Synthesis, biological evaluation and molecular modeling of benzofuran piperidine derivatives as Aβ antiaggregant

Article abstract of DOI:10.1016/j.ejmech.2021.113541

A series of benzofuran piperidine derivatives were designed, synthesized and evaluated as multifunctional Aβ antiaggregant to treat Alzheimer's disease (AD). In vitro results revealed that all of them are very good Aβ antiaggregants and some of the compounds are potent acetylcholinesterase (AChE) inhibitors with moderate antioxidant property. Selected compounds were also tested for neuroprotection activity, LDH release, ATP production and inhibitory activity to prevent Aβ peptides binding to the cell membrane. The different modifications introduced in the structure of our lead compound 3 (hAChE IC₅₀ = 61 μM and self induced Aβ _25-35 aggregation 45.45%), to increase its activity toward AD related targets. The most interesting multifunctional Aβ antiaggregants were compounds 3a, 3h and 3i, highlighting 3h as potent Aβ antiaggregant and good antiacetylholinesterase inhibitor (self induced Aβ _25-35 aggregation 57.71% and hAChE IC₅₀ = 21 μM), with good neuroprotective and antioxidant activity. In addition, these three most promising compounds prevent intracellular reactive oxygen species (ROS) formation and cell apoptosis induced by Aβ_25-35 peptides in SH-SY5Y cells. Molecular docking studies were also accomplished to understand the binding interaction of these compounds on Aβ monomer, Aβ fibril and AChE. Based on all data, compounds 3a, 3h and 3i were concluded as potent multifunctional Aβ antiaggregant, useful candidate for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2021.113541

European Journal of Medicinal Chemistry 222 (2021) 113541
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, biological evaluation and molecular modeling of
benzofuran piperidine derivatives as Ab antiaggregant
Sharmin Reza Chowdhury a, Jinxin Gu a, Yixin Hu a, Juntao Wang a, Shuwen Lei a,
, b, c, *
Mojdeh S. Tavallaie a, Celine Lam a, Dan Lu a, Faqin Jiang a, Lei Fu a
a Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University (SJTU), Shanghai, China
b SJTU-Agilent Technologies Joint Laboratory for Pharmaceutical Analysis, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China
c Academy of Pharmacy, Xi'an Jiaotong e Liverpool University, Suzhou, 215123, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzofuran piperidine derivatives were designed, synthesized and evaluated as multifunc-
Received 5 April 2021
Received in revised form
3 May 2021
Accepted 8 May 2021
Available online 25 May 2021
tional A
very good A
hibitors with moderate antioxidant property. Selected compounds were also tested for neuroprotection
activity, LDH release, ATP production and inhibitory activity to prevent A peptides binding to the cell
membrane. The different modifications introduced in the structure of our lead compound 3 (hAChE
IC50 ¼ 61 M and self induced A aggregation 45.45%), to increase its activity toward AD related
targets. The most interesting multifunctional A
lighting 3h as potent A antiaggregant and good antiacetylholinesterase inhibitor (self induced A
M), with good neuroprotective and antioxidant activity. In
b
antiaggregant to treat Alzheimer's disease (AD). In vitro results revealed that all of them are
b
antiaggregants and some of the compounds are potent acetylcholinesterase (AChE) in-
b
m
b
25-35
Keywords:
Benzofuran piperidine
Alzheimer's disease
Ab antiaggregant
Anti acetylcholinesterase
Neuroprotective
b
antiaggregants were compounds 3a, 3h and 3i, high-
b
b
25-35
aggregation 57.71% and hAChE IC50 ¼ 21
m
addition, these three most promising compounds prevent intracellular reactive oxygen species (ROS)
formation and cell apoptosis induced by Ab25-35 peptides in SH-SY5Y cells. Molecular docking studies
were also accomplished to understand the binding interaction of these compounds on A
fibril and AChE. Based on all data, compounds 3a, 3h and 3i were concluded as potent multifunctional A
b
monomer, A
b
b
antiaggregant, useful candidate for the treatment of AD.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
tangles in the brain, mainly due to the combination of
b-amyloid
(A ) aggregation and changes in tau ( ) protein leading to the
b
t
Alzheimer's disease (AD) has become a global problem on hu-
man wellbeing, with a rising incidence as people's longevity in-
creases [1]. AD is the most common cause of dementia and is a still
incurable, progressive neurodegenerative disorder [2]. AD was first
described by German psychiatrist and pathologist Alois Alzheimer
in 1906. It is a chronic neurodegenerative disease and impairment
in cognition accompanied by abnormal behavior and personality
changes, ultimately leading to full dementia [3]. Scientists describe
AD as a multi-factorial disease in which a complex of proteins,
enzymes, or receptors is involved. The pathogenesis of AD is not
completely clear, however neuro-pathological studies have shown
that the disease process is related with plaques and neurofibrillary
disintegration of microtubules and decreased levels of acetylcho-
line (ACh) in the brain [1e4].
Current treatment of AD is symptomatic, based on three
cholinesterase inhibitors (donepezil, galantamine, and riva-
stigmine) and memantine, affecting the glutamatergic system
(Fig. 1A). Unfortunately, none of these drugs stop the progressive
loss of neurons and so far, there is no treatment that can halt the
progressive deterioration of cognitive faculties in AD patients. As
the incidence rate of AD doubles every 5 years from age 65,
therefore, global burden of AD is expected to 106.8 million people
by 2050. In this consequence, it's become a greatest challenge for
modern medicine to the development of novel therapy to treat AD
[5,6].
The current strategy to discover novel anti-AD agents, aims to
use a multi-targeted approach where a specially designed com-
pound exhibit ability to target multiple pathological factors
* Corresponding author. Shanghai Key Laboratory for Molecular Engineering of
Chiral Drugs.
E-mail address: leifu@sjtu.edu.cn (L. Fu).
https://doi.org/10.1016/j.ejmech.2021.113541
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Fig. 1. Marketed drugs for the treatment of AD and design strategy for benzofuran piperidine derivatives.
involved in AD. This new concept is more rational approach,
especially for the complex and multifactorial disease like AD [7e9].
moiety, a dramatic decrease in potency for AChE inhibition was
observed [34].
Researchers believe that the A
and fundamental for the pathogenesis of AD. The development of
AD is driven by the accumulation and deposition of A peptide
aggregates (in oligomers and protofibrils form) in the brain
[10e18]. The neurotoxic forms of amyloid peptides (A oligomers)
b
hypothesis is the most important
For many years, our research group has been working in syn-
thesis and evaluation of different types of benzofuran derivatives
for long times [35e37]. In terms of AD research, we intent to design
and develop multifunctional small molecules, which are able to
b
b
stop Ab aggregation, restore acetylcholine (ACh) level and reduce
play various roles in AD pathology including, oxidative stress and
cellular metabolism [19,20]. Oxidative stress is included in all the
pathophysiological hypotheses for AD. The free radical and oxida-
tive stress theory of aging indicates that oxidative damage plays a
vital role in neuronal degeneration, and several studies have
demonstrated that the oxidative stress is an early occurring con-
dition in AD [21,22].
oxidative stress in AD brain. Specifically, we design a group of small
molecules include benzofuran ring with substitution at C-2 and C-3
positions since benzofurans were identified as A
b antiaggregants
and site interactions with A were recognized [30e32,38e40]
b
(Fig. 1B). First of all, C-2 position of Benzofuran ring was substituted
with piperidine ring and carry out the biological evaluation toward
AD related targets. We consider this 4-(Benzofuran-2-yl) piperidine
as a lead compound, which exhibited moderate activity toward AD
related targets. During designing and synthesizing the small library
of Benzofuran piperidine derivatives, we also consider the polar
surface area of the compounds which should be less than 70 Å2, so
that the molecule could pass through the blood brain barrier (BBB).
Considering this, further structural modifications of our lead
compound 4-(Benzofuran-2-yl) piperidine were carried out, to in-
crease its activity toward AD related targets. Substitution of C-2
position of Benzofuran was done to increase the inhibitory activity
towards AChE [33,34] and substitution of C-3 position of Benzo-
The benzofuran scaffold represents
a privileged structure
because of its presence in many biologically active natural and
therapeutic products. Biological activities of Benzofuran derivatives
includes antimicrobial, anti-inflammatory, antitubercular, anti-
tumor, cytotoxic, antiplasmodial, antioxidant, AChE inhibitory and
Ab antiaggregation activities [23e32]. Among various compounds
developed as AChE inhibitors, donepezil analogs have been studied
more extensively. The X-ray crystallography and docking studies
demonstrated that the presence of benzyl piperidine contributes its
inhibitory activity by interacting with the catalytic active site (CAS)
[33] and substituting N-benzylpiperidine with N-benzylpiperazine
furan was done to increase the inhibitory activity towards A
b
2

