Novel 18F-labeled dibenzylideneacetone derivatives as potential positron emission tomography probes for in vivo imaging of β-amyloid plaques

Article abstract of DOI:10.1016/j.ejmech.2014.07.070

A series of dibenzylideneacetones were synthesized and evaluated as imaging probes for β-amyloid plaques. They displayed high binding affinity to Aβ_1-42 aggregates (K_i = 6.4 for 8, K_i = 3.0 for 9), and the high binding wer

Full text of DOI:10.1016/j.ejmech.2014.07.070

European Journal of Medicinal Chemistry 84 (2014) 628e638
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
18
Novel F-labeled dibenzylideneacetone derivatives as potential
positron emission tomography probes for in vivo imaging of b-amyloid
plaques
,
, b,
Zijing Li a, Mengchao Cui a *, Jinming Zhang a **, Jiapei Dai c, Xiaojun Zhang b,
Peng Chen 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
c Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for Nationalities, Wuhan 430074, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dibenzylideneacetones were synthesized and evaluated as imaging probes for
b-amyloid
Received 22 April 2014
Received in revised form
17 July 2014
Accepted 19 July 2014
Available online 21 July 2014
plaques. They displayed high binding affinity to Ab1e42 aggregates (Ki ¼ 6.4 for 8, Ki ¼ 3.0 for 9), and the
high binding were confirmed by in vitro autoradiography with AD human and transgenic mouse brain
sections. Two of them were selected for 18F-labeling directly on the benzene ring. In biodistribution
experiments, [18F]8 and [18F]9 displayed high initial uptakes (9.29 0.41 and 5.38 0.68% ID/g) and rapid
washouts from the normal brain (brain2 min/brain60 min ratios of 21.6 and 13.4). These preliminary results
suggest that [18F]8 and [18F]9 may be used as potential PET imaging agents for the detection of A
b
Keywords:
Dibenzylideneacetone derivatives
Ab plaques
plaques in the brain.
© 2014 Elsevier Masson SAS. All rights reserved.
PET imaging agents
AD
1. Introduction
senile plaques (SPs) and neurofibrillary tangles (NFTs). Further-
more, the extra cellular deposition of b-amyloid (Ab) as SPs leading
Alzheimer's disease (AD) is devastating the elderly population
with dementia, loss of memory, cognitive impairment, and inability
to carry out routine activities [1]. It is not only a profound health
and emotional influence on affected individuals and their families,
but also a substantial economic burden on society. Extraordinary
efforts will be spend on both the prevention and the treatment of
dementia in the following decades, since this illness is going to
affect about 63 million people by 2030, and 114 million by 2050
worldwide [2]. Early treatment depends on early disease diagnosis.
However, the clinical diagnosis of AD is based mainly on the ex-
amination of mental and cognitive status nowadays [3], and the
confirmation of the disease is achieved only by the postmortem
examination of brain tissues [4,5]. It has been well established that
the major neuropathological characteristics of AD is the presence of
to the neuronal pathogenesis is currently considered the major
pathological hallmark of AD brains [6,7]. Therefore, recent research
efforts have been focused on noninvasive early visualization of A
b
plaques in living brain tissues to identify and further monitor in-
dividuals at risk for AD, as well as to assist in the evaluation of new
anti-amyloid therapies currently under development [8e10].
Although several imaging techniques such as magnetic reso-
nance imaging (MRI) [11e13], single photon emission computed
tomography (SPECT) [14], and optical imaging [15e18] have been
explored for the visualization of Ab plaques in the past decade, Ab
plaque-specific positron emission tomography (PET) imaging re-
mains the most successful noninvasive technique to date, which
generated great anticipation and high hopes [10,19,20]. Vast array of
PET ligands based on three categories of chemical groups have been
reported, the thioflavin-T derivatives, the stilbene derivatives, and
the aminonaphthyl derivatives [10,19] (Fig. 1). The first PET tracer
specific for Ab
plaques was [11C]-2-(4-(N-methylamino)phenyl)-6-
* Corresponding author. Key Laboratory of Radiopharmaceuticals, Ministry of
Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR
China.
** Corresponding author. Department of Nuclear Medicine, Chinese PLA General
Hospital, Beijing 100853, PR China.
hydroxybenzothiazol ([11C]PIB) [21,22] through modification of
thioflavin-T, but it has certain limitation that requires an on-site
cyclotron because of short half-life of 11C. An analogue of PIB,
which demonstrates potential utility,
[
18F]-2-(3-fluoro-4-(N-
E-mail addresses: cmc@bnu.edu.cn (M. Cui), zhangjm301@163.com (J. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2014.07.070
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
629
Fig. 1. Chemical structures of reported 18F labeled A
b imaging probes.
methylamino)phenyl)benzo[d]thiazol-6-ol ([18F]GE-067) [23] has
been tested clinically and approved by FDA. The stilbene derivatives
designed a new type of 18F-labeled dibenzylideneacetone deriva-
tive (Fig. 2) for A
imaging using a novel 18F-labeling approach in
b
include
[24,25],
[
11C]-4-N-methylamino-40-hydroxystilbene ([11C]SB-13)
two steps. This pathway labels 18F atom directly on the benzene
ring in order to keep the affinity of dibenzylideneacetone to the
greatest extent. Meanwhile, we also expect this innovation could
minimize the molecular weight and improve the properties of the
tracers, such as high brain uptakes, ineligible defluorination and
better brain pharmacokinetics, compared with using the PEG
pathway.
[
18F]-4-(N-methylamino)-40-(2-(2-(2-fluoroethoxy)
ethoxy)ethoxy)-stilbene ([18F]BAY94-9172) [26] and [18F]-(E)-4-(2-
(6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-
methylaniline ([18F]AV-45) [27,28], which are currently under
commercial development for the mapping of A
b
plaque burden in
tracer
living brain tissue. Among them, [18F]AV-45 was the first A
b
approved by FDA. The aminonaphthyl derivative [18F]-2-(1-(2-(N-
(2-fluoroethyl)-N-methylamino)-naphthalene-6-yl)ethylidene)
malononitrile ([18F]FDDNP) was the first PET ligand put into human
trials and also reported to bind to NFTs [29e31], whose binding site
2. Results and discussion
2.1. Chemistry
on Ab deposits may differ from those of the prior two categories [10].
