(E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives serve as probes for β-amyloid plaques

Article abstract of DOI:10.3390/molecules17044252

We report the synthesis and biological evaluation of novel (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives as probes for imaging β-amyloid (Aβ) plaques. These derivatives showed binding affinities for Aβ_1-40 aggregates with Ki values varying from 4.1 to 288.4 nM. (E)-5-(4-iodostyryl)-1H-indole (8) clearly stained Aβ plaques in the brain sections of Alzheimer's disease (AD) model mice (APP/PS1). Furthermore, autoradiography for [¹²⁵I]8 displayed intense and specific labeling of Aβ plaques in the brain sections mentioned above with low background. In biodistribution experiments using normal mice [ ¹²⁵I]8 showed high initial brain uptake followed by rapid washout (4.27 and 0.64% ID/g at 2 and 30 min post injection, respectively). These findings suggests that [¹²³I]8 may be a potential SPECT imaging agent for detecting Aβ plaques in AD brain.

Full text of DOI:10.3390/molecules17044252

Molecules 2012, 17, 4252-4265; doi:10.3390/molecules17044252
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
(E)-5-Styryl-1H-indole and (E)-6-Styrylquinoline Derivatives
Serve as Probes for β-Amyloid Plaques
Yang Yang, Hong-Mei Jia * and Bo-Li Liu *
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry,
Beijing Normal University, Beijing 100875, China; E-Mail: girlamy@mail.bnu.edu.cn
* Authors to whom correspondence should be addressed; E-Mails: hmjia@bnu.edu.cn (H.-M.J.);
liuboli@bnu.edu.cn (B.-L.L.); Tel./Fax: +86-10-5880-8891 (H.-M.J.).
Received: 20 February 2012; in revised form: 28 March 2012 / Accepted: 29 March 2012 /
Published: 10 April 2012
Abstract: We report the synthesis and biological evaluation of novel (E)-5-styryl-1H-indole
and (E)-6-styrylquinoline derivatives as probes for imaging β-amyloid (Aβ) plaques. These
derivatives showed binding affinities for Aβ1–40 aggregates with Ki values varying from 4.1
to 288.4 nM. (E)-5-(4-iodostyryl)-1H-indole (8) clearly stained Aβ plaques in the brain
sections of Alzheimer’s disease (AD) model mice (APP/PS1). Furthermore, autoradiography
for [125I]8 displayed intense and specific labeling of Aβ plaques in the brain sections
mentioned above with low background. In biodistribution experiments using normal mice
[125I]8 showed high initial brain uptake followed by rapid washout (4.27 and 0.64% ID/g
at 2 and 30 min post injection, respectively). These findings suggests that [123I]8 may be a
potential SPECT imaging agent for detecting Aβ plaques in AD brain.
Keywords: Alzheimer’s disease; β-amyloid plaques; binding affinity; imaging agent;
SPECT
1. Introduction
Alzheimer’s disease (AD) is a kind of irreversible, progressive brain disease characterized by
dementia, cognitive impairment and memory loss. Although currently the pathogenesis of AD is not
completely understood, it is generally accepted that β-amyloid (Aβ) plaques is considered to be one of
the biomarkers for early diagnosis of AD [1–3]. Therefore, in vivo imaging agent for Aβ plaques
applicable for PET (positron emission tomography) or SPECT (single photon emission computed

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tomography) would be very useful for early diagnosis of AD and provide significant information to
evaluate the efficacy of AD therapies [4,5].
To date, several radiolabeled ligands have been developed as imaging probes for Aβ plaques [6].
For example, [11C]SB-13 [7,8], [18F]BAY94-9172 [9] and [18F]AV-45 [10,11] derived from Congo
Red (CR), [11C]PIB [12,13], [18F]GE-067 [14] and [123I]IMPY [15–17] derived from thioflavin T
(ThT) (Figure 1). However, [123I]IMPY, the only SPECT tracer tested in human studies, has failed
because of its low in vivo stability and its insufficient target-to-background ratio. In comparison with
PET, SPECT is a more widely accessible and cost-effective technique in terms of routine diagnostic
use. Consequently, the development of more useful imaging agents for Aβ plaques labeled with 123I
(T1/2, 13 h, 159 keV) or 99mTc (T1/2, 6 h, 140 keV) for SPECT has been a critical issue.
Figure 1. Chemical structures of Aβ imaging probes for clinical study.
11CH3
NH
N
NH
18F
NH
18F
O
O
HO
3
3
[11C]SB-13
[18F]BAY94-9172
18F]AV-45
N
N
S
11CH3
NH
N
N
S
NH
N
123I
HO
18F
HO
[11C]PIB
[18F]GE-067
[
123I]IMPY
Previously, we successfully developed a series of novel imaging agents for β-amyloid plaques based
on the N-benzoylindole core which showed high binding affinities with Ki values in the nM range [18].
The brain uptake of these derivatives was encouraging, but their washout from the brain in normal
mice appeared to be relatively slow. Qu et al. have developed indolylphenylacetylenes as potential Aβ
plaques imaging agent, and the use of indolyl groups may improve the brain kinetics for β-amyloid
imaging agents [19]. Recently, Watanabe et al. have developed phenylindoles for image
β-amyloid in brain, these derivatives demonstrated high binding affinities to Aβ1–42 aggregates [20].
Following these successful results, we applied highly conjugated (E)-5-styryl-1H-indole as a core
structure for Aβ imaging agents to explore more useful candidates with favorable pharmacokinetics as
Aβ imaging probes, and developed (E)-6-styrylquinoline derivatives for further studies (Figure 2).
Reported herein are the synthesis and biological evaluation of novel (E)-5-styryl-1H-indole and
(E)-6-styrylquinoline derivatives and especially, two radioiodinated derivatives as potential SPECT
tracers for imaging β-amyloid plaques in the brain.
2. Results and Discussion
2.1. Chemistry and Radiochemistry
The synthetic route to the (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives is shown in
Scheme 1.

