Synthesis and evaluation of donepezil-ferulic acid hybrids as multi-target-directed ligands against Alzheimer's disease

Article abstract of DOI:10.1039/c6md00053c

A novel family of donepezil-ferulic acid hybrids were designed, synthesized and biologically evaluated as multi-target-directed ligands against Alzheimer's disease by fusing a fragment of donepezil and ferulic acid. The in vitro assay indicated that some

Full text of DOI:10.1039/c6md00053c

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RESEARCH ARTICLE
Synthesis and evaluation of donepezil–ferulic acid
hybrids as multi-target-directed ligands against
Alzheimer's disease†‡
Cite this: DOI: 10.1039/c6md00053c
Wei Xu, Xiao-Bing Wang, Zhi-Min Wang, Jia-Jia Wu, Fan Li, Jin Wang and Ling-Yi Kong*
A novel family of donepezil–ferulic acid hybrids were designed, synthesized and biologically evaluated as
multi-target-directed ligands against Alzheimer's disease by fusing a fragment of donepezil and ferulic acid.
The in vitro assay indicated that some of these molecules exhibited potent cholinesterase inhibitory activi-
ties, outstanding radical scavenging activities and good neuroprotective effects on PC12 cells, and could
penetrate into the central nervous system. Compound 5c especially showed moderate acetylcholinesterase
inhibitory activity (IC50 values of 0.398 μM for electric eel acetylcholinesterase) and butyrylcholinester-
ase inhibitory activity (IC50 = 0.976 μM for equine serum butyrylcholinesterase). It also showed signifi-
cant antioxidant activity (1.78 trolox equivalents by the ABTS method, IC50 values of 24.9 μM by the DPPH
method). The kinetic study and molecular docking indicated that compound 5c interacted with both the
peripheral anionic site and the catalytic binding site of acetylcholinesterase. Overall, these results indicated
that compound 5c is a promising drug candidate with balanced properties for the treatment of Alzheimer's
disease.
Received 25th January 2016,
Accepted 3rd March 2016
DOI: 10.1039/c6md00053c
www.rsc.org/medchemcomm
proach has been the major focus of attention, and a variety of
compounds acting on various targets were developed.
1
. Introduction
9–11
Alzheimer's disease (AD) is a progressive neurodegenerative
disease characterized by memory loss and cognitive decline
and affects over 46 million people worldwide. Although con-
Based on the cholinergic hypothesis, the degeneration of cho-
linergic neurons, reduced cholinergic neurotransmission and the
deterioration of the cognitive function of patients were the major
1
12
siderable efforts have been made to investigate the patho-
physiology of AD, the exact etiology of AD remains a mystery.
Several hypotheses, including low levels of acetylcholine,
dyshomeostasis of biometals, neuroinflammation and oxida-
tive stress, are demonstrated to play significant roles in the
symptoms of AD. Therefore, sustaining or recovering the cho-
linergic function was supposed to be clinically beneficial. It was
shown that the use of cholinesterase (ChE) inhibitors has been
13
the most effective treatment for AD hitherto. Actually, AChE
and butyrylcholinesterase (BuChE), two types of ChEs, are all able
2
–5
14
pathogenesis of AD. Currently, the primary therapeutic op-
tions for treatment of this disease are limited to three acetyl-
cholinesterase (AChE) inhibitors (rivastigmine, donepezil and
galantamine) and one N-methyl-D-aspartate (NMDA) receptor
to hydrolyze acetylcholine (Ach). It was demonstrated by X-ray
crystallography that AChE has a 20 Å deep narrow gorge which
consists of two binding sites: the catalytic active site (CAS) at the
bottom and the peripheral anionic site (PAS) near the entrance
6
,7
15,16
antagonist (memantine). However, these drugs are effective
in improving the symptoms for only a short period of time
and cannot cure the disease. Recent studies have pointed out
that drugs impacted on multiple targets can provide a more
effective treatment strategy for AD than single-target-directed
of the gorge.
