Phthalimide-(N-alkylbenzylamine) cysteamide hybrids as multifunctional agents against Alzheimer’s disease: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1111/cbdd.13905

The complex pathogenesis of Alzheimer's disease (AD) calls for multi-target approach for disease treatment. Herein, based on the MTDLs strategy, a series of phthalimide-(N-alkylbenzylamine) cysteamide hybrids were designed, synthesized, and investigated i

Full text of DOI:10.1111/cbdd.13905

Received: 21 December 2020
DOI: 10.1111/cbdd.13905
Revised: 13 April 2021
Accepted: 6 June 2021
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R E S E A R C H A R T I C L E
Phthalimide-(N-alkylbenzylamine) cysteamide hybrids as
multifunctional agents against Alzheimer’s disease: Design,
synthesis, and biological evaluation
Heng Zhang
Xiuxiu Liu
Qing Song
Yong Deng
Guangjun Yu
Zhongcheng Cao
Xiaoming Qiang
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Department of Medicinal Chemistry, Key
Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry
and Sichuan Province, Sichuan Engineering
Laboratory for Plant-Sourced Drug and
Sichuan Research Center for Drug Precision
Industrial Technology, West China School
of Pharmacy, Sichuan University, Chengdu,
China
Abstract
The complex pathogenesis of Alzheimer's disease (AD) calls for multi-target ap-
proach for disease treatment. Herein, based on the MTDLs strategy, a series of
phthalimide-(N-alkylbenzylamine) cysteamide hybrids were designed, synthesized,
and investigated in vitro for the purpose. Most of the target compounds were found
to be potential multi-target agents. In vitro results showed that compound 9e was the
representative compound in this series, endowed with high EeAChE and HuAChE in-
hibitory potency (IC50 = 1.55 µm and 2.23 µm, respectively), good inhibitory activity
against self-induced Aβ1-42 aggregation (36.08% at 25 µm), and moderate antioxidant
capacity (ORAC-FL value was 0.68 Trolox equivalents). Molecular docking stud-
ies rationalized the binding mode of 9e in both PAS and CAS of AChE. Moreover,
9e displayed excellent ability to against H2O2-induced PC12 cell injury and pen-
etrate BBB. Overall, these results highlighted that compound 9e was an effective and
promising multi-target agent for further anti-AD drug development.
Correspondence
Xiuxiu Liu and Yong Deng, Department
of Medicinal Chemistry, Key Laboratory
of Drug-Targeting and Drug Delivery
System of the Education Ministry and
Sichuan Province, Sichuan Engineering
Laboratory for Plant-Sourced Drug and
Sichuan Research Center for Drug Precision
Industrial Technology, West China School
of Pharmacy, Sichuan University, Chengdu,
China.
K E Y W O R D S
Emails: liuxiuxiu@scu.edu.cn; dengyong@
scu.edu.cn
acetylcholinesterase inhibition, Alzheimer's disease, anti-Aβ aggregation, antioxidant, multi-target
agents, neuroprotective effect, phthalimide-(N-alkylbenzylamine)cysteamide hybrids
Funding information
National Natural Science Foundation of
China
1
INTRODUCTION
reverse the progression of the disease due to the riddled na-
ture of AD (Long and Holtzman, 2019).
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Alzheimer's disease (AD), a convoluted and multilayered
neurological disorder, is considered the leading cause of
dementia and characterized by progressive deterioration in
cognitive functions. Currently, 46.8 million people suffer
from AD and its prevalence is forecasted to be tripled by
2050 (Stopschinski and Diamond 2017). AD patients suffer
frequently from impairment in learning and memory, which
impose tremendous burden on caregivers and public health
sector (Perez-Areales et al., 2020). Regrettably, the present
available treatments are just symptomatically and not able to
Multiple pathways and hypotheses have been indicated
in the pathology of the disease such as the low levels of
neurotransmitter acetylcholine (ACh), the aggregation of
β-amyloid (Aβ) peptide, and oxidative stress (Long and
Holtzman 2019). Among several proposed mechanisms,
the cholinergic hypothesis is the most widely accepted.
Acetylcholinesterase inhibitors (AChEIs), which prevent the
hydrolyzation of acetylcholine into choline (Ch) and acetic
acid, and thus increase acetylcholine levels in the synapse
of CNS, could alleviate cognition deficits associated with
Chem Biol Drug Des. 2021;00:1–8.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons A/S.
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ZHANG et Al.
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AD. To this end, striking strides have been made in devel-
oping AChEIs like donepezil, rivastigmine, and galantamine
(Peauger et al., 2017). However, these drugs could not reverse
the onset and process of the malady, only offer time-limited
symptomatic relief for patients. Therefore, AChEIs per se
might not be sufficient to treat AD with highly complex pa-
thologies. Moreover, Aβ deposition has been linked to AchE
expression. The PAS of AchE can bind to Aβ, accelerating
the formation of amyloid fibrils. Therefore, inhibition of PAS
of AchE may affect the aggregation of Aβ.
