Development of genistein-O-alkylamines derivatives as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2020.104602

The multi-target-directed ligands have been regarded as the promising multifunctional agents for the treatment of Alzheimer's disease (AD). Based on our previous work, a series of genistein-O-alkylamines derivatives was developed to further explore the st

Full text of DOI:10.1016/j.bioorg.2020.104602

Bioorganic Chemistry 107 (2021) 104602
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Development of genistein-O-alkylamines derivatives as multifunctional
agents for the treatment of Alzheimer’s disease
,
*
,
,
Zhipei Sanga 1, Jian Shia 1, Yi Zhoua, Keren Wanga, Yiyang Zhaoa, Qingfeng Lib,
Zhanpin Qiaoa, Anguo Wuc *, Zhenghuai Tand *, Wenmin Liua
,
,
,*
a College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China
b Foreign Languages School, Nanyang Normal University, Nanyang 473061, China
c Sichuan Key Medical Laboratory of New Drug Discovery and Drugability Evaluation, Southwest Medical University, Luzhou 646000, China
d Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan Academy of Chinese Medicine Sciences, Chengdu 610041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The multi-target-directed ligands have been regarded as the promising multifunctional agents for the treatment
of Alzheimer’s disease (AD). Based on our previous work, a series of genistein-O-alkylamines derivatives was
developed to further explore the structure-activity-relationship. The results showed that compound 7d indicated
Alzheimer’s disease
Multi-function agents
AChE inhibition
reversible and highly selective hAChE inhibitory activity with IC50 value of 0.53 μM. Compound 7d also dis-
Antioxidant activity
Aβ aggregation inhibition
Metal chelator
played good antioxidant activity (ORAC = 1.1 eq.), promising neuroprotective effect and selective metal che-
lation property. Moreover, compound 7d significantly inhibited self-induced, hAChE-induced and Cu2+-induced
Aβ aggregation with 39.8%, 42.1% and 74.1%, respectively, and disaggregated Cu2+-induced Aβ1-42 aggregation
(67.3%). In addition, compound 7d was a potential autophagy inducer and improved the levels of GPX4 protein.
Furthermore, compound 7d presented good blood-brain-barrier permeability in vitro. More importantly, com-
pound 7d did not show any acute toxicity at doses of up to 1000 mg/kg and presented good precognitive effect
on scopolamine-induced memory impairment. Therefore, compound 7d was a promising multifunctional agent
for the development of anti-AD drugs.
Autophagy inducer
Neuroprotectant
Activation of GPX4
1. Introduction
exact pathogenesis of AD remain unclear. Current research identifies 4
typical progression of AD: preclinical stage, mild cognitive impairment
Alzheimer’s disease (AD) is a slowly progressive and ultimately fatal
degenerative brain disorder that primarily affects the elderly [1]. Newly
available data displays that over 50 million dementia patients, and the
number will be projected to 152 million in 2050. This disease also
produces significant social and economic implications. In 2018, the total
global societal cost of dementia was estimated to be over US$ a trillion,
increasing to US$ 2 trillion by 2030 [2]. Currently, the acetylcholines-
terase inhibitors (AChEs) (donepezil, galantamine and rivastigmine)
were approved by food and drug administration (FDA) for the mild-
moderate AD, and the N-methyl-D-aspartate (NMDA) receptor antago-
nist (memantine) was approved for the moderate-severe AD. Although
these drugs are effective in controlling the AD symptoms to a certain
extent during the period of long-term clinical use, they cannot stop,
prevent, or reverse the progression of AD [3].
(MCI) stage, moderate stage and severe stage. The pathogenesis of AD is
not fully understood [4,5]. Aβ aggregates is considered to be the trig-
gering event in the pathogenesis of AD [6]. The increased fibrillogenic
Aβ prone to aggregate into soluble oligomers, contributing to the dam-
age and death of neurons at synapses. In addition, accumulation of Aβ in
the brain triggers the spread of tau-related neurofibrillary tangles,
neuroinflammation, and neuronal degeneration and death, causing AD
[7]. Therefore, reducing Aβ levels or clearing accumulation of Aβ olig-
omers and protecting neuronal cells in the brain will be beneficial in
treating AD.
The metal ion hypothesis states that high levels and dysregulation of
Cu2+, Fe2+, and Zn2+ are observed in the AD brains, accelerating for-
mation of Aβ plaques as well as neurotoxic oxidative processes [8]. Thus,
biometal chelators would be a promising therapy to reduce Aβ aggre-
gates for treating AD. Moreover, oxidative stress is also a major
AD is a progression degenerative disorder in brain, up to now, the
* Corresponding authors.
E-mail addresses: sangzhipei@126.com (Z. Sang), wuanguo@swmu.edu.cn (A. Wu), tanzhh616@163.com (Z. Tan), liuwm1969@163.com (W. Liu).
1
These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2020.104602
Received 23 August 2020; Received in revised form 1 November 2020; Accepted 24 December 2020
Available online 5 January 2021
0045-2068/© 2020 Elsevier Inc. All rights reserved.

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
contributor to accelerate the accumulation of Aβ oligomers [9]. So,
antioxidant agents are also a promising approach to treat AD.
R1
O
N
OH
O
O
R2
Recently, phase III trials of two monoclonal antibodies against Aβ,
bapineuzumab and solanezumab, failed to significantly improve clinical
outcomes in patients with mild to moderate AD [10]. The failures of
current and previous trials of immunotherapy suggest that targeting of
Aβ alone might not be enough to prevent or slow AD progression, as
multiple mechanisms are involved in AD pathogenesis [10]. Recently,
the multi-target-directed ligands (MTDLs) strategy has been widely
applied and presents the best pharmacological option for halting the
progression of the disease [11–14].
n
R1
N
O
n
R2
Genistein-O-alkylamines derivatives
n = 2, 3, 4, 5, 6
N
NR1R2
=
In addition, autophagy is a vesicle and lysosome-mediated degra-
dative pathway that is essential for protein homeostasis and cell health
[15]. The autophagy-lysosomal system is a complex chain of events
leading to clearance of misfolded or damaged proteins and dysfunctional
organelles. Furthermore, the autophay-initiating Beclin 1 protein is
reduced in early AD, which indicates that initiation of autophay is
decreased in AD [16]. Therefore, autophagy-inducers has been identi-
fied as a promising strategy for the managing of early stage of AD.
Ferroptosis, a novel form of cell death characterized by intracellular
iron overload, may contribute to the neuronal loss of AD [17]. Moreover,
the hallmarks of ferroptosis (iron dysregulation, lipid peroxidation,
inflammation) are recognized as important preclinical signs of AD and
cognitive impairment. Glutathione peroxidase 4 (GPX4) is a unique anti-
peroxidant enzyme, which inhibits lipid peroxidation through directly
reducing membrane lipid hydroperoxides to lipid alcohols. Research
displays that ablation of GPX4 induces ferroptosis of spinal motor neu-
rons, thereby resulting in rapid onset and progression of paralysis and
death in adult mice [18]. Further research determines that ferroptosis
triggers the major cell death in hippocampal neurodegeneration by
ablation of forebrain neuron GPX4, which directly relates to cognitive
impairment [19]. Therefore, improving the levels of GPX4 protein might
present a valuable strategy to delay the pathogenesis of AD.
N
N
N
(A)
(B)
(C)
N
N
N
N
H3C
(F)
Fig. 1. The design strategy of genistein-O-alkylamines derivatives.
CH3
(D)
(E)
(G)
Finally, the key intermediates 3a ~ 3e were treated with different sec-
ondary amine NR1R2 (A ~ G) to get the target compounds 4 ~ 8.
Moreover, these target compounds were confirmed by 1H NMR, 13C
NMR and HR-ESI–MS spectroscopy.
2.2. Pharmacology
2.2.1. AChE and BuChE inhibition
The Ellman’s method was used to evaluate the genistein-O-alkyl-
amines derivatives using donepezil as reference compound [26]. Firstly,
the target compounds were tested using AChE from electrophorus elec-
tricus (eeAChE) and BuChE from equine serum (eqBuChE). The results
were listed in Table 1, expressed as IC50 values. The screening data
displayed that parent compound genistein did not show any eeAChE and
eqBChE inhibitory activity, while most the target derivatives presented
mild to good eeAChE inhibitory activities and weak eqBuChE inhibitory
potency, indicating that introducing O-alkylamines fragments into
genistein skeleton significantly increased eeAChE inhibitory potency.
Particularly, compound 6e presented the best eeAChE inhibitory activity
Genistein is a natural isoflavone with diverse biological activities
including antioxidant activity, neuroprotective property, estrogen ac-
tivity, metal chelating property, anti-inflammatory property, and
amelioration of Aβ-induced impairment, which are beneficial in treating
AD, while the low bioavailability of genistein restricts its clinical use as
anti-AD drug [20–23]. Based on these advantages and disadvantages of
genistein, many genistein-based multifunctional agents have been
developed and applied for the treatment of AD [24–28]. Our previous
work demonstrated that introducing O-alkylbenzylamine fragments into
genistein skeleton significantly increased AChE inhibitory activity,
meanwhile, the candidate agent showed good antioxidant activity,
metal chelating property and significant Aβ aggregates inhibitory ac-
tivities [24,25]. In addition, the N-benzylpiperidine fragment has been
widely applied to develop AChE inhibitor [29–31]. Thus, a series of
novel genistein-O-alkylamines derivatives are designed and developed
to further explore the structure-activity-relationship (SAR) by intro-
ducing different methylene linker and different secondary amines frag-
ments (Fig. 1). The target derivatives are evaluated for the biological
activities including ChEs inhibitory activities, antioxidant activity, ef-
fects on Aβ aggregation, metal chelating property, neuroprotective ef-
fect, inducing autophagy activity, activating GPX4 protein and in vivo
efficacy.
with IC50 value of 0.34 μM. As a whole, both the methylene chain and
secondary amines remarkably influenced eeAChE inhibitory potency.
When the secondary amines fragment was 4-benzylpiperidine (A), the
eeAChE inhibitory activity significantly increased as methylene chain
lengthened, but the optimal length of methylene chain was 4, such as 4a
(IC50 = 30.6
= 6.2 M) > 8a (IC50 = 7.8
was 1-benzylpiperazine (B), the optimal length of methylene chain was
4, such as 4b (IC50 = 26.8 M) < 5b (IC50 = 16.3 M) < 6b (IC50 = 14.8
M) > 7b (IC50 = 15.8 M) > 8b (IC50 = 22.8 M). When the secondary
amines fragment was N-ethylbenzylamine (F) fragment, the optimal
length of methylene chain was 5, such as 6f (IC50 = 2.3 M) < 7d (IC50
= 0.95 M) > 8c (IC50 = 1.6 M). In addition, when the length of
methylene chain was 4, compound 6a with 4-benzylpiperidine group
μ
M) < 5a (IC50 = 13.9
μM) < 6a (IC50 = 3.4 μM) > 7a (IC50
μ
μ
M). When the secondary amines fragment
μ
μ
μ
μ
μ
μ
μ
μ
2. Results and discussion
showed good eeAChE inhibitory activity with IC50 value of 3.4 μM.
When replacing 4-benzylpiperidine of 6a with 1-benzylpiperazine (B) to
get compound 6b, the eeAChE inhibitory activity sharply decreased to
2.1. Chemistry
14.8 μM. The similar results were also observed when the length of
The synthetic strategy of genistein-O-alkylbenzylamines derivatives
4 ~ 8 were depicted in Scheme 1 [25]. The starting material genistein
(1) was treated with excessive amounts of 1,2-dibromoethane (2a), 1,3-
dibromopropane (2b), 1,4-dibromobutane (2c), 1,5-dibromopentane
(2d) or 1,6-dibromohexane (2e) in the presence of anhydrous K2CO3
in CH3CN at 65 ◦C to get the key dibromide intermediates 3a ~ 3e.
methylene chain were 3, 5, 6, respectively. When replacing 4-benzylpi-
peridine of 6a with 4-phenylpiperidine (C) to get compound 6c, the
eeAChE inhibitory activity sharply decreased to 19.8 μM, and replacing
4-benzylpiperidine of 6a with 1,2,3,4-tetrahydroisoquinoline (D) to
obtain compound 6d, the eeAChE inhibitory activity significantly
declined to 16.5 μM. While, when removing the benzyl group of 4-
2

