Synthesis and biological evaluation of 18F-labled 2-phenylindole derivatives as PET imaging probes for β-amyloid plaques

Article abstract of DOI:10.1016/j.bmc.2013.04.028

A novel series of fluorinated 2-phenylindole derivatives were synthesized and evaluated as β-amyloid imaging probes for PET. The in vitro inhibition assay demonstrated that their binding affinities for Aβ_1-42 aggregates ranged from 28.4 to 1097.8 nM. One ligand was labeled with ¹⁸F ([¹⁸F]1a) for its high affinity (K_i = 28.4 nM), which was also confirmed by in vitro autoradiography experiments on brain sections of transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old, male). In vivo biodistribution experiments in normal mice showed that this radiotracer displayed high initial uptake (5.82 ± 0.51% ID/g at 2 min) into and moderate washout (2.77 ± 0.31% ID/g at 60 min) from the brain. [ ¹⁸F]1a could be developed as a promising new PET imaging probe for Aβ plaques although necessary modifications are still needed.

Full text of DOI:10.1016/j.bmc.2013.04.028

Bioorganic & Medicinal Chemistry 21 (2013) 3708–3714
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 18F-labled 2-phenylindole
derivatives as PET imaging probes for b-amyloid plaques
b
b
a
a
Hualong Fu a, Lihai Yu a, Mengchao Cui a, , Jinming Zhang , Xiaojun Zhang , Zijing Li , Xuedan Wang ,
⇑
Jianhua Jia a, Yanping Yang a, Pingrong Yu a, Hongmei Jia a, Boli Liu a,
a Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
⇑
b Department of Nuclear Medicine, Chinese PLA General Hospital, Beijing 100853, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of fluorinated 2-phenylindole derivatives were synthesized and evaluated as b-amyloid
imaging probes for PET. The in vitro inhibition assay demonstrated that their binding affinities for
Ab1–42 aggregates ranged from 28.4 to 1097.8 nM. One ligand was labeled with 18F ([18F]1a) for its high
affinity (Ki = 28.4 nM), which was also confirmed by in vitro autoradiography experiments on brain
sections of transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old, male). In vivo biodistribution
experiments in normal mice showed that this radiotracer displayed high initial uptake (5.82 0.51%
ID/g at 2 min) into and moderate washout (2.77 0.31% ID/g at 60 min) from the brain. [18F]1a could
be developed as a promising new PET imaging probe for Ab plaques although necessary modifications
are still needed.
Received 14 March 2013
Revised 13 April 2013
Accepted 15 April 2013
Available online 24 April 2013
Keywords:
Alzheimer’s disease
b-Amyloid plaque
PET
Autoradiography
Phenylindole
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
backbones for dozens of radio-labeled Ab imaging agents. In virtue
of the high efficiency in crossing the blood–brain barrier (BBB) and
Alzheimer’s disease (AD) is a form of dementia which gradually
worsens with the lapse of time in middle or late life. AD is related
with many clinical symptoms, including cognitive decline, irre-
versible memory loss, disorientation, language impairment, etc.
Major neuropathological features of postmortem AD brain are
the presence of senile plaques and neurofibrillary tangles, which
contain primarily b-amyloid (Ab) aggregates and highly phosphor-
ylated tau proteins, respectively.1 Ab hypothesis, which states that
the Ab plaques play a critical role in the deterioration of AD, is con-
sidered the most important and pivotal theory for the pathogenesis
of AD.2,3 Currently, donepezil, rivastigmine, galantamine, tacrine,
and memantine are the five medications approved by the U.S. Food
and Drug Administration (FDA) and used to treat moderate to se-
vere AD patients. But these medications can only provide tempo-
rary relief from symptoms and act helplessly in halting the
progression or curing AD.4 In addition, the early diagnosis of AD
keeps a difficult problem currently and needs further research
urgently.
selective binding to Ab plaques, [11C]PIB5,6 ([11C]6-OH-BTA-1)
and [11C]AZD21847,8 (2-(6-([11C]methylamino)pyridin-3-yl)benzo
[d]thiazol-6-ol), two analogues of Th-T with a benzothiazole ring
system, have been widely used for clinical trials in AD patients or
under promising commercial development, respectively. IMPY (2-
(40-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine, Ki = 15 nM)
with an imidazopyridine ring system is often used as the compet-
ing radioligand in competition binding assays when labeled by
125I.9–11 A benzofuran derivative [18F]AZD4694 (2-(2-[18F]fluoro-
6-methylamino-pyridin-3-yl)-benzofuran-6-ol) was also evaluated
as an Ab imaging agent and displayed excellent properties.12
Besides, our group has done some meaningful research based on
the benzoxazole derivatives recently, and two 18F-labeled probes
with high binding affinities to Ab aggregates and excellent
in vivo brain pharmacokinetics were selected for further evalua-
tion.13 Benzothiophene, imidazopyridazine and benzoimidazole
derivatives were also explored as Ab imaging agents.14–16 The Ab
imaging probes mentioned above were all belonged to the benzo-
heterocyclic ring system, comprising a phenyl ring fused to a
five-membered heterocyclic ring (Fig. 1).17 In addition to the
Th-T scaffold, stilbene was selected as another promising scaffold
for the development of Ab imaging probes. One of these probes,
The last decade has seen a promising progress in the research
field of imaging probes for Ab plaques. Congo Red (CR) and Thiofla-
vin-T (Th-T), which are the most common used dyes staining for Ab
plaques in human brain postmortem, have provided molecular
[
18F]BAY94-917218,19 ((E)-4-(2-(4-(2-(2-(2-[18F]fluoroethoxy)ethoxy)
ethoxy)phenyl)vinyl]-N-methylaniline, Ki = 2.22 nM), was under a
Phase III clinical trial currently. The styrylpyridine derivative,
⇑
Corresponding authors. Tel./fax: +86 10 58808891.
