Synthesis and evaluation of ethyleneoxylated and allyloxylated chalcone derivatives for imaging of amyloid β plaques by SPECT

Article abstract of DOI:10.1016/j.bmc.2014.03.032

We report radioiodinated chalcone derivatives as new SPECT imaging probes for amyloid β (Aβ) plaques. The monoethyleneoxy derivative 2 and allyloxy derivative 8 showed a high affinity for Aβ(1-42) aggregates with K_i values of 24 and 4.5 nM, res

Full text of DOI:10.1016/j.bmc.2014.03.032

Bioorganic & Medicinal Chemistry 22 (2014) 2622–2628
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of ethyleneoxylated and allyloxylated
chalcone derivatives for imaging of amyloid b plaques by SPECT
a
a
b
a
Takeshi Fuchigami a, , Yuki Yamashita , Mamoru Haratake , Masahiro Ono , Sakura Yoshida ,
⇑
Morio Nakayama a,
⇑
a Department of Hygienic Chemistry, 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:
We report radioiodinated chalcone derivatives as new SPECT imaging probes for amyloid b (Ab) plaques.
The monoethyleneoxy derivative 2 and allyloxy derivative 8 showed a high affinity for Ab(1–42) aggre-
gates with Ki values of 24 and 4.5 nM, respectively. Fluorescent imaging demonstrated that 2 and 8
clearly stained thioflavin-S positive Ab plaques in the brain sections of Tg2576 transgenic mice. In vitro
autoradiography revealed that [125I]2 displayed no clear accumulation toward Ab plaques in the brain
sections of Tg2576 mice, whereas the accumulation pattern of [125I]8 matched with the presence of Ab
plaques both in the brain sections of Tg2576 mice and an AD patient. In biodistribution studies using nor-
mal mice, [125I]2 showed preferable in vivo pharmacokinetics (4.82%ID/g at 2 min and 0.45%ID/g at
60 min), while [125I]8 showed only a modest brain uptake (1.62%ID/g at 2 min) with slow clearance
(0.56%ID/g at 60 min). [125I]8 showed prospective binding properties for Ab plaques, although further
structural modifications are needed to improve the blood brain barrier permeability and washout from
brain.
Received 10 February 2014
Revised 15 March 2014
Accepted 17 March 2014
Available online 27 March 2014
Keywords:
Alzheimer’s disease
Amyloid b plaque
Chalcone
Single photon emission computed
tomography (SPECT)
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
from healthy subjects.5 Recently, [18F]florbetapir (Amyvid) and
[
18F]flutemetamol (Vizamyl) were launched in the United States
Alzheimer’s disease (AD) is the most common form of dementia
in the elderly, characterized by memory loss and cognitive dys-
function. Amyloid b (Ab) plaques and intracellular neurofibrillary
tangles in the postmortem brain have been reported to be the
key pathological hallmarks of AD.1,2 According to the amyloid
hypothesis, disruption of the balance between the production
and clearance of Ab leads to aggregation and accumulation of Ab
in the brain. This process may initiate various factors such as syn-
aptic dysfunction for cognitive impairment in AD.3 Non-invasive
diagnostic methods, such as single photon emission computed
tomography (SPECT) and positron emission tomography (PET),
are expected to be powerful tools for the detection of Ab plaques
in the early stage of AD and monitoring the efficacy of therapeutic
treatments for AD patients. For these purposes, a number of Ab pla-
ques targeted in vivo imaging probes based on the dye staining for
amyloid fibrils including thioflavin-T and Congo Red have been
developed.4 It has been demonstrated that several PET ligands
could detect human brain Ab plaques and distinguish AD patients
and/or Europe for the clinical evaluation of Ab neuritic plaque den-
sity when evaluating patients with cognitive impairment.6,7 SPECT
imaging can provide a cost-effective and convenient approach for
the visualization of Ab plaques in the brain. Considerable effort
has been devoted to the development of SPECT probes labeled with
99mTc or 123/125I for the Ab plaques.8–13 However, none of them
have been approved by the Food and Drugs Administration (FDA)
and/or the European Medicines Agency (EMA). [123I]IMPY is the
only SPECT imaging probe that has been used in clinical studies;
however, the signal-to-noise ratio obtained for plaque labeling
has been unsatisfactory.14 Accordingly, there is real need for clini-
cally useful SPECT probes for the detection of Ab plaques.
We have previously reported Ab imaging agents based on flavo-
noid derivatives, such as flavones,15,16 chalcones,17–20 aurones,21,22
and styrylchromones,23 for nuclear medicine imaging as shown in
Figure 1. Although several compounds have been demonstrated to
be promising Ab imaging probes, clinical studies of the compounds
have not yet been conducted. Our previous studies demonstrated
that chalcone derivatives fluoro-pegylated at the p-position of
the 1-phenyl ring showed a sufficient brain uptake with a good
binding capacity to Ab plaques.19 In addition, radioiodinated flavo-
noids, such as flavone and aurone derivatives, with a methoxy or
⇑
Corresponding authors. Tel.: +81 95 819 2442; fax: +81 95 819 2893 (T.F.).