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Scheme 1. Synthesis of genistein-O-alkylbenzylamines derivatives 4 ~ 8. Reagents and conditions: (i) Br(CH2)nBr (2a ~ 2e), K2CO3, CH3CN, 65 ◦C, 8–10 h; (ii) NR1R2
(A ~ G), K2CO3, CH3CN, 65 ◦C, 6–10 h.
benzylpiperidine in 6a to obtain compound 6e with piperidine (E), the
When the length of methylene chain was 5, the potency to inhibit hAChE
eeAChE inhibitory potency significantly increased to 0.34
μM. More-
were in the order N-ethylbenzylamine (F) (7d, IC50 = 0.53
μ
M) > 4-
1,2,3,4-tetrahy-
μM) > 1-benzylpiperazine (B) (7b,
over, When ring-oping of 1,2,3,4-tetrahydroisoquinoline in compound
benzylpiperidine (A) (7a, IC50
droisoquinoline (D) (7c, IC50 = 8.1
=
1.8
μ
M)
>
6d to get compound 6f with N-ethylbenzylamine (F) group, the eeAChE
inhibitory potency significantly increased to 2.3
μ
M. The similar results
IC50 = 20.2 μM), and compound 7d with N-ethylbenzylamine fragment
were also presented when the length of methylene chain was 5, such as
7c with 1,2,3,4-tetrahydroisoquinoline (IC50 = 8.9 M) < 7d with N-
presented the best hAChE inhibitory potency. In general, compound 7d
was a highly selective hAChE inhibitor and worth further study.
μ
ethylbenzylamine (IC50 = 0.95 μM). Furthermore, replacing 1,2,3,4-tet-
rahydroisoquinoline of 6d with 1-methyl-1,2,3,4-tetrahydroisoquino-
line (G) to obtain compound 6 g, the eeAChE inhibitory potency
2.2.2. Enzyme kinetics studies
Based on the above results, compound 7d was selected to perform a
enzyme kinetic study using AChE [25,26]. Lineweaver-Burk plots and
replots of the slope versus concentration were utilized to obtain the
inhibition constants (Ki). Graphical analysis of the reciprocal
Lineweaver-Burk plots (Fig. 2A) represented both increasing slopes
(decreased Vmax) and intercepts (higher Km) at increasing concentration
of the inhibitor 7d, indicating a mixed-type inhibition. The obtained Ki
sharply increased to 1.2 μM.
Further, the target compounds 4 ~ 8 were re-tested using human
AChE (hAChE) and human BuChE (hBuChE). As described in Table 1,
most of the target derivatives showed mild to good hAChE inhibitory
activity and weak hBuChE inhibition, displaying that most of the de-
rivatives were highly selective hAChE inhibitors. Among these com-
pounds, compound 7d presented the best hAChE inhibitory activity
was 0.50
value.
μM (Fig. 2B), which was consistent with the measured IC50
(IC50 = 0.53 μM). Similarly, both the methylene chain (n = 2, 3, 4, 5 and
6) and secondary amines (A ~ G) remarkably influenced hAChE inhib-
itory potency. When the secondary amines fragment was 4-benzylpiper-
idine (A), the hAChE inhibitory potency remarkably increased as
methylene chain increased, but the optimal length of methylene chain
2.2.3. Molecular modeling study
In order to further explore the possible interacting mechanism of
compound 7d, a molecular docking study was performed using the
crystal structure of hAChE (PDB code: 4ey7) [26,32]. As reported,
Trp86, Asp74, Tyr72, Tyr124, Trp286, Phe297, Phe295, and Tyr337
were the key active sites in hAChE [33]. In the 7d-hAChE complex
(Fig. 3), the 5-hydroxychromen-4-one skeleton interacted with the key
was 5, such as 4a (IC50 = 23.5
6.1 M) < 7a (IC50 = 1.8 M) > 8a (IC50 = 3.2
amines fragment was 1-benzylpiperazine (B), compound 5b displayed
μ
M) < 5a (IC50 = 20.2
μM) < 6a (IC50 =
μ
μ
μ
M). When the secondary
the best hAChE inhibitory potency (IC50 = 1.4 μM) and the optimal
length of methylene chain was 3. When the secondary amines fragment
active residue Tyr341 via two π-π interactions. Moreover, the 5-hydroxy
was N-ethylbenzylamine (F) fragment, compounds 6f, 7d and 8c
and 4-carbonyl group at the 5-hydroxychromen-4-one skeleton formed
one intramolecular hydrogen bond and the 5-hydroxy group could
interact with important active residue Phe338 via one intermolecular
hydrogen bond. The O atom of pyran interacted with key residue Tyr124
via one intermolecular hydrogen bond. The benzene ring of the N-eth-
ylbenzylamine fragment was observed to interact with the key residue
possessed good hAChE inhibitory potency with IC50 values of 0.89 μM,
0.53 μM and 2.8 μM, respectively, and the optimal length of methylene
chain was 5. Furthermore, under the condition with the length of
methylene chain was 4, the potency to inhibit hAChE were in the order
piperidine (E) (6e, IC50 = 0.78 μM) > N-ethylbenzylamine (F) (6f, IC50
= 0.89
2.5 M) > 4-benzylpiperidine (A) (6a, IC50 = 6.1
hydroisoquinoline (D) (6d, IC50 = 11.8 M) > 4-phenylpiperidine (C)
(6c, IC50 = 20.7 M) > 1-benzylpiperazine (B) (8a, IC50 = 25.3 M).
μ
M) > 1-methyl-1,2,3,4-tetrahydroisoquinoline (G) (6 g, IC50
=
Trp286 via two π-π interactions. In addition, compound 7d could also
μ
μ
M) > 1,2,3,4-tetra-
interact with Trp86, Trp286, Tyr124, Tyr341 and Phe338 through hy-
drophobic interactions. The observed interactions provided rational
explanation for the high AChE inhibitory activity towards compound 7d.
μ
μ
μ
3

