Multifunctional 3-Schiff base-4-hydroxycoumarin derivatives with monoamine oxidase inhibition, anti-β-amyloid aggregation, metal chelation, antioxidant and neuroprotection properties against Alzheimer's disease

Article abstract of DOI:10.1039/c5ra13594j

A series of 3-Schiff base-4-hydroxycoumarin derivatives (1-20) have been designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease. In vitro studies indicated that most of the derivatives exhibited significant abilities to inhibit monoamine oxidase (MAO), self-induced and Cu²⁺-induced β-amyloid (Aβ_1-42) aggregati006Fn, as well acting as potential biometal chelators and antioxidants. Moreover, these derivatives were capable of decreasing reactive oxygen species (ROS) and showed good neuroprotective effects in PC12 cells and could penetrate the central nervous system (CNS). In particular, compound 4 exhibited high potency to monoamine oxidase (IC₅₀, 0.673 μM for hMAO-A, 0.711 μM for hMAO-B), good antioxidant activity (1.34 trolox equivalents by ABTS method, 45.8 μM of IC₅₀ by DPPH method), and it also displayed a significant ability to inhibit self-induced and Cu²⁺-induced Aβ_1-42 aggregation (60.1%, 20 μM and 45.7%, 50 μM). Taken together, these results suggested that compound 4 might be a promising lead compound with balanced properties for AD treatment.

Full text of DOI:10.1039/c5ra13594j

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PAPER
Multifunctional 3-Schiff base-4-hydroxycoumarin
derivatives with monoamine oxidase inhibition,
anti-b-amyloid aggregation, metal chelation,
antioxidant and neuroprotection properties against
Alzheimer's disease†
Cite this: RSC Adv., 2015, 5, 70395
*
Zhi-Min Wang, Sai-Sai Xie, Xue-Mei Li, Jia-Jia Wu, Xiao-Bing Wang
*
and Ling-Yi Kong
A series of 3-Schiff base-4-hydroxycoumarin derivatives (1–20) have been designed, synthesized and
evaluated as multifunctional agents against Alzheimer's disease. In vitro studies indicated that most of the
derivatives exhibited significant abilities to inhibit monoamine oxidase (MAO), self-induced and
Cu2+-induced b-amyloid (Ab1–42) aggregati006Fn, as well acting as potential biometal chelators and
antioxidants. Moreover, these derivatives were capable of decreasing reactive oxygen species (ROS) and
showed good neuroprotective effects in PC12 cells and could penetrate the central nervous system (CNS).
In particular, compound 4 exhibited high potency to monoamine oxidase (IC50, 0.673 mM for hMAO-A,
0.711 mM for hMAO-B), good antioxidant activity (1.34 trolox equivalents by ABTS method, 45.8 mM of IC50
by DPPH method), and it also displayed a significant ability to inhibit self-induced and Cu2+-induced
Ab1–42 aggregation (60.1%, 20 mM and 45.7%, 50 mM). Taken together, these results suggested that
compound 4 might be a promising lead compound with balanced properties for AD treatment.
Received 11th July 2015
Accepted 12th August 2015
DOI: 10.1039/c5ra13594j
www.rsc.org/advances
pathogenesis and progression. To date, a wide variety of anti-
oxidants have been examined as neuroprotectants.11 As an
1. Introduction
important factor involved in oxidative stress, monoamine
oxidases (MAOs) have attracted more and more attention in
recent years. MAOs are avin adenine dinucleotide (FAD) con-
taining enzymes, which exist as MAO-A and MAO-B, two
different isoforms.12 Selective inhibitors of MAO-A have been
shown to be effective antidepressants, whereas MAO-B inhibi-
tors are useful in several neurodegenerative disorders.13 It is
well known that MAO-B activity is increased in association with
gliosis, which can result in higher hydrogen peroxide (H2O2)
level and oxidative free radicals which are a possible source of
oxidative stress for vulnerable neurons affected by AD.14 At the
same time, AD patients commonly present depressive symptom,
which suggests that the dual inhibition of MAO-A and MAO-B
could be benecial to AD treatment.15
In addition, the “amyloid cascade hypothesis” of AD states
that the Ab peptide is generated via cleavage of the trans-
membrane amyloid precursor protein (APP) by b- and g-secre-
tases.16,17 Generated Ab peptides can aggregate into oligomers,
protobrils and brils that adopt the well-integrated b-sheet
structure. The progressive accumulation of Ab is accompanied
by oxidative stress and inammation, which leads to neuro-
degeneration.18 Therefore, prevention of Ab aggregation in the
brain is currently being considered as a potential target for
AD therapy. Increasing evidence supports metal ions
Alzheimer's disease (AD) is a fatal neurodegenerative disorder,
which is characterized by memory loss, decline in language
skills and other cognitive impairments.1,2 To date there is no
effective approach for the treatment of AD and its diagnosis
with high accuracy demands a detailed examination of the
brain.3 The main pathological changes in the AD brain are the
abnormal formation of b-amyloid (Ab) plaques and neuro-
brillary tangles (NFTs).4 Although the exact etiology of AD is not
well-known, there are several factors including b-amyloid (Ab)
deposits,5,6 dyshomeostasis of biometals,7 oxidative stress,8
s-protein hyperphosphorylation,9 inammation and decit of
acetylcholine (ACh),10 which seem to play important roles in this
disease. Therefore, a more appropriate approach to addressing
the multifactorial nature of AD may be the development of
multifunctional ligands (ML).
Among the multiple factors inducing AD, oxidative stress
damage is considered to be a major determinant of AD
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; xbwang@cpu.edu.cn;
Fax: +86-25-83271405; Tel: +86-25-83271405
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra13594j
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dyshomeostasis and miscompartmentalization which are
thought to play a signicant part in Ab production/aggregation
and neurotoxicity. Ab shows high affinity for metal ions such as
Cu2+ and Zn2+ which are able to accelerate the formation of Ab
aggregates and neurobrillary tangles.19,20 Several ndings have
indicated that two avenues for metal–Ab species are linked to
neurotoxicity: the facilitation of Ab aggregation and the induc-
tion of oxidative stress through reactive oxygen species (ROS)
generation, leading to neuronal cell death and cognitive
impairment. The rst neurotoxic effects that could be induced
by metal–Ab species may be related to the disruption of
membranes leading to apoptosis and/or aggregate accumula-
tion at and around the synapse resulting in weakened cell
signaling and neuronal death.21 The second avenue of neuro-
toxicity is associated with oxidative stress. When metal ions are
surrounded by Ab species, ROS such as hydrogen peroxide and
hydroxyl radicals can be generated. Cu(I)–Ab oligomers have
been suggested to contain a highly O2 reactive Cu(I) center,
which may also assist in the ROS formation.22,23 Overall, self-
and metal-induced Ab aggregation could be attributed to a high
degree of oxidative stress leading to neurotoxicity, neuronal
death and ultimately, cognitive impairment. Thus, the modu-
lation of metal–Ab interaction and the appropriate protection of
neuronal cells from oxidative damage becomes a promising
method to inhibit the Ab aggregation and thereby reduce the
neurotoxicity of Ab deposits.18,24
Fig.
1
Design strategy of the multifunctional 3-Schiff base-4-
hydroxycoumarin derivatives.
In this work, we described the design, synthesis and pharma-
cological evaluation of a series of 3-Schiff base-4-hydroxycoumarin
derivatives as multifunctional anti-AD reagents. The pharmaco-
logical evaluations of these compounds include MAOs inhibition,
self-induced and Cu2+-induced Ab aggregation, metal chelation,
antioxidant, ROS induced by Cu2+–Ab, neuroprotection and the
blood–brain barrier permeation. Molecular docking studies were
also performed on MAO-A, MAO-B and Ab1–42 to illuminate the
binding modes of the highlighted compounds.
Coumarins and their natural and synthetic derivatives
display a variety of biological properties including anti-
inammatory, enzyme-inhibitory and antioxidant properties
due to their structural diversity.25–27 It was shown that
hydroxycoumarins could counteract Ab toxicity due to its
antioxidant properties in cellular models, and could also
inhibit Ab aggregation and MAOs in vitro.28–32 (E)-N1,N1-Di-
methyl-N4-(pyridin-2-ylmethylene)benzene-1,4-diamine (L1),
can chelate metal ions, modulate metal-induced Ab aggrega-
tion and prevent ROS production, leading to reduction of
metal–Ab neurotoxicity in living cells.33 The metal chelator
clioquinol (CQ) has moved into clinical trials and showed
improved cognition. Long term usage of CQ is oen accom-
panied by subacute myelo-optic neuropathy.34 Although these
metal chelators have adverse effects, some studies using these
compounds showed the involvement of metal ions in AD
pathogenesis.35 Previously, our group has reported different
kinds of multifunctional agents against AD.36–40 To develop a
chemical molecule capable of targeting and modulating the
reactivity of multiple AD pathological factors in biological
systems, we designed a series of novel multifunctional ligands
with the potential properties for inhibition of MAOs, interac-
tion with Ab, metal chelation, control of ROS generation and
antioxidant activity (Fig. 1). For the sake of Ab interaction and
2. Result and discussion
2.1. Chemistry
The synthetic route for 3-Schiff base-4-hydroxycoumarin deriv-
atives 1–20 was shown in Scheme 1. Commercially available
4-hydroxy-3-nitrocoumarin (1a) was dissolved in ethanol and a
catalytic amount of Pd/C was added to the mixture. The solution
was stirred, at room temperature, under H2 atmosphere, for
5 h.41 Aer the completion of the reaction, the mixture was
ltered to eliminate the catalyst. The obtained crude product
was then puried to give the desired intermediate 1b. Then a
mixture of 1b was reacted with different substituted aromatic
aldehydes in ethanol for 6 h at room temperature and the
solvent was removed under reduced pressure. The resulting
crude compounds were washed with cold water and recrystal-
lized by using appropriate solvents to afford the target products
1–20. Structures of all synthesized compounds were character-
ized by comparison with 1H and 13C nuclear magnetic reso-
nance (NMR) spectroscopy data reported in the literature.
