Synthesis and characterization of novel phenylindoles as potential probes for imaging of β-amyloid plaques in the brain

Article abstract of DOI:10.1016/j.bmc.2010.05.013

We synthesized a novel series of phenylindole (PI) derivatives and evaluated their biological activities as probes for imaging Aβ plaques in vivo. The affinity for Aβ plaques was assessed by an in vitro-binding assay using pre-formed synthetic Aβ aggregates. 2-Phenyl-1H-indole (2-PI) derivatives showed high affinity for Aβ42 aggregates with K_i values ranging from 4 to 32 nM. 2-PI derivatives clearly stained Aβ plaques in an animal model of AD. In biodistribution experiments using normal mice, 2-PI derivatives displayed sufficient uptake for imaging, ranging from 1.1% to 2.6% ID/g. Although additional modifications are necessary to improve uptake by and clearance from the brain, 2-PI derivatives may be useful as a backbone structure to develop novel Aβ imaging agents.

Full text of DOI:10.1016/j.bmc.2010.05.013

Bioorganic & Medicinal Chemistry 18 (2010) 4740–4746
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of novel phenylindoles as potential probes
for imaging of b-amyloid plaques in the brain
a
a
b
Hiroyuki Watanabe a, Masahiro Ono a,b, , Mamoru Haratake , Nobuya Kobashi , Hideo Saji ,
*
Morio Nakayama a,
*
a Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a novel series of phenylindole (PI) derivatives and evaluated their biological activities as
probes for imaging Ab plaques in vivo. The affinity for Ab plaques was assessed by an in vitro-binding
assay using pre-formed synthetic Ab aggregates. 2-Phenyl-1H-indole (2-PI) derivatives showed high
affinity for Ab42 aggregates with Ki values ranging from 4 to 32 nM. 2-PI derivatives clearly stained Ab
plaques in an animal model of AD. In biodistribution experiments using normal mice, 2-PI derivatives dis-
played sufficient uptake for imaging, ranging from 1.1% to 2.6% ID/g. Although additional modifications
are necessary to improve uptake by and clearance from the brain, 2-PI derivatives may be useful as a
backbone structure to develop novel Ab imaging agents.
Received 6 April 2010
Revised 1 May 2010
Accepted 4 May 2010
Available online 13 May 2010
Keywords:
Alzheimer’s disease
b-amyloid
Ó 2010 Elsevier Ltd. All rights reserved.
Imaging
Phenylindole
1. Introduction
develop probes for PET/SPECT derived from ThT including
[
11C]PIB,9,10 11C]AZD2184,11,12 TZDM,13 IBOX,14 123I]IMPY,15,16 phen-
[ [
Alzheimer’s disease (AD) is the most common neurodegenera-
tive disorder of the elderly and is characterized clinically by
dementia, cognitive impairment, and memory loss. Postmortem
brains of AD patients reveal neuropathological features: the pres-
ence of senile plaques and neurofibrillary tangles, which contain
b-amyloid (Ab) peptides and highly phosphorylated tau proteins.1,2
The formation and deposition of Ab plaques is considered one of
the most significant factors in AD. Currently, the only definitive
diagnosis of AD is by pathological examination of postmortem
staining of affected brain tissue. Therefore, non-invasive tech-
niques such as positron emission tomography (PET) and single
photon emission computed tomography (SPECT) are useful for
the diagnosis of AD and new anti-amyloid therapies.3–5
Many radiolabeled probes based on the core structure of Congo
Red (CR) and thioflavin T (ThT) have been developed as imaging
agents for Ab plaques. Although CR has large molecular size, some
truncated CR type molecules such as stilbene and styrylpyridine
derivatives have been reported.6–8 Because ThT has a lower molec-
ular weight than CR, implying greater blood–brain penetration and
easier organic synthesis, a number of groups have worked to
ylbenzofuran derivatives,17,18 phenylbenzothiophene derivatives,19
imidazopyridine derivatives,20,21 and imidazopyridazine derivatives22
(Fig. 1). Clinical trials in AD patients have been conducted with
[
11C]PIB and [11C]AZD2184, and indicated the PET-based imaging
of Ab plaques in the living human brain to be useful for the diagno-
sis of AD. Radioiodinated probes for SPECT such as TZDM, IBOX, and
phenylbenzofuran derivatives have shown high affinity for Ab
aggregates in vitro and high initial uptake, but a slow washout from
the brain.13,14,17 Since the slow washout leads to a low signal/noise
ratio in the imaging of Ab plaques in vivo, a molecular design that
facilitates the clearance of the radiolabeled probes from normal
areas of the brain is needed. Several reports have shown the lipo-
philicity of probes to play an important role in uptake by and clear-
ance from brain tissue.9,23 As this may partly explain the slow
washout from the brain, we planned to select a ThT-derived scaffold
with less lipophilicity. In the search for such a scaffold, we focused
on phenylindole, never before applied to the development of Ab
imaging probes, and calculated its log D value to be 3.97, lower than
that of phenylbenzofuran (4.34) or phenylbenzothiophene (4.94)
(calculated with the Sparc On-Line Calculator).