E-mail addresses: cmc@bnu.edu.cn (M. Cui), liuboli@bnu.edu.cn (B. Liu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.028

H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
3709
11CH3
NH
N
N
N
S
N
R2
N
N
123I
R1
HO
HO
N
S
X
[
123I]IMPY
18F
R2
[
11C]PIB
Imidazopyridine derivatives
R1
N
11CH3
NH
N
N
N
Benzothiazole derivatives
R2
NH
S
O
O
R1
HO
O
[
11C]AZD2184
Benzofuran derivatives
[
18F]AZD4694
X = C: [18F]BAY-94-9172
X
O
N
N
O
N
R
R2
X = N: [18F]AV-45
O
18F
HN
O
R1
n
X
O
18F
Benzoxazole derivatives
n = 1, 2, 3; R = NHCH3, N(CH3)2
Stilbene derivatives
N
N
R2
R2
R2
R2
N
N
S
N
H
N
H
R1
R1
R1
R1
Imidazopyridazine derivatives
Benzothiophene derivatives
Benzoimidazole derivatives
Indole derivatives
Figure 1. Structures of Ab imaging agents based on the benzoheterocyclic and stilbene scaffolds.
binding affinity were not reported. In the present study, we re-
ported the synthesis and evaluation of these fluorinated 2-phe-
nyl-1H-indole derivatives as PET radioligands for Ab plaque
imaging.
B
A
I
R2
N
R2
F
N
N
R1
n
R1
: R1 = H
2a: R1 = H
NHCH3
1a
1b
2b
3b
4b
5b
R2 = CH3 n = 1
R2 = CH3 n = 2
: R1 = H
: R1 = H
: R1 = H
: R1 = H
R2 =
R2 =
R2 =
R2 =
R2 = H
2. Results and discussion
2.1. Chemistry
N(CH3)2
OCH3
3a: R1 = CH3 R2 = CH3 n = 1
: R1 = CH3 R2 = CH3 n = 2
5a: R1 = CH3 R2 = H
: R1 = CH3 R2 = H
4a
n = 1
n = 2
The synthetic routes of 18F-labeled derivatives were shown in
Scheme 1 and the other fluorinated 2-phenyl-1H-indolederivatives
except 2a and 6a (see Supplementary data Scheme S1) have been
reported previously.24 The skeleton of compound 1 was obtained
by reacting 1-(4-methylamino-phenyl)-ethanone with phen-
ylhydrazine in polyphosphoric acid (PPA) at 130 °C for 10 min fol-
lowing the classic Fischer indole synthesis method;25 PPA acts as
both catalyst and solvent in this reaction (yield, 49.0%). Compound
1a, used as a non-radioactive ligand of [18F]1a, was obtained by N-
alkylation of 1 with 1-bromo-2-fluoro-ethane using Na2CO3 as base
for about 24 h. Following the same reaction for 1a, compound 2a
was synthesized by reaction of 1 with 1-fluoro-3-iodo-propane
for 3 h. As iodide is a better living group compared with bromine,
2a was made with shorter reaction time and higher yield (62.0% vs
47.0%). In order to synthesize the precursor, the hydroxyl com-
pound 2 was prepared through N-alkylation reaction of 1 with 2-
bromo-ethanol using BuOK as base and KI as catalyst (yield,
59.0%). The hydroxyl group in 2 was subsequently protected by
6a
OCH2CH2OH
N
: R1
=
Figure 2. Chemical structures of fluorinated 2-phenyl-1H-indole derivatives (A)
and iodinated phenylindole derivatives (B).
[
18F]AV-4520 ((E)-4-(2-(6-(2-(2-(2-[18F]fluoroethoxy) ethoxy)eth-
oxy)pyridin-3-yl)vinyl)-N-methylaniline, Ki = 2.87 nM) was the
first Ab imaging probe approved by FDA. Although great successes
have been achieved in the development of Ab imaging agents,
there is still a need for novel molecular motif with better proper-
ties as Ab probes.
Based on these facts, a novel molecular motif phenylindole, also
a benzoheterocyclic ring system, was explored as Ab imaging
probes. The biological activity of indole ring is a key structural
component that can be found ubiquitously in organisms, suggest-
ing that the indole motif should have a proper biocompatibility.21
In 2010, Watanabe et al. reported a series of 125I labeled 2-phenyl-
1H-indole and 1-phenylindole derivatives (Fig. 2B) as Ab imaging
probes.22 2-Phenyl-indole derivatives displayed high affinities for
Ab aggregates in binding experiments in vitro with the Ki values
of 27, 4, 20, 26 nM for 1b, 2b, 3b, 4b (Fig. 2B), respectively. As a
1-phenylindole derivative, 5b displayed a disillusionary result with
the Ki value >10,000 nM, suggesting that the substituent location
on the indole ring is very important for the binding affinity. Several
reports have suggested the lipophilicity of probes may have a sig-
nificant influence on the pharmacokinetics in vivo.6,23 125I atoms
introduced in the above radiotracers may promote the lipophilic-
ity, which may subsequently result in the slow washout from the
brain (brain2 min/brain60 min ratio varies from 1.17 to 1.82 for 1b–
4b) and undesirable target to non-target ratio (brain/blood ratio
less than 1.0) (Table 3).
tert-butyldimethylsilyl chloride (TBDMSCl) to give
3 (yield,
60.0%), and the nitrogen atom in the indole ring (indole-N) was
protected by butyloxycarbonyl (BOC) group subsequently to give
4 (yield, 100.0%). After removal of the TBS-protecting group of 4
by tetrabutyl ammonium fluoride (TBAF), the free hydroxy group
of 5 was converted into tosylate by reacting with p-toluenesulfonyl
chloride (TsCl) in pyridine to give precursor 6 (yield, 59.0%) at
room temperature.
