Development of chalcone-O-alkylamine derivatives as multifunctional agents against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.111737

A series of novel chalcone-O-alkylamine derivatives were designed, synthesized and evaluated as multifunctional anti-Alzheimer's disease agents. Based on the experimental results, compound 23c exhibited good inhibitory potency on both acetylcholinesterase

Full text of DOI:10.1016/j.ejmech.2019.111737

Journal Pre-proof
Development of chalcone-O-alkylamine derivatives as multifunctional agents against
Alzheimer's disease
Ping Bai, Keren Wang, Pengfei Zhang, Jian Shi, Xinfeng Cheng, Qi Zhang, Cheng
Zheng, Yao Cheng, Jian Yang, Xiaoxia Lu, Zhipei Sang
PII:
S0223-5234(19)30889-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111737
EJMECH 111737
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2019
Revised Date: 23 September 2019
Accepted Date: 24 September 2019
Please cite this article as: P. Bai, K. Wang, P. Zhang, J. Shi, X. Cheng, Q. Zhang, C. Zheng, Y.
Cheng, J. Yang, X. Lu, Z. Sang, Development of chalcone-O-alkylamine derivatives as multifunctional
agents against Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111737.
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Development of chalcone-O-alkylamine derivatives as multifunctional agents
against Alzheimer's disease
Ping Baia,d, #, Keren Wang b,#, Pengfei Zhangc,#, Jian Shib, Xinfeng Chengb, Qi Zhanga, Cheng
Zhenga, Yao Chenga, Jian Yanga, Xiaoxia Lua, * Zhipei Sangb, *
a Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China
b College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang,
473061, PR China
c School of Medicine, Henan University Minsheng College, Kaifeng, 475000, PR China
d University of Chinese Academy of Sciences, Beijing 100049, PR China
*Corresponding authors.
E-mail: luxx@cib.ac.cn (Xiaoxia Lu)
E-mail: sangzhipei@126.com (Zhipei Sang)
#These authors contributed equally.

Abstract
A series of novel chalcone-O-alkylamine derivatives were designed, synthesized and evaluated
as multifunctional anti-Alzheimer’s disease agents. Based on the experimental results, com-
pound 23c exhibited good inhibitory potency on both acetylcholinesterase (IC50 = 1.3 ± 0.01
µM) and butyrylcholinesterase (IC50 = 1.2 ± 0.09 µM). Besides, 23c exhibited selective MAO-
B inhibitory activity with IC50 value of 0.57 ± 0.01 µM. Compound 23c was also a potential
antioxidant and neuroprotectant. In addition, compound 23c could inhibit self-induced Aβ1-42
aggregation. Moreover, compound 23c was a selective metal chelator, and could inhibit and
disaggregate Cu2+-induced Aβ1-42 aggregation, which was supported by the further transmission
electron microscopy images. Furthermore, 23c could cross the blood-brain barrier in vitro, and
improved scopolamine-induced memory impairment in vivo assay. Molecular modeling studies
showed that 23c could bind to the active site of AChE, BuChE, Aβ1-42 and MAO-B. Taken to-
gether, these results suggested that compound 23c might be a potential multifunctional agent
for the treatment of AD.
Keywords: Alzheimer’s disease; Chalcone-O-alkylamine derivatives; Multi-functional agents;
Rational design; Synthesis; Precognitive effect.
Abbreviations: AD, Alzheimer’s disease; NMDA, N-methyl-D-aspartate receptor; ACh, ace-
tylcholine; AChE, acetylcholinesterase; BuChE, Butyrylcholinesterase; Aβ, β-amyloid; APP,
amyloid precursor protein; MAO-B, monoamine oxidase B; MAO-A, monoamine oxidase A;
FAD, flavin adenine dinucleotide; PD, Parkinson disease; FDA, Food and Drug Administration;
MTDLs, multi-target-directed ligands; ORAC-FL, Oxygen Radical Absorbance Capacity; ThT,
thioflavin T; PDB, protein data bank; BBB, blood-brain barrier; CNS, central nervous system;
TEM, transmission electron microscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylte-
trazolium; PAMPA, parallel artificial membrane permeation.

1. Introduction
Alzheimer's disease (AD) is characterized by deterioration of memory, decline in language
skills, and other cognitive impairments in elder people [1]. To date, more than 46.8 million
people worldwide are diagnosed with AD and this number will continue to increase. With the
increasing of its incidence, AD has caused a huge economic impact on both individuals and
society [2]. Currently, despite enormous efforts made in the discovery of anti-AD drugs, there
are only three acetylcholinesterase inhibitors (donepezil, rivastigmine and galantamine) and
one N-methyl-D-aspartate receptor (NMDA) antagonist (memantine) that are approved for AD
treatment. Though those drugs can relieve some symptoms of AD patients, they are not able to
reverse the disease fundamentally [3, 4].
The pathogenic mechanism of AD is highly complicated, the exact etiology remains un-
known. In recent years, with the continuous research on the neurochemical, physiological and
pharmacological, the pathogenesis of AD has made progress [5]. Several hypotheses, such as
cholinergic hypothesis [6], amyloid hypothesis [7], have been proposed to explain the mecha-
nism of AD pathogenesis. The cholinergic hypothesis suggests that low level of acetylcholine
(ACh) is the main cause of memory and cognitive impairment in AD patients. Accumulation of
studies suggests that the acetylcholinesterase (AChE) plays an important role in the regulation
of several physiological reactions by hydrolyzing the neurotransmitter ACh in cholinegic syn-
apses [8]. Butyrylcholinesterase (BuChE) is an enzyme closely related to AChE and serves as a
co-regulator of cholinergic neurotransmission by hydrolyzing ACh. In addition, with the pro-
gress of AD, the level of AChE in the brain decreases progressively, but BuChE activity re-
mains the same or increases up to 165% of the normal level. In the advanced stage, BuChE
takes over the hydrolysis of ACh in the AChE deficient brain. Specific BuChE inhibition re-
sults in a 5-fold increase in ACh levels and could circumvent the classical cholinergic toxicity
that is a common side effect of AChE inhibition [9, 10]. Therefore, dual-inhibition of AChE
and BuChE may be a better therapeutic strategy in the treatment of AD.

The “amyloid hypothesis” suggests that the β-amyloid (Aβ) plaques in the brain play a
pivotal role in AD pathogenesis. The amyloid precursor protein (APP) is hydrolyzed by secre-
tase enzymes to produce Aβ peptides that can aggregate into monomers, oligomers and Aβ
plaques which can trigger the pathogenic cascade and finally cause the neuronal loss and de-
mentia [7]. Aβ1-40 and Aβ1-42 are two key types of Aβ isoforms, Aβ1-40 is present in larger
amounts in the AD brain, yet Aβ1-42 is more dissolutive and more prone to aggregate into fibrils
and is more neurtoxic than Aβ1-40 [11]. Additionally, excessive biometals including Cu2+, Fe2+,
Zn2+ and Al3+ are found in the AD brains. These excessive biometals are known to interact with
Aβ peptides and promote their aggregation [12]. Therefore, preventing the aggregation of Aβ1-
42 may provide a potent therapeutic strategy for AD treatment.
Oxidative stress also plays a crucial role in the development of AD [13]. Oxidative stress
is often abnormal in the early stage of AD, and it is involved in the occurrence and develop-
ment of AD. In the brains of the elder people, oxidized substances including nucleic acids, pro-
teins, lipids and carbohydrates, cannot be eliminated, which lead to excessive oxidation of neu-
ron cells [14]. Furthermore, oxidative stress can promote Aβ aggregation and further form neu-
rofibrillary tangels [15]. Therefore, antioxidant agents could be an effective strategy for the
treatment of AD.
Monoamine oxidases A and B (MAO-A and MAO-B) are important flavin adenine dinu-
cleotide (FAD)-dependent enzymes that catalyze the oxidative deamination of monoamines
[16]. MAO inhibitors have been proved to possess therapeutic effects on CNS-related diseases
including depression, Parkinson disease (PD), AD, and so on [17]. Studies have shown that se-
lective MAO-B inhibitors contribute to improve learning and memory [18]. Therefore, selec-
tive MAO-B inhibition is also deemed as a potential approach to treat AD.
So far, the drugs approved by the Food and Drug Administration (FDA) for clinical treat-
ment of AD can only hit single target and have limited effects on mitigate or halt the progres-
sion of AD. In view of the multiple pathogenesis of AD, drugs which can simultaneously hit
multiple targets could be a better strategy for AD treatment. Therefore, the multi-target-directed