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Table 1
ChEs (AChE/BChE) inhibition by 4 ~ 8 and positive compounds.
Comp.
n
NR1R2
IC50 ± SD (
EeAChEb
μ
M)a
ORACf
EqBChEc
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
hAChEd
hBuChEe
4a
4b
5a
5b
6a
6b
6c
2
2
3
3
4
4
4
30.6 ± 2.7
26.8 ± 1.6
23.5 ± 0.91
28.6 ± 0.79
20.2 ± 0.87
1.4 ± 0.08
6.1 ± 0.16
25.3 ± 0.79
20.7 ± 1.3
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
1.1 ± 0.02
1.6 ± 0.03
1.2 ± 0.03
1.4 ± 0.02
0.98 ± 0.02
1.5 ± 0.03
1.1 ± 0.03
13.9 ± 0.97
16.3 ± 0.76
3.4 ± 0.81
14.8 ± 2.1
19.8 ± 0.88
6d
6e
6f
4
4
4
16.5 ± 0.59
0.34 ± 0.02
2.3 ± 0.85
n.a.g
n.a.g
n.a.g
11.8 ± 0.28
0.78 ± 0.06
0.89 ± 0.04
n.a.g
n.a.g
n.a.g
1.0 ± 0.02
0.96 ± 0.02
1.1 ± 0.03
6g
4
1.2 ± 0.06
n.a.g
2.5 ± 0.14
n.a.g
1.0 ± 0.02
7a
7b
7c
7d
8a
8b
8c
5
5
5
5
6
6
6
6.2 ± 0.95
15.8 ± 0.83
8.9 ± 0.41
0.95 ± 0.03
7.8 ± 0.59
22.8 ± 0.34
1.6 ± 0.07
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
1.8 ± 0.69
20.2 ± 0.83
8.1 ± 0.32
0.53 ± 0.37
3.2 ± 0.44
24.1 ± 0.29
2.8 ± 0.29
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
1.1 ± 0.03
1.4 ± 0.04
0.97 ± 0.02
1.0 ± 0.03
0.99 ± 0.02
1.4 ± 0.02
0.97 ± 0.03
Genistein
Donepezil
>100 ± 3.6
>100 ± 5.2
5.21 ± 0.02
>100 ± 4.7
>100 ± 5.9
7.1 ± 0.14
5.6 ± 0.05
0.019 ± 0.0005
0.012 ± 0.0003
NTh
a
IC50 values: 50% inhibitory concentration (means ± SD of three experiments).
EeAChE from electrophorus electricusc.
EqBChE from equine serum.
b
c
d
e
f
hAChE from human erythrocytes.
hBuChE from human serum.
Results are expressed as μM of Trolox equivalent/μM of tested compound.
g
h
n.a. = no active. Compounds defined “no active” means percent inhibition less than 5.0% at a concentration of 12.5
NT = not tested.
μ
M in the assay conditions.
2.2.4. hAChE reversibility of inhibition by compound 7d
data in Table 1, the parent compound genistein indicated excellent
antioxidant activity (ORAC = 4.8 eq), all the target compounds exhibi-
ted good antioxidant potency with ORAC values of 0.9 ~ 1.5 eq., indi-
cating that introducing O-alkylamines fragments into genistein skeleton
significantly decreased antioxidant activity. In addition, the length of
methylene chain did not produce positive effect on the antioxidant ac-
tivity. The secondary amines fragments did not significantly change the
antioxidant activity, except the compounds with 1-benzylpiperazine
fragment, such as 4b (ORAC = 1.6 eq), 5b (ORAC = 1.4 eq), 6b
(ORAC = 1.5 eq), 7b (ORAC = 1.4 eq), 8b (ORAC = 1.4 eq). Among these
derivatives, compound 7d presented good antioxidant activity with
ORAC value of 1.1 eq.
In order to evaluate the reversibility of inhibition by compound 7d,
we assessed the recovery of huAChE inhibition after dilution (Fig. 4A)
with time monitoring (Fig. 4B) [26,34]. As reference reported, donepezil
was a reversible huAChE inhibitor, which was supported by the tested
data in Fig. 4A by the quick recovery of huAChE level. Moreover, the 0.1
× IC50 of compound 7d increased hAChE enzyme activity to 9.7%
compared with control. The further recovery of compound 7d inhibition
after dilution with time monitoring was displayed in Fig. 4B, the hAChE
activity towards 7d exhibited a obvious increase at the first 10 min,
which was consistent with donepezil, and the hAChE activity restored to
95.4% with 0.1 × IC50 compound 7d at 120 min. The results displayed
that compound 7d was a reversible huAChE inhibitor.
2.2.6. Metal-chelating property of 7d
2.2.5. Antioxidant activity
The chelation property of compound 7d was evaluated by UV–visual
spectrometry using Cu2+, Fe2+, Zn2+ and Al3+ [26,34]. As presented in
Fig. 5A, when compound 7d was treated with Cu2+ and Al3+, the elec-
tronic spectra indicated a red shift (the peak shifted from 334 nm to 376
The antioxidant activity of target derivatives 4 ~ 8 were assessed by
oxygen radical absorbance capacity fluorescein (ORAC-FL) method
using Trolox as a referenced compound [26]. According to the screening
4

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Fig. 2. (A) Lineweaver-Burk plot of 7d for AChE hydrolysis. (B) Slope replot vs 7d concentration.
nm and 384 nm, respectively). While, when treating with Fe2+ and Zn2+
,
(Fig. 6) using the transmission electron microscopy (TEM).
respectively, the electronic spectra did not present obvious shift. The
results showed that compound 7d was a selective metal chelator by
selectively chelating Cu2+ and Al3+. The stoichiometry of the 7d-Cu2+
complex was determined using the molar ratio assay at 376 nm. As
presented in Fig. 5B, the absorbance linearly increased initially and then
plateaued. The two straight lines intersected at a mole fraction of 0.98,
2.2.8. Neuroprotective effects against H2O2–induced PC12 cell injury
The neuroprotective effects of 7d against H2O2–induced PC12 cell
injury were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylte-
trazolium (MTT) assays and lactate dehydrogenase (LDH) assay using
Vitamine E (VE) as the referenced compound [26,34]. As presented in
revealing a 1:1 stoichiometry for the complex 7d-Cu2+
.
Fig. 7A, when PC12 cells were exposed to 100
sharply declined to 50.9% (p < 0.01) compared with the untreated
group. When treating with 100 M VE, the cell viability added up to
65.8% (p < 0.01). Under the same condition, when the PC12 cells were
μM H2O2, the cell viability
2.2.7. Effects on the Aβ aggregation
μ
Studies showed that Aβ aggregates is the triggering event in the
pathogenesis of AD [6]. Aβ deposition in the brain of AD patients had
been linked to AChE expression, and the PAS of AChE bond to the Aβ and
accelerated the formation of Aβ fibrils [35]. Moreover, Cu2+ could
induce and aggravate Aβ aggregation [9]. So, inhibiting and dis-
aggregating Aβ aggregates would be beneficial for the treatment of AD.
In order to investigate the effects on Aβ aggregation, we performed self-
induced Aβ1-42 aggregation, hAChE-induced Aβ1-40 aggregation and
treated with 10 μM and 100 μM compound 7d, respectively, the cell
viability increased to 70.3% (p < 0.05) and 79.6% (p < 0.01), respec-
tively, which was better than VE. Furthermore, the neuroprotective ef-
fect of compound 7d were also assessed by LDH assay [26]. As displayed
in Fig. 7B, the PC12 cells were treated with 100 μM H2O2, the LDH vi-
tality suddenly increased to 624.7 (p < 0.01) compared with vehicle
group (544.5), the LDH vitality decreased to 608.4 (p < 0.01) after
Cu2+-induced Aβ
aggregation using ThT fluorescence assay [26].
adding 100
μM VE. Under the same conditions, when treating with 10
According to the 1s-c4r2eening data in Table 2, compound 7d significantly
μ
M and 100 μM compound 7d, respectively, the LDH vitality declined to
inhibited self-induced Aβ
aggregation with 39.8% inhibition rate,
577.8 (p < 0.05) and 529.8 (p < 0.05), respectively. Therefore, the re-
sults reveled that compound 7d exhibited good neuroprotective effect
against H2O2-induced PC12 cell injury.
which was similar with cu1r-c4u2min (42.6%). Compound 7d also inhibited
hAChE-induced Aβ
aggregation (42.1%), which was better than
1-40
donepezil (20.1%). Furthermore, compound 7d could significantly
inhibit and disaggregate Cu2+-induced Aβ
aggregation with 74.1%
2.2.9. The activity of inducing-autophagy
1-42
inhibition rate and 67.3% disaggregation rate, respectively, which were
The stable GFP-RFP-LC3 U87 cells were employed to evaluate the
effect of inducing-autophagy by compound 7d [26]. As displayed in
better than curcumin. Further, the ThT results of Cu2+-induced Aβ
1-42
aggregation were also supplemented by the observed phenomenon
Fig. 8, compound 7d (50 μM, 25 μM and 12.5 μM) presented significant
5