2.2. Radiolabeling
[
18F]1a was prepared from the corresponding tosylate precursor
6 (Scheme 1). After the nucleophilic replacement of the tosylate
leaving group by 18Fꢀ, the N-Boc-protecting group was removed
by treatment of aqueous HCl (1 M). After purification by high per-
formance liquid chromatography (HPLC), the radiochemical purity
of [18F]1a was greater than 98%, and the specific activity was esti-
Simultaneously, our group has reported a series of fluorinated
2-phenyl-1H-indole derivatives (Fig. 2A) as Ab imaging probes.24
However, 18F-labeling and further biological evaluations including
mated approximately 136 GBq/lmol. The overall synthesis time is

3710
H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
O
NHNH2
a
b
NH
N
+
N
H
N
H
N
H
n
F
c
1a: n = 1
2a: n = 2
1
e
d
N
N
N
N
H
N
H
N
Boc
OH
OTBS
OTBS
N
f
4
3
2
h
g
N
N
N
N
Boc
N
Boc
H
18F
OTs
OH
18
1a
F]
[
6
5
Scheme 1. Reagents and conditions: (a) PPA, 130 °C, 10 min; (b) 1-bromo-2-fluoro-ethane (for 1a) or 1-fluoro-3-iodo-propane (for 2a), Na2CO3, acetonitrile, 80 °C, 2 days (for
1a) or 3 h (for 2a); (c) 2-bromo–ethanol, BuOK, KI, acetonitrile, 80 °C, 3 days; (d) TBDMSCl, imidazole, CH2Cl2, 40 °C, 10 min; (e) (Boc)2O, DMAP, CH2Cl2, rt, 2.5 h; (f) TBAF, THF,
rt, 30 min; (g) TsCl, pyridine, rt, 3 h; (h) (1) Kryptofix-222, K2CO3, 18Fꢀ, acetonitrile, 120 °C, 5 min; (2) HCl (1 M), 100 °C, 5 min; (3) NaHCO3, rt.
Table 1
about 60 min, and the total radiochemical yield is about 25–30%
(no decay corrected, n = 5). The radiochemical identity of the 18F-
Inhibition constants (Ki) for binding of fluorinated 2-phenyl-1H-indole derivatives to
Ab1–42 aggregates
labeled ligand was verified by comparing the retention time with
nonradioactive compound after co-injection (see Supplementary
data Fig. S1).
a
Compd
Ki SEM (nM)
1a
2a
28.4 7.4
31.5 11.3
3a
4a
116.8 35.9
79.7 31.2
2.3. In vitro binding studies
5a
6a
IMPY
1030.1 375.2
1097.8 171.6
10.5 1.0
Experiments in vitro to evaluate the affinities of these fluori-
nated 2-phenyl-1H-indole derivatives for Ab1–42 aggregates were
carried out with competition binding assays using [125I]IMPY as
the competing radioligand. IMPY was also measured using the
same system for comparison. As shown in Table 1, 1a and 2a
showed high affinity to Ab1–42 aggregates with the Ki values of
28.4 and 31.5 nM, respectively. However, 3a (Ki = 116.8 nM), 4a
(Ki = 79.8 nM), 5a (Ki = 1030.1 nM) and 6a (Ki = 1097.8 nM) showed
moderate or poor affinities. The comparisons of 1a versus 2a, 3a
versus 4a, and 5a versus 6a showed the length of the fluorinated
alkyl chain has slight influence on the affinity. On the contrary,
the methylation of the indole-N gave negative effects on the affin-
ity of the probes, approving by the higher Ki values of 3a, 4a, 5a,
and 6a compared with those of 1a and 2a. This is in accordance
with the results of 5-iodinated-1-phenylindole derivative.22 Since
the tertiary N,N-dialkylamino group can bring about a positive pro-
motion to the affinity of Ab probes,26 5a and 6a, lack of this impor-
tant group together with the single methyl substitutions at the
indole-N, displayed poor affinity to Ab1–42 aggregates. On the basis
of the high binding affinity to Ab1–42 aggregates, 1a was selected
for 18F labeling and further evaluation.
a
The Ki values were determined in three independent experiments (n = 3).
kind of transgenic mouse model of AD (Fig. 3, D and F), which
was in accord with the results reported previously.27
2.5. In vivo biodistribution studies
To evaluate the uptake and washout kinetics of [18F]1a in brain,
a biodistribution experiment was performed in normal ICR mice
(5 weeks, male) (Table 2). Since a logD value in the range of
0.9–2.5 guarantees
a radiotracer free penetration across the
blood–brain barrier,23 a higher logD value (3.61) for [18F]1a was
important for the brain penetration and may result in high brain
uptake directly. [18F]1a displayed high initial brain uptake (5.82%
ID/g) at 2 min post-injection and the radioactivity in the brain dis-
played moderate washout rate (2.77% ID/g at 60 min) with a
brain2 min/brain60 min ratio of 2.10. In addition, a preferable signal
to noise ratio was achieved with the brain2 min/blood2 min ratio of
1.72. Those values are better compared with those of radio-
iodinated 2-phenyl-1H-indole derivatives reported previously
(Table 3).22 However, in vivo defluorination likely occurs with
2.4. In vitro autoradiography studies
To confirm the specific and high binding affinity of [18F]1a to Ab
plaques, in vitro autoradiography experiments were performed on
the brain sections of Tg mouse (C57BL6, APPswe/PSEN1, 11 months
old, male) and age-matched wild-type mouse (C57BL6, 11 months
old, male). As shown in Figure 3, the distinctive labeling of Ab pla-
ques in the hippocampus and cortical regions of the Tg mouse
brain section was observed (Fig. 3, A and C), while wild-type mouse
brain displayed no such labeling (Fig. 3, G). The positions (or pat-
terns) of the labeled Ab plaques with [18F]1a were also visible by
co-staining the same sections with thioflavin-S (Fig. 3, B, E and
F), confirming that there is a good correlation between the two dif-
ferent techniques in identifying Ab plaques. In addition, some Ab
plaques in the cerebellum region can be clearly observed in this
[
18F]1a, because substantial bone uptake of radioactivity was in-
creased with time (2.05% ID/g at 2 min and 9.71% ID/g at 1 h) after
the administration of [18F]1a to normal mice. This observation was
in accord with the 18F-labeled IMPY derivatives, which may due to
the in vivo N-dealkylation of the N-2-[18F]fluoroethyl group.28 Fur-
thermore, [18F]1a distributed to several other organs simulta-
neously. The radiotracer distributed to liver and kidneys with a
high initial uptake (10.47 1.35 and 9.66 1.30% ID/g at 2 min,
respectively) and then was washed out with a moderate rate
(4.15 0.27 and 3.54 0.33% ID/g at 60 min, respectively). But
the radioactivity was observed to accumulate within the intestines
over time (the uptake ranged from 6.50 1.04% ID/g at 2 min to
11.04 1.54% ID/g at 60 min).

