Design, synthesis, in-silico and biological evaluation of novel chalcone derivatives as multi-function agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.07.021

A series of novel chalcone derivatives was designed, synthesized and evaluated as multifunctional agents for the treatment of AD. Among of these synthesized compounds, compound TM-2 was a selective BuChE inhibitor (IC₅₀ = 2.6 μM) and selective

Full text of DOI:10.1016/j.ejmech.2019.07.021

Accepted Manuscript
Design, synthesis, in-silico and biological evaluation of novel chalcone derivatives as
multi-function agents for the treatment of Alzheimer's disease
Zhipei Sang, Keren Wang, Pengfei Zhang, Jian Shi, Wenmin Liu, Zhenghuai Tan
PII:
S0223-5234(19)30642-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.021
EJMECH 11518
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2019
Revised Date: 3 July 2019
Accepted Date: 6 July 2019
Please cite this article as: Z. Sang, K. Wang, P. Zhang, J. Shi, W. Liu, Z. Tan, Design, synthesis, in-
silico and biological evaluation of novel chalcone derivatives as multi-function agents for the treatment
of Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.07.021.
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ACCEPTED MANUSCRIPT

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Design, synthesis, in-silico and biological evaluation of novel
chalcone derivatives as multi-function agents for the treatment
of Alzheimer’s disease
Zhipei Sanga,#*, Keren Wanga,#, Pengfei Zhangb, Jian Shia, Wenmin Liua,*, Zhenghuai
Tanc,*.
a College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University,
Nanyang, 473061, China
b School of Medicine, Henan University Minsheng College, Kaifeng, 475000, China
c Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan
Academy of Chinese Medicine Sciences, Chengdu, 610041, China
Abbreviations: AD, Alzheimer’s disease; AChEs acetylcholinesterase inhibitors;
NMDA, N-methyl- -aspartate; ACh, acetylcholine; AChE, acetylcholinesterase;
D
BuChE, butyrylcholinesterase; Aβ, β-amyloid; ROS, reactive oxygen species; MAO,
monoamine oxidase; DA, dopamine; 5-HT, serotonin; NE, noradrenaline; MTDLs,
multitarget-directed ligands; EeAChE, Electrophorus electricus AChE; eqBuChE,
equine serum BuChE; PDB, Protein Data Bank; TcAChE, Torpedo californica AChE;
CAS, catalytic active site; PAS, peripheral anionic site; ORAC-FL, Oxygen Radicals
Absorbance Capacity by Fluorescence; ThT, thioflavin T; TEM, transmission electron
microscopy;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;
PAMPA-BBB, parallel artificial membrane permeation assay of the blood−brain
barrier; TPSA, topological polar surface area; HPLC, high-performance liquid
chromatography;
PBS,
phosphate-buffered
saline;
DTNB,
5,5’-dithiobis-2-nitrobenzoic acid.
#These authors contributed equally.

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*Corresponding Author.
E-mail: sangzhipei@126.com (Zhipei Sang)
E-mail: liuwm1969@163.com (Wenmin Liu)
E-mail: tanzhh616@163.com (Zhenghuai Tan)
Abstract
A series of novel chalcone derivatives was designed, synthesized and evaluated as
multifunctional agents for the treatment of AD. Among of these synthesized
compounds, compound TM-2 was a selective BuChE inhibitor (IC50 = 2.6 µM) and
selective MAO-B inhibitor (IC50 = 5.3 µM), which were supported by docking study.
Compound TM-2 also showed good antioxidant activity, and was a selective metal
chelator, as well as a neuroprotectant. Moreover, compound TM-2 could significantly
inhibit self-induced and Cu2+-induced Aβ1-42 aggregation with 70.2% and 80.7%
inhibition rate, respectively, and could disaggregate Cu2+-induced Aβ1-42 aggregation
(73.5%), the further TEM images observed provided rational explanation. Besides,
compound TM-2 displayed good PAMPA-BBB permeability and conformed to the
Lipinski’s rule of five. Further, compound TM-2 presented precognitive effect on
scopolamine-induced memory impairment in vivo assay. Therefore, compound TM-2
might be a promising multifunctional hit compound for the treatment of AD, and the
further structure optimization are in progress.
Keywords: Alzheimer’s disease; chalcone derivatives; Multifunctional agents;
BuChE inhibitors; Antioxidant agents; Monoamine oxidase B inhibitor; Aβ
aggregation inhibitors; Biometals chelator; Neuroprotective effect; Precognitive effect

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1. Introduction
Alzheimer’s disease (AD) is a progressive and fatal neurodegenerative disorder
affecting 46 million people. It is characterized by the progressive deterioration of
various cognitive domains, including a decline in memory and thinking [1]. The
current clinical drugs for the treatment of AD include four acetylcholinesterase
inhibitors (AChEIs) such as tacrine, donepezil, galantamine and rivastigmine and one
N-methyl-D-aspartate (NMDA) receptor antagonists, namely memantine. Although
these single-target drugs could temporarily delay the progression of cognitive decline
in AD, their effects are modest. In addition, AChEIs produce several undesirable side
effects, such as gastrointestinal disturbances, bradycardia, and excessive salivation,
which are associated with peripheral cholinergic receptors [2]. Up to now, the
pathogenesis of AD is still not known, with several competing hypotheses, such as the
cholinergic hypothesis, amyloid hypothesis, oxidative stress hypothesis and metal
chelation hypothesis, attempting to explain it [3, 4].
According to the “cholinergic hypothesis”, cognitive decline in AD is closely
related to the level of acetyl choline (ACh) [5, 6]. Normally, ACh can be hydrolyzed
by both AChE and BuChE. The recent studies show that AChE plays the major role in
the hydrolysis of ACh in the healthy brain, while BuChE takes over the hydrolysis of
AChE in the AChE deficient brain. As AD developed, the AChE level in the brain
decreases progressively, but BuChE activity increases up to 165% of the normal level
[7]. In addition, selective BuChE inhibition is potentially advantageous because it
may circumvent the classical cholinergic toxicity that is a common side effect of
AChE inhibition [8]. Therefore, BuChE is a promising target for the treatment of the
advanced stages of AD.
The production and accumulation of soluble amyloid oligomers of Aβ are a
central event in the neuropathology of AD based on the ''amyloid hypothesis'' [9, 10],
as they are thought to be able to initiate the pathogenic cascade, ultimately leading to
neuronal loss and dementia. Aβ40 and Aβ42 are the main isoforms of Aβ peptides,
increasing evidences suggest that Aβ42 shows more toxic than Aβ40. In addition, Aβ42

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could form protofibrils and fibrillar aggregates at lower concentrations. Thus,
preventing the formation and aggregation of Aβ1-42 is a potential therapeutic approach
for AD.
Oxidative damage is one of the major causes of AD [11], affecting every class of
biological macromolecules, such as nucleic acids, proteins, carbohydrates and lipids.
It also triggers amyloid plaques and neurofibrillary tangles. Therefore, antioxidant
protection is an important way to treat AD. Moreover, transition metals, such as Cu2+,
Zn2+, Al3+ and Fe2+, could promote Aβ aggregation, and might contribute to the
production of reactive oxygen species (ROS) and oxidative stress, implying that these
biometals are closely related to several critical aspects of AD [12,13]. So, modulation
of these biometals in the brain might be a potential therapeutic strategy against AD.
Monoamine oxidase (MAO), an enzyme of the protein family of
flavin-containing amine oxidoreducatases, is responsible for the metabolism of
neurotransmitters such as dopamine (DA), serotonin (5-HT), and noradrenaline (NE).
There are two types of MAO, hMAO-A and hMAO-B. Studies show that hMAO-A
catalyzes the oxidative deamination of serotonin, adrenaline, and noradrenaline, while,
hMAO-B preferentially leads to deamination of β-phenethylamine and benzylamine
[14]. Recently, the selective MAO-B inhibitor, such as selegiline, has been shown to
significantly improve learning and memory deficits in animal models associated with
AD and to slow the disease progression in AD patients [15]. So, selective MAO-B
inhibitors seem to be an effective treatment of AD.
Due to the complexity of AD and the involvement of multi-systems in its
progression, multi-target-directed ligands (MTDLs) strategy is considered to be an
effective way to treat this disease [16-18]. These multi-functional agents,
simultaneously possessing two or more complementary biological activities, may
present an important clinical advance in the future.
Chalcones (α-phenyl-β-benzoylethylene) are prominent secondary-metabolite
precursors of flavonoids and isoflavonoids, and could be found widely in plants.