In the present study, we synthesized a novel series of 2-phenyl-
1H-indole (2-PI) and 1-phenyl-1H-indole (1-PI) derivatives and
evaluated their potential as probes for imaging Ab in vivo. This is
the first time that PI derivatives have been used for SPECT to detect
Ab plaques.
* Corresponding authors. Tel.: +81 75 753 4608; fax: +81 75 753 4568 (M.O.); tel./
fax: +81 95 819 2441 (M.N.).
E-mail addresses: ono@pharm.kyoto-u.ac.jp (M. Ono), morio@nagasaki-u.ac.jp
(M. Nakayama).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.013

H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
4741
11CH3
NH
11CH3
NH
N
N
S
N
N
N
S
N
N
N
N
N
S
123I
O
HO
I
HO
I
11C]PIB
[
11C]AZD2184
[
123I]IMPY
TZDM
IBOX
[
N
N
R'
R
R'
R'
R
N
N
O
S
R
R
N
Benzothiophene derivatives
Imidazopyridine derivatives
Imidazopyridazine derivatives
Benzofuran derivatives
Figure 1. Chemical structure of thioflavin T analogs.
bromo-to-trimethyltin exchange reaction catalyzed by Pd(0). Tri-
methyltin derivatives (4 and 13) were readily reacted with iodine
in ethyl acetate at room temperature to give the iodo derivatives,
5 and 14. The tributyltin derivative 9 was prepared from the corre-
sponding bromo compound by protecting the tert-butoxycarbonyl
(Boc) group. Compound 9 was readily reacted with iodine in ethyl
acetate at room temperature to give the iodo derivative 10 and
deprotected by TFA. Compounds 12 and 14 were converted to 17
and 15 by demethylation with BBr3 in CH2Cl2 (13.0% and 19.5%
yields), respectively. Direct alkylation of 17 and 15 with ethylene
chlorohydrin and potassium carbonate in DMF resulted in 18 and
16, respectively. The synthesis of 1-PI derivatives is outlined in
Scheme 5. Compound 20 was prepared by the copper-mediated
coupling of a substituted indole with 4-(dimethylamino)phenylbo-
ronic acid in a yield of 44.2%.25 The tributyltin derivative 21 was
readily reacted with iodine in ethyl acetate at room temperature
to give the iodo derivative 22. The trimethyltin derivatives were
CH3I
K2CO3
NH2
NH
DMSO
1
Scheme 1.
2. Results and discussion
The synthesis of 2-PI derivatives is outlined in Schemes 1–4. We
used a one-pot method producing 2-PI.24 Compounds 3, 6, and 12
were prepared from 2 and terminal alkynes (1, 4-ethynyl-N,N-
dimethylaniline and p-ethynylanisole) using a palladium catalyst
in the presence of tetrabutylammonium fluoride (TBAF) (27.2–
49.5% yields). Trimethyltin derivatives (4, 7, and 13) were prepared
from the corresponding bromo compounds (3, 6, and 12) using a
CuI
PdCl2(PPh3)2
Br
I
Br
Br
I
Ac2O
+
R
R
NH
N
H
TBAF
THF
NH2
O
CH3
1
3 : R = NHCH3
6 : R = N(CH3)2
12 : R = OCH3
R = N(CH3)2
OCH3
2
(Ph3P)4Pd
(Me3Sn)2
I2
Me3Sn
I
R
R
N
N
Dioxane
H
H
4 : R = NHCH3
7 : R = N(CH3)2
13 : R = OCH3
5 : R = NHCH3
14 : R = OCH3
BBr3
OH
I
I
Cl
OH
O
OH
CH2Cl2
N
H
N
H
DMF
16
15
Scheme 2.
(Ph3P)4Pd
(Bu3Sn)2
Br
(Boc)2O
DMAP
Bu3Sn
Br
N
N
N
N
Boc
N
Boc
N
Acetonitrile
Dioxane
Et3N
H
6
9
8
I
I2
I
TFA
N
N
N
Boc
N
H
Dioxane
10
11
Scheme 3.

4742
H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
BBr3
Br
Br
OH
Cl
OCH3
OH
N
H
CH2Cl2
N
H
DMF
12
17
Me3Sn
(Ph3P)4Pd
(Me3Sn)2
Br
OH
OH
O
O
N
H
N
H
Dioxane
18
19
Scheme 4.
Br
Table 1
Inhibition constants (Ki) for binding of PI derivatives determined using [125I]IMPY as
the ligand in Ab(1–42) aggregates
Cu(OAc)2
Et3N
HO
HO
Br
N
B
N
+
N
H
Compound
Ki (nM)a
CH2Cl2
5
27.0 0.18
4.24 0.71
20.2 5.15
32.9 2.93
25.9 5.13
>10,000
N
20
11
14
15
16
22
Bu3Sn
I
(Ph3P)4Pd
(Bu3Sn)2
I2
N
N
Dioxane
Et3N
a
Values are means standard error of the mean for 3–6 independent
experiments.
