Development of Phenothiazine-Based Theranostic Compounds That Act Both as Inhibitors of β-Amyloid Aggregation and as Imaging Probes for Amyloid Plaques in Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.6b00380

Early detection of Alzheimer's disease (AD) is imperative in enabling the understanding and clinical treatment of this disorder, as well as in preventing its progression. Imaging agents specifically targeting Aβ plaques in the brain and the retina may lead to the early diagnosis of AD. Among them, near-infrared fluorescent (NIRF) imaging has emerged as an attractive tool to noninvasively identify and monitor diseases during the preclinical and early stages. In the present study, we report the design, synthesis, and evaluation of a series of new near-infrared fluorescent probes. Most of these probes displayed maximum emission in PBS (>650 nm), which falls in the good range for NIRF probes. Among them, 4a1 showed the highest affinity toward Aβ aggregates (K_d = 7.5 nM) and an excellent targeting ability for Aβ plaques in slices of brain and retina tissue from double transgenic mice. These compounds are also found to effectively prevent Aβ fibril formation and disaggregate preformed Aβ fibrils, showing a promising potential as theranostic agents for the diagnosis and therapy of AD.

Full text of DOI:10.1021/acschemneuro.6b00380

Subscriber access provided by Fudan University
Article
Development of phenothiazine-based theranostic compounds
that act both as inhibitors of #-amyloid aggregation and
imaging probes for amyloid plaques in Alzheimer's disease
Pascal Dao, Feifei Ye, Yan Liu, Zhiyun Du, Kun Zhang,
Chang-Zhi Dong, Bernard Meunier, and Huixiong CHEN
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00380 • Publication Date (Web): 18 Jan 2017
Downloaded from http://pubs.acs.org on January 19, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Development of phenothiazineꢀbased theranostic compounds that act both
as inhibitors of βꢀamyloid aggregation and imaging probes for amyloid
plaques in Alzheimer's disease
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pascal Dao,
†
Feifei Ye,
†
Yan Liu,
†
Zhi Yun Du, Kun Zhang, Chang Zhi Dong,†,⊥Bernard
†
†
Meunier,†,‡ Huixiong Chen.
*
,†,§
†
Faculty of Light Industry and Chemical Engineering, Guang Dong University of Technology, Guang Dong,
510006, China, CNRS, UMR8601, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
CBNIT, Université Paris Descartes, PRES Sorbonne Paris Cité, UFR Biomédicale, 45 rue des SaintsꢀPères,
75270 Paris Cedex 06, France, Université Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15
§
⊥
rue JꢀA de Baïf, 75205 Paris Cedex 13, France,‡Laboratoire de Chimie de Coordination du CNRS, 205 Route
de Narbonne, 31077 Toulouse Cedex, France
Abstract: Early detection of Alzheimer’s disease (AD) is imperative in enabling the
understanding and clinical treatment of this disorder, as well as in preventing its progression. Imaging
agents specifically targeting Aβ plaques in the brain and the retina may lead to the early diagnosis of
AD. Among them, near infrared fluorescent imaging has emerged as an attractive tool to
noninvasively identify and monitor diseases during the preclinical and early stages. In the present
study, we report the design, synthesis and evaluation of a series of new nearꢀinfrared fluorescent
probes. Most of these probes displayed maximum emission in PBS (> 650 nm), which falls in the good
range for NIRF probes. Among them, 4a1 showed the highest affinity towards to Aβ aggregates (Kd =
7.5 nM) and an excellent targeting ability for Aβ plaques in slices of brain and retina tissue from
double transgenic mice. These compounds are also found to effectively prevent A
β
fibrils formation
and disaggregate preformed A
diagnosis and therapy of AD.
β fibrils, showing a promising potential as theranostic agents for the
Keywords: Alzheimer’s disease, Betaꢀamyloid, Cytotoxicity, NIRF imging, Inhibitors,
Theranostic agents
INTRODUCTION
Alzheimer’s disease (AD) is an ageꢀrelated devastating neurodegenerative disorder, which
severely impacts on the global economic development and healthcare system. The number of
patients increases every year and it is expected to expand to 36 million to 115 million in
1,2
worldwide around the year of 2050. Despite numerous efforts and exponential growth in
investment, nearly all “diseaseꢀmodifying” treatment for AD has failed to show clinical
3
benefits in individuals with symptomatic AD to date. One of the most likely explanations for
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
these failures is that the drugs were administered too late in the course of the AD
4
neuropathological processes. It is a reasonable assumption that these therapies will be more
effective when applied before major brain damage has occurred. Therefore, the development
of novel biomarkers sensitive to preclinical or early clinical stages of AD is crucial for this
5
devastating disease.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
According to the amyloid cascade hypothesis, Aβ aggregates in the brain play a key role
6
in the cascade of event leading to AD. Their formation arises from the aggregation of
peptides Aβ40 and Aβ42, which are generated from amyloid peptide precursor (APP) by
7
cleavage with βꢀ and γꢀsecretases. Recent evidence indicates that Aβ plaques are a critical
8
mediator of neuritic pathology. These extracellular plaques and deposits of Aβ and
intracellular NFT became over the years the pathological hallmark of AD and drug targets.
Recently the pathology data support a model of AD in which Aβ depositions precede clinical
9,10
symptoms by several years. In the second phase of illness, neurofibrillary tangles develop,
neurons die, brain atrophy occurs, and cognition declines. Consequently, in vivo imaging of
Aβ is particularly suitable for identifying individuals at risk and in the early stages of AD.
Therefore, several strategies have been developed for the imaging of amyloid, namely,
radiolabeled amyloidꢀꢀ peptide (Aꢀ) antibodies and peptide fragments, small molecules for
11
PET and SPECT imaging, and compounds for MRI. To date, [ C]PIB is the most extensively
11
studied PET imaging probe for Aβ plaques in humans, while[18F]AVꢀ45 is the first Aβ
12
imaging probe approved by the FDA.
Compared with nuclear imaging modalities, NIRF imaging has emerged as an attractive
tool for optical imaging. NIR light is particularly suitable to be used for in vivo imaging of
molecular processes due to its acceptable depth of penetration, nonꢀinvasive operation,
minimal interferences from autoꢀfluorescence of biological matter and minimal photoꢀdamage
13
to biological samples. Furthermore, the optical imaging modality possesses many
advantages, as it enables safe detection without radiation exposure and realꢀtime imaging
without a timeꢀconsuming data acquisition process and requires readily available instruments
at moderate cost. So far, NIR fluorescent probes have been mainly used in the oncology field
1
4,15
for imaging tumors, both in vitro and in vivo.
Recently, a number of NIR fluorophores
16
have also been developed and employed for AD imaging in vivo, including NIAD,
17
18
19
20
21
CRAND, AOIꢀ987, DANIR, BAP and THKꢀ265 (figure 1). AOIꢀ987 is a charged
molecule with a small Stokes shift and moderate affinity (Kd = 220 nM), which implies
limited BBB permeability and obscured signal increase upon binding with Aβ plaques. Ran et
2
ACS Paragon Plus Environment

Page 3 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
al. have exploited curcumin derivatives as NIR probes for soluble or insoluble Aβ species.
One of these compounds, CRANADꢀ2 was reported as a NIR probe for Aβ plaques with
advantages over other candidates such as its ideal binding constant (Kd = 38.7 nM) and
optimal fluorescent properties (emission maximum at 805 nm). However, most of these
probes are unable to satisfy the divers and stringent requirements for cerebral Aβ species
imaging including good BBB permeability, high selectivity and strong binding affinity
towards Aβ species, low neurotoxicity and good bioꢀstability. On the other hand, few of NIR
probes used as molecular theranostics, which could simultaneously perform imaging of Aβ
species and Aβ aggregation inhibition have been described. It is difficult to predict the full
impact of molecular theranostics in the clinical arena. However, they represent a powerful
emerging platform that could optimize the efficacy and safety of therapy, as well as yield
information about the entire drug development process.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. NIRF probes reported previously for the detection of Aβ plaques.
