Novel salicylamide derivatives as potent multifunctional agents for the treatment of Alzheimer's disease: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.11.022

A series of salicylamide derivatives were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease. In vitro assays demonstrated that most of the derivatives were selective AChE inhibitors. They showed good inhibitory activities of self- and Cu²⁺-induced Aβ_1–42 aggregation, and significant antioxidant activities. Among them, compound 15b exhibited good inhibitory activity toward RatAChE and EeAChE with IC₅₀ value of 10.4 μM and 15.2 μM, respectively. Moreover, 15b displayed high antioxidant activity (2.46 Trolox equivalents), good self- and Cu²⁺-induced Aβ_1–42 aggregation inhibitory potency (42.5% and 31.4% at 25.0 μM, respectively) and moderate disaggregation ability to self- and Cu²⁺-induced Aβ_1–42 aggregation fibrils (23.4% and 27.0% at 25 μM, respectively). Furthermore, 15b also showed biometal chelating abilities, anti-neuroinflammatory ability and BBB permeability. These multifunctional properties indicated compound 15b was worthy of being chosen for further pharmacokinetics, toxicity and behavioral researches to test its potential for AD treatment.

Full text of DOI:10.1016/j.bioorg.2018.11.022

Accepted Manuscript
Novel salicylamide derivatives as potent multifunctional agents for the treat-
ment of Alzheimer's disease: design, synthesis and biological evaluation
Qing Song, Yan Li, Zhongcheng Cao, Xiaoming Qiang, Zhenghuai Tan, Yong
Deng
PII:
S0045-2068(18)31141-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.022
YBIOO 2630
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 October 2018
14 November 2018
17 November 2018
Please cite this article as: Q. Song, Y. Li, Z. Cao, X. Qiang, Z. Tan, Y. Deng, Novel salicylamide derivatives as
potent multifunctional agents for the treatment of Alzheimer's disease: design, synthesis and biological evaluation,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel salicylamide derivatives as potent multifunctional agents for the treatment
of Alzheimer's disease: design, synthesis and biological evaluation
Qing Songa,1, Yan Lia,1, Zhongcheng Caoa, Xiaoming Qianga, Zhenghuai Tanb, Yong Denga,*
aDepartment of Medicinal Chemistry, Key Laboratory of Drug-Targeting and Drug Delivery System
of the Education Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan
Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan
University, Chengdu, 610041, P. R. China
bInstitute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, P. R. China
1 These authors contributed equally to this work.
*Corresponding author.
E-mail: dengyong@scu.edu.cn (Yong Deng)
1

Abstract
A series of salicylamide derivatives were designed, synthesized and evaluated as multifunctional
agents for the treatment of Alzheimer’s disease. In vitro assays demonstrated that most of the
derivatives were selective AChE inhibitors. They showed good inhibitory activities of self- and
Cu2+-induced Aβ1–42 aggregation, and significant antioxidant activities. Among them, compound 15b
exhibited good inhibitory activity toward RatAChE and EeAChE with IC50 value of 10.4 μM and
15.2 μM, respectively. Moreover, 15b displayed high antioxidant activity (2.46 Trolox equivalents),
good self- and Cu2+-induced Aβ1-42 aggregation inhibitory potency (42.5% and 31.4% at 25.0 μM,
respectively) and moderate disaggregation ability to self- and Cu2+-induced Aβ1-42 aggregation fibrils
(23.4% and 27.0% at 25 μM, respectively). Furthermore, 15b also showed biometal chelating
abilities, anti-neuroinflammatory ability and BBB permeability. These multifunctional properties
indicated compound 15b was worthy of being chosen for further pharmacokinetics, toxicity and
behavioral researches to test its potential for AD treatment.
Keywords:
Alzheimer’s disease; Salicylamide derivatives; Acetylcholinesterase inhibitors; Antioxidant; Aβ
aggregation inhibitors; Anti-neuroinflammatory agents.
2

1. Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder in elderly population and the leading
cause of dementia. It is generally characterized by memory loss and cognitive impairments due to the
progressive and irreversible damage to the brain [1, 2]. Over 47 million people lived with dementia
worldwide in 2015, and it is estimated that this number will increase to more than 131 million by
2050 [3]. AD is one of the most enigmatic and intractable issues in medical science, and there are
currently multiple interconnected factors, including β-amyloid (Aβ) deposits, dyshomeostasis of
biometals, oxidative stress, neuroinflammation and degeneration of cholinergic neurons in the central
nervous system (CNS) [4].
The decreased levels of acetylcholine (ACh) resulting in the learning and memory
dysfunctioning, is considered to be a critical determinant of AD pathogenesis [5].
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are key enzymes that play
important roles in cholinergic transmission by hydrolyzing the ACh. AChE was validated as a
therapeutic target to increase cholinergic levels, and AChE inhibitors are effective in temporarily
restoring cholinergic function. But BuChE is mainly distributed in the peripheral areas, and the
inhibition of BuChE may bring certain peripheral side effects [6]. Therefore, the development of
AChE inhibitors may be a more rational therapeutic strategy for AD.
The increased production and accumulation of Aβ oligomer in brain also plays a pivotal role in
AD pathogenesis. The Aβ peptide is generated after enzymatic cleavage of the amyloid precursor
protein (APP) [7]. APP follows two distinct cleavage pathways. In the non-amyloidogenic one, APP
is first cleaved by α- and γ-secretase to form interceptive Aβ17–40/42 peptides or Aβ1–16 peptide. In the
amyloidogenic one, which occurs to a minor extent, APP is cleaved consecutively by β- and
γ-secretases leading to the formation of full-length Aβ peptides (mainly Aβ1–40/42) [8]. Although
Aβ1-40 presents in larger amounts in brain, Aβ1-42 displays lower solubility and higher tendency to
aggregate, leading to the formation of oligomers species that are reorganized into protofibrils and
fibrils, found in amyloid plaques [9]. Therefore, preventing the Aβ1-42 aggregation in the brain may
be a potent therapeutic strategy for AD.
Some evidence suggests that oxidative stress plays an important role in the pathogenesis of AD
[10]. Generally, reactive oxygen species (ROS) are kept at a low level but not fully eliminated.
3

Accumulation of too high levels of ROS is dangerous and defined as oxidative stress [11]. Oxidative
stress occurs early in the initiation of AD, and is associated with the presence of Aβ [12, 13].
Moreover, it has been proposed that oxidative stress acts as aberrant signaling pathways, resulting in
progressive damage to the neuronal network [14]. In turn, both toxic Aβ and excessive neuronal
excitation can induce ROS production [15]. The vicious cycle makes things worse. According to
these, molecules with antioxidant abilitiy may be a good therapy for AD.
In recent years, with the emphasis on the inflammatory mechanism of AD, it’s suggested that
neuroinflammation may be another important pathogenesis of AD [16]. Epidemiological studies have
identified a link between chronic use of NSAIDs and reduced risk of AD [17]. They showed the
protective effect of the long-term use of NSAIDs in AD patients, reducing or delaying the
development of the disease, although the mechanism of this protective effect is still unclear.
Neuroinflammation of AD is mainly manifested by activation of microglial cells in the brain.
Microglia cells are widely distributed in brain, spinal cord, retina and optic nerve, but mainly in the
hippocampus and substantia nigra [18]. Activated microglia can produce proinflammatory cytokines
like TNF-α, IL-1, IL-6 and chemokines. These inflammatory factors will promote the formation of
chronic neuroinflammation, lead to neuron death, and aggravate neurodegenerative lesions [19-21].
Therefore, the use of NSAIDs to relieve neuroinflammation has brought hope to patients with AD.
Salicylic acid (SA) has been widely used for years as an anti-inflammatory drug. As the
discovery and further studies of the effect of neuroinflammation on AD, biological activities of SA
and its derivatives drew people's concern. The studies indicated that SA had certain
anticholinesterase activity (IC50 = 346.0 μM) [22]. In addition, salicylic acid derivatives could inhibit
LPS-induced activation of microglia cells and astrocytes, and reduce the production of relevant
pro-inflammatory factors [23]. In vivo experiments, salicylic acid derivatives could significantly
improve the cognitive and memory function in a transgenic mouse model of AD [24]. The above
research results showed that SA can be used as a leading compound to design new anti-AD drugs.
Because of the complex etiology of AD, the single-target directed drugs which have reached
clinical trials have failed. Recently, more researches in AD have been paid attention to the
development of ‘‘multi-target-directed ligands’’ (MTDLs), by which new scaffolds aiming at two or
more disease targets are designed. Based on previous research of our group, the introduction of the
alkylbenzylamine group to chromone significantly increased the inhibitory activity of AChE, and the
4