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Chalcone and its derivatives are important bioactive molecules with multi biological
activities including antioxidant activity, metal chelation property, anti-inflammatory,
MAO-B inhibition and neuroprotective properties [19, 20]. In our previous work, the
multi-functional agent compound EJ-7f showed significantly selective BuChE
inhibitory activity, and the O-alkylamines fragment was the important pharmacophore
group [21]. However, compound EJ-7f exhibited weak antioxidant activity, moderate
inhibition effect on Aβ aggregation, and compound EJ-7f did not show metal
chelating ability and hMAO-B inhibitory activity. Therefore, according to the MTDLs
drug design strategy, we plan to fuse chalcone and EJ-7f to obtain novel chalcone
derivatives (Figure 1), to test whether these novel molecules possess complementary
multi-functional activities.
In this paper, a series of chalcone derivatives were designed and synthesized
based on MTDLs. These target compounds were evaluated by ChEs/MAOs inhibition,
effect on Aβ1-42 aggregation, antioxidant activity, biometals chelation ability,
neuroprotective effect, the ability of cross blood-brain barrier in vitro and
precognitive effect in vivo assay. Besides, docking studies were also performed to
provide rational explanation for their biological activities.
<Insert Figure 1>
2. Results and discussion
2.1. Chemistry
The synthetic route of target compounds TM-1~TM-8 was depicted in Scheme 1 and
Scheme 2. In the Scheme 1, the starting material 3-hydroxybenzaldehyde 1 was
treated with excessive amounts of 1,3-dibromopropane (2a), 1,4-dibromobutane (2b),
1,5-dibromopentane (2c) or 1,6-dibromohexane (2d), respectively, in the presence of
K2CO3 in anhydrous acetonitrile at 65 °C to obtain the intermediates 3a-3d, and then
the intermediates 3a-3d were reacted with cyclic secondary amines 4a and 4b to get
intermediates 5a~5e. Finally, the target compounds TM-1~TM-5 were obtained by

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the condensation of intermediates 5a~5e with 1-hydroxy acetophenone 6 in alcoholic
50% KOH solution.
<Insert Scheme 1>
Additionally, as for the synthesis of TM-6~TM-8, 2’,4’-dihydroxyacetophenone 7
was used as raw material, the key intermediates 9a~9c were obtained under the same
experiments condition described in Scheme 2. Finally, benzaldehyde derivatives
10a~10c were reacted with intermediates 9a~9c in alcoholic 50% KOH solution to get
the target compounds TM-6~TM-8.
<Insert Scheme 2>
<Insert Scheme 3>
Further, as shown in Scheme 3, TM-9 was obtained by the reduction reaction of
TM-2 in the presence of Pd/C and H2. These target compounds TM-1~TM-9 have
13
not been reported in the literature. Their structures were confirmed by 1H NMR, C
NMR and ESI–MS spectroscopy.
2.2. Pharmacology
2.2.1 Inhibition studies on AChE and BuChE. The inhibitory activities of target
compounds TM-1~TM-9 against eeAChE (from electric eel) and eqBuChE (from
equine serum) were evaluated using the modified Ellman’s method [22, 23]. Chalcone
and donepezil were tested as the positive control. The results were listed in Table 1,
expressed as IC50 values. As expected, the target compounds TM-1~TM-9 showed
moderate to good inhibitory activity against AChE and BuChE compared with
chalcone, implying that introduction of O-alkylamines fragment could increase the
AChE/BuChE inhibitory activity, which was consistent with our design strategy.
According to the screening data in Table 1, the terminal groups NR1R2 and methylene
chain length of side chain significantly affected the inhibitory activities of eqBuChE.
Firstly, the BuChE inhibitory activity presented positive trend as the methylene chain
length increased, and the optimal methylene chain length was 4, such as TM-1 (IC50 =

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11.2 µM) > TM-2 (IC50 = 2.6 µM) < TM-3 (IC50 = 3.7 µM) < TM-4 (IC50 = 4.1 µM).
Replacing the 1,2,3,4-tetrahydroisoquinoline of TM-2 with 4-benzylpiperidine to
obtain compound TM-5, the BuChE inhibitory activity decreased to 4.9 µM, it
showed that 1,2,3,4-tetrahydroisoquinoline fragment contributed to BuChE inhibition.
Secondly, in order to further explore the structure-activity relationship, we changed
the position of side chain of TM-1 (IC50 = 11.2 µM) to get compound TM-6, the
BuChE inhibitory activity decreased to 13.4 µM, and then changing the position of
side chain of TM-2 to get compound TM-7, the BuChE inhibitory activity increased
to 5.2 µM, but it showed good AChE inhibitory activity with IC50 value of 8.3 µM.
Moreover, changing the position of side chain of TM-4 to obtain compound TM-8,
which was a selective AChE inhibitor (IC50 = 6.6 µM) and displayed moderate BuChE
inhibitory potency (IC50 = 10.4 µM). According to the phenomenon observed,
changing the position of side chain could decrease the BuChE inhibitory activity, but
increased the AChE inhibitory potency. Besides, the electron-donating groups, such
as OCH3 in TM-7 and N(CH3)2 in TM-8, were beneficial to AChE/BuChE inhibition,
while the electron-withdrawing group (such as F in TM-6) decreased the BuChE
inhibitory activity. Thus, compound TM-2 exhibited the best BuChE inhibitory
activity with IC50 value of 2.6 µM, and compound TM-8 indicated the best AChE
inhibitory potency (IC50 = 6.6 µM). More interestingly, when compound TM-2 was
reduced to TM-9 in the presence of Pd/C and H2, the BuChE inhibitory activity
increased to 0.64 µM, but TM-9 showed weak AChE inhibitory activity with IC50
value of 23.1 µM, suggesting that the reduction reaction of olefinic bond contributed
to BuChE inhibitory activity.
<Insert Table 1>
2.2.2 Molecular modeling studies of TM-2. According to the above results, compound
TM-2 would be the optimal compound to further study. The further computational
study was performed to explore possible binding mechanism of AChE and BuChE for
compound TM-2 using the docking program, AutoDock 4.2 package with Discovery
Studio 2.5, based on the X-ray crystal structure of AChE (PDB code: 1EVE) and

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BuChE (PDB code: 4tpk) [21, 24]. The results indicated that TM-2 could occupy the
entire AChE enzymatic catalytic active site (CAS), the mid-gorge sites and the
peripheral anionic site (PAS) (Figure 2). The hydroxyl group and carbonyl group of
TM-2 formed one intramolecular hydrogen bonding interaction, and the hydroxyl
interacted with Phe331 via intermolecular hydrogen bonding, moreover, the benzene
ring of chalcone skeleton interacted with key amino acid Tyr334 via σ-π interaction.
Meanwhile, hydrophobic interactions could be observed between the ligand and
amino acid residues Asp72, Trp84, Gly118, Gly123, Tyr121, Phe290, Phe288, Tyr334,
and Phe331.
<Insert Figure 2>
In the TM-2-BuChE complex, we found that compound TM-2 interacted with
BuChE via multiple sites (Figure 3), the hydroxyl group and carbonyl of chalcone
skeleton interacted via intramolecular hydrogen bonding, and further the hydroxyl
interacted with Ser287 via intermolecular hydrogen bonding. In addition, the benzene
ring of 1,2,3,4-tetrahydroisoquinoline interacted with the benzene ring of chalcone
skeleton via π-π interaction, and the benzene ring of 1,2,3,4-tetrahydroisoquinoline
interacted with key amino acid Tyr332 via π-π interaction, and one intermolecular
hydrogen bonding was observed between ligand TM-2 and key amino acid Trp82.
Besides, hydrophobic interactions could be observed between the ligand and
important amino acid residues Trp82, Phe329, Tyr332, Ser287, Trp231 and His438.
Therefore, the phenomenon observed might provide the rational explanation for the
highly selective BuChE inhibition of TM-2.
<Insert Figure 3>
2.2.3 BuChE reversibility of inhibition by TM-2. The recovery of eqBuChE inhibitory
activities after dilution with time monitoring were tested to evaluate the eqBChE
reversibility inhibition by TM-2 [25]. As shown in Figure 4A, the 0.1 × IC50 of
standard donepezil increased eqBChE activity to 9.4% compared with control, that
implying partial recovery of the eqBChE from donepezil. While the 0.1 × IC50 of
standard rivastigmine increased eqBChE activity to -0.7%, indicating no recovery of

