Synthesis and biological evaluation of novel radioiodinated benzimidazole derivatives for imaging α-synuclein aggregates

Article abstract of DOI:10.1016/j.bmc.2017.10.010

α-Synuclein (α-syn) aggregates are commonly found in the brains of patients with Parkinson's disease (PD), dementia with Lewy bodies (DLB), and some other diseases. Therefore, in vivo imaging of α-syn aggregates would aid in drug development, early diagno

Full text of DOI:10.1016/j.bmc.2017.10.010

Accepted Manuscript
Synthesis and biological evaluation of novel radioiodinated benzimidazole de-
rivatives for imaging α-synuclein aggregates
Hiroyuki Watanabe, Taisuke Ariyoshi, Akihiko Ozaki, Masafumi Ihara,
Masahiro Ono, Hideo Saji
PII:
S0968-0896(17)31522-5
https://doi.org/10.1016/j.bmc.2017.10.010
BMC 14015
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2017
10 October 2017
13 October 2017
Please cite this article as: Watanabe, H., Ariyoshi, T., Ozaki, A., Ihara, M., Ono, M., Saji, H., Synthesis and biological
evaluation of novel radioiodinated benzimidazole derivatives for imaging α-synuclein aggregates, Bioorganic &
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.10.010
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Watanabe et al., 1/35
Synthesis and biological evaluation of novel radioiodinated benzimidazole
derivatives for imaging α-synuclein aggregates
Hiroyuki Watanabe1, Taisuke Ariyoshi1, Akihiko Ozaki2, Masafumi Ihara3,
Masahiro Ono*,1, Hideo Saji1
1Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical
Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto
606-8501, Japan.
2Department of Neurology, Osaka Saiseikai Nakatsu Hospital, 2-10-39 Shibata, Kita-ku,
Osaka 530-0012, Japan.
3Department of Stroke and Cerebrovascular Diseases, National Cerebral and
Cardiovascular Center, 5-7-1 Fujishirodai, Suita-shi, Osaka 565-8565, Japan.
*To whom correspondence should be addressed. Phone: +81-75-753-4608, Fax:
+81-75-753-4568, e-mail: ono@pharm.kyoto-u.ac.jp for M. Ono.

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Abstract
α-Synuclein (α-syn) aggregates are commonly found in the brains of patients with
Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and some other diseases.
Therefore, in vivo imaging of α-syn aggregates would aid in drug development, early
diagnosis, and monitoring of disease status. In order to develop imaging probes
targeting α-syn aggregates, we synthesized and evaluated three novel radioiodinated
benzoimidazole (BI) derivatives for selective imaging of α-syn aggregates. In binding
experiments, BI-2 exhibited the highest selective binding affinity for α-syn aggregates
among the BI derivatives. In addition, BI-2 clearly stained Lewy bodies in PD brain
sections, but did not label senile plaques deposited in AD brain sections. However, in
the biodistribution study using normal mice, [125I]BI-2 did not demonstrate high brain
uptake (0.56%ID/g at 2-min post-injection). Further structural modifications of the BI
derivatives are needed, but the BI scaffold may be an attractive candidate for
developing α-syn imaging probes.
Keywords; α-synuclein, imaging probe, Parkinson’s disease, Lewy body

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1. Introduction
α-Synuclein (α-syn), a 140-amino acid presynaptic protein, is the major fibrillary
component of Lewy bodies, which are commonly found in the brains of patients with
Parkinson’s disease (PD), dementia with Lewy bodies (DLB), Parkinson’s disease
dementia (PDD), and multiple system atrophy (MSA).1 PD is the most common
movement disorder, and DLB is the second most common form of neurodegenerative
dementia in elderly people.2, 3 However, there are no effective methods for early
diagnosis or basic remedy for these diseases. In addition, α-syn plays an important role
in the pathogenesis of the diseases because α-syn aggregates form early in the disease
and spread with time in the brain.4 Therefore, in vivo imaging of α-syn aggregates
would aid in drug development, early diagnosis, and monitoring of disease status.5, 6
Nuclear medical techniques, such as positron emission tomography (PET) and
single photon emission computed tomography (SPECT), are valuable tools for in vivo
imaging because of their high sensitivity and quantitative capability in deep tissues. As
β-amyloid (Aβ) and tau aggregates are the neuropathological hallmark of Alzheimer’s
disease (AD), a number of PET and SPECT probes targeting these aggregates have been