E-mail address: t-fuchi@nagasaki-u.ac.jp (T. Fuchigami).
http://dx.doi.org/10.1016/j.bmc.2014.03.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
2623
R
R
R
O
O
O
O
I
I
I
R
I
O
O
O
Chalcones
Styrylchromones
Aurones
Flavones
R= NMe2, NHMe, NH2, OMe, OH
Figure 1. Chemical structures of flavonoid derivatives reported as amyloid imaging probes.
oligoethyleneoxy group at the p-position of the 3-phenyl ring have
the properties of high brain uptake and rapid clearance from the
brain without significant decrease in the binding affinity for
Ab.15,22 On the basis of the results of these studies, we hypothe-
sized that the introduction of ethyleneoxy groups at the p-position
of the 3-phenyl ring or iodoallyloxy group at the p-position of the
1-phenyl ring in the chalcone derivatives could improve the bind-
ing affinity for the Ab plaques and/or brain distribution pattern. In
this paper, we report the additional structural modification of chal-
cone derivatives to discover useful imaging probes for Ab plaques.
oxylated chalcone derivative 8 was prepared as previously
reported.20 The p-toluenesulfonate ester of (E)-3-(tri-n-butylstan-
nyl)prop-2-enol24 was coupled with the phenol 6 to produce the
tributyltin derivative 7 in a yield of 45%. Compound 7 was re-
acted with iodine in CHCl3 to form the target 8 (62% yield).
2.2. In vitro binding assays using Ab1–42 aggregates
In vitro binding experiments to evaluate the binding affinity of
the chalcones for Ab were performed in suspensions of Ab (1–42)
aggregates with [125I]4-dimethylamino-40-iodo-chalcone (DMIC)
which is a high-affinity Ab ligand with a chalcone backbone as de-
scribed in our previous papers.17–20 The inhibition constant (Ki)
values of 2–4 indicated that the binding affinity of these com-
pounds to Ab aggregates was affected by the length of the ethyl-
eneoxy group in the chalcone backbone. The rank order of their
binding affinity for Ab aggregates was as follows: monoethylene-
oxylated derivative 2 (Ki = 24.0 nM) > triethyleneoxylated deriva-
2. Results and discussion
2.1. Chemistry
The target chalcone derivatives were synthesized according to
the procedure shown in Scheme 1. 4-Iodoacetophenone was re-
acted with 4-methoxybenzaldehyde in the presence of a basic
catalyst (10% KOH) in ethanol at room temperature to form
40-iodo-4-methoxy-chalcone 1 in quantitative yield. Direct alkyl-
ation of 1 with ethylene chlorohydrin, ethylene glycol mono-2-
chloroethyl ether, or 2-[2-(2-chloroethoxy)ethoxy]ethanol with
potassium carbonate in DMF provided 2, 3 and 4 for yields of
52, 49 and 17%, respectively. The tributyltin derivative 5 was
tive
4
(Ki = 87.8 nM) > diethyleneoxylated
derivative
3
(Ki = 127.1 nM). The Ki value of 2 was significantly lower than those
of 3 (P <0.001; ANOVA, Bonferroni t test) and 4 (P <0.001). The Ki
value of 4 was significantly lower than that of 3 (P <0.05). The rela-
tionship between the binding affinity and length of the oligoethyl-
eneoxy group of the chalcones was similar to that of the aurone
derivatives.22 On the other hand, the allyloxy derivative 8 showed
excellent binding affinity for Ab (Ki = 4.5 nM), which is stronger
than that of DMIC (Ki = 10.8 nM). Based on these in vitro results,
prepared from the corresponding iodo derivative
4 using a
iodo-to-tributyltin exchange reaction catalyzed by Pd(0) for a
yield of 48%. The starting material 6 for the synthesis of the ally-
O
O
O
O
a
b
H
+
OH
n
OMe
I
OH
I
O
I
2 (n = 1)
3 (n = 2)
1
4
(n = 3)
O
O
c
OH
OH
Bu3Sn
O
I
O
5
2
O
O
O
d
e
HO
N
Bu3Sn
O
N
I
O
N
6
7
8
Scheme 1. Synthesis of chalcone derivatives. Reagents and conditions: (a) 10% KOH, EtOH, rt, 4 h; (b) H(OCH2CH2)nCl, K2CO3, DMF, 80 °C, 13–15 h; (c) dioxane, (Bu3Sn)2,
(Ph3P)4Pd, Et3N, reflux, 11 h; (d) (E)-Bu3SnCH = CHCH2OTs, NaH, DMF, rt, 4 h; (e) I2, CHCl3, rt, 30 min.