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Figure 2. Design considerations of (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives.
I
R3
N
H
R
R
R1
N
O
HN
O
O
N
H
R2
O
18F
N
Scheme 1. Synthetic route of (E)-5-styryl-1H-indole and (E)-6-Styrylquinoline derivatives.
R
125I
R
R
a
b
Br
P(Ph)3Br
2a.R=H, 2b.R=F
N
H
N
125I]8
4.R=H
5.R=F
6.R=Cl
7.R=Br
8.R=I
H
[
1a.R=H, 1b.R=F
1c.R=Cl, 1d.R=Br
1e.R=I, 1f.R=CH3
1g.R=OCH3
d
2c.R=Cl, 2d.R=Br
2e.R=I, 2f.R=CH3
2g.R=OCH3
c
11.R=SnBu3
9.R=CH3
10.R=OCH3
R
R
a
Br
P(Ph)3Br
1b. R=F, 1c. R=Cl
1d. R=Br, 1e. R=I
1f. R=CH3
2b. R=F, 2c. R=Cl
2d. R=Br, 2e. R=I
2f. R=CH3
125I
R
f
OHC
N
N
d
e
14. R=F
[
125I]17
15. R=Cl
16. R=Br
17. R=I
N
N
c
19. R=SnBu3
12
13
18. R=CH3
Reagents and Conditions: (a) PPh3, xylene, reflux; (b) 1H-indole-5-carbaldehyde, CH3ONa,
CH3OH; (c) (Bu3Sn)2, (Ph3P)4Pd, toluene, reflux; (d) [125I]NaI, H2O2, HCl, rt; (e) SeO2, 160 °C, 12 h;
(f) CH3ONa, CH3OH.
The key step was the base-catalyzed Wittig reaction between substituted triphenyl phosphonium
ylides 2a–g and 1H-indole-5-carbaldehyde or quinoline-6-carbaldehyde. The tributyltin derivatives 11,
19 were prepared in yields of 22.3% and 28.6%, respectively, from the bromo-precursors 7, 16 using
an exchange reaction catalyzed by Pd(0). [125I]8 and [125I]17 were prepared via a iodo-destannylation
reaction using hydrogen peroxide as the oxidant. The products were purified by radio-HPLC using a
reverse-phase column and mobile phase consisting of acetonitrile with a flow rate of 1 mL/min. In
order to identify the radiotracer, the non-radioactive 8 and 17 were co-injected and co-eluted with the

Molecules 2012, 17
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corresponding radioactive product, respectively. Their HPLC profiles using acetonitrile and water
(90:10 v/v) as mobile phase at a flow rate of 1 mL/min are present in Figure 3.
Figure 3. HPLC profiles of 8 (A, top), [125I]8 (A, bottom) and 17 (B, top), [125I]17 (B, bottom).
From Figure 3, the retention times of non-radioactive 8 and [125I]8 were observed to be 6.45 min
and 6.89 min, respectively. The retention times of non-radioactive 17 and [125I]17 were observed to be
19.56 min and 19.94 min, respectively. The differences in retention time were in good agreement with
the time lag which corresponds with the volume and flow rate within the distance between the UV and
radioactive detector of our HPLC system. After purification by HPLC, the radiochemical purities of
both [125I]8 and [125I]17 were greater than 98%. The radiochemical yields of [125I]8 and [125I]17 were
48–67% and 61–78%, respectively. The log D values of [125I]8 and [125I]17 were 2.52 ± 0.04 and
2.73 ± 0.03, respectively, which are in the appropriate range for brain imaging agents indicative of
good permeability through the blood-brain barrier (BBB).
2.2. In Vitro Binding Studies Using the Aggregated Aβ1–40
The affinity of (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives for Aβ1–40 aggregates
was determined by competition binding assay using [125I]TZDM as radio-ligand. TZDM was also
screened using the same competition assay for comparison. The Ki values shown in Table 1 were
varied from 4.1 to 288.4 nM suggesting that all these compounds share the same binding site with
ThT. The Ki value of TZDM was 4.2 nM, which is comparable to that of previously reported in the
literature (Ki = 0.9 nM) [21]. (E)-5-styryl-1H-indole (4) without any substituents showed moderate
binding affinity (Ki = 25.1 nM). Introducing a F, Cl or OCH3 group at the para-position of the phenyl
ring decreased the binding affinity (Ki = 89.3, 51.5 and 32.4 nM for compounds 5, 6 and 10, respectively),
while introducing a Br or CH3 group at the same position increased the affinity (Ki = 16.3 and 15.8 nM
for compounds 7 and 9, respectively). It is noteworthy that compound 8 with a iodo group showed
Ki value of 4.1 nM, which is comparable with that of TZDM. In general, (E)-5-styryl-1H-indole
derivatives showed slightly better potency in binding to Aβ1–40 aggregates than (E)-6-styrylquinoline
derivatives. Since derivatives 8 and 17 with iodine at the para-position of the phenyl ring displayed