However, almost all 18F-labeled tracers currently in late phases of
clinical development have high nonspecific white matter uptakes
that give a distinctive white matter pattern in scans of healthy
subjects. Furthermore, in PET scan images of AD patients, those 18F-
labeled tracers frequently show the gray matterewhite matter
demarcation loss, and consequent loss of the normal white matter
pattern, which are the predominant evidence of cortical amyloid
plaques [32]. In the cortical ribbon typical of a positive [11C]PIB scan,
it less often shows the clearly intense binding, too [32]. So, new
probes beyond the above three categories with improved properties
The dibenzylideneacetone derivatives used in the binding as-
says and characterization of the corresponding 18F-labeled ligands
were produced following the synthetic route shown in Scheme 1.
The two substituted (E)-4-phenylbut-3-en-2-one derivatives 4 and
5 were prepared according to the method described previously
[33]. The dibenzylideneacetone derivatives were obtained through
base-catalyzed Claisen condensation reactions that coupled the
suitably substituted (E)-4-phenylbut-3-en-2-one with 4-
flourobenzaldehyde or 2-flourpyridine-5-carboxaldehyde. The un-
symmetrically substituted dibenzylideneacetones 7, 8 and 9 were
obtained in 85.0e90.5% yields, and their corresponding pyridine
substituted compounds 10, 11 and 12 were obtained in 78.3e88.9%
yields. It should be mentioned here that we first used ethanol as the
solvent for Claisen condensation reaction when preparing the
pyridine derivatives 10e12, but it turned out that the fluorine atom
of the aimed product was displaced by ethoxy group (compounds
13e15) at ambient temperature using K2CO3 as base and ethanol as
solvent. The aromatic halides usually undergo nucleophilic sub-
stitution by various nucleophiles, such as secondary amines and
alcoholate ion (e.g. ethanol as nucleophilic reagent at base condi-
tion), and the reaction rate will be accelerate when the fluoro atom
was substituted at the ortho position of the pyridine ring. Therefore,
we tried several different aprotic solvents such as acetonitrile, 1,4-
dioxane, diethyl ether, DMF and THF to proceed this reaction, and
we found DMF is an efficient solvent in this kind of condensation
reaction, compound 10e12 were obtained with high yields
(78.3e88.9%). It means that we should be very careful to choose the
solvent when labeling the pyridine array compounds.
including higher affinity for Ab plaques and less nonspecific binding
in the white matter of the brain are always expected.
In 2010, an extensive set of dibenzylideneacetone derivatives
was synthesized and screened for affinity toward Ab1e42 aggregates
by Cui et al. [33] Structure activity relationships revealed the
binding of dibenzylideneacetones at the para position was highly
tolerant of sterically demanding substitutions (Ki ranging from 0.9
to 7.0 nM). Two of them were labeled with 18F through a short
length of polyethylene glycol chain (PEG). In biodistribution ex-
periments, the radiofluorinated ligands [18F]1 and [18F]2 exhibited
good initial penetration (4.13 and 5.15% ID/g, respectively, at 2 min)
of and ordinary clearance from the brain. The brain2 min/brain60 min
ratio of them is acceptable (4.59, and 4.06 for [18F]1, and [18F]2,
respectively). These preliminary results suggested that the diben-
zylideneacetone structure to be a potential new scaffold for the
development of A
Therefore, based on the backbone of dibenzylideneacetones and
their known affinities for A aggregates, in the present study we
b imaging probes.
b

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Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
Fig. 2. Chemical structures of the designed 18F labeled ligands in this work.
Scheme 1. Reagents and conditions: a. SnCl2$2H2O, EtOH, HCl, reflux; b. K2CO3, CH3I, r.t.; c. NaOH, EtOH, r.t. for 7e9 and 13e15; NaOH, DMF, r.t. for 10e12.
Fig. 3. In vitro fluorescent staining of compound 7e12 (A, C, E, G, I, K, respectively, with filter set of GFP) on brain sections of a Tg model mouse (C57BL6, APPswe/PSEN1, 11 months
old, male), and thioflavin-S on the adjacent sections (B, D, F, H, J, L, respectively, with filter set of DAPI). Stained plaques are indicated by red arrows. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
631
2.2. In vitro fluorescent staining
To evaluate the binding affinities of compounds 7e12 to A
b
plaques, in vitro fluorescent staining of A plaques on sections of
b
brain tissue from Tg model mice (C57BL6, APPswe/PSEN1, 11
months old) were conducted as stated. As shown in Fig. 3 A, C, E, G, I
and K, specific staining of plaques was clearly observed on the brain
section of Tg mice for compound 7e12. The presence and distri-
bution of Ab plaques were perfectly consistent with the results of
staining applying thioflavin-S (a common dye for staining of A
b
plaques) on the adajacent sections (B, D, F, H, J and L).
2.3. In vitro binding studies
Fig. 4. Inhibition curves for the binding of [125I]IMPY to Ab1e42 aggregates.
The quantitative affinities of these dibenzylideneacetone de-
rivatives (7e15) for Ab1e42 aggregates were examined in compe-
tition binding assays, using [125I]-6-iodo-2-(40-dimethylamino-)
phenyl-imidazo[1,2]pyridine ([125I]IMPY) as the competing radio-
ligand. IMPY was also screened employing the same system for
comparison. As illustrated in Table 1, all compounds inhibited the
binding of [125I]IMPY in a dose-dependent manner (Fig. 4) and
displayed high binding affinities to Ab1e42 aggregates with Ki values
varied from 3.0 to 135.2 nM. Consistently to the previously reported
findings, the tertiary N,N-dimethylamino analogues have higher
affinities than the corresponding secondary N-methylamino ana-
logues [34] (e.g., 8 vs. 9,11 vs.12). On the basis of the higher binding
affinities to Ab1e42 aggregates, compounds 8 (Ki ¼ 6.4 3.2 nM) and
9 (Ki ¼ 3.0 0.6 nM) were selected for 18F labeling.