In an attempt to develop theranostic agents for NIRF imaging of βꢀamyloid species and
inhibition of βꢀamyloid aggregation, the donorꢀacceptor architecture bridged by a conjugated
πꢀelectron chain was selected as the backbone structure to produce compounds possessing a
suitable spectral range of the absorption and emission bands along with a potential to satisfy
the necessary requirements for cerebral Aβ species imaging. Here we report the synthesis,
characteristics, and biological evaluations of these theranostic agents as smart NIRF imaging
probes for Aβ plaques with a therapeutic intervention in AD.
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS AND DISCUSSION
The synthetic pathway for the preparation of compounds 4ꢀ8 is shown in Scheme 1. The
compound 1aꢀb was synthesized by reacting phenothiazine with methyl iodide in DMF in the
presence of NaH. Nucleophilic displacement of the chloride of 1b with dimethylamine was
performed by Buchwald–Hartwig amination, leading to 1c. Nꢀmethyl phenothiazine
derivatives 1aꢀc were formylated to their carboxaldehyde derivatives 2aꢀc by Vilsmeir
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
formylation procedure by the reaction of the respective compounds with POCl in DMF. The
3
rhodanine core 3 was produced by the reaction of glycine ester or 3ꢀaminopropanoate (ester)
with bis(carboxymethyl)trithiocarbonate in the presence of triethylamine. The key step in the
formation of the target compounds 4 and 7 were achieved by a Knoevenagel condensation
reaction between the phenothiazineꢀ3ꢀcarbaldehyde derivatives 2aꢀc and the rhodanines 3 in
the presence of piperidine. The esters 4a2, 4b2 and 7a2 can be hydrolyzed to the
corresponding carboxylic acids 5, 8. Finally, the acid 5a was converted to the amide 6, using
BOP as a coupling reagent and dimethylamine as a base.
Scheme 1. Conditions and reagents: a) CH I, NaH, DMF; b) POCl , DMF; c) rhodanine derivatives 3,
piperidine, DCM; d) TFA, DCM; e) BOP, DIPEA.
3
3
First, the fluorescent properties, including excitation and emission wavelengths of these
compounds in PBS (0.2 M, pH = 7.4) were evaluated. As shown in table 1, except for 5a and
4
ACS Paragon Plus Environment

Page 5 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
8, the emission wavelengths for these compounds were ≥ 650 nm, which fall in the good
range for NIRF probes. A significant fluorescence intensity increase in the emission of these
compounds was observed upon association with Aβ1−42 aggregates, especially for the
compounds 6, 5b, 8 (table 1), which have more than 10ꢀfold intensity increase and suggested
that these compounds would be “turned on” upon interacting with a host. However, they did
not exhibit the fluorescence enhancement response to Aβ1−42 monomer (Figure 2). It
suggested that these compounds could binds well to the hydrophobic pockets of Aβ1−42
aggregates, resulting in the restricted rotation upon binding to its substrate. This is further
reflected by a grand increase in quantum yield upon binding to aggregate β–amyloid proteins,
which is a desirable feature for a probe, in order for its fluorescence to be clearly
distinguished from that of the background.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Molecular weight, fluorescence profile, and fold increase of phenothiazineꢀbased compounds
upon interaction with Aβ1−42 aggregates
λem
λ
abs
λ
em
(nm)
with
Aβ
670
Fold
ε
ꢀ1
N°
R1
R2
n
ꢀ1
Φ（%）
(
nm)
(nm)
increase (M cm )
a
4
a1
a2
H
H
CO
2
Et
tBu
1
1
500
490
680
670
4.89
2.48
8377.0
6061.7
5.3, 25.1
6.6, 18.0
a
4
CO
2
670
1
0.4,a
4
b1
Cl
Cl
CO
2
Et
1
1
1
470
470
510
660
660
680
660
660
670
6.2
2.9
2.3
6888.6
7532.2
8575.7
4
9.8
a
4b2
c2
CO
CO
2
2
tBu
tBu
7.1, 17.8
5.0
6
4
4
N(CH
3
)
)
2
a
.9
.6
5a
H
Cl
CO
CO
CO
2
2
2
H
H
H
1
1
1
470
470
500
650
640
680
640
640
670
7.3
11.1
3.2
5756.7
7089.1
6098.6
a
1
0
6.5
.3
5
b
a
1.2
0.1
0
5
c
N(CH
3
2
a
.9
a
a
6
a1
H
H
CO
CO
2
N(CH
Et
3
)
2
1
2
480
490
680
670
680
670
13.1
6.4
4565.7
6377.0
2.0, 12.1
4.3, 57.6
7
7
2
1
0.1,a
a2
H
H
CO
2
tBu
2
2
480
470
660
640
660
640
3.9
5671.8
6061.3
6
6.1
0.2
8
CO
2
H
11.0
a
5.9
a
with Aβ1ꢀ42 aggregates
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A useful NIRF Aβꢀbinding probe should have weak interactions with serum albumin
and low cytotoxicity. Firstly, we compared the fluorescent properties of these free probes in
aqueous solution to their fluorescence properties in the presence of Aβ1−42 aggregates and
bovine serum albumin (BSA). As shown in Figure 2, no significant change in fluorescence
was observed during incubation with BSA, suggesting that there is little or no interaction
between 4a1 (or 5a) and BSA. Then, a cytotoxicity study of 4a1 was carried out by MTT
assays with or without exposure to light using a human neuronal cell line (SHꢀSY5Y) at
different concentrations. As shown in Figure 3, probe 4a1 did not show marked toxicity to
this human neuronal cell line at 50 ꢁM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Emission spectra of 4a1and 5a (1µM) upon interaction with Aβ aggregates or monomer and
BSA.
Figure 3. Cell viability after incubation of 4a1 at different concentrations with a human neuronal cell
line (SHꢀSY5Y) by MTT assay, at 37 °C for 24 h with or without exposure to light (each sample was
tested using three replicates, and the results are reported as the mean ± standard deviation).
To quantitatively evaluate the binding affinities of these probes to Aβ1−42 aggregates,
an in vitro saturation binding assay using Aβ1ꢀ42 aggregates was performed according to
conventional methods. In the results shown in table 2, these compounds showed moderate to
high affinity to Aβ1−42 aggregates, which were significantly higher than that of ThT, wildly
used for detecting protein and peptide aggregation such as Aβ aggregation (Kd = 1.7 ꢀM).
The lipophilicity (logP with 2ꢀ3.5 range) and molecular weight (less than 600 Da) of the
probes are two primary factors affecting the capability of BBB penetration. As shown in table
6
ACS Paragon Plus Environment

Page 7 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
2, several compounds were found to have calculated logP values between 2.0 and 3.5,
suggesting that they can potentially cross the BBB efficiently.