H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
3711
Figure 3. Autoradiography of [18F]1a ex vivo using Tg model mouse (C57BL6, APPswe/PSEN1, 11 months old, male) (A, C, D) and wild-type controls (C57BL6, 11 months old)
(G). Plaques were also confirmed by the staining of the same sections with thioflavin-S (B, E, F and H).
Table 2
3. Conclusions
a
Biodistribution in normal ICR mice after intravenous injection of the [18F]1a
Organ
Time after injection (min)
In conclusion, on the basis of our previous work, we evaluated a
series of fluorinated 2-phenyl-1H-indole derivatives (1a, 2a, 3a, 4a,
5a, 6a) as Ab imaging probe compounds. 1a and 2a displayed high
affinity to Ab aggregates in binding experiments in vitro. In addi-
tion, [18F]1a can clearly and selectively bind to Ab plaques on Tg
mouse brain sections, which confirmed its affinity to Ab aggregates
in vitro. The in vivo biodistribution experiments in normal mice
indicated that [18F]1a displayed excellent uptake into and moder-
ate washout from the brain. However, [18F]1a was unstable for
in vivo defluorination, thus additional structure modifications of
these fluorinated 2-phenyl-1H-indole derivatives were needed,
and may bring potential Ab imaging probes for PET.
2
10
30
60
[
18F]1a (logD = 3.61)
Blood
Brain
Heart
Liver
Spleen
Lung
3.38 0.20
5.82 0.51
6.76 0.62
10.47 1.35
2.73 0.65
6.57 1.33
9.66 1.30
2.05 0.39
1.38 0.16
6.50 1.04
3.81 0.44
5.07 0.18
4.95 0.24
8.38 0.79
3.97 0.56
5.81 1.07
5.62 0.34
4.13 1.27
1.54 0.11
9.06 0.54
4.21 0.09
3.41 0.10
3.71 0.06
4.86 0.37
3.23 0.12
4.27 0.19
4.17 0.30
5.95 0.95
1.43 0.25
9.00 1.52
4.37 0.24
2.77 0.31
3.81 0.59
4.15 0.27
2.71 0.46
3.57 0.50
3.54 0.33
9.71 1.00
1.09 0.17
11.04 1.54
Kidney
Bone
Stomachb
Intestineb
a
Expressed as% injected dose per gram. Average for five mice standard
deviation.
b
4. Experimental section
4.1. General remarks
Expressed as% injected dose per organ.
All reagents used in the synthesis were commercial products
and were used without further purification unless otherwise indi-
cated. The 1H NMR spectra were obtained at 400 MHz on Bruker
spectrometer in CDCl3 solutions at room temperature with TMS
as an internal standard. Chemical shifts were reported as d values
relative to the internal TMS. Coupling constants were reported in
Hertz. Multiplicity is defined by s (singlet), d (doublet), t (triplet),
and m (multiplet). Mass spectra were acquired under Surveyor
MSQ Plus (ESI) instrument. Reactions were monitored by TLC (Sil-
ica gel 60 F254 aluminum sheets, Merck) and compounds were
visualized by illumination with a short wavelength UV lamp
Table 3
Comparison of brain kinetics between [18F]1a and radio-iodonated tracersa
Compd
Brain uptake at
2 minb
Brain2 min/brain60 min
ratio
Brain2 min/blood2 min
ratio
[
[
[
[
[
125I]1b 1.10 0.27
1.33
1.68
1.82
1.17
2.10
0.29
0.19
0.59
0.52
1.72
125I]2b 1.19 0.34
125I]3b 2.11 0.69
125I]4b 2.13 0.54
18F]1a
5.82 0.51
a
The data for [125I]1b–4b are original from Ref. 22.
Expressed as% injected dose per gram.