2.2. Pharmacological evaluation
2.2.1. Monoamine oxidase inhibitory activity. MAO is an
metal chelation, these derivatives were constructed by important target for the treatment of multifactorial diseases,
combining 4-hydroxycoumarin with L1, a molecule previously which suggested that dual inhibition of hMAO-A and hMAO-B
reported to target and regulate metal–Ab and CQ. could be benecial to AD therapy.15 Compounds 1–20 were
For enhanced metal binding ability and antioxidant evaluated the inhibiting ability to human MAOs. It could be
activity, substituents (i.e., phenolic groups, Fig. 1) known to seen from Table 1 that all the tested compounds showed
have metal coordination and antioxidant capability were moderate to good inhibitory activities towards hMAO-A and
integrated into ML.
hMAO-B with IC50 values ranging from sub-micromolar to
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Scheme 1 Synthesis of the designed compounds 1–20.
nanomolar. Some of them exhibited inhibition of hMAOs equal
or better than both the precursors (1a, 1b) and the reference
compounds (pargyline, iproniazid). Among compounds 1–17, 4
(IC50, 0.673 mM for hMAO-A, 0.711 mM for hMAO-B), 14 (IC50,
4.97 mM for hMAO-A, 0.851 mM for hMAO-B) and 17 (IC50,
3.78 mM for hMAO-A, 1.32 mM for hMAO-B) exhibited strong and
balanced hMAOs inhibitory activities. In order to further
improve the activity of the designed compounds against hMAOs,
we replaced the phenyl ring in Schiff base moiety with different
naphthalene ring (18), pyridine ring (19), and quinoline ring
(20). However, there was no clear trend for hMAO-B inhibitory
activities, but a slightly decrease in hMAO-A inhibition.
Table 1 Inhibition of human MAO-A and MAO-B activities of the
synthesized compounds
IC50a (mM)
Compounds
hMAO-A
hMAO-B
Selectivity indexb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
21.7 ꢀ 2.3
4.42 ꢀ 0.43
28.4 ꢀ 1.64
0.673 ꢀ 0.011
4.68 ꢀ 0.66
6.81 ꢀ 0.36
9.43 ꢀ 1.09
9.17 ꢀ 0.83
6.24 ꢀ 0.67
8.23 ꢀ 1.21
9.56 ꢀ 0.44
11.2 ꢀ 0.7
9.81 ꢀ 0.51
4.97 ꢀ 0.41
8.65 ꢀ 0.28
8.31 ꢀ 0.15
3.78 ꢀ 0.62
8.94 ꢀ 0.58
12.3 ꢀ 0.9
31.9 ꢀ 1.7
Nc
2.36 ꢀ 0.07
2.92 ꢀ 0.11
5.51 ꢀ 0.24
0.711 ꢀ 0.013
17.4 ꢀ 1.2
11.2 ꢀ 0.8
4.25 ꢀ 0.63
1.61 ꢀ 0.15
3.18 ꢀ 0.26
3.94 ꢀ 0.27
3.82 ꢀ 0.19
13.7 ꢀ 1.2
18.7 ꢀ 0.8
0.851 ꢀ 0.047
1.79 ꢀ 0.09
3.28 ꢀ 0.14
1.32 ꢀ 0.18
2.41 ꢀ 1.13
4.96 ꢀ 0.37
2.84 ꢀ 0.13
N
9.20
1.51
5.15
0.95
0.27
0.61
2.22
5.70
1.96
2.09
2.50
0.81
0.52
5.84
4.83
2.53
2.86
3.71
2.48
11.2
—
17
18
19
20
1a
1b
5.78 ꢀ 0.33
4.27 ꢀ 0.06
0.214 ꢀ 0.036
8.54 ꢀ 0.64
1.35
—
0.84
Pargyline
Iproniazid
—
7.14 ꢀ 0.38
Fig. 2 Reversibility studies of hMAO-B inhibition by compounds 4, 14
and 17. Compounds 4, 14, 17 and pargyline were preincubated 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 4, 14, 17 and pargyline equal to IC50, the
inhibitory rates were recorded.
a
IC50
:
50% inhibitory concentration (means
ꢀ
SEM of three
b
experiments). Selectivity index
¼
IC50 (hMAO-A)/IC50 (hMAO-B).
c
Inactive at 100 mM (highest concentration tested), at higher
concentrations the compounds precipitate.
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assay was examined.42 Compounds 4, 14 and 17 were selected as
representative inhibitors for their stronger hMAO-B inhibitory
activity. Pargyline, a known irreversible hMAO-B inhibitor, was
used as a reference compound.43 Compounds 4, 14, 17 and
pargyline were pre-incubated with hMAO-B over different time
periods (0, 15, 30, 60 min) at the concentration of about two-fold
IC50. Data shown in Fig. 2 indicated that 4, 14 and 17 were
reversible hMAO-B inhibitors as evidenced by the time-
dependent decrease of their inhibitory activity. In contrast,
Fig. 3 Kinetic study on the mechanism of hMAO-B inhibition by 4. aer treatment of hMAO-B with pargyline, the enzyme inhibi-
Overlaid Lineweaver–Burk reciprocal plots of hMAO-B initial velocity
at increasing p-tyramine concentration (0.05–1 mM) in the absence of
inhibitor and in the presence of 4 are shown. Lines were derived from a
weighted least-squares analysis of the data points.
tory activity was increased.
Subsequently, compound 4 was also used to further inves-
tigate the mode of hMAO-B inhibition. The type of hMAO-B
inhibition was determined by Michaelis–Menten kinetic
experiments.42 The catalytic rates were measured at ve
different p-tyramine concentrations (0.1–1 mM), and each plot
was constructed at four different concentrations of 4 (0, 0.5, 1.0
and 2.0 mM). The overlaid reciprocal Lineweaver–Burk plots
2.2.2. Reversibility and kinetic studies of hMAO-B inhibi-
tion. To evaluate whether these compounds are reversible or
irreversible hMAO-B inhibitors, the time-dependent inhibition
Fig. 4 (A) 3D and (B) 2D docking models of compound 4 with hMAO-A generated with MOE. (C) 3D and (D) 2D docking models of compound 4
with hMAO-B generated with MOE. Atom colors: yellow – carbon atoms of 4, gray – carbon atoms of residues of hMAO-B, dark blue – nitrogen
atoms, red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand.
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2.2.4. Inhibition of self-induced and Cu2+-induced Ab1–42
aggregation. All compounds were tested for their ability to
inhibit self-induced Ab1–42 aggregation by using a thioavin-T
(ThT) based uorometric assay.47 Curcumin (Cur) and resvera-
trol (Res) were used as reference compounds and the results are
summarized in Table 2. From the results, it could be seen that
these compounds exhibited moderate to good potencies (20.2%
to 82.3% at 20 mM) compared to those of resveratrol (67.3% at 20
mM) and curcumin (50.2% at 20 mM). Markedly, 5, 12 and 15
were the most effective compounds, with Ab1–42 aggregation
inhibition potency of 82.3%, 82.0% and 80.2% at 20 mM.
According to the inhibition values of compounds 1–17, different
kinds of substituents did not seem to inuence dramatically to
the Ab1–42 self-aggregation inhibitory activity. From the inhibi-
tion values of compounds 18–20, it appeared that introduction
of large moiety (naphthalene ring and quinoline ring) into
4-hydroxycoumarin seemed to be unfavorable to the inhibition
of self-induced Ab1–42 aggregation.
To investigate the ability of these derivatives to inhibit
Cu2+-induced Ab1–42 aggregation, we selected compounds 2, 4,
5, 12, 15, 17, 19 and 20 for studying by the ThT-binding assay.47
Resveratrol and clioquinol were used as reference compounds.
It could be seen from Fig. 5, the uorescence of Ab1–42 treated
with Cu2+ is 136% that of Ab1–42 alone, which indicated that
Cu2+ accelerates Ab1–42 aggregation. By contrast, the uores-
cence of Ab1–42 treated with Cu2+ and the tested compounds
decreased dramatically (CQ, 56.1% inhibition; Res, 51.7%
(Fig. 3) showed that the plots for different concentrations of 4
were linear and intersected at the x-axis. This pattern indicated
that 4 was a noncompetitive hMAO-B inhibitor, and these results
further
proved
that
6-substituted
3-Schiff
base-4-
hydroxycoumarin derivatives were reversible hMAO-B inhibitors.
2.2.3. Docking studies of compound 4 with hMAO-A and
hMAO-B. The binding modes of compound 4 with hMAO-A and
hMAO-B were investigated based on the X-ray crystallographic
structures (hMAO-A, PDB 2Z5X; hMAO-B, PDB 2V61).44,45 The
relevant difference between the two isoforms is the replacement
of the pair Phe 208/Ile 335 in MAO-A by Ile 199/Tyr 326 in
MAO-B, since it alters the shape of the substrate cavity, leading
to the distinction between “substrate” and “entrance” sites in
MAO-B.46 As shown in Fig. 4A and B, the phenyl ring of
coumarin established a cation-p interaction with Tyr 407 and
Tyr 444. Besides, 2- and 4-position two hydroxyls in benzalde-
hyde moiety formed two hydrogen bonds with residues Phe
208 and Thr 336. It can be seen from the Fig. 4C and D, the
coumarin moiety of 4 was occupied the entrance cavity away
from the FAD cofactor and interacted with lipophilic residues
Pro 102, Pro 104, Trp 119 and Ile 316. Additionally, 4-hydroxy in
the coumarin interacted with Ile 199 and Tyr 326 via two
hydrogen bonds. Meanwhile, another two hydrogen-binding
interactions were seen between 2-position hydroxyl in benzal-
dehyde with residues Ile 198 and Tyr 435. As disclosed above,
the interactions may explain the good hMAO-A and hMAO-B
inhibitory activity of compound 4.
