Fluorescent 1,4-Naphthoquinones to Visualize Diffuse and Dense-Core Amyloid Plaques in APP/PS1 Transgenic Mouse Brains

Article abstract of DOI:10.1021/acschemneuro.9b00093

Recent clinical approvals of brain imaging radiotracers targeting amyloid-β provided clinicians the tools to detect and confirm Alzheimer's disease pathology without autopsy or biopsy. While current imaging agents are effective in postsymptomatic Alzheimer's patients, there is much room for improvement in earlier diagnosis, hence prompting a need for new and improved amyloid imaging agents. Here we synthesized 41 novel 1,4-naphthoquinone derivatives and initially discovered 14 antiamyloidogenic compounds via in vitro amyloid-β aggregation assay; however, qualitative analyses of these compounds produced conflicting results and required further investigation. Follow-up docking and biophysical studies revealed that four of these compounds penetrate the blood-brain barrier, directly bind to amyloid-β aggregates, and enhance fluorescence properties upon interaction. These compounds specifically stain both diffuse and dense-core amyloid-β plaques in brain sections of APP/PS1 double transgenic Alzheimer's mouse models. Our findings suggest 1,4-naphthoquinones as a new scaffold for amyloid-β imaging agents for early stage Alzheimer's.

Full text of DOI:10.1021/acschemneuro.9b00093

Research Article
pubs.acs.org/chemneuro
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Fluorescent 1,4-Naphthoquinones To Visualize Diffuse and Dense-
Core Amyloid Plaques in APP/PS1 Transgenic Mouse Brains
Naewoo Neo Shin,† Hanna Jeon,‡ Youngeun Jung,‡ Seungyeop Baek,‡,∥ Sejin Lee,‡ Hee Chan Yoo,‡
Gi Hun Bae,‡ Keunwan Park,⊥ Seung-Hoon Yang,§ Jung Min Han,‡ Ikyon Kim,*,‡
and YoungSoo Kim*,†,‡
†Integrated Science and Engineering Division, Yonsei University, Incheon 21983, Republic of Korea
‡Department of Pharmacy and Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 21983, Republic of Korea
∥Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
⊥Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung 25451, Republic of Korea
§Department of Medical Biotechnology, College of Life Science and Biotechnology, Dongguk University, Seoul 04620, Republic of
Korea
S
* Supporting Information
ABSTRACT: Recent clinical approvals of brain imaging radiotracers targeting
amyloid-β provided clinicians the tools to detect and confirm Alzheimer’s
disease pathology without autopsy or biopsy. While current imaging agents are
effective in postsymptomatic Alzheimer’s patients, there is much room for
improvement in earlier diagnosis, hence prompting a need for new and
improved amyloid imaging agents. Here we synthesized 41 novel 1,4-
naphthoquinone derivatives and initially discovered 14 antiamyloidogenic
compounds via in vitro amyloid-β aggregation assay; however, qualitative
analyses of these compounds produced conflicting results and required further
investigation. Follow-up docking and biophysical studies revealed that four of
these compounds penetrate the blood-brain barrier, directly bind to amyloid-β
aggregates, and enhance fluorescence properties upon interaction. These
compounds specifically stain both diffuse and dense-core amyloid-β plaques in
brain sections of APP/PS1 double transgenic Alzheimer’s mouse models. Our
findings suggest 1,4-naphthoquinones as a new scaffold for amyloid-β imaging
agents for early stage Alzheimer’s.
KEYWORDS: Alzheimer’s disease, amyloid, diffuse plaques, 1,4-naphthoquinone, imaging agent, Aβ
abundant than dense-core plaques during the early stages and
produce false positives due to the imaging agents having low
affinity toward diffuse plaques.3 To enhance accuracy for early
diagnosis of AD patients, it would be ideal to develop an
amyloid imaging agent that specifically labels diffuse plaques in
addition to dense-core plaques.
Naphthoquinones (NQ) are colored chemical compounds
that exist in nature as secondary metabolites, or organic
products that are not directly involved in growth or
development.4,5 Generally, secondary metabolites play a vital
role in environmental adaptation, defense mechanisms, photo-
protection, and structure stabilization.6 First discovered as a
major component of plants used in many traditional medicines
in Asian countries, NQs have garnered considerable interests
from researchers due to their antibacterial, antifungal,
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common neuro-
degenerative disorder accompanied by extracellular deposition
of amyloid-β (Aβ) plaques in the brain.1 Aβ monomers begin
to aggregate into fibrils and eventually plaques in AD patients
least a decade before symptomatic onset. Thus, Aβ aggregation
has become one of the main targets of AD diagnostic and
therapeutic development.2 Previous reports suggest that the
buildup of diffuse Aβ plaques, amorphous deposits that lack
dense cores or dystrophic neurites, in the cortex is considered
the first pathological change in presymptomatic AD. Although
the precise mechanism is still unclear, it is widely believed that
diffuse plaques are precursors of dense-core plaques, or
amyloid deposits with a compact core surrounded by
dystrophic neurites.3 Current clinically approved amyloid
imaging radiotracers are utilized to primarily detect dense-
core plaques in patients with mild cognitive impairment and
AD; however, this approach encounters limitations when used
for presymptomatic diagnosis, as diffuse plaques are more
Received: February 11, 2019
Accepted: April 15, 2019
© XXXX American Chemical Society
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Figure 1. Chemical structures of novel 41 derivatives of 1,4-NQ. A chemical library of 41 novel fluorescent 1,4-naphthoquinone derivatives was
synthesized. See also Figure S4 for NMR spectrum data.
antitumor, and insecticidal properties.7−10 The diverse set of
pharmacological activities displayed by this small molecule
makes it very attractive as a building block for synthesis of
novel drugs. Recent studies also discovered that several
derivatives of its most stable isomeric form, 1,4-naphthoqui-
nones (1,4-NQ) can exhibit neuroprotective effects and disrupt
Aβ aggregation.11−15 This suggests that 1,4-NQ derivatives
may play a key role in AD treatment.
The original aim of this study was to synthesize and screen
novel 1,4-NQ derivatives against Aβ aggregation to identify
possible drug candidates for AD. First, we synthesized 41 novel
compounds and evaluated the effects of them on Aβ aggregates
by conducting Thioflavin T (ThT) fluorescence assay for
inhibition of Aβ fibrilization; we then selected 14 compounds
to check for dissociation of preformed Aβ aggregates. We
discovered discrepancies in our data after performing gel
electrophoresis for qualitative analysis, as it was not consistent
with our primary results. We suspected that these compounds
might interfere with ThT, which binds to β-sheet structures of
Aβ plaques, in these studies; as such, we measured the
fluorescence spectrum of the selected compounds and found
that four emit fluorescence in the presence of Aβ. We also
performed histological staining in aged B6C3Tg (APPswe,
PSEN 1dE9)85Dbo/J (APP/PS1) double transgenic AD
mouse model brains using these compounds and found that
they specifically stain Aβ plaques. We further assessed their
potential as possible amyloid imaging agents by screening the
compounds through an artificial blood-brain barrier (BBB)
membrane by parallel artificial membrane permeability assay
(PAMPA). Surface plasmon resonance analysis and docking
model studies further confirms the strong Aβ-binding affinity
of an imaging agent candidate.
B
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Figure 2. ThT assay for anti-Aβ aggregation activity of 1,4-NQ derivatives. ThT fluorescence assay was conducted for (A) inhibition of Aβ42
aggregation and (B) disaggregation of Aβ42 aggregates using 50 μM Aβ42 with 0.5, 5, or 50 μM YQ compounds as shown. Fluorescence intensities
(FI) of all samples were normalized to Aβ aggregates that were incubated for 3 days, or 3 d (100%). Data represents the mean of three repeated
experiments SDs, and one-way ANOVA was performed followed by Bonferroni’s posthoc comparisons test (*P < 0.033, **P < 0.002, ***P <
0.001).
to induce inhibition. We selected 14 compounds, YQ-001,
YQ-002, YQ-003, YQ-004, YQ-010, YQ-011, YQ-019, YQ-
029, YQ-030, YQ-031, YQ-032, YQ-033, YQ-034, and YQ-
036, with the highest inhibitory effects on Aβ aggregates
(below 50% fluorescence intensity) for the next disaggregation
assay to see if they can dissociate pre-existing Aβ42 aggregates
(Figure 2A). For the disaggregation assay, Aβ42 aggregates
were prepared before mixing with the 1,4-NQ compounds.
Among the 14 compounds, we found that 10 compounds, YQ-
RESULTS
■
ThT Assay for Anti-Aβ Aggregation Activity of 1,4-NQ
Derivatives. Here we examined whether the synthesized 41
derivatives of 1,4-NQ can inhibit Aβ42 aggregates formation
and dissociate preformed Aβ aggregates by conducting ThT
fluorescence assay (Figure 1). ThT is a dye that undergoes
conformation and fluoresces after binding to cross-β-sheet-rich
structures of Aβ aggregates.16 In inhibition assays, each
compound was added to monomeric Aβ42 prior to incubation
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Figure 3. SDS-PAGE analysis to confirm inhibition and disaggregation effects of 1,4-NQ derivatives on Aβ42. Candidate compounds were selected
for SDS-PAGE analysis on (A) inhibition of Aβ42 aggregation and (B) disaggregation of Aβ42 aggregates. Samples were incubated for 3 days (+)
or 6 days (++) total. Aβ42 samples were prepared by carrying out PICUP chemistry to stabilize oligomer populations and analyzed on 15%
gradient polyacrylamide gels. Samples are distributed by monomers (5 kDa), dimers (10 kDa), oligomers (15 to 75 kDa), and larger aggregates or
fibrils (trapped at the top of the gels). The products were observed by using silver staining. See also Figure S1.
001, YQ-003, YQ-010, YQ-011, YQ-019, YQ-030, YQ-031,
YQ-032, YQ-033, and YQ-036, reduced β-sheet-rich Aβ42
aggregates significantly at 50 μM (Figure 2B). Taken together,
ThT fluorescence assay reveals 14 compounds that inhibit
aggregation of Aβ and 10 compounds that disaggregate Aβ
aggregates.
Fluorescence Spectral Scan of 1,4-NQ Derivatives.