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developed and utilized for the diagnosis of AD.7-10 Notably, some PET probes for in
vivo imaging of Aβ aggregates, such as flutemetamol, florbetaben, and florbetapir, were
approved by the FDA. On the other hand, there have been no reports on the
development of clinically useful PET and SPECT probes to detect α-syn aggregates in
vivo.
Recently, a few α-syn imaging probes that can be used only for in vitro evaluation
have been reported (Figure 1). Phenothiazine derivatives, including SIL5, SIL23, and
SIL26, were reported as the first candidate probes for selective imaging of α-syn
aggregates.11, 12 SIL23 exhibited the moderate binding affinity for α-syn aggregates (Kd =
148 nM), but the selectivity for α-syn aggregates against Aβ and tau aggregates was
insufficient (2.5- and 1.6- fold, respectively). In addition, the biodistribution and
pharmacokinetics of [125I]SIL23 were not evaluated. SIL5 and SIL26 also showed
binding affinity for α-syn aggregates (66 and 16 nM, respectively). The selectivity of
SIL5 for α-syn aggregates against Aβ and tau aggregates was low (1.7- and 2.1- fold,
respectively), but SIL26 showed moderate selectivity for these aggregates (6.6- and 8.1-
fold selectivity versus Aβ and tau aggregates). In a biodistribution study, [11C]SIL5 and

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[18F]SIL26 were able to cross the blood-brain barrier. Therefore, further structural
optimization may lead to development of useful α-syn imaging probes.
3-(Benzylidine)indolin-2-one derivatives were reported as second generation lead
compounds for development of α-syn selective imaging probes.13 Among them, 46a
showed high affinity for α-syn aggregates (Ki = 2 nM) and favorable selectivity against
Aβ and tau aggregates (70- and 40-fold, respectively). 18F labeling of 46a succeeded,
but the brain uptake of [18F]46a was not evaluated. More recently, we also developed
radioiodinated diphenyl (IDP) derivatives as novel ligands for α-syn aggregates.14
IDP-4 exhibited high affinity for α-syn aggregates (Kd = 5.4 nM), but the selectivity of
IDP-4 against Aβ aggregates was not high (4.3-fold). In addition, [125I]IDP-4 showed
low brain uptake (0.45%ID/g at 2-min postinjection in mice). Due to such situations, the
development of useful α-syn imaging probes is strongly desired.

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Figure 1. Chemical structures of previously reported α-syn imaging probes.
In this study, we searched for new scaffolds that could selectively detect α-syn
aggregates. As a recent study found that styryl benzimidazole could bind to α-syn
aggregates in vitro, we selected the benzoimidazole (BI) scaffold as the backbone of the
α-syn imaging probes developed in this study.15 Another recent report demonstrated that
the presence of the diene moiety is important for the affinity for α-syn aggregates.13, 16
In addition, bulky substitution groups, such as benzyl groups, enhanced the selectivity
of α-syn versus Aβ.14 Based on these findings, we designed and synthesized three novel
BI derivatives and evaluated their potential as probes for in vivo imaging of α-syn
aggregates.

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2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, compounds 3 and 4 were prepared according to methods
reported previously.17 The BI scaffold was obtained by an intermolecular cyclization
reaction between 1,2-phenylenediamine with 3 or 4 using Na2S2O5 as an oxidant.18
Following the reaction, (bromomethyl)benzene, 1-(chloromethyl)-4-methoxybenzene,
or iodomethane was added to obtain 5, 6, 7, and 11. The tributyltin derivatives (8, 9, and
10) were prepared from the corresponding bromo compounds using the exchange
reaction of bromine by tributyltin catalyzed by Pd(0). These tributyltin compounds were
also used as the starting materials for radioiodination in the preparation of the
corresponding 125I-labeled derivatives. We could not obtain enough of 8 that is used as a
precursor for the synthesis of 11. Therefore, the synthetic route of 11 was different from
that of 12 and 13.

