6-Methoxy-indanone derivatives as potential probes for β-amyloid plaques in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.07.069

A series of 6-methoxy indanone derivatives was synthesized and evaluated as potential probes for β-amyloid plaque imaging in Alzheimer's disease (AD). Two derivatives (5d and 5k) displayed significant binding abilities in fluorescent staining experiments

Full text of DOI:10.1016/j.ejmech.2016.07.069

Accepted Manuscript
6-Methoxy-indanone derivatives as potential probes for β-amyloid plaques in
Alzheimer's disease
Dou-Dou Nan, Chang-Sheng Gan, Chen-Wei Wang, Jin-Ping Qiao, Xin-Meng Wang,
Jiang-Ning Zhou
PII:
S0223-5234(16)30634-1
10.1016/j.ejmech.2016.07.069
EJMECH 8787
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 May 2016
Revised Date: 26 July 2016
Accepted Date: 27 July 2016
Please cite this article as: D.-D. Nan, C.-S. Gan, C.-W. Wang, J.-P. Qiao, X.-M. Wang, J.-N. Zhou,
6-Methoxy-indanone derivatives as potential probes for β-amyloid plaques in Alzheimer's disease,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.069.
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6-Methoxy-Indanone Derivatives as Potential Probes for β-Amyloid Plaques in
Alzheimer’s Disease
Dou-Dou Nana, Chang-Sheng Ganb,.Chen-Wei Wanga, Jin-Ping Qiaoc, Xin-Meng Wanga, and
Jiang-Ning Zhou*a
a CAS Key laboratory of Brain Function and Disease, School of Life Science, University of Science and Technology of China, Hefei,
Anhui, 230027 (P. R. China)
b Engineering Research Centre of Bio-process of Ministry of Education, Hefei University of Technology, Hefei, Anhui, 230009 (P. R.
China)
c Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230022 (P. R. China)
* Corresponding author. Jiang-Ning Zhou E-mail: jnzhou@ustc.edu.cn.
Abstract: A series of 6-methoxy indanone derivatives was synthesized and evaluated
as potential probes for β-amyloid plaque imaging in Alzheimer’s disease (AD). Two
derivatives (5d and 5k) displayed significant binding abilities in fluorescent staining
experiments using the brain sections of AD patients. Two derivatives showed high
binding affinities to β-amyloid aggregates (5j, Ki = 5.82 ± 0.19 nM) and brain
homogenates of AD patients (5j, Ki = 18.96 ± 0.28 nM) in in vitro binding assay. With
a log P value of 3.45, [125I]5k exhibited an excellent initial brain uptake (5.29 %ID g−1,
2 min after i.v.) and a fast clearance from the brain in biodistribution experiments in
normal mice. In autoradiography, [125I]5k exhibited an obvious binding ability to
β-amyloid plaques and a relatively low nonspecific binding in the brain sections of
AD patients (in vitro) and APP/PS1 transgenic mice (in vitro and ex vivo). Results
suggest that 5k is a potential probe for detecting β-amyloid plaques in vivo.
Keywords: Alzheimer’s disease; β-amyloid plaques; amyloid probes; indanone;
molecular imaging.

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1. Introduction
AD is the most common form of dementia that afflicts millions of people[1, 2].
An epidemiological investigation predicted the number of AD patients to increase to
107 million by 2050[3]. Along with the rise of the aging population worldwide, AD
represents serious economic and medical pressure to society[4]. To date, no drugs or
treatment could entirely cure AD. Therefore, early clinical detection of AD is crucial to
improve the quality of life, eliminate the treatment costs, and extend the lives of
patients[5, 6]. Currently, the noninvasive detection of amyloid aggregates by using
PET or SPECT provides a direct way to diagnose AD at its preliminary stage[7]. With
the advantage of low chemical concentration (pM to nM) in clinics and the high
popularity of scanners in hospitals, designing agents for PET or SPECT is practical
[8].
Primarily histopathological features of AD are β-amyloid plaques which
composed of β-amyloid peptide aggregates and neurofibrillary tangles which
entwisted by abnormal phosphorylated tau protein[5, 9, 10]. Based on conventional
dyes, such as Congo Red and Thioflavin-T (ThT), some radioligands have been
developed for binding to β-amyloid plaques in vivo specifically and noninvasively for
the diagnosis of AD[11, 12] (Scheme 1). [11C]PIB, derived from ThT, is an
extensively investigated PET probe in a clinical setting; this probe displays significant
sensitivity in distinguishing AD subjects and controls[13, 14]. A considerable amount
of 11C-labeled PET probes, such as [11C]PIB and [11C]SB-13, exhibit excellent
binding affinity to β-amyloid plaques; however, the short radioactive decay half-life
18
of 11C (20 min) limits its routine clinical application[15, 16]. By contrast, F-labeled
11
probes are more widely used in clinical PET imaging examinations than C-labeled
PET probes because of its 110 min physical half-life. As the first PET probe used in a
human trial, [18F]FDDNP occupies a different binding site of β-amyloid plaques from
PIB, and it binds to neurofibrillary tangles simultaneously[17-19]. In addition, other
18F-labeled probes have been developed and have showed high binding affinities and
specificities to β-amyloid plaques, these probes include styrylpyridines derivative
[18F]Florbetapir (AV-45), stilbene derivative [18F]Florbetaben (BAY 94-9127), and a

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3’-fluoro-derivative of PIB called [18F]Flutemetamol (GE-067)[20-24].
[18F]Florbetapir and [18F]Flutemetamol were approved by the FDA, and their
evaluation in human trials revealed their potential application with PET [25-27].
Recently, a series of molecules with different skeletal formulas has been found to
have the potential to detect β-amyloid plaques in the experimental phase; these
molecules
include
dibenzylideneacetone
derivatives,
fluoro-pegylated
benzyloxybenzenes, and homoisoflavonoids[28-30]. Barbiturate derivative
([18F]Barbiturate), a novel amyloid imaging probe, demonstrates fast brain penetration,
high affinity, and rapid non-specific clearance[31]. Among SPECT probes,
[125I]IMPY exhibits a high binding affinity to β-amyloid plaques; however, this probe
failed in human trials because of its poor signal-to-noise ratio[32-35].
In our previous research of β-amyloid probes, we reported a series of indanone
derivatives (compounds 1 and 2 in Scheme 1) based on donepezil, a clinical drug for
AD patients[36]. With a hydrophobic and planarized π-π system, indanone derivatives
displayed obvious affinities and specificities to β-amyloid peptide aggregates in brain
homogenates AD patients, fast brain-blood barrier (BBB) penetration, and rapid
washout from the brain in normal ICR mice. However, the indanone derivatives in our
published work exhibited relatively low binding affinities in in vitro binding assay.
Meanwhile, the 125I radiolabeled derivatives showed limited brain uptake in ICR mice
and failed in an ex vivo autoradiography experiment on transgenic mice. Apparently,
the methoxy groups on the aromatic ring of indanone play a critical role in binding
affinities. In the current research, we synthesized a series of indanone derivatives with
6-mehtoxy and evaluated their biological activities to provide a novel probe with
improved properties.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 2, compounds 5a-5b, 5d-i and 5k-n were prepared
following the method described in previous literature[36]. Compounds 5c or 5j were
prepared by heating 5b or 5i with paraformaldehyde and sodium methoxide in

