Synthesis and evaluation of styrylpyran fluorophores for noninvasive detection of cerebral β-amyloid deposits

Article abstract of DOI:10.1016/j.bmc.2016.01.004

The development of amyloid-specific fluorophores allows the visualization of cerebral β-amyloid deposits using optical imaging technology. In the present study, a series of smart styrylpyran fluorophores with compact donor-acceptor architecture were designed and evaluated for noninvasive detection of cerebral β-amyloid deposits. Spectral behavior of the fluorophores changed significantly (optical turn-on) upon binding to β-amyloid aggregates. Computational studies were conducted to correlate the experimental K_d values with calculated binding energies, speculating the relationship between fluorophore structure and β-amyloid affinity. In vivo studies demonstrated that PAD-2 could discriminate APP/PS1 transgenic mice from wild type controls, with specific labeling of cerebral β-amyloid deposits confirmed by ex vivo observation. Collectively, these styrylpyran fluorophores could provide a new scaffold for the development of optical imaging probes targeting cerebral β-amyloid deposits.

Full text of DOI:10.1016/j.bmc.2016.01.004

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of styrylpyran fluorophores for noninvasive
detection of cerebral b-amyloid deposits
a
a,
⇑
b
b
a
Bi-yue Zhu , Yan Cheng , Guo-bo Li , Sheng-yong Yang , Zhi-rong Zhang
a
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of amyloid-specific fluorophores allows the visualization of cerebral b-amyloid deposits
using optical imaging technology. In the present study, a series of smart styrylpyran fluorophores with
compact donor–acceptor architecture were designed and evaluated for noninvasive detection of cerebral
b-amyloid deposits. Spectral behavior of the fluorophores changed significantly (optical turn-on) upon
binding to b-amyloid aggregates. Computational studies were conducted to correlate the experimental
Received 16 October 2015
Revised 3 December 2015
Accepted 5 January 2016
Available online xxxx
d
K values with calculated binding energies, speculating the relationship between fluorophore structure
Keywords:
and b-amyloid affinity. In vivo studies demonstrated that PAD-2 could discriminate APP/PS1 transgenic
mice from wild type controls, with specific labeling of cerebral b-amyloid deposits confirmed by ex vivo
observation. Collectively, these styrylpyran fluorophores could provide a new scaffold for the develop-
ment of optical imaging probes targeting cerebral b-amyloid deposits.
b-Amyloid deposits
Optical imaging
Fluorophore
Styrylpyran
Ó 2016 Published by Elsevier Ltd.
1
. Introduction
Alzheimer’s disease (AD) is the most common progressive neu-
biological research, would be an attractive alternative to PET imag-
ing of b-amyloid deposits in animal models.
To visualize cerebral b-amyloid deposits by in vivo optical
rodegenerative disease that poses an increasingly prevalent global
health care challenge. The lack of effective therapeutics has been
attributed to unspecified etiology of AD and late-stage administra-
tion. Cerebral b-amyloid deposits, composed of extracellular aggre-
gated b-amyloid peptides, have been widely viewed as an
important pathological hallmark of AD.1 As b-amyloid deposition
in the brain occurs prior to the earliest clinical symptoms, monitor-
ing cerebral b-amyloid deposits in vivo would facilitate the eluci-
dation of AD pathogenesis, prediction of AD progression, and
evaluation of the efficacy of anti-amyloid therapeutics.
imaging, fluorescence probes are required to obtain high target-
1
7,18
to-background ratio.
Congo red (CR) and thioflavin T (ThT)
are two well-known fluorescent dyes for b-amyloid deposits, pro-
viding most standardized way of post mortem histological analysis
of AD brains; however, the bulky and charged molecules are not
capable of penetrating blood brain barrier, which is critical for
in vivo cerebral imaging. To detect cerebral b-amyloid deposits
in vivo, design criteria of fluorescence probes are more stringent,
including: rapid uptake in the brain (small molecular size with
suitable lipophilicity), specific labeling of b-amyloid deposits
(high-affinity binding), as well as substantial changes in fluores-
cence properties of bound forms of the probe (large Stokes shift,
and change in fluorescence intensity). In the past few years, several
advances have been made in the development of noninvasive fluo-
–3
Neuroimaging techniques present immense potential for visual-
izing b-amyloid deposits in living brains.4,5 Over the past few years,
positron emission tomography (PET) imaging of cerebral b-amyloid
deposits using amyloid-specific probes has been most widely stud-
6
–16
19
17
20
ied.
Despite its tremendous success in human studies, the use
rescence probes, such as AOI987, NIAD-4, THK-265, DANIR-
2
1
22,23
of PET imaging in preclinical animal models for AD remains con-
strained by considerations of limited availabilities of radioisotopes
and complexity of experimental procedures in radiolabeling and
data acquisition. In contrast, simple and convenient optical imag-
ing technique, which has been one of the most important tools in
2c,
CRANAD series (CRANAD-2, CRANAD-58),
ANCA ser
2
4,25
26–28
ies,
and BODIPY series (BODIPY-7, BAP-1, BAP-2).
