18F-Labeled 2-phenylquinoxaline derivatives as potential positron emission tomography probes for in vivo imaging of β-amyloid plaques

Article abstract of DOI:10.1016/j.ejmech.2012.08.031

In continuation of our study on the 2-phenylquinoxaline scaffold as potential β-amyloid imaging probes, two [¹⁸F]fluoro-pegylated 2-phenylquinoxaline derivatives, 2-(4-(2-[¹⁸F]fluoroethoxy)phenyl)-N- methylquinoxalin-6-amine ([¹⁸F]4a) and 2-(4-(2-(2-(2-[ ¹⁸F]fluoroethoxy)ethoxy)ethoxy)phenyl)-N-methylquinoxalin-6-amine ([¹⁸F]4b) were prepared. Both of them displayed high binding affinity to Aβ_1-42 aggregates (K_i = 10.0 ± 1.4 nM for 4a, K_i = 5.3 ± 3.2 nM for 4b). The specific and high binding of [¹⁸F]4a and [¹⁸F]4b to Aβ plaques was confirmed by in vitro autoradiography on brain sections of AD human and transgenic mice. In biodistribution in normal mice, [¹⁸F]4a displayed high initial brain uptake (8.17% ID/g at 2 min) and rapid washout from the brain. These preliminary results suggest [¹⁸F]4a may be a potential PET imaging agent for Aβ plaques in the living human brain.

Full text of DOI:10.1016/j.ejmech.2012.08.031

European Journal of Medicinal Chemistry 57 (2012) 51e58
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
18F-Labeled 2-phenylquinoxaline derivatives as potential positron emission
tomography probes for in vivo imaging of -amyloid plaques
b
,
a
b
a
a
a
Pingrong Yu a, Mengchao Cui a , Xuedan Wang , Xiaojun Zhang , Zijing Li , Yanping Yang , Jianhua Jia ,
*
Jinming Zhang b, Masahiro Ono c, Hideo Saji c, 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
c Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 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:
In continuation of our study on the 2-phenylquinoxaline scaffold as potential b-amyloid imaging probes,
two [18F]fluoro-pegylated 2-phenylquinoxaline derivatives, 2-(4-(2-[18F]fluoroethoxy)phenyl)-N-meth-
ylquinoxalin-6-amine ([18F]4a) and 2-(4-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)phenyl)-N-methyl-
quinoxalin-6-amine ([18F]4b) were prepared. Both of them displayed high binding affinity to Ab1-42
aggregates (Ki ¼ 10.0 ꢀ 1.4 nM for 4a, Ki ¼ 5.3 ꢀ 3.2 nM for 4b). The specific and high binding of [18F]4a
Received 16 June 2012
Received in revised form
14 July 2012
Accepted 23 August 2012
Available online 1 September 2012
and [18F]4b to A
b plaques was confirmed by in vitro autoradiography on brain sections of AD human and
transgenic mice. In biodistribution in normal mice, [18F]4a displayed high initial brain uptake (8.17% ID/g
Keywords:
Alzheimer’s disease
at 2 min) and rapid washout from the brain. These preliminary results suggest [18F]4a may be a potential
PET imaging agent for A
b
plaques in the living human brain.
Ó 2012 Elsevier Masson SAS. All rights reserved.
b
-Amyloid plaque
2-Phenylquinoxaline
Autoradiography
Biodistribution
1. Introduction
2-(4-([11C]methylamino)phenyl)-6-hydroxybenzothiazol ([11C]PIB,
Ki ¼ 0.87 ꢀ 0.18 nM) [9,10], a neutral analog of thioflavin-T. After
Alzheimer’s disease (AD) is the most common senile dementia.
AD patients suffer from growing dementia and disability including
cognitive decline, irreversible memory loss, disorientation, and
language impairment, which are because of the progressive neu-
rodegeneration in the brain. Till now, there is no effective thera-
peutic method for AD, however, the rapid growing of AD population
has brought a ruin to not only the AD patients but also their
families. AD is histopathologically characterized by b-amyloid (Ab)
plaques and neurofibrillary tangles, which presents in the gray
matter of AD patient even before the dementia [1e3]. In addition,
that, other 11C-labeled A imaging agents such as 4-N-[11C]meth-
b
ylamino-40-hydroxystilbene ([11C]SB-13, Ki ¼ 6.0 ꢀ 1.5 nM) [11,12]
and 2-[6-([11C]methylamino)pyridin-3-yl]-1,3-benzothiazol-6-ol
([11C]AZD2184, Ki ¼ 1.70 ꢀ 0.54 nM) [13e15] had also been re-
ported under clinical trials and displayed effective result in differ-
encing AD patients with controls by measuring the brain uptake
and retention. As a 11C-labeled tracer, the chemical structure is
retained, which completely maintained the biological property of
molecular. But the short half-life of 11C (t1/2 ¼ 20 min) leads to that
the supply of 11C-labeled tracers will be limited to centers equipped
with an on-site cyclotron, which may be the most important reason
for preventing 11C-labeled tracers to be more widely used. Mean-
while, 18F (t1/2 ¼ 110 min) has a longer radioactive decay half-life,
A
b
plaques had not been found in other kinds of dementia such as
frontotemporal dementia or pure vascular dementia [4]. At this
point of view, in vivo detection of A plaques in the brain by posi-
b
tron emission tomography (PET) should be useful for early diag-
nosis of AD [5e8].