The amyloid cascade hypothesis suggests that the aggre-
gation of Aβ is one of the driving forces behind AD develop-
ment (Tycko 2016). One of the neuropathological hallmarks
of AD is the deposition of extracellular Aβ peptide, a primary
constituent of senile plaques, which lead a heightened propen-
sity to the aberrance of the neuronal transit system and cessa-
tion of signal transduction between neurons (De et al., 2019).
Aβ40 and Aβ42 are two major variants of Aβ, since Aβ42 is
far more aggregative than Aβ40, inhibition of Aβ42 aggre-
gation is expected to be a viable option to treat AD (Tycko
2016). Furthermore, Aβ exhibits the ability to enhance the
formation of reactive oxygen species (ROS) and vice versa
(Ojala et al., 2018). ROS and reactive nitrogen oxide spe-
cies (RNOS), the important sources of oxidative stress, are
prone to react with macromolecules such as lipids, nucleic
acids, and proteins in brain and cause a detrimental oxida-
tive damage coupled with the abnormal deposition of both
Aβ aggregates and neurofibrillary tangles in AD (Cheignon
et al., 2018). Therefore, antioxidants have also drawn much
attention and postulated to be an effective therapeutic treat-
ment of AD (Nesi et al., 2017).
These concurrent mechanisms mentioned above contrib-
ute to the progression of AD and are likely to be highly inter-
related (Savelieff et al., 2019). Therefore, on account of the
multifactorial nature of AD, a new paradigm, named multi-
target directed ligands (MTDLs), has been developed (Alcaro
et al., 2019). This creative strategy proposes the design of
new scaffolds with at least two pharmacophore subunits con-
nected in a single molecule that could, in turn, modulate the
interaction and biological response of multiple molecular tar-
gets simultaneously (Zhou et al., 2019).
FIGURE 1 Chemical structures of (N-benzylaminealkyl)
phthalimides, donepezil, N-acetylcysteine, and designed phthalimide-
(N-alkylbenzylamine) cysteamide hybrids
target molecule may also be beneficial to inhibit the aggrega-
tion of Aβ according to subsequent research. Inspired from
these discoveries, our team chose phthalimide as one of the
pharmacophores to interact with PAS. In addition, due to the
peculiar architecture of AChE with two target sites at the top
and the bottom of a gorge, respectively, a molecule could able
to interact with PAS and catalytic anionic site (CAS) may be
promising at some extent (Cheung et al., 2012). Donepezil
(Figure 1) is often considered as the first-line agent to
treat patients with mild to moderate AD. The structure–
activity relationship of donepezil revealed that N-benzyl
group participates in aromatic interactions with Trp84 and
forms hydrogen bonding interactions with Gly118, Gly119,
Gly201, and the oxygen of Ser200 at CAS. The piperidine
participates in a cation-π interaction with Phe330 (Brewster
et al., 2019). Therefore, we designed the N-benzylalkylamine
fragment in target hybrids to expect the interaction effect at
CAS could be manifested. In order to simulate the structure
of donepezil, methoxy groups, which form hydrogen bond
interaction between Glu185 via a bridging water molecule
(Brewster et al., 2019), were also introduced to the phthalim-
ide moiety. Moreover, N-acetylcysteine (NAC), the active
agent in Mucomyst, was used as an adjunct to the manage-
ment of cystic fibrosis (Zollinger and Williams 1964). In
recent year, NAC was widely known as effective scavenger
of free radicals and a major contributor to minimize the
oxidative effect of ROS (Prakash et al., 2015). Besides, it
has been well recorded in document that chemical entities
containing mercapto group such as NAC may facilitate the
neuroprotective function (Wang et al., 2012). Interestingly,
NAC-amide (NACA), an active derivative of NAC, has been
measured in brain after oral or interperitoneal administra-
tion, but not NAC itself (Shahripour et al., 2014). Therefore,
we designed a series of phthalimide-(N- alkylbenzylamine)
cysteamide hybrids (Figure 1) to expect these novel com-
pounds could act as promiscuous ligands with AChE inhi-
bition, anti- Aβ1-42 aggregation, antioxidant property, and
neuroprotective effect.
Phthalimide, a multifunctional pharmacophore endowed
with diverse biological activity, has stolen the limelight
from research groups for its function with regard to neuro-
degenerative disease. Ishihara group has designed a series
of (N-benzylaminealkyl) phthalimide compounds (Figure 1)
as acetylcholinesterase inhibitors, and the optimum activity
was found to be associated with a five carbon chain length
separating the benzylamino group from the phthalimide
moiety (Ishihara et al., 1991). Moreover, the study revealed
that phthalimide could form π-π stacking and hydrophobic
interactions with the peripheral anion site (PAS) of AChE,
which means the introduction of phthalimide fragments into

ZHANG et Al.