ligands (MTDLs) strategy has been considered as an effective way for the treatment of AD, and
some multifunctional agents have been developed and are in clinic trails [19-22]. These candi-
dates, which could hit two or more AD-related targets, may represent a clinic advantage in the
future.
Chalcones (1,3-diphenyl-2-propen-1-ones) (Fig. 1) are prominent secondary-metabolite
precursors of flavonoids and isoflavonoids in plants. Chalcones and their derivatives have been
well studied for drug design because of the multiple pharmacological activities (such as radi-
cal-scavenging, anti-tumor, anti-inflammatory, and neuroprotective properties) [23, 24]. Recent
studies have shown that chalcones and their synthetic analogs exhibit potential bioactivities
associated with neurological diseases especially for AD. For example, Choi, J. W. et al. report-
ed that compounds with a chalcone scaffold exert MAO inhibitory activities for the treatment
18
of AD [25]. One F-radiolabeled chalcone analog is used as an Aβ imaging agent [26]. How-
ever, the lack of ChEs inhibition and metal chelation limited its application in AD therapy.
Therefore, design and synthesis of novel chalcone derivatives based on the MTDLs strategy
will increase pharmacological activities for the development of anti-AD drugs.
<Insert Fig. 1>
In our previous work, a series of genistein-O-alkylamines and scutellarin-O-alkylamine
derivatives was designed and synthesized. Results showed that the O-alkylamine pharmaco-
phore played the key role in cholinesterase inhibition [27, 28]. Moreover, it is reported that
numerous anti-AD compounds with two symmetrical pharmacophoric units can exert a signifi-
cantly higher ChE inhibitory potency [29-31]. Therefore, the O-alkylamine fragment is intro-
duced into both aromatic rings of chalcone to obtain a series of novel chalcone-O-alkylamine
derivatives (Fig. 2), in the hope of acquiring novel molecules possessing various multifunc-
tional activities.
Based on the MTDLs strategy, a series of chalcone-O-alkylamine derivatives was designed
and synthesized. The biological activities of these novel compounds were tested in vitro includ-
ing AChE and BuChE inhibition, MAOs inhibition, effect on Aβ1-42 aggregation, antioxidant ac-
tivity, biometals chelation, neuroprotective effect, the ability of cross blood-brain barrier (BBB)

in vitro and precognitive effect in vivo. Besides, docking studies were also performed to provide
rational explanation for their biological activities.
<Insert Fig. 2>
2. Result and discussion
2.1. Chemistry
The synthetic route of the chalcone-O-alkylamine derivatives was indicated in Scheme 1
and Scheme 2. Due to the presence of intramolecular hydrogen bonding, the 4'-OH of 2', 4'-
dihydroxyacetophenone was more reactive than that of the 2'-OH, compounds 3-6 were easily
synthesized from 2', 4'-dihydroxyacetophenone by using dibromo alkane in the presence of
K2CO3 in anhydrous acetone under refluxing. Subsequently, 3-6 were reacted with correspond-
ing secondary amines a-d and anhydrous K2CO3 in CH3CN at 65 °C to obtain compounds 7-10.
Under the same condition, p-hydroxybenzaldehyde 11 was used as raw material to obtain com-
pounds 16-19. Finally, the target compounds 20-23 were prepared by condensation of the ap-
propriate acetophenones 7-10 with the appropriate aldehydes 16-19 in ethanolic KOH solution.
In addition, compounds 26a and 26b were obtained by the condensation of 24 with 10c and 25
with 19c, respectively. All new compounds were confirmed by 1H NMR and HR-ESIMS, and
parts of them were further characterized by 13C NMR.
<Insert Scheme 1>
<Insert Scheme 2>
2.2. Pharmacology
2.2.1. Inhibition of AChE and BuChE. The modified Ellman’s method was used to evaluate
AChE and BuChE inhibitory activities of target compounds [27]. AChE was from electrophorus
electricus, and BuChE was from equine serum. Donepezil was used as a reference compound.
As shown in Table 1, most of the target compounds displayed moderate to good inhibitory
activity against AChE and BuChE without obvious selectivity, showing that the introduction of
O-alkylamines could significantly increase ChEs inhibitory capacity, which was consistent with
our design strategy. According to the screening data in Table 1, both the length of the meth-
ylene chain and the structure of terminal groups NR1R2 of side chain significantly affected the

AChE/BuChE inhibitory activities. For AChE inhibition, the AChE inhibitory activity gradual-
ly increased as the methylene extended, and the optimal methylene was 6, such as 20a (n =
2) > 21a (n = 3) > 22a (n = 4) > 23a (n =6); 20b (n = 2) > 21b (n = 3) > 22b (n = 4) = 23b (n
=6); 20c (n = 2) > 21c (n = 3) < 22c (n = 4) > 23c (n =6). In most conditions, when the length
of the methylene chain remained the same, the potency to inhibit AChE was in the order: pyr-
role > diethylamine > piperidine > benzylpiperidine, such as 20c < 20d < 20b < 20a; and 21c <
21d < 21b < 21a. Among these compounds, compound 21c showed the best AChE inhibitory
activity with IC50 value of 0.79 µM. For BuChE inhibition, when the terminal groups NR1R2
were benzylpiperidine, piperidine and pyrrole, the BuChE inhibitory activity increased as
methylene chain extended, such as 20a > 21a > 22a > 23a, 20b > 21b > 22b > 23b, and 20c >
21c < 22c > 23c. When the methylene chain was 6, the tendency to inhibit BuChE was in the
order: benzylpiperidine > pyrrole > piperidine > diethylamine, such as 23a < 23c < 23b < 23d.
Particularly, compound 23a showed the best BuChE inhibitory activity with IC50 value of 0.8
µM.
According to the above results, compound 23c presented the balanced and high-efficiency
dual AChE/BuChE inhibitory activity with IC50 values of 1.3 µM and 1.2 µM, respectively. In
order to further evaluate the structure-activity relationship (SAR) of compound 23c, com-
pounds 26a and 26b were synthesized and tested the AChE/BChE inhibitory activity. As shown
in Table 1, compound 26a showed good AChE inhibitory activity with IC50 value of 4.2 µM
and moderate BuChE inhibitory potency with IC50 value of 12.3 µM, and compound 26b inhib-
ited AChE/BuChE inhibitory activities with IC50 values of 8.9 µM and 50.2 µM, respectively,
implying that compounds 26a and 26b were selective AChE inhibitors. In addition, compounds
26a and 26b exhibited lower ChEs inhibitory activity than compound 23c, indicating that the
symmetrical O-alkylamine fragment could contribute to AChE/BuChE inhibitory activity,
which was consist with our design strategy. Further, compounds 23c, 26a and donepezil were
investigated using huAChE and huBuChE, the results in Table 2 showed that compound 23c
displayed good huAChE and huBuChE inhibitory activity with IC50 values of 0.78 µM and

0.91µM, respectively, and compound 26a showed good huAChE inhibitory activity with IC50
value of 3.2 µM, implying that AChE inhibitory activity using human AChE was better than
that using eeAChE.
<Insert Table 1>
<Insert Table 2>
2.2.2. Inhibition of human MAOs. The inhibitory activities toward MAO-A and MAO-B (recom-
binant human enzyme) were evaluated to further investigate the multi-potent biological profiles
of the chalcone-O-alkylamine derivatives [32]. Iproniazid and rasagiline were also tested as ref-
erenced compounds. As shown in Table 1, most target compounds showed good MAO-B inhibi-
tory activities with weak MAO-A inhibitory activities, indicating that the target compounds were
selective MAO-B inhibitors, which contributed to AD treatment. In general, both methylene
chain and terminal groups NR1R2 significantly influenced MAO-B inhibitory activity. When the
NR1R2 was fixed, the MAO-B inhibitory activity increased as methylene chain extended, such as
20a > 21a > 22a > 23a, 20b > 21b > 22b < 23b, 20c = 21c > 22c < 23c, 20d > 21d > 22d < 23d.
Moreover, when the methylene chain was determined, the compounds with cyclic amines (pyr-
role and piperidine) showed better MAO-B inhibitory activity than compounds with benzylpiper-
idine. Especially, compound 23c showed the best MAO-B inhibitory activity with IC50 value of
0.57 µM. Further, the mono-substituted O-alkylamine derivatives 26a and 26b showed lower
MAO-B inhibitory activity than disubstituted O-alkylamine derivative 23c, implying that the di-
substituted O-alkylamine fragment contributed to MAO-B inhibitory activity. Therefore, the re-
sults revealed that compound 23c was a potent selective MAO-B inhibitor and presented good
AChE and BuChE inhibitory activities, which was selected to perform further study.
2.2.3. Antioxidant activity in vitro. Next, we evaluated the antioxidant activity of the synthetic
compounds using the ORAC-FL (Oxygen Radical Absorbance Capacity by Fluorescein) assay
[27, 28]. As show in Table 3, most of the synthetic compounds exhibited good antioxidant activi-
ties in vitro with ORAC values of 0.96-1.4 Trolox (a synthetic analog of vitamin E) equivalents.
The structure-activity relationship analysis indicated that the different substituent moiety NR1R2
and the length of methylene chain did not significantly affect the antioxidant activity. Compound
23c showed good antioxidant activity with an ORAC value of 1.3 eq.
2.2.4. Inhibition of the self-mediated Aβ1-42 aggregation. To gain further insight into the self-
induced Aβ1-42 aggregation inhibitory activity of synthetic chalcone-O-alkylamines derivatives,