Given the conflicting results of the ThT assay and SDS-PAGE
analysis, we suspected that the 1,4-NQ derivatives may be
fluorescent and thus affected the ThT assay results of each
sample. To verify our hypothesis, we conducted fluorescence
spectroscopy to measure the fluorescent property of the
selected 14 compounds with and without presence of Aβ
aggregates. We recorded the absorbance spectra of the selected
14 of the 1,4-NQ derivatives to obtain the fluorescent
excitation wavelength, or the highest peak of the spectrum
(Figure S2A). To obtain the emission point, we measured the
fluorescence emission spectra by exciting each compound
according to their excitation wavelengths respectively (Figure
S2B). The spectral activities of the compounds overlap with
the range of the ThT dye, which supports our hypothesis that
these compounds may have induced false positives in ThT
assay due to fluorescence interference.19 Afterward, we selected
nine candidates suitable for fluorescence detection of Aβ
aggregates in vitro and ex vivo: YQ-002, YQ-003, YQ-004,
YQ-030, YQ-031, YQ-032, YQ-033, YQ-034, and YQ-036.
If our fluorescent compounds interact with Aβ, there could
be a noticeable change in the fluorescence spectrum or
intensity. The selected nine YQ compounds were added to Aβ
monomers or Aβ aggregates. Of the nine compounds, YQ-002,
YQ-003, YQ-030, and YQ-036 have increased fluorescence
when mixed with Aβ monomers or aggregates (Figure 4A).
When in the presence of Aβ monomers or aggregates, YQ-030
produces a drastic increase in fluorescence by nearly 2-fold at
its emission peak, whereas YQ-002, YQ-003, and YQ-036 did
not show a significant increase fluorescence intensity at their
emission peaks. The compounds YQ-002 and YQ-003
exhibited higher fluorescence intensities when in the presence
of Aβ monomers and aggregates, while YQ-036 enhanced
fluorescence in the presence of Aβ aggregates between specific
spectrum regions (YQ-002: 480−670 nm, YQ-003: 580−700
nm, YQ-036: 500−680 nm). Among these four compounds,
Gel Electrophoresis To Confirm Anti-Aβ Aggregation
Effects of 1,4-NQ Derivatives on Aβ42. Even though ThT
is a fluorescent dye regularly used to quantify Aβ fibrils, this
colorimetric assay cannot monitor changes in oligomerization.
Furthermore, the assay could produce false-positive data as the
reduction due to its heavily reliance on ThT binding to β-
sheet-rich structures, as reduced fluorescence intensity may be
attributed to interference by the 1,4-NQ compounds.17
Abnormal behaviors of most of chemicals in disaggregation
assays support such a possibility by showing levels of fibrils
increased in lower dosage (0.5 μM) but reduced in higher
dosage (50 μM) (Figure 2B). As such, we performed SDS-
PAGE analysis with photoinduced cross-linking of unmodified
proteins (PICUP) to visualize Aβ monomers, oligomers, and
fibrils and confirm the effects of 1,4-NQ derivatives YQ-001,
YQ-002, YQ-003, YQ-004, YQ-010, YQ-011, YQ-019, YQ-
029, YQ-030, YQ-031, YQ-032, YQ-033, YQ-034, and YQ-
036 on Aβ aggregation. Due to the metastable nature of
oligomers (oligomers dynamically disaggregate into monomers
and aggregate into fibrils over time) PICUP chemistry is a
necessary procedure that covalently stabilizes oligomers to
enable clear visualization of the different distributions of Aβ in
SDS-PAGE.18 The SDS-PAGE analysis results revealed that
none of the compounds have inhibited aggregation of Aβ
(Figure 3A) or reduced Aβ fibrils (Figure 3B), which is
inconsistent with the previous ThT assay results. We
determined that all 41 1,4-NQ derivatives did not regulate
Aβ aggregate formation and that the ThT assay may have
produced false-positive data.
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Figure 4. Fluorescence spectral scan of selected 1,4-NQ derivatives and histochemistry of AD model mouse brain tissue by selected 1,4-NQ
derivatives. (A) Emission spectra of YQ compounds (50 μM) with synthesized Aβ42 (50 μM) in 3:1 ratio were measured by using their respective
excitation wavelengths to show the fluorescence intensity (FI) between two ranges. The excitation wavelengths for the compounds are as follows:
YQ-002, 400 nm; YQ-003, 470 nm; YQ-004, 400 nm; YQ-030, 490 nm; YQ-031, 510 nm; YQ-032, 470 nm; YQ-033, 460 nm; YQ-034, 470 nm;
YQ-036, 410 nm. These graphs contain four control groups (Aβ monomer, Aβ aggregates, blank, YQ) and two experimental groups (YQ + Aβ
monomer, YQ + Aβ aggregates). Spectra of all samples were measured using an Infinite 200 PRO plate reader. See also Figure S2. (B)
Histochemistry of aged male APP/PS1 double transgenic mouse brains was conducted by staining brain slices with 6E10 or YQ compounds (YQ-
002, YQ-003, YQ-030, and YQ-036). The images of 6E10 and YQ compounds were merged to confirm that both demonstrate colocalization on
both diffuse (arrow) and dense-core (arrowhead) Aβ42 plaques. Scale bars = 100 μm.
YQ-003 is the only compound that enhances fluorescence by
up to 50-fold when emission is collected from 680 to 750 nm.
Altogether, we found four compounds that enhance or alter
their fluorescence properties in the presence of Aβ aggregates.
The fluorescent enhancements of 1,4-NQ derivatives in the
presence of Aβ imply that these compounds may have
potential to be amyloid imaging agents.
Histochemistry of AD Model Mouse Brain Tissue by
1,4-NQ Derivatives. To ascertain whether these 1,4-NQ
derivatives can detect Aβ plaques ex vivo, we conducted
histochemistry on fixed mouse brain tissues of aged APP/PS1
double transgenic AD mouse models, which are reported to
develop amyloid plaques in the cortex at around the age of
6months.20 We performed brain staining using four YQ
compounds and anti-Aβ monoclonal antibody 6E10. Thio-
flavin S only labels dense-core plaques whereas 6E10 can stain
both dense-core and diffuse plaques. We observed that these
1,4-NQ derivatives are colocalized with 6E10 on plaques
containing dense cores with or without surrounding diffuse Aβ
assemlies in the hippocampal and cortical regions (Figure 4B).
Of these compounds, YQ-002, YQ-003, and YQ-036 were
deemed as promising imaging agents for staining Aβ plaques in
APP/PS1 mouse brains as they demonstrate the highest
fluorescence contrast and overlap with 6E10 more effectively
than YQ-030 does.
Molecular Docking Models of the Fluorescent
Compounds to Aβ. The molecular docking models for
YQ-002, YQ-003, YQ-030, and YQ-036 were investigated due
to their increased fluorescence when mixed with Aβ and their
histochemical plaque-staining function. Since there was no
prior knowledge on binding sites for the new compounds, we
first performed an exhaustive global (low-resolution) docking
conformation search by using PatchDock software21 to predict
potential binding pockets in Aβ. As a result, center of mass
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Figure 5. Molecular docking models for selected 1,4-NQ derivatives. Molecular docking models of (A) YQ-002, (B) YQ-003, (C) YQ-030, and
(D) YQ-036 are presented by docking simulations using PatchDock and Autodock vina. To clarify the binding modes, the zoomed binding pocket
in Aβ aggregates is represented by surface and the docked ligands (carbon in yellow) are shown by sticks. (E) Predicted binding energy values by
Autodock vina of YQ-002, YQ-003, YQ-030, and YQ-036. All figures were created with PyMOL software.
coordinates of the bound ligands in the top 100 docking
conformations were used as an initiate position for the
subsequent docking simulation by Autodock vina to generate
more refined docking models.22 After the docking refinements,
the docking conformation with the lowest binding energy was
considered as the final binding model for each fluorescent
compound (Figure 5A−D).
All the compounds (Figure 5A−C) except YQ-036 (Figure
5D) were docked into the same binding pocket formed
between β-sheets. The predicted binding energy values by
Autodock vina were −8.7, −8.5, −7.8, and −6.9 kcal/mol for
YQ-002, YQ-003, YQ-030, and YQ-036, respectively (Figure
5E). YQ-002 showed the lowest binding energy, and, in the
docking model of YQ-002, the aromatic rings fit into the
hydrophobic pocket formed by Ile31 and Val40 (Figure 5A
and E). Similar interactions were also found in YQ-003 and
YQ-030 docking models, but YQ-036 was docked into the
opposite binding site inside the β-strands of Aβ with the least
predicted binding affinity. All the docking models suggest that
the four fluorescent compounds bind to Aβ with favorable
hydrophobic interactions and high shape complementarity.
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PAMPA for Prediction of Blood-Brain Barrier Perme-
ability of 1,4-NQ Derivatives. The ability to cross the BBB
is one of the main criteria required for amyloid imaging agent
candidates. PAMPA determines the permeability of com-
pounds by passively diffusing compounds from donor plates
across a BBB-like artificial lipid membrane into acceptor plates.
The UV−vis spectra of the reference compound standard,
acceptor, and donor plates were measured and effective
permeability (Pe) of the compounds were calculated by the
PAMPA Explorer software (pION Inc.). Compounds are
classified as BBB permeable (CNS+) or BBB impermeable
(CNS−) based on their measured effective permeability
(−log Pe). In accordance with previous reports, −log Pe values
of compounds less than 5.4 are classified as CNS+, and CNS−
for values greater than 5.7.23 The −log Pe values of YQ-002,
YQ-003, YQ-030, and YQ-036 are 4.2, 4.1, 4.1, and 4.6,
respectively, suggesting that all compounds are predicted to be
highly permeable (Table 1).
manifestations of dense-core plaques, detection of both forms
of Aβ plaques in the brain will significantly contribute to
clinical diagnosis of early AD.25−27 The compound YQ-003
shows the most promising results as it shows diffuse and dense-
core Aβ plaques in APP/PS1 AD mouse model brain sections
and can penetrate through the BBB based on PAMPA analysis.
YQ-003 also enhances fluorescence by 50-fold when its
emission is collected from 680 to 750 nm; considering that
biological tissues have lower absorption of NIR light and NIR
enables higher signal-to-background ratio than visible wave-
lengths, the NIR emission property of YQ-003 is very suitable
for an in vivo AD imaging agent candidate.28 The surface
plasmon resonance analyses showed that the binding property
of YQ-002 is unfavorable for clinical applications due to its low
dissociation rate; current imaging agents are labeled with
radioisotopes and must be cleared out from the brain within a
short time frame to minimize risks of radiation.29,30 However,
the strong affinity to Aβ aggregates suggest that YQ-003 or
other derivatives of the 1,4-NQ scaffold could have potential as
amyloid imaging candidates and will require further studies.
