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

Article abstract of DOI:10.1016/j.ejmech.2019.06.026

To discover multifunctional agents for the treatment of Alzheimer's disease (AD), a series of chalcone-O-carbamate derivatives was designed and synthesized based on the multitarget-directed ligands strategy. The in vitro biological activities were evaluat

Full text of DOI:10.1016/j.ejmech.2019.06.026

Accepted Manuscript
Design, synthesis, in-silico and biological evaluation of novel chalcone-O-carbamate
derivatives as multifunctional agents for the treatment of Alzheimer's disease
Zhipei Sang, Keren Wang, Jian Shi, Wenmin Liu, Zhenghuai Tan
PII:
S0223-5234(19)30551-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.026
EJMECH 11429
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 April 2019
Revised Date: 25 May 2019
Accepted Date: 9 June 2019
Please cite this article as: Z. Sang, K. Wang, J. Shi, W. Liu, Z. Tan, Design, synthesis, in-silico and
biological evaluation of novel chalcone-O-carbamate derivatives as multifunctional agents for the
treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.026.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, in-silico and biological evaluation of novel
chalcone-O-carbamate derivatives as multifunctional agents
for the treatment of Alzheimer’s disease
a,#*
a,#
a
a,*
c,*
Zhipei Sang , Keren Wang , Jian Shi , Wenmin Liu , Zhenghuai Tan .
a
College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University,
Nanyang, 473061, China
b
Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan
Academy of Chinese Medicine Sciences, Chengdu, 610041, China
Abbreviations: AD, Alzheimer’s disease; FDA, Food and Drug Administration;
AChEs acetylcholinesterase inhibitors; Aβ, β-amyloid; MTDLs, multitarget-directed
ligands; ACh, acetylcholine; ROS, reactive oxygen species; MAO, monoamine
oxidase; BChE, butylcholinesterase; 4-DMAP, 4-dimethylaminopyridine; PDB,
Protein Data Bank; 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; TLC, thin-layer chromatography; HPLC, high-performance liquid
chromatography; PBL, procine brain lipid.
*
Corresponding Author.
E-mail: sangzhipei@126.com (Zhipei Sang)
E-mail: liuwm1969@163.com (Wenmin Liu)
E-mail: tanzhh616@163.com (Zhenghuai Tan)

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Abstract
To discover multifunctional agents for the treatment of Alzheimer’s disease (AD), a
series of chalcone-O-carbamate derivatives was designed and synthesized based on
the multitarget-directed ligands strategy. The in vitro biological activities were
evaluated including AChE/BChE inhibition, MAO-A/MAO-B inhibition, antioxidant
activities, Aβ1-42 aggregation inhibition, metal-chelating properties and
neuroprotective effects against H O -induced PC12 cell injury. The results showed
2
2
compounds 5b and 5h indicated highly selective BChE inhibitory activity with IC50
values of 3.1 µM and 1.2 µM, respectively and showed highly selective MAO-B
inhibitory potency with IC values of 1.3 µM and 3.7 µM, respectively. In addition,
50
compounds 5b and 5h could inhibit self-induced Aβ1-42 aggregation with 63.9% and
3.1% inhibition percent rate, respectively. Particularly, compound 5b was a potent
antioxidant agent and neuroprotectant, as well as a selective metal chelator by
5
2
+
3+
chelating Cu and Al . Moreover, compound 5b could inhibit and disaggregate
2
+
Cu -induced Aβ
aggregation, which was further supported by the TEM images.
-42
1
Furthermore, compounds 5b and 5h could cross the blood-brain barrier (BBB) in vitro
and conformed to the Lipinski’s rule of five. Finally, the in vivo assay exhibited that
compound 5b could improve scopolamine-induced cognitive impairment. Taken
together, these results revealed that compound 5b might be a potential multifunctional
agent for the treatment of AD, and deserved to do further structure optimization.
Keywords:
Alzheimer’s
disease;
Chalcone-O-carbamate
derivatives;
Butylcholinesterase inhibitors; Monoamine oxidase B inhibitor; Antioxidant agents;
Neuroprotective effect; Aβ aggregation inhibitors; Biometals chelator; Precognitive
effect

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1
. Introduction
Alzheimer’s disease (AD) is an age-related neurodegenerative disease featuring
progressive memory loss, language skills decline, and other cognitive impairments [1].
Nearly 36 million people are affected by Alzheimer’s disease today, and three times
more are projected to be affected in 2050 [2]. Currently, the clinical drugs approved
by FDA for AD patients are focused on acetylcholinesterase inhibitors (AChEs) such
as donepezil, galantamine and rivastigmine. 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 [3]. Up to now, the etiology of AD is not fully
understood, but several factor, such as cholinergic dysfunction, amyloid-β (Aβ)
deposits, tau protein aggregation, oxidative stress and metal ion disorder are
considered to serve as important roles in the pathophysiology of AD [4]. Therefore,
an appropriate strategy to achieve better therapeutic efficacy for AD is proposed by
development of multitarget-directed ligands (MTDLs) that can simultaneously
modulate different targets or mechanisms involved in the neurodegenerative AD
cascade [5-7].
The classical “cholinergic hypothesis” has been widely accepted by researchers
and many efforts have been made to seek selective AChE inhibitors for the treatment
of AD [8-10]. The recent studies show that AChE is selectively responsible for
hydrolyzing neurotransmitter acetylcholine (ACh) in the healthy brain and the early
stages of AD, while BChE, possessing wider substrate specific than AChE, acts as the
major ACh degrading enzyme in AD progression [11]. Additionally, in the brain of
healthy human, both AChE and BChE are found in the ration of 4:1. However, AChE
activity in the AD brain decline progressively by up to 45%, leading to the loss of
neurons and axons, while BChE activity enhanced by up to two-fold in cortex and
hippocampus [12]. This suggests that BChE takes over the hydrolysis of ACh in AD
brain. Moreover, selective BChE inhibition might circumvent the classical cholinergic
toxicity that is a common side effect of AChE inhibition [13]. Therefore, selective

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BChE inhibitors are proposed strategy for the treatment of AD, especially the
advanced stages of AD.
According to the “amyloid hypothesis”, the production and accumulation of
oligomeric aggregates of Aβ are thought to initiate the pathogenic cascade, ultimately
leading to neuronal loss and dementia, which are considered as a central event in the
pathogenesis of AD [14, 15]. In addition, Aβ can efficiently generate reactive oxygen
species (ROS) in the presence of some transition metals. Antioxidant enzymes have
been observed to be increased in AD brain regions and oxidative damage is also
present in the brain of AD patients, damaging biological macromolecules including
proteins, lipids and nucleic acids [16-18]. In addition, redox-active metal ions like
2
+
2+
3+
2+
Cu , Zn , Al and Fe contribute to the production of ROS, which promotes
oxidative stress, leading to AD pathogenesis [19, 20]. Therefore, antioxidants and
modulation of such biometals in the brain have been considered as a potential
therapeutic strategy to treat AD.
Monoamine oxidase (MAO) is a FAD-dependent enzyme, responsible for
oxidative deamination of amine neurotransmitters, serves as an important target for
the treatment of specific features of AD. MAO exists as two distinct enzymatic
isoforms, MAO-A and MAO-B. Selective MAO-A inhibitors are used in the
treatment of depression, while selective MAO-B inhibitors are useful in the treatment
of AD because an increased level of MAO-B has been detected in the brain of AD
patients [21-23]. Therefore, selective MAO-B inhibitors might be another way against
AD.
Chalcone (1,3-diary-2-propen-1-one) is a simple chemical scaffold of many
naturally occurring compounds and has a widespread distribution in vegetables, fruits,
teas, and other plants [24]. Chalcone compounds are prominent secondary-metabolite
precursors of flavonoids and isoflavonoids, and also presented multiple biological
activities including anti-inflammatory, MAO-B inhibition, radical-scavenging and
neuroprotective properties, which would contribute to the treatment of AD [25]. But,
the lack of ChEs inhibition and metal chelation limited its application in AD therapy.
Rivastigmine is a unique cholinesterase inhibitor with both AChE and