the Thioflavin T (ThT) fluorescence method was used, and curcumin was also tested as the refer-
ence compound [27]. The results listed in Table 3 indicated that most of the tested compounds
showed good potencies (51.3 ~ 71.8%) compared with curcumin (47.3 %). From the test results
we concluded that the length of methylene chain and different tertiary amine units did not make
much contribution to the self-induced Aβ1-42 aggregation.
<Insert Table 3>
2.2.5. Molecular modeling studies of 23c. In order to investigate the interaction mode of promis-
ing compound 23c for AChE and BuChE, further molecular docking study was performed using
the docking program, AutoDock 4.2 package with Discovery Studio 2.0, based on the X-ray
crystal structure of AChE (PDB code: 1eve) and BuChE (PDB code: 4tpk) [27, 28]. The results
showed that compound 23c occupied the entire (catalytic active site) CAS, the mid-gorge sites
and the peripheral anionic site (PAS) of AChE (Fig. 3), the benzene ring of chalcone skeleton
interacted with key amino acid Phe330 via π-π interaction, two σ-π interactions with Trp84and
one intermolecular hydrogen bonding interaction with Phe288. Moreover, hydrophobic interac-
tions could be observed between the ligand and Asp72, Trp84, Gly118, Gly117, Phe330, Tyr334,
Phe290, Phe331, Phe288, Trp279. Besides, compound 23c interacted with BuChE via multiple
sites (Fig. 4), the hydroxyl group and carbonyl of chalcone skeleton was involved in a hydrogen
bond with Pro285. In addition, the key amino acid Tyr332 could interact with chalcone skeleton
via one σ-π interaction and one π-π interaction, respectively. And the pyrrole ring could interact
with Thr120 via one intermolecular hydrogen bonding. Besides, hydrophobic interactions could
be observed between the ligand and Thr120, Asp70, Gly116, Phe329, Ala328, Tyr332, Trp82,
His438. Based on these results observed, it could provide rational explanation for the high activi-
ty in inhibiting BuChE and AChE of 23c.
<Insert Fig. 3>
<Insert Fig. 4>
Further molecular docking experiment was conducted on 23c to explore the possible inter-
acting mode with Aβ. The protein complex was downloaded from the Protein Data Bank (PDB:
1BA4). As shown in Fig. 5, the benzene ring of chalcone skeleton interacted with key amino acid
Phe20 via π-π interaction, and the hydroxyl group of 23c interacted with ASP1 via two intermo-
lecular hydrogen bonding. Moreover, hydrophobic interactions were observed between the lig-
and and Asp1, Phe20, Asp23, and Phe19. These interactions might be favorable for the binding

of Aβ and compound 23c.
<Insert Fig. 5>
In addition, molecular docking simulations of 23c with MAO-B were carried out based on
the X-ray crystal structures of human MAO-B (PDB code: 2V60). As shown in Fig. 6, the O-
alkylamine side chain of 23c was located in the well-known binding pocket of huMAO-B, in
close proximity to the enzymatic cofactor FAD. The hydroxyl group of chalcone skeleton inter-
acted with Ile198 and Gln206 via intermolecular hydrogen bonding interaction, respectively, and
the carbonyl group could also interact with Ile199 and Cys172 via intramolecular hydrogen
bonding interaction, respectively. Moreover, the benzene ring of chalcone skeleton interacted
with Tyr398 and Tyr435 via π-π interaction, respectively. Meanwhile, hydrophobic interactions
could be observed between the ligand and Cys172, Tyr398, Tyr435, Gln206, Ile198, Ile199,
Ile316, Pro102, Leu88. Thus, the results observed might provide the rational explanation for the
MAO-B inhibitory activity of 23c.
<Insert Fig. 6>
2.2.6. Metal-chelating properties of compounds 23c. We further tested the ability of compound
23c to chelate biometals including Cu2+, Zn2+, Al3+ and Fe2+. The experiment was conducted by
UV-vis spectrophotometer [33]. The results showed that the maximum absorption wavelength of
23c shifted from 361 nm to 422 nm after adding CuCl2, indicating the formation of 23c-Cu2+
complex (Fig. 7). Conversely, the maximum absorption exhibited no significant shift when AlCl3,
ZnCl2 and FeSO4 was added. These results indicated that the compound 23c could selectively
chelate with Cu2+.
<Insert Fig. 7>
Next, we determined the stoichiometry of the 23c-Cu2+complex by the molar ratio method.
The absorbance of the 23c and CuCl2 complexes at different concentrations at 422 nm was de-
termined by ultraviolet spectroscopy. As seen from Fig. 8, the absorbance first increased linearly
and then tended to be stable. These two lines intersected at a mole fraction of 1.06, showing a
stoichiometric ratio of the composite 23c-Cu2+ complex of 1:1.
<Insert Fig. 8>
2.2.7. Effects on Cu2+-induced Aβ1−42 aggregation and disaggregation. To evaluate the Aβ aggre-
gation ability of chalcone-O-alkylamines derivatives, Cu2+-induced Aβ1-42 aggregation and the
disaggregation assay were carried out using ThT binding assay [27]. Some of the target com-

pounds 20c, 20d, 21b, 21c, 21d, 22b, 22c, 23b, 23c and 23d were selected to test inhibition po-
tency on Cu2+-induced Aβ1-42 aggregation, and curcumin.was also tested as positive compound.
As shown in Table 2, most of the tested compounds showed better inhibition rate than curcumin
(76.5%). The methylene chain and terminal group NR1R2 did not obviously affect the inhibition,
and compound 23c demonstrated good inhibition potency (81.2%).
For disaggregation effects on Cu2+-induced Aβ1−42 aggregation. According to the data, the
representative compound 23c displayed better disaggregation potency with 70.4% disaggregation
ratio than curcumin (56.5%). The results displayed that compound 23c could inhibit and dis-
aggregate Cu2+-induced Aβ1−42 aggregation
2.2.8. Effect on abundance of Aβ fibrils by compound 23c. To complement the ThT assay, Aβ1-42
aggregation was further probed by transmission electron microscopy (TEM) (Fig. 9). For inhibi-
tion assay in Fig. 9B, the fresh Aβ1-42 had aggregated into amyloid fibrils after adding 25 µM
Cu2+ during 24 h incubation, while the fresh Aβ1-42 was treated with 23c and Cu2+, under the
same experiments, small bulk aggregates were observed, implying that compound 23c could in-
hibit Cu2+-induced Aβ1-42 aggregation. As for disaggregation assay, Fig. 9D displayed that the
well-defined Aβ1-42 fibrils obviously decreased after adding 23c for another 24 h incubation, sug-
gesting that 23c could decompose the structure of Cu2+-mediated Aβ aggregation fibrils. There-
fore, the TEM phenomenon further supported the conclusion that 23c could inhibit and disaggre-
gate Cu2+-induced Aβ1-42 aggregation fibrils.
<Insert Fig. 9>
2.2.9. Neuroprotective effects on H2O2-induced PC12 cell injury. The protective effects of 23c
against H2O2 injury were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoli-
um (MTT) assay [33]. According to the test results, 23c did not show modified cell viability up
to the concentration of 10 µM. With increased concentration to 100 µM, 23c induced a decrease
of cell viability (84.7%). The cell viability as determined by MTT reduction was remarkably de-
clined after 100 µM H2O2 exposure, indicating significant sensitivity to H2O2-induced injury
(Fig. 10A). However, treatment with compound 23c, a significant dose-dependent protection was
observed on H2O2-induced PC12 cell injury. Compound 23c showed significant neuroprotective
effect with 87.1% cell viability at 10.0 µM, and when the concentration of inhibitor 23c was 1.0
µM, the cell viabilities dropped to 63.8% (Fig. 10B). This result indicated that compound 23c
could protected H2O2-induced PC12 cell injury.