We have two sound reasons to favor this two-step 18F labeling
strategy. First, the Claisen condensation for coupling was proved to
be fast enough for 18F labeling and a pretty convenient way to label
18F on a benzene ring, a very important component in the core
structure of dibenzylideneacetone. Second, this strategy is ready to
be applied when labeling many other different molecules con-
taining one ketone group in the future studies.
2.5. In vitro stability studies
[
18F]8 and [18F]9 were found not only stable (>90%) in saline
solution after storage for at least 1 h at room temperature during
in vitro stability study, but also having high in vitro stability in mice
plasma. After incubating for 60 min with mice plasma at 37 ꢀC,
more than 90% of the activity was identified as intact compound
2.4. Radiolabeling
As shown in Scheme 2, in the first step of this two-step reaction,
the 18F-labled intermediate [18F]-4-fluorobenzaldehyde ([18F]17)
was prepared with an average radiochemical yield of 40e60% (no
decay corrected) after Sep-Pak C18 cartridge (Waters) separation.
Then [18F]17 was eluted out with ethanol to go through a base-
catalyzed Claisen condensation in the ethanol with two corre-
sponding substituted (E)-4-phenylbut-3-en-2-one separately to
yield [18F]8 and [18F]9. The radiochemical yields of [18F]8 and [18F]9
in the second step were about 50% (no decay corrected), resulting
an average of 25% total yield with radiochemical purity greater than
95% after high performance liquid chromatography (HPLC) purifi-
cation. The specific activities were estimated at approximately
[
18F]8 or [18F]9, respectively.
2.6. In vitro autoradiography studies
Brain sections of a Tg mouse (C57BL6, APPswe/PSEN1, 11 months
old) with Ab plaques confirmed as well as an age-matched control
mouse (C57BL6, 11 months old) were selected as staining material
in the in vitro autoradiographic studies. As shown in Figs. 6 and 7,
A
b
plaques of both hippocampus and cortical regions of the Tg
mouse were selectively labeled by the 18F-labeled tracers [18F]8 and
18F]9, separately, while no notable A
plaques was noticed on the
[
b
160 GBq/mmol (at the end of synthesis), high enough for the
brain of the control mouse. The consistent results of fluorescent
staining stained by thioflavin-S further confirmed the distribution
following in vitro and in vivo studies. The chemical identities of the
18F-labeled tracers were verified by comparison of the retention
times with those of the nonradioactive standard compounds
(Fig. 5).
and pattern of those A
that the configuration/folding of A
different from the tertiary/quaternary structure of A
brains [35], we consider the binding information of new tracers for
b
plaques. Since a previous report suggested
plaques in Tg mice might be
plaques in AD
b
b
Ab plaques in human AD brain even more important. As illustrated
Table 1
in Fig. 8, tracer [18F]9 with a dimethylamino group was tested for
Inhibition constants (Ki, nM) for binding to aggregates of
A
b1e42 versus [125I]IMPY.a
autoradiographic studies in sections of human brain tissue, and as
we supposed, typical labeling pattern of A
observed on the AD brain section. For no apparent labeling was
observed in normal adult brain section in contrast, this result
b plaques was also
Compound
Ki (nM)
7
8
9
17.1 4.3
6.4 3.2
3.0 0.6
further proved its affinity to Ab plaques from AD brain.
10
11
12
13
14
15
IMPY
135.2 58.1
36.5 23.9
8.5 2.0
65.8 17.8
14.2 5.2
7.6 3.9
2.7. In vivo biodistribution studies
The log D values (2.69 0.19 for [18F]8 and 3.89 0.21 for [18F]9,
respectively) indicate that tracer [18F]8 and [18F]9 have moderate
lipophilicity which is suitable for brain imaging. Here, we sum-
marized the time-activity data on the brain permeation as well as
the other organ distribution of the 18F-labeled tracers acquired in
11.5 2.5
a
Measured in triplicate with results given as the
mean SD.

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Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
Scheme 2. Reagents and conditions: a. CH2Cl2, ice-salt bath; b. K18F, K222, DMSO, 160 ꢀC, 6 min; c. NaOH, EtOH, r.t.
Fig. 5. (A) HPLC profiles of 8 and [18F]8. (B) HPLC profiles of 9 and [18F]9. HPLC conditions: Venusil MP C18 column (Agela Technologies, 10 ꢁ 250 mm), CH3CN/H2O ¼ 70/30, 4 mL/
min, UV ¼ 254 nm.
biodistribution study in normal ICR mice in Tables 2 and 3. In this
often used test to measure the initial brain uptake and washout
kinetics from the normal brain, [18F]8 and [18F]9 displayed high
uptakes into the brain (9.29 0.41 and 5.38 0.68% ID/g, respec-
tively) at 2 min post-injection and fast washout kinetics from the
in vivo detection of Ab plaques as potential PET imaging agents.
Compared with the 18F-labeled dibenzylideneacetone derivatives
with PEG chains, the 18F-labeled dibenzylideneacetone derivatives
without PEG chains had greatly improved pharmacokinetics and
may lower nonspecific white matter uptake.
healthy brain (0.43
0.14 and 0.40
0.07% ID/g at 60 min,
respectively), which are comparable to those reported for the 18F-
labeled dibenzylideneacetone derivatives with PEG chains [33]. The
ratio of brain2 min/brain60 min is also considered as an important
index to select tracers with appropriate kinetics in vivo. Based on
this point, the washout of [18F]AV-45 in healthy mice, with a
3. Conclusions
In conclusion, a series of dibenzylideneacetone derivatives was
prepared using straightforward chemistry and evaluated as new
PET imaging tracers for A
deneacetone derivatives tracers displayed various binding affinities
to A aggregates varied from 3.0 to 135.2 nM in the binding studies.