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butyrylcholinesterase (BChE) inhibitory activity and is also available as transdermal
patch that has been approved by FDA for the treatment of mild, moderate, and severe
AD [26]. It has been shown that the carbamate moiety in rivastigmine is the
cholinesterase-inhibitory pharmacophore and that it can covalently bind to the enzyme
and remains attached until it is slowly removed by hydrolysis [27]. In addition, in our
previous work, the derivatives possessing adjacent hydroxyl group and carbonyl
group could be selective metal chelators [28-30]. Therefore, we plan to use the
MTDLs strategy to fuse chalcone, rivastigmine and metal chelation fragment and
create a series of chalcone-O-carbamate derivatives (Figure 1), hoping these
compounds possess complementary multi-functional activities.
<
Insert Figure 1>
In this work, a series of chalcone-O-carbamate derivatives was designed based
on MTDLs. These target compounds were synthesized and evaluated by AChE/BChE
inhibition, antioxidant activity, MAO-A/MAO-B inhibition, Aβ1-42 aggregation
inhibition and disaggregation, biometals chelation properties, neuroprotective effect
and the ability of cross blood-brain barrier in vitro. Further, the optimized molecule
was evaluated in vivo assay. Moreover, docking studies were also used to provide
rational explanation for their biological activities.
2
2
. Results and discussion
.1. Chemistry
The synthetic route of target compounds 5a~5l were depicted in Scheme 1, 2-hydroxy
acetophenone 1 as starting material was treated with 3-hydroxybenzaldehyde 2 in
alcoholic 50% KOH solution to obtain the key intermediate chalcone 3. Finally,
compound 3 was treated with excessive amounts of N,N-disubstituted carbamoyl
chlorides (4a-4f) in the presence of anhydrous K CO and a catalytic amount
2
3
4
-dimethylaminopyridine (4-DMAP) in CH CN at 60–65°C to afford the target
3
1
13
compounds 5a-5l. Their structures were confirmed by H NMR, C NMR and HR–
MS spectroscopy.
<
Insert Scheme 1>

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2
.2. Pharmacology
.2.1. eeAChE and eqBChE Inhibition. The inhibition activity towards eeAChE and
2
eqBChE for the target compounds 5a-5l were evaluated using modified Ellman assay
31]. The parental compound 3 and positive compound rivastigmine and donepezil
were also tested as control purpose. The IC values for AChE and BChE inhibition
[
5
0
were summarized in Table 1. The results revealed that introduction of carbamate
fragment significantly increased eqBChE inhibitory activity with IC values ranging
5
0
from 56.2 µM to 1.2 µM, particularly, compounds 5h, 5i and 5l indicated excellent
eqBChE inhibitory activity compared with positive compound rivastigmine (eqBChE,
IC = 1.3 µM), which was consistent with our design strategy. In addition, in Table 1
5
0
almost all the target compounds were selective BChE inhibitors because they
exhibited weak eeAChE inhibitory potency, except compounds 5e and 5k with the
carbamate moieties containing N,N-diphenylamine group.
The carbamate substitution significantly affected eqBChE inhibitory activity.
When the carbamate moieties at the 3 position of chalcone, target compound 5a with
N,N-dimethylamine group showed good eqBChE inhibitory activity with IC value
5
0
was 2.51 µM, changing N,N-dimethylamine group of 5a with N-ethyl-N-methylamine
and N-methoxymethylamine to obtain 5b and 5c, respectively, the eqBChE inhibitory
activity showed slightly decreased with IC50 value was 3.1 µM and 3.2µM,
respectively. Replacing N,N-dimethylamine group of 5a with N,N-diethylamine to get
5
d, the eqBChE inhibitory activity decreased to 4.8 µM, and then compound 5f with
morpholine group was obtained by means of ringclosure from N,N-dimethylamine
group, 5f displayed good eqBChE inhibitory activity with IC value was 5.1 µM.
5
0
While, replacing N,N-dimethylamine group of 5a with N,N-diphenylamine group to
get 5e, the eqBChE sharply decreased to 56.2 µM. The phenomenon revealed that the
aliphatic series N,N-disubstituted carbamoyl contributed to the eqBChE inhibitory
activity, while the aromatic N,N-disubstituted carbamoyl resulted in negative effect on
eqBChE inhibitory potency.
Furthermore, the carbamate moieties at both 2’ and 3 position of chalcone also

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had significantly effects on eqBChE inhibitory activity. Generally, disubstituted
carbamate derivatives exhibited better eqBChE inhibitory activities that that of
monosubstituted carbamate derivatives, such as 5b < 5h, 5c < 5i, 5d < 5j, 5e < 5k and
5
f < 5l. Moreover, for the disubstituted carbamate derivatives, the potencies to inhibit
eqBChE were in the order N-ethyl-N-methylamine (5h) > N-methoxymethylamine
5i) > morpholine (5l) > N,N-diethylamine (5j) > N,N-dimethylamine (5g) >
(
N,N-diphenylamine (5k). The results observed revealed that carbamate moieties
containing aliphatic amine groups and cyclic amine contributed to the eqBChE
inhibitory activities, but the arylamine produced an adverse effect on eqBChE
inhibitory activity. As for the eeAChE inhibition, the carbamate moieties did not show
significant effect on AChE inhibitory activity. Therefore, the target compounds were
promising selective eqBChE inhibitors, and compounds 5b and 5h were selected to
further study.
<
Insert Table 1>
2
.2.2 EqBChE reversibility of inhibition by 5b and 5h. To further test the eqBChE
reversibility inhibition by 5b and 5h, the recovery of eqBChE inhibitor inhibition
after dilution (Figure 2A), with time monitoring (Figure 2B) were performed [32, 33].
As shown in Figure 2A, compared to controls, the 0.1 × IC of standard donepezil
5
0
increased eqBChE activity to 8.8%, that is an indication of possible slow, partial
recovery of the eqBChE from donepezil. While the 0.1 × IC50 of standard
rivastigmine increased eqBChE activity to -2.9%, implying an indication of possible
no recovery of the eqBChE from rivastigmie. In addition, under the same condition,
the 0.1 × IC of compounds 5b and 5h increased eqBChE activity to 0.7% and
5
0
1
.0%, respectively, showing on recovery of the eqBChE, as similarly with
rivastigmine. Furthermore, as shown in Figure 2B, the eqBChE activity restored to
03.9% with 0.1× IC50 donepezil at 120 min, the eqBChE activity of 0.1× IC50
1
rivastigmine gradually increased to 71.6% at 120 min. As expected, and the eqBChE
activity of 0.1× IC50 compounds 5b and 5h added up to 75.1% and 70.9%,
respectively, at 120 min, like rivastigmine. Therefore, the observed results indicated
that compounds 5b and 5h would be pseudo reversible inhibitors of BChE.

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<
Insert Figure 2>
.2.3 Enzyme kinetic of compound 5b. Compound 5b was selected to further assess
kinetic study for testing the inhibitory mechanism of BChE [34]. As shown in Figure
, graphical analysis showed both increasing slopes (decreased Vmax) and intercepts at
2
2
Y axe. It implied that compound 5b was a reversible competitive inhibitor, and 5b
could compete with substrate BCh, binding with the active site of BChE.
<
Insert Figure 3>
.2.4 Molecular modeling studies of 5b and 5h. The further computational study was
performed to explore possible binding mechanism of AChE and BChE for compounds
2
5
b and 5h using the docking program, AutoDock 4.2 package with Discovery Studio
2
.5, based on the X-ray crystal structure of BChE (PDB code: 4tpk) and AChE (PDB
code: 1EVE) [28, 35]. The results indicated that 5b bind with BChE via multiple sites
Figure 4). The hydroxyl group and carbonyl group of 5b formed one intramolecular
(
hydrogen bonding, and the hydroxyl group and the carbonyl group interacted with key
amino acid Trp82 and important amino acid Gly121 via intermolecular hydrogen
bonding, respectively. In addition, the benzene ring of chalcone skeleton interacted
with key amino acid Trp82 via two π-π interactions, contributing to the BChE
inhibitory activity. Meanwhile, hydrophobic interactions could be observed between
the ligand and Trp82, Asp70, Ala328, Thr120, Gly116, His438. Furthermore,
compound 5h was also observed multiple interactions with BChE (Figure 5), the two
benzene rings of chalcone skeleton simultaneously formed π-π interactions with key
amino acid Trp82 and important amine acid Tyr332, respectively. The carbonyl group
of chalcone skeleton interacted with important amine acid Tyr332 via intermolecular
hydrogen bonding, moreover, the oxygen atom of O-carbamate moiety could interact
with key Thr120 through intermolecular hydrogen bonding. Besides, there were some
hydrophobic interactions were observed between the ligand and Trp82, Asp70, Ala328,
Thr120, Gly116, His438, Tyr332. The multiple interactions observed between ligands
(5b and 5h) and BChE provided reasonable explanation for their potent BChE
inhibitory activity.
<
Insert Figure 4>