Although most of the probes are proven to show selective labeling
of b-amyloid deposits in vivo, the intrinsic properties (short emis-
sion wavelength, moderate amyloid affinity, low washout from the
brain, no bathochromic shift) may limit the potential use of these
reported probes. More recently, fluorescence probes with push–
pull architecture, such as BBTOM-3, DANIR-3b, and DTM-2 have
been developed with improved emission wavelengths and proven
⇑
Corresponding author. Tel.: +86 28 85501566; fax: +86 28 85501615.
E-mail address: yancheng@scu.edu.cn (Y. Cheng).
http://dx.doi.org/10.1016/j.bmc.2016.01.004
968-0896/Ó 2016 Published by Elsevier Ltd.
0
Please cite this article in press as: Zhu, B.-y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.004

2
B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
to be efficient for the discrimination between transgenic and wild-
derivatives showed emission wavelengths beyond 600 nm in
dichloromethane, which were much longer than that of PAD-1
(571 nm) (Table 1). PAD-2, PAD-4, and PAD-6 with lengthened
vinyl chains showed obvious emission redshift, compared to the
emission spectra of PAD-1, PAD-3, and PAD-5, respectively. PAD-
2 to PAD-5 exhibited good fluorescence quantum yields, which
2
9–31
type mice in vivo.
Although BBTOM-3 and DTM-2 were
reported as promising fluorescence probes for cerebral b-amyloid
deposits, the low fluorescence quantum yields may limit spatial
resolution for in vivo imaging. Moreover, DTM-2 exhibited moder-
ate affinity for b-amyloid aggregates. DANIR-3b displayed high
sensitivity in detecting b-amyloid deposits and could differentiate
transgenic mice from wild-type controls at an early time postinjec-
tion, which is beneficial for high-throughout screening of anti-
amyloid therapies.
3
4
were considerably higher than that of ThT (0.01). Besides, excel-
lent fluorescence response was observed upon mixing with aggre-
gated b-amyloid fibrils: PAD-2, PAD-3, and PAD-4 showed brighter
fluorescence (23, 15, and 17 fold increase in fluorescence intensity
respectively) than that of PAD-1 (7 fold), accompanied by signifi-
Recently, we have reported styrylpyran derivative PAD-1 as a
potential fluorescence probe targeting cerebral b-amyloid deposits.
3
5
cant blue shift in emission. This may be caused by the restriction
The fluorophore contains a conjugated
p
system with electron-
of conformational freedom upon binding to b-amyloid fibrils,
1
7
donor (D) and electron-acceptor (A) groups as terminal moieties
decreasing the vibrational–rotational processes. No significant
change in fluorescence was observed during incubating PAD-5
(or PAD-6) with b-amyloid fibrils, indicating that there is little or
no interaction.
3
2
(
Fig. 1). The fluorophore displayed good binding affinity for b-
amyloid fibrils, indicating that styrylpyran may serve as a new
scaffold for the development of fluorescence probes for visualizing
b-amyloid deposits in vivo. In the present study, a series of styryl-
pyran derivatives were designed and evaluated to study the bind-
ing affinity for cerebral b-amyloid deposits and kinetics in the
brain. Herein we report the biological evaluation and structure–ac-
tivity relationship of these styrylpyran derivatives as potential flu-
orescence probes for noninvasive detection of cerebral b-amyloid
deposits.
2.3. Binding ability for b-amyloid deposits
To investigate the specificity and affinity for b-amyloid deposits,
fluorescence staining was performed using brain slices from a 12-
month-old APP/PS1 transgenic mouse. APP/PS1 transgenic mice
have been commonly used for in vitro and in vivo evaluations of
b-amyloid specific probes because of marked cerebral b-amyloid
3
6
deposition. As shown in Figure 2, microscopic images of brain
slices with PAD-2, PAD-3, and PAD-4 staining showed extensive
fluorescent labeling of b-amyloid deposits, while brain images with
PAD-5 and PAD-6 staining displayed no specific labeling. The dis-
tribution of b-amyloid deposits was confirmed by staining of the
adjacent brain slices with thioflavin S (ThS), which is a common
2
2
. Results and discussion
.1. Chemistry
Molecular design of styrylpyran derivatives was aimed to
obtain compact amyloid-specific fluorophores with suitable emis-
sion wavelengths. Dimethylamino and cyano group have been
widely used as optimal D and A group, respectively, and were
selected for the molecular design. Donor–acceptor (D–A) and
3
7
dye for b-amyloid deposits. Despite of its common use for histo-
logical demonstration of b-amyloid fibrils, ThS is not suitable for
quantitative analysis: ThS is a complex mixture of methylated, sul-
fonated polymerized primulin preparation with uncharacterized
structure; it exhibits brighter fluorescence without changes in
the excitation or emission spectra upon binding to b-amyloid fib-
donor–acceptor–donor (D–A–D) architecture with extended
p con-
jugated bridges were designed to reduce the HOMO–LUMO gap for
desired long emission wavelength to avoid autofluorescence. The
key step in the formation of styrylpyran backbone was achieved
by the condensation of pyran or benzopyran with appropriate aro-
matic aldehydes (Scheme 1).33
3
8
rils, resulting in high background fluorescence. Saturation assay
was then performed to quantify the binding affinity (Table 2).
