Discovery and Characterization of Novel Naphthalimide Analogs as Potent Multitargeted Directed Ligands against Alzheimer's Disease

Article abstract of DOI:10.1002/ddr.21708

Current therapeutic drugs for Alzheimer's disease (AD) can only offer limited symptomatic benefits and do not halt disease progression. Multitargeted directed ligands (MTDLs) have been considered to be a feasible way to treat AD due to the multiple neuropathological processes in AD. Previous studies proposed that compounds containing two aromatic groups connected by a carbon chain should act as effective amyloid β (Aβ) aggregation inhibitors although the optimal length of the carbon chain has not been explored. In the current study, a series of naphthalimide analogs were designed and synthesized based on the proposed structure and multiple bioactivities beneficial to the AD treatment were reported. In vitro studies showed that compound 8, which has two aromatic groups connected by a two-carbon chain, exhibited significant inhibition of Aβ aggregation through the prevention of elongation and association of Aβ fibril growth. Furthermore, this compound also displayed antioxidative activities and neuroprotection from Aβ monomer induced toxicity in primary cortical neurons. The results of the present study highlight a novel naphthalimide-based compound 8 as a promising MTDL against AD. Its structural elements can be further explored for enhanced therapeutic capabilities.

Full text of DOI:10.1002/ddr.21708

Received: 27 January 2020
DOI: 10.1002/ddr.21708
Revised: 20 April 2020
Accepted: 29 May 2020
R E S E A R C H A R T I C L E
Discovery and Characterization of Novel Naphthalimide
Analogs as Potent Multitargeted Directed Ligands against
Alzheimer's Disease
Jie Gao1
| James Chapman2
1Department of Clinical and Diagnostic
Science, University of Alabama at Birmingham,
Birmingham, Alabama, USA
Abstract
Current therapeutic drugs for Alzheimer's disease (AD) can only offer limited symp-
tomatic benefits and do not halt disease progression. Multitargeted directed ligands
(MTDLs) have been considered to be a feasible way to treat AD due to the multiple
neuropathological processes in AD. Previous studies proposed that compounds con-
taining two aromatic groups connected by a carbon chain should act as effective
amyloid β (Aβ) aggregation inhibitors although the optimal length of the carbon chain
has not been explored. In the current study, a series of naphthalimide analogs were
designed and synthesized based on the proposed structure and multiple bioactivities
beneficial to the AD treatment were reported. In vitro studies showed that compound
8, which has two aromatic groups connected by a two-carbon chain, exhibited signifi-
cant inhibition of Aβ aggregation through the prevention of elongation and associa-
tion of Aβ fibril growth. Furthermore, this compound also displayed antioxidative
activities and neuroprotection from Aβ monomer induced toxicity in primary cortical
neurons. The results of the present study highlight a novel naphthalimide-based com-
pound 8 as a promising MTDL against AD. Its structural elements can be further
explored for enhanced therapeutic capabilities.
2Department of Discovery and Biomedical
Sciences, University of South Carolina,
Columbia, South Carolina, USA
Correspondence
Jie Gao, Department of Clinical and Diagnostic
Science, University of Alabama at Birmingham,
1716 9th Ave. S, Birmingham, AL 35294.
Email: gaoj@uab.edu
Funding information
University of South Carolina
K E Y W O R D S
Alzheimer's disease, amyloid aggregation, amyloid-β protein, antioxidation, multitargeted
directed ligands, naphthalimide, neuroprotection, oxidative stress
1
|
INTRODUCTION
are ꢀ4.3 kDa proteins produced by normal brain metabolism. Two
major Aβ variants, Aβ40 and Aβ42, have been identified as the principal
Alzheimer's disease (AD) is a widely known to be a result of many
pathological factors such as amyloid plaques and oxidative stress
(Hardy & Allsop, 1991; Su et al., 2008). Aβ aggregation is one of the
most widely studied pathological factors involved in AD. Aβ peptides
pathological species in amyloid plaques deposited in the AD brain, and
Aβ40 is the most plentiful isoform in amyloid plaques (Guntert, Dobeli,
& Bohrmann, 2006; Walsh & Selkoe, 2007). The aggregation process
of Aβ monomers has been described as three steps: nucleus forma-
tion, soluble aggregates, and fibril formation (Goedert
&
Spillantini, 2006). The soluble Aβ aggregates can grow into fibrils
through elongation or association. The elongation growth increases
aggregate size through the addition of Aβ monomers to the ends of
the aggregates and results in fibrils with a mass-per-unit length similar
to the staring material. The association growth increases Aβ
ABBREVIATIONS: AD, Alzheimer's disease; Aβ, amyloid β; BSA, bovine serum albumin; DLS,
dynamic light scattering; DMSO, dimethyl sulfoxide; MTDL, multitargeted directed ligands;
mtDNA, mitochondrial DNA; ORAC, oxygen radical absorbance capacity; PBS, phosphate
buffered saline; Phe, phenylalanine; RH, hydrodynamic radius; ROS, reactive oxygen species;
SEC, size exclusion chromatography; TEM, transmission electron microscopy; ThT,
thioflavin T.
Drug Dev Res. 2020;1–10.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, Inc.