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methanol and subsequent NaBH4 added following a method previously reported[37].
As shown in Scheme 3, the tributyltin precursor of radioiodination 5o, was
synthesized from 5d by reacting with bis(tributyltin) and Pd(PPh3)4 in Et3N at 80 °C.
The trans-configuration of compounds 5b-g, 5i-n were confirmed by the chemical
shift of the vinyl proton in 1H NMR. Because of the deshielding effect resulting from
the neighboring carbonyl group, the vinyl proton gave a higher chemical shift in the
trans-isomer (> 7 ppm) than the cis-isomer[38].
2.2. In vitro binding assay
The binding affinities of the synthesized derivatives were measured by
radioligand competition binding assays with Aβ1–40 aggregates and AD patients
brain homogenates respectively as showed in Table 1.
As mentioned in our previous study, these series of indanone derivatives
probably bind to β-amyloid plaques via the ThT binding site, and methoxy groups at
substituents of indanone ring provide great efforts to binding affinities to AD patients
brain homogenates[36]. In compounds 5a-5n, methoxy group was kept consistently
on the 6-position of indanone ring, and substituent R1 as halogen. The Ki value of 5k
(Ki=28.63 ± 0.1nM to AD patients brain homogenates) designed with methoxy group
was relatively lower than these series of compounds without methoxy group (1, Ki=99
± 5nM; 2, Ki=237 ± 105nM)[36]. This results suggested methoxy group at 6-position
had an influence on binding affinity[39]. In this series of compounds, two aromatic
rings and a double bond linker between them constituted a π-π system that gives the
stably planarity to this structure and ability to insert into β-sheets of β-amyloid
aggregates. The most significant Ki values (5j, 5.82 nM to Aβ1–40 aggregates, and
18.96 nM to AD patients brain homogenates) confirmed the great binding affinities of
those compounds. Comparing compounds 5c, 5d and 5j, 5k to compounds 5a, 5b and
5h, 5i, great changes of binding affinities were contributed by the substitution of
electron-donating group at R2 position which reduced Ki values in a huge range. The
monomethyl amino group increased binding affinities significantly (5c, 5j), but the
dimethyl amino group had a limited effect (5d, 5k) as compared to monomethyl

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amino because of the lipophilicity changes at this position. With the influence of
different substitution (methoxy, F, H), compounds 5f-h and 5l-n exhibited low
binding affinities to either Aβ1–40 aggregates or AD patients brain homogenates.
Even more, limited changes of Ki value between compounds with bromine and iodine
at 5-position may depend on different molecular geometry. Comparing with different
receptors, Aβ1–40 aggregates exhibited a relatively lower Ki value than AD patients
brain homogenates[11]. Thus, considered with both structural stability and binding
affinity, 5k was selected for 125I labeling and other further studies.
2.3. AD brain tissue sections fluorescent staining
This series of indanone derivatives showed green fluorescence and significant
affinities to β-amyloid plaques in fluorescent staining experiment in our previous
research[36]. Synthesized compounds stained β-amyloid plaques in vitro as showed in
Figure 1 (5k) and Figure S1 (5d). Compared with the silver staining (Figure 1A,) and
Thioflavin-S (ThS) staining (Figure 1B) in adjacent sections, 5k labeled β-amyloid
plaques in the same positions (Figure 1C), while the negative control was vehicle
stained adjacent section (Figure 1D). The enlarged images in fluorescence microscope
displayed the same results, and the white arrows indicated β-amyloid plaques (Figure
1 A2-C2, A4-C4). In vitro fluorescent staining experiment could exam the affinities of
candidate compounds directly and provide solid evidence for the following research.
2.4. Radioiodination
As showed in Scheme 3, the tributyltin precursor of radioiodination was
synthesized from 5d through substitution reaction. The [125I]5k was obtained through
standard iododestannylation reactions with [125I]sodium iodide, hydrochloric acid and
hydrogen peroxide. The overall radiochemical yield of [125I]5k was 60% after
purification by HPLC. The 125I-labeled compound was identified by co-injection with
nonradioactive 5k through HPLC and performed a retention time of 8.2min (Figure 2).
The [125I]5k after purification showed radiochemical purity greater than 97% and high
specific activity (approx. 2200Ci mM-1).

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2.5. Partition coefficient measurement
The partition coefficient of [125I]5k was measured by octanol/buffer partitioning.
5k showed a higher partition coefficient of 3.45 than indanone derivatives synthesized
previously (1, 2.80; 2, 2.57)[36]. This increase of partition coefficient may be due to
the adding of methoxy group. Increased lipophilicity would provide a stronger
binding affinity and a better BBB permeability[11].
2.6. In vitro stability study
The stability of [125I]5k was evaluated in ICR mouse and human tissues
respectively in vitro. As shown in Figure 3 and Figure S2, the percent of unchanged
[125I]5k were 9.9% in mouse brain homogenates and 56.0% in whole blood after
30min of incubation. Moreover, the [125I]5k was 98% unchanged in AD patient brain
homogenates for 60min of incubation and 95% for 120 min, while in normal human
brain homogenates the percent decreased to 92% and 90% respectively. The statistical
analysis of unchanged [125I]5k after 2-120min incubation in mouse whole blood, brain
and liver homogenates were shown in Figure 5D.
2.7. Biodistribution in normal mice
The kinetic property of [125I]5k was evaluated by biodistribution experiment in
normal mice (Table 2). [125I]5k shows a brain uptake value of 5.29%IDg-1 at 2 min
post-injection, which indicates its great effect of the BBB penetration. Compared with
1 (2.74%ID g-1) and 2 (4.58%ID g-1), the increase of initial brain uptake may be
results from the rise of lipophilicity of [125I]5k. Furthermore, the value of radioactive
at 60min in brain divide to 2min is considered to be an important parameter to
evaluate the clearance ability and in vivo potential of a candidate. Here, the value of
15.9% indicated a fast clearance of [125I]5k in normal mice brain. Relatively higher
lipophilicity of [125I]5k than previous compounds provides a higher initial
permeability through BBB which suggests its potential of a novel probe in vivo.

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2.8. In vitro autoradiography
The selective binding of [125I]5k to β-amyloid plaques was measured by in vitro
autoradiography with AD patients brain sections, APP/PS1 transgene mice brain
sections and normal human brain sections. The adjacent sections were incubated with
[125I]IMPY as positive control. In hippocampus area of AD patients, the [125I]5k
showed significant ability to stain β-amyloid plaques with a low background noise
(Figure 4 A1) at the same position stained by [125I]IMPY (Figure 4 B1). Likewise,
either of them showed negative staining and low non-specific binding in normal
human brain sections (Figure 4 A3 and B3). The same experiment was performed on
APP/PS1 transgenic mouse brain sections. [125I]5k and [125I]IMPY consistently
stained β-amyloid plaques distributed in the neocortex of APP/PS1 transgenic mice
(Figure 4 A2 and B2).
2.9. In situ micro-autoradiography
In order to overcome the limitation of the scanning resolution of X-ray film, the
in situ micro-autoradiography was performed by nuclear track emulsion in vitro to
detect the details of radioactive ligands binding to β-amyloid plaques. In the optical
microscope, the silver particles in the sections (Figure 5 B1 and C1) were observed
clearly at the same position as the positive fluorescent staining with ThS in the
adjacent sections (Figure 5 A1). The enlarged pictures (Figure 5 A2, B2 and C2)
shows the β-amyloid plaques stained by ThS, [125I]IMPY and [125I]5k accurately in
the same positions. The binding affinity and specificity of [125I]5k to β-amyloid
plaques were confirmed by X-ray film autoradiography and in situ
micro-autoradiography in vitro, independently.
2.10.
Ex vivo autoradiography of transgenic mice brain and wild-type mice brain
The ex vivo experiments were carried out respectively in APP/PS1 transgenic
mice and wild-type mice to further characterize the potential of [125I]5k in living mice
brain tissue. Compared to ThS fluorescent staining (Figure 6A1), the autoradiography
of X-film showed that [125I]5k labeled Aβ deposits in the cortex of transgenic mouse

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brain (Figure 6 B1), while the enlarged picture showed the labeled details (Figure 6
B2). There was no specific labeling in wild-type mouse brain (Figure 6 C and D).
3. Conclusion
We successfully synthesized a series of indanone derivatives with a methoxy
group at 6-position and evaluated their potential as β-amyloid probes. Compounds
with dimethyl and monomethyl amino showed improvement of binding affinities to
previous indanone derivatives in the binding assay in vitro. Compound 5k stained
β-amyloid plaques obviously in fluorescent staining and autoradiography experiment
in vitro. In biodistribution experiment in normal mice, [125I]5k exhibited a higher
initial uptake in brain than previous indanone derivatives and a rapid clearance from
the brain. Furthermore, [125I]5k exhibited the capacity of binding to Aβ deposits in
living transgenic mouse brain in the ex vivo autoradiography experiment. In
conclusion, with a structural optimization, we get a series of indanone compounds
with higher binding affinities to β-amyloid plaques and better brain uptake than
previous study. Compound [125I]5k shows great potential to become an in vivo
imaging probe for β-amyloid plaques.
4. Experimental Section
4.1. Materials and instruments
The reagents used in the synthesis were purchased from Alfa Aesar (UK),
Sigma-Aldrich (USA) and Sinopharm Chemical Reagent Co. Ltd (China) and were
used without further purification unless otherwise indicated. AD patients brain frozen
tissue and paraffin brain sections were obtained from the Netherlands brain bank
(coordinator: Dr. Inge Huitinga) with the permission for the use of tissue and clinical
information for research purposes. [125I]NaI (2200Ci mmol-1) was purchased from
Perkin-Elmer Life and Analytical Science (USA). The 1H NMR spectra were obtained
on a Bruker Avance-500 (500MHz) spectrometer with TMS as the internal standard.
High-resolution MS were obtained with Micromass GCT (EI-MS). FT-IR spectra
were obtained on a Thermo Scientific Nicolet 8700 system. HPLC system consisted