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2
RESULTS AND DISCUSSION
Chemistry
than rivastigmine (IC50 = 9.94 μm) under our experimental
condition. Moreover, 9e showed HuAChE inhibitory potency
(IC50 = 2.23 μm) as well, which is believed to be beneficial
for anti-AD function.
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2.1
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The synthetic route for the designed phthalimide-(N-
alkylbenzylamine) cysteamide hybrids was outlined in
Scheme 1. Commercially available 5,6-dimethoxyisoben
zofuran-1(3H)-one (1) was used as original material, and
4,5-dimethoxyphthalic acid (2) was synthesized by oxidation
of compound 1 by KMnO4 under alkaline condition and then
combined with acetic anhydride via intramolecular dehy-
dration to afford 5,6-dimethoxyisobenzofuran-1,3-dione (3)
(Woodhead et al., 2010). Subsequently, a condensation reac-
tion involving compound 3 with ( )-cysteine was performed,
resulting in the formation of key intermediate 4. Treatment
of compound 4 with 1-hydroxy benzotriazole (HOBt) in the
presence of condensing agent 1-ethyl-3-(3-dimethylpropyla
mine) carbodiimide (EDCI) generated active ester 5 (Ibrahim
et al., 2012). Finally, the target molecules were prepared by
the reaction of 5 with N-alkylbenzylamines (6–8) (Contreras
et al., 1999). The structure of final products was character-
2.2.2
Molecular docking studies
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To provide a reasonable explanation toward the role of
chirality played in the interaction, a docking study of (S)-
9e, (R)-9e with TcAChE was conducted via AutoDock 4.2
package (Lu et al., 2013). Given that EeAChE and TcAChE
share a high degree of homology, the X-ray crystal struc-
ture of TcAChE (PDB code: 1EVE) was chosen for this
study. The docking result was shown in Figure 2. Clearly,
in the complex of TcAChE and (S)-9e, the phthalimide
moiety occupied the PAS and the N-alkylbenzylamine moi-
ety interacted with amino acid residues in the CAS, indi-
cating a mixed-type inhibitory behavior for (S)-9e, which
consisted with our design conception. In addition, the
methoxy group at 5-position and the amido linkage were
involved in hydrogen bond interactions with the Arg289
and Tyr121, respectively. Furthermore, the benzene ring
in N-alkylbenzylamine moiety was observed to adopt π-σ
interaction with Trp84. Besides, (S)-9e also induced hydro-
phobic interactions with the amino acid residues Ser286,
Ile 287, Phe288, Try334, Phe330, Leu282, Trp279, Phe331,
Phe290, Asp72, Trp84, Ser200, Ser122, Glu199, Gly117,
Gly118, Gly123, and Try130. Finally, the calculated bind-
ing energy of (S)-9e was −10.78 kcal/mol and the calculated
Ki value was 12.56 nm. On the other hand, the docking re-
sults of (R)-9e with TcAChE also indicated that compound
(R)-9e could bound perfectly to TcAChE and occupied the
entire enzymatic CAS, the mid-gorge site, and the PAS. In
the TcAChE-(R)-9e complex, similar main intermolecu-
lar interactions with (S)-configuration were observed. For
instance, the benzene ring in phthalimide interacted with
Phe331 via π-σ interaction, the amido linkage was involved
in hydrogen bond interaction with Tyr121, and the benzene
ring in N-alkylbenzylamine formed parallel π-π stacking in-
teraction with Trp84. Furthermore, the calculated binding
energy of (R)-9e was −10.47 kcal/mol and the calculated Ki
value was 20.98 nm. Therefore, based on the above docking
results, it could be concluded that despite the chiral center
changed the spatial configuration of compound 9e, there
was no significant difference in the interaction between the
two configurations and TcAChE.
1
ized by H NMR, ESI-MS, and most of them were further
characterized by 13C NMR.
2.2
Biological evaluation
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2.2.1
Cholinesterase inhibition assay
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The inhibition activity of target compounds toward AChE
(from electric eel, EeAChE and human AChE, HuAChE)
and BuChE (from rat serum, RatBuChE) was performed ac-
cording to modified Ellman's method (Sang et al., 2015), and
the test results were shown in Table 1 with compound 12
(Ishihara et al., 1991), donepezil and rivastigmine as con-
trol compounds. Good inhibitory potency toward EeAChE
was observed for most test compounds with IC50 values
ranged from 1.55 to 38.64 μm except 10c and 11f, and all
synthesized target compounds showed potent and selective
inhibitory potencies toward EeAChE over BuChE. Through
the structure–activity relationship (SAR) analysis, it could
be found that when the length of carbon chain was 3, the
EeAChE inhibitory activity of the test compound was equiv-
alent to compound 12. After the carbon chain was extended,
the inhibitory activity reduced significantly. In addition, gen-
erally speaking, the introduction of O-methoxy groups on N-
alkylbenzylamine fragment (9c, 9d, 10c, 11c, 11d) reduces
the activity of inhibiting EeAChE compared with corre-
sponding compounds without substituents (9a, 9b, 10a, 11a,
11b). Interestingly, compound 9e with O-dimethylamino
substituent determined to be the most potent AChE inhibitor
within this series with IC50 value of inhibiting EeAChE was
1.55 μm, lower than donepezil (IC50 = 0.015 μm) but better
Moreover, to further clarify the structure–activity rela-
tionship of phthalimide-(N-alkylbenzyl amine) cysteam-
ide derivatives, such as the difference in AChE inhibitory
activity due to the change of substituent position of dime-
thylamine group and the change of carbon chain length, we
also selected compound 9f and 11f to conduct the molecular

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TABLE 1 The chemical structures, inhibitory activities toward AChE and BuChE by target compounds and reference compounds
IC50 (μm)a
EeAChE
Inhibition (%)b
IC50 (μm)a
HuAChE
Compd.