Among them, two ligands with high affinities (Ki ¼ 6.4 3.2 nM for
b plaques successfully. These dibenzyli-
brain2 min/brain60
ratios of 3.90 (7.33
1.54% ID/g at 2 min,
min
1.88 0.14% ID/g at 60 min) [27], is still not ideal, so as the recently
reported 18F labeled 2-pyridinyl benzoxazole and benzothiazole
derivatives [36], with brain2 min/brain60 min ratios ranged from 1.77
to 4.95. However, these two tracers showed brain2 min/brain60 min
ratios of 21.6 and 13.4, respectively, indicating that these new type
of 18F-labeled dibenzylideneacetone derivatives without the PEG
chain may lower nonspecific white matter uptake. We also
observed that in vivo defluorination which is an annoying problem
with many 18F-labeled tracers was very slightly occurring with [18F]
8 and [18F]9, because the increase of the bone uptakes with time
were minimal, thus the interference of defluorination with the
imaging is expected to be relatively minor. Additionally, these 18F-
labeled tracers also distributed to several other organs like the liver
and the kidneys, However, their high initial uptakes were followed
by moderate washouts. In summary, these two 18F-labeled de-
rivatives ([18F]8 and [18F]9) meet the preliminary requirements for
b
8 and Ki ¼ 3.0 0.6 nM for 9) to the aggregated A
b were selected for
18F labeling and further evaluation. At first, the high affinities of
these two tracers were again confirmed in the in vitro autoradi-
ography on sections of postmortem AD brain and Tg mouse brain.
Then, in biodistribution experiments, both radiotracers [18F]8 and
[
18F]9 displayed high initial uptakes into (9.29
0.41 and
5.38 0.68% ID/g at 2 min, respectively) and very rapid washouts
from the brain in normal mice (brain2 min/brain60 min ratios of 21.6
and 13.4, respectively), indicating the pharmacokinetics had been
greatly improved by these 18F-labeled dibenzylideneacetone de-
rivatives without PEG chains. All the above results tell those two
components, 8 and 9, are highly desirable for Ab imaging because
they may have much lower nonspecific white matter uptake.
Furthermore, more than 90% of the activity was identified as the

Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
633
Fig. 6. (A, B) In vitro autoradiography of [18F]8 on a Tg model mouse (C57BL6, APPswe/PSEN1, 11 months old, male). (C, D) The presence and distribution of plaques were confirmed
by fluorescence staining using thioflavin-S on the same section with a filter set for GFP. (E) In vitro autoradiography of [18F]8 on a brain section of a age matched normal mouse as
control. (F) Fluorescence staining using thioflavin-S on the same section of control mouse.
intact tracer respectively after 60 min incubating with either saline
performed on silica gel (54e74 mm, Qingdao Haiyang Chemical Co.,
Ltd.). Radiochemical purity was evaluated by HPLC analysis per-
formed on a Shimadzu system SCL-20 AVP equipped with a SPD-
solution or mice plasma at 37 ꢀC, indicating that both [18F]8 and
[
18F]9 displayed high in vitro stability. Overall, [18F]8 and [18F]9 with
18F directly labeled on the benzene ring of dibenzylideneacetone
derivatives are promising PET agents for imaging cerebral A pla-
ques, and further evaluation in model animal and human subjects
20A UV detector (
3200 NaI/PMT -radiation scintillation detector. HPLC separations
were achieved on a Venusil MP C18 reverse phase column (Agela
Technologies, 5
l
¼ 254 nm) as well as a Bioscan Flow Count
b
g
with PET would provide valuable data for further A
nation and development.
b
tracer desig-
m
m, 10 mm ꢁ 250 mm) eluted with a binary
gradient system at a flow rate of 4.0 mL/min on the Shimadzu
HPLC; and HPLC analysis were achieved on a Venusil MP C18
reverse
phase
column
(Agela
Technologies,
5
mm,
4. Experimental
4.6 mm ꢁ 250 mm) eluted with a binary gradient system at a flow
rate of 1.0 mL/min also with the Shimadzu HPLC. Mobile phase A
was deionized water while mobile phase B was HPLC grade
acetonitrile purchased from Amethyst Chemicals. The purities of
the synthesized standard compounds (8 and 9) was determined
using analytical HPLC and was found to be higher than 98%. Fluo-
rescent observation was performed on the Observer Z1 (Zeiss,
Germany) equipped with a DAPI filter set (excitation, 375 nm) and a
GFP filter set (excitation, 488 nm). The normal ICR mice (five weeks,
male) we used for biodistribution experiments were purchased
from the Department of Laboratory Animal Science, Peking Uni-
versity Health Science Center. The Alzheimer's animal model, a
transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old) and an
age-matched control mouse (C57BL6, 11 months old) were pur-
chased from the Institute of Laboratory Animal Science, Chinese
Academy of Medical Sciences. All protocols in this project requiring
the use of mice were under approval by the animal care committee
4.1. General information
All the reagents we used in the synthesis were commercially
available and were used without further purification unless
otherwise indicated. The 1H NMR spectra and 13C NMR were ob-
tained at a 400 MHz on Bruker spectrometer with samples dis-
solved in CDCl3 solutions at room temperature. TMS was used as an
internal standard, and all the chemical shifts were reported as
d
values relative to the internal TMS. The coupling constants were
reported in Hertz. Multiplicity is defined by s (singlet), d (doublet), t
(triplet), and m (multiplet). Mass spectrometry was acquired under
the Surveyor MSQ Plus (ESI) (Waltham, MA, USA) instrument. The
synthesis reactions were carefully monitored by TLC (TLC Silica gel
60 F254 aluminum sheets, Merck), because compounds were all
visualized when illuminated with a short wavelength UV lamp
(l
¼
254 nm). Column chromatography purifications were

634
Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
Fig. 7. (A, B) In vitro autoradiography of [18F]9 on a Tg model mouse (C57BL6, APPswe/PSEN1, 11 months old, male). (C, D) The presence and distribution of plaques were confirmed
by fluorescence staining using thioflavin-S on the same section with a filter set for GFP. (E) In vitro autoradiography of [18F]9 on a brain section of a age matched normal mouse as
control. (F) Fluorescence staining using thioflavin-S on the same section of control mouse.