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
2.2.10. Modulation of GPX4 protein
The effects of compound 7d on GPX4 protein was performed on PC12
cells using MTT assay [36]. As shown in Fig. 9, when the PC12 cells were
treated with RSL3 (GPX4 inhibitor), the cell viability sharply decreased
to 45.1% (p < 0.01) compared with control group. While, when com-
pound 7d (20
μM, 10 μM and 5 μM) was added to the PC12 cells,
respectively, the cell viability was 73.9%, 18.4% and 21%, respectively.
The data displayed 20 μM compound 7d significantly increased cell
viability and improved the levels of GPX4 protein.
2.2.11. In vitro blood-brain barrier permeation assay of 7d
The blood-brain barrier permeability of 7d was assessed by the
parallel artificial membrane permeation assay of the blood-brain barrier
(PAMPA-BBB) using genistein as positive compound [37,38]. This
method was verified using 11 commercial drugs (Table S1), and a good
linear correlation, Pe(exp) = 0.9163Pe(lit.) ꢀ 0.2247 (R2 = 0.9558)
(Fig. S1), was produced by the experimental data versus the reported
values. Finally, the compounds with permeability above 3.44 × 10ꢀ 6
cm/s were deemed to cross the BBB (Table S2). As shown in Table 3,
compound 7d presented 16.92 × 10ꢀ 6 cm/s permeability, exhibiting
good BBB permeability in vitro.
2.2.12. Precognitive effects in vivo by compound 7d
Acute Toxicity. In order to further evaluate the safety profile of
compound 7d, the acute toxicity experiment at doses of 100, 250, 500
and 1000 mg/kg (n = 10 per group) was carried out by oral adminis-
tration of compound 7d using Kunming mice at body weight of 18–22 g.
The results revealed that the animals treated with compound 7d did not
show any acute toxicity and well tolerated at doses of up to 1000 mg/kg.
Effect of 7d on scopolamine-induced mice memory impairment. The step-
down passive avoidance task was performed to evaluate the effects of 7d
on scopolamine-induced memory impairment [26,34]. As displayed in
Fig. 10, the mice were treated with 3 mg/kg scopolamine alone, the step-
down latency (128.7 sec, p < 0.01) was significantly shorter than un-
treated mice (229.3 sec). While treating with 5 mg/kg donepezil, the
latency time significantly increased to 210.3 sec (p < 0.01) and clearly
improved cognitive deficit induced by scopolamine. Furthermore,
treatment with compound 7d at concentration of 4.5 mg/kg, 9 mg/kg
and 18 mg/kg, respectively, the latency time gradually increased to
176.8 sec (p < 0.05), 196.7 sec (p < 0.01) and 217.3 sec (p < 0.05),
respectively in a dose-dependent manner. The results revealed that
compound 7d with high dose could improve scopolamine-induced
memory impairment, deserving to further study.
3. Conclusion
AD is a slowly progressive and ultimately fatal degenerative brain
disorder that primarily affects the elderly. The exact pathogenesis of AD
remains unclear due to the complex and multifactorial causes. At pre-
sent, the MTDLs strategy might present the best pharmacological option
for tackling the multifactorial nature of AD. Based on our previous work,
a series of genistein-O-alkylamines derivatives was rationally developed
and further explored the structure-activity-relationship. The in vitro re-
sults displayed that compound 7d was a reversible and highly selective
Fig. 3. (A) Representation of compound 7d (green stick) interacting with res-
idues in the binding site of hAChE (PDB code: 4ey7), highlighting the protein
residues that participate in the main interactions with the inhibitor. (B) 3D
docking model of compound 7d (green stick) with hAChE. (C) 2D schematic
diagram of docking model of compound 7d with hAChE. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)
hAChE inhibitor (IC50 = 0.53 μM). Both the kinetic study and molecular
docking showed that 7d was a mixed-type AChE inhibitor and could
bind simultaneously to the catalytic active site and peripheral anionic
site of AChE. Compound 7d also displayed good antioxidant activity
(ORAC = 1.1 eq.), neuroprotective effects and metal chelation property.
Furthermore, compound 7d significantly inhibited self-induced, hAChE-
induced and Cu2+-induced Aβ aggregation with 39.8%, 42.1% and
74.1%, respectively, and remarkably disaggregated Cu2+-induced Aβ
1-42
activity of inducing-autophagy in a dose-dependent manner compared
with control group, indicating that compound 7d was a promising
autophagy inducer.
aggregation (67.3%). In addition, compound 7d significantly induced
autophagy and improved the levels of GXP4 protein. More importantly,
compound 7d cross blood-brain-barrier in vitro. Further, in vivo assay
revealed that 7d significantly reversed scopolamine-induced memory
6

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Fig. 4. (A) hAChE recovery after preincubation of 7d diluted to 1× or 0.1 × IC50, compared to standard donepezil diluted, and undiluted inhibition. (B) huAChE
recovey of donepezil and 7d diluted to 0.1 × IC50, were monitored with time at room temperature. Data points, representing the average of three experiments, at
least, were expressed as the mena ± SEM, n = 3.
deficit. Therefore, compound 7d was a promising multi-function agent
with CH2Cl2 (20 mL × 3). The combined organic phases were washed
with saturated aqueous NaCl, dried over Na2SO4, and filtered. The sol-
vent was evaporated to dryness under reduced pressure. The residue was
purified on a silica gel chromatography using petroleum ether/acetone
(15:1) as eluent to get the target compounds 4 ~ 8, and compounds 6a
~ 6 g had been presented in our previous work.
for the development of anti-AD drugs and deserved further study.
4. Experiment section
4.1. Chemistry
7-(2-(4-Benzylpiperidin-1-yl)ethoxy)-3-(4-(2-(4-benzylpiperidin-1-
yl)ethoxy)phenyl)-5-hydroxy-4H-chromen-4-one (4a). Yellow oil,
86.7% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.83 (s,
All the reactions were monitored by thin-layer chromatography
(TLC) with an UV lamp (254 nm). The 1H NMR and 13C NMR spectra
were recorded in CDCl3 solutions using TMS as the internal standard on
a Varian INOVA at 400 and 100 MHz, respectively. The high-resolution
mass spectra (HR-MS) were obtained using a Waters Xevo G2-XS-Qtof
mass spectrometer. The purity of all final compounds was determined
by high-performance liquid chromatography (HPLC) analysis to be over
97%.
–
–
1H, OH), 7.81 (s, 1H, Ar H), 7.41 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.27
–
–
(t, J = 8.0 Hz, 4H, 4 × Ar H), 7.18 (t, J = 8.0 Hz, 2H, 2 × Ar H), 7.13
–
–
(d, J = 7.2 Hz, 4H, 4 × Ar H), 6.93 (d, J = 8.8 Hz, 2H, 2 × Ar H), 6.35
–
(dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 4.15–4.10 (m, 4H, 2 ×
OCH2), 2.99 (t, J = 11.2 Hz, 4H, 2 × NCH2), 2.81–2.77 (m, 4H, 2 ×
NCH2), 2.53 (d, J = 7.2 Hz, 4H, 2 × phCH2), 2.07 (t, J = 11.6 Hz, 4H, 2
× NCH2), 1.65 (d, J = 12.8 Hz, 4H, 2 × CH2), 1.57–1.50 (m, 2H, 2 × CH),
1.41–1.31 (m, 4H, 2 × CH2). HR-ESI-MS: Calcd. for C43H48N2O5 [M +
H]+: 673.3597, found: 673.3643.
4.1.1. General procedure for the synthesis of compounds 3a ~ 3e
The starting material genistein (1) (3.0 mmol) was treated with
excessive amounts (9.0 mmol) of 1,2-dibromoethane (2a), 1,3-dibromo-
propane (2b), 1,4-dibromobutane (2c), 1,5-dibromopentane (2d) or 1,6-
dibromohexane (2e), respectively. And compounds 3a ~ 3e were ob-
tained according to our previous detailed procedure [25,26].
7-(2-(4-Benzylpiperazin-1-yl)ethoxy)-3-(4-(2-(4-benzylpiperazin-1-
yl)ethoxy)phenyl)-5-hydroxy-4H-chromen-4-one (4b). Yellow oil,
80.2% yield, 98.5% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.83 (s,
–
–
1H, OH), 7.84 (s, 1H, Ar H), 7.42 (d, J = 8.8 Hz, 2H, 2 × Ar H),
–
–
7.32–7.30 (m, 8H, 8 × Ar H), 7.27–7.24 (m, 2H, 2 × Ar H), 6.95 (d, J
–
4.1.2. General procedure for the synthesis of target compounds 4 ~ 8
To a solution of Compounds 3a ~ 3e (1.0 mmol) were treated with
the corresponding secondary amines (A ~ G) (3.0 mmol), respectively,
in anhydrous CH3CN (10 mL). And then the reaction mixture was heated
to 65 ◦C for 8–10 h under an argon atmosphere. After reaction
completed, the organic solvent was evaporated under reduced pressure.
Water (20 mL) was added to the residue, and the mixture was extracted
= 8.4 Hz, 2H, 2 × Ar H), 6.37 (dd, J1 = 7.6 Hz, J2 = 2.0 Hz, 2H, 2 ×
–
Ar H), 4.16–4.12 (m, 4H, 2 × OCH2), 3.53 (s, 4H, phCH2), 2.84 (t, J =
5.6 Hz, 4H, 2 × NCH2), 2.65–2.62 (m, 8H, 4 × NCH2), 2.55–2.51 (m, 8H,
4 × NCH2). HR-ESI-MS: Calcd. for C41H46N4O5 [M + H]+: 675.3502,
found: 675.3557.
7-(3-(4-Benzylpiperidin-1-yl)propoxy)-3-(4-(3-(4-benzylpiperidin-1-
yl)propoxy)phenyl)-5-hydroxy-4H-chromen-4-one (5a). Yellow oil,
7

