Pd(OAc)2
Et3N
Cl
+
N
Br
Br
N
O
O
Dioxane
(Bu3Sn)2
Na125
I
H2O2
HCl
(Ph3P4)Pd
Et3N
125
Bu3Sn
I
N
N
[
125I]1
6
Scheme 3.
is the absence of geometrical isomers, a problem often encountered
with their double-bonded counterparts.24 Diphenylacetylenes are
relatively rigid; therefore, the degree of freedom around the triple
bond is limited, leading to a tight fit with the binding pocket at the
b-sheet. Previous papers have reported that diphenylacetylenes
displayed excellent binding to Ab plaques and could be potentially
useful for in vivo imaging of Ab plaques in living human brain.21–23
Based on the positive results reported previously, we applied the
same molecular design to chalcone derivatives which we have re-
cently reported to be useful as probes for the diagnosis of AD.25–27
In this study, we designed and synthesized a new series of diphenyl-
propynone (DPP) derivatives by replacing the double bond in the
chalcone scaffold with a triple bond, and evaluated their usefulness
as probes for imaging Ab (Fig. 1). To our knowledge, this is the first
time the use of DPP derivatives in vivo as probes to image Ab plaques
in the AD brain has been proposed.
The synthesis of the DPP derivatives is outlined in Schemes 1–3.
Various strategies have been developed for the synthesis of a DPP
Table 1
Inhibition by DPP derivatives of ligand binding to Ab (1–42)
aggregates
Compound
Kia (nM)
6.0 0.15
1
2
3
20.4 1.3
13.7 5.0
4
325.8 13.8
45.6 11.5
IMPY
a
Figure 2. Competitive curves of [125I]DMIC against the DPP derivatives Ab (1–42)
Values are the mean and standard error of the mean for three
independent experiments.
aggregates.

M. Ono et al. / Bioorg. Med. Chem. Lett. 21 (2011) 117–120
119
Figure 3. Fluorescent staining of 1 in 10-lm sections of the Tg2576 mouse brain (A). Labeled plaques were confirmed by staining of the adjacent sections with thioflavin S
(B). No apparent staining of 1 was observed in the age-matched control mouse brain (C).
scaffold.28–30 We selected a simple and efficient ligand-less, cop-
per- and solvent-free palladium-catalyzed synthesis of ynones by
coupling acyl chlorides with terminal alkynes which makes use
of only 1 equiv of acid chloride and 1 equiv of Et3N, and a relatively
small amount of catalyst, reported previously.31 With this process,
the DPP derivatives (1, 2, 3, and 5) were prepared by the coupling
of benzoyl chloride (p-iodobenzoyl chloride, p-fluorobenzoyl
chloride, or p-bromobenzoyl chloride) with phenyl acetylene (4-
ethynyl-N,N-dimethylaniline or p-ethynyl anisole) in the presence
of Pd(OAc)2 as the catalyst and Et3N as the base. 3 was converted to
4 by demethylation with BBr3 in CH2Cl2 (32% yield) (Scheme 2).
The tributyltin derivative 6 was prepared from the bromo com-
pound 5 using a bromo to tributyltin exchange reaction catalyzed
by Pd(0) in a yield of 54% (Scheme 3). The tributyltin derivative
was used as the starting material for radioiodination in the prepa-
ration of [125I]1. The novel radioiodinated DPP derivative [125I]1
was obtained by iododestannylation using hydrogen peroxide as
the oxidant which produced the desired radioiodinated ligand. It
was anticipated that the non-carrier-added preparation would re-
sult in a final product bearing a theoretical specific activity similar
to that of 125I (2200 Ci/mmol). The radiochemical identity of the
radioiodinated ligand was verified by co-injection with non-radio-
active 1 from its HPLC profile. The final radioiodinated compound,
have been frequently used to evaluate the specific binding of Ab
plaques in experiments in vitro and in vivo.19,25,33–35 Many Ab pla-
ques were clearly stained with 1 (Fig. 3A), as reflected by the high
affinity for Ab aggregates in in vitro competition assays, while no
labeling was observed in the wild-type mouse brain (Fig. 3C).
The labeling pattern was consistent with that observed with thio-
flavin S, a pathological dye commonly used for staining Ab plaques
in the brain (Fig. 3B). These results suggest that 1 can bind to Ab
plaques in the mouse brain in addition to having affinity for syn-
thetic Ab (1–42) aggregates.