Table 2 Radical scavenging capacity and inhibition of self-induced Ab1–42 aggregation activities of the synthesized compounds
Compounds
ABTS assaya (trolox equivalent)
DPPH scavenging activities IC50b (mM)
Self-induced Ab1–42 aggregation inhibitionc (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.12
1.27
1.07
1.34
1.36
0.82
0.29
0.42
1.19
0.34
0.32
0.80
0.37
0.30
0.64
0.41
1.57
0.39
0.28
0.95
0.31
0.67
n.t.e
1.46
536 ꢀ 44
48.5 ꢀ 2.7
71.3 ꢀ 4.6
45.8 ꢀ 1.2
55.3 ꢀ 6.6
84.3 ꢀ 5.5
354 ꢀ 27
59.4 ꢀ 3.1
78.5 ꢀ 2.1
197 ꢀ 16
87.9 ꢀ 3.7
58.3 ꢀ 1.3
Nd
54.2 ꢀ 4.5
66.0 ꢀ 2.7
76.5 ꢀ 7.6
60.1 ꢀ 4.0
82.3 ꢀ 6.6
47.7 ꢀ 5.5
61.2 ꢀ 3.4
33.2 ꢀ 2.0
57.3 ꢀ 1.1
46.1 ꢀ 4.8
54.3 ꢀ 7.6
82.0 ꢀ 3.3
57.3 ꢀ 2.1
51.1 ꢀ 7.9
80.2 ꢀ 9.1
53.4 ꢀ 7.7
73.5 ꢀ 6.1
41.5 ꢀ 3.8
58.2 ꢀ 4.1
20.2 ꢀ 1.3
47.3 ꢀ 3.2
56.8 ꢀ 6.7
67.3 ꢀ 3.4
50.2 ꢀ 5.9
N
73.1 ꢀ 2.8
268 ꢀ 23
38.6 ꢀ 2.0
122 ꢀ 11
101 ꢀ 4
17
18
19
20
1a
1b
70.8 ꢀ 3.7
752 ꢀ 45
84.6 ꢀ 5.8
137 ꢀ 4
Resveratrol
Curcumin
39.6 ꢀ 2.1
a
b
Data are expressed as (mmol trolox)/(mmol tested compound). IC50: 50% inhibitory concentration (means ꢀ SEM of three experiments).
Inhibition of self-induced Ab1–42 aggregation, the measurements were carried out in the presence of 20 mM compounds (means ꢀ SEM of
c
three experiments). d Inactive at 1000 mM (highest concentration tested), at higher concentrations the compounds precipitate. e n.t. ¼ not tested.
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Fig. 5 Inhibition of Cu2+-induced Ab1–42 aggregation by compounds 2, 4, 5, 12, 17, 19 and 20 comparing with those of resveratrol (Res) and
clioquinol (CQ) ([Ab1–42 ¼ 25 mM, [2, 4, 5, 12, 17, 19 and 20] ¼ 50 mM, [Res] ¼ 50 mM, [CQ] ¼ 50 mM, [Cu2+] ¼ 25 mM, 37 ꢁC, 24 h). Values are
reported as the mean ꢀ SD of three independent experiments. **p < 0.01, ***p < 0.001.
inhibition; 4, 40.3% inhibition; 17, 49.3% inhibition). These ethylbenzthiazoline-6-sulfonicacid))
radical
scavenging
results suggested that the tested compounds could inhibit method and DPPH (diphenyl-1-picrylhydrazyl) radical scav-
Cu2+-induced Ab1–42 aggregation effectively.
enging method.51,52
2.2.5. Docking study of compound 4 with Ab1–42. To
2.2.7.1. ABTS radical cation scavenging method. All
compounds were tested for their antioxidant activities by using
further study the interaction mode of compound 4 for Ab1–42
,
molecular docking study was performed using soware package the ABTS method.51 Trolox, a water-soluble vitamin E analog,
MOE 2008.10. The X-ray crystal structure of the protein Ab1–42 was used as a reference standard. Curcumin, 1a and 1b were
structure (PDB 1IYT) used in the docking study was obtained also determined for comparison. Their antioxidant activities
from the Protein Data Bank.48 As shown in Fig. 6, the benzal- were provided as a trolox equivalent, with their relative potency
dehyde ring of compound 4 interacted with the His 6 via p–p at 25 mM compared with trolox. As shown in Table 2,
stacking interaction. A hydrogen bond interaction was also compounds 2, 3, 4, 5, 9 and 17 had the ability to scavenge the
found between 4-hydroxy group of compound 4 and His 6. ABTS radical with 1.27, 1.07, 1.34, 1.36, 1.19, 1.57 trolox
These results indicated that the p–p stacking and hydrogen equivalents respectively. All of these selected compounds
bond interactions played important roles in the stability of the demonstrated moderate to good antioxidant activities ranging
4/Ab1–42 complex.
from 0.12- to 1.57-fold of the trolox value. The best activity was
2.2.6. Cu2+ chelating effect. The chelating effect of observed for 17 with a nitro group in 5-position of the benzal-
compound 4 for Cu2+ in methanol was studied by UV-vis spec- dehyde ring. Interestingly, ortho- or para-position hydroxyl
trometry with wavelength ranging from 200 to 500 nm.49,50 In groups on the ring of benzaldehyde were also favorable to the
Fig. 7a, UV-vis spectra of compound 4 at increasing Cu2+ ABTS radicals scavenging activity.
concentrations were shown. The increase in absorbance, which
2.2.7.2. DPPH radical scavenging method. DPPH radicals can
could be better estimated by an inspection of the differential be used in preliminary screening of compounds capable of
spectra (Fig. 7b), indicated that there was an interaction scavenging reactive oxygen species, since these nitrogen radi-
between Cu2+ and compound 4. These observations displayed cals are much more stable and easier to handle than oxygen free
that the target compounds could effectively chelate Cu2+, and
thereby could serve as metal chelators for AD therapy.
The stoichiometry of the 4–Cu2+ complex was investigated
using the molar ratio method, by mixing the xed amount metal
ion with increasing ligand. The results showed that absorbance
linearly increased initially and then plateaued (Fig. 8). The two
straight lines intersected at a mole fraction of 0.8, revealing
about a 1 : 1 stoichiometry for complex 4–Cu2+.
2.2.7. In vitro antioxidant activity assays. ROS has been
associated with AD and other neurodegenerative disorders.
Thus, the antioxidant might be an important therapeutic
Fig. 6 Docking study of compound 4 with Ab1–42 generated with
MOE. The interactions between the ligand and residue His 6 are
indicated by the dashed lines.
strategy for AD treatment.22 The antioxidant activities of these
derivatives were examined by using the ABTS (2,20-azino-bis(3-
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Fig. 7 (a) UV-vis (200–500 nm) absorption spectra of compound 4 (25 mM) in methanol after addition of ascending amounts of CuCl2 (5–60 mM).
(b) The differential spectra due to 4–Cu2+ complex formation obtained by numerical subtraction from the above spectra of those of Cu2+ and 4
at the corresponding concentrations.
radicals.52 For comparison purpose, resveratrol and curcumin 14, 18, 19 and 1a had low radical scavenging activities. Among
were used as reference compounds. The IC50 values of all tested them, compounds 4 (IC50, 45.8 mM) and 17 (IC50, 38.6 mM)
compounds are summarized in Table 2. From the table, it could exhibited the most potent scavenging activities. These results
be seen that most of the compounds had much better scav- indicated that the number and position of the OH or other
enging activities than resveratrol, while compounds 1, 7, 10, 13, groups were critical in determination of scavenging activity. In
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However, methoxyl- and methyl-substituted compounds (6–11)
showed slightly reduce the ability of scavenging the DPPH free
radical. Replacement of the hydroxyl groups on benzaldehyde
ring by halogen atoms were also found to reduced activities (13,
14 and 16). This observation indicated that the halogen groups
were negative for the activity compared to the hydroxyl group.
2.2.8. Inhibition of Cu2+–Ab1–42 H2O2 production. Binding
of redox active metal ions such as Cu2+ to Ab1–42 species is
known to be involved in generation of ROS such as H2O2 and
subsequent facilitation of Ab1–42 aggregation and neurotox-
icity.23 The effect of compound 4 on H2O2 production by
Cu2+–Ab1–42 species was examined using the HRP/Amplex Red
assay.53 Under reducing conditions, the Cu2+–Ab1–42 reacted
with O2 to generate H2O2. Addition of compound 4 to such a
solution reduced the production of H2O2 by about 81% for the
Cu2+–Ab1–42 species. By comparison, the strong chelator ethyl-
enediaminetetraacetic acid (EDTA) showed an even more
pronounced effect, almost complete eliminating (>89%), and
CQ showed the reducing effect by about 74%. These results
showed that compound 4 was able to chelate Cu2+ as well as
regulated ROS production, which showed promise for further
applications (Fig. 9).
Fig. 8 Determination of the stoichiometry of complex 4–Cu2+ by
molar ratio method.
