Synthesis and evaluation of 4-hydroxyl aurone derivatives as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2016.04.012

A series of 4-hydroxyl aurone derivatives were designed synthesized and evaluated as potential multifunctional agents for the treatment of Alzheimer's disease. The results demonstrated that most of the derivatives exhibited good multifunctional properties. Among them, compound 14e displayed good inhibitory activities of self- and Cu²⁺-induced Aβ_1-42 aggregation with 99.2% and 84.0% at 25 μM, respectively, and high antioxidant activity with a value 1.90-fold of Trolox. In addition, 14e also showed remarkable inhibitory activities of both monoamine oxidase A and B with IC₅₀ values of 0.271 μM and 0.393 μM, respectively. However the 6-methoxyl aurones 15a-c revealed excellent selectivity toward MAO-B. Furthermore, the representative compounds 14e and 15b displayed good metal-chelating abilities and blood-brain barrier (BBB) permeabilities in vitro.

Full text of DOI:10.1016/j.bmc.2016.04.012

Accepted Manuscript
Synthesis and evaluation of 4-hydroxyl aurone derivatives as multifunctional
agents for the treatment of Alzheimer’s disease
Yan Li, Xiaoming Qiang, Li Luo, Yuxing Li, Ganyuan Xiao, Zhenghuai Tan,
Yong Deng
PII:
S0968-0896(16)30246-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.012
BMC 12926
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
10 March 2016
5 April 2016
5 April 2016
Please cite this article as: Li, Y., Qiang, X., Luo, L., Li, Y., Xiao, G., Tan, Z., Deng, Y., Synthesis and evaluation
of 4-hydroxyl aurone derivatives as multifunctional agents for the treatment of Alzheimer’s disease, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.012
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Synthesis and evaluation of 4-hydroxyl aurone derivatives as multifunctional
agents for the treatment of Alzheimer’s disease
a
a
a
a
a
b,*
a,*
Yan Li , Xiaoming Qiang , Li Luo , Yuxing Li , Ganyuan Xiao , Zhenghuai Tan , Yong Deng
a
Department of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
b
Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, China
*
Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
1

Abstract
A series of 4-hydroxyl aurone derivatives were designed synthesized and evaluated as potential
multifunctional agents for the treatment of Alzheimer’s disease. The results demonstrated that most
of the derivatives exhibited good multifunctional properties. Among them, compound 14e displayed
2
+
good inhibitory activities of self- and Cu -induced Aβ1-42 aggregation with 99.2% and 84.0% at 25
μM respectively, and high antioxidant activity with a value 1.90-fold of Trolox. In addition, 14e also
showed remarkable inhibitory activities of both monoamine oxidase A and B with IC50 values of
0
.271 μM and 0.393 μM respectively. However the 6-methoxyl aurones 15a-c revealed excellent
selectivity toward MAO-B. Furthermore, the representative compounds 14e and 15b displayed good
metal-chelating abilities and blood-brain barrier (BBB) permeabilities in vitro.
Keywords:
Alzheimer’s disease; aurone derivatives; multifunctional agents; β-amyloid aggregation; monoamine
oxidase.
2

1
. Introduction
Alzheimer’s disease (AD), an age-related neurodegenerative disorder of the central nervous
system (CNS), is characterized by insidious onset of memory loss, progressive cognitive
1
deterioration, impairment of activities of daily living and loss of independent function. In addition,
5
0-80% of AD patients suffer from behavioral and psychological symptoms such as depression,
2
psychosis and agitation. Although the etiology of AD remains elusive, multiple factors such as
β-amyloid (Aβ) deposits, τ-protein aggregation, oxidative stress, dyshomeostasis of biometals and
decreased levels of acetylcholine (ACh) are considered to play vital roles in the pathophysiology of
3
AD. According to the amyloid cascade hypothesis, the increased production and accumulation of Aβ
oligomeric aggregates in the brain are considered as a central event in the pathogenesis of AD,
4
initiating the pathogenic cascade and ultimately leading to neuronal loss and dementia. The Aβ1-40
and Aβ1-42 are the major isoforms of Aβ peptides. Aβ1-40 is expressed in larger amounts in the brain,
5
,6
yet Aβ1-42 displays lower solubility and more neurotoxic and has a higher tendency to aggregate.
Therefore, preventing the Aβ1-42 aggregation is a potent therapeutic strategy for AD treatment.
2
+
2+
2+
3+
In addition, the dyshomeostasis of metal ions such as Cu , Fe , Zn and Al clearly occurs in
7
AD brains. Studies suggest that excess of these metal ions have been found in Aβ plaques and they
8
are closely associated with the formation of Aβ plaques and neurofibrillary tangles. Moreover, the
abnormally high levels of these redox-active metals could promote the production of reactive oxygen
9
species (ROS) and oxidative stress. However, oxidative stress is one of the earliest events in the
pathogenesis of AD and may occur before the onset of memory loss, the appearance of senile plaques
1
0
and neurofibrillary tangles. And oxidative damage plays a crucial role in neuronal degeneration,
11,12
which can harm biological molecules such as proteins, DNA, and lipids.
It is demonstrated that
2
+
3+
Aβ catalyses the reduction of Cu and Fe , which could lead to the production of ROS. In turn,
oxidative stress may promote Aβ accumulation by generating the modified Aβ species which are
1
3
prone to aggregate and resistant to clearance. Thus, biometals chelators and antioxidants have been
proposed as potential therapeutic agents against AD.
Monoamine oxidase A and B (MAO-A and -B) are flavin adenine dinucleotide
(
FAD)-containing enzymes, bound to the membrane surface of mitochondria and involved in the
degradation of neurotransmitters and xenobiotic amines. These enzymes are responsible for
3

regulation and metabolism of major monoamine neurotransmitters such as serotonin, dopamine and
1
4
noradrenaline. Selective MAO-A inhibitors are used in clinical practice for the treatment of
depression and anxiety, while MAO-B inhibitors are used to slow down the progression of
Parkinson’s disease (PD) and symptoms associated with AD. However, the biochemical activity of
MAO generates hydroxyl radicals, which are harmful members of the oxygen free radical group
1
5
involved in many neurodegenerative disorders like AD. Recently, MAO inhibitors have been
re-evaluated as potential therapeutics against many age-related pathologies, for example AD and PD.
And in these diseases, neurotoxicity, protein misfolding and/or aggregation, iron accumulation,
1
6,17
mitochondrial damage, and oxidative stress have been considered as major downstream causes.
As mentioned above, MAO could be a potential therapeutic target of AD and other psychiatric and
neurological diseases. In fact, selective MAO-B inhibitors such as selegiline and rasagiline are
beneficial for the treatment of PD and AD, and selective MAO-A inhibitors such as clorgyline and
moclobemide are useful for the treatment of neurological disorders, for example depression and
1
5
anxiety.
Because of the complicated and multifactorial etiology, the “one-molecule, one-target”
paradigm seems not so effective in treating complex diseases like AD. It can only improve clinical
symptoms but cannot mitigate progression of the disease. Therefore, with the development of
multi-target-directed ligands (MTDLs), it is significant to develop novel multifunctional drugs with
1
8-20
two or more complementary bioactivities for the treatment of complex diseases as AD.
Aurones, 2-benzylidenebenzofuran-3(2H)-ones, which are structural isomerides of flavones, are
21
present in vegetables and flowers (Figure 1, 1). Aurones have attracted considerable attentions in
recent years because they possess a wide range of bioactivities associated with neurological diseases
especially for AD. Many studies have shown that naturally occurring aurones as well as the
2
2-24
chemically synthesized analogs exhibited high affinities toward Aβ aggregates
and good MAO
2
5,26
inhibitory activities
. These results suggest that aurone could be an excellent leading scaffold to
design multifunctional drugs against AD. Recently, Masahiro Ono et al. reported an aurone analog,
which possess a radioiodine at 5-position and a dimethylamino group 4’-position (Figure 1, 2). It
2
3
could serve as a probe of Aβ plaques in AD, with high binding affinity (K
i
= 6.82 nM). In addition,
some studies have shown that electron-donating groups like dimethylamino group are closely
27
associated with the binding affinity to Aβ aggregates. Collectively, we designed a series of aurone
4

derivatives that are expected to be multifunctional agents with anti-Aβ aggregation, MAO inhibition,
antioxidant and biometal chelating properties (Figure 2). Our derivatives reported in this study are
2
8-31
either novel compounds, or although known substances
agents has not been reported.
their testing as multifunctional anti-AD
Figure 1. Aurone scaffolds (1) and aurone derivative (2) as Aβ plaques probes.
Figure 2. Structures of the designed multifunctional aurone derivatives 14-17.
. Results and discussion
.1. Chemistry
The synthetic pathways of target derivatives were summarized in Scheme 1 and 2. Substituted
2
2
32
benzaldehydes 8b-h were synthesized according to well-established method. The key intermediate
5
7
was produced from phloroglucinol 3 after Fries rearrangement and cyclization reaction. Compound
was obtained by the dimethylation of 5 and subsequent selective deprotection due to the difficulty
3
3
of direct transformation from 5 to 7. Target compounds 14a-h and 15a-c were afforded by the
condensation of 5 or 7 with corresponding substituted benzaldehydes 8a-h in ethanolic 50% KOH
solution. As reported in many literatures, only Z stereoisomers were isolated. The stereochemistry of
the aurone diastereomers has been elucidated by NMR spectroscopic measurements and by X-ray
2
9,34-36
diffraction analysis.
Compound 16a and 16b were obtained through catalytic hydrogenation of
5