Fig. 8. (A) In vitro autoradiography of [18F]9 on an AD patient (64 years old, female) and (B) a normal person (74 years old, male) as control.
of Beijing Normal University. Post-mortem brain tissues from an AD
case (64 years old, female) and a control case (74 years old, male)
were obtained from the Chinese Brain Bank Center.
yellow solid (yield 85.0%). 1H NMR (CDCl3, 400 MHz):
d 4.07 (s, 2H),
7.68 (d, J ¼ 8.0 Hz, 2H), 6.87 (d, J ¼ 15.6 Hz, 1H), 7.00 (d, J ¼ 16.0 Hz,
1H), 7.09 (t, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 8.4 Hz, 2H), 7.59 (dd,
J1 ¼ 8.7 Hz, J2 ¼ 2.3 Hz, 2H), 7.67 (d, J ¼ 16.0 Hz, 1H), 7.68 (d,
J ¼ 15.6 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
d 114.9, 115.9, 116.2,
4.2. (1E, 4E)-1-(4-Aminophenyl)-5-(4-fluorophenyl)penta-1,4-
dien-3-one (7)
121.6, 124.9, 125.5, 130.1, 130.5, 141.0, 144.1, 149.2, 162.6, 165.1, 188.7.
HRMS (TOF MS ESþ): m/z calcd for C17H15NOFþ 268.1132 [MþH]þ;
found 268.1139.
Dissolve compound
4
(161 mg,
1
mmol) and 4-
fluorobenzaldehyde (124 mg, 1 mmol) in 5 mL ethanol and add
NaOH (40 mg, 1 mmol). The solution was stirred for 10 min at room
temperature, and yellow solids precipitated from the solution. Keep
the reaction for another 1 h. Final product was collected by filtra-
tion and washing by petroleum ether. The residue was purified by
chromatography (ethylacetate/petroleum ether ¼ 1:1, v/v) to afford
4.3. (1E, 4E)-1-(4-Fluorophenyl)-5-(4-(methylamino)phenyl)
penta-1,4-dien-3-one (8)
Compound 8 was prepared following the procedure used for 7.
The residue was purified by chromatography (ethylacetate/

Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
635
Table 2
yellow solids precipitated from the solution. Keep the reaction for
another 1 h. Final product was collected by filtration and washing
by petroleum ether. The residue was purified by silica gel chro-
matography (ethylacetate/petroleum ether ¼ 1:1, v/v) to afford
Biodistribution of [18F]8 in male ICR mice.a
Organ
Time after injection
2 min
10 min
30 min
60 min
yellow solid (yield 78.3%). 1H NMR (CDCl3, 400 MHz):
d 4.07 (s, 2H),
Blood
Brain
Heart
Liver
Spleen
Lung
5.08 1.49
9.29 0.41
8.57 0.67
3.75 0.81
1.65 0.23
2.97 0.34
3.71 1.24
0.56 0.06
1.77 0.28
2.53 0.84
0.43 0.14
1.27 0.31
6.68 (d, J ¼ 8.4 Hz, 2H), 6.87 (d, J ¼ 15.8 Hz, 1H), 7.00 (dd, J1 ¼8.5 Hz,
J2 ¼ 2.7 Hz, 1H), 7.07 (d, J ¼ 15.9 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.68
(t, J ¼ 15.3 Hz, 2H), 8.01e8.06 (m, 1H), 8.43 (s, 1H). 13C NMR (CDCl3,
15.72 1.21 21.29 1.90 15.26 1.94 10.74 0.80
4.29 0.72
2.20 0.28
1.64 0.30
8.99 1.93
1.27 0.38
6.32 0.99
100 MHz): d 111.7,114.9,121.6,124.7,130.5,130.6,136.5,138.8,144.0,
148.8, 149.2, 165.1, 188.5. HRMS (TOF MS ESþ): m/z calcd for
23.90 3.21 12.90 1.63
17.19 1.11 18.19 4.68 16.07 4.42 12.57 0.58
Kidney
Pancreas
Bone
C
16H13N2OFþ 269.1085 [MþH]þ; found 269.1107.
9.95 0.39
1.87 0.33
2.93 0.48
3.49 0.54
0.42 0.08
5.17 0.55
2.12 0.28
1.30 0.70
5.22 1.19
1.43 0.39
2.20 0.72
2.28 0.63
Stomachb
Small intestineb
4.6. (1E, 4E)-1-(6-Fluoropyridin-3-yl)-5-(4-(methylamino)phenyl)
penta-1,4-dien-3-one (11)
9.74 1.01 10.25 5.62 31.22 5.02 55.10 10.58
a
Expressed as % injected dose per gram. Average for 5 mice standard deviation.
Expressed as % injected dose per organ.
b
Compound 11 was prepared following the procedure used for
10. The residue was purified by chromatography (ethylacetate/pe-
troleum ether ¼ 1:2, v/v) to afford yellow solid (yield 83.2%). 1H
Table 3
NMR (CDCl3, 400 MHz):
d
2.90 (s, 3H), 4.24 (s, 1H), 6.60 (d, J ¼ 8.4
Biodistribution of [18F]9 in male ICR mice.a
Hz, 2H), 6.84 (d, J ¼ 15.7 Hz, 1H), 6.99 (dd, J1 ¼ 8.5 Hz, J2 ¼ 2.6 Hz,
1H), 7.08 (d, J ¼ 15.9 Hz, 1H), 7.48 (d, J ¼ 8.4 Hz, 2H), 7.66 (d,
J ¼ 15.9 Hz, 1H), 7.72 (d, J ¼ 15.8 Hz, 1H), 8.01e8.05 (m, 1H), 8.42 (s,
Organ
Time after injection
2 min
10 min
30 min
60 min
1H). 13C NMR (CDCl3, 100 MHz):
d 30.3, 109.9, 112.3, 123.3, 127.3,
Blood
Brain
Heart
Liver
Spleen
Lung
6.89 1.52
5.38 0.68
9.86 1.81
4.44 0.77
1.27 0.14
2.65 0.23
5.56 0.91
0.55 0.09
1.98 0.58
6.94 0.89
1.16 0.32
3.79 1.00
2.80 0.42
0.40 0.07
1.16 0.18
7.94 0.11
0.61 0.18
2.51 0.35
130.3, 130.7, 136.6, 139.4, 145.2, 148.2, 151.7, 165.1, 188.1. HRMS (TOF
MS ESþ): m/z calcd for C17H15N2OFþ 283.1241 [MþH]þ; found
283.1260.