The affinity for aggregated b-amyloid fibrils varied: PAD-2 and
PAD-4 showed higher affinity than that of PAD-1, with dissociation
constant (K
played moderate affinity with K
and PAD-6 showed very poor affinity with K
These quantitative data were in accordance with fluorescent stain-
ing study.
d
) values of 39.7 and 26.6 nM respectively; PAD-3 dis-
value of 94.0 nM, while PAD-5
values over 10,000.
2
.2. Spectroscopic profile
d
Long emission wavelengths and high fluorescence quantum
d
yields are crucial for in vivo cerebral imaging with sufficient spatial
integration of fluorescence intensity.30 As expected, all of the
Figure 1. Chemical structure of styrylpyran derivatives.
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B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
NC
CN
O
O
a
O
1
NC
CN
NC
CN
OHC
b
+
N
O
O
N
PAD-2
NC
CN
NC
CN
OHC
b
m
+
O
N
n
O
m = 0, 1
PAD-3: n = 1
PAD-4: n = 2
N
NC
CN
NC
CN
OHC
m
b
+
O
N
n
n
O
m = 0, 1
N
N
PAD-5: n = 1
PAD-6: n = 2
Scheme 1. Reagents and conditions: (a) malononitrile, acetic anhydride, 140 °C; (b) piperidine, acetonitrile, reflux.
Table 1
Fluorescence profile of styrylpyran derivatives
LogPa
k
abs (nm)
b
e
b
(M cm
ꢀ1
ꢀ1
)
k
ex (nm)
b
k
em (nm)
b
U
F
b
Fluorescence increase foldc
Emission blueshiftc
Compd
Mw
PAD-1
PAD-2
PAD-3
PAD-4
PAD-5
PAD-6
ThTd
303
329
339
365
434
486
319
2.06
2.58
3.79
4.31
4.26
5.29
/
467
487
515
523
485
502
432
43,180
39,860
67,244
68,516
75,530
44,440
/
478
492
519
524
492
507
/
571
636
646
657
613
661
504
0.48
0.29
0.19
0.10
0.64
0.02
0.01
7
46
50
50
40
/
23
15
17
/
/
/
/
1000
a
LogP values were calculated using ChemDraw Ultra 8.0.
b
Maximum absorbance wavelength (kabs), molar absorption coefficient (
) measured in CH Cl
M Ab1–42 aggregates.
e), maximum excitation wavelength (kex), maximum emission wavelength (kem), and fluorescence
quantum yield (
U
F
2
2
.
c
Data measured in PBS with 2.75
Data from Ref. 34.
l
d
2
.4. Molecular docking analysis
To get insight into possible interaction mechanism for b-amy-
For PAD-5 or PAD-6, the molecule is partially inserted into the
binding pocket. The binding modes of these compounds revealed
that hydrophobic interactions and hydrogen bonds are important
contributions for their binding affinity, and the hydrophobic
microenvironment with a lower dielectric constant may lead to
an increase in fluorescence intensity and blue shift in emission.
Besides, small and nearly planar molecules (PAD-1 to PAD-4)
insert into the binding site easily, while large stereo–hindrance
hampers the interaction with b-amyloid fibril (PAD-5 and PAD-6).
loid binding, molecular docking analysis was carried out using
GOLD software on 2-fold Ab1–40 fibril structure (PDB ID:
3
9–41
2
LMO).
Ab1-42 shares the same residues as Ab1–40 with only
one difference of two extra residues at the C-terminus. GoldScore
was used as the scoring function for binding affinity prediction
(Table 2). Since GoldScore yielded excellent correlation with the
experimental binding affinity data, the interaction of these deriva-
tives with active site residues was studied using the top-ranked
binding pose of each compound from GoldScore (Fig. 3). For
PAD-1 or PAD-2, both donor and acceptor parts of the molecule
are located within the hydrophobic binding pocket of the aggre-
gated Ab1–40 fibril: the benzene ring forms hydrophobic interaction
with PHE19, and N-methyl group forms hydrophobic interactions
with PHE19 and ALA21; the cyano group has a hydrogen-bonding
interaction with ILE31. For PAD-3 or PAD-4, the benzene ring of
benzopyran forms hydrophobic interaction with PHE19, and the
cyano group forms a hydrogen-bonding interaction with ILE31.