1

2
GAO AND CHAPMAN
aggregates in size direct lateral aggregate–aggregate interactions and
leads to increase in aggregate size with a larger mass-per-unit length
ratio than the starting aggregate (Nichols et al., 2002).
most useful strategies. Various compounds such as curcumin, rifampi-
cin, benzofuran, and bis-styrylbenzene analogs have been explored as
Aβ aggregation inhibitors (Byun et al., 2008; Lee et al., 2008;
Narlawar, Baumann, Schubenel, & Schmidt, 2007; Ono, Hasegawa,
Naiki, & Yamada, 2004). In particular, the dipeptide (Phe19-Phen20) in
the Aβ monomer was reported to play a critical role in the formation
of soluble aggregates (Tjernberg et al., 1996), therefore, the analogic
structure of this dipeptide, two aromatic end groups connected by a
linker, has been proposed as an ideal scaffold of Aβ aggregation inhibi-
tors. These analogs are hypothesized to inhibit the Aβ aggregation
through the interactions to the phenylalanine residues 19 and 20,
which disrupt the π–π stacking between Aβ monomers (Reinke &
Gestwicki, 2007). According to this proposed structure, numerous
potential inhibitors have been studied and demonstrated potent inhi-
bition of Aβ aggregation (Kroth et al., 2012; McKoy, Chen, Schupbach,
& Hecht, 2012; Nie, Du, & Geng, 2011).
Oxidative stress has also been identified as an early event in the
pathogenesis of AD (Padurariu et al., 2010). The important sources of
oxidative stress, reactive oxygen species (ROS) and reactive nitrogen
oxide species (RNOS), can particularly react with lipids, proteins,
nucleic acid, and other molecules in brain and cause a significant
extent of oxidative damage associated with the abnormal deposition
of both Aβ aggregates and neurofibrillary tangles in AD
(Mattson, 2004). Therefore, antioxidants have been suggested to be a
viable option to not only prevent Aβ aggregation, but also address
some other pathological factors such as mitochondrial dysfunction,
neuronal inflammation, and so on. Some commonly used antioxidants
have been studied for their useful indications in the therapeutic treat-
ment of AD (Feng & Wang, 2012).
These concurrent mechanisms contribute to the progression of
AD and are likely to be highly interrelated (Carreiras, Mendes, Perry,
Francisco, & Marco-Contelles, 2013). Current AD therapies are single
targeted drugs such as acetylcholinesterase inhibitors (donepezil,
rivastigmine, and galantamine) and the NMDA receptor antagonist
(memantine). However, the interactions among the different patholog-
ical factors preclude the effectiveness of these single targeted drugs,
which only offer symptomatic benefits for mild to moderate AD but
without halting or delaying disease progression (Yiannopoulou &
Papageorgiou, 2013). Therefore, multitarget-directed ligands (MTDL),
which can address different pathological targets simultaneously with-
out the risk of drug–drug interactions, have been proposed to be one
of the ideal treatments for AD (Cavalli et al., 2008) and have also been
exploited at both academic and industrial levels. (Goyal, Kaur, &
Goyal, 2018; Kaur et al., 2019; Kaur, Shuaib, Goyal, & Goyal, 2020;
Patil, Thakur, Sharma, & Flora, 2019; Yang et al., 2020).
Naphthalimides have been reported to serve as core scaffold for
many antitumor, anti-inflammatory, antidepressant, antiprotozoal, and
antiviral agents, and some of which were found without obvious sys-
temic toxicities at the therapeutic dose (Kamal, Bolla, Srikanth, &
Srivastava, 2013; Xie et al., 2012). Therefore, in the current study, we
rationally designed and developed a series of naphthalimide com-
pounds, which combine Aβ aggregation inhibition, neutralization of
oxidative stress and neuroprotection, as MTDLs against AD.
2
|
RESULTS AND DISCUSSION
2.1
|
Inhibition of Aβ monomer aggregation
The inhibitory activities of the naphthalimide analogs to Aβ aggrega-
tion are summarized in Table 1 regarding both lag time extension and
plateau reduction. Synthetic Aβ40 was used in this study because the
structure of Aβ40 fibrils formed using synthetic monomers is similar to
During the development of disease modifying drugs for AD, the
prevention of Aβ aggregation at different steps has become one of
TABLE 1
The structure of naphthalimide analogs and their inhibition capability on Aβ monomer aggregation
R
R1
O
N
O
R2
n
Compound
n
1
1
2
3
3
4
2
2
R
R1
H
R2
H
Plateau reduction (% inhibition)
17.4 2.9
Lag extension (fold increase)
ORAC activity
1
2
3
4
5
6
7
8
H
NE
NT
NO2
H
H
H
24.4 5.6
NE
NT
H
H
39.7 8.6
2.4 0.7
NE
NT
H
H
H
22.9 9.7
NT
NO2
H
H
H
37.4 2.9
NE
NT
H
H
34.0 5.2
NE
NT
H
H
OH
OH
35.2 5.1
NE
0.52 0.22
1.22 0.33
H
OH
85.2 2.9
4.9 1.6
Abbreviations: NE, the results of compounds at 100 μM are not significantly different from the ones of control samples; NT, not tested.

GAO AND CHAPMAN
3
FIGURE 1 Assessment of inhibition of Aβ monomer aggregation by naphthalimide analogs. Monomer aggregation assays were conducted in
the absence (solid line, control) and presence (dotted line, experimental) of the naphthalimide analogs. Aggregate formation was monitored using
ThT fluorescence to evaluate the ability of each compound to alter the lag time and plateau. Extension of lag time was proved as an increase in
the experimental lag time, T2, relative to the lag time for the control, T1, and evaluated as a fold increase to the control, T2/T1 (left panel).