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of a Waters 1525 binary high-pressure gradient pump, a 2489 variable dual
wavelength ultraviolet detector, a Bioscan Flow-Count FC-3100 radioactivity detector,
and Hamilton manual glass injectors.
4.2. Chemistry
4.2.1. (E)-5-bromo-6-methoxyl-2-(4-nitrobenzylidene)indan-1-one (5a)
To a solution of 5-bromo-6-methoxy-indan-1-one 3a (169 mg, 0.7 mmol) and
4-nitrobenzaldehyde 4a (106 mg, 0.7 mmol) in ethanol (10 mL) was added 5% NaOH
aqueous solution (120 ꢀL) dropwise with stirring. The reaction mixture was carried
out for 1 hour at room temperature. The precipitate was filtered to give a yellow solid
and washed with ethanol to give 5a (76%). IR γ (cm-1) 1691, 1627, 1517, 1478, 1405,
1343, 1096, 847; HRMS: calcd for C17H1379BrNO4 [M+H]+ 374.0023; found:
374.0021; HRMS: calcd for C17H1381BrNO4 [M+H]+ 376.0004; found: 376.0002.
4.2.2. (E)-5-bromo-6-methoxyl-2-(4-aminobenzylidene)indan-1-one (5b)
To a solution of compound 5a (224 mg, 0.60 mmol) in ethanol (25 mL) was
added saturated hydrochloride (1 mL) and tin (II) chloride dehydrate (678 mg, 3.0
mmol). The reaction mixture was heated at 60℃ for 4 hours under nitrogen and then
cooled to room temperature. After ethanol was evaporated, 1M NaOH was added until
the mixture became alkaline. After extraction with ethyl acetate (50 mL×2), the
combined organic layers were washed with brine, dried over anhydrous Na2SO4, and
filtered. The solvent was removed, and the residue was purified by silica gel
1
chromatography (3:1 petroleum ether/ethyl acetate) to give 5b (56%). H NMR
(d6-DMSO): δ = 7.89 (s, 1H, =CHPh), 7.47 (d, 2H, J = 8.4 Hz, H-2’, H-6’), 7.41 (s,
1H, H-4), 7.32 (s, 1H, H-7), 6.66 (d, 2H, J = 8.4 Hz, H-3’, H-5’), 5.98 (br, 2H, NH2),
13
3.93 (s, 3H, OCH3), 3.87 (s, 2H, H-3); C NMR (CDCl3): δ=192.7, 156.3, 147.9,
142.4, 141.4, 138.5, 133.0, 131.4, 131.0, 124.2, 121.3, 115.0, 105.8, 56.7, 31.3. ppm;
HRMS: calcd for C17H1579BrNO2 [M+H]+ 344.0281; found: 344.0264; HRMS: calcd
for C17H1581BrNO2 [M+H]+ 346.0261; found: 346.0243.

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4.2.3. (E)-5-bromo-6-methoxyl-2-(4-(methylamino)benzylidene)indan-1-one (5c)
To a mixture of 5b (104 mg, 0.3 mmol) and paraformaldehyde (36 mg, 1.2 mmol)
in methanol (8 mL) was added a solution of sodium methoxide (81 mg, 1.5 mmol) at
0°C with stirring. The mixture was heated at 60 °C for 3 hours. After cooling to room
temperature, NaBH4 (68 mg, 1.8 mmol) was added and the solution was then refluxed
for 4h. After cooling the reaction mixture to 0 °C, the cold mixture was added 1M
NaOH aqueous solution and extracted with ethyl acetate. The organic phase was dried
over Na2SO4 and filtered. The filtrate was concentrated and the residue was
chromatographed on a silica gel column (eluted with 4: 1 hexane / ethyl acetate) to
give 5c (46%). 1H NMR (CDCl3): δ = 7.74 (s, 1H, =CHPh), 7.62 (s, 1H, H-4), 7.53 (d,
2H, J = 8.4 Hz, H-2’, H-6’), 7.35 (s, 1H, H-7), 6.64 (d, 2H, J = 8.4 Hz, H-3’, H-5’),
3.96 (s, 3H, OCH3), 3.90 (s, 2H, H-3), 2.91 (s, 3H, NHCH3); HRMS: calcd for
C18H1779BrNO2 [M+H]+ 358.0437; found: 358.0435; HRMS: calcd for C18H1781BrNO2
[M+H]+ 360.0417; found: 360.0416.
4.2.4. (E)-5-bromo-6-methoxyl-2-(4-(dimethylamino)benzylidene)indan-1-one (5d)
5-Bromo-6-methoxy-indan-1-one
3a
(241
mg,
1.0
mmol)
and
4-dimethylaminobenzaldehyde 4d (149 mg, 1.0 mmol) were dissolved in ethanol (10
mL). To this was added 10% NaOH aqueous solution (2.0 mL) dropwise with stirring.
The reaction was carried out for 6 hours at room temperature. The precipitate was
1
filtered and recrystallized from ethanol to give 5d (82%). H NMR (CDCl3): δ=7.74
(s, 1H, =CHPh), 7.63 (s, 1H, H-4), 7.56 (d, 2H, J = 8.7Hz, H-2’, H-6’), 7.35 (s, 1H,
H-7), 6.73 (d, 2H, J = 8.7 Hz, H-3’, H-5’), 3.96 (s, 3H, OCH3), 3.90 (s, 2H, H-3), 3.05
(s, 6H, N(CH3)2); 13C NMR (CDCl3): δ=193.2, 155.8, 151.3, 142.5, 139.2, 135.5,
132.9, 130.7, 130.0, 123.1, 119.2, 112.0, 105.6, 56.6, 40.1, 31.7 ppm; HRMS: calcd
for C19H1979BrNO2 [M+H]+ 372.0594; found: 372.0589; HRMS: calcd for
C19H1981BrNO2 [M+H]+ 374.0573; found: 374.0570.
4.2.5. (E)-5-bromo-6-methoxyl-2-(4-methoxybenzylidene)indan-1-one (5e)
Compound 5e was prepared from 5-bromo-6-methoxy-indan-1-one 3a (241 mg,