2.2.9. Cell viability and neuroprotective effect against
H2O2-induced toxicity. To gain insight into the therapeutic
potential of these derivatives, cell viability and neuroprotective
capacity against oxidative stress were assayed using the PC12
cells.54 Compounds 2, 4, 5, 17 and 18 were selected as
representative compounds of different types and potential
neuroprotective activities. First, the colorimetric MTT [3-(4,5-
Fig. 9 Production of H2O2 from reactions of Ab1–42, Cu2+ and
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide]
assay was performed to examine the potential cytotoxic effects
of these compounds. As indicated in Fig. 10, compounds 2, 4, 5,
17 and 18 did not show signicant effect on cell viability at
1–50 mM aer incubation for 24 h.
compounds upon addition of ascorbate, as determined by
a
HRP/Amplex-Red assay. [Ab1–42] ¼ 200 nM, [Cu2+] ¼ 400 nM,
[chelator] ¼ 800 nM, [ascorbate] ¼ 10 mM, [Amplex Red] ¼ 50 nM,
[HRP] ¼ 0.1 U per mL, and lex/em ¼ 530/590 nm. Values are reported as
the mean ꢀ SD of three independent experiments.
Compounds 2, 4, 5 and 17 were also tested for their capacity
to protect PC12 cells against oxidative stress-associated death
induced by H2O2.54,55 Trolox was used as the reference
compound. In this assay, addition of 200 mM H2O2 to the growth
medium reduced cell viability to 56.8% compared to control.
The tested compounds were added to the media at different
concentrations immediately prior to the H2O2 insult. As can be
seen in Fig. 11, all of the compounds exhibited neuroprotective
effects at concentrations ranging from 1 to 20 mM. Compounds
2, 4, 5 and 17 had showed strong protective capability almost
the same as trolox at the concentration of 10 mM and much
better than trolox at 5 mM. Phase-contrast micrographs showed
H2O2-induced neurotoxicity and neuroprotection in PC12 cells
for the tested compounds (Fig. 12). These observations further
indicated that these novel derivatives had the potential to be
efficient multifunctional agents, including antioxidant activity,
for the treatment of AD.
Fig. 10 Effects of compounds on cell viability in PC12 cells. The cell
viability was determined by the MTT assay after 24 h of incubation with
various concentrations of 2, 4, 5, 17 and 18. The results were expressed
as a percentage of control cells. Values are reported as the mean ꢀ SD
of three independent experiments.
2.2.10. In vitro blood–brain barrier permeation assay. In
the suitable central nervous system (CNS) drugs for AD treat-
ment, blood–brain barrier (BBB) penetration is of particular
importance. So BBB permeability properties of CNS drug
candidates should be determined as early as possible in the
discovery process. Filter for prediction of BBB penetration
compounds 1–5 especially the combination of OH group
substitutions at the ortho- or para-position of the benzaldehyde
ring were correlated with the high scavenging properties.
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Fig. 11 Neuroprotection against H2O2 toxicity. Compounds 2, 4, 5 and 17 were tested for neuroprotective activity against H2O2 toxicity in PC12
cells. Trolox was used as the reference compound. Results are expressed as percent viability compared to cells not treated with H2O2. Data
represent the mean ꢀ SD of three observations. ***p (###p) < 0.001, *p < 0.05, **p < 0.01.
Fig. 12 Phase-contrast micrographs showing H2O2-induced neurotoxicity and neuroprotection of compounds 4 and 17 in PC12 cells. (A) Cells
without compound and H2O2 treatment showed healthy shapes. (B–D) Compounds trolox, 4 and 17 (20 mM) treatment did not induce
neurotoxicity. (E) H2O2 alone (200 mM) induced neurotoxicity. (D–F) Compounds trolox, 4 and 17 (5 mM) were given for 24 h with H2O2 (200 mM)
at 37 ꢁC and co-treatment showed neuroprotection (original magnification, 200).
involves calculation of log BB by means of the equation of the shown higher Pe values than 4.5 were able to cross the
restrictive terms of Lipinski's rules shown in the footnote of blood–brain barrier, but compound 4 was uncertain for BBB
Table S1 (ESI†).56 To determine whether the present permeation (CNSꢀ).
compounds could cross BBB, we also used a parallel arti-
cial membrane permeation assay for BBB (PAMPA-BBB),
Table 3 Permeability (Pe ꢃ 10ꢂ6 cm sꢂ1) in the PAMPA-BBB assay for
which was described by Di et al.57,58 Assay validation was
performed by comparing experimental permeability of 9
commercial drugs with reported values (Table 3). A plot of
experimental data versus bibliographic values gave a good
11 commercial drugs, used in the experiment validation
Commercial drugs
Bibliographya
Experiment b
linear correlation, Pe(exp) ¼ 1.2084Pe(bibl.) ꢂ 0.3055 (R2
¼
Testosterone
Verapamil
b-Estradiol
Clonidine
Corticosterone
Piroxicam
Hydrocortisone
Lomeoxacin
Ooxacin
17
16
12
5.3
5.1
2.5
1.9
1.1
0.8
16.8 ꢀ 1.2
21.0 ꢀ 0.7
17.1 ꢀ 0.4
8.14 ꢀ 0.26
3.11 ꢀ 0.32
1.32 ꢀ 0.07
1.41 ꢀ 0.14
3.72 ꢀ 1.75
1.31 ꢀ 0.01
0.9427). From this equation and taking into account the
limits established by Di et al. for blood–brain barrier
permeation, we classied compounds as follows:
compounds with Pe (10ꢂ6 cm sꢂ1) > 4.5 for high BBB
permeation (CNS+), compounds with Pe (10ꢂ6 cm sꢂ1) < 2.1
for low BBB permeation (CNSꢂ), and compounds with 4.5 >
Pe (10ꢂ6 cm sꢂ1) > 2.1 for uncertain BBB permeation (CNSꢀ).
The Pe values of these selected compounds are summarized
in Table 4. It can be indicated that compounds 7, 17 and 20
a
b
Taken from ref. 57. Data are the mean ꢀ SD of three independent
experiments.
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Table 4 Permeability results (Pe ꢃ 10ꢂ6 cm sꢂ1) from the PAMPA-BBB
assay for selected compounds with their predicted penetration into
the CNS
H2 atmosphere, for 5 h. Aer the completion of the reaction, the
mixture was ltered to eliminate the catalyst. The obtained
crude product was then puried by recrystallization to give the
desired intermediate 1b, in a yield of 78%. Then a mixture of 1b
was reacted with different substituted aromatic aldehydes in
ethanol for 6 h at room temperature and the solvent was
removed under reduced pressure. The resulting crude
compounds were washed with cold water and recrystallized by
using appropriate solvents to afford the target products 1–20.
4.1.2. 3-Amino-4-hydroxy-2H-chromen-2-one (1b). Yield
78%, white powder, m.p. 220–222 ꢁC. 1H NMR (500 MHz,
DMSO) d 8.27 (s, 2H), 7.86 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.52–7.45 (m,
1H), 7.33–7.18 (m, 2H). 13C NMR (126 MHz, DMSO) d 160.71,
152.00, 130.54, 124.12, 123.50, 123.50, 121.57, 116.45, 116.45.
ESI-MS m/z: 178.0 [M + H]+.
Compounds
Permeabilitya (Pe ꢃ 10ꢂ6 cm sꢂ1
)
Prediction
4
7
17
20
4.16 ꢀ 0.33
10.5 ꢀ 0.7
9.95 ꢀ 0.21
8.52 ꢀ 0.15
CNSꢀ
CNS+
CNS+
CNS+
a
Data are the mean ꢀ SD of three independent experiments.
3. Conclusion
In summary, a series of 3-Schiff base-4-hydroxycoumarin deriv-
atives were designed and synthesized as multifunctional agents
for the treatment of AD. Among the synthesized compounds,
most of them exhibited moderate inhibition of both MAO-A and
MAO-B. Kinetic and molecular modeling studies revealed that 4
was a reversible and noncompetitive MAO-B inhibitor. In addi-
tion, these derivatives displayed potential inhibition of self-
induced and Cu2+-induced Ab1–42 aggregation, antioxidant
activity, metal-chelating ability, control of Cu2+–Ab1–42 H2O2
production and low PC12 cells toxicity. Furthermore,
compounds 2, 4, 5 and 17 showed neuroprotective capability
much better than trolox at the concentration of 5 mM, and most
of them were able to cross the blood–brain barrier. These
4.1.3. (E)-4-Hydroxy-3-((2-hydroxybenzylidene)amino)-2H-
chromen-2-one (1). Yield 88%, yellow solid, m.p. > 250 C, IR
ꢁ
(KBr) n 3442, 1689, 1604, 1537, 1487, 1459, 1367, 1264, 1228,
1083, 1036, 928, 909, 756, 735 cmꢂ1. 1H NMR (500 MHz, DMSO)
d 13.06 (s, 1H), 9.93 (s, 1H), 7.99 (d, J ¼ 7.1 Hz, 1H), 7.80 (d, J ¼
7.5 Hz, 1H), 7.63 (t, J ¼ 7.3 Hz, 1H), 7.56 (t, J ¼ 7.6 Hz, 1H), 7.36
(d, J ¼ 5.9 Hz, 2H), 7.09 (t, J ¼ 9.2 Hz, 2H). 13C NMR (126 MHz,
DMSO) d 169.36, 166.61, 159.64, 153.25, 150.33, 136.89, 132.71,
129.89, 125.45, 123.79, 119.99, 117.72, 117.09, 116.97, 116.26,
100.00. ESI-MS m/z: 379.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 280.0613
[M ꢂ H]ꢂ (calcd for 280.0615, C16H9NO4).