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
aggregation [32,38e40]. Our findings show that all synthesized
AChE-induced A
b
aggregation were also determined by the ThT
benzofuran piperidine derivatives demonstrate excellent A
aggregation activity and moderate AChE inhibitory and antioxidant
activities. These multifunctional A antiaggregation small mole-
b
anti
method, and a dual binding site inhibitor of AChE, donepezil, was
chosen as reference compound [44]. As shown in Table 1, most test
b
compounds exhibit effectively A
AChE-induced Ab25-35 aggregation with ranging from 17.02% to
56.49% at 100 M in comparison to donepezil (18.9%). The test
Compounds 3h, 3i and 3j showed stronger inhibitory activity with
b25-35 aggregation inhibition 53.91%, 52.12% and 56.49%
b anti-aggregating activities for
cules represent a new class of compounds to treat AD.
m
2. Results and discussion
A
respectively.
2.1. Chemistry
The synthesis of studied compounds 3a-3q was illustrated in
Scheme 1. Selective lithiation at the
a-position of commercially
2.3. Cholinesterase inhibition assay
available benzofuran 1 was achieved using n-BuLi at ꢀ78 ꢁC in the
presence of dry THF. The resulting benzofuran-2-yllithium reacted
with tert-butyl 4-oxopiperidine-1-carboxylate to afford 2. Treat-
ment of 2 with Et3SiH and TFA under reflux conditions resulted in
the removal of the benzylic hydroxyl group and the deprotection of
the Boc group, yielding 3 [41]. Compound 3 was the lead com-
pound. It was then reacted with - a) various aldehydes, presence of
NaBH(OAc)3 and TEA to form compound 3a-3f; and b) various
benzyl bromide, presence of dry DCM and TEA to form compound
3g-3h. To get Benzofuran piperidine derivatives with substitution
in C-3 position, compound 3a, 3b, 3c and 3e further treated with
Lewis acid SnCl4 and various benzoyl chloride using Friedel-Crufts
acylation to synthesized compound 3i-3q [42].
In vitro inhibitory activities of the test compounds 3-3q against
AChE and BChE enzymes were evaluated using Ellman's method
[45,46] (Table 1). As reported in Table 1, it indicates that all
designed derivatives were able to inhibit AChE with IC50 values in
the range of 5.07e98.55
inhibitory effect of donepezil (IC50 of 0.01
(IC50 of 6.5 M) used as reference drugs. The compounds demon-
strated much weaker inhibitory effects (almost an order of
magnitude) on BChE with the minimum IC50 values of 26.50
and 35.47 M observed for compounds 3m and 3e, respectively.
mM. The results were compared with the
mM) and rivastigmine
m
mM
m
Based on the inhibitory effects on AChE and BChE, a selectivity
index was calculated for those compounds demonstrating IC50
values less than 100
Benzofuran derivative with C-2 substitution with benzyl piperidine
3a (IC50 ¼ 5.07 M) and the second most potent inhibitor was C-2
M).
Substitution to the benzyl group led to a decrease in inhibitory
potency. Also benzofuran derivative with C-3 substitution with
mM. The most potent AChE inhibitor was the
2.2. Self-induced and AChE-induced A
b
anti aggregation
aggregation
m
substitution with 4-fluorobenzyl piperidine 3h (IC50 ¼ 21.30
m
The test compounds were examined for their A
inhibition activity. The result is summarized in Table 1. A thioflavin
T (ThT) fluorometric assay was carried out to assess the inhibitory
b
phenyl 3i (IC50
¼
41.27
mM) and 4-methoxyphenyl 3j
effects of the test compounds on self-induced A
b aggregation
(IC50 ¼ 48.30 M) led to decrease in inhibitory potency.
m
[43,44]. Curcumin, a well-known natural inhibitor for Ab25-35 self-
aggregation, was taken as reference compound. From the results,
it can be seen that these test compounds exhibit good inhibitory
activity (61.91%e19.9% at 100
(50.02% at 100 M). Among these test compounds, compounds 3a,
3i and 3h (55.4%, 61.91% and 57.71% at 100 M, respectively)
exhibited the best A anti-aggregating activity, which were com-
parable to that of curcumin. Current studies also suggested that the
PAS of AChE can bind to A and promote the formation of A fibrils.
Therefore, inhibition of AChE, especially for the inhibition of PAS of
AChE, may reduce A aggregation. A anti-aggregating activities for
m
M) compared with curcumin
2.4. Cytotoxicity
m
m
Cytotoxicity of the most interesting Ab anti-aggregating com-
b
pound (3a,3b,3e,3h,3i,3j) with AChE good inhibitory activities
(IC50 < 50 M) were determined by MTT assay in SH-SY5Y cells
[43,47]. At 10 M concentration, no cytotoxicity was observed with
m
b
b
m
any of the compounds except 3b. Percentage growth of the cells is
reported in Table 2.
b
b
Scheme 1. Synthesis of the studied compounds.
3

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Table 1
Inhibition of self-induced and AChE induced Ab25-35 aggregation and in vitro Cholinesterase inhibition data for test compounds.
Comp
R1
H
R2
Inhibition % SE
Cholinesterase inhibitionc
hAChE IC50 hBChEIC50
Self induced A
b
AChE induced Ab 25-35
Selectivity for
AChEd
25-35
aggregation (%) SEa
aggregation (%) SEb
(
m
M) SE M) SE
(m
3
3a
H
H
45.45 1.3
55.40 2.8
15.22 2.46
40.41 2.95
61.18 3.22
5.07 0.12
64.390 5.600 1
54.260 1.273 10.8
3b
3c
H
H
48.28 1.29
14.67 1.39
34.82 0.89
11.35 3.9
34.89 2.12
52.78 0.77
64.620 2.390 1.8
64.595 2.694 1.2
3d
3e
H
H
8.040 3.2
51.80 5.2
49.73 2.3
92.81 2.16
23.90 2.33
> 100
-
49.79 5.42
35.470 6.70 0.04
3f
H
H
H
19.90 2.7
24.40 3.5
57.71 1.3
61.91 2.3
22.79 4.7
17.04 8.6
53.91 2.76
52.12 2.3
64.23 2.82
98.55 5.72
21.30 1.76
41.27 0.47
> 100
> 100
-
-
3g
3h
3i
55.755 2.058 2.6
> 100
-
3j
47.43 2.3
56.49 4.8
48.30 0.16
45.31 2.08
52.105
33.870
1.07
-
3k
42.93 1.89
37.03 5.15
> 100
3l
54.37 2.8
39.41 4.5
13.24 1.6
28.55 1.5
67.96 3.04
58.19 0.34
62.230 1.923 0.9
3m
26.508
33.350
0.45
4

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Table 1 (continued)
Comp
R1
R2
Inhibition % SE
Cholinesterase inhibitionc
Self induced A
b
AChE induced Ab 25-35
hAChE IC50
M) SE
hBChEIC50
M) SE
Selectivity for
AChEd
25-35
aggregation (%) SEa
aggregation (%) SEb
(
m
(m
3n
37.41 4.5
21.03 3.14
55.41 0.24
66.310
20.223
1.19
3o
45.95 2.08
19.36 3.8
60.98 1.22
61.659 1.386 1.01
3p
3q
42.22 1.2
45.03 1.7
17.02 5.6
97.13 10.1
59.73 0.66
62.595
12.975
0.6
29.41 0.43
59.655 3.118 0.99
Curcumin
Donepezil
Rivastigmie
49.90 1.2
nd
nd
nd
18.9 3.9
nd
nd
nd
0.01 0.006
6.5 0.21
0.117 0.080 11.7
0.30 0.01
0.36
nd ¼ not determined.
a
Self-induced A
presence of 100 M test compound.
AChE-induced A 25-35 aggregation (means SD of three experiments). The thioflavin-T fluorescence method was used, and the measurements were carried out in the
presence of 100 Mtest compound.
b
aggregation (means SD of three experiments). The thioflavin-T fluorescence method was used, and the measurements were carried out in the
25-35
m
b
b
m
c
IC50 values of test compounds represent the concentration that caused 50% enzyme activity loss.
Selectivity for AChE is defined as IC50 (BChE)/IC50 (AChE).
d
Table 2
Cytotoxicity, Neuroprotection, LDH release, Luminescent Cell Viability (ATP%) and DPPH scavenging activity of compounds.
Comp
Cell viability (%)b
Neuronal viability (%)b
LDH release (%)c
ATP (%)c
% DPPH Scavengingd
3a
3b
3e
3h
105.0 0.78
96.4 2.4
102.3 2.4
101.00 0.56
106.6 0.27
106.8 2.6
100
88.91 3.81
nd
294.64 7.8
nd
324.83 4.8
287.37 7.7
316.05 9.8
285 4.7
100
101.59 0.41
nd
102.53 0.45
104.56 0.71
95.49 0.41
100.79 0.41
100
18.19 1.0
12.18 1.06
11.86 0.64
10.81 1.08
21.64 1.4
17.62 0.31
nd
79.09 4.6
80.8 5.5
76.06 4.0
75.17 2.19
100
3i
3j
Controla
A
b25-35
nd
68.66 0.11
45.95 1.5
60.82 4.1
nd
nd
nd
364.06 2.15
90.95 0.82
nd
nd
nd
Curcumin
Donepezil
Trolox
Ferulic acid
Resveratrol
60.04 1.8
101.11 1.1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
55.54 0.6
50.04 0.58
45.71 0.77
a
Control of each assay was individual as untreated.
b
c
All the compound, Ab25-35peptide and the positive controls were tested at a concentration of 10
All the compound and Ab25-35 peptide were tested at a concentration of 10 M.
All the compound and the positive controls were tested at a concentration of 50
mM.
m
d
mM.
2.5. Neuroprotection
and donepezil (60.82%). Also as reported in Fig. 2, treatment of SH-
SY5Y cells with compounds 3a and 3h at 1e10 M significantly
m
Neuroprotective effects of the most interesting
aggregating compound (3a, 3b, 3e, 3h, 3i and 3j) at 10
A
m
b
anti-
reduced the neuronal viability loss induced by Ab25-35 peptide, in a
dose dependent manner.
M con-
centration were determined against the Ab25-35 peptide induced
toxicity in human neuronal SH-SY5Y cells by the MTT assay (Table 2
and Fig. 2) [32,48]. The result in Table 2 showed that compound 3a
and 3h each had the highest capability protective (88.91% and
80.8%) with cell viability of 105% and 101%, respectively. The pro-
tective effect of these compounds against Ab25-35 peptide induced
cell death was significantly higher compared to curcumin (45.95%)
2.6. Determination of cytoplasmic lactate dehydrogenase (LDH)
release induced by Ab25-35 peptideDetermination of cytoplasmic
lactate dehydrogenase (LDH) release induced by Ab25-35
The protective effect of most interesting compounds (3a, 3b, 3e,
3h, 3i, 3j) at 10 mM concentration against Ab25ꢀ35 induced cell death
5

