Synthesis and biological evaluation of novel styryl benzimidazole derivatives as probes for imaging of neurofibrillary tangles in Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2013.02.054

This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer's disease (AD). SBIM derivatives were prepared with 4-iodobenzene-1,2-dia

Full text of DOI:10.1016/j.bmc.2013.02.054

Bioorganic & Medicinal Chemistry 21 (2013) 3356–3362
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Bioorganic & Medicinal Chemistry
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Synthesis and biological evaluation of novel styryl benzimidazole
derivatives as probes for imaging of neurofibrillary tangles in
Alzheimer’s disease
a
a
a
Kenji Matsumura a, Masahiro Ono a, , Masashi Yoshimura , Hiroyuki Kimura , Hiroyuki Watanabe ,
⇑
Yoko Okamoto b, Masafumi Ihara b, Ryosuke Takahashi b, Hideo Saji a
a Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as
agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer’s disease (AD). SBIM deriva-
tives were prepared with 4-iodobenzene-1,2-diamine and substituted cinnamaldehydes. In binding
experiments using recombinant tau and Ab1–42 aggregates, SBIM-3 showed higher affinity for the tau
aggregates than Ab1–42 aggregates (ratio of Kd values was 2.73). In in vitro autoradiography and fluores-
cent staining, [125I]SBIM-3 (or SBIM-3) bound NFT in sections of AD brain tissue. In biodistribution exper-
iments using normal mice, all [125I]SBIM derivatives showed high initial uptake into (3.20–4.11%ID/g at
2 min after the injection) and rapid clearance from (0.12–0.33%ID/g at 60 min after the injection) the
brain. In conclusion, appropriate structural modifications of SBIM derivatives could lead to more useful
agents for the in vivo imaging of NFT in AD brains.
Received 9 January 2013
Revised 14 February 2013
Accepted 15 February 2013
Available online 29 March 2013
Keywords:
Alzheimer’s disease
Neurofibrillary tangles
Tau
Imaging
Ó 2013 Elsevier Ltd. All rights reserved.
Styryl benzimidazole
1. Introduction
9172,11,12
[ [
18F]AV-45,13,14 123I]IMPY15 have been developed and
succeeded in imaging SP in AD brains. Among these compounds,
Alzheimer’s disease (AD), the most common neurodegenerative
disorder, is characterized by memory loss and language impair-
ment and its prevalence is increasing together with life expec-
tancy. Although the etiology of AD is not completely understood,
the progressive accumulation of senile plaques (SP) composed of
amyloid b (Ab) peptide and neurofibrillary tangles (NFT) composed
of hyperphosphorylated tau protein are two neuropathological
hallmarks of the disease.1 Currently, a post-mortem histopatholo-
gical examination of SP and NFT is the only way to confirm AD.
Since these markers probably appear many years prior to the cog-
nitive symptoms of AD,2,3 detecting SP and/or NFT in vivo may lead
to an early diagnosis. Additionally, monitoring these targets in vivo
may also support the development of new medical techniques such
as immunotherapy.
[
11C]PIB has been used in thousands of clinical studies and proved
its utility.6,16 However, a positive [11C]PIB scan identifies the pres-
ence of SP in nearly all AD patients, 60% of individuals with mild
cognitive impairment (MCI), and 20–30% of cognitively normal el-
derly subjects,17 indicating that SP deposit not only in AD brains
but also in healthy brains with age.
The accumulation of NFT corresponds with the severity of clin-
ical symptoms of AD.18–20 There are several PET/SPECT imaging
agents targeting NFT such as
[
11C]BF-158,21
[
[
18F]THK-523,22
[
125I]TH2,23 125I]PDB-3,24 18F]FPPDB,25 and 18F]T808.26 In
[
[
in vitro experiments, these agents have exhibited higher affinity
for tau than Ab. For example, autoradiographic analysis using AD
brain sections showed that positive [18F]THK-523-binding corre-
sponded with the accumulation of NFT. Although [18F]THK-523
has been investigated clinically so far, its accumulation in the
white matter of the brain suggests interference with specific imag-
ing.27 Therefore, further research into the development of NFT-spe-
cific tracers is needed.
We recently developed radioiodinated compounds based on a
phenyldiazenylbenzothiazole (PDB) scaffold as NFT imaging
agents. In in vitro autoradiographic and fluorescent staining exper-
iments, [125I]PDB-3 (or PDB-3) bound to NFT in AD brain sections.24
However, PDB derivatives showed low uptake into (0.94–1.03%ID/g
at 2 min after the injection) and persistent localization in normal
Positron emission tomography (PET) and single photon emis-
sion computed tomography (SPECT) are useful for imaging SP
and/or NFT noninvasively in living brain tissue. Since deposits of
SP have been demonstrated at the earliest stages of the disease
process, PET/SPECT imaging agents targeting SP such as [11C]SB-
13,4,5
[
11C]PIB,6,7
[
11C]BF-227,8
[
18F]FDDNP,9,10
[
18F]BAY94-
⇑
Corresponding author. Tel.: +81 75 753 4608; fax: +81 75 753 4568.