The biodistribution of the radioiodinated compound [125I]
1 in vivo was tested in normal mice (Table 2). A biodistribution study
provides important information on brain uptake. The ideal Ab imag-
ing probe should have good blood–brain penetration to deliver a suf-
ficient dose into the brain while achieving rapid clearance from
normal regions to result in a higher signal to noise ratio in the AD
brain. The initial brain uptake of [125I]1 was 1.55% of injected dose/
gram at 2 min postinjection, whereas the radioactivity accumulated
in the brain was rapidly eliminated (0.76% of injected dose/gram at
60 min postinjection), indicating highly desirable properties for Ab
imaging probes. The radioactivity profile of [125I]1 in the brain was
similar to that of chalcones and related derivatives reported previ-
ously.25,26 The conversion of the double bond in the chalcone scaf-
fold to the triple bond in the DPP scaffold affected neither the
affinity for Ab aggregates nor the pharmacokinetics of the radioac-
tivity in the brain, indicating that the introduction of appropriate
substituted groups into the DPP scaffold can lead to the develop-
ment of new useful PET/SPECT probes for Ab plaques like chalcone
derivatives.
[
125I]1, showed a single peak of radioactivity at a retention time of
14.4 min. [125I]1 was obtained in ca. 50% radiochemical yield with
a radiochemical purity of >95% after purification by HPLC.
(E)-4-Dimethylamino-40-[125I]iodo-chalcone ([125I]DMIC) was
synthesized and used as the radioligand for competition experi-
ments (Kd value of [125I]DMIC is 4.2 nM).25 In vitro competitive
binding experiments to evaluate the affinity of the DPP derivatives
for Ab aggregates were carried out in solutions with [125I]DMIC as a
competitive ligand. The DPP derivatives inhibited the binding of
In conclusion, we successfully designed and synthesized a new
series of DPP derivatives as probes for the imaging of Ab plaques in
the brain in vivo. Some of the derivatives displayed excellent affin-
[
125I]DMIC in a dose-dependent manner (Fig. 2), and the affinity
of DPP derivatives for Ab (1–42) aggregates varied from 6 to
326 nM (Table 1). The DPP derivatives had affinity for Ab (1–42)
aggregates in the following order: 1 > 3 > 2 > 4. The Ki values indi-
cated that the affinity for Ab (1–42) aggregates was affected by the
substituted group in the DPP scaffold, and compounds 1 and 3
showed higher affinity than IMPY, a well known Ab imaging probe
(Ki = 45 nM). We have previously reported that the Ki value of a
chalcone with the dimethylamino group was 2.9 nM,25 indicating
that the DPP derivative 1 has the same binding affinity as this
chalcone derivative. We selected 1 with the highest affinity for
Ab (1–42) aggregates for radiolabeling and additional experiments.
Next, 1 was investigated for its affinity for Ab plaques by in vitro
neuropathological fluorescent staining in Tg2576 transgenic
mouse brain sections as shown in Figure 3. Tg2576 mice show
marked Ab deposition in the cingulated cortex, entorhinal cortex,
dentate gyrus, and hippocampus by 11–13 months of age32 and
Table 2
Biodistribution of radioactivity after injection of [125I]1 in normal micea
Tissue
Time after injection (min)
10 30
3.94 (0.46)
2
60
3.07 (0.20)
Blood
Liver
6.19 (1.05)
5.44 (0.36)
15.29 (1.81)
9.81 (1.31)
8.44 (1.67)
5.19 (0.93)
2.97 (2.55)
3.39 (0.31)
7.53 (1.14)
1.23 (0.09)
19.96 (2.47)
10.45 (1.82)
2.73 (0.79)
4.41 (0.95)
0.75 (0.16)
3.90 (0.44)
10.00 (1.19)
1.55 (0.26)
12.02 (1.21)
8.34 (0.77)
12.04 (1.44)
5.47 (0.27)
2.35 (1.38)
2.57 (0.30)
6.03 (0.46)
0.93 (0.06)
10.06 (1.07)
7.75 (0.71)
14.02 (1.77)
4.43 (1.64)
1.46 (0.38)
2.09 (0.13)
4.94 (0.54)
0.76 (0.07)
Kidney
Intestine
Spleen
Stomachb
Pancreas
Heart
Brain
a
Expressed as percent injected dose per gram. Each value represents the mean
(SD) for 4-6 animals.
b
Expressed as percent injected dose per organ.

120
M. Ono et al. / Bioorg. Med. Chem. Lett. 21 (2011) 117–120
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Supplementary data
Supplementary data (procedures for the preparation of DPP
derivatives, in vitro binding assay, in vitro fluorescent staining
using brain sections from Tg2576 mice, and biodistribution exper-
iments) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.11.058.
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