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Fig. 2. Effects of selected compounds on Ab25-35 peptide-induced neurotoxicity in SH-SY5Y cells. The neuronal viability in SH-SY5Y cells was determined by MTT assay (as described
in the Experimental section), after 24 h of incubation with Ab25-35 peptide (10 M) in the presence or absence of various concentrations of compounds (1e10 M). The values are
reported as mean SD of three independent experiments (p < 0.05 vs. untreated cells, ANOVA).
m
m
in SH-SY5Y cells was further confirmed by the LDH release assay
[49]. Generally damaged cells released LDH from the cells into
(DPPH) [53]. The known antioxidants resveratrol, ferulic acid and
trolox were used as reference compounds (Table 2). These studies
indicated that compounds 3a, 3i and 3j exhibit moderate ability to
culture media. As reported in Table 2,
Ab25ꢀ35 significantly
increased LDH release (364.06%), while all the compounds specially
compounds 3a, 3e and 3j significantly decreased the percentage of
LDH release (294.64%, 287.37% and 285.09% respectively) from SH-
SY5Y cells.
scavenge DPPH radical (17.6e21.64% at 50
idant activity was observed with 3i (DPPH scavenging ¼ 21.64% at
50 M).
mM). Maximum antiox-
m
2.9. Ab25-35 peptide binding to the cell surface
2.7. Determination of Luminescent Cell Viability induced by Ab25-35
peptide
Studies suggested that unaggregated Ab25-35 and Ab31-35 pep-
tides could initiate a cascade of events leading to neurotoxicity
exclusively after their internalization within the neuronal cells [32].
Due to this we evaluated the ability of compounds 3a, 3h and 3i to
prevent the binding between the Ab25-35 peptide and the plasma
membrane surface of SH-SY5Y cells by the CR assay. The binding of
The protective effects of the compounds against Ab25-35 peptide
induced cell death were further confirmed by Luminescent Cell
Viability assay (Table 2) [50,51]. Ab25ꢀ35 significantly decrease
percentage of ATP (90.95%) in SH-SY5Y cells, while all the com-
pounds particularly compounds 3e and 3h increased the percent-
age of ATP (104.56% and 102.53% respectively) in SH-SY5Y cells.
A
b25-35 peptide (10
2 h and was significantly reduced by co-treatment with compounds
3a, 3h and 3i (10 M) showed in Fig. 3.
mM) with SH-SY5Y cells reached maximum in
m
2.8. Antioxidant capacity (DPPH scavenging)
There is variety of evidence that oxidative stress plays a signif-
icant role in inducing and activating multiple cell signaling path-
ways that contribute to production of toxic substances and then
promotes the development of AD [54]. Thereby, the oxidative stress
in SH-SY5Y cells, in terms of intracellular ROS formation induced by
Oxygen containing heterocycles, benzofuran-based compounds
were reported as antioxidant agents [52]. Antioxidant capacity of
compounds (3a, 3b, 3e, 3h, 3i, 3j) was evaluated by UV spectro-
photometry using the stable radical 2,2-diphenyl-1-picrylhydrazyl
A
b25-35 peptide (10 mM) was also investigated. Remarkably,
6

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
[55,56]. The results of flow cytometry assay showed that the
apoptotic ration of the Ab25-35 peptide induced SH-SY5Y cells
markedly increased as compared with that of untreated cells
(control). Co-treatment with compounds 3a, 3h and 3i (10
mM)
significantly decreased the apoptotic cell (Fig. 5), indicating that
these compounds inhibited Ab25-35 peptide induced SH-SY5Y cells
death.
2.11. Molecular docking studies
All three selected compounds were in agreement with the Lip-
inski's ‘‘rule of five” [57] which is developed for predicting the drug
likeness property of a small molecule. In addition, the calculated
polar surface area (PSA) of the compounds 3a, 3h and 3i were found
to be about <50 Å2 (Table 3). A PSA less than 70 Å2 is suitable for a
compound to pass through the blood brain barrier (BBB) [58e60].
To understand whether and how the binding and inhibition
profile of synthesized test compounds are influenced by the
structure, molecular docking was accomplished. All the compounds
3-3q were investigated computationally to define their binding
profile. The most promising compounds 3a, 3h and 3i were
investigated for further study.
Fig. 3. Effects of selected compounds on the binding of Ab25-35 peptide with SH-SY5Y
cells. SH-SY5Y cells were treated with Ab25-35 peptide (10
and 120 min) in the presence or absence of compounds 3a, 3h and 3i (10
m
M) for different time (30, 60
M). At the
m
end of incubation, the Ab25-35 peptide binding to plasma membrane was determined
by CR assay and the results are expressed as percentage of control cells (**p < 0.01, vs.
untreated cells, ANOVA). Determination of intracellular ROS formation induced by
A
b25-35 peptide.
compounds 3a, 3h and 3i (10 mM) significantly inhibited the Ab25-35
peptide-induced ROS formation (Fig. 4). Taken collectively, these
results showed the ability of compounds 3a, 3h and 3i to neutralize
the neurotoxicity of Ab25-35 peptides, suggesting their ability to
prevent the interaction between Ab25-35 peptides, the cell mem-
brane of SH-SY5Y cells, and also antioxidant capability together
responsible for the ROS formation as well as neuronal viability loss.
The binding interactions of the most potent A
hibitors compound 3i (self and AChE induced A
b
b
aggregation in-
aggregation in-
hibition 61.91% and 52.12%, respectively) and compound 3h (self
and AChE induced A aggregation inhibition 57.71% and 53.91%,
respectively) were investigated using A monomer and A fibril.
Studies reveal that A monomer are nontoxic and prevents
neuronal cell death caused by oxidative stress. The A monomer
consist of aromatic residues, phenylalanine (Phe4, Phe19, and Phe20
and trytophan (Tyr10) Pi-stacking interactions between aromatic
residues were suggested to play a significant role in A self-
assembly process by enhanced A fibril formation [61].
Also the A region cosisting of amino acids 12e24 and 30e40
known as the fibril-forming A fragment, is responsible for their
native states self-assembling into a -sheet aggregates. Another key
site within the A
b
b
b
b
b
)
2.10. Detection of apoptotic cells induced by Ab25-35 peptide
b
Apoptosis is a form of programmed cell death, which lead to
characteristic cell changes (morphology) and death. Studies
b
b
showed that, neurotoxic fragment
Ab25-35 peptide, caused
b
apoptosis in a concentration-dependent mode to neuron cells
b
b
-fragment, considered as the nucleation site of
aggregation, is known as the ‘steric zipper’16KLVFFA21 region. This
hexa-peptide sequence is believed to facilitate the monomer dy-
namics, acting as a steric zipper, that lead to dimer formation and
eventually, larger aggregates [62]. In the A
peiperidine ring and benzyl group from both compounds 3i and 3h
were interacted with the Val24 (distance̴ 3 - 5 Å), with amide e
stacked and -alkyl interaction. Furthermore, phenyl group of
compound 3i interacted with Phe20 (distance̴ 4 Å) from the
16KLVFFA21 region with amide-
stacked(Fig. 6- A and Fig. 7-A).The
binding modes in A fibril models also suggested that compound 3i
b monomer assembly,
p
p
p
b
was interlocked between a pair of Ile32 and two pairs of Phe19 and
Phe20. More interastingly, compound 3i was stacked in parallel
between the ‘steric zipper’ 16KLVFFA21 region of Phe20 pairs (dis-
tance̴ 4 - 5 Å). This conformation was unable for the compound 3h
which was interlocked between a pair of Phe19, Ala21and Ile32(-
Fig. 6-B and Fig. 7-B). These studies indicate that C-2 and C-3
substituted benzofuran derivative 3i serves as a suitable template
to design Ab aggregation inhibitors.
The binding interactions of AChE with good activity identified
from the benzofuran piperidine derivatives compounds 3a and 3h
(AChE IC50 5.07 and 21.30
highest docking score revealed that, benzofuran ring of compound
3a(Fig. 8-A) interacted with Trp82, Asp70 and Tyr327 with
shaped interaction (distance̴ 3e5 Å). Piperidine ring engaged with
Phf287 and Tyr120 with -alkyl interaction (distance̴ 5 Å). Benzyl
group of compound 3a form -lone pair interaction with Ser199
and T shaped interaction with Trp232 (distance̴2e4 Å). On the
other hand, benzofuran ring of compound 3h (Fig. 8- B) contributed
mM respectively). Binding mode of
Fig. 4. Effects of selected compounds on Ab25-35 peptide induced intracellular ROS
p-p T
formation in SH-SY5Y cells. SH-SY5Y cells were treated with Ab25-35 peptide (10
mM) in
the presence or absence of compounds 3a, 3h and 3i (10 M). At the end of incubation,
m
ROS formation was determined using a fluorescence probe, DHE, as described in the
Experimental section. The cells were collected by centrifuging; the pellet was re-
suspended and then analyzed by flow cytometry. 10,000 events were collected on
each sample. Data are expressed as the mean SD from three experiments. (*p < 0.05,
**p < 0.01, vs. untreated cells, ANOVA).
p
p
p-p
7