It was shown that PAS is closely interacted with
17
hydrolysis of Ach. Besides, BuChE is another target of interest
in the research and development of anti-Alzheimer's drugs, since
this enzyme exerts a compensatory effect in response to a large
decrease in AChE activity when AD progresses. Donepezil (2,
Fig. 1), an AChE inhibitor, is the second drug approved by the U.
S. Federal Drug Agency (FDA) for the treatment of mild-to-
moderate AD. It shows high selectivity for AChE as opposed to
BuChE. The safety and efficacy of donepezil in the treatment of
AD have encouraged active research in the development of
donepezil-like agents for AD therapy.
On the other hand, oxidative stress also plays a critical
18
8
drugs. Thus, the multi-target-directed ligand (MTDL) ap-
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People's Republic of China. E-mail: cpu_lykong@126.com; Fax: +86 25 8327 1405;
Tel: +86 25 8327 1405
19
20,21
†
‡
The authors declare no competing interests.
Electronic supplementary information (ESI) available. See DOI: 10.1039/
2
2
c6md00053c
role in the AD pathological cascade. Converging lines of
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Considering these criteria, we report the design and syn-
thesis of hybrids of ferulic acid and donepezil as a valuable
strategy to develop effective neuroprotective compounds as
potential AD drugs (Fig. 1). The significant pharmacophores
of donepezil and FA were fused to obtain novel compounds
acting on multiple targets. We then report the biological ac-
tivity studies of these compounds including the inhibition of
ChE activity, antioxidant properties, neuroprotection, metal
chelation and blood–brain barrier permeation.
2
. Results and discussion
2
.1 Chemistry
The synthetic routes for the designed compounds 4a–4i and
a–5n are shown in Scheme 1. Commercially available ferulic
acid derivatives 1a–1g as starting materials were reacted with
1-benzylpiperidin-4-yl)amine (3a) or 4-(2-aminoethyl)-1-
Fig. 1 Drug design strategy for multi-target-directed ligands.
5
evidence demonstrated that oxidative damage in cellular
structures is augmented during aging and it occurs in the
early stages of AD and promotes the formation of other path-
ological hallmarks of the disease, such as amyloid plaques
(
benzylpiperidine (3b) in a mixture of dichloromethane (DCM)
and N,N-dimethylformamide (DMF) to give the target com-
pounds in good yields. To further evaluate the role of the
double bond between the phenyl ring and the amide, com-
pounds 6 and 7 were synthesized (Scheme 2) through a Pd–C
3
4
2
3
and neurofibrillary tangles. Moreover, a multinational study
involving 23 developed countries suggested that higher con-
sumption of dietary flavonoids is associated with lower popu-
3
5
2
4
reduction process. The syntheses of the target compounds
1a–11c are presented in Scheme 3 to explore the effect of
lation rates of dementia. Therefore, drugs which can pre-
vent oxygen free radical damage are helpful for the treatment
of AD.
1
conjugated chains. Interestingly, the FA moiety possessing a
dicarbonyl group happened to be part of curcumin, which is
another natural product and exhibits great potential as a
Due to the important effects of cholinesterase inhibitors
and antioxidants in AD treatment, some agents were
designed and synthesized, which could not only inhibit AChE
but also exhibit neuroprotective effects by decreasing oxida-
3
6
therapeutic agent for AD. Briefly, ethyl 4-chloroacetoacetate,
, was reacted with Ph P to give ylide 9 in good yield, upon a
Wittig reaction with the corresponding aldehyde in tetrahy-
8
3
2
5
tive damage in the brain. A series of tacrine–ferulic acid hy-
brids designed by Benchekroun et al. exhibited excellent inhi-
3
7
drofuran (THF) in the presence of NaH at 0 °C. Finally, con-
densation of 10a–10c with 3a or 3b in xylene under refluxing
conditions yielded 11a–11c. All compounds were purified
2
6
bition towards ChEs and strong antioxidant activity. Be-
sides, 2H-chromen-2-one derivatives and donepezil–ferulic
acid hybrids also showed good ChE inhibitory activities and
3
5
by column chromatography. The structures were verified by
1
13
2
7,28
H-NMR, C-NMR and mass spectrometry as cited in the ex-
perimental section.
potent antioxidant effects.