N
N
22
21
120
100
80
60
40
20
0
Scheme 5.
used as the starting materials for radioiodination in the prepara-
tion of [125I]5, [125I]11, [125I]14, and [125I]16. Novel radioiodinated
2-PI derivatives were obtained by iododestannylation reactions
using NCS as an oxidant (Scheme 6). It was anticipated that not
adding a carrier would result in a final product bearing a theoret-
ical specific activity similar to that of 125I (2200 Ci/mmol). The
radiochemical identity of the radioiodinated ligands was verified
by co-injection with non-radioiodinated compounds from HPLC
profiles. The HPLC retention times are shown in Supplementary
data. [125I]5, [125I]11, [125I]14, and [125I]16 were each obtained in
a radiochemical yield of 14–56% with a radiochemical purity of
>95% after purification by HPLC.
5
15
16
22
11
14
0
1
2
3
4
5
6
7
8
log [inhibitor] (pM)
The affinity of PI derivatives (5, 11, 14, 15, 16, and 22) was eval-
uated based on inhibition of the binding of [125I]IMPY to Ab42
aggregates. The 2-PI derivatives (5, 11, 14, 15, and 16) showed
inhibitory activity toward Ab aggregates, while the 1-PI derivative
22 did not (Table 1 and Fig. 2). The Ki values of 5, 11, 14, 15, and 16
were 27, 4, 20, 33, and 26 nM, respectively, suggesting high affinity
for Ab(1–42) aggregates and considerable tolerance of structural
modifications. They also suggested that the position of the substi-
tuted phenyl group in the PI molecule plays an important role in
the affinity for Ab aggregates.
Figure 2. Curves of [125I]IMPY against 2-PI (5, 11, 14, 15, and 16) and 1-PI (22).
transgenic mice (female, 28 months old) (Fig. 3A), while none were
observed in wild-type mice (female, 22 months old) (Fig. 3B). The
pattern of labeling was consistent with that observed with thiofla-
vin S (Fig. 3C). Compound 11 should therefore show specific bind-
ing to Ab plaques in the mouse brain. Compound 14 also clearly
stained Ab plaques in the Tg2576 mouse brain (data not shown).
A slight difference in Ki of 11 and 14 did not significantly affect
the staining in mouse brain sections.
To confirm the affinity of the 2-PI derivatives for Ab plaques in
the brain, fluorescent staining of sections of brain tissue from an
animal model of AD was carried out with 11 (Fig. 3). Many specks
of fluorescence were observed in brain sections of Tg2576
To evaluate the uptake into the brain of the PI derivatives, bio-
distribution experiments were performed in normal mice with four
radioiodinated PI derivatives; [125I]5, [125I]11, [125I]14, and [125I]16
(Table 2). Radioactivity penetrated the blood–brain barrier with the
rate of uptake ranging from 1.2% to 2.6% ID/g brain at
2–10 min postinjection. But the washout of these probes from the
brain in normal mice appears to be relatively slow. The uptake of
Me3Sn
125I
[
125I]NaI
R
R
N
H
N
H
NCS
[
[
[
[
125I]5 (R = NHCH3)
125I]11 (R = N(CH3)2)
125I]14 (R = OCH3)
[
125I]11 was higher in the stomach than in any other organ, possibly
4
7
due to deiodination. The brain uptake and clearance is similar to
that of radioiodinated ThT analogs such as TZDM and IBOX.13,14
More recently, we have developed 11C-labeled phenylbenzofuran
derivative, which are less lipophilic by replacing the iodine with a
13
19
125I]16 (R = OCH2CH2OH)
Scheme 6.

H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
4743
Figure 3. Neuropathological staining of 11 in 10-lm AD model mouse sections (A) and wild-type mouse sections (B). Labeled plaques were confirmed by staining of the
adjacent sections with thioflavin S (C).
Table 2
the lipophilicity by introducing a hydrophilic group, are necessary
to improve the properties of 2-PI derivatives.
Biodistribution of radioactivity after injection of [125I]2-PI derivatives in normal micea
Tissue
Time after injection (min)
10 30
3. Conclusion
2
60
[
125I]5
We developed PI derivatives as novel SPECT ligands for imaging
Ab plaques in the AD brain. 2-PI derivatives (5, 11, 14, 15, and 16)
displayed excellent affinity for Ab in binding experiments in vitro.
They clearly stained Ab plaques in Tg2576 mouse brain, reflecting
their affinity for Ab aggregates in vitro. The degree to which the
2-PI derivatives penetrated the brain was very encouraging. How-
ever, non-specific binding in vivo reflected by a slow washout from
the normal mouse brain may make them unsuitable for the imag-
ing of Ab plaques. In in vivo biodistribution results in normal mice
indicate that there is a critical need to fine-tune the kinetics of
brain uptake and washout. Additional changes to 2-PI may lead
to useful probes for detecting Ab plaques in the AD brain.