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the eqBChE from rivastigmie. In addition, under the same condition, the 0.1 × IC50
of TM-2 eqBChE activity to 8.2%, as similar with donepezil. Furthermore, according
to the screening data in Figure 4B, the eqBChE activity restored to 126.8% with 0.1×
IC50 donepezil at 240 min, and the eqBChE activity of 0.1× IC50 rivastigmine
gradually increased to 73.2% at 240 min. As expected, and the eqBChE activity of
0.1× IC50 TM-2 increased to 103.5% at 240 min, similar with donepezil. So, the
observed phenomenon revealed that compound TM-2 was a reversible BuChE
inhibitor.
<Insert Figure 4>
2.2.4 Inhibition of MAOs in vitro. As shown in Table 1, the MAO-A and MAO-B
(recombinant human enzyme) inhibitory activities of target compounds TM-1~TM-9
were determined, iproniazid and rasagiline were also tested as referenced compounds
[26]. The results showed that all the target compounds indicated selective MAO-B
inhibitory activities, which contributed to the treatment of AD. Among of these target
compounds, compound TM-3 showed the best MAO-B inhibitory activity with IC50
value of 4.8 µM. According to the structure-active-relationship, the MAO-B
inhibitory activity improved as methylene chain increased, while the optimal
methylene chain was 5, such as TM-1 (IC50 = 10.7 µM) > TM-2 (IC50 = 5.3 µM) >
TM-3 (IC50 = 4.8 µM) < TM-4 (IC50 = 6.7 µM), in addition, the side chain terminal
NR1R2 fragment did not display obvious effect on MAO-B inhibitory activity, such as
TM-5 (IC50 = 6.4 µM) vs TM-2. Moreover, the position of side chain also affected the
inhibitory activity, compounds TM-6~TM-8 was obtained by changing the position
of O-alkylamines, the results showed that compounds TM-7 and TM-8 with
electron-donating groups indicated better MAO-B inhibitory potency than compound
TM-6 with electron-withdrawing group. Further, the ethylenic bond of compound
TM-2 was reduced to TM-9, the MAO-B inhibitory activity sharply decreased to 26.7
µM, and MAO-A inhibitory activity did not show obvious change, suggesting that the
α,β-unsaturated ketone fragment was conducive to MAO-B inhibitory activity.
2.2.5 Molecular modeling study of MAO-B. To further study the binding mode of

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TM-2 with MAO-B, molecular docking study based on the X-ray crystal structures of
human MAO-B (PDB code: 2V60) was performed [26]. As shown in Figure 5, the
2-hydroxy acetophenone moiety of TM-2 was located in the well-known binding
pocket of huMAO-B, in close proximity to the enzymatic cofactor FAD, and formed
one intermolecular hydrogen bonding interaction. Moreover, the benzene ring of
chalcone skeleton interacted with Tyr398 via π-π interaction, and the benzene ring of
1,2,3,4-tetrahydroisoquinoline interacted with Ile316 via σ-π interaction. Meanwhile,
hydrophobic interactions could be observed between the ligand and residues Ile316,
Phe168, Ile199, Pro102, Phe103, Tyr326, Phe343, Tyr435 and Leu88. Thus, the
phenomenon observed could explain why TM-2 could decrease the MAO-B
inhibitory activity.
<Insert Figure 5>
2.2.6 Antioxidant Activity in Vitro. The antioxidant activities of the target compounds
TM-1~TM-9 were tested by the ORAC-FL method (Oxygen Radicals Absorbance
Capacity by Fluorescence) [27, 28]. The vitamin E analogue Trolox was used as a
standard. According to the screening data in Table 1, all the compounds exhibited
good ORAC-FL values of 0.92−1.6 Trolox equivalents. The terminal groups NR1R2
and methylene chain length of side chain showed no obvious influence on the
antioxidant activity, such as TM-1~TM-5. When changing the position of side chain,
the data showed that the compounds with electron-donating groups (TM-7 and TM-8)
displayed better antioxidant activity than compound TM-6 with electron-withdrawing
group. It might be that the electron-donating groups help antioxidant activity. In
addition, compound TM-9 showed good antioxidant activity with ORAC value of 1.4
eq.
2.2.7 Inhibition of Self-induced Aβ1−42 Aggregation. The inhibitory activities of the
target compounds against self-induced Aβ1−42 aggregation were evaluated using a
thioflavin T (ThT) fluorescence assay [29]. Curcumin was used as reference
compound. As summarized in Table 2, all the target compounds TM-1~TM-8
displayed more inhibition of self-induced Aβ1−42 aggregation than curcumin (43.1% at

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25 µM). Taken together, the O-alkylamines side chain showed no meaningful impact
on self-induced Aβ1−42 aggregation inhibition. While, changing the O-alkylamines
side chain of TM-1~TM-5 to obtain TM-6~TM-8, the inhibitory activity remarkably
improved, and further the electron-donating groups (TM-7 and TM-8) in chalcone
skeleton displayed better inhibitory activity than compound TM-6 with
electron-withdrawing group in chalcone skeleton, especially, compound TM-8 with
dimethylamino group exhibited the best inhibitory activity with 97.5% inhibition rate
at 25 µM. It might be that the electron-donating groups served as an important role in
the inhibition of self-induced Aβ1-42 aggregation.
<Insert Table 2>
2.2.8 Molecular modeling studies of Aβ. To further explore the binding mode of the
compound TM-2 with Aβ (PDB: 1BA4), a molecular docking experiment was
performed [30]. As shown in Figure 6, compound TM-2 was locked at the C-terminal
hydrophobic area of Aβ. The hydroxyl group and carbonyl group of chalcone skeleton
formed one intramolecular hydrogen bonding, and further the carbonyl interacted with
Asp1 via intermolecular hydrogen bonding. In addition, the benzene ring of chalcone
skeleton interacted with Lys 16 via positive ion-π interaction, and the methylene chain
interacted with Phe19 via two σ-π interactions. Moreover, compound TM-2 formed
the hydrophobic interactions with the residues ASP1, PHE20, LYS28, ASN27 and
ILE31. These interactions might be favorable for the binding of Aβ and compound
TM-2.
<Insert Figure 6>
2.2.9 Metal-chelating properties of compound TM-2. The ability of compound
TM-2 to chelate biometals such as Cu2+, Zn2+, Al3+, Fe2+, Cu+ and Fe3+ was studied by
UV-vis spectrometry [31]. As shown in Figure 7, when compound TM-2 was treated
with CuCl2 and AlCl3, the maximum absorption wavelength shifted from 361 nm to
418 nm and 426 nm, respectively, indicating the formation of TM-2-Cu2+ and
TM-2-Al3+complex. Conversely, the maximum absorption exhibited no significant

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shift after adding Zn2+, Fe2+, Cu+ and Fe3+. These results indicated that the compound
TM-2 was a selective metal chelator.
<Insert Figure 7>
The stoichiometry of the TM-2-Cu2+ complex was determined using molar ratio
method by preparing solutions of compound TM-2 with increasing amounts of CuCl2.
According to the Figure 8, the absorbance linearly increased at first and then tended
to be stable at 426 nm. The two straight lines intersected at a mole fraction of 1.01,
revealing a 1:1 stoichiometry for complex TM-2-Cu2+.
<Insert Figure 8>
2.2.10 Effects on Cu2+-induced Aβ1−42 aggregation and disaggregation. Thioflavin T
(ThT) binding assay was used to test inhibition and disaggregation effect of TM-2 on
Cu2+-induced Aβ1-42 aggregation [32, 33]. As shown in Table 2, compound TM-2
could inhibit Cu2+-induced Aβ1-42 aggregation with 80.7% inhibition rate, which was
better than that of curcumin (76.5%). And transmission electron microscopy (TEM)
supported the data, Figure 9B illustrated that the sample of Aβ1-42 aggregated into
amyloid fibrils after adding 25 µM Cu2+ for 24 h incubation, while only small bulk
aggregations were observed in the sample of Aβ1-42 in the presence of TM-2. In
addition, compound TM-2 remarkably disaggregated Cu2+-induced Aβ1-42 aggregation
(73.5% at 25 µM), which was supported by TEM in Figure 9D. Thus, both the data
and TEM revealed that TM-2 could inhibit and disaggregate Cu2+-induced Aβ1-42
aggregation fibrils.
<Insert Figure 9>
2.2.11 Neuroprotective effects on H2O2-induced PC12 cell injury. The
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) assay was performed to
test the protective effects of TM-2 against H2O2–induced injury [34]. As shown in
Figure 10A, TM-2 had a wide therapeutic safety range. In addition, cell viability was