Table 2. Dissociation constant of phenothiazineꢀbased compounds to Aβ1−42 aggregates and
inhibition of Aβ1−42 aggregation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inhibition of
a
Kd
(nM)
Aβ
N°
R
1
R
2
m
logP
M
aggregation
IC50 (µM)
0.67 ± 0.02
1.42 ± 0.06
1.52 ± 0.04
1.63 ± 0.02
3.77 ± 0.08
1.07 ± 0.02
1.85 ± 0.06
4.62 ± 0.20
7.32 ± 0.90
7.29 ± 1.0
8.44 ± 1.1
1.72 ± 0.2
ND
4
a1
a2
b1
b2
H
H
Cl
Cl
N(CH
H
Cl
N(CH
H
CO
CO
CO
2
Et
tBu
Et
1
1
1
1
1
1
1
1
1
2
2
2
442.6
470.6
477.0
505.1
513.7
414.5
448.9
457.6
441.6
456.6
484.7
428.6
3.68
4.22
4.24
4.78
4.5
3.08
3.64
3.37
3.19
3.98
4.51
3.38
7.5 ± 0.4
41.5 ± 3.5
74.2 ± 5.4
117.3 ± 8.7
237.6± 6.2
146.9 ± 12.6
695.8 ± 14.9
204.8 ± 20.1
96.4 ± 8.3
28.5 ± 2.5
14.9 ± 1.3
212.5 ± 24.3
1942 ± 32.1
ND
4
4
4
2
2
CO
CO
2
tBu
tBu
4c2
3
3
)
)
2
2
2
5a
CO
CO
CO
2
2
2
H
H
H
5
b
5c
6
CO
CO
CO
CO
2
N(CH
Et
tBu
3 2
)
7
a1
H
H
H
2
7a2
8
ThT
2
2
H
Curcumin
17.4 ± 3.4
a
logP values were calculated using chemdraw program
To validate the feasibility of our compounds as a NIR imaging probes to Aβ plaques,
the in vitro neuropathological fluorescent staining of Aβ plaques in slices of brain tissue from
a double transgenic (APPwse/PSEN1, 9ꢀmonth old) mouse overexpressing Aβ was performed
with 4a1. As shown in Figure 4K and 4L, specific staining of Aβ plaques was clearly
observed in the brain slices of the Tg mouse, which displayed intense and distinct red
fluorescence clusters. The presence and distribution of Aβ was similar to the observation of
staining adjacent slices using ThioflavinꢀT (ThT) (Figure 4G and 4H). These results were
consistent with those observed in a 10ꢀmonth old double transgenic (APPwse/PSEN1), which
showed slightly larger plaques in slices of brain (Figure Sꢀ1). However, there were no plaques
found in the ageꢀmatched wildꢀtype control mouse (Figure 4A, 4B and 4I, 4J). In vitro staining
of Aβ plaques in slices of brain tissue from a double transgenic (APPwse/PSEN1, 9ꢀmonth
old) mouse was also performed by IHC with the commercially available Aβ antibody (Figure
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4). There is no plaque deposition in the ageꢀmatched wildꢀtype control mouse (Figure 4I, 4G),
but distinct fluorescence signals were observed in a double transgenic (APPwse/PSEN1, 9ꢀ
month old) mouse (Figure 4K, 4L). These results clearly demonstrated that 4a1 can
effectively bind to and label Aβ plaque in the brain slice.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Fluorescence staining of 4a1 on brain slices of cortex regions of a double Tg mouse (K and
L, magnification: 5X). Adjacent slices from Tg mouse were stained with ThT (C and D,
magnification: 5X) and with Aβ antibody ab2454 then secondary antibody with fluorophore Alexa 488
(G and H, magnification: 5X). Brain slices from wildꢀtype control mouse was also used in the same
conditions with 4a1 (I and J), ThT (A and B) and Aβ antibody ab2454 then secondary antibody with
fluorophore Alexa 488 (E and F).
Then, the fluorescent staining of Aβ plaques in slices of the eye containing retina tissue
from the same double transgenic mouse was also performed. The retina can serve as an
excellent alternative system for AD research because it can faithfully reflect the changes in
the brain and offers unique advantages that can simplify the investigation of AD. Indeed,
being a part of the central nervous system (CNS), the retina contains a high density of
neuronal cells with a laminar structure outside the brain. Multiple transgenic mouse lines have
22,23
elicited ADꢀlike pathological hallmarks in the retina as disease progresses.
Aβ plaques
were detected in the retina as early as at 2.5 months of age in ADꢀTg mice, but not in the
22
brain, suggesting that Aβ plaques in the retina preceded brain plaques. Furthermore, recent
advances of imaging technologies for the retina have given the opportunity for the
24
development of a more definitive and nonꢀinvasive diagnostic tool for AD pathology. As
anticipated, retinal Aβ plaques were absent in the slices of nonꢀTg (wt) mice, shown in Figure
5
, whereas they were clearly identified in the slices of eye containing retina of ADꢀTg mice
Figure 5J, 5K). The similar results were confirmed using ThT (Figure 5C and 5D) and Aβ
antibody (Figure 5G and 5H).
(
8
ACS Paragon Plus Environment

Page 9 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Fluorescence staining of 4a1 on the slices of the eye containing retina tissue of a double Tg
mouse (J and L, magnification: 5X). Adjacent slices from Tg mouse were stained with ThT (C and D,
magnification: 5X) and with Aβ antibody ab2454 then secondary antibody with fluorophore Alexa 488
(
G and H). Eyes slices from wildꢀtype control mouse was also used in the same conditions with 4a1 (I
and K), ThT (A and B) and Aβ antibody ab2454 then secondary antibody with fluorophore Alexa 488
E and F).
(
To explore the potential therapeutic application of our target molecules, we further
studied whether these compounds can interfere with Aβ1−42 aggregation and its
disaggregation. One reported natural product, curcumin which was shown to inhibit the
2
5
formation of amyloid oligomers and fibrils and to reduce Amyloid in vivo, was included to
validate the screening conditions. Under the experimental conditions, curcumin inhibited
Aβ1−42 aggregation with IC value of 17.4 ꢀM (Table 2), which was similar to previously
50
reported data. Firstly, the seedꢀmediated growth of Aβ1−42 aggregate was carried out in the
presence of compound 4a1 (1 and 10 ꢀM) or without 4a1. The resulting aggregates were
visualized under the fluorescence microscope. As shown in Figure 6, compound 4a has
significantly slowed down the aggregation rate of Aβ1−42 in a doseꢀdependent manner, as
compared with the control. Furthermore, the inhibitory effect of these compounds on the
aggregation of Aβ1−42 was also investigated. As shown in table 2, all these compounds can
effectively inhibit the aggregation of Aβ1−42. The IC of these compounds was found to be
5
0
in the range of 0.67ꢀ8.44 ꢀΜ. Among them, compound 4a1 displayed a strong inhibition on
the aggregation of Aβ1−42. This results was consistent with the fluorescence microscopy
experiments.
9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Inhibition of self aggregation of Aβ1ꢀ42 followed by fluorescence microscopy using 4a1.
Four random fields were presented for each treatment.
Next, we investigated whether these compounds could further disaggregate preformed
Aβ1ꢀ42 fibrils. Aβ1ꢀ42 fibrils that were generated by 3ꢀdays incubation of monomeric Aβ1ꢀ
42 at 37°C were incubated with tested compounds for another 3 days. It was shown that the
samples coꢀincubated with 4a1 or 5a or curcumin as the standard decreased ThT fluorescence
and showed IC50 values of 0.82 ± 0.10, 1.28 ± 0.35 and 9.9 ± 1.2 ꢀM, respectively. The target
compounds 4a1 and 5a exhibited markedly higher inhibitory activity than curcumin,
suggesting that our target compounds able to promote disassembly preformed Aβ1ꢀ42 fibrils
at low concentrations and are of high potential to be used as therapeutic agents to treat AD.
These results were also confirmed by fluorescence microscopy analysis of 4a1 promoting
fibril disaggregation (Figure S2). Indeed, the results show that 4a1 has an impact on fibril
maturation or dissolution.