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Fig. 5. (A) The UV spectrum of compound 7d (37.5
μ
M, in methanol) alone or in the presence of CuCl2, FeSO4, ZnCl2 and AlCl3 (37.5
μ
M, in methanol); (B)
M) with ascending
Determination of the stoichiometry of complex-Cu2+ by using molar ratio method through titrating the methanol solution of compound 7d (37.5
μ
amounts of CuCl2 (ranging from 3.75 to 93.75 μM).
76.9% yield, 97.7% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.84 (s,
NCH2), 2.00–1.96 (m, 4H, 2 × CH2). 13C NMR (100 MHz, CDCl3) 180.8,
165.0, 162.7, 159.3, 157.9, 152.6, 138.1, 130.1, 129.2, 128.2, 127.1,
123.6, 122.9, 114.7, 106.2, 98.6, 92.9, 66.9, 66.4, 63.1, 55.1, 54.9, 53.2,
53.0, 26.7, 26.5. HR-ESI-MS: Calcd. for C43H50N4O5 [M + H]+:
703.3815, found: 703.3867.
–
–
1H, OH), 7.85 (s, 1H, Ar H), 7.43 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.28
–
–
(t, J = 7.6 Hz, 6H, 6 × Ar H), 7.19 (t, J = 7.2 Hz, 2H, 2 × Ar H), 7.14
–
–
(d, J = 8.8 Hz, 4H, 4 × Ar H), 6.95 (d, J = 8.8 Hz, 2H, 2 × Ar H), 6.37
–
(dd, J1 = 7.2 Hz, J2 = 1.6 Hz, 2H, 2 × Ar H), 4.09–4.02 (m, 4H, 2 ×
OCH2), 2.98 (t, J = 12.0 Hz, 4H, 2 × NCH2), 2.57–2.50 (m, 8H, 2 ×
phCH2 + 2 × NCH2), 2.05–1.92 (m, 8H, 2 × NCH2 + 2 × CH2), 1.67 (d, J
= 12.8 Hz, 4H, 2 × CH2), 1.60–1.52 (m, 2H, 2 × CH), 1.44–1.32 (m, 4H,
2 × CH2). 13C NMR (100 MHz, CDCl3) 180.8, 165.0, 162.7, 159.2, 157.9,
152.6, 140.6, 140.5, 130.1, 129.1, 128.2, 125.8, 123.7, 123.0, 114.7,
106.2, 98.7, 92.8, 67.0, 66.4, 55.4, 55.2, 53.9, 53.8, 43.1, 43.0, 37.8,
37.7, 31.9, 31.7, 29.7, 26.5, 26.4. HR-ESI-MS: Calcd. for C45H52N2O5 [M
+ H]+: 701.3910, found: 701.3962.
7-(4-(4-Benzylpiperidin-1-yl)butoxy)-3-(4-(4-(4-benzylpiperidin-1-
yl)butoxy)phenyl)-5-hydroxy-4H-chromen-4-one (6a). Yellow oil,
77.5% yield, 97.5% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.77 (s,
–
–
1H, OH), 7.76 (s, 1H, Ar H), 7.35 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.19
–
–
(t, J = 4.0 Hz, 4H, 4 × Ar H), 7.11 (t, J = 7.2 Hz, 2H, 2 × Ar H), 7.06
–
–
(d, J = 8.8 Hz, 4H, 4 × Ar H), 6.86 (d, J = 8.8 Hz, 2H, 2 × Ar H), 6.28
–
(dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 3.96–3.90 (m, 4H, 2 ×
OCH2), 2.95 (t, J = 12.0 Hz, 4H, 2 × NCH2), 2.47 (d, J = 6.8 Hz, 4H, 2 ×
phCH2), 2.44–2.36 (m, 4H, 2 × NCH2), 1.97–1.87 (m, 4H, 2 × NCH2),
1.74–1.64 (m, 8H, 4 × CH2), 1.59 (d, J = 12.4 Hz, 4H, 2 × CH2),
1.53–1.44 (m, 2H, 2 × CH), 1.39–1.32 (m, 4H, 2 × CH2). HR-ESI-MS:
Calcd. for C47H56N2O5 [M + H]+: 729.4223, found: 729.4263.
7-(3-(4-Benzylpiperazin-1-yl)propoxy)-3-(4-(3-(4-benzylpiperazin-
1-yl)propoxy)phenyl)-5-hydroxy-4H-chromen-4-one (5b). Yellow oil,
80.6% yield, 97.8% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.84 (s,
–
–
1H, OH), 7.84 (s, 1H, Ar H), 7.43 (d, J = 8.4 Hz, 2H, 2 × Ar H),
–
–
7.32–7.30 (m, 8H, 8 × Ar H), 7.27–7.24 (m, 2H, 2 × Ar H), 6.96 (d, J
7-(4-(4-Benzylpiperazin-1-yl)butoxy)-3-(4-(4-(4-benzylpiperazin-1-
yl)butoxy)phenyl)-5-hydroxy-4H-chromen-4-one (6b). Yellow oil,
70.4% yield, 97.3% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.85 (s,
–
–
= 8.8 Hz, 2H, 2 × Ar H), 6.37 (d, J = 9.2 Hz, 2H, 2 × Ar H), 4.08–4.01
(m, 4H, 2 × OCH2), 3.52 (s, 4H, 2 × phCH2), 2.56–2.49 (m, 20H, 10 ×
8