RatBuChE
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
4.52 0.06
5.40 0.15
8.45 1.41
10.38 1.56
1.55 0.17
20.00 0.74
33.12 0.29
27.35 0.81
85.83 4.39
28.80 1.36
38.64 1.76
21.19 0.65
17.95 0.43
22.78 0.58
28.47 1.02
30.86 1.50
30.80 1.03
105.41 5.31
5.28 0.72
0.015 0.002
9.94 0.83
<5.0%
<5.0%
<5.0%
11.30 0.21%
20.90 0.29%
<5.0%
<5.0%
<5.0%
<5.0%
<5.0%
11.80 0.13%
<5.0%
<5.0%
8.89 0.11
7.66 0.08
NTd
NTd
2.23 0.04
NTd
NTd
NTd
NTd
NTd
NTd
10f
NTd
11a
11b
11c
11d
11e
NTd
<5.0%
NTd
10.12 0.10%
17.86 0.31%
33.15 1.12%
8.01 0.15%
<5.0%
NTd
NTd
NTd
11f
12
NTd
NTd
c
Donepezil
Rivastigmine
20.70 1.36 μm
2.86 0.22 μm
0.032 0.001
10.90 0.23
c
aIC50 values represent the concentration of inhibitor required to decrease EeAChE activity by 50% and are the mean of three independent experiments, each performed
in triplicate (SD = standard deviation).
bBuChE from rat serum was used, and tested compounds were used at 50 μm.
cIC50 of the reference compound.
dNT = not tested.
docking study (Figure S1). Compound 9f showed higher
calculated binding energy and Ki value (−8.84 kcal/mol and
332.06 nm, respectively) compared with 9e. In addition, the
benzene ring in N-alkylbenzylamine moiety was observed
to adopt π-π stacked interactions with Trp84, and only one
hydrogen bond interaction between sulfhydryl with Phe288
was observed, which may explain the worse AChE inhib-
itory effect compared with 9e. Moreover, though the cal-
culated binding energy of compound 11f (−8.41 kcal/mol)
was lower than 9f, its calculated Ki value was the highest
(689.14 nm), and only π-π stacked interaction was observed
between the benzene ring of N-benzylalkylamine subunit
and Ser81, which may explain the worst inhibitory bioac-
tivity against AChE.
2.2.3
Inhibition of self-induced Aβ1-42
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Aggregation
It is envisaged that a ligand that can prevent the aggrega-
tion of Aβ1-42 is critical for the development of potential
therapeutic treatment. Hence, in this study, we evaluated
all target ligands and compound 12 for inhibitory poten-
tial against Aβ1-42 aggregation with Thioflavin T (ThT)
fluorescence method (Bartolini et al., 2010), curcumin was
used as positive control, and the results were summarized
in Table 2. Apparently, most compounds showed a moder-
ate to good inhibitory potency of Aβ1-42 aggregation from
12.81% to 50.90%. The structure–activity relationship re-
vealed that the length of linker had no significant effect on

ZHANG et Al.
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SCHEME 1 Synthesis of phthalimide-
(N-alkylbenzylamine) cysteamide
derivatives 9–11. Reagents and conditions:
(i) KMnO4, NaOH, H2O, reflux for 5 hr;
(ii) Ac2O, THF, reflux for 5 hr; (iii)
( )-Cysteine, AcOH, reflux for 10 hr;
(iv) EDCI, HOBt, THF, r.t. for 18 hr;
(v) R1R2N(CH2)nNH2 (6–8), r.t. for
2–4 hr
inhibitory capacity. Compared with compounds owned the
same linkage, the compound 9f (37.40%), 10f (34.67%),
and 11f (50.90%) endowed with p-dimethylamino substitu-
ent showed the strongest inhibitory activity, indicating that
the dimethylamino group helps to increase the affinity of
the compound with Aβ1-42 and enhance the inhibitory activ-
ity, which was consistent with the previous research results
of our group (Li et al., 2016). And the representative com-
pound 9e (36.08%) possessed almost the same inhibitory
activity as curcumin (39.00%) and better than compound
12 (16.31%).