chromone derivative (II) is multifunctional agent for AD treatment [25]. But the molecular weights
(MW) of chromone derivatives are a little high. MW is one of the most important physicochemical
properties that affect membrane permeability, for its reduction often has coincidental beneficial
effects on other parameters, such as rotatable bonds, PSA, and cLogP [26]. Studies showed CNS
drugs tend to be smaller than non-CNS therapeutics, and the mean MW derived from an analysis of
marketed CNS drugs is 310, which offers useful guidance when defining a desirable CNS candidate
[27]. Lipinski and Levin et al. have also suggested that MW should be kept below 400 [28, 29].
Therefore we replaced chromone nucleus with salicylic to reduce their MW for better membrane
permeability. According to these, we designed a series of N-alkylbenzylamine salicylamide
derivatives (Figure 1) by using multi-target-directed drug design strategy to combine salicylic acid
with appropriate secondary amines using carbon spacers of different lengths. Furthermore, we found
that the inhibitory activity of chromone derivatives toward AChE increased a little and even began to
decrease when the linker was up to 6. So based on this trend of structure-activity relationship, we
designed the salicylamide derivatives with the longest linker of 6. These new molecules may
simultaneously possess membrane permeability, AChE inhibition, antioxidative and metal chelating
effects, anti-inflammatory and anti-β-amyloid aggragation properties.
Figure 1. Design strategy of the N-alkylbenzylamine salicylamide derivatives.
5

2. Results and discussion
2.1. Chemistry
The synthetic pathways of target derivatives were summarized in Scheme 1. Compound 2 was
synthesized from phtalimide (1) reacted with KOH. Then compound 2 was reacted with excessive
amounts of 1,3-dibromopropane, 1,4-dibromobutane or 1,6-dibromohexane in the presence of
PEG-400 in acetone to obtain intermediates 3-5 [30]. In turn, these intermediates were reacted with
the corresponding secondary amines 6a-h [25], to provide the intermediates 7-9 respectively. The
latter were reacted with hydrazine hydrate to obtain the key primary amine intermediates 10-12 [31].
Finally, salicylic acid (I) was condensed with the corresponding primary amine 10-12 in the presence
of 1-(3-dimethylaminopropyl)-3-ethyl carbon imine hydrochloride (EDCI) in THF to provide the
N-alkylbenzylamine salicylamide derivatives 13-15. All of the target compounds were characterized
1
13
by H NMR and ESI-MS, and most of them were further characterized by C NMR. The purity of
all target compounds was determined by high-performance liquid chromatography (HPLC) analysis
to be over 97.0 %.
Scheme 1. Synthesis of salicylamide derivatives 13-15. Reagents and conditions: (a) KOH, EtOH,
reflux; (b) Br(CH2)nBr, PEG400, acetone, reflux for 5-8 h; (c) R1R2NH (6a-h), K2CO3, CH3CN,
reflux for 6-8 h; (d) H2NNH2•H2O, EtOH, reflux for 6 h; (e) Salicylic acid (I), EDCI, THF, r.t.,
overnight.
2.2. Pharmacology
2.2.1. Evaluation of AChE and BuChE inhibitory activities
6

All the synthesized salicylamide derivatives were evaluated for their inhibitory activities toward
AChE and BuChE in vitro according to modified Ellman’s method [32, 33]. AChE activity was
measured from both rat cortex homogenate and Electrophorus electricus, and BuChE activity was
measured from rat serum. Rivastigmine and donepezil were used as reference compounds. The ChE
inhibitory results are summarized in Table 1.
As shown in Table 1, salicylamide derivatives (13-15) showed different AChE inhibitory
activities with IC50 values ranging from 10 μM to 200 μM. The structure-activity relationship
analysis showed that the AChE inhibitory activity of the compound was closely related to its
methylene side chain length and terminal benzylamine species. The conversion of these two factors
can improve the interaction between the molecule and the CAS and PAS sites of the enzyme. From
the results of compounds 14a-h, fixing the side chain length, we could find benzylamine moiety had
a large effect on the activity. In general, compounds having benzylamine moieties with N-ethyl (14b,
14d and 14e) or N-propargyl (14h) substitutions were more active than those with N-methyl
substitution (14a and 14c). Compounds with 2-methoxybenzylamine (14c-d and 14h) or
2-dimethylaminobenzylamine (14e) were more active than the compounds (14a-b) with
unsubstituted benzylamine moiety. In conclusion, the (2-methoxybenzyl) ethylamine moiety was
optimal for the inhibition of AChE. Then, we found the introduction of (4-dimethylaminobenzyl)
ethylamine and the benzoxazepine moiety (14f and 14g) did not significantly increase or even
decrease the activity. Especially, compound 14g almost had no AChE inhibitory activity, which
indicated that rigid structure of the terminal benzylamine may be not conducive to its interaction with
the enzyme activity center. In addition, the activities of compounds 15a-b (n = 6) were significantly
higher than those of corresponding 13a-b (n = 3), 14a and 14d (n = 4). Therefore, a 6-methylene
linker was beneficial for AChE inhibitory activity. Among the tested derivatives, 15b exhibited the
most potent inhibitory activity for both RatAChE and EeAChE (IC50 = 10.4 μM and 15.2 μM,
respectively), which was more excellent than rivastigmine (IC50 = 37.1 μM and 23.2 μM,
respectively) but less than donepezil (IC50 = 0.015 μM and 0.021μM, respectively) under our
experimental condition. Compared with chromone derivative (II) (IC50 = 0.07 μM and 0.55 μM,
respectively) [25], the activity of compound 15b is relatively lower. Camparing the molecular
docking results of these two compounds, a strong hydrogen bond was observed between the
4-carbonyl group at the chromone moiety and Tyr121, which may contribute to the better inhibitory
7

activity of chromone derivative (II) toward AChE.
All salicylamide derivatives were inactive or weak on BuChE inhibitory activity. These results
showed the target compounds were potent AChE inhibitors with high selectivity. It may be beneficial
to diminish peripheral cholinergic side effects and provide lower toxicity. Based on the above results,
we selected 15b for kinetic analysis of AChE inhibition.
Table 1. In vitro inhibition of AChE and BuChE and oxygen radical absorbance capacity (ORAC,
Trolox Equivalents) by salicylamide derivatives (13-15) and reference compounds.
IC50 (μM) ± SDa
Compd.
n
NR1R2
ORACe
RatAChEb
RatBuChEc
EeAChEd
3
3
13a
13b
200.0 ± 5.12 175.0 ± 6.06
>200
2.00 ± 0.05
2.80 ± 0.02
2.55 ± 0.07
2.32 ± 0.03
68.5 ± 1.22
>200
>200
>200
>200
4
14a
190.0 ± 3.48 177.0 ± 7.63
4
14b
160.0 ± 2.57
103.0 ± 1.18
31.4 ± 0.73
65.0 ± 2.90
170.0 ± 3.39
n.a.f
>200
>200
4
14c
118.0 ± 2.45 2.90 ± 0.07
68.2 ± 0.99 1.74 ± 0.05
4
14d
>200
4
14e
98.5 ± 2.77
>200
>200
>200
>200
>200
2.49 ± 0.09
2.91 ± 0.03
2.82 ± 0.06
1.24 ± 0.06
4
14f
4
14g
134.0 ± 4.82
>200
4
14h
71.1 ± 4.22
21.2 ± 1.10
10.4 ± 0.478
37.1 ± 1.22
0.015 ± 0.002
6
15a
191.0 ± 12.70 17.7 ± 0.20 2.20 ± 0.04
6
15b
106.0 ± 5.01
9.87 ± 0.23
20.7 ± 0.36
15.2 ± 0.33 2.46 ± 0.03
23.2 ± 0.44
n.t.g
n.t.g
—
—
—
—
Rivastigmine
Donepezil
0.021 ± 0.003
8

a IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean
of 3 independent experiments, each performed in triplicate (SD = standard deviation).
b From 5% rat cortex homogenate.
c BuChE from rat serum and tested compounds were used at 50 μM.
d From Electrophorus electricus.
e The mean ± SD of the three independent experiments. The data are expressed as μM of Trolox equiv/μM of tested
compound.
f n.a. = no active. Compounds defined “no active” means that percent inhibition is less than 5.0% at a concentration
of 50 μM in the assay conditions.
g n.t. = not tested.
2.2.2. Kinetic study for the inhibition of AChE
To gain further insight into the inhibitory mechanism of this family of compounds, 15b, the
most potent AChE inhibitor among these compounds, was chosen for a kinetic study using EeAChE
[34]. The graphical presentation of the reciprocal Lineweaver-Burk plots (Figure 2) showed that
with the concentrations of 15b increasing, both the slopes (decreased Vmax) and intercepts (higher Km)
were increased. This pattern indicated a mixed-type inhibitory behavior for 15b as the consequence
of binding to both the CAS and PAS of AChE.
Figure 2. Kinetic study on the mechanism of EeAChE inhibition by compound 15b. Overlaid
Lineweaver-Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.1-0.4 mM) in the absence and presence of 15b are shown. Lines were derived from a weighted
least-squares analysis of data points.
9