2.5. Brain uptake
Permeability of blood–brain barrier (BBB) is crucial for in vivo
imaging of cerebral b-amyloid deposits. To investigate BBB pene-
tration, brain uptake of high-affinity compounds (PAD-2, -3, and
-4) were evaluated in normal mice. The compounds have small
molecules (<400 Da) and suitable lipophilicity (calculated LogP
values between 2 and 5), indicating that they could potentially
5
cross BBB by passive diffusion (Table 1). As expected, all com-
pounds penetrated BBB rapidly and washed out from the brain
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B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 2. Neuropathological staining of brain slices from an APP/PS1 transgenic mouse (male, 12-month-old). (a) Staining with styrylpyran derivatives (upper). (b) Adjacent
slices were stained with thioflavin S to confirm the presence of b-amyloid plaques (below).
Table 2
Dissociation constant (K
d
) and GoldScore of styrylpyran derivatives
limited BBB permeability and high autofluorescence from biologi-
cal matter during in vivo imaging, making PAD-2 a potentially use-
ful fluorescence probe for noninvasive detection of cerebral b-
amyloid deposits.
a
Compd
K
d
(nM)
GoldScore
PAD-1
PAD-2
PAD-3
PAD-4
PAD-5
PAD-6
58.9 ± 10.2
39.7 ± 9.1
94.0 ± 20.6
26.6 ± 6.3
>10,000
66.5
67.8
61.8
70.6
45.9
33.3
3
. Conclusions
>10,000
In conclusion,
a series of styrylpyran fluorophores were
a
Values are the means ± standard errors of the mean of
determinations.
6
independent
designed and evaluated for targeted detection of cerebral b-amy-
loid deposits. In vitro evaluation showed that styrylpyran motif
has specificity towards b-amyloid deposits and fluorescence
changes upon b-amyloid binding, which could provide a new scaf-
fold for the development of optical probes targeting cerebral b-
amyloid deposits. Among these derivatives, PAD-2 displayed
improved characteristics compared to PAD-1. PAD-2 could dis-
criminate APP/PS1 transgenic mice from wild type controls, with
specific labeling of cerebral b-amyloid deposits in vivo, suggesting
its potential application for targeted detection of b-amyloid depos-
its both in vitro and in vivo.
with time (Fig. 4). The molecular weight and cLogP of PAD-2 were
a little higher than those of PAD-1, and lower than those of PAD-3
and PAD-4. Notably, the clearance of PAD-2 from the brain was
similar to that of PAD-1, and much faster than that of PAD-3 and
PAD-4, suggesting that PAD-2 could contribute minimally to the
background signal. Thus, PAD-2, with rapid BBB penetration and
fast washout from the brain, was selected for further cerebral
imaging in transgenic mice.
4
4
. Experimental section
2
.6. In vivo imaging
.1. Material and methods
Finally, imaging in APP/PS1 transgenic mice and wild type con-
trol mice was conducted to assess the potential of PAD-2 for non-
invasive detection of cerebral b-amyloid deposits. As shown in
Figure 5, fluorescence signals were captured in the brain area, indi-
cating that PAD-2 could penetrate BBB effectively. Fluorescence
intensity of brains in APP/PS1 transgenic mice decreased more
slowly than in wild type controls, which may be contributed to
the specific labeling of cerebral b-amyloid deposits. Ex vivo histo-
logical observation was performed to further confirm the binding
of PAD-2 to cerebral b-amyloid deposits in vivo. Microscopic
images of APP/PS1 brain slices showed distinct labeling of b-amy-
loid plaques, while no labeling was observed in wild type controls
owing to the absence of b-amyloid plaques (Fig. 6). PAD-2 has
slightly lower quantum yield than that of PAD-1, but makes up
for this deficiency by displaying longer emission wavelength,
higher affinity to b-amyloid deposits, and intensively brighter flu-
orescence upon b-amyloid binding. In vivo and ex vivo studies
demonstrated that PAD-2 could specifically label cerebral b-amy-
loid deposits in vivo. Also, the improved properties of PAD-2 over-
came the difficulties of charged Thioflavin dyes (ThT/ThS), such as
All chemicals and solvents were commercial products and were
used without further purification. H NMR spectra were recorded
1
at 400 MHz on a varian INOVA 400 M NMR spectrometer in CDCl
3
solutions with tetramethylsilane (TMS) as an internal standard at
room temperature. Chemical shifts are reported as d values relative
to the internal TMS. Coupling constants are reported in Hertz. Mul-
tiplicity is defined by s (singlet), d (doublet), t (triplet), or m (mul-
tiplet). Mass spectra were acquired using an Agilent 6410B Triple
Quadrupole LC/MS system (ESI). HPLC was performed with a Shi-
madzu system (a LC-10AT pump with a SPD-10A UV detector,
k = 254 nm) with a Cosmosil C18 column (Nakalai Tesque, 5C18
-
AR-II, 4.6 mm ꢁ 150 mm) using a mobile phase (water/acetoni-
trile: 3/7) delivered at a flow rate of 1.0 mL/min. UV/vis and fluo-
rescence spectra were measured by UV-3600 and RF5301PC
spectrophotometers (Shimadzu, Japan), respectively. Fluorescent
observation was performed using Aviovert 40 CFL microscope
(
Zeiss, Germany). All key compounds were proven to show P99%
purity by analytical HPLC. Animal studies were carried out in full
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B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 3. Computational binding models of compounds (PAD-1 to PAD-6) with 2-fold Ab fibrils. Side chains of PHE19, ALA21, ALA30, ILE31, and ILE32 are shown in stick
form.
compliance with the regulations of Animal Care and Use Commit-
tee of Sichuan University.
lected by filtration and recrystallized from ethanol or purified by
silica gel chromatography to afford the final products.