2624
T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
Figure 2. Neuropathological staining of chalcone derivatives 2 (A) and 8 (D) in 10
lm sections of Tg2576 mice brain. Labeled plaques were confirmed by staining of the
adjacent sections with thioflavin-S (B and E). Fluorescence images of 2 (C) and 8 (F) in the age-matched control mouse brain sections.
O
O
a
OH
OH
125I
O
Bu3Sn
O
125
2
I]
5
[
O
O
a
125
I
O
N
Bu3Sn
O
N
125
8
I]
7
[
Scheme 2. Radiosynthesis of 125I labeled chalcone derivatives. Reagents and conditions: (a) Na[125]I, H2O2, HCl, EtOH, rt, 3 min.
Figure 3. In vitro autoradiograms of chalcone derivatives [125I]2 (A) and [125I]8 (B) in the brain sections of Tg2576 mouse brain. Fluorescence image of thioflavin-S in the
adjacent section (C).
we selected the monoethyleneoxy derivative 2 and allyloxy deriv-
ative 8 for further experiments.
wild-type mice brain, there was no significant fluorescence of 2
and 8 (Figs. 2C and F). The autofluorescence of the adjacent brain
slices used in the assays was also examined. Significant fluores-
cence was not observed in any of the slices. These results indicated
that 2 and 8 have specific binding potential to Ab plaques in the
mice brain.
2.3. Fluorescence staining on Tg2576 mice brain sections
Fluorescence imaging experiments of the chalcones 2 and 8
were performed using brain slices from Ab deposition model trans-
genic mice (Tg2576 mice). A number of fluorescence spots of 2 and
8 were observed in the brain slices of the Tg2576 mice (Figs. 2A and
D). The labeling patterns of these compounds corresponded to the
fluorescence signals obtained by thioflavin-S (Figs. 2B and E). In the
2.4. In vitro autoradiography
The radioiodinated chalcones [125I]2 and [125I]8 were prepared
by an iododestannylation reaction using hydrogen peroxide as

T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
2625
Table 1
single-digit nanomolar Ki values for the Ab aggregates successfully
stained the Ab plaques in brain sections of the Tg2576 mice by
in vitro autoradiography.25,26 In addition, it has been shown that
chalcone derivatives with a dimethylamino group at the third po-
sition can be used to visualize Ab plaques by in vitro autoradiogra-
phy.19,27 Thus, chalcone derivatives with a dimethylamino group in
the 3-phenyl ring may be more suitable than those with an alkoxy
group with respect to binding of the Ab plaques in the brain.
To further characterize the binding property of [125I]8 for the Ab
plaques, the in vitro autoradiography of human AD brain sections
was conducted. The autoradiogram of [125I]8 displayed a distinct
accumulation of the radioactivity in the hippocampal section of
the human brain (Fig. 4A), while no significant signals of [125I]8
were observed in the frontal lobe section of the brain (Fig. 4B).
The labeled regions of [125I]8 corresponded to the immunohisto-
chemical staining of Ab in the hippocampal sections (Fig. 4C). Con-
sistent with the autoradiographic image of [125I]8, no Ab plaques
were detected in the frontal lobe section (Fig. 4D). These results
strongly suggested that [125I]8 was bound to the Ab plaques in
the brain tissues.
Inhibition constant (Ki) of chalcone derivatives for Ab aggregates
Compounds
Kia (nM)
2
3
4
8
24.0 10.4
127.1 27.3
87.8 20.3
4.5 1.5
DMIC
10.8 0.9
a
Inhibition constant (Ki) of chalcone derivatives were deter-
mined using [125I]DMIC as a radioligand for Ab aggregates. Each
value (mean SEM) was determined by 3–6 independent
experiments.
the oxidant (Scheme 2). [125I]2 and [125I]8 were obtained in radio-
chemical yields of 66–75% with radiochemical purities of >95%
after purification by HPLC. In vitro autoradiography of [125I]2 and
[
[
125I]8 was carried out using brain sections of the Tg2576 mice.
125I]2 displayed no clear accumulation toward the Ab plaques
(Fig. 3A), while [125I]8 showed a distinctive plaque labeling with
a low background (Fig. 3B). Labeled plaques by [125I]8 were
matched with the thioflavin-S positive regions of the adjacent sec-
tions (Fig. 3C). It is possible that the differing concentration of 2
caused the discrepancy observed between the results of the fluo-
2.5. In vivo biodistribution stuides
Todevelopprospectivein vivoimagingprobesforAb, ahighinitial
brain uptake and rapid clearance from the brain are two important
requirements. Therefore, biodistribution studies of [125I]2 and
rescence imaging and autoradiography studies: 100 lM of 2 was
used for fluorescence staining, whereas only 0.3 nM of [125I]2
was used for autoradiography. On the other hand, this difference
might be related to the binding affinity of the chalcone derivatives
for the Ab aggregates; 8 showed a 5.3-fold lower Ki value than 2
(Table 1). Indeed, it was reported that several Ab probes with
[
125I]8 were performed in normal mice and expressed as% of injected
dose per gram (%ID/g) (Table 2). [125I]2 showed a high initial uptake
(4.82%ID/gat2 min)andrapidclearancefromthebrain(0.45%ID/gat
Figure 4. In vitro autoradiographic images of [125I]8 in the hippocampal (A) and frontal lobe, (B) sections of AD patient (73-year-old male). Immunohistochemical staining of
each section using a monoclonal anti-Ab antibody (C and D, respectively).