E-mail address: ono@pharm.kyoto-u.ac.jp (M. Ono).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.054

K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
3357
mine or 4-iodobenzene-1,2-diamine and substituted cinnamalde-
hydes using Na2S2O5 as an oxidant agent (yield, 67.1–86.7%). The
monomethyl amino derivatives, 2b and 4b, were produced by first
reducing the nitro group to an amino group with powdered iron,
and subsequent monomethylation of the amino group using para-
formaldehyde, sodium borohydride, and sodium methoxide (yield,
69.8–76.5%). Compounds 5a, 5b, and 5c were obtained by protect-
ing the aminogroups of 4a, 4b, and 4c with a tert-butoxycarbonyl
(Boc) group (yield, 79.4–88.3%). The tributyltin precursors (6a,
6b, and 6c) were prepared from corresponding bromo compounds
using a bromo to tributhyltin exchange reaction catalyzed by
Pd(Ph3P)4 (yield, 30.3–59.0%). The radioiodinated ligands, [125I]2a
([125I]SBIM-1), [125I]2b ([125I]SBIM-2), and [125I]2c ([125I]SBIM-3)
were prepared from corresponding tributyltin precursors through
an iododestannylation reaction using hydrogen peroxide as an oxi-
dant followed by stirring with TFA to remove the Boc protecting
groups with a radiochemical yield of 24.0–45.0%. After purification
by HPLC, the specific activity of the no-carrier-added preparation
was comparable to that of [125I]NaI, 81.4 TBq/mmol. Finally, the
identity of [125I]2a ([125I]SBIM-1), [125I]2b ([125I]SBIM-2), and
Figure 1. Structures of PDB derivatives and SBIM derivatives.
mouse brains (2.89–3.23%ID/g at 60 min after the injection), which
might lead to a low signal-to-noise ratio in AD brains. This nonspe-
cific binding to normal areas in the brains may be due to the high
lipophilicity of PDB derivatives.28 Consequently, we planned to re-
place the benzothiazole scaffold in PDB with a benzimidazole scaf-
fold with less lipophilicity. Furthermore, we changed the diazo
moiety to a styryl moiety since the diazo moiety of PDB derivatives
is typically thought to be toxic in humans.29 Based on this knowl-
edge, we designed styrylbenzimidazole derivatives with less lipo-
philicity and less toxicity as candidates for in vivo imaging of
NFT in AD brains (Fig. 1).
In this study, we synthesized three styrylbenzimidazole (SBIM)
derivatives and evaluated their utility as NFT imaging agents. To
our knowledge, this is the first time radioiodinated styrylbenzimi-
dazole derivatives have been used as NFT imaging agents.
[
125I]2c ([125I]SBIM-3) was verified by coinjection with a nonradio-
active compound from HPLC data. HPLC analysis suggested that
these derivatives existed as tautomers. All [125I]SBIM derivatives
were obtained with radiochemical purity of >99% after purification
by HPLC.
2. Results and discussion
2.2. Binding assay with SBIM derivatives using recombinant tau
and Ab1–42 aggregates
2.1. Chemistry and radiolabeling
The synthesis of SBIM derivatives was carried out according to
Schemes 1A, B, and C. Compounds 1, 2c, 3, and 4c were obtained
by intermolecular cyclization between 4-bromobenzene-1,2-dia-
To quantify the affinity for NFT, we carried out binding experi-
ments using recombinant tau aggregates (Table 1). The fluorescent
Scheme 1. (A) Reagents and conditions: (i) Na2S2O5, DMF, 105 °C; (ii) powdered iron, HCl, 80% EtOH, 90 °C; (iii) NaOMe, (HCHO)n, NaBH4, MeOH, 75 °C. (B) Reagents and
conditions: (i) Na2S2O5, DMF, 105 °C; (ii) powdered iron, HCl, 80% EtOH, 90 °C; (iii) NaOMe, (HCHO)n, NaBH4, MeOH, 75 °C; (iv) (Boc)2O, guanidine hydrochloride, EtOH, 40 °C;
(v) (Bu3Sn)2, (Ph3P)4Pd, Et3N, dioxane, 95 °C. (C) Reagents and conditions: (i) (1) [125I]NaI, 3% H2O2, 1 N HCl, rt, (2) TFA, rt.

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K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
Table 1
derivatives were 0.38, 0.93, and 2.73, respectively. Although SBIM-
1 and SBIM-2 had lower affinity for tau aggregates than Ab1–42
aggregates, SBIM-3 had higher affinity for tau aggregates than
Ab1–42 aggregates. These results suggested that minor changes of
substituted groups in the SBIM scaffold might influence the selec-
tive binding between tau and Ab1–42 aggregates. Therefore, further
appropriate modification of SBIM derivatives may lead to useful
agents with much higher affinity for NFT than SP.
Kd Values of SBIM derivatives for recombinant tau and Ab1–42 aggregates
a
Kd
(
l
M)
Kd Ratios of Ab1–42/tau
Compound
Tau
Ab1–42
SBIM-1
SBIM-2
SBIM-3
2.36 0.09
0.45 0.08
0.30 0.02
0.90 0.27
0.42 0.05
0.82 0.10
0.38
0.93
2.73
a
Values are the mean standard error of the mean for six independent
experiments.
2.3. In vitro autoradiography
[
125I]SBIM-3, which showed the best ratio of Kd values for tau
aggregates to Ab1–42 aggregates, was investigated for its binding
to NFT by in vitro autoradiography in an AD brain section. The
autoradiographic image showed a remarkable accumulation of
radioactivity in gray matter (Fig. 2A). The level of radioactivity in
white matter was relatively low, indicating the nonspecific binding
of [125I]SBIM-3 in the AD brain section to be low. [125I]SBIM-3
showed almost no accumulation in the control brain section
(Fig. 2D). The accumulation of radioactivity in the AD brain section
corresponded with the results of immunohistochemical staining
with both the anti phosphorylated tau antibody (AT8) and the anti
Ab1–42 antibody (BC05) (Fig. 2B and C, respectively). Compared
with the results of immunohistochemical staining, [125I]SBIM-3
bound both NFT and SP, which reflected SBIM-3’s relatively low
selective affinity (the ratio of Kd values of SBIM-3 is 2.73). To
achieve high contrast between NFT and SP, it is necessary to devel-
op agents with higher affinity for NFT and lower affinity for SP by
introducing another substituted group such as a methoxy group
into SBIM derivatives.