b

3712
H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
(k = 254 nm or 365 nm). Column chromatography purification
were performed on silica gel (54–74 m) from Qingdao Haiyang
Chemical Co., Ltd. Radiochemical purity was determined by HPLC
performed on a Shimadzu system SCL-20 AVP equipped with a
SPD-20A UV detector (k = 254 nm) and Bioscan Flow Count 3200
4.1.4. 2-[4-(1H-Indol-2-yl)-phenyl]-methyl-amino-ethanol (2)
To the solution of compound 1 (191 mg, 0.9 mmol) and 2-bro-
mo-ethanol (513 mg, 4.1 mmol) in acetonitrile (15 mL) was added
BuOK (223 mg, 2.0 mmol) and KI (catalytic amount). The reaction
mixture was stirred at 80 °C for 3 days. Water was added and the
mixture was extract with ethyl acetate (3 ꢁ 50 mL). In the follow-
ing, the organic phase was washed with water (100 mL) and satu-
rated brine (100 mL), and then dried over anhydrous magnesium
sulfate. Remove the solvent in vacuum and the residue was puri-
fied by flash column chromatography to give compound 2 as a yel-
low solid (55 mg, 59.0%). 1H NMR (400 MHz, CDCl3) d: 3.03 (s, 3H),
3.53 (t, J = 5.6 Hz, 2H), 3.85 (t, J = 5.6 Hz, 2H), 6.67 (d, J = 1.2 Hz,
1H), 6.87 (d, J = 8.6 Hz, 2H), 7.09 (dd, J = 7.1 Hz, 6.9 Hz, 1H), 7.14
(dd, J = 7.7 Hz, 6.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.54 (d,
J = 8.8 Hz, 2H), 7.58 (d, J = 7.7 Hz, 1H), 8.25 (s, 1H). MS (ESI): m/z
calcd for C17H18N2O 266.1, found 267.2 (M+H)+.
l
NaI/PMT
achieved on a Venusil MP C18 reverse phase column (Agela Tech-
nologies, 5
c-radiation scintillation detector. HPLC separations were
l
m, 10 mm ꢁ 250 mm) eluted with a binary gradient
system at a flow rate of 4.0 mL/min and HPLC analysis were
achieved on a Venusil MP C18 reverse phase column (Agela Tech-
nologies, 5
l
m, 4.6 mm ꢁ 250 mm) eluted with a binary gradient
system at a flow rate of 1.0 mL/min. Mobile phase A was water
while mobile phase B was acetonitrile. The purity of the synthe-
sized key compounds was determined using analytical HPLC and
was found to be more than 95%. Fluorescent observation was per-
formed on the Observer Z1 (Zeiss, Germany) equipped with GFP fil-
ter set (excitation, 505 nm). Normal ICR mice (five weeks, male)
were used for biodistribution experiments. Transgenic mice
(C57BL6, APPswe/PSEN1, 11 months old, male), used as an Alzhei-
mer’s model, were purchased from the Institute of Laboratory Ani-
mal Science, Chinese Academy of Medical Sciences. All protocols
requiring the use of mice were approved by the animal care com-
mittee of Beijing Normal University.
4.1.5. 2-[(tert-Butyl-dimethyl-silanyloxy)-ethyl]-[4-(1H-indol-2-
yl)-phenyl]-methyl-amine (3)
To the solution of compound 2 (45 mg, 0.2 mmol) and TBDMSCl
(92 mg, 0.6 mmol) in CH2Cl2 (10 mL) was added imidazole (35 mg,
0.5 mmol). The mixture was stirred for 10 min at 40 °C. Solvent
was removed in vacuum and the residue was purified by flash col-
umn chromatography to give compound 3 as a white solid (39 mg,
60.0%). 1H NMR (400 MHz, CDCl3) d: 0.03 (s, 6H), 0.85 (s, 9H), 3.02
(s, 3H), 3.49 (t, J = 6.0 Hz, 2H), 3.78 (t, J = 5.9 Hz, 2H), 6.63 (s, 1H),
6.76 (s, 2H), 7.05 (dd, J = 7.0 Hz, 6.8 Hz, 1H), 7.10 (dd, J = 6.9 Hz,
7.1 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.55
(d, J = 7.6 Hz, 1H), 8.21 (s, 1H). MS (ESI): m/z calcd for C23H32N2OSi
380.2, found 380.5 (M+).
4.1.1. 4-[(1H-Indol-2-yl)-phenyl]-methyl-amine (1)
1-(4-Methylamino-phenyl)-ethanone (937 mg, 6.3 mmol) and
phenyl-hydrazine (750 mg, 7.0 mmol) were mixed in jelled poly-
phosphoric acid (PPA, 15.0 g). The reaction mixture was kept at
130 °C for 10 min. Ice water (50 mL) was added and the mixture
was neutralized by ammonia water. The gray precipitate was col-
lected by filtration, washed with hot water and then dried at
80 °C. The residue was purified by flash column chromatography
to give compound 1 as a white solid (684 mg, 49.0%). 1H NMR
(400 MHz, CDCl3) d: 2.88 (s, 3H), 6.64 (s, 1H), 6.66 (d, J = 8.8 Hz,
2H), 7.07–7.15 (m, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.6 Hz,
2H), 7.58 (d, J = 7.7 Hz, 1H), 8.19 (s, 1H). MS (ESI): m/z calcd for
4.1.6. 2-(4-{[2-(tert-Butyl-dimethyl-silanyloxy)-ethyl]-methyl-
amino}-phenyl)-indole-1-carboxylic acid tert-butyl ester (4)
To the solution of compound 3 (38 mg, 0.1 mmol) and (Boc)2O
(170 mg, 0.8 mmol) in CH2Cl2 (10 mL) was added DMAP (catalytic
amount). The reaction mixture was stirred at room temperature for
2.5 h. Solvent was removed in vacuum and the residue was puri-
fied by flash column chromatography to give compound 4 as a yel-
lowish oily liquid (56 mg, 100.0%). 1H NMR (400 MHz, CDCl3) d:
0.04 (s, 6H), 0.89 (s, 9H), 1.39 (s, 9H), 3.05 (s, 3H), 3.51 (t,
J = 6.0 Hz, 2H), 3.81 (s, 2H), 6.48 (s, 1H), 6.76 (s, 2H), 7.20–7.28
(m, 4H), 7.52 (d, J = 7.3 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H). MS (ESI):
m/z calcd for C28H40N2O3Si 480.3, found 481.4 (M+H)+.
C
15H14N2 222.1, found 223.1 (M+H)+.