which permits a more widespread application, and allowed
multiple injections from a single production batch. In this case, 18F
may be the better radionuclide for PET imaging. Great efforts have
In the past decades, a number of PET imaging probes for A
plaques have been reported, several of which have been
reported for clinical trials. The first A plaques tracer for PET is
b
been focused on the development of 18F-labeled A
b plaques tracers.
b
Some of them like 4-(N-methylamino)-40-(2-(2-(2-[18F]fluo-
roethoxy)ethoxy)ethoxy)-stilbene ([18F]BAY94-9172, florbetaben,
Ki
¼
2.22
ꢀ
0.54
nM) [16,17], 2-(3-[18F]fluoro-4-
methyaminophenyl)benzothiazol-6-ol ([18F]GE-067, flutemetamol,
Ki ¼ 0.74 ꢀ 0.38 nM) [18] had already been reported under clinical
* Corresponding authors. Tel./fax: þ86 10 58808891.
E-mail addresses: cmc@bnu.edu.cn (M. Cui), liuboli@bnu.edu.cn (B. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.031

52
P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
trials. In April 2012, (E)-4-(2-(6-(2-(2-(2-[18F]fluoroethoxy)ethoxy)
ethoxy)pyridin-3-yl)vinyl)-N-methylaniline ([18F]AV-45, florbeta-
pir, Ki ¼ 2.87 ꢀ 0.17 nM) [19e21], had been approved by the U.S.
Food and Drug Administration (FDA) as a radioactive diagnostic
agent indicated for brain imaging of A
being evaluated for AD and other causes of cognitive impairment.
However, clinical trials for 18F-labeled A
imaging tracers show
b plaques in patients who are
Fig. 2. Chemical structure of 2-phenylquinoxaline derivative [125I]QN-1.
b
that there is greater white matter retention compared with 11C-
labeled agents, and high non-specific white matter retention may
limit the sensitivity of PET imaging. The mechanism of white
matter retention seems to be owing to non-specific binding [22],
besides further modifications in order to decrease lipophilicity are
needed. In addition, almost all of the tracers evaluated in humans
are thioflavin-T or stilbene derivatives. Thus, development of
tracers with a new scaffold may lead a new way to improve the
ethanol in which Pd/C was added as catalyzer (yield 95.6%). Mon-
omethylation of derivative 2 was achieved with paraformaldehyde,
sodium methoxide and sodium borohydride to obtain derivative 3
(yield 88.7%). The corresponding fluoropegylated derivative 4a, 4b
were prepared by 3 with K2CO3, 1-bromo-2-fluoroethane or 2-(2-
(2-fluoroethoxy)ethoxy)ethyl 4-methylbenzenesulfonate in DMF
(yield 51.8%, 35.0%). Ethane-1,2-diyl bis(4-methylbenzenesu-
lfonate) or (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) bis(4-
methylbenzenesulfonate) was coupled with the hydroxy group of
3 with K2CO3 and 18-crown-6 in acetone to obtain 5a, 5b (yield
27.5%, 73.6%). The tosylate precursor 6a and 6b were obtained by
protecting the methylamino groups of 5a and 5b with di-tert-
butyldicarbonate in THF (yield 43.1%, 52.6%).
in vivo properties including higher affinity to A
nonspecific binding in the white matter of the brain.