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Fig. 5. Effects of selected compounds on Ab25-35 peptide induced apoptosis in SH-SY5Y cells. (A) SH-SY5Y cells were treated with Ab25-35 peptide (10
mM) in the presence or absence
of compounds 3a, 3h and 3i (10 M). At the end of incubation, apoptotic ratio was determined using fluorescein-labeled Annexin V and PI probes, as described in the Experimental
m
section. The cells were collected by centrifuging; the pellet was re-suspended and then analyzed by flow cytometry. 10,000 events were collected on each sample. Data are
expressed as the mean SD from three experiments. (*p < 0.05, **p < 0.01, vs. untreated cells (control), ANOVA). (B) Annexin V/PI staining of apoptosis of SH-SY5Y cells treated
with treated with Ab25-35 peptide (10 mM) in the presence or absence of compounds 3a, 3h and 3i (10 mM).
in the forming of T shape
and Ala200 (distance̴ 4e5 Å). Piperidine ring engaged with Phe328
with -alkaly interaction. Furthermore, benzyl group form -anion
p
-
p
interaction with Phe 287, Phe 232
interaction with Asp70 (distance̴5 Å) and Florin molecule inter-
acted with Asn83, Trp82 and Thr79 (distance̴ 2e3 Å). From the
results, it showed that the compound 3h was well fitted in the
p
p
8

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Table 3
active site of the AChE and displayed more favorable interactions,
involving more the aromatic interactions.
Physicochemical parameters of selected compounds.
Compounds
MW
log Pa
PSA (Å2)a
Surface Areaa
3a
3h
3i
291.39
309.38
395.48
4.19
4.33
5.65
36.17
35.73
47.83
455.97
463.05
592.09
3. Experimental section
3.1. Chemistry
a
log P and PSA calculated using Marvin (ChemAxon) software.
Melting points were determined by RY-1G melting point appa-
ratus and are uncorrected. All chemical reactions were monitored
by thin-layer chromatography (TLC) on silica gel plates. Column
Fig. 6. Binding modes of compound 3i (green) with A
b
monomer (PDB ID: 1BA4) and A
b
fibril (PDB ID: 2LMN). The key residues of A
b
monomer for the interaction are showed
(name and ID). Dotted lines represent compound 3i eA
b
interaction. A) Compound 3i docked in A
b
monomer model, B) Compound 3i docked in A fibril model.
b
Fig. 7. Binding modes of compound 3h (green) with A
b
monomer (PDB ID: 1BA4) and A
b
fibril (PDB ID: 2LMN). The key residues of A
b
monomer for the interaction are showed
(name and ID). Dotted lines represent compound 3heA
b
interaction. A) Compound 3h docked in Ab monomer model, B) Compound 3h docked in Ab fibril model.
9

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Fig. 8. Binding modes of compounds with hAChE (PDB: 1B41). . The key residues are showed (name and ID). Dotted lines represent compound -AChE interaction. A) Compound 3a
docked in hAChE model, B) Compound 3h docked in hAChE model.
chromatography was performed on silica gel 200e300
mesh.1HNMR (400 MHz) and 13C NMR (100 MHz) was recorded on
a Brucker 400 MHz. The spin multiplicities are indicated by the
symbols, s (singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet) and brs (broad singlet). The chemical shifts were given in
(s, 2H), 6.45 (s, 1H), 3.12 (d, J ¼ 12.52 Hz, 2H), 2.92 (s, 1H), 2.74 (s,
2H), 2.07 (d, J ¼ 15.26 Hz, 2H), 1.71 (d, J ¼ 16.82 Hz, 2H). ES-MS m/z
calcd for C13H15NO [MþH]þ202.1226, found 202.1221.
3.1.2. General procedure for 4-(benzofuran-2-yl)-1-
benzylpiperidine (3a)
d
(ppm) refer to the signal of CDCl3
(
d
7.26, 1H NMR and
d
77.16, 13
C
NMR) and the signal of (CD3)2SO (
d
2.54, 1H NMR and
d
39.52, 13C
1 eq 4-(Benzofuran-2-yl) piperidine (com 3) and 1 eq benzal-
dehydewas mixed in1.5 eq TEA and 5% AcOH in dry DMF for 30 min
2eq NaBH(OAc)3 was then added and the reaction stirred for 4 h.
The reaction was quenched with 3 N NaOH and extracted with
3XCH2Cl2.The organic layer was washed with dist H2O and brine.
The organic layer was dried over anhydrous sodium sulfate, filtered
and evaporated under pressure. Purification was performed by
flash chromatography (DCM: MeOH ¼ 10:1) to obtained 3a.White
NMR). Chemical shift values are given in parts per million and
coupling constants (J) in Hertz. High resolution mass spectroscopy
was conducted using Agilent 6230 LC-MS. For flow cytometer BD
LSRFortessa was used and BD FACSD software was used to analyze
the data. SYNERGY/LX multimode reader (Biotek Instruments Inc.)
was used for UV visible Spectrometric analysis and VARIOSKAN
FLASH (Thermo scientific) was used for fluorescence measurement.
solid powder (68%). Mp 63 ꢁC. 1H NMR (400 MHz, CDCl3)
d 7.48 (d,
J ¼ 6.65 Hz, 1H), 7.40 (d, J ¼ 7.83 Hz, 1H), 7.28e7.37 (m, 4H),
7.21e7.28 (m, 1H), 7.12e7.21 (m, 2H), 3.54 (s, 2H), 2.97 (d,
J ¼ 11.74 Hz, 2H), 2.69e2.79 (m, 1H), 2.15 (s, 1H), 2.06 (d,
J ¼ 18.39 Hz, 3H), 1.82 (d, J ¼ 16.43 Hz, 2H). 13C NMR (101 MHz,
3.1.1. 4-(Benzofuran-2-yl) piperidine (3)
Benzofuran (1 mL, 0.008 mol) and dry THF were stirred
at ꢀ78 ꢁC under N2 environment for 30 min 5.28 mL n-BuLi (1.6 M)
was added in the reaction mixture slowly. After mixing the reaction
CDCl3)
d 162.7, 154.4, 138.3, 129.1, 128.7, 128.2, 127.0, 123.1, 122.3,
mixtures for
1
h
tert-butyl-4-oxopiperidin-1-carboxylate
120.3, 110.7, 100.2, 63.4, 53.3, 35.7, 30.5. ES-MS m/z calcd for
(1.687 gm) was added slowly at ꢀ78 ꢁC and continue the reaction
for 6hr at tem ꢀ78 ꢁC to ꢀ40 ꢁC. 1 mL MeOH was added to quench
the reaction. After evaporated the reaction mixture under pressure,
EtOAc poured into it and washed with dist H2O and brine. The
organic layer was dried over anhydrous sodium sulfate, filtered and
evaporated under pressure. Purification was performed by flash
chromatography (PE: EA ¼ 5:1) and light-yellow oil was obtained
(compound 2). Yield 90e95%. 3 was obtained by reacting this in-
termediate 2 (0.300 gm, 0.996 mol), TFA (4 eq) and Et3SiH (4eq) in
10 mL dry DCM at rt to 40 ꢁC under N2 gas for 6 h, then overnight at
room temperature. The pH of the reaction mixture was adjusted
basic with the help of Na2HCO3. After evaporated the reaction
mixture under pressure, EtOAc poured into it and washed with dist
H2O and brine. The organic layer was dried over anhydrous sodium
sulfate, filtered and evaporated under pressure. Purification was
performed by flash chromatography (DCM: MeOH ¼ 10:1) and off
white solid powder (compound 3) was obtained. Yield 85e90%. Mp
C
20H21NO [MþH]þ 292.1696, found 292.1955.
3.1.3. 4-((4-(benzofuran-2-yl)piperidin-1-yl)methyl)phenol (3b)
The title compound was obtained by the treatment of 4-
(Benzofuran-2-yl) piperidine (com 3) and 1 eq 4-hydroxy benzal-
dehyde as described in 3a. White solid powder (55%). Mp 62 ꢁC. 1H
NMR (400 MHz, DMSO)
d
7.53 (d, J ¼ 6.65 Hz,1H), 7.47 (s,1H), 7.21 (d,
J ¼ 7.43 Hz, 2H), 7.09 (d, J ¼ 7.83 Hz, 1H), 6.73 (s, 1H), 6.76 (s, 1H),
6.66 (s, 1H), 6.56 (s, 1H), 3.10 (br. s., 1H), 2.88 (s, 2H), 2.73 (s, 2H),
2.01e2.11 (m, 2H), 1.97 (br. s., 2H), 1.55e1.75 (m, 2H). 13C NMR
(101 MHz, DMSO)
d 162.4, 157.3, 153.8, 129.0, 128.4, 123.3, 122.6,
120.5,119.3,115.5,113.8,110.7,100.4, 62.4, 52.7, 35.0, 30.2. ES-MS m/
z calcd for C20H21NO2 [MþH]þ 308.1645, found 308.1606.
3.1.4. 4-(benzofuran-2-yl)-1-(3,4-dimethoxybenzyl)piperidine (3c)
The title compound was obtained by the treatment of 4-
55e60 ꢁC. 1H NMR (400 MHz, CD3OD)
d
7.47 (s, 1H), 7.36 (s, 1H), 7.14
(Benzofuran-2-yl) piperidine (com 3) and
1
eq 2,3-
10