Ferulic acid (FA, 1, Fig. 1) is one of the ubiquitous com-
pounds in nature, which is especially abundant in cereals.
FA, which belongs to the class of phenylpropanoid deriva-
tives, is also identified as one of the main effective compo-
nents of several Chinese medicines such as Ferulic, Angelica
sinensis and Rhizoma ligustici (Chuanxiong). It was revealed
that FA protected the progression of a variety of age-related
2.2 In vitro inhibition of ChEs
In clinical practice, it is well known that ChE inhibitors are
effective in improving the behavior and well-being and
slowing down cognitive decline in patients with dementia.
Thus, the inhibitory activities of the novel donepezil–ferulic
acid hybrids and the reference compounds against AChE and
2
9
diseases due to its antioxidant properties. FA can greatly at-
tenuate neuronal cell death caused by reactive oxygen species
3
8
BuChE were evaluated using the method of Ellman et al.
(
ROS) and protect the brain from amyloid-beta peptide (Aβ)
Considering their lower cost and high degree of sequence
identity, AChE from electric eel and BuChE from equine se-
rum were initially used. The results shown in Table 1 indi-
cated that all the tested compounds showed moderate to
good inhibitory activities towards AChE and BuChE with IC50
values ranging from micromolar to sub-micromolar and all
target compounds were more potent than the parent com-
pounds 3a, 3b and FA. The length of the alkyl spacer between
the amide and the pyridine ring could significantly influence
the inhibitory activity towards both ChEs. The series of com-
pounds 5 which bear a longer alkyl linker between the
N-benzylpiperidine moiety and FA demonstrated better
3
0,31
neurotoxicity.
A recent isolated study on rat hepatocytes
suggested that FA and similar structures are effective in
inhibiting or decreasing glyoxal or methylglyoxal-induced cy-
totoxicity and oxidative stress. Additionally, FA protected
PC12 cells against 2,2′-azobisIJ2-amidinopropane) dihydro-
chloride (AAPH)-induced oxidative stress by increasing cata-
lase and superoxide dismutase activity and reducing cellular
3
2
3
3
lactate dehydrogenase release and malondialdehyde levels.
Consequently, FA could act as the beneficial antioxidant frag-
ment in the designed multi-target donepezil–antioxidant
hybrids.
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Scheme 1 Synthesis of 4a–4i and 5a–5n. Reagents and conditions: EDCI, HOBt, DMF-DCM (1 : 1), rt, 12 h. *(1 : 1).
2
Scheme 2 Synthesis of 6 and 7. Reagents and conditions: H , Pd/C, MeOH, rt, 8 h.
Scheme 3 Synthesis of 11a–11c. Reagents and conditions: (i) Ph3P, toluene, 50 °C, 24 h; (ii) xylene, reflux; (iii) NaH, THF, 40 °C, 3 h, rt, overnight.
activities than the series of compounds 4. Compounds with-
out a hydroxyl group (5g–5k) on the phenylpropanoid moiety
showed better inhibition of both ChEs than compounds with
hydroxyl groups (5d–5f). For example, 5k (IC50 = 0.130 μM for
AChE; IC50 = 0.416 μM for BuChE) possessing three methoxy
droxyl groups were between the IC50 values of the other two
series of compounds, and the location of the hydroxyl and
methoxy groups (e.g., 5a: IC50 = 0.651 μM; 5b: IC50 = 1.06 μM)
could significantly influence the inhibitory activity towards
both ChEs. Compound 5c bearing two methoxy groups on
groups was better than 5e (IC50 = 5.17 μM for AChE; IC50
=
2 4 3
the R and R positions and one hydroxyl group on the R po-
1
65.1 μM for BuChE) with two hydroxyl groups. The IC50
sition has better ChE inhibitory activity (IC50 = 0.398 μM for
AChE; IC50 = 0.976 μM for BuChE).