<Insert Fig. 10>
2.2.10. In vitro blood−brain barrier permeation assay. One of the key requirements for success-
ful central nervous system (CNS) drugs is the ability to cross BBB and having high brain pene-
tration. For further evaluating the BBB penetration of compound 23c, the parallel artificial mem-
brane permeation assay of BBB (PAMPA-BBB) was performed [34, 35]. We compared the per-
meability of 11 commercial drugs with the reported values and verified the effectiveness of the
assay (Table S1). The results showed that our experimental versus literature values have a good
linear correlation (Figure S1). Taking into consideration of the limits established by Di et al's for
the BBB penetration [35], we determined that compounds with effective permeation (Pe) values
above 3.44 × 10−6 cm/s could cross the BBB (Table S2). The measured permeability showed that
the Pe value of 23c was 9.68 ± 0.57 × 10−6 cm/s, which could cross the BBB in vitro.
2.2.11 In vivo assay. The acute toxicity of 23c was tested using Kunming mice at doses of 100,
250 and 500 mg/kg (n = 10 per group). The mice were observed continuously for the first 4 h
through 14 days after oral administration of 23c. The results showed compound 23c did not show
any acute toxicity and well tolerated at doses of up to 500 mg/kg. On this basis, the step-down
passive avoidance task was carried out to evaluate whether 23c could improve scopolamine-
induced memory impairment [28, 36]. Results showed in Fig. 11, treatment with scopolamine (3
mg/kg) alone, the step-down latency remarkably declined to 65.8 sec (p < 0.01) compared with
normal group (162.1 sec), while treatment with 5 mg/kg donepezil, the latency time rose to 156.5
sec (p < 0.01) compared with model group. Moreover, administration of compound 23c (14.8,
7.4 and 3.7 mg/kg), the latency time gradually increased in a dose-dependent manner. In addition,
the high dose group (14.8 mg/kg) of 23c showed the highest latency time (141.9 s, p < 0.05)
among the three dose groups, but displayed lower latency time than that of donepezil group (5
mg/kg, 156.5 sec). Therefore, compound 23c could improve scopolamine-induced memory im-
pairment to some extent.
<Insert Fig. 11>
3. Conclusion
In conclusion, we had designed and synthesized a series of chalcone-O-alkylamine deriva-
tives as multifunctional agents for the treatment of AD. All the synthesized compounds were
evaluated by AChE and BuChE inhibition, antioxidant activity, MAO-A/MAO-B inhibition
and self-induced Aβ1-42 aggregation inhibition in vitro. Among these synthesized compounds,

compound 23c exhibited the best inhibitory potency of AChE and BuChE inhibition, good an-
tioxidant activity, potential selective MAO-B inhibitory activities, and was able to inhibit self-
induced Aβ1-42 aggregation. In addition, compound 23c was a selective metal chelator, and
could inhibit and decompose Cu2+-induced Aβ1-42 aggregation. Both the TEM images and
docking studies provided reasonable explain. Moreover, 23c could cross the BBB in vitro. The
further in vivo assay displayed that compound 23c could improve scopolamine-induced
memory impairment. Taken together, these results suggested that compound 23c might be a
potential multifunctional agent for the treatment of AD. Further studies to evaluate 23c in vivo
are in progress.
4. Experiment section
4.1. Chemistry
Unless otherwise noted, all materials (reagent grade) were purchased from commercial sup-
pliers and used without further purification. The 1H NMR and 13C NMR spectra were recorded in
CDCl3 at 25 °C and referenced to tetramethylsilane (TMS) using a Bruker 400 NMR spectrome-
ter. Mass spectra were obtained on an Agilent-6210 TOF LC-MS Spectrometer. The purities of
all final compounds used for biological evaluation were determined by waters 2695 High Per-
formance Liquid Chromatography (HPLC) with a Waters XBridge C18 column (4.6mm × 150
mm, 5 µm) at a flow rate of 1.0 mL/min. Mobile phase: A: 0.1%TFA in H2O, B: 0.1% TFA in
acetonitrile. The purity of all compounds was higher than 95%. Reaction progress was monitored
by thin-layer chromatography (TLC) on silica gel GF254 plates from Qingdao Haiyang Chemi-
cal Co. Ltd. (China), the spots were detected under an UV lamp (254 nm).
4.1.1. General procedures for the synthesis of compounds 3-6.
Compounds 3-6 were prepared according to our previously described procedure [37].
4.1.2. General procedure for the synthesis of compounds 7-10.
Compounds 7-10 were prepared according to our previously described procedure [37].
4.1.3. General procedure for the synthesis of compounds 12-15.
Compounds 12-15 were prepared refer to the literature and according to our previously described
procedure [27, 38].
4.1.4. General procedure for the synthesis of compounds 16-19.

Compounds 16-19 were prepared refer to the literature and according to our previously described
procedure [27].
4.1.5. General procedure for the synthesis of compounds 20-23, 26a and 26b.
To a mixture of 7-10 or 25 (2 mmol) and the compounds 16-19 or 24 (2.5 mmol) in EtOH (7 mL)
was added an aqueous solution of potassium hydroxide (50%, 6.5 mmol) at 0 °C. The mixture
was stirred at room temperature for 48 hours, the reaction was monitored by TLC, then ice-cold
water was poured into the solution and acidified with 10% HCl to pH = 3, and then the mixture
was adjusted pH =8 using NaHCO3 power. The mixture was extracted with CH2Cl2 (30 mL × 3),
the organic phase was evaporated under reduced pressure. The residue was purified using mix-
tures of CH2Cl2/acetone (100:1) as eluent on a silica gel chromatography to obtain the products
20-23, 26a and 26b.
(E)-1-(4-(2-(diethylamino)ethoxy)-2-hydroxyphenyl)-3-(4-(2-(diethylamino)ethoxy)phenyl)prop-2
-en-1-one (20a). Light yellow oil; Yield: 59.1%; Purity: 97.25% (HPLC). 1H NMR (400 MHz, C
DCl3) 1H NMR (400 MHz, CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.92 – 7.81 (m, 2H, C=CH, Ar–H),
7.66 – 7.59 (m, 2H, 2 × Ar–H), 7.48 (d, J = 15.4 Hz, 1H, C=CH), 7.00 – 6.93 (m, 2H, 2 × Ar–H),
6.54 – 6.46 (m, 2H, 2 × Ar–H), 4.13 (t, J = 6.2 Hz, 4H, 2 × OCH2), 2.93 (t, J = 6.1 Hz, 4H,
2 × CH2), 2.74 – 2.62 (m, 8H, 4 × CH2), 1.16 – 1.06 (m, 12H, 4 × CH3). 13C NMR (100 MHz, C
DCl3) δ 191.8, 166.5, 165.3, 161.1, 144.36, 131.1, 130.46, 127.5, 117.8, 115.0, 114.26, 108.0, 10
1.7, 66.9, 66.7, 51.6, 51.4, 47.9, 11.8, 11.8. HRMS (ESI): Cacld for (C43H50N2O4) requires: m/z 6
59.3804, found: 659.3847.
(E)-1-(2-hydroxy-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pr
op-2-en-1-one (20b). Light yellow oil; Yield: 57.2%; Purity: 97.21% (HPLC).1H NMR (400 MH
z, CDCl3) δ 13.56 (s, 1 H, Ar–OH), 7.88 (d, J = 15.4 Hz, 1H, C=CH), 7.85 (d, J = 4.0 Hz, 1H, Ar
–H), 7.65 – 7.58 (m, 2H, 2 × Ar–H), 7.47 (d, J = 15.4 Hz, 1H, C=CH), 7.35 – 7.25 (m, 4 H,
4 × Ar–H), 7.25 – 7.14 (m, 6H, 6 × Ar–H), 6.99 – 6.92 (m, 2 H, 2 × Ar–H), 6.55 – 6.44 (m, 2H,
2 × Ar–H), 4.20 – 4.16 (m, 4H, 2 × OCH2), 3.09 – 2.96 (m, 4H, 2 × phCH2), 2.84 (q, J = 5.6 Hz,
4H, 2 × CH2), 2.57 (d, J = 7.0 Hz, 4H, 2 × CH2), 2.16 – 2.03 (m, 4 H, 2 × CH2), 1.74 – 1.64 (m,
4H, 2 × CH2), 1.63 – 1.53 (m, 2 H, 2 × CH), 1.48 – 1.31 (m, 4 H, 2 × CH2). 13C NMR (100 MHz,
CDCl3) δ 191.8, 166.5, 165.3, 161.0, 144.2, 131.1, 130.3, 130.28, 127.7, 127.6, 117.8, 115.0, 11
4.87, 114.28, 108.07, 101.79, 67.4, 67.26, 54.9, 54.7, 54.7, 23.5. HRMS (ESI): Cacld for (C29H38
N2O4) requires: m/z 479.2865, found: 479.2896.