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sharply declined to 51.3 % (p < 0.01 vs control) after 100 µM H2O2 exposure in
Figure 10B, while compound TM-2 showed protective effects in a dose-dependent
manner against H2O2-induced PC12 cell injury. At concentrations of 10.0 µM, the cell
viability of compound TM-2 was 82.6%, exhibiting significantly neuroprotective
effects, and the cell viability decreased to 61.9% as the concentration reduced to 1.0
µM. Therefore, the results revealed that compound TM-2 possessed potent
neuroprotective effect on H2O2–induced cell injury.
<Insert Figure 10>
2.2.12 In vitro blood−brain barrier permeation assay. The parallel artificial
membrane permeation assay of the blood−brain barrier (PAMPA-BBB) was
performed to evaluate the BBB permeability of target compounds TM-1~TM-9 [35,
36]. 11 commercial drugs were selected to validate the assay (Table 3), a good linear
correlation was produced, Pe(exp) = 0.9163Pe(bibl.) − 0.2247 (R2 = 0.9558) (Figure
11). From the expression and considering the limit established by Di et al.,
compounds with permeability above 3.44 × 10−6 cm/s were considered to cross the
blood−brain barrier (Table 4). According to the measured permeability in Table 5, all
the target compounds could cross the BBB in vitro.
<Insert Table 3>
<Insert Figure 11>
<Insert Table 4>
<Insert Table 5>
2.2.13 Theoretical evaluation of ADME properties. To evaluate the druglike properties
of chalcone derivatives TM-1~TM-9, we adopted the Molinspiration property
program to calculate the items including milog P, pKa, topological polar surface area
(TPSA), the number of hydrogen-bond acceptors, and the number of hydrogen-bond

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donors [37]. From the data listed in Table 6, we determined that compound TM-2
abided by the Lipinski’s rule of five, acting as a promising drug candidate.
<Insert Table 6>
2.2.14 In vivo assay
Acute Toxicity. The acute toxicity of TM-2 was assessed in Kunming mice at
doses of 250, 500 and 1000 mg/kg (n = 10 per group). After oral administration of
compound TM-2, mice were observed continuously for the first 4 h through 14 days
for any abnormal behavior and mortality changes. The results exhibited that the
animals treated with compound TM-2 did not show any acute toxicity or mortality
immediately.
Effect on scopolamine-induced memory impairment. The step-down passive
avoidance task was performed to further assess whether TM-2 could improve
scopolamine-induced memory impairment [38, 39]. As shown in Figure 12, treatment
with scopolamine alone, the step-down latency time sharply decreased to 70.4 sec (p
< 0.01) compared with normal group (159.7 sec). While after treatment with
donepezil (5 mg/kg), the latency time significantly increased to 151.7 sec (p < 0.01)
compared with scopolamine group, thus implying that the model worked well. In
addition, treatment with compound TM-2 (16.8, 5.6 and 1.9 mg/kg) increased the
latency time in a dose-dependent manner, and the high dose group (16.8 mg/kg)
presented the longest latency time (126.4 sec., p < 0.05) of the three dose groups, but
the high dose (16.8 mg/kg) of compound TM-2 showed lower latency time than the
drug control group with donepezil (5 mg/kg, 151.7 sec). These results showed that
compound TM-2 could improve scopolamine-induced memory impairment, and the
optimal dose was 16.8 mg/kg.
<Insert Figure 12>
3. Conclusion

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In summary, a series of chalcone derivatives was designed and synthesized as
multifunctional agents for the treatment of AD. All the target compounds were
evaluated by ChEs/MAOs inhibitory activities, antioxidant activity, effect on Aβ1-42
aggregation. Among of these synthetic compounds, compound TM-2 indicated the
best BuChE inhibitory activity with IC50 value of 2.6 µM, and showed selective
MAO-B inhibitory activity (IC50 = 5.3 µM), which were supported by molecular
docking study. TM-2 was also an antioxidant and could significantly inhibit
self-induced Aβ1-42 aggregation (70.2%). In addition, compound TM-2 was a
selective metal chelator, and could inhibit (80.7%) and disaggregate (73.5%)
Cu2+-induced Aβ1-42 aggregation. Moreover, TM-2 acted as a neuroprotectant and
could cross the blood-brain barrier (BBB) in vitro, as well as abidance by the
Lipinski’s rule of five. Further, compound TM-2 presented precognitive effect on
scopolamine-induced memory impairment. Therefore, compound TM-2 might be a
promising multifunctional hit compound for the treatment of AD.
4. Experiment section
4.1. Chemistry
All materials were obtained from commercial suppliers and used without further
13
purification. 1H NMR and C NMR spectra were recorded using a Bruker 400 NMR
spectrometer. Mass spectra were recorded on Agilent-6210 TOF LC-MS Spectrometer.
The purity of compounds was determined by high-performance liquid chromatography
(HPLC) analysis to be over 96%. HPLC analysis was carried out on a Shimadzu
LC-10Avp plus system with the use of a Kromasil C18 column (4.6 mm × 250 mm,
5um). All the final compounds were obtained by column chromatography.
4.1.1. General procedures for the synthesis of intermediates 3a-3d and 8a-8c. The
appropriate dibromoalkane derivative 2a-2d (25 mmol) were added to a mixture of
the starting material 3-hydroxybenzaldehyde (1) or 2,4-dihydroxyacetophenone (7)
(10.0 mmol), and the mixture was heated at 65℃ for 8-12h in the presence of
anhydrous K2CO3 (12.0 mmol) in 30 mL CH3CN. The reaction was detected by TLC.
The solvent was evaporated under reduced pressure after complete reaction. The