In a search for novel A imaging probes, we have recently re-
ported a lipophilic 125I-labeled 2-phenylquinoxaline derivative
([125I]QN-1) [23], which displayed high affinity to A
aggregates
(Ki ¼ 4.1 ꢀ 0.7 nM). Film autoradiography and fluorescent staining
confirmed the specific binding to A plaques on postmortem AD
b plaques and less
b
b
b
brain sections. In biodistribution experiment, this probe exhibited
high initial uptake into the brain (6.03% ID/g at 2 min), but unsat-
isfactory rate of washing out (2.98% ID/g at 60 min). This may be
caused by its high lipophilicity (LogD ¼ 4.02) due to the existence of
iodine atom. To improve the pharmacokinetic profile of [125I]QN-1
for using as a PET imaging agent, the iodine atom was replaced by
a short fluorine end-capped polyethylene glycol chain (n ¼ 1 or 3)
[24]. Furthermore, aiming of circumventing the problem of the
rapid in vivo N-demethylation for dimethylamino group which
appeared in metabolism of tracers like 6-[123I]iodo-2-(40-dime-
thylamino-)phenyl-imidazo [1,2] pyridine ([123I]IMPY) [25e27] and
(E)-4-(2-(6-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)pyridin-3-
yl)vinyl)-N,N-dimethylaniline ([18F]AV-19) [7], most of the radio-
2.2. Radiolabeling
To get the radiofluorinated ligands [18F]4a, [18F]4b, the N-BOC-
protected tosylate precursor 6a, 6b was mixed with [18F]fluoride,
potassium carbonate and Kryptofix 222 in acetonitrile under
heating at 110 ꢁC for 5 min. The mixtures were treated with
aqueous hydrochloric acid to remove the N-BOC-protecting group,
and neutralized by sodium dicarbonate. The mixture was loaded on
a Sep-Pak Plus-C18 cartridge (Waters), and the elution was
concentrated and the residue was purified by HPLC. The 18F-labeled
[
18F]4a and [18F]4b were prepared with an average radiochemical
yield of 20% and 52% (no decay corrected), and radiochemical purity
of >98%. The identity of [18F]4a and [18F]4b was verified by
a comparison of the retention time with that of the nonradioactive
compound (Fig. 3), and their specific activity was estimated at
tracers for Ab plaques in clinical trials have a monomethylamino
group. Accounting for the same reason, the dimethylamino group
in [125I]QN-1 was changed to monomethylamino group.
approximately 200 GBq/
mmol.
In the present study, we reported the synthesis and evaluation
of novel 18F-labeled 2-phenylquinoxaline probes for A
b plaques
2.3. Biological evaluation
with fluoro-pegylated side chains of different length (n ¼ 1 or 3)
and a monomethylamino group, expecting of better in vivo prop-
erties comparing with the radioiodinated ligand (Figs. 1 and 2).
To evaluate the binding affinity of the two 2-phenylquinoxaline
derivatives (4a and 4b) to Ab1e42 aggregates, in vitro inhibition
assay was carried out in solutions with [125I]IMPY as the competing
radioligand according to conventional methods [28]. The result is
shown in Table 1. As expected, the two derivatives showed good
binding affinity to Ab1e42 aggregates (Ki ¼ 10.0 ꢀ 1.4 nM for 4a,
Ki ¼ 5.3 ꢀ 3.2 nM for 4b) comparable to the value determined under
the same assay system for IMPY (Ki ¼ 10.5 ꢀ 1.0 nM). Compared
with the iodinated tertiary N,N-dimethylamino analog QN-1
2. Results and discussion
2.1. Chemistry
The synthesis was shown in Scheme 1 and Scheme 2. The
derivative 1 was formed by a one-pot tandem oxide condensation
procedure in DMSO (yield 93.6%). The amino derivative 2 was ob-
tained from 1 by reduction with excess hydrazine hydrate in
(Ki
¼
4.1
ꢀ
0.7 nM), these fluorinated secondary mono-
methylamino analogs still kept the high affinity.
Fig. 1. Chemical structure of reported Ab imaging probes for clinical trials.

P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
53
Scheme 1. Synthesize of the fluoropegylated phenylquinoxaline derivatives 4a and 4b. Reagents and conditions: a. DMSO, rt; b. N2H4$H2O, Pd/C, EtOH, reflux; c. (1) (HCHO)n,
CH3ONa, reflux; (2) NaBH4, reflux; d. K2CO3, DMF, 110 ꢁC.
The logD values (3.14 ꢀ 0.23 for [18F]4a, 2.79 ꢀ 0.14 for [18F]4b
and 4.02 ꢀ 0.12 for [125I]QN-1) showed in Table 3 confirm that the
importing of fluoropegylated chain is an effective path to decrease
the lipophilicity of the tracers. The lipophilicity was reduced as the
length of the fluoro-pegylated chain being increased, which may
lower the non-specific binding in brain (Fig. 4).
Compared with [125I]QN-1 (6.03% ID/g at 2 min) and [18F]AV-
45(7.33% ID/g at 2 min), the initial brain uptake of [18F]4a is supe-
rior. High initial brain uptake and high brain2 min/brain60 min ratio in
normal mouse brain are considered to be important as in vivo
pharmacokinetic indexes for selecting appropriate A
b imaging
tracers. As shown in Table 3, the brain2 min/brain60 min ratio is 2.58,
3.89, 2.07, 3.90 for [18F]4a, [18F]4b, [125I]QN-1, and [18F]AV-45,
respectively. The ratios of [18F]4a and [18F]4b are obviously higher
than that of [125I]QN-1, this may due to the introduction of fluoro-
pegylated chains which reduce the lipophilicity of these two
probes. [18F]4a and [18F]4b also distributed to several other organs.