Figure 2. Autoradiogram of [125I]SBIM-3 in an AD brain section (A) and immuno-
histochemical staining with an antibody against phosphorylated tau (AT8: B) and
Ab1–42 (BC05: C) in adjacent brain sections of A. Autoradiogram of [125I]SBIM-3 in a
control brain section (D).
2.4. Fluorescent staining of AD brain sections
To further confirm the affinity of SBIM-3 for NFT in the AD
brain, a fluorescent staining experiment was carried out in the
brain sections of the same AD patient (Fig. 3A). A number of fluo-
rescent spots were clearly stained in the entorhinal cortex. Then,
the same patient’s brain section was stained by Thioflavin S (ThS)
(Fig. 3B). The fluorescent spots obtained with ThS corresponded
to the spots obtained with SBIM-3, suggesting that SBIM-3 bound
to NFT in the AD brain section.
intensity of SBIM derivatives increased in the presence of tau
aggregates. We calculated the apparent binding dissociation con-
stant (Kd) by utilizing this property of SBIM derivatives. The satu-
ration curves of fluorescent intensity revealed the apparent Kd
values of SBIM-1, 2, and 3 for tau aggregates to be 2.36, 0.45,
and 0.30 lM, respectively. We assumed that SBIM derivatives pos-
sibly bind to Ab1–42 aggregates as well as tau aggregates since Ab1–
42 aggregates also form a b-sheet structure. Then we carried out the
same experiment using Ab1–42 aggregates to evaluate the selective
binding affinity between tu and Ab1–42 aggregates. The apparent Kd
values of SBIM-1, 2, and 3 for Ab1–42 aggregates were 0.90, 0.42,
2.5. In vivo biodistribution in normal mice
To evaluate the uptake of SBIM derivatives into brains, a biodis-
tribution study in normal mice was performed with [125I]SBIM-1,
and 0.82 lM, respectively. The ratios of apparent Kd values of SBIM
Figure 3. Fluorescent staining of the AD brain sections (entorhinal cortex) using SBIM-3 (A) and ThS (B).

K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
3359
Table 2
Biodistribution of radioactivity after injection of SBIM derivatives in normal micea
Time after injection (min)
Tissue
2
10
30
60
[
125I]2a ([125I]SBIM-1)
Blood
Liver
5.94 (0.34)
29.4 (5.46)
10.6 (1.36)
4.09 (0.55)
5.50 (1.46)
4.99 (1.25)
8.31 (1.12)
9.09 (2.74)
1.70 (0.25)
3.20 (0.31)
2.72 (0.72)
29.9 (2.52)
5.04 (0.38)
13.7 (2.43)
4.87 (0.89)
1.93 (0.48)
2.84 (0.33)
3.55 (0.37)
4.13 (1.07)
1.40 (0.22)
1.57 (0.41)
19.3 (1.18)
2.54 (0.38)
22.1 (3.36)
2.71 (0.27)
0.69 (0.13)
1.48 (0.17)
1.62 (0.26)
3.60 (0.88)
0.26 (0.02)
1.24 (0.22)
15.6 (2.38)
3.25 (1.63)
27.4 (5.55)
2.46 (0.53)
1.43 (2.11)
1.13 (0.29)
1.44 (0.14)
4.37 (1.54)
0.12 (0.03)
Kidney
Intestine
Spleen
Pancreas
Heart
Lung
Stomachb
Brain
[
125I]2b ([125I]SBIM-2)
Blood
Liver
3.65 (0.54)
19.9 (1.97)
11.2 (1.24)
4.96 (1.18)
5.19 (0.36)
6.80 (2.51)
6.68 (0.53)
7.13 (1.17)
2.31 (0.68)
4.11 (0.37)
2.76 (0.43)
23.2 (1.97)
5.85 (1.26)
11.5 (2.17)
4.90 (0.51)
2.58 (0.11)
2.80 (0.14)
3.55 (0.30)
4.88 (2.31)
1.42 (0.08)
2.19 (0.33)
16.0 (2.38)
3.07 (0.54)
28.4 (5.49)
3.13 (0.99)
0.92 (0.14)
1.28 (0.15)
1.93 (0.27)
4.65 (1.48)
0.36 (0.05)
1.37 (0.31)
11.0 (2.05)
2.02 (0.65)
30.0 (7.54)
1.74 (0.37)
0.51 (0.08)
0.74 (0.14)
1.32 (0.18)
2.97 (1.03)
0.14 (0.02)
Kidney
Intestine
Spleen
Pancreas
Heart
Lung
Figure 4. Comparison of brain uptake of
[
125I]PDB derivatives and
[
125I]SBIM
Stomachb
Brain
derivatives.