ACCEPTED MANUSCRIPT
residue was resolved with water (100 mL) and was extracted with CH2Cl2 (50 mL ×
3). The combined solvents were treated with saturated aqueous NaCl, dried over
Na2SO4, and filtered. The organic phase was evaporated to obtain crude product,
which was purified on a silica gel chromatography using petroleum ether/acetone
(30:1) as eluent to give the intermediates 3a-3d and 8a-8c.
1
4.1.1.1 3-(3-bromopropoxy)benzaldehyde (3a). Colorless oil, 65.7% yield. H NMR
(400 MHz, CDCl3) δ 9.86 (s, 1H, CHO), 7.33 (d, J = 5.6 Hz, 2H, 2 × Ar-H), 7.28 (s,
1H, Ar-H), 7.09-7.05 (m, 1H, Ar-H), 3.93 (t, J = 6.4 Hz, 2H, OCH2), 1.75 (t, J = 6.0
Hz, 2H, BrCH2), 1.47-1.43 (m, 2H, CH2).
1
4.1.1.2 3-(4-bromobutoxy)benzaldehyde (3b). Colorless oil, 70.2% yield. H NMR
(400 MHz, CDCl3) δ 9.89 (s, 1H, CHO), 7.38-7.30 (m, 2H, 2 × Ar-H), 7.30-7.31 (m,
1H, Ar-H), 7.11-7.07 (m, 1H, Ar-H), 3.98 (t, J = 5.6 Hz, OCH2), 3.42 (t, J = 5.6 Hz,
BrCH2), 2.04-1.97 (m, 2H, CH2), 1.93-1.86 (m, 2H, CH2).
4.1.1.3 3-((6-bromohexyl)oxy)benzaldehyde (3d). Colorless oil, 53.9% yield. 1H NMR
(400 MHz, CDCl3) δ 9.97 (s, 1H, CHO), 7.45-7.43 (m, 2H, 2 × Ar-H), 7.38 (d, J = 2.0
Hz, 1H, Ar-H), 7.19-7.15 (m, 1H, Ar-H), 4.02 (t, J = 6.4 Hz, 2H, OCH2), 3.43 (t, J =
6.8Hz, 2H, BrCH2), 1.93-1.86 (m, 2H, CH2), 1.85-1.79 (m, 2H, CH2), 1.56-1.49 (m,
4H, 2 × CH2).
4.1.1.4 1-(4-(3-bromopropoxy)-2-hydroxyphenyl)ethan-1-one (8a). Colorless oil,
1
78.4% yield. H NMR (400 MHz, CDCl3) δ 12.64 (s, 1H, OH), 7.50 (d, J = 8.8 Hz,
1H, Ar-H), 6.31 (d, J = 9.2 Hz, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 4.00 (t, J = 5.6 Hz, 2H,
OCH2), 3.47 (t, J = 6.0 Hz, 2H, BrCH2), 2.42 (s, 3H, CH3), 2.25-2.17 (m, 2H, CH2).
4.1.1.5 1-(4-(4-bromobutoxy)-2-hydroxyphenyl)ethan-1-one (8b). Colorless oil, 80.5%
1
yield. H NMR (400 MHz, CDCl3) δ 12.75 (s, 1H, OH), 7.64 (d, J = 8.8 Hz, 1H,
Ar-H), 6.44 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H, Ar-H), 6.41 (d, J = 2.0 Hz, 1H, Ar-H),
4.04 (t, J = 6.0 Hz, 2H, OCH2), 3.50 (t, J = 6.4 Hz, 2H, BrCH2), 2.57 (s, 3H, CH3),
2.11-2.04 (m, 2H, CH2), 2.01-1.94 (m, 2H, CH2).
4.1.1.6 1-(4-((6-bromohexyl)oxy)-2-hydroxyphenyl)ethan-1-one (8c). Colorless oil,

ACCEPTED MANUSCRIPT
68.5% yield. 1H NMR (400 MHz, CDCl3) δ 1H NMR 12.73 (s, 1H, OH), 7.61 (d, J =
8.8 Hz, 1H, Ar-H), 6.42 (d, J = 9.2Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 3.99 (t, J = 6.0
Hz, 2H, OCH2), 3.42 (t, J = 6.4 Hz, 2H, BrCH2), 2.54 (s, 3H, CH3), 1.91-1.84 (m, 2H,
CH2), 1.83-1.76 (m, 2H, CH2), 1.52-1.45 (m, 4H, 2 × CH2).
4.1.2. General procedures for the synthesis of intermediates 5a-5e and 9a-9c.
A mixture of the intermediates 3a-3d (3 mmol) or 8a-8c, anhydrous K2CO3 (4.0
mmol) and corresponding secondary amine NR1R2 4a-4b (3.5 mmol) was heated at
65 °C in CH3CN (15 ml). The reaction was treated referenced the detail of 4.1.1. The
residue was purified on a silica gel chromatography using mixtures of
petroleum/acetone (15: 1) as eluent to obtain compounds 5a-5e and 9a-9c.
4.1.2.1 3-(4-(3,4-dihydroisoquinolin-2(1H)-yl)butoxy)benzaldehyde (5b). Colorless oil,
85.9% yield. 1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H, CHO), 7.44-7.42(m, 2H, 2 ×
Ar-H), 7.38 (s, 1H, Ar-H), 7.19-7.15 (m, 1H, Ar-H), 7.13-7.08 (m, 3H, 3 × Ar-H),
7.03-7.01 (m, 1H, Ar-H), 4.07 (t, J = 6.0 Hz, OCH2), 3.67 (s, 2H, phCH2), 2.92 (t, J
= 5.6 Hz, phCH2), 2.77 (t, J = 5.6 Hz, phCH2), 2.61 (t, J = 6.8 Hz, phCH2), 1.91-1.84
(m, 2H, CH2), 1.83-1.78 (m, 2H, CH2).
4.1.2.2
3-((6-(3,4-dihydroisoquinolin-2(1H)-yl)hexyl)oxy)benzaldehyde
(5d).
1
Colorless oil, 81.6% yield. H NMR (400 MHz, CDCl3) δ 9.91 (s, 1H, CHO),
7.40-7.36 (m, 2H, 2 × Ar-H), 7.35 (s, 1H, Ar-H), 7.15-7.11 (m, 1H, Ar-H), 7.09-7.04
(m, 2H, 2 × Ar-H), 6.99-6.97 (m, 1H, Ar-H), 3.96 (t, J = 6.4 Hz, 2H, OCH2), 3.59 (s,
2H, phCH2), 2.87 (t, J = 6.0 Hz, 2H, NCH2), 2.68 (t, J = 6.0 Hz, 2H, NCH2), 2.48 (t, J
= 7.6 Hz, 2H, NCH2), 1.83-1.76 (m, 2H, CH2), 1.65-1.58 (m, 2H, CH2), 1.53-1.46 (m,
2H, CH2), 1.44-1.39 (m, 2H, CH2).
4.1.2.3 3-(4-(4-benzylpiperidin-1-yl)butoxy)benzaldehyde (5e). Colorless oil, 88.3%
yield. 1H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H, CHO), 7.43-7.41 (m, 2H, 2 × Ar-H),
7.36 (s, 1H, Ar-H), 7.27 (t, J = 7.2 Hz, 2H, 2 × Ar-H), 7.18 (t, J = 7.6 Hz, 1H, Ar-H),
7.14 (t, J = 7.6 Hz, 3H, 3 × Ar-H), 4.02 (t, J = 4.0 Hz, 2H, 2 × Ar-H), 3.03 (d, J = 11.2
Hz, 2H, phCH2), 2.54 (d, J = 7.2 Hz, 2H, NCH2), 2.48 (t, J = 7.6 Hz, 2H, NCH2), 2.00
(t, J = 11.2 Hz, 2H, NCH2), 1.83-1.74 (m, 4H, 2 × CH2), 1.67 (d, J = 12.8 Hz, 2H,
CH2), 1.59-1.53 (m, 1H, CH), 1.44 (t, J = 11.6 Hz, 2H, CH2).

ACCEPTED MANUSCRIPT
4.1.2.4
1-(4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propoxy)-2-hydroxyphenyl)ethan-1-one (9a).
Colorless oil, 87.2% yield. 1H NMR (400 MHz, CDCl3) δ 12.73 (s, 1H, OH), 7.62 (d,
J = 8.8 Hz, 1H, Ar-H), 7.14-7.08 (m, 3H, 3 × Ar-H), 7.03-7.01 (m, 1H, Ar-H), 6.44
(dd, J1 = 6.4 Hz, J2 = 2.4 Hz, 1H, Ar-H), 6.42 (d, J = 2.4 Hz, 1H, Ar-H), 4.11 (t, J =
6.4 Hz, 2H, OCH2), 3.66 (s, 2H, phCH2), 2.92 (d, J = 5.6 Hz, 2H, phCH2), 2.76 (d, J =
6.0 Hz, 2H, NCH2), 2.69 (t, J = 7.2 Hz, 2H, NCH2), 2.55 (s, 3H, COCH3), 2.12-2.05
(m, 2H, CH2).`
4.1.2.5
1-(4-(4-(3,4-dihydroisoquinolin-2(1H)-yl)butoxy)-2-hydroxyphenyl)ethan-1-one (9b).
Colorless oil, 80.9% yield. 1H NMR (400 MHz, CDCl3) δ 12.75 (s, 1H, OH), 7.60 (d,
J = 9.2 Hz, 1H, Ar-H), 7.13-7.08 (m, 3H, 3 × Ar-H), 7.02-7.00 (m, 1H, Ar-H), 6.43
(dd, J1 = 6.4 Hz, J2 = 2.4 Hz, 1H, Ar-H), 6.39 (d, J = 2.4 Hz, 1H, Ar-H), 4.03 (t, J =
6.0 Hz, 2H, OCH2), 3.64 (s, 2H, phCH2), 2.91 (t, J = 6.0 Hz, 2H, NCH2), 2.74 (t, J =
6.0 Hz, 2H, NCH2), 2.58 (t, J = 7.6 Hz, 2H, NCH2), 2.53 (s, 3H, COCH3), 1.90-1.80
(m, 2H, CH2), 1.77-1.73 (m, 2H, CH2).
4.1.2.6
1-(4-((6-(3,4-dihydroisoquinolin-2(1H)-yl)hexyl)oxy)-2-hydroxyphenyl)ethan-1-one
1
(9c). Colorless oil, 76.9% yield. H NMR (400 MHz, CDCl3) δ 12.76 (s, 1H, OH),
7.61 (d, J = 8.8 Hz, 1H, Ar-H), 7.13-7.08 (m, 3H, 3 × Ar-H), 7.02-7.00 (m, 1H, Ar-H),
6.42 (dd, J1 = 6.4 Hz, J2 = 2.4 Hz, 1H, Ar-H), 6.40 (d, J = 2.4 Hz, 1H, Ar-H), 3.99 (t, J
= 6.4 Hz, 2H, OCH2), 3.62 (s, 2H, phCH2), 2.90 (t, J = 5.6 Hz, 2H, NCH2), 2.73 (t, J =
6.0 Hz, 2H, NCH2), 2.51 (t, J = 7.6 Hz, 2H, NCH2), 1.83-1.77 (m, 2H, CH2),
1.65-1.59 (m, 2H, CH2), 1.52-1.45 (m, 2H, CH2), 1.43-1.39 (m, 2H, CH2).
4.1.3. General procedure for the synthesis of compounds TM-1~TM-8.
A mixture of benzaldehyde derivatives 5a-5e, 10a, 10b and 10c (5.0 mmol) with
the corresponding acetophenone derivatives 6 and 9a-9c (5.5 mmol) was added slowly
to an amount of 30% aqueous KOH (20 mmol) solution in EtOH (7 mL). The mixture
was stirred for 72 h at room temperature, and then poured into ice-cold water. Acidified
with 10% HCl, and was extracted with CH2Cl2 (50 mL × 3), the organic phase was
evaporated under reduced pressure. The residue was purified using mixtures of