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
(m, 4H, 2 × OCH2), 3.67 (s, 4H, 2 × phCH2), 2.92–2.86 (m, 4H, 2 ×
phCH2), 2.78–2.75 (m, 4H, 2 × NCH2), 2.63–2.59 (m, 4H, 2 × NCH2),
1.88–1.80 (m, 8H, 4 × CH2). HR-ESI-MS: Calcd. for C41H44N2O5 [M +
H]+: 645.3284, found: 645.3319.
Table 2
Inhibition of self-induced Aβ1-42 aggregation, Cu2+-induced Aβ1-42 aggregation,
hAChE-induced Aβ1-40 aggregation and disaggregation of Cu2+-induced Aβ1-42
aggregation by compound 7d and referenced compounds.
% Inhibition of Aβ Aggregation a
% Disaggregation of Cu2+
induced Aβ1-42
-
5-Hydroxy-7-(4-(piperidin-1-yl)butoxy)-3-(4-(4-(piperidin-1-yl)
butoxy)phenyl)-4H-chromen-4-one (6e). Light yellow oil, 73.2% yield,
97.9% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.85 (s, 1H, OH), 7.86
Compound
self-
hAChE-
Cu2+
-
aggregation e
induced b
induced c
induced d
–
–
(s, 1H, Ar H), 7.44 (d, J = 8.4 Hz, 2H, 2 × Ar H), 6.95 (d, J = 8.4 Hz,
7d
39.8 ±
0.86
42.1 ±
0.79
74.1 ± 1.2
67.3 ± 0.59
58.3 ± 0.88
NT f
–
–
2H, 2 × Ar H), 6.36 (d, J = 13.2 Hz, 2H, 2 × Ar H), 4.04–4.00 (m, 4H,
2 × OCH2), 2.59–2.52 (m, 12H, 6 × NCH2), 1.82–1.78 (m, 8H, 4 × CH2),
1.74–1.71 (m, 8H, 4 × CH2), 1.51–1.49 (m, 4H, 2 × CH2). 13C NMR (100
MHz, CDCl3) δ 180.8, 164.9, 162.6, 159.1, 157.9, 152.7, 130.1 (2C),
123.6, 122.9, 114.6 (2C), 106.2, 98.6, 92.7, 68.2, 67.5, 58.3 (2C), 54.1
(2C), 54.0 (2C), 27.1, 26.9, 25.0 (2C), 24.7 (2C), 23.8, 23.7, 22.6, 22.5.
HR-ESI-MS: Calcd. for C33H44N2O5 [M + H]+: 549.3284, found:
549.3299.
Curcumin
Donepezil
42.6 ±
0.51
NT f
56.2 ±
0.94
NT f
20.1 ±
NT f
0.38
a
For inhibition of Aβ aggregation using ThT assay, data are presented as the
mean ± SEM of three independent experiments.
b
Inhibition of self-induced Aβ1-42 aggregation, the concentration of 7d and
Aβ1-42 were 25
μM.
7-(4-(Benzyl(ethyl)amino)butoxy)-3-(4-(4-(benzyl(ethyl)amino)
butoxy)phenyl)-5-hydroxy-4H-chromen-4-one (6f). Yellow oil, 68.9%
yield, 97.5% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.86 (s, 1H,
c
Inhibition of human AChE-induced Aβ1-40 aggregation, the concentration of
7d and Aβ1-40 was 100 and 230
μ
M, respectively.
d
e
Inhibition of Cu2+-induced Aβ1-42 aggregation by compound 7d at 25
μM.
–
–
Disaggregating Cu2+-induced Aβ1-42 aggregation using ThT assay. The con-
OH), 7.85 (s, 1H, Ar H), 7.44 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.35–7.28
–
–
(m, 8H, 8 × Ar H), 7.25–7.23 (m, 2H, 2 × Ar H), 6.94 (d, J = 8.8 Hz,
centration of 7d and Aβ1-42 were 25
μ
M.
f
–
–
2H, 2 × Ar H), 6.34 (dd, J1 = 4.0 Hz, J2 = 2.4 Hz, 2H, 2 × Ar H),
3.98–3.93 (m, 4H, 2 × OCH2), 3.60 (s, 2H, phCH2), 3.59 (s, 2H, phCH2),
2.57–2.47 (m, 8H, 4 × NCH2), 1.85–1.77 (m, 4H, 2 × CH2), 1.70–1.61
(m, 4H, 2 × CH2), 1.06 (t, J = 7.2 Hz, 6H, 2 × CH3). 13C NMR (100 MHz,
CDCl3) δ 180.9, 165.1, 162.7, 159.3, 157.9, 152.6, 130.1 (3C), 129.0
(3C), 128.2 (4C), 126.9 (3C), 123.7, 122.8, 114.7 (3C), 106.2, 98.6,
92.8, 68.4, 67.8, 58.0, 57.9, 52.5, 52.4, 47.2 (2C), 27.0, 26.7, 23.3 (2C),
11.7, 11.6. HR-ESI-MS: Calcd. for C41H48N2O5 [M + H]+: 649.3597,
found: 649.3622.
NT = not tested.
–
–
–
1H, OH), 7.82 (s, 1H, Ar H), 7.42 (d, J = 8.4 Hz, 2H, 2 × Ar H),
–
7.32–7.28 (m, 8H, 8 × Ar H), 7.25–7.20 (m, 2H, 2 × Ar H), 6.94 (d, J
–
= 8.8 Hz, 2H, 2 × Ar H), 6.34 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz, 2H, 2 ×
–
Ar H), 4.02–3.97 (m, 4H, 2 × OCH2), 3.51 (s, 4H, phCH2), 2.53–2.46
(m, 16H, 8 × NCH2), 2.43–2.37 (m, 4H, 2 × NCH2), 1.83–1.78 (m, 4H, 2
× CH2), 1.69–1.63 (m, 4H, 2 × CH2). HR-ESI-MS: Calcd. for C45H54N4O5
[M + H]+: 731.4128, found: 731.4173.
5-Hydroxy-7-(4-(1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)
7-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)butoxy)-3-(4-(4-(3,4-dihy-
droisoquinolin-2(1H)-yl)butoxy)phenyl)-5-hydroxy-4H-chromen-4-one
(6d). Yellow oil, 68.9% yield, 98.0% HPLC purity. 1H NMR (400 MHz,
butoxy)-3-(4-(4-(1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)butoxy)
phenyl)-4H-chromen-4-one (6 g). Yellow oil, 59.8% yield, 97.3% HPLC
purity. 1H NMR (400 MHz, CDCl3) δ 12.85 (s, 1H, OH), 7.84 (s, 1H,
–
CDCl3) δ 12.85 (s, 1H, OH), 7.84 (s, 1H, Ar H), 7.43 (d, J = 8.4 Hz, 2H,
–
–
Ar H), 7.43 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.15–7.12 (m, 4H, 4 ×
–
–
2 × Ar H), 7.21–7.01 (m, 8H, 8 × Ar H), 6.97 (d, J = 8.4 Hz, 2H, 2 ×
–
–
Ar H), 7.10–7.06 (m, 4H, 4 × Ar H), 6.95 (d, J = 8.4 Hz, 2H, 2 ×
–
–
Ar H), 6.38 (dd, J1 = 11.2 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 4.06–4.01
Fig. 6. (A) The inhibition experiment of Cu2+-induced Aβ1-42 aggregation, TEM images of Aβ species from inhibition experiments. (B) The disaggregation experi-
ments of Cu2+-induced Aβ1-42 aggregation, TEM images of samples from disaggregation experiments.
9

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
Fig. 7. (A) The cell viability (%) of compound 7d on H2O2-induced PC12 cell injury by MTT assay. (B) The LDH vitality of compound 7d on H2O2-induced PC12 cell
injury. Three independent experiments were carried out in triplicate. Data were expressed as mean ± SD and percentage of control value. ##p < 0.01 vs control; **p
< 0.01, *p < 0.05 vs H2O2 group.
Fig. 8. The activation of autophagy by 7d (50 µM, 25 µM and 12.5 µM) on stable GFP-LC3 U87 cells using fluorescence microscopy.
–
–
– –
Hz, 5H, 5 × Ar H), 6.97 (d, J = 8.8 Hz, 2H, 2 × Ar H), 6.55 (s, 1H,
Ar H), 6.37 (dd, J1 = 11.2 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 4.07–4.00
(m, 4H, 2 × OCH2), 3.96–3.89 (m, 2H, 2 × phCH), 3.13–3.08 (m, 2H,
phCH2), 2.98–2.90 (m, 2H, phCH2), 2.85–2.78 (m, 2H, NCH2),
2.75–2.73 (m, 2H, NCH2), 2.71–2.63 (m, 4H, 2 × NCH2), 1.89–1.82 (m,
4H, 2 × CH2), 1.81–1.74 (m, 4H, 2 × CH2), 1.37 (d, J = 3.2 Hz, 3H, CH3),
1.35 (d, J = 3.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 180.8,
165.0, 162.7, 159.3, 152.6, 130.1 (4C), 128.9 (4C), 127.4 (4C), 126.0
(2C), 125.8 (2C), 114.7 (3C), 98.6, 92.8, 68.4, 67.8, 56.5 (2C), 53.3,
53.2, 43.7 (2C), 27.1 (2C), 26.9 (2C), 23.9 (2C), 19.3 (2C). HR-ESI-MS:
Calcd. for C43H48N2O5 [M + H]+: 673.3597, found: 673.3635.
–
–
–
Ar H), 6.45 (s, 1H, Ar H), 6.32 (s, 1H, Ar H), 4.02–3.98 (m, 4H, 2 ×
OCH2), 2.99 (d, J = 10.8 Hz, 4H, 2 × phCH2), 2.54 (d, J = 6.8 Hz, 4H, 2
× NCH2), 2.41 (t, J = 8.0 Hz, 4H, 2 × NCH2), 1.95 (t, J = 11.6 Hz, 4H, 2
× NCH2), 1.84–1.80 (m, 4H, 2 × CH2), 1.68–1.58 (m, 8H, 4 × CH2),
1.57–1.52 (m, 2H, 2 × CH), 1.52–1.38 (m, 2H, 2 × CH). 13C NMR (100
MHz, CDCl3), 182.4, 164.9, 164.0, 162.1, 162.0, 157.7, 140.5, 129.1,
129.0, 128.2, 128.0, 125.9, 123.3, 114.9, 105.4, 104.2, 98.5, 93.0, 68.4,
68.1, 58.7, 54.1, 53.8, 53.5, 43.0, 37.8, 31.7, 29.0, 28.8, 26.4, 26.3,
24.1, 24.0. HR-ESI-MS: Calcd. for C49H60N2O5 [M + H]+: 757.4536,
found: 757.4582.
7-((5-(4-Benzylpiperidin-1-yl)pentyl)oxy)-3-(4-((5-(4-benzylpiper-
idin-1-yl)pentyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (7a). Yel-
low oil, 66.7% yield, 97.6% HPLC purity. 1H NMR (400 MHz, CDCl3)
7-((5-(4-Benzylpiperazin-1-yl)pentyl)oxy)-3-(4-((5-(4-benzylpiper-
azin-1-yl)pentyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (7b). Yel-
low oil, 71.9% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3)
–
12.80 (s, 1H, OH), 7.80 (d, J = 8.4 Hz, 2H, 2 × Ar H), 7.27 (t, J = 7.6
–
–
–
12.80 (s, 1H, OH), 7.81 (d, J = 8.4 Hz, 2H, 2 × Ar H), 7.32–7.29 (m,
Hz, 3H, 3 × Ar H), 7.18 (t, J = 4.0 Hz, 2H, 2 × Ar H), 7.14 (d, J = 7.6
10