values of compounds 5a–5c bearing both methoxy and hy-
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Table 1 Inhibition of eeAChE, eqBuChE, DPPH and ABTS of the synthesized compounds
a
IC50 (μM)
DPPH assay
Selectivity
index
ABTSc
assay
b
Compounds
eeAChE
eqBuChE
IC50 (μM)
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
7
1
1
1
3
3
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
i
29.3 ± 1.1
22.0 ± 1.6
31.6 ± 1.3
62.1 ± 2.1
70.8 ± 1.0
59.3 ± 2.1
67.7 ± 2.5
7.12 ± 0.55
28.2 ± 1.2
0.651 ± 0.036
1.06 ± 0.05
0.398 ± 0.028
0.874 ± 0.07
5.17 ± 1.04
2.16 ± 0.667
0.543 ± 0.079
0.290 ± 0.031
0.285 ± 0.026
0.383 ± 0.032
0.130 ± 0.017
0.815 ± 0.080
0.521 ± 0.039
0.444 ± 0.034
33.6 ± 1.7
49.2 ± 1.5
17.3 ± 0.8
1.25 ± 0.20
15.0 ± 0.89
145.8 ± 3.0
90.8 ± 1.9
51.4 ± 0.8
9.01 ± 0.87
7.34 ± 0.30
1.22 ± 0.15
2.47 ± 0.22
0.976 ± 0.102
1.24 ± 0.20
165.1 ± 2.8
1.06 ± 0.17
0.390 ± 0.091
0.670 ± 0.063
1.55 ± 0.18
0.244 ± 0.034
0.416 ± 0.061
1.98 ± 0.26
2.77 ± 0.27
3.10 ± 0.43
31.9 ± 0.9
4.40 ± 0.48
1.08 ± 0.16
53.2 ± 0.9
3.10 ± 0.54
N
1.68
0.786
0.038
0.241
2.06
1.53
0.759
1.26
0.260
1.87
2.33
2.20
1.42
31.9
0.491
0.718
2.31
5.43
0.642
3.20
2.43
5.32
6.98
0.949
0.588
0.415
1.99
0.053
—
32.5 ± 0.9
N
N
10.6 ± 0.8
980 ± 20
N
N
N
22.3 ± 1.1
34.1 ± 1.8
764 ± 18
24.9 ± 1.4
936 ± 31
10.7 ± 1.2
977 ± 28
N
N
N
N
N
1.41
0.35
—
1.81
0.10
—
—
—
1.65
1.39
0.27
1.78
2.10
0.09
—
0.34
—
—
—
—
—
j
k
l
m
n
N
N
N
—
55.1 ± 2.1
48.3 ± 2.3
76.8 ± 4.0
88.7 ± 3.8
890 ± 26
n.t.
n.t.
n.t.
30.6 ± 1.6
23.3 ± 1.5
n.t.
0.76
1.28
0.65
0.46
0.07
n.t.
n.t.
n.t.
1.21
1.53
1
7.48 ± 0.96
2.60 ± 0.37
26.3 ± 0.8
58.3 ± 1.2
N
1a
1b
1c
a
b
158.6 ± 1.8
0.035 ± 0.002
N
—
Donepezil
Ferulic acid
Curcumin
Trolox
4.17 ± 0.27
N
N
0.251
4.95
4.43
—
d
N
N
n.t.
e
n.t.
a
b
c
IC50: 50% inhibitory concentration (means ± SD of three experiments). Selectivity index = IC50 (eqBuChE)/IC50 (eeAChE). Data are
d
expressed as (mmol of trolox)/(mmol of tested compound). Inactive at 1000 μM (highest concentration tested); at higher concentrations, the
compounds precipitate. n.t. = not tested.
e
Table 2 Inhibition of human ChE activitiesa
To further evaluate the role of the double bond between
b
IC50 (μM)
the phenyl ring and the amide, compounds 6 and 7 were syn-
thesized (Scheme 2) and evaluated. Notably, 6 and 7
exhibited lower inhibitory activities for both ChEs, suggesting
that the double bond and the conjugation system with the phenyl
ring are essential to induce inhibitory effects for these analogues.