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
dimethoxybenzaldehyde as described in 3a. White solid powder
MS m/z calcd for C20H20ClNO [MþH]þ326.1306, found 326.1327.
(45%). Mp 61 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.50 (d, J ¼ 7.04 Hz,1H),
7.42 (d, J ¼ 7.83 Hz, 1H), 7.14e7.25 (m, 2H), 6.95 (s, 1H), 6.85 (q,
J ¼ 8.22 Hz, 2H), 6.38 (s, 1H), 3.89 (s, 3H), 3.91 (s, 3H), 3.51 (s, 2H),
3.00 (d, J ¼ 11.74 Hz, 2H), 2.78 (t, J ¼ 11.54 Hz, 1H), 2.05e2.18 (m,
3.1.9. 4-(benzofuran-2-yl)-1-(4-fluorobenzyl)piperidine (3h)
The title compound was obtained by the treatment of 4-
(Benzofuran-2-yl) piperidine (com 3) and 1.5 eq4-bromo benzyl
chlorides as described in 3g. White solid powder (70%). Mp 55 ꢁC.
4H), 1.80e1.91 (m, 2H). 13C NMR (101 MHz, CDCl3)
d162.5, 154.4,
148.8,148.1,130.7,128.7,123.2,122.4,121.3,120.3,112.2,110.7,100.2,
1H NMR (400 MHz, CHLOROFORM-d)
d
7.45 (d, J ¼ 7.04 Hz, 1H), 7.36
65.2, 63.0, 55.8, 53.2, 35.7, 30.3. ES-MS m/z calcd for
(d, J ¼ 7.43 Hz, 1H), 7.25 (br. s., 2H), 7.15 (quin, J ¼ 7.34 Hz, 2H), 6.97
(t, J ¼ 8.41 Hz, 2H), 6.33 (s, 1H), 3.47 (s, 2vvH), 2.92 (d, J ¼ 10.96 Hz,
2H), 2.73 (t, J ¼ 11.74 Hz, 1H), 1.97e2.17 (m, 4H), 1.74e1.85 (m, 2H).
C
22H25NO3[MþH]þ 352.1907, found 352.1894.
3.1.5. 4-(benzofuran-2-yl)-1-(4-methoxybenzyl)piperidine (3d)
13C NMR (101 MHz, CDCl3)
d 163.2, 162.5, 160.8, 154.4, 130.5, 128.7,
The title compound was obtained by the treatment of 4-
123.2, 122.4, 120.3, 114.9, 110.7, 100.3, 62.5, 53.2, 35.6, 30.4. ES-MS
(Benzofuran-2-yl) piperidine (com 3) and
methoxybenzaldehyde as described in 3a. Yellow wish oil. 1H
NMR (400 MHz, CDCl3)
1
eq 4-
m/z calcd for C20H20FNO [MþH]þ 310.1602, found 310.1541.
d
7.51 (d, J ¼ 6.26 Hz, 1H), 7.42 (d,
3.1.10. General procedure for (2-(1-benzylpiperidin-4-yl)
benzofuran-3-yl) (phenyl)methanone (3i)
J ¼ 7.83 Hz, 1H), 7.29 (d, J ¼ 5.09 Hz, 2H), 7.16e7.25 (m, 2H), 6.89 (d,
J ¼ 8.61 Hz, 2H), 6.38 (s, 1H), 3.83 (s, 3H), 3.52 (s, 2H), 3.00 (d,
J ¼ 11.74 Hz, 2H), 2.78 (t, J ¼ 11.54 Hz, 1H), 2.06e2.17 (m, 4H),
SnCl4 (1.2 eq) was added dropwise to a mixture of 3a (1 eq) and
benzoyl chloride (1.2 eq) in dry DCM and the resulting solution was
stirred at room temperature overtime. The reaction was quenched
with ice and stirred for 1 h. The pH of the reaction mixture was
adjusted basic with the help of Na2HCO3. The organics extracted
with 3XCH2Cl2, washed with dist H2O and brine. The organic layer
was dried over anhydrous sodium sulfate, filtered and evaporated
under pressure. Purification was performed by flash chromatog-
raphy (DCM: MeOH ¼ 10:1) to obtained 3i. White solid powder
1.80e1.90 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 162.9, 159.7, 154.4,
132.0, 129.8, 128.0, 123.5, 122.5, 120.5, 114.0, 113.3, 110.8, 101.0,
100.9, 60.5, 55.3, 51.1, 28.2. ES-MS m/z calcd for C21H23NO2
[MþH]þ322.1802, found 322.1802.
3.1.6. 4-(benzofuran-2-yl)-1-(2,5-dimethoxybenzyl)piperidine (3e)
The title compound was obtained by the treatment of 4-
(Benzofuran-2-yl) piperidine (com 3) and 1 eq 2, 5-dimethoxy
benzaldehyde as described in 3a. White solid powder (55%). Mp
(68%). Mp 62 ꢁC. 1H NMR (400 MHz, CHLOROFORM-d)
d 8.13 (d,
J ¼ 7.04 Hz, 3H), 7.81 (d, J ¼ 7.04 Hz, 1H), 7.54 (t, J ¼ 7.24 Hz, 2H),
7.46 (d, J ¼ 7.83 Hz, 5H), 7.37 (d, J ¼ 5.87 Hz, 3H), 4.10 (s, 1H), 4.05 (s,
1H), 3.44 (d, J ¼ 11.35 Hz, 2H), 2.93 (br. s., 1H), 2.68 (br. s., 1H), 2.62
40 ꢁC. 1H NMR (400 MHz, CDCl3)
d
7.51 (d, J ¼ 7.04 Hz, 1H), 7.43 (d,
J ¼ 7.83 Hz, 1H), 7.21 (s, 2H), 7.06 (s, 1H), 6.82 (s, 2H), 6.39 (s,1H),
3.81 (s, 6H), 3.61 (s, 2H), 3.05 (d, J ¼ 11.74 Hz, 2H), 2.78 (s, 1H),
(br. s., 1H), 2.12e2.31 (m, 4H)$13C NMR (101 MHz, CDCl3)
d 196.3,
2.17e2.28 (m, 2H), 2.10 (d, J ¼ 12.52 Hz, 2H), 1.81e1.95 (m, 2H). 13
C
191.9, 167.0, 165.4, 153.8, 153.6, 139.2, 138.3, 132.8, 132.0, 129.9,
129.1, 128.4, 128.0, 124.4, 121.3, 113.2, 111.3, 101.1, 62.3, 52.2, 35.0,
31.4, 30.2, 29.7, 29.3, 22.7. ES-MS m/z calcd for C27H25NO2 [MþH]þ
396.1958, found 396.1962.
NMR (101 MHz, CDCl3) d162.8,154.4,153.6,152.1, 128.8,123.1,122.3,
120.3, 116.3,111.6, 110.7, 100.2, 56.2, 55.7, 53.3, 35.7, 30.5. ES-MS m/z
calcd for C22H25NO3 [MþH]þ 352.1907, found 352.1894.
3.1.7. 4-(benzofuran-2-yl)-1-(4-ethylbenzyl)piperidine (3f)
3.1.11. (2-(1-benzylpiperidin-4-yl)benzofuran-3-yl) (4-
methoxyphenyl)methanone (3j)
The title compound was obtained by the treatment of 4-
(Benzofuran-2-yl) piperidine (com 3) and
ethylbenzaldehyde as described in 3a. White solid powder (55%).
Mp 66 ꢁC. 1H NMR (400 MHz, CDCl3)
1
eq 4-
The title compound was obtained by the treatment of 3a (1 eq)
and 4-methoxybenzoyl chloride as described for 3i. Yellow wish oil.
d
7.60 (d, J ¼ 7.83 Hz, 1H), 7.51
1H NMR (400 MHz, CDCl3)
d
7.85 (d, J ¼ 8.61 Hz, 1H), 7.82 (d,
(d, J ¼ 8.22 Hz, 1H), 7.37 (t, J ¼ 7.63 Hz, 1H), 7.26 (s, 3H), 6.97 (s, 1H),
6.57 (s,1H), 5.92 (br. s.,1H), 3.60 (s, 2H), 3.00 (br. s.,1H), 2.79 (s, 2H),
2.05 (br. s., 1H), 1.72 (br. s., 2H), 1.43 (s, 1H), 1.26 (s, 6H), 0.89 (s, 1H).
J ¼ 8.61 Hz, 1H), 7.49 (d, J ¼ 8.22 Hz, 1H), 7.43 (d, J ¼ 8.61 Hz, 1H),
7.37 (s, 4H), 7.17 (d, J ¼ 5.87 Hz, 1H), 6.89e6.98 (m, 4H), 4.01 (s, 2H),
3.88 (s, 3H), 3.82 (s, 2H), 3.44 (d, J ¼ 11.35 Hz, 2H), 3.29 (br. s., 1H),
2.50 (d, J ¼ 11.35 Hz, 2H), 2.35 (d, J ¼ 11.35 Hz, 2H). 13C NMR
13C NMR (101 MHz, CDCl3)
d 166.8, 160.8, 154.4, 128.4, 127.7, 126.2,
123.6, 122.6, 120.5, 118.0, 111.4, 110.8, 107.4, 100.8, 45.4, 39.3, 35.9,
30.9, 29.4, 24.3. ES-MS m/z calcd for C22H25NO [MþH]þ 320.2009,
found 320.2023.
(101 MHz, CDCl3) d 190.3, 170.5, 165.3, 163.6, 160.7, 153.5, 131.8,
129.0, 128.5, 124.4, 123.4, 121.2, 113.7, 111.3, 55.5, 51.8, 35.6, 28.7. ES-
MS m/z calcd for C28H27NO3 [MþH]þ 426.2064, found 426.2060.
3.1.8. General procedure for 4-(benzofuran-2-yl)-1-(4-
chlorobenzyl)piperidine (3g)
3.1.12. (2-(1-(3,4-dimethoxybenzyl)piperidin-4-yl)benzofuran-3-
yl) (phenyl)methanone (3k)
1 eq of 4-(Benzofuran-2-yl) piperidine (com 3) and 1.5 eq of 4-
choro benzyl chlorides was mixed in the presence of 1.2 eq trie-
thylamine and dry DCM. After stirring the mixture at room tem-
perature overnight, it was then diluted with H2O. The pH of the
reaction mixture was adjusted basic with the help of Na2HCO3. The
organics extracted with 3XCH2Cl2, washed with dist H2O and brine.
The organic layer was dried over anhydrous sodium sulfate, filtered
and evaporated under pressure. Purification was performed by
flash chromatography (DCM: MeOH ¼ 10:1) to obtained 3g. White
solid powder (68%). Mp 46 ꢁC. 1H NMR (400 MHz, CHLOROFORM-d)
The title compound was obtained by the treatment of 3c (1 eq)
and benzoyl chloride as described for 3i. Yellow wish oil. 1H NMR
(400 MHz, CDCl3)
(m, 2H), 7.16 (s, 1H), 6.70e7.04 (m, 6H), 3.91 (s, 4H), 3.88 (s, 2H),
d
7.86 (d, J ¼ 9.00 Hz, 2H), 7.47 (s, 1H), 7.21e7.34
3.47 (s, 2H), 3.15 (br. s., 1H), 3.01 (br. s., 2H), 2.10 (br. s., 3H), 2.01 (br.
s., 1H), 1.89 (d, J ¼ 13.30 Hz, 2H)$13C NMR (101 MHz, CDCl3)
d 190.4,
166.8, 163.5, 153.4, 148.8, 148.0, 132.4, 131.7, 127.0, 124.1, 123.2,
121.2, 115.8, 113.6, 112.1, 111.1, 110.7, 63.0, 55.8, 55.5, 53.4, 35.6, 30.4.
ES-MS m/z calcd for C29H29NO4 [MþH]þ 456.2169, found 456.2222.
d
7.47 (d, J ¼ 7.04 Hz,1H), 7.39 (d, J ¼ 7.43 Hz,1H), 7.24 (s, 4H), 7.17 (s,
3.1.13. (2-(1-(3,4-dimethoxybenzyl)piperidin-4-yl)benzofuran-3-
yl) (3,4dimethoxyphenyl) (3l)
2H), 6.35 (s, 1H), 3.48 (s,2H), 2.93 (d, J ¼ 11.35 Hz, 2H), 2.74 (br. s.,
1H), 2.13 (br. s., 1H), 2.05 (d, J ¼ 16.04 Hz, 3H), 1.75e1.86 (m, 2H). 13
C
The title compound was obtained by the treatment of 3c (1 eq)
and 3,4-dimethoxybenzoyl chloride as described for 3i. Yellow
NMR (101 MHz, CDCl3) d 160.5,154.4,134.1,131.6,128.7, 128.1,123.4,
122.5, 120.5, 110.8, 107.2, 105.2, 100.9, 60.9, 55.5, 51.8, 34.5, 28.7.ES-
wish oil. 1H NMR (400 MHz, CDCl3)
d 7.40e7.51 (m, 3H), 7.29 (s, 1H),
11