2.2.3. Molecular modeling study
To explore the possible interacting mode of the salicylamide derivatives with TcAChE (PDB
code: 1EVE), a molecular modeling study was performed using the docking program, AutoDock 4.2
package with Discovery Studio 2.5 [35]. The results showed in Figure 3 indicated that compound
15b occupied the entire enzymatic CAS, the mid-gorge sites and the PAS, and binds simultaneously
to both the catalytic site and peripheral site. In the TcAChE-15b complex, the secondary amine
moiety of the amide of 15b interacted with residue Phe331 via hydrogen bond interaction. In
addition, the N-(2-methoxybenzyl) ethylamine moiety in the side chain of 15b was observed to bind
to the CAS via parallel π-π interactions with Trp84 and exhibited a potential hydrophobic interaction
with residues Asp72, Phe330, His440 and Gly441. Furthermore, the conformation formed by the
folding methylene side chain and the salicylamide ring in the gorge allowed them to interact with the
residues Gly118, Tyr121, Trp279, Ser286, Ile287, Phe288, Phe290, Phe331, Tyr334 and Gly335 via
the hydrophobic interaction. The docking results, consistent with the kinetic analysis, indicated that
compound 15b could simultaneously bind to the CAS and PAS of AChE, and explained its high
AChE inhibitory activities.
Figure 3. Representation of compound 15b (grey stick) interacting with residues in the binding site
10

of TcAChE (PDB code: 1EVE), highlighting the protein residues that participate in the main
interactions with the inhibitor.
2.2.4. Antioxidant activity assay
The antioxidant activities of the salicylamide derivatives were evaluated by the well-established
ORAC-FL method (oxygen radical absorbance capacity by fluorescein) [36] and the results were
shown in Table 1. The activities of tested compounds to scavenge radicals were expressed as Trolox
(a water-soluble vitamin E analog) equivalent. And their relative activity at concentration of 5 μM
was compared with the highly potent compound Trolox. All the tested compounds displayed high
peroxyl radical absorbance capacities, ranging from 1.24 to 2.91-fold of Trolox. Among them,
compound 14f exhibited the strongest free radical scavenging activity with ORAC-FL value of 2.91
Trolox equivalents due to the introduction of (4-dimethylaminobenzyl) ethylamino moiety. However,
the introduction of propyl (Compound 14h) reduced the activity of the compound with an ORAC-FL
value of 1.24 Trolox equivalents. Representative compound 15b showed good antioxidant activity
with an ORAC-FL value of 2.46 Trolox equivalents, which was significantly better than chromone
derivative (II) (0.83 Trolox equivalents) [25].
2.2.5. Metal-chelating studies
The chelating ability of compound 15b with biometals: Cu2+, Zn2+, Fe2+ and Al3+, was evaluated
by UV-visual spectrometry [37, 38], and the results were shown in Figure 4A. We could find the
UV-Vis spectra of 15b changed significantly after the addition of CuCl2 and AlCl3, with a shoulder
peak appearing at 342 nm and 330 nm, respectively, which indicated the formation of 15b-Cu2+ and
15b-Al3+ complexes. In addition, the absorption increased significantly when CuCl2, FeSO4 and
AlCl3 were added, indicating a possible interaction between the compound 15b and these biometals.
However, the addition of ZnCl2 to 15b solution showed no significant change in the spectra.
To further determine the stoichiometry of 15b-Cu2+ complex, molar ratio method was used by
preparing the methanol solutions of compound 15b with increasing amounts of CuCl2. The UV
spectra were used to obtain the absorbance of the 15b complex and different concentrations of CuCl2
at 342 nm. As indicated in Figure 4B, the absorbance linearly increased initially and then plateaued.
The two straight lines intersected at a mole fraction of 0.97, revealing a 1:1 stoichiometry for
15b-Cu2+ complex.
11

Figure 4. (A) UV spectra of compound 15b (37.5 μM in methanol) alone or in the presence of CuCl2,
ZnCl2, AlCl3 or FeSO4 (37.5 μM, in methanol). (B) Determination of the stoichiometry of
complex-Cu2+ by using the molar ratio method titrating the methanol solution of compound 15b with
ascending amounts of CuCl2. The final concentration of tested compound was 37.5 μM, and the final
concentration of Cu2+ ranged from 3.75 to 150 μM.
2.2.6. Inhibition of self- and Cu2+-induced Aβ1-42 aggregation
The activities of the target compounds on inhibiting self- and Cu2+-induced Aβ1-42 aggregation
were evaluated by a thioflavin T (ThT) fluorescence assay, with curcumin as a reference compound
[39-42]. The results summarized in Table 2 indicated that all the target compounds showed moderate
to good inhibitory activities of self-induced Aβ1-42 aggregation with inhibition ratios ranging from
12

25.1% to 45.1% at 25 μM, compared with that of curcumin (40.2% at 25 μM). However, most of
compounds showed poor inhibitory activity against Cu2+-induced Aβ1-42 aggregation with the
percentage ranging from 8.6% to 31.4%. Furthermore, the representative compound 15b exhibited
moderate inhibitory potency of self- and Cu2+-induced Aβ1-42 aggregation with the percentage of
42.5% and 31.4%, respectively, which had relatively weaker activities compared with chromone
derivative (II) (59.2% and 48.3%, respectively) [25]. For the inhibition of self-induced Aβ1-42
aggregation, compound 14d (n = 4) exhibited the highest inhibitory activity with inhibition ratios of
45.1%. Interestingly, compound 13b (37.1%) and 15b (42.5%) respectively exhibited the highest
inhibitory activity among the derivatives with a 3-methylene linker and 6-methylene linker. It
showed an N-(2-methoxybenzyl) ethylamine group on the terminal side of carbon chain among the
compounds 13b, 14d and 15b may be optimal for the inhibition of self-induced Aβ1-42 aggregation,
which was same as the conclusion resulting from the AChE inhibition assay. As for the Cu2+-induced
Aβ1-42 aggregation, the most potent inhibitor was 15b. But the length of the alkyl chain and terminal
benzylamine species had no significant effect against Cu2+-induced Aβ1-42 aggregation.
Table 2. In vitro inhibition of self- and Cu2+-induced Aβ1-42 aggregation by salicylamide derivatives
and reference compound.
% Inhibition of Aβ1-42 aggregationa
% Disaggregation of Aβ1-42 aggregatione
Compd.
n
Self-inducedb,d
34.0 ± 1.0
37.1 ± 0.9
32.4 ± 1.2
36.2 ± 1.3
31.6 ± 1.1
45.1 ± 2.0
25.1 ± 0.6
29.1 ± 0.8
26.2 ± 0.6
25.3 ± 1.0
33.7 ± 1.4
42.5 ± 0.9
40.2 ± 0.9
Cu2+-inducedc,d
11.5 ± 0.2
14.1 ± 0.3
22.6 ± 0.7
15.6 ± 0.9
13.8 ± 0.8
10.9 ± 0.1
9.8 ± 0.3
Self-inducedf,d
n.t.h
Cu2+-inducedg,d
n.t.h
13a
13b
3
3
25.4 ± 0.8
n.t.h
40.5 ± 1.2
n.t.h
14a
4
14b
4
n.t.h
n.t.h
14c
4
n.t.h
n.t.h
14d
4
29.4 ± 0.6
n.t.h
35.6 ± 1.3
n.t.h
14e
4
14f
4
11.1 ± 0.5
23.6 ± 1.3
8.6 ± 0.1
n.t.h
n.t.h
14g
4
n.t.h
n.t.h
14h
4
n.t.h
n.t.h
15a
6
17.6 ± 0.9
31.4 ± 1.0
66.0 ± 1.3
n.t.h
n.t.h
15b
6
23.4 ± 0.6
n.t.h
27.0 ± 0.9
54.7 ± 1.0
Curcumin
—
13