16.32 4.34 15.37 0.58
3.32 0.43
8.96 1.74
1.47 0.19
4.82 0.07
4.7. (1E, 4E)-1-(4-(Dimethylamino)phenyl)-5-(6-fluoropyridin-3-
yl)penta-1,4-dien-3-one (12)
Kidney
Pancreas
Bone
17.02 1.00 13.96 2.38 15.98 3.64 12.23 4.31
7.16 0.86
2.42 0.51
2.04 0.25
6.52 2.77
2.83 0.48
1.23 0.49
2.54 0.46
2.40 0.33
0.92 0.46
3.11 0.73
1.08 0.16
1.09 0.27
2.28 0.13
Stomachb
Small intestineb
Compound 12 was prepared following the procedure used for
10. The residue was purified by silica gel chromatography (ethyl-
acetate/petroleum ether ¼ 1:3, v/v) to afford yellow solid (yield
7.41 2.83 12.67 1.76 31.18 9.47
a
Expressed as % injected dose per gram. Average for 5 mice standard deviation.
Expressed as % injected dose per organ.
b
88.9%). 1H NMR (CDCl3, 400 MHz):
d
3.07 (s, 6H), 6.73 (d, J ¼ 7.9 Hz,
2H), 6.86 (d, J ¼ 15.7 Hz, 1H), 7.01 (dd, J1 ¼ 8.5 Hz, J2 ¼ 2.8 Hz, 1H),
7.10 (d, J ¼ 15.9 Hz, 1H), 7.54 (d, J ¼ 8.8 Hz, 2H), 7.67 (d, J ¼ 15.9 Hz,
1H), 7.75 (d, J ¼ 15.7 Hz, 1H), 8.03e8.07 (m, 1H), 8.44 (s, 1H). 13C
petroleum ether ¼ 1:2, v/v) to afford yellow solid (yield 87.6%). 1H
NMR (CDCl3, 400 MHz):
d
2.90 (s, 3H), 6.60 (d, J ¼ 8.4 Hz, 2H), 6.85
NMR (CDCl3, 100 MHz): d 40.1, 111.6, 111.9, 120.8, 122.5, 124.4, 124.7,
(d, J ¼ 15.6 Hz, 1H), 7.01 (d, J ¼ 16.0 Hz, 1H), 7.09 (t, J ¼ 8.6 Hz, 2H),
7.48 (d, J ¼ 8.8 Hz, 2H), 7.60 (dd, J1 ¼ 8.8 Hz, J2 ¼ 5.6 Hz, 2H), 7.67 (d,
J ¼ 15.6 Hz, 1H), 7.70 (d, J ¼ 15.60 Hz, 1H). 13C NMR (CDCl3,
130.4, 136.5, 138.4, 144.3, 148.7, 165.0, 188.4. HRMS (TOF MS ESþ):
m/z calcd for C18H17N2OFþ 297.1398 [MþH]þ; found 297.1431.
100 MHz): d 30.3, 112.2, 115.9, 116.1, 120.9, 123.5, 125.5, 130.1, 130.6,
140.7, 144.5, 151.5, 162.8, 164.8, 188.7. HRMS (TOF MS ESþ): m/z
calcd for C18H17NOFþ 282.1289 [MþH]þ; found 282.1291.
4.8. (1E, 4E)-1-(4-aminophenyl)-5-(6-ethoxypyridin-3-yl)penta-
1,4-dien-3-one (13)
Compound 13 was prepared following the procedure used for 7.
The residue was purified by silica gel chromatography (ethyl-
acetate/petroleum ether ¼ 1:1, v/v) to afford yellow solid (yield
4.4. (1E, 4E)-1-(4-(Dimethylamino)phenyl)-5-(4-fluorophenyl)
penta-1,4-dien-3-one (9)
73.6%). 1H NMR (CDCl3, 400 MHz):
d
1.41 (t, J ¼ 7.1 Hz, 3H), 4.06 (s,
Compound 9 was prepared following the procedure used for 7.
The residue was purified by chromatography (ethylacetate/petro-
leum ether ¼ 1:3, v/v) to afford yellow solid (yield 90.5%). 1H NMR
2H), 4.40 (dd, J1 ¼ 14.1 Hz, J2 ¼ 7.0 Hz, 2H), 6.67 (d, J ¼ 8.4 Hz, 2H),
6.76 (d, J ¼ 8.7 Hz, 1H), 6.87 (d, J ¼ 15.8 Hz, 1H), 6.96 (d, J ¼ 15.9 Hz,
1H), 7.44 (d, J ¼ 8.5 Hz, 2H), 7.65 (d, J ¼ 15.8 Hz, 1H), 7.67 (d,
J ¼ 15.8 Hz, 1H), 7.85 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.4 Hz, 1H), 8.32 (d,
(CDCl3, 400 MHz):
d
3.06 (s, 3H), 6.72 (d, J ¼ 8.9 Hz, 2H), 6.88 (d,
J ¼ 15.7 Hz, 1H), 7.04 (d, J ¼ 15.9 Hz, 1H), 7.11 (t, J ¼ 8.6 Hz, 2H), 7.54
(d, J ¼ 8.9 Hz, 2H), 7.62 (dd, J1 ¼ 8.7 Hz, J2 ¼ 5.4 Hz, 2H), 7.69 (d,
J ¼ 15.9 Hz, 1H), 7.74 (d, J ¼ 15.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
J ¼ 2.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
d 14.6, 62.3, 111.7, 114.9,
121.5, 124.7, 130.2, 130.4, 130.5, 136.5, 138.8, 144.1, 148.8, 149.2,
165.0, 188.5. HRMS (TOF MS ESþ): m/z calcd for C18H19N2Oþ2
295.1441 [MþH]þ; found 295.1436.
d
40.1, 111.9, 115.9, 116.1, 120.9,122.5,125.6, 130.1, 130.5, 140.6, 144.4,
152.1, 162.6, 165.1, 188.6. HRMS (TOF MS ESþ): m/z calcd for
C
19H19NOFþ 296.1445 [MþH]þ; found C19H19NOFþ 296.1450.