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
7.19e7.26 (m, 1H), 7.09e7.18 (m, 1H), 6.90 (s, 1H), 6.75e6.86 (m,
3H), 3.93 (s, 3H), 3.84 (s, 4H), 3.88 (s, 5H), 3.45 (s, 2H), 3.06e3.17 (m,
1H), 2.97 (d, J ¼ 10.96 Hz, 2H), 2.11 (br. s., 2H), 1.99 (br. s., 2H), 1.87
and 4-methoxybenzoyl chloride as described for 3i. Yellow wish oil.
1H NMR (400 MHz, CDCl3)
d
7.82e7.87 (m, J ¼ 8.61 Hz, 2H), 7.47 (d,
J ¼ 9.00 Hz, 1H), 7.25 (s, 2H), 7.29 (s, 2H), 7.16 (d, J ¼ 8.22 Hz, 1H),
6.95 (d, J ¼ 8.61 Hz, 2H), 6.83e6.91 (m, J ¼ 8.61 Hz, 2H), 3.89 (s, 3H),
3.80 (s, 3H), 3.54 (s, 2H), 3.16 (t, J ¼ 11.15 Hz, 1H), 3.04 (d,
(d, J ¼ 12.13 Hz, 2H). 13C NMR (101 MHz, CHLOROFORM-d)
d 190.3,
166.7, 153.4, 149.0, 148.0, 131.7, 126.9, 124.5, 124.2, 123.2, 121.2,
115.8, 112.2, 111.3, 111.1, 110.7, 109.9, 100.2, 62.9, 55.8, 53.3, 35.6,
30.3. ES-MS m/z calcd for C31H33NO6 [MþH]þ 516.2381, found
516.2323.
J ¼ 10.96 Hz, 2H), 2.05e2.22 (m, 4H), 1.91 (d, J ¼ 12.91 Hz, 2H). 13
C
NMR (101 MHz, CHLOROFORM-d) d 162.9, 159.7, 154.4, 132.0, 129.8,
128.0, 123.5, 122.5, 120.5, 114.0, 113.3, 110.8, 101.0, 100.9, 60.5. ES-
MS m/z calcd for C29H29NO4 [MþH]þ 456.2169, found 456.2172.
3.1.14. (2-(1-(2,5-dimethoxybenzyl)piperidin-4-yl)benzofuran-3-
yl) (phenyl)methanone (3 m)
3.2. Biological assay
The title compound was obtained by the treatment of 3e (1 eq)
and benzoyl chloride as described for 3i. Yellow wish oil. 1H NMR
3.2.1. Amyloid peptide preparation
(400 MHz, CDCl3)
d
7.89 (br. s., 1H), 7.80 (d, J ¼ 7.04 Hz, 1H), 7.71 (d,
Commercially available Ab25-35 protein fragment (A4559, Sig-
maeAldrich) was dissolved in hexafluoroisopropanol(HFIP) to
1 mg/mL. After sonicating it was incubated at room temperature for
24 h and lyophilized. These unaggregated Ab25-35 film was then
dissolved with dimethylsulfoxide to 1 mM. The Ab25-35 stock so-
lution was then aliquoted and stored at ꢀ20 ꢁuntil for further use
[32]. For the biological assays, the Ab25-35 stock solution was diluted
directly into cell culture media.
J ¼ 8.22 Hz, 1H), 7.56 (br. s., 2H), 7.48 (br. s., 2H), 7.18 (br. s., 1H), 7.04
(br. s., 1H), 6.73e6.84 (m, 2H), 6.46 (s, 1H), 3.78 (s, 6H), 3.61 (br. s.,
2H), 3.05 (d, J ¼ 10.17 Hz, 2H), 2.75e2.85 (m, 1H), 2.21 (br. s., 2H),
2.09 (d, J ¼ 12.13 Hz, 2H), 1.82e1.97 (m, 2H). 13C NMR (101 MHz,
CDCl3)
d 196.4, 153.6, 152.1, 138.4, 131.9, 129.9, 128.2, 125.0, 119.9,
116.4, 113.2, 111.7, 110.7, 100.7, 56.2, 55.7, 53.2, 35.9, 30.3. ES-MS m/z
calcd for C29H29NO4 [MþH]þ 456.2169, found 456.2172.
3.1.15. (2-(1-(2, 5-dimethoxybenzyl) piperidin-4-yl) benzofuran-3-
yl) (3,4 dimethoxyphenyl) methanone (3n)
3.2.2. Inhibition of Ab1-42 self-induced aggregation
Inhibition of self-induced Ab25-35 aggregation of test com-
pounds were evaluated by a Thioflavin T (ThT)-binding assay [44].
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and Thioflavin T (ThT)
The title compound was obtained by the treatment of 3e (1 eq)
and 3,4-dimethoxybenzoyl chloride as described for 3i. Yellow
wish oil. 1H NMR (400 MHz, CDCl3)
d 7.40e7.49 (m, 3H), 7.22e7.28
were purchased from MACKLIN (Shanghai). Amyloid
b (Ab25-35),
(m, 2H), 7.10e7.17 (m, 1H), 6.98e7.04 (m, 1H), 6.84 (d, J ¼ 8.22 Hz,
1H), 6.70e6.77 (m, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.75 (s, 3H), 3.76
(s, 3H), 3.53 (br. s., 2H), 3.10 (br. s., 1H), 3.01 (d, J ¼ 6.65 Hz, 2H), 2.11
(br. s., 4H), 1.86 (d, J ¼ 9.78 Hz, 2H). 13C NMR (101 MHz, CDCl3)
was obtained from SIGMA (Shanghai, China). Ab25-35 samples were
pretreated with HFIP and then resolubilized with a DMSO to give a
10
mM solution. The test compound was dissolved in DMSO. The
test compound (100
m
M) and Ab25-35 (10 M) were incubated in the
m
d
190.4, 166.8, 153.6, 152.1, 149.0, 131.8, 127.0, 124.6, 124.2, 123.2,
assay medium containing 0.01 M NaCl in 0.05 M potassium phos-
phate buffer (pH 7.4) at 37 ꢁC for 48 h. After the incubation, ThT
121.2, 116.4, 115.8, 111.1, 109.9, 56.1, 55.7, 53.4, 35.6, 30.4. ES-MS m/z
calcd for C31H33NO6 [MþH]þ 516.2381, found 516.2407.
(200
100
m
M) in 50 mM glycine-NaOH buffer (pH 8.0) was added to the
mL Ab2-35 test compound and the fluorescence was measured
3.1.16. (2-(1-(2,5-dimethoxybenzyl)piperidin-4-yl)benzofuran-3-
yl) (4-methoxyphenyl) methanone (3o)
The title compound was obtained by the treatment of 3e (1 eq)
and 4-methoxybenzoyl chloride as described for 3i. Yellow wish oil.
on a multi-mode plate reader with excitation and emission wave-
lengths at 446 nm and 490 nm, respectively. Each assay was run in
triplicate and the solvent control was also included. The fluores-
cence intensities were compared and the percent inhibition of
aggregation due to the presence of the test compound was calcu-
lated by the following formula:
1H NMR (400 MHz, CDCl3)
d
7.80e7.90 (m, J ¼ 8.22 Hz, 2H), 7.48 (d,
J ¼ 8.22 Hz, 1H), 7.22e7.31 (m, 2H), 7.12e7.18 (m, 1H), 7.06 (d,
J ¼ 2.35 Hz, 1H), 6.91e6.98 (m, J ¼ 8.22 Hz, 2H), 6.70e6.81 (m, 2H),
3.87 (s, 3H), 3.77 (s, 3H), 3.78 (s, 3H), 3.56 (s, 2H), 3.13 (t,
J ¼ 10.17 Hz, 1H), 3.03 (d, J ¼ 9.00 Hz, 2H), 2.05e2.22 (m, 4H), 1.89
IFo ꢀ IFi
Inhibition ð%Þ ¼
X 100%
IFo
(d, J ¼ 10.56 Hz, 2H) 13C NMR (101 MHz, CDCl3)
d 190.4, 166.8, 163.5,
Here, IFi and IFo are the fluorescence intensities obtained for Ab25-
in the presence and in the absence of test compounds,
153.5, 152.1, 131.7, 127.0, 124.1, 123.2, 121.2, 116.3, 113.6, 111.6, 111.1,
35
55.7, 53.4, 35.6, 30.4. ES-MS m/z calcd for
C30H31NO5
[MþH]þ486.2275, found 486.2300.
respectively.
3.2.3. Inhibition of AChE-induced Ab25-35 aggregation
3.1.17. (2-(1-(3-methoxybenzyl)piperidin-4-yl)benzofuran-3-yl)
(phenyl)methanone (3p)
Inhibition of AChE-induced Ab25-35 aggregation of test com-
pounds were also evaluated by the ThT assay. The eeAChE were
purchased from Sigma-Aldrich (St. Louis, MO, USA). For co incu-
bation experiments, aliquots of Ab25-35 peptide and AChE in pres-
ence or absence of the test compounds were incubated for 48 h at
The title compound was obtained by the treatment of 3d (1 eq)
and benzoyl chloride as described for 3i. Yellow wish oil. 1H NMR
(400 MHz, CDCl3)
d
8.08 (s, 1H), 7.82 (d, J ¼ 6.65 Hz, 2H), 7.60 (d,
J ¼ 7.43 Hz, 2H), 7.45e7.52 (m, 4H), 7.25 (s, 2H), 6.86 (d, J ¼ 8.61 Hz,
2H), 3.80 (s, 3H), 3.48 (s, 2H), 3.11 (br. s., 1H), 3.00 (d, J ¼ 10.96 Hz,
2H), 2.24 (br. s., 1H), 2.14 (br. s., 2H), 2.08 (br. s., 1H), 1.93e2.03 (m,
37 ꢁC. The final concentrations of A
(dissolved in 0.215 M sodium phosphate buffer, pH 8.0) and test
compounds were 100 M, 2 M and 100 M, respectively. After
incubation, After the incubation, ThT (200 M) in 50 mM glycine-
b (dissolved in DMSO), eeAChE
2H). 13C NMR (101 MHz, CHLOROFORM-d)
d 162.9, 159.7, 154.4,
m
m
m
m
132.0, 129.8, 128.0, 123.5, 122.5, 120.5, 114.0, 113.3, 110.8, 101.0,
100.9, 60.5, 55.3, 51.1, 28.2. ES-MS m/z calcd for C28H27NO3 [MþH]þ
426.2064, found 426.2172.
NaOH buffer (pH 8.0) was added and the inhibition percent of ag-
gregation was calculated by the method same as that of self-
induced Ab25-35 experiment [44].
3.1.18. (2-(1-(3-methoxybenzyl) piperidin-4-yl)benzofuran-3-yl)
(4-methoxyphenyl)methanone (3q)
3.2.4. AChE and BChE inhibitory activity
The title compound was obtained by the treatment of 3d (1 eq)
Acetylcholinesterase
human
recombinant
(C1682),
12