4.1.4. (E)-3-((2,3-Dihydroxybenzylidene)amino)-4-hydroxy-
ꢁ
2H-chromen-2-one (2). Yield 86%, red solid, m.p. > 250 C, IR
properties
highlighted
that
the
3-Schiff
base-4-
(KBr) n 3421, 1606, 1542, 1461, 1417, 1228, 1109, 901, 754, 474
hydroxylcoumarin derivatives could serve as new multifunc-
tional drug candidates in the treatment of Alzheimer's disease.
1
cmꢂ1. H NMR (500 MHz, DMSO) d 16.23 (s, 1H), 9.85 (s, 1H),
7.95 (d, J ¼ 7.7 Hz, 1H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.24 (dd, J ¼ 14.2,
7.6 Hz, 2H), 6.75 (d, J ¼ 7.8 Hz, 1H), 6.69 (d, J ¼ 7.4 Hz, 1H), 6.44
(t, J ¼ 7.7 Hz, 1H). 13C NMR (126 MHz, DMSO) d 169.36, 160.72,
158.00, 153.01, 152.15, 147.77, 131.20, 125.37, 123.39, 123.07,
121.21, 118.00, 116.37, 114.72, 114.39, 104.93. ESI-MS m/z: 295.8
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino- [M ꢂ H]ꢂ; HRMS (ESI) m/z 296.0562 [M ꢂ H]ꢂ (calcd for
pharm Chemical Reagent Co., Ltd. (China). Reaction progress 296.0564, C16H10NO5).
was monitored using analytical thin layer chromatography
4.1.5. (E)-3-((2,4-Dihydroxybenzylidene)amino)-4-hydroxy-
(TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical 2H-chromen-2-one (3). Yield 75%, yellow solid, m.p. > 250 ꢁC, IR
Plant, Qingdao, China) plates and the spots were detected (KBr) n 3427, 1619, 1538, 1392, 1316, 1234, 1136, 1031, 759, 445
under UV light (254 nm). Melting point was measured on an cmꢂ1. 1H NMR (500 MHz, DMSO) d 12.79 (s, 2H), 10.94 (s, 1H),
XT-4 micromelting point instrument and uncorrected. IR 9.80 (s, 1H), 7.97 (d, J ¼ 7.3 Hz, 1H), 7.61 (dd, J ¼ 18.6, 7.9 Hz,
(KBr-disc) spectra were recorded by Bruker Tensor 27 spec- 2H), 7.40–7.29 (m, 3H), 6.53 (s, 1H). 13C NMR (126 MHz, DMSO)
1
trometer. H NMR and 13C NMR spectra were measured on a d 168.74, 165.39, 161.62, 159.35, 153.15, 150.41, 137.12, 132.51,
ꢁ
Bruker ACF-500 spectrometer at 25 C and referenced to TMS. 125.30, 123.93, 121.88, 116.95, 110.20, 109.01, 103.26, 102.10.
Chemical shis were reported in ppm (d) using the residual ESI-MS m/z: 295.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 296.0562 [M ꢂ H]ꢂ
solvent line as internal standard. Splitting patterns were (calcd for 296.0564, C16H10NO5).
designed as s, singlet; d, doublet; t, triplet; m, multiplet. Mass
4.1.6. (E)-4-Hydroxy-3-((2,3,4-trihydroxybenzylidene)amino)-
spectra were obtained on a MS Agilent 1100 Series LC/MSD Trap 2H-chromen-2-one (4). Yield 78%, red solid, m.p. 210–212 ꢁC, IR
mass spectrometer (ESI-MS) and a Mariner ESI-TOF spectrom- (KBr) n 3396, 1616, 1546, 1421, 1324, 1228, 1101, 1023, 786, 754,
eter (HRESI-MS), respectively.
461 cmꢂ1. 1H NMR (500 MHz, DMSO) d 10.84 (s, 1H), 9.80 (s, 1H),
4.1.1. General procedures for the preparation of 9.11 (s, 1H), 7.97 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.67–7.53 (m, 1H), 7.32
compounds 1b and 1–20. Compound 1b was synthesized (dd, J ¼ 7.7, 5.8 Hz, 2H), 7.14 (d, J ¼ 8.7 Hz, 1H), 6.59 (d, J ¼ 8.6
according to a previously reported method.41 The commercially Hz, 1H). 13C NMR (126 MHz, DMSO) d 168.62, 159.40, 153.69,
available 4-hydroxy-3-nitrocoumarin (1a) was dissolved in 153.11, 151.27, 149.62, 133.70, 132.48, 126.99, 125.29, 123.92,
ethanol and a catalytic amount of Pd/C was added to the 121.92, 116.93, 109.89, 109.61, 102.11. ESI-MS m/z: 311.9 [M ꢂ
mixture. The solution was stirred, at room temperature, under
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H]ꢂ; HRMS (ESI) m/z 312.0511 [M ꢂ H]ꢂ (calcd for 312.0514, 293.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 294.0773 [M ꢂ H]ꢂ (calcd for
C16H10NO6).
294.0772, C17H12NO4).
4.1.7. (E)-4-Hydroxy-3-((2,4,6-trihydroxybenzylidene)amino)-
4.1.12. (E)-4-Hydroxy-3-((2-hydroxy-4-methylbenzylidene)-
2H-chromen-2-one (5). Yield 67%, yellow solid, m.p. > 250 ꢁC, IR amino)-2H-chromen-2-one (10). Yield 74%, light yellow solid,
(KBr) n 3332, 1616, 1537, 1403, 1296, 1235, 1172, 1136, 1067, 824, m.p. > 250 ꢁC, IR (KBr) n 3057, 1686, 1611, 1527, 1410, 1362,
758, 446 cmꢂ1. 1H NMR (500 MHz, DMSO) d 12.52 (s, 1H), 11.78 1229, 1135, 1083, 903, 748, 443 cmꢂ1 1H NMR (500 MHz,
.
(s, 1H), 10.76 (s, 1H), 10.09 (s, 1H), 7.93 (dd, J ¼ 7.9, 1.4 Hz, 1H), DMSO) d 13.13 (s, 1H), 9.89 (s, 1H), 7.98 (d, J ¼ 7.5 Hz, 1H), 7.67
7.63–7.48 (m, 1H), 7.29 (dd, J ¼ 7.7, 6.0 Hz, 2H), 6.00 (s, 2H). 13
C
(d, J ¼ 7.8 Hz, 1H), 7.62 (t, J ¼ 7.5 Hz, 1H), 7.39–7.30 (m, 2H),
NMR (126 MHz, DMSO) d 168.42, 166.59, 162.13, 162.13, 159.40, 6.92 (d, J ¼ 8.3 Hz, 2H), 2.37 (s, 3H). 13C NMR (126 MHz, DMSO)
153.06, 145.97, 132.21, 125.23, 123.80, 122.08, 116.86, 102.26, d 161.18, 159.78, 153.75, 153.21, 144.87, 137.85, 133.88, 130.29,
99.22, 95.50, 95.50. ESI-MS m/z: 311.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 125.43, 123.83, 121.24, 120.54, 117.74, 117.30, 116.87, 99.93,
312.0516 [M ꢂ H]ꢂ (calcd for 312.0514, C16H10NO6).
14.57. ESI-MS m/z: 293.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 294.0770 [M
4.1.8. (E)-4-Hydroxy-3-((2-hydroxy-3-methoxybenzylidene)- ꢂ H]ꢂ (calcd for 294.0772, C17H12NO4).
amino)-2H-chromen-2-one (6). Yield 77%, yellow solid, m.p. >
4.1.13. (E)-4-Hydroxy-3-((2-hydroxy-5-methylbenzylidene)-
ꢁ
250 C, IR (KBr) n 1619.96, 1589.59, 1472.84, 1399.63, 1326.60, amino)-2H-chromen-2-one (11). Yield 77%, yellow solid, m.p. >
1251.65, 1176.42, 968.39, 884.26, 820.45, 736.91 cmꢂ1. 1H NMR 250 ꢁC, IR (KBr) n 3449, 1688, 1599, 1535, 1364, 1265, 1238,
(500 MHz, DMSO) d 9.93 (s, 1H), 7.99 (dd, J ¼ 7.9, 1.4 Hz, 1H), 1085, 899, 755, 637, 487, 454 cmꢂ1. 1H NMR (500 MHz, DMSO) d
7.68–7.56 (m, 1H), 7.48–7.19 (m, 4H), 7.04 (t, J ¼ 8.0 Hz, 1H), 13.01 (s, 2H), 9.86 (s, 1H), 8.02–7.91 (m, 1H), 7.61 (dd, J ¼ 11.1,
3.91 (s, 3H). 13C NMR (126 MHz, DMSO) d 168.91, 159.36, 4.3 Hz, 1H), 7.56 (s, 1H), 7.37–7.26 (m, 3H), 6.98 (d, J ¼ 8.4 Hz,
153.21, 150.68, 148.67, 148.51, 133.02, 125.60, 125.41, 124.17, 1H), 2.28 (s, 3H). 13C NMR (126 MHz, DMSO) d 169.30, 159.33,
121.42, 120.84, 117.62, 117.07, 103.11, 99.99, 56.90. ESI-MS m/z: 157.06, 153.26, 150.33, 141.24, 136.42, 134.06, 132.85, 129.45,
309.8 [M ꢂ H]ꢂ; HRMS (ESI) m/z 310.0719 [M ꢂ H]ꢂ (calcd for 125.45, 124.04, 121.50, 117.02, 116.95, 96.92, 20.21. ESI-MS m/z:
310.0721, C17H12NO5).