2626
T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
imaging. Although the in vivo property of [125I]8 was unsuitable
for the in vivo imaging, [125I]8 showed excellent binding affinity
for the Ab aggregates and detected Ab plaques in the brain sections
of both the Tg2576 mouse and AD patient by fluorescence micros-
copy and autoradiography. Thus, it has been demonstrated that
introduction of the iodoallyloxy group into the chalcone backbone
effectively improved its binding affinity for Ab plaques. Based on
this study, further chemical modifications could provide useful
Ab imaging probes based on the chalcone backbone.
Table 2
Biodistribution of radioactivity after i.v. injection of 125I labeled chalcone derivatives
in normal micea
Time after injection (min)
Organ
125I]2
2
10
30
60
[
Blood
Liver
3.95(0.30)
14.76(2.88)
10.34(1.72)
3.98(0.66)
2.69(0.54)
7.14(1.34)
2.22(0.41)
6.12(1.26)
6.20(1.19)
4.82(1.19)
3.01(0.58)
20.11(4.81)
9.05(1.72)
10.55(3.41)
2.35(0.74)
4.63(1.37)
3.38(2.38)
3.37(1.00)
3.13(0.88)
2.86(0.87)
2.60(0.42)
18.83(1.20)
10.23(1.96)
26.39(6.69)
1.21(0.10)
3.21(1.24)
4.27(3.43)
1.07(0.44)
1.54(0.13)
1.00(0.05)
2.27(0.60)
15.57(3.17)
9.89(3.07)
25.16(6.54)
0.81(0.14)
1.94(0.42)
4.01(0.38)
0.90(0.54)
1.05(0.32)
0.45(0.09)
Kidney
Intestine
Spleen
Lung
Stomach
Pancreas
Heart
4. Experimental
4.1. General information
Brain
[
125I]8
All reagents were commercial products and used without fur-
ther purification unless otherwise indicated. Na[125]I was obtained
from MP Biomedicals (Costa Mesa, CA, USA). The 1H NMR spectra
were obtained using a Varian Gemini 300 spectrometer with TMS
as the internal standard. The mass spectra were obtained using
JMS-700 N or JMS-T100TD instruments (JEOL, Japan). The HPLC
analysis was performed by a Shimadzu HPLC system (LC-10AT
pump with SPD-10A UV detector, k = 254 nm). An automated gam-
ma counter with a NaI(Tl) detector (Perkin–Elmer, 2470 WIZARD2)
was used to measure the radioactivity. 4-Dimethylamino-40-iodo-
chalcone (DMIC) and [125I]DMIC were prepared by the method in
the literature.10 The AD transgenic mice (Tg2576, C57BL6, APPsw)
were purchased from Taconic Farms, Inc. All animals were supplied
by the Tagawa Experimental Animal Laboratory (Nagasaki, Japan).
The experiments with animals were conducted in accordance with
our institutional guidelines and were approved by the Nagasaki
University Animal Care Committee.
Blood
Liver
2.65(0.21)
23.21(2.52)
8.98(1.60)
1.74(0.59)
4.43(1.11)
8.08(1.39)
2.49(1.07)
4.02(1.48)
8.70(1.66)
1.62(0.30)
2.29(0.19)
19.84(6.55)
6.06(0.57)
2.38(0.88)
5.34(2.03)
4.48 (0.38)
5.19(2.16)
3.27(0.51)
3.39(0.38)
1.63(0.22)
1.97(0.55)
16.91(0.62)
4.52(0.59)
4.45(1.70)
4.68(1.31)
3.16(0.42)
8.98(5.05)
2.35(0.63)
2.20(0.13)
0.87 (0.18)
2.43(1.18)
10.26(0.55)
4.17(1.12)
6.34(2.57)
3.92(1.52)
3.28(1.01)
16.44(6.82)
2.14(1.12)
2.10(0.85)
0.56(0.16)
Kidney
Intestine
Spleen
Lung
Stomach
Pancreas
Heart
Brain
a
Expressed as % of injected dose per gram (%ID/g). Each value represents mean
(SD) for 5 or 6 mice at each interval.