The liver and kidney showed an initial uptake with washout,
continuous gastrointestinal accumulation of the radiotracers
resulted in an intestine uptake (18.75% ID/g and 39.02% ID/g at
60 min). In addition, accumulation of radioactivity in the bone,
2.46% ID/g observed already at 2 min pi and steadily increased up to
6.20% ID/g during the course of the experiment, suggesting little
defluorination in vivo of [18F]4a, while the bone uptake of [18F]4b
remained almost constant, indicating no defluorination in vivo.
In vitro autoradiography in sections of brain tissue from AD
patients or Tg model mice (C57BL6, APPswe/PSEN1, 12 months old)
was carried out to confirm the high binding affinity of [18F]4a and
[
18F]4b to A
b
plaques. As shown in Fig. 5A and C, specific labeling of
plaques was observed in the brain sections of transgenic mice. The
presence and distribution of A plaques was consistent with the
b
results of fluorescent staining using thioflavin-S on the same
sections (Fig. 5B and D). Furthermore, intense labeling of plaques
and low non-specific background were observed in the brain
sections of AD patients (Fig. 6A and B). In contrast, no apparent
labeling was observed in normal adult brain sections (Fig. 6C and
D).
Biodistribution experiments in normal male ICR mice were
carried out to evaluate the ability of radiofluorinated tracers ([18F]
4a and [18F]4b) to penetrate the bloodebrain barrier (BBB) and
properties of clearance from the brain. As shown in Tables 2 and 3,
3. Conclusion
[
18F]4a with a short (n ¼ 1) fluoro-pegylated side chain displayed
In conclusion, two novel 2-phenylquinoxaline derivatives con-
a higher initial brain uptake (8.17% ID/g at 2 min) than that of [18F]
4b with a long (n ¼ 3) side chain (2.49% ID/g at 2 min). The lower
brain uptake for [18F]4b may be due to more hydrogen bonds
formed between the longer fluoro-pegylated chain and water or
other biomoleculars in vivo which decrease the initial brain uptake.
taining an end-capped fluoro-pegylated chains (n ¼ 1, 3) had been
successfully prepared and evaluated as PET imaging tracers for A
b
plaques. These two compounds appeared to have good binding
affinities to A
aggregates. [18F]4a with a short fluoro-pegylated
chain (n ¼ 1) displayed a high initial brain uptake and good ratio
b
Scheme 2. Synthesize of the precursors and radiolabeling. Reagents and conditions: a. K2CO3, acetone, 18-crown-6, reflux; b. (Boc)2O, THF; c. (1) Kryptofix 222, K2CO3, 18Fꢂ, CH3CN,
100 ꢁC; (2) 1 M HCl, 100 ꢁC.

54
P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
Fig. 3. HPLC profiles of 4a, [18F]4a (A) and 4b, [18F]4b (B). HPLC conditions: Venusil MP C18 column (Agela Technologies, 10 ꢃ 250 mm), CH3CN/H2O ¼ 80/20 for 4a, CH3CN/
H2O ¼ 70/30 for 4b, 4 mL/min, UV, 254 nm.
of brain2 min/brain60 min, and the in vitro autoradiography studies
confirmed its specific binding to A plaques and low non-specific
binding, which suggests [18F]4a may be a potential PET imaging
Transgenic mice brain tissues (C57BL6, APPswe/PSEN1, 12 months
old, male) were purchased from Institute of Laboratory Animal
Sciences, Chinese Academy of Medical Sciences.
b
agent for in vivo detection of Ab plaques.
4.2. 4-(6-Nitroquinoxalin-2-yl)phenol (1)
4. Experimental
A mixture of 2-bromo-1-(4-hydroxyphenyl)ethanone (1 mmol)
and 4-nitrobenzene-1,2-diamine (1 mmol) was stirred in 5 mL
DMSO at room temperature. The reaction mixture was poured into
water, and quickly filtered over a buchner funnel. The filter residue
was washed with water, oven dried and purified by silica gel
chromatography (petroleum ether/ethylacetate ¼ 2:1), to give
250 mg of 1 in a yield of 93.6%. 1H NMR (400 MHz, [D6]-DMSO)
4.1. General information
All reagents used in the synthesis were commercial products
and were used without further purification unless otherwise indi-
cated. 18Fꢂ was obtained from the Chinese PLA General Hospital.
The 1H NMR spectra were obtained at 400 MHz on Bruker spec-
trometer in CDCl3 or [D6]-DMSO at room temperature with TMS as
d
10.27 (s, 1H), 9.68 (d, J ¼ 4.5 Hz, 1H), 8.81 (dd, J ¼ 13.2, 2.5 Hz, 1H),
an internal standard. Chemical shifts were reported as
d
values with
8.50 (dd, J ¼ 9.2, 2.6 Hz, 1H), 8.29 (d, J ¼ 8.8 Hz, 2H), 8.23 (t,
J ¼ 9.0 Hz, 1H), 6.98 (d, J ¼ 8.7 Hz, 2H), 5.37 (s, 1H). MS (ESI) m/z
calcd for C14H9N3O3 267.1, found 267.7 [M þ H]þ.