To further explore the effect of conjugated chains on ChE
inhibitory activities and mimic the structure of curcumin, the
other series of compounds (11a–11c) which contain
β-diketone moieties were synthesized (Scheme 3) and evalu-
ated. Obviously, 11a–11c exhibited moderate inhibitory activi-
Selectivity
index
c
Compounds
hAChE
hBuChE
5a
0.729 ± 0.133
1.66 ± 0.29
0.321 ± 0.012
2.20 ± 0.28
1.45 ± 0.18
2.68 ± 0.30
0.411 ± 0.018
0.339 ± 0.032
0.526 ± 0.028
0.690 ± 0.039
0.234 ± 0.015
3.30 ± 0.55
0.362 ± 0.027
0.337 ± 0.032
5.32 ± 0.41
1.12 ± 0.15
3.24 ± 0.50
1.22 ± 0.20
0.784 ± 0.112
145.1 ± 2.6
0.988 ± 0.069
1.41 ± 0.32
0.245 ± 0.048
2.54 ± 0.27
1.44 ± 0.20
0.669 ± 0.101
7.51 ± 0.64
2.07 ± 0.23
4.23 ± 0.48
0.286 ± 0.078
8.28 ± 0.91
1.54
1.95
3.80
0.36
100
0.37
3.43
0.72
4.83
2.09
2.86
2.28
5.72
12.5
0.054
268
5b
5
5
5
c
d
e
5f
5g
5
5
5
h
i
j
ties towards both ChEs (11a, IC50 = 2.60 μM for AChE; IC50
=
5k
5
5
5
7
l
m
n
1
.08 μM for BuChE), suggesting that the β-diketone bond is
not needed to induce inhibition for these analogues. Then, a
selection of the compounds (5a–5n and 7) were evaluated as
inhibitors of human ChEs with an aim to further evaluate
them and the results are listed in Table 2. From the table, it
can be seen that all the compounds are also potent inhibitors
of human ChEs.
Donepezil
0.0308 ± 0.0025
a
AChE from human erythrocytes and BuChE from human serum
were used. IC50: 50% inhibitory concentration (means ± SD of three
experiments). Selectivity index = IC50 (hBuChE)/IC50 (hAChE).
b
c
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2
.3 In vitro antioxidant activities
Lineweaver–Burk plots of 5c (Fig. 2) showed both increasing
slopes and increasing intercepts at increasing inhibitor con-
centration, which suggested that 5c was a mixed-type inhibi-
tor for both enzymes. This pattern indicated that compound
ABTS radical cation scavenging method. The antioxidant
activities of all target compounds were evaluated using an
ABTS (2,2′-azino-bisIJ3-ethylbenzothiazoline-6-sulfonic acid))
radical scavenging assay with a water-soluble vitamin E ana-
5c might be a dual binding site inhibitor of AChE.
3
9
log (trolox) as the reference compound (Table 1). Ferulic
acid and curcumin were also analyzed for comparison. The
antioxidant activities of the compounds were provided in
trolox equivalents, with their relative potency at 25 μM com-
pared with that of trolox. As shown in Table 1, some of these
selected compounds demonstrated outstanding antioxidant
activities. Compounds 4a, 4d, 4i, 5a, 5c, 5e, 6 and 7 had the
ability to scavenge the ABTS radical with 1.41, 1.81, 1.65,
2
.5 Molecular docking study with AChE
In order to further demonstrate the dual-site binding mode
and get an insight into the interaction mechanism of 5c with
AChE, a molecular docking study based on the X-ray crystal
structure of recombinant human acetylcholinesterase in com-
plex with donepezil (hAChE, PDB code 1EVE) was carried out
using the Molecular Operating Environment (MOE) soft-
41,42
ware.
These results shown in Fig. 3 indicated that com-
1
.39, 1.78, 2.10, 0.76 and 7.
pound 5c covered the binding gorge in a satisfactory orienta-
tion and conformation thus generating high inhibitory activity.