[
125I]2c ([125I]SBIM-3)
3. Conclusion
Blood
Liver
3.83 (0.34)
17.4 (1.18)
11.7 (0.87)
3.11 (0.56)
3.48 (0.71)
5.40 (1.57)
8.56 (0.92)
7.39 (0.34)
1.67 (0.25)
3.28 (0.25)
3.24 (0.30)
19.2 (1.48)
7.28 (0.57)
9.22 (1.66)
3.69 (0.78)
3.31 (0.35)
3.46 (0.34)
3.98 (0.22)
2.82 (0.87)
1.61 (0.18)
1.88 (0.23)
14.9 (3.19)
4.32 (0.73)
18.2 (3.12)
2.66 (0.65)
1.67 (0.25)
2.00 (0.28)
2.20 (0.32)
3.83 (0.86)
0.68 (0.11)
1.28 (0.29)
10.1 (0.98)
2.26 (0.28)
26.5 (6.59)
1.42 (0.43)
0.89 (0.13)
1.12 (0.21)
1.39 (0.22)
3.56 (1.49)
0.33 (0.06)
Kidney
Intestine
Spleen
Pancreas
Heart
In conclusion, we designed, synthesized, and evaluated a new
series of SBIM derivatives as potential NFT imaging agents. In bind-
ing experiments, SBIM-3 showed higher affinity for tau aggregates
than Ab1–42 aggregates. In vitro autoradiography and fluorescent
staining revealed that NFT were stained with [125I]SBIM-3 (or
SBIM-3). In biodistribution experiments using normal mice, all
Lung
Stomachb
Brain
[
125I]SBIM derivatives displayed a better initial uptake into and ra-
a
pid clearance from brains after the injection. The brain2 min
/
Each value represents the mean (SD) for 5 animals.
Expressed as % injected dose per organ.
brain60 min ratios of [125I]SBIM derivatives were much higher than
that of any other NFT imaging agent reported previously, perhaps
due to lower lipophilicity. Appropriate structural modifications of
SBIM derivatives may lead to more useful agents for the in vivo
imaging of NFT in AD brains.
b
2, and 3 (Table 2). The uptake of [125I]SBIM-1, 2, and 3 into brains
at 2 min after the injection (brain2 min) was 3.20%, 4.11%, and
3.28%ID/g, respectively. Since there are neither NFT nor SP in nor-
mal brains, the injected agents should washout rapidly. All
4. Experimental
All reagents were commercial products and used without further
purification unless indicated otherwise. 1H NMR spectra were re-
corded on a JEOL JNM-LM400 with TMS as an internal standard. Cou-
pling constants are reported in Hertz. Multiplicity was defined as
singlet (s), doublet (d), triplet (t), and multiplet (m). Mass spectra
were obtained on a SHIMADZU LCMS-2010 EV. HPLC was performed
with a Shimadzu system (a LC-20AT pump with a SPD-20A UV detec-
[
125I]SBIM derivatives showed a rapid clearance from the brain
(brain60 min of [125I]SBIM-1, 2, and 3 were 0.12%, 0.14%, and
0.33%ID/g, respectively). The brain2 min/brain60 min ratio of
[
125I]SBIM-1, 2, and 3 was 26.7, 29.4, and 9.94, much higher than
that of [125I]PDB derivatives (0.30, 0.33, and 0.33 for [125I]PDB-1,
2, and 3, respectively). Compared to
125I]PDB derivatives,
125I]SBIM derivatives showed much better pharmacokinetics in
normal mouse brains (Fig. 4). Moreover, the brain2 min/brain60 min
ratios of
125I]SBIM derivatives were higher than those of
11C]BF158,21 18F]THK-523,22 125I]TH2,23 18F]FPPDB,25 and
18F]T80826 reported previously as NFT imaging agents (5.38,
[
[
tor, k = 254 nm) using a Cosmosil C18 column (Nacalai Tesque, 5C18
-
MS-II, 4.6 ꢀ 150 mm) and acetonitrile/water (containing 0.1%
Et3N) = 6/4 or 5/5 as the mobile phase at a flow rate of 1.0 mL/min.
All key compounds were proven by thismethod to show >99% purity.
[
[
[
[
[
[
approximate 1.83, 6.16, 1.69 (the ratios of brain2 min/brain60 min),
and 2.92 (the ratios of brain2.5 min/brain20 min), respectively). The
log P values for [125I]SBIM-1, 2, and 3 were 1.88, 2.00, and 2.23,
much lower than that for [125I]PDB-3 (3.84). These results suggest
that reducing lipophilicity might lead to better pharmacokinetics
of [125I]SBIM derivatives in the brain. The initial uptake was high-
4.1. Chemistry
4.1.1. (E)-6-Iodo-2-(4-nitrostyryl)-1H-benzimidazole (1)
A mixture of 4-iodobenzene-1,2-diamine (1.17 g, 5.00 mmol),
4-nitrocinnamaldehyde (886 mg, 5.00 mmol), and Na2S2O5
(951 mg, 5.00 mmol) dissolved in 5 mL of dimethylformamide
(DMF) was heated to 105 °C and stirred for 2 h. Ice water (50 mL)
was added, and the precipitate formed was collected by filtration,
washed with water and dried under vacuum to obtain 1.66 g of 1
est in the liver (liver2 min, 17.4–29.4%ID/g) and kidney (kidney2 min
,
10.6–11.7%ID/g) followed by a rapid clearance (liver60 min, 10.1–
15.6%ID/g and kidney60 min, 2.02–3.25%ID/g), whereas the intes-
tines showed an accumulation of radioactivity over time (intes-
tine60 min was 26.5–30.0%ID/g). Since the accumulation in the
stomach was low (2.97–4.37%ID), all [125I]SBIM derivatives were
stable in vivo by 60 min after the injection.
(85.0%) as
a brown solid. (400 MHz, DMSO-d6) d 8.25 (d,
J = 8.9 Hz, 2H), 7.90–7.93 (m, 3H), 7.78 (d, J = 16.5 Hz, 1H), 7.42–
7.50 (m, 3H). MS (APCI) m/z 392 [MH+].