Further investigation is also warranted to in vivo studies
including micro positron emission tomography of AD mouse
model, pharmacokinetic profiling, and isotope labeling
kinetics.31 We hypothesized that these compounds interfered
with ThT by preventing it from binding to the β-sheets of Aβ
aggregates or by altering its fluorescence intensity due to
overlapping spectral activity. The overall binding mechanism of
ThT consists of the chemical dye approaching the surfaces of
cross-β-sheets formed by continuous chains of tyrosine and
leucine before undertaking a twisted, chiral conformation to
bind to the cross-β-sheets.32 Considering that the wavelengths
of several 1,4-NQ derivatives overlap with ThT and the
compounds fluoresce in the presence of Aβ, our evidence
suggests that the dramatic decrease in fluorescence intensity
from ThT assay could be due to the 1,4-NQ derivatives
directly competing with ThT for the same binding sites on the
β-sheets.17,19 To further validate this claim, additional
biochemical, biophysical, and structural investigations must
be carried out in follow up studies to explicate the underlying
mechanisms of 1,4-NQ derivatives on Aβ.
Table 1. PAMPA for Prediction of Blood-Brain Barrier
a
Permeability of Selected 1,4-NQ Derivatives
compd
Pe (10−6 cm s−1
)
−log Pe
CNS prediction
theophylline
lidocaine
progesterone
YQ-002
YQ-003
YQ-030
4.4
82.9
111.0
69.9
73.6
72.7
26.2
5.4
4.1
4.0
4.2
4.1
4.1
4.6
CNS
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
YQ-036
a
The UV−vis spectra of the acceptor and donor plates were measured
after the compounds passively diffused across a BBB-like membrane.
Compounds with −log Pe below values 5.4 are regarded as CNS+, and
higher than 5.7 are regarded as CNS−. Theophylline is a negative
control, and lidocaine and progesterone are positive controls.
Surface Plasmon Resonance Analysis to Confirm Aβ
Binding Property. To confirm direct interaction between
1,4-NQs and Aβ aggregates, surface plasmon resonance
analysis was employed to investigate biomolecular binding
kinetics using a Biacore T200 system. In accordance with the
positive histochemistry results, we selected YQ-002 as the
candidate for this assay. Aβ42 aggregates were fixed onto a
CM5 sensor chip by amine coupling, and YQ-002 in various
concentrations (from 100 to 500 μM) was passed through the
chip surface as an analyte. During the dissociating phase, YQ-
002 was found to not dissociate from the Aβ aggregates,
indicating strong binding to the target; thus, we conducted
single-cycle kinetics analysis instead of the common multicycle
kinetic analysis. The binding response continuously increased
relative to the concentration of YQ-002 (Figure S3),
suggesting that YQ-002 directly binds to immobilized Aβ
aggregates in a dose-dependent manner. The result is
consistent to the docking model results with the lowest
binding energy of the compound. The KD value could not be
calculated properly due to the heterogeneous nature of
immobilized Aβ aggregates.24
Current clinically approved amyloid imaging agents were
developed based on the chemical structures of the common Aβ
histologic dyes ThT and Congo Red, both of which consisting
of amyloid-binding benzothiazole moieties.33,34 Given that
these imaging agents exhibit low binding affinity toward diffuse
plaques, our study suggests that 1,4-NQ can be used as a novel
scaffold that can target both dense-core and diffuse plaques to
develop imaging agents for early AD patients. Overall, our data
indicates that 1,4-NQ scaffold can bind to Aβ and serve as a
basis for future designs of drug and diagnostic tool develop-
ment for AD.
METHODS
■
Chemical Syntheses of 1,4-Naphthoquinone Derivatives.
The synthetic procedure for YQ-001 and YQ-002 was adapted from
DISCUSSION
■
Our study reveals that 1,4-NQ derivatives can exhibit
fluorescence when bound to Aβ aggregates ex vivo.
Considering that dense-core plaques are highly correlated
with pathogenesis of AD and diffuse plaques are the early
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the literature procedure.35,36 To a stirred mixture of 5-hydroxy-1,4-
naphthoquinone (45 mg, 0.258 mmol, 1 equiv) and CeCl3·7H2O (4.8
mg, 0.05 equiv) in EtOH (2 mL) was added 3-methoxybenzylamine
(40 μL, 1.2 equiv) at rt. After being stirred at rt for 2 h, the reaction
mixture was concentrated in vacuo, suspended in water, suction-
filtered, and dried. The resulting solid was purified by silica gel
column chromatography (hexanes/ethyl acetate/dichloromethane =
20:1:2 to 10:1:2) to give YQ-001 (upper spot) and YQ-002 (lower
spot). The following compounds (YQ-003, YQ-004, YQ-005, YQ-
006, YQ-007, YQ-008, YQ-009, YQ-010, YQ-011, YQ-012, YQ-013,
YQ-014, YQ-015, YQ-016, YQ-017, YQ-018, YQ-019, YQ-020, YQ-
021, YQ-022, YQ-023, YQ-024, YQ-032, YQ-033, YQ-034, and YQ-
035) were prepared by the same procedure used for the synthesis of
YQ-001 and 002. Isoquinoline-5,8-dione was synthesized by
following the literature procedure.37,38
being stirred at 60 °C for 6 h, the reaction mixture was quenched with
H2O, concentrated under reduced pressure, diluted with H2O, and
extracted with CH2Cl2. The organic layer was dried over MgSO4 and
concentrated in vacuo to give the crude residue, which was purified by
silica gel column chromatography (hexanes/ethyl acetate/dichloro-
methane = 7:1:2 to 1:1:1) to give YQ-037.
Compounds YQ-038 and YQ-039 were synthesized by following
the procedures for the synthesis of YQ-036. Quinazoline-5,8-dione
was prepared by following the literature procedure.44
The synthetic procedure for YQ-025 was adapted from the
literature procedure.39,40 To a stirred mixture of 6,7-dichloroisoquino-
line-5,8-dione (20 mg, 0.088 mmol, 1 equiv) and CeCl3·7H2O (1.63
mg, 0.05 equiv) in EtOH (1.2 mL) was added aniline (12 μL, 1.5
equiv) at rt. After being stirred at rt for 3 h, the reaction mixture was
concentrated in vacuo and purified by silica gel column chromatog-
raphy (hexanes/ethyl acetate/dichloromethane = 3:1:2 to dichloro-
methane only to dichloromethane/MeOH = 10:1) to give YQ-025.
6,7-Dichloroisoquinoline-5,8-dione was prepared by following the
known procedure.41 The following compounds (YQ-026, YQ-027,
YQ-028, YQ-029, YQ-030, and YQ-031) were prepared by the same
procedure for the synthesis of YQ-025.
The synthetic procedure for YQ-040 was adapted from the
literature procedure.45 A mixture of 1,4-naphthoquinone (50 mg,
0.316 mmol, 1 equiv) and ethyl azidoacetate (102 mg, 0.79 mmol, 2.5
equiv) was heated at 120 °C for 24 h. After evaporation of the solvent,
the residue was purified by silica gel column chromatography
(hexanes/ethyl acetate/dichloromethane = 20:1:2 to 10:1:2 to 5:1:2
to 1:1:1) to give YQ-040. YQ-041 was synthesized by following the
same procedure for the synthesis of YQ-040.
5-Hydroxy-2-((3-methoxybenzyl)amino)naphthalene-1,4-dione
1
(YQ-001). Orange solid, mp: 114.8−118.9 °C; H NMR (400 MHz,
CDCl3) δ 13.0 (s, 1H), 7.6 (d, J = 7.2 Hz, 1H), 7.48 (t, J = 7.8 Hz,
1H), 7.32−7.24 (m, 3H), 6.90−6.84 (m, 3H), 6.39 (s, 1H), 5.67 (s,
1H), 4.37 (d, J = 5.2 Hz, 2H), 3.82 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 189.21, 181.17, 161.23, 160.25, 148.53, 137.18, 134.14,
130.56, 130.30, 126.10, 119.88, 119.23, 114.98, 113.53, 113.50,
100.85, 55.43, 46.96; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C18H16NO4 310.1074, found 310.1081.
8-Hydroxy-2-((3-methoxybenzyl)amino)naphthalene-1,4-dione
1
The synthetic procedure for YQ-036 was adapted from the
literature procedure.42,43 To a stirred mixture of isoquinoline-5,8-
dione (94 mg, 0.59 mmol, 1 equiv) in THF/AcOH (1/1, 3 mL) was
added a solution of NaN3 (50 mg, 0.77 mmol, 1.3 equiv) in H2O (1.5
mL) at rt. After being stirred at 40 °C for 3 h, the reaction mixture
was concentrated under reduced pressure, diluted with H2O, and
extracted with CH2Cl2. The organic layer was washed with aq.
NaHCO3, dried over MgSO4, and concentrated in vacuo to give the
crude residue, which was used for the next step without further
purification. A mixture of 7-aminoisoquinoline-5,8-dione (65 mg,
0.373 mmol, 1 equiv), acetaldehyde diethyl acetal (218 μL, 1.53
mmol, 4.1 equiv), and Mn(OAc)3 (500 mg, 1.866 mmol, 5 equiv) in
AcOH (6 mL) was heated at 60 °C for 3 h. The reaction mixture was
concentrated under reduced pressure, diluted with H2O, and
extracted with ethyl acetate. The organic layer was washed with aq.
NaHCO3, dried over MgSO4, and concentrated in vacuo to give YQ-
036.
(YQ-002). Orange solid, mp: 169.6−170.3 °C; H NMR (400 MHz,
CDCl3) δ 11.53 (s, 1H), 7.61(d, J = 4.4 Hz, 2H), 7.30 (t, J = 8.0 Hz,
1H), 7.14 (t, J = 4.8 Hz, 1H), 6.90 (d, J = 7.2 Hz, 1H), 6.88−6.84 (m,
2H), 6.18 (s, 1H), 5.75 (s, 1H), 4.35 (d, J = 5.6 Hz, 2H), 3.82 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 186.15, 182.47, 161.82, 160.27,
147.63, 137.85, 137.41, 133.54, 130.30, 122.48, 119.91, 118.95,
114.20, 113.53, 113.50, 102.54, 55.45, 46.95; HRMS (ESI-QTOF)
m/z [M + H]+ calcd for C18H16NO4 310.1074, found 310.1074.
2-(Benzylamino)-5-hydroxynaphthalene-1,4-dione (YQ-003).
Red solid, mp: 150−151.3 °C; 1H NMR (400 MHz, CDCl3) δ
12.99 (s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H),
7.41−7.30 (m, 6H), 7.24 (s, 1H), 7.41−7.34 (m, 5H), 7.24 (s, 1H),
6.37 (s, 1H), 5.69 (s, 1H), 4.39 (d, J = 6.0 Hz, 2H); 13C NMR (100
MHz, CDCl3) δ 189.25, 181.23, 161.27, 148.54, 147.52, 135.62,
134.17, 130.60, 129.25, 128.44, 127.79, 126.14, 119.26, 100.83, 47.06;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C17H14NO3 280.0968,
found 280.0970.