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<
Insert Figure 5>
On the other hands, the possible binding mechanism of AChE for compounds 5b
and 5h was also explored. In the 5b-AChE complex (Figure 6A), there were two
characteristic intermolecular hydrogen bonding were observed between ligand and
amine acid. While, in the 5h-AChE complex (Figure 6B), only one characteristic
interaction was found between the Phe288 and the carbonyl group of 5h. Therefore,
the limited characteristic interaction explained the weak AChE inhibitory activity for
compounds 5b and 5h, and further exhibited that compounds 5b and 5h were
promising selective BChE inhibitors.
<
Insert Figure 6>
2
.2.5 Antioxidant Activity in Vitro. Oxygen Radicals Absorbance Capacity by
Fluorescence (ORAC-FL) method was used to test the antioxidant activity of target
compounds 5a-5l [29]. The vitamin E analogue Trolox was used as a standard, and
the parent compound 3 was also tested as control purpose. The screening data were
listed in Table 1, all the compounds showed exhibited potent antioxidant activity with
ORAC-FL values of 0.31−1.3 Trolox equivalents (eq), but presented lower
antioxidant potency than parent compound 3 (2.6 eq Trolox). In addition,
monosubstituted carbamate derivatives (5a-5f) showed better antioxidant activity than
that of disubstituted carbamate derivatives (5g-5l). As expected, the introduction of
carbamate moieties in the chalcone nucleus decreased the radical capture capacity,
while the different tertiary amine unit on the carbamate fragment produced little
influence on the radical capture capacity. The results observed revealed that the
hydroxyl group played crucial role in the antioxidant activity, and the compound 5b
(
1.3 eq) possessing mono-N-ethyl-N-methyl carbamate showed better antioxidant
activity than that of 5h (0.35 eq) with disubstituted carbamate.
.2.6 huMAO-A and huMAO-B inhibition. The MAO-A and MAO-B (from
2
recombinant human enzyme) inhibitory potency of 5a-5l were determined using
fluorescence assay [36, 37]. Iproniazid and rasagiline were tested as referenced
compounds. The parent compound 3 was also assessed as control purpose, which was
a selective huMAO-B inhibitor with IC value was 1.1 µM. The screening data in
5
0

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Table 2 revealed that all the target compounds showed good huMAO-B inhibitory
potency, and weak huMAO-A inhibitory activity compared with compound 3. So, the
synthetic target compounds were selective huMAO-B inhibitors, which was consistent
with our design strategy. In addition, introduction of carbamate fragment weakened
MAO-B inhibitory activities, and the tertiary amine unit in carbamate moieties
showed potent effect on huMAO-B inhibitory activities. Generally, the target
compounds 5a-5d possessing aliphatic amine displayed better huMAO-B inhibitory
activity than the compounds 5e and 5f with cyclamine and arylamine, respectively.
The huMAO-B inhibitory potency were in order N,N-dimethylamine (5g) >
N-ethyl-N-methylamine (5b) > N,N-diethylamine (5j) >N-methoxymethylamine (5i) >
morpholine (5l) > N,N-diphenylamine (5k). Moreover, the compounds (5g-5l) with
disubstituted carbamate fragment indicated lower huMAO-B inhibitory activity than
the compounds (5a-5f) with monosubstituted carbamate. According to the results
observed, compound 5a exhibited the best huMAO-B inhibitory activity with IC50
value was 0.8 µM, and the representative compounds 5b and 5h were potent selective
huMAO-B inhibitors with IC values were 1.3 µM and 3.7 µM, respectively.
5
0
<
Insert Table 2>
2
.2.7 Molecular modeling study of MAO-B. To further study the binding mode of 5b
and 5h with MAO-B, molecular docking studies based on the X-ray crystal structures
of human MAO-B (PDB code: 2V60) were performed [25]. In 5b-MAO-B complex
(Figure 7A), the hydroxyl group and carbonyl group of 5b formed intramolecular
hydrogen bonding interaction, and the hydroxyl group and the carbonyl group
interacted with important amino acid Ile199 and Tyr326 via intermolecular hydrogen
bonding, respectively. The O atom and the carbonyl of O-carbamate moiety could
simultaneously interact with Tyr398 via intermolecular hydrogen bonding. In addition,
the benzene ring of chalcone skeleton interacted with Cys172 via σ-π interaction.
While, in the 5h-MAO-B complex (Figure 7B), some characteristic interactions were
observed, the carbonyl of O-carbamate moiety interacted with amine acid Tyr435
intermolecular hydrogen bonding. Moreover, the benzene ring of chalcone skeleton
interacted with Ile199 forming σ-π interaction. Besides, some hydrophobic

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interactions could be observed between the ligands (5b and 5h) and MAO-B. Thus,
the phenomenon observed might provide possible explanation that compounds 5b and
5
h could decrease the MAO-B inhibitory activity.
Insert Figure 7>
.2.8 Inhibition of Self-induced Aβ1−42 Aggregation. The potent selective BChE
<
2
inhibitors 5a, 5b, 5c, 5h, 5i, 5j and 5l were selected to evaluate the inhibitory activity
toward self-induced Aβ1−42 aggregation using thioflavin T (ThT) fluorescence assay
[
35]. Curcumin was used as reference compound, and the parent compound 3 was
also tested as control purpose. According to the screening data in Table 2, the parent
compound 3 (70.6%) showed more inhibitory activity toward self-induced Aβ1−42
aggregation than curcumin (43.1%). All the tested compounds displayed better
inhibitory potency than curcumin, but indicated lower inhibition than compound 3. In
addition, the monosubstituted carbamate derivatives (5a-5c) exhibited better
inhibition than the disubstituted carbamate compounds (5h-5j, 5l). The possible
reason was that the hydroxyl group served as important role in inhibition of
self-induced Aβ1−42 aggregation. Moreover, the tertiary amine unit produced no
obvious effect on self-induced Aβ1−42 aggregation inhibition.
2
.2.9 Metal-chelating properties of compounds 5b and 5h. The chelation ability of
compounds 5b and 5h was assessed by UV-visual spectrometry using biometals such
2
+
2+
2+
3+
as Cu , Fe , Zn and Al [28, 29]. The results were depicted in Figure 8. The
electronic spectra of 5b indicated a red shift (the peak at 359 nm shifted to 435 nm
and 428 nm, respectively) after adding CuCl and AlCl . While, compound 5b was
2
3
treated with FeSO and ZnCl , respectively, the electronic spectra presented no
4
2
obvious shift. The results revealed that compound 5b was a selective metal chelator
2
+
3+
and could selectively chelate Cu and Al . In addition, the electronic spectra of 5h
showed no significant changes after adding CuCl , AlCl , FeSO and ZnCl ,
2
3
4
2
2+
3+
2+
2+
suggesting little or no complex formation between 5h with Cu , Al , Fe or Zn .
The phenomenon observed exhibited that the 2’-hydroxyl and carbonyl group of the
chalcone nucleus contributed to the chelation.
2
+
3+
The above results showed that compound 5b could chelate Cu and Al , which

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have been promising therapeutic applications in AD patients. In addition, several
2
+
studies had shown that Cu significantly contributed to the formation of senile
2+
plaques and the complex Aβ1-42-Cu produced more reactive oxygen species. Thus,
2
+
compound 5b could selective chelate Cu and could simultaneously stop the
formation of amyloid plaques and relieved oxidative stress.
<
Insert Figure 8>
2
+
The stoichiometry of the 5b-Cu complex was determined using the molar ratio
method. The UV spectra were recorded and treated by numerical subtraction of CuCl2
and 16d at corresponding concentrations at 435nm. As shown in Figure 9, the
absorbance linearly increased initially and then plateaued. The two straight lines
intersected at a mole fraction of 0.97, indicating a 1:1 stoichiometry for the complex
2
+
5
b-Cu .
<
Insert Figure 9>
2+
2
.2.10 Effects on Cu -induced Aβ
aggregation. Compound 5b was a selective
−42
1
2
+
2+
metal chelator, and could chelate Cu to form the 5b-Cu complex. So, thioflavin T
(ThT) binding assay was performed to evaluate inhibition and disaggregation effect of
2
+
5
b on Cu -induced Aβ
aggregation [38, 39]. The data from Table 2 showed that
-42
1
2+
compound 5b significantly inhibit Cu -induced Aβ
aggregation (78.3%), which
-42
1
was higher than positive compound curcumin (73.4%). Moreover, 5b could
2
+
decompose Cu -induced Aβ aggregation with 83.1% disaggregation rate at 25 µM
,
1
-42
and displayed better disaggregation than curcumin (58.2%).
To further complement the ThT binding assay, transmission electron microscopy
(TEM) was used to observe the Aβ1-42 aggregation phenomenon. For inhibition assay,
as shown in Figure 10B, the fresh Aβ1-42 had aggregated into amyloid fibrils after
2
+
adding 25 µM Cu during 24 h incubation, while the fresh Aβ was treated with 5b,
1
-42
under the same experiments, small bulk aggregates were observed. It implied that
2+
compound 5b could inhibit Cu -induced Aβ
aggregation. As for disaggregation
-42
1
assay, Figure 10D displayed that the well-defined Aβ1-42 fibrils obviously decreased
after adding 5b for another 24 incubation, suggesting that 5b could decompose the
2
+
structure of Cu -mediated Aβ aggregation fibrils. Therefore, the TEM phenomenon