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K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
4.1.2. (E)-4-[2-(6-Iodo-1H-benzimidazol-2-yl)ethenyl]aniline
(2a)
from 4a. 1H NMR (400 MHz, CD3OD) d 7.84 (s, 1H), 7.59 (s, 1H),
7.44 (d, J = 16.3 Hz, 1H), 7.31–7.34 (m, 3H), 7.24 (dd, J = 1.8,
8.5 Hz, 1H), 6.75 (d, J = 16.5 Hz, 1H), 6.53 (d, J = 8.7 Hz, 2H), 2.76
(s, 3H). MS (APCI) m/z 328 [MH+].
To a mixture of 1 (1.96 g, 5.00 mmol) and concentrated HCl
(1.20 mL) in 80% EtOH (60 mL) was added powdered iron (1.12 g,
20.0 mmol). The reaction mixture was stirred for 24 h under reflux,
and then cooled to room temperature. The precipitate of iron oxides
and hydroxy salts was removed by filtration. The solvent was re-
moved and the residue was neutralized with saturated NaHCO3(aq)
and extracted with ethyl acetate. The organic phase was dried over
Na2SO4 and filtered. The filtrate was concentrated and the residue
was purified by silica gel chromatography (chloroform/metha-
nol = 10/1) to give 1.53 g of 2a (84.6%) as a yellow solid. mp: 116–
118 °C 1H NMR (400 MHz, CD3OD) d 7.80 (s, 1H), 7.44 (d, J = 16.6 Hz,
1H), 7.40 (dd, J = 1.6, 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H) 7.22 (d,
J = 8.4 Hz, 1H), 6.78 (d, J = 16.5 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H). HRMS
(EI) m/z calcd for C15H12IN3 (M+) 361.0076, found 361.0080.
4.1.8. (E)-4-[2-(6-Bromo-1H-benzimidazol-2-yl)ethenyl]-N,N-
dimethylaniline (4c)
The same reaction described above to prepare 1 was used, and
340 mg of 4c was obtained in a yield of 86.7% as a yellow solid from
4-bromo-1,2-diaminobenzene and 4-dimethylaminocinnamalde-
hyde. 1H NMR (400 MHz, CD3OD)
d 7.62 (s, 1H), 7.51 (d,
J = 16.5 Hz, 1H), 7.45 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 8.5 Hz, 1H),
7.29 (dd, J = 1.8, 8.5 Hz, 1H), 6.84 (d, J = 16.4 Hz, 1H), 6.74 (d,
J = 8.9 Hz, 2H), 2.99 (s, 6H). MS (APCI) m/z 342 [MH+].
4.1.9. (E)-4-[2-(6-Bromo-1-tert-butoxycarbonyl-benzimidazo-2-
yl)ethenyl]-N-tert-butoxycarbonylaniline (5a)
4.1.3. (E)-4-[2-(6-Iodo-1H-benzimidazol-2-yl)ethenyl]-N-
methylaniline (2b)
Compound 4a (682 mg, 2.00 mmol) was added to a stirred solu-
tion of guanidine hydrochloride (15 mol %) and (Boc)2O (437 mg,
2.00 mmol) in EtOH (5 mL), at 40 °C and stirred for 2 h. The solvent
was removed and extracted with ethyl acetate. The organic phase
was dried over Na2SO4 and filtered. The solvent was removed,
and the residue was purified by silica gel chromatography (ethyl
acetate/hexane = 1/4) to give 700 mg of 5a (79.4%) as a yellow so-
lid. 1H NMR (400 MHz, CDCL3) d 8.12 (s, 1H), 7.92 (d, J = 16.0 Hz,
1H), 7.78–7.84 (m, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.39–7.45 (m,
3H), 1.75 (s, 9H), 1.57 (s, 9H). MS (APCI) m/z 514 [MH+].
A solution of NaOMe (28 wt % in MeOH, 3.67 mL) was added to
a
mixture of 2a (1.65 g, 4.58 mmol) and paraformaldehyde
(741 mg, 24.7 mmol) in methanol (20 mL) dropwise. The mixture
was stirred under reflux for 1 h. After NaBH4 (866 mg, 22.9 mmol)
was added, the solution was heated under reflux for 2 h. 1 M NaOH
(30 mL) was added to the cold mixture and extracted with ethyl
acetate. The organic phase was dried over Na2SO4 and filtered.
The solvent was removed, and the residue was purified by silica
gel chromatography (chloroform/methanol = 49/1) to give 1.20 g
of 2b (69.8%) as
a
yellow solid. mp: 117-119 °C 1H NMR
4.1.10. (E)-4-[2-(6-Bromo-1-tert-butoxycarbonyl-benzimidazo-
2-yl)ethenyl]-N-tert-butoxycarbonyl-N-methylaniline (5b)
The same reaction described above to prepare 5a was used, and
1.50 g of 5b was obtained in a yield of 81.3% as a yellow solid from
4b. 1H NMR (400 MHz, acetone-d6) d 7.91–7.92 (m, 2H), 7.67 (d,
J = 8.5 Hz, 2H), 7.58–7.60 (m, 1H), 7.48–7.51 (m, 1H), 7.41 (d,
J = 8.5 Hz, 2H), 7.20–7.31 (m, 1H), 3.28 (s, 3H), 1.78–1.80 (m, 9H),
1.48 (s, 9H) MS (APCI) m/z 528 [MH+].