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
2.84 (t, J = 6.0 Hz, 4H, 2 × phCH2), 2.68 (t, J = 7.6 Hz, 4H, 2 × NCH2),
2.48 (t, J = 7.6 Hz, 4H, 2 × NCH2), 1.82–1.74 (m, 4H, 2 × CH2),
1.66–1.56 (m, 4H, 2 × CH2), 1.51–1.43 (m, 4H, 2 × CH2). HR-ESI-MS:
Calcd. for C43H48N2O5 [M + H]+: 673.3597, found: 673.3636.
7-((5-(Benzyl(ethyl)amino)pentyl)oxy)-3-(4-((5-(benzyl(ethyl)
amino)pentyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (7d). Yellow
oil, 88.9% yield, 98.5% HPLC purity. 1H NMR 12.86 (s, 1H, OH), 7.84 (s,
–
–
1H, Ar H), 7.43 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.35–7.28 (m, 8H, 8 ×
–
–
Ar H), 7.24 (d, J = 7.6 Hz, 2H, 2 × Ar H), 6.95 (d, J = 8.8 Hz, 2H, 2 ×
–
–
Ar H), 6.35 (dd, J1 = 6.4 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 3.99–3.94
(m, 4H, 2 × OCH2), 3.59 (s, 4H, 2 × phCH2), 2.57–2.51 (m, 4H, 2 ×
NCH2), 2.50–2.45 (m, 4H, 2 × NCH2), 1.81–1.74 (m, 4H, 2 × CH2),
1.60–1.52 (m, 4H, 2 × CH2), 1.49–1.42 (m, 4H, 2 × CH2), 1.06 (t, J =
7.2 Hz, 6H, 2 × CH3). 13C NMR 180.8, 165.1, 162.7, 159.3, 157.9, 152.6,
139.5, 139.4, 130.7 (2C), 129.0 (2C), 128.9 (2C), 128.2 (4C), 126.9
(2C), 123.6, 122.8, 114.7 (2C), 106.1, 98.6, 92.8, 68.6, 68.0, 58.0, 57.9,
52.9, 52.8, 47.3, 47.2, 31.0, 29.1, 28.8, 26.7, 23.9, 23.8, 11.6, 11.5. HR-
ESI-MS: Calcd. for C43H52N2O5 [M + H]+: 677.3910, found: 677.3944.
7-((6-(4-Benzylpiperidin-1-yl)hexyl)oxy)-3-(4-((6-(4-benzylpiper-
idin-1-yl)hexyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (8a). Yellow
oil, 70.6% yield, 98.2% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.84
Fig. 9. Cell viability was tested by MTT assay. Three independent experiments
were performed. Data were expressed as mean ± SD. ##p < 0.01 vs. control;
**p < 0.01, *p < 0.05 vs RSL3 inhibitor group.
Table 3
Permeability Pe (×10ꢀ 6 cm/s) of compound 7d and its predictive penetration in
the CNS.
–
–
(s, 1H, OH), 7.85 (s, 1H, Ar H), 7.44 (d, J = 8.8 Hz, 2H, 2 × Ar H),
Compounda
Pe (×10ꢀ 6 cm/s)b
16.92 ± 0.97
Prediction
–
–
–
–
7.29–7.25 (m, 5H, 5 × Ar H), 7.18 (t, J = 6.8 Hz, 2H, 2 × Ar H), 7.13
7d
CNS+
(d, J = 6.8 Hz, 4H, 4 × Ar H), 6.95 (d, J = 8.8 Hz, 2H, 2 × Ar H), 6.36
a
–
(dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 4.03–3.96 (m, 4H, 2 ×
Compound 7d was dissolved in DMSO at 5 mg/mL and diluted with PBS/
OCH2), 3.01–2.98 (m, 4H, 2 × phCH2), 2.54 (d, J = 6.8 Hz, 4H, 2 ×
NCH2), 2.42–2.36 (m, 4H, 2 × NCH2), 2.00–1.93 (m, 4H, 2 × NCH2),
1.82–1.77 (m, 4H, 2 × CH2), 1.66 (d, J = 12.8 Hz, 4H, 2 × CH2),
1.61–1.53 (m, 4H, 2 × CH2), 1.53–1.42 (m, 6H, 2 × CH2 + 2 × CH),
1.40–1.33 (m, 4H, 2 × CH2). 13C NMR 180.8, 165.1, 162.7, 159.3, 158.0,
152.6, 140.5, 130.1, 129.1, 128.2, 125.8, 123.7, 122.8, 114.7, 106.2,
98.6, 92.8, 68.5, 67.9, 58.8, 53.8, 53.7, 43.0, 37.7, 31.7, 31.6, 30.9,
29.1, 28.8, 27.3, 27.2, 25.9, 25.8. HR-ESI-MS: Calcd. for C47H58N4O5 [M
+ H]+: 784.4815, found: 784.4862.
EtOH (70:30). The final concentration of compounds was 100
μ
g/mL.
b
Values are expressed as the mean ± SD of three independent experiments.
–
–
8H, 8 × Ar H), 7.27–7.24 (m, 2H, 2 × Ar H), 6.98 (d, J = 8.4 Hz, 2H,
–
–
–
2 × Ar H), 6.56 (s, 1H, Ar H), 6.45 (s, 1H, Ar H), 6.33 (s, 1H,
–
Ar H), 4.02–3.98 (m, 4H, 2 × OCH2), 3.52 (s, 4H, 2 × phCH2),
2.56–2.44 (m, 16H, 8 × NCH2), 2.41 (t, J = 8.0 Hz, 4H, 2 × NCH2),
1.85–1.82 (m, 4H, 2 × CH2), 1.62–1.57 (m, 4H, 2 × CH2), 1.52–1.45 (m,
4H, 2 × CH2). 13C NMR (100 MHz, CDCl3) 182.4, 164.9, 164.0, 162.1,
157.7, 137.9, 129.3, 128.2, 128.0, 127.1, 123.4, 114.9, 105.4, 104.2,
98.5, 93.0, 68.4, 68.1, 63.0, 58.5, 58.4, 53.2, 52.8, 29.0, 28.9, 26.5,
26.4, 24.0, 23.9. HR-ESI-MS: Calcd. for C47H58N4O5 [M + H]+:
759.4441, found: 759.4498.
7-((6-(4-Benzylpiperazin-1-yl)hexyl)oxy)-3-(4-((6-(4-benzylpiper-
azin-1-yl)hexyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (8b). Yellow
oil, 59.2% yield, 97.8% HPLC purity. 1H NMR (400 MHz, CDCl3) 12.86
–
–
(s, 1H, OH), 7.85 (s, 1H, Ar H), 7.44 (d, J = 8.8 Hz, 2H, 2 × Ar H),
–
–
7.32–7.27 (m, 10H, 10 × Ar H), 6.95 (d, J = 8.8 Hz, 2H, 2 × Ar H),
7-((5-(3,4-Dihydroisoquinolin-2(1H)-yl)pentyl)oxy)-3-(4-((5-(3,4-
dihydroisoquinolin-2(1H)-yl)pentyl)oxy)phenyl)-5-hydroxy-4H-chro-
men-4-one (7c). Yellow oil, 62.7% yield, 97.7% HPLC purity. 1H NMR
–
6.37 (dd, J1 = 7.6 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 4.03–3.96 (m, 4H, 2
× OCH2), 3.52 (s, 4H, 2 × phCH2), 2.99 (t, J = 4.8 Hz, 8H, 4 × NCH2),
2.54–2.51 (m, 8H, 4 × NCH2), 2.39–2.32 (m, 4H, 2 × NCH2), 1.83–1.77
(m, 4H, 2 × CH2), 1.56–1.46 (m, 8H, 4 × CH2), 1.41–1.35 (4H, 2 × CH2).
HR-ESI-MS: Calcd. for C47H58N4O5 [M + H]+: 787.4754, found:
787.4798.
–
–
12.79 (s, 1H, OH), 7.77 (s, 1H, Ar H), 7.36 (d, J = 8.8 Hz, 2H, 2 ×
–
–
Ar H), 7.05–7.01 (m, 6H, 6 × Ar H), 6.96–6.93 (m, 2H, 2 × Ar H),
–
6.89 (d, J = 8.4 Hz, 2H, 2 × Ar H), 6.29 (dd, J1 = 6.4 Hz, J2 = 2.0 Hz,
–
2H, 2 × Ar H), 3.97–3.91 (m, 4H, 2 × OCH2), 3.57 (s, 4H, 2 × phCH2),
Fig. 10. Effects of compound 7d on scopolamine-induced memory deficit in the step-down passive avoidance test. Values are expressed as mean ± SEM (n = 10). #
p
< 0.05 vs normal group. *p < 0.05 and **p < 0.01 vs scopolamine-treated control group.
11