Reduction of control plateau was evidenced as an decrease in the experimental plateau, P2, relative to the plateau for the control, P1, and
evaluated as the percentage decrease to the control, [(P1 ‑ P2)/P1] × 100% (right panel)
TABLE 2
The lengths and angles of the two aromatic groups
within compound 1, 3, 4, and 6
Length between two
aromatic groupsa
Angles between two
aromatic groupsa
Compound
Phe-Phe
8.525 Å
6.470 Å
8.752 Å
8.826 Å
8.976 Å
173.81ꢁ
112.66ꢁ
179.70ꢁ
167.83ꢁ
170.86ꢁ
1
3
4
6
aThe length of the linkers and the angles of the two aromatic groups are
measured by Discovery Studio 2.5 (Accelrys, San Diego, CA).
aromatic rings, significantly extended the lag time by 2.4 0.7 folds
over the control sample, which indicated that the two-carbon chain
might be a proper length for 1,8-naphthalimide analogs as Aβ inhibi-
tors. The lag time extension of compound 3 might be due to its con-
formational compatibility to Phe19-Phe20 in Aβ peptide, which
interfered the initial binding of Aβ monomers at the early stage.
Therefore, compound 1, 3, 4, and 6 in their most energetically favor-
able computed conformations were aligned to one of the phenyl rings
in Phe19-Phe20 with the help of the Discovery Studio 2.5, a software
that simulates small molecules and macromolecules systems. The
aligned result is shown in Figure 2 and the lengths and angles of the
two aromatic rings within the compounds are listed in Table 2. As
shown in the Table 2, the two aromatic rings in compound 3 possess a
length of 8.752 Å and an angle of 179.70ꢁ, both of which are similar
to the ones of Phe19-Phe20 (8.525 Å and 173.81ꢁ, respectively). This
conformation of compound 3 leads the 1,8-naphthalimide ring to a
similar direction to the Phe19-Phe20 peptide (green, Figure 2). These
molecular modeling results supported the experimental data that com-
pound 3 can hinder the Phe19-Phe20 binding between Aβ monomers
and effectively delay the Aβ aggregate formation.
FIGURE 2 The alignment of compound 1, 3, 4, and 6 (blue) and
the Phe-Phe peptide (green) in the most energetically favorable
computed conformations
fibrils isolated from amyloid plaques (Goedert & Spillantini, 2006). The
production of Aβ aggregates had been measured through the equilib-
rium plateau (indicating the amount of aggregated forms) and the lag
time (indicating the delay of Aβ aggregate production) (Figure 1),
which are normally monitored by thioflavin T (ThT), a fluorescent
compound specifically binding to the β-sheet structures in the Aβ
aggregates. The lag time extension, which occurs in the early stages of
Aβ aggregation, was determined as the fold increase of the time at
which ThT fluorescence is first increased in the presence and absence
of inhibitor, T1 and T2, respectively (Figure 1, left panel). The plateau
reduction, which occurs in the late points of Aβ aggregation, was
defined as the percentage decrease of the plateau ThT fluorescence
in the presence of inhibitor, P2, compared to the plateau observed in
the absence of inhibitor, P1 (Figure 1, right panel). It should be noted
that compound 3, containing a two-carbon chain between the two
In order to increase the plateau inhibition of Aβ aggregation and
the antioxidant activity of compound 3, hydroxyl groups were added

4
GAO AND CHAPMAN
to its structure and offered compound 7 and 8. The compound 8 with
two hydroxyl groups presents significant Aβ aggregation inhibition
with a plateau reduction of 85.2 2.9% and a lag time extension of
4.9 1.6 folds over the control sample. The results of compound 7
and 8 indicated that 3-hydroxyl substitution may be more critical for
potent inhibitory activities than 4-hydroxyl substitution on phenyl
rings. It might also be conceivable that at least two hydroxyl groups
are required to exhibit potent Aβ aggregation inhibition.
2.2
|
The mechanistic-specific inhibition of
compound 8
In order to explore the inhibitory mechanisms of compound 8, the sol-
uble Aβ aggregates increase in size via association or elongation was
monitored as the change in hydrodynamic radius (RH). As shown in
Figure 3a, whereas Aβ monomer or soluble aggregates incubated
alone exhibited negligible changes in RH, a steady increase in RH was
observed when soluble Aβ aggregates (2 μM) were incubated in the
presence of Aβ monomer. However, when the same soluble Aβ aggre-
gates were incubated with compound 8 (80 μM) before the addition
of Aβ monomer, the observed elongation rate, or rate of increase in
RH, was reduced by 75.3 9.5%, which meant that compound 8 sig-
nificantly inhibited the elongation of soluble Aβ aggregates. Similarly,
Figure 3b showed a steady increase in RH when the aggregates were
incubated in the presence of NaCl. When 2 μM soluble Aβ aggregates
were incubated with compound 8 (10 μM) before the addition of
Based on these compounds evaluated as Aβ aggregation inhibi-
tors, the preliminary structure activity relationship could be summa-
rized as: (a) the addition of a nitro group on the naphthalimide phenyl
ring does not potentiate the Aβ aggregation inhibition; (b) a two-car-
bon chain might possess the optimal length in these 1,8-
naphthalimide molecules for lag time extension instead of plateau
reduction; and (c) The presence of two or more phenol groups in the
naphthalimide structure can significantly potentiate the molecular
activity to inhibit Aβ aggregation.