4.1.2. 4-(1H-Indol-2-yl)-N-(2-fluoroethyl)-N-
methylbenzenamine (1a)
A solution of compound 1 (101 mg, 0.5 mmol), 1-bromo-2-fluo-
roethane (437 mg, 2.6 mmol) and Na2CO3 (0.22 g, 2 mmol) in ace-
tonitrile (10 mL) was stirred at 80 °C for 2 days. Water was added
and the mixture was extract with ethyl acetate (3 ꢁ 50 mL). The
organic layer was washed with saturated brine (50 mL) and dried
over anhydrous magnesium sulfate. After the ethyl acetate was re-
moved in vacuum, the residue was purified by flash column chro-
matography to give compound 1a as a yellowish solid (58 mg,
47.0%). 1H NMR (400 MHz, CDCl3) d: 3.08 (s, 3H), 3.70 (dt,
J = 24.4 Hz, 4.9 Hz, 2H), 4.65 (dt, J = 47.1 Hz, 5.1 Hz, 2H), 6.67 (s,
1H), 6.82 (d, J = 8.2 Hz, 2H), 7.09 (dd, J = 7.2 Hz, 7.1 Hz, 1H), 7.14
(dd, J = 7.2 Hz, 7.0 Hz, 1H) 7.37 (d, J = 7.9 Hz, 1H), 7.56 (d,
J = 8.8 Hz, 2H), 7.58 (d, J = 8.0 Hz), 8.24 (s, 1H). MS (ESI): m/z calcd
for C17H17N2F 269.2; found 269.1 (M+).
4.1.7. 2-{4-[(2-Hydroxy-ethyl)-methyl-amino]-phenyl}-indole-
1-carboxylic acid tert-butyl ester (5)
To the solution of compound 4 (56 mg, 0.1 mmol) in THF
(10 mL) was added TBAF (2 mL, 1 M in THF, 2 mmol). The reaction
mixture was stirred for 30 min at room temperature, and then oily
liquid was given after the solvent was removed in vacuum. Wash
the oily liquid with water (50 mL) and extract the mixture with
CH2Cl2 (3 ꢁ 25 mL); in the following, the organic phase was
washed with water (3 ꢁ 50 mL) and dried over anhydrous magne-
sium sulfate. Solvent was removed in vacuum and the residue was
purified by flash column chromatography to give compound 5 as a
yellowish oily liquid (33 mg, 89.0%). 1H NMR (400 MHz, CDCl3) d:
1.40 (s, 9H), 3.03 (s, 3H), 3.53 (s, 2H), 3.85 (s, 2H), 6.49 (s, 1H),
6.85 (d, J = 8.2 Hz, 2H), 7.20–7.32 (m, 4H), 7.52 (d, J = 7.4 Hz, 1H),
8.14 (d, J = 8.2 Hz, 1H). MS (ESI): m/z calcd for C22H26N2O3 366.2,
found 367.2 (M+H)+.
4.1.3. (3-Fluoro-propyl)-[4-(1H-indol-2-yl)-phenyl]-amine (2a)
The same reaction as described above to prepare 1a was used,
and a yellowish solid 2a was obtained (79 mg, 62.0%). 1H NMR
(300 MHz, CDCl3) d: 1.97 (m, 1H), 2.04 (m, 1H), 3.02 (s, 3H), 3.55
(t, J = 5.4 Hz, 2H), 4.48 (t, J = 4.2 Hz, 1H), 4.60 (t, J = 4.2 Hz, 1H),
6.67 (s, 1H), 6.78 (d, J = 6.6 Hz, 2H), 7.10 (t, J = 5.4 Hz, 1H), 7.15
(t, J = 5.4 Hz, 1H), 7.37 (d, J = 6.3 Hz, 1H), 7.54 (d, J = 6.9 Hz, 2H),
7.59 (d, J = 5.7 Hz, 1H), 8.20 (b, 2H). MS (ESI): m/z calcd for
4.1.8. 2-(4-{Methyl-[2-(toluene-4-sulfonyloxy)-ethyl]-amino}-
phenyl)-indole-1-carboxylic acid tert-butyl ester (6)
To the solution of compound 5 (33 mg, 0.09 mmol) in anhy-
drous pyridine (1 mL) was added TsCl (43 mg, 0.2 mmol) after it
cooled to 0 °C in ice-water. The reaction mixture was stirred for
C
19H19N2F 282.2; found 283.0 (M+).

H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
3713
3 h with the temperature rising slowly from 0 °C to room temper-
4.4. Determination of the partition coefficient
The determination of partition coefficients of radiofluorinated
ature. Water was added and the water phase was extract with
ethyl acetate (3 ꢁ 50 mL). In the following, wash the combined or-
ganic phase with water (100 mL) and dry over anhydrous magne-
sium sulfate, then remove the solvent in vacuum. The residue
was purified by flash column chromatography to give compound
6 as a yellowish oily liquid (27 mg, 59.0%). 1H NMR (400 MHz,
CDCl3) d: 1.39 (s, 9H), 2.41 (s, 3H), 2.94 (s, 3H), 3.65 (t, J = 6.0 Hz,
2H), 4.19 (t, J = 6.0 Hz, 2H), 6.47 (s, 1H), 6.61 (d, J = 8.8 Hz, 2H),
7.20–7.31 (m, 6H), 7.52 (d, J = 7.3 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H),
8.14 (d, J = 8.2 Hz, 1H). MS (ESI): m/z calcd for C29H32N2O5S
520.2, found 521.2 (M+H)+.
tracers was performed using the method previously reported.30
A
solution of 18F-labeled tracer (1110 kBq) was added to a premixed
suspension containing 3.0 g n-octanol and 3.0 g PBS (0.05 M,
pH = 7.4) in a test tube, vortexed for 3 min at room temperature,
and then centrifuged for 5 min at 3000 rpm. Two aliquots from
the n-octanol (50 lL) and water (500 lL) layers were measured.