0.68 Trolox equivalents, which showed a moderate antioxi-
dant capacity.
2.2.5
Neuroprotective activity of H2O2-
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induced PC12 cell injury
As a kind of ROS, H2O2 participates in the pathogenesis of
many neurological diseases. Therefore, we used the H2O2-
mediated oxidative damage model of PC12 cell to determine
the neuroprotective effect of selected compounds (Yuan
et al., 2014) with Trolox as positive compound, and the test
result was shown in Figure 3. It is suggested that the survival
rate of PC12 cell in the presence of 150 μm H2O2 (the Model
group) was only 44.65%, indicating that H2O2 can signifi-
cantly induce PC12 cell injury. And the test results indicated
that the survival rate of PC12 cell was 61.32% and 72.53%
when Trolox was added at the concentration of 1 and 10 μm,
respectively. When the selected compounds 9e, 9f, 10f, and
11f were added at a concentration of 1.0 μm, the viability of
PC12 was 60.83%, 62.58%, 66.30%, and 64.32%. Moreover,
at the concentration of 10.0 μm, the viability of PC12 was dra-
matically increased to 71.42%, 73.56%, 83.64%, and 80.27%,
respectively. It could be concluded that compound 9f, 10f,
and 11f had a better significant protective effect on the oxida-
tive injury of PC12 cell induced by H2O2 than Trolox, and the
test results also showed that the compounds containing the
p-dimethylamino moiety had the strongest protective effect
on PC12 cell, which was consistent with the results measured
by ORAC-FL method. Representative compound 9e, with the
strongest AChE inhibitory activity, also exhibited good neu-
roprotective effect based on our experimental results.
2.2.4
In vitro antioxidant capacity assay
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Since oxidative stress plays a major role in the progres-
sion of AD, we evaluated the antioxidant activities of all
designed compounds through ORAC-FL method (Dávalos
et al., 2004). As shown in Table 2, all target compounds dem-
onstrated moderate to excellent antioxidant activity ranging
from 0.30- to 2.63-fold of the Trolox value. The results indi-
cated that the NAC fragment contained in the molecule plays
a great role in absorbing free radicals compared with the
compound 12. Furthermore, dimethylamino group enhance
the antioxidant activity of target compounds compared with
other substituent. Interestingly, dimethylamino substituent at
para-situation leaded to a significant improvement of anti-
oxidant capacity such as compounds 9f, 10f, and 11f, which
exhibited the most potent antioxidant activity of this family
with ORAC-FL values of 1.44, 2.63, and 2.20 trolox equiva-
lents, respectively. In addition, the ORAC-FL value of repre-
sentative compound 9e bearing o-dimethylamino group was

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ZHANG et Al.
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FIGURE 2 Representation of
compound (S)-9e and (R)-9e (colored by
atom type) interacting with residues in
the binding site of TcAChE (PDB code:
1EVE), highlighting the protein residues that
participate in the main interactions with the
inhibitor
TABLE 2 Inhibition of self-induced Aβ1-42 aggregation and oxygen radical absorbance capacity (ORAC, Trolox Equivalents) by target
compounds and control compounds
% inhibition of Aβ1–42
aggregationa
% inhibition of Aβ1–42
aggregationa
Compd.
ORACb
Compd.
ORACb
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
30.82 1.34
34.31 1.17
29.50 0.75
26.16 1.00
36.08 1.09
37.40 1.57
25.00 0.59
17.69 0.38
16.23 0.47
12.81 0.32
0.44 0.02
0.50 0.02
0.38 0.01
0.30 0.04
0.68 0.03
1.44 0.08
0.45 0.02
0.45 0.01
0.39 0.03
0.47 0.02
10e
10f
11a
11b
11c
11d
11e
11f
28.45 1.39
34.67 1.58
28.91 1.09
33.25 0.86
30.90 1.31
39.47 1.54
47.75 2.18
50.90 2.37
16.31 1.12
39.00 0.65
0.61 0.02
2.63 0.11
0.47 0.03
0.48 0.02
0.46 0.01
0.53 0.02
0.69 0.02
2.20 0.13
0.14 0.08
not tested
12
Curcumin
aInhibition of self-induced Aβ1-42 aggregation (25 μm) produced by tested inhibitor at 25 μm.
bThe mean SD of the three independent experiments. Data are expressed as μm of Trolox equivalent/μm of tested compound.