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Scheme 1 Synthetic route for BI derivatives. Reagents and conditions: (i) 1) (1,
3-dioxolan-2-yl)methyl-triphenylphosphonium bromide, 18-crown-6, NaH, THF, rt; 2)
20% HCl, THF, rt; (ii) Na2S2O5, DMF, 105°C; (iii) 1) NaH, DMF, 0°C; 2)
(bromomethyl)benzene, 1-(chloromethyl)-4-methoxybenzene, or iodomethane, DMF, rt;
(iv) (SnBu3)2, (Ph3P)4Pd, dioxane, Et3N, 95°C; (v) I2, CHCl3, rt; (vi) 1) NaH, DMF,
0°C; 2) (bromomethyl)benzene, DMF, rt.

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2.2. Radiolabeling
125I-labeled BI derivatives were obtained by an iododestannylation reaction using
125I]NaI and hydrogen peroxide as the oxidants under acidic conditions (Scheme 2).
[
After the reaction was completed, the radioiodinated compounds were purified by
HPLC, as detailed in the experimental section. The no-carrier-added derivatives were
expected to be produced as a final product with a theoretical specific activity of 81.4
TBq/mmol, similar to that of 125I. The radiochemical identity of the radioiodinated
ligands was confirmed by co-injection with non-radioiodinated compounds from their
HPLC profiles (Table S1). [125I]BI-1, [125I]BI-2, and [125I]BI-3 were obtained in a
radiochemical yield of 37.5, 52.0, and 81.0%, respectively, with a radiochemical purity
of >99%.
Scheme 2. Radiosynthesis of [125I]BI derivatives. Reagents and conditions: (i) [125I]NaI,

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3% H2O2, 1 N HCl, rt.
2.3. Binding assay using recombinant α-syn and Aβ1–42 aggregates
It is well known that α-syn aggregates often colocalize with Aβ aggregates in the
PDD and DLB brain, thus α-syn imaging probes need to have selective binding affinity
for α-syn aggregates versus Aβ aggregates.10, 19 After confirming that the fluorescence
intensity of BI derivatives increased in the presence of α-syn and Aβ1-42 aggregates, we
performed saturation binding assays to calculate the apparent binding dissociation
constant (Kd) of these aggregates. Table 1 summarizes the Kd values of the BI
derivatives for α-syn and Aβ1-42 aggregates. The Kd value of BI-1, BI-2, and BI-3 for
α-syn aggregates was 485, 99.5, and 874 nM, respectively. For Aβ1-42 aggregates, BI-1,
BI-2, and BI-3 had Kd values of 179, 727, and 271 nM, respectively. The selectivity for
α-syn aggregates versus Aβ1-42 aggregates (Kd ratio of Aβ1-42/α-syn) was 0.37, 7.31, and
0.31 for BI-1, BI-2, and BI-3, respectively. These results suggested that the substituent
of the BI scaffold affected the affinity and selectivity for α-syn aggregates. Among the
BI derivatives, BI-2 exhibited the highest selective binding affinity for α-syn aggregates.

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The selectivity of BI-2 for α-syn aggregates versus Aβ aggregates was higher than or
comparable with that of IDP derivatives and phenothiazine derivatives, such as SIL5,
SIL23, and SIL26, but lower than some 3-(benzylidine)indolin-2-one derivatives,
including 46a,12-14 indicating that BI-2 has the potential to selectivity detect α-syn
aggregates.
Table 1. Kd values of BI derivatives for recombinant α-syn and Aβ1-42 aggregates
Kd (nM)a
Compounds
BI-1
α-syn
Aβ1-42
485 160
99.5 20.8
874 169
179 59.1
727 227
271 67.3
BI-2
BI-3
aValues are the mean standard error of the mean for three independent experiments.
2.3. Fluorescent staining of PD and AD brain sections
To confirm the selective binding affinity for Lewy bodies, we carried out

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fluorescent staining experiments of PD and AD brain sections using BI-2. Several
fluorescent spots were observed in the brain section of a PD patient (Figure 2A). When
the same section was immunostained with an antibody against α-syn, the fluorescent
spots obtained with BI-2 corresponded with the results of immunohistochemical
staining (Figure 2B). In the immunohistochemical staining of AD brain sections with
anti-Aβ antibody, we observed extensive accumulation of Aβ aggregates, but marked
fluorescence derived from BI-2 was not observed (Figure 2C and 2D). These results
suggested that BI-2 selectively bound α-syn aggregates deposited in PD brain sections
in vitro.