Blood
Liver
3.77 (1.25)
1.71 (0.15)
26.83 (1.28)
4.31 (0.44)
3.57 (0.34)
19.67 (8.82)
2.87 (0.33)
3.59 (0.29)
1.34 (0.53)
1.68 (0.13)
1.36 (0.15)
0.80 (0.13)
22.85 (9.60)
5.53 (1.77)
0.97 (0.33)
7.83 (2.24)
2.31 (0.70)
5.68 (1.27)
0.52 (0.03)
1.10 (0.27)
23.07 (2.33)
3.84 (0.67)
8.54 (1.80)
10.34 (1.26)
2.12 (0.32)
2.96 (0.42)
2.55 (2.12)
1.42 (0.03)
15.89 (6.13)
2.61 (0.86)
9.48 (1.88)
5.89 (3.27)
1.27 (0.23)
1.88 (0.57)
1.68 (0.53)
0.83 (0.17)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
[
125I]11
Blood
Liver
6.18 (0.65)
10.20 (1.92)
9.49 (1.61)
1.80 (0.24)
4.13 (0.64)
5.21 (2.50)
8.08 (1.59)
4.03 (0.65)
1.19 (0.34)
4.53 (0.37)
5.14 (1.01)
4.46 (0.71)
3.09 (0.40)
3.28 (0.57)
4.13 (0.67)
4.82 (0.67)
10.65 (2.18)
1.19 (0.30)
3.68 (0.41)
3.55 (0.62)
4.58 (1.24)
4.52 (0.58)
2.60 (0.41)
3.10 (0.41)
1.87 (0.21)
17.77 (2.04)
0.96 (0.17)
3.29 (1.00)
3.24 (0.76)
4.71 (2.44)
6.08 (1.58)
2.24 (0.64)
2.30 (0.54)
1.84 (0.76)
16.26 (3.52)
0.71 (0.19)
Kidney
Intestine
Spleen
Pancreas
Heart
4. Experimental
4.1. General
Stomachb
Brain
[
125I]14
All reagents were commercial products and used without fur-
ther purification unless otherwise indicated. 1H NMR spectra were
obtained on a Varian Gemini 300 spectrometer with TMS as an
internal standard. Coupling constants are reported in hertz. Multi-
plicity was defined by s (singlet), d (doublet), t (triplet), and m
(multiplet). Mass spectra were obtained on a JEOL IMS-DX.
Blood
Liver
3.59 (1.41)
17.81 (5.73)
8.76 (2.19)
1.90 (0.66)
4.22 (0.78)
4.54 (0.44)
7.72 (1.94)
0.72 (0.39)
2.11 (0.69)
1.97 (0.54)
13.96 (4.21)
4.96 (1.44)
6.85 (1.86)
3.43 (1.15)
3.94 (1.46)
2.97 (1.26)
1.38 (1.42)
2.07 (0.71)
1.38 (0.25)
10.38 (2.64)
2.89 (0.44)
12.32 (3.65)
2.96 (0.41)
1.95 (0.33)
1.47 (0.20)
2.67 (3.24)
1.36 (0.37)
0.74 (0.37)
8.22 (1.87)
2.05 (0.54)
20.35 (5.86)
2.02 (0.92)
1.29 (0.37)
1.06 (0.42)
3.06 (1.28)
1.16 (0.32)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
4.1.1. 4-Ethynyl-N-methylbenzenamine (1)
[
125I]16
To a solution of 4-ethynylaniline (819 mg, 7 mmol) in DMSO
were added methyl iodide (1.3 mL, 21 mmol) and anhydrous
K2CO3 (4.8 g, 35 mmol). The reaction mixture was stirred at room
temperature for 3 h. After it was poured into water, the mixture
was extracted with ethyl acetate. The organic layers were dried
over Na2SO4. The solvent was removed and the residue was purified
by silica gel chromatography (hexane/ethyl acetate = 2:1) to give
313 mg of 1 (34.1%). 1H NMR (300 MHz, CDCl3) d 2.49 (s, 1H),
2.83 (s, 3H), (s, 1H), 6.50 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H).
Blood
Liver
4.07 (0.30)
17.69 (2.64)
11.93 (1.83)
1.97 (0.22)
7.56 (1.24)
5.55 (1.00)
12.91 (2.16)
0.97 (0.31)
2.13 (0.54)
1.60 (0.30)
16.77 (2.20)
9.35 (0.46)
5.76 (0.66)
7.09 (1.60)
5.59 (0.56)
5.84 (0.36)
1.97 (1.05)
2.62 (0.21)
1.26 (0.26)
13.42 (1.70)
6.77 (0.83)
11.32 (1.65)
4.75 (0.82)
3.57 (0.40)
2.97 (0.62)
2.11 (0.26)
1.93 (0.18)
0.80 (0.20)
8.17 (1.28)
2.76 (0.45)
18.98 (3.21)
2.39 (0.52)
2.22 (0.29)
1.36 (0.17)
1.62 (0.44)
1.82 (0.35)
Kidney
Intestine
Spleen
Pancreas
Heart
Stomachb
Brain
a
Expressed as % injected dose per gram. Each value represents the mean (SD) for
3–5 animals.
b
4.1.2. N-(4-Bromo-2-iodophenyl)acetamide (2)
Expressed as % injected dose per organ.