2.2.6
Blood brain barrier permeation assay
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The ability to cross blood brain barrier (BBB) is a requisite
investigation in pharmacological studies in respect to CNS
drugs for the purpose of interacting with their specific tar-
gets. Therefore, permeability of representative compound
9e was evaluated by PAMPA-BBB assay. First, we meas-
ured the BBB permeability of 11 commercially available
drugs (Table S1, Supplementary Material), compared with
literature values to establish the correlation between experi-
mental Pe(exp) and described Pe(bibl.), and a good linear cor-
relation was obtained: Pe(exp) = 0.9163Pe(bibl.) − 0.2247
(R2 = 0.9558) (Figure S2). According to the evaluation
conditions established by Di et al. (2003), we defined that
compounds with permeability (Pe) over 3.44 × 10−6 cm/s−1
suggested excellent brain penetrability (Table 3). The result
FIGURE 3 Effects of compounds 9e, 9f, 10f, and 11fand Trolox
on H2O2-induced PC12 cell injury (n = 3, mean SD; ###p < .0001
vs. control, ***p < .001 vs. model)

ZHANG et Al.
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TABLE 3 Ranges of permeability of PAMPA-BBB assays
(Pe × 10–6 cm/s)
ORCID
Yong Deng
https://orcid.org/0000-0002-6565-6073
High BBB permeation predicted (CNS+)
Uncertain BBB permeation (CNS )
Low BBB permeation predicted (CNS−)
Pe > 3.44
3.44 > Pe > 1.61
Pe < 1.61
REFERENCES
Alcaro, S., Bolognesi, M. L., García-Sosa, A. T., & Rapposelli, S.
(2019). Editorial: Multi-target-directed ligands (MTDL) as chal-
lenging research tools in drug discovery: From design to phar-
macological evaluation. Frontiers in Chemistry, 7, 71. https://doi.
org/10.3389/fchem.2019.00071
Bartolini, M., Bertucci, C., Bolognesi, M. L., Cavalli, A., Melchiorre,
C., & Andrisano, V. (2010). Insight into the kinetic of amyloid beta
(1–42) peptide self-aggregation: Elucidation of inhibitors' mech-
anism of action. ChemBioChem, 8(17), 2152–2161. https://doi.
org/10.1002/cbic.200700427
Brewster, J. T., Dell'Acqua, S., Thach, D. Q., & Sessler, J. L. (2019).
Classics in chemical neuroscience: Donepezil. ACS Chemical
Neuroscience, 10(1), 155–167. https://doi.org/10.1021/acschemneu
ro.8b00517
Cheignon, C., Tomas, M., Bonnefont-Rousselot, D., Faller, P., Hureau,
C., & Collin, F. (2018). Oxidative stress and the amyloid beta pep-
tide in Alzheimer's disease. Redox Biology, 14, 450–464. https://doi.
org/10.1016/j.redox.2017.10.014
TABLE 4 Permeability result Pe (×10–6 cm/s) from the PAMPA-
BBB assay for representative compound 9e with its predicted ability to
penetrate into the CNS
Compd.a
Pe (×10–6 cm/s)b
Prediction
9e
8.24 0.36
CNS+
aCompound was dissolved in DMSO at 5 mg/ml and diluted with PBS/EtOH
(70:30). The final concentration of compound was 100 μg/ml.
bData are the mean SD of three independent experiments.
in Table 4 demonstrated that compound 9e could cross the
BBB and penetrate into CNS.
Cheung, J., Rudolph, M. J., Burshteyn, F., Cassidy, M. S., Gary, E. N.,
Love, J., Franklin, M. C., & Height, J. J. (2012). Structures of human
Acetylcholinesterase in complex with pharmacologically important
ligands. Journal of Medicinal Chemistry, 55(22), 10282–10286.
https://doi.org/10.1021/jm300871x
Contreras, J. M., Rival, Y. M., Chayer, S., Bourguignon, J. J., &
Wermuth, C. G. (1999). Aminopyridazines as acetylcholinesterase
inhibitors. Journal of Medicinal Chemistry, 42(4), 730–741. https://
doi.org/10.1021/jm981101z
Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending
applicability of the oxygen radical absorbance capacity (ORAC-
Fluorescein) assay. Journal of Agricultural and Food Chemistry,
52(1), 48–54. https://doi.org/10.1016/10.1021/jf0305231
De, S., Wirthensohn, D. C., Flagmeier, P., Hughes, C., Aprile, F. A.,
Ruggeri, F. S., Whiten, D. R., Emin, D., Xia, Z., Varela, J. A.,
Sormanni, P., Kundel, F., Knowles, T. P. J., Dobson, C. M., Bryant,
C., Vendruscolo, M., & Klenerman, D. (2019). Different soluble
aggregates of Aβ42 can give rise to cellular toxicity through differ-
ent mechanisms. Nature Communications, 10(1), 1–11. https://doi.
