Synthesis and pharmacological evaluation of multi-functional homoisoflavonoid derivatives as potent inhibitors of monoamine oxidase B and cholinesterase for the treatment of Alzheimer's disease

Article abstract of DOI:10.1039/c7md00199a

A series of homoisoflavonoid derivatives was designed, synthesized and evaluated as potential multi-functional anti-Alzheimer's agents for their inhibitory activity on cholinesterase and monoamine oxidase. Among them, compound 16 showed moderate acetylcho

Full text of DOI:10.1039/c7md00199a

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Synthesis
Homoisoflavonoid Derivatives as Potent Inhibitors of Monoamine Oxidase B
and Cholinesterase for the Treatment of Alzheimer’s Disease
and
Pharmacological
Evaluation
of
Multi-Functional
†
QiaoꢀHong Liu, JiaꢀJia Wu, Fan Li, Pei Cai, XueꢀLian Yang, LingꢀYi Kong*,
XiaoꢀBing Wang*.
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China.
† The authors declare no competing interests.
* Corresponding Authors. Tel/Fax: +86ꢀ25ꢀ83271405; Eꢀmail:cpu_lykong@126.com;
xbwang@cpu.edu.cn

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Abstract
A series of homoisoflavonoid derivatives were designed, synthesized and evaluated as
potential multiꢀfunctional antiꢀAlzheimer’s agents for their inhibitory activity on
cholinesterase and monoamine oxidase. Among them, compound 16 showed moderate
acetylcholinesterase (AChE) inhibitory activity (eeAChE IC50 = 0.89 ± 0.02 ꢁM;
hAChE IC50 = 0.657 ± 0.002 ꢁM) and significant monoamine oxidase B (MAOꢀB) (h
MAOꢀB IC50 = 0.0372 ± 0.0002 ꢁM) inhibitory activity. Both kinetic analysis of
AChE, MAOꢀB inhibition and molecular modeling study revealed that compound 16
is a dual binding site inhibitor of AChE and noncompetitive inhibitor of MAOꢀB.
Furthermore, 16 could penetrate through the bloodꢀbrain barrier (BBB) in vitro. Most
importantly, oral administration of 16 demonstrated no marked signs of acute toxicity
and it could significantly reverse scopolamineꢀinduced memory impairment in mice.
These results suggested that compound 16 is a promising multifunctional drug
candidate with potential effect for the treatment of Alzheimer’s disease.
Keywords: Alzheimer’s disease, homoisoflavonoid derivatives, acetylcholinesterase,
monoamine oxidase, multifunctional agents.
Introduction
With the development of society, the mankind begins to focus on healthy of oneself,
dementia is one of the most serious health issues.1 Alzheimer’s disease (AD), one
form of dementia, which is marked mainly by progressive memory loss, cognitive
decline and behavior disorder.2,3 Senile plaques and neurofibrillary tangles are the two
leading pathologic hallmarks of AD.4
Due to complexity of the causative factors, several hypotheses were put forward to
illuminate AD, including cholinergic system dysfunction, βꢀamyloid peptide (Aβ)
deposits, tau protein aggregation, calcium mishandling and so on.5 From 1984 to 2014,
considerable investments have been devoted to clinical trials and drug development
against AD, only several drugs were approved for AD include cholinesterase
inhibitors (donepezil, galantamine, rivastigmine and tacrine) and an Nꢀmethylꢀ