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
butyrylcholinesterase human recombinant (B4186), acetylth-
iocholineiodide (A5751), and S-butyrylthiocholine iodide (B3253)
were purchasedfrom Sigma-Aldrich and 5, 50-Dithiobis(2-
nitrobenzoic acid) (DTNB) was purchased from Calbiochem (Los
Angeles CA, USA).
saline (PBS) and incubated with MTT (5 mg/mL) in PBS for 2 h at
37 ꢁC in 5% CO2. After again washing, the formazan crystals were
dissolved with DMSO. The amount of formazan was measured
(570 nm, ref. 690 nm) with UVevisible microplate reader [32,48].
The neuronal viability was expressed as a percentage of control
cells and calculated by the formula: (absorbance of treated neu-
rons/absorbance of untreated neurons) X100.
Cholinesterase activity of AChE and BChE was determined by the
Ellman's assay. Reactions were performed in a medium containing
substrate (1e100 mM) combined with 0.125 mMDTNB in 100 mM 3-
(N-morpholino)propanesulfonic acid buffer, pH 8.0 at 25 ꢁC, initi-
ated by the addition of enzyme, and monitored spectrophotomet-
rically at 412 nm in a UVevisible spectrophotometer. The activity
was determined by measuring the increase in absorbance at
412 nm for 7 min. Activities of the tested compound were
compared to control to estimate percentage of inhibition (I%) [45].
Dose-response curves were plotted using the GraphPad Prism 5
software and IC50 values were determined. Data are expressed as
mean SEM. Each data point was the average of experiments in
triplicate. Donepezil (Sigma) and Rivastigmine (Sigma) were tested
as a reference compound.
3.2.8. Determination of cytoplasmic lactate dehydrogenase (LDH)
release induced by Ab25-35 peptide
Cytotoxicity was quantitatively evaluated by measuring LDH
released from the damaged cells into culture media. This assay is
based on a coupled enzymatic conversion from 2-p-(iodophenyl)ꢀ
3-(p-nitrophenyl)ꢀ 5-phenyltetrazolium chloride (INT, a tetrazo-
lium salt) into a formazan product, and the enzymatic reaction is
catalyzed by LDH released from cells and diaphorase in the assay
substrate mixture. LDH cytotoxicity assay Kit (Beyotime Biotech-
nology, China) was used to evaluate the protective effects of com-
pounds against Ab25-35 peptide induced damage [49]. The SH-SY5Y
cells were seeded in 96 well plates at 5 ꢂ 104 cells/well, incubated
for 24 h. After incubation SH-SY5Y cells were washed with phos-
phate buffered saline (PBS), added serum free medium and sub-
3.2.5. Cell culture
For cell toxicity and neuroprotection assays, Human neuronal
SH-SY5Y cells were routinely grown at 37 ꢁC in a humidified
incubator with 5% CO2 in Dulbecco's modified Eagle's medium
supplemented with 10% foetal bovine serum, 2 mM glutamine, 50
U/mL penicillin and 50 mg/mL streptomycin [43,47].
sequently treated with 10
m
M of unaggregated Ab25-35peptide for
24 h at 37 ꢁC in 5% CO2, in presence or absence 10
mM of test
compounds. After removal of the treatment, SH-SY5Y cells were
again washed with phosphate buffered saline (PBS) and LDH
releasing agent from the kit was added into the cells before 1hr of
3.2.6. Cytotoxicity
detection. Then the plate was centrifuged at 400g for 5 min. 120 ml
Cell viability was determined using a thiazolyl blue tetrazo-
liumromide [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide] (MTT) assay [43,47]. SH-SY5Y cells were seeded
into 96-well plates at 5000 cells per well and cultured in DMEM
supplemented with 10% FBS at 37 ꢁC, 5% CO2. Cells were treated
of cells culture medium was taken into a new plate and LDH
detecting working solution from the kit was added. The readings
were taken at 490 nm by a micro plate reader prior to the 1 h, light
restrictive, incubation period at room temperature [49]. LDH
release (%) was calculated with the following equation:
with test compounds at a concentration of 1e10
mM and incubated
for48 h. After the incubation, 10 L of the MTT reagent (5 mg/mL)
m
OD value of treated well ꢀ Od value of the blank
OD value of untrated well ꢀ OD value of the blank
LDH release ð%Þ ¼
X100
was added to each well. Formazan crystals were solubilized with
100 lL of DMSO. Absorbance values were measured at 690 and
570 nm using a UVevisible microplate reader. Cell viability was
expressed as a percentage of control cells and calculated by the
formula: (absorbance of treated neurons/absorbance of untreated
neurons) X100.
3.2.9. Determination of Luminescent Cell Viability induced by Ab25-
peptide
35
Luminescent Cell Viability Assay was used to evaluate the pro-
tective effects of the test compounds against Ab25-35 peptide induced
cell death. The homogeneous “add-mix-measure” format causes cell
lysis and generates a luminescent signal which is proportional to the
amount of ATP present and the amount of ATP is directly propor-
tional to the number of cells present in culture. CellTiter-Glo®
Luminescent Cell Viability Assay Kit (Promega Corporation, USA) was
used [50,51]. The SH-SY5Y cells were seeded in 96 well plates at
5 ꢂ 104 cells/well, incubated for 24 h and subsequently treated with
3.2.7. Neuroprotective effects against Ab25-35 peptide
To evaluate the protective effects of compounds against Ab25-35
peptide induced neurotoxicity, the SH-SY5Y cells were used. Cells
were seeded in 96 well plates at 3 ꢂ 104 cells/well, incubated for
10
presence or absence of 10
tion the plate was equilibrated in the room temperature for 30 min
and then 100 l CellTiter reagent from the kit was added and mixed
m
M of unaggregated Ab25-35 peptide for 24 h at 37 ꢁC in 5% CO2, in
24 h and subsequently treated with 10 mM of unaggregated Ab25-35
mM of test compounds. After the incuba-
peptide for 24 hat 37 ꢁC in 5% CO2, in presence or absence of various
concentrations of test compounds (1e10 M). The neuronal
m
m
viability in terms of mitochondrial metabolic function was evalu-
ated by the reduction of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-
diphenyl-2H-tetrazolium bromide MTT to formazan. The process
was described in previous section. Briefly, after removal of the
treatment, SH-SY5Y cells were washed with phosphate buffered
for 2 min on an orbital shaker to induce cell lysis. The plate allowed
incubating at room temperature for 10 min to stabilize luminescent
signal before recording luminescence. Relative Luminescence (%)
calculated with the following equation:
13