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1.0 mmol) and 4-methoxybenzaldehyde 4e (136 mg, 1.0 mmol) with the similar
1
procedure described for compound 5d (85%). H NMR (CDCl3): δ=7.76 (s, 1H,
=CHPh), 7.64 (s, 1H, H-4), 7.62 (d, 2H, J = 9.0 Hz, H-2’, H-6’), 7.35 (s, 1H, H-7),
6.98 (d, 2H, J = 9.0 Hz, H-3’, H-5’), 3.97 (s, 3H, OCH3), 3.94 (s, 2H, H-3), 3.87 (s,
3H, OCH3); 13C NMR (CDCl3): δ=193.4, 161.0, 155.9, 142.6, 138.6, 134.4, 132.6,
132.4, 130.8, 127.9, 119.9, 114.5, 105.6, 56.6, 55.4, 31.5 ppm.; HRMS: calcd for
C18H1679BrO3 [M+H]+ 359.0277; found: 359.0256; HRMS: calcd for C18H1681BrO3
[M+H]+ 361.0257; found: 361.0235.
4.2.6. (E)-5-bromo-6-methoxyl-2-(4-fluorobenzylidene)indan-1-one (5f)
Compound 5f was prepared from 5-bromo-6-methoxy-indan-1-one 3a (241 mg,
1.0 mmol) and 4-fluorobenzaldehyde 4f (124 mg, 1.0 mmol) with the similar
1
procedure described for compound 5d (86%). H NMR (CDCl3): δ=7.76 (s, 1H,
=CHPh), 7.66-7.63 (m, 3H, H-4, H-2’, H-6’), 7.34 (s, 1H, H-7), 7.17-7.14 (m, 2H,
13
H-3’, H-5’), 3.97 (s, 3H, OCH3), 3.94 (s, 2H, H-3); C NMR (d6-DMSO): δ=192.4,
164.0, 161.6, 155.4, 143.3, 137.7, 134.9, 133.1, 132.1, 131.0, 119.4, 116.2, 105.6,
55.6, 30.8 ppm; HRMS: calcd for C17H1379BrFO2 [M+H]+ 347.0078; found: 347.0075;
calcd for C17H1381BrFO2 [M+H]+ 349.0057; found: 349.0054.
4.2.7. (E)-5-bromo-6-methoxyl-2-benzylidene-indan-1-one (5g)
Compound 5g was prepared from 5-bromo-6-methoxy-indan-1-one 3a (241 mg,
1.0 mmol) and benzaldehyde 4g (106 mg, 1.0 mmol) with the similar procedure
1
described for compound 5d (86%). H NMR (CDCl3): δ=7.78 (s, 1H, =CHPh),
7.69-7.66 (m, 3H, H-4, H-2’, H-6’), 7.48 (t, 2H, J = 7.8 Hz, H-3’, H-5’), 7.44-7.41 (m,
13
1H, H-4’), 7.37 (s, 1H, H-7), 3.99 (s, 2H, H-3), 3.98 (s, 3H, OCH3); C NMR
(d6-DMSO): δ=193.4, 156.0, 142.8, 138.4, 135.2, 134.8, 134.6, 130.9, 130.8, 129.9,
129.0, 120.4, 105.7, 56.6, 31.5 ppm; HRMS: calcd for C17H1479BrO2 [M+H]+
329.0172; found: 329.0155; HRMS: calcd for C17H1481BrO2 [M+H]+ 331.0152; found:
331.0134.

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4.2.8. (E)-5-iodo-6-methoxyl-2-(4-nitrobenzylidene)indan-1-one (5h)
Compound 5h was prepared from 5-iodo-6-Methoxy-indan-1-one 3h (288 mg,
1.0mmol) and 4-nitrobenzaldehyde 4h (151 mg, 1.0 mmol) with the similar procedure
described for compound 5a (78%). IR γ (cm-1) 1693, 1629, 1516, 1471, 1401, 1314,
1098, 845; HRMS: calcd for C17H13INO4 [M+H]+ 421.9988; found: 421.9979.
4.2.9. (E)-5-iodo-6-methoxyl-2-(4-aminobenzylidene)indan-1-one (5i)
Compound 5i was prepared from compound 5h (252 mg, 0.6 mmol) with the
1
similar procedure described for compound 5b (52%). H NMR (CDCl3): δ=7.72 (s,
1H, =CHPh), 7.58 (s, 1H, H-4), 7.53-7.51 (m, 2H, H-2’, H-6’), 7.19 (s, 1H, H-7),
6.77-6.76 (d, 2H, J = 7.8 Hz, H-3’, H-5’), 3.91 (s, 2H, H-3), 3.87 (s, 3H, OCH3);
HRMS: calcd for C17H15INO2 [M+H]+ 392.0142; found: 392.0123.
4.2.10. (E)-5-iodo-6-methoxyl-2-(4-(methylamino)benzylidene)indan-1-one (5j)
Compound 5j was prepared from 5i (117 mg, 0.3 mmol) with the similar
1
procedure described for compound 5c (40%). H NMR (CDCl3): δ=7.61 (s, 1H,
=CHPh), 7.55 (d, 2H, J = 8.4 Hz, H-2’, H-6’), 7.43 (m, 1H, H-4), 7.35 (m, 1H, H-7),
6.65 (d, 2H, J = 8.4 Hz, H-3’, H-5’), 3.92 (s, 2H, H-3), 3.87 (s, 3H, OCH3), 2.91 (s,
3H, NHCH3); 13C NMR (CDCl3): δ=194.3, 159.5, 150.6, 142.2, 139.9, 134.8, 132.9,
131.0, 126.7, 124.5, 123.1, 112.3, 105.8, 55.7, 32.1, 30.3 ppm; HRMS: calcd for
C18H17INO2 [M+H]+ 406.0299; found: 406.0277.
4.2.11. (E)-5-iodo-6-methoxyl-2-(4-(dimethylamino)benzylidene)indan-1-one (5k)
Compound 5k was prepared from 5-iodo-6-methoxy-indan-1-one 3h (288 mg,
1.0 mmol) and 4-dimethylamino-benzaldehyde 4k (149 mg, 1.0 mmol) with the
1
similar procedure described for compound 5d (83%). H NMR (CDCl3): δ=8.00 (s,
1H, =CHPh), 7.64 (s, 1H, H-4), 7.56 (d, 2H, J = 8.7Hz, H-2’, H-6’), 7.26 (s, 1H, H-7),
6.73 (d, 2H, J = 8.7 Hz, H-3’, H-5’), 3.95 (s, 3H, OCH3), 3.89 (s, 2H, H-3), 3.05 (s,
13
6H, NMe2); C NMR (CDCl3): δ=193.4, 157.8, 151.3, 143.0, 140.4, 136.9, 135.6,
132.8, 130.0, 123.1, 112.0, 104.3, 94.8, 56.7, 40.1, 31.5 ppm; HRMS: calcd for

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C19H19INO2 [M+H]+ 420.0455; found: 420. 0450.
4.2.12. (E)-5-iodo-6-methoxyl-2-(4-methoxybenzylidene)indan-1-one (5l)
Compound 5l was prepared from 5-iodo-6-methoxy-indan-1-one 3h (288 mg, 1.0
mmol) and 4-methoxybenzaldehyde 4l (136 mg, 1.0 mmol) with the similar procedure
described for compound 5d (83%). 1H NMR (CDCl3): δ=8.03 (s, 1H, =CHPh), 7.66
(s, 1H, H-4), 7.62 (d, 2H, J = 9.0 Hz, H-2’, H-6’), 7.27 (s, 1H, H-7), 6.99 (d, 2H, J =
13
9.0 Hz, H-3’, H-5’), 3.96 (s, 3H, OCH3), 3.93 (s, 2H, H-3), 3.88 (s, 3H, OCH3); C
NMR (CDCl3): δ=192.5, 160.7, 157.5, 143.6, 138.9, 137.1, 133.3, 132.9, 132.7, 127.4,
114.6, 104.0, 96.4, 56.7, 55.4, 30.7 ppm; HRMS: calcd for C18H16IO3 [M+H]+
407.0139; found: 407.0116.
4.2.13. (E)-5-iodo-6-methoxyl-2-(4-fluorobenzylidene)indan-1-one (5m)
Compound 5m was prepared from 5-iodo-6-Methoxy-indan-1-one 3h (288 mg,
1.0 mmol) and 4-fluorobenzaldehyde 4m (124 mg, 1.0 mmol) with the similar
1
procedure described for compound 5d (85%). H NMR (CDCl3): δ=8.04 (s, 1H,
=CHPh), 7.67-7.65 (m, 3H, H-4, H-2’, H-6’), 7.27 (s, 1H, H-7), 7.18-7.15 (m, 2H,
13
H-3’, H-5’), 3.96 (s, 3H, OCH3), 3.94 (s, 2H, H-3); C NMR (CDCl3): δ=193.4,
164.7, 162.2, 158.1, 143.1, 139.5, 137.2, 133.4, 132.7, 132.6, 116.4, 104.3, 96.2, 56.7,
31.1 ppm; HRMS: calcd for C17H13FIO2 [M+H]+ 394.9939; found: 394.9939.
4.2.14. (E)-5-iodo-6-methoxyl-2-benzylidene-indan-1-one (5n)
Compound 5n was prepared from 5-iodo-6-methoxy-indan-1-one 3h (288 mg,
1.0 mmol) and benzaldehyde 4n (106 mg, 1.0 mmol) with the similar procedure
described for compound 5d (82%). 1H NMR (CDCl3): δ=8.03 (s, 1H,), 7.69 (s, 1H,
H-4), 7.65 (d, 2H, J = 7.2 Hz, H-2’, H-6’), 7.46 (t, 2H, J = 7.2 Hz, H-3’, H-5’),
7.43-7.41 (m, 1H, H-4’), 7.27 (s, 1H, H-7), 3.97 (s, 2H, H-3), 3.95 (s, 3H, OCH3); 13C
NMR (d6-DMSO): δ=192.7, 157.6, 143.8, 138.6, 137.2, 135.5, 134.8, 133.3, 130.7,
130.0, 129.0, 104.1, 96.9, 56.7, 30.7; HRMS: calcd for C17H14IO2 [M+H]+ 377.0033;
found: 377.0031.