The donepezil moiety occupied the CAS interacting by π–π
stacking with Trp84 and Phe330 of 4.37 Å and 4.36 Å, respec-
tively. The phenylpropanoid moiety established a polar contact
with Phe288 and Ser286 in the PAS (3.10 Å and 2.65 Å). After
analyzing the docking results and taking the kinetic study into
consideration, it was confirmed that compound 5c was a dual
binding site inhibitor that could interact simultaneously with
the PAS and CAS of AChE.
DPPH radical scavenging method. DPPH (2,2-diphenyl-1-
picrylhydrazyl) radicals can be used in preliminary screening of
scavenging reactive oxygen species, since these nitrogen radicals
are much more stable and easier to handle than oxygen free rad-
40
icals. The IC50 values of all the compounds in Table 1 indi-
cated that compounds 4b, 4c, 4f, 4g, 4h and 5g–5n without a
phenolic hydroxyl had lower radical scavenging activities. Obvi-
ously, compounds 4e and 5d, which contain one phenolic hy-
droxyl at the R
low radical scavenging activities. Compounds 4a, 4d, 5a and 5e
which bear a phenolic hydroxyl group or methoxy group on R
demonstrated that the group at the ortho position of the pheno-
lic hydroxyl is crucial. Moreover, the IC50 of compound 5c (IC50
4.9 μM) showed that the locations of the phenolic hydroxyl and
3
position of the phenylpropanoid moiety, showed
2
2.6 Neuroprotection study
Motivated by the promising in vitro results of the antioxidant
assay, target compounds 4a–4i, 5a–5n, 6 and 7 were tested
using the hydrogen peroxide (H O ) model on PC12 cells to
=
2
2
2
methoxy groups are non-adjustable. After all these biological
evaluations, 5c was chosen as the most promising compound for
further study because of its strong and balanced inhibition for
both ChEs and antioxidant activity close to that of trolox.
further confirm the antioxidant properties in neural cells.
Toxic free radicals formed from H O results in oxidative
2
2
damage to lipids, proteins and DNA, which finally cause
mitochrondrial dysfunction, calcium imbalance and apopto-
4
3
sis in neuronal cells. PC12 cells were used as a screening
model for studying neurodegenerative diseases and trolox
2
.4 Kinetic study of ChE inhibition
4
4
To further uncover the mechanism of inhibition and the
binding site of target compounds on ChEs, a kinetic study
was selected as the positive control in this test.
Firstly, the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide] assay was carried out to
9
was performed with the most promising compound 5c. The
Fig. 2 Kinetic study on the mechanism of eeChEs. (A) Kinetic study on the mechanism of eeAChE inhibition by compound 5c. Overlaid
Lineweaver–Burk reciprocal plots of AChE initial velocity at increasing substrate concentration (0.05–0.50 mM) in the absence of an inhibitor and
in the presence of 5c are shown. Lines were derived from a weighted least-squares analysis of the data points. (B) Kinetic study on the mechanism
of eqBuChE inhibition by compound 5c. Overlaid Lineweaver–Burk reciprocal plots of BuChE initial velocity at increasing substrate concentration
(
0.05–0.50 mM) in the absence of an inhibitor and in the presence of 5c are shown. Lines were derived from a weighted least-squares analysis of
the data points.
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Fig. 3 (A) 3D docking model of compound 5c with hAChE. Atom colors: yellow – carbon atoms of 5c, gray – carbon atoms of residues of hAChE,
dark blue – nitrogen atoms, red – oxygen atoms. (B) 2D schematic diagram of the docking model of compound 5c with hAChE. The figure was
prepared using the ligand interaction application in MOE. The dashed lines represent the interactions between the protein and the ligand.
3
,45
evaluate the activities of the target compounds at higher con-
centrations without the risk of inducing cytotoxicity in nor-
mal cells. As indicated in Fig. 4, all compounds except 5m
did not show potential cytotoxic effects on PC12 cells at 20
μM after incubation for 24 h.
higher in an AD brain compared to a healthy brain.