CONCLUSION
We successfully designed and synthesized a series of novel smart NIRF probes with
donorꢀacceptor architecture for detection of Aβ plaques. Most of these probes displayed
maximum emission in PBS (≥ 650 nm). Upon binding to Aβ1−42 aggregates, these
1
0
ACS Paragon Plus Environment

Page 11 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
compounds displayed a significant fluorescence intensity increase in the emission spectra,
which is very close to the good range for NIRF imaging. In binding experiments in vitro, they
showed good affinity for Aβ1−42 aggregates. Among them, 4a1 showed the highest affinity
(Kd = 7.5 nM). Fluorescence staining of Aβ plaques in brain and eye slices in vitro
demonstrated an excellent targeting ability. From a medicinal chemistry perspective 4a1
offers advantages of a smallꢀmolecule scaffold which is easily amenable to further
manipulation to improve fluorescence response and amyloidꢀbinding properties. Remarkably,
it offers the additional advantage of a concomitant promising profile of Aβ aggregation
inhibition and disaggregation of preformed Aβ fibrils, together with a high stability in mouse
serum and a low toxicity to human neuronal cells. 4a1 will be selected for further studies in
the laboratory.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MATERIALS AND METHODS
All commercial materials were used without further purification. Melting points were
determined on a Kofler apparatus as uncorrected values. Analytical thinꢀlayer
chromatography was performed on precoated 250 ꢁm layer thickness silica gel 60 F254 plates
and visualized with UV light. Column chromatography was performed using silica gel 60
1
13
(
40−63 ꢁm). H NMR and C NMR spectra were measured on 400MHz spectrometer in
DMSOꢀd6 or CDCl with chemical shift (δ) given in parts per million (ppm) relative to TMS
3
as internal standard and recorded at 23 °C. MS (ESI) was determined by using a QTRAP
spectrometer with ion source. The purity of the compounds (>95%) was established by
elemental analysis. Fluorescence and ultraviolet−visible spectra were measured using a
spectrofluorophotometer (RFꢀ5301PC, Shimadzu, Japan) and a UV spectrophotometer (UVꢀ
2450, Shimadzu, Japan), respectively. The fluorescence properties, including absorbance
spectrum, excitation and emission wavelengths of the synthesized probes in PBS were
evaluated on a multiꢀmode spectrophotometer (Spectramax Paradigm, Molecular Devices,
San Francisco, CA, USA). Fluorescence QYs were measured using an aqueous solution of
rhodamine 6G as a standard (Φ = 0.76).
Monomeric Aβ1ꢀ42 was purchased from GL Biochem. Transgenic mice
(APPswe/PSEN1dE9, 9ꢀ10 months old, male) and male C57BL/6 counterparts were provided
from GDMLAC (certification number: 44007200000555).The animals were sacrificed by
decapitation. The brain and eyes of each mouse was removed carefully and cut into slice
11
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 12 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
along the sagittal plane. The protocols of animal test were approved by the Institutional
Animal Care and Use Committee of GDMLAC.
General Method for the Synthesis of compounds 1a and 1b. A solution of the
3
corresponding compound phenothiazine (10 mmol, 1 equiv) in DMF with CH I (20 mmol, 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
eq) and NaH (15 mmol, 1.5 equiv) was stirred at RT for 4 h. The solvent was evaporated
under vacuum. The crude residue was diluted in ethyl acetate, washed with NaHCO , dried
3
2 4
over Na SO , filtered and concentrated. The crude product was purified by silica gel column
chromatography eluted with a mixture of petroleum ether and ethyl acetate.
1
10-methyl-10H-phenothiazine (1a). 2.05 g (96%); H NMR (400 MHz, Acetone) δ
7
.21 (td, J = 8.0, 1.5 Hz, 1H), 7.21 (d, J 2.0 Hz, 1H), 7.14 (dd, J = 8.0, 1.5 Hz, 2H), 6.96 (d,
J = 7.0 Hz, 2H),6.95 (td, J=7.0 Hz, 1.5 Hz, 1H),3.39 (s, 3H).
2-chloro-10-methyl-10H-phenothiazine (1b). 2.40 g (97%); H NMR (400 MHz,
) δ 7.16 (td, J = 7.8, 1.3 Hz, 1H), 7.12 (dd, J = 7.8, 1.3 Hz, 1H), 6.99 (d, J = 8.5Hz,
H), 6.94 (t, J = 7.5Hz, 1H), 6.88 (dd, J = 8.5, 2.0 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.73 (d, J
1
CDCl
3
1
= 2.0 Hz, 1H), 3.29 (s, 3H).
N,N,10-trimethyl-10H-phenothiazin-2-amine (1c). A solution of 1b (2 mmol, 1eq) in
dioxane with tBuONa (4 mmol, 2eq) was purged with argon before the addition of Pd
(0.08 mmol, 0.04 eq) and Xphos (0.16 mmol, 0.04 eq). The mixture was stirred at reflux for
h. The mixture was filtered. The solvent was evaporated. Purification by silica gel column
2 3
(dba)
6
chromatography eluted with a mixture of petroleum ether and ethyl acetate provided the title
1
compound as a yellow oil; 328 mg (64%); H NMR (400 MHz, CDCl
3
) δ 7.16ꢀ7.10 (m, 2H),
6.97 (d, J=8.5, 1H), 6.89 (td, J= 8.0, 1.0 Hz, 1H), 6.8 (dd, J= 8.0, 1.0 Hz, 1H), 6.33 (dd, J =
8.0, 2.5 Hz, 1H), 6.21 (d, J = 2.5, 1H), 3.36(s, 3 H), 2.92 (s, 6H).
General Method for the Synthesis of compounds 2aꢀc. A solution of the
corresponding compound (1a, 1b or 1c, 2.5 mmol, 1 equiv) in DMF was added to a mixture
of POCl (7.5 mmol, 3 eq) and DMF (7.5 mmol, 3 eq) at 0°C. The mixture was stirred at RT
3
for 30 min before heating at 100°C for 7h. The mixture was diluted in DCM and washed with
4
brine in three times. The combined organic phase was dried over MgSO , filtered and
concentrated in vacuum. The crude product was purified by silica gel column chromatography
eluted with a mixture of petroleum ether and ethyl acetate.
1
1
0-methyl-10H-phenothiazine-3-carbaldehyde (2a). 350 mg (58%); H NMR (400
MHz, CDCl ) δ 9.78 (s, 1H), 7.64 (dd, J = 8.7, 2.0 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.17 (td,
3
1
2
ACS Paragon Plus Environment

Page 13 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
J=7.8, 1.6 Hz, 1H), 7.11 (dd, J = 7.8 , 1.6 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 5.4
Hz, 1H), 6.82 (d, J = 5.4 Hz, 1H), 3.41(s, 3H).
1
8
-chloro-10-methyl-10H-phenothiazine-3-carbaldehyde (2b). 262 mg (38%); H NMR
(400 MHz, CDCl3) δ 7.16 (td, J= 7.8, 1.4 Hz, 1H), 7.11 (dd, J = 7.8, 1.4 Hz, 1H), 7.01 (d, J =
8
.2 Hz, 1H), 6.93 (td, J = 7.6, 0.7 Hz, 1H), 6.88 (dd, J = 8.2, 2.0 Hz, 1H), 6.8 (d, J = 8.2 Hz,
H), 6.75 (d, J = 2.0 Hz, 1H), 3.35 (s, 3H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8-(dimethylamino)-10-methyl-10H-phenothiazine-3-carbaldehyde (2c). 305 mg
1
(
43%); H NMR (400 MHz, CDCl
3
) δ9.97 (s, 1H), 7.49 (s, 1H), 7.15 (td, J=8.0, 1.5Hz, 1H),
7
.12 (dd, J = 8.0, 1.5 Hz, 1H), 6.95 (td, J = 7.5, 1.0Hz, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.32 (s,
1H), 3.42 (s, 3H), 2.91 (s, 6H).
General Method for the Synthesis of compounds 3. A mixture of glycine ethyl ester
or tertꢀbutyl ester hydrochloride (3.6 mmol, 1 eq) and bis(carboxymethyl)trithiocarbonate
(7.2 mmol, 1.2 eq) in a mixed solvent of 2ꢀpropanol (4 mL) and triethylamine (1 mL, 2eq)
was stirred for 1 hunder reflux. The solvent was removed, and the residue was purified
bysilica gel chromatography eluted with mixture of petroleum ether and ethyl acetate.
Ethyl 2-(4-oxo-2-thioxothiazolidin-3-yl)acetate (3a1). 639 mg (81%); yellow solid; mp
1
5
5°C; H NMR (400 MHz, CDCl
3
) δ =4.65 (s, 2H), 4.19 (q, J = 7.8 Hz, 2H), 4.01 (s, 2H),
1
.22 (t, J = 7.8 Hz, 3H).
tert-butyl 2-(4-oxo-2-thioxothiazolidin-3-yl)acetate (3a2). 605 mg (68%); yellow solid;
1
mp 56°C; H NMR (400 MHz, CDCl
3
) δ =4.62 (s, 2H), 4.07 (s, 2H), 1.46 (s, 9H).