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4.2.15.
(E)-5-tributylstannyl-6-methoxyl-2-(4-(dimethylamino)benzylidene)indan-1-one (5o)
A mixture of 5d (45mg, 0.12 mmol), bis(tributyltin) (417 mg, 0.73 mmol) and
Pd(PPh3)4 (28mg, 0.024 mmol) in toluene (5 mL) was heated at 110°C under nitrogen
atmosphere for 12 hours. The solvent was removed, and the residue was purified by
silica gel chromatography (eluted with 6:1 petroleum ether/ethyl acetate) to give 5o
(26%). 1H NMR (CDCl3): δ=7.62-7.60 (m, 3H, =CHPh, H-2’, H-6’), 7.53 (s, 1H, H-4),
7.24 (m, 1H, H-7), 6.74 (d, 2H, J = 9.0 Hz, H-3’, H-5’), 3.93 (s, 2H, H-3), 3.84 (s, 3H,
OCH3), 3.05 (s, 6H, NMe2), 1.55-1.51 (m, 6H, -CH2Sn), 1.35-1.32 (m, 6H,
-CH2CH2Sn), 1.10-1.07 (m, 6H, -CH2CH2CH2Sn), 0.91-0.88 (m, 9H, CH3).
4.3. Preparation of radioiodinated ligands
The radioiodinated ligands [125I]5k and [125I]IMPY were prepared from the
tributyltin precursor through an iododestannylation reaction according to the
procedure described in the literatures with some modifications[40]. 100µg precursor
(5o) was dissolved in ethanol at a concentration of 1µg µL-1, and sealed in a vial.
Then 1mCi [125I]NaI was quantified by a radioactivity calibrator (HD-175, Beijing,
China) and added into the vial via a syringe. The reaction was started by adding of
100µL hydrochloric acid solution (3% w w-1) and 100µL hydrogen peroxide solution
(3% w w-1) at the same time. The reaction was stopped by adding of 150µL saturated
sodium bisulfite solution 10 minutes later. After replacement the radioactive gas in the
vial, saturated sodium bicarbonate solution was used to regulate the pH value to
7.2-7.6. The product was washed with 10mL 20% ethanol solution and washed out
with 5mL 100% ethanol by a C18 sep-pek column (Waters, USA). The product
solution was concentrated by nitrogen and further purified with a reversed-phase
HPLC column (column: Waters Symmetry C18, 4.6×250mm, 5µm; mobile phase:
methanol/water 9:1 for [125I]5k or 8:2 for [125I]IMPY). The radioiodinated ligands
were identified by co-injection with unlabeled ligands according to previous methods.

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4.4. In vitro binding assays
The in vitro radioligand competition binding assays were followed the methods
described before with some modifications[36, 41]. Aβ1-40 was dissolved in a buffer
solution (pH 7.4) containing 10mM sodium phosphate and 1mM EDTA to a final
concentration of 0.5mg mL-1, incubated at 37°C for 48 hours, stored at -80°C, and
diluted to a final concentration of 0.2 µg mL-1. Frozen AD brain tissues (85-year-old
male, Braak 5) were dissected into gray and white matters on ice. Gray matters were
weighted and homogenized by a motor-driven homogenizer (GlasCol, USA) in
ice-cooled 10mM phosphate-buffered solution (PBS) to a final concentration of 50mg
mL-1. AD patients brain homogenates were stored at -80°C, and dissolved to a
concentration of 0.2mg mL-1 in the final mixture when used. The competition binding
studies were carried out in 12mm×75mm borosilicate glass tubes (VWR International,
USA). Radioactive ligand ([125I]IMPY, 0.06µCi mL-1 in the final assay mixture),
competitive ligands (3µ
M to 0.1nM in the final assay mixture), and receptors (Aβ1-40
aggregates or AD patients brain gray homogenates) were mixed on ice in the solution
of 50mM PBS (pH 7.4) including bovine serum albumin (0.1%, w v-1) to a final
volume of 1 mL. Nonspecific binding was defined in the presence of unlabeled IMPY
with a final concentration of 600nM according to previous methods. Then the mixture
was incubated in 37°C water bath shaker with a speed of 60rpm for 2 hours. The free
and bound radioactive ligands were separated by an M-24 cell harvester (Brandel,
USA) with 15mL cold PBS (pH 7.4, 50mM) through Whatman GF/B filters
(immersed with 1% polyethylenimine). Filters containing bound radioactive ligands
were counted in a γ counter (Zonkia GC-1200, Hefei, China) with a counting
efficiency of 65%. The inhibition constant (Ki) was calculated using the
Cheng-Prusoff equation: Ki=IC50 (1+[L]Kd)-1 by GraphPad Prism 5.0[42].
4.5. AD patients brain tissue sections staining
AD patients paraffin tissue was sliced into 6µm by a microtome (Leica RM2235,
Germany). Bielschowsky sliver staining and fluorescent staining was implemented
following the methods described in literature with some modifications[43]. The

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paraffin brain sections were washed sequentially in xylene, gradient ethanol solutions,
and PBS (10mM, pH=7.4) at first. In order to quench the auto-fluorescence, sections
were blanched in potassium permanganate solution (0.05%, w v-1) for 20 minutes,
washed in PBS (10mM, pH 7.4), then faded in oxalic acid and potassium
metabisulfite mixed solution (0.2% oxalic acid, 0.2% potassium metabisulfite, 10mM
PBS) for 30 seconds. After washing in PBS (10mM, pH 7.4) again, the sections were
stained respectively by ThS (0.1% in ddH2O) for 10minutes at room temperature or
5k (10µM in 20% ethanol) for 2 hours at 37°C. Then the sections were differentiated
in 70% ethanol (for ThS, 5minutes) or 10% ethanol (for 5k, 5minutes) in dark. At last,
the sections were washed in PBS (10mM, pH 7.4) and sealed with 80% glycerin and
coverslips. The sections were photographed by a fluorescence microscope (Olympus
IX81, Tokyo, Japan) and a SPOT digital camera (Diagnostic Instruments, USA).
4.6. Partition coefficient measurement
4g 1-octanol, 4g PBS (10mM, pH 7.4) and 6.5µCi [125I]5k were mixed
completely into a 15mL centrifuge tube. Then centrifuged 3000rpm for 10 minutes,
the tube was placed statically at room temperature for a while. After the initial
distribution, gently remove 5g 1-octanol which contained [125I]5k to a new 15mL
centrifuge tube, and mixed another 5g PBS (10mM, pH 7.4) completely. The tube was
centrifuged 3000rpm for 10 minutes and placed statically at room temperature for a
while again. At last, the lipid phase and aqueous phase was removed out gently and
respectively, and then measured the weight and counted by a γ counter (five times
each phase). The partition coefficient value was calculated from the following
formula. Log P=Log [(CLP/WLP) (CAP/WAP)-1]; CLP: count of lipid phase; WLP:
weight of lipid phase; CAP: count of aqueous phase; WAP: weight of the aqueous
phase.
4.7. In vitro stability study
The in vitro stability of [125I]5k was evaluated with the method described in the
literature with certain modifications[44]. The mouse tissues were obtained from ICR