Com-
pounds with metal-chelating effects might provide an addi-
tional and therapeutic strategy for the treatment of AD.
To evaluate the chelation ability of compound 5c, a UV–vis
spectroscopy assay was carried out with the wavelength ranging
46
The protective efficacy of the target compounds against
H O at 5 μM is reported in Fig. 5. Some of the target com-
from 200 to 400 nm (Fig. 7). The absorption spectra of 5c
(75 μM) alone or in the presence of CuSO , FeSO , FeCl or ZnCl
2
2
2
4
4
3
pounds significantly showed protective effects against dam-
age induced by H at 5 μM as shown in Fig. 5. On the basis
(150 μM) for 30 min in methanol were recorded. It can be seen
that new optical bands were detected at 272 nm after the addi-
O
2 2
of the screening results above, compounds with good antioxi-
dant activity were selected and tested to further evaluate the
neuroprotective effect at 1, 5 and 10 μM (Fig. 6). Notably,
compounds bearing a phenolic hydroxyl group on the
phenylpropanoid moiety exhibited much higher activities
than compounds without a hydroxyl group, which is consis-
tent with the result of the DPPH test.
tion of CuSO to the solution of compound 5c, which demon-
4
strated the production of the corresponding complex via metal
chelation. The chelating ability of 5c was attributed to the pres-
ence of the dimethoxy group and hydroxyl group on the
phenylpropanoid moiety and amide moiety in the core of the
47,48
compound.
3 4
However, with the addition of FeCl , FeSO and
ZnCl , there was no significant change.
2
2
.7 Metal-chelating properties of 5c
2.8 In vitro blood–brain barrier permeation assay
The destruction of the balance of metal ions in CNS could re-
sult in neurodegenerative disorders. Studies have shown that
the levels of biometals such as Cu , Zn , Fe and Fe are
In AD treatment, the ability of a drug to permeate the blood–
brain barrier (BBB) is a critical property for central nervous
system (CNS) drugs. The parallel artificial membrane
2
+
2+
2+
3+
Fig. 4 Effects of compounds on cell viability in PC12 cells. The cell viability was determined by the MTT assay after 24 h. The viability of untreated
cells is defined as 100%. Data are expressed as the mean ± SD, n = 3. Statistical significance was analyzed by ANOVA: ***p < 0.001.
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Fig. 5 Protective effects of 4a–4i and 5a–5n against H
2
O
2
-induced injury in PC12 cells at 5 μM. PC12 cells were pretreated with the tested
compounds for 4 h. Then, the cells were treated with 100 μM H
untreated cells is defined as 100%. Data are expressed as the mean ± SD, n = 3. Statistical significance was analyzed by ANOVA:
compared to control, *P < 0.05, **P < 0.01, ***P < 0.001 compared to H
2 2
O for 20 h. Cell viability was determined by the MTT assay. The viability of
#
##
P < 0.001
2
2
O .
permeability assay for BBB (PAMPA-BBB), which was de-
scribed by Di et al., was used to assess the BBB penetration
4
9
of the target compounds. Assay validation was performed
by comparing the experimental permeabilities of 9 reference
drugs with their reported values (Table 3), which gave a good
2
e e
linear correlation: P (exp) = 1.0416; P (Bibl.) = 0.8567 (R
=
0
.9443). For blood–brain barrier permeation, we classified
−
6
−1
e
the compounds as follows: compounds with P (10 cm s )
>
4.5 for high BBB permeation (CNS+), compounds with P
e
−
6
−1
(
10 cm s ) < 2.1 for low BBB permeation (CNS−) and com-
−
6
−1
pounds with 4.5 > P
e
(10 cm s ) > 2.1 for uncertain BBB
values of these selected com-
permeation (CNS±). The P
e
pounds are summarized in Table 4. It can be seen that com-
pounds 5a, 5c, 5e and 5k might be able to cross the BBB.