293.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 294.0771 [M ꢂ H]ꢂ (calcd for
4.1.9. (E)-4-Hydroxy-3-((2-hydroxy-4-methoxybenzylidene)- 294.0772, C17H12NO4).
amino)-2H-chromen-2-one (7). Yield 87%, yellow solid, m.p. >
4.1.14. (E)-3-((4-(Diethylamino)-2-hydroxybenzylidene)-
250 ꢁC, IR (KBr) n 3445, 1677, 1519, 1411, 1360, 1290, 1231, amino)-4-hydroxy-2H-chromen-2-one (12). Yield 69%, yellow
1
1211, 1119, 1031, 906, 755, 679, 555, 474 cmꢂ1. H NMR (500 solid, m.p. > 250 C, IR (KBr) n 3447, 2976, 1672, 1610, 1520,
ꢁ
MHz, DMSO) d 13.05 (s, 2H), 9.85 (s, 1H), 7.97 (dd, J ¼ 7.9, 1.4 1423, 1350, 1237, 1140, 1079, 753, 690 cmꢂ1. 1H NMR (500 MHz,
Hz, 1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.62–7.58 (m, 1H), 7.33 (dd, J ¼ DMSO) d 12.20 (s, 2H), 9.63 (s, 1H), 7.92 (d, J ¼ 7.4 Hz, 1H), 7.54
7.7, 4.4 Hz, 2H), 6.70 (dd, J ¼ 8.7, 2.1 Hz, 1H), 6.59 (d, J ¼ 2.1 Hz, (t, J ¼ 7.5 Hz, 1H), 7.46 (d, J ¼ 8.9 Hz, 1H), 7.28 (t, J ¼ 6.6 Hz,
1H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO) d 168.90, 166.47, 2H), 6.44 (d, J ¼ 8.5 Hz, 1H), 6.25 (s, 1H), 3.46–3.41 (m, 4H), 1.15
163.54, 159.63, 153.11, 150.58, 135.94, 132.90, 125.32, 123.79, (t, J ¼ 6.8 Hz, 6H). 13C NMR (126 MHz, DMSO) d 168.11, 161.61,
122.23, 116.83, 116.67, 107.93, 101.89, 101.29, 56.15. ESI-MS m/ 159.40, 153.93, 152.98, 149.89, 132.03, 125.18, 123.75, 122.13,
z: 309.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 310.0720 [M ꢂ H]ꢂ (calcd for 116.80, 106.15, 105.05, 102.12, 97.37, 96.47, 44.87, 44.87, 12.94,
310.0721, C17H12NO5).
12.94. ESI-MS m/z: 351.0 [M ꢂ H]ꢂ; HRMS (ESI) m/z 351.1347 [M
4.1.10. (E)-4-Hydroxy-3-((2-hydroxy-5-methoxybenzylidene)- ꢂ H]ꢂ (calcd for 351.1350, C20H19N2O4).
amino)-2H-chromen-2-one (8). Yield 72%, light yellow solid,
4.1.15. (E)-3-((5-Bromo-2-hydroxybenzylidene)amino)-4-
m.p. > 250 ꢁC, IR (KBr) n 3444, 1679, 1597, 1538, 1511, 1447, hydroxy-2H-chromen-2-one (13). Yield 77%, yellow solid, m.p. >
1381, 1257, 1224, 1090, 1037, 899, 752, 713, 479 cmꢂ1. 1H NMR 250 ꢁC, IR (KBr) n 3446, 1698, 1612, 1531, 1491, 1461, 1416,
(500 MHz, DMSO) d 12.63 (s, 1H), 9.92 (s, 1H), 8.08–7.94 (m, 1H), 1372, 1316, 1282, 1196, 1076, 900, 824, 755, 663, 498 cmꢂ1. H
1
7.64 (t, J ¼ 7.7 Hz, 1H), 7.44 (d, J ¼ 2.8 Hz, 1H), 7.35 (dd, J ¼ 7.6, NMR (500 MHz, DMSO) d 12.99 (s, 1H), 9.87 (s, 1H), 8.13 (d, J ¼
4.4 Hz, 2H), 7.17 (dd, J ¼ 9.0, 3.0 Hz, 1H), 7.03 (d, J ¼ 9.0 Hz, 1H), 2.1 Hz, 1H), 8.00 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.65 (dd, J ¼ 11.0, 5.0
3.79 (s, 3H). 13C NMR (126 MHz, DMSO) d 168.87, 158.95, Hz, 2H), 7.36 (dd, J ¼ 7.6, 4.7 Hz, 2H), 7.03–6.98 (m, 1H). 13C
153.24, 152.90, 150.01, 142.46, 132.98, 127.69, 125.43, 124.11, NMR (126 MHz, DMSO) d 172.79, 158.95, 158.28, 153.16, 149.22,
121.40, 121.14, 118.01, 117.06, 116.71, 99.98, 56.26. ESI-MS m/z: 136.78, 135.25, 133.11, 125.65, 125.37, 124.24, 120.48, 119.21,
312.2 [M + H]+; HRMS (ESI) m/z 310.0718 [M ꢂ H]ꢂ (calcd for 117.05, 111.17, 99.03. ESI-MS m/z: 391.2 [M + Na]+; HRMS (ESI)
310.0721, C17H12NO5).
m/z 357.9717 [M ꢂ H]ꢂ (calcd for 357.9720, C16H9BrNO4).
4.1.16. (E)-3-((3,5-Dibromo-2-hydroxybenzylidene)amino)-
4.1.11. (E)-4-Hydroxy-3-((2-hydroxy-3-methylbenzylidene)-
amino)-2H-chromen-2-one (9). Yield 75%, yellow solid, m.p. > 4-hydrꢁoxy-2H-chromen-2-one (14). Yield 76%, yellow solid, m.p.
250 ꢁC, IR (KBr) n 3446, 1681, 1650, 1600, 1545, 1487, 1412, > 250 C, IR (KBr) n 3444, 1692, 1617, 1555, 1485, 1420, 1384,
1
1373, 1326, 1240, 1212, 1109, 1036, 899, 754, 472 cmꢂ1. 1H NMR 1288, 1204, 1149, 896, 752, 695, 477 cmꢂ1. H NMR (500 MHz,
(500 MHz, DMSO) d 12.44 (s, 1H), 9.81 (s, 1H), 7.99 (dd, J ¼ 8.0, DMSO) d 11.28 (s, 1H), 9.77 (s, 1H), 7.97 (d, J ¼ 7.8 Hz, 1H), 7.67
1.4 Hz, 1H), 7.66–7.59 (m, 1H), 7.56 (d, J ¼ 7.4 Hz, 1H), 7.40 (d, J (dd, J ¼ 10.0, 4.5 Hz, 2H), 7.56 (t, J ¼ 7.6 Hz, 1H), 7.30 (t, J ¼ 6.4
¼ 7.3 Hz, 1H), 7.34 (dd, J ¼ 7.4, 6.4 Hz, 2H), 6.98 (t, J ¼ 7.6 Hz, Hz, 2H). 13C NMR (126 MHz, DMSO) d 166.12, 160.78, 159.76,
1H), 2.29 (s, 3H). 13C NMR (126 MHz, DMSO) d 159.54, 157.51, 156.44, 152.71, 136.45, 132.36, 125.17, 124.78, 123.91, 121.52,
152.90, 138.13, 136.10, 132.78, 131.98, 131.38, 126.38, 125.21, 116.69, 114.13, 113.45, 111.73, 99.94. ESI-MS m/z: 437.7 [M +
124.15, 120.63, 120.05, 118.30, 116.94, 99.44, 16.45. ESI-MS m/z:
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H]+; HRMS (ESI) m/z 435.8825 [M ꢂ H]ꢂ (calcd for 435.8826,
4.1.22. (E)-4-Hydroxy-3-((quinolin-2-ylmethylene)amino)-2H-
chromen-2-one (20). Yield 65%, brown solid, m.p. 215–217 ꢁC, IR
(KBr) n 3442, 1675, 1604, 1561, 1530, 1503, 1461, 1392, 1331, 1298,
C
16H8Br2NO4).
4.1.17. (E)-3-((5-Fluoro-2-hydroxybenzylidene)amino)-4-
1
1213, 1199, 1179, 1145, 1124, 1064, 901, 831, 759, 456 cmꢂ1. H
hydroxy-2H-chromen-2-one (15). Yield 69%, yellow solid, m.p. >
250 ꢁC, IR (KBr) n 3075, 1683, 1603, 1544, 1513, 1453, 1372,
NMR (500 MHz, DMSO) d 9.90 (s, 1H), 8.60 (d, J ¼ 8.2 Hz, 1H),
8.54 (d, J ¼ 8.5 Hz, 1H), 8.12 (d, J ¼ 8.4 Hz, 1H), 8.04 (t, J ¼ 8.8 Hz,
2H), 7.85 (t, J ¼ 7.6 Hz, 1H), 7.69 (t, J ¼ 7.8 Hz, 2H), 7.43–7.35 (m,
2H). 13C NMR (126 MHz, DMSO) d 164.39, 160.71, 153.38, 151.79,
149.35, 146.41, 138.73, 132.72, 131.36, 130.99, 129.71, 129.01,
128.68, 127.07, 124.88, 121.08, 119.23, 116.74, 98.74. ESI-MS m/z:
317.1 [M + H]+; HRMS (ESI) m/z 339.0743 [M + Na]+ (calcd for
339.0740, C19H12N2NaO3).