4
4
.2. Chemistry
4.2.2. General procedure B
To a solution of compound 1 (1 mmol) and substituted aromatic
aldehydes (1 mmol) in acetonitrile (10 mL) was added piperidine
.2.1. General procedure A
To a solution of (2,6-dimethyl-4H-pyran-4-ylidene)malononi-
(200 lL). The reaction mixture was stirred under reflux for 10 h.
trile (1 mmol) and substituted aromatic aldehydes (1–2 mmol) in
acetonitrile (10 mL) was added piperidine (200 L). The reaction
mixture was stirred under reflux for 12 h. The precipitate was col-
The precipitate was collected by filtration and recrystallized from
ethanol or purified by silica gel chromatography to afford the final
products.
l
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6
B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1
7
H), 7.49 (d, 2H, J = 8.4 Hz), 7.54 (d, 1H, J = 6.0 Hz), 7.60 (s, 1H),
.69–7.73 (m, 1H), 8.90 (d, 1H, J = 8.4 Hz). MS: m/z 340 (M +H).
+
4
.2.6. 2-(2-((1E,3E)-4-(4-(Dimethylamino)phenyl)buta-1,3-
dienyl)-4H-chromen-4-ylidene)malononitrile (PAD-4)
PAD-4 was prepared following general procedure B (yield:
1
2
1
2
6.6%). H NMR (400 MHz, CDCl
3
, ppm): d 3.06 (s, 6H), 6.58 (d,
H, J = 10.8 Hz), 6.74 (d, 2H, J = 4.8 Hz) 6.79 (s, 1H), 7.41–7.43 (m,
H), 7.50 (d, 2H, J = 6.0 Hz), 7.53 (d, 1H, J = 5.6 Hz), 7.58 (d, 1H,
J = 10.8 Hz), 7.69–7.72 (m, 2H), 8.91 (d, 1H, J = 6.0 Hz). MS: m/z
+
3
66 (M +H).
4
.2.7. 2-(2,6-Bis((E)-4-(dimethylamino)styryl)-4H-pyran-4-
ylidene)malononitrile(PAD-5)
PAD-5 was prepared following general procedure A (yield:
1
2
0.5%). H NMR (400 MHz, CDCl
3
, ppm): d 3.06 (s, 12H), 6.56 (d,
1
H, J = 8.4 Hz), 6.77 (d, 4H, J = 6.8 Hz), 7.44 (s, 2H), 7.48 (d, 2H,
+
Figure 4. Comparison of brain uptake of PAD-1, -2, -3, and -4 in normal mice (n = 5
at each time point).
J = 8.4 Hz). MS: m/z 435 (M +H).
4
.2.8. 2-(2,6-Bis((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-
dienyl)-4H-pyran-4-ylidene)malononitrile (PAD-6)
4
.2.3. 2-(2-Methyl-4H-chromen-4-ylidene)malononitrile (1)
Malononitrile (1.44 g, 21.8 mmol) was added to a stirring solu-
PAD-6 was prepared following general procedure A (yield:
1
1
2
7.2%). H NMR (400 MHz, CDCl
3
, ppm): d 3.09 (s, 12H), 6.24 (d,
tion of 2-methylchromone (3.10 g, 19.4 mmol) in acetic anhydride
H, J = 15.6 Hz), 6.54 (s, 2H), 6.81–6.88 (m, 2H), 6.94 (d, 4H,
(
4 mL). The reaction mixture was stirred at 140 °C for 20 h. The
J = 15.2 Hz), 7.30–7.33 (m, 2H), 7.48 (d, 4H, J = 8.0 Hz). MS: m/z
reaction mixture was cooled to room temperature and filtered.
The crude solid was recrystallized from ethanol to give 1.58 g of
1
+
4
4
4
87 (M +H).
1
(39.3%). H NMR (400 MHz, CDCl
3
): d 2.44 (s, 3H), 6.73 (s, 1H),
.3. Biological evaluation
7
.25–7.45 (m, 2H), 7.62–7.70 (m, 1H), 8.92 (d, 1H, J = 8.4 Hz).