150
400
(a)
(b)
100
50
0
200
0
0
5
10
15
0
5
10
15
Time (min)
Time (min)
FIGURE 3 Effect of compound 8 on soluble Aβ soluble aggregate growth. (a) soluble Aβ aggregates (2 μM) with positive control (0 μM, □), or
compound 8 (80 μM,▲), and Aβ monomer (30 μM) was added to induce aggregate growth. As negative controls, the soluble Aβ aggregates (◇) or
Aβ monomer (ꢁ) were incubated alone. Increases in aggregate size were monitored continuously as changes in RH. (b) soluble Aβ aggregates (2 μM)
with positive control (0 μM, □), or compound 8 (10 μM, ▲), and 150 mM NaCl was added to induce aggregate association. As a negative control,
the soluble Aβ aggregates (ꢁ) were incubated alone and in the absence of NaCl. Increases in aggregate size were monitored continuously as
changes in RH. Linear regression (solid lines) was performed to determine association growth rates (r2). Results are representative of three
independent experiments
FIGURE 4 Morphology of Aβ1-40
aggregates. SEC-isolated Aβ
monomers in 40 mM Tris–HCl,
pH 8.0, were aggregated alone
(control, left panel) or with
compound 8 (100 μM, right panel).
After control reached plateau, the
reactions were terminated and
samples were gridded and visualized
by TEM as described under
experimental section. Results are
representative of two independent
experiments. Images are shown
relative to a scale bar of 0.5 μm

GAO AND CHAPMAN
5
NaCl, the observed association rate, or rate of increase in RH, was
reduced by 75.2 11.4%, which indicates a potent inhibition on the
association of Aβ aggregate. The presence of 2% DMSO has a negligi-
ble effect on the growth of the soluble Aβ aggregates by association
(data not shown).
compound 8 (right). The fibrils are shorter and thinner in this picture
than the ones in the left picture. This observation backed up the fact
that compound 8 prevents the production of Aβ fibrils from mono-
mers through the inhibition of both elongation and association.
In order to visualize the formation and morphological changes of
Aβ fibrils, the transmission electron microscopy (TEM) was employed
to acquire direct images of Aβ aggregates. Figure 4 depicted the Aβ
fibril images when Aβ monomers were incubated with NaCl alone
(left) or in the presence of NaCl and compound 8 (right). Both pictures
showed fibrillar aggregates but with some notable differences. For
example, there are less fibrils were formed in the presence of
2.3
|
Antioxidative activity of compound 7 and
compound 8
The antioxidative activity of compound 7 and 8 was evaluated by the
oxygen radical absorbance capacity (ORAC) assay and the inducible
nitric oxygen synthase (iNOS) induction assay. In the ORAC assay, the
antioxidative activity was evaluated based on the ORAC values. The
ORAC value of the control agent Trolox is designed as 1 and the
values of the testing compound will compare to Trolox. As shown in
Table 1, compound 8 with the ORAC value of 1.22 0.33 demon-
strated a more potent antioxidative activity than Trolox. Consistent
with the ORAC data, compound 8 can also significantly suppress the
production of iNOS induced by IFN-γ at 8 hr (Figure 5). These data
indicated that compound 8 has a potent antioxidative activity against
oxidative damages in addition to the Aβ aggregation inhibition.
2.4
|
Neuroprotective activity of compound 8
Compound 8 was further tested on primary cortical neuronal cultures
to assess the potential neuroprotective effects against the com-
promised neuronal viability induced by the Aβ monomers. As shown
in Figure 6, 24 hr incubation with the Aβ monomers (200 nM)
decreased cell survival by about 40% in each series of experiments.
Compound 8 significantly increased the cell viability around 80% of
control values at the concentrations of 100 nM and 1 μM although
extremely high concentration (10 μM) of the compound demonstrates
toxic activity. It has been reported that phenolic compounds can pro-
tect neurons from amyloid challenges through multiple mechanisms
such as the inhibition of NADPH oxidase and subsequent reactive
oxygen species generation, the reduction of inflammation, and so on
(Kovacsova, Andrej, Jana, Stanislava, & Olga, 2010). Effectiveness of
compound 8 might also attribute to these mechanisms. Its structural
features may aid the rational drug design approaches in future.
FIGURE 5 Effects of compounds 7 (100 μM) and compound 8
(100 μM) on the induced expression of iNOS. C+ (positive control)
was an archived ANA-1 cell lysate known to have iNOS induction.
Numbers under each band represent the densitometric quantification
of iNOS bands, and adjusted for Actin levels
100
*
80
*
60
40
*
0
0nM
1uM
10uM
100uM
10nM
100nM
2.5
|
Drug likeness of compound 8
Neg. Ctrl.
Compound 8
Several physiochemical properties of compound 8 related drug like-
ness were calculated by QikProp, version 3.5, Schrödinger, LLC
FIGURE 6 Neuroprotective effects of compound 8 against Aβ
monomer (200 μM) as determined in a cell viability assay in rat
cortical neuron primary culture
(2015). The virtual screening results indicate that compound
8
TABLE 3
Compliance of Compound 8 with computational parameters of drug likeness
Compound
Molecular weight (kD)
CLogP
acceptHB
donorHB
Rule of 5 violation
8
333
2.8
4.5
2
0
Abbreviations: CLogP, calculated logP; acceptHB, estimated number of hydrogen bonds acceptor; donorHB, estimated number of hydrogen bonds donor.