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2
+
further supported the conclusion that 5b could inhibit and disaggregate Cu -induced
Aβ1-42 aggregation fibrils.
<
Insert Figure 10>
2
.2.11 Neuroprotective effects on H O -induced PC12 cell injury. Compounds 5b and
2
2
5
h were selected to assess the neuroprotective effects against H O –induced PC12
2 2
cell injury using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) assays
29]. As shown in Figure 11, treatment with 100 µM H O , the cell viability sharply
[
2
2
declined to 49.6 % (p < 0.01) compared with the untreated group. It meant that the
model worked well. Under the same experiments condition, the cell viabilities
increased to 78.3% and 63.2% after adding compounds 5b and 5h, respectively at
1
0.0 µM. While, when the concentration of 5b and 5h reduced to 1.0 µM, the cell
viabilities were 62.9% and 53.7%, respectively. Therefore, the phenomenon displayed
that compound 5b showed potent neuroprotective effect by capturing the hydroxyl
radical, generated by H O .
2
2
<
Insert Figure 11>
2
.2.12 In vitro blood−brain barrier permeation assay. The parallel artificial
membrane permeation assay of the blood−brain barrier (PAMPA-BBB) was used to
assess the BBB permeability of 5b and 5h [35, 40]. We selected 11 commercial drugs
to validate the assay (Table S1), and a good linear correlation was produced through
the experimental data versus the reported values, P (exp) = 0.9163P (bibl.) − 0.2247
e
e
2
(
R = 0.9558) (Figure S1). In view of the limit established by Di et al. we pointed out
−
6
that compounds with permeability above 3.44 × 10 cm/s could cross the BBB
Table S2). According to the measured permeability (Table 3), both 5b and 5h could
cross the BBB in vitro.
(
<
Insert Table 3>
2
.2.13 Theoretical evaluation of ADME properties. Molinspiration property program

ACCEPTED MANUSCRIPT
was used to evaluate the druglike properties of 5b and 5d, the items including log P,
topological polar surface area (TPSA), the number of hydrogen-bond acceptors, and
the number of hydrogen-bond donors [41]. The data were listed in Table 4, both 5b
and 5d complied with the Lipinski’s rule of five, serving as a potent drug candidate.
<
Insert Table 4>
From the above results, compound 5b was chosen to evaluate the
effect in vivo. Primarily, Kunming mice were treated with 5b at doses of 20, 100, and
00 mg/kg (n = 10 per group), mice were observed continuously for the first 4 h
2
.2.14 In vivo assay.
5
through 14 days and did not observe any abnormal behavior and mortality changes.
Secondly, the Y-maze (three arms) test was performed to further assess whether 5b
could improve scopolamine-induced memory impairment [29, 42]. As shown in
Figure 12A, the total arm entries indicated that there was no obvious difference
between the untreated group, model group (scopolamine alone), 5b (2.6, 7.8 and 23.4
mg/kg) group and Riva. (rivastigmine, 6 mg/kg) group, suggesting that compound 5b
and rivastigmine presented no effect on spontaneous alternation behavior. From the
results in Figure 12B, treatment with scopolamine (3 mg/kg, i.p.) alone, the
alternation obviously decreased to 45.6% (p<0.01) compared with the untreated group
(72.1%). However, the spontaneous alternation behavior remarkably increased after
treating with rivastigmine (6 mg/kg) (p< 0.01) or 5b (2.6, 7.8 and 23.4 mg/kg; p >
0
6
.05, p < 0.01, and p < 0.05, respectively). In addition, the medium dose (7.8 mg/kg,
5.1%) showed the highest alternation among the three dose groups and displayed
similar potency compared with rivastigmine (6 mg/kg) (65.3%). The high dose group
(
23.4 mg/kg, 59.5%) of 5b showed lower alternation than that of medium dose group
7.8 mg/kg, 65.1%), the reason might be that the high dose group (23.4 mg/kg) of 5b
(
showed some neurotoxicity and the medium dose would be the optimal dose.
Therefore, the results indicated that compound 5b could improve short-term working
memory in mice.
<
Insert Figure 12>
3
. Conclusion

ACCEPTED MANUSCRIPT
In summary, a series of chalcone-O-carbamate derivatives was designed using the
multi-target-directed ligands strategy to discover multifunctional agents for the
treatment of Alzheimer’s disease (AD). All the target compounds were synthesized
and evaluated by multiple biological activities including AChE/BChE inhibition,
MAO-A/MAO-B inhibition, antioxidant activities, Aβ1-42 aggregation inhibition,
metal-chelating properties and neuroprotective effects against H O -induced PC12
2
2
cell injury. The results displayed that compounds 5b and 5h were considered as
optimal multifunction agents, 5b and 5h were highly selective BChE inhibitors with
IC values of 3.1 µM and 1.2 µM, respectively, and showed highly selective MAO-B
5
0
inhibitory potency with IC values of 1.3 µM and 3.7 µM, respectively. In addition,
50
compounds 5b and 5h could inhibit self-induced Aβ1-42 aggregation with 63.9% and
3.1% inhibition, respectively. Furthermore, compounds 5b and 5h could cross the
5
blood-brain barrier (BBB) in vitro and conformed to the Lipinski’s rule of five. In
particular, compound 5b showed potent antioxidant activity (ORAC = 1.3 eq) and
neuroprotective effect. Compound 5b served as selective metal chelator by chelating
2
+
3+
2+
Cu and Al , and could inhibit and disaggregate Cu -induced Aβ
aggregation,
-42
1
which was further supported by the TEM images. Further, compound 5b could
improve scopolamine-induced cognitive impairment in vivo assay. Therefore, the
results indicated that compound 5b could be a promising multi-functional agent for
the treatment of AD, and the further study and the structure optimization are in
progress.
4
. Experiment section
4
.1. Chemistry. All materials were obtained from commercial suppliers. The reaction
was monitored by thin-layer chromatography (TLC) on silica gel GF254 plates. The
crude products were purified by column chromatography using silica gel (230-400
mesh), and 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 C column (4.6 mm ×
1
8
1
13
2
50 mm, 5um). The H NMR and C NMR spectra of target compounds were recorded

ACCEPTED MANUSCRIPT
using a Bruker 400 NMR spectrometer, and mass spectra were recorded on
Agilent-6210 TOF LC-MS Spectrometer.
4
.1.1. The synthesis of intermediate 3. The starting material 2-hydroxy acetophenone
(1.0 mol) was treated with 3-hydroxybenzaldehyde 2 (1.0 mol) in the presence of an
1
amount of a 30% aqueous KOH (4.0 mol) in in EtOH (25 mL) for 3 days, the reaction
was monitored by TLC. After the reaction completed, the mixture was poured into
ice-cold water. Acidified with 10% HCl, overnight, a yellow precipitate formed and
filtrated, the residue was recrystallized with 80% ethanol to get golden yellow solid 3.
(E)-3-(2-Hydroxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). Golden yellow
1
solid, yield: 78.2%. H NMR 12.78 (s, 1H, OH), 7.92 (d, J = 8.0 Hz, 1H, Ar-H), 7.86
(d, J = 15.6 Hz, 1H, CH=CH), 7.63 (d, J = 15.6 Hz, 1H, CH=CH), 7.51 (t, J = 8.0 Hz,
1
H, Ar-H), 7.32 (t, J = 7.6 Hz, 1H, Ar-H), 7.25 (d, J = 9.2 Hz, 1H, Ar-H), 7.14 (s, 1H,
Ar-H), 7.04 (d, J = 8.0 Hz, 1H, Ar-H), 6.95 (t, J = 8.0 Hz, 1H, Ar-H), 6.92 (d, J = 8.4
Hz, 1H, Ar-H), 5.06 (s, 1H, OH).
4
.1.2 General procedures for the synthesis of target compounds 5a-5f. To a mixture
of intermediate 3 (1.0 mmol), anhydrous K CO (1.2 mmol), and 4-DMAP (0.03
2
3
mmol) in 4 mL MeCN, N,N-disubstituted carbamoyl chlorides (4a-f) (1.1 mmol)was
added dropwise at -5°C. The mixture was heated at 65°C for 6–8 h, the solvent was
evaporated under reduced pressure. Water (25 ml) was added to the residue and the
mixture was extracted with CH Cl (2 × 25 mL). The combined organic phases were
2
2
washed saturated aqueous sodium chloride (30 mL), dried over anhydrous Na SO ,
2
4
and filtered. The solvent was evaporated under a vacuum to give the crude product
which was purified by column chromatography on silica gel (petroleum/acetone = 30:
1
as eluent) to get the desired products 5a-5f.
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl dimethylcarbamate (5a).
1
Pale yellow oil, yield: 56.2%, 97.6% HPLC purity. H NMR (400 MHz, CDCl ) δ
3
1
7
1
8
2.78 (s, 1H, OH), 7.91 (d, J = 8.4 Hz, 1H, Ar-H), 7.87 (d, J = 16.0 Hz, 1H, CH=CH),
.62 (d, J = 16.0 Hz, 1H, CH=CH), 7.52-7.46 (m, 3H, 3 × Ar-H), 7.42 (t, J = 7.6 Hz,
H, Ar-H), 7.19 (t, J = 7.2 Hz, 1H, Ar-H), 7.03 (d, J = 8.0 Hz, 1H, Ar-H), 6.94 (t, J =
.0 Hz, 1H, Ar-H), 3.09 (s, 3H, NCH ), 3.00 (s, 3H, NCH ). HR-ESI-MS: Calcd. For
3
3