Ethyl 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoate (3b1). 781 mg (93%); yellow
1
solid; mp 53°C; H NMR (400 MHz, CDCl
3
) δ 4.30 (t, J = 7.8 Hz, 2H), 4.18 (q, J = 7.8 Hz,
2
H), 4.00 (s, 2H), 2.68 (t, J = 7.8 Hz, 2H), 1.27 (t, J = 7.7 Hz, 3H).
tert-butyl3-(4-oxo-2-thioxothiazolidin-3-yl)propanoate (3b2). 583 mg (62%); yellow
1
solid; mp 52°C; H NMR (400 MHz, CDCl
3
) δ 4.25 (t, J = 7.8 Hz, 2H), 3.98 (s, 2H), 2.61 (t, J
=
7.8 Hz, 2H), 1.45 (s, 9H).
General Method for the Synthesis of compounds 4 and 7. A solution of the
corresponding compound (2aꢀc, 0.1 mmol, 1 eq) in DCM with the corresponding Nꢀ
substituted rhodanines derivatives 3 (0.11 mmol, 1.1 eq) and piperidine (0.2 mmol, 2 eq) was
stirred at RT for 10h. The mixture was neutralized with acetic acid, washed with brine, dried
over MgSO
eluted with mixture of petroleum ether and ethyl acetate.
E)-ethyl 2-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-thioxothiazoli
4
and concentrated. The crude was purified by silica gel column chromatography
(
1
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 14 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
din-3-yl)acetate (4a1). 30 mg (68%); Red solid; mp 162°C; H NMR (400 MHz, CDCl
3
) δ
.62 (s, 1H), 7.3 (dd, J = 8.5, 2.0 Hz, 1H), 7.22 (dd, J = 2.0Hz, 1H), 7.17 (td, J = 7.6, 1.4 Hz,
H), 7.12 (dd, J = 7.6, 1.2 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.82 (d,
7
1
J = 8.2 Hz, 1H), 4.8 (s, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.40 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H).
1
3
3
C NMR (400 MHz, CDCl ) δ 192.71, 167.16, 165.95, 148.18, 144.18, 133.12, 131.14,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
28.94, 127.84, 127.47, 127.35, 124.56, 123.55, 122.32, 119.77, 114.70, 114.39, 62.04, 44.91,
+
30.93, 14.13. MS (ESI) m/z: 443.1 [M + H] . Anal. Calcd for C21
H
18
2
N O
3
S
3
: C, 56.99; H,
4.10; N, 6.33. Found: C, 57.16; H, 4.06; N, 6.26.
(E)-tert-butyl
2-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)acetate (4a2). 37 mg (78%); Red solid; mp 132°C; H NMR (400
MHz, CDCl
3
) δ 7.61 (s, 1H), 7.3 (dd, J = 8.0, 2.0 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.17 (td, J
=
7.5, 1.5 Hz, 1H), 7.12 (dd, J = 8.0, 1.5 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 8.0 Hz,
13
1H), 6.82 (d, J = 8.0 Hz, 1H), 4.73 (s, 2H), 3.40 (s, 3H), 1.44 (s, 9H). C NMR (400 MHz,
CDCl ) δ 192.99, 167.40, 165.05, 148.28, 144.38, 133.08, 131.25, 129.10, 127.99, 127.70,
27.51, 124.71, 123.69, 122.51, 120.11, 114.85, 114.54, 83.30, 45.79, 31.10, 20.17. MS (ESI)
3
1
+
m/z: 471.2 [M + H] . Anal. Calcd for C23
8.82; H, 4.65; N, 5.98.
E)-ethyl
22 2 3 3
H N O S : C, 58.70; H, 4.71; N, 5.95. Found: C,
5
(
2-(5-((8-chloro-10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)acetate (4b1). 37 mg (78%); Red solid; mp 173°C; H NMR (400
MHz, CDCl
3
) δ 7.61 (s, 1H), 7.31 (dd, J = 8.5, 2.0 Hz, 1H), 7.2 (d, J = 2.0 Hz, 1H), 7.01 (d, J
=
8.5 Hz, 1H), 6.94 (dd, J = 8.0, 2.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 2.0 Hz,
1
3
1H), 4.83 (s, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.38 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H). C NMR
400 MHz, CDCl ) δ 191.53, 166.68, 164.90, 146.33, 144.37, 132.78, 131.70, 130.10, 129.88,
27.83, 126.76, 123.29, 122.23, 119.78, 119.32, 114.08, 113.66, 61.03, 43.88, 34.72, 13.15.
(
3
1
+
MS (ESI) m/z: 477.2 [M + H] . Anal. Calcd for C21
Found: C, 52.72; H, 3.63; N, 5.90.
2 3 3
H17ClN O S : C, 52.88; H, 3.59; N, 5.87.
(
E)-tert-butyl 2-(5-((8-chloro-10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)acetate (4b2). 41 mg (83%); Red solid; mp 208°C; H NMR (400
MHz, CDCl ) δ 7.60 (s, 1H), 7.3 (dd, J = 8.5, 2.0 Hz, 1H), 7.2 (d, J = 2.0 Hz, 1H), 7.02 (d, J =
3
8
.0 Hz, 1H), 6.94 (dd, J = 8.5, 2.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H),
13
4.73 (s, 2H), 3.38 (s, 3H), 1.23 (s, 9H). C NMR (400 MHz, CDCl
3
) δ 192.68, 167.16,
1
1
64.83, 147.30, 145.43, 132.52, 132.35, 130.90, 128.86, 128.01, 127.82, 124.32, 123.24,
+
20.84, 120.52, 115.10, 114.68, 83.15, 45.62, 35.73, 28.00. MS (ESI) m/z: 505.1 [M + H] .
1
4
ACS Paragon Plus Environment

Page 15 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Anal. Calcd for C23
.49.
2 3 3
H21ClN O S : C, 54.70; H, 4.19; N, 5.55. Found: C, 54.84; H, 4.13; N,
5
(E)-tert-butyl 2-(5-((8-(dimethylamino)-10-methyl-10H-phenothiazin-3-yl)methylene)-
1
4-oxo-2-thioxothiazolidin-3-yl)acetate (4c2). 35 mg (69%); Red solid; mp 155°C; H NMR
(400 MHz, CDCl
3
) δ 7.77 (s, 1H), 7.15 (t, J = 8.0 Hz, 1H), 7.13ꢀ7.09 (m, 2H), 6.94 (t, J = 7.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.45 (s,1H), 4.72 (s, 2H), 3.40 (s, 3H), 2.71 (s, 6H), 1.44 (s,
13
9H). C NMR (400 MHz, CDCl
3
) δ 195.24, 167.95, 165.13, 154.54, 149.55, 144.32, 132.42,
129.97, 127.56, 127.32, 123.35, 123.10, 120.45, 119.33, 116.61, 114.65, 104.71, 82.93, 45.64,
+
4
4.40, 35.67, 20.01. MS (ESI) m/z: 514.2 [M + H] . Anal. Calcd for C H N O S : C, 58.45;
25 27 3 3 3
H, 5.30; N, 8.18. Found: C, 58.62; H, 5.26; N, 8.21.