6
GAO AND CHAPMAN
TABLE 4
The standard computational parameters of pharmacokinetics for Compound 8
Compound
8
LogS (mol/ml)
−4.3
LogKhsa
0.24
CNS
Caco
LogBB
−1.3
−2
259
Standard rangea
−6.5–0.5
−1.5–1.5
−2 (inactive)
<25 poor
−3.0–1.2
+2 active)
>500 great
Abbreviations: Caco, predicted apparent Caco-2 cell permeability in nm/sec; CNS, Predicted central nervous system activity; LogBB, predicted brain/blood
partition coefficient; LogS, prediction of aqueous solubility; LogKhsa, prediction of binding to human serum albumin.
aFor 95% of known drugs based on Schrödinger—Qikprop v3.5 (2015) software results.
followed the modified Lipinski's “Rule of Five” for CNS drug likeness
properties (Table 3). In addition, its pharmacokinetics parameters such
as aqueous solubility (LogS), serum protein binding (LogKhsa), and
gut-blood barrier (Caco-2 cell permeability) were within the standard
ranges of 95% known drugs (Table 4), although its distribution to the
central nervous system was predicted to be poor based on the logBB
and the predicted central nervous system activity (CNS). It has been
documented that the estimated number of hydrogen bond donors
should be less than 1 for CNS drugs (Ghose, Herbertz, Hudkins,
Dorsey, & Mallamo, 2012). This poor distribution to CNS might be
due to the phenolic groups, which provided two active hydrogen bond
donors and significantly decreased its penetration to BBB. A number
of studies reported various approaches to make prodrugs in order to
improve phenolic drug deliveries (Placzek et al., 2016; Thomas,
Majumdar, & Sloan, 2009; Valiveti et al., 2005). Therefore, as a prom-
ising leading compound, the CNS activity of compound 8 could be
improved through prodrugs.
Thioflavin T (ThT) was obtained from Sigma (St. Louis, MO). Bovine
serum albumin (BSA) and dimethyl sulfoxide (DMSO) were purchased
from EMD Biosciences (San Diego, CA). Tris–HCl buffer was made of
tris base, purchased from Promega (Madison, WI) and tris hydrochlo-
ride, purchased from EMD (Philadelphia, PA) with a pH value of 8.0.
4.2
|
Chemical synthesis
Melting points were determined on an Electrothermal® melting point
apparatus (capillary method) and are uncorrected. 1H-NMR spectra
were obtained on Mercury VX-300 spectrometer (Varian), using deu-
terochloroform (CDCl3) as the solvent. Most chemicals were pur-
chased from Acros Organics and TCI America chemicals. Mass
spectrometry was performed utilizing a VG Analytical 70S magnetic
sector mass spectrometer (waters, Milford Mass). Chromatography
was performed using Geduran® silica gel (40-63 μm).
1,8-naphthalimide analogs (compound 1–8).
To a suspension of 1,8-naphthalic anhydride (3.0 g, 15 mmol) in
toluene (100 mL), the corresponding reactants (15 mmol) were added
dropwise. The reaction mixture was refluxed with a condenser and a
Dean-Starke trap for 5 hr and then evaporated to yellow solid, which
was recrystallized with toluene (60 mL) to yield products.
3
|
CONCLUSIONS
AD is set to become even more prevalent as the average age of the
population continues to increase. Valid AD treatment discovery is a
crucial issue for both pharmaceutical industries and academia. In this
study, a series of 1,8-naphthalimide analogs were designed and syn-
thesized as a new type of structural scaffold for multiple potent bio-
logical activities including Aβ aggregation inhibition, antioxidant effect
and neuroprotectivity, which might be favorable for further structural
modification and drug development against AD. Of these compounds
evaluated herein, compound 8 was found to effectively inhibit Aβ
aggregation and oxidative damages; and also associated with modest
neuroprotective effects from toxicity induced by Aβ monomers in the
primary neuronal culture model. Together, compound 8 provided
structural information for an optimal scaffold of MTDL in the drug
development of AD.
2-Benzyl-1H-benzo[de]isoquinoline-1,3(2H)-dione(1) mp:194–
196ꢁC. 1H NMR(300 MHz, CDCl3) δ 5.32 (s, 2H, CH2N), 7.16–7.26(m,
4H, ArH), 7.48(d, 1H, J = 7.2, ArH), 7.68(t, J = 8.4, 2H, ArH), 8.13(d,
J = 8.4, 2H, ArH), 8.54(d, J = 7.2, 2H, ArH). HRMS: Obs. M + H,
287.0942. Calc. M + H, 287.0946.
2-Benzyl-5-nitro-1H-benzo[de]isoquinoline-1,3(2H)-dione(2)
mp:194–196ꢁC.1H NMR(300 MHz, CDCl3) δ 5.40 (s, 2H, CH2N),
7.28–7.34(m, 3H, ArH), 7.55(d, J = 7.2, 2H, ArH), 7.94(t, J = 7.8, 1H,
ArH), 8.42(d, J = 8.4, 1H, ArH), 8.79(d, J = 7.2, 1H, ArH), 9.13(s, 1H,
ArH), 9.34(s, 1H, ArH). HRMS: Obs. M + H, 332.0797. Calc. M + H,
332.0797.