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
Luminescence value of treated well ꢀ Luminescence value of the blank
Relative Luminescence ð%Þ ¼
X100
Luminescence value of untrated well ꢀ Luminescence value of the blank
3.2.10. Antioxidant capacity (DPPH scavenging)
The capability of Benzofuran piperidine derivatives to scavenge
the DPPH radical was utilized as a measure of antioxidant capacity.
colorflow cytometry analysis using fluoresce-labeled AnnexinV and
PI was performed. Cells were fluoresce-labeled according to the
Annexin V-FITC Apoptosis Detection kit (SIGMA, USA). Briefly, after
treatment, SH-SY5Y cells were harvested by trypsinization, and
washed once with PBS. The cells were collected by centrifuging at
1000 rpm for 3 min; the pellet was re-suspended in binding buffer
with FITC-conjugated AnnexinV and PI for 15 min at room tem-
perature, and then analyzed by flow cytometry (BD LSR For-
tessa).10,000 events were collected on each sample [55,56].
Benzofuran piperidine derivatives (500
mM) and DPPH (56 mM)
stock solutions were prepared in anhydrous methanol. The exper-
iment was carried out in a 96-well clear, flat bottom plate and
added as follows: 90
(50 M) final concentration. Control solutions contained 90
anhydrous methanol and 10 L test compound. DPPH control
contained 90 L of DPPH, and 10 L anhydrous methanol. The
mL DPPH, 10 mL test compound solution
m
mL
m
m
m
readings were taken at 517 nm with 30 s shaking (double orbital at
530 cpm). After light restrictive 1 h incubation at room temperature
again readings were taken at 517 nm after another round of 30 s
shaking (double orbital at 530 cpm) using a UV-microplate reader
[52]. The antioxidant capacity (DPPH scavenging activity) was
evaluated as the percentage of inhibition according to the following
equation:
3.3. Molecular docking studies
3.3.1. GOLD (Genetic Optimization for Ligand Docking)
Docking studies of the designed compound is conceded using
GOLD (Genetic Optimization for Ligand Docking) 5.1 program from
the Cambridge Crystallographic Data Center, UK. GOLD uses a ge-
netic algorithm for docking flexible ligands into a protein binding
Absorbance of control ꢀ Absorbance of sample
DPPH scavenging ð%Þ ¼
X100
Absorbance of control
site to determine the full variety of ligand conformational flexibility
with the partial flexibility of the protein. Bound conformations are
predicted and assessed using Goldscore [62]. Discovery Studio was
used for peptide-ligand and protein-ligand binding visualization.
3.2.11. Determination of Ab25-35peptide binding to the cell surface
SH-SY5Y cells were seeded in 96-well plates at 5 ꢂ 103 cells/
wellfor 24 h. At the end of incubation, the medium was changed
with a fresh one with Ab25-35 peptide (10
mM) and various com-
3.3.2. Docking study with Ab peptide
pounds (10 M) for 30, 60 and 120 min and then washed twice with
m
A
b42 monomer (PDB ID: 1BA4) and Ab fibril (PDB ID: 2LMN) is
PBS. The residual Ab25-35 peptide cell complex was stained with CR
in PBS for 20 min and measured with a UVevisible microplate
reader at 540 nm (bound CR). CR values were reported as percent
increases in treated cells vs. untreated cells (taken as 100%) [32].
used as the receptor for docking. The ligand binding site in docking
is defined as a collection of residues placed within a sphere of 20 A ꢁ
diameters around the coordinates of F20 [63]. All other parameters
were kept default values. Here we discussed one of the highest
GOLD scored docking.
3.2.12. Determination of intracellular ROS formation induced by
A
b25-35 peptide
Intracellular ROS was evaluated using the fluorescent probe
3.3.3. Docking study with AChE
DCFH-DA [54]. The SH-SY5Y cells were seeded in 96 well plates at
The X-ray crystallographic structure of the mAChE was used
(PDB: 1B41). The active site was defined as all atoms within a radius
of 12 Å around some specific residue atoms: OH of Tyr337 for AChE
[64]. All water molecules in the PDB files were removed, and all
hydrogen atoms were then added to the protein with the help of
Discovery Studio.
1 ꢂ 106 cells/well, incubated for 24 h and subsequently treated with
10 m
M of unaggregated Ab25-35 peptide for 24 h at 37 ꢁC in 5% CO2,
in presence or absence of 10
cubation cells was washed with PBS three times. The cells were
incubated with DCFH-DA (10 M) in the loading media for 30 min.
mM of test compounds. After the in-
m
After DCFH-DA was removed, the cells were rinsed once with PBS.
The cells were collected by centrifuging at 1500 rpm for 5 min; the
pellet was re-suspended in PBS and then analyzed by flow
cytometry (BD LSR Fortessa). 10,000 events were collected on each
sample.
3.4. Statistical analysis
The data obtained were analyzed using the Prism GraphPad
software program version 4.0 and expressed as mean
S.E.M.
3.2.13. Flow cytometric detection of apoptotic cells induced by Ab25-
Statistical significances of differences among groups were evalu-
ated by analysis of variance (ANOVA) followed by the Bonferroni
test. P values less than 0.05 (p < 0.05) were considered significant.
peptide
35
To distinguish the apoptotic cells from the necrotic cells, two
14

S.R. Chowdhury, J. Gu, Y. Hu et al.
European Journal of Medicinal Chemistry 222 (2021) 113541
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Declaration of competing interest
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Acknowledgements
This research was supported by the Chinese Government
Scholarship (CSC Scholarship), Medical-Engineering Joint Fund of
Shanghai Jiao Tong University (No. YG2017MS29, No.
ZH2018ZDB07), and Agilent research fund (Agilent Grant ID
#4608). The author would like to also thank the Hong Kong Uni-
versity of Science and Technology (HKUST) for the support for AChE
and BChE inhibitory activity assay.
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