ACCEPTED MANUSCRIPT
+
C H NO [M + H] : 312.1191, found: 312.1233.
18
17
4
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl ethyl(methyl)carbamate (5b).
1
Pale yellow oil, yield: 60.7%, 98.8% HPLC purity. H NMR (400 MHz, CDCl ) δ
3
1
7
1
8
2.79 (s, 1H, OH), 7.91 (d, J = 8.4 Hz, 1H, Ar-H), 7.88 (d, J = 15.6 Hz, 1H, CH=CH),
.63 (d, J = 15.6 Hz, 1H, CH=CH), 7.51-7.45 (m, 3H, 3 × Ar-H), 7.41 (t, J = 8.0 Hz,
H, Ar-H), 7.20 (t, J = 5.6 Hz, 1H, Ar-H), 7.02 (d, J = 8.4 Hz, 1H, Ar-H), 6.94 (t, J =
.0 Hz, 1H, Ar-H), 3.50 (q, J = 7.2 Hz, 1H, 1/2 NCH ), 3.43 (q, J = 7.2 Hz, 1H, 1/2
2
NCH ), 3.10 (s, 3/2H, 1/2 NCH ), 3.02 (s, 3/2H, 1/2 NCH ), 1.27 (t, J = 6.8 Hz, 3/2 H,
2
3
3
1
3
1
1
4
/2 CH ), 1.21 (t, J = 7.2 Hz, 3/2 H, 1/2 CH ). C NMR 193.6, 163.6, 152.0, 144.6,
3
3
36.5, 136.0, 129.8, 129.7, 126.0, 124.4, 124.3, 121.3, 120.9, 120.0, 118.9, 118.6,
4.2, 34.3 (1/2 C), 33.9 (1/2 C), 13.3 (1/2 C), 12.5 (1/2 C). HR-ESI-MS: Calcd. For
+
C H NO [M + H] : 326.1348, found: 326.1379.
19
19
4
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl methoxy(methyl)carbamate
1
(
5c). Pale yellow oil, yield: 36.5%, 97.4% HPLC purity. H NMR (400 MHz, CDCl )
3
δ 12.78 (s, 1H, OH), 7.91 (d, J = 8.0 Hz, 1H, Ar-H), 7.86 (d, J = 15.6 Hz, 1H,
CH=CH), 7.62 (d, J = 15.6 Hz, 1H, CH=CH), 7.52-7.48 (m, 3H, 3 × Ar-H), 7.44 (t, J
=
8.0 Hz, 1H, Ar-H), 7.19 (d, J = 7.6 Hz, 1H, Ar-H), 7.02 (d, J = 7.6 Hz, 1H, Ar-H),
6
.94 (t, J = 8.0 Hz, 1H, Ar-H), 3.84 (s, 3H, OCH ), 3.33 (s, 3H, NCH ). HR-ESI-MS:
3
3
+
Calcd. For C H NO [M + H] : 328.1140, found: 328.1182.
1
8
17
5
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl diethylcarbamate (5d). Pale
1
yellow oil, yield: 50.9%, 98.6% HPLC purity. H NMR (400 MHz, CDCl ) δ 12.79 (s,
3
1
H, OH), 7.93 (dd, J = 6.8 Hz, J = 1.2 Hz, 1H, Ar-H), 7.89 (d, J = 15.6 Hz, 1H,
1 2
CH=CH), 7.65 (d, J = 15.6 Hz, 1H, CH=CH), 7.53-7.46 (m, 3H, 3 × Ar-H), 7.43 (t, J
7.6 Hz, 1H, Ar-H), 7.21 (d, J = 8.0 Hz, 1H, Ar-H), 7.03 (d, J = 8.4 Hz, 1H, Ar-H),
.95 (t, J = 8.0 Hz, 1H, Ar-H), 3.50-3.38 (m, 4H, 2 × NCH ), 3.19-3.13 (m, 6H, 2 ×
=
6
2
13
CH ). C NMR 193.6, 163.6, 153.9, 152.1, 144.6, 136.5, 136.0, 129.8, 125.9, 124.3,
3
1
21.3, 120.9, 120.0, 118.9, 118.6, 42.4, 42.0, 14.3, 13.4. HR-ESI-MS: Calcd. For
+
C H NO [M + H] : 340.1504, found: 340.1549.
20
21
4
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl diphenylcarbamate (5e).
1
Pale yellow oil, yield: 42.3%, 97.3% HPLC purity. H NMR (400 MHz, CDCl ) δ
3

ACCEPTED MANUSCRIPT
1
7
1
7
1
1
4
2.75 (s, 1H, OH), 7.91 (d, J = 8.0 Hz, 1H, Ar-H), 7.86 (d, J = 15.6 Hz, 1H, CH=CH),
.62 (d, J = 15.2 Hz, 1H, CH=CH), 7.52-7.48 (m, 2H, 2 × Ar-H), 7.45-7.34 (m, 10H,
0 × Ar-H), 7.29-7.20 (m, 3H, 3 × Ar-H), 7.02 (d, J = 8.4 Hz, 1H, Ar-H), 6.94 (t, J =
1
3
.6 Hz, 1H, Ar-H). C NMR 193.6, 163.6, 155.7, 151.6, 149.0, 148.6, 144.4, 137.0,
36.5, 133.8, 131.5, 129.9, 129.8, 128.7, 126.9, 126.8, 126.3, 123.9, 121.1, 120.9,
+
20.8, 120.0, 118.9, 118.7, 110.8. HR-ESI-MS: Calcd. For C H NO [M + H] :
2
8
21
4
36.1504, found: 436.1549
(E)-3-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl morpholine-4-carboxylate
1
(
5f). Pale yellow oil, yield: 61.2%, 98.7% HPLC purity. H NMR (400 MHz, CDCl )
3
δ 12.72 (s, 1H, OH), 7.83 (d, J = 8.0 Hz, 1H, Ar-H), 7.80 (d, J = 16.0 Hz, 1H,
CH=CH), 7.56 (d, J = 15.6 Hz, 1H, CH=CH), 7.42 (t, J = 8.4 Hz, 2H, 2 × Ar-H), 7.35
(t, J = 7.6 Hz, 2H, 2 × Ar-H), 7.12 (d, J = 8.0 Hz, 1H, Ar-H), 6.95 (d, J = 8.4 Hz, 1H,
Ar-H), 6.86 (t, J = 7.6 Hz, 1H, Ar-H), 3.69-3.67 (m, 4H, 2 × NCH ), 3.63-3.62 (m, 2H,
2
1
3
OCH ), 3.52-3.51 (m, 2H, OCH ). C NMR 193.5, 163.6, 153.4, 151.7, 144.4, 136.6,
2
2
1
4
4
36.0, 130.0, 129.8, 126.3, 124.3, 121.2, 120.9, 119.9, 119.0, 118.7, 66.7, 66.5, 44.9,
+
4.2. HR-ESI-MS: Calcd. For C H NO [M + H] : 354.1297, found: 354.1346
2
0
19
5
.1.3 General procedures for the synthesis of target compounds 5g-5l. The detailed
procedure referenced the To a mixture of intermediate 3 (1.0 mmol), anhydrous
K CO (3.0 mmol), and 4-DMAP (0.03 mmol) in 5 mL MeCN, The appropriate
2
3
N,N-disubstituted carbamoyl chlorides (4a-f) (2.5 mmol) was added dropwise at -5°C.
The mixture was heated at 65°C for 10-15 h, the solvent was treted according to
synthesis of 5a-5l. The target compounds 5g-5l were obtained using column
chromatography on silica gel (petroleum/acetone = 50: 1 as eluent)
(
E)-3-(3-(2-((Dimethylcarbamoyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
dimethylcarbamate (5g). Pale yellow oil, yield: 30.2%, 98.7% HPLC purity. H NMR
400 MHz, CDCl ) δ 7.65 (dd, J = 6.4 Hz, J = 1.2 Hz, 1H, Ar-H), 7.54 (d, J = 16.0
1
(
3
1
2
Hz, 1H, CH=CH), 7.53-7.50 (m, 1H, Ar-H), 7.38 (d, J = 4.8 Hz, 2H, 2 × Ar-H),
7
7
3
.33-7.30 (m, 2H, 2 × Ar-H), 7.22 (d, J = 8.0 Hz, 1H, Ar-H), 7.17-7.14 (m, 1H, Ar-H),
.12 (d, J = 16.0Hz, 1H, CH=CH), 3.10 (s, 3H, NCH ), 3.01 (s, 3H, NCH ), 2.97 (s,
3
3
13
H, NCH ), 2.89 (s, 3H, NCH ). C NMR 192.2, 154.6, 154.2, 151.9, 149.4, 143.9,
3
3