Molecules 2012, 17
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nanomolar affinities for Aβ1–40 aggregates, we prepared [125I]8 and [125I]17 for further evaluation as
potential ligands for 123I-labeled SPECT imaging agents.
Table 1. Ki values of (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives for Aβ1–40
aggregates against [125I]TZDM.
Compound Ki (nM) a
Compound Ki (nM) a
4
25.1 ± 2.1 10
89.3 ± 2.6 14
32.4 ± 1.9
5
270.4 ± 1.5
45.0 ± 1.3
23.5 ± 1.3
8.6 ± 1.2
6
51.5 ± 1.0 15
16.3 ± 1.7 16
7
8
4.1 ± 0.2
15.8 ± 1.5 18
4.2 ± 0.4
TZDM b
17
9
288.4 ± 1.3
TZDM
0.9 ± 0.2
a Measured in triplicate with results given as the mean ± SD; b Data from [21].
2.3. In Vitro Fluorescent Staining of Amyloid Plaques in Brain Sections from Transgenic Mouse
To confirm the binding affinities of these derivatives for Aβ plaques in the brain, in vitro fluorescent
staining of brain sections (8 µm) from a transgenic model mouse (APP/PS1, 12 months, male) was
carried out with compound 8. As shown in Figure 4, many fluorescence spots were observed in the
brain sections of transgenic mice (Figure 4B). The fluorescent labeling pattern was consistent with that
observed with thioflavin-S (Figure 4A). These results suggested that 8 show specific binding to Aβ
plaques in the transgenic model mouse brain.
Figure 4. The labeled plaques were confirmed by staining of the adjacent sections by
thioflavin-S (A); Fluorescence staining of compound 8 on AD model mouse sections from
the cortex (B).
2.4. In Vitro Labeling of Brain Sections from Transgenic Mouse by Autoradiography
The results of in vitro autoradiography of [125I]8 in the brain sections of a transgenic model mouse
(APP/PS1, 12 months, male) are shown in Figure 5. [125I]8 showed excellent labeling of Aβ plaques in
the cortex region of the brain sections, and no remarkable accumulation of radioactivity were observed
in white matter. The same sections were also stained with thioflavin-S and the localizations of Aβ
plaques were in accord with the results of autoradiography. These results demonstrated that [125I]8

Molecules 2012, 17
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was specific for Aβ plaques, which were consistent with the high binding affinity of compound 8 to
Aβ1–40 aggregates.
Figure 5. The presence and distribution of plaques in the sections were confirmed with
thioflavin-S staining (A, C) (red arrows); Autoradiography of [125I]8 in vitro in Tg model
mouse (APP/PS1, 12 months, male) brain sections (B).
2.5. In Vivo Biodistribution Studies
In vivo biodistribution studies of [125I]8 and [125I]17 were performed in normal mice. The uptake of
radiotracer in the organs of interest at different time points after intravenous administration of [125I]8
and [125I]17 is summarized in Table 2. [125I]8 showed high initial brain uptake followed by rapid
clearance (4.27 and 0.28% ID/g at 2 and 60 min post injection, respectively). On the other hand,
[125I]17 showed relatively low brain uptake and slow washout (2.05 and 0.55% ID/g at 2 and 60 min
post injection, respectively). As compared with previously reported radioiodinated N-benzoylindole
derivatives [18], radioiodinated (E)-5-styryl-1H-indole derivative [125I]8 showed greatly improved
brain uptake. Because there are no plaques in normal brain, potential Aβ-specific probe should possess
high brain uptake followed by fast washout in normal mice. The brain2min/brain60min ratio has been used
as an index to compare the washout rate from normal brain and select candidate tracers with
appropriate kinetics in vivo. It was reported that [123I]IMPY showed a high initial brain uptake and fast
washout in normal mice (2.88% ID/organ and 0.21% ID/organ at 2 min and 60 min postinjection,
respectively) [16]. The brain2min/brain60min ratio of [125I]8 (15.3) is higher than that of [123I]IMPY
(13.7), indicating [125I]8 may possess suitable pharmacokinetic properties for imaging Aβ plaques in
AD brain. Accordingly, [125I]8 may be comparable or even better for detecting Aβ plaques. Therefore,