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
7-((6-(Benzyl(ethyl)amino)hexyl)oxy)-3-(4-((6-(benzyl(ethyl)
uranyl acetate (1% w/v ddH2O). Upon removal of excess uranyl acetate,
the grids were dried for 15 min at room temperature. Images from each
sample were taken on a transmission electron microscope [34].
amino)hexyl)oxy)phenyl)-5-hydroxy-4H-chromen-4-one (8c). Yellow
oil, 64.1% yield, 98.1% HPLC purity. 1H NMR 12.86 (s, 1H, OH), 7.85 (s,
–
–
1H, Ar H), 7.43 (d, J = 8.8 Hz, 2H, 2 × Ar H), 7.35–7.28 (m, 6H, 8 ×
–
–
Ar H), 7.24 (d, J = 7.6 Hz, 2H, 2 × Ar H), 6.95 (d, J = 8.8 Hz, 2H, 2 ×
4.2.7. Autophagy assay
–
–
Ar H), 6.34 (dd, J1 = 6.4 Hz, J2 = 2.0 Hz, 2H, 2 × Ar H), 3.98–3.94
(m, 4H, 2 × OCH2), 3.59 (s, 4H, 2 × phCH2), 2.57–2.45 (m, 8H, 2 ×
NCH2), 1.84–1.76 (m, 4H, 2 × CH2), 1.58–1.50 (m, 8H, 4 × CH2),
1.50–1.44 (m, 4H, 2 × CH2), 1.06 (t, J = 7.2 Hz, 6H, 2 × CH3). HR-ESI-
MS: Calcd. for C45H56N2O5 [M + H]+: 705.4223, found: 705.4260.
Stable GFP-LC3 U87 cells cultured onto the coverslips in 6-well
plates, and then compound 7d was added and incubated for 24 h.
After treatment, the cells were fixed by 4% paraformaldehyde (PFA) for
20 min at room temperature, then washed with PBS for 2 times. The
slides were air dried and mounted by FluorSave™ mounting media
(Calbiochem, San Diego, CA, USA), which were then examined by
fluorescence microscopy [26].
4.2. Biological activity
4.2.8. The activity of GPX4 protein
4.2.1. AChE and BuChE inhibition
The test drugs used in this study were dissolved with DMSO and
stored at ꢀ 20 ◦C until further use. The viability of the cells treated as the
indicated drugs was measured by MTT assay using the 3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich,
MO, USA) reagent [36]. Briefly, PC-12 cells seeded in 96-well micro-
plates were treated RSL3 or cotreated RSL3 with the test compounds as
the indicated concentrations for 24 h. After treatments, the medium was
removed and 100 uL of fresh medium with 10 µL of MTT solution (5 µg/
mL) was added into each well, followed by a further incubation for 4 h.
The formed formazan representing the viability of cells was then dis-
solved by 100 µL DMSO. The solution was then subjected to the spec-
trophotometer and the colorimetric reading was determined at OD 570
nm. The percentage of cell viability by these two methods was calculated
using the following formula: Cell viability (%) = Cells number treated/
Cells number DMSO control × 100. Data were obtained from three in-
dependent experiments.
The AChE and BuChE inhibitory capacity were assessed by Ellman
assay using eeAChE, huAChE, eqBuChE and human serum BuChE). The
detailed procedure referenced our previous work [25,26].
4.2.2. Molecular modeling studies
The crystal structure of AChE complexed with donepezil (code ID:
4ey7) was obtained from the Protein Data Bank [32]. The 3D Structure
of 7d 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 in-
hibitor was accomplished. Docking studies were performed using the
AUTODOCK 4.2.6 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. 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 =
ꢀ 13.988, y = ꢀ 43.906, z = 27.109. 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). A
cluster analysis was performed on the docking results using a root mean
square (RMS) tolerance of 1.0 Å and the lowest energy conformation of
the highest populated cluster was selected for analysis. Graphic ma-
nipulations and visualizations were done by Autodock Tools or Dis-
covery Studio 3.0 software [26].
4.2.9. Step-down passive avoidance test
Kunming mice at body weight of 18–22 g (six weeks old, either
gender) were supplied by the Center of Experimental Animals of Sichuan
Academy of Chinese Medicine Sciences (eligibility certification no.
SCXK-Chuan2018-19). Mice were maintained under standard conditions
with a 12 h:12 h light–dark cycle. Prior to each experiment, mice were
fasted overnight and allowed free access to water. After the adminis-
tration of the compound, the mice were observed continuously for the
first 4 h for any abnormal behavior and mortality changes, intermit-
tently for the next 24 h, and occasionally thereafter for 14 days for the
onset of any delayed effects.
4.2.3. Antioxidant activity assay
A step-down passive avoidance test was used to assess learning and
memory in mice [26,34]. The apparatus consisted of a grid floor with a
wooden block placed in the center. The block served as a shock free
zone. The mice underwent two separate trials: a training trial and a test
trial 24 h later. For training trial, mice were initially placed on the block
and were given an electrical foot shock (0.5 mA, 2 s) through the grid
floor on stepping down. Compounds 7d (4.5, 9.0 and 18.0 mg/kg, p.o.)
or donepezil (5.0 mg/kg, p.o.) as a positive control were orally given 1 h
before each training trial. After 30 min, memory impairment was
induced by administering scopolamine (3 mg/kg, i.p.). Twenty-four
hours after the training trial, mice were placed on the block and the
time for the animal to step down was measured as latency time for test
trial. An upper cut-off time was set at 300 s. All data are expressed as
mean ± SEM. Differences between groups were examined for statistical
significance using one-way ANOVA with Student’s t test. A P value<0.05
denoted the presence of a statistically significant difference.
The antioxidant activity was assessed by the oxygen radical absor-
bance capacity fluorescein (ORAC-FL) assay. The detailed procedure
referenced our previous work [26,34].
4.2.4. Neuroprotective effect
The neuroprotective effects were evaluated with 3-(4,5-dimethylth-
iazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and LDH
assay [34]. The detailed procedure referenced our previous work
[26,34].
4.2.5. Effects on Aβ1-42 aggregation
The inhibition and disaggregation towards self-induced Aβ
ag-
1-42
gregation, hAChE-induced Aβ1-40 aggregation, Cu2+-induced Aβ1-42 ag-
gregation were evaluated using ThT fluorescence assay. The detailed
procedure detection method was the same as described previous work
[26].
Declaration of Competing Interest
4.2.6. Transmission electron microscopy (TEM) assay
For the Cu2+-induced experiment, the Aβ stock solution was diluted
with HEPES buffer (20 mM, pH 6.6, 150 mM NaCl). The sample prep-
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
aration was the same as for the ThT assay. Aliquots (10
μL) of the
samples were placed on a carbon-coated copper/rhodium grid for 2 min
at room temperature. Excess sample was removed using filter paper
followed by washing twice with ddH2O. Each grid was incubated with
12

Z. Sang et al.
Bioorganic Chemistry 107 (2021) 104602
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