ACCEPTED MANUSCRIPT
1
3
3
35.9, 132.6, 132.4, 129.8, 129.6, 126.5, 125.5, 125.4, 124.1, 123.8, 121.2, 36.7, 36.6,
+
6.5, 36.4. HR-ESI-MS: Calcd. For C H N O [M + H] : 383.1562, found:
2
1
22
2
5
83.1605
(E)-3-(3-(2-((Ethyl(methyl)carbamoyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
1
ethyl(methyl)carbamate (5h). Pale yellow oil, yield: 35.1%, 97.7% HPLC purity. H
NMR (400 MHz, CDCl ) δ 7.63 (t, J = 8.0 Hz, 1H, Ar-H), 7.54 (d, J = 14.8 Hz, 1H,
3
CH=CH), 7.52-7.50 (m, 1H, Ar-H), 7.38 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.31 (t, J =
6
.4 Hz, 2H, 2 × Ar-H), 7.23 (t, J = 7.6 Hz, 1H, Ar-H), 7.17-7.15 (m, 1H, Ar-H), 7.11
(
d, J = 16.0Hz, 1H, CH=CH), 3.48 (q, J = 7.2 Hz, 1H, 1/2 NCH ), 3.41 (q, J = 7.2 Hz,
2
1
H, 1/2 NCH ), 3.36 (q, J = 7.2 Hz, 1H, 1/2 NCH ), 3.30 (q, J = 7.2 Hz, 1H, 1/2
2 2
NCH ), 3.08 (s, 3/2H, 1/2 NCH ), 3.00 (s, 3/2H, 1/2 NCH ), 2.96 (s, 3/2H, 1/2 NCH ),
2
3
3
3
2
.89 (s, 3/2H, 1/2 NCH ), 1.25 (t, J = 6.8 Hz, 3/2 H, 1/2 CH ), 1.20 (t, J = 6.8 Hz, 3/2
3 3
H, 1/2 CH ), 1.12 (t, J = 6.8 Hz, 3/2 H, 1/2 CH ), 1.05 (t, J = 6.8 Hz, 3/2 H, 1/2 CH ).
3
3
3
+
HR-ESI-MS: Calcd. For C H N O [M + H] : 411.1875, found: 411.1909.
23
26
2
5
(E)-3-(3-(2-((Methoxy(methyl)carbamoyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
methoxy(methyl)carbamate (5i). Pale yellow oil, yield: 40.9%, 97.3% HPLC purity.
1
H NMR (400 MHz, CDCl ) δ 7.69 (d, J = 6.4 Hz, 1H, Ar-H), 7.57 (d, J = 15.6 Hz,
3
1
H, CH=CH), 7.55 (dd, J = 13.6 Hz, J = 2.0 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H),
1 2
7
.40-7.36 (m, 2H, 2 × Ar-H), 7.34 (d, J = 6.8 Hz, 1H, Ar-H), 7.25 (d, J = 6.8 Hz, 1H,
Ar-H), 7.22-7.20 (m, 1H, Ar-H), 7.18 (d, J = 16.0 Hz, 1H, CH=CH), 3.81 (s, 3H,
1
3
OCH ), 3.71 (s, 3H, OCH ), 3.30 (s, 3H, NCH ), 3.21 (s, 3H, NCH ). C NMR 193.2,
3
3
3
3
1
1
4
64.6, 161.8, 149.0, 144.1, 132.5, 129.9, 129.8, 126.5, 126.0, 125.9, 123.8, 123.6,
+
21.1, 35.6, 35.5, 29.7, 29.6. HR-ESI-MS: Calcd. For C H N O [M + H] :
2
1
22
2
7
15.1461, found: 415.1510.
(
E)-3-(3-(2-((Diethylcarbamoyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
diethylcarbamate (5j). Pale yellow oil, yield: 40.9%, 97.3% HPLC purity. H NMR
400 MHz, CDCl ) δ 7.61 (d, J = 7.2 Hz, 1H, Ar-H), 7.54 (d, J = 16.0 Hz, 1H,
1
(
3
CH=CH), 7.50-7.45 (m, 1H, Ar-H), 7.37-7.36 (m, 2H, 2 × Ar-H), 7.31-7.26 (m, 2H, 2
Ar-H), 7.22 (d, J = 8.0 Hz, 1H, Ar-H), 7.16 (d, J = 3.6 Hz, 1H, Ar-H), 7.10 (d, J =
6.0Hz, 1H, CH=CH), 3.46-3.31 (m, 4H, 2 × NCH ), 1.25-1.19 (m, 6H, 2 × CH ).
×
1
2
3

ACCEPTED MANUSCRIPT
+
HR-ESI-MS: Calcd. For C H N O [M + H] : 439.2188, found: 439.2246.
25
30
2
5
(
E)-3-(3-(2-((Diphenylcarbamoyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
diphenylcarbamate (5k). Pale yellow oil, yield: 34.7%, 97.4% HPLC purity. H NMR
400 MHz, CDCl ) δ 7.59 (d, J = 7.6 Hz, 1H, Ar-H), 7.50 (d, J = 16.0 Hz, 1H,
1
(
3
CH=CH), 7.46 (t, J = 7.6 Hz, 1H, Ar-H), 7.35-7.29 (m, 12H, 12 × Ar-H), 7.26-7.20
m, 12H, 12 × Ar-H), 7.11 (t, J = 8.0 Hz, 2H, 2 × Ar-H), 7.03 (d, J = 16.0 Hz, 1H,
(
1
3
CH=CH). C NMR 192.8, 191.9 (2C), 152.8, 152.6, 151.6, 149.0, 144.1, 142.3,
1
1
42.2, 142.1, 136.1, 132.7, 132.3, 129.8, 129.7, 129.2 (6C), 129.1 (6C), 126.9, 126.8,
26.7 (2C), 126.6 (2C), 126.5, 125.9, 125.8, 123.8 (2C), 123.3, 121.2 (2C).
+
HR-ESI-MS: Calcd. For C H N O [M + H] : 631.2188, found: 631.2227.
41
30
2
6
(E)-3-(3-(2-((Morpholine-4-carbonyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl
1
morpholine-4-carboxylate (5l). Pale yellow oil, yield: 50.6%, 98.3% HPLC purity. H
NMR (400 MHz, CDCl ) δ 7.57 (d, J = 7.6 Hz, 1H, Ar-H), 7.47 (d, J = 15.2 Hz, 1H,
3
Ar-H), 7.44 (d, J = 16.8 Hz, 1H, CH=CH), 7.34-7.32 (m, 2H, 2 × Ar-H), 7.26 (t, J =
6
.8 Hz, 2H, 2 × Ar-H), 7.15 (d, J = 8.4 Hz, 1H, Ar-H), 7.10-7.07 (m, 1H, Ar-H), 7.04
(
d, J = 16.4 Hz, 1H, CH=CH), 3.69-3.65 (m, 4H, 2 × NCH ), 3.61-3.58 (m, 2H,
2
13
NCH ), 3.51-3.48 (m, 8H, 4 × OCH ), 3.38-3.36 (m, 2H, NCH ). C NMR 192.2,
2
2
2
1
1
53.3, 153.0, 151.7, 149.1, 144.3, 135.9, 132.4, 132.3, 130.0, 126.5, 125.8, 125.6,
24.0, 123.7, 121.4, 66.6, 66.5 (2C), 66.4, 44.9 (2C), 44.1 (2C). HR-ESI-MS: Calcd.
+
For C H N O [M + H] : 467.1774, found: 467.1821.
2
5
26
2
7
4
4
.2 Biological evaluation
.2.1 AChE and BuChE inhibition assay. The modified Ellman method was performed
to test the AChE and BChE inhibitory ability of the compounds, using eeAChE and
eqBChE (Sigma-Aldrich Co.). The detailed procedure referenced our previous work
[
28.31].
.2.2. Antioxidant activity assay
4
The antioxidant activity was tested by the oxygen radical absorbance capacity f
luorescein (ORAC-FL) assay. The details could reference our previous work [2
8
, 29].
4
.2.3. Recombinant human MAO-A and MAO-B inhibition studies. Recombinant