60 min). On the other hand, [125I]8 showed a modest brain uptake
(1.62%ID/g at 2 min) and relatively slow clearance from the brain
(0.56%ID/g at 60 min). The ratio brain2 min/brain60 min is considered
to be an important index for the selection of the candidate com-
pounds. The brain2 min/brain60 min ratios of [125I]2 and [125I]8 were
10.7 and 2.9, respectively. It should be noted that [125I]2 had a com-
parable brain2 min/brain60 min ratio in the normal mice brain with the
clinically used Ab probes, such as [123I]IMPY (13.7)12,13 and [11C]BF-
227 (12.3).28,29 The difference in the in vivo properties among these
probes may be partly explained by lipophilicity. Because the lipo-
philicity of 2 (clogP = 4.3, calculated using a ChemBioDraw Ultra
13.0) was between those of BF-227 (clogP = 3.85) and IMPY
(clogP = 4.64), 2 may have preferable lipophilicity for penetration
of the blood–brain barrier, whereas 8 has higher lipophilicity
(clogP = 5.64), which might reduce the brain uptake and washout
from brain. The initial uptake of [125I]8 in the liver was much higher
than that of [125I]2, while a comparable accumulation of [125I]2 and
4.1.1. (E)-1-(4-Iodophenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (1)
4-Iodoacetophenone (2.46 g, 10 mmol) and 4-anisaldehyde
(1.2 mL, 10 mmol) were dissolved in 20 mL of ethanol. The mixture
was allowed to stir for 15 min in an ice bath. A 30 mL aliquot of
10% aqueous potassium hydroxide solution was then slowly added
dropwise into the reaction mixture. The reaction solution was al-
lowed to stir at room temperature for 4 h. A precipitate was col-
lected and washed with ethanol to provide 3.69 g of 1 (quant).
1H NMR (300 MHz, CDCl3) d ppm: 7.85 (d, J = 8.1 Hz, 2H), 7.80 (d,
J = 15.7 Hz, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H),
7.34 (d, J = 15.7 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 3.86 (s, 3H).
[
125I]8 was observed in the lung and kidney. Both tracers showed in-
crease of radioactivity in the stomach with time, but the uptake of
125I]8 was higher than that of [125I]2. These results indicated that
4.1.2. (E)-3-(4-(2-Hydroxyethoxy)phenyl)-1-(4-
iodophenyl)prop-2-en-1-one (2)
To a solution of compound 1 (120 mg, 0.34 mmol) in DMF
(2 mL) was added 2-chloroethanol (45 lL, 0.34 mmol) and K2CO3
[
thedeionidation of[125I]8 was faster thanthat of[125I]2 in vivo. How-
ever, no significant increase in blood radioactivitylevel of [125I]8 was
observed over time. In addition, the mouse plasma samples obtained
at 10 and 60 min post-injection of [125I]8 were analyzed by radio-
TLC; the metabolites of [125I]8 were much more hydrophilic than
the parent [125I]8 (data not shown). In general, blood–brain barrier
penetration by very polar compounds is limited.30 Therefore, the
deiodination could have minimal effects on the brain accumulation
of [125I]8. However, it should be considered that the [125I]iodoallyl-
oxy group of [125I]8 was unstable in vivo. Novel 125I labeled iodoalke-
nyl-substituted chalcone derivatives with improved in vivo
pharmacokinetics and metabolic stability could be prospective Ab
imaging probes.
(138 mg, 1.02 mmol). The mixture was heated at 80 °C for 13 h.
After cooling the reaction mixture, the solvent was quenched by
H2O and extracted three times with EtOAc. The combined organic
layer was washed with brine. After drying over Na2SO4, the solvent
was removed and the crude product was chromatographed on sil-
ica gel with hexane/EtOAc = 1:1 to give 5 (70 mg, 52%) as a yellow
solid. 1H NMR (300 MHz, CDCl3) d ppm: 4.01 (t, J = 4.5 Hz, 2H), 4.13
(t, J = 4.8 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 7.38 (d, J = 15.6 Hz, 1H),
7.60 (d, J = 9.0 Hz, 2H), 7.74 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 15.6 Hz,
1H), 7.85 (d, J = 8.7 Hz, 2H). HRMS (FAB) m/z calcd for C17H16IO3
(M+) 395.014, found 395.014.
3. Conclusion
4.1.3. (E)-3-(4-(2-(2-Hydroxyethoxy)ethoxy)phenyl)-1-(4-
iodophenyl)prop-2-en-1-one (3)
Using the above procedure for 2 starting from compound 1 and
2-(2-Chloroethoxy)ethanol (58 lL, 0.56 mmol), the title compound
[
125I]2 had a modest binding affinity for the Ab aggregates and
showed an unclear in vitro autoradiogram for the Ab plaque, while
its pharmacokinetic property was preferable for the in vivo brain

T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
2627
3 (60 mg, 49%) was obtained as a yellow solid. 1H NMR (300 MHz,
4.2. Radioiodination
CDCl3) d ppm: 3.69 (t, J = 5.1 Hz, 2H), 3.78 (t, J = 5.1 Hz, 2H), 3.92 (t,
J = 4.5 Hz, 2H), 4.20 (t, J = 4.5 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 7.38
(d, J = 15.6 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H),
7.75 (d, J = 15.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H). HRMS (FAB) m/z
calcd for C19H20IO4 (M+) 439.041, found 439.040.