org/10.1038/s41467-019-09477-3
3
CONCLUSION
|
In this work, we have reported the synthesis and in vitro
biological evaluation of eighteen new phthalimide-(N-
alkylbenzylamine) cysteamide hybrids as potential multifunc-
tional anti-Alzheimer's disease agents. Most of the bioactive
compounds were selective and highly potent AChE inhibitors
with IC50 values at micromolar level and exhibited a good
inhibition of Aβ1-42 as well as moderate to excellent antioxi-
dant capacity. Moreover, neuroprotective activity was also
performed in a cell-based assay to indicate that the synthetic
compounds owned good neuroprotective effects in the range
of concentrations studied. Among them, compound 9e was the
most potent and balanced ligand with excellent EeAChE and
HuAChE inhibitory potency (IC50 = 1.55 and 2.23 μm, respec-
tively), good efficacy to inhibit Aβ1-42 aggregation (36.08% at
25 μm), and a moderate antioxidant capacity (ORAC-FL value
was 0.68 Trolox equivalents). Furthermore, the ability of 9e to
against H2O2-induced PC12 cell injury and cross BBB sug-
gested that it could be considered as a promising multi-target
compound for the treatment of AD. Further studies to develop
structural refinements are underway and will be reported in
due course.
Di, L., Kerns, E. H., Fan, K., Mcconnell, O. J., & Carter, G. T. (2003).
High throughput artificial membrane permeability assay for blood-
brain barrier. European Journal of Medicinal Chemistry, 38(3),
223–232. https://doi.org/10.1016/S0223-5234(03)00012-6
Ibrahim,T.S.,Tala,S.R.,El-Feky,S.A.,Abdel-Samii,Z.K.,&Katritzky,
A. R. (2012). Cysteinoyl- and cysteine-containing dipeptidoylben-
zotriazoles with free sulfhydryl groups: Easy access to N-terminal
and internal cysteine peptides. Chemical Biology & Drug Design,
80(2), 194–202. https://doi.org/10.1111/j.1747-0285.2011.01303
ACKNOWLEDGMENTS
This work was supported by Chinese National Natural
Science Foundation (22077091), Sichuan Science and
Technology Program (2020YFS0067), and the Fundamental
Research Funds for the Central Universities.
Ishihara, Y., Kato, K.,
& Goto, G. (1991). Central cholin-
ergic agents. I. Potent acetylcholinesterase inhibitors,
2-[ω-[N-alkyl-N-(ω-phenyl-alkyl)amino]alkyl]-1H-isoindole-
1,3(2H)-diones, based on a new hypothesis of the enzyme's active
site. Chemical Pharmaceutical Bulletin, 39(12), 3225–3235. https://
doi.org/10.1248/cpb.39.3225
CONFLICT OF INTEREST
The authors disclose that there is no potential conflict of
interest.
Li, Y., Qiang, X., Luo, L. I., Yang, X., Xiao, G., Liu, Q. I., Ai, J., Tan, Z.,
& Deng, Y. (2016). Aurone mannich base derivatives as promising

8
ZHANG et Al.
|
multifunctional agents with acetylcholinesterase inhibition, anti-β-
amyloid aggragation and neuroprotective properties for the treatment
of Alzheimer's disease. European Journal of Medicinal Chemistry,
126, 762–775. https://doi.org/10.1016/j.ejmech.2016.12.009
action and therapeutic opportunities. Brain and Behavior., 4(2),
108–122. https://doi.org/10.1002/brb3.208
Stopschinski, B. E., & Diamond, M. I. (2017). The prion model for
progression and diversity of neurodegenerative diseases. Lancet
Neurology, 16(4), 323–332. https://doi.org/10.1016/S1474
-4422(17)30037-6
Long, J. M., & Holtzman, D. M. (2019). Alzheimer Disease: An update
on pathobiology and treatment strategies. Cell, 179(2), 312–399.
https://doi.org/10.1016/j.cell.2019.09.001
Lu, C., Guo, Y., Yan, J., Luo, Z., Luo, H.-B., Yan, M., Huang, L., & Li,
X. (2013). Design, synthesis, and evaluation of multitarget-directed
resveratrol derivatives for the treatment of Alzheimer's disease.
Journal of Medicinal Chemistry, 56(14), 5843–5859. https://doi.
org/10.1021/jm400567s
Nesi, G., Sestito, S., Digiacomo, M., & Rapposelli, S. (2017). Oxidative
stress, mitochondrial abnormalities and proteins deposition:
Multitarget approaches in Alzheimer's disease. Current Topics in
Medicinal Chemistry, 17, 3062–3079. https://doi.org/10.2174/15680
26617666170607114232
Ojala, J., Timonen, A., Sutinen, E., Anderson, G., Kangas, L., &
Thornell, A. (2018). Oxidative stress and neuronal damages:
Estrogenic compounds, anti-Apoptotic factors, and amyloidogenesis.
Reactive Oxygen Species, 5(15), 176–198. https://doi.org/10.20455/
ros.2018.831
Peauger, L., Azzouz, R., Gembus, V., Ţînţaş, M.-L., Sopková-de
Oliveira Santos, J., Bohn, P., Papamicaël, C., & Levacher, V. (2017).