(400 MHz, CD3OD) d 7.75 (s, 1H), 7.41 (d, J = 16.3 Hz, 1H), 7.39
(dd, J = 1.7, 8.4 Hz, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.4 Hz,
1H), 6.73 (d, J = 16.3 Hz, 1H), 6.52 (d, J = 8.7 Hz, 2H), 2.73 (s, 3H).
HRMS (EI) m/z calcd for C16H14IN3 (M+) 375.0233, found 375.0235.
4.1.4. (E)-4-[2-(6-Iodo-1H-benzimidazol-2-yl)ethenyl]-N,N-
dimethylaniline (2c)
The same reaction described above to prepare 1 was used, and
320 mg of 2c was obtained in a yield of 82.3% as a yellow solid from
4-iodobenzene-1,2-diamine and 4-dimethylaminocinnamalde-
hyde. mp: 245 °C (decomposition) 1H NMR (400 MHz, DMSO-d6)
d 7.82 (s, 1H), 7.56 (d, J = 16.2 Hz, 1H), 7.49 (d, J = 9.0 Hz, 2H),
7.41 (dd, J = 1.7, 8.4 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 6.90 (d,
J = 16.5 Hz, 1H), 6.75 (d, J = 8.7 Hz, 2H), 2.97(s, 6H). HRMS (EI) m/
z calcd for C17H16IN3 (M+) 389.0389, found 389.0386.
4.1.11. (E)-4-[2-(6-Bromo-1-tert-butoxycarbonyl-benzimidazo-
2-yl)ethenyl]-N,N-dimethylaniline (5c)
The same reaction described above to prepare 5a was used, and
150 mg of 5c was obtained in a yield of 88.3% as a yellow solid from
4c. 1H NMR (400 MHz, CDCL3) d 8.10 (s, 1H) 7.93 (d, J = 15.9 Hz,
1H), 7.67 (d, J = 15.9 Hz, 1H), 7.51–7.55 (m, 3H), 7.42 (dd, J = 1.8,
8.5 Hz, 1H), 6.70 (d, J = 8.8 Hz, 2H), 3.02 (s, 6H), 1.76 (s, 9H). MS
(APCI) m/z 442 [MH+].
4.1.5. (E)-6-Bromo-2-(4-nitrostyryl)-1H-benzimidazole (3)
The same reaction described above to prepare 1 was used, and
921 mg of 3 was obtained in a yield of 67.1% as a yellow solid from
4-bromo-1,2-diaminobenzene and 4-nitrocinnamaldehyde. 1H
NMR (400 MHz, DMSO-d6) d 8.27 (d, J = 7.3 Hz, 2H), 7.95 (d,
J = 7.8 Hz, 2H), 7.78–7.82 (m, 2H), 7.54 (d, J = 8.2 Hz, 1H), 7.45 (d,
J = 16.5 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), MS (APCI) m/z 344 [MH+].
4.1.12. (E)-4-[2-(1-tert-Butoxycarbonyl-6-tributylstannyl-
benzimidazo-2-yl)ethenyl]-N-tert-butoxycarbonylaniline (6a)
A mixture of 5a (494 mg, 1.10 mmol), bis(tributyltin) (1.12 mL,
2.20 mmol), and (Ph3P)4Pd (555 mg, 0.480 mmol) in a mixed sol-
vent (15 mL, dioxane/Et3N = 2/1) was stirred under reflux for 5 h.
The solvent was removed, and the residue was purified by silica
gel chromatography (hexane/ethyl acetate = 4/1) to give 432 mg
of 6a (59.0%) as a yellow oil. 1H NMR (400 MHz, CDCL3) d 7.93 (d,
J = 16.0 Hz, 1H) 7.83–7.90 (m, 3H), 7.56 (d, J = 8.7 Hz, 2H), 7.37–
7.41 (m, 3H), 6.70 (s,1H), 1.75 (s, 9H), 1.52 (s, 9H), 0.87–1.66 (m,
27H). MS (APCI) m/z 726 [MH+].
4.1.6. (E)-4-[2-(6-Bromo-1H-benzimidazol-2-yl)ethenyl]aniline
(4a)
The same reaction described above to prepare 2a was used, and
545 mg of 4a was obtained in a yield of 81.0% as a yellow solid
from 3. 1H NMR (400 MHz, CD3OD) d 7.65 (s, 1H), 7.52 (d,
J = 16.5 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H),
7.32 (dd, J = 1.8, 8.5 Hz, 1H), 6.85 (d, J = 16.4 Hz, 1H), 6.71 (d,
J = 8.5 Hz, 2H). MS (APCI) m/z 314 [MH+].
4.1.13. (E)-4-[2-(1-tert-Butoxycarbonyl-6-tributylstannyl-
benzimidazo-2-yl)ethenyl]-N-tert-butoxycarbonyl-N-
methylaniline (6b)
The same reaction described above to prepare 6a was used, and
210 mg of 6b was obtained in a yield of 30.3% as a yellow oil from
5b. 1H NMR (400 MHz, CD3OD) d 7.78–7.84 (m, 2H), 7.66–7.71 (m,
2H), 7.51 (d, J = 8.7 Hz, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.21 (d,
4.1.7. (E)-4-[2-(6-Bromo-1H-benzimidazol-2-yl)ethenyl]-N-
methylaniline (4b)
The same reaction described above to prepare 2b was used, and
500 mg of 4b was obtained in a yield of 76.5% as a yellow solid

K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
3361
J = 8.5 Hz, 2H), 3.16 (s, 3H), 1.66 (s, 9H), 1.37 (s, 9H), 0.78–1.59 (m,
27H). MS (APCI) m/z 740 [MH+].
transferred into two test tubes for counting. The remainder of
the 1-octanol phase was transferred (1 mL) into a new test tube.