1
1320, 1248, 1096, 1039, 903, 820, 754, 487 cmꢂ1. H NMR (500
MHz, DMSO) d 12.98 (s, 2H), 9.86 (s, 1H), 7.97 (d, J ¼ 7.5 Hz, 1H),
7.79 (d, J ¼ 7.0 Hz, 1H), 7.62 (t, J ¼ 7.3 Hz, 1H), 7.35 (t, J ¼ 6.7 Hz,
3H), 7.04 (dd, J ¼ 8.6, 3.8 Hz, 1H). 13C NMR (126 MHz, DMSO) d
160.72, 159.36, 155.52, 153.19, 149.45, 136.81, 133.05, 129.61,
125.41, 124.18, 122.61, 119.57, 118.31, 117.04, 116.46, 104.14.
ESI-MS m/z: 300.1 [M + H]+; HRMS (ESI) m/z 298.0524 [M ꢂ H]ꢂ
(calcd for 298.0521, C16H9FNO4).
4.1.18. (E)-3-((5-Chloro-2-hydroxybenzylidene)amino)-4-
hydroxy-2H-chromen-2-one (16). Yield 74%, yellow solid, m.p. >
250 ꢁC, IR (KBr) n 3058, 1679, 1601, 1537, 1500, 1442, 1390,
4.2. Pharmacology
4.2.1. In vitro inhibition of monoamine oxidase. Human
MAO-A and MAO-B were purchased from Sigma-Aldrich. The
1364, 1322, 1273, 1221, 1088, 1040, 919, 895, 816, 751, 734 cmꢂ1
.
1H NMR (500 MHz, DMSO) d 9.87 (s, 1H), 7.95 (d, J ¼ 7.5 Hz, capacity of the test compounds to inhibit hMAO-A and hMAO-B
1H), 7.77 (s, 1H), 7.56 (s, 1H), 7.38 (s, 1H), 7.28 (d, J ¼ 6.9 Hz, activities was assessed by Amplex Red MAO assay.15 Briey, 0.1
2H), 7.01–6.92 (m, 1H). 13C NMR (126 MHz, DMSO) d 168.98, mL of sodium phosphate buffer (0.05 mM, pH 7.4) containing
159.95, 158.23, 153.17, 149.61, 136.20, 132.55, 131.42, 128.03, the test drugs at various concentrations and adequate amounts
125.45, 123.85, 121.54, 120.02, 118.90, 116.83, 98.65. ESI-MS m/ of recombinant hMAO-A or hMAO-B required and adjusted to
z: 313.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 314.0224 [M ꢂ H]ꢂ (calcd for obtain in our experimental conditions the same reaction
314.0226, C16H9ClNO4).
velocity, i.e., to oxidize (in the control group) the same
4.1.19. (E)-4-Hydroxy-3-((2-hydroxy-5-nitrobenzylidene)amino)- concentration of substrate: 165 pmol of p-tyramine per min
2H-chromen-2-one (17). Yield 69%, yellow solid, m.p. > 250 ꢁC, IR (hMAO-A: 1.1 mg protein; specic activity: 150 nmol of p-tyra-
(KBr) n 3276, 1691, 1607, 1528, 1482, 1426, 1368, 1261, 1227, 1098, mine oxidized to p-hydroxyphenylacetaldehyde per min per mg
1026, 901, 762, 736, 472 cmꢂ1. 1H NMR (500 MHz, DMSO) d 9.96 (s, protein; hMAO-B: 7.5 mg protein; specic activity: 22 nmol of
1H), 8.53 (s, 1H), 8.08 (d, J ¼ 9.3 Hz, 1H), 7.98 (d, J ¼ 7.2 Hz, 1H), p-tyramine transformed per min per mg protein) were incu-
7.88 (d, J ¼ 8.0 Hz, 1H), 7.59 (t, J ¼ 7.7 Hz, 1H), 7.37 (d, J ¼ 8.3 Hz, bated for 15 min at 37 ꢁC in a at-black-bottom 96-well
1H), 7.31 (d, J ¼ 7.8 Hz, 1H). 13C NMR (126 MHz, DMSO) d 168.69, microtest plate placed in a dark uorimeter chamber. Aer this
159.36, 155.52, 153.19, 149.46, 137.03, 133.05, 130.54, 129.50, incubation period, the reaction was started by adding 200 mM
125.41, 124.18, 122.47, 118.31, 117.04, 113.57, 104.21. ESI-MS m/z: (nal concentrations) Amplex Red reagent, 1 U per mL horse-
324.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 425.0464 [M ꢂ H]ꢂ (calcd for radish peroxidase, and 1 mM p-tyramine. The production of
325.0466, C16H9N2O6).
H2O2 and consequently, of resorun, was quantied at 37 ꢁC in
4.1.20. (E)-4-Hydroxy-3-(((2-hydroxynaphthalen-1-yl)methy- a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA)
lene)amino)-2H-chromen-2-one (18). Yield 79%, yellow solid, multi-mode detection platform reader based on the uores-
m.p. > 250 ꢁC, IR (KBr) n 3443, 1688, 1619, 1534, 1444, 1422, cence generated (excitation, 545 nm; emission, 590 nm). The
1373, 1282, 1224, 1088, 1027, 903, 819, 754, 466 cmꢂ1. 1H NMR specific uorescence emission was calculated aer subtraction
(500 MHz, DMSO) d 10.88 (s, 1H), 8.15 (s, 2H), 8.03 (d, J ¼ 7.0 Hz, of the background activity. The background activity was deter-
1H), 7.97 (d, J ¼ 6.6 Hz, 1H), 7.76 (s, 1H), 7.63 (s, 1H), 7.51 (s, mined from wells containing all components except the MAO
1H), 7.37 (d, J ¼ 7.3 Hz, 2H), 7.31 (d, J ¼ 6.7 Hz, 1H). 13C NMR isoforms, which were replaced by a sodium phosphate buffer
(126 MHz, DMSO) d 167.95, 160.98, 158.87, 152.19, 144.70, solution (0.05 mM, pH 7.4). The percent inhibition was calcu-
136.40, 131.79, 131.73, 128.99, 128.69, 127.29, 124.44, 123.89, lated by the following expression: (1 ꢂ IFi/IFc) ꢃ 100% in which
123.18, 120.95, 119.52, 118.31, 116.12, 106.91, 102.41. ESI-MS IFi and IFc are the uorescence intensities obtained for hMAOs
m/z: 329.9 [M ꢂ H]ꢂ; HRMS (ESI) m/z 330.0771 [M ꢂ H]ꢂ in the presence and absence of inhibitors aer subtracting the
(calcd for 330.0772, C20H12NO4).
respective background.
4.1.21. (E)-4-Hydroxy-3-((pyridin-2-ylmethylene)amino)-2H-
4.2.2. Reversibility and kinetic studies of hMAO-B inhibi-
chromen-2-one (19). Yield 77%, red solid, m.p. > 250 ꢁC, IR (KBr) tion. To determine whether the inhibition of hMAO-B by the
n 3424, 1648, 1604, 1527, 1460, 1403, 1348, 1313, 1213, 1150, 3-Schiff base-4-hydroxycoumarin derivatives were reversible or
1
1106, 1059, 946, 908, 759 cmꢂ1. H NMR (500 MHz, DMSO) d irreversible, the time-dependence of inhibition of the selected
9.79 (s, 1H), 8.49 (s, 1H), 7.91 (d, J ¼ 54.9 Hz, 2H), 7.67 (s, 1H), inhibitors 4, 14, 17 and reference compound pargyline were
7.48 (s, 1H), 7.25 (d, J ¼ 44.5 Hz, 3H). 13C NMR (126 MHz, DMSO) examined.42 Compounds were allowed to pre-incubate with
d 153.41, 150.29, 146.66, 137.94, 131.50, 130.41, 125.85, 125.85, recombinant human hMAO-B for various periods of time (0, 15,
ꢁ
124.43, 123.55, 122.95, 119.73, 116.31, 110.79, 79.66. ESI-MS m/z: 30, 60 min) at 37 C in potassium phosphate buffer (0.05 mM,
267.1 [M + H]+; HRMS (ESI) m/z 265.0616 [M ꢂ H]ꢂ (calcd for pH 7.4). The concentrations of the compounds were about two-
265.0619, C15H9N2O4).
fold the measured IC50 values for the inhibition of hMAO-B. The
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reactions were subsequently diluted two-fold to yield a nal pH. The X-ray crystal structure of the hMAO-A (PDB 2Z5X),
enzyme concentration of 0.015 mg mLꢂ1 and concentrations of hMAO-B (PDB 2V61) and Ab1–42 (PDB 1IYT) were applied to
the inhibitors that are about equal to the IC50 values. The build the starting model, which were obtained from the Protein
reactions were incubated at 37 ꢁC for a further 15 min. All Data Bank (http://www.rcsb.org). Heteroatoms and water
measurements were carried out in triplicate and are expressed molecules in the PDB les were removed and all hydrogen
as mean ꢀ SD.
atoms were subsequently added to the proteins. Compound 4
To obtain of the mechanism of action 4, reciprocal plots of was drawn in MOE. The compound was then protonated using
1/velocity versus 1/substrate were constructed at different the protonate 3D protocol and energy was minimized using the
concentrations of the substrate p-tyramine (50–500 mM) by MMFF94x force eld in MOE. Aer the enzymes and compound
using Ellman's method. Four different concentrations of 4 (0, 4 were ready for the docking study, 4 was docked into the active
0.5, 1.0 and 2.0 mM) were selected for the kinetic analysis of site of the protein by the “Triangle Matcher” method. The dock
hMAO-B inhibition. The plots were assessed by a weighted least- scoring in MOE soware was done using ASE scoring function
squares analysis that assumed the variance of velocity (v) to be a and forceeld was selected as the renement method. The best
constant percentage of v for the entire data set. Slopes of these 10 poses of molecules were retained and scored. Aer docking,
reciprocal plots were then plotted against the concentration of 4 the geometry of resulting complex was studied using the MOE's
in a weighted analysis. Data analysis was performed with Graph pose viewer utility.