1
4a and 14f with 10% Pd/C as catalyst in THF. The synthesis of 11 was reported by our group
6
previously. The condensation of 11 with p-dimethylaminobenzaldehyde (8a) in ethanolic 50% KOH
solution afforded chalcone 12. Subsequently, cyclization of 12 with mercuric acetate in pyridine
produced compound 13, deprotection of which afforded the target compound 17. All the aurone
1
derivatives were characterized by H NMR and HR-ESI-MS, and parts of them were further
1
3
characterized by C NMR.
Scheme 1. Synthesis of aurone derivatives 14-16. Reagents and conditions: (a) ClCH
2
COCl, AlCl
3
,
EtOAc, r.t., overnight; then 50 °C, 6 h; (b) NaOAc, MeOH, reflux for 4 h; (c) Me
2
SO , K CO ,
4
2
3
dimethoxyethane, reflux for 5 h; (d) AlCl
3
, CH
2
Cl , reflux for 5 h; (e) 2- or 4-substituted
2
benzaldehyde (8a-h), 50% KOH, EtOH, r.t., overnight; (f) H
2
, 10% Pd/C, THF, r.t., overnight.
Scheme 2. Synthesis of aurone derivatives 17. Reagents and conditions: (g) Ac
2
O, BF
3
•Et O, EtOAc,
2
6

5
5
0 °C, 10 h; (h) HN(CH
3
)
2
•HCl, NaHCO
3
, H
2
O, 70 °C, 10 h; (i) Me
2
SO
4
, K
2
CO
3
, acetone, reflux for
, pyridine, reflux
h; (j) (4-Me N)benzaldehyde (8a), 50% KOH, EtOH, r.t., overnight; (k) Hg(OAc)
2
2
for 15 h; (l) 40% HBr, 100 °C, 2 h.
2
2
.2. Pharmacology
.2.1. Inhibition of self- and Cu -induced Aβ1-42 aggregation
2
+
2
+
The inhibition of self- and Cu -induced Aβ1-42 aggregation by our synthetic derivatives was
3
7,38
determined by using thioflavin T (ThT) assay, with curcumin as a reference compound.
The
results summarized in Table 1 indicated that these aurone derivatives exhibited moderate to good
2
+
inhibitory activities of self- and Cu -induced Aβ1-42 aggregation (22.3-99.8% and 20.9-90.1% at 25
μM respectively) compared with that of curcumin (41.3 ± 0.9% and 67.2 ± 1.3% respectively).
Noticeably, the potencies of dihydroxy aurones 14a-h were much higher than those of corresponding
monohydroxy aurones and the reduction products. It revealed that 6-hydroxy group in aurone may
strengthen the interaction between the derivatives and Aβ1-42 protein and the α, β-unsaturated ketone
skeleton of aurone was essential for the potency. From the comparison between the potencies of 14a
and 14h, we could find that the substitution position of amino groups may have no obvious impact
on the inhibitory activities of Aβ1-42 aggregation. The results of 14a-h demonstrated that, unlike
hydroxyl group, diverse amino groups showed no significant difference between the effects on
potencies (except for 14f and 14g). Compared the potencies of 14a and 17, we could find that the
transformation of 6-OH to 6-N(CH
importance of 6-OH group.
3
) lowered the inhibitory activity, further indicating the
2
2
+
Table 1. In vitro inhibition of self- and Cu -induced Aβ1-42 aggregation, MAO inhibitory activities
and oxygen radical absorbance capacity (ORAC, Trolox equivalents) of aurone derivatives and
reference compounds.
a
%
Inhibition of Aβ1-42 aggregation
IC50 ± SD (μM)
e
f,g
Compd.
SI
ORAC
b,g
2+
c,g
d,g
d,g
Self-induced
97.4 ± 1.3
95.3 ± 2.0
91.7 ± 2.1
99.8 ± 1.5
99.2 ± 1.1
80.7 ± 1.7
77.7 ± 2.4
Cu -induced
MAO-A
MAO-B
5.940 ± 0.021
1
4a
4b
61.4 ± 2.2
86.6 ± 1.7
88.9 ± 3.1
84.8 ± 1.6
84.0 ± 1.5
50.1 ± 3.1
52.0 ± 2.8
0.233 ± 0.005
0.039
0.035
0.344
1.169
0.690
3.176
2.90 ± 0.03
1.97 ± 0.07
2.04 ± 0.05
1.56 ± 0.02
1.90 ± 0.09
2.50 ± 0.05
1
0.0279 ± 0.004 0.808 ± 0.023
1
4c
4d
4e
4f
4g
0.115 ± 0.009
0.457 ± 0.010
0.271 ± 0.013
1.08 ± 0.024
15.8% ± 0.9%
7
0.334 ± 0.008
0.391 ± 0.011
0.393 ± 0.025
0.34 ± 0.031
10.8 ± 0.102
1
1
1
1
> 0.926 2.50 ± 0.05

1
1
1
4h
5a
5b
98.2 ± 1.6
53.7 ± 1.3
40.3 ± 1.0
44.1 ± 2.1
42.7 ± 2.2
38.0 ± 2.4
22.3 ± 1.2
41.3 ± 0.9
—
90.1 ± 1.3
75.7 ± 1.2
46.0 ± 1.9
47.5 ± 2.2
40.5 ± 2.6
20.9 ± 2.1
71.6 ± 1.3
67.2 ± 1.3
—
13.5% ± 1.0%
20.7% ± 1.5%
18.3% ± 1.1%
18.4% ± 1.4%
23.5% ± 2.5%
13.9% ± 1.2%
16.8% ± 2.1%
—
0.88% ± 0.3%
—
2.91 ± 0.02
0.703 ± 0.043 > 14.225 1.62 ± 0.08
0.226 ± 0.017 > 44.248 1.00 ± 0.06
1
5c
6a
1.50 ± 0.098
27.2% ± 2.9%
8.7% ± 1.0%
13.5% ± 1.2%
—
> 6.667 1.66 ± 0.04
1
—
—
3.07 ± 0.17
1
6b
3.56 ± 0.14
1
7
—
2.43 ± 0.12
Curcumin
Clorgyline
Rasagiline
Iproniazid
—
—
—
—
—
0.0027 ± 0.0006
1.42 ± 0.015
2.56 ± 0.023
4.19 ± 0.101
0.0825 ± 0.002
1.95 ± 0.074
0.00064
17.21
1.31
—
—
—
—
a
b
c
For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
Inhibition of self-induced Aβ1-42 aggregation (25 μM) by tested inhibitors at 25 μM.
2+
2+
Inhibition of Cu -induced Aβ1-42 aggregation. The concentration of tested compounds and Cu
were 25 μM.
d
Percentages are the percent inhibition of MAO by tested compounds at 10 μM.
e
SI = IC50 (MAO-A) / IC50 (MAO-B)
f
The data are expressed as μM of Trolox equiv/μM of tested compound.
g
The mean ± SD of the three independent experiments.
2
.2.2. Recombinant human MAO-A and –B inhibition studies
Subsequently, we evaluated these compounds for their potential MAO-A and -B inhibitory
properties. Recombinant human MAO-A and -B were employed as enzyme sources, with clorgyline,
rasagiline and iproniazid as reference compounds. Kynuramine, the common substrate for MAO-A
and -B, was used for the enzyme activity measurement of both isozymes. Kynuramine is oxidized by
the isozymes to yield 4-hydroxyquinoline which fluoresces in the alkaline medium used to terminate
3
9-41
the enzymatic reactions.
The concentrations of 4-hydroxyquinoline in the reactions were
measured using fluorescence spectrophotometry without interference from kynuramine or test
compounds. We first tested our derivatives at the concentration of 10 μM for preliminary screening.
The samples with the inhibition ratios greater than 50% would be retested for the IC50 values at
various concentrations. All determinations were carried out in triplicate. The results were
summarized in Table 1. As it shown, most of the aurone derivatives were effective in inhibiting
MAO in submicromolar range. Compound 14b was the most potent MAO-A inhibitor with an IC50 of
0
.0279 ± 0.004 μM. And the most potent MAO-B inhibitor was 15b (IC50 = 0.226 ± 0.017 μM).
8