The 125I-labeled compounds ([125I]2, [125I]8) were prepared
from the corresponding tributyltin derivatives (2 and 6, respec-
tively) by iododestannylation. Briefly, 3% H2O2 (50
to a mixture of the corresponding tributyltin derivative (50
50
L EtOH), Na[125]I (3.7–7.4 MBq, specific activity 81.4 GBq/
mol), and 1 M HCl (50 L) in a sealed vial. The reaction was al-
lowed to proceed at room temperature for 3 min and terminated
by addition of satd. NaHSO3aq (100 L). After alkalization with
100 L of satd. NaHCO3aq and extraction with ethyl acetate, the
l
L) was added
lg/
l
4.1.4. (E)-3-(4-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)phenyl)-
1-(4-iodophenyl)prop-2-en-1-one (4)
l
l
Using the above procedure for 2 starting from compound 1 and
l
2-[2-(2-chloroethoxy)ethoxy]ethanol (50
l
L, 0.34 mmol), the title
l
compound 6 (28 mg, 17%) was obtained as a yellow solid. 1H
NMR (300 MHz, CDCl3) d ppm: 7.85 (d, J = 8.7 Hz, 2H), 7.76 (d,
J = 15.6 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H),
7.37 (d, J = 15.6 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H), 4.18 (t, J = 5.1 Hz,
2H), 3.89 (t, J = 5.1 Hz, 2H), 3.70–3.76 (m, 4H), 3.62–3.66 (m, 4H).
HRMS (FAB) m/z calcd for C21H24IO5 (M+) 483.067, found 484.065.
extract was dried by passing through an anhydrous Na2SO4 column
and evaporated to dryness. The crude products were purified by
HPLC on a Cosmosil C18 column (Nacalai Tesque, 5C18-AR-II,
4.6 ꢀ 250 mm) with an isocratic solvent of CH3CN/H2O (5:5 for
[
125I]2 and 6:4 for [125I]8) at the flow rate of 1.0 mL/min. The reten-
tion time of
respectively.
[ [
123I]2 and 123I]8 were 18 min and 30 min,
4.1.5. (E)-3-(4-(2-Hydroxyethoxy)phenyl)-1-(4-
tributylstannylphenyl)prop-2-en-1-one (5)
4.3. Binding assays using the aggregated Ab peptide in solution
A mixture of 2 (58 mg, 0.15 mmol), bis(tributyltin) (0.12 mL,
0.24 mmol) and Pd(PPh3)4 (8.4 mg, 7.27
l
mol) in a mixed solvent
Binding assays by using filtration techniques were carried out
as described previously.10 A solid form of Ab(1–42) 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 incubated at 37 °C for 42 h with gentle and con-
(9.0 mL, 2:1 dioxane/triethylamine mixture) was stirred for 11 h
under reflux. The solvent was removed, and the residue was puri-
fied by silica gel chromatography hexane/EtOAc = 2: 1, which gave
5 (40 mg, 0.0717 mmol, 48%) as an orange-colored oil. 1H NMR
(300 MHz, CDCl3)
d ppm: 7.92 (d, J = 7.8 Hz, 2H), 7.75 (d,
J = 15.9 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 15.9 Hz, 1H),
6.98 (d, J = 9.0 Hz, 2H), 4.13 (t, J = 4.8 Hz, 2H), 4.01 (t, J = 4.8 Hz,
2H), 1.52–1.58 (m, 9H), 1.31–1.35 (m, 9H), 1.07–1.13 (m, 9H),
0.87–0.92 (m, 9H), MS (FAB) m/z 559 [M+].
stant shaking. A mixture containing 50
(8 pM–12.5 M in 10% ethanol). 50
L of 0.02 nM [125I]DMIC,
50 L of the Ab aggregates, and 850 L of 10% ethanol was incu-
lL of test compounds
l
l
l
l
bated at room temperature for 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 measured by
an automatic gamma counter (PerkinElmer, 2470 WIZARD2). Val-
ues for the half-maximal inhibitory concentration (IC50) were
determined from displacement curves of three independent exper-
iments using GraphPad Prism 4.0, and those for the inhibition con-
stant (Ki) were calculated using the Cheng–Prusoff equation.