2-Phenethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione(3)
mp:127–128ꢁC. 1H NMR(300 MHz, CDCl3) δ 2.96 (m, 2H, CH2CH2N),
4.34(m, 2H, CH2CH2N), 7.14–7.33(m, 5H, ArH), 7.70(t, J = 7.5, 2H,
ArH), 8.16(d, J = 8.1, 2H, ArH), 8.54(d, J = 7.2, 2H, ArH). HRMS: Obs.
M + H, 301.1100. Calc. M + H, 301.1103.
4
|
EXPERIMENTAL
4.1
|
Materials and general methods
2-(3-Phenylpropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione(4)
mp:88–89ꢁC.1HNMR (300 MHz, CDCl3)
δ
1.99 (m, 2H,
Unlabeled and N-terminally biotinylated Aβ were purchased from Ana-
Spec (Fremont, CA) and were stored in lyophilized formed at −20ꢁC.
CH2CH2CH2N), 2.71(t, J = 7.5, 2H, CH2CH2CH2N), 4.19(t, J = 7.5, 2H,
CH2CH2CH2N), 7.05–8.00(m, 1H, ArH), 7.17–7.19(m, 4H, ArH), 7.69

GAO AND CHAPMAN
7
(t, J = 7.5, 2H, ArH), 8.14(d, J = 8.1, 2H, ArH), 8.53(d, J = 7.5, 2H, ArH).
HRMS: Obs. M + H, 315.1255. Calc. M + H, 315.1259.
aggregation was monitored by ThT fluorescence. Soluble aggregates
were separated from fibrils via centrifugation (10 min, 18,000 g) and
from unreacted monomers via SEC on a Superdex 75 column, where
soluble aggregates eluted in the void volume (8–10 mL). Concentra-
tions of Aβ soluble aggregates, expressed in monomer units, were
determined from UV absorbance at 276 nm corrected for light scat-
tering, and equivalent ThT fluorescence measurements were assessed.
Measurement of soluble aggregate size via dynamic light scattering
(DLS) using a DynaPro-MSX instrument (Wyatt Technology Corpora-
tion, Santa Barbara, CA) revealed that this procedure consistently
yielded soluble Aβ soluble aggregates displaying an average hydrody-
namic radius (RH) of ꢀ80 nm. Aβ soluble aggregates were stored at
4ꢁC for up to 3 days. Following storage, ThT fluorescence measure-
ments were used to correct for any change in soluble Aβ aggregate
concentration, and DLS measurements were used to ensure that solu-
ble aggregates maintained their initial size.
5-Nitro-2-(3-phenylpropyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione(5) mp:128–129ꢁC. 1H NMR(300 MHz, CDCl3) δ 2.04 (m, 2H,
CH2CH2CH2N), 2.72(t, J = 8.1, 2H, CH2CH2CH2N), 4.20(t, J = 7.5, 2H,
CH2CH2CH2N), 7.04(m, 1H, ArH), 7.15–7.19(m, 4H, ArH), 7.87(t,
J = 7.8, 1H, ArH), 8.35(d, J = 8.1, 1H, ArH), 8.70(d, J = 7.1, 1H, ArH),
9.06(d, J = 2.1, 1H, ArH), 9.24(d, J = 2.1, 1H, ArH). HRMS: Obs. M
+ H, 360.1105. Calc. M + H, 360.1110.
2-(4-Phenylbutyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione(6)
mp:108–109ꢁC. 1H NMR(300 MHz, CDCl3) δ 1.67–1.74 (m, 4H,
CH2CH2CH2CH2N), 2.62(t, J = 6.8, 2H, CH2CH2CH2CH2N), 4.16(t,
J = 6.6, 2H, CH2CH2CH2CH2N), 7.06–7.16(m, 5H, ArH), 7.69(t,
J = 7.1, 2H, ArH), 8.16(d, J = 7.4, 2H, ArH), 8.53(dd, J = 7.2, 2H, ArH).
HRMS: Obs. M + H, 329.1413. Calc. M + H, 329.1416.
2-(4-Hydroxyphenethyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione(7) mp:216–217ꢁC; 1H NMR(300 MHz, CDCl3) δ 2.96 (m, 2H,
CH2CH2N), 4.36(m, 2H, CH2CH2N), 4.62(s, 1H, OH), 6.80(d, J = 7.5,
2H, ArH), 7.21(d, J = 7.5, 2H, ArH), 7.77(t, J = 7.2, 2H, ArH), 8.23(d,
J = 7.2, 2H, ArH), 8.61(d, J = 7.2, 2H, ArH). HRMS: Obs. M + H,
318.1135. Calc. M + H, 318.1130.
4.5
|
Aβ monomer aggregation assay
Reaction mixtures of 400 μL containing 20 μM SEC-isolated Aβ mono-
mer, 150 mM NaCl, 40 mM Tris–HCl (pH 8.0), 5% (v/v) DMSO, and
selected concentrations of naphthalimide analogs were prepared in
1500 μL nonstick microtubes at room temperature (25ꢁC). Reactions
containing 0 μM naphthalimide analogs served as the positive control.