Molecules 2012, 17
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(E)-5-styryl-1H-indole derivative [125I]8, with nanomolar affinity to Aβ1–40 aggregates, excellent BBB
permeability as well as fast washout from the normal brain, may be suitable for development as a
novel Aβ imaging agent.
Table 2. Biodistribution in normal mice after iv injection of [125I]8 and [125I]17 (% ID/g,
avg of 5 mice ± SD) and its partition coefficient (D).
Organ
2 min
15 min
30 min
60 min
120 min
240 min
[
125I]8 (log D = 2.52 ± 0.04)
Blood
Brain
Heart
Liver
11.91 ± 0.62 12.29 ± 1.30 7.64 ± 0.62
4.49 ± 0.28
0.28 ± 0.06
1.59 ± 0.28
4.45 ± 0.23
2.44 ± 0.12
3.28 ± 0.10
4.65 ± 0.98
1.72 ± 0.21
0.81 ± 0.12
2.93 ± 0.30
0.20 ± 0.08
1.47 ± 0.48
4.19 ± 0.61
1.87 ± 0.28
2.30 ± 0.20
1.93 ± 0.76
3.48 ± 0.79
0.61 ±0.24
1.50 ± 0.13
0.10 ± 0.02
0.65 ± 0.16
3.06 ± 0.36
1.37 ± 0.09
1.43 ± 0.52
1.34 ± 0.36
2.31 ± 1.44
0.35 ± 0.09
4.27 ± 0.49
5.76 ± 0.38
1.37 ± 0.16
3.70 ± 0.40
0.64 ± 0.11
2.55 ± 0.52
14.73 ± 0.66 10.66 ± 0.31 7.14 ± 1.13
Spleen
Lung
4.38 ± 0.33
4.21 ± 0.26
3.44 ± 0.21
5.39 ± 0.83
10.76 ± 0.63 8.22 ± 0.88
Kidney
Stomach a
Muscle
11.66 ± 1.52 14.89 ± 4.23 9.46 ± 1.95
1.23 ± 0.56
2.64 ± 0.40
4.82 ± 0.46
2.01 ± 0.24
3.50 ± 0.19
1.25 ± 0.04
[
125I]17 (log D = 2.73 ± 0.03)
Blood
Brain
Heart
Liver
11.39 ± 1.56 6.31 ± 0.51
5.80 ± 0.37
0.93 ± 0.13
3.21 ± 0.11
9.12 ± 0.53
4.66 ± 0.52
5.64 ± 0.41
6.57 ± 0.50
3.86 ± 0.74
0.55 ± 0.11
2.55 ± 0.21
6.97 ± 0.28
4.71 ± 0.97
4.55 ± 0.49
4.72 ± 0.69
6.75 ± 0.43
1.40 ± 0.34
1.97 ± 0.35
0.26 ± 0.03
1.33 ± 0.06
3.93 ± 0.46
2.25 ± 0.36
2.01 ± 0.18
2.20 ± 0.34
1.38 ± 0.24
0.14 ± 0.02
0.80 ± 0.19
2.96 ± 0.28
1.59 ± 0.17
1.43 ± 0.28
1.64 ± 0.23
2.05 ± 0.25
7.70 ± 0.86
1.18 ± 0.17
3.80 ± 0.12
22.45 ± 1.79 9.95 ± 0.18
5.88 ± 0.30 5.91 ± 0.58
Spleen
Lung
13.56 ± 1.71 6.42 ± 0.47
15.01 ± 1.56 7.47 ± 1.01
Kidney
Stomach a
Muscle
4.05 ± 0.09
2.78 ± 0.42
15.84 ± 0.78 8.11 ± 1.21
1.68 ± 0.20 2.30 ± 0.32
a Expressed as % ID/organ.
11.12 ± 2.48 7.03 ± 1.51
0.72 ± 0.21 0.61 ± 0.08
3. Experimental
3.1. General
Unless otherwise indicated, all chemicals used in synthesis were commercial products and were
used without further purification. Na125I (2200 Ci/mmol) was obtained from PerkinElmer Life and
Analytical Sciences, USA. The double transgenic (APP/PS1) AD model mouse was obtained from
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative
1
Medicine Center of Peking Union Medical College (Beijing, China). H-NMR spectra were obtained
on Bruker (400 MHz) NMR spectrometer at room temperature with TMS as an internal standard.
Chemical shifts are reported as δ values relative to internal TMS. Coupling constants are reported in
hertz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), and m (multiplet). Mass spectra were
acquired using the Surveyor MSQ Plus (ESI) (Waltham, MA, USA) instrument. HPLC was performed
on a Shimadzu SCL-10AVP system (Shimadzu Corporation, Kyoto, Japan) which consisted of a
binary pump with on-line degasser, a model SPD-10AVP UV-VIS detector operating at a wavelength
of 254 nm, and a Packard 500TR series flow scintillation analyzer (Packard BioScience Co.,