ACCEPTED MANUSCRIPT
CH2Cl2/acetone (30:1) as eluent on a silica gel chromatography to obtain the products
TM-1~TM-8.
(E)-3-(3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propoxy)phenyl)-1-(2-hydroxyphenyl)pr
op-2-en-1-one (TM-1).Yellow oil, 46.8% yield, 97.8% HPLC purity. 1H NMR (400
MHz, CDCl3) δ 12.81 (s, 1H, OH), 8.08 (d, J = 7.2 Hz, 1H, Ar-H), 7.94 (d, J = 7.6 Hz,
1H, Ar-H), 7.87 (d, J = 15.6 Hz, CH=CH), 7.64 (d, J = 15.6 Hz, 1H, CH=CH), 7.50 (t,
J = 8.0 Hz, 1H, Ar-H), 7.41 (t, J = 7.6 Hz, 1H, Ar-H), 7.33 (t, J = 8.0 Hz, 2H, 2 ×
Ar-H), 7.24 (d, J = 7.2 Hz, 1H, Ar-H), 7.18 (d, J = 8.0 Hz, 2H, 2 × Ar-H), 7.03 (d, J =
8.0 Hz, 1H, Ar-H), 6.96 (q, J = 7.6 Hz, 2H, 2 × Ar-H), 4.07 (t, J = 5.2 Hz, 2H, OCH2),
3.67 (s, 2H, phCH2), 3.59 (t, J = 5.6 Hz, 2H, phCH2), 3.00 (t, J = 6.0 Hz, 2H, NCH2),
1.89-1.86 (m, 4H, 2 × CH2). 13C NMR (100 MHz, CDCl3) 193.8, 163.6, 159.4, 145.4,
137.9, 136.4, 136.0, 131.6, 130.0, 129.8, 129.5, 128.3, 127.1, 126.8, 121.5, 120.4,
120.0, 118.9, 118.6, 117.1, 114.2, 67.6, 46.9, 46.1, 28.2, 26.6, 24.4. MS (ESI) m/z:
414.2 [M + H] +
(E)-3-(3-(4-(3,4-dihydroisoquinolin-2(1H)-yl)butoxy)phenyl)-1-(2-hydroxyphenyl)pro
p-2-en-1-one (TM-2). Yellow oil, 50.7% yield, 98.5% HPLC purity. 1H NMR (400
MHz, CDCl3) δ 12.80 (s, 1H, OH), 7.87 (d, J = 7.6 Hz, 1H, Ar-H), 7.75 (d, J = 15.2
Hz, 1H, CH=CH), 7.56 (d, J = 15.2 Hz, 1H, CH=CH), 7.39 (t, J = 8.0 Hz, 1H, Ar-H),
7.23 (t, J = 8.0 Hz, 1H, Ar-H), 7.20-7.13 (m, 1H, Ar-H), 7.09-7.06 (m, 3H, 3 × Ar-H),
7.04 (t, J = 7.2 Hz, 1H, Ar-H), 6.98-6.90 (m, 3H, 3 × Ar-H), 6.88-6.84 (m, 1H, Ar-H),
4.02 (s, 2H, phCH2), 3.94 (t, J = 5.2 Hz, 2H, OCH2), 3.15-3.13 (m, 2H, phCH2),
3.01-2.99 (m, 2H, NCH2), 2.94-2.91 (m, 2H, NCH2), 1.97-1.95 (m, 2H, CH2),
1.82-1.78 (m, 2H, CH2). MS (ESI) m/z: 428.2 [M + H] +
(E)-3-(3-((5-(3,4-dihydroisoquinolin-2(1H)-yl)pentyl)oxy)phenyl)-1-(2-hydroxyphenyl
)prop-2-en-1-one (TM-3). Yellow oil, 42.1% yield, 97.6% HPLC purity. 1H NMR
(400 MHz, CDCl3) δ 12.81 (s, 1H, OH), 7.96 (d, J = 7.6 Hz, 1H, Ar-H), 7.88 (d, J =
15.6 Hz, 1H, CH=CH), 7.67 (d, J = 15.2 Hz, 1H, CH=CH), 7.54-7.48 (m, 1H, Ar-H),
7.36-7.31 (m, 2H, 2 × Ar-H), 7.29-7.23 (m, 2H, 2 × Ar-H), 7.20-7.16 (m, 2H, 2 ×
Ar-H), 7.10 (d, J = 7.2 Hz, 1H, Ar-H), 7.03 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 6.96 (t, J =
5.6 Hz, 1H, Ar-H), 4.04 (s, 2H, phCH2), 3.92 (t, J = 5.2 Hz, 2H, OCH2), 3.15-3.13 (m,