A mixture of 4-bromo-2-iodoaniline (586 mg, 2 mmol) and ace-
tic anhydride (0.19 mL, 2 mmol) in toluene (5 mL) was stirred at
room temperature for 3.5 h. The solid that formed was filtered
and washed with hexane to give 464 mg of 2 (69.4%). 1H NMR
(300 MHz, CDCl3) d 2.24 (s, 3H), 7.39 (s, 1H), 7.46 (dd, J = 2.4,
2.1 Hz, 1H), 7.90 (d, J = 2.1 Hz, 1H), 8.13 (d, J = 8.7 Hz, 1H).
hydroxy group.18 11C]Phenylbenzofuran, which has less lipophilic-
[
ity than [125I]phenylbenzofuran, showed a higher and faster peak of
brain uptake and faster washout in normal mice. The improved
properties of
[
11C]phenylbenzofuran derivatives could make
them a better candidate for the imaging of Ab plaques. Similar
to [125I]phenylbenzofuran, the 2-PI derivatives had unfavorable
in vivo pharmacokinetics in normal mice, despite their good affinity
for Ab aggregates. Additional structural changes, that is, reducing
4.1.3. 4-(5-Bromo-1H-indol-2-yl)-N-methylbenzenamine (3)
A mixture of 1 (313 mg, 2.4 mmol), 2 (396 mg, 1.2 mmol),
PdCl2(PPh3) (60 mg, 0.06 mmol), CuI (50 mg, 0.22 mmol), THF

4744
H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
(5 mL), and TBAF (1 M solution in THF, 5 mL) was stirred under re-
flux for 5 h. After removal of the THF, the residue was diluted with
water and extracted with ethyl acetate, and the organic phase was
dried over Na2SO4. The solvent was removed and the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 2:1)
to give 179 mg of 3 (49.5%). 1H NMR (300 MHz, CDCl3) d 2.90 (s,
3H), 6.57 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 8.7 Hz, 2H), 7.21 (s, 2H),
7.49 (d, J = 8.7 Hz, 2H), 7.69 (s, 1H) 8.25 (s, 1H).
1,4-dioxane/Et3N) was stirred under reflux for 11 h. The solvent
was removed, and the residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate = 9:1) to give 20 mg of 9 (12.4%). 1H
NMR (300 MHz, CDCl3) d 0.84–1.61 (m, 36H), 2.99 (s, 6H), 6.46 (s,
1H), 6.75 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.35 (d,
J = 8.1 Hz, 1H), 7.61 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H).
4.1.10. tert-Butyl 2-(4-(dimethylamino)phenyl)-5-iodo-1H-
indole-1-carboxylate (10)
4.1.4. N-Methyl-4-(5-(trimethylstannyl)-1H-indol-2-yl)
benzenamine (4)
The same reaction as described above to prepare 5 was used,
and 10 mg of 10 was obtained in a 67.6% yield from 9 (20 mg,
0.03 mmol). 1H NMR (300 MHz, CDCl3) d 1.37 (s, 9H), 3.00 (s,
3H), 6.39 (s, 1H), 6.74 (d, J = 8.7 Hz, 2H), 7.29 (s, 2H), 7.53 (dd,
J = 1.8, 1.8 Hz, 1H) 7.84 (d, J = 2.1 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H).
A mixture of 3 (179 mg, 0.59 mmol), Pd(PPh3)4 (88 mg,
0.077 mmol) and (Me3Sn)2 (198 mg, 0.6 mmol) in 1,4-dioxane
(5 mL) was stirred under reflux for 3.5 h. The solvent was removed
and the residue was purified by silica gel chromatography (hexane/
ethyl acetate = 3:1) to give 6 mg of 4 (2.1%). 1H NMR (300 MHz,
CDCl3) d 0.30 (s, 9H), 2.88 (s, 3H), 3.84 (s, 1H), 6.62 (s, 1H), 6.67
(d, J = 8.7 Hz, 2H), 7.22 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H),
7.50 (d, J = 9.0 Hz, 2H), 7.70 (s, 1H), 8.18 (s, 1H).
4.1.11. 4-(5-Iodo-1H-indol-2-yl)-N,N-dimethylbenzenamine
(11)
To a solution of 10 (71 mg, 0.15 mmol) in CH2Cl2 (2 mL) was
added TFA (300 lL) at room temperature. After the mixture was
stirred for 3 h, the solvent was removed. The residue was purified
by preparative TLC (hexane/ethyl acetate = 3:1) to give 13 mg of 11
(27.0%). 1H NMR (300 MHz, CDCl3) d 3.02 (s, 6H), 6.57 (s, 1H), 6.79
(d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.7 Hz, 1H), 7.36 (dd, J = 1.5, 1.5 Hz,
1H), 7.53 (d, J = 9.0 Hz, 1H), 7.89 (s, 1H), 8.28 (s, 1H). HRMS m/z
C16H15N2I found 362.0278/calcd 362.0280 (M+).
4.1.5. 4-(5-Iodo-1H-indol-2-yl)-N-methylbenzenamine (5)
To a solution of 4 (9 mg, 0.019 mmol) in ethyl acetate (1 mL)
was added a solution of iodine in ethyl acetate (1 mL, 0.25 M) at
room temperature. The mixture was stirred for 15 s, and NaHSO3
solution (1 mL) was added. The organic phase was separated and
dried over Na2SO4. The solvent was removed and the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 3:1)
to give 5 mg of 5 (77.4%). 1H NMR (300 MHz, CDCl3) d 2.89 (s, 3H),
6.56 (s, 1H), 6.67 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.1 Hz, 1H), 7.37 (dd,
J = 1.8, 1.8 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H) 7.90 (s, 1H), 8.23 (s, 1H).