From the results of 14a-g, we could find that cyclic amine substituted aurones exhibited MAO-B
selective inhibitory activities, while the non-cyclic amine substituted aurones preferred MAO-A.
Different from the conclusion of Aβ1-42 aggregation assays, the comparison between the potencies of
1
4a and 14h derived that the substituted sites were of great importance and the 4’-substituted aurones
were more effective. Interestingly, when the hydroxyl group at the 6-position of aurone (14a, 14e and
4f) was replaced by a methoxy group (15a-c), the inhibitory activity to MAO-A decreased
1
dramatically. On the contrary, the MAO-B inhibitory potencies increased or showed no significant
change. As a result, the selectivity to MAO-B was remarkably elevated. It indicated that the hydroxyl
group at 6-position of aurone was critical to the MAO-A inhibitory activity, while 6-methoxy group
was beneficial to the MAO-B selectively inhibition. The low potency of 17 suggested that only
hydroxyl or methoxy group at 6-position was profitable. From the results of 16a and 16b, we
concluded that the α,β-unsaturated ketone skeleton of aurone was indispensable, consistent with the
above conclusion of Aβ1-42 aggregation assays.
2
.2.3. Molecular modeling study
In order to explore the hypothetical binding modes of the aurone derivatives with respect to both
isoforms of human MAO, docking simulations were carried out using the docking program,
AutoDock 4.2 package with Discovery Studio 2.5 based on the X-ray crystal structures of human
MAO-A (PDB code: 2Z5X) and MAO-B (PDB code: 2V60). Taking into account the experimental
hMAO inhibition data and the resulting structure-activity relationship, compounds 14e and 15b were
chosen for our molecular modeling investigation. And the most stable binding modes of both
compounds in the hMAO-A and -B active sites were graphically inspected (Figure 3 and 4). As
shown in Figure 3, the benzofuran-3(2H)-one moieties of 14e and 15b were close to the FAD
cofactor, adopting parallel π-π interactions with Tyr407. And the B-rings of both 14e and 15b
adopted parallel π-π interactions with Phe208. In Figure 3A, the 6-OH of 14e was involved in two
hydrogen bonds between Asn181 and Tyr444 (Asn181=O • • • HO-14e; Tyr444-OH • • • O-14e).
While in Figure 3B, one hydrogen bond was generated between the 6-OCH
3
of 15b and Tyr444
(
Tyr444-OH • • • O-15b). This could explain the critical function of hydroxyl group at the 6-position
and the significant difference between the MAO-A inhibitory activities of 14e and 15b. Meanwhile,
both 14e and 15b interacted with many other amino acids, such as Tyr69, Leu97, Ala111, Ile180,
Val210, Gln215, Cys323, Ile325, Ile335, Leu337, Phe352 and so on.
9

Figure 3. Representation of compound 14e (A) and 15b (B) docked into the binding site of hMAO-A,
highlighting the protein residues that participate in the main interactions with the inhibitor.
Hydrogen-bonds are shown with the green dotted lines. Pictures were generated with Discovery
Studio 2.5. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
In both 14e- and 15b-hMAO-B complexes, the interaction pattern was similar (Figure 4). From
Figure 4A, it could be found that four hydrogen bonds were generated between the ligand 14e and
the active site of hMAO-B: two between the hydroxyl group at 4-position and Tyr435-OH
(
Tyr435-OH • • • O-14e; Tyr435-O • • • HO-14e), one between the carbonyl group at 3-position and
Tyr435-OH (Tyr435-OH • • • O=14e) and one between the hydroxyl group at 6-position and the FAD
N5 atom (FAD-N5 • • • OH-14e). Similarly in 15b-hMAO-B complex (Figure 4B), the hydroxyl
group at 4-position and carbonyl group at 3-position interacted with Cys172 and Tyr435 via four
hydrogen bonds in total (Cys172=O • • • HO-15b; Cys172-SH • • • O=15b; Tyr435-OH • • • O=15b;
Tyr435-O • • • HO-15b). Moreover, in both 14e- and 15b-hMAO-B complexes, the
benzofuran-3(2H)-one moieties and B-rings interacted with Tyr398 and Tyr326 via parallel π-π
interactions, respectively. Other interactions could be observed between the ligands (14e and 15b)
and Pro102, Pro104, Phe168, Leu171, Ile198, Ile199, Gln206, Ile316, Phe343 and so on. The
docking studies, consistent with the conclusion resulting from the hMAO inhibition assay, explained
the high MAO inhibitory activity and the MAO-B selectivity of these aurone derivatives.
10

Figure 4. Representation of compound 14e (A) and 15b (B) docked into the binding site of hMAO-B,
highlighting the protein residues that participate in the main interactions with the inhibitor.
Hydrogen-bonds are shown with the green dotted lines. Pictures were generated with Discovery
Studio 2.5. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
2
.2.4. Antioxidant activity assay
The antioxidant activities of aurone derivatives were evaluated by the well-established
4
2
ORAC-FL method (oxygen radical absorbance capacity by fluorescein) with the results shown in
Table 1. Trolox, a water-soluble vitamin E analog, was used as the standard. And the activities of
tested compounds to scavenge radicals were expressed as Trolox equivalent. Their relative activities
at concentration of 5 μM were compared with the highly potent compound Trolox. All the tested
derivatives exhibited high peroxyl radical absorbance capacities, ranging from 1.00 to 3.56-fold of
Trolox. Upon the comparison of the results of 14a-h with that of 15a-c, it was apparent that the
dihydroxy aurone derivatives (except for 14d) possessed higher antioxidant potencies than the
monohydroxy aurones. It could be found that the reduction products 16a and 16b displayed excellent
antioxidant activities, higher than other derivatives. The results of 14a, 15a and 17 (ORAC-FL values
of 2.90 ± 0.03, 1.62 ± 0.08 and 2.43 ± 0.12 equiv, respectively) demonstrated that the introduction of
dimethylamino group at 6-position was important to the improvement of antioxidant activity,
although the potency of 17 was slightly inferior to that of 14a. Among the tested compounds, 16b
showed the most potent antioxidant activity with ORAC-FL values of 3.56 ± 0.14 Trolox
equivalents.
11

Figure 5. UV spectra of compound 14e (A) and 15b (B) (37.5 μM in methanol) alone or in the
presence of CuCl , ZnCl , FeSO or AlCl (37.5 μM, in methanol). Determination of the
stoichiometry of complex-Cu by using the molar ratio method of titrating the methanol solution of
compound 14e (C) and 15b (D) with ascending amounts of CuCl . The final concentration of tested
compound was 37.5 μM, and the final concentration of Cu ranged from 3.75 to 150 μM.
2
2
4
3
2
+
2
2
+
2
.2.5. Metal-chelating studies
In addition, 14e and 15b were chosen as representatives to evaluate the metal chelating abilities
2
+
2+
2+
3+
toward biometals such as Cu , Zn , Fe and Al using UV-visual spectrometry. The results were
shown in Figure 5. It could be seen that, the spectrum of 14e was significantly altered upon the
addition of CuCl
absorption from 450 nm to 498 nm occurred, indicating the formation of a 14e-Cu complex. The
similar red shifts were also observed when ZnCl and AlCl were added. However, no significant
shift occurred with the addition of FeSO . The chelating effect could be reasonably attributed to the
-carbonyl and 4-hydroxyl groups in aurone. Similar results were observed for 15b (Figure 5B). The
2
, ZnCl
2
and AlCl
3
(Figure 5A). When CuCl was added, a red shift in the maximum
2
2
+
2
3
4
3
2
+
2+
molar ratio method was performed to determine the stoichiometry of 14e-Cu and 15b-Cu
complexes, by preparing the methanol solutions of 14e and 15b with ascending amounts of CuCl
2
.
12