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mice (18-22g, SPF). After peritoneal injection of pentobarbital sodium (50mg kg-1),
plasma samples were drawn from cardiac puncture and mixed with heparin (Heaplink,
China) to prevent clotting. Then, myocardial perfusion was performed with cold
saline (0.9%NaCl solution, 40mL). The brain and liver tissues were excised and
homogenized by an electric homogenizer to a concentration of 100mg mL-1 with cold
saline (0.9%NaCl solution). AD patients and control human brain homogenates were
prepared similarly to the method above and with a final concentration of 100mg mL-1
without separating white and gray matter.
Respectively, purified [125I]5k (100µCi) was dissolved in 50µl 0.9% sodium
chloride solution, and mixed with 300µl mouse whole blood, brain and liver
homogenates or AD patient and normal human brain homogenates. The mixture was
incubated at 37°C. The radioactive compounds were extracted by removing out 50µL
of the mixture and mixing with 200µL methanol and 50µL 0.9% sodium chloride
solution in each time point (10 minutes, 30minutes, 60minutes, and 120minutes).
After 13000rpm centrifugation for 20 minutes, the supernatant was detected by a
reversed-phase HPLC system (column: Waters Symmetry C18, 4.6×250mm, 5µm;
mobile phase: methanol/water 9:1).
4.8. Biodistribution in normal ICR mice
The experimental animals were 8 weeks male normal ICR mice (18-22g, SPF),
and the protocol was approved by the ethics committee of the School of Life Science,
University of Science and Technology of China. The [125I]5k was dissolved in sodium
chloride solution (0.9%NaCl solution) with a final concentration of 20µCi mL-1 and
injected 0.1mL to each male ICR mouse through the tail vein. There were at least
three mice killed by severing cervical quickly in each time point (2, 10, 30, 60, 120,
360, and 1440 minutes). Blood, brain, and organs interested were weighed and
measured the γ counts with a γ ray detector. %ID g-1= CO WO-1 1%CI-1, CO: counts
of organs (unit: cpm), WO: weight of organs (unit: g), 1%CI: counts of 1% injected
radioactive solution (unit: cpm).

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4.9. In vitro sections autoradiography (film and in situ micro-autoradiography)
The pretreatment of AD patients brain sections was followed the methods mentioned
in the part of sections staining above. The frozen mouse brain tissue was obtained
from an APP/PS1 transgenic mouse (C57BL/6, APPswe, PSEN1dE9, 14 months old,
male) purchased from Model Animal Research Center of Nanjing University, which
encodes a double mutant form of APP and PS1. The transgenic mice brain frozen
tissue was sliced into 25 µm by a Leica CM1900 cryostat microtome (Leica,
Germany). After washing in 50 nM PBS, all the sections were incubated with 50 mM
PBS (pH 7.4) contained 0.1% BSA at 37°C for 20 minutes. Then the BSA solution
was removed, and [125I]5k or [125I]IMPY (1.25µCi dissolved in 1 mL 50mM PBS
contained 0.1% BSA respectively) was dropped to the tissues. After incubation for 2
hours at 37°C, the sections were orderly washed in 50mM PBS (pH 7.4) for 5 minutes,
50 mM PBS contained 10% ethanol solution for 5 minutes, and 50mM PBS (pH 7.4)
for 5 minutes. The sections were washed out in water and dried at room temperature.
Using X-ray films (Kodak BioMax MR, USA) to exposure to the radioactive sections
in dark, and the images were scanned by a scanner. The in situ micro-autoradiography
was proceeded by covering the radio labeled sections with nuclear emulsion
(Amersham Hypercoat Emulsions, PRN40, USA) gently. The exposure times were
referred to the X-ray films autoradiography performed before. Finally, all the sections
were stained with ThS as the positive control. The optical images were obtained by an
optical microscopy (ZEISS Axioskop2 plus, Germany), and the fluorescent images
were acquired by a fluorescence microscopy (Olympus IX81, Tokyo, Japan).
4.10.
Ex vivo autoradiography of transgenic mouse brain and wild-type mouse
brain
The experiments were performed by using APP/PS1 transgenic mice (C57BL/6,
APPswe, PSEN1dE9, 14 months old, male) and wild-type mice (C57BL/6, age
matched, male) purchased from Model Animal Research Center of Nanjing University.
After purification, [125I]5k was concentrated with a C18 sep-pek column (Waters,
USA) to replace the methanol. 100µL solution of [125I]5k (0.9%NaCl solution

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contained 5% ethanol and 200µCi [125I)5k) were injected to each mouse through the
tail vein. After 30 minutes, the animals were killed by severing cervical quickly and
brains were frozen in liquid nitrogen. Sections of 25µm were cut in a Leica CM1900
cryostat microtome (Leica, Germany) and exposed to X-ray films (Kodak BioMax
MR) in dark. At last, sections were stained by ThS to confirm the deposition of
Aβ, and the images were obtained using a confocal laser scanning microscope (ZEISS
LSM510, Germany).
Acknowledgements
This work was supported by the Natural Science Foundation of China (91432305,
91132304, 81501187)，the Strategic Priority Research Program of the Chinese
Academy of Science (XDB02030001) and the Anhui Provincial Natural Science
Foundation (1508085QH173). The authors have declared no conflict of interest.

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References
[1] A. Alzheimer's, 2014 Alzheimer's disease facts and figures, Alzheimer's & dementia : the journal of
the Alzheimer's Association, 10 (2014) e47-92.
[2] R. Stewart, Comment: How personal is Alzheimer disease?, Neurology, 83 (2014) 1542.
[3] R. Brookmeyer, E. Johnson, K. Ziegler-Graham, H.M. Arrighi, Forecasting the global burden of
Alzheimer's disease, Alzheimer's & dementia : the journal of the Alzheimer's Association, 3 (2007)
186-191.
[4] I. Campbell-Taylor, Contribution of Alzheimer disease to mortality in the United States, Neurology,
83 (2014) 1302.
[5] E.D. Roberson, L. Mucke, 100 years and counting: prospects for defeating Alzheimer's disease,
Science, 314 (2006) 781-784.
[6] A. Nordberg, Amyloid imaging in Alzheimer's disease, Neuropsychologia, 46 (2008) 1636-1641.
[7] A. Nordberg, PET imaging of amyloid in Alzheimer's disease, The Lancet. Neurology, 3 (2004)
519-527.
[8] L. Zhu, K. Ploessl, H.F. Kung, PET/SPECT imaging agents for neurodegenerative diseases, Chemical
Society reviews, 43 (2014) 6683-6691.
[9] D. Square, Amyloid beta protein and Alzheimer disease, CMAJ : Canadian Medical Association
journal = journal de l'Association medicale canadienne, 156 (1997) 1040-1042.
[10] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer's disease: progress and problems on
the road to therapeutics, Science, 297 (2002) 353-356.
[11] L. Cai, R.B. Innis, V.W. Pike, Radioligand development for PET imaging of beta-amyloid
(Abeta)--current status, Current medicinal chemistry, 14 (2007) 19-52.
[12] Y.P. Yang, M.C. Cui, Radiolabeled bioactive benzoheterocycles for imaging beta-amyloid plaques in
Alzheimer's disease, Eur J Med Chem, 87 (2014) 703-721.
[13] C.A. Mathis, W.E. Klunk, J.C. Price, S.T. DeKosky, Imaging technology for neurodegenerative
diseases: progress toward detection of specific pathologies, Archives of neurology, 62 (2005) 196-200.
[14] J.C. Price, W.E. Klunk, B.J. Lopresti, X. Lu, J.A. Hoge, S.K. Ziolko, D.P. Holt, C.C. Meltzer, S.T. DeKosky,
C.A. Mathis, Kinetic modeling of amyloid binding in humans using PET imaging and Pittsburgh
Compound-B, Journal of cerebral blood flow and metabolism : official journal of the International
Society of Cerebral Blood Flow and Metabolism, 25 (2005) 1528-1547.
[15] M.P. Kung, C. Hou, Z.P. Zhuang, D. Skovronsky, H.F. Kung, Binding of two potential imaging agents
targeting amyloid plaques in postmortem brain tissues of patients with Alzheimer's disease, Brain
research, 1025 (2004) 98-105.
[16] N.P. Verhoeff, A.A. Wilson, S. Takeshita, L. Trop, D. Hussey, K. Singh, H.F. Kung, M.P. Kung, S. Houle,
In-vivo imaging of Alzheimer disease beta-amyloid with [11C]SB-13 PET, The American journal of
geriatric psychiatry : official journal of the American Association for Geriatric Psychiatry, 12 (2004)
584-595.
[17] E.D. Agdeppa, V. Kepe, J. Liu, G.W. Small, S.C. Huang, A. Petric, N. Satyamurthy, J.R. Barrio,
2-Dialkylamino-6-acylmalononitrile substituted naphthalenes (DDNP analogs): novel diagnostic and
therapeutic tools in Alzheimer's disease, Molecular imaging and biology : MIB : the official publication
of the Academy of Molecular Imaging, 5 (2003) 404-417.
[18] E.D. Agdeppa, V. Kepe, J. Liu, S. Flores-Torres, N. Satyamurthy, A. Petric, G.M. Cole, G.W. Small, S.C.
Huang, J.R. Barrio, Binding characteristics of radiofluorinated 6-dialkylamino-2-naphthylethylidene