4.9. (1E, 4E)-1-(6-ethoxypyridin-3-yl)-5-(4-(methylamino)phenyl)
penta-1,4-dien-3-one (14)
4.5. (1E, 4E)-1-(4-Aminophenyl)-5-(6-fluoropyridin-3-yl)penta-
1,4-dien-3-one (10)
Compound 14 was prepared following the procedure used for 7.
The residue was purified by silica gel chromatography (ethyl-
acetate/petroleum ether ¼ 1:2, v/v) to afford yellow solid (yield
Dissolve compound 4 (161 mg, 1 mmol) and 6-fluoropyridine-3-
carbaldehyde (125 mg,1 mmol) in 5 mL DMF and add NaOH (40 mg,
1 mmol). The solution was stirred for 1 h at room temperature, and
83.4%). 1H NMR (CDCl3, 400 MHz):
d
1.41 (t, J ¼ 7.0 Hz, 3H), 2.89 (s,
3H), 4.40 (dd, J1 ¼ 14.1 Hz, J2 ¼ 7.0 Hz, 2H), 6.59 (d, J ¼ 8.6 Hz, 2H),

636
Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
6.76 (d, J ¼ 8.7 Hz, 1H), 6.84 (d, J ¼ 15.8 Hz, 1H), 6.97 (d, J ¼ 15.8 Hz,
1H), 7.47 (d, J ¼ 8.6 Hz, 2H), 7.69 (d, J ¼ 16.0 Hz, 1H), 7.65 (d,
J ¼ 16.0 Hz, 1H), 7.84 (dd, J1 ¼ 8.6 Hz, J2 ¼ 2.4 Hz, 1H), 8.32 (d,
Inhibition experiments were carried out according to procedures
described previously [37] with some modification. To be brief,
100
was added to a mixture which contains 100
IMPY, 100,000 cpm/100 L), 100
L of inhibitors (10ꢂ4e10ꢂ8.5 M in
ethanol), and 700
L of PBS (0.2 M, pH ¼ 7.4) in a final volume of
mL of aggregated A
b
fibrils (28 nM in the final assay mixture)
J ¼ 2.1 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
d
14.6, 30.3, 62.3, 111.6,
m
L of radioligand ([125I]
112.2, 120.8, 123.6, 124.3, 124.7, 130.6, 136.5, 138.5, 144.4, 148.7,
151.5, 165.0, 188.5. HRMS (TOF MS ESþ): m/z calcd for C19H21N2Oþ2
309.1603 [MþH]þ; found 309.1542.
m
m
m
1 mL in 12 mm ꢁ 75 mm borosilicate glass tubes. The mixture was
incubated at room temperature for 3 h, and then, the bound and
free radioactive fractions were separated by vacuum filtration
through borosilicate glass fiber filters (Whatman GF/B) performed
by a Mp-48T cell harvester (Brandel, Gaithersburg, MD). The
radioactivity caught by the filters containing the bound 125I-ligand
4.10. (1E, 4E)-1-(4-(dimethylamino)phenyl)-5-(6-ethoxypyridin-3-
yl)penta-1,4-dien-3-one (15)
Compound 15 was prepared following the procedure used for 7.
The residue was purified by silica gel chromatography (ethyl-
acetate/petroleum ether ¼ 1:3, v/v) to afford yellow solid (yield
was measured by a g-counter (WALLAC/Wizard1470, United States)
with about 70% efficiency. Under the assay conditions, the specif-
ically bound fraction accounted for about 10% of total radioactivity.
Nonspecific binding was defined in the presence of IMPY (1 mM) in
the same way. The inhibitory concentration (IC50) was determined
using Graph Pad Prism 4.0 software. The inhibition constant (Ki)
was calculated according to the Cheng-Prusoff inhibition constant
equation: Ki ¼ IC50/(1þ[L]/Kd).
85.8%). 1H NMR (CDCl3, 400 MHz):
d
1.40 (t, J ¼ 7.1 Hz, 3H), 3.02 (s,
6H), 4.40 (dd, J1 ¼ 14.2 Hz, J2 ¼ 7.1 Hz, 2H), 6.67 (d, J ¼ 8.8 Hz, 2H),
6.75 (d, J ¼ 8.6 Hz, 1H), 6.84 (d, J ¼ 15.7 Hz, 1H), 6.97 (d, J ¼ 15.8 Hz,
1H), 7.50 (d, J ¼ 8.8 Hz, 2H), 7.64 (d, J ¼ 15.9 Hz, 1H), 7.70 (d,
J ¼ 15.7 Hz, 1H), 7.84 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.3 Hz, 1H), 8.32 (d,
J ¼ 2.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz):
d 14.6, 40.1, 62.2, 111.6,
111.9, 120.7, 122.4, 124.4, 124.7, 130.4, 136.4, 138.4, 144.3, 148.7,
152.0, 165.0, 188.4. MS (TOF MS ESþ): m/z calcd for C20H23N2Oþ2
323.1760 [MþH]þ; found 323.1804.