(E)-1-(2-hydroxy-4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)prop
-2-en-1-one (20c). Light yellow oil; Yield: 51.1%; Purity: 97.05% (HPLC). 1H NMR (400 MHz,
CDCl3) δ 13.53 (s, 1 H, Ar–OH), 7.87 – 7.77 (m, 2 H, C=CH, Ar–H), 7.58 (d, J = 8.5 Hz, 2 H,
2 × Ar–H), 7.43 (d, J = 15.4 Hz, 1 H, C=CH), 6.94 (dd, J = 8.7, 6.6 Hz, 2 H, 2 × Ar–H), 6.51 – 6.
43 (m, 2 H, 2 × Ar–H), 4.14 (t, J = 6.1 Hz, 4 H, 2 × OCH2), 2.75 – 2.80 (m, 4 H, 2 × CH2), 2.51
(q, J = 5.4 Hz, 8H, 4 × CH2), 1.64 – 1.59 (m, 8H, 4 × CH2), 1.48 – 1.51 (m, 4H, 2 × CH2).13C N
MR (100 MHz, CDCl3) δ 191.8, 190.8, 166.5, 165.2, 161.0, 144.2, 131.1, 130.45, 128.7, 128.5, 1
27.7, 127.6, 117.8, 115.1, 114.8, 114.6, 114.2, 110.6, 107.9, 101.8, 101.5, 79.7, 66.4, 66.2, 66.1,
65.9, 57.7, 57.6, 57.5, 55.0, 55.0, 55.0, 44.1, 29.7, 25.8, 25.8, 25.8, 25.7, 24.1, 24.1. HRMS (ES
I): Cacld for (C27H34N2O4) requires: m/z 451.2552, found: 451.2598
(E)-1-(4-(2-(4-benzylpiperidin-1-yl)ethoxy)-2-hydroxyphenyl)-3-(4-(2-(4-benzylpiperidin-1-yl)et
hoxy)phenyl)prop-2-en-1-one (20d). Light yellow oil; Yield: 49.1%; Purity: 97.82% (HPLC).1H
NMR (400 MHz, CDCl3) δ 13.56 (s, 1H, Ar–OH), 7.88 (d, J = 15.4 Hz, 1H, C=CH), 7.85 (d, J =
4.0 Hz, 1H, Ar–H), 7.65 – 7.58 (m, 2H, 2 × Ar–H), 7.47 (d, J = 15.4 Hz, 1H, C=CH), 7.35 – 7.25
(m, 4H, 4 × Ar–H), 7.25 – 7.14 (m, 6H, 6 × Ar–H), 6.99 – 6.92 (m, 2H, 2 × Ar–H), 6.55 – 6.44
(m, 2H, 2 × Ar–H), 4.18 (m, 4H, 2 × OCH2), 3.09 – 2.96 (m, 4H, 2 × phCH2), 2.84 (q, J = 5.6 Hz,
4H, 2 × CH2), 2.57 (d, J = 7.0 Hz, 4H, 2 × CH2), 2.16 – 2.03 (m, 4H, 2 × CH2), 1.74 – 1.64 (m,
4H, 2 × CH2), 1.63 – 1.53 (m, 2H, 2 × CH), 1.48 – 1.31 (m, 4H, 2 × CH2). 13C NMR (100 MHz,
CDCl3) δ 191.8, 166.5, 165.3, 161.0, 144.2, 131.1, 130.3, 130.2, 127.7, 127.6, 117.8, 115.0, 114.
8, 114.2, 108.0, 101.7, 67.4, 67.2, 54.9, 54.7, 54.7, 23.5. HRMS (ESI): Cacld for (C27H38N2O4) r
equires: m/z 455.2865, found: 455.2911.
(E)-1-(4-(3-(diethylamino)propoxy)-2-hydroxyphenyl)-3-(4-(3-(diethylamino)propoxy)phenyl)pro
p-2-en-1-one (21a). Light yellow oil; Yield: 49.5%; Purity: 98.77% (HPLC).1H NMR (400 MHz,
CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.90 – 7.80 (m, 2H, C=CH, Ar–H), 7.65 – 7.58 (m, 2H, 2 × Ar–
H), 7.47 (d, J = 15.4 Hz, 1H, C=CH), 6.97 – 6.93 (m, 2H, 2 × Ar–H), 6.49 (d, J = 8.7 Hz, 2H,
2 × Ar–H), 4.09 (t, J = 6.2 Hz, 4H, 2 × OCH2), 2.73 – 2.59 (m, 12H, 6 × CH2), 2.06 – 1.95 (m, 4
H, 2 × CH2), 1.12 – 1.05 (m, 12H, 4 × CH3). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.6, 165.5,
161.2, 144.3, 131.1, 130.4, 127.4, 117.7, 114.9, 114.1, 107.8, 101.6, 66.6, 66.4, 49.3, 49.2, 47.0,
31.9, 31.5, 29.7, 29.4, 26.6, 26.5, 22.7, 14.2, 11.5, 11.4. HRMS (ESI): Cacld for (C45H54N2O4) re
quires: m/z 687.4117, found: 687.4149.

(E)-1-(2-hydroxy-4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-3-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)
prop-2-en-1-one (21b). Light yellow oil; Yield: 55.3%; Purity: 96.81% (HPLC). 1H NMR (400 M
Hz, CDCl3) δ 13.55 (s, 1H, Ar–OH), 7.87 – 7.79 (m, 2H, C=CH, Ar–H), 7.59 (dt, J = 8.9, 2.2 Hz,
2H, 2 × Ar–H), 7.45 (d, J = 15.4 Hz, 1H, C=CH), 6.99 – 6.91 (m, 2H, 2 × Ar–H), 6.54 – 6.44 (m,
2H, 2 × Ar–H), 4.20 – 4.14 (m, 4H, 2 × OCH2), 2.97 – 2.90 (m, 4H, 2 × CH2), 2.71– 2.58 (m, 12
H, 6 × CH2), 1.88– 1.77 (m, 12H, 6 × CH2). 13C NMR (100 MHz, CDCl3) δ 191.8, 168.5, 166.5,
165.2, 161.0, 144.2, 131.1, 130.4, 130.2, 127.7, 127.5, 117.8, 115.0, 114.8, 114.8, 114.2, 108.0, 1
01.7, 101.5, 67.3, 67.1, 67.0, 55.0, 54.9, 54.7, 54.7, 44.1, 29.7, 23.5. HRMS (ESI): Cacld for (C31
H42N2O4) requires: m/z 507.3178, found: 507.3216.
(E)-1-(2-hydroxy-4-(3-(piperidin-1-yl)propoxy)phenyl)-3-(4-(3-(piperidin-1-yl)propoxy)phenyl)p
rop-2-en-1-one (21c). Light yellow oil; Yield: 56.4%; Purity: 96.47% (HPLC). 1H NMR (400 M
Hz, CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.92 – 7.81 (m, 2H, C=CH, Ar–H), 7.62 (d, J = 8.4 Hz, 2H,
2 × Ar–H), 7.47 (d, J = 15.3 Hz, 1H, C=CH), 6.95 (d, J = 8.4 Hz, 2H, 2 × Ar–H), 6.49 (d, J = 8.5
Hz, 2H, 2 × Ar–H), 4.09 (t, J = 6.3 Hz, 4H, 2 × OCH2), 2.62 – 2.47 (m, 12H, 6 × CH2), 2.15 – 2.
01 (m, 4H, 2 × CH2), 1.73 – 1.73 (m, 8H, 4 × CH2). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.6,
165.4, 161.2, 144.3, 131.1, 130.4, 127.5, 117.7, 115.0, 114.1, 107.9, 101.6, 66.7, 66.5, 55.8, 55.7,
54.5, 54.5, 29.7, 26.3, 26.3, 25.6, 25.5, 24.2, 24.1. HRMS (ESI): Cacld for (C29H38N2O4) require
s: m/z 479.2865, found: 479.2899.
(E)-1-(4-(3-(4-benzylpiperidin-1-yl)propoxy)-2-hydroxyphenyl)-3-(4-(3-(4-benzylpiperidin-1-yl)p
ropoxy)phenyl)prop-2-en-1-one (21d). Light yellow oil; Yield: 58.6%; Purity: 97.22% (HPLC).1
H NMR (400 MHz, CDCl3) δ 13.53 (s, 1H, Ar–OH), 7.88 (d, J = 15.4 Hz, 1H, C=CH), 7.85 (d, J
= 4.0 Hz, 1H, Ar–H), 7.65 – 7.59 (m, 2H, 2 × Ar–H), 7.47 (d, J = 15.4 Hz, 1H, C=CH), 7.35 – 7.
25 (m, J = 7.3 Hz, 4H, 4 × Ar–H), 7.23 (dd, J = 7.4, 1.6 Hz, 2H, 2 × Ar–H), 6.96 – 6.91 (m, 2H,
2 × Ar–H), 6.50 – 6.44 (m, 2H, 2 × Ar–H), 4.15 – 6.05 (m, 4H, 2 × OCH2), 3.28 – 3.11 (m, 4H,
2 × phCH2), 2.85 – 2.68 (m, 4H, 2 × CH2), 2.60 (q, J = 2.6 Hz, 4H, 2 × CH2), 2.30 – 2.10 (m, 8
H), 1.80 – 1.71 (m, 4H, 2 × CH2), 1.69 – 1.53 (m, 6H, 3 × CH2). 13C NMR (100 MHz, CDCl3) δ
191.9, 144.3, 131.3, 130.4, 129.1, 128.4, 127.8, 126.2, 117.9, 114.9, 114.4, 101.8, 42.3, 31.9, 31.
7, 31.5, 31.5, 30.3, 30.2, 30.2, 29.7, 29.7, 29.5, 29.4, 27.2, 22.7, 14.14. HRMS (ESI): Cacld for
(C29H42N2O4) requires: m/z 483.3178, found: 483.3221.
(E)-1-(4-(4-(diethylamino)butoxy)-2-hydroxyphenyl)-3-(4-(4-(diethylamino)butoxy)phenyl)prop-
2-en-1-one (22a). Light yellow oil; Yield: 54.9%; Purity: 98.07% (HPLC).1H NMR (400 MHz,