Fig. 6 Protective effects of target compounds with prominent activities
at 5 μM (4d, 5a, 5c, 5e and 7) against H -induced injury in PC12 cells at
2 2
O
1, 5 and 10 μM. PC12 cells were pretreated with the tested compounds for
4
2 2
h. Then, the cells were treated with 100 μM H O for 20 h. Cell viability
was determined using the MTT assay. The viability of untreated cells is
−
6
−1
defined as 100%. Data are expressed as the mean ± SD, n = 3. Statistical
e
Table 3 Permeability (P × 10 cm s ) in the PAMPA-BBB assay for 9
###
significance was analyzed by ANOVA:
P < 0.001 compared to control,
commercial drugs used in the experiment validation
*
P < 0.05, **P < 0.01, ***P < 0.001 compared to H O .
2 2
a
b
Commercial drugs
Bibl.
PBS/EtOH (70 : 30)
Dopamine
Hydrocortisone
Piroxicam
Corticosterone
Clonidine
Progesterone
β-Estradiol
Verapamil
0.2
1.9
2.5
5.1
5.3
9.3
12
0.24
1.89
1.39
4.26
4.56
5.91
12.02
18.6
15.6
16
17
Testosterone
a
b
Taken from ref. 49. Data are the mean ± SD of three independent
experiments.
−6
−1
Table 4 Permeability (P
e
× 10 cm s ) in the PAMPA-BBB assay for
donepezil–ferulic acid hybrids and their predictive penetration in the CNS
−
6
−1
Compound
P
e
× 10 cm s
Prediction
5
a
5.16 ± 0.32
7.68 ± 0.59
5.38 ± 0.35
7.43 ± 0.60
CNS+
CNS+
CNS+
CNS+
Fig.
presence of CuSO
150 μM) in MeOH.
7
UV absorbance spectrum of 5c (75 μM) alone or in the
5c
5e
5k
4
(150 μM), ZnCl (150 μM), FeSO (150 μM) or FeCl
2
4
3
(
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3
. Conclusion
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In summary, this study involved the design, synthesis and
biological evaluation of a novel series of MTDLs against AD
by fusing the pharmacophores of ferulic acid and donepezil.
The biological screening results indicated that most of the
derivatives showed potent ChE inhibitory activity. Specifically,
the target compounds displayed excellent potency in scaveng-
ing reactive free radicals. The optimal candidate compound,
7
8
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5
c, exhibited moderate ChE inhibitory activities (0.398 μM
for eeAChE, 0.321 μM for hAChE, 0.976 μM for eqBuChE and
.22 μM for hBuChE), good biometal-chelating ability and
2
015, 93, 42–50.
1
1
1
0 A. Cavalli, M. L. Bolognesi, A. Minarini, M. Rosini, V.
antioxidant activity (1.78 trolox equivalents). Kinetic and mo-
lecular modeling studies indicated that 5c was a mixed-type
inhibitor, binding simultaneously to the active and periph-
eral sites of AChE. Above all, due to improvement of the ac-
tivity, and BBB permeability, 5c could thus be considered as
a potential multifunctional neuroprotective agent and serve
as new a lead candidate for the treatment of AD.
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1
1
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Abbreviations
AD
MTDL Multi-target-directed ligand
FA Ferulic acid
AChE Acetylcholinesterase
BuChE Butyrylcholinesterase
Alzheimer's disease
CNS
BBB
MTT
Central nervous system
Blood–brain barrier
Methyl thiazolyl tetrazolium
16 M. Harel, L. K. Sonoda, I. Silman, J. L. Sussman and T. L.
Rosenberry, J. Am. Chem. Soc., 2008, 130, 7856–7861.
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19 S. L. Rogers, R. S. Doody, R. C. Mohs, L. T. Friedhoff and G.
Donepezil Study, Arch. Intern. Med., 1998, 158, 1021–1031.
DPPH 2,2-Diphenyl-1-picrylhydrazyl
ABTS 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
Acknowledgements
We gratefully acknowledge the financial support of the Pro-
gram 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) and the National Natural Science Founda-
tion of China (81573313).
2
2
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