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Figure 2. Fluorescence staining of BI-2 in midbrain sections from a PD patient (A) and
cortical sections from an AD patient (C). Immunohistochemical staining with
fluorescent antibody against α-syn (B) and Aβ (D).
2.4. In vivo biodistribution in normal mice
To evaluate the brain uptake and washout of [125I]BI derivatives, we performed
biodistribution experiments using normal mice. The results of the biodistribution study

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are shown in Table S2 in Supplementary Information. [125I]BI-3 exhibited moderate
uptake (1.89%ID/g) at 2-min post-injection and clearance from brain over time (1.0 and
0.53%ID/g at 30- and 60-min post-injection, respectively). In contrast, [125I]BI-1 and
[
125I]BI-2 displayed low initial uptake at 2-min post-injection (0.52 and 0.56%ID/g
respectively) and retention in the brain (Figure 3). The bulky substitution groups of
125I]BI-1 and [125I]BI-2 may have reduced penetration of the blood-brain barrier. In
[
general, the criteria for useful Aβ and tau imaging probes are high initial brain uptake
(>4% ID/g at 2-min post-injection in mice) and rapid clearance from normal brains
(<1% ID/g at 30-min post-injection in mice).20, 21 Useful imaging probes targeting
α-syn aggregates have not been reported, but the criteria for α-syn imaging probes may
be the same as those for probes targeting Aβ and tau aggregates. Considering brain
uptake, the BI derivatives do not meet the criteria for clinical use. To develop useful
α-syn imaging probes, additional structural changes are needed to improve the brain
uptake of the compounds without impairing selective affinity for α-syn aggregates.

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Figure 3. Comparative brain uptake of radioactivity after intravenous injection of
125I]BI derivatives.
[
3. Conclusion
In conclusion, we designed and synthesized three novel radioiodinated BI
derivatives for imaging α-syn aggregates in the brain. Among the derivatives tested in
this study, [125I]BI-2 exhibited the highest selective binding affinity for α-syn
aggregates. In addition, BI-2 selectively stained Lewy bodies in the brain sections, but
the brain uptake and clarance of [125I]BI-2 did not meet the criteria for the in vivo
imaging of α-syn aggregates in the brain. Taken together, further structural

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modifications of the BI derivatives may lead to useful α-syn imaging probes.
4. Experimental Part
Materials and Methods
All reagents were commercial products and used without further purification
unless indicated otherwise. Smart Flash EPCLC W-Prep 2XY (Yamazen Corp., Osaka,
1
Japan) was used for silica gel chromatography. H and 13C NMR spectra were recorded
using a JEOL JNM-ECS400 (JEOL, Tokyo, Japan), and chemical shifts were recorded
in δ (ppm) relative to TMS as an internal standard. Coupling constants are reported in
Hertz. Multiplicity was defined as singlet (s), doublet (d), or multiplet (m). Mass spectra
were obtained on a SHIMADZU LCMS-2020 EV (SHIMADZU, Kyoto, Japan) and
JEOL JMS-SX 102A QQ. HPLC was performed with the Shimadzu system (LC-20AD
pump with an SPD-20A UV detector, λ = 254 nm) using a Cosmosil C18 column
(Nacalai Tesque, Kyoto, Japan, 5C18-AR-II, 4.6 × 150 mm) and CH3CN/H2O as the
mobile phase at a flow rate of 1.0 mL/min. All key compounds were proven by this
method to be 95% pure.

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4.1. Chemistry
4.1.1. (E)-4-(2-Bromovinyl)benzaldehyde (1)
To a stirred solution of 4-bromobenzaldehyde (2000 mg, 10.81 mmol),
18-crown-6 (25 mg) and (1,3-dioxolan-2-ylmethyl)-triphenylphosphoniumbromide
(7000 mg, 16.3 mmol) in THF (90 mL) under N2, NaH (1038 mg, 86.5 mmol) was
added in one portion. After 2-h stirring at room temperature, the reaction mixture was
quenched with water and extracted with EtOAc. After evaporation of EtOAc, the
residue was dissolved in the mixture of THF (90 mL) and 20% hydrochloric acid, and
stirred for 2 h at room temperature. After the mixture was turned basic with 2 N
NaOHaq and aqueous saturated solution of NaHCO3, the organic solvent was
evaporated, and the mixture extracted with EtOAc. The combined EtOAc extracts were
washed with brine and dried over Na2SO4. The solvent was removed, and the residue
was purified by silica gel chromatography (EtOAc/hexane = 17/83) to give 2 (1568 mg,
1
68.8%). H NMR (400 MHz, CDCl3) δ 9.71 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 7.6 Hz,
2H), 7.44 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 16.0 Hz, 1H), 6.71 (dd, J1 = 8.0 Hz, J2 = 16.8