CDCl3) δ 13.58 (s, 1H, Ar–OH), 7.91 – 7.82 (m, 2H, C=CH, Ar–H), 7.65 – 7.59 (m, 2H, 2 × Ar–
H), 7.48 (d, J = 15.4 Hz, 1H, C=CH), 6.98 – 6.92 (m, 2H, 2 × Ar–H), 6.51 – 6.46 (m, 2H, 2 × Ar
–H), 4.05 (t, J = 6.4 Hz, 4H, 2 × OCH2), 2.64 – 2.51 (m, 12H, 6 × CH2), 1.88 – 1.79 (m, 4H,
2 × CH2), 1.71 – 1.65 (m, 4H, 2 × CH2), 1.09 – 1.05 (m, 12H, 4 × CH3). 13C NMR (100 MHz, C
DCl3) δ 191.8, 166.6, 165.6, 161.3, 144.3, 131.1, 130.4, 127.7, 127.4, 117.7, 115.0, 114.7, 114.1,
108.0, 101.5, 68.2, 68.0, 52.5, 52.4, 46.8, 46.2, 31.9, 31.7, 31.5, 31.5, 30.3, 30.2, 29.7, 29.6, 29.4,
29.0, 27.2, 27.1, 23.4, 23.4, 22.7, 14.1, 11.5, 11.5, 10.7. HRMS (ESI): Cacld for (C47H58N2O4) re
quires: m/z 715.4430, found: 715.4465.
(E)-1-(2-hydroxy-4-(4-(pyrrolidin-1-yl)butoxy)phenyl)-3-(4-(4-(pyrrolidin-1-yl)butoxy)phenyl)pr
op-2-en-1-one (22b). Light yellow oil; Yield: 57.4%; Purity: 96.77% (HPLC).1H NMR (400 MH
z, CDCl3) δ13.58 (s, 1H, Ar–OH), 7.90 – 7.83 (m, 2H, C=CH, Ar–H), 7.62 (d, J = 8.3 Hz, 2H,
2 × Ar–H), 7.48 (d, J = 15.3 Hz, 1H, C=CH), 6.95 (d, J = 8.3 Hz, 2H, 2 × Ar–H), 6.49 (d, J = 10.
7 Hz, 2H, 2 × Ar–H), 4.06 (t, J = 6.3 Hz, 4H, 2 × OCH2), 2.69 – 2.51 (m, 12H, 6 × CH2), 1.94 –
1.67 (m, 16H, 8 × CH2). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.6, 165.5, 161.3, 144.3, 131.1,
130.4, 127.4, 117.7, 115.0, 114.1, 107.9, 101.6, 68.1, 67.9, 56.0, 56.0, 54.1, 31.9, 31.7, 31.5, 31.
4, 30.3, 30.2, 30.2, 29.7, 29.7, 29.6, 29.5, 29.4, 29.2, 27.2, 27.1, 25.3, 25.3, 23.4, 22.7, 14.1. HR
MS (ESI): Cacld for (C33H46N2O4) requires: m/z 535.3491, found: 535.3516.
(E)-1-(2-hydroxy-4-(4-(piperidin-1-yl)butoxy)phenyl)-3-(4-(4-(piperidin-1-yl)butoxy)phenyl)prop
-2-en-1-one (22c). Light yellow oil; Yield: 51.1%; Purity: 97.26% (HPLC). 1H NMR (400 MHz,
CDCl3) δ 13.58 (s, 1H, Ar–OH), 7.91 – 7.81 (m, 2H, C=CH, Ar–H), 7.66 – 7.59 (m, 2H, 2 × Ar–
H), 7.47 (d, J = 15.3 Hz, 1H, C=CH), 6.97 – 6.92 (m, 2H, 2 × Ar–H), 6.51 – 6.44 (m, 2H, 2 × Ar
–H), 4.05 (t, J = 6.1 Hz, 4H, 2 × OCH2), 2.60 – 2.44 (m, 12H, 6 × CH2), 1.90 – 1.73 (m, 8H,
4 × CH2), 1.71 – 1.65 (m, 8H, 4 × CH2), 1.53 – 1.46 (m, 4H, 2 × CH2). 13C NMR (100 MHz, CD
Cl3) δ 191.8, 166.6, 165.5, 161.2, 144.3, 131.1, 130.4, 127.4, 117.7, 114.9, 114.1, 107.9, 101.5, 6
8.0, 67.8, 58.7, 54.4, 54.4, 29.7, 27.2, 27.1, 25.4, 25.4, 24.1, 24.1, 23.0. HRMS (ESI): Cacld for
(C31H42N2O4) requires: m/z 507.3178, found: 507.3224.
(E)-1-(4-(4-(4-benzylpiperidin-1-yl)butoxy)-2-hydroxyphenyl)-3-(4-(4-(4-benzylpiperidin-1-yl)bu
toxy)phenyl)prop-2-en-1-one (22d). Light yellow oil; Yield: 60.1%; Purity: 97.73% (HPLC). 1H
NMR (400 MHz, CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.92 – 7.81 (m, 2H, C=CH, Ar–H), 7.66 – 7.5
8 (m, 2H, Ar–H), 7.48 (d, J = 15.4 Hz, 1H, C=CH), 7.31 (d, J = 7.5 Hz, 4H, 4 × Ar–H), 7.25 – 7.
14 (m, 6H, 4 × Ar–H), 6.97 – 6.91 (m, 2H, 2 × Ar–H), 6.48 (d, J = 8.2 Hz, 2H, 2 × Ar–H), 4.05 (t,

J = 6.3 Hz, 4H, 2 × OCH2), 3.05 – 2.87 (m, 4H, 2 × CH2), 2.56 (d, J = 7.0 Hz, 4H, 2 × CH2), 2.4
6 – 2.35 (m, 4H, 2 × CH2), 1.91 (q, J = 9.4, 7.2 Hz, 4H, 2 × CH2), 1.83 (q, J = 6.9 Hz, 4H, 2 × C
H2), 1.77 – 1.62 (m, 8H, 4 × CH2), 1.60 – 1.51 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 191.
8, 166.6, 165.5, 161.3, 144.3, 140.7, 140.6, 131.2, 130.4, 129.1, 128.2, 127.4, 125.8, 125.8, 117.7,
115.0, 114.1, 108.0, 101.6, 68.1, 67.9, 58.5, 58.5, 53.9, 43.2, 37.9, 32.1, 31.7, 29.7, 27.3, 27.1, 2
3.5, 23.4, 22.7, 14.2. HRMS (ESI): Cacld for (C31H46N2O4) requires: m/z 511.3491, found: 511.3
534.
(E)-1-(4-((6-(diethylamino)hexyl)oxy)-2-hydroxyphenyl)-3-(4-((6-(diethylamino)hexyl)oxy)pheny
l)prop-2-en-1-one (23a). Light yellow oil; Yield: 55.1%; Purity: 96.92% (HPLC).1H NMR (400
MHz, CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.90 – 7.80 (m, 2H, C=CH, Ar–H), 7.60 (dd, J = 8.8, 2.4
Hz, 2H, 2 × Ar–H), 7.46 (d, J = 15.4 Hz, 1H, C=CH), 6.93 (dd, J = 8.7, 2.4 Hz, 2H, 2 × Ar–H), 6.
51 – 6.41 (m, 2H, 2 × Ar–H), 4.00 (t, J = 6.4 Hz, 4H, 2 × OCH2), 2.73 (q, J = 7.2 Hz, 8H, 4 × CH
2), 2.67 – 2.58 (m, 4H, 2 × CH2), 1.88 – 1.74 (m, 4H, 2 × CH2), 1.61 (q, J = 8.2 Hz, 4H, 2 × CH2),
1.50 (ddt, J = 14.0, 10.5, 5.9 Hz, 4H, 2 × CH2), 1.41 (t, J = 8.6 Hz, 4H, 2 × CH2), 1.31– 1.26 (m,
4H, 2 × CH2), 1.16 (t, J = 7.2 Hz, 12H, 4 × CH3). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.5, 1
65.5, 161.3, 144.3, 131.2, 130.4, 127.7, 127.3, 123.9, 117.7, 114.9, 114.0, 107.9, 101.5, 68.1, 67.
9, 52.1, 46.6, 31.9, 31.7, 31.4, 30.3, 30.2, 29.7, 29.6, 29.6, 29.3, 29.0, 28.8, 27.1, 27.0, 25.8, 25.8,
25.3, 22.7, 14.1, 10.3, 10.3, 10.2. HRMS (ESI): Cacld for (C51H66N2O4) requires: m/z 771.5056,
found: 771.5089.
(E)-1-(2-hydroxy-4-((6-(pyrrolidin-1-yl)hexyl)oxy)phenyl)-3-(4-((6-(pyrrolidin-1-yl)hexyl)oxy)ph
enyl)prop-2-en-1-one (23b). Light yellow oil; Yield: 54.3%; Purity: 97.39% (HPLC). 1H NMR (4
00 MHz, CDCl3) δ 13.57 (s, 1H, Ar–OH), 7.92 – 7.81 (m, 2H, C=CH, Ar–H), 7.62 (d, J = 8.6 Hz,
2H, 2 × Ar–H), 7.48 (d, J = 15.4 Hz, 1H, C=CH), 6.95 (d, J = 8.5 Hz, 2H, 2 × Ar–H), 6.54 – 6.4
4 (m, 2H, 2 × Ar–H), 4.10– 4.01 (m, 4H, 2 × OCH2), 2.70 – 2.51 (m, 12H, 6 × CH2), 1.93 – 1.70
(m, 14H, 2 × CH2), 1.68 – 1.57 (m, 2H, CH2), 1.56 – 1.47 (m, 2H, CH2), 1.47 – 1.39 (m, 2H, CH
2). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.5, 165.6, 161.3, 144.2, 131.1, 130.3, 127.3, 117.7,
114.9, 114.0, 107.9, 101.5, 68.2, 67.9, 56.4, 56.0, 54.1, 29.7, 28.9, 28.4, 27.3, 27.2, 25.9, 25.3, 23.
4, 23.4. HRMS (ESI): Cacld for (C37H54N2O4) requires: m/z 591.4117, found: 591.4139.
(E)-1-(2-hydroxy-4-((6-(piperidin-1-yl)hexyl)oxy)phenyl)-3-(4-((6-(piperidin-1-yl)hexyl)oxy)phen
1
yl)prop-2-en-1-one (23c). Light yellow oil; Yield: 57.9%; Purity: 98.24% (HPLC). H NMR (40
0 MHz, CDCl3) δ13.58 (s, 1H, Ar–OH), 7.92 – 7.80 (m, 2H, C=CH, Ar–H), 7.62 (d, J = 8.4 Hz,