.3.1. Preparation of aggregated b-amyloid fibrils
4
.2.4. 2-(2-((1E,3E)-4-(4-(Dimethylamino)phenyl)buta-1,3-
Ab1–42 peptide (Amresco, USA) was gently dissolved in PBS solu-
tion (pH 7.4) containing 1 mM EDTA. The solution (0.25 mg/mL)
was incubated at 37 °C for 42 h with gentle and constant shaking.
dienyl)-6-methyl-4H-pyran-4-ylidene)malononitrile (PAD-2)
PAD-2 was prepared following general procedure A (yield:
1
2
6
0.5%). H NMR (400 MHz, CDCl
.14 (d, 1H, J = 14.8 Hz), 6.49 (s, 1H), 6.55 (s, 1H), 6.75 (d, 2H,
3
): d 2.33 (s, 3H), 3.04 (s, 6H),
4
.3.2. Spectroscopic characterization
J = 8.4 Hz), 6.78 (d, 2H, J = 11.2 Hz), 6.86 (d, 1H, J = 15.6 Hz), 7.39
+
Maximum absorbance wavelength (kabs), molar absorption
(
d, 2H, J = 8.4 Hz). MS: m/z 330 (M +H).
coefficient (e), maximum excitation wavelength (kex), maximum
emission wavelength (kem), and fluorescence quantum yield (
were measured in dichloromethane. Rhodamine B (Sigma, USA)
was taken as a reference for determining fluorescence quantum
F
U )
4
.2.5. (E)-2-(2-(4-(Dimethylamino)styryl)-4H-chromen-4-
1
9
ylidene)malononitrile (PAD-3)
PAD-3 was prepared following general procedure B (yield:
yields. Each compound (1.0
in PBS solution alone or a prepared solution of Ab1–42 aggregates
2.75 M, final concentration) in PBS to test interactions of each
lM, final concentration) was dissolved
3
1
0.7%). 1H NMR (400 MHz, CDCl
3
, ppm): d 3.08 (s, 6H), 6.58 (d,
H, J = 16.0 Hz), 6.75 (d, 2H, J = 8.4 Hz) 6.78 (s, 1H), 7.40–7.42 (m,
(
l
Figure 5. In vivo imaging of APP/PS1 transgenic mice and wild type controls after intravenous injection of PAD-2. (A) Images at 60 min postinjection; (B) quantitative analysis
of fluorescence intensity at different timepoint.
Please cite this article in press as: Zhu, B.-y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.004

B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Figure 6. Ex vivo histological observation of brain slices from APP/PS1 transgenic mice and wild type controls after intravenous injection of PAD-2 (upper). The adjacent brain
slices were stained with thioflavin S to localize b-amyloid plaques (below).
compound with b-amyloid aggregates (fluorescence increase and
blueshift).
(BioReal, Austria). Intensity of brain fluorescence at each timepoint
[F(t)] was calculated from the photon counts and was normalized
by the subtraction of preinjected background signals [F(pre)]. Rel-
ative fluorescence intensity (%) at each timepoint was calculated
by the following equation:
4
.3.3. In vitro fluorescence staining of APP/PS1 brain tissue
Brain sections from an aged APP/PS1 transgenic mouse (male,
1
2 months, 15 lm) were incubated with each compound (1.0 lM
Relative fluorescence intensity
in 30% ethanol) for 5–10 min at room temperature. The localization
of b-amyloid plaques was confirmed by staining the adjacent brain
sections with thioflavin S (0.125%). The sections were then washed
with 40% ethanol (two 2-min washes), rinsed with water for 30 s.
After drying at room temperature, fluorescent observation was
then performed with FITC and GFP filter sets.
0
¼ ½FðtÞ ꢀ FðpreÞꢂ=½Fðt Þ ꢀ FðpreÞꢂ ꢁ 100%
ðIÞ
0
F(t ) represents the highest intensity for each compound.
4
.3.6. In vivo analysis of PAD-2 on APP/PS1 transgenic mice
The QuickView 3000 imaging system was used to perform
in vivo imaging. Images were obtained with a customized 534-
nm excitation filter and 716-nm emission filter. QuickView 3000
software was used for data analysis. 16-Month-old APP/PS1 trans-
genic mice and corresponding wild-type control mice were shaved
before background imaging. PAD-2 (2.0 mg/kg, 20% DMSO, 80%
propylene glycol) was injected into the mice through tail vein.
Real-time images were recorded and Regions of Interest (ROIs)
were drawn covering the whole brain. The data were analyzed
using the Eq. I.