4.1.6. (E)-3-(4-(Dimethylamino)phenyl)-1-(4-(((E)-3-
(tributylstannyl)allyl)oxy)phenyl) prop-2-en-1-one (7)
To a solution of compound 613 (25.0 mg, 0.094 mmol) in DMF
was added NaH (24.6 mg, 1.05 mmol). To the mixture was added
(E)-Bu3SnCH = CHCH2OTs17 (83 mg, 0.174 mmol) in DMF and stir-
red at room temperature for 4 h. The mixture was quenched by
saturated NH4Cl and extracted three times with CHCl3. The com-
bined organic layer was washed with brine. The solvent was re-
moved and the crude product was chromatographed on silica gel
with hexane/EtOAc = 2:1 to give 7 (25 mg, 45%) as a yellow-
ocher oil. 1H NMR (300 MHz, CDCl3) d ppm: 8.01 (d, J = 9.0 Hz,
2H), 7.79 (d, J = 15.6 Hz, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.36 (d,
J = 15.6 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H),
6.38 (dt, J = 18.8, 3.1 Hz, 1H), 6.17(d, J = 18.0 Hz, 2H), 4.64 (d,
J = 3.3 Hz, 2H), 3.04 (s, 6H), 1.57–0.864 (27H, m). MS (FAB) m/z
598 (M+).
4.4. Fluorescence staining on Tg2576 mice brain sections
The Tg2576 mice (female, 22-month-old) and wild-type mice
(female, 22-month-old) were used as the Alzheimer’s model and
control mice, respectively. After the mice were sacrificed by decap-
itation, the brains were immediately removed and frozen in pow-
dered dry ice. The frozen blocks were sliced into serial sections,
10
(100
DMSO for 5 min twice. The fluorescence images were collected
by BZ8100 (Keyence) using DAPI-BP filter set (excitation,
l
m thick. Each slide was incubated with a 50% DMSO solution
lM) of 2 and 8 for 3 h. The sections were washed in 50%
4.1.7. (E)-3-(4-(Dimethylamino)phenyl)-1-(4-(((E)-3-
a
iodoallyl)oxy)phenyl)prop-2-en-1-one (8)
360 nm; diachromic mirror, 400 nm; longpass filter, 460 nm) or a
GFP-BP filter set (excitation, 470 nm; diachromic mirror, 495 nm;
longpass filter, 535 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 the micro-
scope in the same condition with that of chalcones.
To a solution of 7 (22.2 mg, 0.037 mmol) in CHCl3 (5.0 mL) was
added a solution of iodine in CHCl3 (2.0 mL, 0.25 M) at room tem-
perature. The mixture was stirred at room temperature for 30 min
and a saturated NaHSO3 solution (10 mL) was added. The mixture
was stirred for 5 min and the organic phase was separated. The
aqueous layer was extracted three times with CHCl3. The organic
layer was successively washed with saturated aqueous NaHCO3
and brine, then dried over Na2SO4. The crude product was chro-
matographed on silica gel with hexane/EtOAc = 3:1 to give 8
(10.0 mg, 62%) as a yellow powder. 1H NMR (300 MHz, CDCl3) d
ppm: 8.02 (d, J = 6.4 Hz, 2H), 7.78 (d, J = 11.6 Hz, 1H), 7.53 (d,
J = 5.9 Hz, 2H), 7.34 (d, J = 11.7 Hz, 1H), 6.95 (d, J = 6.6 Hz, 2H),
6.80 (dt, J = 13.6, 6.0 Hz, 1H), 6.70 (d, J = 6.2 Hz, 2H), 6.59 (d,
J = 11.2 Hz, 1H), 4.54 (d, J = 5.7 Hz, 2H), 3.05 (s, 6H). HRMS (FAB)
m/z calcd for C20H21INO2 (M+) 434.0617, found 434.0598.
4.5. In vitro autoradiography on Tg2576 mice brain sections
The brain sections of Tg2576 mice (female, 22-month-old) were
incubated in the 50% DMSO solution containing [125I]2 or [125I]8
(3.7 kBq/150
l
L, 0.3 nM) for 1 h. The slices were rinsed 5 min ꢀ 3
each with a 60% DMSO solution, and subsequently dipped into cold
water (30 s). The sections were dried under a stream of cold air and
placed in contact with the imaging plates (BAS-MS 2040; Fuji Film)
for 24 h. The distributions of the radioactivity on the plates were

2628
T. Fuchigami et al. / Bioorg. Med. Chem. 22 (2014) 2622–2628
7. Williams, S. C. P. Nat. Med. 2013, 19, 1551.
analyzed by a Fluoro Image Analyzer (FLA5100; Fuji Film). Thereaf-
ter, the serial sections were also stained with thioflavin S. In vitro
autoradiography of the postmortem brain slices were performed
according to the above described method. The postmortem brain
tissues from an autopsy-confirmed case of AD (73-year-old male)
were obtained from BioChain Institute, Inc. The presence and local-
ization of plaques on the sections were confirmed by immunohis-
tochemical staining using the monoclonal antibody BC05 (Wako)
as already reported.31
8. Cheng, Y.; Ono, M.; Kimura, H.; Ueda, M.; Saji, H. J. Med. Chem. 2012, 55, 2279.
9. Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu, H.; Ono, M.; Jia, H.;
Saji, H.; Liu, B. J. Med. Chem. 2013, 56, 471.