ACCEPTED MANUSCRIPT
derivatives as positron emission tomography imaging probes for beta-amyloid plaques in Alzheimer's
disease, The Journal of neuroscience : the official journal of the Society for Neuroscience, 21 (2001)
RC189.
[19] K. Shoghi-Jadid, G.W. Small, E.D. Agdeppa, V. Kepe, L.M. Ercoli, P. Siddarth, S. Read, N.
Satyamurthy, A. Petric, S.C. Huang, J.R. Barrio, Localization of neurofibrillary tangles and beta-amyloid
plaques in the brains of living patients with Alzheimer disease, The American journal of geriatric
psychiatry : official journal of the American Association for Geriatric Psychiatry, 10 (2002) 24-35.
[20] K.T. Ong, V.L. Villemagne, A. Bahar-Fuchs, F. Lamb, N. Langdon, A.M. Catafau, A.W. Stephens, J.
Seibyl, L.M. Dinkelborg, C.B. Reininger, B. Putz, B. Rohde, C.L. Masters, C.C. Rowe, Abeta imaging with
18F-florbetaben in prodromal Alzheimer's disease: a prospective outcome study, Journal of neurology,
neurosurgery, and psychiatry, 86 (2015) 431-436.
[21] D.F. Wong, In Vivo Imaging of Amyloid Deposition in Alzheimer Disease Using the Radioligand
18F-AV-45 (Flobetapir F 18) (vol 51, pg 913, 2010), J Nucl Med, 51 (2010) 1327-1327.
[22] H.F. Kung, S.R. Choi, W.C. Qu, W. Zhang, D. Skovronsky, F-18 Stilbenes and Styrylpyridines for PET
Imaging of A beta Plaques in Alzheimer's Disease: A Miniperspective, J Med Chem, 53 (2010) 933-941.
[23] M. Koole, D.M. Lewis, C. Buckley, N. Nelissen, M. Vandenbulcke, D.J. Brooks, R. Vandenberghe, K.
Van Laere, Whole-body biodistribution and radiation dosimetry of 18F-GE067: a radioligand for in vivo
brain amyloid imaging, J Nucl Med, 50 (2009) 818-822.
[24] W. Zhang, M.P. Kung, S. Oya, C. Hou, H.F. Kung, F-18-labeled styrylpyridines as PET agents for
amyloid plaque imaging, Nucl Med Biol, 34 (2007) 89-97.
[25] C. Curtis, J.E. Gamez, U. Singh, C.H. Sadowsky, T. Villena, M.N. Sabbagh, T.G. Beach, R. Duara, A.S.
Fleisher, K.A. Frey, Z. Walker, A. Hunjan, C. Holmes, Y.M. Escovar, C.X. Vera, M.E. Agronin, J. Ross, A.
Bozoki, M. Akinola, J. Shi, R. Vandenberghe, M.D. Ikonomovic, P.F. Sherwin, I.D. Grachev, G. Farrar, A.P.
Smith, C.J. Buckley, R. McLain, S. Salloway, Phase 3 trial of flutemetamol labeled with radioactive
fluorine 18 imaging and neuritic plaque density, JAMA neurology, 72 (2015) 287-294.
[26] K. Adamczuk, J. Schaeverbeke, N. Nelissen, V. Neyens, M. Vandenbulcke, K. Goffin, J. Lilja, K.
Hilven, P. Dupont, K. Van Laere, R. Vandenberghe, Amyloid imaging in cognitively normal older adults:
comparison between F-flutemetamol and C-Pittsburgh compound B, European journal of nuclear
medicine and molecular imaging, (2015).
[27] K. Garber, First FDA-approved beta-amyloid diagnostic hits the market, Nature biotechnology, 30
(2012) 575.
[28] Z.J. Li, M.C. Cui, J.M. Zhang, J.P. Dai, X.J. Zhang, P. Chen, H.M. Jia, B.L. Liu, Novel F-18-labeled
dibenzylideneacetone derivatives as potential positron emission tomography probes for in vivo
imaging of beta-amyloid plaques, Eur J Med Chem, 84 (2014) 628-638.
[29] Y.P. Yang, H.L. Fu, M.C. Cui, C. Peng, Z.G. Liang, J.P. Dai, Z.Y. Zhang, C.P. Lin, B.L. Liu, Preliminary
evaluation of fluoro-pegylated benzyloxybenzenes for quantification of beta-amyloid plaques by
positron emission tomography, Eur J Med Chem, 104 (2015) 86-96.
[30] C.S. Gan, Z.Z. Zhao, D.D. Nan, B.B. Yin, J.Y. Hu, Homoisoflavonoids as potential imaging agents for
beta-amyloid plaques in Alzheimer's disease, Eur J Med Chem, 76 (2014) 125-131.
[31] E. Calamai, S. Dall'Angelo, D. Koss, J. Domarkas, T.J. McCarthy, M. Mingarelli, G. Riedel, L.F.
Schweiger, A. Welch, B. Platt, M. Zanda, 18F-barbiturates are PET tracers with diagnostic potential in
Alzheimer's disease, Chemical communications, 49 (2013) 792-794.
[32] P.J. Song, S. Bernard, P. Sarradin, J. Vergote, C. Barc, S. Chalon, M.P. Kung, H.F. Kung, D. Guilloteau,
IMPY, a potential beta-amyloid imaging probe for detection of prion deposits in scrapie-infected mice,

ACCEPTED MANUSCRIPT
Nucl Med Biol, 35 (2008) 197-201.
[33] A.B. Newberg, N.A. Wintering, K. Plossl, J. Hochold, M.G. Stabin, M. Watson, D. Skovronsky, C.M.
Clark, M.P. Kung, H.F. Kung, Safety, biodistribution, and dosimetry of I-123-IMPY: a novel amyloid
plaque-imaging agent for the diagnosis of Alzheimer's disease, J Nucl Med, 47 (2006) 748-754.
[34] M.P. Kung, Z.P. Zhuang, C. Hou, H.F. Kung, Development and evaluation of iodinated tracers
targeting amyloid plaques for SPECT Imaging, J Mol Neurosci, 24 (2004) 49-53.
[35] M.P. Kung, C. Hou, Z.P. Zhuang, B. Zhang, D. Skovronsky, J.Q. Trojanowski, V.M.Y. Lee, H.F. Kung,
IMPY: an improved thioflavin-T derivative for in vivo labeling of beta-amyloid plaques, Brain research,
956 (2002) 202-210.
[36] J.P. Qiao, C.S. Gan, C.W. Wang, J.F. Ge, D.D. Nan, J. Pan, J.N. Zhou, Novel Indanone Derivatives as
Potential Imaging Probes for ss-Amyloid Plaques in the Brain, Chembiochem, 13 (2012) 1652-1662.
[37] M. Ono, A. Wilson, J. Nobrega, D. Westaway, P. Verhoeff, Z.P. Zhuang, M.P. Kung, H.F. Kung,
11C-labeled stilbene derivatives as Abeta-aggregate-specific PET imaging agents for Alzheimer's
disease, Nucl Med Biol, 30 (2003) 565-571.
[38] D.N. Kevill, N.H. Cromwell, E.D. Weiler, Cis-Trans Isomerism of Exocyclic Alpha,Beta-Unsaturated
Indanones + Tetralones, J Org Chem, 29 (1964) 1276-&.
[39] C.A. Mathis, Y. Wang, D.P. Holt, G.F. Huang, M.L. Debnath, W.E. Klunk, Synthesis and evaluation of
11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents, J Med Chem, 46 (2003)
2740-2754.
[40] Z.P. Zhuang, M.P. Kung, A. Wilson, C.W. Lee, K. Plossl, C. Hou, D.M. Holtzman, H.F. Kung,
Structure-activity relationship of imidazo[1,2-a]pyridines as ligands for detecting beta-amyloid plaques
in the brain, J Med Chem, 46 (2003) 237-243.
[41] X.H. Duan, J.P. Qiao, Y. Yang, M.C. Cui, J.N. Zhou, B.L. Liu, Novel anilinophthalimide derivatives as
potential probes for beta-amyloid plaque in the brain, Bioorganic & medicinal chemistry, 18 (2010)
1337-1343.
[42] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and the concentration
of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochemical
pharmacology, 22 (1973) 3099-3108.
[43] T. Uchihara, Silver diagnosis in neuropathology: principles, practice and revised interpretation,
Acta neuropathologica, 113 (2007) 483-499.
[44] M.C. Cui, Z.J. Li, R.K. Tang, B.L. Liu, Synthesis and evaluation of novel benzothiazole derivatives
based on the bithiophene structure as potential radiotracers for beta-amyloid plaques in Alzheimer's
disease, Bioorganic & medicinal chemistry, 18 (2010) 2777-2784.