HRMS m/z C15H13N2I found 348.0109/calcd 348.0123 (M+).
4.1.12. 5-Bromo-2-(4-methoxyphenyl)-1H-indole (12)
The same reaction as described above to prepare 3 was used,
and 54 mg of 12 was obtained in a 30.7% yield from 2 (198 mg,
0.6 mmol) and p-ethynylanisole (116
(300 MHz, CDCl3) 3.87 (s, 3H), 6.50 (s, 1H), 6.99 (d, J =
l
L, 0.9 mmol). 1H NMR
d
8.7 Hz, 2H), 7.25 (s, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.12 (s, 1H), 8.30
4.1.6. 4-(5-Bromo-1H-indol-2-yl)-N,N-dimethylbenzenamine (6)
The same reaction as described above to prepare 3 was used,
and 25 mg of 6 was obtained in a 27.2% yield from 2 (99 mg,
0.3 mmol) and 4-ethynyl-N,N-dimethylaniline (65 mg, 0.45 mmol).
1H NMR (300 MHz, CDCl3) d 3.02 (s, 6H), 6.59 (d, J = 1.8 Hz, 1H),
6.78 (d, J = 9.0 Hz, 2H), 7.21–7.22 (m, 2H), 7.53 (d, J = 9.0 Hz, 2H),
7.68 (s, 1H) 8.27 (s, 1H).
(s, 1H).
4.1.13. 2-(4-Methoxyphenyl)-5-(trimethylstannyl)-1H-indole (13)
The same reaction as described above to prepare 4 was used,
and 15 mg of 13 was obtained in a 23.9% yield from 12 (49 mg,
0.16 mmol). 1H NMR (300 MHz, CDCl3) d 0.30 (s, 9H), 3.86 (s,
3H), 6.68 (s, 1H), 6.98 (d, J = 9.0 Hz, 2H), 7.26 (d, J = 7.2 Hz, 1H),
7.39 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 9.0 Hz, 2H), 7.73 (s, 1H), 8.22
(s, 1H).
4.1.7. N,N-Dimethyl-4-(5-(trimethylstannyl)-1H-indol-2-yl)
benzenamine (7)
The same reaction as described above to prepare 4 was used,
and 2 mg of 7 was obtained in a 7.1% yield from 6 (22 mg,
0.07 mmol). 1H NMR (300 MHz, CDCl3) d 0.31 (s, 9H), 3.01 (s,
6H), 6.64 (s, 1H), 6.79 (d, J = 9.3 Hz, 2H), 7.23 (d, J = 6.9 Hz, 1H),
7.38 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.71 (s, 1H), 8.21
(s, 1H).
4.1.14. 5-Iodo-2-(4-methoxyphenyl)-1H-indole (14)
The same reaction as described above to prepare 5 was
used, and 9 mg of 14 was obtained in a 66.3% yield from 13
(15 mg, 0.039 mmol). 1H NMR (300 MHz, CDCl3) d 3.86 (s, 3H),
6.63 (s, 1H), 6.99 (d, J = 9.0 Hz, 2H), 7.16 (d, J = 8.4 Hz, 1H), 7.41
(dd, J = 1.8, 1.5 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.93 (s, 1H),
8.29 (s, 1H). HRMS m/z C15H12NOI found 348.9962/calcd
348.9964 (M+).
4.1.8. tert-Butyl 5-bromo-2-(4-(dimethylamino)phenyl)-1H-
indole-1-carboxylate (8)
(Boc)2O (159 mg, 0.73 mmol) was added to a solution of 7
(41 mg, 0.13 mmol) and 4-(N,N-dimethylamino)pyridine (DMAP)
(4.8 mg, 0.004 mmol) in acetonitrile. The reaction mixture was stir-
red at room temperature for 3 h. After it was poured into water, the
mixture was extracted with ethyl acetate. The organic layers were
dried over Na2SO4. The solvent was removed and the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 4:1)
to give 54 mg of 8 (100%). 1H NMR (300 MHz, CDCl3) d 1.38 (s, 9H),
3.00 (s, 6H), 6.41 (s, 1H), 6.75 (d, J = 8.4 Hz, 2H), 7.29 (s, 2H), 7.36
(d, J = 9.0 Hz, 1H), 7.63 (s, 1H) 8.02 (d, J = 8.7 Hz, 1H).
4.1.15. 4-(5-Iodo-1H-indol-2-yl)phenol (15)
BBr3 (0.7 mL, 1 M solution in CH2Cl2) was added to a solution of
14 (80 mg, 0.23 mmol) in CH2Cl2 (5 mL) dropwise in an ice bath.
The mixture was allowed to warm to room temperature and stirred
for 24 h. Water was added while the reaction mixture was cooled
in an ice bath. The mixture was extracted with CHCl3 and the water
layer was 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 (hexane/
ethyl acetate = 4:1) to give 15 mg of 15 (19.5%). 1H NMR
(300 MHz, CD3OD) d 6.57 (s, 1H), 6.76 (d, J = 8.7 Hz, 2H), 7.16 (d,
J = 8.7 Hz, 1H), 7.29 (dd, J = 1.5, 1.5 Hz, 1H), 7.60 (d, J = 8.7 Hz,
2H), 7.81 (s, 1H). HRMS m/z C14H10NOI found 334.9808/calcd
334.9807 (M+).