Reactions were incubated under continuous agitation to enhance Aβ
aggregation. During the time course of aggregation, 20 μL aliquots
were periodically taken from the reaction solution and combined with
140 μL of 10 μM ThT for ThT fluorescence measurement. When asso-
ciated with β-sheet structures of Aβ, ThT will give rise to a new excita-
tion maximum at 450 nm and enhanced emission at 482 nm, in
comparison with the excitation at 385 nm and emission at 445 nm of
unbound ThT. Measurements were monitored using an LS-45 lumi-
nescence spectrometer (PerkinElmer Inc., Waltham, MA) with excita-
tion at 450 nm and emission from 470 to 500 nm. Results were
reported by calculating the area under the fluorescence emission
spectrum curve (F) and were corrected by subtracting the ThT back-
ground. Data are reported as the change in normalized F (NF) with
time, where NF is the ratio of the experimental value of fluorescence
to the value of plateau fluorescence in the absence of inhibitor.
2-(3,4-Dihydroxyphenethyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione(8) mp:239–240ꢁC; 1H NMR(300 MHz, CDCl3) δ 2.92(m, 2H,
CH2CH2N), 4.36(m, 2H, CH2CH2N), 6.81(s, 2H, ArH), 6.88(shoulder
peak, 2H, OH), 6.91(s, 1H, ArH), 7.77(t, J = 7.2, 2H, ArH), 8.23(d,
J = 8.1, 2H, ArH), 8.61(d, J = 7.2, 2H, ArH). HRMS: Obs. M + H,
334.1080. Calc. M + H, 334.1079.
4.3
|
Preparation of Aβ monomer
Lyophilized Aβ peptide (Anaspec, Fremont, CA) was stored desiccated
at −20ꢁC until reconstitution in 50 mM NaOH at a concentration of
2 mg/mL to minimize the formation of small aggregates. Remaining
aggregates were excluded via size-exclusion chromatography (SEC) on
a Superdex 75 HR 10/300 column (GE Healthcare, Piscataway, NJ).
Bovine serum albumin of 2 mg/mL (EMD Biosciences, San Diego, CA)
was employed as a pretreatment to reduce nonspecific Aβ interaction
with the dextran matrix of the column. Using a running buffer of
40 mM Tris–HCl (pH 8.0), the elution profile showed a characteristic
peak corresponding to monomeric Aβ. Aβ monomer concentrations
were determined with a calculated extinction coefficient of 1,450 M-
1 cm‑1 at 276 nm. Purified Aβ monomer samples were used fresh or
stored at 4ꢁC for up to 4 days.
4.6
|
Molecular modeling
All the ligands were generated using the sketcher in the Accelrys Dis-
covery Studio Client 2.5 molecular modeling package. All the hydro-
gen atoms were added to define the correct ionization and tautomeric
states. The CHARMm force field was then applied to the each mole-
cule to assign atom types and partial charges prior to saving in .dsv
format. The “Smart Minimizer” energy minimization algorithm was
used for the minimization procedure with a 0.1 kcal/(mol × Å) energy
gradient during a maximum 2000-step cycle of minimization. The
4.4
|
Preparation of soluble Aβ aggregate
To produce Aβ soluble aggregates, purified monomeric Aβ (up to
100 μM) was incubated with 40 mM Tris–HCl (pH 8.0) containing
3 mM NaCl at room temperature. Solutions were agitated vigorously
by continuous vortexing (800 rpm) to promote assembly, and

8
GAO AND CHAPMAN
“AlignToSelectedSubstructure” protocol was utilized to align the com-
manner until the control grid contained a sufficient quantity of sample
for visualization. The gridded sample was then stained with 2% fil-
tered uranyl acetate (Electron Microscope Sciences, Hatfield, PA) for
8 min. The staining solution was wicked away from the grid edge, and
the grid as allowed to air dry overnight. The dried grid was visualized
using a JEOL 200CX transmission electron microscope (JEOL, Pea-
body, MA) at 120 kV.
pounds based the common substructure.
4.7
|
Soluble Aβ aggregate elongation assay
SEC-isolated Aβ soluble aggregates of 2 μM in 40 mM Tris–HCl
(pH 8.0) were combined with 80 μM of compound 8 and incubated at
room temperature without agitation for 15 min to allow binding of
compound 8 and soluble aggregates. Soluble aggregate elongation
was initiated by the addition of 30 μM SEC-isolated monomer and
periodically monitored using DLS. Reactions conducted in the absence
of compound 8 served as the positive control. Experiments in which
RH of soluble aggregates was monitored in the absence of added
monomers or in which RH of monomers was monitored in the absence
of added soluble aggregates served as negative controls and reflected
the stability of Aβ soluble aggregate and monomer, respectively. Solu-
ble aggregate growth was monitored as an increase in RH using DLS.
Results are reported as the change in RH with time. Elongation rates
were determined by regression of the linear portion of this data. The
percentage inhibition was calculated from the ratio of the experimen-
tal elongation rate to the elongation rate observed in the absence of
compound 8.
4.10
(ORAC) assay
|
Oxygen radical absorbance capability
The OxiSelect™ Oxygen Radical Antioxidant Capacity (ORAC) Activity
Assay was used to determine the antioxidant capacity of
naphthalimide analogs. Briefly, 25 μL of the sample or standard and
150 μL of the ×1 fluorescein probe, prepared in assay diluent, were
added to each well and incubated at 37ꢁC for 30 min. After incuba-
tion, 25 μL of free radical initiator solution was added. A Synergy 2
plate reader measured the fluorescence of the samples at an excita-
tion wavelength of 480 nm and an emission wavelength of 520 nm
every minute for 90 min at a constant temperature of 37ꢁC. The area
under the curve (AUC) of the blank was subtracted from all other
AUC values to get the net AUC. Using the equation from the trend
line the net AUC value of each sample could be used to obtain the
equivalent Trolox concentration of the sample. This value was then
divided by the concentration of the sample and reported as the ORAC
value where the standard, Trolox, has an ORAC value of 1.