a For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
b Inhibition of self-induced Aβ1-42 aggregation (25 μM) by tested inhibitors at 25 μM.
c Inhibition of Cu2+-induced Aβ1-42 aggregation. The concentration of tested compounds and Cu2+ were 25 μM.
d The mean ± SD of the three independent experiments.
e For disaggregation of Aβ aggregation fibrils, the thioflavin-T fluorescence method was used.
f For disaggregation of self-induced Aβ1-42 aggregation fibrils, the concentration of tested compounds and Aβ1-42
were 25 μM.
g For disaggregation of Cu2+-induced Aβ1-42 aggregation fibrils, the concentration of tested compounds and Cu2+
were 25 μM.
h n.t. = not tested.
2.2.7. Effect on the disaggregation of Aβ1-42 aggregation fibrils
We selected some relatively high active salicylamide derivatives (13b, 14d and 15b) to test their
disaggregation ability to the formed Aβ fibers. The results were summarized in Table 2. Compounds
13b, 14d and 15b manifested the disaggregation potency of self- induced Aβ1-42 aggregation fibrils
with the percentage of 25.4%, 29.4% and 23.4%, respectively. As for those of disaggregation of
Cu2+-induced Aβ1-42 aggregation fibril, their disaggregation ratios were 40.5%, 35.6% and 27.0%,
respectively. It revealed that these compounds could not only inhibit the aggregation of Aβ1-42, but
also depolymerize the Aβ1-42 aggregation fibrils, with the potency of inhibiting and eliminating Aβ
plaques in AD brains.
2.2.8. In vitro blood-brain barrier permeation assay
For the drug targeting on central nervous system, it is an important prerequisite for its efficacy
to penetrate the blood-brain barrier (BBB). A parallel artificial membrane permeation assay of the
blood-brain barrier (PAMPA-BBB) was performed to determine the BBB penetration of the target
compounds [43]. First, the experimental permeabilities of 11 commercial drugs were compared with
reported values (Table S1, Supplementary Material). A plot of experimental data versus
bibliographic values produced a good linear correlation: Pe (exp.) = 0.9163 × Pe (bibl.) − 0.2247 (R2
= 0.9558) (Figure S1, Supplementary Material). Combined with the evaluation conditions
established by Di et al. [44] for BBB permeation, we determined that compounds with Pe values
above 3.44 × 10-6 cm/s could cross the BBB (Table S2, Supplementary Material). The representative
compound 15b was selected for this assay. As the result was shown in Table 3, compound 15b
exhibited good BBB permeabilities and would be able to reach the therapeutic targets in central
14

nervous system (CNS).
Table 3. Permeability results Pe (× 10-6 cm/s) from the PAMPA-BBB assay for 15b with its predicted
penetration into the CNS.
Compd.a
Pe (× 10-6 cm/s)b
Prediction
CNS +
15b
6.27 ± 0.12
a Compound 15b was dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration
of each compound was 100 μg/mL.
b Data are the mean ± SD of three independent experiments.
2.2.9 In vitro anti-neuroinflammatory activity assay
Neuroinflammation in AD is mainly characterized by the activation of microglia in the brain
and the release of multiple proinflammatory cytokines. To evaluate the anti-neuroinflammatory
activity of the target compounds, representative compound 15b was tested with the inflammatory
BV-2 microglial stimulated by lipopolysaccharide (LPS). Flurbiprofen and salicylic acid were used
as reference compound. In vitro neurocytotoxicity, inhibition of LPS induced NO and TNF-α
production were investigated [45-48].
The in vitro cytotoxicity assay was carried out by the MTT method. As the results shown in
Figure 5, whether adding LPS (0.1 μg/mL) or not, cell viability did almost not change after treatment
with various concentrations of all the compounds (0.5, 2.5 and 10.0 μM). It indicated compound 15b
was not toxic toward the BV-2 cell at concentrations below 10 μM, and LPS (0.1 μg/mL) could not
alter cell viability also.
Inhibition of LPS-induced NO production was measured by using Griess reaction method. First,
we examined the effect of compounds themselves on NO release from BV-2 cells without LPS. As
shown in Figure 6, NO release volume didn’t change a lot compared with the control group, so
compounds had no effect on it. Then in LPS-treated cells, we observed a massive induction of NO
production, which was significantly reduced by treatment with 15b in a dose-dependent manner. 15b
displayed NO inhibitory activity with the respective percentages of 13.3%, 30.0% and 55.0% when
the cells were treated with 0.5, 2.5 and 10.0 μM, which was much better than flurbiprofen (11.8%
and 23.0% when the cells were treated with 2.5 and 10.0 μM) and SA (10.0% and 18.5% when the
cells were treated with 2.5 and 10.0 μM).
15

Inhibition of LPS-induced TNF-α production was measured by the enzyme-linked
immunosorbent assay (ELISA). The results were shown in Figure 7. First we could also find that
15b had no effect on TNF-α production without LPS up to the concentration of 10 μM. Then in
LPS-treated cells, 15b showed moderate inhibitory activities on LPS-induced TNF-α production with
the respective inhibition ratios of 13.5%, 27.1% and 45.3% when the cells were treated with 0.5, 2.5
and 10.0 μM, which was still much better than flurbiprofen (9.6% and 30.3% when the cells were
treated with 2.5 and 10.0 μM) and SA (7.6% and 20.6% when the cells were treated with 2.5 and
10.0 μM).
The above results indicate that the introduction of the alkylbenzylamine group to salicylic acid
can improve its anti-inflammatory activity, which may partly due to the enhancement of its lipid
solubility. All these suggest that 15b is a promising antiinflammatory and neuroprotective agent.
16

Figure 5. Effects of SA, 15b and flurbiprofen without LPS (A) or with LPS (B) on the cell viability
of microglia BV-2 cells. BV-2 cells were pre-incubated with indicated concentrations of compounds
for 30 min followed by 1.0 μg/mL LPS treatment for 24 h. Cell viability was determined by MTT
assay. The data are expressed as the mean ± SD from three independent experiments.
Figure 6. Effects of SA, 15b and flurbiprofen on NO release in BV-2 cells and LPS-stimulated BV-2
cells. The data are expressed as the mean ± SD from three independent experiments.
17

Figure 7. Effects of SA, 15b and flurbiprofen on TNF-α release in LPS-stimulated BV-2 cells. The
data are expressed as the mean ± SD from three independent experiments.
3. Conclusion
In summary, a series of salicylamide derivatives were designed, synthesized and evaluated as
multifunctional agents for the treatment of AD. In vitro assays demonstrated that some compounds
possessed moderate AChE inhibitory activity and displayed high selectivity for AChE over BuChE.
Most compounds showed good inhibitory activities of self- and Cu2+-induced Aβ1–42 aggregation, and
significant antioxidant activities. Among them, compound 15b exhibited a useful inhibitory activity
toward RatAChE and EeAChE with IC50 value of 10.4 μM and 15.2 μM, respectively. The kinetic
analysis suggested that 15b showed mixed-type inhibition, and could bind to both CAS and PAS of
AChE, which was consistent with the molecular modeling study. In addition, 15b displayed good
self- and Cu2+-induced Aβ1-42 aggregation inhibitory potency (42.5% and 31.4% at 25 μM,
respectively), and moderate disaggregation ability to self- and Cu2+-induced Aβ1-42 aggregation
fibrils (23.4% and 27.0% at 25 μM, respectively). 15b also exhibited significant antioxidant activity
and good biometal chelating ability. 15b also possessed the prospective property of acting as
anti-neuroinflammatory agent. At last, 15b could cross the BBB and penetrate into brain. Under the
effective treatment concentration, 15b is worthy of being chosen for further in vivo assays to study its
18