Molecules 2012, 17
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Wallingford, CT, USA) with a Alltech Alltima RPC-18 column (5 μm, ID = 4.6 mm, length = 250 mm).
The samples were analyzed using acetonitrile and water (90:10 v/v) as mobile phase at a flow rate of
1 mL/min. The sample was separated using acetonitrile as mobile phase at a flow rate of 1 mL/min. All
key compounds were proven by analytical HPLC analysis to show ≥95% purity (Supporting information).
3.1.1. General Procedure for Preparing Substituted Triphenyl Phosphonium Ylide 2 (2a–g)
The suitable 4-substituted-1-(bromomethyl)benzene 1a–g (1 mmol) and triphenylphosphine (1 mmol)
was heated to reflux in xylene (10 mL) for about 6 h. The mixture was filtered and crude materials
were purified by recrystallization with toluene.
3.1.2. General Procedure for Preparing 4–11, 13
The appropriate substituted compounds 2a–g (1 mmol), 1H-indole-5-carbaldehyde (3, 1 mmol), and
CH3ONa (1 mmol) was heated to reflux in CH3OH (12 mL) for about 5 h. The organic solvent was
removed under vacuum. Crude materials were purified by column chromatography on silica gel
(petroleum ether/AcOEt, 4/1).
(E)-5-Styryl-1H-indole (4). Yield 53.6%, 1H-NMR (DMSO-d6) δ: 7.73 (1H, s), 7.58 (2H, d, J = 7.5 Hz),
7.43–7.38 (5H, m), 7.32 (1H, d, J = 16.5 Hz), 7.22 (1H, t, J = 7.2 Hz), 7.12 (1H, d, J = 16.4 Hz), 6.44
(1H, d, J = 3.0 Hz). HRMS m/z C16H13N found 220.1120/calcd 220.1126 ([M+H]+). m.p. 162–163 °C.
(E)-5-(4-Fluorostyryl)-1H-indole (5). Yield 71.4%, 1H-NMR (DMSO-d6) δ: 7.72 (1H, s), 7.62 (2H, dd,
J1 = 8.6 Hz, J2 =5.7 Hz), 7.40 (2H, dd, J1 = 11.4 Hz, J2 = 8.6 Hz), 7.34 (1H, d, J = 3.0 Hz), 7.27 (1H, d,
J = 16.4 Hz), 7.19 (2H, t, J = 8.8 Hz), 7.12 (1H, d, J = 16.4 Hz), 6.44 (1H, d, J = 3.0 Hz). HRMS m/z
C16H12FN found 238.0878/calcd 238.0876 ([M+H]+). m.p. 179–180 °C.
(E)-5-(4-Chlorostyryl)-1H-indole (6). Yield 70.2%, 1H-NMR (DMSO-d6) δ: 7.74 (1H, s), 7.60 (2H, d,
J = 8.5 Hz), 7.44–7.38 (4H, m), 7.35 (1H, d, J = 3.0 Hz), 7.34 (1H, d, J = 16.4 Hz), 7.12 (1H, d,
J = 16.4 Hz), 6.44 (1H, d, J = 3.0 Hz). HRMS m/z C16H12ClN found 254.0733/calcd 254.0737
([M+H]+). m.p. 205–206 °C.
(E)-5-(4-Bromostyryl)-1H-indole (7). Yield 68.3%, 1H-NMR (DMSO-d6) δ: 7.74 (1H, s), 7.54 (4H, s),
7.43–7.40 (2H, m), 7.36 (1H, d, J = 16.4 Hz), 7.35 (1H, d, J = 3.1 Hz), 7.11 (1H, d, J = 16.4 Hz), 6.44
(1H, d, J = 3.0 Hz). HRMS m/z C16H12BrN found 298.0235/calcd 298.0231 ([M+H]+). m.p. 215–216 °C.
1
(E)-5-(4-Iodostyryl)-1H-indole (8). Yield 55.3%, H-NMR (DMSO-d6) δ: 7.73 (1H, s), 7.70 (2H, d,
J = 8.4 Hz), 7.43–7.39 (4H, m), 7.36 (1H, d, J = 16.4 Hz), 7.34 (1H, d, J = 3.1 Hz), 7.08 (1H, d,
J = 16.5 Hz), 6.44 (1H, d, J = 2.9 Hz). HRMS m/z C16H12IN found 246.1283/calcd 246.1299
([M+H]+). m.p. 211–212 °C.
1
(E)-5-(4-Methoxystyryl)-1H-indole (9). Yield 67.7%, H-NMR (DMSO-d6) δ: 7.70 (1H, s), 7.47 (2H,
d, J = 8.0 Hz), 7.39 (2H, dd, J1 = 11.5 Hz, J2 = 8.6 Hz), 7.33 (1H, d, J = 3.0 Hz), 7.25 (1H, d, J = 16.4 Hz),
7.17 (2H, d, J = 8.0 Hz), 7.08 (1H, d, J = 16.4 Hz), 6.44 (1H, d, J = 3.0 Hz), 2.31 (3H, s). HRMS m/z
C17H15N found 234.1126/calcd 234.1126 ([M+H]+). m.p. 176–177 °C.