The UV spectra were used to obtain the absorbance of the 14e and 15b complexes and different
concentrations of CuCl at 498 nm and 488 nm respectively. The results showed that absorbance
2
linearly increased initially and then plateaued (Figure 5C and 5D). The points for the straight lines to
intersect were determined to be at a mole proportion of 0.95 and 1.11 respectively, revealing a 1:1
2
+
2+
stoichiometry for 14e-Cu and 15b-Cu complexes.
2
.2.6. In vitro blood-brain barrier permeation assay
An important prerequisite of any compound to act on neurodegenerative processes is to cross
the blood-brain barrier (BBB) and penetrate into the brain. To predict the possible in vivo BBB
permeability of the aurone derivatives, the parallel artificial membrane permeation assay of the
4
3,44
blood-brain barrier (PAMPA-BBB) was performed
and 14e and 15b were still chosen as
representatives. Assay validation was made by comparing experimental permeabilities of 11
commercial drugs with reported values (Table 2). A plot of experimental data versus bibliographic
2
values gave a good linear correlation, P
e
(exp.) = 0.9163 × P
e
(bibl.) − 0.2247 (R = 0.9558) (Figure
4
3
6
). From this equation and taking into account the limit established by Di et al. for BBB permeation,
-6
we determined that compounds with P values above 3.44 × 10 cm/s could cross the BBB (Table 3).
e
The result in Table 4 indicated that 14e and 15b would be able to penetrate the BBB and reach the
therapeutic targets in central nervous system (CNS).
-6
Table 2. Permeability P
the experiment validation.
Commercial drugs
e
(× 10 cm/s) in the PAMPA-BBB assay for 11 commercial drugs used in
a
b
Bibl
16
10
PBS/EtOH (70:30)
16.90 ± 0.36
9.60 ± 0.21
Verapamil
Oxazepam
Diazepam
Clonidine
Imipramine
Testosterone
Caffeine
16
5.3
13
11.86 ± 0.23
5.10 ± 0.16
10.10 ± 0.22
16.30 ± 0.25
1.28 ± 0.05
0.47 ± 0.01
0.72 ± 0.02
0.42 ± 0.01
0.10 ± 0.003
17
1.3
0.9
2.5
0.1
0.12
Enoxacine
Piroxicam
Norfloxacin
Theophylline
43
a
Taken from Ref.
b
Data are the mean ± SD of three independent experiments.
13

Figure 6. Lineal correlation between experimental and reported permeability of commercial drugs
2
using the PAMPA-BBB assay. P
e
(exp) = 0.9163 × P
e
(bibl.) − 0.2247 (R = 0.9558).
-6
Table 3. Ranges of permeability of PAMPA-BBB assays (P
e
× 10 cm/s).
> 3.44
3.44 > P
< 1.61
High BBB permeation predicted (CNS +)
Uncertain BBB permeation (CNS +/-)
Low BBB permeation predicted (CNS -)
P
e
e
> 1.61
P
e
-6
Table 4. Permeability results P
e
(× 10 cm/s) from the PAMPA-BBB assay for selected aurone
derivatives with their predicted penetration into the CNS.
a
-6
b
Compd.
P
e
(× 10 cm/s)
Prediction
CNS +
1
1
4e
6.68 ± 0.29
7.24 ± 0.22
5b
CNS +
a
Compound 14e and 15b were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30).
The final concentration of each compound was 100 μg/mL.
b
Data are the mean ± SD of three independent experiments.
3
. Conclusion
In conclusion, a series of aurone derivatives were designed, synthesized and evaluated as
multifunctional agents for the treatment of AD. We observed that all the compounds exhibited
2
+
moderate to good inhibitory activities of self- and Cu -induced Aβ1-42 aggregation. And most of
them were effective in inhibiting MAO in submicromolar range. Furthermore, 15a-c exhibited high
selectivity to MAO-B over MAO-A, which may serve as potential MAO-B selective inhibitors. All
14

of the tested derivatives showed high antioxidant activities, ranging from 1.00 to 3.56-fold of Trolox,
revealing their potential abilities to decrease the oxidative damage. Moreover, the representative
compounds 14e and 15b showed good metal-chelating properties and BBB permeabilities. These
properties indicate that the aurone derivatives may provide a good template for the development of
new multifunctional anti-AD agents.
4
4
. Experimental section
.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
1
13
without further purification. The H NMR and C NMR spectra were recorded using TMS as the
internal standard in DMSO-d on a Varian INOVA spectrometer at 25 °C. Coupling constants are
6
given in Hz. MS spectra data were obtained on an Agilent-6210 TOF LC-MS spectrometer. Melting
points (uncorrected) were measured on YRT-3 melting-point apparatus (China). Reaction progress
was monitored by thin-layer chromatography (TLC) using silica gel GF254 plates from Qingdao
Haiyang Chemical Co. Ltd. (China) and the spots were detected under UV light (254 nm). Column
chromatography was performed with silica gel (230–400 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. (China).
4
.1.1. Synthesis of 2-chloro-1-(2,4,6-trihydroxyphenyl)ethanone (4)
Anhydrous phloroglucinol (3, 5.02 g, 39.8 mmol) was dissolved in EtOAc (75 mL). AlCl (21.1
3
g, 158 mmol) was added at room temperature to give a pale brown suspension. The reaction mixture
was stirred at room temperature for 30 min. Chloroacetyl chloride (3.28 mL, 43.6 mmol) was added,
and the reaction mixture was stirred at room temperature overnight and then heated at 50 °C for 6 h.
The reaction mixture was quenched by pouring into ice-water and the product was extracted with
EtOAc (6 × 60 mL), then washed with brine, dried over Na
2
SO , filtered and concentrated under
4
reduced pressure to give compound 4 (6.25 g) as a pale yellow solid, which was used without further
purification.
4
.1.2. Synthesis of 4,6-dihydroxybenzofuran-3(2H)-one (5)
Compound 4 (6.25 g) was dissolved in methanol (94 mL) and anhydrous NaOAc (5.34 g, 65.1
mmol) was added. The reaction mixture was then refluxed under argon atmosphere for 4 h. The
methanol was evaporated and the residue was recrystallized from water to give compound 5 (4.54 g,
15

1
8
8.2%) as a pale yellow solid. H NMR (400 MHz, DMSO-d ) δ 10.62 (brs, 2H), 5.91 (s, 2H), 4.54
6
2
8
(
s, 2H). (Data are in agreement with references .)
4
.1.3. Synthesis of 4-hydroxy-6-methoxybenzofuran-3(2H)-one (7)
Compound 7 was obtained by the dimethylation of 5 and subsequent selective deprotection
3
3
33
using the method reported previously. mp 139-141 °C (lit. 140-142 °C).
.1.4. General procedure for the synthesis of 8a-h
Compounds 8b-h were prepared as previously described. Compound 8a was obtained from
commercial supplier.
4
3
2
4
.1.5. General procedure for the synthesis of 14a-h and 15a-c
To a solution of 5 or 7 in ethanol (3 mL/mmol) were added an aqueous solution of potassium
hydroxide (50%, 5 mL/mmol) and a benzaldehyde derivative (8a-h, 1.5 equiv.) The solution was
stirred overnight until TLC showed complete disappearance of the starting material. Ethanol was
removed under reduced pressure. The residue was diluted into distilled water and acidified with an
aqueous solution of hydrochloric acid (10%), then the mixture was basified with saturated NaHCO
3
solution to adjust the pH to 7-8. The precipitate was filtered, washed with water and dried at room
temperature to afford the corresponding crude aurone derivative as orange to dark red solid.
4
.1.5.1. (Z)-2-(4-(dimethylamino)benzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (14a)
Compound 14a was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8a according to the general procedure. After purification by
chromatagraphy on silica gel using petroleum ether/acetone (2:1) as eluent, the pure product 14a was
1
obtained as a red solid, yield 60.7%. H NMR (600 MHz, DMSO-d
6
) δ 10.77 (s, 1H), 10.75 (s, 1H),
7
6
2
4
.74 (d, J = 7.8 Hz, 2H), 6.79 (d, J = 7.8 Hz, 2H), 6.54 (s, 1H), 6.20 (s, 1H), 6.07 (s, 1H), 3.00 (s,
2
+
H). (Data are in agreement with references .) HR-ESI-MS: Calcd. for C17
H
16NO [M + H] :
4
98.1079, found : 298.1078.
.1.5.2. (Z)-2-(4-(ethyl(methyl)amino)benzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (14b)
Compound 14b was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8b according to the general procedure. After purification by
chromatagraphy on silica gel using petroleum ether/acetone (2:1) as eluent, the pure product 14b was
1
obtained as a dark red solid, yield 38.1%. H NMR (600 MHz, DMSO-d
6
) δ 10.82 (brs, 2H), 7.72 (d,
16