potential effect of multi-target collaborative treatment of AD.
4. Experimental section
4.1 Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. Melting points (uncorrected) were measured on YRT-3 melting-point
apparatus (China). HPLC analysis was carried out on a Shimadzu LC-10Avp plus system with the
13
use of a Kromasil C18 column (4.6 mm × 250 mm, 5 μm). The 1H NMR and C NMR spectra were
recorded in CDCl3 on a Varian INOVA spectrometer at 25 °C with TMS as the internal standard.
Coupling constants are given in Hz. Mass spectra were recorded on Agilent-6210 TOF LC-MS
Spectrometer. All the reactions were monitored by thin-layer chromatography (TLC) using silica gel
GF254 plates from Qingdao Haiyang Chemical Co. Ltd. (China) and the spots were detected under
UV light (254 nm). Column chromatography was performed using silica gel (230-400 mesh)
purchased from Qingdao Haiyang Chemical Co. Ltd. (China).
4.1.1 Potassium 1,3-dioxoisoindolin-2-ide (2)
Compound 2 was prepared as previously described [30].
4.1.2 General procedure for the synthesis of 3-5
To a mixture of dibromoalkane (32.0 mmol), PEG400 (0.23 mL) in acetone (60 mL), compound
2 (2.96 g, 16.0 mmol) was added in portions under refluxing condition. The resulting mixture was
heated for 5-8 h. After the reaction was finished, the solvent was evaporated under reduced pressure.
Then water (50 mL) was added to the residue and the mixture was extracted with dichloromethane
(50 mL×3). The combined organic phases were washed with brine, dried over sodium sulfate, filtered
and concentrated under reduced pressure. The obtained residue was recrystallized from ethanol to
afford compound 3-5.
4.1.2.1 2-(3-Bromopropyl)isoindoline-1,3-dione (3)
Compound 3 was synthesized from 2 and 1,3-dibromopropane according to the general
procedure; white solid; yield: 75.3%; mp 70-71 °C (Lit. [49] 72-75 °C).
4.1.2.2 2-(4-Bromobutyl)isoindoline-1,3-dione (4)
Compound 4 was synthesized from 2 and 1,4-dibromobutane according to the general procedure;
19

white solid; yield: 75.9%; mp 79-80 °C (Lit. [30] 79-81 °C).
4.1.2.3 2-(6-Bromohexyl)isoindoline-1,3-dione (5)
Compound 5 was synthesized from 2 and 1,6-dibromohexane according to the general
procedure; white solid; yield: 70.0%; mp 57-58 °C (Lit. [30] 57-58 °C).
4.1.3 General procedure for the synthesis of 7-9
To a suspension of the corresponding secondary amine (3.22mmol) and anhydrous K2CO3
(765mg, 5.48mmol) in CH3CN (20 mL) was added the intermediates 3-5 (0.60 mmol). The mixture
was refluxed for 6-8 h. The solvent was removed under reduced pressure. The residue was diluted
with water (30mL) and the mixture was extracted with dichloromethane (30mL×3). The combined
organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated under
reduced pressure. The residue was purified on a silica gel chromatography in petroleum
ether/acetone (20/1, v/v) to afford compound 7-9.
4.1.3.1 2-(3-(Benzyl(methyl)amino)propyl)isoindoline-1,3-dione (7a)
Compound 7a was synthesized from 3 and 6a according to the general procedure; colorless oil;
1
yield: 80.0%; H NMR (400 MHz, CDCl3) δ 7.84-7.82 (m, 2H), 7.72-7.69 (m, 2H), 7.29-7.23 (m,
5H), 3.76 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.17 (s, 3H), 1.92-1.88 (m, 2H).
4.1.3.2 2-(4-(Ethyl(2-methoxybenzyl)amino)propyl)isoindoline-1,3-dione (7b)
Compound 7b was synthesized from 3 and 6d according to the general procedure; colorless oil;
1
yield: 85.3%; H NMR (400 MHz, CDCl3)  7.84-7.82 (m, 2H), 7.72-7.69 (m, 2H), 7.43 (d, J = 7.2
Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 6.90 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.72
(t, J = 6.8 Hz, 2H), 3.70 (s, 2H), 2.65-2.57 (m, 4H), 1.96-1.91 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H).
4.1.3.3 2-(4-(Benzyl(methyl)amino)butyl)isoindoline-1,3-dione (8a)
Compound 8a was synthesized from 4 and 6a according to the general procedure; colorless oil;
1
yield: 71.5%; H NMR (400 MHz, CDCl3)  7.85-7.83 (m, 2H), 7.71-7.70 (m, 2H), 7.29-7.22 (m,
5H), 3.69 (t, J = 7.2 Hz, 2H), 3.51 (s, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.19 (s, 3H), 1.72-1.68 (m, 2H),
1.60-1.56 (m, 2H).
4.1.3.4 2-(4-(Benzyl(ethyl)amino)butyl)isoindoline-1,3-dione (8b)
Compound 8b was synthesized from 4 and 6b according to the general procedure; colorless oil;
1
yield: 81.8%; H NMR (400 MHz, CDCl3)  7.85-7.83 (m, 2H), 7.73-7.71 (m, 2H), 7.33 (d, J = 6.8
Hz, 2H), 7.29 (t, J = 6.8 Hz, 2H), 7.22 (t, J = 6.8 Hz, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.58 (s, 2H),
20

2.57-2.46 (m, 4H), 1.71-1.66 (m, 2H), 1.56-1.52 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H).
4.1.3.5 2-(4-((2-Methoxybenzyl)(methyl)amino)butyl)isoindoline-1,3-dione (8c)
Compound 8c was synthesized from 4 and 6c according to the general procedure; colorless oil;
1
yield: 71.8%; H NMR (400 MHz, CDCl3)  7.85-7.83 (m, 2H), 7.73-7.71 (m, 2H), 7.30 (d, J = 7.2
Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.71
(t, J = 6.8 Hz, 2H), 3.49 (s, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.20 (s, 3H), 1.74-1.69 (m, 2H), 1.62-1.59
(m, 2H).
4.1.3.6 2-(4-(Ethyl(2-methoxybenzyl)amino)butyl)isoindoline-1,3-dione (8d)
Compound 8d was synthesized from 4 and 6d according to the general procedure; colorless oil;
1
yield: 87.2%; H NMR (400 MHz, CDCl3)  7.84-7.81 (m, 2H), 7.72-7.69 (m, 2H), 7.39 (d, J = 7.2
Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.68
(t, J = 6.8 Hz, 2H), 3.57 (s, 2H), 2.53-2.47 (m, 4H), 1.73-1.66 (m, 2H), 1.56-1.51 (m, 2H), 1.04 (t, J
= 7.2 Hz, 3H).
4.1.3.7 2-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butyl)isoindoline-1,3-dione(8e)
Compound 8e was synthesized from 4 and 6e according to the general procedure; colorless oil;
1
yield: 84.0%; H NMR (400 MHz, CDCl3) δ 7.84-7.82 (m, 2H), 7.71-7.69 (m, 2H), 7.56 (d, J = 7.2
Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.06-7.00 (m, 2H), 3.67 (t, J = 7.2 Hz, 2H), 3.60 (s, 2H), 2.66 (s,
6H), 2.50-2.45 (m, 4H), 1.70-1.67 (m, 2H), 1.53-1.49 (m, 2H), 1.02 (t, J = 6.8 Hz, 3H).
4.1.3.8 2-(4-((4-(Dimethylamino)benzyl)(ethyl)amino)butyl)isoindoline-1,3-dione(8f)
Compound 8f was synthesized from 4 and 6f according to the general procedure; colorless oil;
1
yield: 77.1%; H NMR (400 MHz, CDCl3) δ 7.84-7.82 (m, 2H), 7.71-7.69 (m, 2H), 7.18 (d, J = 8.8
Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 3.68 (t, J = 7.2 Hz, 2H), 3.52 (s, 2H), 2.92 (s, 6H), 2.53-2.46 (m,
4H), 1.70-1.66 (m, 2H), 1.57-1.53 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H).
4.1.3.9 2-(4-(2,3-Dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)butyl)isoindoline-1,3-dione (8g)
Compound 8g was synthesized from 4 and 6g according to the general procedure; colorless oil;
1
yield: 81.3%; H NMR (400 MHz, CDCl3) δ 7.85-7.82 (m, 2H), 7.73-7.70 (m, 2H), 7.19-7.11 (m,
2H), 7.00-6.95 (m, 2H), 4.04 (t, J = 4.4 Hz, 2H), 3.81 (s, 1H), 3.71 (t, J = 7.2 Hz, 2H), 3.07 (t, J = 4.4
Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H), 1.73-1.67 (m, 2H), 1.60-1.54 (m, 2H).
4.1.3.10 2-(4-((2-Methoxybenzyl)(prop-2-yn-1-yl)amino)butyl)isoindoline-1,3-dione (8h)
Compound 8h was synthesized from 4 and 6h according to the general procedure; colorless oil;
21