4.8
|
Soluble Aβ aggregate association assay
2 μM SEC-isolated Aβ1–40 soluble aggregates in 40 mM Tris–HCl
(pH 8.0) were combined with 10 μM filtered compound 8 and incu-
bated without agitation at 25ꢁC for 15 min to allow binding. Soluble
aggregate growth via association was initiated by addition of concen-
trated NaCl for a final concentration of 150 mM NaCl and periodically
monitored by measurement of the increase in soluble aggregates RH
using DLS. Reactions containing 0 μM compound 8 served as the posi-
tive control. Experiments in which RH of Aβ1–40 soluble aggregates
was monitored in the absence of added NaCl served as a negative
control and reflected the stability of the Aβ1–40 soluble aggregates.
Results are reported as the increase in RH with time. Association rates
were determined by regression of the linear portion of this data. The
percentage inhibition was calculated from the ratio of the experimen-
tal association rate to the association rate observed in the absence of
inhibitory.
4.11
|
The expression of iNOS assay
The murine macrophage cell line (ANA-1 cells) was incubated for
12 hr vehicle (1% DMSO), or compound 7 (100 μM), or compound 8
(100 μM), washed, then exposed to IFN-γ (100 U/mL) for 0, 2, 4 and
8 hr. Cell lysates were analyzed by western blot analysis as described
previously (Ying et al., 2005).
4.12
|
MTT assay for neuroprotective activities
Cortical neuronal cultures were derived from the cerebral cortex of
Sprague–Dawley rat embryos (E17-18). Briefly, cells dissociated from
the cerebral cortex of embryos were seeded at a density of 5 × 105
cells/mL onto poly-D-lysine pre-coated 96-well plates for neuronal
MTT assay. In the experiments, neurons were pre-treated with com-
pound 8 alone (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) for 24 hr.
The cells were washed and then challenged with 200 nM Aβ for
another 24 hr. For each condition described above, a total of 2–4
independent experiments were performed with seven replicates per
drug concentration evaluated. Cell viability was determined using a
commercially available MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl
tetrazolium bromide) assay (Invitrogen Vybrant® MTT Cell Prolifera-
tion Assay Kit). Differences were analyzed for statistical significance
4.9
|
Transmission electron microscopy
Aβ monomers of 20 μM were incubated with 150 mM NaCl in the
presence of 100 μM compound 8 under agitation. After aggregation
reached a plateau, which was recognized by ThT fluorescence of the
control reaction, gridded samples were prepared and imaged. A 20 μL
sample containing aggregated Aβ was placed upon
a formvar-
supported nickel grid (Electron Microscope Sciences, Hatfield, PA) and
allowed to adsorb for 2 min. Sample application was repeated in this

GAO AND CHAPMAN
9
formation. The Journal of Biological Chemistry, 287(41), 34786–34800.
https://doi.org/10.1074/jbc.M112.357665
using one-way ANOVA, followed by Holm-Sidak post hoc compari-
sons. Significance was set at p < .05.
Lee, J. H., Byeon, S. R., Lim, S. J., Oh, S. J., Moon, D. H., Yoo, K. H., …
Kim, D. J. (2008). Synthesis and evaluation of stilbenylbenzoxazole
and stilbenylbenzothiazole derivatives for detecting beta-amyloid
fibrils. Bioorganic & Medicinal Chemistry Letters, 18(4), 1534–1537.
Mattson, M. P. (2004). Pathways towards and away from Alzheimer's dis-
ease. Nature, 430(7000), 631–639.
ACKNOWLEDGMENT
The authors would like to thank Dr Melissa Moss, Dr Lorne Hofseth,
and Dr Campbell McIness from University of South Carolina who pro-
vided insight and expertise that greatly assisted the research.
McKoy, A. F., Chen, J., Schupbach, T., & Hecht, M. H. (2012). A novel
inhibitor of amyloid beta (Abeta) peptide aggregation: From high
throughput screening to efficacy in an animal model of Alzheimer dis-
ease. The Journal of Biological Chemistry, 287(46), 38992–39000.
https://doi.org/10.1074/jbc.M112.348037
ORCID
Jie Gao
https://orcid.org/0000-0001-5951-1399
Narlawar, R., Baumann, K., Schubenel, R., & Schmidt, B. (2007). Curcumin
derivatives inhibit or modulate beta-amyloid precursor protein metab-
olism. Neurodegenerative Diseases, 4(2–3), 88–93.
Nichols, M. R., Moss, M. A., Reed, D. K., Lin, W. L., Mukhopadhyay, R.,
Hoh, J. H., & Rosenberry, T. L. (2002). Growth of beta-amyloid (1-40)
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Nie, Q., Du, X. G., & Geng, M. Y. (2011). Small molecule inhibitors of amy-
loid beta peptide aggregation as a potential therapeutic strategy for
Alzheimer's disease. Acta Pharmacologica Sinica, 32(5), 545–551.
https://doi.org/10.1038/aps.2011.14
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How to cite this article: Gao J, Chapman J. Discovery and
Characterization of Novel Naphthalimide Analogs as Potent
Multitargeted Directed Ligands against Alzheimer's Disease.
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