2-(Benzylamino)-8-hydroxynaphthalene-1,4-dione (YQ-004).
1
Red solid, mp: 207.8−208.2 °C; H NMR (400 MHz, CDCl3) δ
11.53 (s, 1H), 7.62 (d, J = 4.4 Hz, 2H), 7.41−7.31 (m, 5H), 7.15 (t, J
= 4.8 Hz, 1H), 6.19 (s, 1H), 5.76 (s, 1H), 4.38 (d, J = 5.6 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ 186.16, 182.46, 161.82, 147.63,
137.86, 135.85, 133.55, 129.22, 128.38, 127.78, 122.48, 118.95,
114.20, 102.49, 47.00; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C17H14NO3 280.0968, found 280.0966.
8-Hydroxy-2-((4-nitrophenyl)amino)naphthalene-1,4-dione
(YQ-005). Red solid, mp: 332−333 °C; 1H NMR (400 MHz, DMSO-
d6) δ 11.5 (s, 1H), 9.67 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 7.77 (t, J =
8.0 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7,50 (d, J = 7.2 Hz, 1H), 7.31
The synthetic procedure for YQ-037 was adapted from the
literature procedure.43 To a stirred mixture of YQ-036 (20 mg, 0.1
mmol, 1 equiv) in THF (1.5 mL) were added 60% NaH (12 mg, 0.3
mmol, 3 equiv) and MeI (18 μL, 0.3 mmol, 3 equiv) at 0 °C. After
H
DOI: 10.1021/acschemneuro.9b00093
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
(d, J = 8.4 Hz, 1H), 6.46 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
160.86, 137.94, 125.58, 123.21, 122.52, 118.19, 106.78; HRMS (ESI-
QTOF) m/z [M + H]+ calcd for C16H11N2O5 311.0662, found
311.0663.
2-(p-Tolylamino)naphthalene-1,4-dione (YQ-014). Pink solid,
mp: 192.4−195.1 °C; H NMR (400 MHz, CDCl3) δ 8.11 (t, J =
1
6.2 Hz, 2H), 7.76 (t, J = 7.6 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.51 (s,
1H), 7.25−7.13 (m, 4H), 6.36 (s, 1H), 2.37 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 184.00, 182.30, 145.16, 135.79, 135.04, 134.87,
133.46, 132.40, 130.54, 130.38, 126.63, 126.29, 122.87, 103.17, 21.15;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C17H14NO2 264.1019,
found 264.1015.
8-Hydroxy-2-(p-tolylamino)naphthalene-1,4-dione (YQ-006).
1
Red solid, mp: 193.4−196.4 °C; H NMR (400 MHz, CDCl3) δ
11.57 (s, 1H), 7.67−7.62 (m, 2H), 7.47 (s, 1H), 7.23 (d, J = 8.0 Hz,
2H), 7.20−7.15 (m, 3H), 6.31 (s, 1H), 2.37 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 186.39, 183.19, 161.87, 145.08, 137.97, 136.05,
134.61, 133.34, 130.44, 123.02, 122.68, 118.88, 114.17, 103.70, 21.16;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C17H14NO3 280.0968,
found 280.0968.
2-((4-Bromophenyl)amino)naphthalene-1,4-dione (YQ-015).
1
Red solid, mp: 269.9−271.5 °C; H NMR (400 MHz, DMSO-d6) δ
9.30 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.87
(t, J = 7.6 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H),
7.37 (d, J = 8.4 Hz, 2H) 6.15 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 82.74, 181.46, 145.79, 137.63, 134.94, 132.77, 132.49, 132.16,
130.44, 126.19, 125.48, 125.32, 117.11, 102.66; HRMS (ESI-QTOF)
m/z [M + H]+ calcd for C16H11BrNO2 327.9968, found 327.9968.
2-((4-Chlorophenyl)amino)-8-hydroxynaphthalene-1,4-dione
(YQ-016). Red solid, mp: 267.8−270 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.30 (s, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.96 (d, J = 7.2
Hz, 1H), 7.87 (t, J = 7.4 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.49 (d, J =
8.4 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 6.13 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ 182.72, 181.46, 145.90, 137.19, 134.94, 132.76,
132.50, 130.44, 129.25, 128.96, 126.19, 125.32, 125.20, 102.56;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C16H11ClNO2
284.0473, found 284.0472.
2-((4-Bromophenyl)amino)-8-hydroxynaphthalene-1,4-dione
(YQ-007). Brown solid, mp: 225−227 °C; 1H NMR (400 MHz,
DMSO-d6) δ 11.52 (s, 1H), 9.32 (s, 1H), 7.74 (t, J = 8.0 Hz, 1H),
7.62 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.8 Hz,
2H), 7.27 (d, J = 8.4 Hz, 1H), 6.08 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 185.78, 182.45, 160.89, 146.25, 137.97, 137.92, 133.26,
132.59, 126.05, 122.80, 118.07, 117.68, 114.74, 103.25; HRMS (ESI-
QTOF) m/z [M + H]+ calcd for C16H11BrNO3 343.9917, found
343.9917.
Ethyl 3-((8-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)benzoate (YQ-008). Brown solid, mp: 169.6−172 °C; 1H
NMR (400 MHz, CDCl3) δ 11.53 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H),
7.68−7.63 (m, 2H), 7.57 (s, 1H), 7.53−7.47 (m, 2H), 7.20 (dd, J = 2,
2.2 Hz, 1H), 6.36 (s, 1H), 4.41 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 186.01, 183.23, 165.73,
161.90, 144.62, 138.05, 137.63, 133.08, 132.42, 129.94, 126.92,
123.94, 122.93, 119.01, 114.05, 104.48, 61.59, 29.83, 14.45; HRMS
(ESI-QTOF) m/z [M + H]+ calcd for C19H16NO5 338.1023, found
338.1024.
2-((4-Chlorophenyl)amino)-8-hydroxynaphthalene-1,4-dione
(YQ-009). Brown solid, mp: 228−229 °C; 1H NMR (400 MHz,
DMSO-d6) δ 11.48 (s, 1H), 9.29 (s, 1H), 7.71 (t, J = 8.0 Hz, 1H),
7.41−7.37 (m, 5H), 7.34 (d, J = 8.4 Hz, 1H), 6.03 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 185.79, 160.88, 146.37, 137.98, 129.68,
125.78, 122.79, 118.07, 103.16; HRMS (ESI-QTOF) m/z [M + H]+
calcd for C16H11ClNO3 300.0422, found 300.0423.
8-Hydroxy-2-(phenylamino)naphthalene-1,4-dione (YQ-010).
Orange solid, mp: 204−206 °C; 1H NMR (400 MHz, CDCl3) δ
11.57 (s, 1H), 7.66−7.63 (m, 2H), 7.53 (s, 1H), 7.44 (t, J = 7.6 Hz,
2H), 7.28 (d, J = 7.6 Hz, 2H), 7.24−7.19 (m, 2H), 6.38 (s, 1H); 13C
NMR (100 MHz, CDCl3) δ 186.30, 183.29, 161.90, 144.81, 138.01,
137.31, 133.24, 129.91, 126.02, 122.94, 122.80, 118.93, 114.15,
104.08; HRMS (ESI-QTOF) m/z [M + H]+ calcd for C16H12NO3
266.0812, found 266.0811.
Ethyl 3-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzoate
(YQ-017). Orange solid, mp: 138−194 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.39 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 6.0
Hz, 2H), 7.86 (t, J = 7.4 Hz, 1H), 7.79 (t, J = 7.8 Hz, 2H), 7.68 (d, J =
7.6 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 6.12 (s, 1H), 4.33 (q, J = 6.8
Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ
182.77, 181.35, 165.25, 146.07, 138.68, 134.89, 132.75, 132.47,
130.99, 130.44, 129.81, 128.11, 126.17, 125.59, 125.30, 124.03,
102.43, 61.01, 14.16; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C19H16NO4 322.1074, found 322.1076.
2-((3,4,5-Trimethoxyphenyl)amino)naphthalene-1,4-dione (YQ-
018). Red solid, mp: 1432.-144.5 °C; 1H NMR (400 MHz, CDCl3) δ
8.11 (dd, J = 2.4, 7.6 Hz, 2H), 7.76 (t, J = 7.4 Hz, 1H), 7.67 (t, J = 7.4
Hz, 1H), 7.46 (s, 1H), 6.49 (s, 2H), 6.34 (s, 1H), 3.87 (s, 6H), 3.86
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 183.96, 182.15, 154.08,
145.26, 136.18, 135.12, 133.38, 133.29, 132.50, 130.46, 126.66,
126.34, 103.54, 100.85, 61.19, 56.45; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C19H18NO5 340.1179, found 340.1180.
2-((4-Methoxyphenyl)amino)naphthalene-1,4-dione (YQ-019).
1
Brown solid, mp: 145.4−147.2 °C; H NMR (400 MHz, CDCl3) δ
8.12−8.09 (m, 2H), 7.75 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H),
7.44 (s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 5.2 Hz, 2H), 6.22
(s, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 183.89,
182.32, 157.80, 145.81, 135.03, 133.53, 132.35, 130.55, 130.14,
126.58, 126.29, 124.99, 115.05, 102.66, 55.71; HRMS (ESI-QTOF)
m/z [M + H]+ calcd for C17H14NO3 280.0968, found 280.0967.
7-(Phenylamino)isoquinoline-5,8-dione (YQ-020). Orange solid,
8-Hydroxy-2-((3,4,5-trimethoxyphenyl)amino)naphthalene-1,4-
1
dione (YQ-011). Brown solid, mp: 167.1−168.9 °C; H NMR (400
MHz, CDCl3) δ 11.55 (s, 1H), 7.68−7.63 (m, 2H), 7.42 (s, 1H), 7.20
(dd, J = 2.4, 6.8 Hz, 1H), 6.49 (s, 2H), 6.30 (s, 1H), 3.87 (s, 6H),
3.86 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 186.21, 183.17, 161.89,
154.13, 145.23, 138.06, 136.37, 133.25, 133.00, 122.79, 118.97,
114.09, 104.07, 101.04, 61.19, 56.46; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C19H18NO6 356.1129, found 356.1130.
1
mp: 190.2−193 °C; H NMR (400 MHz, CDCl3) δ 9.34 (s, 1H),
9.05 (d, J = 4.8 Hz, 1H), 7.91 (d, J = 4.8 Hz, 1H), 7.70−7.52 (m,
2H), 7.44 (t, J = 7.6 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 6.47 (s, 1H);
13C NMR (100 MHz, CDCl3) δ 182.18, 181.61, 156.59, 148.25,
145.03, 138.79, 136.87, 129.97, 126.47, 124.23, 123.16, 118.90,
103.72; HRMS (ESI-QTOF) m/z [M + H]+ calcd for C15H11N2O2
251.0815, found 251.0816.