1
yield: 81.2%; H NMR (400 MHz, CDCl3) δ 7.85-7.83 (m, 2H), 7.72-7.70 (m, 2H), 7.35 (d, J = 7.2
Hz, 1H ), 7.22 (t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H),
3.71 (t, J = 7.2 Hz, 2H), 3.65 (s, 2H), 3.36 (s, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.23 (s, 1H), 1.78-1.71
(m, 2H), 1.63-1.57 (m, 2H).
4.1.3.11 2-(6-(Benzyl(methyl)amino)hexyl)isoindoline-1,3-dione (9a)
Compound 9a was synthesized from 5 and 6a according to the general procedure; colorless oil;
1
yield: 67.5%; H NMR (400 MHz, CDCl3)  7.85-7.82 (m, 2H), 7.73-7.70 (m, 2H), 7.29-7.23 (m,
5H), 3.67 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 2.36 (q, J = 7.2 Hz, 2H), 2.18 (s, 3H), 1.69-1.66 (m, 2H),
1.55-1.49 (m, 2H), 1.38-1.31 (m, 4H).
4.1.3.12 2-(6-(Ethyl(2-methoxybenzyl)amino)hexyl)isoindoline-1,3-dione (9b)
Compound 9b was synthesized from 5 and 6d according to the general procedure; colorless oil;
1
yield: 67.5%; H NMR (400 MHz, CDCl3)  7.85-7.83 (m, 2H), 7.72-7.70 (m, 2H), 7.40 (d, J = 8.0
Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 3.81 (s, 3H), 3.66
(t, J = 7.2 Hz, 2H), 3.58 (s, 2H), 2.52 (q, J = 6.8 Hz, 2H), 2.44 (t, J = 6.4 Hz, 2H), 1.68-1.65 (m, 2H),
1.50-1.47 (m, 2H), 1.33-1.31 (m, 4H), 1.04 (t, J = 6.8 Hz, 3H)
4.1.4 General procedure for the synthesis of 10-12
To a solution of N-aminoalkylphthalimide derivatives 7-9 (4mmol) in ethanol (50ml) was
added hydrazine hydrate (1.23ml, 20 mmol). The mixture was refluxed for 6 h. Then the solvent was
removed under reduced pressure. The residue was diluted with 5% aqueous sodium hydroxide
solution (20mL) and the mixture was stirred for 0.5 h. Then the water (30mL) was added and the
mixture was extracted with dichloromethane (30mL×3). The combined organic phases were washed
with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give
compounds 10-12 as colorless oil, which were used without further purification.
4.1.5 General procedure for the synthesis of (N-aminoalkyl) salicylamide derivatives 13-15
The mixture of salicylic acid (50.0 mg, 0.362 mmol), the corresponding (N-aminoalkyl)
salicylamide derivatives 10-12 (0.434 mmol) and EDCI (104 mg, 0.543 mmol) in THF (2 mL) was
stirred at room temperature overnight. The solvent was removed under reduced pressure and the
residue was diluted with dichloromethane (5mL). The organic phase was washed with saturated
solution of NaHCO3 and brine, dried over sodium sulfate, filtered and concentrated under reduced
pressure to give the corresponding crude product, which was purified on a silica gel chromatography
22

using mixtures of CH2Cl2/acetone as eluent, obtaining the corresponding (N-aminoalkyl)
salicylamide derivatives 13-15.
4.1.5.1 N-(3-(Benzyl(methyl)amino)propyl)-2-hydroxybenzamide (13a)
Compound 13a was synthesized from salicylic acid and 10a according to the general procedure;
purification: CH2Cl2/acetone (1/4, v/v); colorless oil; yield: 63.0%; MS (ESI) m/z 299.1 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 12.73 (brs, 1H), 8.83 (brs, 1H), 7.35-7.26 (m, 6H), 7.13-7.11 (m, 1H),
6.95 (d, J = 8.4 Hz, 1H), 6.63 (t, J = 7.6 Hz, 1H), 3.61-3.57 (m, 4H), 2.70 (t, J = 5.2 Hz, 2H), 2.31 (s,
13
3H), 1.85-1.80 (m, 2H); C NMR (100 MHz, CDCl3) δ 169.8, 161.5, 137.2, 133.6, 129.6, 128.5,
127.6, 125.8, 118.4, 118.1, 114.4, 63.1, 57.2, 41.2, 40.1, 24.5.
4.1.5.2 N-(3-(Ethyl(2-methoxybenzyl)amino)propyl)-2-hydroxybenzamide (13b)
Compound 13b was synthesized from salicylic acid and 10b according to the general procedure;
purification: CH2Cl2/acetone (1/1, v/v); colorless oil; yield: 64.2%; MS (ESI) m/z 343.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 12.88 (brs, 1H), 9.30 (brs, 1H), 7.34-7.26 (m, 4H), 6.95-6.90 (m, 2H),
6.80 (d, J = 8.0 Hz, 1H), 6.49 (brs, 1H), 3.70 (s, 2H), 3.61 (s, 3H), 3.56 (t, J = 5.2 Hz, 2H), 2.80-2.70
(m, 4H), 2.00-1.85 (m, 2H), 1.08 (brs, 3H); 13C NMR (100 MHz, CDCl3) δ 169.8, 161.4, 157.8,
133.3, 131.9, 129.3, 126.2, 124.4, 120.2, 118.2, 117.9, 114.3, 110.5, 54.8, 53.1, 52.7, 46.7, 40.6, 23.8,
9.8.
4.1.5.3 N-(4-(Benzyl(methyl)amino)butyl)-2-hydroxybenzamide (14a)
Compound 14a was synthesized from salicylic acid and 11a according to the general procedure;
purification: CH2Cl2/acetone (3/1, v/v); colorless oil; yield: 46.2%; MS (ESI) m/z 313.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 12.52 (brs, 1H), 7.85 (brs, 1H), 7.41-7.28 (m, 7H), 6.96 (d, J = 8.4 Hz,
1H), 6.71 (t, J = 7.6 Hz, 1H), 3.65 (s, 2H), 3.50-3.44 (m, 2H), 2.58-2.52 (m, 2H), 2.27 (s, 3H),
13
1.78-1.70 (m, 4H); C NMR (100 MHz, CDCl3) δ 170.1, 161.3, 136.6, 133.7, 129.4, 128.4, 127.6,
125.9, 118.4, 118.1, 114.5, 61,5, 56.3, 41.9, 39.1, 26.6, 24.3.
4.1.5.4 N-(4-(Benzyl(ethyl)amino)butyl)-2-hydroxybenzamide (14b)
Compound 14b was synthesized from salicylic acid and 11b according to the general procedure;
purification: CH2Cl2/acetone (3/1, v/v); colorless oil; yield: 56.8%; MS (ESI) m/z 327.3 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 12.47 (brs, 1H), 7.37-7.26 (m, 7H), 7.17 (brs, 1H), 6.96 (d, J = 8.0 Hz,
1H), 6.77 (t, J = 7.6 Hz, 1H), 3.65 (s, 2H), 3.46-3.40 (m, 2H), 2.62-2.52 (m, 4H), 1.72-1.62 (m, 4H),
13
1.08 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 169.9, 161.2, 137.6, 133.8, 129.2, 128.3,
23