Pad Prism 4.03 soware (Graph Pad Soware Inc.).
4.2.5. Spectrophotometric measurement of complex with
4.2.3. Inhibition of self-induced and Cu2+-induced Ab1–42 Cu2+. The study of Cu2+ was performed in methanol at 25 ꢁC
aggregation. Inhibition of self-induced Ab1–42 aggregation was using UV-vis spectrophotometer (SHIMADZU UV-2450PC) with
measured using a ThT-binding assay.47 HFIP pretreated Ab1–42 wavelength ranging from 200 to 500 nm.49,50 The difference
samples (Anaspec Inc) were resolubilized with a 50 mM phos- UV-vis spectra due to complex formation was obtained by
phate buffer (pH 7.4) to give a 25 mM solution. Each tested numerical subtraction of the spectra of the Cu2+ alone and the
compound was rstly prepared in DMSO at a concentration of compound alone (at the same concentration used in the
200 mM and 1 mL of each was added to the well of black, opaque mixture) from the spectra of the mixture. A xed amount of 4
Corning 96-well plates such that the nal solvent concentration (25 mmol Lꢂ1) was mixed with growing amounts of Cu2+
was 10%. The nal concentration of each compound was 20 mM (5–60 mmol Lꢂ1) and tested the difference UV-vis spectra to
and was prepared in independent triplicates. The solvent investigate the ratio of 4/Cu2+ in the complex.
control was also included. Then, 9 mL of 25 mM Ab1–42 sample
4.2.6. In vitro antioxidant activity assays.51,52 ABTS radical
was added to each well and the samples mixed by gentle trap- cation scavenging method: 2,20-azino-bis-2-ethybenz-thiazoline-
ping. Plates were covered to minimize evaporation and incu- 6-sulfonic acid (ABTS) was dissolved in puried water to a 7 mM
bated in dark at room temperature for 46–48 h with no concentration. ABTS radical cation (ABTSc+) was produced by
agitation. Aer the incubation period, 200 mL of 5 mM ThT in reacting ABTS stock solution with 2.45 mM potassium persul-
50 mM glycine–NaOH buffer (pH 8.0) was added to each well. fate (nal concentration) and allowing the mixture to stand in
Fluorescence was measured on a SpectraMax M5 (Molecular the dark at room temperature for at least 18 h before use. The
Devices, Sunnyvale, CA, USA) multi-mode plate reader with stock solution of ABTS was serially diluted with sodium phos-
excitation and emission wavelengths at 446 nm and 490 nm, phate buffer (50 mM, pH 7.4) to 100 mM. Trolox and all target
respectively. The uorescence intensities were compared and compounds at different concentrations (total volume of 50 mL)
the percent inhibition due to the presence of the inhibitor was were added to 150 mL of 100 mM ABTS solution, respectively.
calculated by the following formula: (1 ꢂ IFi/IFo) ꢃ 100% where Aer the addition of either trolox or another antioxidant to the
IFi and IFo are the uorescence intensities obtained for Ab1–42 in ABTS solution, complete mixing of reactants was achieved by
the presence and in the absence of inhibitor, respectively.
bubbling three to four times using plastic pipettes. The optical
For the inhibition of Cu2+-induced Ab1–42 aggregation absorbance of ABTS at 415 nm was measured at 6 min aer
ꢁ
experiment, the Ab1–42 was diluted in 20 mM HEPES (pH 6.6) addition and equilibrated at 30 C. Each individual treatment
with 150 mM NaCl. The mixture of the peptide (10 mL, 25 mM, was repeated for three times and the results of the experiments
nal concentration) with or without copper (10 mL, 25 mM, nal were compared.
concentration) and the test compound (10 mL, 50 mM, nal
DPPH radical scavenging method: DPPH is one of the few
concentration) was incubated at 37 ꢁC for 24 h. The 20 mL of the stable and commercially available organic nitrogen radicals and
sample was diluted to a nal volume of 200 mL with 50 mM has a UV-vis absorption maximum at 517 nm. Upon reduction,
glycine–NaOH buffer (pH 8.0) containing thioavin T (5 mM). the solution color fades; the reaction progress is conveniently
The detection method was the same as that of self-induced monitored by a spectrophotometer. To test free radical-
Ab1–42 aggregation experiment.
scavenging effects, compounds 1–20, 1a–b and resveratrol
4.2.4. Molecular docking studies. To study the binding were adjusted with methanol solution to nal concentrations of
details of compound 4 with hMAO-A and hMAO-B, docking 0–200 mM. Methanolic DPPH (400 mM) was used in the reaction
studies were performed using the Molecular Operating Envi- mixture. Serial dilutions of the test sample (20 mL) were
ronment (MOE 2008.10) soware package, and the Marvin combined with the DPPH (180 mL, 400 mM) solution in a 96-well
soware package (http://www.chemaxon.com) was carried out microtitre plate. MeOH was used as a negative control and
to calculate the protonation level of the ligand in physiological resveratrol was used as positive control. The reaction mixtures
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were incubated for 30 min at 37 ꢁC in the dark and the change in
absorbance at 517 nm was measured. Mean values were
obtained from triplicate experiments. Inhibition percent was
calculated using the equation: DPPH radical-scavenging rate ¼
[1 ꢂ (A ꢂ C)/B] ꢃ 100%, where A is the absorbance of the sample
(DPPH + compounds), B is the absorbance of the DPPH radical–
methanol solution, and C is the absorbance of the sample
(compounds) alone. Percent inhibition was plotted against
concentration, and the equation for the line was used to obtain
the IC50 value. The IC50 values of samples were compared
against the standard, resveratrol, and the lower the IC50 of
synthesized compounds, the better it is as an antioxidant.
4.2.7. Hydrogen peroxide production assay. Hydrogen
peroxide production was determined using a HRP/Amplex Red
assay. A general protocol from Invitrogen's Amplex Red
Hydrogen Peroxide/Peroxidase assay as followed.53 Reagents
were added directed to a 96-well plate in the following order to
give a 100 mL nal solution: CuCl2 (400 nM), phosphate buffer,
Ab1–42 peptide (200 mM), compounds (800 nM, 1% v/v DMSO),
sodium ascorbate (10 mM). The reaction was allowed to incubate
for 30 min at room temperature. Aer this incubation, 50 mM of
freshly prepared working solution containing 100 nM Amplex
Red (Sigma) and 0.2 U per mL HRP (Sigma) in phosphate buffer
was added to each well, and the reaction was allowed to incu-
bate for 30 min at room temperature. Fluorescence was
4.2.9. In vitro blood–brain barrier permeation assay. Brain
penetration of compounds was evaluated using a parallel arti-
cial membrane permeation assay (PAMPA) in a similar manner
as described by Di et al.57 Commercial drugs were purchased
from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was
obtained from Avanti Polar Lipids. The donor microplate (PVDF
membrane, pore size 0.45 mm) and the acceptor microplate
were both from Millipore. The 96-well UV plate (COSTAR®) was
from Corning Incorporated. The acceptor 96-well microplate
was lled with 300 mL of PBS/EtOH (7 : 3), and the lter
membrane was impregnated with 10 mL of PBL in dodecane
(20 mg mLꢂ1). Compounds were dissolved in DMSO at
5 mg mLꢂ1 and diluted 50-fold in PBS/EtOH (7 : 3) to achieve a
concentration of 100 mg mLꢂ1, 200 mL of which was added to
the donor wells. The acceptor lter plate was carefully placed on
the donor plate to form a sandwich, which was le undisturbed
for 16 h at 25 ꢁC. Aer incubation, the donor plate was carefully
removed and the concentration of compound in the acceptor
wells was determined using a UV plate reader (Flexstation® 3).
Every sample was analyzed at ve wavelengths, in four wells, in
at least three independent runs, and the results are given as the
mean ꢀ standard deviation. In each experiment, 9 quality
control standards of known BBB permeability were included to
validate the analysis set.
measured using a SpectraMax Paradigm plate reader (lex/lem
530/590). Error bars represent standard deviations for at least
three measurements.
¼
Acknowledgements
This research work was nancially supported by Innovative
Research Team in University (IRT1193), A Project Funded by the
Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD), Project of Graduate Education
Innovation of Jiangsu Province (No. CXZZ13_0320).
4.2.8. Cell viability and neuroprotection activity assay.54,55
PC12 cells (rat pheochromocytoma) was obtained from the Cell
Bank of the Chinese Academy of Sciences (Shanghai, China)
and cultured in RPMI-1640 medium containing 10% (v/v) foetal
bovine serum, 100 U penicillin per mL and 100 mg streptomycin
per mL under 5% CO2 at 37 ꢁC. The culture media were changed
every other day. Cells were subcultured in 96-well plates at a
seeding density of 2 ꢃ 104 cells per well and allowed to adhere
and grow. When cells reached the required conuence, they
were placed into serum-free medium and treated with
compounds 2, 4, 5, 17 and 18. Twenty-four hours later the
survival of cells was determined by MTT assay. Briey, aer
incubation with 20 mL of MTT at 37 ꢁC for 4 h, living cells
containing MTT formazan crystals were solubilized in 200 mL
DMSO. The absorbance of each well was measured using a
microculture plate reader with a test wavelength of 570 nm and
a reference wavelength of 630 nm. Results are expressed as the
mean ꢀ SD of three independent experiments.
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