respect to residual solvents. The multiplicity is defined by s
(singlet), d (doublet), t (triplet), m (multiplet). Mass spectrometry
was acquired under the Surveyor MSQ Plus (ESI) (Waltham, MA,
USA) instrument. Reactions were monitored by TLC (precoated
silica gel plate F254, Merck). Radiochemical purity was determined
by HPLC performed on a Shimadzu SCL-20 AVP equipped with
4.3. 4-(6-Aminoquinoxalin-2-yl)phenol (2)
A mixture of compound 1 (2 mmol) and hydrazine hydrate
(8 mmol) in ethanol (20 mL) was refluxed over night, in which Pd/C
a Bioscan Flow Count 3200 NaI/PMT
detector. Separations were achieved on a Venusil MP C18 column
(Agela Technologies, 10
m, 10 mm ꢃ 250 mm) eluted with a binary
g-radiation scintillation
m
gradient system at a 4.0 mL/min flow rate. Mobile phase A was
water while mobile phase B was acetonitrile. Fluorescent obser-
vation was performed by the LSM 510 META (Zeiss, Germany)
equipped with a LP 505 filter set (excitation, 405 nm; long-pass
filter, 505 nm). The purity of the synthesized key compounds was
determined using analytical HPLC and was found to be more than
95%. ICR Mice (five weeks, 20e22 g, male) were used for bio-
distribution experiments. All protocols requiring the use of mice
were approved by the animal care committee of Beijing Normal
University. Postmortem brain tissues from an autopsy-confirmed
Table 2
Biodistribution of in ICR normal mice after iv injections of [18F]tracers (% ID/g,
Mean ꢀ SD, n ¼ 5).
2 min
10 min
30 min
60 min
[
18F]4a (LogD ¼ 3.14 ꢀ 0.23)
Blood
Brain
Heart
Liver
Spleen
Lung
4.93 ꢀ 0.43
8.17 ꢀ 1.33
6.08 ꢀ 0.37
9.50 ꢀ 1.04
4.71 ꢀ 0.17
6.38 ꢀ 0.79
9.37 ꢀ 1.11
2.46 ꢀ 0.73
1.81 ꢀ 0.42
9.63 ꢀ 1.10
4.68 ꢀ 0.32
5.07 ꢀ 0.71
4.27 ꢀ 0.60
7.80 ꢀ 1.33
3.87 ꢀ 0.57
4.36 ꢀ 0.42
5.96 ꢀ 0.42
2.84 ꢀ 0.54
2.55 ꢀ 0.54
12.87 ꢀ 2.61
5.09 ꢀ 0.30
3.45 ꢀ 0.13
4.28 ꢀ 0.54
5.37 ꢀ 0.51
3.35 ꢀ 0.50
3.78 ꢀ 0.56
4.25 ꢀ 0.43
5.73 ꢀ 0.29
1.84 ꢀ 0.36
17.15 ꢀ 1.23
5.24 ꢀ 0.45
3.17 ꢀ 0.22
4.24 ꢀ 0.22
5.00 ꢀ 0.84
3.04 ꢀ 0.64
3.77 ꢀ 0.36
3.79 ꢀ 0.66
6.20 ꢀ 0.25
2.50 ꢀ 1.19
18.75 ꢀ 3.90
case of AD (5
mm, temporal lobe) and a control subject (5 mm,
Kidney
Bone
temporal lobe) were obtained from BioChain Institute Inc.
Stomacha
Intestinea
Table 1
[
18F]4b (LogD ¼ 2.79 ꢀ 0.14)
Inhibition constants (Ki) for binding to aggregates of
Blood
5.51 ꢀ 0.34
2.49 ꢀ 0.22
3.21 ꢀ 0.46
14.18 ꢀ 1.66
2.07 ꢀ 0.97
4.07 ꢀ 0.45
5.52 ꢀ 0.46
1.49 ꢀ 0.34
1.55 ꢀ 0.12
6.06 ꢀ 0.64
3.32 ꢀ 0.32
1.44 ꢀ 0.11
1.76 ꢀ 0.19
14.09 ꢀ 1.49
1.65 ꢀ 0.11
2.40 ꢀ 0.33
3.91 ꢀ 0.27
1.00 ꢀ 0.40
2.81 ꢀ 1.31
12.26 ꢀ 2.94
2.38 ꢀ 0.21
0.83 ꢀ 0.17
1.19 ꢀ 0.19
7.90 ꢀ 1.07
1.21 ꢀ 0.21
1.76 ꢀ 0.25
3.27 ꢀ 0.74
1.12 ꢀ 0.23
3.21 ꢀ 1.62
32.83 ꢀ 6.30
1.54 ꢀ 0.12
0.64 ꢀ 0.07
1.03 ꢀ 0.15
4.89 ꢀ 0.74
0.94 ꢀ 0.39
1.15 ꢀ 0.11
1.65 ꢀ 0.94
1.78 ꢀ 0.06
6.53 ꢀ 2.03
39.02 ꢀ 6.98
A
b1-42 versus [125I]IMPY.a
Brain
Heart
Liver
Spleen
Lung
Compound
Ki (nM)
4a
4b
10.0 ꢀ 1.4
5.3 ꢀ 3.2
4.1 ꢀ 0.7
10.5 ꢀ 1.0
QN-1b
IMPYb
Kidney
Bone
Stomacha
Intestinea
a
Measured in triplicate with results given as the
mean ꢀ SD.
b
a
Data from literature [23].