Molecules 2012, 17
4260
(E)-5-(4-Methoxystyryl)-1H-indole (10). Yield 65.2%, 1H-NMR (DMSO-d6) δ: 7.68 (1H, s), 7.51 (2H,
d, J = 8.6 Hz), 7.40–7.35 (2H, m), 7.33 (1H, d, J = 3.0 Hz), 7.16 (1H, d, J = 16.4 Hz), 7.06 (1H, d,
J = 16.4 Hz), 6.93 (2H, d, J = 8.5 Hz), 6.42 (1H, d, J = 3.0 Hz), 3.77 (3H, s). HRMS m/z C17H15NO
found 250.1241/calcd 250.1232 ([M+H]+). m.p. 163–164 °C.
(E)-5-(4-(Tributylstannyl)styryl)-1H-indole (11). A mixture of 7 (29.8 mg, 0.1 mmol), bis(tributyltin)
(290 mg, 0.5 mmol), and Pd(Ph3P)4 (12.0 mg, 0.01 mmol) in toluene (15 mL) was stirred at 110 °C
overnight. After removing the solvent in vacuo, the crude products were purified by column
chromatography (petroleum ether/AcOEt, 20/1) to give 11 as a light-yellow-colored solid with a yield
of 22.3%. 1H-NMR (CDCl3) δ: 8.87 (1H, d, J = 2.8 Hz), 8.15 (1H, d, J = 8.1 Hz), 8.08 (1H, d, J = 8.7 Hz),
7.98 (1H, dd, J1 = 8.9 Hz, J2 = 1.6 Hz), 7.82 (1H, s), 7.52 (4H, dd, J1 = 19.6 Hz, J2 = 8.5 Hz), 7.40
(1H, dd, J1 = 8.2 Hz, J2 = 4.2 Hz),7.28 (2H, dd, J1 = 16.4 Hz, J2 = 8.0 Hz), 1.64–1.55 (6H, m),
1.40–1.35 (6H, m), 1.16–1.11 (6H, m), 0.97–0.90 (9H, m). ESI-MS m/z C28H39NSn found 510.4/calcd
509.2 ([M+H]+).
Quinoline-6-carbaldehyde (13). Quinoline-6-carbaldehyde (13) was prepared from 6-methylquinoline
(12) according to the previously reported procedure [22]. 6-Methylquinoline (12, 4.0 g, 27.6 mmol)
was heated to 160 °C and selenium dioxide (2.0 g, 18.4 mmol) was added. The mixture was stirred for
16 h, cooled to room temperature, and diluted with ethyl acetate (30 mL). The solution was decanted,
and the residue was extracted with ethylacetate (20 mL × 2). The combined organic phase was
concentrated, and the residue was purified by column chromatography on silica gel (petroleum
ether/AcOEt, 4/1) to give 13 as a light gray solid (1.3 g, 30%). 1H-NMR (CDCl3) δ: 10.21 (1H, s), 9.06
(1H, dd, J1 = 4.2 Hz, J2 = 1.6 Hz), 8.38–8.35 (2H, m), 8.27–8.24 (2H, m), 7.55 (1H, dd, J1 = 8.3 Hz,
J2 = 4.3 Hz).
3.1.3. General procedure for preparing 14–19
The suitable substituted compounds 2b–f (1 mmol), 13 (1 mmol), CH3ONa (1 mmol) was heated to
reflux in CH3OH (12 mL) for about 6 h. The organic solvent was removed under vacuum. Crude
materials were washed by water and purified by column chromatography on silica gel (petroleum
ether/AcOEt, 6/1).
(E)-6-(4-Fluorostyryl)quinoline (14). Yield 66.8%, 1H-NMR (DMSO-d6) δ: 8.86 (1H, dd, J1 = 8.9 Hz,
J2 = 1.4 Hz), 8.35 (1H, d, J = 8.0 Hz), 8.11 (1H, dd, J1 = 8.8 Hz, J2 = 1.5 Hz), 8.07 (1H, s), 8.01 (1H,
d, J = 8.8 Hz), 7.73(2H, dd, J1 = 8.7 Hz, J2 = 5.7 Hz), 7.54 (1H, dd, J1 = 8.2 Hz, J2 = 4.2 Hz), 7.45 (2H,
dd, J1 = 16.5 Hz, J2 = 7.7 Hz), 7.26 (1H, t, J = 8.8 Hz). HRMS m/z C17H12FN found 250.1039/calcd
250.1032 ([M+H]+). m.p. 120–121 °C.
1
(E)-6-(4-Chlorostyryl)quinoline (15). Yield 72.3%, H-NMR (DMSO-d6) δ: 8.86 (1H, d, J = 3.4 Hz),
8.35 (1H, d, J = 7.9 Hz), 8.12 (1H, d, J = 8.8 Hz), 8.08 (1H, s), 8.01 (1H, d, J = 8.7 Hz), 7.70 (2H, d,
J = 8.2 Hz), 7.53 (2H, dd, J1 = 8.4 Hz, J2 = 4.1 Hz), 7.49–7.47 (4H, m). HRMS m/z C17H12ClN found
266.0732/calcd 266.0737 ([M+H]+). m.p. 129–130 °C.