J = 7.8 Hz, 2H), 6.77 (d, J = 7.8 Hz, 2H), 6.52 (s, 1H), 6.17 (s, 1H), 6.06 (s, 1H), 3.46 (s, 2H), 2.95 (s,
+
3
H), 1.07 (s, 3H). HR-ESI-MS: Calcd. for C18
H18NO
4
[M + H] : 312.1236, found : 312.1238.
4
.1.5.3. (Z)-2-(4-(diethylamino)benzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (14c)
Compound 14c was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8c according to the general procedure. After purification by chromatagraphy
on silica gel using dichloromethane/methanol (10:1) as eluent, the pure product 14c was obtained as
1
a dark red solid, yield 50.0%. H NMR (400 MHz, DMSO-d
6
) δ 10.75 (brs, 2H), 7.71 (d, J = 8.8 Hz,
2
7
4
H), 6.73 (d, J = 8.8 Hz, 2H), 6.51 (s, 1H), 6.18 (s, 1H), 6.06 (s, 1H), 3.41-3.36 (m, 4H), 1.12 (t, J =
+
.2 Hz, 6H). HR-ESI-MS: Calcd. for C19
H
20NO [M + H] : 326.1392, found : 326.1399.
4
.1.5.4. (Z)-4,6-dihydroxy-2-(4-(pyrrolidin-1-yl)benzylidene)benzofuran-3(2H)-one (14d)
Compound 14d was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8d according to the general procedure. After purification by
chromatagraphy on silica gel using petroleum ether/acetone (2:1) as eluent, the pure product 14d was
1
obtained as a dark red solid, yield 36.3%. H NMR (400 MHz, DMSO-d
6
) δ 10.90 (brs, 2H), 7.72
(
brs, 2H), 6.61 (brs, 2H), 6.51 (s, 1H), 6.15 (s, 1H), 6.02 (s, 1H), 3.36 (brs, 4H), 1.96 (brs, 4H).
+
HR-ESI-MS: Calcd. for C19
H
18NO [M + H] : 324.1236, found : 324.1234.
4
4
.1.5.5. (Z)-4,6-dihydroxy-2-(4-(piperidin-1-yl)benzylidene)benzofuran-3(2H)-one (14e)
Compound 14e was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8e according to the general procedure. After purification by chromatagraphy
on silica gel using dichloromethane/methanol (15:1) as eluent, the pure product 14e was obtained as
1
a dark red solid, yield 47.1%. H NMR (400 MHz, DMSO-d
6
) δ 10.78 (brs, 2H), 7.73 (d, J = 8.8 Hz,
2
H), 6.98 (d, J = 8.8 Hz, 2H), 6.52 (s, 1H), 6.19 (s, 1H), 6.06 (s, 1H), 3.34 (s, 4H), 1.59 (s, 6H).
2
8
+
(
Data are in agreement with references .) HR-ESI-MS: Calcd. for C20
H
20NO [M + H] : 338.1392,
4
found : 338.1398.
4
.1.5.6. (Z)-4,6-dihydroxy-2-(4-morpholinobenzylidene)benzofuran-3(2H)-one (14f)
Compound 14f was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8f according to the general procedure. After purification by chromatagraphy
on silica gel using dichloromethane/methanol (15:1) as eluent, the pure product 14f was obtained as
1
an orange red solid, yield 19.6%. H NMR (600 MHz, DMSO-d
6
) δ 10.86 (brs, 2H), 7.77 (d, J = 8.4
Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.55 (s, 1H), 6.21 (s, 1H), 6.08 (s, 1H), 3.75 (t, J = 4.8 Hz, 4H),
17

3
0
3
.24 (t, J = 4.8 Hz, 4H). (Data are in agreement with references .) HR-ESI-MS: Calcd. for
+
C
19
H18NO
5
[M + H] : 340.1185, found : 340.1192.
4
.1.5.7.
14g)
Compound 14g was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
(Z)-4,6-dihydroxy-2-(4-(4-methylpiperazin-1-yl)benzylidene)benzofuran-3(2H)-one
(
benzaldehyde derivative 8g according to the general procedure. After purification by
chromatagraphy on silica gel using dichloromethane/methanol (8:1) as eluent, the pure product 14g
1
was obtained as an orange red solid, yield 10.1%. H NMR (400 MHz, DMSO-d
6
) δ 10.80 (brs, 2H),
7
4
3
4
.75 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 6.52 (s, 1H), 6.21 (s, 1H), 6.10 (s, 1H), 3.51 (brs,
+
H), 2.51 (brs, 4H), 2.27 (s, 3H). HR-ESI-MS: Calcd. for C20
53.1512.
H
21
N
2
O [M + H] : 353.1501, found :
4
.1.5.8. (Z)-2-(2-(dimethylamino)benzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (14h)
Compound 14h was synthesized from 4,6-dihydroxybenzofuran-3(2H)-one (5) and
benzaldehyde derivative 8h according to the general procedure. After purification by
chromatagraphy on silica gel using dichloromethane/methanol (10:1) as eluent, the pure product 14h
1
was obtained as an orange red solid, yield 64.8%. H NMR (400 MHz, DMSO-d
6
) δ 10.94 (brs, 2H),
8
.03 (d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.17-7.11 (m, 2H), 6.86 (s, 1H), 6.20 (s, 1H), 6.08
1
3
(
s, 1H).2.71 (s, 6H). C NMR (150 MHz, DMSO-d
6
) δ 179.5, 168.0, 167.6, 158.7, 154.1, 147.7,
1
31.1, 130.2, 125.6, 122.7, 119.0, 105.1, 103.0, 97.9, 90.7, 45.1 (2C). HR-ESI-MS: Calcd. for
+
C
17
H16NO
4
[M + H] : 298.1079, found : 298.1078.
4
.1.5.9.
15a)
Compound 15a was synthesized from 4-hydroxy-6-methoxybenzofuran-3(2H)-one (7) and
(Z)-2-(4-(dimethylamino)benzylidene)-4-hydroxy-6-methoxybenzofuran-3(2H)-one
(
benzaldehyde derivative 8a according to the general procedure. After purification by
chromatagraphy on silica gel using dichloromethane/methanol (20:1) as eluent, the pure product 15a
1
was obtained as an orange red solid, yield 15.6%. H NMR (400 MHz, DMSO-d
6
) δ 10.96 (brs, 1H),
7
3
4
.76 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 6.48 (s, 1H), 6.12 (s, 1H), 3.83 (s,
+
H), 2.99 (s, 6H). HR-ESI-MS: Calcd. for C18
H18NO
4
[M + H] : 312.1236, found : 312.1241.
.1.5.10. (Z)-4-hydroxy-6-methoxy-2-(4-(piperidin-1-yl)benzylidene)benzofuran-3(2H)-one
(
15b)
18

Compound 15b was synthesized from 4-hydroxy-6-methoxybenzofuran-3(2H)-one (7) and
benzaldehyde derivative 8e according to the general procedure. After purification by chromatagraphy
on silica gel using dichloromethane/methanol (20:1) as eluent, the pure product 15b was obtained as
1
an orange red solid, yield 53.3%, mp 200-201 °C. H NMR (400 MHz, DMSO-d
6
) δ 11.00 (brs, 1H),
7
3
1
.75 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.58 (s, 1H), 6.48 (s, 1H), 6.11 (s, 1H), 3.83 (s,
13
H), 3.32 (s, 4H), 1.59 (s, 6H). C NMR (150 MHz, DMSO-d ) δ 179.4, 168.0, 167.5, 158.2, 151.7,
6
45.8, 132.7 (2C), 121.2, 114.7 (2C), 110.4, 104.5, 97.0, 88.8, 56.2, 48.3 (2C), 25.2 (2C), 24.2.
+
HR-ESI-MS: Calcd. for C21
H22NO
4
[M + H] : 352.1549, found : 352.1553.
4
.1.5.11. (Z)-4-hydroxy-6-methoxy-2-(4-morpholinobenzylidene)benzofuran-3(2H)-one (15c)
Compound 15c was synthesized from 4-hydroxy-6-methoxybenzofuran-3(2H)-one (7) and
benzaldehyde derivative 8f according to the general procedure. After purification by chromatagraphy
on silica gel using dichloromethane/methanol (20:1) as eluent, the pure product 15c was obtained as
1
an orange red solid, yield 61.6%, mp 228 °C (decomposed). H NMR (400 MHz, DMSO-d
6
) δ 11.10
(
brs, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.60 (s, 1H), 6.48 (s, 1H), 6.10 (s, 1H),
1
3
3
1
.83 (s, 3H), 3.75 (s, 4H), 3.25 (s, 4H). C NMR (150 MHz, DMSO-d ) δ 179.4, 168.1, 167.6,
6
58.3, 151.6, 146.0, 132.5 (2C), 122.4, 114.5 (2C), 110.1, 104.3, 97.0, 89.0, 66.1 (2C), 56.3, 47.4
+
(
2C). HR-ESI-MS: Calcd. for C20
H
20NO
5
[M + H] : 354.1341, found : 354.1346.
4
.1.6. General procedure for the synthesis of 16a and 16b
A mixture of compound 14a or 14f (100 mg) in THF (2 mL) and 10% palladium on carbon (10
mg) was stirred under hydrogen (balloon) overnight. The mixture was then filtered and the filtrate
was concentrated to give crude product 16a or 16b as brown oil. Purification by chromatagraphy on
silica gel using dichloromethane/methanol as eluent afforded the pure product 16a or 16b.
4
.1.6.1. 2-(4-(dimethylamino)benzyl)-4,6-dihydroxybenzofuran-3(2H)-one (16a)
Compound 16a was synthesized from Compound 14a according to the general procedure. After
purification by chromatagraphy on silica gel using dichloromethane/methanol (15:1) as eluent, the
1
pure product 16a was obtained as an almost white solid, yield 52.5%, mp 193-195 °C. H NMR (400
MHz, DMSO-d
6
) δ 10.57 (brs, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 5.86 (s, 1H),
.85 (s, 1H), 4.73 (dd, J = 7.6 Hz, J = 3.6 Hz, 1H), 3.04 (dd, J = 14.8 Hz, J = 3.6 Hz, 1H), 2.83 (s,
H), 2.73 (dd, J = 14.8 Hz, J = 7.6 Hz, 1H). C NMR (100 MHz, DMSO-d ) δ 195.0, 174.4, 168.0,
5
6
1
2
1
2
13
1
2
6
19