127.3, 125.9, 118.4, 118.1, 114.5, 57.5, 52.1, 47.0, 39.1, 26.9, 24.1, 10.7.
4.1.5.5 2-Hydroxy-N-(4-((2-methoxybenzyl)(methyl)amino)butyl)benzamide (14c)
Compound 14c was synthesized from salicylic acid and 11c according to the general procedure;
purification: CH2Cl2/acetone (2/1, v/v); colorless oil; yield: 43.1%; MS (ESI) m/z 343.0 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 8.59 (brs, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.34-7.28 (m, 3H), 6.97-6.86 (m,
3H), 6.56 (t, J = 7.2 Hz, 1H), 3.76 (s, 3H), 3.71 (s, 2H), 3.45 (t, J = 5.6 Hz, 2H), 2.62 (t, J = 6.0 Hz,
2H), 2.26 (s, 3H), 1.84-1.78 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 170.0, 161.2, 157.8, 133.5,
132.0, 129.5, 126.3, 123.4, 120.3, 118.2, 117.8, 114.6, 110.4, 56.4, 55.2, 55.0, 42.0, 38.7, 26.2, 23.9.
4.1.5.6 N-(4-(Ethyl(2-methoxybenzyl)amino)butyl)-2-hydroxybenzamide (14d)
Compound 14d was synthesized from salicylic acid and 11d according to the general procedure;
purification: CH2Cl2/acetone (1/1, v/v); colorless oil; yield: 46.5%; MS (ESI) m/z 357.1 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 8.22 (brs, 1H), 7.57 (brs, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.32 (t, J = 8.0
Hz, 2H), 6.98-6.87 (m, 3H), 6.69 (t, J = 7.2 Hz, 1H), 3.88 (s, 2H), 3.80 (s, 3H), 3.46 (t, J = 6.0 Hz,
2H), 2.80-2.72 (m, 4H), 1.86-1.73 (m, 4H), 1.16 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
170.1, 161.2, 157.8, 133.6, 131.8, 129.8, 126.6, 122.4, 120.5, 118.3, 117.8, 114.5, 110.5, 55.2, 51.9,
51.0, 47.3, 38.5, 26.4, 23.2, 9.6.
4.1.5.7 N-(4-((2-(Dimethylamino)benzyl)(ethyl)amino)butyl)-2-hydroxybenzamide (14e)
Compound 14e was synthesized from salicylic acid and 11e according to the general procedure;
purification: CH2Cl2/acetone (3/2, v/v); colorless oil; yield: 73.7%; MS (ESI) m/z 370.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 7.93 (brs, 1H), 7.73 (d, J = 6.4 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H),
7.34-7.26 (m, 2H), 7.15 (d, J = 8.0 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.79 (t,
J = 7.6 Hz, 1H), 3.98 (s, 2H), 3.45 (t, J = 6.0 Hz, 2H), 2.80-2.75 (m, 4H), 2.64 (s, 6H), 1.80-1.77 (m,
2H), 1.69-1.66 (m, 2H), 1.15 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.0, 161.0, 153.1,
133.5, 130.8, 129.0, 128.7, 126.8, 124.0, 119.7, 118.4, 117.7, 114.6, 52.1, 51.8, 47.1, 45.1, 38.3, 26.3,
22.7, 9.6.
4.1.5.8 N-(4-((4-(Dimethylamino)benzyl)(ethyl)amino)butyl)-2-hydroxybenzamide (14f)
Compound 14f was synthesized from salicylic acid and 11f according to the general procedure;
purification: CH2Cl2/acetone (3/2, v/v); pale yellow oil; yield: 41.4%; MS (ESI) m/z 370.1 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 10.20 (brs, 1H), 8.20 (brs, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.34 (t, J =
8.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.80 (t, J = 8.0 Hz, 1H), 6.66 (d, J =
24

8.4 Hz, 2H), 3.79 (s, 2H), 3.46 (t, J = 6.0 Hz, 2H), 2.93 (s, 6H), 2.81-2.70 (m, 4H), 1.84-1.77 (m, 2H),
1.73-1.67 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.1, 161.2, 150.4,
133.6, 131.0, 126.7, 119.9, 118.5, 117.9, 114.6, 112.2, 56.3, 51.2, 46.3, 40.3, 40.2, 38.4, 26.4, 22.7,
9.6.
4.1.5.9 N-(4-(2,3-Dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)butyl)-2-hydroxybenzamide (14g)
Compound 14g was synthesized from salicylic acid and 11g according to the general procedure;
1
purification: CH2Cl2/acetone (3/1, v/v); colorless oil; yield:50.4%; MS (ESI) m/z 341.2 [M+H]+; H
NMR (400 MHz, CDCl3) δ 12.44 (brs, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.20 (t,
J = 8.0 Hz, 1H), 7.13 (d, J = 6.4 Hz, 1H), 7.03-6.98 (m, 3H), 6.78 (t, J = 8.0 Hz, 1H), 4.07 (t, J = 4.4
Hz, 2H), 3.88 (s, 2H), 3.50-3.42 (m, 2H), 3.12 (t, J = 4.4 Hz, 2H), 2.53 (t, J = 6.8 Hz, 2H), 1.76-1.62
(m, 4H); 13C NMR (100 MHz, CDCl3) δ 169.9, 161.2, 159.6, 133.8, 130.7, 130.6, 128.7, 125.6,
123.4, 120.7, 118.5, 118.2, 114.4, 69.2, 57.9, 57.8, 52.7, 39.3, 26.9, 24.7.
4.1.5.10 2-Hydroxy-N-(4-((2-methoxybenzyl)(prop-2-yn-1-yl)amino)butyl)benzamid (14h)
Compound 14h was synthesized from salicylic acid and 11h according to the general procedure;
purification: CH2Cl2/acetone (25/1, v/v); colorless oil; yield:33.1%; MS (ESI) m/z 367.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 12.61 (brs, 1H), 7.82 (brs, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.31-7.25 (m,
2H), 7.09 (d, J = 8.0 Hz, 1H), 6.97-6.90 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.49 (t, J = 7.6 Hz, 1H),
3.75 (s, 3H), 3.69 (s, 2H), 3.44 (q, J = 5.6 Hz, 2H), 3.26 (d, J = 2.4 Hz, 2H), 2.67 (t, J = 6.0 Hz, 1H),
2.26 (t, J = 2.4 Hz, 1H), 1.83-1.69 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 169.9, 161.4, 157.8,
133.6, 131.4, 129.0, 125.6, 125.5, 120.3, 118.2, 118.2, 114.5, 110.4, 78.1, 73.8, 55.0, 52.9, 51.1, 42.4,
39.2, 26.5, 24.8.
4.1.5.11 N-(6-(Benzyl(methyl)amino)hexyl)-2-hydroxybenzamide (15a)
Compound 15a was synthesized from salicylic acid and 12a according to the general procedure;
1
purification: CH2Cl2/acetone (2/1, v/v); colorless oil; yield:65.0%; MS (ESI) m/z 341.3 [M+H]+; H
NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.6 Hz, 1H), 7.39-7.26 (m, 6H), 6.97 (d, J = 8.4 Hz, 1H),
6.84 (t, J = 7.6 Hz, 1H), 6.59 (brs, 1H), 3.61 (s, 2H), 3.43 (q, J = 6.4 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H),
13
2.28 (s, 3H), 1.64-1.59 (m, 4H), 1.42-1.34 (m, 4H); C NMR (100 MHz, CDCl3) δ 169.7, 160.9,
137.4, 133.8, 129.3, 128.2, 127.2, 126.0, 118.6, 118.2, 114.8, 61.8, 56.8, 41.6, 39.4, 29.2, 26.7, 26.5.
4.1.5.12 N-(6-(Ethyl(2-methoxybenzyl)amino)hexyl)-2-hydroxybenzamide (15b)
Compound 15b was synthesized from salicylic acid and 12b according to the general procedure;
25

1
purification: CH2Cl2/acetone (2/1, v/v); colorless oil; yield:43.0%; MS (ESI) m/z 385.2 [M+H]+; H
NMR (400 MHz, CDCl3) δ 7.46 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H), 7.26-7.23 (m, 1H),
6.98-6.91 (m, 2H), 6.88-6.82 (m, 2H), 6.69 (brs, 1H), 3.82 (s, 3H), 3.74 (s, 2H), 3.46-3.41 (m, 2H),
2.67-2.63 (m, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.64-1.59 (m, 4H), 1.42-1.32 (m, 4H), 1.13 (t, J = 6.8 Hz,
13
3H); C NMR (100 MHz, CDCl3) δ 169.7, 161.1, 157.7, 133.7, 130.9, 128.6, 126.2, 125.0, 120.4,
118.5, 118.2, 114.9, 110.3, 55.3, 52.7, 50.9, 47.3, 39.4, 29.1, 26.7, 26.5, 25.8, 10.8.
4.2 Biological evaluation
4.2.1 Inhibition experiments of AChE and BuChE
To determine the in vitro cholinesterase activities of the compounds, the spectrophotometric
method of Ellman was performed [32, 33], using AChE from 5% rat cortex homogenate, purified
AChE from Electrophorus electricus (Sigma-Aldrich Co.) and BuChE from rat serum. The brain
homogenate was preincubated with tetraisopropyl pyrophosphoramide (iso-OMPA, selective
inhibitor of BuChE, 4.0 mmol/L, Sigma-Aldrich Co.) for 5 min before use. For rat AChE or BuChE
inhibition assays, the reaction mixture (100 µL) consisted of acetylthiocholine iodide (1 mmol/L, 30
µL) (J&K Scientific) or butyrylthiocholine iodide (1 mmol/L, 30 µL) (TCI Shanghai Development),
phosphate-buffered solution (0.1 mmol/L, pH = 7.40, 40 µL), 5% homogenate or 25% serum (10 µL)
and different concentrations of test compounds (20 µL). The mixture was incubated at 37 °C for 15
min, followed by the addition of 5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) (J&K
Scientific) to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Changes in absorbance were
detected at 405 nm in a Varioskan Flash Multimode Reader (Thermo Scientific). For EeAChE
inhibition assay, EeAChE (0.05 U/mL, final concentration) was used and the assay was carried out in
a phosphate buffer (0.01 mmol/L, pH = 8.00). Changes in absorbance were detected at 412 nm. The
other procedure was the same as above. The inhibitory activities of compounds toward AChE or
BuChE would reduce the color generation and IC50 values were calculated as the concentration of
compound that produces 50% AChE or BuChE activity inhibition. Donepezil was applied as the
positive drug. All samples were assayed in triplicate.
4.2.2 Kinetic study for the inhibition of AChE
Kinetic characterization of AChE inhibition was carried out based on a reported method using
purified AChE from E. Electricus (EeAChE) [34]. The assay solution (100 μL) consists of 0.1 M
26