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Hz, 1H). MS (ESI) m/z 211 and 213 [MH+].
4.1.2. (E)-4-(2-Iodovinyl)benzaldehyde (2)
The same reaction as described above to prepare 1 was used and 1784 mg of 2
was obtained in a 78.7% yield from 4-iodobenzaldehyde. 1H NMR (400 MHz, CDCl3) δ
9.71 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.29 (d, J =
8.4 Hz, 2H), 6.72 (dd, J1 = 7.6 Hz, J2 = 16.0 Hz, 1H). MS (ESI) m/z 258.9 [MH+].
4.1.3. 4-((1E,3E)-4-Bromobuta-1,3-dien-1-yl)benzaldehyde (3)
The same reaction as described above to prepare 1 was used and 659 mg of 3
was obtained in a 37.4% yield from 1.1H NMR (400 MHz, DMSO-d6) δ 9.60 (d, J = 8.4
Hz, 1H), 7.57-7.63 (m, 4H), 7.50 (dd, J1 = 10.4 Hz, J2 = 15.2 Hz, 1H), 7.31 (dd, J1 =
10.8 Hz, J2 = 15.6 Hz, 1H), 7.17 (d, J = 15.2 Hz, 1H), 6.30 (dd, J1 = 8.0 Hz, J2 = 16.8
Hz, 1H). MS (ESI) m/z 237 and 239 [MH+].
4.1.4. 4-((1E,3E)-4-Iodobuta-1,3-dien-1-yl)benzaldehyde (4)

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The same reaction as described above to prepare 1 was used and 1192 mg of 4
was obtained in a 61.9% yield from 2. 1H NMR (400 MHz, CD3OD) δ 9.57 (d, J = 7.6
Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.46 (dd, J1 = 10.8 Hz, J2 = 15.2 Hz, 1H), 7.36 (d, J =
8.4 Hz, 2H), 7.21 (dd, J1 = 12.0 Hz, J2 = 15.2 Hz, 1H), 7.09 (d, J = 16.0 Hz, 1H), 6.30
(dd, J1 = 8.0 Hz, J2 = 16.8 Hz, 1H). MS (ESI) m/z 284.9 [MH+].
4.1.5.
1-Benzyl-2-((1E,3E)-4-(4-bromophenyl)buta-1,3-dien-1-yl)-1H-benzo[d]imidazole
(5)
A mixture of 1,2-phenylenediamine (108 mg, 1.0 mmol), 3 (237 mg, 1.0
mmol), and Na2S2O5 (190 mg, 1.0 mmol) in DMF (5 mL) was stirred at 105˚C under N2
for 2.5 h. After the mixture was cooled to 0˚C,NaH (48 mg, 2.0 mmol) was added and
stirred for 30 min at 0˚C. Then, (bromomethyl)benzene (342 mg, 2.0 mmol) was added
and stirred at room temperature for 18 h. The solution was quenched with water and
extracted with EtOAc, The combined EtOAc extracts were washed with brine and dried
over Na2SO4. The solvent was removed, and the residue was purified by silica gel

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1
chromatography (EtOAc/hexane = 26/74) to give 5 (79.4 mg, 19.0%). H NMR (400
MHz, DMSO-d6) δ 7.60-7.64 (m, 2H), 7.55-7.57 (m, 3H), 7.49 (d, J = 8.4 Hz, 2H), 7.31
(d, J = 8.4 Hz, 2H), 7.25-7.27 (m, 1H), 7.21-7.23 (m, 1H), 7.19-7.20 (m, 2H), 7.14 (d, J
= 7.2 Hz, 2H), 7.06 (d, J = 15.2 Hz, 1H), 6.95 (d, J = 15.2 Hz, 1H), 5.61 (s, 2H). MS
(ESI) m/z 415 and 417 [MH+].
4.1.6.
2-((1E,3E)-4-(Bromophenyl)buta-1,3-dien-1-yl)-1-(4-methoxybenzyl)-1H-benzo[d]i
midazole (6)
The same reaction as described above to prepare 5 was used with
1-(chloromethyl)-4-methoxybenzene as a substitute for (bromomethyl)benzene and 215
1
mg of 6 was obtained in a 24.2% yield from 3. H NMR (400 MHz, DMSO-d6)
δ
7.57-7.63 (m, 4H), 7.51 (d, J = 8.0 Hz, 2H), 7.19-7.29 (m, 4H), 7.14 (d, J = 8.0 Hz, 2H),
7.11 (d, J = 14.0 Hz, 1H), 6.96 (d, J = 15.6 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 5.53 (s,
2H), 3.69 (s, 3H). MS (ESI) m/z 445 and 447 [MH+].