Expressed as % ID.

P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
55
give 54 mg of 4a in a yield of 51.8%. 1H NMR (400 MHz, CDCl3)
d
9.10
Table 3
Comparison of inhibition constants (Ki) and brain kinetics between radiolabeled 2-
(s, 1H), 8.08 (d, J ¼ 8.8 Hz, 2H), 7.87 (d, J ¼ 9.1 Hz, 1H), 7.13 (dd,
J ¼ 9.1, 2.6 Hz, 1H), 7.08 (d, J ¼ 8.8 Hz, 2H), 6.98 (d, J ¼ 2.5 Hz, 1H),
4.89e4.85 (m, 1H), 4.77e4.73 (m, 1H), 4.35e4.33 (m, 1H), 4.28 -
phenylquinoxaline derivatives and [18F]AV-45.
a
Compound
Ki (nM)
Brain2 min
Ratio2 min/60 min
LogD
4.25 (m, 1H), 3.00 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 159.50,
[
[
[
[
18F]4a
10.0
5.3
4.1
8.17
2.49
6.03
7.33
2.58
3.89
2.07
3.90
3.14
2.79
4.02
2.41
18F]4b
149.80, 147.20, 143.75, 142.77, 137.12, 130.77, 130.02, 128.27, 122.07,
115.17, 103.34, 82.70, 81.00, 67.37, 67.17, 30.53. HRMS (ESI) m/z calcd
for C17H16FN3O 298.1356, found 298.1360 [M þ H]þ.
125I]QN-1b
18F]AV-45c
2.9
a
b
c
Expressed as % ID/g.
Data from literature [23].
Data from literature [19].
4.6. 2-(4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)phenyl)-N-methyl
quinoxalin-6-amine (4b)
was added as catalyzer. The reaction mixture was cooled to room
temperature and then filtered. The filtrate was concentrated, and
purified by silica gel chromatography (petroleum ether/
ethylacetate ¼ 1:1) to give 453 mg of 2 in a yield of 95.6%. 1H NMR
The reaction described for 4a was used, and 27 mg of 4b was
obtained in a yield of 35.0%. 1H NMR (400 MHz, CDCl3)
d 9.09 (s,1H),
8.06 (d, J ¼ 8.6 Hz, 2H), 7.86 (d, J ¼ 9.0 Hz, 1H), 7.12 (dd, J ¼ 9.1,
2.1 Hz,1H), 7.06 (d, J ¼ 8.6 Hz, 2H), 6.98 (d, J ¼ 1.7 Hz,1H), 4.65e4.62
(m, 1H), 4.53e4.50 (m, 1H), 4.24e4.20 (m, 2H), 3.93e3.90 (m, 2H),
3.82e3.79 (m, 1H), 3.78e3.72 (m, J ¼ 12.5, 4.9 Hz, 6H), 3.00 (s, 3H).
(400 MHz, [D6]-DMSO)
d
9.86 (s, 1H), 9.21 (s, 1H), 8.10 (d, J ¼ 8.7 Hz,
2H), 7.78 (d, J ¼ 9.0 Hz, 1H), 7.28 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.99 (d,
J ¼ 2.4 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H), 6.03 (s, 2H). MS (ESI) m/z
calcd for C14H11N3O 237.1, found 237.7 [M þ H]þ.
13C NMR (100 MHz, CDCl3)
d 158.85, 148.76, 146.25, 142.55, 141.65,
136.08, 129.22, 128.93, 127.12, 121.08, 114.11, 102.12, 82.96, 81.29,
69.90, 69.86, 69.55, 69.35, 68.77, 66.55, 29.49. HRMS (ESI) m/z calcd
for C21H25FN3O3 386.1880, found 386.1891 [M þ H]þ.