(E)-ethyl
3-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)propanoate (7a1). 40 mg (87%); Red solid; mp 177°C; H NMR (400
MHz, CDCl
3
) δ 7.58 (s, 1H), 7.28 (dd, J = 8.5, 2.0 Hz, 1H), 7.2 (d,J = 2 Hz,1H), 7.17 (td, J =
.5, 1.5 Hz, 1H), 7.12 (dd, J = 7.5, 1.5 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.84ꢀ6.80 (m, 2H),
.4 (t, J = 7 Hz, 1H), 4.13 (q, J = 7 Hz, 1H), 3.40 (s, 3H), 2.73.4 (t, J = 7.5 Hz, 2H), 1.54 (s,
7
4
1
3
4H), 1.24 (t, J = 7.5 Hz, 3H). C NMR (400 MHz, CDCl
3
) δ 192.86, 170.62, 167.63, 148.19,
1
1
44.33, 132.72, 131.22, 129.04, 127.98, 127.48, 124.67, 123.67, 122.46, 120.02, 114.82,
+
14.50, 61.11, 40.18, 31.62, 31.07, 14.30. MS (ESI) m/z: 457.1 [M + H] . Anal. Calcd for
: C, 57.87; H, 4.42; N, 6.14. Found: C, 57.62; H, 4.50; N, 6.21.
22 20 2 3 3
C H N O S
(E)-tert-butyl
3-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)propanoate (7a2). 39 mg (80%); Red solid; mp 182°C; H NMR (400
MHz, CDCl
3
) δ 7.57 (s, 1H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.2 (d, J = 2 Hz, 1H), 7.17 (td, J =
7
4
.5, 1.5 Hz, 1H), 7.12 (dd, J = 7.5, 1.5 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.84ꢀ6.80 (m, 2H),
13
.36 (t, J = 7 Hz, 2H), 3.39 (s, 3H), 2.65 (t, J = 7.5 Hz, 2H), 1.42 (s, 9H). C NMR (400
) δ 192.73, 170.47, 167.51, 148.07, 144.20, 132.57, 131.06, 128.90, 127.56,
27;35, 124.57, 123.52, 122.34, 119.92, 114.66, 114.34, 60.96, 40.03, 35.65, 31.47, 14.13.
MHz, CDCl
3
1
+
MS (ESI) m/z: 485.2 [M + H] . Anal. Calcd for C24
Found: C, 59.72; H, 4.86; N, 5.61.
24 2 3 3
H N O S : C, 59.48; H, 4.99; N, 5.78.
General Method for the Synthesis of compounds 5 and 8. A solution of the
corresponding compound (4 or 7, 0.05 mmol, 1 equiv) was stirred at RT in a mixture of
DCM/TFA at a ratio of 2:1 for 4 h. The solvent was concentrated under vacuum before the
addition of diethyl ether, the solid was filtrate and wash with diethyl ether.
(E)-2-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
1
yl)acetic acid (5a). 19 mg (92%); Red solid; mp 253°C; H NMR (400 MHz, Acetone) δ 7.71
1
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 16 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(s, 1H), 7.52 (dd, J = 8.5, 2 Hz, 1H), 7.4 (d, J = 2 Hz, 1H), 7.25 (td, J = 7.5, 1 Hz, 1H), 7.17
(dd, J = 7.5, 1 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.05ꢀ7.00 (m, 2H), 4.87 (s, 2H), 3.48 (s, 3H).
1
3
C NMR (400 MHz, DMSO) δ 192.68, 167.23, 166.25, 147.67, 143.71, 133.14, 130.97,
129.08, 128.05, 126.98, 126.90, 123.43, 122.86, 120.98, 118.67, 115.30, 115.18, 44.96, 30.63.
+
MS (ESI) m/z: 415.1 [M + H] . Anal. Calcd for C19
Found: C, 54.87; H, 3.49; N, 6.61.
14 2 3 3
H N O S : C, 55.05; H, 3.40; N, 6.76.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(E)-2-(5-((8-chloro-10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)acetic acid (5b). 20 mg (91%); Red solid; mp 242°C; H NMR (400
MHz, CDCl ) δ 7.79 (s, 1H), 7.53 (dd, J = 8.5, 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.20 (d,
3
1
3
J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.09ꢀ7.05 (m, 2H), 4.71 (s, 2H), 3.38 (s, 3H). C
NMR (400 MHz, CDCl ) δ 189.19, 150.45, 149.89, 144.33, 131.35, 130.00, 129.88, 128.98,
27.84, 126.92, 126.66, 124.26, 112.76, 107.07, 99.43, 39.81, 28.70. MS (ESI) m/z: 449.1 [M
3
1
+
+ H] . Anal. Calcd for C19
N, 6.32.
2 3 3
H13ClN O S : C, 50.83; H, 2.92; N, 6.24. Found: C, 50.99; H, 2.86;
(E)-2-(5-((8-(dimethylamino)-10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
1
thioxothiazolidin-3-yl)acetic acid (5c). 21 mg (93%); Red solid; mp 289°C; H NMR (400
MHz, DMSO) δ 7.58 (s, 1H), 7.31 (s, 1H), 7.24 (td, J = 8.0, 1.5Hz, 1H), 7.17 (dd, J = 8.0, 1
Hz,1H), 7.02 (d, J = 8.0 Hz, 1H), 7.0 (d, J = 8.0Hz, 1H), 6.67 (s,1H), 4.70 (s, 2H), 3.41 (s,
1
3
3H), 2.70 (s, 6H). C NMR (400 MHz, DMSO) δ 195.56, 167.94, 167.63, 154.52, 149.70,
144.37, 133.09, 130.76, 128.34, 127.33, 123.77, 122.21, 119.98, 118.50, 115.81, 115.55,
+
1
06.05, 45.46, 44.26, 31.16. MS (ESI) m/z: 458.1 [M + H] . Anal. Calcd for C21
H
19
N
3
O
3
3
S :
C, 55.12; H, 4.19; N, 9.18. Found: C, 55.72; H, 4.12; N, 9.21.
(E)-3-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-
1
yl)propanoic acid (8). 19 mg (89%); Red solid; mp 250°C; H NMR (400 MHz, DMSO)
δ12.51 (bs, 1H), 7.72 (s, 1H), 7.48 (dd, J = 8.5, 2.0 Hz, 1H), 7.42 (d, J = 2 Hz,1H), 7.25 (td, J
=
7.5, 1.5 Hz, 1H), 7.19 (dd, J = 7.5, 1.5 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.05ꢀ6.99 (m, 2H),
13
4
.22 (t, J = 7 Hz, 2H), 3.38 (s, 3H), 2.62 (t, J = 7.5 Hz, 2H). C NMR (400 MHz, CDCl
3
) δ
169.68, 166.71, 147.58, 144.59, 143.71, 133.23, 129.92, 128.10, 127.70, 127.27, 125.84,
124.14, 123.18, 123.07, 122.46, 121.08, 114.46, 114.17, 81.25, 40.12, 35.61, 32.54, 28.04.
+
MS (ESI) m/z: 429.1 [M + H] . Anal. Calcd for C20
Found: C, 55.87; H, 3.86; N, 6.45.
16 2 3 3
H N O S : C, 56.06; H, 3.76; N, 6.54.
(E)-N,N-dimethyl-2-(5-((10-methyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)acetamide (6). A solution of 4c (0.05 mmol, 1 eq) in DCM and
Dimethylamine (0.1 mmol, 2 eq) and DIPEA (0.13 mmol, 2.5 eq) was added BOP (0.1 mmol,
1
6
ACS Paragon Plus Environment

Page 17 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
2 eq). The mixture was stirred for 3h. The mixture was washed with brines, dried over MgSO
4
and concentrated under vacuum. Purified by silica gel column chromatography eluted with a
mixture of petroleum ether and ethyl acetate afforded the title compound as a red solid, 20
1
mg (90 %); mp 217°C; H NMR (400 MHz, CDCl
3
) δ 7.61 (s, 1H), 7.3 (dd, J = 8.5, 2 Hz,
1H), 7.21 (d, J = 2 Hz,1H), 7.17 (td, J = 7.5, 1.5 Hz, 1H), 7.12 (dd, J = 7.5, 1.5 Hz, 1H), 6.96
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
1
td, J = 7.5, 1 Hz 1H), 6.85ꢀ6.80 (m, 2H), 4.91 (s, 2H), 3.40 (s, 3H), 3.11 (s, 3H), 2.97 (s, 3H).