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4.1.7.
2-((1E,3E)-4-(Bromophenyl)buta-1,3-dien-1-yl)-1-methyl-1H-benzo[d]imidazole (7)
The same reaction as described above to prepare 5 was used with
iodomethane as a substitute for (bromomethyl)benzene and 237 mg of 7 was obtained in
a 35.0% yield from 3. 1H NMR (400 MHz, DMSO-d6) δ 7.62 (d, J = 11.6 Hz, 1H), 7.59
(d, J = 8.0 Hz, 2H), 7.57 (d, J = 6.8 Hz, 1H), 7.51-7.55 (m, 3H), 7.27-7.31 (m, 1H), 7.22
(dd, J1 = 6.4 Hz, J2 = 13.2 Hz, 2H), 7.03 (d, J = 15.2 Hz, 1H), 6.97 (d, J = 15.6 Hz, 1H),
3.86 (s, 3H). MS (ESI) m/z 339 and 341 [MH+].
4.1.8.
1-Benzyl-2-((1E,3E)-4-(4-(tributhylstinnyl)phenyl)buta-1,3-dien-1-yl)-1H-benzo[d]i
midazole (8)
A mixture of 5 (75 g, 0.18 mol), (SnBu3)2 (314 g, 0.54 mol), and (Ph3P)4Pd
(42 mg, 0.036 mmol) in a mixed solvent (15 mL, 2:1 dioxane/Et3N mixture) was stirred
at 95˚C for 1.5 h. The solvent was removed, and the residue was purified by silica gel
1
chromatography (EtOAc/hexane = 1/3) to give 13.7 mg of 8 (13.7%). H NMR (400

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MHz, CD3OD) δ 7.61-7.67 (m, 2H), 7.41-7.44 (m, 5H), 7.24-7.30 (m, 5H), 7.12 (d, J =
8.4 Hz, 3H), 6.88 (d, J = 15.2 Hz, 1H), 6.85 (d, J = 15.2 Hz, 1H), 5.56 (s, 2H),
0.87-1.58 (m, 27H). MS (ESI) m/z 627 [MH+].
4.1.9.
1-(4-Methoxybenzyl)-2-((1E,3E)-4-(4-(tributhylstinnyl)phenyl)buta-1,3-dien-1-yl)-1
H-benzo[d]imidazole (9)
The same reaction as described above to prepare 8 was used and 39 mg of 9
1
was obtained in a 13.7% yield from 6. H NMR (400 MHz, CD3OD) δ 7.61-7.67 (m,
2H), 7.43-7.48 (m, 5H), 7.21-7.28 (m, 2H), 7.16 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 8.8 Hz,
2H), 6.91 (d, J = 6.0 Hz, 1H), 6.86 (d, J = 8.4 Hz, 3H), 5.50 (s, 2H), 3.73 (s, 3H),
0.85-1.61 (m, 27H). MS (ESI) m/z 657 [MH+].
4.1.10.
1-Methyl-2-((1E,3E)-4-(4-(tributhylstinnyl)phenyl)buta-1,3-dien-1-yl)-1H-benzo[d]
imidazole (10)