Molecules 2012, 17
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(E)-6-(4-Bromostyryl)quinoline (16). Yield 73.9%, 1H-NMR (DMSO-d6) δ: 8.87 (1H, dd, J1 = 4.1 Hz,
J2 = 1.5 Hz), 8.36 (1H, d, J = 8.0 Hz), 8.14 (1H, dd, J1 = 8.8 Hz, J2 = 1.6 Hz), 8.09 (1H, s), 8.01 (1H,
d, J = 8.8 Hz), 7.63(4H, dd, J1 = 13.3 Hz, J2 = 8.8 Hz), 7.54 (1H, dd, J1 = 8.1 Hz, J2 = 4.2 Hz), 7.48
(2H, dd, J1 = 16.6 Hz, J2 = 13.3 Hz). HRMS m/z C17H12BrN found 310.0217/calcd 310.0231
([M+H]+). m.p. 143–144 °C.
1
(E)-6-(4-Iodostyryl)quinoline (17). Yield 70.2%, H-NMR (DMSO-d6) δ: 8.87 (1H, d, J = 4.1 Hz),
8.35 (1H, d, J = 8.0 Hz), 8.12 (1H, d, J = 8.9 Hz), 8.09 (1H, s), 8.01 (1H, d, J = 8.9 Hz), 7.78 (2H, d,
J = 8.0 Hz), 7.56–7.40 (5H, m). HRMS m/z C17H12IN found 358.0100/calcd 358.0093 ([M+H]+). m.p.
173–174 °C.
1
(E)-6-(4-Methylstyryl)quinoline (18). Yield 71.7%, H-NMR (DMSO-d6) δ: 8.85 (dd, J1 = 4.0 Hz,
J2 = 1.4 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.11 (dd, J1 = 8.8 Hz, J2 = 1.6 Hz, 1H), 8.06 (s, 1H), 8.01
(d, J = 8.8 Hz, 1H), 7.57(d, J = 8.0 Hz, 2H), 7.54 (dd, J1 = 8.3 Hz, J2 = 4.3 Hz, 1H), 7.41 (s, 2H), 7.23
(d, J = 7.9 Hz, 2H). HRMS m/z C17H12IN found 246.1299/calcd 246.1283 ([M+H]+). m.p. 125–126 °C.
(E)-6-(4-(Tributylstannyl)styryl)quinolin (19). The same reaction described above to prepare 11 was
1
used, and a primrose yellow-colored solid of 19 was obtained in a yield of 28.6% from 16. H-NMR
(CDCl3) δ: 8.87 (1H, d, J = 2.8 Hz), 8.15 (1H, d, J = 8.1 Hz), 8.08 (1H, d, J = 8.7 Hz), 7.98 (1H, dd,
J1 = 8.9 Hz, J2 = 1.6 Hz), 7.82 (1H, s), 7.52 (4H, dd, J1 = 19.6 Hz, J2 = 8.5 Hz), 7.40 (1H, dd, J1 = 8.2 Hz,
J2 = 4.2 Hz),7.28 (2H, dd, J1 = 16.4 Hz, J2 = 8.0 Hz), 1.64–1.55 (6H, m), 1.40–1.34 (6H, m), 1.16–1.13
(6H, m), 0.96–0.91 (9H, m). ESI-MS m/z C29H39NSn found 522.6/calcd 521.2 ([M+H]+).
3.1.4. Preparation of Radioiodinated Ligands
The radioiodinated compounds [125I]8 and [125I]17 were prepared from the corresponding tributyltin
derivatives by an iododestannylation according to the procedure described previously [21]. Briefly,
H2O2 (3%, 100 µL) was added to a mixture of a tributyltin derivative (0.1 mg/100 µL in ethanol),
sodium [125I]iodide (specific activity 2,200 Ci/mmol), and 1 M HCl (100 µL) in a sealed vial. The
reaction was allowed to proceed at room temperature for 15 min and then quenched by addition
of saturated NaHSO3 solution (50 µL). The reaction mixture, after neutralization with 1 M NaOH, was
purified by HPLC using a Alltech Alltima RPC-18 column (250 mm  4.6 mm, 5 µm) and mobile
phase consisting of acetonitrile with a flow rate of 1.0 mL/min. Finally, the radiochemical identity of
the radioiodinated ligands were verified by co-injection and co-elution with non-radioactive 8 and 17
from HPLC profiles (Alltech Alltima RPC-18 column, 250 × 4.6 mm, 5 μm, CH3CN/H2O = 9/1 at the
flow rate of 1.0 mL/min). The desired fractions containing the product were collectd and evaporated to
dryness and redissolved in 100% ethanol. The final products were stored at −20 °C for further studies.
3.2. Partition Coefficient Determination
The determination of partition coefficients of [125I]8 and [125I]17 was performed according to the
procedure previously reported with some modifications [23]. Ligand [125I]8 or [125I]17 (~5 μCi) was
mixed with 3 mL each of n-octanol and PBS (0.02 M, pH 7.4) in a test tube. The test tube was
vortexed for 5 min at room temperature, followed by centrifugation for 10 min at 3,000 rpm. Two