3
3
C NMR (400 MHz, CDCl ) δ 192.15, 166.42, 164.90, 147.60, 144.59, 143.92, 133.69,
1
29.89, 128.14, 127.70, 127.27, 125.89, 124.13, 123.17, 121.03, 114.46, 114.17, 45.57, 35.61,
+
2
7.98. MS (ESI) m/z: 442.1 [M + H] . Anal. Calcd for C H N O S : C, 57.12; H, 4.34; N,
21
19
3
2 3
9.52. Found: C, 57.47; H, 4.45; N, 9.57.
Preparation of Aβ monomer and aggregates. Aβ1ꢀ42monomer was prepared by
1
1
dissolving Aβ1ꢀ42 powder in HFIP, which is sonicated and stirred for 24h at RT before dried
under argon to give a clear film. Aβ1ꢀ42 monomer was reꢀsuspended in DMSO (200 µM), and
stored at ꢀ20°C prior to use. Aβ1ꢀ42 aggregates were prepared using the solution of Aβ1ꢀ42
monomer (200 µM in DMSO), which was diluted with PBS buffer (pH 7.4) at 20 µM, stirred
at 37°C for 2 days, and store at ꢀ20°C.
Tissue Preparation. Brain and eyes were collected from each mouse, formalinꢀfixed
and embedded in paraffin. Then, the brain was coronally sectioned (4 µm) and the eyes was
sagitally sectioned (4µm).
Fluorescence studies upon binding to Aβ1ꢀ42 aggregates and BSA. A solution of 10
ꢀL of aggregated Aβ1ꢀ42 (2 µM) or BSA (40 ꢁg/mL) was added to the fluorescent probes (10
ꢁ
L, 1 ꢁM in PBS, pH 7.4) in a final volume of 20 µL. The mixture was incubated for 30 min
at RT, and the fluorescent parameters (fluorescence excitation/emission wavelength and
intensity) were measured by a multiꢀmode spectrophotometer (Spectramax Paradigm,
Molecular Devices, San Francisco, CA, USA).
Measurement of cell viability. Cell viability was determined by a quantitative
colorimetric assay using MTT. SHꢀSY5Y cells were seeded at 5000 cells per well in 96ꢀwell
plates and incubated overnight. Then the cells were treated with the compounds at different
concentrations for 1h before exposed to a laser of an spectrum imaging system or not. After
further incubation for 48 h, MTT solution (2 mg/mL) was added, and the cells were incubated
for another 30 min. The absorbance of each well was measured at 570 nm.
1
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 18 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In vitro Aβ1ꢀ42 aggregates binding constant measurement. A solution of aggregated
Aβ1ꢀ42 aggregates (1 µM in the final assay mixture) was mixed with different concentrations
ꢀ5
ꢀ10
of the fluorescent probes (10 to 10 in PBS) and incubated for 30 min at RT. The
fluorescent intensity was measured by a multiꢀmode spectrophotometer (Spectramax
Paradigm, Molecular Devices, San Francisco, CA, USA). The resulting intensities, corrected
where for background fluorescence were plotted in Grafpad Prism. Kd values were
determined using the singleꢀsite ligand binding module. Each measurement was run in
triplicate.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In vitro staining of Aβ plaques in transgenic mouse brain and eye. Paraffinꢀ
embedded brain and eye tissue from double transgenic mouse and control mouse were used
for in vitro fluorescent staining. The brain (4 ꢁm) and eye (4 ꢁm) sections were deparaffinized
with 2x20 min washes in xylene, 2x5 min washes in 80% ethanol/H O, a 5 min wash in 60%
2
2
ethanol/H O and running tap water for 10 min and then incubated in PBS for 30 min.
For fluorescent staining of Aβ plaques, they were incubated with fluorescent probe 4a1
(10 nM) for 30 min. The location of plaques was confirmed by staining adjacent section with
ThT. Finally, the sections were washed with 40% ethanol and PBS for 10 min.
For in vitro staining of Aβ plaques with Aβ antibody, they were incubated with primary
Aβ antibody (ab2454, Cell signaling, dilution ratio A: 500) at 4°C overnight. The sections
were washed with 3x5 min PBS and incubated with secondary antibody with fluorophore
Alexa 488 (Invitrogen, dilution ratio 1:200) for 60 min and then washed with 3x5 min PBS.
Fluorescent observation was performed using a fluorescence microscope (Olympus
IX71 inverted fluorescence microscope).
In vitro inhibition and disaggregation of Aβ 1ꢀ42 Aggregation. Inhibition of Aβ 1ꢀ42
aggregation was performed by incubation of a mixture of Aβ 1ꢀ42 monomer (10 µL, 20 µM)
with or without the tested compound (10 µL, 2 µM) at 37°C for 48 h. 20 µL of thioflavinꢀT at
5
µM was added. The fluorescence intensities were recorded 5 min later (excitation 450 nm;
emission 485 nm). The percent inhibition of aggregation was calculated by the expression (1ꢀ
IFi/IFc)*100, in whichIFi and IFc are the fluorescence intensities obtained for Aβ in the
1
8
ACS Paragon Plus Environment

Page 19 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
presence and absence of inhibitors after subtracting the background, respectively. Each
measurement was run in triplicate.
Disaggregation of preformed Aβ aggregates was carried out as following: Aβ 1ꢀ42
monomer (20 µM) was incubated for 3 days at 37°C to generate aggregates. Then the solution
was mixed with or without the tested compound at various concentrations and incubated for 3
days at 37°C. Finally, ThT at 5 µM was added and the fluorescence intensities were recorded
(excitation 440 nm, emission 480 nm).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fluorescence staining of inhibition of selfꢀaggregation. The inhibition of Aβ 1ꢀ42
selfꢀaggregation was followed by fluorescence microscopy. Aβ1ꢀ42 monomer (50 µL, 20 µM)
with or without the tested compound (50 µL, 2 µM or 20 µM) at 37°C for 48 h. The formation
of aggregate was observed by fluorescence microscope (Olympus IX71 inverted fluorescence
microscope). Digital images of four random fields were taken at 10x magnification. The
fluorescence intensity was calculated by measuring the average pixel intensity per field with
the program ImageJ.
Statistical Analysis. Data were analyzed using the InStat software program (GraphPad
Software, San Diego, CA). Student’s t test was used. All values are expressed as mean ± SD.
p ≤ 0.05 was considered statistically significant.
AUTHOR INFORMATION
Corresponding Author
*Permanent address: CNRS, UMR8601, Laboratoire de Chimie et Biochimie
Pharmacologiques et Toxicologiques, CBNIT, Université Paris Descartes, PRES Sorbonne
Paris Cité, UFR Biomédicale, 45 rue des SaintsꢀPères, 75270 Paris Cedex 06, France. Eꢀmail:
huixiong.chen@parisdescartes.fr. Phone: 33ꢀ1ꢀ42864085, Fax: 33ꢀ1ꢀ42864082.
1
9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 20 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Author contributions
HC and PD conceived and designed the experiments. PD and FY performed the experiments.
HC, PD, ZYD, KZ, CZD and BM analyzed the data. YL contributed reagents, materials, and
analysis tools. HC and PD wrote the paper.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Funding
This study is supported by China Postdoctoral Science Foundation (grant no.2015M572280)
and China Science Foundation (grant no. 21672043).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The technical assistance of Guo Q. H. and Wang G. H. is gratefully acknowledged.
REFERENCE
1
. Thies W, Bleiler L. (2016) 2016 Alzheimer’s disease facts and figures. Alzheimers Dement.
J. Alzheimers Assoc.12, 459–509.
. Shatenstein, B., BarbergerꢀGateau, P., and Mecocci, P. (2015) Prevention of AgeꢀRelated
2
Cognitive Decline: Which Strategies, When, and for Whom? J. Alzheimers Dis. JAD48, 35–
53.
3. Lang, A. E. (2010) Clinical trials of diseaseꢀmodifying therapies for neurodegenerative
diseases: the challenges and the future. Nat. Med. 16, 1223–1226.