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The same reaction as described above to prepare 8 was used and 68 mg of 10
was obtained in a 21 % yield from 10. 1H NMR (400 MHz, DMSO-d6) δ 7.56-7.64 (m,
2H), 7.42-7.54 (m, 5H), 7.12-7.27 (m, 2H), 7.00 (d, J = 15.2 Hz, 1H), 6.95 (d, J = 16.0
Hz, 1H), 3.86 (s, 3H), 0.84-1.56 (m, 3H). MS (ESI) m/z 551 [MH+].
4.1.11.
1-Benzyl-2-((1E,3E)-4-(4-iodophenyl)buta-1,3-dien-1-yl)-1H-benzo[d]imidazole
(11; BI-1)
The same reaction as described above to prepare 5 was used and 186 mg of 11
1
was obtained in a 20.1% yield from 4. H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J =
8.4 Hz, 2H), 7.61-7.63 (m, 2H), 7.55 (dd, J1 = 3.6 Hz, J2 = 6.0 Hz, 1H), 7.31-7.36 (m,
4H), 7.27 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 4.4 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.20 (d,
J = 3.6 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 7.07 (d, J = 14.8 Hz, 1H), 6.93 (d, J = 15.6 Hz,
1H) 5.61(s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 150.6, 143.0, 137.6, 137.3 136.6,
136.2, 135.5, 135.2, 129.1, 128.8, 128.7, 127.6, 126.6, 122.43, 122.38, 118.7, 118.3,
110.5, 94.4, 45.7. HRMS (FAB) m/z calcd for C18H16N2I 387.0358; found 387.0361.

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4.1.12.
2-((1E,3E)-4-(Iodophenyl)buta-1,3-dien-1-yl)-1-(4-methoxybenzyl)-1H-benzo[d]imi
dazole (12; BI-2)
A solution of 9 (60 mg, 0.092 mmol) and I2 (34.7 mg, 0.137 mmol) in CHCl3
(10 mL) was stirred for 30 min at room temperature. The reaction mixture was
quenched with aqueous saturated NaHSO3 and extracted with CHCl3. The combined
CHCl3 extracts were washed with brine and dried over Na2SO4. The solvent was
removed, and the residue was purified by silica gel chromatography (EtOAc/hexane =
1
1:2) to give 32 mg of 12 (71%). H NMR (400 MHz, CD3OD) δ 7.66 (d, J = 8.4 Hz,
2H), 7.57-7.63 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.20 (m, 4H), 7.13 (d, J = 15.6 Hz,
1H), 7.07 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 15.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 6.82
(d, J = 8.0 Hz, 1H), 5.46 (s, 2H), 3.71 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.6,
150.5, 143.1, 137.6, 136.5, 136.2, 135.4, 135.1, 129.2, 129.1, 128.7, 128.1, 122.34,
122.30, 118.7, 118.5, 114.1, 110.5, 94.4, 55.1, 45.3. HRMS (FAB) m/z calcd for
C25H22N2OI 493.0777; found 493.0780.

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4.1.13.
2-((1E,3E)-4-(Iodophenyl)buta-1,3-dien-1-yl)-1-methyl-1H-benzo[d]imidazole (13;
BI-3)
The same reaction as described above to prepare 12 was used and 20 mg of 13
was obtained in a 94% yield from 10. 1H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J = 8.8
Hz, 2H), 7.53-7.63 (m, 3H), 7.37 (d, J = 8.4 Hz, 2H), 7.19-7.31 (m, 3H), 7.03 (d, J =
15.6 Hz, 1H), 6.94 (d, J = 15.6 Hz, 1H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 150.7, 142.9, 137.6, 136.2, 136.1, 136.0, 134.9, 129.2, 128.7, 122.1, 118.6, 118.5,
110.1, 94.3, 29.5. HRMS (FAB) m/z calcd for C24H20N2I 463.0671; found 463.0675.
4.2. Radiolabeling
The radioiodinated compounds [125I]11 ([125I]BI-1), [125I]12 ([125I]BI-2), and
[
125I]13 ([125I]BI-3) were prepared from the corresponding tributyltin derivatives by
iododestannylation. Briefly, to initiate the reaction, 50 µL of H2O2 (3%) was added to a
mixture of a tributyltin derivative (100 µg/100 µL EtOH), [125I]NaI (3.7-14.8 MBq,