7-((4-Methoxyphenyl)amino)isoquinoline-5,8-dione (YQ-012).
Black solid, mp: 227.6−228 °C; 1H NMR (400 MHz, CDCl3) δ
9.33 (s, 1H), 9.04 (d, J = 4.8 Hz, 1H), 7.91 (d, J = 4.8 Hz, 1H), 7.51
(s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.29 (s,
1H), 3.84 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 181.95, 181.73,
158.26, 156.59, 148.20, 145.87, 139.02, 129.39, 125.26, 124.32,
118.95, 115.18, 103.01, 55.73; HRMS (ESI-QTOF) m/z [M + H]+
calcd for C16H13N2O3 281.0921, found 281.0920.
7-((4-Fluorophenyl)amino)isoquinoline-5,8-dione (YQ-021).
1
Brown solid, mp: 261.2−263.8 °C; H NMR (400 MHz, DMSO-
d6) δ 9.49 (s, 1H), 9.20 (s, 1H), 9.06 (d, J = 4.8 Hz, 1H), 7.81 (d, J =
4.8 Hz, 1H), 7.44−7.39 (m, 2H), 7.33−7.28 (m, 2H), 6.05 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 181.11, 158.44, 156.10, 147.25,
146.96, 138.15, 133.97, 126.37, 124.51, 118.13, 116.32, 116.09,
102.00; HRMS (ESI-QTOF) m/z [M + H]+ calcd for C15H10FN2O2
269.0721, found 269.0726.
2-((4-Fluorophenyl)amino)-8-hydroxynaphthalene-1,4-dione
1
(YQ-013). Red solid, mp: 235.8−236.7 °C; H NMR (400 MHz,
CDCl3) δ 8.12 (t, J = 7.2 Hz, 2H), 7.77 (t, J = 7.2 Hz, 1H), 7.68 (t, J
= 7.4 Hz, 1H), 7.45 (s, 1H), 7.24 (s, 2H), 7.13 (t, J = 7.8 Hz, 2H),
6.26 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 183.98, 182.09, 145.42,
135.13, 133.44, 132.56, 130.47, 126.68, 126.36, 125.19, 125.11,
116.90, 116.67, 103.26; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C16H11FNO2 268.0768, found 268.0768.
7-((4-Iodophenyl)amino)isoquinoline-5,8-dione (YQ-022). Pur-
1
ple solid, mp: 239.2−240.1 °C; H NMR (400 MHz, CDCl3) δ 9.35
(s, 1H), 9.06 (d, J = 5.2 Hz, 1H), 7.92 (d, J = 4.8 Hz, 1H), 7.76 (d, J =
I
DOI: 10.1021/acschemneuro.9b00093
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
8.4 Hz, 2H), 7.56 (s, 1H), 7.05 (d, J = 8.4 Hz, 3H), 6.45 (s, 1H); 13
C
6-Chloro-7-((3,4,5-trimethoxyphenyl)amino)isoquinoline-5,8-
1
dione (YQ-031). Purple solid, mp: 166.2−169.8 °C; H NMR (400
NMR (100 MHz, CDCl3) δ 182.21, 181.41, 156.73, 148.35, 144.47,
139.05, 138.63, 136.76, 124.67, 124.12, 118.93, 104.29, 90.28; HRMS
(ESI-QTOF) m/z [M + H]+ calcd for C15H10IN2O2 376.9781, found
376.9782.
MHz, CDCl3) δ 9.33 (s, 1H), 9.06 (d, J = 4.8 Hz, 1H), 7.99 (d, J =
4.8 Hz, 1H), 7.78 (s, 1H), 6.34 (s, 2H), 3.86 (s, 3H), 3.84 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 180.03, 176.24, 156.79, 153.07,
148.68, 141.58, 138.25, 136.75, 132.55, 123.52, 119.54, 114.51,
102.94, 61.23, 56.38; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C18H16ClN2O5 375.0742, found 375.0742.
7-(p-Tolylamino)isoquinoline-5,8-dione (YQ-023). Black solid,
1
mp: 218.4−223.6 °C; H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H),
9.04 (d, J = 4.8 Hz, 1H), 7.91 (d, J = 4.8 Hz, 1H), 7.58 (s, 1H), 7.24
(d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.41 (s, 1H), 2.38 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 182.08, 181.70, 156.58, 148.23,
145.28, 138.93, 136.58, 134.17, 130.53, 124.28, 123.21, 118.92,
103.42, 21.19; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C16H13N2O2 265.0972, found 265.0971.
7-((4-Chlorophenyl)amino)isoquinoline-5,8-dione (YQ-032).
1
Purple solid, mp: 258−261.2 °C; H NMR (400 MHz, CDCl3) δ
9.35 (s, 1H), 9.06 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 4.4 Hz, 1H), 7.57
(s, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.8 Hz, 2H), 6.41 (s,
1H); 13C NMR (100 MHz, CDCl3) δ 182.19, 181.42, 156.71, 148.33,
144.79, 138.67, 135.48, 131.89, 130.17, 124.39, 124.14, 118.94,
104.06; HRMS (ESI-QTOF) m/z [M + H]+ calcd for C15H10ClN2O2
285.0425, found 285.0426.
2-(Phenylamino)naphthalene-1,4-dione (YQ-024). Red solid,
1
mp: 192.7−193.3 °C; H NMR (400 MHz, CDCl3) δ 8.12 (t, J =
7.4 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.57 (s,
1H), 7.42 (t, J = 7.8 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.22 (t, J = 7.4
Hz, 1H), 6.42 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 184.10,
182.23, 144.88, 137.57, 135.08, 133.37, 132.51, 130.51, 129.85,
126.69, 126.32, 125.79, 122.77, 103.54; HRMS (ESI-QTOF) m/z [M
+ H]+ calcd for C16H12NO2 250.0863, found 250.0864.
4-((5,8-Dioxo-5,8-dihydroisoquinolin-7-yl)amino)benzonitrile
1
(YQ-033). Brown solid, mp: 392 °C (decomposed); H NMR (400
MHz, CDCl3) δ 9.38 (s, 1H), 9.10 (d, J = 5.2 Hz, 1H), 7.93 (d, J =
4.8 Hz, 1H), 7.74 (d, J = 8.4 Hz, 3H), 7.39 (d, J = 8.4 Hz, 2H), 6.63
(s, 1H); 13C NMR (100 MHz, CDCl3) δ 156.73, 148.37, 113.91,
121.84, 118.75, 105.80; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C16H10N3O2 276.0768, found 276.0767.
6-Chloro-7-(phenylamino)isoquinoline-5,8-dione (YQ-025). Pur-
1
ple solid, mp: 252.4−254.2 °C; H NMR (400 MHz, CDCl3) δ 9.35
Ethyl 3-((5,8-dioxo-5,8-dihydroisoquinolin-7-yl)amino)benzoate
(YQ-034). Red solid, mp: 157.9−161 °C; 1H NMR (400 MHz,
CDCl3) δ 9.35 (s, 1H), 9.06 (d, J = 4.4 Hz, 1H), 7.94−7.91 (m, 3H),
7.69 (s, 1H), 7.55−7.47 (m, 2H), 6.45 (s, 1H), 4.40 (q, J = 7.2 Hz,
2H), 1.41 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
182.24, 181.40, 165.62, 156.67, 148.32, 144.86, 138.65, 137.21,
132.52, 130.04, 127.39, 127.18, 124.18, 118.91, 118.12, 104.11, 61.65,
14.45; HRMS (ESI-QTOF) m/z [M + H]+ calcd for C18H15N2O4
323.1026, found 323.1024.
(s, 1H), 9.07 (d, J = 4.8 Hz, 1H), 8.00 (d, J = 5.2 Hz, 1H), 7.80 (s,
1H), 7.38 (t, J = 7.6 Hz, 2H), 7.28 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 8
Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 180.08, 176.24, 156.77,
155.01, 148.70, 141.66, 138.22, 136.93, 128.70, 126.53, 124.86,
124.58, 119.55; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C15H10ClN2O2 285.0425, found 285.0423.
6-Chloro-7-((4-iodophenyl)amino)isoquinoline-5,8-dione (YQ-
026). Brown solid, mp: 267.6−271.1 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.56 (s, 1H), 9.17 (s, 1H), 9.07 (d, J = 4.8 Hz, 1H) 7.89
(d, J = 5.2 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) δ 179.63, 175.83, 155.90, 147.54,
143.28, 138.62, 137.56, 136.63, 126.12, 125.77, 124.35, 118.61, 89.07;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C15H9ClIN2O2
410.9392, found 410.9391.
2-((4-Iodophenyl)amino)naphthalene-1,4-dione (YQ-035). Pur-
1
ple solid, mp: 234.4−237.1 °C; H NMR (400 MHz, CDCl3) δ 8.12
(t, J = 6.6 Hz, 2H), 7.79−7.66 (m, 4H), 7.51 (s, 1H), 7.04 (d, J = 8.4
Hz, 2H), 6.40 (s, H); 13C NMR (100 MHz, CDCl3) δ 184.02, 181.99,
144.29, 138.87, 137.47, 135.18, 133.22, 132.67, 130.41, 126.76,
126.39, 124.27, 104.18, 89.27; HRMS (ESI-QTOF) m/z [M + H]+
calcd for C16H11INO2 375.9829, found 375.9829.
6-Chloro-7-(p-tolylamino)isoquinoline-5,8-dione (YQ-027). Pur-
1
ple solid, mp: 263.3−265.6 °C; H NMR (400 MHz, CDCl3) δ 9.34
1H-Pyrrolo[3,2-g]isoquinoline-4,9-dione (YQ-036). Yellow solid,
(s, 1H), 9.06 (d, J = 4.8 Hz, 1H), 7.99 (d, J = 4.8 Hz, 1H), 7.77 (s,
1H), 7.17 (d, J = 7.6 Hz, 2H), 7.01 (d, J = 7.6 Hz, 2H), 2.38 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 180.13, 156.74, 154.91, 149.09,
1
mp: 295.6−296.3 °C; H NMR (400 MHz, DMSO-d6) δ 13.14 (s,
1H), 9.20 (s, 1H), 9.04 (d, J = 4.4 Hz, 1H), 7.90 (d, J = 4.8 Hz, 1H),
7.44 (s, 2H), 6.76 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
179.87, 174.77, 155.74, 147.71, 139.73, 132.50, 128.93, 127.43,
119.27, 108.81; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C11H7N2O2 199.0502, found 199.0501.
148.67, 141.79, 138.31, 136.62, 134.34, 129.28, 124.89, 124.65,
119.54, 21.24; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C16H12ClN2O2 299.0582, found 299.0583.