4. Salloway, S., Sperling, R., and Brashear, H. R. (2014) Phase 3 trials of solanezumab and
bapineuzumab for Alzheimer’s disease. N. Engl. J. Med. 370, 1460.
5. Jack, C. R., Albert, M. S., Knopman, D. S., McKhann, G. M., Sperling, R. A., Carrillo, M.
C., Thies, B., and Phelps, C. H. (2011) Introduction to the recommendations from the
National Institute on AgingꢀAlzheimer’s Association workgroups on diagnostic guidelines for
Alzheimer’s disease. Alzheimers Dement. J. Alzheimers Assoc. 7, 257–262.
6. Hardy, J., and Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer’s disease:
progress and problems on the road to therapeutics. Science 297, 353–356.
2
0
ACS Paragon Plus Environment

Page 21 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
7. Selkoe, D. J. (1999) Translating cell biology into therapeutic advances in Alzheimer’s
disease. Nature 399, A23ꢀ31.
8
. MeyerꢀLuehmann, M., SpiresꢀJones, T. L., Prada, C., GarciaꢀAlloza, M., de Calignon, A.,
Rozkalne, A., KoenigsknechtꢀTalboo, J., Holtzman, D. M., Bacskai, B. J., and Hyman, B. T.
2008) Rapid appearance and local toxicity of amyloidꢀbeta plaques in a mouse model of
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Alzheimer’s disease. Nature 451, 720–724.
9. Bolognesi, M. L. (2013) Amyloid Chemical Probes and Theranostics: Steps Toward
Personalized Medicine in Neurodegenerative Diseases, in Medicinal Chemistry Approaches to
Personalized Medicine (Lackey, K., and Roth, B. D., Eds.), pp 211–226. WileyꢀVCH Verlag
GmbH & Co. KGaA.
10. Staderini, M., Martín, M. A., Bolognesi, M. L., and Menéndez, J. C. (2015) Imaging of βꢀ
amyloid plaques by near infrared fluorescent tracers: a new frontier for chemical
neuroscience. Chem. Soc. Rev. 44, 1807–1819.
11. Kadir, A., Marutle, A., Gonzalez, D., Schöll, M., Almkvist, O., Mousavi, M., Mustafiz,
T., DarrehꢀShori, T., Nennesmo, I., and Nordberg, A. (2011) Positron emission tomography
imaging and clinical progression in relation to molecular pathology in the first Pittsburgh
Compound B positron emission tomography patient with Alzheimer’s disease. Brain J.
Neurol. 134, 301–317.
12. Camus, V., Payoux, P., Barré, L., Desgranges, B., Voisin, T., Tauber, C., La Joie, R.,
Tafani, M., Hommet, C., Chételat, G., Mondon, K., de La Sayette, V., Cottier, J. P., Beaufils,
E., Ribeiro, M. J., Gissot, V., Vierron, E., Vercouillie, J., Vellas, B., Eustache, F., and
Guilloteau, D. (2012) Using PET with 18FꢀAVꢀ45 (florbetapir) to quantify brain amyloid load
in a clinical environment. Eur. J. Nucl. Med. Mol. Imaging 39, 621–631.
13. Raymond, S. B., Kumar, A. T. N., Boas, D. A., and Bacskai, B. J. (2009) Optimal
parameters for near infrared fluorescence imaging of amyloid plaques in Alzheimer’s disease
mouse models. Phys. Med. Biol. 54, 6201.
1
4. Yi, X., Wang, F., Qin, W., Yang, X., and Yuan, J. (2014) Nearꢀinfrared fluorescent probes
in cancer imaging and therapy: an emerging field. Int. J. Nanomedicine 9, 1347–1365.
5. Kosaka, N., Ogawa, M., Choyke, P. L., and Kobayashi, H. (2009) Clinical implications of
1
nearꢀinfrared fluorescence imaging in cancer. Future Oncol. Lond. Engl.5, 1501–1511.
16. Nesterov, E. E., Skoch, J., Hyman, B. T., Klunk, W. E., Bacskai, B. J., and Swager, T. M.
(2005) In Vivo Optical Imaging of Amyloid Aggregates in Brain: Design of Fluorescent
Markers. Angew. Chem. Int. Ed. 44, 5452–5456.
2
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 22 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
17. Ran, C., Xu, X., Raymond, S. B., Ferrara, B. J., Neal, K., Bacskai, B. J., Medarova, Z.,
and Moore, A. (2009) Design, synthesis, and testing of difluoroboronꢀderivatized curcumins
as nearꢀinfrared probes for in vivo detection of amyloidꢀbeta deposits. J. Am. Chem. Soc.131,
15257–15261.
18. Hintersteiner, M., Enz, A., Frey, P., Jaton, A.ꢀL., Kinzy, W., Kneuer, R., Neumann, U.,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rudin, M., Staufenbiel, M., Stoeckli, M., Wiederhold, K.ꢀH., and Gremlich, H.ꢀU. (2005) In
vivo detection of amyloidꢀbeta deposits by nearꢀinfrared imaging using an oxazineꢀderivative
probe. Nat. Biotechnol. 23, 577–583.
19. Fu, H., Tu, P., Zhao, L., Dai, J., Liu, B., and Cui, M. (2016) Amyloidꢀβ Deposits Target
Efficient NearꢀInfrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo
Imaging. Anal. Chem.88, 1944–1950.
2
0. Ono, M., Watanabe, H., Kimura, H., and Saji, H. (2012) BODIPYꢀbased molecular probe
for imaging of cerebral βꢀamyloid plaques. ACS Chem. Neurosci. 3, 319–324.
1. Okamura, N., Mori, M., Furumoto, S., Yoshikawa, T., Harada, R., Ito, S., Fujikawa, Y.,
2
Arai, H., Yanai, K., and Kudo, Y. (2011) In vivo detection of amyloid plaques in the mouse
brain using the nearꢀinfrared fluorescence probe THKꢀ265. J. Alzheimers Dis. JAD23, 37–48.
22. KoronyoꢀHamaoui, M., Koronyo, Y., Ljubimov, A. V., Miller, C. A., Ko, M. K., Black,
K. L., Schwartz, M., and Farkas, D. L. (2011) Identification of amyloid plaques in retinas
from Alzheimer’s patients and noninvasive in vivo optical imaging of retinal plaques in a
mouse model. NeuroImage 54 Suppl 1, S204ꢀ217.
23. Frost, S., Kanagasingam, Y., Sohrabi, H., Vignarajan, J., Bourgeat, P., Salvado, O.,
Villemagne, V., Rowe, C. C., Macaulay, S. L., Szoeke, C., Ellis, K. A., Ames, D., Masters, C.
L., RaineyꢀSmith, S., Martins, R. N., and AIBL Research Group, null. (2013) Retinal
vascular biomarkers for early detection and monitoring of Alzheimer’s disease. Transl.
Psychiatry 3, e233.
24. Cordeiro, M. F., Guo, L., Coxon, K. M., Duggan, J., Nizari, S., Normando, E. M., Sensi,
S. L., Sillito, A. M., Fitzke, F. W., Salt, T. E., and Moss, S. E. (2010) Imaging multiple phases
of neurodegeneration: a novel approach to assessing cell death in vivo. Cell Death Dis.1, e3.
25. Yang F., Lim G. P., Begum A. N., Ubeda O. J., Simmons M. R., Ambegaokar S. S., Chen
P. P., Kayed R., Glabe C. G., Frautschy S. A., Cole G. M. (2005) Curcumin inhibits formation
of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J. Biol.
Chem. 280(7):5892ꢀ901.
2
2
ACS Paragon Plus Environment

Page 23 of 24
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 24 of 24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4a
1
50000
100000
λem : 670 nm
5
0000
Kd : 7.5 nM
0
M
M
M
µ
µ
µ
1
1
0
2
T
1
h
a
4
4a1
T
ACS Paragon Plus Environment