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specific activity 81.4 TBq/mmol), and 50 µL of 1 N HCl in a sealed vial. The reaction
was allowed to proceed at room temperature for 5 min and terminated by the addition of
saturated NaHSO3 aq. (100 µL). After neutralization with sodium hydrogen carbonate
and extraction with EtOAc, the extract was dried by passing through an anhydrous
Na2SO4 column. The solution was blown dry with a stream of nitrogen gas. The
radioiodinated ligand was purified by HPLC on a Cosmosil C18 column with an
isocratic elution using CH3CN/H2O (7/3) as the solvent, at a flow rate of 1.0 mL/min.
4.3. In vitro binding assay using α-syn and Aβ1-42 aggregates
α-Syn and Aβ1-42 were purchased from rPeptide (Bogart, GA, USA) and the
Peptides Institute, Inc. (Osaka, Japan), respectively. α-Syn aggregates were prepared by
incubating recombinant α-syn monomers (1.67 mg/mL in 50 mM Tris-HCl, 250 mM
NaCl, pH 7.48) at 37˚C for 6 days with stirring at 1000 rpm. Aβ1-42 aggregates were
prepared by incubating the Aβ1-42 peptides (0.25 mg/mL in PBS, pH 7.4) at 37˚C for 2
days with gentle and constant shaking. The formation of the α-Syn or Aβ1-42 aggregates
was confirmed with the measurement of the fluorescence intensity of thioflavin-S. The

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reaction mixtures containing BI-1, BI-2, or BI-3 (final conc. 0-20 µM) and α-syn or
Aβ1-42 aggregates (final conc. 2.2 µM) were incubated at room temperature for 30 min.
Then, fluorescence intensities were recorded using Infinite M200PRO (TECAN,
Männedorf, Switzerland) and Kd binding curves were generated by GraphPad Prism 5.0
(GraphPad Software, Inc., La Jolla, CA, USA).
4.4. Fluorescence staining of human PD and AD brain sections
The experiments involving human tissues were performed in accordance with
relevant guidelines and regulations, and were approved by the ethics committee of
Osaka Seiseikai Nakatsu Hospital and the National Cerebral and Cardiovascular Center.
Informed consent for the experiments using post-mortem brain tissues was obtained
from all subjects in this study-antemortem. Postmortem brain tissues from
autopsy-confirmed cases of PD (female, 69 years old) were obtained from Osaka
Seiseikai Nakatsu Hospital and those of AD (male, 76 years old) were obtained from the
National Cerebral and Cardiovascular Center. Eight- and six- micrometer-thick serial
sections of paraffin-embedded blocks for PD and six- for AD, respectively, were used

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for staining. The sections were subjected to two 15-min incubations in xylene, two
1-min incubations in 100% EtOH, one 1-min incubation in 90% EtOH, and one 1-min
incubation in 70% EtOH, followed by two 2.5-min washes in water. The sections were
incubated with BI-2 (200 µM, 50% EtOH) for 15 min. The sections were observed with
a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) equipped with a U-2A
filter set and multispectral imaging system (Nuance FX, Caliper Life Sciences Inc.,
Hopkinton, MA, USA).
4.5. Immunohistochemical staining of α-syn in human PD brain sections
After fluorescence staining, the midbrain sections were activated by 90%
formic acid and incubated with anti-phosphorylated α-syn primary antibody (pSyn#64,
Wako) at 4˚C for 3 h. After three 5-min incubations in PBS-Tween 20, they were
incubated with a fluorescent secondary antibody labeled with Alexa Fluor 647 at 4˚C
for 1 h. After three 5-min incubations in PBS-Tween 20, the sections were observed
with a fluorescence microscope (FSX100, Olympus Corp., Tokyo, Japan) equipped with
a U-DM-CY5-3 filter set.

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4.6. Immunohistochemical staining of Aβ in human AD brain sections
After fluorescence staining, the brain sections were activated by 90% formic
acid and incubated with Aβ1-42 (BC05, Wako) primary antibody at room temperature for
1 h. After three 5-min incubations in PBS containing 0.02% Tween 20 (PBS-Tween20),
they were incubated with fluorescent secondary antibody labeled with Alexa Fluor 488
at room temperature for 1 h. After three 5-min incubations in PBS-Tween 20, the
sections were observed with a fluorescence microscope (FSX100) equipped with a
U-MWIBA3 filter set.
4.7. In vivo biodistribution in normal mice
The experiments with animals were conducted in accordance with our
institutional guidelines and approved by Kyoto University. A saline solution (100 µL,
20 kBq) of each [125I]BI derivative containing EtOH (10 µl) was injected directly into
the tail vein of ddY mice (5 weeks old, male). The mice were sacrificed at 2, 10, 30, and
60 min post-injection. The organs of interest were removed and weighed, and