4.4. 4-(6-(Methylamino)quinoxalin-2-yl)phenol (3)
A solution of compound 3 (2.3 mmol) in methanol (20 mL) was
added with sodium methoxide (4.5 mmol) and paraformaldehyde
(9 mmol). The reaction mixture was refluxed for 2 h then cooled to
4.7. 2-(4-(6-(Methylamino)quinoxalin-2-yl)phenoxy)ethyl 4-methy
lbenzenesulfonate (5a)
0
ꢁC in an ice bath. Sodium borohydride (9 mmol) was added
A mixture of compound 3 (0.4 mmol), ethane-1,2-diyl bis(4-
methylbenzenesulfonate) (0.6 mmol) and K2CO3 (1.2 mmol) in
acetone (10 mL) was refluxed for 10 h, in which 18-Crown-6 was
added as catalyzer. After the solvent being removed in vacuo, the
residue was purified by silica gel chromatography (petroleum
ether/ethylacetate ¼ 1:2) to give 49.4 mg of 5a in a yield of 27.5%. 1H
carefully. The mixture was refluxed again for 1 h and cooled to
room temperature. After solvent being removed in vacuo, water
(10 mL) was added, and then ethylacetate (3 ꢃ 50 mL) was used for
extraction. The organic phase was dried over MgSO4, and concen-
trated latter. The residue was purified by silica gel chromatography
(petroleum ether/ethylacetate ¼ 1:1) to give 512 mg of 3 in a yield
of 88.7%. 1H NMR (400 MHz, [D6]-DMSO)
d 9.80 (s, 1H), 9.18 (s, 1H),
NMR (400 MHz, CDCl3)
d
9.08 (s, 1H), 8.04 (d, J ¼ 8.8 Hz, 2H), 7.85 (t,
J ¼ 8.4 Hz, 3H), 7.36 (d, J ¼ 8.0 Hz, 2H), 7.13 (dd, J ¼ 9.1, 2.6 Hz, 1H),
6.98 (d, J ¼ 2.4 Hz, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 4.42 (dd, J ¼ 5.5,
3.9 Hz, 2H), 4.24 (d, J ¼ 4.9 Hz, 2H), 3.00 (s, 3H), 2.45 (s, 3H). MS
(ESI) m/z calcd for C24H23N3O4S 449.1, found 449.6 [M þ H]þ.
8.05 (d, J ¼ 8.7 Hz, 2H), 7.74 (d, J ¼ 9.1 Hz, 1H), 7.25 (dd, J ¼ 9.1,
2.5 Hz, 1H), 6.91 (d, J ¼ 8.6 Hz, 2H), 6.74 (d, J ¼ 2.4 Hz, 1H), 2.83 (s,
3H). MS (ESI) m/z calcd for C15H13N3O 251.1, found 251.8 [M þ H]þ.
4.5. 2-(4-(2-Fluoroethoxy)phenyl)-N-methylquinoxalin-6-amine
(4a)
4.8. 2-(2-(2-(4-(6-(Methylamino)quinoxalin-2-yl)phenoxy)ethoxy)
ethoxy)ethyl 4-methylbenzenesulfonate (5b)
A
mixture of compound 3 (0.35 mmol) and 1-bromo-2-
fluoroethane (0.5 mmol) in DMF (5 mL) was refluxed for 2 h.
After being cooled to room temperature, water (10 mL) and CH2Cl2
(20 mL) were added to the mixture. The organic layer was sepa-
rated, washed with water (3 ꢃ 10 mL), and dried over MgSO4. The
solvent was removed in vacuo, and the residue was purified by
silica gel chromatography (petroleum ether/ethylacetate ¼ 2:1) to
The reaction described for 5a was used, and 210 mg of 5b was
obtained in a yield of 73.6%. 1H NMR (400 MHz, CDCl3)
d 9.09 (s,1H),
8.06 (d, J ¼ 8.7 Hz, 2H), 7.85 (d, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 8.2 Hz,
2H), 7.32 (d, J ¼ 8.1 Hz, 2H), 7.13 (dd, J ¼ 9.1, 2.5 Hz, 1H), 7.05 (d,
J ¼ 8.8 Hz, 2H), 6.97 (d, J ¼ 2.4 Hz, 1H), 4.21e4.15 (m, J ¼ 6.4, 5.0 Hz,
4H), 3.89e3.84 (m, 2H), 3.73e3.67 (m, 4H), 3.66e3.62 (m, J ¼ 5.9,
3.1 Hz, 2H), 2.99 (s, 3H), 2.42 (s, 3H). MS (ESI) m/z calcd for
C28H32N3O6S 538.2, found 538.5 [M þ H]þ.
120
4a
4b
100
4.9. 2-(4-(6-(tert-Butoxycarbonyl)quinoxalin-2-yl)phenoxy)ethyl
4-methylbenzenesulfonate (6a)
80
60
40
20
0
Compound 5a (0.1 mmol) was added to a solution of di-tert-
butyldicarbonate (0.4 mmol) and DIEA (0.2 mmol) in THF, and the
reaction mixture was refluxed over night. After the solvent being
removed in vacuum, the residue was purified by silica gel chro-
matography (petroleum ether/ethylacetate ¼ 1:1) to give 24 mg of
6a in a yield of 43.1%. 1H NMR (400 MHz, CDCl3)
d 9.24 (s, 1H), 8.12
(d, J ¼ 8.6 Hz, 2H), 8.03 (d, J ¼ 8.9 Hz,1H), 7.90e7.76 (m, 4H), 7.36 (d,
J ¼ 8.0 Hz, 2H), 6.96 (d, J ¼ 8.6 Hz, 2H), 4.46e4.39 (m, 2H), 4.29e
4.21 (m, 2H), 3.43 (s, 3H), 2.46 (s, 3H), 1.50 (s, 9H). MS (ESI) m/z
calcd for C29H31N3O6S 549.2, found 549.7 [M þ H]þ.