1
57.9, 149.4, 130.1 (2C), 124.1, 112.5 (2C), 102.7, 96.3, 90.1, 86.0, 40.4 (2C), 36.1. HR-ESI-MS:
+
Calcd. for C17
H18NO
4
[M + H] : 300.1236, found : 300.1240.
4
.1.6.2. 4,6-dihydroxy-2-(4-morpholinobenzyl)benzofuran-3(2H)-one (16b)
Compound 16b was synthesized from Compound 14f according to the general procedure. After
purification by chromatagraphy on silica gel using dichloromethane/methanol (20:1) as eluent, the
1
pure product 16b was obtained as a pale yellow solid, yield 20.5%, mp 197-199 °C. H NMR (400
MHz, DMSO-d
6
) δ 10.59 (brs, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.87 (s, 1H),
.85 (s, 1H), 4.76 (dd, J = 8.4 Hz, J = 3.6 Hz, 1H), 3.71 (s, 4H), 3.09-3.04 (m, 5H), 2.75 (dd, J
4.8 Hz, J = 8.4 Hz, 1H). C NMR (150 MHz, DMSO-d ) δ 194.9, 174.4, 168.0, 157.9, 149.9,
5
1
1
1
2
1
=
1
3
2
6
30.1 (2C), 127.2, 115.1 (2C), 102.7, 96.3, 90.1, 85.8, 71.5 (2C), 48.7 (2C), 36.1. HR-ESI-MS:
+
Calcd. for C19
H20NO
5
[M + H] : 342.1341, found : 342.1345.
4
.1.7.(E)-1-(4-(dimethylamino)-2-hydroxy-6-methoxyphenyl)-3-(4-(dimethylamino)phenyl)prop
-
2-en-1-one (12)
The synthesis of 11 starting from anhydrous phloroglucinol (4) was reported by our group
6
previously. Compound 12 was synthesized from compound 11 and p-dimethylaminobenzaldehyde
(
8a) in the same manner to 14a-h and 15a-c as a brown oil, and the crude product was used without
further purification.
.1.8.(Z)-6-(dimethylamino)-2-(4-(dimethylamino)benzylidene)-4-methoxybenzofuran-3(2H)-on
e (13)
To a solution of chalcone 12 (204 mg) in pyridine (6 mL) was added Hg(OAc)
4
2
(191 mg, 0.600
mmol) at room temperature, and the mixture was refluxed for 15 h. The cooled reaction mixture was
poured into ice-cold water (20 mL) and extracted with EtOAc (5 × 20 mL), then washed with brine,
dried over Na
2
4
SO , filtered and concentrated under reduced pressure to give crude compound 13
(
249 mg) as a dark red solid, which was used without further purification.
4
.1.9.(Z)-6-(dimethylamino)-2-(4-(dimethylamino)benzylidene)-4-hydroxybenzofuran-3(2H)-on
e (17)
Compound 13 (100 mg) was dissolved in an aqueous solution of hydrobromic acid (40%, 2 mL)
and the solution was heated at 100 °C for 2 h. The cooled mixture was basified with saturated
NaHCO solution to adjust the pH to 7 and extracted with CH Cl (5 × 10 mL), then washed with
brine, dried over Na SO , filtered and concentrated under reduced pressure to give the crude product
3
2
2
2
4
20

as a brown solid. After purification by chromatagraphy on silica gel using dichloromethane/methanol
1
(
30:1) as eluent, the pure product 17 (15 mg) was obtained as a dark red solid. H NMR (400 MHz,
DMSO-d ) δ 10.45 (brs, 1H), 77.2 (d, J = 7.6 Hz, 2H), 6.76 (d, J = 7.6 Hz, 2H), 6.42 (s, 1H), 6.12 (s,
6
1
3
1
1
H), 5.83 (s, 1H), 3.03 (s, 6H), 2.98 (s, 6H). C NMR (150 MHz, DMSO-d ) δ 178.0, 167.4, 157.6,
6
50.6, 146.2, 132.2 (2C), 120.2, 112.2 (2C), 108.6, 100.3, 93.0, 86.0, 40.3 (2C), 39.9 (2C).
+
HR-ESI-MS: Calcd. for C19
H
21
N
2
O
3
[M + H] : 325.1552, found : 325.1558.
4
4
.2. Biological activity
2
+
.2.1. Inhibition of self- and Cu -induced Aβ1-42 aggregation
A Thioflavin T-based fluorometric assay was performed to investigate the self-induced Aβ1-42
3
7,45-46
aggregation.
Thioflavin T (Basic Yellow 1, ThT) was purchased from TCI (Shanghai)
Development. β-Amyloid1-42 (Aβ1-42), supplied as trifluoroacetate salt, was purchased from
ChinaPeptides Co., Ltd. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from Energy
Chemical. Briefly, Aβ1-42 was dissolved in HFIP (1 mg/mL) and after incubation for 24 h at room
temperature, the solvent was evaporated. Then the HFIP pretreated Aβ1-42 was dissolved in dry
DMSO to a final stock concentration of 200 μM and was kept frozen at –80 °C until use. Solutions of
test compounds were prepared in DMSO in 2.5 mM for storage and diluted with phosphate buffer
solution (pH 7.4) before use. For the self-induced assay, Aβ1-42 (20 μL, 25 μM, final concentration)
was incubated with 20 μL of test compounds (25 μM, final concentration) in 50 mM phosphate
buffer solution (pH 7.40) at 37 °C for 24 h. To minimize evaporation effect the wells were sealed by
a transparent heat-resistant plastic film. After incubation, 160 μL of 5 μM ThT in 50 mM
glycine-NaOH buffer (pH 8.5) was added. Each assay was run in triplicate. Fluorescence was
measured on a Varioskan Flash Multimode Reader (Thermo Scientific) with excitation and emission
wavelengths at 446 nm and 490 nm, respectively. The fluorescence intensities were calculated after
subtraction of the background activity. The background activity was determined from wells
containing all components except Aβ1-42, which were replaced by a phosphate buffer solution (pH
7
.40). The percent inhibition due to the presence of the inhibitor was calculated by the following
expression: (1-IF /IF ) × 100, in which IF and IF are the fluorescence intensities obtained for Aβ1-42
in the presence and in the absence of inhibitors after subtracting the background, respectively.
i
c
i
c
2
+
4,47
2+
As for the inhibition of Cu -induced Aβ1-42 aggregation , solutions of Cu were prepared
21

from standards to concentration of 75 μM using the HEPES buffer (20 mM, pH 6.60, 150 mM NaCl)
and the Aβ1-42 stock solution was diluted in HEPES buffer (20 mM, pH 6.60, 150 mM NaCl). The
2
+
mixture of the peptide (20 μL, 25 μM, final concentration) and Cu (20 μL, 25 μM, final
concentration), with or without the tested compound (20 μL, 25 μM, final concentration) was
incubated at 37 °C for 24h. After the incubation, 190 μL of 5 μM ThT in 50 mM glycine-NaOH
buffer (pH 8.50) was added. Each assay was run in triplicate. The detection method was the same as
that of self-induced Aβ1-42 experiment.
4
.2.2. Recombinant human MAO-A and –B inhibition studies
Recombinant human MAO-A and –B (5 mg/mL) were purchased from Sigma-Aldrich,
pre-aliquoted and stored at -80 °C. Solutions of test compounds were prepared in DMSO in 2.5 mM
for storage and diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM)
before use. All the enzymatic reactions were conducted in potassium phosphate buffer (100 mM, pH
7
.40, containing KCl 20.2 mM) to a final volume of 500 μL containing kynuramine (45 μM for
MAO-A and 30 μM for MAO-B) and various concentrations of test compounds (0-100 μM) with the
concentration of DMSO lower than 4%. The reactions were initiated by the addition of MAO-A or
MAO-B (7.5 μg/mL) and the solutions were incubated at 37 °C for 30 min. The enzymatic reactions
were terminated by the addtion of 400 μL NaOH (2N) and then 1000 μL water, centrifuged for 10
min at 16000 g. The concentrations of the MAO generated 4-hydroxyquinoline in the reactions were
determined by measuring the fluorescence of the supernatant on a Varioskan Flash Multimode
Reader (Thermo Scientific) with excitation and emission wavelengths at 310 nm and 400 nm,
3
9,41
respectively.
A linear calibration curve was constructed by preparing samples containing
4
-hydroxyquinoline (0.047–1.56 μM) dissolved in 500 μL potassium phosphate buffer. To each
calibration standard, 400 μL NaOH (2N) and 1000 μL water were added. The appropriate control
samples were included to confirm that the test compounds do not fluoresce or quench the
fluorescence of 4-hydroxyquinoline under the assay conditions. IC50 values were calculated from
sigmoidal dose-response curves (graphs of the initial rate of kynuramine oxidation versus the
logarithm of inhibitor concentration). Each sigmoidal curve was constructed from six different
compound concentrations spanning at least three orders of magnitude. Data analyses were carried out
with GraphPad Prism 5 employing the one site competition model. IC50 values were determined in
triplicate and expressed as mean ± SD.
22