ACCEPTED MANUSCRIPT
2H, phCH2), 2.34 (t, J = 7.6 Hz, 2H, NCH2), 2.13-2.08 (m, 2H, NCH2), 1.91-1.83 (m,
4H, 2 × CH2), 1.64-1.59 (m, 2H, CH2). MS (ESI) m/z: 442.2 [M + H] +
(E)-3-(3-((6-(3,4-dihydroisoquinolin-2(1H)-yl)hexyl)oxy)phenyl)-1-(2-hydroxyphenyl)
prop-2-en-1-one (TM-4). Yellow oil, 45.3% yield, 98.3% HPLC purity. 1H NMR (400
MHz, CDCl3) δ 12.79 (s, 1H, OH), 7.91 (d, J = 8.4 Hz, 1H, Ar-H), 7.87 (d, J = 16.0
Hz, 1H, CH=CH), 7.62 (d, J = 15.6 Hz, 1H, CH=CH), 7.49 (t, J = 7.6 Hz, 1H, Ar-H),
7.32 (t, J = 8.0 Hz, 1H, Ar-H), 7.23 (d, J = 7.6 Hz, 1H, Ar-H), 7.17 (s, 1H, Ar-H),
7.11-7.08 (m, 3H, 3 × Ar-H), 7.03-7.01 (m, 2H, 2 × Ar-H), 6.97 (d, J = 9.2 Hz, 1H,
Ar-H), 6.92 (d, J = 7.6 Hz, 1H, Ar-H), 4.01 (t, J = 6.0 Hz, 2H, OCH2), 3.66 (s, 2H,
phCH2), 2.91 (t, J = 5.2 Hz, 2H, phCH2), 2.77 (t, J = 5.6 Hz, 2H, NCH2), 2.55 (t, J =
7.6 Hz, 2H, NCH2), 1.85-1.80 (m, 2H, CH2), 1.70-1.62 (m, 2H, CH2), 1.56-1.51 (m,
2H, CH2), 1.46-1.39 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) 193.7, 163.6, 159.6,
145.5, 136.4, 136.0, 134.4, 134.2, 130.0, 129.7, 128.7, 126.6, 126.2, 125.7, 121.3,
120.4, 120.1, 118.9, 117.1, 114.4, 68.1, 58.1, 55.9, 50.8, 29.2, 28.8, 27.3, 26.9, 26.0.
MS (ESI) m/z: 456.2 [M + H] +
(E)-3-(3-(4-(4-benzylpiperidin-1-yl)butoxy)phenyl)-1-(2-hydroxyphenyl)prop-2-en-1-o
ne (TM-5). Yellow oil, 40.8% yield, 97.8% HPLC purity. 1H NMR (400 MHz, CDCl3)
δ 12.81 (s, 1H, OH), 8.00-7.91 (m, 1H, Ar-H), 7.87 (d, J = 15.2 Hz, 1H, CH=CH),
7.66 (d, J = 15.2 Hz, 1H, CH=CH), 7.54-7.49 (m, 2H, 2 × Ar-H), 7.35-7.28 (m, 3H, 3
× Ar-H), 7.24-7.19 (m, 2H, 2 × Ar-H), 7.16-7.11 (m, 2H, 2 × Ar-H), 7.07-7.00 (m, 2H,
2 × Ar-H), 6.96 (t, J = 8.4 Hz, 1H, Ar-H), 4.04 (t, J = 6.0 Hz, 2H, OCH2), 3.51 (d, J =
5.6 Hz, 2H, phCH2), 2.98-2.95 (m, 2H, NCH2), 2.60 (d, J = 6.8 Hz, 2H, NCH2),
2.56-2.51 (m, 2H, NCH2), 2.05-2.01 (m, 4H, 2 × CH2), 1.86-1.80 (m, 5H, 2 × CH2 +
CH). MS (ESI) m/z: 470.3 [M + H] +
(E)-1-(4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propoxy)-2-hydroxyphenyl)-3-(4-fluoro
phenyl)prop-2-en-1-one (TM-6). Yellow oil, 52.1% yield, 97.5% HPLC purity. 1H
1
NMR (400 MHz, CDCl3) δ H NMR 12.34 (s, 1H, OH), 7.85 (d, J = 15.2 Hz, 1H,
Ar-H), 7.82 (d, J = 8.0 Hz, 1H, Ar-H), 7.66 (d, J = 5.6 Hz, 1H, Ar-H), 7.64 (d, J = 5.6
Hz, 1H, Ar-H), 7.49 (d, J = 15.2 Hz, 1H, CH=CH), 7.29-7.26 (m, 2H, 2 × Ar-H), 7.19

ACCEPTED MANUSCRIPT
(d, J = 6.8 Hz, 1H, Ar-H), 7.15-7.09 (m, 3H, 3 × Ar-H), 6.46 (d, J = 8.8 Hz, 1H,
Ar-H), 6.43 (d, J = 1.6 Hz, 1H, Ar-H), 4.23 (s, 2H, phCH2), 4.16 (t, J = 5.6 Hz, 2H,
OCH2), 3.37-3.35 (m, 2H, phCH2), 3.22-3.20 (m, 4H, 2 × NCH2), 2.46 (m, 2H, CH2).
MS (ESI) m/z: 432.2 [M + H] +
(E)-1-(4-(4-(3,4-dihydroisoquinolin-2(1H)-yl)butoxy)-2-hydroxyphenyl)-3-(2-methoxy
phenyl)prop-2-en-1-one (TM-7). Yellow oil, 56.4% yield, 98.6% HPLC purity. 1H
NMR (400 MHz, CDCl3) δ 13.58 (s, 1H, OH), 8.18 (d, J = 15.6 Hz, 1H, CH=CH),
7.79 (d, J = 8.8 Hz, 1H, Ar-H), 7.67 (d, J = 15.6 Hz, 1H, CH=CH), 7.61 (d, J = 7.6 Hz,
1H, Ar-H), 7.36 (t, J = 7.6 Hz, 1H, Ar-H), 7.12-7.09 (m, 3H, 3 × Ar-H), 7.02-6.95 (m,
2H, 2 × Ar-H), 6.92 (d, J = 8.4 Hz, 1H, Ar-H), 6.47-6.43 (m, 2H, 2 × Ar-H), 4.02 (t, J
= 5.6 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.67 (s, 2H, phCH2), 2.91 (t, J = 5.6 Hz,
2H, phCH2), 2.77 (t, J = 5.6 Hz, 2H, NCH2), 2.60 (t, J = 6.8 Hz, 2H, NCH2), 1.87-1.78
(m, 4H, 2 × CH2). 13C NMR (100 MHz, CDCl3) 192.4, 166.6, 165.6, 158.9, 140.0,
134.3, 134.1, 132.0, 131.4, 129.4, 128.7, 126.6, 126.3, 125.7, 123.8, 120.9, 120.8,
114.2, 111.3, 107.9, 101.6, 68.1, 57.6, 55.9, 55.6, 50.8, 29.8, 28.8, 27.0, 23.5. MS
(ESI) m/z: 458.2 [M + H] +
(E)-1-(4-((6-(3,4-dihydroisoquinolin-2(1H)-yl)hexyl)oxy)-2-hydroxyphenyl)-3-(4-(dim
ethylamino)phenyl)prop-2-en-1-one (TM-8). Yellow oil, 32.7% yield, 98.2% HPLC
purity. 1H NMR (400 MHz, CDCl3) δ 13.77 (s, 1H, OH), 7.86 (d, J = 15.2 Hz, 1H,
CH=CH), 7.81 (d, J = 8.8 Hz, 1H, Ar-H), 7.55 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.36 (d,
J = 15.6 Hz, 1H, CH=CH), 7.12-7.10 (m, 3H, 3 × Ar-H), 7.02 (d, J = 5.2 Hz, 1H,
Ar-H), 6.68 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 6.45 (d, J = 9.6 Hz, 2H, 2 × Ar-H), 4.00 (t,
J = 6.4 Hz, 2H, OCH2), 3.68 (s, 2H, NCH2ph), 3.04 (s, 6H, 2 × NCH3), 2.93 (t, J = 5.2
Hz, 2H, phCH2), 2.79 (t, J = 5.2 Hz, 2H, NCH2), 2.56 (t, J = 7.6 Hz, 2H, NCH2),
1.83-1.79 (m, 2H, CH2), 1.70-1.64 (m, 2H, CH2), 1.55-1.47 (m, 2H, CH2), 1.46-1.42
(m, 2H, CH2). 13C NMR (100 MHz, CDCl3) 191.8, 166.5, 165.3, 152.2, 145.3, 134.1,
134.0, 130.9, 130.6 (2C), 128.7, 126.6, 126.3, 125.7, 122.6, 114.7, 114.3, 111.9 (2C),
107.7, 101.6, 68.2, 58.0, 55.8, 50.7, 40.1 (2C), 29.0, 28.6, 27.2, 26.8, 25.9. MS (ESI)
m/z: 499.3 [M + H] +