7-((4-Bromophenyl)amino)-6-chloroisoquinoline-5,8-dione (YQ-
028). Purple solid, mp: 293.8−294.7 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.57 (s, 1H), 9.17 (s, 1H), 9.07 (d, J = 4.8 Hz, 1H), 7.89
(d, J = 5.2 Hz, 1H). 7.51 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) δ 179.62, 175.85, 155.91, 147.54,
143.40, 138.17, 137.56, 130.80, 125.96, 124.36, 118.62, 116.84,
115.08; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C15H9BrClN2O2 362.9530, found 362.9533.
1-Methyl-1H-pyrrolo[3,2-g]isoquinoline-4,9-dione (YQ-037). Yel-
low solid, mp: 193.7−195.4 °C; H NMR (400 MHz, DMSO-d6) δ
9.22 (s, 1H), 9.04 (d, J = 4.8 Hz, 1H), 7.90 (d, J = 4.8 Hz, 1H), 7.49
(d, J = 2.8 Hz, 1H), 6.74 (d, J = 2.8 Hz, 1H), 4.05 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 176.57, 175.25, 155.60, 147.90, 139.29,
134.16, 128.14, 118.99, 107.86, 36.96; HRMS (ESI-QTOF) m/z [M
+ H]+ calcd for C12H9N2O2 213.0659, found 213.0660.
1
6-Aminoquinazoline-5,8-dione (YQ-038). Brown solid, mp: 304
1
6-Chloro-7-((4-methoxyphenyl)amino)isoquinoline-5,8-dione
°C (decomposed); H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H),
1
9.25 (s, 1H), 5.97 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
181.20, 179.42, 162.90, 155.55, 154.81, 150.74, 124.41, 104.61;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C8H6N3O2 176.0455,
found 176.0456.
(YQ-029). Black solid, mp: 225.6−227.6 °C; H NMR (400 MHz,
CDCl3) δ 9.33 (s, 1H), 9.05 (d, J = 5.2 Hz, 1H), 7.99 (d, J = 4.8 Hz,
1H), 7.75 (s, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H),
3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 180.12, 176.10, 158.41,
156.74, 148.64, 141.97, 138.37, 129.78, 126.73, 126.56, 123.59,
119.54, 113.87, 55.66; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C16H12ClN2O3 315.0531, found 315.0531.
7-Methyl-5H-pyrrolo[2,3-g]quinazoline-5,9(6H)-dione (YQ-039).
Yellow solid, mp: 284 °C (decomposed); 1H NMR (400 MHz,
DMSO-d6) δ 9.57 (s, 1H), 9.38 (s, 1H), 6.54 (s, 1H), 2.34 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 178.45, 172.47, 161.97, 156.10,
155.54, 140.61, 131.10, 128.90, 126.09, 107.76, 13.12; HRMS (ESI-
QTOF) m/z [M + H]+ calcd for C11H7N3O2 214.0611, found
214.0614.
Ethyl 2-(4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d][1,2,3]triazol-
1-yl)acetate (YQ-040). Yellow solid, mp: 141.4−143.4 °C; 1H
NMR (400 MHz, CDCl3) δ 8.33 (d, J = 7.6 Hz, 1H), 8.18 (d, J = 7.6
Hz, 1H), 7.87−7.78 (m, 2H), 5.59 (s, 2H), 4.29 (q, J = 6.8 Hz, 2H),
6-Chloro-7-((2-iodophenyl)amino)isoquinoline-5,8-dione (YQ-
030). Purple solid, mp: 216.8−218.6 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.55 (s, 1H), 9.17 (s, 1H), 9.06 (d, J = 4.8 Hz, 1H), 7.89
(d, J = 4.4 Hz, 1H), 7.33 (t, J = 7.6 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) δ 179.72, 175.70, 155.94, 147.55,
143.55, 138.59, 137.68, 136.65, 128.00, 126.15, 124.92, 124.37,
124.02, 118.65, 114.03; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C15H9ClIN2O2 410.9392, found 410.9392.
J
DOI: 10.1021/acschemneuro.9b00093
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ACS Chemical Neuroscience
Research Article
1.31 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 176.50,
175.42, 165.19, 145.32, 135.35, 134.30, 134.14, 133.51, 132.54,
127.94, 127.27, 62.80, 50.98, 14.06; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C14H12N3O4 286.0822, found 286.0821.
mM) and diluted with deionized water (100 μM). One stock was
stored immediately at −80 °C to represent Aβ monomers and the
other stock was incubated for 3 days to produce Aβ aggregates. 1,4-
NQ derivatives were dissolved in DMSO (1 mM) followed by a serial
dilution with deionized water (50 μM). Then 25 μL of Aβ monomers
or aggregates and 75 μL of 1,4-NQ derivatives were added to each
well in a 96-well opaque round-bottom plate. The emission spectra of
the compounds with Aβ were recorded following the same process as
the emission scan.
Histochemistry of APP/PS1 Double Transgenic Mouse
Brains. Brain tissues were fixed in 4% paraformaldehyde overnight
at 4 °C and immersed in 30% sucrose for 48 h to induce
cryoprotection. Sections of 35 μm were cut with a cryostat
(CM1860, Leica). For brain staining, antigen retrieval on aldehyde-
fixed brain sections was performed with 1% SDS in PBS. Sections
were blocked with 20% horse serum in PBS for 1 h before overnight
incubation at 4 °C with mouse monoclonal antibody 6E10 (1:200),
the primary antibody that detects Aβ plaques. Following an overnight
incubation, a secondary goat anti-mouse IgG conjugated with Alexa
fluor 555 (1:200) was added for 1 h. 1,4-NQ derivatives stocks in
DMSO (10 mM) were diluted 500 μM in PBS and added to the
sections overnight. The images of amyloid plaques in the cortex and
the hippocampus were captured on a fluorescence microscope
(DM500, Leica) equipped with filter cubes containing excitation
and emission filters: A4 filter cube for Hoechst staining detection
(data not shown, excitation filter: BP 360/40; dichromatic mirror:
400; emission filter: BP 470/40), N2.1 filter cube for 6E10 staining
detection (excitation filter: BP 515−560; dichromatic mirror: 580;
emission filter: LP 590), and L5 filter cube for YQ compound
detection (excitation filter: BP 480/40; dichromatic mirror: 505;
emission filter: BP 527/30).
1-(4-Methoxybenzyl)-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione
1
(YQ-041). Brown solid, mp: 179.4−181.9 °C; H NMR (400 MHz,
CDCl3) δ 8.31 (d, J = 7.2 Hz, 1H), 8.22 (d, J = 7.2 Hz, 1H), 7.81 (p, J
= 7.6 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.0 hz, 2H), 5.95
(s, 2H), 3.77 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 176.93,
175.51, 160.23, 145.83, 135.31, 134.41, 133.54, 132.99, 130.39,
127.98, 127.51, 126.17, 114.46, 55.44, 53.56; HRMS (ESI-QTOF)
m/z [M + H]+ calcd for C16H12N5O3 322.0935, found 322.0939.
Animals. Male APP/PS1 double transgenic mice (B6C3-Tg-
(APPswe,PSEN 1dE9)85Dbo/Mmjax) were housed in a laboratory
animal breeding room in Yonsei University under regulated
conditions with 12 h/12 h light−dark phase. Food and water were
provided ad libitum. PCR analysis with DNA from tail biopsy
following the Jackson Laboratory protocol was carried out to confirm
the genotypes of APP/PS1 mice. Using the standard protocol
recommended by the Jackson Laboratory, mouse brain tissues were
collected at the age of 24 months for histology study. Animal care was
done in accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and authorized by The
Committee for the Care and Use of Laboratory Animals at Yonsei
University.
ThT Fluorescence Assay. ThT fluorescence assay was performed
to quantify the β-sheet complex of protein aggregates. Aβ42 peptides
were synthesized by DMSO-incorporated Fmoc solid phase peptide
synthesis protocol as previously reported.46 For Aβ aggregation
inhibition assay, in-house synthetic Aβ42 peptides and 1,4-NQ
derivatives were dissolved in DMSO to prepare stocks (1 mM) and
diluted with deionized water (100 μM). Aβ42 peptides were then
incubated with 1,4-NQ derivatives at 37 °C for 3 days; final
concentration of Aβ42 was 50 μM and that of 1,4-NQ derivatives
were 0.5, 5, and 50 μM. For Aβ disaggregation assay, Aβ42 stock (100
μM) was incubated at 37 °C for 3 days to prepare Aβ aggregates. Aβ
aggregates were then diluted with 1,4-NQ derivatives solutions (same
concentrations as above) and reincubated for an additional 3 days at
37 °C. Twenty-five μL of the incubated samples and 75 μL of ThT (5
μM ThT in 50 mM glycine buffer, pH 8.9) were added in a 96-well
half area black plate. Fluorescence intensities of ThT bound to Aβ
were measured at 450 nm (excitation) and 485 nm (emission) using a
microplate reader (Infinite 200 PRO, Tecan).
SDS-PAGE with PICUP. SDS-PAGE and PICUP chemistry were
conducted to evaluate Aβ species by size distribution.47,48 Because Aβ
aggregates are naturally metastable, all samples were irradiated thrice
(each session for 1 s) with Tris(2,2′-bipyridyl)dichlororuthenium(II)
hexahydrate (1 mM) and ammonium persulfate (20 mM) to induce
cross-linking before adding 5× sample buffer with β-mercaptoethanol
and incubating at 90 °C for 5 min. These Aβ samples were loaded
onto 1.0 mm-thick 15% gradient polyacrylamide gel (Gradi-Gel II,
ELPIS) for peptide separation, and the Aβ bands were then visualized
with silver staining in accordance with the PlusOne Silver Staining Kit
protocol (GE Healthcare).
Fluorescent Spectral Scan. Fluorescent spectral scans were
performed using a microplate reader (Infinite 200 PRO, Tecan). For
excitation scans, 1,4-NQ derivatives were dissolved in DMSO to
prepare 1 mM stocks before serial dilution is carried out with
deionized water. Then 150 μL of the samples (50 μM) was then
aliquoted to a 96-well clear round-bottom plate and the data points of
the compound’s excitation spectra was measured at 10 nm increments
from 230 to 900 nm. The peaks for each compound were recorded as
the excitation wavelengths. For emission scans, 150 μL of the same
samples prepared above were transferred to a 96-well opaque round-
bottom plate. The excitation wavelengths were set according to their
respective compounds and the emissions were scanned at 10 nm
increments at varying spectra ranges. Next, the emission spectra of the
candidate compounds mixed with Aβ is needed to determine whether
these compounds exhibit a fluorescence shift in the presence of Aβ.