phosphate buffer (pH 8.00), with the addition of 30 μL of 0.2% DTNB, 10 μL of 0.5 U/mL EeAChE,
and 20 μL of substrate (ATCh). Three different concentrations of inhibitors were added to the assay
solution and pre-incubated with the EeAChE for 15 min at 37°C, followed by the addition of
substrate in different concentrations. Kinetic characterization of the hydrolysis of ATCh catalyzed by
EeAChE was done spectra-metrically at 274 nm. The parallel control experiments were performed
without inhibitor. The plots were assessed by a weighted least square analysis that assumed the
variance of v to be a constant percentage of v for the entire data set. Slopes of reciprocal plots were
then plotted against the concentration of 15b in a weighted analysis, and Ki was determined as the
intercept on the negative x-axis.
4.2.3 Molecular modeling study
The crystal structure of AChE complexed with donepezil (PDB code: 1EVE) was obtained from
the Protein Data Bank after removing the original inhibitors and water molecules [35]. The 3D
Structure of 15b was built and performed geometry optimization by molecular mechanics. After
addition of Gasteiger charges, removal of hydrogen atoms, addition of their atomic charges to
skeleton atoms, and the assignment of proper atomic types, the further preparation of the inhibitor
was accomplished. Autotors was then used to define the rotatable bonds in the ligands. Docking
studies were performed using the AUTODOCK 4.2 program. By using Autodock Tools (ADT;
version 1.5.6), polar hydrogen atoms were added to amino acid residues, and Gasteiger charges were
assigned to all atoms of the enzyme. The resulting enzyme structure was used as an input for the
AUTOGRID program. AUTOGRID performed a pre-calculated atomic affinity grid maps for each
atom type in the ligand, plus an electrostatics map and a separate desolvation map presented in the
substrate molecule. All maps were calculated with 0.375 Å spacing between grid points. The center
of the grid box was placed at the center of donepezil with coordinates x = 2.023, y = 63.295, z =
67.062. The dimensions of the active site box were set at 60 × 60 × 60 Å. Flexible ligand docking
was performed for the compounds. Each docked system was performed by 100 runs of the
AUTODOCK search by the Lamarckian genetic algorithm (LGA). Other than the referred
parameters above, the other parameters were accepted as default. A cluster analysis was performed
on the docking results using a root mean square (RMS) tolerance of 1.0 and the lowest energy
conformation of the highest populated cluster was selected for analysis. Graphic manipulations and
visualizations were done by Autodock Tools or Discovery Studio 2.5 software.
27

4.2.4 Antioxidant activity assay
The antioxidant potency was evaluated by the oxygen radical absorbance capacity-fluorescein
(ORAC-FL) assay [36]. Fluorescein (FL) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid (Trolox) were purchased from TCI (Shanghai) Development. 2,2’-Azobis(amidinopropane)
dihydrochloride (AAPH) was purchased from Accela ChemBio Co. Ltd. All the assays were
performed with 75 mM phosphate buffer (pH = 7.40), and the final reaction mixture was 200 μL. FL
(120 μL, 150 nM final concentration) and antioxidant (20 μL) were added in the wells of a black
96-well plate using Trolox as a standard (1-8 μM, final concentration). The plate was incubated for
15 min at 37 °C and then placed in a Varioskan Flash Multimode Reader (Thermo Scientific). AAPH
solution (60 μL, 12 mM final concentration) was added rapidly using an autosampler and the
fluorescence recorded every minute for 90 min with excitation at 485 nm and emission at 535 nm.
The plate was automatically shaken prior to each reading. A blank (FL + AAPH) using phosphate
buffer instead of antioxidant and Trolox calibration were carried out in each assay. The samples were
measured at different concentrations (1-10 μM). All the reaction mixture was prepared in duplicate,
and at least three independent assays were performed for each sample. Antioxidant curves
(fluorescence versus time) were normalized to the curve of the blank in the same assay, and then the
area under the fluorescence decay curve (AUC) was calculated. The net AUC of a sample was
obtained by subtracting the AUC of the blank. ORAC-FL values were expressed as Trolox
equivalents by using the standard curve calculated for each sample, where the ORAC-FL value of
Trolox was taken as 1, indicating the antioxidant potency of the tested compounds.
4.2.5 Metal-chelating studies [37, 38]
The chelating studies were performed using a Varioskan Flash Multimode Reader (Thermo
Scientific). The UV absorption spectra of tested compound alone or in the presence of CuCl2, ZnCl2,
AlCl3, and FeSO4 were recorded with wavelength ranging from 200 to 600 nm after incubating for
30 min in methanol at room temperature. The final volume of reaction mixture was 200 μL, and the
final concentrations of tested compound and metals were 37.5 μM. The difference UV-vis spectra
due to complex formation were obtained by numerical subtraction of the spectra of the metal alone
and the compound alone from the spectra of the mixture.
The stoichiometry of the compound-Cu2+ complex was determined by titrating the methanol
solution of tested compound with ascending of CuCl2. The final concentration of tested compound
28

was 37.5 μM, and the final concentration of CuCl2 ranged from 3.75 to 150 μM. The UV spectra
were recorded and treated by numerical subtraction of CuCl2 and tested compound at corresponding
concentrations, plotted versus the mole fraction of tested compound.
4.2.6 Inhibition of self- and Cu2+-induced Aβ1-42 aggregation
In order to investigate the self-induced Aβ1-42 aggregation, a Thioflavin T-based fluorometric
assay was performed [39-42]. Thioflavin T (ThT) was purchased from TCI (Shanghai) Development.
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from Energy Chemical. β-Amyloid1-42
(Aβ1-42), supplied as trifluoroacetate salt, was purchased from GL Biochem Ltd. Briefly, Aβ1-42 was
dissolved in HFIP (1 mg/mL) and incubation for 24 h at room temperature, then the solvent was
evaporated. And it was dissolved in dry DMSO to a final stock concentration of 200 μM and kept
frozen at -80 °C. Tested compounds were dissolved in DMSO in 2.5 mM for storage and diluted with
phosphate buffer solution (pH 7.4) before use. For the self-induced aggregation assay, Aβ1-42 (20 μL,
25 μM, final concentration) together with test compounds (20 μL, 25 μM, final concentration) were
incubated in 50 mM phosphate buffer solution (pH 7.40) at 37 °C for 24 h. The plate was sealed with
a transparent heat-resistant plastic film to minimize evaporation effect. After the incubation, 160 μL
of 5 μM ThT in 50 mM glycine-NaOH buffer (pH 8.5) was added. Each assay was run in triplicate.
Fluorescence was measured on a Varioskan Flash Multimode Reader (Thermo Scientific) with
excitation and emission wavelengths at 446 nm and 490 nm, respectively. The percent inhibition due
to the presence of the inhibitor was calculated by the following expression: (1-IFi/IFc) × 100, in
which IFi and IFc are the fluorescence intensities obtained for Aβ1-42 in the presence and in the
absence of inhibitors after subtracting the background, respectively.
For the Cu2+-induced Aβ1-42 aggregation assay, solutions of Cu2+ were prepared from standards
to concentration of 75 μM using the HEPES buffer (20 mM, pH 6.60, 150 mM NaCl). The Aβ1-42
stock solution was diluted with HEPES buffer (20 mM, pH 6.60, 150 mM NaCl). The mixture of
Aβ1-42 (20 μL, 25 μM, final concentration) and Cu2+ (20 μL, 25 μM, final concentration), with or
without the tested compound (20 μL, 25 μM, final concentration) was incubated at 37 °C for 24h.
Then, 190 μL of 5 μM ThT in 50 mM glycine-NaOH buffer (pH 8.50) was added. Each assay was
run in triplicate. The detection method was the same as that of self-induced Aβ1-42 aggregation assay.
4.2.7 Effect on the disaggregation of Aβ1-42 aggregation fibrils
For the disaggregation of self-induced Aβ fibrils experiment, the Aβ1-42 stock solution was
29