4
.2.3. Molecular modeling study
The simulation systems were built based on the X-ray crystal structures of hMAO-A (PDB code:
4
8
2
Z5X) and hMAO-B (PDB code: 2V60), both from the Protein Data Bank. The original ligands
and water molecules were removed and hydrogen atoms were added onto both proteins and cofactors.
Docking studies were performed using Autodock 4.2 program and each docked system was
performed by 200 runs of the Autodock search by the Lamarckian genetic algorithm (LGA). A
cluster analysis was performed on the docking results using a root mean square (RMS) tolerance of
1
.0 and the lowest energy conformation of the highest populated cluster was selected for analysis.
Graphic manipulations and visualizations were done by Autodock Tools or Discovery Studio 2.5
software.
4
.2.4. Antioxidant activity assay
The antioxidant activity was determined by the oxygen radical absorbance capacity-fluorescein
4
9
(
ORAC-FL) assay. 2,2’-Azobis(amidinopropane) dihydrochloride (AAPH) was purchased from
Accela ChemBio Co., Ltd. Fluorescein (FL) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (Trolox) were purchased from TCI (Shanghai) Development. All the assays were
performed with 75 mM phosphate buffer (pH 7.40), and the final reaction mixture was 200 μL.
Antioxidant (20 μL) and fluorescein (120 μL, 150 nM final concentration) were added in the wells of
a black 96-well plate with Trolox as a standard (1-8 μM, final concentration). The mixture was
pre-incubated for 15 min at 37 °C and then placed in a Varioskan Flash Multimode Reader (Thermo
Scientific). AAPH solution (60 μL, 12 mM final concentration) was added rapidly using an
autosampler and the fluorescence recorded every minute for 90 min with excitation at 485 nm and
emission at 535 nm. The plate was automatically shaken prior to each reading. A blank (FL + AAPH)
using phosphate buffer instead of antioxidant and Trolox calibration were carried out in each assay.
The samples were measured at different concentrations (1-10 μM). All the reaction mixture was
prepared in duplicate, and at least three independent assays were performed for each sample.
Antioxidant curves (fluorescence versus time) were normalized to the curve of the blank in the same
assay, and then the area under the fluorescence decay curve (AUC) was calculated. The net AUC of a
sample was obtained by subtracting the AUC of the blank. ORAC-FL values were expressed as
Trolox equivalents by using the standard curve calculated for each sample, where the ORAC-FL
value of Trolox was taken as 1, indicating the antioxidant potency of the tested compounds.
23

4
,50
4
.2.5. Metal-chelating studies
The chelating studies were performed with a Varioskan Flash Multimode Reader (Thermo
Scientific). The UV absorption spectra of compound (14e or 15b) alone or in the presence of CuCl
ZnCl , AlCl , and FeSO were recorded with wavelength ranging from 200 to 600 nm after
2
,
2
3
4
incubating for 30 min at room temperature. The final volume of reaction mixture was 200 μL, and
the final concentrations of tested compound and metals were 37.5 μM. Numerical subtraction of the
spectra of the metal alone and the compound alone from the spectra of the mixture gave the
difference UV-vis spectra due to complex formation.
2
+
The stoichiometry of the compound-Cu complex was determined by titrating the methanol
solution of tested compound with ascending of CuCl . The final concentration of tested compound
was 37.5 μM, and the final concentration of Cu ranged from 3.75 to 150 μM. The UV spectra were
recorded and treated by numerical subtraction of CuCl and tested compound at corresponding
concentrations, plotted versus the mole fraction of tested compound.
2
2
+
2
4
.2.6. In vitro blood-brain barrier permeation assay
The blood-brain barrier penetration of compounds was evaluated using the parallel artificial
4
3
membrane permeation assay of the blood-brain barrier (PAMPA-BBB) described by Di et al.
Commercial drugs were purchased from Sigma and Alfa Aesar. And porcine brain lipid (PBL) was
purchased from Avanti Polar Lipids. The donor plate (MATRNPS50) and the acceptor plate (PVDF
membrane, pore size 0.45 μm, MAIPN4550) were purchased from Millipore. Filter PDVF membrane
units (diameter 25 mm, pore size 0.45 µm) from Pall Corporation were used to filter the samples.
Test compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (70:30) to a
final concentration of 100 μg/mL. Then 350 μL of the diluted compound solution (100 μg/mL) were
added to the donor wells. The filter membrane was coated with PBL in dodecane (selected
empirically as 4 μL volume of 20 μg/mL PBL in dodecane) and the acceptor wells were filled with
2
00 μL of PBS/EtOH (70:30). The acceptor filter plate was carefully put on the donor plate to form a
sandwich (consisting of the aqueous donor with test compound on the bottom, lipid membrane in the
middle and the aqueous acceptor on the top), which was left undisturbed for 18 h at 25 °C. After
incubation, the donor and acceptor plates were separated carefully and the concentration of drug in
the donor and acceptor wells was determined using the Varioskan Flash Multimode Reader (Thermo
Scientific). Every sample was analyzed at ten wavelengths in four wells and in at least three
24

independent runs. P
e
was calculated using the following expression:
+ 1/V ) × t]
P
e
A
= – ln [1 – C (t)/Cequilibrium]/[A × (1/V
D
A
C
equilibrium = [C
D
(t) × V
D
+ C
A
(t) × V
A
]/( V
D
+ V )
A
Where P
e
is permeability in the unit of cm/s. A is effective filter area and t is the permeation time. V
is the volume of acceptor well. C (t) is the compound
concentration in acceptor well at time t, and C (t) is the compound concentration in donor well at
D
is the volume of donor well and V
A
A
D
time t. Results are given as the mean ± SD. In the experiment, 11 quality control drugs of known
BBB permeability were included to validate the analysis set. A plot of the experimental data versus
2
literature values gave a strong linear correlation, P
e
(exp.) = 0.9163 × P
e
(bibl.)−0.2247 (R = 0.9558).
4
3
From this equation and taking into account the limit established by Di et al. for BBB permeation,
-6
we determined that compounds with P values above 3.44 × 10 cm/s could cross the BBB.
e
Acknowledgments
This work was supported in part by the Chinese National Natural Science Foundation
20872099), the Research Fund for the Doctoral Program of Higher Education (20110181110079)
(
and the National Science and Technology Major Project on “Key New Drug Creation and
Manufacturing Program”(2013ZX09301304-002).
References and notes
1
2
. Salloway, S.; Mintzer, J.; Weiner, M. F.; Cummings, J. L. Alzheimer's Dementia 2008, 4, 65.
. Hamilton, G.; Evans, K. L.; MacIntyre, D. J.; Deary, I. J.; Dominiczak, A.; Smith, B. H.; Morris,
A. D.; Porteous, D. J.; Thomson, P. A. Neurosci. Lett. 2012, 510, 6.
3
4
. Schelterns, P.; Feldman, H. Lancet Neurol. 2003, 2, 539.
. Huang, L.; Lu, C.; Sun, Y.; Mao, F.; Luo, Z.; Su, T.; Jiang, H.; Shan, W.; Li, X. J. Med. Chem.
2
012, 55, 8483.
5
6
. Sharma, A. K.; Pavlova, S. T.; Kim, J.; Finkelstein, D.; Hawco, N. J.; Rath, N. P.; Kim, J.; Mirica,
L. M. J. Am. Chem. Soc. 2012, 134, 6625.
. Liu, Q.; Qiang, X. M.; Li, Y.; Sang, Z. P.; Li, Y. X.; Tan, Z. H.; Deng, Y. Bioorg. Med. Chem.
2
015, 23, 911.
25