The partition coefficient was expressed as the logarithm of the ra-
tio of the count per gram from n-octanol versus PBS. Samples from
the n-octanol layer were repartitioned until the partition coeffi-
cient values tend to stabilization. The measurement was conducted
in triplicate and repeated five times.
4.2. Radiolabeling
[
18F]Fluoride on the QMA cartridge was eluted into a 10 mL
4.5. Biodistribution studies
reaction vial with 1 mL of Kryptofix222/K2CO3 solution (13 mg of
Kryptofix222 and 1.1 mg of K2CO3 in CH3CN/H2O, 4/1, v/v). The sol-
vent was removed by continuously blowing of nitrogen at 120 °C.
Subsequently, the residue was azeotropically dried with 1 mL of
anhydrous acetonitrile three times at 120 °C under a stream of
nitrogen gas. Tosylate precursor 6 (4–5 mg) was added to the reac-
tion vessel after being dissolved in 1 mL anhydrous acetonitrile.
The reaction mixture was heated to 100 °C and kept for 5 min. A
The biodistribution experiments were performed in normal ICR
mice (5 weeks, male) and approved by the Animal Care Committee
of Beijing Normal University. A saline solution containing the
[
18F]1a (740–1110 kBq/mL) with 10% EtOH was intravenously in-
jected into the tail, and the mice (n = 5 for each time point) were
sacrificed at 2, 10, 30 and 60 min post injection. The organs of
interest were removed, weighed and counted with an automatic
gamma counter. Data are expressed as the percent injected dose
per gram of tissues (% ID/g).
solution of HCl (200 lL, 1 M) was added to the mixture after it
cooled down to room temperature, and then keep the reaction vial
at 100 °C for additional 5 min. Subsequently, the reaction mixture
was neutralized with anhydrous sodium hydrogen carbonate and
then 10 mL of water was added. The mixture flowed past a precon-
ditioned Sep-Pak C18 (Waters) cartridge and wash the cartridge
with 20 mL of water afterwards to remove the inorganic salt. The
labeled compound [18F]1a was eluted with 1.5 mL of acetonitrile
and then the solvent was removed with a stream of nitrogen.
The residue was redissolved in acetonitrile and then purified with
4.6. In vitro autoradiography using brain sections from Tg
mouse
Paraffin-embedded Tg mouse and a wild type mouse brain sec-
tions were treated following the previous method reported.13 The
brain sections were incubated with [18F]1a (185–370 kBq) for 1 h
at room temperature. Finally, the sections were washed with 40%
EtOH. After drying, the 18F-labeled sections were exposed to an
imaging plate (PerkinElmer, USA) for 1 h. Autoradiographic images
were obtained using a scanner system (Cyclone, Packard). The
presence and location of plaques were confirmed with fluorescent
staining using thioflavin-S (0.125%) on the same brain sections.
HPLC (Agela Technologies,
water = 7/3; flow rate = 4.0 mL/min). The retention time of
18F]1a was 9.57 min in this HPLC system. The specific activity
5
lm, 10 mm ꢁ 250 mm, CH3CN/
[
was estimated by comparing the UV peak intensity of purified
18F-labeled compounds with reference nonradioactive compounds.
4.3. Binding assays in vitro using Ab1–42 aggregates
Acknowledgments
The affinity of these fluorinated 2-phenylindoles derivatives
were first evaluated by in vitro competitive binding assays with
The authors thank Dr. Jin Liu (College of Life Science, Beijing
Normal University) for assistance in the in vitro neuropathological
staining. This work was funded by National Natural Science Foun-
dation of China (No. 21201019) and Doctoral Fund of Ministry of
Education of China (No. 20120003120013) and Fundamental Re-
search Funds for the Central Universities (No. 2012LYB19).
[
[
125I]IMPY for Ab1-42 aggregates in solution. The radio-ligand
125I]IMPY was prepared according to procedures described previ-
ously.11 After HPLC purification, the radiochemical purity of
[
125I]IMPY was greater than 95%. A solid form of Ab42 was pur-
chased from Osaka Peptide Institute and the Ab1–42 aggregates
were prepared following the procedures described previously.22
The inhibition experiments were carried out in 12 ꢁ 75 mm boro-
Supplementary data
silicate glass tubes with the reaction mixture containing 100
l
L of
125I]IMPY
L of test compound (10ꢀ4 to 10ꢀ8.5
L of 0.05% bovine serum albumin solution
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.028.
aggregated Ab1-42 fibrils, 100
(100,000 cpm/100 L), 100
in ethanol) and 700
lL of radioligand [
l
l
M
l
References and notes
in a total volume of 1 mL. The mixture was incubated at room tem-
perature for 3 h before it was filtered through Whatman GF/B fil-
ters using a Brandel Mp-48T cell harvester. Filters containing the
bound [125I]IMPY were measured by gamma counter (WALLAC/
Wizard 1470, USA) with 70% counting efficiency. The half maximal
inhibitory concentration (IC50) was determined from displacement
curves of three independent experiments using GraphPad Prism
4.0 and the inhibition constant (Ki) was calculated using the
Cheng–Prusoff equation:29 Ki = IC50/(1 + [L]/Kd), where [L] is the
concentration of [125I]IMPY used in the assay and Kd is the dissoci-
ation constant of IMPY.9
1. Jucker, M.; Walker, L. C. Ann. Neurol. 2011, 70, 532.
2. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
3. Hardy, J. A.; Higgins, G. A. Science 1992, 256, 184.
4. Kung, H. F. ACS Med. Chem. Lett. 2012, 3, 265.
5. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D. P.;
Bergström, M.; Savitcheva, I.; Huang, G.-F.; Estrada, S.; Ausén, B.; Debnath, M.
L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni,
G.; Mathis, C. A.; Långström, B. Ann. Neurol. 2004, 55, 306.
6. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G.-F.; Debnath, M. L.; Klunk, W. E. J.
Med. Chem. 2003, 46, 2740.
7. Johnson, A. E.; Jeppsson, F.; Sandell, J.; Wensbo, D.; Neelissen, J. A.; Jureus, A.;
Strom, P.; Norman, H.; Farde, L.; Svensson, S. P. J. Neurochem. 2009, 108, 1177.

3714
H. Fu et al. / Bioorg. Med. Chem. 21 (2013) 3708–3714
8. Nyberg, S.; Jonhagen, M. E.; Cselenyi, Z.; Halldin, C.; Julin, P.; Olsson, H.;
Freund-Levi, Y.; Andersson, J.; Varnas, K.; Svensson, S.; Farde, L. Eur. J. Nucl. Med.
Mol. Imaging 1859, 2009, 36.
9. Kung, M.-P.; Hou, C.; Zhuang, Z.-P.; Zhang, B.; Skovronsky, D.; Trojanowski, J.
Q.; Lee, V. M. Y.; Kung, H. F. Brain Res. 2002, 956, 202.
10. Newberg, A. B.; Wintering, N. A.; Plössl, K.; Hochold, J.; Stabin, M. G.; Watson, M.;
Skovronsky, D.; Clark, C. M.; Kung, M.-P.; Kung, H. F. J. Nucl. Med. 2006, 47, 748.
11. Zhuang, Z.-P.; Kung, M.-P.; Wilson, A.; Lee, C.-W.; Plössl, K.; Hou, C.; Holtzman,
D. M.; Kung, H. F. J. Med. Chem. 2002, 46, 237.
12. Schou, M.; Peter, J.; Varnas, K.; Anders, J.; Sandell, J.; Cselenyi, Z.; Swahn, B. M.;
Svensson, S.; Lindstrom-Boo, E.; Farde, L.; Halldin, C. J. Labelled Compd.
Radiopharm. 2011, 54, S41.
Krause, S.; Friebe, M.; Lehman, L.; Lindemann, S.; Dinkelborg, L. M.; Masters, C.
L.; Villemagne, V. L. Lancet Neurol. 2008, 7, 129.
19. Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung, H. F. J. Med. Chem.
2005, 48, 5980.
20. Zhang, W.; Kung, M. P.; Oya, S.; Hou, C.; Kung, H. F. Nucl. Med. Biol. 2007, 34, 89.
21. Wei, Y.; Deb, I.; Yoshikai, N. J. Am. Chem. Soc. 2012, 134, 9098.
22. Watanabe, H.; Ono, M.; Haratake, M.; Kobashi, N.; Saji, H.; Nakayama, M.
Bioorg. Med. Chem. 2010, 18, 4740.
23. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E. J. Nucl. Med. 1983,
24, 1030.
24. Yu, L. H.; Scheunemann, M.; Deuther-Conrad, W.; Hiller, A.; Fischer, S.; Sorger,
D.; Sabri, O.; Jia, H. M.; Steinbach, J.; Brust, P.; Liu, B. L. Bull. Korean Chem. Soc.
2010, 31, 177.
13. Cui, M.; Wang, X.; Yu, P.; Zhang, J.; Li, Z.; Zhang, X.; Yang, Y.; Ono, M.; Jia, H.;
Saji, H.; Liu, B. J. Med. Chem. 2012, 55, 9283.
25. Robinson, B. Chem. Rev. 1963, 63, 373.
14. Chang, Y. S.; Jeong, J. M.; Lee, Y.-S.; Kim, H. W.; Ganesha, R. B.; Kim, Y. J.; Lee, D.
S.; Chung, J.-K.; Lee, M. C. Nucl. Med. Biol. 2006, 33, 811.
26. Ono, M.; Cheng, Y.; Kimura, H.; Cui, M.; Kagawa, S.; Nishii, R.; Saji, H. J. Med.
Chem. 2011, 54, 2971.
15. Cui, M.; Ono, M.; Kimura, H.; Kawashima, H.; Liu, B. L.; Saji, H. Nucl. Med. Biol.
2011, 38, 313.
27. Kung, M. P.; Hou, C.; Zhuang, Z. P.; Cross, A. J.; Maier, D. L.; Kung, H. F. Eur. J.
Nucl. Med. Mol. Imaging 2004, 31, 1136.
16. Zeng, F.; Alagille, D.; Tamagnan, G. D.; Ciliax, B. J.; Levey, A. I.; Goodman, M. M.
ACS Med. Chem. Lett. 2010, 1, 80.
17. Cui, M.; Ono, M.; Kimura, H.; Liu, B.; Saji, H. Bioorg. Med. Chem. Lett. 2011, 21,
4193.
28. Cai, L.; Chin, F. T.; Pike, V. W.; Toyama, H.; Liow, J.-S.; Zoghbi, S. S.; Modell, K.;
Briard, E.; Shetty, H. U.; Sinclair, K.; Donohue, S.; Tipre, D.; Kung, M.-P.;
Dagostin, C.; Widdowson, D. A.; Green, M.; Gao, W.; Herman, M. M.; Ichise, M.;
Innis, R. B. J. Med. Chem. 2004, 47, 2208.
18. Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan, R.; Pike, K. L.; O’Keefe, G.;
Tochon-Danguy, H.; Chan, G.; Berlangieri, S. U.; Jones, G.; Dickinson-Rowe, K.
L.; Kung, H. P.; Zhang, W.; Kung, M. P.; Skovronsky, D.; Dyrks, T.; Hall, G.;
29. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
30. Cui, M.; Ono, M.; Kimura, H.; Ueda, M.; Nakamoto, Y.; Togashi, K.; Okamoto, Y.;
Ihara, M.; Takahashi, R.; Liu, B.; Saji, H. J. Med. Chem. 2012, 55, 9136.