4
.3.4. In vitro binding assays using aggregated b-amyloid fibrils
Binding affinity of each compound was assessed by saturation
assays with Ab1–42 aggregates in PBS solution. A mixed PBS solu-
tion containing each compound (1 nM–10 M) and Ab1–42 aggre-
gates (1.5 M) was incubated at room temperature for 1 h and
transferred to a black 96-well plate to measure fluorescent inten-
sity by a multifunction microplate reader (Thermo Scientific Var-
l
l
ioskan Flash, USA). Dissociation constant (K
using GraphPad Prism 5.0 (GraphPad Software, Inc., USA).
d
) was calculated
4
.3.7. Ex vivo examination of b-amyloid labeling
After in vivo imaging at 120 min postinjection, the mice were
4
.3.5. Biodistribution experiments with normal mice
Kunming mice (8 weeks, male) were intravenously injected
immediately sacrificed by decapitation. For microscopic observa-
tion, the brains were sliced (15- m-thick) using a CM1950 cryostat
Leica, Germany). After drying, the brain slices were observed using
with 50 lL of each compound (2.0 mg/kg, 20% DMSO, 80% propy-
l
lene glycol). The mice were sacrificed at 5, 30, 60, 120 min postin-
jection and whole brains were taken out (n = 5 at each time point).
Fluorescence signals were then recorded and analyzed using a
QuickView 3000 imaging system with QuickView 3000 software
(
a microscope equipped with a FITC filter set. The adjacent slices
were stained with thioflavin S to localize b-amyloid plaques, and
were observed using the same microscope with a GFP filter set.
Please cite this article in press as: Zhu, B.-y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.004

8
B.-y. Zhu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11. Choi, S. R.; Golding, G.; Zhuang, Z. P.; Zhang, W.; Lim, N.; Hefti, F.; Benedum, T.
4
.4. Computational study
E.; Kilbourn, M. R.; Skovronsky, D.; Kung, H. F. J. Nucl. Med. 2009, 50, 1887.
2. Clark, C. M.; Schneider, J. A.; Bedell, B. J.; Beach, T. G.; Bilker, W. B.; Mintun, M.
A.; Pontecorvo, M. J.; Hefti, F.; Carpenter, A. P.; Flitter, M. L.; Krautkramer, M. J.;
Kung, H. F.; Coleman, R. E.; Doraiswamy, P. M.; Fleisher, A. S.; Sabbagh, M. N.;
Sadowsky, C. H.; Reiman, E. P.; Zehntner, S. P.; Skovronsky, D. M. JAMA 2011,
1
All of the docking process was carried out using GOLD 5.0 pro-
gram, which is based on genetic algorithm.24 A segment of Ab1–40
fibril was extracted from RCSB Protein Data Bank (PDB ID: 2LMO)
and used as the reference receptor. Prior to molecular docking,
the preparation of Ab1–40 fibril structure, including the addition
of hydrogen atoms, deletion of water molecules, and assigning of
Charmm force field, was performed using Discovery Studio 3.1
software package. A sphere including residues ALA30, ILE31,
ILE32, PHE19, PHE20, and ALA21 were defined as the binding site.
GoldScore scoring function was used to identify docking poses as
well as to rank these poses. 30 docking runs were set for each com-
pound. The best docking pose was preserved and corresponding
scoring values was extracted.
3
05, 275.
13. Ono, M.; Cheng, Y.; Kimura, H.; Cui, M.; Kagawa, S.; Nishii, R.; Saji, H. J. Med.
Chem. 2011, 54, 2971.
1
1
1
4. J. Nucl. Med. 2012, 53, 15N.
5. Sabri, O.; Seibyl, J.; Rowe, C.; Barthel, H. Clin. Transl. Imaging 2015, 3, 13.
6. Thal, D. R.; Beach, T. G.; Zanette, M.; Heurling, K.; Chakrabarty, A.; Ismail, A.;
Smith, A. P.; Buckley, C. Alzheimers Dement. 2015, 8, 975.
7. Nesterov, E. E.; Skoch, J.; Hyman, B. T.; Klunk, W. E.; Bacskai, B. J.; Swager, T. M.
Angew. Chem., Int. Ed. 2005, 44, 5452.
1
18. Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580.
19. Hintersteiner, M.; Enz, A.; Frey, P.; Jaton, A. L.; Kinzy, W.; Kneuer, R.; Neumann,
U.; Rudin, M.; Staufenbiel, M.; Stoeckli, M.; Wiederhold, K. H.; Gremlich, H. U.
Nat. Biotechnol. 2005, 23, 577.
20. Okamura, N.; Mori, M.; Furumoto, S.; Yoshikawa, T.; Harada, R.; Ito, S.;
Fujikawa, Y.; Arai, H.; Yanai, K.; Kudo, Y. J. Alzheimers Dis. 2011, 23, 37.
Acknowledgments
21. Cui, M.; Ono, M.; Watanabe, H.; Kimura, H.; Liu, B.; Saji, H. J. Am. Chem. Soc.
014, 136, 3388.
2
2
2. Ran, C.; Xu, X.; Raymond, S. B.; Ferrara, B. J.; Neal, K.; Bacskai, B. J.; Medarova,
The study was supported by the National Natural Science Foun-
dation of China (81402891), Ph.D. Programs Foundation of Ministry
of Education of China (20130181120114), and China Postdoctoral
Science Foundation Grant (2014M560723).
Z.; Moore, A. J. Am. Chem. Soc. 2009, 131, 15257.