7
8
9
1
. Huang, M.; Xie, S. S.; Jiang, N.; Lan, J. S.; Kong, L. Y.; Wang, X. B. Bioorg. Med. Chem. Lett.
015, 25, 508.
2
. Bonda, D. J.; Wang, X.; Perry, G.; Nunomura, A.; Tabaton, M.; Zhu, X.; Smith, M. A.
Neuropharmacology 2010, 59, 290.
. Braidy, N.; Poljak, A.; Marjo, C.; Rutlidge, H.; Rich, A.; Jayasena, T.; Inestrosa, N. C.; Sachdev,
P. Front. Aging Neurosci. 2014, 6.
0. Yanovsky, I.; Finkin-Groner, E.; Zaikin, A.; Lerman, L.; Shalom, H.; Zeeli, S.; Weill, T.;
Ginsburg, I.; Nudelman, A.; Weinstock, M. J. Med. Chem. 2012, 55, 10700.
11. Lee, S.; Zheng, X.; Krishnamoorthy, J.; Savelieff, M. G.; Park, H. M.; Brender, J. R.; Kim, J. H.;
Derrick, J. S.; Kochi, A.; Lee, H. J. J. Am. Chem. Soc. 2013, 136, 299.
1
1
2. Modak, M. A.; Parab, P.; Ghaskadbi, S. S. Diabetes/Metab. Res. Rev. 2014, 30, 31.
3. Maynard, C. J.; Bush, A. I.; Masters, C. L.; Cappai, R.; Li, Q. X. Int. J. Exp. Pathol. 2005, 86,
1
47.
1
1
1
1
4. El-Faham, A.; Al Marhoon, Z.; Abdel-Megeed, A.; Khattab, S. N.; Bekhit, A. A.; Albericio, F.
Bioorg. Med. Chem. Lett. 2015, 25, 70.
5. Patil, P. O.; Bari, S. B.; Firke, S. D.; Deshmukh, P. K.; Donda, S. T.; Patil, D. A. Bioorg. Med.
Chem. Lett. 2013, 21, 2434.
6. Pisani, L.; Barletta, M.; Soto-Otero, R.; Nicolotti, O.; Mendez-Alvarez, E.; Catto, M.; Introcaso,
A.; Stefanachi, A.; Cellamare, S.; Altomare, C. J. Med. Chem. 2013, 56, 2651.
7. Pisani, L.; Catto, M.; Leonetti, F.; Nicolotti, O.; Stefanachi, A.; Campagna, F.; Carotti, A. Curr.
Med. Chem. 2011, 18, 4568.
1
1
8. Dias, K. S.; Jr, V. C. Curr. Neuropharmacol. 2014, 12, 239.
9. Agis-Torres, A.; Sölhuber, M.; Fernandez, M.; Sanchez-Montero, J. M. Curr. Neuropharmacol.
2
014, 12, 2.
2
2
0. Prati, F.; Uliassi, E.; Bolognesi, M. L. Med.chem.commun. 2014, 5, 853.
1. Okombi, S.; Rival, D.; Bonnet, S.; Mariotte, A.-M.; Perrier, E.; Boumendjel, A. J. Med. Chem.
2
006, 49, 329.
2. Maya, Y.; Ono, M.; Watanabe, H.; Haratake, M.; Saji, H.; Nakayama, M. Bioconjugate Chem.
008, 20, 95.
3. Ono, M.; Maya, Y.; Haratake, M.; Ito, K.; Mori, H.; Nakayama, M. Biochem. Biophys. Res.
2
2
2
26

Commun. 2007, 361, 116.
2
2
2
4. Watanabe, H.; Ono, M.; Kimura, H.; Kagawa, S.; Nishii, R.; Fuchigami, T.; Haratake, M.;
Nakayama, M.; Saji, H. Bioorg. Med. Chem. Lett. 2011, 21, 6519.
5. Geldenhuys, W. J.; Funk, M. O.; Van der Schyf, C. J.; Carroll, R. T. Bioorg. Med. Chem. Lett.
2
012, 22, 1380.
6. Morales-Camilo, N.; Salas, C. O.; Sanhueza, C.; Espinosa-Bustos, C.; Sepúlveda-Boza, S.;
Reyes-Parada, M.; Gonzalez-Nilo, F.; Caroli-Rezende, M.; Fierro, A. Chem. Biol. Drug Des.
2
015, 85, 685.
2
2
7. Ono, M.; Yoshida, N.; Ishibashi, K.; Haratake, M.; Arano, Y.; Mori, H.; Nakayama, M. J. Med.
Chem. 2005, 48, 7253.
8. Meguellati, A.; Ahmed-Belkacem, A.; Yi, W.; Haudecoeur, R.; Crouillère, M.; Brillet, R.;
Pawlotsky, J. M.; Boumendjel, A.; Peuchmaur, M. Eur. J. Med. Chem. 2014, 80c, 579.
9. Zhang, M.; Xu, X. H.; Cui, Y.; Xie, L. G.; Kong, C. H. Pest Manage. Sci. 2012, 68, 1512.
0. Haudecoeur, R.; Ahmed-Belkacem, A.; Yi, W.; Fortuné, A.; Brillet, R.; Belle, C.; Nicolle, E.;
Pallier, C.; Pawlotsky, J.-M.; Boumendjel, A. J. Med. Chem. 2011, 54, 5395.
2
3
3
3
1. Liu, B.; Zhang, M.; Xu, X. H.; Xie, L. G.; Chinese Patent CN 101914081 A, 2010.
2. Lu, C.; Guo, Y.; Li, J.; Yao, M.; Liao, Q.; Xie, Z.; Li, X. Bioorg. Med. Chem. Lett. 2012, 22,
7
683.
3
3
3. Buechi, G.; Weinreb, S. M. J. Am. Chem. Soc. 1971, 93, 746.
4. Manjulatha, K.; Srinivas, S.; Mulakayala, N.; Rambabu, D.; Prabhakar, M.; Arunasree, K. M.;
Alvala, M.; Rao, M. V. B.; Pal, M. Bioorg. Med. Chem. Lett. 2012, 22, 6160.
5. Lee, C. Y.; Chew, E. H.; Go, M. L. Eur. J. Med. Chem. 2010, 45, 2957.
3
3
3
6. King, T. J.; Hastings, J. S.; Heller, H. G. J. Chem. Soc., Perkin Trans. 1975, 1455.
7. Manuela, B.; Carlo, B.; Laura, B. M.; Andrea, C.; Carlo, M.; Vincenza, A. Chembiochem 2007, 8,
2
152.
3
8. Sang, Z. P.; Qiang, X. M.; Li, Y.; Yuan, W.; Liu, Q.; Shi, Y. K.; Ang, W.; Luo, Y. F.; Tan, Z. H.;
Deng, Y. Eur. J. Med. Chem. 2015, 94, 348.
3
4
9. Legoabe, L. J.; Petzer, A.; Petzer, J. P. Bioorg. Chem. 2012, 45, 1.
0. Novaroli, L.; Reist, M.; Favre, E.; Carotti, A.; Catto, M.; Carrupt, P. A. Bioorg. Med. Chem.
2
005, 13, 6212.
27

4
1. Strydom, B.; Malan, S. F.; Castagnoli, N.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. 2010, 18,
018.
1
4
4
4
2. Dávalos, A.; Gómez-Cordovés, C.; Bartolomé, B. J. Agric. Food Chem. 2004, 52, 48.
3. Di, L.; Kerns, E. H.; Fan, K.; Mcconnell, O. J.; Carter, G. T. Eur. J. Med. Chem. 2003, 38, 223.
4. Sang, Z. P.; Li, Y.; Qiang, X. M.; Xiao, G. Y.; Liu, Q.; Tan, Z. H.; Deng, Y. Bioorg. Med. Chem.
2
015, 23, 668.
4
5. Michela, R.; Elena, S.; Manuela, B.; Andrea, C.; Luisa, C.; Nicoleta, P.; David W, M.; Andrea, T.;
Maria L, B.; Anna, M. J. Med. Chem. 2008, 51, 4381.
4
4
4
6. Li, R. S.; Wang, X. B.; Hu, X. J.; Kong, L. Y. Bioorg. Med. Chem. Lett. 2013, 23, 2636.
7. Geng, J.; Li, M.; Wu, L.; Ren, J.; Qu, X. J. Med. Chem. 2012, 55, 9146.
8. Nicoletta, D.; Adriana, B.; Rossella, F.; Luca Proietti, M.; Matilde, Y. E.; Francesco, O.; Stefano,
A. J. Med. Chem. 2011, 54, 2155.
4
5
9. Sang, Z. P.; Qiang, X. M.; Li, Y.; Wu, B.; Zhang, H.; Zhao, M. G.; Deng, Y. Chem. Biol. Drug
Des. 2015, 86, 1168.
0. He, Y.; Yao, P. F.; Chen, S. B.; Huang, Z. H.; Huang, S. L.; Tan, J. H.; Li, D.; Gu, L. Q.; Huang,
Z. S. Eur. J. Med. Chem. 2013, 63, 299.
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Graphical Abstract
29