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Dꢀaspartate (NMDA) receptor antagonist (memantine).6 Apart from memory and
cognitive improvement, these marketed drugs have shown only a little efficacy to
prevent the progression of the disease.7 Thus, the pattern of ‘oneꢀmoleculeꢀoneꢀtarget’
might not be applicable to the complex nature of AD. Accordingly, the
‘multiꢀtargetꢀdirected ligand’ (MTDL) strategy has been developed in recent years.8,9
The combination of pharmacophore molecules with different biological activity is one
of the most common design strategies. Many researches have described the
underlying therapeutic application of multimodal cholinesterase inhibitors with
additional activities, such as amyloid plaques inhibition,10,11 antioxidant,12,13 and
monoamine oxidase (MAO) inhibition.14,15
Cholinesterase (ChE), one of glycoprotein, is classified into two types: AChE and
butyrylcholinesterase (BuChE).16 AChE (EC 3.1.1.7) is responsible for the hydrolysis
of acetycholine (ACh) in central and peripheral nervous systems,5 and the inactivation
of AChE induced by diverse inhibitors can result in the increasing of ACh.17 AChE
can hydrolyze the natural neurotransmitter ACh faster than the synthetic substrate,
butyrylcholine (BuCh), conversely, BuChE demonstrates similar activity towards the
two substrates.18 The cholinergic hypothesis suggests that cognitive impairment is
mainly caused by the deficits of ACh.19 Similarly, alterations in the dopaminergic and
monoaminergic are also thought to be responsible for the abnormal behavior of
AD.15,20 This evidence has led to the suggestion that MAO is also an effective target
for the AD therapy.21,22 Monoamine oxidase (MAO, EC 1.4.3.4) is a flavoenzyme that
catalyzes the oxidative deamination of biogenic and dietary amines. Owing to the
difference in substrate and inhibitor selectivity, MAO exists as two isoforms, MAOꢀA
and MAOꢀB. 5ꢀHydroxytryptamine (5ꢀHT) and βꢀphenylethylamine (PEA) are
preferentially catabolized by MAOꢀA and MAOꢀB, separately.23,24 Except the
increased amines neurotransmission, the beneficial properties of MAO inhibitors
(IMAOs) are associated with the decrease of the end products like hydrogen peroxide
and aldehydes, which promote the formation of reactive oxygen species (ROS) and
may contribute to neuronal damage.25 MAOꢀA inhibitors are used in depressive
disorder, while the MAOꢀB inhibitors are employed to treat neurodegenerative disease,

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such as AD.26 Furthermore, MAOꢀB activity is obviously increased in the selected
brain region of AD.27 Overall, these observations suggest that the inhibitor of MAOꢀB
seem to be valuable for AD.
Homoisoflavanoids are specific flavonoids found in a variety of roots, bulbs and
leaves of Fabaceae and Asparagaceae families.28 Considerable attentions have been
paid for their diverse biological activities, including antioxidant,28,29 antifungal,30 and
antiꢀinflammatory,31 Recent research indicates that natural homoisoflavonoid could
obviously inhibit the MAOꢀB activity.32 Moreover, increasing evidences suggest that
homoisoflavoniond derivatives, modified based on para hydroxyl, exhibit better
activities against AD.33,34
Based on the homoisoflavanoid potentiality against AD, we designed, synthesized
and evaluated a new series of suitably substituted compounds. Starting from a
multitarget directed ligand, the new molecular framework was projected following a
hybridization strategy (Figure 1).35 A variety of hybrids was designed by connecting
the 3ꢀbenzylidenechromanꢀones to different alkyamine, alkybenzylamine or
alkylpiperazine groups both were characterized by many AChE inhibitors (like
donepezil) through a flexible spacer. The 3ꢀbenzylidenechromanꢀone was chosen as a
molecular fragment because it could inhibit MAOꢀB by fitting the MAOꢀB enzymatic
cleft32 and this scaffold was also presumably to interact at the peripheral anionic site
(PAS) of AChE. Considering the important functions of the nitrogen atom from the
amine side chain which interact with the catalytic site (CAS) of AChE,14,36,37 we
introduced these groups to acquire a dual AChEꢀMAO B inhibitor. In order to
diversify the compounds and analyze the structureꢀactivity relationship (SAR), the
position of the amine chain was designed to vary from the 2'ꢀOH to 3'ꢀOH and 4'ꢀOH
of 3ꢀbenzy ꢀlidenechromanꢀones. The designed compounds were evaluated with their
ChEs and MAOs inhibition activity in vitro. The kinetics of the enzyme inhibition
activity and bloodꢀbrain barrier (BBB) permeation were evaluated. Moreover,
molecular docking was performed to get insight into the mechanism of the selected
compound. Finally, the optimal compound 16 was further assessed the acute toxicity

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in vivo and the memory improvement in the mouse model of scopolamineꢀinduced
impairment.
Results and discussion
Chemistry
The synthesis of target compounds 16ꢀ45 were accomplished along with the
summarized in Scheme 1. The baseꢀcatalyzed condensation of compound 1 with
substituted benzaldehydes 2ꢀ4 was carried out to afford the key intermediate
(E)ꢀ3ꢀbenzylidenechromanꢀ4ꢀones 5ꢀ7.38 Subsequently, treatment of 5ꢀ7 with different
α,ωꢀdibromoalkanes in acetonitrile produced compounds 8ꢀ15.39 These compounds
were then treated with dimethylamine and other secondary amines in the presence of
potassium carbonate to afford the target products 16ꢀ45 in 15ꢀ82% yields.
Cholinesterase inhibitory activity
The AChE inhibitory activities of all target compounds 16ꢀ45 were listed in Table 1.
Donepezil and compound 5 were used as reference compounds. The results showed
that all target compounds showed moderate activities with IC50 values ranging from
submicromolar to micromolar. Compound 16 was the most potential one having
inhibitory activity to eeAChE (IC50 = 0.89 ± 0.02 ꢁM). Most of the synthetic
compounds had better inhibitory activity against eeAChE compared with the
precursor 5, which indicated the amine chain is necessary for the AChE inhibition.
The results also showed that the general trend for eeAChE inhibition was
4'ꢀOꢀmodified > 2'ꢀOꢀmodified > 3'ꢀOꢀmodified compounds. To optimize the
inhibitory of compound, the linker length and the terminal amines groups were
introduced to the leading compound. In contrast, the compound with twoꢀcarbon
spacer between the amine groups and hydroxyl of compound 5 exhibited better
inhibition than those with a threeꢀcarbon, fourꢀcarbon or sixꢀcarbon linker. For
example, the eeAChE inhibitory activity of compound 16 (IC50 = 0.89 ± 0.02 ꢁM) was
fiveꢀfold higher than that of 17 (IC50 = 5.21 ± 0.18 ꢁM) with threeꢀcarbon linker, and

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twelveꢀfold higher than that of 18 (IC50 = 12.29 ± 0.12 ꢁM) with fourꢀcarbon linker
and that of 19 (IC50 = 12.81 ± 0.11 ꢁM) with sixꢀcarbon linker. As an exception, the
eeAChE inhibitory activity of 27 with 6ꢀmethylene chain was better than that of 24.
The results revealed that suitable length of linker played an important role in eeAChE
inhibitory activity. The terminal amine group on the side chain also was crucial for
AChE inhibition. Most of compounds possessing an Nꢀmethyl or Nꢀethyl group
(16ꢀ23, 42ꢀ45) displayed better eeAChE inhibitory activity than those possessing
benzylamine group (24ꢀ30) and piperazine group (32ꢀ37). It was because that the
alkyamino side chain can interact with catalytic active site (CAS) of AChE,40 and this
was also supported by the molecular docking study.
Almost all compounds exhibited moderate or very weak eqBuChE inhibitory
activity and showed higher selectivity for eeAChE over eqBuChE. This selectivity
profile may be beneficial to lessen the peripheral cholinergic side effects and provide
lower toxicity e.g. tacrine, an AChE inhibitor, was proposed some side effects which
may be attributed their poor selectivity.41
The good biological profiles of many inhibitors against eeAChE (16, 20, 22, 23, 27,
28, 36, 42, 43 and 44) promoted us to extend our inhibition assays to the human
enzymes, AChE (hAChE) and BuChE (hBuChE). The bioactivity of the selected
compounds was listed in Table 2. All the test compounds showed moderate inhibition
against hAChE and they were relatively lower than eeAChE. Unexpectedly,
compound 16 (hAChE IC50 = 0.657 ± 0.002 ꢁM) and 23 (hAChE IC50 = 1.85 ± 0.05
ꢁM), bearing the Nꢀmethyl and Nꢀethyl group, exhibited better inhibitory activity by
comparing with eeAChE. This phenomenon occurred may be result from the
conformational difference between hAChE and eeAChE.42 Taking all these into
account, compound 16 was selected for the relatively potent ChEs inhibitor among all
the synthetic target compounds.
Kinetic study of eeAChE inhibition
To explore inꢀdepth the mechanism of eeAChE inhibition, compound 16 was selected
for enzyme kinetic study. The results displayed that both slopes and intercepts were

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increased accordingly with the concentration of compound 16 (Figure 2). This pattern
indicated 16 was a mixedꢀtype inhibitor to eeAChE, thus supporting a dual binding
site AChE inhibitor. And Replots of the slope versus different concentration of
compound 16 gave an inhibition constant, Ki of 0.404 ꢁM.
Monoamine oxidase inhibitory activity
To clarify the study of the multitarget profile of the synthetic compounds, the
inhibitory activity against hMAOꢀB and hMAOꢀA was determined and compared
with the lead compound 5 and pargyline by the Amplex Red MAO assay.43 As seen in
Table 2, compounds 16ꢀ18, 20, 21, 25, 42ꢀ44, showed potent hMAOꢀB inhibitory
activity, acting in the nanomolar (IC50 ranging from 0.0372 ± 0.0002 to 0.880 ± 0.005
ꢁM). On the contrary, they were less potent against hMAOꢀA. The most potent
hMAOꢀB inhibitor 16 (hMAOꢀB IC50 = 0.0372 ± 0.0002 ꢁM) also showed a good
selectivity to hMAOꢀB and hMAOꢀA (SI = 1024). Compounds 32ꢀ37, bearing a
piperazine group, and 26ꢀ31, bearing a benzylamine group, exhibited both relatively
moderate or poor hMAOꢀB and hMAOꢀA inhibitory activity. Similarly,
2'ꢀOꢀmodified compounds 38ꢀ41 and 3'ꢀOꢀmodified compound 45 also showed
moderate hMAOꢀB and hMAOꢀA inhibitory. Altogether, these results showed that
compounds bearing Nꢀmethyl or Nꢀethyl groups were the relatively better hMAOꢀB
inhibitor. Among them, compound 16 was the most potent inhibitor, even better than
pargyline and the lead compound 5.
Reversibility and kinetic study of MAO-B inhibition
To explore the reversibility/irreversibility toward hMAOꢀB inhibitor, the most potent
hMAOꢀB inhibitor compound 16 was selected to study by a timeꢀdependent inhibition
assay.14 Compound 16 was preꢀincubated with hMAOꢀB over different time periods
(0, 15, 30, 45, 60 min) at a concentration of twofold IC50, 0.003 ꢁM. Irreversibility
hMAOꢀB inhibitor pargyline was used as reference (Figure 3A). These findings

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demonstrated that 16 was not a timeꢀdependent inhibitor but was reversible hMAOꢀB
inhibitor.
To further study the type of hMAOꢀB inhibition, the kinetic behavior of 16 toward
hMAOꢀB was determined by plotting of LineweaverꢀBurk (Figure 3B). The catalytic
rates were measured at five different pꢀtyramine concentrations (0.05ꢀ0.25 mM) with
different concentrations of 16. The results showed that the Vmax decreased as the
concentration of 16 was increased, whereas the Km value keeps constant. Therefore,
compound 16 is a noncompetitive inhibitor, Ki of 0.551ꢁM.
In vitro blood-brain barrier permeation study
The bloodꢀbrain barrier (BBB) is the significant criterion of the successful central
nervous system (CNS) drugs, To evaluate whether the synthesized compounds could
penetrate this barrier, a parallel artificial membrane permeation assay (PAMPA) was
adopted.44 Compared with the permeability of 8 commercial drugs reported in
literatures (Supporting Information, Table S1), a plot of experimental data versus
reported values generated a good linear correlation, Pe (exp.) = 0.6022 Pe (Bibl.) +
0.8073 (R2 = 0.9749) (Figure S1). From this equation and considering the limitation
established by Di et al. for BBB permeation, the following ranges of permeability
were established: compounds with Pe (10ꢀ6 cm sꢀ1) > 3.22 for high BBB permeation
(CNS+), compound with Pe (10ꢀ6 cm sꢀ1) < 2.31 for low BBB permeation (CNSꢀ), and
compound with 3.22 > Pe (10ꢀ6 cm sꢀ1) > 2.31 for uncertain BBB permeation (CNS±).
From the results shown in Table 3, compound 16 show higher Pe value than 3.22 (Pe
= 10.56×10ꢀ6 cm sꢀ1), which indicated that 16 can cross the BBB and may reach the
therapeutic targets in the CNS.
Molecular modeling studies
To study the possible interaction mode of compound 16 with AChE and hMAOꢀB, a
molecular modeling was performed using the Molecular Operating Environment
(MOE) software. The Xꢀray crystal structures of hAChE and hMAOꢀB acquired from

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the PDB. As shown in Figure 4A and B, compound 16 can simultaneously interact
with PAS and CAS of hAChE (PDB 4EY7). The phenyl of chromanꢀ4ꢀone was
located into PAS of hAChE through a πꢀπ stacking interaction with Trp 286 (3.76 Å).
In the bottom of the gorge, the protonated nitrogen of Nꢀdimethylamino can bind to
CAS with the indole ring of Trp 86 (4.07 Å) via a cationꢀπ interaction. The docking
results implied that 16 was a dual site binding inhibitor for hAChE. All these results
accorded with our molecular design for AChE inhibition.
For MAOꢀB studies, the Xꢀray crystal structure with FAD of hMAOꢀB (PDB 2V60)
was used. We docked compound 16 into the binding site of hMAOꢀB observed that
16 can suitably fit into the pocket. In close to the FAD cofactor, a πꢀπ stacking was
seen between Tyr 398 (3.97Å) and the phenyl ring of chromanꢀ4ꢀone. Meanwhile,
another πꢀπ interaction was established between the substituted phenyl ring and
residues Tyr 326 (4.45 Å) (Figure 5A and 5B). Beside, a hydrogen bond was also
formed between the oxygen of chromanꢀ4ꢀone and Tyr 435ꢀOH (3.06 Å). Above all
of these results further confirmed that 16 is a potent hMAOꢀB inhibitor.
Acute toxicity studies
The test compound 16 was given in single oral doses of 0, 677, 1333 or 2000 mg/kg
to investigate the acute toxicity in vivo. The Kunming mice were randomly divided
into 4 groups (5 mice/group): 0, 677, 1333, and 2000. Mice were observed the first 4
h for fatality and abnormal behavior after oral administration of the compound. The
abnormal effect was monitored for the next 14 days, including body weight changes,
fatality changes and mental behavior. All mice were executed on the 14th day and
examined for the possible damage to the heart, liver or kidneys. There was no acute
toxicity during the experimental period. The phenomenon indicated that the
compound 16 was safe at doses up to 2000 mg/kg within two weeks.
Cognitive and memory assays in vivo

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Scopolamine induces memory impairment has been used widely to assess the
potential agents for AD therapy.3,37,45 To determine whether the treatment of 16
improve memory in scopolamineꢀinduced mice, the passive avoidance test were
performed.46
As the results shown in Figure 6, the latency of mice treated with scopolamine
alone (3 mg/kg) showed was significantly shorter (p < 0.05) than that of
vehicleꢀtreated mice (normal group). Treatment with compounds 16 (3, 9, 27 mg/kg)
increased the latency time in a doseꢀdependent manner, and the maximum dose group
(27 mg/kg) exhibited the longer latency (241.2 s) (p < 0.001) than the other dose
groups, even better than the donepezil group (5 mg/kg) (215.3 s). Therefore, the
optimal dose of 16 might be 27 mg/kg. These results revealed that compound 16 may
have reversed scopolamineꢀinduced memory impairment in the stepꢀdown passive
avoidance test by increasing brain cholinergic activity due to the inhibition of AChE.
Conclusion
In conclusion, our study involved the synthesis of a new series of homoisoflavonoid
derivatives as multitarget antiꢀAlzheimer agents. These compounds were estimated
the inhibitory activity of AChE and MAO in vitro. Among them, compound 16
exhibited moderate inhibitory activity toward AChE and significant hMAOꢀB
inhibitory activity. Kinetic and molecular docking studies demonstrated that
compound 16 was a dual binding site AChE inhibitor and noncompetitive inhibitor of
MAOꢀB. Moreover, compound 16 could cross the bloodꢀbrain barrier by
PAMPAꢀBBB assay. In vivo assays demonstrated that 16 did not show signs of acute
toxicity at doses of up to 2000mg/kg and could reverse cognitive deficit in the
stepꢀdown passive avoidance test. Above all, compound 16 might be a promising lead
as the multitarget agents for the further study in the treatment of AD.
Abbreviations
AD
Alzheimer’s disease
AChE
Acetylcholinesterase

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Aβ
βꢀamyloid protein
IMAOs
ROS
MAO inhibitors
NMDA NꢀmethylꢀDꢀaspartate
Reactive oxygen species
Peripheral anionic site
Catalytic site
MTDL
ChE
Multiꢀtargetꢀdirected ligand
PAS
CAS
SAR
BBB
SI
Cholinesterase
BuChE
ACh
Butyrylcholinesterase
Acetycholine
Structureꢀactivity relationship
Bloodꢀbrain barrier
BuCh
MAO
5ꢀHT
PEA
Butyrylcholine
Selectivity index
Monoamine oxidase
5ꢀHydroxytryptamine
βꢀphenylethylamine
CNS
Central nervous system
PAMPA Parallel artificial membrane
permeation assay
Acknowledgments
We gratefully acknowledge the financial support of the National Natural Science
Foundation of China (81573313), the Program for Changjiang Scholars and
Innovative Research Team in University (IRT_15R63), the Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions
(PAPD).
All procedures were approved by the China Pharmaceutical University Animal
Care and Use Committee (IACUC) and were in compliance with the National Institute
of Health (NIH) guidelines
Conflict of Interest
The authors no declare competing interests.
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Legends
Scheme 1. Reagents and conditions: (i) piperidine, EtOH, room temperature,
overnight; (ii) K2CO3, KI, CH3CN, 65°C, overnight; (iii) K2CO3, KI, acetone, 56 °C,
overnight.
Table 1. MAOs and ChEs inhibition data of the synthesized compounds.
Table 2. Inhibition of human AChE activity.
Table 3. Permeability results (Pe × 10-6 cm s-1) from the PAMPA-BBB assay for
selected compounds with their predicted penetration into the CNS.
Figure 1. The rational design strategy for the multi-functional potential anti-
Alzheimer’s agents.
Figure 2. Kinetic study on the mechanism of eeAChE inhibition by compound 16.
Overlaid Lineweaver-Burk reciprocal plots of AChE initial velocity at increasing
substrate concentration (0.05-0.25 mM) in the absence of inhibitor and in the presence
of 16 are shown. Lines were derived from a weighted least-squares analysis of the
data points.
Figure 3. (A) Reversibility studies of hMAO-B inhibition by compound 16.
Compound 16 and pargyline were pre-incubated for various periods of time (0-60 min)
with hMAO-B at concentrations equal to twofold the IC50 values for the inhibition of
the enzyme. After dilution to concentrations of 16 and pargyline equal to IC50, the
inhibitory rates were recorded. (B) Kinetic study on the mechanism of hMAO-B
inhibition by 16. Overlaid Lineweaver–Burk reciprocal plots of hMAO-B initial
velocity at increasing p-tyramine concentration (0.05-0.25 mM) in the absence of
inhibitor and in the presence of 16 are shown. Lines were derived from a weighted
least-squares analysis of the data points.
Figure 4. (A) 3D docking model of compound 16 with hAChE. Atom colors: yellow-
carbon atoms of 16, gray-carbon atoms of residues of hAChE, dark blue-nitrogen
atoms, red-oxygen atoms. The dashed lines represent the interactions between the
protein and the ligand. (B) 2D schematic diagram of docking model of compound 16
with AChE. The figure was prepared using the ligand interactions application in

Page 17 of 28
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DOI: 10.1039/C7MD00199A
MOE.
Figure 5. (A) 3D docking model of compound 16 with hMAO-B. Atom colors:
yellow-carbon atoms of 16, gray-carbon atoms of residues of hMAO-B, green-carbon
atoms of FAD, dark blue-nitrogen atoms, red-oxygen atoms. The dashed lines
represent the interactions between the protein and the ligand. (B) 2D schematic
diagram of docking model of compound 16 with hMAO-B. The figure was prepared
using the ligand interactions application in MOE.
Figure 6. Effects of compound 16 on scopolamine-induced memory deficit in the
step-down passive avoidance test. Compound 16 (at 3, 9, 27 mg/kg, p.o.), or
donepezil (5 mg/kg, p.o) were orally given 1 h before treatment with scopolamine.
After 30 min, the mice were treated with scopolamine (3 mg/kg, i.p.) and test in the
#
step-down passive avoidance. Values are expressed as the mean SEM (n = 10). p <
*
0.05 vs normal group. p < 0.05, **p < 0.01 and ***p < 0.001 vs scopolamine-treated
control group.

MedChemComm
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DOI: 10.1039/C7MD00199A
Scheme 1. Reagents and conditions: (i) piperidine, EtOH, room temperature,
overnight; (ii) K2CO3, KI, CH3CN, 65°C, overnight; (iii) K2CO3, KI,
acetone, 56 °C, overnight.

Page 19 of 28
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DOI: 10.1039/C7MD00199A
Table 1. MAOs and ChEs inhibition data of the synthesized compounds.
IC50a (µM) + SD
Selectivity index
IC50 (µM) + SD
Selectivity index
Compound
R
Position
n
eeAChE
eqBuChE
eqBuChE/eeAChE
hMAO-B
hMAO-A hMAO-A/hMAO-B
16
17
A
A
A
A
B
B
B
B
C
C
C
C
D
D
D
D
E
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
o-OH
o-OH
o-OH
o-OH
m-OH
m-OH
m-OH
m-OH
﹣
2
3
0.89±0.02
5.21±0.18
12.29±0.12
12.81±0.11
1.03±0.01
3.20±0.06
6.10±0.10
3.75±0.24
19.33±1.30
52.26±0.31
19.72±0.22
1.77±0.09
1.29±0.07
29.42±0.15
43.34±0.59
16.96±0.25
10.11±0.06
13.02±0.11
32.34±0.19
21.02±0.17
1.02±0.07
60.78±0.14
65.26±0.82
75.08±1.21
65.30±1.25
54.79±0.26
1.04±0.02
4.97±0.06
4.16±0.17
14.07±0.28
44.4+0.12
28.71±1.21
20.34±0.89
18.91±0.23
9.12±0.14
28.88±0.73
8.27±0.15
4.61±0.14
4.83±0.33
41.46±0.14
19.29±0.80
18.41±0.35
50.74±1.11
49.39±0.28
17.16±0.07
10.85±0.16
40.00±0.60
15.45±0.10
10.40±0.27
27.27±0.18
31.45±0.13
89.55±0.99
N
32.3
3.9
0.0372±0.0002 38.1±0.14
1024
>2283
>132
12.1
1223
379
0.0438±0.0003
0.756±0.002
3.22±0.11
Nb
18
4
1.54
N
19
6
0.712
28
39.1±0.17
20
2
0.0381±0.001 46.6±0.08
21
3
2.58
0.166±0.010
2.86±0.22
2.90±0.17
2.28±0.14
0.880±0.005
8.61±0.04
>100
62.9±0.20
22
4
0.756
1.29
52.9±0.32
18.5
13.1
>43.8
>114
>11.6
>1
23
6
37.8±0.21
24
2
2.14
N
25
3
0.369
0.934
28.7
N
26
4
N
27
6
N
28
2
38.3
4.29±0.03
1.59±0.04
3.68±0.13
>100
N
>23.3
﹣
29
3
0.583
0.25
﹣
30
4
N
>27.2
>1
31
6
2.36
N
32
3
1.53
22.7±1.3
16.4±0.2
6.24±0.4
97.5±2.9
38.6±0.54
>100
N
>4.41
>6.1
>16.1
>1.03
>2.59
>1
33
E
4
0.799
0.843
1.5
N
34
E
6
N
35
F
2
N
36
F
3
87.8
N
37
F
6
>1.65
0.154
0.116
0.176
0.0892
8.05
N
38
A
A
B
B
A
A
B
B
﹣
﹣
﹣
2
10.03±0.17
8.74±0.18
11.50±0.34
4.89±0.35
8.37±0.19
2.16±0.01
1.68±0.06
1.03±0.03
N
>100
N
>1
39
3
>100
N
>1
40
2
43.8±2.1
N
N
N
>2.64
﹣
41
3
42
2
0.571±0.022
38.0±0.44
66.5
1935
97.1
3.55
>413
n.t.
43
3
0.435
0.403
0.0732
>2.25
120
0.0484±0.0002 93.7±1.11
44
2
0.824±0.021
18.2±0.21
0.242±0.002
n.t.
80.0±0.46
64.7±0.05
N
45
3
5
﹣
﹣
﹣
Donepezil
Pargyline
﹣
0.0163±0.0003 1.96±0.10
n.t.c
n.t.
n.t.
﹣
n.t.
0.0752±0.0004 1.10±0.01
14.6
a IC50: 50% inhibitory concentration (means ± SD of three experiments).
b
Inactive at 100 µM (highest concentration tested), at higher concentrations the
compounds precipitate.
c n.t.=not tested

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DOI: 10.1039/C7MD00199A
Table 2. Inhibition of human AChE activity.
IC50 a (µM)
hAChE
Selectivity index
Compound
hBuChE
9.31±0.02
23.6±1.1
11.3±0.3
4.25±0.06
20.0±0.3
74.24±0.2
Nb
hBuChE/hAChE
14.2
16
0.657±0.002
3.29±0.01
14.1±0.4
20
7.17
22
0.801
2.30
23
1.85±0.05
40.9±1.6
27
0.489
33.3
28
22.3±0.7
36
4.72±0.08
1.41±0.01
25.72±0.10
30.3±0.1
>21.2
3.15
42
4.44±0.03
39.3±0.6
10.3±0.4
N
43
1.53
44
5
0.340
1.01
99.9±2.12
0.0247±0.0003
Donepezil
5.58±0.04
226
a IC50: 50% inhibitory concentration (means ± SD of three experiments).
b
Inactive at 100 µM (highest concentration tested), at higher concentrations the
compounds precipitate.

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Table 3. Permeability results (Pe × 10-6 cm s-1) from the PAMPA-BBB assay for
selected compounds with their predicted penetration into the CNS.
Compounds Permeability (Pe × 10-6 cm s-1) a
10.56 ± 0.41
Prediction
CNS+
16
a Data are the mean ± SD of three independent experiments.

MedChemComm
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DOI: 10.1039/C7MD00199A
Figure 1. The rational design strategy for the multi-functional potential
anti-Alzheimer’s agents

Page 23 of 28
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DOI: 10.1039/C7MD00199A
Figure 2. Kinetic study on the mechanism of eeAChE inhibition by compound 16.
Overlaid Lineweaver-Burk reciprocal plots of AChE initial velocity at
increasing substrate concentration (0.05-0.25 mM) in the absence of
inhibitor and in the presence of 16 are shown. Lines were derived from a
weighted least-squares analysis of the data points.

MedChemComm
Page 24 of 28
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DOI: 10.1039/C7MD00199A
Figure 3. (A) Reversibility studies of hMAO-B inhibition by compound 16.
Compound 16 and pargyline were pre-incubated for various periods of time
(0-60 min) with hMAO-B at concentrations equal to twofold the IC50 values
for the inhibition of the enzyme. After dilution to concentrations of 16 and
pargyline equal to IC50, the inhibitory rates were recorded. (B) Kinetic study
on the mechanism of hMAO-B inhibition by 16. Overlaid Lineweaver–Burk
reciprocal plots of hMAO-B initial velocity at increasing p-tyramine
concentration (0.05-0.25 mM) in the absence of inhibitor and in the
presence of 16 are shown. Lines were derived from a weighted least-squares
analysis of the data points.

Page 25 of 28
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DOI: 10.1039/C7MD00199A
Figure 4. (A) 3D docking model of compound 16 with hAChE. Atom colors: yellow-
carbon atoms of 16, gray-carbon atoms of residues of hAChE, dark
blue-nitrogen atoms, red-oxygen atoms. The dashed lines represent the
interactions between the protein and the ligand. (B) 2D schematic diagram
of docking model of compound 16 with AChE. The figure was prepared
using the ligand interactions application in MOE.

MedChemComm
Page 26 of 28
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DOI: 10.1039/C7MD00199A
Figure 5. (A) 3D docking model of compound 16 with hMAO-B. Atom colors:
yellow-carbon atoms of 16, gray-carbon atoms of residues of hMAO-B,
green-carbon atoms of FAD, dark blue-nitrogen atoms, red-oxygen atoms.
The dashed lines represent the interactions between the protein and the
ligand. (B) 2D schematic diagram of docking model of compound 16 with
hMAO-B. The figure was prepared using the ligand interactions application
in MOE.

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DOI: 10.1039/C7MD00199A
Figure 6. Effects of compound 16 on scopolamine-induced memory deficit in the
step-down passive avoidance test. Compound 16 (at 3, 9, 27 mg/kg, p.o.), or
donepezil (5 mg/kg, p.o) were orally given 1 h before treatment with
scopolamine. After 30 min, the mice were treated with scopolamine (3
mg/kg, i.p.) and test in the step-down passive avoidance. Values are
#
*
expressed as the mean SEM (n = 10). p < 0.05 vs normal group. p < 0.05,
**p < 0.01 and ***p < 0.001 vs scopolamine-treated control group.

MedChemComm
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13x10mm (300 x 300 DPI)