ACCEPTED MANUSCRIPT
human MAO-A and MAO-B were purchased from Sigma-Aldrich and stored at
-80 °C [36]. 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
(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
terminated the enzymatic reactions and the mistures were centrifuged at 16000g for
5
1
0 min. 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
5
00 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. IC values were determined in triplicate and expressed as
5
0
mean ± SD.
.2.4 Molecular docking. The crystal structure of BChE (PDB code: 4tpk), ACh
4
E (PDB code: 1EVE) and MAO-B (PDB code: 2V60) were obtained from the
Protein Data Bank after eliminating the original inhibitors and water molecules.
[
30] Docking studies were performed using the AUTODOCK 4.2 program. Gr
aphic manipulations and visualizations were done by Autodock Tools or Discov
ery Studio 2.5 software.

ACCEPTED MANUSCRIPT
4
.2.5. Metal binding studies. The metal binding studies were carried out in a
Varioskan Flash Multimode Reader (Thermo Scientific) [28, 29]. The UV absorption
of the tested compounds 5b and 5h, in the absence or presence of CuCl , ZnCl , AlCl
3
2
2
and FeSO was recorded with wavelength ranging from 200 to 600 nm after
4
incubating for 30 min in methanol at room temperature. The final volume of reaction
mixture was 200 µL, and the final concentrations of tested compound and metals were
2
+
3
7.5 µM. The stoichiometry of the compound-Cu complex was determined by
titrating the methanol solution of tested compound with ascending of CuCl . The final
2
2+
concentration of tested compound was 37.5 µM, and the final concentration of Cu
ranged from 3.75 to 93.75 µM. The UV spectra were recorded and treated by
numerical subtraction of CuCl and tested compound at corresponding concentrations,
2
plotted versus the mole fraction of tested compound.
4
.2.6. Effect of test compounds on metal-induced Aβ1-42 aggregation experiments and
2
+
disaggregation by ThT method. For the inhibition of Cu -induced Aβ aggregation
1
-42
experiment [30, 38]. HEPES buffer solutions (20 mM, pH 6.6) containing 150 µM
2
+
NaCl were prepared with distilled water. Solutions of Cu were prepared from
standards to concentration of 75 µM using the HEPES buffer at pH = 6.6. The Aβ1-42
stock solution was diluted in HEPES buffer. The mixture of the peptide (20 µL, 25
2
+
µM, final concentration) and Cu (20 µL, 25 µM, final concentration), was incubated
at 37 °C for 24h with or without the tested compound at different concentrations (20
µL, 25 µM, final concentration). After incubated, 190 µL of 5 µM Thioflavin T in 50
mM glycine-NaOH buffer (pH 8.5) was added. Each assay was run in triplicate. The
detection method was the same as that of self-induced Aβ1-42 experiment.
For the disaggregation of copper-induced Aβ fibrils experiment, the Aβ1-42 stock
solution was diluted in HEPES buffer (20 mM, pH 6.6, 150 mM NaCl). The mixture
2
+
of the Aβ1-42 (20 µL, 25 µM, final concentration) with Cu (20 µL, 25 µM, final
concentration) was incubated 37 °C for 24 h. The tested compound (20 µL, 25 µM,
final concentration) was then added and incubated at 37 °C for another 24 h. To
minimize evaporation effect the wells were sealed by a transparent heat-resistant
plastic film. After incubation, 190 µL of 5 µM thioflavin T in 50 mM glycine-NaOH

ACCEPTED MANUSCRIPT
buffer (pH 8.5) was added. Each assay was run in triplicate. The detection method
was the same as above.
4
.2.7. Transmission Electron Microscopy (TEM) Assay. The preparation of samples
2
+
could reference the self-induced and Cu -induced Aβ
aggregation experiments.
-42
1
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 ddH O. Each grid was incubated with uranyl acetate
2
(
1% w/v ddH O). Upon removal of excess uranyl acetate, the grids were dried for 15
2
min at room temperature. Images from each sample were taken on a Field Emission
Transmission Electron Microscope (JEM-2100F).
4
.2.8. In vitro blood−brain barrier permeation assay. The blood-brain barrier
penetration of compounds was evaluated using the parallel artificial membrane
permeation assay (PAMPA) described by Di et al [40]. Commercial drugs were
purchased from Sigma and Alfa Aesar. Porcine brain lipid (PBL) was obtained from
Avanti Polar Lipids. The donor microplate (PVDF membrane, pore size 0.45 mm) and
acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR) was
from Corning Incorporated. The acceptor 96-well microplate was filled with 350 µL
of PBS/EtOH (70:30), and the filter membrane was impregnated with 4 µL of PBL in
dodecane (20 mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted
5
0-fold in PBS/EtOH (70:30) to a final concentration of 100 µg/mL. The detailed
procedure refers to our previous work.
.2.9. The Y–maze task. Rivastigmine was purchased from Shanghai Titan Technology
4
Co., Ltd. Eisai China Inc. Scopolamine was purchase from J&K Scientific. 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-Sichuan 2015-030). Mice were maintained under
standard conditions with a 12 h:12 h light–dark cycle, a temperature and humidity
controlled environment with access to food and water ad libitum..
The Y-maze test was performed to assess learning and memory in mice as
previously described [29, 42]. Sixty mice were random divide into six groups. They

ACCEPTED MANUSCRIPT
were i.g. compound 5b (2.6 mg/kg, 7.8 mg/kg and 23.4 mg/kg), rivastigmine (6
mg/kg, as a positive control), same volume of water (untreated group or or model
group) once respectively. After 30 min, memory impairment was induced by
administering scopolamine (3 mg/kg, i.p.) or the same volume of normal saline
(untreated group). Then 30 min later, the learning and memory capacitys of mouse
were measured by the Y-maze test. The maze was made of black-colored acryl and
positioned at equal angles. Rats were placed at the end of the arm and allowed to
move freely through the maze during 8 min sessions. Arm entry sessions were
recorded when the hind paws of the rat were completely placed in the arm.
Consecutive entry into three arms in alternative order was defined as successive
entries on overlapping triplet sets and the alternation percentage was calculated as the
ratio of actual to possible alternations (defined as the total number of arm entries
minus 2), multiplied by 100.
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

ACCEPTED MANUSCRIPT
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).
Supporting Information Available:
1
13
The Tables and Figures in PAMPA-BBB experiments. The representative H,
C
NMR and HR-MS spectra for the synthesized compounds are available as
supplementary material.
References
1
. Barnett R. Alzheimer’s disease. Lancet. 2019, 393 (10181): 1589.
2
. Marešová P, Mohelská H, Dolejš J, Kuča K. Socio-economic aspects of
Alzheimer’s disease. Curr Alzheimer Res. 2015, 12(9): 903-911.
3
4
5
. Lane CA, Hardy J, Schott JM. Alzheimer’s disease. Eur J Neurol. 2018, 25(1):
5
9-70.
. Graham WV, Bonito-Oliva A, Sakmar TP. Update on Alzheimer’s disease therapy
and prevention strategies. Annu Rev Med. 2017, 68: 413-430.
. de Freitas Silva, M.; Dias, K.S.T.; Gontijo, V.S.; Ortiz, C.J.C.; Viega, C.
Multi-target directed drugs as a modern approach for drug design
towards Alzheimer's disease: an update. Curr Med Chem. 2018, 25(29):
3
491-3525.
6
. Umar, T.; Hoda, N. Alzheimer's disease: A systemic review of substantial
therapeutic targets and the leading multi-functional molecules. Curr Top Med
Chem. 2017, 17(31): 3370-3389.
7
. Ibrahim MM, Gabr MT. Multitarget therapeutic strategies for Alzheimer's disease.
Neural Regen Res. 2019, 14(3): 437-440.
8
. Li Q, He S, Chen Y, Feng F, Qu W, Sun H. Donepezil-based multi-functional
cholinesterase inhibitors for treatment of Alzheimer's disease. Eur J Med Chem,
2
018, 158:463-477.
9
. Sameem B, Saeedi M, Mahdavi M, Shafiee A. A review on tacrine-based
scaffolds as multi-target drugs (MTDLs) for Alzheimer's disease. Eur J Med