Two separate stocks of Aβ42 peptides were dissolved in DMSO (1
Generation of Molecular Docking Models. Three-dimensional
conformers for each compound were generated using BEST algorithm
̀
in Discovery Studio 2018 software (Dassault Systemes). The
conformers were energy minimized by CHARMM force field and
clustered with RMSD 0.5 Å, which resulted in 36, 8, 2, and 1 distinct
conformers for YQ-002, YQ-003, YQ-030, and YQ-036, respectively.
All the conformers were separately used to run the global docking
search by PatchDock program.21
The top 100 output docking structures for each compound were
used to define a docking starting position in the subsequent docking
simulation by Autodock vina by calculating ligand center of mass
coordinates.22 The input chemical structures were prepared by
minimizing CHARMM force field in Discover Studio (full
minimization function). After the docking simulations by Autodock
vina for 100 different starting positions, the docking conformation
with the lowest binding energy was finally selected as the most
plausible docking model for each compound. The structure of Aβ
(PDB ID: 2LMO) was prepared as described in our previous work.49
All figures were created with PyMOL software (The PyMOL
̈
Molecular Graphics System, version 1.2r3pre, Schrodinger, LLC).
PAMPA Assay. PAMPA-BBB studies were performed using the
PAMPA Explorer kit (pION Inc.) according to the manufacturer’s
instructions. Stock solutions of four 1,4-NQ derivatives were prepared
in DMSO (10 mM). Each stock solution was diluted with Prisma HT
buffer (pH 7.4) to a final concentration of 50 μM. Then, 200 μL of
the resulting stock solution was added to each well of donor plates (n
= 3). The PVDF (0.45 μm) filter membrane on the acceptor plate was
coated with 5 μL of the BBB-1 lipid. Each well of the acceptor plate
was filled with 200 μL of brain sink buffer. The acceptor plate was
placed on the donor plate to form a sandwich; the sandwich was
incubated at 37 °C for 4 h without stirring. The UV−vis spectra of the
solutions for reference, acceptor, and donor plates were measured
using plate reader (Infinite 200 PRO, Tecan). Effective permeability
(Pe) for each compound was calculated using the PAMPA Explorer
software v.3.5 (pION). Theophylline is used as a negative control, and
lidocaine and progesterone are used as positive controls for this assay.
Designations of CNS were assigned by comparison with compounds
that were identified in previous reports.23
K
DOI: 10.1021/acschemneuro.9b00093
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ACS Chemical Neuroscience
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Surface Plasmon Resonance Analysis. The surface plasmon
resonance analysis was performed using a Biacore T200 instrument
and CM5 sensor chips (GE Healthcare). Aβ42 stock was prepared in
DMSO (10 mM) and diluted with PBS (pH 7.4) to make 1 mg/mL.
The diluted Aβ42 was incubated for 1 day at 37 °C to make
aggregates. PBS (pH 7.4) was used as a running buffer at 25 °C.
Incubated Aβ42 aggregates were diluted 25-fold with 10 mM sodium
acetate solution (pH 4.0) and fixed onto the sensor chip surface using
amine coupling chemistry. The remaining activated surface groups
were blocked with 1 M ethanolamine (pH 8.5) to prevent covalent
binding of the analyte to the surface; the immobilization value was
1576.5 RU and the theoretical Rmax was 108 RU. Typically, one
binding cycle comprised an association phase, followed by a
dissociation phase, while the responses for different concentrations
of the analyte were collected for multiple cycles. Removing the analyte
from the ligand must be ensured after each cycle, but in this study, the
high binding affinity of the analyte to the ligand made it difficult to
maintain a proper surface regeneration condition. As such, single-
cycle kinetics was applied by increasing the concentrations of analyte
onto the ligand surface in one cycle.50,51 YQ-002 stock was 10 mM in
DMSO and diluted with PBS-P (pH 7.4) containing 5% DMSO as a
series of samples: 100, 200, 300, 400, and 500 μM. The analyte
contact and dissociation time were 120 and 600 s, respectively, and
the flow rate was 20 μL/min.
gram for Brain Science NRF-2018M3C7A1021858, and NRF-
2017R1A2A2A05069364), and Yonsei University (2018-22-
0022).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AD, Alzheimer’s disease; Aβ, amyloid-β; NQ, naphthoqui-
nones; 1,4-NQ, 1,4-naphthoquinones; ThT, Thioflavin T;
B6C3Tg, APPswe, PSEN1dE9; 85Dbo/J, APP/PS1; BBB,
blood-brain barrier; PAMPA, parallel artificial membrane
permeability assay; PICUP, photoinduced cross-linking of
unmodified proteins
REFERENCES
■
(1) Haass, C., and Selkoe, D. J. (2007) Soluble protein oligomers in
neurodegeneration: lessons from the Alzheimer’s amyloid beta-
peptide. Nat. Rev. Mol. Cell Biol. 8 (2), 101−12.
(2) Sevigny, J., Chiao, P., Bussiere, T., Weinreb, P. H., Williams, L.,
Maier, M., Dunstan, R., Salloway, S., Chen, T., Ling, Y., O’Gorman, J.,
Qian, F., Arastu, M., Li, M., Chollate, S., Brennan, M. S., Quintero-
Monzon, O., Scannevin, R. H., Arnold, H. M., Engber, T., Rhodes, K.,
Ferrero, J., Hang, Y., Mikulskis, A., Grimm, J., Hock, C., Nitsch, R. M.,
and Sandrock, A. (2016) The antibody aducanumab reduces Aβ
plaques in Alzheimer’s disease. Nature 537 (7618), 50−6.
(3) Abner, E. L., Neltner, J. H., Jicha, G. A., Patel, E., Anderson, S.
L., Wilcock, D. M., Van Eldik, L. J., and Nelson, P. T. (2018) Diffuse
amyloid-beta plaques, neurofibrillary tangles, and the impact of APOE
in elderly persons’ brains lacking neuritic amyloid plaques. J.
Alzheimer's Dis. 64 (4), 1307−1324.
(4) Babula, P., Adam, V., Havel, L., and Kizek, R. (2009)
Noteworthy secondary metabolites naphthoquinones - their occur-
rence, pharmacological properties and analysis. Curr. Pharm. Anal. 5,
47−68.
(5) Widhalm, J. R., and Rhodes, D. (2016) Biosynthesis and
molecular actions of specialized 1,4-naphthoquinone natural products
produced by horticultural plants. Hortic. Res. 3, 16046.
Statistical Analysis. All graphs were obtained with GraphPad
Prism 7.0 software, and all statistical analyses were conducted with
one-way ANOVA followed by Bonferroni’s posthoc comparisons (*P
< 0.033, **P < 0.002, ***P < 0.001). The error bars represent the
standard deviation (SD).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.9b00093.
Full images of SDS-PAGE analysis with PICUP
chemistry; excitation and emission spectral scan of
selected 1,4-NQ derivatives; surface plasmon resonance
analysis to confirm Aβ binding property; structural
spectra of YQ compounds (PDF)
(6) Edreva, A., Velikova, V., Tsonev, T., Dagnon, S., Gu
Aktas, L. (2007) Stress-protective role of secondary metabolites:
̈
rel, A. L., and
̧
Diversity of functions and mechanisms. Gen. Appl. Plant Physiol., 34.
(7) Kim, B. H., Yoo, J., Park, S. H., Jung, J. K., Cho, H., and Chung,
Y. (2006) Synthesis and evaluation of antitumor activity of novel 1,4-
naphthoquinone derivatives (IV). Arch. Pharmacal Res. 29 (2), 123−
30.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: ikyonkim@yonsei.ac.kr (I.K.), for medicinal chem-
istry.
*E-mail: y.kim@yonsei.ac.kr (Y.K.), for amyloid investigation.
ORCID
Ikyon Kim: 0000-0002-0849-5517
YoungSoo Kim: 0000-0001-5029-7082
Author Contributions
N.N.S., H.J., and Y.J. contributed equally. N.N.S., H.J., Y.J.,
I.K., and Y.K. designed the experiments. N.N.S. and H.J.
performed the in vitro and ex vivo experiments. Y.J., G.H.B.,
and I.K. designed and synthesized the YQ compounds. H.C.Y.
and J.M.H. performed PAMPA assay. S.L. and S.B. prepared
synthetic Aβ42. K.P. and S.H.Y. performed molecular docking
model experiments. N.N.S., H.J., I.K., and Y.K. wrote the
manuscript. All images were created by the authors for this
manuscript.
■
́
́
(8) Lopez, L. I., Nery Flores, S. D., Silva Belmares, S. Y., and Saenz
Galindo, A. (2014) Naphthoquinones: biological properties and
synthesis of lawsone and derivatives - a structured review. Vitae 21,
248−258.
(9) Tandon, V. K., Chhor, R. B., Singh, R. V., Rai, S., and Yadav, D.
B. (2004) Design, synthesis and evaluation of novel 1,4-
naphthoquinone derivatives as antifungal and anticancer agents.
Bioorg. Med. Chem. Lett. 14 (5), 1079−83.
(10) Vukic, M. D., Vukovic, N. L., Djelic, G. T., Popovic, S. L., Zaric,
M. M., Baskic, D. D., Krstic, G. B., Tesevic, V. V., and Kacaniova, M.
M. (2017) Antibacterial and cytotoxic activities of naphthoquinone
pigments from Onosma visianii Clem. Exp. Clin. Sci. J. 16, 73−88.
(11) Bermejo-Bescos, P., Martin-Aragon, S., Jimenez-Aliaga, K. L.,
Ortega, A., Molina, M. T., Buxaderas, E., Orellana, G., and Csaky, A.
G. (2010) In vitro antiamyloidogenic properties of 1,4-naphthoqui-
nones. Biochem. Biophys. Res. Commun. 400 (1), 169−74.
(12) Castagnoli, K. P., Steyn, S. J., Petzer, J. P., Van der Schyf, C. J.,
and Castagnoli, N., Jr. (2001) Neuroprotection in the MPTP
Parkinsonian C57BL/6 mouse model by a compound isolated from
tobacco. Chem. Res. Toxicol. 14 (5), 523−7.
Funding
This work was supported by Korea Health Industry Develop-
ment Institute (KHIDI, HI18C0836010018), National Re-
search Foundation (Basic Science Research Program NRF-
2018R1A6A1A03023718, Original Technology Research Pro-
(13) Nam, K. N., Son, M. S., Park, J. H., and Lee, E. H. (2008)
Shikonins attenuate microglial inflammatory responses by inhibition
L
DOI: 10.1021/acschemneuro.9b00093
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Research Article
of ERK, Akt, and NF-kappaB: neuroprotective implications. Neuro-
pharmacology 55 (5), 819−25.