Donepezil-based central acetylcholinesterase inhibitors by means of
a "bio-oxidizable" prodrug strategy: Design, synthesis and in vitro
biological evaluation. Journal of Medicinal Chemistry, 60, 5909–
5926. https://doi.org/10.1021/acs.jmedchem.7b00702
Pérez-Areales, F. J., Garrido, M., Aso, E., Bartolini, M., De Simone,
A., Espargaró, A., Ginex, T., Sabate, R., Pérez, B., Andrisano, V.,
Puigoriol-Illamola, D., Pallàs, M., Luque, F. J., Loza, M. I., Brea, J.,
Ferrer, I., Ciruela, F., Messeguer, A., & Muñoz-Torrero, D. (2020).
Centrally active multitarget anti-Alzheimer agents derived from the
antioxidant lead CR-6. Journal of Medicinal Chemistry, 63(17),
9360–9390. https://doi.org/10.1021/acs.jmedchem.0c00528
Prakash, A., Kalra, J. K., & Kumar, A. (2015). Neuroprotective ef-
fect of N-acetyl cysteine against streptozotocin-induced memory
dysfunction and oxidative damage in rats. Journal of Basic and
Clinical Physiology and Pharmacology, 26(1), 13–23. https://doi.
org/10.1515/jbcpp-2013-0150
Tycko, R. (2016). Alzheimer's disease: Structure of aggregates revealed.
Nature, 37(7621), 492–493. https://doi.org/10.1038/nature19470
Wang, Y., Guan, X.-L., Wu, P.-F., Wang, C.-M., Cao, H., Li, L., Guo,
X.-J., Wang, F., Xie, N. A., Jiang, F.-C., & Chen, J.-G. (2012).
Multifunctional mercapto-tacrine derivatives for treatment of age-
related neurodegenerative diseases. Journal of Medicinal Chemistry,
55(7), 3588–3592. https://doi.org/10.1021/jm300124p
Woodhead, A. J., Angove, H., Carr, M. G., Chessari, G., Congreve,
M., Coyle, J. E., Cosme, J., Graham, B., Day, P. J., Downham,
R., Fazal, L., Feltell, R., Figueroa, E., Frederickson, M., Lewis,
J., McMenamin, R., Murray, C. W., O’Brien, M. A., Parra, L., …
Woolford, A.-A. (2010). Discovery of (2,4-dihydroxy-5-isopropylp
henyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-y
l] methanone (AT13387), a novel inhibitor of the molecular chap-
erone HSP90 by fragment based drug design. Journal of Medicinal
Chemistry, 53(16), 5956–5969. https://doi.org/10.1021/jm100060b
Yuan, W., Shang, Z. P., Qiang, X. M., Tan, Z. H., & Deng, Y. (2014).
Synthesis of pterostilbene and resveratrol carbamate derivatives as
potential dual cholinesterase inhibitors and neuroprotective agents.
Research on Chemical Intermediates, 40(2), 787–800. https://doi.
org/10.1007/s11164-012-1003-1
Zhou, J., Jiang, X., He, S., Jiang, H., Feng, F., Liu, W., Qu, W., & Sun, H.
(2019). Rational design of multi-target-directed ligands: Strategies
and emerging paradigms. Journal of Medicinal Chemistry, 62(20),
8881–8914. https://doi.org/10.1021/acs.jmedchem.9b00017
Zollinger, R. M., & Williams, R. D. (1964). A mucolytic agent:
Acetylcysteine (Mucomyst). Journal of the American
Medical Association, 190, 147–148. https://doi.org/10.1001/
jama.1964.03070150057013
SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
Sang, Z.-P., Qiang, X.-M., Li, Y., Wu, B., Zhang, H., Zhao, M.-G.,
& Deng, Y. (2015). Design, synthesis, and biological evaluation
of scutellarein carbamate derivatives as potential multifunctional
agents for the treatment of Alzheimer's disease. Chemical Biology
How to cite this article: Zhang, H., Song, Q., Yu, G.,
Cao, Z., Qiang, X., Liu, X., & Deng, Y. (2021).
Phthalimide-(N-alkylbenzylamine) cysteamide
hybrids as multifunctional agents against Alzheimer’s
disease: Design, synthesis, and biological evaluation.
Chemical Biology & Drug Design, 00, 1–8.
https://doi.org/10.1111/cbdd.13905
&
Drug Design, 86(5), 1168–1177. https://doi.org/10.1111/
cbdd.12580
Savelieff, M. G., Nam, G., Kang, J., Lee, H. J., Lim, M., & Lim, M. H.
(2019). Development of multifunctional molecules as potential ther-
apeutic candidates for Alzheimer's disease, Parkinson's disease, and
amyotrophic lateral sclerosis in the last decade. Chemical Reviews,
119(2), 1221–1322. https://doi.org/10.1021/acs.chemrev.8b00138
Shahripour, R. B., Harrigan, M. R., & Alexandrov, A. V. (2014). N-
acetylcysteine (NAC) in neurological disorders: Mechanisms of