1-Octanol (2.0 mL) and PBS (ꢁ) (3.0 mL) were pipetted into the test
tube. The vortexing, centrifuging, and counting were repeated. The
4.1.14. (E)-4-[2-(1-tert-Butoxycarbonyl-6-tributylstannyl-
benzimidazo-2-yl)ethenyl]-N,N-dimethylaniline (6c)
The same reaction described above to prepare 6a was used, and
210 mg of 6c was obtained in a yield of 40.3% as a yellow oil from
5c. 1H NMR (400 MHz, CDCL3) d 7.94 (d, J = 16.0 Hz, 1H), 7.87 (d,
J = 7.9 Hz, 1H), 7.83 (s, 1H) 7.74 (d, J = 15.9 Hz, 1H), 7.53 (d,
J = 8.5 Hz, 2H), 7.34 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.9 Hz, 2H),
3.01 (s, 6H) 1.74 (s, 9H), 0.86–1.67 (m, 27H). MS (APCI) m/z 654
[MH+].
amount of radioactivity in each tube was measured with a
c coun-
ter (Perkin Elmer, Wizard 1470). The partition coefficient was cal-
culated using Eq. 1:
ðcounts=lL in 1-octanolÞ=ðcounts=lL in bufferÞ ¼ r
ð1Þ
4.5. In vitro autoradiography
Postmortem brain tissues from an autopsy-confirmed case of
AD (a 93-year-old, female) and a control (a 73-year-old, female)
were obtained from the Graduate School of Medicine, Kyoto Uni-
versity and BioChain Institute Inc., respectively. The presence and
location of NFT and SP in the sections were confirmed with immu-
nohistochemical staining using anti-phosphoylated tau antibody
(AT8) and Ab1–42 antibody (BC05), respectively. Six-micrometer-
thick serial sections of paraffin-embedded blocks were used for
staining. The sections were subjected to two 15-min incubations
in xylene, two 1-min incubations in 100% EtOH, one 1-min incuba-
tion in 90% EtOH, one 1-min incubation in 80% EtOH, and one 1-
min incubation in 70% EtOH to completely deparaffinize them, fol-
lowed by two 2.5-min washes in water. The sections were incu-
4.2. Binding assay with SBIM derivatives using recombinant tau
and Ab1–42 aggregates
The 441-aa isoform of human tau was expressed from a cDNA
clone in Escherichia coli and purified as described previously.30
Tau aggregates were prepared by incubating tau protein (1 mg/
mL in MES buffer, pH 6.8) at 37 °C for 3 days with gentle and con-
stant shaking in the presence of 0.1 mg/mL heparin. A solid form of
Ab1–42 was purchased from Peptide Institute (Osaka, Japan). Aggre-
gation was achieved by gently dissolving the peptide (0.25 mg/mL)
in PBS solution (pH 7.4). The solutions were incubated at 37 °C for
42 h with gentle and constant shaking. The binding experiments
were carried out in Protein LoBind Tubes (Eppendorf). A mixture
bated with [125I]2c ([125I]SBIM-3) (92.5 kBq/100
lL) for 1 h at
room temperature. They were then dipped in 50% EtOH for 2 h
and washed with water for 30 s. After drying, the 125I-labeled sec-
tions were exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan)
of tau aggregates (final conc. 10
conc. 10 g/mL) were incubated at room temperature for 30 min in
the presence of SBIM derivatives (final conc. 0.02–10 M), dis-
lg/mL) or Ab1–42 aggregates (final
l
l
overnight. Autoradiographic images were obtained using
BAS5000 scanner system (Fuji Film).
a
pensed to MULTI WELL PLATE (0.4 mL ꢀ 96 wells flatbottom,
SUMITOMO BAKELITE CO., LTD, Japan), and subjected to fluores-
cence spectroscopy (kex = 440 nm; kem = 480 nm for Ab1–42 aggre-
gates and kex = 440 nm; kem = 510 nm for tau aggregates). The
fluorescence intensity was plotted and the apparent Kd values of
SBIM derivatives for recombinant tau and Ab1–42 aggregates were
calculated from the fluorescent saturation curves using GraphPad
Prism software (Graph Pad software, San Diego, CA).
4.6. Immunohistochemical staining of NFT and SP in human AD
brain sections
Postmortem brain tissues from an autopsy-confirmed case of
AD (a 93-year-old, female) were obtained from the Graduate
School of Medicine, Kyoto University. Deparaffinization was car-
ried out according to the procedure described above. They were
then autoclaved for 15 min in 0.01 M citric acid buffer (pH 6.8)
to activate the antigen. After two 5-min incubations in PBS-
Tween20, the sections were incubated at room temperature with
anti phosphorylated tau (AT8) or Ab1–42 primary antibody (BC05)
overnight. After three 2-min incubations in PBS-Tween20, they
were incubated with biotinylated goat anti-mouse IgG at room
temperature for 1 h. After three 5-min. incubations in PBS-
Tween20, the sections were incubated with Streptavidin–Peroxi-
dase complex at room temperature for 30 min. After three 2-min
incubations in PBS-Tween20, they were incubated with DAB as a
chromogen for 30 min. After being washed with water, the sections
were observed under a microscope.