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3-(3-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)butoxy)phenyl)-1-(2-hydroxyphenyl)propan
-1-one (TM-9). To a mixture of TM-2 (1.5 mmol) in EtOH, Pd/C (0.075 mmol) was
added to the mixture in the presence of H2. The reaction was stirred for 28h at room
temperature, the mixture was treated and the organic phase was evaporated under
reduced pressure. The residue was purified using mixtures of petroleum ether/acetone
(50:1) as eluent on a silica gel chromatography to obtain TM-9. Light yellow oil,
40.1% yield, 98.2% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 1H NMR (400 MHz,
CDCl3) δ 12.79 (s, 1H, OH), 7.52 (d, J = 7.6 Hz, 1H, Ar-H), 7.20-7.15 (m, 4H, 4 ×
Ar-H), 7.13-7.11 (m, 1H, Ar-H), 7.04 (d, J = 7.6 Hz, 2H, 2 × Ar-H), 7.00 (d, J = 4.4
Hz, 1H, Ar-H), 6.95 (d, J = 8.0 Hz, 1H, Ar-H), 6.91 (s, 1H, Ar-H), 6.87-6.83 (m, 1H,
Ar-H), 4.03 (t, J = 6.0 Hz, 2H, OCH2), 3.99 (t, J = 6.0 Hz, 2H, phCH2), 3.89-3.86 (m,
4H, 2 × phCH2), 3.02-3.00 (m, 4H, 2 × NCH2), 2.80-2.77 (m, 2H, COCH2), 1.93-1.85
(m, 4H, 2 × CH2). MS (ESI) m/z: 430.2 [M + H] +
4.2 Biological activity
4.2.1 Inhibition assay of AChE and BuChE. The inhibitory potency of synthetic
compounds against AChE and BuChE was assessed by modified Ellman assay [22,
39]. For AChE inhibition assays, a reaction mixture (100 µL) containing ATC (1
mmol/L, 30 µL), pH = 8.0 phosphate-buffered solution, 10 µL EeAChE (0.45 U/mL)
and different concentrations of test compounds (20 µL) was incubated at 37°C for 15
min. Then 5,5’-dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) was added. The
inhibitory activities were determined by using a Varioskan Flash Multimode Reader at
412 nm. The inhibition percentage was calculated by the following formula: (1−Ai/Ac)
× 100, where Ai and Ac are the absorbance obtained for AChE in the presence and
absence of inhibitors, respectively. In vitro BuChE assay was performed using a
method similar as described above.
4.2.2 Recombinant human MAO-A and MAO-B inhibition studies. Recombinant
human MAO-A and MAO-B were purchased from Sigma-Aldrich and stored at
-80 °C [26]. Solutions of test compounds were prepared in DMSO (2.5 mM) and
diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM)
to a final volume of 500 µL containing various concentrations of test compounds

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(0-100 µM) and kynuramine (45 µM for MAO-A and 30 µM for MAO-B). The
reactions were initiated by the addition of the enzyme (7.5 µg/mL) and then incubated
for 30 min at 37. Then 400 µL NaOH (2N) and 1000 µL water were added to
terminate the enzymatic reactions and the mixtures were centrifuged at 16000g for 10
min.5 The concentrations of the MAOs generated 4-hydroxyquinoline were
determined by measuring the fluorescence of the supernatant on a Varioskan Flash
Multimode Reader (PerkinElmer) with excitation and emission wavelengths at 310
nm and 400 nm, respectively. A linear calibration curve was constructed by
preparing samples containing 4-hydroxyquinoline (0.047–1.56 µM) dissolved in
500 µL potassium phosphate buffer. To each calibration standard, 400 µL NaOH (2 N)
and 1000 µL water were added. The appropriate control samples were included to
confirm that the test compounds do not fluoresce or quench the fluorescence of
4-hydroxyquinoline under the assay conditions. IC50 values were calculated from
sigmoidal dose-response curves (graphs of the initial rate of kynuramine oxidation
versus the logarithm of inhibitor concentration). Each sigmoidal curve was
constructed form six different compound concentrations spanning at least three orders
of magnitude. Data analyses were performed with GraphRad Prism 5 employing the
one site competition model. IC50 values were determined in triplicate and expressed as
mean ± SD.
4.2.3. Antioxidant activity assay
The antioxidant activity was tested by the oxygen radical absorbance capacity
fluorescein (ORAC-FL) assay. The details referenced our previous work [24].
4.2.4. Metal binding study
The metal binding studies were performed in a Varioskan Flash Multimode Reader
(Thermo Scientific). The CuCl2, ZnCl2, AlCl3, FeSO4, CuCl, and Fe(NO3)3 were used
to test the metal binding assay. In addition, the stoichiometry of the compound-Cu2+
complex could reference our previous work [24, 34].

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4.2.5. Effect of test compounds on self-induced Aβ1-42 aggregation assay. Thioflavin
T-based flurometric assay was performed to investigate the self-induced Aβ1-42
aggregation [24, 29]. Briefly, the Aβ1-42 treated (20 µL, 25 µM, final concentration)
was incubated at 37 °C for 24 h with or without 20 µL of test compounds at different
concentrations ranging from 1-50 µM in 50 mM pH = 7.4. After incubated, 160 µL of
5 µM thioflavin T in 50 mM glycine-NaOH buffer (pH 8.5) was added. Fluorescence
was measured by a Varioskan Flash Multimode Reader (Thermo Scientific) with
excitation and emission wavelengths at 446 and 490 nm, respectively. Each assay was
run in triplicate. The presence of the inhibitor was calculated by the following
expression (1-IFi/IFc) × 100, in which IFi and IFc are the fluorescence intensities
obtained for Aβ1-42 in the presence and absence of inhibitors after subtracting the
background, respectively.
4.2.6. Effect of test compounds on Cu2+-induced Aβ1-42 aggregation. The mixture of
Aβ1-42 (20 µL, 25 µM) and Cu2+ (20 µL, 25 µM) was incubated at 37 °C for 24h with
or without the tested compound. After incubated, 190 µL of 5 µM Thioflavin T in 50
mM glycine-NaOH buffer (pH 8.5) was added. The detection method was performed
according to the self-induced Aβ1-42 experiment [24, 32].
As for the disaggregation of Cu2+-induced Aβ fibrils experiment, the mixture of the
Aβ1-42 (20 µL, 25 µM) with Cu2+ (20 µL, 25 µM) was incubated 37 °C for 24 h. The
tested compound (20 µL, 25 µM) was then added and incubated at 37 °C for another
24 h. After incubation, 190 µL of 5 µM thioflavin T in 50 mM glycine-NaOH buffer
(pH 8.5) was added. The detection method was the same as above.
4.2.7. Transmission Electron Microscopy (TEM) Assay. The preparation of samples
could reference the self-induced and Cu2+-induced Aβ1-42 aggregation experiments.
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 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 Field Emission

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Transmission Electron Microscope (JEM-2100F).
4.2.8. H2O2 induced PC12 cell injury. MTT reduction assay was used to evaluate the
protection effect of TM-2 [34]. The cells were grown at 37 °C in a humidified
atmosphere of 5% CO2. Neuronal PC12 cells were plated at a density of 105 cells/well
on 96-well plates in 100 µL of DEME. The cells were pre-incubated with TM-2 for
24 h before H2O2 (100 µM) was added. The cells were treated with or without H2O2
for two hours, and then replaced with fresh DMEM medium. Assays for cell viability
were performed 24 h after cultured at 37 °C in fresh medium. The cells were treated
with 25 µL MTT (5 mg/mL in PBS) for 4 h at 37 °C and then were lysed in a buffered
solution containing N, N-dimethylformamide (pH 4.5, 50% (aq, v/v)) and sodium
dodecyl sulfate (SDS, 20% (w/v)) overnight at room temperature in the dark. The
absorbance (A590 nm) was measured using Elx800 microplate reader (Bio-Tek). %
inhibition = [1-(Asample/Acontrol)]×100.
4.2.9. In vitro blood−brain barrier permeation assay
The blood-brain barrier penetration of compounds was evaluated using the paral
lel artificial membrane permeation assay (PAMPA) described by Di et al [36].
The detailed procedure refers to our previous work.
4.2.10. 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[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.
A step-down passive avoidance test was performed to test the ability of memory in
mice [39]. 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, 2s) through the grid

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floor on stepping down. We used a total of 60 mice in the passive avoidance test with
10 mice were used per treatment. Compounds TM-2 (1.9, 5.6 and 161.8 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 less
than 0.05 denoted the presence of a statistically significant difference.
AUTHOR INFORMATION
Corresponding Author
* For Zhipei Sang: E-mail: sangzhipei@126.com
* For Wenmin Liu: E-mail: liuwm1969@163.com
* For Zhenghuai Tan: E-mail: tanzhh616@163.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 Foundation and Frontier Projects of Nanyang
Science and Technology Bureau (2017JCQY020 and 2017 JCQY021), the Special
Project of Nanyang Normal University (SYKF2018081 and STP2018004).

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Supporting Information Available:
1
13
The representative H, C NMR and HPLC spectra for the synthesized compounds
are available as supplementary material.
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