23. Zhang, X.; Tian, Y.; Li, Z.; Tian, X.; Sun, H.; Liu, H.; Moore, A.; Ran, C. J. Am. Chem.
Soc. 2013, 135, 16397.
2
2
2
4. Chang, W. M.; Dakanali, M.; Capule, C. C.; Sigurdson, C. J.; Yang, J.; Theodorakis,
E. A. ACS Chem. Neurosci. 2011, 2, 249.
5. Cao, K.; Farahi, M.; Dakanali, M.; Chang, W. M.; Sigurdson, C. J.; Theodorakis, E.
A.; Yang, J. J. Am. Chem. Soc. 2012, 134, 17338.
6. Ono, M.; Ishikawa, M.; Kimura, H.; Hayashi, S.; Matsumura, K.; Watanabe, H.;
Shimizu, Y.; Cheng, Y.; Cui, M.; Kawashima, H.; Saji, H. Bioorg. Med. Chem. Lett.
2010, 20, 3885.
7. Ono, M.; Watanabe, H.; Kimura, H.; Saji, H. ACS Chem. Neurosci. 2012, 3, 319.
8. Watanabe, H.; Ono, M.; Matsumura, K.; Yoshimura, M.; Kimura, H.; Saji, H. Mol.
Imaging 2013, 12, 338.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.01.004.
2
2
References and notes
29. Zhou, K.; Fu, H.; Feng, L.; Cui, M.; Dai, J.; Liu, B. Chem. Commun. 2015, 11665.
30. Fu, H.; Cui, M.; Zhao, L.; Tu, P.; Zhou, K.; Dai, J.; Liu, B. J. Med. Chem. 2015, 58,
6
972.
1
2
3
4
5
6
.
.
.
.
.
.
Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res. Commun. 1984, 122, 1131.
Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333.
Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
3
3
3
1. Watanabe, H.; Ono, M.; Saji, H. Chem. Commun. 2015, 17124.
2. Cheng, Y.; Zhu, B.; Deng, Y.; Zhang, Z. Anal. Chem. 2015, 87, 4781.
3. Tolmachev, A. I.; Kachkovskii, A. D.; Kudinova, M. A.; Kurdiukov, V. V.; Ksenzov,
S.; Schrader, S. Dyes Pigments 2007, 74, 348.
4. Voropai, E. S.; Samtsov, M. P.; Kaplevskii, K. N.; Maskevich, A. A.; Stepuro, V. I.;
Povarova, O. I.; Kuznetsova, I. M.; Turoverov, K. K.; Fink, A. L.; Uverskii, V. N. J.
Appl. Spectrosc. 2003, 70, 868.
5. For more details, see the Supporting information.
6. Bilkei-Gorzo, A. Pharmacol. Ther. 2014, 142, 244.
7. Cheng, Y.; Ono, M.; Kimura, H.; Kagawa, S.; Nishii, R.; Kawashima, H.; Saji, H.
ACS Med. Chem. Lett. 2010, 1, 321.
8. LeVine, H., III Methods Enzymol. 1999, 309, 274.
Selkoe, D. J. Nat. Biotechnol. 2000, 18, 823.
Mathis, C. A.; Wang, Y.; Klunk, W. E. Curr. Pharm. Des. 2004, 10, 1469.
Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.; Zhuang, Z. P.;
Kung, M. P.; Kung, H. F. Nucl. Med. Biol. 2003, 30, 565.
Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G. F.; Debnath, M. L.; Klunk, W. E. J.
Med. Chem. 2003, 46, 2740.
Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D. P.;
Bergstrom, M.; Savitcheva, I.; Huang, G. F.; Estrada, S.; Ausen, B.; Debnath, M.
L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni,
G.; Mathis, C. A.; Langstrom, B. Ann. Neurol. 2004, 55, 306.
Koole, M.; Lewis, D. M.; Buckley, C.; Nelissen, N.; Vandenbulcke, M.; Brooks, D.
J.; Vandenberghe, R.; Van Laere, K. J. Nucl. Med. 2009, 50, 818.
3
7
.
.
3
3
3
8
3
3
4
9. Xu, W.; Lucke, A. J.; Fairlie, D. P. J. Mol. Graph. Model. 2015, 57, 76.
0. Petkova, A. T.; Ishii, Y.; Balbach, J. J.; Antzutkin, O. N.; Leapman, R. D.; Delaglio,
F.; Tycko, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742.
9
.
1
0. Villemagne, V. L.; Ong, K.; Mulligan, R. S.; Holl, G.; Pejoska, S.; Jones, G.;
O’Keefe, G.; Ackerman, U.; Tochon-Danguy, H.; Chan, J. G.; Reininger, C. B.; Fels,
L.; Putz, B.; Rohde, B.; Masters, C. L.; Rowe, C. C. J. Nucl. Med. 2011, 52, 1210.
4
1. Cook, N. P.; Ozbil, M.; Katsampes, C.; Prabhakar, R.; Martí, A. A. J. Am. Chem. Soc.
2
013, 135, 10810.
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