4.3. Radiolabeling
The radioiodinated forms of compounds [125I]2a ([125I]SBIM-1),
[
125I]2b ([125I]SBIM-2), and [125I]2c ([125I]SBIM-3) were prepared
from the corresponding tributyltin derivatives by iododestannyla-
tion. Briefly, to initiate the reaction, 100 L of H2O2 (3%) was added
to a mixture of a tributyltin derivative (150 g/150 L EtOH),
125I]NaI (3.7–7.4 MBq, specific activity 81.4 TBq/mmol), and
l
l
l
[
100 lL of 1 N HCl in a sealed vial. The reaction was allowed to pro-
ceed at room temperature for 15 min and terminated by addition
of saturated NaHSO3(aq) (200 L). After neutralization with so-
l
dium hydrogen carbonate and extraction with ethyl acetate, the
extract was dried by passing through an anhydrous Na2SO4 col-
umn. TFA (80 lL) was added to the solution and blown dry with
4.7. Fluorescent staining of AD brain sections
a stream of nitrogen gas. The radioiodinated ligand was purified
by HPLC on a Cosmosil C18 column with an isocratic solvent of ace-
tonitrile/H2O (containing 0.1% Et3N) = 6/4 or 5/5 at a flow rate of
1.0 mL/min.
Postmortem brain tissues from an autopsy-confirmed case of
AD (a 93-year-old, female) were obtained from the Graduate
School of Medicine, Kyoto University. Deparaffinization was car-
ried out according to the procedure described above. The brain tis-
4.4. Measurement of logP values
sue was incubated with a 50% ethanol solution (300 lM) of 2c
(SBIM-3) for 30 min. Finally, the section was washed in ethanol
for 30 s. Fluorescent observation was performed with the Keyence
system (excitation filter, 450–490 nm; emission filter, 510–
560 nm; DM filter; 495 nm). The same patient’s brain section
was stained with ThS, a pathological dye used for staining NFT in
the brain, and observed with the Keyence system (excitation filter,
450–490 nm; emission filter, 510–560 nm; DM filter; 495 nm).
The experimental determination of partition coefficients of
[
125I]2a ([125I]SBIM-1),
[
125I]2b ([125I]SBIM-2), and
[
125I]2c
([125I]SBIM-3) was performed in 1-octanol and PBS (ꢁ) at a pH of
7.4. 1-Octanol (3.0 mL) and PBS (ꢁ) (3.0 mL) were pipetted into a
12-mL-test tube containing 0.37 MBq of test compounds. The test
tube was vortexed for 2 min, and centrifuged (10 min, 2000 rpm).
Aliquots (500
l
L) from the 1-octanol and PBS (ꢁ) phases were

3362
K. Matsumura et al. / Bioorg. Med. Chem. 21 (2013) 3356–3362
12. Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan, R.; Pike, K. L.; O’Keefe, G.;
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(BIOREVO BZ-9000, Keyence Corp., Osaka, Japan).
4.8. In vivo biodistribution in normal mice
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The experiments with animals were conducted in accordance
with our institutional guidelines and approved by the Kyoto Uni-
versity.
125I]2b ([125I]SBIM-2), and
20.6 kBq) containing ethanol (10
A
saline solution (100
l
L) of
125I]2c ([125I]SBIM-3) (13.2–
L) was injected intravenously
[
125I]2a ([125I]SBIM-1),
[
[
l
directly into the tail of ddY mice (5-week-old, male). The mice
were sacrificed at various time points post-injection. The organs
of interest were removed and weighed, and the radioactivity was
measured with an automatic g counter.
19. Arriagada, P. V.; Growdon, J. H.; Hedley-Whyte, E. T.; Hyman, B. T. Neurology
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Acknowledgments
20. Nelson, P. T.; Alafuzoff, I.; Bigio, E. H.; Bouras, C.; Braak, H.; Cairns, N. J.;
Castellani, R. J.; Crain, B. J.; Davies, P.; Del Tredici, K.; Duyckaerts, C.; Frosch, M.
P.; Haroutunian, V.; Hof, P. R.; Hulette, C. M.; Hyman, B. T.; Iwatsubo, T.;
Jellinger, K. A.; Jicha, G. A.; Kovari, E.; Kukull, W. A.; Leverenz, J. B.; Love, S.;
Mackenzie, I. R.; Mann, D. M.; Masliah, E.; McKee, A. C.; Montine, T. J.; Morris, J.
C.; Schneider, J. A.; Sonnen, J. A.; Thal, D. R.; Trojanowski, J. Q.; Troncoso, J. C.;
Wisniewski, T.; Woltjer, R. L.; Beach, T. G. J. Neuropathol. Exp. Neurol. 2012, 71,
362.
This study was supported by the Funding Program for Next
Generation World-Leading Researchers and JSPS Research Fellow-
ships for Young Scientists. We thank Professor Hiroshi Mori for
kindly providing human tau cDNA for the in vitro binding assays.
21. Okamura, N.; Suemoto, T.; Furumoto, S.; Suzuki, M.; Shimadzu, H.; Akatsu, H.;
Yamamoto, T.; Fujiwara, H.; Nemoto, M.; Maruyama, M.; Arai, H.; Yanai, K.;
Sawada, T.; Kudo, Y. J. Neurosci. 2005, 25, 10857.
22. Fodero-Tavoletti, M. T.; Okamura, N.; Furumoto, S.; Mulligan, R. S.; Connor, A.
R.; McLean, C. A.; Cao, D.; Rigopoulos, A.; Cartwright, G. A.; O’Keefe, G.; Gong,
S.; Adlard, P. A.; Barnham, K. J.; Rowe, C. C.; Masters, C. L.; Kudo, Y.; Cappai, R.;
Yanai, K.; Villemagne, V. L. Brain 2011, 134, 1089.
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