10. Qu, W.; Kung, M. P.; Hou, C.; Oya, S.; Kung, H. F. J. Med. Chem. 2007, 50, 3380.
11. Qu, W.; Kung, M. P.; Hou, C.; Benedum, T. E.; Kung, H. F. J. Med. Chem. 2007, 50,
2157.
12. 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.
13. Zhuang, Z. P.; Kung, M. P.; Wilson, A.; Lee, C. W.; Plössl, K.; Hou, C.; Holtzman,
D. M.; Kung, H. F. J. Med. Chem. 2003, 46, 237.
14. Kung, M. P.; Weng, C. C.; Lin, K. J.; Hsiao, I. T.; Yen, T. C.; Wey, S. P. Chang Gung
Med. J. 2012, 35, 211.
15. Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.; Arano, Y.; Mori, H.; Nakayama,
M. J. Med. Chem. 2005, 48, 7253.
4.6. In vivo biodistribution in normal mice
16. Ono, M.; Watanabe, R.; Kawashima, H.; Kawai, T.; Watanabe, H.; Haratake, M.;
Saji, H.; Nakayama, M. Bioorg. Med. Chem. 2009, 17, 2069.
17. Ono, M.; Hori, M.; Haratake, M.; Tomiyama, T.; Mori, H.; Nakayama, M. Bioorg.
Med. Chem. 2007, 15, 6388.
18. Ono, M.; Haratake, M.; Mori, H.; Nakayama, M. Bioorg. Med. Chem. 2007, 15,
6802.
19. Ono, M.; Watanabe, R.; Kawashima, H.; Cheng, Y.; Kimura, H.; Watanabe, H.;
Haratake, M.; Saji, H.; Nakayama, M. J. Med. Chem. 2009, 52, 6394.
20. Ono, M.; Ikeoka, R.; Watanabe, H.; Kimura, H.; Fuchigami, T.; Haratake, M.; Saji,
H.; Nakayama, M. ACS Chem. Neurosci. 2010, 1, 598.
A saline solution (100 l
L) of [125I]2 or [125I]8 (7.4 kBq) was di-
rectly injected intravenously into the tail of ddY mice (5-week-
old, 20–25 g). The mice were sacrificed at various times post-injec-
tion. The organs of interest were removed and weighed, and the
radioactivity was measured by the gamma counter.
4.7. Statistical analysis
21. Ono, M.; Maya, Y.; Haratake, M.; Ito, K.; Mori, H.; Nakayama, M. Biochem.
Biophys. Res. Commun. 2007, 361, 116.
22. Maya, Y.; Ono, M.; Watanabe, H.; Haratake, M.; Saji, H.; Nakayama, M.
Bioconjugate Chem. 2009, 20, 95.
23. Ono, M.; Maya, Y.; Haratake, M.; Nakayama, M. Bioorg. Med. Chem. 2007, 15,
444.
Statistical significance was determined at p <0.05 using the one
way analysis of variance for comparison of more than two means,
followed by post hoc tests using Bonferroni’s correction.
24. Zea-Ponce, Y.; Mavel, S.; Assaad, T.; Kruse, S. E.; Parsons, S. M.; Emond, P.;
Chalon, S.; Giboureau, N.; Kassiou, M.; Guilloteau, D. Bioorg. Med. Chem. 2005,
13, 745.
Acknowledgments
25. Cui, M.; Ono, M.; Kimura, H.; Ueda, M.; Nakamoto, Y.; Togashi, K.; Okamoto, Y.;
Ihara, M.; Takahashi, R.; Liu, B.; Saji, H. J. Med. Chem. 2012, 55, 9136.
26. Cui, M.; Ono, M.; Kimura, H.; Liu, B.; Saji, H. J. Med. Chem. 2011, 54, 2225.
27. Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu, H.; Ono, M.; Jia, H.;
Saji, H.; Liu, B. J. Med. Chem. 2013, 56, 471.
Financial support was provided by a Grant-in-Aid for Scientific
Research (B) (Grant No. 21390348) from Japan Society for the Pro-
motion of Science (JSPS).
28. 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.
29. Okamura, N.; Shiga, Y.; Furumoto, S.; Tashiro, M.; Tsuboi, Y.; Furukawa, K.;
Yanai, K.; Iwata, R.; Arai, H.; Kudo, Y.; Itoyama, Y.; Doh-ura, K. Eur. J. Nucl. Med.
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30. Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376.
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