4.1.9. tert-Butyl 5-(tributylstannyl)-2-(4-(dimethylamino)
phenyl)-1H-indole-1-carboxylate (9)
A mixture of 8 (54 mg, 0.13 mmol), (Bu3Sn)2 (0.3 mL) and
(Ph3P)4Pd (16 mg, 0.01 mmol) in a mixed solvent (6 mL, 1:1 =

H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
4745
4.1.16. 2-(4-(5-Iodo-1H-indol-2-yl)phenoxy)ethanol (16)
A mixture of 15 (13 mg, 0.039 mmol), potassium carbonate
(48 mg, 0.12 mmol) and ethylene chlorohydrin (4 L, 0.06 mmol)
J = 1.2 Hz, 1H). HRMS m/z
362.0280 (M+).
C
16H15N2I found 362.0287/calcd
l
in anhydrous DMF (3 mL) was stirred under reflux for 10.5 h. After
cooling to room temperature, water was added, and the reaction
mixture was extracted with CHCl3. The organic layer was sepa-
rated, dried over Na2SO4 and evaporated. The resulting residue
was purified by preparative TLC (hexane/ethyl acetate = 1:1) to
give 3 mg of 16 (20.4%). 1H NMR (300 MHz, CD3OD) d 3.89 (t,
J = 9.6 Hz, 2H), 4.09 (t, J = 9.3 Hz, 2H), 6.62 (s, 1H), 7.02 (d,
J = 8.7 Hz, 2H), 7.17 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 1.8, 1.5 Hz,
1H), 7.70 (d, J = 9.0 Hz, 2H), 7.82 (s, 1H), 11.0 (s, 1H). HRMS m/z
C16H14NO2I found 379.0078/calcd 379.0069 (M+).
4.2. Iododestannylation reaction
The radioiodinated forms of compounds 5, 11, 14, and 16 were
prepared from the corresponding trimethyltin derivatives by
iododestannylation using the previously described N-chlorosuccin-
imide (NCS) method, with some modifications.26 Briefly, a 80
lL
solution of 5, 11, 14, and 16 in methanol containing 1% acetic
acid (0.56 mg/mL) was mixed with 20 L of NCS in methanol
l
(0.5 mg/mL) in a sealed vial, and [125I]NaI (0.1–0.2 mCi, specific
activity 2200 Ci/mmol) was added. The reaction was allowed to
proceed at room temperature for 20 s and terminated by addition
of NaHSO3. After extraction with ethyl acetate, the extract was
dried by passing through an anhydrous Na2SO4 column and blown
dry with a stream of nitrogen gas. The radioiodinated ligand was
purified by HPLC on a Cosmosil C18 column with an isocratic sol-
vent of H2O/acetonitrile (4:6–1:1) at a flow rate of 1.0 mL/min.
4.1.17. 4-(5-Bromo-1H-indol-2-yl)phenol (17)
The same reaction as described above to prepare 15 was used,
and 13 mg of 17 was obtained in a 13.0% yield from 13 (105 mg,
0.347 mmol). 1H NMR (300 MHz, CD3OD) d 6.58 (s, 1H), 6.84 (d,
J = 9.0 Hz, 2H), 7.11 (dd, J = 1.8, 1.5 Hz, 1H), 7.25 (d, J = 8.7 Hz,
1H), 7.60 (d, J = 9.0 Hz, 3H).
4.3. Binding assays using the aggregated Ab peptide in solution
4.1.18. 2-(4-(5-Bromo-1H-indol-2-yl)phenoxy)ethanol (18)
The same reaction as described above to prepare 16 was used,
and 1.8 mg of 18 was obtained in a 14.2% yield from 17 (11 mg,
0.038 mmol). 1H NMR (300 MHz, CD3OD) d 3.89 (t, J = 8.7 Hz, 2H),
4.09 (t, J = 9.6 Hz, 2H), 6.64 (s, 1H), 7.02 (d, J = 8.7 Hz, 2H), 7.13
(dd, J = 1.5, 1.5 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 7.61 (s, 1H), 7.70
(d, J = 8.7 Hz, 2H).
A solid form of Ab42 was purchased from Peptide Institute
(Osaka, Japan). Aggregation was carried out by gently dissolving
the peptide (0.25 mg/mL) in a buffer solution (pH 7.4) containing
10 mM sodium phosphate and 1 mM EDTA. The solution was
incubated at 37 °C for 42 h with gentle and constant shaking. Bind-
ing assays were carried out as described previously.27 125I]IMPY
[
(6-iodo-2-(40-dimethylamino)phenyl-imidazo[1,2]pyridine) with
2200 Ci/mmol specific activity and greater than 95% radiochemical
purity was prepared using the standard iododestannylation reac-
tion as described previously.15 Binding assays were carried out in
4.1.19. 2-(4-(5-(Trimethylstannyl)-1H-indol-2-yl)phenoxy)
ethanol) (19)
The same reaction as described above to prepare 4 was used,
and 9 mg of 19 was obtained in a 89.8% yield from 18 (8 mg,
0.02 mmol). 1H NMR (300 MHz, CDCl3) d 0.31 (s, 9H), 3.99 (t,
J = 8.7 Hz, 2H), 4.14 (t, J = 9.3 Hz, 2H), 6.71 (d, J = 11.1 Hz, 1H),
6.76 (d, J = 9.3 Hz, 1H), 6.98–7.08 (m, 2H), 7.39 (s, 1H), 7.57–7.62
(m, 2H), 7.88 (d, J = 9.3 Hz, 1H), 8.25 (s, 1H).