(31) Klunk, W. E., and Mathis, C. A. (2008) The future of amyloid-
beta imaging: a tale of radionuclides and tracer proliferation. Curr.
Opin. Neurol. 21 (6), 683−687.
(14) Scherzer-Attali, R., Pellarin, R., Convertino, M., Frydman-
Marom, A., Egoz-Matia, N., Peled, S., Levy-Sakin, M., Shalev, D. E.,
Caflisch, A., Gazit, E., and Segal, D. (2010) Complete phenotypic
recovery of an Alzheimer’s disease model by a quinone-tryptophan
hybrid aggregation inhibitor. PLoS One 5 (6), No. e11101.
(15) Tomiyama, T., Shoji, A., Kataoka, K., Suwa, Y., Asano, S.,
Kaneko, H., and Endo, N. (1996) Inhibition of amyloid beta protein
aggregation and neurotoxicity by rifampicin. Its possible function as a
hydroxyl radical scavenger. J. Biol. Chem. 271 (12), 6839−44.
(16) Munishkina, L. A., and Fink, A. L. (2007) Fluorescence as a
method to reveal structures and membrane-interactions of amyloido-
genic proteins. Biochim. Biophys. Acta, Biomembr. 1768 (8), 1862−
1885.
(32) Biancalana, M., and Koide, S. (2010) Molecular mechanism of
Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta,
Proteins Proteomics 1804 (7), 1405−1412.
(33) Groenning, M. (2010) Binding mode of Thioflavin T and other
molecular probes in the context of amyloid fibrils-current status.
Journal of Chemical Biology 3 (1), 1−18.
(34) Teoh, C. L., Su, D., Sahu, S., Yun, S.-W., Drummond, E., Prelli,
F., Lim, S., Cho, S., Ham, S., Wisniewski, T., and Chang, Y.-T. (2015)
Chemical fluorescent probe for detection of Aβ oligomers. J. Am.
Chem. Soc. 137 (42), 13503−13509.
(35) Bhasin, D., Chettiar, S. N., Etter, J. P., Mok, M., and Li, P. K.
(2013) Anticancer activity and SAR studies of substituted 1,4-
naphthoquinones. Bioorg. Med. Chem. 21 (15), 4662−9.
(36) Koller, A., Rudi, A., Gravalos, M. G., and Kashman, Y. (2001)
Synthesis and electrophilic substitution of pyrido[2,3,4-kl]- acridines.
Molecules 6 (4), 300−322.
(37) Anderson, W. P., Connarty, B., and El-Hajj, T. (1986) A simple
efficient synthesis of 5,8-Isoquinolinedione. Synth. Commun. 16,
1557−1560.
(17) Hudson, S. A., Ecroyd, H., Kee, T. W., and Carver, J. A. (2009)
The thioflavin T fluorescence assay for amyloid fibril detection can be
biased by the presence of exogenous compounds. FEBS J. 276 (20),
5960−72.
(18) Bitan, G. (2006) Structural study of metastable amyloidogenic
protein oligomers by photo-induced cross-linking of unmodified
proteins. Methods Enzymol. 413, 217−236.
(38) Kitahara, Y., Nakai, T., Nakahara, S., Akazawa, M., Shimizu, M.,
and Kubo, A. (1991) Synthesis of 5, 6-, 5, 8- and 7, 8-
isoquinolinediones from the corresponding isoquinolinols and
dimethoxyisoquinolines. Chem. Pharm. Bull. 39 (9), 2256−2263.
(39) Ryu, C.-K., Lee, I. K., Jung, S. H., Kang, H. Y., and Lee, C. O.
(2000) 7-(Substituted-phenyl)amino-5,8-isoquinolinediones: Synthe-
sis and cytotoxic activities on cancer cell lines. Med. Chem. Res. 10,
40−49.
(40) Ryu, C.-K., Jeong, H.-J., Lee, S. K., You, H.-J., Choi, K. U.,
Shim, J.-Y., Heo, Y. H., and Lee, C.-O. (2001) Effects of 6-arylamino-
5,8-quinolinediones and 6-chloro-7-ary-lamino-5,8-isoquinolinediones
on NAD(P)H: quinone oxidoreductase (NQO1) activity and their
cytotoxic potential. Arch. Pharmacal Res. 24 (5), 390−396.
(41) Leepasert, T., Shahabi, M., Shanab, K., Schirmer, E., Holzer,
(19) Jameson, L. P., Smith, N. W., and Dzyuba, S. V. (2012) Dye-
binding assays for evaluation of the effects of small molecule
inhibitors on amyloid (abeta) self-assembly. ACS Chem. Neurosci. 3
(11), 807−19.
(20) Meyer-Luehmann, M., Spires-Jones, T. L., Prada, C., Garcia-
Alloza, M., de Calignon, A., Rozkalne, A., Koenigsknecht-Talboo, J.,
Holtzman, D. M., Bacskai, B. J., and Hyman, B. T. (2008) Rapid
appearance and local toxicity of amyloid-beta plaques in a mouse
model of Alzheimer’s disease. Nature 451 (7179), 720−4.
(21) Schneidman-Duhovny, D., Inbar, Y., Nussinov, R., and
Wolfson, H. J. (2005) PatchDock and SymmDock: servers for rigid
and symmetric docking. Nucleic Acids Res. 33, W363−7.
(22) Trott, O., and Olson, A. J. (2009) AutoDock Vina: improving
the speed and accuracy of docking with a new scoring function,
efficient optimization, and multithreading. J. Comput. Chem. 31 (2),
455−61.
(23) Di, L., Kerns, E. H., Fan, K., McConnell, O. J., and Carter, G. T.
(2003) High throughput artificial membrane permeability assay for
blood-brain barrier. Eur. J. Med. Chem. 38 (3), 223−232.
(24) O’Hare, E., Scopes, D. I. C., Kim, E.-M., Palmer, P., Jones, M.,
Whyment, A. D., Spanswick, D., Amijee, H., Nerou, E., McMahon, B.,
Treherne, J. M., and Jeggo, R. (2013) Orally bioavailable small
molecule drug protects memory in Alzheimer’s disease models.
Neurobiol. Aging 34 (4), 1116−1125.
(25) Liscic, R. M. (2013) Immunological aspects and anti-amyloid
strategy for Alzheimer’s dementia. Arh. Hig. Rada Toksikol. 64 (4),
603−8.
(26) Sundaram, G. S. M., Dhavale, D. D., Prior, J. L., Yan, P., Cirrito,
J., Rath, N. P., Laforest, R., Cairns, N. J., Lee, J.-M., Kotzbauer, P. T.,
and Sharma, V. (2016) Fluselenamyl: a novel benzoselenazole
derivative for PET detection of amyloid plaques (Aβ) in Alzheimer’s
disease. Sci. Rep. 6, 35636.
(27) Viola, K. L., and Klein, W. L. (2015) Amyloid beta oligomers in
Alzheimer’s disease pathogenesis, treatment, and diagnosis. Acta
Neuropathol. 129 (2), 183−206.
(28) Zhang, X., Bloch, S., Akers, W., and Achilefu, S. (2012) Near-
infrared molecular probes for in vivo imaging. Curr. Protoc. Cytom. 60,
12.27.1.
W., Spreitzer, H., Aicher, B., Muller, G., and Gunther, E. (2013)
̈ ̈
Synthesis and antiproliferative activity of new cytotoxic tri- and
tetraazabenzo[3,2-a]fluorene-5,6-dione derivatives. Bioorg. Med.
Chem. Lett. 23 (19), 5264−5266.
(42) Wu, Y.-L., Chuang, C.-P., and Lin, P.-Y. (2001) Oxidative free
radical reactions between 2-amino-1,4-naphthoquinones and carbonyl
compounds. Tetrahedron 57 (26), 5543−5549.
(43) Shanab, K., Schirmer, E., Knafl, H., Wulz, E., Holzer, W.,
Spreitzer, H., Schmidt, P., Aicher, B., Muller, G., and Gunther, E.
̈ ̈
(2010) Synthesis and biological evaluation of new cytotoxic
azanaphthoquinone pyrrolo-annelated derivatives. Bioorg. Med.
Chem. Lett. 20 (13), 3950−3952.
(44) Kitahara, Y., Nakahara, S., Tanaka, Y., and Kubo, A. (1992)
Synthesis of 5,8-quinoxalinediones and 5,8-quinazolinediones. Hetero-
cycles 34, 1623−1630.
(45) Fosso, M. Y., Chan, K. Y., Gregory, R., and Chang, C.-W. T.
(2012) Library synthesis and antibacterial investigation of cationic
anthraquinone analogs. ACS Comb. Sci. 14 (3), 231−235.
(46) Lee, S., and Kim, Y. (2015) Anti-amyloidogenic approach to
access amyloid-β(1−42) in Fmoc solid-phase synthesis. Bulletin of the
Korean Chemical Society 36 (8), 2147−2149.
(47) Bitan, G., and Teplow, D. B. (2004) Rapid photochemical
cross-linking–a new tool for studies of metastable, amyloidogenic
protein assemblies. Acc. Chem. Res. 37 (6), 357−64.
(48) Rahimi, F., Maiti, P., and Bitan, G. (2009) Photo-induced
cross-linking of unmodified proteins (PICUP) applied to amyloido-
genic peptides. J. Visualized Exp. No. 23, e1071.
́
̈
(29) Friden, M., Wennerberg, M., Antonsson, M., Sandberg-Stall,
M., Farde, L., and Schou, M. (2014) Identification of positron
emission tomography (PET) tracer candidates by prediction of the
target-bound fraction in the brain. EJNMMI Res. 4 (1), 50−50.
(30) Pike, V. W. (2009) PET radiotracers: crossing the blood-brain
barrier and surviving metabolism. Trends Pharmacol. Sci. 30 (8), 431−
40.
(49) Yang, S. H., Lee, D. K., Shin, J., Lee, S., Baek, S., Kim, J., Jung,
H., Hah, J. M., and Kim, Y. (2017) Nec-1 alleviates cognitive
impairment with reduction of Abeta and tau abnormalities in APP/
PS1 mice. EMBO Mol. Med. 9 (1), 61−77.
M
DOI: 10.1021/acschemneuro.9b00093
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
(50) Karlsson, R., Katsamba, P. S., Nordin, H., Pol, E., and Myszka,
D. G. (2006) Analyzing a kinetic titration series using affinity
biosensors. Anal. Biochem. 349 (1), 136−147.
(51) Myszka, D. G., Jonsen, M. D., and Graves, B. J. (1998)
Equilibrium analysis of high affinity interactions using BIACORE.
Anal. Biochem. 265 (2), 326−330.
N
DOI: 10.1021/acschemneuro.9b00093
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX