Chromone and donepezil hybrids as new multipotent cholinesterase and monoamine oxidase inhibitors for the potential treatment of Alzheimer's disease

Article abstract of DOI:10.1039/c9md00441f

A series of chromone and donepezil hybrids were designed, synthesized, and evaluated as multipotent cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for the potential therapy of Alzheimer's disease (AD). In vitro studies showed that the great majority of these compounds exhibited potent inhibitory activity toward BuChE and AChE and clearly selective inhibition for hMAO-B. In particular, compound 5c presented the most balanced potential for ChE inhibition (BuChE: IC₅₀ = 5.24 μM; AChE: IC₅₀ = 0.37 μM) and hMAO-B selectivity (IC₅₀ = 0.272 μM, SI = 247). Molecular modeling and kinetic studies suggested that 5c was a mixed-type inhibitor, binding simultaneously to peripheral and active sites of AChE. It was also a competitive inhibitor, which occupied the substrate and entrance cavities of MAO-B. Moreover, compound 5c could penetrate the blood-brain barrier (BBB) and showed low toxicity to rat pheochromocytoma (PC12) cells. Altogether, these results indicated that compound 5c might be a hopeful multitarget drug candidate with possible impact on Alzheimer's disease therapy.

Full text of DOI:10.1039/c9md00441f

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RESEARCH ARTICLE
Chromone and donepezil hybrids as new
multipotent cholinesterase and monoamine
oxidase inhibitors for the potential treatment of
Alzheimer's disease†
Cite this: DOI: 10.1039/c9md00441f
Xiao-Bing Wang,
Fu-Cheng Yin, Ming Huang,
Neng Jiang,
*
Jin-Shuai Lan and Ling-Yi Kong
A series of chromone and donepezil hybrids were designed, synthesized, and evaluated as multipotent
cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for the potential therapy of Alzheimer's dis-
ease (AD). In vitro studies showed that the great majority of these compounds exhibited potent inhibitory
activity toward BuChE and AChE and clearly selective inhibition for hMAO-B. In particular, compound 5c
presented the most balanced potential for ChE inhibition (BuChE: IC50 = 5.24 μM; AChE: IC50 = 0.37 μM)
and hMAO-B selectivity (IC50 = 0.272 μM, SI = 247). Molecular modeling and kinetic studies suggested that
5c was a mixed-type inhibitor, binding simultaneously to peripheral and active sites of AChE. It was also a
competitive inhibitor, which occupied the substrate and entrance cavities of MAO-B. Moreover, compound
5c could penetrate the blood–brain barrier (BBB) and showed low toxicity to rat pheochromocytoma
(PC12) cells. Altogether, these results indicated that compound 5c might be a hopeful multitarget drug
candidate with possible impact on Alzheimer's disease therapy.
Received 16th September 2019,
Accepted 8th November 2019
DOI: 10.1039/c9md00441f
rsc.li/medchem
and recovering or sustaining ACh can diminish these symp-
toms of AD.4,5 It is rational and effective to increase the
1. Introduction
Alzheimer's disease (AD) is the most prominent form of de-
mentia among the elderly that results in tremendous emo-
tional and financial burden on society, individuals and their
caregivers.1 Data from 2018 indicated that approximately 50
million people around the world suffered from dementia, and
the figure is going to increase sharply up to 132 million by
2050.2 Although 100 years have passed since its discovery, the
etiology of AD is still unclear. Many hypotheses have been de-
veloped to elucidate the molecular mechanism of AD based
on several factors involved in its pathogenesis including
β-amyloid (Aβ) deposits, τ-protein aggregation, oxidative
stress, inflammation, dyshomeostasis of biometals and low
levels of acetylcholine.3
amount of ACh through inhibition of cholinesterases (ChEs)
that are responsible for the hydrolysis of ACh in pre-synaptic
areas for the treatment of AD's symptoms.6 There are two
types of ChEs in the central nervous system, butyrylcholin-
esterase (BuChE) and acetylcholinesterase (AChE). At present,
several AChE inhibitors have been approved by the FAD for
the treatment of AD including rivastigmine, donepezil, and
galantamine. However, these single target drugs just have
limited and transient impact on this disease in clinical ther-
apy because of the multifactorial nature of AD.7
Many research studies have indicated that monoamine oxi-
dase B (MAO-B) also played a pivotal role in the pathogenesis
and development of AD. Its high level in the brain may cause
a cascade of biochemical events, eventually leading to neuro-
nal dysfunction.8,9 Indeed, the age-related increase of brain
MAO-B activity would produce higher levels of H2O2 and oxi-
dative free radicals, which have been correlated with the de-
velopment of oxidative stress.10,11 On the other hand, recent
biochemical and clinical studies indicated that MAO-B inhibi-
tors, such as ladostigil and rasagiline, also have shown great
antiapoptotic activities and neuroprotective and inhibitory ef-
fects on Aβ aggregation.12–14 Thus, the development of MAO-B
inhibitors has been considered as an attractive approach for
the therapeutic strategy for AD.
Currently, the therapeutic treatments for AD are mainly
focusing on the classical cholinergic hypothesis, which
suggests that the low level of choline, especially ACh in the
brain, is the key factor in its cognitive and memory deficits,
Jiangsu Key Laboratory of Bioactive Natural Product Research and, State Key
Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry,
School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing 210009, People's Republic of China.
E-mail: cpu_lykong@126.com; Fax: +86 25 83271405; Tel: +86 25 83271405
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9md00441f
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In recent years, the multi-target-directed ligand (MTDL)
strategy has shown a great advantage in the treatment of
this multifaceted disease.15–17 With the development of the
“one molecule, multiple targets” paradigm, donepezil, which
is the most effective pharmacological agent for AD treat-
ment, has attracted more and more attention recently. A
study of a donepezil–TcAChE complex by X-ray crystallogra-
phy has revealed that donepezil is a dual binding site AChE
inhibitor, and the indanone and benzylpiperidine moieties
of donepezil could interact with the peripheral (non-cata-
lytic) and central (catalytic) binding sites of AChE, respec-
tively.18 According to the MTDL strategy, it is advantageous
to replace the indanone fragment of donepezil with addi-
tional bioactive molecules to produce multifunctional ChE
inhibitors. For example, donepezil–ebselen hybrids have
been designed as potent ChE inhibitors with various other
(4H-1-benzopyran-4-one) structure is the pharmacophore of a
great number of biologically active compounds. The ability
to inhibit hMAO enzymes is one of the most developed
method in AD research. The chromone core is found in fla-
vones and isoflavones, which are molecules containing also
the chalcone moiety; all these structures are preferential
scaffolds for the development of hMAO inhibitors.29
In this paper, we are focused on studying chromone which
has been recognized as a privileged scaffold for the develop-
ment of monoamine oxidase inhibitors.30,31 Considering that
the combination of inhibitors targeting ChE and MAO-B is
also an effective strategy for developing new multi-functional
anti-AD drugs,32–34 we combined chromone and the
benzylpiperidine moieties of donepezil using an amide linker
to obtain a series of new hybrids that are expected to be dual-
acting AChE and MAO-B inhibitors (Fig. 1).
In this study, a series of new donepezil–chromone hybrids
4a–4h and 5a–5h were designed, synthesized and evaluated
for their biological activity, including ChE and MAO inhibi-
tion as well as CNS permeation in vitro. Last, molecular
modeling studies are performed to investigate the interaction
mechanism, the structure–activity relationships and the bind-
ing mode of new hybrid compounds.
ebselen-related
pharmacological
properties.19
N-[(5-
(Benzyloxy)-1-methyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-
1-amine and donepezil hybrids have been designed as
multifunctional agents capable of inhibiting MAOs and
ChEs,20 and a donepezil–RS67333 hybrid has been designed
to be a dual serotonin subtype 4 receptor agonist/acetylcho-
linesterase inhibitor.21 On the other hand, flavonoids, a
large family of naturally occurring compounds, have been
receiving increasingly widespread attention in present-day
society because they exhibit a wide range of biological activ-
ities associated with neurological disorders especially for
AD.22,23 Indeed, several flavonoids have been shown to in-
hibit the development of AD and reverse cognitive deficits
in rodent models, indicating that new effective flavonoid de-
rivatives are promising candidates for the research on anti-
AD drugs.24–26 Recently, our group has reported the synthe-
sis of tacrine–flavonoid and flavonoid–basic side-chain hy-
brids as multifunctional ChE inhibitors against AD.27,28
Among them, the framework of chromone contributes
greatly to the activities of flavonoid hybrids. The chromone
2. Results and discussion
2.1. Chemistry
Scheme 1 depicts the general procedure for the synthesis of
donepezil–chromone hybrids 4a–4h and 5a–5h. 4-Oxo-4H-
chromene-2-carboxylic acid (3h), 1-benzylpiperidin-4-amine (4)
and 2-(1-benzylpiperidin-4-yl)ethanamine (5) are commercially
available, whereas the 4-oxo-4H-chromene-3-carboxylic acids
(3a–3g) were synthesized according to a described route.
Initially, 2-hydroxyacetophenones (1a–1g) were converted
to 3-formylchromones (2a–2g) by a modified Harnisch proce-
dure with phosphorus oxychloride and DMF. Then
Fig. 1 Design strategy for donepezil and chromone hybrids.
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Scheme 1 Reagents and conditions: (i) POCl3, DMF, 0 °C, 2 h; (ii) NaClO2, NH2HSO3, CH2Cl2, 0 °C, 3 h; (iii) thionyl chloride, reflux;
1-benzylpiperidin-4-amine (4), K2CO3, CH2Cl2, rt, 8 h; (iv) thionyl chloride, reflux; 2-(1-benzylpiperidin-4-yl)ethanamine (5), K2CO3, CH2Cl2, rt, 8 h.
intermediates (2a–2g) were oxidized with sodium chlorite
(NaClO2) and sulfamic acid (NH2SO3H) to afford the corre-
sponding acids (3a–3g).35 Finally, 3a–3h were treated with
acetyl chloride and a catalytic amount of DMF in CH2Cl2 to
produce the acyl chloride and subsequently reacted with
1-benzylpiperidin-4-amine (4) or 2-(1-benzylpiperidin-4-
yl)ethanamine (5) in CH2Cl2, and the target compounds
4a–4h and 5a–5h were obtained in good yields.36
Table 1 Cholinesterases and human recombinant MAO isoforms inhibitory activity of tested compounds and reference compounds
eeAChE
eqBuChE
Selectivity
BuChE/AChE
hMAO-A
hMAO-B
IC50 (μM)
Selectivity
hMAO-A/hMAO-B
Compd.
R
IC50a (μM)
IC50a (μM)
inhibitionb (%)
a
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
H
2.96 0.22
10.14 0.54
1.85 0.16
1.74 0.14
1.57 0.12
1.58 0.23
9.23 0.65
5.69 0.43
0.069 0.03
0.033 0.02
0.37 0.05
0.11 0.01
0.104 0.09
0.145 0.08
0.154 0.02
0.31 0.01
0.032 0.02
nt.
18.67 0.74
24.36 0.92
12.43 0.65
15.27 0.63
9.45 0.82
9.78 0.86
18.24 0.78
27.86 0.92
0.60 0.05
0.45 0.04
5.24 0.45
0.91 0.07
1.17 0.08
0.52 0.04
1.24 0.07
1.54 0.12
2.47 0.32
nt.
6.3
2.4
6.7
8.8
6.1
6.2
1.2
7.6
8.7
13.7
14.2
8.3
11.3
3.7
11.9
4.9
77
63.8 5.7
7.5 0.7
17.68 0.75
46.27 1.78
0.035 0.003
19.46 0.44
22.82 0.74
28.64 0.68
19.73 0.24
33.29 0.83
35.29 0.76
11.23 0.44
0.272 0.04
52.45 1.68
26.13 0.86
30.93 0.76
29.46 0.82
20.03 0.24
nt
<5.6
>2.1
210.9
>5.1
>4.4
>3.5
>5.1
>3.1
<2.8
<8.9
247
>1.9
>3.8
>3.2
>3.4
>4.9
—
6-OCH3
6-OBn
6-CH3
6-Br
7-OCH3
7-Br
H
11.6 0.5 μMa
48.1 2.7
35.5 3.4
37.4 2.9
31.6 2.5
29.7 2.2
68.6 6.7
72.4 8.4
67.2 0.7μMa
30.6 2.8
48.2 4.5
32.4 2.7
30.3 1.4
29.5 2.7
ntc
H
6-OCH3
6-OBn
6-CH3
6-Br
7-OCH3
7-Br
H
—
—
—
Donepezil
Pargyline
Iproniazid
—
—
nt.
0.12 0.01
6.93 0.34
—
0.85
nt.
nt.
5.89 0.74 μMa
a
c
Data are the mean SD of three independent experiments. b Test concentration is 100 μM. nt. = not tested.
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2.2. Inhibitory activity against BuChE and AChE
The inhibitory activity of hybrids 4a–4h and 5a–5h against
BuChE (from equine serum) and AChE (from electric eel) was
evaluated with donepezil as the reference compound using
Ellman's method.37 The IC50 values and AChE selectivity ra-
tios for inhibitory effects of all the test compounds are shown
in Table 1. The results showed that all new target compounds
showed moderate to good potent inhibitory activity to both
ChEs with IC50 values ranging from micromolar to nano-
molar. In particular, compound 5b, with a 6-methoxy group
substituent at the chromone moiety, provided the most po-
tent inhibitory activity for ChEs (BuChE: IC50 = 0.45 μM,
AChE: IC50= 0.033 μM), quite similar to that obtained for
donepezil (BuChE: IC50 = 2.47 μM, AChE: IC50 = 0.032 μM).
For the sake of clarity, all the hybrids were divided into
two series according to the length of the carboxamide (com-
pounds 4a–4h) and N-ethylcarboxamide (compounds 5a–5h)
linkage. As seen in Table 1, the length of the linkage of the
new compounds has great effects on the inhibitory activities
of both AChE and BuChE. Generally, compounds 5a–5h with
N-ethylcarboxamide linkages between the benzylpiperidine
and chromone manifest higher activity than their counter-
parts (4a–4h) with carboxamide linkages. On the other hand,
the activity of chromones substituted at the 2-position (4h
and 5h) was slightly lower than those of the corresponding
hybrids 4a and 5a. These results showed that the inhibitory
potency against ChEs is also closely related to the position
and length of the linkage.
In an effort to explore the effect of substitution at the
benzene ring of the chromone moiety on the ChE inhibitory
activities, we also have introduced different substituents
with varying properties to chromone. From the IC50 values
of compounds 5a–5e, it appeared that the activity of substit-
uent chromones against AChE was slightly less than that of
corresponding non-substituted chromone 5a. In particular,
connecting the OCH3 group to the 6-position of the chrom-
one ring (5b) improves the inhibitory activity against AChE.
Conversely, connecting the bulky OBn group to the 6-posi-
tion (5c) led to a large reduction of efficacy against AChE
isoforms. Moreover, compounds 5f and 5g possessing OCH3
(5f) and Br (5g) groups at the 7-position of chromone
showed a decreased AChE inhibitory activity compared to
Fig. 3 Lineweaver–Burk plots resulting from the subvelocity curve of
the BuChE activity with different substrate concentrations (0.05–0.50
mM) in the absence and presence of 0.5 × IC50, 1 × IC50, and 2 × IC50 5c.
the corresponding homologues 5b and 5e with the same
substituent at the 6-position of chromone. These results in-
dicated that both the steric hindrance and the electronic ef-
fects of the chromone enhance AChE inhibition. Interest-
ingly, the opposite trend was observed for the effect of
substituents on compounds 4a–4g. For example, compound
4b with 6-OCH3 on the chromone exhibited the worst inhib-
itory activity, while compounds 4c–4f with the other groups
showed augmented AChE inhibitory activity.
In addition, the influence of the substitution at the ben-
zene ring of the chromone moiety on BuChE inhibitory activ-
ity has the same trend as that on AChE inhibitory activity.
Some evidence suggests that the inhibition of BuChE may
also represent a treatment for Alzheimer's disease, with ac-
tions on memory and learning, possibly mediated through el-
evated ACh and lower Aβ levels.38,39 Consequently, most of
the hybrids exhibited good balanced AChE/BuChE inhibitory
activity, and may represent good ChE inhibitors to treat
Alzheimer's disease.
2.3. Inhibitory activity against hMAO-A and hMAO-B
To confirm the multipotent biological profile of the target
compounds, inhibitory activity against MAOs was determined
Fig. 4 Recovery of enzyme activity after dilution. MAO-B was pre-
incubated with compounds 5c and pargyline at concentrations equal
to 10 × IC50 and 100 × IC50 for 30 min and then diluted to 0.1 × IC50
and 1 × IC50, respectively. The residual enzyme activities were subse-
quently measured.
Fig. 2 Lineweaver–Burk plots resulting from the subvelocity curve of
the AChE activity with different substrate concentrations (0.05–0.50
mM) in the absence and presence of 0.5 × IC50, 1 × IC50, and 2 × IC50 5c.
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hMAO-A. A structure–activity relationship analysis showed
that hybrids with the benzyloxy group at the 6 position of
the chromone were favorable for inhibitory activity against
hMAO-B. In particular, compound 4c, featuring the
benzyloxy group at the
6 position of the chromone,
displayed the most potent inhibitory activity with an IC50
value of 0.035 μM, being about 3-fold and 200-fold more ac-
tive than the references, pargyline (IC50 = 0.12 μM) and
iproniazid (IC50 = 6.93 μM), respectively. All other analogues
gave weaker activities when the benzyloxy group was
substituted by other groups. Dramatically, compound 5d,
which featured a methyl group at the same position, pro-
vided the weakest. Moreover, MAO-B inhibitory potency was
also closely related to the length of the alkylene chain. For
example, compound 5c with the N-ethylcarboxamide linkage
between benzylpiperidine and chromone is also a great MAO-
B inhibitor with an IC50 value of 0.272 μM, being about 7.8-
fold less potent than its counterpart 4c with a carboxamide
linker. These results indicate that steric factors changed the
hMAO-B inhibitory activity.
Fig. 5 Lineweaver–Burk plots resulting from the subvelocity curve of
the MAO-B activity with different substrate concentrations (0.05–1.0
mM) in the absence and presence of 0.5 × IC50, 1 × IC50, and 2 × IC50 5c.
and compared with those of pargyline and iproniazid.40 The
results are depicted in Table 1. Most of these hybrids are
moderate inhibitors of hMAO-B with IC50 values in the
micromolar range, and exhibited weak or no inhibition of
Fig. 6 Molecule docking of compound 5c with AChE generated with MOE: (a) the 2D picture of binding is depicted; (b) the 3D picture of binding
is depicted.
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On account of the MAO and ChE inhibitory activity re-
2.5. Kinetic studies for hMAO-B
sults, compound 5c, which showed the most balanced poten-
tial to inhibit ChEs (AChE: IC50 = 0.37 μM; BuChE: IC50
=
To study the inhibitory activity of 5c toward hMAO-B, firstly,
we analyzed the reversibility/irreversibility of the binding of
compound 5c by the recovery assay of the enzymatic activities
after dilution of enzyme–inhibitor complexes.41 Pargyline,
with known irreversibility for hMAO-B, was used. The hMAO-
B enzyme was firstly incubated with compound 5c at concen-
trations of 10 × IC50 and 100 × IC50 for 30 min. These incuba-
tions were subsequently diluted 100-fold to yield inhibitor
concentrations of 0.1 × IC50 and 1 × IC50, respectively, and
the residual enzyme catalytic rates were measured. In con-
trast to pargyline, we observed that 5c reversibly inhibited
MAO-B, since its MAO-B activities were recovered after dilut-
ing the enzyme–inhibitor complexes (Fig. 4). Then we have
explored the possibility that 5c acts as a competitive inhibitor
of MAOB in accordance with a previous report.30 For this pur-
pose, the kinetic study of 5c against hMAO-B was performed.
As shown in Fig. 5, the Lineweaver–Burk plots for different
concentrations of 5c are linear and each set has a common
y-intercept. This behaviour indicated that 5c is a competitive
5.24 μM) and MAO-B selectively (IC50 = 0.272 μM, SI = 247), is
considered to be a promising multi-functional inhibitor for
further study.
2.4. Kinetic studies for ChE
To gain information on the mechanism of action of compound
5c on ChEs, kinetic studies were carried out. The Lineweaver–
Burk reciprocal plots of 5c against AChE (Fig. 2) showed in-
creasing slopes and intercepts with higher inhibitor concentra-
tions, indicating a mixed-type inhibitory behaviour for com-
pound 5c in the presence of AChE. Therefore this supports
that compound 5c could simultaneously bind to the PAS and
CAS of AChE. In contrast, a different plot for BuChE was
obtained, showing different Km and constant Vmax values in dif-
ferent inhibitor concentrations (Fig. 3). This suggested compet-
itive inhibition, revealing that these compounds compete for
the same binding site (CAS) as the substrate butyrylcholine.
Fig. 7 Molecule docking of compound 5c with MAO-B generated with MOE: (a) the 2D picture of binding is depicted; (b) the 3D picture of binding
is depicted; (c) the 2D picture of binding of compound 5c with MAO-A is depicted; (d) the 3D picture of binding of compound 5c with MAO-A is
depicted.
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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
inhibitor of MAO-B isozymes and offers further support for
the finding that 5c is a reversible MAO-B inhibitor.
Compounds
Permeabilitya (Pe × 10−6 cm s−1
)
Prediction
2.6. Molecular modeling studies of compound 5c with AChE
4a
4c
5b
5c
15.6 1.2
4.6 0.6
16.7 0.4
5.4 0.3
CNS+
CNS+
CNS+
CNS+
To obtain more insights into the interaction mode of com-
pound 5c against AChE, molecular docking study was
performed using software package MOE 2008.10. The X-ray
crystal structure of the TcAChE complex with donepezil (PDB
ID: 1EVE) was applied to build the starting model of AChE.
As shown in Fig. 6, the N-benzylpiperidine moiety of 5c was
oriented towards the CAS of AChE, via π–cation interactions
between the quaternary nitrogen of the piperidine ring with
Trp84 and Phe330. Besides, its benzene ring could also inter-
act with Trp84 via π–π stacking interactions. The chromone
moiety interacted with the indole ring from Trp279 of the
PAS via π–π stacking interactions. In addition, the 6-OBn of
5c is exposed outside the active site of AChE, which may also,
in part, explain the decreased inhibitory activity of AChE
compared to that of 5a. All these results clearly indicated that
compound 5c was a dual binding site (DBS) AChE inhibitor
in agreement with the kinetic study, which demonstrated the
rationality of our molecular design.
a
Data are the mean SD of three independent experiments.
2.8. In vitro blood–brain barrier permeation assay
Since brain penetration is essential for successful anti-AD
drugs,42,43 we have evaluated the potential of these hybrids to
cross the blood–brain barrier (BBB). For this purpose, a paral-
lel artificial membrane permeation assay for BBB (PAMPA-
BBB) was used, which was described by Di et al.44 Assay vali-
dation was conducted by comparing experimental permeabil-
ities of 9 commercial drugs with reported values (Table 2). A
plot of experimental data versus bibliographic values gave a
good linear correlation, Pe (exp.) = 1.2084 Pe (bibl.) − 0.3055
(R2 = 0.9263). From this equation and considering the limit
established by Di et al. for blood–brain barrier permeation,
we established that compounds with permeability values over
4.5 × 10−6 cm s−1 should be able to cross the BBB. Four com-
pounds (4a, 4c, 5b and 5c) that exhibited good activity
against AChE or MAO-B were chosen as the test compounds.
The results summarised in Table 3 indicated that all of the
selected hybrids (4a, 4c, 5b and 5c) could penetrate the BBB
to target the enzyme in the central nervous system.
2.7. Molecular modeling studies for compound 5c with
MAO-B and MAO-A
To evaluate the binding modes of the most potent compound
5c with MAO-B and MAO-A, docking studies were employed
with MOE 2008.10, based on the protein crystal structure of
MAO-B (2V60) and MAO-A (2Z5Y). As can be seen from Fig. 7,
the chromone moiety of 5c is located within the substrate
cavity of the enzyme, in close proximity of the flavin adenine
dinucleotide (FAD) cofactor, and the π–π stacking interactions
are seen between its benzyloxy group with Tyr435 and
Tyr398. Meanwhile, Tyr326 is involved in a hydrogen bond
interaction with the amide carbonyl of 5c. In contrast, the
benzylpiperidine moiety of 5c occupies the hydrophobic
pocket in the entrance cavity, formed by Pro102, Ser200,
Leu88, Gly101, Thr201, Glu84, Ile199 and Ile316. Further-
more, a hydrogen bond was detected for the quaternary nitro-
gen of the piperidine ring with Glu84.
2.9. Rat pheochromocytoma (PC12) cell toxicity
To gain insight into the therapeutic potential of these deriva-
tives, the most potent compounds 4c, 5b and 5c were selected
as the candidates to further study the potential toxicity effect
on the rat pheochromocytoma (PC12) cells (PC12 cells came
from Cell Resource Center, Shanghai Institute of Life Sci-
ences, Chinese Academy of Sciences). After exposing the cells
to compounds 4c, 5b and 5c for 24 h, cell viability was tested
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) assay. As indicated in Fig. 8, these compounds did not
Table 2 Permeability (Pe × 10−6 cm s−1) in the PAMPA-BBB assay for 9
commercial drugs, used in the experiment validation
Commercial drugs
Bibliographya
Experimentb
Testosterone
Verapamil
β-Estradiol
Clonidine
Corticosterone
Piroxicam
Hydrocortisone
Lomefloxacin
Ofloxacin
17
16
12
5.3
5.1
2.5
1.9
1.1
0.8
16.81 1.23
21.01 0.75
17.03 0.44
8.14 0.26
3.11 0.32
1.32 0.07
1.41 0.14
1.81 0.22
1.31 0.01
a
b
Fig. 8 The cell viability of compounds 4c, 5b and 5c on PC12 cells at
0–50 μM.
Taken from ref. 44. Data are the mean SD of three independent
experiments.
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show significant effects on cell viability at 1–50 μM. This
suggested that compounds 4c, 5b and 5c were nontoxic to
PC12 cells and might be suitable multifunctional agents for
treating AD.
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3. Conclusion
In summary, a series of donepezil and chromone hybrids have
been designed, synthesized and evaluated as multi-functional
anti-AD agents with cholinesterase and MAO inhibitory activi-
ties. Most of the compounds were potent ChE inhibitors with
IC50 values ranging from micromolar to nanomolar. These hy-
brids were also potent, exhibited high MAO-B selectivity and
interacted reversibly with hMAO-B. Among the synthesized
compounds, compound 5c was the most attractive derivative,
being able to inhibit ChEs (AChE: IC50 = 0.37 μM; BuChE: IC50
= 5.24 μM) and having high MAO-B selectivity (IC50 = 0.272
μM, SI = 247). Meanwhile, compound 5c could penetrate the
blood–brain barrier (BBB) and showed low cell toxicity to rat
pheochromocytoma (PC12) cells in vitro. Altogether, the
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Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This research work was financially supported by the National
Natural Science Foundation of China (81573313), the Innova-
tive Research Team in University (IRT_15R63), a project
funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD), and the Project
of Graduate Education Innovation of Jiangsu Province (No.
CXZZ13_0320).
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