Molecules 2012, 17
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weighed samples from the n-octanol (50 µL) and buffer layers (400 µL) were counted in a γ-counter.
The partition coefficient was expressed as the logarithm of the ratio of the counts per gram from
n-octanol versus that of PBS. Samples from the n-octanol layer were repartitioned until consistent
partition coefficient values were obtained. The measurements were done in triplicate and repeated
three times.
3.3. In Vitro Binding Studies Using the Aggregated Aβ1–40
The lyophilized white powder of β-amyloid(1–40) were purchased from AnaSpec (San Jose, CA, USA).
After reconstituted by adding basic buffer (1% NH4OH. 60–70 μL) to β-amyloid (1–40) (1 mg)
aggregation of Aβ1–40 was carried out by gently dissolving Aβ1–40 (0.25 mg/mL) in a PBS buffer
(pH 7.4). The solution was incubated at 37 °C for 72 h with gentle and constant shaking. Binding
studies were carried out according to the procedure described previously with some modifications
using [125I]TZDM as the radiolabeled standard [24]. Briefly, the competition binding assays were
performed by mixing Aβ1–40 aggregates (100 µL), and [125I]TZDM (100 µL) in appropriate
concentration (0.02 nM, diluted in 10% EtOH), test ligand (10−5–10−10 M, 100 µL) and PBS (0.02 M,
pH 7.4, 700 µL) in a final volume of 1 mL. The mixture was incubated at 37 °C for 2 h. Then the
bound and free radioactivities were separated by vacuum filtration through Whatman GF/B glass filters
via a Brandel Mp-48T cell harvester followed by 3 × 4 mL washes with PBS (0.02 M, pH 7.4, 4 °C)
containing 10% ethanol at room temperature. Filters containing the bound 125I ligand were counted in a
γ-counter (WALLAC Wizard 1470, PerkinElmer Life Sciences, Waltham, MA, USA) with 75%
counting efficiency. The IC50 values were determined using GraphPad Prism 5.0, and those for the
inhibition constant (Ki) were calculated using the Cheng Prusoff equation: Ki = IC50/(1 + [L]/Kd) [25].
3.4. In Vitro Fluorescent Staining of Amyloid Plaques in Brain Sections from Transgenic Mouse
Paraffin-embedded brain sections of transgenic model mouse (8 µm, APP/PS1, 12 months, male)
were used for the in vitro fluorescent staining of amyloid plaques. The brain sections were
deparaffinized with xylene, ethanol and distilled water. After immersion in PBS (0.02 M, pH 7.4) for
30 min, the brain sections were incubated with 20% ethanol solution (1 µM) of compound 8 for 10 min.
The localization of plaques was confirmed by staining with thioflavin-S on the adjacent sections.
Finally, the sections were washed with 40% ethanol and PBS (0.02 M, pH 7.4). Fluorescent
observation was performed by a Stereo Discovery V12 instrument (Zeiss, Oberkochen, Germany)
equipped with a LP 505 filter set (excitation, 405 nm).
3.5. In Vitro Labeling of Brain Sections from Transgenic Mouse by Autoradiography
The brain sections mentioned above were incubated with [125I]8 (5 µCi/100µL) for about 20 min at
room temperature. Then the sections were washed with saturated Li2CO3 in 40% EtOH for 3 min and
40% EtOH for 3 min, followed by rinsing with water for 30 s. After drying, the 125I-labeled sections
were exposed to phosphorus film for 8 h and then scanned with the phosphor imaging system
(Cyclone, Packard) at the resolution of 600 dpi. The presence and localization of plaques were

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confirmed by the fluorescent staining with thioflavin-S on the same sections using a Stereo Discovery
V12 (Zeiss) instrument equipped with a LP 505 filter set (excitation: 405 nm).
3.6. In Vivo Biodistribution in Normal Mice
In vivo biodistribution studies were performed in KunMing normal mice (female, average weight
18–22 g) and in accordance with the national laws related to the care and experiments on laboratory
animal. A saline solution (100 µL) containing [125I]8 or [125I]17 (1 µCi) was injected directly into the
tail vein of mice. The mice (n = 5 for each time point) were sacrificed at designated time points
post-injection. The organs of interest were removed and weighed, and the radioactivity was counted
with an automatic γ-counter (WALLAC Wizard 1470).
4. Conclusions
A series of (E)-5-styryl-1H-indole and (E)-6-styrylquinoline based compounds have been synthesized
and evaluated as novel imaging probes for Aβ plaques. Compound 8 was found to possess nanomolar
affinity for β-amyloid plaques. In autoradiography, [125I]8 clearly labeled amyloid plaques in the cortex
region of AD model mice. Moreover, [125I]8 displayed high initial brain uptake and fast clearance in
biodistribution studies in normal mice. The findings suggest that the (E)-5-styryl-1H-indole derivative
[123I]8 may be a potential probe for detecting β-amyloid plaques in the AD brain.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/4/4252/s1.
Acknowledgments
This work was funded by NSFC (20871021 and 21071023). The authors thank Mengchao Cui
(College of Chemistry, Beijing Normal University, Beijing, China) for his kindness in providing the
paraffin-embedded brain sections of AD model mice. The authors also thank Jin Liu (College of Life
Science, Beijing Normal University) for providing the Zeiss Oberver Z1 equipment.
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Sample Availability: Samples of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).