2H, 2 × Ar–H), 7.48 (d, J = 15.3 Hz, 1H, C=CH), 6.94 (d, J = 8.5 Hz, 2H, 2 × Ar–H), 6.53 – 6.44
(m, 2H, 2 × Ar–H), 4.04 (t, J = 6.2 Hz, 4H, 2 × OCH2), 2.64 – 2.46 (m, 12H, 6 × CH2), 1.88 – 1.
64 (m, 20H, 10 × CH2), 1.55 – 1.46 (m, 6H, 3 × CH2), 1.42– 1.37 (m, 2H, CH2). 13C NMR (100
MHz, CDCl3) δ 191.8, 166.6, 165.6, 161.3, 144.3, 131.1, 130.4, 127.4, 117.7, 114.9, 114.0, 107.9,
101.5, 68.2, 67.8, 58.8, 58.7, 54.4, 54.2, 31.9, 29.7, 28.8, 27.2, 27.2, 25.8, 25.4, 24.9, 24.1, 23.8,
23.0, 22.7, 14.1. HRMS (ESI): Cacld for (C35H50N2O4) requires: m/z 563.3804, found: 563.3847.
(E)-1-(4-((6-(4-benzylpiperidin-1-yl)hexyl)oxy)-2-hydroxyphenyl)-3-(4-((6-(4-benzylpiperidin-1-
yl)hexyl)oxy)phenyl)prop-2-en-1-one (23d). Light yellow oil; Yield: 51.1%; Purity: 97.36% (HPL
C).1H NMR (400 MHz, CDCl3) δ 13.53 (s, 1H, Ar–OH), 7.90 – 7.79 (m, 2H, C=CH, Ar–H), 7.6
0 (dd, J = 8.7, Hz, 2H, 2 × Ar–H), 7.47 (d, J = 15.4 Hz, 1H, C=CH), 6.92 (dd, J = 8.7, 2.5 Hz, 2
H, 2 × Ar–H), 6.52 – 6.40 (m, 2H, 2 × Ar–H), 4.12 – 3.95 (m, 4H, 2 × OCH2), 3.53 – 3.40 (m, 4
H, 2 × CH2), 3.00 – 2.75 (m, 4H, 2 × CH2), 2.67 – 2.46 (m, 10H, 5 × CH2), 2.11 – 1.61 (m, 20H,
4 × CH2, 4 × CH3), 1.58 – 1.36 (m, 4H, 2 × CH2). 13C NMR (100 MHz, CDCl3) δ 191.8, 166.5, 1
65.2, 161.0, 144.2, 131.1, 130.3, 130.2, 127.5, 117.8, 115.0, 114.8, 114.7, 114.2, 107.9, 101.7, 67.
3, 67.1, 54.9, 54.9, 54.7, 54.7, 23.5, 22.7, 14.2. HRMS (ESI): Cacld for (C35H54N2O4) requires:
m/z 567.4117, found: 567.4153.
(E)-1-(2-hydroxy-4-((6-(pyrrolidin-1-yl)hexyl)oxy)phenyl)-3-phenylprop-2-en-1-one (26a). Light
yellow oil; Yield: 43.7%; Purity: 98.11% (HPLC). 1H NMR (400 MHz, CDCl3) δ 13.42 (s, 1H, A
r–OH), 8.07 (d, J = 7.2 Hz, 1H, Ar-H), 7.87 (d, J = 15.6 Hz, 1H, CH=CH), 7.83 (d, J = 9.2 Hz, 1
H, Ar-H), 7.66-7.64 (m, 2H, 2 × Ar-H), 7.58 (d, J = 15.6 Hz, 1H, CH=CH), 7.43-7.38 (m, 2H,
2 × Ar-H), 6.47 (d, J = 8.8 Hz, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 3.98 (t, J = 6.0 Hz, 2H, OCH2), 3.
30-3.27 (m, 4H, 2 × NCH2), 3.04 (t, J = 8.0 Hz, 2H, NCH2), 2.13-2.09 (m, 4H, 2 × CH2), 1.93-1.
87 (m, 2H, CH2), 1.82-1.75 (m, 2H, CH2), 1.52-1.44 (m, 4H, 2 × CH2). 13C NMR (100 MHz, CD
Cl3) δ 191.8, 166.6, 165.7, 144.4, 134.7, 131.5, 131.4, 130.7, 129.6, 129.0, 128.5, 128.0, 120.3, 1
14.0, 108.0, 101.6, 68.0, 55.3, 53.5 (2C), 28.6, 26.5, 25.6, 25.4, 23.3 (2C). HRMS (ESI): Cacld f
or (C25H31NO3) requires: m/z 394.2337, found: 394.2382.
(E)-1-(2-hydroxyphenyl)-3-(4-((6-(pyrrolidin-1-yl)hexyl)oxy)phenyl)prop-2-en-1-one (26b). Ligh
t yellow oil; Yield: 49.2%; Purity: 97.92% (HPLC).1H NMR (400 MHz, CDCl3) δ 12.94 (s, 1H,
Ar–OH), 7.93 (d, J = 8.8 Hz, 1H, Ar-H), 7.90 (d, J = 16.0 Hz, 1H, CH=CH), 7.62 (d, J = 8.4 Hz,
2H, 2 × Ar-H), 7.55 (d, J = 15.6 Hz, 1H, CH=CH), 7.49 (t, J = 7.6 Hz, 1H, Ar-H), 7.02 (d, J = 8.0
Hz, 1H, Ar-H), 6.98 (d, J = 9.2 Hz, 1H, Ar-H), 6.93 (d, J = 8.0 Hz, 2H, 2 × Ar-H), 4.01 (t, J = 6.0