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Scheme 1. Chemical structures of β-amyloid plaques probes.
Scheme 2. Synthesis of compounds 5a-5n. Reagents and conditions: i) SnCl2ꢀ2H2O;
ii) (HCHO)n, CH3ONa, NaBH4.
Scheme 3. Synthesis of compounds 5o and [125I]5k.
Figure 1. Fluorescent staining of adjacent AD patients brain tissue sections
(83-year-old female, Braak 5, hippocampus, 6µm thickness). A) silver staining, B)
ThS, C) 5k, D) Blank control. The images A2-D2 and A4-D4 are magnified images of
the boxed area in A1-D1 and A3-D3 respectively. The white arrows indicate
β-amyloid plaques, and the scale bars are 100 µm.
Figure 2. HPLC chromatograms of A) 5k, B) [125I]5k and C) Merged. Flow: 1mL
minute-1, 90%methanol. Retention times: 8.1 minutes for 5k (UV), 8.2 minutes for
[125I]5k (γ).
Figure 3. In vitro stabilities of [125I]5k in plasma, brain homogenate and liver
homogenate of ICR mouse, and in AD and control human brain homogenates. The
contents of the images are as follow: A) purified [125I]5k, B) [125I]5k incubated in
mouse brain homogenate for 30 minutes, C) [125I]5k incubated in mouse plasma for
30 minutes, D) the statistical curves of unchanged [125I]5k in the mouse plasma, brain
homogenate and liver homogenate at 2 to 120 minutes after incubation, E) [125I]5k
incubated in AD patients brain homogenate for 60 minutes, F) [125I]5k incubated in
AD patients brain homogenate for 120 minutes, G) [125I]5k incubated in control
human brain homogenate for 60 minutes, H) [125I]5k incubated in control human
brain homogenate for 120 minutes. Flow: 1 mL per minute, 90%methanol. Retention
times: 8.8 minutes for [125I]5k (γ).
Figure 4. In vitro autoradiography of [125I]IMPY and [125I]5k binding to AD patients
brain sections (83-year-old female, Braak 5, hippocampus, 6µm thickness),
APP/PSEN1 transgenic mouse brain sections (14 months old, female, 20µm
thickness), and control human brain sections (72-year-old female, Braak 0,
hippocampus, 6µm thickness). A1-A3 is [125I]IMPY binding to AD patient brain
section, APP/PSEN1 transgenic mouse brain section and control human brain section

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respectively. As above, B1-B3 is [125I]5k. A1 and B1, A2 and B2, A3 and B3 is
adjacent section to each other. The black dots indicate the radioactive compounds
binding to β-amyloid plaques.
Figure 5. In situ micro-autoradiography of [125I]IMPY and [125I]5k binding to
adjacent AD patients brain sections (83-year-old female, Braak 5, hippocampus, 6 µm
thickness). A) ThS staining obtained from a fluorescent microscope, B) [125I]IMPY
and C) [125I]5k micro-autoradiography obtained from an optical microscope. The
images A2-C2 are magnified images of A1-C1 respectively The scale bar is 100µm.
Figure 6. Autoradiography of [125I]5k ex vivo using APP/PS1 transgenic mouse
(C57BL/6, APPswe, PSEN1dE9, 14 months old, male) and wild-type mouse
(C57BL/6, age marched, male). A) ThS staining of transgenic mouse brain section, B)
autoradiography of transgenic mouse brain section, C) ThS staining of wild-type
mouse brain section, D) autoradiography of wild-type mouse brain section. The image
A2 and B2 was magnified image of the boxed area in A1 and B1 respectively. The red
arrows indicate deposition of Aβ in APP/PS1 transgenic mouse brain. The scale bars
are 1000 µm.

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Table 1. The Ki value of compounds competed to [125I]IMPY with Aβ 1-40 aggregates and AD patients
brain homogenates.
O
O
R1
R2
R1
R2
Ki ± SEM (nM)[a]
Ki ± SEM(nM) [a]
(AD patients brain
homogenates)
>1000
(Aβ 1-40)
5a
5b
Br
Br
Br
Br
Br
Br
Br
I
NO2
NH2
88.31 ± 7.14
66.27 ± 0.91
27.32 ± 1.45
6.67 ± 0.70
189.76 ± 12.48
>1000
675.98 ± 27.47
66.64 ± 1.25
68.06 ± 3.1
>1000
5c
NHCH3
N(CH3)2
OCH3
F
5d
5e
5f
>1000
5g
H
211.47 ± 28.62
120.46 ± 17.50
25.36 ± 0.16
5.82 ± 0.19
11.85 ± 1.25
37.14 ± 1.28
>1000
>1000
5h
NO2
>1000
5i
I
NH2
>1000
5j
I
NHCH3
N(CH3)2
OCH3
F
18.96 ± 0.28
28.63 ± 0.66
>1000
5k
I
5l
I
5m
5n
I
>1000
I
H
354.15 ± 22.5
15[b]
>1000
IMPY
5[c]
[a] Each value was determined in three separate experiments with duplicate measurements each time.
[b] Accroding to the literature[40]. [c] Accroding to the literature[15].

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Table 2. Biodistribution of [125I]5k in normal male ICR mice, each point for three animals at
least. %ID g-1 (means ± SDs). The log P is partition coefficient measurement
.
organ
2 min
10 min
30 min
1h
2h
6h
24h
[
125I]5k (log P =3.45 )
blood
brain
heart
liver
15.23 ± 2.35
5.29 ± 1.03
28.91 ± 4.95
70.72 ± 11.66
12.31 ± 5.60
50.83 ± 16.20
31.84 ± 4.09
3.95 ± 0.40
7.90 ± 1.51
4.93 ± 0.33
6.89 ± 1.19
3.51 ± 0.57
5.97 ± 1.39
40.26 ± 6.00
17.52 ± 4.06
14.36 ± 2.51
19.31 ± 3.08
4.10 ± 0.82
3.00 ± 0.51
2.75 ± 0.44
4.29 ± 0.86
1.58 ± 0.34
2.81 ± 0.62
24.08 ± 7.40
15.95 ± 5.97
10.20 ± 5.80
11.14 ± 3.55
4.09 ± 1.61
1.83 ± 0.66
2.51 ± 0.96
3.46 ± 0.98
0.84 ± 0.13
1.92 ± 0.43
20.21 ± 4.83
15.13 ± 6.23
8.17 ± 2.10
8.22 ± 3.19
3.87 ± 1.42
2.05 ± 1.32
1.72 ± 0.34
2.51 ± 0.86
0.50 ± 0.20
1.55 ± 0.59
12.64 ± 2.59
3.18 ± 0.90
4.66 ± 1.60
6.17 ± 2.47
3.69 ± 1.84
3.38 ± 2.78
1.94 ± 0.15
1.17 ± 0.26
0.26 ± 0.06
0.61 ± 0.26
6.16 ± 1.39
0.87 ± 0.27
1.61 ± 0.37
2.40 ± 1.00
1.87 ± 0.26
0.84 ± 0.28
1.08 ± 0.42
0.72 ± 0.21
0.15 ± 0.03
0.37 ± 0.05
4.76 ± 0.76
1.02 ± 0.22
1.13 ± 0.25
0.61 ± 0.09
0.60 ± 0.04
0.21 ± 0.06
0.57 ± 0.21
spleen
lung
kidney
skin
muscle
bone

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