12 ꢀ 75 mm borosilicate glass tubes. A mixture containing 50
of test compound (0.2 pM–400 nM in 10% EtOH), 50
125I]IMPY (0.02 nM diluted in 50% EtOH), 50
L of Ab42 aggre-
gates, and 850 L of 10% ethanol was incubated at room tempera-
lL
lL of
[
l
l
ture for 3 h. The mixture was then filtered through Whatman GF/B
filters using a Brandel M-24 cell harvester, and the filters contain-
ing the bound 125I ligand were placed in a gamma counter (Aloka,
ARC-380). Values for the half-maximal inhibitory concentration
(IC50) were determined from displacement curves of three inde-
pendent experiments using GraphPad Prism 4.0, and those for
the inhibition constant (Ki) were calculated using the Cheng–
Prusoff equation.28
4.1.20. 4-(5-Bromo-1H-indol-1-yl)-N,N-dimethylbenzenamine
(20)
A mixture of 5-bromoindole (100 mg, 0.51 mmol), 4-(dimethyl-
amino)-phenylboronic acid (84 mg, 0.51 mmol), Cu(OAc)2 (200 mg,
1.00 mmol), triethylamine (0.18 mL), and powdered molecular
sieves 3 Å was suspended in CH2Cl2 (10 mL), and stirred for 1 h.
The solvent was removed, and the residue was purified by silica
gel chromatography (hexane/ethyl acetate = 9:1) to give 71 mg of
20 (44.2%). 1H NMR (300 MHz, CDCl3) d 3.02 (s, 6H), 6.55 (d,
J = 2.7 Hz, 1H), 6.82 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 1.8 Hz, 1H) 7.78
(d, J = 1.2 Hz, 1H).
4.4. Neuropathological staining of mouse brain sections
The experiments with animals were conducted in accordance
with our institutional guidelines and approved by the Nagasaki
University Animal Care Committee. Tg2576 transgenic mice (fe-
male, 28 months old) and wild-type mice (female, 22 months
old) were used as the Alzheimer’s model and control, respectively.
After the mice were sacrificed by decapitation, the brain was
immediately removed and frozen in powdered dry ice. The frozen
4.1.21. 4-(5-(Tributylstannyl)-1H-indol-1-yl)-N,N-dimethylben-
zenamine (21)
The same reaction as described above to prepare 9 was used,
and 21 mg of 21 was obtained in a 12.4% yield from 20 (102 mg,
0.32 mmol). 1H NMR (300 MHz, CDCl3) d 0.87–1.56 (m, 27H),
3.02 (s, 6H), 6.61 (d, J = 3.3 Hz, 1H), 6.82 (d, J = 9.0 Hz, 2H), 7.24
(d, J = 3.0 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 3.0 Hz, 1H),
7.77 (s, 1H).
blocks were sliced into serial sections, 10
incubated with a 50% EtOH solution (100
l
m thick. Each slide was
M) of compounds 11
l
and 14 for 10 min. The sections were washed in 50% EtOH for
1 min two times, and examined using a microscope (KEYENCE
BZ-8100) equipped with a DAPI-BP filter set (excitation, 360 nm;
diachromic mirror, 400 nm; longpass filter, 460 nm). Thereafter,
the sections were also stained with thioflavin S, a pathological
dye commonly used for staining Ab plaques in the brain, and
examined using a microscope (KEYENCE BZ-8100) equipped with
4.1.22. 4-(5-Iodo-1H-indol-1-yl)-N,N-dimethylbenzenamine
(22)
The same reaction as described above to prepare 5 was used,
and 8 mg of 22 was obtained in a 55.3% yield from 21 (21 mg,
0.04 mmol). 1H NMR (300 MHz, CDCl3) d 3.02 (s, 6H), 6.54 (d,
J = 3.3 Hz, 1H), 6.81 (d, J = 6.6 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H),
7.29 (d, J = 9.0 Hz, 2H), 7.41 (dd, J = 1.5, 1.8 Hz, 1H), 7.99 (d,
a
GFP-BP filter set (excitation, 470 nm; diachromic mirror,
495 nm; longpass filter, 535 nm).

4746
H. Watanabe et al. / Bioorg. Med. Chem. 18 (2010) 4740–4746
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Acknowledgments
The study was supported by a Grants-in Aid for Young Scientists
(A) and Exploratory Research from the Ministry of Education, Cul-
ture, Sports, Science and Technology, the Program for Promotion of
Fundamental Biomedical Innovation (NIBIO), and a Health Labor
Sciences Research Grant.
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