ACCEPTED MANUSCRIPT
Chem. 2017, 128:332-345.
1
1
1
0. McHardy SF, Wang HL, McCowen SV, Valdez MC. Recent advances in
acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature
(
2012-2015). Expert Opin Ther Pat. 2017, 27(4):455-476.
1. Namdaung U, Athipornchai A, Khammee T, Kuno M, Suksamrarn S.
-Arylbenzofurans from Artocarpus lakoocha and methyl ether analogs with
2
potent cholinesterase inhibitory activity. Eur J Med Chem. 2018, 143: 1301-1311.
2. Furukawa-Hibi Y, Alkam T, Nitta A, Matsuyama A, Mizoguchi H, Suzuki K,
Moussaoui S, Yu QS, Greig NH, Nagai T, Yamada K. Butyrylcholinesterase
inhibitors ameliorate cognitive dysfunction induced by amyloid-β peptide in mice.
Behav Brain Res. 2011, 225(1): 222-229.
1
3. Greig NH, Utsuki T, Ingram DK, Wang Y, Pepeu G, Scali C, Yu QS, Mamczarz J,
Holloway HW, Giordano T, Chen D, Furukawa K, Sambamurti K, Brossi A,
Lahiri DK. Selective butyrylcholinesterase inhibition elevates brain acetylcholine,
augments learning and lowers Alzheimer beta-amyloid peptide in rodent. Proc
Natl Acad Sci U S A. 2005, 102(47): 17213-17218.
1
1
1
4. Paroni G, Bisceglia P, Seripa D. Understanding the amyloid hypothesis in
Alzheimer's disease. J Alzheimers Dis. 2019, 68(2): 493-510.
5. Ricciarelli R, Fedele E. The amyloid cascade hypothesis in Alzheimer’s disease:
it’s time to change our mind. Curr Neuropharmacol. 2017, 15(6): 926-935.
6. Bonda DJ, Wang X, Perry G, Nunomura A, Tabaton M, Zhu X, Smith MA.
Oxidative stress in Alzheimer disease: a possibility for prevention.
Neuropharmacology. 2010, 59(4-5):290-294.
1
7. Butterfield DA, Halliwell B. Oxidative stress, dysfunctional glucose metabolism
and Alzheimer disease. Nat Rev Neurosci. 2019, 20(3):148-160.
1
8. Cheignon C, Tomas M, Bonnefont-Rousselot D, Faller P, Hureau C, Collin F.
Oxidative stress and the amyloid beta peptide in Alzheimer’s disease. Redox Biol.
2
018, 14: 450-464.
1
9. Spinello A, Bonsignore R, Barone G, Keppler BK, Terenzi A. Metal ions and
metal complexes in Alzheimer’s disease. Curr Pharm Des. 2016, 22(26):

ACCEPTED MANUSCRIPT
3
996-4010.
2
2
2
0. Drew SC. The case for abandoning therapeutic chelation of copper ions in
Alzheimer's disease. Front Neurosci. 2017,11:317.
1. Kumar B, Gupta VP, Kumar V. A perspective on monoamine oxidase enzyme as
drug target: challenges and opportunities. Curr Drug Targets. 2017, 18(1): 87-97.
2. Pazini AM, Gomes GM, Villarinho JG, da Cunha C, Pinheiro F, Ferreira AP,
Mello CF, Ferreira J, Rubin MA. Selegiline reverses aβ25-35-induced cognitive
deficit in male mice. Neurochem Res. 2013, 38(11): 2287-2294.
2
3. Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M,
Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ. A
controlled trial of selegiline, alpha-tocopherol, or both as treatment for
Alzheimer's disease. The Alzheimer's Disease Cooperative Study. N Engl J Med.
1
997, 336(17):1216-22.
2
4. Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z. Chalcone: a privileged
structure in medicinal chemistry. Chem Rev 2017, 117(12): 7762-7810.
2
5. Zhang X, Rakesh KP, Bukhari SNA, Balakrishna M, Manukumar HM, Qin HL.
Multi-targetable chalcone analogs to treat deadly Alzheimer's disease: Current
view and upcoming advice. Bioorg Chem. 2018, 80: 86-93.
2
6. Kandiah N, Pai MC, Senanarong V, Looi I, Ampil E, Park KW, Karanam AK,
Christopher S. Rivastigmine: the advantages of dual inhibition of
acetylcholinesterase and butyrylcholinesterase and its role in subcortical vascular
dementia and Parkinson's disease dementia. Clin Interv Aging. 2017, 12: 697-707.
7. Chaudhaery SS, Roy KK, Shakya N, Saxena G, Sammi SR, Nazir A, Nath C,
Saxena AK. Novel carbamates as orally active acetylcholinesterase inhibitors
2
found
to
improve
scopolamine-induced
cognition
impairment:
pharmacophore-based virtual screening, synthesis, and pharmacology. J Med
Chem. 2010, 51(17): 6490-6505.
2
8. Sang Z, Qiang X, Li Y, Xu R, Cao Z, Song Q, Wang T, Zhang X, Liu H, Tan Z,
Deng
Y.
Design,
synthesis
and
evaluation
of
scutellarein-O-acetamidoalkylbenzylamines as potential multifunctional agents for

ACCEPTED MANUSCRIPT
the treatment of Alzheimer's disease. Eur J Med Chem. 2017, 135: 307-323.
9. Sang Z, Li Y, Qiang X, Xiao G, Liu Q, Tan Z, Deng Y. Multifunctional
scutellarin-rivastigmine hybrids with cholinergic, antioxidant, biometal chelating
and neuroprotective properties for the treatment of Alzheimer's disease. Bioorg
Med Chem. 2015, 23(4): 668-680.
2
3
0. Qiang X, Sang Z, Yuan W, Li Y, Liu Q, Bai P, Shi Y, Ang W, Tan Z, Deng Y.
Design, synthesis and evaluation of genistein-O-alkylbenzylamines as potential
multifunctional agents for the treatment of Alzheimer's disease. Eur J Med Chem.
2
014, 76: 314-331.
3
3
3
3
1. Sang Z, Wang K, Han X, Cao M, Tan Z, Liu W. Design, synthesis, and evaluation
of novel ferulic acid derivatives as multi-target-directed ligands for the treatment
of Alzheimer's disease. ACS Chem Neurosci. 2019, 10(2):1008-1024.
2. Zarmouh, N.O.; Eyunni, S.K.; Soliman, K.F. The Benzopyrone Biochanin-A as a
reversible, competitive, and selective monoamineoxidase B inhibitor. BMC
Complement Altern. Med. 2017, 17(1): 34.
3. Martini, F.; Bruning, C.A.; Soares, S.M.; Nogueira, C.W.; Zeni, G. Inhibitory
effect of ebselen on cerebral acetylcholinesterase activity in vitro: kinetics and
reversibility of inhibition. Curr. Pharm. Des. 2015, 21(7): 920-924.
4. Rosenberry, TL; Brazzolotto, X; Macdonald, IR; Wandhammer, M;
Trovaslet-Leroy, M; Darvesh, S; Nachon, F. Comparison of the binding of
reversible inhibitors to human butyrylcholinesterase and acetylcholinesterase: a
crystallographic, kinetic and calorimetric study. Molecules. 2017, 22(12). Pii:
E2098.
3
5. Sang, Z.; Pan, W.; Wang, K.; Ma, Q.; Yu, L.; Yang, Y.; Bai, P.; Leng, C.; Xu, Q.;
Li, X.; Tan, Z.; Liu, W. Design, synthesis and evaluation of novel ferulic
acid-O-alkylamine derivatives as potential multifunctional agents for the treatment
of Alzheimer's disease. Eur. J. Med. Chem. 2017, 130, 379-392.
3
6. Li Y, Qiang X, Luo L, Yang X, Xiao G, Zheng Y, Cao Z, Sang Z, Su F, Deng Y.
Multitarget drug design strategy against Alzheimer's disease: Homoisoflavonoid
Mannich base derivatives serve as acetylcholinesterase and monoamine oxidase B