Hz, 2H, OCH2), 3.28-3.23 (m, 4H, 2 × NCH2), 3.01 (t, J = 8.0 Hz, 2H, NCH2), 2.15-2.12 (m, 4H,
2 × CH2), 1.94-1.89 (m, 2H, CH2), 1.84-1.80 (m, 2H, CH2), 1.56-1.51 (m, 2H, CH2), 1.50-1.45
(m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 193.7, 163.5, 161.5, 145.4, 136.2, 132.0, 130.6, 12
9.6, 127.3, 118.8, 118.6, 117.5, 115.0, 114.7, 67.8, 55.5, 53.6 (2C), 29.7, 28.8, 26.5, 25.7, 23.4 (2
C). HRMS (ESI): Cacld for (C25H31NO3) requires: m/z 394.2337, found: 394.2374.
4.2. Biological evaluation
4.2.1. AChE and BuChE inhibition assay
The modified Ellman method was used to determine the AChE and BuChE inhibitory ability of
the compounds, using AChE from electric eel (Sigma-Aldrich Corporation) and human erythro-
cytes (Sigma-Aldrich Corporation) or BuChE from equinum serum (Sigma-Aldrich Corporation)
and human serum (Sigma-Aldrich Corporation). Acetylthiocholine iodide (1 mmol/L, 30 µL),
phosphate-buffered solution (0.1 mmol/L, pH = 8.0, 40 lL), ChE (0.05 U/mL, final concentration,
10 µL) and different concentrations of test compounds (20 µL) were used for the ChE inhibition
assay. Blanks containing all components except ChE were carried out. IC50 values were calculat-
ed as the concentration of compound that produces more than 50% AChE or BuChE activity in-
hibition. The detailed procedure refers to the literature and our previous work [28, 36].
4.2.2. In vitro inhibition of MAOs
To evaluate the MAOs inhibition activity of target compounds, recombinant human MAO-A and
-B purchased from Sigma–Aldrich Co. were used. The detailed procedure refers to the literature
and our previous work [39].
4.2.3. Molecular docking
The X-ray crystal structure of AChE (PDB code: 1eve), BuChE (PDB code: 4tpk), Aβ (PDB:
1BA4) and human MAO-B (PDB code: 2V60) was download from Protein Data Bank
(www.rcsb.org) [39]. The AUTODOCK 4.2 program was used for the docking study of 23c.
Each docked system was performed by 200 runs of the Autodock search by the Lamarckian ge-
netic algorithm (LGA). A cluster analysis was performed on the docking results using a root
mean square (RMS) tolerance of 1.0 and the lowest energy conformation of the highest populat-
ed cluster was selected for analysis. Graphic manipulations and visualizations were done by Au-
todock Tools (ADT; version 1.5.6) or Discovery Studio 2.5 software.
4.2.4. Metal binding studies

The metal binding studies were performed with Varioskan Flash Multimode Reader (Thermo
Scientific) [40]. After 30-minutes incubation in methanol at room temperature, the UV spectrum
value with a wavelength in the range of 200-600 nm of 23c was recorded with or without CuCl2,
ZnCl2, AlCl3 and FeSO4. The final concentration of test compound and metal was 37.5 µM.
The stoichiometry of the compound-Cu2+ complex was determined by titrating the methanol so-
lution of the test compounds with an increasing concentration of CuCl2. The final concentration
of the test compound was 37.5 µM and the final concentration of Cu2+ ranged from 3.75 to 93.75
µM. The molar fraction relative to the test compound was investigated by subtracting CuCl2 and
the corresponding concentration of test compound for recording and processing.
4.2.5 Antioxidant activity assay
The antioxidant activity of target compounds was devalued by the oxygen radical absorbance
capacity fluorescein (ORAC-FL) assay [41]. All the assays were performed with 75mM phos-
phate buffer (pH = 7.40), and the final reaction mixture was 200 µL. FL (120 µL, 150 nM final
concentration) and antioxidant (20 µL) were added in the wells of a black 96-well plate using
Trolox as a standard (1-8 µM, final concentration). The details could reference our previous
work [27, 28].
4.2.6 Self-induced Aβ1-42 aggregation assay
The self-induced Aβ1-42 aggregation was measured by a Thioflavin T-based (ThT) binding assay
[42, 43]. Thioflavin T (Basic Yellow 1, ThT) was purchased from TCI (Shanghai) Development.
β-Amyloid1–42 (Aβ1-42), supplied as trifluoroacetate salt, was purchased from ChinaPeptides Co.,
Ltd. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from Energy Chemical. Briefly,
Aβ1-42 was dissolved in 1 mg/mL of HFIP and incubated for 24 hours at room temperature. The
dried Aβ1-42 was then re-dissolved in DMSO to make a solution of 200 µM and maintained at -
80 °C. Solutions of test compounds were prepared in DMSO in 2.5 mM for storage and diluted
with phosphate buffer solution (pH 7.4) before use. Aβ1–42 (20 µL, 25 µM, final concentration)
was incubated with 20 µL of test compounds (25 µM, final concentration) in 50 mM phosphate
buffer solution (pH 7.40) at 37 ° C for 24 h. After the incubation, 160 µL of 5 µM ThT in 50mM
glycine-NaOH buffer (pH 8.0) was added to each well. Fluorescence was measured by a Vari-
oskan Flash Multimode Reader (Thermo Scientific) with excitation and emission wavelengths of
446 and 490 nm, respectively.
4.2.7 Metal-induced Aβ1-42 aggregation and disaggregation

Cu2+-induced Aβ1-42 aggregation was determined using ThT method [28]. Solutions of Cu2+ were
prepared from standards to concentration of 75 µM HEPES buffer solution (20 mM, pH 6.6, 150
µM NaCl). The Aβ1-42 stock solution was diluted in HEPES buffer (20 mM, pH 6.6, 150 µM
NaCl). The mixture of Aβ1-42 (20 µL, 25 µM, final concentration) and Cu2+ (20 µL, 25 µM, final
concentration) with or without the tested compound (20 µL, 25 µM, final concentration) was in-
cubated at 37 ° C for 24 h. After which, 190 µL of 5 µM ThT in 50 mM glycine-NaOH buffer
(pH 8.50) was added. Each assay was performed in triplicate. The detection method was the
same as the self-induced Aβ1-42 experiment.
In the disaggregation of Cu+-induced Aβ1-42 fibrils experiment, Aβ1-42 stock solutions were dilut-
ed in HEPES buffer (20 mM, pH = 6.6, 150 mM NaCl). A mixture of Aβ1-42 (20 µL, 25 µM, final
concentration and Cu2+ (20 µL, 25 µM, final concentration) was incubated for 24 hours at 37° C.
20 µL inhibitor (final concentration was 25 µM) was then added into the mixture of Aβ1-42 with
Cu2+ and incubated at 37 °C for another 24 hours. Heat-resistant plastic film was used to mini-
mize evaporation effect. After which, glycine-NaOH buffer with 190 µL of 5 µM thioflavin T
was added. Each assay was run in triplicate. The detection method was the same as the self-
induced Aβ1-42 experiment.
4.2.8 Hydrogen peroxide induced PC12 cell injury [27]
PC-12 cells were seeded into 96-well plates at a density of 1 × 105 cells per mL in DMEM medi-
um (GIBCO.) supplemented with 10% heat-inactivated bovine calf serum (Hyclone). After incu-
bation overnight, the medium was replaced with fresh DMEM medium without calf serum and
phenol red. And tested compounds at various concentrations were added and incubated with the
cells for further 2 h. Then the cells were treated with 100 µM H2O2 for 2 hours. For the control
group, the cells were treated without H2O2 for 2 hours. At the end of the assay, the cells were in-
cubated with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
for 4 h at 37 ° C. The supernatants were carefully removed, and 150 µL DMSO was added into
each well. The absorbance at 570 nm of the formazan was determined using a Varioskan Flash
Multimode Reader (Thermo Scientific). Data were expressed as mean ± SD. To determine the
cytotoxicity of the compound, tested compound was incubated with PC-12 cells for 48 h without
the addition of H2O2, and other procedures were similar to the above mentioned.
4.2.9 In vitro blood−brain barrier permeation assay

As described by the literature [35], parallel artificial membrane permeation assay was used to
evaluate the blood-brain barrier penetration of target compounds. The detailed procedure refers
to our previous work [36].
4.2.10 In vivo assay
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[chuan] 2015-030). Mice were maintained under standard conditions with
a 12 h:12 h light–dark cycle at 20−22 °C with a relative humidity of 60−70%. Sterile food and
water were provided according to institutional guidelines.
Acute toxicity. Compound 23c was suspended in 0.5% carboxymethylcellulose sodium
(CMC-Na) salt solution (100, 250, 500 mg/kg) and given via oral administration according to the
divided experimental groups. After the administration of the compound 23c, the mice were ob-
served 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 ef-
fects. All animals were sacrificed on the 14th day after drug administration and were macroscop-
ically examined for possible damage to the heart, liver, and kidneys.
A step-down passive avoidance test was performed to test the ability of memory in mice [27,
36]. 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 elec-
trical foot shock (0.5 mA, 2s) through the grid floor on stepping down. Compound 23c (14.8, 7.4
and 3.7 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 sco-
polamine (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 less than 0.05 denoted the
presence of a statistically significant difference.
AUTHOR INFORMATION

Corresponding Author
* For Xiaoxia Lu: E-mail: luxx@cib.ac.cn
* For Zhipei Sang: E-mail: sangzhipei@126.com
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. #These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by the Special Project of Nanyang Normal University
(SYKF201808 and 12019QN001. Graduate Innovation Fund Project of Nanyang Normal Uni-
versity (NO.02). The Foundation and Frontier Projects of Nanyang Science and Technology Bu-
reau (2017JCQY020 and 2017 JCQY021). The China Scholarship Council (NO.201808410456).
The Key Scientific Research Project of Colleges and Universities in Henan Province
(NO.20A350006). National Science Foundation of China (No. 21861142007) and the Biological
Resources Network of Chinese Academy of Sciences (No. ZSTH-030).
Supporting Information Available:
1
Figures and Tables in PAMPA-BBB, and some representative H, 13C NMR, HR-ESI-MS and
HPLC spectra for the synthesized compounds are available as supplementary material.
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