4.14. Radiolabeling
[
18F]Fluoride trapped on a QMA Light cartridge (Waters) was
4.11. 4-(N, N, N-trimethylamino)benzaldehyde triflate (16)
eluted with 1 mL of Kryptofix222/K2CO3 solution (13 mg of Kryp-
tofix222 and 1.1 mg of K2CO3 in CH3CN/H2O, 4: 1 (v/v) into a clean
glass vial. The solvent was quickly removed by heating at 120 ꢀC
under a stream of nitrogen gas. Then, the residue was azeotropi-
cally dried with 1 mL of anhydrous acetonitrile by three times
(about 0.3 mL for each) at 120 ꢀC under a stream of nitrogen gas. 4-
(N, N, N-trimethylamino)benzaldehyde triflate (5.0 mg) dissolved
in 1.0 mL of dry DMSO was added to the reaction vessel containing
the 18Fꢂ activity and heated at 160 ꢀC for 6 min. After the reaction
vial was cooled by running cold water to room temperature,
10.0 mL of water were added to this orange solution. We passed the
mixture with a syringe through a Sep-Pak C18 cartridge (Agela
Technologies). The cartridge was washed with 10 mL of water, and
the intermediate [18F]-4-fluorobenzaldehyde was eluted with 1 mL
of EtOH the [18F]-4-fluorobenzaldehyde was washed out by 1 mL
EtOH. The appropriate ketones (5 or 6, 1.0 mg) were added into the
EtOH solution containing [18F]-4-fluorobenzaldehyde, as well as
4.0 mg of NaOH to catalyze the Claisen condensation, the mixture
was kept for 30 min with gentle shaking under room temperature.
The longer the reaction was, the higher the condensation yields are
found. But taking the decay into consideration, 30 min is better. The
solvent was evaporated under nitrogen gas and the residue was
Dissolve 4-dimethylaminobenzaldehyde (124 mg, 1 mmol) in
5 mL CH2Cl2 and add methyl trifluoromethanesulfonate (150 mL,
1.2 mmol) dropwise in ice salt bath. The solution was stirred for 3 h
in the ice salt bath, and white solid precipitated from the solution.
After filtration, washing with CH2Cl2, 75.7 mg white solid was ob-
tained (yield 24.2%). 1H NMR (D2O, 400 MHz):
d
3.66 (s, 9H), 8.02 (d,
J ¼ 9.0 Hz, 2H), 8.13 (d, J ¼ 9.0 Hz, 2H), 9.99 (s, 1H).
4.12. In vitro fluorescent staining of Ab plaques on transgenic
mouse brain sections
Before use, paraffin-embedded brain sections of Tg mouse
(C57BL6, APPswe/PSEN1, 11 months old, male, 6 m) were depar-
m
affinized according to the following procedure: 2 ꢁ 20 min washes
with xylene, 2 ꢁ 5 min washes with 100% ethanol, 5 min washes
with 90% ethanol/H2O, 5 min wash with 80% ethanol/H2O, 5 min
wash with 60% ethanol/H2O, running tap water for 10 min, and
then incubated in PBS buffer (0.2 M, pH ¼ 7.4) for 30 min. The brain
sections were then incubated with 10% (v/v) ethanol solution of
7e12 (1
mM) for 10 min at room temperature just before examining
by microscope. The localization and patterns of A
b
plaques was
dissolved in HPLC eluent and neutralized with 6
mL acetic acid
confirmed by staining with thioflavin-S on the adjacent sections as
contrast. For the staining of thioflavin-S, the deparaffinized sections
were immersed in a thioflavin-S solution (0.125%, w/w) containing
10% EtOH (v/v) for 3 min and then washed with 40% EtOH. Finally,
before examining by the microscope, the sections were washed
again with 40% ethanol and PBS (0.2 M, pH ¼ 7.4) for 10 min.
Fluorescent observation was performed by microscope Observer Z1
(Zeiss, Germany) equipped with DAPI (excitation, 375 nm) and GFP
filter sets (excitation, 488 nm).
before subjecting to HPLC for purification (Agela Technologies,
5
m
m, 10 mm ꢁ 250 mm, CH3CN/H2O ¼ 70/30; flow rate ¼ 4 mL/
min). After purification by HPLC, the radiochemical purity of [18F]8
and [18F]9 was greater than 95%. The retention time of [18F]8 and
[
18F]9 were 11.05 and 17.02 min in this HPLC system, respectively.
The preparation took 90 min and the total radiochemical yield was
about 25% (at the end of synthesis, no decay corrected).
4.15. In vitro autoradiography on brain sections of human and
transgenic model mouse
4.13. In vitro binding assay to Ab aggregates
The paraffin-embedded sections of human and transgenic
model mouse brain were deparaffinized first with 2 ꢁ 20 min
washes in xylene; then 2 ꢁ 5 min washes in 100% ethanol; 5 min
wash in 90% ethanol/H2O; 5 min wash in 80% ethanol/H2O; 5 min
wash in 60% ethanol/H2O and finally 10 min wash in running tap
water before incubating in PBS (0.2 M, pH ¼ 7.4) for 30 min. The
sections were then incubated with [18F]8 and [18F]9 (1.85 MBq/
The trifluoroacetic acid salt forms of peptides Ab1e42 were
purchased from Osaka Peptide Institute (Osaka, Japan). Aggregation
reaction was carried out by first gently dissolving the peptide
(0.56 mg/mL for Ab1e42) in a buffer solution (pH ¼ 7.4) which
contained 10 mM sodium phosphate and 1 mM EDTA. The solution
was incubated with gentle and constant shaking at 37 ꢀC for 42 h.

Z. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 628e638
637
200
mL) solution in saline for 1 h at room temperature. After they
Appendix A. Supplementary data
were rinsed with water for 1 min, washed with 40% EtOH, and dried
with blowing air, the sections were exposed to a phosphorus plate
(Perkin Elmer, USA) for 2 h in dark. In vivo autoradiographic images
were obtained by a phosphor imaging system (Cyclone, Packard).
After the in vitro autoradiographic examination, the same mouse
brain sections were stained again by thioflavin-S solution the same
way in in vitro fluorescent staining to confirm the presence and
pattern of Ab plaques. The fluorescent observation was performed
on the Observer Z1 (Zeiss, Germany) equipped with GFP filter sets
(excitation, 488 nm).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.070.
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[
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m
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This work was funded by National Natural Science Foundation of
China (No: 21201019 and 81371593), Research Fund for the
Doctoral Program of Higher Education of China (No:
20120003120013) and Fundamental Research Funds for the Central
Universities (No: 2012LYB19). The authors present special thanks to
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