-11
-10
-9
-8
Log C
-7
-6
-5
Fig. 4. Inhibition curves for the binding of [125I]IMPY to Ab1e42 aggregates.

56
P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
Fig. 5. In vitro autoradiography of [18F]4a and [18F]4b (A, C) on a Tg model mouse (C57BL6, APPswe/PSEN1, 12 months old, male). The presence and distribution of plaques in the
sections were confirmed by fluorescence staining using thioflavin-S on the same sections with a filter set for GFP (B, D).
4.10. 2-(2-(2-(4-(6-(tert-Butoxycarbonyl)quinoxalin-2-yl)phenoxy)
ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6b)
(1 mg) in 1 mL CH3CN was added to the reaction tube containing
dried 18Fꢂ activities. The mixture was heated at 110 ꢁC under
nitrogen protecting for 5 min. 200 mL HCl (1 M) was added into the
The reaction described for 6a was used, and 67 mg of 6b was
tube and the mixture was heated for another 5 min at 110 ꢁC under
nitrogen protecting for deprotection, and neutralized by NaHCO3
after being cooled down to room temperature. Water (5 mL) was
added, and the mixture was passed through a preconditioned Sep-
Pak Plus-C18 cartridge (Waters), which was washed with 10 mL
water later. The 18F-labeled compound was eluted with 2 mL of
acetonitrile, and purified by HPLC. The 18F-labeled [18F]4a and [18F]
4b were prepared with an average radiochemical yield of 20% and
52% (no decay corrected), and radiochemical purity of >98%.
obtained in a yield of 52.6%. 1H NMR (400 MHz, CDCl3)
d 9.25 (s,1H),
8.15 (d, J ¼ 8.8 Hz, 2H), 8.03 (d, J ¼ 9.0 Hz, 1H), 7.85 (d, J ¼ 2.3 Hz,
1H), 7.83e7.77 (m, 3H), 7.32 (d, J ¼ 8.0 Hz, 2H), 7.08 (d, J ¼ 8.9 Hz,
2H), 4.22e4.16 (m, 4H), 3.90e3.85 (m, 2H), 3.73e3.68 (m, 4H),
3.66e3.61 (m, 2H), 3.43 (s, 3H), 2.42 (s, 3H), 1.50 (s, 9H). MS (ESI) m/
z calcd for C33H39N3O6S 637.2, found 637.7 [M þ H]þ.
4.11. Radiolabeling
[
18F]Fluoride trapped on a QMA cartridge was eluted with 1 mL
4.12. Binding assay in vitro using Ab1-42 aggregates
of Kryptofix 222/K2CO3 solution. The solvent was removed at
110 ꢁC under a stream of nitrogen gas. The residue was azeo-
tropically dried with 1 mL of anhydrous acetonitrile twice at 110 ꢁC
under a stream of nitrogen gas. A solution of tosylate precursor
Inhibition experiments were carried out in 12 ꢃ 75 mm boro-
silicate glass tubes according to procedures described previously
with some modifications. The radio-ligand [125I]IMPY was prepared
Fig. 6. In vitro autoradiography of [18F]4a and [18F]4b on AD human brain sections (A, B) and control human brain sections (C, D).

P. Yu et al. / European Journal of Medicinal Chemistry 57 (2012) 51e58
57
according to procedures described previously [25], after HPLC
purification, the radiochemical purity was greater than 95%. For the
inhibition assay, 100 L Ab1-42 aggregates solution, 100
L [125I]
IMPY solution, 100 L BSA solution (1%) and 100 L inhibitors
Acknowledgments
m
m
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 NSFC (21071023).
m
m
(10ꢂ4e10ꢂ9.5 M) were added into borosilicate glass tubes. The
mixture was incubated at 37 ꢁC for 2 h, and the free radioactivity
were separated by vacuum filtration through Whatman GF/B filters
using a Brandel Mp-48T cell harvester followed by 3 ꢃ 4 mL washes
with PBS (0.02 M, pH 7.4) at room temperature. Filters with the
bound [125I]IMPY were counted in gamma counter (WALLAC/
Wizard 1470, USA) with 70% efficiency. Inhibition experiments
were repeated three times, and the half maximal inhibitory
concentration (IC50) was determined usingGraphPad Prism 4.0, and
the inhibition constant (Ki) was calculated using the ChengePrusoff
equation: Ki ¼ IC50/(1 þ [L]/Kd) [29]
Appendix A. Supplementary information
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.08.031.
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